ABSTRACT
BACKGROUND: Association of HLA-B27 with spondyloarthritis (SpA) has been known for 50 years, but still remains unexplained. We recently showed that HLA-B27 expressed in wing imaginal disc from HLA-B27/human-ß2 microglobulin (hß2m) transgenic Drosophila deregulated bone morphogenetic protein (BMP) pathway by interacting physically with type I BMP receptor (BMPR1) Saxophone (Sax), leading to crossveinless phenotype. METHODS: Genetic interaction was studied between activin/transforming growth factor ß (TGFß) pathway and HLA-B27/hß2m in transgenic Drosophila wings. The HLA-B27-bound peptidome was characterized in wing imaginal discs. In mesenteric lymph node (mLN) T cells from HLA-B27/hß2m rat (B27 rat), physical interaction between HLA-B27 and activin receptor-like kinase-2 (ALK2), ALK3 and ALK5 BMPR1s, phosphorylation of small mothers against decapentaplegic (SMADs) and proteins of the non-canonical BMP/TGFß pathways induced by its ligands, and the transcript level of target genes of the TGFß pathway, were evaluated. RESULTS: In HLA-B27/hß2m transgenic Drosophila, inappropriate signalling through the activin/TGFß pathway, involving Baboon (Babo), the type I activin/TGFß receptor, contributed to the crossveinless phenotype, in addition to deregulated BMP pathway. We identified peptides bound to HLA-B27 with the canonical binding motif in HLA-B27/hß2m transgenic Drosophila wing imaginal disc. We demonstrated specific physical interaction, between HLA-B27/hß2m and mammalian orthologs of Sax and Babo, i.e. ALK2 and ALK5 (i.e. TGFß receptor I), in the mLN cells from B27 rat. The magnitude of phosphorylation of SMAD2/3 in response to TGFß1 was increased in T cells from B27 rats, showing evidence for deregulated TGFß pathway. Accordingly, expression of several target genes of the pathway was increased in T cells from B27 rats, in basal conditions and/or after TGFß exposure, including Foxp3, Rorc, Runx1 and Maf. Interestingly, Tgfb1 expression was reduced in naive T cells from B27 rats, even premorbid, an observation consistent with a pro-inflammatory pattern. CONCLUSIONS: This study shows that HLA-B27 alters the TGFß pathways in Drosophila and B27 rat. Given the importance of this pathway in CD4 + T cells differentiation and regulation, its disturbance could contribute to the abnormal expansion of pro-inflammatory T helper 17 cells and altered regulatory T cell phenotype observed in B27 rats.
Subject(s)
Animals, Genetically Modified , HLA-B27 Antigen , Signal Transduction , Spondylarthritis , Transforming Growth Factor beta , Animals , Signal Transduction/physiology , Spondylarthritis/metabolism , Spondylarthritis/immunology , Humans , HLA-B27 Antigen/genetics , HLA-B27 Antigen/metabolism , HLA-B27 Antigen/immunology , Transforming Growth Factor beta/metabolism , Rats , Drosophila , Drosophila melanogaster , Wings, Animal/metabolismABSTRACT
Introduction: In spondyloarthritis (SpA), an increased type 3 immune response, including T helper cells (Th) 17 excess, is observed in both human and SpA animal models, such as the HLA-B27/human ß2-microglobulin transgenic rat (B27-rat). Methods: To investigate this unexplained Th17-biased differentiation, we focused on understanding the immunobiology of B27-rat naive CD4+ T cells (Tn). Results: We observed that neutrally stimulated B27-rat Tn developed heightened Th17 profile even before disease onset, suggesting an intrinsic proinflammatory predisposition. In parallel with this observation, transcriptomic and epigenomic analyses showed that B27-rat Tn exhibited a decreased expression of Interferon/Th1- and increased expression of Th17-related genes. This molecular signature was predicted to be related to an imbalance of STAT1/STAT3 transcription factors activity. Stat1 mRNA and STAT1 protein expression were decreased before disease onset in Tn, even in their thymic precursors, whereas Stat3/STAT3 expression increased upon disease establishment. Confirming the relevance of these results, STAT1 mRNA expression was also decreased in Tn from SpA patients, as compared with healthy controls and rheumatoid arthritis patients. Finally, stimulation of B27-rat Tn with a selective STAT1 activator abolished this preferential IL-17A expression, suggesting that STAT1-altered activity in B27-rats allows Th17 differentiation. Discussion: Altogether, B27-rat Tn harbor a STAT1 deficiency preceding disease onset, which may occur during their thymic differentiation, secondarily associated with a persistent Th17 bias, which is imprinted at the epigenomic level. This early molecular phenomenon might lead to the persistent proinflammatory skew of CD4+ T cells in SpA patients, thus offering new clues to better understand and treat SpA.
