Progenitor Cells Derived From Injured Cartilage Surface Respond to Damage Associated Molecular Patterns

doi:10.21203/rs.3.rs-120535/v1

Download PDF

Research article

Progenitor Cells Derived From Injured Cartilage Surface Respond to Damage Associated Molecular Patterns

https://doi.org/10.21203/rs.3.rs-120535/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objective: To elucidate how chondrogenic progenitor cells (CPCs) originated from mechanically injured cartilage surface respond to proinflammatory endogenous damage-associated-molecular-patterns (DAMPs).

Design: Passage 1 bovine CPCs and non-CPCs isolated from injured articular cartilage either by blunt impaction or by scratches were treated with mitochondrial DAMPs (MTDs) composed of fMLF and CpG DNA, or HMGB1 (a nuclear DAMP), or IL-1b for 24 hrs. At the end of the experiments, the expression levels of matrix metalloproteinases (MMPs), chemokines, and cytokines that are associated with cartilage degeneration was examined with Western blotting and quantitative PCR.

Results: Both HMGB1 and MTDs remarkably up-regulated expression level of pro-MMP-13 protein in CPCs while showed weak effect on non-CPCs. Compared to non-CPCs, CPCs expressed significantly higher baseline mRNA levels of MMP-13, CXCL12, and IL-6. MTDs further increased the expression levels of MMP-13 and IL-6 in CPCs while HMGB1 did not show such effect. When treated with MTDs, CPCs also expressed significantly higher levels of IL-8 mRNA than did non-CPCs. However, compared to non-CPCs, CPCs expressed much lower levels of MMP-3 and active MMP-13 proteins as well as lower mRNA levels of CCL2 and IL-6 in response to IL-1b.

Conclusions: CPCs were more sensitive than non-CPCs in response to DAMPs, especially MTDs, to up-regulate expression of MMP-13, IL-6 and -8. When IL-1b was present, CPCs were less responsive than non-CPCs in terms of up-regulating MMP-3, CCL2, and IL-6 expression. The proinflammatory nature of CPCs implied their critical role in the early phase of PTOA development.

Rheumatology

Chondrogenic progenitor cells

cartilage injury

DAMPs

inflammation

cartilage degradation

Trauma to weight-bearing joint leads to post-traumatic osteoarthritis (PTOA). Focal mechanical damage in articular cartilage results in irreversible degradation of the tissue. Cartilage degeneration is partially responsible for the typical symptoms including joint pain and stiffness observed in patients diagnosed with PTOA. Extracellular matrix (ECM) destruction and cell death are the cause of cartilage degeneration [1-7]. How mechanical insults induce catabolic changes in cartilage cells remains poorly understood.

Cells termed as “chondrocytes” have long been thought as the only cell type in articular cartilage [2, 8]. However, studies have showed that cartilage harbored chondrogenic progenitor cells that could be induced to differentiate into mature chondrocytes. Dowthwaite and colleagues reported that there was 1-2% of chondrocyte population residing in the surface zone of articular cartilage of 7-day-old calves were progenitor-like cells which possessed a high colony-forming efficiency and could differentiate into chondrocytes [9]. Furthermore, they discovered chondrogenic progenitor cells (CPCs) in normal human femoral condyle cartilage which accounted for 0.7% of total cell population [10].

Correspondingly, our group discovered that injury to cartilage surface could induce migration of CPCs into the injury site and those cells could repopulate the damaged area. Migrating CPCs expressed high level of PRG4 indicating that they originated from the superficial zone. However, compared to mesenchymal stem cells (MSCs) or normal chondrocytes, CPCs expressed higher levels of cytokines, chemokines and MMPs involved in cartilage inflammation [11].

Instant cell death, a critical form of cartilage injury, immediately follows trauma to the joint, which may create pro-inflammatory environments. Several ex vivo studies revealed that significant amount of cell death quickly following mechanical insults was observed in the superficial tangential zone and then turned into a slower propagating “wave of cell death” [12-15]. Those observations were later confirmed in in vivo studies that revealed even more profound effect. They showed complete loss of cells spanning the full-thickness of injured cartilage only weeks after the insult [7, 16, 17]. Dead cells spill intracellular contents, some of which are proinflammatory and capable of priming immune cells including dendritic cells, T cells and macrophages [18]. Those derived endogenously from stressed or injured tissues are termed as “alarmins” or endogenous danger/damage-associated molecular patterns (DAMPs) [19, 20].

DAMPs may come from ruptured mitochondria. Hajizadeh et al. reported that mitochondrial DNA (mtDNA) containing unmethylated CpG motif was detected in synovial fluids of 70% patients diagnosed with rheumatoid arthritis (RA). Rheumatoid factor (RF) was detected with significantly higher frequency in patients whose synovial fluid samples were mtDNA-positive [21]. Moreover, Dong et al. and Collins et al. reported that mtDNA was inflammatogenic both in vitro and in vivo and its proinflammatory effect was mediated by monocytes/macrophages, NF-kB, and TNF-a [22, 23]. Zhang and colleagues further discovered that mtDNA and formyl peptides formed mitochondrial DAMPs (MTDs) which could trigger innate immunity leading to neutrophil-mediated organ injury usually observed in a systemic inflammatory response syndrome (SIRS) [24].

