Reduced Neutrophil Infiltration Research Articles

Caffeic acid modulates activation of neutrophils and attenuates sepsis-induced organ injury by inhibiting 5-LOX/LTB4 pathway

BackgroundSepsis is a critical systemic inflammatory syndrome which usually leads to multiple organ dysfunction. Caffeic acid (CA), a phenolic compound derived from various plants, has been proved to be essential in neuroprotection, but its role in septic organ damage is unclear. This research aimed to investigate whether CA protects against organ injury in a mouse model of cecal ligation and puncture (CLP). MethodsCA (30 mg/kg) or vehicle was administered by intraperitoneal injection immediately after CLP. The samples of blood, lungs, and livers were collected 24 h later. Organ injury was assessed by histopathological examination (HE staining), neutrophil infiltration (myeloperoxidase fluorescence), oxidative stress levels (MDA, SOD, HO-1), and inflammatory cytokines (TNF-α, IL-1β, and IL-6) release in lung and liver tissues. Neutrophil extracellular trap (NET) formation was analyzed by immunofluorescence. In vitro experiments were performed to investigate the potential mechanisms of CA using small interfering RNA (siRNA) techniques in neutrophils, and the effect of CA on neutrophil apoptosis was analyzed by flow cytometry. ResultsResults showed that CA treatment improved the 7-day survival rate and attenuated the histopathological injury in the lung and liver of CLP mice. CA significantly reduced neutrophil infiltration in the lungs and livers of CLP mice. TNF-α, IL-1β, IL-6 and LTB4 were reduced in serum, lung, and liver of CA-treated CLP mice, and phosphorylation of MAPK (p38, ERK, JNK) and p65 NF-κB was inhibited in lungs and livers. CA treatment further increased HO-1 levels and enhanced superoxide dismutase (SOD) activity, but reduced malondialdehyde (MDA) levels and NET formation. Similarly, in vitro experiments showed that CA treatment and 5-LOX siRNA interference inhibited inflammatory activation and NET release in neutrophils, suppressed MAPK and NF-κB phosphorylation in LPS-treated neutrophils, and decreased LTB4 and cfDNA levels. Flow cytometric analysis revealed that CA treatment reversed LPS-mediated delayed apoptosis in human neutrophils, and Western blot also indicated that CA treatment inhibited Bcl-2 expression but increased Bax expression. CA treatment did not induce further changes in neutrophil apoptosis, inflammatory activation, and NET release when 5-LOX was knocked down by siRNA interference. ConclusionsCA has a protective effect on lung and liver injury in a murine model of sepsis, which may be related to inhibition of the 5-LOX/LTB4 pathway.

The anti-myotoxic effects and mechanisms of Sinonatrix annularis serum and a novel plasma metalloproteinase inhibitor towards Deinagkistrodon acutus envenomation

Non-venomous snakes commonly evolve natural resistance to venom to escape predators. Sinonatrix annularis serum has been shown to inhibit Deinagkistrodon acutus venom-induced hemorrhage and upregulation of serum CK, CK-MB, LDH, AST and ALT levels. Using TMT-labeled proteomics analysis, 168 proteins were found to be altered significantly in the envenomed gastrocnemius muscle and categorized into pathways such as complement and coagulation cascades, leukocyte transendothelial migration, and JAK/STAT signaling. These alterations were mitigated by S. annularis serum. Subsequently, a novel metalloproteinase inhibitor, SaMPI, was isolated from S. annularis serum by two-step chromatography. It showed strong antidotal effects against D. acutus envenomation, including inhibition of subcutaneous bleeding caused by crude venom and DaMP (a metalloproteinase derived from D. acutus) activity in a 1:1 ratio. Histology and immunoblotting analyses demonstrated that SaMPI mitigated myonecrosis, reduced neutrophil infiltration and local inflammatory factor release, and retarded JAK/STAT and MAPK signaling activation. Analysis of the SaMPI gene cloned by 5′-RACE revealed a shared sequence identity of 58–79% with other SVMP inhibitors. These findings demonstrate the protective effects of SaMPI and indicate its potential value as a candidate for viper bite adjuvant therapy.

