MDA5

Abstract: ABSTRACT Germline-encoded pattern recognition receptors [PRRs] in mammalian cells function in the detection of molecular patterns associated with pathogen invasion or cellular damage. A PRR subset is activated by the atypical presence and location of double-stranded RNA [dsRNA] or its synthetic analogue polyinosinic-polycytidylic acid [poly(I:C)], triggering pro-inflammatory signalling and death in many cell types. Poly(I:C) has been tested as a sole or combination cancer therapy in preclinical studies and clinical trials. The purpose of this study was to evaluate the effects of poly(I:C) transfection via electroporation on cell lines from a cancer of epithelial origin, 4T1 mammary carcinoma, and a cancer of mesenchymal origin, WEHI 164 fibrosarcoma. The effects of the poly(I:C) delivery on cell metabolism implicate the induction of cell death. A pro-inflammatory response was demonstrated by mRNA upregulation and the secretion of Type I interferon and several cytokines and chemokines. The mRNAs of dsRNA sensor DExD/H-box helicase 58/retinoic acid-inducible gene I protein [Ddx58/RIG-I] and sensor/co-sensor DEAH-box helicase 9 [Dhx9] were not regulated, but the mRNAs of RNA sensors toll-like receptor 3 [TLR3], interferon-induced with helicase C domain 1/melanoma differentiation-associated protein 5 [Ifih1/MDA5] and Z-DNA binding protein 1 [Zbp1] and co-sensors DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 [Ddx60] and interferon-inducible protein 204 [Ifi204] were upregulated in both cell lines. The mRNAs encoding signalling pathways components were present or upregulated in both cell types. These data demonstrate that RNA sensing effects can be amplified by electroporation delivery, potentially expanding the practicality of this immunotherapeutic approach.

Abstract: In mammals, heterogeneous ribonucleoprotein U (hnRNPU), also named as nuclear matrix protein-nuclear scaffold attachment factor (SAFA), was originally identified as a DNA/RNA interactor protein. It has been reported that human hnRNPU facilitates IFN-β generation after vesicular stomatitis virus (VSV) infection. Nevertheless, the role of chicken hnRNPU (chhnRNPU) in IFN-β regulation as well as in infectious bursal diseases virus (IBDV) replication is still unclear. Here, we found that chhnRNPU inhibits IFN-β production via interacting with MDA5 and MAVS, and facilitates IBDV replication via associating with genomic dsRNA of IBDV. Firstly, chicken hnRNPU (chhnRNPU) was widely expressed in different tissues of chickens and was distributed in the nucleus of DF-1 cells. Overexpression of chhnRNPU significantly suppresses IFN-β promoter activities induced by MDA5 and MAVS. Additionally, immunoprecipitated by dsRNA antibodies, which followed LC-MS analysis demonstrate that chhnRNPU is a partner of viral genomic dsRNA. chhnRNPU is translocated from nucleus to cytosol to co-localize with replication complex of IBDV after IBDV infection. Over-expression of chhnRNPU significantly promotes IBDV replication, which was determined by western blotting, qRT-PCR and TCID50 assay. Furthermore, knock down chhnRNPU by siRNA remarkably facilitates IFN-β production, and inhibits IBDV proliferation. These data collectively reveal that chhnRNPU positively regulates IBDV replication via negatively regulating IFN-β response.

Abstract: C-X-C motif chemokine 10 (CXCL10) is an inflammatory chemokine and a key molecule in the pathogenesis of rheumatoid arthritis (RA). Melanoma differentiation-associated gene 5 (MDA5) is an RNA helicase that plays a role in innate immune and inflammatory reactions. The details of the regulatory mechanisms of CXCL10 production and the precise role of MDA5 in RA synovitis have not been fully elucidated. The aim of this study was to examine the role of MDA5 in regulating CXCL10 expression in cultured human rheumatoid fibroblast-like synoviocytes (RFLS). RFLS was stimulated with Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA mimetic. Expression of interferon beta (IFN-β), MDA5, and CXCL10 was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay. A neutralizing antibody of IFN-β and siRNA-mediated MDA5 knockdown were used to determine the role of these molecules in regulating CXCL10 expression downstream of TLR3 signaling in RFLS. Poly I:C induced IFN-β, MDA5, and CXCL10 expression in a concentration- and time-dependent manner. IFN-β neutralizing antibody suppressed the expression of MDA5 and CXCL10, and knockdown of MDA5 decreased a part of CXCL10 expression (p < 0.001). The TLR3/IFN-β/CXCL10 axis may play a crucial role in the inflammatory responses in RA synovium, and MDA5 may be partially involved in this axis.

Abstract: As one of the most prevalent inflammatory myopathy disease in childhood, the patients with juvenile dermatomyositis (JDM) suffer from a great heterogeneity in clinical manifestations. Among them, antiMDA5 presented with a a greater risk with interstitial lung disease (ILD), which may lead to poor prognosis. In clinical practice, some patients are refractory to immunosuppressive treatments. JAK inhibitors (JAKi) can regulate JAKSTAT pathway and decrease the interferoninduced STAT1 phosphorylation, and has been applied into both adult DM and patients with JDM. Here, we share the experience of JAKi therapy in nine antiMDA5 positive refractory JDM cases and tried to identify the risk factors for poor response. The nine patients were diagnosed and classified according to Bohan and Peter’s criteria and received treatment between July 2017 and November 2022. We obtained written informed consent from the guardians of all patients before initiating JAKi therapy. Among the nine patients, 33% (3/9) patients were female, the mean age of onset was 5.1±3.6 years, the mean age to start addon JAKi therapy was 8.3±3.5 years. Three cases of them progressed rapidly in ILD. All the nine cases suffered from refractory ILD with poor response to glucocorticoids combined with two or more immunosuppressants (closporin, methotrexate or mycophenolate mofetil), and in the subsequent JAKi treatment, all the patients received tofacitinib with an initial dosage of 2.5 mg two times per day in patients of <25 kg (n=4) and initial dosage of 5 mg two times per day in patients of ≥25 kg (n=5). The nine patients were followed in a period of a median of 14.5 months (range: 4–59 months). ILD alleviated or evanished in five patients (55.5%), which were defined as responded group, and ILD retained or deteriorated in four patients (44.5%), which were defined as nonresponded group. Then we compared the clinical information (online supplemental materials) before we initiated JAKi therapy. Figure 1A shows the therapeutic effect in a typical patient. The radar plots from five aspects (figure 1B) showed that the nonresponded group tended to show higher serum ferritin, serum IgG level and higher proportion of total T cells, CD4+T cells and CD8+T cells. Then, we performed a MannWhitney test which showed that nonresponded group have an elevated serum IgG level and higher proportion of total T cells with significant difference(p=0.021, Z=−2.309 and p=0.021. Z=−2.309, respectively). Besides ILD, the rash and muscle weakness resolved in 1 year of all the nine patients. As far as we know, it is the first case series research summarising the efficiency of tofacitinib on antiMDA5 positive JDM patients with refractory ILD. Though tofacitinib was proved to be effective in treating children with refractory JDM by several researches, we found limitation existing in treating refractory ILD among antiMDA5 positive JDM patients, even combined with all the available mainstream immunosuppressants alternately. Compared with those who responded well, the elevated level of serum IgG and increased proportion of total T cells before tofacitinib therapy may help to identify patients who respond poorly to this pharmaceutical. However, our study was retrospectively conducted in a limited number of case series, perspective cohort should be established to testify the efficiency and safety of tofacitinib in treating refractory ILD among antiMDA5 positive JDM patients.