Despite knowledge the gut microbiota regulates bone mass, mechanisms governing the normal gut microbiota's osteoimmunomodulatory effects on skeletal remodeling and homeostasis are unclear in the healthy adult skeleton. Young adult specific-pathogen-free and germ-free mice were used to delineate the commensal microbiota's immunoregulatory effects on osteoblastogenesis, osteoclastogenesis, marrow T-cell hematopoiesis, and extra-skeletal endocrine organ function. We report the commensal microbiota has anti-anabolic effects suppressing osteoblastogenesis and pro-catabolic effects enhancing osteoclastogenesis, which drive bone loss in health. Suppression of Sp7(Osterix) and Igf1 in bone, and serum IGF1, in specific-pathogen-free mice suggest the commensal microbiota's anti-osteoblastic actions are mediated via local disruption of IGF1-signaling. Differences in the RANKL/OPG Axis in vivo, and RANKL-induced maturation of osteoclast-precursors in vitro, indicate the commensal microbiota induces sustained changes in RANKL-mediated osteoclastogenesis. Candidate mechanisms mediating commensal microbiota's pro-osteoclastic actions include altered marrow effector CD4 + T-cells and a novel Gut-Liver-Bone Axis. The previously unidentified Gut-LiverBone Axis intriguingly implies the normal gut microbiota's osteoimmunomodulatory actions are partly mediated via immunostimulatory effects in the liver. The molecular underpinnings defining commensal gut microbiota immunomodulatory actions on physiologic bone remodeling are highly relevant in advancing the understanding of normal osteoimmunological processes, having implications for the prevention of skeletal deterioration in health and disease.Gut microbiota interactions with the host modulates gastrointestinal processes, metabolism and immunity 1-5 , having implications for the development and homeostasis of host tissues 6,7 . Extensive research has focused on the commensal gut microbiota immunoregulatory effects in the context of resistance to pathogenic microbes and intestinal homeostasis 8,9 , and more recently investigations have begun to define the normal gut microbiota's role in the pathophysiology of metabolic and autoimmune disease states 4,6,9,10 . Central to this investigation, the commensal gut microbiota's influence on physiologic tissue remodeling and homeostasis at extra-gastrointestinal sites is largely unknown 11 . The study of osteoimmunology has elucidated that innate-immunity, marrow effector T-cells, and diverse endocrine organs regulate osteoclast-osteoblast mediated bone remodeling, both in health and disease [12][13][14][15][16][17] . Bone remodeling is a continuous dynamic skeletal renewal process in which monocyte-myeloid derived osteoclast cells resorb old bone matrix, and mesenchymal derived osteoblast cells subsequently form new bone matrix. Skeletal

Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1−/− mice we show that STAT1 is critical for mediating the recruitment of Ly6Chi iMOs into organs during L. donovani infection, and adaptive transfer of wild type Ly6Chi iMOs into STAT1−/− recipients renders them susceptible to disease. Our findings reveal an unexpected pathogenic role for Ly6Chi iMOs in promoting parasite survival in VL and open the possibility of targeting this population for host-directed therapy during VL.

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Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts.