Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia.

During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation ex vivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.

The TOX-RAGE axis mediates inflammatory activation and lung injury in severe pulmonary infectious diseases.

Thymocyte selection-associated high-mobility group box (TOX) is a transcription factor that is crucial for T cell exhaustion during chronic antigenic stimulation, but its role in inflammation is poorly understood. Here, we report that TOX extracellularly mediates drastic inflammation upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by binding to the cell surface receptor for advanced glycation end-products (RAGE). In various diseases, including COVID-19, TOX release was highly detectable in association with disease severity, contributing to lung fibroproliferative acute respiratory distress syndrome (ARDS). Recombinant TOX-induced blood vessel rupture, similar to a clinical signature in patients experiencing a cytokine storm, further exacerbating respiratory function impairment. In contrast, disruption of TOX function by a neutralizing antibody and genetic removal of RAGE diminished TOX-mediated deleterious effects. Altogether, our results suggest an insight into TOX function as an inflammatory mediator and propose the TOX-RAGE axis as a potential target for treating severe patients with pulmonary infection and mitigating lung fibroproliferative ARDS.

Pyruvate kinase M2 sustains cardiac mitochondrial quality surveillance in septic cardiomyopathy by regulating prohibitin 2 abundance via S91 phosphorylation.

The endogenous mitochondrial quality control (MQC) system serves to protect mitochondria against cellular stressors. Although mitochondrial dysfunction contributes to cardiac damage during many pathological conditions, the regulatory signals influencing MQC disruption during septic cardiomyopathy (SC) remain unclear. This study aimed to investigate the involvement of pyruvate kinase M2 (PKM2) and prohibitin 2 (PHB2) interaction followed by MQC impairment in the pathogenesis of SC. We utilized LPS-induced SC models in PKM2 transgenic (PKM2TG) mice, PHB2S91D-knockin mice, and PKM2-overexpressing HL-1 cardiomyocytes. After LPS-induced SC, cardiac PKM2 expression was significantly downregulated in wild-type mice, whereas PKM2 overexpression in vivo sustained heart function, suppressed myocardial inflammation, and attenuated cardiomyocyte death. PKM2 overexpression relieved sepsis-related mitochondrial damage via MQC normalization, evidenced by balanced mitochondrial fission/fusion, activated mitophagy, restored mitochondrial biogenesis, and inhibited mitochondrial unfolded protein response. Docking simulations, co-IP, and domain deletion mutant protein transfection experiments showed that PKM2 phosphorylates PHB2 at Ser91, preventing LPS-mediated PHB2 degradation. Additionally, the A domain of PKM2 and the PHB domain of PHB2 are required for PKM2-PHB2 binding and PHB2 phosphorylation. After LPS exposure, expression of a phosphorylation-defective PHB2S91A mutant negated the protective effects of PKM2 overexpression. Moreover, knockin mice expressing a phosphorylation-mimetic PHB2S91D mutant showed improved heart function, reduced inflammation, and preserved mitochondrial function following sepsis induction. Abundant PKM2 expression is a prerequisite to sustain PKM2-PHB2 interaction which is a key element for preservation of PHB2 phosphorylation and MQC, presenting novel interventive targets for the treatment of septic cardiomyopathy.

Heterogeneity of Benefit from Earlier Time-to-Antibiotics for Sepsis.

