Expression and Regulation of the Cell Surface Proteins CD47 and SIRPα in Resident Periodontal Cells

Expression and Regulation of the Cell Surface Proteins CD47 and SIRPα in Resident Periodontal Cells

Student: Sara Engman

Tutors: Post-doc Cecilia Koskinen Holm, DDS, PhD Associate professor Pernilla Lundberg, DDS, PhD Department of Molecular Periodontology

901 87 Umeå University

ABSTRACT

Periodontal disease is an inflammatory disorder affecting the supporting tissues of the tooth. The inflammation triggers a destruction of the connective- and bone tissue surrounding the tooth, a process that is not fully elucidated. It is known that

periodontitis shares features with other inflammatory disease like Crohn's disease and rheumatoid arthritis. The cell surface proteins signal regulatory protein alpha (SIRPα) and cluster of differentiation 47 (CD47) are important for the progression of the inflammation in rheumatoid arthritis and Crohn's disease. It has also been shown that lack of SIRPα or CD47 render in reduced number of osteoclast. The aim of this study was to investigate if cells from the periodontium (human gingival fibroblasts) from periodontally healthy individuals express SIRPα and CD47 and if the expression of these membrane proteins is regulated under inflammatory conditions.

We demonstrate, by using quantitative rt-qPCR, that human gingival fibroblasts express both CD47 and SIRPα mRNA. The expression of SIRPα was positively regulated (2- fold) by the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1-beta (IL-1β). TNF-α caused a 2-fold up-regulation of CD47 in human gingival fibroblasts. Neither CD47 nor SIRPα were time-dependently regulated by the two pro-inflammatory cytokines.

We here conclude that SIRPα and CD47 gene expression are up-regulated in human

gingival fibroblasts cultured under inflammatory conditions. These findings indicate

that SIRPα and CD47 may play a role in periodontal disease.

INTRODUCTION

The periodontium consists of gingiva, periodontal ligament and alveolar bone. Its function is to support the tooth and enable attachment of the tooth to the jawbone. The most superficial part is the gingiva, covering the alveolar bone and the cervical portion of the teeth. The gingiva consists of epithelium and the underlying connective tissue.

The most common cell found in the connective tissue is the gingival fibroblast, a spindle-shaped cell known to produce different types of collagen fibers and to

participate in synthesis of the extracellular matrix. Other cells present in the connective tissue are mast cells, macrophages and few inflammatory cells. The periodontal

ligament is the cellular connective tissue situated between the root cementum and the socket of the alveolar bone, and it attaches the tooth to the alveolar bone. The main component of the periodontal ligament is collagen fibers, also known as Sharpey´s fibers, produced by fibroblasts (periodontal ligament cells) (Lindhe and P. Lang, 2015).

Periodontal disease is a chronic inflammatory disorder affecting the periodontium resulting in tissue degradation. Commensal microorganisms that colonise the tooth surface trigger an immune response and initiate the disease. The early phase of disease is known as gingivitis; inflammation in the gum (gingiva), and this state is fully reversible by proper removal of the biofilm covering the tooth. In some individuals, untreated gingivitis proceeds to periodontitis, inflammation in the periodontium, and consequently jaw bone destrucion (Lindhe and P. Lang, 2015). If left untreated, periodontitis will eventually result in tooth loss. Epidemiological studies show that more severe forms of periodontitis affect approximately 10 % of the population (Hugoson et al., 2008).

