TNF-α Response Research Articles

DOP095 IL-23-activated cells as response biomarkers for risankizumab in Crohn’s disease patients

Abstract Background The interleukin (IL)-23 signaling is a vital pathway in Crohn’s disease (CD). IL-23 activates T-helper (Th) 17 cells, IL-17-secreting CD8 T (Tc)17 cells, γδ T cells, natural killer (NK) T cells, and group 3 innate lymphoid cells (ILC3) to secrete inflammatory cytokines including IL-17, IFN-γ, and TNF-α. Risankizumab is the first selective IL-23 antagonist approved for moderate-to-severe CD; however, no biomarker currently exists to predict response to this medication. Here, we characterized the phenotypes of IL-23-activated cells that predict response to risankizumab in CD patients. Methods Adult patients with active ileal/colonic CD were included (n = 28). Blood samples were collected before and after initiation of risankizumab. Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved for batch analysis. After stimulation with IL-23, PBMCs were stained using a pre-optimized and validated antibody panel for 37 lineage markers and cytokines for CyTOF. Cell clusters were resolved using UMAP (Figure 1), opt-SNE and FlowJo. Patients’ responses were evaluated 12-40 weeks after initiation of risankizumab based on clinical symptoms (e.g., CDAI) and endoscopic/radiographic assessment. Results Among the IL-23-activated cells, increased frequency of NKT cells was identified in the peripheral blood of non-responders before initiation of risankizumab (not shown). In pre-risankizumab PBMCs, we identified significantly elevated TNF-α in Th17 cells, Tc17 cells, NKT cells, and MAIT cells in non-responders vs. responders (Figure 2A). Similarly, in PBMCs 4 weeks after 1st risankizumab infusion, Tc17 cells, γδ T cells, NKT cells, and MAIT cells in non-responders produced significantly more TNF-α compared to those cells from responders. In addition, we observed increased IL-17A in multiple IL-23-activated cells in non-responders 4 weeks after starting risankizumab (Figure 2B). Peripheral and mucosal IL-23-activated cells from later non-responders before and 4 weeks after treatment spontaneously secreted TNF-α and IL-17A without stimulation. A similar correlation between therapeutic response and expression of TNF-α or IL-17A was not observed in ustekinumab, an anti-IL-12/IL-23 antibody (not shown). Conclusion CyTOF of patients’ PBMCs before and after starting risankizumab revealed previously unknown molecular signatures, especially augmented TNF-a production in multiple IL-23-activated cells, which predict non-response to risankizumab in CD patients. Those findings identified a novel mechanism of anti-IL-23 resistance, may help identify response biomarkers for IL-23 antagonists and provide a rationale for anti-IL-23/anti-TNF combination therapy in a subset of patients with refractory CD.

Ginkgo biloba extract EGb 761® ameliorates cognitive impairment and alleviates TNFα response in 5xFAD Alzheimer's disease model mice.

Ginkgo biloba leaf extract EGb 761® has shown clinical efficacy in patients with mild cognitive impairment and dementia. However, the pharmacological action of EGb 761® in Alzheimer's disease (AD) remains unclear and molecular mechanisms targeted in the brain are not completely understood. We aimed to investigate 1) the potential sex-dependent effects of oral administration of EGb 761® in 5xFAD mice, an AD mouse model, and 2) the underlying microglial subtype responsible for the observed anti-inflammatory effects in the brain. Eight-week old 5xFAD and wild type mice received EGb 761®-supplemented diet or control diet for eight weeks. The study investigated changes in cognitive function as well as amyloid plaque load, expression of AD-related genes, and anti-inflammatory effects. Moreover, we used organotypic brain slices for confirmation and assessment of concentration-dependency of the observed EGb 761® effects and performed single cell RNA sequencing on the prefrontal cortex of male mice with focus on microglia. We demonstrate that EGb 761® treatment improves cognitive function in 5xFAD mice in several behavioral tests. Analysis of the brain tissue from these animals indicated a reduction in amyloid plaque load in the prefrontal cortex (PFC). This brain area was further investigated to assess the molecular changes that occurred following EGb 761® intake. Alterations in the expression of genes related to AD were highly sex-specific with effects on the cholinergic system, the γ-secretase complex, and neuroinflammation. Anti-inflammatory effects of EGb 761® with a particularly pronounced reduction of the TNFα-response could be shown for the PFC but also peripherally in the serum of 5xFAD mice of both sexes. Single-cell RNA sequencing revealed that EGb761® mainly affected disease-associated microglia stage 2 (DAM2), which are thought to have a detrimental role in AD. EGb 761® shows efficacy in the treatment of cognitive deficits in the 5xFAD mouse model via multimodal activity, including sex-specific and sex-unrelated mechanisms including the normalization of neuroinflammatory parameters.

Nopalea cochenillifera Regulates the Immune Response and Gut Microbiota in Mice.

