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Open Access

Peer-reviewed

Research Article

NGF Modulates trkANGFR/p75NTR in αSMA-Expressing Conjunctival Fibroblasts from Human Ocular Cicatricial Pemphigoid (OCP)

Abstract

Objective

In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkANGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure.

Materials and Methods

Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkANGFR/p75NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkANGFR/p75NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters.

Results

OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75NTR and lower trkANGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75NTR. NGF exposure did not affect trkANGFR levels in early OCP-FBs while decreased both αSMA/p75NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs.

Conclusions

Taken together, these data are suggestive for a NGF/p75NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75NTR might be viewed as a potential therapeutic target.

Citation: Micera A, Stampachiacchiere B, Di Zazzo A, Sgrulletta R, Cortes M, Normando EM, et al. (2015) NGF Modulates trkANGFR/p75NTR in αSMA-Expressing Conjunctival Fibroblasts from Human Ocular Cicatricial Pemphigoid (OCP). PLoS ONE 10(11): e0142737. https://doi.org/10.1371/journal.pone.0142737

Editor: Andrea Caporali, University of Edinburgh, UNITED KINGDOM

Received: January 16, 2015; Accepted: October 26, 2015; Published: November 16, 2015

Copyright: © 2015 Micera et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper.

Funding: This work was supported by the Italian Ministry of Health (MoH) and by the intramural financial support (Fondazione Roma). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. In addition, the authors would like to underline that the results reported in the present manuscript are original and not submitted elsewhere.

Introduction

The Ocular Cicatricial Pemphigoid (OCP) is an immune-mediated chronic inflammatory disease of the eye, characterized by chronic-recurrent conjunctival inflammation, progressive sub-epithelial fibrosis and tissue remodeling [1–3]. Inflammatory infiltrates and activated Fibroblasts (FBs) contribute actively to the uncontrolled extracellular matrix (ECM) deposition (remodeling process), leading to structural and functional changes (keratinization and blindness) [3,4]. Several pro-inflammatory/fibrogenic cytokines and growth factors, including Transforming Growth Factor β1 (TGFβ1) and Interleukin 4 (IL4), have shown the ability to modulate the survival of activated FBs and their collagen deposition [2,5–7].

An involvement of Nerve Growth Factor (NGF) pathway in OCP has been previously reported by our group: an increased trkANGFR immunoreactivity has been observed in OCP conjunctival stroma and a consistent NGF release has been quantified in OCP tears [8,9]. The effect of NGF in tissue remodelling and fibroblast activity is actually controversial: NGF might exert pro/anti-inflammatory effects or profibrogenic activity, acting as a “modulator” of the local immune/inflammatory response, in a receptor expression dependent manner [10–16]. In the last decade, the NGF modulatory effect on Fibroblasts (FBs) and their activated myofibroblast counterpart (myoFBs) has been prospected in view of the surface trkANGFR/p75NTR rate expression and the NGF ability to trigger apoptosis in FBs from different tissues as well as TGFβ1-induced myoFBs [17–21].

To address the question as whether NGF might modulate OCP-fibrosis, activated FBs and NGF immunoreactivity were verified both in tissues and cultures. Next, the study was extended to the in vitro characterization of OCP-FBs and the potential NGF influence on OCP-FB phenotype by monitoring αSMA, trkANGFR/p75NTR and TGFβ1/IL4 in NGF-exposed OCP sub-cultures.

Materials and Methods

Ethics Statement

The study followed the guidelines of the Declaration of Helsinki for research involving human subjects and was approved by the intramural Ethical committee (UCBM). Informed written consent was signed by each patient adhering to the study.

Patients and conjunctival specimens

Conjunctival biopsies were obtained from 7 patients with clinical and histological (Hematoxylin & Eosin, HE; Bio-Optica, Milan, Italy) diagnosis of OCP (2M/5F; mean age, SD, range 55–88 years) and from 6 healthy age matched patients (control group), during routine cataract surgery (5M/1F; mean age, SD, range 59–81 years).

Two fragments were produced from each biopsy: one conjunctival fragment was included in paraffin and sectioned to provide 5μm-sections for light/confocal microscopy, while the other fragment was used to achieve primary culture of conjunctival OCP FBs.

OCP specimens were classified according to the stage of the disease [22,23] and grouped as follows: early group comprising 3 patients (stages I or II; Foster) and advanced group including 4 patients (stages III or IV). The immunofluorescent analysis was performed for identifying the presence of a linear immunoglobulin deposition alongside the Basament Membrane Zone (BMZ), according to the specific immunoreactivity (FC-coupled IgGAM antibodies; OBT0119F; Oxford Biotech., Oxford, UK), univocally present in OCP positive sections. Basal histology included Giemsa (48900; Fluka, Milan, Italy), Haematoxylin and Eosin (HE; 05-M06014/05-M10002;Bio-Optica; Milan, Italy) as well as the Periodic Acid Schiff (PAS; 04-130802/05-M06002; Bio-Optica; Milan, Italy) stainings.

All sterile tissue culture plastic-ware and reagents were from NUNC (Roskilde, Denmark) and Serva (Heidelberg, Germany). Culture media and supplements were from Euroclone (Milan, Italy). Ultrapure/RNAse free water was provided by Direct-Q5 Apparatus (Millipore, Vimodrone, Milan, Italy).

Explants, FB subcultures and NGF studies

Conjunctival fragments were put as explants in 24-well plates and left to attach for 10min before adding DMEM supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 1mM sodium Pyruvate, 2mM glutamine, 100U/mL penicillin and 100μg/mL streptomycin (37°C, 5% CO2 in air) [17,18]. Outgrowing FBs were quickly harvested (0.2% trypsin-0.025% EDTA; HyQ trypsin; HyClone, Waltham, MA) and directly used or sub-cultured in T-21cm2/T-75cm2 flasks (3rd-5th passage) for NGF exposure. The cell culture purity was estimated by the CK19 exclusion test (anti-cytokeratin 19 conjunctival epithelial marker, 1/100; Dako, Hamburg, Germany).

For stimulation studies, serum starved confluent monolayers were exposed to increasing NGF concentrations (0 to 100ng/mL β-NGF Grade I; Alomone Labs Ltd, Jerusalem, Israel) or 1ng/mL TGFβ1 (positive control; R&D system, Minneapolis, MN) performed in 0.5% FBS-DMEM for 24hrs.

Conditioned media were collected for ELISA/Western Blot analysis, while monolayers were directly processed for confocal analysis or treated with trypsin-EDTA solution to obtain single cells for molecular (105 cells) / biochemical (106 cells) analysis.

Confocal Analysis

Conjunctival sections and monolayers were subjected to fluorescent immunostaining. Briefly, de-waxed sections and confluent monolayers on round coverslips (Mierfield, USA) were washed in Hank’s Balanced Sodium Salt (HBSS), fixed in 2% buffered ρ-Formaldehyde (PFA), equilibrated in PBS [10mM phosphate buffer and 137mM NaCl; pH 7.5], briefly permeabilized with 0.5% Triton X100 in PBS (TX-PBS) and probed with the specific antibodies, either alone or in combination: mouse anti-human αSMA antibodies (1/60; Novocastra, Newcastle, UK), goat anti-human NGF antibody (sc-549; 1/100); rabbit anti-human trkANGFR (sc-118; 1/150) and goat anti-human p75NTR (sc-6188; 1/75) antibodies (all from Santa Cruz Biotech., Santa Cruz, CA). The specific binding was detected by using Cy2/Cy3-conjugated specie-specific secondary antibodies, depending on the specific staining (1/500-1/700; Jackson ImmunoResearch Labs., Europe Ltd, Suffolk, UK). Nuclei were counterstained with Propidium Iodide (5μg/mL; ICN, Milan, Italy). Acquisitions were carried out using the E2000U confocal microscope equipped with C1 software (Nikon, Tokyo, Japan). Control sections were stained in parallel (control irrelevant IgGs; Vector Laboratories, Inc. Burlingame, CA) and used for the channel-series acquisitions (Nikon).

