In SM/J Mice Albicans Candida Genetic Control of Susceptibility To

Genetic Control of Susceptibility to Candida albicans in SM/J Mice Irena Radovanovic, Vicki Leung, Alexandra Iliescu, Silayuv E. Bongfen, Alaka Mullick, David Langlais and Philippe This information is current as Gros of September 25, 2021. J Immunol 2014; 193:1290-1300; Prepublished online 27 June 2014; doi: 10.4049/jimmunol.1400783

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Genetic Control of Susceptibility to Candida albicans in SM/J Mice

Irena Radovanovic,*,† Vicki Leung,†,‡ Alexandra Iliescu,*,† Silayuv E. Bongfen,*,† Alaka Mullick,x David Langlais,*,† and Philippe Gros*,†

In the immunocompromised host, invasive infection with the fungal pathogen Candida albicans is associated with high morbidity and mortality. Sporadic cases in otherwise normal individuals are rare, and they are thought to be associated with genetic predisposition. Using a mouse model of systemic infection with C. albicans, we identified the SM/J mouse strain as unusually susceptible to infection. Genetic linkage studies in informative [C57BL/6JxSM/J]F2 mice identified a major locus on distal chromosome 15, given the appellation Carg5, that regulates C. albicans replication in SM/J mice. Cellular and molecular immunophenotyping experiments, as well as functional studies in purified cell populations from SM/J and C57BL/6J, and in [C57BL/

6JxSM/J]F2 mice fixed for homozygous or heterozygous Carg5 alleles, indicate that Carg5-regulated susceptibility in SM/J is Downloaded from associated with a complex defect in the myeloid compartment of these mice. SM/J neutrophils express lower levels of Ly6G, and importantly, they show significantly reduced production of reactive oxygen species in response to stimulation with fMLF and PMA. Likewise, CD11b+Ly6G2Ly6Chi inflammatory monocytes were present at lower levels in the blood of infected SM/J, recruited less efficiently at the site of infection, and displayed blunted oxidative burst. Studies in F2 mice establish strong correlations between Carg5 alleles, Ly6G expression, production of serum CCL2 (MCP-1), and susceptibility to C. albicans.

Genomic DNA sequencing of chromatin immunoprecipitated for myeloid proinﬂammatory transcription factors IRF1, IRF8, http://www.jimmunol.org/ STAT1 and NF-kB, as well as RNA sequencing, were used to develop a “myeloid inﬂammatory score” and systematically analyze and prioritize potential candidate genes in the Carg5 interval. The Journal of Immunology, 2014, 193: 1290–1300.

andida albicans is a commensal microbe that resides in invasive dermatophytic disease has been reported in individuals the gastrointestinal and genitourinary tracts of healthy deficient in CARD9 (6, 8), and more recently, in IL12Rb1 (9). C individuals (1). Breaching of the host barrier or immu- Besides CARD9 deficiency (6), the genetic component of sus- nologic defenses results in C. albicans infections, which can range ceptibility to systemic C. albicans infection in humans is still from superficial and benign to disseminated infections with sig- unknown. Parallel genetic studies in inbred and mutant mouse by guest on September 25, 2021 nificant morbidity and mortality. Notably, Candida species con- strains have identified the complement system as playing a critical stitute the fourth most common cause of nosocomial bloodstream and determining role in early innate defenses against blood borne infections (2), with C. albicans accounting for more than half of Candida (10, 11). Both arms of the complement system (classical all Candida infections, and for ∼37% of the associated mortality. and lectin pathway) are engaged in this response. Activation of the In the immunocompetent host, cases of chronic mucocutaneous classical pathway relies on C1q-dependent recognition of fungal candidiasis are sporadic, but multiplex families with dominant (3– Ags alone or in association with host cells, or complexed with Igs 5) and recessive (5–7) inheritance have been described. The study (12). Alternatively, Candida surface carbohydrate structures such of these rare familial cases has identified genes with critical roles as mannoproteins interact with the mannan-binding lectin, thereby in host response to Candida, including recognition of fungal Ags, triggering the lectin pathway and associated activation of C2 and as well as Th1 and Th17 polarized immune responses. In parallel, C4 (12). Both pathways converge at the formation of a C3 con- vertase, which produces the potent chemotactic factors C3a and C5a. Their release causes recruitment of inflammatory cells such *Biochemistry Department, McGill University, Montreal, Quebec H3G 0B1, Canada; as neutrophils and inflammatory monocytes to the site of infec- †Complex Traits Group, McGill University, Montreal, Quebec H3G 0B1, Canada; tion. On the other hand, cleavage of C5 into C5a (activator) and ‡Department of Human Genetics, McGill University, Montreal, Quebec H3G 0B1, Canada; and xNational Research Council-Biotechnology Research Institute, Montreal, C5b initiates the formation of the membrane attack complex Quebec H4P 2R2, Canada (MAC), a macromolecular lytic complex composed of C5b and Received for publication March 27, 2014. Accepted for publication May 26, 2014. other complement subunits (12). Studies in mice deficient in factor This work was supported by a Team Grant in Fungal Pathogenesis from the Canadian B, mannan-binding lectin, C1q, C2, C3, and C5 have validated the Institutes of Health Research. The funders had no role in study design, data collection role of both arms of the complement pathway in defenses against and analysis, decision to publish, or preparation of the manuscript. C. albicans infection (11, 13–16). Address correspondence and reprint requests to Dr. Philippe Gros, Department of In humans and mouse models, efficient production of inflam- Biochemistry, McGill University, 3649 Promenade Sir William Osler, Room 366, Montreal, QC H3G 0B1, Canada. E-mail address: [email protected] matory leukocytes (neutrophils, inflammatory monocytes) and The online version of this article contains supplemental material. timely recruitment of these cells to the site of infection is required Abbreviations used in this article: BM, bone marrow; BMDM, BM-derived macro- for protection during C. albicans infection. Indeed, increased phage; CGD, chronic granulomatous disease; ChIP-seq, chromatin immunoprecipitation vulnerability to invasive fungal infection is observed in clinical sequencing; LOD, logarithm of odds; MFI, mean fluorescence intensity; MOI, multi- neutropenia (17, 18), in mutant mice deficient in the cell adhesion plicity of infection; RLU, relative luminescence unit; ROS, reactive oxygen species. molecule ICAM-1 (19), and in the neutrophil chemokine receptor Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 CXCR2 (20). NADPH oxidase–dependent production of reactive www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400783 The Journal of Immunology 1291 oxygen species (ROS) by neutrophils is essential for fungicidal Infection with C. albicans activity, and deficiency in NADPH oxidase components in patients C. albicans strain SC5314 was grown overnight in YPD medium (1% yeast suffering from chronic granulomatous disease (CGD) causes re- extract, 2% Bacto Peptone, and 2% dextrose) at 30˚C with shaking. Blas- current and severe infections with bacterial and fungal pathogens tospores were harvested by centrifugation, washed twice in PBS, counted (21, 22). The early egress of monocytes to the site of infection, and using a hemacytometer, and resuspended in PBS at the required density. For the ultimate contribution of these inflammatory monocytes to an- experimental infections, mice were inoculated via the tail vein with 200 mlof a suspension containing 5 3 104 C. albicans blastospores in PBS. Forty- tifungal defenses, is poorly understood. In humans, monocytopenia eight hours after infection, target organs were harvested, homogenized in caused by deficiency in GATA2 (23, 24) and IRF8 (25) predisposes PBS, serially diluted, and plated on YPD-agar Petri dishes containing 34 mg/ to mycobacterial infections (following Bacillus Calmette–Gue´rin ml of chloramphenicol. The plates were incubated at 30˚C for 24–48 h, and vaccination), although invasive infections with fungal pathogens the CFUs were counted and expressed as log10 CFU per organ. such as Aspergillus and Cryptococcus,aswellasoralcandidiasis, Genotyping and linkage mapping have been reported in some of these patients. During systemic in- hi As described previously (34), genomic DNA was isolated from tail clips of fection with C. albicans in mice, Ly6C inflammatory monocytes individual F2 mice collected at the time of sacrifice. A total of 150 rapidly infiltrate infected kidneys, potentiating innate antifungal [C57BL/6JxSM/J]F2 male mice were genotyped at the McGill University immunity to reduce fungal burden (26). In line with these results, and Genome Quebec Innovation Centre (Montreal, QC, Canada) using monocytopenic mice exhibit exacerbated infection parameters (27). Sequenome iPlex Gold technology and a custom panel that contained 126 We have previously shown that roughly half of the inbred labo- informative SNPs distributed evenly across the genome. Nine additional microsatellite markers annotated in the Mouse Genome Informatics Da- ratory mouse strains carry a naturally occurring 2-bp deletion in the tabase (http://www.informatics.jax.org) were used for gap filling and fine C5 gene (Hc allele) that causes C5 deficiency (28), which renders mapping (29). An additional cohort of 204 female and 40 male [C57BL/ Downloaded from the affected strains susceptible to acute infection with C. albicans 6JxSM/J]F2 mice were subsequently genotyped for markers underlying (10, 16, 29). In addition to the major effect of the C5 status on loci of interest near the Carg5 locus. Linkage analysis was performed using Haley-Knott multiple regression analysis assuming a normal model, fungal replication in target organs (kidney, brain) and overall sur- a nonparametric analysis, or a two-part model (35, 36) with the qtl vival from C. albicans infection (10), early studies in AcB/BcA implementation of the R software (37). Empirical genome-wide signifi- recombinant congenic mouse strains suggested that additional C5- cance was calculated by permutation testing (1000 tests).

