Activation of leukotriene B₄ receptor 1 is a prerequisite for complement receptor 3-mediated antifungal responses of neutrophils

Abstract

Invasive fungal infections are life-threatening, and neutrophils are vital cells of the innate immune system that defend against them. The role of LTA4H-LTB₄-BLT1 axis in regulation of neutrophil responses to fungal infection remains poorly understood. Here, we demonstrated that the LTA4H-LTB₄-BLT1 axis protects the host against Candida albicans and Aspergillus fumigatus, but not Cryptococcus neoformans infection, by regulating the antifungal activity of neutrophils. Our results show that deleting Lta4h or Blt1 substantially impairs the fungal-specific phagocytic capacity of neutrophils. Moreover, defective activation of the spleen tyrosine kinase (Syk) and extracellular signal-related kinase (ERK1/2) pathways in neutrophils accompanies this impairment. Mechanistically, BLT1 regulates CR3-mediated, β-1,3-glucan-induced neutrophil phagocytosis, while a physical interaction with CR3 with slight influence on its dynamics is observed. Our findings thus demonstrate that the LTA4H-LTB₄-BLT1 axis is essential for the phagocytic function of neutrophils in host antifungal immune response against Candida albicans and Aspergillus fumigatus.

Introduction

The opportunistic fungal infection poses a life-threatening risk, particularly to immunocompromised individuals such as those with AIDS, patients receiving myeloablative chemotherapy and those undergoing organ or hematopoietic stem cell transplantation (HSCT) [1–3]. The global coronavirus pandemic has increased the incidence of fungal disease in recent years [4, 5]. In addition, advances in diagnostic techniques and a greater understanding of invasive fungi have led to reports of fungal infections in immunocompetent populations [6–8]. Candida, Aspergillus and Cryptococcus are the main pathogenic fungi that cause candidiasis, aspergillosis, and cryptococcosis, respectively, accounting for more than 90% of invasive fungal infections [9]. These fungi have been identified as major threats to public health and are included in fungal priority pathogen list by the World Health Organization (WHO) as of October 2022 (https://www.who.int/publications/i/item/9789240060241) [10]. Despite the availability of antifungal drugs for clinical treatment, they have not substantially reduced mortality rates and have instead led to the emergence of drug-resistant and/or multidrug-resistant fungi, presenting new threats to public health [3].

The host immune system is essential for defending against fungal infections. Although different invasive fungi have varying environmental sources and pathogenesis, multiple types of immune cells typically sense and effectively eliminate them [11, 12]. Innate immune cells, such as macrophages/monocytes, neutrophils, and dendritic cells (DCs), are considered the most effective cell types as the first line of host defense. They directly recognize and clear invading fungi [13]. Phagocytes recognize fungi through the interplay between soluble or membrane-bound pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) on fungal cell wall, such as mannan, β-1,3-glucan, and chitin [14, 15]. Once fungal cells are phagocytized, the engulfed microorganisms are processed through fusion with lysosomes into phagolysosomes. Intracellular fungicidal responses, which depend on hydrolases, antimicrobial peptides, and reactive oxygen intermediates (ROI)/reactive nitrogen intermediates (RNI), are then initiated, further regulating downstream signaling pathways and gene transcription [16, 17]. Neutrophils can also kill hyphal Candida extracellularly by forming neutrophil extracellular traps (NETs) [18].

Leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme that comprises zinc-dependent epoxide hydrolase activity and aminopeptidase activity. The enzyme catalyzes the conversion of leukotriene A₄ (LTA₄), which is generated from phospholipids through sequential action of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and 5-lipoxygenase-activating protein (FLAP), into pro-inflammatory leukotriene B₄ (LTB₄) through its epoxide hydrolase activity [19]. LTB₄ has been shown to be a potent chemotactic lipid mediator that mediates leukocyte adhesion and aggregation to the endothelium, especially for neutrophils, via its high-affinity receptor BLT1 [20]. The enzyme’s aminopeptidase activity is involved in the biodegradation of the chemotactic tripeptide Pro-Gly-Pro (PGP) and is activated by albumin and monovalent anions such as chloride [21, 22]. Because LTB₄ possesses chemotactic mediator properties, the LTB₄-BLT1 axis has been shown to be involved in the pathogenesis of chronic inflammatory diseases [20].

In a study by Tobin et al., Lta4h mutations were found to increase susceptibility to Mycobacterium tuberculosis, highlighting the importance of balanced eicosanoid production in vertebrate resistance to mycobacterial infection [23]. In this present study, we found that LTA4H plays a protective role against C. albicans and A. fumigatus infections in a BLT1-dependent manner. Mice lacking Lta4h or Blt1 showed higher susceptibility to those infection, accompanied by higher inflammation and fungal burden. The phagocytosis of invading fungi by neutrophils was significantly attenuated in mice lacking Lta4h or Blt1. The complement receptor 3 (CR3), one of the main pattern recognition receptors on neutrophils that mediated the phagocytosis of β-1,3-glucan-exposed fungi, was found to be regulated by the LTA4H-LTB₄-BLT1 axis possibly through physical interaction with BLT1.

Results

Lta4h deficiency leads to increased susceptibility to fungal infection

Through their analysis of clinical data from patients with non-HIV cryptococcal meningitis, Zhou et al. identified multiple single nucleotide polymorphisms (SNPs) in Lta4h gene that were significantly associated with abnormal expression of inflammatory cytokines in the cerebrospinal fluid of patients [24]. Based on this finding, we generated Lta4h knockout mice using the CRISPR-Cas9 technology to investigate the immunomodulatory function of LTA4H in fungal infections (Fig. S1a). As expected, LTA4H deficiency was confirmed at both transcriptional and protein levels (Fig. S1b, c).

In our initial investigation, we sought to determine if the deletion of Lta4h impacted host’s susceptibility to C. neoformans infection (Fig. S1d). However, the absence of Lta4h did not impact the survival rate of mice at the infection dose of 10⁶ and 10⁴ CFU (Fig. S1e, g). Moreover, there were no significant differences in fungal burden in brain and lung tissues between control and Lta4h^−/− groups (Fig. S1f, h). Additionally, despite the significant elevation of cytokine (TNFα and IFNγ) expression levels in response to C. neoformans infection, Lta4h deletion did not alter their expression levels (Fig. S1i). Therefore, no significant involvement of LTA4H in C. neoformans infection had been observed, at least at the doses we examined.

Next, we investigated the impact of Lta4h deficiency on host susceptibility to Candida albicans. Following systemic challenge, Lta4h^−/− mice rapidly succumbed to C. albicans infection, while Lta4h^+/− mice had a 50% survival rate at 15 days post-infection (Fig. 1A). The fungal load in the kidneys of infected Lta4h^−/− mice was significantly higher than in the control group (Fig. 1B), whereas such difference was not observed in spleens (Fig. S2a), showing an organ-specific difference. PAS staining results were consistent with these findings (Fig. 1C), while HE staining suggested severer tissue damage than the control mice (Fig. 1C). The proportion of immune cell populations was comparable between two genotypes at naïve state, excluded the possible influence of LTA4H on immune cell development (Fig. S2d). However, in response to C. albicans infection, the percentage and cell number of kidney-infiltrating neutrophils in Lta4h^−/− mice were significantly higher than in the control group after 72 h of infection (Figs. 1D and S2d), while the mean fluorescence intensity of CD11b in Lta4h^−/− mice displayed a slight decrease (Fig. S2e). Immunohistochemistry against Ly6G confirmed these results (Fig. 1C). Additionally, Lta4h deficient mice showed significantly elevated level of pro-inflammatory cytokines in both kidney homogenate and serum samples (Figs. 1E, F and S2b, c). Many chemokines, including Ccl2, Ccl3, Ccl20, Cxcl2, and Cxcl3, were upregulated in the kidneys of Lta4h^−/− mice after 48 and 72 h of infection (Fig. S2c). Overall, the loss of Lta4h gene resulted in higher levels of fungal load and inflammation, as well as more neutrophil infiltration in the kidneys, in accordance with increased mortality in mice.

