A Mouse Model of Adoptive Immunotherapeutic Targeting of Autoimmune Arthritis Using Allo-Tolerogenic Dendritic Cells

Objective

Tolerogenic dendritic cells (tDCs) are immunosuppressive cells with potent tolerogenic ability and are promising immunotherapeutic tools for treating rheumatoid arthritis (RA). However, it is currently unknown whether allogeneic tDCs (allo-tDCs) induce tolerance in RA, and whether the numbers of adoptively transferred allo-tDCs, or the requirement for pulsing with relevant auto-antigens are important.

Methods

tDCs were derived from bone marrow precursors of C57BL/B6 mice, which were induced in vitro by GM-CSF, IL-10 and TGF-β1. Collagen-induced arthritis (CIA) was modeled in D1 mice by immunization with type II collagen (CII) to test the therapeutic ability of allo-tDCs against CIA. Clinical and histopathologic scores, arthritic incidence, cytokine and anti-CII antibody secretion, and CD4⁺Th subsets were analyzed.

Results

tDCs were characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability. Following adoptive transfer of low doses (5×10⁵) of CII-loaded allo-tDCs, a remarkable anti-arthritic activity, improved clinical scores and histological end-points were found. Serological levels of inflammatory cytokines and anti-CII antibodies were also significantly lower in CIA mice treated with CII-pulsed allo-tDCs as compared with allo-tDCs. Moreover, treatment with allo-tDCs altered the proportion of Treg/Th17 cells.

Conclusion

These findings suggested that allo-tDCs, especially following antigen loading, reduced the severity of CIA in a dose-dependent manner. The dampening of CIA was associated with modulated cytokine secretion, Treg/Th17 polarization and inhibition of anti-CII secretion. This study highlights the potential therapeutic utility of allo-tDCs in autoimmune arthritis and should facilitate the future design of allo-tDC immunotherapeutic strategies against RA.     

Type II collagen antibody response is enriched in the synovial fluid of rheumatoid joints and directed to the same major epitopes as in collagen induced arthritis in primates and mice

Introduction

Antibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collagen induced arthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA.

Methods

Analysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice.

Results

Many CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group.

Conclusion

CII conformational dependent antibody responses are common in RA and are likely to originate from rheumatoid joints but did not show a correlation with ACPA response. Importantly, the fine specificity of the anti-CII response is similar with CIA in monkeys and rodents where the recognized epitopes are conserved and have a major pathogenic role. Thus, anti-CII antibodies may both contribute to, as well as be the consequence of, local joint inflammation.     

Impact of Schistosoma japonicum infection on collagen-induced arthritis in DBA/1 mice: a murine model of human rheumatoid arthritis

Background

The hygiene hypothesis suggests that helminth infections prevent a range of autoimmune diseases.

Methodology/Principal Findings

To investigate the effects of S. japonicum infection on collagen-induced arthritis (CIA), male DBA/1 mice were challenged with unisexual or bisexual S. japonicum cercariae two weeks prior to bovine type II collagen (CII) immunization or at the onset of CIA. S. japonicum infection prior to CII immunization significantly reduced the severity of CIA. ELISA (enzyme linked immunosorbent assay) showed that the levels of anti-CII IgG and IgG2a were reduced in prior schistosome-infected mice, while anti-CII IgG1 was elevated. Splenocyte proliferation against both polyclonal and antigen-specific stimuli was reduced by prior schistosome infection as measured by tritiated thymidine incorporation (³H-TdR). Cytokine profiles and CD4⁺ T cells subpopulation analysis by ELISA and flow cytometry (FCM) demonstrated that prior schistosome infection resulted in a significant down-regulation of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β and IL-6) and Th1 cells, together with up-regulation of the anti-inflammatory cytokine IL-10 and Th2 cells. Interestingly, the expansion of Treg cells and the reduction of Th17 cells were only observed in bisexually infected mice. In addition, prior schistosome infection notably reduced the expression of pro-inflammatory cytokines and receptor activator of NF-κB ligand (RANKL) in the inflamed joint. However, the disease was exacerbated at one week after infection when established CIA mice were challenged with bisexual cercariae.

