Th1 Phenotype Research Articles

The T-box transcription factors T-bet and Eomes repress protective Th17 CD4 T cell responses against Influenza A Virus

Abstract Promoting strong CD4 cell immunity is an attractive strategy to generate a universal Influenza A Virus (IAV) vaccine. Though most CD4 cell responses against IAV are classified as Th1, Th17 and cytotoxic CD4 cells (ThCTL) may also contribute to viral clearance. Interestingly, we find the transcription factor T-bet (Tbx21), the ‘master regulator’ of Th1 differentiation, to be dispensable for protective CD4 responses against IAV. Surprisingly, Tbx21−/− cells develop Th17 characteristics but retain Th1 functionality, highlighted by strong IFN-γ production. While the transcription factor Eomesodermin (Eomes) can compensate for loss of T-bet to promote antiviral CD8 responses, its roles in CD4 cells is still unclear. Here we analyze Eomes−/−, Tbx21−/− and Tbx21−/−Eomes−/− CD4 cell responses against IAV using an adoptive transfer model to determine the extent to which Eomes regulates antiviral CD4 functions. Eomes−/− cells mirrored the prototypical Th1 responses of WT CD4 cells. Strikingly, Tbx21−/−Eomes−/− cells exhibited near complete loss of both Th1 responses and ThCTL function. In contrast, Tbx21−/− Eomes−/− developed stronger Th17 attributes than Tbx21−/− cells. We assessed protection provided by transfer of in vitro Th17-primed WT, Eomes−/−, Tbx21−/− and Tbx21−/−Eomes−/− cells to unprimed WT mice infected with lethal IAV. While WT and Eomes−/− effectors gained Th1 characteristics in vivo, Tbx21−/−Eomes−/− cells strengthened their Th17 phenotype. Remarkably, all of the Th17 effectors were equally protective. Our observations reinforce the concept that Th17 cells can provide strong protection against IAV, independently of Th1 and ThCTL responses, and suggest Th17 cells use unique pathways to do so.

Classical splenic dendritic cell subsets, cDC1 and cDC2, from C57BL/6 mice are more potent in stimulating the Th1 immune response than those from BALB/c mice during mousepox

Abstract Classical (conventional) dendritic cells (cDCs) specialize in presenting antigen to naïve T cells, and are divided into cDC1 (CD8α+) and cDC2 (CD11b+) subpopulations based on their lineage, surface phenotype and characteristics. Despite differences, both cDC subsets can stimulate CD4+ T cells and promote polarization toward a T helper 1 (Th1) phenotype. Many studies have shown that C57BL/6 (H-2b) and BALB/c (H-2d) mice readily mount vigorous Th1 or Th2 responses, respectively, upon infection with different infectious agents, including ectromelia virus (ECTV), responsible for mousepox in mice. In this work, we compared the ability of cDC subsets from mouse strains with different susceptibility to mousepox (C57BL/6 – resistant and BALB/c – susceptible) to stimulate the Th1 cytokine immune response during ECTV infection. Results showed that splenic cDC1 and cDC2 from BALB/c mice highly express MHC class II, CD83 and/or CD86 than those from C57BL/6 mice at 5 days post infection with ECTV. Despite higher activation status, both subsets of cDCs from BALB/c mice produce low amounts of Th1-polarizing cytokines, including IL-12 and IFN-γ, than those from C57BL/6 mice. Moreover, splenic cDC1 and cDC2 cells, from ECTV-infected C57BL/6 mice, stimulated higher proliferation and production of IFN-γ and IL-2 by allogeneic CD4+ T cells of C3H (H-2k) mice in a mixed leukocyte reaction than BALB/c mice. Both subsets of BALB/c cDCs up-regulated genes engaged in cell maturation and activation compared to C57BL/6 cDCs. Overall, our data indicate that both, cDC1 and cDC2 promote CD4+ T cell differentiation into IFN-γ-producing Th1 subset, and so ensure development of a strong cell-mediated immune response and recovery of C57BL/6 mice from mousepox.

