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

Abstract

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 IL1β. 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.

Osteocytes regulate senescence of bone and bone marrow

“…To corroborate the results observed from scRNA-seq, flow cytometry and further analysis were performed after removing adherent cells as previously reported ( Ding et al, 2022 ; Figure 5—figure supplement 1A and B ). Although there was no significant change in HSC (Lin - c-Kit + Sca1 + , LSK + cell) numbers between DTA het and WT mice ( Figure 5—figure supplement 2A and B ), DTA het mice demonstrated significantly increased number of short-term HSCs (ST-HSCs) with decreased number of long-term HSCs (LT-HSCs), indicating that HSCs in DTA het mice bone marrow were mobilized ( Figure 5—figure supplement 2C and D ).…”

“…In support of this, previous studies have suggested that progenitor cells can receive a long-lasting impact from the in vivo local microenvironment, where these cells are situated. Isolation of cells for in vitro cell differentiation or even transferring cells to healthy mice would not alter their original in vivo phenotypes ( Cao et al, 2020 ; Isaac et al, 2014 ; Ding et al, 2022 ; Edgar et al, 2021 ; Li et al, 2022 ).…”

“…Mice femurs and L3 lumbar were stripped of soft tissue and fixed in 4% PFA overnight at 4°C, then stored in 70% ethanol until scanned using the μCT instrument (SkyScan 1176). Relevant structure parameters of the μCT instrument were as previously reported ( Ding et al, 2022 ): scanning voxel size, 9 × 9 × 9 µm 3 ; X-ray tube potential, 50 kV and 450 μA; integration time, 520 ms; and rotation step, 0.4° for 180° scanning. CTAn micro-CT software version 1.13 (Bruker) was used to analyze the images.…”

Osteocytes regulate organismal senescence of bone and bone marrow

“…Mice femurs and L3 lumbar were stripped of soft tissue and fixed in 4% PFA overnight at 4 °C, then stored in 70% ethanol until scanned using the μCT instrument (SkyScan 1176). Relevant structure parameters of the μCT instrument were as previous reported(Ding et al 2022): scanning voxel size, 9×9×9 um 3 ; X-ray tube potential, 50 kV and 450 uA; integration time, 520 ms; rotation Step, 0.4° for 180° scanning. CTAn micro-CT software version 1.13 (Bruker) was used to analyze the images.…”

Roles of Mitochondrial DNA Damage in Kidney Diseases: A New Biomarker

“…TLR9 can act as a DNA recognition receptor and participate in the natural immune response in human body. A growing number of studies suggest that TLR9 plays an important role in the development of autoimmune diseases [ 40 , 41 ]. TLR9 is a member of the TLR family of proteins that are most closely related to mtDNA [ 42 ].…”