Aberrant innate immune profile associated with COVID-19 mortality in Pretoria, South Africa.

The African continent reported the least number of COVID-19 cases and deaths of all the continents, although the exact reasons for this are still unclear. In addition, little is known about the immunological profiles associated with COVID-19 mortality in Africa. The present study compared clinical and immunological parameters, as well as treatment outcomes in patients admitted with COVID-19 in Pretoria, South Africa, to determine if these parameters correlated with mortality in this population. The in-hospital mortality rate for the cohort was 15.79%. The mortality rate in people living with HIV (PLWH) was 10.81% and 17.16% in people without HIV (p = 0.395). No differences in age (p = 0.099), gender (p = 0.127) or comorbidities were found between deceased patients and those who survived. All four of the PLWH who died had a CD4+ T-cell count <200 cells/mm3, a significantly higher HIV viral load than those who survived (p = 0.009), and none were receiving antiretroviral therapy. Seven of 174 (4%) patients had evidence of auto-antibodies neutralizing Type 1 interferons (IFNs). Two of the them died, and their presence was significantly associated with mortality (p = 0.042). In the adjusted model, the only clinical parameters associated with mortality were: higher fraction of inspired oxygen (FiO2) (OR: 3.308, p = 0.011) indicating a greater need for oxygen, high creatinine (OR: 4.424, p = 0.001) and lower platelet counts (OR: 0.203, p = 0.009), possibly secondary to immunothrombosis. Overall, expression of the co-receptor CD86 (p = 0.021) on monocytes and percentages of CD8+ effector memory 2 T-cells (OR: 0.45, p = 0.027) was lower in deceased patients. Decreased CD86 expression impairs the development and survival of effector memory T-cells. Deceased patients had higher concentrations of RANTES (p = 0.003), eotaxin (p = 0.003) and interleukin (IL)-8 (p < 0.001), all involved in the activation and recruitment of innate immune cells. They also had lower concentrations of transforming growth factor (TGF)-ß1 (p = 0.40), indicating an impaired anti-inflammatory response. The immunological profile associated with COVID-19 mortality in South Africa points to the role of aberrate innate immune responses.

Feasibility of Using a Type I IFN-Based Non-Animal Approach to Predict Vaccine Efficacy and Safety Profiles.

Animal-based tests are used for the control of vaccine quality. However, because highly purified and safe vaccines are now available, alternative approaches that can replace or reduce animal use for the assessment of vaccine outcomes must be established. In vitro tests for vaccine quality control exist and have already been implemented. However, these tests are specifically designed for some next-generation vaccines, and this makes them not readily available for testing other vaccines. Therefore, universal non-animal tests are still needed. Specific signatures of the innate immune response could represent a promising approach to predict the outcome of vaccines by non-animal methods. Type I interferons (IFNs) have multiple immunomodulatory activities, which are exerted through effectors called interferon stimulated genes (ISGs), and are one of the most important immune signatures that might provide potential candidate molecular biomarkers for this purpose. This paper will mainly examine if this idea might be feasible by analyzing all relevant published studies that have provided type I IFN-related biomarkers for evaluating the safety and efficacy profiles of vaccines using an advanced transcriptomic approach as an alternative to the animal methods. Results revealed that such an approach could potentially provide biomarkers predictive of vaccine outcomes after addressing some limitations.

Type I interferon promotes the fate of Toll-like receptor 9-stimulated follicular B cells to plasma cell differentiation.

The activation and differentiation of B cells into plasma cells (PCs) play critical roles in the immune response to infections and autoimmune diseases. Toll-like receptor 9 (TLR9) responds to bacterial and viral DNA containing unmethylated CpG motifs and triggers immune responses in B cells; however, abnormal recognition of self-DNA by TLR9 can cause autoimmune diseases. When stimulated with TLR9 agonists, follicular (FO) B cells, a subset of B cells residing in the FO regions of secondary lymphoid organs, exhibit a propensity for activation but fail to give rise to PCs. The factors that enable the transition of TLR9-activated FO B cells from activation to differentiation into PCs remain unclear. In this study, we show that type I interferon-alpha (IFNα) signaling causes FO B cells activated by CpG stimulation to differentiate into PCs. Although CpG stimulation alone only temporarily increased interferon regulatory factor 4 (IRF4) expression in FO B cells, co-stimulation with both CpG and IFNα enhanced and maintained high IRF4 expression levels, ultimately enabling the cells to differentiate into PCs. Overexpression of IRF4 in FO B cells results in CpG-induced PC transition without IFN signaling. Furthermore, co-stimulation of TLR9 and IFNα receptors significantly enhanced mammalian target of rapamycin (mTOR) signaling, which regulates IRF4 expression and PC generation. These findings suggest that IFNα may play a key role in promoting the fate of PC differentiation in FO B cells activated by TLR9 stimulation.

cGLRs Join Their Cousins of Pattern Recognition Receptor Family to Regulate Immune Homeostasis.