Conclusion/Significance

Our data provide direct evidence that the Th2 response evoked by prior S. japonicum infection can suppress the Th1 response and pro-inflammatory mediator and that bisexual infection with egg-laying up-regulates the Treg response and down-regulates the Th17 response, resulting in an amelioration of autoimmune arthritis. The beneficial effects might depend on the establishment of a Th2-dominant response rather than the presence of the eggs. Our results suggest that anti-inflammatory molecules from the parasite could treat autoimmune diseases.     

Anthocyanin Extracted from Black Soybean Seed Coats Prevents Autoimmune Arthritis by Suppressing the Development of Th17 Cells and Synthesis of Proinflammatory Cytokines by Such Cells,via Inhibition of NF-κB

Introduction

Oxidative stress plays a role in the pathogenesis of rheumatoid arthritis (RA). Anthocyanin is a plant antioxidant. We investigated the therapeutic effects of anthocyanin extracted from black soybean seed coats (AEBS) in a murine model of collagen-induced arthritis (CIA) and human peripheral blood mononuclear cells (PBMCs) and explored possible mechanisms by which AEBS might exert anti-arthritic effects.

Material and Methods

CIA was induced in DBA/1J mice. Cytokine levels were measured via enzyme-linked immunosorbent assays. Joints were assessed in terms of arthritis incidence, clinical arthritis scores, and histological features. The extent of oxidative stress in affected joints was determined by measuring the levels of nitrotyrosine and inducible nitric oxide synthase. NF-κB activity was assayed by measuring the ratio of phosphorylated IκB to total IκB via Western blotting. Th17 cells were stained with antibodies against CD4, IL-17, and STAT3. Osteoclast formation was assessed via TRAP staining and measurement of osteoclast-specific mRNA levels.

Results

In the CIA model, AEBS decreased the incidence of arthritis, histological inflammation, cartilage scores, and oxidative stress. AEBS reduced the levels of proinflammatory cytokines in affected joints of CIA mice and suppressed NF-κB signaling. AEBS decreased Th17 cell numbers in spleen of CIA mice. Additionally, AEBS repressed differentiation of Th17 cells and expression of Th17-associated genes in vitro, in both splenocytes of naïve DBA/1J mice and human PBMCs. In vitro, the numbers of both human and mouse tartrate-resistant acid phosphatase⁺ (TRAP) multinucleated cells fell, in a dose-dependent manner, upon addition of AEBS.

Conclusions

The anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling. Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.     

Effect of sclerostin-neutralising antibody on periarticular and systemic bone in a murine model of rheumatoid arthritis: a microCT study

Introduction

Patients with chronic inflammatory diseases have increased bone loss and bone fragility and are at increased risk of fracture. Although anti-resorptive drugs are effective in blocking inflammation-induced bone loss, they are less effective at rebuilding bone. We have previously shown that treatment with sclerostin antibody (Scl-AbI) builds bone and can prevent or restore bone loss in a murine model of inflammatory bowel disease. In this study, we tested the effect of Scl-AbI in a murine model of rheumatoid arthritis (the collagen-induced arthritis model, CIA). We hypothesised that sclerostin blockade can protect and restore bone both locally and systemically without affecting progression of inflammation.

Methods

CIA was induced in male DBA/1 mice, which were treated with either PBS or Scl-AbI (10 mg/kg, weekly) prophylactically for 55 days or therapeutically for 21 days (starting 14 days post onset of arthritis). Systemic inflammation was assessed by measuring the serum concentration of anti-CII IgG1, IgG2a and IgG2b by ELISA. Changes in bone mass and structure, either at sites remote from the joints or at periarticular sites, were measured using DEXA and microCT. Bone focal erosion was assessed in microCT scans of ankle and knee joints.

