BackgroundMast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs).MethodCultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 μg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 μg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 μg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex.ResultsTryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients.ConclusionMC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.

BackgroundCircular RNA circFADS2 plays protective roles in LPS-induced inflammation, which promotes sepsis, suggesting its involvement in sepsis.MethodsExpression of circFADS2, mature miR-15a-5p, and miR-15a-5p precursor in plasma samples from sepsis patients and healthy controls was determined by RT-qPCR. The circFADS2 expression vector was transfected in lung cells, followed by the measurement of the expression levels of mature miR-15a-5p and miR-15a-5p precursor to study the role of circFADS2 in miR-15a-5p maturation. Cell apoptosis was analyzed by cell apoptosis assay.ResultsCircFADS2 was upregulated in sepsis and inversely correlated with mature miR-15a-5p, but not miR-15a-5p precursor. In lung cells, circFADS2 overexpression decreased the level of mature miR-15a-5p, but not miR-15a-5p precursor. LPS treatment decreased miR-15a-5p expression and increased circFADS2 level. Cell apoptosis analysis showed that circFADS2 overexpression reduced miR-15a-5p overexpression-induced apoptosis of LPS-treated lung cells.ConclusionsCircFADS2 is upregulated in sepsis to suppress LPS-induced lung cell apoptosis by inhibiting miR-15a-5p maturation.

BackgroundThe spread of a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in China and other countries is of great concern worldwide with no effective vaccine. This study aimed to design a novel vaccine construct against SARS-CoV-2 from the spike S protein and orf1ab polyprotein using immunoinformatics tools. The vaccine was designed from conserved epitopes interacted against B and T lymphocytes by the combination of highly immunogenic epitopes with suitable adjuvant and linkers.ResultsThe proposed vaccine composed of 526 amino acids and was shown to be antigenic in Vaxigen server (0.6194) and nonallergenic in Allertop server. The physiochemical properties of the vaccine showed isoelectric point of 10.19. The instability index (II) was 31.25 classifying the vaccine as stable. Aliphatic index was 84.39 and the grand average of hydropathicity (GRAVY) was − 0.049 classifying the vaccine as hydrophilic. Vaccine tertiary structure was predicted, refined and validated to assess the stability of the vaccine via Ramachandran plot and ProSA-web servers. Moreover, solubility of the vaccine construct was greater than the average solubility provided by protein sol and SOLpro servers indicating the solubility of the vaccine construct. Disulfide engineering was performed to reduce the high mobile regions in the vaccine to enhance stability. Docking of the vaccine construct with TLR4 demonstrated efficient binding energy with attractive binding energy of − 338.68 kcal/mol and − 346.89 kcal/mol for TLR4 chain A and chain B respectively. Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells and INF-γ. Upon cloning, the vaccine protein was reverse transcribed into DNA sequence and cloned into pET28a(+) vector to ensure translational potency and microbial expression.ConclusionA unique vaccine construct from spike S protein and orf1ab polyprotein against B and T lymphocytes was generated with potential protection against the pandemic. The present study might assist in developing a suitable therapeutics protocol to combat SARSCoV-2 infection.

BackgroundBacillus ancthracis causes cutaneous, pulmonary, or gastrointestinal forms of anthrax. B. anthracis is a pathogenic bacterium that is potentially to be used in bioterrorism because it can be produced in the form of spores. Currently, protective antigen (PA)-based vaccines are being used for the prevention of anthrax, but it is necessary to develop more safe and effective vaccines due to their prolonged immunization schedules and adverse reactions.MethodsWe selected the lipoprotein GBAA0190, a potent inducer of host immune response, present in anthrax spores as a novel potential vaccine candidate. Then, we evaluated its immune-stimulating activity in the bone marrow-derived macrophages (BMDMs) using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Protective efficacy of GBAA0190 was evaluated in the guinea pig (GP) model.ResultsThe recombinant GBAA0190 (r0190) protein induced the expression of various inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in the BMDMs. These immune responses were mediated through toll-like receptor 1/2 via activation of mitogen-activated protein (MAP) kinase and Nuclear factor-κB (NF-κB) pathways. We demonstrated that not only immunization of r0190 alone, but also combined immunization with r0190 and recombinant PA showed significant protective efficacy against B. anthracis spore challenges in the GP model.ConclusionsOur results suggest that r0190 may be a potential target for anthrax vaccine.

