Abstract
Abdominal surgery is linked with peritoneal adhesions. We investigated that the anti‐fibrotic agent pirfenidone (PFD) has immune modulating activities and evaluated its effects on the function of T helper type 1 (Th1), Th2 and T regulatory (Treg) cells, which may play important roles in peritoneal adhesions. Eighteen female Wistar rats underwent right‐sided parietal peritoneal and right uterine horn adhesion model. Rats were randomized into 3 groups as group 1 (control) (closure of midline abdominal incision without any agent administrations), group 2 (closure of incision after intraperitoneal administration of PFD) and group 3 (closure of incision and only oral administration of PFD for 14 days). Relaparotomy was performed 14 days after the first surgery. Effect of PFD on adhesion formation was assessed on Th1, Th2 and Treg cells counts using Anti‐T‐bet, Anti‐GATA‐3 Anti‐FOXP3 antibodies respectively. Th1 counts were moderate in the control group, and didn't show a significant difference between all groups. Th2 cell counts were very high in the control group, but both intraperitoneal and oral administration of PFD resulted in a significant reduction in Th2 cell counts. Treg cell counts were low in number in the control group. In the intraperitoneal administration of PFD group, Treg cell counts were significantly lower than control group. There was no difference of the Treg cells between control groups and the oral administration of PFD group. PFD has prevention effect on intraperitoneal adhesions. This prevention effect seems to be related with the reduction in the numbers of Th2 and Treg cells.
Keywords: Adhesion, Pirfenidone, Th1, Th2, Treg
Introduction
Peritoneal adhesions develop postoperatively in most patients, and lead to significant clinical morbidity and mortality, such as small bowel obstruction, infertility, abdominopelvic pain [1]. Following trauma, polymorphonuclear cells (predominantly neutrophils), monocytes, and leukocytes migrate into the peritoneum and synthesize several mediators (e.g., interleukin (IL)‐17 and transforming growth factor‐beta (TGF‐β)) which lead to forming peritoneal adhesions.
Peritoneal adhesions are highly cellular tissues with a high proliferation rate. Macrophages and neutrophils play an important role in triggering adhesions, whereas the role of T cells in this process is not known completely. However, it was demonstrated that the immunopathogenesis of adhesion formation is controlled by T cells and that T cell‐derived cytokines and chemokines play an important role in the development of this deleterious host response [2]. Tzianabos et al. suggested that modulating the activity of these T cells by blockade of costimulatory and coinhibitory molecules significantly affects the severity of adhesions that eventually develop [3].
T cells in the peritoneum are divided into several subpopulations, including Th cells, cytotoxic T lymphocytes, γδ T cells, memory T cells, and regulatory T cells. Th cells are one of the most important cell populations in the peritoneum and are composed of two main subpopulations, defined as Th1 and Th2 [4]. Th1 cells induce cellular immunity, granuloma formation and protection against intracellular pathogens [5]. The Th2 subset favors production of the various immunoglobulin classes that shape or help humoral immunity [6]. Another cell type called Treg cells, participate in the development of self‐tolerance; and play an important role in the suppression of autoreactive T cells and downregulate the proinflammatory responses of both Th1 and Th2 cells [7].
PFD (5‐methyl‐1‐phenyl‐2‐[1H]‐pyridone) has well‐documented anti‐fibrotic and anti‐inflammatory properties in both in vitro and in vivo models of fibrosis and, also one of two recently FDA‐approved medications for the treatment of idiopathic pulmonary fibrosis. PFD exerts its effects by reducing fibroblast proliferation and inhibiting TGF‐β‐stimulated collagen production. In addition, PFD has been shown to modulate multiple signaling pathways including tumor necrosis factor α (TNF‐α), IL‐1‐β, and platelet derived growth factor [8]. We also reported that both intraperitoneal and oral administration of PFD reduced the tissue levels of inflammatory markers (TGF‐β and IL‐17) in both parietal and visceral peritoneum, and concluded that PFD is an effective agent on the prevention of postoperative fibrosis [9].
