Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells.

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity-the condensates in oral epithelium disassembled within 1-2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4-7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic "macromolecular crowding") confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm-the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid-liquid phase separation (LLPS) in cells of the oral mucosa.

Changes in the innate immune response to SARS-CoV-2 with advancing age in humans.

BACKGROUND: Advancing age is a major risk factor for respiratory viral infections. The infections are often prolonged and difficult to resolve resulting hospitalizations and mortality. The recent COVID-19 pandemic has highlighted this as elderly subjects have emerged as vulnerable populations that display increased susceptibility and severity to SARS-CoV-2. There is an urgent need to identify the probable mechanisms underlying this to protect against future outbreaks of such nature. Innate immunity is the first line of defense against viruses and its decline impacts downstream immune responses. This is because dendritic cells (DCs) and macrophages are key cellular elements of the innate immune system that can sense and respond to viruses by producing inflammatory mediators and priming CD4 and CD8 T-cell responses. RESULTS: We investigated the changes in innate immune responses to SARS-CoV-2 as a function of age. Our results using human PBMCs from aged, middle-aged, and young subjects indicate that the activation of DCs and monocytes in response to SARS-CoV-2 is compromised with age. The impairment is most apparent in pDCs where both aged and middle-aged display reduced responses. The secretion of IL-29 that confers protection against respiratory viruses is also decreased in both aged and middle-aged subjects. In contrast, inflammatory mediators associated with severe COVID-19 including CXCL-8, TREM-1 are increased with age. This is also apparent in the gene expression data where pathways related host defense display an age dependent decrease with a concomitant increase in inflammatory pathways. Not only are the inflammatory pathways and mediators increased after stimulation with SARS-CoV-2 but also at homeostasis. In keeping with reduced DC activation, the induction of cytotoxic CD8 T cells is also impaired in aged subjects. However, the CD8 T cells from aged subjects display increased baseline activation in accordance with the enhanced baseline inflammation. CONCLUSIONS: Our results demonstrate a decline in protective anti-viral immune responses and increase in damaging inflammatory responses with age indicating that dysregulated innate immune responses play a significant role in the increased susceptibility of aged subjects to COVID-19. Furthermore, the dysregulation in immune responses develops early on as middle-aged demonstrate several of these changes.

Evaluation of the serum levels of CCL2, CCL3, and IL-29 after first and second administrations of the COVID-19 vaccine (Oxford-AstraZeneca).

BACKGROUND: Previous studies show that chemokines and cytokines play a very important role in eliciting an appropriate response against viruses. Vaccination causes inflammation in the person receiving the vaccine, accompanied with production of inflammatory molecules by immune cells. The more and better the production and expression of chemokines and cytokines by immune cells, the better the response of the acquired immune system. Chemokines and cytokines are critical in promoting the innate immune response against the COVID-19. Here we intended to assess serum levels of CCL2, CCL3, and interleukin (IL)-29 in patients received COVID-19 vaccine. METHODS: In this study, 40 subjects vaccinated with the Oxford-AstraZeneca COVID-19 vaccine were selected. Blood samples were collected before injection of the vaccine, 3-5 days after the first dose injection, and 3-5 days subsequent to the second vaccination. To check the serum level of CCL2, CCL3, and IL-29, ELISA technique was used. RESULTS: Our results indicated that the serum levels of CCL2, CCL3, and IL-29 were significantly higher after first and second dose of vaccination compared to before vaccine administration. Furthermore, serum levels of all these mediators were higher after second dose of vaccine compared to the first vaccine administration. CONCLUSIONS: Oxford-AstraZeneca COVID-19 vaccine is able to induce inflammatory CCL2 and CCL3 chemokines as well as protective interferon lambda (IL-29).

Up and away with cervical cancer: IL-29 is a promising cytokine for immunotherapy of cervical cancer due to its powerful upregulation of p18, p27, and TRAILR1.

