Iris Chen

Program: Molecular Microbiology and Microbial Pathogenesis

Current advisor: Meng Wu, PhD

Undergraduate university: China Pharmaceutical University, 2020

Enrollment year: 2023

Research summary
Studies complement C3’s role in vaginal Candida infections, examining fungal clearance, morphogenesis, neutrophil recruitment, and tissue damage using wild-type vs C3 knockout mice to determine if complement activation aids defense or drives immunopa

Aim 2: Characterize the role of local complement in vaginal antifungal immunity against Candida albicans
Rationale: Vulvovaginal candidiasis (VVC) represents a unique immunopathological disease affecting 75% of women globally, with 8% experiencing recurrent infections that significantly impact quality of life and healthcare costs1. Unlike other candidal infections that primarily affect immunocompromised individuals, VVC occurs in healthy, immunocompetent women, suggesting distinct pathogenic mechanisms that remain incompletely understood. The disease is characterized by a paradoxical immune response where robust inflammation and neutrophil recruitment correlate with symptom severity rather than fungal clearance, indicating that host immune activation contributes to pathology rather than protection2-4.
Recent mechanistic insights have identified candidalysin, a cytolytic peptide toxin secreted by hyphal forms of Candida albicans, as a key driver of VVC immunopathology through direct epithelial damage and NLRP3 inflammasome activation5-8. This discovery has fundamentally altered understanding of VVC pathogenesis, revealing that the yeast-to-hypha transition and subsequent candidalysin production drive inflammatory cascades that correlate with symptoms9. This discovery has fundamentally altered understanding of VVC pathogenesis, shifting focus from simple host defense failure to recognizing immune activation as a contributor to disease pathology.
The complement system represents a critical component of innate immunity that bridges pathogen recognition with effector mechanisms through opsonization, anaphylatoxin production, and membrane attack complex formation. In VVC, complement activation occurs through multiple pathways, with C3 serving as the central convergence point generating C3a for neutrophil recruitment and C3b for opsonization10-13. However, the functional consequences of complement activation in the vaginal environment remain undefined, particularly given the unique microenvironmental factors that distinguish vaginal immunity from systemic or other mucosal sites. Estrogen-dependent immunomodulation significantly affects complement regulation, with recent evidence demonstrating that estrogen promotes innate immune evasion of C. albicans through inactivation of the alternative complement system14. Additionally, the acidic vaginal pH in humans influences both complement protein stability and fungal virulence factor expression, creating complex interactions between host defense mechanisms and pathogen adaptation strategies15,16.
C. albicans has evolved sophisticated complement evasion mechanisms that may be particularly relevant in the persistent colonization characteristic of VVC. The fungus binds factor H through multiple surface proteins including Gpm117, Pra118, Hgt119, and Gpd220, recruiting negative regulatory proteins to its surface and locally downregulating complement activation10. These evasion strategies, combined with the secretion of complement-degrading proteases such as secreted aspartyl proteinases, suggest that complement dysfunction rather than simple complement deficiency may contribute to VVC pathogenesis21,22.
The massive neutrophil infiltration observed in VVC represents another paradox where recruited effector cells fail to clear infection and may contribute to tissue damage4,23. Recent work has identified neutrophil anergy in the vaginal environment, potentially mediated by heparan sulfate inhibition of Mac1-mediated fungal recognition24,25. Understanding how complement fragments, particularly C5a, affect neutrophil recruitment versus function, and whether complement activation amplifies neutrophil-mediated immunopathology, is essential for comprehending VVC disease mechanisms13,26.
