Jean-Pierre Levesque

Background: Neurogenic heterotopic ossifications (NHOs) are heterotopic bones that develop in periarticular muscles after traumatic brain (TBI) and spinal cord injuries (SCI). The mechanisms leading to NHO are incompletely understood and the only effective treatment to-date remains surgical resection. We previously established that several inflammatory pathways drive NHO pathogenesis in injured muscles in a mouse model of NHO and in humans. We also demonstrated a functional association between gram-negative bacterial infections and NHO development via lipopolysaccharide (LPS), a pathogen associated molecular pattern (PAMP), which exacerbated NHO in a Toll-like receptor-4 (TLR4)-dependent manner in mice. Methods: Using our mouse model of NHO induced by SCI and muscle injury in mice, we tested the effect of a large array of purified PAMPs post-surgery to mimic fungal, viral and bacterial infections and measured NHO bone volumes by micro-computerized tomography (microCT). The effect of PAMPs was also tested in vitro on human muscle progenitors and monocyte/macrophage populations. Results: Muscle progenitors and monocyte/macrophage populations from humans and mice express numerous pattern recognition receptors. In mice, numerous PAMPs produced by bacteria, viruses and fungi exacerbated NHO formation, and the majority of these PAMPs indirectly stimulated fibro-adipogenic progenitor (FAP) calcium mineralization in vitro via macrophages. Likewise, in humans, some PAMPs, particularly those binding to TLR2, directly and indirectly increased the calcium mineralization and osteogenic differentiation of human FAPs isolated from muscles surrounding human NHO. Finally, we established that the indirect stimulation of human FAP mineralization was mediated by inflammatory cytokines IL-1 and oncostatin M secreted by monocytes in response to PAMPs. Overall, our findings suggest that numerous types of infection have the potential to exacerbate NHO development and further highlight the role of oncostatin M and IL-1 signaling pathways in NHO pathophysiology.

The oncostatin M receptor (OSMR) has recently emerged as an adverse prognostic factor in acute myeloid leukemia (AML) and several non-hematological malignancies. In this perspective, we discuss how oncostatin M (OSM) and its receptor OSMR regulate tumor cells as well as mesenchymal and endothelial cells, which are key components of hematopoietic stem cell and tumor stem cell niches, and how these mechanisms could explain the poor prognosis associated with high expression of OSM and OSMR in hematological and non-hematological malignancies.

Purpose of review: The skeletal system is one of the main targets of endocrine hormone dysregulation. After briefly summarizing the endocrinal regulation of the skeleton, this review focuses on the recent advances in our understanding of how endocrine dysregulations promote the development of heterotopic bones after trauma. Recent findings: Endocrinal regulation of skeletal fetal development, and growth is well established. However, whether trauma such as central nervous system insults, hip arthroplasty and severe burns trigger heterotopic ossifications via hormonal dysregulation has just begun to be investigated. Recent findings in mouse models of trauma-induced and most particularly spinal cord injury-induced heterotopic ossifications suggest that spinal cord injury leads to stimulation of catecholamine and glucocorticoid secretion by adrenal glands early post-injury, which triggers pathological bone formation in injured muscles. Summary: This review summarizes the recently identified roles of adrenal hormones in driving pathological heterotopic ossification development after spinal cord injury. Further research into how endocrine hormones regulate heterotopic bone formation after trauma is essential and likely to reveal new targetable pathways to prevent pathological heterotopic ossifications.

Purpose of the Review: The bone and hematopoietic tissues coemerge during development and are functionally intertwined throughout mammalian life. Oncostatin M (OSM) is an inflammatory cytokine of the interleukin-6 family produced by osteoblasts, bone marrow macrophages, and neutrophils. OSM acts via two heterodimeric receptors comprising GP130 with either an OSM receptor (OSMR) or a leukemia inhibitory factor receptor (LIFR). OSMR is expressed on osteoblasts, mesenchymal, and endothelial cells and mice deficient for the Osm or Osmr genes have both bone and blood phenotypes illustrating the importance of OSM and OSMR in regulating these two intertwined tissues. Recent Findings: OSM regulates bone mass through signaling via OSMR, adaptor protein SHC1, and transducer STAT3 to both stimulate osteoclast formation and promote osteoblast commitment; the effect on bone formation is also supported by action through LIFR. OSM produced by macrophages is an important inducer of neurogenic heterotopic ossifications in peri-articular muscles following spinal cord injury. OSM produced by neutrophils in the bone marrow induces hematopoietic stem and progenitor cell proliferation in an indirect manner via OSMR expressed by bone marrow stromal and endothelial cells that form hematopoietic stem cell niches. OSM acts as a brake to therapeutic hematopoietic stem cell mobilization in response to G-CSF and CXCR4 antagonist plerixafor. Excessive OSM production by macrophages in the bone marrow is a key contributor to poor hematopoietic stem cell mobilization (mobilopathy) in people with diabetes. OSM and OSMR may also play important roles in the progression of several cancers. Summary: It is increasingly clear that OSM plays unique roles in regulating the maintenance and regeneration of bone, hematopoietic stem and progenitor cells, inflammation, and skeletal muscles. Dysregulated OSM production can lead to bone pathologies, defective muscle repair and formation of heterotopic ossifications in injured muscles, suboptimal mobilization of hematopoietic stem cells, exacerbated inflammatory responses, and anti-tumoral immunity. Ongoing research will establish whether neutralizing antibodies or cytokine traps may be useful to correct pathologies associated with excessive OSM production.

