CXCL9 inhibition does not ameliorate disease in murine models of both primary and secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic Lymphohistiocytosis (HLH) is a group of disorders culminating in systemic inflammation and multi-organ failure with high incidence of hepatic dysfunction. Overproduction of IFN-γ is the main immunopathological driver in this disorder. Monokine induced by IFN-γ (CXCL9) serves as a biomarker for disease activity and response to treatment in this disorder. However, very little is understood about the actual functional role of CXCL9 in pathogenesis in HLH. In the current study, we sought to determine the role of CXCL9 in pathogenesis in murine models of both Familial HLH (prf1-/-) and Toll Like Receptor (TLR) 9 repeated stimulation induced Macrophage Activation Syndrome (MAS), a form of secondary HLH. FHL and MAS were induced in both CXCL9 genetically deficient mice (cxcl9-/-) and controls as well as using AMG487, a pharmacological antagonist of the CXCL9 receptor, CXCR3. Results showed that CXCL9 genetic deficiency did not improve disease parameters or hepatitis in both models. Consistent with genetic ablation of CXCL9, inhibition of its receptor, CXCR3, by AMG487 did not show any significant effects in the FHL model. Taken together, inhibition of CXCL9-CXCR3 interaction does not ameliorate HLH physiology in general, or hepatitis as a classical target organ of disease.

BRAF-V600E utilizes posttranscriptional mechanisms to amplify LPS-induced TNFα production in dendritic cells in a mouse model of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is an inflammatory disease characterized by abnormal dendritic cells (DCs) with hyperactive ERK signaling, called "LCH cells." Since DCs rely on ERK signaling to produce inflammatory molecules in response to pathogenic cues, we hypothesized that hyperactive ERK enhances DCs inflammatory responses. We specifically investigated TLR4-induced TNFα production in LCH cells by utilizing the BRAF-V600Efl/+ :CD11c-Cre mouse model of LCH, which hyperactivates ERK in DCs. We measured LPS-induced TNFα production both in vivo and in vitro using splenic CD11c+ cells and bone marrow-derived DCs with or without pharmacologic BRAFV600E inhibition. We observed a reversible increase in secreted TNFα and a partially reversible increase in TNFα protein per cell, despite a decrease in TLR4 signaling and Tnfa transcripts compared with controls. We examined ERK-driven, posttranscriptional mechanisms that contribute to TNFα production and secretion using biochemical and cellular assays. We identified a reversible increase in TACE activation, the enzyme required for TNFα secretion, and most strikingly, an increase in protein translation, including TNFα. Defining the translatome through polysome-bound RNA sequencing revealed up-regulated translation of the LPS-response program. These data suggest hyperactive ERK signaling utilizes multiple posttranscriptional mechanisms to amplify inflammatory responses in DCs, advancing our understanding of LCH and basic DC biology.

Familial hemophagocytic lymphohistiocytosis hepatitis is mediated by IFN-γ in a predominantly hepatic-intrinsic manner.

Interferon gamma (IFN-γ) is the main cytokine driving organ dysfunction in Familial Hemophagocytic Lymphohistiocytosis (FHL). Blockade of IFN-γ pathway ameliorates FHL hepatitis, both in animal models and in humans with FHL. Hepatocytes are known to express IFN-γ receptor (IFN-γ-R). However, whether IFN-γ induced hepatitis in FHL is a lymphocyte or liver intrinsic response to the cytokine has yet to be elucidated. Using a IFNgR-/- bone marrow chimeric model, this study showed that non-hematopoietic IFN-γ response is critical for development of FHL hepatitis in LCMV-infected Prf1-/- mice. Lack of hepatic IFN-γ responsiveness results in reduced hepatitis as measured by hepatomegaly, alanine aminotransferase (ALT) levels and abrogated histologic endothelial inflammation. In addition, IFN-γ non-hematopoietic response was critical in activation of lymphocytes by soluble interleukin 2 receptor (sIL-2r) and recruitment of CD8+ effector T lymphocytes (CD8+ CD44hi CD62Llo) (Teff) and inflammatory monocytes. Lastly, non-hematopoietic IFN-γ response results in increased hepatic transcription of type 1 immune response and oxidative stress response pathways, while decreasing transcription of genes involved in extracellular matrix (ECM) production. In summary, these findings demonstrate that there is a hepatic transcriptional response to IFN-γ, likely critical in the pathogenesis of FHL hepatitis and hepatic specific responses could be a therapeutic target in this disorder.

