Osteoclasts modulate the balance between immunosuppression and inflammation through antigen presentation and interactions with CD4+ T cells

Poster presentationInternational audienc

A Novel Reliable and Efficient Procedure for Purification of Mature Osteoclasts Allowing Functional Assays in Mouse Cells

Osteoclasts (OCLs) are multinucleated phagocytes of monocytic origin responsible for physiological and pathological bone resorption including aging processes, chronic inflammation and cancer. Besides bone resorption, they are also involved in the modulation of immune responses and the regulation of hematopoietic niches. Accordingly, OCLs are the subject of an increasing number of studies. Due to their rarity and the difficulty to isolate them directly ex vivo, analyses on OCLs are usually performed on in vitro differentiated cells. In this state, however, OCLs represent a minority of differentiated cells. Since up to date a reliable purification procedure is still lacking for mature OCLs, all cells present in the culture are analyzed collectively to answer OCL-specific questions. With the development of in-depth transcriptomic and proteomic analyses, such global analyses on unsorted cells can induce severe bias effects in further results. In addition, for instance, analysis on OCL immune function requires working on purified OCLs to avoid contamination effects of monocytic precursors that may persist during the culture. This clearly highlights the need for a reliable OCL purification procedure. Here, we describe a novel and reliable method to sort OCLs based on cell multinucleation while preserving cell viability. Using this method, we successfully purified multinucleated murine cells. We showed that they expressed high levels of OCL markers and retained a high capacity of bone resorption, demonstrating that these are mature OCLs. The same approach was equally applied for the purification of human mature OCLs. Comparison of purified OCLs with mononucleated cells or unsorted cells revealed significant differences in the expression of OCL-specific markers at RNA and/or protein level. This exemplifies that substantially better outcomes for OCLs are achieved after the exclusion of mononucleated cells. Our results clearly demonstrate that the in here presented procedure for the analysis and sorting of pure OCLs represents a novel, robust and reliable method for the detailed examination of bona fide mature OCLs in a range that was previously impossible. Noteworthy, this procedure will open new perspectives into the biology of osteoclasts and osteoclast-related diseases

Saccharomyces boulardii Strain CNCM I-745 Modifies the Mononuclear Phagocytes Response in the Small Intestine of Mice Following Salmonella Typhimurium Infection

Intestinal mononuclear phagocytes (MPs) comprise dendritic cells (DCs) and macrophages (Mφs) that play different roles in response to Salmonella infection. After phagocytosis, DCs expressing CD103 transport Salmonella from the intestinal tract to the mesenteric lymph nodes (MLN) and induce adaptive immune responses whereas resident Mφs expressing CX3CR1 capture bacteria in the lumen and reside in the lamina propria (LP) where they induce a local immune response. CX3CR1+ Mφs are generated from Ly6Chi monocytes that enter the colonic mucosa and differentiate locally. We previously demonstrated that the probiotic yeast Saccharomyces boulardii CNCM I-745 (S.b) prevents infection by Salmonella enterica serovar Typhimurium (ST), decreases ST translocation to the peripheral organs and modifies the pro-and anti-inflammatory cytokine profiles in the gut. In the present study, we investigated the effect of S.b on the migratory CD103+ DCs and the resident CX3CR1+ Mφs. MPs were isolated from the LP of streptomycin-treated mice infected by ST with or without S.b treatment before or during the infection. In S.b-pretreated mice, we observed a decrease of the CD103+ DCs in the LP that was associated with the drop of ST recovery from MLN. Interestingly, S.b induced an infiltration of LP by classical Ly6Chi monocytes, and S.b modified the monocyte-Mφ maturation process in ST-infected mice. Our results showed that S.b treatment induced the expansion of Ly6Chi monocytes in the blood as well as in the bone marrow (BM) of mice, thus contributing to the Mφ replenishment in LP from blood monocytes. In vitro experiments conducted on BM cells confirmed that S.b induced the expansion of CX3CR1+ Mφs and concomitantly ST phagocytosis. Altogether, these data demonstrate that Saccharomyces boulardii CNCM I-745 modulates the innate immune response. Although here, we cannot explicitly delineate direct effects on ST from innate immunity, S. b-amplified innate immunity correlated with partial protection from ST infection. This study shows that S.b can induce the expansion of classical monocytes that are precursors of resident Mφs in the LP

Identification of an osteoclastogenic CD4+ T cell population producing IL-17 and TNFα

Poster presentationInternational audienc

Unravelling the sex-specific diversity and functions of adrenal gland macrophages

Despite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.</p

Emerging Roles of Osteoclasts in the Modulation of Bone Microenvironment and Immune Suppression in Multiple Myeloma

Multiple myeloma (MM) is one of the most common forms of hematologic malignancy resulting from cancerous proliferation of mature malignant plasma cells (MPCs). But despite the real improvement in therapeutics in the past years, it remains largely incurable. MM is the most frequent cancer to involve bone due to the stimulation of osteoclast (OCL) differentiation and activity. OCLs have a unique capacity to resorb bone. However, recent studies reveal that they are not restrained to this sole function. They participate in the control of angiogenesis, medullary niches, and immune responses, including in MM. Therefore, therapeutic approaches targeting OCLs probably affect not only bone resorption but also many other functions, and OCLs should not be considered anymore only as targets to improve the bone phenotype but also to modulate bone microenvironment. In this review, we explore these novel contributions of OCLs to MM which reveal their strong implication in the MM physiopathology. We also underline the therapeutic interest of targeting OCLs not only to overcome bone lesions, but also to improve bone microenvironment and anti-tumoral immune responses

Characterization of a novel bipotent hematopoietic progenitor population in normal and osteopetrotic mice.

Several reports indicate that osteoclasts and B-lymphocytes share a common progenitor. This study focuses on the characterization of this bipotent progenitor from the bone marrow of the osteopetrotic oc/oc mouse, where the bipotent progenitor population is amplified, and of normal mice. INTRODUCTION: Osteoclasts have a myelomonocytic origin, but they can also arise in vitro from pro-B-cells, suggesting that a subset of normal pro-B-cells is uncommitted and may reorient into the myeloid lineage representing a B-lymphoid/osteoclastic progenitor. The aim of this study was to characterize this progenitor population. MATERIALS AND METHODS: The osteopetrotic oc/oc mouse was used as a choice model because it displays an increased number of both osteoclasts and pro-B-cells in the bone marrow. Our results have been confirmed in normal littermates. Bone marrow cells from these animals were analyzed by flow cytometry. After sorting, the cells were cultured under different conditions to assess their differentiation capacity. RESULTS: Pro-B-cells from oc/oc and normal mice include an unusual biphenotypic population expressing markers from the B-lymphoid (CD19, CD43, CD5) and the myeloid (F4/80) lineages. This population also expresses progenitor markers (CD34 and Flt3) and is uncommitted. After sorting from the oc/oc bone marrow, this population is able to differentiate in vitro into osteoclast-like cells in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), into dendritic-like cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and TNFalpha, and into immature B-cells when seeded onto ST2 cells in the presence of IL-7. CONCLUSION: Our results show the existence of a novel bipotent biphenotypic hematopoietic progenitor population present in the bone marrow that has retained the capacity to differentiate into myeloid and B-lymphoid cells

Gut microbiome and bone

International audienc

Interleukin-7 partially rescues B-lymphopoiesis in osteopetrotic oc/oc mice through the engagement of B220+ CD11b+ progenitors.

OBJECTIVE: We recently identified in the mouse bone marrow a B-lymphoid/myeloid B220+ CD11b+ progenitor population. This population is accumulated in the osteopetrotic oc/oc mouse, which suggests that it could be controlled by bone marrow factors whose expression varies in this pathologic bone environment. Among the possible factors, interleukin (IL)-7 is involved in the control of B lymphopoiesis and osteoclastogenesis. Therefore, we hypothesized that IL-7 could regulate the accumulation of the B220+ CD11b+ population in oc/oc mice. METHODS: B220+ CD11b+ cells sorted from oc/oc mice were treated with IL-7 and their phenotype was analyzed by flow cytometry and real-time reverse transcriptase polymerase chain reaction (RT-PCR). In vivo, IL-7 was injected in oc/oc mice, and B220+ CD11b+ and B cells, as well as B-cell proliferation and apoptosis, were analyzed by flow cytometry. The expression of B lymphopoiesis and myelopoiesis markers was analyzed by real-time RT-PCR. RESULTS: In vitro, IL-7 induced the differentiation of B220+ CD11b+ cells into B lymphocytes through the induction of Pax5 and the inhibition of myeloid markers. In vivo, IL-7 injections in oc/oc mice induced a decrease of the B220+ CD11b+ population and the partial restoration of B-cell population, which was reduced in oc/oc mice. In parallel, upon IL-7 injections, Pax5 expression was induced in B220+ cells and B-cell apoptosis was reduced. CONCLUSIONS: Our results demonstrate that IL-7 injection can partially rescue B lymphopoiesis in oc/oc mice through the engagement of the B220+ CD11b+ population in the B-lymphoid pathway. Therefore, IL-7 delivery could represent a new therapeutic perspective to circumvent the lymphopenia observed in infantile malignant osteopetrosis patients