Primary deep digital flexor tendon (DDFT) injuries within the navicular bursa and those accompanying navicular bone fibrocartilage erosions are among the common causes of performance-limiting forelimb lameness in horses.1–4 Intrabursal DDFT heals mainly through fibrosis due to its poor intrinsic healing capacity and is prone to repeat tearing/rupture.3–5 The torn tendon fibers within the navicular bursa are a source of persistent inflammation and contribute to fibrous tissue/adhesion formation which compromises the normal gliding function of DDFT; therefore, these factors negatively influence the long-term functional outcome of affected horses.1–3,6–8 Endogenous deep digital flexor (DDF) tenocytes play a vital role in the healing of DDFT as they secrete the tendon extracellular matrix (ECM).5 DDF tenocytes are gaining attention for their healing attributes and delineating cellular processes specific to intrasynovial DDF tenocyte biosynthesis is fundamental to identifying and developing effective therapies for enhanced intrasynovial tendon healing.
Circulating monocytes and macrophages that become localized within the tendon after following injury persist throughout the inflammatory, proliferative, and remodeling stages of healing.9,10 Dynamic shift of proinflammatory and regulatory macrophages regulate inflammation during tissue healing via secretions of inflammatory cytokines and mediators, IL-1β, IL-6, IL-8, IL-10, IL-17, TNF-α, and IFN-γ, and IL-10, TGF-β, IL-4, and IL-6, respectively.11–13 Although these cytokines/mediators share overlapping functions, the sequential influx of proinflammatory and regulatory macrophages has been shown to be vital for promoting a regenerative tissue healing response.9,12 The secreted cytokines facilitate macrophage recruitment of endogenous fibroblasts for extracellular matrix (ECM) synthesis and influence subsequent tissue remodeling.9,10,14 An imbalance in ECM synthesis and degradation from tissue-resident cells and both tissue-resident cells and extrinsic immune cells, respectively, during healing are key reasons implicated in the development of fibrosis.15,16 In both acute and chronic tendon injuries, inflammatory cytokines IL-1β, IL-6, TNF-α, and matrix metalloproteinases (MMPs) such as MMP-1, -3, and -13 are up-regulated.15,16 However, there is minimal information on how macrophage-induced inflammatory cytokines impact the bioactivities of ECM remodeling enzymes, MMPs, and their inhibitors tissue inhibitor matrix metalloproteinases (TIMPs) in tendon-derived cells, which are vital for tendon ECM remodeling. In the context of equine DDFT injuries, determining the biological mechanisms controlling macrophages and DDF tenocytes may shed light on DDFT fibrosis that occurs during the healing process.
The impact of macrophage-fibroblast co-culture has been extensively investigated in the fields of cancer metastasis and wound healing.17,18 Both direct (cell-to-cell) and in-direct (noncontact mediated) co-culture methods have demonstrated crosstalk and influence the ECM gene expression and cytokine secretion of both fibroblasts and macrophages.19,20 In this study, donor-matched peripheral blood CD14+ monocyte-derived macrophages were co-cultured with DDF tenocytes in direct and transwell systems to determine the underlying mechanisms of macrophage-induced inflammation on DDF tenocyte ECM remodeling enzyme bioactivities. Undifferentiated, granulocyte-macrophage colony-stimulating factor (GM-CSF) pretreated and lipopolysaccharide + interferon gamma (LPS+IFN-γ)-primed or proinflammatory, and interleukin-4 and interleukin-10 (IL-4+IL-10)-primed or regulatory macrophages, recently characterized by our group,21 were co-cultured with DDF tenocytes and MMP and TIMP gene expression and culture media concentrations were quantified. This study hypothesized that direct contact between macrophages and DDF tenocytes was not necessary for inducing MMP and TIMP gene expression and secretion.
Methods
Peripheral blood monocyte-derived macrophages and DDF tenocytes used in the experiments were autologously matched from all horses included in this study. The horses included in this study consisted of 2 mares and 3 geldings (2 Thoroughbreds and 3 Quarter Horses) between 9–11 years of age. All study procedures were performed in compliance with the university's Institutional Animal Care and Use Committee guidelines for research on animals.
