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.

Expression of TFH Markers and Detection of RHOA p.G17V and IDH2 p.R172K/S Mutations in Cutaneous Localizations of Angioimmunoblastic T-Cell Lymphomas.

Skin biopsies of 41 angioimmunoblastic T-cell lymphoma patients were retrospectively analyzed for the expression of follicular helper T-cell (TFH) markers, Epstein-Barr virus (EBV), and the presence of RHOA (p.G17V) and IDH2 (p.R172K/S) mutations using allele-specific polymerase chain reaction. We categorized cases into 4 distinctive patterns: (1) low-density lymphocytic perivascular infiltrates (n=11), (2) dense perivascular infiltrates with atypical cells and occasional inflammatory cells (n=13), (3) diffuse infiltrates reminiscent of angioimmunoblastic T-cell lymphoma (n=4), or (4) other aspects (n=13). Two EBV and 2 plasmacytoid lymphoproliferative disorders were seen. We observed variable expression of TFH markers (CD10 [50%], BCLB6 [84%], PD1 [94%], CXCL13 [84%], and ICOS [97.5%]), and EBV B-blasts (26%). A TFH phenotype was identified in 82% and 73%, respectively, of cases with the most challenging patterns 1 and 2. TFH markers and EBV can thus help for diagnosis and are detected in samples with low-density infiltrates. We found RHOA G17V and IDH2 R172K/S mutations in the skin in 14/18 (78%) and 3/16 (19%) cases, respectively. The RHOA G17V mutation was identified in a proportion of biopsies with patterns 1 and 2, which represent a diagnostic challenge. The RHOA G17V mutation was detected both in the skin and lymph node (LN) biopsies in 7/9 (64%) cases, and in only the skin or the LN of 1 sample each. The frequency of RHOA G17V mutation was similar to that reported in LNs. It may represent a sensitive diagnostic marker in the skin, helpful in cases with low-density infiltrates.

Effect of tyrosine hydroxylase overexpression in lymphocytes on the differentiation and function of T helper cells.

The aim of the present study was to examine the effect of the overexpression of tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines (CAs), in lymphocytes on the differentiation and function of T helper (Th) cells. A recombinant TH overexpression plasmid (pEGFP-N1-TH) was constructed and transfected into mesenteric lymphocytes using nucleofection technology. These cells were stimulated with concanavalin A (Con A) for 48 h and then examined for TH expression and CA content, as well as for the percentage of Th1 and Th2 cells, cytokine concentrations and for the levels of signaling molecules. The lymphocytes overexpressing TH also expressed higher mRNA and protein levels of TH, and synthesized more CAs, including norepinephrine (NE), epinephrine (E) and dopamine (DA) than the mock-transfected control cells. TH gene overexpression in the lymphocytes reduced the percentage of interferon-γ (IFN-γ)-producing CD4+ cells and the ratio of CD4+IFN-γ+/CD4+IL-4+ cells, as well as the percentages of CD4+CD26+ and CD4+CD30+ cells and the ratio of CD4+CD26+/CD4+CD30+ cells. TH overexpression also reduced the secretion of IFN-γ and tumor necrosis factor (TNF) from lymphocytes. Moreover, NE inhibited the Con A-induced lymphocyte proliferation and decreased both cyclic adenosine monophosphate (cAMP) levels and p38 mitogen-activated protein kinase (MAPK) expression in the lymphocytes. Our findings thus indicate that TH gene overexpression promotes the polarization and differentiation of CD4+ cells towards Th2 cells, and this effect is mediated by the cAMP and p38 MAPK signaling pathways.

