Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model

Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by chronic joint inflammation and synovial hyperplasia leading to bone and joint destruction. The life expectancy is lowered and quality of life is decreased in RA patients. So far little is known about the actual disease initiating stimulus; however, extensive research over the last decades have shown that multiple genetic as well as environmental factors interact and trigger the onset of RA [1, 2]. The autoimmune inflammation of RA is maintained by inappropriate action of macrophages, B-cells, T-cells, and other types of cells leading to dysregulated cytokine/chemokine production. The synovial inflammation is caused by infiltration and proliferation of activated immune cells including neutrophils, macrophages, fibroblasts, mast cells, NK cells, NKT cells, T-cells as well as plasma cells [3]. Progressive joint and bone destruction is mediated through the activities of osteoclasts, chondrocytes, synovial fibroblasts and cytokine induction of destructive enzymes, chiefly matrix metalloproteinases (MMP) [4]. Current therapy mainly aims to inhibit the biological function of tumor necrosis factor-alpha (TNF-α) and lymphocyte proliferation. Due to ineffectiveness of anti-TNF-α therapy in certain patients and various side effects of methotrexate which inhibits lymphocytes proliferation, there is still the need to identify new target molecules/pathways and to develop new treatment [5]. Immunoregulatory and anti-inflammatory strategies that affect B-cell activation, T-cell activation or inhibit proinflammatory cytokines have recently shown great potential for the treatment of RA [5, 6].

Human alpha-1 antitrypsin (hAAT) is a 52 kDa serum glycoprotein, synthesized primarily in the liver. It is also expressed in other types of cells including neutrophils, monocytes, macrophages, alveolar macrophages, intestinal epithelial cells, carcinoma cells and the cornea [7‐10]. The normal serum level of hAAT is 1-2mg/ml. During inflammation, hAAT level, as an acute phase reactant, can increase 3-4 folds, suggesting an important role in responding to inflammation in the human body. Increasing evidence indicates that hAAT is immunoregulatory, anti-inflammatory and may be used for the treatment of RA. It inhibits neutrophil elastase and proteinase 3 with high efficiency, as well as cathepsin G, thrombin, trypsin and chymotrypsin with lower efficiency [11]. Most of these proteases target receptor proteins, involved in proinflammatory cytokine expression and cell signaling [12]. It also has been reported that neutrophil elastase inhibitors reduce incidence as well as severity of collagen-induced arthritis (CIA) in both rats and mice [13]. Human AAT is able to completely eliminate the acute inflammatory infiltration and connective tissue breakdown in the lung in a cigarette smoke-induced emphysema mouse model [14]. It also inhibits lipopolysaccharide (LPS)-stimulated release of TNF-α and interleukin (IL) -1β, and enhances the production of anti-inflammatory cytokine IL-10 [15‐17]. Human AAT significantly protects against the lethality induced by TNF-α or endotoxin in mice [18]. It can also induce expression of IL1-Ra in human peripheral blood mononuclear cells (PBMC's) [19] and reduces ischemia-induced apoptosis and inflammation [20]. We have recently shown, that combination therapy using doxycycline and hAAT gene therapy reduces arthritis development in mice, suggesting a therapeutic effect of hAAT in an arthritis mouse model [21].

Recombinant adeno-associated virus vectors (rAAV) have been widely used for gene therapy in animal models and human clinical trials [22], because of their unique features in safety and efficiency. It has been reported that rAAV mediated long-term and high levels of transgene expression in a wide variety of tissues, including muscle [23], lung [24], liver [25], brain [26] and eye [27]. Recently developed rAAV vectors including new serotypes of AAV, mutants AAV and double stranded AAV have provided more opportunities and challenges for their application [28‐31]. Previously, we have shown hAAT gene therapy using rAAV2 and rAAV1 vectors prevented type 1 diabetes. However, the immune response to the transgene product (hAAT) complicated the therapeutic effect [32, 33]. We have recently discovered that rAAV8 vector fail to transduce dendritic cells and induce immune tolerance to transgene product entailing rAAV8 as a promising vector used for therapeutic intervention [34].

In the present study we further investigated the feasibility of hAAT with its anti-inflammatory and immunoregulatory properties for the treatment of RA using both, protein therapy and rAAV8 mediated gene therapy.

RA is a complex systemic autoimmune disease of unknown etiology. Although recently developed biologics that target TNF-alpha have provided dramatic improvement in controlling disease activity in many patients, continued searches for more efficient and safer treatments are still needed. In the present study we showed that hAAT, administered as protein or through rAAV8 mediated gene therapy, reduced levels of serum anti-CII auto-antibodies and B-cell activating factor (BAFF) and significantly delayed arthritis development in a mouse model.