During the periodontal inflammatory process different cytokines, chemokines and other inflammatory mediators like prostaglandins are released, affecting and controlling the progression of the disease (Cekici et al., 2014). The pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1-beta (IL-1β) are well known to be involved in periodontal disease and are secreted from leukocytes and resident

periodontal cells (i.e. gingival fibroblasts, periodontal ligament fibroblasts and vascular

endothelial cells). These cytokines contribute to the destruction of both connective- and

bone tissue resulting in loss of tooth attachment (Graves and Cochran, 2003)

Both TNF-α and IL-1β enhance the expression of other cytokines in periodontal lesions (e.g.

human gingival fibroblasts), amongst those are interleukin-6 (IL-6) and interleukin-34 (Palmqvist et al., 2008; Bostrom and Lundberg, 2013). IL-6 and IL-34 have been shown to stimulate the differentiation of osteoclasts and may therefore also contribute to bone resorption in periodontal disease (Ohsaki et al., 1992). TNF-α, IL-1β and IL-6 are also found in gingival crevicular fluid from pathological gingival pockets (Rossomondo et al.,1990; Masada et al., 1990; Geivelis et al.,1993). Recent findings have demonstrated that human gingival fibroblasts produce the chemokines monocyte chemoattractant protein-1 (MCP-1) and eotaxin, and the production is regulated by pro-inflammatory cytokines (Bostrom et al., 2015).

The inflammatory process in periodontitis shares many features with other

inflammatory diseases, among them rheumatoid arthritis. Patients with rheumatoid arthritis have multiple inflamed joints, which over time manifests in joint tissue destruction. Studies of rheumatoid arthritis have suggested that the disease is a

multifactorial, relapsing disease, leading to an increased systemic load, which are also seen in periodontal disease (Kallberg et al., 2007; Vaudo et al., 2004; Kalburgi et al., 2014; Cullinan et al., 2001). It has also been suggested that patients with rheumatoid arthritis have more severe periodontitis (reviewed in Fuggle et al., 2016). Crohn's disease, displayed by inflammation in the intestines, is also a relapsing inflammatory disease and like periodontitis, caused by the commensal bacterial flora and dependent of an individual susceptibility (Khor et al., 2011).

The cell surface proteins cluster of differentiation 47 (CD47) and signal regulatory protein alpha (SIRPα) are known to contribute to progression of inflammatory diseases.

Both proteins belong to the immunoglobulin-like super family and all cell types express

CD47, whereas SIRPα is mostly expressed by myeloid cells (Barclay and Van den

Berg, 2014). The interaction between CD47 and SIRPα was initially discovered as an

important ”marker of self” for the host immune system to recognize endogenous from

non-endogenous molecules (Oldenborg et al., 2000), as well as controlling phagocytosis

(Okazawa et al., 2005) and formation of macrophages (Han et al., 2000). Moreover,

CD47 and SIRPα play an important role in leukocyte-cell migration (Stenberg et al., 2014) as well as in the differentiation of bone resorbing osteoclasts (Lundberg et al., 2007; Koskinen et al., 2013).

Interestingly in rheumatoid arthritis, SIRPα mutant mice (lacking the intracellular signalling domain) have a reduced susceptibility to arthritis induced by type-II-collagen (Okuzawa et al., 2008). Studies of experimental colitis in mice and Crohn's disease in humans shows an accumulation of SIRPα-positive dendritic cells (DCs) in the inflamed intestine and that the different DCs are able to secrete pro-inflammatory cytokines (e.g.

TNF-α, IL-6 and IL-1β). Lack of CD47 in mice with colitis resulted in a less severe form of colitis. Moreover, the SIRPα-positive DCs in tissues from patients with Crohn's disease was targeted by CD47. This indicates a considerably role of CD47 and SIRPα in animal models and intestinal inflammation in humans (Baba et al., 2013;

Fortin et al., 2009). Therefore, it is of interest to investigate if CD47 and SIRPα may be of importance in periodontal disease.

The aim of this study was to analyse if SIRPα and CD47 are present in gingival

fibroblasts and if the expression of each membrane protein is regulated by inflammatory cytokines seen in periodontitis.

