CD4⁺Foxp3⁺T_regs maintain immune homeostasis, but distinct mechanisms underlying their functional heterogeneity during infections are driven by specific cytokine milieu. Here we show that MyD88 deletion in Foxp3⁺ cells altered their function and resulted in increased fungal burden and immunopathology during oral Candida albicans (CA) challenge. Excessive inflammation due to the absence of MyD88 in T_regs coincided with a reduction of the unique population of IL-17A expressing Foxp3⁺ cells (T_reg17) and an increase in dysfunctional IFN-γ⁺/Foxp3⁺ cells (T_regIFN-γ) in infected mice. Failure of MyD88^-/- T_regs to regulate effector CD4⁺ T cell functions correlated with heightened levels of IFN-γ in CD4⁺ T cells, as well as increased infiltration of inflammatory monocytes and neutrophils in oral mucosa in vivo. Mechanistically, IL-1β/MyD88 signaling was required for the activation of IRAK-4, Akt, and mTOR, which led to the induction and proliferation of T_reg17 cells. In the absence of IL-1 receptor signaling, T_reg17 cells were reduced, but IL-6-driven expansion of T_regIFN-γ cells was increased. This mechanism was physiologically relevant during Candida infection in aged mice, as they exhibited IL-1 receptor/MyD88 defect in Foxp3⁺ cells, loss of p-mTOR^highT_reg17 cells and reduced levels of IL-1β in oral mucosa, which coincided with persistent tongue inflammation. Concurrent with T_reg dysfunction, aging was associated with increased CD4⁺ T cell hyperactivation and heightened levels of IL-6 in mice and humans in oral mucosa in vivo. Taken together, our data identify IL-1β/MyD88/T_reg axis as a new component that modulates inflammatory responses in oral mucosa. Also, dysregulation of this axis in an aging immune system may skew host defense towards an immunopathological response in mucosal compartments.

An increased accumulation of immune-dysfunction-associated CD4⁺Foxp3⁺ regulatory T cells (T_regs) is observed in aging oral mucosa during infection. Here we studied the function of T_regs during oral cancer development in aging mucosa. First, we found heightened proportions of T_regs and myeloid-derived suppressor cells (MDSC) accumulating in mouse and human oral squamous cell carcinoma (OSCC) tissues. Using the mouse 4-Nitroquinoline 1-oxide(4-NQO) oral carcinogenesis model, we found that tongues of aged mice displayed increased propensity for epithelial cell dysplasia, hyperplasia, and accelerated OSCC development, which coincided with significantly increased abundance of IL-1β, T_regs, and MDSC in tongues. Partial depletion of T_regs reduced tumor burden. Moreover, fungal abundance and dectin-1 signaling were elevated in aged mice suggesting a potential role for dectin-1 in modulating immune environment and tumor development. Confirming this tenet, dectin-1 deficient mice showed diminished IL-1β, reduced infiltration of T_regs and MDSC in the tongues, as well as slower progression and reduced severity of tumor burden. Taken together, these data identify an important role of dectin-1 signaling in establishing the intra-tumoral immunosuppressive milieu and promoting OSCC tumorigenesis in the context of aging.

Metastasis is responsible for more than 90% of prostate cancer-associated mortality in the United States. One of the distinctive reason for metastasis has been the imbalance of matrix metalloproteinases (MMPs) as a result of reduced expression of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) a novel tumor suppressor. RECK has been shown to be a potent inhibitor of MMP-2 and MMP-9. Loss of RECK has been frequently identified in clinical prostate cancer specimens and prostate cancer cell lines, therefore mechanistic understanding of its loss and approaches to restore its expression would facilitate inhibition of metastasis. Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) has been shown to suppress prostate cancer metastasis, however the mechanism has not been fully elucidated. We determined whether treatment with GTP has ability to restore induction of RECK and play a key role in suppressing invasiveness in prostate cancer. Human prostate cancer LNCaP tumor were implanted in the ventral prostate of athymic nude mice for 2 weeks followed by per-oral intake of GTP at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Other groups were treated with DNA methyltransferase inhibitor-5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor-trichostatin A (TSA) and histone methyltransferase inhibitor-3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed RECK hypermethylation and significantly enhanced its expression in the tumor tissue. Similar findings were noted in cell culture where treatment of PC-3 and LNCaP cells with 20µM EGCG and 10µg/mL GTP for 72 h significantly induced RECK expression at mRNA and protein levels along with decrease invasiveness in these cells. Our findings suggest that induction of RECK is a key epigenetic event reactivated by GTP/EGCG that results in suppression of MMP-2/MMP-9, matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression. Citation Format: Eswar Shankar, Omair Iqbal, Natarajan Bhaskaran, Gauri Deb, Gregory T. MacLennan, Pingfu Fu, Sanjay Gupta. Epigenetic modifications involving reactivation of RECK inhibiting MMP-9 and MMP-2 in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5084.