BackgroundKawasaki disease is an autoimmune disease characterized by systemic vasculitis of unknown aetiology and most commonly occurs in children under 5 years old. Previous studies have found that the over-activation of lymphocytes is an important mechanism of Kawasaki disease. Activin A, also known as immunosuppressive factor P, is a multifunctional growth and transforming factor. However, whether activin A is involved in the regulation of peripheral lymphocytes activity in Kawasaki disease is unclear. Thus, we aimed to investigate the effect of activin A on the activity of peripheral lymphocytes in acute-phase Kawasaki disease.MethodsSeven patients with Kawasaki disease and seven healthy controls were studied. Peripheral blood lymphocytes were isolated by Ficoll density gradient centrifugation. The activation of CD4+ and CD8+ T cells and CD19+ B cells was investigated by flow cytometry. The expression of activin type IIA receptors was investigated by flow cytometry.ResultsImmune imbalance in CD4 and CD8 lymphocytes were detected in acute-phase Kawasaki disease. The expression of activin type IIA receptors on CD8+ T cells and CD19+ B cells was increased in acute-phase Kawasaki disease and decreased following treatment with activin A. Activin A suppressed the expression of CD25 and CD69 on CD8+ T cells and the expression of CD69 on CD19+ B cells.ConclusionsThe expression of activin type IIA receptor was increased on CD8+ T cells and CD19+ B cells in Kawasaki disease. Activin A suppressed the expression of CD25, CD69 and activin type IIA receptors on peripheral CD8+ T lymphocyte. Activin A plays different roles in different lymphocyte subsets and suppresses peripheral CD8+ T lymphocyte activity in acute-phase Kawasaki disease.

BackgroundNET (neutrophil extracellular trap) has been shown to directly influence inflammation; in SLE (systemic lupus erythematosus), it is reportedly a plausible cause for the broken self-tolerance that contributes to this pathology. Meanwhile, the role of NET is not easily explicable, and there is a serious discrepancy in the role of NET in SLE pathology and generally inflammation; in particular, the interactions of neutrophils with NET have been rarely inspected. This study evaluates the effect of NET on neutrophils in the context of SLE. The neutrophils were incubated by the collected NET (from SLE patients and healthy controls) and their expression of an activation marker, viability and oxidative burst ability were measured.ResultsThe level of cell mortality, CD11b expression and the oxidative burst capacity were elevated in NET-treated neutrophils. Also, the elevation caused by the SLE NET was higher than that produced by the healthy NET.ConclusionThe decreased neutrophil viability was not due to the increase in apoptosis; rather, it was because of the augmentation of other inflammatory cell-death modes. The upregulation of CD11b implies that NET causes neutrophils to more actively contribute to inflammation. The increased oxidative burst capacity of neutrophils can play a double role in inflammation. Overall, the effects induced by NET on neutrophils help prolong inflammation; accordingly, the NET collected from SLE patients is stronger than the NET from healthy individuals.

BackgroundInterleukin-17A (IL17A) is a proinflammatory cytokine critically involved in autoimmune diseases, and monoclonal antibodies of IL17A have been approved for clinical treatment of psoriasis. However, a usable psoriatic animal model has been always required for preclinical evaluation of IL17A antagonists. Imiquimod (IMQ)-induced psoriasis model is widely used in fundamental research, but it’s not able to accurately show anti-psoriatic effect of IL17A antagonists with conventional modelling condition.ResultsOn female C57BL/6 mice, with optimization on the usage of IMQ, positive control reagent and anti-mIL17A antibody, a 7-day model with proper testing window, acceptable disease severity as well as high repeatability was developed, and the efficacy of IL17A antagonist can be objectively evaluated by several qualitative and quantitative indices. Meanwhile, we validated the detailed involvement of IL17A signaling in disease progression, confirmed that the expression levels of IL17A and its related cytokines were induced by IMQ application, and its downstream cytokines can be inhibited by IL17A antagonist treatment. In further study, we revealed that IL17A was transient induced by IMQ and directly caused downstream signaling activation. This finding on the kinetical change of IL17A signaling will manifest the pharmacokinetics-pharmacodynamics investigation of IL17A antagonists.ConclusionsOur work presents the application of a convenient psoriatic animal model in the research and development of IL17A antagonists, meanwhile providing extra evidence for understanding IL17A’s role in the progression of IMQ-induced psoriasis model, which manifest the research and development of IL17A antagonists.

