Alox15 restrains glycolysis to support M2 macrophage effector function during helminth immunity

Abstract

Macrophage polarization by type-2 cytokines is central to anti-helminth immunity and tissue repair. While some hallmark changes in macrophages are well-characterized and associated with protection against helminths, it is still unclear how macrophages exert their anti-helminth effects. In this context, we investigated Arachidonate 15-lipoxygenase (Alox15), a lipoxygenase well known for its role in macrophage polarization in the context of metabolic diseases, and a hallmark of type-2 macrophage (M2) human polarization. We show that in the absence of Alox15, M2 cannot trap and kill helminths. Surprisingly, expression of M2 markers was normal despite a loss of function. Instead, we found a concomitant increase in pro-inflammatory responses due to an uncontrolled activation of glycolysis. We further show that activation of Peroxisome proliferator-activated receptor-delta (PPAR-δ) by lipids downstream of Docosapentaenoic acid (DPA) can restore normal glycolysis control, highlighting a novel role of lipids in the fine-tuning of the metabolic support required for optimal macrophage polarization.

Introduction

Helminths are large multicellular organisms that have co-evolved with their host displaying very complex host-pathogen relationships. Because of this, despite rarely causing mortality, helminths cause a high morbidity level, especially in tropical and subtropical areas with lower economic status, where burdens are still high (Diemert et al., 2018). Despite intensive research, to date, there is no vaccine for helminth infections of humans and our understanding of protective responses against re-infection are still incomplete. Laboratory models of the three main types of helminthiases (soil transmitted helminth (STH), filariasis and schistosomiasis) have shown that communication between the adaptive and innate immune systems is required for such protection to occur. Macrophages, in particular, have been shown to be central to protective immune responses against helminths in various murine models, such as filariae and STH (Coakley and Harris, 2020, Lechner et al., 2021). Alternative or “M2” activation triggered by the type 2 cytokines IL-4 and IL-13, by the parasite itself, or by specific antibodies, have been shown to be required to control parasite motility and survival (Bouchery et al., 2015, Chen et al., 2022, Esser-von Bieren et al., 2013). These M2 macrophages express several STAT6-induced effector molecules such as Arginase 1 (Arg1), Resistin-like molecule alpha (Relm-α, Retnla) and chitinase-like protein Ym-1 (Chil3) that have been suggested to contribute to host defense against helminths. Our group and others have described a role for Arginase-1 in protection in rodent hookworm models (Bouchery et al., 2015, Chen et al., 2022, Esser-von Bieren et al., 2013). In other parasites, such as Leishmania spp. Arg1/nitric oxide synthase 2 (Nos2) balance is thought to be crucial for protection (Goldman-Pinkovich et al., 2016, Muxel et al., 2017). Interestingly, a recent paper challenged the importance of Arginase-1 in protection against helminths (Westermann et al., 2023) and this is in line with our own observations that treatment blocking Arginase-1 activity was only partially reversing protection (Bouchery et al., 2015). Altogether, this prompted us to evaluate other key metabolic molecules in M1/M2 polarization. In an effort to make the finding more translatable (due to controversy on the importance of Arginase-1 in human macrophages (Thomas and Mattila, 2014)), we looked for key molecules both highly expressed in human and mouse macrophages polarized by type 2 cytokines.

In humans, the lipoxygenase Alox15 is highly expressed in response to type 2 cytokines in macrophages both in vitro and in vivo (Ackermann et al., 2017, Czimmerer et al., 2012, Gundra et al., 2014, Schif-Zuck et al., 2011, Snodgrass et al., 2018, Uderhardt et al., 2012, Werz et al., 2018, Henkel et al., 2019). Alox15 is a lipoxygenase that catalyzes the stereo-specific peroxidation of polyunsaturated fatty acids (PUFAs). Alox15 functions to generate phospholipid oxidation products crucial for orchestrating the removal of apoptotic cells as well as synthesizing precursor lipids required for the production of specialized pro-resolving mediators (SPMs) that facilitate the resolution of inflammation (Snodgrass and Brune, 2019). In mice, Alox15 has been implicated in many models of metabolic syndrome such as atherosclerosis, diabetes, obesity, and hypertension (Bleich et al., 1999, Cyrus et al., 2001, Cyrus et al., 1999) in which macrophage polarization also plays a key role. Recently, Alox15 has also been shown to be required for the maintenance of alveolar macrophages (Pernet et al., 2023). Alox15 downstream products are endogenous ligands for the transcription factors PPAR-γ and PPAR-δ (Snodgrass and Brune, 2019, Huang et al., 1999).

In the context of helminth infection, Alox15 and its products have been shown to be upregulated in lung macrophages 7 days post-infection with N. brasiliensis and in intestinal tissue during secondary infection with Heligmosomoides polygyrus (Bosurgi et al., 2017, Entwistle et al., 2017, Henkel et al., 2019). Furthermore, lipid pathways associated with Alox15 activity have been shown to be increased in macrophages after filarial infection (Thomas et al., 2012). However, the role of Alox15 in M2 polarization and in protection against helminths has never been addressed to date. In the current study, we demonstrate the crucial role of the Alox15 pathway in M2 polarization through control of a glycolysis:oxidative phosphorylation (OXPHOS) balance and its associated protective role in the context of parasitic infection.

Results

Alox15 expression is required for protection against hookworm re-infection

N. brasiliensis (Nb) is a rodent hookworm model that causes long-term activation of IL-4 polarised macrophages in the lungs, through which the parasite migrates on its way to the intestine. Alox15 upregulation in the lungs of Nb infected mice has previously been reported at day 7 (D7) post-infection (Bosurgi et al., 2017). We confirmed this upregulation of Alox15 in lung tissue and saw a low but persistent expression of Alox15 transcripts up to D40 post-infection and 2 days post re-infection (Fig. S1 A). Because Alox15 can be expressed by many cell types, we performed single cell sequencing of CD45⁺ lung cells to determine which cell type was principally expressing Alox15 after Nb infection and reinfection (Fig. 1, A and B, Fig. S1 B). Consistent with the literature, macrophages and granulocytes (eosinophils and neutrophils) are the main innate cell type that increase in number 40 days post-infection and 2 days post-reinfection. Alox15 expression was specifically increased upon infection in interstitial macrophages (IMs), pro-alveolar macrophages (pro-AMs) and granulocytes, with the highest expression in IMs. Of note, a transitional population of monocytes that seems to give rise to IMs was constitutively expressing Alox15 and continued doing so after infection. We further validated this increase in interstitial macrophage-specific expression of Alox15 by qPCR 7 days post-infection (Fig. S1 A).

Figure 1. Alox15 deficiency leads to impaired protection against rodent hookworm and reduced wound healing in the mouse lung.

(A) Wild-type (WT) C57BL/6/J mice were infected with 550 Nb L3s subcutaneously of left untreated (D0). 40 days post infection, mice were either left untreated (D40) or reinfected subcutaneously with 550 Nb L3s (D40 +D2) for 2 days. Lungs were harvested and CD45⁺ cells were isolated and prepared for single-cell sequencing on the BD Rhapsody platform (n= 4 mice per condition). UMAP visualization of scRNA-seq data of D0, D40 and D40+D2 identifying 20 distinct cell clusters or visualized by treatment conditions. (B) Normalized expression of Alox15 transcripts across all cell types and shown by treatment conditions. (C) Alox15^-/- and WT controls were infected twice with 250 Nb L3. Two days after secondary infection, larvae were collected and enumerated from BALF and Baermann of lung tissue. N=5 mice per group, 2 independent experiments (D) Haemorrhage was assessed 2 days post-primary infection in BALF. N=5 mice per group, 2 independent experiments. (E) Haemorrhage was assessed 5 days after a secondary infection. (F) Intestinal worm burden was assessed 5 days post-primary infection by Baermann. N=4-5 mice per group, 3 independent experiments. (G) Prussian blue staining from WT and Alox15^-/- mice 5 days post-infection. N=4-5 mice per group, 2 independent experiments. Scale bar 100 µm. NS, non-significant (p>0.05); *, p≤0.05; ** p≤0.05; *** p≤0.001.

To explore the role of Alox15 in macrophage polarization in response to a type-2 environment, we utilized Alox15 knock-out mice (Alox15^-/-) in which a neomycin resistance cassette has been inserted in exon 3 (Green-Mitchell et al., 2013). As previously reported, alveolar macrophages number are decreased in Alox15^-/- in healthy state (Fig. S1 C, (Pernet et al., 2023)), but other Alox15 expressing cells in the lungs are not (Fig. S1 D). It is well-established that upon reinfection with Nb, IL-4 polarized macrophages are able to trap and kill the parasite, resulting in a decreased worm burden in the lungs. We thus assessed the consequence of Alox15 deletion on the protective response against Nb. At 2 days post-reinfection, Alox15^-/- mice could not control their infection, with a higher burden of lung larvae as compared to wild-type (WT) controls (Fig. 1 C). The lung immune response was largely unaffected, with no change in Th2 cells, neutrophils, alveolar macrophages, interstitial macrophages (Fig. S1 E and F). Of note we observed an increase in eosinophils during re-infection in Alox15^-/- mice. Overall lung cytokines characterization did reveal only an increase in IL-17, but no significant change in type 2 cytokines (Fig. S1 G). During pulmonary migration, Nb causes extensive damage to the lungs that results in short-term hemorrhage. IL-4 polarized macrophages have been shown to control hemorrhage in both primary and secondary infection (Chen et al., 2012). We thus assessed pulmonary hemorrhage and found that hemorrhage 2 days post-reinfection was higher in Alox15^-/- mice than in WT counterparts (Fig. 1 D).

During a primary infection, inflammatory foci and elevated red blood cell levels in bronchoalveolar lavage fluid are resolved by D5 in WT mice. With a classical infective dose of 550 L3, Alox15^-/- mice suffered a heightened level of hemorrhage from D3 post-infection, requiring us to lower the dose of infection to avoid adverse events (Fig. S1 H). With a low dose of infection (250 L3), equivalent numbers of larvae and hemorrhage were found between WT and Alox15^-/- D2 post-infection (Fig. S1 H and I). Interestingly, the hemorrhage was similar at D2 post infection (Fig. S1 J) but not resorbed at D5 post-infection in Alox15^-/- mice as compared to WT counterparts (Fig. 1 E). This suggests that the sustained hemorrhage observed 5 days post-infection results from decreased repair rather than enhanced pulmonary damage. IL-4 polarized macrophages contribute to timely wound repair but not the trapping of lung larvae following primary infection (Chen et al., 2012). To validate this hypothesis, we performed Prussian blue staining on lung sections of WT and Alox15^-/- mice at D5 post-infection (Fig. 1 G). While the proportion of hemosiderin-laden macrophages (to total macrophage) was similar between genotypes (Fig. 1 F), Alox15^−/− lungs exhibited macrophages with visibly stronger iron staining together with larger areas of iron deposition within the tissue. Morphologically, these macrophages appeared enlarged, and darker consistent with increased intracellular iron burden. Importantly, we did not observe increased tissue damage, inflammatory infiltrates, or architectural disruption in Alox15^−/− lungs. The intestinal worm burden D5 post-infection was not significantly different between WT and Alox15^-/- mice (Fig. 1 F). Altogether, this suggests that Alox15 is important for both the lung tissue repair and for killing of helminth larvae.

Macrophage Alox15 expression is required for larvicidal activity and tissue repair

Alox15 is expressed in many cells in the lungs and the impaired tissue repair and larval trapping observed in Alox15^-/- mice after primary or secondary Nb infection respectively, could be due to defects in macrophages or other cell types and involve extrinsic signaling (such as modified eicosanoids in the lung micro-environment) or intrinsic signaling (due to the loss of Alox15 expression in cells themselves). Furthermore, Alox15 is a hallmark of M2 polarization both in human and mice (Fig. S2 A) (Derlindati et al., 2015, Shinoda et al., 2023, Snodgrass et al., 2021). To determine whether the loss of Alox15 in macrophages alone can recapitulate the loss of protective immunity seen in Alox15^-/- mice, we used intranasal adoptive transfer of IL-4 polarised macrophages (Fig. 2 A) into naïve mice to mimic the protection usually observed during secondary infection (Bouchery et al., 2015). Wild-type or Alox15^-/- bone-marrow derived macrophages were harvested and polarized with IL-4 in vitro to generate M(IL-4) that were transferred intranasally into naïve wild-type animals, which were infected with Nb 1 day later. Mice that received wild-type M(IL-4) had lower worm burdens than those that received Alox15^-/- M(IL-4), while the worm burden was similar between untreated mice (no macrophage transfer) and mice that received Alox15^-/- M(IL-4) (Fig. 2 A). The viability of M(IL-4) macrophages from Alox15^-/- or Wild-type was not significantly different before transfer (Fig. S2 B), nor was the viability of interstitial macrophages after transfer (Fig. S2 C). Overall, the immune profile in the lungs was not affected by the treatment, with no difference between WT and Alox15^-/- mice for any of the cell type studied (Fig. S2 D and E). Altogether, these results directly implicate intrinsic Alox15 signaling in the protective response of macrophages to Nb.

