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. Author manuscript; available in PMC: 2012 Feb 24.
Published in final edited form as: Curr Mol Med. 2012 Feb 1;12(2):113–125. doi: 10.2174/156652412798889090

Kruppel-Like Factor 2 (KLF2) Regulates Monocyte Differentiation and Functions in mBSA and IL-1β-Induced Arthritis

M Das 1, J Lu 1, M Joseph 1, R Aggarwal 1, S Kanji 1, BK McMichael 2, BS Lee 2, S Agarwal 3, A Ray-Chaudhury 4, OH Iwenofu 4, P Kuppusamy 1, VJ Pompili 1, MK Jain 5,*, H Das 1,*
PMCID: PMC3286012  NIHMSID: NIHMS356879  PMID: 22280353

Abstract

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b+F4/80+Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Keywords: Arthritis, KLF2, inflammation, monocytes, osteoclasts

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic, systemic and inflammatory disease associated with progressive destructions of synovial joints, bones and ligaments [1]. A large number of female populations is affected by RA, and is significantly increasing by year. The patients become permanently work disabled during the first 2–3 years of the disease and more than half of patients show disability during the first 10 years of the disease [2]. Even though many drugs are available for the treatment of RA, they are effective only at the early stage of disease to relieve pain, the long-term effect of the current treatments is also associated with undesired side effects [3]. Thus, a better understanding on the molecular mechanisms of RA pathogenesis is warranted for development of more specific therapeutic strategies.

Various cells play an active role in pathogenesis of RA. The initiation of pathogenesis occurs presumably by antigen-dependent activation of T cells followed by subsequent recruitment of B cells and monocytes [4]. Once monocytes are recruited to the inflammatory sites, the secretion of proinflammatory effectors leads to tissue destruction and contributes significantly to the pathogenesis of RA [5, 6]. These accumulated monocytes differentiate into two different types of synovial tissue macrophages. The type A synoviocytes reside on the synovial-lining layer, while sibling interstitial macrophages are diffusely distributed within synovium. These macrophages produce a number of inflammatory mediators and interact with other surrounding cells, immune cells and extracellular matrix macromolecules [7, 8]. Those macrophages can also differentiate into osteoclasts, which are mainly responsible for subchondrial bone destruction in RA [9]. Other than differentiation and secretion of inflammatory molecules, monocytes have also been reported to be able to rescue RA-derived synovial T cells from glucocorticoid-induced apoptosis [10]. The use of methotrexate, a common treatment for RA, was reported to down-regulate the activating Fc gamma receptor I and IIa on monocytes [11]. All of these above reports indicated that monocyte is a key regulator in the pathogenesis of RA and current therapy for RA is also targeted to the monocytes. A better understanding of the regulatory mechanisms of monocytes in RA will provide new insights for better management of disease using more specific targets.

Kruppel-like factor 2 (KLF2) is a member of the zinc-finger family of transcription factors, called Kruppel-like factor. The first member of the family, known as erythroid KLF (EKLF, KLF1), is a key regulator of β-globin gene synthesis and erythropoiesis [12]. The lung KLF (LKLF, KLF2) plays an important regulatory role in hematopoietic cell biology including cell quiescence, cell proliferation, differentiation and survival. It has been shown that KLF2 regulates T cell activation and is induced during the maturation of single-positive T cells and is rapidly extinguished after single positive T cell activation [13]. Forced overexpression of KLF2 induced a quiescent T cell phenotype whereas KLF2-null T cells exhibited a spontaneously activated phenotype [14]. Recently, it has been shown that KLF2 regulates myeloid cell activation. The transcription factor, NF-κB and hypoxia negatively regulate KLF-2 expression during inflammation [15]. However, KLF2 deficient mice are embryonic lethal due to the leaky blood vessels and hemorrhages [16].

Previously, we have shown that KLF2 is a negative regulator of monocyte activation and function and is mediated by the reduction of cytokine secretions and attenuation of phagocytic capacity modulating NF-κB and AP-1 promoter activities [17]. The regulatory role of KLF2 in the proinflammatory activation of monocytes, and the wide influence of KLF2 on other inflammatory cells spurred our interest in exploring the relationship between KLF2 and RA. The current investigation was undertaken to evaluate the regulatory role of KLF2 in vivo, in the context of induced arthritis. Herein, we show that mBSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice, which inherit an increased number of inflammatory monocytes. The severity of arthritis was associated with the higher expression levels of proteins, such as HSP 60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow of arthritic KLF2 hemizygous mice. The severity is also partly due to the matured differentiation of monocytes towards osteoclasts, which mediate severe damage of cartilage and bone in arthritic mice. These data provide new insights and evidences of transcription (KLF2)-mediated regulation of arthritis via modulation of monocyte differentiation and function.