Flow cytometry

Single-cells were washed in HBSS containing Ca++/Mg++ and fixed in 3.7% buffered PFA. After washing, cells were either directly immunostained or further incubated in 70% methanol in PBS (20°C, 24hrs). For staining, cells were equilibrated in FACS buffer [0.1% saponin and 0.1% NaN3 in PBS; pH 7.5] and incubated with the above reported primary antibodies diluted in 0.1% BSA-FACS buffer. The specific binding was detected with Cy2/PE-conjugated specie-specific secondary antibodies (1/600-1/700; Jackson). Cells (104 events) were analysed using the MACSQuant flow cytometer and cell plots were arranged using the manufacturers’ provided software (Miltenyi Biotech., Gladbach, Germany). Changes in Mean Fluorescence Intensity (MFI) were calculated as follows: ΔMFI = [(MFIspecific/ MFInot-specific] and ΔMFI values ≥1 were used for statistical analysis.

Relative Real-Time PCR analysis

Total RNA was extracted from confluent monolayers using the Puregene RNA purification kit (Gentra Systems, Minnesota, USA). Total RNA samples were spectrophotometrically analysed (ND-1000; NanoDrop, Wilmington, DE; λ260/λ280>1.8). Total RNA (3 μg) samples were reverse transcribed to a final volume of 21μlL, using 50 pM oligo dT21-primer, 1 mM dNTP mix and 200 U reverse transcriptase (IMPROM; Promega, Milan, Italy) in a programmable PTC100 thermocycler (MJ Research, Watertown, MA) and 3μL were run for amplification with the specific target/referring primers in an Opticon2 MJ thermocycler (MJ Research), according to the manufacturers’ instructions (Table 1). PCR experiments were carried out in a final volume of 20μL containing 3μL cDNA for target genes (or 1μL/3μL for GAPDH/H3 reference genes) and 17μL of master mix [10μL SYBR Green PCR Mix (Applied Biosystems, Foster City, CA), 0.5μL of each primer (10 pM; MWG, Biotech, Ebersberg, Germany) and milliQ-water]. Amplicons were verified for their specificity according to the Southern blotting analysis. Negative controls (without template or with total RNA) were carried out for each run, to rule out any genome contamination. Single threshold cycle values (Cts) were run in the REST 384–2006 software [24] to get increase or decrease difference in target gene expression, with respect to reference genes and compared to controls. Data are gene expression ratio provided in log2 scale.

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Table 1. Primers and amplification program used in the study.

https://doi.org/10.1371/journal.pone.0142737.t001

ELISA

To evaluate NGF in the culture media, a two site NGF-ELISA (0.5pg/mL sensitivity and no cross-reactivity) was carried out in Maxisorp NUNC 96 well ELISA plates precoated with mouse anti-NGF antibodies (0.4μg/mL; MAB256, R&D) and incubated with standards (0.15pg/mL to 1ng/mL β-NGF; Alomone) or prediluted samples (1:3). The following steps included as follows: the biotinylated polyclonal anti-NGF antibodies (0.15μg/mL; BAF256, R&D), the streptavidin solution (Biosource International, Camarillo, CA) and the TMB substrate (Biosource). Optical density was detected by an ELISA plate reader (λ450–550; Sunrise; Tecan Systems, Inc., San Jose, CA) and calculations were carried out according to the protein normalization (A280 Nanodrop) [16]. The biological activity of NGF released by OCP-FBs was verified separately by using a PC12 bioassay (see [18]).

TGFβ1 and IL4 were measured in the conditioned media by commercially available ELISA kits, according to the manufacturers’ instructions (Biosource).

