Levels Of IFN-β Research Articles

PMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production.

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.

Acinetobacter baumannii up-regulates LncRNA-GAS5 and promotes the degradation of STX17 by blocking the activation of YY1

ABSTRACT Acinetobacter baumanniitriggers autophagy, affects the degradation of autophagy, and causes severe inflammatory injury. LncRNA growth arrest-specific transcript 5 (LncRNA-GAS5) and Yin and Yang 1 (YY1) are known to play an important role in the regulation of autophagy, however, the precise role of LncRNA-GAS5 and YY1 in the damage to autophagy caused by Acinetobacter baumanniiremains unclear. The aim of this study was to investigate the role of LncRNA-GAS5 and YY1 in the regulation of autophagy induced by Acinetobacter baumannii. We found that LncRNA-GAS5 was up-regulated following infection with Acinetobacter baumannii, thus resulting in the degradation of STX17, autophagy disorders, and the aggravated replication of Acinetobacter baumannii. We also analyzed the mechanism of interaction between LncRNA-GAS5 and YY1 and found that YY1 regulated its expression in a negative manner by binding to the promoter of LncRNA-GAS5. LncRNA-GAS5 and YY1 had opposite effects on the expression of STX17, this process maintained the stable expression of STX17. Following Acinetobacter baumannii infection, YY1 was down regulated and then separated from the binding region of LncRNA-GAS5, thus resulting in the activation of LncRNA-GAS5 transcription and reduction in STX17 protein expression. Finally, we infected LncRNA-GAS5 knockdown mice with Acinetobacter baumannii, the expression levels of IFN-β in the lungs increased significantly, this alleviated lung injury. In conclusion, our work demonstrated the mechanism by which Acinetobacter baumannii infection can cause the degradation of STX17. We also demonstrated that LncRNA-GAS5 may be a potential therapeutic target for the treatment of lung injury induced by Acinetobacter baumannii.

The fowl adenovirus serotype 4 (FAdV-4) induce cellular pathway in chickens to produce interferon and antigen-presented molecules (MHCI/II)

FAdV-4 is the major strain of adenovirus that responsible for hydro-pericardial syndrome (HPS) in poultry. In this study, the virus's specific gene fragments were isolated from clinically suspected cases and amplified by PCR. Finally, after a viral infection to investigate the immune response of the host, the gene expression of MHC (major histo-compatible) molecules (MHCIα, MHCIIβ), Ii (Invariant Chain) gene, inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), and transcription factors (MDA5, STING, IRF7, and NF-kB) were detected by real-time PCR (fluorescence technology). The results of sequence comparison showed that the clinically isolated virus was 100% homologous to a virulent strain of avian adenovirus group C serotype 4 (FAdV-4), which were named AH-FAdV-4. The TCID50 and pathogenicity of the virus were determined that was 106.52/0.1 mL with a mortality rate of 100% in chickens and 0% in ducks. Furthermore, results showed that the expression level of MHCIα, MHCIIβ, and Ii genes in chicken embryo kidney cells significantly (P < 0.01) upregulated (increased) after infection, which was 43, 5.2, and 2.5 times higher than the control group. With the addition of PDTC, an inhibitor of NF-kB, then the expression level of MHCIα, MHCIIβ, and Ii was decreased significantly (P < 0.01) than the control group. The transcription levels of these genes were decreased 0.64, 0.27, and 0.26 respectively. Simultaneously, the expression levels of IFN-β, IFN-γ, and IL-1β were also significantly (P < 0.01) up-regulated (increased) 7.8, 22.7, and 5 times higher than the control group. It was found that up-regulation of STING and NF-κB pathways are directly involved in the regulation of inflammatory cytokines (IFN-β, IFN-γ, and IL-1β), MHC molecules (MHCIα, MHCIIβ), and Ii gene. The results also showed that the gene regulation pathways consecutively increased the expression levels of MDA5, STING, IRF7, and NF-kB. It is conducted that the expression levels of cytokines, MHC molecules, and li gene were increased by STING and NF-kB pathways.

Endothelial Activation in Orientia tsutsugamushi Infection Is Mediated by Cytokine Secretion From Infected Monocytes.