Pattern recognition receptors (PRRs) recognize danger signals such as PAMPs/MAMPs and DAMPs to initiate a protective immune response. TLRs, NLRs, CLRs, and RLRs are well-characterized PRRs of the host immune system. cGLRs have been recently identified as PRRs. In humans, the cGAS/STING signaling pathway is a part of cGLRs. cGAS recognizes cytosolic dsDNA as a PAMP or DAMP to initiate the STING-dependent immune response comprising type 1 IFN release, NF-κB activation, autophagy, and cellular senescence. The present article discusses the emergence of cGLRs as critical PRRs and how they regulate immune responses. We examined the role of cGAS/STING signaling, a well-studied cGLR system, in the activation of the immune system. The following sections discuss the role of cGAS/STING dysregulation in disease and how immune cross-talk with other PRRs maintains immune homeostasis. This understanding will lead to the design of better vaccines and immunotherapeutics for various diseases, including infections, autoimmunity, and cancers.

Relationships between Type 1 interferon signatures and clinical features of the new-onset lupus patients in Japan.

OBJECTIVES: The objective of the study is to investigate the relationships between Type 1 interferon (T1-IFN) signatures and clinical characteristics of lupus patients. METHODS: We examined 49 new-onset lupus patients who were diagnosed between 1999 and 2017. The patients treated with >10 mg of prednisolone or hydroxychloroquine were excluded from this study. Serum T1-IFN signatures were revealed by a functional reporter assay and standardized by recombinant IFN-α. Patient backgrounds, clinical findings, and treatments were retrospectively extracted from their electrical medical records. Clinical data were also available, including SLE Disease Activity Index of SLE patients on admission. RESULTS: T1-IFN signatures of lupus patients closely correlated with lupus disease activities, such as SLE Disease Activity Index-2K, white blood cell, C3 levels, and the titre of double-strand DNA antibody. We found fever and acute lupus dermatitis closely associated with T1-IFN signature. CONCLUSIONS: In lupus patients, fever and acute lupus dermatitis are good indicators of a strong T1-IFN signature.

Functional evaluation of rare OASL variants by analysis of SLE patient-derived iPSCs.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by genetic heterogeneity and an interferon (IFN) signature. The overall landscapes of the heritability of SLE remains unclear. OBJECTIVES: To identify and elucidate the biological functions of rare variants underlying SLE, we conducted analyses of patient-derived induced pluripotent stem cells (iPSCs) in combination with genetic analysis. METHODS: Two familial SLE patient- and two healthy donor (HD)-derived iPSCs were established. Type 1 IFN-secreting dendritic cells (DCs) were differentiated from iPSCs. Genetic analyses of SLE-iPSCs, and 117 SLE patients and 107 HDs in the ImmuNexUT database were performed independently. Genome editing of the variants on iPSCs was performed with the CRISPR/Cas9 system. RESULTS: Type 1 IFN secretion was significantly increased in DCs differentiated from SLE-iPSCs compared to HD-iPSCs. Genetic analyses revealed a rare variant in the 2'-5'-Oligoadenylate Synthetase Like (OASL) shared between SLE-iPSCs and another independent SLE patient, and significant accumulation of OASL variants among SLE patients (HD 0.93%, SLE 6.84%, OR 8.387) in the database. Genome editing of mutated OASL 202Q to wild-type 202 R or wild-type OASL 202 R to mutated 202Q resulted in reduced or enhanced Type 1 IFN secretion of DCs. Three other OASL variants (R60W, T261S and A447V) accumulated in SLE patients had also capacities to enhance Type 1 IFN secretion in response to dsRNA. CONCLUSIONS: We established a patient-derived iPSC-based strategy to investigate the linkage of genotype and phenotype in autoimmune diseases. Detailed case-based investigations using patient-derived iPSCs provide information to unveil the heritability of the pathogenesis of autoimmune diseases.