Results

Circulating anti-CII immunoglobulins were significantly elevated in mice with CIA and there were no significant differences in the levels of anti-CII immunoglobulins in mice treated with PBS or Scl-ABI. Prophylactic Scl-AbI treatment prevented the decrease in whole body bone mineral density (BMD) and in the bone volume fraction at axial (vertebral body) and appendicular (tibial proximal metaphysis trabecular and mid-diaphysis cortical bone) sites seen in PBS-treated CIA mice, but did not prevent the formation of focal bone erosions on the periarticular bone in the knee and ankle joints. In the therapeutic study, Scl-AbI restored BMD and bone volume fraction at all assessed sites but was unable to repair focal erosions.

Conclusions

Sclerostin blockade prevented or reversed the decrease in axial and appendicular bone mass in the murine model of rheumatoid arthritis, but did not affect systemic inflammation and was unable to prevent or repair local focal erosion.     

Bile Salt-Stimulated Lipase Plays an Unexpected Role in Arthritis Development in Rodents

Objective

The present study aimed to explore the hypothesis that bile salt-stimulated lipase (BSSL), in addition to being a key enzyme in dietary fat digestion during early infancy, plays an important role in inflammation, notably arthritis.

Methods

Collagen-induced arthritis (CIA) and pristane-induced arthritis (PIA) in rodents are commonly used experimental models that reproduce many of the pathogenic mechanisms of human rheumatoid arthritis, i.e. increased cellular infiltration, synovial hyperplasia, pannus formation, and erosion of cartilage and bone in the distal joints. We used the CIA model to compare the response in BSSL wild type (BSSL-WT) mice with BSSL-deficient ‘knock-out’ (BSSL-KO) and BSSL-heterozygous (BSSL-HET) littermates. We also investigated if intraperitoneal injection of BSSL-neutralizing antibodies affected the development or severity of CIA and PIA in mice and rats, respectively.

Results

In two consecutive studies, we found that BSSL-KO male mice, in contrast to BSSL-WT littermates, were significantly protected from developing arthritis. We also found that BSSL-HET mice were less prone to develop disease compared to BSSL-WT mice, but not as resistant as BSSL-KO mice, suggesting a gene-dose effect. Moreover, we found that BSSL-neutralizing antibody injection reduced both the incidence and severity of CIA and PIA in rodents.

Conclusion

Our data strongly support BSSL as a key player in the inflammatory process, at least in rodents. It also suggests the possibility that BSSL-neutralizing agents could serve as a therapeutic model to reduce the inflammatory response in humans.     

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.     

Enforced Expression of Roquin Protein in T Cells Exacerbates the Incidence and Severity of Experimental Arthritis

To investigate the role of Roquin, a RING-type ubiquitin ligase family member, we used transgenic mice with enforced Roquin expression in T cells, with collagen-induced arthritis (CIA). Wild-type (WT) and Roquin transgenic (Tg) mice were immunized with bovine type II collagen (CII). Arthritis severity was evaluated by clinical score; histopathologic CIA severity; proinflammatory and anti-inflammatory cytokine levels; anti-CII antibody levels; and populations of Th1, Th2, germinal center B cells, and follicular helper T cells in CIA. T cell proliferation in vitro and cytokine levels were determined to assess the response to CII. Roquin Tg mice developed more severe CIA and joint destruction compared with WT mice. Production of TNF-α, IFN-γ, IL-6, and pathogenic anti-collagen CII-specific IgG and IgG2a antibodies was increased in Roquin Tg mice. In addition, in vitro T cell assays showed increased proliferation and proinflammatory cytokine production in response to CII as a result of enforced Roquin expression in T cells. Furthermore, the Th1/Th2 balance was altered by an increased Th1 and decreased Th2 population. These findings suggest that overexpression of Roquin exacerbates the development of CIA and that enforced expression of Roquin in T cells may promote autoimmune diseases such as CIA.     

Antibodies Specific for Carbamylated Proteins Precede the Onset of Clinical Symptoms in Mice with Collagen Induced Arthritis

Objective

The immune response to post-translationally modified antigens is a key characteristic of rheumatoid arthritis. Carbamylation is such a posttranslational modification. Recently, we demonstrated that autoantibodies recognizing carbamylated proteins are present in sera of rheumatoid arthritis. The molecular mechanisms underlying the break of tolerance and hence the induction of anti-CarP antibody responses are unknown as well as their appearance in mouse models for systemic arthritis. Therefore we analyzed their appearance in the mouse collagen-induced arthritis model.

