Immunohistochemical study of Krüppel-like factor 4 in the spinal cords of rats with experimental autoimmune encephalomyelitis
Introduction
Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune central nervous system (CNS) disease that is used as a model of human multiple sclerosis (MS) (Shin et al., 2012). EAE is characterized by the infiltration of autoimmune T cells and reactive gliosis (Shin et al., 2012, Shin et al., 1995). There is a general agreement that encephalitogenic T cells play a key role in the induction of EAE (Matsumoto, 2005, Swanborg, 2001), and that suppressor T cells and alternatively activated M2 macrophages are involved in its remission paralysis (Shin et al., 2012). Several transcription factors, including nuclear factor kappa B (NF-κB) (Ellrichmann et al., 2012), peroxisome proliferator-activated receptor (PPAR) (Aleshin et al., 2013) and other signaling and transcription molecules (Chen and Shannon, 2013), have been associated with the progression of EAE via the activation of inflammatory cells and glial cells, or EAE remission by counteracting the effects of pro-inflammatory mediators.
The Krüppel-like factors (KLFs), a class of zinc-finger transcription factors, play a role in diverse biological processes including proliferation, differentiation, growth, development, and survival (McConnell and Yang, 2010, Moore et al., 2011). KLF4, which is also known as gut KLF (GKLF), is expressed in the epithelia of several organs including the skin, lungs, gastrointestinal tract, heart, lymphoid tissues, and neuronal cells (Evans and Liu, 2008, Katz et al., 2002, Moore et al., 2011). KLF4 is a key cell cycle regulator and an important mediator of p53 during the response to DNA damage (McConnell and Yang, 2010, Yoon et al., 2003). Functionally, KLF4 is a bi-directional transcriptional factor that can either activate or repress transcription via various mechanisms in epithelial cells, pluripotent stem cells, and cancers, depending on the target gene (Evans and Liu, 2008).
Recently, it was reported that KLF4 regulates proliferation and IL-17 expression directly during the development of double negative, CD4−CD8−, thymocyte development reducing the severity of EAE (An et al., 2011), suggesting that KLF4 is a negative regulator of autoimmune diseases. KLF4 is also involved in the pro-inflammatory activation of classically activated M1 macrophages by increasing the expression of inducible nitric oxide synthase (iNOS) (Feinberg et al., 2005). A similar activation of KLF4 in response to lipopolysaccharide (LPS) was observed in microglial cells (Kaushik et al., 2010). In addition to the pro-inflammatory activation of macrophages, KLF4 is a critical regulator of the polarization of macrophages from pro-inflammatory M1 to anti-inflammatory M2 (Liao et al., 2011). Taken together, these findings suggest that KLF4 is involved in the suppression of T cell proliferation, and the phenotypic switching of macrophages.
Since KLF4 plays important roles in cellular signaling in T cells, macrophages, and other cell types, it is worthwhile to study the expression pattern of KLF4 during EAE, which is characterized by the infiltration of T cells and macrophages, reactive microgliosis, astrogliosis, and the phenotypic switching of macrophages (Ahn et al., 2012, Shin et al., 2012).
Section snippets
Animals
Lewis rats were obtained from OrientBio Inc. (OrientBio, Kyunggido, Korea), and were bred in our animal facility. Rats of both genders (7–8 weeks old; 160–200 g, n = 25) were used. All animal experiments were conducted in accordance with the Jeju National University Guide for the Care and Use of Laboratory Animals.
EAE induction
EAE induction and all experiments were performed as described in our previous studies (Ahn et al., 2012, Shin et al., 2012, Shin et al., 1995). Briefly, the footpads of both hind feet of
EAE histopathology
Histological examinations revealed that there were no inflammatory cells and tissue damage in the spinal cords of normal rats (Fig. 1A) and complete Freud's adjuvant (CFA)-immunized control rats (data not shown). At the early EAE disease stage (G.1, day 10 post-immunization (PI)), inflammatory cells were present in the subarachnoid space (Fig. 1B, arrows) and had infiltrated into the spinal cord parenchyma. At the peak stage of EAE (G.3, day 14 PI), a large number of inflammatory cells were
Discussion
This study provides the first data to suggest that KLF4 expression is increased significantly in the spinal cords of rats with peak stage EAE, and declines thereafter. The specific cellular localization of KLF4 in rat spinal cord tissues was also identified.
The current study identified KLF4 expression in T cells in the meninges and spinal cord parenchyma in EAE rats. This suggests that some T cells might be suppressed via the activation of KLF4 because KLF4 regulates the proliferation and
Conclusion
Collectively, the present study reveals that KLF4 is expressed constitutively in glial cells and neurons in the spinal cords of normal rats, and that the increased expression of KLF4 is associated with the regulation of the inflammation caused by autoimmune T cells in EAE. We postulate that KLF4 regulates rat EAE via its expression in multiple cell types, including T cells, macrophages, and reactive astrocytes.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (Grant number NRF-2013R1A1A3009982) and by the Ministry of Education (grant number 2014R1A1A2055965).
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