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

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1. LPS-CIA model.

(A) Mice (25 mice/group) were immunized at the base of the tail with 100 µg of chicken type II collagen on day “−27” and then injected i.p. with LPS (25 µg in PBS) on day 0. Two milligrams of neutralizing Abs or isotype control Abs were injected into mice 24 h before LPS injection. During the protocol, five injections were given, at 2, 5, 8, 12, and 16 days post-LPS injection. On day 5 and 19, mice were sacrificed to collect the paws and the serum for further analysis. (B) Murine monoclonal anti-S100A9 (Clone 2A5) was tested for specificity by western blot against murine protein mS100A9, mS100A8, a lysate of murine neutrophils, human protein hS100A12, hS100A9, hS100A8 and a lysate of human neutrophils.

Figure 2. Effect of anti-S100A9, TNFα, or isotype control Abs on the LPS-CIA clinical score.

(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.

Figure 3. Histological assessment of joint infiltration and destruction in anti-S100A9-, anti-TNFα-, and isotype control-treated animals.

(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.

Figure 4. Decreased neutrophil and monocyte antigen expression in the paws of anti-S100A9-treated mice.

(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).

Figure 5. S100A9 treatment decreases the secretion of pro-inflammatory cytokines (A) 5 days after LPS injection, sera were collected and tested for IL-6 by ELISA.

Values are the mean ± SEM of 10 mice. *p<0.05, t test (n = 10 serum samples/group) (B) Thirty micoliters from a pool of 5 paw homogenates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and tested for the presence of IL-6 and TNF-α. F: forepaw, H: hind paw.

Figure 6. Neutrophil migration across HUVECs in response to S100A9.

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.

Figure 7. S100A9 increases neutrophil adhesion to fibrinogen.

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.

Figure 8. The effect of S100A9 on neutrophil migration is abolished by anti-CD11b and anti-CD18 antibodies.

S100A9 (40 µg/ml) +/− antibodies against CD11a, CD11b, and CD18, or the isotype control were added to 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. 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<0.05, **p<0.01, one-way ANOVA, Dunnett’s multiple comparison test.

Figure 9. S100A9 induces the secretion of cytokines by monocytes.

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).