Figure 2. Alox15 expression in macrophages is required for larvicidal activity and tissue repair.

Murine bone marrow-derived macrophages were grown from WT or Alox15^-/- mice and polarized with recombinant IL-4 for 24 hours. (A) 2. 10⁶ BMDM(IL-4) cells were intranasally administered to naïve C57BL/6/J WT mice. The next day, mice were infected with 550 L3 of Nb. Two days after infection, larvae were collected and enumerated from BALF and Baermann of lung tissue. N=4-5 mice per group, pool of 3 independent experiments. (B-E) In vitro and ex vivo co-cultures of macrophages and Nippostrongylus brasiliensis (Nb) larvae were used to assess the requirement of Alox15 for anti-helminth functions. (B) BMDM activated with IL-4 for 24hr were co-cultured with 100 third-stage N. brasiliensis larvae (Nb L3). The percentage of larvae with more than 10 cells attached was assessed for each genotype. Each point represent a single mouse or pool of mouse. N=12, pooled from 4 independent experiments. (C) Representative brightfield microscopy of IL-4 stimulated BMDM bound to Nb L3 as described in B. (D) Viability of larvae from B., was measured using the cell impermeable fluorescent dye, Sytox Green. Each point represent a single mouse or pool of mouse. N=6, pooled from 3 independent experiments (E) Lungs of D7 infected WT or Alox15^-/- mice were digested enzymatically and interstitial macrophages, alveolar macrophages, transitional macrophages were co-cultured with Nb L3 for 4 days without further stimulation. Each point represent a single mouse or pool of mouse or a technical duplicate for a given mouse. N=2-4 mice, pool of 4 independent experiments. (F) Viability of larvae co-cultured with interstitial macrophages was measured using the cell impermeable fluorescent dye, Sytox Green. Each point represent a single mouse or pool of mouse. N=6, pooled from 3 independent experiments

Because the mechanisms by which macrophages exert larvicidal effects are incompletely understood, we further explored the importance of Alox15 in larval killing by measuring macrophage-mediated trapping in vitro [20]. We set up in vitro co-cultures of bone marrow-derived M(IL-4) with Nb third-stage larvae (L3). After 5 days of co-culture, as previously reported (Chen et al., 2014), wild-type M(IL-4) were bound to Nb L3 (Fig. 2 B and C). In contrast, Nb L3 trapping by M(IL-4) generated from Alox15^-/- mice was markedly reduced with less macrophages found bound to each larva (Fig. 2 B and C). We further assessed if the larvicidal activity of Alox15^-/- macrophages was compromised by measuring larval cuticle permeability with the cell impermeant SYTOX Green dye. In line with the trapping, we observed a marked increase in the percentage of dead larvae trapped by WT BMDM as compared to Alox15^-/- (Fig. 2 D). As we previously observed Alox15 expression in alveolar macrophages (AMs), interstitial macrophages (IMs) as well as transitional monocytes after Nb infection (Fig. S1 A), we sort isolated those macrophages subpopulation 7 days with Nb (Fig. S2 F) to refine which subpopulation had a predominant role in the observed phenotype As previously reported, we found AMs bind larvae with low efficiency in vitro in WT, but binding was enhanced in Alox15^-/- AMs (Fig. 2 E). Less than 20% binding was observed for transitional monocytes from both WT and Alox15^-/- mice. As for BMDM, fewer larvae were bound by IMs when isolated from infected Alox15^-/- mice compared to WT (Fig. 2 E). In line with reduced trapping, we also observed a decrease larval killing by Alox15^-/- macrophages (Fig. 2 F).

Altogether, our results show that Alox15 deficiency affects macrophage capability to trap helminths parasites, particularly in the lung parenchyma.

Alox15 is required for larvicidal activity in an Arginase-1 independent manner

We further explored the role of Alox15 expression in regulating the phenotype of IL-4 activated macrophages. Wild-type or Alox15^-/- bone marrow-derived macrophages were generated and polarized with or without IL-4. The expression of canonical M2 signature genes was studied by real-time quantitative PCR (Fig. 3 A). Despite a strong literature pointing towards the importance of Arginase-1 (Bouchery et al., 2015, Chen et al., 2022, Esser-von Bieren et al., 2013) in the anti-helminth role of macrophages, Arginase-1 expression was surprisingly higher in Alox15^-/- than in WT M(IL-4) (Fig. 3 A). MMP-9 gene expression was significantly lower in Alox15^-/- M(IL-4) than WT, while other genes typically associated with M(IL-4) polarization (Retnla, Ym1, Fn1, MMP12, MRC1, TIMP-1) were mostly unaffected by Alox15 deletion. We further confirmed that the protein level of RELMα, a well-known molecule associated with M2 induced protection in helminth was not affected by Alox15 deficiency (Fig. 3 B). Due to the surprising higher gene expression of Arginase-1 in Alox15^-/- M(IL-4), despite a loss of functional parasite killing, we next examined the protein expression and activity of Arginase-1. Arginase-1 expression was slightly higher in Alox15^-/- M(IL-4) than WT control (Fig. 3 C), whilst Arginase-1 activity, measured by urea production, was similar in both WT and Alox15^-/- mice (Fig. 3 D). To assess the relative importance of Arginase-1 and Alox15 expression in the anti-helminth function of M(IL-4), we once again set up a co-culture assay of M(IL-4) either from WT or Alox15^-/- mice, with or without the addition of the Arginase-1 inhibitor BEC (S-(2-Boronoethyl)-l-Cysteine). As previously reported, binding of larvae by wild-type M(IL-4) was reduced by Arginase-1 inhibition (Fig. 3 E). However, BEC treatment did not further reduce parasite binding by Alox15^-/- M(IL-4) (Fig. 3 E).

Figure 3. Alox15 deficiency cause a loss of function in M(IL-4) independently of Arginase-1 activity.

Murine bone marrow-derived macrophages were grown from C57BL/6/J (WT) or Alox-15^-/- mice and polarized or not (M0) with recombinant IL-4 (M(IL4)) for 24 hours. (A) Gene expression of hallmark genes associated with M(IL-4) polarization obtained by multiplex RT-qPCR and expressed as z-score. N=3 (B) Alox15^-/- and WT controls were infected with 250 Nb L3. Two days after secondary infection, lungs were collected and RELM-α protein expression was analysed by immune-histofluorescence. Representative results from 2 independent experiment with N=4. (C) Mean Fluorescence Intensity of Arginase-1 analyzed by flow cytometry. N=3, representative of two experiments (D) Arginase-1 activity measured by urea production. N=3, 2 independent experiments. (E) BMDM(IL-4) were co-cultured with 100 third-stage N. brasiliensis larvae (Nb L3). The percentage of larvae with more than 10 cells attached was assessed for each genotype and condition. N=4-6, pool of 2 independent experiments. (F) BMDM were grown from C57BL/6/J (WT) or LysMcre Arg1 flox mice and polarized with recombinant IL-4 (M(IL4)) for 24 hours. Immunofluorescence against Alox15 was performed and acquired on a confocal microscope. Intensity of staining was analyzed using Fiji. N=2-3, pool of 2 independent experiments (G) RNA-sequencing was performed on BMDM(IL-4) from WT or Alox15^-/- with or without BEC blocade. Heatmap of M2 gene set. N=4. (H) Enrichment plots of M2 gene signature comparing IL-4 treatment plus BEC blocade to IL-4 only (FDR = 7.58^-01). Enrichment plots of M2 gene signature comparing IL-4 treatment in WT and Alox15^-/- BMDM (FDR = 8.54^-01).

While Arginase-1 expression is directly triggered by IL-4 stimulation, it has previously been shown that efferocytosis is required for Alox15 gene expression to increase in response to IL-4 stimulation (Bosurgi et al., 2017, Snodgrass and Brune, 2019). This suggests that Arginase-1 expression could be independent, or upstream, of Alox15 expression. We thus assessed the impact of Arginase-1 blockade on Alox15 expression levels in WT M(IL-4) by immunohistochemistry. We found that Alox15 expression was reduced when Arginase-1 was absent (Fig. 3 F), suggesting that Arginase-1 expression is upstream of Alox15 and the role of Arginase-1 in M(IL-4) mediated trapping and killing of helminth parasites may occur through indirect pathways (Chen et al., 2022).

Despite several reports of Arginase-1 being required for parasite killing or trapping (Bouchery et al., 2015, Chen et al., 2022, Esser-von Bieren et al., 2013), its role in macrophage polarization following IL-4 activation has not been studied in depth. We performed bulk RNA sequencing of WT BMDM treated for 24 hours with IL-4 and/or BEC and compared it to their Alox15^-/- counterparts. Surprisingly, no differentially expressed genes were found when comparing M(IL-4+BEC) to M(IL-4). Despite BEC treatment and Alox15 depletion having similar functional consequences both in vitro and in vivo in terms of loss of parasite trapping/killing, we could not find any genes that were commonly differentially downregulated between the two treatments as compared to M(IL-4) from WT suggesting that Alox15 and Arginase-1 might fall into two distinct anti-helminth pathways or modalities, rather than Alox15 being downstream of Arginase-1. Of note, Alox15 expression was 1.26 times upregulated after BEC treatment as compared to WT (log fold change, p-value = 0.06) suggesting a loop of regulation between Alox15 and Arginase-1 expression. We used a gene set enrichment approach to assess how the overall M2 polarization was affected both in Alox15^-/- BMDM or in BEC treated M(IL-4) as compared to WT M(IL-4). Intriguingly given the loss of function, the M2 polarization was not affected by either treatment at least at the time point tested (24 hours post-polarization) (Fig. 3 G and H).

Alox15 deficiency in macrophages causes upregulation of pro-inflammatory pathways and metabolic rewiring

To refine our understanding of the role of Alox15 in IL-4 activation of macrophages, we analyzed transcriptomic changes during the transition from resting (M0) to IL-4 stimulated (M(IL-4)) BMDM for both WT and Alox15^-/- mice. As expected, IL-4 stimulation caused a large number of genes to be up- and down-regulated in WT mice (Fig. S3 A, Table S1). In contrast, direct comparison of M(IL-4) from WT and Alox15^-/- mice revealed a relatively restricted transcriptional signature associated with Alox15 deficiency with only 87 statistically significant differentially regulated genes (Fig. S3 B), with 23 downregulated and 64 upregulated. Of note, one hallmark M2 gene, namely Chil3, was identified as downregulated (fold change -0.8). Differentially expressed genes were predominantly upregulated in Alox15^-/- macrophages, including Tom1l1, Acap1, and the interferon-responsive gene Xaf1, suggesting alterations in intracellular signalling, membrane trafficking, and inflammatory/stress-response pathways. Metascape (Zhou et al., 2019) enrichment pathway analysis revealed a clear association with “mitotic cell cycle process” (GO:1903047) as well as in “lung development” and “intracellular receptor signaling pathway” (Fig. S3 C). Association between cell cycle and Alox15 deficiency in macrophages has recently been reported (Pernet et al., 2023), although the authors showed that at resting state this defect is limited to alveolar macrophages and that absence of Alox15 decreased cell proliferation.

Optimal activation of ‘M2’ macrophages, in particular in the context of helminth infection, has previously been reported to require both IL-4 and/or IL-13 stimulation as well as apoptotic cell recognition (Bosurgi et al., 2017) and is further enhanced by surfactant produced in the lungs (Minutti et al., 2017). Nb infection causes important damage to the lungs and efferocytosis has been shown to be essential in this model for optimal activation of M2 macrophages (Bosurgi et al., 2017, Chen et al., 2012). Furthermore, macrophages have been shown to have tissue specificity, with lung macrophages being markedly different from bone marrow-derived macrophages (Gordon and Pluddemann, 2017). To validate our findings in the context of helminth infection we assessed the role of Alox15 in IL-4 polarization in vivo and performed bulk RNA sequencing on total CD45⁺ adherent fraction of the lungs purified 7 days post-infection with N. brasiliensis either in WT or in Alox15^-/- mice. Deconvolution of the data confirmed the presence of alveolar macrophages, interstitial macrophages, dendritic cells and monocytes (Fig. S3 D). Marked changes were observed with 1653 genes found to be upregulated and 1073 downregulated in Alox15^-/- macrophages as compared to WT counterparts (Fig. 4 A, Table S2). We next used Metascape pathway enrichment analysis of genes specifically upregulated and downregulated in Alox15^-/- lung macrophages. Interestingly, upregulated genes were associated with immune response pathways characteristic of type 1 response (‘response to virus’ (GO: 0009615) and ‘response to interferon-beta’ (GO:0035456)) (Fig. 4 B). We thus further looked at hallmark genes typical of ‘M2’ and ‘M1’ polarization. As reported earlier, the changes to the M2 phenotype were minor with enhancement of Arginase-1 expression and MMP-9 (Fig. 4 C). In contrast, genes typically associated with M1 polarization were expressed at markedly higher levels in Alox15^-/- macrophages than in WT controls (Fig. 4 C).

