CD200Rhigh neutrophils with dysfunctional autophagy establish systemic immunosuppression by increasing regulatory T cells.

Distinct neutrophil populations arise during certain pathological conditions. The generation of dysfunctional neutrophils during sepsis and their contribution to septicemia-related systemic immune suppression remain unclear. In this study, using an experimental sepsis model that features immunosuppression, we identified a novel population of pathogenic CD200Rhigh neutrophils that are generated during the initial stages of sepsis and contribute to systemic immune suppression by enhancing regulatory T (Treg) cells. Compared to their CD200Rlow counterparts, sepsis-generated CD200Rhigh neutrophils exhibit impaired autophagy and dysfunction, with reduced chemotactic migration, superoxide anion production, and TNF-α production. Increased soluble CD200 blocks autophagy and neutrophil maturation in the bone marrow during experimental sepsis, and recombinant CD200 treatment in vitro can induce neutrophil dysfunction similar to that observed in CD200Rhigh neutrophils. The administration of an α-CD200R antibody effectively reversed neutrophil dysfunction by enhancing autophagy and protecting against a secondary infection challenge, leading to increased survival. Transcriptome analysis revealed that CD200Rhigh neutrophils expressed high levels of Igf1, which elicits the generation of Treg cells, while the administration of an α-CD200R antibody inhibited Treg cell generation in a secondary infection model. Taken together, our findings revealed a novel CD200Rhigh neutrophil population that mediates the pathogenesis of sepsis-induced systemic immunosuppression by generating Treg cells.

Sphingosylphosphorylcholine inhibits plasma cell differentiation and ameliorates experimental autoimmune encephalomyelitis.

Introduction: Multiple sclerosis (MS) is a potentially disabling disease that damages the brain and spinal cord, inducing paralysis of the body. While MS has been known as a T-cell mediated disease, recent attention has been drawn to the involvement of B cells in its pathogenesis. Autoantibodies from B cells are closely related with the damage lesion of central nervous system and worse prognosis. Therefore, regulating the activity of antibody secreting cell could be related with the severity of the MS symptoms. Methods: Total mouse B cells were stimulated with LPS to induce their differentiation into plasma cells. The differentiation of plasma cells was subsequently analyzed using flow cytometry and quantitative PCR analysis. To establish an experimental autoimmune encephalomyelitis (EAE) mouse model, mice were immunized with MOG35-55/CFA emulsion. Results: In this study, we found that plasma cell differentiation was accompanied by upregulation of autotaxin, which converts sphingosylphosphorylcholine (SPC) to sphingosine 1-phosphate in response to LPS. We observed that SPC strongly blocked plasma cell differentiation from B cells and antibody production in vitro. SPC downregulated LPS-stimulated IRF4 and Blimp 1, which are required for the generation of plasma cells. SPC-induced inhibitory effects on plasma cell differentiation were specifically blocked by VPC23019 (S1PR1/3 antagonist) or TY52159 (S1PR3 antagonist), but not by W146 (S1PR1 antagonist) and JTE013 (S1PR2 antagonist), suggesting a crucial role of S1PR3 but not S1PR1/2 in the process. Administration of SPC against an EAE mouse model significantly attenuated the symptoms of disease, showing decreased demyelinated areas of the spinal cord and decreased numbers of cells infiltrated into the spinal cord. SPC markedly decreased plasma cell generation in the EAE model, and SPC-induced therapeutic effects against EAE were not observed in µMT mice. Conclusion: Collectively, we demonstrate that SPC strongly inhibits plasma cell differentiation, which is mediated by S1PR3. SPC also elicits therapeutic outcomes against EAE, an experimental model of MS, suggesting SPC as a new material to control MS.

Unique characteristics of lung-resident neutrophils are maintained by PGE2/PKA/Tgm2-mediated signaling.

Lung-resident neutrophils need to be tightly regulated to avoid degranulation- and cytokine-associated damage to fragile alveolar structures that can lead to fatal outcomes. Here we show that lung neutrophils (LNs) express distinct surface proteins and genes that distinguish LNs from bone marrow and blood neutrophils. Functionally, LNs show impaired migratory activity toward chemoattractants and produce high levels of interleukin-6 (IL-6) at steady state and low levels of tumor necrosis factor-α in response to lipopolysaccharide (LPS) challenge. Treating bone marrow neutrophils with bronchoalveolar lavage fluid or prostaglandin E2 induces LN-associated characteristics, including the expression of transglutaminase 2 (Tgm2) and reduced production of inflammatory cytokines upon LPS challenge. Neutrophils from Tgm2-/- mice release high levels of inflammatory cytokines in response to LPS. Lung damage is significantly exacerbated in Tgm2-/- mice in an LPS-induced acute respiratory distress syndrome model. Collectively, we demonstrate that prostaglandin E2 is a key factor for the generation of LNs with unique immune suppressive characteristics, acting through protein kinase A and Tgm2, and LNs play essential roles in protection of the lungs against pathogenic inflammation.