In-depth proteomic analysis of juvenile dermatomyositis serum reveals protein expression associated with muscle-specific autoantibodies.

OBJECTIVES: The clinical symptoms and complications of JDM differ depending on the type of muscle-specific autoantibodies (MSAs) present. We aimed to identify protein expression profiles specific for MSAs that characterize various clinical features by comprehensively analyzing the proteins present in the serum of patients with JDM. METHODS: We analysed sera from patients with JDM that were positive for anti-melanoma differentiation-associated protein 5 (MDA5) antibodies (n = 5), anti-nuclear matrix protein 2 (NXP2) antibodies (n = 5) and anti-transcriptional intermediary factor 1 alpha or gamma subunit (TIF1-γ) antibodies (n = 5), and evaluated healthy controls (n = 5) via single-shot liquid chromatography-tandem mass spectrometry (MS) in data-independent acquisition mode, which is superior for comparative quantitative analysis. We identified different protein groups based on MSAs and performed pathway analysis to understand their characteristics. RESULTS: We detected 2413 proteins from serum MS analysis; 508 proteins were commonly altered in MSAs, including many myogenic enzymes and IFN-regulated proteins. Pathway analysis using the top 50 proteins that were upregulated in each MSA group revealed that the type I IFN and proteasome pathways were significantly upregulated in the anti-MDA5 antibody group alone. CONCLUSION: Although JDM serum contains many proteins commonly altered in MSAs, the pathways associated with clinical features of MSAs differ based on protein accumulation. In-depth serum protein profiles associated with MSAs may be useful for developing therapeutic target molecules and biomarkers.

Mild COVID-19 in an APECED Patient with Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) and High Titer of Type 1 IFN-Abs: A Case Report.

Autoimmune-Poly-Endocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED), caused by mutations in the Autoimmune Regulator (AIRE) gene, is an autosomal recessive multi-organ autoimmunity syndrome usually defined by high serum titers of type I Interferon Autoantibodies (Type 1 IFN-Abs). These antibodies have recently been found in individuals in the general population who develop life-threatening Coronavirus Disease 2019 (COVID-19), but the significance of pre-existing Type 1 IFN-Abs in APECED patients with COVID-19 remains unclear. Previous reports of COVID-19 outcomes in APECED patients have been divergent, and protective roles have been proposed for female sex, age <26 years, and immunomodulatory medications including intravenous immunoglobulin (IVIg). We report the case of a 30-year-old male APECED patient who experienced a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with mild symptoms of fatigue and headache without respiratory distress and did not require hospitalization. He received a stress dose of hydrocortisone for adrenal insufficiency and continued on his baseline medications, including subcutaneous administration of Immunoglobulins (SCIgs) for chronic inflammatory demyelinating polyneuropathy (CIDP). Mild COVID-19 in a 30-year-old male patient with APECED and pre-existing Type 1 IFN-Abs was unexpected. Younger age and management of autoimmunity may have played a role.

Imidazonaphthyridine effects on Chikungunya virus replication: Antiviral activity by dependent and independent of interferon type 1 pathways.

The Chikungunya virus (CHIKV) causes Chikungunya fever, a disease characterized by symptoms such as arthralgia/polyarthralgia. Currently, there are no antivirals approved against CHIKV, emphasizing the need to develop novel therapies. The imidazonaphthyridine compound (RO8191), an interferon-α (IFN-α) agonist, was reported as a potent inhibitor of HCV. Here RO8191 was investigated for its potential to inhibit CHIKV replication in vitro. RO8191 inhibited CHIKV infection in BHK-21 and Vero-E6 cells with a selectivity index (SI) of 12.3 and 37.3, respectively. Additionally, RO8191 was capable to protect cells against CHIKV infection, inhibit entry by virucidal activity, and strongly impair post-entry steps of viral replication. An effect of RO8191 on CHIKV replication was demonstrated in BHK-21 through type-1 IFN production mechanism and in Vero-E6 cells which has a defective type-1 IFN production, also suggesting a type-1 IFN independent mode of action. Molecular docking calculations demonstrated interactions of RO8191 with the CHIKV E proteins, corroborated by the ATR-FTIR assay, and with non-structural proteins, supported by the CHIKV-subgenomic replicon cells assay.

Macrophage inflammatory state influences susceptibility to lysosomal damage.