In Silico Modeling of Metabolic State in Single Th17 Cells Reveals Novel Regulators of Inflammation and Autoimmunity

Abstract Cellular metabolism is a major regulator of immune response, but it is difficult to study the metabolic status of an individual immune cell using current technologies. Here, we present Compass, an algorithm to characterize the metabolic landscape of single cells in silico based on single-cell RNA-Seq and flux balance analysis. We used Compass to study the landscape of metabolic heterogeneity in Th17 cells and search for novel metabolic regulators of their inflammatory function. In central carbon metabolism, Compass predicted a metabolic switch between glycolysis and fatty acid oxidation that mirrors the Th17 vs. Treg phenotype, which we validated through transcriptomic, metabolic and functional assays. The TCA cycle was predicted to break at two points, both of which have been independently identified by other groups in M1 macrophage polarization. Surprisingly, and contrary to common immunometabolic understanding, Compass predicted that glycolysis too was divided into functional modules, and that one of them supported an anti-inflammatory phenotype. We validate the paradoxical prediction and demonstrate that inhibition of this module promotes a pro-inflammatory transcriptional program in Th17 cells, resulting in neuroinflammation in an adoptive transfer model of autoimmune disease. In conclusion, Compass is a widely applicable algorithm to characterize metabolic states at single cell resolution. It allows associating cellular metabolic states with effector functions and detection of metabolic targets that regulate effector phenotypes. We expect it to become a widely used tool with the increasing availability of single-cell RNA-Seq data, spurred by efforts such as the human cell atlas.

Immunomodulation by dimethyl fumarate treatment improves mouse sciatic nerve regeneration

Traumatic injury to the peripheral nervous system (PNS) often generates sensorimotor deficits that impair the quality of life of the patient. The success of nerve regeneration is related to tissue clearance and the formation of a microenvironment that sustains and stimulates axon growth up to the target. In this sense, macrophages are important for axon and myelin debris removal, neovascularization and the production of neurotrophic factors. Macrophage activation is improved by T helper (Th) lymphocytes, whose role remains few explored upon traumatic nerve injuries. Dimethyl fumarate (DMF) is the first-line drug for the treatment of multiple sclerosis due to its neuroprotective, anti-inflammatory and immunomodulatory properties. DMF improves nerve regeneration via antioxidant and cytoprotective cell signaling pathways. However, the direct activity on the cell immune response following nerve axotomy requires further investigation. In the present study, we evaluated DMF activity on Th cells and macrophage polarization, axonal regeneration and motor recovery following sciatic nerve crush in mice. For this aim, operated animals received DMF or vehicle once a day, starting at 3 days postinjury (dpi). Using an in vivo cell migration assay, we observed reduced lymphocyte infiltration in the nerves of DMF-treated mice at 7 dpi. Flow cytometry revealed DMF-responsive lymphocyte polarization from the pro- (Th1) to anti-inflammatory (Th2) phenotype at 7 dpi but not at 14 dpi. No effect was observed on macrophage polarization (from M1 to M2), although DMF reduced the frequency of the proinflammatory M1 subset from 7 to 14 dpi. Quantification of neurofilament (axon marker) and growth-associated protein 43 (GAP-43) immunolabeling showed improved axonal regeneration under DMF treatment at 14 dpi. Better motor recovery was observed in the DMF-treated group, as verified by an automated walking track test. Overall, our data reinforce the pro-regenerative capacity of DMF after traumatic nerve injury based on downmodulation of the proinflammatory immune response.

Single-cell transcriptomics identifies an effectorness gradient shaping the response of CD4+ T cells to cytokines

Naïve CD4+ T cells coordinate the immune response by acquiring an effector phenotype in response to cytokines. However, the cytokine responses in memory T cells remain largely understudied. Here we use quantitative proteomics, bulk RNA-seq, and single-cell RNA-seq of over 40,000 human naïve and memory CD4+ T cells to show that responses to cytokines differ substantially between these cell types. Memory T cells are unable to differentiate into the Th2 phenotype, and acquire a Th17-like phenotype in response to iTreg polarization. Single-cell analyses show that T cells constitute a transcriptional continuum that progresses from naïve to central and effector memory T cells, forming an effectorness gradient accompanied by an increase in the expression of chemokines and cytokines. Finally, we show that T cell activation and cytokine responses are influenced by the effectorness gradient. Our results illustrate the heterogeneity of T cell responses, furthering our understanding of inflammation.

CD4+ T Cells in the Blood of MS Patients Respond to Predicted Epitopes From B cell Receptors Found in Spinal Fluid.

B cells are important pathogenic players in multiple sclerosis (MS), but their exact role is not known. We have previously demonstrated that B cells from cerebrospinal fluid (CSF) of MS patients can activate T cells that specifically recognize antigenic determinants (idiotopes) from their B cell receptors (BCRs). The aim of this study was to evaluate whether in silico prediction models could identify antigenic idiotopes of immunoglobulin heavy-chain variable (IGHV) transcriptomes in MS patients. We utilized a previously assembled dataset of CSF IGHV repertoires from MS patients. To guide selection of potential antigenic idiotopes, we used in silico predicted HLA-DR affinity, endosomal processing, as well as transcript frequency from nine MS patients. Idiotopes with predicted low affinity and low likelihood of cathepsins cleavage were inert controls. Peripheral blood mononuclear cells from these patients were stimulated with the selected idiotope peptides in presence of anti-CD40 for 12 h. T cells were then labeled for activation status with anti-CD154 antibodies and CD3+CD4+ T cells phenotyped as memory (CD45RO+) or naïve (CD45RO−), with potential for brain migration (CXCR3 and/or CCR6 expression). Anti-CD14 and -CD8 were utilized to exclude monocytes and CD8+ T cells. Unstimulated cells or insulin peptides were negative controls, and EBNA-1 peptides or CD3/CD28 beads were positive controls. The mean proportion of responding memory CD4+ T cells from all nine MS patients was significantly higher for idiotope peptides with predicted high HLA-DR affinity and high likelihood of cathepsin cleavage, than toward predicted inert peptides. Responses were mainly observed toward peptides affiliated with the CDR3 region. Activated memory CD4+ T cells expressed the chemokine receptor CCR6, affiliated with a Th17 phenotype and allowing passage into the central nervous system (CNS). This in vitro study suggests that that antigenic properties of BCR idiotopes can be identified in silico using HLA affinity and endosomal processing predictions. It further indicates that MS patients have a memory T cell repertoire capable of recognizing frequent BCR idiotopes found in endogenous CSF, and that these T cells express chemokine receptors allowing them to reach the CSF B cells expressing these idiotopes.

Abstract B45: NFAT5/TonEBP mediated anti-tumor efficiency of high salt activated CD4+T lymphocytes

Abstract Cell-based immunotherapy is rapidly emerging as a promising alternative to conventional chemotherapy-based treatment for cancer. Previous studies have shown that high salt treatment induces naive CD4+T lymphocytes to differentiate into a highly proinflammatory Th17 phenotype. In our current study, we analyzed the ability of salt treatment to induce cancer-specific Th17 lymphocytes. Naive CD4+T cells isolated by immunomagnetic bead isolation from splenocytes of 10-week-old BALB/c mice were treated in vitro with high salt (Δ0.05 M NaCl) along with CD3 monoclonal antibodies (mAbs), CD28 mAbs and IL-2 for 4 days along with heat killed 4T1 breast cancer cells. The isolated CD4+CCR6+CD45RA-CXCR3- cells were tested for IL-17 expression by flow cytometry and determined to be >95% pure. Orthotopic murine breast tumor models established by injection of 4T1 cells into the mammary fat pad of Nu/J mice were administered two doses of (1 × 106) salt induced Th17 cells on day 7 and day 12, post-tumor implantation. By day 28, the high salt induced Th17 cells cohort demonstrated significantly reduced tumor growth (97 ± 34 mm3 vs 512 ± 91 mm3, untreated control, p<0.05) and lung metastasis. However, no effect on tumor growth was noted when these 4T1 sensitized high salt treated Th17 cells were injected into Nu/J mice with prostrate (RM1) and lung tumors (KLN205), thus suggesting that the specificity of salt induced Th17 cells to breast tumors. Similarly, in vitro high salt mediated differentiation of Th17 cells when co-treated with heat killed RM1 and KLN205 cancer cells were able to induce respective murine tumor regression. Mechanistic studies demonstrated that the high salt induction to the Th17 phenotype was mediated by the tonicity-dependent transcription factor, TonEBP/NFAT5. The shRNA-based knockdown of TonEBP abrogated the induction of the Th17 phenotype and expression of proinflammatory cytokines IL-17, IL-1β and IL-6. Taken together, our data suggests that high salt mediated in vitro differentiation of Th17 cells could induce tumor specific anticancer CD4+T lymphocytes, which may give rise to a cell-based anticancer immunotherapy application. Citation Format: Suneetha Amara, Pooja Yenuga, Roy Zent, Venkataswarup Tiriveedhi. NFAT5/TonEBP mediated anti-tumor efficiency of high salt activated CD4+T lymphocytes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B45.

