Bruno Raposo

Monoclonal Anti-CD8 Therapy Induces Disease Amelioration in the K/BxN Mouse Model of Spontaneous Chronic Polyarthritis

OBJECTIVE CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis. METHODS CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy. RESULTS CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L-CD27+ T cells expressing proinflammatory cytokines (interferon-γ [IFNγ], tumor necrosis factor α [TNFα], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 mAb-treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb-treated mice. In anti-CD8 mAb-treated mice, the serologic levels of TNFα, IFNγ, IL-6, and IL-5 normalized. The levels of the disease-related anti-glucose-6-phosphate isomerase antibodies did not change. CONCLUSION These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.

Epitope-specific antibody response is controlled by immunoglobulin VH polymorphisms

Epitope-specific antibody responses recognized by germline-encoded structures are of significant relevance for the development of autoantibody-mediated autoimmune diseases.

Deficient Production of Reactive Oxygen Species Leads to Severe Chronic DSS-Induced Colitis in Ncf1/p47phox-Mutant Mice

Background Colitis is a common clinical complication in chronic granulomatous disease (CGD), a primary immunodeficiency caused by impaired oxidative burst. Existing experimental data from NADPH-oxidase knockout mice propose contradictory roles for the involvement of reactive oxygen species in colitis chronicity and severity. Since genetically controlled mice with a point-mutation in the Ncf1 gene are susceptible to chronic inflammation and autoimmunity, we tested whether they presented increased predisposition to develop chronic colitis. Methods Colitis was induced in Ncf1-mutant and wild-type mice by a 1st 7-days cycle of dextran sulfate sodium (DSS), intercalated by a 7-days resting period followed by a 2nd 7-days DSS-cycle. Cytokines were quantified locally in the colon inflammatory infiltrates and in the serum. Leukocyte infiltration and morphological alterations of the colon mucosa were assessed by immunohistochemistry. Results Clinical scores demonstrated a more severe colitis in Ncf1-mutant mice than controls, with no recovery during the resting period and a severe chronic colitis after the 2nd cycle, confirmed by histopathology and presence of infiltrating neutrophils, macrophages, plasmocytes and lymphocytes in the colon. Severe colitis was mediated by increased local expression of cytokines (IL-6, IL-10, TNF-α, IFN-γ and IL-17A) and phosphorylation of Leucine-rich repeat kinase 2 (LRRK2). Serological cytokine titers of those inflammatory cytokines were more elevated in Ncf1-mutant than control mice, and were accompanied by systemic changes in functional subsets of monocytes, CD4+T and B cells. Conclusion This suggests that an ineffective oxidative burst leads to severe chronic colitis through local accumulation of peroxynitrites, pro-inflammatory cytokines and lymphocytes and systemic immune deregulation similar to CGD.

Genetic control of antibody production during collagen‐induced arthritis development in heterogeneous stock mice

OBJECTIVE To identify genetic factors driving pathogenic autoantibody formation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), in order to better understand the etiology of RA and identify possible new avenues for therapeutic intervention. METHODS We performed a genome-wide analysis of quantitative trait loci controlling autoantibody to type II collagen (anti-CII), anti-citrullinated protein antibody (ACPA), and rheumatoid factor (RF). To identify loci controlling autoantibody production, we induced CIA in a heterogeneous stock-derived mouse cohort, with contribution of 8 inbred mouse strains backcrossed to C57BL/10.Q. Serum samples were collected from 1,640 mice before arthritis onset and at the peak of the disease. Antibody concentrations were measured by standard enzyme-linked immunosorbent assay, and linkage analysis was performed using a linear regression-based method. RESULTS We identified loci controlling formation of anti-CII of different IgG isotypes (IgG1, IgG3), antibodies to major CII epitopes (C1, J1, U1), antibodies to a citrullinated CII peptide (citC1), and RF. The anti-CII, ACPA, and RF responses were all found to be controlled by distinct genes, one of the most important loci being the immunoglobulin heavy chain locus. CONCLUSION This comprehensive genetic analysis of autoantibody formation in CIA demonstrates an association not only of anti-CII, but interestingly also of ACPA and RF, with arthritis development in mice. These results underscore the importance of non-major histocompatibility complex genes in controlling the formation of clinically relevant autoantibodies.

Heterogeneous stock mice are susceptible to encephalomyelitis and antibody-initiated arthritis but not to collagen- and G6PI-induced arthritis.

The strategy of using heterogeneous stock (HS) mice has proven to be successful in fine mapping of quantitative trait loci in complex diseases. However, whether these mice can be used for arthritis, encephalomyelitis and autoimmune phenotypes has not been addressed. Here, we screened the Northport HS mice for arthritis phenotypes using three different models: collagen‐induced arthritis (CIA), using rat, bovine or chicken collagen type II (CII); recombinant human glucose‐6‐phosphate isomerase (G6PI)‐induced arthritis; and collagen antibody–induced arthritis (CAIA). Irrespective of the origin of collagen, we found HS mice to be fairly resistant to CIA and G6PI‐induced arthritis, despite the development of antibodies against the respective antigens. On the other hand, HS mice were found to be susceptible for CAIA. Similarly, these mice developed encephalomyelitis (EAE) induced either with mouse or rat spinal cord homogenate (SCH), or with recombinant rat myelin oligodendrocyte glycoprotein, with elevated antibody levels against CNS proteins. Accordingly, we conclude that the use of HS mice for fine mapping and positional cloning of gene(s) involved in CAIA and EAE is possible, but not for collagen‐ and G6PI‐induced arthritis.