Subject(s)
Arthritis, Rheumatoid , Spondylarthritis , Animals , Humans , Rats , Arthritis, Rheumatoid/metabolism , CD4-Positive T-Lymphocytes , Rats, Transgenic , RNA, Messenger/metabolism , STAT1 Transcription Factor/metabolismABSTRACT
Introduction: Spondylarthritis (SpA) development in HLA-B27/human ß2-microglobulin transgenic rat (B27-rat) is correlated with altered conventional dendritic cell (cDC) function that promotes an inflammatory pattern of CD4+T cells, including a biased expansion of pro-inflammatory Th17 population and imbalance of regulatory T cells cytokine profile. Transcriptomic analysis revealed that cDCs from B27-rats under express IL-27, an anti-inflammatory cytokine which induces the differentiation of IL-10+ regulatory T cells and inhibits Th17 cells. Methods: Here, we first investigated whether in vitro addition of exogenous IL-27 could reverse the inflammatory pattern observed in CD4+ T cells. Next, we performed preclinical assay using IL-27 to investigate whether in vivo treatment could prevent SpA development in B27-rats. Results: in vitro addition of IL-27 to cocultures of cDCs and CD4+ T cell subsets from B27-rats reduced IL-17 and enhanced IL-10 production by T cells. Likewise, IL-27 inhibited the production of IL-17 by CD4+ T cells from SpA patients. Interestingly, in vivo treatment with recombinant IL-27 starting before SpA onset, inhibited SpA development in B27-rats through the suppression of IL-17/TNF producing CD4+ T cells. Discussion: Overall, our results reveal a potent inhibitory effect of IL-27 and highlight this cytokine as a promising new therapeutic target in SpA, especially for SpA patients non responders to currently approved biotherapies.
Subject(s)
Interleukin-27 , Spondylarthritis , Animals , Humans , Rats , Cytokines , Interleukin-10 , Interleukin-17 , Rats, Transgenic , Th17 CellsABSTRACT
INTRODUCTION: Axial spondyloarthritis (AxSpA) is an inflammatory disorder that affects the joints, entheses, and bone tissues and is sometimes associated with psoriasis, anterior uveitis, and gut inflammation. Its pathogenesis is not wholly understood and treatment strategies require optimization. Data concerning AxSpA pathogenesis support a critical role of abnormal CD4+ T cell differentiation and exacerbated type 3 immune response. This knowledge boosted the development of interleukin (IL)-17 and Janus kinase inhibitors for AxSpA treatment beyond tumor necrosis factor-α inhibition. AREAS COVERED: Emerging drug targets in animal and cellular models and with phase-II clinical trials have been evaluated. We also reflect on key issues for preclinical and clinical research going forward. EXPERT OPINION: Some of the most promising approaches include: (i) modulation of transforming growth factor-ß family that could exert a specific role on bone formation; (ii) blockade of granulocyte-macrophage colony-stimulating factor that could reduce type 3 immune responses, and (iii) rebalancing of biased immune response by cytokines such as IL-2 or IL-27 that could favor anti-inflammatory response and sustained drug-free remission. Multiomics tools and artificial intelligence could contribute to identification of optimal targets and help stratify patients for the most appropriate treatment options.
Subject(s)
Axial Spondyloarthritis , Pharmaceutical Preparations , Spondylarthritis , Animals , Artificial Intelligence , Humans , Spondylarthritis/drug therapy , Spondylarthritis/metabolism , Tumor Necrosis Factor-alphaABSTRACT
Bone marrow adipose tissue (BMAT) has recently been recognized as a distinct fat depot with endocrine functions. However, if and how it is regulated by chronic inflammation remains unknown. Here, we investigate the amount of white fat and BMAT in HLA-B27 transgenic rats and curdlan-challenged SKG mice, two well-established models of chronic inflammatory spondyloarthritis (SpA). Subcutaneous and gonadal white adipose tissue and BMAT was reduced by 65-70% and by up to 90% in both experimental models. Consistently, B27 rats had a 2-3-fold decrease in the serum concentrations of the adipocyte-derived cytokines adiponectin and leptin as well as a 2-fold lower concentration of triglycerides. The bone marrow of B27 rats was further characterized by higher numbers of neutrophils, lower numbers of erythroblast precursors, and higher numbers of IL-17 producing CD4+ T cells. IL-17 concentration was also increased in the serum of B27 rats. Using a cell culture model, we show that high levels of IL-17 in the serum of B27 rats negatively impacted adipogenesis (-76%), an effect that was reversed in the presence of neutralizing anti-IL-17 antibody. In summary, these findings show BMAT is severely reduced in two experimental models of chronic inflammatory SpA and suggest that IL-17 is involved in this process.
