Therapeutic effect of adipose-derived mesenchymal stem cells in a porcine model of abdominal sepsis.

BACKGROUND: The term sepsis refers to a complex and heterogeneous syndrome. Although great progress has been made in improving the diagnosis and treatment of this condition, it continues to have a huge impact on morbidity and mortality worldwide. Mesenchymal stem cells are a population of multipotent cells that have immunomodulatory properties, anti-apoptotic effects, and antimicrobial activity. We studied these capacities in a porcine model of peritoneal sepsis. METHODS: We infused human adipose-derived mesenchymal stem cells (ADSCs) into a porcine model of peritoneal sepsis. Twenty piglets were treated with antibiotics alone (control group) or antibiotics plus peritoneal infusion of ADSCs at a concentration of 2 × 106 cells/kg or 4 × 106 cells/kg (low- and high-dose experimental groups, respectively). The animals were evaluated at different time points to determine their clinical status, biochemical and hematologic parameters, presence of inflammatory cytokines and chemokines in blood and peritoneal fluid, and finally by histologic analysis of the organs of the peritoneal cavity. RESULTS: One day after sepsis induction, all animals presented peritonitis with bacterial infection as well as elevated C-reactive protein, haptoglobin, IL-1Ra, IL-6, and IL-1b. Xenogeneic ADSC infusion did not elicit an immune response, and peritoneal administration of the treatment was safe and feasible. One day after infusion, the two experimental groups showed a superior physical condition (e.g., mobility, feeding) and a significant increase of IL-10 and TGF-ß in blood and a decrease of IL-1Ra, IL-1b, and IL-6. After 7 days, all animals treated with ADSCs had better results concerning blood biomarkers, and histopathological analysis revealed a lower degree of inflammatory cell infiltration of the organs of the peritoneal cavity. CONCLUSIONS: Intraperitoneal administration of ADSCs as an adjuvant therapy for sepsis improves the outcome and diminishes the effects of peritonitis and associated organ damage by regulating the immune system and reducing intra-abdominal adhesions in a clinically relevant porcine model of abdominal sepsis.

Significance of Serum Procalcitonin and C-Reactive Protein in the Diagnosis and Prediction of Spontaneous Bacterial Peritonitis in Decompensated Chronic Liver Disease.

The role of serum procalcitonin (PCT) and C-reactive protein (CRP) levels in the diagnosis of spontaneous bacterial peritonitis (SBP) with decompensated chronic liver disease (CLD) has been a subject of debate. The purpose of this cross-sectional, observational study was to evaluate the significance of CRP and PCT for the diagnosis and prediction of SBP in decompensated CLD patients. Fifty patients with ascites due to decompensated CLD were enrolled conveniently from the department of Gastrointestinal, Hepatobiliary and Pancreatic disorders (GHPD), BIRDEM General Hospital, Bangladesh from July 2019 to July 2020. Of these decompensated CLD patients with SBP were enrolled as the case group and without SBP as control group. Diagnostic and predictive value of PCT and CRP were calculated using the different statistical analysis. Among 50 patients, SBP was diagnosed in 9 patients (18.0%). The ROC analysis results yielded that the optimum cut off value for PCT was 0.67ng/ml and sensitivity, specificity, positive predictive value, negative predictive value, accuracy, AUC were 88.9%, 90.2%, 66.6%, 97.3, 90%, 0.947 respectively. On the contrary the optimum cut off value for CRP was 57.4mg/L and sensitivity, specificity, positive predictive value, negative predictive value, accuracy, AUC were 77.8%, 85.4%, 53.8%, 94.5%, 84%, 0.859 respectively. Our results indicate that the value of serum PCT and CRP were reliable to diagnose SBP in ascites due to decompensated CLD. Serum PCT and CRP level measurements may provide an early good diagnostic test for SBP in decompensated CLD patients.

Ascitic Fluid Calprotectin And Ratio Of Calprotectin To Total Protein In Spontaneous Bacterial Peritonitis.

