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. 2022 May 16;23(7):e54339. doi: 10.15252/embr.202154339

Nlrp3 inflammasome activation in macrophages suffices for inducing autoinflammation in mice

Ulrika C Frising 1,2, Silvia Ribo 1,2, M Giulia Doglio 1,2, Bernard Malissen 3, Geert van Loo 2,4, Andy Wullaert 1,2,5,✉
PMCID: PMC9253760  PMID: 35574994

Abstract

Cryopyrin‐associated periodic syndromes (CAPS) are a spectrum of autoinflammatory disorders caused by gain‐of‐function NLRP3 mutant proteins that form hyperactive inflammasomes leading to overproduction of the pro‐inflammatory cytokines IL‐1β and IL‐18. Expressing the murine gain‐of‐function Nlrp3A350V mutant selectively in neutrophils recapitulates several autoinflammatory features of human CAPS, but the potential contribution of macrophage inflammasome hyperactivation to CAPS development is poorly defined. Here, we show that expressing Nlrp3A350V in macrophages is sufficient for driving severe multi‐organ autoinflammation leading to perinatal lethality in mice. In addition, we show that macrophages contribute to autoinflammation also in adult mice, as depleting macrophages in mice ubiquitously expressing Nlrp3A350V significantly diminishes splenic and hepatic IL‐1β production. Interestingly, inflammation induced by macrophage‐selective Nlrp3A350V expression does not provoke an influx of mature neutrophils, while neutrophil influx is still occurring in macrophage‐depleted mice with body‐wide Nlrp3A350V expression. These observations identify macrophages as important cellular drivers of CAPS in mice and support a cooperative cellular model of CAPS development in which macrophages and neutrophils act independently of each other in propagating severe autoinflammation.

Keywords: cryopyrin‐associated periodic syndromes, inflammasome, interleukin‐1β, macrophage, Nlrp3

Subject Categories: Immunology, Molecular Biology of Disease, Signal Transduction

Selectively expressing the Nlrp3A350V mutant in mouse macrophages is sufficient to drive severe multi‐organ autoinflammation reminiscent of cryopyrin‐associated periodic syndromes (CAPS) that are caused by such Nlrp3 gain‐of‐function mutations in humans.

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Introduction

Cryopyrin‐associated periodic syndromes (CAPS) are a spectrum of autoinflammatory disorders associated with gain‐of‐function mutations in the gene encoding cryopyrin, more commonly known as NLRP3. CAPS patients suffer from systemic inflammation including fever and cutaneous, musculoskeletal, and ocular involvements to varying degrees. Depending on the symptoms and disease severity, CAPS encompass three clinical entities: familial cold‐induced autoinflammatory syndrome (FCAS), Muckle‐Wells syndrome (MWS), and neonatal‐onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (Booshehri & Hoffman, 2019). Genetic analyses of families inheriting FCAS or MWS in an autosomal dominant pattern led to the identification of disease‐causative heterozygous mutations in the NLRP3 gene (Hoffman et al, 2001a, 2001b). Clinical similarities between MWS and NOMID subsequently allowed the identification of homozygous NLRP3 mutations in NOMID patients (Aksentijevich et al, 2002; Feldmann et al, 2002). Meanwhile, over 100 CAPS‐associated NLRP3 gain‐of‐function mutations were reported (Infevers database: https://infevers.umai‐montpellier.fr/) (Touitou et al, 2004; Booshehri & Hoffman, 2019). CAPS‐associated NLRP3 mutations cause excessive activation of the NLRP3 inflammasome, a protein complex in which caspase‐1 mediates maturation of the IL‐1β and IL‐18 cytokines, allowing secretion of their active forms. Consistent with the inflammasome activating function of NLRP3 leading to IL‐1β secretion, most CAPS patients can be treated efficiently with IL‐1β blocking therapies (Booshehri & Hoffman, 2019). While this illustrates the crucial pathogenic role of IL‐1β downstream of NLRP3 inflammasome activation in CAPS patients, the contributions of various possible cell types driving this disease have not been fully elucidated.

Brydges et al (2009) generated an elegant mouse model allowing cell type‐specific expression of a murine Nlrp3A350V gain‐of‐function protein. In this mouse model, wild‐type (WT) exon 3 of the endogenous Nlrp3 gene was replaced by an exon 3 containing the A350V mutation, while inserting a floxed neomycin cassette (NeoR) in intron 2 of the Nlrp3 gene. The resulting homozygous Nlrp3NeoR‐A350V/NeoR‐A350V mice were deficient for endogenous Nlrp3 expression but also did not express the Nlrp3A350V mutant because of transcriptional silencing by the intronic NeoR gene. Breeding Nlrp3NeoR‐A350V/NeoR‐A350V mice with hemizygous Cre‐transgenic mice results in Nlrp3NeoR‐A350V/WT offspring in which the NeoR cassette of the Nlrp3NeoR‐A350V allele will be excised in Cre‐expressing cell type(s), resulting in Nlrp3A350V expression specifically in these cells (Brydges et al, 2009).

Several studies using this mouse model provided important insights into the cellular mechanisms driving CAPS pathogenesis. Mice expressing Nlrp3A350V in both macrophages and neutrophils by using a LysM driven Cre transgene displayed severe autoinflammation leading to perinatal lethality (Brydges et al, 2009), indicating an important role for these myeloid cells in driving CAPS in mice. Interestingly, a very recent study showed that Mrp8Cre‐driven neutrophil‐specific expression of Nlrp3A350V was sufficient to induce a similar CAPS phenotype as induced by LysMCre‐driven Nlrp3A350V expression (Stackowicz et al, 2021). This demonstrated a crucial cell‐intrinsic pathogenic role for neutrophils in Nlrp3A350V‐mediated autoinflammation in mice. In contrast, given the dual specificity of the LysMCre transgene for both neutrophils and macrophages (Abram et al, 2014), the specific cell‐intrinsic role of macrophages in provoking CAPS in mice remains to be elucidated.

Here, we show that Nlrp3A350V expression in macrophages was sufficient to provoke lethal CAPS in mice. Moreover, depleting macrophages in a CAPS model induced by ubiquitous Nlrp3A350V expression demonstrated a crucial contribution for macrophages in producing IL‐1β and IL‐18 in CAPS. Interestingly, autoinflammation induced by macrophage‐driven Nlrp3A350V expression was not associated with the influx of Ly6G+ neutrophils while macrophage‐depleted Nlrp3A350V‐expressing mice still exhibited this characteristic CAPS feature, indicating that inflammasome hyperactivation in macrophages and neutrophils can independently drive CAPS in mice.

