Persistence of candida albicans in the oral mucosa induces a curbed inflammatory host response that is independent of immunosuppression

Controlled immune activation in response to commensal microbes is critical for the maintenance of stable colonization and prevention of microbial overgrowth on epithelial surfaces. Our understanding of the host mechanisms that regulate bacterial commensalism has increased substantially, however, much less data exist regarding host responses to members of the fungal microbiota on colonized surfaces. Using a murine model of oropharyngeal candidiasis, we have recently shown that differences in immune activation in response to diverse natural isolates of Candida albicans are associated with different outcomes of the host-fungal interaction. Here we applied a genome-wide transcriptomic approach to show that rapid induction of a strong inflammatory response characterized by neutrophil-associated genes upon C. albicans colonization inversely correlated with the ability of the fungus to persist in the oral mucosa. Surprisingly, persistent fungal isolates showed no signs of a compensatory regulatory immune response. By combining RNA-seq data, genetic mouse models, and co-infection experiments, we show that attenuation of the inflammatory response at the onset of infection with a persistent isolate is not a consequence of enhanced immunosuppression. Importantly, depletion of regulatory T cells or deletion of the immunoregulatory cytokine IL-10 did not alter host-protective type 17 immunity nor did it impair fungal survival in the oral mucosa, indicating that persistence of C. albicans in the oral mucosa is not a consequence of suppressed antifungal immunity

Neutrophil-recruiting chemokines in the oral mucosa are reduced in absence of IL-1R signaling.

<p>(<b>A</b>) Chemokine expression in the tongues of naïve (n) and infected (inf) WT mice was measured by qRT-PCR 24 hours post-infection. (<b>B</b>) Chemokine expression in the tongues of naïve and infected WT and infected <i>Il1r1</i>^-/- mice 24 hours post-infection. (<b>C</b>) CD45⁺ leukocytes, CD45^- EpCAM⁺ CD31^- epithelial cells, and CD45^- EpCAM^- CD31⁺ endothelial cells were isolated from the tongues of naïve and infected WT mice by FACS sorting 24 hours post-infection, and <i>Cxcl1</i> mRNA was quantified by qRT-PCR. (<b>D</b>) The tongue-derived keratinocyte (TDK) cell line was treated with recombinant IL-1α (20 ng/ml), anakinra (250 μg/ml), or PBS, and CXCL1 levels in the culture supernatant were determined by ELISA. Bar graphs show the group mean + SD. Data are representative of two (A, C–D) or pooled from two (B) independent experiments, with the exception of the naïve group in B, which is from one experiment. Statistical analysis was performed using log₁₀ transformation and Student’s t-test with Welch’s correction (A) or one-way ANOVA with Dunnett’s test (B, D).</p

Endothelial cell-derived G-CSF in the oral mucosa is IL-1-dependent.

<p>(<b>A</b>) VE-cadherin-cre x ROSA26-RFP mice were infected with <i>C</i>. <i>albicans</i> strain pACT1-GFP or left uninfected. The images show each a 3D reconstruction of a Z stack of a 20x fields of view acquired from whole mount samples of naïve or infected tongues 24 hours post-infection. Fungal hyphae are identified in the avascular tongue epithelium by their morphology and green fluorescence (white arrow). The papillae appear yellow due to both green and red autofluorescence. Muscle cells display green autofluorescence. (<b>B</b>) <i>Csf3</i> mRNA was quantified in the tongues of naïve WT and infected WT and <i>Il1r1</i>^-/- mice by qRT-PCR 24 hours post-infection. (<b>C</b>) CD45⁺ leukocytes, CD45^- EpCAM⁺ CD31^- epithelial cells and CD45^- EpCAM^- CD31⁺ endothelial cells were isolated from the tongues of naïve WT and infected WT and <i>Il1r1</i>^-/- mice by FACS sorting 24 hours post-infection. Cell lysates were prepared from the sorted populations and G-CSF protein was quantified in the lysates by ELISA. The data of the WT mice are identical with those shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005882#ppat.1005882.g004" target="_blank">Fig 4B</a>. (<b>D</b>) G-CSF levels were determined in the serum of naïve (open bars) and infected (closed bars) WT and <i>Il1r1</i>^-/- mice by ELISA 24 hours post-infection. (<b>E–F</b>) Quantification of Ly6G^hi mature neutrophils (E) and Ly6G^lo immature neutrophils (F) in the bone marrow of infected WT and <i>Il1r1</i>^-/- mice analyzed 24 hours post-infection. (<b>G–H</b>) As in (E–F), but mice were treated with recombinant G-CSF or left untreated as indicated. Each symbol represents an individual mouse and the lines represent the mean (E–H) or geometric mean (B) of each group. Bar graphs in C–D show the group mean + SD. Data are pooled from two (B–C, G–H) or representative of two (A, D–F) independent experiments, with the exception of the naive group in B, which is from one experiment. Statistical analysis was performed using log₁₀ transformation (B) and Student’s t-test with Welch’s correction (C, E–F), a one-way ANOVA with Tukey’s test (G—H) or Dunnett’s test (B), or two way ANOVA with Tukey’s test (D).</p

Endothelial cells are the primary source of G-CSF production during OPC.