System A amino acid transporters regulate glutamine uptake and attenuate antibody-mediated arthritis

Proliferation of rapidly dividing bone marrow‐derived cells is strongly dependent on the availability of free glutamine, whose uptake is mediated through different amino acid transporters. The sodium‐coupled neutral amino acid transporter (SNAT) family was previously reported to be associated with the development of collagen‐induced arthritis in mice. Here, we tested the hypothesis whether impairment of SNAT proteins influences immune cell function and in turn alters arthritis development. The 2‐(methylamino)isobutyric acid (MeAIB), a SNAT‐specific substrate, was used to modulate the function of SNAT proteins. We demonstrate that glutamine uptake by murine naive lymphocytes, and consequent cell proliferation, is strongly associated with system A transporters. Physiological impairment of SNAT proteins reduced the antibody‐initiated effector phase of arthritis, mainly by affecting the levels of circulating monocytes and neutrophils. MeAIB was also shown to affect the proliferation of immortalized cells, through trans‐inhibition of SNAT proteins. Based on our observations, we conclude that SNAT proteins regulate the initial stages of lymphocyte activation by regulating glutamine uptake, and that the effector phase of arthritis can be affected by non‐metabolized SNAT substrates. Most probably, metabolically active cells within both the adaptive and the innate immune systems are regulated by SNAT proteins and play a role in modifying arthritis development.

Disease amelioration in the K/BxN mouse model of spontaneous chronic arthritis after CD8 T cell depletion

CD8 T cells are part of the T cell pool infiltrating the rheumatoid synovial membrane. Moreover, CD8 T cells have been linked to the formation of synovial ectopic germinal centres in rheumatoid arthritis (RA), and they produce proinflammatory cytokines. Hence, CD8 T cells appear to be intimately involved in initiating and maintaining the chronic inflammation in the RA synovium. Nevertheless, studies on animal models of …

Increased salt exposure affects both lymphoid and myeloid effector functions, influencing innate-associated disease but not T-cell-associated autoimmunity

High salt consumption has since long been associated with elevated blood pressure and cardiovascular disease. Recently, mouse studies suggested that a high dietary salt intake exacerbates the clinical manifestations of autoimmunity. Using naïve cells ex vivo after pre‐exposure of mice to high salt intake, we showed that increased salt exposure affects the viability and effector functions of immune cells. CD4+ T‐cells evidenced a pro‐inflammatory phenotype characterized by increased secretion of IFNγ and IL‐17A, when exposed to high salt concentrations in vitro. Interestingly, this phenotype was associated with osmotic pressure, as replacing salt for d‐mannitol resulted in similar observations. However, high salt intake did not alter the development of T‐cell‐dependent autoimmunity. Instead, recruitment of peritoneal macrophages was increased in mice pre‐exposed to high salt concentrations. These cells had an increased production of both TNFα and IL‐10, suggesting that salt stimulates expansion and differentiation of different subsets of macrophages. Moreover, mice pre‐exposed to high salt intake developed exacerbated symptoms of colitis, when induced by dextran sulphate sodium. The aggravated colitis in salt‐exposed animals was associated with a higher frequency of CD4+ T‐cells and CD11b+ CD64+ macrophages producing TNFα. These phenotypes correlated with elevated titres of faecal IgA and higher lymphocytic cellularity in the colon, mesenteric lymph nodes and spleen. In conclusion, we report here that high salt intake affects both lymphoid and myeloid cells ex vivo. However, the effects of high salt intake in vivo seem less pronounced in terms of CD4+ T‐cell responses, whereas macrophage‐dependent pathologies are significantly influenced.

Genetic influence of specific anticollagen antibody production in a mouse model of rheumatoid arthritis

Backgroundand objectives Rheumatoid arthritis (RA) is an autoimmune disease affecting about 1% of the population. Though of unknown aetiology, it is considered to have a strong genetic influence, resulting in the activation of different immune cells and consequent bone and cartilage destruction of the joints. The production of anticollagen type II (CII) antibodies is a common feature between RA patients and the collagen-induced arthritis (CIA) mouse model. Understanding how distinct anti-CII epitopes are genetically selected could benefit the development of new RA therapies. Here, the authors assess the genetic influence of specific anti-CII antibody production in a mouse model of RA. Material and methods In this particular study, the authors have used as genetic variance tool a heterogeneous stock (HS) mice cohort of eight inbred mouse strains. Three of the major anti-CII B cell epitopes (C1, U1 and J1) were analysed by ELISA. Sequencing of the genes coding for the M2139 monoclonal antibody (J1-specific mAb) was additionally done for all the HS strains. Results A genome wide study of antibody response during CIA development suggested that specific anti-CII antibodies are regulated by the immunoglobulin heavy chain locus (IgH). The haplotype reconstruction analysis showed a preference for the anti-J1 antibody production by certain mouse strains, in detriment of anti-C1 antibodies. The authors thus hypothesised that the selection of particular IgHv genes is responsible for this J1 versus C1 epitope selection. Preliminary sequencing data of the genes coding for the M2139 monoclonal antibody in all the HS strains, supports the initial hypothesis. The difference relies on an amino acid change from a hydrophobic tryptophan to a polar amino acid (eg, threonine) in the CDR1 region. Three-dimensional structure of M2139 Fab with triple helical J1 peptide shows that the tryptophan in the CDR1 region of the heavy chain is contacting with a hydrophobic leucine amino acid in the J1 epitope. This interaction may be essential for recognition of the J1 epitope. Replacement of Trp by Thr in this position could abrogate the binding to J1 epitope. Conclusions CIA development in HS mice allowed for a genome wide study on anti-CII specific responses. The data revealed a genetic association with the production of anti-J1 antibodies. Sequencing of the genes coding for the anti-J1 mAb reveal an amino acid substitution influencing the polarity within the CDR1 region. Cloning and expression of these mutations and consequent anti-J1 binding properties will further on confirm or reject our hypothesis.