Elevated Levels Of TNF Research Articles

RIP1 inhibition protects retinal ganglion cells in glaucoma models of ocular injury

AbstractReceptor-interacting protein 1 (RIP1, RIPK1) is a critical mediator of multiple signaling pathways that promote inflammatory responses and cell death. The kinase activity of RIP1 contributes to the pathogenesis of a number of inflammatory and neurodegenerative diseases. However, the role of RIP1 in retinopathies remains unclear. This study demonstrates that RIP1 inhibition protects retinal ganglion cells (RGCs) in preclinical glaucoma models. Genetic inactivation of RIP1 improves RGC survival and preserves retinal function in the preclinical glaucoma models of optic nerve crush (ONC) and ischemia–reperfusion injury (IRI). In addition, the involvement of necroptosis in ONC and IRI glaucoma models was examined by utilizing RIP1 kinase-dead (RIP1-KD), RIP3 knockout (RIP3-KO), and MLKL knockout (MLKL-KO) mice. The number of RGCs, retinal thickness, and visual acuity were rescued in RIP1-kinase-dead (RIP1-KD) mice in both models, while wild-type (WT) mice experienced significant retinal thinning, RGC loss, and vision impairment. RIP3-KO and MLKL-KO mice showed moderate protective effects in the IRI model and limited in the ONC model. Furthermore, we confirmed that a glaucoma causative mutation in optineurin, OPTN-E50K, sensitizes cells to RIP1-mediated inflammatory cell death. RIP1 inhibition reduces RGC death and axonal degeneration following IRI in mice expressing OPTN-WT and OPTN-E50K variant mice. We demonstrate that RIP1 inactivation suppressed microglial infiltration in the RGC layer following glaucomatous damage. Finally, this study highlights that human glaucomatous retinas exhibit elevated levels of TNF and RIP3 mRNA and microglia infiltration, thus demonstrating the role of neuroinflammation in glaucoma pathogenesis. Altogether, these data indicate that RIP1 plays an important role in modulating neuroinflammation and that inhibiting RIP1 activity may provide a neuroprotective therapy for glaucoma.

The roles of genetic mutation and cytokines/chemokines in immune response and their association with uveal melanoma patient outcome

The impact of tumor mutations and the interplay of cytokines and chemokines on the immune response and clinical outcomes in uveal melanoma (UM) warrants further exploration. In our study, we delved into the correlation between genetic alterations and survival rates in a cohort of 188 UM patients, utilizing data from cBioPortal. We assessed the composition of immune cell populations within 80 UM tumors by examining RNA sequence-based gene expression data from The Cancer Genome Atlas (TCGA). Furthermore, we scrutinized the relationship between genetic mutations and the expression of cytokines and chemokines, as well as their influence on various immune cell subsets. Our investigation revealed a significant association between the presence of mutated GNAQ or SF3B1 genes and improved progression-free survival (PFS), disease-specific survival (DSS), and overall survival (OS) when compared to patients with non-mutated counterparts. In contrast, the presence of immune response gene mutations was associated with a detrimental effect on PFS, DSS, and OS. We also observed that the expression levels of cytokines and chemokines were positively linked to the infiltration of immune killer cells and inversely related to the populations of B cells and dendritic cells. Elevated expression levels of PDCD1, TNF, IL6, CXCL9, and CXCL10 were found to be correlated with reduced OS. Intriguingly, an increase in CD8+ T cell populations was associated with a poorer OS, a finding that warrants further investigation. These findings underscore the potential utility of cytokines/chemokines expression levels, immune cell subsets, and mutation status as critical biomarkers for the selection of patients who are most likely to benefit from immunotherapeutic interventions. Our research provides valuable insights that could guide the development of more targeted and effective treatment strategies for UM patients.

Diagnostic markers of acute encephalitis syndrome and COVID-associated multisystem inflammatory syndrome in children from Southern India.

