Antiviral Cytokine Response in Neuroinvasive and Non-Neuroinvasive West Nile Virus Infection

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50; serum median 24.48, IQR 11.93–49.81; p < 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.     

SARS-CoV-2 Nucleocapsid Protein Interacts with RIG-I and Represses RIG-Mediated IFN-β Production

SARS-CoV-2 is highly pathogenic in humans and poses a great threat to public health worldwide. Clinical data shows a disturbed type I interferon (IFN) response during the virus infection. In this study, we discovered that the nucleocapsid (N) protein of SARS-CoV-2 plays an important role in the inhibition of interferon beta (IFN-β) production. N protein repressed IFN-β production induced by poly(I:C) or upon Sendai virus (SeV) infection. We noted that N protein also suppressed IFN-β production, induced by several signaling molecules downstream of the retinoic acid-inducible gene I (RIG-I) pathway, which is the crucial pattern recognition receptor (PRR) responsible for identifying RNA viruses. Moreover, our data demonstrated that N protein interacted with the RIG-I protein through the DExD/H domain, which has ATPase activity and plays an important role in the binding of immunostimulatory RNAs. These results suggested that SARS-CoV-2 N protein suppresses the IFN-β response through targeting the initial step, potentially the cellular PRR–RNA-recognition step in the innate immune pathway. Therefore, we propose that the SARS-CoV-2 N protein represses IFN-β production by interfering with RIG-I.     

Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways

Hepatitis B virus (HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon (IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors (TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.     

Inhibition of interferon γ induced interleukin 12 production: A potential mechanism for the anti-inflammatory activities of tumor necrosis factor

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) γ production. IFN-γ, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-γ inhibits production of chemokines (macrophage inflammatory proteins MIP-1α and MIP-1β). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-γ priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-γ-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF^+/+ and TNF^−/− mice injected with Corynebacterium parvum were compared. TNF^−/− mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF^+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF^−/− mice compared with TNF^+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF^−/− mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-γ inhibition of chemokine production and inhibition of IFN-γ-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).     

Galectin-9 and Interferon-Gamma Are Released by Natural Killer Cells upon Activation with Interferon-Alpha and Orchestrate the Suppression of Hepatitis C Virus Infection

Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role of these molecules in HCV suppression. We speculated that galectin-9 might act extracellularly to inhibit HCV binding to host cells and downstream infection. In silico approaches predicted the binding of HCV envelope protein E2 to galectin-9 carbohydrate-recognition domains, and co-immunoprecipitation assays confirmed physical interaction. IFN-γ, on the other hand, triggered the intracellular expressions of two antiviral gate-keepers in target cells, namely, myxovirus-1 (MX1) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Collectively, our data add more complexity to the antiviral innate response mediated by NK cells and highlight galectin-9 as a key molecule that might be exploited to neutralize productive viral infection.     

COPD Is Associated with Elevated IFN-β Production by Bronchial Epithelial Cells Infected with RSV or hMPV

IFN treatment may be a viable option for treating COPD exacerbations based on evidence of IFN deficiency in COPD. However, in vitro studies have used primarily influenza and rhinoviruses to investigate IFN responses. This study aims to investigate the susceptibility to infection and IFN response of primary bronchial epithelial cells (BECs) from COPD donors to infection with RSV and hMPV. BECs from five COPD and five healthy donors were used to establish both submerged monolayer and well-differentiated (WD) cultures. Two isolates of both RSV and hMPV were used to infect cells. COPD was not associated with elevated susceptibility to infection and there was no evidence of an intrinsic defect in IFN production in either cell model to either virus. Conversely, COPD was associated with significantly elevated IFN-β production in response to both viruses in both cell models. Only in WD-BECs infected with RSV was elevated IFN-β associated with reduced viral shedding. The role of elevated epithelial cell IFN-β production in the pathogenesis of COPD is not clear and warrants further investigation. Viruses vary in the responses that they induce in BECs, and so conclusions regarding antiviral responses associated with disease cannot be made based on single viral infections.     

Interferon β transduction of peripheral blood lymphocytes from HIV-infected donors increases Th1-type cytokine production and improves the proliferative response to recall antigens

We are developing a gene therapy method of HIV infection based on the constitutive low production of interferon (IFN) β. Peripheral blood lymphocytes (PBL) from HIV-infected patients at different clinical stages of infection were efficiently transduced with the HMB-H^bHuIFNβ retroviral vector. The constitutive low production of IFN-β in cultured PBL from HIV-infected patients resulted in a decreased viral production and an enhanced survival of CD4⁺ cells, and this protective effect was observed only in the PBL derived from donors having a CD4⁺ cell count above 200 per mm³. In IFN-β-transduced PBL from healthy and from HIV-infected donors, the production of the Th1-type cytokines IFN-γ and interleukin (IL)-12 was enhanced. In IFN-β-transduced PBL from HIV-infected donors, the production of IL-4, IL-6, IL-10, and tumor necrosis factor α was maintained at normal levels, contrary to the increased levels produced by the untransduced PBL. The proliferative response to recall antigens was partially restored in IFN-β-transduced PBL from donors with an impaired antigen response. Thus, in addition to inhibiting HIV replication, IFN-β transduction of PBL from HIV-infected donors improves several parameters of immune function.     