The erythroblastic island (EBI) is a multicellular functional erythropoietic unit comprising a central macrophage nurturing a rosette of maturing erythroblasts. Since the discovery of EBIs more than half a century ago, EBIs are still studied by traditional microscopy methods after enrichment by sedimentation. These isolation methods are not quantitative and do not enable precise quantification of EBI numbers or frequency in the bone marrow or spleen tissues. Conventional flow cytometric methods have enabled quantification of cell aggregates co-expressing macrophage and erythroblast markers; however, it is unknown whether these aggregates contain EBIs as these aggregates cannot be visually assessed for EBI content. Combining the strengths of both microscopy and flow cytometry methods, in this chapter we describe an imaging flow cytometry method to analyze and quantitatively measure EBIs from the mouse bone marrow. This method is adaptable to other tissues such as the spleen or to other species provided that fluorescent antibodies specific to macrophages and erythroblasts are available. Key words Erythroblastic island Macrophage Imaging flow cytometry Erythropoiesis

The cells of origin of neurogenic heterotopic ossifications (NHOs), which develop frequently in the periarticular muscles following spinal cord injuries (SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells (SCs), which are myogenic, and fibroadipogenic progenitors (FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα⁺ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56⁺ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.

Neurogenic heterotopic ossifications (NHOs) are incapacitating complications of traumatic brain and spinal cord injuries (SCI) that manifest as abnormal bone formation in periarticular muscles. Using a unique model of NHO after SCI in genetically unmodified mice, we have previously established that the innate immune system plays a key driving role in NHO pathogenesis. The role of adaptive immune cells in NHO pathogenesis, however, remains unexplored in this model. Here we established that B lymphocytes were reduced in the spleen and blood after SCI and increased in muscles of mice in which NHO develops, whereas minimal changes in T cell frequencies were noted. Interestingly, Rag1 ^-/- mice lacking mature T and B lymphocytes, developed NHO, similar to wild-type mice. Finally, mice that underwent splenectomy before SCI and muscle damage also developed NHO to the same extent as non-splenectomized SCI controls. Overall, our findings show that functional T and B lymphocytes have minimal influence or dispensable contributions to NHO development after experimental SCI in mice.

This article answers an old question of great clinical significance: What genetic determinants control the number of hematopoietic stem and progenitor cells present in an individual? Indeed, successful engraftment of transplanted hematopoietic stem cells is largely dependent on the dose of CD34+ HSPC transplanted. Below the threshold of 2x10⁶ CD34⁺cells/kg, engraftment is not warranted while above that threshold, the higher the number of CD34⁺ cells transplanted, the faster, more complete hematopoietic reconstitution is with higher survival {1,2}. The most common source of these HSPC is mobilized blood because daily injections of granulocyte colony-stimulating factor (G-CSF) in healthy allogenic donors or autologous patients mobilizes HSPC from the bone marrow where they normally reside into the blood. Mobilized blood leukocytes collected by apheresis and CD34⁺ cells selected by large-scale magnetism activated cell sorting before being transplanted. Between individuals, there is also a strong positive correlation between the number of CD34⁺ HSPC mobilized with G-CSF and the number of circulating CD34⁺ in basal condition prior to mobilization {3,4} reflecting that the level of HSPC mobilization is correlated with the initial number of HSPC present in the body (and thus available for mobilization). Studies into the mechanisms that maintain HSPC within the bone marrow have highlighted the importance of the chemotactic interaction between the chemokine CXCL12 and its receptor CXCR4 expressed by HSPC {5}, which led to the development of CXCR4 antagonists currently approved to boost HSPC mobilization {6} particularly in the autologous setting {7,8}. However, another strategy would be to find treatments that increase HSPC pool size prior to mobilization {9}. This paper is an important step in this direction. Although studies in different inbred mouse strains evidenced the genetic dependence of HSC pool size and mobilization in response to G-CSF in the late 1990s {10,11}, technical limitations of the time-limited the scope of these studies. Using a powerful genome-wide association study (GWAS) in 13,167 healthy blood donors residing in Sweden, the authors identified 7 genes in which single nucleotide polymorphisms (SNPs) are significantly associated with a higher number of circulating CD34⁺ HSPC in basal conditional. These genes are CXCR4 (not surprisingly as CXCR4 SNPs had already been associated with higher/lower mobilization), CEBPA a transcription factor controlling myelopoiesis, ITGA9 encoding integrin α9 which plays a role in HSPC mobilization {12}, and 5 genes with unknown roles in hematopoiesis: PPM1H, ENO1, RERE, and ARHGAP45. Surprisingly the gene with SNP variants most significantly associated with high basal level CD34⁺ HSPC was PPM1H. PPM1H encodes protein phosphatase Mg²⁺/Ca²⁺-dependent 1 which is expressed by most immature HSPC. Intriguingly the variants of this gene found in donors with high CD34+ HSPC counts corresponded to low PPM1H expression. It was then found that these SNPs are all located in the 1^st intron of the PPM1H gene in a region that loops between two promoters of the gene and is a target of the hematopoietic transcription factor MYB. Finally, in reporter assays and knock-down experiments, the authors show that the G>A variant allele in the rs772557 SNP abolishes binding of MYB and down-regulates PPM1H expression in adequation with lower PPM1H expression in carriers of the rs772557A allele. This study is noteworthy because it is the first to identify SNPs associated with higher CD34⁺ HSPC counts at basal levels and suggest that specific inhibitors of PPM1H phosphatase activity may be useful drugs to expand HSPC numbers in patients requiring autologous HSPC transplantation.