Macrophages possess mechanisms for reinforcing the integrity of their endolysosomes against damage. This property, termed inducible renitence, was previously observed in murine macrophages stimulated with LPS, peptidoglycan, IFNγ, or TNFα, which suggested roles for renitence in macrophage resistance to infection by membrane-damaging pathogens. This study analyzed additional inducers of macrophage differentiation for their ability to increase resistance to lysosomal damage by membrane-damaging particles. Renitence was evident in macrophages activated with LPS plus IFNγ, PGE2 , or adenosine, and in macrophages stimulated with IFN-ß, but not in macrophages activated with IL-4 or IL-10. These responses indicated roles for macrophage subtypes specialized in host defense and suppression of immune responses, but not those involved in wound healing. Consistent with this pattern, renitence could be induced by stimulation with agonists for TLR, which required the signaling adaptors MyD88 and/or TRIF, and by infection with murine norovirus-1. Renitence induced by LPS was dependent on cytokine secretion by macrophages. However, no single secreted factor could explain all the induced responses. Renitence induced by the TLR3 agonist Poly(I:C) was mediated in part by the type I IFN response, but renitence induced by Pam3CSK4 (TLR2/1), LPS (TLR4), IFNγ, or TNFα was independent of type 1 IFN signaling. Thus, multiple pathways for inducing macrophage resistance to membrane damage exist and depend on the particular microbial stimulus sensed.

Early IFN-α signatures and persistent dysfunction are distinguishing features of NK cells in severe COVID-19.

Longitudinal analyses of the innate immune system, including the earliest time points, are essential to understand the immunopathogenesis and clinical course of coronavirus disease (COVID-19). Here, we performed a detailed characterization of natural killer (NK) cells in 205 patients (403 samples; days 2 to 41 after symptom onset) from four independent cohorts using single-cell transcriptomics and proteomics together with functional studies. We found elevated interferon (IFN)-α plasma levels in early severe COVD-19 alongside increased NK cell expression of IFN-stimulated genes (ISGs) and genes involved in IFN-α signaling, while upregulation of tumor necrosis factor (TNF)-induced genes was observed in moderate diseases. NK cells exert anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) activity but are functionally impaired in severe COVID-19. Further, NK cell dysfunction may be relevant for the development of fibrotic lung disease in severe COVID-19, as NK cells exhibited impaired anti-fibrotic activity. Our study indicates preferential IFN-α and TNF responses in severe and moderate COVID-19, respectively, and associates a prolonged IFN-α-induced NK cell response with poorer disease outcome.

SARS-CoV-2 Spike Protein-Directed Monoclonal Antibodies May Ameliorate COVID-19 Complications in APECED Patients.

Patients with the monogenic immune dysregulatory syndrome autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene, uniformly carry neutralizing autoantibodies directed against type-I interferons (IFNs) and many develop autoimmune pneumonitis, both of which place them at high risk for life-threatening COVID-19 pneumonia. Bamlanivimab and etesevimab are monoclonal antibodies (mAbs) that target the SARS-CoV-2 spike protein and block entry of SARS-CoV-2 in host cells. The use of bamlanivimab and etesevimab early during infection was associated with reduced COVID-19-associated hospitalization and death in patients at high risk for progressing to severe disease, which led the US Food and Drug Administration to issue an emergency use authorization for their administration in non-hypoxemic, non-hospitalized high-risk patients. However, the safety and efficacy of these mAbs has not been evaluated in APECED patients. We enrolled two siblings with APECED on an IRB-approved protocol (NCT01386437) and admitted them prophylactically at the NIH Clinical Center for evaluation of mild-to-moderate COVID-19. We assessed the safety and clinical effects of early treatment with bamlanivimab and etesevimab. The administration of bamlanivimab and etesevimab was well tolerated and was associated with amelioration of COVID-19 symptoms and prevention of invasive ventilatory support, admission to the intensive care, and death in both patients without affecting the production of antibodies to the nucleocapsid protein of SARS-CoV-2. If given early in the course of COVID-19 infection, bamlanivimab and etesevimab may be beneficial in APECED and other high-risk patients with neutralizing autoantibodies directed against type-I IFNs.

Enhancing innate immunity against virus in times of COVID-19: Trying to untangle facts from fictions.