Viral Glycoproteins Induce NLRP3 Inflammasome Activation and Pyroptosis in Macrophages

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1β. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1β release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.     

Prothymosin-α inhibits HIV-1 via Toll-like receptor 4-mediated type I interferon induction

Induction of type I interferons (IFN) is a central feature of innate immune responses to microbial pathogens and is mediated via Toll-like receptor (TLR)-dependent and -independent pathways. Prothymosin-α (ProTα), a small acidic protein produced and released by CD8⁺ T cells, inhibits HIV-1, although the mechanism for its antiviral activity was not known. We demonstrate that exogenous ProTα acts as a ligand for TLR4 and stimulates type I IFN production to potently suppress HIV-1 after entry into cells. These activities are induced by native and recombinant ProTα, retained by an acidic peptide derived from ProTα, and lost in the absence of TLR4. Furthermore, we demonstrate that ProTα accounts for some of the soluble postintegration HIV-1 inhibitory activity long ascribed to CD8⁺ cells. Thus, a protein produced by CD8⁺ T cells of the adaptive immune system can exert potent viral suppressive activity through an innate immune response. Understanding the mechanism of IFN induction by ProTα may provide therapeutic leads for IFN-sensitive viruses.     

FMDV Leader Protein Interacts with the NACHT and LRR Domains of NLRP3 to Promote IL-1β Production

Foot-and-mouth disease virus (FMDV) infection causes inflammatory clinical symptoms, such as high fever and vesicular lesions, even death of animals. Interleukin-1β (IL-1β) is an inflammatory cytokine that plays an essential role in inflammatory responses against viral infection. The viruses have developed multiple strategies to induce the inflammatory responses, including regulation of IL-1β production. However, the molecular mechanism underlying the induction of IL-1β by FMDV remains not fully understood. Here, we found that FMDV robustly induced IL-1β production in macrophages and pigs. Infection of Casp-1 inhibitor-treated cells and NOD-, LRR- and pyrin domain-containing 3 (NLRP3)-knockdown cells indicated that NLRP3 is essential for FMDV-induced IL-1β secretion. More importantly, we found that FMDV L^pro associates with the NACHT and LRR domains of NLRP3 to promote NLRP3 inflammasome assembly and IL-1β secretion. Moreover, FMDV L^pro induces calcium influx and potassium efflux, which trigger NLRP3 activation. Our data revealed the mechanism underlying the activation of the NLRP3 inflammasome after FMDV L^pro expression, thus providing insights for the control of FMDV infection-induced inflammation.     

Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.     

Genetic and Shared Environmental Influences on Interferon-γ Production in Response to Mycobacterium tuberculosis Antigens in a Ugandan Population

Interferon-γ (IFN-γ) is a key cytokine in the immune response to Mycobacterium tuberculosis (Mtb). Many studies established IFN-γ responses are influenced by host genetics, however differed widely by the study design and heritability estimation method. We estimated heritability of IFN-γ responses to Mtb culture filtrate (CF), ESAT-6, and Antigen 85B (Ag85B) in 1,104 Ugandans from a household contact study. Our method separately evaluates shared environmental and genetic variance, therefore heritability estimates were not upwardly biased, ranging from 11.6% for Ag85B to 22.9% for CF. Subset analyses of individuals with latent Mtb infection or without human immunodeficiency virus infection yielded higher heritability estimates, suggesting 10–30% of variation in IFN-γ is caused by a shared environment. Immunosuppression does not negate the role of genetics on IFN-γ response. These estimates are remarkably close to those reported for components of the innate immune response. These findings have implications for the interpretation of IFN-γ response assays and vaccine studies.     

Mast cells elicit proinflammatory but not type I interferon responses upon activation of TLRs by bacteria

Balanced induction of proinflammatory and type I IFN responses upon activation of Toll-like receptors (TLRs) determines the outcome of microbial infections and the pathogenesis of autoimmune and other inflammatory diseases. Mast cells, key components of the innate immune system, are known for their debilitating role in allergy and autoimmunity. However, their role in antimicrobial host defenses is being acknowledged increasingly. How mast cells interact with microbes and the nature of responses triggered thereby is not well characterized. Here we show that in response to TLR activation by Gram-positive and -negative bacteria or their components, mast cells elicit proinflammatory but not type I IFN responses. We demonstrate that in mast cells, bound bacteria and TLR ligands remain trapped at the cell surface and do not undergo internalization, a prerequisite for type I IFN induction. Such cells, however, can elicit type I IFNs in response to vesicular stomatitis virus which accesses the cytosolic retinoic acid-inducible gene I receptor. Although important for antiviral immunity, a strong I IFN response is known to contribute to pathogenesis of several bacterial pathogens such as Listeria monocytogenes. Interestingly, we observed that the mast cell-dependent neutrophil mobilization upon L. monocytogenes infection is highly impaired by IFN-β. Thus, the fact that mast cells, although endowed with the capacity to elicit type I IFNs in response to viral infection, elicit only proinflammatory responses upon bacterial infection shows that mast cells, key effector cells of the innate immune system, are well adjusted for optimal antibacterial and antiviral responses.     