Results

NLRP3A350V expression in macrophages promotes severe CAPS in mice

To investigate the contribution of macrophage‐intrinsic Nlrp3A350V expression in CAPS development, we bred Nlrp3NeoR‐A350V/NeoR‐A350V mice to hemizygous transgenic mice expressing Cre recombinase under the macrophage‐specific Fcgr1 promoter (Fcgr1CreTg/WT) (Scott et al, 2018). While resulting Fcgr1CreTg/WT offspring allowed Nlrp3A350V expression in macrophages along with body‐wide expression of the WT Nlrp3 allele (Nlrp3Mac‐A350V/WT), Fcgr1CreWT/WT control littermates only expressed Nlrp3 from their WT allele (Nlrp3NeoR‐A350V/WT) (Fig 1A). Both genotypes were born in approximate Mendelian ratios (Fig 1B), indicating that macrophage Nlrp3A350V expression did not compromise in utero development of Nlrp3Mac‐A350V/WT mice. However, unlike their Nlrp3NeoR‐A350V/WT littermates, Nlrp3Mac‐A350V/WT pups started dying from day 2 on, with all of them succumbing within 11 days after birth (Fig 1C). This impaired perinatal development of Nlrp3Mac‐A350V/WT mice was accompanied by a failure to gain body weight from day 4 onwards (Fig 1D). Besides their impaired growth, Nlrp3Mac‐A350V/WT pups were clearly distinguishable from Nlrp3NeoR‐A350V/WT littermates by a persistent lack of fur growth and the appearance of scaling skin erythema (Fig 1E). Next, we measured circulating serum cytokines to evaluate whether the Nlrp3Mac‐A350V/WT phenotype was associated with a systemic inflammatory signature. These analyses showed significantly upregulated levels of the inflammasome‐generated cytokines IL‐1β and IL‐18 (Fig 1F and G) and significantly increased amounts of IL‐6 and TNF in serum of Nlrp3Mac‐A350V/WT pups (Fig 1H and I). Overall, these observations showed a similar phenotypic appearance and serum cytokine profile in Nlrp3Mac‐A350V/WT pups compared with previous reports on pups expressing Nlrp3A350V in both macrophages and neutrophils (Brydges et al, 2009; McGeough et al, 2012, 2017). This suggests that Nlrp3A350V expression in macrophages suffices to elicit the proinflammatory cytokine cocktail needed for driving systemic CAPS pathogenesis in mice.

Figure 1. NLRP3A350V expression in macrophages promotes severe CAPS in mice.

Figure 1

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  • A
    Breeding scheme and mouse nomenclature.
  • B
    Number of pups obtained from Nlrp3NeoR‐A350V/NeoR‐A350V x Fcgr1CreTg/WT matings, with expected and observed Mendelian ratios of the indicated genetic offspring.
  • C
    Survival curves of Nlrp3NeoR‐A350V/WT (n = 14) and Nlrp3Mac‐A350V/WT littermates (n = 14).
  • D
    Growth curves of Nlrp3NeoR‐A350V/WT (n = 8) and Nlrp3Mac‐A350V/WT littermates (n = 10).
  • E
    Representative pictures of Nlrp3NeoR‐A350V/WT and Nlrp3Mac‐A350V/WT pups at indicated days after birth. Scale bar 1 cm.
  • F–I
    (F) IL‐1β, (G) IL‐18, (H) IL‐6, and (I) TNF analyses in serum from 6‐ to 9‐day‐old pups (Nlrp3NeoR‐A350V/WT n = 7, Nlrp3Mac‐A350V/WT n = 6).

Data information: Statistics were analyzed by the (C) log‐rank (Mantel–Cox) test, (D) student t‐test, or (F‐I) Mann–Whitney test. Dots represent individual biological replicates. Error bars represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

CAPS in Nlrp3Mac‐A350V/WT mice is mediated by both IL‐1β and IL‐18 inflammasome‐generated cytokines

We next evaluated the effects of macrophage Nlrp3A350V expression in the spleen, liver, and kidney of Nlrp3Mac‐A350V/WT pups to determine the inflammasome‐mediated inflammatory capacities of macrophages in each of these organs. Histopathological analyses revealed signs of inflammation in the spleen, liver, and kidney from Nlrp3Mac‐A350V/WT pups (Fig 2A). Nlrp3Mac‐A350V/WT spleens displayed a disorganized tissue architecture of red and white pulps, while Nlrp3Mac‐A350V/WT livers showed inflammatory foci and large necrotic lesions containing dead cells (Fig 2A), similar to those described after Tamoxifen‐induced ubiquitous Nlrp3A350V expression in adult mice (Schuster‐Gaul et al, 2020). In addition, Nlrp3Mac‐A350V/WT kidneys showed signs of tubulointerstitial inflammation (Fig 2A), consistent with the role of Nlrp3 in this type of renal inflammation during chronic kidney disease (Vilaysane et al, 2010). We next evaluated inflammasome activity in these pathologies by measuring IL‐1β and IL‐18 in the respective organ homogenates. The spleen, liver, and kidney from Nlrp3Mac‐A350V/WT pups displayed significantly increased IL‐1β levels, indicative of ongoing inflammasome activation (Fig 2B–D). However, IL‐18 levels were upregulated in the spleen but not in the liver or kidney of Nlrp3Mac‐A350V/WT pups (Fig 2B–D). To monitor inflammasome activation on a molecular level we evaluated caspase‐1 activation in organ homogenates. The active p10 subunit of caspase‐1 was readily detected in spleen and liver lysates of Nlrp3Mac‐A350V/WT pups (Fig 3A and B). In contrast, despite their increased IL‐1β production, kidneys of Nlrp3Mac‐A350V/WT pups did not contain detectable amounts of active caspase‐1 (Fig 3C), likely reflecting lower macrophage numbers in kidneys. Consistent with caspase‐1 cleavage, both the spleen and liver of Nlrp3Mac‐A350V/WT pups displayed processing of pro‐IL‐1β to the mature IL‐1β cytokine as assessed by Western blotting (Fig 3D and E). Further supporting proteolytic caspase‐1 activity in these organs, the spleen and liver of Nlrp3Mac‐A350V/WT mice also showed cleavage of the caspase‐1 substrate Gasdermin D (GSDMD) (Fig 3F and G). These Western blots also revealed that Nlrp3Mac‐A350V/WT organs harbored increased levels of full‐length GSDMD as compared to healthy Nlrp3NeoR‐A350V/WT mice, perhaps reflecting inflammatory infiltrates in the Nlrp3Mac‐A350V/WT organs (Fig 3F and G). The observed GSDMD cleavage in both the spleen and liver of Nlrp3Mac‐A350V/WT mice suggests ongoing pyroptosis in these mice. Although CAPS in a mouse model expressing the Nlrp3D301N mutant was shown to depend on GSDMD (Xiao et al, 2018), future genetic studies will be needed to address the causal role of GSDMD‐driven pyroptosis for CAPS development in Nlrp3Mac‐A350V/WT mice.

