Defective IL-17- and IL-22-dependent mucosal host response to Candida albicans determines susceptibility to oral candidiasis in mice expressing the HIV-1 transgene

Phenotyping of cervical lymph node (CLN) CD4+ T-cells, harvested ex vivo from Tg mice 7 or 70 days after infection or not with C. albicans, revealed significantly enhanced percentages of Th17, Th1Th17 and Treg subsets but strikingly depleted absolute numbers of Th1, Th2, Th17, Th1Th17 and Treg cell populations compared to non-Tg mice (Figure 1). Furthermore, a significant expansion in absolute numbers of the Th2 subset observed in non-Tg mice 7 days after infection with C. albicans was absent in the Tg mice (Figure 1). Interestingly, mean surface expression of CCR6 by Th17 cells (CD4+ CXCR3+ CCR4+ CCR6+) was not significantly altered (p > 0.05) by HIV-1 transgene expression, indicating that this determinant of Th17 cell migration was preserved in the Tg mice.

To determine if expression of the HIV-1 transgene alters the differentiation of naive CD4+ T-cells into specific subsets, we next assessed expression of signature CD4+ T-cell subset genes and production of cytokines after differentiation of naive splenic cells in vitro. Numbers of Naïve CD4+ T-cells recovered per spleen were sharply diminished in Tg compared to non-Tg mice (1.5±0.2 versus 7.90.4±10⁵, p < 0.0001; N = 11), consistent with previous findings in CLNs of Tg mice [40]. The predicted up- or down-regulation of signature gene expression [43],[44] was found after incubation of naive cells from both non-Tg and Tg mice with cocktails of differentiating cytokines and blocking antibodies specific to the Th1, Th2, Th17 and Treg subsets (Figure 2). However, HIV-1 transgene expression nevertheless altered gene expression profiles of the polarized subsets (Figure 2). Compared to non-transgenic controls, polarized Th17 cells from CD4C/HIV^MutA Tg mice displayed increased expression of Ahr, Il22 and Foxp3, and polarized Treg cells had enhanced expression of Ahr and Il17a (Figure 2), suggesting that expression of the HIV-1 transgene in CD4+ T-cells may produce an intermediate Th17-Treg phenotype under these differentiating conditions. Although Gata3 expression was lower in differentiated Th1 cells from Tg mice (Figure 2), the most relevant finding was that expression of this Th2 signature gene was unaffected by transgene expression in Th2 differentiating conditions (Figure 2). These findings demonstrate that Naïve CD4+ cells from Tg mice maintain the overall capacity to differentiate into specific subsets in vitro. Furthermore, HIV-1 transgene expression did not significantly alter cytokine production in supernatants of cells differentiated or not into specific subsets (p > 0.05) (Figure 3). Production of IFN-γ and IL-17A by naive CD4+ cells from Tg and non-Tg mice increased comparably in response to Th1 and Th17 differentiating conditions (Figure 3). Therefore, using identical numbers of Naïve CD4+ T-cells from Tg and non-Tg mice, in vitro differentiated CD4+ T-cell lineages from Tg mice maintained their capacity to produce the critical cytokines required for a protective adaptive immune response to C. albicans.

Oral burdens of C. albicans were significantly increased (p < 0.05) in Tg compared to non-Tg mice on days 3-17 after inoculation, as reported previously [39], and treatment of the Tg mice with the combination of IL-17 and IL-22 reduced oral burdens on days 5-12 compared to untreated Tg controls (Figure 4A). Nevertheless, on days 3-17 after inoculation, oral burdens of C. albicans in Tg mice treated with the combination of IL-17 and IL-22 remained significantly greater (p < 0.05) than in untreated control non-Tg mice, showing that this cytokine treatment did not fully restore resistance to oral candidiasis. Interestingly, treatment with IL-17 alone only produced a transient reduction (p < 0.05) of oral burdens on day 7 (Figure 4B), while IL-22 alone was without significant effect (p > 0.05) (Figure 4C), showing that IL-17 and IL-22 are both required and non-redundant for mucosal host defense against C. albicans.

Histopathology of tongues of untreated control Tg mice, conducted 7 days after oral inoculation of C. albicans, showed the expected dense hyphal penetration of the epithelium of the entire dorsum of the tongue, accompanied by occasional inflammatory cell infiltrates [39] (Figure 5B1,2). In contrast, in Tg mice treated with the combination of IL-17 and IL-22, the density of Candida hyphae was sharply diminished, and, in most of the epithelium, hyphae were entirely absent (Figure 5A1,2). Compared to untreated Tg controls (Figure 5B1), this cytokine treatment did not induce an additional influx of polymorphonuclear leukocytes (PMNs) or other inflammatory cells to the epithelium of these Tg mice at this time point of infection (Figure 5A1). In the control non-Tg mice, which at day 7 are resolving primary Candida infection (Figure 4A) [39], only one or two small foci of Candida hyphae were found in the keratinized layer of the epithelium of three of the six mice examined, with an underlying epithelial inflammatory infiltrate composed of PMNs (Figure 5C1,2). Uninfected non-Tg and Tg mice showed an absence of Candida hyphae and inflammatory cell response (Additional file 1).