In addition to MTDs, high mobility group box 1 protein (HMGB1) is a nuclear DAMP (NuD) . HMGB1 is a non-histone protein and involved in DNA organization and regulation of transcription. Recent study showed that HMGB1 not only mediated sepsis but also played a critical role in pathophysiology of trauma, autoimmunity, ischemia perfusion injury, and cancer [25]. HMGB1 was demonstrated as a systemic inflammation mediator in a murine fracture model. It signaled through membrane toll-like receptor 4 (TLR4) located on the apical surface of intestinal cells to elicit inflammatory response and end-organ injury following bilateral femur fracture [26]. Our previous study revealed that HMGB1 synergized with IL-1b or Fn-f on up-regulation of ECM degrading enzymes including MMP-3, -13, ADAMTS-5, ADAM-8, and iNOS in articular chondrocytes [27].

However, it still remains unclear whether CPCs respond differently from remaining chondrocytes to DAMPs released from dead cells following joint trauma. We hypothesized that in injured cartilage CPCs were more responsive to DAMPs than the remaining cell population in terms of up-regulating expression of MMPs, chemokines, or cytokines involved in cartilage inflammatory degradation. To test this hypothesis, we employed an ex vivo cartilage injury model and established cultures of CPCs and non-CPCs, respectively. We then compared the expression levels of MMP-3, -13, CXCL12, CCL2, IL-6, and IL-8 in those cultures in response to MTDs or HMGB1 stimulation.

Establishment of cartilage surface injury ex vivo model

Bovine osteochondral explants (2.0–2.5 cm W X 2.0–2.5 cm L X 0.5–1.0 cm H) were sawed under sterile conditions from medial or lateral tibial plateau of stifle joints of 18-month-old cows obtained from a local abattoir (Bud's Custom Meats, Inc., Riverside, IA, USA). Explants were pre-equilibrated for 48 hrs in serum free DMEM/F12 supplemented with 50 U/ml Penicillin, 50 mg/l Streptomycin, and 2.5mg/l Amphotericin B inside a humidified 37 ̊C incubator supplied with 5% CO₂ and 5% O₂.

To create an impact injury, cartilage surface was subjected to a single blunt impaction with a customized drop-tower reported in our previous studies [11, 28]. Briefly, an osteochondral explant was rigidly fixed in a chamber with a 5.0 mm diameter brass rod resting on the surface of cartilage. A 2-kg mass was dropped from 14 cm height onto the brass rod resulting in impact energy density of 14 J/cm². This high energy impact created cracks and instant cell death in cartilage surface. To create a scratch injury, a sterile 26 G1/2 needle was dragged over cartilage surface. An X-shaped matrix tear in the superficial zone was generated.

Harvest and monolayer culture of CPCs, non-CPCs and normal articular chondrocytes

As previously reported [11, 29], an impacted or scratched osteochondral explant was firstly incubated with 0.25% trypsin-EDTA for 10 min with cartilage side facing down. CPCs were then collected, counted, and cultured in DMEM/F12 supplemented with 10% FBS, 50 U/ml Penicillin, 50 mg/l Streptomycin, and 2.5mg/l Amphotericin B inside a 37 ̊C incubator supplied with 5% CO₂, 5% O₂and 100% humidity.

To harvest non-CPCs, full thickness cartilage slices were shaved from osteochondral explants immediately after CPCs were harvested, and were weighed, minced, and subjected to 0.4% protease (Sigma-Aldrich^®, St. Louis, MO) for 90 mins and then to 0.02% collagenase (Sigma-Aldrich^®, St. Louis, MO) for 16 hrs to release chondrocytes. Same method was used in isolation of normal articular chondrocytes (ACs) from uninjured cartilage. Passage 1 CPCs, or non-CPCs, or ACs were seeded at a high density (3.0 X 10⁵cells/cm²). The experimental design was summarized and illustrated in Figure 1.

Light microscopy and calculation of cell population doubling time (PDT)

The growth status of CPCs, or non-CPCs, or ACs was recorded with an Olympus CKX53 light microscope (Olympus Corporation of the Americas, PA, USA) equipped with a camera (Olympus Soft Imaging Solutions GMBH, Olympus LC30, Munster, Germany). PDT of CPCs, or non-CPCs, or ACs was calculated from total cell counts obtained at the seeding time of passage 0 and 1 cells, respectively.

Treating CPCs or non-CPCs with DAMPs or IL-1b

At Day 4 post-seeding, passage 1 CPCs or non-CPCs were switched to serum free media in order to synchronize cell cycle. After 24-hr serum deprivation, cultures were replenished with equal volume of fresh serum free media immediately prior to treatments with DAMPs or IL-1b. Cultures were divided into four treatment groups (each group in triplicates): 1) non-treated control; 2) 10 nM rhHMGB1/HMG-1 (R&D Systems, Minneapolis, MN); 3) MTDs: 10 nM N-formyl-Met-Leu-Phe (fMLF) (Tocris Bioscience, Bristol, UK) + 10 µg/mL CpG DNA (a 22-mer oligonucleotides containing CpG motifs) (InvivoGen, San Diego, CA); 4) 10 ng/mL rhIL-1b (R&D Systems, Minneapolis, MN). After 24-hr stimulation, conditioned media were collected for determination of MMP expression with Western blotting and cells were lysed for total RNA extraction and real-time PCR analysis.