Coptis chinensis Franch. extract up-regulate type I helper T-cell cytokine through MAPK activation in MOLT-4 T cell.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried rhizome of Coptis chinensis Franch. (Huanglian) has been widely used in Asian traditional medicine. It was already known that Coptis chinensis Franch. rhizome has various pharmacological properties including its anti-oxidant, anti-cancer, and anti-inflammatory activity. AIM OF THE STUDY: To evaluate the immune-enhancement effect of the Coptis chinensis Franch. rhizome extract on helper T cells and its signaling mechanism. MATERIALS AND METHODS: MOLT-4 human T cell line was used to investigate the effect of the Coptis chinensis Franch. rhizome extract. Cell viability was measured by the MTT assay and cytokine expression level was analyzed by ELISA and qRTPCR. MAPKs signal molecule's activation level was detected by immunoblotting. RESULTS: The expression of IFN-γ, a cytokine of type I helper T (Th1) cell, increased; however, IL-4 was not affected by the Coptis chinensis Franch. rhizome extract. Other Th1 cytokines, such as IL-1ß, IL-2, and IL-6, also increased. These data suggest that the Coptis chinensis Franch. rhizome extract activates MOLT-4 cell to Th1 cell, not type II helper T cell. Furthermore, the Coptis chinensis Franch. rhizome extract activates the Mitogen-activated protein kinase (MAPKs) signaling pathways. CONCLUSION: The results obtained from this study suggest that the Coptis chinensis Franch. rhizome extract should be used as an immune enhancer in anti-inflammatory medicine, adjuvant materials, and as a supplement to treat weakened immune system.

T Follicular Helper Cell-Dependent Clearance of a Persistent Virus Infection Requires T Cell Expression of the Histone Demethylase UTX.

Epigenetic changes, including histone methylation, control T cell differentiation and memory formation, though the enzymes that mediate these processes are not clear. We show that UTX, a histone H3 lysine 27 (H3K27) demethylase, supports T follicular helper (Tfh) cell responses that are essential for B cell antibody generation and the resolution of chronic viral infections. Mice with a T cell-specific UTX deletion had fewer Tfh cells, reduced germinal center responses, lacked virus-specific immunoglobulin G (IgG), and were unable to resolve chronic lymphocytic choriomeningitis virus infections. UTX-deficient T cells showed decreased expression of interleukin-6 receptor-α and other Tfh cell-related genes that were associated with increased H3K27 methylation. Additionally, Turner Syndrome subjects, who are predisposed to chronic ear infections, had reduced UTX expression in immune cells and decreased circulating CD4(+) CXCR5(+) T cell frequency. Thus, we identify a critical link between UTX in T cells and immunity to infection.

Dysfunctional BLK in common variable immunodeficiency perturbs B-cell proliferation and ability to elicit antigen-specific CD4+ T-cell help.

Common Variable Immunodeficiency (CVID) is the most prevalent primary antibody deficiency, and characterized by defective generation of high-affinity antibodies. Patients have therefore increased risk to recurrent infections of the respiratory and intestinal tract. Development of high-affinity antigen-specific antibodies involves two key actions of B-cell receptors (BCR): transmembrane signaling through BCR-complexes to induce B-cell differentiation and proliferation, and BCR-mediated antigen internalization for class-II MHC-mediated presentation to acquire antigen-specific CD4(+) T-cell help.We identified a variant (L3P) in the B-lymphoid tyrosine kinase (BLK) gene of 2 related CVID-patients, which was absent in healthy relatives. BLK belongs to the Src-kinases family and involved in BCR-signaling. Here, we sought to clarify BLK function in healthy human B-cells and its association to CVID.BLK expression was comparable in patient and healthy B-cells. Functional analysis of L3P-BLK showed reduced BCR crosslinking-induced Syk phosphorylation and proliferation, in both primary B-cells and B-LCLs. B-cells expressing L3P-BLK showed accelerated destruction of BCR-internalized antigen and reduced ability to elicit CD40L-expression on antigen-specific CD4(+) T-cells.In conclusion, we found a novel BLK gene variant in CVID-patients that causes suppressed B-cell proliferation and reduced ability of B-cells to elicit antigen-specific CD4(+) T-cell responses. Both these mechanisms may contribute to hypogammaglobulinemia in CVID-patients.