Monocyte isolation and macrophage differentiation
Approximately 150 mL of jugular venous blood was collected from each horse. Peripheral blood mononuclear cells (PBMC) were isolated using 1.073 Ficoll density centrifugation as previously described.21–24 The resultant PBMC suspension was incubated with 1:1000 CD14 antibody clone Big10 (Enzo Life Sciences) for 30 minutes at a concentration of 10 uL/1 X 106 cells.23,25 Cell viability was determined using a Trypan blue dye exclusion test and >98% of live cells were confirmed for subsequent experiments. Next, the cell suspension was incubated with secondary IgG1 magnetic beads (Miltenyi Biotec) and processed through an LS column using the quadroMACSTM magnetic separator (Miltenyi Biotec) according to the manufacturer's instructions. On average, 150 mL of blood yielded 20 X 106 monocytes that were 95% to 98% immunopositive for CD14+ as confirmed via flow cytometry.21
The CD14+ monocytes were seeded in 9.5 cm2 tissue culture plates (Falcon) at 0.2 X 106 cells/cm2 density and cultured in a basal medium consisting of RPMI (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) for 7 days to generate undifferentiated or control macrophages. For proinflammatory macrophage differentiation, CD14+ monocytes were maintained in basal medium containing 50 ng/mL of recombinant equine GM-CSF (Kingfisher Biotech) for 6 days and then primed with 50 ng/mL IFNγ26,27 (Kingfisher Biotech) and 100 ng/mL of LPS26–30 (Escherichia coli strain O55:B5; Enzo Life Sciences) for 24 hours. For regulatory macrophage differentiation, CD14+ monocytes were maintained in basal medium for 6 days and then primed with 20 ng/mL of recombinant equine IL-426,27,29 (Kingfisher Biotech) and 20 ng/mL of recombinant equine IL-1026,27,31 (Kingfisher Biotech) for 24 hours. The undifferentiated, proinflammatory, and regulatory macrophages used in the co-culture experiments have been characterized via phenotype, flow cytometry, and cytokine profile.21
DDFT harvesting and cell isolation
Horses donated to the university's postmortem research efforts for reasons unrelated to musculoskeletal disease were euthanized with sodium pentobarbital (150 mg/kg IV) in compliance with AVMA guidelines. The forelimbs were harvested via disarticulation at the metacarpophalangeal joint. The hoof was cleaned, hair in the pastern region was clipped, and the digits were scrubbed with a disinfectant solution. The limbs were then disarticulated at the level of the distal interphalangeal joint, exposing the proximal aspect of the distal sesamoid bone without severing the DDFT. The DDFT opposing the distal sesamoid bone was determined to be normal based on gross assessment at the time of harvest.
The DDFT segment within the podotrochlear bursa was dissected and the tendinous region was separated from the fibrocartilaginous region5 and diced into 0.25 cm3 segments and digested in 0.15% collagenase type I (Worthington) in DMEM (Gibco) supplemented with 2% fetal bovine serum (FBS) and 2% penicillin-streptomycin (Gibco) at 37 °C overnight (16 hours). After digestion, cells were filtered through a 40 μm filter (thermo-fisher) and centrifuged at 300 X g for 10 minutes. The supernatant was removed, and the cell pellet was resuspended in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cell yield was determined by counting a representative aliquot using a hemocytometer and an inverted light microscope. Cell viability was determined using a Trypan blue exclusion dye (Sigma). Cells were seeded at 5,000 cells/cm2 and incubated at 37 °C, 95% air 5% CO2 in a humidified incubator, and allowed to form colonies. Resulting colony-forming units were detached with 0.02% EDTA and 0.05% trypsin (Gibco) and seeded at 10,000 cells/cm2 and expanded in monolayer for 2 passages.
Macrophage and DDF tenocyte co-culture
Passage 2 DDF tenocytes (referred to as “DDF tenocytes” henceforth) were detached with 0.02% EDTA and 0.05% trypsin (Gibco) and seeded at 20,000 cells/cm2 in ultralow attachment plates (Corning) and allowed to form aggregates for 72 hours.32 For direct co-culture, DDF tenocyte aggregates were added to tissue culture plates containing 20,000/cm2 control, GM-CSF pretreated, and LPS+IFN-γ-primed, or IL-4+IL-10-primed macrophages and allowed to equilibrate and initiate cell-to-cell contact. For transwell co-culture, a transwell insert with 0.4 μm pore size (Corning) containing 20,000/cm2 macrophages was introduced to DDF tenocyte aggregates in ultralow attachment plates so the cells could not intermix, but cytokine/soluble factor exchange was still permissible. Both direct and transwell co-cultures were maintained for 72 hours with one media change at 36 hours. At the end of 72 hours, all cell-media suspensions were collected in 15 mL centrifuge tubes (Corning) and centrifuged at 300 X g for 10 minutes. The media were aspirated, aliquoted, and stored at −80 °C for MMP and TIMP quantification. The cell pellets were snap-frozen in liquid nitrogen and stored at −80 °C until RNA isolation.