Cervical cancer is one of the most common types of female cancers worldwide. IL-29 is an interesting cytokine in the IFNλ family. Its role in the pathogenesis of neoplasia is complicated and has been studied in other cancers, such as lung cancer, gastric cancer, and colorectal cancer. IL-29 has been previously reported to promote the growth of pancreatic cancer. However, the direct role of IL-29 in cervical cancer has not been studied yet. This study was performed to investigate the direct effect on cervical cancer cell growth. Clonogenic survival assay, cell proliferation, and caspase-3 activity kits were used to evaluate the effects of IL-29 on cell survival, proliferation, and apoptosis of a well-studied cervical cancer cell line, SiHa. We further investigated the potential molecular mechanisms by using RT-PCR and IHC. We found that the percentage of colonies of SiHa cells was decreased in the presence of IL-29. This was consistent with a decreased OD value of cancer cells. Furthermore, the relative caspase-3 activity in cancer cells increased in the presence of IL-29. The anti-proliferative effect of IL-29 on cancer cells correlated with increased expression of the anti-proliferative molecules p18 and p27. The pro-apoptotic effect of IL-29 on cancer cells correlated with increased expression of the pro-apoptotic molecule TRAILR1. IL-29 inhibits cervical cancer cell growth by inhibiting cell proliferation and promoting cell apoptosis. Thus, IL-29 might be a promising cytokine for immunotherapy of cervical cancer.

Importance of screening severe COVID-19 patients for IFN-λ1, IL-6 and anti-S1 IgG levels.

Cytokine storm is an important cause of death in COVID-19 patients. A recent clinical study showed that administration of recombinant interferon lambda 1 (IFN-λ1 or IL-29) may prevent severe COVID-19. On the other hand, IL-6 has been associated as a prognostic marker of worsening for COVID-19 patients. The objective of this study is to screen IFN-λ1, IL-6 and antibody levels in consecutive serum sample sets of COVID-19 patients. A total of 365 serum samples collected from 208 hospitalized COVID-19 patients were analyzed for IFN-λ1 and IL-6 levels as well as SARS-CoV-2 neutralizing antibodies and anti-S1 IgG antibodies. Analyses of serum samples for cytokine levels showed that IFN-λ1 (>8 pg/mL) and IL-6 (>2 pg/mL) were detected in approximately 64% and 21% patients, respectively. A decrement in IFN-λ1 levels and IL-6 levels above 35 pg/mL can be sign of clinical severity and upcoming dead. An increment in IL-6 levels wasn't detected in every COVID-19 patient but a decrement in IL-6 levels was related to clinical improvement. Importantly, the detection of IFN-λ1 level together with an increase in anti-S1 IgG antibody response were observed in clinically improved patients. Screening severe COVID-19 patients for IFN-λ1, IL-6, and anti-S1 IgG antibody levels during their hospital stay especially in intensive care units may be beneficial to monitor the clinical status and management of treatment strategies. Importantly, detection of IFN-λ1 together with protective IgG antibody response can be an indication of clinical improvement in severe COVID-19 patients and these patients may be discharged from the hospital soon.

The Cooperation of IL-29 and PLGA Nanoparticles Improves the Protective Immunity of the gD-1 DNA Vaccine Against Herpes Simplex Virus Type 1 in Mice.

In clinical practice, the low immunogenicity and low stability of the DNA plasmid vaccine candidates are two significant shortcomings in their application against infectious diseases. To overcome these two disadvantages, the plasmid expressing IL-29 (pIL-29) as a genetic adjuvant and polylactic-co-glycolic acid (PLGA) as a non-viral delivery system were used, respectively. In this study, the pIL-29 encapsulated in PLGA nanoparticles (nanoIL-29) and the pgD1 encapsulated in PLGA nanoparticles (nanoVac) were simultaneously applied to boost immunologic responses against HSV-1. We generated spherical nanoparticles with encapsulation efficiency of 75 ± 5% and sustained the release of plasmids from them. Then, Balb/c mice were subcutaneously immunized twice with nanoVac+nanoIL-29, Vac+IL-29, nanoVac, Vac, nanoIL-29, and/or IL-29 in addition to negative and positive control groups. Cellular immunity was evaluated via lymphocyte proliferation assay, cytotoxicity test, and IFN-γ, IL-4, and IL-2 measurements. Mice were also challenged with 50X LD50 of HSV-1. The nanoVac+nanoIL-29 candidate vaccine efficiently enhances CTL and Th1-immune responses and increases the survival rates by 100% in mice vaccinated by co-administration of nanoVac and nanoIL-29 against the HSV-1 challenge. The newly proposed vaccine is worth studying in further clinical trials, because it could effectively improve cellular immune responses and protected mice against HSV-1.