INTRODUCTION: In light of the current COVID-19 pandemic, during which the world is confronted with a new, highly contagious virus that suppresses innate immunity as one of its initial virulence mechanisms, thus escaping from first-line human defense mechanisms, enhancing innate immunity seems a good preventive strategy. METHODS: Without the intention to write an official systematic review, but more to give an overview of possible strategies, in this review article we discuss several interventions that might stimulate innate immunity and thus our defense against (viral) respiratory tract infections. Some of these interventions can also stimulate the adaptive T- and B-cell responses, but our main focus is on the innate part of immunity. We divide the reviewed interventions into: 1) lifestyle related (exercise, >7 h sleep, forest walking, meditation/mindfulness, vitamin supplementation); 2) Non-specific immune stimulants (letting fever advance, bacterial vaccines, probiotics, dialyzable leukocyte extract, pidotimod), and 3) specific vaccines with heterologous effect (BCG vaccine, mumps-measles-rubeola vaccine, etc). RESULTS: For each of these interventions we briefly comment on their definition, possible mechanisms and evidence of clinical efficacy or lack of it, especially focusing on respiratory tract infections, viral infections, and eventually a reduced mortality in severe respiratory infections in the intensive care unit. At the end, a summary table demonstrates the best trials supporting (or not) clinical evidence. CONCLUSION: Several interventions have some degree of evidence for enhancing the innate immune response and thus conveying possible benefit, but specific trials in COVID-19 should be conducted to support solid recommendations.

Type I IFN signaling in T regulatory cells modulates chemokine production and myeloid derived suppressor cells trafficking during EAE.

Interferon-ß has therapeutic efficacy in Multiple Sclerosis by reducing disease exacerbations and delaying relapses. Previous studies have suggested that the effects of type I IFN in Experimental Autoimmune Encephalomyelitis (EAE) in mice were targeted to myeloid cells. We used mice with a conditional deletion (cKO) of the type I IFN receptor (IFNAR) in T regulatory (Treg) cells to dissect the role of IFN signaling on Tregs. cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice were more activated, the activation status and effector cytokine production of CD4+Foxp3- T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase. Production of chemokines (CCL8, CCL9, CCL22) by CD4+Foxp3- T cells and LN resident cells from cKO mice was suppressed. Suppression of chemokine production was accompanied by a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs and recruitment to peripheral organs was comparable. This study demonstrates that signaling by type I IFNs in Tregs reduces their capacity to suppress chemokine production, with resultant alteration of the entire microenvironment of draining lymph nodes leading to enhancement of MDSC homing, and beneficial effects on disease outcome.

Innate immune stimulation of whole blood reveals IFN-1 hyper-responsiveness in type 1 diabetes.

AIMS/HYPOTHESIS: Self-antigen-specific T cell responses drive type 1 diabetes pathogenesis, but alterations in innate immune responses are also critical and not as well understood. Innate immunity in human type 1 diabetes has primarily been assessed via gene-expression analysis of unstimulated peripheral blood mononuclear cells, without the immune activation that could amplify disease-associated signals. Increased responsiveness in each of the two main innate immune pathways, driven by either type 1 IFN (IFN-1) or IL-1, have been detected in type 1 diabetes, but the dominant innate pathway is still unclear. This study aimed to determine the key innate pathway in type 1 diabetes and assess the whole blood immune stimulation assay as a tool to investigate this. METHODS: The TruCulture whole blood ex vivo stimulation assay, paired with gene expression and cytokine measurements, was used to characterise changes in the stimulated innate immune response in type 1 diabetes. We applied specific cytokine-induced signatures to our data, pre-defined from the same assays measured in a separate cohort of healthy individuals. In addition, NOD mice were stimulated with CpG and monocyte gene expression was measured. RESULTS: Monocytes from NOD mice showed lower baseline vs diabetes-resistant B6.g7 mice, but higher induced IFN-1-associated gene expression. In human participants, ex vivo whole blood stimulation revealed higher induced IFN-1 responses in type 1 diabetes, as compared with healthy control participants. In contrast, neither the IL-1-induced gene signature nor response to the adaptive immune stimulant Staphylococcal enterotoxin B were significantly altered in type 1 diabetes samples vs healthy control participants. Targeted gene-expression analysis showed that this enhanced IFN response was specific to IFN-1, as IFN-γ-driven responses were not significantly different. CONCLUSIONS/INTERPRETATION: Our study identifies increased responsiveness to IFN-1 as a feature of both the NOD mouse model of autoimmune diabetes and human established type 1 diabetes. A stimulated IFN-1 gene signature may be a potential biomarker for type 1 diabetes and used to evaluate the effects of therapies targeting this pathway. DATA AVAILABILITY: Mouse gene expression data are found in the gene expression omnibus (GEO) repository, accession GSE146452 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146452 ). Nanostring count data from the human experiments were deposited in the GEO repository, accession GSE146338 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146338 ). Data files and R code for all analyses are available at https://github.com/rodriguesk/T1D_truculture_diabetologia . Graphical abstract.

