CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model.

Th17 cells and CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are thought to promote and suppress inflammatory responses, respectively. Here we explore why under Th17 cell polarizing conditions, Treg cells did not suppress, but rather upregulated, the expression of interleukin-17A (IL-17A), IL-17F, and IL-22 from responding CD4(+) T cells (Tresp cells). Upregulation of IL-17 cytokines in Tresp cells was dependent on consumption of IL-2 by Treg cells, especially at early time points both in vitro and in vivo. During an oral Candida albicans infection in mice, Treg cells induced IL-17 cytokines in Tresp cells, which markedly enhanced fungal clearance and recovery from infection. These findings show how Treg cells can promote acute Th17 cell responses to suppress mucosal fungus infections and reveal that Treg cells have a powerful capability to fight infections besides their role in maintaining tolerance or immune homeostasis.

Innate immune mechanisms to oral pathogens in oral mucosa of HIV-infected individuals.

A crucial aspect of mucosal HIV transmission is the interaction between HIV, the local environmental milieu and immune cells. The oral mucosa comprises many host cell types including epithelial cells, CD4 + T cells, dendritic cells and monocytes/macrophages, as well as a diverse microbiome predominantly comprising bacterial species. While the oral epithelium is one of the first sites exposed to HIV through oral-genital contact and nursing infants, it is largely thought to be resistant to HIV transmission via mechanisms that are still unclear. HIV-1 infection is also associated with predisposition to secondary infections, such as tuberculosis, and other diseases including cancer. This review addresses the following questions that were discussed at the 8th World Workshop on Oral Health and Disease in AIDS held in Bali, Indonesia, 13 September -15 September 2019: (a) How does HIV infection affect epithelial cell signalling? (b) How does HIV infection affect the production of cytokines and other innate antimicrobial factors, (c) How is the mucosal distribution and function of immune cells altered in HIV infection? (d) How do T cells affect HIV (oral) pathogenesis and cancer? (e) How does HIV infection lead to susceptibility to TB infections?

CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells.

A key issue in mammalian immunology is how CD4+CD25+Foxp3+ regulatory T cells (T(reg) cells) suppress immune responses. Here we show that T(reg) cells induced apoptosis of effector CD4+ T cells in vitro and in vivo in a mouse model of inflammatory bowel disease. T(reg) cells did not affect the early activation or proliferation of effector CD4+ T cells. Cytokines that signal through the common gamma-chain suppressed T(reg) cell-induced apoptosis. T(reg) cell-induced effector CD4+ T cell death required the proapoptotic protein Bim, and effector CD4+ T cells incubated with T(reg) cells showed less activation of the prosurvival kinase Akt and less phosphorylation of the proapoptotic protein Bad. Thus, cytokine deprivation-induced apoptosis is a prominent mechanism by which T(reg) cells inhibit effector T cell responses.

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6.

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

Origin and functions of pro-inflammatory cytokine producing Foxp3+ regulatory T cells.

CD4(+)CD25(+)Foxp3(+) regulatory cells (Tregs) are a special lineage of cells central in the maintenance of immune homeostasis, and are targeted for human immunotherapy. They are conventionally associated with the production of classical anti-inflammatory cytokines such as IL-10, TGF-ß and IL-35, consistent to their anti-inflammatory functions. However, emerging evidence show that they also express effector cytokines such as IFN-γ and IL-17A under inflammatory conditions. While some studies reveal that these pro-inflammatory cytokine producing Foxp3(+) regulatory cells retain their suppressive ability, others believe that these cells are dys-regulated and are associated with perpetuation of immunopathology. Therefore the development of these cells may challenge the efficacy of human Treg therapy. Mechanistically, toll-like receptor (TLR) ligands and the pro-inflammatory cytokine milieu have been shown to play important roles in the induction of effector cytokines in Tregs. Here we review the mechanisms of development and the possible functions of pro-inflammatory cytokine producing Foxp3+ Tregs.

Isolation of T cells from mouse oral tissues.

BACKGROUND: Utilizing mouse models provides excellent immunological and experimental tools to study oral immune responses. However for functional assays, isolating T lymphocytes from the oral tissues has proved to be challenging due to the absence of reliable methods that yield viable cells with consistency. To study adaptive immune cell interactions in the oral mucosal tissues, it is necessary to isolate T cells with a good viability and study them at the single cell level. FINDINGS: We have established an improved method to isolate immune cells, including Tregs and Th17 cells from intra-epithelial niches and lamina propria of the tongue, gingival and palatal tissues in the oral mucosa of mice. CONCLUSION: This new method of isolating immune cells from oral tissues will enable us to further our understanding of oral tissue immune cells and their role during oral infections and oral inflammation.

