T-bet Expression Research Articles

Perforin production by T cells and Natural Killer cells in CMV-reactivated allogeneic cell transplant patients is associated with early post-transplant death

Abstract Introduction: Cytomegalovirus (CMV) is a prevalent herpesvirus that poses a serious risk to immunocompromised patients, particularly hematopoietic stem cell transplant (HSCT) recipients. CMV reactivation increases the risk of mortality due to secondary infections. CMV infection has been shown to epigenetically induce immune exhaustion and decrease non-specific immunity. Methods: We reviewed flow cytometry data from CMV seropositive HSCT recipients to determine if CMV reactivation predicted mortality via modulation of lymphocyte function. Peripheral blood mononuclear cells (PBMCs) were collected from recipients 30 days following transplant and stimulated with CMV-specific peptide pools of pp65 and staphylococcal enterotoxin B (SEB) to assess CMV-specific and non-specific immune function. Results: Non-specific, SEB-induced, polyfunctionality of CD4 and CD8 T cells was diminished in deceased CMV seropositive HSCT recipients. In deceased individuals, T-bet and perforin expression were elevated, with decreased IFNγ, TNFα, or IL2 SEB-induced production. Discussion: Mortality in CMV reactivated HSCT recipients was associated with non-specific immune exhaustion phenotypes, but it remains unclear whether this was a result of a higher pathogenic burden, recurrent CMV reactivation, or chemotherapy and myeloablation.

Rheumatoid arthritis autologous synovial fluid affects the plasticity and function of peripheral and induced T regulatory cells in vitro

The synovial fluid (SF) microenvironment in rheumatoid arthritis (RA) may alter the stability and function of Tregs. In the present study, we assessed cytokine levels and percentage of Tregs, Tregs expressing CXCR3 (Th1-like Treg), CCR6 (Th17-like Treg) in RA peripheral blood (PB) and RA-SF using fluorescence cytometry. Effect of autologous SF on plasticity and function of RA-PB Tregs (pTregs; CD4+CD25hiCD127Lo/−) and induced vimentin-pulsed Tregs (iTregsVIM) was assessed in vitro. Cytokines and percentage of Th1-like and Th17-like Tregs were higher in RA-PB than OA-PB; higher in RA-SF than osteoarthritis (OA)-SF. Compared to OA-SF exposed OA-pTregs, RA-SF exposed RA-pTregs showed higher percentage of Th1-like (11% vs 20%) and Th17-like (16% vs 36%) Tregs; higher T-bet (p = 0.0001), RORγ (p = 0.0001) and lower FOXP3 (p = 0.0001) gene expression; and diminished percentage suppression of autologous T effector cells (36% vs 74%). RA-SF exposed iTregsVIM showed increased percentage of Th1-like and Th17-like Tregs compared to iTregsVIM exposed to AB serum (8% vs 0.1%; 21% vs 0.1%). IL-2, Tocilizumab and 5-azacytidine reduced the conversion of iTregsVIM (8% vs 2.4%; 21% vs 6.9%), when used in combination. To conclude, microenvironment in the RA synovial fluid is possibly responsible for conversion of pTregs into Th-like Tregs and their functional loss. A blockade of cytokine receptors and methyl transferases could inhibit Tregs conversion, providing clinical relevance for future Tregs targeting therapies.

T-bet Regulates Ion Channels and Transporters and Induces Apoptosis in Intestinal Epithelial Cells.

T-bet, encoded by TBX21, is extensively expressed across various immune cell types, and orchestrates critical functions in their development, survival, and physiological activities. However, the role of T-bet in non-immune compartments, notably the epithelial cells, remains obscure. Herein, a Tet-O-T-bet transgenic mouse strain is generated for doxycycline-inducible T-bet expression in adult animals. Unexpectedly, ubiquitous T-bet overexpression causes acute diarrhea, intestinal damage, and rapid mortality. Cell-type-specific analyses reveal that T-bet-driven pathology is not attributable to its overexpression in CD4+ T cells or myeloid lineages. Instead, inducible T-bet overexpression in the intestinal epithelial cells is the critical determinant of the observed lethal phenotype. Mechanistically, T-bet overexpression modulates ion channel and transporter profiles in gut epithelial cells, triggering profound fluid secretion and subsequent lethal dehydration. Furthermore, ectopic T-bet expression enhances gut epithelial cell apoptosis and markedly suppresses colon cancer development in xenograft models. Collectively, the findings unveil a previously unrecognized role of T-bet in intestinal epithelial cells for inducing apoptosis, diarrhea, and local inflammation, thus implicating its potential as a therapeutic target for the treatment of cancer and inflammatory diseases.