Figure 4. Alox15 deficiency enables development of a M1/M2 hybrid after IL-4 polarization.

(A) mRNA expression profiles (volcano plots) as determined by RNA-sequencing isolated from total lung macrophages from WT or Alox-15^-/- mice 7 days post-infection with Nb L3. Red, significantly differentially expressed (DE) genes with a P value <0.01 (n=4-5, representative of 2 independent experiments). (B) Pathway enrichment using Metascape of DE genes upregulated in Alox-15^-/- lung macrophages. (C) Gene expression of hallmark genes associated with macrophages M1 or M2 polarization obtained from RNA sequencing and expressed as z-score. * Corrected P-value <0.05. (D) Pathway enrichment using Metascape of DE genes downregulated in Alox-15^-/- lung macrophages. (E) Pathway enrichment using Metascape of a cluster of DE genes (cluster 7) downregulated in Alox-15^-/- lung macrophages with a strong signature in metabolism.

As no decrease in hallmark ‘M2’ genes was observed in M2 from Alox15^-/- mice, we conducted pathway enrichment analysis on downregulated genes specifically associated with Alox15 deficiency. Those were linked to cell cycle and cell metabolism (Fig. 4 D). Reduced proliferation of alveolar macrophages, but not of other macrophage populations, has previously been reported in Alox15^-/- mice (Pernet et al., 2023). We have previously shown that Alox15^-/- alveolar macrophages have no defect in Nb L3 trapping (Fig. 2 E), suggesting the defect in proliferation of AMs is unlikely to affect our phenotype. To rule out a role of proliferation in the phenotype we have described, we assessed proliferation of BMDM from WT or Alox15^-/- mice stimulated or not with IL-4. We observed that BMDM stimulated with IL-4 from Alox15^-/- mice had no difference in Ki67 expression after one day of IL-4 stimulation (Fig. S3 E). We thus concluded that a defect in cell cycle was unlikely to be sufficient to explain the lack of larvicidal activity observed in Alox15^-/- M(IL-4), thus we decided to further focus on the metabolic changes observed in Alox15^-/- lung macrophages. To separate the effect of cell cycle and metabolism we conducted cluster enrichment analysis (Fig. S3 F). The genes downregulated in Alox15^-/- lung macrophages formed three separate clusters enriched for cell cycle (cluster 1) or metabolic (cluster 7) and to a less significant extent RNA metabolism (cluster 10) responses respectively (Fig. S3 F, Table S3). Pathway enrichment analysis on the cluster 7 revealed a strong downregulation of lipid metabolic and biosynthetic pathways in Alox15^-/- lung macrophages (Fig. 4 E). The genes upregulated in Alox15^-/- lung macrophages formed three separate clusters enriched for inflammatory/anti-bacterial responses (cluster 3), anti-viral/IFN-β response (cluster 6) and repair processes (cluster 9).

Taken together, Alox15 deficiency does not abrogate M2 polarization globally but rather selectively modulates discrete transcriptional programs, such as pro-inflammatory and metabolic responses.

Alox15 deficient macrophages have a high energetic profile after IL-4 polarization

Polarization of macrophages by IL-4 or IFN-γ has been shown to induce different metabolic requirements (Van den Bossche et al., 2017). As the main change observed by RNAseq of lung macrophages was linked to metabolic pathways, we further explored the transcriptomic signature of enzymes involved in glycolysis and OXPHOS in lung macrophages of WT or Alox15^-/- background. In line with the increased expression of M1 polarization genes, genes associated with glycolysis were upregulated in Alox15^-/- lung M(IL-4) (Fig. 5 A). In contrast, analysis of mitochondrial metabolic pathways revealed limited and selective transcriptional changes, with a small subset of oxidative phosphorylation–associated genes showing reduced expression in Alox15^-/- macrophages (Fig. S3G). Similarly, modest changes were observed in genes linked to fatty acid oxidation and lipolysis (Fig. S3 H and I).

Figure 5. Alox15 deficient M1/M2 hybrid have a heightened energetic profile after IL-4 stimulation.

(A) Total lung macrophages from WT or Alox-15^-/- mice 7 days post-infection with Nb L3 were isolated and RNA sequenced. (A) Gene expression of hallmark genes associated with the glycolytic pathway obtained from RNA sequencing and expressed as z-score. DEGs are marked with a * and were identified in cluster 7 from Fig. S3 G. (B) Energy map obtained by Seahorse flux analysis on BMDM derived from WT or Alox-15^-/- mice and polarized or not (M0) with recombinant IL-4 (M(IL4)) for 24 hours or LPS (M(LPS)) for 12 hours. (n=3-4, representative of 6 independent experiments). (C) OCR and SRC (Spare respiratory Capacity) of BMDM stimulated or not with IL-4 or LPS from both WT or Alox-15^-/- mice at baseline and after sequential treatment (vertical lines) with glucose (Gluc.), oligomycin (OM), FCCP and Pyruvate, Antimycin-A (AA) and Rotenone (rot) as a combined Mito and Glycolytic stress test [79]. Data representative of 3-4 independent experiments, n = 5 wells per condition, each condition with 1-3 mice per experiment. Statistical post-hoc test compared same polarization between genotypes or between polarization within genotypes. Non-reported comparisons are non-significant. (D) ECAR and Glycolytic capacity of BMDM stimulated or not with IL-4 or LPS from both WT and Alox-15^-/- mice as described in C. Statistical post-hoc test compared same polarization between genotypes or between polarization within genotypes. Non-reported comparisons are non-significant. (E) BMDM stimulated with IL-4 in normal medium (IL-4), with IL-4 in Seahorse medium in absence of glucose (IL-4 low glucose) or in Seahorse medium with glucose (IL-4 high glucose) were co-cultured with 100 third-stage N. brasiliensis larvae (Nb L3). The percentage of larvae with more than 10 cells attached was assessed for each genotype. (F) Interstitial macrophages were MACS isolated from naive WT or Alox15^-/- mice and stimulated for 4 days with IL-4 and Nb L3. Each point represents a single mouse. N=4-8 mice, pool of 2 independent experiments. (G) Gene expression of key genes affected by Alox15 deficiency (M1, M2 or metabolic hallmark) measured by qPCR on Alox15^-/- BMDM stimulated with IL-4 for IL-24hr in presence or absence of glucose. The macrophages were culture in presence or absence of glucose. Data are expressed as z-score. N=2-3, pool of 2 independent experiments.

We next wanted to validate whether these gene expression changes translated to altered metabolic phenotypes. Using a Seahorse bioanalyser we compared resting BMDM (M0) to M(IL-4) and M(LPS) from a WT or Alox15^-/- background. At resting state, Alox15^-/- macrophages had a slightly higher energetic status with a higher ECAR, as compared to WT (Fig. 5B). This difference was markedly stronger after IL-4 polarization whereas WT macrophages showed little change as compared to resting cells (Fig. 5B). At baseline, there was no difference in spare respiratory capacity (SRC) between WT and Alox15^-/- macrophages. However, after IL-4, but not LPS stimulation, there was a marked difference in SRC between the two genotypes, suggesting that Alox15^-/- macrophages were still receptive to IL-4-mediated induction of OXPHOS as has been reported previously for WT mice (Fig. 5 C).

Interestingly, even from baseline, Alox15^-/- BMDM exhibited a higher glycolytic capacity than their WT counterparts (Fig. 5 D). As expected, stimulation with LPS enhanced glycolysis in WT mice, but not in Alox15^-/- macrophages (Fig. 5 D). Instead, a marked increase in glycolysis could be seen after IL-4 polarization in Alox15^-/- macrophages, suggesting a combination of metabolic features typically associated respectively with M1 and M2 polarization.

To investigate if increased glycolysis in Alox15-deficient macrophages was responsible for their lack of anti-helminth function, we polarized BMDM with IL-4 in medium containing either a lower or higher glucose content and examined worm binding as before. Interestingly, when Alox15^-/- macrophages were polarized in glucose-free conditions, cells were able to trap larvae to the same extent as their WT counterparts (Fig. 5 E). A similar phenotype is observed with interstitial macrophages (Fig. 5F). In contrast, WT macrophages polarized in glucose free media were less efficient at trapping larvae in line with a previous report of poor M2 polarization in low glucose settings (Fig. 5 E) (Svedberg et al., 2019). Associated with this increase in larval trapping by Alox15^-/- M2 macrophages polarized in glucose free media, we observed a decrease in pro-inflammatory genes, an increase in Ym1 and PPAR-δ (Fig. 5 G).

Alox15 is central to the anti-helminth activity of macrophages but not in anti-Leishmania immunity

We found it striking that after LPS polarization, OXPHOS was heightened in Alox15^-/- macrophages but that the high baseline glycolysis rate of Alox15 macrophages remained unchanged. We thus decided to further explore the importance of Alox15 in M1/M2 polarization.

First, we polarized bone marrow derived macrophages with M2 or M1 stimulants (IL-4, IL-13, LPS and IFN-γ) and then co-cultured these cells with Nb. As previously described (Bouchery et al., 2022), M0 macrophages can bind to some extent to Nb larvae and IL-4 polarization enhances this binding (Fig. 6 A). The reduced binding of Alox15^-/- macrophages was similar for IL-4, IL-13 or IL-4+IL-13 stimulation. In line with the literature, M1 polarized macrophages, stimulated with LPS or LPS+IFN-γ, were unable to bind to Nb larvae when derived from WT mice (Fig. 6 A). Alox15^-/- mice exhibited increased binding as compared to M1 macrophages from WT mice and instead looked similar to M0 macrophages from either strain (Fig. 6 A). This indicates an overall alteration in macrophage polarization in absence of Alox15.

Figure 6. Alox15 deficiency do not impair macrophage control of Leishmania major.

(A) Murine bone marrow-derived macrophages were grown from C57BL/6/J (WT) or Alox-15^-/- mice and polarized or not (M0) with recombinant IL-4, with IL-13, with a combination of IL-4 and IL-13, with LPS or LPS and IFN-γ for 24 hours. Then those were co-cultured with 100 third-stage N. Brasiliensis larvae (Nb L3). The percentage of larvae with more than 10 cells attached was assessed for each genotype. N=3-4, pool of 3 independent experiments. (B) Parasitic index relative to control of a high dose of L. Mexicana infection of BMDM grown from C57BL/6/J (WT) or Alox-15^-/- mice. The proportion of macrophages infected with L. mexicana-TurboRFP was measured 4 h to 144h later by flow cytometry. N=3, 3 independent experiments. (C) Parasitic index as in B after a low dose of infection. N=3, 3 independent experiments. (D) Nitrite concentration measure by Griess assay N=3, pooled 2 independent experiments.

Unlike helminths, Leishmania spp. are obligate intracellular parasites. Macrophages act as the replicative niche or, depending on their activation state, control and clear the intracellular infection. Increased ARG1 activity is thought to be beneficial to the parasite in several ways. Firstly, ARG1 competes with NOS2 for L-arginine substrate, thereby diminishing NO and parasite death (Pessenda and da Silva, 2020, Schleicher et al., 2016, Wanasen and Soong, 2008). Secondly, increased availability of L-arginine in non-inflammatory macrophages is diverted into polyamine synthesis. Polyamines are required for parasite growth and production of the antioxidant trypanothione, which assists in withstanding the oxidative environment within the parasitophorous vacuole (Pessenda and da Silva, 2020). ARG1 catalyzes the conversion of L-arginine to urea and L-ornithine, a precursor for polyamine species required for Leishmania spp. replication (Pessenda and da Silva, 2020, Schleicher et al., 2016, Wanasen and Soong, 2008, Tomiotto-Pellissier et al., 2018).

Given the altered polarization of Alox15 macrophages stimulated with IL-4 and higher Arginase-1 expression, we set out to assess if control of this protozoan parasite is similarly affected by Alox15 deficiency. Intriguingly, in a high dose model of infection with L. mexicana, no difference between genotypes could be seen at any of the time points (Fig. 6 B). Polarization with LPS or with IL-4+IL-13 gave similar results (Fig. 6 B). However, the high multiplicity of infection used here, leading to highly efficient infection rates (usually >80% infection), may have overwhelmed and obscured host responses that occur under more physiological conditions. Interestingly, in a low dose model of infection with L. mexicana, Alox15 deficiency caused a slight decrease in parasite index at late time points indicating better macrophage control (Fig. 6 C). Despite this, we did not observe a difference in nitric oxide concentration even at late time points between the two genotypes (Fig. 6 D). No difference was observed in the initial levels of infection, suggesting Alox15 is not required for phagocytosis, but may play an important role in facilitating subsequent parasite replication.