Acute encephalitis syndrome (AES) in children poses a significant public health challenge in India. This study aims to explore the utility of host inflammatory mediators and neurofilament (NfL) levels in distinguishing etiologies, assessing disease severity, and predicting outcomes in AES. We assessed 12 mediators in serum (n = 58) and 11 in cerebrospinal fluid (CSF) (n = 42) from 62 children with AES due to scrub typhus, viral etiologies, and COVID-associated multisystem inflammatory syndrome (MIS-C) in Southern India. Additionally, NfL levels in serum (n = 20) and CSF (n = 18) were examined. Clinical data, including Glasgow coma scale (GCS) and Liverpool outcome scores, were recorded. Examining serum and CSF markers in the three AES etiology groups revealed notable distinctions, with scrub typhus differing significantly from viral and MIS-C causes. Viral causes had elevated serum CCL11 and CCL2 compared with scrub typhus, while MIS-C cases showed higher HGF levels than scrub typhus. However, CSF analysis showed a distinct pattern with the scrub typhus group exhibiting elevated levels of IL-1RA, IL-1β, and TNF compared with MIS-C, and lower CCL2 levels compared with the viral group. Modeling the characteristic features, we identified that age ≥3 years with serum CCL11 < 180 pg/mL effectively distinguished scrub typhus from other AES causes. Elevated serum CCL11, HGF, and IL-6:IL-10 ratio were associated with poor outcomes (p = 0.038, 0.005, 0.02). Positive CSF and serum NfL correlation, and negative GCS and serum NfL correlation were observed. Median NfL levels were higher in children with abnormal admission GCS and poor outcomes. Measuring immune mediators and brain injury markers in AES provides valuable diagnostic insights, with the potential to facilitate rapid diagnosis and prognosis. The correlation between CSF and serum NfL, along with distinctive serum cytokine profiles across various etiologies, indicates the adequacy of blood samples alone for assessment and monitoring. The association of elevated levels of CCL11, HGF, and an increased IL-6:IL-10 ratio with adverse outcomes suggests promising avenues for therapeutic exploration, warranting further investigation.

The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

An unwelcome third party: the role of inflammation in the relationship between depression and Peyronie's disease.

The article "The Association Between Peyronie's Disease and Depression: A Scoping Review" explores the intricate relationship between Peyronie's disease (PD) and depression. Authored by Rune Kraglund, Emil Durukan, Annamaria Giraldi, and Mikkel Fode, the scoping review delves into shared risk factors such as obesity, smoking, and diabetes, potentially justifying the higher prevalence of depression in patients with PD. Interestingly, data suggest an increased risk of depression even before PD symptoms manifest, hinting at a complex interplay. The review emphasizes the relevance of systemic inflammation in the pathophysiology of both disorders. While bidirectional links between depression and inflammation are established in literature, PD's association with systemic inflammation adds a layer of complexity. Conditions such as cancer, linked to systemic inflammation, correlate with PD and depression, highlighting common pathways. Examining specific inflammatory markers, the article notes elevated levels of TNF, IL-6, platelet-derived growth factor, TNF-α, and fibroblast growth factor in PD and depressive disorders. The discussion extends to the scoping review's significance, emphasizing the often-overlooked correlation between psychiatric and sexual health disorders. Understanding the psychological aspects of PD is deemed crucial, considering that depressive conditions may affect treatment adherence and outcomes. The article acknowledges the challenge of establishing a causal link between PD's psychological impact and depression. However, it suggests a potential intertwined relationship where PD may lead to depression due to its impact on sexual function and self-esteem, while depression, in turn, enhances inflammatory responses and aggravates PD. In conclusion, the article proposes that the involvement of inflammation complicates the relationship between PD and depression. This complex interplay, though not fully elucidated in the current literature, provides a compelling theme for future studies, offering insights into the shared pathophysiology and potential paths for improved understanding and treatment of these interconnected disorders.

The Pharmacological Mechanisms Underlying the Protective Effect of Ginsenoside Rg3 against Heart Failure.

Heart failure represents the terminal stage of various cardiovascular diseases. This study aims to explore the pharmacological mechanisms underlying the protective effect of Ginsenoside Rg3 against heart failure. Potential targets of Ginsenoside Rg3 were identified using SwissTargetPrediction and the Comparative Toxicogenomics Database, while heart failure-related genes were retrieved from the Comparative Toxicogenomics Database, Therapeutic Target Database, DisGeNET, and PharmGKB. Overlapping of Ginsenoside Rg3 targets with heart failure-related genes identified drug-disease interaction genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted on the drug-disease interaction genes to elucidate their biological functions. A protein-protein interaction network was constructed using the drug-disease interaction genes, and the hub genes were identified by topological analysis. Additionally, we validate the expression of IL-6 and TNF by real-time PCR. The intersection of Ginsenoside Rg3 targets and heart failure-related genes yielded 15 drug-disease interaction genes. Enrichment analysis highlighted the involvement of inflammation-related GO terms and KEGG pathways, such as positive regulation of interleukin-8 and -6 production, regulation of immune effector process, cytokine receptor binding, cytokine activity, adipocytokine signaling pathway, and IL-17 signaling pathway, which are implicated in the cardioprotective effect. Topological analysis revealed four hub genes: STAT3, CASP3, TNF, and IL-6. The application of Ginsenoside Rg3 significantly reversed the elevated levels of IL-6 and TNF in the isoproterenol-treated H9c2 cell line. Our findings suggest that the cardioprotective effect of Ginsenoside Rg3 may be mediated through its anti-inflammation properties. Further research is required to elucidate and validate the detailed cardioprotective mechanisms of Ginsenoside Rg3.