The Transcription Factor Zfp335 Promotes Differentiation and Survival of Effector Th1 Cells by Directly Regulating Lmna Expression.

Ag-specific effector CD4+ T cells play a crucial role in defending against exogenous pathogens. However, the mechanisms governing the differentiation and function of IFN-γ-producing effector CD4+ Th1 cells in immune responses remain largely unknown. In this study, we elucidated the pivotal role of zinc finger protein 335 (Zfp335) in regulating effector Th1 cell differentiation and survival during acute bacterial infection. Mice with Zfp335 knockout in OT-II cells exhibited impaired Ag-specific CD4+ T cell expansion accompanied by a significant reduction in resistance to Listeria infection. Furthermore, Zfp335 deficiency restricted the effector CD4+ Th1 cell population and compromised their survival upon Listeria challenge. The expression of T-bet and IFN-γ was accordingly decreased in Zfp335-deficient Th1 cells. Mechanistically, Zfp335 directly bound to the promoter region of the Lmna gene and regulated its expression. Overexpression of Lmna was able to rescue the survival and function of Zfp335-deficient effector Th1 cells. Therefore, our study provides novel insights into the mechanisms governing effector Th1 cell differentiation and survival during acute infection.

Tumor-associated macrophages impair NK cell IFN-γ production and contribute to tumor progression in clear cell renal cell carcinoma.

Tumor-associated macrophages (TAM) are abundant in several tumor types and usually correlate with poor prognosis. Previously, we demonstrated that anti-inflammatory macrophages (M2) inhibit NK cell effector functions. Here, we explored the impact of TAM on NK cells in the context of clear-cell renal cell carcinoma (ccRCC). Bioinformatics analysis revealed that an exhausted NK cell signature strongly correlated with an M2 signature. Analysis of TAM from human ccRCC samples confirmed that they exhibited an M2-skewed phenotype and inhibited IFN-γ production by NK cells. Moreover, human M0 macrophages cultured with conditioned media from ccRCC cell lines generated macrophages with an M2-skewed phenotype (TAM-like), which alike TAM, displayed suppressive activity on NK cells. Moreover, TAM depletion in the mouse Renca ccRCC model resulted in delayed tumor growth and reduced volume, accompanied by an increased frequency of IFN-γ-producing tumor-infiltrating NK cells that displayed heightened expression of T-bet and NKG2D and reduced expression of the exhaustion-associated co-inhibitory molecules PD-1 and TIM-3. Therefore, in ccRCC, the tumor microenvironment polarizes TAM toward an immunosuppressive profile that promotes tumor-infiltrating NK cell dysfunction, contributing to tumor progression. In addition, immunotherapy strategies targeting TAM may result in NK cell reinvigoration, thereby counteracting tumor progression.

Exploring the immune-modulating properties of boswellic acid in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a condition where the central nervous system loses its myelin coating due to autoimmune inflammation. The experimental autoimmune encephalomyelitis (EAE) simulates some aspects of human MS. Boswellic acids are natural compounds derived from frankincense extract, known for their anti-inflammatory properties. The purpose of this research was to investigate therapeutic potential of boswellic acids. Mice were divided into three groups: low-dose (LD), high-dose (HD), and control groups (CTRL). Following EAE induction, the mice received daily doses of boswellic acid for 25 days. Brain tissue damage, clinical symptoms, and levels of TGF-β, IFN-γ, and IL-17 cytokines in cell cultured supernatant of lymphocytes were assessed. Gene expression of transcription factors in brain was measured using real-time PCR. The levels of brain demyelination were significantly lower in the treatment groups compared to the CTRL group. Boswellic acid reduced the severity and duration of EAE symptoms. Furthermore, boswellic acid decreased the amounts of IFN-γ and IL-17, also the expression of T-bet and ROR-γt in brain. On the contrary, it increased the levels of TGF-β and the expression FoxP3 and GATA3. Our findings suggest that boswellic acids possess therapeutic potential for EAE by modulating the immune response and reducing inflammation.