Proteomic Changes in Intracranial Blood During Human Ischemic Stroke

IntroductionSince 2015, mechanical thrombectomy is the standard treatment for emergent large vessel occlusion stroke. The previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) protocol (clinicaltrials.gov NCT03153683) utilizes thrombectomy to isolate distal blood within the artery immediately downstream from the clot, peripheral blood proximal to the clot, and the thrombus.MethodsTissue samples were obtained in accordance with the established BACTRAC protocol and immediately frozen at −80°C. For this study, we compared the intracranial intraluminal blood distal to the thrombus, using each subject’s systemic arterial blood as an internal comparative control. 40μl aliquots of plasma were thawed and randomized to 96‐well plates and shipped on dry ice to Olink Proteomics (Olink Proteomics, Boston, MA). Cardiometabolic and inflammatory panels were run on 1μl aliquots using a proximity extension assay (PEA) with proximal blood plasma as an internal control for each subject and distal blood plasma as the injury site sample. Protein data were analyzed as average protein expression levels in distal plasma as compared to average protein expression levels in proximal plasma.Results25 adult subjects (>18yrs) were included in the study, of which 15 (60%) were female. Median age was 64 (24–91). 16 subjects (64%) had hypertension, 15 (60%) had BMI > 25, 10 (40%) were active smokers, 6 (24%) had previous stroke, 4 (16%) had type II diabetes, and 4 (16%) had hyperlipidemia. Mean infarct time was 513 ± 246 minutes and mean infarct volume was 58,172 ± 82,284 mm3. Proteins with the greatest change in expression in the cardiometabolic panel were superoxide dismutase 1 (SOD1), CD59, lipocalin‐2 (LCN2), and defensin alpha 1 (DEFA1). Proteins with greatest change in expression in the inflammatory panel were interleukin 4 (IL‐4), AXIN1, interleukin 1 alpha (IL‐1a), sirtuin 2 (SIRT2), interleukin 5 (IL‐5) and leukemia inhibitory factor (LIF). There was a significant sex difference in expression levels of interleukin 20 receptor subunit alpha (IL‐20RA) (p=0.04) and IL‐4 (p=0.03).ConclusionsThese protein data show a complex set of intraluminal responses occurring distal to the thrombus in acute ischemic stroke. Changes in SOD1, LIF, and SIRT2 demonstrate neuroprotective and anti‐inflammatory activity, LCN2 and DEFA1 suggest a response similar to antimicrobial activity, and proteins such as IL‐4 and IL‐5 suggest eosinophil activation and a Th2 phenotype. Future studies will focus on the relationships of proteins with infarct time, infarct volume, and functional outcomes. Such analyses will provide insight into the initial molecular and cell signaling in response to ischemic stroke in the human condition.Support or Funding InformationThe project described was supported by the National Center for Advancing Translational Sciences, through Grant UL1TR001998 and UKHealthCare. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Grant through Center for Clinical and Translational Science

IL-9 exerts biological function on antigen-experienced murine Tcells and exacerbates colitis induced by adoptive transfer.

IL-9 is involved in various Tcell-dependent inflammatory models including colitis, encepahlitis, and asthma. However, the regulation and specificity of IL-9 responsiveness by Tcells during immune responses remains poorly understood. Here, we addressed this question using two different models: experimental colitis induced by transfer of naive CD4+ CD45RBhigh Tcells into immunodeficient mice, and OVA-specific Tcell activation. In the colitis model, constitutive IL-9 expression exacerbated inflammation upon transfer of CD4+ CD45RBhigh Tcells from WT but not from Il9r-/- mice, indicating that IL-9 acts directly on Tcells. Suprisingly, such naïve CD4+ CD45RBhigh Tcells failed to express the Il9r or respond to IL-9 in vitro, in contrast with CD4+ CD45RBlow Tcells. By using OVA-specific Tcells, we observed that Tcells acquired the capacity to respond to IL-9 along with CD44 upregulation, after long-lasting (5 to 12days) in vivo antigenic stimulation. Il9r expression was associated with Th2 and Th17 phenotypes. Interestingly, in contrast to the IL-2 response, antigen restimulation downregulated IL-9 responsiveness. Taken together, our results demonstrate that IL-9 does not act on naïve Tcells but that IL-9 responsiveness is acquired by CD4+ Tcells after in vivo activation and acquisition of memory markers such as CD44.

