IL-1β Levels Research Articles

Exosomes from Limosilactobacillus fermentum Ameliorate Benzalkonium Chloride-Induced Inflammation in Conjunctival Cells.

Dry eye is characterized by persistent instability and decreased tear production, which are accompanied by epithelial lesions and inflammation on the surface of the eye. In our previous paper, we reported that supplementation with Limosilactobacillus fermentum HY7302 (HY7302) could inhibit corneal damage in a benzalkonium chloride (BAC)-induced mouse model of dry eye, through its effects in gut microbiome regulation. The aim of this study was to determine what functional extracellular substances can alter the inflammatory response of conjunctival cells. We isolated exosomes from HY7302 probiotic culture supernatant, analyzed their morphological characteristics, and found that their average size was 143.8 ± 1.1 nm, which was smaller than the exosomes from the L. fermentum KCTC 3112 strain. In addition, HY7302-derived exosomes significantly reduced the levels of genes encoding pro-inflammatory cytokines, including interleukin (IL)-20, IL-8, IL-6, and IL-1B, in BAC-treated human conjunctival cells. Moreover, HY7302-derived exosomes significantly increased the levels of genes encoding tight junction proteins, including TJP1, TJP2, and occludin-1, in Caco-2 cells. Lastly, the HY7302 exosomes reduced mRNA expression levels of IL1B, IL20, IL6, IL8, and NFAT5 in a transwell coculture system. Our findings indicate that HY7302 exosomes have potential for use in the treatment of ocular inflammation-related dry eye disease, through gut-eye axis communication via exosomes.

Soluble Urokinase-Type Plasminogen Activator Receptor and Inflammatory Biomarker Response with Prognostic Significance after Acute Neuronal Injury - a Prospective Cohort Study.

Aneurysmal subarachnoid hemorrhage (aSAH), ischemic stroke (IS), and traumatic brain injury (TBI) are severe conditions impacting individuals and society. Identifying reliable prognostic biomarkers for predicting survival or recovery remains a challenge. Soluble urokinase type plasminogen activator receptor (suPAR) has gained attention as a potential prognostic biomarker in acute sepsis. This study evaluates suPAR and related neuroinflammatory biomarkers in serum for brain injury prognosis. This prospective study included 31 aSAH, 30 IS, 13 TBI, and three healthy controls (n = 77). Serum samples were collected on average 5.9days post-injury, analyzing suPAR, IL-1β, cyclophilin A, and TNFα levels using ELISA. Outcomes were assessed 90days post-injury with the modified Rankin Scale (mRS), categorized as favorable (mRS 0-2) or unfavorable (mRS 3-6). Statistical analyses included 2-tailed t-tests, Pearson's correlations, and machine learning linear discriminant analysis (LDA) for biomarker combinations. Elevated suPAR levels were found in brain injury patients compared to controls (p = 0.017). Increased suPAR correlated with unfavorable outcomes (p = 0.0018) and showed prognostic value (AUC = 0.66, p = 0.03). IL-1β levels were higher in the unfavorable group (p = 0.0015). LDA combinatory analysis resulted a fair prognostic accuracy with canonical equation = 0.775[suPAR] + 0.667[IL1-β] (AUC = 0.77, OR 0.296, sensitivity 93.1%, specificity 53.1%, p = 0.0007). No correlation was found between suPAR and CRP or infection status. Elevated suPAR levels in acute brain injury patients were associated with poorer outcomes, highlighting suPAR's potential as a prognostic biomarker across different brain injury types. Combining IL-1β with suPAR improved prognostic accuracy, supporting a multimodal biomarker approach for predicting outcomes.

Rosmarinic acid alleviated intestinal barrier damage caused by Escherichia coli by regulating the gut microbiota and inhibiting the NF-κB signalling pathway in mice.

Escherichia coli (E. coli) is a common zoonotic foodborne pathogen that poses a major threat to public health and economic development. Rosmarinic acid (RA) can inhibit intestinal inflammation; however, the protective effect of RA against the intestinal barrier damage induced by E. coli in mice and the underlying mechanism have not been elucidated. In this study, mice were orally administered with RA (20 mg kg-1) by gavage for one week and then were intraperitoneally challenged with E. coli. Mouse colonic epithelial cells (MCECs) were pretreated with RA for 6 h and challenged with E. coli (MOI = 1000) for 3 h. The results revealed that RA alleviated E. coli-induced weight loss in mice; reduced the increase in the levels of TNF-α, IL-6 and IL-1β in the serum; alleviated the decrease in ZO-1 protein expression; and increased intestinal permeability by inhibiting the NF-κB signalling pathway both in vivo and in vitro. Moreover, RA relieved the increase in intestinal permeability, reversed the structural damage to the mouse gut microbiota caused by E. coli, and increased the abundance of beneficial bacteria, including Lachnospiraceae_NK4136_group. Additionally, RA lost its protective function against E. coli infection in a pseudosterile mouse model, suggesting that the protection induced by RA was dependent on the gut microbiota. In conclusion, these results indicate that RA alleviates E. coli-induced inflammatory damage to the intestinal barrier by inhibiting the NF-κB signalling pathway and maintaining gut microbiota homeostasis. These findings provide new ideas and foundations for the application of RA as protection against E. coli.