Evaluation of exosomes encapsulated recombinant Interleukin-29 for its in vitro anticancer studies.

Exosomes have recently been considered ideal biotherapeutic nanocarriers that broaden the frontiers of current drug delivery systems to overcome the shortcomings associated with cytokine-based immunotherapy. Using this approach, the current study aimed to assess anti-proliferative activity of purified IL-29 and exosomes encapsulated IL-29. The IL-29+pET-28a construct was transformed into Rosetta 2(DE3) cells which was used for the large-scale production of IL-29. Exosomes isolated from H1HeLa, and SF-767 cells using Total Exosome Isolation reagent were loaded with IL-29 via sonication. Isolation of exosomes was validated using their core protein signature by western blotting and specific miRNA profiles by RT-PCR. The drug loading efficiency of exosomes derived from H1HeLa cells was higher than that of SF-767-derived exosomes. The drug release kinetics of IL-29 encapsulated exosomes exhibited stable release of the recombinant drug. Around 50% of all cancer cell lines survived when IL-29 was administered at a concentration of 20 µg/mL. A survival rate of less than 10% was observed when cells were treated with 20 µg/mL IL-29 loaded exosomes. It was concluded that IL-29 loaded exosomes had a more significant cytotoxic effect against cancer cells, which might be attributed to sustained drug release, improved half-life, superior targeting efficacy, capacity to harness endogenous intracellular trafficking pathways, and heightened biocompatibility of exosomes.

Systematic levels of IL-29 and microRNA185-5p were not associated with severe COVID-19 in the Iranian population.

BACKGROUND: Increased systematic pro-inflammatory cytokines is the main cause of the inflammatory conditions of the hospitalized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. In this project, serum levels of IL-29 and whole blood levels of microRNA-185-5p (miR-185-5p) were evaluated in the hospitalized SARS-CoV-2 infected patients. METHODS: This project was performed on the 60 hospitalized SARS-CoV-2 infected patients and 60 healthy controls to evaluate IL-29 and miR185-5p expression levels. IL-29 expression was explored using enzyme linked immunoassay (ELISA), while miR185-5p was evaluated using Real-Time PCR techniques. RESULTS: The results demonstrated that neither IL-29 serum levels nor relative expressions of miR-185-5p were significantly different between patients and healthy controls. CONCLUSION: Due to the results that are presented here, systematic levels of IL-29 and miR-185-5p cannot be considered as the main risk factors for induction of inflammation in the hospitalized SARS-CoV-2 infected patients.

Cloning and expression of an anti-cancerous cytokine: human IL-29 gene in Chlamydomonas reinhardtii.

Green algae, Chlamydomonas reinhardtii, with low cultivation cost, absence of endotoxins and insusceptibility to human pathogens is emerging as a potential system for the future production of recombinant proteins. The recent development of molecular tools enabling recombinant protein expression in algae chloroplast has provided new research and advance opportunities for developing low-cost therapeutic proteins. In the present study, algae chloroplast expression system was evaluated for the recombinant production of an anti-cancerous therapeutic protein, Interleukin 29 (IL29). The IL29 gene was cloned into algae chloroplast expression vector (pSRSapI). After the transformation, the positive clones were screened for homoplasmy and the presence of the IL29 gene by spot test and PCR analysis, respectively. The expressed SDS-PAGE and western blotting assay characterized IL-29. The algae expressed IL-29 was biologically active in an anti-proliferating bioassay using HepG2 cells. The results suggest that the Chlamydomonas reinhardtii expression system is convenient, low-cost, eco-friendly, and safe to express IL29.

Association between Psoriasis Disease and IFN-λ Gene Polymorphisms.

Psoriasis is one of the most common chronic immune-mediated skin diseases, having a strong genetic predisposition. Psoriasis is a T-cell-mediated disease with a mixed Th1/Th17 cytokines environment. IL-23/IL-17 axis hyperactivation is the primary pathogenesis. Psoriasis lesions have been known to exhibit high IFN-λ1 and IFN-stimulated genes (ISGs) expression, which appears to be driven by Th17 cells. However, the role and mechanism of IFN-λs in psoriasis disease remains unknown. The study aimed to investigate the relationship between IL-28B and IL-29 gene polymorphisms with psoriasis disease and clinical severity. We performed single-nucleotide polymorphisms (SNPs) of IL-28B rs12979860 (IL-28 C/T), rs8099917 (IL-28 T/G), and IL-29 rs30461 (IL-29 T/C) in 140 patients with psoriasis disease and 159 healthy controls using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The genotype and allele frequency distributions of the rs12979860 (IL-28 C/T) and rs30461 (IL-29 T/C) polymorphisms were similar in the patient and control groups and were not statistically significant. The TG genotype of rs8099917 was statistically significantly different in patients from both groups. The TG genotype increased the risk of disease1.9-fold. The G allele may be associated with the pathogenesis of psoriasis.

