Protectin Conjugates in Tissue Regeneration 1 Inhibits Macrophage Pyroptosis by Restricting NLRP3 Inflammasome Assembly to Mitigate Sepsis via the cAMP-PKA Pathway.

Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel anti-inflammatory and proresolving lipid mediator biosynthesized from docosahexaenoic acid. Excessive activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and consequent pyroptosis are involved in diverse inflammatory diseases. However, how PCTR1 affects NLRP3 inflammasome activation and pyroptosis are still unclear. Here, we demonstrated that PCTR1 inhibited NLRP3 inflammasome activation and pyroptosis. These results show that PCTR1 dose-dependently inhibited gasdermin D cleavage in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin stimulation. Additionally, PCTR1 treatment after LPS priming inhibited caspase-1 activation and subsequent mature interleukin-1ß release independent of the nuclear factor-kappa B pathway. PCTR1 exerted its inhibitory effects by blocking NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC) interaction and ASC oligomerization, thereby restricting NLRP3 inflammasome assembly. However, the inhibitory effect of PCTR1 could be reversed by KH7 and H89, which are the inhibitors of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Moreover, PCTR1 treatment alleviated lung tissue damage and improved mouse survival in LPS-induced sepsis. Our study unveils the molecular mechanism of negative regulation of NLRP3 inflammasome activation and pyroptosis by a novel lipid mediator and suggests that PCTR1 may serve as a potential treatment option for NLRP3-inflammasome driven diseases.

RvD1 accelerates the resolution of inflammation by promoting apoptosis of the recruited macrophages via the ALX/FasL-FasR/caspase-3 signaling pathway.

The uncontrolled inflammatory response caused by a disorder in inflammation resolution is one of the reasons for acute respiratory distress syndrome (ARDS). The macrophage pool markedly expands when inflammatory monocytes, known as recruited macrophages, migrate from the circulation to the lung. The persistent presence of recruited macrophages leads to chronic inflammation in the resolution phase of inflammation. On the contrary, elimination of the recruited macrophages at the injury site leads to the rapid resolution of inflammation. Resolvin D1 (RvD1) is an endogenous lipid mediator derived from docosahexaenoic acid. Mice were administered RvD1 via the tail vein 3 and 4 days after stimulation with lipopolysaccharide. RvD1 reduced the levels of the inflammatory factors in the lung tissue, promoted the anti-inflammatory M2 phenotype, and enhanced the phagocytic function of recruited macrophages to alleviate acute lung injury. We also found that the number of macrophages was decreased in BAL fluid after treatment with RvD1. RvD1 increased the apoptosis of recruited macrophages partly via the FasL-FasR/caspase-3 signaling pathway, and this effect could be blocked by Boc-2, an ALX/PRP2 inhibitor. Taken together, our findings reinforce the concept of therapeutic targeting leading to the apoptosis of recruited macrophages. Thus, RvD1 may provide a new therapy for the resolution of ARDS.

Resolvin Conjugates in Tissue Regeneration 1 Promote Alveolar Fluid Clearance by Activating Alveolar Epithelial Sodium Channels and Na, K-ATPase in Lipopolysaccharide-Induced Acute Lung Injury.

Acute respiratory distress syndrome (ARDS), a common and fatal clinical condition, is characterized by the destruction of epithelium and augmented permeability of the alveolar-capillary barrier. Resolvin conjugates in tissue regeneration 1 (RCTR1) is an endogenous lipid mediator derived from docosahexaenoic acid , exerting proresolution effects in the process of inflammation. In our research, we evaluated the role of RCTR1 in alveolar fluid clearance (AFC) in lipopolysaccharide-induced ARDS/acute lung injury (ALI) rat model. Rats were injected with RCTR1 (5 µg/kg) via caudal veins 8 hours after lipopolysaccharide (LPS) (14 mg/kg) treatment, and then AFC was estimated after 1 hour of ventilation. Primary type II alveolar epithelial cells were incubated with LPS (1 ug/ml) with or without RCTR1 (10 nM) for 8 hours. Our results showed that RCTR1 significantly enhanced the survival rate, promoted the AFC, and alleviated LPS-induced ARDS/ALI in vivo. Furthermore, RCTR1 remarkably elevated the protein expression of sodium channels and Na, K-ATPase and the activity of Na, K-ATPase in vivo and in vitro. Additionally, RCTR1 also decreased neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) level via upregulating Ser473-phosphorylated-Akt expression. Besides this, inhibitors of receptor for lipoxin A4 (ALX), cAMP, and phosphatidylinositol 3-kinase (PI3K) (BOC-2, KH-7, and LY294002) notably inhibited the effects of RCTR1 on AFC. In summary, RCTR1 enhances the protein levels of sodium channels and Na, K-ATPase and the Na, K-ATPase activity to improve AFC in ALI through ALX/cAMP/PI3K/Nedd4-2 pathway, suggesting that RCTR1 may become a therapeutic drug for ARDS/ALI. SIGNIFICANCE STATEMENT: RCTR1, an endogenous lipid mediator, enhanced the rate of AFC to accelerate the resolution of inflammation in the LPS-induced murine lung injury model. RCTR1 upregulates the expression of epithelial sodium channels (ENaCs) and Na, K-ATPase in vivo and in vitro to accelerate the AFC. The efficacy of RCTR1 on the ENaC and Na, K-ATPase level was in an ALX/cAMP/PI3K/Nedd4-2-dependent manner.