Immunomodulatory effects of pevonedistat, a NEDD8-activating enzyme inhibitor, in chronic lymphocytic leukemia-derived T cells.

Novel targeted agents used in therapy of lymphoid malignancies, such as inhibitors of B-cell receptor-associated kinases, are recognized to have complex immune-mediated effects. NEDD8-activating enzyme (NAE) has been identified as a tractable target in chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma. We and others have shown that pevonedistat (TAK-924), a small-molecule inhibitor of NAE, abrogates NF-κB signaling in malignant B cells. However, NF-κB pathway activity is indispensable in immune response, and T-cell function is altered in patients with CLL. Using T cells derived from patients with CLL, we demonstrate that although targeting NAE results in markedly differential expression of NF-κB-regulated genes and downregulation of interleukin (IL)-2 signaling during T-cell activation, T cells evade apoptosis. Meanwhile, NAE inhibition favorably modulates polarization of T cells in vitro, with decreased Treg differentiation and a shift toward TH1 phenotype, accompanied by increased interferon-γ production. These findings were recapitulated in vivo in immunocompetent mouse models. T cells exposed to pevonedistat in washout experiments, informed by its human pharmacokinetic profile, recover NAE activity, and maintain their response to T-cell receptor stimulation and cytotoxic potential. Our data shed light on the potential immune implications of targeting neddylation in CLL and lymphoid malignancies.

Evidence for Associations Between Th1/Th17 "Hybrid" Phenotype and Altered Lipometabolism in Very Severe Graves Orbitopathy.

The purpose of this article is to investigate the characteristics of Th1-cell and Th17-cell lineages for very severe Graves orbitopathy (GO) development. Flow cytometry was performed with blood samples from GO and Graves disease (GD) patients and healthy controls, to explore effector T-cell phenotypes. Lipidomics was conducted with serum from very severe GO patients before and after glucocorticoid (GC) therapy. Immunohistochemistry and Western blotting were used to examine orbital-infiltrating Th17 cells or in vitro models of Th17 polarization. In GD, Th1 cells predominated in peripheral effector T-cell subsets, whereas in GO, Th17-cell lineage predominated. In moderate-to-severe GO, Th17.1 cells expressed retinoic acid receptor-related orphan receptor-γt (RORγt) independently and produced interleukin-17A (IL-17A), whereas in very severe GO, Th17.1 cells co-expressed RORγt and Tbet and produced interferon-γ (IFN-γ). Increased IFN-γ-producing Th17.1 cells positively correlated with GO activity and were associated with the development of very severe GO. Additionally, GC therapy inhibited both Th1-cell and Th17-cell lineages and modulated a lipid panel consisting of 79 serum metabolites. However, in GC-resistant, very severe GO, IFN-γ-producing Th17.1 cells remained at a high level, correlating with increased serum triglycerides. Further, retro-orbital tissues from GC-resistant, very severe GO were shown to be infiltrated by CXCR3+ Th17 cells expressing Tbet and STAT4 and rich in triglycerides that promoted Th1 phenotype in Th17 cells in vitro. Our findings address the importance of Th17.1 cells in GO pathogenesis, possibly promoting our understanding of the association between Th17-cell plasticity and disease severity of GO.

CD4+ Memory Stem T Cells Recognizing Citrullinated Epitopes Are Expanded in Patients With Rheumatoid Arthritis and Sensitive to Tumor Necrosis Factor Blockade.

