Candida albicans Promotes Oral Cancer via IL-17A/IL-17RA-Macrophage Axis

ABSTRACT The association between Candida albicans (C. albicans) and oral cancer (OC) has been noticed for a long time, but the mechanisms for C. albicans promoting OC are rarely explored. In this study, we determined that C. albicans infection promoted OC incidence in a 4-nitroquinoline 1-oxide (4NQO)-induced mouse tongue carcinogenesis model as well as promoted OC progression in a tongue tumor-bearing mouse model (C3H/HeN-SCC VII). We then demonstrated that tumor-associated macrophage (TAMs) infiltration was elevated during C. albicans infection. Meanwhile, the attracted TAMs polarized into M2-like macrophages with high expression of programmed death ligand 1 (PD-L1) and galectin-9 (GAL-9). Further analysis suggested that the interleukin (IL)-17A/IL-17RA pathway activated in OC cells was a contributor to the excessive TAMs infiltration in C. albicans-infected mice. Thus, we constructed IL-17A neutralization and macrophage depletion experiments in C3H/HeN-SCC VII mice to explore the role of IL-17A/IL-17RA and TAMs in OC development caused by C. albicans infection. The results showed that both IL-17A neutralization and macrophage depletion tended to reduce the TAMs number and tumor size in mice with C. albicans infection. Collectively, our finding revealed that C. albicans promoted OC development via the IL-17A/IL-17RA-macrophage axis, opening perspectives for revealing C. albicans-tumor immune microenvironment links.


RESULTS
C. albicans infection increased oral cancer incidence in 4NQO mice. During the experiment (Fig. 1A), C. albicans infection caused weight loss in the 4NQO mice, which was reversed by the administration of fluconazole (FCZ) (Fig. 1B). In week 16, the incidence of oral tumor in the 4NQO 1 CA group was approximately three times higher than in the 4NQO group (45.5% versus 16.7%, Fig. 1C and Fig. S1A). In week 20, the tumor incidence in the 4NQO 1 CA group reached 100%, which was still higher than the 4NQO group (83.3%) ( Fig. 1D and Fig. S1B). Interestingly, the tumor incidence in the 4NQO 1 CA 1 FCZ group was lower than both the 4NQO 1 CA and 4NQO groups ( Fig. 1D and E and Fig. S1B), which suggested that antifungal treatment effectively reversed the tumor development caused by C. albicans infection. Additionally, the Ki-67 (a proliferation marker) expression of tongue epithelium in the 4NQO 1 CA group was higher than the 4NQO group and the 4NQO 1 CA 1 FCZ group (Fig. 1F), which also means the higher risk of tumorigenesis in mice with C. albicans infection.
Tumor-associated macrophage accumulation was associated with IL-17A/IL-17RA signaling pathway during C. albicans infection. RNA sequencing (RNA-seq) was used to screen the possible mechanisms for C. albicans promoting OC. One hundred twenty-eight differentially expressed genes (DEGs) were filtered between the 4NQO and 4NQO 1 CA group (Fig. S2A), 239 DEGs were found between the 4NQO 1 CA and 4NQO 1 CA 1 FCZ group (Fig. S2B), and only 27 DEGs were found between the 4NQO and 4NQO 1 CA 1 FCZ group (Fig. S2C). The administration of FCZ reversed some of the changed genes in tumors caused by C. albicans infection (Fig. S2D and E).
Among the DEGs, the interleukin (IL)-17 signaling pathway was noticeable from the Kyoto Encyclopedia Genes and Genomes (KEGG) enrichment analysis, which was upregulated in 4NQO 1 CA mice ( Fig. 2A) and downregulated after FCZ treatment (Fig. 2B). Meanwhile, the upregulation of downstream molecules (S100A9, S100A8, MMP3, and CXCL10) of IL-17RA activation in the 4NQO 1 CA mice was confirmed by quantitative real-time PCR (RT-qPCR) ( Fig. 2C and Fig. S2F). To identify the IL-17RA expression cells, immunofluorescence (IF) was performed and found that epithelial cells and tumor cells were the main cells expressing IL-17RA (Fig. 2D). Thus, an in vitro experiment was designed and showed that SCC VII cells expressed more S100A9 when treated with rmIL-17A (Fig. 2E), which confirmed activation of the IL-17A/IL-17RA signaling pathway in oral tumor cells.