The temporal relationship between complement activation and candidalysin-mediated tissue damage represents a critical knowledge gap with therapeutic implications. Complement fragments may modulate epithelial cell activation and inflammatory mediator production, potentially amplifying tissue damage initiated by candidalysin. Alternatively, complement activation may occur secondary to tissue damage, representing a consequence rather than cause of immunopathology. Early complement activation may provide initial antifungal defense through opsonization and neutrophil recruitment, but persistent activation in the context of fungal evasion mechanisms and candidalysin-mediated damage may transition to predominantly harmful effects. This biphasic model would explain the apparent contradiction between complement’s established antimicrobial functions and the immunopathological nature of VVC.
Characterizing complement’s role in VVC has significant translational implications. If complement activation contributes primarily to immunopathology, targeted complement inhibition could represent a novel therapeutic approach. Conversely, if complement provides essential antifungal defense that is subverted by fungal evasion mechanisms, strategies to enhance complement function might prove beneficial. The proposed research addresses these critical knowledge gaps by systematically characterizing complement C3’s role in fungal clearance, morphogenesis, inflammatory responses, and tissue damage during experimental VVC, with the goal of informing evidence-based therapeutic strategies for this prevalent disease.
Aim 2.1: Role of complement C3 in fungal clearance and morphogenesis during murine VVC
We will establish the murine VVC model by intravaginally inoculating estrogen-pretreated C57BL/6 wild-type (WT) and C3 knockout (C3KO) female mice with Candida albicans SC5314 (1 x 107 CFU), alongside PBS-treated sham controls. To capture acute complement responses before extensive hyphal formation, vaginal lavage collection will occur at days 1, 3, 5, and 7 post-infection. Fungal burden will be assessed through CFU enumeration and morphological analysis by hyphal scoring under microscopy. Beyond simple hyphal scoring, we will implement quantitative measures of candidalysin expression through RT-PCR to determine whether C3 deficiency affects this key virulence factor.
Expected Outcomes: C3KO mice will likely exhibit increased fungal burden due to impaired opsonization and reduced neutrophil recruitment. Without C3’s pathogen-tagging and immune cell recruitment functions, initial fungal clearance will be significantly compromised. Longitudinal analysis will likely reveal a biphasic infection pattern. First, poor early clearance allows persistent fungal colonization. Second, prolonged infection pressure drives morphological changes as yeasts transition to invasive hyphal forms. Additionally, candidalysin expression may be altered in C3KO mice, potentially revealing how complement proteins influence fungal virulence factor production and tissue invasion dynamics. These findings will illuminate complement’s dual role in both pathogen elimination and immune response coordination.
Potential Challenges and Alternative Approaches: If WT and C3 KO mice show no phenotypic differences, we will first confirm knockout efficacy through C3 protein measurement by ELISA and functional hemolytic assays. If the knockout is validated but no phenotype emerges, alternative strategies include: (1) higher inoculum doses to overwhelm residual immunity, (2) extended infection duration (14 days) to capture chronic responses, (3) alternative C. albicans strains with varying virulence profiles, and (4) earlier sampling timepoints (6-24 hours) to detect acute complement responses. Should these modifications fail to reveal complement involvement, this would suggest either redundant pathway compensation, species-specific differences between human and murine complement systems, or fundamental limitations of the mouse model in recapitulating human VVC pathophysiology. The research would then pivot toward human-relevant systems including primary vaginal epithelial cell cultures, analysis of complement activation in clinical VVC samples, or development of three-dimensional organoid models that better recapitulate human vaginal architecture and the acidic microenvironment critical for C. albicans pathogenesis.
Aim 2.2: Role of complement C3 in host inflammatory response, neutrophil function, and tissue damage during murine VVC