The effect of quercetin, a flavonoid, on lung injury caused by sepsis

Background/Aim: Lung injury is frequently observed in cases with sepsis, which can lead to conditions that progress to acute respiratory distress syndrome (ARDS) causing mortality. There is no specific treatment for sepsis or sepsis-induced lung injury. Antioxidant therapy has been one of the most prominent options for treatment, according to pathophysiological studies. The aim of this study was to investigate the effects of quercetin, a powerful antioxidant, on sepsis and sepsis-related lung injury. Methods: Thirty-two adult male Sprague Dawley rats were divided into five groups. The control group (CNRL) received 1.5 ml saline via the intragastric route. The quercetin group (QUER [n=5]) underwent no sepsis procedure and received 20 mg/kg quercetin via the intragastric route starting 15 days before the procedure. The sham group (SHAM [n=6]) underwent a surgical incision and received 1.5 ml intragastric olive oil (quercetin dissolves in oil). The sepsis group (SEPS [n=7]) underwent the sepsis procedure. The sepsis and quercetin group (SEPS+QUER [n=7]) underwent the sepsis procedure and received 20 mg/kg quercetin via the intragastric route for 15 days before the procedure. Cecal ligation and puncture methods were used to induce sepsis. While ALT, AST, LDH, GGT and CRP values were analyzed from rat blood, MDA and GSH levels were analyzed from lung tissue. Results: The results showed that quercetin reduced neutrophil infiltration (TLIS 3.5 [0.26] in the SEPS group vs TLIS 2.75 [0.29] in the SEPS+QUER group [P=0.01]), intra-alveolar macrophage count (SEPS vs SEPS+QUER [P=0.01]) and cell proliferation (SEPS vs SEPS+QUER [P=0.01]), and that it helped to preserve lung anatomy during sepsis. It was observed that MDA levels in the lung tissue decreased with the treatment of quercetin to septic rats (SEPS vs SEPS+QUER [P=0.046]). Conclusion: These findings suggest that quercetin may be a potential treatment option for sepsis. However, more studies are needed to determine whether quercetin is a viable option as a therapeutic strategy in patients.

Crisaborole Inhibits Itch and Pain by Preventing Neutrophil Infiltration in a Mouse Model of Atopic Dermatitis.

Crisaborole, a phosphodiesterase 4 (PDE4) inhibitor, has been approved for the treatment of mild to moderate atopic dermatitis. Atopic dermatitis is often associated with increased pain. Using a mouse model, this study investigated whether crisaborole suppresses pain associated with atopic dermatitis and the potential mechanisms underlying it. The mouse model for atopic dermatitis was developed by repeatedly applying MC903. MC903-treated mice had increased spontaneous scratching (itch-related behaviour) and wiping behaviour (pain-related behaviour). Crisaborole was topically applied to the cheek skin of MC903-treated mice, and it reduced both itch- and pain-related behaviours in these mice. Immunofluorescence staining revealed that crisaborole reduced neutrophil infiltration and interaction of neutrophils with sensory neurones. Intradermal injection of S100A8/A9, proinflammatory neutrophil mediator, enhanced not only itch-related behaviours evoked by histamine or chloroquine, but also pain-related behaviours evoked by capsaicin. Calcium imaging of mouse dorsal root ganglion neurones revealed that pretreatment with S100A8/A9 significantly increased calcium responses to histamine and capsaicin, and the proportion of chloroquine-sensitive neurones. These findings suggest that the PDE4 inhibitor reduces itch and pain, in part by inhibiting infiltration of S100A8/A9-containing neutrophils in a mouse model of MC903-induced atopic dermatitis.

Apoptotic neutrophil-mediated inflammatory microenvironment regulation for the treatment of ARDS

Acute respiratory distress syndrome (ARDS) is a critical respiratory disease characterized by uncontrolled acute lung inflammation with high mortality. At present, effective therapeutic drugs for clinical practice are still lacking. Neutrophil apoptosis and subsequent efferocytosis by macrophages can modulate the lung inflammatory microenvironment of ARDS, thereby promoting the repair of injured lungs. Inspired by the spontaneous process and immunomodulatory properties of apoptotic neutrophils, an apoptotic neutrophil-mediated drug delivery system (T-NP@ApoNEs) for ARDS treatment was constructed. After intravenous injection, T-NP@ApoNEs could adhere to the inflammatory vascular endothelial cells and thus accumulate in the inflamed lung, which were then efficiently phagocytized by macrophages through efferocytosis. Apoptotic neutrophils and pre-loaded anti-inflammatory drug TPCA-1 could synergistically regulate the inflammatory microenvironment of ARDS lungs. It exhibited significant effects on reducing neutrophil infiltration, inhibiting cytokine storm, and promoting macrophage polarization into anti-inflammatory phenotype. In summary, the apoptotic neutrophil-mediated drug delivery strategy may provide an effective therapeutic modality for ARDS.