Rationale: Shorter time-to-antibiotics improves survival from sepsis, particularly among patients in shock. There may be other subgroups for whom faster antibiotics are particularly beneficial.Objectives: Identify patient characteristics associated with greater benefit from shorter time-to-antibiotics.Methods: Observational cohort study of patients hospitalized with community-onset sepsis at 173 hospitals and treated with antimicrobials within 12 hours. We used three approaches to evaluate heterogeneity of benefit from shorter time-to-antibiotics: 1) conditional average treatment effects of shorter (⩽3 h) versus longer (>3-12 h) time-to-antibiotics on 30-day mortality using multivariable Poisson regression; 2) causal forest to identify characteristics associated with greatest benefit from shorter time-to-antibiotics; and 3) logistic regression with time-to-antibiotics modeled as a spline.Measurements and Main Results: Among 273,255 patients with community-onset sepsis, 131,094 (48.0%) received antibiotics within 3 hours. In Poisson models, shorter time-to-antibiotics was associated with greater absolute mortality reduction among patients with metastatic cancer (5.0% [95% confidence interval; CI: 4.3-5.7] vs. 0.4% [95% CI: 0.2-0.6] for patients without cancer, P < 0.001); patients with shock (7.0% [95% CI: 5.8-8.2%] vs. 2.8% [95% CI: 2.7-3.5%] for patients without shock, P = 0.005); and patients with more acute organ dysfunctions (4.8% [95% CI: 3.9-5.6%] for three or more dysfunctions vs. 0.5% [95% CI: 0.3-0.8] for one dysfunction, P < 0.001). In causal forest, metastatic cancer and shock were associated with greatest benefit from shorter time-to-antibiotics. Spline analysis confirmed differential nonlinear associations of time-to-antibiotics with mortality in patients with metastatic cancer and shock.Conclusions: In patients with community-onset sepsis, the mortality benefit of shorter time-to-antibiotics varied by patient characteristics. These findings suggest that shorter time-to-antibiotics for sepsis is particularly important among patients with cancer and/or shock.

Geo-economic Influence on the Effect of Fluid Volume for Sepsis Resuscitation: A Meta-Analysis.

Rationale: Sepsis management relies on fluid resuscitation avoiding fluid overload and its related organ congestion. Objectives: To explore the influence of country income group on risk-benefit balance of fluid management strategies in sepsis. Methods: We searched e-databases for all randomized controlled trials on fluid resuscitation in patients with sepsis or septic shock up to January 2023, excluding studies on hypertonic fluids, colloids, and depletion-based interventions. The effect of fluid strategies (higher versus lower volumes) on mortality was analyzed per income group (i.e., low- and middle-income countries [LMICs] or high-income countries [HICs]). Measurements and Main Results: Twenty-nine studies (11,798 patients) were included in the meta-analysis. There was a numerically higher mortality in studies of LMICs as compared with those of HICs: median, 37% (interquartile range [IQR]: 26-41) versus 29% (IQR: 17-38; P = 0.06). Income group significantly interacted with the effect of fluid volume on mortality: Higher fluid volume was associated with higher mortality in LMICs but not in HICs: odds ratio (OR), 1.47; 95% confidence interval (95% CI): 1.14-1.90 versus 1.00 (95% CI: 0.87-1.16), P = 0.01 for subgroup differences. Higher fluid volume was associated with increased need for mechanical ventilation in LMICs (OR, 1.24 [95% CI: 1.08-1.43]) but not in HICs (OR, 1.02 [95% CI: 0.80-1.29]). Self-reported access to mechanical ventilation also significantly influenced the effect of fluid volume on mortality, which increased with higher volumes only in settings with limited access to mechanical ventilation (OR: 1.45 [95% CI: 1.09-1.93] vs. 1.09 [95% CI: 0.93-1.28], P = 0.02 for subgroup differences). Conclusions: In sepsis trials, the effect of fluid resuscitation approach differed by setting, with higher volume of fluid resuscitation associated with increased mortality in LMICs and in settings with restricted access to mechanical ventilation. The precise reason for these differences is unclear and may be attributable in part to resource constraints, participant variation between trials, or other unmeasured factors.

Stratification of Sepsis Patients on Admission into the Intensive Care Unit According to Differential Plasma Metabolic Phenotypes.

Delayed diagnosis of patients with sepsis or septic shock is associated with increased mortality and morbidity. UPLC-MS and NMR spectroscopy were used to measure panels of lipoproteins, lipids, biogenic amines, amino acids, and tryptophan pathway metabolites in blood plasma samples collected from 152 patients within 48 h of admission into the Intensive Care Unit (ICU) where 62 patients had no sepsis, 71 patients had sepsis, and 19 patients had septic shock. Patients with sepsis or septic shock had higher concentrations of neopterin and lower levels of HDL cholesterol and phospholipid particles in comparison to nonsepsis patients. Septic shock could be differentiated from sepsis patients based on different concentrations of 10 lipids, including significantly lower concentrations of five phosphatidylcholine species, three cholesterol esters, one dihydroceramide, and one phosphatidylethanolamine. The Supramolecular Phospholipid Composite (SPC) was reduced in all ICU patients, while the composite markers of acute phase glycoproteins were increased in the sepsis and septic shock patients within 48 h admission into ICU. We show that the plasma metabolic phenotype obtained within 48 h of ICU admission is diagnostic for the presence of sepsis and that septic shock can be differentiated from sepsis based on the lipid profile.

The role of TREM-1 in septic myocardial pyroptosis and septic cardiomyopathy in vitro and in vivo.

Septic cardiomyopathy (SCM) is an acute cardiac dysfunction involving myocardial cell pyroptosis. TREM-1 is a known receptor on cell membrane that can amplify the inflammatory response. Our previous studies have shown that TREM-1 in cardiomyocytes is involved in the activation of NLRP3 through the SMC4/NEMO pathway. Here, we aimed to use Trem-1 and Nlrp3 knockout mice to verify the effect of TREM-1 through NLRP3 on cardiac function in septic mice. The results showed that TREM-1 knockout resulted in a decrease in the release of downstream cell signals, including SMC4 and NLRP3, resulting in a decrease in cytokine release and improvement of cardiac dysfunction. Knockout of NLRP3 also reduced cardiomyocyte pyroptosis and increased survival rate. The therapeutic targeting of TREM-1 activation of NLRP3 and its pathway may contribute to the treatment or prevention of SCM.

Improving Sepsis Outcomes in the Era of Pay-for-Performance and Electronic Quality Measures: A Joint IDSA/ACEP/PIDS/SHEA/SHM/SIDP Position Paper.

The Centers for Medicare & Medicaid Services (CMS) introduced the Severe Sepsis/Septic Shock Management Bundle (SEP-1) as a pay-for-reporting measure in 2015 and is now planning to make it a pay-for-performance measure by incorporating it into the Hospital Value-Based Purchasing Program. This joint IDSA/ACEP/PIDS/SHEA/SHM/SIPD position paper highlights concerns with this change. Multiple studies indicate that SEP-1 implementation was associated with increased broad-spectrum antibiotic use, lactate measurements, and aggressive fluid resuscitation for patients with suspected sepsis but not with decreased mortality rates. Increased focus on SEP-1 risks further diverting attention and resources from more effective measures and comprehensive sepsis care. We recommend retiring SEP-1 rather than using it in a payment model and shifting instead to new sepsis metrics that focus on patient outcomes. CMS is developing a community-onset sepsis 30-day mortality electronic clinical quality measure (eCQM) that is an important step in this direction. The eCQM preliminarily identifies sepsis using systemic inflammatory response syndrome (SIRS) criteria, antibiotic administrations or diagnosis codes for infection or sepsis, and clinical indicators of acute organ dysfunction. We support the eCQM but recommend removing SIRS criteria and diagnosis codes to streamline implementation, decrease variability between hospitals, maintain vigilance for patients with sepsis but without SIRS, and avoid promoting antibiotic use in uninfected patients with SIRS. We further advocate for CMS to harmonize the eCQM with the Centers for Disease Control and Prevention's (CDC) Adult Sepsis Event surveillance metric to promote unity in federal measures, decrease reporting burden for hospitals, and facilitate shared prevention initiatives. These steps will result in a more robust measure that will encourage hospitals to pay more attention to the full breadth of sepsis care, stimulate new innovations in diagnosis and treatment, and ultimately bring us closer to our shared goal of improving outcomes for patients.

Effect of Preoperative Antibiotic Therapy on Operative Culture Yield for Diagnosis of Native Joint Septic Arthritis.

BACKGROUND: Native joint septic arthritis (NJSA) is definitively diagnosed by a positive Gram stain or culture, along with supportive clinical findings. Preoperative antibiotics are known to alter synovial fluid cell count, Gram stain, and culture results and are typically postponed until after arthrocentesis to optimize diagnostic accuracy. However, data on the impact of preoperative antibiotics on operative culture yield for NJSA diagnosis are limited. METHODS: We retrospectively reviewed adult cases of NJSA who underwent surgery at Mayo Clinic facilities from 2012 to 2021 to analyze the effect of preoperative antibiotics on operative culture yield through a paired analysis of preoperative culture (POC) and operative culture (OC) results using logistic regression and generalized estimating equations. RESULTS: Two hundred ninety-nine patients with NJSA affecting 321 joints were included. Among those receiving preoperative antibiotics, yield significantly decreased from 68.0% at POC to 57.1% at OC (P < .001). In contrast, for patients without preoperative antibiotics there was a non-significant increase in yield from 60.9% at POC to 67.4% at OC (P = .244). In a logistic regression model for paired data, preoperative antibiotic exposure was more likely to decrease OC yield compared to non-exposure (odds ratio [OR] = 2.12; 95% confidence interval [CI] = 1.24-3.64; P = .006). Within the preoperative antibiotic group, additional antibiotic doses and earlier antibiotic initiation were associated with lower OC yield. CONCLUSIONS: In patients with NJSA, preoperative antibiotic exposure resulted in a significant decrease in microbiologic yield of operative cultures as compared to patients in whom antibiotic therapy was held prior to obtaining operative cultures.

Francisella tularensis Bone and Joint Infections: United States, 2004-2023.

Tularemia is caused by the highly infectious bacterium Francisella tularensis, which is recognized as a Tier 1 bioterrorism agent. Tularemia has a range of recognized clinical manifestations, but fewer than 20 bone or joint infections from 6 countries have been reported in the literature to date. This series includes 13 cases of F. tularensis septic arthritis or osteomyelitis in the United States during 2004-2023 and describes exposures, clinical presentation, diagnosis, and outcomes for this rare but severe form of tularemia. Clinicians should consider F. tularensis in patients with compatible exposures or a history of joint replacement or immunosuppression.

Formation of Single-Species and Multispecies Biofilm by Isolates from Septic Transfusion Reactions in Platelet Bag Model.

During 2018-2021, eight septic transfusion reactions occurred from transfusion of platelet units contaminated with Acinetobacter spp., Staphylococcus saprophyticus, Leclercia adecarboxylata, or a combination of those environmental organisms. Whether biofilm formation contributed to evasion of bacterial risk mitigations, including bacterial culture, point-of-care testing, or pathogen-reduction technology, is unclear. We designed a 12-well plate-based method to evaluate environmental determinants of single-species and multispecies biofilm formation in platelets. We evaluated bacteria isolated from septic transfusion reactions for biofilm formation by using crystal violet staining and enumeration of adherent bacteria. Most combinations of bacteria had enhanced biofilm production compared with single bacteria. Combinations involving L. adecarboxylata had increased crystal violet biofilm production and adherent bacteria. This study demonstrates that transfusion-relevant bacteria can produce biofilms well together. More work is needed to clarify the effect of biofilms on platelet bacterial risk control strategies, but US Food and Drug Administration-recommended strategies remain acceptable.

Association of the social disorganization index with time to first septic shock event in children with acute myeloid leukemia.

