RESUMO
Osteoarthritis (OA) is the most common joint disease, affecting over 300 million people world-wide. Accumulating evidence attests to the important roles of the immune system in OA pathogenesis. Understanding the role of various immune cells in joint degeneration or joint repair after injury is vital for improving therapeutic strategies for treating OA. Post-traumatic osteoarthritis (PTOA) develops in ~50% of individuals who have experienced an articular trauma like an anterior cruciate ligament (ACL) rupture. Here, using the high resolution of single-cell RNA sequencing, we delineated the temporal dynamics of immune cell accumulation in the mouse knee joint after ACL rupture. Our study identified multiple immune cell types in the joint including neutrophils, monocytes, macrophages, B cells, T cells, NK cells and dendritic cells. Monocytes and macrophage populations showed the most dramatic changes after injury. Further characterization of monocytes and macrophages reveled 9 major subtypes with unique transcriptomics signatures, including a tissue resident Lyve1hiFolr2hi macrophage population and Trem2hiFcrls+ recruited macrophages, both showing enrichment for phagocytic genes and growth factors such as Igf1, Pdgfa and Pdgfc. We also identified several genes induced or repressed after ACL injury in a cell type-specific manner. This study provides new insight into PTOA-associated changes in the immune microenvironment and highlights macrophage subtypes that may play a role in joint repair after injury.
Assuntos
Lesões do Ligamento Cruzado Anterior , Receptor 2 de Folato , Osteoartrite , Animais , Lesões do Ligamento Cruzado Anterior/complicações , Lesões do Ligamento Cruzado Anterior/genética , Humanos , Articulação do Joelho/patologia , Glicoproteínas de Membrana , Camundongos , Osteoartrite/genética , Osteoartrite/patologia , RNA-Seq , Receptores ImunológicosRESUMO
Recent studies in mouse models have shown that gut microbiota significantly influences bone health. We demonstrated that 2-week oral treatment with broad spectrum antibiotics followed by 4 weeks of recovery of the gut microbiota results in dysbiosis (microbiota imbalance)-induced bone loss in mice. Because gut microbiota is critical for the development of the immune system and since both microbiota and the immune system can regulate bone health, in this study, we tested the role of the immune system in mediating post-antibiotic dysbiosis-induced bone loss. For this, we treated wild-type (WT) and lymphocyte deficient Rag2 knockout (KO) mice with ampicillin/neomycin cocktail in water for 2 weeks followed by 4 weeks of water without antibiotics. This led to a significant bone loss (31% decrease from control) in WT mice. Interestingly, no bone loss was observed in the KO mice suggesting that lymphocytes are required for dysbiosis-induced bone loss. Bray-Curtis diversity metrics showed similar microbiota changes in both the WT and KO post-antibiotic treated groups. However, several operational taxonomic units (OTUs) classified as Lactobacillales were significantly higher in the repopulated KO when compared to the WT mice, suggesting that these bacteria might play a protective role in preventing bone loss in the KO mice after antibiotic treatment. The effect of dysbiosis on bone was therefore examined in the WT mice in the presence or absence of oral Lactobacillus reuteri treatment for 4 weeks (post-ABX treatment). As hypothesized, mice treated with L. reuteri did not display bone loss, suggesting a bone protective role for this group of bacteria. Taken together, our studies elucidate an important role for lymphocytes in regulating post-antibiotic dysbiosis-induced bone loss.
Assuntos
Antibacterianos , Reabsorção Óssea , Disbiose , Microbioma Gastrointestinal , Animais , Reabsorção Óssea/microbiologia , Osso Esponjoso , Disbiose/induzido quimicamente , Linfócitos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Glucocorticoids (GCs) are potent immune-modulating drugs with significant side effects, including glucocorticoid-induced osteoporosis (GIO). GCs directly induce osteoblast and osteocyte apoptosis but also alter intestinal microbiota composition. Although the gut microbiota is known to contribute to the regulation of bone density, its role in GIO has never been examined. To test this, male C57/Bl6J mice were treated for 8 weeks with GC (prednisolone, GC-Tx) in the presence or absence of broad-spectrum antibiotic treatment (ABX) to deplete the microbiota. Long-term ABX prevented GC-Tx-induced trabecular bone loss, showing the requirement of gut microbiota for GIO. Treatment of GC-Tx mice with a probiotic (Lactobacillus reuteri [LR]) prevented trabecular bone loss. Microbiota analyses indicated that GC-Tx changed the abundance of Verrucomicobiales and Bacteriodales phyla and random forest analyses indicated significant differences in abundance of Porphyromonadaceae and Clostridiales operational taxonomic units (OTUs) between groups. Furthermore, transplantation of GC-Tx mouse fecal material into recipient naïve, untreated WT mice caused bone loss, supporting a functional role for microbiota in GIO. We also report that GC caused intestinal barrier breaks, as evidenced by increased serum endotoxin level (2.4-fold), that were prevented by LR and ABX treatments. Enhancement of barrier function with a mucus supplement prevented both GC-Tx-induced barrier leakage and trabecular GIO. In bone, treatment with ABX, LR or a mucus supplement reduced GC-Tx-induced osteoblast and osteocyte apoptosis. GC-Tx suppression of Wnt10b in bone was restored by the LR and high-molecular-weight polymer (MDY) treatments as well as microbiota depletion. Finally, we identified that bone-specific Wnt10b overexpression prevented GIO. Taken together, our data highlight the previously unappreciated involvement of the gut microbiota and intestinal barrier function in trabecular GIO pathogenesis (including Wnt10b suppression and osteoblast and osteocyte apoptosis) and identify the gut as a novel therapeutic target for preventing GIO. © 2019 American Society for Bone and Mineral Research.