DNAM-1 Immunoreceptor Protects Mice from Concanavalin A-Induced Acute Liver Injury by Reducing Neutrophil Infiltration.

DNAX accessory molecule-1 (DNAM-1; CD226) is an activating immunoreceptor on T cells and NK cells. The interaction of DNAM-1 with its ligand CD155 expressed on hematopoietic and nonhematopoietic cells plays an important role in innate and adaptive immune responses. In this study, we investigated the role of the DNAM-1-CD155 axis in the pathogenesis of T cell-mediated Con A-induced acute liver injury. Unexpectedly, DNAM-1-deficient (Cd226-/-) mice exhibited more severe acute liver injury and higher concentrations of IL-6 and TNF-α than did wild-type (WT) mice after Con A injection. We found that a larger number of neutrophils infiltrated into the liver of Cd226-/- mice compared with WT mice after Con A injection. Depletion of neutrophils ameliorated liver injury and decreased IL-6 and TNF-α in Cd226-/- mice after Con A injection, suggesting that neutrophils exacerbate the liver injury in Cd226-/- mice. Hepatocytes produced more significant amounts of CXCL1, a chemoattractant for neutrophils, in Cd226-/- mice than in WT mice after Con A injection. In the coculture of hepatocytes with liver lymphocytes, either DNAM-1 deficiency in liver lymphocytes or CD155 deficiency in hepatocytes promoted CXCL1 production by hepatocytes. These results suggest that the interaction of DNAM-1 with CD155 inhibits CXCL1 production by hepatocytes, leading to ameliorating acute liver injury.

Inhibition of protein arginine deiminase 4 prevents inflammation-mediated heart failure in arthritis.

Rheumatoid arthritis is a prototypic inflammatory condition with affected patients being at greater risk of incident heart failure (HF). Targeting innate immune cell function in the pathogenesis of HF bears the potential to guide the development of future therapies. A collagen-induced arthritis (CIA) model in DBA/1 J mice was used to generate arthritis. Mice with CIA developed concentric hypertrophic myocardial remodeling, left ventricular (LV) diastolic dysfunction, and HF with elevated plasma B-type natriuretic peptide levels but preserved LV ejection fraction. Key features of HF in CIA were increased infiltration of activated neutrophils, deposition of neutrophil extracellular traps in the myocardium, and increased tissue levels of the proinflammatory cytokine IL-1β. Specific inhibition of protein arginine deiminase 4 (PAD4) by an orally available inhibitor (JBI-589), administered after the onset of clinical arthritis, prevented HF with reduced neutrophil infiltration. We identify PAD4-mediated neutrophil activation and recruitment as the key thromboinflammatory pathway driving HF development in arthritis. Targeting PAD4 may be a viable therapeutic approach for the prevention of HF secondary to chronic inflammation.

Dissecting the Therapeutic Mechanisms of Sphingosine-1-Phosphate Receptor Agonism during Ischaemia and Reperfusion.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia-reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)-cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE-cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

Type 2 innate lymphoid cells are protective against hepatic ischaemia/reperfusion injury