Subject(s)
Adipose Tissue/pathology , Bone Marrow/pathology , HLA-B27 Antigen/genetics , Interleukin-17/blood , Spondylarthritis/pathology , Animals , CD4-Positive T-Lymphocytes/metabolism , Disease Models, Animal , Female , Mice , Mice, Inbred BALB C , Mice, Transgenic , Rats , Rats, Inbred F344 , Rats, Transgenic , Spondylarthritis/genetics , Spondylarthritis/immunologyABSTRACT
Understanding the complex mechanisms underlying a disorder such as spondyloarthritis (SpA) may benefit from studying animal models. Several suitable models have been developed, in particular to investigate the role of genetic factors predisposing to SpA, including HLA-B27, ERAP1, and genes related to the interleukin (IL)-23/IL-17 axis. One of the best examples of such research is the HLA-B27 transgenic rat model that fostered the emergence of original theories regarding HLA-B27 pathogenicity, including dysregulation of innate immunity, contribution of the adaptive immune system to chronic inflammation, and influence of the microbiota on disease development. Very recently, a new model of HLA-B27 transgenic Drosophila helped to expand further some of those theories in an unexpected direction involving the TGFß/BMP family of mediators. On the other hand, several spontaneous, inducible, and/or genetically modified mouse models-including SKG mouse, TNFΔARE mouse and IL-23-inducible mouse model of SpA-have highlighted the importance of TNFα and IL-23/IL-17 axis in the development of SpA manifestations. Altogether, those animal models afford not only to study disease mechanism but also to investigate putative therapeutic targets.
Subject(s)
Spondylarthritis , Aminopeptidases , Animals , Disease Models, Animal , HLA-B27 Antigen/genetics , Humans , Mice , Minor Histocompatibility Antigens , Rats , Rats, TransgenicABSTRACT
OBJECTIVES: The human leucocyte antigen (HLA)-B27 confers an increased risk of spondyloarthritis (SpA) by unknown mechanism. The objective of this work was to uncover HLA-B27 non-canonical properties that could explain its pathogenicity, using a new Drosophila model. METHODS: We produced transgenic Drosophila expressing the SpA-associated HLA-B*27:04 or HLA-B*27:05 subtypes, or the non-associated HLA-B*07:02 allele, alone or in combination with human ß2-microglobulin (hß2m), under tissue-specific drivers. Consequences of transgenes expression in Drosophila were examined and affected pathways were investigated by the genetic interaction experiments. Predictions of the model were further tested in immune cells from patients with SpA. RESULTS: Loss of crossveins in the wings and a reduced eye phenotype were observed after expression of HLA-B*27:04 or HLA-B*27:05 in Drosophila but not in fruit flies expressing the non-associated HLA-B*07:02 allele. These HLA-B27-induced phenotypes required the presence of hß2m that allowed expression of well-folded HLA-B conformers at the cell surface. Loss of crossveins resulted from a dominant negative effect of HLA-B27 on the type I bone morphogenetic protein (BMP) receptor saxophone (Sax) with which it interacted, resulting in elevated mothers against decapentaplegic (Mad, a Drosophila receptor-mediated Smad) phosphorylation. Likewise, in immune cells from patients with SpA, HLA-B27 specifically interacted with activin receptor-like kinase-2 (ALK2), the mammalian Sax ortholog, at the cell surface and elevated Smad phosphorylation was observed in response to activin A and transforming growth factor ß (TGFß). CONCLUSIONS: Antagonistic interaction of HLA-B27 with ALK2, which exerts inhibitory functions on the TGFß/BMP signalling pathway at the cross-road between inflammation and ossification, could adequately explain SpA development.