It is worth mentioning that our ongoing work found that there was no difference in the IL-17 signaling pathway of the tongue epithelium between long-term (8 month) C. albicans-infected mice and normal mice (Fig. S2G). Additionally, a publicly available RNAseq data set (GSE101469) (24) was used to find that the IL-17 signaling pathway was significantly upregulated in carcinoma (C57BL/6 mice drank 50 mg/L 4NQO water for 28 weeks) compared with normal lingual mucosa (4NQO for week 0) (Fig. S2H), while there was no significant difference between 4NQO for weeks 0 and 12 (hyperplasia and mild and moderate dysplasia) (Fig. S2I). The above results may indicate an activation of the IL-17A/IL-17RA signaling pathway in the tumor microenvironment (TME) during oral cancer development, rather than just the mucosal immune response caused by C. albicans infection.
Furthermore, the tumor immune cell infiltration was estimated from the RNA-seq data set by the use of ImmuCellAI-mouse (http://bioinfo.life.hust.edu.cn/ImmuCellAI -mouse/#!). The results showed that the macrophage infiltration level was upregulated in the 4NQO 1 CA group, compared with the 4NQO group and the 4NQO 1 CA 1 FCZ group (Fig. 2F), which was validated by immunohistochemistry (IHC) of F4/80 (a mouse macrophage marker) (Fig. 2G).
In addition, a positive correlation was also found between IL-17A expression level and macrophage infiltration level in head and neck squamous cell carcinoma (HNSC) (Fig. 2I) from the TCGA data in TIMER web server (https://cistrome.shinyapps.io/timer/). Meanwhile, the association between C. albicans detected in HNSC (4) and IL-17A in matched samples from TCGA data was analyzed. When the samples were divided into undetected (C. albicans = 0, n = 72), low abundance (0 ; 0.01, n = 24), and high abundance (.0.01, n = 14) groups according to the abundance of C. albicans, it was found that IL-17A expression in the high-abundance group was higher than low-abundance group (P , 0.05) and undetected group (P . 0.05) ( Fig. 2J and Table S1).
It should be noted that macrophage level was estimated from the RNA-seq data, the calculation of which may include the expression of IL-17A, IL-17RA, and their downstream molecules. Thus, IF was performed and showed that more macrophages surrounded the epithelial cells and tumor cells with higher IL-17RA expression (Fig. 2K). Thus, excessive infection of C. albicans may cause more IL-17A production and release, which further activates the IL-17A/IL-17RA signaling pathway in OC cells to induce macrophage infiltration.