Determination of MMP expression with Western blotting

Conditioned media were dialyzed against MilliQ water and then concentrated to dry powder with a speed vacuum. Equal volume of prepared reduced medium samples was resolved by 10% SDS-PAGE. Proteins were then blotted onto a nitrocellulose membrane. After been blocked with 3% BSA/TBS, blots were incubated with 1% BSA/TBST containing 1:3,000 diluted anti-MMP-3 antibody (Biomol International, Kelayres, PA) or 1:1,000 diluted anti-MMP-13 antibody (Abcam^®, Cambridge, MA) overnight at 4 °C. After being washed three times, those blots were then incubated with 1% BSA/TBST containing 1:7,600 diluted goat anti-rabbit IgG antibody conjugated with HRP (Sigma-Aldrich^®, St. Louis, MO). Chemiluminescent signals were generated with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific Inc., Rockford, IL) and captured with Kodak BioMax MR film (Sigma-Aldrich^®, St. Louis, MO) or Blue Classic Autoradiography film (RPI, Mount Prospect, IL).

Expression of MMPs, chemokines, and cytokines determined with real-time PCR

CPCs or non-CPCs were lysed with TRIZOL^® (Invitrogen, Waltham, MA) and total RNAs were extracted with Qiagen RNEasy Kits (Qiagen Inc, Valencia, CA). A 50 ng of each RNA sample was firstly reverse transcribed into cDNA with TaqMan™ Reverse Transcription Reagents (Applied Biosystem, Grand Island, NY). Quantitative PCR reactions were performed with SYBR™ Green PCR Master Mix (Applied Biosystem, Grand Island, NY). Primers for bovine β-actin, MMP-3, -13, CCL2, CXCL12, IL-6 & 8, were provided by Integrated DNA Technologies (Coralville, IA) (sequences available upon request). Data were obtained using an ABI Prism 7700 Sequence detection System (Applied Biosystems, Foster City, CA).

Statistical analysis

Relative gene expression to β-actin was calculated using a comparative Ct method (2^−ΔΔCt). Results are reported as mean ± standard deviation (SD). Relative expression of each target gene in CPCs from impact or scratch injury was compared to that in non-CPCs with Welch's t-test. P (T<=t) two-tail value was reported. P < 0.05 was considered statistically significant. The 95% confidence interval (CI) was also calculated by using this formula: CI = Mean ± t X (SD/√sample size). The sample size was determined by n = (z² × σ²)/MOE².

Compared to non-CPCs, CPCs displayed remarkably different morphology and much shorter PDT

At day 1 post-seeding, passage 0 CPCs isolated either from blunt impacted or scratched cartilage displayed elongated spindle shape while corresponding non-CPCs showed rounded shape. CPCs was also notably larger than non-CPCs (Figure 2A-C). PDT was 1.7 days for CPCs from impact injury and 1.9 days for CPCs from scratch injury, both which was much shorter than that for non-CPCs (6 days) or ACs (3.6 days) (Figure 2D). In cartilage of an osteochondral explant, CPCs only accounted for 9.5% while non-CPCs for 90.5% of the whole cell population.

CPCs were more responsive than non-CPCs to DAMPs stimulation in terms of up-regulation of MMP-13 expression while non-CPCs expressed moderately more MMP-3 mRNA than did CPCs

When left untreated, CPCs from impact injury expressed more MMP-13 proform (pro-MMP-13) than did non-CPCs. When either DAMPs were present, CPCs secreted significantly more pro-MMP-13 than did non-CPCs with CPCs from impact injury showing stronger response than those from scratch injury (Figure 3A, top blot).

Consistently, in non-treated cultures, CPCs from impact injury expressed 98.1 ± 3.4 (95% CI: 89.659, 106.541) fold more MMP-13 mRNA than did non-CPCs while this fold increase reduced to 21.8 ± 2.6 (95% CI: 15.345, 28.255) for CPCs from scratch injury. Although HMGB1 stimulation did not further increase this expression difference, MTDs induced significantly more MMP-13 mRNA expression in CPCs from scratch injury (201.4 ± 5.6; 95% CI: 187.497, 215.303; P = 0.0004) and in CPCs from impact injury (128.5 ± 0.8; 95% CI: 126.514, 130.486; P = 0.00002) than in non-CPCs (1.8 ± 0.1; 95% CI: 1.552, 2.048) (Figure 3B). Nonetheless, CPCs and non-CPCs responded similarly to IL-1b stimulation in spite of over 400-fold increase of MMP-13 mRNA expression over the reference gene (Figure 3C). At protein level, IL-1b induced more MMP-13 active form in non-CPCs than in CPCs (Figure 3A, top blot).