Memory stem T (Tscm) cells are long-lived, self-renewing T cells that play a relevant role in immunologic memory. This study was undertaken to investigate whether Tscm cells accumulate in rheumatoid arthritis (RA). The polarization and differentiation profiles of circulating T cells were assessed by flow cytometry. Antigen-specific T cells were characterized by staining with major histocompatibility complex class II tetramers. The T cell receptor (TCR) repertoire was analyzed by high-throughput sequencing using an unbiased RNA-based approach in CD4+ T cell subpopulations sorted by fluorescence-activated cell sorting. We analyzed the dynamics of circulating Tscm cells (identified as CD45RA+CD62L+CD95+ T cells) by flow cytometry in 27 RA patients, 16 of whom were also studied during treatment with the anti-tumor necrosis factor (anti-TNF) agent etanercept. Age-matched healthy donors were used as controls. CD4+ Tscm cells were selectively and significantly expanded in RA patients in terms of frequency and absolute numbers, and significantly contracted upon anti-TNF treatment. Expanded CD4+ Tscm cells displayed a prevalent Th17 phenotype and a skewed TCR repertoire in RA patients, with the 10 most abundant clones representing up to 53.7% of the detected sequences. CD4+ lymphocytes specific for a citrullinated vimentin (Cit-vimentin) epitope were expanded in RA patients with active disease. Tscm cells accounted for a large fraction of Cit-vimentin-specific CD4+ cells. Our results indicate that Tscm cells, including expanded clones specific for relevant autoantigens, accumulate in RA patients not exposed to biologic agents, and might be involved in the natural history of the disease. Further analysis of Tscm cell dynamics in autoimmune disorders may have implications for the design and efficacy assessment of innovative therapies.

Protection against Trichinella spiralis in BALB/c mice via oral administration of recombinant Lactobacillus plantarum expressing murine interleukin-4

Interleukin-4 (IL-4), an immunomodulatory cytokine derived from activated T lymphocytes were shown to regulate Th2-type immune responses. It plays an important role in anti-parasitic infections. In this study, a recombinant plasmid was designed using murine IL-4 co-expressed with pgsA anchor system of Lactobacillus plantarum NC8 and was delivered by live Lactobacillus plantarum NC8, which exhibited an enhanced immunogenicity in protection of BALB/c mice from infection with Trichinella spiralis. The results showed that the levels of serum IgG1 and mucosal secretory IgA (sIgA) were both increased significantly in mice orally inoculated with NC8-pSIP409-pgsA-mIL-4, and the Th2 phenotype immune response was up-regulated. A 29.9 % reduction in adult worm burden at 7 days post-infection (dpi) and 83.3 % reduction in muscle larvae burden at 28 dpi were observed in immune-stimulated mice with NC8-pSIP409-pgsA-mIL-4. Moreover, weight loss and pathological changes were also improved in mice of NC8-pSIP409-pgsA-mIL-4 group. Taken together, it suggests that NC8-pSIP409-pgsA-mIL-4 could improve the intestinal mucosal immunity and promoted the elimination of the adult worm in Trichinella-infected mice. This study laid the foundation for the development of a novel vaccines against Trichinellosis.

Leishmania donovani infection induce differential miRNA expression in CD4+ T cells

Visceral leishmaniasis is characterized by mixed production of Th1/2 cytokines and the disease is established by an enhanced level of Th2 cytokine. CD4+ T cells are main cell type which produces Th1/2 cytokine in the host upon Leishmania infection. However, the regulatory mechanism for Th1/2 production is not well understood. In this study, we co-cultured mice CD4+ T cells with Leishmania donovani infected and uninfected macrophage for the identification of dysregulated miRNAs in CD4+ T cells by next-generation sequencing. Here, we identified 604 and 613 known miRNAs in CD4+ T cells in control and infected samples respectively and a total of only 503 miRNAs were common in both groups. The expression analysis revealed that 112 miRNAs were up and 96 were down-regulated in infected groups, compared to uninfected control. Nineteen up-regulated and 17 down-regulated miRNAs were statistically significant (p < 0.05), which were validated by qPCR. Further, using insilco approach, we identified the gene targets of significant miRNAs on the basis of CD4+ T cell biology. Eleven up-regulated miRNAs and 9 down-regulated miRNAs were associated with the cellular immune responses and Th1/2 dichotomy upon Leishmania donovani infection. The up-regulated miRNAs targeted transcription factors that promote differentiation of CD4+ T cells towards Th1 phenotype. While down-regulated miRNAs targeted the transcription factors that facilitate differentiation of CD4+ T cells towards Th2 populations. The GO and pathway enrichment analysis also showed that the identified miRNAs target the pathway and genes related to CD4+ T cell biology which plays important role in Leishmania donovani infection.

