S-allylmercapto-N-acetylcysteine ameliorates pulmonary fibrosis in mice via Nrf2 pathway activation and NF-κB, TGF-ß1/Smad2/3 pathway suppression.

Pulmonary fibrosis (PF) is a chronic lung disease characterised by alveolar inflammatory injury, alveolar septal thickening, and eventually fibrosis. Patients with severe Coronavirus Disease 2019 (COVID-19) may have left a certain degree of pulmonary fibrosis. PF is commonly caused by oxidative imbalance and inflammatory damage. S-allylmercapto-N-acetylcysteine (ASSNAC) exhibits anti-oxidative and anti-inflammatory effects in other diseases. However, the pharmacodynamics of ASSNAC remain unclear for PF. This investigation aimed to evaluate the efficacy and mechanism of ASSNAC against PF. The PF model was established by TGF-ß1 stimulating HFL-1 cells in vitro. ASSNAC exhibited the potential to inhibit fibroblast transformation into myofibroblasts. Also, in the PF mice model with bleomycin (BLM), the sodium salt of ASSNAC (ASSNAC-Na) inhalation was treated. ASSNAC remarkably improved mice's lung tissue structure and collagen deposition. The important indicator proteins of PF, collagen Ⅰ, collagen Ⅲ, and α-SMA significantly decreased in the ASSNAC treated groups. Besides, ASSNAC attenuated oxidative stress by reversing glutathione (GSH), superoxide dismutase (SOD) levels and interfering with Nrf2/NOX4 signaling pathways. ASSNAC showed an anti-inflammatory effect by reducing the number of inflammatory cells and inflammatory cytokines, such as TNF-α and IL-6, and blocking the NF-κB signaling pathway. ASSNAC inhibited fibroblast differentiation by blocking the TGF-ß1/Smad2/3 signaling pathway. This study implicates that ASSNAC alleviates pulmonary fibrosis through fighting against oxidative stress, reducing inflammation and inhibiting fibroblast differentiation.

Sirt1 overexpression improves senescence-associated pulmonary fibrosis induced by vitamin D deficiency through downregulating IL-11 transcription.

Determining the mechanism of senescence-associated pulmonary fibrosis is crucial for designing more effective treatments for chronic lung diseases. This study aimed to determine the following: whether Sirt1 and serum vitamin D decreased with physiological aging, promoting senescence-associated pulmonary fibrosis by activating TGF-ß1/IL-11/MEK/ERK signaling, whether Sirt1 overexpression prevented TGF-ß1/IL-11/MEK/ERK signaling-mediated senescence-associated pulmonary fibrosis in vitamin D-deficient (Cyp27b1-/- ) mice, and whether Sirt1 downregulated IL-11 expression transcribed by TGF-ß1/Smad2 signaling through deacetylating histone at the IL-11 promoter in pulmonary fibroblasts. Bioinformatics analysis with RNA sequencing data from pulmonary fibroblasts of physiologically aged mice was conducted for correlation analysis. Lungs from young and physiologically aged wild-type (WT) mice were examined for cell senescence, fibrosis markers, and TGF-ß1/IL-11/MEK/ERK signaling proteins, and 1,25(OH)2 D3 and IL-11 levels were detected in serum. Nine-week-old WT, Sirt1 mesenchymal transgene (Sirt1Tg ), Cyp27b1-/- , and Sirt1Tg Cyp27b1-/- mice were observed the pulmonary function, aging, and senescence-associated secretory phenotype and TGF-ß1/IL-11/MEK/ERK signaling. We found that pulmonary Sirt1 and serum vitamin D decreased with physiological aging, activating TGF-ß1/IL-11/MEK/ERK signaling, and promoting senescence-associated pulmonary fibrosis. Sirt1 overexpression improved pulmonary dysfunction, aging, DNA damage, senescence-associated secretory phenotype, and fibrosis through downregulating TGF-ß1/IL-11/MEK/ERK signaling in Cyp27b1-/- mice. Sirt1 negatively regulated IL-11 expression through deacetylating H3K9/14ac mainly at the region from -871 to -724 of IL-11 promoter, also the major binding region of Smad2 which regulated IL-11 expression at the transcriptional level, and subsequently inhibiting TGF-ß1/IL-11/MEK/ERK signaling in pulmonary fibroblasts. This signaling in aging fibroblasts could be a therapeutic target for preventing senescence-associated pulmonary fibrosis induced by vitamin D deficiency.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Mediates Transforming Growth Factor Beta (TGF-b) Hijacking and Immune Dysregulation Through A Novel Gain of Function Mutation in Its Nonstructural 15 (NSP15) Protein