Western Blotting

Total proteins were extracted from single-cells lysed in modified RIPA buffer [50 mM Tris-HCl (pH 7.7), 150mM NaCl, 1%Triton X-100 and 0.1% SDS] freshly supplemented with phosphatase/protease inhibitors (Pierce). Equivalent amounts of protein (30μg) were subjected to 7–15% SDS-PAGE electrophoresis (Miniprotean3; Bio-Rad, Hercules, CA) and resolved proteins were transferred to Hy-bond membranes (semi-dry blotting apparatus; Bio-Rad). The membranes were washed in 0.05% Tween 20 in PBS (TW-PBS), blocked in 5% non-fat dry milk-PBS and probed with trkANGFR (1/700), p75NTR(1/500), αSMA (1/500) and GAPDH (1/1000) primary antibodies, followed by secondary POD-conjugated specie-specific antibodies (1/10000; Jackson Immuneresearch). Developing was performed according to the ECL technique (SuperSignal West Pico Trial; Pierce, Rockford, IL). Bands were acquired/analysed in a Kodak Image station equipped with a 1D Image analysis software (110F; Kodak, Tokyo, Japan).

Statistical Analysis

All experimental procedures were conducted in triplicate, each one repeated three times. Both molecular and biochemical data (means±SD) were analysed for significant differences (p < .05), by the ANOVA-Tukey-Kramer coupled post-hoc analysis (StatView II for PC; Abacus Concepts. Inc., Barkley, CA). REST-ANOVA analysis followed by Tukey Kramer posthoc comparisons was used to validate molecular data.

Results

Only conjunctival biopsies from patients’ with confirmed ophthalmic and microscopic diagnosis of OCP were included in this study. Immunofluorescent staining revealed the presence of a clear linear fluorescence alongside the BMZ, representative of autoantibodies deposition in both early (A) and advanced (B) OCP sections (Fig 1). No significant difference in IgGAM immunoreactivity was observed between early and advanced OCP, except for an increasing fluorescent signal in the epithelium and stroma (asterisk; Fig 1B). Contiguous to those IgGAM staining, basal histology was also performed by using Giemsa (Fig 1C and 1D), HE (Fig 1E and 1F) and PAS (Fig 1G and 1H). A depletion of goblet cells was observed in early OCP (arrowheads) while the absence of goblet cells was detected in advanced OCP, as pointed in Giemsa, HE and particularly PAS panels (Fig 1C–1H). The presence of squamous metaplasia (pluristratified non-keratinised epithelial with absence of goblet cells and homogenization of connective tissue) was particularly evident in advanced OCP (see § in Fig 1D and 1F). A marked vessel ectasia was particularly evident in early OCP (see asteriks in Fig 1E). Edema and mild infiltrates with prevalence of plasmacells (arrows) were observed in early with respect to advanced OCP (Fig 1C).

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Fig 1. Histological characterization of OCP conjunctivas.

Representative confocal (A-B) and light microscopy (C-H) images, including Giemsa (CD), HE (EF) and PAS (GH) stainings, from dewaxed conjunctival sections. A-B. Linear fluorescent-coupled IgGAM reactivity in the Basement Membrane Zone of early (white arrows; A) and advanced (B; the white asterisk indicates the stromal immunoreactivity) OCP sections. Fluorescent linear immunoreactivity is absent in normal sections. Depletion of goblet cells (pointed by black arrowheads) in early (C,E,G) with respect to the complete absence in advanced OCP (D,F,H); the presence of a squamous metaplasia (↔) particularly in advanced OCP (D,F); vessel ectasia (*) more prominent in early (E) than advanced (F); edema with mild infiltrates (arrows) in early (C) and to a less extend in advanced (D) OCP, particularly plasmacells and some granulocytes; and finally superficial homogenization of connective tissue (§), particularly evident in advanced OCP (D,F). Magnifications: x400.

https://doi.org/10.1371/journal.pone.0142737.g001

Characterization of activated FBs

Adjacent conjunctival sections and primary cultures of FBs were analysed for αSMA expression by confocal microscopy and flow cytometry (FCM) analysis. As shown in Fig 2, αSMA immunoreactivity in outgrowth of OCP-tissue (B) and outgrew FBs (D), as compared to control specimens (respectively A and C; p < .05). FCM analysis confirmed the higher αSMA protein expression in OCP-FBs (Fig 2E; p < .05). FCM results were corroborated at the molecular level (+14.00±4.80 expression ratio in OCP-FBs vs. controls; p < .05).