MATERIALS AND METHODS

Isolation and Culture of Monocytes from Bone Marrow

Flushing the femurs of 6–8 weeks old male KLF2 hemizygous mice (KLF2+/−) or littermate WT (KLF2+/+) control mice of C57/BL6 background isolated bone marrow (BM) cells, flushed BM cells were filtered through a 70 µm nylon cell strainer (BD Labware), and depleted red blood cells by 5-minute incubation with RBC lysis buffer (Sigma Chemical Co., St Louis, MO). Cells were then cultured in DMEM medium (Sigma) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin, at 37°C in a 5% CO2 atmosphere for two hours for plastic adherence. Adhered monocytes were either stimulated with lipopolysaccharide (LPS, 25 ng/ml) for 6 hours or without stimulus used as a control.

Real Time RT-PCR Analysis

Total RNA was isolated from BM-derived monocytes after 6 hours of either stimulation with LPS or without stimulus as a control using RNeasy Kit (Qiagen, USA). One microgram of RNA was used for the synthesis of cDNA using oligo dT (Invitrogen, USA) primer. Real-time RT-PCR was performed using one micro liter of cDNA for the inflammatory gene specific primers such as MCP-1, PAI-1 and COX2, keeping β-actin as an internal control using a standard SYBR green Taqman protocol and real-time PCR machine (Stratagene, MX3000P, Santa Clara, CA). KLF2 gene was also evaluated for the verification of hemizygocity. Relative fold-expression levels of stated genes were measured considering unstimulated BM-derived monocytes isolated from WT (KLF2+/+) mice as a basal level.

Induced Inflammation and Flow Cytometric Analyses

Six to eight weeks old male KLF2 hemizygous mice (KLF2+/−) or littermate WT (KLF2+/+) control mice of C57/BL6 background were injected i.p. with 1 ml of 5% thioglycollate (Sigma Inc). The peritoneal cavity was lavaged with 10 ml of buffer (PBS with 1.0% BSA) after 24 h of thioglycollate injection. Similarly, peripheral blood was collected in a heparinized tube by cardiac puncture from a separate set of similar experiments. After centrifugation, pellet was collected and washed with PBS and using RBC lysis buffer red blood cells were lysed. Mononuclear cells were subjected to 11-color flow cytometric analysis (markers used are follows: CD90.2-PE =T lymphocytes, B220-PE=B lymphocytes, CD49-PE=NK cells, NK1.1-PE=NK cells, Ly6G-PE=Granulocytes, F4/80-PerCP=Resident macrophage, CD11c-PerCP=Dendritic cells, IAB-PerCP= MHC-II, Mac-3-PerCP=Mononuclear phagocytes, Ly6C-FITC=Inflammatory monocyte/macrophage, CD11b-APC=Monocytes/macropage) using a FACS machine (BD Biosciences, CA) and analyzed by using FlowJo software (version 7.5, Tree Star Inc. OR).

Induction of Arthritis

Both, wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) male mice (6–8 weeks), of C57Bl/6 background were used for all in vivo induction of arthritis. Following previously published protocols [18, 19], under anesthesia, a single intra-articular (i.a.) injection of 6 µl (20 mg /ml) of mBSA (Sigma) was injected into the right knee on day 0 of the experiment, followed by daily subcutaneous (s.c.) injection of 20 µl of either saline or IL-1β (250 ng) into the ipsilateral footpad on days 0–2. Mice were sacrificed on day 8 following mBSA administrations. Contra lateral knee joints received saline which was considered as a vehicle control.

Histological Assessment of Arthritis

Arthritis was assessed by histological examination as described in [22], with some modifications. Knee joints were exposed by removal of the overlying skin and then excised. Limbs were fixed in periodate-lysine-paraformaldehyde for overnight and decalcified in 10% EDTA (BDH Chemicals, Victoria, Australia) and 7.5% polyvinylpyrolidone (Sigma) in Tris buffer (pH 6.95) for 7–10 days [25]. A transverse cut was made when the bones were fully decalcified and processed for paraffin embedding. Tissues were cut into 5 µm sections, placed on aminoalkylsilane-coated slides, and stained for routine histology with hematoxylin and eosin. Five defined pathologic features were graded for severity from 0 (normal) to 5 (severe), according to Staite et al. [17], and in a blinded manner, as follows. Soft tissue inflammation, assessed in the infrapatellar fat pads, joint capsule, and the area adjacent to the periosteal sheath, was graded according to the extent of cellular infiltration and angiogenesis. Joint space exudate was identified as leukocytes scattered discretely or in aggregates in the joint space. Synovitis (synovial hyperplasia) was defined as hyperplasia of the synovium, but did not include pannus formation. Pannus was defined as hypertrophic synovial tissue forming a tight junction with the articular surface. Evaluation of cartilage and bone damage was based on the loss of cartilage matrix, disruption and loss of cartilage surface, and the extent and depth of the subchondral bone erosion [19]. Histomorphometric analyses were performed from multiple H&E sections using two different magnifications (40× and 100×) of three animals per group by the trained pathologist, department of pathology, the Ohio State University Medical Center.

