Mast Cell Induction of Fibrotic Signaling
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Mast Cell Induction of Fibrotic Signaling Following Severe Burn Injury
Jayson W. Jay1,3, Amina El Ayadi2, Michael D. Wetzel2, Anesh Prasai2, David N. Herndon2,3, Celeste C. Finnerty1,2,3
Institute for Translational Sciences1, Department of Surgery2, Shriners Hospital for Children3,
University of Texas Medical Branch, Galveston, Texas 77555
Over 450,000 burn injuries are treated annually in the US, with approximately 25,000 cases requiring intensive inpatient care. Painful and pruritic hypertrophic scars (HTS), occurring subsequent to delayed wound healing, are a major sequela in up to 90% of severely burned patients, reducing function and quality of life. It is well-understood that transforming growth factor-beta (TGF-²) signaling serves as a potent profibrotic stimulus, contributing to proliferation in dermal fibroblasts (Fb) and excessive collagen deposition during HTS progression. Current therapeutic options, including pressure garments and surgical interventions, are inadequate for reducing post burn HTS fibrosis. Scar contractures persist and functional restoration is incomplete, limiting the post burn quality of life. despite growing evidence of mast cell involvement in fibrotic pathologies (disorders?), investigations into MC roles in post-burn scarring pathogenesis remains relatively
To identify novel therapies to improve wound healing, we have studied the role of mast cells (MCs) in the innate immune response as moderators of pathologic scarring. Proteinases released by MCs contribute to both extracellular matrix (ECM) remodeling and a fibrotic phenotype through an MC-Fb paracrine axis,(Dong, Chen et al. 2012) though the mechanism remains poorly understood. The MC-Fb axis points to a role for protease activated receptors (PARs) in dermal Fbs. Physiologically, limited and distinct serine proteinases are known to activate PAR-2. Moreover, the only MC granule constituent known to activate PAR-2 signaling is tryptase, which may have consequences in scarring pathologies. PAR-2 stimulation is known to initiate cellular proliferation in pulmonary Fbs,(Akers, Parsons et al. 2000) yet PAR-2 expression, activation, and subsequent signaling mechanisms have yet to be investigated in post burn HTSs.
In this investigation we examined the paracrine signaling axis between major MC secreted mediators and dermal fibroblast PAR-2 involvement in the progression of the hypertrophic scar phenotype following a severe burn injury.
MATERIALS AND METHODS
- Mast Cell density in post burn hypertrophic scar over time
Tissue procurement and paraffin embedding: Hypertrophic scar (HTS) and bordering non-burned skin (NBS) biopsies were obtained during normal revision surgeries at 6 months, 12 months, 24 months, and 48 months following burn injury from pediatric burn patients in this IRB approved study. Samples were rinsed in sterile PBS and immediately transferred to 10% neutral buffered formalin for a minimum of 48 hours for fixation. Following, tissues were transferred to 70% ethanol for 24 hours then processed and embedded in paraffin wax blocks.
- Toluidine blue staining
- [Quantitative; blinded observer counts (at low power)]
- [Need new staining and quantification at high power (recent literature utilizes "MCs per high-powered field", but we can stick with MCs per mm2]
- Dual Immunofluorescence; targets: Tryptase & Chymase
- [Qualitative and Quantitative; corrected total cell fluorescence (CTCF) calculation via ImageJ: 5 randomly selected regions of interest per slide per time point measured]
Immunostaining and Immunofluorescence Quantification: Tissues were cut to 4¼M sections and positioned onto glass slides. Sections were then cleared in washes of xylene and rehydrated through 100% and 95% ethanol to water. Antigen retrieval proceeded by incubating tissues in heated target retrieval solution (1X, DAKO). Sections were then washed and blocked in 5% horse serum for 1 hour prior to incubating in primary antibody overnight at 4oC. Isotyped antibody (IgG) served as primary negative controls for all tissues. Following overnight incubation, sections were washed in PBS then sections were covered fluorescent-conjugated secondary antibody for 1 hour at room temperature. Following secondary incubation, all sections were stained with nuclear label DAPI (1:5000) diluted in PBS for 20 minutes prior to imaging. Imaging was completed on a NIKON (insert device info here). Fluorescence intensity was quantified through corrected total cell fluorescence (CTCF) with ImageJ utilizing 5 randomly selected dermal regions of interest per image at high magnification (20X). Five separate tissues sections per time point were analyzed. One-way ANOVA analysis compared CTCF values between groups.
- PAR-2 expression in post burn hypertrophic scar fibroblasts
- [Immunocytochemistry for PAR-2 expression between PCS and HTS / no treatment, just basal expression]
Immunocytochemistry: PCS, NBS and primary HTS cells were expanded in DMEM FBS-supplemented media and passaged once prior to fixation and fluorescent immunostaining. (This method section needs a little work)
- MC Tryptase and Chymase effect on dermal fibroblast fibrotic signaling gene expression
- [NBS and HTS in vitro (cell culture), treatment with synthetic PAR-2 inhibitor, then treat with recombinant MC Tryptase, MC Chymase, both, or neither]
Cell Culture and Treatment: 2.50 x 105 PCS (ATCC) fibroblasts, primary human fibroblasts from NBS and HTS were cultured in Dulbecco's Minimal Essential Media supplemented with 10% fetal bovine serum for 24 hours in 12-well plates. After 24 hours of attachment, media was refreshed and cells were treated with either media only (control), recombinant human (rh) Tryptase-²II (rhTryp) (200 ng/mL, Enzo Life Sciences), or rhChymase (200 ng/mL, R&D Systems). In parallel, each treatment group was pretreated with the PAR-2 antagonist FSLLRY (10 ¼M, Tocris Bioscience) for 1 hour before being treated with either media only, rhTryptase (200 ng/mL), or rhChymase as before. After 24 hours, media was removed and cells were washed in PBS prior to RNA isolation. All experiments were performed in triplicate.
- [RT-qPCR; targets: PAR-2, ERK-1, ERK-2, Collagen-I (²-actin as housekeeping control)]
RNA Isolation: RNA isolation via the RNEasy Mini Kit (Qiagen) proceeded according to manufacturer's instructions. Breifly, cells were lysed in RLT buffer and RNA was allowed to precipitate in 80% ethanol before transfer to the RNA mini column. Following centrifugation, the column was washed twice in wash buffer and once in RPE buffer. Finally isolated RNA was eluted in approximately 20 ¼L RNase-free water. RNA concentration and purity was assessed by UV spectroscopy (NanoDrop 1000); absorbance was measured at 260 nm and 280 nm.
qRT-PCR: Isolated RNA was then reverse transcribed to yield pure complimentary DNA (cDNA) via iScript Reverse Transcriptase kit (Biorad). Thermocycling was set to 5 minutes at 25oC, 30 minutes at 42oC, then 5 minutes at 85oC. Once cycling was complete, cDNA was held at 4oC until gene expression assessment via PCR. Prior to PCR, cDNA was diluted in DNase-free water 1:10. Transcripts were then amplified using the (insert machine name here) with SYBR Green Master Mix along with primer pairs for PAR-2 (F: 5'-GCAGGTGAGAGGCTGACTTT-3', R: 5'-CAGTCGGTTCCGGTCTAACCGG-3', Hamilton et al., 2001), ERK-1 (F: 5'-TACAAGCTTAGCTCGGCCTATGACCACGTG-3', R: 5'-TACGAATTCGGCTTTAGATCTCGGTGGAGC-3', Akers et al., 2000), ERK-2 (F: 5'-TACAAGCTTCGAGCACCAACCATCGAGCAA-3', R: ACGAATTCCCCTGTACCAAGGTGTGGCCA-3', Akers et al., 2000), and Collagen-I (F: 5'-CCCAAGGACAAGAGGCATGT-3', R: 5'-CACGTGGGAGTGATGGAGAG-3', NCBI Primer Blast). Gene expression was then normalized to ²-Actin and fold change was calculated utilizing the delta-delta Ct method. Gene expression between treatment groups was assessed by one-way ANOVA with a post-hoc Tukey's test. Significant differences in expression were accepted at p 0.05.
- MC released mediator effect on dermal fibroblast proliferation
- [Same in vitro setup as (B) above].
- [MTT Assay for each condition].
MTT Proliferation Assay: An MTT assay (ATCC Life Sciences, Manassas, VA) determined the cellular proliferation rate per manufacturer's instructions. Briefly, after culture treatment, 10 ¼L MTT Reagent was added per well. The plate was then incubated at 37oC in 5% CO2 for 3 hours until a purple precipitate was visible within cells. Detergent reagent then lysed the cells at ambient temperature for 2 hours in the dark. Optical density (OD) absorbance was recorded at 570nm. Mean OD readings per treatment were normalized to controls and assessed by a two-tailed student's t test for comparisons between NBS and HTS groups, as well as one-way analysis of variance (ANOVA) for assessments between treatments. Significant differences in proliferation were accepted at p0.05.