Unlike MMP-13, MMP-3 protein expression was only detected in the group treated with IL-1b. Compared to non-CPCs, CPCs secreted much less MMP-3 (Figure 3, bottom blot). Consistently, the fold increase of MMP-3 mRNA expression in groups either untreated or treated with DAMPs was less than 3 while in the group treated with IL-1b the fold increase was over 200. CPCs expressed much less MMP-3 mRNA than did non-CPCs when treated with IL-1b (Figure 3D&E).

CPCs expressed higher levels of CXCL12 than did non-CPCs in response to DAMPs; however, CPCs expressed less CCL2 than did non-CPCs in response to IL-1b

When DAMPs were added into cultures, CPCs from either source expressed more CXCL12 mRNA than did non-CPCs [93.8 ± 25.9 or 95.1 ± 10.4 (95% CI: 29.501, 158.099 or 69.281, 120.919) vs. 1.0 ± 0.1 (95% CI: 0.752, 1.248) when HMGB1 was present; 70.8 ± 4.1 or 59.4 ± 10.8 (95% CI: 60.621, 80.979 or 32.588, 86.212) vs. 1.1 ± 0.1 (95% CI: 0.852, 1.348) when MTDs were present]. Nonetheless, similar expression difference was detected in non-treated group (P = 0.001 for CPCs from scratch injury vs. non-CPCs; P = 0.004 for CPCs from impact injury vs. non-CPCs). This baseline expression difference was not enhanced by IL-1b (Figure 4A).

Unlike CXCL12, mRNA expression of another chemokine CCL2 was low and did not show much difference between CPCs and non-CPCs with or without DAMPs treatment. However, CCL expression was drastically increased by IL-1b. The expression in CPCs was only half of that in non-CPCs [126.0 ± 14.9 (95% CI: 89.009, 162.991) in CPCs from scratch injury or 155.0 ± 7.3 (95% CI: 136.877, 173.123) in CPCs from impact injury vs. 303.0 ± 33.0 (95% CI: 221.074, 384.926) in non-CPCs; P = 0.003 or 0.017 for CPCs from scratch injury or from impact injury vs. non-CPCs] (Figure 4B&C).

CPCs expressed more IL-6 or -8 mRNA than did non-CPCs in response to MTDs

CPCs expressed significantly more baseline IL-6 mRNA than did non-CPCs: 6.4 ± 0.4 (95% CI: 5.407, 7.393) or 4.1 ± 0.5 (95% CI: 2.859, 5.341) in CPCs from scratch or impact injury vs. 1.0 ± 0.1 (95% CI: 0.752, 1.248) in non-CPCs (P = 0.002 or 0.008 for CPCs from scratch or impact injury vs. non-CPCs). In response to MTDs, CPCs expressed 10 times more IL-6 mRNA over the baseline while non-CPCs did minimally [186.5 ± 4.3 (95% CI: 175.825, 197.175) or 83.6 ± 6.8 (95% CI: 66.718, 100.482) for CPCs from scratch or impact injury vs 2.0 ± 0.1 (95% CI: 1.752, 2.248) for non-CPCs; P = 0002 or 0.0022]. However, HMGB1 did not further elevate IL-6 mRNA expression in CPCs (Figure 5 A&B). Surprisingly, when IL-1b was present, CPCs expressed significantly lower IL-6 mRNA than did non-CPCs (P = 0.016 or 0.031 for CPCs from scratch or impact injury vs. non-CPCs) although 6,000 times more IL-6 expression over the baseline was induced in both types of cells (Figure 5C).

Similar to IL-6, IL-8 expression was greatly induced by MTDs in CPCs and the expression difference from that in non-CPCs was statistically significant [26.3 ± 1.6 (95% CI: 22.328, 30.272) or 13.9 ± 1.2 (95% CI: 10.921, 16.879) in CPCs vs. 3.7 ± 0.1 (95% CI: 3.452, 3.948) in non-CPCs; P = 0.0017 or 0.0021]. Unlike IL-6, this expression pattern sustained when IL-1b was present. Also, IL-8 expression was less in CPCs than in non-CPCs when HMGB1 was present and the baseline expression difference was moderate (Figure 5 D-F).

We previously reported a group of CPCs in the superficial zone of mechanically injured articular cartilage in weight-bearing joint. Compared to normal chondrocytes, those cells were migratory in response to defined or undefined chemotactic factors resulted from cartilage damage and cell death. One defined chemotactic factor examined in that study was HMGB1 that is a NuD and can be passively released from the dead cells in damaged cartilage [11]. In order to test our hypothesis that CPCs were more responsive to DAMPs than the remaining cell population in terms of up-regulating expression of MMPs, chemokines, or cytokines involved in cartilage inflammatory degradation, we examined and compared the expression levels of MMPs, chemokines, and cytokines involved in PTOA pathogenesis between CPCs and non-CPCs in response to MTDs or NuD, two major proinflammatory DAMPs. Our study revealed that CPCs were morphologically different from non-CPCs in monolayer cultures, more active in proliferation, and more responsive to DAMPs, especially MTDs, than non-CPCs to up-regulate inflammatory genes including MMP-13, IL-6, and IL-8. To our knowledge, this is the first investigation into the inflammatory response to DAMPs by CPCs emerged from mechanically injured articular cartilage.

