# Streptococcus sanguinis-induced cytokine and matrix metalloproteinase-1 release from platelets

Fabrice Cognasse, Hind Hamzeh-Cognasse, Adrien Chabert, Elke Jackson, Charles-Antoine Arthaud, Olivier Garraud, Archie Mcnicol

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Fabrice Cognasse, Hind Hamzeh-Cognasse, Adrien Chabert, Elke Jackson, Charles-Antoine Arthaud, et al.. Streptococcus sanguinis-induced cytokine and matrix metalloproteinase-1 release from platelets. BMC Immunology, 2014, 15 (1), pp.15. 10.1186/1471-2172-15-15. http://hal-04995256

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# Streptococcus sanguinis-induced cytokine and matrix metalloproteinase-1 release from platelets

Fabrice Cognasse<fnref n="1" />,<fnref n="2" />*†, Hind Hamzeh-Cognasse<fnref n="2" />†, Adrien Chabert<fnref n="2" />, Elke Jackson³,⁴, Charles-Antoine Arthaud<fnref n="1" />, Olivier Garraud<fnref n="1" />,<fnref n="2" /> and Archie McNicol³,⁴

**Abstract**

**Background:** *Streptococcus sanguinis* (*S.sanguinis*), a predominant bacterium in the human oral cavity, has been widely associated with the development of infective endocarditis. Platelets play both a haemostatic function and can influence both innate and adaptive immune responses. Previous studies have shown that *S.sanguinis* can interact with, and activate, platelets.

**Results:** The aim of this study was to determine whether *S.sanguinis* stimulates the release of matrix metalloproteinases (MMPs) 1, 2 and 9 and the pro-inflammatory mediators SDF-1, VEGF and sCD40L, from platelets and to subsequently pharmacologically address the release mechanism (s). *S.sanguinis* stimulated the release of MMP-1, SDF-1, VEGF and sCD40L from platelets and inhibitors of cyclooxygenase and phosphatidylinositol 3-kinase, and antagonists of the αIIbβ3 integrin and glycoprotein Ib, each inhibited the secretion of all factors.

**Conclusions:** Therefore the release of MMP-1, SDF-1, VEGF and sCD40L occurs late in the platelet response to *S.sanguinis* and highlights the complex intracellular signalling pathways stimulated in response to *S.sanguinis* which lead to haemostasis, MMP and pro-inflammatory mediator secretion.

**Keywords:** Platelets, Cytokines, Signalling, Oral cavity, Inflammation, *Streptococcus sanguinis*

**Background**

The oral cavity is one of the most common gateways of entry of bacteria, including *Streptococcus sanguinis* (*S. sanguinis*) an organism which has been implicated in the development of infective endocarditis [1], into the blood [2]. A correlation exists between periodontitis, where there is an increased incidence of bacteremias [3], and the development of cardiovascular disease [4], although the causality is controversial [5]. Multiple studies have demonstrated that *S. sanguinis* can activate human platelets in a strain and donor dependent manner [6], and therefore it is possible that platelets play a central role in any relationship between periodontitis and cardiovascular pathologies [6].

Traditionally platelets have been considered solely as components of haemostasis and by extension the pathological process of thrombosis [7,8]. However, it is now clear that platelets are innate immune cells and are

associated with the early stages of atherosclerosis and other inflammatory conditions [9-11]. Platelets contain in, and secrete from, alpha granules a range of soluble immunomodulatory factors such as Stromal cell-derived factor (SDF)-1/CXCL12, a member of the CXC chemokine family and Vascular Endothelial Growth Factor (VEGF), a sub-family of growth factors, stimulate vasculogenesis and angiogenesis [12]. Furthermore, CD40 ligand (CD40L, CD154), of which approximately 95% of its soluble form (sCD40L) is generated from platelets, is a modulator of humoral and cellular immunity, has pro-inflammatory properties and provides a link between the immune system and atherothrombosis [13].