VU0155069 inhibits inflammasome activation independent of phospholipase D1 activity.

The inflammasome is a specialized multiprotein oligomer that regulates IL-1ß production. Although regulation of the inflammasome is related to crucial inflammatory disorders such as sepsis, pharmacological inhibitors that effectively inhibit inflammasome activity are limited. Here, we evaluated the effects of a phospholipase D1 (PLD1)-selective inhibitor (VU0155069) against sepsis and inflammasome activation. VU0155069 strongly enhances survival rate in cecal ligation and puncture (CLP)-induced sepsis by inhibiting lung inflammation, leukocyte apoptosis, and the production of proinflammatory cytokines, especially IL-1ß. VU0155069 also significantly blocked IL-1ß production, caspase-1 activation, and pyroptosis caused by several inflammasome-activating signals in the bone marrow-derived macrophages (BMDMs). However, VU0155069 did not affect LPS-induced activation of signaling molecules such as MAPK, Akt, NF-κB, and NLRP3 expression in the BMDMs. VU0155069 also failed to affect mitochondrial ROS generation and calcium increase caused by nigericin or ATP, and subsequent ASC oligomerization caused by several inflammasome-activating signals. VU0155069 indirectly inhibited caspase-1 activity caused by LPS + nigericin in BMDMs independent of PLD1 activity. We demonstrated that a PLD1 inhibitor, VU0155069, shows anti-septic activity as well as inflammasome-inhibiting effects. Our results suggest that VU0155069 can be considered a novel inflammasome inhibitor.

A phospholipase D2 inhibitor, CAY10594, ameliorates acetaminophen-induced acute liver injury by regulating the phosphorylated-GSK-3ß/JNK axis.

We examined the role of phospholipase D2 (PLD2) on acetaminophen (APAP)-induced acute liver injury using a PLD2 inhibitor (CAY10594). 500 mg/kg of APAP challenge caused acute liver damage. CAY10594 administration markedly blocked the acute liver injury in a dose-dependent manner, showing almost complete inhibition with 8 mg/kg of CAY10594. During the pathological progress of acute liver injury, GSH levels are decreased, and this is significantly recovered upon the administration of CAY10594 at 6 hours post APAP challenge. GSK-3ß (Serine 9)/JNK phosphorylation is mainly involved in APAP-induced liver injury. CAY10594 administration strongly blocked GSK-3ß (Serine 9)/JNK phosphorylation in the APAP-induced acute liver injury model. Consistently, sustained JNK activation in the cytosol and mitochondria from hepatocytes were also decreased in CAY10594-treated mice. Many types of immune cells are also implicated in APAP-induced liver injury. However, neutrophil and monocyte populations were not different between vehicle- and CAY10594-administered mice which are challenged with APAP. Therapeutic administration of CAY10594 also significantly attenuated liver damage caused by the APAP challenge, eliciting an enhanced survival rate. Taken together, these results indicate that PLD2 is involved in the intrinsic response pathway of hepatocytes driving the pathogenesis of APAP-induced acute liver injury, and PLD2 may therefore represent an important therapeutic target for patients with drug-induced liver injury.

Lysophosphatidic acid protects against acetaminophen-induced acute liver injury.

We investigated the effect of lysophosphatidic acid (LPA) in experimental acetaminophen (APAP)-induced acute liver injury. LPA administration significantly reduced APAP-challenged acute liver injury, showing attenuated liver damage, liver cell death and aspartate aminotransferase and alanine aminotransferase levels. APAP overdose-induced mortality was also significantly decreased by LPA administration. Regarding the mechanism involved in LPA-induced protection against acute liver injury, LPA administration significantly increased the glutathione level, which was markedly decreased in APAP challenge-induced acute liver injury. LPA administration also strongly blocked the APAP challenge-elicited phosphorylation of JNK, ERK and GSK3ß, which are involved in the pathogenesis of acute liver injury. Furthermore, LPA administration decreased the production of TNF-α and IL-1ß in an experimental drug-induced liver injury animal model. Mouse primary hepatocytes express LPA1,3-6, and injection of the LPA receptor antagonist KI16425 (an LPA1,3-selective inhibitor) or H2L 5765834 (an LPA1,3,5-selective inhibitor) did not reverse the LPA-induced protective effects against acute liver injury. The therapeutic administration of LPA also blocked APAP-induced liver damage, leading to an increased survival rate. Collectively, these results indicate that the well-known bioactive lipid LPA can block the pathogenesis of APAP-induced acute liver injury by increasing the glutathione level but decreasing inflammatory cytokines in an LPA1,3,5-independent manner. Our results suggest that LPA might be an important therapeutic agent for drug-induced liver injury.

Identification of novel peptides that stimulate human neutrophils.

Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we identified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified peptides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-dependent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leukocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimulated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide receptor (FPR)1 while the other peptide (MMHWAM) acted on FPR2. Since the three novel peptides were specific agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling.