IFN Production Research Articles

Schisandrin C enhances cGAS-STING pathway activation and inhibits HBV replication.

Schisandra Chinensis (Turcz.) Baill. is a long-term used traditional Chinese medicine with the functions of tonifying the kidney and calming the heart, tonifying qi and engendering fluid. It can be used to treat insomnia and dreaminess, spermatorrhea, coughs, as well as liver and kidney deficiency of Yin or Yang Syndrome. Modern pharmacological studies have shown that Schisandra Chinensis regulates host immunity and exhibits anti-cancer, antiviral and liver-protecting effects. However, the specific mechanism by which Schisandra Chinensis modulates antiviral immunity is unknown. We sought to explore the therapeutic effect of the active components of Schisandra Chinensis on anti-viral immunity and further investigate the underlying mechanism. Immunoblotting, quantitative real-time PCR, enzyme-linked immunosorbent assay, immunofluorescence, and immunoprecipitation were used to investigate the effect of schisandrin C (SC), one of the most abundant and biologically active components of Schisandra Chinensis, on the activation of cGAS-STING signaling pathway and the underlying mechanism. In addition, CMA-mediated STING activation and hydrodynamic injection-mediated HBV-replicating mouse model were used to investigate the effect of SC on the activation of STING signaling pathway and its antiviral effect in vivo. SC promoted cGAS-STING pathway activation, accompanied by increased production of interferon β (IFN β) and downstream gene expression. Moreover, SC also exerted anti-HBV effects, reducing HBeAg, HBcAg, HBsAg, and HBV DNA levels in hydrodynamic injection-mediated HBV-replicating mouse model and elevating the production of IFN β and expression of interferon-stimulated genes (IFIT1, ISG15, and CXCL10). Mechanistically, SC could facilitate the interaction between TANK-binding kinase 1 (TBK1) and STING, which is important for IRF3 phosphorylation and production of IFN β. Our study confirmed that SC enhances cGAS-STING pathway activation and inhibits HBV replication, as well as provides clues for chronic hepatitis B and other infectious diseases treated by SC.

PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.

Novel nanoadjuvants balance immune activation with modest inflammation: implications for older adult vaccines

BackgroundAge-associated impairments of immune response and inflammaging likely contribute to poor vaccine efficacy. An appropriate balance between activation of immune memory and inflammatory response may be more effective in vaccines for older adults; attempts to overcome reduced efficacy have included the addition of adjuvants or increased antigenic dose. Next generation vaccine formulations may also use biomaterials to both deliver and adjuvant vaccine antigens. In the context of aging, it is important to determine the degree to which new biomaterials may enhance antigen-presenting cell (APC) functions without inducing potent inflammatory responses of APCs or other immune cell types (e.g., T cells). However, the effect of newer biomaterials on these cell types from young and older adults remains unknown.ResultsIn this pilot study, cells from young and older adults were used to evaluate the effect of novel biomaterials such as polyanhydride nanoparticles (NP) and pentablock copolymer micelles (Mi) and cyclic dinucleotides (CDN; a STING agonist) on cytokine and chemokine secretion in comparison to standard immune activators such as lipopolysaccharide (LPS) and PMA/ionomycin. The NP treatment showed adjuvant-like activity with induction of inflammatory cytokines, growth factors, and select chemokines in peripheral blood mononuclear cells (PBMCs) of both young (n = 6) and older adults (n = 4), yet the degree of activation was generally less than LPS. Treatment with Mi or CDN resulted in minimal induction of cytokines and chemokine secretion with the exception of increased IFN-α and IL-12p70 by CDN. Age-related decreases were observed across multiple cytokines and chemokines, yet IFN-α, IL-12, and IL-7 production by NP or CDN stimulation was equal to or greater than in cells from younger adults. Consistent with these results in aged humans, a combination nanovaccine composed of NP, Mi, and CDN administered to aged mice resulted in a greater percentage of antigen-specific CD4+ T cells and greater effector memory cells in draining lymph nodes compared to an imiquimod-adjuvanted vaccine.ConclusionsOverall, our novel biomaterials demonstrated a modest induction of cytokine secretion with a minimal inflammatory profile. These findings suggest a unique role for biomaterial nanoadjuvants in the development of next generation vaccines for older adults.

