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.

Chemerin triggers migration of a CD8 T cell subset with natural killer cell functions.

The recruitment of cells with effector functions into the tumor microenvironment holds potential for delaying cancer progression. We show that subsets of human CD28-effector CD8 T cells, CCR7- CD45RO+ effector memory, and CCR7- CD45RO- effector memory RA phenotypes, express the chemerin receptor CMKLR1 and bind chemerin via the receptor. CMKLR1-expressing human CD8 effector memory T cells present gene, protein, and cytotoxic features of NK cells. Active chemerin promotes chemotaxis of CMKLR1-expressing CD8 effector memory cells and triggers activation of the α4ß1 integrin. In an experimental prostate tumor mouse model, chemerin expression is downregulated in the tumor microenvironment, which is associated with few tumor-infiltrating CD8+ T cells, while forced overexpression of chemerin by mouse prostate cancer cells leads to an accumulation of intra-tumor CD8+ T cells. Furthermore, α4 integrin blockade abrogated the chemerin-dependent recruitment of CD8+ T effector memory cells into implanted prostate tumors in vivo. The results identify a role for chemerin:CMKLR1 in defining a specialized NK-like CD8 T cell, and suggest the use of chemerin-dependent modalities to target effector CMKLR1-expressing T cells to the tumor microenvironment for immunotherapeutic purposes.

Endothelial CCRL2 induced by disturbed flow promotes atherosclerosis via chemerin-dependent ß2 integrin activation in monocytes.

AIMS: Chemoattractants and their cognate receptors are essential for leucocyte recruitment during atherogenesis, and atherosclerotic plaques preferentially occur at predilection sites of the arterial wall with disturbed flow (d-flow). In profiling the endothelial expression of atypical chemoattractant receptors (ACKRs), we found that Ackr5 (CCRL2) was up-regulated in an endothelial subpopulation by atherosclerotic stimulation. We therefore investigated the role of CCRL2 and its ligand chemerin in atherosclerosis and the underlying mechanism. METHODS AND RESULTS: By analysing scRNA-seq data of the left carotid artery under d-flow and scRNA-seq datasets GSE131776 of ApoE-/- mice from the Gene Expression Omnibus database, we found that CCRL2 was up-regulated in one subpopulation of endothelial cells in response to d-flow stimulation and atherosclerosis. Using CCRL2-/-ApoE-/- mice, we showed that CCRL2 deficiency protected against plaque formation primarily in the d-flow areas of the aortic arch in ApoE-/- mice fed high-fat diet. Disturbed flow induced the expression of vascular endothelial CCRL2, recruiting chemerin, which caused leucocyte adhesion to the endothelium. Surprisingly, instead of binding to monocytic CMKLR1, chemerin was found to activate ß2 integrin, enhancing ERK1/2 phosphorylation and monocyte adhesion. Moreover, chemerin was found to have protein disulfide isomerase-like enzymatic activity, which was responsible for the interaction of chemerin with ß2 integrin, as identified by a Di-E-GSSG assay and a proximity ligation assay. For clinical relevance, relatively high serum levels of chemerin were found in patients with acute atherothrombotic stroke compared to healthy individuals. CONCLUSIONS: Our findings indicate that d-flow-induced CCRL2 promotes atherosclerotic plaque formation via a novel CCRL2-chemerin-ß2 integrin axis, providing potential targets for the prevention or therapeutic intervention of atherosclerosis.

Development of a rapid image-based high-content imaging screening assay to evaluate therapeutic antibodies against the monkeypox virus.

Antibody-based therapy is emerging as a critical therapeutic countermeasure to treat acute viral infections by offering rapid protection against clinical disease. The advancements in structural biology made it feasible to rationalize monoclonal antibodies (mAbs) by identifying key and, possibly, neutralizing epitopes of viral proteins for therapeutic purposes. A critical component in assessing mAbs during pandemics requires the development of rapid but detailed methods to detect and quantitate the neutralization activity. In this study, we developed and optimized two high-content image (HCI)-based assays: one to detect viral proteins by staining and the second to quantify cytopathic viral effects by a label-free phenotypic assay. These assays were employed to screen for therapeutic antibodies against the monkeypox virus (MPXV) using surrogate poxviruses such as vaccinia virus (VACV). Plaque-based neutralization results confirmed the HCI data. The phenotypic assay found pox virus-induced syncytia formation in various cells, and we were able to quantitate and use this phenotype to screen mAbs. The HCI identified several potent VACV-neutralizing antibodies that showed in vitro efficacy against both clades of MPXV. In addition, a combination study of ST-246/tecovirimat/TPOXX a single neutralizing antibody Ab-40, showed synergistic activity against VACV in an in-vitro neutralization assay. This rapid high-content method utilizing state-of-the-art technologies enabled the evaluation of hundreds of mAbs quickly to identify several potent anti-MPXV neutralizing mAbs for further development.

