IFN-γ Transcripts Research Articles

TGF-β1 suppresses the T-cell response in teleost fish by initiating Smad3- and Foxp3-mediated transcriptional networks

Transforming growth factor-β1 (TGF-β1) can suppress the activation, proliferation, and function of many T-cell subsets, protecting organisms from inflammatory and autoimmune disease caused by an overexuberant immune response. However, whether and how TGF-β1 regulates T-cell immunity in early vertebrates remain unknown. Here, using a Nile tilapia (Oreochromis niloticus) model, we investigated suppression of the T-cell response by TGF-β1 in teleost species. Tilapia encodes an evolutionarily conserved TGF-β1, the expression of which in lymphocytes is significantly induced during the immune response following Edwardsiella piscicida infection. Once activated, tilapia T cells increase TGF-β1 production, which in turn suppresses proinflammatory cytokine expression and inhibits T-cell activation. Notably, we found administration of TGF-β1 cripples the proliferation of tilapia T cells, reduces the potential capacity of Th1/2 differentiation, and impairs the cytotoxic function, rendering the fish more vulnerable to bacterial infection. Mechanistically, TGF-β1 initiates the TGF-βR/Smad signaling pathway and triggers the phosphorylation and nuclear translocation of Smad2/3. Smad3 subsequently interacts with several transcriptional partners to repress transcription of cytokines IL-2 and IFN-γ but promote transcription of immune checkpoint regulator CTLA4 and transcription factor Foxp3. Furthermore, TGF-β1/Smad signaling further utilizes Foxp3 to achieve the cascade regulation of these T-cell genes. Taken together, our findings reveal a detailed mechanism by which TGF-β1 suppresses the T cell-based immunity in Nile tilapia and support the notion that TGF-β1 had already been employed to inhibit the T-cell response early in vertebrate evolution, thus providing novel insights into the evolution of the adaptive immune system.

Pyruvate: Ferredoxin oxidoreductase is involved in IgA-related microbiota dysbiosis and intestinal inflammation.

Inflammatory bowel diseases (IBDs) are associated with both immune abnormalities and dysbiosis, characterized by a loss of Faecalibacterium prausnitzii (F. prausnitzii). However, the reason for F. prausnitzii deficiency remains unclear. 16S rDNA seque-ncing and IgA enzyme-linked immunosorbent assay (ELISA) were applied to identify bacterial community and IgA changes in ulcerative colitis (UC) patients. Forced immunization with F. prausnitzii in rabbits was conducted. To screen for potential IgA-reactive proteins in F. prausnitzii lysates, we performed western blotting and mass spectrometry analyses. Pyruvate: ferredoxin oxidoreductase (PFOR) was cloned and purified, then the immunoreactivity of PFOR was verified in peripheral blood mononuclear cells (PBMCs) through PCR, ELISpot assay and single-cell sequencing (scRNA-seq). Finally, the UC fecal dysbiosis was re-analyzed in the context of the phylogenetic tree of PFOR. F. prausnitzii was underrepresented in UC patients with elevated F. prausnitzii-reactive IgA in the fecal supernatant. Forced immunization with F. prausnitzii in rabbits led to high interferon-γ (IFN-γ) transcription in the colon, along with beta diversity disturbance and intestinal inflammation. PFOR was identified as an IgA-binding antigen of F. prausnitzii and the immunoreactivity was validated in PBMCs, which showed elevated expression of inflammatory cytokines. The scRNA-seq revealed enhanced signals in both T regulatory cells (Tregs) and monocytes after PFOR incubation. Furthermore, phylogenetic analysis revealed that PFOR was a common but conserved protein among the gut bacteria. Our results collectively suggest that PFOR is a bioactive protein in the immune system and may contribute to host-microbial crosstalk. Conserved but bioactive microbial proteins, such as PFOR, warrant more attention in future host-microbial interaction studies.