BACKGROUND: Pediatric acute myeloid leukemia (AML) chemotherapy increases the risk of life-threatening complications, including septic shock (SS). An area-based measure of social determinants of health, the social disorganization index (SDI), was hypothesized to be associated with SS and SS-associated death (SS-death). METHODS: Children treated for de novo AML on two Children's Oncology Group trials at institutions contributing to the Pediatric Health Information System (PHIS) database were included. The SDI was calculated via residential zip code data from the US Census Bureau. SS was identified via PHIS resource utilization codes. SS-death was defined as death within 2 weeks of an antecedent SS event. Patients were followed from 7 days after the start of chemotherapy until the first of end of front-line therapy, death, relapse, or removal from study. Multivariable-adjusted Cox regressions estimated hazard ratios (HRs) comparing time to first SS by SDI group. RESULTS: The assembled cohort included 700 patients, with 207 (29.6%) sustaining at least one SS event. There were 233 (33%) in the SDI-5 group (highest disorganization). Adjusted time to incident SS did not statistically significantly differ by SDI (reference, SDI-1; SDI-2: HR, 0.84 [95% confidence interval (CI), 0.51-1.41]; SDI-3: HR, 0.70 [95% CI, 0.42-1.16]; SDI-4: HR, 0.97 [95% CI, 0.61-1.53]; SDI-5: HR, 0.72 [95% CI, 0.45-1.14]). Nine patients (4.4%) with SS experienced SS-death; seven of these patients (78%) were in SDI-4 or SDI-5. CONCLUSIONS: In a large, nationally representative cohort of trial-enrolled pediatric patients with AML, there was no significant association between the SDI and time to SS.

Integrated multi-omics analysis and machine learning developed diagnostic markers and prognostic model based on Efferocytosis-associated signatures for septic cardiomyopathy.

Septic cardiomyopathy (SCM) is characterized by an abnormal inflammatory response and increased mortality. The role of efferocytosis in SCM is not well understood. We used integrated multi-omics analysis to explore the clinical and genetic roles of efferocytosis in SCM. We identified six module genes (ATP11C, CD36, CEBPB, MAPK3, MAPKAPK2, PECAM1) strongly associated with SCM, leading to an accurate predictive model. Subgroups defined by EFFscore exhibited distinct clinical features and immune infiltration levels. Survival analysis showed that the C1 subtype with a lower EFFscore had better survival outcomes. scRNA-seq analysis of peripheral blood mononuclear cells (PBMCs) from sepsis patients identified four genes (CEBPB, CD36, PECAM1, MAPKAPK2) associated with high EFFscores, highlighting their role in SCM. Molecular docking confirmed interactions between diagnostic genes and tamibarotene. Experimental validation supported our computational results. In conclusion, our study identifies a novel efferocytosis-related SCM subtype and diagnostic biomarkers, offering new insights for clinical diagnosis and therapy.

m6A methylation in myocardial tissue of septic mice analyzed using MeRIP/m6A-sequencing and RNA-sequencing.

Septic cardiomyopathy is a secondary myocardial injury caused by sepsis. N6-methyl-adenosine (m6A) modification is involved in the pathological progression of septic cardiomyopathy; however, the pathological mechanism remains unclear. In this study, we identified the overall m6A modification pattern in septic myocardial injury and determined its potential interactions with differentially expressed genes (DEGs). A sepsis mouse model exhibiting septic symptoms and myocardial tissue damage was induced by lipopolysaccharide (LPS). LPS-induced septic myocardial tissues and control myocardial tissues were subjected to methylated RNA immunoprecipitation sequencing and RNA sequencing to screen for differentially expressed m6A peaks and DEGs. We identified 859 significantly m6A-modified genes in septic myocardial tissues, including 432 upregulated and 427 downregulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to explore the biological importance of differentially expressed m6A methylated genes and DEGs. Differentially expressed m6A methylated genes were enriched in immune- and inflammation-related pathways. Conjoint analysis revealed co-expression of differentially expressed m6A genes and DEGs, including genes that were upregulated or downregulated and those showing opposite trends. High expression of m6A-related genes (WTAP and IGF2BP2), interleukin-17, and interleukin-17 pathway-related genes (MAPK11 and TRAF3IP2) was verified using reverse transcription-quantitative PCR. We confirmed the presence of m6A modification of the transcriptome and m6A-mediated gene expression in septic myocardial tissues.

MMP-8 causes leftward shift in end-diastolic pressure-volume relationship and may explain the development of diastolic dysfunction in septic cardiomyopathy.

