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.

The role and regulation of phospholipase D in metabolic disorders.

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and free choline. In mammals, PLD exists in two well-characterized isoforms, PLD1 and PLD2, and it plays pivotal roles as signaling mediators in various cellular functions, such as cell survival, differentiation, and migration. These isoforms are predominantly expressed in diverse cell types, including many immune cells, such as monocytes and macrophages, as well as non-immune cells, such as epithelial and endothelial cells. Several previous studies have revealed that the stimulation of these cells leads to an increase in PLD expression and its enzymatic products, potentially influencing the pathological responses in a wide spectrum of diseases. Metabolic diseases, exemplified by conditions, such as diabetes, obesity, hypertension, and atherosclerosis, pose significant global health challenges. Abnormal activation or dysfunction of PLD emerges as a potential contributing factor to the pathogenesis and progression of these metabolic disorders. Therefore, it is crucial to thoroughly investigate and understand the intricate relationship between PLD and metabolic diseases. In this review, we provide an in-depth overview of the functional roles and molecular mechanisms of PLD involved in metabolic diseases. By delving into the intricate interplay between PLD and metabolic disorders, this review aims to offer insights into the potential therapeutic interventions.

WKYMVm ameliorates obesity by improving lipid metabolism and leptin signalling.

Obesity is a metabolic disorder that results from an imbalance of energy intake and consumption. As low-grade chronic inflammation caused by obesity can lead to various complications, it is important to develop effective treatments against obesity. In this study, we investigate the effects of WKYMVm, a strong anti-inflammatory agent, against obesity. Administration of WKYMVm into high fat diet (HFD)-induced obese mice significantly attenuated body weight gain, food intake and increased insulin sensitivity. HFD-induced hepatic steatosis and adipose tissue hypertrophy were also markedly ameliorated by WKYMVm. During the maturation of adipocytes, WKYMVm improves lipid metabolism by increasing lipolysis, adipogenesis, mitochondrial biogenesis and fat browning. WKYMVm administration also elicited a decrease in leptin levels, but an increase in leptin sensitivity via regulation of hypothalamic endoplasmic reticulum stress and the leptin receptor cascade. Taken together, our results show that WKYMVm ameliorates obesity by improving lipid metabolism and leptin signalling, suggesting that WKYMVm can be a useful molecule for the development of anti-obesity agents.

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.

Functional roles of sphingolipids in immunity and their implication in disease.

Sphingolipids, which are components of cellular membranes and organ tissues, can be synthesized or degraded to modulate cellular responses according to environmental cues, and the balance among the different sphingolipids is important for directing immune responses, regardless of whether they originate, as intra- or extracellular immune events. Recent progress in multiomics-based analyses and methodological approaches has revealed that human health and diseases are closely related to the homeostasis of sphingolipid metabolism, and disease-specific alterations in sphingolipids and related enzymes can be prognostic markers of human disease progression. Accumulating human clinical data from genome-wide association studies and preclinical data from disease models provide support for the notion that sphingolipids are the missing pieces that supplement our understanding of immune responses and diseases in which the functions of the involved proteins and nucleotides have been established. In this review, we analyze sphingolipid-related enzymes and reported human diseases to understand the important roles of sphingolipid metabolism. We discuss the defects and alterations in sphingolipid metabolism in human disease, along with functional roles in immune cells. We also introduce several methodological approaches and provide summaries of research on sphingolipid modulators in this review that should be helpful in studying the roles of sphingolipids in preclinical studies for the investigation of experimental and molecular medicines.

Emerging roles of neutrophils in immune homeostasis.