The role of IL-15 in activating STAT5 and fine-tuning IL-17A production in CD4 T lymphocytes.

IL-15 is an important IL-2-related cytokine whose role in Th17 cell biology has not been fully elucidated. In this study, we show that exogenous IL-15 decreased IL-17A production in Th17 cultures. Neutralization of IL-15 using an Ab led to increases in IL-17A production in Th17 cultures. Both Il15(-/-) and Il15r(-/-) T cell cultures displayed higher frequency of IL-17A producers and higher amounts of IL-17A in the supernatants compared with those of wild-type (WT) cells in vitro. IL-15 down-modulated IL-17A production independently of retinoic acid-related orphan receptor-γt, Foxp3, and IFN-γ expression. Both Th17 cells and APCs produced IL-15, which induced binding of STAT5, an apparent repressor to the Il17 locus in CD4 T cells. Also, in a model of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE), Il15(-/-) mice displayed exacerbated inflammation-correlating with increased IL-17A production by their CD4(+) T cells-compared with WT controls. Exogenous IL-15 administration and IL-17A neutralization reduced the severity of EAE in Il15(-/-) mice. Taken together, these data indicate that IL-15 has a negative regulatory role in fine-tuning of IL-17A production and Th17-mediated inflammation.

The power and the promise of restimulation-induced cell death in human immune diseases.

Controlled expansion and contraction of lymphocytes both during and after an adaptive immune response are imperative to sustain a healthy immune system. Both extrinsic and intrinsic pathways of lymphocyte apoptosis are programmed to eliminate cells at the proper time to ensure immune homeostasis. Genetic disorders of apoptosis described in mice and humans have established Fas and Bim as critical pro-apoptotic molecules responsible for T-cell death in response to T-cell receptor restimulation and cytokine withdrawal, respectively. Emerging evidence prompts revision of this classic paradigm, especially for our understanding of restimulation-induced cell death (RICD) and its physiological purpose. Recent work indicates that RICD employs both Fas and Bim for T-cell deletion, dispelling the notion that these molecules are assigned to mutually exclusive apoptotic pathways. Furthermore, new mouse model data combined with our discovery of defective RICD in X-linked lymphoproliferative disease (XLP) patient T cells suggest that RICD is essential for precluding excess T-cell accumulation and associated immunopathology during the course of certain infections. Here, we review how these advances offer a refreshing new perspective on the phenomenon of T-cell apoptosis induced through antigen restimulation, including its relevance to immune homeostasis and potential for therapeutic interventions.

Beta-defensin index: A functional biomarker for oral cancer detection.

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

Conditional ablation of the Notch2 receptor in the ocular lens.

Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation.

Distinct SARS-CoV-2 specific NLRP3 and IL-1ß responses in T cells of aging patients during acute COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19) that presents with varied clinical manifestations ranging from asymptomatic or mild infections and pneumonia to severe cases associated with cytokine storm, acute respiratory distress syndrome (ARDS), and even death. The underlying mechanisms contributing to these differences are unclear, although exacerbated inflammatory sequelae resulting from infection have been implicated. While advanced aging is a known risk factor, the precise immune parameters that determine the outcome of SARS-CoV-2 infection in elderly individuals are not understood. Here, we found aging-associated (age ≥61) intrinsic changes in T cell responses when compared to those from individuals aged ≤ 60, even among COVID-positive patients with mild symptoms. Specifically, when stimulated with SARS-CoV-2 peptides in vitro, peripheral blood mononuclear cell (PBMC) CD4+ and CD8+ T cells from individuals aged ≥61 showed a diminished capacity to produce IFN-γ and IL-1ß. Although they did not have severe disease, aged individuals also showed a higher frequency of PD-1+ cells and significantly diminished IFN-γ/PD-1 ratios among T lymphocytes upon SARS-CoV-2 peptide stimulation. Impaired T cell IL-1ß expression coincided with reduced NLRP3 levels in T lymphocytes. However, the expression of these molecules was not affected in the monocytes of individuals aged ≥61. Together, these data reveal SARS-CoV-2-specific CD4+ and CD8+ T-cell intrinsic cytokine alterations in the individuals older than 61 and may provide new insights into dysregulated COVID-directed immune responses in the elderly.