Linagliptin ameliorated interleukin-29-induced reduction of extracellular matrix genes through the nuclear factor erythroid 2-related factor 2 (Nrf2)/sry-type high-mobility-group box (SOX)-9 axis in an in vitro study on C-28/I2 chondrocytes.

Osteoarthritis (OA) is a severe orthopedic disease commonly observed in the elderly population and is closely related to the degradation of extracellular matrix (ECM) in cartilage tissues. Interleukin-29 (IL-29) is a cytokine that has been recently linked with the progression of OA. However, the physiological roles of IL-29 in ECM genes and function are unknown. Linagliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor recently reported to exert significant anti-inflammatory properties. In this study, we used IL-29 to stimulate C-28/I2 chondrocytes to build an inflammatory injury model. We aimed to investigate the protective effect of Linagliptin on IL-29-induced degradation of ECM. We found that IL-29 stimulation reduced the expressions of Col2a1 and Acan in C-28/I2 chondrocytes, and this effect was mediated by SRY-related high-mobility group box gene-9 (SOX-9), as we showed that overexpression of SOX-9 could rescue the reduction of Col2a1 and Acan. Interestingly, we found that IL-29 stimulation pronouncedly promoted the expression of DPP-4. Treatment with 100 nM of the DPP-4 inhibitor Linagliptin ameliorated IL-29-induced expressions of SOX-9, Col2a1, and Acan. Lastly, the nuclear level of nuclear factor erythroid 2-related factor 2 (Nrf2) was dramatically declined in IL-29-challenged chondrocytes and the protective effects of Linagliptin on the expressions of SOX-9, Col2a1, and Acan were abolished by the knockdown of Nrf2. Taken together, our data reveal that Linagliptin ameliorated IL-29-induced reduction of ECM genes partially through the Nrf2/SOX-9 axis in C-28/I2 chondrocytes. Further in vivo and clinical studies will be done to clarify the protective benefits of Linagliptin in OA.

The protective effects of Olmesartan against interleukin-29 (IL-29)-induced type 2 collagen degradation in human chondrocytes.

Osteoarthritis (OA) is a cartilage degenerative disease commonly observed in the elderly population and is pathologically characterized by the degradation of the cartilage extracellular matrix (ECM). Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are critical enzymes involved in the degradation of ECM. Olmesartan is an inhibitor of the angiotensin II receptor developed for the treatment of hypertension, and recent studies show that it exerts anti-inflammatory effects in arthritis. The present study aimed to investigate the mechanism of the protective effect of Olmesartan on cartilage ECM degradation. Interleukin-29 (IL-29) is a novel inflammatory mediator involved in the inflammation and degradation of cartilage in OA, and human T/C-28a2 cells treated with it were the inflammatory model in vitro. We found that the degradation of type 2 collagens and aggrecans was induced by IL-29, accompanied by the upregulation of MMPs and ADAMTSs, but the presence of Olmesartan significantly ameliorated these increases. In addition, Olmesartan abolished IL-29- induced oxidative stress and elevated the expression level of TNF receptor-associated factor 6 (TRAF-6). Mechanistically, we showed that Olmesartan suppressed IL-29- caused inhibitor kappa B α (IκBα) expression and nuclear translocation of nuclear factor kappa-B (NF-κB) p65, indicating it suppressed the activation of the NF-κB pathway. Collectively, our data reveal that Olmesartan exerted a protective function on IL-29- induced type 2 collagen degradation in human chondrocytes.

De novo developed protein binders mimicking Interferon lambda signaling.

