Probes for TNF

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.

Chemotherapy-induced neuropathy (CIPN) is a common complication of paclitaxel. CIPN affects the quality of life of cancer survivors and frequently leads to discontinuation of treatment. Paclitaxel affects neuronal microtubules and induces neuronal mitochondrial dysfunction. However, there is limited clinical information regarding paclitaxel's effects on monocytes. Preclinical studies suggest that paclitaxel-induced neuronal damage is driven by monocytes/macrophages. Therefore, we evaluated whether paclitaxel selectively induces mitochondrial dysfunction and a pro-inflammatory phenotype in human circulating monocytes. We conducted studies in human primary peripheral blood mononuclear cells (PBMCs) from cancer patients being treated with paclitaxel, and in vitro analysis in PBMC cells and monocytes, and THP-1 monocytes in the presence of paclitaxel (0.1, 1, 10 uM). We used flow cytometric markers to study mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential, namely MitoSox and DIOC6(3) respectively. We also measured mRNA levels of pro- and anti-inflammatory molecules using qRT-PCR. In vitro paclitaxel induced a depolarization state in mitochondria in THP-1, human primary monocytes, and primary human PBMCs, but it did not change MitoSox. Monocytes in PBMCs cells from patients treated with paclitaxel showed significative depolarization state in mitochondria when compared to cells from control patients. In THP-1 cells, paclitaxel enhanced mRNA levels of the pro-inflammatory cytokines IL-8 and TNF alpha. In human primary PBMCs, paclitaxel reduced the anti-inflammatory factors CD163 and IL-10, and enhanced the TNF alpha, COX-2 and MCP-1 mRNA levels. Our study provides evidence that paclitaxel can induce mitochondrial dysfunction in isolated human monocytes and in monocytes present in total PBMCs cells. The observed depolarizing changes are indicative of a pro-mitophagy state, which is in accordance with the paclitaxel-induced pro-inflammatory phenotype in these cells. Early detection of mitochondria dysfunction in human monocytes could be a predictable sign to CIPN development in cancer patients. Our research was supported by the Early-Career Investigator Award W81XWH-16-1-0438 of the Department of Defense, The Pershing Square Sohn Cancer Research Alliance, Weill Cornell Medicine Funds, Department of Anesthesiology-Wake Forest School of Medicine Funds, Comprehensive Cancer Center-Wake Forest School of Medicine Funds, NIDA R21CA248106, National Center for Advancing Translational Sciences (NCATS)-NIH through Grant Award Number UL1TR001420. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Porcine Epidemic Diarrhea Virus (PEDV) is the causative agent of swine epidemic diarrhea. In order to study the pathogenic mechanism of PEDV, PEDV was inoculated into Vero cells cultured in vitro, and the total RNA of Vero cells was extracted to construct a library for Illumina high-throughput sequencing and screening of differentially expressed genes (p < 0.05). Five differentially expressed genes for qRT-PCR verification analysis were randomly selected, and the verification results were consistent with the transcriptome sequencing results. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis was performed on the differentially expressed genes screened above. The results showed that the target gene annotations of differentially expressed genes in the African green monkey genome were mainly enriched in the TNF signaling pathway, the P53 signaling pathway, the Jak-STAT signaling pathway, the MAPK signaling pathway, and immune inflammation. In addition, it has been reported that Puma can promote apoptosis and is a key mediator of P53-dependent and non-dependent apoptosis pathways. However, there is no report that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. It was found by flow cytometry that PEDV infection induced apoptosis, and by Western Blotting detection, PEDV infection significantly increased the expression of p53, BAX, and Puma apoptosis-related proteins. Treatment Vero cells with the p53 inhibitor, PFT-α, could significantly inhibit PEDV-induced apoptosis. Studies have shown that PEDV infection can activate Puma and induce apoptosis in a P53-dependent pathway. These findings provide data support for further elucidating the pathogenic mechanism of PEDV and developing an effective vaccine against PEDV.

Although the cardioprotective benefits of sodium-glucose cotransporter 2 (SGLT2) inhibitors are now widely appreciated, the mechanisms underlying these benefits remain unresolved. Tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a) is a receptor for tumor necrosis factor superfamily member 12 (Tnfsf12). Tnfrsf12a is highly inducible and plays a key role in the development of cardiac hypertrophy and heart failure. Here we set out to determine if SGLT2 inhibition affects the Tnfsf12/Tnfrsf12a system in the stressed myocardium. C57BL/6N mice that had undergone sham or transverse aortic constriction (TAC) surgery were treated with either the SGLT2 inhibitor empagliflozin (400 mg/kg diet; 60-65 mg/kg/day) or standard chow alone and were followed for 8 weeks. Tnfrsf12a expression in mouse hearts was assessed by in situ hybridization, qRT-PCR, and immunoblotting. Left ventricular (LV) mass, end-systolic volume, and end-diastolic volume were all increased in TAC mice and were significantly lower with empagliflozin. Myocyte hypertrophy and interstitial fibrosis in TAC hearts were similarly attenuated with empagliflozin. Tnfrsf12a expression was upregulated in mouse hearts following TAC surgery but not in the hearts of empagliflozin-treated mice. In cultured cardiomyocytes, Tnfrsf12a antagonism attenuated the increase in cardiomyocyte size that was induced by phenylephrine. Empagliflozin attenuates LV enlargement in mice with hypertrophic heart failure. This effect may be mediated, at least in part, by a reduction in loading conditions which limits upregulation of the inducible, proinflammatory, and prohypertrophic TNF superfamily receptor, Tnfrsf12a. Disruption of the Tnfsf12/Tnfrsf12a feed forward system may contribute to the cardioprotective benefits of SGLT2 inhibition.

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for PRRSV, but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystanders PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP) and GFP+ (PRRSV infected) and GFP- (bystander) cells were sorted for RNA-sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP+ and 0.06% reads from GFP- PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP+ as compared to GFP- PAMs. Importantly, negative immune regulators including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3), and T-cell exhaustion markers (PD-L1, PD-L2, IL10, IDO1, and TGFB2) were highly upregulated in GFP+ cells as compared to GFP- cells. By using in situ hybridization assay, RNA transcripts of TNF and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. Importance PRRSV is widespread in many swine producing countries, causing substantial economic loses to the swine industry. PAM is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAM from an acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV-GFP strain to infect pigs and sorted infected- and bystander- PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of and NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.