Delayed-type Hypersensitivity Arthritis Research Articles

NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis

ObjectiveRecent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like...

Examining methotrexate’s safety and efficacy in combination with immune checkpoint inhibitors to control immune related inflammatory arthritis in cancer

Abstract Immune checkpoint inhibitors (ICIs) are associated with high rates of toxic side effects known as immune related adverse events such as inflammatory arthritis (ir-RA). ir-RA is empirically treated with drugs for rheumatoid arthritis such as methotrexate (MTX) and hydroxychloroquine which was recently shown to reduce the anti-tumor benefit of ICIs in mouse models of cancer. We have set out to determine the safety of MTX use in combination with ICIs, its effect on the anti-tumoral immune response and its efficacy in treating ir-RA in a novel arthritis-B16 PD-L1 tumor mouse model. We observed that MTX did not diminish the anti-tumor benefit of ICIs in our melanoma model and significantly altered the intra-tumoral CD8 +T cell population by promoting the formation of central memory cells and diminishing PD1 expression. We have identified that MTX acts on neuropilin-1 (NRP1), a receptor identified as a CD8 +T cell exhaustion marker promoting terminal exhaustion and limiting memory cell formation in tumors. We have shown that MTX treatment in vitro reduces the expression of NRP1 on stimulated CD8 +T cells. We have also advanced in the development of a novel arthritis-tumor model showing that MTX can control and diminish inflammation in the delayed type hypersensitivity arthritis (DTHA) and reduced the frequency of Th1 and Th17 cells in the popliteal lymph nodes of mice, with Th17 cells known to have a crucial role in ir-RA. Together these results show that MTX is safe for use in combination with ICIs in our tumor model and is effective in our DTHA model that mimics the immune profile of ir-RA. With the development of our combined arthritis-tumor model we will be able to assess MTX’s efficacy in controlling arthritis in ICIs treated tumor bearing mice.

Impact of delayed type hypersensitivity arthritis on development of heart failure by aortic constriction in mice.

Patients with rheumatoid arthritis (RA) have increased risk of heart failure (HF). The mechanisms and cardiac prerequisites explaining this association remain unresolved. In this study, we sought to determine the potential cardiac impact of an experimental model of RA in mice subjected to HF by constriction of the ascending aorta. Aorta was constricted via thoracotomy and placement of o-rings with inner diameter 0.55 mm or 0.66 mm, or sham operated. RA-like phenotype was instigated by delayed-type hypersensitivity arthritis (DTHA) two weeks after surgery and re-iterated after additional 18 days. Cardiac magnetic resonance imaging (MRI) was performed before surgery and at successive time points throughout the study. Six weeks after surgery the mice were euthanized, blood and tissue were collected, organ weights were documented, and expression levels of cardiac foetal genes were analysed. In a supplemental study, DTHA-mice were euthanized throughout 14 days after induction of arthritis, and blood was analysed for important markers and mediators of RA (SAP, TNF-α and IL-6). In order to put the latter findings into clinical context, the same molecules were analysed in serum from untreated RA patients and compared to healthy controls. Significant elevations of inflammatory markers were found in both patient- and murine blood. Furthermore, the DTHA model appeared clinically relevant when compared to the inflammatory responses observed in three prespecified RA severity disease states. Two distinct trajectories of cardiac dysfunction and HF development were found using the two o-ring sizes. These differences were consistent by both MRI, organ weights and cardiac foetal gene expression levels. Still, no difference within the HF groups, nor within the sham groups, could be found when DTHA was induced. DTHA mediated systemic inflammation did not cause, nor modify HF caused by aortic constriction. This indicates other prerequisites for RA-induced cardiac dysfunction.

Activated, Pro-Inflammatory Th1, Th17, and Memory CD4+ T Cells and B Cells Are Involved in Delayed-Type Hypersensitivity Arthritis (DTHA) Inflammation and Paw Swelling in Mice.

Delayed-type hypersensitivity arthritis (DTHA) is a recently established experimental model of rheumatoid arthritis (RA) in mice with pharmacological values. Despite an indispensable role of CD4+ T cells in inducing DTHA, a potential role for CD4+ T cell subsets is lacking. Here we have quantified CD4+ subsets during DTHA development and found that levels of activated, pro-inflammatory Th1, Th17, and memory CD4+ T cells in draining lymph nodes were increased with differential dynamic patterns after DTHA induction. Moreover, according to B-cell depletion experiments, it has been suggested that this cell type is not involved in DTHA. We show that DTHA is associated with increased levels of B cells in draining lymph nodes accompanied by increased levels of circulating IgG. Finally, using the anti-rheumatoid agents, methotrexate (MTX) and the anti-inflammatory drug dexamethasone (DEX), we show that MTX and DEX differentially suppressed DTHA-induced paw swelling and inflammation. The effects of MTX and DEX coincided with differential regulation of levels of Th1, Th17, and memory T cells as well as B cells. Our results implicate Th1, Th17, and memory T cells, together with activated B cells, to be involved and required for DTHA-induced paw swelling and inflammation.