An inclusive study on cytokine gene expression in Parkinson's disease: Advanced analysis using Bayesian regression model

BackgroundParkinson's disease (PD) is the second most prevalent neurodegenerative disease throughout the globe whose specific pathophysiology is unknown. Researchers believe that inflammation and oxidative stress contribute to PD development. Also, alterations in cytokines production appear to have a key role in the pathogenesis of PD. The aim of the current study was to evaluate gene expression levels of nine cytokines in the peripheral blood of PD patients compared to a healthy control group. MethodsReal-time PCR was used to analyze cytokines gene expression followed by advanced statistical analysis performed using Bayesian regression model in R (version 4.1.0) statistical software. ResultsTNF-α, IL-1β, IL-2, IL-4, IFN-γ, IL-17 and IL-6 transcript levels were upregulated in patients compared to healthy controls. However, CXCL8 expression was downregulated in patients compared to controls and IFN-β expression was not statistically different between the two groups. While we found no significant difference between the groups based on gender and age regarding TNF-α, IL-1β, CXCL8, IL-2, IL-4, IFN-γ and IFN-β gene expression, IL-6 and IL-17 transcript levels showed significant upregulations in older subjects and in females, respectively. In addition, we found that the interaction effects between gender and group on gene expression levels were not significant. In this way, the subgroup analysis within gender revealed that in each gender, expression levels of TNF-α, IL-2, IL-4, IL-6, IFN-γ and IL-17 were significantly higher in patients than controls. However, IFN-β expression level did not show any significant difference between groups and subgroups. ConclusionThe present study provides evidence on significant alterations in cytokine expression with different patterns and points to immune system dysregulation in PD.

Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1-/- Mice.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.

Systematic single-cell pathway analysis to characterize early Tcell activation.

Pathway analysis is a key analytical stage in the interpretation of omics data, providing a powerful method for detecting alterations in cellular processes. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package single-cell pathway analysis (SCPA). We demonstrate the effectiveness of SCPA over commonly used methods, generate a scRNA-seq Tcell dataset, and characterize pathway activity over early cellular activation. This reveals regulatory pathways in Tcells, including an intrinsic type I interferon system regulating Tcell survival and a reliance on arachidonic acid metabolism throughout Tcell activation. A systems-level characterization of pathway activity in Tcells across multiple tissues also identifies alpha-defensin expression as a hallmark of bone-marrow-derived Tcells. Overall, this work provides a widely applicable tool for single-cell pathway analysis and highlights regulatory mechanisms of Tcells.

IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish

Utilization of specialized Th1 cells to resist intracellular pathogenic infection represents an important innovation of adaptive immunity. Although transcriptional evidence indicates the potential presence of Th1-like cells in some fish species, the existence of CD3+CD4+IFN-γ+ T cells, their detailed functions, and the mechanism determining their differentiation in these early vertebrates remain unclear. In the present study, we identified a population of CD3+CD4-1+IFN-γ+ (Th1) cells in Nile tilapia upon T-cell activation in vitro or Edwardsiella piscicida infection in vivo. By depleting CD4-1+ T cells or blocking IFN-γ, Th1 cells and their produced IFN-γ were found to be essential for tilapia to activate macrophages and resist the E. piscicida infection. Mechanistically, activated T cells of tilapia produce IL-2, which enhances the STAT5 and mTORC1 signaling that in turn trigger the STAT1/T-bet axis-controlled IFN-γ transcription and Th1 cell development. Additionally, mTORC1 regulates the differentiation of these cells by promoting the proliferation of CD3+CD4-1+ T cells. Moreover, IFN-γ binds to its receptors IFNγR1 and IFNγR2 and further initiates a STAT1/T-bet axis-mediated positive feedback loop to stabilize the Th1 cell polarization in tilapia. These findings demonstrate that, prior to the emergence of tetrapods, the bony fish Nile tilapia had already evolved Th1 cells to fight intracellular bacterial infection, and support the notion that IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to determine Th1 cell fate, which is an ancient mechanism that has been programmed early during vertebrate evolution. Our study is expected to provide novel perspectives into the evolution of adaptive immunity.