Using the same experimental model, we will assess host immune responses and tissue pathology in WT and C3 KO mice. Neutrophil recruitment will be quantified by flow cytometric analysis of vaginal lavage using neutrophil-specific markers (CD11b+Ly6G+), as neutrophil influx serves as the primary correlate of symptomatic infection. Inflammatory mediators including IL-1beta (the major NLRP3 inflammasome effector) and the alarmin S100A8 will be measured by ELISA, as these represent key immunopathological markers identified in transcriptomic studies3,27. Tissue damage will be evaluated through lactate dehydrogenase (LDH) release as a quantitative marker of epithelial cell death and histopathological scoring of vaginal epithelial integrity and inflammatory infiltrates3. This will determine whether C3 deficiency affects the balance between protective immunity and immunopathological tissue damage, revealing complement’s dual role in antifungal defense versus inflammatory injury.
Expected Outcomes: C3KO mice will likely show reduced neutrophil recruitment and decreased levels of inflammatory markers including IL-1beta and S100A8, potentially revealing complement’s contribution to VVC immunopathology. However, the temporal dynamics may be complex, with early protective effects of complement transitioning to predominantly inflammatory contributions as infection progresses. Epithelial barrier function may be better preserved in C3KO mice if complement contributes to tissue damage, though this could be offset by increased fungal burden.
Potential Challenges and Alternative Approaches: If complement deficiency shows no effect on inflammatory parameters, alternative approaches include: (1) Modified timepoints – earlier sampling (12-24 hours) for acute responses or extended duration (14-21 days) for chronic effects, (2) Detailed neutrophil functional assessment including activation markers (CD66b), degranulation products (myeloperoxidase), and NET formation, and (4) Additional tissue damage markers such as tight junction proteins and advanced histopathological scoring.
If these modifications fail to reveal complement involvement, this would suggest pathway redundancy, species-specific differences, or that complement’s role is limited to early pathogen recognition. Research would then pivot to: (1) Complement receptor-specific knockouts (C3aR, C5aR1), (2) Pharmacological complement inhibition in WT mice, (3) Ex vivo studies with primary human vaginal epithelial cells, or (4) Analysis of complement activation in clinical VVC samples to bridge experimental findings with human disease pathophysiology.
1. Swidsinski A, Guschin A, Tang Q, et al. Vulvovaginal candidiasis: histologic lesions are primarily polymicrobial and invasive and do not contain biofilms. Am J Obstet Gynecol. 2019;220(1):91.e1-91.e8. doi:10.1016/j.ajog.2018.10.023
2. Fidel PL, Barousse M, Espinosa T, et al. An intravaginal live Candida challenge in humans leads to new hypotheses for the immunopathogenesis of vulvovaginal candidiasis. Infect Immun. 2004;72(5):2939-2946. doi:10.1128/IAI.72.5.2939-2946.2004
3. Peters BM, Palmer GE, Nash AK, Lilly EA, Fidel PL, Noverr MC. Fungal morphogenetic pathways are required for the hallmark inflammatory response during Candida albicans vaginitis. Infect Immun. 2014;82(2):532-543. doi:10.1128/IAI.01417-13
4. Black CA, Eyers FM, Russell A, Dunkley ML, Clancy RL, Beagley KW. Acute neutropenia decreases inflammation associated with murine vaginal candidiasis but has no effect on the course of infection. Infect Immun. 1998;66(3):1273-1275. doi:10.1128/IAI.66.3.1273-1275.1998
5. Moyes DL, Wilson D, Richardson JP, et al. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature. 2016;532(7597):64-68. doi:10.1038/nature17625
6. Richardson JP, Willems HME, Moyes DL, et al. Candidalysin Drives Epithelial Signaling, Neutrophil Recruitment, and Immunopathology at the Vaginal Mucosa. Infect Immun. 2018;86(2):e00645-17. doi:10.1128/IAI.00645-17
7. Kasper L, König A, Koenig PA, et al. The fungal peptide toxin Candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. Nat Commun. 2018;9(1):4260. doi:10.1038/s41467-018-06607-1
8. Rogiers O, Frising UC, Kucharíková S, et al. Candidalysin Crucially Contributes to Nlrp3 Inflammasome Activation by Candida albicans Hyphae. mBio. 2019;10(1):e02221-18. doi:10.1128/mBio.02221-18
9. Ho J, Yang X, Nikou SA, et al. Candidalysin activates innate epithelial immune responses via epidermal growth factor receptor. Nat Commun. 2019;10(1):2297. doi:10.1038/s41467-019-09915-2
