Search

Neutrophils are polymorphonuclear leukocytes that play a central role in host defense against infection and tissue injury. They are rapidly recruited to the inflamed site and execute a variety of functions to clear invading pathogens and damaged cells. However, many of their defense mechanisms are capable of inflicting collateral tissue damage. Neutrophil-driven inflammation is a unifying mechanism underlying many common diseases. Efficient removal of neutrophils from inflammatory loci is critical for timely resolution of inflammation and return to homeostasis. Accumulating evidence challenges the classical view that neutrophils represent a homogeneous population and that halting neutrophil influx is sufficient to explain their rapid decline within inflamed loci during the resolution of protective inflammation. Hence, understanding the mechanisms that govern neutrophil functions and their removal from the inflammatory locus is critical for minimizing damage to the surrounding tissue and for return to homeostasis. In this review, we briefly address recent advances in characterizing neutrophil phenotypic and functional heterogeneity and the molecular mechanisms that determine the fate of neutrophils within inflammatory loci and the outcome of the inflammatory response. We also discuss how these mechanisms may be harnessed as potential therapeutic targets to facilitate resolution of inflammation.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81−/− mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection., Lactate is a by-product of glycolysis that can function via its G protein receptor GPR81. Here the authors show that LPS or Salmonella infection enhances glycolytic metabolism in bone marrow neutrophils, resulting in lactate production, which increases endothelial barrier permeability and mobilization of these neutrophils by targeting endothelial GPR81.

ARTS (Sept4_i2) is a pro-apoptotic protein and a product of the Sept4 gene. ARTS acts upstream of mitochondria to initiate caspase activation. ARTS induces apoptosis by specifically binding XIAP and allowing de-repression of active caspases required for Mitochondrial Outer Membrane Permeabilzation (MOMP). Moreover, ARTS promotes apoptosis by inducing ubiquitin-mediated degradation of both major anti-apoptotic proteins XIAP and Bcl-2. In the resolution phase of inflammation, the infiltrating leukocytes, which execute the acute innate response, undergo apoptosis and are subsequently cleared by phagocytic macrophages (i.e. efferocytosis). In this course, macrophages undergo reprogramming from inflammatory, to anti-inflammatory, and eventually to resolving macrophages that leave the injury sites. Since engulfment of apoptotic leukocytes is a key signaling step in macrophage reprogramming and resolution of inflammation, we hypothesized that a failed apoptosis in leukocytes in vivo would result in an impaired resolution process. To test this hypothesis, we utilized the Sept4/ARTS-/- mice, which exhibit resistance to apoptosis in many cell types. During zymosan A-induced peritonitis, Sept4/ARTS-/- mice exhibited impaired resolution of inflammation, characterized by reduced neutrophil apoptosis, macrophage efferocytosis and expression of pro-resolving mediators. This was associated with increased pro-inflammatory cytokines and reduced anti-inflammatory cytokines, secreted by resolution-phase macrophages. Moreover, ARTS overexpression in leukocytes in vitro promoted an anti-inflammatory behavior. Overall, our results suggest that ARTS is a key master-regulator necessary for neutrophil apoptosis, macrophage efferocytosis and reprogramming to the pro-resolving phenotype during the resolution of inflammation.

The resolution of inflammation facilitates proper wound healing and limits tissue repair short of exaggerated fibrotic scarring. The atypical chemokine receptor (ACKR)2/D6 scavenges inflammatory chemokines, while IFN-β is a recently unveiled pro-resolving cytokine. Both effector molecules limit acute inflammatory episodes and promote their resolution in various organs. Here, we found fibrotic skin lesions from ACKR2-/- mice presented increased epidermal and dermal thickening, atrophy of the subcutaneous adipose tissue, augmented disorientation of collagen deposition, and enhanced deformation and loss of hair follicles compared to WT counterparts. In addition, affected skin sections from ACKR2-/- mice contained reduced levels of the pro-resolving mediators IFN-β and IL-10, but increased levels of the pro-inflammatory chemokines CCL2 and 3, the pro-fibrotic cytokine TGF-β, and the immune-stimulating cytokine IL-12. Notably, treatment with exogenous IFN-β rescued, at least in part, all the pro-fibrotic outcomes and lesion size in ACKR2-/- mice and promoted expression of the pro-resolving enzyme 12/15-lipoxygenase (LO) in both ACKR2-/- and WT mice. Moreover, Ifnb-/- mice displayed enhanced pro-fibrotic indices upon exposure to bleomycin. These findings suggest ACKR2 is an important mediator in limiting inflammatory skin fibrosis and acts via IFN-β production to promote the resolution of inflammation and minimize tissue scaring.