Methods

collagen induced arthritis was induced by immunization with type II collagen in complete Freund''s adjuvant. Arthritis severity was monitored by clinical scoring and anti-CarP antibody levels were determined by ELISA.

Results

Anti-CarP antibodies were detectable in mice with collagen induced arthritis. We did not detect ACPA in mice with collagen induced arthritis. The specificity of the antibodies for carbamylated proteins was confirmed by inhibition assays and immunoblotting. Injection with complete Freund''s adjuvant without type II collagen could also induce anti-CarP antibodies, however, in mice with arthritis, the anti-CarP antibody response was stronger and developed more rapidly. The onset of collagen induced arthritis was preceded by an increase of anti-CarP IgG2a levels in the serum.

Conclusion

In mice with collagen induced arthritis we did not observe an immune response against citrullinated antigens, but we did observe an immune response against carbamylated antigens. This anti-CarP response already appeared before disease onset, indicating that collagen induced arthritis can be used as an in vivo model to study anti-CarP antibodies. Our data also indicate that the tolerance to carbamylated proteins, in contrast to the response to citrullinated proteins, is easily broken and that arthritis boosts the immune response against these proteins. The anti-CarP response in mice with CIA can be used as a model for immune responses to post-translationally modified proteins.     

AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis

Objective

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods

We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results

AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions

AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory arthritis.     

The Additive Inflammatory In Vivo and In Vitro Effects of IL-7 and TSLP in Arthritis Underscore the Therapeutic Rationale for Dual Blockade

Introduction

The cytokines interleukin (IL)-7 and thymic stromal lymphopoietin (TSLP) signal through the IL-7R subunit and play proinflammatory roles in experimental arthritis and rheumatoid arthritis (RA). We evaluated the effect of inhibition of IL-7R- and TSLPR-signalling as well as simultaneous inhibition of IL-7R- and TSLPR-signalling in murine experimental arthritis. In addition, the effects of IL-7 and TSLP in human RA dendritic cell (DC)/T-cell co-cultures were studied.

Methods

Arthritis was induced with proteoglycan in wildtype mice (WT) and in mice deficient for the TSLP receptor subunit (TSLPR-/-). Both mice genotypes were treated with anti-IL-7R or phosphate buffered saline. Arthritis severity was assessed and local and circulating cytokines were measured. Autologous CD1c-positive DCs and CD4 T-cells were isolated from peripheral blood of RA patients and were co-cultured in the presence of IL-7, TSLP or both and proliferation and cytokine production were assessed.

Results

Arthritis severity and immunopathology were decreased in WT mice treated with anti-IL-7R, in TSLPR-/- mice, and the most robustly in TSLPR-/- mice treated with anti-IL-7R. This was associated with strongly decreased levels of IL-17, IL-6 and CD40L. In human DC/T-cell co-cultures, TSLP and IL-7 additively increased T-cell proliferation and production of Th17-associated cytokines, chemokines and tissue destruction factors.

Conclusion

TSLP and IL-7 have an additive effect on the production of Th17-cytokines in a human in vitro model, and enhance arthritis in mice linked with enhanced inflammation and immunopathology. As both cytokines signal via the IL-7R, these data urge for IL-7R-targeting to prevent the activity of both cytokines in RA.     

Anti-T cell immunoglobulin and mucin domain-2 monoclonal antibody exacerbates collagen-induced arthritis by stimulating B cells

Introduction

T cell immunoglobulin and mucin domain-2 (TIM-2) has been shown to regulate CD4 T cell activation. However, the role of TIM-2 in the autoimmune disease models has not been clarified yet. In this study, we investigated the effects of anti-TIM-2 monoclonal antibodies (mAbs) in collagen-induced arthritis (CIA) to determine whether TIM-2 contributes to the development of T helper (Th) 1 or Th17 cells and joint inflammation.