Compared to non-CPCs in mechanically injured cartilage, CPCs expressed significantly more MMP-13 at both mRNA and protein levels in response to HMGB1 or MTD stimulation. MMP-13 is a zinc-dependent collagenase that is a major cartilage ECM degrading metalloproteinase implicated in OA pathogenesis. The ECM of articular cartilage is mainly composed of type II collagen and proteoglycan. MMP-13 also named as collagenase-3 can specifically cleave type II collagen triple helix into characteristic ¾ and ¼ fragments [30-32]. Besides type II collagen, MMP-13 degrades other types of collagen, such as type IV and IX, and proteoglycan in cartilage ECM as well [33]. Secreted as a zymogen by chondrocytes, synoviocytes, or immune cells in a normal or an inflamed joint, the proform of MMP-13 consists of 471 amino acids and is 53.8 kDa in size. The removal of the N-terminal 84 amino acids by other MMPs, such as MMP-3, exposes the catalytic domain of MMP-13 and turns it into the active form that is 48 kDa in size [34]. Due to glycosylation modification, pro-MMP-13 displayed a MW around 64 kDa and the active form was around 54 kDa when their expressions were examined with Western blotting under reducing conditions as seen in our study [35].

Although we observed much higher levels of pro-MMP-13 produced by CPCs than non-CPCs when either DAMPs were present in the cultures, we did not detect any active form of this metalloprotease in the conditioned media of CPCs. This result was well backed up by observations that MMP-3 protein secretion by CPCs was undetectable when either DAMPs were present in the cultures. On the other hand, MMP-3 expression could be strongly induced by IL-1b in either CPCs or non-CPCs cultures accompanied by the expression of both pro- and active MMP-13. This implied that MMP-3 might be the major protease responsible for the removal of the N-terminal peptide from pro-MMP-13 structure and generation of the active form of MMP-13 in CPCs and non-CPCs. Moreover, the observation of MMP-3 expression in either CPCs or non-CPCs could only be significantly upregulated by IL-1b not by HMGB1 or MTDs was consistent with our previous discovery showing that those types of DAMPs could not upregulate expression of MMP-3 in normal chondrocytes but either of them could synergize with proinflammatory cytokines in up-regulating MMP-3 and other cartilage-degrading proteases [27]. Interestingly, we also observed that CPCs expressed much lower levels of MMP-3 mRNA and protein than did non-CPCs when IL-1b was present in the cultures. This may suggest that the majority of MMP-3 was secreted by non-CPCs rather than CPCs whose cell population was only one tenth of that of non-CPCs and mainly resided in the surface zone of the injured cartilage.

Studies have shown that chemokine CXCL12 or stromal cell-derived factor-1a (SDF-1a) is one of the up-stream effectors of MMPs in OA pathogenesis. Kanbe et al. reported that in OA patients CXCL12 secreted by synovial fibroblasts could up-regulate expression of MMP-3 in chondrocytes via interaction with its specific receptor CXCR4 [36]. Wang and colleagues further showed that MMP-3 up-regulation and cartilage degradation induced by CXCL12-CXCR4 axis could be attenuated by AMD3100, a CXCR4 antagonist [37]. Our data showed that in mechanically injured cartilage CPCs expressed ~110-fold higher baseline levels of CXCL12 mRNA than did non-CPCs. This result was consistent with our previous findings that CPCs expressed 28-fold higher CXCL12 than did normal chondrocytes or 12-fold higher than did mesenchymal stem cells (MSCs) [11]. However, the relatively higher CXCL12 mRNA expression in CPCs we observed was not accompanied by higher MMP-3 mRNA level. This might imply that chemokine CXCL12 was not a main inducer of MMP-3 expression in CPCs emerged from mechanically injured cartilage, i.e. CXCL12 could not up-regulate MMP-3 in CPCs via autocrine mode. We also observed that neither HMGB1 nor MTDs further enhanced the fold increase of CXCL12 mRNA expression in CPCs over non-CPCs. The fold increase of CXCL12 mRNA expression was only slightly enhanced by IL-1b. This indicated that those two types of DAMPs or IL-1b might have little effect on further up-regulating CXCL12 mRNA expression in CPCs which already showed much higher baseline expression than did non-CPCs.

In addition to CXCL12, chemokine CCL2 was implicated in OA pathogenesis since it could recruit monocytes to cause inflammatory damage of cartilage [38]. In our study, MTDs enhanced CCL2 mRNA expression in CPCs but to a much lesser extent than did IL-1b which greatly increased the expression by more than 100-fold. Moreover, when IL-1b was present, CPCs expressed significantly less CCL2 mRNA than did non-CPCs, which was comparable to MMP-3 expression. This concurrent up-regulation of CCL2 and MMP-3 by IL-1b observed mainly in non-CPCs was consistent with what Robert et al. reported in hepatic stellate cells involved in chronic liver inflammation [39]. Interestingly, Zarebska and colleagues discovered that CCL2 ^(-/-) mice displayed strongly suppressed MMP-3 expression in joint tissue 6 hrs post-destabilization of the medial meniscus (DMM) surgical procedure which is frequently employed to create PTOA in an animal model [40]. However, more evidence is required to verify whether MMP-3 could be up-regulated by CCL2 in CPCs in mechanically injured cartilage via autocrine pathway.

As one of the main proinflammatory cytokines involved in OA pathophysiology, IL-6 level elevates in synovial fluids and serum of OA patients and this elevation is correlated with up-regulation MMP-1 and -13 expression and with reduction of type II collagen synthesis which result in cartilage damage [41]. Our data indicated that CPCs expressed significantly higher baseline level of IL-6 mRNA than did non-CPCs. This result was consistent with our previous findings made from comparing baseline IL-6 expression between CPCs and normal chondrocytes [11]. Furthermore, we observed that MTDs remarkably enhanced IL-6 expression in CPCs. Similarly, Schwacha et al. and Hu et al. reported that MTDs could induce IL-6 expression in gammadelta T cells which play critical role in sterile inflammation like OA and could stimulate hepatocytes to increasingly produce IL-6 leading to liver injury, respectively [42, 43]. Similar to CCL2 and MMP-3, when IL-1b was present, CPCs transcribed less IL-6 mRNA than did non-CPCs. Those results implied that, in response to IL-1b, different signaling mechanisms were employed by CPCs and non-CPCs to up-regulate expression of IL-6, CCL2 and MMP-3 genes.

Studies showed that chondrocytes could secret IL-8, another member of interleukins, which attracted neutrophils to joint tissue and caused degranulation of neutrophils leading to cartilage degradation [44, 45]. A recent study further demonstrated that serum IL-8 level positively associated with knee OA symptoms, with levels of cartilage degradation markers, with up-regulation of MMP-3 & -13, and with radiographic characteristics [46]. Our data indicated MTDs could significantly up-regulate IL-8 mRNA expression in CPCs, not in non-CPCs. This suggested that in the early phase of cartilage injury CPCs might be the major player that attracts neutrophils to the injury site via IL-8 production upon stimulation mainly by MTDs released from dead cells. IL-8 might act on CPCs via autocrine pathway to up-regulate MMP-13 expression in response to MTD stimulation.

Taken together, our study demonstrated that CPCs emerged from the superficial zone of mechanically injured articular cartilage and accounted for only ~10% of total cell population not only divided two times faster than did non-CPCs but also expressed significantly higher baseline mRNA levels of MMP-13, CXCL12, and IL-6. More importantly, CPCs responded to endogenous DAMPs differently from non-CPCs. Between two types of DAMPs examined in this study, MTDs were more stimulatory than HMGB1 to CPCs in terms of up-regulating expression of MMP-13, IL-6, and -8. Those findings shed light on the dynamics of inflammatory response inside mechanically injured articular cartilage which may lead to PTOA. Future studies are needed to elucidate the complicated role of CPCs playing in the early phase of PTOA development.

Mechanical injury to articular cartilage surface stimulated migration of CPCs to the lesion. Due to this migratory property, CPCs could be easily separated from non-CPCs in injured cartilage with trypsin treatment. In addition to shorter PDT and more elongated shape in monolayer cultures, CPCs displayed were more sensitive than non-CPCs in response to DAMPs, especially to MTDs, that were released from damaged mitochondria and nuclei. Compared to non-CPCs, significant up-regulation of MMP-13, IL-6 and -8 was detected in CPCs when challenged by MTDs. Nonetheless, CPCs were less responsive to IL-1b than did non-CPCs in terms of up-regulating MMP-3, CCL2, and IL-6 expression. The proinflammatory nature of CPCs implied their critical role in the early phase of PTOA development.

PTOA: Post-traumatic osteoarthritis

CPCs: Chondrogenic progenitor cells

DAMPs: Damage-associated molecular patterns

MTDs: Mitochondrial damage-associated molecular patterns

NuD: Nuclear damage-associated molecular patterns

HMGB1: High mobility group box 1 protein

SDF-1a: Stromal cell-derived factor-1a

MSCs: Mesenchymal stem cells

Ethics declarations

Ethical Approval and Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of supporting data

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors have nothing to declare.

Funding

Supported by US DHHS, National Institutes of Health/NIAMS grant P50 AR055533, by a Merit Review Award from the Department of Veterans Affairs, and by the Jiangsu Provincial Natural Science Foundation of China grant awarded to Lei Ding (BK20171143).

Authors' contributions

Conception and design: L. D., J. A. M., analysis and interpretation of the data: L. D., Z. C., H. Z., J. A. M., drafting of the article: L. D., critical revision of the article for important intellectual content: Q. W., H. S., J. A. B., final approval of the article: L. D., Z. C., H. Z., Q. W., H. S., J. A. M., J. A. B., obtaining of funding: L. D., J. A. M., J. A. B., collection and assembly of data: L. D, Z. C., H. Z.

Acknowledgements

We thank Barbara Laughlin, Abigail Lehman, John Bierman for preparing osteochondral explants and culture reagents.