Scrub typhus, caused by Orientia tsutsugamushi, is a common systemic infection in Asia. Delay in diagnosis and treatment can lead to vasculitis in the visceral organs and other complications. The mechanisms that drive endothelial activation and the inflammatory response in O. tsutsugamushi infection remain unknown. In addition, the interaction between monocytes and endothelial cells is still unclear. Here we demonstrate that O. tsutsugamushi-infected human dermal microvascular endothelial cells produced moderate levels of chemokines and low levels of IL-6 and IFN-β, but not TNF or IL-1β. Recombinant TNF and cytokine-rich supernatants from infected monocytes markedly enhanced chemokine production in infected endothelial cells. We also show that TNF and monocyte supernatants, but not O. tsutsugamushi infection of endothelial cells per se, upregulated the endothelial cell surface expression of ICAM-1, E-selectin, and tissue factor. This finding was consistent with the inability of O. tsutsugamushi to induce cytokine secretion from endothelial cells. The upregulation of surface molecules after stimulation with monocyte supernatants was significantly reduced by neutralizing anti-TNF antibodies. These results suggest that endothelial cell activation and response are mainly mediated by inflammatory cytokines secreted from monocytes.

Chai Hu oral liquid enhances the immune functions of both spleen and bursa of Fabricius in heat-stressed broilers through strengthening TLR4-TBK1 signaling pathway

Heat stress can affect the poultry production and immune status of broilers. Heat stress disrupts intestinal integrity and increases intestinal inflammation, which is related with body immune dysfunction. Chai Hu oral liquid used as an antipyretic and anti-inflammatory drug is widely used in exogenous fever of poultry, but its resistance to heat stress and the mechanism is still unclear. In this study, a chronic heat stressed broilers model was established to explore the mechanisms of broilers’ immune function changes and the effects of Chai Hu oral liquid. In this study, a total of 480 broilers were randomly divided into 6 groups with 80 replicates. Heat stress (HS) group broilers were stressed at 35 ± 2°C for 5 or 10 consecutive d with 6 h/d. Heat stressed (for 5 or 10 d) broilers were given with Jieshu KangreSan (Positive), Chai Hu oral liquid high, middle and low dosage (CH-High, CH-Mid, CH-Low) by oral administration. Birds in control group were treated with the same volume of PBS only in 25 ± 2°C. All birds were sacrificed at last heat stress challenged day. Changes in immune function were assessed by immune organs index, serum IFN-γ level, gene and protein expressions of immune factors in spleen and bursa of Fabricius. Results from this experiment showed that heat stress enhanced the immune organs’ edema by directly increased the organs indexes of spleen and bursa of Fabricius in broilers. Heat stress for 10 d also increased bursa of Fabricius HSP70 protein level and significantly lowered the spleen and bursa of Fabricius proteins expressions of IFN-α, IFN-β, and IFN-γ in broilers. The IFN-β and IFN-γ protein levels in spleen and bursa of Fabricius also decreased in heat stressed broilers for 5 d. The gene and protein expressions of TLR4 and TBK1 markedly decreased in spleen and bursa of Fabricius of broilers treated with chronic heat stress. Chai Hu oral liquid reduced edema of immune organs and elevated TLR4-TBK1 signaling pathway to release immune factors. Above results indicated that chronic heat stress induced impaired immune function by inhibiting TLR4-TBK1 signaling pathway, and Chai Hu oral liquid had effective protection of body's immune ability by enhancing this signaling pathway.

Changes in Cerebrospinal Fluid Balance of TNF and TNF Receptors in Naïve Multiple Sclerosis Patients: Early Involvement in Compartmentalised Intrathecal Inflammation

An imbalance of TNF signalling in the inflammatory milieu generated by meningeal immune cell infiltrates in the subarachnoid space in multiple sclerosis (MS), and its animal model may lead to increased cortical pathology. In order to explore whether this feature may be present from the early stages of MS and may be associated with the clinical outcome, the protein levels of TNF, sTNF-R1 and sTNF-R2 were assayed in CSF collected from 122 treatment-naïve MS patients and 36 subjects with other neurological conditions at diagnosis. Potential correlations with other CSF cytokines/chemokines and with clinical and imaging parameters at diagnosis (T0) and after 2 years of follow-up (T24) were evaluated. Significantly increased levels of TNF (fold change: 7.739; p < 0.001), sTNF-R1 (fold change: 1.693; p < 0.001) and sTNF-R2 (fold change: 2.189; p < 0.001) were detected in CSF of MS patients compared to the control group at T0. Increased TNF levels in CSF were significantly (p < 0.01) associated with increased EDSS change (r = 0.43), relapses (r = 0.48) and the appearance of white matter lesions (r = 0.49). CSF levels of TNFR1 were associated with cortical lesion volume (r = 0.41) at T0, as well as with new cortical lesions (r = 0.56), whilst no correlation could be found between TNFR2 levels in CSF and clinical or MRI features. Combined correlation and pathway analysis (ingenuity) of the CSF protein pattern associated with TNF expression (encompassing elevated levels of BAFF, IFN-γ, IL-1β, IL-10, IL-8, IL-16, CCL21, haptoglobin and fibrinogen) showed a particular relationship to the interaction between innate and adaptive immune response. The CSF sTNF-R1-associated pattern (encompassing high levels of CXCL13, TWEAK, LIGHT, IL-35, osteopontin, pentraxin-3, sCD163 and chitinase-3-L1) was mainly related to altered T cell and B cell signalling. Finally, the CSF TNFR2-associated pattern (encompassing high CSF levels of IFN-β, IFN-λ2, sIL-6Rα) was linked to Th cell differentiation and regulatory cytokine signalling. In conclusion, dysregulation of TNF and TNF-R1/2 pathways associates with specific clinical/MRI profiles and can be identified at a very early stage in MS patients, at the time of diagnosis, contributing to the prediction of the disease outcome.

