Response Sepsis Research Articles

A Transcriptomic Approach to Understand Patient Susceptibility to Pneumonia After Abdominal Surgery.

To describe immune pathways and gene networks altered following major abdominal surgery and to identify transcriptomic patterns associated with postoperative pneumonia. Nosocomial infections are a major healthcare challenge, developing in over 20% of patients aged 45 or over undergoing major abdominal surgery, with postoperative pneumonia associated with an almost 5-fold increase in 30-day mortality. From a prospective consecutive cohort (n=150) undergoing major abdominal surgery, whole-blood RNA was collected preoperatively and at 3 time-points postoperatively (2-6, 24, and 48h). Twelve patients diagnosed with postoperative pneumonia and 27 matched patients remaining infection-free were identified for analysis with RNA-sequencing. Compared to preoperative sampling, 3639 genes were upregulated and 5043 downregulated at 2 to 6 hours. Pathway analysis demonstrated innate-immune activation with neutrophil degranulation and Toll-like-receptor signaling upregulation alongside adaptive-immune suppression. Cell-type deconvolution of preoperative RNA-sequencing revealed elevated S100A8/9-high neutrophils alongside reduced naïve CD4 T-cells in those later developing pneumonia. Preoperatively, a gene-signature characteristic of neutrophil degranulation was associated with postoperative pneumonia acquisition ( P =0.00092). A previously reported Sepsis Response Signature (SRSq) score, reflecting neutrophil dysfunction and a more dysregulated host response, at 48 hours postoperatively, differed between patients subsequently developing pneumonia and those remaining infection-free ( P =0.045). Analysis of the novel neutrophil gene-signature and SRSq scores in independent major abdominal surgery and polytrauma cohorts indicated good predictive performance in identifying patients suffering later infection. Major abdominal surgery acutely upregulates innate-immune pathways while simultaneously suppressing adaptive-immune pathways. This is more prominent in patients developing postoperative pneumonia. Preoperative transcriptomic signatures characteristic of neutrophil degranulation and postoperative SRSq scores may be useful predictors of subsequent pneumonia risk.

Tamibarotene targets heparin-binding protein for attenuating lung injury in sepsis.

Excessively active pulmonary inflammation is a hallmark of sepsis-induced lung damage. A synthetic retinoid drug called tamibarotene reduces inflammation in a variety of conditions, including acute promyelocytic leukemia (APL), renal fibrosis, and neuroinflammation. Its effect on sepsis-related lung injury, however, has not been explained. The purpose of the study was to investigate how tamibarotene affected lung damage induced by cecal ligation and puncture (CLP) procedure. A CLP sepsis mouse model was developed, and tamibarotene was pretreated to determine whether it improved lung injury and survival. The degree of lung injury was evaluated using the Hematoxylin and eosin staining and lung injury score. In order to determine pulmonary vascular permeability, measurements were taken for total protein and cell content of bronchoalveolar lavage fluid (BALF), wet/dry ratio of the lung, and Evans blue stain. The BALF inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β, and IL-17A were discovered by enzyme-linked immunosorbent serologic assay (ELISA). Then, the levels of heparin-binding protein (HBP), and phospho-nuclear factor kappa-B (p-NF-κB) P65, and NF-κB P65 were determined using ELISA and Western blot analysis, respectively. Tamibarotene considerably increases survival and lessens lung damage stimulated by sepsis. Specifically, tamibarotene significantly relieves pulmonary vascular permeability and inhibits inflammation response in sepsis. Moreover, we further confirmed that these ameliorating effects of tamibarotene on sepsis may be exerted by targeting HBP and regulating the activation of NF-κB signaling pathway. These findings demonstrated that tamibarotene lessens sepsis-induced lung injury, and the effect could be exerted by targeting HBP and thereby deregulating the NF-κB signaling pathway.

Macrophage-targeted Nanomedicine for Sepsis: Diagnosis and Therapy.