Subject(s)
Gene Expression Regulation , HLA-B27 Antigen/genetics , RNA/genetics , Spondylarthritis/genetics , Transforming Growth Factor beta/genetics , Activin Receptors, Type I/biosynthesis , Activin Receptors, Type I/genetics , Animals , Animals, Genetically Modified , Blotting, Western , Cells, Cultured , Disease Models, Animal , Drosophila melanogaster , HLA-B27 Antigen/biosynthesis , Humans , Signal Transduction , Spondylarthritis/metabolism , Spondylarthritis/pathology , Transforming Growth Factor beta/metabolismABSTRACT
BACKGROUND: Spondyloarthritis (SpA) is a chronic inflammatory disease affecting primarily axial and peripheral joints and sometimes also extra-articular organs, such as the gut. Rats transgenic for HLA-B27 and human ß2-microglobulin (B27-Tg rat) develop clinical manifestations resembling human disease. In this model, it has been shown that CD103+ conventional dendritic cells (cDCs) exhibited altered functions, likely promoting SpA development. CD4- cDC subpopulation expressing XCR1, a chemokine receptor involved in their migration, have been described to be tolerogenic in steady state. Thus, in this study, we wished to examine the fate of XCR1+ cDCs in this animal model of SpA. METHODS: cDC populations were isolated from the spleen, mesenteric lymph nodes (MLN), and colonic lamina propria from B27-TG and control nontransgenic (NTG) and/or HLA-B7 transgenic rats after collagenase digestion and density gradient and characterized with flow cytometry or real-time PCR. Migration of cDCs from intestinal mucosa to MLN was assessed, using TLR-7 stimulation with Resiquimod. RESULTS: We observed a reduced frequency of cCD4- DCs in B27-Tg rats, as compared to control rats. Furthermore, such decrease was not due to excessive death of CD4- cDCs in B27-Tg rats. Interestingly, we observed a decrease frequency of the XCR1+ subpopulation among CD4- cDCs in the spleen, MLN, and lamina propria from B27-Tg rats. Finally, after TLR-7 stimulation, the migration of XCR1+ cDCs to MLN was proportionally reduced in B27-Tg rats. CONCLUSION: Our results demonstrate for the first time a decreased proportion of the tolerogenic XCR1+ cDC subpopulation in SpA target organs in B27-Tg rat, which may affect the maintenance of self-tolerance and control of inflammation.
Subject(s)
Dendritic Cells/immunology , HLA-B27 Antigen/immunology , Receptors, Chemokine/immunology , Spondylarthritis/immunology , beta 2-Microglobulin/immunology , Animals , Antigens, CD/immunology , Cell Movement/genetics , Cell Movement/immunology , Cells, Cultured , Dendritic Cells/metabolism , Disease Models, Animal , HLA-B27 Antigen/genetics , HLA-B27 Antigen/metabolism , Humans , Immune Tolerance/genetics , Immune Tolerance/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Integrin alpha Chains/immunology , Rats, Inbred F344 , Rats, Transgenic , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Spondylarthritis/genetics , Spondylarthritis/metabolism , beta 2-Microglobulin/genetics , beta 2-Microglobulin/metabolismABSTRACT
Mice transgenic for the RET oncogene provide a remarkable model for investigating the mechanisms underlying the promotion and the development of melanoma. This model was established on the C57BL/6 genetic background. In the present study, we investigated an effect of the strongly proinflammatory and autoimmune genetic makeup of the non-obese diabetic (NOD) strain. We bred (NODxB6)F1 mice and backcrossed them with NOD mice. F1 mice and mice at subsequent generations of backcrossing showed marked acceleration of tumor development, in particular with a more frequent and earlier extension of the primary uveal melanoma. In close relation with this severe evolution, we observed a profound drop in Dectin-1 expression on CD11b(+)Ly6G(+) granulocytic myeloid cells correlating with an expansion of CD4(+)Foxp3(+) T regulatory cell and of interferon(IFN)γ-producing CD8(+) T cell subsets in tumors. IFNγ is a major inducer of the type 2 nitric-oxide synthase (Nos2) gene whose products are known to be tumorigenic. Germline inactivation of the Nos2 gene was associated with a dramatically improved tumor prognosis and a restoration of Dectin-1 expression on myeloid cells. Moreover, in vivo treatment of (NODxB6)F1.RET(+) mice with curdlan, a glucose polymer that binds Dectin-1, prevented tumor extension and was associated with marked reduction of the CD4(+)Foxp3(+) T cell subset. These observations highlight the (NODxB6)F1.RET(+) mice as a new model to investigate the role of the immune system in the host-tumor relationship and point to Dectin-1 and Nos2 as potentially promising therapeutic targets.
