In situ neutrophil efferocytosis shapes T cell immunity to influenza infection.

Early recruitment of neutrophils from the blood to sites of tissue infection is a hallmark of innate immune responses. However, little is known about the mechanisms by which apoptotic neutrophils are cleared in infected tissues during resolution and the immunological consequences of in situ efferocytosis. Using intravital multiphoton microscopy, we show previously unrecognized motility patterns of interactions between neutrophils and tissue-resident phagocytes within the influenza-infected mouse airway. Newly infiltrated inflammatory monocytes become a chief pool of phagocytes and play a key role in the clearance of highly motile apoptotic neutrophils during the resolution phase. Apoptotic neutrophils further release epidermal growth factor and promote the differentiation of monocytes into tissue-resident antigen-presenting cells for activation of antiviral T cell effector functions. Collectively, these results suggest that the presence of in situ neutrophil resolution at the infected tissue is critical for optimal CD8+ T cell-mediated immune protection.

Long-Term Microgliosis Driven by Acute Systemic Inflammation.

Severe sepsis, a systemic inflammatory response to infection, is an increasing cause of morbidity in intensive care units. During sepsis, the vasculature is profoundly altered, leading to release of microbial virulence factors and proinflammatory mediators to surrounding tissue, causing severe systemic inflammatory responses and hypoxic injury of multiple organs. To date, multiple studies have explored pathologic conditions in many vital organs, including lungs, liver, and kidneys. Although data suggest that sepsis is emerging as a key driver of chronic brain dysfunction, the immunological consequence of severe inflammatory responses in the brain remain poorly understood. In this study, we used C57BL/6 sepsis mouse models to establish a disease phenotype in which septic mice with various degrees of severity recover. In the early phases of sepsis, monocytes infiltrate the brain with significantly elevated proinflammatory cytokine levels. In recovered animals, monocytes return to vehicle levels, but the number of brain-resident microglia is significantly increased in the cortex, the majority of which remain activated. The increase in microglia number is mainly due to self-proliferation, which is completely abolished in CCR2 knockout mice. Collectively our data suggest that early monocyte infiltration causes permanent changes to microglia during sepsis, which may ultimately dictate the outcome of future infections and neuropathological diseases.

Activated Protein C Attenuates Severe Inflammation by Targeting VLA-3high Neutrophil Subpopulation in Mice.

The host injury involved in multiorgan system failure during severe inflammation is mediated, in part, by massive infiltration and sequestration of hyperactive neutrophils in the visceral organ. A recombinant form of human activated protein C (rhAPC) has shown cytoprotective and anti-inflammatory functions in some clinical and animal studies, but the direct mechanism is not fully understood. Recently, we reported that, during endotoxemia and severe polymicrobial peritonitis, integrin VLA-3 (CD49c/CD29) is specifically upregulated on hyperinflammatory neutrophils and that targeting the VLA-3high neutrophil subpopulation improved survival in mice. In this article, we report that rhAPC binds to human neutrophils via integrin VLA-3 (CD49c/CD29) with a higher affinity compared with other Arg-Gly-Asp binding integrins. Similarly, there is preferential binding of activated protein C (PC) to Gr1highCD11bhighVLA-3high cells isolated from the bone marrow of septic mice. Furthermore, specific binding of rhAPC to human neutrophils via VLA-3 was inhibited by an antagonistic peptide (LXY2). In addition, genetically modified mutant activated PC, with a high affinity for VLA-3, shows significantly improved binding to neutrophils compared with wild-type activated PC and significantly reduced neutrophil infiltration into the lungs of septic mice. These data indicate that variants of activated PC have a stronger affinity for integrin VLA-3, which reveals novel therapeutic possibilities.

Cutting Edge: Selective Oral ROCK2 Inhibitor Reduces Clinical Scores in Patients with Psoriasis Vulgaris and Normalizes Skin Pathology via Concurrent Regulation of IL-17 and IL-10.

Targeted inhibition of Rho-associated kinase (ROCK)2 downregulates the proinflammatory T cell response while increasing the regulatory arm of the immune response in animals models of autoimmunity and Th17-skewing human cell culture in vitro. In this study, we report that oral administration of a selective ROCK2 inhibitor, KD025, reduces psoriasis area and severity index scores by 50% from baseline in 46% of patients with psoriasis vulgaris, and it decreases epidermal thickness as well as T cell infiltration in the skin. We observed significant reductions of IL-17 and IL-23, but not IL-6 and TNF-α, whereas IL-10 levels were increased in peripheral blood of clinical responders after 12 wk of treatment with KD025. Collectively, these data demonstrate that an orally available selective ROCK2 inhibitor downregulates the Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via a defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.

Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism.

Chronic graft-versus-host disease (cGVHD) remains a major complication following allogeneic bone marrow transplantation (BMT). The discovery of novel therapeutics is dependent on assessment in preclinical murine models of cGVHD. Rho-associated kinase 2 (ROCK2) recently was shown to be implicated in regulation of interleukin-21 (IL-21) and IL-17 secretion in mice and humans. Here, we report that the selective ROCK2 inhibitor KD025 effectively ameliorates cGVHD in multiple models: a full major histocompatibility complex (MHC) mismatch model of multiorgan system cGVHD with bronchiolitis obliterans syndrome and a minor MHC mismatch model of sclerodermatous GVHD. Treatment with KD025 resulted in normalization of pathogenic pulmonary function, which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. Spleens of mice treated with KD025 had decreased frequency of T follicular helper cells and increased frequency of T follicular regulatory cells, accompanied by a reduction in signal transducer and activator of transcription 3 (STAT3) and concurrent increase in STAT5 phosphorylation. The critical role of STAT3 in this cGVHD model was confirmed by data showing that mice transplanted with inducible STAT3-deficient T cells had pulmonary function comparable to the healthy negative controls. The therapeutic potential of targeted ROCK2 inhibition in the clinic was solidified further by human data demonstrating the KD025 inhibits the secretion of IL-21, IL-17, and interferon γ along with decreasing phosphorylated STAT3 and reduced protein expression of interferon regulatory factor 4 and B-cell lymphoma 6 (BCL6) in human peripheral blood mononuclear cells purified from active cGVHD patients. Together these data highlight the potential of targeted ROCK2 inhibition for clinical cGVHD therapy.

CD109, a novel TGF-ß antagonist, decreases fibrotic responses in a hypoxic wound model.

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF-ß co-receptor and TGF-ß antagonist shown to inhibit TGF-ß-induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild-type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post-wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild-type mice showed increased levels of HIF-1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post-wounding as compared to those in wild-type mice and to non-hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.

CD109 overexpression ameliorates skin fibrosis in a mouse model of bleomycin-induced scleroderma.

OBJECTIVE: Transforming growth factor ß (TGFß) is a profibrotic cytokine, and its aberrant function is implicated in several types of fibrotic pathologies including scleroderma (systemic sclerosis [SSc]). Multiple lines of evidence show that increased TGFß signaling contributes to progressive fibrosis in SSc by promoting fibroblast activation, excessive extracellular matrix (ECM) deposition, and dermal thickening. We have previously identified CD109 as a TGFß coreceptor and have shown that it antagonizes TGFß signaling and TGFß-induced ECM expression in vitro in human keratinocytes and fibroblasts. The aim of the present study was to examine the ability of CD109 to prevent skin fibrosis in a mouse model of bleomycin-induced SSc. METHODS: Transgenic mice overexpressing CD109 in the epidermis and their wild-type (WT) littermates were injected with bleomycin in phosphate buffered saline (PBS) or with PBS alone every other day for 21 days or 28 days. Dermal thickness and collagen deposition were determined histologically using Masson's trichrome and picrosirius red staining. In addition, collagen and fibronectin content was analyzed using Western blotting, and activation of TGFß signaling was examined by determining phospho-Smad2 and phospho-Smad3 levels using Western blotting and immunohistochemistry. RESULTS: Transgenic mice overexpressing CD109 in the epidermis showed resistance to bleomycin-induced skin fibrosis, as evidenced by a significant decrease in dermal thickness, collagen crosslinking, collagen and fibronectin content, and phospho-Smad2/3 levels, as compared to their WT littermates. CONCLUSION: Our findings suggest that CD109 inhibits TGFß signaling and fibrotic responses in experimental murine scleroderma. They also suggest that CD109 regulates dermal-epidermal interactions to decrease extracellular matrix synthesis in the dermis. Thus, CD109 is a potential molecular target for therapeutic intervention in scleroderma.

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing.

Transforming growth factor-ß (TGF-ß) is a multifunctional growth factor involved in all aspects of wound healing. TGF-ß accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol-anchored protein, as a novel TGF-ß coreceptor and inhibitor of TGF-ß signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild-type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin-1α and monocyte chemoattractant protein-1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.

Metabolic adaptation supports enhanced macrophage efferocytosis in limited-oxygen environments.

Apoptotic cell (AC) clearance (efferocytosis) is performed by phagocytes, such as macrophages, that inhabit harsh physiological environments. Here, we find that macrophages display enhanced efferocytosis under prolonged (chronic) physiological hypoxia, characterized by increased internalization and accelerated degradation of ACs. Transcriptional and translational analyses revealed that chronic physiological hypoxia induces two distinct but complimentary states. The first, "primed" state, consists of concomitant transcription and translation of metabolic programs in AC-naive macrophages that persist during efferocytosis. The second, "poised" state, consists of transcription, but not translation, of phagocyte function programs in AC-naive macrophages that are translated during efferocytosis. Mechanistically, macrophages efficiently flux glucose into a noncanonical pentose phosphate pathway (PPP) loop to enhance NADPH production. PPP-derived NADPH directly supports enhanced efferocytosis under physiological hypoxia by ensuring phagolysosomal maturation and redox homeostasis. Thus, macrophages residing under physiological hypoxia adopt states that support cell fitness and ensure performance of essential homeostatic functions rapidly and safely.

WNK1 enforces macrophage lineage fidelity.

The appropriate development of macrophages, the body's professional phagocyte, is essential for organismal development, especially in mammals. This dependence is exemplified by the observation that loss-of-function mutations in colony stimulating factor 1 receptor (CSF1R) results in multiple tissue abnormalities owing to an absence of macrophages. Despite this importance, little is known about the molecular and cell biological regulation of macrophage development. Here, we report the surprising finding that the chloride-sensing kinase With-no-lysine 1 (WNK1) is required for development of tissue-resident macrophages (TRMs). Myeloid-specific deletion of Wnk1 resulted in a dramatic loss of TRMs, disrupted organ development, systemic neutrophilia, and mortality between 3 and 4 weeks of age. Strikingly, we found that myeloid progenitors or precursors lacking WNK1 not only failed to differentiate into macrophages, but instead differentiated into neutrophils. Mechanistically, the cognate CSF1R cytokine macrophage-colony stimulating factor (M-CSF) stimulates macropinocytosis by both mouse and human myeloid progenitors and precursor cells. Macropinocytosis, in turn, induces chloride flux and WNK1 phosphorylation. Importantly, blocking macropinocytosis, perturbing chloride flux during macropinocytosis, and inhibiting WNK1 chloride-sensing activity each skewed myeloid progenitor differentiation from macrophages into neutrophils. Thus, we have elucidated a role for WNK1 during macropinocytosis and discovered a novel function of macropinocytosis in myeloid progenitors and precursor cells to ensure macrophage lineage fidelity. Highlights: Myeloid-specific WNK1 loss causes failed macrophage development and premature deathM-CSF-stimulated myeloid progenitors and precursors become neutrophils instead of macrophagesM-CSF induces macropinocytosis by myeloid progenitors, which depends on WNK1Macropinocytosis enforces macrophage lineage commitment.

Early life high fructose exposure disrupts microglia function and impedes neurodevelopment.

Despite the success of fructose as a low-cost food additive, recent epidemiological evidence suggests that high fructose consumption by pregnant mothers or during adolescence is associated with disrupted neurodevelopment 1-7 . An essential step in appropriate mammalian neurodevelopment is the synaptic pruning and elimination of newly-formed neurons by microglia, the central nervous system's (CNS) resident professional phagocyte 8-10 . Whether early life high fructose consumption affects microglia function and if this directly impacts neurodevelopment remains unknown. Here, we show that both offspring born to dams fed a high fructose diet and neonates exposed to high fructose exhibit decreased microglial density, increased uncleared apoptotic cells, and decreased synaptic pruning in vivo . Importantly, deletion of the high affinity fructose transporter SLC2A5 (GLUT5) in neonates completely reversed microglia dysfunction, suggesting that high fructose directly affects neonatal development. Mechanistically, we found that high fructose treatment of both mouse and human microglia suppresses synaptic pruning and phagocytosis capacity which is fully reversed in GLUT5-deficient microglia. Using a combination of in vivo and in vitro nuclear magnetic resonance- and mass spectrometry-based fructose tracing, we found that high fructose drives significant GLUT5-dependent fructose uptake and catabolism, rewiring microglia metabolism towards a hypo-phagocytic state. Importantly, mice exposed to high fructose as neonates exhibited cognitive defects and developed anxiety-like behavior which were rescued in GLUT5-deficient animals. Our findings provide a mechanistic explanation for the epidemiological observation that early life high fructose exposure is associated with increased prevalence of adolescent anxiety disorders.

Neutrophil heterogeneity in complement C1q expression associated with sepsis mortality.

Sepsis is a life-threatening systemic inflammatory condition causing approximately 11 million annual deaths worldwide. Although key hyperinflammation-based organ dysfunctions that drive disease pathology have been recognized, our understanding of the factors that predispose patients to septic mortality is limited. Due to the lack of reliable prognostic measures, the development of appropriate clinical management that improves patient survival remains challenging. Here, we discovered that a subpopulation of CD49chigh neutrophils with dramatic upregulation of the complement component 1q (C1q) gene expression arises during severe sepsis. We further found that deceased septic patients failed to maintain C1q protein expression in their neutrophils, whereas septic survivors expressed higher levels of C1q. In mouse sepsis models, blocking C1q with neutralizing antibodies or conditionally knocking out C1q in neutrophils led to a significant increase in septic mortality. Apoptotic neutrophils release C1q to control their own clearance in critically injured organs during sepsis; thus, treatment of septic mice with C1q drastically increased survival. These results suggest that neutrophil C1q is a reliable prognostic biomarker of septic mortality and a potential novel therapeutic target for the treatment of sepsis.

First we eat, then we do everything else: The dynamic metabolic regulation of efferocytosis.

Clearance of apoptotic cells, or "efferocytosis," is essential for diverse processes including embryonic development, tissue turnover, organ regeneration, and immune cell development. The human body is estimated to remove approximately 1% of its body mass via apoptotic cell clearance daily. This poses several intriguing cell metabolism problems. For instance, phagocytes such as macrophages must induce or suppress metabolic pathways to find, engulf, and digest apoptotic cells. Then, phagocytes must manage the potentially burdensome biomass of the engulfed apoptotic cell. Finally, phagocytes reside in complex tissue architectures that vary in nutrient availability, the types of dying cells or debris that require clearance, and the neighboring cells they interact with. Here, we review advances in our understanding of these three key areas of phagocyte metabolism. We end by proposing a model of efferocytosis that integrates recent findings and establishes a new paradigm for testing how efferocytosis prevents chronic inflammatory disease and autoimmunity.

Biomarkers and Associated Immune Mechanisms for Early Detection and Therapeutic Management of Sepsis.

Sepsis is conceptually defined as life-threatening organ dysfunction that is caused by a dysregulated host response to infection. Although there has been significant advancement in recent decades in defining and understanding sepsis pathology, clinical management of sepsis is challenging due to difficulties in diagnosis, a lack of reliable prognostic biomarkers, and treatment options that are largely limited to antibiotic therapy and fundamental supportive measures. The lack of reliable diagnostic and prognostic tests makes it difficult to triage patients who are in need of more urgent care. Furthermore, while the acute inpatient treatment of sepsis warrants ongoing attention and investigation, efforts must also be directed toward longer term survival and outcomes. Sepsis survivors experience incomplete recovery, with long-term health impairments that may require both cognitive and physical treatment and rehabilitation. This review summarizes recent advances in sepsis prognosis research and discusses progress made in elucidating the underlying causes of prolonged health deficits experienced by patients surviving the early phases of sepsis.

Increased generation of Foxp3(+) regulatory T cells by manipulating antigen presentation in the thymus.

Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases.

ROCK2 signaling is required to induce a subset of T follicular helper cells through opposing effects on STATs in autoimmune settings.

Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.