The COVID-19 pandemic is shining a spotlight on the field of immunology like never before. To appreciate the diverse ways in which immunologists have contributed, Nature Reviews Immunology invited the president of the International Union of Immunological Societies and the presidents of 15 other national immunology societies to discuss how they and their members responded following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)., For this Viewpoint, Nature Reviews Immunology asked the presidents of 16 immunology societies from around the world to discuss how their society and its members responded to the COVID-19 pandemic. Their answers highlight the incredible contributions that immunologists around the globe have made following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The neutrophil granule protein lactoferrin is cleaved and accumulates in efferocytic macrophages as inflammation is resolved. Two peptides present within a resolution-associated 17 kDa fragment of lactoferrin promote the termination of inflammation in vivo by enhancing murine macrophage reprogramming. Here, we report that these two bioactive tripeptides, phenylalanine-lysine-aspartic acid and phenylalanine-lysine-glutamic acid (FKD and FKE, respectively), inhibit ERK and cJun activation following human macrophage exposure to LPS. In addition, these peptides at low concentrations (1&ndash, 10 &mu, M) modulate human macrophage reprogramming to an anti-inflammatory/pro-resolving phenotype. This was reflected by inhibition of LPS-induced TNF-&alpha, and IL-6 secretion and increased IL-10 levels. Moreover, we found naturally occurring FKE analogs (FKECH and FKECHLA) can recapitulate the activity of the short peptide in regulating macrophage cytokine secretion, whereas a reversed EKF peptide was inert in this respect. Curiously, FKD and FKE also regulated cytokine production by bone marrow-derived mouse macrophages, but in a very different fashion than their effect on human macrophages. Thus, lactoferrin peptides limit pro-inflammatory signaling and cytokine production by LPS-activated human macrophages and thereby enhance the resolution of inflammation.

During the resolution of acute inflammation, macrophages undergo reprogramming from pro-inflammatory, to anti-inflammatory/reparative, and eventually to pro-resolving macrophages. Galectin-1 (Gal-1) is a bona fide pro-resolving lectin while interferon β (IFN-β) was recently shown to facilitate macrophage reprogramming and resolution of inflammation. In this study, we found Gal-1null mice exhibit a hyperinflammatory phenotype during the resolution of zymosan A-induced peritonitis but not during the early inflammatory response. This phenotype was characterized by reduced macrophage numbers, increased secretion of pro-inflammatory cytokines, such as interleukin-12 (IL-12), and reduced secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10). In addition, we found a delayed expression of the pro-resolving enzyme 12/15-lipoxygenase in macrophages and heightened levels of the inflammatory protease proteinase-3 (PR3) in peritoneal fluids from Gal-1null mice. Moreover, we observed sex-dependent differences in the inflammatory profile of Gal-1null mice. Notably, we found that IFN-β levels were reduced in resolution-phase exudates from Gal-1null mice. Administration of IFN-β in vivo or ex vivo treatment was able to rescue, at least in part, the hyperinflammatory profile of Gal-1null mice. In particular, IFN-β recovered a subset of F4/80+GR-1+ macrophages, restored IL-12 and IL-10 secretion from macrophages to WT values and diminished abnormal peritoneal PR3 levels in Gal-1null mice. In conclusion, our results revealed a new Gal-1-IFN-β axis that facilitates the resolution of inflammation and might restrain uncontrolled inflammatory disorders. Fil: Yaseen, Hiba. University of Haifa; Israel Fil: Butenko, Sergei. University of Haifa; Israel Fil: Polishuk Zotkin, Irina. University of Haifa; Israel Fil: Schif Zuck, Sagie. University of Haifa; Israel Fil: Pérez Sáez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ariel, Amiram. University of Haifa; Israel

Atherosclerosis and cardiovascular complications are prevalent among patients undergoing chronic hemodialysis (HD). In this population, peripheral polymorphonuclear leukocytes (PMNLs) are primed, releasing proinflammatory mediators such as elastase. Elastase is normally inhibited by a specific inhibitor, avoiding undesirable degradation of cellular and extracellular components. This study tested the hypothesis that in states of noninfectious inflammation, elastase is released by PMNLs and acts in an uncontrolled manner to inflict vascular damage. Blood was collected from patients undergoing HD and healthy controls (HC). PMNL intracellular and surface expressions of elastase were determined by quantitative real-time PCR, Western blotting, and flow cytometry. The elastase activity was evaluated using a fluorescent substrate. The levels of serum α1-antitrypsin (α1-AT), the natural elastase inhibitor, were determined by Western blot. Free active elastase was elevated in HD sera, whereas the levels of α1-AT were decreased compared with HC. The levels of the intracellular elastase enzyme and its activity were lower in HD PMNLs despite similar expression levels of elastase mRNA. Elastase binding to PMNL cell surface was higher in HD compared with HC. The increased circulating levels of free active elastase released from primed HD PMNLs together with the higher cell surface-bound enzymes and the lower levels of α1-AT result in the higher elastase activity in HD sera. This exacerbated elastase activity could lead to the endothelial dysfunction, as hypothesized. In addition, it suggests that free circulating elastase can serve as a new biomarker and therapeutic target to reduce inflammation and vascular complications in patients on hemodialysis.