Inflammation, Immune Senescence, and Dysregulated Immune Regulation in the Elderly.

An optimal immune response requires the appropriate interaction between the innate and the adaptive arms of the immune system as well as a proper balance of activation and regulation. After decades of life, the aging immune system is continuously exposed to immune stressors and inflammatory assaults that lead to immune senescence. In this review, we will discuss inflammaging in the elderly, specifically concentrating on IL-6 and IL-1b in the context of T lymphocytes, and how inflammation is related to mortality and morbidities, specifically cardiovascular disease and cancer. Although a number of studies suggests that the anti-inflammatory cytokine TGF-b is elevated in the elderly, heightened inflammation persists. Thus, the regulation of the immune response and the ability to return the immune system to homeostasis is also important. Therefore, we will discuss cellular alterations in aging, concentrating on senescent T cells and CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) in aging.

Regulation of IL-17A-Producing Cells in Skin Inflammatory Disorders.

This review focuses on the IL-17A family of cytokines produced by T lymphocytes and other immune cells and how they are involved in cutaneous pathogenic responses. It will also discuss cutaneous dysbiosis and FOXP3+ regulatory T cells in the context of inflammatory conditions linked to IL-17 responses in the skin. Specifically, it will review key literature on chronic mucocutaneous candidiasis and psoriasis.

Fungal Colonization and Infections-Interactions with Other Human Diseases.

Candida albicans is a commensal fungus that asymptomatically colonizes the skin and mucosa of 60% of healthy individuals. Breaches in the cutaneous and mucosal barriers trigger candidiasis that ranges from asymptomatic candidemia and mucosal infections to fulminant sepsis with 70% mortality rates. Fungi influence at least several diseases, in part by mechanisms such as the production of pro-carcinogenic agents, molecular mimicking, and triggering of the inflammation cascade. These processes impact the interactions among human pathogenic and resident fungi, the bacteriome in various organs/tissues, and the host immune system, dictating the outcomes of invasive infections, metabolic diseases, and cancer. Although mechanistic investigations are at stages of infancy, recent studies have advanced our understanding of host-fungal interactions, their role in immune homeostasis, and their associated pathologies. This review summarizes the role of C. albicans and other opportunistic fungi, specifically their association with various diseases, providing a glimpse at the recent developments and our current knowledge in the context of inflammatory-bowel disease (IBD), cancers, and COVID-19. Two of the most common human diseases where fungal interactions have been previously well-studied are cancer and IBD. Here we also discuss the emerging role of fungi in the ongoing and evolving pandemic of COVID-19, as it is relevant to current health affairs.

The Role of Dectin-1 Signaling in Altering Tumor Immune Microenvironment in the Context of Aging.

An increased accumulation of immune-dysfunction-associated CD4+Foxp3+ regulatory T cells (Tregs) is observed in aging oral mucosa during infection. Here we studied the function of Tregs during oral cancer development in aging mucosa. First, we found heightened proportions of Tregs 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-1ß, Tregs, and MDSC in tongues. Partial depletion of Tregs 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-1ß, reduced infiltration of Tregs 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.

Naïve CD4+ T Cell Lymphopenia and Apoptosis in Chronic Hepatitis C Virus Infection Is Driven by the CD31+ Subset and Is Partially Normalized in Direct-Acting Antiviral Treated Persons.