Neutrophils, the most abundant innate immune cells, play essential roles in the innate immune system. As key innate immune cells, neutrophils detect intrusion of pathogens and initiate immune cascades with their functions; swarming (arresting), cytokine production, degranulation, phagocytosis, and projection of neutrophil extracellular trap. Because of their short lifespan and consumption during immune response, neutrophils need to be generated consistently, and generation of newborn neutrophils (granulopoiesis) should fulfill the environmental/systemic demands for training in cases of infection. Accumulating evidence suggests that neutrophils also play important roles in the regulation of adaptive immunity. Neutrophil-mediated immune responses end with apoptosis of the cells, and proper phagocytosis of the apoptotic body (efferocytosis) is crucial for initial and post resolution by producing tolerogenic innate/adaptive immune cells. However, inflammatory cues can impair these cascades, resulting in systemic immune activation; necrotic/pyroptotic neutrophil bodies can aggravate the excessive inflammation, increasing inflammatory macrophage and dendritic cell activation and subsequent TH1/TH17 responses contributing to the regulation of the pathogenesis of autoimmune disease. In this review, we briefly introduce recent studies of neutrophil function as players of immune response. [BMB Reports 2022; 55(10): 473-480].

2-Undecanone derived from Pseudomonas aeruginosa modulates the neutrophil activity.

Pseudomonas aeruginosa (P. aeruginosa) is a well-known Gramnegative opportunistic pathogen. Neutrophils play key roles in mediating host defense against P. aeruginosa infection. In this study, we identified a metabolite derived from P. aeruginosa that regulates neutrophil activities. Using gas chromatography-mass spectrometry, a markedly increased level of 2-undecanone was identified in the peritoneal fluid of P. aeruginosa-infected mice. 2-Undecanone elicited the activation of neutrophils in a Gαi-phospholipase C pathway. However, 2-undecanone strongly inhibited responses to lipopolysaccharide and bactericidal activity of neutrophils against P. aeruginosa by inducing apoptosis. Our results demonstrate that 2-undecanone from P. aeruginosa limits the innate defense activity of neutrophils, suggesting that the production of inhibitory metabolites is a strategy of P. aeruginosa for escaping the host immune system. [BMB Reports 2022; 55(8): 395-400].

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.

Sphingosylphosphorylcholine ameliorates experimental sjögren's syndrome by regulating salivary gland inflammation and hypofunction, and regulatory B cells.

Sjögren syndrome (SS) is an autoimmune disease in which immune cells infiltrate the exocrine gland. Since SS is caused by a disorder of the immune system, treatments should regulate the immune response. Sphingosylphosphorylcholine (SPC) is a sphingolipid that mediates cellular signaling. In immune cells, SPC has several immunomodulatory functions. Accordingly, this study verifies the immunomodulatory ability and therapeutic effect of SPC in SS. To understand the function of SPC in SS, we treated SPC in female NOD/ShiJcl (NOD) mice. The mice were monitored for 10 weeks, and inflammation in the salivary glands was checked. After SPC treatment, we detected the expression of regulatory B (Breg) cells in mouse splenocytes and the level of salivary secretion-related genes in human submandibular gland (HSG) cells. Salivary flow rate was maintained in the SPC-treated group compared to the vehicle-treated group, and inflammation in the salivary gland tissues was relieved by SPC. SPC treatment in mouse cells and HSG cells enhanced Breg cells and salivary secretion markers, respectively. This study revealed that SPC can be considered as a new therapeutic agent against SS.

IM156, a new AMPK activator, protects against polymicrobial sepsis.

IM156, a novel biguanide with higher potency of AMP-activated protein kinase activation than metformin, has inhibitory activity against angiogenesis and cancer. In this study, we investigated effects of IM156 against polymicrobial sepsis. Administration of IM156 significantly increased survival rate against caecal ligation and puncture (CLP)-induced sepsis. Mechanistically, IM156 markedly reduced viable bacterial burden in the peritoneal fluid and peripheral blood and attenuated organ damage in a CLP-induced sepsis model. IM156 also inhibited the apoptosis of splenocytes and the production of inflammatory cytokines including IL-1ß, IL-6 and IL-10 in CLP mice. Moreover, IM156 strongly inhibited the generation of reactive oxygen species and subsequent formation of neutrophil extracellular traps in response to lipopolysaccharide in neutrophils. Taken together, these results show that IM156 can inhibit inflammatory response and protect against polymicrobial sepsis, suggesting that IM156 might be a new treatment for sepsis.