Fig. 1.

LTA4H regulates the host immune response against C. albicans. A Survival of Lta4h^−/− (n = 11) and Lta4h^+/− (n = 10) mice following systemic infection with 2 × 10⁵ CFU of C. albicans (strain SC5314). B Fungal load in kidneys of infected Lta4h^−/− (n = 5) and Lta4h^+/− (n = 6) mice at 72 h post-infection. C Kidney histopathological examination of Lta4h^−/− mice and Lta4h^+/− mice (hematoxylin and eosin staining, periodic acid-Schiff staining and Ly6G staining, scale bars, 1000 μm) at 72 h post-infection with 2 × 10⁵ CFU of C. albicans. Representative images of at least three replicates are shown. White arrows highlight C. albicans hyphae in renal parenchyma. D Representative flow cytometry plots, statistical results (both percentage and total number) of kidney-infiltrating neutrophils (Ly6G⁺CD11b⁺) in Lta4h^−/− (n = 7) and Lta4h^+/− (n = 7) at 72 h post-infection with 2 × 10⁵ CFU of C. albicans. E Protein levels of IL6 and IL1β in kidney homogenates of Lta4h^−/− (n = 6) and Lta4h^+/− (n = 7) at 72 h post-infection. F Gene expression analysis of Il6 and Il1b in kidney tissue at 24–72 h post-infection. Each experiment was performed at least 2–3 times. Data are expressed as mean ± SEM. In (A), statistical analysis by log-rank (Mantel–Cox) test. In (B, D–F), Student’s t test or multiple t-test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figs. S1 and S2

Lta4h deficiency affects neutrophil phagocytosis

To determine the role of Lta4h in immune and non-immune cells during infection, we conducted bone marrow transfer experiment followed by systemic C. albicans infection. As depicted in Fig. S3a, there was no difference in the survival rate of mice after the transfer of wild-type bone marrow into Lta4h^+/− and Lta4h^−/− recipients. However, when Lta4h^+/− and Lta4h^−/− bone marrow was transferred into wild-type recipients, the chimeric mice displayed a phenotype similar to that of Lta4h^−/− mice (Fig. S3b), suggesting that Lta4h in hematopoietic cells might play a crucial role in C. albicans infection. However, depletion of T and B cells did not affect the phenotype (Fig. S3c), indicating that the function of LTA4H might be more prominent in myeloid leukocytes than in lymphoid leukocytes in response to C. albicans infection.

After isolating myeloid leukocytes with high expression level of Lta4h (including macrophage/monocyte, dendritic cell, and neutrophil) from Lta4h^−/− mice, their antifungal responses were evaluated following C. albicans stimulation in vitro. Although Lta4h deficiency slightly increased TNFα and IL6 at the transcriptional level (Fig. S4a, b), their protein levels in BMDM and BMDC were comparable to controls except for IL6 in Lta4h^−/− BMDM (Fig. 2A, D). For IL1β, the protein levels were too low to be detected in both cells after stimulation, and their transcription did not differ between two groups (Fig. S4a, b). Except for Ccl2 (BMDC) and Cxcl3 (BMDM), the expression of other chemokines and Nos2 was not significantly altered by Lta4h deletion (Fig. S4a, b). NO release and phagocytosis in BMDM were found to be unaffected (Fig. 2B, C). However, the deletion of Lta4h significantly dampened antifungal responses of neutrophils to C. albicans. Images of swarming behavior that coordinate neutrophil accumulation and form tight cell aggregates to isolate the site of infection showed that Lta4h^+/− neutrophils rapidly aggregated on hyphal-form C. albicans whereas Lta4h^−/− neutrophils failed to form the corresponding clusters, showing an impaired swarming potential (Fig. S4c). Besides, loss of Lta4h significantly decreased ROS production, phagocytosis and killing of neutrophils, with particularly pronounced effect on phagocytosis (Figs. 2E, F and S4d). The results of in vivo phagocytosis experiment further demonstrated that the deletion of Lta4h substantially impaired the phagocytosis of neutrophils in vivo without affecting macrophages (Fig. 2G, H). Collectively, LTA4H is primarily involved in the antifungal function of neutrophils, but not of macrophages and DCs. Since Lta4h deficiency exhibits the most pronounced effect on neutrophil phagocytosis and the underlying mechanism remains poorly understood, we therefore explore the role of Lta4h in neutrophil phagocytosis in the following experiments.

Fig. 2.

LTA4H is essential for anti-Candida function of neutrophil. A Protein levels of TNFα and IL6 in supernatant of Lta4h^+/− and Lta4h^−/− BMDMs after stimulation for 24 h with heat-inactivated C. albicans (MOI = 10). B NO production in supernatant of Lta4h^+/− and Lta4h^−/− BMDMs after stimulation for 24 h or 48 h at MOI5 or MOI10. C Phagocytosis of live GFP-expressing C. albicans (MOI = 1) by Lta4h^+/− and Lta4h^−/− BMDMs after 15, 30 or 60 min. D Protein levels of TNFα and IL6 levels in supernatant of Lta4h^+/− and Lta4h^−/− BMDCs after stimulation for 24 h with heat-inactivated C. albicans at MOI10. E Phagocytosis and (F) killing potential of Lta4h^+/− or Lta4h^−/− neutrophils toward live GFP-expressing C. albicans (MOI = 5) and C. albicans (MOI = 1) at indicated time points. In vivo phagocytosis of (G) neutrophils (Ly6G⁺CD11b⁺) and (H) macrophages (F4/80⁺CD11b^+/−) within kidney tissue of Lta4h^+/− or Lta4h^−/− mice at 2.5 h post-infection with 1 × 10⁷ CFU of Ruby-expressing C. albicans. Each experiment was performed at least 2–3 times. Data are expressed as mean ± SEM. Student’s t test or multiple t-test was performed. ns nonsignificant as p > 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figs. S3 and S4

Intact neutrophil function relies on LTA4H-LTB₄-BLT1 axis

As described in the introduction, LTA4H functions by catalyzing LTA₄ to generate LTB₄, which exerts its biological function by binding to the high-affinity receptor BLT1. To investigate if the regulation of neutrophil phagocytosis by LTA4H is dependent on the BLT1 receptor, exogenous LTB₄ was added to the phagocytosis assay system. The results depicted in Fig. 3A showed that the addition of exogenous LTB₄ completely restored the phagocytosis defect observed in Lta4h-deficient neutrophils, indicating that LTA4H could regulate neutrophil function in a BLT1-dependent manner.

Fig. 3.

The function of LTA4H in neutrophils is dependent on BLT1. A Phagocytosis of GFP-expressing C. albicans by Lta4h^+/− or Lta4h^−/− neutrophils in the presence or absence of exogenous LTB₄ (10 ng/ml) at indicated time points. B Survival of Blt1^−/− (n = 14) and Blt1^+/− (n = 12) mice following systemic infection with 2 × 10⁵ CFU of C. albicans (strain SC5314). C Kidney histopathological examination of Blt1^−/− and Blt1^+/− mice (scale bars, 1000 μm) at 72 h post-infection with 2 × 10⁵ C. albicans. Representative images of at least three replicates are shown. White arrows highlight C. albicans hyphae in renal parenchyma. D Representative flow cytometry plots and bar graphs on kidney-infiltrating neutrophils in Blt1^−/− (n = 7) and Blt1^+/− (n = 7) mice at 72 h post-infection with 2 × 10⁵ CFU C. albicans. E Phagocytosis of GFP-expressing C. albicans by Blt1^+/− or Blt1^−/− neutrophils in the presence or absence of exogenous LTB₄ (10 ng/ml) at indicated time points. F In vivo neutrophil phagocytosis within kidney tissue of Blt1^−/− (n = 6) or Blt1^+/− (n = 4) mice at 2.5 h post-infection with 1 × 10⁷ CFU Ruby-expressing C. albicans. G Experimental scheme to obtain mixed BM chimeric mice and evaluate the in vivo phagocytosis of Lta4h^−/−, Blt1^−/−, and WT neutrophils during Ruby-expressing C. albicans infection. Comparison of in vivo phagocytosis between (H) Lta4h^−/−, (I) Blt1^−/− and WT neutrophils within kidneys of mixed BM chimeric mice at 2.5 h post-infection. In (A, D–F, H, I) each experiment was performed at least three independent times. Data are expressed as mean ± SEM. Student’s t test or multiple t-test was performed. In (B), statistical analysis by log-rank (Mantel–Cox) test. ns nonsignificant as p > 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figs. S5 and S6