Altered T cell plasticity favours Th17 cells in early arthritis.

The predominance of differentiated Th17 cells has been implied as a key driver of autoimmune arthritis, including early RA. Because accumulating evidence suggests that Th cell differentiation is a plastic process, we investigated plasticity and underlying molecular mechanisms to address the shift towards the Th17 phenotype in early RA. A cohort of 61 patients with early, active, untreated RA and 45 age- and sex-matched healthy controls were studied. Viable in vitro- and in vivo-generated Th1, Th2 and Th17 cells were FACS-sorted and transdifferentiated under Th1-, Th2- or Th17-inducing conditions. The cytokine Th profile of the transdifferentiated cells was assessed by flow cytometry. Th cell-associated cytokine and transcription factor gene loci were analysed by chromatin immunoprecipitation assay and their expression by quantitative real-time PCR. In vitro-generated Th cells showed substantial plasticity, which was similar between RA and healthy controls, whereas in vivo-derived Th1 and Th2 cells from RA patients demonstrated an enhanced plasticity towards IL-17-expressing phenotypes compared with healthy controls. Further, in vivo-generated Th17 cells from RA patients showed a resistance to transdifferentiate into Th1 or Th2 cells. The serum/glucocorticoid-regulated kinase 1-forkhead box protein O1-IL-23 receptor (SGK1-FOXO1-IL-23R) axis together with increased RORC expression was associated with the predominant Th17 phenotype in early RA. Our data indicate that in vivo-originated Th subsets are prone to Th17 cell transdifferentiation in early RA, while Th17 cells are resistant to changes in their phenotype. Together, the data imply that an altered plasticity contributes to the Th17 shift in early RA.

Neuroinflammation after Intracerebral Hemorrhage and Potential Therapeutic Targets

Spontaneous intracerebral hemorrhage (ICH) is a catastrophic illness causing significant morbidity and mortality. Despite advances in surgical technique addressing primary brain injury caused by ICH, little progress has been made treating the subsequent inflammatory cascade. Pre-clinical studies have made advancements identifying components of neuroinflammation, including microglia, astrocytes, and T lymphocytes. After cerebral insult, inflammation is initially driven by the M1 microglia, secreting cytokines (e.g., interleukin-1β [IL-1β] and tumor necrosis factor-α) that are involved in the breakdown of the extracellular matrix, cellular integrity, and the blood brain barrier. Additionally, inflammatory factors recruit and induce differentiation of A1 reactive astrocytes and T helper 1 (Th1) cells, which contribute to the secretion of inflammatory cytokines, augmenting M1 polarization and potentiating inflammation. Within 7 days of ICH ictus, the M1 phenotype coverts to a M2 phenotype, key for hematoma removal, tissue healing, and overall resolution of inflammation. The secretion of anti-inflammatory cytokines (e.g., IL-4, IL-10) can drive Th2 cell differentiation. M2 polarization is maintained by the secretion of additional anti-inflammatory cytokines by the Th2 cells, suppressing M1 and Th1 phenotypes. Elucidating the timing and trigger of the anti-inflammatory phenotype may be integral in improving clinical outcomes. A challenge in current translational research is the absence of an equivalent disease animal model mirroring the patient population and comorbid pathophysiologic state. We review existing data and describe potential therapeutic targets around which we are creating a bench to bedside translational research model that better reflects the pathophysiology of ICH patients.