Construction of constitutive expression of Eimeria tenella eukaryotic initiation factor U6L5H2 on the surface of Lactobacillus plantarum and evaluation of its immunoprotective efficiency against chicken coccidiosis

Lactobacillus strains exhibit preferable properties that make them attractive candidates for vaccine delivery systems because of their ability to regulate intestinal mucosal immunity in the body. To date, live Lactobacillus delivery vaccines reported for the defense against Eimeria tenella have been inducer-dependent systems whose applications are significantly limited due to their unattainable induction conditions in vivo. Here, a constitutive expression of Lactobacillus plantarum NC8 surface display system was constructed. Then, this system was used to prepare a live oral vaccine to constitutively express the E. tenella U6L5H2 (EtU6) protein on the NC8 surface and to evaluate its protective efficacy against E. tenella challenge in chickens. The results showed that the heterologous protein (EGFP or EtU6) was successfully expressed on the surface of L. plantarum NC8 without any inducer. The immunoprotection of EtU6 with constitutive expression in L. plantarum NC8 system (NC8/Pc-EtU6) was significantly stronger than that of EtU6 with induced expression of L. plantarum NC8 system (NC8/Pi-EtU6) (ACI: 168.28 vs. 152.74) as evidenced by increased body weight, decreased oocyst output and lesion scores. Furthermore, the constitutive system NC8/Pc-EtU6 produced higher levels of specific cecal SIgA, serum IgG, transcription of cytokines IFN-γ and IL-2, and lymphocyte proliferation than the induced system NC8/Pi-EtU6. These results indicate that, compared to the inducible system, the constitutive surface display system of L. plantarum has the advantages of continuously expressing antigens in vivo and stimulating the host immune system. It could be an ideal platform for vaccine expression. The live vector vaccine for coccidiosis constructed by this constitutive system greatly improves the application potential in chicken production and provides a novel platform for the prevention of coccidiosis in chickens.

The Ifng antisense RNA 1 (IFNG-AS1) and growth arrest-specific transcript 5 (GAS5) are novel diagnostic and prognostic markers involved in childhood ITP.

Background/aim: IFNG-AS1 is a long noncoding RNA that works as an enhancer for the Interferon-gamma (IFN-γ) transcript. GAS5 (growth arrest-specific 5) is a lncRNA that is associated with glucocorticoid resistance. Aberrant expressions of IFNG-AS1 and GAS5 are directly linked to numerous autoimmune disorders but their levels in childhood ITP are still obscure. This study aims to elucidate expressions of target lncRNAs in childhood ITP and their association with pathophysiology and clinical features of the disease as well as their association with types and treatment responses. Method: The fold changes of target lncRNAs in blood samples from children with ITP and healthy controls were analyzed using quantitative real-time PCR (qRT-PCR). Results: There were overexpressed lncRNAs IFNG-AS1 and GAS5 in serum of childhood ITP patients [(median (IQR) = 3.08 (0.2–22.39) and 4.19 (0.9–16.91) respectively, Also, significant higher IFNG-AS1 and GAS5 (p < 0.05) were present in persistent ITP (3–12 months) [ median (IQR) = 4.58 (0.31–22.39) and 3.77 (0.87–12.36) respectively] or chronic ITP (>12 months) [ median (IQR) = 5.6 (0.25–12.59) and 5.61 (1.15–16.91) respectively] when compared to newly diagnosed <3 months patients [IFNG-AS1 median (IQR) = 1.21 (0.2–8.95), and GAS5 median (IQR) = 1.07 (0.09–3.55)]. Also, significant higher lncRNAs IFNG-AS1 and GAS5 were present in patients with partial response to treatment [IFNG-AS1 median (IQR) = 4.15 (0.94–19.25), and GAS5 (median (IQR) = 4.25 (0.81–16.91)] or non-response [IFNG-AS1 median (IQR) = 4.19 (1.25–22.39) and GAS5 median (IQR) = 5.11 (2.34–15.27)] when compared to patients who completely responded to treatment (IFNG-AS1 median (IQR) = 2.09 (0.2–14.58) and GAS5 (median (IQR) = 2.51 (0.09–10.33). In addition, following therapy, the expressions of IFNG-AS1 and GAS5 are significantly negatively correlated with platelet count. Conclusion: Findings suggest that lncRNAs IFNG-AS1 and GAS5 are novel diagnostic and prognostic genetic markers for childhood ITP that can aid in a precise prediction of the disease’s progress at the time of diagnosis and could be a useful tool for treatment planning.