Cactus contains dietary fiber and minerals and is expected to have preventive effects against diabetes, arteriosclerosis, and other diseases. Additionally, cactus intake induces the production of short-chain fatty acids derived from the gut microbiota, which might influence immune functions. In this study, we examined the effects of a cactus (Nopalea cochenillifera: NC)-supplemented diet on lipopolysaccharide (LPS)-induced immune responses and intestinal barrier function. Male C3H/HeN mice were randomly divided into three groups-no fiber (NF), cellulose-containing fiber (Cellu), and cactus-added (NC) diets-for 6 weeks. The TNF-α and IL-10 responses to LPS, antibody titers, and intestinal barrier function, as well as the fecal microbiota, were analyzed. The plasma TNF-α but not the IL-10 concentrations were significantly higher in the NC group than in the NF and Cellu groups. Furthermore, the plasma IgG antibody titers were significantly higher in the NC group than in the other groups. The NC group showed higher mucin content and IgA antibody titers in their feces compared with the Cellu group. The succinate and lactate contents, which induce a reduction in TNF-α secretion by macrophages, in the cecum of the NC group were significantly lower than those in the Cellu and NF groups. In contrast, the butyrate content was significantly higher in the cecum of the NC group compared to that of the Cellu group, with a significantly higher relative abundance of butyrate-producing bacteria. Taken together, we found that cactus intake regulates innate and adaptive immune function via the gut microbiota in mice. Therefore, cactus supplementation might serve as a strategy to develop novel functional foods with dietary fiber.

Influence of Biological Sex and Congenital Iron Deficiency on Neonatal Cytokine Responses.

Stimulated cord blood mononuclear cell (CBMC) cytokine responses were previously shown to predict the risk of childhood atopic disease. Iron deficiency (ID) at birth may also program atopic disease. Males are at a higher risk of pediatric atopic disease, but it is not known whether congenital ID impacts CBMC immune responses differentially by sex. Cord blood (CB) samples were collected from healthy term or near-term neonates after elective cesarean deliveries. A transferrin saturation ≤ 25% defined congenital ID. CBMCs were stimulated with either phytohemagglutinin (PHA) or PHA plus an iron chelator. Of the 85 neonates, the 26 neonates with congenital ID exhibited lower plasma tumor necrosis factor-α (TNF-α), as well as higher CBMC TNF-α and IL-8 responses than iron-sufficient neonates (p = 0.017, p = 0.013, and p = 0.007, respectively). Higher CBMC TNF-α responses were seen in both males and females with congenital ID. However, females with congenital ID also had lower plasma IL-6, lower plasma TNF-α, and higher CBMC interleukin (IL)-8 responses. Additionally, iron chelation during culture influenced stimulated CBMC IFN-γ and CBMC TNF-α responses. Congenital ID may influence stimulated CBMC cytokine responses, but results point to a sex-specific regulation of immune balance at birth. Because males are more prone to infantile ID and more likely to develop early childhood asthma, future studies should further investigate how fetal sex and congenital iron status impacts childhood immune responsiveness to infections and antigenic stimulation from the rearing environment.

Systemic Cancer Hallmarks as Novel Markers Associated with Progression-Free Survival in Gastroenteropancreatic Neuroendocrine Tumor Patients Undergoing Peptide Receptor Radionuclide Therapy

Introduction: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are a heterogeneous group of tumors often detected at the metastatic stage. The aim of this study was to profile the peripheral blood transcriptome through RNA-Seq and investigate the association of the systemic cancer hallmarks with progression-free survival, in peptide receptor radionuclide therapy (PRRT)-treated GEP-NET patients. Methods: The cohorts were discovery cohort [PRRT-naïve well-differentiated GEP-NETs, n = 59; age- and sex-matched healthy individuals, n = 38], and independent evaluation cohort [GEP-NETs, n = 66]. Peripheral blood transcriptomes were profiled through RNA sequencing and cancer hallmarks were identified via Gene Set Enrichment Analysis (GSEA). Activities of cancer hallmarks in each sample were calculated using Gene Set Variation Analysis. Differentially expressed genes were identified with DESeq2. Progression-free survival was used as a primary endpoint and prognostic association was evaluated using univariate and multivariate Cox proportional hazard (COXPH) analyses. Results: RNA-Seq captured global changes in the peripheral blood transcriptome of GEP-NET patients. Peripheral blood transcriptome of NET patients showed differential enrichment of 30 systemic cancer hallmarks viz., TNF-α signaling via NF-κB, IL2/STAT5 signaling, TNF-α response, TNF-γ response, IL6/JAK/STAT signaling, TGF-β signaling, heme metabolism, etc. In the univariate analyses, two cancer hallmarks were prognostically significant (p < 0.05) in GEP-NETs. Heme metabolism and IL2/STAT5 signaling were statistically significant in the discovery cohort (n = 58) and independent evaluation cohort (n = 66). In multivariate COXPH analyses, heme metabolism and IL2/STAT5 signaling were independently associated with PFS in GEP-NET patients undergoing PRRT. Conclusions: This study provides comprehensive coverage of the peripheral blood transcriptome of GEP-NET patients via RNA-Seq and identifies systemic cancer hallmarks as independent prognostic factors in NETs.