MATERIALS AND METHODS

Collection of human gingival fibroblasts

Gingival biopsies were collected from six periodontally healthy individuals (3 women

and 3 males). The biopsies were surgically removed under anaesthesia (Xylocain

Adrenalin 20 mg/mL + 12.5 µg/mL injection fluid), from the premolar region of the

mandible. The area was probed and visually examined to ensure no signs of clinical

inflammation. Each biopsy measured 4x4 mm and was removed with a scalpel to bone

contact. The biopsies were coded and kept in sodium chloride water. After collection,

the biopsies were dissected into smaller pieces and seeded in 25cm

culture bottle

(Nunclon™ Delta Surface/Thermo Fisher Scientific, USA) (Figure 1A), in alpha

minimum essential medium (α-MEM), 10 % fetal calf serum (FCS) (GIBCO-BRL/Life

Technologies, Paisley, UK), 10 x antibiotics (Sigma Aldrich, USA) and L-glutamine (Life Technology, USA), in 5 % CO

at 38C°.

Human gingival fibroblast cell culture

The cells were seeded in a concentration of 8

-10

cells/well in 24-wells culturing plates (Nunclon™ Delta Surface/Thermo Fisher Scientific, USA) with α-MEM, 10 % fetal calf serum, antibiotics and L-glutamine and stimulated with IL-1β (100 pg/mL) or TNF- α (50 ng/mL) (R&D Systems, USA) for 6-48 hours in 5 % CO

at 38C°.

RNA isolation and cDNA synthesis

mRNA from human gingival fibroblasts was isolated using RNAqueous

-Micro Kit (Ambion, Austin, Texas) according to the manufacturer´s instructions. The mRNA was reversed transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).

Gene expression analysis

The gene expression of IL-6, SIRPα and CD47 (Thermo Fisher Scientific, USA) was analysed with quantitative real-time RT-qPCR technique using Taq-man (API PRISM 7900HT Sequence Detection System). By simultaneous analysis of the housekeeping gene ribosomal protein L13a (RPL13a) (Invitrogen, USA) (Applied Biosystems, Warrington, UK), the amplification diversity of each gene was controlled.

Statistical analysis

The statistical analyses were performed in GraphPad Prism 7.0a using one-way analysis (ANOVA) with Levene’s homogenicity test, and post-hoc Turkey’s test. Student’s t-test was used when appropriate. All experiments were performed at least twice with

comparable results and all data are represented as the means ± SEM/SD. The significance levels were set to *p<0.05, **p<0.01 and ***p<0.001.

Collecting literature

Relevant literature was collected from PubMed using the search terms/MeSH terms:

periodontitis, gingivitis, inflammation, macrophages, dendritic cells, fibroblasts,

membrane proteins, signal regulatory protein alpha, CD47, interleukin-6, pro- inflammatory cytokines, tumor necrosis factor alpha, interleukin-1-beta, rheumatoid arthritis, colitis and Crohn's disease. Some of the literature was collected from references in previously used articles and articles selected by the supervisor.

Ethical consideration

The study was approved by the Regional Research Ethics Committee at the Department of Odontology, Umeå University. All participants gave their informed consent. Possible complications from collecting the biopsies for gingival fibroblasts were considered to be modest to the possible benefit of widening the knowledge about periodontal inflammation.

RESULTS

Pro-inflammatory cytokines stimulate SIRPα and CD47 gene expression in human gingival fibroblasts

Gingival fibroblasts from six periodontally healthy individuals were cultured for 24 hours (Figure 1A), and the gingival fibroblasts from all individuals were shown to exhibit a basal mRNA expression of both SIRPα and CD47 (Figure 1B and C, control groups). Inflammatory conditions were induced in the cell cultures from each

individual, by addition of the pro-inflammatory cytokines TNF-α (50 ng/mL) or IL- 1β (100 pg/mL). Both TNF-α and IL-1β caused an approximate 2-fold increase of SIRPα mRNA expression (ranging between 1.7-2.1-fold and 1.8-2.3-fold,

respectively) (p<0.05 and p<0.01) (Figure 1B). The mRNA expression of CD47 was significantly increased by addition of TNF-α (1.2-2.1-fold) (p<0.001). IL-1β did not increase the mRNA expression of CD47 (Figure 1C). IL-6 was used as a positive control for IL-1β and ΤΝF-α’s ability to alter the gingival fibroblast phenotype. The results showed that the mRNA expression of IL-6 was significantly increased when stimulated with TNF-α (10.6-14.7-fold) and IL-1β (15.4-29.8-fold) compared to unstimulated control (p<0.001) (Figure 1D).