unrelated genetic loci could also contribute to host response to http://www.jimmunol.org/ Histology C. albicans infection (10, 30, 31). Other studies by Ashman et al. (32, 33) suggested that severity of infection evaluated by type and Whole kidneys were decapsulated, fixed in 4% paraformaldehyde, dehy- extent of histopathologic damage in target organs is regulated by drated in ethanol/xylene, and embedded in paraffin as described previously (38). Histologic 5-mm sections were cut longitudinally with a microtome other loci (Carg1, Carg2), whose map location and identity have and fixed onto glass slides. Sections were deparaffinized, then stained with remained unknown. We recently surveyed a representative cohort of periodic acid–Schiff reagent (Sigma-Aldrich, Oakville, ON, Canada) to 23 phylogenetically distant inbred mouse strains for susceptibility to detect C. albicans elements, and counterstained with hematoxylin (Sigma- disseminated C. albicans infection using kidney fungal load as the Aldrich). Stained sections were examined with a light microscope using 103 and 403 objectives and photographed. phenotypic measure (29). These studies confirmed the critical role of the C5 locus in susceptibility to infection and identified two Single-cell suspension and flow cytometry by guest on September 25, 2021 discordant strains: AKR/J (C5-deficient, resistant) and SM/J C57BL/6J and SM/J male mice were sacrificed 48 h after C. albicans (C5-sufficient, susceptible) (29). Additional haplotype and linkage infection, exsanguinated, and perfused with PBS containing 2 mM EDTA, mapping studies identified two loci Carg3 (Chr 8) and Carg4 (Chr and their organs were harvested. Noninfected control mice underwent the 11) as regulating resistance to C. albicans infection in the context of same procedure. Anticoagulated peripheral blood (∼100–125 ml) was C5 deficiency (29). lysed with ACK lysis buffer and centrifuged, and the resulting leukocyte pellet was washed and suspended in FACS buffer (PBS, 2% FBS, 2 mM In this study, we have examined the genetic basis and phenotypic EDTA). Whole blood was collected from a subset of [C57BL/6JxSM/J]F2 expression of C5-independent susceptibility to C. albicans infec- animals 48h postinfection and leukocytes prepared as described. Spleens tion in the SM/J strain. We show that a locus on Chr 15, which we were finely minced, and single-cell suspensions were obtained through designate Carg5, regulates susceptibility expressed as increased triturating. RBCs were eliminated by incubation with ACK lysis buffer. The resulting leukocyte pellet was washed, filtered through a 40-mm cell fungal replication in the kidney. Detailed cellular immunopheno- strainer, and suspended in FACS buffer. Femoral bone marrow (BM) was typing and molecular studies strongly suggest that Carg5-regu- flushed with RPMI supplemented with 0.5% FBS, followed by RBC lysis. lated susceptibility to C. albicans infection in SM/J is caused by The pellet was washed, filtered through a 40-mm cell strainer, and sus- a pleiotropic myeloid defect, characterized by an altered number pended in FACS buffer. Infiltrating kidney leukocytes were enriched by and function of neutrophils and inflammatory monocytes, in- Percoll gradient centrifugation, as described previously (39). Decapsulated kidneys were finely minced and digested at 37˚C in RPMI containing cluding altered expression of Ly6G, and aberrant production of Liberase TL and grade II DNAse I for 30 min. Digested tissue was washed, reactive oxygen radicals in both cell types. Studies in [C57BL/ and the remaining red cells were lysed briefly with ACK lysis buffer. The 6JxSM/J]F2 mice fixed for different allelic combinations at Carg5 cells were then passed through a 40-mm filter, washed, and separated over further establish a strong correlations among neutrophil Ly6G a discontinuous 40/70% Percoll gradient. The leukocytes at the interphase were isolated, washed, and suspended in FACS buffer. expression, production of serum CCL2, and susceptibility to Cells were first stained with the Zombie Aqua viability dye (BioLegend, C. albicans (fungal load). San Diego, CA) and then surface stained for 30 min in the dark at 4˚C with the following Abs: APC-eFluor780 anti-CD45 (30-F11), PE-Cy7 anti- CD11b (M1/70), PE anti-Ly6C (HK1.4), and BV421 anti-Ly6G (1A8; Materials and Methods BioLegend). Acquisition was performed using an eight-color FACS Canto Mice II flow cytometer (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). Cell aggregates were gated out based on the forward A/J, C57BL/6J, and SM/J inbred strains were obtained from the Jackson scatter (FSC)-height versus FSC-area plot, and the live cell gate was Laboratories (Bar Harbor, ME) as pathogen-free 8–12 wk-old mice. established based on the exclusion of viability dye. Leukocytes were gated [C57BL/6JxSM/J] F2 progeny were bred by systematic brother-sister as CD45+ cells; neutrophils were further identified as CD11b+Ly6G+, mating of [C57BL/6JxSM/J] F1 mice. Animal housing and experimental and CD11b+Ly6G2Ly6Chi denoted inflammatory monocytes. The gating procedures were conducted following the guidelines of the Canadian strategy is also illustrated in Supplemental Fig. 2. Data were expressed as Council of Animal Care and were approved by the Animal Care Com- the percentage of total CD45+ cells. All Abs are from eBioscience, unless mittee of McGill University. stated otherwise. 1292 MYELOID DEFECT IN C. ALBICANS–SUSCEPTIBLE SM/J MICE