A few reports describe a role for T lymphocytes in this surgical adhesion process; however, the inhibitor role of PFD on T cell function in the peritoneal adhesion process is not known. Therefore, in this study, we designed to investigate the prevention effects of both intraperitoneal and oral administration of PFD in postoperative intraabdominal adhesions on an experimental rat model. To test this hypothesis, we focused on the T helper cells functions as main representatives of peritoneal adhesion process.
Material and methods
The research protocol was approved on May 12th, 2015 with the project number of 77.637.435–47 by the local institutional review board for experimental animal studies, in adherence with the National Institute of Health guidelines (Washington, DC, USA) for the care and use of laboratory animals. Eighteen 4 to 5 month‐old female Wistar rats weighing 250 ± 20 g were housed in a climate‐controlled (relative humidity of 40 ± 5% and temperature of 21–24 °C) animal care facility, with a 12‐h light/dark cycle. Before and after surgical procedures, the animals were provided with standard rat chow and water. Anesthesia was induced by injection of ketamine (45 mg/kg i.m. of Ketalar; Eczacibasi, Istanbul, Turkey) and xylazine (5 mg/kg). The surgical procedures were performed under sterile conditions and powder‐free sterile surgical gloves were used during procedures. All operations were performed by the same author (PSH). The rats were randomly assigned into three groups as group 1 (control) (closure of midline abdominal incision without any agent administrations), group 2 (closure of incision after intraperitoneal administration of PFD) and group 3 (closure of incision and only oral administration of PFD for 14 days). Each group consisted of 6 rats and not sequentially operated in order to minimize bias. The operation was limited to 15 min for each rat and antibiotic prophylaxis was not given. A 3‐cm vertical midline incision was performed and both uterine horns were exposed. Punctate serosal hemorrhages were generated by scraping with a No.15 scalpel blade until petechial bleeding emerged at the right abdominal sidewall and anti‐mesenteric surface of the right uterine horn to create adhesions. The abdominal incision was closed in two layers using a simple interrupted polyglactin 910 (4.0 Vicryl®) suture for the peritoneal fascia and the skin. Closure of abdominal incision was performed without any agent administration after right visceral peritoneum and right uterine horn were damaged in group 1 and group 3 animals. Abdominal incisions were closed after intraperitoneal PFD (Pirfenex, Cipla, India) administrations in 150 mg doses in each animal in group 2. Oral PFD (25 mg/kg daily) was administered in group 3 animals with nasogastric gavage for 14 days. The second laparotomy was performed 15 days after the first surgery by the same author (PSH), blinded to the groups. Approximately 2 mm3 of tissues were biopsied from the scarred peritoneal and uterine areas.
Immunohistochemistry
Formaline‐fixed, paraffin‐embedded periton and uterus sections were used for immunohistochemical staining. Tissue samples were stored at 60 °C overnight and then dewaxed with xylene for 30 min. After dehydration of the sections with ethanol, they were washed with distilled water. Subsequently, the samples were treated with 2% trypsin (ab970, Abcam, Cambridge, UK) at 37 °C for 15 min and incubated in 3% H2O2 solution for 15 min to inhibit endogenous peroxidase activity. Then, the sections were incubated with Anti‐GATA‐3 Primer Antibody (sc‐268, Santa Cruz Biotechnology, Inc. Dallas, Texas), Anti‐FOXP3 Primer Antibody (sc‐80792, Santa Cruz Biotechnology, Inc. Dallas, Texas), and Anti‐T‐bet Primer Antibody (sc‐21003, Santa Cruz Biotechnology, Inc. Dallas, Texas) in a 1/100 dilution for 18 h at +4 °C. Incubation with biotinylated IgG and administration of streptavidin peroxidase (Histostain Plus kit, Zymed 87–9999; Zymed, San Francisco, CA) performed. After washing the secondary antibody with PBS three times, for 5 min, the sections were stained with DAB substrate system containing diaminobenzidine (DAB, K007, DBS, Pleasanton, CA, USA) to detect the immunoreactivity, and then stained with Mayer's hematoxylin (72804E, Microm, Walldorf, Germany) for counterstaining. They were covered with a mounting medium (01730 Surgipath, Cambridge, UK) and observed with a light microscopy (Olympus BX‐40, Tokyo, Japan).