To demonstrate that the phenotype we reported is not just specific to IL-4-mediated polarization of macrophages, we utilized another rodent helminth parasite, namely Heligmosomoides polygyrus bakeri (Hpb). Like Nb, protection against secondary infection requires Arginase-1 expressing macrophages (Esser-von Bieren et al., 2013). As Hpb is an enteric nematode, granulomas rich in macrophages are typically formed around the larvae within the submucosa of the proximal small intestine. However, in contrast to Nb, activation of macrophages able to kill Hpb larvae requires antibody, rather than IL-4. At 4 and 14 days post-secondary infection, Alox15 deficient animals had a higher worm burden than their WT counterparts (Fig. 7 A and C). During reinfection, granulomas rich in macrophages are typically formed around the larvae in the duodenum of the small intestine. We therefore assessed granuloma number and composition in WT and Alox15^-/- mice. There was a marked delay in granuloma formation in Alox15^-/- mice, with significantly fewer granulomas in Alox15^-/- mice than their WT counterparts 4 days post-reinfection (Fig. 7 B and D). Of note, parasite burden in primary infection was not different between genotypes (Fig. S4 A). In line with our findings for Nb infection, the granulomas were still rich in macrophages and expressed heightened level of Arginase-1 in Alox15^-/- mice (Fig. 7 E). As Alox15 can be expressed by other cells that could be involved in granuloma formation (eosinophils and epithelial cells), we next used an in vitro system to assess the role of Alox15 in macrophage trapping of Hpb larvae. We again generated BMDM and co-incubated them with Hpb L3 in the presence of serum from infected mice, but without IL-4 activation. In line with the phenotype of decreased trapping with Nb, the motility of the larvae co-incubated with (immune serum-activated) Alox15^-/- macrophages as compared to WT was higher, suggesting a less efficient trapping (Fig. 7 F). It has previously been shown that the decreased motility of Hpb larvae trapped by macrophages can be reversed by Arginase-1 blockade (Esser-von Bieren et al., 2013). We first assessed Arginase-1 activity after Hpb and immune serum incubation in Alox15^-/- macrophages, and as in our Nb L3 and IL-4 assays, Arginase-1 activity was markedly enhanced in KO macrophages (Fig. 7 G). We further determined that despite a notable difference in the drivers of macrophage polarization (antibodies versus IL-4), Arginase-1 blockade was not able to further increase motility in Alox15^-/- macrophages (Fig. 7 F).

Figure 7. Alox15 is required for macrophage mediated protection against H. polygyrus.

(A-D) Alox15^-/- and WT controls were infected twice with 200 Hpb L3. 4 or 14 days after secondary infection, parasites and granulomas were enumerated from the small intestine. N=4-5 mice per group, 2 independent experiments. (E) Immunofluorescence staining of Arginase and F4/80 in intestinal granulomas surrounding Hpb larvae at 4 days post-reinfection. N= 5-6, Quantification from 8-10 granulomas for each genotype. (F) Murine bone marrow-derived macrophages were grown from C57BL/6/J (WT) or Alox-15^-/- mice. Differentiated BMDM were co-cultured with 100 third-stage Hpb larvae in presence of immune serum (IS) and with or without the arginase inhibitor BEC. The percentage of motile larvae was assessed for each genotype by microscopy. n=3-9 mice, pool of 3 independent experiments. (G) Arginase-1 activity of BMDM stimulated for 24 hours with IS and 100 Hpb L3 measured by urea production. (n=6, representative of 2 independent experiments).

To further generalize our findings, we took advantage of a recently published single cell RNA sequencing dataset comparing BALB/c versus B6 pleural immune cells in their response against the filarial pathogen Litomosoides sigmodontis (Finlay et al., 2023). The authors showed that in BALB/c susceptible mice, the presence of Type 1 cytokines prevents incoming monocytes from fully differentiating into tissue resident cells which in turn polarize to M2. Interestingly, by exploring their dataset, we found that Alox15 expression was enriched in tissue macrophages in resistant C57BL/6 mice after infection, but not in transitional hybrid M1/M2 macrophages in susceptible BALB/c mice (Fig. S4 B). Furthermore, as in the case of infection with Nb and Hpb, Arg1 and Rtnla expression were similar between the two macrophage populations.

Altogether, these observations show that Alox15 is a crucial enzyme involved in macrophage functional M2 commitment, thus affecting anti-helminth immunity.

PPAR-δ activation can overcome Alox15 deficiency in helminth mediated trapping/killing

Alox15 is a lipid metabolizing enzyme, with a broad role in the production and metabolism of lipid mediators important in health and diseases (Snodgrass and Brune, 2019). Recently, Alox15 has been proposed to be a key metabolizing enzyme in T regulatory cell differentiation and regulatory phenotypes (Marques et al., 2021). In the absence of Alox15, Tregs had low FOXP3 expression and a high glycolytic profile. Supplementation with resolvins, lipid mediators downstream of DPA, EPA or DHA, rescued the altered T regulatory phenotype. Due to the parallels between our findings, we reasoned that Alox15 deficiency in M2 could be rescued by lipid supplementation.

To identify such lipids of interest, we performed lipidomic analysis of Hpb-infected intestines 4 days after secondary infection in WT and in Alox15^-/- mice. Larval products of Hpb have previously been shown to trigger a broad anti-inflammatory eicosanoid shift after primary infection by suppressing the 5-lipoxygenase pathway and inducing the cyclooxygenase (COX) pathway (de Los Reyes Jimenez et al., 2020). In secondary infection, arachidonic acid as well as linoleic acid were found in a similar quantity in WT and Alox15^-/- animals (Fig. 8 A). However, 12- and 15-HETE, two downstream products of Arachidonic acid were significantly decreased in Alox15^-/- intestine as compared to WT counterparts. Similarly, 17-HDHA, downstream of DHA, was also reduced in Alox15^-/- tissue. Although not significant, a similar trend could be observed for 9- and 13-HODEs downstream of Linoleic acid. We found no significant difference between genotypes in any other eicosanoids tested or in RvD1 (Fig. 8 A).

Figure 8. Heightened glycolysis cause loss of anti-helminth killing in Alox15^-/- M2.

(A) LC-MS/MS analysis of LOX metabolites of Arachidonic acid (AA), of Linoleic acid (LA) and of Docosahexaenoic acid (DHA) in intestinal culture supernatants from Hpb infected mice N=4 per genotype, baseline for WT N=2. (B and C) Murine bone marrow-derived macrophages were grown from C57BL/6/J (WT) or Alox-15^-/- mice. Differentiated BMDM were co-cultured with 100 third-stage Hpb larvae in presence of immune serum (IS) and with or without the Docosapentaenoic acid (DPA) or a PPAR-δ agonist. The percentage of motile larvae was assessed for each genotype by microscopy. N= 3-6 per condition, pool from 3 independent experiments. Of note B&C share some data points for the DMSO conditions. (D) Glycolytic capacity of BMDM stimulated as with IL-4 and/or PPAR-δ agonist for 24 hours prior to the assay. Data representative of 3 independent experiments, N= 5 wells per condition, each condition with 1-3 mice per experiment. (E) BMDM from Alox15^-/- or WT were stimulated with IL-4 and DPA or a PPAR-δ agonist (GW) or the arginase inhibitor BEC for 24 hours. RNA sequencing was performed. Bars show significance of enrichment scores for IFN-β response or M2 signature gene sets, respectively. Dashed line indicates an FDR-threshold of 0.05.

Omega-6 PUFAs are the most abundant polyenoic fatty acids in mammalian cells and are thus major lipoxygenase (LOX) substrates. We thus focused first on arachidonic acid and linoleic acid metabolism. Using the Hpb trapping assay, we performed rescue experiments by supplementing 9-HODE, 13-HODE and 15-HETE the most common downstream mediator products of Alox15. However, none of those reversed the increase Hpb motility observed in co-culture assay with Alox15 deficient macrophages (Fig. S5 A).

Given we also observed a decrease in the Omega-3 (n-3) PUFA, 17-HDHA, downstream products of Alox15, we further considered if those could be involved in our helminth-specific phenotype. It has recently been shown that n-3 Docosapentaenoic acid (DPA) supplementation can enhance macrophage efferocytosis and phagocytosis (Pistorius et al., 2018). DPA acts as a metabolic intermediate and reservoir between Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). We thus assessed if n-3 DPA supplementation could rescue the absence of trapping/killing observed in Alox15 deficient macrophages. Indeed, n-3 DPA supplementation restored the ability of Alox15-deficient macrophages to trap Hpb larvae (Fig. 8 B). To confirm this phenotype reversal was not specific to Hpb trapping or BMDM, we performed an ex vivo binding assay with IL-4 polarized interstitial macrophages and Nb larvae. Similarly, DPA was able to rescue the binding as well as killing of Nb larvae by Alox-15 deficient macrophages (Fig. S5 B and C).

Given Omega-3 PUFAs and downstream metabolites are ligands, or partial-ligands, of peroxisome proliferator-activated receptors (PPAR), we next used an agonist of PPAR-γ and PPAR-δ, Rosiglitazone and GW501516 respectively as another method to validate that enhancing lipid metabolism could reverse the Alox15^-/- phenotype. Interestingly, both PPAR agonists rescued the macrophage larvicidal activity against Hpb larvae (Fig. S5 D). GW501516, the PPAR-δ agonist led to a significant reversal in the impaired ability of Alox15^-/- macrophages to reduce Hp larval motility as well as Nb L3 binding and killing by interstitial macrophages, similar to the effect observed with exogenous DPA (Fig. 8 C, Fig. S5C). To further investigate the role of PPAR-δ in macrophage anti-helminth activation, we validated increased expression of PPAR-δ in BMDM after IL-4 stimulation as well as in interstitial macrophages 7 days post- N. brasiliensis infection (Fig. S5 E and F). Furthermore, bone marrow derived macrophages treated with the PPAR-δ inhibitor GSK3787 were unable to bind to Nb L3, similarly to Alox15^-/- macrophages (Fig. S5 G) and had an enhanced pro-inflammatory profile (Fig. S5 H)

To further confirm that Alox15 downstream lipid metabolites control anti-helminth activity through PPAR mediated control of glycolysis, we performed Seahorse XF analysis of glycolysis and OXPHOS after rescuing Alox15^-/- M(IL-4) with PPAR-δ stimulation. As expected, both glycolysis (Fig. 8 D) and OXPHOS (Fig. S5 I) normalized to the level obtained for WT. We performed GSEA-like analysis on RNA signatures of Alox15^-/- and WT BMDM macrophages treated with BEC, DPA, and the PPAR-δ agonist GW501516, to assess the enrichment of a pro-inflammatory type I interferon signature, consistent with Alox15^-/- lung macrophages (cluster 6, Fig. S2 E) and for M2 signatures. The analysis confirmed that both DPA and GW501516, but not BEC decreased the heightened type I interferon signature observed in Alox15^-/- macrophages, while the M2 signature was not affected (Fig. 8 E, Fig. S5 J, Table S4). Altogether, our results show that Alox15 metabolism of DPA activates PPAR-δ that in turn controls glycolysis activation to ensure a functional anti-helminth M2 commitment.

Discussion

Macrophages are central to anti-helminth immunity in the vast majority of mouse models but are largely unstudied in human helminthiasis. In rodents, macrophages require a so-called ‘alternative’ or “M2” activation mediated by type 2 cytokines or antibodies (Bouchery et al., 2015, Bouchery et al., 2022, Chen et al., 2022, Chen et al., 2012, Esser-von Bieren et al., 2013). This M2 signature is also enhanced by collectins and stress signals (Bosurgi et al., 2017, Minutti et al., 2017). However, macrophage M2 polarization differs between mouse and human, at least in terms of hallmark genes. With the long-term goal of elucidating the anti-helminth role of macrophage in humans, we investigated Alox15, an arachidonate lipoxygenase that is a hallmark of human M2 polarization (Czimmerer et al., 2012, Snodgrass and Brune, 2019). Upon genetic deletion of Alox15, we found that macrophages cannot efficiently trap and kill hookworm larvae in mice, nor could they repair tissue damage. Interestingly, hallmark genes of M2 were not compromised, raising the question of why function was lost. We further found that Alox15^-/- macrophages activated with IL-4 displayed a hybrid phenotype of classical (IFN-β signature) and M2 activation. This was associated with an uncontrolled switch to glycolysis. We finally show that to ensure a full M2 commitment, Alox15-derived metabolites of DPA are required to activate PPAR-δ, which keeps glycolysis in check.

For several decades, hallmark M2 genes have been used as a readout of alternative activation. Our study demonstrates that upregulation of such genes was not sufficient for functional activation; i.e. trapping helminth parasites or repairing tissue damaged by parasite migration. Notably, Arginase-1 has previously been proposed to participate in macrophage-mediated protection against helminths as both a polarization marker and enzymatic effector molecule (Bouchery et al., 2015, Chen et al., 2022, Chen et al., 2012, Esser-von Bieren et al., 2013). Despite high Arginase-1 expression and robust urea formation, Alox15 deficient macrophages displayed impaired anti-helminth immunity with both N. brasiliensis and H. polygyrus. These findings indicate that Arginase-1 induced urea formation alone is insufficient for anti-helminth effector function and identify ALOX15 as a critical component of the macrophage program required for parasite trapping. Together with our observation that Alox15 expression is lost in Arg1-deficient macrophages, these data further suggest that ALOX15 functions downstream of, or in concert with, Arginase-1-dependent macrophage activation pathways.