Terminalia chebula Retz. extract ameliorates the symptoms of atopic dermatitis by regulating anti-inflammatory factors in vivo and suppressing STAT1/3 and NF-ĸB signaling in vitro

BackgroundTerminalia chebula (TC) is a traditional medicinal plant used for treating various diseases in humans. However, pharmacological mechanisms underlying the effects of TC in atopic treatment remain unelucidated. Hypothesis/purposeWe investigated the therapeutic effects of TC extract in a mouse model of atopic dermatitis (AD) in vivo and the anti-inflammatory mechanism in vitro. Study design/methodsFor the in vivo study, AD was induced by Dermatophagoides farinae extract (Dfe) in NC/Nga mice. After 14 days of oral administration, the effects of TC concentrations of 30, 100, and 300 mg/kg were analyzed by assessing morphological changes visually; measuring serum levels of inflammatory chemokines/cytokines, IgE, histamine, MDC, TARC, RANTES, and TSLP using ELISA kits; and counting infiltrated mast cells. For in vitro analyses, we used IFNγ/TNF-α-stimulated human keratinocyte cell lines to study the mechanism of action. The production of chemokines/cytokines in the IFNγ/TNF-α-stimulated HaCaT cells was measured using ELISA and a bead array kit. The signaling pathways were analyzed by western blotting and the expression of the transcriptional factors using RT-PCR and luciferase assay. ResultsAdministration of TC significantly alleviated AD-like symptoms in vivo and decreased the ear thickness, dermatitis score, keratinization, and mast cell infiltration. It also resulted in decreased serum levels of IgE, histamine, and inflammation-related mediators MDC, TARC, RANTES, and TSLP compared with those in the Dfe treatment group. Moreover, TC downregulated the expression of the inflammatory chemokines RANTES and MDC in IFNγ/TNF-α-stimulated HaCaT cells. TC inhibited phosphorylated STAT1/3 and NK-κB subunits and nuclear translocation of NF-κB. It also suppressed the transcription of IFNγ, IL-6, IL-8 and MCP-1 in the IFNγ/TNF-α-stimulated HaCaT cells. TC and its constituents, chebulic acid, gallic acid, corlagin, chebulanin, chbulagic acid, ellagic acid, and chebulinic acid, strongly inhibited the nuclear translocation of NF-κB, STAT1, and STAT3 and decreased the expression of inflammatory cytokines at the mRNA level. ConclusionsOverall, TC extract alleviated AD-like symptoms by regulating anti-inflammatory factors in vivo and suppressing STAT1/3 and NF-κB signaling in vitro. In addition, our results show the in vivo effect of partial improvements in AD, as well as the in vitro effect on inflammatory factors by the constituents of TC. This finding provides that TC extract and its components could be potential therapeutic drugs for AD.

Moringa oleifera Leaf Powder Dietary Inclusion Differentially Modulates the Antioxidant, Inflammatory, and Histopathological Responses of Normal and Aeromonas hydrophila-Infected Mono-Sex Nile Tilapia (Oreochromis niloticus).

This study aimed to detect the impact of Moringa oleifera leaf powder dietary inclusion on the antioxidant and innate immune responses of mono-sex Nile tilapia fingerlings. A total of 180 fingerlings were allocated in a random method into three groups with triplicate each. One group (1st group) received the control diet (basal diet (BD) free of moringa) and the other groups (2nd and 3rd) fed BD containing M. oleifera leaf powder at 5 and 10% of the diet, respectively. After 6 weeks of feeding, fish were randomly redistributed into four replicates and rested for 24 h. Then, each fish in the first two replicates was injected with 0.2 mL of PBS, while the others were injected with 0.2 mL of A. hydrophila suspension (1.8 × 106 CFU/mL). Healthy fish fed on M. oleifera leaf powder showed enhanced immune response manifested by significant increases in phagocytic and lysozyme activities with a marked H/L ratio (P < 0.05). In addition, significant alterations of the lymphocytic and heterophilic population in circulation with increasing infiltration in tissue such as the spleen were noticed. Also, M. oleifera significantly upregulated the antioxidants, CAT and GPx, proinflammatory cytokines, IL1-β, IL-8, and IFN-γ relative mRNA levels. On the other hand, following A. hydrophila challenging conditions, M. oleifera caused downregulations of IL1-β, IL-8, and IFN-γ transcription levels, and also lowered the CAT and GPx mRNA levels. In addition, a marked reduction of leukocytic infiltration plus a significant improvement of the degenerative changes in intestinal architecture has occurred. So, M. oleifera leaf powder can be included in the fish diet to enhance immune response under normal health conditions and lower the infection-associated inflammatory response.