Upregulated Intrathecal Expression of VEGF-A and Long Lasting Global Upregulation of Proinflammatory Immune Mediators in Vaccine Breakthrough Tick-Borne Encephalitis.

Although anti-TBE vaccines are highly effective, vaccine breakthrough (VBT) cases have been reported. With increasing evidence for immune system involvement in TBE pathogenesis, we characterized the immune mediators reflecting innate and adaptive T and B cell responses in neurological and convalescent phase in VBT TBE patients. At the beginning of the neurological phase, VBT patients have significantly higher serum levels of several innate and adaptive inflammatory cytokines compared to healthy donors, reflecting a global inflammatory state. The majority of cytokines, particularly those associated with innate and Th1 responses, are highly concentrated in CSF and positively correlate with intrathecal immune cell counts, demonstrating the localization of Th1 and proinflammatory responses in CNS, the site of disease in TBE. Interestingly, compared to unvaccinated TBE patients, VBT TBE patients have significantly higher CSF levels of VEGF-A and IFN-β and higher systemic levels of neutrophil chemoattractants IL-8/CXCL8 and GROα/CXCL1 on admission. Moreover, serum levels of IL-8/CXCL8 and GROα/CXCL1 remain elevated for two months after the onset of neurological symptoms, indicating a prolonged systemic immune activation in VBT patients. These findings provide the first insights into the type of immune responses and their dynamics during TBE in VBT patients. An observed systemic upregulation of neutrophil derived inflammation in acute and convalescent phase of TBE together with highly expressed VEGF-A could contribute to BBB disruption that facilitates the entry of immune cells and supports the intrathecal localization of Th1 responses observed in VBT patients.

Abstract 524: The novel STING agonist VB-85247 induces robust durable antitumor immune responses by intravesical administration in a non-muscle invasive bladder cancer model

Abstract Introduction: Bacillus Calmette-Guerin (BCG) unresponsive non-muscle invasive bladder cancer (NMIBC) patients are in great need of effective immunotherapies. STING (Stimulator of Interferon Genes) plays a central role in mounting innate and adaptive immune responses to tumor cells. Activation of the STING pathway leads to the induction of inflammatory cytokines (IFN-α/β, TNF-α, IL-6 and CXCL10), maturation and activation of dendritic cells (DC), and induction of anti-tumor T cells. Our group has developed a novel STING agonist, VB-85247. Here we use a murine NMIBC model to report the potent antitumor effect of VB-85247 compared to standard of care BCG and anti-PD1 checkpoint inhibitor therapy currently used in the management of NMIBC. Methods: We developed a mouse model of Non-Muscle Invasive Bladder Cancer (NMIBC) utilizing orthotopically implanted MB49-Luc cells, permitting BLI (Bioluminescence) measurement of tumor growth by In Vivo Imaging Systems (IVIS). Results: VB-85247 was found to bind to mouse STING and all major variants of human STING protein. VB-85247 induced high levels of IFN-β and other cytokines across cells from different species, including primary human bladder epithelial cells. VB-85247 treatment by intravesical instillation in the mouse model of NMIBC resulted in dose dependent tumor regression starting after the first treatment, achieving up to a 100% complete response rate at the 40 µg dose level after 5 weekly treatments. The treatment was well tolerated, eliciting strong and durable anti-tumor immune responses without any mortality. All cured mice rejected a re-challenge with MB49-Luc cells with no further treatment, demonstrating long-lasting anti-tumor immunity. By contrast, BCG treatment in the same model was not efficacious. Combination with anti-PD1 treatment reduced the dose of VB-85247 needed to achieve 100% complete responses to 20 µg, whereas anti-PD1 treatment alone resulted in only 25% complete responses. In addition, a single dose of 40 µg VB-85247 by bladder instillation in the NMIBC model induced systemic immune responses including serum cytokines demonstrating Type I IFN responses plus DC mobilization and activation in the blood, draining lymph nodes, and spleen within 24 hours. Conclusions: The STING agonist VB-85247 was well tolerated and displayed robust efficacy by bladder instillation in a mouse orthotopic tumor model of NMIBC, achieving up to 100% complete responses. All cured mice rejected fresh inoculations of tumor cells with no further treatment, demonstrating induction of immunologic memory. Treatment with BCG was not efficacious in the model. These results suggest the potential utility of the VB-85247 STING agonist in the treatment of BCG unresponsive NMIBC patients. Based on these data, VB-85247 is being advanced to clinical development. Citation Format: Miglena Prabagar, Venu Bommireddy, Rachael Siegel, Haihua Zheng, Lee Pellegrino, Stanley Nawoschik, Albert Uveges, Steven Paget, Ku Lu, Krista Saufler, Axel Metzger, David Diller, Jason Trama, Grant Gallagher, Chia-Yu Huang, Brian McGuinness, James R. Beasley, Michael McQueney, Martin Adelson, Gary L. Schieven, Eli Mordechai, Rukiye-Nazan Eraslan. The novel STING agonist VB-85247 induces robust durable antitumor immune responses by intravesical administration in a non-muscle invasive bladder cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 524.