Анализ ответа клеток микроглии мыши линии SIM-A9 на действие γ-излучения

Purpose: Characterization of the response of mouse microglia cells of the SIM-A9 line to the γ-irradiation. Material and methods: Irradiation of the cells in suspension was carried out using a GUT-200M installation (cobalt-60 γ-radiation source). The radiosensitivity of cells was assessed by the number of surviving cells and their clonogenic activity. The effect of γ-radiation on the phenotype and expression of colony-stimulating growth factor receptor-1 and of epidermal growth factor, which are required to stimulate microglial cells proliferation, was studied using flow cytometry after staining the cells with appropriate fluorescently labeled CD11b, CD45, TMEM119, CSF-1R и EGFR antibodies. Analysis of the relative expression of mRNA genes for the cytokines IL-1β, IL-6, and TNFα in response to γ-radiation was performed using RT-PCR. Statistical analysis was carried out using Student’s t-test in the Origin program. Results: High radiosensitivity of SIM-A9 cells has been demonstrated. When analyzing the dependence of the clonogenic activity of cells on the radiation dose, it was shown that the D37 value for these cells was equal to 1 Gy. Irradiation caused a cell cycle block in the G0/G1 phase with a decrease in the proportion of cells in the S– and G2/M-phases. The cell death of irradiated SIM-A9 cells occurred by apoptosis. The peculiarity of SIM-A9 cells compared to brain microglia is their phenotype of activated microglia CD11b+/CD45high with an insignificant content of CD11b+/CD45-/low cells and no change in it after irradiation. An increase in the level of mRNA expression of the proinflammatory cytokine genes IL-1β, IL-6 and TNFα in response to γ-irradiation of SIM-A9 cells was shown, which reflects their activation and corresponds to the response of brain microglia cells during total mice irradiation and local cranial irradiation. Conclusion: The obtained patterns indicate the possibility of using the SIM-A cell line in model radiobiological studies, including the study of intercellular interactions of brain cells of different types with microglia cells.

7820 Immune system adaptation during gender-affirming testosterone treatment

Abstract Disclosure: T. Lakshmikanth: None. C. Consiglio: None. F. Sardh: None. R. Forlin: None. J. Wang: None. Z. Tan: None. H. Barencilla: None. L. Rodriguez: None. J. Sugrue: None. P. Noori: None. M. Ivanchenko: None. L. Piñero Páez: None. L. Gonzalez: None. C. Habimana Mugabo: None. A. Johnsson: None. H. Ryberg: None. Å. Hallgren: None. C. Pou: None. Y. Chen: None. J. Mikeš: None. A. James: None. P.M. Dahlqvist: None. J. Wahlberg Hughes: None. A. Hagelin: None. M. Holmberg: None. M. Degerblad: None. M. Isaksson: None. D. Duffy: None. O. Kampe: None. N. Landegren: None. P. Brodin: None. Immune system adaptation during gender-affirming testosterone treatment Infectious, inflammatory and autoimmune conditions present differently in males and females. Following SARS-CoV-2 infection, male sex is associated with increased risk of death, while female sex is associated with increased risk of the postinfectious disorder, long COVID. Similar trends are seen in other infections. Female immune responses to vaccines are typically stronger, and adverse reactions are more frequent, like most autoimmune diseases. Immunological sex-differences stem from genetic, hormonal and behavioural factors but their relative importance is poorly understood. To understand the consequences of changing sex-hormones in vivo, we performed longitudinal, systems-level analyses in 22 adults, assigned female at birth, and undergoing gender-affirming testosterone treatment (trans-men). We find that sex hormones modulate a cross-regulation axis of type-I interferon (IFN-I) and TNFα in humans. This is mediated by a reduced frequency of plasmacytoid dendritic cells (pDC), and functional attenuation of IFN-I responses in both pDCs and monocytes. Conversely, testosterone potentiates monocyte responses to lipopolysaccharide (LPS) leading to increased TNFα, IL-6 and IL-15 production and downstream epigenetic opening of NFκB regulated genes and potentiation of IFNγ responses, primarily by NK cells. IFNγ in turn feeds back onto monocytes leading to upregulation of SLAMF7 and further enhanced TNFα responses by myeloid cells. These results are corroborated by sex-divergent responses in multiple public datasets and collectively illustrate the dynamic recalibration of human immune systems by sex hormones with implications for individuals undergoing gender-affirming hormone therapy, but also better understanding of divergent responses to infection, vaccines and self-antigens in cisgender individuals. Presentation: 6/2/2024