Endodontic treatment modifies circulatory inflammatory mediator levels: A systematic review with meta-analysis.

There is limited and conflicting data on the reduction of circulatory inflammatory mediators in patients with apical periodontitis (AP) following endodontic treatment. To answer the following research question: in adult healthy patients with AP [Population (P)], is there a difference before [Comparator (C)] and after various endodontic treatments (nonsurgical, surgical or retreatment) [Intervention (I)] on systemic levels of inflammatory biomarkers [Outcome (O)] in the follow-up period[Time (T)]? An electronic literature search was conducted in the databases Scopus, PubMed, Clarivate Analytics' Web of Science, Cochrane Database of Systematic Reviews and Grey literature from inception to July 2024 with no language restrictions. Observational studies examining changes in serum levels of inflammatory mediators were included. Two independent reviewers selected studies, extracted data and critically appraised the included studies. Qualitative and quantitative (meta-analysis) data synthesis methods were employed. The Newcastle-Ottawa Scale was used to assess the quality of the included studies. Sixteen studies met the inclusion criteria, of which six were included in the meta-analysis. These studies were published between 1992 and 2024, involving a total of 596 patients (54% females) aged between 16 and 75 years. The meta-analysis of pooled data showed a significant decrease in high-sensitive C-reactive protein (hs-CRP) levels in the serum of patients with AP 6 months after treatment [2.26 ± 1.76 versus 1.28 ± 1.06 mg/L, (Z = 2.03, p = .04)] and a decrease in interleukin-1β (IL-1β) levels 12 months after treatment [13.01 ± 5.95 versus 10.86 ± 3.52 pg/mL, (Z = 3.72, p < .01)]. One study was assessed as poor quality, while all others were considered high quality. Despite the differences in methodologies across the included studies, it has been established that effective endodontic treatment leads to a reduction in systemic inflammatory biomarkers in the body. Following effective endodontic treatment in patients with AP, the systemic levels of hs-CRP and IL-1β exhibit a significant reduction at 6 and 12 months, respectively. Further clinical studies should investigate whether effective endodontic treatment and reduced levels of investigated biomarkers may change the clinical presentation of systemic diseases. PROSPERO database (CRD42024559271).

Deranged cytokine levels are linked to cancer-related cognitive impairment in lymphoma patients receiving R-CHOP chemotherapy

Cancer-related cognitive impairment (CRCI) is a significant issue commonly observed following chemotherapy treatment. The study aimed to investigate the changes in cognitive function and their association with IL-6, IL-1β, and IL-10 levels before and after R-CHOP chemotherapy over six cycles. Seventy chemotherapy naïve, newly diagnosed lymphoma patients were enrolled. Cognitive functions and inflammatory cytokines were assessed at baseline (TP1), after 3rd cycle (TP2), and after 6th cycle (TP3). Patients, with mean age of 44.17 ± 13.67 years, showed significantly increased levels of IL-6 and IL-1β and decreased IL-10 levels over time (p < .001). On the Montreal Cognitive Assessment (MoCA), scores of domains such as executive functioning (p = .002), attention (p < .001), language (p < .001), recall (p = .005), and orientation (p < .001) significantly decreased post six cycles of R-CHOP chemotherapy. Correlation analysis at TP2 indicated a positive association between elevated IL-6 levels with a decrease in MoCA scores indicating a decline in cognitive function (ρ = 0.68, p < .001). At TP3, no association of MoCA scores with IL-6 and IL-1β was observed. Decreased IL-10 levels showed a weak association with decreased MoCA scores at TP2 and TP3 ( ρ = 0.2, p = .09; for TP3, ρ = 0.16, p = .17), but this was not significant. In summary, the findings of the present study highlight significant cognitive decline and changes in inflammatory cytokine levels following six cycles of R-CHOP. Objective cognitive assessments may be done to detect CRCI in patients treated with R-CHOP.

