From genes to function in autoimmunity - a salty story

Autoimmunity is a condition in which the ability to tolerate self breaks down, resulting in immune responses against the body’s own healthy cells and tissues. Autoimmune diseases are complex and multifactorial, and both genetic and environmental factors are known to play a crucial role. Using animal models, it is possible to study different aspects of arthritis disease development in an environmentally and genetically controlled setting. In this thesis, I have investigated the effect of genetic risk loci and the single environmental factor sodium chloride (NaCl) on immune cell function and the development of autoimmune diseases using mouse models. In Paper I, using four congenic sub-loci within the arthritis susceptible Cia9 locus on chromosome 1, we found that the NOD.Q polymorphic Fc gamma receptor gene (FcγR) cluster located within sub-loci Cia9i and Cia9k, regulated arthritis. Polymorphic FcγR2b and FcγR4 were contained in both Cia9i and Cia9k, whereas Cia9i mice also carried polymorphic FcγR3. FcγR2b gene and protein expression were downregulated in Cia9i and Cia9k mice, whereas FcγR3 was upregulated in Cia9i mice and found downregulated in Cia9k mice compared to littermate control mice. This difference in FcγR3 expression affected killing by NK cells and phagocytosis by macrophages in vitro and PC61 antibody induced regulatory T cell depletion in vivo. Interestingly, arthritis development was regulated by interaction between FcγR2b and FcγR3 without affecting anti-collagen type II antibody secretion. These results show that polymorphisms in both FcγR2b and FcγR3 regulate the severity of inflammatory responses. In paper II, we investigated the importance of the system A family of amino acid transporters (SNAT), based on an identified congenic locus, as mediators of immune cell function and arthritis development. We demonstrated that SNAT proteins affect the initial stages of lymphocyte activation by regulating glutamine uptake in the presence of Na+, and that the effector phase of arthritis could be suppressed by blocking SNAT proteins. Paper III describes the effect of salt (e.g. NaCl) on the development of autoimmunity. Here we show that a moderate salt intake affects both T cell and macrophage phenotypes in vitro and ex vivo. However, these moderate levels of salt intake did not alter the development of T cell-dependent autoimmunity, whereas the dextran sulphate sodium (DSS)-induced colitis was exacerbated in mice pre-exposed to salt. Taken together, I have shown that the interplay between two genes enhances arthritis disease development, whereas a single environmental factor has no impact on arthritis despite triggering the immune system. These results contribute to the understanding of the mechanism behind complex multifactorial diseases as a small building block towards therapeutic intervention

The oncoprotein TBX3 is controlling severity in experimental arthritis

Abstract Background Development of autoimmune diseases is the result of a complex interplay between hereditary and environmental factors, with multiple genes contributing to the pathogenesis in human disease and in experimental models for disease. The T-box protein 3 is a transcriptional repressor essential during early embryonic development, in the formation of bone and additional organ systems, and in tumorigenesis. Methods With the aim to find novel genes important for autoimmune inflammation, we have performed genetic studies of collagen-induced arthritis (CIA), a mouse experimental model for rheumatoid arthritis. Results We showed that a small genetic fragment on mouse chromosome 5, including Tbx3 and three additional protein-coding genes, is linked to severe arthritis and high titers of anti-collagen antibodies. Gene expression studies have revealed differential expression of Tbx3 in B cells, where low expression was accompanied by a higher B cell response upon B cell receptor stimulation in vitro. Furthermore, we showed that serum TBX3 levels rise concomitantly with increasing severity of CIA. Conclusions From these results, we suggest that TBX3 is a novel factor important for the regulation of gene transcription in the immune system and that genetic polymorphisms, resulting in lower expression of Tbx3, are contributing to a more severe form of CIA and high titers of autoantibodies. We also propose TBX3 as a putative diagnostic biomarker for rheumatoid arthritis

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

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 naïve lymphocytes, and consequent cell proliferation is strongly associated with system A transporters. Physiological impairment of SNAT proteins reduced 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, via 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 likely, metabolically active cells both within the adaptive and the innate immune system are regulated by SNAT proteins and play a role in modifying arthritis development. This article is protected by copyright. All rights reserved

Genetic dissection of a major haplotype associated with arthritis reveal FcγR2b and FcγR3 to act additively

A haplotype with tightly linked Fc gamma receptor (FcγR) genes is known as a major locus controlling immune responses and autoimmune diseases, including arthritis. Here, we split a congenic fragment derived from the NOD mouse (Cia9) to study its effect on immune response and arthritis in mice. We found that arthritis susceptibility was indeed controlled by the FcγR gene cluster and a recombination between the FcγR2b and FcγR3 loci gave us the opportunity to separately study their impact. We identified the NOD-derived FcγR2b and FcγR3 alleles as disease-promoting for arthritis development without impact on antibody secretion. We further found that macrophage-mediated phagocytosis was directly correlated to FcγR3 expression in the congenic mice. In conclusion, we positioned FcγR2b and FcγR3 alleles as disease regulatory and showed that their genetic polymorphisms independently and additively control innate immune cell activation and arthritis

Genetic dissection of a major haplotype associated with autoimmune disease, FcγR2b and FcγR3 act additively

A haplotype with tightly linked Fc gamma receptor (FcγR) genes is known as a major locus controlling immune responses and autoimmune diseases, including arthritis. Here, we split a congenic fragment derived from the NOD mouse (Cia9) to study its effect on immune response and arthritis in mice. We found that arthritis susceptibility was indeed controlled by the FcγR gene cluster and a recombination between the FcγR2b and FcγR3 loci gave us the opportunity to separately study their impact. We identified the NOD‐derived FcγR2b and FcγR3 alleles as disease‐promoting for arthritis development without impact on antibody secretion. We further found that macrophage‐mediated phagocytosis was directly correlated to FcγR3 expression in the congenic mice. In conclusion, we positioned FcγR2b and FcγR3 alleles as disease regulatory and showed that their genetic polymorphisms independently and additively control innate immune cell activation and arthritis