7820 Immune system adaptation during gender-affirming testosterone treatment

Abstract Disclosure: T. Lakshmikanth: None. C. Consiglio: None. F. Sardh: None. R. Forlin: None. J. Wang: None. Z. Tan: None. H. Barencilla: None. L. Rodriguez: None. J. Sugrue: None. P. Noori: None. M. Ivanchenko: None. L. Piñero Páez: None. L. Gonzalez: None. C. Habimana Mugabo: None. A. Johnsson: None. H. Ryberg: None. Å. Hallgren: None. C. Pou: None. Y. Chen: None. J. Mikeš: None. A. James: None. P.M. Dahlqvist: None. J. Wahlberg Hughes: None. A. Hagelin: None. M. Holmberg: None. M. Degerblad: None. M. Isaksson: None. D. Duffy: None. O. Kampe: None. N. Landegren: None. P. Brodin: None. Infectious, inflammatory and autoimmune conditions present differently in males and females. Following SARS-CoV-2 infection, male sex is associated with increased risk of death, while female sex is associated with increased risk of the postinfectious disorder, long COVID. Similar trends are seen in other infections. Female immune responses to vaccines are typically stronger, and adverse reactions are more frequent, like most autoimmune diseases. Immunological sex-differences stem from genetic, hormonal and behavioural factors but their relative importance is poorly understood. To understand the consequences of changing sex-hormones in vivo, we performed longitudinal, systems-level analyses in 22 adults, assigned female at birth, and undergoing gender-affirming testosterone treatment (trans-men). We find that sex hormones modulate a cross-regulation axis of type-I interferon (IFN-I) and TNFα in humans. This is mediated by a reduced frequency of plasmacytoid dendritic cells (pDC), and functional attenuation of IFN-I responses in both pDCs and monocytes. Conversely, testosterone potentiates monocyte responses to lipopolysaccharide (LPS) leading to increased TNFα, IL-6 and IL-15 production and downstream epigenetic opening of NFκB regulated genes and potentiation of IFNγ responses, primarily by NK cells. IFNγ in turn feeds back onto monocytes leading to upregulation of SLAMF7 and further enhanced TNFα responses by myeloid cells. These results are corroborated by sex-divergent responses in multiple public datasets and collectively illustrate the dynamic recalibration of human immune systems by sex hormones with implications for individuals undergoing gender-affirming hormone therapy, but also better understanding of divergent responses to infection, vaccines and self-antigens in cisgender individuals. Presentation: 6/2/2024

Levels of Tumor Necrosis Factor Alfa and Iron Status in Pediatric Patients with Newly Diagnosed Solid Tumors

Abstract Background Tumor necrosis factor alfa (TNF-α) plays a crucial role in the etiopathogenesis of cancer related anemia. Aim: To assess the levels of TNF-α, erythropoietin (EPO), and iron status in relation to anemia in children with newly diagnosed solid tumors. Methods we measured serum levels of TNF-α, EPO and assessed iron status using enzyme linked immunosorbent assay in 42 patients (20 non-anemia and 22 anemic) with newly diagnosed solid malignancies and compared them to 25 age- and sex-matched healthy controls. Results Among the 42 patients enrolled, 14 (33.3%) patients had brain tumors, seven (16.6%) were diagnosed with neuroblastoma, and four (9.5%) with Wilms tumor. The mean hemoglobin levels were 12.39±0.8 g/dl and 9.57±1.3 g/dl in non-anemic and anemic patients respectively. We found significantly higher levels of serum TNF-α and lower EPO levels among patients when compared to the control group (p &amp;lt; 0.001). A post hoc analysis showed a significantly higher TNF-α and lower EPO levels among anemic versus controls (p &amp;lt; 0.001) and non-anemic versus control (p &amp;lt; 0.001). A significant positive correlation was found between both EPO and TNF-α in both anemic and non-anemic group (r = 0.48, p = 0.024) and (r = 0.44, p = 0.021) respectively. However, the median (IQR) levels of both TNF- α and EPO were comparable between both anemic and non-anemic patients (306 (237.5) pg/ml versus 353 (156.2) pg/ml, p = 0.47) and (93 (46) mIU/ml versus 87 (51) mIU/ml, p = 0.48) respectively. The median (IQR) levels of serum ferritin and transferrin saturation index (TSI), as well as; serum TNF-α did not correlate with any of the red cell indices yet; directly correlated with platelet counts (r = 0.491, p = 0.02). Conclusion Children with solid malignancies have significantly higher levels of TNF-α and impaired EPO responses than normal children, however, no significant effect was observed for either on hemoglobin and red cell indices. Whether the positive relationship between TNF- α and EPO suggest other mechanisms of TNF- α related anemia needs further studies.

Relevance of antigen-induced IL-6 and mitogen-induced or spontaneous IFN-γ secretions in whole blood cultures for detection of Mycobacterium tuberculosis infection and disease.

