TLR9-mediated inflammation drives a Ccr2-independent peripheral monocytosis through enhanced extramedullary monocytopoiesis.

Monocytes are innate immune cells that interact with their environment through the expression of pattern recognition receptors, including Toll-like receptors (TLRs). Both monocytes and TLRs are implicated in driving persistent inflammation in autoimmune diseases. However, cell-intrinsic mechanisms to control inflammation, including TLR tolerance, are thought to limit inflammatory responses in the face of repeated TLR activation, leaving it unclear how chronic TLR-mediated inflammation is maintained in vivo. Herein, we used a well-characterized model of systemic inflammation to determine the mechanisms allowing sustained TLR9 responses to develop in vivo. Monocytes were identified as the main TLR9-responsive cell and accumulated in peripherally inflamed tissues during TLR9-driven inflammation. Intriguingly, canonical mechanisms controlling monocyte production and localization were altered during the systemic inflammatory response, as accumulation of monocytes in the liver and spleen developed in the absence of dramatic increases in bone marrow monocyte progenitors and was independent of chemokine (C-C motif) receptor 2 (Ccr2). Instead, TLR9-driven inflammation induced a Ccr2-independent expansion of functionally enhanced extramedullary myeloid progenitors that correlated with the peripheral accumulation of monocytes in both wild-type and Ccr2(-/-) mice. Our data implicate inflammation-induced extramedullary monocytopoiesis as a peripheral source of newly produced TLR9 responsive monocytes capable of sustaining chronic TLR9 responses in vivo. These findings help to explain how chronic TLR-mediated inflammation may be perpetuated in autoimmune diseases and increase our understanding of how monocytes are produced and positioned during systemic inflammatory responses.

Hyperinflammation, rather than hemophagocytosis, is the common link between macrophage activation syndrome and hemophagocytic lymphohistiocytosis.

PURPOSE OF REVIEW: Macrophage activation syndrome is the rheumatic disease-associated member of a group of hyperinflammatory syndromes characterized by uncontrolled cytokine storm. In this review, we highlight recent publications related to the pathoetiology of hyperinflammatory syndromes with an emphasis on how this new knowledge will guide our diagnosis, treatment, and future research efforts to better understand these deadly conditions. RECENT FINDINGS: The heterogeneity of clinical manifestations seen in patients with hyperinflammatory syndromes continues to grow as novel genetic and immunotherapeutic triggers of cytokine storm have been identified. Recent studies characterize unique cytokine and gene expression profiles from patients with different hyperinflammatory syndromes, whereas novel murine models begin to define networks of immune dysregulation thought to drive excessive inflammation in cytokine storm. SUMMARY: Emerging evidence suggests hypercytokinemia is the driving cause of immunopathology and morbidity/mortality in hyperinflammatory syndromes. Therefore, approaches to block cytokine function may be fruitful in treating hyperinflammatory syndromes with less toxicity than current therapies. However, not all hyperinflammatory syndromes result in the same pathogenic cytokine profile, implying that a personalized approach will be required for effective use of anticytokine therapies in the treatment of hyperinflammatory syndromes.

Improving Outcomes of Pediatric Lupus Care Delivery With Provider Goal-Setting Activities and Multidisciplinary Care Models.

OBJECTIVE: The present study was undertaken to evaluate high-quality care delivery in the context of provider goal-setting activities and a multidisciplinary care model using an electronic health record (EHR)-enabled pediatric lupus registry. We then determined associations between care quality and prednisone use among youth with systemic lupus erythematosus (SLE). METHODS: We implemented standardized EHR documentation tools to autopopulate a SLE registry. We compared pediatric Lupus Care Index (pLCI) performance (range 0.0-1.0; 1.0 representing perfect metric adherence) and timely follow-up 1) before versus during provider goal-setting activities and population management, and 2) in a multidisciplinary lupus nephritis versus rheumatology clinic. We estimated associations between pLCI and subsequent prednisone use adjusted for time, current medication, disease activity, clinical features, and social determinants of health. RESULTS: We analyzed 830 visits by 110 patients (median 7 visits per patient [interquartile range 4-10]) over 3.5 years. The provider-directed activity was associated with improved pLCI performance (adjusted ß 0.05 [95% confidence interval (95% CI) 0.01, 0.09]; mean 0.74 versus 0.69). Patients with nephritis in multidisciplinary clinic had higher pLCI scores (adjusted ß 0.06 [95% CI 0.02, 0.10]) and likelihood of timely follow-up than those in rheumatology (adjusted relative risk [RR] 1.27 [95% CI 1.02, 1.57]). A pLCI score of ≥0.50 was associated with 0.72-fold lower adjusted risk of subsequent prednisone use (95% CI 0.53, 0.93). Minoritized race, public insurance, and living in areas with greater social vulnerability were not associated with reduced care quality or follow-up, but public insurance was associated with higher risk of prednisone use. CONCLUSION: Greater attention to quality metrics is associated with better outcomes in childhood SLE. Multidisciplinary care models with population management may additionally facilitate equitable care delivery.