Abstract 2091: Macrophage PI3Kg control of chromatin accessibility and transcription factor activity promotes immune suppression and tumor progression

Abstract Tumor development and progression are associated with immune suppression and evasion as well as angiogenesis, fibrosis, and metastasis. Macrophages play critical roles in tumor progression by controlling these events and are the focus of efforts to develop novel cancer therapeutics. We previously showed that PI(3)Kinase γ, a class Ib phosphatidyl inositol 3, 4, 5 kinase that is expressed primarily in myeloid lineage cells, controls tumor progression by serving as a critical switch between immune stimulation and suppression. PI3Kγ also promotes angiogenesis, fibrosis and metastasis. PI3Kγ signaling in macrophages promotes transcription of tumor-promoting cytokines and factors such as Pdgf, Tgfb, Ccl2, Plau, Mmps and Arg-1 and inhibits expression of Il12, Tnfa, Nos2 and other pro-inflammatory factors to stimulate tumor progression and immune evasion. Genetic or pharmaceutical inhibition of PI3Kγ reverses these events and suppress tumor growth. In order to develop novel strategies to control macrophage contributions to cancer progression, we investigated in detail how this kinase controls gene expression. We found that PI3Kγ deletion or inhibition with a selective inhibitor IPI-549, which is currently in late stage clinical development for cancer therapy, stimulates the expression and secretion of IL12, IL6, IL1β, NOS, IFNα, and TNFα, while reducing the secretion and expression of IL10, CCL2, PDGFβ, ARG-1, TGFβ and other factors. Gene expression analysis indicates that PI3Kγ inhibition in macrophages rapidly stimulates transcription of NFκB and IFNγ driven pro-inflammatory pathways as well as nuclear translocation of transcription factors STAT1, IRF1, IRF3, TBK1 and NFκB and inhibition of CEBPβ nuclear activity. To further understand the impact of PI3Kγ on transcription in macrophages, we performed ATAC seq on WT and PI3Kγ null macrophages stimulated with LPS/IFNγ in order to determine the impact of PI3Kγ activity and inhibition on chromatin accessibility. We found that PI3Kγ deletion promoted more open chromatin sites for IRF, FRA I, Pu.1, and NFκB binding, as well as fewer open chromatin sites for AP1 (FOS/JUN), IRF8, and CEBP sites. Importantly, we identified overall chromatin accessibility in the promoters of some NFκB regulated genes, such as Tnfα and Il6, as well as novel open chromatin sites in the promoters for Il12b and Il1b in PI3Kγ null macrophages. We also identified novel closed chromatin sites in the promoters of Il10, Plau and Ccl2, genes that are downregulated upon PI3Kγ inhibition. Further studies to identify the mechanisms by which PI3Kγ regulates transcription are investigating PI3Kγ controls of epigenetic modifiers and transcription factors are ongoing. These studies should lead to the identification of new approaches to regulate tumor associated macrophages to inhibit tumor progression. Citation Format: Hui Chen, Jingwen Liao, Christina Flores, Diana Hargreaves, Judith Varner. Macrophage PI3Kg control of chromatin accessibility and transcription factor activity promotes immune suppression and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2091.

