Intrasynovial tendon injuries are common debilitating conditions characterized by persistent inflammation and tissue degeneration during healing, and are associated with poorer outcomes than extrasynovial tendon injuries. Changes in tendon structure and vascularity following injury facilitate circulating immune cell influx, of which, monocyte-derived macrophages dominate and persist throughout healing. Macrophages are emerging targets for modulating/manipulating tendon healing as they play a fundamental role by initiating and/or resolving inflammation and recruiting fibroblasts via secreted signals for extracellular matrix (ECM) synthesis. Macrophages respond to local stimuli, exhibit pro-inflammatory or anti-inflammatory/regulatory phenotypes, and exert differential effects at the tissue healing site. Therefore, the aim of this research was to investigate macrophage-derived inflammatory and regulatory signals, and subsequently examine their effects on intrasynovial flexor tendon cell bioactivity. Our specific objectives included (1) optimizing peripheral blood monocyte-derived macrophage in vitro differentiation protocols, and subsequently determining their inflammatory and regulatory cytokine secretion, and (2) investigating intrasynovial flexor tenocyte-macrophage interactions in direct (cell-to-cell contact) and transwell (non-contact mediated) in vitro co-culture model systems.
Peripheral blood CD14+ monocytes were isolated from 5 horses (IACUC approved) via sequential density gradient centrifugation and magnetic bead-based positive selection. Forelimb intrasynovial deep digital flexor tendons opposing the distal sesamoid bone were harvested from 4 of those horses immediately following euthanasia, collagenase digested to obtain single cell suspensions, and monolayer passaged twice to isolate intrasynovial flexor tenocytes (CD90+105+27-45-). (1) Monocytes were maintained in basal medium (RPMI + 10% FBS + 1% penicillin-streptomycin) and ± 50ng/mL equine GM-CSF for 6 days for macrophage differentiation. Subsequently, regulatory or inflammatory phenotypes were induced via 24-hour priming with 20ng/mL IL-4 and 20ng/mL IL-10, or 50ng/mL IFNg and 100ng/mL LPS respectively. Macrophage morphology, proliferation (tetrazolium-based MTT assay), flow cytometry-based CD206 and 86 surface expression, and secretory profiles (ELISA quantification of TNFa, IL-1β, IL-1Ra, IL-6, and TGFβ-1) were assessed. (2) Donor-matched intrasynovial flexor tenocyte and inflammatory or regulatory macrophage co-cultures were established in direct and transwell systems. Tenocyte proliferation and migration were accessed in monolayer co-culture. Tenocytes were allowed to aggregate in ultra-low attachment plates for 72 hours prior to beginning co-culture to evaluate transcriptional and secretory changes. The co-cultures were maintained for 72 hours, and the tenocytes and co-culture media were stored in -80C for mRNA, qRT-PCR, and ELISA quantification. All data were assessed for normality (Shapiro-Wilk) and comparative differences between treatment groups were assessed with one-way analysis of variance or its non-parametric equivalent. Significance was set at p≤0.05.
Monocytes cultured in basal medium with GM-CSF yielded macrophages with a rounded morphology. IL-4+IL-10 resulted in elongate, spindle-shaped macrophages, whereas LPS+IFNg gave rise to rounded, granular macrophages. These morphologies were not impacted by GM-CSF treatment. GM-CSF significantly (p=0.03) increased macrophage proliferation compared to those cultured in basal medium alone by 15-fold, with no differences between inflammatory or regulatory priming. CD86 expression increased during macrophage differentiation but was not statistically significant (p=0.6), and there was no difference in CD206 or 86 expression in regulatory or inflammatory macrophages. Macrophages cultured in basal medium alone secreted low levels of IL-1Ra, IL-10, TNFa, and IL-1β. Macrophages primed with LPS+IFNg showed a significant (p=0.012) increase in IL-1β secretion (median 136.1;100.4-198.3) pg/mL. Both regulatory and inflammatory priming increased TNFa (p=0.034) secretion. GM-CSF followed by LPS+IFNg and IL-4+IL-10 priming yielded a 6-fold increase in IL-1Ra secretion (p=0.029, p=0.014). IL-6 was not detected in macrophage differentiation culture medium irrespective of the treatment. (2) Tenocytes cultured with IL-4 + IL-10 (± GM-CSF) macrophage conditioned media exhibited a 1.5-fold increase in proliferation (p=0.003, p=0.02). Tenocytes co-cultured with IL-4+ IL-10 primed macrophages with GM-CSF pre-treatment had significantly (p=0.0005) increased migration compared to tenocytes cultured without macrophages. Direct co-culture with LPS+IFNg primed macrophages downregulated COL1A1 mRNA 5.2-fold (p=0.01), SOX-9 and ACAN mRNA by 3-fold (p=0.003, p=0.004) and COL2A1 mRNA by 15-fold (p=0.04). No significant differences were seen in transwell co-culture. MMP-3 mRNA was upregulated with both direct and transwell co-culture with IL-4 + IL-10 (+ GM-CSF) primed macrophages, and MMP-13 was upregulated 32-fold (p=0.01) in direct co-culture. IL-6 mRNA was upregulated 352-fold (p=0.04) with LPS+IFNg macrophage direct co-culture. IL-6 secretion was significantly increased in both direct and transwell co-culture whereas IL-6 was not quantified in the media of macrophage or tenocyte culture alone.
Overall, when discussing our results specifically with respect to M1 and M2 macrophages differential effects of DDF tenocyte ECM gene expression, our results demonstrate that co-culture with M1 macrophages downregulates tenocyte matrix gene expression, whereas the matrix gene expression of DDF tenocytes under basal conditions and those co-cultured with M2 macrophages are largely equivalent. From a mechanistic standpoint, while elevated IL-6 concentrations during healing has been attributed to pro-resolving effects as well as towards tendon degeneration, the specific timeline for IL-6 impact on tenocyte ECM synthesis, and in turn, overall tendon healing outcomes are key areas of future work in the field of intrasynovial tendon immunomodulation.