Ruxolitinib Rescues Mice from Lethal Interferon Beta Toxicity and TLS

Introduction. Oncolytic Vesicular Stomatitis Virus expressing human interferon beta and the sodium iodide symporter reporter gene (VSV-hIFNβ-NIS) is in Phase 1 clinical testing for systemic administration in patients with relapsed multiple myeloma, acute myeloid leukemia and T cell lymphoma (NCT03017820). Local production of interferon β enhances virotherapy by activating the immune system, and restricts viral replication in normal cells by activating IFN induced antiviral responses. Thus, the virus propagates efficiently in IFN non-responsive cancer cells but is constrained in IFN responsive cells. Cancer cells that are defective in their viral sensing and IFN response pathways are exquisitely sensitive to VSV-IFNβ-NIS oncolysis, cumulating in tumor lysis syndrome and IFNβ induced toxicity in the MPC-11 mouse plasmacytoma model. Here, we evaluated the feasibility of using ruxolitinib, a clinically approved Jak1/2 inhibitor, to rescue mice from virotherapy induced TLS and IFNβ toxicity.Methods. To characterize IFNβ toxicity in the absence of TLS , varying levels of mIFNβ were achieved in the blood of mice by intramuscular injection of an AAV1 vector encoding mIFNβ. For the virotherapy-associated TLS and IFNβ toxicity model, Balb/c mice with subcutaneous MPC-11 murine plasmacytoma were given one intravenous administration of VSV-mIFNβ-NIS. To determine whether the Jak/STAT inhibitor ruxolitonib could impact IFNβ toxicity, mice were treated with ruxolitinib (2 mg/dose by oral gavage) given twice a day for 10 days. All mice were monitored for adverse clinical signs andtumor size. Plasma IFNβ levels, CBC and comprehensive clinical chemistry panels were analyzed at necropsy.Results. There was a dose dependent increase in plasma IFNβ levels with increasing doses of AAV1-mIFNβ given intramuscularly to mice. Adverse clinical signs were seen in mice given high doses of AAV1-mIFNβ. There was a good correlation between IFNβ and AST or ALT levels. High circulating levels of IFNβ resulted in elevation in transaminases, thrombocytopenia, anemia and lymphopenia. IFN receptor defective mice were completely resistant to these toxic effects even with blood levels of >80,000 pg/ml IFNβ. Nontumor bearing Balb/c mice were shown to tolerate high IV doses of VSV-mIFNβ-NIS with transient lymphopenia and transaminases. In Balb/c mice with IFN nonresponsive MPC-11 tumors that support robust viral replication, VSV-mIFNβ-NIS therapy was highly effective for tumor control, but resulted in TLS and IFNβ toxicity, requiring early euthanasia of mice. Mice given VSV and a neutralizing anti-IFNR antibody did not show any adverse clinical signs due to IFNβ toxicity but control of tumor growth was significantly impaired in comparison to virus treatment alone. In contrast combination treatment of mice VSV and ruxolitinib resulted in reversal of lethal IFNβ toxicity without loss of effective tumor control.Conclusions. Our data demonstrate that ruxolitinib reverses the toxicity of IFNβ seen following highly efficacious VSV-IFNβ-NIS virotherapy in the MPC-11 plasmacytoma model. Clinical evaluation of this promising combination is warranted. [Display omitted] DisclosuresPeng:Vyriad: Equity Ownership; Imanis Life Sciences: Employment, Equity Ownership. Russell:Vyriad: Equity Ownership; Imanis Life Sciences: Equity Ownership.

Type I Interferon Dependent hsa-miR-145-5p Downregulation Modulates MUC1 and TLR4 Overexpression in Salivary Glands From Sjögren's Syndrome Patients.