Preparation of neutrophils and macrophages from BM Statistical analysis BM neutrophils were isolated by Percoll density gradient centrifugation as All data were analyzed using GraphPad Prism 6.0 statistical software. An described previously (40). BM from 8–16 wk-old male mice was flushed unpaired, two-tailed Student t test was used to establish significance of from tibiae and femurs with HBSS-Prep (Ca2+-/Mg2+-free HBSS [Wisent] differences, and these were deemed significant when p , 0.05. supplemented with 0.5% FBS, 20 mM HEPES). RBCs were lysed by hy- potonic lysis, and BM cells were resuspended in HBSS-Prep, filtered through Computation of the myeloid inflammatory score a70-mm cell strainer, gently layered over a 62.5% Percoll layer, and centrifuged at 1000 3 g for 30 min. Mature neutrophils were harvested from Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA se- the bottom layer of the gradient, and the purity was evaluated with flow quencing (RNA-seq) from myeloid cells (C57BL/6 BMDMs) before and cytometry. Standard preparations yielded neutrophils of .85% purity, de- after IFN-g stimulation were performed to identify and quantify binding termined by characteristic forward and side scatter and high expression of of the proinflammatory transcription factors IRF1, IRF8 (D. Langlais, Gr-1 or Ly6G. In some experiments, cytospin preparations were made and L.B. Barreiro, and P. Gros, unpublished observations), STAT1 (42), and NF-kB stained with Diff-Quick to visualize neutrophil morphology. BM-derived (p65) (43), in the context of IFN-g stimulation. This analysis was con- macrophages (BMDMs) were isolated from femurs of 8–16 wk-old male ducted for the 231 genes located under the Carg5 minimal confidence mice and were cultured in DMEM (Thermo Scientific) containing 10% heat- interval. We computed a “ChIP-seq score” for each gene to summarize inactivated FBS, 20% L-cell-conditioned medium as a source of M-CSF, 100 gene-to-DNA-binding association of the core inflammatory transcription U/ml penicillin, and 100 mg/ml streptomycin in bacteriologic-grade dishes at factors IRF8, IRF1, STAT1, and NF-kB (p65). This score was weighed with two thirds coming from peak proximity to gene transcriptional start 37˚C in a humidified atmosphere containing 5% CO2. Seven days later, cells were harvested by gentle washing of the monolayer with PBS Citrate. site using a 20-kb window, and one third from peak height relative to the median of peak heights for each transcription factor. ROS production di 1 2 þðPip50%Þ Downloaded from ROS production was measured in neutrophils and BMDM by luminol-ECL n w 5 ChIP-seq score : S ¼ + as described previously (22, 41) with slight modifications. Briefly, 10 neu- tf;g i¼1 1:5 trophils were resuspended in HBSS containing 100 mM luminol in a 96-well hi luminometer plate (Costar). Following 10 min incubation at 37˚C, neu- where Pi ¼ ; if Pi.1; then Pi ¼ 1; trophils were activated with 3 mM fMLF (Sigma-Aldrich) or 100 nM PMA 2H (Sigma-Aldrich) delivered by the injector port of EnSpire multiplate reader, where tf = transcription factor; g = gene; w = genomic window size (20 kbp); and luminescence was measured every 10s for 45 min. BMDMs were plated d = distance of peak from transcriptional start site; n = number of peaks in 5 at 2 3 10 cells/well in a 96-well luminometer plate and primed with IFN-g the window; h = peak height; and H = median of peak height. Genes showing http://www.jimmunol.org/ (50 U/ml) for 18 h. BMDMs were washed gently and incubated with HBSS detectable mRNA expression (from RNA-seq data; FPKM $ 20) were at- supplemented with 100 mM luminol for 10 min at 37˚C. ROS production tributed an “Expression score” of 1. These expressed genes were also given was induced with 200 nM PMA and acquired as above. a “Responsive score” of 1 if they were significantly regulated by a 3-h IFN-g treatment (Fold change $ |2| and p value # 0.05). The ChIP-seq score and BMDM phagocytosis assay and immunofluorescence RNA-seq scores were added to attribute each gene a myeloid inflammation score. This myeloid inflammation score was used as an indicator of a pos- 3 5 BMDMs (2 10 ) were seeded onto glass coverslips in a 24-well plate sible participation of the gene in inflammatory processes. and allowed to incubate overnight. The cells were washed gently, and an appropriate suspension of opsonized CAI4-GFP Candida yeast cells in culture media was added at a multiplicity of infection (MOI) of 10, and brought into contact with BMDM by brief centrifugation. After 30 min of Results by guest on September 25, 2021 Genetic analysis of susceptibility of SM/J mice to coincubation at 37˚C and 5% CO2, cells were gently washed twice with C. albicans PBS to remove nonphagocytosed Candida yeast cells. Cells were fixed C5-sufficient C57BL/6J (B6) and SM/J mice were challenged i.v. with 4% paraformaldehyde (Mecalab) for 20 min at room temperature. 3 4 After being blocked with 5% BSA in PBS, the cells were incubated with with 5 10 C. albicans blastospores, and the fungal growth was rabbit anti-Candida Ab (Accurate Chemical and Scientific Corp.). Finally, determined in the kidney by enumeration of CFUs 48 h later the coverslips were incubated with the Cy3-conjugated anti-rabbit sec- (Fig. 1A). Compared to resistant C57BL/6J controls, SM/J dis- ondary Ab, followed by DAPI staining (Molecular Probes) and mounting played significantly higher fungal load, particularly in female of coverslips on glass slides. Phagocytosis was analyzed using a 633 6 6 immersion oil objective, and images were acquired with a Zeiss LSM5 mice (Fig. 1A, left panel; log10CFU: 4.8 0.1 versus 2.9 0.2) Pascal laser scanning confocal microscope. The percentage of phagocy- although this difference was still visible and significant in male tosis was determined by counting the number of macrophages containing mice (Fig. 1A, right panel; log10CFU: 5.0 6 0.1 versus 4.4 6 0.1). at least one Candida cell, and divided by the total number of macrophages, Susceptibility of the SM/J strain was not limited to the kidney, but using ImageJ. The percentage of phagocytosis was calculated from a total was also evident in secondary target organs, such as the brain of 12 images from two independent experiments. (Supplemental Fig. 1A; log10CFU: 2.8 6 0.2 versus 2.2 6 0.1, p = 6 BMDM killing assay 0.0056) and the heart (Supplemental Fig. 1B; log10CFU: 2.6 0.3 versus 2.0 6 0.1, p = 0.0341). Histologic analysis of infected 3 5 BMDMs (2 10 ) were seeded in 96-well plates and allowed to create kidney sections (Fig. 1B) showed extensive fungal invasion and a monolayer at 37˚C. The cells were washed gently, and an appropriate suspension of opsonized SC5314 blastospores in culture media was added numerous infection foci in SM/J, despite noticeable recruitment of to achieve an MOI of 10, followed by a brief centrifugation. After 30 min leukocytes. Kidney sections from C57BL/6J mice were charac- of coincubation at 37˚C, nonphagocytosed Candida were removed by terized by an attenuated presence of fungal elements that consisted gentle washing with PBS, and killing was allowed to proceed during an mostly of pseudohyphal morphology. These results indicate that additional 2 h. At the end of incubation, BMDM were lysed with distilled water, and the wells were rinsed gently with PBS. Liberated yeast cells although SM/J mice are C5 sufficient, they are unable to control were loaded with 5 mM FUN1 yeast viability dye (Molecular Probes) for C. albicans replication in target organs. 30 min at 30˚C, and the red (viable yeast) fluorescence was acquired on the Inheritance and genetic control of C5-independent susceptibility EnSpire multiplate reader. The percentage of live Candida cells was cal- to C. albicans in SM/J was investigated in informative F1 and F2 culated by dividing the fluorescence of the sample incubated for 2 h by its crosses generated between resistant C57BL/6J and susceptible corresponding phagocytosis control. SM/J, in which all mice bear wild type C5 alleles. A total of 190 CCL2 determination male and 204 female [C57BL/6JxSM/J]F2 animals, along with F1 hybrids, were infected with C. albicans, and kidney fungal load was Whole blood was collected from C. albicans infected [C57BL/6JxSM/J]F2 animals 48 h after infection. Serum was separated by centrifugation, and used as the phenotypic measure of susceptibility (Fig. 2A). [C57BL/ circulating CCL2 levels were determined with a commercial ELISA kit, 6JxSM/J]F1 were as resistant to the infection as C57BL/6J controls according to the manufacturer’s instructions (R&D Systems). (Fig. 2A), indicating that susceptibility to C. albicans in SM/J is The Journal of Immunology 1293 Downloaded from http://www.jimmunol.org/ FIGURE 1. SM/J mice display increased fungal burden following C. albicans infection. (A) C57BL/6J (B6) and SM/J mice (n = 9–10) were infected with 5 3 104 C. albicans blastospores, and CFUs were enumer- ated 48 h later in the kidney and segregated according to gender. ***p , 0.0001. (B) Histologic analysis (periodic acid–Schiff staining) shows increased fungal infiltration and growth in SM/J male mice, where hyphae FIGURE 2. A novel locus on distal chromosome 15, Carg5, confers and tissue damage are more prominent compared with the resistant susceptibility to C. albicans infection in the SM/J strain. A total of 190 C57BL/6J (B6) strain. Dashed rectangles identify the area of magnification male and 204 female [C57BL/6JxSM/J]F2 animals, along with [C57BL/ 3 4 (original magnification 40). 6JxSM/J]F1 controls, were infected intravenously with 5 3 10 C. albi- by guest on September 25, 2021 cans blastospores and sacrificed 48 h later. (A) Fungal load was assessed in the kidney and segregated according to gender. Fungal burden from inherited in a recessive manner, whereas the distribution of CFU in C57BL/6J (B6) and SM/J parental controls from Fig. 1 is included for the [C57BL/6JxSM/J]F2 population extending between and beyond comparative purposes. Whole genome scan was performed in 150 [C57BL/ the extremes of parental values suggested a complex genetic con- 6JxSM/J]F2 male progeny using 134 informative markers, and using trol. Although CFU counts in the male F2 population appeared to kidney fungal load (regressed to gender-specific mean) as the phenotypic follow a Gaussian distribution, D’Agostino and Pearson omnibus trait. (B) Genome-wide LOD score traces identify a significant linkage normality test revealed that fungal distribution (log10CFU) in peak on chromosome 15 (LOD = 3.93). The addition of 204 female and 40 neither male nor female F2 animals passed the normality test male [C57BL/6JxSM/J]F2 mice genotyped for eight markers underlying this locus augmented the significance of the chromosome 15 QTL (solid (p =0.0016andp , 0.0001, respectively). The log10CFU were thus regressed to an experiment-specific and gender-specific mean, line; LOD = 6.09). The horizontal line designating genome-wide threshold yielding the “corrected log CFU” approaching normal distribution for the entire F2 population (LOD = 3.59; p = 0.05) was calculated by 10 performing 1000 permutations. (C) Minimal Bayesian interval for Carg5 is and amenable for linkage analysis. The [C57BL/6JxSM/J]F2 male located between 97.8 and 103.1 Mb. (D) Effect of allele combination at population was genotyped for 134 informative SNPs, and micro- marker D15Mit77 on kidney fungal loads (regressed to gender-specific satellite markers dispersed evenly across the entire genome. Link- mean). ***p , 0.0001 when comparing B versus S, and p 5 0.0001 when age analysis in R/qtl was performed with Haley-Knott regression comparing H to S. B, homozygote C57BL/6J; H, heterozygote; S, homo- using corrected log10CFU (Fig. 2B) or a non-parametric method zygote SM/J. using crude log10CFU (data not shown). Both analyses identified a highly significant locus on distal chromosome 15 (Fig. 2B, dashed line; logarithm of odds [LOD] = 3.93) that regulates fungal load in higher kidney fungal load, whereas homozygosity or heterozygosity the kidney, and that was given the temporary designation Carg5 for C57BL/6J alleles resulted in reduced fungal load (Fig. 2D, p , (C. albicans resistance gene 5). Studies in additional F2 progeny 0.0001). These results identify Carg5 as a novel locus that regulates (204 female, 40 male) validated the effect of Carg5 (peak LOD = C. albicans proliferation in the SM/J mouse strain in a C5- 6.09; Fig. 2B, solid line), and delineated a minimal Bayesian in- independent manner, with susceptibility alleles being inherited in terval (95% confidence limit) situated between 97.8 and 103.1 Mb a recessive fashion. (Fig. 2C). It was estimated that Carg5 explains 11.3% of the phenotypic variance, suggesting additional genetic regulators of the Neutrophil recruitment and function in SM/J host response to C. albicans infection in the two parental strains. A Early engagement of the myeloid compartment, including mobili- pairwise scan, however, failed to detect any significant interacting zation of neutrophils to the site of infection, is required for protection or additive loci. Segregation analysis indicated that homozygosity against C. albicans (44, 45). Hence, flow cytometry was used to for SM/J alleles at the peak marker D15Mit77 is associated with monitor blood and BM populations of CD11b+Ly6G+ neutrophils 1294 MYELOID DEFECT IN C. ALBICANS–SUSCEPTIBLE SM/J MICE