Immunohistochemistry

For identification of synovial macrophages, deparaffinized sections were incubated with specific mAb against Mac-3 and F4/80, and subsequently stained for 1 hour using a peroxidase-conjugated rabbit anti-rat IgG (Dako, Carpinteria, CA). Endogenous peroxidase was blocked with 0.3% H2O2 (30% weight/volume, Sigma) in methanol. Peroxidase activity was demonstrated by incubation with 3, 39-diaminobenzidine tetrahydrochloride (DAB, Sigma)-H2O2 solution. A peroxidase-conjugated rabbit anti-mouse IgG was incubated for 1 hour, and color was developed with DAB-H2O2 solution as described above. The color was also developed by BCIP/nitroblue tetrazolium substrate detection kit (Zymed Laboratories, South San Francisco, CA). Tartarate resistant acid phosphatase (TRAP) staining was also performed with the formalin fixed paraffin embedded tissues with the help of histopathology core facility, dept of pathology, College of Veterinary Medicine, The Ohio State University.

Micro Computed Tomography (Micro CT)

Formalin fixed femur bones was encased in a tight fitted plastic tube to prevent any motion during scanning. Femurs were scanned using a high-resolution micro computed tomography (Micro CT) scanner (SkyScan1172-D, Kontich, Belgium) at 16 µm resolution. The scanned images were reconstructed using Skyscan Nrecon software and analyzed with the CTan software (Kontich, Belgium). Analyses of tarsi, phalanges and ankle were carried out using a fixed area. A threshold was established and the same threshold value was kept constant for all samples. The analyses were performed for the bone volume and surface area in animals from all groups using the Skyscan software and following manufacturer’s protocol. The results were reported in mean ± SEM (n=6/ per group). Three-dimensional (3D) models were reconstructed using CTVOL software from SkyScan.

Osteoclast Differentiation

To determine the role of KLF2 in osteoclastic differentiation and function, bone marrow cells were collected from femurs of KLF hemizygous mice and littermate WT control mice after termination of experiments and were induced for osteoclastic differentiation in vitro. In, brief, bone marrow cells were cultured overnight at 37°C incubator with 5% CO2 in αMEM containing 10% heat inactivated fetal bovine serum in the presence of 20 ng/ml M-CSF (R & D Systems, Minneapolis, MN). Next day, non-adherent cells were collected and incubated for an additional 5–8 days in αMEM medium with 20 ng/ml M-CSF, and 50 ng/ml GST-RANKL (20). Fresh medium was replaced on every alternate day. At day 2, 4 and 6 of differentiation, the cells were stained for TRAP staining using an acid phosphatase, leukocyte, TRAP staining kit (Sigma Aldrich, USA) and was viewed and imaged with a fluorescense microscope (Nikon, Axioplan2, Carl Zeiss). After TRAP staining, TRAP-positive multinucleated cells (3 nuclei, DAPI was used for nuclear staining) were counted as osteoclast-like cells.

Osteoclast Cytoskeleton Structure and Functionality

Osteoclasts were cultured on either glass coverslips or thinly cut ivory slices. Cells were fixed at various time points of culture with 1% formaldehyde in pH 6.5 (30 minutes at room temp), stabilization buffer (127 mM NaCl, 5 mM KCl, 1.1 mM NaH2PO4, 0.4 mM KH2PO4, 2 mM MgCl2, 5.5 mM glucose, 1 mM EGTA, 20 mM Pipes), and subsequently fixed and permeabilized with 2% formaldehyde, 0.2% Triton X-100, and 0.5% deoxycholate in the same stabilization buffer. Cells were stained with F-actin specific Ab and visualized using a Zeiss 510 META laser scanning confocal microscope (Campus Microscopy and Imaging Facility, The Ohio State University). Actin ring and podosome thicknesses were determined by generating Z-stack images of randomly selected cells and these structures were measured at their thickest points [20]. Bone resorption was assessed using ivory slices and osteoclasts were gently removed with cotton swabs and washed with water. The ivory slices were then stained with hematoxylin stain for 5 minutes at room temperature and excess stain was removed by washing with water and pits were imaged with a confocal microscope.

Statistical Analysis

Values were expressed as mean±SEM and statistical analysis was performed by ANOVA. Students t-test was performed and the results were considered significant when p values were <0.05.

RESULTS

Elevated Induction of Inflammatory Genes in Bone Marrow-Derived Monocytes of KLF2 Hemizygous Mice

To investigate the in vivo regulatory effect of KLF2 gene on monocytes in the context of inflammation, KLF2 hemizygous mice and littermate WT control mice were sacrificed and bone marrow was collected from the femur bone by flushing with PBS. Bone marrow was subjected to plastic adherence for two hours. Non-adherent cells were removed and adherent cells were subjected to the LPS stimulus (25 ng/ml) for 6 hours. Non-stimulated cells were used as a control. Real time RT-PCR analysis revealed that the levels of inflammatory genes in monocytes were not significantly different in basal level when compared between WT vs hemizygous mice. However, upon stimulation, the levels of all inflammatory genes were elevated. The levels of inflammatory genes, MCP-1, COX-2 and PAI-1 were significantly higher in KLF2 hemizygous mice compared to the WT littermate controls (Fig. 1A). These data indicate that KLF2 regulates inflammatory genes of monocytes in ex vivo culture.