Oral tumor cells treated with IL-17A attracted macrophages and induced macrophages into immunosuppressive phenotype. Based on the above relationship between IL-17A/IL-17RA signaling activation and macrophage infiltration in oral tumors, a Transwell migration experiment was designed (Fig. 3A) and found that SCC VII cells treated with rmIL-17A attracted more RAW 264.7 cells into the lower chamber (Fig. 3B). Then, among the chemokines associated with macrophage attraction, the chemokine (C-C motif) ligand 2 (CCL2) gene level in SCC VII cells (Fig. 3C) and CCL2 protein concentration in cell extract (Fig. 3D) and cultivate supernatant (Fig. 3E) were upregulated when SCC VII cells were treated with rmIL-17A. Thus, the OC cells with IL-17RA activation released more CCL2 to attract macrophages into the tumor environment. Furthermore, the influence of OC cells with IL-17RA activation on the phenotype of macrophages was explored. When RAW 264.7 cells were directly treated with rmIL-17, the M1-like markers (NOS2 and TNF-a) were upregulated, but M2-like markers (ARG-1 and IL-10) were not influenced (Fig. 3F). When RAW 264.7 cells were cultured with supernatant from SCC VII cells, both M1-and M2-like markers (ARG-1, NOS2, and TNF-a) were upregulated, with M1-like markers being dominant (Fig. 3F). Interestingly, the supernatant from SCC VII cells pretreated with rmIL-17A caused more significant upregulation of M2-like markers (ARG-1 and IL-10) in macrophages compared with supernatant from SCC VII cells alone (Fig. 3F). With flow cytometry (FCM), a similar trend could be found in the expression of CD86 (M1) and CD163 (M2) on the macrophages (Fig. 3G). The above results indicated that the separate existence of IL-17A or OC cells induced a polarization dominated by M1-like macrophages, but the OC cells pretreated with IL-17A could promote M2 polarization.
In vivo experiments further demonstrated the above in vitro results. C. albicans infection caused the upregulation of CCL2 (Fig. S3C) concentration as well as CD163, PD-L1, and GAL-9 expression on tumor-associated macrophages (TAMs) (Fig. S3D) in oral tumors isolated from C3H/HeN-SCC VII mice.
The neutralization of IL-17A alleviated the tumor progression promoted by C. albicans infection. An in vivo experiment was designed to confirm the role of IL-17A in C. albicans promoting OC (Fig. 4A). Although there was no statistical difference, the oral tumor in the CA group was bigger than the CON group (P = 0.3144) and was reduced after the neutralization of IL-17A (P = 0.1173) ( Additionally, more M2-like macrophages (CD163 1 ) accumulated into the tumor from mice with C. albicans infection and reduced after IL-17A neutralization (Fig. 4F). The PD-L1 expression on macrophages was also upregulated in the CA group than CON group and seemed to be reversed after the neutralization of IL-17A (Fig. 4G).
The depletion of macrophages alleviated the tumor progression promoted by C. albicans infection. Furthermore, the role of macrophages in C. albicans promoting OC was confirmed in vivo (Fig. 5A). When without C. albicans infection, there was no significant difference in tumor size between Mw depletion and CON group ( Fig. 5B and C and Fig. S5A). However, when accompanied by C. albicans infection, the depletion of   S5A). The depletion of tumor-infiltrated macrophages was validated by IHC (Fig. 5D). The above results indicated that the decreased tumor volume in the CA 1 Mw depletion group may be an effect of C. albicans infection and macrophage depletion together rather than macrophage depletion alone.

DISCUSSION
The association between C. albicans infection and OC development has been recognized for decades, but the mechanisms for C. albicans promoting OC still need to be explored. This study confirmed the role of C. albicans in promoting OC and further systematically explored the underlying mechanisms from the perspective of TIME. Specifically, the infection of C. albicans caused the activation of the IL-17A/IL-17RA signaling pathway in OC cells. Then, the activated OC cells released more CCL2 to attract macrophages. Meanwhile, the attracted macrophages changed into an immunosuppressive phenotype in the TME. As a result, an immunosuppressive microenvironment facilitating OC development was shaped with the participation of C. albicans, IL-17A, and TAMs (Fig. 5E).
Elevated IL-17 signature genes can be found in multiple human cancers such as cervical cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, and colorectal cancer (26). It is interesting to note that mucosal sites with microbial colonization seem to be the main victims of the above cancers with IL-17A/IL-17RA hyperactivation. Coincidentally, the relationships between C. albicans infection and esophageal cancer (22), gastric cancer (27), hepatocellular carcinoma (28), and colorectal cancer (29) have been revealed. Thus, it is speculated that microbial dysbiosis including the overgrowth of C. albicans may be an important contributor to the unrestrained IL-17 signaling pathway and the subsequent cancer progression.