Effects of nanocapsules containing lumefantrine and artemether in an experimental model of cerebral malaria

BackgroundMalaria, a tropical neglected disease, imposes a significant burden on global health, leading to the loss of thousands of lives annually. Its gold standard treatment is a combination therapy of lumefantrine (LUM) and artemether (ART). Nanotechnology holds significant potential for improving drug bioavailability and potency while reducing adverse effects.ObjectivesThis study aimed to develop lipid-core nanocapsules containing ART and LUM and evaluate their effects in an experimental cerebral malaria model (ECM).MethodsThe polymeric interfacial deposition method was used to develop lipid-core nanocapsules (LNCs) containing ART and LUM (LNCARTLUM) and were characterized using micrometric and nanometric scales. Male C57BL/6 mice were infected with Plasmodium (P.)berghei ANKA (PbA, 1 × 105 PbA-parasitized red blood cells, intraperitoneally). On day 5 post-infection, PbA-infected mice were orally administered with ART + LUM, LNCARTLUM, blank nanocapsules (LNCBL), or ethanol as a control. Parasitemia, clinical scores, and survival rates were monitored throughout the experiment. Organ-to-body weight ratios, cytokine quantification, and intravital microscopy analyses were conducted on day 7 post-infection.ResultsLNCs were successfully developed and characterized. The treatment with LNCARTLUM in ECM resulted in complete clearance of parasitemia at 10 dpi, decreased clinical scores, and maintained 100% survival rates. Thereated mice exhibited splenomegaly and reduced TNF-α, IL-1β, and MCP1 levels in the brain. Furthermore, the LNCARTLUM treatment protected the brain microvasculature, reducing the number of cells in the rolling process and adherent to the microvasculature endothelium.ConclusionNanoformulations can potentially improve the efficacy of antimalarial drugs and be considered a promising approach to treat malaria.

Associations Between Plasma Levels of NLRP3 Protein, Interleukin-1 Beta and Features of Acute ST-Elevation Myocardial Infarction.

Phenotyping inflammation in ST-elevation myocardial infarction (STEMI) is a challenge for modern cardiology. NLRP3 inflammasome is a proven predictor of adverse outcomes in cardiovascular disease, but its specificity in stratifying inflammatory activity in patients with myocardial infarction (MI) has not been demonstrated. The aim of this paper is to describe the levels of NLRP3 protein and IL-1β concentrations and their changes in dynamics and associations with clinical, laboratory and instrumental characteristics of patients with STEMI. A total of 45 patients with STEMI were enrolled. Concentrations of NLRP3 and IL-1β were evaluated in arterial and venous EDTA blood from the infarct-related coronary and peripheral arteries and veins on days 1, 3 and 7 after MI. The concentrations of markers were higher on the first day after MI with a maximum decrease on the third day. The levels of both markers in venous plasma correlated with those in arterial blood, allowing their routine determination in venous plasma on the first day after MI. IL-1β levels correlated directly with the wall motion index and inversely with left ventricular ejection fraction and stroke volume, which characterize the potential contribution to adverse myocardial remodeling. There were two multidirectional trends in changes in NLRP3 and IL-1β levels during hospitalization. Initially higher levels with a gradual decrease by day 7 were associated with a longer duration of myocardial ischemia and higher plasma troponin I levels. Further evaluation of the long-term outcomes of MI will allow identifying inflammatory factors that input to the development of secondary major adverse cardiac events and will provide a new step in the understanding of inflammatory phenotyping.

Exercise rescues cognitive impairment through inhibiting the fibrinogen neuroinflammative pathway in diabetes.

Fibrinogen is a pivotal factor in the activation of neuroinflammation and cognitive impairment. While exercise, especifically swimming, has demonstrated cognitive benefits, the molecular protective mechanisms orchestrated by exercise in response to blood-brain barrier (BBB) leakage in diabetes remain elusive. This study systematically investigates the impact of fibrinogen on neuroinflammation and the role of exercise in diabetic rats. Diabetic rats underwent an 8-week swimming exercise regimen, and subsequent assessments included changes in interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), astroglia activation, BBB permeability, and key epithelial tight junction proteins such as zona occludins (ZO)-1, Claudin-5, and matrix metalloproteinase-9 (MMP-9). Spatial learning and memory were evaluated using the Morris water maze test and the novel object recognition test. The study revealed that exercise significantly improved cognitive function, potentially by suppressing fibrinogen levels and astroglia activation. Intriguingly, heightened fibrinogen expression markedly attenuated the protective effects of exercise on BBB integrity. Fibrinogen emerged as a potential compromise to exercise protective effect by increasing expression levels of inflammatory factors IL-1β and TNF-α. In summary, our findings elucidate that fibrinogen may contribute to the deterioration of cognition and diminish the protective effects of exercise by amplifying the neuroinflammatory process through damaged BBB in diabetes.

Novel Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling.