To further confirm the results of the above in vitro experiments, we generated Blt1 knockout mice, in which the translation of BLT1 stopped at E55 (Fig. S5a, b). We found that Blt1-deficient mice were more susceptible to C. albicans infection, similar to Lta4h-deficient mice (Fig. 3B). Additionally, Blt1^−/− mice had significantly higher fungal load and neutrophil infiltration in kidneys after infection for 72 h (Fig. 3C, D), while a similar decrease in CD11b MFI was observed (Fig. S6a). Most importantly, the results of in vitro phagocytosis showed that Blt1 deletion also significantly impaired neutrophil phagocytosis, which could not be rescued by the addition of exogenous LTB₄ (Fig. 3E), thus confirming the regulation of LTB₄-BLT1 on neutrophil phagocytosis. The results of in vivo experiment also demonstrated the effect of Blt1 on neutrophil phagocytosis (Fig. 3F).

To further ensure whether the effect of Lta4h or Blt1 on neutrophil phagocytosis was cell-intrinsic, we conducted competitive bone marrow transfer experiments as shown in Fig. 3G. The results showed that bone marrow cells derived from CD45.1⁺ wild-type mice and CD45.2⁺ Lta4h^−/− mice or CD45.2⁺ Blt1^−/− mice were successfully reconstituted in recipients with a ratio of 1:1 (Fig. S6b, c). In chimeric mice that were reconstituted from mixed bone marrow from CD45.2⁺ Lta4h^−/− and CD45.1⁺ wild-type mice, there was no difference in the proportion of Ruby⁺ neutrophils between the two genotypes (Fig. 3H), demonstrating a restored phagocytosis activity of Lta4h^−/− neutrophils. Following infection with Ruby-expressing C. albicans, the proportion of Ruby⁺ neutrophils were still significantly lower in Blt1^−/− neutrophils than in CD45.1⁺ wild-type neutrophils (Fig. 3I). These results further illustrated that BLT1, but not LTA4H, functioned intrinsically in neutrophil phagocytosis.

Lta4h/Blt1-deficient neutrophils display equivalent chemotactic responses in systemic C. albicans infection

As previously described, C. albicans systemic infection for 72 h resulted in a higher recruitment of neutrophils into the kidneys of Lta4h^−/− or Blt1^−/− mice compared to control mice (Figs. 1D and 3D). Based on the role of LTB₄-BLT1 on chemotactic properties of neutrophils, we analyzed neutrophil infiltration during the early phase of infection at 3, 6, 24 and 48 h. LTB₄ secretion analysis confirmed that Lta4h deletion essentially abolished LTB₄ production in response to C. albicans stimulation (Fig. S6d). Flow cytometry results showed that at an infection dose of 2 × 10⁵ CFU, there was no significant increase in the number of kidney-infiltrating neutrophils at 3–6 h post-infection (Fig. S6e). Although neutrophil recruitment was significantly upregulated after 24–48 h post-infection, it was not affected by Lta4h deletion (Fig. S6e). When the infection dose was increased to 1 × 10⁷ CFU, both Lta4h^−/− and Blt1^−/− mice exhibited a similar level of neutrophil chemotaxis as the controls, even though neutrophils were recruited at 2.5 h post-infection (Fig. S6g, h). Accompanied with these results, MFI of CD11b was not affected by loss of Lta4h or Blt1 after C. albicans infection for 2.5–24 h (Fig. S6f–h).

In addition, in competitive bone marrow transfer experiments, we further compared the level of renal infiltration between CD45.2⁺ Lta4h^−/− or CD45.2⁺ Blt1^−/− neutrophils and CD45.1⁺ wild-type neutrophils. The results showed that CD45.2⁺ Lta4h^−/− and CD45.2⁺ Blt1^−/− neutrophils exhibited comparable chemotactic properties to wild-type neutrophils in the same Candida-infected recipient mice (Fig. S6b, c), which was consistent with the in vivo results of Blt1^−/− and Lta4h^−/− mice.

Lta4h/Blt1 deficiency attenuates the activation of Syk and ERK1/2 pathway in neutrophils

On exploring the detailed mechanism of how LTA4H-LTB₄-BLT1 axis regulated neutrophil antifungal responses, we searched for the pivotal downstream joint that BLT1 could build connection with. As a multistate GPCR, BLT1 could engage diverse signaling molecules, thereby modulating activation of essential intracellular cascades such as calcium flux and the mitogen associated protein kinases (MAPKs) signaling pathway [25]. Since the intracellular calcium mobilization plays a crucial role in phagocytes, we compared the calcium flux in C. albicans-stimulated Blt1^+/− and Blt1^−/− neutrophils. The results showed that calcium mobilization in neutrophils induced by C. albicans stimulation was significantly attenuated by loss of BLT1 (Fig. S7a), suggesting the importance of BLT1 in supporting neutrophil activities. To determine whether MAPK pathway is involved in neutrophil phagocytosis, we then assessed the phosphorylation of extracellular signal-related kinase (ERK1/2), p38, c-Jun N-terminal kinase (JNK1/2), as well as Syk (spleen tyrosine kinase) and Src in vitro. We found that loss of Lta4h or Blt1 significantly attenuated the phosphorylation of ERK1/2, partially weakened the phosphorylation of p38, and did not affect the phosphorylation of JNK1/2 (Fig. 4A, B). Interestingly, Lta4h- or Blt1-deficient neutrophils also exhibited a lower level of Syk phosphorylation without significant changes in Src phosphorylation compared to the control group (Fig. 4C, D). Furthermore, we generated a BLT1-overexpressing NIH3T3 cell line and stimulated it with C. albicans in vitro. We found that BLT1 overexpression could not effectively activate ERK1/2 (Fig. S7b), indicating that BLT1 itself did not directly recognize or interact with C. albicans. Taken together with the result that deletion of BLT1 attenuates the phosphorylation of Syk, which acts through interaction between its SRC homology 2 (SH2) domains and receptor-associated immunoreceptor tyrosine-based activation motifs (ITAMs), it is reasonable to assume that BLT1 may regulate phagocytosis by assisting phagocytic receptors on neutrophils.

Fig. 4.

LTA4H-BLT1 regulates neutrophil phagocytosis through Syk signaling. Representative images of Western blot analysis on the phosphorylation of MAPK and Src, Syk signaling in Ltah^−/− (A, C) or Blt1^−/− (B, D) neutrophils stimulated with live C. albicans (MOI = 1) at indicated time points. β-tubulin was used as the internal controls. E Western blot analysis on the phosphorylation of ERK1/2 signaling in WT neutrophils stimulated with live C. albicans (MOI = 1) in the presence of Saracatinib (Src inhibitor), Piceatannol (Syk inhibitor), and PD98059 (ERK1/2 inhibitor), respectively. F Representative flow cytometry plots and bar graphs on phagocytosis of WT neutrophils at indicated time points in the presence of 50 μM Saracatinib (Src inhibitor), 50 μM Piceatannol (Syk inhibitor), or 10 μM PD98059 (ERK1/2 inhibitor), respectively. Each experiment was performed at least 2–3 times. In (A–E), data are representative of three independent experiments (n = 3). Fold change = IntDen_{other band}/IntDen_{first band}. In (F), data are expressed as mean ± SEM. Multiple t-test was performed. ****p < 0.0001. See also Fig. S7

Furthermore, we conducted experiments using specific chemical inhibitors to validate the role of Src and Syk in neutrophil phagocytosis. We found that inhibition of Src and Syk significantly reduced the phagocytosis of C. albicans by neutrophils (Figs. 4E, F and S7c, d), confirming their crucial role in neutrophil phagocytosis. However, inhibition of ERK1/2 did not affect neutrophil phagocytosis, despite previous studies suggesting that the ERK1/2 pathway could regulate neutrophil phagocytosis in some circumstances (Figs. 4F and S7e, f) [26, 27]. Additionally, we conducted a quantitative analysis of gene expression and found that, similar to Lta4h-deficient neutrophils, inhibition of ERK1/2 in wild-type neutrophils significantly down-regulated the expression of chemokines (Fig. S7g, h), indicating that the ERK1/2 pathway might be involved in regulation of chemokine expression.

