Carlos A. Estrada

CCR2 Plays a Critical Role in Dendritic Cell Maturation: Possible Role of CCL2 and NF-κB

We postulated that CCR2-driven activation of the transcription factor NF-κB plays a critical role in dendritic cell (DC) maturation (e.g., migration, costimulation, and IL-12p70 production), necessary for the generation of protective immune responses against the intracellular pathogen Leishmania major. Supporting this notion, we found that CCR2, its ligand CCL2, and NF-κB were required for CCL19 production and adequate Langerhans cell (LC) migration both ex vivo and in vivo. Furthermore, a role for CCR2 in upregulating costimulatory molecules was indicated by the reduced expression of CD80, CD86, and CD40 in Ccr2−/− bone marrow-derived dendritic cells (BMDCs) compared with wild-type (WT) BMDCs. Four lines of evidence suggested that CCR2 plays a critical role in the induction of protective immunity against L. major by regulating IL-12p70 production and migration of DC populations such as LCs. First, compared with WT, Ccr2−/− lymph node cells, splenocytes, BMDCs, and LCs produced lower levels of IL-12p70 following stimulation with LPS/IFN-γ or L. major. Second, a reduced number of LCs carried L. major from the skin to the draining lymph nodes in Ccr2−/− mice compared with WT mice. Third, early treatment with exogenous IL-12 reversed the susceptibility to L. major infection in Ccr2−/− mice. Finally, disruption of IL-12p70 in radioresistant cells, such as LCs, but not in BMDCs resulted in the inability to mount a fully protective immune response in bone marrow chimeric mice. Collectively, our data point to an important role for CCR2-driven activation of NF-κB in the regulation of DC/LC maturation processes that regulate protective immunity against intracellular pathogens.

The complex role of the chemokine receptor CCR2 in collagen-induced arthritis: implications for therapeutic targeting of CCR2 in rheumatoid arthritis.

CCR2 has been widely considered as a potential therapeutic target for autoimmune disease, particularly rheumatoid arthritis, and various CCR2 blocking agents have been developed, some of which have entered clinical trials. In this review, we examine the relevant information regarding the role of CCR2, and to a lesser extent of the closely related chemokine receptor CCR5, in the immunopathogenesis of collagen-induced arthritis, an animal model of rheumatoid arthritis. Experimental evidence showing that CIA is accelerated and exacerbated when CCR2 is genetically inactivated (knockout mice) or blocked with specific antibodies warrant additional investigations before the relevance of the findings in rodent models can be applied to human patients with RA.

Role of Astrocytes and Chemokine Systems in Acute TNFα induced Demyelinating Syndrome: CCR2-dependent Signals promote Astrocyte Activation and Survival via NF-κB and Akt

Chemotactic factors known as chemokines play an important role in the pathogenesis of multiple sclerosis (MS). Transgenic expression of TNFalpha in the central nervous system (CNS) leads to the development of a demyelinating phenotype (TNFalpha-induced demyelination; TID) that is highly reminiscent of MS. Little is known about the role of chemokines in TID but insights derived from studying this model might extend our current understanding of MS pathogenesis and complement data derived from the classic autoimmune encephalomyelitis (EAE) model system. Here we show that in TID, chemokines and their receptors were significantly increased during the acute phases of disease. Notably, the CCL2 (MCP-1)-CCR2 axis and the closely related ligand-receptor pair CCR1-CCL3 (MIP-1alpha) were among the most up-regulated during disease. On the other hand, receptors like CCR3 and CCR4 were not elevated. This significant increase in the levels of chemokines/receptors correlated with robust immune infiltration of the CNS by inflammatory cells, i.e., macrophages, and immune cells particularly T and B cells. Immunostaining and confocal microscopy, along with in vitro studies revealed that astrocytes were a major source of locally produced chemokines and expressed functional chemokine receptors such as CCR2. Using an in vitro system we demonstrate that expression of CCR2 was functional in astrocytes and that signaling via this receptor lead to activation of NF-kB and Akt and was associated with increased astrocyte survival. Collectively, our data suggests that transgenic murine models of MS are useful to dissect mechanisms of disease and that in these models, up-regulation of chemokines and their receptors may be key determinants in TID.

CCL2‐independent role of CCR2 in immune responses against Leishmania major

The chemokine CCL2 (MCP‐1) and its receptor CCR2 modulate leucocyte migration and T helper differentiation. CCL2 or CCR2 knockout (KO) mice have divergent phenotypes following infection with the intracellular parasite Leishmania major (L. major). Compared to wild‐type (WT) mice, intradermally infected CCR2 KO mice in the L. major‐resistant C57BL/6j background become susceptible and fail to generate protective Th1 responses. In contrast, subcutaneously infected CCL2 KO mice in the L. major‐susceptible BALB/c background are resistant and exhibit reduced pathogenic Th2 responses. Here we explore two variables that may account for this contrasting outcome, namely background strain and route of infection. We found that the CCR2‐null state, both in the BALB/c and the C57BL/6j background, was associated with increased susceptibility to intradermal or subcutaneous L. major infection. Notably, the CCL2‐null state did not change the ability of C57BL/6j mice to mount protective responses following intradermal infection. Dual genetic inactivation of CCR2 and CCL2 in the L. major‐resistant C57BL/6j background resulted in a shift to a susceptible phenotype analogous to that of CCR2 KO in the C57BL/6j background. We concluded that CCL2‐independent effects of CCR2 are indispensable for the control of L. major infection and the generation of protective immune responses.