48 h after infection with C. albicans, using the gating strategy de- around higher fungal counts ($log104.25). Conversely, F2 mice scribed in Supplemental Fig. 2. Compared with resistant C57BL/6J exhibiting higher levels of Ly6G (MFI . 1) generally display controls, SM/J mice displayed lower numbers of granulocytes lower fungal burden (.log104.25); however, this hypothesis could (expressed as fraction of CD45+ cells) in BM at steady state and not be validated with Fisher exact test (p = 0.2443). These results after C. albicans challenge (Supplemental Fig. 3A, right panel; p = show that Carg5 modulates Ly6G expression on neutrophils and 0.0022 and p , 0.0001, respectively). Nevertheless, infection in- fungal replication in the kidney, suggesting a possible role of duced a strong recruitment of CD11b+Ly6G+ neutrophils in the Ly6G in neutrophil antifungal effector function. blood and spleen of both strains (Supplemental Fig. 3A). Interest- Ly6G is a small, GPI-linked protein expressed specifically on the ingly, flow cytometric analysis indicated that blood and BM neu- surface of neutrophils (46, 47). Although its functional role in trophils from SM/J expressed lower levels of Ly6G compared with these cells is still debated, its expression is regulated during their C57BL/6J counterparts (Fig. 3A). Indeed, quantification of maturation, with the highest expression detected in the most ma- Ly6G fluorescence indicates an ∼50% reduction in mean fluores- ture cells that bear segmented nuclei and have lost colony-forming cence intensity (MFI) of SM/J neutrophils compared with C57BL/ potential (48). Examination of neutrophil cytospins from SM/J and 6J in blood and BM (Fig. 3B, p , 0.0001); this difference in MFI C57BL/6J (Fig. 4A) did not detect major morphologic anomalies, was noted at steady state and following infection (data not shown). suggesting that reduced expression of Ly6G is not associated with We next investigated the relationship between Ly6G expression an obvious block in maturation of these cells. In addition to the level (MFI) on SM/J and C57BL/6J neutrophils and Carg5-regu- alleged role for Ly6G in granulocyte maturation, Wang et al. (49) lated susceptibility to C. albicans in vivo. For this investigation, reported that transient Ab ligation of Ly6G attenuates neutrophil a cohort of [C57BL/6JxSM/J]F2 mice was infected with C. albi- migration to sites of inflammation. We did not, however, detect Downloaded from cans; blood was collected 48 h later for cellular immunopheno- differences in the proportions of neutrophils recruited (% of typing, and kidneys were harvested for fungal load determination. CD45+ cells) to C. albicans–infected kidneys from SM/J versus We assessed the effect of homozygosity for SM/J (S) or C57BL/6J C57BL/6J mice (Supplemental Fig. 3B). Hence, we examined (B) alleles or heterozygosity (H) at Carg5 on blood cellular functional aspects of neutrophils isolated from SM/J and C57BL/ immunophenotypes (Ly6G MFI calculated for individual mice) 6J. The importance of ROS production by neutrophils is evidenced