Effects of Lta4h/Blt1 on neutrophils are fungal-specific, based on β-1,3-glucan recognition

We next investigated whether the regulation of neutrophil phagocytosis by Lta4h or Blt1 is specific for fungal pathogens. When GFP-expressing C. albicans was replaced with APC⁺ beads, Lta4h^−/− neutrophils showed an equivalent level of phagocytosis as controls (Fig. 5A). Unlike fungal stimulation, the deletion of Blt1 did not affect the phosphorylation of ERK1/2 in response to lipopolysaccharide (LPS) stimulation, even though it could effectively increase phosphorylation of ERK1/2 in a dose-dependent manner (Figs. 5B and S8a). Consistent with the dispensable role of BLT1 in LPS stimulation, the phagocytosis of S. aureus or S. pneumoniae by neutrophils was unaffected by Blt1 deletion (Fig. 5C). Moreover, the survival rate of Lta4h knockout mice after lung infection with S. pneumoniae was not significantly different from that of control mice (Fig. S8b). Based on these results, we can conclude that the role of Lta4h or Blt1 in neutrophil phagocytosis is not pathogen-wide but rather fungal-specific.

Fig. 5.

BLT1 regulates the neutrophil phagocytosis against β-1,3-glucan-exposed fungi. A Representative flow cytometry plots and bar graphs on phagocytosis of Lta4h^+/− or Lta4h^−/− neutrophils toward APC⁺ beads at indicated time points. B Western blot analysis on the phosphorylation of ERK1/2 signaling in Blt1^+/− and Blt1^−/− neutrophils after stimulation with 50 ng/ml LPS at indicated time points. C Comparison of phagocytosis between Blt1^+/− and Blt1^−/− neutrophils toward GFP-expressing Staphylococcus aureus or FITC-labeled Streptococcus pneumoniae (MOI = 10). D Western blot analysis on the phosphorylation of ERK1/2 signaling in Blt1^+/− and Blt1^−/− neutrophils after stimulation with 100 μg/ml mannan or curdlan at indicated time points. E Comparison of phagocytosis between Blt1^+/− and Blt1^−/− neutrophils toward FITC-zymosan particles. F Survival of Blt1^−/− (n = 7) and Blt1^+/− (n = 6) mice following intratracheal instillation with 4 × 10⁷ CFU A. fumigatus (strain CEA10). G Phagocytosis of Blt1^+/− and Blt1^−/− neutrophils toward AF633-labeled A. fumigatus (MOI = 10). Each experiment was performed at least 2–3 times. In (B) and (D), data are representative of three independent experiments (n = 3). Fold change = IntDen_{other band}/IntDen_{first band}. β-tubulin was used as the internal controls. In (A, C, E, and G), data are expressed as mean ± SEM. Multiple t-test was performed. In (F), statistical analysis by log-rank (Mantel–Cox) test. ns nonsignificant as p > 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figs. S8 and S9

We investigated the predominant phagocytic pathway in neutrophils, including opsonization-mediated pathways or direct recognition of fungal PAMPs. To this end, we used Fcgr2b/3/4^−/− mice and observed that simultaneous deletion of Fcgr2b/3/4 did not affect the phagocytosis of neutrophils (Fig. S8c), ruling out the effect of LTA4H or BLT1 on FcγRs-mediated phagocytosis.

Furthermore, we compared the phagocytosis of C. albicans in FBS-free medium with that of mouse serum-opsonized C. albicans and found that the deficiency of Blt1 impaired the phagocytosis of C. albicans only in FBS-free medium but not in opsonized system (Fig. S8d), thus confirming that BLT1 was not involved in neutrophil phagocytosis against opsonized C. albicans.

To determine which cell wall component is involved in the process of LTA4H-BLT1-mediated phagocytosis, we investigated ERK1/2 activation in Blt1^−/− neutrophils stimulated with purified mannan and curdlan. Our results showed that curdlan, but not mannan, effectively activated the phosphorylation of ERK1/2 in neutrophils (Fig. 5D). Consistent with the results of C. albicans stimulation, Blt1^−/− neutrophils stimulated with curdlan also exhibited lower levels of ERK1/2 phosphorylation compared with the control group, indicating the critical role of BLT1 in curdlan-induced ERK1/2 activation. Additionally, we used FITC-zymosan particles to mimic fungi for phagocytosis experiments. Similar to the phagocytosis of C. albicans, Lta4h- or Blt1-deficient neutrophils were also impaired in the phagocytosis of zymosan particles (Figs. 5E and S8e), thus confirming that the regulation of phagocytosis by Lta4h or Blt1 was dependent on the activation of β-1,3-glucan.

Based on this recognition of β-1,3-glucan-exposed fungi, we speculate that the LTA4H-LTB₄-BLT1 axis also functions similarly in Aspergillus infection. As expected, Blt1^−/− mice were more susceptible to A. fumigatus pulmonary infection (Fig. 5F), infected Blt1^−/− mice displayed severer lung tissue damage and higher levels of neutrophil infiltration (Fig. S9a–c). Blt1-deficient neutrophils also exhibited decreased phagocytosis when co-cultured with AF633-labeled A. fumigatus (Fig. 5G). These findings collectively indicate that LTA4H-LTB₄-BLT1 axis regulates β-1,3-glucan-triggered fungal phagocytosis in neutrophils.

BLT1 regulates CR3-mediated neutrophil phagocytosis

To identify the phagocytic receptor responsible for recognizing β-1,3-glucan and mediating fungal phagocytosis on neutrophils, and to investigate its regulation by the LTA4H-LTB₄-BLT1 axis, we examined two candidate receptors: C-type lectin receptor Dectin-1 and complement receptor 3, both of which have been shown to participate in fungal phagocytosis [28, 29]. The cell surface display of CD11b (CR3) was found to be much higher than that of Dectin-1 on neutrophils (Fig. S10a). As shown in Fig. 6A, B, loss of Dectin-1 did not affect phagocytosis or ERK1/2 phosphorylation in response to C. albicans in neutrophils. However, CR3 knockout resulting from deletion of the CD11b subunit substantially impaired phagocytosis of C. albicans and FITC-zymosan particles (Figs. 6C and S10b). These results collectively suggest that CR3 might be the primary phagocytic receptor on neutrophils responsible for phagocytosis of β-1,3-glucan-exposed fungi. Therefore, we hypothesize that in neutrophils, CR3-mediated fungal phagocytosis is regulated by the LTA4H-LTB₄-BLT1 axis.

Fig. 6.

