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Krüppel-Like Factor 4: New Target for Rheumatoid Arthritis Treatment

Genome Wide Association Studies (GWAS) have allowed researcher to quickly identify numerous disease risk loci in a single experiment. Efficient follow-up studies using transgenic mice and well-characterized disease models, such as the Collagen Induced Arthritis (CIA) and Collagen Antibody Induced Arthritis (CAIA) model, are essential for determining potential pathological roles of these risk loci. To highlight how the CIA and CAIA models can be used for genetic function studies, we introduce a study analyzing the role of Krüppel-Like Factor 4 (KLF4) in experimental autoimmune arthritis.

KLF4 is part of the Sp/KLF family of transcription factors, each of which contains three zinc finger motifs within the C-terminal of the protein. Originally identified in 1996 as a gut-enriched transcription factor which arrests cell growth, KLF4 has since been isolated from numerous cell types with an array of highly context-specific effects (1). More importantly, Takahashi and Yamanaka reported that pluripotent stem cells (iPS) could be induced from mouse embryonic cells and adult fibroblasts by overexpression of KLF4 and three other transcription factors (2). KLF4 has also been implicated in numerous diseases, including cancer and colitis (3). To elucidate the role of KLF4 in rheumatoid arthritis, Choi et al. manipulated KLF4 function/expression in the CIA and CAIA mouse models of RA and observed the effect of KLF4 manipulation on inflammatory responses using several inflamamtory markers. 

Expression of KLF4 in Autoimmune Arthritis
Using Immunization Grade Bovine Type II Collagen and Freund’s adjuvants, Choi et al. induced experimental arthritis in DBA1/J mice by following Chondrex, Inc.’s recommended mouse CIA protocol. Compared to control non-arthritic control mice, joints of CIA mice had increased levels of KLF4 protein and KLF4 mRNA, along with increased mRNA of other inflammatory markers (IL-6, IL-1β, and MMP13). Fibroblast-like synoviocytes (FLS) isolated from joints of CIA mice showed high KLF4 expression via immunofluorescence analysis, leading the researchers to conclude that FLS are the major producer of KLF4 protein in CIA mouse joints.

Modifying KLF4 Expression and Function in Autoimmune Arthritis

To further elucidate the role of KLF4 in autoimmune arthritis, Chondrex, Inc.'s Arthrogen-CIA Antibody Cocktail Kit was used to induce arthritis in CRISPER/cas9 generated KLF4 knockout mice with a C57BL/6 background. Compared to CAIA mice with a functional KLF4 gene, CAIA-KLF4 knockout (KO) mice had significantly reduced arthritis scores (0-16 scale) and decreased swelling of the hindlimbs and forelimbs, as well as low cellular infiltration of mouse synovium and decreased cartilage destruction. Furthermore, CAIA-KLF4 KO mice had reduced transcription of IL-6, IL-1β, TNFα, and MMP13 mRNA, indicating a reduced inflammatory response in these mice.

The CAIA experiment implicates KLF4 in inflammatory responses during experimental autoimmune arthritis. To corroborate this finding, the researchers turned again to the CIA model and conducted two more experiments in which they manipulated KLF4 function: 

1)    Using minicircle vectors to induce overexpression of KLF4 at the time of CIA immunizations of DBA1/J mice 

2)    Using Kenpaullone (a KLF4 inhibitor) to downregulate KLF4 expression at the time of CIA immunization of DBA1/J mice 

CIA mice with induced overexpression of KLF4 had increased arthritis severity in the hindlimbs and forelimbs, and increased transcription of IL-6 and MMP13 mRNA, as compared to control mice. In addition to increases in these inflammatory markers, cellular infiltration of mouse synovium and cartilage was also increased by KLF4 overexpression in CIA mice.  Consistent with the earlier CAIA experiment, Kenpaullone treatment resulted in reduced arthritis scores compared to CIA mice treated with DMSO. Additionally, Kenpaullone treated CIA mice had decreased transcription of IL-6, IL-1β, MMP13, and MMP9 mRNA.

In vitro Studies Elucidate Role of KLF4 in Cellular Immune Responses

The results of these CIA and CAIA experiments indicate that KLF4 plays a role in the progression of experimental autoimmune arthritis. However, the mechanism underlying the modulatory role of KLF4 could not be deduced with these methods alone. With previous studies finding that KLF4 can directly regulate Th17 cell differentiation (5) and the reported role of Th17 in rheumatoid arthritis progression (6), Choi et al. sought to evaluate how KLF4 affects Th17 cell populations in experimental autoimmune arthritis models. 

To do so, CD4+ T cells were isolated from CAIA-KLF4 KO mice, CAIA control mice, and normal control mice and the differentiation of Th17 cells and expression of IL-17 mRNA was measured. CD4+ T cells from CAIA-KLF4 KO mice had decreased Th17 cell numbers and decreased IL-17A mRNA transcripts as compared to CAIA control mice (although both were higher than the normal control group). 

Since KLF4 was found to be released primarily from FLS, Choi et al. compared FLS proliferation from CAIA-KLF4 KO mice and CAIA control mice. FLS from CAIA-KLF4 KO mice showed reduced proliferation and increased apoptosis compared to FLS isolated from normal CAIA mice. This increase in apoptosis was supported by reduced mRNA expression of BCL2, an anti-apoptotic factor, in FLS from CAIA-KLF4 KO mice.

To evaluate the applicability of these findings to human disease, FLS were isolated from RA patients and osteoarthritis (OA) patients. FLS from RA patients showed higher expression of KLF4 than FLS from OA patients. Furthermore, Choi et al. knockdowned KLF4 expression using a lentiviral vector to study how KLF4 functions in human FLS. KLF4 knockdown resulted in decreased proliferation and increased apoptosis of FLS, as well as decreased expression of BCL2, IL-6, MMP13, and IFN-γ mRNA.

KLF4 Modulates Autoimmune Arthritis Through FLS and Th17 Cells

Choi et al. showed that inhibition/knockout of KLF4 alleviated the severity and progression of experimental autoimmune arthritis, while also decreasing inflammatory markers in mouse joint tissue. Moreover, KLF4 knockdown of FLS isolated from human RA patients also resulted in decreased inflammatory markers, suggesting that KLF4 could be a promising new therapeutic target for patients whose symptoms are not well managed with current RA treatments. Given the heterogeneity of RA etiology and the speed with which new disease risk loci can be identified, efficient evaluation of genetic factors for therapeutic value is vital. Well characterized disease models, such as the CIA and CAIA models, and transgenic mice are therefore essential tools for assessing the efficacy of novel RA treatments targeting GWAS identified genetic factors.



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  6. J. van Hamburg et al., Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin‐17A production. Arthritis & Rheumatism 63  73-83 (2010).