and on fungal replication, exploring possible correlations between by increased susceptibility to fungal infections in individuals di- http://www.jimmunol.org/ the two. No effect of Carg5 alleles was detected for the level of agnosed with CGD, which is caused by reduced or absent gen- blood neutrophils in infected F2 mice (Fig. 3C). However, there eration of ROS (21, 50). Neutrophils were purified from BM of was a significant effect of Carg5 alleles on neutrophil Ly6G MFI, SM/J and C57BL/6J mice and production of ROS in response to with Carg5S homozygotes showing significantly less expression stimulation with fMLF (Fig. 4B) or PMA (Fig. 4C) was assessed than Carg5B6 homozygotes or heterozygotes (Fig. 3D; p = 0.0008 by chemiluminescence. SM/J neutrophils showed a maximal ROS and p = 0.0001, respectively). In addition, linear regression burst in response to fMLF that was lower than that of C57BL/6J analysis of Ly6G MFI versus kidney fungal load in individual F2 neutrophils (8.499 3 103 relative luminescence units [RLU] versus mice identified a modest but significant correlation between the 1.337 3 104 RLU). Similarly, reduced total ROS (area under the two phenotypes (Fig. 3E; R2 = 0.1482; p = 0.0075). The distri- curve) in SM/J neutrophils was also noted after stimulation with by guest on September 25, 2021 bution of susceptible versus resistant genotypes (Fig. 3E) may also PMA (3.878 3 106 RLU versus 8.384 3 106 RLU). In both be indicative of a threshold effect. That is, the dot cloud of F2 instances, the initial burst kinetics were identical, but the peak animals that possess lower levels of Ly6G (MFI , 1) clusters amount of ROS produced was significantly reduced in SM/J. These

