A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis

Rheumatoid arthritis (RA) is marked by de-regulated recruitment, activation, and retention of inflammatory white blood cells in affected joints [1]. Subsequent autoantibody production, release of cytokines, and cell-cell contacts may perpetuate inflammation and lead to joint destruction through activation of stromal fibroblast-like synoviocytes (FLSs) and osteoclasts [2]. Many of the cellular processes required for perpetuation of inflammation and joint destruction in RA are regulated by Rac GTPases, members of the Rho-like family of small GTPase signaling proteins [3].

Rac1 is ubiquitously expressed in mammalian tissues, whereas expression of Rac2 is limited to cells of hematopoietic lineage [4, 5]. Rac GTPases are activated by a broad array of extracellular stimuli relevant to RA, including chemokines, lymphocyte antigen receptor ligation, inflammatory cytokines, and cell-cell adhesion [6‐11]. Following activation, Rac proteins initiate multiple signaling pathways that regulate cytoskeletal rearrangements, kinase cascades needed for gene transcription, and assembly of the NADPH oxidase [6, 12]. Transfection of active and dominant-negative mutants of Rac1 as well as genetic studies have demonstrated that lymphocytes and neutrophils require Rac1 signaling for efficient polarized chemotactic responses and trafficking in vivo [13‐19]. Although macrophages do not require Rac1 and Rac2 function for chemotactic responses, macrophage invasion of tissue is dependent upon Rac1 and Rac2 [20]. Rac signaling is also important for productive interactions between lymphocytes and antigen-presenting cells (APCs). After antigen recognition by T cells, ezrin-radixin-moesin proteins are dephosphorylated through a Rac1-dependent pathway, favoring relaxation of the cytoskeleton and subsequently promoting T cell-APC conjugate formation [21]. Reciprocally, Rac activity in dendritic cells (DCs) is required for effective antigen presentation to T cells and subsequent T-cell priming [22]. Antigen receptor-dependent activation of Rac signaling also stimulates activation of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and nuclear factor-kappa-B signaling pathways important for lymphocyte activation, proliferation, and survival [7‐9]. Many of these downstream signaling pathways are now being explored as potential therapeutic targets in RA [23]. Rac proteins also serve additional important functions in cells of myeloid lineage which contribute to inflammation and joint destruction in RA. Oxidative bursts of macrophages and neutrophils rely upon Rac1-dependent assembly of the NADPH oxidase machinery [12]. Additionally, in vitro studies of osteoclasts transfected with plasmid encoding dominant-negative Rac and in vivo studies in Rac-deficient mice have identified essential but redundant roles for Rac1 and Rac2 proteins in osteoclastogenesis, osteoclast motility, and bone resorption [24, 25].

Together, these studies indicate that therapeutic strategies targeting Rac1 function may be of clinical benefit in RA. However, preclinical assessment of Rac1 inhibition has been hampered by a lack of compounds specifically targeting Rac1 and by limited analyses of Rac1 in animal models of arthritis, a consequence of early findings demonstrating that genetic deletion of Rac1 in mice results in early embryonic lethality [26]. NSC23766, a pharmacological compound that inhibits Rac GTPases via targeting of the activating guanine nucleotide exchange factors Tiam1 and Trio, suppresses RA FLS proliferation and invasiveness in vitro, effects mimicked by siRNA (short interfering RNA) silencing of Rac1 expression in these cells [27]. This may indicate that specific inhibition of Rac1 may be therapeutically beneficial in RA. However, mice in which Rac1 has been conditionally deleted in mature neutrophils and macrophages on a Rac2-deficient background show a complex phenotype in a Chlamydia-induced infection model of arthritis [28]. In these animals, Rac1 has a bimodal effect on disease progression. In the acute phase, Rac1 deficiency delays recruitment and activation of inflammatory neutrophils in the joint, whereas in the chronic phase, disease is exacerbated due to an inability of neutrophils to clear the pathogen. In this study, we targeted Rac1 in mice with collagen-induced arthritis (CIA), using a Rac1-specific cell-permeable carboxy-terminal inhibitory peptide that we have previously shown to block Rac1 function in human lymphocytes, endothelial cells, and epithelial cells [11, 29, 30].

In this report, we provide evidence that the inhibitory Rac1 carboxy-terminal peptide suppresses anti-collagen antibody production associated with reduced paw swelling in mice with CIA. We have found that the administration of Rac1 peptide in vivo, either as an early treatment or as treatment of mice with more chronic arthritis, results in a significant reduction of circulating levels of anti-bCII IgG1 and IgG2a or IgG2a and IgG2b antibodies, respectively, depending on the stage of the disease. In both early and chronic arthritis, there was an effect on IgG2a levels. In many murine in vivo model systems, IgG2a and IgG2b antibodies display greater pro-inflammatory properties than IgG1 and IgG3 [37]. Initial evidence presented here indicates that Rac1 inhibitory peptide exerts its protective effect at least in part through interference with T-cell receptor (TCR)/CD28 signals needed for T-cell expression of inflammatory cytokines as well as for expression of costimulatory proteins required for B-cell activation.