Rationale: The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions via the acute respiratory distress syndrome (ARDS). The early immune suppression of SARS-CoV-2 then subsequent inflammation suggests an unusual ability to cause immune dysregulation. Host transforming growth factor beta (TGF-β) is an immunesuppressing and profibrotic cytokine frequently “hijacked” by microbes to evade immune detection. We discovered a KRFK domain (a potent activating motif for latent TGF-β) in the SARS-CoV-2 nonstructural 15 (NSP15) protein. We hypothesized that this NSP15 protein causes immune dysregulation by activation of latent TGF-β and subsequent activation of immunosuppressive Tregulatory (Treg) cells, and that substantial TGF-β is present in the lungs of COVID-19 ARDS patients. Methods: We evaluated TGF-β1 concentrations in endotracheal aspirates (ETA) of 27 COVID-19 ARDS patients by ELISA. We produced recombinant SARS-CoV-2 NSP15 protein in E. coli and tested its ability to activate latent TGF-β1 using in vitro assays. TGF-β inhibitors were assessed for their ability to block effects. We obtained blood mononuclear cells from healthy subjects and isolated Tregs to assess their activation state via intracellular smad-2 phosphorylation (pSMAD2) using flow cytometry. Results: The KRFK domain was present in all SARS-CoV-2 variants. High concentrations of both active and total TGF-β1 were detected in ETA of COVID-19 ARDS patients (150 +/- 34 pg/ml active;1,819 +/- 304 pg/ml total) in a range previously shown to affect T cell function. NSP15 at 2.4 nM increased activation of latent TGF-β 12-fold (P < .001 vs. vehicle), compared to an 11% activation with the positive control thrombospondin-1 (TSP1;10 nM) (Figure). TGF-β receptor inhibitors blocked NSP15 effects on latent TGF-β activation and intracellular TGF-β1 signaling in a bioassay by over 95% (p<.01). At tested concentrations (25, 50, 100 nM) NSP15 increased Treg pSMAD2 levels via activation of latent TGF-β1, exceeding levels seen in Tregs stimulated with 400 pM of active TGF-β1 (+ control) (pSMAD2 + cells: vehicle 1.1%, active TGF-β1 43%, NSP15/latent TGF-β1 49-56%). Conclusions: High concentrations of active and total TGF-β1 are present in the lungs of COVID-19 ARDS patients, suggesting SARS-CoV-2 uses host TGF-β hijacking as a mechanism for immune evasion. The NSP15 protein of SARSCoV- 2 potently activates latent TGF-β, leading to Treg activation. TGF-β inhibitors are potent inhibitors of these NSP15 effects. A strategy to block NSP15-mediated effects with TGF-β inhibitors is an innovative therapy worthy of testing in animal models of COVID-19.

Computationally predicted SARS-COV-2 encoded microRNAs target NFKB, JAK/STAT and TGFB signaling pathways.

Recently an outbreak that emerged in Wuhan, China in December 2019, spread to the whole world in a short time and killed >1,410,000 people. It was determined that a new type of beta coronavirus called severe acute respiratory disease coronavirus type 2 (SARS-CoV-2) was causative agent of this outbreak and the disease caused by the virus was named as coronavirus disease 19 (COVID19). Despite the information obtained from the viral genome structure, many aspects of the virus-host interactions during infection is still unknown. In this study we aimed to identify SARS-CoV-2 encoded microRNAs and their cellular targets. We applied a computational method to predict miRNAs encoded by SARS-CoV-2 along with their putative targets in humans. Targets of predicted miRNAs were clustered into groups based on their biological processes, molecular function, and cellular compartments using GO and PANTHER. By using KEGG pathway enrichment analysis top pathways were identified. Finally, we have constructed an integrative pathway network analysis with target genes. We identified 40 SARS-CoV-2 miRNAs and their regulated targets. Our analysis showed that targeted genes including NFKB1, NFKBIE, JAK1-2, STAT3-4, STAT5B, STAT6, SOCS1-6, IL2, IL8, IL10, IL17, TGFBR1-2, SMAD2-4, HDAC1-6 and JARID1A-C, JARID2 play important roles in NFKB, JAK/STAT and TGFB signaling pathways as well as cells' epigenetic regulation pathways. Our results may help to understand virus-host interaction and the role of viral miRNAs during SARS-CoV-2 infection. As there is no current drug and effective treatment available for COVID19, it may also help to develop new treatment strategies.