Tryptophan Ameliorates Barrier Integrity and Alleviates the Inflammatory Response to Enterotoxigenic Escherichia coli K88 Through the CaSR/Rac1/PLC-γ1 Signaling Pathway in Porcine Intestinal Epithelial Cells

Background Impaired intestinal barrier integrity plays a crucial role in the development of many diseases such as obesity, inflammatory bowel disease, and type 2 diabetes. Thus, protecting the intestinal barrier from pathological disruption is of great significance. Tryptophan can increase gut barrier integrity, enhance intestinal absorption, and decrease intestinal inflammation. However, the mechanism of tryptophan in decreasing intestinal barrier damage and inflammatory response remains largely unknown. The objective of this study was to test the hypothesis that tryptophan can enhance intestinal epithelial barrier integrity and decrease inflammatory response mediated by the calcium-sensing receptor (CaSR)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/phospholipase Cγ1 (PLC-γ1) signaling pathway. Methods IPEC-J2 cells were treated with or without enterotoxigenic Escherichia coli (ETEC) K88 in the absence or presence of tryptophan, CaSR inhibitor (NPS-2143), wild-type CaSR overexpression (pcDNA3.1-CaSR-WT), Rac1-siRNA, and PLC-γ1-siRNA. Results The results showed that ETEC K88 decreased the protein concentration of occludin, zonula occludens-1 (ZO-1), claudin-1, CaSR, total Rac1, Rho family member 1 of porcine GTP-binding protein (GTP-rac1), phosphorylated phospholipase Cγ1 (p-PLC-γ1), and inositol triphosphate (IP3); suppressed the transepithelial electrical resistance (TEER); and enhanced the permeability of FITC-dextran compared with the control group. Compared with the control group, 0.7 mM tryptophan increased the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; elevated the TEER; and decreased the permeability of FITC-dextran and contents of interleukin-8 (IL-8) and TNF-α. However, 0.7 mM tryptophan+ETEC K88 reversed the effects induced by 0.7 mM tryptophan alone. Rac1-siRNA+tryptophan+ETEC K88 or PLC-γ1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. NPS2143+tryptophan+ETEC K88 decreased the TEER and the protein concentration of CaSR, total Rac1, GTP-rac1, p-PLC-γ1, ZO-1, claudin-1, occludin, and IP3; increased the permeability of FITC-dextran; and improved the contents of IL-8 and TNF-α compared with tryptophan+ETEC K88. pcDNA3.1-CaSR-WT+Rac1-siRNA+ETEC K88 and pcDNA3.1-CaSR-WT+PLC-γ1-siRNA+ETEC K88 decreased the TEER and enhanced the permeability in porcine intestine epithelial cells compared with pcDNA3.1-CaSR-WT+ETEC K88. Conclusion Tryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-γ1 signaling pathway.

Tryptophan, one of the functional amino acids, has been reported to improve the growth, decrease stress-induced injury, improve appetite and mitochondrial function, enhance antioxidant status, increase immunity, enhance the diversity of the intestinal microbiome, change anabolism, and improve intestinal wound restitution in animals (10)(11)(12)(13)(14)(15)(16). In particular, tryptophan plays a vital role in protecting intestinal integrity by regulating the expression of TJ proteins (17)(18)(19). The transepithelial electrical resistance (TEER) and permeability reflect the integrity and function of the intestinal epithelium layer and are utilized to evaluate pathogenic microorganisms' challenges (18). Nevertheless, the effects of tryptophan supplementation on intestinal TEER and permeability in ETEC K88-induced intestinal epithelial cells have not been investigated. Lack of tryptophan can change the gut microbial ecosystem and lead to intestinal inflammation (20). Additionally, tryptophan supplementation reduces the mRNA levels of proinflammatory cytokines interleukin-8 (IL-8) and IL-1b in the gut (21). However, the exact molecular mechanisms by which tryptophan contributes to intestinal barrier integrity and inflammation response of intestinal epithelial cells remain unknown.
The calcium-sensing receptor (CaSR) plays critical roles in the regulation of intestinal inflammation, intestinal epithelium restitution, and intestinal TJ protein expression (21)(22)(23)(24). Tryptophan induces the activation of CaSR, which decreases the mRNA levels of proinflammatory cytokines IL-8 and IL-1b in piglets, suggesting that the CaSR signaling pathway may be involved in intestinal inflammatory response (21,25). A research in mice reported that the suppression of CaSR could improve FITCconjugated dextran and decrease the TEER in the intestine (26). CaSR overexpression can enhance IPEC-J2 cell migration (24). CaSR stimulation increased zonula occludens-1 (ZO-1) and Factin-binding protein interaction in Madin-Darby canine kidney (MDCK) cells (27). However, whether tryptophan influences intestinal barrier permeability and TJ proteins through CaSR signaling remains unknown. The activation of CaSR results in the activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and phosphorylation of phospholipase Cg1 (PLC-g1), which are involved in inflammatory response and intestinal epithelial cell migration (24,(28)(29)(30).