Involvement of Tumor Necrosis Factor Receptor Type II in FoxP3 Stability and as a Marker of Treg Cells Specifically Expanded by Anti-Tumor Necrosis Factor Treatments in Rheumatoid Arthritis.

To study the involvement of Treg cells expressing tumor necrosis factor receptor type II (TNFRII) in exerting control of inflammation in experimental models and in the response to anti-TNF treatments in patients with rheumatoid arthritis (RA) or spondyloarthritis (SpA). The role of TNFRII in Treg cells was explored using a multilevel translational approach. Treg cell stability was evaluated by analyzing the methylation status of the Foxp3 locus using bisulfite sequencing. Two models of inflammation (imiquimod-induced skin inflammation and delayed-type hypersensitivity arthritis [DTHA]) were induced in TNFRII-/- mice, with or without transfer of purified CD4+CD25+ cells from wild-type (WT) mice. In patients with RA and those with SpA, the evolution of the TNFRII+ Treg cell population before and after targeted treatment was monitored. Foxp3 gene methylation in Treg cells was greater in TNFRII-/- mice than in WT mice (50% versus 36.7%). In cultured Treg cells, TNF enhanced the expression, maintenance, and proliferation of Foxp3 through TNFRII signaling. Imiquimod-induced skin inflammation and DTHA were aggravated in TNFRII-/- mice (P < 0.05 for mice with skin inflammation and P < 0.0001 for mice with ankle swelling during DTHA compared to WT mice). Adoptive transfer of WT mouse Treg cells into TNFRII-/- mice prevented aggravation of arthritis. In patients with RA receiving anti-TNF treatments, but not those receiving tocilizumab, the frequency of TNFRII+ Treg cells was increased at 3 months of treatment compared to baseline (mean ± SEM 65.2 ± 3.1% versus 49.1 ± 5.5%; P < 0.01). In contrast, in anti-TNF-treated patients with SpA, the frequency of TNFRII+ Treg cells was not modified. TNFRII expression identifies a subset of Treg cells that are characterized by stable expression of Foxp3 via gene hypomethylation, and adoptive transfer of TNFRII-expressing Treg cells ameliorates inflammation in experimental models. Expansion and activation of TNFRII+ Treg cells may be one of the mechanisms by which anti-TNF agents control inflammation in RA, but not in SpA.

Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models.

Genetic absence of the urokinase-type plasminogen activator (uPA) reduces arthritis progression in the collagen-induced arthritis (CIA) mouse model to an extent just shy of disease abrogation, but this remarkable observation has not been translated into therapeutic intervention. Our aim was to test the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes mouse uPA significantly reduced arthritis progression in the CIA and delayed-type hypersensitivity arthritis models. In the CIA model, the impact of anti-uPA treatment was on par with the effect of blocking TNF-α by etanercept. A pharmacokinetics evaluation of the therapeutic Ab revealed target-mediated drug disposition consistent with a high turnover of endogenous uPA. The cellular expression patterns of uPA and uPAR were characterized by double immunofluorescence in the inflamed synovium from patients with rheumatoid arthritis and compared with synovium from healthy donors. The arthritic synovium showed expression of uPA and uPAR in neutrophils, macrophages, and a fraction of endothelial cells, whereas there was little or no expression in synovium from healthy donors. The data from animal models and human material provide preclinical proof-of-principle that validates uPA as a novel therapeutic target in rheumatic diseases.

Pharmacological Value of Murine Delayed-type Hypersensitivity Arthritis: A Robust Mouse Model of Rheumatoid Arthritis in C57BL/6 Mice.

