PAD4 is not essential for disease in the K/BxN murine autoantibody-mediated model of arthritis

Introduction

Both murine and human genome-wide association studies have implicated peptidyl arginine deiminase (PAD4) as a susceptibility gene in rheumatoid arthritis (RA). In addition, patients with RA commonly have autoantibodies which recognize PAD4 or and/or citrullinated peptides. This study aims to evaluate the role of PAD4 in the effector phase of arthritis.

Methods

PAD4 knock out (KO) and wild type (WT) C57BL/6J mice were injected with K/BxN sera to induce disease. Progression of disease was monitored by measuring paw and ankle swelling and clinical indexes of disease, and pathogenesis was assessed by indexing of clinical progression on paws collected from WT and PAD4 KO mice injected with K/BxN serum. PAD4 activity was determined by visualization of neutrophil extracellular traps (NETs) and immunohistological analysis of histone citrullination.

Results

PAD4 activity is readily detectable in the inflamed synovium of WT but not PAD4 deficient animals, as demonstrated by histone citrullination and NET formation. However, PAD4 WT and KO animals develop K/BxN serum transfer disease with comparable severity and kinetics, with no statistically significant differences noted in clinical scores, swelling, joint erosion or joint invasion.

Conclusions

PAD4 WT and KO mice develop disease in the K/BxN serum transfer model of arthritis with similar severity and kinetics, indicating that PAD4 is dispensable in this effector phase model of disease.     

Matrix metalloproteinase-8 deficiency increases joint inflammation and bone erosion in the K/BxN serum-transfer arthritis model

Introduction  

Rheumatoid arthritis is an autoimmune disease in which joint inflammation leads to progressive cartilage and bone erosion. Matrix metalloproteinases (MMPs) implicated in homeostasis of the extracellular matrix play a central role in cartilage degradation. However, the role of specific MMPs in arthritis pathogenesis is largely unknown. The aim of the present study was to investigate the role of Mmp-8 (collagenase-2) in an arthritis model.     

Spermatogenesis in mice is not affected by histone H1.1 deficiency

The linker histone subtype H1.1 belongs to the group of main-type histones and is synthesized in somatic tissues as well as in germ cells during the S phase of the cell cycle. In adult mice the histone gene H1.1 is expressed mainly in thymus, spleen, and testis. The single-copy gene coding for the H1.1 protein was eliminated by homologous recombination in mouse embryonic stem cells. Mice homozygous for the deficient H1.1 gene developed normally until the adult stage without H1.1 mRNA and H1.1 protein. No anatomic abnormalities could be detected. In addition, mice lacking the H1.1 gene were fertile and they showed normal spermatogenesis and testicular morphology.     

A complicated liaison: IL-33 and IL-33R in arthritis pathogenesis

Interruption of cytokine signaling, by targeting either the cytokine itself or its cellular receptor, is a mainstay in the therapy for patients with rheumatic diseases. Interleukin (IL)-33, a member of the IL-1 cytokine family, has emerged as an important mediator of inflammatory responses. In a side-by-side examination of IL-33-deficient and IL-33 receptor (IL-33R)-deficient mice in the K/BxN serum transfer model, arthritis was ameliorated in the IL-33R knockout (KO) mice but not in the IL-33 KO mice. These findings complement previous knowledge on IL-33R signaling, demonstrating that the IL-33R cross-activates other signaling pathways in addition to IL-33-mediated signals. The results reported by Martin and colleagues in a previous issue of Arthritis Research & Therapy underline the clinical relevance of IL-33R cross-signaling and further illustrate that targeting a cytokine receptor (IL-33R) can have completely different clinical outcomes than targeting the respective cytokine.     

Influenza virus infection is not affected by serum amyloid P component

BACKGROUND: Binding of serum amyloid P component (SAP) to its ligands, including bacteria, chromatin and amyloid fibrils, protects them from degradation, is anti-opsonic and anti-immunogenic. SAP thereby enhances the virulence of pathogenic bacteria to which it binds. However SAP also contributes to host resistance against bacteria to which it does not bind. Human SAP has been reported to bind to the influenza virus and inhibit viral invasion of cells in tissue culture. We therefore investigated a possible role of SAP in either host resistance or viral virulence during influenza infection in vivo. MATERIALS AND METHODS: The clinical course of mouse adapted influenza virus infection, the host antibody response, and viral replication, were compared in wild type mice, mice with targeted deletion of the SAP gene, and mice transgenic for human SAP. The effects of reconstitution of SAP deficient mice with pure human SAP, and of a drug that specifically blocks SAP binding in vivo, were also studied. Binding of mouse and human SAP to immobilized influenza virus was compared. RESULTS: The presence, absence, or availability for binding of SAP in vivo had no significant or consistent effect on the course or outcome of influenza infection, or on either viral replication or the anti-viral antibody response. Mouse SAP bound much less avidly than human SAP to influenza virus. CONCLUSIONS: In marked contrast to the dramatic effects of SAP deficiency on host resistance to different bacterial infections, mouse SAP apparently plays no significant role during infection of mice with influenza virus. Human SAP binds much more avidly than mouse SAP to the virus, but also had no effect on any of the parameters measured and is therefore unlikely to be involved in human influenza infection.     