ABSTRACT
OBJECTIVE: Although osteopenia is frequent in spondyloarthritis (SpA), the underlying cellular mechanisms and association with other symptoms are poorly understood. This study aimed to characterize bone loss during disease progression, determine cellular alterations, and assess the contribution of inflammatory bowel disease (IBD) to bone loss in HLA-B27 transgenic rats. METHODS: Bones of 2-, 6-, and 12-month-old non-transgenic, disease-free HLA-B7 and disease-associated HLA-B27 transgenic rats were examined using peripheral quantitative computed tomography, µCT, and nanoindentation. Cellular characteristics were determined by histomorphometry and ex vivo cultures. The impact of IBD was determined using [21-3 x 283-2]F1 rats, which develop arthritis and spondylitis, but not IBD. RESULTS: HLA-B27 transgenic rats continuously lost bone mass with increasing age and had impaired bone material properties, leading to a 3-fold decrease in bone strength at 12 months of age. Bone turnover was increased in HLA-B27 transgenic rats, as evidenced by a 3-fold increase in bone formation and a 6-fold increase in bone resorption parameters. Enhanced osteoclastic markers were associated with a larger number of precursors in the bone marrow and a stronger osteoclastogenic response to RANKL or TNFα. Further, IBD-free [21-3 x 283-2]F1 rats also displayed decreased total and trabecular bone density. CONCLUSIONS: HLA-B27 transgenic rats lose an increasing amount of bone density and strength with progressing age, which is primarily mediated via increased bone remodeling in favor of bone resorption. Moreover, IBD and bone loss seem to be independent features of SpA in HLA-B27 transgenic rats.
Subject(s)
Bone Remodeling/physiology , Bone and Bones/pathology , Inflammatory Bowel Diseases/pathology , Osteoclasts/cytology , Spondylarthropathies/pathology , Animals , Cell Differentiation/physiology , Disease Models, Animal , HLA-B27 Antigen/genetics , Inflammatory Bowel Diseases/complications , Inflammatory Bowel Diseases/genetics , Male , Rats , Rats, Inbred F344 , Rats, Transgenic , Spondylarthropathies/complications , Spondylarthropathies/genetics , Tomography, X-Ray ComputedABSTRACT
OBJECTIVE: HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic (B27-transgenic) rats develop an inflammatory disorder resembling spondyloarthritis, with accumulation of proinflammatory Th17 cells. Because Treg cells and Th17 cells have opposing effects in inflammatory disorders, we sought to determine whether biased expansion of Th17 cells could result from altered Treg cell frequency and/or function in B27-transgenic rats. METHODS: We characterized the phenotype and function of Treg cells from B27-transgenic rats in comparison with those from control rats, by examining their expression of cell surface markers, suppressive activity, cytokine production, and differentiation pattern. RESULTS: In B27-transgenic rats, the preferential accumulation of CD4+ Teff cells over Treg cells was not associated with a defect in Treg cell differentiation or suppressive activity. The expression of Treg cell markers was similar between B27-transgenic and control rats, with the exception of the inducible costimulator (ICOS) molecule, which was overexpressed in B27-transgenic rats. High levels of ICOS are considered to be a hallmark of Treg cells with heightened suppressive activity and interleukin-10 (IL-10) expression. Paradoxically, the production of IL-10 by Treg cells was reduced in B27-transgenic rats, whereas the production of IL-17 was enhanced. Moreover, the addition of anti-ICOS monoclonal antibodies during Treg cell differentiation in the presence of dendritic cells from B27-transgenic rats reversed this cytokine profile, restoring the balance between IL-10 and IL-17 in Treg cells from B27-transgenic rats. CONCLUSION: We observed dysregulated production of IL-10 and IL-17 by Treg cells from B27-transgenic rats, which may contribute to disease development. Moreover, our data highlight a key role for ICOS signaling in the generation of imbalanced production of IL-10 and IL-17 by Treg cells in this experimental model of spondyloarthritis.