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Fig 2. aSMA expression in both OCP conjunctiva and derived FBs.

A-D. Confocal analysis for α-SMA in both control (A) and OCP (B) conjunctival biopsies, as well as in control (C) and OCP (D) FB. Arrows point to specific α-SMA immunoreactivity in OCP tissue (B; red staining; see M&M for details). In monolayers, nuclei were counterstained with propidium iodide (C-D). [Magnifications: AB, x400; CD, x600] E-F. Flow cytometry analysis specific for α-SMA in control (E) and OCP (F) FBs. iso = fluorescence intensity related to isotypes.

https://doi.org/10.1371/journal.pone.0142737.g002

Expression of NGF and trkANGFR/p75NTR

OCP conjunctiva and OCP-FBs were examined for NGF-trkANGFR/p75NTR expression. As observed by confocal analysis, NGF expression was decreased in the epithelium and specifically increased in OCP stroma, as compared to controls (Fig 3A and 3B; p < .05). Indeed, confocal analysis showed higher cytoplasmic and perinuclear NGF immunoreactivity in outgrew OCP-FBs, supporting those data observed in OCP stroma (see arrows; Fig 3C). In addition, the conditioned media from OCP-FBs showed higher NGF levels than those of control counterparts (420.00±90.00 vs. 220.00±64.00 pg/mL; p < .05), as detected by NGF ELISA. In line, NGFmRNA was increased in these OCP-FBs, as compared to their control FBs (+5.00±1.30 expression ratio; p < .05). No significant difference was quantified between early and advanced specimens.

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Fig 3. NGF immunoreactivity in OCP conjunctiva and FBs.

Confocal analysis for NGF (green) in control (A) and OCP (B,C) conjunctiva. C. Immunofluorescence for NGF (red) and αSMA (green) expression in the conjunctival stroma. Note the presence of intracytoplasm and perinuclear staining. Nuclei were counterstained with toto3. Magnifications: AB, x400; C, x600 (oil immersion).

https://doi.org/10.1371/journal.pone.0142737.g003

Confocal analysis showed a high p75NTR expression associated with a weak trkANGFR expression in OCP-FBs (Fig 4B). By contrary, control FBs showed a huge trkANGFR expression with respect to p75NTR slightly expressed (Fig 4A). To support, the related FCM analysis for trkANGFR and p75NTR are provided in Fig 4C and 4D respectively.

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Fig 4. trkANGFR and p75NTR in OCP conjunctiva and FBs.

AB. Confocal analysis of control (A) and OCP (B) FBs double (overlays; x400) and single stained for trkANGFR and p75NTR (see below). Relevant single immunoreactions are shown below and cross-reactivity of trkANGFR and p75NTR are marked with white arrows (overlays). CD: Flow cytometry analysis of control (left) and OCP (right) FBs showing expression of trkANGFR (C) and p75NTR (D). Related isotype fluorescence intensity data are shown (iso).