RNA isolation and quantitative RT-PCR
Total RNA was isolated using a previously described protocol.38,39 In the direct co-cultures, total RNA was isolated from DDF tenocytes and macrophages, whereas, in the transwell co-cultures only DDF tenocytes were used. The samples were homogenized in guanidinium thiocyanate-phenol-chloroform solution (TRIzol, ThermoFisher Scientific) according to the manufacturer's suggested protocol. The resultant pellet was purified using RNeasy silica columns that included on-column DNase digestion. The concentration of RNA was determined by measuring the absorbance at 260 nM (A260) and 320 nM (A320) in NanoDrop OneC (ThermoFisher Scientific). One microgram of RNA from each sample was reverse transcribed (SuperscriptIV, ThermoFisher Scientific) using oligo(dT) primers. Equine-specific primers (Table 1) were designed from published sequences in Genebank using ClustalW multiple sequence alignment (available at www.ebi.ac.uk). Primer specificity was confirmed by cloning and sequencing the amplicons during optimization experiments. PCR amplifications were catalyzed by Taq DNA polymerase (ABI QuantStudio 3, Applied Biosystems ThermoFisher Scientific) in the presence of SYBR green. Relative gene expression was quantified using the 2-ΔΔCT method and normalized to the expression of the reference gene, elongation factor-1a (EF1a),40 and expressed as fold change from DDF tenocytes maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin alone.
rtPCR primer sequences
| Gene | Accession number | Sequence | Amplicon (bp) | |
|---|---|---|---|---|
| MMP-1 | XM_023452130 | S | 5’ GGT GAA GGA AGG TCA AGT TCT GAT | 304 |
| A | 5’ AGT CTT CTA CTT TGG AAA AGA GCT TCT C | |||
| MMP-2 | XM_023637007.1 | S | 5’ ACC CCA CTA CGG TTT TCT CG | 212 |
| A | 5’ TAC TTC ACA CGG ACC ACT TGC | |||
| MMP-3 | NM_001081764.1 | S | 5’ GGC AAC GTA GAG CTG AGT AAA GCC | 223 |
| A | 5’ CAA CGG ATA GGC TGA GCA CGC | |||
| MMP-9 | NM001111302.1 | S | 5’ GGC CAG TTC CAG ACC TTT GA | 221 |
| A | 5’ CCA TCT CCG TGC TCC CTA AC | |||
| MMP-13 | AW261123 | S | 5’ AAG CCA CTT TGT GCT TCC TGA T | 215 |
| A | 5’ GGA TCG CAT TTG TCT GGT GTT | |||
| TIMP-1 | NM_001082515.1 | S | 5’ AAC CAG ACC TTA CAG CG | 186 |
| A | 5’ GTC CAA TAG TTG TCC GGC GA | |||
| TIMP-2 | XM_023651899.1 | S | 5’ CCC CAT CAA GCG GAT TCA GT | 156 |
| A | 5’ GCC TTT CCT GCG ATG AGG TA | |||
| EF1α | NM_001081781.1 | S | 5’ CCC GGA CAC AGA GAC TTC AT | 328 |
| A | 5’ AGC ATG TTG TCA CCA TTC CA |
MMP = Matrix metalloproteinase. TIMP = Tissue inhibitor matrix metalloproteinase.
ELISA quantification
Equine-specific MMP-3 (R&D Quantikine), -9 (ThermoFischer Scientific), and TIMP-1 (Cloud Clone) and -2 (ThermoFischer Scientific) concentrations in the media of direct and transwell DDF tenocyte-macrophage co-cultures were quantified via manufacturer's directions. There were 100 uL of each sample analyzed in duplicate following manufacturer instructions and absorbance was measured at 450 nm using a microplate reader (BioTek ELx808).
Statistical analysis
Normal distribution of all data was confirmed using Shapiro-Wilk's test. Univariate general linear model analyses were conducted to determine the impact on individual dependent variables (MMP, TIMP mRNA, and MMP, TIMP secretion) with co-culture (direct or transwell) and macrophage (undifferentiated, proinflammatory or GM-CSF pretreated and LPS+IFN-γ-primed, or, regulatory or IL-4+IL-10-primed) types as fixed factors. Post hoc multiple comparisons were conducted with Tukey's test. Significance was set at P < .05. All statistics were conducted using IBM® SPSS version 28.0.1.1, and the graphs were generated using Prism GraphPad version 9.1.0.
Results
Matrix metalloproteinase (MMP) mRNA expression
Means and SD of MMP mRNA in direct and transwell co-cultures of DDF tenocytes and macrophages are depicted (Figure 1).