Methods

DBA/1 mice were treated with anti-TIM-2 mAbs during the early or late phase of CIA. Type II collagen (CII)-specific CD4 T-cell proliferative response and cytokine production were assessed from lymph node cell culture. The serum levels of CII-specific antibody were measured by ELISA. The expression of TIM-2 on CD4 T cells or B cells was determined by flow cytometric analysis.

Results

Administration of anti-TIM-2 mAbs in early phase, but not late phase, significantly exacerbated the development of CIA. Although anti-TIM-2 mAbs treatment did not affect the development of Th1 or Th17 cells in the draining lymph node, the serum levels of anti-CII antibodies were significantly increased in the anti-TIM-2-treated mice. TIM-2 expression was found on splenic B cells and further up-regulated by anti-immunoglobulin (Ig)M, anti-CD40, and interleukin(IL)-4 stimulation. In contrast, CD4 T cells did not express TIM-2 even when stimulated with both anti-CD3 and anti-CD28 mAbs. Interestingly, anti-TIM-2 mAbs enhanced proliferation and antibody production of activated B cells in vitro.

Conclusions

TIM-2 signaling influences both proliferation and antibody production of B cells during the early phase of CIA, but not induction of Th1 or Th17 cells.     

Inhibition of TREM-1 and Dectin-1 Alleviates the Severity of Fungal Keratitis by Modulating Innate Immune Responses

Purpose

To explore the possibility that inhibiting triggering receptor expressed on myeloid cells-1 (TREM-1) and Dendritic cell-associated C-type lectin-1(Dectin-1) could modulate the innate immune response and alleviate the severity of corneal fungal keratitis.

Method

TREM-1 and Dectin-1 expression was detected in fungus-infected human corneal specimens by real-time PCR. C57BL/6 (B6) mice were injected with Aspergillus fumigatus and divided into 4 groups that received subconjunctival injections of PBS and IgG as a control (group I), mTREM-1/IgG fusion protein (group II), the soluble β-glucan antagonist laminarin (group III), or mTREM-1/Fc and laminarin (group IV). Corneal virulence was evaluated based on clinical scores. TREM-1 and Dectin-1 mRNA levels were assayed using real-time PCR. The distribution patterns of TREM-1, Dectin-1 and cellular infiltrates in fungus-infected corneas were examined by immunohistochemistry. Moreover, changes in T Helper Type1 (Th1)-/ T Helper Type1 (Th2)- type cytokines and proinflammatory cytokines were measured.

Results

The expression of TREM-1 and Dectin-1 increased significantly and correlated positively with the progression of fungal keratitis. Most infiltrated cells were neutrophils and secondarily macrophages in infected cornea. The clinical scores decreased after interfering with TREM-1 and Dectin-1 expression in infected mouse corneas. Levels of Th1-type cytokines including interleukin-12 (IL-12), IL-18 and interferon-γ (IFN-γ) were decreased in the cornea, while the levels of Th2-type cytokines, including IL-4, IL-5 and IL-10, showed obvious increases.

Conclusion

TREM-1 and Dectin-1 function concurrently in the corneal innate immune response by regulating inflammatory cytokine expression in fungal keratitis. Inhibition of TREM-1 and Dectin-1 can alleviate the severity of corneal damage by downregulating the excessive inflammatory response.     

Changes in Soluble CD18 in Murine Autoimmune Arthritis and Rheumatoid Arthritis Reflect Disease Establishment and Treatment Response

Introduction

In rheumatoid arthritis (RA) immune activation and presence of autoantibodies may precede clinical onset of disease, and joint destruction can progress despite remission. However, the underlying temporal changes of such immune system abnormalities in the inflammatory response during treat-to-target strategies remain poorly understood. We have previously reported low levels of the soluble form of CD18 (sCD18) in plasma from patients with chronic RA and spondyloarthritis. Here, we study the changes of sCD18 before and during treatment of early RA and following arthritis induction in murine models of rheumatoid arthritis.

Methods

The level of sCD18 was analyzed with a time-resolved immunoflourometric assay in 1) plasma from early treatment naïve RA patients during a treat-to-target strategy (the OPERA cohort), 2) plasma from chronic RA patients, 3) serum from SKG and CIA mice following arthritis induction, and 4) supernatants from synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from 6 RA patients cultured with TNFα or adalimumab.