Clinicopathological and ultrastructural study of African swine fever outbreak in North-East India

The present investigation focuses on examining the clinical, histopathological, and ultrastructural changes that occurred in pig, during an outbreak of African swine fever (ASF) in 2022 in Assam, India. The disease initially manifested as a per-acute case with high mortality but without any evident clinical signs. Subsequently, some animals exhibited an acute form of the disease characterized by high fever (104–106 °F), anorexia, vomiting, respiratory distress, and bleeding from the anal and nasal orifices. During acute African swine fever virus (ASFV) infections, elevated levels of pro-inflammatory IL-1α, IL-1β, IL-6, TNF, CCL2, CCL5, and CXCL10 were detected in the palatine tonsil, lymph nodes, spleen, and kidney using qPCR assay. These molecular changes were associated with haemorrhages, edemas, and lymphoid depletion. Postmortem examinations revealed prominent features such as splenomegaly with haemorrhages, haemorrhagic lymphadenitis, severe petechial haemorrhage in the kidney, pneumonia in the lungs, and necrotic palatine tonsil. Histopathological analysis demonstrated lymphocyte depletion in lymphoid organs, multi-organ haemorrhages, and interstitial pneumonia in the lungs. Scanning electron microscopy (SEM) further confirmed lymphocyte depletion in lymphoid organs through lymphocyte apoptosis and kidney damage with distorted tubules due to red blood cell destruction. Transmission electron microscopy reaffirmed lymphocyte apoptosis by observing chromatin condensation and nucleus margination in lymphocytes of lymphoid organs. These findings provide comprehensive insights into the clinical, histopathological, and ultrastructural aspects of ASF outbreak in pigs. Understanding the pathological changes associated with ASF can contribute to improved diagnosis, prevention, and control measures for this highly contagious and economically devastating viral disease.

Repetitive mild traumatic brain injury-induced neurodegeneration and inflammation is attenuated by acetyl-L-carnitine in a preclinical model.

Repetitive mild traumatic brain injuries (rmTBI) may contribute to the development of neurodegenerative diseases through secondary injury pathways. Acetyl-L-carnitine (ALC) shows neuroprotection through anti-inflammatory effects and via regulation of neuronal synaptic plasticity by counteracting post-trauma excitotoxicity. This study aimed to investigate mechanisms implicated in the etiology of neurodegeneration in rmTBI mice treated with ALC. Adult male C57BL/6J mice were allocated to sham, rmTBI or ALC + rmTBI groups. 15 rmTBIs were administered across 23 days using a modified weight drop model. Neurological testing and spatial learning and memory assessments via the Morris Water Maze (MWM) were undertaken at 48h and 3months. RT-PCR analysis of the cortex and hippocampus was undertaken for MAPT, GFAP, AIF1, GRIA, CCL11, TDP43, and TNF genes. Gene expression in the cortex showed elevated mRNA levels of MAPT, TNF, and GFAP in the rmTBI group that were reduced by ALC treatment. In the hippocampus, mRNA expression was elevated for GRIA1 in the rmTBI group but not the ALC + rmTBI treatment group. ALC treatment showed protective effects against the deficits displayed in neurological testing and MWM assessment observed in the rmTBI group. While brain structures display differential vulnerability to insult as evidenced by location specific postimpact disruption of key genes, this study shows correlative mRNA neurodegeneration and functional impairment that was ameliorated by ALC treatment in several key genes. ALC may mitigate damage inflicted in the various secondary neurodegenerative cascades and contribute to functional protection following rmTBI.