Figure 2. NLRP3A350V expression in macrophages provokes multiorgan autoinflammation.

Figure 2

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  • A
    Representative H&E stained spleen, liver, and kidney sections from 9‐day‐old Nlrp3NeoR‐A350V/WT and Nlrp3Mac‐A350V/WT littermates. Scale bars 100 μm.
  • B–D
    IL‐1β and IL‐18 protein levels measured in (B) spleen, (C) liver, and (D) kidney homogenates from 6‐ to 9‐day‐old indicated mice.

Data information: Statistics in (B‐D) were analyzed by the student t‐test (IL‐18 graphs in B, C, D and IL‐1β graph in D) or Mann–Whitney test (IL‐1β graphs in B and C). Dots represent individual biological replicates. Error bars represent mean ± SD. ns—not significant; *P < 0.05.

Figure 3. CAPS in Nlrp3Mac‐A350V/WT mice is associated with inflammasome activity in the spleen and liver and is mediated by both IL‐1β and IL‐18 cytokines.

Figure 3

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  • A–C
    Tissue lysates from (A) spleen, (B) liver, and (C) kidney from 6‐ to 9‐day‐old indicated mice were immunoblotted to determine cleavage of caspase‐1. Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (−) are respective organ homogenates from caspase‐1−/− mice.
  • D–G
    Tissue lysates from (D, F) spleen and (E, G) liver from 6‐ to 9‐day‐old indicated mice were immunoblotted to determine pro‐IL‐1β cleavage to IL‐1β (D‐E) or GSDMD cleavage to its p30 and p20 fragments (F‐G). Each lane represents a lysate from a different mouse. Positive control lanes (+) are BMDMs stimulated with LPS/ATP; negative control lanes (−) are respective organ homogenates from (D‐E) caspase‐1−/− or (F‐G) Gsdmd−/− mice.
  • H–K
    Growth (H, J) and survival (I, K) curves of Nlrp3NeoR‐A350V/WT and Nlrp3Mac‐A350V/WT littermates injected subcutaneously with PBS or Anakinra (H‐I) or with anti‐IL‐18 or isotype antibodies (J‐K). Injection days are indicated with arrows.

Data information: Statistics in (H, J) were analyzed by the student t‐test, error bars represent mean ± SD of indicated numbers of biological replicates. Statistics in (I, K) were analyzed by the log‐rank Mantel–Cox test. ns—not significant; *P < 0.05; **P < 0.01.

Source data are available online for this figure.

Finally, upon obtaining these data supporting genuine inflammasome activity in Nlrp3Mac‐A350V/WT mice, we sought to identify the inflammasome‐generated cytokine(s) responsible for eliciting CAPS in these mice. To address the role of IL‐1β, we treated Nlrp3Mac‐A350V/WT pups with the IL‐1 receptor (IL‐1R) antagonist Anakinra. Anakinra‐treated Nlrp3Mac‐A350V/WT pups showed an improved growth curve but eventually did not live significantly longer than PBS‐treated Nlrp3Mac‐A350V/WT pups (Fig 3H and I). Next, we used anti‐IL‐18 neutralizing antibodies to investigate the potential role of IL‐18 in the CAPS pathology of Nlrp3Mac‐A350V/WT pups. While anti‐IL‐18 treatment did not ameliorate weight gain, anti‐IL‐18‐treated Nlrp3Mac‐A350V/WT pups survived significantly longer than Nlrp3Mac‐A350V/WT pups treated with control isotype antibodies (Fig 3J and K). Together, these experiments showed contributions for both IL‐1β and IL‐18 to autoinflammation in Nlrp3Mac‐A350V/WT mice. However, IL‐18 seemed to play a more important role in driving perinatal lethality, consistent with genetic experiments addressing this question in mice with LysMCre‐driven Nlrp3A350V expression (Brydges et al, 2013).

CAPS in Nlrp3Mac‐A350V/WT mice develops independently of mature neutrophils

To gain more detailed insights into the role of macrophages in eliciting CAPS in various Nlrp3Mac‐A350V/WT organs we performed immunohistochemical F4/80 macrophage staining in the spleen, liver, and kidney of these mice. We observed clustering of F4/80+ macrophages around necrotic lesions in Nlrp3Mac‐A350V/WT livers and the appearance of F4/80+ macrophage foci in kidneys of Nlrp3Mac‐A350V/WT mice (Fig 4A). Flow cytometry myeloid cell analyses (Fig EV1) to more rigorously and more specifically quantify CD11b+F4/80+CD64+ macrophages subsequently revealed increased macrophage numbers in the spleen and kidney but not in the liver of Nlrp3Mac‐A350V/WT pups when compared to control littermates (Fig 4B–D). Thus, consistent with inflammasome activation in macrophages of Nlrp3Mac‐A350V/WT mice, flow cytometric and immunohistochemical analyses provided evidence for increased infiltration and/or pronounced clustering of macrophages, respectively, in distinct organs of Nlrp3Mac‐A350V/WT mice. In addition to macrophages, we observed a prominent increase in CD11b+Ly6C+Ly6G− monocytes in all organs examined from Nlrp3Mac‐A350V/WT mice (Fig 4E–G). However, Nlrp3Mac‐A350V/WT mice did not display increased numbers of CD11b+Ly6C+Ly6G+ neutrophils in any organs examined and even showed significantly lower CD11b+Ly6C+Ly6G+ neutrophil numbers in the liver (Fig 4H–J).

Figure 4. CAPS in Nlrp3Mac‐A350V/WT mice is associated with macrophage infiltrations without infiltration of Ly6G+ neutrophils.

Figure 4

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  • A
    Representative F4/80 immunohistochemical macrophage (brown) staining of spleen, liver, and kidney sections from 9‐day‐old Nlrp3NeoR‐A350V/WT and Nlrp3Mac‐A350V/WT littermates. Scale bars 100 μm.
  • B–D
    Flow cytometric CD11b+F4/80+CD64+ macrophage quantifications in (B) spleen, (C) liver, and (D) kidney from indicated 6‐day‐old mice.
  • E–G
    Flow cytometric CD11b+Ly6C+Ly6G− monocyte quantifications in (E) spleen, (F) liver, and (G) kidney from 6‐day‐old indicated mice.
  • H–J
    Flow cytometric CD11b+Ly6C+Ly6G+ neutrophil quantifications in (H) spleen, (I) liver, and (J) kidney from 6‐day‐old indicated mice.

Data information: Statistics in (B‐J) were analyzed by the student t‐test (B, D, E, F, G, and J) or Mann–Whitney test (C, H and I). Dots represent individual biological replicates. Error bars represent mean ± SD. ns—not significant; *P < 0.05; **P < 0.01.