Dental follicle mesenchymal stem cells regulate responses in sepsis

Objective: Sepsis-induced immune alterations are associated with secondary infections and increased risk of death. The use of mesenchymal stem cells (MSCs) has been described as a novel therapeutic strategy. We evaluated the immunomodulatory effects of human dental follicle (DF-MSCs) on lymphocytes of sepsis and septic shock patients. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood samples of sepsis, septic shock and healthy subjects. PBMCs were co-cultured in the presence and absence of DF-MSCs with or without interferon-gamma (IFN-γ) for 72 hours. CD4+CD25+FoxP3+regulatory T (Treg) cell frequency, lymphocyte proliferation, cytokine levels and apoptosis were evaluated via flow cytometry. Results: DF-MSCs significantly suppressed proliferation of lymphocytes in sepsis group compared to septic shock group (p&amp;lt;0.005). DF-MSCs remarkably increased Treg ratio in sepsis compared to control group (p&amp;lt;0.05). Reduction of lymphocyte apoptosis in cocultures of DF-MSCs and PBMC was significant in both sepsis and septic shock groups. IFN-γ stimulation of DF-MSCs ameliorated shift in the T-cell subsets from Th2 to Th1 phenotype in septic shock. Conclusion: Our findings revealed that DF-MSCs have immunoregulatory effects both in sepsis and septic shock, by reducing interleukin-4 (IL-4) and increasing IFN-γ levels. This immunoreactivity regulation may open new therapeutic approaches for septic shock patients.

Synthetic Abortive HIV-1 RNAs Induce Potent Antiviral Immunity.

Strong innate and adaptive immune responses are paramount in combating viral infections. Dendritic cells (DCs) detect viral infections via cytosolic RIG-I like receptors (RLRs) RIG-I and MDA5 leading to MAVS-induced immunity. The DEAD-box RNA helicase DDX3 senses abortive human immunodeficiency virus 1 (HIV-1) transcripts and induces MAVS-dependent type I interferon (IFN) responses, suggesting that abortive HIV-1 RNA transcripts induce antiviral immunity. Little is known about the induction of antiviral immunity by DDX3-ligand abortive HIV-1 RNA. Here we synthesized a 58 nucleotide-long capped RNA (HIV-1 Cap-RNA58) that mimics abortive HIV-1 RNA transcripts. HIV-1 Cap-RNA58 induced potent type I IFN responses in monocyte-derived DCs, monocytes, macrophages and primary CD1c+ DCs. Compared with RLR agonist poly-I:C, HIV-1 Cap-RNA58 induced comparable levels of type I IFN responses, identifying HIV-1 Cap-RNA58 as a potent trigger of antiviral immunity. In monocyte-derived DCs, HIV-1 Cap-RNA58 activated the transcription factors IRF3 and NF-κB. Moreover, HIV-1 Cap-RNA58 induced DC maturation and the expression of pro-inflammatory cytokines. HIV-1 Cap-RNA58-stimulated DCs induced proliferation of CD4+ and CD8+ T cells and differentiated naïve T helper (TH) cells toward a TH2 phenotype. Importantly, treatment of DCs with HIV-1 Cap-RNA58 resulted in an efficient antiviral innate immune response that reduced ongoing HIV-1 replication in DCs. Our data strongly suggest that HIV-1 Cap-RNA58 induces potent innate and adaptive immune responses, making it an interesting addition in vaccine design strategies.

Hepatic ILC2 activity is regulated by liver inflammation-induced cytokines and effector CD4+ T cells

In immune-mediated hepatitis, type 2 innate lymphoid cells (ILC2) as well as effector CD4+ T cells have been shown to drive disease pathology. However, less is known about mechanisms involved in the regulation of ILC2 function during liver inflammation. We showed that in homeostasis, hepatic ILC2 constituted a very small population with a naive, inactive phenotype. During immune-mediated hepatitis, the cytokines IL-33 and IFNγ were expressed in liver tissue. IL-33 induced strong activation and expression of type 2 cytokines as well as IL-6 by hepatic ILC2 while IFNγ suppressed cytokine production. Interestingly, this inhibitory effect was overcome by IL-33. The phenotype of activated hepatic ILC2 were stable since they did not show functional plasticity in response to liver inflammation-induced cytokines. Moreover, hepatic ILC2 induced a Th2 phenotype in activated CD4+ T cells, which increased ILC2-derived cytokine expression via IL-2. In contrast, Th1 cells inhibited survival of ILC2 by production of IFNγ. Thus, hepatic ILC2 function is regulated by IL-33, IL-2, and IFNγ. While IL-33 and IL-2 support hepatic ILC2 activation, their inflammatory activity in immune-mediated hepatitis might be limited by infiltrating IFNγ-expressing Th1 cells.

Differentiation of Pathogenic Th17 Cells Is Negatively Regulated by Let-7 MicroRNAs in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1β-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.