Lactobacillus gasseri LGV03 isolated from the cervico-vagina of HPV-cleared women modulates epithelial innate immune responses and suppresses the growth of HPV-positive human cervical cancer cells

Persistent human papillomavirus (HPV) infections is necessary for the development of cervical cancers. An increasing number of retrospective studies have found the depletion of Lactobacillus microbiota in the cervico-vagina facilitate HPV infection and might be involved in viral persistence and cancer development. However, there have been no reports confirming the immunomodulatory effects of Lactobacillus microbiota isolated from cervico-vaginal samples of HPV clearance in women. Using cervico-vaginal samples from HPV persistent infection and clearance in women, this study investigated the local immune properties in cervical mucosa. As expected, type I interferons, such as IFN-α and IFN-β, and TLR3 globally downregulated in HPV+ persistence group. Luminex cytokine/chemokine panel analysis revealed that L. jannaschii LJV03, L. vaginalis LVV03, L. reuteri LRV03, and L. gasseri LGV03 isolated from cervicovaginal samples of HPV clearance in women altered the host's epithelial immune response, particularly L. gasseri LGV03. Furthermore, L. gasseri LGV03 enhanced the poly (I:C)-induced production of IFN by modulating the IRF3 pathway and attenuating poly (I:C)-induced production of proinflammatory mediators by regulating the NF-κB pathway in Ect1/E6E7 cells, indicating that L. gasseri LGV03 keeps the innate system alert to potential pathogens and reduces the inflammatory effects during persistent pathogen infection. L. gasseri LGV03 also markedly inhibited the proliferation of Ect1/E6E7 cells in a zebrafish xenograft model, which may be attributed to an increased immune response mediated by L. gasseri LGV03.

Poly(ADP-ribose) polymerase 9 mediates early protection against Mycobacterium tuberculosis infection by regulating type I IFN production.

The ADP ribosyltransferases (PARPs 1-17) regulate diverse cellular processes, including DNA damage repair. PARPs are classified on the basis of their ability to catalyze poly-ADP-ribosylation (PARylation) or mono-ADP-ribosylation (MARylation). Although PARP9 mRNA expression is significantly increased in progressive tuberculosis (TB) in humans, its participation in host immunity to TB is unknown. Here, we show that PARP9 mRNA encoding the MARylating PARP9 enzyme was upregulated during TB in humans and mice and provide evidence of a critical modulatory role for PARP9 in DNA damage, cyclic GMP-AMP synthase (cGAS) expression, and type I IFN production during TB. Thus, Parp9-deficient mice were susceptible to Mycobacterium tuberculosis infection and exhibited increased TB disease, cGAS and 2'3'-cyclic GMP-AMP (cGAMP) expression, and type I IFN production, along with upregulation of complement and coagulation pathways. Enhanced M. tuberculosis susceptibility is type I IFN dependent, as blockade of IFN α receptor (IFNAR) signaling reversed the enhanced susceptibility of Parp9-/- mice. Thus, in sharp contrast to PARP9 enhancement of type I IFN production in viral infections, this member of the MAR family plays a protective role by limiting type I IFN responses during TB.

Comparison of different techniques for evaluation of cellular immunity to SARS-CoV-2 virus

