Study Of The Effects And Crosstalk Of IL-17 And TGF-β During Peritoneal Adhesion Formation After Surgery

Part I Analysis of the intraperitoneal drainage fluid from patients who had undergone laparotomyObjectives:To study the change patterns of inflammatory cytokines and growth factors in the intraperitoneal drainage fluid.Methods:In consideration of the normal performance in which a peritoneal drainage pipe was left right after the laparotomy was done, our study group take advantage of it for collecting the drainage fluid from the patients without making additional trauma. Just before the operation, fluid inside the peritoneal cavity was taken. Then every morning after the surgery, fresh fluid from the drainage pipe was collected. The collected fluid would be centrifuged at 14000 rpm at 4℃ for 10 minutes. The supernatant was collected and stored in-80 ℃ until further use. After enough qualified samples were collected, ELISA test was performed to analyze the concentration of IFN-y, IL-17 and TGF-β.The time course patterns of these cytokines and factors were compared and analyzed. ANOVA (analysis of variance) was used to detect differences of the concentrations in different time points.Results:From October 2012 to April 2014, clinical data and samples from 60 cases of patients who had undergone Iaparotomy for colectomies for the first time were collected. Prior to this step, patients who had suspicious infections or anastomotic leakage had already been excluded. Before the operations were done, there was little IL-17 or IFN-y in abdominal cavity. However IL-17 began to accumulate as soon as 12 hours after surgery and would reach the peak value around 24 hours afterwards (68.18±12.36 pg/ml). Then it would decrease very soon to half of the maximum value 2 days after surgery (33.16±7.24 pg/ml).4 days later, IL-17 already decreased to the base line. Similar with the pattern of IL-17, concentration of IFN-y soon increased 12 hours after surgery and reached the peak point 2 days later (75.77 ± 11.21pg/ml). It would stay at a relatively higher concentration until the fourth day. Then it would soon decrease and got back to base level at the sixth day. On the other hand, pattern of TGF-β was different. It would reach the peak value 12 hours after surgery (82.42±10.12 pg/ml), then it began to decrease and got back to 37.50± 7.22pg/ml at the third day. Then again concentration of TGF-P slowly increased and reached to the second peak value at the sixth day (95.12+16.19pg/ml).Conclusions:(1) After the laparotomy, in the abdominal cavity, cytokine IL-17 and IFN-y increased very soon and reached peak value around 1-2 days. Then it would soon decrease (without any potential infections or other complications). Basically the cytokines would get back to normal level around 5 to 7 days after surgery. (2) After laparotomy, there would be two peak values for TGF-β. The first value appeared 12-24 hours after surgery and the second one 6-7 days afterwards.Part Ⅱ Evaluation and construction of abdominal adhesion models on miceObjectives:To optimize a suitable protocol for constructing the stable and safe peritoneal adhesion model on mice.Methods:By referring to previous published data, a laparotomy was performed to construct the adhesion model. Detailed protocol was as follows:BALB/c mice were used for the model.3% sterilized chloral hydrate was injected via intraperitoneal injection for anesthesia. The dose of the drug was regulated according to the weight of each mouse (8μl/g).5 minutes after the injection, mice became anesthetic. Before the surgery started, the toe of the mouse was clamped slightly by the hemostatic forceps to test whether anesthesia effect works well. To ensure similar trauma was made on each mouse, the protocols were restricted to the same standard. An anterior midline incision through the abdominal wall and peritoneum was made. The length of the incision was 2 cm. Wet surgical gauze was used to abrade the surface of the abdominal wall and the intestine. To find the suitable strategy, different stroke power was tested as follows: A:Stroke on the abdominal cavity 20 times