We hereby describe the process of design and selection of nonantibody protein binders mimicking cytokine signaling. We chose to mimic signaling of IFN-λ1, type 3 interferon (also known as IL-29) for its novelty and the importance of its biological functions. All four known interferons λ signal through binding to the extracellular domains of IL-28 receptor 1 (IL-28R1) and IL-10 receptor 2 (IL-10R2). Our binders were therefore trained to bind both receptors simultaneously. The bifunctional binder molecules were developed by yeast display, a method of directed evolution. The signaling capacity of the bivalent binders was tested by measuring phosphorylation of the JAK/STAT signaling pathway and production of mRNA of six selected genes naturally induced by IFN- λ1 in human cell lines. The newly developed bivalent binders offer opportunities to study cytokine-related biological functions and modulation of the cell behavior by receptor activation on the cell surfaces alternative to the use of natural IFN-λ.

IFN-λ Modulates the Migratory Capacity of Canine Mammary Tumor Cells via Regulation of the Expression of Matrix Metalloproteinases and Their Inhibitors.

Interactions between neoplastic and immune cells taking place in tumors drive cancer regulatory mechanisms both in humans and animals. IFN-λ, a potent antiviral factor, is also secreted in the tumor; however, its role in tumor development is still unclear. In our study, we investigate the influence of IFN-λ on the canine mammary tumor (CMT) cell survival and their metastatic potential in vitro. First, we examined, by Western blot, the expression of the IFN-λ receptor complex in three CMT cell lines (P114, CMT-U27 and CMT-U309). We showed that only two cell lines (P114 and CMT-U27) express both (IL-28RA and IL-10Rb) receptor subunits and respond to IFN-λ treatment by STAT phosphorylation and the expression of interferon-stimulated genes. Using MTT, crystal violet and annexin-V assays, we showed a minimal role of IFN-λ in CMT viability. However, IFN-λ administration had a contradictory effect on cell migration in the scratch test, namely, it increased P114 and decreased CMT-U27 motility. Moreover, we demonstrated that this process is related to the expression of extracellular matrix metalloproteinases and their inhibitors; furthermore, it is independent of Akt and ERK signaling pathways. To conclude, we showed that IFN-λ activity is reliant on the expression of two receptor subunits and tumor type, but further investigations are needed.

Association Between IL28B, IL29 Gene Polymorphisms and Clinical Manifestations of Behçet's Disease.

BACKGROUND: Behçet's disease (BD) is a chronic, multisystemic, inflammatory disease characterized by relapsing episodes of a wide spectrum of clinical findings. The role and mechanism of IFN-λs in BD remain unknown. The aim of this study was to investigate the relationship between IL29 and IL28B gene polymorphisms and BD or clinical manifestations. METHODS: Using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, single-nucleotide polymorphisms of IL28B rs8099917 (IL28 G/T), rs12979860 (IL28 C/T) and IL29 rs30461 (IL29 T/C) were studied in 94 patients with BD and 90 healthy controls. RESULTS: Our study did not show any relationship between Behçet Disease and genotype or allele frequencies of IL28B (rs8099917, rs12979860) and IL29 (rs30461) gene polymorphisms (p > .05). We found that the TT genotype of rs12979860 (IL28 C/T) polymorphism is higher in healthy controls and patients without central nervous system (CNS) involvement compared to patients with CNS involvement (p = .014 and p = .022). CONCLUSIONS: As a result, although the relationship was found between IL28 and IL29 gene polymorphisms with some clinical manifestations of BD, it was not directly related to the predisposition of the disease. The relationship between IL-28 and IL-29 which act as regulators in inflammatory processes, with Behçet disease, needs to be investigated in further studies.

IL-29 Exhibits Anti-Tumor Effect on Pan-48 Pancreatic Cancer Cells by Up-regulation of P21 and Bax.