Subject(s)
Inducible T-Cell Co-Stimulator Protein/metabolism , Interleukin-10/biosynthesis , Interleukin-17/biosynthesis , Spondylarthritis/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Male , Rats , Rats, Transgenic , Spondylarthritis/immunology , T-Lymphocytes, Regulatory/immunologyABSTRACT
OBJECTIVE: In HLA-B27-transgenic rats, the development of a disorder that mimics spondyloarthritis (SpA) is highly correlated with dendritic cell (DC) dysfunction. The present study was undertaken to analyze the underlying mechanisms of this via transcriptome analysis. METHODS: Transcriptome analysis of ex vivo-purified splenic CD103+CD4+ DCs from B27-transgenic rats and control rats was performed. Transcriptional changes in selected genes were confirmed by quantitative reverse transcriptase-polymerase chain reaction. A meta-analysis of our rat data and published data on gene expression in macrophages from ankylosing spondylitis (AS) patients was further performed. RESULTS: Interferon (IFN) signaling was the most significantly affected pathway in DCs from B27-transgenic rats; the majority of genes connected to IFN were underexpressed in B27-transgenic rats as compared to controls. This pattern was already present at disease onset, persisted over time, and was conserved in 2 disease-prone B27-transgenic rat lines. In DCs from B27-transgenic rats, we further found an up-regulation of suppressor of cytokine signaling 3 (which may account for reverse IFN signaling) and a down-regulation of interleukin-27 (a cytokine that opposes Th17 differentiation and promotes Treg cells). The meta-analysis of data on conventional DCs from rats and data on monocyte-derived macrophages from humans revealed 7 IFN-regulated genes that were negatively regulated in both human and rat SpA (i.e., IRF1, STAT1, CXCL9, CXCL10, IFIT3, DDX60, and EPSTI1). CONCLUSION: Our results suggest that expression of HLA-B27 leads to a defect in IFNγ signaling in antigen-presenting cells in both B27-transgenic rats and SpA patients, which may result in Th17 expansion and Treg cell alteration (as shown in B27-transgenic rats) and contribute to disease pathogenesis.
Subject(s)
Antigen-Presenting Cells/immunology , HLA-B27 Antigen/genetics , Interferon-gamma/genetics , Macrophages/immunology , Spondylarthritis/immunology , Animals , Antigen-Presenting Cells/pathology , Gene Expression Profiling , HLA-B27 Antigen/immunology , Humans , Macrophages/pathology , Rats , Rats, Transgenic , Spondylarthritis/genetics , Spondylarthritis/pathology , Spondylitis, Ankylosing/genetics , Spondylitis, Ankylosing/immunology , Spondylitis, Ankylosing/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathologySubject(s)
Disease Models, Animal , Genomics , Spondylarthritis/genetics , Spondylarthritis/physiopathology , Animals , Arthritis, Psoriatic/genetics , Arthritis, Psoriatic/immunology , Arthritis, Psoriatic/physiopathology , Arthritis, Reactive/genetics , Arthritis, Reactive/immunology , Arthritis, Reactive/physiopathology , Humans , Mice , Rats , Spondylarthritis/immunologyABSTRACT
Proteins belonging to the P(II) family coordinate cellular nitrogen metabolism by direct interaction with a variety of enzymes, transcriptional regulators and transporters. The sensing function of P(II) relies on its ability to bind the nitrogen/carbon signalling molecule 2-oxoglutarate (2-OG). In Proteobacteria, P(II) is further subject to reversible uridylylation according to the intracellular levels of glutamine, which reflect the cellular nitrogen status. A number of P(II) proteins have been shown to bind ADP and ATP in a competitive manner, suggesting that P(II) might act as an energy sensor. Here, we analyse the influence of the ADP/ATP ratio, 2-OG levels and divalent metal ions on in vitro uridylylation of the Azospirillum brasilense P(II) proteins GlnB and GlnZ, and on interaction with their targets AmtB, DraG and DraT. The results support the notion that the cellular concentration of 2-OG is a key factor governing occupation of the GlnB and GlnZ nucleotide binding sites by ATP or ADP, with high 2-OG levels favouring the occupation of P(II) by ATP. Both P(II) uridylylation and interaction with target proteins responded to the ADP/ATP ratio within the expected physiological range, supporting the concept that P(II) proteins might act as cellular energy sensors.