Rat myometrial Na/K ATPase is increased by serum but not by isoproterenol and relaxin

1. Na/K ATPase activity in rat myometrial cells in culture exhibited a Kapp of 0.93 mM for Rb+ and a Ki of 31 microM for ouabain with respect to Rb+. 2. 86Rb+ uptake was stimulated by serum and monensin but was not affected by the uterine relaxants isoproterenol and relaxin in 0.5-7.5 mM Rb+. Nonetheless, these relaxants elicited significant increases in 45Ca2+ efflux under similar conditions. 3. These data suggest that increased Na/Ca exchange resulting from a stimulation of Na/K ATPase is not involved in the mechanism of action of relaxin and isoproterenol in the uterus.     

The severity of experimental arthritis is independent of IL-36 receptor signaling

Introduction

Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.

Methods

Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.

Results

IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.

Conclusions

The development and severity of experimental arthritis are independent of IL-36R signaling.     

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33

Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.     

Endogenous IL-11 is pro-inflammatory in acute methylated bovine serum albumin/interleukin-1-induced (mBSA/IL-1)arthritis

OBJECTIVE: To evaluate the role of interleukin-11 (IL-11) in acute mBSA/IL-1-induced inflammatory arthritis. METHODS: IL-11 was administered via intra-articular (IA) injection into knee joints of C57BL/6 mice and joint histology was assessed. The mitogenic response to IL-11 was measured in wild-type (WT) synovial fibroblasts. IL-1 was used as a comparator in both the studies. The severity of acute methylated bovine serum albumin (mBSA)/IL-1 arthritis was determined in WT and IL-11 receptor null (IL-11Ra1-/-) mice. In parallel experiments, a neutralising antibody to IL-11 was administered to WT mice throughout this model. RESULTS: IA injections of IL-11 resulted in mild-to-moderate joint inflammation which was less than that due to IA IL-1. IL-11 had a dose-dependent mitogenic effect on WT synovial fibroblasts (P<0.01). mBSA/IL-1 acute arthritis was reduced in IL-11Ra1-/- versus WT mice (histological arthritis score: 10.1+/-0.5 versus 12.8+/-0.7, respectively; P=0.01). Administration of an IL-11 neutralising antibody to WT mice reduced mBSA/IL-1 acute arthritis scores compared to control antibody (10.6+/-0.7 versus 13.3+/-0.6, respectively; P=0.02). CONCLUSIONS: These data demonstrate that endogenous IL-11 exerts relatively mild but consistent pro-inflammatory effects in acute inflammatory arthritis.     

Soluble IL-6 receptor governs IL-6 activity in experimental arthritis: blockade of arthritis severity by soluble glycoprotein 130

Studies in IL-6-deficient (IL-6(-/-)) mice highlight that IL-6 contributes to arthritis progression. However, the molecular mechanism controlling its activity in vivo remains unclear. Using an experimental arthritis model in IL-6(-/-) mice, we have established a critical role for the soluble IL-6R in joint inflammation. Although intra-articular administration of IL-6 itself was insufficient to reconstitute arthritis within these mice, a soluble IL-6R-IL-6 fusion protein (HYPER-IL-6) restored disease activity. Histopathological assessment of joint sections demonstrated that HYPER-IL-6 increased arthritis severity and controlled intrasynovial mononuclear leukocyte recruitment through the CC-chemokine CCL2. Activation of synovial fibroblasts by soluble IL-6R and IL-6 emphasized that these cells may represent the source of CCL2 in vivo. Specific blockade of soluble IL-6R signaling in wild-type mice using soluble gp130 ameliorated disease. Consequently, soluble IL-6R-mediated signaling represents a promising therapeutic target for the treatment of rheumatoid arthritis.     