Assuntos
Microbioma Gastrointestinal , Osteoporose , Animais , Densidade Óssea , Glucocorticoides/toxicidade , Masculino , Camundongos , Osteoblastos , Osteoporose/induzido quimicamente , Osteoporose/tratamento farmacológicoRESUMO
Oral treatment with probiotic bacteria has been shown to prevent bone loss in multiple models of osteoporosis. In previous studies we demonstrated that oral administration of Lactobacillus reuteri in healthy male mice increases bone density. The host and bacterial mechanisms of these effects however are not well understood. The objective of this study was to understand the role of lymphocytes in mediating the beneficial effects of L. reuteri on bone health in male mice. We administered L. reuteri in drinking water for 4 weeks to wild type or Rag knockout (lack mature T and B lymphocytes) male mice. While L. reuteri treatment increased bone density in wild type, no significant increases were seen in Rag knockout mice, suggesting that lymphocytes are critical for mediating the beneficial effects of L. reuteri on bone density. To understand the effect of L. reuteri on lymphocytes in the intestinal tissues, we isolated mesenteric lymph node (MLN) from naïve wild type mice. In ex vivo studies using whole mesenteric lymph node (MLN) as well as CD3+ T-cells, we demonstrate that live L. reuteri and its secreted factors have concentration-dependent effects on the expression of cytokines, including anti-inflammatory cytokine IL-10. Fractionation studies identified that the active component of L. reuteri is likely water soluble and small in size (<3 kDa) and its effects on lymphocytes are negatively regulated by a RIP2 inhibitor, suggesting a role for NOD signaling. Finally, we show that T-cells from MLNs treated with L. reuteri supernatants, secrete factors that enhance osterix (transcription factor involved in osteoblast differentiation) expression in MC3T3-E1 osteoblasts. Together, these data suggest that L. reuteri secreted factors regulate T-lymphocytes which play an important role in mediating the beneficial effects of L. reuteri on bone density.
Assuntos
Densidade Óssea , Interações entre Hospedeiro e Microrganismos/imunologia , Limosilactobacillus reuteri/metabolismo , Osteoblastos/metabolismo , Probióticos/farmacologia , Linfócitos T/imunologia , Animais , Diferenciação Celular , Linhagem Celular , Proteínas de Homeodomínio/genética , Interleucina-10/metabolismo , Intestinos , Linfonodos/citologia , Masculino , Mesentério , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Probióticos/administração & dosagem , Fator de Transcrição Sp7/metabolismoRESUMO
Antibiotic treatment, commonly prescribed for bacterial infections, depletes and subsequently causes long-term alterations in intestinal microbiota composition. Knowing the importance of the microbiome in the regulation of bone density, we investigated the effect of postantibiotic treatment on gut and bone health. Intestinal microbiome repopulation at 4-weeks postantibiotic treatment resulted in an increase in the Firmicutes:Bacteroidetes ratio, increased intestinal permeability, and notably reduced femoral trabecular bone volume (approximately 30%, p < 0.01). Treatment with a mucus supplement (a high-molecular-weight polymer, MDY-1001 [MDY]) prevented the postantibiotic-induced barrier break as well as bone loss, indicating a mechanistic link between increased intestinal permeability and bone loss. A link between the microbiome composition and bone density was demonstrated by supplementing the mice with probiotic bacteria. Specifically, Lactobacillus reuteri, but not Lactobacillus rhamnosus GG or nonpathogenic Escherichia coli, reduced the postantibiotic elevation of the Firmicutes:Bacteroidetes ratio and prevented femoral and vertebral trabecular bone loss. Consistent with causing bone loss, postantibiotic-induced dysbiosis decreased osteoblast and increased osteoclast activities, changes that were prevented by both L. reuteri and MDY. These data underscore the importance of microbial dysbiosis in the regulation of intestinal permeability and bone health, as well as identify L. reuteri and MDY as novel therapies for preventing these adverse effects. © 2018 American Society for Bone and Mineral Research.