Subject(s)
Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Azospirillum brasilense/metabolism , Bacterial Proteins/metabolism , Cations, Divalent/metabolism , Ketoglutaric Acids/metabolism , PII Nitrogen Regulatory Proteins/metabolism , Azospirillum brasilense/genetics , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , PII Nitrogen Regulatory Proteins/genetics , Signal TransductionABSTRACT
OBJECTIVE: HLA-B27/human ß2-microglobulin-transgenic (B27-transgenic) rats, a model of spondylarthritis (SpA), develop spontaneous colitis and arthritis under conventional conditions. CD4+ T cells are pivotal in the development of inflammation in B27-transgenic rats. This study was undertaken to characterize the phenotype of CD4+ T cells in this model and to determine whether dendritic cells (DCs) induce proinflammatory T cells. METHODS: The phenotype of CD4+ T cells from rat lymph nodes (LNs) draining the sites of inflammation was analyzed by flow cytometry. Immunostaining was used to detect interleukin-17 (IL-17)-producing cells in the rat joints. DCs from B27-transgenic or control rats (transgenic for HLA-B7 or nontransgenic) were cocultured with control CD4+ T cells and stimulated with anti-T cell receptor α/ß. RESULTS: IL-17A- and tumor necrosis factor α (TNFα)-producing CD4+ T cells were expanded in mesenteric and popliteal LNs from B27-transgenic rats. The accumulation of Th17 cells correlated with disease development, in contrast to Th1 or Treg cells. IL-17-positive mononuclear cells were detected in the arthritic joints of B27-transgenic rats but not in the joints of control rats. Finally, in vitro cocultures demonstrated that Th17 cells were preferentially induced and expanded by DCs from B27-transgenic rats, by a process that may involve defective engagement of costimulatory molecules. CONCLUSION: Our findings indicate that expanded CD4+ T cells in B27-transgenic rats exhibit a proinflammatory Th17 phenotype characterized by IL-17A and TNFα production. Furthermore, this population is preferentially induced by DCs from B27-transgenic rats. These data point toward an induction of Th17 cells as a possible pathogenic mechanism in this model of SpA. However, their pathogenic role still needs to be shown.
Subject(s)
Dendritic Cells/immunology , HLA-B27 Antigen/immunology , Spondylarthritis/immunology , Th17 Cells/immunology , Animals , Cell Proliferation , Cells, Cultured , Coculture Techniques , Dendritic Cells/pathology , Disease Models, Animal , Female , Genetic Predisposition to Disease , HLA-B27 Antigen/genetics , Joints/immunology , Joints/pathology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Lymph Nodes/immunology , Lymph Nodes/pathology , Male , Rats , Rats, Inbred F344 , Rats, Transgenic , Spondylarthritis/genetics , Spondylarthritis/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathology , Th1 Cells/immunology , Th1 Cells/pathology , Th17 Cells/pathology , beta 2-Microglobulin/geneticsABSTRACT
The P(II) proteins comprise a family of widely distributed signal transduction proteins that integrate the signals of cellular nitrogen, carbon and energy status, and then regulate, by protein-protein interaction, the activity of a variety of target proteins including enzymes, transcriptional regulators and membrane transporters. We have previously shown that the P(II) proteins from Azospirillum brasilense, GlnB and GlnZ, do not alter their migration behavior under native gel electrophoresis following incubated for a few minutes at 95°C. This data suggested that P(II) proteins were either resistant to high temperatures and/or that they could return to their native state after having been unfolded by heat. Here we used (1)H NMR to show that the A. brasilense GlnB is stable up to 70°C. The melting temperature (Tm) of GlnB was determined to be 84°C using the fluorescent dye Sypro-Orange. P(II) proteins from other Proteobacteria also showed a high Tm. We exploited the thermo stability of P(II) by introducing a thermal treatment step in the P(II) purification protocol, this step significantly improved the homogeneity of A. brasilense GlnB and GlnZ, Herbaspirillum seropedicae GlnB and GlnK, and of Escherichia coli GlnK. Only a single chromatography step was necessary to obtain homogeneities higher than 95%. NMR(1) and in vitro uridylylation analysis showed that A. brasilense GlnB purified using the thermal treatment maintained its folding and activity. The purification protocol described here can facilitate the study of P(II) protein family members.
Subject(s)
Bacterial Proteins/chemistry , Chromatography, Affinity/methods , PII Nitrogen Regulatory Proteins/chemistry , Azospirillum brasilense/enzymology , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Nuclear Magnetic Resonance, Biomolecular , PII Nitrogen Regulatory Proteins/isolation & purification , PII Nitrogen Regulatory Proteins/metabolism , Protein Conformation , Protein Multimerization , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transition TemperatureABSTRACT
The ammonium transport family Amt/Rh comprises ubiquitous integral membrane proteins that facilitate ammonium movement across biological membranes. Besides their role in transport, Amt proteins also play a role in sensing the levels of ammonium in the environment, a process that depends on complex formation with cytosolic proteins of the P(II) family. Trimeric P(II) proteins from a variety of organisms undergo a cycle of reversible posttranslational modification according to the prevailing nitrogen supply. In proteobacteria, P(II) proteins are subjected to reversible uridylylation of each monomer. In this study we used the purified proteins from Azospirillum brasilense to analyze the effect of P(II) uridylylation on the protein's ability to engage complex formation with AmtB in vitro. Our results show that partially uridylylated P(II) trimers can interact with AmtB in vitro, the implication of this finding in the regulation of nitrogen metabolism is discussed. We also report an improved expression and purification protocol for the A. brasilense AmtB protein that might be applicable to AmtB proteins from other organisms.