Sepsis is a syndrome involving complex pathophysiological and biochemical dysregulation. Nanotechnology can improve our understanding of the pathophysiology of sepsis and contribute to the development of novel diagnostic and therapeutic strategies to further reduce the risk of sepsis. Macrophages play a key role in the progression of sepsis, thus, macrophage-associated pathological processes are important targets for both diagnostic and treatment of sepsis. In this paper, we reviewed efforts in the past decade of nanotechnologybased solutions for manipulate macrophages in sepsis diagnosis and management according to the type of nanomaterial. We addressed the latest progress of nanoparticles targeting macrophages for early sepsis detection. Additionally, we summarized the unique advantages of macrophage-targeted nanoparticles in the treatment of sepsis. These nanoparticles can improve the dysregulation of inflammatory response in sepsis by inhibiting the release of inflammatory factors and regulating macrophage apoptosis, activity and polarization. Finally, we present future opportunities as well as challenges of novel diagnostic and therapeutic strategies with the aim of accelerating the clinical translation of nanomedicine for sepsis treatment.

The Angiopoietin-2/Angiopoietin-1 ratio increases early in burn patients and predicts mortality

BackgroundAngiopoietin-2 (Angpt-2) is involved in the pathogenesis of the capillary leak syndrome in sepsis and has been shown to be associated with worse outcomes in diverse critical illnesses. It is however unclear whether Angpt-2 plays a similar role in severely burned patients during the early phase characterized by massive capillary leakage. Our aim was to analyze the Angiopoietin-2/Angiopoietin-1 ratio (Angpt-2/Angpt-1 ratio) over the first two days in critically ill burn patients and examine its association with survival and further clinical parameters. MethodsAdult burn patients with a total burn surface area (TBSA) ≥ 20% treated in the burn intensive care unit (ICU) of the University Hospital of Zurich, Switzerland, were included. Serum samples were collected prospectively and serum Angpt-1 and Angpt-2 were measured by enzyme-linked immunosorbent assay (ELISA) over the first two days after burn insult and stratified according to survival status, TBSA and the abbreviated burn severity index (ABSI). Due to hemodilution in the initial resuscitation phase, the Angpt-2/Angpt-1 ratio was normalized to albumin. ResultsFifty-six patients were included with a median age of 51.5 years. Overall mortality was 14.3% (8/56 patients). The total amount of infused crystalloids was 12́902 ml (IQR 9́362–16́770 ml) at 24 h and 18́461 ml (IQR 13́024–23́766 ml) at 48 h. The amount of substituted albumin was 20 g (IQR 10–50 g) at 24 h and 50 g (IQR 20–60 g) at 48 h.The albumin-corrected Angpt-2/Angpt-1 ratios increased over the first 48 h after the burn insult (d0: 0.5 pg*l/ml*g [IQR 0.24 – 0.80 pg*l/ml*g]; d1: 0.83 pg*l/ml*g [IQR 0.29 – 1.98 pg*l/ml*g]; d2: 1.76 pg*l/ml*g [IQR 0.70 – 3.23 pg*l/ml*g]; p < 0.001) and were significantly higher in eventual ICU non-survivors (p = 0.005), in patients with a higher TBSA (p = 0.001) and in patients with a higher ABSI (p = 0.001). ConclusionsIn analogy to the pathological host response in sepsis, the Angpt-2/Angpt-1 ratio steadily increases in the first two days in critically ill burn patients, suggesting a putative involvement in the pathogenesis of capillary leakage in burns. A higher Angpt-2/Angpt-1 ratio is associated with mortality, total burn surface area and burn scores.

Mortality Risk Prediction in Abdominal Septic Shock Treated with Polymyxin-B Hemoperfusion: A Retrospective Cohort Study.

Endotoxin, a component of the cell membrane of gram-negative bacteria, is a trigger for dysregulated inflammatory response in sepsis. Extracorporeal purification of endotoxin, through adsorption with polymyxin B, has been studied as a therapeutic option for sepsis. Previous studies suggest that it could be effective in patients with high endotoxin levels or patients with septic shock of moderate severity. Here, we perform a retrospective, single-centre cohort study of 93 patients suffering from abdominal septic shock treated with polymyxin-B hemoperfusion (PMX-HP) between 2015 and 2020. We compared deceased and surviving patients one month after the intervention using X2 and Mann-Whitney U tests. We assessed the data before and after PMX-HP with a Wilcoxon single-rank test and a multivariate logistic regression analysis. There was a significant reduction of SOFA score in the survivors. The expected mortality using APACHE-II was 59.62%, whereas in our sample, the rate was 40.9%. We found significant differences between expected mortality and real mortality only for the group of patients with an SOFA score between 8 and 13. In conclusion, in patients with abdominal septic shock, the addition of PMX-HP to the standard therapy resulted in lower mortality than expected in the subgroup of patients with intermediate severity of illness.