Recently, Westermann and colleagues proposed that neither macrophages nor macrophage Arginase-1 activity are essential for trapping H. polygyrus larvae, instead identifying a STAT6-dependent contribution of intestinal epithelial cells to granuloma formation and parasite containment (Westermann et al., 2023). Importantly, the same study demonstrated that macrophage-intrinsic STAT6 signaling remains necessary for protection against N. brasiliensis, consistent with our previous findings (Bouchery et al., 2015). In the present study, we found that macrophage Alox15 expression was required for trapping of both H. polygyrus and N. brasiliensis in vitro, including under conditions where H. polygyrus larvae were trapped in the absence of exogenous IL-4. Furthermore, macrophage-specific deletion of Alox15 was sufficient to impair protection in vivo after adoptive transfer, establishing a cell-intrinsic requirement for ALOX15 in macrophage-mediated anti-helminth immunity.

For both Nb and Hpb, macrophages binding/trapping to the larvae was compromised by Alox15 deficiency, suggesting that parasite recognition might be affected by Alox15 deficiency. However, comparing Arginase-1 blockade (by BEC) with Alox15 genetic deficiency did not lead to any common genes being downregulated. It is important to note here that this transcriptomic analysis was performed after 24 hours of IL-4 stimulation, but Alox15 expression is known to peak at 48 hours, therefore it is possible we simply missed the optimal window to assess this response (Han et al., 2014).

N. brasiliensis infection in mice typically gives a rapid, and strong Th2 response. This textbook anti-helminth immunity is distinct from that observed outside the laboratory, as human or wild animal helminthiasis is characterized by a ‘modified type 2’ response thought to be a hybrid of Th2/Th1/ Tregs (Diaz and Allen, 2007, Loke et al., 2022). Although there are discrepancies in the literature, chronic human hookworm infection has been associated with a mixed cytokine response; IFN-γ, TNFa, and IL-10 are elevated in the serum in addition to IL-5 (de Ruiter et al., 2020, Quinnell et al., 2004, Pit et al., 2001). Recently, CyToF of helminth-exposed patients has deepened our understanding of the immune response in humans and confirmed this hybrid response is due to ILC2s, CD4+, CD8+, and γδ T cells (de Ruiter et al., 2020). To date, we could not find any reports of mixed polarization of macrophages in helminth infected individuals, however this is likely due to the fact that peripheral, and not tissue responses are studied in the context of human helminth infection. In future, flow cytometry of, for example, sputum samples from infected patients could give insights into macrophage polarization. We speculate that in this mixed cytokine environment, macrophages might not commit to a purely ‘alternative’ phenotype and instead develop an altered pro-inflammatory/M2 commitment as recently shown for sequential IL-4/LPS stimulation (Dang et al., 2023). Given the central role of M2 macrophages in protection against reinfection in rodent helminth models, such a defect in M2 polarization could explain why hookworm-infected individuals are highly susceptible to re-infection for life. In line with this idea, Finlay et al. recently compared two mouse genotypes with different susceptibility to filarial infection and reported that in the presence of Type 1 cytokines, tissue resident macrophages do not properly adopt the M2 phenotype and instead display lower OXPHOS and heightened phagocytosis. (Finlay et al., 2023) Interestingly, despite an overall decrease in an IL-4-associated gene signature, the authors report that the hallmark M2 genes were not strongly affected, similar to our model. By reanalyzing the single cell sequencing data published by Finlay and colleagues, we have further shown that Alox15 expression, but not Arginase-1, was correlated with parasite clearance and efficient M2 polarization, highlighting that Alox15 is likely to be a generic anti-helminth feature of macrophage polarization.

As highly plastic cells, the polarization of macrophage activation has been described as a continuum with ‘classical’ and ‘alternative’ states at either end. While comparisons of in vivo and in vitro datasets have begun to challenge the simplicity of this model (Kieler et al., 2021, Van den Bossche et al., 2017), the terms are still widely used. It has been proposed in recent years that M1 enhance their glycolysis to meet their energetic needs, while M2 use the more energy efficient oxidative phosphorylation. Yet there is some conflict over the requirement for glycolysis in alternative activation (Kieler et al., 2021, Van den Bossche et al., 2017), which is thought to be due, in part, to the use of the inhibitor 2-DG to block glycolysis; 2-DG appears to have off-target effects on OXPHOS activity. There is reason to suspect that intact glycolysis is required for efficient anti-helminth immunity. For example, inhibiting glucose utilization by intraperitoneal injection of 2-DG during H. polygyrus infection perturbs M2 polarization, significantly reducing expression of RELMa (Huang et al., 2016). Furthermore, it has been noted that alveolar macrophages are not protective against the lung phase of N. brasiliensis infection as they are hyporesponsive to IL-4 due to the glucose-restricted environment of the alveolar niche (Svedberg et al., 2019).

While M1 polarization clearly requires metabolic reprogramming, M2 polarization may be more subtle. In our own study, we noted that OXPHOS activity was not different between basal ‘M0’ and IL-4 activated ‘M2’ bone marrow-derived macrophages. Here, we argue that glycolysis needs to be actively restrained to obtain a functional anti-helminth/pro-repair M2 commitment.

While PPAR-δ is classically viewed as a regulator of lipid metabolism and fatty acid oxidation, accumulating evidence suggests that it can more broadly control the balance between glycolysis and oxidative metabolism. In both CD8 T cells and mesenchymal stem cells, PPAR-δ activity promotes oxidative metabolism while limiting commitment to glycolysis, indicating that regulation of cellular metabolic state may represent a conserved feature of the PPAR-δ transcriptional program (Bevilacqua et al., 2024, Contreras-Lopez et al., 2020). Moreover, PPAR-δ has been reported to function not only as a transcriptional activator but also as a context-dependent cis transcriptional repressor (Krogsdam et al., 2002, Martin-Martin et al., 2018), raising the possibility that PPAR-δ activation in macrophages may directly suppress glycolytic gene expression or upstream regulators of glycolysis.

Failure of that control results in the development of a “pro-inflammatory” M2 signature which lacks anti-helminth activity, but retains anti-Leishmania control. This suggests that alternative effector functions are more tightly regulated than classical effector functions. In line with our work, a hybrid M1/M2 signature has recently been reported after sequential exposure of BMDM to IL-4 and LPS in vitro, with a metabolic profile matching the one we described for Alox15^-/- M(IL-4), i.e. a heightened glycolysis together with enhanced OXPHOS (Dang et al., 2023). The authors then prove that the pro-inflammatory genes expressed by hybrid M1/M2 macrophages require HIF-1α stabilization as in the case of ‘classical’ polarization. Whether this also occurs in the absence of Alox15^-/- should be further investigated. Other hybrid M1/M2 macrophage phenotypes have recently been reported in response to IL-33 stimulation which elicits the co-expression at the protein and mRNA level of alternative markers (Arg1, Retnla, Chil3, Alox15) as well as pro-inflammatory markers (IL-6, IL-1b) (Faas et al., 2021). Interestingly, IL-33-induced hybrid polarization did not display enhanced glycolysis but rather full integrity of the TCA cycle. IL-33 is very strongly released in the lungs after Nb infection and given we observed stronger transcriptional differences in vivo that in vitro between WT and Alox15^-/- macrophages, it could be the case that Alox15 is also important to the fine tuning of IL-33 activation of macrophages.

Despite a staggering number of publications on Alox15, its role in cellular metabolism has not been well-studied to date. Namgaladze et al. first established the possible involvement of Alox15 in cellular immunometabolism in 2015 (Namgaladze et al., 2015). Indeed, the authors showed that AMPK inhibition blocked Alox15 upregulation after IL-4 stimulation but did not further characterize the activation profile of said macrophages. Instead, they investigated the role of AMPK and Alox15 in the IL-4 priming effect prior to LPS activation. In this context, they could show that Alox15 deficiency decreased the pro-inflammatory signature of IL-4 primed macrophages. Treatment with 12- or 15- HETE instead of IL-4 could similarly potentiate pro-inflammatory cytokine production in macrophages. In our model, 12- and 15-HETE supplementation was not sufficient to restore the anti-helminth activity of Alox15^-/- M(IL-4). We have unfortunately not assessed cytokine production after this supplementation. However, n-3 DPA supplementation, another lipid substrate for Alox15, was able to restore a full M2 commitment in Alox15^-/- M(IL-4), both functionally and metabolically. A current limitation of our study is that we have not identified the responsible downstream metabolite(s) of DPA.

Interestingly a similar finding has been reported in regulatory T cells(Marques et al., 2021), where Alox15 deficiency has been shown to dampen Treg activation and cause upregulation of pro-inflammatory factors. Supplementation with resolvins - downstream products of Alox15 activity on DPA/DHA – was shown to rescue Treg activation. Of note, the functional relevance of resolvins is a highly debated topic in the field due to their production in very low quantity and the associated challenge of detecting them with current technology. Using metabolomics, the authors reported heightened glycolysis and TCA in Alox15^-/- Tregs. Together with our results, this suggests that the SPMs/Alox15 pathway might be crucial as a ‘confirmation signal’ to allow the development of a pro-repair, anti-helminth M2 commitment. Failure to deliver this secondary signal may then push the immune cell towards a pro-inflammatory phenotype as a ‘fail-safe’ or ‘default’ pathway.

Materials and Methods

Reagents

The following reagents were used in various assays: IL-4 10 ng/mL (Peprotech, Gibco, Cat. # 214-14-500UG), IL-13 10 ng/mL (Peprotech, Gibco, Cat. # 210-13-10UG), LPS 10 ng/ml (Thermofisher, Cat. # 00-4976-93), IFN-γ: 10 ng/mL (Peprotech, Gibco, Cat. # 315-05-100UG) and were reconstituted in PBS (BioConcept, Cat. # 3-05F29-I).

BEC, Hydrochloride 1 uM (Sigma, Cat. # 197900), Rosiglitazone 0.1, 0.5, 1uM (PPAR-γ agonist, Sigma, Cat. # 557366-M), GW501516 0.1, 1, 5uM (PPAR-δ agonist, Sigma, Cat. # SML1491), n-3 Docosapetaenoic acid sodium salt DPA 100uM (Cayman Chemical, Cat. # 21907), 15-HETE (Cayman Chemical, Cat. # 34700), 13-HODE (Cayman Chemical, Cat. # 38600), 9-HODE (Cayman Chemical, Cat. # 38400), GSK3787 (Sigma, Cat. # 516567) were reconstituted in DMSO (Sigma, Cat. # D4540 or Applichem Cat. # A3672,0100).

The following media or media supplements were used: RPMI 1640 (Gibco Cat. # A1049101 or BioConcept, Cat. # 1-41F01-I), DMEM Gibco (Cat. # 35050061).

Mice

C57BL/6J (WT) mice were obtained from Charles River Laboratories (Germany) or from Animal Resources Center (Australia). Alox15-KO mice were originally bought from the Jackson’s laboratory or originated from the Research Institute of McGill University. All mice were maintained under specific pathogen-free (SPF) conditions at École Polytechnique Fédérale de Lausanne (EPFL), Switzerland or by the Monash Intensive Care Unit Facility at Monash University, AMREP campus Melbourne, Australia or by the Animal Facility, Swiss TPH, Switzerland. All mice were age and sex-matched and used between 8-14 weeks of age. Wild-type C57BL6/J mice, bred separately, and of the same sex were randomly assigned to experimental groups. Mice were maintained at 3-5 animals per cage and ad libitum access to water and food. All animal experiments were approved by the Service de la consommation et des affaires vétérinaires (1066 Épalinges, Canton of Vaud, Switzerland) with the authorization number VD 3001, or by the AEC committee of the Alfred campus, Melbourne Australia with authorization number E/1846/2018/M or by Basel-land animal welfare authorities under the authorization numbers BL/543/2022 and BL/560/2025.

Preparation and Isolation of N. brasiliensis Larvae

Nb was maintained by monthly passage in Lewis rats. Infective larvae (L3) were prepared from 2-week-old rat fecal cultures as previously described (Camberis et al., 2003). 550 L3 or 250 L3 were delivered by subcutaneous injection in 100 μL of sterile PBS to the scruff of the neck.

Viable larvae were recovered from tissue (lung or gut) by baermann at 37 °C for at least 2 h as previously reported(Bouchery et al., 2017). Viable worms follow a thermal gradient and migrate out of the bag to collect at the bottom for counting. Larvae present in the large airways were harvested by bronchoalveolar lavage, with 3 × 1 ml of PBS, followed by filtering through a 40-µm mesh. For larvae count and red blood cell count, the 3ml of wash were pooled together. Larvae were manually counted under brightfield on a stereomicroscope. Red blood cells were counted with a haemocytometer, after centrifugation of the BALF and resuspended in 1 ml to account for the difference of BALF volume harvested in each mouse. D0 marks the day of infection. If not explicitly mentioned, the time between primary and reinfection was 30 days.