Diet prevents the expansion of segmented filamentous bacteria and ileo-colonic inflammation in a model of Crohn’s disease

BackgroundCrohn’s disease (CD) is associated with changes in the microbiota, and murine models of CD-like ileo-colonic inflammation depend on the presence of microbial triggers. Increased abundance of unknown Clostridiales and the microscopic detection of filamentous structures close to the epithelium of TnfΔARE mice, a mouse model of CD-like ileitis pointed towards segmented filamentous bacteria (SFB), a commensal mucosal adherent bacterium involved in ileal inflammation.ResultsWe show that the abundance of SFB strongly correlates with the severity of CD-like ileal inflammation in two mouse models of ileal inflammation, including TnfΔARE and SAMP/Yit mice. SFB mono-colonization of germ-free TnfΔARE mice confirmed the causal link and resulted in severe ileo-colonic inflammation, characterized by elevated tissue levels of Tnf and Il-17A, neutrophil infiltration and loss of Paneth and goblet cell function. Co-colonization of SFB in human-microbiota associated TnfΔARE mice confirmed that SFB presence is indispensable for disease development. Screening of 468 ileal and colonic mucosal biopsies from adult and pediatric IBD patients, using previously published and newly designed human SFB-specific primer sets, showed no presence of SFB in human tissue samples, suggesting a species-specific functionality of the pathobiont. Simulating the human relevant therapeutic effect of exclusive enteral nutrition (EEN), EEN-like purified diet antagonized SFB colonization and prevented disease development in TnfΔARE mice, providing functional evidence for the protective mechanism of diet in modulating microbiota-dependent inflammation in IBD.ConclusionsWe identified a novel pathogenic role of SFB in driving severe CD-like ileo-colonic inflammation characterized by loss of Paneth and goblet cell functions in TnfΔARE mice. A purified diet antagonized SFB colonization and prevented disease development in TnfΔARE mice in contrast to a fiber-containing chow diet, clearly demonstrating the important role of diet in modulating a novel IBD-relevant pathobiont and supporting a direct link between diet and microbial communities in mediating protective functions.8ntX7JCRAmgBnFgN24WWkMVideo

Ureaplasma urealyticum and Mycoplasma hominis urogenital infections associate with semen inflammation and decreased sperm quality.

Ureaplasma urealyticum and Mycoplasma hominis are among the most prevalent sexually transmitted infections proposed to induce urogenital inflammation and impair sperm quality. However, the topic remains controversial since contradictory findings have been reported. Herein, we performed a comprehensive analysis of U. urealyticum and M. hominis urogenital infections and their association with urogenital inflammation (i.e., leukocyte subsets and inflammatory cytokines in semen,) and sperm quality parameters in a cohort of men with couple's primary infertility undergoing initial infertility evaluation or with lower urinary tract symptoms and no infertility-related complaints. Overall, U. urealyticum and M. hominis infection was detected in 17.0% and 23.6% of patients, respectively, whereas the coinfection was detected in 3.8% of patients only. Remarkably, similar infection frequencies were found in the different patient subpopulations analyzed. Moreover, infections were associated with elevated semen levels of TNF, IL-1β, and IL-6 and/or increased counts of total leukocytes and their subsets, including CD4 and CD8 T lymphocytes and neutrophils. In addition, M. hominis infection and the coinfection with U. urealyticum were associated with impairments in sperm quality variables. Our results indicate that U. urealyticum and M. hominis male urogenital infections induce urogenital inflammation and decrease sperm quality, thus impairing male fertility potential. Screening for U. urealyticum and M. hominis infections and performing a comprehensive analysis of different leukocyte subsets and inflammatory cytokines in semen may be clinically helpful in the diagnosis and follow-up of male urogenital infection.