CC chemokine receptor (CCR)-2 prevents arthritis development following infection by Mycobacterium avium

The host factors that influence autoimmune arthritides such as rheumatoid arthritis have not been fully elucidated. We previously found that genetic inactivation of CC chemokine receptor 2 (CCR2) in the arthritis-prone DBA/1j mouse strain significantly increases the susceptibility of this strain to autoimmune arthritis induced by immunization with collagen type II (CII) and complete Freund’s adjuvant (CFA). Here, we show that following intradermal infection with Mycobacterium avium, a similar arthritis phenotype was detected in Ccr2-null mice in the DBA/1j, but not in the BALB/c background. The failure to develop arthritis in Ccr2-null BALB/c mice occurred in the face of high bacterial burdens and low interferon gamma (IFNγ) production. By contrast, Ccr2-null DBA/1j mice had low bacterial burdens, produced normal amounts of IFNγ, and had high titers of autoantibodies against CII. Thus, the Ccr2-null state in an arthritic-prone genetic background leads to increased arthritis susceptibility following infectious (M. avium) and noninfectious (CII/CFA) challenges. Because CCR2 serves as a negative regulator of murine arthritis, caution might need to be exercised while testing CCR2 blockers in human arthritis or other diseases. These findings also indicate that Ccr2-null DBA/1j mice might serve as a valuable model system to uncover the immunological determinants of arthritis and to test novel antiarthritic agents.

Important role of CCR2 in a murine model of coronary vasculitis

BackgroundChemokines and their receptors play a role in the innate immune response as well as in the disruption of the balance between pro-inflammatory Th17 cells and regulatory T cells (Treg), underlying the pathogenesis of coronary vasculitis in Kawasaki disease (KD).ResultsHere we show that genetic inactivation of chemokine receptor (CCR)-2 is protective against the induction of aortic and coronary vasculitis following injection of Candida albicans water-soluble cell wall extracts (CAWS). Mechanistically, both T and B cells were required for the induction of vasculitis, a role that was directly modulated by CCR2. CAWS administration promoted mobilization of CCR2-dependent inflammatory monocytes (iMo) from the bone marrow (BM) to the periphery as well as production of IL-6. IL-6 was likely to contribute to the depletion of Treg and expansion of Th17 cells in CAWS-injected Ccr2+/+ mice, processes that were ameliorated following the genetic inactivation of CCR2.ConclusionCollectively, our findings provide novel insights into the role of CCR2 in the pathogenesis of vasculitis as seen in KD and highlight novel therapeutic targets, specifically for individuals resistant to first-line treatments.

Microscopic Computed Tomography–Based Virtual Histology for Visualization and Morphometry of Atherosclerosis in Diabetic Apolipoprotein E Mutant Mice

Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the arteries and is a leading cause of heart disease and stroke in developed and developing countries.1 Animal models have become increasingly important tools for addressing key mechanistic and therapeutic questions that cannot be answered from human studies of atherosclerosis. However, the small-scale vascular structures in genetically engineered mice require labor-intensive histomorphometric techniques to quantify lesions. Recently, a new technique has emerged to image ex vivo blocks of soft tissue by staining tissue with metal solutions, then scanning with a microscopic computed tomography (microCT) instrument (Figure I in the online-only Data Supplement).2 This technique was originally applied to the study of the developing heart in embryos3 and fetuses (Figure II in the online-only Data Supplement) but can also be applied to the en bloc imaging of the heart, great vessels, and lesions thereof. By this method, tissues are left intact, but one can employ image analysis to create “virtual” histological …

Critical role of chemokine (C-C motif) receptor 2 (CCR2) in the KKAy + Apoe / mouse model of the metabolic syndrome

Aims/hypothesisChemokines and their receptors such as chemokine (C-C motif) receptor 2 (CCR2) may contribute to the pathogenesis of the metabolic syndrome via their effects on inflammatory monocytes. Increased accumulation of CCR2-driven inflammatory monocytes in epididymal fat pads is thought to favour the development of insulin resistance. Ultimately, the resulting hyperglycaemia and dyslipidaemia contribute to development of the metabolic syndrome complications such as cardiovascular disease and diabetic nephropathy. Our goal was to elucidate the role of CCR2 and inflammatory monocytes in a mouse model that resembles the human metabolic syndrome.MethodsWe generated a model of the metabolic syndrome by backcrossing KKAy+ with Apoe−/− mice (KKAy+Apoe−/−) and studied the role of CCR2 in this model system.ResultsKKAy+Apoe−/− mice were characterised by the presence of obesity, insulin resistance, dyslipidaemia and increased systemic inflammation. This model also manifested two complications of the metabolic syndrome: atherosclerosis and diabetic nephropathy. Inactivation of Ccr2 in KKAy+Apoe−/− mice protected against the metabolic syndrome, as well as atherosclerosis and diabetic nephropathy. This protective phenotype was associated with a reduced number of inflammatory monocytes in the liver and muscle, but not in the epididymal fat pads; circulating levels of adipokines such as leptin, resistin and adiponectin were also not reduced. Interestingly, the proportion of inflammatory monocytes in the liver, pancreas and muscle, but not in the epididymal fat pads, correlated significantly with peripheral glucose levels.Conclusions/interpretationCCR2-driven inflammatory monocyte accumulation in the liver and muscle may be a critical pathogenic factor in the development of the metabolic syndrome.