Gingival fibroblasts from one representative individual of the six previously analysed

individuals were selected for proceeding analysis described below.

Time-independent effect of pro-inflammatory cytokine regulation of the SIRPα and CD47 gene expression, in human gingival fibroblasts

Human gingival fibroblasts were cultured in the absence and presence of IL-1β (100 pg/mL) and TNF-α (50 ng/mL) for 6, 12, 24 and 48 hours to investigate if the mRNA expression of SIRPα and CD47 was time-dependently regulated. No time dependent regulation of the analysed genes was seen after stimulation with IL-1β (100 pg/mL) for 6 to 48 hours of culture (Figure 2A). Neither did TNF-α (50 ng/mL) time-dependently regulate the mRNA expression of SIRPα and CD47 (Figure 2B), after 6 to 48 hours of culture. Moreover, the mRNA expression of IL-6 was not regulated over time by IL- 1β. However, TNF-α up-regulated IL-6 over time (Figure 2A, B).

SIRPα expression in human gingival fibroblasts is concentration dependently regulated by IL-1β

The gingival fibroblasts were stimulated with IL-1β (concentrations of 10, 30, 100 and 300 pg/mL) for 24 hours in order to investigate if the mRNA expression correlated with the concentration of IL-1β. Results show a small but significant concentration

dependent increase in mRNA expression of SIRPα compared to control. 30 pg/mL of IL-1β was the lowest concentration to give a significant mRNA increase (1.3-fold, p<0.05) and 300 pg/mL gave the highest mRNA expression of SIRPα (1.7-fold) (Figure 3A). The effect of IL-1β on CD47 mRNA expression was not concentration dependent (Figure 3B). The positive control cytokine, IL-6 was significantly increased in a concentration dependent manner, when IL-1β was added to the cultures 10 pg/mL (p<0.05) to 300 pg/mL of IL-1β (Figure 3C).

SIRPα expression in human gingival fibroblasts is concentration dependently regulated by TNF-α

The gingival fibroblasts were stimulated with TNF-α (concentrations of 10, 25, 50 and 100 ng/mL) for 24 hours, followed by analysis of mRNA expression of SIRPα and CD47. Results show a significant concentration dependent increase of SIRPα mRNA expression compared to un-stimulated cells. TNF-α 10 ng/mL was the lowest

concentration to induce a significant increase in SIRPα expression (2.5-fold, p<0.001),

and the maximum mRNA expression was reached at 50 ng/mL (3.2-fold) (Figure 4A).

The mRNA expression of CD47 was also positively regulated in a concentration

dependent manner, although not to the same extent as SIRPα. TNF-α 10 ng/mL was the lowest concentration to increase the expression of CD47 1.3-fold (p<0.01) compared to control, and the highest mRNA expression was detected when stimulating with TNF-α 25 ng/mL (1.5-fold) (Figure 4B). The positive control cytokine IL-6 was also

concentration dependently up-regulated by TNF-α, reaching the first significant effect at 10 ng/mL (p<0.001) and highest mRNA expression effect at TNF-α 50 ng/mL (Figure 4C).

DISCUSSION

In the present study we analysed if human gingival fibroblasts express mRNA for the cell surface proteins CD47 and SIRPα and if pro-inflammatory cytokines can effect the expression.

CD47 is known to be ubiquitously expressed whereas SIRPα is foremost expressed by myeloid cells (Barclay and Van den Berg , 2014). Our findings demonstrate, as

expected, that human gingival fibroblasts express CD47. Moreover, we are, to the best of our knowledge, the first to show that SIRPα is expressed by human gingival

fibroblasts. These results are consistent with our previous findings that SIRPα expression is detected in osteoblasts, which also are, as fibroblasts, cells of

mesenchymal origin (Koskinen et al., 2013). The fact that gingival fibroblasts, from six different periodontally healthy individuals, display equal basal mRNA expression of both SIRPα and CD47, make our results valid not only at an individual level.