Blockade of vascular endothelial growth factor receptor I (VEGF-RI), but not VEGF-RII, suppresses joint destruction in the K/BxN model of rheumatoid arthritis

It was recently shown that vascular endothelial growth factor (VEGF), a growth factor for endothelial cells, plays a pivotal role in rheumatoid arthritis. VEGF binds to specific receptors, known as VEGF-RI and VEGF-RII. We assessed the physical and histological effects of selective blockade of VEGF and its receptors in transgenic K/BxN mice, a model of rheumatoid arthritis very close to the human disease. Mice were treated with anti-mouse VEGF Ab, anti-mouse VEGF-RI and -RII Abs, and an inhibitor of VEGF-RI tyrosine kinase. Disease activity was monitored using clinical indexes and by histological examination. We found that synovial cells from arthritic joints express VEGF, VEGF-RI, and VEGF-RII. Treatment with anti-VEGF-RI strongly attenuated the disease throughout the study period, while anti-VEGF only transiently delayed disease onset. Treatment with anti-VEGF-RII had no effect. Anti-VEGF-RI reduced the intensity of clinical manifestations and, based on qualitative and semiquantitative histological analyses, prevented joint damage. Treatment with a VEGF-RI tyrosine kinase inhibitor almost abolished the disease. These results show that VEGF is a key factor in pannus development, acting through the VEGF-RI pathway. The observation that in vivo administration of specific inhibitors targeting the VEGF-RI pathway suppressed arthritis and prevented bone destruction opens up new possibilities for the treatment of rheumatoid arthritis.     

Induction of Krüppel‐like factor 2 reduces K/BxN serum‐induced arthritis

Krüppel‐like factor 2 (KLF2) critically regulates activation and function of monocyte, which plays important pathogenic role in progressive joint destruction in rheumatoid arthritis (RA). It is yet to be established the molecular basis of KLF2‐mediated regulation of monocytes in RA pathogenesis. Herein, we show that a class of compound, HDAC inhibitors (HDACi) induced KLF2 expression in monocytes both in vitro and in vivo. KLF2 level was also elevated in tissues, such as bone marrow, spleen and thymus in mice after infusion of HDACi. Importantly, HDACi significantly reduced osteoclastic differentiation of monocytes with the up‐regulation of KLF2 and concomitant down‐regulation of matrixmetalloproteinases both in the expression level as well as in the protein level. In addition, HDACi reduced K/BxN serum‐induced arthritic inflammation and joint destruction in mice in a dose‐dependent manner. Finally, co‐immunoprecipitation and overexpression studies confirmed that KLF2 directly interacts with HDAC4 molecule in cells. These findings provide mechanistic evidence of KLF2‐mediated regulation of K/BxN serum‐induced arthritic inflammation.     

Synovial expression of IL-15 in rheumatoid arthritis is not influenced by blockade of tumour necrosis factor

Blockade of tumour necrosis factor (TNF) is an effective treatment in rheumatoid arthritis (RA), but both non-responders and partial responders are quite frequent. This suggests that other pro-inflammatory cytokines may be of importance in the pathogenesis of RA and as possible targets for therapy. In this study we investigated the effect of TNF blockade (infliximab) on the synovial expression of IL-15 in RA in relation to different cell types and expression of other cytokines, to elucidate whether or not IL-15 is a possible target for therapy, independently of TNF blockade. Two arthroscopies with multiple biopsies were performed on nine patients with RA and knee-joint synovitis before and after three infusions of infliximab (3 mg/kg). Synovial biopsies were analysed with immunohistochemistry for expression of IL-15, TNF, IL-1α, IL-1ß and IFN-γ, and for the cell surface markers CD3, CD68 and CD163. Stained synovial biopsy sections were evaluated by computerized image analysis. IL-15 expression was detected in all synovial biopsies taken at baseline. After infliximab therapy, the expression of IL-15 was increased in four patients and reduced in five. Synovial expression of IL-15 was not correlated with any CD marker or with the presence of any other cytokine. Synovial cellularity was decreased after 8 to 10 weeks of treatment with a significant reduction of the CD68-positive synovial cells, whereas no significant change was seen in the number of CD3-positive T cells and CD163-expressing macrophages. The number of TNF-producing cells in the synovial tissue at baseline was correlated with a good response to therapy. Thus, in this study the synovial expression of IL-15 in RA was not consistently influenced by TNF blockade, being apparently independent of TNF expression in the synovium. Consequently, we propose that IL-15 should remain as a therapeutic target in RA, regardless of the response to TNF blockade.     

