Potential Probiotic Lactobacillus Rhamnosus (MTCC-5897) Attenuates Escherichia Coli Induced Inammatory Response in Intestinal Cells

Probiotics are microbes having tremendous potential to prevent gastrointestinal disorders. In current investigation, immunomodulatory action of probiotic Lactobacillus rhamnosus (LR:MTCC-5897) was studied during exclusion, competition and displacement of Escherichia coli on intestinal epithelial (Caco-2) cells. The incubation of intestinal cells with E. coli, enhanced downstream signalling and activated nuclear factor kappa B (NF-κB). This signicantly increased (p<0.01) the pro-inammatory cytokines (IL-8, TNF-α and IFN-ϒ) expression. While, incubation of epithelial cells with L. rhamnosus during exclusion and competition with E. coli, counteracted these enhanced expressions. The immunomodulatory feature of L rhamnosus was also highlighted with increased (p<0.05) transcription of toll like receptor-2 (TLR-2) and single Ig IL-1-related receptor (SIGIRR) along with diminished expression of TLR-4. Likewise, attenuation (p<0.05) of E.coli-mediated enhanced nuclear translocation of NF-κB p-65 subunit by L. rhamnosus during exclusion was conrmed with western blotting. Thus, present nding establishes the prophylactic potential of L. rhamnosus against exclusion of E. coli in intestinal cells.


Introduction
The  Kim et al. 2005). Previous evidences displayed that E. coli K12 which is generally referred as "safe" strain, have some virulence genes and regulatory process similar to pathogenic bacteria and could switch to invasive and pathogenic life style without any major change in genetic ux (Koli et al. 2011). These bacteria further translocated to extra intestinal tissue sites like mesenteric lymph nodes, spleen and liver by disruption of gut barrier in immunocompromised aged mice (Sharma et al. 2014). Similarly, under in vitro conditions, E. coli K12 enhanced the intestinal permeability by altering the barrier integrity through decreasing the expression of tight junctions (Bhat et al. 2019, a). Furthermore, E. coli K12 derived lipopolysaccharides (LPS) also caused enhanced T-helper (Th1/Th17) immune response which resulted in severe intestinal in ammation in mice model (Gronbach et al. 2014).
Thus, balancing the deregulated bacterial ecosystem is only a substitute for the prevention of various gutrelated diseases and conferring health bene ts. Nowadays, nutritionists and researchers are looking forward to probiotics as a healthier alternative to preserve gut immune homeostasis. Probiotics have been known to have many bene cial effects on metabolism, junctional integrity and regulation of mucosal or systemic immune response (Galdeano et al. 2019;Bron et al. 2017). Therefore, probiotic consumption as a versatile functional food has increased tremendously due to its enormous health effects. Though, the number of indirect evidences depicted the health bene ts of probiotics through immune-modulations that displayed changes in expressions of immunoglobulins and pro-in ammatory cytokines in pre-clinical and clinical trials (Oh et al. 2018;Groeger et al. 2013;Milajerdi et al. 2019;Horvath et al. 2016). However, scanty direct evidences are available which can suggest undergoing molecular events to establish microbe's potential individually because probiotic microbes have highly complex and strain speci c mode of action (Chiu et al. 2013). Hence, the present study was designed to understand the immunomodulatory signals released from host intestinal epithelial cells, being the rst site of interaction after ingestion, in response to probiotic bacteria L. rhamnosus in presence of E. coli. Earlier, this potential probiotic strain of L. rhamnosus (MTCC:5897) restored Th1/Th2 immune homeostasis, anti-oxidative status and antagonize translocation of pathogenic E. coli in aging mice (Sharma et al. 2014). Likewise, its feeding in the form of fermented milk in ovalbumin allergen sensitized weaning mice also alleviated symptoms of allergies and depicted its immunomodulatory potential (Saliganti et al. 2015). This potential probiotic also maintained junctional integrity in E. coli induced in ammatory response in intestinal cells (Bhat et

