Studies on the role of KLF4 in mouse models of autoimmune arthritis and in patients with rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes mild to severe joint inflammation of knees and fingers due to genetic factors, infection, environmental factors, epigenetic factors and immune system abnormalities. But the exact mechanism that can give the answers for pathogenesis of RA is not fully elucidated. In recent studies, auto-antibodies such as ACPA (anti-citrullinated protein antibody) from auto-reactive B cells and IL-17 from pro-inflammatory T helper cells Th17 are known to be major factors that work during RA pathogenesis. Also, fibroblast-like synoviocytes (FLS) are reported to play major roles in RA by changing into a tumor-like phenotype and mediating cartilage destruction both directly and indirectly. In the progress of pathogenesis of RA, FLS secretes proinflammatory cytokines such as IL-1β, IL-6, GM-CSF, CXCL-10, IL-8 and CCL2 and matrix metalloproteinases (MMPs). Kruppel-like factor 4 (KLF4), a member of the KLF family, plays significant roles in cell survival, proliferation, and differentiation. Especially, KLF4 is implicated in regulation of inflammation. KLF4 is known to bind to late inflammatory cytokine promoter, and promotes its expression, translocation, and release in RAW 264.7 macrophages in response to LPS stimulation. In addition, KLF4 plays a role in secretion of inflammatory cytokines by monocytes and dendritic cells. A recent study reported increased expression of KLF4 in synovial tissue from RA patients. However, its precise role in rheumatoid arthritis in different models, including mouse autoimmune disease models, remains unclear. In this study, I examined the role of KLF4 during development of autoimmune arthritis in mouse models. For the purpose, I used KLF4 knockout mice rendered by Ribonucleic acid (RNA)-guided endonuclease (RGEN). Clusters of regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CAS9) was used to delete 13 nucleotides from the KLF4 exon 2 region. The resulting frame-shift mutation in the DNA sequence led to a marked alteration in the amino acid codons and a production of non-functional KLF4 proteins. To test whether KLF4 regulates the inflammation of autoimmune arthritis in a mouse model, collagen antibody-induced arthritis (CAIA) model was performed. It was found that deletion of KLF4 reduces inflammation induced by CAIA. Inflammation factors such as IL-6, IL-1β, TNF-, and MMP13 are downregulated on KLF4 knockout mice. In addition, I assessed collagen-induced arthritis (CIA) in control mice and KLF4-overexpressing mice generated by a minicircle vector injection. Severity of CIA in mice overexpressing KLF4 was greater than that in mice injected with control vector. Finally, I verified the inflammatory roles of KLF4 in CIA by treating Kenpaullone which is used as KLF4 inhibitor. Consistent with CAIA results, severity of CIA in mice treating Kenpaullone was relieved in contrast to treating DMSO group. mRNA expression of inflammation factors was also reduced on Kenpaullone treated mice. In brief, KLF4 regulates the severity of arthritis in both of autoimmune arthritis mouse model, CIA and CAIA. <br/> Next, I examined the autoimmune-related cell subsets involved in autoimmune arthritis regulated by KLF4. Since KLF4 is reported to regulate Th17 cell differentiation directly on KLF4 conditional knockout mice, Th17 cell differentiation of KLF4 knockout mice was performed. When isolated CD4+ T cells were cultured in Th17 differentiation-inducing medium, a reduction in the number of RORγt+ T cells (Th17 cells) was observed in KLF4Rg-/- mice. These data demonstrated that KLF4 RGEN knockout mice and KLF4 conditional knockout mice specific for CD4 T cells have common traits for Th17 differentiation. Then, I focused on human/mouse FLS to discover the cellular process involved in RA pathogenesis including proliferation, apoptosis and inflammation including MMPs. In FLS, KLF4 was upregulated by lipopolysaccharide (LPS) induction and downregulated when p38 or NF-κB inhibitor was co-treated with LPS. KLF4 upregulated expression of mRNA encoding proinflammatory cytokines IL-1β and IL-6. KLF4 also regulated expression of MMP13 in the synovium. I found that blockade of KLF4 in FLS increased apoptosis and suppressed proliferation followed by downregulation of anti-apoptotic factor BCL2. These results indicate that KLF4 plays a crucial role in pathogenesis of inflammatory arthritis in vitro, by regulating apoptosis, MMP expression, and cytokine expression by FLS. In addition, as MMP13 among the MMP family has the ability to destroy collagen, the level of collagen degradation was evaluated. When treated with LPS, higher degradation of type I collagen was observed in the culture media of si-KLF4-transduced human FLS. <br/> In summary, I showed here that KLF4 regulates development of experimental arthritis in CIA and CAIA models. Also, KLF4 regulates proliferation and apoptosis of FLS, along with expression of genes encoding MMPs and proinflammatory cytokines. Thus, KLF4 might be a novel transcription factor for generating RA by modulating cellular process of FLS.<br/>