Lipoteichoic Acid from Lacticaseibacillus rhamnosus GG Modulates Dendritic Cells and T Cells in the Gut

Lipoteichoic acid (LTA) from Gram-positive bacteria exerts different immune effects depending on the bacterial source from which it is isolated. Lacticaseibacillus rhamnosus GG LTA (LGG-LTA) oral administration reduces UVB-induced immunosuppression and skin tumor development in mice. In the present work, we evaluate the immunomodulatory effect exerted by LGG-LTA in dendritic cells (DC) and T cells, both in vitro and in the gut-associated lymphoid tissue (GALT). During cell culture, LTA-stimulated BMDC increased CD86 and MHC-II expression and secreted low levels of pro and anti-inflammatory cytokines. Moreover, LTA-treated BMDC increased T cell priming capacity, promoting the secretion of IL-17A. On the other hand, in orally LTA-treated mice, a decrease in mature DC (lamina propria and Peyer’s patches) was observed. Concomitantly, an increase in IL-12p35 and IFN-γ transcription was presented (lamina propria and Peyer’s Patches). Finally, an increase in the number of CD103+ DC was observed in Peyer’s patches. Together, our data demonstrate that LGG-LTA activates DC and T cells. Moreover, we show that a Th1-biased immune response is triggered in vivo after oral LTA administration. These effects justify the oral LTA activity previously observed.

Leishmania infantum UBC1 in Metacyclic Promastigotes from Phlebotomus perniciosus, a Vaccine Candidate for Zoonotic Visceral Leishmaniasis.

Leishmania parasites cause outstanding levels of morbidity and mortality in many developing countries in tropical and subtropical regions. Numerous gene expression profiling studies have been performed comparing different Leishmania species’ life-cycles and stage forms in regard to their distinct infective ability. Based on expression patterns, homology to human orthologues, in silico HLA-binding predictions, and annotated functions, we were able to select several vaccine candidates which are currently under study. One of these candidates is the Leishmania infantum ubiquitin-conjugating enzyme E2 (LiUBC1), whose relative levels, subcellular location, in vitro infectivity in the U937 myeloid human cell model, and protection levels in Syrian hamsters against L. infantum infection were studied herein. LiUBC1 displays a low level of similarity with the mammalian orthologs and relevant structure differences, such as the C-terminal domain, which is absent in the human ortholog. LiUBC1 is present in highly infective promastigotes. Knock-in parasites overexpressing the enzyme increased their infectivity, according to in vitro experiments. Syrian hamsters immunized with the recombinant LiUBC1 protein did not show any parasite burden in the spleen, unlike the infection control group. The IFN-γ transcript levels in splenocytes were significantly higher in the LiUBC1 immunized group. Therefore, LiUBC1 induced partial protection against L. infantum in the Syrian hamster model.

Epigenetic drug ameliorated type 1 diabetes via decreased generation of Th1 and Th17 subsets and restoration of self-tolerance in CD4+ T cells

Female NOD mice develop autoimmune diabetes spontaneously without extrinsic manipulation. Previously, we have shown that weekly administration of the prediabetic female NOD mice with the histone modifier Trichostatin A (TSA) prevented diabetes onset. Herein we show that T lymphocytes from diabetic mice transferred diabetes into immunodeficient NOD.scid recipients while those isolated from drug-treated mice displayed reduced disease-causing ability. Drug treatment also repressed T cell receptor-mediated IFN-γ transcription. Splenic CD4+ T-cells purified from prediabetic mice could be polarized into IFN-γ -producing Th1 and IL-17A-expressing Th17 subsets ex vivo. Adoptive transfer of these cells into immunocompromised NOD.scid mice caused diabetes comparably. Polarized Th1 cells were devoid of IL-17A-producing cells and did not transdifferentiate into Th17 cells in the spleen of immunodeficient recipients. However, polarized Th17 cell preparation had a few contaminant Th1 cells. Adoptive transfer of polarized Th17 cells into NOD.scid recipients led to IFN-γ transcription in recipient splenocytes. Notably, TSA treatment of prediabetic mice abolished the ability of CD4+ T-cells to differentiate into diabetogenic Th1 and Th17 cells ex vivo. This was accompanied by the absence of Ifng and Il17a transcription in the spleen of NOD.scid recipients receiving cells, respectively cultured under Th1 and Th17 polarizing conditions. Significantly, the histone modifier restored the ability of CD4+ but not CD8+ T-cells to undergo CD3-mediated apoptosis ex vivo in a caspase-dependent manner. These results indicate that the histone modifier bestowed protection against type 1 diabetes via negative regulation of signature lymphokines and restitution of self-tolerance in CD4+ T cells.

