Oral Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Bone marrow neutrophil precursors inhibit osteoclast differentiation in vivo and in vitro (#185)

Blessing Crimeen-Irwin 1 , Tsuyoshi Isojima 1 2 , Narelle E McGregor 1 , Ryan C Chai 3 , Mriga Dutt 4 , Ingrid J Poulton 1 , Emma C Walker 1 , Benjamin L Parker 4 , Natalie A Sims 1 5 6
  1. St Vincent's Institute, Fitzroy, VIC, Australia
  2. Department of Pediatrics, Toronomon Hospital, Tokyo
  3. Garvan Institute of Medical Research, Darlinghurst
  4. Department of Anatomy & Physiology, The University of Melbourne, Melbourne
  5. Department of Medicine at St. Vincent's Hospital, The University of Melbourne, Melbourne, VIC, Australia
  6. Mary Mackillop Institute for Health Research, Australian Catholic University, Melbourne

Activated circulating neutrophils stimulate pathogenic bone destruction, but whether they control bone structure without inflammation is unclear. People with congenital neutropenia inconsistently exhibit osteopenia, and mice with neutropenia due to G-CSF (Granulocyte Colony Stimulating Factor) or G-CSF receptor (G-CSFR) deletion have no bone phenotype. Surprisingly, when G-CSFR null mice were crossed with mice with STAT3 hyperactivation in osteoblasts and osteocytes (Dmp1Cre.Socs3f/f mice) they had extremely high osteoclast numbers and lacked dense cortical bone. We hypothesized that neutrophils or their progenitors in marrow inhibit osteoclast formation in high bone remodelling states. To test this, Dmp1Cre.Socs3f/fand control mice were subjected to neutrophil depletion for 2 weeks by anti-Ly6G antibody treatment, or for 6 weeks by combining anti-Ly6G with a mouse IgG2a anti-rat antibody to maintain long-term depletion.

The protocols depleted neutrophils in the circulation, but had opposing effects on marrow neutrophil progenitors and bone mass. Anti-Ly6G treatment for two weeks increased marrow immature neutrophils by 25%. In Dmp1Cre.Socs3f/fbone, this lowered mRNA markers of osteoclasts (Dcstamp, Acp5) and osteoblasts (Col1a1), suggesting reduced remodeling. In contrast, 6 weeks of treatment reduced marrow neutrophil progenitors by 50%, doubled osteoclast surface without changing osteoblasts, and halved trabecular bone mass in control and Dmp1Cre.Socs3f/f mice. Both results suggest bone marrow neutrophil progenitors inhibit osteoclast formation in vivo.

Since pre-neutrophils, unlike mature neutrophils, are mitotic, we tested their direct action on osteoclastogenesis by co-culturing RAW264.7 osteoclast progenitors with pre-neutrophils FACS-purified from C57BL/6 mice. Pre-neutrophils dose-dependently inhibited osteoclast differentiation. This effect was halved by preventing cell-cell contact of the two populations with a membrane, suggesting close proximity or direct contact is needed.

In summary, neutrophil progenitors inhibit osteoclast differentiation in vivo and in vitro. This identifies a new cell population in the bone marrow environment that limits bone remodelling and supports the formation of cortical bone.