IFN-γ Transcripts Research Articles

Arctiin Alleviates Atopic Dermatitis Against Inflammation and Pyroptosis Through Suppressing TLR4/MyD88/NF-κB and NLRP3/Caspase-1/GSDMD Signaling Pathways.

Atopic dermatitis (AD) is a prevalent skin condition worldwide. The immune response plays a crucial role in the pathogenesis of AD. Arctiin (ARC), a natural lignan, has been extensively investigated because of its anti-inflammatory, antioxidant, and anticancer properties. However, the impact of ARC on AD remains uncertain. Therefore, this study investigated the therapeutic effects of ARC in AD. AD-like lesions were induced in mice by applying 2,4-dinitrochlorobenzene (DNCB). The efficacy of ARC in AD was assessed by measuring skin lesion scores and thickness, pathological observation, and serum IgE concentrations. The expression of relevant proteins and genes in the back skin of the mice was assessed. Moreover, the TLR4/MyD88/NF-κB and NLRP3/Caspase-1/GSDMD signaling pathways were assessed in HaCaT cells stimulated with TNF-α and IFN-γ. ARC effectively alleviated AD-like dermatitis induced by DNCB in mice, reducing the skin thickness, mast cell infiltration in skin tissue, and serum total IgE levels. In addition, the expression of IL-1β and the mRNA transcription of TSLP and IFN-γ were downregulated. ARC also suppressed the TLR4/MyD88/NF-κB pathway, and molecular docking confirmed that ARC had exceptional binding properties with TLR4. Moreover, ARC ameliorated pyroptosis by inhibiting the activation of the nod-like receptor protein-3/Caspase-1/GSDMD cascade. ARC has remarkable anti-AD effects by inhibiting inflammation and pyroptosis through the TLR4/MyD88/NF-κB and NLRP3/Caspase-1/GSDMD signaling pathways. This suggests that ARC has potential as a new drug candidate for treating AD, which provides a novel approach to the clinical management of AD.

Physiologically Achievable Concentration of 2-Deoxy-D-Glucose Stimulates IFN-γ Secretion in Activated T Cells In Vitro.

2-deoxy-D-glucose (2DG) is a glycolysis and protein N-glycosylation inhibitor with promising anti-tumor and immunomodulatory effects. However, 2DG can also suppress T cell function, including IFN-γ secretion. Few human T cell studies have studied low-dose 2DG, which can increase IFN-γ in a Jurkat clone. We therefore investigated 2DG's effect on IFN-γ in activated human T cells from PBMCs, with 2DG treatment commenced either concurrently with activation or 48 h after activation. Concurrent 2DG treatment decreased IFN-γ secretion in a dose-dependent manner. However, 2DG treatment of pre-activated T cells had a hormetic effect on IFN-γ, with 0.15-0.6 mM 2DG (achievable in vivo) increasing and >2.4 mM 2DG reducing its secretion. In contrast, IL-2 levels declined monotonously with increasing 2DG concentration. Lower 2DG concentrations reduced PD-1 and increased CD69 expression regardless of treatment timing. The absence of increased T-bet or Eomes expression or IFNG transcription suggests another downstream mechanism. 2DG dose-dependently induced the unfolded protein response, suggesting a possible role in increased IFN-γ secretion, possibly by increasing the ER folding capacity for IFN-γ via increased chaperone expression. Overall, low-dose, short-term 2DG exposure could potentially improve the T cell anti-tumor response.

Expression of Hormones' Receptors in Human Corneal Endothelium from Fuchs' Dystrophy: A Possible Gender' Association.

Background: Age and sex are the most significant risk of factors for advanced Fuchs dystrophy. Nevertheless, few data are available on the hormone's receptor pattern expressed in adult and advanced fuchs endothelial corneal dystrophy (FECD). We investigated the impact of gender, growth factors and extracellular matrix (ECM) regulatory proteins expressed by the dystrophic endothelia. Methods: Ten dystrophic endothelial tissues and 10 normal endothelial sheets (corneoscleral specimens; Eye Bank) were used for this characterization study. Hormones' receptors (ERα, AR, PR, SHBG), few growth factors (VEGFA, βNGF, TGFβ1), some ECM regulators (MMP1, MMP7) and few inflammatory cytokines (IFNγ, IL10) were analyzed by real-time RT-PCR. Results: ERα transcripts were significantly increased, AR and SHBG transcripts were decreased in Fuchs endothelia from female patients, and no changes were detected for PR transcripts. VEGFA, βNGF and TGFβ1 transcripts were upregulated in Fuchs' endothelia, but not significantly linked to gender. High MMP1 and low MMP7 transcripts' expression were detected in Fuchs' specimens, mainly in males than females. An increased IFNγ (Th1) transcript expression was observed in females than males, and a trend to increase for IL10 (Th2) transcripts was detected in males than females. Conclusions: Our findings clearly indicate that hormone receptors, growth factors and matrix mediators as well as a Th1 pathway are predominant in Fuchs' dystrophy, displaying a pattern of expression specific for the female phenotype. The differential expression of hormones' receptors and the Th1/Th2 ratio might prompt to new theories to be tested in vitro and in vivo models, such as the use of hormonal substitute for counteracting this endothelial cell lost.

