Jennifer L. Auger

Absence of β2 Integrins Impairs Regulatory T Cells and Exacerbates CD4+ T Cell-Dependent Autoimmune Carditis

The immunopathogenic mechanisms mediating inflammation in multiorgan autoimmune diseases may vary between the different target tissues. We used the K/BxN TCR transgenic mouse model to investigate the contribution of CD4+ T cells and β2 integrins in the pathogenesis of autoimmune arthritis and endocarditis. Depletion of CD4+ T cells following the onset of arthritis specifically prevented the development of cardiac valve inflammation. Genetic absence of β2 integrins had no effect on the severity of arthritis and unexpectedly increased the extent of cardiovascular pathology. The exaggerated cardiac phenotype of the β2 integrin-deficient K/BxN mice was accompanied by immune hyperactivation and was linked to a defect in regulatory T cells. These findings are consistent with a model in which the development of arthritis in K/BxN mice relies primarily on autoantibodies, whereas endocarditis depends on an additional contribution of effector T cells. Furthermore, strategies targeting β2 integrins for the treatment of systemic autoimmune conditions need to consider not only the role of these molecules in leukocyte recruitment to sites of inflammation, but also their impact on the regulation of immunological tolerance.

Incomplete TCR-β allelic exclusion accelerates spontaneous autoimmune arthritis in K/BxN TCR transgenic mice.

Allelic exclusion of antigen receptor loci is a fundamental mechanism of immunological self‐tolerance. Incomplete allelic exclusion leads to dual T‐cell receptor (TCR) expression and can allow developing autoreactive αβ T lymphocytes to escape clonal deletion. Because allelic exclusion at the TCR‐β locus is more stringent than at the TCR‐α locus, dual TCR‐β expression has not been considered a likely contributor to autoimmunity. We show here that incomplete TCR‐β allelic exclusion permits developing thymocytes bearing the autoreactive, transgene‐encoded KRN TCR to be positively selected more efficiently, thereby accelerating the onset of spontaneous autoimmune arthritis. Our findings highlight dual TCR‐β expression as a mechanism that can enhance the maturation of autoreactive pathogenic T cells and lead to more rapid development of autoimmune disease.

Autoantibody-mediated arthritis in the absence of C3 and activating Fcγ receptors: C5 is activated by the coagulation cascade

IntroductionThe effector functions of immunoglobulin G (IgG) are mediated by interaction of its Fc region with Fc receptors (FcγRs) and/or the complement system. The three main pathways of complement activation converge at C3. However, C3-independent pathways can activate C5 and other downstream complement components during IgG-initiated inflammatory responses. These C3-independent pathways of C5 activation are triggered by activating FcγRs in some systems or can be activated by factors of the coagulation cascade such as thrombin. Here we studied the interplay of C3, C5, and activating FcγRs in a model of spontaneous autoantibody-driven arthritis.MethodsWe utilized the K/BxN TCR transgenic mouse model of arthritis. We bred K/BxN mice bearing targeted or naturally-occurring mutations in one or more of the genes encoding complement components C3, C5, and FcRγ, the cytoplasmic signaling chain shared by the activating FcγRs. We measured arthritis development, the production of arthritogenic autoantibodies, T cell activation status and cytokine synthesis. In addition, we treated mice with anti-C5 monoclonal antibodies or with the thrombin inhibitor argatroban.ResultsWe have previously shown that genetic deficiency of C5 protects K/BxN mice from the development of arthritis. We found here that C3-deficient K/BxN mice developed arthritis equivalent in severity to C3-sufficient animals. Arthritis also developed normally in K/BxN mice lacking both C3 and FcRγ, but could be ameliorated in these animals by treatment with anti-C5 monoclonal antibody or by treatment with argatroban. Production of arthritogenic autoantibodies, T cell activation, and T cell cytokine production were not affected by the absence of C3, C5, and/or FcRγ.ConclusionsIn K/BxN mice, C5-dependent autoantibody-driven arthritis can occur in the genetic absence of both complement C3 and activating FcγRs. Our findings suggest that in this setting, thrombin activates C5 to provoke arthritis.

Arthritis in KRN T cell receptor transgenic mice does not require interleukin‐17 or Th17 cells

Th17 cells and interleukin‐17 (IL‐17) cytokine family members are implicated in the pathogenesis of many rheumatic diseases. Most studies in mouse models of inflammatory arthritis have demonstrated a key role for the proinflammatory cytokine IL‐17A and its receptor, the IL‐17 receptor (IL‐17R) A/C heterodimer. The aim of this study was to use a rigorous genetic approach to evaluate the contribution of Th17 cells and IL‐17 in the autoantibody‐dependent KRN T cell receptor–transgenic mouse model of arthritis.

Brief Report: Arthritis in KRN T Cell Receptor–Transgenic Mice Does Not Require Interleukin‐17 or Th17 Cells

Th17 cells and interleukin‐17 (IL‐17) cytokine family members are implicated in the pathogenesis of many rheumatic diseases. Most studies in mouse models of inflammatory arthritis have demonstrated a key role for the proinflammatory cytokine IL‐17A and its receptor, the IL‐17 receptor (IL‐17R) A/C heterodimer. The aim of this study was to use a rigorous genetic approach to evaluate the contribution of Th17 cells and IL‐17 in the autoantibody‐dependent KRN T cell receptor–transgenic mouse model of arthritis.