CR3 is regulated by LTA4H-BLT1 axis and shows physical interaction with BLT1. A Western blot analysis on the phosphorylation of ERK1/2 signaling in Clec7a^+/+ and Clec7a^−/− (Dectin1^−/−) neutrophils after in vitro stimulation with live C. albicans (MOI = 1) for 0, 5, 15, 30, and 45 min. Data are representative of three independent experiments (n = 3). Fold change = IntDen_{other band}/IntDen_{first band}. B Phagocytosis of live GFP-expressing C. albicans (MOI = 5) by Clec7a^+/+ and Clec7a^−/− (Dectin1^−/−) neutrophils at indicated time points. C Representative flow cytometry plots and statistical results on phagocytosis of Itgam^+/+ or Itgam^−/− (CR3 KO) neutrophils toward live GFP-expressing C. albicans (MOI = 5). D Representative flow cytometry plots and statistical results on phagocytosis of BLT1-overexpressing (BLT1-OE) RAW264.7 cells or RAW264.7 cells (Control) (with or without 1 or 5 ng/ml LTB₄) toward live GFP-expressing C. albicans (MOI = 5). E Comparison on phagocytosis of BLT1-overexpressing RAW264.7 cells (BLT1-OE), BLT1-overexpressing & Itgam-deficient RAW264.7 cells (BLT1-OE&CR3 KO) toward live GFP-expressing C. albicans (MOI = 5) in the presence or absence of 5 ng/ml LTB₄. F CCR2-overexpressing, CXCR2-overexpressing and BLT1-overexpressing RAW264.7 cells were stimulated with live C. albicans (MOI = 10) in the presence or absence of 5 ng/ml LTB₄. Endogenously expressed CD11b and CD18 in RAW264.7 were immunoprecipitated using anti-Flag antibody. The immunocomplexes were analyzed by Western blot with indicated antibodies. β-tubulin was used as the internal controls. Each experiment was performed at least 2–3 times. In (B–E), data are expressed as mean ± SEM. Student’s t test or multiple t-test was performed. ns nonsignificant as p > 0.05. *p < 0.05, ****p < 0.0001. See also Fig. S10

mRNA expression analysis of Lta4h, Blt1 and Blt2 indicated that the relatively high expression of Blt1 in neutrophils might contribute to the cell specificity of its effect on phagocytosis (Fig. S10c). To test our hypothesis about the regulatory role of BLT1 on CR3-mediated phagocytosis, we generated a BLT1-overexpressing RAW264.7 cell line. The high level of inherent CR3 expression and absence of BLT1 expression on RAW264.7 cells made this cell line appropriate for exploring the functional regulation of CR3 by BLT1 (Fig. S10d). Our phagocytosis experiments showed that overexpression of BLT1 greatly enhanced phagocytic capacity in the presence of LTB₄ (Fig. 6D) while exogenous LTB₄ could not promote the phagocytosis of RAW264.7 control cells, confirming the critical role of BLT1 in phagocytosis of phagocytic effector cells. Loss of CR3 in the original RAW264.7 cell line significantly weakened its phagocytic capacity (Fig. S10e), indicating that RAW264.7 cells also phagocytized C. albicans in a CR3-dependent manner. Importantly, under the premise that LTB₄ stimulation can significantly promote the phagocytosis activity of BLT1-overexpressing cells, CR3-deficient BLT1-OE cells could no longer respond to LTB₄ (Fig. 6E). The fact that knocking out CR3 deprived these cells of their ability to respond to LTB₄ clearly demonstrated a substantial functional link between LTB₄-BLT1 axis and CR3.

We next wondered whether BLT1 directly interacted with CR3 or indirectly regulated CR3-mediated phagocytosis. Immunoprecipitation results showed that endogenously expressed CD11b and CD18 subunits in RAW264.7 cells were constitutively immunoprecipitated with or without C. albicans stimulation while there was no obvious interaction in the negative control groups represented by CCR2 and CXCR2 overexpression (Fig. 6F), indicating a physical interaction between receptor CR3 and BLT1. Remarkably, exogenous LTB₄ could significantly enhance the binding between BLT1 and CR3 (Fig. 6F). Additionally, we also analyzed the cell surface display of CD11b in Blt1^−/− neutrophils to evaluate the effect of Blt1 deficiency on CR3 dynamics. The results showed that without C. albicans stimulation, Blt1 deletion did not affect cell surface display of CR3, while Blt1^−/− neutrophils showed a slight decrease after stimulation for 30 or 60 min (Fig. S10f). Combined with the results of phagocytosis experiment using BLT1-overexpressing RAW264.7, we can conclude that CR3-mediated phagocytosis is regulated by BLT1, and this regulation is possibly affected by physical interaction between these two receptors.

Discussion

LTA4H-LTB₄-BLT1 axis has been shown to be implicated in the pathogenesis of various inflammatory diseases [20], Histoplasma capsulatum [30], and Paracoccidioides brasiliensis infections [31]. In this study, we found that LTA4H regulates host susceptibility to C. albicans and A. fumigatus infection by regulating CR3-mediated phagocytosis of neutrophils, but not macrophages, in a BLT1-dependent manner. LTA4H-BLT1 regulates neutrophil phagocytosis initiated by the recognition of fungal cell wall component β-1,3-glucan.

Invasive fungal disease poses a serious threaten to human health, especially in immunocompromised or immunosuppressed individuals. Candida, Aspergillus, and Cryptococcus infections account for more than 90% of invasive fungal infections and are prioritized by the WHO in the Fungal Pathogen Priority List [9, 10]. Similar to the their significance against bacterial infections, such as Staphylococcus, Streptococcus, and Mycobacteria, neutrophils are crucial first-line responders in the clearance of fungal infections even though excessive inflammatory response caused by neutrophil infiltration and activation can also lead to tissue damage [13]. Their phagocytic activity is essential for controlling the progression of pulmonary aspergillosis caused by inhalation of airborne conidia. In addition, neutrophil phagocytosis plays a crucial role in the early stages of commensal C. albicans dissemination as well as in iatrogenic Candida infections [32]. Abnormal neutrophil phagocytosis can result in high levels of fungal load and uncontrolled immune responses, as observed in patients with neutropenia, showing higher incidence of pulmonary aspergillosis than in immunocompetent populations [33, 34]. Therefore, it is of great significance to focus on the effect of neutrophil phagocytosis during fungal infection.

The fungal load in the kidneys of Lta4h^−/− mice in response to systemic C. albicans infection was significantly higher than in the control group, whereas there was no difference in the spleens, showing an organ-specific difference. Previous study has revealed that kidneys are target organ of murine disseminated candidiasis [35]. It has been validated that in a fatal mouse model of invasive candidiasis, neutrophil infiltration in the kidney was much lower than those in the spleen and liver during first 24 h after infection, which limited the effective control of C. albicans growth and promoted C. albicans filamentation in the kidney [36]. Continuous accumulation of neutrophils in the kidney at later time points enhanced the progression of immunopathology. Therefore, the kidney-specific differences in response to systemic C. albicans infection are representative characteristics of this model and support our rationale in investigating the function of kidney-infiltrating neutrophils.

Numerous studies have shown that the LTB₄-BLT1 axis functions as a signal-amplifying mechanism to enhance chemotactic responses in neutrophils [37]. While BLT1 has been shown to induce chemotaxis of eosinophils [38], various types of T cells [39, 40], and dendritic cells [41], its chemotactic properties in neutrophils have been extensively studied in different disease models, such as peritonitis [38, 42, 43] and allergic skin inflammation [44]. These studies have demonstrated that deletion or inhibition of BLT1 significantly reduced neutrophil recruitment. However, in our systemic candidemia model, neither Lta4h nor Blt1 deficiency affected neutrophil recruitment in kidneys at time points of 2.5–24 h. Similarly, Lee’s study showed that Blt1^−/− mice had equivalent numbers of PMN infiltrated into lungs [45] after intravenous infection. Caffrey-Carr’s study revealed that both Blt1^−/− mice and Alox5^−/− mice (5-lipoxygenase-deficient mice, lack all leukotriene synthesis) were more susceptible to respiratory A. fumigatus (strain CEA10) infection and showed a significant defect in neutrophil and eosinophil recruitment at the early infection stage (12 hpi) [46]. It seems that the contribution of LTA4H-LTB₄-BLT1 axis to neutrophil trafficking is not completely identical in different targeted organs and may be closely related to infection mode. It is worth mentioning that in our pulmonary A. fumigatus (strain CEA10) infection model, lung-infiltrating neutrophils in Blt1^−/− mice were significantly lower than those in control mice at 24 h post-infection. However, at 48 h post-infection, Blt1^−/− mice exhibited higher levels of neutrophil infiltration than controls (Fig. S9c). Nonetheless, our results, as well as Lee’s and Hopke’s studies, supported the idea that Lta4h/Blt1 deficiency impairs neutrophil clustering and swarming function [45, 47].