Soluble CD4 effectively prevents excessive TLR activation of resident macrophages in the onset of sepsis

T lymphopenia, occurring in the early phase of sepsis in response to systemic inflammation, is commonly associated with morbidity and mortality of septic infections. We have previously shown that a sufficient number of T cells is required to constrain Toll-like receptors (TLRs) mediated hyperinflammation. However, the underlying mechanisms remains unsolved. Herein, we unveil that CD4+ T cells engage with MHC II of macrophages to downregulate TLR pro-inflammatory signaling. We show further that the direct contact between CD4 molecule of CD4+ T cells or the ectodomain of CD4 (soluble CD4, sCD4), and MHC II of resident macrophages is necessary and sufficient to prevent TLR4 overactivation in LPS and cecal ligation puncture (CLP) sepsis. sCD4 serum concentrations increase after the onset of LPS sepsis, suggesting its compensatory inhibitive effects on hyperinflammation. sCD4 engagement enables the cytoplasmic domain of MHC II to recruit and activate STING and SHP2, which inhibits IRAK1/Erk and TRAF6/NF-κB activation required for TLR4 inflammation. Furthermore, sCD4 subverts pro-inflammatory plasma membrane anchorage of TLR4 by disruption of MHC II-TLR4 raft domains that promotes MHC II endocytosis. Finally, sCD4/MHCII reversal signaling specifically interferes with TLR4 but not TNFR hyperinflammation, and independent of the inhibitive signaling of CD40 ligand of CD4+ cells on macrophages. Therefore, a sufficient amount of soluble CD4 protein can prevent excessive inflammatory activation of macrophages via alternation of MHC II-TLR signaling complex, that might benefit for a new paradigm of preventive treatment of sepsis.

A pharmacoproteomic landscape of organotypic intervention responses in Gram-negative sepsis

Sepsis is the major cause of mortality across intensive care units globally, yet details of accompanying pathological molecular events remain unclear. This knowledge gap has resulted in ineffective biomarker development and suboptimal treatment regimens to prevent and manage organ dysfunction/damage. Here, we used pharmacoproteomics to score time-dependent treatment impact in a murine Escherichia coli sepsis model after administering beta-lactam antibiotic meropenem (Mem) and/or the immunomodulatory glucocorticoid methylprednisolone (Gcc). Three distinct proteome response patterns were identified, which depended on the underlying proteotype for each organ. Gcc enhanced some positive proteome responses of Mem, including superior reduction of the inflammatory response in kidneys and partial restoration of sepsis-induced metabolic dysfunction. Mem introduced sepsis-independent perturbations in the mitochondrial proteome that Gcc counteracted. We provide a strategy for the quantitative and organotypic assessment of treatment effects of candidate therapies in relationship to dosing, timing, and potential synergistic intervention combinations during sepsis.

#3436 EFFICACY OF ADJUNCT HEMOPERFUSION VERSUS STANDARD MEDICAL THERAPY ON 28-DAY MORTALITY IN LEPTOSPIROSIS PATIENTS WITH RENAL FAILURE AND SHOCK

