Toll-like receptor 9 deficiency induces osteoclastic bone loss via gut microbiota-associated systemic chronic inflammation.

Toll-like receptors (TLRs) play pivotal roles in inflammation and provide important links between the immune and skeletal systems. Although the activation of TLRs may affect osteoclast differentiation and bone metabolism, whether and how TLRs are required for normal bone remodeling remains to be fully explored. In the current study, we show for the first time that TLR9-/- mice exhibit a low bone mass and low-grade systemic chronic inflammation, which is characterized by the expansion of CD4+ T cells and increased levels of inflammatory cytokines, including TNFα, RANKL, and IL1ß. The increased levels of these cytokines significantly promote osteoclastogenesis and induce bone loss. Importantly, TLR9 deletion alters the gut microbiota, and this dysbiosis is the basis of the systemic inflammation and bone loss observed in TLR9-/- mice. Furthermore, through single-cell RNA sequencing, we identified myeloid-biased hematopoiesis in the bone marrow of TLR9-/- mice and determined that the increase in myelopoiesis, likely caused by the adaptation of hematopoietic stem cells to systemic inflammation, also contributes to inflammation-induced osteoclastogenesis and subsequent bone loss in TLR9-/- mice. Thus, our study provides novel evidence that TLR9 signaling connects the gut microbiota, immune system, and bone and is critical in maintaining the homeostasis of inflammation, hematopoiesis, and bone metabolism under normal conditions.

IL-10 Deficiency Accelerates Type 1 Diabetes Development via Modulation of Innate and Adaptive Immune Cells and Gut Microbiota in BDC2.5 NOD Mice.

Type 1 diabetes is an autoimmune disease caused by T cell-mediated destruction of insulin-producing ß cells. BDC2.5 T cells in BDC2.5 CD4+ T cell receptor transgenic Non-Obese Diabetic (NOD) mice (BDC2.5+ NOD mice) can abruptly invade the pancreatic islets resulting in severe insulitis that progresses rapidly but rarely leads to spontaneous diabetes. This prevention of diabetes is mediated by T regulatory (Treg) cells in these mice. In this study, we investigated the role of interleukin 10 (IL-10) in the inhibition of diabetes in BDC2.5+ NOD mice by generating Il-10-deficient BDC2.5+ NOD mice (BDC2.5+Il-10-/- NOD mice). Our results showed that BDC2.5+Il-10-/- NOD mice displayed robust and accelerated diabetes development. Il-10 deficiency in BDC2.5+ NOD mice promoted the generation of neutrophils in the bone marrow and increased the proportions of neutrophils in the periphery (blood, spleen, and islets), accompanied by altered intestinal immunity and gut microbiota composition. In vitro studies showed that the gut microbiota from BDC2.5+Il-10-/- NOD mice can expand neutrophil populations. Moreover, in vivo studies demonstrated that the depletion of endogenous gut microbiota by antibiotic treatment decreased the proportion of neutrophils. Although Il-10 deficiency in BDC2.5+ NOD mice had no obvious effects on the proportion and function of Treg cells, it affected the immune response and activation of CD4+ T cells. Moreover, the pathogenicity of CD4+ T cells was much increased, and this significantly accelerated the development of diabetes when these CD4+ T cells were transferred into immune-deficient NOD mice. Our study provides novel insights into the role of IL-10 in the modulation of neutrophils and CD4+ T cells in BDC2.5+ NOD mice, and suggests important crosstalk between gut microbiota and neutrophils in type 1 diabetes development.

Impairment of circulating endothelial progenitor cells (EPCs) in patients with glucocorticoid-induced avascular necrosis of the femoral head and changes of EPCs after glucocorticoid treatment in vitro.

BACKGROUND: Avascular necrosis of the femoral head (ANFH) is a severe complication after high-dose glucocorticoid (GC) administration. The pathogenesis of GC-induced ANFH remains unclear. Though the important role of endothelial progenitor cells (EPCs) in the progression of GC-induced ANFH has been noticed, the effects of GCs on EPCs and the underlying mechanism still need further study. METHODS: Circulating EPCs were obtained from the peripheral blood of ANFH patients and healthy controls by Ficoll-density gradient centrifugation. CD133+CD34+ cells with DiI-Ac-LDL uptake and FITC-UEA-1 binding were considered as EPCs. Number and functions of EPCs were analyzed by flow cytometry, chemotaxis assay, and tube formation assay. EPCs from healthy controls were also treated by different concentrations of methylprednisolone and prednisolone in vitro, and cell growth and angiogenic function were evaluated. Expression of CXCR7 and its downstream Akt/GSK-3ß/Fyn pathway were also analyzed by western blots after cells treated by methylprednisolone in vitro. RESULTS: The number and functions of EPCs in patients with GC-induced ANFH were significantly decreased. In vitro study showed for the first time that except extremely high concentrations, low to medium concentrations of GCs did not have significant effects on EPCs' growth. Methylprednisolone and prednisolone both inhibited angiogenesis of EPCs even at low concentrations. Mechanism studies found CXCR7 was downregulated in EPCs after methylprednisolone treatment in vitro. Expression and phosphorylation of Akt and GSK-3ß were also decreased with an upregulation of Fyn expression after steroid treatment. CONCLUSIONS: Our study showed that GC-induced ANFH patients have reduced the number and impaired functions of circulating EPCs. GCs did not show a significant effect on the growth of EPCs in vitro except extremely high concentrations of GCs. However, GCs significantly impaired EPC angiogenic function in vitro, even at low concentrations. Our study also suggested that downregulation of CXCR7 and its downstream Akt/GSK-3ß/Fyn pathway in EPCs might be a novel mechanism of how GCs suppress EPCs' angiogenesis.