Sjögren’s syndrome (SS) is an autoimmune disease that mainly affects salivary glands (SG) and is characterized by overactivation of the type I interferon (IFN) pathway. Type I IFNs can decrease the levels of hsa-miR-145-5p, a miRNA with anti-inflammatory roles that is downregulated in SG from SS-patients. Two relevant targets of hsa-miR-145-5p, mucin 1 (MUC1) and toll-like receptor 4 (TLR4) are overexpressed in SS-patients and contribute to SG inflammation and dysfunction. This study aimed to evaluate if hsa-miR-145-5p modulates MUC1 and TLR4 overexpression in SG from SS-patients in a type I IFN dependent manner. Labial SG (LSG) biopsies from 9 SS-patients and 6 controls were analyzed. We determined hsa-miR-145-5p levels by TaqMan assays and the mRNA levels of MUC1, TLR4, IFN-α, IFN-β, and IFN-stimulated genes (MX1, IFIT1, IFI44, and IFI44L) by real time-PCR. We also performed in vitro assays using type I IFNs and chemically synthesized hsa-miR-145-5p mimics and inhibitors. We validated the decreased hsa-miR-145-5p levels in LSG from SS-patients, which inversely correlated with the type I IFN score, mRNA levels of IFN-β, MUC1, TLR4, and clinical parameters of SS-patients (Ro/La autoantibodies and focus score). IFN-α or IFN-β stimulation downregulated hsa-miR-145-5p and increased MUC1 and TLR4 mRNA levels. Hsa-miR-145-5p overexpression decreased MUC1 and TLR4 mRNA levels, while transfection with a hsa-miR-145-5p inhibitor increased mRNA levels. Our findings show that type I IFNs decrease hsa-miR-145-5p expression leading to upregulation of MUC1 and TLR4. Together, this suggests that type I interferon-dependent hsa-miR-145-5p downregulation contributes to the perpetuation of inflammation in LSG from SS-patients.

Milk Kefir as a Natural Probiotic, Individually or in Combination with Organic Acids in Broiler Chickens: Influence on the Immune-Related Gene Expression, Intestinal Morphology, Microbiota Activity, and Serum Biochemistry

The objective of this study was to examine the influence of the addition of milk kefir (MK), as a natural probiotic to drinking water, individually or in combination with organic acids (OA), on the growth performance, intestinal microbial counts and morphology, serum metabolites and immune status in broiler chickens. Two hundred and fifty one-day-old broiler chicks (Ross 308) were randomly distributed into 4 treatment groups including a control group, and the control group with MK (2% of drinking water), OA (350 mL/1000 L of water), or MK+OA. At d 35, Body weight gain, feed intake, and feed conversion ratio were recorded. At the end of the experiment (d 35), five birds per treatment were selected to determine the bacterial population, serum biochemical metabolites, and intestinal morphology. According to the results, water supplemented with MK increased the mRNA levels of IFN-γ and IFN-β at 35 d in the jejunum (P < 0.05). OA supplementation significantly increased the gene expression of IL-6 (P < 0.05). On the other hand, the gene expression of IL-12 increased in broilers fed the control diet (P < 0.05). The broiler chickens that received MK+OA showed an improvement in feed conversion ratio (P < 0.05) compared with OA and control groups. The addition of MK+OA resulted in a higher relative weight of thigh in broiler chickens compared with the MK group (P < 0.05), while the relative weight of spleen was lower in OA and MK treatments compared with the MK+OA group (P < 0.05). The results also indicated that the addition of MK+OA decreased the ileocaecal E. coli population compared to other treatments (P < 0.05). Conversely, the birds that received MK+OA had a higher viable count of ileocaecal lactobacilli (P < 0.05) compared with the OA group. In conclusion, the combination of OA with MK had beneficial effects on the performance, intestinal immune-related genes, and gut microbiota activity of broiler chickens.

Altered Immune Phenotypes and HLA-DQB1 Gene Variation in Multiple Sclerosis Patients Failing Interferon β Treatment.