Objectives: To evaluate the diagnostic and prognostic role of ascitic fluid calprotectin and its ratio to total protein in spontaneous bacterial peritonitis cases. Method: The prospective study was conducted at Kafrelsheikh University Hospital, Egypt, from November 2019 to December 2020, and comprised cirrhotic patients of either gender with ascites. Diagnostic abdominal paracentesis was performed for all patients and ascetic fluid calprotectin was measured. Patients were followed for development of spontaneous bacterial peritonitis or mortality. Data was analysed using SPSS 20. RESULTS: Of the 90 patients, 61(67.7%) were males and 29(32.2%) were females. There were 67(74.4%) patients with spontaneous bacterial peritonitis; 48(71.6%) males and 19(28.3%) females with mean age 60.42±8.3 years. The remaining 23(25.5%) did not have spontaneous bacterial peritonitis; 13(56.5%) males and 10(43.4%) females with mean age 59.7±7.4 years. The patients had significantly higher calprotectin, and calprotectin/total protein ratio (p<0.05). Logistic regression identified ascitic fluid calprotectin as a significant predictor of mortality (p=0.05). The non-survivors had significantly higher ascitic fluid calprotectin and calprotectin/total protein ratio compared to the survivors (p<0.05). CONCLUSIONS: Ascites calprotectin level and itsratio to total protein wasfound to be accurate diagnostic and predictive biomarkers for spontaneous bacterial peritonitis.

Discovery of a selective NLRP3-targeting compound with therapeutic activity in MSU-induced peritonitis and DSS-induced acute intestinal inflammation.

The aberrant activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is known to contribute to the pathogenesis of various human inflammation-related diseases. However, to date, no small-molecule NLRP3 inhibitor has been used in clinical settings. In this study, we have identified SB-222200 as a novel direct NLRP3 inhibitor through the use of drug affinity responsive target stability assay, cellular thermal shift assay, and surface plasmon resonance analysis. SB-222200 effectively inhibits the activation of the NLRP3 inflammasome in macrophages, while having no impact on the activation of NLRC4 or AIM2 inflammasome. Furthermore, SB-222200 directly binds to the NLRP3 protein, inhibiting NLRP3 inflammasome assembly by blocking the NEK7 - NLRP3 interaction and NLRP3 oligomerization. Importantly, treatment with SB-222200 demonstrates alleviation of NLRP3-dependent inflammatory diseases in mouse models, such as monosodium urate crystal-induced peritonitis and dextran sulfate sodium-induced acute intestinal inflammation. Therefore, SB-222200 holds promise as a lead compound for the development of NLRP3 inhibitors to combat NLRP3-driven disease and serves as a versatile tool for pharmacologically investigating NLRP3 biology.

GILZ Modulates the Recruitment of Monocytes/Macrophages Endowed with a Resolving Phenotype and Favors Resolution of Escherichia coli Infection.

Macrophages are important effectors of inflammation resolution that contribute to the elimination of pathogens and apoptotic cells and restoration of homeostasis. Pre-clinical studies have evidenced the anti-inflammatory and pro-resolving actions of GILZ (glucocorticoid-induced leucine zipper). Here, we evaluated the role of GILZ on the migration of mononuclear cells under nonphlogistic conditions and Escherichia coli-evoked peritonitis. TAT-GILZ (a cell-permeable GILZ-fusion protein) injection into the pleural cavity of mice induced monocyte/macrophage influx alongside increased CCL2, IL-10 and TGF-ß levels. TAT-GILZ-recruited macrophages showed a regulatory phenotype, exhibiting increased expression of CD206 and YM1. During the resolving phase of E. coli-induced peritonitis, marked by an increased recruitment of mononuclear cells, lower numbers of these cells and CCL2 levels were found in the peritoneal cavity of GILZ-deficient mice (GILZ-/-) when compared to WT. In addition, GILZ-/- showed higher bacterial loads, lower apoptosis/efferocytosis counts and a lower number of macrophages with pro-resolving phenotypes. TAT-GILZ accelerated resolution of E. coli-evoked neutrophilic inflammation, which was associated with increased peritoneal numbers of monocytes/macrophages, enhanced apoptosis/efferocytosis counts and bacterial clearance through phagocytosis. Taken together, we provided evidence that GILZ modulates macrophage migration with a regulatory phenotype, inducing bacterial clearance and accelerating the resolution of peritonitis induced by E. coli.