BCMA is essential for the survival of long-lived bone marrow plasma cells.

Long-lived humoral immunity is manifested by the ability of bone marrow plasma cells (PCs) to survive for extended periods of time. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes. We show that BLyS can sustain PC survival in vitro, and this survival can be further enhanced by interleukin 6. Selective up-regulation of Mcl-1 in PCs by BLyS suggests that this alpha-apoptotic gene product may play an important role in PC survival. Blockade of BLyS, via transmembrane activator and cyclophilin ligand interactor-immunoglobulin treatment, inhibited PC survival in vitro and in vivo. Heightened expression of B cell maturation antigen (BCMA), and lowered expression of transmembrane activator and cyclophilin ligand interactor and BAFF receptor in PCs relative to resting B cells suggests a vital role of BCMA in PC survival. Affirmation of the importance of BCMA in PC survival was provided by studies in BCMA-/- mice in which the survival of long-lived bone marrow PCs was impaired compared with wild-type controls. These findings offer new insights into the molecular basis for the long-term survival of PCs.

Brief Report: Interferon-γ-Mediated Immunopathology Potentiated by Toll-Like Receptor 9 Activation in a Murine Model of Macrophage Activation Syndrome.

OBJECTIVE: Macrophage activation syndrome (MAS) is a life-threatening cytokine storm syndrome that occurs in patients with underlying rheumatic diseases. Preclinical and clinical data suggest that interferon-γ (IFNγ) is pathogenic in MAS, but how IFNγ may be linked to disease pathogenesis remains unknown. This study was undertaken to determine whether IFNγ signals synergize with systemic innate immune responses to drive the cytokine storm in a murine model of MAS. METHODS: IFNγ-deficient mice were treated with 5 doses of the Toll-like receptor 9 (TLR-9) agonist CpG 1826, IFNγ, or a combination of the 2 stimuli over the course of 10 days. Immunopathologic features of MAS, including cytopenias, hepatitis, hepatosplenomegaly, and induction of inflammatory myelopoiesis, were assessed. Mixed bone marrow chimeras were created to determine whether TLR-9- and IFNγ receptor 1 (IFNγR1)-dependent signals induce enhanced myelopoiesis in a cell-intrinsic or cell-extrinsic manner. RESULTS: IFNγ-deficient mice did not develop features of MAS when treated with repeated doses of either the TLR-9 agonist or IFNγ alone. In contrast, IFNγ-deficient mice treated with both the TLR-9 agonist and IFNγ developed cytopenias, hepatitis, and hepatosplenomegaly, reproducing major clinical features of MAS. TLR-9- and IFNγR1-dependent signals synergized to enhance myeloid progenitor cell function and induce myelopoiesis in vivo, which occurred through cell-extrinsic mechanisms and correlated with the induction of disease. CONCLUSION: These findings demonstrate that TLR-9-driven signals potentiate the effects of IFNγ to initiate murine MAS, and provide evidence that induction of inflammatory myelopoiesis is a common TLR-9- and IFNγ-dependent pathway that may contribute to the pathogenesis of MAS.

Myeloid Folliculin balances mTOR activation to maintain innate immunity homeostasis.