FIGURE 3. Carg5 regulation of Ly6G expression on neutrophils of SM/J mice. Single-cell suspensions from blood and BM of C57BL/6J and SM/J male mice 48 h after C. albicans infection were stained for CD45, CD11b, and Ly6G and analyzed by flow cytometry. (A) Representative contour plots showing neutrophils (boxed), defined as CD11b+Ly6G+, are provided for both C57BL/6J (B6) and SM/J strains. (B) Gating on neutrophils, Ly6G intensity profiles were constructed for C57BL/6J (B6, gray) and SM/J (dashed line). The Ly6G MFI of SM/J cells quantified relative to the C57BL/6J strain and illustrates a reduced expression of the Ly6G Ag in the SM/J strain. (C) In [C57BL/6JxSM/ J]F2 mice of both sexes, Carg5 alleles (marker D15Mit77) have no effect on the number of circulating Ly6G+ cells in blood, (D) but they have a strong effect on Ly6G MFI (p =0.0008).(E) In these F2 progeny, linear regression analysis identifies a significant correlation between Ly6G MFI and kidney fungal load (R2 = 0.1482; p = 0.0075); the 95% confidence interval is identified. B = C57BL/6J, black circles; H = heterozygote, gray circles; S = SM/J, white circles. ***p , 0.001. The Journal of Immunology 1295

Inflammatory monocyte recruitment and function in SM/J CD11b+Ly6G-Ly6Chi inflammatory monocytes are essential for protection against systemic candidiasis (27). This population was investigated in naive and C. albicans–infected SM/J and C57BL/ 6J mice (Fig. 5A). While no significant interstrain difference in the levels of inflammatory monocytes could be discerned at steady state (Fig. 5A), their numbers were severely reduced in blood 48 h after C. albicans challenge (Fig. 5A, left panel; p , 0.0001) and BM (Fig. 5A, right panel; p = 0.0044) of SM/J mice compared with C57BL/6J, with a similar trend in the spleen (albeit not reaching statistical significance; Fig. 5A, middle panel; p = 0.0606). Upon exposure to microbial products or proinflammatory factors, Ly6Chi monocytes can migrate to tissues and differentiate into professional phagocytes (51). Hence, interstrain differences in functional properties of BMDM from SM/J and C57BL/6J macrophages were investigated. Phagocytosis of C. albicans was measured using a GFP-expressing C. albicans CAI4 strain (Fig. 5B, 5C), and an anti–C. albicans Ab which allows discrimination of intracellular and extracellular yeast cells and permits calculation of Downloaded from a phagocytic index (fraction of macrophages with at least one internalized yeast cell). SM/J and C57BL/6J macrophages phagocy- tosed C. albicans with similar avidity (Fig. 5C). However, C57BL/ FIGURE 4. SM/J neutrophils display diminished production of ROS. 6J macrophages displayed a modest but significantly enhanced A ( ) Cystospin images of neutrophils from male SM/J and C57BL/6J (B6) fungicidal activity compared with their SM/J counterpart (measured show similar morphology (segmented nuclei). Production of reactive ox- asyeastmetabolicactivity2hafterincubation)(Fig.5D;p = http://www.jimmunol.org/ ygen radicals measured by luminescence and following stimulation with 0.0233). As such, the capacity of IFN-g–primed macrophages to 3mM fMLF (B) or 100 nM PMA (C) by SM/J (white circles) and C57BL/6J (B6) neutrophils (black circles). Gray hatching identifies confidence limit produce ROS in response to PMA was investigated (Fig. 5E). calculated from quadruplicate technical replicates and two to three mice Strikingly, peak and total (area under the curve) ROS production by per strain. The results are representative of two independent experiments. SM/J macrophages was significantly reduced by a factor of ∼3-fold compared with C57BL/6J, suggesting that SM/J macrophages have diminished antimicrobial function. Together, these results point to results demonstrate reduced ROS production by neutrophils from a myeloid defect in SM/J mice, expressed as inadequate mobiliza- C. albicans–susceptible SM/J compared with their C. albicans–resis- tion of inflammatory monocytes and weakened antimicrobial by guest on September 25, 2021 tant C57BL/6J counterpart. defenses (oxidative burst) of macrophages.

FIGURE 5. Mononuclear phagocyte numbers and function in response to C. albicans infection in SM/J. (A) Forty-eight hours after C. abicans infection, single- cell suspensions were prepared from blood, spleen, and BM, stained for CD45, CD11b, Ly6C and Ly6G, and analyzed with flow cytometry. Proportions of inflammatory monocytes, defined as CD11b+Ly6G2Ly6Chi, are shown. SM/J mice display a reduced reservoir of inflammatory monocytes in the BM (p = 0.0044) and in blood (p , 0.0001). **p 5 0.0044, ***p , 0.0001. (B) Phagocytosis of GFP-CAI4 yeast cells by BM derived macrophages from male SM/J and C57BL/6J (B6) mice; green yeast cells are intracellular, whereas or- ange-red yeast cells are extracellular (see Materials and Methods). (C) Phagocytic index was calculated from the number of macrophages having internalized at least one yeast cell. (D) Intracellular viability and growth of yeast cells was determined by FUN1 stain (original magnification 363) after 2 h of incubation at an MOI of 10, which showed significant C. albicans fungal growth in SM/J macrophages (p = 0.0233). *p 5 0.0233. (E) Generation of ROS upon PMA stimulation was significantly decreased in SM/J macrophages (white circles) compared with C57BL/6J (black circles). 1296 MYELOID DEFECT IN C. ALBICANS–SUSCEPTIBLE SM/J MICE