It is unlikely that the suppressive effect of Rac1 peptide on paw swelling and anti-collagen antibody production observed in our studies is due to inhibitory effects on immune cell trafficking. First, we noted no significant differences in cellular infiltration of the synovium in Ctrl and Rac1 peptide-treated mice. Second, the Rac1 peptide did not influence the numbers or percentages of B and T lymphocytes in blood or spleen during CIA. These results are consistent with genetic studies indicating that Rac1 and Rac2 play redundant roles in lymphocyte and myeloid cell trafficking, reflecting the high specificity of the Rac1 peptide for interfering with signaling from Rac1 but not other Rho family GTPases [29]. Murine neutrophils express both Rac1 and Rac2, and genetic deletion of each GTPase has revealed their important but redundant contributions to neutrophil chemotactic responses in vitro and in vivo [18, 19]. Rac1-deficient murine neutrophils retain chemokinetic responses but are unable to orient and migrate toward chemokine gradients. In contrast, Rac2 is required for efficient neutrophil migration [19]. Although similar direct comparative analyses have not been performed on B and T cells in these mice, initial studies indicate that lymphocyte trafficking is regulated primarily by Rac2 [15].

Surprisingly, we observed little if any effect of Rac1 peptide on cartilage and joint destruction in murine arthritis, although a significant decrease in paw swelling was observed in mice when treated at the onset of disease, and a reproducible trend toward reduced paw swelling was noted in mice treated with Rac1 peptide at a more chronic phase of disease. Decreases in paw swelling after treatment with Rac1 peptide which we observed might be due to effects on the formation of edema. Experiments conducted in vitro have demonstrated that exposure of human endothelial cells to reactive oxygen species or engagement of the integrin ligand VCAM (vascular cell adhesion molecule) leads to Rac1-dependent loss of cadherin-mediated endothelial cell-cell adhesion. In the presence of the Rac1 peptide, endothelial cell-cell adhesion is maintained (data not shown) [11, 38]. A role for Rac1 in maintaining vascular endothelial integrity in vivo is also indirectly suggested in studies of c-Jun knockout mice, in which inhibition of c-Jun, a downstream target of Rac signaling, suppresses edema, paw swelling, and inflammation in an experimental model of arthritis [39]. Lack of effect of Rac1 peptide on joint destruction in CIA may reflect a redundant role for Rac2 in supporting osteoclastogenesis [24, 25].

Rac1 inhibitory peptide may inhibit anti-bCII antibody production in CIA via one or more mutually non-exclusive mechanisms. It is unlikely that Rac1 inhibition directly interferes with B-cell activation as Rac1 and Rac2 make critically redundant contributions to the transduction of B-cell receptor signals required for both survival and efficient cell cycle entry [9, 40]. Rac2 deletion in mice results in decreased B-cell maturation and T cell-independent antigen responses [14, 15]. In contrast, conditional deletion of Rac1 in the B-cell compartment has no observable effect on B-cell maturation or function unless Rac2 is simultaneously deleted [40]. Similarly redundant but critical roles for Rac1 and Rac2 are observed during T-cell maturation and proliferative responses to TCR triggering [17]. Consistent with this, we observed no effect of Rac1 inhibitory peptide on TCR/CD28-induced T-cell IL-2 production ex vivo. However, we did note that Rac1 inhibition decreased the ability of T cells to produce the inflammatory cytokines TNFα, IFNγ, and IL-17 in response to TCR/CD28 triggering. These findings bear similarities with a recent report that thionamides interfere with human T-cell TNFα and IFNγ production via inhibition of Rac1 activation [41]. Interference with anti-bCII production in CIA might also be attributable to direct suppression of TCR/CD28 signaling as we observe that ICOS and CD154 expression is compromised in T cells treated with Rac1 peptide. Sequential upregulation of these two proteins plays a requisite role in providing costimulatory signals required for efficient B-cell antibody production and isotype class switching [42, 43]. Blockade of either ICOS or CD154 interaction with their respective ligands is protective in murine models of arthritis and suppresses autoantibody production [33‐36]. In RA, synovial T-cell expression of CD154 can also promote antigen-independent activation of macrophages and stromal cells [44‐46]. Finally, Rac1 inhibition may interfere with anti-bCII antibody production via effects on DCs. Although this possibility is not addressed in our studies, Rac1-deficient CD8α⁺ DCs, but not Rac2-deficient DCs, fail to migrate to secondary lymphoid organs and cannot establish stable contacts with naïve T cells [22]. DCs from mindin^-/- mice, which have reduced expression of Rac1 and Rac2, also have impaired priming capacity due to inefficient engagement with T cells, in turn leading to defective humoral responses to T cell-dependent antigens in these mice [47].

Our studies suggest that Rac1 peptide suppresses T-cell activation and subsequent generation of inflammatory cytokines and costimulatory proteins needed for anti-collagen antibody production in CIA. Future studies will be required not only to further define specific effects on lymphocytes and lymphocyte interactions with APCs but also to consider the potential contributions of Rac2, independently or in conjunction with Rac1, to pathology in CIA. Structure-based studies have recently led to the development of small-molecular-weight compounds that can specifically prevent interaction of Rac1 and Rac2 with activating guanine nucleotide exchange factors [27, 48]. These compounds can block RA FLS growth and matrix invasion in vitro, although their efficacy in the treatment of arthritis in vivo remains to be established.

We demonstrate that a cell-permeable inhibitory Rac1 carboxy-terminal peptide can reduce paw swelling and antibody production during murine experimental arthritis. These protective effects may be attributed at least in part to suppression of T-cell inflammatory cytokine and costimulatory protein expression. Conceivably, Rac1 peptide treatment could augment the pharmacological activity toward B-lineage cells of other immunosuppressive therapies, like rituximab or atacicept, which may theoretically increase therapeutic activity. An alternative approach that might perhaps result in a beneficial effect on both clinical signs and symptoms as well as joint destruction could be the suppression of Rac signaling in RA by compounds targeting both Rac1 and Rac2 signaling. The present study supports the rationale for future studies exploring these approaches.