Amelioration of Murine Macrophage Activation Syndrome by Monomethyl Fumarate in Both a Heme Oxygenase 1-Dependent and Heme Oxygenase 1-Independent Manner.

OBJECTIVE: Macrophage activation syndrome (MAS) is characterized by increased serum levels of ferritin and heme oxygenase 1 (HO-1), and yet no known function is ascribed to these molecules in MAS. Because HO-1 is antiinflammatory, we hypothesized that pharmacologic activation of HO-1 could ameliorate MAS disease activity. Dimethyl fumarate (DMF), a treatment approved by the US Food and Drug Administration for multiple sclerosis, activates HO-1. Monomethyl fumarate (MMF) is the active metabolite of DMF. We therefore evaluated whether MMF could elicit HO-1-dependent therapeutic improvements in a murine model of MAS. METHODS: We induced MAS by repeated activation of Toll-like receptor 9 (TLR-9) in wild-type and myeloid-specific HO-1-deficient mice. MMF was administered twice daily to test its efficacy. We assessed organ weights, serum cytokine levels, histologic features of the spleen and liver tissue, and complete blood cell counts to evaluate disease activity. Statistical testing was performed using Student's t-test or by 2-way analysis of variance as appropriate. RESULTS: The presence of HO-1 was required for the majority of TLR-9-induced interleukin-10 (IL-10). IL-10 production in TLR-9-induced MAS was found to correlate with the myeloid-HO-1 gene dose in myeloid cells (P < 0.001). MMF treatment increased the levels of HO-1 in splenic macrophages by ~2-fold (P < 0.01), increased serum levels of IL-10 in an HO-1-dependent manner in mice with TLR-9-induced MAS (P < 0.005), and improved multiple disease parameters in both an HO-1-dependent and HO-1-independent manner. CONCLUSION: TLR-9-induced production of IL-10 is regulated by HO-1 activity both in vitro and in vivo. Therapeutic enhancement of the HO-1/IL-10 axis in a murine model was able to significantly ameliorate MAS disease activity. These results suggest that HO-1 may be viable as a MAS therapeutic target, and treatment with DMF and MMF should be considered in future investigations of MAS therapy.

Genetic Deficiency of Interferon-γ Reveals Interferon-γ-Independent Manifestations of Murine Hemophagocytic Lymphohistiocytosis.

OBJECTIVE: Familial hemophagocytic lymphohistiocytosis (FHLH) is a complex cytokine storm syndrome caused by genetic abnormalities rendering CD8+ T cells and natural killer cells incapable of cytolytic killing. In murine models of FHLH, interferon-γ (IFNγ) produced by CD8+ T cells has been identified as a critical mediator of disease, and an IFNγ-blocking antibody (emapalumab) has recently been approved by the Food and Drug Administration. However, development of hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) in patients who are genetically unresponsive to IFNγ questions the absolute necessity of IFNγ in driving disease. This study was undertaken to determine the necessity of IFNγ in driving HLH. METHODS: IFNγ-/- Prf1-/- mice were infected with lymphocytic choriomeningitis virus (LCMV), and HLH immunopathologic features, including survival, weight loss, cytopenias, cytokine profiles, and immune cell phenotypes, were assessed. Mixed bone marrow chimeras were created to determine the immune cell-intrinsic role of IFNγ receptor signaling. CD8+ T cell depletion and interleukin-33 (IL-33)/ST2 blockade were performed using monoclonal antibodies. RESULTS: LCMV infection of IFNγ-/- Prf1-/- mice resulted in severe HLH-like disease. CD8+ T cells and the IL-33/ST2 axis remained essential mediators of disease; however, IFNγ-independent HLH immunopathology correlated with a 10-15-fold increase in neutrophilia (P < 0.001) and an altered cytokine milieu dominated by IL-6, IL-1ß, and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05). Furthermore, IFNγ regulated CD8+ T cell expression of GM-CSF and neutrophil survival. CONCLUSION: IFNγ is not necessary for the development of fulminant HLH, requiring physicians to consider case-by-case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL-33/ST2 signaling, may be more universally applicable treatment options that ameliorate both IFNγ-dependent and -independent manifestations of HLH/MAS.