Matrix metalloproteinase (MMP) mRNA in digital flexor tendon (DDF) tenocyte and macrophage co-cultures (n = 5) quantified using the 2-ΔΔCT method and normalized to EF1α. Macrophage phenotypes include undifferentiated (denoted as “control”), proinflammatory or granulocyte-macrophage colony-stimulating factor (GM-CSF) pretreated and LPS+IFNγ-primed (denoted as “LPS+IFNγ (+)”), or regulatory or IL-4+IL-10-primed (denoted as “IL-4+IL-10 (-)”). Solid and checkered bars represent direct and transwell co-culture mean ± SD values, respectively. * and + represent significant differences (*,+ P ≤ .05, ++ P < .01, *** P < .001) within and between direct or transwell co-culture groups, respectively. † Represents significant difference († P ≤ .05) between direct and transwell co-culture.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.05.0106
Direct co-culture of DDF tenocytes with GM-CSF pretreated and LPS+IFN-γ-primed macrophages significantly increased MMP-1 (P = .019), -3 (P < .001), and -13 (P = .026) mRNA, and significantly decreased MMP-9 (P = .016) mRNA compared to co-culture with control macrophages. MMP-1 and -3 mRNA in direct co-culture with GM-CSF pretreated and LPS+IFN-γ-primed macrophages were significantly increased compared to respective transwell co-cultures (P = .02 and P = .008, respectively). Direct or transwell co-culture with control, GM-CSF pretreated and LPS+IFN-γ-primed, or IL-4+IL-10-primed macrophages did not significantly affect MMP-2 mRNA.
Culture media MMP concentrations
Means and SD of culture media MMP-3 and -9 concentrations in direct and transwell co-cultures of DDF tenocytes and macrophages are depicted (Figure 2).
Matrix metalloproteinase (MMP) -3 and -9 media concentrations in digital flexor tendon (DDF) tenocyte and macrophage co-cultures (n = 5) quantified using ELISA. Macrophage phenotypes include undifferentiated (denoted as “control”), proinflammatory or granulocyte-macrophage colony-stimulating factor (GM-CSF) pretreated and LPS+IFNγ-primed (denoted as “LPS+IFNγ (+)”), or regulatory or IL-4+IL-10-primed (denoted as “IL-4+IL-10 (-)”). Solid and checkered bars represent direct and transwell co-culture mean ± SD values, respectively. * and + represent significant differences (*,+ P ≤ .05, **,++ P < .01 *** P < .001) within and between direct or transwell co-culture groups, respectively. † represents significant difference between direct and transwell co-culture.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.05.0106
Media MMP-3 concentrations in direct and transwell DDF tenocyte and GM-CSF pretreated and LPS+IFN-γ-primed macrophage co-cultures were significantly increased compared to respective control (P < .001 and P = .005, respectively) and IL-4+IL-10-primed macrophage (P = .015 and P = .011, respectively) co-cultures. In contrast, media MMP-9 concentrations in direct and transwell DDF tenocyte and GM-CSF pretreated and LPS+IFN-γ-primed macrophage co-cultures were significantly decreased compared to respective control (P = .002 and P < .001, respectively) or IL-4+IL-10-primed macrophage co-cultures (P = .005 and P = .003, respectively).
Tissue inhibitors of MMP (TIMP) mRNA expression
Means and SD of TIMP mRNA in direct and transwell co-cultures of DDF tenocytes and macrophages are depicted (Figure 3).
Tissue inhibitor matrix metalloproteinase (TIMP) -1 and -2 mRNA in digital flexor tendon (DDF) tenocyte and macrophage co-cultures (n = 5) quantified using the 2-ΔΔCT method and normalized to EF1α. Macrophage phenotypes include undifferentiated (denoted as “control”), proinflammatory or granulocyte-macrophage colony-stimulating factor (GM-CSF) pretreated and LPS+IFNγ-primed (denoted as “LPS+IFNγ (+)”), or regulatory or IL-4+IL-10-primed (denoted as “IL-4+IL-10 (-)”). Solid and checkered bars represent direct and transwell co-culture mean ± SD values, respectively. * and + represent significant differences within and between direct or transwell co-culture groups, respectively.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.05.0106
Direct co-culture of DDF tenocytes with IL-4+IL-10-primed macrophages significantly increased TIMP-1 mRNA, compared to co-culture with control macrophages (P = .021). TIMP-1 mRNA in transwell co-culture with IL-4+IL-10-primed macrophages was significantly increased compared to respective direct co-cultures (P = .022). Direct or transwell co-culture of DDF tenocytes with control, GM-CSF pretreated, and LPS+IFN-γ-primed or IL-4+IL-10-primed macrophages did not significantly affect TIMP-2 mRNA.
TIMP secretion
Means and SD of culture media TIMP-1 and -2 concentrations in direct and transwell co-cultures of DDF tenocytes and macrophages are depicted (Figure 4).