The role of LTB₄ in promoting killing of phagocytes has been previously reported in several studies [31, 48–51]. The results of the current study showed that deletion of LTA4H and BLT1 resulted in loss of phagocytic capacity of neutrophil against Candida and Aspergillus. Interestingly, Lta4h-/Blt1-deficient macrophage (both BMDMs and peritoneal macrophages) showed equivalent phagocytic capacity against C. albicans compared to the control group. The previous studies investigating the effect of BLT1 on macrophage phagocytosis have shown conflicting results. Okamoto et al. found that BLT1-deficient BMDMs exhibit lower phagocytic capability of IgG-opsonized zymosan but not of non-opsonized zymosan [51]. The study by Morato-Marques reported a reduction in phagocytosis of C. albicans by LT-deficient AMs and peritoneal macrophages [52]. The differences in BLT1 function on macrophage phagocytosis could presumably due to differences in functional pattern recognition receptors (PRRs) and C. albicans strains. Un-opsonized live C. albicans strain SC5314 was used in our study whereas Okamoto’s study and Morato-Marques’s study utilized opsonized/non-opsonized zymosan particles and C. albicans strain CHN1 (a human pulmonary clinical isolate), respectively. The results in Okamoto’s study clearly showed that phagocytosis of non-opsonized zymosan by Blt1-deficient macrophages was comparable with phagocytosis by wild-type macrophages, which was similar to our results using the non-opsonized C. albicans. Besides, several previous studies have established that different C. albicans isolates display a wide range of phenotypic properties both in vitro and in models of infection [53–56]. Intraspecies variation can therefore exert a major impact on C. albicans strain behavior and determine the outcome of host-fungal interactions [53]. More importantly, in our study, the results of in vivo phagocytosis experiments further supported the in vitro results. Additionally, the expression level of BLT1 was found to be much lower in macrophages (BMDMs, PMs and AMs) compared to neutrophils, suggesting the significance of LTB₄-BLT1 being dominant in neutrophils rather than macrophages.

Furthermore, the study constructed a BLT1-overexpressing RAW264.7 cell line and showed that exogenous addition of LTB₄ significantly enhanced the phagocytosis of BLT1-overexpressing cells but not primitive cells against Candida. This finding suggests that the difference in BLT1 expression might contribute to cell sensitivity in its effect on fungal phagocytosis.

The precise mechanism by which BLT1 regulates phagocytosis, especially in fungal infections, remains unclear. Although the interaction between BLT1 and FcγRI within lipid rafts (LRs) has been shown to regulate FcγRI-mediated phagocytosis in AMs, this study also revealed that BLT1 failed to redistribute upon AMs stimulation with C. albicans [48]. While the cross-talk between LTB₄-BLT1 and IgG-FcγRs signaling or dectin-1/mannose receptor has been found to mediate the enhancement of phagocytosis of opsonized zymosan and C. albicans in macrophages [51, 52] respectively, neither FcγRs KO nor Dectin-1 KO affected the phagocytosis of neutrophil against non-opsonized C. albicans (Figs. 6B and S8c). Our present results revealed that in neutrophils, it was CR3 that participated in the phagocytosis of C. albicans and A. fumigatus and was regulated by LTA4H-LTB₄-BLT1 axis. Increasing evidence has shown that CR3, which is abundantly expressed in neutrophils, regulates gene expression, cytoskeletal rearrangements, and ultimately mediates cell adherence, migration, ROS production and phagocytic activity. As a phagocytic receptor, CR3 has been shown to mediate phagocytosis of complement-opsonized pathogens as well as non-opsonized microbes through the I domain and lectin-like binding domain, respectively [57]. Given the central role of CR3 in neutrophils, studies showed that adherence of neutrophils to different substrates via CR3 plays a crucial role in the activation state of neutrophils, which mainly correlates with the mechanical traction force generated between neutrophils and substrates [58–60]. In terms of the regulatory role of the LTA4H-LTB₄-BLT1 axis on neutrophil phagocytosis, our results showed that it displays fungal-specificity and β-1,3-glucan-dependency. Since C. neoformans-infected Lta4h^−/− mice did not phenocopy the susceptibility to C. albicans infection, we speculate that the encapsulation of C. neoformans evaded the antifungal function of neutrophils as it is determined by the interaction of PRRs with cell wall components. Therefore, we conclude that in neutrophils, LTA4H-LTB₄-BLT1 primarily regulates CR3-mediated phagocytosis of non-opsonized β-1,3-glucan-exposed fungi such as C. albicans, A. fumigatus. Furthermore, we observed a physical interaction between BLT1 and CR3, suggesting a possible regulation pattern by BLT1. The substantial reduction in phagocytosis of CR3-intact Blt1-deficient neutrophils suggests that BLT1 activation is a prerequisite for phagocytic process in neutrophils.

The surface display of CD11b in neutrophils (represented by CD11b MFI) from naïve mice was not affected by BLT1 deficiency (Fig. S10f), it was also comparable between LTA4H or BLT1 KO neutrophils and their counterparts at the early stage of infection including 2.5 h (Fig. S6g, h) and 3/6/24 h (Fig. S6f) as we detected, while it exhibited a decreased tendency till the time point of 48 h (Fig. S6f). However, the MFI of CD11b within neutrophils in the kidney was significantly lower in Lta4h^−/− and Blt1^−/− mice at 72 h post-infection (Figs. S2e and S6a), suggesting that LTA4H-LTB₄-BLT1 axis could regulate CD11b expression in neutrophils during C. albicans infection. Similarly, in several previous studies, a reduction in LTB₄-induced CD11b expression on neutrophils was also observed after treatment with leukotriene B₄ receptor antagonists (LY293111, BIIL 284) [61–64]. These findings further extended the regulatory impact of BLT1 on CR3.

Since the discovery of LTA4H and BLT1, growing evidence had shown the importance of the LTA4H-LTB₄-BLT1 axis in neutrophil-mediated inflammation and immune responses [20, 25, 37]. In current study, our results were derived from Lta4h or Blt1 total knockout mice rather than neutrophil-specific deletion mice. Although human and mouse neutrophils share many of the same characteristics, such as phagocytosis, killing and chemotaxis, the conclusions also need to be validated in human neutrophils due to human and murine neutrophils differ with respect to representation in blood, receptors, nuclear morphology, signaling pathways, granule proteins, NADPH oxidase regulation, magnitude of oxidant and hypochlorous acid production, and their repertoire of secreted molecules [65–68].

In summary, our study revealed that mice lacking LTA4H or BLT1 were more vulnerable to fungal infections, including C. albicans and A. fumigatus, with significantly higher fungal load and inflammation. Mechanistically, the deficiency of LTA4H or BLT1 greatly impairs CR3-mediated neutrophil phagocytosis based on the recognition of β-glucan in non-opsonized fungi as shown in schematic model (Fig. 7). These results shed new light on the regulatory role of the LTA4H-LTB₄-BLT1 axis in modulating neutrophil function during Candida and Aspergillus infection. While our results imply that agonists targeting LTA4H/BLT1 may be able to modulate the antifungal activity of neutrophils, further explorations are required from a clinical perspective due to the complexity of the host immune system.

Fig. 7.