Although the exact mechanisms underlying the therapeutic effect remain to be further investigated, several mechanisms may be involved. One is through the inhibition of proinflammatory cytokine production. It is well known that various proinflammatory cytokines, including TNF-α and IL1-β, play major roles in the pathogenesis of RA [3]. Strategies targeting these cytokines have proven to be effective in treatment of RA [38]. Previous work done by Janciauskiene and her colleagues clearly demonstrated that hAAT inhibited LPS-induced TNF-α, IL-6 and IL-1β production by human monocytes [15, 16]. In addition, hAAT completely suppressed macrophage inflammatory protein-2 (MIP-2)/monocyte chemotactic protein-1 (MCP-1) gene expression in lung [39]. Human AAT also enhanced anti-inflammatory cytokine IL-10 production from monocytes [15]. As a consequence of interfering with the cytokine/chemokine network, hAAT may also inhibit polymorphonuclear leukocyte (PMN) invasion into the joint. Churg et al. demonstrated that hAAT inhibited silica-induced PMN influx into the lung and partially suppressed nuclear transcription factor B (NF-κB) translocation and increased inhibitor of NF-κB (I-⊠κB) levels in a mouse model of acute PMN mediated inflammation [39]. Thus, it is possible that the effects of hAAT on pro-inflammatory cytokine production contribute to suppression of autoimmune-mediated inflammation.

In previous studies we showed that hAAT reduced anti-insulin auto-antibodies (IAA) and attenuated cell-mediated autoimmunity [32, 33]. Consistent with these results, the present study showed that hAAT reduced the levels of anti-CII auto-antibodies and the IgG2a/IgG1 ratios of anti-CII auto-antibodies (mCII and bCII). We have observed that the effect of hAAT to suppress arthritis development is more profound in early stage of arthritis development. This is supported by the effect of hAAT on pathognomonic IgG2a antibody development at early time points (Fig.2) as well as the observation that mice eventually develop arthritis overtime. Therefore, hAAT maybe especially suitable for combination therapies. We did not observe significant effect of AAT on T-cell proliferation and cytokine production in vitro (Figure 6A and 6B) indicating that AAT may have limited direct effect on T-cells. These data also suggest that AAT may more directly affect B-cell activity. Indeed, we have shown that AAT therapies significantly reduced B-cell activating factor of the TNF-α family (BAFF) in vitro and in vivo. BAFF is an important factor that modulates B-cell tolerance and homeostasis. It has been shown that soluble BAFF is elevated in serum and target organs of CIA model [40] and BAFF antagonists suppressed arthritis development in murine models of rheumatoid arthritis [41]. In addition, increased BAFF levels were found in serum of RA patients which correlated with serum levels of rheumatoid factor [42]. The exact mechanism that AAT suppresses BAFF production remains to be elucidated.

Another possible mechanism of hAAT suppressing arthritis development is through inhibition of proteinases to prevent tissue injury and joint destruction. Human AAT is well known as a serine proteinase inhibitor (serpin). It inhibits proteinase 3, neutrophil elastase, and cathepsin G. These serine proteases are released by joint invading neutrophils following inflammatory stimuli and have shown to be involved in arthritis development [12, 13, 43, 44]. Human AAT can also reduce ischemia-induced apoptosis, inflammation, and acute phase response in the kidney [20]. We have recently shown that hAAT directly inhibits caspase 3 activity and protects islet cells from cytokine and chemically-induced apoptosis [45].

In the protein therapy studies, we used Prolastin^®, which is clinical grade hAAT purified from human plasma. Repeated IP injection of hAAT induced strong humoral immune response against hAAT in DBA/1 mice (Figure 1B), similar to what has been observed in previous studies [46, 47]. It is possible that non-specific inflammation caused by repeated IP injection is responsible for inhibition of arthritis. In order to rule out this possibility, we performed rAAV8 mediated hAAT gene therapy. AAV serotype 8 vector is unique for this purpose because it can mediate long term and high levels of transgene expression in the liver and muscle, but is not able to transduce dendritic cells and has low immunogenicity [48, 49]. Indeed, after a single injection of rAAV8-CB-hAAT vector, sustained high levels of hAAT were detected in the circulation, while no detectable levels of anti-hAAT antibodies were present (Figure 3B) in contrast to mice that received hAAT protein therapy. These results are consistent with our recent observations in NOD mice and imply new applications of rAAV8 vectors [34]. The detailed mechanism that rAAV8 vector mediates no immune response to the transgene product remains elusive. Importantly, we have observed protective effects and reductions of auto-antibodies by hAAT gene therapy. These results strongly support our hypothesis that hAAT is able to reduce inflammation in autoimmune diseases, such as RA and type 1 diabetes.

Our results from protein and gene therapy showed that hAAT is effective in delaying arthritis development in a mouse model of CIA. They indicate that hAAT has immunoregulatory and immunomodulatory effects and has great potential as a new treatment for RA. We also have shown that rAAV8 mediated gene therapy resulted in a reduced immune response to the transgene product. Future studies will focus on improvement of the therapeutic effect by optimizing the dose and timing of hAAT or rAAV8 vector delivery, and by combination therapy with other anti-arthritic drugs.