Tissue inhibitor matrix metalloproteinase (TIMP) -1 and -2 media concentrations in digital flexor tendon (DDF) tenocyte and macrophage co-cultures (n = 5) quantified using ELISA. Macrophage phenotypes include undifferentiated (denoted as “control”), proinflammatory or granulocyte-macrophage colony-stimulating factor pretreated and LPS+IFNγ-primed (denoted as “LPS+IFNγ (+)”), or regulatory or IL-4+IL-10-primed (denoted as “IL-4+IL-10 (-)”). Solid and checkered bars represent direct and transwell co-culture mean ± SD values, respectively. + represents significant difference between direct or transwell co-culture groups. † represents significant difference between direct and transwell co-culture.
Citation: American Journal of Veterinary Research 84, 12; 10.2460/ajvr.23.05.0106
There were no significant differences in media TIMP-1 concentrations of direct or transwell co-cultures of DDF tenocytes and control, GM-CSF pretreated and LPS+IFN-γ-primed, or IL-4+IL-10-primed macrophages. Media TIMP-2 concentration of direct control macrophage co-culture was significantly higher than the respective transwell co-culture (P = .045).
Discussion
This study investigated the cellular interactions between donor-matched tenocytes harvested from DDFT within the navicular bursa and control (7-day adherent culture), proinflammatory (6-day GM-CSF pretreated and 24-hour LPS+IFN-γ priming) and regulatory (6-day adherent culture and 24-hour IL-4+IL-10 priming) macrophages differentiated from peripheral blood CD14+ monocytes, specifically with regards to MMP and TIMP bioactivities. DDF tenocyte and macrophage co-cultures were maintained in direct and transwell co-culture platforms to determine if cell-to-cell contact was essential or if the interactions were paracrine mediated (noncontact), respectively. Direct co-culture of DDF tenocytes with GM-CSF pretreated and LPS+IFN-γ-primed macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with GM-CSF pretreated and LPS+IFN-γ-primed and IL-4+IL-10-primed macrophages resulted in a corresponding MMP-3 increase and MMP-9 decrease in media concentrations. While direct co-culture with IL-4+IL-10-primed macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and co-culture media concentrations were largely unchanged.
Matrix metalloproteases are critical for ECM degradation and play a key role in tissue remodeling and repair.33 MMP-1, -3, and -9 overlap in their breakdown of collagen type I, II, and III.33,34 MMP-1, -3, and -13 are also involved in aggrecan and proteoglycan breakdown within connective tissues.33,34 MMP-9 is a collagenase and a gelatinase MMP, playing a role in the breakdown of collagen type IV, V, VIII, X, XI, and XIV.33,34 In this study direct co-culture with GM-CSF pretreated and LPS+IFNγ-primed macrophages upregulated MMP-1, -3, and -13 mRNA. Although similar trends in MMP mRNA were seen in direct and transwell co-cultures, MMP-1 and -3 mRNA in direct co-cultures were significantly higher than the corresponding transwell co-culture mRNA values. This is likely because direct co-culture total RNA was isolated from DDF tenocytes and macrophages, whereas the transwell co-culture contained total RNA from just the DDF tenocytes. The results suggest that direct and transwell co-culture with GM-CSF pretreated and LPS+IFN-γ-primed macrophages upregulate DDF tenocyte MMP-1, 3, and -13 mRNA. While there is negligible information on macrophage phenotype and the impact on tenocyte and tendon MMP gene expression profiles; macrophages are key producers of MMPs and the significance of MMP mRNA upregulation by GM-CSF pretreated and LPS+IFN-γ-primed macrophages will require concurrent evaluation of tendon ECM gene expression in DDF tenocytes.
The MMP-3 and MMP-9 transcriptional changes noted in direct co-culture with GM-CSF pretreated and LPS+IFN-γ-primed macrophages were corroborated with culture media MMP-3 increase and MMP-9 decrease in both direct and transwell co-culture with GM-CSF pretreated and LPS+IFN-γ-primed macrophages. These results support that direct cell-to-cell contact between DDF tenocytes and GM-CSF pretreated and LPS+IFN-γ-primed macrophages is not essential. This interaction, where direct cell-to-cell contact is not essential, is consistent in macrophage interactions with palmar fascia myofibroblast,35 tumor fibroblasts,36 and biomaterial-mediated fibrosis model with fibroblasts.18 Our previous study characterizing GM-CSF pretreated and LPS+IFN-γ- and IL-4+IL-10-primed equine macrophages has demonstrated that IL-1β is the key secreted proinflammatory cytokine.21 In that regard, MMP-1, -3, and -13 transcriptional and translational increases in GM-CSF pretreated and LPS+IFN-γ-primed macrophage co-cultures seen in this study are consistent with those where tenocytes supplemented with exogenous Il-1β exhibit a proinflammatory profile with increased MMP mRNA.37 Further, Vinhas et al (2020) has shown that IL-1β-primed tenocytes interact with macrophages via connexin 43 and increase tenocyte proliferation and migration.37 MMP-9 mRNA concentrations were first noted to increase at day 7 in humans with pulmonary fibrosis.38 While the decrease in MMP-9 mRNA and culture media concentrations seen with GM-CSF pretreated and LPS+IFN-γ-primed macrophage co-cultures in this study is unclear, further research assessing temporal changes over an extended timeframe is warranted.