For an effective control of tuberculosis (TB), there is a persistent need for biomarkers that can report true estimates of TB infection (TBI) and predict its progression towards active TB disease. We investigated whether the cell-mediated immune responses to Mycobacterium tuberculosis (Mtb) antigens could provide such biomarkers. The study subjects (n = 174) comprised a cohort of smear-positive, drug-sensitive, HIV-negative pulmonary TB patients (n = 54) and their household contacts (HC, n = 120). Whole blood cultures, in the presence or absence of Mtb antigens- membrane (MtM), purified protein derivative (PPD) and alpha-crystallin (Acr), or the mitogen PHA were subjected to determinations, by flow cytometry, for T cell proliferative and, by ELISA, for IFN-γ, TNF-α, and IL-6 cytokine responses. Additionally, serum levels of the three cytokines were also estimated. The strongest cell-proliferative and cytokine responses were induced by MtM and IL-6 was the most abundantly produced cytokine. While none of the responses induced by Mtb antigens or the serum cytokines levels could discriminate between TB and HC, the exvivo cytokine responses induced by PHA or 'spontaneously' could apparently do so. The concentrations of IFN-γ induced by PHA in TB blood cultures were significantly lower than in HC cultures (AUC = 0.72). Conversely, the spontaneous IFN-γ or TNF-α secretions in TB cultures were significantly higher than in HC cultures (AUC = 0.66). Our results suggest that IL-6 responses to MtM could be a sensitive indicator of TBI, and low levels of PHA-induced or high levels of spontaneous IFN-γ secretions in HC blood cultures may indicate a progressive infection.

Prenatal blood metals, per- and polyfluoroalkyl substances and antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion

BackgroundEvidence suggests that prenatal per- and polyfluoroalkyl substances (PFAS) and metals, two classes of chemicals found ubiquitously in human populations, influence immune system development and response. ObjectiveWe evaluated whether first trimester blood PFAS and metals were associated with antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion. MethodsWe measured six PFAS, as well as six nonessential and four essential metals, in first trimester blood from participants in the longitudinal pre-birth Project Viva cohort, recruited between 1999 and 2000 in eastern Massachusetts. We measured antigen- or mitogen-stimulated cord blood mononuclear cell proliferation responses (n = 269–314) and cytokine secretion (n = 217–302). We used covariate-adjusted least absolute shrinkage and selection operator (LASSO) for variable selection and multivariable regression to estimate associations with the immune markers. ResultsEach ng/mL of MeFOSAA was associated with a 3.6% (1.4, 5.8) higher lymphocyte proliferation response after stimulation with egg antigen, as well as 0.8 (0.7, 1.0) reduced odds of having IFN-γ detected in response to dust mite. Each ng/g increment of cesium was associated with 27.8% (−45.1, −4.9) lower IL-10 levels in response to dust mite. Each ng/g increment of mercury was associated with 12.0% (1.3, 23.8) higher IL-13 levels in response to mitogen PHA. Each ng/g increment of selenium and zinc was associated with 0.2% (0.01, 0.4) and 0.01% (0.002, 0.02) higher TNF-α in response to mitogen PHA, respectively. ConclusionsPrenatal metals and PFAS influence cord blood lymphocyte proliferation and cytokine secretion in ways that may increase risk for atopic disease in childhood.

The relationship between TNF-α, IL-1, IL-12, IL-17, IL-23, IL-36 expression and treatment response in psoriasis histopathologically and immunohistochemically

Aim There is no marker that can predict whether there is resistance to treatment in patients with psoriasis. In this study, we investigated the relationship between the staining rates of TNF-α, IL-1, IL-12, IL-17, IL-23, and IL-36 markers immunohistochemically from cutaneous biopsy and the treatment success. Methods The patients who were followed up in the dermatology clinic with the diagnosis of plaque-type psoriasis vulgaris and received biological treatment and previously had cutaneous biopsy were included in the study. The cutaneous biopsies of the cases that met the conditions were re-sectioned and subjected to immunohistochemical examination for TNF-α, IL-1, IL-12, IL-17, IL-23, and IL-36. Results Comparing the staining scores with psoriasis area severity index (PASI); A statistically significant positive correlation was found between PASI and TNF-α staining score (p = 0.034). A statistically significant positive correlation was found between PASI and IL-17 staining score (p = 0.004). When the staining scores and PASI response rates of psoriasis treatment were evaluated in terms of correlation; there was a positive correlation between TNF-α, IL-17, and IL-23 immunohistochemical staining rates and PASI response rates. Conclusions In line with the data obtained from our study, we think that making immunohistochemical scoring before the biological treatment decision in psoriasis patients will be beneficial in treatment selection. In this respect, our study may open a new era in the selection of biological treatments for psoriasis.

Respiratory SARS-CoV-2 Infection Causes Skeletal Muscle Atrophy and Long-Lasting Energy Metabolism Suppression.