Soluble mediators in the function of the epidermal-immune-neuro unit in the skin.

Skin is the largest, environmentally exposed (barrier) organ, capable of integrating various signals into effective defensive responses. The functional significance of interactions among the epidermis and the immune and nervous systems in regulating and maintaining skin barrier function is only now becoming recognized in relation to skin pathophysiology. This review focuses on newly described pathways that involve soluble mediator-mediated crosstalk between these compartments. Dysregulation of these connections can lead to chronic inflammatory diseases and/or pathologic conditions associated with chronic pain or itch.

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.

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.

Chemerin-Derived Peptide Val66-Pro85 Is Effective in Limiting Methicillin-Resistant S. aureus Skin Infection.

Chemerin-derived peptide Val66-Pro85 (p4) restricts the growth of a variety of skin-associated bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). To better understand the antimicrobial potential of chemerin peptide, we compared p4 activity against MRSA in vitro to cathelicidin LL-37, one of the key endogenous peptides implicated in controlling the growth of S. aureus. The efficacy of p4 was also validated in relevant experimental models of skin pathology, such as topical skin infection with community-acquired MRSA, and in the context of skin inflammatory diseases commonly associated with colonization with S. aureus, such as atopic dermatitis (AD). We showed that p4 collaborates additively with LL-37 in inhibiting the growth of S. aureus, including MRSA, and that p4 was effective in vivo in reducing MRSA burden. p4 was also effective in reducing levels of skin-infiltrating leukocytes in S. aureus-infected AD-like skin. Taken together, our data suggest that p4 is effective in limiting S. aureus and, in particular, MRSA skin infection.

CCR2 deficiency alters activation of microglia subsets in traumatic brain injury.

In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2-/- mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2-/- TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets.

Redox Active Antimicrobial Peptides in Controlling Growth of Microorganisms at Body Barriers.

Epithelia in the skin, gut and other environmentally exposed organs display a variety of mechanisms to control microbial communities and limit potential pathogenic microbial invasion. Naturally occurring antimicrobial proteins/peptides and their synthetic derivatives (here collectively referred to as AMPs) reinforce the antimicrobial barrier function of epithelial cells. Understanding how these AMPs are functionally regulated may be important for new therapeutic approaches to combat microbial infections. Some AMPs are subject to redox-dependent regulation. This review aims to: (i) explore cysteine-based redox active AMPs in skin and intestine; (ii) discuss casual links between various redox environments of these barrier tissues and the ability of AMPs to control cutaneous and intestinal microbes; (iii) highlight how bacteria, through intrinsic mechanisms, can influence the bactericidal potential of redox-sensitive AMPs.

Bacteria Modify Their Sensitivity to Chemerin-Derived Peptides by Hindering Peptide Association With the Cell Surface and Peptide Oxidation.

Chronic inflammatory skin diseases like psoriasis alter the local skin microbiome and lead to complications such as persistent infection with opportunistic/pathogenic bacteria. Disease-associated changes in microbiota may be due to downregulation of epidermal antimicrobial proteins/peptides, such as antimicrobial protein chemerin. Here, we show that chemerin and its bioactive derivatives have differential effects on the viability of different genera of cutaneous bacteria. The lethal effects of chemerin are enhanced by bacterial-derived ROS-induced chemerin peptide oxidation and suppressed by stationary phase sigma factor RpoS. Insight into the mechanisms underlying changes in the composition of cutaneous bacteria during autoreactive skin disease may provide novel ways to mobilize chemerin and its peptide derivatives for maximum antimicrobial efficacy.

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.

Novel CMKLR1 Inhibitors for Application in Demyelinating Disease.

Small molecules that disrupt leukocyte trafficking have proven effective in treating patients with multiple sclerosis (MS). We previously reported that chemerin receptor chemokine-like receptor 1 (CMKLR1) is required for maximal clinical and histological experimental autoimmune encephalomyelitis (EAE); and identified CMKLR1 small molecule antagonist 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) that significantly suppressed disease onset in vivo. Here we directly compared α-NETA versus FDA-approved MS drug Tecfidera for clinical efficacy in EAE; characterized key safety/toxicity parameters for α-NETA; identified structure-activity relationships among α-NETA domains and CMKLR1 inhibition; and evaluated improved α-NETA analogs for in vivo efficacy. α-NETA proved safe and superior to Tecfidera in suppressing clinical EAE. In addition, we discovered structurally differentiated α-NETA analogs (primarily ortho- or para-methoxy substitutions) with significantly improved target potency in vitro and improved efficacy in vivo. These findings suggest that α-NETA-based CMKLR1 inhibitors may prove safe and effective in treating demyelinating diseases and potentially other autoimmune disorders.

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.

Chemerin Suppresses Breast Cancer Growth by Recruiting Immune Effector Cells Into the Tumor Microenvironment.