In vivo MMPs are regulated by activation of precursor zymogens and are inhibited by endogenous inhibitors TIMP.10,34,39 In normal tissue environments MMPs are co-secreted with TIMP-1 in a 1:1 ratio.33,34 The balance of MMP and TIMP is critical for reparative or anti-fibrotic ECM remodeling, with ratio imbalances being implicated in the development of fibrosis.34,40 Four TIMPs have been identified in vertebrates (TIMP-1, 2, 3, and 4), with expression being regulated by tissue remodeling and MMP activities.40 In addition to MMP inhibition, TIMP-1 and -2 have erythroid-potentiating and cell growth-promoting activities, as well as antiapoptotic activity.38,40 Because of these beneficial effects, and their universal inhibition of all MMPs, TIMP-1 and -2 concentrations were assessed in this study. In this in vitro study, direct co-culture of DDF tenocytes with IL-4+IL-10-primed macrophages significantly, albeit marginally, increased TIMP-1 mRNA, or in other words, IL-4+IL-10-primed macrophages increased TIMP-1 mRNA. However, the overall upregulation of MMP with GM-CSF pretreated and LPS+IFN-γ-primed, and IL-4+IL-10-primed macrophages was not accompanied by concurrent changes to TIMP-1 and 2 mRNA and secretions. Additional analyses beyond the 72-hour co-culture timepoint are warranted to determine the validity of MMP increase without a concurrent TIMP induction.
Although the primary objective of this study was to investigate the cellular interactions between DDF tenocytes and macrophages involved in MMP and TIMP bioactivities, there are limitations of this in vitro study that need to be considered. Media concentrations of MMP and TIMP include contributions from both macrophages and DDF tenocytes in both direct and transwell co-cultures. Further evaluation of MMP and TIMP gene expression data of the differentiated macrophages and tenocytes individually in co-culture would allow for further delineation of the source of MMP or TIMP changes seen in this study. The macrophage differentiation protocol aimed at generating proinflammatory and proregulatory phenotypes, but the lack of a commercially available equine macrophage colony-stimulating factor (M-CSF) before IL-4+IL-10 priming impeded the ability to accurately interpret the effect of regulatory macrophages on MMP/TIMP bioactivity. A single timepoint of 72 hours of co-culture in this study does not take into consideration the temporally regulated activities of MMP and TIMP. A longer-duration study evaluating the temporal concentrations of MMP and TIMP is warranted. Finally, the absence of tendon tissue in this study did not allow for the evaluation of MMP and TIMP activities at the tissue level, with innate immune responses that would occur in vivo.
Macrophage concentrations remain increased within the damaged tendon for up to 14 to 28 days postinjury.41 A timely transition from a proinflammatory to regulatory macrophage response controls the tissue healing environment and ECM synthesis capacity, promoting a regenerative tissue healing response.9,14 Therefore, understanding the mechanisms that control macrophage and tenocyte activities can be used to leverage immunomodulatory therapies for enhanced tendon healing. This study analyzed the in vitro MMP and TIMP activity of equine peripheral blood CD14+ monocyte-derived macrophages co-cultured with DDF tenocytes. We observed upregulation of MMP mRNA and secretions primarily with GM-CSF pretreated and LPS+IFNγ-primed macrophage co-cultures, and upregulation of TIMP-1 mRNA under direct co-culture with IL-4+IL-10-primed macrophages, and no significant changes to TIMP secretion under any other co-culture condition. There were no significant differences between direct and transwell co-cultures, suggesting that DDF tenocytes and macrophage interactions do not require direct cell-to-cell contact.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funding was obtained from the Ohio State University Equine Research Fund by the Ohio State Racing Commission Grant 2020-30. This publication was supported, in part, by the National Center for Advancing Translational Sciences of the National Institutes of Health under Grant Number KL2TR002734. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
References
- 1.↑
Wright IM, Kidd L, Thorp BH. Gross, histological and histomorphometric features of the navicular bone and related structures in the horse. Equine Vet J. 1998;30:220–234. doi:10.1111/j.2042-3306.1998.tb04491.x
- 2.