Fig. (1). Elevated levels of inflammatory genes and higher recruitment of inflammatory monocytes in peripheral blood and peritoneum of KLF2 hemizygous mice.

Fig. (1)

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A. Real time RT-PCR was performed to analyze the levels of inflammatory genes as stated from bone marrow-derived monocytes in the presence or absence of LPS for six hours. Four mice in each group were assessed for each gene. The level of specific gene expression in KLF2 +/+ mice without stimulus was considered as a base line of expression for a particular gene and relative fold expression was graphically presented. B & C. The peritoneal cavity was lavaged after 24 h of injection. Peripheral blood was collected by cardiac puncture from a separate set of experiments after injection of thioglycollate to the mice. Mononuclear cells were subjected to 11-color flowcytometric analysis and recruitment of Ly6C positive inflammatory cells was evaluated in peripheral blood (B) and in peritoneal lavage (C). Left panels show the representative flowcytometric analyses and right panels show the cumulative graphical data of three independent experiments.

Recruitment of Inflammatory Monocytes in Peripheral Blood and Peritoneum

In response to the inflammatory stimuli, monocytes express increased levels of proinflammatory factors. We next investigated whether subsets of monocytes were being specifically recruited into the inflammatory site in vivo. To investigate that, KLF2 hemizygous mice and littermate WT controls were injected with 1 ml of 5% thioglycollate (i.p.) to mediate inflammation. The peritoneal cavity was lavaged with 10 ml of buffer (PBS with 1.0% BSA) after 24 h of injection. Peripheral blood was collected by cardiac puncture from a separate set of experiments after (i.p.) injecting thioglycollate to the mice. Isolated mononuclear cells were subjected to 11-color flowcytometric analysis and recruitment of CD11b+F4/80+Ly6C+ positive inflammatory cells was evaluated in peripheral blood and in peritoneal lavage. Flow cytometric analysis revealed that recruitment of inflammatory CD11b+F4/80+Ly6C+ monocytes was significantly higher in KLF2 hemizygous mice (29%) compared to WT littermate controls (15%) in peripheral blood (Fig. 1B) a 2-fold increment. Similar observation was also noticed in peritoneal cavity, where recruitment of inflammatory cell in KLF2 hemizygous mice was 47% compared to WT littermate controls, 30% (Fig. 1C), a 1.5-fold increment.

Severe Arthritic Damage in Cartilage and Bones of KLF2 Hemizygous Mice

Since inflammatory cells are highly recruited in the KLF2 hemizygous mice, we sought to determine whether this higher recruitment of inflammatory cells has any effect in the development or severity of RA in mice. Both wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) mice were challenged with intra-articular injection of mBSA and footpad injection of IL-1β for the development of arthritis. Mice were evaluated for arthritis on every alternate day for 8 days and were sacrificed at termination of experiment. The ankle thickness values were as follows at day 4, WT = 0.104± 0.0007 inch vs KLF2+/− = 0.109±0.0010 inch, p<0.05; and at day 8 WT = 0.106± 0.0011 inch vs KLF2+/− = 0.114±0.0028 inch, p<0.05. Limbs were formalin fixed, decalcified, paraffin blocked and sectioned. Hematoxylin and eosin (H & E) staining was performed to the sections. Joints (ankle to toe) were evaluated for damage and inflammation. Histological analyses demonstrated that induction of arthritis was mediated by mBSA and IL-1β in both WT and KLF2 hemizygous mice. However, the severity of arthritic inflammation (inflammation surface area / total bone surface area X 100, in WT =7.40 ± 0.82 vs KLF+/− = 14.98 ± 2.80, p<0.05), cartilage erosion (damaged cartilage / total cartilage X 100, in WT =5.40 ± 0.47 vs KLF+/− = 10.11 ± 1.87, p<0.05), and bone erosion (eroded bone surface / total bone surface X 100, in WT =6.40 ± 1.63 vs KLF+/− = 19.13 ± 8.75, p<0.05) were more prominent in KLF2 hemizygous mice (Fig. 2A, upper panels) compared to the WT littermate control mice (Fig. 2A, lower panels). Clinical scores for the arthritic damage in KLF2 hemizygous mice (4.32 ± 0.185) and WT littermate control (1.76 ± 0.092) were also assessed and found to be significantly higher (p<0.001). Damage in KLF2 hemizygous mice (Fig. 2A, right, upper panel).

Fig. (2). Arthritis is more prominent in bone joints of KLF2 hemizygous mice associated with higher recruitment of monocytes.

Fig. (2)

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A. Both wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) mice were challenged with intra-articular injection of mBSA and footpad injection of rIL-1β for the development of acute arthritis. Mice were evaluated for arthritis (external measurement) at every alternate day for 8 days. Hematoxylin and eosin (H&E) staining was performed to the sections. Joints (ankle to toe) were evaluated for inflammation and damage in bone and cartilage (arrow heads). Four mice in each group and three sections from each mouse were evaluated. Cumulative graphical presentation of clinical scores of arthritic damage (n = 5/group, and five different points were considered for scoring (right panel). B. Arthritic joint tissue sections were evaluated for recruitment of immune cells (arrow heads) such as CD45 for leukocyte, Mac3 for mononuclear phagocytes and F4/80 for resident macrophage in the wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) mice after termination of experiment. Four mice in each group and three sections from each mouse were evaluated. C. TRAP staining was performed to evaluate osteoclasts in the arthritic bones from KLF2 hemizygous (KLF2+/−) and WT littermate controls (KLF2+/+) mice. Arrowheads indicate the TRAP+ osteoclasts in the bone.