CD8α⁺ dendritic cells improve collagen-induced arthritis in CC chemokine receptor (CCR)-2 deficient mice.

OBJECTIVE Dendritic cells (DCs) have long been recognized as potential therapeutic targets of rheumatoid arthritis (RA). Increasing evidence has showed that DCs are capable of suppressing autoimmunity by expanding FoxP3⁺ regulatory T cells (T(reg)), which in turn exert immunosuppression by increasing TGFβ-1. In the SKG mice, activated DC prime autoreactive T cells causing autoantibody production and an inflammatory arthritic response. Recently, we reported that CC-chemokine receptor-2 deficient (Ccr2⁻/⁻) mice had impaired DCs migration and reduced CD8α⁺ DCs in the C57Bl/6J mice strain and that these mice were more susceptible to collagen antibody-induced arthritis (CAIA), compared to wild type mice. To examine the mechanism by which DCs contribute to the increased susceptibility of arthritis in Ccr2⁻/⁻ mice, we tested the hypothesis that CD8α⁺ DCs are protective (tolerogenic) against autoimmune arthritis by examining the role of CD8α⁺ DCs in Ccr2⁻/⁻ and SKG mice. METHODS To examine the mechanism by which DCs defects lead to the development of arthritis, we used two murine models of experimental arthritis: collagen-induced arthritis (CIA) in DBA1/J mice and zymosan-induced arthritis in SKG mice. DBA1/J mice received recombinant fms-like tyrosine kinase 3 ligand (Flt3L) injections to expand endogenous DCs populations or adoptive transfers of CD8α⁺ DCs. RESULTS Flt3L-mediated expansion of endogenous CD8α⁺ DCs resulted in heightened susceptibility of CIA. In contrast, supplementation with exogenous CD8α⁺ DCs ameliorated arthritis in Ccr2⁻/⁻ mice and enhanced TGFβ1 production by T cells. Furthermore, SKG mice with genetic inactivation of CCR2 did not affect the numbers of DCs nor improve the arthritis phenotype. CONCLUSION CD8α⁺ DCs were tolerogenic to the development of arthritis. CD8α⁺ DCs deficiency heightened the sensitivity to arthritis in Ccr2⁻/⁻ mice. Ccr2 deficiency did not alter the arthritic phenotype in SKG mice suggesting the arthritis in Ccr2⁻/⁻ mice was T cell-independent.

Crosstalk Between Cytotoxic T-Lymphocyte–Associated Antigen-4 and Interleukin-12 in Cytotoxic T-Lymphocyte–Mediated Myocarditis: Adding Another Link to the Chain

See related article, pages 248–257 Myocarditis, or inflammation of the myocardium, may arise from immune-mediated destruction of myocardium harboring foreign viral or microbial antigens or from a dysregulated immune system that is unable to distinguish self myocardial antigens from foreign antigens. Of these causes, immune-mediated myocarditis is a primary etiology. Although myocarditis remains a relatively rare event, prognosis following diagnosis remains poor, and 6 year mortality rates approaching 30% have been reported.1 T lymphocytes mediate acquired immunity against a foreign antigen via humoral or cell-mediated mechanisms and hence may affect the outcome of myocarditis. T-lymphocyte involvement was first shown in the 1970s, when T-lymphocyte depletion decreased the inflammatory response and increased survival in a murine model of Coxsackie virus B3–induced myocarditis.2 Since then, research has focused on delineating the roles of individual lymphocyte subtypes. In this issue, Love et al examine the role of cytotoxic T-lymphocyte–associated antigen (CTLA)-4 in CD8+ T-cell regulation by using a transgenic model of CD8+ T-lymphocyte–mediated myocarditis.3 One subset of T lymphocytes is the CD8+ T lymphocytes, which are antigen-specific cytotoxic cells that normally protect against infection by killing infected cells via effector molecules such as perforins and granzymes. In the myocardium, CD8+ lymphocytes regulate both myocarditis and allograft rejection by differentiating into cytotoxic T lymphocytes (CTLs).4,5 Differentiation involves several effector functions, including increased proliferation and cytokine production.6 The CTL response to an infection can be divided into 4 phases. The first is the activation phase, in which naive CTL precursors are primed, undergo cell expansion, acquire effector function, travel to sites …