In this MiniReview, we summarize the body of knowledge on the delayed-type hypersensitivity arthritis (DTHA) model, a recently developed arthritis model with 100% incidence, low variation and synchronized onset in C57BL/6 (B6) mice, and compare it to other murine arthritis models. It is desirable to have robust arthritis models in B6 mice, as many transgene strains are bred on this background. However, several of the most widely used mouse model of arthritis cannot be induced in B6 mice without the drawback of lower incidence, reduced severity and higher variation, if at all. DTHA is induced by modifying a classical methylated bovine serum albumin (mBSA)-induced DTH response by administering a cocktail of anti-type II collagen antibodies (anti-CII) between immunization and challenge. Arthritis affects one, predefined paw in which acute inflammation and severe arthritis rapidly develop and peak after 4-7 days. Disease is self-resolving over the course of around 3 weeks. Disease manifestations resemble those seen in other arthritis models and include bone erosion, cartilage destruction, oedema, pannus and new bone formation. Induction of DTHA is dependent on CD4+ T cells while B cells are dispensable. The DTHA model is set apart from other murine arthritis models in that it can be induced in B6 mice with 100% incidence and with high and consistent severity. This is the clearest advantage of the model, as the mechanisms of disease and clinical manifestations can be found in other arthritis models. The model holds potential for future modifications that may improve the lack of chronicity.

Anti-RANKL treatment inhibits erosive joint destruction and lowers inflammation but has no effect on bone formation in the delayed-type hypersensitivity arthritis (DTHA) model.

BackgroundThe aims of the present study were to determine the relationship between bone destruction and bone formation in the delayed-type hypersensitivity arthritis (DTHA) model and to evaluate the effect of receptor activator of nuclear factor κB ligand (RANKL) blockade on severity of arthritis, bone destruction, and bone formation.MethodsDTHA was induced in C57BL/6 mice. Inflammation, erosive joint damage, and new bone formation were semiquantitatively scored by histology. Osteoclast activity was assessed in vivo, and messenger RNA (mRNA) expression of mediators of bone destruction and bone formation were analyzed by mRNA deep sequencing. Serum concentrations of tartrate-resistant acid phosphatase 5b, carboxy-terminal telopeptide I (CTX-I), matrix metalloproteinase 3 (MMP3), and serum amyloid P component (SAP) were determined by enzyme-linked immunosorbent assay. Anti-RANKL monoclonal antibody treatment was initiated at the time of immunization.ResultsBone destruction (MMP3 serum levels, cathepsin B activity, and RANKL mRNA) peaked at day 3 after arthritis induction, followed by a peak in cartilage destruction and bone erosion on day 5 after arthritis induction. Periarticular bone formation was observed from day 10. Induction of new bone formation indicated by enhanced Runx2, collagen X, osteocalcin, MMP2, MMP9, and MMP13 mRNA expression was observed only between days 8 and 11. Anti-RANKL treatment resulted in a modest reduction in paw and ankle swelling and a reduction of serum levels of SAP, MMP3, and CTX-I. Destruction of the subchondral bone was significantly reduced, while no effect on bone formation was seen.ConclusionsAnti-RANKL treatment prevents joint destruction but does not prevent new bone formation in the DTHA model. Thus, although occurring sequentially during the course of DTHA, bone destruction and bone formation are apparently not linked in this model.

Depletion of regulatory T cells leads to an exacerbation of delayed-type hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade.

ABSTRACTRodent models of arthritis have been extensively used in the elucidation of rheumatoid arthritis (RA) pathogenesis and are instrumental in the development of therapeutic strategies. Here we utilise delayed-type hypersensitivity arthritis (DTHA), a model in C57BL/6 mice affecting one paw with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregs and the role of IL-17 in connection with Treg depletion. Given the relevance of Tregs in RA, and the possibility of developing Treg-directed therapies, this approach could be relevant for advancing the understanding of Tregs in inflammatory arthritis. Selective depletion of Tregs was achieved using a Foxp3-DTR-eGFP mouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of the Foxp3 gene. Anti-IL-17 monoclonal antibody (mAb) was used for IL-17 blockade. Numbers and activation of Tregs increased in the paw and its draining lymph node in DTHA, and depletion of Tregs resulted in exacerbation of disease as shown by increased paw swelling, increased infiltration of inflammatory cells, increased bone remodelling and increased production of inflammatory mediators, as well as increased production of anti-citrullinated protein antibodies. Anti-IL-17 mAb treatment demonstrated that IL-17 is important for disease severity in both the presence and absence of Tregs, and that IL-17 blockade is able to rescue mice from the exacerbated disease caused by Treg depletion and caused a reduction in RANKL, IL-6 and the number of neutrophils. We show that Tregs are important for the containment of inflammation and bone remodelling in DTHA. To our knowledge, this is the first study using the Foxp3-DTR-eGFP mouse on a C57BL/6 background for Treg depletion in an arthritis model, and we here demonstrate the usefulness of the approach to study the role of Tregs and IL-17 in arthritis.

Treatment with anti-C5aR mAb leads to early-onset clinical and mechanistic effects in the murine delayed-type hypersensitivity arthritis model

Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.