Synovial expression of IL-15 in rheumatoid arthritis is not influenced by blockade of tumour necrosis factor

Blockade of tumour necrosis factor (TNF) is an effective treatment in rheumatoid arthritis (RA), but both non-responders and partial responders are quite frequent. This suggests that other pro-inflammatory cytokines may be of importance in the pathogenesis of RA and as possible targets for therapy. In this study we investigated the effect of TNF blockade (infliximab) on the synovial expression of IL-15 in RA in relation to different cell types and expression of other cytokines, to elucidate whether or not IL-15 is a possible target for therapy, independently of TNF blockade. Two arthroscopies with multiple biopsies were performed on nine patients with RA and knee-joint synovitis before and after three infusions of infliximab (3 mg/kg). Synovial biopsies were analysed with immunohistochemistry for expression of IL-15, TNF, IL-1alpha, IL-1ss and IFN-gamma, and for the cell surface markers CD3, CD68 and CD163. Stained synovial biopsy sections were evaluated by computerized image analysis. IL-15 expression was detected in all synovial biopsies taken at baseline. After infliximab therapy, the expression of IL-15 was increased in four patients and reduced in five. Synovial expression of IL-15 was not correlated with any CD marker or with the presence of any other cytokine. Synovial cellularity was decreased after 8 to 10 weeks of treatment with a significant reduction of the CD68-positive synovial cells, whereas no significant change was seen in the number of CD3-positive T cells and CD163-expressing macrophages. The number of TNF-producing cells in the synovial tissue at baseline was correlated with a good response to therapy. Thus, in this study the synovial expression of IL-15 in RA was not consistently influenced by TNF blockade, being apparently independent of TNF expression in the synovium. Consequently, we propose that IL-15 should remain as a therapeutic target in RA, regardless of the response to TNF blockade.     

Drosophila longevity is not affected by heterochromatin-mediated gene silencing

Two highly conserved histone deacetylases, Sir2 and Rpd3, have been linked to caloric restriction and the extension of longevity. Because the Drosophila forms of each protein can silence genes in either euchromatin or heterochromatin, we determined whether longevity extension is mediated by silencing in the latter domain. When silencing was increased and decreased using mutations that affect heterochromatin protein 1 (HP1), but have no direct effect upon Sir2 or Rpd3, lifespan was unaffected. Heterochromatin-mediated gene silencing was then modulated without directly influencing HP1 as well as the deacetylases, again yielding no effect on lifespan. Mortality rates were unchanged by all manipulations, indicating that euchromatic targets are likely to be the effectors of deacetylase-mediated longevity extension in Drosophila [corrected]     

Intestinal tumorigenesis is not affected by progesterone signaling in rodent models

Clinical data suggest that progestins have chemopreventive properties in the development of colorectal cancer. We set out to examine a potential protective effect of progestins and progesterone signaling on colon cancer development. In normal and neoplastic intestinal tissue, we found that the progesterone receptor (PR) is not expressed. Expression was confined to sporadic mesenchymal cells. To analyze the influence of systemic progesterone receptor signaling, we crossed mice that lacked the progesterone receptor (PRKO) to the Apc(Min/+) mouse, a model for spontaneous intestinal polyposis. PRKO-Apc(Min/+) mice exhibited no change in polyp number, size or localization compared to Apc(Min/+). To examine effects of progestins on the intestinal epithelium that are independent of the PR, we treated mice with MPA. We found no effects of either progesterone or MPA on gross intestinal morphology or epithelial proliferation. Also, in rats treated with MPA, injection with the carcinogen azoxymethane did not result in a difference in the number or size of aberrant crypt foci, a surrogate end-point for adenoma development. We conclude that expression of the progesterone receptor is limited to cells in the intestinal mesenchyme. We did not observe any effect of progesterone receptor signaling or of progestin treatment in rodent models of intestinal tumorigenesis.     

Reduced incidence and severity of collagen-induced arthritis in mice lacking IL-18

We have recently reported the presence and a potential proinflammatory role of IL-18 in the synovium of patients with rheumatoid arthritis. To obtain direct evidence that IL-18 plays an influential role in articular inflammation, we investigated the development of collagen-induced arthritis in a strain of mice lacking IL-18 (IL-18(-/-)) of DBA/1 background. IL-18(-/-) mice developed markedly reduced incidence of arthritis compared with heterozygous or wild-type mice. Of the IL-18(-/-) mice that developed arthritis, the severity of the disease was significantly reduced compared with the intact mice. This was accompanied by reduced articular inflammation and destruction evident on histology. IL-18(-/-) mice also had significantly reduced Ag-specific proliferation and proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-6, and IL-12) production by spleen and lymph node cells in response to bovine type II collagen (CII) in vitro compared with wild-type mice, paralleled in vivo by a significant reduction in serum anti-CII IgG2a Ab level. Treatment with rIL-18 completely reversed the disease of the IL-18(-/-) mice to that of the wild-type mice. These data directly demonstrate a pivotal role of IL-18 in the development of inflammatory arthritis and suggest that antagonists to IL-18 may have therapeutic potential in rheumatic diseases.