INFLAMMATORY CYTOKINES PERTURB NK CELL FUNCTION AND GENE SIGNATURE IN CHRONIC MYELOID LEUKEMIA

Natural killer (NK) cells have the capacity to eliminate malignant cells by releasing cytotoxic granules, which makes them a potent anti-cancer immunotherapeutic. NK cells'mature phenotype and counts positively correlate with prolonged treatment-free survival in chronic myeloid leukemia (CML), while dysfunctional NK cells are predictive of relapse in CML patients. The molecular drivers of this impairment during leukemic progression remain unclear. Given the established role of inflammatory cytokines (namely IL1β, IL-6, GM-CSF, TNFα) in driving myeloid malignancies, we aimed to define their effect on NK cell phenotype and function using a unique pre-clinical chimeric mouse model of CML. We hypothesize that leukemic cytokines impede NK cell maturation and cytotoxicity and shift NK cells toward a pro-tumor cytokine-producing phenotype. We establish a chimeric mouse model of CML by transplanting bone marrow BCR-ABL+ CD45.2 cells into sub-lethally irradiated CD45.1 C57BL/6 wild-type mice. For controls, mice are transplanted with non-mutant cells. Such chimeras represent a robust model to study the exposure of non-transformed host cells to leukemia. At the endpoint, splenic, blood, and bone marrow NK cells are harvested and profiled by flow cytometry, RT-qPCR, scRNA-seq, or used for degranulation assay. Blood samples are collected to obtain serum. Consistent with clinical observations, NK cells are severely reduced in numbers and frequencies and display an immature phenotype and diminished expression of activating receptors (Nkp46, Ly49D) while upregulating inhibitory molecules (Lag-3, TIGIT, NKG2A) during leukemia. Moreover, CML-exposed NK cells have a higher proliferation rate and a greater cell death indicating perturbed homeostasis. We revealed a preferential loss of terminally mature CD27−CD11b+ NK subset in CML mice, suggesting a functional shift from cytotoxic phenotype toward the immature cytokine-producing. In agreement, CML-exposed NK cells show impaired cytotoxicity as measured by target-specific degranulation ex vivo . Next, we mapped the transcriptional landscape of CML-exposed NK cells using single-cell RNA-sequencing. We confirmed decreased gene expression of NK maturation and cytotoxicity markers ( Itgam, Cx3cr1, Prf1, Gzma ) and upregulation of inhibitory molecules ( Tigit, Lag-3, Pdcd1lg1/2) and proteins involved in the cell cycle and senescence ( Mki67, Ccna, Ccnb ). Importantly, CML-exposed NK cells had significantly increased mRNA levels of inflammatory cytokines including IL1β, TNF, GM-CSF; cytokine receptors Il6st, Il-2Ra; and negative regulators of STAT signaling SOCS1/2/3 and CIS. Furthermore, IL-6-STAT3 and IL1β/TNF-signaling gene signatures were enriched in CML-exposed NK cells – an effect that is likely to be triggered by inflammatory cytokines. Thus, we next found that peripheral blood serum from CML mice dampens healthy NK cell degranulation ex vivo , compared to serum from control mice. Further ELISA analysis revealed elevated levels of IL-6, IL-1, TNF, and GM-CSF in CML serum samples. Some of these cytokines might be secreted by functionally skewed NK cells, as suggested by gene expression analysis. Altogether, our data suggest that leukemic cytokines contribute to NK cell dysfunction and polarize them toward a pro-inflammatory phenotype, representing optimal targets for NK-boosting CML immunotherapies.

Monocyte subsets display age-dependent alterations at fasting and undergo non-age-dependent changes following consumption of a meal

BackgroundMonocytes are a heterogenous population of immune cells whose subsets and functions become substantially dysregulated with advanced age. Although much of our current understanding of the age-related changes in monocytes is derived from fasting blood samples, most people are predominately in the postprandial state during waking hours. As hormonal, metabolic, and immunological changes in response to the consumption of a meal are manifested in postprandial blood, it’s unclear how age-dependent changes in peripheral monocytes at fasting are impacted by a dietary challenge.ObjectiveWe investigated the impact of age and meal consumption on circulating monocyte frequencies and subsets defined as classical (CD14 + CD16-), intermediate (CD14 + CD16 +), or non-classical (CD14dim CD16 +) in a cohort of 349 healthy adult volunteers grouped into categories based on their age: young adults (18–33 y, n = 123), middle adults (34–49 y, n = 115), and older adults (50–66 y, n = 111).ResultsFollowing 12-h fast total monocyte counts inversely correlated with subject age. Older adults had significantly fewer circulating monocytes along with elevated levels of TGs, cholesterol, glucose, IL-6, IL-8, TNF, neopterin, and CCL2 compared with young adults. Circulating monocyte pools in older adults consisted of smaller proportions of classical but larger proportions of intermediate and non-classical monocytes. Proportions of classical monocytes were inversely correlated with plasma TNF, IL-8, and neopterin while intermediate monocytes were positively correlated with plasma IL-6, TNF, and neopterin. Three hours after consuming a fat-containing meal postprandial monocyte counts increased in all age groups. Despite age-dependent differences in monocyte subsets at fasting, consumption of a meal induced similar changes in the proportions of classical and non-classical monocytes across age groups. Within the circulating postprandial monocyte pool, percentages of classical monocytes decreased while non-classical monocytes increased. However no change in precursory intermediate monocytes were detected. Our study confirms that ageing is associated with changes in monocyte frequencies and subsets and shows that consuming a fat-containing meal induces temporal changes in monocyte frequency and subsets independently of subject age.Clinical trialRegistered on ClincialTrials.gov (Identifier: NCT02367287)

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ’s mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ’s effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC50 values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.