The endogenous neurosteroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP) modulates inflammatory and neuroinflammatory signaling through toll-like receptors (TLRs) in human and mouse macrophages, human blood cells and alcohol-preferring (P) rat brains. Although it is recognized that 3α,5α-THP inhibits TLR4 activation by blocking interactions with MD2 and MyD88, the comprehensive molecular mechanisms remain to be elucidated. This study explores additional TLR4 activation sites, including TIRAP binding to MyD88, which is pivotal for MyD88 myddosome formation, as well as LPS interactions with the TLR4:MD2 complex. Both male and female P rats (n = 8/group) received intraperitoneal administration of 3α,5α-THP (15 mg/kg; 30 min) or a vehicle control, and their hippocampi were analyzed using immunoprecipitation and immunoblotting techniques. 3α,5α-THP significantly reduces the levels of inflammatory mediators IL-1β and HMGB1, confirming its anti-inflammatory actions. We found that MyD88 binds to TLR4, IRAK4, IRAK1, and TIRAP. Notably, 3α,5α-THP significantly reduces MyD88-TIRAP binding (Males: -31 ± 9%, t-test, p < 0.005; Females: -53 ± 15%, t-test, p < 0.005), without altering MyD88 interactions with IRAK4 or IRAK1, or the baseline expression of these proteins. Additionally, molecular docking and molecular dynamic analysis revealed 3α,5α-THP binding sites on the TLR4:MD2 complex, targeting a hydrophobic pocket of MD2 usually occupied by Lipid A of LPS. Surface plasmon resonance (SPR) assays validated that 3α,5α-THP disrupts MD2 binding of Lipid A (Kd = 4.36 ± 5.7 μM) with an inhibition constant (Ki) of 4.5 ± 1.65 nM. These findings indicate that 3α,5α-THP inhibition of inflammatory mediator production involves blocking critical protein-lipid and protein-protein interactions at key sites of TLR4 activation, shedding light on its mechanisms of action and underscoring its therapeutic potential against TLR4-driven inflammation.

Oxidative stress and mitochondrial dysfunction contributes to postoperative cognitive dysfunction in elderly rats dependent on NLRP3 activation.

Postoperative cognitive dysfunction (POCD), a complication following procedures such as orthopedic surgery, is associated with a worsened prognosis, especially in the elderly population. Several mechanisms have been proposedfor communication between the immune system and the brain after surgery. In an experimental tibial fracture (TF) model, we aimed to understand the role of the NLR family pyrin domain containing 3 (NLRP3) on oxidative stress and mitochondrial dysfunction as mechanisms underlying POCD in aged and adult rats.Adult or aged male Wistar rats were subjected to the TF model and receivedintracerebroventricularsaline or MCC950 (140ng/kg), a specific small-molecule inhibitor that selectively blocks activation of the NLRP3 inflammasome.We followed the control (sham) and TF groups treated with MCC950 or saline for seven days to determine cognitive function and survival.The prefrontal cortex and hippocampus were isolated for NLRP3 evaluation, cytokine analysis, oxidative stress measurements, myeloperoxidase activity, nitric oxide formation, mitochondrial respiratory chain enzymes, and succinate dehydrogenase (SDH) activity. Seven days after TF induction, NLRP3 levels increased in the hippocampus and prefrontal cortex in both ages, showed an enhancement in aged rats compared to adults, and experienced a reversal with MCC950 administration.The administration of MCC950 restored memory, IL-1β and IL-10 levels, nitrite/nitrate, lipid peroxidation in the hippocampus and prefrontal cortex, and preserved catalase activity in the prefrontal cortex in aged rats. At the same age, the complex I activity alteration in both regions and complex II, IV, and SDH in the prefrontal cortex werereversed. In conclusion, NLRP3 activation contributes to POCD development because it is intrinsically involved in mitochondrial dysfunction and oxidative stress after orthopedic surgery in aged rats.

Abstract 4137337: Targeting Macrophage NCOR1 to Alleviate Inflammation in Heart Failure with Preserved Ejection Fraction