The time- and concentration studies of the effect of pro-inflammatory cytokine

stimulation of SIRPα and CD47 expression were performed on gingival fibroblasts

from one of the six individuals. We used IL-6 as a marker of the phenotype switch in

gingival fibroblasts stimulated with the two pro-inflammatory cytokines TNF-α or IL-

1β. Besides the increased IL-6 mRNA expression, significant concentration-dependent

regulation of SIRPα was seen after stimulation with TNF-α. A tendency of

concentration-dependent regulation of CD47 mRNA was also seen after stimulation with TNF-α, but the differences between the doses were not significant. Similarly, stimulation with IL-1β did show significant concentration-dependent increase of SIRPα, but not of CD47. The mRNA expression of IL-6 was concentration dependent after stimulation with both TNF-α and IL-1β.

Time-course studies of SIRPα and CD47 expression in gingival fibroblasts displayed no time-dependent effect of neither TNF-α nor IL-1β during 6 to 48 hours of incubation. A draw back of this study is that we did not include time points before 6h. This would have given us the possibility to detect an early time-dependent increase of SIRPα and CD47 in response to pro-inflammatory cytokines. TNF-α, induced a time-dependent increase of IL-6 in gingival fibroblasts, but no time-dependent correlation was seen after stimulation with IL-1β.

There is no published data regarding pro-inflammatory cytokines ability to regulate SIRPα expression in fibroblasts or other cells of mesenchymal origin. CD47 and its binding partner thrombospondin-1 have been investigated in relation to sporadic inclusion body myositis, an inflammatory muscle disease (Salajegheh et al., 2007). It was demonstrated that the mRNA expression of CD47 was up-regulated in muscle fibers from diseased individuals compared to healthy controls (Salajegheh et al., 2007).

Moreover, they showed an enhanced CD47 mRNA expression in a myoblast cell line

stimulated with TNF-α compared to un-stimulated control. Interestingly, a skin

fibroblast cell line was also used as a control for the myoblast cell line, and no TNF-α

dependent increase of CD47 mRNA expression was detected in that cell line. The

findings regarding skin fibroblasts are contradictory to our demonstrated data that TNF-

α up-regulate CD47 in gingival fibroblasts, whilst the myoblast data is reminiscent with

our findings. It is know that fibroblasts from different regions of the body have various

profiles of gene expression, which indicates their different tissue-specific functions and

interactions with other cells (Hakkinen et al., 2014). This might explain the different

TNF-α induced CD47 expression seen between the skin fibroblasts and gingival

fibroblasts.

Knowing that, together with human ginigival fibroblasts, the periodontal ligament cells are the most common cell types found in the periodontium (Lindhe and P. Lang, 2015) it would be interesting to investigate and compare the basal gene expression of SIRPα and CD47 in these cells. Moreover, the periodontal ligament cells are key cells in regulating tooth to jaw-bone attachment. It would be interesting to evaluate the SIRPα and CD47 expression in periodontal ligament cells under inflammatory conditions.

In periodontitis the inflammatory response renders in a dysregulated activity between bone producing osteoblasts and bone degrading osteoclasts, resulting in loss of tooth supporting bone tissue. Both under healthy and inflammatory conditions, the bone resorption process is regulated by various cytokines, where macrophage colony- stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) are the major cytokines. Different cell types, including osteoblasts

(Yamashita et al., 2012) and fibroblasts, express M-CSF (Bostrom and Lundberg, 2013) and RANKL (Belibasakis et al., 2014). Our research group have previously shown that SIRPα and CD47 are important for differentiation of bone resorbing osteoclasts

(Lundberg et al., 2007; Koskinen et al., 2013). Lack of either CD47 or SIRPα result in reduced number of osteoclast. This is partly explained by the findings that CD47 activates intracellular SIRPα signaling in osteoblasts, followed by M-CSF and RANKL production. If lack of signaling occur, osteoclast differentiation will be impaired

(Koskinen et al., 2013). Hypothetically, an initial inflammation in the periodontal tissue may induce increased expression of SIRPα in gingival fibroblasts, which could result in increased formation of M-CSF and RANKL. M-CSF and RANKL will enhance

osteoclast differentiation and thereby increase the degradation of tooth supporting jaw- bone.

