Le rôle des protéines S100 dans la migration des neutrophiles au site inflammatoire

La réaction inflammatoire est un mécanisme de défense de l’hôte, toutefois, dans certaines circonstances, il arrive que cette réaction se retourne contre l’organisme. Il est donc important de comprendre l’origine de ce problème afin d’y trouver des solutions. Une étape majeure de la réaction inflammatoire est la migration des leucocytes du sang vers la région affectée. De ces cellules, le neutrophile est le premier à se rendre au site inflammatoire. Outre son rôle de défense de première ligne, le neutrophile a également la capacité de libérer divers médiateurs qui lui permettent d’orchestrer la réponse immunitaire. Le neutrophile est un réservoir majeur de protéines S100A8, S100A9 et S100A12. Depuis une vingtaine d’années, ces protéines ne cessent d’être associées à diverses pathologies infectieuses ou inflammatoires. Elles sont en effet détectées à de fortes concentrations dans le sérum de patients atteints d’arthrites, de maladies inflammatoires de l’intestin, de tuberculose, etc. Elles sont au demeurant des marqueurs sensibles de l’activité de ces maladies. Des études indiquent que ces protéines sont sécrétées dans des conditions inflammatoires et qu’elles exercent dans le milieu extracellulaire des fonctions pro-inflammatoires. Plus particulièrement, elles sont associées à la migration des leucocytes. Toutefois, les mécanismes d’action précis de ces protéines restent à élucider. Dans ces travaux de thèse, nous nous sommes intéressés aux rôles des protéines S100 dans la migration transendothéliale et tissulaire des neutrophiles. Nous avons observé que l’ajout de la protéine S100A9, dans des modèles in vitro, favorise l’adhésion des neutrophiles d’une part à l’endothélium et d’autre part à la fibronectine par l’activation des intégrines β2 et favorise ainsi la migration de ces cellules au site inflammatoire. S100A9 joue donc un rôle d’amplificateur de la réponse inflammatoire. Ces travaux nous aident à mieux comprendre le rôle des protéines S100 dans l’inflammation et plus particulièrement dans la migration des neutrophiles. Ces protéines constituant des cibles thérapeutiques potentielles, il est donc important de détailler leurs rôles dans le contexte inflammatoire.Inflammation is one of the body’s defence mechanisms. In some cases, this reaction can be detrimental to the host it is supposed to protect. It is thus important to understand the origin of such reactions in order to find solutions. A key step of inflammatory reactions is leukocyte migration from blood to the injured area. Among leukocytes, neutrophils are the first to reach the inflammatory site. In addition to their role as the body’s first line of defence, neutrophils also help orchestrate the immune response through the release of inflammatory mediators. The neutrophils are a major reservoir of S100A8, S100A9, and S100A12 proteins. In the past 20 years, these proteins have been increasingly associated with various infectious and inflammatory diseases as they were detected at high concentrations in the serum of patients suffering from arthritis, inflammatory bowel diseases and tuberculosis to name a few. Moreover, they were shown to be sensitive markers of these diseases’ activity. Some studies have indicated that these proteins are secreted in inflammatory conditions and that they have pro-inflammatory activities in the extracellular space. Specifically, they were associated with leukocyte migration. However, the precise mechanisms through which these proteins affect the inflammatory response remain to be elucidated. In this work, we investigated the roles of S100 proteins in neutrophil transendothelial and tissue migration. We observed that S100A9 protein increased neutrophil adhesion to the endothelium and fibronectin by activating β2 integrins which led to the stimulation of cell migration to the inflammatory site in in vitro models. This work provided insight on the roles of S100 proteins in inflammation and more specifically in neutrophil migration. As these proteins represent potential therapeutic targets, it remains important to further understand their roles in inflammatory conditions

An inflammation loop orchestrated by S100A9 and calprotectin is critical for development of arthritis.

The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.In this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.Treatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα, IL-1β and IL-6, and of chemokines like MIP-1α and MCP-1.The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed

S100A9 induces the secretion of cytokines by monocytes.