Insights into hidradenitis suppurativa

Hidradenitis suppurativa (HS) is a chronic, debilitating, inflammatory skin disorder with a prevalence of around 1% and a profound impact on patients' quality of life. Characteristic lesions such as inflammatory nodules, abscesses, and sinus tracts develop in the axillae, inguinal, and gluteal areas, typically during or after puberty. Acomplex interplay of genetic predisposition, hormonal factors, obesity, and smoking contributes to development and maintenance of the disease. HS is considered to arise from an intrinsic defect within the hair follicle, leading to follicular plugging, cyst formation, and subsequent rupture that in turn induce an acute inflammatory response characterized by elevated levels of IL-1β, IL-17, and TNF. Over time, acute lesions transition into chronic disease, with active draining sinus tracts accompanied by extensive fibrosis. HS is associated with other immune-mediated inflammatory diseases, metabolic and cardiovascular disorders, and psychiatric comorbidities. Treatment of HS often requires a combination of antibiotic or immunosuppressing therapies and surgical intervention. Nonetheless, the currently available treatments are not universally effective, and many drugs, which are often repurposed from other inflammatory diseases, are under investigation. Studies into the early stages of HS may yield treatments to prevent disease progression; yet, they are hampered by a lack of appropriate invitro and animal models.

Circulating cardiomyocyte-derived extracellular vesicles reflect cardiac injury during systemic inflammatory response syndrome in mice

The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).

DAMPs are able to skew CD4+ T cell subsets and increase the inflammatory profile in pregnant women with preeclampsia

Preeclampsia (PE) is characterized by abnormal activation of the immune system. The intense systemic inflammatory reaction, could be related to the presence of molecules released after cell stress or death, that are capable of inducing inflammation and are known as damage-associated molecular patterns (DAMP). This study evaluated the profile of T cells through the analysis of transcription factors and the cytokines produced after culture with or without DAMPs: heat shock protein 70 (Hsp70), hyaluronan (HA) and monosodium urate (MSU). Twenty pregnant women with PE, 20 normotensive (NT) pregnant women and 20 non-pregnant (NP) women were studied. The results showed polarization toward Th1/Th17 and a decrease in Th2/Treg profiles in preeclamptic women associated with elevated levels of TNF, IFN-γ, and IL-17A and diminished levels of TGF-β1 and IL-10 when compared to the normotensive group. In addition, preeclamptic women had a higher percentage of cells co-expressing T-bet/GATA-3 and T-bet/RORγt and fewer T-bet/FoxP3 cells when compared to normotensive group. MSU induced an increase in IFN-γ and IL-22 in all studied groups. MSU, HA, and Hsp70 induced significant higher production of TNF in the PE and NP groups. The PE group showed elevated levels of TGF-β1 after incubation with MSU, HA, and Hsp70, whereas HA and Hsp70 decreased TGF-β1 production in NT group. The results suggest that these alarmins may play a role in the activation of innate and adaptive immune systems by skewing CD4 + T cells and increasing the release of inflammatory cytokines, thereby contributing to the pathogenesis of this important syndrome.

Impact of data extraction errors in meta-analyses on the association between depression and peripheral inflammatory biomarkers: an umbrella review.