The engulfment of apoptotic polymorphonuclear cells (PMN) during the resolution of inflammation leads to macrophage reprogramming culminating in reduced proinflammatory and increased anti-inflammatory mediator secretion. The atypical chemokine receptor D6/ACKR2 is expressed on apoptotic PMN and plays an important role in regulating macrophage properties during and after engulfment. In this study, we found that the inflammatory chemokine CCL5 is mostly retained (75%) during the resolution of zymosan A peritonitis in mice. Moreover, this chemokine is secreted by resolution-phase macrophages (2.5 ng/ml) and promotes their reprogramming in vivo in D6+/+ mice (2-fold increase in IL-10/IL-12 ratio) but not their D6−/− counterparts. In addition, CCL5 enhanced macrophage reprogramming ex vivo exclusively when bound to D6+/+ apoptotic PMN. Signaling through p38MAPK and JNK in reprogrammed macrophages was enhanced by CCL5-bound apoptotic PMN (3.6–4 fold) in a D6-dependent manner, and was essential for reprogramming. Thus, CCL5 exerts a novel proresolving role on macrophages when acting in concert with apoptotic PMN-expressed D6.

The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-β-related gene signature in mice. We also report elevated levels of IFN-β in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-β impairs, whereas treatment with exogenous IFN-β enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-β promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-β produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation., Clearance of apoptotic neutrophils by macrophages is important for the resolution of inflammation. Here, the authors show that interferon-β produced by resolution phase macrophages promotes neutrophil apoptosis and efferocytosis and induces macrophage reprogramming to a pro-resolving phenotype, thereby identifying IFN-β as a multi-pronged pro-resolution cytokine.

Innate immune neutrophils provide the first line of host defense against bacterial infections. Neutrophils under steady state rely almost entirely on glycolysis and exhibit very low levels of oxidative phosphorylation. The metabolite lactate has long been considered a "waste byproduct" of cell metabolism which accumulates during inflammation and sepsis. Increased plasma lactate levels in human patients is used as a marker for sepsis diagnosis. However, the direct effector actions of lactate, particularly in regulating neutrophil mobilization and function during inflammation has remained obscure. To better understand the metabolic consequences of BM neutrophil activation during the onset of inflammation, we tested how bacterial lipopolysaccharides (mimicking gram negative bacterial inflammation) introduced intraperitoneally (i.p.) affect neutrophil metabolism and mobilization. RNAseq of sorted BM neutrophils revealed that LPS-activated neutrophils upregulate enzymes catalyzing the first part of glycolysis (hexokinase and PFKL) and downregulate the expression of TCA cycle enzymatic genes. In addition, LPS enhanced neutrophil lactate production and release as indicated by higher levels of BM lactate and higher expression of LDHA and MCT4. In addition, LPS increased NADPH oxidase (NOX)-mediated reactive oxygen species and HIF-1α levels in BM neutrophils, which are up-stream of glycolytic enzymes and lactate production and release. Recently, we reported that i.p. lactate administration rapidly activated and mobilized neutrophils from BM to the circulation (ASH, 2017). To test if lactate acts preferentially on neutrophils, we also examined other types of hematopoietic cells. Interestingly, we found that lactate specifically and rapidly (i.e., within 4 hrs) mobilized neutrophils to the circulation whereas the levels of peripheral blood (PB) monocytes, lymphocytes, granulocyte monocyte progenitors (GMPs) and hematopoietic progenitor stem cells (LSK) were reduced following lactate administration. LPS treatment failed to mobilize activated ROShigh neutrophils to the PB in NOX-/- mice, while lactate administration partially rescued this defect following LPS treatment. Our data also reveal that the NOX/ROS axis operates upstream of lactate production in BM neutrophils since abnormal metabolic rates were found in NOX-/- neutrophils during the onset of the acute inflammatory responses. Moreover, we found that BM endothelial cells (BMEC) abundantly express the highly selective lactate receptor GPR81, and that neutrophil-released lactate increased BM vascular permeability via BMEC GPR81 signaling (ASH, 2017). Consistent with a role of the lactate/GPR81 axis in enhanced vascular permeability, we find that i.p. injected LPS reduced VE-Cadherin expression on highly permeable sBMECs in GPR81 dependent manner. Notably, neutralizing VE-Cadherin in GPR81-/- mice can rescue and elevate PB neutrophil levels, similarly to wild-type (WT) mice, suggesting that VE-Cadherin is downstream of GPR81 signaling and plays a role in neutrophil mobilization. Finally, to examine the potential clinical relevance of our findings, we infected WT, NOX-/- and GPR81-/- mice with Salmonella Typhimurium and found out that this pathogen drove high generation of ROS, elevated HIF-1αlevels, and triggered lactate production and release in WT BM neutrophils. In contrast, BM neutrophils of infected NOX-/- mice exhibited significantly lower HIF-1αand impaired lactate production and release. Consequently, WT mice infected with Salmonella had a higher levels of neutrophils in the blood, as compared to their NOX-/- or GPR81-/- mice counterparts. Altogether, our data reveal that the same regulatory mechanisms by which neutrophils respond to LPS challenges are used during bacterial infection with Salmonella. Our study highlights lactate released by BM neutrophils as a key pro-inflammatory stimulus of a novel immune-metabolic crosstalk which is triggered by infection and locally opens the BM vascular barrier to facilitate neutrophil mobilization and recruitment to sites of inflammation. Targeting this immune-metabolic crosstalk between lactate-producing neutrophils and the BM endothelium could be useful for the control of pathological neutrophil activation and mobilization during bacterial infections and help treatments of neutrophil related immune disorders. Disclosures No relevant conflicts of interest to declare.