We count the T cell subgroups according to immunostaining for T‐Bet (Th1), GATA‐3 (Th2) FOXP3 (Treg) expressions in the peritoneal and uterine samples of each group were evaluated semiquantitatively using HSCORE analysis [10]. The immunostaining intensities were categorized by the following scores: 0 (no staining), 1 (weak, but detectable staining), 2 (moderate staining), and 3 (intense staining). A HSCORE value was derived for each specimen by calculating the sum of the percentage of cells for both parietal peritoneum and visceral peritoneum of the uterus that stained at each intensity category, multiplied by its respective score, using the formula H‐score = ∑Pi (i+1), where “i” is the intensity of staining with a value of 1, 2 or 3 corresponding to weak, moderate or strong staining, respectively. Pi is the percentage of stained cells for each intensity, varying from 0 to 100%. For each slide, five different fields were evaluated microscopically at 200× magnification. HSCORE evaluation was performed independently by at least two investigators (MO and AU) blinded to the source of the samples as well as to each other's results; the average score of both was then used.
Ethical consent
The research protocol was approved by the institutional review board on May 12th, 2015 and supported by the Scientific Research Fund of Celal Bayar University with the project number of 77.637.435–47.
Statistical analysis
Statistical analysis was performed using the Statistical Package for Social Science (SPSS for Windows, Version 15.0, Chicago, Illinois). The descriptive statistics for the normally distributed variables were expressed as the mean ± standard deviation. Kruskal–Wallis test was used for statistical comparisons of the groups which variables did not fit the normal distribution and the Mann–Whitney U test with Bonferronni correction was used for post‐hoc tests. P‐value of <0.05 is considered as statistically significant.
Results
We reported in the previous study that the macroscopic adhesions in the control group were prominent than the both intraperitoneal and oral administration of PFD groups. Both intraperitoneal and oral administration of PFD reduced the tissue levels of inflammatory markers (TGF‐β and IL‐17) in both parietal and visceral peritoneum compared to control group. We examined if PFD has any effect on Th1, Th2 and Treg cells during peritoneal adhesion pathogenesis (Suppl).
Th1 counts were moderate in the control group, compared to both intraperitoneal and oral administration of PFD groups (Figure 1a–c). Th1 counts didn't show a significant difference between all groups (Table 1). Th2 cell counts were very high in the control group, but both intraperitoneal and oral administration of PFD resulted in a significant reduction in Th2 cell counts (Figure 1d–f) (Table 1).
Figure 1.
Immunohistochemical analyses of parietal peritoneal cells' for Th1, Th2 and Treg cells. Many Th1 cell were seen in the control group (a), compared to both intraperitoneal (b) and oral administration of pirfenidone groups (c). Th2 cell counts were very high in the control group (d), but both intraperitoneal (e) and oral administration (f) of pirfenidone resulted in a significant reduction in Th2 cell counts. Treg cell counts were low in number in the control group (g) compared the Th1 and Th2 groups. In the intraperitoneal administration of pirfenidone group, Treg cell counts (h) were significantly lower than control group. There was no difference of the Treg cells between control groups and the oral administration of pirfenidone group (i). Original magnification ×200; arrows: T lymphocytes.
Table 1.
H‐score values of Th1, Th2 and Treg in the peritoneum.
Treg cell counts were low in number in the control group. In the intraperitoneal administration of PFD group, Treg cell counts were significantly lower than control group (Figure 1g–i). There was no difference of the Treg cells between control groups and the oral administration of PFD group (Table 1).
Discussion
After pelvic or abdominal surgery, post‐surgical adhesions are still a critical problem despite a multitude of studies dedicated to find modalities to prevent their occurrence. PFD administration promotes an anti‐fibrotic response in a surgical mouse model of adhesion induction, but the mechanisms mediating this effect have not been established. In the current study, we assessed that effect of intraperitoneal administration of PFD on the T helper cells functions for prevention of postoperative intra‐abdominal adhesion formation in animal model of rat.
The peritoneum has a normal repair mechanism which is a complex process and includes inflammation, angiogenesis, cell migration, and turnover of the extracellular matrix. Injured peritoneal surface exudates a high‐protein consisting fluid, known as the provisional matrix, that coagulates within 3 h, creating fibrinous bands between corresponding surfaces to maintain their contact [11]. The peritoneal fibroblasts invade the wound area and perform the collagen synthesis and extracellular matrix deposition [12]. It is known that alterations in the local concentration of cytokines, growth factors, and proteases are key factors in adhesion formation which are synthesized by macrophages, neutrophils and lymphocytes. CD4+ αβ T cells are required for surgical and postinfectious adhesion formation. Activated CD4+ T cells enter the peritoneal cavity, orchestrate chemokine production and leukocyte trafficking, contributing to adhesion development [3].
The peritoneum is a special environment where T cells react and all three major subpopulations, defined as Th1, Th2, and Treg are found in it. We also observed the lymphocytes in the adhesion area, and counted these T cells using H score technique. The numbers of Th1 cells are moderate in the control group and there are no significant differences between the groups. It's known that Th1 cells and their secreted cytokines play a prominent role in the initiation and progression of fibrosis. The cells express Tim‐3 and IFN‐γ for adhesion formation. Tzianabos et al. demonstrated that the blockade to the Th1 immune response by Tim‐3 Ig should lead to exacerbation of adhesion formation [3].
We also observed that the number of Th2 cell is increased in the adhesion area compared to the Th1 cells and Treg cells. We suggest that Th2 cells are linked to peritoneal adhesion. Th2 cells secrete many cytokines such as IL‐4, IL‐5, IL‐13 and IL‐21 and they have distinct roles in the regulation of tissue remodeling and fibrosis. When TA also reported that IL‐13 secreting Th2 cells induce the production of latent TGF‐β1 in macrophages and can also serve as an indirect activator of TGF‐β. IL‐4 was found to have increased levels in many fibrotic disease [13]. Klínger et al. reported that patients on peritoneal dialysis with or without peritonitis showed immune activation per se and high production of pro‐inflammatory cytokines accompanied by a strong pattern of Th2 cytokines and a deficiency of IFN‐γ production. This indicated immunodeviation towards a Th2 response and Th1 immunodeficiency [6].
T‐bet is a key regulator of adhesion formation and T‐bet‐deficient mice showed significantly reduced adhesion formation. The effect correlated with fewer IFN‐ γ ‐producing CD4+ T cells in the peritoneal cavity and draining mesenteric LN [3]. In our study we observed that the number of Treg cells is low in adhesion group. It's known that Treg cells secrete IL‐10 and TGF‐β. TGF‐β plays an important role in development of adhesions at the site of peritoneal injury [14]. TGF‐β stimulates fibroblast activation and extracellular matrix synthesis. It also carries out transcriptional activation of pro‐fibrotic genes, via the TGF‐β/Smads signaling pathway or through alternative pathways such as the p38 MAPK signaling and RAS/ERK MAPK signaling pathways [15]. In addition to this, intraperitoneal injection of high doses of TGF‐β3, increased adhesion formation after injury of the peritoneum with enhanced collagen deposition and fibroblastic proliferation [16].
