Spleen-derived circulating intercellular adhesion molecule 2 promotes osteosclerosis in chronic myelofibrosis

Abstract

Mutations in the gene coding G6b-B, Mpig6b in mice, and MPIG6B in humans lead to myelofibrosis, progressive splenomegaly and osteosclerosis. Aberrant megakaryocyte function in bone marrow and spleen may affect bone cells through cell-cell contact or soluble factors. We aimed to assess the role of circulating factors produced by splenic cells in mediating osteosclerosis in Mpig6b^−/− mice using splenectomy, a procedure commonly used in clinics. Splenectomy or sham surgery was performed on 32-week-old female Mpig6b^−/− and littermate wildtype (WT) mice, when splenomegaly and long bone osteosclerosis were established. Twenty weeks later, the hematopoietic parameters and bone structure of the femur and lumbar vertebra were analyzed. Splenectomy significantly improved platelet count, but the larger platelet size persisted in Mpig6b^−/−mice. The Mpig6b^−/− femurs with established osteosclerosis were not affected by splenectomy. In contrast, splenectomy prevented the development of osteosclerosis in vertebrae of Mpig6b^−/− mice. Significant vertebral bone loss and increased circulating TRACP 5b were demonstrated in splenectomized Mpig6b^−/− mice compared to sham-operated and WT mice. Using quantitative plasma proteomics, we identified intercellular adhesion molecule 2 (ICAM2) as significantly increased in Mpig6b^−/− mice compared to WT and reversed to normal levels after splenectomy. The interaction of membrane-bound ICAM2 and MAC1 was reported to be critical for osteoclast fusion. Our in vitro study revealed that soluble recombinant ICAM2 dose-dependently inhibited osteoclast fusion, suggesting that competitive binding of MAC1 with soluble ICAM2 prevented its interaction with membrane-bound ICAM2. Thus, we identified the soluble ICAM2 as a novel inhibitor of osteoclast fusion and mediator of osteosclerosis in Mpig6b^−/− mice.

References

[1] Tefferi A. Primary myelofibrosis: 2023 update on diagnosis, risk-stratification, and management. Am. J. Hematol., 2023, 98: 801-821.

[2] Mascarenhas J, et al.Biological drivers of clinical phenotype in myelofibrosis. Leukemia, 2023, 37: 255-264.

[3] Smith CJ, et al.A population-based study of outcomes in polycythemia vera, essential thrombocythemia, and primary myelofibrosis in the United States from 2001 to 2015: comparison with data from a Mayo Clinic single institutional series. Am. J. Hematol., 2021, 96: E464-E468.

[4] Roaldsnes C, Holst R, Frederiksen H, Ghanima W. Myeloproliferative neoplasms: trends in incidence, prevalence and survival in Norway. Eur. J. Haematol., 2017, 98: 85-93.

[5] Chornenki NLJ, et al.Characterizing frailty in myeloproliferative neoplasms: results from the ORCHID study. Leuk. Res., 2022, 113106788

[6] Santos FP, et al.Splenectomy in patients with myeloproliferative neoplasms: efficacy, complications and impact on survival and transformation. Leuk. Lymphoma, 2014, 55: 121-127.

[7] Mesa RA. How I treat symptomatic splenomegaly in patients with myelofibrosis. Blood, 2009, 113: 5394-5400.

[8] Mesa RA, Nagorney DS, Schwager S, Allred J, Tefferi A. Palliative goals, patient selection, and perioperative platelet management: outcomes and lessons from 3 decades of splenectomy for myelofibrosis with myeloid metaplasia at the Mayo Clinic. Cancer, 2006, 107: 361-370.

[9] Mughal TI, Vaddi K, Sarlis NJ, Verstovsek S. Myelofibrosis-associated complications: pathogenesis, clinical manifestations, and effects on outcomes. Int. J. Gen. Med., 2014, 7: 89-101.

[10] Malara A, Abbonante V, Zingariello M, Migliaccio A, Balduini A. Megakaryocyte contribution to bone marrow fibrosis: many arrows in the quiver. Mediterr. J. Hematol. Infect. Dis., 2018, 10: e2018068.

[11] Stavnichuk M, Komarova SV. Megakaryocyte-bone cell interactions: lessons from mouse models of experimental myelofibrosis and related disorders. Am. J. Physiol. Cell Physiol., 2022, 322: C177-C184.