International audience; Neutrophils are critically involved in host defence and they also modulate the inflammatory process. Turning the inflammatory response towards a resolutive outcome requires a dialogue between apoptotic neutrophils and proresolving macrophages through complex key molecular interactions controlling efferocytosis, anti-inflammatory reprogramming and ultimately immune regulation. In this review, we will first focus on recent molecular analyses aiming at characterizing the role of proteins expressed on apoptotic neutrophils and their cognate partners expressed on macrophages in the resolution of inflammation. These will include chemokine receptors and their ligands and annexin A1 and its receptor FPR2. We will next depict how the structural and enzymatic properties of proteinase 3 (PR3), the autoantigen in vasculitis, allow its expression on apoptotic neutrophils, which in turn affects efferocytosis and immune response associated with the clearance of apoptotic cells. This example illustrates that the fate of apoptotic neutrophils directly influences the resolution of inflammation and immune responses thereby potentially contributing to systemic and nonresolving inflammation as well as autoimmunity.

During the resolution of inflammation macrophages undergo functional changes upon exposure to pro-resolving agents in their microenvironment. Primarily, engulfment of apoptotic polymorphonuclear (PMN) cells promotes conversion of macrophages toward a pro-resolving phenotype characterized by reduced CD11b expression. These macrophages are not phagocytic, do not respond to TLR ligands, and express relatively high levels of the pro-resolving enzyme 12/15-lipoxygenase (LO). Here, we report that the immuno-regulatory lectin galectin-1 is selectively expressed by CD11bhigh, but not CD11blow macrophages. Upon exposure in vivo and ex vivo, galectin-1 directly promoted macrophage conversion from a CD11bhigh to a CD11blow phenotype and up-regulated the expression and activity of 12/15-LO. Moreover, galectin-1 treatment in vivo promoted the loss of phagocytic capacity (efferocytic satiation) in peritoneal macrophages and down-regulated secretion of TNF-α, IL-1β, and IL-10 upon LPS exposure. Our results suggest that galectin-1 could be an essential mediator in the control of macrophage function during the resolution of inflammation. Fil: Rostoker, Ran. University of Haifa. Faculty of Natural Sciences. Departament of Biology; Israel; Fil: Yaseen, Hiba. University of Haifa. Faculty of Natural Sciences. Departament of Biology; Israel; Fil: Schif Zuck, Sagie. University of Haifa. Faculty of Natural Sciences. Departament of Biology; Israel; Fil: Lichtenstein, Rachel G.. Ben-Gurion University. Department of Biotechnology Engineering; Israel; Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Quimica Biologica; Argentina; Fil: Amiram, Ariel. University of Haifa. Faculty of Natural Sciences. Departament of Biology; Israel