Authors' information

Affiliations

Jiangnan University Wuxi College of Medicine, Department of Basic Medical Sciences, Wuxi, Jiangsu, China

Lei Ding

University of Iowa Hospitals and Clinics, Department of Orthopaedics and Rehabilitation, Iowa City, Iowa, USA

Lei Ding, Cheng Zhou, Hongjun Zheng, Joseph A. Buckwalter & James A. Martin

Jiangnan University Affiliated Hospital, Department of Orthopaedic Surgery, Wuxi, Jiangsu, China

Quanming Wang

The Second Affiliated Hospital of Harbin Medical University, Dept. of Endocrinology and Metabolism, Harbin, China

Haiyan Song

Veterans Affairs Medical Center, Iowa City, Iowa, USA

Joseph A. Buckwalter

Anderson DD, Chubinskaya S, Guilak F, Martin JA, Oegema TR, Olson SA, et al. Post-traumatic osteoarthritis: improved understanding and opportunities for early intervention. J Orthop Res 2011; 29: 802-809.
Bhosale AM, Richardson JB. Articular cartilage: structure, injuries and review of management. Br Med Bull 2008; 87: 77-95.
Brown TD, Johnston RC, Saltzman CL, Marsh JL, Buckwalter JA. Posttraumatic osteoarthritis: a first estimate of incidence, prevalence, and burden of disease. J Orthop Trauma 2006; 20: 739-744.
Buckwalter JA, Brown TD. Joint injury, repair, and remodeling: roles in post-traumatic osteoarthritis. Clin Orthop Relat Res 2004: 7-16.
Martin JA, Brown T, Heiner A, Buckwalter JA. Post-traumatic osteoarthritis: the role of accelerated chondrocyte senescence. Biorheology 2004; 41: 479-491.
Furman BD, Olson SA, Guilak F. The development of posttraumatic arthritis after articular fracture. J Orthop Trauma 2006; 20: 719-725.
Mrosek EH, Lahm A, Erggelet C, Uhl M, Kurz H, Eissner B, et al. Subchondral bone trauma causes cartilage matrix degeneration: an immunohistochemical analysis in a canine model. Osteoarthritis Cartilage 2006; 14: 171-178.
Akkiraju H, Nohe A. Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration. J Dev Biol 2015; 3: 177-192.
Dowthwaite GP, Bishop JC, Redman SN, Khan IM, Rooney P, Evans DJ, et al. The surface of articular cartilage contains a progenitor cell population. J Cell Sci 2004; 117: 889-897.
Williams R, Khan IM, Richardson K, Nelson L, McCarthy HE, Analbelsi T, et al. Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage. PLoS One 2010; 5: e13246.
Seol D, McCabe DJ, Choe H, Zheng H, Yu Y, Jang K, et al. Chondrogenic progenitor cells respond to cartilage injury. Arthritis Rheum 2012; 64: 3626-3637.
Jeffrey JE, Gregory DW, Aspden RM. Matrix damage and chondrocyte viability following a single impact load on articular cartilage. Arch Biochem Biophys 1995; 322: 87-96.
Bush PG, Hodkinson PD, Hamilton GL, Hall AC. Viability and volume of in situ bovine articular chondrocytes-changes following a single impact and effects of medium osmolarity. Osteoarthritis Cartilage 2005; 13: 54-65.
Martin JA, McCabe D, Walter M, Buckwalter JA, McKinley TO. N-acetylcysteine inhibits post-impact chondrocyte death in osteochondral explants. J Bone Joint Surg Am 2009; 91: 1890-1897.
Natoli RM, Scott CC, Athanasiou KA. Temporal effects of impact on articular cartilage cell death, gene expression, matrix biochemistry, and biomechanics. Ann Biomed Eng 2008; 36: 780-792.
Milentijevic D, Rubel IF, Liew AS, Helfet DL, Torzilli PA. An in vivo rabbit model for cartilage trauma: a preliminary study of the influence of impact stress magnitude on chondrocyte death and matrix damage. J Orthop Trauma 2005; 19: 466-473.
Isaac DI, Meyer EG, Kopke KS, Haut RC. Chronic changes in the rabbit tibial plateau following blunt trauma to the tibiofemoral joint. J Biomech 2010; 43: 1682-1688.
Lieberthal J, Sambamurthy N, Scanzello CR. Inflammation in joint injury and post-traumatic osteoarthritis. Osteoarthritis Cartilage 2015; 23: 1825-1834.
Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 1994; 12: 991-1045.
Seong SY, Matzinger P. Hydrophobicity: an ancient damage-associated molecular pattern that initiates innate immune responses. Nat Rev Immunol 2004; 4: 469-478.
Hajizadeh S, DeGroot J, TeKoppele JM, Tarkowski A, Collins LV. Extracellular mitochondrial DNA and oxidatively damaged DNA in synovial fluid of patients with rheumatoid arthritis. Arthritis Res Ther 2003; 5: R234-240.
Dong C, Liu Y, Sun C, Liang H, Dai L, Shen J, et al. Identification of Specific Joint-Inflammatogenic Cell-Free DNA Molecules From Synovial Fluids of Patients With Rheumatoid Arthritis. Front Immunol 2020; 11: 662.