Deletion of the mRNA endonuclease Regnase-1 promotes NK cell anti-tumor activity via OCT2-dependent transcription of Ifng

Limited infiltration and activity of natural killer (NK) and Tcells within the tumor microenvironment (TME) correlate with poor immunotherapy responses. Here, we examined the role of the endonuclease Regnase-1 on NK cell anti-tumor activity. NK cell-specific deletion of Regnase-1 (Reg1ΔNK) augmented cytolytic activity and interferon-gamma (IFN-γ) production invitro and increased intra-tumoral accumulation of Reg1ΔNK-NK cells invivo, reducing tumor growth dependent on IFN-γ. Transcriptional changes in Reg1ΔNK-NK cells included elevated IFN-γ expression, cytolytic effectors, and the chemokine receptor CXCR6. IFN-γ induced expression of the CXCR6 ligand CXCL16 on myeloid cells, promoting further recruitment of Reg1ΔNK-NK cells. Mechanistically, Regnase-1 deletion increased its targets, the transcriptional regulators OCT2 and IκBζ, following interleukin (IL)-12 and IL-18 stimulation, and the resulting OCT2-IκBζ-NF-κB complex induced Ifng transcription. Silencing Regnase-1 in human NK cells increased the expression of IFNG and POU2F2. Our findings highlight NK cell dysfunction in the TME and propose that targeting Regnase-1 could augment active NK cell persistence for cancer immunotherapy.

IFIH1 loss of function predisposes to inflammatory and SARS-CoV-2-related infectious diseases.

The IFIH1 gene, encoding melanoma differentiation-associated protein 5 (MDA5), is an indispensable innate immune regulator involved in the early detection of viral infections. Previous studies described MDA5 dysregulation in weakened immunological responses, and increased susceptibility to microbial infections and autoimmune disorders. Monoallelic gain-of-function of the IFIH1 gene has been associated with multisystem disorders, namely Aicardi-Goutieres and Singleton-Merten syndromes, while biallelic loss causes immunodeficiency. In this study, nine patients suffering from recurrent infections, inflammatory diseases, severe COVID-19 or multisystem inflammatory syndrome in children (MIS-C) were identified with putative loss-of-function IFIH1 variants by whole-exome sequencing. All patients revealed signs of lymphopaenia and an increase in inflammatory markers, including CRP, amyloid A, ferritin and IL-6. One patient with a pathogenic homozygous variant c.2807+1G>A was the most severe case showing immunodeficiency and glomerulonephritis. The c.1641+1G>C variant was identified in the heterozygous state in patients suffering from periodic fever, COVID-19 or MIS-C, while the c.2016delA variant was identified in two patients with inflammatory bowel disease or MIS-C. There was a significant association between IFIH1 monoallelic loss of function and susceptibility to infections in males. Expression analysis showed that PBMCs of one patient with a c.2016delA variant had a significant decrease in ISG15, IFNA and IFNG transcript levels, compared to normal PBMCs, upon stimulation with Poly(I:C), suggesting that MDA5 receptor truncation disrupts the immune response. Our findings accentuate the implication of rare monogenic IFIH1 loss-of-function variants in altering the immune response, and severely predisposing patients to inflammatory and infectious diseases, including SARS-CoV-2-related disorders.

Th1/Th2 associated transcription factors and cytokines in pregnancies with systemic lupus erythematosus.

Th1/Th2 shift occurs during pregnancy. Systemic lupus erythematosus (SLE) flares may induce the dysregulation of Th1 and Th2 cells. We aimed to investigate the dynamic changes of Th1/Th2 associated transcription factors and cytokines in patients with SLE during pregnancy. Twenty-five pregnant patients with SLE and twenty-two healthy age-matched women served as controls from September 2021 to March 2022 were enrolled in the study. Real-time quantitative reverse transcription polymerase chain reaction analysis of peripheral blood mononuclear cells were performed to measure the expression of Th1 specific transcription factors T-bet, cytokine IFN-γ, and Th2 specific transcription factors GATA3, cytokine IL-4. The main statistical analysis methods were t test, Mann-Whitney U-test, Pearson correlation and Spearman rank correlation analysis. The mRNA level of IFN-γ and the relative expression of T-bet/GATA3 and IFN-γ/IL-4 in SLE patients were significantly higher than those in healthy individuals, whereas the GATA3 expression is lower in pregnant patients with SLE (p<0.001, p<0.05, p<0.05 and p<0.01 during the whole pregnancy, respectively; p<0.05, p<0.01, p<0.05 and p<0.05 specifically for the 3rd trimester, respectively). There were significant correlations between T-bet and IFN-γ (r=0.492, p<0.05), and between T-bet/GATA3 and IFN-γ/IL-4 (r=0.482, p<0.05). Our work indicates that in SLE patients Th1/Th2 shift is blocked with up-regulation of Th1 cell function and insufficient Th2 polarisation during pregnancy, which may be involved in adverse pregnancy outcomes.

