High Dimensional Analyses of Circulating Immune Cells in Psoriatic Arthritis Detects Elevated Phosphorylated STAT3

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.

Macrophages induce inflammation by efferocytosis of apoptotic prostate cancer cells via HIF-1α stabilization

Clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment of a cellular response to hypoxia. Here we show that efferocytic macrophages promote HIF-1α stabilization under normoxic conditions through interaction with phosphorylated STAT3. Inflammatory cytokine gene expression analysis of efferocytic HIF-1α-mutant macrophages revealed a reduced expression of the pro-tumorigenic Mif. Furthermore, stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in macrophages. Finally, macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by tumor-associated macrophages triggers p-STAT3/HIF-1α/MIF signaling to enhance tumor-promoting inflammation in bone, suggesting this axis as a target for metastatic prostate cancer.

Effect of (−)-Epigallocatechin Gallate on Activation of JAK/STAT Signaling Pathway by Staphylococcal Enterotoxin A

Staphylococcal enterotoxin A (SEA), which is a superantigen toxin protein, binds to cytokine receptor gp130. Gp130 activates intracellular signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. The effects of SEA on the JAK/STAT signaling pathway in mouse spleen cells were examined. After treatment with SEA, mRNA expression levels of interferon gamma (IFN-γ) and suppressor of cytokine-signaling 1 (SOCS1) increased. SEA-induced IFN-γ and SOCS1 expression were decreased by treatment with (−)-epigallocatechin gallate (EGCG). The phosphorylated STAT3, Tyr705, increased significantly in a SEA concentration-dependent manner in mouse spleen cells. Although (−)-3″-Me-EGCG did not inhibit SEA-induced phosphorylated STAT3, EGCG and (−)-4″-Me-EGCG significantly inhibited SEA-induced phosphorylated STAT3. It was thought that the hydroxyl group at position 3 of the galloyl group in the EGCG was responsible for binding to SEA and suppressing SEA-induced phosphorylation of STAT3. Through protein thermal shift assay in vitro, the binding of the gp130 receptor to SEA and the phosphorylation of STAT3 were inhibited by the interaction between EGCG and SEA. As far as we know, this is the first report to document that EGCG inhibits the binding of the gp130 receptor to SEA and the associated phosphorylation of STAT3.

The Gain-of-Function p53 R248W Mutant Promotes Migration by STAT3 Deregulation in Human Pancreatic Cancer Cells

Missense p53 mutations (mutp53) occur in approx. 70% of pancreatic ductal adenocarcinomas (PDAC). Typically, mutp53 proteins are aberrantly stabilized by Hsp90/Hsp70/Hsp40 chaperone complexes. Notably, stabilization is a precondition for specific mutp53 alleles to acquire powerful neomorphic oncogenic gain-of-functions (GOFs) that promote tumor progression in solid cancers mainly by increasing invasion and metastasis. In colorectal cancer (CRC), we recently established that the common hotspot mutants mutp53R248Q and mutp53R248W exert GOF activities by constitutively binding to and hyperactivating STAT3. This results in increased proliferation and invasion in an autochthonous CRC mouse model and correlates with poor survival in patients. Comparing a panel of p53 missense mutations in a series of homozygous human PDAC cell lines, we show here that, similar to CRC, the mutp53R248W protein again undergoes a strong Hsp90-mediated stabilization and selectively promotes migration. Highly stabilized mutp53 is degradable by the Hsp90 inhibitors Onalespib and Ganetespib, and correlates with growth suppression, possibly suggesting therapeutic vulnerabilities to target GOF mutp53 proteins in PDAC. In response to mutp53 depletion, only mutp53R248W harboring PDAC cells show STAT3 de-phosphorylation and reduced migration, again suggesting an allele-specific GOF in this cancer entity, similar to CRC. Moreover, mutp53R248W also exhibits the strongest constitutive complex formation with phosphorylated STAT3. The selective mutp53R248W GOF signals through enhancing the STAT3 axis, which was confirmed since targeting STAT3 by knockdown or pharmacological inhibition phenocopied mutp53 depletion and reduced cell viability and migration preferentially in mutp53R248W-containing PDAC cells. Our results confirm that mutp53 GOF activities are allele specific and can span across tumor entities.

Shikonin Derivatives from Onsoma visianii Decrease Expression of Phosphorylated STAT3 in Leukemia Cells and Exert Antitumor Activity

Antitumor effects of shikonins on chronic lymphocytic leukemia (CLL) and B-cell prolymphocytic leukemia (B-PLL) are mostly unexplored. The antitumor activity of shikonins, isolated from Onosma visianii Clem (Boraginaceae), in BCL1, mouse CLL cells and JVM-13, human B-PLL cells was explored in this study. The cytotoxicity of shikonin derivatives was measured by an MTT test. Cell death, proliferation, cell cycle, and expression of molecules that control these processes were analyzed by flow cytometry. Expression of STAT3-regulated genes was analyzed by real-time q-RT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The antitumor effects of shikonin derivatives in vivo were analyzed, using flow cytometry, by detection of leukemia cells in the peripheral blood and spleens of mice intravenously injected with BCL1 cells. The two most potent derivatives, isobutyrylshikonin (IBS) and α-methylbutyrylshikonin (MBS), induced cell cycle disturbances and apoptosis, inhibited proliferation, and decreased expression of phospho-STAT3 and downstream-regulated molecules in BCL1 and JVM-13 cells. IBS and MBS decreased the percentage of leukemia cells in vivo. The link between the decrease in phosphorylated STAT3 by MBS and IBS and BCL1 cell death was confirmed by detection of enhanced cell death after addition of AG490, an inhibitor of Jak2 kinase. It seems that IBS and MBS, by decreasing STAT3 phosphorylation, trigger apoptosis, inhibit cell proliferation, and attenuate leukemia cell stemness.