In the present study, IL-17A signature gene levels were correlated with OC progression in mice with C. albicans infection. Moreover, the neutralization of IL-17A partly alleviated the cancer progression caused by C. albicans infection. Some studies have also revealed that bacteria might promote the progression of colon cancer and gastric cancer by IL-17 induction (30,31). This study further demonstrated the role of IL-17 in OC progression caused by C. albicans infection, which adds evidence for the fungi/ bacteria-IL-17-cancer axis.
Th17 cells, Tc17 cells (IL-17-producing CD8 1 T cells), and some innate immune subsets such as gd -T cells, natural killer T (NKT) cells, and type 3 "innate lymphoid cells" (ILC3) can express IL-17 (25). Among them, IL-17-producing gd T cells have been shown to promote breast cancer metastasis (32) and colorectal cancer development (33). Our further work will determine which IL-17-producing cell types are the key actors for C. albicans promoting OC development.
TAMs are also double-edged swords in TME with the ability to promote cancer or inhibit cancer (34). TAMs have been shown to promote tumor cell survival and proliferation, angiogenesis, and suppression of immune responses (34,35). Our data showed  A recent single-cell-RNA-sequencing study reported that certain clusters of macrophages might participate in OC carcinogenesis with C. albicans infection (36). However, it is still undetermined which clusters of TAMs participate in this process. Our data showed that C. albicans infection induced M2-like TAMs with PD-L1 and GAL-9 upregulation in TME. Both OC cells with IL-17RA activation and C. albicans are contributors to this kind of TAMs. It is noteworthy that M1-and M2-macrophages in vitro are not representative of their in vivo states, and their associated gene expression is widely distributed with overlapping populations and heterogeneous patterns (37). With recent progress in genomics, single-cell-RNA-sequencing, and time-of-flight technologies, macrophages can be classified into more distinct clusters beyond M1-and M2-polarized phenotypes (38). Thus, more specific TAM phenotypes in C. albicans promoting OC need to be identified.
The polarization mechanisms of macrophages are complex and involved with multiple cytokines and metabolic pathways (38). It has been reported that cancer cells (HeLa, A549, and Myc-Cap/CR) treated with IL-17A induced more M2 polarization of RAW264.7 and THP-1 cells, which may be a result of the cyclooxygenase 2/prostaglandin E 2 (COX-2/PGE 2 ) pathway in the cancer cells (39). In addition, IL-17 was reported to directly induce THP-1-derived macrophages and mouse peritoneal macrophages into M2-like phenotypes via NF-k B activation (40). In our study, IL-17 or OC cells directly induced more M1-like macrophage phenotypes, respectively. However, when OC cells were pretreated with IL-17, M1-like phenotypes induced by OC cells alone polarized into more M2-like phenotypes. Thus, the coexistence of OC cells and IL-17A may produce synergistic benefits to induce M2 polarization of macrophages.
Despite some valuable phenomena that have been discovered in this work, limitations should not be ignored. First, there was no statistically significant difference in tumor size between C. albicans infected and uninfected C3H mice, which may be due to the limited number of mice. Second, this study mainly focused on the IL-17A/IL-17RA-macrophage axis but ignored other immune cell subtypes in C. albicans promoting OC. As we have discovered, the CD4 1 T cells and B cells were downregulated, but the MDSCs in peripheral blood were upregulated when 4NQO mice were infected with C. albicans. Especially, MDSCs are accepted as T-cell immunosuppressive cells (41). Thus, our further work will explore the role of T cells and MDSCs in C. albicans promoting OC. Finally, more clinical evidence is needed to demonstrate the correlations between C. albicans, IL-17A/IL-17-RA, macrophage, and cancer.
In summary, we demonstrated that C. albicans infection promoted OC incidence and progression in mice models. Further data found that OC cells with IL-17A/IL-17RA activation during C. albicans infection attracted macrophages into TME. Meanwhile, the attracted macrophages polarized into M2-like TAMs with PD-L1 and GAL-9 upregulation, which finally induced an immunosuppressive microenvironment to promote OC development. Thus, the treatment of C. albicans infection in oral potentially malignant disorders and oral cancer patients will be beneficial in delaying cancer development.