Uncovering the anti-inflammatory mechanisms of phenolic-enriched maple syrup extract in lipopolysaccharide-induced peritonitis in mice: insights from data-independent acquisition proteomics analysis.

Our group has previously reported on the phytochemical composition and biological activities of a phenolic-enriched maple syrup extract (MSX), which showed promising anti-inflammatory effects in several disease models including diabetes and Alzheimer's disease. However, the efficacious doses of MSX and its molecular targets involved in the anti-inflammatory effects are not fully elucidated. Herein, the efficacy of MSX in a peritonitis mouse model was evaluated in a dose-finding study and the underlying mechanisms were explored using data-independent acquisition (DIA) proteomics assay. MSX (at 15, 30 and 60 mg kg-1) alleviated lipopolysaccharide-induced peritonitis by reducing the levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α) in the serum and major organs of the mice. Furthermore, DIA proteomics analyses identified a panel of proteins that were significantly altered (both up- and down-regulated) in the peritonitis group, which were counteracted by the MSX treatments. MSX treatment also modulated several inflammatory upstream regulators including interferon gamma and TNF. Ingenuity pathway analysis suggested that MSX may modulate several signaling pathways in the processes of initiation of cytokine storm, activation of liver regeneration, and suppression of hepatocyte apoptosis. Together, these proteomic and in vivo findings indicate that MSX could regulate inflammation signaling pathways and modulate inflammatory markers and proteins, providing critical insight to its therapeutic potential.

ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis.

Peritoneal inflammation remains a major cause of treatment failure in patients with kidney failure who receive peritoneal dialysis. Peritoneal inflammation is characterized by an increase in neutrophil infiltration. However, the molecular mechanisms that control neutrophil recruitment in peritonitis are not fully understood. ELMO and DOCK proteins form complexes which function as guanine nucleotide exchange factors to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. In the current study, we found that deletion of the Elmo1 gene causes defects in chemotaxis and the adhesion of neutrophils. ELMO1 plays a role in the fMLP-induced activation of Rac1 in parallel with the PI3K and mTORC2 signaling pathways. Importantly, we also reveal that peritoneal inflammation is alleviated in Elmo1 knockout mice in the mouse model of thioglycollate-induced peritonitis. Our results suggest that ELMO1 functions as an evolutionarily conserved regulator for the activation of Rac to control the chemotaxis of neutrophils both in vitro and in vivo. Our results suggest that the targeted inhibition of ELMO1 may pave the way for the design of novel anti-inflammatory therapies for peritonitis.

Narirutin exerts anti-inflammatory activity by inhibiting NLRP3 inflammasome activation in macrophages.

Citrus peel has long been used in traditional medicine in Asia to treat common cold, dyspepsia, cough, and phlegm. Narirutin-a flavanone-7-O-glycoside-is the major flavonoid in citrus peel, and has anti-oxidative, anti-allergic, and anti-inflammatory activities. However, the anti-inflammatory mechanism of narirutin has not been fully elucidated. This study is aimed to investigate the effects of narirutin on the Nod-like receptor protein 3 (NLRP3)-mediated inflammatory response in vitro and in vivo, and determine the underlying mechanism. THP-1 differentiated macrophages and bone marrow-derived macrophages (BMDMs) were used for in vitro experiments, while dextran sulfate sodium (DSS)-induced colitis and alum-induced peritonitis mouse models were constructed to test inflammation in vivo. Narirutin suppressed secretion of interleukin (IL)-1ß and pyroptosis in lipopolysaccharide (LPS)/ATP-stimulated macrophages. Narirutin decreased the expression of NLRP3 and IL-1ß in the LPS-priming step through inhibition of NF-κB, MAPK and PI3K /AKT signaling pathways. Narirutin inhibited NLRP3-ASC interaction to suppress NLRP3 inflammasome assembly. Furthermore, oral administration of narirutin (300 mg/kg) alleviated inflammation symptoms in mice with peritonitis and colitis. These results suggest that narirutin exerts its anti-inflammatory activity by suppressing NLRP3 inflammasome activation via inhibition of the NLRP3 inflammasome priming processes and NLRP3-ASC interaction in macrophages.