Most techniques for evaluation of T-cell immunity are laborious and unsuitable for routine laboratory diagnostics, thus encouraging researchers to look for accessible and reproducible tests. The purpose of our study is to compare three methods aimed for evaluation of cellular immune response levels to the SARS-CoV-2 viral antigens in patients who have been ill and vaccinated against a new coronavirus infection. We have examined 26 persons who experienced mild or moderate COVID-19 (group 1); 19 people vaccinated twice with Sputnik V, who did not have clinical COVID-19 (group 2); 21 subjects who had COVID-19 and were twice vaccinated with Sputnik V (group 3), and 14 persons who had COVID-19 twice (group 4). Peripheral blood mononuclear cells were isolated by gradient centrifugation. The first tested technique was performed as follows: the mononuclear cells were incubated with the S-protein of the SARS-CoV-2 virus, and stained with fluorescently labeled antibodies. The percentage of CD8highCD107a was counted by means of BD FACS Canto II flow cytometer. When assessed by the ELISpot method with “Human IFN-γ ELISpot” kit, IFNγ production was stimulated by SARS-CoV-2 S-protein, or a mixture of SARS-CoV-2 protein peptides in the “Corona-T-test” kit. There were no significant differences in the levels of CD107a expression on CD8high cells between the groups 1, 2, 3, and 4, as well as in amounts of IFNγ producers against SARS-CoV-2 S-protein when using “Human IFN-γ ELISpot” kit. Production of IFN was significantly lower in group 3 (hybrid immunity), i.e., 317.29±19.04 pg/ml compared to groups 1 and 2 (post-infection and post-vaccination immunity), i.e., 454.95±20.32 and 470.77±26.24 pg /ml, respectively. The relative level of IFNγ -producing cells in group 2 was higher (22.34±3.77) versus 16.83±2.35 in group 1, and 15.46±1.83 in group 3, whereas the relative levels of IFNγ did not differ in these groups. Stimulation with full-length S-protein showed a significant reduction in the number of spots in group 4 (breakthrough immunity), i.e., 30.59±2.29 vs 58.97±4.47 in group 3. Stimulation with a mixture of SARS-CoV-2 peptides in group 4 vs group 3 revealed a significantly increased number of IFNγ -producing cells (86.72±7.20 versus 69.38±5.53) and higher IFNγ production (991.25±65.18 pg/ml versus 760.76±50.70 pg/ml). Appropriate relative values were as follows: 10.30±2.77 versus 8.61±2.66, and 68.10±9.41 versus 48.35±8.15, respectively. The results of three methods for evaluation of cellular immune response correlate positively with each other, but at different significance levels.

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is not well understood. In this study, we screened 80 vaccinia genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS. E5 is responsible for abolishing cGAMP production during vaccinia virus (Western Reserve strain) infection of dendritic cells. E5 localizes to the cytoplasm and nucleus of infected cells. Cytosolic E5 triggers ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, and thereby improves antigen-specific T cell responses.

MEK Inhibition Sensitizes Pancreatic Cancer to STING Agonism by Tumor Cell-intrinsic Amplification of Type I IFN Signaling.

Stimulator of interferon genes (STING) agonists are currently in development for treatment of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Response rates to STING agonists alone have been promising yet modest, and combination therapies will likely be required to elicit their full potency. We sought to identify combination therapies and mechanisms that augment the tumor cell-intrinsic effect of therapeutically relevant STING agonists apart from their known effects on tumor immunity. We screened 430 kinase inhibitors to identify synergistic effectors of tumor cell death with diABZI, an intravenously administered and systemically available STING agonist. We deciphered the mechanisms of synergy with STING agonism that cause tumor cell death in vitro and tumor regression in vivo. We found that MEK inhibitors caused the greatest synergy with diABZI and that this effect was most pronounced in cells with high STING expression. MEK inhibition enhanced the ability of STING agonism to induce type I IFN-dependent cell death in vitro and tumor regression in vivo. We parsed NFκB-dependent and NFκB-independent mechanisms that mediate STING-driven type I IFN production and show that MEK signaling inhibits this effect by suppressing NFκB activation. Our results highlight the cytotoxic effects of STING agonism on PDAC cells that are independent of tumor immunity and that these therapeutic benefits of STING agonism can be synergistically enhanced by MEK inhibition.

An immunomodulatory antibody-drug conjugate targeting BDCA2 strongly suppresses plasmacytoid dendritic cell function and glucocorticoid responsive genes.