Siglec-F-expressing neutrophils are essential for creating a profibrotic microenvironment in renal fibrosis.

The roles of neutrophils in renal inflammation are currently unclear. On examining these cells in the unilateral ureteral obstruction murine model of chronic kidney disease, we found that the injured kidney bore a large and rapidly expanding population of neutrophils that expressed the eosinophil marker Siglec-F. We first verified that these cells were neutrophils. Siglec-F+ neutrophils were recently detected in several studies in other disease contexts. We then showed that a) these cells were derived from conventional neutrophils in the renal vasculature by TGF-ß1 and GM-CSF; b) they differed from their parent cells by more frequent hypersegmentation, higher expression of profibrotic inflammatory cytokines, and notably, expression of collagen 1; and c) their depletion reduced collagen deposition and disease progression, but adoptive transfer increased renal fibrosis. These findings have thus unveiled a subtype of neutrophils that participate in renal fibrosis and a potentially new therapeutic target in chronic kidney disease.

A unique population of neutrophils generated by air pollutant-induced lung damage exacerbates airway inflammation.

BACKGROUND: Diesel exhaust particles (DEPs) are the main component of traffic-related air pollution and have been implicated in the pathogenesis and exacerbation of asthma. However, the mechanism by which DEP exposure aggravates asthma symptoms remains unclear. OBJECTIVE: This study aimed to identify a key cellular player of air pollutant-induced asthma exacerbation and development. METHODS: We examined the distribution of innate immune cells in the murine models of asthma induced by house dust mite and DEP. Changes in immune cell profiles caused by DEP exposure were confirmed by flow cytometry and RNA-Seq analysis. The roles of sialic acid-binding, Ig-like lectin F (SiglecF)-positive neutrophils were further evaluated by adoptive transfer experiment and in vitro functional studies. RESULTS: DEP exposure induced a unique population of lung granulocytes that coexpressed Ly6G and SiglecF. These cells differed phenotypically, morphologically, functionally, and transcriptionally from other SiglecF-expressing cells in the lungs. Our findings with murine models suggest that intratracheal challenge with DEPs induces the local release of adenosine triphosphate, which is a damage-associated molecular pattern signal. Adenosine triphosphate promotes the expression of SiglecF on neutrophils, and these SiglecF+ neutrophils worsen type 2 and 3 airway inflammation by producing high levels of cysteinyl leukotrienes and neutrophil extracellular traps. We also found Siglec8- (which corresponds to murine SiglecF) expressing neutrophils, and we found it in patients with asthma-chronic obstructive pulmonary disease overlap. CONCLUSION: The SiglecF+ neutrophil is a novel and critical player in airway inflammation and targeting this population could reverse or ameliorate asthma.

Targeting PLD2 in adipocytes augments adaptive thermogenesis by improving mitochondrial quality and quantity in mice.

Phospholipase D (PLD)2 via its enzymatic activity regulates cell proliferation and migration and thus is implicated in cancer. However, the role of PLD2 in obesity and type 2 diabetes has not previously been investigated. Here, we show that during diet-induced thermogenesis and obesity, levels of PLD2 but not PLD1 in adipose tissue are inversely related with uncoupling protein 1, a key thermogenic protein. We demonstrate that the thermogenic program in adipose tissue is significantly augmented in mice with adipocyte-specific Pld2 deletion or treated with a PLD2-specific inhibitor and that these mice are resistant to high fat diet-induced obesity, glucose intolerance, and insulin resistance. Mechanistically, we show that Pld2 deletion in adipose tissue or PLD2 pharmacoinhibition acts via p62 to improve mitochondrial quality and quantity in adipocytes. Thus, PLD2 inhibition is an attractive therapeutic approach for obesity and type 2 diabetes by resolving defects in diet-induced thermogenesis.