Background: Inflammatory response is a key player in heart failure with preserved ejection fraction, however the underlying mechanisms remain unclear. Recent evidence indicates that macrophage inflammation driven by the transcriptional coregulatory protein NCOR1 could play a pivotal role in disease states. Hypothesis: NCOR1 orchestrates macrophage inflammation and cardiac dysfunction in HFpEF. Methods: HFpEF was induced in mice using a validated model combining high-fat diet and L-NAME for 15 weeks. Cardiac function was assessed using high-resolution ultrasound imaging and LV samples were used to study macrophage polarization and inflammation by FACS. We generated myeloid-specific NCOR1-KO mice to investigate the in vivo effects of macrophage NCOR1 depletion on HFpEF. In vitro experiments included RAW 264.7 cells polarization and the assessment of macrophage phenotype switching in the presence or the absence of NCOR1. To study the crosstalk of macrophages with the surrounding cardiac cells, cardiomyocytes (H9c2) and endothelial cells (HAECS) were exposed to conditioned media from cultured RAW. Results: The expression of macrophage markers in human LV specimens showed an increase in pro-inflammatory macrophages (M1) and a decrease in regulatory macrophages (M2) as compared to age-matched control mice. HFpEF mice showed increased expression of vascular adhesion molecules (ICAM1, ICAM2, E-selectin) and a significant recruitment of M1 macrophages as proved by qPCR, WB, and flow cytometry analysis. Of interest, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) – and lung congestion were significantly reduced in mice with macrophage NCOR1 deletion as compared to WT littermates, while exercise tolerance was significantly improved. These findings were associated with a reduction of myocardial inflammation (TNF-a, IL6, IL-1b) in Ncor1-KO mice. In vitro assays showed that stimulation of macrophages with PA induced M1 type switch and increased NCOR1, TNF-a, IL-6, and IL-1b levels, whereas NCOR1 depletion prevented detrimental changes of macrophage conditional medium (Fig.1). Conclusion: Targeting macrophage NCOR1 could represent a promising approach to blunt myocardial inflammation in HFpEF.

Melatonin attenuates cardiac dysfunction and inflammation in dilated cardiomyopathy via M2 macrophage polarization.

Melatonin is a neuroendocrine hormone that exerts protective effects on the heart. Increasing evidence suggests that macrophage M2-type polarization improves myocardial regeneration and repair. Therefore, this study investigated whether melatonin ameliorates dilated cardiomyopathy (DCM) by modulating M2-type polarization. DCM mice were established by induction with doxorubicin and then treated with melatonin. Cardiac dysfunction was determined by measuring left ventricular ejection fraction (LVEF) and left ventricular internal dimensions at end-diastole and end-systole. Heart injury and fibrosis were determined by hematoxylin and eosin staining and Sirius Red staining, respectively. Serum concentrations of melatonin, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) were measured through enzyme-linked immunosorbent assays. M2-type macrophages were analyzed by flow cytometry. Relative mRNA and protein levels were determined by reverse transcription quantitative polymerase chain reaction and western blotting, respectively. Circulating melatonin levels were significantly decreased in DCM mice and were associated with LVEF. Treatment with melatonin markedly ameliorated cardiac dysfunction, improved survival, and alleviated pathological changes and collagen deposition in DCM mice. Furthermore, melatonin-treated DCM mice displayed lower serum and cardiac levels of IL-1β, IL-6, and TNF-α, as well as higher numbers of M2-type macrophages in cardiac tissue, indicating that melatonin treatment could decrease inflammatory responses and facilitate M2 macrophage polarization in DCM mice. Thus, melatonin treatment alleviated cardiac dysfunction and inflammatory responses by promoting M2 macrophage polarization in the DCM mouse model.

Abstract 4134539: Hybrid Cardiomyocyte Targeting Peptide Down-regulates NF-κB Pathway in Human Cardiomyocytes

Introduction: Role of NF-κB driven inflammation leading to heart failure with preserved ejection fraction has received little attention. Our previous in vivo work with cardiac targeting peptide (CTP) in guinea pigs identified suppression of NF- κB pathway by CTP. In the current work we sought to further study the effects of a hybrid version of CTP (hCTP: amino acids at positions 3 and 11 substituted with D-amino acids) on TNF-α mediated NF-κB activation, as well as expression of its downstream inflammatory markers IL-1β and IL-8. Methods: Human cardiomyocytes (CMCs) were transfected using Lipofectamine 3000 with a reporter luciferase plasmid carrying an NF-κB promoter site upstream of the luciferase gene. Cells were treated with various concentrations of hCTP for 24 hours, followed by a TNF-α challenge (20ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to above manipulations was assessed by FACS using live-dead stain. CMCs were treated as above, and cell culture supernatants analyzed for IL-8 levels using human ELISA kit. In another set of experiments, CMCs were transfected with NF-κB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, incubated with 10nM angiotensin 2 (Ang2) and 100nM phenylephrine (PE) for 24hrs to assess levels of IL-1β in the cell culture media using an ELISA assay. Results: hCTP inhibited TNF-α mediated NF-κB activation with a dose-response curve, with maximum inhibition with hCTP seen at 10µM concentrations (p&lt;0.001). Treatment with hCTP inhibited the TNF-α induced IL-8 secretion with significant decreases seen even at the 1µM dose. Similarly, secretion of IL-1β production by CMCs in response to Ang2/PE stimulation was suppressed by hCTP even at the lowest tested dose (1µM; p&lt;0.5). Conclusions: Our data suggests that hCTP inhibited NF-κB pathway transcriptional activity in response to TNF-α stimulation in a dose-dependent fashion in human CMCs. Furthermore, treatment with hCTP inhibited TNF-α induced IL-8 and IL-1β secretion even at doses as low as 1µM. The ability of hCTP to efficiently targeting this inflammatory pathway suggests that it has therapeutic potential in the treatment of heart failure. Further in vivo studies are ongoing to test this hypothesis.

Small heterodimer partner-interacting leucine zipper protein suppresses pain and cartilage destruction in an osteoarthritis model by modulating the AMPK/STAT3 signaling pathway

ObjectiveOsteoarthritis (OA) is a degenerative joint disease caused by the breakdown of joint cartilage and adjacent bone. Joint injury, being overweight, differences in leg length, high levels of joint stress, abnormal joint or limb development, and inherited factors have been implicated in the etiology of OA. In addition to physical damage to the joint, a role for inflammatory processes has been identified as well. Small heterodimer partner-interacting leucine zipper protein (SMILE) regulates transcription and many cellular functions. Among the proteins activated by SMILE is the peroxisome proliferator-activated receptor (PPAR) γ, which mediates the activities of CD4 + T helper cells, including Th1, Th2, and Th17, as well as Treg cells. PPAR-γ binds to STAT3 to inhibit its transcription, thereby suppressing the expression of the NF-κB pathway, and in turn, the expression of the inflammatory cytokines interferon (IFN), interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which are sub-signals of STAT3 and NF-κB.MethodsOA was induced in control C57BL/6 mice and in C57BL/6-derived SMILE-overexpressing transgenic (SMILE Tg) mice. The protein expression levels in the joint and spleen tissues were analyzed by immunohistochemistry and immunofluorescence images. In addition, flow cytometry was performed for detecting changes of the changes of immune cells.ResultsLess cartilage damage and significantly reduced levels of OA biomarkers (MMP13, TIMP3 and MCP-1) were observed in SMILE Tg mice. Immunohistochemistry performed to identify the signaling pathway involved in the link between SMILE expression and OA revealed decreased levels of IL-1β, IL-6, TNF-α, and phosphorylated AMPK in synovial tissues as well as a significant decrease in phosphorylated STAT3 in both cartilage and synovium. Changes in systemic immune cells were investigated via flow cytometry to analyze splenocytes isolated from control and SMILE Tg mice. SMILE Tg mice had elevated proportions of CD4 + IL-4 + cells (Th2) and CD4 + CD25 + Foxp3 + cells (Treg) and a notable decrease in CD4 + IL-17 + cells (Th17).ConclusionOur results show that overexpressed SMILE attenuates the symptoms of OA, by increasing AMPK signaling and decreasing STAT3, thus reducing the levels of inflammatory immune cells.

Abstract 4132409: Caspase-4/11 promotes a high-fat diet and chronic kidney disease-accelerated vascular inflammation via caspase-4/11-IL-1b two-tier trained immunity

Introduction: Chronic kidney disease (CKD) is a common inflammatory disease affecting &gt;15% of the adult population and 50% of all patients with advanced/end stages (4 to 5) of CKD have cardiovascular disease. Research Questions: How multiple disease risk factors interplay to accelerate vascular inflammation in CKD. Aim: To determine whether hyperlipidemia and CKD have a synergy in accelerating vascular inflammation via trained immunity (TI). Methods: we performed aortic pathological analysis and RNA-sequencing in high-fat diet (HFD)-fed 5/6 nephrectomy CKD (HFD+CKD) mice. Results: 1) HFD+CKD increases aortic cytosolic lipopolysaccharide (LPS) levels, caspase-11 (casp11) activation, and 998 gene expressions of TI pathways in the aorta (first-tier TI mechanism); 2) Casp11 —/— decreases aortic neointima hyperplasia, aortic recruitment of macrophages, and casp11-gasdermin D-mediated cytokine secretion; 3) Casp11 —/— decreases N-terminal gasdermin D (N-GSDMD) membrane expression on aortic endothelial cells and aortic IL-1b levels; 4) LPS transfection into human aortic endothelial cells results in casp4 (human)/casp11 (mouse) activation and increases N-GSDMD membrane expression; 5) IL-1b serves as the second-tier mechanism underlying HFD+CKD-promoted TI. Conclusion: Hyperlipidemia and CKD accelerate vascular inflammation by promoting two-tier trained immunity

Electroacupuncture alleviates neuropathic pain in a rat model of CCD via suppressing P2X3 expression in dorsal root ganglia

BackgroundSciatica and low back pain are prevalent clinical types of neuropathic pain that significantly impair patients' quality of life. Conventional therapies often lack effectiveness, making these conditions challenging to treat. Electroacupuncture (EA) is an effective physiotherapy for pain relief. Prior research has demonstrated a relationship between the frequency of neuropathic pain and the analgesic impact of EA stimulation. This work aimed to assess the analgesic effects of EA in a rat model of chronic compression of the dorsal root ganglion (CCD) and to understand the underlying processes.MethodsWe established a rat CCD model to simulate sciatica and low back pain. EA was applied to rats with CCD at various frequencies (2 Hz, 100 Hz, and 2/100 Hz). The paw withdrawal threshold (PWT) was measured to assess analgesic effects. Additionally, protein levels of the purinergic receptor P2X3 (P2X3) and the expression of nociceptive neuronal markers were analyzed using immunohistochemistry and western blot (WB) techniques. The study also measured levels of proinflammatory cytokines TNF-α and IL-1β in the dorsal root ganglion (DRG). The involvement of P2X3 receptors was further investigated using the P2X3 agonist, α,β-methylene ATP (α,β-meATP).ResultsCCD rats developed pronounced mechanical allodynia. EA stimulation at all tested frequencies produced analgesic effects, with 2/100 Hz showing superior efficacy compared to 2 Hz and 100 Hz. The expression of P2X3 was increased in ipsilateral DRG of CCD model rats. P2X3 were co-labeled with isolectin B4 (IB4) and transient receptor potential vanilloid (TRPV1), indicating their role in nociception. 2/100 Hz EA treatment significantly reduced mechanical allodynia and inhibited the overexpression of P2X3, TRPV1, substance P (SP), and calcitonin gene-related peptide (CGRP) in the ipsilateral DRG of CCD model rats. Additionally, EA reduced the levels of proinflammatory cytokines TNF-α and IL-1β in the ipsilateral DRG, indicating an anti-inflammatory effect. The P2X3 agonist α,β-me ATP attenuated the analgesic effect of 2/100 Hz EA in CCD rats. The WB and immunofluorescence results consistently demonstrated P2X3 inhibition contributed to the analgesic effects of 2/100 Hz EA on CCD-induced neuropathic pain.ConclusionsOur findings suggest that 2/100 Hz EA alleviates neuropathic pain in rats by inhibiting the upregulation of P2X3 receptors in the ipsilateral DRG. This study backs up EA as a viable treatment option for sciatica and low back pain in clinical settings.

IMMU-37. PRE-TREATMENT INNATE INFLAMMATION ASSOCIATES WITH SURVIVAL AFTER POLIO VIROTHERAPY AND CAN BE MODULATED TO SENSITIZE GLIOMAS TO IN SITU VACCINATION

Abstract Early-stage clinical trials in glioblastoma (GBM) have tested various virotherapy strategies, yet only a subset of patients appear to benefit. We previously demonstrated that a recombinant poliovirus (PVSRIPO) functions by inducing MDA5-dependent, type I interferon (IFN) dominant innate inflammation in myeloid cells to provoke durable and functional antitumor T cell responses in mice. Long-term survival after intratumoral PVSRIPO infusion was observed in a subset of patients with recurrent GBM (rGBM) in two clinical trials. Survival after PVSRIPO was associated with higher pre-treatment intratumoral neutrophil density and MHC-class II gene expression, but also higher pre-treatment inflammatory cytokines in blood. Post-treatment induction of type I/III IFNs detected in blood one day after PVSRIPO infusion also associated with survival of patients with rGBM, suggesting that the proficiency of IFN responses to PVSRIPO may dictate therapy outcome. In human ex vivo glioma tissue assays, only a subset of gliomas mounted type I IFN responses to PVSRIPO and STING agonist treatment, in a manner linked with higher pre-treatment IL-1b levels, and lower CXCL10 induction after treatment. Thus, baseline innate inflammation either influences or reflects the capacity of glioma tumors to respond to virotherapy. Strikingly, in glioma bearing mice, peripheral vaccination against tumor irrelevant vaccines in alum adjuvant (i.e., against antigens not expressed by the tumor) induced bone marrow, spleen and intratumoral innate inflammation; rescued the antitumor efficacy of PVSRIPO in an otherwise refractory glioma model; and improved survival after intratumor STING agonist therapy. Notably, peripheral vaccination induced intratumoral neutrophil and CD4+ T cell inflammation, and increased MHC-class II expression on myeloid cells. These data imply a role for pre-treatment systemic innate immune training in the antitumor efficacy of virotherapy and indicate that modulating either systemic innate inflammation and/or the inflammatory status of bone marrow myeloid progenitors may sensitize gliomas to virotherapy.