Abstract 2685: Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers

Abstract Innate lymphoid cells (ILCs) and natural killer (NK) cells are a heterogeneous family of innate immune cells that regulate inflammation, immune tolerance and tissue homeostasis in infection, chronic inflammation, metabolic diseases and cancer. The ILC family lacks the expression of antigen-specific receptors and demonstrate effector function by generating cytokines and secreted proteins. In tumor microenvironment, ILCs play a dual role including both tumor regression and tumor promotion, suggesting their diverse phenotypes and functional states. Among the ILC family, ILC1s and NK cells share some features, including the expression of the transcription factor T-bet and the ability to produce IFN-γ. In the present study, we analyzed single-cell RNA sequencing (scRNAseq) data to investigate the landscape and functional status of ILC families in patients with head and neck squamous cell carcinoma (HNSCC). The GSE164690 dataset, including pre-processed scRNAseq data and clinical data, was obtained from Gene Expression Omnibus. A total of 17 pairs of tumor-derived CD45-positive cells and peripheral blood lymphocytes, consisting of 6 HPV-positive HNSCCs and 11 HPV-negative HNSCCs, were analyzed using the Seurat v4 R package. After quality control, a total of 95,809 cells were obtained and clustered into 16 immune cell clusters. Next, a total of 7,278 NK cells were extracted and sub-clustered into 11 clusters. To characterize the 11 clusters, the abundances of NK subsets were estimated by CIBERSORTx. This approach identified eight NK cell clusters, two intraepithelial ILC1 (ieILC1) clusters, and one ieILC1-NK-intermediate (ieILC1-NK-int) cluster. Single-sample GSEA (ssGSEA) analysis was performed to further characterize each subset. ieILC1 clusters expressed tissue-resident markers ITGA1, CD69, and CXCR6, whereas ieILC1-NK-int cluster expressed ILC1 markers CCR7, IL7R, and SELL. Principle component analysis (PCA) based on ssGSEA indicated that ieILC1 clusters and ieILC1-NK-int showed the different PCA distribution from NK clusters. Inflammation and effector function-related pathways, including inflammatory response, interferon-gamma response, and interferon-alpha response, were enriched in ieILC1 clusters compared with the others. Among ieILC1 clusters, several pathways, including hypoxia, TNFA signaling via NFKB, allograft rejection, TGF beta signaling, were more enriched in ieILC1-1 than in ieILC1-2, suggesting that ieILC1-1 is the most immunologically active phenotype among NK subclusters. Of note, ieILC1-2 mainly comprised cells isolated from HPV-negative tumors, whereas ieILC1-1 mainly comprised cells isolated from HPV-positive tumors. In conclusion, we have demonstrated the diverse landscape of NK and ieILC1 cells in patients with HNSCC. Further characterization of the ILC family would lead to a novel therapeutic approach against HNSCC. Citation Format: Hideyuki Takahashi, Shota Ida, Hiroe Tada, Kazuaki Chikamatsu. Activated phenotype of intraepithelial type 1 innate lymphoid cells is enriched in HPV-positive head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2685.

Lack of ATP2B1 in CD4+ T Cells Causes Colitis.