Carg5-regulated serum Ccl2 levels predict renal fungal burden on antifungal defenses. For this, we computed a “myeloid inflam- in C. albicans–infected mice matory score” for each gene in the Carg5 interval (Supplemental During systemic C. albicans infection, rapid recruitment of Table I, Fig. 6D). Using chromatin immunoprecipitation and DNA CD11b+Ly6G2Ly6Chi inflammatory monocytes to the kidney sequencing (ChIP-seq), we identified and quantified the intensity of occurs within 48 h and is required for protection in this organ (26, binding peaks for the myeloid transcription factors IRF1, IRF8, 27). Flow cytometric analysis of renal infiltrating leukocytes in STAT1, and NF-kB (p65) in the vicinity of all the genes in the SM/J and C57BL/6J mice demonstrated reduced accumulation interval, and detected following treatment of macrophages with of inflammatory monocytes in SM/J compared with C57BL/6J IFN-g (IRF8, IRF1, STAT1) or LPS (NF-kB p65). The number of (Fig. 6A; p = 0.0008). Because CCL2 (MCP-1) is a critical peaks, the peak height and proximity of the peaks to the tran- proinflammatory myeloid chemokine that is required for inflam- scription start site were used to give each gene a ChIP-seq score. matory monocyte egress from BM and into affected tissues (52), RNA-seq was used to establish baseline and IFN-g–responsive we investigated a possible effect of Carg5 alleles on CCL2 serum mRNA expression in myeloid cells and to produce an expression levels (measured 48 h after infection) in a subset of [C57BL/ score for each gene. The ChIP-seq score and expression scores were 6JxSM/J]F2 mice (Fig. 6B). Interestingly, F2 animals bearing tabulated to produce a myeloid inflammatory score for each gene in the SM/J allele at Carg5 showed higher levels of serum CCL2 the interval (Fig. 6D, Supplemental Table I). This analysis allowed than those homozygous or heterozygous for C57BL/6J alleles did us to generate a list of the 10 most significant positional candidates (Fig. 6B; p = 0.0018). This result suggests that reduced or delayed (Fig. 6D). These candidates include Rnd1, Tuba1b/Tuba1c, Fmnl3, leukocyte recruitment into infected kidneys occurs in the context Tmbim6, Atf1, Nr4a1, Itgb7, Itga5,andNckap1l. of robust CCL2 serum production in SM/J mice. Furthermore, we Downloaded from observed a highly significant positive correlation (Fig. 6C; R2 = Discussion 0.4957; p , 0.0001) between serum CCL2 levels and kidney We have used genetic analysis in the mouse model of systemic in- fungal load, with higher levels of CCL2 being associated with fection with C. albicans to understand the early host defense higher fungal load in [C57BL/6JxSM/J]F2 mice segregating mechanisms that are required for protection against pathogenic Carg5 alleles. Nevertheless, we cannot exclude the possibility that fungi. Earlier studies in inbred mouse strains have documented the heightened circulating CCL2 levels occur as a consequence of critical role of the C5 complement component, whose deficiency in http://www.jimmunol.org/ higher CFU in susceptible [C57BL/6JxSM/J]F2 mice. the form of a naturally occurring 2-bp mutation (28), causes strong susceptibility to C. albicans infection (fungal proliferation, overall Candidate gene analysis of the Carg5 interval survival) (10, 14, 16, 30, 53). In addition, we and others have Lastly, we scrutinized the Carg5 interval for the presence of can- mapped Carg loci, namely Carg1 through Carg4, that additionally didate genes that could play a role in host defenses to infection in regulate susceptibility to C. albicans infectioninaC5-independent general, and in myeloid cell function in particular. The Carg5 fashion (29, 32, 33). We characterized the genetic component and minimal physical interval (97–103.4 Mb) is highly gene rich and the mechanistic basis of unique susceptibility to C. albicans in the contains .200 genes, including clusters of keratin and olfactory “phenodeviant” SM/J strain that is C5 sufficient and expected to be receptor gene families. Considering the noted effect of Carg5 alleles resistant to infection. Susceptibility in SM/J is expressed as increased by guest on September 25, 2021 on the function of the myeloid compartment, we inspected the in- fungal replication in target organs (Fig. 1, Supplemental Fig. 1), with terval for “myeloid” genes involved in early inflammatory response a noticeable gender bias, where male mice showed increased sus- that could be prioritized as morbid candidates for the Carg5 effect ceptibility compared with females (54–56). Subsequent linkage

FIGURE 6. Carg5 regulation of serum CCL2 levels and correlation with kidney fungal load. (A) Two days after infection, kidney infiltrating leukocytes were analyzed by flow cytometry, showing reduced recruitment of inflammatory monocytes in SM/J (***p = 0.0008). (B)In [C57BL/6JxSM/J]F2 progeny of both genders, Carg5 alleles (peak marker D15Mit77; **p = 0.0018) have a strong influence on CCL2 serum levels. (C) Linear regression analysis illustrates a highly significant positive correlation between serum CCL2 levels and renal fungal burden (R2 = 0.4957; p , 0.0001) in the F2 progeny. (D)Two hundred thirty-one genes mapping to the minimal Carg5 genetic interval were given a myeloid inflammatory score computed based on the inte- gration of ChIP-Seq and RNA-Seq data, as described in Materials and Methods.The10 highest scoring genes are identified. B, C57BL/ 6J, black circles; H, heterozygote, gray circles; S, SM/J, white circles. The Journal of Immunology 1297 mapping in [C57BL/6JxSM/J]F2 progeny mapped a novel locus on with higher fungal loads (Fig. 6C). Similarly, the hyper-susceptible distal chromosome 15 that we term Carg5 (cumulative peak LOD of A/J strain exhibits elevated levels of CCL2 following infection with 6.09; Fig. 2), and whose alleles regulate fungal replication in the C. albicans (16, 67), prompting us to postulate that the effect of kidney. The Carg5 locus was not detected in our previous haplotype Carg5 on CCL2 serum levels, albeit highly correlated with fungal association (EMMA analysis) studies in 23 inbred mouse strains load, may be secondary and adaptive to a primary myeloid defect. (29), implying that the Carg5 effect is unique to the SM/J strain. Whether SM/J mice possess impaired emergency myelopoiesis Comparative immunophenotyping of the innate immune com- remains to be ascertained, and it will entail challenging this strain partment, and antimicrobial functional analysis of immune cells with other pathogens. Thus far, SM/J mice have been deemed from SM/J and C57BL/6J mice and from [C57BL/6JxSM/J]F2, susceptible to blood-stage malaria caused by Plasmodium chabaudi demonstrate that Carg5 alleles have a pleiotropic effect on sev- (68), and they are considered a non-healer strain when the ear eral aspects of myeloid cell function. Neutrophils play a key role pinnae are wounded (69). The wound-healing trait is controlled by in defenses against fungal infections (17, 44). Compared with their multiple loci, including a locus Heal4 mapping to distal chromo- C57BL/6J counterpart, SM/J mice show lower proportions of neu- some 15 and overlapping with Carg5. Taken together, our findings trophils in the BM, and mature neutrophils produced in SM/J show strongly suggest that susceptibility of SM/J mice to C. albicans a lower level of surface expression of the Ly6G Ag (Fig. 3A, 3B). infection is associated with a weakened myeloid compartment in Studies in [C57BL/6JxSM/J]F2 mice demonstrate that Carg5 this strain, affecting both neutrophils and inflammatory monocytes, alleles are associated with expression of Ly6G (Fig. 3D) and es- with the mapped Carg5 locus contributing significantly to several tablish a correlation between the level of Ly6G expression and aspects of this myeloid phenotype. replication of C. albicans in kidneys, with the major effect con- Because Carg5 affects the myeloid compartment of SM/J mice, Downloaded from tributed by homozygosity for SM/J alleles in these mice (Fig. 3E). we scrutinized the minimal confidence interval (97–103.8 Mb) for Hence, it is tempting to speculate that Carg5 alleles have an effect putative positional candidates underlying these effects. Initial rec- on functional maturation of Ly6G+ neutrophils in SM/J mice. ognition of Candida yeast and filamentous forms occurs through Interestingly, the effect of Carg5 on neutrophil Ly6G expression pathogen-associated molecular patterns and corresponding pattern does not appear to affect recruitment of these cells to different recognition receptors, such as C-type lectin receptors and TLRs,