Antigen-Independent Activation of CD8 T Cells Promotes Salt Sensitive Hypertension

While CD8+ T cells (CD8Ts) have recently emerged as key players in hypertension, the precise mechanisms underlying their involvement remain incompletely understood. Our previous research uncovered the critical role of CD8T activation during the initial phase of blood pressure elevation, facilitating their direct interaction with the distal convoluted tubule (DCT) via the interferon-gamma (IFNγ)-PDL1 pathway. Importantly, we have now discovered that CD8T activation during this early phase occurs in an antigen-independent manner. In this study, we delve into the contribution of the P2X7 receptor to CD8T activation and its subsequent promotion of sodium retention in the kidney. We hypothesize that blocking this receptor on CD8Ts will disrupt CD8T-tubular interaction and lead to a reduction in blood pressure. To investigate whether CD8T activation in hypertension necessitates T cell receptor (TCR) stimulation, we employed Rag2−/−/OT1mice, which exclusively harbor transgenic OT1 CD8Ts, recognizing only ovalbumin peptides but not other antigens. These mice, without any exposure to ovalbumin, were induced to develop hypertension through the DOCA + salt (DOCA) model. Blood pressure was monitored using radio-biotelemetry, and CD8T activation was confirmed using flow cytometry. In our in-vitro studies, CD8Ts were obtained from both WT and P2X7 KO mice and stimulated with BZATP or ATP. The endpoints include calcium influx (measured via the intracellular calcium indicator Fluo-4), IFNγ transcription, and glycolytic activity (assessed by Seahorse metabolic flux analyzer) as indicators of activation. To validate our findings in an in-vivo context, we measured the activation profiles of CD8Ts obtained from WT and P2X7 KO mice subjected to sham or DOCA treatment, employing RT-PCR and flow cytometry. Additionally, we conducted an adoptive transfer experiment, transferring naïve WT CD8Ts to P2X7 KO mice undergoing DOCA treatment. Blood pressure was consistently monitored via radio-biotelemetry. Our observations revealed that CD8Ts from Rag2−/−/OT1 mice become activated in response to the DOCA pressor, leading to increased IFNγ production and contributing to salt-sensitive hypertension, even in the absence of TCR-mediated antigen stimulation. Agonists of the P2X7 receptor, such as BZATP and ATP, elicited calcium influx in CD8Ts, upregulated IFNγ transcription, and enhanced glycolytic activity, all indicative of T cell activation. Furthermore, CD8Ts pre-stimulated with the P2X7 agonist BZATP displayed increased tubular PDL1 expression and sodium retention in our co-culture model when compared to naïve CD8Ts. Notably, DOCA-treated P2X7 KO CD8Ts were unable to induce an increase in blood pressure in our adoptive transfer model. However, transferring sham WT CD8Ts to DOCA-treated P2X7 KO mice successfully restored hypertension to WT levels. Our findings suggest a novel mechanism in which P2X7 receptor stimulation contributes to CD8T activation in the pathogenesis of hypertension. The deficiency of this receptor on CD8Ts prevents their activation by ATP, resulting in attenuated blood pressure elevation. As such, the CD8T P2X7 receptor presents itself as a promising target for the improvement of blood pressure management. Funding: NIH R01-HL146713, AHA 23TPA1076467. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

Arginase inhibitor reduces fungal dissemination in murine pulmonary cryptococcosis by promoting anti-cryptococcal immunity

Elevation of arginase enzyme activity in the lung contributes to the pathogenesis of various chronic inflammatory diseases and infections. Inhibition of arginase expression and activity is able to alleviate those effects. Here, we investigated the immunomodulatory effect of arginase inhibitor in C. neoformans infection. In the pulmonary cryptococcosis model that was shown to recapitulate human infection, we found arginase expression was excessively induced in the lung during the late stage of infection. To inhibit the activity of arginase, we administered a specific arginase inhibitor, nor-NOHA, during C. neoformans infection. Inhibition of arginase reduced eosinophil infiltration and level of IL-13 secretion in the lungs. Whole lung transcriptome RNA-sequencing analysis revealed that treatment with nor-NOHA resulted in shifting the Th2-type gene expression patterns induced by C. neoformans infection to the Th1-type immune profile, with higher expression of cytokines Ifng, Il6, Tnfa, Csf3, chemokines Cxcl9 and Cxcl10 and transcription factor Stat1. More importantly, mice treated with arginase inhibitor had more infiltrating brain leukocytes and enhanced gene expression of Th1-associated cytokines and chemokines that are known to be essential for protection against C. neoformans infection. Inhibition of arginase dramatically attenuated spleen and brain infection, with improved survival. Taken together, these studies demonstrated that inhibiting arginase activity induced by C. neoformans infection can modulate host immune response by enhancing protective type-1 immune response during C. neoformans infection. The inhibition of arginase activity could be an immunomodulatory target to enhance protective anti-cryptococcal immune responses.