https://doi.org/10.1371/journal.pone.0142737.g004

αSMA and p75NTRco-expression in OCP-derived activated FBs

The higher αSMA and p75NTR and the lower trkANGFR protein expressions in OCP-FBs were also detected by WB analysis, as compared to control FBs (Fig 5A; p<0.05). As shown in Fig 5B, αSMA protein co-localized with p75NTR but not with trkANGFR, as detected by confocal analysis. This would suggest the existence of a specific trkANGFR/p75NTR expression ratio during the progress of fibrosis. Particularly, basal histology showed a strong infiltration of inflammatory cells (arrows pointing to plasmacells in Fig 1C), the presence of connective tissue homogenization (asterisk in 1D and 1F) in the underlying stroma (§) and a significant decrease (absence) of goblet cells in the epithelium of advanced OCP sections (see arrowheads pointing residual goblet cells in early OCP) (Fig 1C and 1E). Activated fibroblast were also visible in early OCP (Fig 1C). No significant difference in αSMA, p75NTR and trkANGFR transcripts was detected in early OCP-FBs compared to controls (respectively +1.05±05, +1.30±0.50 and +2.00±1.00 expression ratio; p>.05). In advanced OCP-FBs, αSMA and p75NTR transcripts were significantly increased (+9.00±0.40 and +5.00±0.50 expression ratio; p < .001) while trkANGFR transcript was significantly decreased (-4.00±1.00 expression ratio; p < .05), as compared to controls. By FCM analysis, αSMA protein was increased in early OCP-FBs (2.8±0.58 vs. 0.22±0.06 MFI, early vs. controls; p>.05) and significantly increased in advanced OCP-FBs (27.05±3.6 vs. 0.22±0.06 MFI, advanced vs. controls; p < .001). Indeed, p75NTR protein was increased in both early (15.52±5.97 vs. 1.44±0.6 MFI, early vs. controls; p < .05) and advanced (17.0±3.0 vs.1.44±0.6 MFI, advanced vs. controls; p < .01) OCP-FBs. Finally, trkANGFR protein expression in early OCP-FBs was comparable to those of control-FBs while it decreased in advanced OCP-FBs (2.41±1.27 vs. 15.5±1.00 MFI, advanced vs. controls; p < .01).

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Fig 5. αSMA and trkANGFR/p75NTR expression in OCP-FBs.

A. Representative Western blot analysis specific for αSMA, p75NTR and trkANGFR proteins in control (left) and OCP (right) FBs (n = 2/each group). Normalization was checked by GAPDH reprobing on the same gels. BC. Confocal analysis for α-SMA/trkANGFR (B) and α-SMA/p75NTR(C) in OCP-FBs (overlays, x600). Respective single immunoreactions are shown below. p75NTR and α-SMA cross-reactivity is highlighted with white arrows (C).

https://doi.org/10.1371/journal.pone.0142737.g005

NGF modulation of αSMA and trkANGFR/p75NTRexpression in OCP-FBs

NGF effect on αSMA and trkANGFR/p75NTR modulation was thereafter investigated by exposure to 10ng/mL NGF over 24hrs. A preliminary dose response study was carried out on OCP-FBs (0–100 ng/mL NGF over 24hrs), highlighting the 10 ng/mL NGF dosage. In early OCP-FBs, decreased αSMA and p75NTR (respectively 0.83±0.19 vs. 2.8±0.58 MFI and 1.08±0.56 vs. 15.52±5.97 MFI; p < .05) as well as unchanged trkANGFR (20.33±8.52 vs. 20.93±4.87 MFI; p>.05) protein expressions were observed upon NGF exposure. In advanced OCP-FBs, no significant changes of αSMA and p75NTR (respectively 17.60±1.15 vs. 17.00±6.12 and 31±4.77 vs. 26.83±3.84 MFI; p>.05) as well as trkANGFR (2.93±0.83 vs. 2.41±1.27 MFI; p>.05) protein expressions were detected upon NGF exposure. With respect to trkANGFR/p75NTR expression, FCM analysis showed that 94.91% early OCP-FBs were trkANGFR positive, with 57.24% co-expressing p75NTR. Upon NGF exposure, 96.88% early OCP-FBs were still trkANGFR positive, with 24.12% co-expressing p75NTR and 72.76% expressing only trkANGFR. The statistical analysis showed that a decrease of 57% in trkANGFR/p75NTR co-expressing cells occurred in association with a shift to trkANGFR expressing cells. The trkANGFR/p75NTR immunoreactivity in NGF exposed early OCP-FBs is shown (Fig 6).

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Fig 6. trkANGFR/p75NTR expression in NGF exposed early OCP-FBs.