BACKGROUND/AIM: Pancreatic cancer is the most lethal cancer of the digestive system. IL-29 is a new member of the IFNλ family and well-known for its strong antiviral activity. However, its direct effect on pancreatic cancer is still unclear. This study was performed to investigate if IL-29 has any direct effect on Pan-48 pancreatic cancer cells. MATERIALS AND METHODS: Clonogenic survival assay, cell proliferation, and caspase-3 activity kits were used to evaluate the effects of IL-29 on cell survival, proliferation, and apoptosis of Pan-48 pancreatic cancer cells. RT-PCR and IHC were subsequently performed to explore IL-29's potential molecular mechanisms. RESULTS: The percentage of colonies of Pan-48 cells was decreased following the addition of IL-29. This was consistent with a decreased optical density (OD) value of cancer cells. Furthermore, the relative caspase-3 activity in cancer cells was increased after the addition of IL-29, indicating increased apoptosis of cancer cells. The anti-proliferative effect of IL-29 on cancer cells correlated with increased expression of the anti-proliferative molecule p21. The pro-apoptotic effect of IL-29 on cancer cells correlated with an increased expression of the pro-apoptotic molecule Bax. CONCLUSION: IL-29 constrains Pan-48 pancreatic cell growth via up-regulation of p21 and Bax. Our study suggests a potential use of IL-29 in immunotherapy for pancreatic cancer treatment.

IL-27 in patients with Chikungunya fever: A possible chronicity biomarker?

BACKGROUND/PURPOSE: Although many patients with chikungunya virus disease (CHIKVD), an arboviral disease characterized by sudden fever and incapacitating poliartralgia, develop chronic articular symptoms, the mechanisms involved in CHIKVD's chronification and its possible biomarkers remain poorly understood. Interleukin (IL)-17A, IL-21, IL-22, IL-29, and transforming growth factor (TGF)-ß have been implicated in the pathogenesis of other inflammatory joint diseases, including rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. Since chronic manifestations of CHIKVD share many clinical and immunological characteristics with those diseases, we assessed the serum levels of those cytokines and analyzed their associations with clinical manifestations in patients with CHIKVD. METHODS: We evaluated 45 patients (36 female, mean age: 55.2 ± 13.8 years) with CHIKVD serologically confirmed by enzyme-linked immunosorbent assay (ELISA), articular manifestations upon evaluation, and no previous history of inflammatory rheumatologic diseases, along with 49 healthy age- and sex-matched controls. We tested anti-Chikungunya IgM and IgG antibodies and measured IL-17A, IL-21, IL-22, IL-27, IL-29, and TGF-ß serum levels with specific ELISA kits. RESULTS: IL-27, IL-17A, and IL-29 appeared in most patients but not in controls. IL-27 serum levels were higher in patients with chronic symptoms (median: 523.0 pg/mL [62.5-1,048]) than in ones in the acute or subacute stage (median: 62.5 pg/mL [62.5-483.8], p = .008). In patients with CHIKVD, we found significant correlations between IL-27 levels and tender joint counts (r = .32, p = .006), along with associations between IL-17A levels and swollen joint counts (r = .315, p = .0352). Furthermore, patients with arthritis had higher IL-17A levels (median: 23.14 pg/mL [20.6-25.86]) than ones without (median: 20.29 pg/mL [3.91-22.43], p = .0352). We did not detect IL-22 in either group or IL-21 in patients with CHIKVD. CONCLUSION: Serum levels of IL-17A, IL-27, and IL-29 were high in patients with CHIKVD and had important associations with articular manifestations, which might indicate the inflammatory nature of Chikungunya infection in patients with joint symptoms and the roles of those cytokines in the disease's pathophysiology.

IL28B, IL29 and micro-RNA 548 in subacute sclerosing panencephalitis as a rare disease.

Subacute sclerosing panencephalitis (SSPE) is a progressive neurodegenerative disease which affects children and young adults, caused by a persistent infection of defective measles virus. IFN-λs (IL-28A, IL-28B and IL-29) are a group of cytokines mediating antiviral responses. It has been shown that IL-29 levels are significantly higher in infected cells with defective measles virus. IL-29 expression is thought to be regulated at post-transcriptional level and miRNA-548 family targets the 3'UTR of the IFNL1 gene. Impaired immune system has an important role as well as viral factors in SSPE. The aim of our study investigates whether IL-28B, IL-29 levels and gene polymorphisms contribute to the damaged immune response leading to the development of SSPE. Also possible association of miR-548 family with IL-29 and SSPE is explored. Frequencies of rs12979860, rs8099917, rs30461, serum levels of IL-28B, IL-29 and expression levels of miR-548b, miR-548c, miR-548i are determined at 64 SSPE patients and 68 healthy controls. Serum IL-29 levels are statistically significant higher in SSPE patients. Allele frequencies of rs8099917 are statistically significant higher in SSPE patients and resulted G allele is found to increase 2.183-fold risk of SSPE. The expression levels of miR-548b-5p, miR-548c-5p and miR-548i are found to be statistically significant higher in SSPE patients. Dramatically increased level of IL-29 seen in patient group indicates that the elevated miR-548 expression is compensatory result of the over-activated immune system response. Further studies referred to IL28, IL29 and related miRNA's will be enlightened the pathogenesis of SSPE.