The ATP2B1 gene encodes for a calcium pump, which plays a role in removing Ca2+ from cells and maintaining intracellular Ca2+ homeostasis. Reduction of the intracellular Ca2+ concentration in CD4+ T cells is thought to reduce the severity of colitis, while elevation of Ca2+ in CD4+ T cells induces T cell hyperactivity. Our aim was to clarify the role of ATP2B1 in CD4+ T cells and in inflammatory bowel disease development. A murine CD4+ T cell-specific knockout (KO) of ATP2B1 was created using a Cre-loxP system. CD4+ T cells were isolated from thymus, spleen, and blood using fluorescence-activated cell sorting. To quantify messenger RNA levels, quantitative real-time polymerase chain reaction was performed. Although the percentages of CD4+ T cells in both KO mouse spleen and blood decreased compared with those of the control samples, both T-bet (a T helper 1 [Th1] activity marker) and GATA3 (a Th2 activity marker) expression levels were further increased in KO mouse blood CD4+ T cells (vs control blood). Diarrhea and colonic wall thickening (with mucosal changes, including crypt distortion) were seen in KO mice but not in control mice. Prior to diarrhea onset, the KO mouse colon length was already noted to be shorter, and the KO mouse stool water and lipid content were higher than that of the control mice. Tumor necrosis factor α and gp91 expressions were increased in KO mouse colon. Lack of ATP2B1 in CD4+ T cells leads to Th1 and Th2 activation, which contributes to colitis via elevation of tumor necrosis factor α and oxidative stress.

Mechanisms of action of Shizhenqing granules for eczema treatment: Network pharmacology analysis and experimental validation

BackgroundJiuwan decoction has been used to treat chronic eczema since the Qing Dynasty. According to clinical experience, Shizhenqing granules (SZQG), derived from the Jiuwan decoction, exert beneficial clinical effects on acute eczema and reduce recurrence. Therefore, we elucidated the underlying mechanisms of SZQG through network pharmacology, molecular docking, and experimental validation. MethodsThe main chemical components of SZQG were identified by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). And the targets of SZQG against eczema were screened out through online databases. Then, the regulatory network map of the “herbal compound–potential target” and the target protein-protein interaction (PPI) network was constructed. The Gene Ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted using by R language. Additionally, the interaction between the active compounds and the targets was verified by molecular docking technology. Finally, an experiment in vivo was used to verify the effect and mechanism of SZQG on eczema. ResultsUsing UHPLC-MS/MS, 158 main chemical compounds of SZQG were identified, and 72 compounds were selected according to the criteria for further analysis. All 237 potential targets of SZQG in eczema were explored using multiple online databases. The network with 14 core targets was screened out, including STAT3, RELA, TNF, JUN, MAPK3, IL-6, PIK3CA, STAT1, MAPK14, MAPK1, IL-4, NFKBIA, IL1B, and MYC. KEGG analyses indicated that the therapeutic effects of SZQG on eczema were predominantly associated with cytokine-cytokine receptor interaction, TNF, MAPK, NF-κB, toll-like receptor, T cell receptor, and Th1 and Th2 cell differentiation signaling pathways. Furthermore, the good affinity between the core compounds and core targets was verified by molecular docking technology, particularly for RELA and MAPK. Animal experiments revealed that SZQG downregulated MAPK14, RELA, T-bet, and GATA3 mRNA expression, reduced immunoglobulin E (IgE) and interleukin-4 (IL-4) serum concentrations, and improved eczema-like lesions in model rats. ConclusionThis study identified potential targets and signaling pathways of SZQG in the treatment of eczema, whereby RELA and MAPK14 may constitute the main therapeutic targets of SZQG in cytokine regulation and reduction of inflammatory responses.

Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota

Ethnopharmacological relevanceChinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. Aim of the studyThis study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. Materials and methods61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1β, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iβ, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. ResultsGQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1β, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1β. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. ConclusionsGQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

Elotuzumab Enhances CD16-Independent NK Cell-Mediated Cytotoxicity against Myeloma Cells by Upregulating Several NK Cell-Enhancing Genes.