The relationship between type 1 IFN and vasculopathy in anti-MDA5 antibody-positive dermatomyositis patients.

OBJECTIVE: Based on the antibody profiles of inflammatory myositis patients, we investigated the type 1 IFN (T1-IFN) signature in serum and DM skin to determine the relationship between T1-IFN and vasculopathy in anti-melanoma differentiation-associated 5 gene (MDA5) antibody-positive DM patients. METHODS: We examined 47 patients with new-onset inflammatory myositis. We divided them into three groups: the anti-MDA5 antibody-positive patients (MDA5 group, n = 16), the anti-aminoacyl-tRNA synthetase antibody-positive patients (aminoacyl-tRNA synthetase group, n = 12), and the double-negative patients (n = 19). Serum T1-IFN signatures were revealed by a functional reporter assay, and we evaluated the T1-IFN signatures of skin based on Mx1 expression by immunohistochemistry. RESULTS: The numbers of patients with classical DM, clinically amyopathic DM and interstitial lung disease were 1, 15 and 13 in the MDA5 group, 2, 3 and 11 in the aminoacyl-tRNA synthetase group, and 10, 1 and 4 in the double-negative group, respectively. The signs of vasculopathies (i.e. palmer papules, skin ulcers and mononeuritis multiplex) were identified only in the MDA5 patients. Most of the MDA5 group showed the highest serum T1-IFN signatures among the three groups. In the histological analysis of DM skin, perivascular inflammations were significant in the MDA5 group. The MDA5 group's Mx1 expression was significantly strong, distributed in blood vessels and interstitial fibroblasts, and had spread to deep dermis. CONCLUSION: Anti-MDA5 antibody-positive DM patients showed high T1-IFN signatures in serum and affected skin. The high T1-IFN signatures of the MDA5 antibody-positive DM patients in serum and deep vasculatures suggested that T1-IFN may have important roles in the vasculopathy of these patients.

Type-1 interferons prolong the lifespan of neutrophils by interfering with members of the apoptotic cascade.

Polymorphonuclear Neutrophils (PMNs) are metabolically highly active phagocytes, present in abundant numbers in the circulation. These active cells take the onus of clearing invading pathogens by crowding at inflammatory sites in huge numbers. Though PMNs are extremely short living and die upon spontaneous apoptosis, extended lifespan has been observed among those cells arrive at the inflammation sites or tackle intracellular infections or face any microbial challenges. The delay/inhibition of spontaneous apoptosis of these short-living cells at the inflammatory core rather helps in combating pathogens. Like many candidates, type-1 interferons (type-1 IFNs) is a group of cytokines predominant at the inflammation site. Although there are some isolated reports, a systematic study is still lacking which addresses the impact of the predominant type of interferon on the spontaneous apoptosis of neutrophils. Here in, we have observed that exposure of these IFNs (IFN-ß, IFN-α & IFN-ω etc) on human neutrophils prevents the degradation of the Bfl1, an important anti-apoptotic partner in the apoptotic cascade. Treatment showed a significant reduction in the release of cytochrome-C in the cytosol, a critical regulator in the intrinsic apoptotic pathway. We also noticed a reduction in the conversion of procaspase -3 to active caspase-3, a crucial executioner caspase towards initiation of apoptosis. Taken together our results show that exposure to interferon interferes with apoptotic pathways of neutrophils and thereby delay its spontaneous apoptosis. These findings would help us further deciphering specific roles if these inflammatory agents are causing any immune-metabolomic changes on PMNs at the inflammatory and infection core.

Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5.