TRIM21 Promotes Innate Immune Response to RNA Viral Infection through Lys27-Linked Polyubiquitination of MAVS.

Human innate immunity responds to viral infection by activating the production of interferons (IFNs) and proinflammatory cytokines. The mitochondrial adaptor molecule MAVS plays a critical role in innate immune response to viral infection. In this study, we show that TRIM21 (tripartite motif-containing protein 21) interacts with MAVS to positively regulate innate immunity. Under viral infection, TRIM21 is upregulated through the IFN/JAK/STAT signaling pathway. Knockdown of TRIM21 dramatically impairs innate immune response to viral infection. Moreover, TRIM21 interacts with MAVS and catalyzes its K27-linked polyubiquitination, thereby promoting the recruitment of TBK1 to MAVS. Specifically, the PRY-SPRY domain of TRIM21 is the key domain for its interaction with MAVS, while the RING domain of TRIM21 facilitates the polyubiquitination chains of MAVS. In addition, the MAVS-mediated innate immune response is enhanced by both the PRY-SPRY and RING domains of TRIM21. Mutation analyses of all the lysine residues of MAVS further revealed that Lys325 of MAVS is catalyzed by TRIM21 for the K27-linked polyubiquitination. Overall, this study reveals a novel mechanism by which TRIM21 promotes the K27-linked polyubiquitination of MAVS to positively regulate innate immune response, thereby inhibiting viral infection.IMPORTANCE Activation of innate immunity is essential for host cells to restrict the spread of invading viruses and other pathogens. MAVS plays a critical role in innate immune response to RNA viral infection. In this study, we demonstrated that TRIM21 targets MAVS to positively regulate innate immunity. Notably, TRIM21 targets and catalyzes K27-linked polyubiquitination of MAVS and then promotes the recruitment of TBK1 to MAVS, leading to upregulation of innate immunity. Our study outlines a novel mechanism by which the IFN signaling pathway blocks RNA virus to escape immune elimination.

Ankyrin repeat domain 1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum.

BACKGROUND: Atopic dermatitis (AD) is a common inflammatory skin disease. A subset of patients with AD are susceptible to disseminated herpes simplex virus (HSV) infection, a complication termed eczema herpeticum (ADEH+). The immune mechanisms causing ADEH+ remain elusive. Using RNA sequencing, we recently found that ankyrin repeat domain 1 (ANKRD1) was significantly induced in human PBMCs upon HSV-1 stimulation, and its induction in patients with ADEH+ was significantly reduced compared with that seen in AD patients without a history of eczema herpeticum (ADEH-). OBJECTIVE: We sought to validate ANKRD1 gene expression in nonatopic (NA) subjects, patients with ADEH-, and patients with ADEH+ and to delineate the biological function of ANKRD1 and the signaling pathway or pathways involved. METHODS: Purification of human PBMCs, monocytes, B cells, dendritic cells, T cells, and natural killer cells; RNA extraction and quantitative RT-PCR; small interfering RNA technique; co-immunoprecipitation; and Western blot assays were used. RESULTS: ANKRD1 expression was significantly reduced in PBMCs from patients with ADEH+ after HSV-1 stimulation compared with PBMCs from patients with ADEH-. We found that the induction of ANKRD1 by HSV-1 and multiple pattern recognition receptor agonists are mediated by inflammatory cytokines. Silencing ANKRD1 gene expression in antigen-presenting cells led to increased viral load and reduced IFNB1 and IL29 production. Using co-immunoprecipitation methods, we demonstrated that ANKRD1 formed protein complexes with interferon regulatory factor (IRF) 3 and IRF7, which are important transcription factors regulating signaling transduction of pattern recognition receptors. Overexpression of ANKRD1 enhanced the IRF3-mediated signaling pathways. CONCLUSION: ANKRD1 is involved in IRF3-mediated antiviral innate immune signaling pathways. Its reduced expression in patients with ADEH+ might contribute to the pathogenesis of ADEH+.