Schematic model for the mechanism by which LTA4H-LTB₄-BLT1 axis regulates CR3-mediated neutrophil phagocytosis. In response to systemic C. albicans infection, LTB₄ derived from LTA4H hydrolysis binds to its high-affinity receptor BLT1 on neutrophils, which could facilitate the CR3-mediated neutrophil phagocytosis against C. albicans via direct interaction with CR3, thereby protecting mice from candidiasis. In the absence of Lta4h or Blt1, the LTA4H-LTB₄-BLT1 axis is unable to be activated, thus severely impairing CR3-mediated neutrophil phagocytosis. Dysfunctional neutrophils are unable to clear the infected C. albicans quickly and efficiently. Lta4h- or Blt1-deficient mice eventually die due to tissue abscesses caused by the increased fungal load

Materials and methods

Mice

Both Lta4h^−/− and Blt1^−/− mice were generated using CRISPR-Cas9 technology in the Laboratory Animal Resources Center at Tsinghua University (as described in Figs. S1 and S5) and bred on a C57BL/6 background. Wild-type C57BL/6 mice were obtained from Jackson Laboratory. To generate Lta4h^−/−Rag1^−/− mice, Lta4h^−/− mice were crossed with Rag1^−/− mice (from Jackson Laboratory), and their offspring were crossed for several generations. Itgam^−/− mice and Fcgr2b/3/4^−/− mice were provided by Dr. Jingren Zhang (Tsinghua university) [69]. Clec7a^−/− mice were provided by Dr. Xinming Jia [70]. All mice were housed in the specific-pathogen-free animal facilities at Tsinghua University, and genotyped according to standard protocol before being used for experiment after 8–10 weeks of age. All mouse experiments were performed in compliance with the institutional guidelines and according to the protocol approved by the Institutional Animal Care and Use Committee of Tsinghua University.

Bone marrow-chimeric mice generation

Four-week-old recipient mice of CD45.2⁺ wild-type, CD45.2⁺ Lta4h^+/−, and CD45.2⁺ Lta4h^−/− genotypes were subjected to lethal irradiation by X-ray (550 rad × 2), and intravenously administered with 2 × 10⁶ bone marrow cells obtained from either CD45.2⁺ Lta4h^+/−, CD45.2⁺ Lta4h^−/−, or CD45.2⁺ wild-type donor mice.

In competitive bone marrow transplantation, a 1:1 mixture of CD45.1⁺ wild-type and CD45.2⁺ Lta4h^−/− or a 1:1 mixture of CD45.1⁺ wild-type and CD45.2⁺ Blt1^−/− were transferred into CD45.2⁺ wild-type recipients after being irradiation by X-ray (550 rad × 2). All recipients were given drinking water containing antibiotics for 21 days to prevent bacterial infections. These chimeras were used for further experiments 6–8 weeks after the initial reconstitution.

Fungal culture, bacterial culture and infection

Cryopreserved C. neoformans strain H99, C. albicans strain SC5314, as well as GFP-expressing and Ruby-expressing C. albicans, were cultured in Yeast Extract Peptone Dextrose Medium (YPD) at 30 °C and 200 rpm for 12–16 h. The cultured C. neoformans and C. albicans were collected by centrifugation at 4000 rpm for 5 min, washed with 1 × PBS, and counted using a hemocytometer. For mouse infection, suspension containing 2 × 10⁵ CFU C. albicans in 200 μl, or 1 × 10⁴/1 × 10⁶ CFU C. neoformans in 50 μl were administered via lateral tail veins or intratracheal instillation, respectively.

The Aspergillus fumigatus strain CEA10 (also known as CBS144.89) was cultured on glucose minimal medium (GMM) plates at 37 °C for 7 days. The conidia were harvested and washed three times with PBST (containing 0.025% Tween-20) at 4000 rpm. The washed conidia were resuspended in PBST at a concentration of 8 × 10⁸ CFU/ml, and 50 μl of the suspension was administered to Avertin-anesthetized mice via intratracheal instillation. To generate fluorescent Aspergillus reporter (FLARE) conidia, the method described in Guo’s study [71] was followed.

Streptococcus pneumoniae (strain 23F), and GFP-expressing Staphylococcus aureus were obtained from Dr. Jingren Zhang (Tsinghua University). Both GFP-expressing S. aureus and S. pneumoniae were cultured in brain heart infusion (BHI, Solarbio) broth at 37 °C until the absorbances at 600 nm reached 1.0 for S. aureus and 0.6 for S. pneumoniae. The bacterial suspensions were then frozen in media containing 25% glycerol and stored at −80 °C. After 48 h, cryopreserved bacteria were thawed and counted using BHI agar plates for S. aureus and tryptic soy agar (TSA) plates supplemented with 5% defibrinated sheep blood for S. pneumoniae.

For the mouse infection experiment, the cryopreserved S. pneumoniae were washed once and resuspended in 1 × PBS to a concentration of 2 × 10⁷ CFU/ml. Next, 50 μl of this suspension was administered to Avertin-anesthetized mice via intratracheal instillation. The survival of infected mice was continuously monitored and analyzed after 1 week.

Phenotypic and histopathological analysis of mice

The survival of infected mice was monitored continuously and analyzed using Graphpad Prism 8.0. The fungal load in the kidney and spleen after C. albicans infection or in brain and lung after C. neoformans infection at indicated time points was assessed at indicated time points by plating diluted tissue homogenate on YPD agar plates. All plates were incubated at 30 °C, and individual colonies were counted.

Infected Lta4h^−/− or Blt1^−/− mice were euthanized after anesthesia. Kidneys and lungs were collected and fixed with 4% (w/v) paraformaldehyde (PFA) solution. Histopathological examination was performed by Servicebio. Tissue damage, fungal load, and immune cell infiltration in the kidneys or lungs were characterized by hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS) staining, and immunohistochemistry (IHC), respectively. All sections were imaged using a Pannoramic Scan system (3DHISTECH).

Primary cells preparation

Primary bone marrow-derived macrophage (BMDM), peritoneal macrophage (PM) and bone marrow-derived dendritic cell (BMDC) of different genotypes were prepared as previously described [72]. BMDMs, PMs and BMDCs were cultured in complete DMEM medium at 37 °C.

Bone marrow neutrophils were isolated using anti-Ly6G microbeads (Miltenyi, 130-120-337) following the manufacturer’s protocol. Peritoneal neutrophils were isolated from peritoneal exudates of casein-stimulated mice according to published protocols [73]. Purity of isolated neutrophils was assessed by FACS analysis after staining with antibodies specific for CD11b and Ly-6G, with a purity of over 95%. The purified neutrophils were then cultured in RPMI medium supplemented with 5% (v/v) heat-inactivated FBS and antibiotics.

Generation of cell lines

NIH3T3 cells and RAW264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) and RPMI1640 medium (Gibco), supplemented with 10% (v/v) fetal bovine serum, 1 × non-essential amino acids, 1 × sodium pyruvate, and 1 × antibiotics (penicillin and streptomycin) at 37 °C in a humidified atmosphere composed of 5% CO₂.

CCR2-overexpressing, CXCR2-overexpressing, BLT1-overexpressing or CR3 knockout RAW264.7 cells were generated by lentivirus transduction. These lentivectors carried Ccr2, Cxcr2 and Blt1 coding sequence or Itgam-targeting sgRNA sequence along with blasticidin or puromycin selection marker.

Blt1-overexpressing RAW264.7 cells, CR3^−/− RAW264.7 cells, or Blt1-overexpressing CR3^−/− RAW264.7 cells were seeded into a 48-well plate at a density of 3 × 10⁵ cells per well and co-cultured with GFP-expressing C. albicans at MOI of 5 in the presence or absence of LTB₄ after centrifugation at 300 × g. The proportion of GFP⁺ RAW264.7 cells was quantified by flow cytometry.

NIH3T3 cells were seeded into 24-well plates at a density of 5 × 10⁵ cells/well, and transiently transfected with BLT1-overexpressing vector or control vector after attachment. Following stimulation with live C. albicans (MOI = 1) for 15 or 30 min, cells were washed and harvested for Western blot analysis.

Fluorescent-labeled S. pneumoniae preparation

S. pneumoniae was labeled with fluorescent dye FITC according to published protocols [74]. First, cryopreserved S. pneumoniae were washed once in 1 × PBS at 10,000 × g and then resuspended at 1 × 10⁹ CFU/ml in 200 μl of 1 × PBS containing 200 μg/ml of FITC solution (Sigma). The mixture was incubated in the dark for 20 min at 37 °C with gentle agitation every 5 min. After incubation, the FITC-conjugated S. pneumoniae were washed twice with 1 × PBS and resuspended at a concentration of 2 × 10⁷ CFU/ml in basal RPMI 1640 medium.