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An indigenous probiotic L. rhamnosus (LR:MTCC-5897) used under present investigation was previously isolated from household curd and characterized for probiotic attributes (Sharma et al. 2014). For this study, bacterial culture was activated in sterile de Man, Rogosa and Sharpe (MRS) broth (Hi-Media, Mumbai, Maharashtra, India) for 18 h under aerobic incubation at 37°C before use. Next day, activated culture was used to harvest bacterial pellets by centrifugation at 2000 х g for 10 min, followed by washing with phosphate buffered saline (PBS, pH-7.4). For in vitro treatments, bacterial pellets were resuspended in antibiotic free DMEM media to obtain 10 9 colony forming unit (CFU) ml − 1 . The number of bacteria was determined by plate counting on MRS agar plates after aerobic incubation at 37°C for 24-48 h. This particular dose of L. rhamnosus was selected on the basis of previous investigation in which this probiotic was found safe in Caco-2 cells upto 24 h and displayed immunomodulatory effects in weanling mice (Bhat et

Stimulation of intestinal cells with bacteria
Caco-2 cells with 1х10 5 cells ml − 1 density were seeded in 6-well plate and after obtaining con uency, cells were treated with L. rhamnosus (1х10 9 CFU ml − 1 ) or E. coli (1х10 8 CFU ml − 1 ) for 3 h at 37°C in 5% CO 2 . For further experiments, Caco-2 cells were incubated with probiotic/E. coli under three different challenge modes known as exclusion (Ex: pre-treatment), competition (Com) and displacement (Dis: posttreatment) respectively. In exclusion assay, Caco-2 cells were incubated with probiotic L. rhamnosus for 3 h, then media was removed and cells were washed with PBS followed by 3 h incubation with E. coli containing DMEM medium for in ammatory stimulation. In competition assay, Caco-2 cells were simultaneously incubated with L. rhamnosus and E. coli for 3 h. While, during displacement assay, Caco-2 cells were initially treated with E. coli containing medium which was then removed after 3 h of incubation and cells were washed with PBS. Later, these intestinal cells were incubated with L. rhamnosus for 3 h. In all these experimental assays, Caco-2 cells grown in DMEM media acted as a negative control. All sets of experiments were carried out in triplicate. The treated cells were washed with ice-cold PBS twice and used for RNA extraction and western blotting.

RNA isolation and relative expression of genes associated with immune response
Total RNA was isolated from Caco-2 cells following Trizol method as described in manufacturer protocol and further used for relative quanti cation of genes associated with immune response. Purity of the RNA was con rmed by determining O.D. at 260/280 ratio using microplate spectrophotometer (BioTek Instruments, Winooski, Vermont, USA). RNA integrity was con rmed on 1.5% agarose gel through electrophoresis. Total RNA (1 µg) was reverse transcribed to cDNA using a reverse transcription kit (Thermo Fisher Scienti c, Waltham, Massachusetts, USA) following user manual. The prepared cDNA was stored in -20°C until used further. Quantitative real-time PCR (qRT) analysis, reactions were conducted to determine the relative gene expression by using ABI-fast 7500 thermocycler system (Applied Biosystems, California, USA). For mRNA expressions, qRT-PCR reactions were performed in 10 µl reaction volume containing 1 µl of test sample, 5 µL of syber (Thermo scienti c, USA), 0.5 µl of each primer and 3 µl nuclease free water. Sequences of primers are shown in Table 1. GAPDH was used as a reference gene throughout the experiments. The thermal pro le for reaction was: initial denaturation of 5 min at 94°C, 35 cycles of denaturation (94°C for 30 sec), annealing (60°C for 30 sec) and extension (60°C for 45 sec) and nal extension cycle at 60°C for 5 min. After ampli cation, threshold (Ct) values of both control and treatment groups with reference genes (GAPDH) were used for calculating fold changes in respective target genes expression (Livak and Schmittgen 2001). Table 1 Sequence of primers along with their corresponding amplicon size for mRNA quanti cation immunity related genes using qRT-PCR Gene Sequence of primer Amplicon Length (bp) Accession number Genes related to Interleukins Genes related to pathogen recognition receptor (PRR) Genes related to NF-κB pathway

Statistical analysis
Data were analysed using GraphPad Prism (Version 5.01) software. Experimental results are presented as means ± S.E.M. Data were subjected to analysis of variance (ANOVA) and the Tukey test was used to separate the means (p < 0.05) which were considered statistically signi cant. folds than E. coli (8.12 ± 1.74 folds) stimulated cells (Fig. 1A). Likewise, mRNA levels of pro-in ammatory marker TNF-α, decreased considerably (p ≤ 0.01) during L.rhamnosus incubation in presence of E.coli irrespective of the mode of challenge (Fig. 1B). Pro-in ammatory cytokine IL-23 merely showed signi cantly (p ≤ 0.05) diminished mRNA transcription from intestinal cells during exclusion by probiotic LR than other modes of challenges with E. coli (Fig. 1C). On the other hand, no major alterations occurred in expression of IL-6 irrespective of the type of challenge between E. coli and probiotic bacteria during incubation with intestinal cells.

Modulation in intestinal cytokines
The results of actually released pro-in ammatory (TNF-α, IFN-ϒ) and regulatory cytokines (IL-10 and TGFβ) measured by ELISA are shown in Fig. 2. E. coli challenged Caco-2 cells showed signi cantly (p ≤ 0.05) higher release of in ammatory cytokines (TNF-α and IFN-ϒ) along with diminished secretions of antiin ammatory cytokine (IL-10) as compared to negative control cells. It was also observed that incubation of probiotic L. rhamnosus individually or during challenge with E. coli during exclusion, competition and displacement assays prevented in ammatory response by signi cantly (p ≤ 0.05) reducing the secretions of in ammatory cytokines (TNF-α, IFN-ϒ) than E. coli in amed cells ( Fig. 2A and B). On the other hand, probiotic L. rhamnosus treated intestinal cells showed much higher (p ≤ 0.05) production of TGF-β and IL-10 in comparison to E. coli in amed cells ( Fig. 2C and D). Besides, E. coli exclusion by L. rhamnosus signi cantly (p ≤ 0.05) enhanced the release of IL-10 and TGF-β from intestinal cells, though assays based variations were observed during competition and displacement. Thus, it is clearly depicted that probiotic L. rhamnosus has an inhibitory effect on in ammatory milieu induced by E. coli in intestinal cells by suppressing pro-in ammatory cytokines and modulating anti-in ammatory cytokines more effectively during exclusion assay.

Modulation in expression of Toll like receptors (TLRs)
To get more insight in molecular events related to immunomodulation brought by L. rhamnosus, mRNA expressions of key pathogen recognition receptors (PRR's) were assessed in Caco-2 cells (Fig. 3). Stimulation of Caco-2 cells with E. coli or probiotic L. rhamnosus induced differential expression of these genes. mRNA transcription of TLR-4 was enhanced (p ≤ 0.05) to 2.83 ± 0.13 folds after exposure of E. coli than control cells (Fig. 3A) while it remained near to control levels on incubation with probiotic L. rhamnosus individually or suppressed it signi cantly (p ≤ 0.05) during respective exclusion and competition assays as compared to E. coli in amed cells. On the other hand, exposure of probiotic L. rhamnosus noticeably (p ≤ 0.05) enhanced the TLR-2 expression by 2.42 ± 0.42 folds than E. coli infected cells. Similarly, probiotic L. rhamnosus caused signi cantly higher (p ≤ 0.05) TLR-2 mRNA expression in intestinal cells than E. coli treated epithelial cells during exclusion as well as competition with in ammatory agent E. coli, (Fig. 3B). Though, displacement assays showed its transcriptional expression almost same to control or E. coli treated cells. mRNA expression of adaptor protein MyD-88, which regulates downstream signalling of all PRR's in intestinal cells showed statistically higher (p < 0.05) transcriptional activity in E. coli in amed cells (Fig. 3C) without any major modulations in treatment groups.

Inhibition of NF-κB signalling in intestinal epithelial cells
In present investigation, infection of Caco-2 cells with E. coli resulted in increased mRNA expression of NF-κB to 4.73 ± 0.34 folds (p ≤ 0.05) than control. In contrast, probiotic L. rhamnosus exposure to intestinal cells kept mRNA transcription (1.84 ± 0.14 folds) close to control and signi cantly less (p ≤ 0.05) than E. coli treated cells. Exclusion and competition of E. coli with L. rhamnosus caused 3.05 ± 0.62 and 2.44 ± 0.64 folds of mRNA expression, respectively which differed insigni cantly from control or E. coli treated intestinal epithelial cells (Fig. 4A). The in ammatory challenge of E. coli to epithelial cells suppressed (p ≤ 0.05) transcription of single immunoglobulin IL-1R-related receptor (SIGIRR), a negative regulator of NF-κB, than control (Fig. 4B). While, L. rhamnosus incubation to intestinal epithelial cells individually or during various modes of challenge with E. coli resulted into signi cantly higher (p ≤ 0.05) transcription of SIGIRR than E. coli in amed cells (Fig. 4B). Figure 4C shows western blot analysis of nuclear translocation of NF-κB, p-65 subunit from cytoplasm which was essentially required for activation of in ammatory responses by secretion of various in ammatory cytokines. The nuclear translocation of p-65 subunit increased signi cantly (p < 0.05) in the cells infected with E. coli as compared to unstimulated negative control cells (Fig. 4d & e). In opposition, cells treated with L. rhamnosus showed reduced nuclear translocation of p-65. Likewise, pre-incubation of Caco-2 cells with L. rhamnosus before E. coli during exclusion assay also resulted in much less (p ≤ 0.05) translocation of this factor than E. coli in amed cells (Fig. 4d & e), which was contrary to the observations made during competition or displacement assays.

Discussion
Intestinal epithelial cells, besides acting as a physical barrier, also play crucial role as immune modulator because epithelium is the site of interaction between microbes and host. Although, under present investigation, post-treatment of probiotic L. rhamnosus during displacement assay was not found much effective in controlling in ammatory response but it clearly mitigated in ammatory responses and maintained intestinal homeostasis by achieving balance between pro and anti-in ammatory cytokines (Shadnoush et al. 2013) during exclusion and competition assays. For further insight into plausible mechanism of immunomodulation by probiotic L. rhamnosus in presence of E. coli induced in ammatory response, mRNA expression of NF-κB and its translocation from cytoplasm to nucleus were also explored. It was well established that regulated NF-κB dependent signalling is critical for e cient immune response, but prolonged activation contributes to generation of in ammatory diseases (Yan and Polk 2010

Conclusion
In summary, this study clearly provided direct insight into the mode of action of probiotic L. rhamnosus (LR:MTCC-5897) under in ammatory milieu induced by E. coli in intestinal cells. This potential probiotic strain displayed immunomodulatory and anti-in ammatory functions to varying extent in intestinal cells depending upon the type of E. coli challenge. However, differential expression of TLRs caused effective reduction in NF-κB p-65 nuclear translocation during exclusion of E.coli with L.rhamnosus. Thus immune homeostasis was achieved by reducing the expression of pro-in ammatory and enhancing the release of regulatory cytokines. Thus, it can be concluded that L. rhamnosus (LR:MTCC-5897) may be a potential candidate to produce nutraceuticals products for prevention of E. coli induced intestinal in ammation.