Higher Recruitment of Monocytes in the Arthritic Joints of KLF2 Hemizygous Mice

We suspected that the severe damage of arthritic joints and bones of KLF2 hemizygous mice might be due to the higher number of recruited inflammatory cells. To investigate that arthritic joint tissue sections were evaluated for recruitment of immune cells such as lymphocyte, mononuclear phagocytes and resident macrophage in the wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) arthritic mice. Immuno-histochemical data reveals that there were no major differences in the recruitment of lymphocytes in both groups of animals. However, the recruitment of monocytes and macrophages is higher in KLF2 hemizygous arthritic bone tissues compared to WT littermate arthritic bone tissues (Fig. 2B). TRAP staining (Fig. 2C) revealed that statistically significant (p<0.001) higher number of TRAP positive osteoclasts were present in KLF2 hemizygous mice (11.0 ± 0.84/high power field) compared to the WT littermate controls (6.0 ± 0.53/ high power field).

Micro Computed Tomography (Micro CT) Analyses Reveal Severe Arthritis in KLF2 Hemizygous Mice

Next, we evaluated the severity of arthritic changes by micro computer tomography (micro CT). Total limb bones were harvested from wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) mice after termination of mBSA and IL-1β-induced arthritis experiment and the bones were formalin fixed. Each limb bone was enclosed in a tight fitted plastic tube and was scanned using SkyScan high-resolution micro CT scanner at a fixed resolution. The scanned images were reconstructed using Skyscan Nrecon software and analyzed with the CTan software. Scanned images (Fig. 3, left panels) and analyzed values (Fig. 3, right panels) were graphically presented from tarsi (Fig. 3A), phalanges (Fig. 3B) and ankle (Fig. 3C). Six animals from each group were analyzed. Micro CT analyses revealed that bone and cartilage damage was more severe in KLF2 hemizygous mice compared to WT control mice including tarsi, phalanges and ankle region.

Fig. (3). Micro computed tomography (micro CT) analysis of arthritic limbs.

Fig. (3)

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Total limb bones were harvested after termination of mBSA and IL-1β-induced arthritis experiment. Each bone was scanned using SkyScan high-resolution micro CT scanner at a fixed resolution. Scanned images and analyzed values were graphically presented from tarsi (A), phalanges (B) and ankle (C). Six animals from each group were analyzed and representative data was presented.

Expression Levels of Various Signaling Molecules in Bone Marrow of Mice

To investigate the molecular basis of the differential response in the development of severity in arthritis mediated by the KLF2 gene, total bone marrow was harvested from WT and KLF2 hemizygous mice after induction of arthritis or without induction of arthritis used as a control. Total protein was isolated and analyzed by using Western blots for various relevant markers such as Akt, PTEN, pPTEN, P65, p53, P38, P21, Erk1/2, VEGF, HSP90, HSP70, HSP60, HSP25/27, MMP9 and MMP13 as these molecules have shown to play a critical role in arthritis. Heat shock proteins (HSP) are strong immunogenic molecules, which can modulate autoimmune process [21]. In RA lesion, HSP25/27 was found to play a role in the regulation of osteoblasts function, however, the detailed mechanism is still unknown [22]. HSP70 and 90 were also reported to be important in the pathogenesis in RA [21]. Insufficient apoptosis of synovial macrophages, fibroblasts and lymphocytes are proposed to contribute to the damage in rheumatoid arthritis. Thus, analysis of the signaling molecules, which are important for apoptosis, such as Akt and Erk1/2 are important indicators for illustrating the mechanisms of severe inflammation and damage in KLF2 hemizygous mice [23]. Matrix metalloproteinases (MMPs) are known to play a pivotal role in the destruction of cartilage and bone in RA. It was shown that MMP-13 is important in cartilage degeneration [24]. MAPK (p38) activation is reported to be critical in the activation and induction of chronic inflammation [25]. It has been shown that administration of antisense p65 can also significantly reduce the inflammation in arthritis [26]. Our data demonstrate that the levels of signaling molecules are significantly modulated in KLF2 hemizygous mice compared to the WT control (Fig. 4). For example, expression level of Akt showed a 7-fold increase in KLF2 hemizygous mice compared to WT mice at the basal level, however, after induction of arthritis elevated levels of Akt (23.59±1.06-fold increase in WT vs 21±0.85-fold increase in KLF2 +/− mice), P65 (4.56±0.18-fold in WT vs 5.71±0.42-fold in KLF2 +/− mice) and VEGF (2.11±0.18/ 3.67±0.16-fold in WT vs 2.06±0.15/ 3.06±0.22-fold in KLF2 +/− mice) were observed. The levels of Akt, p65 or VEGF were not changed much. In contrast, expression levels of PTEN, P53 (0.57±0.01-fold in WT vs 0.39±0.03-fold in KLF2 +/− mice), P38 (0.88±0.02-fold in WT vs 0.53±0.01-fold in KLF2 +/− mice), P21 (1.34±0.41-fold in WT vs 1.02±0.34-fold in KLF2 +/− mice), Erk1/2 (0.73±0.02/ 0.90±0.30-fold in WT vs 0.53±0.04/0.69±0.04-fold in KLF2 +/− mice) were either similar or lower in the KLF2 hemizygous mice compared to WT mice in the basal level. Upon induction of arthritis, the levels remained lower in KLF2 hemizygous mice compared to WT littermate control mice. Similarly, heat shock protein molecules did not show any difference in the levels of expression at the basal level among KLF2 hemizygous and WT mice. However, upon arthritis development, molecules such as HSP90, HSP70, HSP60 were elevated in all mice, however, the levels of HSP60 and HSP90 were much higher in KLF2 hemizygous mice compared to WT mates (HSP 60 1.5±0.10-fold in WT vs 1.42±0.12-fold in KLF2 +/− mice, HSP 90 56.87±3.08-fold in WT vs 78.00±6.64-fold in KLF2 +/− mice). In contrast, HSP25/27 showed decreased level after arthritis compared to without arthritis (0.16±0.01-fold in WT vs 0.07±0.01-fold in KLF2 +/− mice). However, the level of HSP25/27 was much lower in KLF2 hemizygous mice compared to WT arthritic mice. There was very low expression in HSP70 in the basal level, however, upon induction of arthritis, elevated level was observed in both groups. There was no significant difference in the expression level of HSP70 in between KLF hemizygous mice and WT littermate controls.