Compared to uninfected controls at day 7, infection of non-Tg mice with C. albicans significantly (p < 0.05) enhanced tongue tissue expression of S100a8, Ccl20, Il17, Il22 and, to a lesser degree, of the Dfb3 gene (Figure 6). In striking contrast, the heightened expression of these genes in response to Candida infection was completely abrogated in untreated control Tg mice, with the exception of Defb3 which showed a modest increase (p < 0.05) comparable to that of the infected non-Tg mice (p > 0.05) (Figure 6). Consistent with the reduced oral burdens of C. albicans, combined treatment with IL-17 and IL-22 restored the ability of the Tg mice to up-regulate expression of S100a8, Ccl20 and Il22 in response to C. albicans infection, to a level not significantly different from infected non-Tg mice (p > 0.05) (Figure 6). Expression of Ccl2 was unaffected by transgene expression, Candida infection or cytokine treatment at this time point after oral inoculation.

The Tg mice expressing Nef, Env, and Rev of HIV-1 in CD4+ T-cells, DCs, and macrophages (CD4C/HIV^MutA Tg mice) have been described elsewhere [52]. CD4C/HIV^MutA mutant DNA harbors mouse CD4 enhancer and human CD4 promoter elements to drive the expression of HIV-1 genes in CD4 + CD8+ and CD4+ thymocytes, in peripheral CD4+ T-cells, and in macrophages and DCs. Founder mouse F21388 was bred on the C3H background. Animals from this line express moderate levels of the transgene, with 50% survival at 3 months [52]. Several HIV-1 genes (Gag, Pol, Vif, Vpr, Tat and Vpu) are mutated in the CD4C/HIV^MutA DNA, whereas Env, Rev and Nef are intact. The generation of CD4C/HIV^MutG mice revealed that selective expression of the Nef gene is required and sufficient to elicit an AIDS-like disease in these Tg mice [52]. This disease is characterized by failure to thrive, wasting, severe atrophy and fibrosis of lymphoid organs, a preferential depletion of CD4+ T-cells, with altered CD4+ T-cell proliferation in vitro, loss of CD4+ T-cell help, CD4+ T-cell and B-cell activation and impaired DC maturation and function [42],[52]-[54],[86],[87]. In addition, disease of the lung (lymphocytic interstitial pneumonitis), heart (myocytolysis, myocarditis), and kidney (segmental glomerulosclerosis, tubulointerstitial nephritis, microcystic dilatation) develop in these Tg mice [52],[88].

Specific-pathogen-free male and female Tg mice and non-Tg littermates were housed in sterilized individual cages equipped with filter hoods, supplied with sterile water, and fed with sterile mouse chow. All animal experiments were approved by the Animal Care Committee of the University of Montreal (protocol 12-088; Additional file 2).

Oral inoculation with C. albicans LAM-1, a clinical isolate, was done as described elsewhere [39],[81]. In brief, mice were inoculated by topical application of 10⁸ pelleted blastoconidia recovered on sterile calcium alginate Calgiswabs (Puritan Medical Products, Guilford, ME). A longitudinal quantification of C. albicans in the oral cavity of individual mice was done from day 1 until euthanizing of the animals. Calgiswabs used for sampling were dissolved in 2 mL of Ringer’s citrate buffer and plated on Sabouraud dextrose agar supplemented with chloramphenicol (0.05 g/L). Plates were incubated for 24 h at 37°C, and data were expressed as oral C. albicans colony forming units recovered.