Leukocyte cathepsin C deficiency attenuates atherosclerotic lesion progression by selective tuning of innate and adaptive immune responses.

OBJECTIVE: The protein degrading activity of cathepsin C (CatC), combined with its role in leukocyte granule activation, suggests a contribution of this cystein protease in atherosclerosis. However, no experimental data are available to validate this concept. APPROACH AND RESULTS: CatC gene and protein expression were increased in ruptured versus advanced stable human carotid artery lesions. To assess causal involvement of CatC in plaque progression and stability, we generated LDLr(-/-)//CatC(-/-) chimeras by bone marrow transplantation. CatC(-/-) chimeras presented attenuated plaque burden in carotids, descending aorta, aortic arch and root, at both the early and advanced plaque stage. CatC was abundantly expressed by plaque macrophages and foam cells. CatC expression and activity were dramatically downregulated in plaques of CatC(-/-) chimeras, supporting a hematopoietic origin of plaque CatC. Our studies unveiled an unexpected feedback of CatC deficiency on macrophage activation programs and T helper cell differentiation in as much as that CatC expression was upregulated in M1 macrophages, whereas its deficiency led to combined M2 (in vitro) and Th2 polarization (in vivo). CONCLUSIONS: Our data implicate CatC has a role in the selective tuning of innate and adaptive immune responses, relevant to a chronic immune disease, such as atherosclerosis.

Critical role of p38 and GATA3 in natural helper cell function.

Natural helper (NH) cells, a member of Lin(-)IL-2R(+)IL-7R(+)IL-25R(+)IL-33R(+)GATA3(+) group 2 innate lymphoid cell subset, are characterized by the expression of transcription factors GATA3 and RORα and production of large amounts of Th2 cytokines such as IL-5, IL-6, and IL-13 upon IL-33 stimulation or a combination of IL-2 and IL-25. We have studied the signal transduction pathways critical for the cytokine expression and development of NH cell. Either stimulation with IL-33 or a combination of IL-2 and IL-25 induced p38 activation and phosphorylation of GATA3 in NH cells, and the phosphorylated form of GATA3 bound to the IL-5 and IL-13 promoters. All these events were blocked by SB203580, a p38 inhibitor. Inhibition of p38 also blocked IL-6 production. The mature NH cells lacking Gata3 were impaired in the proliferation and production of IL-5 and IL-13, but not IL-6, indicating that both p38 and GATA3 are critical for the proliferation and production of IL-5 and IL-13 and that the mechanisms downstream of p38 differ between IL-6 and IL-5/IL-13. In contrast, the NH cells lacking RORα showed no impairment in the proliferation and cytokine production, indicating that GATA3 but not RORα plays a pivotal role in the effector functions of mature NH cell. However, deletion of either GATA3 or RORα in hematopoietic stem cells severely blocked the development into NH cells. Our results demonstrate the important roles of p38 and GATA3 in NH cell functions.

The steroidogenic enzyme Cyp11a1 is essential for development of peanut-induced intestinal anaphylaxis.

BACKGROUND: Cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone. Cyp11a1 expression is increased in activated T cells. OBJECTIVES: We sought to determine the role of Cyp11a1 activation in the development of peanut allergy and TH cell functional differentiation. METHODS: A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on T(H)1, T(H)2, and T(H)17 differentiation were determined. Cyp11a1 gene silencing was performed with Cyp11a1-targeted short hairpin RNA. RESULTS: Peanut sensitization and challenge resulted in diarrhea, inflammation, and increased levels of Cyp11a1, IL13, and IL17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG treatment decreased IL13 and IL17A mRNA expression in the small intestine without affecting Cyp11a1 mRNA or protein levels. In vitro the inhibitor decreased IL13 and IL17A mRNA and protein levels in differentiated T(H)2 and T(H)17 CD4 T cells, respectively, without affecting GATA3, retinoic acid-related orphan receptor γt (RORγt), or T(H)1 cells and IFNG and T-bet expression. Short hairpin RNA-mediated silencing of Cyp11a1 in polarized T(H)2 CD4 T cells significantly decreased pregnenolone and IL13 mRNA and protein levels. CONCLUSION: Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T(H)2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.