For adoptive transfer, 1.7-2 million cells (stimulated for 24 hrs with IL-4) were administered intranasally in 50 μl of PBS, the evening prior to infection with 550L3.

Preparation and Isolation of H. polygyrus bakeri Larvae

The standard Hpb infective stage-three larvae (L3) were generated in the laboratory based on published protocols (Moyat et al., 2022). 200 L3 were administered per os as previously described (Esser-von Bieren et al., 2013). Viable worm burdens were obtained by baermann as previously described (Bouchery et al., 2017). Granulomas were enumerated by manual counting of the small intestinal contents under a dissecting microscope. D0 marks the day of infection. If not explicitly mentioned, the time between primary and reinfection was 30 days.

Leishmania mexicana maintenance and infection

Transgenic L. mexicana stably expressing the fluorescent protein TurboRFP (L. mexicanaTurboRFP) were maintained in promastigote stage at 27°C in RPMI media pH 7.4 supplemented with 10% FCS with biweekly passaging. Only cultures <20 passages since isolation from infected murine tissue were used to generate amastigotes for infections. Amastigotes were generated by incubating stationary phase promastigotes in RPMI media pH 5.5 supplemented with 20% FCS for 4 days at 33°C 5% CO2.

Macrophage generation

For bone marrow-derived macrophages, marrow was flushed from the femur and tibia of C57BL/6 mice and passed through cell strainers (70 µm) in PBS. The cells (10⁶ mL⁻¹) were cultured in 30% M-CSF (supernatant of L929 cell culture) supplemented medium (RPMI, 10% FCS, penicillin/streptomycin) for 7 days as described previously (Bouchery et al., 2022). Macrophages were harvested between day 7 and 10 of culture for experiments.

For lung macrophages, mice were either used naïve or infected with 250 L3 subcutaneously. The lungs were washed by bronchoalveolar lavage with 3 mL of PBS and then digested in media (DMEM (Gibco), 2 mg mL⁻¹ collagenase (Sigma C0130 or Gibco, Cat #17100017), DNase 12.5 U mL⁻¹ (Roche, CAt # 04536282001) at 37°C on an orbital shaker for 45 min. Interstitial macrophages (IMs), alveolar macrophages (AMs) and monocytes were cell sorted. Cells are stained for viability assessment with Zombie NIR fixable dye (BioLegend, Cat #423105) according to manufacturer instructions. The following combination of antibodies were used: CD45-BV605 (1:100, clone 30-F11, RRID AB_2562341, BioLegend Cat # 103139); CD11c-AF700 (1:100, Clone N418, RRID AB_528735, BioLegend Cat #17319); F4/80-BV785 (1:100, clone BM8, RRID AB_2563667, BioLegend Cat #123141); SiglecF-PE (1:200, clone S17007L, RRID AB_2750234, BioLegend Cat # 155505); CD11b-PerCp (1:100, clone M1/70, RRID AB_2129374, BioLegend Cat # 101230); GR1-FITC (1:100, clone RB6-8C5, RRID AB_313371, BioLegend Cat# 108406). Gates were set based on fluorescence minus one (FMO) controls. The cells were sorted on a BD FACSAria Fusion at the cytometry facility of the Biozentrum (Basel). The isolated cells were seeded (2.5 × 10⁵) directly in a flat-bottom 96-well plate (Nunc) and allowed to adhere for at least 20 minutes, before co-culture with the larvae in complete DMEM (10% FCS, 1% penicillin/streptomycin).

For some ex-vivo experiment, interstitial macrophages were enriched from naïve lungs after bronchoalveolar lavage by F4/80+ MACS selection. The cells were rested overnight and washed before addition of IL4 and Nb L3 for 4 days.

Larval binding assay

Nb assay

macrophages were polarized or not with recombinant IL-4 (10 ng mL⁻¹) and various inhibitors or agonists (see reagents) for 24 hours. 0.1×10⁶ cells were then co-cultured with 100 third-stage N. brasiliensis larvae (Nb L3) in 96-well plates, and the number of larvae with >10 cells attached to their surface was manually counted by light microscopy after 4 days of co-culture (Chen et al., 2014), and expressed as a percentage of total larvae per well. Binding is often assessed by 2 independent individuals, normalization is done on control group. Quantifications are performed in a blinded manner. For each animal, 3 replicates are counted.

Hpb assay

0.1×10⁶ BMDM were co-cultured with 100 third-stage H. polygyrus bakeri (Hpb) in presence of immune serum. Inhibitors, supplements or agonists were provided 24 hours prior to adding the larvae. All co-cultures were performed at 37°C, 5% CO₂, for 24 h. The percentage of motile larvae attacked by macrophages were then quantified using a stereo microscope. Quantifications are performed in a blinded manner. For each animal, 3 replicates are counted.

Larval viability assessment

After the co-culture, Sytox green was added (1:100, Life technologies, Cat#S7020) to the wells and viability of the larvae was assessed using fluorescent microscopy. Larvae were considered dead, or to have impaired viability, when staining positive for the dye, and expressed as a percentage of total larvae per well.

Leishmania/macrophage co-culture assays

BMDM infection with L. mexicanaTurboRFP and subsequent flow cytometric analysis were performed as described previously (McGowan et al., 2022). Briefly, BMDMs were seeded into non-treated 96-well plates at 110,000 cells/well. Following overnight incubation at 33°C, BMDMs were co-incubated with L. mexicanaTurboRFP amastigotes at a low (0.5:1) or high (3:1) multiplicity of infection at 33°C 5% CO2 for the indicated period. At the indicated time point, infected macrophages were gently detached from plates and single cell suspensions were stained with Live/dead fixable violet dye (Molecular Probes), anti-F4/80-APCeF780 (1:100, clone BM8, RRID AB_2735036, eBiosciences Cat # 47-4801-82) and anti-CD11b-PerCp (1:100, clone M1/70, RRID AB_2129374, BioLegend Cat # 101230), and analyzed by flow cytometry. Dead cells and extracellular parasites were excluded prior to gating of infected macrophages (SSC^hiFSC^hiF4/80⁺CD11b⁺TurboRFP-H⁺) and uninfected macrophages (SSC^hiFSC^hiF4/80⁺CD11b⁺TurboRFP-H^-). Data was analyzed using FlowJo software where infected macrophages are expressed as the percentage of gated viable, single macrophages. The TurboRFP geometric mean fluorescence intensity (gMFI) of infected macrophages minus that of uninfected macrophages was used as an indicator of average parasitic burden. Parasitic index, the product of the percent infection and parasitic burden, was used as a more general readout of infection.

RNA isolation and RT-qPCR

RNA of 1×10⁶ cells was extracted with a TriPure (Sigma-Aldrich, Cat #11667157001), and reverse transcribed using FIREScript RT cDNA synthesis Kit Solis Biodyne (Lucerna, Cat #06-15-00200) for qPCR analysis. qPCR was performed using PowerUp SYBR green Applied Biosystems (Thermofisher, Cat #A25741) on an Bio-Rad CFX96 touch Real-Time PCR detection system. Data analysis was performed with CFX Maestro, and expression was normalized according to expression of the housekeeping gene GAPDH.

Multiplex PCR

Custom oligonucleotide primers and probes (Table 5) were designed using National Center for Biotechnology Information (Bethesda, Md) primer blast software and were purchased from Microsynth (Balgach, Switzerland). Amplification was carried out using iQ Multiplex Powermix and a CFX96 Real-Time detection system using forward and reverse primers at a concentration of 10μM and probes at a concentration of 50μM. Absolute quantification was performed using the CFX Manager software 2.1 (all from Bio-Rad) based on values obtained with a set of purified amplicons used as standards.

Table 5. oligonucleotide primers and probes for multiplex PCR analysis.

Oligonucleotide name	NCBI reference sequence	5’ label	Sequence
Retnla/Fizzl forward	NM_020509.3	-	5’-TTGCCAATCCAGCTAACTATCCCT -3’
Retnla/Fizzl reverse		-	5’ -AGCACACCCAGTAGCAGTCA -3’
Retnla/Fizz1_probe		TX Red	5’- ACAGATGGGCCTCCTGCCCTGCTGGGA- 3’
Argl forward	NM_007482.3	-	5’ -CCTGCTGGGAAGGAAGAAAAGG-3’
Arg1 reverse		-	5’ -TCTCTGTAAGATAGGCCTCCCA-3’
Arg1 probe		FAM	5’ -TTCACCCCGGCGACCGGCACCCC-3’
Chil3/YM1 forward	NM_009892.3	-	5’ -CAAGTTGAAGGCTCAGTGGCTC-3’
Chil3/YM1 reverse		-	5’ -TGTCAGAGGGAAATGTCTCTGGT-3’
Chil3/YM1_probe		Cy5	5’ -GGAGGTGCCGTGGTCTGGCCCCTGG- 3’
MRC1 forward	NM_008625.2	-	5’ -TCTGGATTGGACTCAACAGTCT-3’
MRC1 reverse		-	5’ -CTCTGATGATGGACTTCCTGGT-3’
MRC1_probe		HEX	5’- ACGCAGTGGTTGGCAGTGGGCTGGAGG- 3’
FN1 forward	NM_010233.2	-	5’ -CAGCAAGCCACAGTTTCTGATA-3’
FN1 reverse		-	5’ -TCTGTAATAGCGCACAGAGACG-3’
FN1_probe		TX Red	5’ -TGCCTCCACCCCCACCAGCCTGCTCA- 3’
TIMP1 forward	NM_001044384.1	-	5’ -GCCCTTCGCATGGACATTTATT-3’
TIMP1 reverse		-	5’ -CATGGTATCTGCTCTGGTGTGT-3’
TIMP1 probe		FAM	5’ -TCCACTGTGCAGCCCCTGCCGCCA-3’
MMP7 forward	NM_001319986.1	-	5’ -CTCTAGGTCATGCCTTCGCA-3’
MMP7 reverse		-	5’ -AAGTTCACTCCTGCGTCCTCA-3’
MMP7 probe		Cy5	5’ -CCTGGGCCAGGCCTAGGCGGAGA-3’
MMP9 forward	NM_013599.4	-	5’ -CCTGGCTCTCCTGGCTTTC-3’
MMP9 reverse		-	5’ -ACAAGTATGCCTCTGCCAGC-3’
MMP9_probe		HEX	5’- TGCCCCTTACCAGCGCCAGCCGACTTT- 3’
HPRT1 forward	NM_013556.2	-	5’ -AGAGCGTTGGGCTTACCTCA-3’
HPRT1 reverse		-	5’ -TGGTTCATCATCGCTAATCACGAC-3’
HPRT1 probe		Cy5.5	5’ -TTTTGCCGCGAGCCGACCGGTCC-3’

The primers and probes specific for Retnla/FIZZ1, ARG1, CHIL3/YM1, MRC1; and FN1, TIMP1, MMP7, and MMP9; were collectively used in multiplex-1, and -2 respectively. The primers and probe specific for HPRT1, our reference gene, were added into each multiplex. Cy, Cyanine; FAM, fluorescein; HEX, hexachloro-fluorescein; Tx Red, Texas Red.

BMDM transcriptomics

Fastq files were processed using the Laxy tool (Powell, 2020) using the RNAsik pipeline (Tsyganov et al., 2018) using STAR aligner (Dobin et al., 2013) with Mus musculus GRCm38. Reads were quantified using featureCounts (Liao et al., 2014) producing the raw genes count matrix and various quality control metrics, summarised in MultiQC report (Ewels et al., 2016). The gene count matrix was analysed with Degust (Powell et al., David R. Powell, Andrew Perry, Michael Milton), a web tool which performs differential expression analysis using limma voom normalisation producing counts per million (CPM) library size normalisation and trimmed mean of M values (TMM) normalisation for RNA composition, and also several quality plots such as classical multidimensional scaling [MDS] and MA plots. Later, we retrieved (gene x sample) count matrices from several Degust analyses (Degust v 4.3, R v3.6.1), combined them ensuring each sample only appeared once, extracted gene-level annotations, parsed the experimental design, and combined everything into a single DGEList object for use with the edgeR package (v4.0.15).

Experiment design

The experimental design contained cell samples derived from mice of 2 genotypes (WT and Alox-15 KO; 4 mice per genotype were available for analysis) submitted to 5 treatments (M0, Il-4 stimulated, Il-4 + BEC treatment, Il-4+DPA treatment, Il-4+GW treatment), but not all (genotype; treatment) combinations were performed (no Il-4 + GW in WT; no Il-4 + BEC in KO). To account for the unequal repeated measures, we created a design matrix with no shared intercept, but one intercept per mouse (to account for mouse-to-mouse variation), and one indicator variable for each (genotype; treatment) combination, leaving out combinations involving M0 to ensure the matrix was of full rank. A heatmap (generated using ComplexHeatmap v2.20.0) of the design matrix is shown in supplementary Table 5, while a table (generated using ExploreModelMatrix v1.16.0) showing how the model terms are combined for each sample is shown in supplementary Table 5.