LTB₄ measurement

Peritoneal neutrophils (2 × 10⁶ cells/well) isolated from Lta4h^−/− mice were co-incubated with live yeast-form C. albicans (MOI = 1) in 800 μl of RPMI1640 medium for 5, 20 and 60 min. Supernatant was collected for LTB₄ measurement. 100 μl supernatant was extracted with 2 volumes of 8:2 acetonitrile/methanol (v/v) containing 10 ng of deuterium-labeled internal standard d8-arachidonic acid (Cayman Chemicals) and centrifuged twice at 14,000 rpm for 10 min. LTB₄ was analyzed and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as described previously [75].

Immunoprecipitation and western blotting

CCR2-, CXCR2- and BLT1-overexpression RAW264.7 cells stimulated with C. albicans in the presence or absence of LTB₄ were washed with cold phosphate-buffered saline (PBS), and lysed in lysis buffer (30 mM Tris-HCl, pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 10% glycerol, and 1% Triton X-100) containing 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM PMSF, and a protease inhibitor mixture (Roche). Cell lysates were centrifugated at 18,000 × g for 10 min, and immunoprecipitated with anti-FLAG agarose beads. The incubated beads were washed four times in lysis buffer and samples were eluted by boiling in 10 μl 5× SDS loading buffer. The input and immunoprecipitated samples were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

After stimulation with different stimuli (including live C. albicans, mannan, curdlan, and LPS) at indicated concentrations and time points, both adherent and suspended neutrophils were collected and pooled, and lysed with lysis buffer for 30 min at 4 °C. After centrifugation, the lysate supernatants were collected and mixed with loading buffer.

All samples were electrophoresed by SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF, Millipore) membranes. The membranes were blocked with 5% (m/v) bovine serum albumin (BSA) and incubated with primary antibodies (1:1000) overnight at 4 °C. Tris-buffered saline buffer (TBS) supplemented with 0.05% (v/v) Tween-20 was used to wash the membrane and dilute the secondary antibody. After washing with TBST, the membrane was incubated with an HRP-conjugated secondary antibody. The abundance of target proteins was detected using a chemiluminescence system with mixed ECL substrate. Integrated density (IntDen) of band was analyzed using FIJI/ImageJ (version 2.14.0/1.54f) software.

Quantitative PCR

Total RNA was extracted manually using TRIzol (Life Technologies) and assessed for quantity and quality using a NanoDrop spectrophotometer (Thermo Scientific). The first-strand cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The abundance of gene expression was quantified with 2 × M5 HiPer Real-time PCR Super mix (Mei5 Biotechnology Co., Ltd) on a Roche 480 Real-Time PCR system (Roche). Gapdh was used as the housekeeping gene in qPCR analysis, and the primer sequences used are listed in Table S1.

Cytokines measurement

The expression of cytokines in C. neoformans-infected brain or C. albicans-infected kidneys was quantified using Ready-SET-GO ELISA kits (eBioscience) following the manufacturer’s instructions. Absorbance at 405 nm was measured in each well using a microplate reader.

FACS analysis

The influx of neutrophil in response to C. albicans infection in the kidney was evaluated using a flow cytometer. Kidneys were collected and digested with collagenase II for 30 min, followed by filtration and lysis with RBC buffer to remove red blood cells. Lymphocytes were then isolated using a two-layer Percoll gradient of 40% and 80%. After Fc blocking, the cell suspensions were stained targeted antibodies, and dead cells were excluded using Live/Dead Ghost Dye Violet 510. The quantification of different lymphocyte populations was performed using a BD LSR Fortessa cytometer, and flow cytometric data were analyzed with the FlowJo software.

ROS and NO detection

The intracellular production of ROS in neutrophils was assessed using 2′,7′-dichlorofluorescein diacetate (DCF-DA). Primary neutrophils were plated at 2 × 10⁵ cells per well in 48-well plate and treated with heat-killed C. albicans at an MOI of 10. After 1, 3, and 6 h of treatment, cells were incubated with 2 μM DCF-DA at 37 °C for 20 min in the dark and washed with 1 × PBS five times. The fluorescence intensity in cells was measured using a flow cytometer at an excitation wavelength 488 nm.

After being stimulated in vitro, the production of nitric oxide (NO) in the cell culture supernatants of BMDM was measured using an NO assay kit (Beyotime Biotechnology) following the manufacturer’s protocol. Specifically, 50 μl of supernatant or a series of diluted standards were added to 96-well flat bottom plates, followed by the addition of 50 μl of Griess I and Griess II to each well. After 10 min, the absorbance at 540 nm was measured using microplate reader.

Ca²⁺ influx measurement

Neutrophils isolated from bone marrow were loaded with 5 μM Indo-1 AM at 37 °C for 30 min in Hank’s Balanced Salt Solution (HBSS) in the dark. The cells were then washed for twice in HBSS. After incubation in 37 °C water bath for more than 10 min in the dark, the baseline was recorded for 30 s on LSR II(BD). By addition of intact C. albicans at the MOI of 10, calcium influx of Blt1^+/– or Blt1^−/− neutrophils were measured. All data were analyzed using kinetics in FlowJo.

Evaluation of neutrophil function

The effect of LTA4H on neutrophil function was assessed through three different assays: swarming, phagocytosis, and killing. Purified neutrophils were plated at a density of 1 × 10⁵/well, 5 × 10⁵/well, and 1 × 10⁶/dish for phagocytosis, killing, and fluorescence imaging assays, respectively. For the phagocytosis assay, live GFP-expressing C. albican (at MOI of 5), APC⁺ beads, FITC-zymosan particles, GFP-expressing S. aureus, or FITC-labeled S. pneumoniae (at MOI of 10) were added to 96-well plates, followed by centrifugation at 300 × g for 5 min. After co-culture for 15, 30, or 60 min at 37 °C, the proportion of GFP⁺ neutrophil, APC⁺ neutrophil or FITC⁺ neutrophil was quantified using flow cytometer. Neutrophils pre-treated with 10 ng/ml LTB₄ were used to evaluate whether the phagocytosis was Blt1-dependent. For the in vitro killing assay, live C. albicans at MOI of 1 were added to each well and co-cultured with neutrophils for different time periods. The mixture of supernatant and lysed neutrophils was serially diluted and plated onto YPD plates to evaluate the killing of neutrophils by calculating the difference between total C. albicans counts and remaining C. albicans counts. For fluorescence imaging, live C. albicans were first cultured in complete RPMI1640 medium for 4 h to form hyphal morphology and then cross-linked by UV treatment. Neutrophils stained with PE-conjugated Ly6G antibody were added to dishes and observed using confocal laser-scanning microscope after co-culture for 1 h.

In vivo phagocytosis evaluation

To evaluate neutrophil phagocytosis of different genotypes in vivo, Ruby-expressing C. albicans were utilized. Briefly, Lta4h^−/− mice, Blt1^−/− mice, and reconstituted chimeras were systemically infected with 1 × 10⁷ CFU Ruby-expressing C. albicans. After 2.5 h of infection, all mice were anesthetized and sacrificed, and perfused with 1 × PBS. The kidneys were collected, digested, homogenized, and stained with a mixture of antibodies. Flow cytometry was used to quantify both the proportion of neutrophils in infected kidneys and Ruby⁺ neutrophils.

Statistical analysis

All data are presented as mean ± SEM and are representative of at least two independent experiments, as indicated. GraphPad Prism 9 (GraphPad Prism Software Inc., San Diego, CA) was used for graphing and statistical analysis. Student’s t test, multiple t-test and log-rank (Mantel–Cox) test were used for comparisons. The significance level was set as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Author contributions

YX and XL conceived and designed this study. YX and SX performed experiments, data analysis, and prepared manuscript. LZ provided the scientific and clinical view for this study. All authors contributed to edit the manuscript and agree to be accountable for all aspects of the work.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41423-024-01130-4.