B:Before and after the cecum, every 2cm,20 times of stoke on the intestine,5 points were selected. C.A+B D:A+B but decreased stroke times (10 times on each point instead) A widely used standard scoring system was taken for evaluation of peritoneal adhesion degree. Details are as follows: 0:No adhesion at all 1:only one thin fimly adhesion 2:>1 thin adhesion 3:thick adhesion with focal point 4:more than one thick adhesion with focal points 5:vascularized adhesionsResults:7 days after surgery, death rate and adhesion score were recorded and compared, the results were as follows: Group A; adhesion score 1.8 ± 0.3, death rate 0% Group B; adhesion score 4.0±0.2, death rate 20% Group C:adhesion score 4.3 ±0.5, death rate 20% Group D.adhesion score 4.2±0.4, death rate 0%Conclusions:Merely abrading on the abdominal wall cannot successfully induce peritoneal adhesion formation. However over-trauma on the surface of intestine would lead to serve damage and leakage of the intestine, resulting in death. Only combination of the abdominal cavity and intestine together with a reduced strength could promote stable adhesion formation and stopped them from dying. The optimized protocol for adhesion induction would be; abrades 10 times at the following points; before and after the cecum, every 2cm,20 times of stoke on the intestine,5 points were selected plus stroke on the abdominal cavity 20 times. B:Before and after the cecum, every 2cPart Ⅲ Expression and origins of IL-17, IFN-γ and TGF-β in the abdominal cavity in mouse modelObjectives:To study the regulation pattern of IL-17, IFN-γ and TGF-β in abdominal cavity in mouse peritoneal adhesion models. Analyze the origins of IL-17 and IFN-γ in the abdominal cavity after the surgery.Methods:Different time points were chosen as follows to test the protein concentrations: normal mice; 2,4,6,8,12,24,48 and 72 hours after surgery. At each time point, the mice were sacrificed.2 ml sterilized saline was injected into abdominal cavity and the washed for 5 minutes. The liquid was withdrawn by the syringe. The fluid was then centrifuged and the supernant was used for ELISA test for IL-17, IFN-y, TGF-β. The splined down cells were collected and sent for FACS staining on CD4, IL-17 and IFN-y.Results:Under normal circumstance, there is nearly no IL-17 and IFN-y in abdominal adhesion. However 4 hours after surgery, IL-17 quickly began to accumulate and reached the peak value 123.56±19.28 pg/ml.48 hours after surgery it already got back to 46.72±7.17pg/ml, then it got back to the base level. IFN-y had a similar pattern with IL-17. 12 hours after surgery it reached peak point with a concentration of 70.95±19.29pg/ml, then 72 hours after surgery it got to the baseline level. For TGF-β it could reach the first peak 4 hours after surgery (66.79±10.18pg/ml). But later on it would decrease to a relatively low level 24 hours after surgery (19.18±5.28 pg/ml). However,72 hours after operation, the abdominal concentration of TGF-P would again rise to a new peak of 103.25±20.14pg/ml. In the abdominal cavity IFN-y secreting cells were mainly CD4+T cells. IL-17 secreting cells were mainly y5T cells and also Th17 cells to a less degree.Conclusions:Data from the mouse model had a similar pattern compared with data from the clinical patients. There is acute increase of the cytokines IL-17 and IFN-y shortly after surgery. For TGF-P there is two peak points. After surgery Thl, Th17 and γδT cells could be detected in the abdominal cavity and considered as the main resource of those quickly increased cytokines.Part IV Validation of the strategies for preventing adhesion formation in mouse modelObjectives:To validate the effects of anti-inflammatory and anti-fibrosis treatment against peritoneal adhesion formation on mice.Methods:By referring to the results from part Ⅲ, different strategies were used for prevention of peritoneal adhesion as follows:(1) Anti-inflammatory treatment:Different does from 10 to 100 p.g were used in different treatment time patterns. The first pattern is the "early treatment"; an injection was given 0,1,2 days post operation. The second pattern is the "late treatment"; an injection was given d4, d5 and d6 after surgery.