Blood dendritic cell antigen 2 (BDCA2) is exclusively expressed on plasmacytoid dendritic cells (pDCs) whose uncontrolled production of type I IFN (IFN-I) is crucial in pathogenesis of SLE and other autoimmune diseases. Although anti-BDCA2 antibody therapy reduced disease activity in SLE patients, its clinical efficacy needs further improvement. We developed a novel glucocorticoid receptor agonist and used it as a payload to conjugate with an anti-BDCA2 antibody to form an BDCA2 antibody-drug conjugate (BDCA2-ADC). The activation of BDCA2-ADC was evaluated in vitro. Inhibitory activity of BDCA2-ADC was evaluated in peripheral blood mononuclear cells or in purified pDCs under ex vivo toll-like receptor agonistic stimulation. The global gene regulation in purified pDCs was analysed by RNA-seq. The antigen-dependent payload delivery was measured by reporter assay. The BDCA2-ADC molecule causes total suppression of IFNα production and broader inhibition of inflammatory cytokine production compared with the parental antibody in human pDCs. Global gene expression analysis confirmed that the payload and antibody acted synergistically to regulate both type I IFN signature genes and glucocorticoid responsive genes in pDCs. Taken together, these data suggest dual mechanisms of BDCA2-ADC on pDCs and the potential for BDCA2-ADC to be the first ADC treatment for SLE in the world and a better treatment option than anti-BDCA2 antibody for SLE patients.

Preventive effects of quercetin against foot-and-mouth disease virus in vitro and in vivo by inducing type I interferon.

Foot-and-mouth disease (FMD) is an acute contagious infectious disease that affects cloven-hoofed animals. Although current emergency FMD vaccines only take effect 7 days after vaccination, antiviral agents, such as quercetin, which is a common flavonoid, could reduce the spread of FMD virus (FMDV) during outbreaks. We investigated the in vitro and in vivo antiviral effects of quercetin against FMDV. Analysis of viral copy numbers showed that quercetin had a dose-dependent inhibitory effect on FMDV at concentrations between 19.5 and 1,250 μM in porcine cells. In addition, we observed a quercetin-induced interferon (IFN)-α protein and interferon-stimulated gene (ISG) upregulation in swine cells. Enzyme-linked immunosorbent assay of sera revealed that quercetin induces the production of IFN-α, IFN-β, IFN-γ, interleukin (IL)-12, and IL-15 in mice. Inoculation of mice with quercetin or a combination of quercetin with an inactivated FMD vaccine enhanced both the survival rate and neutralizing antibody titer. Therefore, we suggest the use of quercetin as a novel and effective antiviral agent for controlling FMDV infection; however, further investigation of its application in livestock is required.

Borrelia burgdorferic-di-AMP induces type I IFN response in macrophage through the activation of STING signaling pathway

Abstract Lyme disease (LD) which is caused by the spirochete Borrelia burgdorferi (Bb) poses a global health threat. Bb infection induces type I IFN response that is associated with arthritis and other symptoms. However, the ligands, PAMPs, and signaling pathways associated with type I IFN production by Bb are poorly characterized. Stimulator of Interferon Genes (STING) signaling pathway is an intracellular surveillance pathway that detects c-di-nucleotides (CDNs) produced by pathogens through STING and induces type I IFN production. Bb produces CDNs and hence it has the potential to activate the STING signaling pathway. In this study, the role of c-di-AMP in the elicitation of type I IFN response to Bb infection was elucidated. The RAW cell line was infected with Bb and activation of STING signaling was measured by detecting the activation of downstream proteins (pSTING, pTBK1, pIRF3 & pNF-κB), proinflammatory cytokines (IL6, TNF-α) and Type I IFN (IFN-β) response using ELISA, western blotting, immunostaining, and Luciferase Reporter assay. Bb infection to macrophage results in c-di-AMP mediated elicitation of type I IFN response. The induction level of type I IFN response was dependent on the level of c-di-AMP as the Bb mutant strain producing higher c-di-AMP level induced higher cytokine production. Interestingly, c-di-AMP released into the BSK-II medium during the culture of Bb could also stimulate the production of type I IFN response. The induction of type I IFN by c-di-AMP was dependent on STING protein as the RAW STING KO cell line showed a reduction in the level of IFN-β. In conclusion, our results show that Bb-produced c-di-AMP is a major PAMP that triggers type I interferon production in macrophage by activation of the STING signaling pathway.