CD4+ T cell effector activities accelerate Alzheimer's disease pathologies.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive impairments linked to pathological deposition of misfolded self-protein amyloid beta (Aβ) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered beginning early and extending throughout the disease. How these occurs and whether they can be harnessed to affect disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teff) serving to drive pro-inflammatory and neurodestructive immunity. Here, we investigated the role of effector immunity could affect cellular level disease pathobiology in an AD animal model. Aβ-specific Th1 and Th17 Teff were induced by Aβ-immunization of donors, CD4+ T cells isolated, expanded by selective culture in the presence of Aβ, and cloned as monoclonal Teff lines. Aβ-specific Th1 and Th17 Teff were adoptively transferred into the APP/PS1 double transgenic mice. Three weeks after transfer, radial arm water maze (RAWM), biochemical, immunohistochemical and transcriptomic tests assessed memory functions, pathologies, and mechanism. Development of stable monoclonal Aβ-Th1 and Aβ-Th17 cells was verified by specific cytokine signatures, nuclear transcription factors and haplotype matched major histocompatibility-II (MHCII)-Aβ tetramer staining. Accelerated memory impairments were observed in APP/PS1 mice that received Aβ-specific Teffs. Both Aβ-Th1 and Aβ-Th17 cells significantly induced pro-inflammatory cytokines TNF-α, IFN-γ and IL-17 and transcription factors Tbet and RORγ compared to controls in the periphery. Aβ load significantly increased while synaptic plasticity decreased in the brain of AD mice received Aβ-Th1 cells. Aβ-Teff driven systemic inflammatory responses were attributed to the decreased numbers and functions of regulatory T cells (Treg) in both the CNS and periphery. Additionally, transferred Aβ-Teff increased microglia reactivity and neuroinflammatory activities in the affected brain regions. Autoreactive Aβ-Teff transform a pro-inflammatory microenvironment accelerating AD pathology. This is sped by limiting Treg activities. Control of this neurodestructive environment represents a potential therapeutic strategy and can be sped by augmenting peripheral Treg numbers and function. Similar therapeutic ends have been shown to be efficacious in both pre-clinical and clinical trials in related neurodegenerative disorders.

Altered Peripheral Immune Profiles in First-Episode, Drug-Free Patients With Schizophrenia: Response to Antipsychotic Medications.

Previous research has demonstrated aberrations in the levels of inflammatory cytokines in patients with schizophrenia (SCZ), but most of the respective studies have tested a narrow set of inflammatory cytokines. Here, we aimed to analyze broad immune profiles in the peripheral blood of the first-episode drug-free (FEDF) patients with SCZ at baseline and after an 8-week treatment with atypical antipsychotics. Serum samples from 24 FEDF patients with SCZ and 25 healthy control (HC) subjects were tested using Luminex multiplex analysis for 30 cytokines, chemokines, and growth factors. Multiple comparison tests demonstrated that interleukin-2 (IL-2), IL-4, interferon-gamma (IFN-γ), monokine induced by IFN-γ, and granulocyte colony-stimulating factor (G-CSF) levels were significantly increased, whereas those of the epidermal growth factor were significantly decreased in the FEDF patients with SCZ. Moreover, the levels of the 6 dysregulated cytokines as well as those of 12 additional soluble factors in FEDF patients with SCZ were significantly decreased after 8 weeks of antipsychotic treatment. Furthermore, the transcription of G-CSF and IFN-γ was significantly increased in FEDF patients with SCZ when compared with controls, and G-CSF and IFN-γ mRNA levels were highly correlated with their respective protein concentrations. Receiver operating characteristic curves showed that G-CSF and IFN-γ had good performance in differentiating between FEDF patients with SCZ and HC subjects. Taken together, our data revealed that FEDF patients with SCZ were accompanied by a unique pattern of immune profile, and antipsychotic medications seemed to suppress the immune function in these patients, which could be used to develop novel targets for the diagnosis and treatment of SCZ.