The mTOR pathway is central to most cells. How mTOR is activated in macrophages and modulates macrophage physiology remain poorly understood. The tumor suppressor Folliculin (FLCN) is a GAP for RagC/D, a regulator of mTOR. We show here that LPS potently suppresses FLCN in macrophages, allowing nuclear translocation of the transcription factor TFE3, leading to lysosome biogenesis, cytokine production, and hypersensitivity to inflammatory signals. Nuclear TFE3 additionally activates a transcriptional RagD positive feedback loop that stimulates FLCN-independent canonical mTOR signaling to S6K and increases cellular proliferation. LPS thus simultaneously suppresses the TFE3 arm and activates the S6K arm of mTOR. In vivo, mice lacking myeloid FLCN reveal chronic macrophage activation, leading to profound histiocytic infiltration and tissue disruption, with hallmarks of human histiocytic syndromes like Erdheim-Chester Disease. Our data thus identify a critical FLCN-mTOR-TFE3 axis in myeloid cells, modulated by LPS, that balances mTOR activation and curbs innate immune responses.

Microbiota-dependent signals are required to sustain TLR-mediated immune responses.

Host-commensal interactions are critical for the generation of robust inflammatory responses, yet the mechanisms leading to this effect remain poorly understood. Using a murine model of cytokine storm, we identified that host microbiota are required to sustain systemic TLR-driven immune responses. Mice treated with broad-spectrum antibiotics or raised in germ-free conditions responded normally to an initial TLR signal but failed to sustain production of proinflammatory cytokines following administration of repeated TLR signals in vivo. Mechanistically, host microbiota primed JAK signaling in myeloid progenitors to promote TLR-enhanced myelopoiesis, which is required for the accumulation of TLR-responsive monocytes. In the absence of TLR-enhanced monocytopoiesis, antibiotic-treated mice lost their ability to respond to repeated TLR stimuli and were protected from cytokine storm-induced immunopathology. These data reveal priming of TLR-enhanced myelopoiesis as a microbiota-dependent mechanism that regulates systemic inflammatory responses and highlight a role for host commensals in the pathogenesis of cytokine storm syndromes.

Up-regulation of human monocyte CD163 upon activation of cell-surface Toll-like receptors.

The hemoglobin (Hb) scavenger receptor, CD163, is a cell-surface glycoprotein that is expressed exclusively on monocytes and macrophages. It binds and internalizes haptoglobin-Hb complexes and has been implicated in the resolution of inflammation. Furthermore, the regulation of CD163 during an innate immune response implies an important role for this molecule in the host defense against infection. LPS, derived from the outer membrane of Gram-negative bacteria, activates TLR4 to cause acute shedding of CD163 from human monocytes, followed by recovery and induction of surface CD163 to higher levels than observed on untreated monocytes. We now report that the TLR2 and TLR5 agonists--Pam3Cys and bacterial flagellin--have similar effects on CD163 surface expression. Up-regulation of CD163 following treatment of human PBMC with TLR2, TLR4, and TLR5 agonists parallels increased production of IL-6 and IL-10, and neutralization of IL-6 and/or IL-10 blocks CD163 up-regulation. Furthermore, simultaneous stimulation of TLR2 or TLR5 in combination with TLR4 activation results in enhanced up-regulation of CD163. It is notable that exogenous recombinant IFN-gamma (rIFN-gamma) suppresses cell-surface, TLR-mediated IL-10 production as well as CD163 up-regulation. Sustained down-regulation of CD163 mediated by rIFN-gamma can be partially rescued with exogenous rIL-10 but not with exogenous rIL-6. This divergent regulation of CD163 by cytokines demonstrates that human monocytes react differently to infectious signals depending on the cytokine milieu they encounter. Thus, surface CD163 expression on mononuclear phagocytes is a carefully regulated component of the innate immune response to infection.

Pivotal advance: activation of cell surface Toll-like receptors causes shedding of the hemoglobin scavenger receptor CD163.