Regulation of plasmacytoid dendritic cells byER stress-mediated metabolic responses

Abstract Plasmacytoid dendritic cells (pDCs) chronically produce type I interferon (IFN-I) in autoimmune diseases, including systemic sclerosis (SSc) and systemic lupus erythematosus (SLE). We report that the IRE1α-XBP1 branch of the unfolded protein response (UPR) inhibits IFN-α production by TLR7- or TLR9-activated pDCs. In SSc patients, UPR gene expression was reduced in pDCs, which inversely correlated with IFN-I–stimulated gene expression. CXCL4, a chemokine highly secreted in SSc patients, downregulated IRE1α-XBP1–controlled genes and promoted IFN-α production by pDCs. Mechanistically, IRE1α-XBP1 activation rewired glycolysis to serine biosynthesis by inducing phosphoglycerate dehydrogenase (PHGDH) expression. This process reduced pyruvate access to the tricarboxylic acid (TCA) cycle and blunted mitochondrial ATP generation, which are essential for pDC IFN-I responses. Notably, PHGDH expression was reduced in pDCs from patients with SSc and SLE, and pharmacological blockade of TCA cycle reactions inhibited IFN-I responses in pDCs from these patients. Hence, modulating the IRE1α-XBP1–PHGDH axis may represent a hitherto unexplored strategy for alleviating chronic pDC activation in autoimmune disorders.

Tumor Progression Locus 2 Promotes Mouse Plasmacytoid Dendritic Cell Cytokine Secretion and Signaling

Abstract The autoimmune disorder systemic lupus erythematosus is thought to be caused by the inefficient clearance of dying cells. This releases nucleic acid debris that stimulates type I interferon (T1 IFN) secretion. Plasmacytoid dendritic cells (pDCs) are the major source of T1 IFNs, which are causally linked to lupus pathogenesis. Both T1 IFNs and pDCs are thus the focus of intense studies for immunotherapies. The serine-threonine kinase, tumor progression locus 2 (TPL2), negatively regulates T1 IFN secretion by conventional dendritic cells (cDCs) and macrophages in response to Toll-like receptor 9 (TLR9) stimulation in vitro. In contrast, we and others have shown that TPL2 is required for T1 IFN production in TLR9-stimulated pDCs. The goal of this study is to validate TPL2-dependent regulation of pDC function in vivoand to delineate the mechanisms responsible. Both cDCs and pDCs develop normally in Tpl2−/−mice, but Tpl2−/−mice show defective secretion of T1 IFNs and proinflammatory cytokines in response to administration of CpG (TLR9) or R848 (TLR7) in vivo. The same defect in T1 IFN and proinflammatory cytokine production was observed in bone marrow-derived pDCs and cDCs in vitro. A modest defect in TLR-induced expression of maturation markers CD80, CD86 and MHC class II was seen in Tpl2−/−pDCs, suggesting impaired NFkB activation. Flow cytometric measurement of phosphoproteins demonstrated that TPL2 is also required for TLR-induced activation of MAPK and ribosomal S6 kinase pathways in pDCs. Ongoing studies are examining the role of TPL2 in TLR trafficking and lupus pathogenesis. These data establish TPL2-dependent regulation of pDC functions, which may be exploited for therapeutic intervention in lupus or other IFN-related diseases. Supported by grants from NIH/NIAMS (R21 AR075317) titled "Tpl2 regulation of pDC function and SLE pathogenesis"