Abstract Background and Aims Leptospirosis, a ubiquitous zoonotic infection in the Philippines, is a classic representation of sepsis-induced multi-organ dysfunction with its life-threatening complications: pulmonary hemorrhage, renal and liver failure. Despite pre-established treatment guidelines, leptospirosis- induced morbidity and mortality continue to rise. Hemoperfusion with HA 330 is a promising adjunct therapy as it removes rogue cytokines responsible for the dysregulated inflammatory response in severe sepsis. However, studies on its survival benefit have yielded conflicting results, so far. This study was designed to assess the efficacy and safety of hemoperfusion among leptospirosis patients in septic shock and renal failure, in terms of improvement in 28-day mortality, SOFA score, level of inflammatory markers, hemodynamics, renal and pulmonary function. Method This is an open-label randomized controlled trial which enrolled a total of 37 adult presumptive leptospirosis patients with early signs of septic shock and acute renal failure. The study participants were randomized into either of the two treatment arms: 1. standard medical therapy (SMT) alone or 2. SMT + HA330 Hemoperfusion (HP). All patients were managed based on the NKTI Leptospirosis treatment protocol (hydration, antibiotics, three-day steroid pulsing, single cyclophosphamide dose and respective organ support) and were placed on daily intermittent hemodialysis. Subjects randomized to the HP group received three hemoperfusion sessions (on top of SMT). Vital signs, urine output, vasopressor dose, P/F ratio and biochemical parameters (including level of inflammatory markers) of patients from each treatment arm were measured at baseline, day 3 and day 7 and compared subsequently. Monitoring of participants continued until 28 days post-randomization. Descriptive statistics was used to summarize the general and clinical characteristics of the participants. Independent Sample T-test, Mann-Whitney U test, and Fisher's Exact/Chi-square test was used to determine the difference of mean, median and frequency between hemoperfusion group and control group, respectively. Mean, median, or risk difference and their corresponding 95% confidence intervals were calculated. Friedman test or Wilcoxon's signed-rank test were used to determine whether differences between groups were significant over time. Intention to treat and per protocol analyses were performed. Null hypothesis was rejected at 0.05α-level of significance. Stata 15.0 (StataCorp LLC, TX) was used for data analysis. Results Hemoperfusion conferred a 36.84% (p = 0.017) risk reduction in 28-day mortality. Serial monitoring of inflammatory markers and SOFA score of patients showed significant improvement in sepsis score (p = 0.018 HP, 0.002 SMT) and levels of procalcitonin (p = 0.013 HP, 0.003 SMT), IL6 (p = 0.033 HP, 0.020 SMT) and lactate (p&amp;lt; 0.001 HP, 0.021 SMT) in both treatment arms, from baseline to Day 7. There is, however, no statistically significant difference in the change in SOFA (p = 0.965) and inflammatory marker levels if we compare HP versus SMT (p = 0.997, 0.451, 0.858, 0.052 for hsCRP, procalcitonin, IL6 and lactate, respectively). Statistically significant improvement in serum creatinine (p = 0.04) and PF ratio (p = 0.045) were observed in the hemoperfusion cohort as early as Day 3. Vasopressor dose did not differ significantly between two groups (p = 0.792). No adverse events were observed in both treatment arms. Conclusion Hemoperfusion is a safe and effective adjunct therapy in managing severe sepsis. It promotes earlier renal and pulmonary function recovery and, in doing so, improves survival of septic shock patients.

Association between Changes in Plasma Metabolism and Clinical Outcomes of Sepsis.

Current prognostic biomarkers for sepsis have limited sensitivity and specificity. This study aimed to investigate dynamic lipid metabolomics and their association with septic immune response and clinical outcomes of sepsis. This prospective cohort study included patients with sepsis who met the Sepsis 3.0 criteria. On hospitalization days 1 (D1) and 7 (D7), plasma samples were collected, and patients underwent liquid chromatography with tandem mass spectrometry. A total of 40 patients were enrolled in the study, 24 (60%) of whom were men. The median age of the enrolled patients was 81 (68-84) years. Thirty-one (77.5%) patients had a primary infection site of the lung. Participants were allocated to the survivor (25 cases) and nonsurvivor (15 cases) groups based on their 28-day survival status. Ultimately, a total of 113 lipids were detected in plasma samples on D 1 and D 7, of which 42 lipids were most abundant in plasma samples. The nonsurvival group had significantly lower lipid expression levels in lysophosphatidylcholine (LysoPC) (16 : 0, 17 : 0,18 : 0) and 18 : 1 SM than those in the survival group (p < 0.05) on D7-D1. The correlation analysis showed that D7-D1 16 : 0 LysoPC (r = 0.367, p = 0.036),17 : 0 LysoPC (r = 0.389, p = 0.025) and 18 : 0 LysoPC(r = 0.472, p = 0.006) levels were positively correlated with the percentage of CD3+ T cell in the D7-D1. Plasma LysoPC and SM changes may serve as prognostic biomarkers for sepsis, and lipid metabolism may play a role in septic immune disturbances.