10. Harpf V, Rambach G, Würzner R, Lass-Flörl C, Speth C. Candida and Complement: New Aspects in an Old Battle. Front Immunol. 2020;11:1471. doi:10.3389/fimmu.2020.01471
11. Kozel TR. Activation of the complement system by pathogenic fungi. Clin Microbiol Rev. 1996;9(1):34-46. doi:10.1128/CMR.9.1.34
12. Cheng SC, Joosten LAB, Kullberg BJ, Netea MG. Interplay between Candida albicans and the mammalian innate host defense. Infect Immun. 2012;80(4):1304-1313. doi:10.1128/IAI.06146-11
13. Hünniger K, Bieber K, Martin R, et al. A second stimulus required for enhanced antifungal activity of human neutrophils in blood is provided by anaphylatoxin C5a. J Immunol. 2015;194(3):1199-1210. doi:10.4049/jimmunol.1401845
14. Kumwenda P, Cottier F, Hendry AC, et al. Estrogen promotes innate immune evasion of Candida albicans through inactivation of the alternative complement system. Cell Rep. 2022;38(1):110183. doi:10.1016/j.celrep.2021.110183
15. Vylkova S, Carman AJ, Danhof HA, Collette JR, Zhou H, Lorenz MC. The fungal pathogen Candida albicans autoinduces hyphal morphogenesis by raising extracellular pH. mBio. 2011;2(3):e00055-00011. doi:10.1128/mBio.00055-11
16. Borg-von Zepelin M, Beggah S, Boggian K, Sanglard D, Monod M. The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol Microbiol. 1998;28(3):543-554. doi:10.1046/j.1365-2958.1998.00815.x
17. Poltermann S, Kunert A, von der Heide M, Eck R, Hartmann A, Zipfel PF. Gpm1p is a factor H-, FHL-1-, and plasminogen-binding surface protein of Candida albicans. J Biol Chem. 2007;282(52):37537-37544. doi:10.1074/jbc.M707280200
18. Luo S, Poltermann S, Kunert A, Rupp S, Zipfel PF. Immune evasion of the human pathogenic yeast Candida albicans: Pra1 is a Factor H, FHL-1 and plasminogen binding surface protein. Mol Immunol. 2009;47(2-3):541-550. doi:10.1016/j.molimm.2009.07.017
19. Lesiak-Markowicz I, Vogl G, Schwarzmüller T, et al. Candida albicans Hgt1p, a multifunctional evasion molecule: complement inhibitor, CR3 analogue, and human immunodeficiency virus-binding molecule. J Infect Dis. 2011;204(5):802-809. doi:10.1093/infdis/jir455
20. Luo S, Hoffmann R, Skerka C, Zipfel PF. Glycerol-3-phosphate dehydrogenase 2 is a novel factor H-, factor H-like protein 1-, and plasminogen-binding surface protein of Candida albicans. J Infect Dis. 2013;207(4):594-603. doi:10.1093/infdis/jis718
21. Gropp K, Schild L, Schindler S, Hube B, Zipfel PF, Skerka C. The yeast Candida albicans evades human complement attack by secretion of aspartic proteases. Mol Immunol. 2009;47(2-3):465-475. doi:10.1016/j.molimm.2009.08.019
22. Luo S, Dasari P, Reiher N, et al. The secreted Candida albicans protein Pra1 disrupts host defense by broadly targeting and blocking complement C3 and C3 activation fragments. Mol Immunol. 2018;93:266-277. doi:10.1016/j.molimm.2017.07.010
23. Peters BM, Palmer GE, Nash AK, Lilly EA, Fidel PL, Noverr MC. Fungal morphogenetic pathways are required for the hallmark inflammatory response during Candida albicans vaginitis. Infect Immun. 2014;82(2):532-543. doi:10.1128/IAI.01417-13
24. Yano J, Noverr MC, Fidel PL. Vaginal Heparan Sulfate Linked to Neutrophil Dysfunction in the Acute Inflammatory Response Associated with Experimental Vulvovaginal Candidiasis. mBio. 2017;8(2):e00211-17. doi:10.1128/mBio.00211-17
25. Yano J, Peters BM, Noverr MC, Fidel PL. Novel Mechanism behind the Immunopathogenesis of Vulvovaginal Candidiasis: Neutrophil Anergy. Infect Immun. 2018;86(3):e00684-17. doi:10.1128/IAI.00684-17
26. Zipfel PF, Skerka C. Complement, Candida, and cytokines: the role of C5a in host response to fungi. Eur J Immunol. 2012;42(4):822-825. doi:10.1002/eji.201242466
27. Bruno VM, Shetty AC, Yano J, Fidel PL, Noverr MC, Peters BM. Transcriptomic analysis of vulvovaginal candidiasis identifies a role for the NLRP3 inflammasome. mBio. 2015;6(2):e00182-15. doi:10.1128/mBio.00182-15

Graduate publications
Krishna Prasad GVR, Grigsby SJ, Erkenswick GA, Portal-Celhay C, Mittal E, Yang G, Fallon SM, Chen F, Klevorn T, Jain N, Li Y, Mitreva M, Martinot AJ, Ernst JD, Philips JA. 2025 Macrophage-T cell interactions promote SLAMF1 expression for enhanced TB defense. Nat Commun, 16(1):6794. PMCID: PMC12287521

Tian X, Zhang L, Qian X, Peng Y, Chen F, Bengtson S, Wang Z, Wu M. 2025 Gut complement system: a new frontier in microbiota-host communication and intestinal homeostasis. J Clin Invest, 135(19):e188349