Septic cardiomyopathy (SCM) with diastolic dysfunction carries a poor prognosis, and the mechanisms underlying the development of diastolic dysfunction remain unclear. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils and degrades collagen I. MMP-8 levels correlate with SCM severity. We scrutinized, for the first time, the direct impact of MMP-8 on cardiac systolic and diastolic functions. Isolated rat hearts were perfused with Krebs-Henseleit solution in a Langendorff setup with computer-controlled filling pressures of both ventricles in an isovolumetric regime. The end-diastolic pressure (EDP) varied periodically between 3 and 20 mmHg. After baseline recordings, MMP-8 (100 µg/mL) was added to the perfusion. Short-axis views of both ventricles were continuously acquired by echocardiography. MMP-8 perfusion resulted in a progressive decline in peak systolic pressures (Psys) in both ventricles, but without significant changes in their end-systolic pressure-area relationships (ESPARs). Counterintuitively, conspicuous leftward shifts of the end-diastolic pressure-area relationships (EDPARs) were observed in both ventricles. The left ventricle (LV) end-diastolic area (EDA) decreased by 32.8 ± 5.7% (P = 0.008) at an EDP of 10.5 ± 0.4 mmHg, when LV Psys dropped by 20%. The decline of Psys was primarily due to the decrease in EDA, and restoring the baseline EDA by increasing EDP recovered 81.33 ± 5.87% of the pressure drop. Collagen I generates tensile (eccentric) stress, and its degradation by MMP-8 causes end-diastolic pressure-volume relationship (EDPVR) leftward shift, resulting in diastolic and systolic dysfunctions. The diastolic dysfunction explains the clinically observed fluid unresponsiveness, whereas the decrease in end-diastolic volume (EDV) diminishes the systolic functions. MMP-8 can explain the development of SCM with diastolic dysfunction.NEW & NOTEWORTHY MMP-8, released from activated neutrophils and macrophages, is markedly elevated in sepsis, correlating with sepsis severity and mortality. MMP-8 targets collagen I of the cardiac ECM and induces diastolic dysfunction with fluid unresponsiveness, associated with decreased EDV, reduced sarcomere length, and diminished systolic function. Unlike other MMPs that predominantly cleave collagen-III and contribute to cardiac dilatation, thereby increasing sarcomere length, MMP-8 leads to a leftward shift in the EDPVR, resulting in diastolic and systolic dysfunctions.

Commonly disrupted pathways in brain and kidney in a pig model of systemic endotoxemia.

Sepsis is a life-threatening state that arises due to a hyperactive inflammatory response stimulated by infection and rarely other insults (e.g., non-infections tissue injury). Although changes in several proinflammatory cytokines and signals are documented in humans and small animal models, far less is known about responses within affected tissues of large animal models. We sought to understand the changes that occur during the initial stages of inflammation by administering intravenous lipopolysaccharide (LPS) to Yorkshire pigs and assessing transcriptomic alterations in the brain, kidney, and whole blood. Robust transcriptional alterations were found in the brain, with upregulated responses enriched in inflammatory pathways and downregulated responses enriched in tight junction and blood vessel functions. Comparison of the inflammatory response in the pig brain to a similar mouse model demonstrated some overlapping changes but also numerous differences, including oppositely dysregulated genes between species. Substantial changes also occurred in the kidneys following LPS with several enriched upregulated pathways (cytokines, lipids, unfolded protein response, etc.) and downregulated gene sets (tube morphogenesis, glomerulus development, GTPase signal transduction, etc.). We also found significant dysregulation of genes in whole blood that fell into several gene ontology categories (cytokines, cell cycle, neutrophil degranulation, etc.). We observed a strong correlation between the brain and kidney responses, with significantly shared upregulated pathways (cytokine signaling, cell death, VEGFA pathways) and downregulated pathways (vasculature and RAC1 GTPases). In summary, we have identified a core set of shared genes and pathways in a pig model of systemic inflammation.