PURPOSE: Hepatitis C virus (HCV) poses a risk of chronic liver disease and threatens a significant number of people worldwide. MicroRNAs (miRNAs) are linked to the regulation of hepatocarcinogenesis. Although miR-373 is required for HCV infection, the underlying mechanisms of miR-373 involvement in HCV replication remain elusive. MATERIALS AND METHODS: Quantitative reverse transcription PCR assays were performed to detect the abundances of miR-373 and HCV RNA either in Huh 7.5 cells or liver biopsy specimens with HCV infection. Luciferase assay was employed to probe the interactions between miR-373 and interferon regulatory factor 5 (IRF5). Western blot was conducted to investigate the effect of miR-373 and IRF5 on HCV replication and activation of type 1 interferon (IFN) response in JFH1-infected Huh 7.5 cells. RESULTS: HCV infection appeared to be caused by increased miR-373 expression. Addition of miR-373 promoted HCV RNA expression, while miR-373 depletion led to an inhibitive effect on HCV replication. Concordantly, IRF5, as a direct target, was limited by miR-373 in JFH1-infected Huh 7.5 cells. In addition, introduction of IRF5 protected HCV replication in the presence of abundant miR-373. Furthermore, the miR-373-mediated inhibitory effect on type 1 IFN response was ablated following IRF5 accumulation. CONCLUSION: miR-373 abrogation reduced HCV replication via activation of type 1 IFN responses by targeting IRF5 in JFH1-infected Huh 7.5 cells, suggesting a promising therapeutic for treating HCV infection.

Plasmacytoid dendritic cell heterogeneity is defined by CXCL10 expression following TLR7 stimulation.

Plasmacytoid dendritic cells (pDCs) play a critical role in bridging the innate and adaptive immune systems. pDCs are specialized type I interferon (IFN) producers, which has implicated them as initiators of autoimmune pathogenesis. However, little is known about the downstream effectors of type I IFN signaling that amplify autoimmune responses. Here, we have used a chemokine reporter mouse to determine the CXCR3 ligand responses in DCs subsets. Following TLR7 stimulation, conventional type 1 and type 2 DCs (cDC1 and cDC2, respectively) uniformly upregulate CXCL10. By contrast, the proportion of chemokine positive pDCs was significantly less, and stable CXCL10+ and CXCL10- populations could be distinguished. CXCL9 expression was induced in all cDC1s, in half of the cDC2 but not by pDCs. The requirement for IFNAR signaling for chemokine reporter expression was interrogated by receptor blocking and deficiency and shown to be critical for CXCR3 ligand expression in Flt3-ligand-derived DCs. Chemokine-producing potential was not concordant with the previously identified markers of pDC heterogeneity. Finally, we show that CXCL10+ and CXCL10- populations are transcriptionally distinct, expressing unique transcriptional regulators, IFN signaling molecules, chemokines, cytokines, and cell surface markers. This work highlights CXCL10 as a downstream effector of type I IFN signaling and suggests a division of labor in pDCs subtypes that likely impacts their function as effectors of viral responses and as drivers of inflammation.

Depletion of MicroRNA-373 Represses the Replication of Hepatitis C Virus via Activation of Type 1 Interferon Response by Targeting IRF5

PURPOSE: Hepatitis C virus (HCV) poses a risk of chronic liver disease and threatens a significant number of people worldwide. MicroRNAs (miRNAs) are linked to the regulation of hepatocarcinogenesis. Although miR-373 is required for HCV infection, the underlying mechanisms of miR-373 involvement in HCV replication remain elusive. MATERIALS AND METHODS: Quantitative reverse transcription PCR assays were performed to detect the abundances of miR-373 and HCV RNA either in Huh 7.5 cells or liver biopsy specimens with HCV infection. Luciferase assay was employed to probe the interactions between miR-373 and interferon regulatory factor 5 (IRF5). Western blot was conducted to investigate the effect of miR-373 and IRF5 on HCV replication and activation of type 1 interferon (IFN) response in JFH1-infected Huh 7.5 cells. RESULTS: HCV infection appeared to be caused by increased miR-373 expression. Addition of miR-373 promoted HCV RNA expression, while miR-373 depletion led to an inhibitive effect on HCV replication. Concordantly, IRF5, as a direct target, was limited by miR-373 in JFH1-infected Huh 7.5 cells. In addition, introduction of IRF5 protected HCV replication in the presence of abundant miR-373. Furthermore, the miR-373-mediated inhibitory effect on type 1 IFN response was ablated following IRF5 accumulation. CONCLUSION: miR-373 abrogation reduced HCV replication via activation of type 1 IFN responses by targeting IRF5 in JFH1-infected Huh 7.5 cells, suggesting a promising therapeutic for treating HCV infection.