Peritoneal transformation shortly after kidney transplantation in pediatric patients with preceding chronic peritoneal dialysis.

BACKGROUND: The unphysiological composition of peritoneal dialysis (PD) fluids induces progressive peritoneal fibrosis, hypervascularization and vasculopathy. Information on these alterations after kidney transplantation (KTx) is scant. METHODS: Parietal peritoneal tissues were obtained from 81 pediatric patients with chronic kidney disease stage 5 (CKD5), 72 children on PD with low glucose degradation product (GDP) PD fluids, and from 20 children 4-8 weeks after KTx and preceding low-GDP PD. Tissues were analyzed by digital histomorphometry and quantitative immunohistochemistry. RESULTS: While chronic PD was associated with peritoneal hypervascularization, after KTx vascularization was comparable to CKD5 level. Submesothelial CD45 counts were 40% lower compared with PD, and in multivariable analyses independently associated with microvessel density. In contrast, peritoneal mesothelial denudation, submesothelial thickness and fibrin abundance, number of activated, submesothelial fibroblasts and of mesothelial-mesenchymal transitioned cells were similar after KTx. Diffuse peritoneal podoplanin positivity was present in 40% of the transplanted patients. In subgroups matched for age, PD vintage, dialytic glucose exposure and peritonitis incidence, submesothelial hypoxia-inducible factor 1-alpha abundance and angiopoietin 1/2 ratio were lower after KTx, reflecting vessel maturation, while arteriolar and microvessel p16 and cleaved Casp3 were higher. Submesothelial mast cell count and interleukin-6 were lower, whereas transforming growth factor-beta induced pSMAD2/3 was similar as compared with children on PD. CONCLUSIONS: Peritoneal membrane damage induced with chronic administration of low-GDP PD fluids was less severe after KTx. While peritoneal microvessel density, primarily defining PD transport and ultrafiltration capacity, was normal after KTx and peritoneal inflammation less pronounced, diffuse podoplanin positivity and profibrotic activity were prevalent.

Skin Interstitial Fluid-Based SERS Tags Labeled Microneedles for Tracking of Peritonitis Progression and Treatment Effect.

Skin interstitial fluid (ISF)-based microneedle (MN) sensing has recently exhibited wide promise for the minimally invasive and painless diagnosis of diseases. However, it is still a great challenge to diagnose more disease types due to the limited in situ sensing techniques and insufficient ISF biomarker sources. Herein, ISF is employed to pioneer the tracking of acute peritonitis progression via surface-enhanced Raman scattering (SERS) tags labeled MNs patch technique. Densely deposited core-satellite gold nanoparticles and 3-mercaptophenylboronic acid as a Raman reporter enable the developed MNs patch with high sensitivity and selectivity in the determination of H2O2, an indicator of peritonitis development. Importantly, the MNs patch not only reliably tracks the different states of peritonitis but also evaluates the efficacy of drugs in the treatment of peritonitis, as evidenced by the altered SERS signal consistent with plasma pro-inflammatory factor (TNF-α) and peritoneum pathological manifestations. Interestingly, the major source of H2O2 in ISF of acute peritonitis investigated may not be through conventional blood capillary filtration pathway. This work provides a new route and technique for the early diagnosis of acute peritonitis and the evaluation of drug therapy effects. The developed MNs patch is promising to serve as a universal sensing tool to greatly enrich the variety and prospect of ISF-based disease diagnosis.

Neutrophil extracellular trap inhibition improves survival in neonatal mouse infectious peritonitis.

