Expansion of Foxp3+ T-cell populations by Candida albicans enhances both Th17-cell responses and fungal dissemination after intravenous challenge

Research Funding Wellcome Trust, UK. Grant Numbers: 086827, 080088 NIH. Grant Number: DE022550Peer reviewedPublisher PD

Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of Tissue Adaptation.

Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation

Treg/Th17 relationship during candidiasis.

<p>Tregs promote Th17 responses as well as acquire Th17 characteristics during disseminated infection. However, Treg/Th17 responses are associated with pathogenicity in this form of candidiasis. Conversely, Treg enhancement of Th17 responses during OPC is protective. Tregs suppress Th17 responses during gastrointestinal candidiasis, leading to decreased <i>C. albicans</i> colonization. Whether Tregs impact Th17 responses during vaginal and cutaneous candidiasis and the resulting outcome of disease remains to be determined.</p

Human genetic defects associated with susceptibility to <i>Candida</i> infections.

<p>Adapted from references <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004456#ppat.1004456-HernndezSantos1" target="_blank">[1]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004456#ppat.1004456-Huppler1" target="_blank">[2]</a> and Casanova and Puel (personal communication). Abbreviations: APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; APS1, autoimmune polyendocrine syndrome type 1; IgE, immunoglobulin E.</p><p>Human genetic defects associated with susceptibility to <i>Candida</i> infections.</p

The IL-17-induced gene Lipocalin 2 is dispensable for immunity to oral candidiasis

Oropharyngeal candidiasis (OPC, thrush) is an opportunistic fungal infection caused by the commensal microbe Candida albicans. Immunity to OPC is strongly dependent on CD4+ T cells, particularly of the Th17 subset. IL-17-deficiency in mice or humans leads to chronic mucocutaneous candidiasis, but the specific downstream mechanisms of IL-17-mediated host defense remain unclear. Lipocalin-2 (Lcn2, 24p3, NGAL) is an antimicrobial host defense protein produced in response to inflammatory cytokines, particularly IL-17. Lcn2 plays a key role in preventing iron acquisition by bacteria that use catecholate-type siderophores, and lipocalin 2-/- mice are highly susceptible to infection by E. coli and Klebsiella pneumonia. The role of Lcn2 in mediating immunity to fungi is undefined. Accordingly, in this study we evaluated the role of 24p3 in immunity to oral infection with C. albicans. Lcn2 is strongly upregulated following oral infection with C. albicans, and its expression is almost entirely abrogated in mice defective in IL-17 signaling. However, Lcn2-/- mice were completely resistant to OPC, comparable to WT mice. Moreover, Lcn 2-deficiency mediated protection from OPC induced by steroid immunosuppression. Therefore, despite its potent regulation during C. albicans infection, Lcn2 is not required for immunity to mucosal candidiasis

IL-17C^-/- and IL-17RE^-/- mice are resistant to oropharyngeal candidiasis.

<p><b>A.</b> WT and IL-17RA^-/- mice (n = 3 per group) were subjected to OPC and IL-17C transcript levels assessed by qPCR. *p<0.05 with error bars indicating SEM. <b>B.</b> OPC was induced in the indicated mice (IL-17C^-/- Sham, n = 2; IL-17RE^-/- Sham, n = 2; Infected: IL-17C^+/+ n = 9; IL-17RE^+/+, n = 6; IL-17C^-/-, n = 9; IL-17RE^-/-, n = 6; IL-17RA^-/- n = 7). Tongue was harvested on day 5, and CFU enumerated 48 h later by plating serial dilutions on YPD agar. Data presented as the geometric mean of CFU. Each data point represents one mouse, and the graph depicts pooled data from two independent experiments (n = 2 for each sham-infected cohort and n≥6 total for each Candida-infected cohort). *p<0.0001 by Mann Whitney U test. <b>C.</b> Weight loss was assessed daily and is presented as a percentage of starting weight. Error bars indicate SEM. <b>D.</b> Indicated mice (n = 3 per group) were subjected to OPC and qPCR performed for Defb3 and Lcn2 genes on day 2. *p<0.05 by ANOVA and post-hoc Tukey’s test. Error bars indicate SEM.</p

IL-17C^-/- and IL-17RE^-/- mice are resistant to systemic candidiasis.

<p><b>A-C.</b> IL-17RE^-/- and IL-17RE^+/+ littermate controls were subjected to systemic candidiasis by tail vein injection of 2x10⁵ C. albicans strain SC5314 (IL-17RE^+/+ n = 11; IL-17RE^-/-, n = 13; IL-17C^+/+; n = 10; IL-17C^-/- n = 11). Mice were evaluated daily and time to sacrifice is presented. ns., not significant. Data are representative of 2 independent experiments. <b>C-D.</b> IL-17RE^-/- mice (n = 7) and IL-17C^-/- (n = 5) mice were injected with 1x10⁵ cells C. albicans as in panel A. WT littermates (IL-17C^+/+, n = 4 and IL-17RE^+/+, n = 5) and IL-17RA^-/- mice (n = 5) served as controls. ns., not significant. Data are representative of one independent experiment. <b>E.</b> Weight change assessments were made in all cohorts after infection with 2x10⁵ C. albicans cells, and presented as a percentage of starting weight. <b>F.</b> Kidneys were harvested from each cohort on day 2 after systeic infection and qperformed for the indicated genes. *p<0.05 by ANOVA and post-hoc Tukey’s test. <b>G.</b> Indicated tissue types were harvested on day 2 and tissue fungal burdens determined. Each data point represents one mouse. (IL-17RE^+/+ Sham, n = 3; IL-17RE^-/- Sham, n = 2; IL-17RE^+/+ n = 9; IL-17RE^-/- n = 10). Pooled data from two independent experiments is shown.</p

IL-17RE^-/- mice are resistant to cutaneous candidiasis.

<p><b>A.</b> The indicated mice were subjected to dermal candidiasis by subcutaneous injection with C. albicans strain CAF2-1 hyphae (IL-17RE^+/+ Sham, n = 6; Infected: IL-17RC^-/-, n = 5; C57BL/6 WT, n = 4; IL-17RE^+/+, n = 21; IL-17RE^-/-, n = 19). The percent of mice positive for lesions over time is presented. *p<0.01 compared to WT by a Log-rank (Mantel Cox) test. Graph depicts pooled data from two independent experiments. <b>B.</b> Ulceration (left panels) and nodule formation (right panels) are depicted in WT, IL-17RE^-/- and IL-17RC^-/- mice on days 4 and 6. Data are representative of two independent experiments.</p