The hemoglobin scavenger receptor (HbSR) CD163 is a monocyte/macrophage-specific glycoprotein that binds and facilitates uptake of haptoglobin-hemoglobin (Hp-Hb) complexes, which are rapidly formed in the circulation upon hemolysis of red blood cells. Hemolysis can be caused by a diverse range of infectious agents and provides pathogens a source of iron to enhance their survival and replication. Previous work demonstrated that lipopolysaccharide (LPS) activates monocytes to cleave cell-bound HbSR into a soluble mediator that retains the capacity to bind Hp-Hb complexes. We report that blocking LPS activation of Toll-like receptor 4 prevents LPS-mediated shedding of CD163. Furthermore, activation of two other cell surface Toll-like receptors (TLR), TLR2 and TLR5, induces shedding of the HbSR from human monocytes. In contrast, treatment of monocytes with intracellular TLR3, TLR7, and TLR9 agonists failed to cause HbSR shedding, suggesting that this shedding event is selective to cell surface TLR activation. These data demonstrate that the soluble HbSR is released from monocytic cells in response to TLR signaling as an acute innate immune response to extracellular pathogen infections.

Weathering the storm: Improving therapeutic interventions for cytokine storm syndromes by targeting disease pathogenesis.

Cytokine storm syndromes require rapid diagnosis and treatment to limit the morbidity and mortality caused by the hyperinflammatory state that characterizes these devastating conditions. Herein, we discuss the current knowledge that guides our therapeutic decision-making and personalization of treatment for patients with cytokine storm syndromes. Firstly, ICU-level supportive care is often required to stabilize patients with fulminant disease while additional diagnostic evaluations proceed to determine the underlying cause of cytokine storm. Pharmacologic interventions should be focused on removing the inciting trigger of inflammation and initiation of an individualized immunosuppressive regimen when immune activation is central to the underlying disease pathophysiology. Monitoring for a clinical response is required to ensure that changes in the therapeutic regimen can be made as clinically warranted. Escalation of immunosuppression may be required if patients respond poorly to the initial therapeutic interventions, while a slow wean of immunosuppression in patients who improve can limit medication-related toxicities. In certain scenarios, a decision must be made whether an individual patient requires hematopoietic cell transplantation to prevent recurrence of disease. Despite these interventions, significant morbidity and mortality remains for cytokine storm patients. Therefore, we use this review to propose a clinical schema to guide current and future attempts to design rational therapeutic interventions for patients suffering from these devastating conditions, which we believe speeds the diagnosis of disease, limits medication-related toxicities, and improves clinical outcomes by targeting the heterogeneous and dynamic mechanisms driving disease in each individual patient.

IL-10 distinguishes a unique population of activated, effector-like CD8+ T cells in murine acute liver inflammation.

Immune-mediated liver injury is a central feature of hyperinflammatory diseases, such as hemophagocytic syndromes, yet the immunologic mechanisms underlying those processes are incompletely understood. In this study, we used the toll-like receptor 9 (TLR9)-mediated model of a hemophagocytic syndrome known as macrophage activation syndrome (MAS) to dissect the predominant immune cell populations infiltrating the liver during inflammation. We identified CD8+ T cells that unexpectedly produce interleukin-10 (IL-10) in addition to interferon-γ (IFN-γ) as a major hepatic population induced by TLR9 stimulation. Despite their ability to produce this anti-inflammatory cytokine, IL-10+ hepatic CD8+ T cells in TLR9-MAS mice did not resemble CD8+ T suppressor cells. Instead, the induction of these cells occurred independently of antigen stimulation and was partially dependent on IFN-γ. IL-10+ hepatic CD8+ T cells demonstrated an activated phenotype and high turnover rate, consistent with an effector-like identity. Transcriptional analysis of this population confirmed a gene signature of effector CD8+ T cells yet suggested responsiveness to liver injury-associated growth factors. Together, these findings suggest that IL-10+ CD8+ T cells induced by systemic inflammation to infiltrate the liver have initiated an inflammatory, rather than regulatory, program and may thus have a pathogenic role in severe, acute hepatitis.

Dissecting the heterogeneity of macrophage activation syndrome complicating systemic juvenile idiopathic arthritis.

