Examination of control asymptomatic cohorts reveals heightened anti-EBV and HHV-6 A/B dUTPase antibodies in the aging populations.

Members of the human Herpesviridae are found in high prevalence in the human virome. While these viruses are known to cause numerous disease pathologies in symptomatic individuals little is known concerning the role that these viruses may have in modulating the host immune system in asymptomatic "healthy" individuals, especially during the aging process. Examination of three cohorts of "healthy asymptomatic" individuals (n = 255) for the presence of antibodies against the herpesviruses deoxyuridine triphosphate nucleotidohydrolase (dUTPase) as a marker for lytic/abortive-lytic replication demonstrated that all cohorts exhibited differential anti-herpesvirus dUTPase antibodies positivity frequencies ranging from 40.4% to 84% with some individuals in these cohorts expressing antibodies to the dUTPases of multiple herpesviruses (17.2%-56%). Furthermore, our results demonstrate that there was a statistically significant difference in anti-human herpesvirus 6 A and 6B (HHV-6 A/B) dUTPase antibodies in Cohort 3 (age = 66.2 ± 15.02 years) versus Cohort 1 (age 46.88 ± 8.61 years), suggesting that reactivation of HHV-6 A/B is not attenuated by aging. It is well established/documented that herpesvirus dUTPases induce immune dysfunction, as such it is of critical importance that additional studies be performed to determine how these viral proteins alter immune responses in asymptomatic individuals.

GM-CSF: An immune modulatory cytokine that can suppress autoimmunity.

GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases such as Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance.

EBV/HHV-6A dUTPases contribute to myalgic encephalomyelitis/chronic fatigue syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, debilitating, multisystem illness of unknown etiology for which no cure and no diagnostic tests are available. Despite increasing evidence implicating EBV and human herpesvirus 6A (HHV-6A) as potential causative infectious agents in a subset of patients with ME/CFS, few mechanistic studies address a causal relationship. In this study we examined a large ME/CFS cohort and controls and demonstrated a significant increase in activin A and IL-21 serum levels, which correlated with seropositivity for antibodies against the EBV and HHV-6 protein deoxyuridine triphosphate nucleotidohydrolase (dUTPases) but no increase in CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation and for the generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH function in ME/CFS. In vitro studies revealed that virus dUTPases strongly induced activin A secretion while in vivo, EBV dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Together, our data indicate abnormal germinal center (GC) activity in participants with ME/CFS and highlight a mechanism by which EBV and HHV6 dUTPases may alter GC and extrafollicular antibody responses.

Murine Models of Alcohol Consumption: Imperfect but Still Potential Source of Novel Biomarkers and Therapeutic Drug Discovery for Alcoholic Liver Disease.

Animal models of liver disease are fundamentally important to strengthen our knowledge and understanding of human liver diseases. Murine models of alcohol consumption are utilized to investigate alcoholic liver injury to develop new therapeutic targets. The well accepted and commonly used murine models of chronic alcohol consumption are Meadows-Cook (MC) and Lieber-DeCarli (LD). LD model is based on an isocaloric high-fat liquid diet, but mice under the MC model fed on a regular chow diet with alcohol added to the drinking water. Alcoholic liver disease in real world is frequently diagnosed in patients with obesity and high fat intake, mirroring LD diet. The overlap of the specific effect of ethanol and obesity is difficult to differentiate by clinician and pathologist. In this commentary, we will further discuss our research findings comparing MC and LD as a tool to dissect early alcohol versus increased fat intake detrimental effects on the liver. The critical analysis of these two models could provide evidence to differentiate the specific impact of alcohol on the liver from the combined influence of alcohol and diet. Ultimately, these investigations could uncover potential biomarkers and therapeutic targets for personalized type of alcoholic liver injury.

Alcohol Consumption Accumulation of Monocyte Derived Macrophages in Female Mice Liver Is Interferon Alpha Receptor Dependent.

Monocytes develop in the bone marrow from the hematopoietic stem cells and represent heterogeneous phagocyte cells in the circulation. In homeostatic and inflammatory conditions, after recruitment into tissues, monocytes differentiate into macrophages and dendritic cells. Alcohol use causes about 3.3 million worldwide deaths per year, which is about 5.9% of all deaths. In the United States and Europe, alcohol use disorders represent the fifth leading cause of death. Females are more susceptible to alcoholic liver injury in both humans and mice. Strikingly, we still do not know how much of this difference in tissue injury is due to the differential effect of alcohol and its toxic metabolites on a) parenchymal or resident cells and/or b) immune response to alcohol. Therefore, we used a model of chronic alcohol exposure in mice to investigate the dynamics of monocytes, an innate immune cell type showed to be critical in alcoholic liver injury, by using immunophenotypic characterization. Our data reveal a sex-dimorphism of alcohol response of hepatic monocytes in female mice that is interferon receptor alpha dependent. This dimorphism could shed light on potential cellular mechanism(s) to explain the susceptibility of females to alcoholic immunopathogenesis and suggests an additional targetable pathway for alcoholic liver injury in females.

Sex specific effect of alcohol on hepatic plasmacytoid dendritic cells.