Carnosol alleviates cisplatin-induced acute kidney injury by regulating apoptosis and pyroptosis.

The use of the common anticancer drug cisplatin (CP) in clinical practice often leads to acute kidney injury (AKI); however, no protective therapy is available. Therefore, new drugs that reduce the nephrotoxicity induced by CP are urgently needed. Carnosol (CA) is an antioxidant found. We investigated the renoprotective effects of CA on CP-induced AKI in male C57BL/6 mice and HK2 cells. CA mitigated renal dysfunction, histopathological changes and tubular injury in vivo, as indicated by the expression of NGAL, KIM1 and HMGB1. Moreover, the numbers of apoptotic cells and the expression of apoptotic proteins were dramatically reduced after CA treatment in mouse kidneys and HK2 cells. CA significantly ameliorated CP-induced inflammation and decreased TNF-α and IL-1β levels in vivo and in vitro and macrophage infiltration in the mouse kidney. CA decreased the expression levels of p-p65/p65, NLRP3 and ASC, which indicates that CA suppressed the activation of the NF-κB/NLRP3 signaling axis induced by CP in vivo and in vitro. In addition, CA decreased the levels of certain protein in pyroptotic cells, as indicated by the expression of cleaved caspase-1, GSDMD, and mature IL-1β and IL-18 in vivo and in vitro. Finally, CA reduced the level of cleaved caspase-1, but those of GSDMD and NLRP3 protein were not significantly different after treatment with the NLRP3 inhibitor MCC950 and were elevated by the NLRP3 activator nigericin. In conclusion, this study revealed that CA protects against CP-induced AKI by decreasing apoptosis and NF-κB/NLRP3/GSDMD-mediated pyroptosis, which provides new insight into the prevention of AKI.

The effect of methylphenidate on the reproductive function of female rats.

Research on the effects of methylphenidate on female fertility is limited. This study evaluated the effects of methylphenidate on reproductive function, oxidants, antioxidants, proinflammatory cytokines, prolactin, and cortisol in female rats. Forty-eight albino Wistar female rats were divided into four groups consisting of 12 rats, which were given pure water orally once daily for 7 days (HG-1), 10 mg/kg methylphenidate orally once daily for 7 days (MP-1), pure water orally once daily for 30 days (HG-2), and 10 mg/kg methylphenidate orally once daily for 30 days (MP-2). At the end of the treatment periods, tail vein blood was collected from six rats per group for prolactin and cortisol determination. Subsequently, euthanasia was performed and the ovaries were removed. Ovaries were analyzed for malondialdehyde (MDA), total glutathione (tGSH), superoxide dismutase (SOD), catalase (CAT), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α), and immunohistochemically. For breeding, the remaining six rats were mated with male rats for 1 month. Rats that failed to give birth were classed as infertile. A comparison of MP-1 and MP-2 groups to healthy controls revealed an elevation in MDA and corticosterone levels, and a decline in tGSH, SOD, and CAT levels (p < 0.001). Methylphenidate did not affect prolactin, IL-1β, and TNF-α levels (p > 0.05). MP-1 and MP-2 exhibited immunopositivity for 8-hydroxy-2'-deoxyguanosine (8-OHDG). MP-2 rats developed 66.7% infertility while MP-1, HG-1, and HG-2 rats did not. In ovaries, methylphenidate caused oxidative stress, but did not induce inflammation. Long-term use of methylphenidate caused increased cortisol levels and infertility.

Selection of optimal extraction and RT-PCR protocols for stool RNA detection of colorectal cancer associated immune genes

There is a growing interest in using fecal mRNA transcripts as biomarkers for non-invasive detection of colorectal cancer (CRC). The following study compares different RNA extraction and reverse transcription PCR (RT-PCR) methods for mRNA detection in stool and identifies a robust and sensitive protocol. A combination of the Stool total RNA purification kit (Norgen) and the Superscript III one-step RT-PCR kit (Invitrogen) provided high RNA purity and sensitive and consistent mRNA detection, making them well-suited candidates for large-scale studies. We tested the protocol by detecting the mRNA of several immune genes (CXCL1, IL8, IL1B, IL6, PTGS2, and SPP1) in 22 CRCs, 24 adenomatous polyps, and 22 control stool samples. All these inflammatory markers, except for CXCL1, showed a strong association with CRC. Cancer stool samples showed increased levels of IL1B, IL8, and PTGS2 transcripts compared to polyp and control groups. Thus, this work supports the potential use of fecal mRNA as biomarkers for CRC detection.