STINGing CD4+ T cells to induce refractoriness to HIV infection

Abstract HIV rapidly infects CD4+ T cells where it can establish latent infection. Identifying immune mechanisms which can render CD4+ T cells refractory to infection represents a critical step in limiting infection and viral spread. Using purified memory CD4+ T cells from healthy donors, we found that activation of the STING signaling pathway with 0.5 mM of the STING agonist diABZI was sufficient to induce a state of refractoriness to in vitro HIV infection in memory CD4+ T cells, a major source of the HIV reservoir. We observed that STING activation in CD4+ T cells induces Type I IFN production and activates p-IRF3. CRISPR KD of STING in memory CD4+ T cells led to a significant increase in HIV infection, demonstrating a cell intrinsic role for STING signaling in limiting HIV infection/replication. Using proteomics, we identified that diABZI significantly and potently induces restriction factors and proteins involved in antiviral immunity and Type 1 IFN signaling. Importantly, proteins that were significantly reduced mapped to pathways including RNA processing and protein translation which are aspects of the host machinery which HIV needs to co-opt to replicate and produce infectious virions. Our data provide mechanistic insight into the STING pathway as a means to render CD4+ T cells refractory to HIV infection and highlights that this pathway lands a “1–2” punch by increasing expression of antiviral proteins that directly target the virus while suppressing the cellular machinery needed for the viral life cycle. The potential to use a STING agonist as a post-exposure therapeutic which directly limits viral infection while promoting global antiviral immunity represents a novel strategy that may stop HIV infection in its tracks.

Hospitalized COVID-19 patients exhibit increased numbers of low density neutrophils expressing the transcription factor ARID3a

Abstract We previously showed that individuals with systemic lupus erythematosus (SLE) and high levels of Type I inflammatory cytokines had increased numbers of low density neutrophils (LDNs) in their peripheral blood compared to healthy donors. Increased numbers of LDNs were associated with increased expression of the transcription factor ARID3a and increased interferon alpha (IFNα) production. Data from other cell types suggest that ARID3a expression occurs upstream of IFNα expression. Therefore, we sought to determine if increases in ARID3a-expressing LDNs might also occur in other severe inflammatory responses, such as infection with SARS-CoV2 where IFNα expression was known to be increased. Subjects admitted to the hospital with a confirmed diagnosis of SARS-CoV2 infection were enrolled in our IRB approved study and peripheral blood mononuclear cells from 24 patients were evaluated via flow cytometry for numbers of LDNs and ARID3a expression at the time of admission and at various time points after hospitalization. Numbers of circulating LDNs were dramatically increased in COVID patient samples at the time of hospitalization, as were percentages of those cells expressing ARID3a protein. IL-6 expression was also high in the LDNs, while IFNα expression was more variable. Over the course of two weeks, IFNα expression appeared to be associated with, or to lag just behind ARID3a expression in most patients, while IL-6 expression levels were not associated with ARID3a or IFNα expression. These data suggest that ARID3a expression is induced in LDNs in association with severe viral infection and may implicate ARID3a as a potential regulator of inflammatory pathways in infection as well as autoimmunity. Supported in part by grant NIH U19 AI06269

Differential role of TLR7 signaling in SARS-CoV-2-induced antiviral and inflammatory responses

Abstract Since its emergence in 2019, SARS-CoV-2 has caused significant morbidity and mortality in humans. SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses drive acute lung injury and severe pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and pathogenic inflammatory responses are not well defined. Since CoVs are single-stranded RNA (ssRNA) viruses that make double-stranded RNA (dsRNA) intermediates during replication, we assessed the antiviral and pro-inflammatory roles of ssRNA and dsRNA mimics in mouse macrophages. We show that R837-TLR7 signaling induced excessive inflammation, while PolyIC-TLR3/MDA activation caused robust IFN production. Since R837-TLR7 induced high levels of inflammatory cytokines, using a novel mouse-adapted SARS-COV-2, we examined the role of TLR7 signaling in COVID-19 pathogenesis. Our results show that TLR7 KO mice were more susceptible to SARS-CoV-2 infection as compared to WT mice. Further investigation of SARS-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral interferons (IFNα/β/λ), ISGs (ISG15 and CXCL10), and several pro-inflammatory cytokine/chemokines in TLR7 KO lungs compared to WT. Interestingly, immunolabelling of cells isolated from infected WT and TLR7 KO lungs showed a high neutrophil count in TLR7 KO mice compared to WT mice. Furthermore, reduced lung IFN levels and increased mortality correlated with high lung viral titer in TLR7 KO mice. In summary, our findings conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite TLR7-mediated robust inflammatory cytokine production, TLR7-induced IFN and ISG responses play a key role in host protection.