Blunden A, Dyson S, Murray R, et al. Histopathology in horses with chronic palmar foot pain and age-matched controls. Part 2: the deep digital flexor tendon. Equine Vet J. 2006;38:23–27. doi:10.2746/042516406775374342
- 3.↑
Blunden A, Dyson S, Murray R, et al. Histopathology in horses with chronic palmar foot pain and age-matched controls. Part 1: navicular bone and related structures. Equine Vet J. 2006;38:15–22. doi:10.2746/042516406775374298
- 4.↑
Blunden A, Murray R, Dyson S. Lesions of the deep digital flexor tendon in the digit: a correlative MRI and post mortem study in control and lame horses. Equine Vet J. 2009;41:25–33. doi:10.2746/042516408X343028
- 5.↑
Quam VG, Altmann NN, Brokken MT, et al. Zonal characterization and differential trilineage potentials of equine intrasynovial deep digital flexor tendon-derived cells. BMC Vet Res. 2021;17:138. doi:10.1186/s12917-021-02793-1
- 6.↑
Jann H, Blaik M, Emerson R, et al. Healing characteristics of deep digital flexor tenorrhaphy within the digital sheath of horses. Vet Surg. 2003;32:421–430. doi:10.1053/jvet.2003.50059
- 7.
Dyson SJ, Murray R, Schramme MC. Lameness associated with foot pain: results of magnetic resonance imaging in 199 horses (January 2001–December 2003) and response to treatment. Equine Vet J. 2005;37:113–121. doi:10.2746/0425164054223804
- 8.↑
Beck S, Blunden T, Dyson S, et al. Are matrix and vascular changes involved in the pathogenesis of deep digital flexor tendon injury in the horse? Vet J. 2011;189:289–295. doi:10.1016/j.tvjl.2010.07.015
- 9.↑
Stolk M, Klatte-Schulz F, Schmock A, et al. New insights into tenocyte-immune cell interplay in an in vitro model of inflammation. Sci Rep. 2017;7:9801. doi:10.1038/s41598-017-09875-x
- 10.↑
Best KT, Nichols AEC, Knapp E, et al. NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival. Sci Signal. 2020;13(658):eabb7209. doi:10.1126/scisignal.abb7209
- 11.↑
Woodell-May JE, Sommerfeld SD. Role of inflammation and the immune system in the progression of osteoarthritis. J Orthop Res. 2020;38:253-257. doi:10.1002/jor.24457
- 12.↑
Sugg KB, Lubardic J, Gumucio JP, et al. Changes in macrophage phenotype and induction of epithelial-to-mesenchymal transition genes following acute Achilles tenotomy and repair. J Orthop Res. 2014;32:944–951. doi:10.1002/jor.22624
- 13.↑
Hamidzadeh K, Christensen SM, Dalby E, et al. Macrophages and the recovery from acute and chronic inflammation. Annu Rev Physiol. 2017;79:567–592. doi:10.1146/annurev-physiol-022516-034348
- 14.↑
Blomgran P, Blomgran R, Ernerudh J, et al. A possible link between loading, inflammation and healing: immune cell populations during tendon healing in the rat. Sci Rep. 2016;6:29824. doi:10.1038/srep29824
- 15.↑
Ackerman JE, Geary MB, Orner CA, et al. Obesity/Type II diabetes alters macrophage polarization resulting in a fibrotic tendon healing response. PLoS ONE. 2017;12:e0181127. doi:10.1371/journal.pone.0181127
- 16.↑
Ackerman JE, Nichols AE, Studentsova V, et al. Cell non-autonomous functions of S100a4 drive fibrotic tendon healing. Elife. 2019;8. doi:10.7554/eLife.45342
- 17.↑
An Y, Liu F, Chen Y, et al. Crosstalk between cancer-associated fibroblasts and immune cells in cancer. J Cell Mol Med. 2020;24:13–24. doi:10.1111/jcmm.14745
- 18.↑
Witherel CE, Abebayehu D, Barker TH, et al. Macrophage and fibroblast interactions in biomaterial-mediated fibrosis. Adv Healthc Mater. 2019;8:e1801451. doi:10.1002/adhm.201801451
- 19.↑
Novak CM, Sethuraman S, Luikart KL, et al. Alveolar macrophages drive lung fibroblast function in cocultures of IPF and normal patient samples. Am J Physiol Lung Cell Mol Physiol. 2023;324:L507–L520. doi:10.1152/ajplung.00263.2022
- 20.↑
Ullm F, Riedl P, Machado de Amorim A, et al. 3D scaffold-based macrophage fibroblast coculture model reveals IL-10 dependence of wound resolution phase. Adv Biosyst. 2020;4:e1900220. doi:10.1002/adbi.201900220
- 21.↑
Bowlby CM, Purmessur D, Durgam SS. Equine peripheral blood CD14+ monocyte-derived macrophages in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-y or IL-4+IL-10 differentiation. Vet Immunol Immunopathol. 2023;255:110534. doi:10.1016/j.vetimm.2022.110534
- 22.