Although type 2 innate lymphoid cells (ILC2s) were originally found to be liver-resident lymphocytes, the role and importance of ILC2 in liver injury remains poorly understood. In the current study, we sought to determine whether ILC2 is an important regulator of hepatic ischaemia/reperfusion injury (IRI). ILC2-deficient mice (ICOS-T or NSG) and genetically modified ILC2s were used to investigate the role of ILC2s in murine hepatic IRI. Interactions between ILC2s and eosinophils or macrophages were studied in coculture. The role of human ILC2s was assessed in an immunocompromised mouse model of hepatic IRI. Administration of IL-33 prevented hepatic IRI in association with reduction of neutrophil infiltration and inflammatory mediators in the liver. IL-33-treated mice had elevated numbers of ILC2s, eosinophils, and regulatory T cells. Eosinophils, but not regulatory T cells, were required for IL-33-mediated hepatoprotection in IRI mice. Depletion of ILC2s substantially abolished the protective effect of IL-33 in hepatic IRI, indicating that ILC2s play critical roles in IL-33-mediated liver protection. Adoptive transfer of exvivo-expanded ILC2s improved liver function and attenuated histologic damage in mice subjected to IRI. Mechanistic studies combining genetic and adoptive transfer approaches identified a protective role of ILC2s through promoting IL-13-dependent induction of anti-inflammatory macrophages and IL-5-dependent elevation of eosinophils in IRI. Furthermore, invivo expansion of human ILC2s by IL-33 or transfer of exvivo-expanded human ILC2s ameliorated hepatic IRI in an immunocompromised mouse model of hepatic IRI. This study provides insight into the mechanisms of ILC2-mediated liver protection that could serve as therapeutic targets to treat acute liver injury. We report that type 2 innate lymphoid cells (ILC2s) are important regulators in a mouse model of liver ischaemia/reperfusion injury (IRI). Through manipulation of macrophage and eosinophil phenotypes, ILC2s mitigate liver inflammation and injury during liver IRI. We propose that ILC2s have the potential to serve as a therapeutic tool for protecting against acute liver injury and lay the foundation for translation of ILC2 therapy to human liver disease.

Activation of aldehyde dehydrogenase-2 improves ischemic random skin flap survival in rats.

Random skin flaps have many applications in plastic and reconstructive surgeries. However, distal flap necrosis restricts wider clinical utility. Mitophagy, a vital form of autophagy for damaged mitochondria, is excessively activated in flap ischemia/reperfusion (I/R) injury, thus inducing cell death. Aldehyde dehydrogenase-2 (ALDH2), an allosteric tetrameric enzyme, plays an important role in regulating mitophagy. We explored whether ALDH2 activated by N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide (Alda-1) could reduce the risk of ischemic random skin flap necrosis, and the possible mechanism of action. Modified McFarlane flap models were established in 36 male Sprague-Dawley rats assigned randomly to three groups: a low-dose Alda-1 group (10 mg/kg/day), a high-dose Alda-1 group (20 mg/kg/day) and a control group. The percentage surviving skin flap area, neutrophil density and microvessel density (MVD) were evaluated on day 7. Oxidative stress was quantitated by measuring the superoxide dismutase (SOD) and malondialdehyde (MDA) levels. Blood perfusion and skin flap angiogenesis were assessed via laser Doppler flow imaging and lead oxide-gelatin angiography, respectively. The expression levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α), vascular endothelial growth factor (VEGF), ALDH2, PTEN-induced kinase 1 (PINK1), and E3 ubiquitin ligase (Parkin) were immunohistochemically detected. Indicators of mitophagy such as Beclin-1, p62, and microtubule-associated protein light chain 3 (LC3) were evaluated by immunofluorescence. Alda-1 significantly enhanced the survival area of random skin flaps. The SOD activity increased and the MDA level decreased, suggesting that Alda-1 reduced oxidative stress. ALDH2 was upregulated, and mitophagy-related proteins (PINK1, Parkin, Beclin-1, p62, and LC3) were downregulated, indicating that ALDH2 inhibited mitophagy through the PINK1/Parkin signaling pathway. Treatment with Alda-1 reduced neutrophil infiltration and expressions of inflammatory cytokines. Alda-1 significantly upregulated VEGF expression, increased the MVD, promoted angiogenesis, and enhanced blood perfusion. ALDH2 activation can effectively enhance random skin flap viability via inhibiting PINK1/Parkin-dependent mitophagy. Moreover, enhancement of ALDH2 activity also exerts anti-inflammatory and angiogenic properties.