Neutralization of TLR2 in combination with either TNF-α or IL-1ß antibody reduces the severity of septic arthritis through STAT3/mTOR signalling in lymphocytes.

Staphylococcus aureus induced Septic arthritis is considered a medical concern. S.aureus binds TLR2 to induce an array of inflammatory responses. Generation of pro-inflammatory cytokines induces T cell responses and control Th17/Treg cell balance. Regulation of T cell-mediated immunity in response to inflammation is significantly influenced by mTOR. Presence of elevated TNF-α, IL-1ß decreases Treg cell activity through STAT3/mTOR, promoting proliferation of T cells towards Th17 cells. Therefore, we postulated, neutralizing TLR2 with either TNF-α or IL-1ß in combination could be useful in modifying Th17/Treg cell ratio in order to treat septic arthritis by suppressing expression of mTOR/STAT3. To date, no studies have reported effects of neutralization of TLR2 along with either TNF-α or IL-1ß on amelioration of arthritis correlating with mTOR/STAT3 expression. Contribution of T lymphocytes collected from blood, spleen, synovial tissues, their derived cytokines IFN-γ, IL-6, IL-17, TGF-ß, IL-10 were noted. Expression of TLR2, TNFR1, TNFR2, NF-κB along with mTOR/STAT3 also recorded. Neutralization of TLR2 along with TNF-α and IL-1ß were able to shift Th17 cells into immunosuppressive Treg cells. Furthermore,elevated expression of IL-10, TNFR2 and demoted expression of mTOR/ STAT3 along with NF-κB in lymphocytes confirms its role in resolution of arthritis. It was also effective in reducing oxidative stress via increasing expression of the antioxidant enzymes. As a result, it can be inferred that Treg-derived IL-10, which may mitigate inflammatory effects of septic arthritis by influencing the mTOR/STAT3 interaction in lymphocytes, may be selected as a different therapeutic strategy for reducing the impact of septic arthritis.

Quantitative microbial risk assessment for ingestion of antibiotic resistance genes from private wells contaminated by human and livestock fecal sources.

We used quantitative microbial risk assessment to estimate ingestion risk for intI1, erm(B), sul1, tet(A), tet(W), and tet(X) in private wells contaminated by human and/or livestock feces. Genes were quantified with five human-specific and six bovine-specific microbial source-tracking (MST) markers in 138 well-water samples from a rural Wisconsin county. Daily ingestion risk (probability of swallowing ≥1 gene) was based on daily water consumption and a Poisson exposure model. Calculations were stratified by MST source and soil depth over the aquifer where wells were drilled. Relative ingestion risk was estimated using wells with no MST detections and >6.1 m soil depth as a referent category. Daily ingestion risk varied from 0 to 8.8 × 10-1 by gene and fecal source (i.e., human or bovine). The estimated number of residents ingesting target genes from private wells varied from 910 (tet(A)) to 1,500 (intI1 and tet(X)) per day out of 12,000 total. Relative risk of tet(A) ingestion was significantly higher in wells with MST markers detected, including wells with ≤6.1 m soil depth contaminated by bovine markers (2.2 [90% CI: 1.1-4.7]), wells with >6.1 m soil depth contaminated by bovine markers (1.8 [1.002-3.9]), and wells with ≤6.1 m soil depth contaminated by bovine and human markers simultaneously (3.1 [1.7-6.5]). Antibiotic resistance genes (ARGs) were not necessarily present in viable microorganisms, and ingestion is not directly associated with infection. However, results illustrate relative contributions of human and livestock fecal sources to ARG exposure and highlight rural groundwater as a significant point of exposure.IMPORTANCEAntibiotic resistance is a global public health challenge with well-known environmental dimensions, but quantitative analyses of the roles played by various natural environments in transmission of antibiotic resistance are lacking, particularly for drinking water. This study assesses risk of ingestion for several antibiotic resistance genes (ARGs) and the class 1 integron gene (intI1) in drinking water from private wells in a rural area of northeast Wisconsin, United States. Results allow comparison of drinking water as an exposure route for antibiotic resistance relative to other routes like food and recreational water. They also enable a comparison of the importance of human versus livestock fecal sources in the study area. Our study demonstrates the previously unrecognized importance of untreated rural drinking water as an exposure route for antibiotic resistance and identifies bovine fecal material as an important exposure factor in the study setting.