Expression analysis of candidate genes in vitiligo patients & effect of oxidative stress on melanocytes

BackgroundVitiligo is an acquired hypomelanotic autoimmune disorder characterized by depigmented macules on the skin. Oxidative stress acts as an initial trigger and autoimmunity leads to vitiligo progression. Cytokines are key mediators of immune system and imbalance between pro- and anti-inflammatory cytokines play a vital role in the pathogenesis of autoimmune diseases. Also, relationship between oxidative stress and oxidative stress induced cytokine secretion is well established. Therefore, the present study aimed to monitor the expression levels of selected cytokine genes (IL1A, IL4, IL1R1, IL1RN and IFNG) from vitiliginous skin along with the effect of oxidative stress on in vitro cultured normal human melanocyte (NHM). MethodsThe gene expression of IL1A, IL1R1, IL1RN, IL4 and IFNG was performed by real-time PCR. Effect of oxidative stress on NHM was measured by MTT assay upon H2O2 treatment. ResultsOutcome of the study revealed significant increase in IFNG transcript levels from the non lesional and lesional skin of vitiligo patients as compared to controls (p = 0.0045 and p = 0.0198, respectively). However, no significant differrence was observed in the transcript levels of IL1A, IL4, IL1R and IL1RN from non lesional and lesional skin of vitiligo patients as compared to controls (p > 0.05). A significant decrease in melanocyte viability was observed upon H2O2 treatment in a dose dependent manner (p < 0.0001). ConclusionThe increased IFNG expression in vitiliginous skin advocates IFN-γ associated melanocyte destruction in vitiligo and decreased melanocyte viability upon H2O2 treatment suggests the vulnerability of melanocytes to oxidative stress which persists in vitiligo microenvironment.

PPARγ ligands modulate the immune response mediators in the pig myometrium — An in vitro study

The current study was conducted with the aim to investigate effects of PPARγ ligands on synthesis of nuclear receptor κB (NF-κB) and selected cytokines (IL-1β, IFNγ, TNFα, IL-4, IL-10, LIF) in the pig myometrium on days 14–15 of the estrous cycle (late-luteal phase) and days 14–15 of the gestational period (beginning of embryonic implantation). The myometrial slices were incubated in vitro for 6 h in medium containing PPARγ ligands, agonists: 15d-prostaglandin J2 or pioglitazone, and antagonist – T0070907. The mRNA transcript and protein abundances were evaluated in tissues and culture medium. During the estrous cycle, PPARγ ligands did not have an effect on the mRNA transcript abundance of the immune response mediators used for treatments. The IL-10 protein abundance in the tissue was less when there was inclusions of pioglitazone in the medium, while the treatment with T0070907 resulted in a larger abundance of NF-κB, IL-1β (in the tissue) and IL-4 (in tissue and culture media). During the gestational period, pioglitazone or PGJ2 suppressed mRNA IFNγ and IL-10 transcript and protein abundances (in the tissue and culture media), whereas there was an enhanced NF-κB protein abundance (in the tissue). Treatment with T0070907 had diverse effects (e.g., for NFκB inhibited mRNA transcript abundance or enhanced protein abundance). The observed changes are related mainly in tissues from pregnant animals. Responses to PPARγ antagonist are indicative of the possible involvement of PPARγ-independent factors as well as ligand-independent activation of the receptor, ligand selectivity/functionality or tissue receptivity to the factors evaluated.

Upregulation of FOXP3 is associated with severity of hypoxia and poor outcomes in COVID-19 patients

The levels of messenger RNA (mRNA) transcription of FOXP3, IFN-γ, TNF, IL-6 and COX-2 from both COVID-19 infected and control subjects were evaluated using SYBRTM green real-time polymerase chain reaction (RT-PCR). Severe/critical cases showed significantly lower lymphocyte counts and higher neutrophil counts than the mild or moderate cases. There were significantly lower levels of mRNA expressions of IFN-γ, TNFα and FOXP3 in COVID-19 patients than in the control group. On the other hand, IL-6 and COX-2 expressions were significantly higher in patients suffering from severe disease. FOXP3 expressions were correlated with the severities of hypoxia and were excellent in predicting the disease severity. This was followed by the IL-6, COX-2 and TNFα expressions. FOXP3 expression was the only biomarker to show a significant correlation with patient mortality. It was concluded that SARS-CoV-2 infection is associated with the downregulation of FOXP3 and upregulations of IL-6 and COX-2.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a ‘cytokine storm.’ In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.