In conclusion, pro-inflammatory cytokines of importance in periodontitis regulate expression of the cell surface proteins SIRPα and CD47 in human gingival fibroblasts.

Further studies are required to analyse if the inflammatory induction of CD47 and

SIRPa in gingival fibroblasts are of importance for the pathogenesis of periodontitis.

ACKNOWLEDGMENTS

We thank Rima Sulniute, Elisabeth Granström and Inger Lundgren for skilful technical assistance.

This work was supported by the Swedish Dental Society, and the County Council of Västerbotten.

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Figure 1. Photomicrograph image (A) show the outgrowth of fibroblasts from the biopsies (10x magnification). SIRPα (B), CD47 (C) and IL-6 (D) mRNA expression of in

unstimulated human gingival fibroblasts (control) and gingival fibroblasts stimulated with TNF-α (50 ng/mL) and IL-1β (100 pg/mL) for 24 hours. The mRNA was quantified by quantitative real-time RT-qPCR and the diversity of the amplification was normalized to RPL13a. Each box represents the collected mean quantity of mRNA (4 samples per group) from each of the six individuals ± SD. *P<0.05, **P<0.01 and ***P<0.001, statistical analysis using One-Way ANOVA.

Control TNF-α IL-1β

0.0 0.1 0.2 0.3 0.4 0.5 2 4 6 8

Quantity mRNA IL-6

IL6

***

***

Control TNF-α IL-1β

0 10 20 30 40 50 60 70 80 90

Quantity mRNA CD47

CD47

ns

***

Control TNF-α IL-1β

0 10 20 30 40 50

Quantity mRNA SIRPα

SIRPα

*

**

A

D C

B

Figure 2. Time-plot of measured quantity of SIPRα, CD47 and IL-6 mRNA show no time- dependent regulation after stimulation with IL-1β (100 pg/mL) for 6, 12, 24 and 48 hours (A). Similarly, no time-dependent regulation of SIPRα and CD47 mRNA after stimulation with TNF-α (50 ng/mL) for 6, 12, 24 and 48 hours were seen. The mRNA expression of IL- 6 did increase over time after stimulation with TNF-α (Β). The mRNA was quantified using real-time RT-qPCR and the diversity of the amplification was normalized to RPL13a. The figures (A and B) show the mean value of 4 samples per group.

Figure 3. Concentration-dependent regulation of SIRPα (A) and IL-6 (C) mRNA in

unstimulated controls and stimulated gingival fibroblasts with IL-1β (10 - 300 pg/mL) for

24 hours. No concentration dependent correlation was seen when analysing the quantity of

CD47 (B). The mRNA was quantified by quantitative real-time RT-qPCR and the diversity

of the amplification was normalized to RPL13a. The figures (A-C) show the mean value of

4 samples per group ± SEM. *P<0.05, **P<0.01 and ***P<0.001, statistical analysis using

One-Way ANOVA.

Figure 4. Concentration-dependent regulation of SIRPα (A) and IL-6 (C) in unstimulated controls and stimulated gingival fibroblasts with TNF-α (10 - 100 ng/mL) for 24 hours.

CD47 (B) showed a positively concentration-dependent increase in mRNA, but not significant as with SIRPα and IL-6. The mRNA was quantified by quantitative real-time RT-qPCR and the diversity of the amplification was normalized to RPL13a. The figures (A- C) show the mean value of 4 samples per group ± SEM. *P<0.05, **P<0.01 and

***P<0.001, statistical analysis using One-Way ANOVA.