BackgroundInterferon beta (IFNβ) has been prescribed as a first-line disease-modifying therapy for relapsing-remitting multiple sclerosis (RRMS) for nearly three decades. However, there is still a lack of treatment response markers that correlate with the clinical outcome of patients.AimTo determine a combination of cellular and molecular blood signatures associated with the efficacy of IFNβ treatment using an integrated approach.MethodsThe immune status of 40 RRMS patients, 15 of whom were untreated and 25 that received IFNβ1a treatment (15 responders, 10 non-responders), was investigated by phenotyping regulatory CD4+ T cells and naïve/memory T cell subsets, by measurement of circulating IFNα/β proteins with digital ELISA (Simoa) and analysis of ~600 immune related genes including 159 interferon-stimulated genes (ISGs) with the Nanostring technology. The potential impact of HLA class II gene variation in treatment responsiveness was investigated by genotyping HLA-DRB1, -DRB3,4,5, -DQA1, and -DQB1, using as a control population the Milieu Interieur cohort of 1,000 French healthy donors.ResultsClinical responders and non-responders displayed similar plasma levels of IFNβ and similar ISG profiles. However, non-responders mainly differed from other subject groups with reduced circulating naïve regulatory T cells, enhanced terminally differentiated effector memory CD4+ TEMRA cells, and altered expression of at least six genes with immunoregulatory function. Moreover, non-responders were enriched for HLA-DQB1 genotypes encoding DQ8 and DQ2 serotypes. Interestingly, these two serotypes are associated with type 1 diabetes and celiac disease. Overall, the immune signatures of non-responders suggest an active disease that is resistant to therapeutic IFNβ, and in which CD4+ T cells, likely restricted by DQ8 and/or DQ2, exert enhanced autoreactive and bystander inflammatory activities.

Multi-Functional MPT Protein as a Therapeutic Agent against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), avoids the host immune system through its virulence factors. MPT63 and MPT64 are the virulence factors secreted by MTB which regulate host proteins for the survival and proliferation of MTB in the host. Here, we found that MPT63 bound directly with TBK1 and p47phox, whereas MPT64 interacted with TBK1 and HK2. We constructed a MPT63/64-derived multifunctional recombinant protein (rMPT) that was able to interact with TBK1, p47phox, or HK2. rMPT was shown to regulate IFN-β levels and increase inflammation and concentration of reactive oxygen species (ROS), while targeting macrophages and killing MTB, both in vitro and in vivo. Furthermore, the identification of the role of rMPT against MTB was achieved via vaccination in a mouse model. Taken together, we here present rMPT, which, by regulating important immune signaling systems, can be considered an effective vaccine or therapeutic agent against MTB.

Chlorogenic acid is a positive regulator of MDA5, TLR7 and NF-κB signaling pathways mediated antiviral responses against Gammacoronavirus infection

Chlorogenic acid (CGA) is a phenolic compound that has been well studied for its antiviral, anti-inflammatory and immune stimulating properties. This research was aimed to focus on the antiviral properties of CGA on infectious bronchitis virus (IBV) in vivo and in vitro for the very first time. The outcome of in vitro experiments validated that, out of five previously reported antiviral components, CGA significantly reduced the relative mRNA expression of IBV-N in CEK cells. At high concentration (400 mg/kg), CGA supplementation reduced IBV-N mRNA expression levels and ameliorated the injury in trachea and lungs. The mRNA expression levels of IL-6, IL-1β, IL-12, and NF-κB were considerably turned down, but IL-22 and IL-10 were enhanced in trachea. However, CGA-H treatment had considerably increased the expression levels of MDA5, MAVS, TLR7, MyD88, IRF7, IFN-β and IFN-α both in trachea and lungs. Moreover, CGA-H notably induced the CD3+, CD3+ CD4+ and CD4+/CD8+ proliferation and significantly increased the IgA, IgG, and IgM levels in the serum. In conclusion, these results showed that at high concentration CGA is a strong anti-IBV compound that can effectively regulate the innate immunity through MDA5, TLR7 and NF-κB signaling pathways and have the potential to induce the cell mediated and humoral immune response in IBV infected chickens.

Alteration of type I interferon response is associated with subclinical atherosclerosis in virologically suppressed HIV-1-infected male patients.

Given human immunodeficiency virus‐1 (HIV‐1)‐infected patients have alterations in the type I interferon (IFN‐I) pathway and are also at elevated risk of atherosclerosis, we evaluated IFN‐I response and subclinical cardiovascular disease (CVD) association in HIV‐1‐infected patients. Transcript levels of IFN‐α/β and IFN‐stimulated gene 56 (ISG56) were evaluated by RT/real‐time PCR in peripheral blood mononuclear cells collected from asymptomatic HIV‐1‐positive male patients at high risk of developing CVD (n = 34) and healthy subjects (n = 21). Stenosis degree (≥ or <50%), calcium volume score, calcium Agatston score, and myocardial extracellular volume were examined by coronary computerized tomography scan. Carotid intima‐media thickness (cIMT), Framingham risk score, atherosclerotic cardiovascular disease (ASCVD) score, and risk score developed by data collection on adverse effects of anti‐HIV drugs (D:A:D) were also measured. Increased IFN‐α, IFN‐β, and ISG56 levels were observed in all HIV‐1‐infected males compared to healthy controls (p < .001 for all genes analyzed). HIV‐1‐infected patients with a stenosis degree ≥50% showed a higher Framingham risk score (p = .019), which was correlated with IFN‐β and ISG56 levels. HIV‐1‐infected males with enhanced IFN‐I levels and stenosis displayed a higher ASCVD calculated risk (p = .011) and D:A:D score (p = .004). Also, there was a trend toward higher IFN‐α and ISG56 mRNA levels in HIV‐1‐positive patients with an increased cIMT (p > .05). Dysregulation of IFN‐I response might participate in the pathogenesis of HIV‐1‐associated CVD.