Multiple myeloma (MM) is an intractable hematological malignancy caused by abnormalities in plasma cells. Combination therapy using antibodies and natural killer (NK) effectors, which are innate immune cells with safe and potent antitumor activity, is a promising approach for cancer immunotherapy and can enhance antitumor effects. Elotuzumab (Elo) is an immune-stimulatory antibody that targets the signaling lymphocytic activation molecule family 7 (SLAMF7) expressed on the surface of MM and NK cells. We confirmed that Elo strongly promoted NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against SLAMF7-positive MM cells in a CD16-dependent NK cell line, and also activated expanded NK cells derived from peripheral blood mononuclear cells of healthy donors and patients with MM in the present study. However, the antitumor effects and genes involved in the direct promotion of NK cell-mediated activation using Elo in CD16-independent NK cells are not clearly known. In this study, we demonstrated that Elo pretreatment significantly enhanced CD16-independent NK cell-mediated cytotoxicity in both SLAMF7-positive MM.1S and SLAMF7-negative K562, U266, and RPMI 8226 tumor cells. Upon direct simulation of CD16-independent NK cells with Elo, increased levels of CD107a degranulation and IFN-γ secretion were observed along with the upregulation of granzyme B, TNF-α, and IL-1α gene expression. The enhanced NK cell function could also be attributed to the increased expression of the transcription factors T-BET and EOMES. Furthermore, the augmentation of the antitumor effects of CD16-independent NK cells upon pretreatment with Elo enhanced the expression of CRTAM, TNFRSF9, EAT-2, and FOXP3 genes and reduced the expression of HSPA6. Our results suggest that Elo directly promotes the cytotoxic function of CD16-independent NK cells against target cells, which is associated with the upregulation of the expression of several NK cell-enhancing genes.

Activin A levels are raised during human tuberculosis and blockade of the activin signaling axis influences murine responses to M. tuberculosis infection.

Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

Vitamin D improves autoimmune diseases by inhibiting Wnt signaling pathway.

In this study, we investigated the development of the Wnt signaling pathway in vitamin D (VitD) to improve systemic lupus erythematosus in mice to breakthrough clinical treatment approaches. Body weight changes were recorded during rearing. Antinuclear antibodies (ANA), anti-dsDNA, and anti-snRNP were detected in the mouse serum using an enzyme-linked immunosorbent assay. Apoptosis of Th1 and Th2 immune cells in mice was detected using flow cytometry. Reverse transcription polymerase chain reaction was used to detect the expression of T-bet, GATA3, and Wnt3a mRNA in the spleens of each group. Western blot analysis was performed to detect the expression of Wnt1, p-β-catenin, β-catenin, glycogen synthase kinsase3β (GSK-3β), Wnt3a, c-myc, and cyclin D1 protein in mice spleens. β-catenin in mice spleen was visualized using immunohistochemistry. VitD did not substantial reduce the body weight of MRL/LPR mice, whereas the inhibitor did. VitD notably decreased the concentrations of ANA, anti-double-stranded DNA, and anti-snRNP in the serum of MRL/LPR mice and alleviated apoptosis of Th1 and Th2 cells. VitD markedly increased the expression of T-bet and GATA mRNA in the spleen of MRL/LPR mice and consequently increased the levels of Wnt3a and β-catenin. Western blot analysis revealed that the levels of GSK-3β, p-β-catenin, Wnt1, Wnt3a, c-myc, and cyclin D1 could be reduced by VitD, compared with MRL/LPR. Immunohistochemistry demonstrated that the expression of β-catenin was the most pronounced in the spleen of MRL/LPR mice, and the expression level of β-catenin decreased substantially after VitD intervention. VitD can further inhibit the nuclear translocation of β-catenin by downregulating the expression of Wnt ligands (Wnt1 and Wnt3a), which reduces the expression of the downstream target gene cyclin D1. Systemic lupus erythematosus in mice was improved by inhibiting the activation of Wnt/β-catenin signal pathway.

Thymoquinone improves experimental autoimmune encephalomyelitis by regulating both pro-inflammatory and anti-inflammatory cytokines.

Introduction Multiple sclerosis (MS) is an autoimmune condition marked by inflammation and the loss of myelin in the central nervous system (CNS). The aim of this research was to understand how Thymoquinone regulate the molecular and cellular processes involved in controlling experimental autoimmune encephalomyelitis (EAE), which is an animal model often used to study MS. MethodsFemale C57BL/6 mice were split into different groups receiving different doses (low, medium, and high) of Thymoquinone simultaneously with EAE induction. Clinical scores and other measurements were observed daily throughout the 25-day post immunization. We assessed lymphocyte infiltration and demyelination in the spinal cord through histological staining, analyzed T-cell profiles using ELISA, and quantified the expression levels of transcription factors in the CNS using Real-time PCR. Results Thymoquinone prevented the development of EAE. Histological experiments revealed only a small degree of leukocyte infiltration into the CNS. Thymoquinone resulted in a notable reduction in the generation of IFN-γ, IL-17, and IL-6, while simultaneously increasing the production of IL-4, IL-10, and TGF-β in Th2 and Treg cells. Results from Real-time PCR suggested Treatment with Thymoquinone decreased the expression of T-bet and ROR-γt while increasing the expression of Foxp3 and GATA3. Conclusion These findings showed that Thymoquinone could decrease both disease incidence and severity.

Naringenin modifies T-helper responses and macrophage activities in BALB/c mice.

Naringenin is a naturally occurring flavonoid found in citrus fruits. This study was done to compare the oral immunomodulatory effects of naringenin and prednisolone. The effect of one-month oral administration of naringenin (10, 20, and 40 mg/kg) and prednisolone (2 mg/kg) on peritoneal macrophage was compared in the first set of experiments. Separate evaluations were conducted on the effects of naringenin on in vivo and ex vivoT-helper (Th) lymphocyte responses and their subsets in mice immunized with ovalbumin (OVA). Animals challenged with OVA received oral doses of naringenin or prednisolone from two days prior to immunization to 28 days after immunization. Naringenin and prednisolone increased macrophages' respiratory burst, and nitric oxide and interleukin (IL)-10 production while decreasing IL-12 production. Macrophages isolated from mice administered with 40 mg/kg naringenin had greater phagocytic potential than those isolated from mice administered with prednisolone. OVA-challenged mice treated with 40 mg/kg naringenin or prednisolone had decreased delayed-type hypersensitivity comparable to control mice.The splenocyte proliferation index was lower in the prednisolone-treated group than the naringenin-treated group, even at 40 mg/kg. In the splenocyte cultures, both agents decreased T-bet expression. Naringenin, in contrast to prednisolone, did not affect GATA3expression. The 40 mg/kg naringenin dose reduced RORγt more effectively than prednisolone. All these findings indicate the potential of naringenin as a modifying agent of immune responses. Consequently, naringenin may be beneficial in controlling some immunopathological conditions.

P027 Involvement of PD-L1 in myenteric plexitis associated with postoperative recurrence of Crohn's disease

Abstract Background Despite a growing therapeutic armamentarium, post-operative recurrence (POR) in Crohn’s disease (CD) remains frequent. The presence of myenteric plexitis at the proximal margin of ileocolonic resection has been recognized as a risk factor for early POR. They are defined by the accumulation of immune cells in contact with the ganglia of the myenteric plexus. In preliminary work, we have shown an increase in the number of T lymphocytes (TL) in contact with enteric glial cells (EGC) in patients with CD depending on ICAM-1/LFA-1 pathway. As CD is classically associated with a Th1 immune response, the aim of this work was to study the impact of EGC/CD4+ TL contacts on Th1-type cytokine production (IFNγ and its transcription factor Tbet) and to identify molecules that may be involved in these regulations. Methods In vitro in rats, the impact of EGC/CD4+ TL co-culture on IFNγ production was studied by ELISA and qPCR of the transcription factor Tbet. The expression and regulation of PD-L1, a candidate molecule, was then analyzed in murine and human EGC by qPCR and immunohisto/cytochemistry. Results While EGC alone produced only a small amount of IFNγ, there was a significant reduction in IFNγ production in EGC/CD4+ TL co-cultures compared with CD4+ TL alone. Yet the proportion of CD4+ TL cells capable of producing IFNγ was significantly increased in contact with EGC, as was their survival. In parallel, Tbet was downregulated in CD4+ TL in contact with EGC. These results suggested the involvement of PD-L1, a molecule known to negatively regulate cytokine production (notably IFNγ) and Tbet expression. Expression of PD-L1 mRNA and its protein was demonstrated in murine and human EGC and was upregulated under pro-inflammatory conditions. Analysis of transmural sections at the proximal margin of ileocolonic resection from patients (n=15 controls, n=14 CD without POR, n=15 CD with POR) showed PD-L1 expression in myenteric ganglia that was restricted to EGC (no neuronal expression) (Figure 1A). PD-L1 fluorescence intensity in myenteric ganglia at the proximal resection margin was significantly lower in CD patients with POR than in controls (∆13.5%; p=0.012) (Figure 1B). Conclusion Lower IFNγ production in EGC/CD4+ TL co-cultures associated with reduced Tbet expression by TL in contact with EGC suggest a role of PD-L1 in myenteric plexitis. PD-L1 expression has been demonstrated in murine and human EGC, with significantly lower expression in myenteric ganglia located at the proximal resection margin of CD patients with POR compared with controls. Blocking the ICAM-1/LFA-1 pathway and/or restoring the PD-1/PD-L1 loop could be of interest for developing strategies to prevent and/or treat POR in CD patients.

High-intensity intermittent training ameliorates methotrexate-induced acute lung injury

Inflammation and oxidative stress are recognized as two primary causes of lung damage induced by methotrexate, a drug used in the treatment of cancer and immunological diseases. This drug triggers the generation of oxidants, leading to lung injury. Given the antioxidant and anti-inflammatory effects of high-intensity intermittent training (HIIT), our aim was to evaluate the therapeutic potential of HIIT in mitigating methotrexate-induced lung damage in rats. Seventy male Wistar rats were randomly divided into five groups: CTL (Control), HIIT (High-intensity intermittent training), ALI (Acute Lung Injury), HIIT+ALI (pretreated with HIIT), and ALI + HIIT (treated with HIIT).HIIT sessions were conducted for 8 weeks. At the end of the study, assessments were made on malondialdehyde, total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (Gpx), myeloperoxidase (MPO), interleukin 10 (IL-10), tumor necrosis factor-alpha (TNF-α), gene expression of T-bet, GATA3, FOXP3, lung wet/dry weight ratio, pulmonary capillary permeability, apoptosis (Caspase-3), and histopathological indices.Methotrexate administration resulted in increased levels of TNF-α, MPO, GATA3, caspase-3, and pulmonary edema indices, while reducing the levels of TAC, SOD, Gpx, IL-10, T-bet, and FOXP3. Pretreatment and treatment with HIIT reduced the levels of oxidant and inflammatory factors, pulmonary edema, and other histopathological indicators. Concurrently, HIIT increased the levels of antioxidant and anti-inflammatory factors.

MicroRNA-194 regulates parasitic load and IL-1β-dependent nitric oxide production in the peripheral blood mononuclear cells of dogs with leishmaniasis.