Differentially methylated circulating DNA: A novel biomarker to monitor beta cell death.

Diabetes mellitus (DM) is a metabolic disorder of glucose homeostasis caused by insufficient secretion or inadequate action of insulin. Nowadays, the increased morbidity of DM is a worldwide issue. Pancreatic beta cell death plays a key role in the progress of DM, especially Type 1 diabetes (T1D). Traditional biomarkers, such as C-peptide and islet autoimmune antibodies are limited to reflect beta cell death and to identify high risk patients in the clinical practice. Recently, a novel biomarker, differentially methylated circulating DNA, has become a research hotspot. It has better sensitivity and specificity in the detection of beta cell death. Assays of beta cell-derived differentially methylated insulin DNA in serum are helpful to predict the possibility to develop T1D in the high risk population. They have also been applied to evaluate beta cell death in Type 2 diabetes (T2D), gestational diabetes mellitus (GDM), islet transplantation and islet specific immune therapy. Although more studies are needed to identify the best methylation target sites in the INS gene, differentially methylated circulating DNA may be a good method to evaluate the progression and prognosis of islet related diseases in the future.

Activation-induced cytidine deaminase deficiency accelerates autoimmune diabetes in NOD mice.

B cells play an important role in type 1 diabetes (T1D) development. However, the role of B cell activation-induced cytidine deaminase (AID) in diabetes development is not clear. We hypothesized that AID is important in the immunopathogenesis of T1D. To test this hypothesis, we generated AID-deficient (AID-/-) NOD mice. We found that AID-/-NOD mice developed accelerated T1D, with worse insulitis and high levels of anti-insulin autoantibody in the circulation. Interestingly, neither maternal IgG transferred through placenta, nor IgA transferred through milk affected the accelerated diabetes development. AID-/-NOD mice showed increased activation and proliferation of B and T cells. We found enhanced T-B cell interactions in AID-/-NOD mice, with increased T-bet and IFN-γ expression in CD4+ T cells in the presence of AID-/- B cells. Moreover, excessive lymphoid expansion was observed in AID-/-NOD mice. Importantly, antigen-specific BDC2.5 CD4+ T cells caused more rapid onset of diabetes when cotransferred with AID-/- B cells than when cotransferred with AID+/+ B cells. Thus, our study provides insights into the role of AID in T1D. Our data also suggest that AID is a negative regulator of immune tolerance and ablation of AID can lead to exacerbated islet autoimmunity and accelerated T1D development.

IRAK-M deficiency promotes the development of type 1 diabetes in NOD mice.

Type 1 diabetes mellitus (T1DM) is an organ-specific autoimmune disease characterized by progressive destruction of insulin-secreting pancreatic ß-cells. Both T-cell-mediated adaptive responses as well as innate immune processes are involved in pathogenesis. Interleukin-1 receptor-associated kinase M (IRAK-M) can effectively inhibit the MyD88 downstream signals in Toll-like receptor pathways, while lack of IRAK-M is known to be associated with autoimmunity. Our study showed that IRAK-M-deficient (IRAK-M(-/-)) nonobese diabetic (NOD) mice displayed early onset and rapid progression of T1DM with impaired glucose tolerance, more severe insulitis, and increased serum anti-insulin autoantibodies. Mechanistic studies showed that the enhanced activation and antigen-presenting function of IRAK-M(-/-) antigen-presenting cells from IRAK-M(-/-) mice were responsible for the rapid progression of disease. Moreover, IRAK-M(-/-) dendritic cells induced enhanced activation of diabetogenic T cells in vitro and the rapid onset of T1DM in vivo in immunodeficient NOD mice when cotransferred with diabetogenic T cells. This study illustrates how the modulation of innate immune pathways through IRAK-M influences the development of autoimmune diabetes.