TEER and Permeability Assay
The TEER and FD4 flux of porcine intestinal epithelial cells were detected according to the method of a previous study (36). Briefly, the IPEC-J2 cells (5 × 10 5 /mL) were seeded in 12-well transwell insert (1.12 cm 2 , 0.4 mm) with collagen-coated PTFE membrane (Corning Inc., NY, USA) with 0.5 mL of 10% FBS medium in transwell inserts and 1.5 mL of 10% FBS medium in the plate well. The medium was replaced daily. When the TEER values reached a plateau, the IPEC-J2 cells were considered to form a monolayer. Then, cells were washed with PBS and treated with different reagents. IPEC-J2 cells in each transwell insert membrane were incubated with different reagents at 37°C for the indicated time and treated with 10 mL of FITC-dextran 4 kDa (10 mg/mL) for 2 h. About 200 mL of the basal medium was utilized for fluorescence analysis in a microplate fluorescence reader (emission, 528 nm; excitation, 485 nm, SpectraMax M2, Molecular Devices, China). The concentrations of FITC-dextran were determined via standard curves generated using serial dilution of FITC-dextran.

Real-Time PCR
The PCR experimental procedure was carried out as previously described (37). Briefly, total RNA from IPEC-J2 cells was extracted by TRIzol reagents (TaKaRa, Chengdu, China). One microliter of total RNA was reverse transcribed to cDNA using the PrimeScript RT reagent Kit (TaKaRa, Chengdu, China) with gDNA Eraser (TaKaRa, Chengdu, China). Samples were run on a real-time PCR system (ABI 7900HT, Applied Biosystems) using SYBR Premix Ex Taq II (TaKaRa, Dalian, China), and the total volume of the system was 10 mL. Samples were thermocycled using the program (41 cycles of 95°C for 10 s, 58°C for 35 s), followed by a melting curve program (65°C for 5 s, 95°C for 15 s), and all PCR reactions were run in triplicate. The gene primers used are listed in Table S1. The relative mRNA expression of Rac1, PLC-g1, and CaSR was calculated using the 2 −DDCt method.

Statistical Analysis
All data was analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). The homogeneity of variances was evaluated by Levene's test. All results were represented as mean ± standard error of mean (SEM). The significance of differences among treatments were identified at P-value <0.05.

Tryptophan Improved TEER and Decreased Permeability in Porcine Intestinal Epithelial Cells Challenged With ETEC K88
The IPEC-J2 cell monolayer was investigated for epithelial barrier function in response to ETEC K88 infection in the absence or presence of different doses of tryptophan (0.3 and 0.7 mM). Compared with the control group, IPEC-J2 cells treated with ETEC K88 alone showed a spontaneous decrease in TEER value and a significant increase in permeability of FITCdextran (P < 0.05, Figures 1A, B). Pretreatment with tryptophan (0.3 and 0.7 mM) reversed the ETEC K88-induced reduction of TER value (compared with 0.3 and 0.7 mM tryptophan-treated cells) (P < 0.05, Figures 1A, B). Moreover, treatment with 0.3 and 0.7 mM tryptophan significantly increased the TEER and significantly decreased the permeability of FITC-dextran in IPEC-J2 cell monolayers after 48 h (compared with untreated cells) (P < 0.05, Figures 1A, B). The best protective effect of tryptophan on TEER and permeability was obtained at 0.7 mM concentration. Therefore, we used 0.7 mM tryptophan in subsequent research.