Inhibiting membrane rupture with NINJ1 antibodies limits tissue injury

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus d-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia–reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia–reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Infusion of GMSCs relieves autoimmune arthritis by suppressing the externalization of neutrophil extracellular traps via PGE2-PKA-ERK axis

IntroductionRheumatoid arthritis (RA) is a systemic autoimmune disease with limited treatment success, characterized by chronic inflammation and progressive cartilage and bone destruction. Accumulating evidence has shown that neutrophil extracellular traps (NETs) released by activated neutrophils are important for initiating and perpetuating synovial inflammation and thereby could be a promising therapeutic target for RA. K/B × N serum transfer-induced arthritis (STIA) is a rapidly developed joint inflammatory model that somehow mimics the inflammatory response in patients with RA. Human gingival-derived mesenchymal stem cells (GMSCs) have been previously shown to possess immunosuppressive effects in arthritis and humanized animal models. However, it is unknown whether GMSCs can manage neutrophils in autoimmune arthritis. ObjectivesTo evaluate whether infusion of GMSCs can alleviate RA by regulating neutrophils and NETs formation. If this is so, we will explore the underlying mechanism(s) in an animal model of inflammatory arthritis. MethodsThe effects of GMSCs on RA were assessed by comparing the symptoms of the K/B × N serum transfer-induced arthritis (STIA) model administered either with GMSCs or with control cells. Phenotypes examined included clinical scores, rear ankle thickness, paw swelling, inflammation, synovial cell proliferation, and immune cell frequency. The regulation of GMSCs on NETs was examined through immunofluorescence and immunoblotting in GMSCs-infused STIA mice and in an in vitro co-culture system of neutrophils with GMSCs. The molecular mechanism(s) by which GMSCs regulate NETs was explored both in vitro and in vivo by silencing experiments. ResultsWe found in this study that adoptive transfer of GMSCs into STIA mice significantly ameliorated experimental arthritis and reduced neutrophil infiltration and NET formation. In vitro studies also showed that GMSCs inhibited the generation of NETs in neutrophils. Subsequent investigations revealed that GMSCs secreted prostaglandin E2 (PGE2) to activate protein kinase A (PKA), which ultimately inhibited the downstream extracellular signal-regulated kinase (ERK) pathway that is essential for NET formation. ConclusionOur results demonstrate that infusion of GMSCs can ameliorate inflammatory arthritis mainly by suppressing NET formation via the PGE2-PKA-ERK signaling pathway. These findings further support the notion that the manipulation of GMSCs is a promising stem cell-based therapy for patients with RA and other autoimmune and inflammatory diseases.

CD301b +DCs orchestrate type 2 immune responses to helminth infection in the lung

Abstract Helminth parasites induce potent host type 2 immune responses (T2I) that promote parasite clearance and prevent reinfection. The parasite-induced T2I involve both innate and adaptive immune cell components, but how the activation of different cell types is coordinated during the initiation of T2I is incompletely understood. Dendritic cells (DCs) are best known for their role in bridging innate and adaptive immunity by sensing immunological insults and presenting antigens to T cells. We previously found that CD301b +DCs, a major subset of migratory type 2 conventional DCs in peripheral organs, are specifically required for the differentiation of T helper type 2 (Th2) cells in the skin- and lung-draining lymph nodes following infection with Nippostrongylus brasiliensis (Nb) in mice, but their overall role in host immune responses against Nb infection remains unclear. Here, we show that the depletion of CD301b +DCs results in reduced T2I in the lung and impaired worm clearance. The reduction in type 2 cytokine production and eosinophil influx was observed as early as 2 days post-infection before the onset of adaptive immunity, as well as in CD4T cell-depleted mice, suggesting that CD301b +DCs play a critical role in the induction of innate T2I independently of their role in Th2 cell priming. Moreover, CD301b +DC-depleted mice exhibited ameliorated lung hemorrhage with reduced neutrophil infiltration and IL-17 production early after infection. These data suggest the role of CD301b +DCs in co-regulating innate type 2 and type 3 inflammation mechanisms during the early phase of Nb infection in the lung. This work was supported by NIH grant R01AI132576.

THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS.