Sterile particle-induced inflammation is mediated by macrophages releasing IL-33 through a Bruton's tyrosine kinase-dependent pathway.

Initiation of the innate sterile inflammatory response that can develop in response to microparticle exposure is little understood. Here, we report that a potent type 2 immune response associated with the accumulation of neutrophils, eosinophils and alternatively activated (M2) macrophages was observed in response to sterile microparticles similar in size to wear debris associated with prosthetic implants. Although elevations in interleukin-33 (IL-33) and type 2 cytokines occurred independently of caspase-1 inflammasome signalling, the response was dependent on Bruton's tyrosine kinase (BTK). IL-33 was produced by macrophages and BTK-dependent expression of IL-33 by macrophages was sufficient to initiate the type 2 response. Analysis of inflammation in patient periprosthetic tissue also revealed type 2 responses under aseptic conditions in patients undergoing revision surgery. These findings indicate that microparticle-induced sterile inflammation is initiated by macrophages activated to produce IL-33. They further suggest that both BTK and IL-33 may provide therapeutic targets for wear debris-induced periprosthetic inflammation.

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.

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.

Disruption of IL-33 Signaling Limits Early CD8+ T Cell Effector Function Leading to Exhaustion in Murine Hemophagocytic Lymphohistiocytosis.

Danger signals mediated through ST2, the interleukin-33 (IL-33) receptor, amplify CD8+ T cell-mediated inflammation in the murine model of familial hemophagocytic lymphohistiocytosis type 2 (FHL2), and blockade of ST2 provides a potential therapeutic strategy in this disease. However, the long-term effects of disrupting IL-33/ST2 signaling on the CD8+ T cell compartment are unknown. Here, we examined the evolution of the T cell response in murine FHL type 2 in the absence of ST2 signaling and found that CD8+ T cells gradually undergo exhaustion, similar to a related nonfatal FHL model. ST2 inhibition indirectly promotes CD8+ T cell exhaustion, and in contrast to other forms of FHL, reversal of exhaustion does not affect mortality. Disruption of IL-33 signaling exerts a more significant impact on the CD8+ T cell compartment early in the course of disease by intrinsically limiting CD8+ T cell proliferative and cytokine production capacity. Our data thus suggest that while ST2 blockade ultimately enables the development of CD8+ T cell exhaustion in late-stage murine FHL2, exhaustion is merely an effect, rather than the cause, of extended survival in these mice. The acute impact of ST2 inhibition on both the quantity and quality of the effector CD8+ T cell response more likely underlies the protective benefits of this treatment. This study provides evidence that redefines the relationship between CD8+ T cell exhaustion and mortality in murine FHL and supports the therapeutic use of ST2 blockade during the acute stage of disease.

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.

ST2 contributes to T-cell hyperactivation and fatal hemophagocytic lymphohistiocytosis in mice.

Cytokine storm syndromes, such as familial hemophagocytic lymphohistiocytosis (FHL), are lethal disorders caused by uncontrolled, systemic immune activation. In the murine model of FHL, in which perforin-deficient (Prf1(-/-)) mice are infected with lymphocytic choriomeningitis virus (LCMV), disease is driven by overabundant interferon (IFN)γ-producing LCMV-specific CD8(+) T cells thought to arise from excessive antigen stimulation through the T-cell receptor. However, this paradigm is insufficient to explain several fundamental aspects of FHL, namely, the inability of many pathogenic antigens to induce hyperinflammation, and the previously identified role of MyD88 in the disease. We now show a novel role for the MyD88-dependent interleukin-33 (IL-33) receptor, ST2, in FHL. Expression of IL-33 and ST2 is upregulated in LCMV-infected Prf1(-/-) mice. Blockade of ST2 markedly improves survival of LCMV-infected Prf1(-/-) mice and reduces the severity of multiple disease parameters, including serum levels of IFNγ. This decrease in IFNγ corresponds to a reduction in both the frequency of IFNγ(+) LCMV-specific CD8(+) and CD4(+) T cells and the magnitude of IFNγ expression in these cells. These findings demonstrate that disruption of ST2 signaling in the murine model of FHL reduces T cell-mediated production of IFNγ and suggest a revised paradigm in which danger signals such as IL-33 are crucial amplifiers of immune dysregulation in FHL. Furthermore, this study provides evidence to support blockade of ST2 as a novel therapeutic strategy for FHL.

Toll-like receptor 9 and interferon-γ receptor signaling suppress the B-cell fate of uncommitted progenitors in mice.

Systemic inflammatory response syndrome describes a heterogeneous group of cytokine storm disorders, with different immunogens and cytokines leading to variations in organ pathology. The severe inflammation generated by the cytokine storm results in widespread organ pathology including alterations in T- and B-lymphocyte counts. This study explores the roles of TLR9 and IFN-γR stimulation in decreasing T- and B-cell lymphopoiesis in a mouse model of hyperinflammation. We demonstrate that early B-cell lymphopoiesis is severely compromised during TLR9- and IFN-γ-driven hyperinflammation from the Ly-6D(+) common lymphoid progenitor stage onwards with different effects inhibiting development at multiple stages. We show that TLR9 signaling directly decreases in vitro B-cell yields while increasing T-cell yields. IFN-γ also directly inhibits B-cell and T-cell differentiation in vitro as well as when induced by TLR9 in vivo. Microarray and RT-PCR analysis of Ly-6D(-) common lymphoid progenitors point to HOXa9 and EBF-1 as transcription factors altered by TLR9-induced inflammation. Our work demonstrates both cellular and molecular targets that lead to diminished B-cell lymphopoiesis in sustained TLR9- and IFN-γ-driven inflammation that may be relevant in a number of infectious and autoimmune/inflammatory settings.

Brief report: alternative activation of laser-captured murine hemophagocytes.

OBJECTIVE: Hemophagocytes (HPCs) are activated macrophages that have engulfed other hematopoietic cells. Although HPCs are rarely identified in normal spleen tissue and bone marrow, an excess of these macrophages characterizes many cytokine storm syndromes, particularly macrophage activation syndrome and hemophagocytic lymphohistiocytosis. This study was undertaken to assess the functions of HPCs and their significance in acute inflammatory conditions. METHODS: HPCs were generated in wild-type mice using repeated stimulation with Toll-like receptor 9 (TLR-9) and interleukin-10 receptor blockade. RNA was extracted from HPCs that had been isolated by lasercaptured microdissection. Transcriptional profiles of the HPCs were then compared to those of resting splenic macrophages. In addition, bone marrow samples were obtained from a diverse cohort of patients in whom excess hemophagocytosis was identified by clinical bone marrow biopsy or aspiration. The bone marrow samples were analyzed by immunohistochemistry for markers of classic (CD64) or alternative (CD163 and CD206) macrophage activation. RESULTS: Differential gene expression and gene set enrichment analyses of murine HPCs identified upregulation of genes and gene sets associated with alternative activation of HPCs. Immunohistochemical analyses of HPCs in human bone marrow samples showed universal staining of HPCs for CD163, but rarely for CD206 or CD64. CONCLUSION: Laser-captured murine TLR-9­ induced HPCs had a transcriptional profile similar to that of alternatively activated macrophages. In addition, HPC expression of CD163 was confirmed in a uniquely diverse cohort of patients with hemophagocytic syndromes. Collectively, these data support the hypothesis that HPCs have both immunoregulatory and clean-up functions.

Interferon-γ mediates anemia but is dispensable for fulminant toll-like receptor 9-induced macrophage activation syndrome and hemophagocytosis in mice.

