Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes
Michael J. Kraakman, Man K.S. Lee, Annas Al-Sharea, Dragana Dragoljevic, Tessa J. Barrett, Emilie Montenont, Debapriya Basu, Sarah Heywood, Helene L. Kammoun, Michelle Flynn, Alexandra Whillas, Nordin M.J. Hanssen, Mark A. Febbraio, Erik Westein, Edward A. Fisher, Jaye Chin-Dusting, Mark E. Cooper, Jeffrey S. Berger, Ira J. Goldberg, Prabhakara R. Nagareddy, Andrew J. Murphy
Michael J. Kraakman, Man K.S. Lee, Annas Al-Sharea, Dragana Dragoljevic, Tessa J. Barrett, Emilie Montenont, Debapriya Basu, Sarah Heywood, Helene L. Kammoun, Michelle Flynn, Alexandra Whillas, Nordin M.J. Hanssen, Mark A. Febbraio, Erik Westein, Edward A. Fisher, Jaye Chin-Dusting, Mark E. Cooper, Jeffrey S. Berger, Ira J. Goldberg, Prabhakara R. Nagareddy, Andrew J. Murphy
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Research Article Inflammation Vascular biology

Neutrophil-derived S100 calcium-binding proteins A8/A9 promote reticulated thrombocytosis and atherogenesis in diabetes

Abstract

Platelets play a critical role in atherogenesis and thrombosis-mediated myocardial ischemia, processes that are accelerated in diabetes. Whether hyperglycemia promotes platelet production and whether enhanced platelet production contributes to enhanced atherothrombosis remains unknown. Here we found that in response to hyperglycemia, neutrophil-derived S100 calcium-binding proteins A8/A9 (S100A8/A9) interact with the receptor for advanced glycation end products (RAGE) on hepatic Kupffer cells, resulting in increased production of IL-6, a pleiotropic cytokine that is implicated in inflammatory thrombocytosis. IL-6 acts on hepatocytes to enhance the production of thrombopoietin, which in turn interacts with its cognate receptor c-MPL on megakaryocytes and bone marrow progenitor cells to promote their expansion and proliferation, resulting in reticulated thrombocytosis. Lowering blood glucose using a sodium-glucose cotransporter 2 inhibitor (dapagliflozin), depleting neutrophils or Kupffer cells, or inhibiting S100A8/A9 binding to RAGE (using paquinimod), all reduced diabetes-induced thrombocytosis. Inhibiting S100A8/A9 also decreased atherogenesis in diabetic mice. Finally, we found that patients with type 2 diabetes have reticulated thrombocytosis that correlates with glycated hemoglobin as well as increased plasma S100A8/A9 levels. These studies provide insights into the mechanisms that regulate platelet production and may aid in the development of strategies to improve on current antiplatelet therapies and to reduce cardiovascular disease risk in diabetes.

Authors

Michael J. Kraakman, Man K.S. Lee, Annas Al-Sharea, Dragana Dragoljevic, Tessa J. Barrett, Emilie Montenont, Debapriya Basu, Sarah Heywood, Helene L. Kammoun, Michelle Flynn, Alexandra Whillas, Nordin M.J. Hanssen, Mark A. Febbraio, Erik Westein, Edward A. Fisher, Jaye Chin-Dusting, Mark E. Cooper, Jeffrey S. Berger, Ira J. Goldberg, Prabhakara R. Nagareddy, Andrew J. Murphy

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Figure 6

Neutrophil-derived S100A8/A9 promotes thrombocytosis in diabetic mice.

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Neutrophil-derived S100A8/A9 promotes thrombocytosis in diabetic mice. (...
(A) Il6 mRNA expression was quantified in BMDMs cultured in high glucose (25 mM) and incubated with S100A8/A9 for 6 hours. n = 4. *P < 0.05 vs. 0. (B) BM progenitor cells were cultured in high glucose with S100A8/A9 for 16 hours. MkP proliferation was quantified via EdU incorporation. n = 3 per group. (C–G) WT and S100a9–/– mice were made diabetic with STZ and analyzed after 4 weeks of diabetes. (C) Blood glucose levels. Livers were digested, and the abundance of Kupffer cells (D) and IL-6 expression (E) were quantified by flow cytometry. Circulating reticulated platelets (F) and BM MkPs (G) were measured by flow cytometry. n = 5 WT, n = 9 STZ, and n = 10 S100a9–/– STZ. *P < 0.05 vs. WT (C and D) and vs. all groups (E–G). (H) Experimental overview: BM from WT or S100a9–/– mice was transplanted into WT recipient mice, and after 5 weeks a subgroup of mice were rendered diabetic with STZ. (I–L) Kupffer cell IL-6 expression (I), reticulated platelets (J), BM MkP abundance (K), and MkP proliferation (L) were quantified by flow cytometry. n = 5 WT control, n = 5 WT STZ, n = 4 S100a9–/– control, n = 6 S100a9–/– STZ. *P < 0.05 for disease effect, #P < 0.05 for genotype effect. (M–Q) Neutrophil depletion studies: Neutrophils were depleted in WT and diabetic (STZ) mice after the onset of diabetes for 4 weeks by i.p. injection of the neutrophil-specific anti–Ly6-G antibody (clone 1A8, 1 mg/injection, every 3 days), and these mice were compared with isotype control–treated mice. (M) Neutrophil levels were assessed by flow cytometry. (N) Plasma S100A8/A9 levels. (O) Platelet levels were quantified by complete blood count. (P and Q) Abundance (P) and proliferation (Q) of BM MEPs were quantified by flow cytometry. n = 5 per group. *P < 0.05 for disease effect, #P < 0.05 for treatment effect. All data represent the mean ± SEM. P values were obtained using 1-way ANOVA (A–G) or 2-way ANOVA (I–Q).