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 published online 27 June 2014 http://www.jimmunol.org/content/early/2014/06/27/jimmun ol.1400783 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/06/27/jimmunol.140078 Material 3.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published June 27, 2014, doi:10.4049/jimmunol.1400783 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 immu- nophenotyping 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: 000–000. 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 2 MYELOID DEFECT IN C. ALBICANS–SUSCEPTIBLE SM/J MICE 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.