Results

Plasma levels of sCD18 were decreased in chronic RA patients compared with early RA patients and in early RA patients compared with healthy controls. After 12 months of treatment the levels in early RA patients were similar to healthy controls. This normalization of plasma sCD18 levels was more pronounced in patients with very early disease who achieved an early ACR response. Plasma sCD18 levels were associated with radiographic progression. Correspondingly, the serum level of sCD18 was decreased in SKG mice 6 weeks after arthritis induction compared with healthy littermates. The sCD18 levels in both SKG and CIA mice exhibited a biphasic course after arthritis induction with an initial increase above baseline followed by a decline. Shedding of CD18 from RA SFMC and RA PBMC cultures was increased by TNFα and decreased by adalimumab.

Conclusions

The plasma sCD18 levels were altered in patients with RA, in mice with autoimmune arthritis and in cell cultures treated with TNFα and adalimumab. Decreased levels of plasma sCD18 could reflect autoimmunity in transition from early to chronic disease and normalization in response to treatment could reflect autoimmunity in remission.     

Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains

Introduction

Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing A^q MHC class II molecules, and B10.DR4.Ncf1^*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401).

Methods

B10.Q and B10.DR4.Ncf1^*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA.

Results

At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1^*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the A^q-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization.

Conclusions

The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.     

Invariant NKT Cells Act as an Adjuvant to Enhance Th2 Inflammatory Response in an OVA-Induced Mouse Model of Asthma

Background

Invariant natural killer T cells (iNKT cells) are a unique subset of T lymphocytes and are considered to play an important role in the development of allergic bronchial asthma. Recently, iNKT cells were shown to play an immunoregulatory role in CD4⁺ and CD8⁺ T cell-mediated adaptive immune response. Allergen-specific Th2 inflammatory responses are an important part of the adaptive immune response in asthma. However, the regulatory functions of the Th2 inflammatory response in asthma have not been studied in detail.

Method

In this study, we have investigated the regulatory functions of iNKT cells on the Th2 inflammatory response in an ovalbumin (OVA)-induced murine model of asthma.

Results

Our results demonstrate that α-Galactosylceramide (α-GalCer) administration activated iNKT cells but could not induce the Th2 inflammatory response in wild-type (WT) mice. In the OVA-induced asthma model, α-GalCer administration and adoptive transfer of iNKT cells significantly augmented the Th2 inflammatory responses, including elevated inflammatory cell infiltration in the lung and bronchoalveolar lavage fluid (BALF); increased levels of IL-4, IL-5, and IL-13 in the BALF and splenocyte culture supernatant; and increased serum levels of OVA-specific IgE and IgG1. In addition, the Th2 inflammatory response was reduced, but not completely abrogated in CD1d^-/- mice immunized and challenged with OVA, compared with WT mice.

Conclusion

These results suggest that iNKT cells may serve as an adjuvant to enhance Th2 inflammatory response in an OVA-induced murine model of asthma.     

Thalidomide Inhibits Alternative Activation of Macrophages In Vivo and In Vitro: A Potential Mechanism of Anti-Asthmatic Effect of Thalidomide

Background

Thalidomide is known to have anti-inflammatory and immunomodulatory actions. However, the effect and the anti-asthmatic mechanism of thalidomide in the pathogenesis of asthmatic airways are not fully understood.

Objective

This study is designed to determine the effect and the potential mechanism of thalidomide in the pathogenesis of asthmatic airways using animal model of allergic asthma.

Methods

Six-week-old female BALB/C mice were sensitized with alum plus ovalbumin (OVA) and were exposed to OVA via intranasal route for 3 days for challenge. Thalidomide 200 mg/kg was given via gavage twice a day from a day before the challenge and airway hyperresponsivenss (AHR), airway inflammatory cells, and cytokines in bronchoalveolar lavage fluids (BALF) were evaluated. The expression levels of pro-inflammatory cytokines and other mediators were evaluated using ELISA, real time (RT)-qPCR, and flow cytometry. CRL-2456, alveolar macrophage cell line, was used to test the direct effect of thalidomide on the activation of macrophages in vitro.