Gcn5 is required for PU.1-dependent IL-9 induction in Th9 cells.

Naive CD4+ T cells differentiate into various effector Th subsets depending on the Ags and cytokine microenvironment they encounter. IL-9-secreting Th9 cells are the most recent Th subset to be described. PU.1, one of the transcription factors required for the development of Th9 cells, binds to the Il9 gene. In this study, we show that PU.1 increases histone acetylation at the Il9 locus through direct interactions with histone acetyltransferases. In the absence of PU.1, there is decreased association of Gcn5 and p300/CBP associated factor and increased association of histone deacetylases at the Il9 locus in Th9 cells. Inhibition of histone deacetylase activity augments PU.1-dependent IL-9 production. PU.1 forms a complex with Gcn5, and inhibition of the expression of Gcn5 results in reduced IL-9 production. Moreover, the effects of Gcn5 on IL-9 production are specific as the production of IL-10 and IL-21, two additional cytokines produced by Th9 cells, is not altered after decreased Gcn5 expression. Together, these data define a PU.1-dependent mechanism for altered histone acetylation and expression of the Il9 locus in Th9 cells.

A selective inhibitor reveals PI3Kγ dependence of T(H)17 cell differentiation.

We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.

Can we see PIP(3) and hydrogen peroxide with a single probe?

A genetically encoded sensor for parallel measurements of phosphatidylinositol 3-kinase activity and hydrogen peroxide (H(2)O(2)) levels (termed PIP-SHOW) was developed. Upon elevation of local phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) concentration, the sensor translocates from the cytosol to the plasma membrane, while a ratiometric excitation change rapidly and simultaneously reports changes in the concentration of H(2)O(2). The dynamics of PIP(3) and H(2)O(2) generation were monitored in platelet-derived growth factor-stimulated fibroblasts and in T-lymphocytes after formation of an immunological synapse. We suggest that PIP-SHOW can serve as a prototype for many fluorescent sensors with combined readouts.

Distinct proteinase 3-induced cytokine patterns in Wegener´s granulomatosis, Churg-Strauss syndrome, and healthy controls.

OBJECTIVES: To analyse whether a specific cytokine pattern is elicited in response to the autoantigen proteinase 3 (PR3) in active Wegener's granulomatosis (WG). METHODS: Six-colour flow cytometry was used to analyse cytokine production and surface markers of the total CD4+ T-cell population ex vivo and in PR3-stimulated T-cell lines of patients with active PR3-ANCA-positive WG, PR3-ANCA-negative Churg-Strauss syndrome (CSS), and healthy controls (HC). RESULTS: The cytokine response of the total PB CD4+ T cell population was skewed towards distinct pro-inflammatory cytokine patterns in WG (Th1-type) and CSS (Th17, Th1-/Th2-type). Th2-type as well as Th17 cell populations including Th17/Th1, Th17/Th2 and Th22 cells were elicited in response to PR3 stimulation in WG. In contrast, CSS patients displayed a Th2-type dominated response following PR3 stimulation. CONCLUSIONS: These data suggest that the cytokine response of the total CD4+ T-cell population and PR3-specific cells is influenced by the underlying disorder.

PI3Kγ deficiency delays the onset of experimental autoimmune encephalomyelitis and ameliorates its clinical outcome.