<p>Cells (1 × 10⁶ cells/ml) were incubated with S100A9 (10 µg/ml) for 24 h. Supernatants were harvested and cytokines were measured by (A) cytokine arrays or (B) ELISA. CTRL: unstimulated cells. Values are the mean ± SEM of 5 different experiments. *p<0.05, t test. (C) Monocytes were stimulated with different concentrations of S100A9 (0.1–40 µg/ml) and IL-6 or TNF-α concentrations were measured in the supernatant. (D) Monocytes were incubated with 10 µg/ml of S100A9 and IL-6 or TNF-α concentrations were assessed as a function of time. Solid line: IL-6 dosage (open circles: unstimulated cells, solid circles: S100A9 stimulated cells), dotted line: TNF-α (white squares: unstimulated cells, solid squares: S100A9 stimulated cells).</p

S100A9 increases neutrophil adhesion to fibrinogen.

<p>Neutrophils were incubated with S100A9 (40 µg/ml) or IL-8 (5 ng/ml) alone or in combination and allowed to adhere to fibrinogen for different incubation periods at 37°C. The number of adhered neutrophils was determined as described in Materials and Methods. Data shown represent the mean ± SEM of at least 3 experiments using neutrophils from different donors.</p

Neutrophil migration across HUVECs in response to S100A9.

<p>A) Increasing concentrations of S100A9 were added in the upper wells with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower wells and neutrophils were allowed to migrate for 2 h at 37°C. Data are the mean ± SEM of 3 experiments using neutrophils from different donors. *p<0.05, **p<0.01, one-way ANOVA, Dunnett’s multiple comparison test. B) S100A9 prolongs the time for neutrophil migration across endothelial cells. S100A9 (40 µg/ml) was added to the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower well and every 30 min for up to 2 h the upper wells were moved to new lower wells. The number of transmigrated cells was determined as described in Materials and Methods. Data shown represent the mean ± SEM of at least 3 experiments using neutrophils from different donors.</p

Effect of anti-S100A9, TNFα, or isotype control Abs on the LPS-CIA clinical score.

<p>(A) Anti-collagen II Abs were measured in mouse serum 4 h after LPS injection. The results are expressed as µg of IgG Ab/ml of serum. (B) S100A8 and S100A9 expression in arthritic paws. Paw tissue sections were stained with rabbit pre-immune serum, rabbit anti-S100A8, or rabbit anti-S100A9 polyclonal IgGs. B: bone, Js: joint space. (Magnification 1000X). (C) Four hours after LPS injection, sera were collected and ELISA S100A8/A9 were performed *p<0.05, one-way ANOVA test (n = 10 paws/group), Tukey’s Multiple Comparison Test (D) Clinical score as assessed by two blinded observers. Data are the mean scores calculated from at least 15 mice per group until day 19.</p

Histological assessment of joint infiltration and destruction in anti-S100A9-, anti-TNFα-, and isotype control-treated animals.

<p>(A) H&E-stained sections of wrist at 40× magnification and safranin/fast green-stained sections of the distal phalange joints at 100× magnification. The enlargement was taken at 1000 × magnification. B: bone, C: cartilage, J: joint. (B) Bone destruction, collagen degradation, and cell infiltration as assessed by two blinded observers (on a scale of 0–3, 0–2, 0–2, respectively). *p<0.05, one-way ANOVA test (n  = 10 paws/group, 5 forepaws and 5 hind paws), Tukey’s multiple comparison test.</p

Decreased neutrophil and monocyte antigen expression in the paws of anti-S100A9-treated mice.

<p>(A) Western blot analysis of 7/4 antigen (Ag), a neutrophil marker (top). Quantification of 7/4 Ag on an immunoblot by densitometry analysis (bottom). (B) Western blot analysis of Gr-1 Ag, a marker of granulocytes and monocytes (top). Quantification of Gr-1 Ag on an immunoblot by densitometric analysis (bottom).</p