Betaglycan (TβRIII) Is Expressed in the Thymus and Regulates T Cell Development by Protecting Thymocytes from Apoptosis

<div><p>TGF-β type III receptor (TβRIII) is a coreceptor for TGFβ family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-β <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044217#pone.0044217-Massague1">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044217#pone.0044217-TenDijke1">[3]</a>, bone morphogenetic proteins (BMP2/4) and inhibins regulate different checkpoints during T cell differentiation. Although TβRIII is expressed on hematopoietic cells, the role of this receptor in the immune system remains elusive. Here, we provide the first evidence that TβRIII is developmentally expressed during T cell ontogeny, and plays a crucial role in thymocyte differentiation. Blocking of endogenous TβRIII in fetal thymic organ cultures led to a delay in DN-DP transition. In addition, <em>in vitro</em> development of TβRIII^−/− thymic lobes also showed a significant reduction in absolute thymocyte numbers, which correlated with increased thymocyte apoptosis, resembling the phenotype reported in Inhibin α ^−/− thymic lobes. These data suggest that Inhibins and TβRIII may function as a molecular pair regulating T cell development.</p> </div

TβRIII deficiency results in increased apoptosis of developing thymocytes.

<p>(A) Left panel, representative CD4 versus CD8 staining dot plots from TβRIII^+/+ and TβRIII^−/− fetal thymic lobes at day 7 of culture. Histograms show the expression of active caspase 3⁺ cells in each gated thymocyte subset. Right panel, graphs represent the percentage of active caspase 3⁺ cells and the levels of expression (MFI values) in each thymocyte subset. Data are representative of three independent experiments. (B) Left panel, representative histograms show the percentage of Annexin V⁺ cells in gated thymocyte subsets. Right panel, graph shows the analysis of the percentage of Annexin V⁺ cells in thymocytes from day 7 TβRIII^+/+ or TβRIII^−/− FTOCs. Data are representative of two independent experiments. Mean values ± SEM are shown (TβRIII^+/+ n = 3 and TβRIII^−/− n = 3). Asterisks indicate statistically significant differences (** p≤0.05).</p

The blocking of TβRIII in FTOCs alters T cell development.

<p>E14 thymic lobes were cultured in the presence of anti-TβRIII antibody or in the presence of pre-immune serum (control lobe). At day 3 and 7 of culture thymic lobes were disaggregated, counted and stained with antibodies to CD4, CD8. (A) Representative CD4 versus CD8 staining dot plots. (B). Comparative graphs represent the percentages of DN, DP, CD4SP and CD8SP thymocytes obtained after 3 and 7 days of culture between both treatments. (C) Analysis of cell numbers in non-treated and anti-TβRIII treated FTOCs at day 3 and 7. Data are representative of two independent experiments. Mean values ± SEM are shown (n  = 7 per group for day 3, and n  = 9 per group for day 7). Asterisks indicate *p≤0.05.</p

TβRIII is developmentally expressed during T cell ontogeny.

<p>Thymocytes from 4 to 6 week old C57BL/6 background mice were stained with antibodies to CD4, CD8, and TβRIII. Pre-immune serum was used as an internal background staining control. (A) Representative histograms showing the percentage of TβRIII⁺ cells in gated DN, DP, CD4⁺ SP and CD8⁺ SP subsets. Graphs show the percentage of TβRIII⁺ thymocytes and geometric MFI calculated after subtracting the background staining. (B) Representative histograms showing the percentage of TβRIII⁺ thymocytes in DN1, DN2, DN3 and DN4 immature subsets. Graph represents the analysis of TβRIII⁺ cells and geometric MFI in gated DN1, DN2, DN3 and DN4 immature subsets. Unstained (filled curve in gray), preimmune serum (gray line) and anti-TβRIII antiserum (black line). Data are representative of 4 independent experiments. (C) Left panel, graph shows the percentage of TβRIII⁺ cells and geometric MFI in gated CD69⁻ and CD69⁺ SP thymocytes as showed in histograms. Right panel, graph represents the analysis of TβRIII⁺ cells and geometric MFI in gated CD62L⁻, CD62L⁺ and CD62L^hi SP thymocytes as showed in upper panel. Mean values ± SEM are shown (n  = 5 per group). Asterisks indicate * p≤0.05, and **p≤0.01.</p

Abnormal PTPN11 enhancer methylation promotes rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and joint inflammation

The PTPN11 gene, encoding the tyrosine phosphatase SHP-2, is overexpressed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) compared with osteoarthritis (OA) FLS and promotes RA FLS invasiveness. Here, we explored the molecular basis for PTPN11 overexpression in RA FLS and the role of SHP-2 in RA pathogenesis. Using computational methods, we identified a putative enhancer in PTPN11 intron 1, which contained a glucocorticoid receptor- binding (GR-binding) motif. This region displayed enhancer function in RA FLS and contained 2 hypermethylation sites in RA compared with OA FLS. RA FLS stimulation with the glucocorticoid dexamethasone induced GR binding to the enhancer and PTPN11 expression. Glucocorticoid responsiveness of PTPN11 was significantly higher in RA FLS than OA FLS and required the differentially methylated CpGs for full enhancer function. SHP-2 expression was enriched in the RA synovial lining, and heterozygous Ptpn11 deletion in radioresistant or innate immune cells attenuated K/BxN serum transfer arthritis in mice. Treatment with SHP-2 inhibitor 11a-1 reduced RA FLS migration and responsiveness to TNF and IL-1β stimulation and reduced arthritis severity in mice. Our findings demonstrate how abnormal epigenetic regulation of a pathogenic gene determines FLS behavior and demonstrate that targeting SHP-2 or the SHP-2 pathway could be a therapeutic strategy for RA

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration

Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation.

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice

OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis. METHODS: Through RPTPα knockdown, we assessed FLS gene expression by quantitative polymerase chain reaction analysis and enzyme-linked immunosorbent assay, invasion and migration by Transwell assays, survival by annexin V and propidium iodide staining, adhesion and spreading by immunofluorescence microscopy, and activation of signaling pathways by Western blotting of FLS lysates. Arthritis development was examined in RPTPα-knockout (KO) mice using the K/BxN serum-transfer model. The contribution of radiosensitive and radioresistant cells to disease was evaluated by reciprocal bone marrow transplantation. RESULTS: RPTPα was enriched in the RA synovial lining. RPTPα knockdown impaired RA FLS survival, spreading, migration, invasiveness, and responsiveness to platelet-derived growth factor, tumor necrosis factor, and interleukin-1 stimulation. These phenotypes correlated with increased phosphorylation of Src on inhibitory Y(527) and decreased phosphorylation of FAK on stimulatory Y(397) . Treatment of RA FLS with an inhibitor of FAK phenocopied the knockdown of RPTPα. RPTPα-KO mice were protected from arthritis development, which was due to radioresistant cells. CONCLUSION: By regulating the phosphorylation of Src and FAK, RPTPα mediates proinflammatory and proinvasive signaling in RA FLS, correlating with the promotion of disease in an FLS-dependent model of RA

TGFβ responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness

In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK, promotes RA FLS invasiveness