Assuntos
Antibacterianos/efeitos adversos , Reabsorção Óssea , Disbiose , Microbioma Gastrointestinal/efeitos dos fármacos , Limosilactobacillus reuteri , Probióticos/farmacologia , Animais , Antibacterianos/farmacologia , Bacteroides/classificação , Bacteroides/crescimento & desenvolvimento , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/microbiologia , Reabsorção Óssea/patologia , Reabsorção Óssea/prevenção & controle , Disbiose/induzido quimicamente , Disbiose/microbiologia , Disbiose/prevenção & controle , Firmicutes/classificação , Firmicutes/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
In recent years a link between the gastrointestinal tract and bone health has started to gain significant attention. Dysbiosis of the intestinal microbiota has been linked to the pathology of a number of diseases which are associated with bone loss. In addition modulation of the intestinal microbiota with probiotic bacteria has revealed to have both beneficial local and systemic effects. In the present chapter, we discuss the intestinal and bone immune systems, explore how intestinal disease affects the immune system, and examine how these pathologic changes could adversely impact bone health.
Assuntos
Osso e Ossos/imunologia , Trato Gastrointestinal/imunologia , Sistema Imunitário/imunologia , Transdução de Sinais/imunologia , Animais , Remodelação Óssea/imunologia , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Trato Gastrointestinal/citologia , Trato Gastrointestinal/metabolismo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Enteropatias/imunologia , Linfócitos/imunologia , Linfócitos/metabolismoRESUMO
The intestinal epithelial barrier plays an essential role in maintaining host homeostasis. The barrier regulates nutrient absorption as well as prevents the invasion of pathogenic bacteria in the host. It is composed of epithelial cells, tight junctions, and a mucus layer. Several factors, such as cytokines, diet, and diseases, can affect this barrier. These factors have been shown to increase intestinal permeability, inflammation, and translocation of pathogenic bacteria. In addition, dysregulation of the epithelial barrier can result in inflammatory diseases such as inflammatory bowel disease. Our lab and others have also shown that barrier disruption can have systemic effects including bone loss. In this chapter, we will discuss the current literature to understand the link between intestinal barrier and bone. We will discuss how inflammation, aging, dysbiosis, and metabolic diseases can affect intestinal barrier-bone link. In addition, we will highlight the current suggested mechanism between intestinal barrier and bone.
Assuntos
Osso e Ossos/fisiologia , Trato Gastrointestinal/fisiologia , Mucosa Intestinal/fisiologia , Transdução de Sinais , Junções Íntimas/fisiologia , Animais , Disbiose/fisiopatologia , Humanos , Inflamação/fisiopatologia , Mucosa Intestinal/metabolismo , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismoRESUMO
BACKGROUND & AIMS: Wnt10b is a crucial regulator of bone density through its ability to promote osteoblastogenesis. Parathyroid hormone has been shown to regulate Wnt10b expression in CD8+ T cells. However, the relative expression and other source(s) of Wnt10b in the bone marrow immune cells (BMICs) is unknown. Sex hormones and cytokines such as, estrogen and TNFα are critical regulators of bone physiology but whether they regulate BMIC Wnt10b expression is unclear. To determine the potential regulation of Wnt10b by estrogen and TNFα, we assessed Wnt10b expression by flow cytometry under estrogen- and TNFα-deficient conditions. METHODS: Effects of TNFα was determined in male and female C57BL/6 wildtype and TNFα knockout mice. Effect of estrogen was investigated 4, 6 and 8 weeks post-surgery in ovariectomized Balb/c mice. Intracellular Wnt10b was detected using goat anti-mouse Wnt10b and a conjugated secondary antibody and analyzed by flow cytometry. RESULTS: Wnt10b expression was sex- and lineage-specific. Females had 1.8-fold higher Wnt10b signal compared to males. Percent of Wnt10b+ myeloid cells was higher in females than males (8.9% Vs 5.4%) but Wnt10b+ lymphoid cells was higher in males than females (6.3% Vs 2.5%). TNFα ablation in males increased total BM Wnt10b expression 1.5-fold but significantly reduced the percentage of BM Wnt10b+ CD4+ T cells (65%), CD8+ T cells (59%), dendritic cells (59%), macrophages (56%) and granulocytes (52%). These effects of TNFα on Wnt10b were observed only in males. In contrast to TNFα, estrogen-deficiency had indirect effects on BMIC Wnt10b levels; reducing the average percentage of BM Wnt10b+ CD8+ T cells (25%) and granulocytes (26%) across an 8-week time course. CONCLUSION: Our results demonstrate unique cell type- and sex-dependent effects on BMIC Wnt10b expression. Together, our results reveal myeloid cells in the bone marrow as an important source of Wnt10b under complex hormonal and cytokine regulation.