Muscle fatigue represents the most prevalent symptom of long-term COVID, with elusive pathogenic mechanisms. We performed a longitudinal study to characterize histopathological and transcriptional changes in skeletal muscle in a hamster model of respiratory SARS-CoV-2 infection and compared them with influenza A virus (IAV) and mock infections. Histopathological and bulk RNA sequencing analyses of leg muscles derived from infected animals at days 3, 30, and 60 post-infection showed no direct viral invasion but myofiber atrophy in the SARS-CoV-2 group, which was accompanied by persistent downregulation of the genes related to myofibers, ribosomal proteins, fatty acid β-oxidation, tricarboxylic acid cycle, and mitochondrial oxidative phosphorylation complexes. While both SARS-CoV-2 and IAV infections induced acute and transient type I and II interferon responses in muscle, only the SARS-CoV-2 infection upregulated TNF-α/NF-κB but not IL-6 signaling in muscle. Treatment of C2C12 myotubes, a skeletal muscle cell line, with combined IFN-γ and TNF-α but not with IFN-γ or TNF-α alone markedly impaired mitochondrial function. We conclude that a respiratory SARS-CoV-2 infection can cause myofiber atrophy and persistent energy metabolism suppression without direct viral invasion. The effects may be induced by the combined systemic interferon and TNF-α responses at the acute phase and may contribute to post-COVID-19 persistent muscle fatigue.

In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-β and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Caspase-8 activity mediates TNFα production and restricts Coxiella burnetii replication during murine macrophage infection.

Coxiella burnetii is an obligate intracellular bacteria that causes the global zoonotic disease Q Fever. Treatment options for chronic infection are limited, and the development of novel therapeutic strategies requires a greater understanding of how C. burnetii interacts with immune signaling. Cell death responses are known to be manipulated by C. burnetii, but the role of caspase-8, a central regulator of multiple cell death pathways, has not been investigated. In this research, we studied bacterial manipulation of caspase-8 signaling and the significance of caspase-8 to C. burnetii infection, examining bacterial replication, cell death induction, and cytokine signaling. We measured caspase, RIPK, and MLKL activation in C. burnetii-infected tumor necrosis factor alpha (TNFα)/cycloheximide-treated THP-1 macrophage-like cells and TNFα/ZVAD-treated L929 cells to assess apoptosis and necroptosis signaling. Additionally, we measured C. burnetii replication, cell death, and TNFα induction over 12 days in RIPK1-kinase-dead, RIPK3-kinase-dead, or RIPK3-kinase-dead-caspase-8-/- bone marrow-derived macrophages (BMDMs) to understand the significance of caspase-8 and RIPK1/3 during infection. We found that caspase-8 is inhibited by C. burnetii, coinciding with inhibition of apoptosis and increased susceptibility to necroptosis. Furthermore, C. burnetii replication was increased in BMDMs lacking caspase-8, but not in those lacking RIPK1/3 kinase activity, corresponding with decreased TNFα production and reduced cell death. As TNFα is associated with the control of C. burnetii, this lack of a TNFα response may allow for the unchecked bacterial growth we saw in caspase-8-/- BMDMs. This research identifies and explores caspase-8 as a key regulator of C. burnetii infection, opening novel therapeutic doors.

Transcriptomic signatures of classical monocytes reveal pro-inflammatory modules and heterogeneity in polyarticular juvenile idiopathic arthritis.

Polyarticular juvenile idiopathic arthritis (pJIA) is a childhood-onset autoimmune disease. Immune cells contribute to persistent inflammation observed in pJIA. Despite the crucial role of monocytes in arthritis, the precise involvement of classical monocytes in the pathogenesis of pJIA remains uncertain. Here, we aimed to uncover the transcriptomic patterns of classical monocytes in pJIA, focusing on their involvement in disease mechanism and heterogeneity. A total of 17 healthy subjects and 18 premenopausal women with pJIA according to ILAR criteria were included. Classical monocytes were isolated, andRNA sequencing was performed. Differential expression analysis was used to compare pJIA patients and healthy control group. Differentially expressed genes (DEGs) were identified, and gene set enrichment analysis (GSEA) was performed.Using unsupervised learning approach, patients were clustered in two groups based on their similarities at transcriptomic level. Subsequently, these clusters underwent a comparative analysis to reveal differences at the transcriptomic level. We identified 440 DEGs in pJIA patients of which 360 were upregulated and 80 downregulated. GSEA highlighted TNF-α and IFN-γ response. Importantly, this analysis not only detected genes targeted by pJIA therapy butalso identified new modulators of immuno-inflammation. PLAUR, IL1B, IL6, CDKN1A, PIM1, and ICAM1 were pointed as drivers of chronic hyperinflammation. Unsupervised learning approach revealed two clusters within pJIA, each exhibiting varying inflammation levels. These findings indicate the pivotal role of immuno-inflammation driven by classical monocytes in pJIA and reveals the existence of two subclusters within pJIA, regardless the positivity of rheumatoid factor and anti-CCP, paving the way to precision medicine.