Acute respiratory distress syndrome (ARDS) is characterized by uncontrolled inflammation, which manifests as leukocyte infiltration and lung injury. However, the molecules that initiate this infiltration remain incompletely understood. We evaluated the effect of the nuclear alarmin IL-33 on lung damage and the immune response in LPS-induced lung injury. We established a LPS-induced lung injury mouse model. We used genetically engineered mice to investigate the relationship among the IL-33/ST2 axis, NKT cells, and ARDS. We found that IL-33 was localized to the nucleus in alveolar epithelial cells, from which it was released 1 h after ARDS induction in wild-type (WT) mice. Mice lacking IL-33 (IL-33 - / - ) or ST2 (ST2 - / - ) exhibited reduced neutrophil infiltration, alveolar capillary leakage, and lung injury in ARDS compared with WT mice. This protection was associated with decreased lung recruitment and activation of invariant nature killer (iNKT) cells and activation of traditional T cells. Then, we validated that iNKT cells were deleterious in ARDS in CD1d - / - and Vα14Τg mice. Compared with WT mice, Vα14Τg mice exhibited increased lung injury in ARDS, and the CD1d - / - mice showed outcomes opposite those of the Vα14Τg mice. Furthermore, we administered a neutralizing anti-ST2 antibody to LPS-treated WT and Vα14Τg mice 1 h before LPS administration. We found that IL-33 promoted inflammation through NKT cells in ARDS. In summary, our results demonstrated that the IL-33/ST2 axis promotes the early uncontrolled inflammatory response in ARDS by activating and recruiting iNKT cells. Therefore, IL-33 and NKT cells may be therapeutic target molecules and immune cells, respectively, in early ARDS cytokine storms.

A 3-O-sulfated heparan sulfate dodecasaccharide (12-mer) suppresses thromboinflammation and attenuates early organ injury following trauma and hemorrhagic shock.

Dysregulated inflammation and coagulation are underlying mechanisms driving organ injury after trauma and hemorrhagic shock. Heparan sulfates, cell surface glycosaminoglycans abundantly expressed on the endothelial surface, regulate a variety of cellular processes. Endothelial heparan sulfate containing a rare 3-O-sulfate modification on a glucosamine residue is anticoagulant and anti-inflammatory through high-affinity antithrombin binding and sequestering of circulating damage-associated molecular pattern molecules. Our goal was to evaluate therapeutic potential of a synthetic 3-O-sulfated heparan sulfate dodecasaccharide (12-mer, or dekaparin) to attenuate thromboinflammation and prevent organ injury. Male Sprague-Dawley rats were pre-treated subcutaneously with vehicle (saline) or dekaparin (2 mg/kg) and subjected to a trauma/hemorrhagic shock model through laparotomy, gut distention, and fixed-pressure hemorrhage. Vehicle and dekaparin-treated rats were resuscitated with Lactated Ringer's solution (LR) and compared to vehicle-treated fresh-frozen-plasma-(FFP)-resuscitated rats. Serial blood samples were collected at baseline, after induction of shock, and 3 hours after fluid resuscitation to measure hemodynamic and metabolic shock indicators, inflammatory mediators, and thrombin-antithrombin complex formation. Lungs and kidneys were processed for organ injury scoring and immunohistochemical analysis to quantify presence of neutrophils. Induction of trauma and hemorrhagic shock resulted in significant increases in thrombin-antithrombin complex, inflammatory markers, and lung and kidney injury scores. Compared to vehicle, dekaparin treatment did not affect induction, severity, or recovery of shock as indicated by hemodynamics, metabolic indicators of shock (lactate and base excess), or metrics of bleeding, including overall blood loss, resuscitation volume, or hematocrit. While LR-vehicle-resuscitated rodents exhibited increased lung and kidney injury, administration of dekaparin significantly reduced organ injury scores and was similar to organ protection conferred by FFP resuscitation. This was associated with a significant reduction in neutrophil infiltration in lungs and kidneys and reduced lung fibrin deposition among dekaparin-treated rats compared to vehicle. No differences in organ injury, neutrophil infiltrates, or fibrin staining between dekaparin and FFP groups were observed. Finally, dekaparin treatment attenuated induction of thrombin-antithrombin complex and inflammatory mediators in plasma following trauma and hemorrhagic shock. Anti-thromboinflammatory properties of a synthetic 3-O-sulfated heparan sulfate 12-mer, dekaparin, could provide therapeutic benefit for mitigating organ injury following major trauma and hemorrhagic shock.

Cannabinoid enhancement of lncRNA MMP25-AS1/MMP25 interaction reduces neutrophil infiltration and intestinal epithelial injury in HIV/SIV infection.