Modelling

We estimated dispersions with edgeR::estimateDisp(…, design=…, robust=TRUE), and fitted the model with edgeR::glmQLFit(…, design=…). We then interrogated the model by defining manual contrasts combining the relevant indicator terms and passing these to edgeR::glmQLFTest(…, coef=…, contrast=…) and edgeR::topTags(…, n=512). The following contrasts were used (Table 6):

Contrast name	WT mic e (x4)	KO mice (x4)	WT_IL4	KO_IL4	WT_DPA	KO_DPA	WT_BE C	KO_G W
IL4_vs_M0_in_WT	(0)	(0)	+1	0	0	0	0	0
KO_vs_WT_in_IL4	(-0.25)	(+0.25)	-1	1	0	0	0	0
nfn_vs_fn*	(0)	(0)	-0.25	+0.5	-0.25	-0.25	+0.5	-0.25
nv_KO-IL4_vs_fn*	(0)	(0)	-0.25	+1	-0.25	-0.25	0	-0.25
GW_vs_IL4_in_KO	(0)	(0)	0	-1	0	0	0	1
DPA_vs_IL4_in_KO	(0)	(0)	0	-1	0	1	0	0
BEC_vs_IL4_in_WT	(0)	(0)	-1	0	0	0	1	0

^*The “nfn_vs_fn” (non-functional versus functional) and “nv_KO-IL4_vs_fn” (naïve KO-IL-4 versus functional) contrasts were defined based on observed phenotypes of the immune cells: were they functionally antihelminthic or not? The splitting of the various (genotype; treatment) combinations into these 2 categories is shown in supplementary Table 5.

Volcano plots for several of these contrasts were generated by plotting the estimated log fold change versus the -log10-transformed p-values (corrected for multiple testing). The top 20 most extreme differentially-expressed genes (in terms of log2 fold change and/or corrected p-value) were labelled.

Custom gene sets of interest

We also curated several lists of genes known to be involved in various immune processes. One manually compiled 10 genes expressed in our dataset known from the literature as key markers of the M2 activation signature (“Field.M2.signature”). Another extracted 11 genes expressed in our dataset with an annotation with GO term GO:0035456 “response to interferon-beta” (“GO_resp_IfnB”) from the org.Mm.eg.db R package (v 3.18.0, as of 23/08/2024, leveraging R package GO.db, of same version).

Enrichment analyses: we performed “competitive” gene set enrichment analyses using limma::cameraPR() (v.3.60.4) on the set of all expressed genes, ranked by the F-test statistic from the calls to edgeR::topTags(), additionally signed by the sign of the concomitant logFC (thus allowing genes with high F but negative logFCs to be ranked towards the end of the gene list), for each of the custom gene sets of interest described above. Barcode plots were generated by showing the genes ranks, and highlighting those belonging to the custom gene set, while reported corrected p-values are those of cameraPR().

Single cell sequencing

Lungs were minced and then digested in media (DMEM (Gibco), 2 mg mL−1 collagenase (Sigma), DNase 12.5 U mL−1 (Roche, SigmaAldrich, Switzerland)) at 37°C on an orbital shaker for 45 min at 105 rpm. Lungs were then passed through a 70µm cell strainer with syringe plungers. Red blood cell lysis was performed (Merck, Cat #11814389001). Whole transcriptome analyses of FACS sorted CD45+ cells were performed using the BD Rhapsody Single Cell Analysis System (BD, Biosciences). Cells were stained with Zombie Aqua live/dead (Biolegend, Cat# 423101) and Fc block (2.4G2, BioXCell, Cat #BE0307) according to manufacturer’s instructions. Prior to FACS staining, cells were labelled with sample tags (Cat # 633793 BD Mouse Single-Cell Multiplexing Kit) according to the manufacturer’s protocol. As the CD45 sample tag clone and FACS antibody clone were the same (30-F11), sample tagging was performed in the presence of a 1:1 (w/w) quantity of anti-CD45 FACS antibody. In brief, for each condition, 10⁶ cells were resuspended in staining buffer (1% BSA, 1% EDTA in PBS) and incubated with the respective Sample Tag:anti-CD45 BUV395 cocktails for 20 min on ice. Cells were then transferred to a 5-ml polystyrene tube, washed twice with 2 ml staining buffer and centrifuged at 400g for 5 min. Samples were stained in a FACS antibody cocktail mix for 20 mins on ice: CD45-AF488 (1:200, clone 30-F11, RRID: AB_312973 BioLegend, Cat # 103108), CD19-APCe780 (1:100, 1D3, eBioscience, Cat #47-0193-80, RRID AB_10853189), Gr-1-PE (1:200, clone RB6-8C5, BioLegend RRID AB_313373, Cat# 108408), SiglecF-PE-CF594 (1:200, clone E50-2440, RRID AB_2687994,BD, Cat# 562757), Ter-119-Biotin-SA-PECy5 (1:100, clone Ly-76, RRID AB_313705, BioLegend, Cat# 116204), CD11c-Pecy7 (1:400, clone HL3, BD, Cat# 564079), CD3e-BUV737(1:100, clone 145-2C11, RRID AB_2870100, BD, Cat# 564618), CD11b-PacBlue (1:400, clone M1/70, RRID AB_755986,BioLegend, Cat# 101224), CD4-BV711 (1:100, clone RM4-5, RRID AB_2921025, eBioscience, 1 Cat# 4-0042-83), CD8-BV786 (1:300, clone H35-17.2, RRID AB_2740577, BD, Cat# 740952). Cells were washed and resuspended in 1 ml staining buffer for counting. Next, an equal number of cells from 4 barcoded samples per group were pooled and the mixture was centrifuged at 400g for 5 min. The BD Rhapsody cartridge was loaded with 40,000 cells. Single cells were isolated with the BD Rhapsody Express Single-Cell Analysis System according to the manufacturer’s recommendations (BD Biosciences). cDNA libraries were prepared using the BD Rhapsody Whole Transcriptome Analysis Amplification Kit (633801 BD Biosciences) following the BD Rhapsody System mRNA Whole Transcriptome Analysis (WTA) and Sample Tag Library Preparation Protocol (BD Biosciences). The final libraries were quantified using a Qubit Fluorometer with the Qubit dsDNA HS Kit (Q32851 Thermo Fisher Scientific). Library size distribution was measured with the Agilent high-sensitivity D5000 assay on a TapeStation 4200 system (5067-5592 Agilent Technologies). Sequencing was performed in paired-end mode (2 × 75 cycles) on a NovaSeq 6000 with NovaSeq 6000 SP Reagent Kit chemistry.

Single cell sequencing analysis

Gene quantification was generated with STARsolo (STAR-2.7.10a, (Dobin et al., 2012)) and aligned to a GENCODE Version M29 reference (Frankish et al., 2019) with sample tags added for de-multiplexing purposes. Parameters were in accordance with DB Rhapsody (version 1) with default settings. Subsequently, the resulting filtered matrix underwent quality control assessments and analytical evaluations within the R environment (version 4.3.2, (Team, 2021)), utilizing various R packages such as SoupX (version 1.6.2, (Young and Behjati, 2020)) for the removal of ambient mRNAs, scater (version 1.30.1, (McCarthy et al., 2017)) to identify outlier metrics and Seurat (version 5.0.1, (Hao et al., 2024)). No other cell filtering criteria were applied regarding total number of UMIs, unique expression genes, or mitochondrial gene proportion. However, genes with cell counts less than 3 were excluded. Seurat’s demultiplexing function was employed to eliminate doublets and ambiguous cells. Additionally, Seurat’s SCTransform was applied using the ‘glmGamPoi’ method, followed by RunPCA, RunUMAP, FindNeighbors and FindClusters for clustering analysis. Default parameters were utilized, except for employing 40 dimensions of input features and proceeding with a resolution of 2 for cluster analysis. Manual annotations were conducted by referencing markers from various published sources (Aegerter et al., 2022, Du et al., 2015, Du et al., 2017, Du et al., 2021, Hu et al., 2022, Program et al., 2023, Travaglini et al., 2020). UMAP figures were customized using ggplot2 (version 3.4.4, 14) (Wickham, 2016).

Lung macrophages transcriptomics

Lungs from Day 7 Nb infection in Alox15^-/- or WT mice were minced and then digested in media (DMEM (Gibco), 2 mg mL−1 collagenase (Sigma), DNase 12.5 U mL−1 (Roche, SigmaAldrich, Switzerland)) at 37°C on an orbital shaker for 45 min at 105 rpm. Total lung cells were enriched in immune cells by CD45⁺ MACS selection, before being seeded in 6 well plates at 1million/mL. After overnight culture, the adherent fraction was used for RNA sequencing. Histological characterization shows a macrophage purity of >90% and deconvolution of the bulk RNA sequencing showed the main cell types were AMs, IMs, monocytes and dendritic cells (Figure S3E). The deconvolution used cybersort website and we used our own single cell sequencing data from Figure 1A&B to create the reference table.

The next day the adherent fraction was lysed in trizol and RNA was extracted. RNA sequencing reads were processed by the UNIL’s Lausanne Genomic Technologies Facility’s pipeline (version 4). Main steps thereof included using cutadapt for adaptor and quality trimming, STAR for aligning to the Mouse genome, and htseq-counts for hit counting in gene exons. Hit count matrices were read into R (v 3.5.0) and analysed using DESeq2 (v.1.20.0). The experimental design passed to DESeq2 aimed to test the effect of Genotype (Alox15^-/- versus WT), with 5 samples per group. Genes with no hits were excluded, retaining 24636 out of >40k mouse genes, and DESeq2 size factors were then calculated for library normalisation. The differential expression analysis was performed using 2 different thresholding approaches: one stringent (FDR cutoff of 5%, log2FoldChange cutofff of 0.10, i.e. fold change cutoff of ~7 and one broad (FDR cutoff of 10%, log2FoldCHange cutoff of 0.01, i.e. ~0.7% to allow the exploration of more borderline cases depending on the biological question. These filters were used in 3 settings: testing for differentially expressed genes (either direction), testing for upregulated genes, and testing for downregulated genes, as each of these lead to slightly different statistical tests. DESeq2 by default applies Benjamini-Hochberg multiple testing correction. Across all 6 test settings combined, 18405 out of 24636 genes were tested for differential expression by DESeq2 (following DESeq2’s internal optimised expression-based filter).

The 6 differentially expressed gene (DEG) lists were tested for enrichment in terms (GO terms/KEGG maps/REACTOME maps) using gProfileR v.0.6.6, specifying as an appropriate background universe the list of all genes tested for differential expression (settings: unordered query, max p-value of 10%, do not exclude IEA annotations, non-regional query, report only GO:BP, KEGG and REACTOME hits, no hierarchical filtering, do not track underrepresentation). As the gProfileR tool sends requests to a webserver, we note that we performed this analysis on 26/10/2018. Network analysis was performed using WGCNA v.1.64.1. Normalised counts were used to perform a light filtering on genes to use for analysis (mean expression >= 1.5 hits, CV >= 0.05), leaving 16300 genes. Shifted log10 (sl10) normalised counts were then examined. Genes without at least 20% of values above 0.10 (i.e. ~0.25 hits) were excluded, removing a further 395 genes. Ultimately, the sl10 expression data for 15905 genes was passed to WGCNA for network (clustering) analysis. In all following analyses, we used signed network types, “bicor” correlations, with a maxPOutliers setting of 0.05.

In the absence of a clear elbow or value that passed the recommended scale-free topology measure of 0.9, a soft thresholding power of 9 was selected as a trade-off between scale-free topology and connectivity. Adjacency and Topological Overlap Matrices (TOM) were calculated following the settings above. The fastcluster package’s hclust() function was called to cluster genes based on the TOM using Ward D2’s criterion. The dynamicTreeCut package function cutreeDynamic() (hybrid approach, cutHeight of 9, deepSplit of 1, minModuleSize of 1000, pamRespectsDendro set to True) was used to split the genes into 11 clusters with concerted variations across the dataset. The lists of genes in each cluster were tested for enrichments in annotation terms from various ontologies (GO:BP, KEGG, REACTOME) using gProfileR. They were also tested for enrichments in lists of DEGs of one direction or the other using one-sided hypergeometric tests. Additionally, the distributions of log2 fold changes between WT and Alox15 were plotted as an indication of module-wide concerted changes between genotypes.

Sample preparation for LC-MS/MS

For intestinal tissue culture, the intestine was removed, extensively flushed with cold PBS. The intestine was isolated, cut longitudinally and placed separately in 100 mm Petri dishes with PBS containing 10 µg/ml Gentamicin, 100 U/ml Penicillin and 100 µg/ml Streptomycin (all from GIBCO). Opened cut intestines were gently scraped to remove mucus, washed 3 times and transferred into a new Petri dish. The anterior 4 cm of the intestine (duodenum) were cut into 1–2 mm pieces and transferred to a 24-well plate with 2 ml/well of RPMI media (PAA Laboratories) containing 10% heat inactivated FCS, 10 µg/ml Gentamicin, 100 U/ml Penicillin and 100 µg/ml Streptomycin (all from GIBCO). 24-well plates were incubated over night at 37°C before supernatants were harvested and analyzed by LC-MS/MS.