STING controls opioid-induced itch and chronic itch via spinal tank-binding kinase 1-dependent type I interferon response in mice

BackgroundPatients receiving epidural or intrathecal opioids administration for neuraxial analgesia frequently suffer from an irritating itch. STING (stimulator of interferon genes), an innate immune modulator, is strongly implicated in pain pathogenesis via neuron-immune modulation. Given that pain and itch share some common neurocircuits, we evaluate the therapeutic potential of STING agonists in opioid-induced itch and chronic itch.MethodsOpioids (morphine, fentanyl and sufentanil) were intrathecally injected to induce acute itch. Chronic itch was induced by dry skin and contact dermatitis. Opioids analgesic effect, itch-induced scratching behavior, spinal expression of STING, phosphorylation of TBK1 (tank-binding kinase 1), IRF3 (interferon regulatory factor-3) and ERK (extracellular signal-regulated kinase), as well as production of IFN-α and IFN-β were examined. STING agonists (DMXAA and ADU-S100), TBK1 inhibitor, recombinant IFN-α and IFN-β elucidated the mechanism and treatment of itch. Whole-brain functional connectivity was evaluated using resting-state fMRI.ResultsWe report the primary expression of STING protein by the spinal dorsal horn neurons. Intraperitoneal injection of DMXAA dose-dependently reduces morphine-induced scratch bouts, without impairing morphine antinociception. Simultaneously, DMXAA alleviates fentanyl- and sufentanil-induced itching-like behavior, and chronic scratching behavior caused by dry skin and contact dermatitis. Furthermore, DMXAA drastically increases spinal phosphorylation of TBK1 and IRF3 following morphine exposure, dry skin and contact dermatitis. DMXAA-induced anti-pruritus effects and spinal productions of IFN-α and IFN-β are compensated by intrathecal delivery of the TBK1 inhibitor. Also, ADU-S100, recombinant IFN-α and IFN-β exhibits remarkable attenuation in scratching behaviors after morphine injection and dermatitis. Recombinant IFN-α inhibits morphine-induced spinal phosphorylation of ERK. Finally, DMXAA prevents dermatitis-induced the increase of cerebral functional connectivity between regions of interests such as primary somatosensory cortex, piriform cortex, retrosplenial cortex, colliculus and ventral thalamus.ConclusionsSTING activation confers protection against opioid-induced itch and chronic itch through spinal up-regulation of TBK1-IRF3-type I interferon cascades in mice, suggesting that STING agonists are promising candidates in translational development for pruritus relief.

Live Mycobacterium paragordonae induces heterologous immunity of natural killer cells by eliciting type I interferons from dendritic cells via STING-dependent sensing of cyclic-di-GMP

Exploiting the heterologous effects of vaccines is a feasible strategy to combat different pathogens. These effects have been explained by enhanced immune responses of innate immune cells. Mycobacterium paragordonae is a rare nontuberculosis mycobacterium that has temperature–sensitive properties. Although natural killer (NK) cells exhibit heterologous immunity features, the cellular crosstalk between NK cells and dendritic cells (DCs) during live mycobacterial infection has remained elusive. We show that live but not dead M. paragordonae enhances heterologous immunity against unrelated pathogens in NK cells by IFN-β of DCs in both mouse models and primary human immune cells. C-di-GMP from live M. paragordonae acted as a viability-associated pathogen-associated molecular pattern (Vita-PAMP), leading to STING-dependent type I IFN production in DCs via the IRE1α/XBP1s pathway. Also, increased cytosolic 2′3′-cGAMP by cGAS can induce type I IFN response in DCs by live M. paragordonae infection. We found that DC-derived IFN-β plays a pivotal role in NK cell activation by live M. paragordonae infection, showing NK cell-mediated nonspecific protective effects against Candida albicans infection in a mouse model. Our findings indicate that the heterologous effect of live M. paragordonae vaccination is mediated by NK cells based on the crosstalk between DCs and NK cells.