Collins LV, Hajizadeh S, Holme E, Jonsson IM, Tarkowski A. Endogenously oxidized mitochondrial DNA induces in vivo and in vitro inflammatory responses. J Leukoc Biol 2004; 75: 995-1000.
Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 2010; 464: 104-107.
Klune JR, Dhupar R, Cardinal J, Billiar TR, Tsung A. HMGB1: endogenous danger signaling. Mol Med 2008; 14: 476-484.
Levy RM, Mollen KP, Prince JM, Kaczorowski DJ, Vallabhaneni R, Liu S, et al. Systemic inflammation and remote organ injury following trauma require HMGB1. Am J Physiol Regul Integr Comp Physiol 2007; 293: R1538-1544.
Ding L, Buckwalter JA, Martin JA. DAMPs Synergize with Cytokines or Fibronectin Fragment on Inducing Chondrolysis but Lose Effect When Acting Alone. Mediators Inflamm 2017; 2017: 2642549.
Ding L, Guo D, Homandberg GA, Buckwalter JA, Martin JA. A single blunt impact on cartilage promotes fibronectin fragmentation and upregulates cartilage degrading stromelysin-1/matrix metalloproteinase-3 in a bovine ex vivo model. J Orthop Res 2014; 32: 811-818.
Zhou C, Zheng H, Seol D, Yu Y, Martin JA. Gene expression profiles reveal that chondrogenic progenitor cells and synovial cells are closely related. J Orthop Res 2014; 32: 981-988.
Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PJ, et al. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest 1996; 97: 761-768.
Wang M, Sampson ER, Jin H, Li J, Ke QH, Im HJ, et al. MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther 2013; 15: R5.
Perez-Garcia S, Carrion M, Gutierrez-Canas I, Villanueva-Romero R, Castro D, Martinez C, et al. Profile of Matrix-Remodeling Proteinases in Osteoarthritis: Impact of Fibronectin. Cells 2019; 9.
Shiomi T, Lemaitre V, D'Armiento J, Okada Y. Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases. Pathol Int 2010; 60: 477-496.
Malemud CJ. Matrix Metalloproteinases and Synovial Joint Pathology. Prog Mol Biol Transl Sci 2017; 148: 305-325.
Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem 1996; 271: 1544-1550.
Kanbe K, Takagishi K, Chen Q. Stimulation of matrix metalloprotease 3 release from human chondrocytes by the interaction of stromal cell-derived factor 1 and CXC chemokine receptor 4. Arthritis Rheum 2002; 46: 130-137.
Wang XY, Chen Y, Tang XJ, Jiang LH, Ji P. AMD3100 Attenuates Matrix Metalloprotease-3 and -9 Expressions and Prevents Cartilage Degradation in a Monosodium Iodo-Acetate-Induced Rat Model of Temporomandibular Osteoarthritis. J Oral Maxillofac Surg 2016; 74: 927 e921-927 e913.
Raghu H, Lepus CM, Wang Q, Wong HH, Lingampalli N, Oliviero F, et al. CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis. Ann Rheum Dis 2017; 76: 914-922.
Robert S, Gicquel T, Bodin A, Lagente V, Boichot E. Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1beta or TNF-alpha Release from Human Hepatic Stellate Cells. PLoS One 2016; 11: e0153118.
Miotla Zarebska J, Chanalaris A, Driscoll C, Burleigh A, Miller RE, Malfait AM, et al. CCL2 and CCR2 regulate pain-related behaviour and early gene expression in post-traumatic murine osteoarthritis but contribute little to chondropathy. Osteoarthritis Cartilage 2017; 25: 406-412.
Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol 2011; 7: 33-42.
Schwacha MG, Rani M, Zhang Q, Nunez-Cantu O, Cap AP. Mitochondrial damage-associated molecular patterns activate gammadelta T-cells. Innate Immun 2014; 20: 261-268.
Hu Q, Wood CR, Cimen S, Venkatachalam AB, Alwayn IP. Mitochondrial Damage-Associated Molecular Patterns (MTDs) Are Released during Hepatic Ischemia Reperfusion and Induce Inflammatory Responses. PLoS One 2015; 10: e0140105.
Elford PR, Cooper PH. Induction of neutrophil-mediated cartilage degradation by interleukin-8. Arthritis Rheum 1991; 34: 325-332.
Lotz M, Terkeltaub R, Villiger PM. Cartilage and joint inflammation. Regulation of IL-8 expression by human articular chondrocytes. J Immunol 1992; 148: 466-473.
Ruan G, Xu J, Wang K, Zheng S, Wu J, Bian F, et al. Associations between serum IL-8 and knee symptoms, joint structures, and cartilage or bone biomarkers in patients with knee osteoarthritis. Clin Rheumatol 2019; 38: 3609-3617.

Download PDF

Version 1

posted

You are reading this latest preprint version

Progenitor Cells Derived From Injured Cartilage Surface Respond to Damage Associated Molecular Patterns

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Version 1