OBJECTIVE: To seek insights into the heterogeneity of macrophage activation syndrome (MAS) complicating systemic juvenile idiopathic arthritis (sJIA) through the analysis of a large patient sample collected in a multinational survey. METHODS: International pediatric rheumatologists and hemato-oncologists entered their patient data, collected retrospectively, in a Web-based database. The demographic, clinical, laboratory, histopathologic, therapeutic, and outcome data were analyzed in relation to (1) geographic location of caring hospital, (2) subspecialty of attending physician, (3) demonstration of hemophagocytosis, and (4) severity of clinical course. RESULTS: A total of 362 patients were included by 95 investigators from 33 countries. Demographic, clinical, laboratory, and histopathologic features were comparable among patients seen in diverse geographic areas or by different pediatric specialists. Patients seen in North America were given biologics more frequently. Patients entered by pediatric hemato-oncologists were treated more commonly with biologics and etoposide, whereas patients seen by pediatric rheumatologists more frequently received cyclosporine. Patients with demonstration of hemophagocytosis had shorter duration of sJIA at MAS onset, higher prevalence of hepatosplenomegaly, lower levels of platelets and fibrinogen, and were more frequently administered cyclosporine, intravenous immunoglobulin (IVIG), and etoposide. Patients with severe course were older, had longer duration of sJIA at MAS onset, had more full-blown clinical picture, and were more commonly given cyclosporine, IVIG, and etoposide. CONCLUSION: The clinical spectrum of MAS is comparable across patients seen in different geographic settings or by diverse pediatric subspecialists. There was a disparity in the therapeutic choices among physicians that underscores the need to establish uniform therapeutic protocols.

Estradiol attenuates lipopolysaccharide-induced CXC chemokine ligand 8 production by human peripheral blood monocytes.

Regulation of the inflammatory response is imperative to the maintenance of immune homeostasis. Activated monocytes elaborate a broad variety of proinflammatory cytokines that mediate inflammation, including CXCL8. Release of this chemokine attracts neutrophils to sites of bacterial invasion and inflammation; however, high levels of CXCL8 may result in excessive neutrophil infiltration and subsequent tissue damage. In this study, we demonstrate that 17beta-estradiol (E2) attenuates LPS-induced expression of CXCL8 in human peripheral blood monocytes. Treatment of monocytes with estradiol before administration of LPS reduces CXCL8 message and protein production through an estrogen receptor-dependent mechanism, and luciferase reporter assays demonstrate that this inhibition is mediated transcriptionally. Importantly, the ability of estradiol-pretreated LPS-activated monocytes to mobilize neutrophils is impaired. These results implicate a role for estradiol in the modulation of the immune response, and may lead to an enhanced understanding of gender-based differences in inflammatory control mechanisms.

The macrophage CD163 surface glycoprotein is an erythroblast adhesion receptor.

Erythropoiesis occurs in erythroblastic islands, where developing erythroblasts closely interact with macrophages. The adhesion molecules that govern macrophage-erythroblast contact have only been partially defined. Our previous work has implicated the rat ED2 antigen, which is highly expressed on the surface of macrophages in erythroblastic islands, in erythroblast binding. In particular, the monoclonal antibody ED2 was found to inhibit erythroblast binding to bone marrow macrophages. Here, we identify the ED2 antigen as the rat CD163 surface glycoprotein, a member of the group B scavenger receptor cysteine-rich (SRCR) family that has previously been shown to function as a receptor for hemoglobin-haptoglobin (Hb-Hp) complexes and is believed to contribute to the clearance of free hemoglobin. CD163 transfectants and recombinant protein containing the extracellular domain of CD163 supported the adhesion of erythroblastic cells. Furthermore, we identified a 13-amino acid motif (CD163p2) corresponding to a putative interaction site within the second scavenger receptor domain of CD163 that could mediate erythroblast binding. Finally, CD163p2 promoted erythroid expansion in vitro, suggesting that it enhanced erythroid proliferation and/or survival, but did not affect differentiation. These findings identify CD163 on macrophages as an adhesion receptor for erythroblasts in erythroblastic islands, and suggest a regulatory role for CD163 during erythropoiesis.

Lipopolysaccharide-induced IL-1 beta production by human uterine macrophages up-regulates uterine epithelial cell expression of human beta-defensin 2.

The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1beta. High doses of estradiol enhance LPS-induced IL-1beta expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1beta induce secretion of antimicrobial human beta-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.