Inflammation is the physiological response to tissue injury caused by pathogens or trauma. Nevertheless, inflammation should be resolved in a timely manner, resulting in elimination of the inflammatory cells and mediators from the injured tissue, to avoid its deleterious consequences. Uncontrolled inflammation can lead to inflammatory, autoimmune, and cancerous disorders that are the result of improper resolution. The healing of the injured tissue during the termination of inflammation must also be tightly controlled since excessive tissue repair can lead to fibrosis and scarring of the affected organ. In the last three decades, it has been revealed that the resolution of inflammation is tightly orchestrated by specific cells, protein, and lipid mediators that are produced at proper timing and distinct locations. The bioactivity of these anti-inflammatory, pro-resolving, and immunoregulatory agents results in clearance of the tissue from inflammatory leukocytes and their products, and the return of homeostatic tissue architecture and function. Here, we will survey the current endogenous mechanisms governing the resolution of inflammation and directing it towards injury healing and halting of acquired immune responses while preventing excessive tissue repair and fibrosis. We focus on the role played by apoptotic polymorphonuclear cells (PMNs), 15-lipoxygenase (LO)-derived lipid mediators, and TGFβ in this macrophage-governed decision-making process and suggest new modes of action for fibrosis prevention and return to homeostasis.

Efferocytosis has been suggested to promote macrophage resolution programs that are dependent on motility and emigration, however, few studies have addressed directed migration in resolving macrophages. In this report, we hypothesized that efferocytosis would induce differential chemokine receptor expression. Polarized macrophage populations, including macrophages actively engaged in efferocytosis, were characterized by PCR array and traditional transwell motility assays. We identified specific up-regulation of chemokine receptor CXCR4 on both mouse and human macrophages and characterized in vivo expression of CXCR4 in a resolving model of murine peritonitis. Using adoptive transfer and AMD3100 blocking, we confirmed a role for CXCR4 in macrophage egress to draining lymphatics. Collectively these data provide an important mechanistic link between efferocytosis and macrophage emigration.

The resolution of acute inflammation is hallmarked by the apoptotic death of inflammatory polymorphonuclear (PMN) cells, followed by their clearance by macrophages. In turn, resolution-phase macrophages exert reduced proinflammatory cytokine production, termed immune silencing. In this study, we found that the atypical chemokine receptor D6 plays an important and chemokine scavenging-independent role in promoting macrophage-mediated resolution. D6(-/-) mice displayed increased numbers of macrophages (2.2-fold increase), but not neutrophils, in their peritonea during the resolution of murine zymosan A-initiated peritonitis, in comparison to D6(+/+) animals. Moreover, D6-deficient macrophages engulfed higher numbers of apoptotic PMN cells in vivo (1.6-fold increase), and secreted higher amounts of TNF-α, CCL3, and CCL5 ex vivo than their wild-type (WT) counterparts. In addition, D6 was found to be expressed on apoptotic neutrophils from healthy humans and rodents. Moreover, the immune silencing of LPS-stimulated macrophages following their incubation with senescent PMN cells ex vivo (in terms of TNF-α, IL-1β, and CCL5 secretion) was diminished (50-65% decrease) when D6(-/-) PMN cells were applied. Accordingly, the adhesive responses induced by macrophage interactions with senescent PMN cells were reduced with D6-deficient PMN cells. Thus, our results indicate a novel mode of action for D6 during the resolution of inflammation that is instrumental to the shaping of resolving macrophage phenotypes and the completion of resolution.

Current research depicts specific modes of immunity and energy metabolism as being interrelated at the molecular, cellular, organ and organism level. Hence, whereas M2 (alternatively-activated) macrophages dominate insulin-sensitive adipose tissue in the lean, M1-skewed (classically-activated) macrophages accumulate in parallel to adiposity in the obese, and promote inflammation and insulin resistance, that is, meta-inflammation. The latest frontier of immuno-metabolism explores the coregulation of energy metabolism and immune function within hematopoietic cells. M1-skewed macrophages are sustained in edematous, hypoxic tissues by anaerobic glycolysis, whereas mitochondrial biogenesis and respiration dominates in M2 cells. We review the underlying mechanisms and the consequences of the transition from M2 to M1 predominance in adipose tissue, as well as the extracellular signals and transcription factors that control macrophage phenotypes and impose distinct metabolic modes.