Abstract 1404: Blood-based transcriptomics provides novel insights into Sjögren's syndrome-associated lymphomagenesis

Abstract Sjögren's syndrome (SS) is a debilitating autoimmune disorder that carries the highest risk of progressing to lymphoma among all autoimmune diseases, with up to 10% of patients developing SS-associated lymphoma. However, the underlying mechanisms are unknown, and there are currently no actionable targets for prevention and therapeutic intervention.To elucidate the mechanisms associated with SS-lymphoma, we performed RNA-Seq in peripheral blood from SS patients without (n=11) or with lymphoma (n=8). Differential gene expression and pathway analysis identified TNF-α signaling via NF-κB, inflammatory response and interferon gamma (IFN-γ) response as the top pathways enriched in SS-lymphoma patients. Interestingly, the enriched IFN-γ response pathway is consistent with our published work identifying elevated IFN-γ transcript levels in salivary gland tissues from SS patients with lymphoma. Central to SS pathogenesis is the elevation of TNF-α signaling, triggering NF-κB pathway activation and subsequent inflammation, along with inflammasome activation, in line with prior observations. Our data reveal significant upregulation of these pathways in SS-lymphoma patients, which together with the inflammatory response and IFN-γ pathway enrichment, support the idea that persistent autoimmune-related chronic inflammation fosters a microenvironment conducive to lymphomagenesis.Next, we sought to assess the impact of SS immune landscape in SS-lymphoma by performing an immune modular analysis using an established framework of 97 whole blood transcriptomics modules (Pascual), derived from transcripts co-expressed in blood across various autoimmune conditions. This analysis revealed a significant downregulation of T-cell and enrichment of B-cell modules in SS-lymphoma compared to SS patients. These findings are consistent with our biopsy results, showing lower T cell counts in high-grade vs. low-grade SS patients. As patients with high-grade SS are more likely to develop SS-lymphoma, these data possibly suggest that suppressed T-cell responses are involved in lymphomagenesis. In addition, the significant enrichment in B-cell transcriptional modules mirrors the observed clinical increase in B cell counts during the transition to SS-lymphoma. Germline mutations in B-cells are thought to drive the clonal expansion of malignant B cells, which may contribute to the observed increase of B cell transcriptomes in SS patients developing lymphoma. We are currently investigating the gene mutation landscape in these datasets to better understand their contribution to SS- lymphomagenesis.This study provides preliminary evidence that chronic inflammation along with dysregulation of T and B compartments in SS significantly contributes to lymphoma progression, emphasizing the need for further research to uncover the molecular basis and develop early diagnostic/intervention tools. Citation Format: Ada Gjyrezi, Charalampos Skarlis, Paul Zumbo, Doron Betel, Ioanna E. Stergiou, Michael Voulgarelis, Clio P. Mavragani, Paraskevi Giannakakou. Blood-based transcriptomics provides novel insights into Sjögren's syndrome-associated lymphomagenesis [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 1404.

Acanthopanax senticosus cultures fermented by Lactobacillus rhamnosus enhanced immune response through improvement of antioxidant activity and inflammation in crucian carp (Carassius auratus)

Lactic acid bacteria (LAB) have been recognized as safe microorganism that improve micro-flora disturbances and enhance immune response. A well-know traditional herbal medicine, Acanthopanax senticosus (As) was extensively utilized in aquaculture to improve growth performance and disease resistance. Particularly, the septicemia, skin wound and gastroenteritis caused by Aeromonas hydrophila threaten the health of aquatic animals and human. However, the effects of probiotic fermented with A. senticosus product on the immune regulation and pathogen prevention in fish remain unclear. Here, the aim of the present study was to elucidate whether the A. senticosus fermentation by Lactobacillus rhamnosus improve immune barrier function. The crucian carp were fed with basal diet supplemented with L. rhamnosus fermented A. senticosus cultures at 2 %, 4 %, 6 % and 8 % bacterial inoculum for 8 weeks. After trials, the weight gain rate (WGR), specific growth rate (SGR) were significantly increased, especially in LGG-6 group. The results confirmed that the level of the CAT, GSH-PX, SOD, lysozyme, and MDA was enhanced in fish received with probiotic fermented product. Moreover, the L. rhamnosus fermented A. senticosus cultures could trigger innate and adaptive immunity, including the up-regulation of the C3, C4, and IgM concentration. The results of qRT-PCR revealed that stronger mRNA transcription of IL-1β, IL-10, IFN-γ, TNF-α, and MyD88 genes in the liver, spleen, kidney, intestine and gills tissues of fish treated with probiotic fermented with A. senticosus product. After infected with A. hydrophila, the survival rate of the LGG-2 (40 %), LGG-4 (50 %), LGG-6 (60 %), LGG-8 (50 %) groups was higher than the control group. Meanwhile, the pathological damage of the liver, spleen, head-kidney, and intestine tissues of probiotic fermentation-fed fish could be alleviated after pathogen infection. Therefore, the present work indicated that L. rhamnosus fermented A. senticosus could be regard as a potential intestine-target therapy strategy to protecting fish from pathogenic bacteria infection.

Evaluation of protective efficacy, serological responses, and cytokine modulation induced by polyvalent Leptospira vaccines in hamsters

Whole-cell inactivated vaccines (bacterins) are the only licensed vaccines available for leptospirosis prevention and control, especially in domestic and farm animals. However, despite their widespread use, inconsistencies in their efficacy have been reported. Because immunity induced by bacterins is mainly mediated by antibodies against leptospiral lipopolysaccharides, the involvement of cellular responses is not well-known. The aim of this study was to investigate the efficacy and characterize the humoral and cellular immune responses induced by whole-cell inactivated leptospirosis bacterin formulations containing serovars Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjoprajitno, and Pomona. For the potency test, hamsters were immunized with one dose of polyvalent bacterins (either commercial or experimental) and then challenged with a virulent Pomona strain. Serological (MAT and IgM and IgG-ELISA) and cellular (cytokine transcription in blood evaluated by RT-qPCR) analyses were performed. The results revealed that vaccination with either bacterin formulation was able to protect 90–100% of the hamsters infected with the Pomona serovar, although most of the surviving animals remained as renal carriers. Specific agglutinating antibodies and significant levels of IgM, IgG, and IgG2 (P < 0.05) that were able to react with the six serovars present in the vaccine formulations were produced, indicating that the vaccines can potentially provide immunity against all strains. The protective immunity of these vaccines was mainly mediated by balanced a Th1/Th2 response, characterized by increased IFN-γ, IL-10 and IL-α transcription. These data support the importance of characterizing immunological responses involved in bacterin efficacy and investing in the improvement of these vaccine formulations.

Implications of IFNγ SNP rs2069705 in primary Sjögren's syndrome: transcriptional activation and B cell infiltration.

Primary Sjögren's syndrome (pSS) is characterized by its autoimmune nature. This study investigates the role of the IFNγ SNP rs2069705 in modulating the susceptibility to pSS. Differential expression of IFNγ and BAFF was analyzed using the GEO database's mRNA microarray GSE84844. Genotyping of the IFNγ SNP rs2069705 was conducted via the dbSNP website. The JASPAR tool was used for predicting transcription factor bindings. Techniques such as dual-luciferase reporter assays, Chromatin immunoprecipitation, and analysis of a pSS mouse model were applied to study gene and protein interactions. A notable increase in the mutation frequency of IFNγ SNP rs2069705 was observed in MNCs from the exocrine glands of pSS mouse models. Bioinformatics analysis revealed elevated levels of IFNγ and BAFF in pSS samples. The model exhibited an increase in both CD20+ B cells and cells expressing IFNγ and BAFF. Knocking down IFNγ resulted in lowered BAFF expression and less lymphocyte infiltration, with BAFF overexpression reversing this suppression. Activation of the Janus kinase (JAK)/STAT1 pathway was found to enhance transcription in the BAFF promoter region, highlighting IFNγ's involvement in pSS. In addition, rs2069705 was shown to boost IFNγ transcription by promoting interaction between its promoter and STAT4. SNP rs2069705 in the IFNγ gene emerges as a pivotal element in pSS susceptibility, primarily by augmenting IFNγ transcription, activating the JAK/STAT1 pathway, and leading to B-lymphocyte infiltration in the exocrine glands.NEW & NOTEWORTHY The research employed a combination of bioinformatics analysis, genotyping, and experimental models, providing a multifaceted approach to understanding the complex interactions in pSS. We have uncovered that the rs2069705 SNP significantly affects the transcription of IFNγ, leading to altered immune responses and B-lymphocyte activity in pSS.

Human metapneumovirus respiratory infection affects both innate and adaptive intestinal immunity.

Respiratory infections are one of the leading causes of morbidity and mortality worldwide, mainly in children, immunocompromised people, and the elderly. Several respiratory viruses can induce intestinal inflammation and alterations in intestinal microbiota composition. Human metapneumovirus (HMPV) is one of the major respiratory viruses contributing to infant mortality in children under 5 years of age worldwide, and the effect of this infection at the gut level has not been studied. Here, we evaluated the distal effects of HMPV infection on intestinal microbiota and inflammation in a murine model, analyzing several post-infection times (days 1, 3, and 5). Six to eight-week-old C57BL/6 mice were infected intranasally with HMPV, and mice inoculated with a non-infectious supernatant (Mock) were used as a control group. We did not detect HMPV viral load in the intestine, but we observed significant changes in the transcription of IFN-γ in the colon, analyzed by qPCR, at day 1 post-infection as compared to the control group. Furthermore, we analyzed the frequencies of different innate and adaptive immune cells in the colonic lamina propria, using flow cytometry. The frequency of monocyte populations was altered in the colon of HMPV -infected mice at days 1 and 3, with no significant difference from control mice at day 5 post-infection. Moreover, colonic CD8+ T cells and memory precursor effector CD8+ T cells were significantly increased in HMPV-infected mice at day 5, suggesting that HMPV may also alter intestinal adaptive immunity. Additionally, we did not find alterations in antimicrobial peptide expression, the frequency of colonic IgA+ plasma cells, and levels of fecal IgA. Some minor alterations in the fecal microbiota composition of HMPV -infected mice were detected using 16s rRNA sequencing. However, no significant differences were found in β-diversity and relative abundance at the genus level. To our knowledge, this is the first report describing the alterations in intestinal immunity following respiratory infection with HMPV infection. These effects do not seem to be mediated by direct viral infection in the intestinal tract. Our results indicate that HMPV can affect colonic innate and adaptive immunity but does not significantly alter the microbiota composition, and further research is required to understand the mechanisms inducing these distal effects in the intestine.

Characterization of immune responses and immunopathology in turkeys experimentally infected with clostridial dermatitis-producing strains of Clostridium septicum

Clostridium septicum is one of the major causative agents of clostridial dermatitis (CD), an emerging disease of turkeys, characterized by sudden deaths and necrotic dermatitis. Despite its economic burden on the poultry industry, the immunopathological changes and pathogen-specific immune responses are poorly characterized. Here, we used three strains of C. septicum, namely Str. A1, Str. B1 and Str. C1, isolated from CD field outbreaks, to experimentally infect turkeys to evaluate local (skin and muscle) and systemic (spleen) pathological and immunological responses. Results showed that while all three strains produced an acute disease, Str. A1 and B1 caused significantly higher mortality when compared to Str. C1. Gross and histopathology evaluation showed that birds infected with Str. A1 and B1 had severe inflammatory, edematous, granulomatous and necrotic lesions in the skin, muscle and spleen, while these lesions produced by Str. C1 were relatively less severe and mostly confined to skin and/or muscle. Immune gene expression in these tissues showed that Str. B1-infected birds had significantly higher expression of interleukin (IL)−1β, IL-6 and interferon (IFN)γ genes compared to uninfected control, suggesting a robust inflammatory response both locally as well as systemically. The transcription of IL-1β and IFNγ in the muscle or spleen of Str. A1-infected birds and IL-1β in the skin of Str. C1-infected group was also significantly higher than control. Additionally, Str. A1 or B1-infected groups also had significantly higher IL-4 transcription in these tissues, while birds infected with all three strains developed C. septicum-specific serum antibodies. Furthermore, splenic cellular immunophenotyping in the infected turkeys showed a marked reduction in CD4+ cells. Collectively, it can be inferred that host responses against C. septicum involve an acute inflammatory response along with antibody production and that the disease severity seem to depend on the strain of C. septicum involved in CD in turkeys.

Transcriptional responses of cytokines, immunoglobulin A, and nitric oxide genes in 1-day-old chicks post Salmonella typhimurium infection: An experimental study.

Salmonella has become one of the hazards prevalent foodborne pathogens causing different diseases in chickens. However, Salmonella typhimurium (ST), a nonhost-specific serovar, is a major avian agent that causes severe disturbance in young chicken wellness. The occurrence of Salmonella in chickens and their antimicrobial resistance were explored in this study. In addition, the immune response of 1-day-old broiler chicks, against multidrug resistant (MDR) ST infection, was also assessed at 4 and 24 hours post infection (pi) in the cecum and spleen, representing their mucosal and systemic immune responses, respectively. A total of 375 samples from 130 diseased and apparently healthy broiler and layer chickens were randomly collected for Salmonella isolation, identification, and resistance profile evaluation, from farms and different clinical laboratories. The immune response of 1-day-old broiler chicks, Ross 308, against in-vivo ST infection was ascertained through the evaluation of heterophile phagocytosis and s expression of cytokines, immunoglobulin A and other immune-regulating genes in the cecum and spleen. Twenty-four, 1-day-old nonvaccinated broiler chicks were used and divided into two groups. The chicks in the infected group were orally inoculated with 0.5 ml of 2 × 108 colony forming units (CFU)/ml of MDR ST suspension, while those in the control group were taken nutrient broth. Seven out of 130 (5.38%) examined chickens were positive for Salmonella. All isolates (100%) were resistant to amoxicillin-clavulanic acid (AMC), cefazolin (CZ), cefoxitin (FOX), ciprofloxacin (CIP), nalidixic acid (NA), tetracycline (TE), fosfomycin (FOS), and colistin (CT) with multiple antimicrobial resistances (MARs) index range of 0.72-0.83, where none of them was resistant to meropenem (MEM). The results of immune response revealed that chicks infected with ST showed significantly different phagocytosis percentages and index values compared to controls. According to the real-time quantitative polymerase chain reaction (RT-qPCR) results, the transcription of IL-8, iNOS, IL-18, IgA, and IFN-γ for chicks infected by ST showed a significantly increased trend (p < 0.01) with increasing chicken age and was higher in the cecum than spleen compared to controls (p < 0.05) during 24 hours after infection. The findings indicated a strong mucosal immune response in the chicks after the ST challenge, which reflects humoral and cellular responses. Our insight recommended the occurrence of a natural immune response stimulator at 1 day age to face the infection, and this can prevent the resistance transfer, with efficient control measures.