Fcγ Receptor III and Fcγ Receptor IV on Macrophages Drive Autoimmune Valvular Carditis in Mice

Arthritis and valvular carditis coexist in several human rheumatic diseases, including systemic lupus erythematosus, rheumatic fever, and rheumatoid arthritis. T cell receptor–transgenic K/BxN mice develop spontaneous autoantibody‐associated arthritis and valvular carditis. The common Fc receptor γ (FcRγ) signaling chain is required for carditis to develop in K/BxN mice. FcRγ pairs with numerous receptors in a variety of cells. The aim of this study was to identify the FcRγ‐associated receptors and Fcγ receptor (FcγR)–expressing cells that mediate valvular carditis in this model.

The neuropeptide neuromedin U promotes autoantibody- mediated arthritis

IntroductionNeuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary goal of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects.MethodsSerum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU.ResultsNMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux.ConclusionsNMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors.

CD301b/MGL2+ Mononuclear Phagocytes Orchestrate Autoimmune Cardiac Valve Inflammation and Fibrosis

Background: Valvular heart disease is common and affects the mitral valve (MV) most frequently. Despite the prevalence of MV disease (MVD), the cellular and molecular pathways that initiate and perpetuate it are not well understood. Methods: K/B.g7 T-cell receptor transgenic mice spontaneously develop systemic autoantibody-associated autoimmunity, leading to fully penetrant fibroinflammatory MVD and arthritis. We used multiparameter flow cytometry, intracellular cytokine staining, and immunofluorescent staining to characterize the cells in inflamed K/B.g7 MVs. We used genetic approaches to study the contribution of mononuclear phagocytes (MNPs) to MVD in this model. Specifically, we generated K/B.g7 mice in which either CX3CR1 or CD301b/macrophage galactose N-acetylgalactosamine–specific lectin 2 (MGL2)–expressing MNPs were ablated. Using K/B.g7 mice expressing Cx3Cr1-Cre, we conditionally deleted critical inflammatory molecules from MNPs, including the Fc-receptor signal-transducing tyrosine kinase Syk and the cell adhesion molecule very late antigen–4. We performed complementary studies using monoclonal antibodies to block key inflammatory molecules. We generated bone marrow chimeric mice to define the origin of the inflammatory cells present in the MV and to determine which valve cells respond to the proinflammatory cytokine tumor necrosis factor (TNF). Finally, we examined specimens from patients with rheumatic heart disease to correlate our findings to human pathology. Results: MNPs comprised the vast majority of MV-infiltrating cells; these MNPs expressed CX3CR1 and CD301b/MGL2. Analogous cells were present in human rheumatic heart disease valves. K/B.g7 mice lacking CX3CR1 or in which CD301b/MGL2-expressing MNPs were ablated were protected from MVD. The valve-infiltrating CD301b/MGL2+ MNPs expressed tissue-reparative molecules including arginase-1 and resistin-like molecule &agr;. These MNPs also expressed the proinflammatory cytokines TNF and interleukin-6, and antibody blockade of these cytokines prevented MVD. Deleting Syk from CX3CR1-expressing MNPs reduced their TNF and interleukin-6 production and also prevented MVD. TNF acted through TNF receptor–1 expressed on valve-resident cells to increase the expression of vascular cell adhesion molecule–1. Conditionally deleting the vascular cell adhesion molecule-1 ligand very late antigen–4 from CX3CR1-expressing MNPs prevented MVD. Conclusions: CD301b/MGL2+ MNPs are key drivers of autoimmune MVD in K/B.g7 mice and are also present in human rheumatic heart disease. We define key inflammatory molecules that drive MVD in this model, including Syk, TNF, interleukin-6, very late antigen–4, and vascular cell adhesion molecule–1.

Cutting Edge: Dual TCRα Expression Poses an Autoimmune Hazard by Limiting Regulatory T Cell Generation

Despite accounting for 10–30% of the T cell population in mice and humans, the role of dual TCR-expressing T cells in immunity remains poorly understood. It has been hypothesized that dual TCR T cells pose an autoimmune hazard by allowing self-reactive TCRs to escape thymic selection. We revisited this hypothesis using the NOD murine model of type 1 diabetes. We bred NOD mice hemizygous at both TCRα and β (TCRα+/− β+/−) loci, rendering them incapable of producing dual TCR T cells. We found that the lack of dual TCRα expression skewed the insulin-specific thymocyte population toward greater regulatory T (Treg) cell commitment, resulting in a more tolerogenic Treg to conventional T cell ratio and protection from diabetes. These data support a novel hypothesis by which dual TCR expression can promote autoimmunity by limiting agonist selection of self-reactive thymocytes into the Treg cell lineage.

Macrophage scavenger receptor 1 (Msr1, SR-A) Influences b cell autoimmunity by regulating soluble autoantigen concentration

The class A macrophage scavenger receptor Msr1 (SR-A, CD204) has been reported to participate in the maintenance of immunological tolerance. We investigated the role of Msr1 in a mouse model of autoantibody-dependent arthritis. Genetic deficiency of Msr1 in K/BxN TCR transgenic mice decreased the incidence and severity of arthritis because of decreased autoantibody production. Despite normal initial activation of autoreactive CD4+ T cells, potentially autoreactive B cells in Msr1−/− K/BxN mice retained a naive phenotype and did not expand. This was not due to an intrinsic B cell defect. Rather, we found that macrophages lacking Msr1 were inefficient at taking up the key autoantigen glucose-6-phosphate isomerase and that Msr1-deficient mice had elevated serum concentrations of glucose-6-phosphate isomerase. Arthritis developed normally when bone marrow from Msr1−/− K/BxN mice was transplanted into hosts whose macrophages did express Msr1. Thus, Msr1 can regulate the concentration of a soluble autoantigen. In this model, the absence of Msr1 led to higher levels of soluble autoantigen and protected mice from developing pathogenic autoantibodies, likely because of altered cognate interactions of autoreactive T and B cells with impaired differentiation of follicular Th cells.