Selection of viral variants with enhanced transmission and reduced neutralization susceptibility alongside lateral introductions may explain the persistence of porcine reproductive and respiratory syndrome virus in vaccinated breeding herds.

This study investigates the long-term evolutionary dynamics of porcine reproductive and respiratory syndrome virus (PRRSV-1) in an endemically infected and vaccinated pig herd. Over a one year and a half period, piglets from seven farrowing batches in a 300-sow PRRSV-vaccinated farm were monitored from birth to nine weeks of age by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Eighty-five PRRSV-positive samples were subjected to whole genome sequencing (Illumina Miseq), and 251 samples to open reading frame 5 (ORF5) sequencing. Farm-specific PRRSV variants' impact on anti-PRRSV antibodies was evaluated using enzyme-linked immunosorbent and neutralizing antibody assays. The replication kinetics and cytokine inhibition capabilities (IFN-α and TNF-α) of these variants were assessed in porcine alveolar macrophages. The study revealed fluctuating PRRSV-1 incidences in farrowing units and nurseries, attributed to two key evolutionary events: an escape variant emergence and a lateral introduction of a new strain. Initially, strain 1 variant α was swiftly replaced within weeks by variant 1β (99.5 per cent genomic similarity), with twenty-five amino acid mutations, primarily in nsp1α, GP2, GP3, and GP5, including an additional glycosylation site and a deletion downstream the neutralization epitope of GP5. This shift to 1β correlated with increased incidence in nurseries and higher viral loads, with sera from 1α-exposed animals showing reduced neutralization against 1β. Consistently for in vitro assays, variant 1β demonstrated enhanced replication in porcine alveolar macrophages but no difference regarding IFN-α or TNF-α responses. Later, a new strain (strain 2, 83.3 per cent similarity to strain 1) emerged and led to incidence resurgence because of the low cross reactivity with the previous antibodies. The study highlights PRRSV's rapid adaptability and challenges in controlling its spread, underscoring the necessity for more effective vaccines and eradication approaches.

Cytokine Response to Resistance Exercise in Children with Excess Fat and Prader-Willi Syndrome

Background: Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine that is secreted from adipose tissue and macrophages; interleukin-6 (IL-6), secreted from adipose tissue, macrophages and skeletal muscle, has both proinflammatory and anti-inflammatory properties. Previous research showed excess adiposity leads to an increased concentration of both cytokines at rest while there is limited research on resistance exercise. Purpose: To compare IL-6 and TNF-α responses to resistance exercise in children with Prader-Willi Syndrome (PWS), a genetic cause for obesity, vs. children with normal fat mass (lean), or excess body fat (obesity) Methods: Nine children with PWS (11.4 ± 3.3 years, 45.6 ± 5.2% body fat), 11 lean children (9.2 ± 1.4 years, 18.6 ± 5.0% bodyfat), and 12 children with obesity (9.6 ± 1.3 years, 40.4 ± 5.4% body fat) participated. Children stepped onto an elevated platform wearing a weighted vest for 6 sets of 10 repetitions per leg (sets separated by 1 min of rest). Children with PWS, stepped to a platform height of 23.0 cm and wore a vest load that was computed as (20% of stature* 50% of lean body mass)/23.0 cm. For the controls, the platform height was 20% of the stature and the vest load was 50% of the lean body mass. Blood samples were obtained before (pre), immediately after (post), and during recovery from exercise (+15 and +60 min). Areas under the curve (AUC) were computed for IL-6 and TNF-α. Results: There was no group by time interaction for IL-6 ( p=0.378) but a group effect ( p=0.011) and a time effect ( p&lt;0.001). Pairwise comparisons showed 60+ (1.065 ± 0.10 pg/mL) was higher than pre (0.660 ± 0.05 pg/mL), post (0.728 ± 0.06 pg/mL), and +15 (0.764 ± 0.066 pg/mL), p&lt;0.001 for all. Children with PWS and children with obesity had higher IL-6 concentrations than lean children (0.943 ± 0.12 pg/mL and 0.951 ± 0.10 pg/mL vs. 0.519 ± 0.11 pg/mL, p≤0.038 for both). For TNF-α, there were no group by time interaction, time or group effects ( p≥0.420 for all). IL-6 AUC ( p=0.013) but not TNF-α AUC ( p=0.404) were different between groups. Children with PWS and children with obesity showed a higher IL-6 AUC than lean children (85.106 ± 41.81 pg/mL, 86.796 ± 33.60 pg/mL vs. 47.611 ± 21.00 pg/mL, p≤ 0.041 for both). Conclusion: There were no differences in IL-6 or TNF-α responses to exercise based on having PWS or having excess body fat in children. However, excess body fat was associated with higher IL-6 concentration and AUC. Peak concentrations for IL-6 were observed at 60 minutes after exercise similar to other studies. Potentially, IL-6 concentrations immediately post-exercise were not elevated because the exercise protocol was not of high intensity or long enough. Study funded by the US Army Medical Research and Material Command Contract W81XWH-08-1-0025 (DAR). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