BACKGROUND: Treatment of neonatal peritonitis and sepsis is challenging. Following infection, neutrophils elaborate neutrophil extracellular traps (NETs)-extracellular lattices of decondensed chromatin decorated with antimicrobial proteins. NETs, however, can augment pathogenic inflammation causing collateral damage. We hypothesized that NET inhibition would improve survival in experimental neonatal infectious peritonitis. METHODS: We induced peritonitis in 7 to 10-day-old mice by intraperitoneal injection with cecal slurry. We targeted NETs by treating mice with neonatal NET-Inhibitory Factor (nNIF), an endogenous NET-inhibitor; Cl-amidine, a PAD4 inhibitor; DNase I, a NET degrading enzyme, or meropenem (an antibiotic). We determined peritoneal NET and cytokine levels and circulating platelet-neutrophil aggregates. Survival from peritonitis was followed for 6 days. RESULTS: nNIF, Cl-amidine, and DNase I decreased peritoneal NET formation and inflammatory cytokine levels at 24 h compared to controls. nNIF, Cl-amidine, and DNase I decreased circulating platelet-neutrophil aggregates, and NET-targeting treatments significantly increased survival from infectious peritonitis compared to controls. Finally, nNIF administration significantly improved survival in mice treated with sub-optimal doses of meropenem even when treatment was delayed until 2 h after peritonitis induction. CONCLUSIONS: NET inhibition improves survival in experimental neonatal infectious peritonitis, suggesting that NETs participate pathogenically in neonatal peritonitis and sepsis. IMPACT: 1. Neutrophil extracellular trap formation participates pathogenically in experimental neonatal infectious peritonitis. 2. NET-targeting strategies improve outcomes in a translational model of neonatal infectious peritonitis. 3. NET inhibition represents a potential target for drug development in neonatal sepsis and infectious peritonitis.

Association of longitudinal high-sensitive C-reactive protein levels with changing membrane characteristics in peritoneal dialysis.

BACKGROUND: Increasing peritoneal permeability with ultrafiltration and solute removal inadequacy is a challenging issue in peritoneal dialysis (PD). Decreasing permeability is less frequent but also results in diminished solute clearance. We evaluated the association between longitudinal high-sensitive C-reactive protein (hs-CRP) values and the change in transport characteristics of the peritoneal membrane in PD patients. METHODS: This is a retrospective, single-center study of incident PD patients. An increase or decrease in peritoneal transport status is defined as two or more categories of a rise or decline in the peritoneal equilibration test (PET) from their baseline during follow-up. The 4-h dialysate/plasma creatinine ratio was used to classify transport characteristics. Hs-CRP values were obtained from the routine annual examinations of the patients. RESULTS: Baseline demographics, residual kidney function, frequency of high glucose-containing dialysate, and icodextrin use were similar between the groups. Total episodes of peritonitis within the first 5 years of follow-up were higher in stable transporters than in increased and decreased transporters (p = 0.009). Stable transporters' mean hs-CRP values did not change within 5 years (Wilks' λ = 0.873, F (2.317, 180.740) = 2.210, p = 0.10). Increased and decreased transporters' hs-CRP values significantly raised over the years (Wilks' λ = 0.422, F (1.979, 77.163) = 3.405, p = 0.04 and Wilks' λ = 0.558, F (3.673, 66.107) = 4.396, p = 0.001, respectively). CONCLUSIONS: Our study shows that the peritoneal membrane may change into different characteristics in many patients over time, despite very low peritonitis frequencies and similar baseline characteristics that may be significantly affected by systemic inflammation.

Resolvin D2 induces anti-microbial mechanisms in a model of infectious peritonitis and secondary lung infection.

In severe bacterial infections, there is a pro-inflammatory response to promote bacterial clearance but this response can cause tissue injury. Later, the immune system becomes dysregulated and the host is unable to clear a secondary or a pre-existing infection. Specialized Pro-resolving Mediators (SPMs) such as resolvin D2 (RvD2) have been shown to be beneficial for inflammation/infection resolution in animal models of sepsis but in vivo mechanisms by which RvD2 may promote bacterial clearance and/or attenuate deleterious effects of a secondary infection have not been fully established. In this study, we used the 2-hit model of cecal ligation and puncture (CLP) induced infectious peritonitis and secondary lung infection with Pseudomonas aeruginosa to find possible antimicrobial and immunomodulatory mechanisms of RvD2. We show that RvD2 given as late as 48h after CLP surgery reduced blood bacterial load without altering plasma cytokines compared to mice given saline vehicle. RvD2 increased splenic neutrophil accumulation as well as average reactive oxygen species (ROS) production. There was also an increase in an immature leukocyte population the myeloid derived suppressor cells (MDSCs) in the spleen of RvD2 treated mice. RvD2 reduced lung lavage bacterial load 24h after P. aeruginosa administration and significantly decreased lung lavage levels of IL-23, a cytokine essential in the Th-17 inflammatory response. In addition, we show that RvD2 increased the number of non-inflammatory alveolar macrophages after P. aeruginosa administration compared to saline treated mice. The study uncovered an antimicrobial mechanism of RvD2 where RvD2 increases mature neutrophil and MDSC accumulation into the spleen to promote blood bacterial clearance. The study showed that in this 2-hit model, RvD2 promotes lung bacterial clearance, increased non-inflammatory alveolar macrophage number and inhibits an adaptive immune pathway providing evidence of its resolution mechanism in secondary pulmonary infection.