Alcoholic liver disease includes a spectrum of clinical and histological entities. They result from the combined direct effect of alcohol and its metabolites on immune cells and resident tissue cells. In humans and mice, females are more susceptible to alcoholic liver injury than males. Despite being involved in sex specific differences of immune mediated tissue injury, plasmacytoid dendritic cells (pDCs) have not been thoroughly assessed as a cellular target of alcohol in humans or mice. Therefore, Meadows-Cook diet was used to study alcohol effect on hepatic dendritic cells. Alcohol consumption for 12 weeks increased hepatic pDCs in female mice. The expression of the C-C chemokine receptor type 2 (CCR2) increased in hepatic pDC of alcohol-fed female mice. Bone marrow transplant chimera showed CCR2 dependent bone marrow egress of pDCs. Chronic alcohol exposure has a sex specific effect on hepatic pDCs population that may explain sex differences to alcoholic liver disease.

PKC-ѳ is dispensable for OX40L-induced TCR-independent Treg proliferation but contributes by enabling IL-2 production from effector T-cells.

We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3+ Tregs, but not Foxp3- effector T-cells (Teff), when CD4+ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4+ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4+ T-cells from WT and OX40-/- mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4+ T-cells from PKC-Ѳ-/- mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40-/-, and PKC-Ѳ-/- mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.

Soluble OX40L and JAG1 Induce Selective Proliferation of Functional Regulatory T-Cells Independent of canonical TCR signaling.

Regulatory T-cells (Tregs) play a pivotal role in maintaining peripheral tolerance. Increasing Treg numbers/functions has been shown to ameliorate autoimmune diseases. However, common Treg expansion approaches use T-Cell Receptor (TCR)-mediated stimulation which also causes proliferation of effector T-cells (Teff). To overcome this limitation, purified patient-specific Tregs are expanded ex vivo and transfused. Although promising, this approach is not suitable for routine clinical use. Therefore, an alternative approach to selectively expand functional Tregs in vivo is highly desired. We report a novel TCR-independent strategy for the selective proliferation of Foxp3+Tregs (without Teff proliferation), by co-culturing CD4+ T-cells with OX40 L+Jagged(JAG)-1+ bone marrow-derived DCs differentiated with GM-CSF or treating them with soluble OX40 L and JAG1 in the presence of exogenous IL-2. Tregs expanded using soluble OX40 L and JAG1 were of suppressive phenotype and delayed the onset of diabetes in NOD mice. Ligation of OX40 L and JAG1 with their cognate-receptors OX40 and Notch3, preferentially expressed on Tregs but not on Teff cells, was required for selective Treg proliferation. Soluble OX40L-JAG1-induced NF-κB activation as well as IL-2-induced STAT5 activation were essential for the proliferation of Tregs with sustained Foxp3 expression. Altogether, these findings demonstrate the utility of soluble OX40 L and JAG1 to induce TCR-independent Treg proliferation.

Age-dependent divergent effects of OX40L treatment on the development of diabetes in NOD mice.

Earlier, we have shown that GM-CSF derived bone marrow (BM) dendritic cells (G-BMDCs) can expand Foxp3(+) regulatory T-cells (Tregs) through a TCR-independent, but IL-2 dependent mechanism that required OX40L/OX40 interaction. While some reports have shown suppression of autoimmunity upon treatment with an OX40 agonist, others have shown exacerbation of autoimmune disease instead. To better understand the basis for these differing outcomes, we compared the effects of OX40L treatment in 6-week-old pre-diabetic and 12-week-old near diabetic NOD mice. Upon treatment with OX40L, 6-week-old NOD mice remained normoglycemic and showed a significant increase in Tregs in their spleen and lymph nodes, while 12-week-old NOD mice very rapidly developed hyperglycemia and failed to show Treg increase in spleen or LN. Interestingly, OX40L treatment increased Tregs in the thymus of both age groups. However, it induced Foxp3(+)CD103(+)CD38(-) stable-phenotype Tregs in the thymus and reduced the frequency of autoreactive Teff cells in 6-week-old mice; while it induced Foxp3(+)CD103(-)CD38(+) labile-phenotype Tregs in the thymus and increased autoreactive CD4(+) T cells in the periphery of 12-week-old mice. This increase in autoreactive CD4(+) T cells was likely due to either a poor suppressive function or conversion of labile Tregs into Teff cells. Using ex vivo cultures, we found that the reduction in Treg numbers in 12-week-old mice was likely due to IL-2 deficit, and their numbers could be increased upon addition of exogenous IL-2. The observed divergent effects of OX40L treatment were likely due to differences in the ability of 6- and 12-week-old NOD mice to produce IL-2.

Dual Role of GM-CSF as a Pro-Inflammatory and a Regulatory Cytokine: Implications for Immune Therapy.

Granulocyte macrophage colony stimulating factor (GM-CSF) is generally recognized as an inflammatory cytokine. Its inflammatory activity is primarily due its role as a growth and differentiation factor for granulocyte and macrophage populations. In this capacity, among other clinical applications, it has been used to bolster anti-tumor immune responses. GM-CSF-mediated inflammation has also been implicated in certain types of autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. Thus, agents that can block GM-CSF or its receptor have been used as anti-inflammatory therapies. However, a review of literature reveals that in many situations GM-CSF can act as an anti-inflammatory/regulatory cytokine. We and others have shown that GM-CSF can modulate dendritic cell differentiation to render them "tolerogenic," which, in turn, can increase regulatory T-cell numbers and function. Therefore, the pro-inflammatory and regulatory effects of GM-CSF appear to depend on the dose and the presence of other relevant cytokines in the context of an immune response. A thorough understanding of the various immunomodulatory effects of GM-CSF will facilitate more appropriate use and thus further enhance its clinical utility.