Accumulating evidence suggests that alterations in inflammatory biomarkers are important in depression. However, previous meta-analyses disagree on these associations, and errors in data extraction may account for these discrepancies. PubMed/MEDLINE, Embase, PsycINFO, and the Cochrane Library were searched from database inception to 14 January 2020. Meta-analyses of observational studies examining the association between depression and levels of tumor necrosis factor-α (TNF-α), interleukin 1-β (IL-1β), interleukin-6 (IL-6), and C-reactive protein (CRP) were eligible. Errors were classified as follows: incorrect sample sizes, incorrectly used standard deviation, incorrect participant inclusion, calculation error, or analysis with insufficient data. We determined their impact on the results after correction thereof. Errors were noted in 14 of the 15 meta-analyses included. Across 521 primary studies, 118 (22.6%) showed the following errors: incorrect sample sizes (20 studies, 16.9%), incorrect use of standard deviation (35 studies, 29.7%), incorrect participant inclusion (7 studies, 5.9%), calculation errors (33 studies, 28.0%), and analysis with insufficient data (23 studies, 19.5%). After correcting these errors, 11 (29.7%) out of 37 pooled effect sizes changed by a magnitude of more than 0.1, ranging from 0.11 to 1.15. The updated meta-analyses showed that elevated levels of TNF- α, IL-6, CRP, but not IL-1β, are associated with depression. These findings show that data extraction errors in meta-analyses can impact findings. Efforts to reduce such errors are important in studies of the association between depression and peripheral inflammatory biomarkers, for which high heterogeneity and conflicting results have been continuously reported.

Defining the Role of Nuclear Factor (NF)-κB p105 Subunit in Human Macrophage by Transcriptomic Analysis of NFKB1 Knockout THP1 Cells.

Since its discovery over 30 years ago the NF-ĸB family of transcription factors has gained the status of master regulator of the immune response. Much of what we understand of the role of NF-ĸB in immune development, homeostasis and inflammation comes from studies of mice null for specific NF-ĸB subunit encoding genes. The role of inflammation in diseases that affect a majority of individuals with health problems globally further establishes NF-ĸB as an important pathogenic factor. More recently, genomic sequencing has revealed loss of function mutations in the NFKB1 gene as the most common monogenic cause of common variable immunodeficiencies in Europeans. NFKB1 encodes the p105 subunit of NF-ĸB which is processed to generate the NF-ĸB p50 subunit. NFKB1 is the most highly expressed transcription factor in macrophages, key cellular drivers of inflammation and immunity. Although a key role for NFKB1 in the control of the immune system is apparent from Nfkb1-/- mouse studies, we know relatively little of the role of NFKB1 in regulating human macrophage responses. In this study we use the THP1 monocyte cell line and CRISPR/Cas9 gene editing to generate a model of NFKB1-/- human macrophages. Transcriptomic analysis reveals that activated NFKB1-/- macrophages are more pro-inflammatory than wild type controls and express elevated levels of TNF, IL6, and IL1B, but also have reduced expression of co-stimulatory factors important for the activation of T cells and adaptive immune responses such as CD70, CD83 and CD209. NFKB1 -/- THP1 macrophages recapitulate key observations in individuals with NFKB1 haploinsufficiency including decreased IL10 expression. These data supporting their utility as an in vitro model for understanding the role of NFKB1 in human monocytes and macrophages and indicate that of loss of function NFKB1 mutations in these cells is an important component in the associated pathology.