Samples were prepared as triplicates in medium/MeOH (1:1) or PBS/MeOH (1:1) with an analyte concentration of 0.1, 1, or 10 ng/mL (10× higher concentrations for PUFAs). Automated solid phase extractions were performed with a Microlab STAR robot (Hamilton, Bonaduz, Switzerland). Prior to extraction, all samples were diluted with H₂O to a MeOH content of 15% and 10 μL of IS stock solution was added. Samples were extracted using Strata-X 96-well plates (30 mg, Phenomenex, Aschaffenburg, Germany) and eluted with MeOH. Samples were evaporated to dryness under N₂ stream and redissolved in 100 μL MeOH/H₂O (1:1).

LC-MS/MS lipid mediator analysis

Chromatographic separation of eicosanoids was achieved with a 1260 Series HPLC (Agilent, Waldbronn, Germany) using a Kinetex C18 reversed phase column (2.6 μm, 100 × 2.1 mm, Phenomenex) with a SecurityGuard Ultra Cartridge C18 (Phenomenex) precolumn. The Sciex QTRAP 5500 mass spectrometer (Sciex, Darmstadt, Germany), equipped with a Turbo-V™ ion source, was operated in negative ionization mode. Identification of metabolites was achieved via retention time and scheduled multiple reaction monitoring (sMRM). Unique Q1/Q3 transitions were selected for each analyte by using single analyte injections and comparison with the literature. Analytes with identical MRM transitions were differentiated by retention time as described in (Henkel et al., 2019)

Flow cytometry

BMDM were polarized or not with recombinant IL-4 (10 ng mL⁻¹, peprotech) for 24 hours. The next day, cells were detached with cold PBS and a cell scraper. Isolated cells were resuspended in FACS buffer, and Fc-receptors were blocked (2.4G2) before staining with monoclonal antibodies before collection on a Fortessa flow cytometer (BD) or on a Cytoflex-S. Fluorochrome-conjugated antibodies to the following cell surface molecules were used: CD45-AF700 (1:400, clone 30-F11,, RRID AB_1645208, BD Cat# 560510), APCeF780 (1:100, clone BM8, RRID AB_2735036, eBiosciences Cat # 47-4801-82), Arginase-AF647 (1:100, clone EPR22033-369, Abcam Cat# ab307468) or Arginase-APC (1:100, polyclonal, RRID RRID: AB_2810265, R&D systems, Cat# IC5868A), Ki-67-PE (1:10, clone SolA15, RRID AB_10854564, Ebiosciences, Cat# 14-5698-82). Cell suspensions were stained with LIVE/DEAD fixable aqua or violet dead cell Stain kit (Invitrogen) when fixation was required for intracellular staining. Analysis was performed with the FlowJo software (Treestar).

Lungs from naïve or Nb infected mice on Alox15^-/- or WT background were minced and then digested in media (DMEM (Gibco), 2 mg mL−1 collagenase (Sigma), DNase 12.5 U mL−1 (Roche, SigmaAldrich, Switzerland)) at 37°C on an orbital shaker for 45 min at 105 rpm. After digestion, samples were counted and 2 million cells were stained as reported for the macrophages or the Th2/ILC2s. For the macrophages panels the following antibodies were used: CD45-BV510 (1:400, clone 30-F11, RRID AB_2561392, BioLegend Cat #103137); F4/80-APCeF780 (1:200, clone BM8, RRID AB_2735036, eBiosciences Cat # 47-4801-82); CD11c-BV785 (1:200, Clone N418, RRID AB_11219204, BioLegend Cat #117335); SiglecF-PE-Dazzle594 (1:200, Clone S17007L, RRID AB_2890716, BioLegend Cat# 155530); CD11b-FITC (1:200, clone M1/70, RRID AB_312789, BioLegend Cat # 101206); CD117-PE (1:200, Clone 2B8, RRID AB_465812, eBiosciences Cat #12-1171-81); Fc epsilon R1 alpha-PE-Cy7 (1:200, Clone MAR-1, RRID AB_10640122, BioLegend Cat # 134318); RELMa-AF700 (1:100, Clone DS8RELM,, RRID AB_2811895, eBiosciences Cat #56-5441-82); iNOS-PerCpCy5.5 (1:100, Clone W16030C, RRID AB_3106057, BioLegend Cat # 696810); Arginase-APC (1:100, polyclonal, RRID RRID: AB_2810265, R&D systems, Cat# IC5868A). Intracellular staining is done using eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, Cat# 00-5523-00). Interstitial macrophages are defined as F4/80⁺ CD11b+SiglecF- (CD11c ^low/int), Alveolar macrophages are defined as F4/80^hi CD11c^high CD11b^-/low SiglecF⁺, Eosinophils are defined as: F4/80int SiglecF⁺ CD11c^- Cd11b⁺ high SSC, Monocytes are defined as CD11b⁺ F4/80^low CD11c^-. Gates were set based on fluorescence minus one (FMO) controls.

For the Th2/ILC2 panel, the following antibodies were used : CD45-BV510 (1:400, clone 30-F11, RRID AB_2561392, BioLegend Cat #103137); CD25-BV785 (1:400, clone PC61, RRID AB_2564131, BioLegend Cat# 102051); CD4-APC/Fire750 (1:400, clone GK1.5, RRID AB_2572111, BioLegend Cat# 100460); SiglecF-PE (1:200, clone S17007L, RRID AB_2750235, BioLegend Cat# 155506); Gr-1-PE (1:200, clone RB6-8C5, RRID AB_313373, BioLegend Cat# 108408); B220-PE (1:200, clone RA3-6B2, RRID AB_312993, BioLegend Cat# 103208); CD11b-PE (1:200, clone M1/70, RRID AB_312791, BioLegend Cat# 101208); CD11c-PE (1:150, clone N418, RRID AB_313777, BioLegend Cat# 117308); NKp46-PE (1:100, clone 29A1.4, RRID AB_2235755, BioLegend Cat# 137604); CD8-PE (1:400, clone 53-6.7, RRID AB_312747, BioLegend Cat# 100708); F4/80-PE (1:400, clone BM8, RRID AB_893486, BioLegend Cat# 123110); KLRG1-BV605 (1:200, clone 2F1, RRID AB_2563357, BioLegend Cat# 138419); CD127-AF700 (1:100, clone A7R34, RRID AB_1937287, BioLegend Cat# 135002); ST2-APC (1:100, clone DIH4, RRID AB_2728175, BioLegend Cat# 146606); Fc epsilon R1 alpha-PE-Cy7 (1:400, Clone MAR-1, RRID AB_10640122, BioLegend Cat # 134318); c-kit-PerCP(1:100, Clone 2B8, RRID AB_2131599, BioLegend Cat # 105822); Gata-3-AF488 (1:100, Clone TWAJ, RRID AB_2574493, eBiosciences Cat # 53-9966-42). Intracellular staining is done using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, Cat# 00-5523-00). Th2 cells are identified as CD45⁺ DUMP-PE^- C-kit^- CD127^- CD4⁺ Gata-3⁺ and ILC2s are identified as CD45⁺ DUMP-PE^- C-kit^- CD127^- CD4^– KLRG-1⁺ Gata-3⁺. Gates were set based on fluorescence minus one (FMO) controls.

The bronchoalveolar lavage cells were counted and stained as described previously. Antibodies include: CD45-BV510 (1:400, clone 30-F11, RRID AB_2561392, BioLegend Cat #103137); GR1-FITC (1:100, clone RB6-8C5, RRID AB_313371, BioLegend Cat# 108406); anti-CD11b-PerCp (1:100, clone M1/70, RRID AB_2129374, BioLegend Cat # 101230); SiglecF-PE-Dazzle594 (1:200, Clone S17007L, RRID AB_2890716, BioLegend Cat# 155530); F4/80-APCeF780 (1:200, clone BM8, RRID AB_2735036, eBiosciences Cat # 47-4801-82). Alveolar macrophages are defined as CD45⁺ SiglecF⁺, F4/80^hi, CD11b ^low–int, Gr1^–, Eosinophils as CD45⁺ SiglecF⁺, F4/80^–/low, MertK^–, CD11b⁺, Gr1 ^low, very high SSC, and neutrophils as CD45⁺ Gr1^hi, CD11b^hi, F4/80^-. Gates were set based on fluorescence minus one (FMO) controls.

Arginase-1 activity assay

Macrophage arginase-1 activity was determined according to previously published methods (Esser-von Bieren et al., 2013). Briefly, 300000 adherent macrophages (48 well plate) were lysed and conversion of L-arginine was quantified indirectly by measuring urea production with a QuantiChrom Urea Assay (BioAssay Systems, Hayward, CA).

Griess assay

Culture media from L. mexicanaTurboRFP infection assays was harvested by centrifuging to remove cells and retaining supernatant. 50 uL supernatant was first incubated at room temperature for 7 min with 50 uL 0.1% sulfanilamide in phosphoric acid prior to addition of 50 uL N-1-napthylethylenediamine dihydrochloride (NEDD) and a further 7 min incubation. Plates were then analyzed by plate reader at 540 nm. Sample nitrite concentration was determined by a nitrite standard dilution series.

Fluorescence and bright-field imaging

BMDM were grown on 8 chambers ibidi slides, fixed in 2% PFA. Prior to staining, cells were permeabilized in PBS, 0.5% triton X100 (Sigma) and blocked in 5% PBS, BSA (Sigma) and then stained with DAPI and anti-Alox15 antibody (1:100, Abb244205, Abcam). Granulomas were identified on paraffin sections by light microscopy and serial sections were used for immunofluorescence staining for Arginase-1-AF568 (1:800) and F4/80-AF647 (BM8, 1:300, Ebiosciences) and DAPI (Sigma). Stained tissue sections were imaged with an inverted point scanning confocal microscope (Zeiss LSM 710) with a Plan-Apochromat (63×/1.4 NA or 40×/1.3 NA) objective. Alox15 or Arginase intensity was measured in Fiji 1.54p. To control for differences in background fluorescence between experiments and antibody/dye batches, the contrast was adjusted to minimize autofluorescence and a minimum brightness threshold was set such that only positive staining could be visualized. The same contrast and threshold values were applied to all images taken across all treatment groups within a single experiment using Fiji 1.54p (Schindelin et al., 2012).

Lungs were fixed in 4% PFA overnight and embedded in paraffin the next day. 5um thick section were stained with Prussian blue staining (Sigma, Cat #HT20) according to manufacturer instructions and imaged on a Zeiss Axio Scan.Z1 Slide Scanner (Imaging core facility Biozentrum). Images were analysed in Fiji 1.54p. 5 areas were randomly counted per section of lungs and 2 sections were counted per animal. In each area, macrophages of each of the following category were counted: no staining, light staining, intermediate staining and dark staining. The proportion was calculated on 100 macrophages per lung section. Quantifications were performed in a blinded manner.

Seahorse flux analysis

BMDM were polarized or not with recombinant IL-4 (10 ng mL⁻¹, peprotech) for 24 hours and with LPS (10 ng/ml, Sigma) for 8 hours. The next day, cells were detached with cold PBS and a cell scraper. Isolated macrophages from individual mice or a pool of 2-3 mice were plated at 50 000 cells per well in 96 well plate (100 ul) or at 150 000 cells per well in a 24 well plate (450 ul) in XF media and allowed to adhere for 30 min at 37C, 5%C02. The cells were then transferred for 30 min to a 37C incubator without C02 just prior to analysis. ECAR and OCR were measured in XF media under basal conditions, in response to 25 mM glucose, 1.5 μM oligomycin, 1.5 μM FCCP, 1mM Sodium Pyruvate, 0.5 μM Antimycin A, 0.5 μM Rotenone (Sigma) using a 96- or 24-well extracellular flux analyzer XFe-96 and XFe-24 (Seahorse Bioscience) as previously described (Van den Bossche et al., 2015).

Data analysis

All data were analyzed using GraphPad Prism 8.2 (GraphPad Software, La Jolla, CA, USA) or R 3.4.3.21. For LC-MS/MS analysis, all samples were normalized to their RNA content. Data were analyzed using Wilcoxon, Friedman, or Kruskal-Wallis test with respective post hoc test as appropriate. Significant differences were defined at P <0.05, **** P<0.0001; *** P<0.001; ** P<0.01; * P <0.05. Tests and n numbers used are indicated in figure legends. Note that unless mentioned in the legend of the figure, only the post-hoc tests shown on the figure have been performed.

Summary.

Ovezgeldiyev et al. reveal a metabolic pathway linking lipid mediator production to macrophage activation during helminth immunity. Alox15 promotes glycolysis and M2 polarization, while downstream DPA-derived lipid mediators activate PPARδ to control macrophage effector function.