PI3Ks control signal transduction triggered by growth factors and G-protein-coupled receptors and regulate an array of biological processes, including cellular proliferation, differentiation, survival and migration. Herein, we investigated the role of PI3Kγ in the pathogenesis of EAE. We show that, in the absence of PI3Kγ expression, clinical signs of EAE were delayed and mitigated. PI3Kγ-deficient myelin oligodendrocyte glycoprotein (MOG)(35-55) -specific CD4(+) T cells appeared later in the secondary lymphoid organs and in the CNS than their WT counterparts. Transfer of WT CD4(+) cells into PI3Kγ(-/-) mice prior to MOG(35-55) immunisation restored EAE severity to WT levels, supporting the relevance of PI3Kγ expression in Th cells for the pathogenesis of EAE; however, PI3Kγ was dispensable for Th1 and Th17 differentiation, thus excluding an altered expression of these pathogenetically relevant cytokines as the cause for ameliorated EAE in PI3Kγ(-/-) mice. These findings demonstrate that PI3Kγ contributes to the development of autoimmune CNS inflammation.

Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells.

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3ß protein levels in Th17 cells, whereas GSK3ß was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.

Phosphoinositide 3-kinase activity in T cells regulates the magnitude of the germinal center reaction.

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (T(FH)) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of T(FH) cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key T(FH) cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in T(FH) cells.

Activating and inhibitory functions for the histone lysine methyltransferase G9a in T helper cell differentiation and function.

Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However, whether G9a plays a similar role in adult cells is still unclear. We identify a critical role for G9a in CD4(+) T helper (Th) cell differentiation and function. G9a-deficient Th cells are specifically impaired in their induction of Th2 lineage-specific cytokines IL-4, IL-5, and IL-13 and fail to protect against infection with the intestinal helminth Trichuris muris. Furthermore, G9a-deficient Th cells are characterised by the increased expression of IL-17A, which is associated with a loss of H3K9me2 at the Il17a locus. Collectively, our results establish unpredicted and complex roles for G9a in regulating gene expression during lineage commitment in adult CD4(+) T cells.

Pharmacologic inhibition of MEK-ERK signaling enhances Th17 differentiation.

The cytokines and transcription factors that promote Th17 cell development have been extensively studied. However, the signaling pathways that antagonize Th17 differentiation remain poorly characterized. In this study, we report that pharmacologic inhibition of MEK-ERK signaling enhances the in vitro differentiation of Th17 cells and increases their gene expression of il-17a, il-17f, il-21, il-22, and il-23r. IL-2, which suppresses Th17 differentiation via STAT5 activation, also acts through ERK signaling to inhibit Th17 generation. In turn, ERK signaling is found to potentiate the production of IL-2 and activate STAT5, suggesting the existence of an autoregulatory loop to constrain Th17 development. Finally, compared with the transfer of untreated Th17 cells, the transfer of ERK-inhibited Th17 cells leads to accelerated onset and exacerbated colitis in immunodeficient mice. Our data indicate that MEK-ERK signaling negatively regulates Th17 differentiation in a Th cell-intrinsic manner.

Distinct metabolism of cyclic adenosine monophosphate in regulatory and helper CD4+ T cells.

Regulatory T cells (Treg) are crucial for the suppression of antigen-specific immune responses by activated conventional T cells (Tcon). It has been recently reported that this suppression is mediated by cyclic adenosine monophosphate (cAMP) transported from Treg to Tcon via gap junctions. However, the underlying biochemical mechanisms of cAMP accumulation in activated Treg are still unclear. Here we reported that although non-activated murine Treg and Tcon displayed similar intracellular cAMP amounts, both subpopulations showed distinct expression of enzymes regulating cAMP metabolism. Thus, in Treg, activities of both anabolic (adenylyl cyclase, AC) and catabolic (phosphodiesterase, PDE) enzymes were lower than in Tcon. Furthermore, we demonstrated for the first time the expression of the PDE11 protein in murine Treg and Tcon. Treg activation by IL-2 induced a strong AC7 activation and cAMP accumulation in Treg. In contrast, Tcon showed a significant decrease in the AC7 activity and cAMP amounts under these conditions. Our data suggest that the mechanism of cAMP accumulation in stimulated Treg involves the AC7 activation and provide new insight into the modulation of Treg activities via AC inhibition or stimulation in various pathological processes like tumor and autoimmune diseases.