Rosuvastatin Enhances Alveolar Fluid Clearance in Lipopolysaccharide-Induced Acute Lung Injury by Activating the Expression of Sodium Channel and Na,K-ATPase via the PI3K/AKT/Nedd4-2 Pathway.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are devastating clinical conditions characterized by pulmonary epithelial damage and protein-rich fluid accumulation in the alveolar spaces. Statins are a class of HMG-CoA reductase inhibitors, which exert cholesterol-lowering and anti-inflammatory effects. METHODS: Rosuvastatin (1 mg/kg) was injected intravenously in rats 12 h before lipopolysaccharide (LPS, 10 mg/kg) administration. Eight hours later after LPS challenge, alveolar fluid clearance (AFC) was detected in rats (n = 6-8). Rosuvastatin (0.3 µmol/mL) and LPS were cultured with primary rat alveolar type II epithelial cells for 8 h. RESULTS: Rosuvastatin obviously improved AFC and attenuated lung-tissue damage in ALI model. Moreover, it enhanced AFC by increasing sodium channel and Na,K-ATPase protein expression. It also up-regulated P-Akt via reducing Nedd4-2 in vivo and in vitro. Furthermore, LY294002 blocked the increase in AFC in response to rosuvastatin. Rosuvastatin-induced AFC was found to be partly rely on sodium channel and Na,K-ATPase expression via the PI3K/AKT/Nedd4-2 pathway. CONCLUSION: In summary, the findings of our study revealed the potential role of rosuvastatin in the management of ALI/ARDS.

LXA4 Inhibits Lipopolysaccharide-Induced Inflammatory Cell Accumulation by Resident Macrophages in Mice.

INTRODUCTION: Alveolar macrophages that regulate the inflammatory response in lungs are the main target cell for the treatment of inflammatory pulmonary pathologies, such as acute respiratory distress syndrome (ARDS). Yolk sac derived alveolar resident macrophages play an important role in the pulmonary inflammatory response. With regards to anti-inflammatory actions, lipoxin A4 (LXA4) has been identified as an inflammatory "braking signal". METHODS: In vivo, LXA4 (0.1 µg/mouse) was injected intraperitoneally after intratracheal (1 mg/kg) lipopolysaccharide (LPS) administration; flow cytometry was used to measure peripheral blood monocyte derived recruited macrophage and neutrophil numbers; resident alveolar macrophage was depleted by liposome clodronate; CXCL2, CCL2, MMP9 level was detected by RT-PCR and ELISA. In vitro, sorted resident macrophages (1×106) were cultured with LPS (1 µg/mL) and LXA4 (100 nmol/mL) with or without BOC-2 (10 µM) for 24 h to gain a better understanding of the mechanisms of LXA4. RESULTS: LXA4 inhibited tumor necrosis factor-a (TNF-a) and interleukin-1ß (IL-1ß) production induced by LPS. LXA4 also mediated LPS-induced macrophage recruitment and showed that this was dependent on CCL2 secretion and release by resident macrophages. LXA4 protects lung tissue by inhibiting neutrophil recruitment, partly through the CXCL2/MMP-9 signaling pathway. CXCL2 and MMP-9 are mainly expressed by resident macrophages and neutrophils, respectively. Finally, LXA4's beneficial effects were abrogated by BOC-2, an LXA4 receptor inhibitor. CONCLUSION: These results suggest that LXA4 may be a promising therapy for preventing and treating ARDS.

PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury.

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

MCTR1 enhances the resolution of lipopolysaccharide-induced lung injury through STAT6-mediated resident M2 alveolar macrophage polarization in mice.

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro-resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post-treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS-induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin-1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS-induced lung injury.

RvD1 ameliorates LPS-induced acute lung injury via the suppression of neutrophil infiltration by reducing CXCL2 expression and release from resident alveolar macrophages.

Acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS) are life-threatening critical syndromes characterized by the infiltration of a large number of inflammatory cells that lead to an excessive inflammatory response. Resolvin D1 (RvD1), an endogenous lipid mediator, is believed to have anti-inflammatory and proresolving effects. In the present study, we examined the impact of RvD1 on the pulmonary inflammatory response, neutrophil influx, and lung damage in a murine model of lipopolysaccharide (LPS)-induced ALI. Treatment with RvD1 protected mice against LPS-induced ALI, and compared to untreated mice, RvD1-treated mice exhibited significantly ameliorated lung pathological changes, decreased tumor necrosis factor-α (TNF-α) concentrations and attenuated neutrophil infiltration. In addition, treatment with RvD1 attenuated LPS-induced neutrophil infiltration via the downregulation of CXCL2 expression on resident alveolar macrophages. Finally, BOC-2, which inhibits the RvD1 receptor lipoxin A4 receptor/formyl peptide receptor 2 (ALX/FPR2), reversed the protective effects of RvD1. These data demonstrate that RvD1 ameliorates LPS-induced ALI via the suppression of neutrophil infiltration by an ALX/FPR2-dependent reduction in CXCL2 expression on resident alveolar macrophages.