Subject(s)
Azospirillum brasilense/chemistry , Bacterial Proteins/chemistry , Cation Transport Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Nitrogen/metabolism , Quaternary Ammonium Compounds/metabolism , Uridine Monophosphate/chemistryABSTRACT
Despite their increasing use in autoimmune, inflammatory, and allergic conditions, the mechanism of action of i.v. Igs (IVIg) is poorly understood. On the basis of the critical role of invariant NKT (iNKT) cells in allergic airway inflammation (AAI) and their constitutive expression of the low-affinity IgG receptor FcγRIIIA, we surmised that IVIg targets iNKT cells to exert their anti-inflammatory effect. We found that IVIg treatment significantly inhibited AAI in OVA-sensitized C57BL/6 mice and downregulated α-galactosylceramide-induced iNKT cell activation and cytokine production. Allergic responses were restored in iNKT cell-deficient mice by transferring iNKT cells from PBS- but not from IVIg-treated mice, suggesting that IVIg acts directly on activated iNKT cells that have a critical role in AAI. The inhibitory effects of IVIg on both iNKT cell activation/function and OVA-driven AAI were lost in FcγRIIIA(-/-) mice. Our data unravel an FcγRIIIA-dependent inhibitory effect of IVIg on activated iNKT cells that confers protection in AAI.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Immunoglobulins, Intravenous/physiology , Inflammation Mediators/physiology , Natural Killer T-Cells/immunology , Natural Killer T-Cells/pathology , Receptors, IgG/physiology , Respiratory Hypersensitivity/immunology , Respiratory Hypersensitivity/prevention & control , Adoptive Transfer , Allergens/immunology , Allergens/toxicity , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/pathology , Bronchial Hyperreactivity/therapy , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Galactosylceramides/antagonists & inhibitors , Galactosylceramides/pharmacology , Immunoglobulins, Intravenous/therapeutic use , Inflammation Mediators/therapeutic use , Lymphocyte Activation/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Natural Killer T-Cells/metabolism , Ovalbumin/immunology , Ovalbumin/toxicity , Receptors, IgG/therapeutic use , Respiratory Hypersensitivity/pathology , Spleen/immunology , Spleen/pathology , Spleen/transplantationABSTRACT
Previous evidence indicated that growth hormone (GH) modulates cell migration in the thymus, and that extracellular matrix and chemokines are involved. Herein, we studied migration of peripheral lymphocytes derived from spleen and lymph nodes of GH-transgenic (GH-Tg) mice. We initially found that the relative cell numbers (normalized per gram of body weight) in lymph nodes and spleens from GH-Tg were higher at all ages tested (2-3, 7 and 12 months), as compared to wild type age-matched controls. Functionally, we found that lymphocyte migration triggered by laminin or fibronectin was enhanced in cells from GH-Tg versus control mice, independent of the organ from which the cells were derived (as ascertained in young adult animals). However, such an enhancement in migration was statistically significant only for CD4+ and CD8+ T cells from mesenteric lymph nodes. Migration of lymphocytes from mesenteric lymph nodes of GH-Tg mice, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was enhanced compared to lymphocytes from control mice. Rather surprisingly, the membrane levels of the corresponding extracellular matrix or chemokine receptors in peripheral lymphoid organs of GH-Tg mice did not necessarily correlate with the changes seen in migratory responses. In conclusion, our data show for the first time that GH alters lymphocyte migration in the periphery of the immune system. Considering that GH is used as an adjuvant therapeutic agent in immunodeficiencies, including AIDS, the concepts defined herein provide relevant background knowledge for future GH-related immune interventions.
Subject(s)
Chemokines/metabolism , Extracellular Matrix/physiology , Growth Hormone/genetics , Growth Hormone/physiology , Lymphocytes/physiology , Animals , B-Lymphocyte Subsets/physiology , Cell Movement , Chemotaxis, Leukocyte , Female , Fibronectins/metabolism , Image Cytometry , Immunohistochemistry , Lymph Nodes/pathology , Male , Mice , Mice, Transgenic , Spleen/pathology , T-Lymphocyte Subsets/physiologyABSTRACT
Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the P(II) family. In this study we have characterized in vitro complex formation between the AmtB and P(II) proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB-P(II) complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg(2+) and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB-P(II) complex defines a new regulatory role for Amt proteins in Prokaryotes.