Intestinal epithelial barrier dysfunction, a hallmark of HIV/SIV infection, persists despite viral suppression by combination antiretroviral therapy (cART). Emerging evidence suggests a critical role for long noncoding RNAs (lncRNAs) in maintaining epithelial homeostasis. We simultaneously profiled lncRNA/mRNA expression exclusively in colonic epithelium (CE) of SIV-infected rhesus macaques (RMs) administered vehicle (VEH) or Δ-9-tetrahydrocannabinol (THC). Relative to controls, fewer lncRNAs were up- or downregulated in CE of THC/SIV compared with VEH/SIV RMs. Importantly, reciprocal expression of the natural antisense lncRNA MMP25-AS1 (up 2.3-fold) and its associated protein-coding gene MMP25 (attracts neutrophils by inactivating alpha-1 anti-trypsin/SERPINA1) (down 2.2-fold) was detected in CE of THC/SIV RMs. Computational analysis verified 2 perfectly matched complementary regions and an energetically stable (normalized binding free energy = -0.2626) MMP25-AS1/MMP25 duplex structure. MMP25-AS1 overexpression blocked IFN-γ-induced MMP25 mRNA and protein expression in vitro. Elevated MMP25 protein expression in CE of VEH/SIV but not THC/SIV RMs was associated with increased infiltration by myeloperoxidase/CD11b++ neutrophils (transendothelial migration) and epithelial CD47 (transepithelial migration) expression. Interestingly, THC administered in combination with cART increased MMP25-AS1 and reduced MMP25 mRNA/protein expression in jejunal epithelium of SIV-infected RMs. Our findings demonstrate that MMP25-AS1 is a potentially unique epigenetic regulator of MMP25 and that low-dose THC can reduce neutrophil infiltration and intestinal epithelial injury potentially by downregulating MMP25 expression through modulation of MMP25-AS1.

INNATE TL1A SIGNALING PROMOTES INTESTINAL NEUTROPHIL ACTIVATION AND COLITIS ASSOCIATED CANCER

Intestinal inflammation associated with chronic inflammatory bowel disease (IBD) increases the risk of developing colitis-associated cancer (CAC), but the cellular mechanisms driving CAC are not well defined. Polymorphisms in TNFSF15 (called TNF-like cytokine 1a or TL1A) are highly associated with IBD and the aim of this work is to evaluate a potential mechanistic role of TL1A in regulating CAC. Data from the human atlas protein database revealed that TL1A protein is present in 50% of colorectal cancer tissue samples and that high expression of TNFSF15 correlated with reduced survival. To test the functional role for TL1A in tumorigenesis, we used the well-established AOM/DSS model of CAC in mice deficient for the TL1A receptor (called death receptor 3 or DR3) and mice deficient for myeloid derived TL1A (CD11C cre+TNFSF15fl/fl). We found that both DR3-deficient mice and myeloid derived TL1A deficient mice had a significant reduction in tumor number compared to heterozygous littermate controls. To evaluate the contribution of innate lymphocytes, we used DR3-deficient mice on a RAG-deficient background in our AOM/DSS model. Even in the absence of B and T cells, DR3-deficient mice still showed significant reduction in tumor burden, suggesting innate lymphoid cell (ILC) contribution. DR3-deficient mice also revealed a significant reduction in neutrophil infiltration. Neutrophil depletion with α-Ly6G similarly resulted in a significant reduction of tumor numbers in our CAC model in a DR3 dependent manner. To test the direct regulation of neutrophils by ILCs, we establish a co-culture model. TL1A activated ILCs triggered neutrophil activation (CD177) and maturation (CD11B) which required GM-CSF. In vivo activation with a DR3 agonist confirmed intestinal ILC3 production of GM-CSF but also impacted bone marrow granulopoiesis by expanding both mature neutrophils and granulocyte-macrophage progenitors (GMPs).

The macrophages regulate intestinal motility dysfunction through the PGE2 Ptger3 axis during Klebsiella pneumonia sepsis.

Gut motility dysfunction, the most common complication of post-septic organ dysfunction, depends on immune and neuronal cells. This study aimed to investigate the mechanisms that activate these cells and the contribution of macrophages to the recovery of intestinal motility dysfunction after sepsis. Postoperative gut motility dysfunction was induced by establishing Klebsiella pneumonia sepsis in mice with selective deletion of neutrophils and macrophages in the gut. The distribution of orally administered fluorescein isothiocyanate-dextran and carmine excretion time was used to determine the severity of small bowel disease. The effect of macrophages on intestinal motility was evaluated after prostaglandin E2 therapy. We found that muscular neutrophil infiltration leading to neuronal loss in the intestine muscle triggered intestinal motility dysfunction after pneumonia sepsis; however, reduced neutrophil infiltration did not improve intestinal motility dysfunction. Moreover, macrophage depletion aggravated gut motility dysfunction. The addition of macrophages directly to a smooth muscle was responsible for the recovery of intestinal motility. Our results suggest that a direct interaction between macrophages and smooth muscle is neurologically independent of the restoration of intestinal dysmotility.