MDA5 expression impacts immune cell activation during T1D development

Abstract Coxsackievirus B3 (CVB3) infection has been associated with triggering Type 1 diabetes (T1D), potentially due to direct β-cell damage and bystander activation of autoreactive T cells. CVB3-infection of Non-Obese Diabetic (NOD) mice accelerates T1D onset, in part due to melanoma differentiation-associated protein 5 (MDA5)-dependent antiviral responses. However, the role of MDA5 on immune cells during T1D remains unclear. By using mice that contain an in-frame deletion within the helicase 1 domain of MDA5 (ΔHel1) or lack MDA5 expression (KO), we can test the hypothesis that reduced MDA5 expression impairs IFN-α/β synthesis, immune cell activation, and T1D onset. NOD and KO mice develop T1D equally, but ΔHel1 mice exhibit delayed spontaneous and CVB3-accelerated T1D. During spontaneous T1D, 12-week-old ΔHel1 mice had a significant reduction in TNF+ F4/80+ macrophages (MΦ) (1.6-fold), perforin+ CD8+ T cells (13.1-fold), and IFNγ+ CD4+ T cells (6.1-fold), compared to NOD and KO mice. ΔHel1 mice had a significant reduction in pancreatic IFN-α (2.2-fold) and IFN-β (3.1-fold) levels compared to NOD mice following CVB3-infection. CVB3-infected ΔHel1 mice had a significant decrease in pancreatic TNF+ F4/80+ MΦ (2-fold) compared to NOD and KO mice. However, ΔHel1 and KO mice had reduced pancreatic perforin+ CD8+ T cells (2.5-fold) and IFNγ+ CD4+ T cells (1.8-fold) compared to NOD mice following CVB3 infection. The ΔHel1 mutation decreased basal and CVB3-responsive MΦ and T cells within the pancreata thereby delaying T1D onset. Yet, loss of MDA5 expression failed to delay T1D indicating MDA5-dependant responses may be required to prevent T1D. Future studies will determine if T1D onset is dictated by MDA5 dsRNA binding and/or ATPase activity.

Antiviral proinflammatory phenotype of monocytes in anti-MDA5 antibody-associated interstitial lung disease.

To evaluate upstream and downstream regulators leading to macrophage activation and subsequent cytokine storm in patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-associated interstitial lung disease (ILD). We conducted an integrated miRNA-mRNA association analysis using circulating monocytes from 3 patients with anti-MDA5-associated ILD and 3 healthy controls and identified disease pathways and a regulator effect network by Ingenuity Pathway Analysis (IPA). The expression of relevant genes and proteins was verified using an independent validation cohort, including 6 patients with anti-MDA5-associated ILD, 5 with anti-aminoacyl tRNA synthetase antibody-associated ILD, and 6 healthy controls. IPA identified 26 matched pairs of downregulated miRNA and upregulated mRNAs and revealed that canonical pathways mediated by type I IFN signalling and C-C motif ligand 2 (CCL2) were responsible for the pathogenic process (P < 0.05 for all pathways). The regulatory network model identified IFN-β; Toll-like receptors 3, 7, and 9; and PU.1 as upstream regulators, while the downstream effect of this network converged at the inhibition of viral infection. mRNA and protein expression analysis using validation cohort showed a trend towards the increased expression of relevant molecules identified by IPA in patients with anti-MDA5-associated ILD compared with those with anti-aminoacyl tRNA synthetase antibody-associated ILD or healthy controls. The expression of all relevant genes in monocytes and serum levels of CCL2 and IFN-β declined after treatment in survivors with anti-MDA5-associated ILD. An antiviral proinflammatory network orchestrated primarily by activated monocytes/macrophages might be responsible for cytokine storm in anti-MDA5-associated ILD.