Cellular and Cytokine Responses to Salmonella enterica Serotype Typhi Proteins in Patients with Typhoid Fever in Bangladesh

We assessed interferon-gamma (IFN-γ) responses via enzyme-linked immunosorbent spot (ELISPOT) to a number of S. Typhi antigens in samples from humans with S. Typhi bacteremia and typhoid fever in Bangladesh. Compared with responses in healthy endemic zone controls, there were significantly increased IFN-γ responses at the time of clinical presentation (acute phase) and at convalescence 14–28 days later. The majority (80–90%) of IFN-γ expressing T cells were CD4+. We observed a significant increase in interleukin-17 (IL-17) positive CD4 + T cells at convalescent versus acute stage of infection using an intracellular cytokine staining assay. We also found that stimulated peripheral blood mononuclear cells (PBMCs) produced significantly increased levels of a number of cytokines at the convalescent versus acute phase of infection, including IFN-γ, MIP-1β, sCD40L, TNF-β, IL-13, and IL-9. These results suggest that S. Typhi antigens induce a predominantly Th1 response, but that elevations in other cytokines may be modulatory.     

Heterogeneity of immune control in chronic hepatitis B virus infection: Clinical implications on immunity with interferon-α treatment and retreatment

Hepatitis B virus (HBV) infection is a global public health issue. Interferon-α (IFN-α) treatment has been used to treat hepatitis B for over 20 years, but fewer than 5% of Asians receiving IFN-α treatment achieve functional cure. Thus, IFN-α retreatment has been introduced to enhance antiviral function. In recent years, immune-related studies have found that the complex interactions between immune cells and cytokines could modulate immune response networks, in-cluding both innate and adaptive immunity, triggering immune responses that control HBV replication. However, heterogeneity of the immune system to control HBV infection, particularly HBV-specific CD8⁺ T cell heterogeneity, has consequ-ential effects on T cell-based immunotherapy for treating HBV infection. Altogether, the host’s genetic variants, negative-feedback regulators and HBV components affecting the immune system''s ability to control HBV. In this study, we reviewed the literature on potential immune mechanisms affecting the immune control of HBV and the clinical effects of IFN-α treatment and retreatment.     

Cytokine Responses to Novel Antigens in a Peri-Urban Population in Brazil Exposed to Leishmania infantum chagasi

Visceral leishmaniasis (VL) is fatal if untreated, and there are no vaccines for this disease. High levels of CD4-derived interferon-γ (IFN-γ) in the presence of low levels of interleukin-10 (IL-10) predicts vaccine success. Tumor necrosis factor-α (TNF-α) is also important in this process. We characterized human immune responses in three groups exposed to Leishmania infantum chagasi in Brazil: 1) drug-cured VL patients (recovered VL); 2) asymptomatic persons with positive Leishmania-specific delayed-type hypersensitivity skin reactions (DTH+); and 3) DTH-negative household contacts. Magnitude of DTH correlated with crude Leishmania antigen–driven IFN-γ, TNF-α, and IL-5, but not IL-10. DTH+ persons showed equivalent levels of IFN-γ, but higher levels of IL-10, to tryparedoxin peroxidase and Leishmania homolog of receptor for activated C kinase compared with recovered VL patients. The IFN-γ:IL-10 and TNF-α:IL-10 ratios were higher in recovered VL patients than in DTH+ persons. Seven of 11 novel candidates (R71, L37, N52, L302.06, M18, J41, and M22) elicited cytokine responses (36–71% of responders) in recovered VL patients and DTH+ persons. This result confirmed their putative status as cross-species vaccine/immunotherapeutic candidates.     

Expansion of human NK-22 cells with IL-7, IL-2, and IL-1β reveals intrinsic functional plasticity

Natural killer-22 (NK-22) cells are a human NK cell subset situated in mucosal-associated lymphoid tissues that specialize in IL-22 secretion in response to IL-23. Here we investigated the cytokine requirements for NK-22 cell expansion. IL-7 maintained the sur-vival of NK-22 cells and IL-22 production in response to IL-23 but was insufficient to induce robust expansion. Proliferation of NK-22 cells was increased markedly by adding either IL-1β or IL-2 to IL-7 and was even stronger in the presence of IL-1β plus IL-2. In contrast to IL-7, continuous culture in IL-1β and IL-2 modified NK-22 cytokine profiles. IL-1β promoted constitutive IL-22 secretion rather than acute IL-22 production in response to IL-23 and induced IL-17 in some cells. IL-2 reduced secretion of IL-22 and IL-17, increasing production of IFN-γ and leukemia inhibitory factor. Functional deviation toward IFN-γ production also was induced by continuous culture in IL-23. These results demonstrate the functional plasticity of NK-22 cells, which may allow flexible responses to different pathogens. Finally, we found that NK-22 cells released the B-cell survival factor, B-cell activating factor belonging to the TNF family (BAFF), suggesting a potential role of NK-22 cells in promoting B-cell–mediated mucosal immunity.