OA01 Cluster medicine: the role of genetics for identifying shared biological pathways between rheumatoid arthritis and common comorbidities for targeting treatments

Abstract Background/Aims Rheumatoid arthritis (RA) is a chronic inflammatory disorder associated with significant pain and disability, as well as increased risk of comorbidities. These comorbidities, such as coronary artery disease, stroke, heart failure and type 2 diabetes, often form predictable clusters highlighting the importance of common disease mechanisms. Identifying these clusters, and the underlying shared mechanisms, is key to the concept of cluster medicine which aims to take a more systemic approach to treating comorbidity. It is hypothesised that inflammatory processes in RA promote the pathogenesis of these comorbid diseases, which typically occur at an earlier stage of life than in the general population. However, few studies have investigated the degree of shared genetic susceptibility implicated across these traits. The present study aimed to identify and characterise pleiotropic loci and shared biological pathways involved in the pathogenesis of RA and cardiometabolic disorders. Methods European-only GWAS summary statistics were collected for RA (seropositive, seronegative, and overall) and seven cardiometabolic traits, including ischemic stroke, any stroke, heart failure, coronary artery disease, hypertension, atrial fibrillation, and type 2 diabetes. Cross-trait linkage disequilibrium score regression was employed to estimate the degree of genetic correlation (rg) between all pairs of traits. Subset-based meta-analysis was performed using the R package ASSET to identify pleiotropic single nucleotide polymorphisms (SNPs). Pleiotropic SNPs were assigned to genes based on positional and eQTL mapping. Prioritised genes were tested for over-representation in functional categories and biological pathways in gene sets obtained from MsigDB and WikiPathways using the hypergeometric test loci. Gene mapping and enrichment analyses were conducted in FUMA. Results RA showed significant positive genetic correlation with all cardiometabolic traits, with the seronegative subtype showing greater correlation than the seropositive subtype. Correlations between RA and cardiometabolic phenotypes ranged from 0.11 - 0.38, the strongest of which was between seronegative RA and heart failure (rg = 0.38, P = 2.6e-07). A total of 67 lead SNPs were identified as potentially pleiotropic. Pleiotropic SNPs were mapped to genes including IL6R, PTPN11, ATXN2, PLCL1 and SOX6. Pathway enrichment analyses revealed that mapped genes were primarily involved in inflammatory signalling pathways, involving interleukins, TNF-α and interferon (gamma and alpha) response. Conclusion This study provides evidence for pleiotropy across RA and cardiometabolic traits, whereby inherited genetic variants predispose to aberrant cytokine signalling, thus conferring risk to multiple diseases where inflammation is a key driver of pathogenesis. Disclosure M. Stoves: None. M. Soomro: None. S. Zhao: None. D. Plant: None. A. Barton: None. J. Bowes: None.

Determination of Early Diagnostic Biomarkers of Renal Dysfunction After Cardiopulmonary Bypass: miR-21 and miR10a Mediated Postoperative Inflammation

Objective: Acute renal failure (ARF) prevalence is high among patients who undergo cardiopulmonary bypass (CPB), and this condition can only be diagnosed via serum creatinine level (sCr) conventionally within 48 hours. Therefore, we need early novel diagnosis biomarkers to start preventive treatment of ARF. For that reason, we aimed to analyze if plasma miR-21 derived from heart, correlates with kidney- enriched miR-10a during inflammatory IL-6, IL-1β, and TNF-α response in terms of acute renal failure 30 minutes after CPB.&#x0D; Methods: Patients (n=46, Female:8 and Male:38), aged 61.08±9.41, who underwent CPB surgery were included. Blood samples were collected during the pre – and post-CPB (30 minutes after CPB). Demographic data of all cases were collected. Quantification of expression levels of miR-21 and miR-10a was done via quantitative PCR (qPCR). Determination of plasma concentration of relevant cytokines, IL-6, IL-1β, and TNF-α was done via ELISA.&#x0D; Results: The circulating level of miR-21 during post-CPB period (-11.78±6.98) was significantly higher (p≤0.05) than pre-CPB period (-6.55±7.11), but there was no significant change (p&gt;0.05) in the circulating level of miR-10a between pre – (-12.22±3.55) and post-CPB (-11.60±3.36) periods. When we compared the mean ΔΔCt values of miR-21 and miR-10a, downregulation was observed in the expression level of miR-10a (0.62±3.77) whilst the expression level of miR-21 (-5.22±7.25) was upregulated (p≤0.05). The levels of plasma concentration of IL-6 (2.74±2.50 ng/l) and TNF-α (83.63±9.33 ng/l) were increased during post-CPB period (both were ***p