We observed the peritoneal adhesions in the control group; however, the adhesions were minimal for both intraperitoneal and oral administrated PFD group. Furthermore, intraperitoneal administration of PFD compared to oral administration was more effective in reducing peritoneal adhesion. The activity of PFD was investigated in several well‐characterized animal models of fibrosis in the lung, liver, heart and kidney. In these studies, treatment‐related reductions in fibrosis are associated with modulation of cytokines and growth factors [17]. In our previous study, we investigated the effect of both oral and intraperitoneal PFD administration for the prevention of the postoperative adhesion formation and interestingly, intraperitoneal administration of PFD was found to be effective compared to controls while this prevention effect was related with suppression of TGF‐β and IL‐17 [9].
PFD has a broad mechanism of action, exerting its anti‐inflammatory action through several pathways. We observed that intraperitoneal PFD administration causes a decrease in number of T lymphocytes especially in Th2 and Treg cells. We suggest that PFD reduced the number of T lymphocytes and thus inflammation was suppressed. PFD was found to inhibit the responder frequency of TCR‐stimulated CD4+ cell total proliferation in vitro and in vivo, whereas both CD4 and CD8 proliferation index were reduced by PFD and claimed that PFD's immune modulating activities encompass inhibitory effects upon DC activation and function [18].
We detected that PFD reduced Th2 cells count both in intraperitoneal and oral administration groups compared to the control group (Table 1 and Figure 1). We claim that PFD suppressed the Th2 cell function and diminishes the peritoneal adhesion. Visner et al. also reported that PFD (400 mg/kg for 7 days) has negative effect on T cell subsets and Treg percentage in spleen and lymph nodes in C57BL/6 mice and PFD was found to significantly inhibit T cell receptor (TCR)‐stimulated CD4+ T cells proliferation in vitro. It may be related with based on proliferation index of CD4+ cells with a reduction of proliferation at the higher division populations [18]. PFD inhibited the antigen‐specific proliferation of lymph node mononuclear cells in OVA sensitized mice, and inhibited production of the profibrotic Th2 cytokines, IL‐4 and IL‐13 [17].
Our study demonstrated that PFD reduced the Th1 cell number slightly in the intraperitoneal and oral administration groups, but the decrease of the Th1 cell number didn't make significant differences from control group. PFD was shown to inhibit the expression of the Th1 cytokines TNF‐α and IFN‐γ [19], but it couldn't be related with T cell number because PFD inhibits a number of proinflammatory cytokines/chemokines from activated lymphocytes including IFN‐γ, IL‐1β, TNF‐α, IL‐4, IL‐17, FGF‐β, IP‐10, MIP‐1, Mig [20]. The inhibition of TGF‐β1 is a key factor in the development of peritoneal fibrosis. However Hisatomi et al. reported that PFD also inhibited over‐expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF‐β1 [21].
We also detected that Treg cell counts were significantly reduced in the intraperitoneal administration of PFD group than control and oral administration of PFD groups Treg cell secretes TGF‐β which is a central mediator in the development of fibrosis. Previously studies have shown that PFD reduced the development of fibrosis in lung and tracheal transplant models [22]. This was associated with an inhibition of the profibrotic cytokine TGF‐β and a reduction in collagen formation. Visner et al. examined whether PFD had effects on Treg cell activity and reported that PFD does not appear to affect Treg function [18].
In conclusion, many different agents have been used heretofore in the effort to arrest the adhesion pathway. We found that PFD's prevention effects on peritoneal adhesion may still be related with a reduction in the Th2 and Treg cell numbers. The prevention effect of PFD is more prominent in the intraperitoneal administration thus, systemic effects occurring are minimal. Therefore, PFD may be used as a prevention agent for peritoneal adhesion, after abdominal surgical operation.
Supporting information
Supplementary data
Supplementary data
Supplementary data related to this article can be found at https://doi.org/10.1016/j.kjms.2017.03.011.
Conflicts of interest: All authors declare no conflicts of interest.
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