Fig. (4). Levels of signaling molecules in bone marrow after induced arthritis development.

Fig. (4)

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Total bone marrow was harvested from wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) mice after termination of mBSA and IL-1β-induced arthritis or without induction of arthritis as a control. Total protein was isolated and analyzed using Western blots for various markers stated. W= KLF2+/+ and H= KLF2+/− mouse.

Among the matrix metal loproteinases tested, MMP13 showed slightly higher basal expression level in KLF2 hemizygous mice compared to WT mice and very low levels of MMP9 were observed in both groups. Upon induction of arthritis, MMP9 and MMP13 levels were remarkably increased in both groups of animals (MMP9 = 5.82±0.22-fold in WT vs 5 .05±0.22-fold in KLF2 +/− mice, MMP13 = 5.19±0.63-fold in WT vs 7.34±0.81-fold in KLF2 +/− mice); however, the level of MMP13 was much higher in KLF2 hemizygous mice compared to WT littermates and there was no significant change in the level of MMP9. These differential levels of signaling molecules might be part of the reasons KLF2 hemizygous mice showed severe arthritis and higher damage in bone and joint tissues.

Elevated Osteoclastic Differentiation of Bone Marrow-Derived Monocytes in KLF2 Hemizygous Mice

We next sought that reason behind the severeness of the bone and joint damage found in KLF2 hemizygous mice is linked with the preferential differentiation of monocytes to the osteoclasts. To investigate that, osteoclast-precursor cells were harvested from femurs of KLF2 hemizygous and WT mice after induction of arthritis. Cells were adhered to the plastic overnight using αMEM containing FBS and M-CSF. Non-adherent cells were collected and incubated for an additional 6 days in αMEM media with M-CSF, and GST-RANKL for osteoclastic differentiation. Cells were stained for TRAP at day 2, 4 and 6 of differentiation. Images revealed that osteoclastic differentiation is significantly higher in KLF2 hemizygous animals compared to WT animals at any given day of differentiation (Fig. 5). Importantly, number of nuclei is significantly higher in D6 differentiated osteoclasts derived from KLF2 hemizygous mice compared to WT mice, indicating mature nature of osteoclasts in KLF2 hemizygous mice.

Fig. (5). Osteoclastic differentiation of bone marrow-derived monocytes.

Fig. (5)

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Osteoclast precursor cells were harvested from femurs of wild type (KLF2+/+) and KLF2 hemizygous (KLF2+/−) mice after termination of mBSA and IL-1β-induced arthritis. At day 2, 4 and 6 of differentiation, the cells were stained for TRAP.

Osteoclasts Derived from KLF2 Hemizygous Mice are Functionally Aggressive

To investigate nature of the osteoclasts and their functionality, osteoclasts derived from KLF2 hemizygous mice and WT mice were stained for their cytoskeleton and their bone destroying capabilities. Actin staining of osteoclast indicates that osteoclasts derived from KLF2 hemizygous mice have prominent actin ring, which is more diffused and aggressive in nature compared to the WT mice where podosomes are more prominent indicating less aggressiveness of the osteoclasts (Fig. 6A). To test the functionality of those osteoclasts, bone resorption activity was performed on ivory slices and stained with F-actin. On ivory slices, osteoclasts also show more diffused nature of actin ring those derived from KLF2 hemizygous mice compared to those derived from WT mice, which show more podosomes (Fig. 6B, upper panel). When analyzed for pit formation by osteoclasts on ivory slices, we found that osteoclasts derived from KLF2 hemizygous mice were more functionally aggressive and generated more pits compared to osteoclasts derived from WT mice (Fig. 6B, lower panel).