Confocal images showing the trkANGFR (FC/green, A) and p75NTR (Cy5/blue, B) immunoreactivity in early OCP-FBs exposed to NGF over 24hrs (overlays, C). Nuclei counterstained with propidium iodide are shown in all panels. The cytoplasmic and perinuclear distribution of both receptors is clearly visible. Magnifications: A-C, x400.

https://doi.org/10.1371/journal.pone.0142737.g006

NGF modulation of OCP-activated FBs derived TGFβ1 and IL4 cytokines

Last, changes in TGFβ1 and IL4 profibrogenic factor release were also detected in the conditioned media from baseline and NGF treated OCP-FBs. TGFβ1 and IL4 levels in the conditioned media from early OCP-FBs were respectively 8-times (101.00±30.00 vs. 12.00±2.10 pg/mL TGFβ1, p < .05) and 6-times (308.00±7.00 vs. 55.00±40.00 pg/mL IL4; p < .001) higher as compared to control counterparts. Conditioned media from advanced OCP-FBs did not show difference in both TGFβ1 and IL4 levels, as compared to controls. Upon 10ng/mL NGF exposure, IL4 protein decreased in the conditioned media from early (26.00±10.00 vs. 101.00±30.00 pg/mL IL4; p < .05) and advanced (1.80±0.30 vs. 23.00±4.80 pg/mL IL4; p < .05) OCP-FBs. By contrary, TGFβ1 levels decreased only in the conditioned media from early OCP-FBs (63.00±40.00 vs. 308.00±7.00 pg/mL TGFβ1; p < .05).

Discussion

Increasing data indicate that the chronic inflammatory process occurring in OCP conjunctiva leads to FB activation and survival, with overt collagen deposition and excessive matrix deposition [3]. As a product of different structural/immune cells and FBs/myoFBs, both cytokines and growth factors actively contribute to subepithelial fibrosis and conjunctival scarring [25]. To date, different proinflammatory and profibrogenic factors have been investigated by different groups, which have focused their attention especially on receptor signalling [26]. While in a previous study we have described the trkANGFR and NGF expression respectively in OCP conjunctivas and tears, herein we hypothesize a possible NGF role in the modulation of cultured OCP-FBs [8,9].

First of all, αSMA expression was detected in OCP conjunctiva and confirmed in primary cultures of FBs obtained from OCP explants. αSMA (α-Smooth Muscle Actin) represents the most reliable phenotypic marker for the majority of fibrotic states. Our findings indicate the presence of activated FBs inside inflamed/fibrotic OCP conjunctiva [7,27,28]. If these activated FBs are αSMA-expressing myofibroblast (myoFBs) remains to be clarified since in previous studies the possible differentiation of OCP-FBs into myoFBs was not reported [29].

An increase in the stroma and a significant decrease in the epithelium were detected for NGF in OCP conjunctival biopsies (n = 7), as compared to control ones. To the best of our knowledge, this data has not been previously described and it is supported by our recent findings showing an increased NGF content in OCP tear fluids [8,9]. The observation of NGF-expressing OCP-FBs might suggest that the NGF increase in OCP stroma as well as the increased NGF levels in OCP tears might be partially due to local activated-FBs. On the other side, the decreased NGF immunoreactivity in OCP epithelium is actually missing of explanation and not investigated/discussed in this study.