Covid-19 Interstitial Pneumonia: Histological and Immunohistochemical Features on Cryobiopsies

Background: The pathogenetic steps leading to Covid-19 interstitial pneumonia remain to be clarified. Most postmortem studies to date reveal diffuse alveolar damage as the most relevant histologic pattern. Antemortem lung biopsy may however provide more precise data regarding the earlier stages of the disease, providing a basis for novel treatment approaches. Objectives: To ascertain the morphological and immunohistochemical features of lung samples obtained in patients with moderate Covid-19 pneumonia. Methods: Transbronchial lung cryobiopsy was carried out in 12 Covid-19 patients within 20 days of symptom onset. Results: Histopathologic changes included spots of patchy acute lung injury with alveolar type II cell hyperplasia, with no evidence of hyaline membranes. Strong nuclear expression of phosphorylated STAT3 was observed in >50% of AECII. Interalveolar capillaries showed enlarged lumen and were in part arranged in superposed rows. Pulmonary venules were characterized by luminal enlargement, thickened walls, and perivascular CD4+ T-cell infiltration. A strong nuclear expression of phosphorylated STAT3, associated with PD-L1 and IDO expression, was observed in endothelial cells of venules and interstitial capillaries. Alveolar spaces macrophages exhibited a peculiar phenotype (CD68, CD11c, CD14, CD205, CD206, CD123/IL3AR, and PD-L1). Conclusions: Morphologically distinct features were identified in early stages of Covid-19 pneumonia, with epithelial and endothelial cell abnormalities different from either classical interstitial lung diseases or diffuse alveolar damage. Alveolar type II cell hyperplasia was a prominent event in the majority of cases. Inflammatory cells expressed peculiar phenotypes. No evidence of hyaline membranes and endothelial changes characterized by IDO expression might in part explain the compliance and the characteristic pulmonary vasoplegia observed in less-advanced Covid-19 pneumonia.

Epigenetic Silencing of Tumor Suppressor miR-124 Directly Supports STAT3 Activation in Cutaneous T-Cell Lymphoma

Increasing evidence supports a potential role for STAT3 as a tumor driver in cutaneous T-cell lymphomas (CTCL). The mechanisms leading to STAT3 activation are not fully understood; however, we recently found that miR-124, a known STAT3 regulator, is robustly silenced in MF tumor-stage and CTCL cells. Objective: We studied here whether deregulation of miR-124 contributes to STAT3 pathway activation in CTCL. Methods: We measured the effect of ectopic mir-124 expression in active phosphorylated STAT3 (p-STAT3) levels and evaluated the transcriptional impact of miR-124-dependent STAT3 pathway regulation by expression microarray analysis. Results: We found that ectopic expression of miR-124 results in massive downregulation of activated STAT3 in different CTCL lines, which resulted in a significant alteration of genetic signatures related with gene transcription and proliferation such as MYC and E2F. Conclusions: Our study highlights the importance of the miR-124/STAT3 axis in CTCL and demonstrates that the STAT3 pathway is regulated through epigenetic mechanisms in these cells. Since deregulated STAT3 signaling has a major impact on CTCL initiation and progression, a better understanding of the molecular basis of the miR-124/STAT3 axis may provide useful information for future personalized therapies.

LncRNA KCNA2-AS regulates spinal astrocyte activation through STAT3 to affect postherpetic neuralgia

Objectives Postherpetic neuralgia (PHN) is the most common complication of herpes zoster, but the mechanism of PHN is still unclear. Activation of spinal astrocytes is involved in PHN. Our study aims to explore whether lncRNA KCNA2 antisense RNA (KCNA2-AS) regulates spinal astrocytes in PHN through signal transducers and activators of transcription 3 (STAT3). Methods Varicella zoster virus (VZV)-infected CV-1 cells were injected into rats to construct a PHN model. Primary spinal cord astrocytes were activated using S-Nitrosoglutathione (GSNO). Glial fibrillary acidic protein (GFAP; marker of astrocyte activation), phosphorylated STAT3 (pSTAT3), and KCNA2-AS were analyzed by immunofluorescence and RNA fluorescence in situ hybridization. RNA pull-down and RNA immunoprecipitation were used to detect binding of KCNA2-AS to pSTAT3. Results KCNA2-AS was highly expressed in the spinal cord tissue of PHN model rats, and was positively correlated with GFAP expression. GFAP was significantly increased in GSNO-induced cells, but the knockdown of KCNA2-AS reversed this result. Meanwhile, pSTAT3 was significantly increased in GSNO-induced cells, but knockdown of KCNA2-AS reduced pSTAT3 within the nucleus while the total pSTAT3 did not change significantly. pSTAT3 bound to KCNA2-AS and this binding increased with GSNO treatment. Furthermore, knockdown of KCNA2-AS in PHN model rats relieved mechanical allodynia. Conclusion Down-regulation of KCNA2-AS alleviates PHN partly by reducing the translocation of pSTAT3 cytoplasm to the nucleus and then inhibiting the activation of spinal astrocytes.