(2) Anti-fibrosis treatment:referring to the treatment pattern mentioned above but uses anti-TGF-β instead.Results:l00μg antibodies worked best for the prevention treatment for IL-17 and IFN-y. Early blocking of IL-17 (d0-d2) had a significantly higher effect than the late blocking group (d4-d6). (1.7+0.6 vs 4.1+0.9, p=0.001). Similar to this, early blocking of IFN-y had a significantly higher effect than the late blocking group (1.5±0.4 vs 4.0±0.8, p=0.001). However, anti-TGF-β had a opposite readout, with higher efficiency of late blocking compared to the early blocking (4.2±0.8 vs 2.1±1.0, p=0.003).Conclusions:Only early anti-inflammatory and late anti-fibrosis treatment turned out to be effective for preventing adhesion formation. Opposite to this usage of anti-inflammatory antibodies at the late time point or anti-fibrosis antibodies at the early time point have not affections on adhesion formation.PartV Study of the crosstalk between inflammatory and fibrosis during adhesion formationObjectives:To study the effects of acute phase inflammation on the late phase of fibrosis during peritoneal adhesion formation.Methods:After sacrificing the mice,2 ml pre-warmed trypsinization-EDTA solution were injected into the abdominal cavity and shake at 37 ℃ for 15 minutes. Then the solution was withdrawn by the syringe and diluted by 10% FBS in DMEM to stop the digestion. The liquid was centrifuged and splined-down cells were collected and planted in six-well plates and cultured. About 1 week later the cells had a second passage. These cells were used for the experiments. To mimic the pattern observed in part II and III, a complex stimulation strategy was used. IL-17 and/or IFN-y were added to the culture system for 24 hours and then the medium was changed with medium containing TGF-β for another 24 hours. After 48 hours of stimulation, the supernant was collected and the concentration of t-PA/PAI-1 was tested. Western-blot was used for analyzing TGF-β relative signals such as Smad 2/3, p-Smad 2/3. Besides, abdominal adhesion tissues were collected and RNA in the tissue was isolated for qPCR test on chemokine relative to CD4+T cells and y8T cells.Results:In the supernant of the culture system; In the PBS control group, t-PA concentration is 101.32±15.12ng/ml; PAI-1 concentration is 42.85±10.13ng/ml. In the IL-17 pre-stimulated group, t-PA concentration is 51.12±6.02ng/ml; PAI-I concentration is 82.45±11.33ng/ml. In the IFN-y pre-stimulated group, t-PA concentration is 91.12±11.32ng/ml; PAI-1 concentration is 82.45±11.33ng/ml. In the IL-17+IFN-y pre-stimulated group, t-PA concentration is 31.12±802ng/ml; PAI-1 concentration is 162.45±25.93ng/ml. Among different treatments, IL-17+IFN-y pre-stimulating could significantly increase the reaction of the mesothial cells against TGF-β. In the mRNA from the stimulated cells:in the PBS control group, t-PA, PAI-1 mRNA expression was balanced to be 1.0 as the controls. In the IL-17 pre-stimulated group, t-PA expression was 0.42±0.13 and PAI-12.46±0.78. In the IFN-γ pre-stimulated group, t-PA expression was 1.02±0.43 and PAI-10.99±0.18. In the IL-17+IFN-γ group, t-PA expression was 0.22±0.10 and PAI-16.48±2.08. In all the experimental groups, the IL-17+IFN-γ group had lowest t-AP but highest PAI-1. Also western blot results indicate that compared to cells directly stimulated by TGF-P, pre-treatment of IL-17+IFN-γ could activate more Smad 2/3 pathway with stronger phosphorylation. qPCR on abdominal adhesion tissue indicated that the mRNA of the relative chemokine such as CCL-22, KC significantly increased.Conclusions:Compared to the merely TGF-P stimulated group, mesothelium cells which were pre-stimulated with IL-17+IFN-γ had a stronger reaction, with higher secretion of promoting fibrosis factor PAI-1 and lower secretion of inhibiting fibrosis factor t-PA, together with higher phosphorylation of Smad 2/3. Besides, after surgery, secretion of chemokine such as SDF-1, CCL-22 by mesothelium increased significantly, which could further recruit the inflammatory cells.