Protective Role for Smooth Muscle Cell Hepcidin in Abdominal Aortic Aneurysm.

Hepcidin is a liver-derived hormone that controls systemic iron homeostasis, by inhibiting the iron exporter ferroportin in the gut and spleen, respective sites of iron absorption and recycling. Hepcidin is also expressed ectopically in the context of cardiovascular disease. However, the precise role of ectopic hepcidin in underlying pathophysiology is unknown. In patients with abdominal aortic aneurysm (AAA), hepcidin is markedly induced in smooth muscle cells (SMCs) of the aneurysm wall and inversely correlated with the expression of LCN2 (lipocalin-2), a protein implicated in AAA pathology. In addition, plasma hepcidin levels were inversely correlated with aneurysm growth, suggesting hepcidin has a potential disease-modifying role. To probe the role of SMC-derived hepcidin in the setting of AAA, we applied AngII (Angiotensin-II)-induced AAA model to mice harbouring an inducible, SMC-specific deletion of hepcidin. To determine whether SMC-derived hepcidin acted cell-autonomously, we also used mice harboring an inducible SMC-specific knock-in of hepcidin-resistant ferroportinC326Y. The involvement of LCN2 was established using a LCN2-neutralizing antibody. Mice with SMC-specific deletion of hepcidin or knock-in of hepcidin-resistant ferroportinC326Y had a heightened AAA phenotype compared with controls. In both models, SMCs exhibited raised ferroportin expression and reduced iron retention, accompanied by failure to suppress LCN2, impaired autophagy in SMCs, and greater aortic neutrophil infiltration. Pretreatment with LCN2-neutralizing antibody restored autophagy, reduced neutrophil infiltration, and prevented the heightened AAA phenotype. Finally, plasma hepcidin levels were consistently lower in mice with SMC-specific deletion of hepcidin than in controls, indicating that SMC-derived hepcidin contributes to the circulating pool in AAA. Hepcidin elevation in SMCs plays a protective role in the setting of AAA. These findings are the first demonstration of a protective rather than deleterious role for hepcidin in cardiovascular disease. They highlight the need to further explore the prognostic and therapeutic value of hepcidin outside disorders of iron homeostasis.

Nicaraven protects against endotoxemia-induced inflammation and organ injury through modulation of AMPK/Sirt1 signaling in macrophages

Endotoxemia is a disease characterized by systemic inflammatory responses and organ injury caused by lipopolysaccharide (LPS) infection, with high mortality. Nicaraven (AVS), a potent hydroxyl radical scavenger, has been proven to regulate the inflammatory response in tumors. To investigate the protective effects and mechanisms of AVS in endotoxemia, mice were injected intraperitoneally with LPS to induce endotoxemia. AVS treatment significantly decreased the levels of pro-inflammatory cytokines in the serum, reduced neutrophil infiltration, attenuated multiple organ injury, and increased the survival rate in LPS-challenged mice. In the LPS-induced inflammatory model of macrophages, AVS inhibited macrophage activation, suppressed nitric oxide (NO) production, and inhibited the expression and secretion of pro-inflammatory cytokines. Mechanistically, AVS treatment up-regulated silence information regulator transcript-1 (Sirt1) expression in a time- and dose-dependent manner. AVS treatment activated the AMP-dependent protein kinase (AMPK)/Sirt1 signaling pathway and suppressed the activation of nuclear factor kappa B (NF-κB) in macrophages exposed to LPS. However, the anti-inflammatory effects of AVS could be reversed by the AMPK, the Sirt1 inhibitor, or the histone deacetylase inhibitor. We confirmed that the AMPK inhibitor inhibited AVS-mediated AMPK/Sirt1 activation and NF-κB p65 acetylation. These results suggested that AVS alleviated endotoxemia by activating the AMPK/Sirt1 signaling pathway in macrophages.