Figure EV1. Flow cytometric analysis of Nlrp3NeoR‐A350V/WT and Nlrp3Mac‐A350V/WT pups.

Figure EV1

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Representative flow cytometric gating of Nlrp3NeoR‐A350V/WT pups. Doublets were excluded before identifying the immune cells (CD45+ cells). Macrophages were identified as CD11b+F4/80+CD64+ cells, monocytes were identified as CD11b+Ly6C+Ly6G− cells, and neutrophils were identified as CD11b+Ly6C+Ly6G+ cells. A—area; FSC—forward scatter; H—height; L/D—live/dead; SSC—side scatter; W—width.

Since neutrophil infiltration is a prominent feature of CAPS both in humans and mice (Brydges et al, 2009; McGeough et al, 2012; Hoffman & Broderick, 2016) the lack of CD11b+Ly6C+Ly6G+ neutrophil influx in tissues of Nlrp3Mac‐A350V/WT mice suffering from severe CAPS was remarkable. Therefore, to better understand the effect of macrophage‐derived inflammation on neutrophils, we analyzed bone marrow (BM) of Nlrp3Mac‐A350V/WT mice to see whether ongoing CAPS impacted on the neutrophil‐generating granulopoiesis process. To do so, we followed a gating strategy as described (Khoyratty et al, 2021), which starts by excluding CD115‐expressing monocytes, after which in the CD115−CD11b+Ly6C+ population we discriminated cKit+CXCR4− preneutrophils and cKit−CXCR4− neutrophils, subdividing the latter as immature (Ly6G+CD101−) or mature (Ly6G+CD101+) neutrophils (Fig 5A). Strikingly, these analyses showed that Nlrp3Mac‐A350V/WT BM contained significantly less CD115+ monocytes than control Nlrp3NeoR‐A350V/WT BM (Fig 5B). However, although Nlrp3Mac‐A350V/WT BM displayed higher levels of cKit+CXCR4− preneutrophils, BM of these pups showed significantly less Ly6G+CD101+ mature and Ly6G+CD101− immature neutrophils than BM of Nlrp3NeoR‐A350V/WT pups (Fig 5B). In contrast, BM of Nlrp3Mac‐A350V/WT pups contained a unique population of Ly6G‐CD101+ cells (Fig 5B), which were also significantly more present in the spleen, liver, and kidney of Nlrp3Mac‐A350V/WT pups (Fig 5C–E). In addition, Nlrp3Mac‐A350V/WT pups showed a significantly increased population of Ly6G‐CD101− cells in the BM, spleen, liver, and kidney (Fig 5B–E). The Ly6G+CD101+ mature and Ly6G+CD101− immature neutrophil levels in the spleen, liver, and kidney of Nlrp3Mac‐A350V/WT pups showed similar decreases as observed in the BM (Fig 5C–E), with the exception of the ratio of immature neutrophils in the kidney that was higher in Nlrp3Mac‐A350V/WT pups than in their littermate controls (Fig 5E). Together, these observations indicate that macrophage‐derived inflammation in Nlrp3Mac‐A350V/WT mice disturbed the neutrophil maturation process in Nlrp3Mac‐A350V/WT mice, preventing mature Ly6G+CD101+ neutrophil influxes and leading to an accumulation of Ly6G−CD101+ and Ly6G−CD101− cells in these mice. Although revealing the exact identities of these Ly6G− cell types will require further investigation, our more detailed neutrophil flow cytometric analyses indicate that CAPS in Nlrp3Mac‐A350V/WT mice develops independently of mature neutrophil influxes.

Figure 5. CAPS in Nlrp3Mac‐A350V/WT mice develops independently of mature neutrophils.

Figure 5

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  • A
    Representative flow cytometric gating in bone marrow of 6‐day‐old Nlrp3NeoR‐A350V/WT (top) and Nlrp3Mac‐A350V/WT (bottom) pups, with the indication of the populations quantified in B‐E. Analogous gating strategies were used for other organs.
  • B–E
    Quantifications as % of CD45+ cells in (B) bone marrow, (C) spleen, (D) liver, and (E) kidney.

Data information: Statistics in (B–E) were analyzed by the student t‐test (B: preneutrophils, Ly6G+ CD101− immature neutrophils, C: CD115+ monocytes, Ly6G+ CD101+ mature neutrophils, Ly6G+ CD101− immature neutrophils, Ly6G− CD101− cells; D: CD115+ monocytes, Ly6G+ CD101+ mature neutrophils, Ly6G+ CD101− immature neutrophils, Ly6G− CD101− cells; E: preneutrophils, Ly6G+ CD101+ mature neutrophils, Ly6G+ CD101− immature neutrophils) or Mann–Whitney test (B: CD115+ monocytes, Ly6G+ CD101+ mature neutrophils, Ly6G− CD101− cells, Ly6G− CD101+ cells; C: preneutrophils, Ly6G− CD101+ cells; D: preneutrophils, Ly6G− CD101+ cells; E:, CD115+ monocytes, Ly6G− CD101− cells, Ly6G− CD101+ cells). Dots represent individual biological replicates. Error bars represent mean ± SD. ns not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Macrophages contribute to CAPS development in adult mice with ubiquitous Nlrp3A350V expression