<p>(<b>A</b>) CD45⁺ leukocytes, CD45^- EpCAM⁺ CD31^- epithelial cells, and CD45^- EpCAM^- CD31⁺ endothelial cells were isolated from the tongues of naïve and infected WT mice by FACS sorting 24 hours post-infection, and <i>Csf3</i> mRNA was quantified by qRT-PCR. (<b>B</b>) Tongue cell populations were sorted from the tongues of naïve and infected WT mice as in (A). Cell lysates were generated from the sorted populations and G-CSF protein was quantified in the lysates by ELISA. (<b>C</b>) G-CSF levels were determined in the serum of naïve (n) and infected (inf) WT mice by ELISA. Data are pooled from two (B) or representative of two (A) or five (C) independent experiments. Bar graphs show the group mean + SD. Statistical analysis was performed using Student’s t-test with Welch’s correction.</p

IL-1R signaling makes an important contribution to neutrophil recruitment to the oral mucosa.

<p>(<b>A</b>) CD45⁺ Ly6C^int Ly6G⁺ neutrophils were quantified by flow cytometry in the tongues of naïve (n) and <i>C</i>. <i>albicans</i> infected (inf) WT mice 24 hours post-infection. (<b>B</b>) Neutrophils were quantified in the tongues of infected WT and <i>Il1r1</i>^-/- mice 24 hours post-infection. (<b>C</b>) The fungal burden in the tongues of infected WT and <i>Il1r1</i>^-/- mice was determined on day 3 post-infection. Each symbol represents an individual mouse, and the lines represent the geometric mean of each group. The dotted line represents the detection limit. Data are pooled from two (B) or representative of three (A) or two (C) independent experiments. Statistical analysis was performed using log₁₀ transformation and Student’s t-test with Welch’s correction.</p

TDKs release IL-1α in response to <i>C</i>. <i>albicans</i>.

<p>(<b>A—B</b>) TDKs were stimulated with <i>C</i>. <i>albicans</i>, zymosan, curdlan or left untreated (PBS), and IL-1α (A) and IL-1β (B) levels were determined in the supernatant by cytometric bead array. (<b>C</b>) IL-1α release from TDKs was determined as in (A) after stimulation with live or heat-killed (HK) <i>C</i>. <i>albicans</i> strain SC5314, with the yeast-locked strain <i>hgc1</i>Δ/Δ, its revertant <i>hgc1</i>Δ/Δ::<i>hgc1</i>, or with preformed hyphae prepared from strain SC5314. (<b>D</b>) TDKs were stimulated with <i>C</i>. <i>albicans</i> or left unstimulated (PBS). Amphotericin B was added after 8 hours of stimulation to prevent hyphal overgrowth. IL-1α levels were determined in the supernatant and in lysates prepared from the cells in the same wells. As a control, triton was added to separate wells containing cells and supernatant to quantify total amounts of IL-1α per well (total). Bar graphs show the group mean + SD. Data are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (A–B) or Tukey’s test (C).</p

Keratinocyte-derived IL-1α regulates G-CSF production during OPC.

<p>(<b>A</b>) <i>Il1a</i> and <i>Il1b</i> mRNA was quantified in the tongues of naïve (n) and infected (inf) WT mice by qRT-PCR 24 hours post-infection. (<b>B–C</b>) WT, <i>Il1a</i>^-/- and <i>Il1b</i>^-/- mice were infected with <i>C</i>. <i>albicans</i>. <i>Csf3</i> mRNA levels in naïve and infected tongue tissue were determined by qRT-PCR (B), and G-CSF serum levels were measured by ELISA (C) before infection (open bars) and 24 hours post-infection (closed bars). (<b>D</b>) Immunofluorescent staining of sagittal tongue sections from naïve and infected WT and <i>Il1a</i>^-/- mice stained for IL-1α (yellow) and DAPI (blue) 24 hours post-infection. (<b>E</b>) Immunofluorescent staining of sagittal tongue sections from infected WT mice stained for keratin-6 (K6, left) or keratin-14 (K14, right) in red, as well as IL-1α (yellow) and DAPI (blue) 24 hours post-infection. Note that the IL-1α signal is absent in neutrophil-rich areas. The white arrow serves as orientation. (<b>F</b>) IL-1α and IL-1β mRNA expression in the tongues of naïve WT and infected WT, <i>Il1a</i>^-/- and <i>Il1b</i>^-/- mice was measured by qRT-PCR 24 hours post-infection. The detection limit, which was calculated using the average Ct (<i>Actb</i>) of all samples and Ct (<i>Il1a</i>) or Ct (<i>Il1b</i>) = 50, is depicted by a dotted line. Each symbol represents an individual mouse (A–B, F) and the lines represent the geometric mean of each group. The bar graph in C shows the group mean + SD. Data are representative of two independent experiments (A, D–E), or pooled from two independent experiments (B–C, F), with the exception of the naïve groups in C, which are the mean + SD of 4 (WT) or 3 (<i>Il1a</i>^-/-, <i>Il1b</i>^-/-) animals from one experiment. Statistical analysis was performed using log₁₀ transformation (A–B, F) and Student’s t-test with Welch’s correction (A), a one-way ANOVA with Dunnett’s test (B, F) or two-way ANOVA with Tukey’s test (C).</p