Activation of formyl peptide receptor 1 elicits therapeutic effects against collagen-induced arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder which shows production of autoantibodies, inflammation, bone erosion, swelling and pain in joints. In this study, we examined the effects of an immune-modulating peptide, WKYMVm, that is an agonist for formyl peptide receptors (FPRs). Administration of WKYMVm into collagen-induced arthritis (CIA) mice, an animal model for RA, attenuated paw thickness, clinical scores, production of type II collagen-specific antibodies and inflammatory cytokines. WKYMVm treatment also decreased the numbers of TH 1 and TH 17 cells in the spleens of CIA mice. WKYMVm attenuated TH 1 and TH 17 differentiation in a dendritic cell (DC)-dependent manner. WKYMVm-induced beneficial effects against CIA and WKYMVm-attenuated TH 1 and TH 17 differentiation were reversed by cyclosporin H but not by WRW4, indicating a crucial role of FPR1. We also found that WKYMVm augmented IL-10 production from lipopolysaccharide-stimulated DCs and WKYMVm failed to suppress TH 1 and TH 17 differentiation in the presence of anti-IL-10 antibody. The therapeutic administration of WKYMVm also elicited beneficial outcome against CIA. Collectively, we demonstrate that WKYMVm stimulation of FPR1 in DCs suppresses the generation of TH 1 and TH 17 cells via IL-10 production, providing novel insight into the function of FPR1 in regulating CIA pathogenesis.

Isovaleric acid ameliorates ovariectomy-induced osteoporosis by inhibiting osteoclast differentiation.

Osteoclasts (OCs) play important roles in bone remodelling and contribute to bone loss by increasing bone resorption activity. Excessively activated OCs cause diverse bone disorders including osteoporosis. Isovaleric acid (IVA), also known as 3-methylbutanoic acid is a 5-carbon branched-chain fatty acid (BCFA), which can be generated by bacterial fermentation of a leucine-rich diet. Here, we find that IVA suppresses differentiation of bone marrow-derived macrophages into OCs by RANKL. IVA inhibited the expression of OC-related genes. IVA-induced inhibitory effects on OC generation were attenuated by pertussis toxin but not by H89, suggesting a Gi -coupled receptor-dependent but protein kinase A-independent response. Moreover, IVA stimulates AMPK phosphorylation, and treatment with an AMPK inhibitor blocks IVA-induced inhibition of OC generation. In an ovariectomized mouse model, addition of IVA to the drinking water resulted in significant decrease of body weight gain and inhibited the expression of not only OC-related genes but also fusogenic genes in the bone tissue. IVA exposure also blocked bone destruction and OC generation in the bone tissue of ovariectomized mice. Collectively, the results demonstrate that IVA is a novel bioactive BCFA that inhibits OC differentiation, suggesting that IVA can be considered a useful material to control osteoclast-associated bone disorders, including osteoporosis.

Sphingosylphosphorylcholine blocks ovariectomy-induced bone loss by suppressing Ca2+ /calmodulin-mediated osteoclast differentiation.

Osteoporosis is a disease in which bone mineral density decreases due to abnormal activity of osteoclasts, and is commonly found in post-menopausal women who have decreased levels of female hormones. Sphingosylphosphorylcholine (SPC) is an important biological lipid that can be converted to sphingosine-1-phosphate (S1P) by autotaxin. S1P is known to be involved in osteoclast activation by stimulating osteoblasts, but bone regulation by SPC is not well understood. In this study, we found that SPC strongly inhibits RANKL-induced osteoclast differentiation. SPC-induced inhibitory effects on osteoclast differentiation were not affected by several antagonists of S1P receptors or pertussis toxin, suggesting cell surface receptor independency. However, SPC inhibited RANKL-induced calcineurin activation and subsequent NFATc1 activity, leading to decrease of the expression of Trap and Ctsk. Moreover, we found that bone loss in an experimental osteoporosis mouse model was recovered by SPC injection. SPC also blocked ovariectomy-induced body weight increase and Nfatc1 gene expression in mice. We also found that SPC inhibits RANKL-induced osteoclast differentiation in human macrophages. Since currently available treatments for osteoporosis, such as administration of female hormones or hormone receptor modulators, show serious side effects, SPC has potential as a new agent for osteoporosis treatment.