MATERIALS AND METHODS
Mice. All animal procedures described in this study were reviewed and approved by the Biomedical Ethics Committee of Peking University (LA2021388). C57BL/6N male mice (6 to 8 weeks) and C3H/HeN  Cell lines and culture conditions. Mouse oral squamous cell carcinoma cell line (SCC VII) and macrophages (RAW 264.7) were used. SCC VII and RAW 264.7 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 100 U/mL penicillin-streptomycin (Invitrogen), respectively. Cells were maintained at 37°C in the presence of 5% CO 2 .
The 4NQO-induced mouse tongue carcinogenesis model. C57BL/6N mice were randomly divided into three groups (n = 6 per group): control group (4NQO), C. albicans infection group (4NQO 1 CA), and antifungal group (4NQO 1 CA 1 FCZ). From week 0, all three groups were fed with carcinogen 4NQO (Sigma) in the drinking water (100 mg/mL), which lasted for 16 weeks. From week 4, the mice in the 4NQO 1 CA group and 4NQO 1 CA 1 FCZ group were infected with C. albicans on the dorsum of the tongue by a cotton swab soaked with 200 mL of C. albicans suspension (6 Â 10 8 CFU/mL), while the mice in the 4NQO group were applied with medium alone, once every 3 days. From week 16, 4NQO water was removed. The mice in the 4NQO group and the 4NQO 1 CA group were fed with normal water, while the mice in the 4NQO 1 CA 1 FCZ group were fed with 0.5 mg/mL fluconazole (FCZ; Sigma) in drinking water and lasted for 4 weeks (Fig. 1A).
The tongue tumor-bearing mouse model (C3H/HeN-SCC VII). C3H/HeN mice were used for the tongue tumor-bearing mouse model. From week 21, the mice were fed with 0.1% (wt/vol) tetracycline hydrochloride (TCN; Sigma) in drinking water for 1 week to suppress the endogenous microflora (18). From week 0, the mice in C. albicans-infected group (CA) were infected with C. albicans on the dorsum of the tongue by a cotton swab soaked with 200 mL of C. albicans suspension (6 Â 10 8 CFU/mL, once every 3 days, 2 weeks); meanwhile, these mice were treated with 10 5 CFU/mL C. albicans in drinking water (changed daily to prevent C. albicans from settling) until the sacrifice. The mice in the uninfected group (CON) were treated with medium alone on the dorsum of the tongue (once every 3 days, 2 weeks) and fed with normal water. After 2 weeks of preinfection, all of the mice were injected with SCC VII cells (2.5 Â 10 5 cells in a 25-mL volume) into the submucosa of the tongue dorsum and lasted for 2 weeks. The tumor volume was calculated using the formula: Tumor RNA extraction, sequencing, and transcriptome analysis. RNA-seq assay was performed by Shanghai Biotechnology Corporation. Briefly, total RNA was extracted from the tumor of the mice in the 4NQO, 4NQO 1 CA, and 4NQO 1 CA 1 FCZ groups by the use of miRNeasy minikit (Qiagen, GmBH, Germany) and purified by RNAClean XP kit (Beckman Coulter, Inc. Kraemer Boulevard Brea, CA, USA) and RNase-Free DNase Set (Qiagen, GmBH, Germany). cDNA library was constructed using VAHTS Universal V6 RNA-seq Library Prep kit for Illumina (Vazyme, USA) and then sequenced with Illumina NovaSeq6000. The sequencing reads were genome mapped using Hisat2 (version: 2.0.4). Stringtie (version: 1.3.0) was used to calculate reads per kilobase per million mapped reads (RPKM) values. The edgeR was used to analyze the differentially expressed genes with the following filter criteria: q value of #0.05 and fold change of $2.
Transwell migration experiments. Transwell chamber (8.0-mm pore membranes; Corning) was used to explore the migration of macrophages. SCC VII cells (1 Â 10 4 cells per well) were seeded in the lower chamber and incubated for 24 h. Then, the culture medium was replaced with 600 mL serum-free medium supplemented with 0, 50, or 100 ng/mL recombinant mouse IL-17A (rmIL-17A, 421-ML, R&D Systems) and cultured for 8 h. After that, 200 mL RAW 264.7 cells (5 Â 10 5 /mL) in serum-free medium were added into the upper chamber. After being cocultured for 24 h, the RAW 264.7 cells remaining on the upper surface of the membrane were removed, and the cells passed onto the lower surface of the membrane were fixed, stained with 0.1% crystal violet solution, and numbered under the microscope.
Coculture experiments. To explore the influence of OC cells stimulated with IL-17A on macrophages, RAW 264.7 cells were cultured in culture supernatants from SCC VII cells treated with 0 or 100 ng/mL rmIL-17A for 8 h. After being cultured for 24 h, the RAW 264.7 cells were collected for RT-qPCR and FCM.
Immune cell phenotyping by flow cytometry. Peripheral blood mononuclear cells were separated by density gradient centrifugation. Tumor-infiltrating immune cells were separated by mechanically homogenized and filtered with a 70-mm cell strainer, followed by CD45 1 staining and gating. The antibodies for leukocyte subpopulations are listed in Table S2. The stained cells were analyzed using a flow cytometer (CytoFlEX S, Beckman Coulter). FlowJo software (v. 10) was used for data analysis. C. albicans infection promoted IL-17A production and the following IL-17RA signal activation in oral tumor cells; then, tumor cells released CCL2 to attract macrophages into TME; the macrophages in TME showed an immunosuppressive phenotype with elevated expressions of PD-L1, GAL-9, and M2-like macrophage markers. **, P , 0.01.
Quantitative real-time PCR. Total RNA was extracted with TRIzol Reagent (Invitrogen) and reversetranscribed into cDNA with ABScript III Reverse Transcriptase (Abclonal, Wuhan, China). Then, the amplification was performed in triplicate using Universal SYBR green Fast qPCR Mix (Abclonal, Wuhan, China). The melting curves were checked and the relative gene expression was analyzed using the 2 2DDCt method with normalization to GAPDH and ACTB. The primer (Sangon Biotech, Shanghai, China) sequences are listed in Table S3.
Enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent assay (ELISA) method was performed according to the instruction manual to detect the concentration of S100A9 and CCL2 in culture supernatants of SCC VII cells and RAW 264.7 cells as well as the tissue homogenization of tumor.
Western blot. Total proteins in cells and tissues were extracted by radioimmunoprecipitation (RIPA)buffer (Thermo Fisher Scientific) added with protease and phosphatase inhibitors (Solarbio, CN). The Western blot (WB) experiment was performed according to the standard procedures. Primary antibodies against CCL2 were used at a 1:1,000 dilution.
Statistics. Each experiment was repeated three times unless otherwise stated. GraphPad Prism (v.8.0) was used for statistical analyses. Data are presented as mean 6 standard deviation (SD). Data were statistically assessed for normality and variance homogeneity using the Shapiro-Wilk test and F test, respectively. Student's two-tailed t test was used to determine the statistical relevance between the two groups. Pearson's correlation was used to analyze the correlation between macrophage infiltration level and IL-17A/IL-17RA signature genes. A P value of ,0.05 was indicative of statistical significance (*, P , 0.05; **, P , 0.01; ***, P , 0.001; ****, P , 0.0001).
Data availability. Sequence data that support the findings of this study have been deposited in NCBI BioProject with the primary accession code PRJNA944176 and in NCBI Sequence Read Archive (SRA) with the primary accession code SRP427315.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.