OBJECTIVE: Macrophage activation syndrome (MAS) is a devastating cytokine storm syndrome complicating many inflammatory diseases and characterized by fever, pancytopenia, and systemic inflammation. It is clinically similar to hemophagocytic lymphohistiocytosis (HLH), which is caused by viral infection of a host with impaired cellular cytotoxicity. Murine models of MAS and HLH illustrate that interferon-γ (IFNγ) is the driving stimulus for hemophagocytosis and immunopathology. This study was undertaken to investigate the inflammatory contributors to a murine model of Toll-like receptor 9 (TLR-9)-induced fulminant MAS. METHODS: Wild-type, transgenic, and cytokine-inhibited mice were treated with an IL-10 receptor blocking antibody and a TLR-9 agonist, and parameters of MAS were evaluated. RESULTS: Fulminant MAS was characterized by dramatic elevations in IFNγ, IL-12, and IL-6 levels. Increased serum IFNγ levels were associated with enhanced IFNγ production within some hepatic cell populations but also with decreased numbers of IFNγ-positive cells. Surprisingly, IFNγ-knockout mice developed immunopathology and hemophagocytosis comparable to that seen in wild-type mice. However, IFNγ-knockout mice did not become anemic and had greater numbers of splenic erythroid precursors. IL-12 neutralization phenocopied disease in IFNγ-knockout mice. Interestingly, type I IFNs contributed to the severity of hypercytokinemia and weight loss, but their absence did not otherwise affect MAS manifestations. CONCLUSION: These data demonstrate that both fulminant MAS and hemophagocytosis can arise independently of IFNγ, IL-12, or type I IFNs. They also suggest that IFNγ-mediated dyserythropoiesis, not hemophagocytosis, is the dominant cause of anemia in fulminant TLR-9-induced MAS. Thus, our data establish a novel mechanism for the acute anemia of inflammation, but suggest that a variety of triggers can result in hemophagocytic disease.

Eliminating murine norovirus by cross-fostering.

Murine norovirus (MNV) is a newly discovered and extremely prevalent pathogen of laboratory mouse colonies. MNV causes severe disease in some immunocompromised mouse strains and can cause persistent infections even in immunocompetent mice. Despite the fact that immunocompetent mice are generally asymptomatic, the possibility that MNV infection might alter immune responses makes its eradication a potentially useful goal for many facilities. Initial attempts by others to use a strategy of testing and culling were unsuccessful, whereas complete depopulation and facility decontamination was successful. However, these measures may be impractical, and finding less drastic approaches seemed prudent. Based on a report that cross-fostering of pups from MNV-positive mothers to MNV-negative ones could be successful in experimental MNV infection, we undertook a comprehensive fostering program using Swiss Webster mothers, careful sanitary measures, and fecal PCR testing to eradicate the virus from a mouse colony recently infected with MNV. We successfully decontaminated 17 of 18 (94%) litters and managed to prevent spread when a new MNV-infected mouse strain entered quarantine at our facility. These results suggest that cross-fostering, when performed in a setting of excellent sanitary procedures, may be practical for the large number of mouse facilities in which MNV is endemic.

IgG1 is pathogenic in Leishmania mexicana infection.

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.

Inducible expression of functional mu opioid receptors in murine dendritic cells.

Opioids are known to exert direct effects on the immune system, and the expression of functional opioid receptors has been reported on several immune cell types. Dendritic cells (DCs) are important inducers and regulators of immune responses. In this study, we investigated whether murine dendritic cells express functional mu opioid receptors (MOR). RT-PCR analysis and double immunofluorescence staining revealed the expression of MOR in activated murine dendritic cells. We also studied the dynamic expression of MOR messenger RNA in murine dendritic cells in response to different Toll-like receptor ligands. Functionally, treatment of DCs with endomorphin 1 (EM1), a specific agonist of MOR, can inhibit the forskolin-induced formation of cyclic adenosine monophosphate level in activated DCs. Moreover, EM1 treatment resulted in less activation of p38 MAPK and more activation of ERK signaling in lipopolysaccharide-stimulated DCs. Consistently, treatment of DCs with EM1 altered cytokine production by increasing IL-10 and decreasing IL-12 and IL-23. Our results suggest that MOR is inducibly expressed on activated DCs and functionally mediates EM1-induced effects on DCs. Thus, dendritic cells might be involved in crosstalk between the neuroendocrine and the immune system.