Background: The mechanisms underlying naïve CD4+ lymphopenia during chronic Hepatitis C Virus (HCV) infection are unclear. Whether direct-acting antiviral (DAA) therapy restores peripheral naïve CD4+ T cell numbers and function is unknown. Methods: We enumerated frequencies and counts of peripheral naïve CD4+, CD4+CD31+ and CD4+CD31- T cells by flow cytometry in a cross sectional analysis comparing chronic HCV infected (n=34), DAA-treated(n=29), and age-range matched controls (n=25), as well as in a longitudinal cohort of HCV DAA treated persons (n=16). The cross-sectional cohort was stratified by cirrhosis state. Cell apoptosis/survival (AnnexinV+7AAD+/BCL-2 labeling) and cell cycle entry (Ki67 expression) of CD31+ and CD31- naïve CD4+ T cells was analyzed directly ex vivo and following 3 and 5 days of in vitro culture with media, interleukin (IL) -7 or CD3/CD28 activator. Results: In the cross-sectional cohort, naïve CD4+ proportions were lower in chronic HCV infected persons compared to controls and DAA-treated persons, an effect in part attributed to cirrhosis. Age was associated with naïve cell counts and proportions in HCV infected and treated persons as well. Naïve CD4+ cell proportions negatively correlated with plasma levels of soluble CD14 following therapy in DAA-treated persons. Naïve CD4+ cells from HCV infected persons exhibited greater direct ex vivo apoptosis and cell-cycling compared to cells from DAA-treated persons and controls, and this was localized to the CD4+CD31+ subset. On the other hand, no remarkable differences in expression of BCL-2 or IL-7 Receptor (CD127) at baseline or following in vitro media or IL7 containing culture were observed. In the longitudinal cohort, naïve CD4+CD31+/CD31- ratio tended to increase 24 weeks after DAA therapy initiation. Conclusions: Activation and apoptosis of peripheral naïve CD4+CD31+ T cells appear to contribute to naïve CD4+ lymphopenia in chronic HCV infection, and this defect is partially reversible with HCV DAA therapy. Age and cirrhosis -associated naïve CD4+ lymphopenia is present both before and after HCV DAA therapy. These findings have implications for restoration of host immune function after DAA therapy.

CD152 (CTLA-4) determines the unequal resistance of Th1 and Th2 cells against activation-induced cell death by a mechanism requiring PI3 kinase function.

Survival of antigen-experienced T cells is essential for the generation of adaptive immune responses. Here, we show that the genetic and antibody-mediated inactivation of CD152 (cytotoxic T lymphocyte antigen 4) in T helper (Th) effector cells reduced the frequency of nonapoptotic cells in a completely Fas/Fas ligand (FasL)-dependent manner. CD152 cross-linking together with stimulation of CD3 and CD28 on activated Th2 cells prevented activation-induced cell death (AICD) as a result of reduced Fas and FasL expression. Apoptosis protection conferred by CD152 correlated with the up-regulation of Bcl-2 and was mediated by phosphatidylinositol 3 kinase, which prevented FasL expression through the inhibitory phosphorylation of Forkhead transcription factor FKHRL1. We show that signals induced by CD152 act directly on activated T lymphocytes and, due to its differential surface expression on activated Th1 and Th2 cells, induce resistance to AICD mainly in Th2 cells.

IL-1ß-MyD88-mTOR Axis Promotes Immune-Protective IL-17A+Foxp3+ Cells During Mucosal Infection and Is Dysregulated With Aging.

CD4+Foxp3+Tregs 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 Tregs coincided with a reduction of the unique population of IL-17A expressing Foxp3+ cells (Treg17) and an increase in dysfunctional IFN-γ+/Foxp3+ cells (TregIFN-γ) in infected mice. Failure of MyD88-/- Tregs 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-1ß/MyD88 signaling was required for the activation of IRAK-4, Akt, and mTOR, which led to the induction and proliferation of Treg17 cells. In the absence of IL-1 receptor signaling, Treg17 cells were reduced, but IL-6-driven expansion of TregIFN-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-mTORhighTreg17 cells and reduced levels of IL-1ß in oral mucosa, which coincided with persistent tongue inflammation. Concurrent with Treg 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-1ß/MyD88/Treg 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.

Microbiome Dependent Regulation of Tregs and Th17 Cells in Mucosa.

Mammals co-exist with resident microbial ecosystem that is composed of an incredible number and diversity of bacteria, viruses and fungi. Owing to direct contact between resident microbes and mucosal surfaces, both parties are in continuous and complex interactions resulting in important functional consequences. These interactions govern immune homeostasis, host response to infection, vaccination and cancer, as well as predisposition to metabolic, inflammatory and neurological disorders. Here, we discuss recent studies on direct and indirect effects of resident microbiota on regulatory T cells (Tregs) and Th17 cells at the cellular and molecular level. We review mechanisms by which commensal microbes influence mucosa in the context of bioactive molecules derived from resident bacteria, immune senescence, chronic inflammation and cancer. Lastly, we discuss potential therapeutic applications of microbiota alterations and microbial derivatives, for improving resilience of mucosal immunity and combating immunopathology.