As our above observations in Nlrp3Mac‐A350V/WT mice established that inflammasome hyperactivation in macrophages was sufficient to cause CAPS in mice, we next aimed to investigate whether inflammasome hyperactivation in macrophages conversely was necessary for provoking CAPS. To be able to address this question, we crossbred Nlrp3NeoR‐A350V/NeoR‐A350V mice with hemizygous transgenic mice expressing a Tamoxifen (Tam)‐inducible CreERT2 recombinase under the ubiquitous Rosa26 promoter (R26CreERT2Tg/WT). This resulted in Nlrp3iR26‐A350V/WT mice enabling Tam‐inducible global Nlrp3A350V expression, which was shown to trigger nonlethal autoinflammation associated with body weight loss, and prominent liver inflammation and fibrosis (McGeough et al, 2012; Vande Walle et al, 2019; Schuster‐Gaul et al, 2020). Next, we used clodronate‐loaded liposomes (Clod‐Lipo) to transiently deplete macrophages (Van Rooijen & Sanders, 1994) in Nlrp3iR26‐A350V/WT mice in order to determine the contribution of macrophages in this Tam‐inducible model of body‐wide Nlrp3A350V‐mediated autoinflammation (Fig 6A). Clod‐Lipo‐ and Tam‐treated Nlrp3iR26‐A350V/WT mice displayed no significant changes in body weight loss when compared to Tam‐treated Nlrp3iR26‐A350V/WT mice that had received control liposomes (Ctrl‐Lipo) (Fig 6B), indicating that the overall clinical pathology was not attenuated by macrophage depletion. However, in contrast to Ctrl‐Lipo‐treated mice, macrophage‐depleted Nlrp3iR26‐A350V/WT mice did not develop splenomegaly upon Tam treatment in comparison with their Vehicle (Veh) treated counterparts (Fig 6C). Since this pointed to organ‐specific beneficial effects of macrophage depletion we proceeded to more closely examine immune cell infiltration (Fig EV2) and cytokine production in various organs of Tam‐treated Nlrp3iR26‐A350V/WT mice. Even though macrophage depletion strategies are not capable of continuously and fully eradicating macrophages due to repopulation dynamics of the macrophage niche (Guilliams et al, 2020), the Clod‐Lipo treatment regime prevented macrophage accumulation in the spleen and liver of Tam‐treated Nlrp3iR26‐A350V/WT mice. Indeed, Tam administration to Ctrl‐Lipo‐treated Nlrp3iR26‐A350V/WT mice caused a significant macrophage influx in the spleen and liver, reflecting ongoing autoinflammation, while identical Tam treatment of Clod‐Lipo‐treated Nlrp3iR26‐A350V/WT mice resulted in significantly less spleen and liver infiltration of macrophages (Fig 6D). In contrast, Clod‐Lipo treatment did not affect macrophage numbers in the kidney of Nlrp3iR26‐A350V/WT mice (Fig 6D). These results showing less macrophages in the spleen and liver but not in the kidney enabled us to directly correlate macrophage prevalence with the production of inflammasome‐generated cytokines in these organs. In line with the observed macrophage abundancies, Clod‐Lipo‐ and Tam‐treated Nlrp3iR26‐A350V/WT mice harbored significantly less IL‐1β in the spleen and liver but not in the kidney when compared to Ctrl‐Lipo‐ and Tam‐treated Nlrp3iR26‐A350V/WT mice (Fig 6E), suggesting that macrophages crucially contribute to IL‐1β production during Nlrp3A350V‐instigated CAPS in mice. In addition, Tam treatment significantly elevated IL‐18 levels in the spleen of Ctrl‐Lipo‐injected but not of macrophage‐depleted Nlrp3iR26‐A350V/WT mice (Fig 6F). In contrast, similar to Nlrp3Mac‐A350V/WT pups, the liver and kidney did not display upregulated IL‐18 levels upon Tam‐induced CAPS in Nlrp3iR26‐A350V/WT mice (Fig 6F). These data suggest that splenic macrophages critically contribute to IL‐18 production, while CAPS in mice does not provoke detectable IL‐18 levels in the liver and kidney. Because macrophage‐depleted Nlrp3iR26‐A350V/WT mice showed residual Tam‐induced IL‐1β production in the spleen and liver (Fig 6E), we next evaluated the influx of other myeloid cells. Interestingly, compared with Ctrl‐Lipo‐injected mice, macrophage‐depleted Nlrp3iR26‐A350V/WT mice did not show decreased splenic CD11b+Ly6C+Ly6G+ neutrophils upon Tam treatment and still showed a significant influx of these cells in the liver and kidney (Fig 6G). Thus, inversely mirroring the lack of Ly6G+ neutrophilic inflammation during macrophage‐driven CAPS in Nlrp3Mac‐A350V/WT pups (Fig 4E–G), macrophage depletion still allowed CD11b+Ly6C+Ly6G+ neutrophil influx during body‐wide CAPS in adult mice (Fig 6G), thereby supporting the notion that neutrophilic inflammation in CAPS develops independently of macrophages. Notably, the absence of IL‐18 production in the liver and kidney of Tam‐treated Nlrp3iR26‐A350V/WT mice despite neutrophilic inflammation is consistent with the inability of neutrophils from CAPS patients to produce IL‐18 (Stackowicz et al, 2021). Finally, in addition to CD11b+Ly6C+Ly6G+ neutrophils, macrophage‐depleted Nlrp3iR26‐A350V/WT mice displayed similar infiltration of CD11b+Ly6C+Ly6G− monocytes in the spleen and a reduced yet significant influx of these cells in the liver (Fig EV3). Although we cannot exclude contributions of remaining macrophages, our observations in macrophage‐depleted Nlrp3iR26‐A350V/WT mice suggest that residual Tam‐induced IL‐1β production in these mice derives from CD11b+Ly6C+Ly6G+ neutrophils and CD11b+Ly6C+Ly6G− monocytes.

Figure 6. Macrophages contribute to NLRP3A350V‐induced production of inflammasome‐generated cytokines in adult mice.

Figure 6

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  • A
    Experimental set‐up for administrating control liposomes (Ctrl‐Lipo) or macrophage‐depleting clodronate liposomes (Clod‐Lipo) and vehicle control (Veh) or Tamoxifen (Tam) for inducing whole‐body NLRP3A350V expression in Nlrp3iR26‐A350V/WT mice.
  • B
    Body weight of Nlrp3iR26‐A350V/WT cohorts with indicated treatments (Ctrl‐Lipo + Veh n = 5; Ctrl‐Lipo + Tam n = 6; Clod‐Lipo + Veh n = 4; Clod‐Lipo + Tam n = 6).
  • C
    Relative spleen weights of Nlrp3iR26‐A350V/WT cohorts with indicated treatments at day 6.
  • D–G
    (D) Flow cytometric quantification of CD11b+F4/80+CD64+ macrophages, (E) IL‐1β and (F) IL‐18 protein levels, and (G) flow cytometric quantification of CD11b+Ly6C+Ly6G+ neutrophils in spleen, liver, and kidney from Nlrp3iR26‐A350V/WT cohorts with indicated treatments at day 6.

Data information: Statistics in (B–G) were analyzed by the student t‐test (C, D, E: spleen and kidney; F: spleen and liver; G: spleen and liver) or Mann–Whitney test (E: liver, F: kidney, G: kidney). Dots represent individual biological replicates. Error bars represent mean ± SD. ns—not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure EV2. Flow cytometric analysis of Nlrp3iR26‐A350V/WT mice .

Figure EV2

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Representative flow cytometric gating of Nlrp3iR26‐A350V/WT mice. Doublets and dead cells were excluded before identifying the immune cells (CD45+ cells). Macrophages were identified as CD11b+F4/80+CD64+ cells, monocytes were identified as CD11b+Ly6C+Ly6G− cells, and neutrophils were identified as CD11b+Ly6C+Ly6G+ cells. A—area; FSC—forward scatter; H—height; SSC—side scatter; W—width.

Figure EV3. Monocyte infiltration in organs of Tamoxifen‐treated control and macrophage‐depleted Nlrp3iR26‐A350V/WT mice.

