GSE185521
Transcriptional signatures of bone marrow mononuclear cell-mediated resolution of synovitis
synovial macrophages through joint injection with bone marrow mononuclear cells (BMNC) induces lasting synovial inflammation  resolution. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of  BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of  co-expressed gene networks associated with traits from our previous in vivo and in vitro studies. We also identified activated  and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of  inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response  characterized by a short spike in the expression of a NF-?B- and mitogen-related gene network. This response was associated  with sustained increased expression of another gene network comprising known drivers of resolution (IL-10, IGF-1, PPARG,  isoprenoid biosynthesis). This network was common to SF and ISF, but more highly expressed in ISF and. Similarly, most highly  activated pathways in ISF included the super pathway of cholesterol biosynthesis and PPAR-? signaling, with pro-resolving functional  annotations that improve mitochondrial metabolism and deactivate NF-?B signaling. Higher expression of the PPAR-? c0-activator  1-? in synovium from inflamed joints treated with BMNC, and equivalent IL-1ß staining between BMNC- and DPBS-treated joints,  emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest  that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are  enhanced following tissue damage. These mechanisms translate into macrophage proliferation optimizing their capacity to  counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while  driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis.  Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further  investigation on the effects of PPAR-? signaling enhancement for the treatment of arthritic conditions. Overall design: Samples used in the current report were obtained from two previous studies using the same horses. These in vitro (Menarim et al, FASEB J, 2020) and in vivo (Menarim et al, FASEB J, 2021) studies were counterparts of a larger project assessing the effects of BMNC on joint inflammation resolution. Briefly, eight skeletally mature Thoroughbred horses (3-9 years old, median 5 years; 2 females and 6 castrated males) free of OA or systemic inflammation were used under IACUC approval and oversight. General and musculoskeletal health were confirmed by clinical, hematological and orthopedic evaluations. Following sternal bone marrow aspiration for BMNC isolation, synovitis was induced in both radiocarpal joints, as a way of producing more homogeneous inflammation and inflamed synovial fluid (ISF)  than could be acquired from naturally occurring OA. Normal synovial fluid (SF) was collected from healthy middle carpal joints. BMNC from each horse were cultured independently (not pooled) in neat (100%) autologous SF or ISF and harvested at 0 (uncultured), 48 and 96 hours, and 6 and 10 days for RNA isolation. RNA-seq was used to identify transcriptional signatures of BMNC in response to acute joint inflammation. The transcriptome of BMNC was assessed over time within the same group (SF or ISF), as well as comparatively between groups at each time point (Figure 1). The expression of 7 potential upstream regulator genes identified following bioinformatical analysis was assessed by immunohistochemistry in the synovium of inflamed joints from the same horses, 6 days after treatment with BMNC or Dulbecco's phosphate buffered saline (DPBS). There are 4 supplementary files and there will be a 5th after reanalysis.