NLRP3 inflammasome activation in neutrophils directs early inflammatory response in murine peritonitis.

NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates caspase-1-dependent processing of inflammatory cytokines such as IL-1ß, an essential endothelial activator, and contributes to the pathology of inflammatory diseases. To evaluate the role of NLRP3 in neutrophils in endothelial activation, which is still elusive, we used the thioglycollate-induced peritonitis model characterized by an early neutrophil influx, on Nlrp3-/- and Nlrp3+/+ mice. Nlrp3-/- mice recruited fewer neutrophils than Nlrp3+/+ into the peritoneum and showed lower IL-1ß in peritoneal lavage fluid. The higher production of IL-1ß in Nlrp3+/+ was neutrophil-dependent as neutrophil depletion prevented the IL-1ß production. The Nlrp3+/+ neutrophils collected from the peritoneal fluid formed significantly more filaments (specks) than Nlrp3-/- neutrophils of ASC (apoptosis-associated speck-like protein containing a caspase activating and recruitment domain), a readout for inflammasome activation. Intravital microscopy revealed that leukocytes rolled significantly slower in Nlrp3+/+ venules than in Nlrp3-/-. Nlrp3-/- endothelial cells isolated from mesenteric vessels demonstrated a lower percentage of P-selectin-positive cells with lower intensity of surface P-selectin expression than the Nlrp3+/+ endothelial cells evaluated by flow cytometry. We conclude that neutrophils orchestrate acute thioglycollate-induced peritonitis by producing IL-1ß in an NLRP3-dependent manner. This increases endothelial P-selectin expression and leukocyte transmigration.

Akkermansia muciniphila Reduces Peritonitis and Improves Intestinal Tissue Wound Healing after a Colonic Transmural Defect by a MyD88-Dependent Mechanism.

Anastomotic leakage is a major complication following colorectal surgery leading to peritonitis, complications, and mortality. Akkermansia muciniphila has shown beneficial effects on the gut barrier function. Whether A. muciniphila reduces peritonitis and mortality during colonic leakage is unknown. Whether A. muciniphila can directly modulate the expression of genes in the colonic mucosa in humans has never been studied. We investigated the effects of a pretreatment (14 days) with live A. muciniphila prior to surgical colonic perforation on peritonitis, mortality, and wound healing. We used mice with an inducible intestinal-epithelial-cell-specific deletion of MyD88 (IEC-MyD88 KO) to investigate the role of the innate immune system in this context. In a proof-of-concept pilot study, healthy humans were exposed to A. muciniphila for 2 h and colonic biopsies taken before and after colonic instillation for transcriptomic analysis. Seven days after colonic perforation, A.-muciniphila-treated mice had significantly lower mortality and severity of peritonitis. This effect was associated with significant improvements of wound histological healing scores, higher production of IL22, but no changes in the mucus layer thickness or genes involved in cell renewal, proliferation, or differentiation. All these effects were abolished in IEC-MyD88 KO mice. Finally, human subjects exposed to A. muciniphila exhibited an increased level of the bacterium at the mucus level 2 h after instillation and significant changes in the expression of different genes involved in the regulation of cell cycling, gene transcription, immunity, and inflammation in their colonic mucosa. A. muciniphila improves wound healing during transmural colonic wall defect through mechanisms possibly involving IL22 signaling and requiring MyD88 in the intestinal cells. In healthy humans, colonic administration of A. muciniphila is well tolerated and changes the expression of genes involved in the immune pathways.