- Mast cell presence increases over time in pediatric post burn hypertrophic scars.
Toluidine blue staining and blinded observer counts show significantly increased presence of mast cells (Fig. 1) in post-burn scars over time. MC density increased in HTS during all time points compared to NBS, significantly at 6 months (p<0.05) and most significantly at 24 months (p<0.0001) following burn injury. Mean mast cell density in NBS is 7.2 MC/mm2 (SD ± 1.29), at 6 months post burn is 218.9 MC/mm2 (SD ± 63.9), at 12 months post burn is 144.3 MC/mm2 (SD ± 48.5), at 18 months 75.7 MC/mm2, at 24 months is 677.5 MC/mm2, and at 48 months is 77.2 MC/mm2.
- Dual immunofluorescence confirms increased tryptase- and chymase-positive MCs in post burn hypertrophic scars over time (Fig. 2):
Quantitative CTCF shows increased presence of MC Tryptase and Chymase in post-burn HTS over time compared to non-burned skin. Mean tryptase/chymase dual stain CTCF values for post-burn HTS at 6 months (5.8 x 104), 24 months (7.8 x 104), and 48 months (1.2 x 105) were significantly elevated (*p<0.05) when compared to non-burned skin (NBS, 2.4 x 104). Mean CTCF for post-burn HTS at 12 months (5.0 x 104) were not significantly different from NBS. Mean CTCF HTS at 48 months was also significantly higher when compared to all other HTS time points (p<0.01) These data show MC localization to burn scars after several years following the original injury, further showing that MC density increases significantly over time. This suggests that MCs play a vital role in the maintenance of the post burn scar tissue and, together with previous data, provides more evidence that MCs may indeed contribute to the fibrotic phenotype seen in hypertrophic scar pathology.
- ICC PAR-2 expression is significantly increased in HTS compared to NBS.
- Mean PAR-2 CTCF for post-burn HTS primary fibroblasts (1.30 x 106 ± 2.93 x 105) was significantly more intense than mean CTCF for neonatal PCS fibroblasts (1.02 x 105 ± 1.17 x 105) in vitro. These data also indicate localization of PAR-2 to HTS membranes as compared to nuclear overlap signals seen in PCS fibroblasts. (Fig. 3)
- Following tryptase treatment, collagen-I mRNA increased significantly in HTS Fbs compared to NBS Fbs after 24 hours. However, when first treated with FSLLRY, collagen-I and ERK 1/2 mRNA levels decreased significantly in HTS Fbs (p<0.05). PAR-2 transcripts did not reach the level for detection. These data may indicate that MC tryptase may contribute to dermal fibroblast proliferation via PAR-2 activation and subsequent ERK signaling in hypertrophic scar progression (Fig. 4)
- HTS proliferation increased significantly compared to NBS after treatment with rhTryp (p=0.003, n=3). However, when cells were pretreated with FSLLRY then treated with rhTryp, cellular proliferation significantly decreased in HTS fibroblasts (p0.05, n=3), whereas, there was no difference in the proliferation of NBS fibroblasts. When treated with rhChym alone, there were no significant differences in cellular proliferation between NBS and HTS or when pre-treated with FSLLRY then with rhChym. For combination treatment with rhTryp and rhChym, there was a significant decrease in HTS proliferation compared to NBS proliferation when pre-treated with FSLLRY (p0.05) (Fig. 5)
Need a discussion wrapup for mast cell presence increasing in HTS (from TB stain and IF dual staining ---- possibly due to increased chemotactic factors within the wound )
Together, these data suggest that MC tryptase has a proliferative effect in HTS-derived fibroblasts and that the fibroproliferative effect can be inhibited by antagonism of the PAR-2 receptor. This indicates that MC degranulation may contribute to HTS progression through continued activation of fibroblast PAR-2 receptors. Interestingly, it appears that MC chymase has a greater proliferative effect in NBS-derived fibroblasts as compared to HTS-derived fibroblasts indicating that MC chymase's effects may be localized to the borders of post-burn HTS.
Future directions will examine post burn wound environment for potential MC chemotactic factors
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