Potential role of EBV and Toll-like receptor 9 ligand in patients with systemic lupus erythematosus.

SLE is a multisystem autoimmune disease characterized by multiple immunological abnormalities including production of autoantibodies. While the etiology of SLE is largely unknown, it is generally accepted that both genetic and environmental factors contribute to disease risk and immune dysregulation. Production of IFN-α is important for protecting the host against infections; however, over stimulation of innate immune pathways can induce autoimmune disease. Environmental factors, particularly Epstein-Barr virus (EBV), have been proposed to play an important role in SLE disease. Improper engagement of Toll-like receptor (TLR) pathways by endogenous or exogenous ligands may lead to the initiation of autoimmune responses and tissue injury. EBV is shown to be a potent stimulant of IFN-α by TLR signaling cascades. Given the highlighted role of IFN-α in SLE pathogenesis and potential role of EBV infection in this disease, the present study is aimed at exploring the in vitro effects of EBV infection and CPG (either alone or in combination) on IFN-α. We also examined the expression level of CD20 and BDCA-4 and CD123 in PBMCs in 32 SLE patients and 32 healthy controls. Our results showed PBMCs treated with CPG-induced higher levels of IFN-α and TLR-9 gene expression fold change compared to cells treated with either EBV or EBV-CPG. Moreover, PBMCs treated with CPG produced significantly higher IFN-α concentration in supernatant compared to cells treated with EBV but not EBV-CPG. Our results further highlight the potential role of EBV infection and TLRs in SLE patients although more studies are warranted to ascertain the global imprint that EBV infection can have on immune signature in patients with SLE.

Assessment of formation and durability of adaptive immunity in COVID-19 convasescents

The study of adaptive immunity in COVID-19 convalescent patients is important, because no consensus on whether the disease severity affects formation and durability of COVID-19 immune response has been achieved. A comparative assessment of emergence and durability of sustained cellular and humoral immunity in convalescent patients with COVID-19 of varying severity was carried out. The study involved volunteers with asymptomatic (n = 30), moderate (n = 21) and severe (n = 12) COVID-19. The average age of the subjects was 47.312.5 years. The formation of cellular immunity was assessed by increased IFN production in response to 1620 hour-long SARS-CoV-2-derived glycoprotein S (RBD) lymphocyte stimulation. To measure IFN level, the Gamma InterferonIFA-BEST test system manufactured by Vector-Best JSC, Russia, was used. The humoral immune response was recorded by detecting SARS-CoV-2RBD-specific class G antibodies using the SARS-CoV-2RBD-ELISA-Gamalei test system (FSBI NITSEM N.F. Gamalei of the Ministry of Health of Russia). It was revealed that humoral and cellular immunity against SARS-CoV-2 proteins was formed in all COVID-19 convalescent patients. However, the number of subjects with adaptive immunity to COVID-19 and the duration of its preservation depends on the severity of the infection. A significant decrease in the number of subjects with cellular immunity was revealed in the group of severe COVID-19. Most of the volunteers in this group had class G immunoglobulins before the end of the follow-up. In this group, unlike the other two, no patients were identified in whom only the cellular arm of the immune response was activated. Volunteers who did not retain adaptive immunity to the COVID-19 pathogen appeared only by the end of the follow-up period. Among those recovered after moderate disease 78 months later there was a decrease in the number of people with cellular and humoral immunity. This process started earlier than in the group of patients who were asymptomatic and continued until the end of the study. The proportion of individuals with cellular immunity increased, and at later timepoint with humoral immune response. By the end of the study, a high percentage of volunteers remained asymptomatically infected, having cellular and humoral immunity to SARS-CoV-2. Their number remained significantly higher than in the group of moderate COVID-19, but lower than in severe COVID-19. By the end of the study, an increased number of volunteers with solely cellular immune response was recorded in this group. At the end of the follow-up period, the number of volunteers with humoral immunity against SARS-CoV-2 remained higher compared to those with a cellular immune response.