Infiltration of immune cells into the tumor microenvironment (TME) can regulate growth and survival of neoplastic cells, impacting tumorigenesis and tumor progression. Correlations between the number of effector immune cells present in a tumor and clinical outcomes in many human tumors, including breast, have been widely described. Current immunotherapies utilizing checkpoint inhibitors or co-stimulatory molecule agonists aim to activate effector immune cells. However, tumors often lack adequate effector cell numbers within the TME, resulting in suboptimal responses to these agents. Chemerin (RARRES2) is a leukocyte chemoattractant widely expressed in many tissues and is known to recruit innate leukocytes. CMKLR1 is a chemotactic cellular receptor for chemerin and is expressed on subsets of dendritic cells, NK cells, and macrophages. We have previously shown that chemerin acts as a tumor suppressive cytokine in mouse melanoma models by recruiting innate immune defenses into the TME. Chemerin/RARRES2 is down-regulated in many tumors, including breast, compared to normal tissue counterparts. Here, using a syngeneic orthotopic EMT6 breast carcinoma model, we show that forced overexpression of chemerin by tumor cells results in significant recruitment of NK cells and T cells within the TME. While chemerin secretion by EMT6 cells did not alter their phenotypic behavior in vitro, it did significantly suppress tumor growth in vivo. To define the cellular effectors required for this anti-tumor phenotype, we depleted NK cells or CD8+ T cells and found that either cell type is required for chemerin-dependent suppression of EMT6 tumor growth. Finally, we show significantly reduced levels of RARRES2 mRNA in human breast cancer samples compared to matched normal tissues. Thus, for the first time we have shown that increasing chemerin expression within the breast carcinoma TME can suppress growth by recruitment of NK and T cells, thereby supporting this approach as a promising immunotherapeutic strategy.

Support of Tumor Endothelial Cells by Chemokine Receptors.

Tumor-associated vascular endothelium comprises a specialized and diverse group of endothelial cells that, although not cancer themselves, are integral to cancer progression. Targeting the tumor vasculature can have significant efficacy in reducing tumor burden, although loss of efficacy due to acquisition of resistance mechanisms is common. Here we review mechanisms by which tumor endothelial cells (TEC) utilize chemokine receptors to support tumor progression. We illustrate how chemokine receptors support and may serve as functional markers of the diverse TEC population. We focus on ACKR1 (DARC), ACKR3 (CXCR7), CXCR4, and CCR2, as these are the best studied chemokine receptors in TEC; and suggest that targeting these receptors on the tumor vasculature may prove efficacious in slowing or reversing tumor growth. We also mention CXCR2 and CXCR3 as important mediators or tumor angiogenesis, given their distinct roles with angiogenic and angiostatic chemokines, respectively.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions.

Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein abundantly produced in the skin epidermis. Despite the fact that most of the bactericidal activity present in human skin exudates is chemerin-dependent, just how chemerin shapes skin defenses remains obscure. Here we demonstrate that p4, a potent antimicrobial human chemerin peptide derivative, displays killing activity against pathogenic methicillin-resistant Staphylococcus aureus strains and suppresses microbial growth in a topical skin infection model. Mechanistically, we show that p4 homodimerization is required for maximal bactericidal activity and that an oxidative environment, such as at the skin surface, facilitates p4 disulfide bridge formation, required for the dimerization. p4 led to rapid damage of the bacterial internal membrane and inhibited the interaction between the membranous cytochrome bc1 complex and its redox partner, cytochrome c These results suggest that a chemerin p4-based defense strategy combats bacterial challenges at the skin surface.

Mechanisms and Functions of Chemerin in Cancer: Potential Roles in Therapeutic Intervention.

Chemerin [RARRES2 [retinoic acid receptor responder 2], TIG2 [tazarotene induced gene 2 (TIG2)]] is a multifunctional cytokine initially described in skin cultures upon exposure to the synthetic retinoid tazarotene. Its secreted pro-form, prochemerin, is widely expressed, found systemically, and is readily converted into active chemerin by various proteases. Subsequent studies elucidated major roles of chemerin as both a leukocyte chemoattractant as well as an adipokine. Chemerin's main chemotactic receptor, the G-protein coupled receptor CMKLR1, is expressed on macrophages, dendritic, and NK cells. With respect to its role in immunology, chemerin mediates trafficking of these cells to sites of inflammation along its concentration gradient, and likely helps coordinate early responses, as it has been shown to have antimicrobial and angiogenic properties, as well. Recently, there has been mounting evidence that chemerin is an important factor in various cancers. As with its role in immune responses-where it can act as both a pro- and anti-inflammatory mediator-the potential functions or correlations chemerin has in or with cancer appears to be context dependent. Most studies, however, suggest a downregulation or loss of chemerin/RARRES2 in malignancies compared to the normal tissue counterparts. Here, we perform a comprehensive review of the literature to date and summarize relevant findings in order to better define the roles of chemerin in the setting of the tumor microenvironment and tumor immune responses, with an ultimate focus on the potential for therapeutic intervention.