Rac1/PLC-g1 Signaling Pathway Contributes to Tryptophan-Induced Upregulation of TEER and Downregulation of Permeability and Inflammatory Response in Porcine Intestinal Epithelial Cells
To explore the molecular mechanism by which tryptophan regulates intestinal barrier integrity, IPEC-J2 cells were transfected with Rac1-siRNA or PLC-g1-siRNA for 24 h before the addition of tryptophan (0.7 mM). Then, the cells were challenged with ETEC K88 for 2 h. The results showed that Rac1-siRNA and PLC-g1- siRNA significantly decreased Rac1 and PLC-g1 mRNA expression, respectively (P < 0.05, Figures S1A, B). Treatment with 0.7 mM tryptophan significantly increased the TEER and significantly decreased the permeability of FITC-dextran and contents of IL-8 and TNF-a in IPEC-J2 cell monolayers compared with untreated cells (P < 0.05, Figures 2A-D). Compared with the control group, ETEC K88 suppressed the TEER, enhanced the permeability of FITC-dextran, and improved the contents of IL-8 and TNF-a (P < 0.05, Figures 2A-D). Compared with cells treated with 0.7 mM tryptophan alone, cells treated with 0.7 mM tryptophan + ETEC K88 showed decreased TEER, increased permeability of FITCdextran, and enhanced contents of IL-8 and TNF-a (P < 0.05, Figures 2A-D). In addition, compared with treatment with 0.7 mM tryptophan + ETEC K88, Rac1-siRNA or PLC-g1-siRNA inhibited the tryptophan-induced upregulation of TEER and downregulation of FITC-dextran permeability and IL-8 and TNF-a contents in IPEC-J2 cells challenged with ETEC K88 (P < 0.05, Figures 2A-D).
Taken together, the results suggest that the regulation of TEER, permeability, and inflammatory response by tryptophan is dependent on the Rac1/PLC-g1 signaling pathway.

Inhibition of CaSR by NPS2143 Disrupts the Effect of Tryptophan on TJ, Inflammatory Response, TEER, and Permeability in ETEC K88-Challenged IPEC-J2 Cells
Compared with ETEC K88, ETEC K88+tryptophan increased the protein concentrations of occludin, ZO-1, claudin-1, and CaSR, but this effect was inhibited by NPS2143 (P < 0.05, Figures 3A-D). Tryptophan+ETEC K88+NPS2143 significantly reduced the protein concentrations of occludin, ZO-1, claudin-1, and CaSR compared with tryptophan+ETEC K88 (P < 0.05, Figures 3A-D). Treatment with 0.7 mM tryptophan significantly increased the TEER and significantly decreased the permeability of FITC-  dextran in IPEC-J2 cell monolayers compared with untreated cells (P < 0.05, Figures 4A, B). Compared with the control group, treatment with ETEC K88 significantly decreased the TEER and increased the permeability of FITC-dextran in IPEC-J2 cells (P < 0.05, Figures 4A, B). Moreover, treatment with 0.7 mM tryptophan+ETEC K88 significantly decreased the TEER and increased the permeability of FITC-dextran compared with 0.7 mM tryptophan alone (P < 0.05, Figures 4A, B). In the tryptophan +ETEC K88+NPS2143 group, the TEER was significantly reduced, and the permeability of FITC-dextran was significantly increased compared with the tryptophan+ETEC K88 group (P < 0.05, Figures 4A, B). Figures 5A, B, ETEC K88 significantly decreased the contents of IL-8 and TNF-a in IPEC-J2 cells compared with the control group. Compared with the control group, cells treated with tryptophan had decreased IL-8 and TNF-a contents. Compared with cells treated with tryptophan alone, cells treated with tryptophan+ETEC K88 showed increased contents of IL-8 and TNF-a. However, the incubation of IPEC-J2 cells with NPS2143 reversed the effects of tryptophan on IL-8 and TNF-a contents.

DISCUSSION
Different cytokines can modify the junctional complex. The proinflammatory roles of TNF-a and IL-8 were linked with ETEC and increased intestinal permeability (38). Thus, TNF-a and IL-8 parameters were selected in this study. We found that ETEC K88 enhanced the contents of TNF-a and IL-8, which is in agreement with a previous article that ETEC K88 induced intestinal proinflammatory response in pigs (8). above-mentioned finding, ETEC K88 significantly decreased the TEER values and increased the permeability of FITC-dextran in ETEC K88-challenged IPEC-J2 cells, which suggests that the cell damage model was successfully constructed. In this study, tryptophan significantly decreased the contents of TNF-a and IL-8 in ETEC K88-challenged IPEC-J2 cells, suggesting that tryptophan can attenuate ETEC K88-induced proinflammatory response. This finding is in line with that of a previous study, which showed that tryptophan reduced the gene expression of IL-8 and IL-1b in the gut (21). Proinflammatory cytokines have been related to pathogen-induced alteration of TJ proteins (39).
Here, tryptophan (0.7 mM) increased the protein concentrations of occludin, ZO-1, and claudin-1 in ETEC K88-challenged and non-challenged IPEC-J2 cells. These findings were consistent with previous studies on pigs (40), Caco-2 cells, and IPEC-1 cells (18,19). The current study also demonstrated that tryptophan (0.3 and 0.7 mM) significantly increased the TEER values and decreased the permeability of FITC-dextran in ETEC K88challenged IPEC-J2 cells. Taken together, our results suggested that tryptophan can improve intestinal barrier integrity and decrease proinflammatory response. The regulation of intestinal barrier integrity and proinflammatory response is complex, involving numerous intracellular molecular signaling and kinases, such as CaSR, PLC signaling, and RHO kinase. These molecules regulate TJ protein expression, TJ assembly, and redistribution by phosphorylation (41)(42)(43)(44). CaSR signaling regulates the TEER in the intestine of mice, TJ protein expression, and proinflammatory immune response (21,26). We found that NPS2143 reversed the enhancement effect of tryptophan on the protein concentrations of ZO-1, occludin, claudin-1, and CaSR and the TEER and decrease of permeability and IL-8 and TNF-a contents. The overexpression of pcDNA3.1-p(CaSR) markedly increased the TEER and decreased the permeability of FITC-dextran. Taken together, these results suggested that tryptophan protects intestinal epithelial barrier integrity and alleviates intestinal inflammation though CaSR signaling. The Rho family of small guanosine triphosphatases, such as Rho, Cdc42, and Rac1, has been reported to regulate the composition and function of TJs (45)(46)(47)(48). The PLC-dependent pathway has been demonstrated in the assembly of TJs in MDCK cells (49,50). In this study, we found that Rac1-siRNA+tryptophan+ETEC K88 or PLC-g1-siRNA+tryptophan+ETEC K88 reduced the TEER, increased the permeability of FITC-dextran, and enhanced the contents of IL-8 and TNF-a compared with tryptophan+ETEC K88. Collectively, these results suggested that tryptophan can improve intestinal barrier integrity and decrease proinflammatory response at least partly through Rac1/PLC-g1 signaling in intestinal epithelial cells. The effects of CaSR on the mRNA expression of inflammatory cytokines and intestinal barrier integrity are associated with two downstream effectors Rac1 and PLC-g1 (28,30). In the present study, our results showed that tryptophan+ETEC K88+NPS2143 decreased the protein concentrations of GTP-rac1, total Rac1, and p-PLC-g1 and contents of IP 3 compared with tryptophan+ETEC K88. Furthermore, we found that the inhibition of Rac1 or PLC-g1 by Rac1-siRNA and PLC-g1-siRNA significantly reduced the TEER and increased the permeability of FITC-dextran in cells treated with pcDNA3.1-p(CaSR) and ETEC K88. These results were consistent with those of previous reports, indicating that tryptophan can enhance IPEC-J2 cell migration through the CaSR/Rac1/PLC-g1 signaling pathway (24). Taken together, these results suggest that CaSR is required for tryptophaninduced activation of Rac1/PLC-g1 signaling, which increases intestinal epithelial TJ and decreases intestinal epithelial permeability and inflammatory response in IPEC-J2 cells after ETEC K88 challenge.
Collectively, the results suggest that tryptophan can improve intestinal epithelial barrier integrity and decrease inflammatory response through the CaSR/Rac1/PLC-g1 signaling pathway. This study not only offers new insights into the function of tryptophan, but also indicates the necessity for further investigating the effect of tryptophan on intestinal health in vivo.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
GL and KG conceived and designed the experiment. KG and GL wrote the paper. GL, KG, and FW performed the research and analyzed the data. GJ, HZ, XC, CW, RZ, GT, JC, JT, and JW contributed to the analysis and manuscript preparation. All authors contributed to the article and approved the submitted version.