sites of infection in SM/J mice (Supplemental Fig. 3). Rather, we whose synergistic interaction can result in augmented downstream http://www.jimmunol.org/ found that SM/J neutrophils produced less ROS upon stimulation immune activation via NF-kB (70, 71). In addition, the protective with fMLF or PMA (Fig. 4B, 4C), both with respect to peak and role for the IFN-g axis in C. albicans infection has been demon- total production. Decreased ROS production by neutrophils is strated by increased susceptibility of mice deficient for IFN-g (72) expected to provide a permissive environment for fungal survival or IFN-g receptor (73). Signaling by IFN-g activates the JAK-STAT and replication in infected organs. Indeed, patients suffering from pathway and results in binding of IFN regulatory factors, IRF1 and CGD caused by mutations in components of the ROS generating IRF8, as well as STAT1 to IFN-g activation site elements and IFN- NADPH enzyme complex are susceptible to fungal infections (21, stimulated response elements, thereby orchestrating and controlling 50). Moreover, Ly6G directly associates with b2-integrins (49); many facets of innate and adaptive immune responses (74). We therefore, it is possible that reduced Ly6G expression on SM/J therefore used data from chromatin immunoprecipitation sequenc- by guest on September 25, 2021 neutrophils leads to decreased interaction with CD11a/CD18 ing for IFN-g–responsive IRF8, IRF1, and STAT1 and LPS- (LFA-1), further contributing to diminished ROS generation (57). responsive NF-kB (p65) DNA-binding, together with RNA-seq Finally, neutrophils release granule proteins, including LL-37, expression profiling before and after IFN-g treatment, to compute azurocidin, and cathepsin G (58, 59), that are important for a myeloid inflammatory score for each gene in the Carg5 interval. further recruitment of ROS-producing inflammatory (Ly6Chi) Among the 10 most significant positional candidates (Fig. 6E, monocytes (60) to the site of infectious or inflammatory insults. Supplemental Table I), Tmbim6, which encodes the anti-apo- In mice, two monocyte subsets can be distinguished by chemo- ptotic BAX inhibitor-1 (BI-1), ranked first. BI-1 was identified kine receptors and adhesion molecule expression. Inflammatory in a functional screen in yeast searching for suppressors of monocytes express Ly6ChiCCR2+ and represent counterparts to BAX-induced apoptosis (75). BI-1 was shown to regulate ER + 2 lo + human CD14 CD16 , whereas mouse Ly6C CX3CR1 resident stress-associated reactive oxygen species negatively in a dose- monocytes correlate with human nonclassical CD14+CD16+ dependent manner, thereby protecting cells from apoptosis (76, monocytes (61, 62). In mice, both subsets play important roles in 77). Cells from mice that are deficient in BI-1 exhibit hyper- the host defense against systemic fungal infection (27, 63), with sensitivity to apoptosis induced by ER stress (78); conversely, inflammatory monocytes conferring protective antifungal activity BAX-deficient neutrophils demonstrated delayed apoptosis (79). during the first 48 h following infection (27). The protective role Therefore, it is tempting to speculate that SM/J neutrophils ex- of inflammatory monocytes and their derivatives is further dem- press higher levels of BI-1 than C57BL/6J, which results in lower onstrated in the pulmonary infection with Cryptococcus neofor- ER stress-induced ROS production and decreased apoptosis. In mans (64) and Aspergillus fumigatus (65), where CCR22/2 and turn, neutrophil apoptosis during infection is essential to convey diphtheria toxin–treated CCR2-DTR mice, respectively, exhibit “eat-me” signals that attract inflammatory monocytes to the site of increased fungal growth, decreased survival, or both. We show inflammation (80). BI-1 also plays a role in actin polymerization that the myeloid defect in the SM/J strain is also expressed as an and cell adhesion (81), mechanisms that are tightly regulated altered number and function of inflammatory monocytes, includ- during leukocyte egress and extravasation (82). The second most ing reduced or delayed recruitment of these cells to the site of significant gene product, ITGA5 (a5), is the a subunit of the fi- infection, and diminished production of ROS in BMDM from these bronectin receptor (a5b1) expressed on various immune cells, mice (Figs. 5, 6). CCL2 is the main ligand for the CCR2 receptor mediating binding to fibronectin and extravasation toward infec- involved in inflammatory monocyte mobilization and recruitment tious stimulus (83–85). Blockade of a5b1 resulted in attenuated (52, 66). In [C57BL/6JxSM/J]F2 mice, we observed a strong effect zymosan-induced recruitment of neutrophils and monocytes to the of Carg5 alleles on the level of serum CCL2, and we detected peritoneum, by reducing cell adhesion and transmigration (86). In a strong correlation between CCL2 serum levels and kidney fungal addition, activation of a5b 1 in human PMNs suppressed b-glu- replication, with higher CCL2 levels (in Carg5S mice) associated can–mediated superoxide production, which could be reverted by 1298 MYELOID DEFECT IN C. ALBICANS–SUSCEPTIBLE SM/J MICE blocking a5b1 (87). We observed that SM/J BMDM displayed features of Candidiasis in patients with inherited interleukin 12 receptor b1 deficiency. Clin. Infect. 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