Several recent data, including our own, demonstrate that platelets have the capacity to sense external signals through a single type of pathogen recognition receptor and differentially adjust the innate immune response by the appropriate secretion of a number of cytokines/chemokines and some of their receptors [14,15]. Italiano and colleagues initially demonstrated that there are two discrete sub-populations of platelet alpha granules, one containing pro-angiogenic factors and one containing anti-angiogenic factors [16,17];

<footnote>* Correspondence: fabrice.cognasse@univ-st-etienne.fr</footnote>

<footnote>†Equal contributors</footnote>

<footnote n="1">¹Etablissement Français du Sang (EFS) Auvergne-Loire, Saint-Étienne, France</footnote>

<footnote n="2">²GIMAP-EA3064, Université de Lyon, Saint-Étienne 42023, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
indeed subsequent studies have suggested more heterogeneity among these granules [18]. This has led to the concept of differential alpha granule release, although the mechanisms remain unknown [17].

Matrix MetalloProteinases (MMPs) constitute a family of zinc- and calcium-dependent proteinases that are involved in the turnover of the extracellular matrix (ECM) of connective tissue. They degrade most components of the ECM and participate in a variety of pathological processes, including atherosclerosis, myocardial infarction and aortic aneurysms, as well as tumour growth and metastasis [19]. MMP-1 is expressed on the surface of resting platelets and, following platelet activation, its levels are upregulated and its activity engaged [20]. There is less MMP-2 than MMP-1 on the platelet surface, and the presence of both MMP-3 and MMP-9 is controversial [20]. In addition to its effects on the extracellular matrix, MMP-1 can regulate outside-in signalling in platelets resulting in the phosphotyrosine phosphorylation, and subsequent redistribution, of β3 integrins as a pre-requisite for platelet aggregation [20].

Interestingly, collagen can activate MMP-1, which in turn cleaves the platelet protease activating receptor, PAR-1, with the resultant engagement of the receptor and enhanced platelet activation [21]. To date there are no reports of the effects of *S. sanguinis* on MMPs in platelets.

The aim of this study was to determine whether *S. sanguinis* releases platelet MMPs (MMP-1, 2 and 9) and to pharmacologically address the mechanism by which the MMPs and pro-inflammatory mediators (SDF-1, VEGF and sCD40L) are released.

## Results and discussion

Kerrigan and colleagues have suggested that the interaction between *S. sanguinis* and GPIb on platelets is not only important for the pathogenesis of infective endocarditis but may also play a contributory role in some cases of myocardial infarction [22]. Studies have shown that plasma levels of sCD40L is a predictor of recurrent cardiovascular disorders (e.g. myocardial infarction and stroke) [23]. As previously observed [24], *S. sanguinis* 2017-78 stimulated the release of significant amounts of sCD40L from platelets (Figure 1A), consistent with Kerrigan and colleagues proposed role for platelets in *S. sanguinis*-induced myocardial infarction [22]. The secretion of sCD40L was accompanied by the secretion of VEGF (Figure 1B), SDF-1 (Figure 1C) and MMP-1 (Figure 1D). In each case, the level of secretion elicited by *S. sanguinis* 2017-78 was statistically similar to that elicited by the soluble positive control collagen (Figure 1A-D).

<figure><img src="image_2.png" /><figcaption>**Figure 1** Streptococcus sanguinis-induced soluble molecule secretion from platelets. Platelets were stirred in with either *S. sanguinis* strain 2017-78 (n = 15), collagen (2 µg/ml; n = 5) or saline control (Unstimulated; n = 11). Release reactions were terminated and the levels of (A) sCD40L, (B) VEGF, (C) SDF-1, (D) MMP-1, (E) MMP-2 and (F) MMP-9 in the releasates determined by multi-plex luminex. Results are mean ± SEM of several individual experiments using the platelets from different donors and subsequently analyzed using the Mann-Whitney U-test (\*P < 0.05 with respect to saline control; \*\*P < 0.05 with respect to the saline control).</figcaption></figure>
In contrast S. sanguinis 2017-78 did not elicit the release of MMP-2 (Figure 1E) or MMP-9 (Figure 1F). This constitutes the first demonstration that the oral microorganism S. sanguinis can induce the secretion of a tissue-destructive molecule (MMP-1) from platelets to the local vasculature, thus potentially participating in the breakdown of the ECM at sites of vascular lesion. The functional significance of the secretion of MMP-1 is unclear. A novel autocrine role for MMP-1 in collagen-stimulated platelets has been proposed, whereby collagen converts inactive MMP-1 to active MMP-1 which in turn cleaves the thrombin receptor PAR-1 exposing the ligand and leading to full platelet activation [20]. A similar mechanism in S. sanguinis-induced platelet activation is attractive as S. sanguinis and collagen utilise many similar intracellular signalling pathways [25]. To date the effects of PAR-1 receptor antagonists, such as SCH 530398, on platelet activation induced by either collagen or S. sanguinis have not been examined. Interestingly, however, certain strains of Porphyromonas gingivalis have been shown to activate platelets via the PAR-1 and PAR-4 thrombin receptors on platelets [26]. Inhibition of this proteolytic action had no

effect on this activation, and therefore it is clear that a complex mechanism is involved [27] and a role for MMP-1 cannot be discounted.

