Cytokine and hormonal regulation of bone marrow immune cell Wnt10b expression

This study is funded by National Center for Complementary and Integrative Health (https://nccih.nih.gov/), U.S National Institutes of Health (Grant Code: 1R01AT007695-01) awarded to LRM and NP and by National Institute of Diabetes and Digestive and Kidney Diseases (www.niddk.nih.gov/), U.S National Institutes of Health (Grant code: R01DK101050) awarded to LRM and NP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Single-cell RNA-Seq reveals changes in immune landscape in post-traumatic osteoarthritis

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

Bone Marrow Wnt10b Expression Flow Cytometry

Analysis of antibody for the intracellular detection of Wnt10b in bone marrow immune cells by flow cytometry. <p><b></b></p

Cell type as percentage of Balb/c bone marrow.

<p>Cell type as percentage of Balb/c bone marrow.</p

Comparison of female WT and TNF^ko Wnt10b⁺ cells as percentage of total bone marrow.

<p>Comparison of female WT and TNF^ko Wnt10b⁺ cells as percentage of total bone marrow.</p

Comparison of male and female C57BL/6 bone marrow immune cell Wnt10b expression.

<p>Bone marrow (BM) was isolated from the femora of male (n = 22) and female (n = 5) C57BL/6 adult mice and analyzed by flow cytometry. Wnt10b MFI, % of cell type and % of total bone marrow was calculated. a) Female total BM expressed significantly higher levels of Wnt10b compared to males with significant cell-type specific sex-differences identified. b) Relative contribution of cell-types investigated to the Wnt10b⁺ population in males and females. c) Female total BM had a higher % of Wnt10b⁺ cells compared to males; cell-type specific differences in Wnt10b expression profiles were observed between the sexes. Statistical analysis performed by student’s t-test; *<i>p</i><0.05 and **<i>p</i><0.01.</p

Wnt10b gating profile.

<p>Gating profile for analyzing Wnt10b expression in bone marrow immune cells. Doublets were removed by singlet gating and debris removed from analysis. T cells were selected by gating on CD3⁺ cells then further refined by gating on CD4⁺ and CD8⁺. Macrophages were gated as F4/80⁺ and F4/80⁺ CD11c⁺. Dendritic cells were classified as F4/80^- CD11c⁺. Granulocytes were gated according to their FSc and SSc.</p

Comparison of male and TNF^ko Wnt10b⁺ cells as percentage of total bone marrow.

<p>Comparison of male and TNF^ko Wnt10b⁺ cells as percentage of total bone marrow.</p

Detection of Wnt10b by flow cytometry.

<p>Bone marrow (BM) was isolated from the femur of a male C57BL/6 mouse (n = 1) and cultured ± 50nM PTH for 3 hours. Cells were stained for Wnt10b and analyzed by flow cytometry, gating on bone marrow and CD8⁺ T cells. In the bone marrow population (a) a shift in peak was observed for the Wnt10b stained cells over the isotype control corresponding to approx. 1.5% of vehicle treated cells. PTH increased the number of Wnt10b⁺ BM cells to 3.5% and the MFI greater than 200-fold. Gating on the CD8⁺ T cells (b) in the vehicle treated group identified approximately 5% were Wnt10b⁺. Following PTH treatment this increased 3-fold.</p

Comparison of male and female Wnt10b⁺ cells as percentage of total bone marrow.

<p>Comparison of male and female Wnt10b⁺ cells as percentage of total bone marrow.</p