Cytokine levels in gingival tissues as an indicator to understand periodontal disease severity

Cytokines regulate periodontal pathogenesis and are relevant estimates of current disease activity. There is sparse information on status of cytokine protein levels in periodontal pocket (gingival) tissues. The current study analysed proteins and transcripts of selected cytokines in varying severity of periodontal disease and elucidated cytokine/cytokine ratios that best indicated periodontal disease severity, in gingival tissues. A total of 92 participants comprising of generalised moderate periodontitis (GMP, n = 18), generalised severe periodontitis (GSP, n = 46) and periodontally healthy controls (PHC, n = 25) were recruited for the study. Interproximal gingival tissue samples were utilised for cytokine protein estimation and mRNA quantification by qRT-PCR and ELISA respectively. Selected key pro and anti-inflammatory cytokines, also representative of various Th subsets were analysed. ROC curve analysis was performed and Youden index was calculated for individual cytokines and pro/anti-inflammatory cytokine ratio to estimate the best indicator of periodontal severity/progression in tissues. IL-1β, TGF-β and IFN-γ cytokine protein levels varied significantly (p ≤ 0.05) with severity of periodontal disease between groups. On comparison between deep and shallow sites within same participant, deep sites showed significant elevation of TGF-β (p ≤ 0.01) and IFN-γ (p ≤ 0.05) and IL-17 cytokines and shallow sites showed elevation of IL-4(p ≤ 0.01) and IL-1β (p ≤ 0.05) cytokines. Analysis of transcripts showed IFN-γ and IL-1β transcript predominance in GSP (p = 0.01) compared to PHC. ROC analysis illustrated 97% sensitivity, 93% specificity with Youden index of 90% for IL-1β cytokine and 81%sensitivity, 79% specificity with a Youden index of 60% for IL-1β/TGF-β ratio In periodontal pocket tissue, a lack of distinct predominance of specific cytokines between study groups or between shallow and deep sites affected by periodontal disease was observed. However, ROC analysis of cytokines revealed IL-1β cytokine and IL-1β/TGF-β ratio as promising indicators of periodontal disease severity in gingival tissues.

Innate immune response to double-stranded RNA in American heritage chicken breeds

Backyard poultry flocks that employ heritage breeds of chicken play a crucial role in the maintenance of poultry pathogens of economic and zoonotic importance. This study examined innate immunity to viral pathogens in heritage chicken breeds using a model of viral double-stranded RNA (dsRNA). Following intraperitoneal injection of high molecular weight (HMW) -poly(I:C)/Lyovec into 4-wk-old chicks, we evaluated gene expression in peripheral blood mononuclear cells (PBMCs) and splenocytes. There was a significant difference across breeds in the expression of IL-4, IL-12p40, IFNγ, and B-cell activating factor (BAFF) in the spleen. In PBMCs, a significant difference in IFN-α expression was seen across breeds. Approximately 57% of IFN-α transcripts in PBMCs was explained by levels of expression of MDA5 transcripts. Using flow cytometry, we showed that only monocytes/macrophages (KUL01+ cells) expressed the scavenger receptor CD163. Regression analysis showed that 42% of fold change in CD163 expression on PBMCs was explained by breed (P < 0.0004). In general, breeds that responded to HMW-poly(I:C) by showing higher upregulation of IFNγ, IL-1β, and IL-12p40 transcripts in the spleen, and higher IFNα transcripts in peripheral blood, expressed less CD163 on blood monocytes. These findings suggest a genetic basis for the response of chickens to double-stranded RNA. Surface expression of the scavenger receptor CD163 in PBMCs following injection of high molecular weight poly(I:C) may be a rapid method to select chickens for breeding based on innate immune response to viral dsRNA.