Absence of TNF-α Promotes Anemia of Inflammation and Inflammatory Bowel Disease

Anemia of inflammation (AI) is a hallmark of a multitude of disorders including infections, autoimmune diseases and malignancies. The pathophysiology of AI is currently under investigation. While the hematological aspect of AI and the involvement of cytokines such as IL6, IL1α, IL1 β has been subject to extensive study, the inflammatory and immunological arm has only recently begun to be explored. This involves a complex interplay of cytokine mediators both pro and anti-inflammatory, mediating a broader innate and ultimately adaptive immune response that could potentially be linked to the hematological deficiencies seen under AI. In particular, highly elevated levels of the cytokine TNF a have been noted in inflammatory conditions, including an established acute mouse model of AI induced by a single injection of the heat killed pathogen Brucella abortus (BA). The latter results observed by us prompted us to furtherinvestigate the role of this cytokine in BA induced AI.To study the above we injected mice lacking TNFα (TNFα-/-) with BA and studied hematological, inflammatory and immunological changes in our model. Following a single injection of BA, we found that TNFα -/- mice develop an irreversible macrocytic, hyperchromic anemia with hemoglobin levels dropping as low as 7 g/dL within two weeks of BA injection and not returning to normal even after eight weeks unlike their WT counterparts. This is further characterized by a complete abolition of Bone Marrow (BM) erythropoiesis and extra medullary erythropoiesis in the spleen (SPL). We also found elevated serum levels of erythropoietin and iron, indicating that the resulting anemia is not caused by a lack of these factors. The anemia was accompanied by a fall in the lifespan of RBCs in peripheral blood and a concurrent increase in production of reactive oxygen species by progenitors and terminally differentiated RBCs. Moreover, our experiments using WT GFP+ RBCs in BA treated TNFα-/- mice indicate that even the healthy GFP+ RBCs are cleared faster in the latter animals. Preliminary data, suggest that macrophages derived from BA treated TNFα-/- express pro-inflammatory markers and that the same cells can consume more RBCs than macrophages from control animals, suggesting increased macrophagocytosis. Subsequently, we looked at changes in serum cytokine levels over time after injection of BA. We found that unlike WT animals injected with BA, TNFα-/- mice show a sustained production of IFNγ, IL12-p40 and IL1β. We also found an expansion of splenic resident macrophages and T cells which are cell types implicated in the production of the cytokines mentioned previously. Finally, to extend the reach of this model to other disorders we treated TNFα-/- mice with low doses of dextran sodium sulfate (DSS) and found that only these animals developed inflammatory bowel disease (IBD) characterized by blood in the feces and severe anemia, unlike WT animals treated the same way.Our observations on the upregulation of IFNγ and IL12 is leading us to study macrophage activation and T helper cell polarization in the BM, SPL and lymph nodes. Moreover, we are also investigating the potential crosstalk between these effectors, cytokines and erythropoiesis looking at oxidative stress, survival and cell differentiation of erythroid progenitors. As a first step, we are investigating the recovery from BA of IFNγ-/- mice, as TNFα-/- mice with AI show a significant upregulation of IFNγ. Preliminary complete blood count of BA treated IFNγ -/- mice indicates that these animals do recover from an inflammatory insult and the anemia is reversible, unlike the TNFα-/- mice. These results suggest that absence of IFNγ could potentially rescue the severely impaired phenotype that we found in our TNFα-/- mice and the recovery in BA treated TNFα/IFNγ double knock out mice.Given the history of TNFα in enabling inflammatory reactions and promoting cell death, the view of this cytokine in supporting recover under AI as inferred from our results is relatively novel and suggest caution in the use of TNFα inhibitors. Furthermore, our data point to an important role in the crosstalk between TNFα and INFγ and in their effect on erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

ErbB4 regulates interferon signaling in classically activated macrophages

Background Classically activated macrophages drive intestinal inflammation, which is necessary to limit infection. However, over-aggressive responses from these cells, including elevated cytokine release, may contribute to inflammatory bowel disease. Previous studies have implicated the receptor tyrosine kinase ErbB4 and its ligands (neuregulins/NRGs) in regulating macrophage activity and survival. However, the influence of ErbB4 and its specific ligand NRG4 on cytokine expression and downstream signaling pathways in these cells remains untested. We hypothesized that ErbB4 regulates pathways associated with cytokine production in macrophages. Methods We generated bone-marrow derived macrophages from mice lacking ErbB4 in cells of myeloid lineage (LysMCre/ErbB4FF) or sex-matched littermate controls (ErbB4FF), and stimulated pro-inflammatory activation with interferon (IFN)-gamma plus lipopolysaccharide (classical activation). Cells were analyzed by RNA sequencing, and gene-set enrichment analysis (GSEA) was performed using publicly available Hallmark gene-sets. To test the role of NRG4, activated cells were treated with recombinant ligand at 100 ng/ml for 24 hours to determine effects on cytokine expression. RNA was collected and analyzed for cytokines by qPCR. Results Classically activated macrophages from LysMCre/ErbB4FF mice displayed an enhanced pro-inflammatory cytokine profile by GSEA (p<0.05) compared to ErbB4FF mice. We confirmed elevated levels of Tnf (32% higher, p<0.05) and Cxcl1 (69% higher, p<0.05) by qPCR. NRG4 treatment limited Tnf (p<0.05) and Cxcl1 (p<0.05) expression in classically activated macrophages from wild-type mice. GSEA showed a reduced type-I and type-II IFN gene expression signature in cells from LysMCre/ErbB4FF mice versus ErbB4FF mice. Conclusion Loss of ErbB4 in macrophages leads to dysregulated IFN signaling and enhanced pro-inflammatory cytokine production in macrophages. NRG/ErbB4 signaling in macrophages drives an anti-inflammatory feedback loop and may limit aberrant intestinal inflammation.