Rheumatoid arthritis and risk of osteoarticular infection and death following Staphylococcus aureus bacteraemia: A nationwide cohort study.

OBJECTIVES: Osteoarticular infection (OAI) is a feared complication of Staphylococcus aureus bacteraemia (SAB) and is associated with poor outcomes. We aimed to explore risk of OAI and death following SAB in patients with and without rheumatoid arthritis (RA) and to identify risk factors for OAI in patients with RA. METHODS: Danish nationwide cohort study of all patients with microbiologically verified first-time SAB between 2006-2018. We identified RA, SAB, comorbidities, and RA-related characteristics (e.g. orthopaedic implants, antirheumatic treatment) in national registries including the rheumatology registry DANBIO. We estimated cumulative incidence of OAI and death and adjusted hazard ratios (HRs, multivariate Cox regression). RESULTS: We identified 18 274 patients with SAB (n = 367 with RA). The 90-day cumulative incidence of OAI was 23.1%(95%CI 18.8; 27.6) for patients with RA and 12.5%(12.1; 13.0) for patients without RA (non-RA) (HR 1.93(1.54; 2.41)). For RA patients with orthopaedic implants cumulative incidence was 29.4%(22.9; 36.2) (HR 1.75(1.08; 2.85), and for current users of tumor necrosis factor inhibitors (TNFi) it was 41.9%(27.0; 56.1) (HR 2.27(1.29; 3.98) compared with non-users). All-cause 90-day mortality following SAB was similar in RA (35.4%(30.6; 40.3)) and non-RA (33.9%(33.2; 34.5), HR 1.04(0.87; 1.24)). CONCLUSION: Following SAB, almost one in four patients with RA contracted OAI corresponding to a doubled risk compared with non-RA. In RA, orthopaedic implants and current TNFi use were associated with approximately doubled OAI risk. One in three died within 90 days in both RA and non-RA. These findings encourage vigilance in RA patients with SAB to avoid treatment delay of OAI.

COX-2 optimizes cardiac mitochondrial biogenesis and exerts a cardioprotective effect during sepsis.

BACKGROUND: Septic cardiomyopathy is a component of multiple organ dysfunction in sepsis. Mitochondrial dysfunction plays an important role in septic cardiomyopathy. Studies have shown that cyclooxygenase-2 (COX-2) had a protective effect on the heart, and prostaglandin E2 (PGE2), the downstream product of COX-2, was increasingly recognized to have a protective effect on mitochondrial function. OBJECTIVE: This study aims to demonstrate that COX-2/PGE2 can protect against septic cardiomyopathy by regulating mitochondrial function. METHODS: Cecal ligation and puncture (CLP) was used to establish a mouse model of sepsis and RAW264.7 macrophages and H9C2 cells were used to simulate sepsis in vitro. The NS-398 and celecoxib were used to inhibit the activity of COX-2. ZLN005 and SR18292 were used to activate or inhibit the PGC-1α activity. The mitochondrial biogenesis was examined through the Mitotracker Red probe, mtDNA copy number, and ATP content detection. RESULTS: The experimental data suggested that COX-2 inhibition attenuated PGC-1α expression thus decreasing mitochondrial biogenesis, whereas increased PGE2 could promote mitochondrial biogenesis by activating PGC-1α. The results also showed that the effect of COX-2/PGE2 on PGC-1α was mediated by the activation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB). Finally, the effect of COX-2/PGE2 on the heart was also verified in the septic mice. CONCLUSION: Collectively, these results suggested that COX-2/PGE2 pathway played a cardioprotective role in septic cardiomyopathy through improving mitochondrial biogenesis, which has changed the previous understanding that COX-2/PGE2 only acted as an inflammatory factor.