Construction of Protein–Protein Interaction Networks on Capsicum Against Sepsis by Regulating of Molecular Mechanisms: Bioinformatics Approaches

We aimed to create the construction of protein–protein interaction networks for target proteins and to analyze the molecular characteristics of capsicum, screen and identify the potential targets of capsicum against sepsis using bioinformatics approaches. The potential anti-sepsis targets of capsicum were screened by chemical protein interaction analysis and matched with the published anti-sepsis related disease targets in the Online Mendelian Inheritance in Man database (OMIM) and the Genecards Database to screen out the anti-sepsis targets of capsicum. The potential targets of capsicum against sepsis were further identified by molecular docking server. Capsicum exhibited good medicinal properties. Overall, 14 potential targets were screened and 3453 disease targets were obtained from OMIM and Genecards databases. Subsequently, we found that there were 14 proteins that were both molecular targets and disease targets. The molecular docking server identified that the active ingredient beta-carotene had the ability to bind to 11 targeted proteins, which was a potential target of capsicum against sepsis. Capsicum can exert its therapeutic effects by interacting with key amino acid residues of the targeted proteins. Capsicum can inhibit tissue and organ damage of sepsis by regulating CAV1 and CTNNB1, and reduce inflammatory response of sepsis by regulating MYC, VEGFA, NR3C1 and DUSP1d, and ultimately prevent and treat sepsis.

Liver proteomic analysis reveals the key proteins involved in host immune response to sepsis.

Sepsis is a serious infection-induced response in the host, which can result in life-threatening organ dysfunction. It is of great importance to unravel the relationship between sepsis and host immune response and its mechanisms of action. Liver is one of the most vulnerable organs in sepsis, however, the specific pathogenesis of septic liver injury has not been well understood at the protein level. A total of 12 healthy Sprague-Dawley (SD) male rats aged from 6 to 8 weeks were adaptively housed in individual cages in the specific pathogen free animal room. These lab rats were grouped into two groups: treatment (N = 9) and control (N = 3) groups; only three mice from the treatment group survived and were used for subsequent experiments. A TMT-based proteomic analysis for liver tissue was performed in the septic rat model. A total of 37,012 unique peptides were identified, and then 6,166 proteins were determined, among which 5,701 were quantifiable. Compared to the healthy control group, the septic rat group exhibited 162 upregulated and 103 downregulated differentially expressed proteins (DEPs). The upregulated and downregulated DEPs were the most significantly enriched into the complement and coagulation cascades and metabolic pathways. Protein-protein interaction (PPI) analysis further revealed that the upregulated and downregulated DEPs each clustered in a PPI network. Several highly connected upregulated and downregulated DEPs were also enriched into the complement and coagulation cascades pathways and metabolic pathways, respectively. The parallel reaction monitoring (PRM) results of the selected DEPs were consistent with the results of the TMT analysis, supporting the proteomic data. Our findings highlight the roles of complement and coagulation cascades and metabolic pathways that may play vital roles in the host immune response. The DEPs may serve as clinically potential treatment targets for septic liver injury.

Therapeutic Strategies to Ameliorate Antibiotic Resistance and Host-Inflammation Response in Sepsis: an Innovative Approach

Therapeutic Strategies to Ameliorate Antibiotic Resistance and Host-Inflammation Response in Sepsis: an Innovative Approach

Mechanism of large-conductance calcium-activated potassium channel involved in inflammatory response in sepsis

To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.

CysLT1R is essential for optimal TLR4 responses and associated macrophage polarization and sepsis in response to LPS

Background: Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4 and LTE4) are arachidonic acid-derived inflammatory lipds, acting mainly via two G-protein-coupled receptors (GPCRs), cysteinyl leukotriene receptor 1 (CysLT1R) and cysteinyl leukotriene receptor 2 (CysLT2R). Cys-LTs are mainly recognized as potent modulators of asthma and allergy with their effects on mast cells. However, their role in other pathophysiological conditions are not well understood. Higher amounts of cys-LTs were reported in the lungs of mice subjected to sepsis than compared to sham mice, suggesting that CysLTs play critical role in sepsis via modulation of macrophage inflammatory responses. Objective: To investigate how cys-LT/CysLTR axis determines the inflammatory fate of macrophages in LPS/TLR4-induced sepsis. Methods: Bone marrow-derived stem cells (BMSC) from wild type (WT) and CysLT1R null ( Cysltr1-/-) mice were differentiated into macrophages and polarized to inflammatory phenotype using LPS via TLR4 pathway. Transcript and protein expression was analyzed by qPCR/RNA seq and western blotting/multiplex ELISA respectively. Results: LPS/TLR4 axis polarized WT macrophages to inflammatory phenotype by activating inflammatory intracellular signals (p38, CREB, and NFkB), upregulating surface expression of inflammatory markers (CD68, CD11b and LFA-1), enhancing phagocytosis and augmenting inflammatory cytokine secretion, in vitro. In contrast, LPS treated Cysltr1–/– macrophages exhibited attenuated TLR4 signaling observed with WT mice, suggesting that inhibition of CysLT1R prevents optimal TLR4 activation in response to LPS. Notably, reporter assays revealed a strong interaction between TLR4 and CysLT1R in HEK blue TLR4 cells overexpressing CysLT1R, further emphasizing a potential CysLT1R-TLR4 cross talk in driving TLR4-dependent inflammatory macrophage phenotype. Finally, Cysltr1–/– mice were protected from LPS-induced sepsis compared to WT mice with comcomitant reduction in the production of inflammatory cytokines. Conclusions: Our results uncover a potential crosstalk between two important innate immune receptors, TLR4-CysLT1R, in mediating macrophage inflammatory phenotype and sepsis in vivo. Understanding this interaction may offer novel potential therapeutic targets for macrophage-associated inflammatory disorders like sepsis. This work was supported by National Institutes of Health (R01AI144115 and R01HL148585) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Myeloid-KLF2 knockout neonatal mice experience mortality from lipopolysaccharide in an age-dependent and cell-specific manner

Abstract Background: Preterm neonates exhibit increased mortality from Gram negative sepsis as compared to term neonates. The innate immune system is at a critical phase of development during the neonatal period. Mechanisms underlying activation of the preterm neonatal innate immune response are poorly understood. Our prior data has shown that Kruppel-like factor (KLF)-2 is a critical transcription factor to maintain murine myeloid cells in a quiescent state. We hypothesize that myeloid KLF2 plays a critical role in the neonatal immune response in Gram negative sepsis. Methods: Preterm neonatal (P4) and term neonatal (P12) myeloid KLF2 knockout (MyK2KO) and age matched Cre mice were given 5ug/gm of E. coli O55:B5 lipopolysachharide (LPS) via intraperitoneal (i.p.) injection. For cell depletion studies, antibodies were injected i.p. 24 hours prior to LPS injection, followed by a repeat dose 24h after LPS. Ly6G antibody was used for neutrophil depletion, CD115 for monocyte depletion and equal dose of both for depletion of both cell lines. Results: P4 MyK2KO mice have significantly increased mortality within 48 h of LPS exposure as compared to P4 Cre mice (81% vs 41%, n=19, p&amp;lt;0.01 χ2test). At P12, there is 100% survival of both groups of mice. Depletion of either neutrophils, monocytes or both together, decreased the mortality in P4 MyK2KO mice from 81% to 55%. Conclusion: Preterm neonatal mice lacking myeloid KLF2 have an exaggerated response to LPS as compared to term neonatal MyK2KO or age-matched control mice. In vivo depletion of neutrophils, monocytes or both prior to LPS attenuates this response. Future studies will focus on the mechanism underlying the postnatal age-specific and cell-specific role of KLF2 in the neonatal innate immune response. Supported by grants from Rainbow Faculty Pilot Grant (Mukherjee PI) and NIH (R01 HL142647 Nayak PI)

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Extracellular vesicles: A promising therapy against SARS-CoV-2 infection

Citrulline, Intestinal Fatty Acid-Binding Protein and the Acute Gastrointestinal Injury Score as Predictors of Gastrointestinal Failure in Patients with Sepsis and Septic Shock.

Gastrointestinal (GI) failure can be both a cause of sepsis and a consequence of the systemic pro-inflammatory response in sepsis. Changes in biomarkers of enterocyte damage, citrulline and I-FABP (intestinal fatty acid binding protein), may indicate altered intestinal permeability and damage. The study group consisted of patients with sepsis (N = 28) and septic shock (N = 30); the control group included patients without infection (N = 10). Blood samples were collected for citrulline and I-FABP and a 4-point AGI score (acute GI injury score) was calculated to monitor GI function on days 1, 3, 5, 7, and 10. Citrulline concentrations in the study group were lower than in the control. Lower values were also noted in septic patients with shock when compared to the non-shock group throughout the study period. I-FABP was higher in the septic shock group than in the sepsis group only on days 1 and 3. Citrulline was lower in patients with GI failure (AGI III) when compared to AGI I/II, reaching significance on days 7 (p = 0.034) and 10 (p = 0.015); moreover, a higher AGI score was associated with an increased 28 day mortality (p = 0.038). The results indicate that citrulline measurements, along with the AGI assessment, have clinical potential in monitoring GI function and integrity in sepsis.