As product of stromal inflammation, it is reasonable to hypothesize that NGF might contribute to tissue remodeling by influencing the FB phenotype, as observed in previous studies conducted on other cell types [14,17,18,20]. With respect to the activated FB phenotype, NGF effects might cover either cell survival and/or soluble mediator release [14,17]. According to literature, NGF activity is driven by trkANGFR and p75NTR receptors, which mediate NGF signal alone or in cooperation [30–35]. As a new finding, NGF, trkANGFR/p75NTR and αSMA (co)expressions were detected in primary cell cultures alongside with sub-cultured OCP-FBs. Of interest, sub-cultured OCP-FBs showed the ability to retain FB phenotype upon few passages and were therefore suitable for stimulation studies. Interestingly, trkANGFR/p75NGF expression was found strictly dependent to the early/advanced grouping of disease as well as αSMA phenotype correlated to p75NTR and paralleled the severity of fibrosis. Particularly, FBs from advanced OCP showed higher αSMA and p75NTR together with lower trkANGFR, as compared to early and control counterparts. This expression would imply a close association of p75NTR with OCP-FB phenotype, and highlight a possible modulation of myoFB apoptosis, as observed in other systems [20,35]. To date, the role of trkANGFR and p75NTR in tissue remodeling remains controversial. As documented, both trkANGFR and p75NTR can mediate either survival or apoptosis, depending on their surface receptor (co)expression and microenvironment [33,36–38]. In early healing process, high levels of trkANGFR might drive both migration and differentiation (as initial matrix remodelling) while in late healing process the trkANGFR down-regulation might allow p75NTR to mediate other biological activities, alone or eventually in cooperation with trkANGFR [33,39–41]. As described, fibrotic tissues appear characterized by low trkANGFR and high p75NTR expression [17,18,20]. In this study, a higher trkANGFR/p75NTR ratio (the outcome of a trkANGFR over-expression) was observed in early OCP-FBs while lower trkANGFR/p75NTR ratio (the outcome of an increased p75NTR expression) was detected in advanced OCP-FBs, according to the clinical and histological features (infiltrates and remodelling features) [2,22,42]. To support our findings, the lower trkANGFR/p75NTR ratio expression in advanced OCP-FBs (the outcome of an increased p75NTR expression) has been also reported in other fibrotic conditions, either in vitro/ex vivo [7,17,18,28,43]. A down-regulation of both αSMA and p75NTR expression was observed in NGF-exposed early OCP-FBs, while no effect was detected in NGF-exposed advanced counterpart. The observation that NGF modulated trkANGFR/p75NTR ratio expression preferentially in early OCP-FBs would suggest that a potential control of activated FBs might be possible in early OCP showing a mild-moderate clinical facet, opening to potential NGF therapeutic applications. As shown, activated FBs disappear alongside “proper repair process” while αSMA-expressing activated FBs survive in pathological remodelling [7,28]. This process might be highly regulated by growth factors and cytokines, including NGF, all known to be increased in OCP tissues and tears [5,6,8,9]. Therefore, a possible cross-talk between NGF and other profibrogenic factors cannot be excluded. In line, TGFβ1 and IL4 were extensively investigated in fibrosis and are widely reported to contribute selectively to tissue remodelling and overt fibrosis in different disorders via an extensive sustaining of myoFBs [2,6,28,44]. Therefore, we wonder whether NGF might influence TGFβ1 and IL4 release from sub-cultures of OCP-FBs. The biochemical analysis highlighted a significant decrease of TGFβ1 and IL4 in the conditioned media from NGF-exposed early OCP-FBs, while only a decrease of IL4 was monitored in advanced counterparts. This selective effect holds up the potential NGF involvement in OCP remodelling, through a modulation of inflammatory/fibrogenic soluble factors, at least in early stage of disease.

Overall, OCP is a chronic inflammatory disease that slowly evolves in severe conjunctival scarring and visual impairments [1,2,45]. Most of the current OCP therapies target the suppression of inflammation, as counteracting the recurrent inflammation represents the main way to reduce progressive remodelling [46–48]. The findings of this in vitro study suggest a possible NGF effect on early OCP-FBs having a low trkANGFR/p75NTR ratio, highlighting the possible NGF effect in the modulation of FB activity during the early stages of disease. Since the topical NGF application has been suggested as a therapeutic tool in some ocular surface disorders [11,49], these findings encourage further studies to understand the underlying NGF mechanism in OCP conjunctiva in order to develop alternative strategies to counteract fibrosis.

Acknowledgments

The data of this study were partially presented at the Annual Meeting of the Association for Research in Vision and Ophthalmology, 2007, Fort-Lauderdale, Florida (Stampachiacchiere et al., Invest. Ophthalmol. Vis. Sci. 2007 48: E-Abstract 5306). This study was partially supported by Ministry of Health (Italy). The authors wish to express gratitude to Fondazione Roma for continuous support. Special thanks to Dr. Eleonora Perrella for her valuable contribution (Pathology, UCBM).

Author Contributions

Conceived and designed the experiments: AM BS AL SB. Performed the experiments: AM BS ADZ RS EMN AL SB. Analyzed the data: AM BS ADZ RS MC AL SB. Contributed reagents/materials/analysis tools: AM SB. Wrote the paper: AM BS AL SB.

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