Fig. (6). Osteoclast cytoskeleton structure and functionality.

Fig. (6)

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Osteoclasts were cultured after termination of mBSA and IL-1β-induced arthritis. Cells were stained with F-actin specific Ab and visualized using laser scanning confocal microscope. Images of F-actin ring and podosome were shown here generated by Z-stack images of randomly selected cells (A). B. Bone resorption activity was performed on ivory slices and stained with F-actin as mentioned above (upper panel). After termination of culture on ivory slices in separate set of experiments, osteoclasts were gently removed with cotton swabs and washed with water. Ivory slices were then stained with hematoxylin and imaged under confocal microscope for pits. Images and measured values were graphically presented (lower panel).

DISCUSSION

Rheumatoid arthritis is an inflammatory disease where multiple mechanisms of immune systems play a critical role in the development and maintenance of its pathology as well as severity of the disease. Numerous cellular mechanisms and signaling pathways drive the inflammation observed in this disease, and current evidences suggest involvement of the innate as well as the adaptive immune systems (Th-1 or Th-17 –driven, and B cells) in the development of RA pathology [27, 28]. Several lines of evidences suggest a strong influence of innate immune cells such as macrophage and synovial fibroblasts in the progression of disease as they produce large amount of proinflammatory mediators leading to the destruction to the cartilage and bone [29]. Among the proinflammatory mediators, cytokines, chemokines and matrix metal loproteinases secreted by innate immune cells are critical in the destruction of cartilage and bone in RA [29]. Other innate immune cells that may have a role in RA include neutrophil, mast cells and natural killer cells [30–32]. Although their presence in high numbers in synovial fluid and tissues was reported, and these cells are capable of secreting cytokines and chemokines and may contribute to the pathogenesis, however, their contributions are not well-defined as monocytes and synoviocytes.

Two distinct subpopulations of monocytes are present in mice; CX3CR1loGr1+ and CX3CR1hiGr1− cells. The CX3CR1loGr1+ subtype is designated as inflammatory monocytes characterized by expression of L6C/G+VLA2+CD62L+CCR2+ and can be activated by inflammation, while CX3CR1hiGr1− monocytes could be influenced only slightly by inflammation and characterized by expression of L6C/G−VLA2−CD62L− CCR2−. During inflammation, CX3CR1loGr1+ population showed a higher potential to migrate and was able to invade the peritoneum, indicates that the CX3CR1low monocytes subpopulation play an important role in inflammation based diseases compared to CX3CR1high subpopulation of monocytes [33]. Targeting this particular subpopulation of monocytes may reduce the inflammatory diseases without affecting the potential homeostatic role of CCR2−CX3CR1high monocytes in the brain or in the bones [33]. Our study has shown that KLF2 has significant impact on the population of these two subsets. Herein, we show a 2-fold increase in the inflammatory subset of monocytes in peripheral blood (CD11b+F4/80+Ly6C+ population), and over 50% increase of the same subset in the peritoneum of KLF2 hemizygous mice compared to the WT littermate control after induction of inflammation. We hypothesize that the recruitment of higher number of inflammatory monocytes in KLF2 hemizygous mice (KLF2+/−) is associated with the severity of inflammation and pathogenesis, thus KLF2 acts as a regulator for inflammation. This is also consistent with our previous finding that injection of KFL2 overexpressed monocytes reduced the carrageenan-induced inflammation in immunocompromised mice [17].

Various cytokines, chemokines and growth factors produced by monocytes/macrophage contribute to the progression and maintenance of inflammation and pathogenesis. These include interleukin (IL)-8/CXCL8, MCP-1/CCL2, MIP-1α/CCL3, IL-1, TNF-α, and IL-6. Of those IL-1 and TNF-α were considered to be the major proinflammatory cytokines and are the main targets of new developing drugs for RA [8, 34]. In the previous study, we have shown that KLF2 inhibits monocyte activation by downregulating various inflammatory cytokines such as CD40L, macrophage inflammatory protein 1(MIP) α, MIP-1β, IL-1β, IL-8, TNF-α, and macrophage chemotactic protein (MCP)-1 [17]. In our current in vivo studies, we have shown that under LPS challenge, the expression levels of MCP-1, COX2 and PAI-1 in monocytes are significantly higher in KLF2 hemizygous mice compared to wild type littermate control. These data provide an evidence of regulatory effects of KLF2, which we have shown previously in in vitro by overexpression and underexpression of KLF2 gene in monocytes and now we show that the effects persist in in vivo condition too. It is well known that tissue recruitment of inflammatory monocytes is dependent on CCR2-MCP-1 interaction. Higher level of expression of MCP-1 facilitates the recruitment of monocytes into the inflammatory site [5, 29]. Up-regulation of COX2 also contributes to the resistance to apoptosis through inhibition of p53, which in turn contributes to the persistence of RA [8, 35]. Accumulating data suggests that PAI-1 antigen and mRNA were increased significantly compared to normal tissues in synovium from RA patients as well as in experimental murine arthritic animals [36]. Other signaling molecules involved in the regulation of arthritis are Akt, p65, Hsp90 and Hsp60. The higher expression level of Akt contributes to the anti-apoptosis of monocytes, whereas higher expressions of HSP60 lead to higher activation of T cells [37]. It has been shown that in human immune response, binding of nuclear factor-κB (NF-κB) to the promoter of HSP90 can induce HSP90 expression [38] and the up-regulation of p65 may in part regulated by Akt [39]. We have previously shown that KLF2 inhibit proinflammatory gene via NF-κB and AP-1 [39]. Due to the critical role of NF-κB in arthritis [40], we propose that KLF2 can reduce RA through regulation of NF-κB via recruitment of co-activators such as PCAF (17). Thus, enhanced KLF-2 expression can lead to multiple effects, which can reduce the inflammation in RA via previously shown mechanisms [17]. Matrix-degrading enzymes, matrix metal loproteinases (MMPs) are responsible for the destruction of cartilages, bones and articular structures in RA. Besides direct degradation of cartilage and bone they are also capable of activating other protease, and thus, mediate a cascade of severe matrix degradation process [41]. Several MMPs are reportedly elevated in the synovial fluid and in the serum of RA patients [42]. Our observation of severe joint destruction in KLF2 hemizygous arthritic mice was associated with the higher level of MMP9 and MMP13 molecules in the bone marrow and is consistent with the previous reports [43, 44].

Furthermore, we show in this study that not only reduced expression of KLF2 can lead to increased recruitment of inflammatory monocytes, also the monocytes of KLF2+/− mice showed an increased potential to differentiate into mature osteoclasts in vitro. It is well established that after entry to the synovial inflammatory site monocytes differentiate to the osteoclastic lineage by fusion of cells, polarization of prokaryons and the acquisition of ruffled membranes a characteristics of osteoclasts and play a crucial role in the subchondral bone destruction in RA [45]. This osteoclastic differentiation occurs under the influence of various signaling molecules such as RANK/RANKL interactions and in the effect of other factors like integrins and M-CSF [8]. Study provided evidences that the CD11b+, CD16− peripheral blood monocytes, corresponding to CX3CR1loGr1+ population, but not CD16+ monocytes, differentiated into osteoclasts by stimulation with RANKL and M-CSF [9, 46], which indicate the capability of inflammatory monocytes to differentiate into osteoclasts efficiently. It is possible that the enhanced capability of bone resorption and more matured osteoclasts differentiation came from the inflammatory monocytes, which is much higher in numbers in the KLF2 hemizygous mice. This finding supports the hypothesis that lack of KLF2 is not only able to increase the number of inflammatory monocytes in RA, but also capable of enhancing the differentiation of monocytes towards osteoclasts.

The pathogenesis of RA involves a complex interaction between T cells, B cells, monocytes, macrophages and fibroblasts along with matrix molecules. The infiltration of CD4+ T cells into the inflammatory site recruits monocytes, macrophages and fibroblasts. And these cells express various cytokines and growth factors such as TNF-α and interleukin-1 within the synovial cavity, which lead to a damaging cascade and trigger the production of osteoclasts [34]. The discovery of the important role of KLF2 genes provides an opportunity to target gene for the treatment of RA. We provide evidences that KLF2+/− mice possess a significant increased number of the inflammatory monocytes and those monocytes display an increased capability to differentiate into mature and functional osteoclasts. Thus, together with our previous study, these results indicate that KLF2 has potential to inhibit inflammatory cytokines such as TNF-α, IL-1β, MCP-1, MIP-1α and IL-8 [17], and to attenuate the inflammatory properties of monocytes, which lead to differentiate into osteoclasts. Previous studies also showed that KLF2 regulate the migratory and regulatory activity of T cells, and differentiation and maturation of B cells [47, 48]. Herein we show that KLF2 regulates monocyte function and differentiation in the context of RA, and thus plays an important role in the pathogenesis in RA. Hence, regulating KLF2 might provide a new avenue to target RA without influencing regular immune responses.

ACKNOWLEDGEMENTS

Authors are thankful to Drs. Jack F. Bukowski (Brigham and Women’s Hospital, Harvard Medical School) and Martin Lubow (The Ohio State University Medical Center) for their critical reading and suggestions for the manuscript.

This work was supported in part by National Institutes of Health grants, K01 AR054114 (NIAMS), SBIR R44 HL092706-01 (NHLBI), R21 CA143787 (NCI), R01 HL086548 (MKJ) and The Ohio State University start-up fund.

Footnotes

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Das had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design: HD and MD. Acquisition of data: HD, MD, JL, MJ, RA, SK, AR and BM. Analysis and interpretation of data: HD, MD, BL, SA, PK, AR, VP and MKJ.

DISCLOSURE

Authors have no conflict of interest. Authors did not receive any fund from commercial sources.

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