Neutrophils and emergency granulopoiesis drive immune suppression and an extreme response endotype during sepsis.

Sepsis arises from diverse and incompletely understood dysregulated host response processes following infection that leads to life-threatening organ dysfunction. Here we showed that neutrophils and emergency granulopoiesis drove a maladaptive response during sepsis. We generated a whole-blood single-cell multiomic atlas (272,993 cells, n = 39 individuals) of the sepsis immune response that identified populations of immunosuppressive mature and immature neutrophils. In co-culture, CD66b+ sepsis neutrophils inhibited proliferation and activation of CD4+ T cells. Single-cell multiomic mapping of circulating hematopoietic stem and progenitor cells (HSPCs) (29,366 cells, n = 27) indicated altered granulopoiesis in patients with sepsis. These features were enriched in a patient subset with poor outcome and a specific sepsis response signature that displayed higher frequencies of IL1R2+ immature neutrophils, epigenetic and transcriptomic signatures of emergency granulopoiesis in HSPCs and STAT3-mediated gene regulation across different infectious etiologies and syndromes. Our findings offer potential therapeutic targets and opportunities for stratified medicine in severe infection.

P120-Catenin suppresses NLRP3 inflammasome activation in macrophages.

Inflammasome activation is of central importance for the process of generation of overwhelming inflammatory response and the pathogenesis of sepsis. The intrinsic molecular mechanism for controlling inflammasome activation is still poorly understood. Here we investigated the role of p120-catenin expression in macrophages in regulating nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)- and pyrin domain-containing proteins 3 (NLRP3) inflammasome activation. Depletion of p120-catenin in murine bone marrow-derived macrophages enhanced caspase-1 activation and secretion of active interleukin (IL)-1β in response to ATP stimulation following LPS priming. Coimmunoprecipitation analysis showed that p120-catenin deletion promoted NLRP3 inflammasome activation by accelerating the assembly of the inflammasome complex comprised of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Depletion of p120-catenin also increased the production of mitochondrial reactive oxygen species. Pharmacological inhibition of mitochondrial reactive oxygen species nearly completely abolished NLRP3 inflammasome activation, caspase-1 activation, and the production of IL-1β in p120-catenin-depleted macrophages. Furthermore, p120-catenin ablation significantly disrupted mitochondrial function, evidenced by decreased mitochondrial membrane potential and lower production of intracellular ATP. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of p120-catenin-deficient macrophages dramatically enhanced the accumulation of IL-1β and IL-18 in bronchoalveolar lavage fluid. These results demonstrate that p120-catenin prevents NLRP3 inflammasome activation in macrophages by maintaining mitochondrial homeostasis and reducing the production of mitochondrial reactive oxygen species in response to endotoxin insult. Thus, inhibition of NLRP3 inflammasome activation by stabilization of p120-catenin expression in macrophages may be a novel strategy to prevent an uncontrolled inflammatory response in sepsis.

Thymol regulates the Endothelin-1 at gene expression and protein synthesis levels in septic rats

Sepsis is a serious systemic inflammatory response to infections. In this study, effects of thymol treatments on sepsis response were investigated. A total of 24 rats were randomly divided into 3 different treatment groups, namely as Control, Sepsis and Thymol. A sepsis model was created with a cecal ligation and perforation (CLP) in the sepsis group. For the treatment group, 100 mg/kg dose of thymol was administered via oral gavage and sepsis was established with a CLP after 1 h. All rats were sacrificed at 12 h post-opia. Blood and tissue samples were taken. ALT, AST, urea, creatinine and LDH were evaluated to assess the sepsis response in separated sera. Gene expression analysis was conducted for ET-1, TNF-α, IL-1 in lung, kidney and liver tissue samples. ET-1 and thymol interactions were determined by molecular docking studies. The ET-1, SOD, GSH-Px and MDA levels were determined by ELISA method. Genetic, biochemical and histopathological results were evaluated statistically. The pro-inflammatory cytokines and ET-1 gene expression revealed a significant decrease in the treatment groups, while there was an increase in septic groups. SOD, GSH-Px and MDA levels of rat tissues were significantly different in the thymol groups as compared to the sepsis groups (p < 0.05). Likewise, ET-1 levels were significantly reduced in the thymol groups. In terms of serum parameters, present findings were consistent with the literature. It was concluded based on present findings that thymol therapy may reduce sepsis-related morbidity, which would be beneficial in the early phase of the sepsis.