During the resolution phase of inflammation apoptotic leukocytes are efferocytosed by macrophages in a nonphlogistic fashion that results in diminished responses to bacterial moieties and production of anti-inflammatory cytokines. Complement receptor 3 (CR3) and pro-resolving lipid mediators promote the engulfment of apoptotic leukocytes by macrophages. Here, we present evidence for the emergence of pro-resolving, CD11blow macrophages in vivo during the resolution of murine peritonitis. These macrophages are distinct from the majority of peritoneal macrophages in terms of their functional protein expression profile, as well as pro-resolving properties, such as apoptotic leukocyte engulfment, indifference to TLR ligands, and emigration to lymphoid organs. Notably, we also found macrophages convert from the CD11bhigh to the CD11blow phenotype upon interaction with apoptotic cells ex vivo. In addition, we found that the pro-resolving lipid mediators resolvin (Rv) E1 and RvD1, and the glucocorticoid dexamethasone (Dex) regulated pro-resolving macrophage functions in vivo. This regulation culminated in a novel pro-resolving function, namely reducing the apoptotic leukocyte ingestion requirement for CD11blow macrophage generation. These new phenotype and molecular pathway markers define the new satiated-macrophage. Thus, we suggest that satisfying-efferocytosis generates CD11blow macrophages that are essential for complete non-phlogistic containment of inflammatory agents and the termination of acute inflammation.

Breast cancer that recurs as metastatic disease many years after primary tumor resection and adjuvant therapy appears to arise from tumor cells that disseminated early in the course of the disease but did not develop into clinically apparent lesions. These long-term surviving, quiescent disseminated dormant tumor cells (QDTC) are resistant to conventional therapies that target actively dividing cells. The mechanisms responsible for maintaining the survival and outgrowth of QDTC remain largely unknown. Recently, we found that fibrotic-like microenvironment with extensive deposition of Type I collagen (Col-I) and fibroectin, established at the site of the residing QDTC, promoted the outbreak of QDTC. Therefore, we hypothesized that promoting resolution—the endogenous mechanism that terminates inflammation and fibrotic responses and actively directs tissue return to homeostasis—at the permissive site will “normalize” it and prevent metastatic outbreak. Here we demonstrate that soluble factors secreted by ex vivo generated pro-resolving CD11blow macrophages can inhibit the establishment of a fibrotic niche resulting in the inhibition of the metastatic outgrowth of QDTC. The inhibition of the fibrotic niche was due to inhibition of TGFβ1-induced differentiation of fibroblasts to myofibroblasts and myofibroblasts’ apoptosis. Interestingly, our data also demonstrate that the blockade of fibroblast differentiation to myofibroblasts is not mediated by inhibition of the canonical signaling of TGFβ1. Taken together our results demonstrate a pioneering conceptual approach to “normalize” the microenvironment that supports the outgrowth of QDTC by promoting the tissue resolution axis. This abstract is also being presented as Poster B36. Citation Format: Odelya Gilon, Yonatan David Feuermann, Sagie Schif-Zuck, Keren Weidenfeld, Palle Von Huth, Edmond Sabo, Amiram Ariel, Dalit Barkan. Preventing the recurrence of breast cancer at the metastatic niche using resolution-phase macrophages [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR05.

The physiological resolution of a well-orchestrated inflammatory response is essential to maintain homeostasis. Therefore, gaining a comprehensive understanding in molecular terms of the events that direct the termination of acute inflammation is imperative. Recently, new families of local-acting mediators were discovered that are biosynthesized from the essential fatty acids eicosapentaenoic acid and docosahexaenoic acid. These new chemical mediators are endogenously generated in inflammatory exudates collected during the resolution phase, and were termed resolvins and protectins because specific members of these families control the magnitude and duration of inflammation in animals. In addition, recent results indicate novel actions of resolvins and protectins in removing chemokines ferried from the tissue by apoptotic neutrophils and T cells during resolution. Here, we review recent advances on the biosynthesis and actions of these novel anti-inflammatory and proresolving mediators.

Pain is a complex sensation that may be protective or cause undue suffering and loss of function, depending on the circumstances. Peripheral nociceptor neurons (PNs) innervate most tissues, and express ion channels, nocisensors, which depolarize the cell in response to intense stimuli and numerous substances. Inflamed tissues manifest inflammatory hyperalgesia in which the threshold for pain and the response to painful stimuli are decreased and increased, respectively. Constituents of the inflammatory milieu sensitize PNs, thereby contributing to hyperalgesia. Polyunsaturated fatty acids undergo enzymatic and free radical-mediated oxygenation into an array of bioactive metabolites, oxygenated polyunsaturated fatty acids (oxy-PUFAs), including the classic eicosanoids. Oxy-PUFA production is enhanced during inflammation. Pioneering studies by Vane and colleagues from the early 1970s first implicated classic eicosanoids in the pain associated with inflammation. Here, we review the production and action of oxy-PUFAs that are not classic eicosanoids, but nevertheless are produced in injured/ inflamed tissues and activate or sensitize PNs. In general, oxy-PUFAs that sensitize PNs may do so directly, by activation of nocisensors, ion channels or GPCRs expressed on the surface of PNs, or indirectly, by increasing the production of inflammatory mediators that activate or sensitize PNs. We focus on oxy-PUFAs that act directly on PNs. Specifically, we discuss the role of arachidonic acid-derived 12S-HpETE, HNE, ONE, PGA2, iso-PGA2 and 15d-PGJ2, 5,6-and 8,9-EET, PGE2-G and 8R,15S-diHETE, as well as the linoleic acid-derived 9-and 13-HODE in inducing acute nocifensive behavior and/or inflammatory hyperalgesia in rodents. The nocisensors TRPV1, TRPV4 and TRPA1, and putative Gαs-type GPCRs are the PN targets of these oxy-PUFAs.

During the resolution phase of inflammation, the ‘corpses’ of apoptotic leukocytes are gradually cleared by macrophages. Here we report that during the resolution of peritonitis, the CCR5 chemokine receptor ligands CCL3 and CCL5 persisted in CCR5-deficient mice. CCR5 expression on apoptotic neutrophils and activated apoptotic T cells sequestered and effectively cleared CCL3 and CCL5 from sites of inflammation. CCR5 expression on late apoptotic human polymorphonuclear cells was downregulated by proinflammatory stimuli, including tumor necrosis factor, and was upregulated by ‘proresolution’ lipid mediators, including lipoxin A4, resolvin E1 and protectin D1. Our results suggest that CCR5+ apoptotic leukocytes act as ‘terminators’ of chemokine signaling during the resolution of inflammation.

Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, β1 integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-γ (INF-γ) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-γ production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites.

The mechanisms underlying the inflammatory and metastatic processes share a number of similar pathways, such as those involving adhesion, migration and extravasation. In this article, the effects of pro-inflammatory cytokines on metastatic-related activities of colon cancer cells were tested. The expression and biological activity of the proteoglycan CD44 in low (LS174T) and high metastatic (HM7) cell lines following exposure to TNFα and IL-8 were assessed. Treated cells expressed more CD44 splice variants (CD44v), while CD44 standard protein (CD44s) expression remained unchanged. Treatment with TNFα induced IL-8 secretion and IL-8 gene transcription in a time-dependent manner. Both cytokines enhanced the ability of the cells to adhere to the CD44-specific ligand hyaluronic acid, an effect that was specifically blocked by an anti-IL-8 antibody. These results suggest that the effect of TNFα on IL-8 is responsible for the regulation of the expression of CD44 isoforms. Additional experiments showed that neither of the cytokines tested regulate the expression of CD44 gene regulation via activation of a well-characterized specific 22-bp epidermal growth factor regulatory element present in the CD44 promoter sequence, suggesting that this is not the mechanism of activation. We conclude that immuno-modulatory mediators can modify the expression of cell-to-cell or cell-to-matrix adhesion proteins, implicated in the determination of phenotypes associated with aggressiveness and metastasis of colon cancer cells.

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology.

The adhesion of leukocytes to the extracellular matrix (ECM) depends on their responses to variations in the chemotactic signals in their milieu, as well as on the functioning of cytoskeletal and context-specific receptors. Ezrin, radixin, and moesin constitute a family of proteins that link the plasma membrane to the actin cytoskeleton. The surface expression of moesin on T cells and its role in cell adhesion has not been fully elucidated. Recently, we found that IL-2 peptides generated by elastase modified the adhesion of activated T cells to ECM ligands. Here, we further examined the adhesion regulatory effects of EFLNRWIT, one of the IL-2 peptides, as well as the existence and putative function of its receptor on T cells. We found that when presented to T cells in the absence of another activator, the EFLNRWIT peptide induced cell adhesion to vessel wall and ECM components. Binding of a radiolabeled peptide to T cells, precipitation with the immobilized peptide, and amino acid sequencing of the precipitated protein revealed that EFLNRWIT exerts its function via a cell surface-expressed moesin-like moiety, whose constitutive expression on T cells was increased after activation. This notion was further supported by our findings that: 1) anti-moesin mAb inhibited the binding of T cells to the immobilized EFLNRWIT peptide, 2) immobilized recombinant moesin bound the IL-2 peptide, and 3) soluble moesin inhibited the EFLNRWIT-induced T cell adhesion to fibronectin. Interestingly, moesin appears to be generally involved in T cell responses to adhesion-regulating signals. Thus, the IL-2 peptide EFLNRWIT appears to exert its modulating capacities via an adhesion-regulating moesin-like receptor.

Summary Migration of T cells into extravascular sites of inflammation is mediated by cell–cell and cell–matrix adhesion receptors, including the hyaluronan-binding glycoprotein, CD44. The biochemical nature of CD44 variants and the ligand specificity, function and the regulation of activation of CD44 expressed on various cell types have been extensively studied. However, little is still known about the short-term influence of cytokines and chemokines on the activation of CD44 on human T cells. Therefore, we studied the role of inflammatory mediators in regulating the adhesion of T cells from human peripheral blood to immobilized hyaluronan under static or shear stress conditions. We found that the CD44-dependent adhesion, under static and shear stress (i.e. relative gradual resistance to flow of 150 and 1500 s−1) conditions, of T cells to hyaluronan requires a T-cell activation of 2–3 hr and is regulated by the cross-linking of CD3, cytokines (e.g. interleukin-2 and tumour necrosis factor-α), and chemokines (e.g. MIP-1β, interleukin-8, and RANTES). This T-cell adhesion was manifested by polarization, spreading and co-localization of cell surface CD44 with a rearranged actin cytoskeleton in hyaluronan-bound T cells. Thus, cytokines and chemokines present in the vicinities of blood vessel walls or present intravascularly in tissues where immune reactions take place, can rapidly activate the CD44 molecules expressed on T cells.

SUMMARY We have found previously that disaccharides (DS) enzymatically generated from heparin or heparan sulphate can modulate tumour necrosis factor-a (TNF-a) secretion from immune cells in vitro and cell-mediated immune reactions in vivo. Here, we show that such DS can modulate the adhesion and migration of human T cells. We found that certain heparin- and heparan sulphatederived DS induced, in a dose-dependent manner, the adhesion of human T cells to both extracellular matrix (ECM) and immobilized fibronectin (FN); maximal T-cell adhesion occurred with 1 ng/ml of DS. The levels of T-cell adhesion to ECM that were induced by the tested DS molecules resembled those induced by the prototypic chemokine, macrophage inflammatory protein 1b (MIP-1b). However, the kinetics of DS-induced T-cell adhesion to FN resembled that induced by phorbol myristate acetate (PMA), but not that induced by MIP-1b. This adhesion appeared to involve b1 integrin recognition and activation, and was associated with specific intracellular activation pathways. Although a first exposure of T cells to certain DS molecules appeared to result in cell adhesion, a subsequent exposure of T cells to pro-adhesive chemokines, such as MIP-1b or RANTES, but not to other pro-adhesive stimuli, for example interleukin-2 or CD3 cross-linking, resulted in inhibition of T-cell adhesion to and chemotactic migration through FN. Hence, we propose that the breakdown products of tissues generated by inflammatory enzymes are part of an intrinsic functional programme, and not necessarily molecular waste. Moreover, because the DS molecules exert their modulatory functions within a limited time, it appears that the historical encounters of the tissue-invading cells with the constituents of inflamed loci may dictate the cells’ behaviour upon subsequent exposure to proinflammatory mediators.

Elevated extracellular K(+) ([K(+)](o)), in the absence of "classical" immunological stimulatory signals, was found to itself be a sufficient stimulus to activate T cell beta1 integrin moieties, and to induce integrin-mediated adhesion and migration. Gating of T cell voltage-gated K(+) channels (Kv1.3) appears to be the crucial "decision-making" step, through which various physiological factors, including elevated [K(+)](o) levels, affect the T cell beta1 integrin function: opening of the channel leads to function, whereas its blockage prevents it. In support of this notion, we found that the proadhesive effects of the chemokine macrophage-inflammatory protein 1beta, the neuropeptide calcitonin gene-related peptide (CGRP), as well as elevated [K(+)](o) levels, are blocked by specific Kv1.3 channel blockers, and that the unique physiological ability of substance P to inhibit T cell adhesion correlates with Kv1.3 inhibition. Interestingly, the Kv1.3 channels and the beta1 integrins coimmunoprecipitate, suggesting that their physical association underlies their functional cooperation on the T cell surface. This study shows that T cells can be activated and driven to integrin function by a pathway that does not involve any of its specific receptors (i.e., by elevated [K(+)](o)). In addition, our results suggest that undesired T cell integrin function in a series of pathological conditions can be arrested by molecules that block the Kv1.3 channels.

The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co-ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly-N-acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin-1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin-2 (IL-2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin-1, or its more stable, related, C2-S molecule obtained by site-directed mutagenesis. The presence of galectin-1 was shown to inhibit T-cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose-dependent manner. This effect was specifically blocked by anti-galectin-1 antibody and was dependent on the lectin's carbohydrate-binding properties. The inhibition of T-cell adhesion by galectin-1 correlates with the ability of this molecule to block the re-organization of the activated cell's actin cytoskeleton. Furthermore, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production was markedly reduced when IL-2-activated T cells were incubated with galectin-1 or its mutant. This effect was prevented by beta-galactoside-related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin-1 subfamily on inflammatory and autoimmune processes.