Inducible IL-23p19 expression in human microglia via p38 MAPK and NF-kappaB signal pathways.

Activated microglia can release a variety of proinflammatory cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). IL-23, a novel proinflammatory cytokine, is required for the induction of experimental autoimmune encephalomyelitis. Previously we demonstrated that IL-23 is expressed in MS lesions and that microglia are one cellular source of IL-23 in MS patients. In the present study we investigated the inducible expression and regulation of p19, a key subunit of IL-23, in human microglia. We demonstrated the inducible expression of IL-23p19 by lipopolysaccharide-stimulated microglial cells. Using signaling pathway-specific inhibitors, we showed that blocking p38 MAP kinase or NF-kappaB signaling pathway significantly reduced p19 expression in microglia. The regulatory role of p38 MAP kinase in p19 expression was further confirmed by decreased expression in microglia transduced with dominant-negative p38. We concluded that the p38 MAP kinase and NF-kappaB signaling pathways play an important role in regulation of IL-23p19 expression on human microglia, and are thus potential therapeutic targets in the treatment of MS.

Increased IL-23p19 expression in multiple sclerosis lesions and its induction in microglia.

IL-12 has long been considered important in the pathogenesis of multiple sclerosis. However, evidence from recent studies strongly supports the critical role of IL-12-related proinflammatory cytokine IL-23, but not IL-12, in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of this disease. The role of IL-23 in the CNS immunity of multiple sclerosis patients has not been elucidated; nor is it known whether human microglia produce this cytokine. In this study we investigated the expression of IL-23p19 and p40, with its key subunit p19 as the focus, in histologically characterized CNS specimens from multiple sclerosis and control cases using in situ hybridization and immunohistochemistry. A significant increase in mRNA expression and protein production of both subunits of IL-23 was found in lesion tissues compared with non-lesion tissues. Double staining showed that activated macrophages/microglia were an important source of IL-23p19 in active and chronic active multiple sclerosis lesions. We also detected IL-23p19 expression in mature dendritic cells which were preferentially located in the perivascular cuff of active lesions. The finding that human microglia produce IL-23 was further confirmed by the inducible production of IL-23p19 and p40 in cultured human microglia in vitro upon different Toll-like receptor stimulations. Taken together, these findings on the expression of IL-23p19 in multiple sclerosis lesions may lead to a better understanding of the events culminating in human multiple sclerosis.

Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentiation in vitro and in vivo.

Dendritic cells (DCs) have been suggested to direct a type of Th differentiation through their cytokine profile, e.g., high IL-12/IL-23 for Th1 (named DC1/immunogenic DCs) and IL-10 for Th2 (DC2/tolerogenic DCs). Suppressor of cytokine signaling (SOCS)-3 is a potent inhibitor of Stat3 and Stat4 transduction pathways for IL-23 and IL-12, respectively. We thus hypothesize that an enhanced SOCS-3 expression in DCs may block the autocrine response of IL-12/IL-23 in these cells, causing them to become a DC2-type phenotype that will subsequently promote Th2 polarization of naive T cells. Indeed, in the present study we found that bone marrow-derived DCs transduced with SOCS-3 significantly inhibited IL-12-induced activation of Stat4 and IL-23-induced activation of Stat3. These SOCS-3-transduced DCs expressed a low level of MHC class II and CD86 on their surface, produced a high level of IL-10 but low levels of IL-12 and IFN-gamma, and expressed a low level of IL-23 p19 mRNA. Functionally, SOCS-3-transduced DCs drove naive myelin oligodendrocyte glycoprotein-specific T cells to a strong Th2 differentiation in vitro and in vivo. Injection of SOCS-3-transduced DCs significantly suppressed experimental autoimmune encephalomyelitis, a Th1 cell-mediated autoimmune disorder of the CNS and an animal model of multiple sclerosis. These results indicate that transduction of SOCS-3 in DCs is an effective approach to generating tolerogenic/DC2 cells that then skew immune response toward Th2, thus possessing therapeutic potential in Th1-dominant autoimmune disorders such as multiple sclerosis.