Age-related decline in the resistance of mice to bacterial infection and in LPS/TLR4 pathway-dependent neutrophil responses.

Host defense against bacterial and fungal infections diminishes with age. In humans, impaired neutrophil responses are thought to contribute to this decline. However, it remains unclear whether neutrophil responses are also impaired in old mice. Here, we investigated neutrophil function in old mice, focusing on responses primed by lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria like E. coli, which signals through toll-like receptor (TLR) 4. We show that old mice have a reduced capacity to clear pathogenic E. coli during septic peritonitis. Neutrophil recruitment was elevated during LPS-induced but not aseptic peritonitis. Neutrophils from old mice showed reduced killing of E. coli. Their reactive oxygen species (ROS) production was impaired upon priming with LPS but not with GM-CSF/TNFα. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas impairment of NET release in response to S. aureus was independent of LPS. Unexpectedly, chemotaxis was normal, as were Rac1 and Rac2 GTPase activities. LPS-primed activation of Erk and p38 Mapk was defective. PIP3 production was reduced upon priming with LPS but not with GM-CSF/TNFα, whereas PIP2 levels were constitutively low. The expression of 5% of neutrophil proteins was dysregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as TLR-pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. The upregulation of CD180 and downregulation of MyD88 likely contribute to the impaired LPS signaling. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS/TLR4 pathway dependence resolves previous controversy regarding effects of age on murine neutrophils and confirms that mice are an appropriate model for the decline in human neutrophil function.

Mitochondrial calcium uniporter promotes phagocytosis-dependent activation of the NLRP3 inflammasome.

Mitochondria, a highly metabolically active organelle, have been shown to play an essential role in regulating innate immune function. Mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) is an essential process regulating mitochondrial metabolism by targeting key enzymes involved in the tricarboxylic acid cycle (TCA). Accumulative evidence suggests MCU-dependent mitochondrial Ca2+ signaling may bridge the metabolic reprogramming and regulation of immune cell function. However, the mechanism by which MCU regulates inflammation and its related disease remains elusive. Here we report a critical role of MCU in promoting phagocytosis-dependent activation of NLRP3 (nucleotide-binding domain, leucine-rich repeat containing family, pyrin domain-containing 3) inflammasome by inhibiting phagolysosomal membrane repair. Myeloid deletion of MCU (McuΔmye) resulted in an attenuated phagolysosomal rupture, leading to decreased caspase-1 cleavage and interleukin (IL)-1ß release, in response to silica or alum challenge. In contrast, other inflammasome agonists such as adenosine triphosphate (ATP), nigericin, poly(dA:dT), and flagellin induced normal IL-1ß release in McuΔmye macrophages. Mechanistically, we demonstrated that decreased NLRP3 inflammasome activation in McuΔmye macrophages was caused by improved phagolysosomal membrane repair mediated by ESCRT (endosomal sorting complex required for transport)-III complex. Furthermore, McuΔmye mice showed a pronounced decrease in immune cell recruitment and IL-1ß production in alum-induced peritonitis, a typical IL-1-dependent inflammation model. In sum, our results identify a function of MCU in promoting phagocytosis-dependent NLRP3 inflammatory response via an ESCRT-mediated phagolysosomal membrane repair mechanism.

Cord Blood Natural Killer Cells Inhibit Sepsis Caused by Feces-Induced Acute Peritonitis via Increasing Endothelium Integrity.

Sepsis is associated with acute peritonitis, which can be induced by lipopolysaccharide exposure and feces. Generally, lipopolysaccharide induces mono-microbial peritonitis, whereas feces cause poly-microbial peritonitis; the latter is a more complicated and closer to the clinical diseases. Although several reports have discussed the mechanism of immune response in peritonitis-induced sepsis, however, the role of natural killer (NK) cells in sepsis, especially the relationship between NK cells and stabilization of the vascular endothelial barrier, is still unclear. Accordingly, in this study, we assessed the roles of NK cells in an acute sepsis model in mice. NK cells were injected via the tail vein into mice with acute sepsis, and nitric oxide (NO), anti-inflammatory cytokine, and angiogenic factors were tested to explore the effects of NK cells on sepsis. The survival rate of septic model mice infused with NK cells was significantly improved compared with the control group. Interestingly, the levels of NO, interleukin-10, and vascular endothelial growth factor (VEGF) decreased in NK cells therapy group. After the injection of NK cells, CD31 positive endothelial cells significantly increased in the kidneys and liver, although the expression of VEGF, ANGPT-1, and ET-1 was downregulated. Consistent with our hypothesis, the transfusion of NK cells into mice with sepsis blocked inflammation and increased endothelium integrity. Overall, these findings suggest that NK cells may block sepsis by modulating the VEGF pathway.