Domestic dogs are the primary urban reservoirs of Leishmania infantum, the causative agent of visceral leishmaniasis. In Canine Leishmaniasis (CanL), modulation of the host's immune response may be associated with the expression of small non-coding RNAs called microRNA (miR). miR-194 expression increases in peripheral blood mononuclear cells (PBMCs) of dogs with leishmaniasis with a positive correlation with the parasite load and in silico analysis demonstrated that the TRAF6 gene is the target of miR-194 in PBMCs from diseased dogs. Here, we isolated PBMCs from 5 healthy dogs and 28 dogs with leishmaniasis, naturally infected with L. infantum. To confirm changes in miR-194 and TRAF6 expression, basal expression of miR-194 and gene expression of TRAF6 was measured using qPCR. PBMCs from healthy dogs and dogs with leishmaniasis were transfected with miR-194 scramble, mimic, and inhibitor and cultured at 37° C, 5% CO2 for 48 hours. The expression of possible targets was measured: iNOS, NO, T-bet, GATA3, and FoxP3 were measured using flow cytometry; the production of cytokines IL-1β, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and TGF-β in cell culture supernatants was measured using capture enzyme-linked immunosorbent assays (ELISA). Parasite load was measured using cytometry and qPCR. Functional assays followed by miR-194 inhibitor and IL-1β blockade and assessment of NO production were also performed. Basal miR-194 expression was increased in PBMC from dogs with Leishmaniasis and was negatively correlated with TRAF6 expression. The mimic of miR-194 promoted an increase in parasite load. There were no significant changes in T-bet, GATA3, or FoxP3 expression with miR-194 enhancement or inhibition. Inhibition of miR-194 increased IL-1β and NO in PBMCs from diseased dogs, and blockade of IL-1β following miR-194 inhibition decreased NO levels. These findings suggest that miR-194 is upregulated in PBMCs from dogs with leishmaniasis and increases parasite load, possibly decreasing NO production via IL-1β. These results increase our understanding of the mechanisms of evasion of the immune response by the parasite and the identification of possible therapeutic targets.

Modulatory Effects of Hydatid Cyst Fluid on a Mouse Model of Experimental Autoimmune Encephalomyelitis.

The reduced burden of helminth parasites in industrialized countries is probably one of the reasons for the increased prevalence of autoimmune disorders such as multiple sclerosis (MS). The current study aimed to evaluate the potential preventive effects of hydatid cyst fluid (HCF) on the disease severity in an EAE mouse model of MS. EAE-induced mice were treated with HCF before and after EAE induction. An RT-PCR-based evaluation of IFN-γ, IL-1β, TNF, T-bet, IL-4, GATA3, IL-17, RoRγ, TGF-β, and FOXP3 expression levels in splenocytes and an ELISA-based analysis of IFN-γ and IL-4 levels in cell culture supernatant of splenocytes were performed. Histopathological examinations of mice during the study were also conducted. The expression levels of T-bet, IL-4, GATA3, TGF-β, and FOXP3 in EAE + HCF mice were significantly higher compared to EAE + PBS mice. In the EAE + HCF group, the expression levels of IFN-γ, IL-1β, and TNF were significantly lower than in the EAE + PBS group. The histopathological results showed significantly reduced inflammation and demyelination in EAE + HCF mice compared to EAE + PBS mice. Our study provides proof-of-concept in the EAE mouse model of MS that helminth-derived products such as HCF have a potential prophylactic effect on MS development and present a novel potential therapeutic strategy.

Nickel Challenge In Vitro Affects CD38 and HLA-DR Expression in T Cell Subpopulations from the Blood of Patients with Nickel Allergy.

Nickel allergy is a major health problem and shows clinical manifestation of contact eczema. The response of specific lymphocyte subpopulations in sensitized patients after new challenge to nickel has until now not been studied in detail. To evaluate if nickel-based elicitation reaction could be objectively identified by multi-parametric flow cytometry, immunophenotyping of specific T cells was applied. White blood cells from 7 patients (4 positive in patch test, 3 negative) were challenged by nickel and in vitro short-term culture. Standardized antibody-dye combinations, specific for T helper(h)1, Th17 and cytotoxic T cell activation, were selected according to the recommendations of Stanford Human Immune Monitoring Center. In cytotoxic CD8+CCR7+CD45RA+ T cells from patients suffering from nickel allergy, CD38 and HLA-DR were elevated comparing to healthy donors. After challenge to nickel in vitro both markers decreased in CD8+CCR7+CD45RA+ T cells but found up-regulated in CD4+CCR7+CD45RA+CCR6-CXCR3+Th1 cells. Intracellular expression of T-bet and RORγt further indicated Th1 and Th17 cells. Finally, CD4+CD25+CCR4- T cells increased after challenge with nickel in PBMCs of patients with nickel allergy. Flow cytometry based quantification of T cell markers might be used as a specific and reliable method to detect chemical induced skin sensitization and confirm diagnostic patch testing in the clinics.