[12] Zingariello M, et al.Characterization of the TGF-beta1 signaling abnormalities in the Gata1low mouse model of myelofibrosis. Blood, 2013, 121: 3345-3363.

[13] Chagraoui H, et al.Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO. Blood, 2003, 101: 2983-2989.

[14] Spampinato M, et al.Focus on osteosclerotic progression in primary myelofibrosis. Biomolecules, 2021, 11: 122.

[15] Tiedemann K, Tsao S, Komarova SV. Platelets and osteoblasts: secretome connections. Am. J. Physiol. Cell Physiol., 2022, 323: C347-C353.

[16] Suva LJ, et al.Platelet dysfunction and a high bone mass phenotype in a murine model of platelet-type von Willebrand disease. Am. J. Pathol., 2008, 172: 430-439.

[17] Coxon CH, Geer MJ, Senis YA. ITIM receptors: more than just inhibitors of platelet activation. Blood, 2017, 129: 3407-3418.

[18] Mazharian A, et al.Mice lacking the ITIM-containing receptor G6b-B exhibit macrothrombocytopenia and aberrant platelet function. Sci. Signal., 2012, 5: ra78.

[19] Melhem M, et al.Novel G6B gene variant causes familial autosomal recessive thrombocytopenia and anemia. Eur. J. Haematol., 2017, 98: 218-227.

[20] Hofmann I, et al.Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice. Blood, 2018, 132: 1399-1412.

[21] Stavnichuk M, et al.Severity of megakaryocyte-driven osteosclerosis in Mpig6b-deficient mice is sex-linked. J. Bone Miner. Res., 2021, 36: 803-813.

[22] Song MK, Park BB, Uhm JE. Understanding splenomegaly in myelofibrosis: association with molecular pathogenesis. Int. J. Mol. Sci., 2018, 19: 898.

[23] Tremblay D, et al.Modern management of splenomegaly in patients with myelofibrosis. Ann. Hematol., 2020, 99: 1441-1451.

[24] Polverelli N, et al.Splenomegaly in patients with primary or secondary myelofibrosis who are candidates for allogeneic hematopoietic cell transplantation: a Position Paper on behalf of the Chronic Malignancies Working Party of the EBMT. Lancet Haematol., 2023, 10: e59-e70.

[25] Alasmari BG, et al.A rare MPIG6B gene mutation in a saudi adolescent male with thrombocytopenia, anemia, and myelofibrosis: a case report. Cureus, 2024, 16: e54042.

[26] Khan AA, Rathod SG, Chozakade A, Jalid A, Geelani SA. Rare MPIG6B gene mutation in an Indian male with anemia and thrombocytopenia. Turk. J. Haematol., 2022, 39: 268-271.

[27] Chen H, et al.Case report of a novel MPIG6B gene mutation in a Chinese boy with pancytopenia and splenomegaly. Gene, 2019, 715143957

[28] Cenariu D, et al.Extramedullary hematopoiesis of the liver and spleen. J. Clin. Med., 2021, 10: 5831.

[29] Yamamoto K, et al.Extramedullary hematopoiesis: elucidating the function of the hematopoietic stem cell niche (Review). Mol. Med. Rep., 2016, 13: 587-591.

[30] Reichard A, Wanner N, Farha S, Asosingh K. Hematopoietic stem cells and extramedullary hematopoiesis in the lungs. Cytom. A, 2023, 103: 967-977.

[31] Wernick B, et al.Temporal changes in hematologic markers after splenectomy, splenic embolization, and observation for trauma. Eur. J. Trauma Emerg. Surg., 2017, 43: 399-409.

[32] Gaither C, Popp R, Zahedi RP, Borchers CH. Multiple reaction monitoring-mass spectrometry enables robust quantitation of plasma proteins regardless of whole blood processing delays that may occur in the clinic. Mol. Cell. Proteom., 2022, 21100212

[33] Li X, et al.Role of intercellular adhesion molecule-2 in osteoclastogenesis. Genes Cells, 2012, 17: 568-575.

[34] Mende N, et al.Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans. Blood, 2022, 139: 3387-3401.

[35] Bucelli C, et al.Osteolytic lesions in primary myelofibrosis and effect of ruxolitinib therapy: report of a case and literature review. Chemotherapy, 2018, 63: 340-344.