Formyl peptide receptors in the mucosal immune system.

Formyl peptide receptors (FPRs) belong to the G protein-coupled receptor (GPCR) family and are well known as chemotactic receptors and pattern recognition receptors (PRRs) that recognize bacterial and mitochondria-derived formylated peptides. FPRs are also known to detect a wide range of ligands, including host-derived peptides and lipids. FPRs are highly expressed not only in phagocytes such as neutrophils, monocytes, and macrophages but also in nonhematopoietic cells such as epithelial cells and endothelial cells. Mucosal surfaces, including the gastrointestinal tract, the respiratory tract, the oral cavity, the eye, and the reproductive tract, separate the external environment from the host system. In mucosal surfaces, the interaction between the microbiota and host cells needs to be strictly regulated to maintain homeostasis. By sharing the same FPRs, immune cells and epithelial cells may coordinate pathophysiological responses to various stimuli, including microbial molecules derived from the normal flora. Accumulating evidence shows that FPRs play important roles in maintaining mucosal homeostasis. In this review, we summarize the roles of FPRs at mucosal surfaces.

Novel Sca-1+ macrophages modulate the pathogenic progress of endotoxemia.

Macrophages are important innate immune cells that play crucial roles in inflammatory responses. Accumulating evidence has demonstrated macrophage heterogeneity based on biomarkers, functions, and localization. Here, we report a novel stem cell antigen-1 (Sca-1)-positive macrophage population induced in the pathological conditions caused by lipopolysaccharide (LPS). Sca-1 is only upregulated in macrophages but not in monocytes and neutrophils upon LPS injection. Sca-1+ macrophages develop from resident peritoneal macrophages. LPS-induced Sca-1+ macrophage generation was partly blocked by anti-IFN-γ antibody, suggesting a role of IFN-γ in the process. LPS-stimulated production of IL-6, TNF-α, and CCL2 is significantly lower in Sca-1+ macrophages compared to their counterpart Sca-1- macrophages. Depletion of Sca-1+ macrophages using anti-Sca-1 antibody significantly increased survival rate and reduced lung and kidney damage in an LPS-induced sepsis model. Taken together, we discovered a novel population of Sca-1+ macrophages in LPS-induced septic conditions.

Novel CD11b+Gr-1+Sca-1+ myeloid cells drive mortality in bacterial infection.

Extreme pathophysiological stressors induce expansion of otherwise infrequent leukocyte populations. Here, we found a previously unidentified CD11b+Gr-1+ myeloid cell population that expresses stem cell antigen-1 (Sca-1) induced upon experimental infection with Staphylococcus aureus. Although CD11b+Gr-1+Sca-1+ cells have impaired migratory capacity and superoxide anion-producing activity, they secrete increased levels of several cytokines and chemokines compared to Sca-1- counterparts. The generation of CD11b+Gr-1+Sca-1+ cells is dependent on IFN-γ in vivo, and in vitro stimulation of bone marrow cells or granulocyte-macrophage progenitors with IFN-γ generated CD11b+Gr-1+Sca-1+ cells. Depletion of CD11b+Gr-1+Sca-1+ cells by administrating anti-Sca-1 antibody strongly increased survival rates in an S. aureus infection model by reducing organ damage and inflammatory cytokines. However, adoptive transfer of CD11b+Gr-1+Sca-1+ cells decreased survival rates by worsening the pathogenesis of S. aureus infection. Together, we found a previously unidentified pathogenic CD11b+Gr-1+Sca-1+ population that plays an essential role in mortality during bacterial infection.