Figure EV3

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Quantification of monocytes (CD11b+Ly6C+Ly6G−) from the different Nlrp3iR26‐A350V/WT cohorts (Ctrl‐Lipo + Veh n = 5; Ctrl‐Lipo + Tam n = 6; Clod‐Lipo + Veh n = 4; Clod‐Lipo + Tam n = 6, experimental layout in Fig 4A) in the spleen (left), liver (middle), and kidney (right).

Data information: Statistics were analyzed by the student t‐test. Dots represent individual biological replicates. Error bars represent mean ± SD. ns—not significant; *P < 0.05; **P < 0.01.

Discussion

In this study, we used two complementary mouse model approaches to investigate whether macrophages were sufficient and necessary as cellular drivers of Nlrp3A350V‐induced CAPS in mice. Collectively, our observations in the Nlrp3Mac‐A350V/WT and the Tam‐inducible Nlrp3iR26‐A350V/WT mouse models provide two lines of evidence establishing macrophages as important cellular drivers of CAPS. Lethal autoinflammation in Nlrp3Mac‐A350V/WT mice showed that inflammasome activation in macrophages is sufficient for provoking CAPS, while macrophage depletion in Nlrp3iR26‐A350V/WT mice showed that macrophages are necessary for eliciting maximal splenic and hepatic output of the inflammasome‐generated cytokines IL‐1β and IL‐18 during CAPS.

Our observations in mice are consistent with CAPS patient observations, as monocytes from CAPS patients that were differentiated into macrophages with GMCSF showed higher IL‐1β production upon LPS/nigericin‐induced NLRP3 activation than macrophages from healthy donors (Camilli et al, 2020). In line with these patient cell observations, the development of severe CAPS in mice with LysMCre‐driven Nlrp3 mutant expression suggested a prominent role for macrophages in CAPS development (Brydges et al, 2009). However, since LysMCre does not target the entire macrophage lineage and on top targets neutrophils (Abram et al, 2014), the recent finding that Mrp8Cre‐driven Nlrp3A350V expression in neutrophils was sufficient for promoting fulminant CAPS (Stackowicz et al, 2021) questioned the specific role for macrophages in this pathology. Moreover, this study also showed that CD11cCre‐driven Nlrp3A350V expression was harmless for mice (Stackowicz et al, 2021). Because CD11cCre targets dendritic cells (DCs) and certain macrophage populations (Abram et al, 2014), this observation suggested that restricting Nlrp3A350V expression to DCs and to a subset of macrophages was not sufficient to induce CAPS (Stackowicz et al, 2021). Here, we directed Nlrp3A350V expression using a Cre‐encoding cDNA inserted into the 3′ UTR of the endogenous Fcgr1 gene (Scott et al, 2018). The latter encodes the CD64 marker that is expressed in all macrophage subsets (Gautier et al, 2012). Accordingly, reporter mice showed that more than 90% of microglia, splenic macrophages, and colonic macrophages were targeted by Fcgr1Cre, while only a negligible portion of the splenic neutrophil population was affected (Scott et al, 2018). Thus, severe CAPS development in Nlrp3Mac‐A350V/WT mice unequivocally identifies macrophages as a causative cell type in triggering Nlrp3A350V‐mediated CAPS in mice. Yet, although this conclusion was supported by our observation that depleting macrophages in mice ubiquitously expressing Nlrp3A350V diminished splenic and hepatic IL‐1β and IL‐18 production, other potential CAPS‐triggering cell types should not be neglected. In this respect, consistent with the findings of Stackowicz et al (2021), we observed substantial neutrophilic infiltration in macrophage‐depleted Nlrp3A350V‐expressing mice. Together with our observation that macrophage‐selective Nlrp3A350V expression in Nlrp3Mac‐A350V/WT mice did not elicit mature neutrophil influxes, while Mrp8Cre‐driven Nlrp3A350V expression in neutrophils sufficed to provoke CAPS, these observations collectively support a cooperative cellular model in which macrophages and neutrophils can independently drive Nlrp3A350V‐mediated CAPS in mice.

Interestingly, we observed that CAPS in Nlrp3Mac‐A350V/WT mice was not associated with influxes of mature Ly6G+CD101+ neutrophils or immature Ly6G+CD101− neutrophils but was characterized by accumulating numbers of Ly6G−CD101− cells and the introduction of Ly6G−CD101+ cells. Although further investigations are required to reveal the identity of the latter Ly6G−CD101+ cells, the reduced presence of Ly6G+ neutrophils in Nlrp3Mac‐A350V/WT mice suggests that perhaps in severe inflammatory conditions certain neutrophils undergo an alternative differentiation trajectory in which CD101 is upregulated before Ly6G. Likewise, identifying the accumulating Ly6G−CD101− cells in Nlrp3Mac‐A350V/WT pups will require future investigation. Several previous studies on healthy adult mice excluded CD115‐expressing monocytes as we did in our analyses and then merged Ly6G+CD101− and Ly6G+CD101− populations as immature neutrophils (Evrard et al, 2018; Kwok et al, 2020; Khoyratty et al, 2021). Thus, the accumulating Ly6G−CD101− cells in Nlrp3Mac‐A350V/WT mice likely include a fraction of immature neutrophils. In addition, we cannot exclude that this fraction of Ly6G−CD101− immature neutrophils takes part also in our CD11b+Ly6C+Ly6G− monocyte analyses. However, although we excluded CD115+ monocytes, CD115lo monocytes are present in steady‐state mice (Rojo et al, 2019) and it is known that severe inflammation leads to emergency monopoiesis generating several specialized monocyte subsets that might escape CD115‐based exclusion (Guilliams et al, 2018). For instance, LPS‐induced inflammation promotes the development of neutrophil‐like monocytes that derive directly from CD115lo granulocyte‐monocyte progenitors in the BM (Yanez et al, 2017). Therefore, we hypothesize that the accumulating Ly6G−CD101− cells represent monocyte subsets induced by severe inflammation in Nlrp3Mac‐A350V/WT pups, and some immature neutrophils that are less maturated than Ly6G+CD101− immature neutrophils. This hypothesis aligns with a recent single‐cell transcriptomic profiling study showing that ubiquitous Nlrp3A350V expression promoted BM granulopoiesis leading to increased monocyte and neutrophil numbers, in which the latter was reflected by increased numbers of Ly6Ghi mature and Ly6Gint immature neutrophils in the liver of these Nlrp3A350V‐expressing mice (Calcagno et al, 2022).

While future studies will thus be needed to fully clarify the exact contributions of monocytes and neutrophils in CAPS development in Nlrp3Mac‐A350V/WT mice, a reporter gene analysis in Fcgr1CreTg/WT mice showed that up to 20% of splenic monocytes exhibited Fcgr1Cre activity (Scott et al, 2018). Therefore, a partial contribution of monocyte‐intrinsic Nlrp3A350V expression to the autoinflammatory pathology in Nlrp3Mac‐A350V/WT mice is possible. In contrast, the lack of Fcgr1Cre activity in Ly6G+ neutrophils (Scott et al, 2018) predicts that neither immature nor mature Ly6G+ neutrophils in Nlrp3Mac‐A350V/WT mice contribute to CAPS by inflammasome activation. Moreover, IL‐1β expression in hepatic neutrophils of ubiquitous Nlrp3A350V‐expressing mice was shown to be restricted to mature neutrophils (Calcagno et al, 2022). Therefore, the fact that Mrp8Cre‐driven expression of Nlrp3A350V in neutrophils was sufficient to cause severe IL‐1β‐driven neutrophilic inflammation (Stackowicz et al, 2021) suggests that the neutrophil maturation process in CAPS depends on neutrophil‐intrinsic inflammasome activation. Together with our observations in Nlrp3Mac‐A350V/WT mice, this supports a cooperative model in which both macrophages and neutrophils contribute to CAPS development in mice. Further supporting this model at the level of cytokine involvement, we showed that CAPS development in Nlrp3Mac‐A350V/WT mice was promoted by both IL‐1β and IL‐18, while similar experiments in Nlrp3Neu‐A350V/WT mice only showed a role for IL‐1β (Stackowicz et al, 2021). Indeed, Anakinra treatment improved the growth of Nlrp3Mac‐A350V/WT pups, while anti‐IL‐18 antibodies prolonged their survival. Although Anakinra blocks the binding of both IL‐1α and IL‐1β to the IL‐1R, the fact that CAPS patients can be treated with anti‐IL‐1β monoclonal antibodies (Lachmann et al, 2009) suggests that the weight gain improvement provided by Anakinra in our model is mediated by blocking IL‐1β activities. The additional role for IL‐18 is in line with a genetic study in mice with LysMCre‐driven Nlrp3A350V expression, in which IL‐18R deletion provided a better protection from perinatal lethality than IL‐1R deletion did (Brydges et al, 2013). Together, these observations suggest that the cooperative actions of neutrophils and macrophages for driving CAPS in mice may relate to combining IL‐1β and IL‐18 cytokine actions.

Taken together, our observations combine with previous reports to identify macrophages next to neutrophils, but not dendritic cells or mast cells (Stackowicz et al, 2021), as cellular drivers of Nlrp3A350V‐mediated CAPS in mice. Nevertheless, it is important to be careful when generalizing our macrophage conclusions in Nlrp3A350V‐expressing mice to all Nlrp3‐causative CAPS mutations. For instance, mast cells were shown to differentially contribute to inflammation in Nlrp3A350V‐ versus Nlrp3R258W‐driven CAPS models (Nakamura et al, 2012; Stackowicz et al, 2021). In addition, CAPS in Nlrp3A350V‐expressing mice was fully prevented by disabling both IL‐1 and IL‐18 cytokine actions, while mice expressing the more pathogenic Nlrp3L351P mutant still developed CAPS in those conditions (Brydges et al, 2013). As these studies illustrate that the cellular and molecular mechanisms of CAPS pathogenesis may depend on the nature of the Nlrp3 gain‐of‐function mutation, also the relative contribution of macrophages to CAPS pathology might vary with the exact genetic cause of disease. In this respect, it is interesting that CD11cCre‐driven expression of Nlrp3A350V did not induce CAPS, while similarly inducing Nlrp3L351P expression did provoke CAPS (Stackowicz et al, 2021). Given the partial targeting of macrophages by CD11cCre (Abram et al, 2014), our identification of macrophages as potent CAPS‐triggering cells suggests that the potential of CD11cCre to differentially drive Nlrp3A350V‐ versus Nlrp3L351P‐mediated CAPS might derive from differential inflammasome activating capacities of these Nlrp3 mutants in limited numbers of CD11cCre‐targeted macrophages. This hypothesis based on more potent Nlrp3 inflammasome responses in macrophages than in DCs is reminiscent of in vitro observations using bone marrow‐derived DCs, in which Nlrp3‐mediated inflammasome responses derive from macrophages rather than DCs (Erlich et al, 2019). Future mouse model studies will be needed to more carefully dissect the individual contributions of various innate immune cell types in CAPS driven by distinct Nlrp3 mutants. In conclusion, our study identifies macrophages next to neutrophils as a CAPS‐triggering cell type in mice, which in the long‐term may direct the development of therapeutic approaches to specifically target both macrophages and neutrophils in CAPS patients.

Materials and Methods

Mice

Nlrp3NeoR‐A350V/NeoR‐A350V mice (B6.129‐Nlrp3tm1Hhf/J) (Brydges et al, 2009) and R26CreERT2 transgenic mice (B6.129‐Gt(ROSA)26Sortm1(cre/ERT2)Tyj /J) (Ventura et al, 2007) mice were originally obtained from the Jackson Laboratories. Fcgr1Cre transgenic mice (B6‐Fcgr1tm3Ciphe) (Scott et al, 2018) were provided by Dr. Bernard Malissen (Centre d'Immunologie de Marseille‐Luminy, France). All mice used in this study were generated on C57BL/6 background or backcrossed at least ten generations to C57BL/6J background. All mice were bred in‐house in individually ventilated cages (IVC) in the Specific Pathogen Free facility at Ghent University. In all experiments, up to five mice were housed per cage in a 12‐h light‐12‐h dark cycle and were fed autoclaved standard rodent feed (Ssniff, Soest, Germany) at libitum with free access to drinking water. Mice were assigned to experimental groups according to genotype and treatment. All animal experiments were performed according to institutionally approved protocols according to national (Belgian Laws 14/08/1986 and 22/12/2003, Belgian Royal Decree 06/04/2010) and European (EU Directives 2010/63/EU, 86/609/EEG) animal regulations. Animal protocols were reviewed and approved by the Ethical Committee Animal Experimentation—Ghent University—Faculty of Medicine and Health Sciences (permit number LA1400536) with approval IDs 2020‐031 and 2022‐001. All necessary efforts were made to minimize the suffering of the animals.

Treatment with Anakinra or IL‐18 neutralizing antibodies

Nlrp3NeoR‐A350V/WT and Nlrp3Mac‐A350V/WT pups were injected subcutaneously every other day beginning on day 3 of life for a total of five doses either with 300 mg/kg Anakinra (Sobi, Kineret) or PBS vehicle, or with 10 mg/kg rat IgG2aκ anti‐mouse IL‐18 (clone YIGIF74‐1G7, Bio X Cell) or 10 mg/kg rat IgG2aκ isotype antibody controls (clone 2A3, Bio X Cell) in PBS. All treatments were injected in a volume of 20 µl/g body weight.

Clodronate‐mediated macrophage depletion and tamoxifen‐induced Nlrp3A350V induction

Clodronate liposomes were obtained from Liposoma BV (Amsterdam, The Netherlands) and were administrated in two intraperitoneally injected doses of 10 μl suspension per g of body weight 5 days apart, according to the manufacturer’s instruction. Induction of CreERT2 activity in Nlrp3iR26‐A350V/WT mice in order to induce ubiquitous Nlrp3A350V expression was achieved by administration of tamoxifen on two consecutive days (T5648, Sigma‐Aldrich, dissolved in 1:9 ethanol/corn oil (C‐8267, Sigma‐Aldrich) at 50 mg/ml) through oral gavage at a dose of 5 mg tamoxifen per mouse per day. Vehicle control animals received oral gavages of 100 µl of 1:9 ethanol/corn oil on two consecutive days.

Cytokine analyses

Serum samples were prepared from fresh blood, which was kept at room temperature for 30 min to clot followed by cold centrifugation for 10 min at 1,400 g. Tissue samples were weighed and were homogenized in 500 μl PBS, after which lysis was completed by the addition of lysis buffer (20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P‐40) and incubation for 10 min on ice. Full speed centrifugation for 30 min cleared the homogenate and supernatant was used for further analysis. TNF, IL‐6, and IL‐18 serum, and tissue levels, and IL‐1β tissue levels were determined by a magnetic bead‐based multiplex assay using Luminex technology (Bio‐Rad) according to the manufacturer’s instructions. IL‐1β serum levels were determined by the Mouse IL‐1 beta/IL‐1F2 Quantikine HS ELISA Kit (R&D systems) according to the manufacturer’s protocol. Cytokines from serum were expressed as a concentration per ml of serum. Cytokines from tissue homogenates were normalized to the weight of tissue.

Western Blotting

Tissue homogenates were incubated with cell lysis buffer (20 mM Tris–HCl (pH 7.4), 200 mM NaCl, 1% Nonidet P‐40) and denatured in Laemmli buffer by boiling for 10 min. Proteins were separated by SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Blocking, antibody incubations, and washing of the membrane were done in PBS supplemented with 0.05% Tween 20 (v/v) and 3% (w/v) nonfat dry milk. Immunoblots were incubated overnight with primary antibody against Caspase‐1 (1:1,000; Santa Cruz Biotechnology; catalog number sc‐514), IL‐1β 1:1,000; R&D System, catalog number AF‐401‐NA) or GSDMD (1:1,000; BioVision, catalog number ab 209845). Appropriate horseradish peroxidase‐conjugated (111‐035‐144, Jackson ImmunoResearch Laboratories) secondary antibodies were used to detect primary antibodies, while β‐actin was detected with the directly labeled primary antibody β‐actin‐HRP (1:10,000; Santa Cruz). Proteins of interest were visualized by the enhanced SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Flow cytometry analyses

Single‐cell suspension of the spleen, liver, and kidney were made by mechanical dissociation. The cells were passaged through 70 µm cell strainers. ACK buffer (Lonza) was used to lyse the red blood cells in all the organs. Liver and kidney suspensions went also through Percoll (37.5%, GEAkta) gradient centrifugation. For flow cytometric staining, the following antibodies and dyes were used: Fixable viability dye, CD101 (Moushi101), CD117 (2B8, eBioscience), CD45 (30‐F11), Ly6C (HK1.4), CD11b (M1/70), F4/80 (BM8), CD115 (AFS98), CD64 (X54‐5/7.1, BioLegend), CD184 (2B11/CXCR4), and Ly6G (1A8, BD Pharmagen). The samples were acquired on a BD LSR Fortessa cytometer (BD). Data were analyzed using FlowJo v.10 (BD Bioscience).

Histological analyses

Tissues were fixed in 4% paraformaldehyde, were embedded in paraffin, and cut in 4 µm sections. For histopathological analysis, haematoxylin and eosin staining was performed according to standard protocol. For immunohistochemical staining, paraffin sections were rehydrated and heat‐induced antigen retrieval was performed in citrate buffer at pH 6.0. A primary antibody against F4/80 (Serotec) was used for detecting macrophages. Biotinylated secondary antibodies were purchased from Perkin Elmer and Dako. Stainings were visualized with ABC Kit Vectastain Elite (Vector Laboratories) or Streptavidin‐HRP (Millipore) and DAB substrate (DAKO and Vector Laboratories). Incubation times with DAB substrate were equal for all samples. Nuclei were counterstained with haematoxylin.

Statistics

GraphPad Prism 9.2 software was used for data analysis. Data are represented as mean with SD. For mouse survival curves, statistical significance was determined by the log‐rank Mantel–Cox test. Other data were analyzed by applying either unpaired student t‐tests or unpaired Mann–Whitney tests in case of no normal distribution of the values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.

Author contributions

Ulrika Cecilia Frising: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Writing—original draft; Writing—review & editing. Silvia Ribo: Formal analysis; Investigation; Methodology; Writing—original draft. M Giulia Doglio: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Writing—original draft; Writing—review & editing. Bernard Malissen: Resources. Geert van Loo: Project administration. Andy Wullaert: Conceptualization; Formal analysis; Funding acquisition; Writing—original draft; Project administration; Writing—review & editing.

In addition to the CRediT author contributions listed above, the contributions in detail are:

UCF, SRG, and MGD performed experiments; UCF, SRG, MGD, and AW designed the experiments and analyzed the data; BM contributed essential materials; GvL and AW supervised the project. UCF, SRG, MGD, and AW wrote the manuscript with input from all other authors.

Disclosure and competing interests statement

The authors declare that they have no conflict of interest.

Supporting information

Expanded View Figures PDF

Click here for additional data file. (1.2MB, pdf)

Source Data for Figure 3

Click here for additional data file. (286.9KB, pdf)

Acknowledgements

We are grateful for the excellent technical support from Sze Men Choi, Amelie Fossoul, and Maarten Verdonckt. All flow cytometry and microscopy were performed using infrastructure from the VIB Flow Core and VIB Bioimaging Core headed by Gert Van Isterdael and Saskia Lippens, respectively. We thank all core personnel for extensive training and assistance. Research in the A.W. lab is supported by the Odysseus grant G.0C49.13N and the research grants 3G.0447.18 and 3G.0448.18 from the Fund for Scientific Research‐Flanders and the BOF UGent grant BOF.24Y.2019.0032.01. U.C.F is a Postdoctoral Research Fellow supported by the Fund for Scientific Research‐Flanders. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

EMBO reports (2022) 23: e54339.

Data availability

No primary datasets have been generated and deposited.

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Associated Data

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    Supplementary Materials

    Expanded View Figures PDF

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    Source Data for Figure 3

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    Data Availability Statement

    No primary datasets have been generated and deposited.

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