Encapsulation in hollow fibres of xenogeneic cells engineered to secrete IL-4 or IL-13 ameliorates murine collagen-induced arthritis (CIA).

A strategy of gene therapy using IL-4 or IL-13 xenogeneic transfected cells encapsulated into permeable hollow fibres (HF) was used to treat CIA. Hydrogel-based hollow fibres were obtained from AN-69 copolymer, already known for its biocompatibility and tolerance in rodents. Permeability to IL-4 and lack of cell leakage from the fibres were ascertained in vitro and in vivo. Chinese hamster ovary (CHO) fibroblasts transfected with mouse IL-4 gene were encapsulated in HF (6.25 x 105 cells/HF). IL-4 was detected in vitro in the culture supernatant of filled fibres for at least 19 days. IL-4 or IL-13 transfected CHO cells encapsulated in HF were implanted in the peritoneum of mice on days 11-13 after immunization with type II collagen. Control mice were treated with fibre containing CHO cells transfected with beta-galactosidase (betagal) gene; a positive control group consisted of mice treated by subcutaneous injection of 106 cells on days 10 and 25. Mice were monitored for signs of arthritis by observers unaware of the status of animals. Results of these experiments indicate that severity of the articular disease was significantly reduced in the groups of mice treated with CHO/IL-4 or CHO/IL-13 cells encapsulated in HF, compared with control groups receiving CHO/betagal cells encapsulated in HF. Histological analysis confirmed these data and extended them to a better inhibitory effect of encapsulated cells compared with free cells on inflammatory and destructive joint disease. Moreover, such long-term treatment with HF was well tolerated; macroscopic and histological aspects of peritoneal cavity were moderately inflammatory. Thus, our results may have important implications for clinical use of gene transfected cells as therapeutic agents in the treatment of autoimmune diseases.     

Blockade of IL-12 during the induction of collagen-induced arthritis (CIA) markedly attenuates the severity of the arthritis

The effect of blocking IL-12, a potent inducer of interferon-gamma (IFN-γ) and promoter of Th1 cell responses, during the induction phase of CIA was investigated. Arthritis was elicited in male DBA/1 mice by immunizing with type II collagen (CII) in Freund's complete adjuvant. Neutralizing anti-IL-12 antibodies were administered twice weekly from CII immunization. It was found that administration of anti-IL-12 from immunization until the onset of clinical arthritis did not lower the incidence of arthritis, but dramatically attenuated the severity of the disease, both clinically and histopathologically. This regime was associated with reduced IFN-γ levels produced by ex vivo CII-stimulated draining lymph node cells, and with diminished spontaneous ex vivo production of tumour necrosis factor (TNF), IL-6 and IL-10 by freshly isolated synovial cells. Total anti-CII antibody serum levels in these mice were lower than in the controls, but there was no change in the IgG2a/IgG1 ratio. These findings confirm that IL-12 has a major role in the induction of murine CIA and suggests that this disease is propagated, in part, by cells of the Th1 phenotype.     

Gene therapy in autoimmune disease

Recent work on gene therapies for autoimmune disease has continued to provide insight into the pathogenesis of autoimmunity. Reliable, effective and targeted gene therapy applications have been achieved by using transduced dendritic cells and antigen-specific T cells as delivery vehicles. Bioluminescence imaging has been implemented to visualize cell trafficking and homing in vivo. As a first step into human gene therapy, a phase I clinical trial for assessing the feasibility and safety of gene transfer has been completed in a group of rheumatoid arthritis patients.     

Systemic gene transfer of binding immunoglobulin protein (BiP) prevents disease progression in murine collagen-induced arthritis

Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production.     

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-α)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13

TNF-α is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-α transgene modified with the 3′ untranslated region of β-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-α-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4 weeks old. Control groups of transgenic mice were engrafted with β-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-α transgene, endogenous mouse TNF-α and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3 weeks of treatment (P < 0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-α transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.     

Reduction of monocyte-macrophage activation markers upon anti-CD4 treatment. Decreased levels of IL-1, IL-6, neopterin and soluble CD14 in patients with rheumatoid arthritis.

Anti-CD4 MoAbs have been successfully used in initial treatment trials of rheumatoid arthritis. One remarkable feature of this therapy was the early reduction of synovitis along with a decrease of the erythrocyte sedimentation rate (ESR) and the C-reactive protein (CRP). Since not only T helper cells, but also monocytes-macrophages bear the CD4 antigen, the question was raised whether the immediate effects observed may have been in part due to an influence on the mononuclear phagocyte system. Immediately after MoAb infusions, a significant reduction of the absolute peripheral blood monocyte count down to 30% (P < 0.001) was noted within the first hour of injection. In contrast to strikingly elevated levels of soluble CD4 after treatment which was indicative of T cell lysis, soluble CD14 levels did not rise, but rather decreased from previously elevated levels. Before treatment, activation of the monocyte-macrophage system had been signified by elevated serum levels of IL-1, IL-6, CRP and neopterin as well as a marked in vitro production of IL-1, tumour necrosis factor-alpha (TNF-alpha) and IL-6. Subsequent anti-CD4 treatment resulted in a rapid and significant reduction of monocyte-derived circulating cytokines and mediators concordant with a reduced capacity to produce IL-1, TNF-alpha, and IL-6 in those patients who demonstrated clinical benefits. Therefore, studies of monocyte activation markers may be useful in identifying subsequent responders to anti-CD4 therapy.     

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL-10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra-articular injection with the IL-10-expressing virus (Ad5E1mIL-10) caused substantial over-expression of IL-10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL-10 was noted for a few days, being maximal at day 1. One intra-articular injection of Ad5E1mIL-10 in the knee joints of collagen type II (CII)-immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL-10 expression by Ad5E1mIL-10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL-10 over-expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall-induced arthritis model revealed that local IL-10 over-expression markedly suppressed the production of tumour necrosis factor-alpha (TNF-alpha) and IL-1alpha, but had no significant effect on IL-1beta and IL-12 production in the inflamed synovium. These data indicate that local over-expression of IL-10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down-regulation of TNF-alpha.     

IL-4 down-regulates the surface expression of CD5 on B cells and inhibits spontaneous immunoglobulin and IgM-rheumatoid factor production in patients with rheumatoid arthritis.

There is evidence to suggest that CD5+ B cells may be associated with autoimmunity, e.g. they are increased in patients with rheumatoid arthritis (RA). In this study, we found that the expression of CD5 on RA B cells increased spontaneously, following culture for up to 4 days in vitro in the absence of T cells, supporting the idea that the CD5+ B cell possesses distinctive features. The spontaneous increase of CD5 expression was down-regulated by recombinant IL-4 (rIL-4). Other cytokines studied (rIL-1 alpha, rIL-2, rIL-5, rIL-6) did not alter CD5 expression. Studies of antibody production showed that rIL-4 could reduce spontaneous production of total IgG and IgM in non-stimulated RA T plus B cell cultures. Spontaneous production of IgM rheumatoid factor (IgM-RF), measured by a newly developed avidin-biotin complex ELISA, was also reduced by rIL-4. Furthermore, rIL-4 reduced the increase in IgM-RF production observed on stimulation with Staphylococcus aureus Cowan I (SAC) or pokeweed mitogen (PWM). Thus, IL-4 might act as a regulator of the development of abnormal B cell differentiation in patients with RA.     

Inhibition of experimental autoimmune uveoretinitis by systemic and subconjunctival adenovirus-mediated transfer of the viral IL-10 gene

Pathological ocular manifestations result from a dysregulation in the balance between proinflammatory type 1 cytokines and regulatory type 2 cytokines. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with potent immunosuppressive effects. We have examined the efficiency of viral IL-10 adenovirus (Ad-vIL-10)-mediated gene transfer on experimental autoimmune uveoretinitis (EAU) induced in mice and rats by purified retinal autoantigens, respectively, interphotoreceptor binding protein (IRBP) and S-antigen (S-Ag). B10-A mice that received a single unilateral injection of Ad-vIL-10 in the retro-orbital sinus venosus performed 1 day before immunization with IRBP in the footpads showed high levels of circulating vIL-10 in their sera and a significant reduction in pathological ocular manifestations. Lower levels of IFN-gamma and IL-2 were found in cellular supernatants from IRBP-stimulated splenic cells in these treated mice. The local effect on ocular disease of vIL-10 was neutralized completely by injection of a monoclonal anti-vIL-10 antibody, demonstrating the specificity of the treatment. To determine whether the transfer of the vIL-10 gene within the periocular tissues of the eye could prevent acute EAU, a subconjunctival injection of Ad-vIL-10 was performed in Lewis rats simultaneously with S-antigen in the footpads. This injection determined in situ vIL-10 expression with very low circulating vIL-10 and led to a significant reduction of EAU without affecting the systemic immune response. The present results suggest that Ad-mediated gene transfer resulting in systemic and local expression of vIL-10 provide a promising approach for the treatment of uveitis.     

Targeted gene therapy of autoimmune diseases: advances and prospects

Idealized gene therapy of autoimmune diseases would mean getting the right drug to the right place at the right time to affect the right mechanism of action. In other words, a specific gene therapy strategy needs to have functional, spatial and temporal specificity. Functional specificity implies targeting the cellular, molecular and/or genetic mechanisms relevant to the disease, without affecting nondiseased organs or tissues through mechanisms that cause adverse effects. Spatial specificity means the delivery of the therapeutic agent exclusively to sites and cells that are relevant to the disease. Temporal specificity is, in principle, synonymous with controlled on-demand expression of the therapeutic gene and thus represents a major safety feature. This article reviews recent advances in strategies to use gene therapy in the treatment of autoimmune diseases.     

Macrophage repolarization with targeted alginate nanoparticles containing IL-10 plasmid DNA for the treatment of experimental arthritis

In this study, we have shown for the first time the effectiveness of a non-viral gene transfection strategy to re-polarize macrophages from M1 to M2 functional sub-type for the treatment of rheumatoid arthritis (RA). An anti-inflammatory (IL-10) cytokine encoding plasmid DNA was successfully encapsulated into non-condensing alginate based nanoparticles and the surface of the nano-carriers was modified with tuftsin peptide to achieve active macrophage targeting. Enhanced localization of tuftsin-modified alginate nanoparticles was observed in the inflamed paws of arthritic rats upon intraperitoneal administration. Importantly, targeted nanoparticle treatment was successful in reprogramming macrophage phenotype balance as ∼66% of total synovial macrophages from arthritic rats treated with the IL-10 plasmid DNA loaded tuftsin/alginate nanoparticles were in the M2 state compared to ∼9% of macrophages in the M2 state from untreated arthritic rats. Treatment significantly reduced systemic and joint tissue pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) expression and prevented the progression of inflammation and joint damage as revealed by magnetic resonance imaging and histology. Treatment enabled animals to retain their mobility throughout the course of study, whereas untreated animals suffered from impaired mobility. Overall, this study demonstrates that targeted alginate nanoparticles loaded with IL-10 plasmid DNA can efficiently re-polarize macrophages from an M1 to an M2 state, offering a novel treatment paradigm for treatment of chronic inflammatory diseases.