[36] Merry GM, Aronowitz PB. Myelofibrosis with massive hepatosplenomegaly and osteolytic bone lesions. J. Hosp. Med., 2010, 5: E27-E28.

[37] Tsoutsou PG, et al.ICAM-1, ICAM-2 and ICAM-3 in the sera of patients with idiopathic pulmonary fibrosis. Inflammation, 2004, 28: 359-364.

[38] Tran D, et al.Plasma proteomic signature predicts myeloid neoplasm risk. Clin. Cancer Res., 2024, 30: 3220-3228.

[39] Bouwens E, et al.Circulating biomarkers of cell adhesion predict clinical outcome in patients with chronic heart failure. J. Clin. Med., 2020, 9: 195.

[40] Conant K, et al.Matrix metalloproteinase-dependent shedding of intercellular adhesion molecule-5 occurs with long-term potentiation. Neuroscience, 2010, 166: 508-521.

[41] Diacovo TG, deFougerolles AR, Bainton DF, Springer TA. A functional integrin ligand on the surface of platelets: intercellular adhesion molecule-2. J. Clin. Investig., 1994, 94: 1243-1251.

[42] Fidanza A, et al.Single-cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs. Blood, 2020, 136: 2893-2904.

[43] Psaila B, et al.Single-cell analyses reveal megakaryocyte-biased hematopoiesis in myelofibrosis and identify mutant clone-specific targets. Mol. Cell, 2020, 78: 477-492 e478.

[44] Haghayegh Jahromi N, et al.Intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 differentially contribute to peripheral activation and CNS entry of autoaggressive Th1 and Th17 cells in experimental autoimmune encephalomyelitis. Front. Immunol., 2019, 10: 3056.

[45] Chong DLW, et al.ICAM-1 and ICAM-2 are differentially expressed and up-regulated on inflamed pulmonary epithelium, but neither ICAM-2 nor LFA-1: ICAM-1 are required for neutrophil migration into the airways in vivo. Front. Immunol., 2021, 12691957

[46] Desterke C, Martinaud C, Ruzehaji N, Le Bousse-Kerdiles MC. Inflammation as a keystone of bone marrow stroma alterations in primary myelofibrosis. Mediat. Inflamm., 2015, 2015415024

[47] Lane SW. Shelter from the cytokine storm in myelofibrosis. Blood, 2023, 141: 2415-2416.

[48] El-Masri BM, et al.Mapping RANKL- and OPG-expressing cells in bone tissue: the bone surface cells as activators of osteoclastogenesis and promoters of the denosumab rebound effect. Bone Res., 2024, 12: 62.

[49] Ono T, Hayashi M, Sasaki F, Nakashima T. RANKL biology: bone metabolism, the immune system, and beyond. Inflamm. Regen., 2020, 40: 2.

[50] Udagawa N, et al.Osteoclast differentiation by RANKL and OPG signaling pathways. J. Bone Miner. Metab., 2021, 39: 19-26.

[51] Gronloh MLB, Tebbens ME, Kotsi M, Arts JJG, van Buul JD. Intercellular adhesion molecule 2 regulates diapedesis hotspots by allowing neutrophil crawling against the direction of flow. Vasc. Biol., 2023, 5: e230005.

[52] Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods, 2012, 9: 671-675.

[53] Suttie AW. Histopathology of the spleen. Toxicol. Pathol., 2006, 34: 466-503.

[54] O’Malley, D. P. Atlas of Anatomic Pathology (Springer New York, 2013).

[55] Bouxsein ML, et al.Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Miner. Res., 2010, 25: 1468-1486.

[56] Dempster DW, et al.Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res., 2013, 28: 2-17.

[57] Hao Y, et al.Integrated analysis of multimodal single-cell data. Cell, 2021, 184: 3573-3587 e3529.

[58] Armstrong S, Pereverzev A, Dixon SJ, Sims SM. Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts. J. Cell Sci., 2009, 122: 136-144.

[59] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25: 402-408.

[60] Tiedemann K, et al.Breast cancer-derived factors stimulate osteoclastogenesis through the Ca²⁺/protein kinase C and transforming growth factor-beta/MAPK signaling pathways. J. Biol. Chem., 2009, 284: 33662-33670.

Funding

Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)(PJT-165939)

Rights & permissions

The Author(s)