The α7 Nicotinic Acetylcholine Receptor Agonist GTS-21 Improves Bacterial Clearance via Regulation of Monocyte Recruitment and Activity in Polymicrobial Septic Peritonitis.

The cholinergic anti-inflammatory pathway has been identified as an effective pathway to modify inflammatory responses. Here, we verified that delayed administration with a selective α7nAChR agonist GTS-21 enables a more efficient elimination of the offending pathogens, diminished inflammatory response and organ injury, and improved survival rates in the polymicrobial septic peritonitis model. We illustrated that the improved bacterial clearance upon GTS-21 stimulation was accompanied by enhanced recruitment of monocytes into the peritoneal cavity and simultaneously increased phagocytic activity and iNOS expression of these recruited monocytes. Mechanically, splenectomy prior to administration of GTS-21 attenuated the recruitment of monocytes into the peritoneal cavity and abolished the protective benefits of GTS-21 treatment. Meanwhile, GTS-21 administration accelerates the deployment of splenic monocytes during septic peritonitis. Collectively, these data suggested that appropriate selective pharmacological α7nAChR activation promotes monocytes trafficking in a spleen-dependent manner and upregulates the antibacterial activity of recruited monocytes during septic peritonitis, which may be utilized as a promising therapeutic modality for patients suffering from septic peritonitis.

Glycoprotein 96 in Peritoneal Dialysis Effluent-Derived Extracellular Vesicles: A Tool for Evaluating Peritoneal Transport Properties and Inflammatory Status.

Background: Extracellular vesicles (EVs) from peritoneal dialysis effluent (PDE), containing molecules such as proteins and microRNAs (miRNAs), may be potential biological markers to monitor peritoneal function or injury. Peritoneal inflammation is an important determinant of peritoneal solute transport rate (PSTR). Thus, the aim of this study is to determine whether the specific proteins capable of evaluating the PSTR could be found in PDE-EVs, and explore the underlying mechanism for the association between PSTR and peritoneal inflammation. Methods: Sixty patients undergoing peritoneal dialysis (PD) were divided into two groups: high/high average transport (H/A) group (PET >0.65) and low/low average transport (L/A) group (PET <0.65). EVs derived from PDE (PDE-EVs) were isolated by ultracentrifugation. Proteomic analysis was performed to explore the differentially expressed proteins and identify the potential biomarkers in PDE-EVs from the two groups, and we focused on glycoprotein 96 (GP96) as it could be involved in the inflammatory process. The expression of GP96 in PDE-EVs and inflammatory cytokines was quantified by real-time PCR and enzyme-linked immunosorbent assay. The infiltration of macrophages and neutrophils into the peritoneum was detected using immunohistochemistry in a PD rat model. Results: The expression of PDE-EVs-GP96 was significantly higher in the H/A group, and was positively correlated with the PSTR and the level of the inflammatory factor interleukin (IL)-6. GP96-enriched EVs enhanced the secretion of proinflammatory cytokines IL-1ß, IL-6, tumor necrosis factor (TNF)-α, and IL-8 in macrophages, which was reversed by a pharmacological GP96-specific inhibitor (PU-WS13). The GP96 inhibitor also reduced local peritoneal inflammation by decreasing the infiltration of inflammatory cells and levels of proinflammatory cytokines (IL-6 and TNF-α) and chemokines (CCL2, CXCL1, and CXCL2) in a PD rat model. Conclusions: PDE-EVs-GP96 is a new promising tool to evaluate the status of peritoneal inflammation and PSTR, and the mechanism may be related to affecting the inflammatory properties of macrophages.