Evaluation of Toll-like receptor 3 (TLR3) signaling pathway genes and its genetic polymorphisms in ectopic and eutopic endometrium of women with endometriosis

ObjectiveToll-like receptors (TLRs, as members of the innate immune system) are expressed in the human endometrium and their aberrant regulation and expression are involved in the pathogenesis of endometrial diseases. This study is aimed at evaluation of TLR3 signaling pathway genes and its genetic changes in endometriosis patients. Materials and methodsBlood samples were collected from 83 endometriosis patients and 93 healthy fertile women and PCR was performed in blood-derived DNA for detection of SNP of TLR3. Also, ectopic (EC) and eutopic (EU) endometrial biopsies were obtained from endometriosis patients (n = 20), as well as endometrium from healthy women (n = 16, CE). Q-PCR was performed for determination of mRNA expression level of TLR3 signaling pathway genes (TLR3, TICAM, NF-kB1A, CXCL10, IRF3, IFN-B1, IL-6 and IL-8). Also, serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were determined using ELISA. ResultsThe mRNA expression levels of TLR3, NF-kB1A, IFN-B1, IRF3, TICAM1, IL-6 and IL-8 were significantly higher in EU compared to ectopic ones and also compared to CE. SNPs frequency (rs3775291 and rs3775290) was not significantly different between patients and controls. Serum protein levels of TLR3, IFN-β, IL-6 and IL-8 were significantly increased in endometriosis patients. ConclusionSignificant changes were observed in the expression of IL-6 and IL-8 cytokines and other genes in TLR3 cascade in diseased EU, demonstrating that EU similarly to EC is in an intensive inflammatory state. These fundamental alterations in the concept of immune response in EU may lead to its activation, escapes from apoptosis, and displaced implantation of the endometrium.

Shenling Baizhu Powder Inhibits RV-SA11-Induced Inflammation and Rotavirus Enteritis via TLR4/MyD88/NF-κB Signaling Pathway.

Rotavirus enteritis (RVE) is a common acute intestinal infectious disease caused by rotavirus infection. It is an important cause of death in children younger than 5 years worldwide. Shenling baizhu powder (SBP), a classic traditional Chinese formulation, is one of the most popularly prescribed medicines for digestive diseases. Clinical studies have revealed the protective effects of SBP on RVE. However, the potential mechanism is still unclear. In this study, we aimed to evaluate the anti-rotavirus effect of SBP and its mechanism, focusing on the TLR4/MyD88/NF-κB signaling pathway. Our results demonstrated that, based on the inhibition of the virus-induced cytopathic effect in Caco-2 cells, the concentration for 50% of maximal effect (EC50) and selectivity index (SI) of SBP for RV-SA11 in the serum were 5.911% and 11.63, respectively. A total of 219 active compounds with oral bioavailability ≥30% and drug-likeness ≥ 0.18 were selected from the 10 ingredients present in the formulation of SBP, which acted on 471 potential targets. A total of 226 target genes of RVE were obtained from the GeneCards database. The protein-protein interaction (PPI) network showed that there was a close interaction between 44 common targets of SBP and RVE. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SBP acted on RVE through various inflammatory pathways and the intestinal immune network. Subsequently, we investigated the effect of SBP on TLR4/MyD88/NF-κB signaling pathway in vitro. After infection with RV- SA11, the expression of TLR4, MyD88, and NF-κB mRNA and protein increased significantly, which could be abolished by SBP treatment. In addition, the IL-1β, TNF-α, IL-6, and IFN-β levels increased markedly in Caco-2 cells infected with RV-SV11. Treatment with SBP partly reversed the changes of IL-1β, TNF-α, and IL-6, while further increased the level of IFN-β. In conclusion, our study revealed that SBP can significantly inhibit rotavirus replication and proliferation in vitro. The antiviral effect may be related to the regulation of the TLR4/MyD88/NF-κB signaling pathway, followed by the down regulation of inflammatory cytokines and up regulation of IFN-β induced by rotavirus.

Type 1 interferon mediates chronic stress-induced neuroinflammation and behavioral deficits via complement component 3-dependent pathway.

Chronic stress is a major risk factor in the pathophysiology of many neuropsychiatric disorders. Further, chronic stress conditions can promote neuroinflammation and inflammatory responses in both humans and animal models. Type I interferons (IFN-I) are critical mediators of the inflammatory response in the periphery and responsible for the altered mood and behavior. However, the underlying mechanisms are not well understood. In the present study, we investigated the role of IFN-I signaling in chronic stress-induced changes in neuroinflammation and behavior. Using the chronic restraint stress model, we found that chronic stress induces a significant increase in serum IFNβ levels in mice, and systemic blockade of IFN-I signaling attenuated chronic stress-induced infiltration of macrophages into prefrontal cortex and behavioral abnormalities. Furthermore, complement component 3 (C3) mediates systemic IFNβ-induced changes in neuroinflammation and behavior. Also, we found significant increases in the mRNA expression levels of IFN-I stimulated genes in the prefrontal cortex of depressed suicide subjects and significant correlation with C3 and inflammatory markers. Together, these findings from animal and human postmortem brain studies identify a crucial role of C3 in IFN-I-mediated changes in neuroinflammation and behavior under chronic stress conditions.