The absence of MMP-2 secretion may reflect the subcellular localisation of MMP-2 within the platelet. Studies by Sawicki and colleagues suggest that MMP-2 is localised to the cytosol rather than to granules [28]. Although in the same studies MMP-2 was secreted in activated platelets [28], it is possible that the weak nature of platelet activation by S. sanguinis cannot stimulate this release.

The presence, secretion and release of MMP-9 from platelets is controversial. Some studies suggest that platelets do not secrete MMP-9 [29,30], whereas others report, not only that MMP-9 is secreted from platelets in vivo, but that it plays a crucial role in sCD40L release in pathological conditions such as Behçet's disease [31] and abdominal sepsis [32].

To determine the potential roles played by individual signalling pathways in the secretion of sCD40L, VEGF, SDF-1 and MMP-1 in response to S.sanguinis, we employed various inhibitors of biochemical pathways at concentrations previously reported to have maximal effects. Such concentrations

<figure><img src="image_3.png" /><figcaption>**Figure 2** Effects of inhibitors on Streptococcus sanguinis induced sCD40L (A), VEGF (B), SDF-1 (C) and MMP-1 (D) secretion from platelets. Platelets were pre-incubated with either aspirin (100 µM; 20 mins.; n = 7), wortmannin (100 nM; 2 mins; n = 5), RGDS (1 mM; 5 mins; n = 5), AN51 (0.165 mg/mL, 5 mins; n = 5) or the saline control then stirred with S. sanguinis strain 2017-78. Release reactions were terminated and the levels of sCD40L in the releasates determined by multi-plex luminex. Results are shown as box plots with whiskers for the data range representing median, upper and lower quartiles of several individual experiments using the platelets from different donors and subsequently analyzed using the Mann-Whitney U-test (*P* < 0.05 with respect to saline control; \*P < 0.05 with respect to S. sanguinis strain 2017-78).</figcaption></figure>
optimise the conditions to identify individual signalling
pathways, although recognising the potential for non-
selective actions.

We have previously shown roles for GPIb, COX, the αIIbβ3 integrin and PI3K in *S. sanguinis*-induced platelet aggregation [25,33,34]. Consistent with the aggregation results [24], pre-treatment of platelets with the GPIb antagonist AN51 (0.165 mg/mL, 5mins.), the COX inhibitor aspirin (100 µM; 20 min), the αIIbβ3 integrin antagonist RGDS (1 mM; 5 mins.) or the PI3K inhibitor wortmannin (100 nM; 2 mins.) each significantly decreased *S. sanguinis* strain 2017-78-induced secretion of sCD40L (Figure 2A).

Pretreatment of platelets with the same inhibitors also
decreased the secretion of VEGF (Figure 2B), SDF-1
(Figure 2C) and MMP-1 (Figure 2D) consistent with roles
for GPIb, COX, the αIIbβ3 integrin and PI3K in the secre-
tion of these proteins. The precise sublocalisation of SDF-1,
VEGF and MMP-1 within the individual heterogenic popu-
lation of granules is unknown. However, the current obser-
vations suggest, either that SDF-1, VEGF and MMP-1 are
contained in the same granule population, or that the gran-
ule populations containing the proteins are regulated by the
same signalling pathways.

Conclusions

The concept of differing signalling pathways regulating the
release of the individual populations of alpha granules was
introduced by Italiano and Batinelli [17]. The pathway(s) in-
volved in secretion has not been established. However as
secretion was sensitive to inhibition by RGDS, it appears
that it occurs distal to the engagement of the αIIbβ3 integ-
rin. Speculatively this could lead to the incorporation, and
activation, of ERK into the cytoskeleton [35], Rac activity,
cytoskeletal rearrangement [36] and the subsequent alpha
granule secretion [37]. Further description of their mecha-
nisms of action will expand our understanding of platelet
activation and oral/mucosal inflammatory process and pro-
duce new therapeutic strategies.