BackgroundSystemic lupus erythematosus is an autoimmune disease characterized by an overproduction of autoantibodies resulting from dysregulation in multiple immune cell types. D-mannose is a C− 2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus.ResultsFirstly, the effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of non-autoimmune C57BL/6 (B6) bone marrow-derived dendritic cells (BMDCs) and their ability to induce antigen-specific CD4+ T cell proliferation and activation. D-mannose inhibited the maturation of BMDCs and their induction of antigen-specific T cell proliferation and activation. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated B6 mice. To assess the effect of D-mannose in mouse models of lupus, we used the graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. In the cGVHD model, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus.ConclusionOverall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

BackgroundThe immunosuppressive effects of regulatory B-cells (Bregs) and their immunosuppressive cytokines on immune responses in autoimmune disorders, mainly systemic lupus erythematosus (SLE), have been recently established. Therefore, the purpose of this article has been the exploration of the expressions of cytokines produced by B cells in newly diagnosed SLE patients.ResultsThe findings demonstrated that the gene expression of IL-10, TGF-β, IL-35, PD-L1, and FasL was significantly up-regulated in SLE patients compared to healthy subjects (P < 0.05). Additionally, the results revealed that serum levels of IL-10, TGF-β, IL-35, PD-L1 were remarkably increased in patients with SLE compared to healthy subjects (P < 0.0001). However, serum levels of IL-10 and TGF-β decreased significantly with increasing SLEDAI score in studied patients (P < 0.05).ConclusionIt was concluded that the release of anti-inflammatory cytokines, particularly IL-10 and TGF-β, might inhibit immune responses and autoreactive immune cells in a compensatory manner in SLE patients with mild to moderate disease activity.

BackgroundPresepsin (soluble CD14 subtype) is a novel biomarker of sepsis used for early diagnosis. Originally, CD14 is known as the pattern recognition receptor for the lipopolysaccharide (LPS)/LBP complexes, and the diagnostic value of this molecule for gram-positive bacteria, which contain less amount of LPS, is uncertain. To confirm its effectiveness in the diagnosis of bacteremia caused by gram positive bacteria, and to evaluate the interspecies difference of presepsin production against various bacterial strains, we conducted an in vitro assay to evaluate presepsin levels in response to five Gram negative and four Gram positive bacteria.ResultsWhole blood was yielded from five healthy volunteers and co-cultured with bacterial strains at 37 °C for 4 h. Twenty seven clinical isolates of nine species (E. coli, K. pneumoniae, E. cloacae, P. aeruginosa, S. maltophilia, S. aureus, S. pyogenes, B. cereus, and C. striatum) derived from blood cultures of non-neutropenic bacteremia patients were used. Culture supernatants were harvested and presepsin levels were measured. The presepsin level in the gram-negative bacteria 273 (218–352) pg/mL was significantly higher than in the gram-positive bacteria 200 (143–275) pg/mL (p = 0.0002). The presepsin levels were significantly lower in C. striatum, in comparison to other bacteria, and S. pyogenes showed similar results. And the presepsin levels in P. aeruginosa were significantly lower compared to E. cloacae, K. pneumoniae, and S. aureus.ConclusionsPresepsin production can also be evoked by gram-positive bacteria, and interspecies differences of presepsin response may exist, which should be considered in the diagnosis of sepsis, especially in certain gram-positive bacteremia such as S. pyogenes or C. striatum.