Th-1 cytotoxic cell-mediated response predominates in the tracheal mucosa following Mycoplasma synoviae infection of MS-H-vaccinated chickens

Mycoplasma synoviae is a pathogen of poultry that causes upper respiratory tract disease. MS-H is a live attenuated temperature-sensitive vaccine that effectively control M. synoviae infection in chickens. However, the mechanisms underpinning protection have not been described previously. In this study, specific-pathogen-free chickens were vaccinated at 3 weeks of age with MS-H vaccine and challenged with field strain M. synoviae 94011/v-18d at 6 weeks of age. Tracheal mucosal inflammation was characterised by the assessment of thickness, histopathological lesions, cellular infiltrates and cytokine transcription. Tracheal lesion scores of unvaccinated-challenged (-V+C) birds were higher than that of vaccinated-challenged (+V+C) birds. +V+C birds displayed early upregulation of IL-4, consistent with a Th-2-skewed response, followed by a later increase in IFN-γ transcription, indicating transition to a Th-1-skewed response. -V+C birds displayed a concurrent early Th-2 and Th-17 response characterised by increase expression of IL-4 and IL-17A respectively, and late T regulatory response characterised by increased IL-10 transcription. +V+C chickens had more cytotoxic T cells (CD8+ T cells) at 7- and 21 days post-challenge (dpc), while -V+C chickens had higher numbers of infiltrating CD4+CD25+ at 7 and 21 dpc. Overall, these observations suggest that the immune response in +V+C chickens had an inflammation characterised by an early Th-2 skewed response followed closely by a Th-1 response and infiltration of cytotoxic T cells, while the response in -V+C chickens was an early Th-2/Th-17-skewed response closely followed by a T regulatory response.

AMPK Controls Glycolytic Compensation in Gvhd-Causing T Cells

Background: The heightened metabolic requirements of alloreactive T cells following allogeneic stem cell transplantation represents a promising area for therapeutic intervention. We have shown that during the initiation of acute graft-versus-host-disease (aGVHD), murine T cells upregulate activity of AMP-activated protein kinase (AMPK), a cellular energy sensor that regulates oxidative and mitochondrial metabolism. Further, transfer of allogeneic T cells deficient in AMPK (AMPK KO) increased recipient survival and decreased disease severity while still preserving leukemia clearance. In the current studies, we demonstrate a novel role for AMPK in controlling glycolytic compensation, both in vitro and vivo, and provide a mechanism as to why cells lacking AMPK are unable to perform this vital function. Together, these data highlight the potential clinical utility in targeting metabolic decision-making to curtail T cell-mediated inflammatory responses, with significant therapeutic implications for the prevention and treatment of aGVHD. Methods: AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients and recovered on day 7 post-transplant. To delete AMPK in primary human T cells, T cells were isolated from healthy donors and electroporated with Cas 9 ribonucleoprotein complexes targeting the AMPKa1 locus. Glycolytic compensation was assessed by measuring extracellular acidification rates (ECAR) using the Agilent Seahorse analyzer and a mitochondrial stress kit. Intracellular cytokine expression for IFNgamma was assessed in day 7 murine T cells following re-stimulation with F1 splenocytes in the presence of Brefeldin A. Aldolase A and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzymatic assays were run on cell lysates with Biovision-Abcam kits according to manufacturer instructions. To measure GAPDH activity, T cells were positively selected for CD45.1 from day 7 recipient spleens, followed by cell lysis and detection of enzymatic activity. Results: AMPK KO murine T cells recovered on day 7 were unable to mediate a compensatory increase in glycolysis following inhibition of the electron transport chain (Figure 1A), (-6.499±5.156 vs 29.11±7.064, p&amp;lt;0.0001). Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following human T cell expansion in vivo in a xenogeneic model of GVHD. Immunoprecipitation of proteins from day 7 murine T cells, using an antibody specific to phosphorylated AMPK targets, recovered lower levels of multiple glycolytic enzymes including aldolase, enolase, pyruvate kinase M, and GAPDH (Figure 1B). Functionally, murine T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation and a decrease in GAPDH activity on day 7 post-transplant. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of IFNγ upon antigenic re-stimulation with F1 splenocytes. Discussion: Our results demonstrate that in addition to influencing oxidative metabolism, AMPK also plays a role in glycolytic metabolism during GVHD in both murine and human T cells. Further, our data suggest that the more prominent lesion in human T cells post-transplant was in glycolysis , a result supported by the novel observation that a set of glycolysis-related proteins are immunoprecipitated together at much lower rates in the absence of AMPK. These findings suggest a model in which AMPK phosphorylation provides a nidus for important interactions among glycolytic enzymes and that in the absence of AMPK, these interactions are lessened, and glycolysis is consequently reduced. Important to our understanding of GVHD pathogenesis, rates of T cell glycolysis can be directly linked to IFNγ production, where reduced glycolytic activity frees GAPDH to sequester IFNγ transcripts and thereby limit IFNγ protein translation. Altogether, these results document a prominent role for AMPK in controlling T cell glycolytic activity during GVHD development and offer an additional mechanistic explanation for the decreased severity of GVHD seen following transplantation of AMPK KO T cells. Consistently similar results in human xenogeneic T cells provides a further strong rationale for development of AMPK inhibition as a clinically translatable strategy for GVHD prevention and treatment.