Mauel S, Steinbach F, Ludwig H. Monocyte-derived dendritic cells from horses differ from dendritic cells of humans and mice. Immunology. 2006;117:463–473. doi:10.1111/j.1365-2567.2005.02319.x
- 23.↑
Steinbach F, Bischoff S, Freund H, et al. Clinical application of dendritic cells and interleukin-2 and tools to study activated T cells in horses–first results and implications for quality control. Vet Immunol Immunopathol. 2009;128:16–23. doi:10.1016/j.vetimm.2008.10.317
- 24.↑
Moyo NA, Marchi E, Steinbach F. Differentiation and activation of equine monocyte-derived dendritic cells are not correlated with CD206 or CD83 expression. Immunology. 2013;139:472–483. doi:10.1111/imm.12094
- 25.↑
Durán MC, Willenbrock S, Carlson R, et al. Enhanced protocol for CD14+ cell enrichment from equine peripheral blood via anti-human CD14 mAb and automated magnetic activated cell sorting. Equine Vet J. 2013;45:249–253. doi:10.1111/j.2042-3306.2012.00616.x
- 26.↑
Fujiwara Y, Hizukuri Y, Yamashiro K, et al. Guanylate-binding protein 5 is a marker of interferon-γ-induced classically activated macrophages. Clin Transl Immunol. 2016;5:e111. doi:10.1038/cti.2016.59
- 27.↑
Iqbal S, Kumar A. Characterization of in vitro generated human polarized macrophages. J Clin Cell Immunol. 2015;6:380. doi:10.4172/2155-9899.1000380
- 28.
Menarim BC, Gillis KH, Oliver A, et al. Autologous bone marrow mononuclear cells modulate joint homeostasis in an equine. FASEB J. 2019;33:14337–14353. doi:10.1096/fj.201901684RR
- 29.↑
Graff JW, Dickson AM, Clay G, et al. Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem. 2012;287:21816–21825. doi:10.1074/jbc.M111.327031
- 30.↑
Naskou MC, Norton NA, Copland IB, et al. Innate immune responses of equine monocytes cultured in equine platelet lysate. Vet Immunol Immunopathol. 2018;195:65–71. doi:10.1016/j.vetimm.2017.11.005
- 31.↑
Orekhov AN, Orekhova VA, Nikiforov NG, et al. Monocyte differentiation and macrophage polarization. Vessel Plus. 2019;3:10. doi:10.20517/2574-1209.2019.04
- 32.↑
Sullivan SN, Altmann NN, Brokken MT, et al. Effects of methylprednisolone acetate on equine deep digital flexor tendon-derived cells. Front Vet Sci. 2020;7:486. doi:10.3389/fvets.2020.00486
- 33.↑
Laronha H, Caldeira J. Structure and function of human matrix metalloproteinases. Cells. 2020;9(5):1076. doi:10.3390/cells9051076
- 34.↑
Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res. 2006;69:562–573. doi:10.1016/j.cardiores.2005.12.002
- 35.↑
Gonga-Cavé BC, Pena Diaz AM, O’Gorman DB. Biomimetic analyses of interactions between macrophages and palmar fascia myofibroblasts derived from Dupuytren's disease reveal distinct inflammatory cytokine responses. Wound Repair Regen. 2021;29:627–636. doi:10.1111/wrr.12928
- 36.↑
Buechler MB, Fu W, Turley SJ. Fibroblast-macrophage reciprocal interactions in health, fibrosis, and cancer. Immunity. 2021;54:903–915. doi:10.1016/j.immuni.2021.04.021
- 37.↑
Vinhas A, Rodrigues MT, Gonçalves AI, et al. Pulsed electromagnetic field modulates tendon cells response in IL-1β-conditioned environment. J Orthop Res. 2020;38:160–172. doi:10.1002/jor.24538
- 38.↑
Sato M, Hwang DM, Guan Z, et al. Regression of allograft airway fibrosis: the role of MMP-dependent tissue remodeling in obliterative bronchiolitis after lung transplantation. Am J Pathol. 2011;179:1287–1300. doi:10.1016/j.ajpath.2011.05.032
- 39.↑
Szóstek-Mioduchowska AZ, Baclawska A, Okuda K, et al. Effect of proinflammatory cytokines on endometrial collagen and metallopeptidase expression during the course of equine endometrosis. Cytokine. 2019;123:154767. doi:10.1016/j.cyto.2019.154767
- 40.↑
Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003;92:827–839. doi:10.1161/01.RES.0000070112.80711.3D
- 41.↑
Sunwoo JY, Eliasberg CD, Carballo CB, et al. The role of the macrophage in tendinopathy and tendon healing. J Orthop Res. 2020;38:1666–1675. doi:10.1002/jor.24667