Methods

Materials

S. sanguinis strain 2017-78 was a gift from Dr M. Herzberg (University of Minnesota) [38]. Blood Agar Base No. 2 was obtained from Oxoid Inc. (Nepean, ON, Canada) and sheep's blood from Atlas Laboratories (Winnipeg, MB, Canada). Collagen, the cyclo-oxygenase (COX) inhibitor aspirin, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, the peptise αIIbβ3 integrin antagonist Argine-Glycine-Aspartic Acid-Serine (RGDS), and the GP (glycoprotein) Ib receptor blocker AN51 were all purchased from Sigma-Aldrich Canada Ltd (Oakville, ON, Canada).

Blood collection

The study was approved by the Research Ethics Board of
the University of Manitoba. Blood was collected after in-
formed consent, from healthy human volunteers who had
denied taking medication known to interfere with platelet
function. Platelet-rich plasma (PRP) was prepared by cen-
trifugation (200 × g; 20 min). *S. sanguinis* strain 2017-78
was prepared as previously reported [24]. PRP was stirred
with *S. sanguinis* strain 2017-78 in the presence of the in-
hibitor or vehicle control, and was aggregation monitored
continuously. At a time corresponding to that which
*S. sanguinis* strain 2017-78 induced maximal aggregation,
the release was terminated, the sample was centrifuged
and the supernatants stored at -80 °C until assayed [24].

Soluble factor detection

The levels of soluble factors MMPs (MMP-1, 2 and 9) and pro-inflammatory mediators (SDF-1, VEGF and sCD40L) were measured in duplicate from the releasates by multiplex luminex kits (Bio-Rad, Marnes-la-Coquette, France). Absorbance at 450 nm was determined using an ELISA reader (Miltiskan EX, Labsystem, Helsinki, Finland), as previously reported [39].

Statistical analysis

Inter-experiment comparisons of *S. sanguinis* strain 2017-78-induced MMPs and cytokines release were analyzed by means of the paired *t*-test. The effects of inhibitors on *S. sanguinis* strain 2017-78-induced secretion were analyzed using the non-parametric Mann–Whitney *U*-test. Results are shown as box plots with whiskers for the data range representing median, upper and lower quartiles and a *P*-value < 0.05 was considered to be significant.

Abbreviations

*S. sanguinis*: *Streptococcus sanguinis*; MMP: Matrix metalloproteinase; SDF1: Stromal cell-derived factor 1; VEGF: Vascular endothelial growth factor; sCD40L: Soluble CD40 ligand; COX: Cyclooxygenase; PI3K: Phosphatidylinositol 3-kinase; RGDS: Argine-glycine-aspartic acid-serine peptide; GP: Glycoprotein; ECM: Extracellular matrix; PRP: Platelet-rich plasma.

Competing interests

The authors declare no competing financial interests and no conflict of
interest regarding this study.

Authors' contributions

FC, HHC, AMcN, OG: Analysis and interpretation of results; FC, CAA, HHC, AC, EJ, AMcN, OG; Preparation of manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank Mrs. Marie-Ange Eyraud for her experimental expertise, and Dr. Sara Israels and Pr. Bruno Pozzetto for helpful discussions and critical reading of this manuscript. This study was funded by grants from the Heart and Stroke Foundation of Manitoba, L'Établissement Français du Sang", La Plaine - Saint-Denis, France, the French Agence Nationale de la Recherche (ANR) under reference ANR-12-JSV1-0012-01 and the Rhône-Alpes Region - ARC 2012-1200870001.
Author details

Received: 30 October 2013 Accepted: 7 April 2014

Published: 22 April 2014

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doi:10.1186/1471-2172-15-15

Cite this article as: Cognasse et al.: Streptococcus sanguinis-induced cytokine and matrix metalloproteinase-1 release from platelets. BMC Immunology 2014 15:15.

<footnote n="1">¹Etablissement Français du Sang (EFS) Auvergne-Loire, Saint-Étienne, France. ²GIMAP-EA3064, Université de Lyon, Saint-Étienne 42023, France.</footnote>

<footnote n="3">³Oral Biology and Pharmacology & Therapeutics, University of Manitoba, Winnipeg, MB, Canada. ⁴International Centre for Oral Systemic Health, University of Manitoba, Winnipeg, MB, Canada.</footnote>