Results

The mice with thalidomide treatment showed significantly reduced levels of allergen-induced BALF and lung inflammation, AHR, and the expression of a number of pro-inflammatory cytokines and mediators including Th2 related, IL-17 cytokines, and altered levels of allergen-specific IgG1/IgG2a. Of interesting note, thalidomide treatment significantly reduced expression levels of allergen- or Th2 cytokine-stimulated alternative activation of macrophages in vivo and in vitro.

Conclusion

These studies highlight a potential use of thalidomide in the treatment of allergic diseases including asthma. This study further identified a novel inhibitory effect of thalidomide on alternative activation of macrophages as a potential mechanism of anti-asthmatic effect of thalidomide.     

Spontaneous Immunity Against the Receptor Tyrosine Kinase ROR1 in Patients with Chronic Lymphocytic Leukemia

Background

ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL) and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients.

Materials and Methods

Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9) and healthy donors (n = 6). IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay.

Results

The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05).

Conclusion

ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy.     

A recombinant vaccine effectively induces c5a-specific neutralizing antibodies and prevents arthritis

Objectives

To develop and validate a recombinant vaccine to attenuate inflammation in arthritis by sustained neutralization of the anaphylatoxin C5a.

Methods

We constructed and expressed fusion protein of C5a and maltose binding protein. Efficacy of specific C5a neutralization was tested using the fusion protein as vaccine in three different arthritis mouse models: collagen induced arthritis (CIA), chronic relapsing CIA and collagen antibody induced arthritis (CAIA). Levels of anti-C5a antibodies and anti-collagen type II were measured by ELISA. C5a neutralization assay was done using a rat basophilic leukemia cell-line transfected with the human C5aR. Complement activity was determined using a hemolytic assay and joint morphology was assessed by histology.

Results

Vaccination of mice with MBP-C5a led to significant reduction of arthritis incidence and severity but not anti-collagen antibody synthesis. Histology of the MBP-C5a and control (MBP or PBS) vaccinated mice paws confirmed the vaccination effect. Sera from the vaccinated mice developed C5a-specific neutralizing antibodies, however C5 activation and formation of the membrane attack complex by C5b were not significantly altered.

Conclusions

Exploitation of host immune response to generate sustained C5a neutralizing antibodies without significantly compromising C5/C5b activity is a useful strategy for developing an effective vaccine for antibody mediated and C5a dependent inflammatory diseases. Further developing of such a therapeutic vaccine would be more optimal and cost effective to attenuate inflammation without affecting host immunity.     

Investigation of the role of endosomal Toll-like receptors in murine collagen-induced arthritis reveals a potential role for TLR7 in disease maintenance

Introduction

Endosomal toll-like receptors (TLRs) have recently emerged as potential contributors to the inflammation observed in human and rodent models of rheumatoid arthritis (RA). This study aims to evaluate the role of endosomal TLRs and in particular TLR7 in the murine collagen induced arthritis (CIA) model.

Methods

CIA was induced by injection of collagen in complete Freund''s adjuvant. To investigate the effect of endosomal TLRs in the CIA model, mianserin was administered daily from the day of disease onset. The specific role of TLR7 was examined by inducing CIA in TLR7-deficient mice. Disease progression was assessed by measuring clinical score, paw swelling, serum anti-collagen antibodies histological parameters, cytokine production and the percentage of T regulatory (T_reg) cells.

Results

Therapeutic administration of mianserin to arthritic animals demonstrated a highly protective effect on paw swelling and joint destruction. TLR7^-/- mice developed a mild arthritis, where the clinical score and paw swelling were significantly compromised in comparison to the control group. The amelioration of arthritis by mianserin and TLR7 deficiency both corresponded with a reduction in IL-17 responses, histological and clinical scores, and paw swelling.

Conclusions

These data highlight the potential role for endosomal TLRs in the maintenance of inflammation in RA and support the concept of a role for TLR7 in experimental arthritis models. This study also illustrates the potential benefit that may be afforded by therapeutically inhibiting the endosomal TLRs in RA.