Single-cell suspensions of CLNs and spleen were prepared as previously described [40]. Cells were surface stained with four fluorochrome-labelled antibodies (CD4-FITC, CXCR3-PERCP-CY5.5, CCR4-APC, CCR6-PE; BioLegend, San Diego, CA) and their respective isotype controls to specifically identify the Th1, Th2, Th1Th17 and Th17 subsets (Additional file 3). Chemokine receptors CCR4, CCR6 and CXCR3 are surface markers for the functionally distinct Th1 (CXCR3 + CCR6-), Th2 (CCR4 + CCR6-), Th17 (CCR4+, CCR6+), and Th1Th17 (CXCR3 + CCR6+) memory CD4+ T-cell subsets [25],[29]. As a positive control for CCR4, CCR6 and CXCR3 identification of these subsets, splenic CD4+ T-cells were enriched to >90% purity by negative selection (Mouse CD4+ T-Cell Enrichment Kit; Stemcell Technologies, Vancouver, BC) and differentiated in vitro into Th1, Th2 and Th17 cells using stimulation with anti-CD3 and anti-CD28 antibodies (eBioscience, San Diego, CA) and combinations of cytokines and anti-cytokine antibodies [89]. Regulatory T-cells were quantitated by staining with anti-mouse anti-CD4, anti-CD25 (both BD Biosciences) and anti-Foxp3 (eBioscience) fluorescence-labeled monoclonal antibodies and their respective isotype controls (Additional file 3). Cell surface marker analysis was conducted on a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest software. Data were acquired for 50,000 events by gating on CD4+ cells.

Single-cell suspensions of spleen were prepared as previously described [40]. Splenic CD4+ T cells were enriched to >90% purity by negative selection (Mouse CD4+ T-Cell Enrichment Kit; Stemcell Technologies, Vancouver, BC) and surface stained with anti-mouse anti-CD4, anti-CD25, anti-CD62L and anti-CD44 fluorescence-labeled monoclonal antibodies and their respective isotype controls (BD Biosciences). Naive CD4+ T-cells (CD4 + CD25- CD62L^hiCD44^lo) were sorted using a FACSVantage SE instrument (BD Biosciences).

To assay the capacity of CD4+ T-cells from Tg mice to differentiate into specific subsets in vitro, 1×10⁵ sorted naive CD4+ T-cells were cultured in 200 l of ISCOVE medium (Wisent) supplemented with 10% fetal bovine serum (Gibco). Cells were activated with anti-CD3 and anti-CD28 (Dynabeads Mouse T-Activator CD3/CD28; Gibco) for 6 days at 37°C and 5% CO₂ in the presence of specific cytokines (eBioscience) and antibodies (BD Biosciences): IL-12, IFN-γ (each 10 ng/ml) and anti-IL-4 (Th1); IL-4 (5 ng/ml) and anti-IFN-γ (Th2); TGF-β (5 ng/ml), IL-6 (20 ng/ml), IL-1β (10 ng/ml), IL-21 (10 ng/ml), IL-23 (10 ng/ml), anti-IFN-γ and anti-IL-4 (Th17); TGF-β (5 ng/ml), IL-2 (10 ng/ml) (Treg). After 6 days of incubation, cells were harvested to assay the expression of the Tbet, Ifng, Tnf, Gata3, Stat6, Il4, Il10, Foxp3, Tgfb, Ahr, Rora, Rorc, Il17a, Il17f, Il21 and Il22 genes. RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol, and qRT-PCR was performed for 40 cycles on a RotorGene 6000 instrument (Qiagen). Ubc, B2m and Atp5b were utilized as reference genes (Primerdesign, UK), and data analysis was done using Qiagen REST 2009 software.

To determine cytokine production by the differentiated cells, culture supernatants were also harvested at day 6 and assayed using the BD cytometric bead array Flex Set (BD Biosciences) according to the manufacturer’s protocol on a FACSCalibur flow cytometer equipped with BD CellQuest software. Data analysis was performed using BD FCAP array software 3.0.

To determine if cytokine treatment can restore resistance to OPC in the Tg mice, Tg and control non-Tg mice were inoculated orally with C. albicans. Beginning at day 1 after inoculation, Tg mice were treated with PBS or 3 g of recombinant IL-17 and/or IL-22 (eBioscience) i.p. every two days for 14 days. Control non-Tg mice were untreated. Oral fungal burdens were determined daily [39] to compare efficacy of treatments.

In separate experiments, Tg and non-Tg mice were inoculated or not with C. albicans, and the inoculated Tg mice were treated or not with the combination of IL-17 and IL-22 on days 1, 3 and 5 post-inoculation. On day 7, the mice were euthanized and the tongues were harvested and bisected longitudinally. qRT-PCR was performed to determine expression of the Defb3, S100a8, Il17, Il22, Ccl2 and Ccl20 genes in tongue tissue, normalized to 18S, or Gapdh and Ubc. Histopathological examination was performed as described [39].

CD4+ T-cell subset populations and cytokine production were analyzed with IBM SPSS Statistics for Windows version 20 software (IBM, Armonk, NY) using analysis of variance. Qiagen REST 2009 software was used to analyze gene expression results in real-time qRT-PCR. Oral burdens of C. albicans were compared using analysis of variance with the Welch correction, followed by the Games-Howell test for multiple comparisons of unequal variances. Differences were considered to be significant at a p value of <0.05.