The Role of Combining Probiotics in Preventing and Controlling Inflammation: A Focus on the Anti-Inflammatory and Immunomodulatory Effects of Probiotics in an In Vitro Model of IBD

Objective IBD is an inflammatory disease with abnormalities such as dysbiosis and abnormal immune system activity. Probiotics, as live beneficial microorganisms, play a role in maintaining health through various mechanisms, including the modulation of the immune system and the control of inflammation. Here, we aimed to investigate the efficacy of a probiotic mixture of Lactobacillus spp. and Bifidobacterium spp. in modulating JAK/STAT and NF-kB inflammatory signaling pathways. Method A quantitative real-time polymerase chain reaction (qPCR) assay was conducted to analyze the expression of JAK/STAT and inflammatory genes after treatment with the probiotic mixture before, after, and simultaneously with the sonicated pathogen in the HT-29 cell line. The production of IL-6 and IL-1β after probiotic treatment was investigated via cytokine assay. Results Treatment with probiotics resulted in downregulation of TIRAP, IRAK4, NEMO, and RIP genes in the NF-kB pathway and JAK/STAT genes compared with sonicat-treated cells as inflammation inducers. The production of IL-6 and IL-1 decreased after probiotic treatment. Conclusions The probiotic mixture of Lactobacillus spp. and Bifidobacterium spp. showed anti-inflammatory effects by modulating JAK/STAT and NF-kB signaling pathways. The use of probiotics could be considered as an appropriate complementary treatment for patients with inflammatory bowel disease.


Introduction
Probiotics as live benefcial microorganisms have various roles in maintaining health. Prevention of gastrointestinal symptoms such as diarrhea, lowering cholesterol levels, and prevention of cancer and allergy are some of the notable efects of probiotics [1]. In addition, one of the most important remarkable efects of probiotics is to infuence the consequences of dysbiosis. An imbalance of the intestinal microbiome can lead to an increase in pathogenic bacteria and a decrease in the healthy microbiota. Tis process eventually leads to abnormal function of immune cells, including T1 and T17 [2]. T1 and T17 could infuence the infammatory response via the production of IL-2 and IL-17. However, the abnormal activity of these cells usually leads to chronic infammation [3]. Probiotics could infuence this process via immunomodulatory functions. Indeed, Tregs suppress the immune response and prevent infammation in the colon by producing IL-10. Probiotics could modulate the immune system and reduce infammatory conditions by causing an efect on Tregs [4]. Lactobacillus spp. and Bifdobacterium spp. are two probiotic members that have anti-infammatory efects. Te antiinfammatory characteristics of these probiotic strains, especially in the intestinal epithelial cells, and the reduction of symptoms in patients with ulcerative colitis have been demonstrated [5]. Also, various studies have been demonstrated that using the combination of probiotic strains compared with individual strains has stronger efects on improving health conditions [6].
One of the infammatory diseases that pose challenges to patients is infammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn's disease (CD) are two subtypes of IBD with some diferences, including the location of the GI tract that is afected. Te exact etiology of IBD is not yet fully understood. However, risk factors include genetics, stress, antibiotic use, dysbiosis of the gut microbiome, and abnormal immune system function [7]. IBD is described as a disease with remission and relapse phases. Te exact timing of relapse is not known, but the use of an agent or strategy that could prolong the duration of the remission phase would be crucial for patients with IBD [8]. As mentioned earlier, probiotics have immunomodulatory efects, and since IBD may be caused by immunologic imbalance, probiotic intake may be suitable to control IBD. Te immunomodulatory properties of probiotics could be diferent. Various molecular signaling pathways, for instance, could be afected by probiotics, so probiotics exert their immunoregulatory and anti-infammatory properties [9]. Te Janus kinase/signal transducer and activator of transcription (JAK/STAT) and the nuclear factor kappalight-chain-enhancer of activated B Cells (NF-kB) are two signaling pathways involved in various human diseases, including infammatory disorders. Te association of these signaling pathways with pre-and anti-infammatory cytokines may elucidate the link with infammatory diseases such as IBD. Since infammatory cytokines play an important role in the progression of IBD, fnding an agent with positive modulatory efects on the aforementioned signaling pathways could be useful for the treatment or prevention of IBD [10,11].
Our laboratory has demonstrated the phenotypic efects of probiotics on the control of infammatory status [12]. Also, the immunomodulatory and anti-infammatory efects of diferent probiotic strains before infammation induction have been shown to clarify the preventive efects of probiotic strains to reduce infammation [13]. Indeed, the identifcation of the exact molecular efects of probiotics on signaling pathways involved in the progression of infammation may clarify the benefcial efects of probiotics. In the present study, we aimed to demonstrate the immunomodulatory and anti-infammatory efects of Lactobacillus spp. and Bifdobacterium spp. as a mixture before, after, and during infammation by causing an efect on JAK/STAT and NF-kB signaling pathways to determine whether they could be used as therapeutic or preventive options for infammatory conditions, including IBD.

Bacterial Strain, Culture Medium, and Growth
Conditions. Here, the in vitro assay was conducted to evaluate the efects of probiotics on NF-kB and JAK/STAT signaling pathways in the HT-29 cell line. Four Lactobacillus spp. including L. plantarum, L. rhamnosus, L. brevis, and L. reuteri and three Bifdobacterium spp. including B. bifdum, B. longum, and B. infantis were isolated from stool samples and breast milk as previously reported [12,14]. Te preparation of probiotic cocktails in addition to pathogenic bacteria, including enterotoxin-producing Escherichia coli (ETEC) and Salmonella typhimurium (ST) were explained [13]. Te experimental protocols were established following the Declaration of Helsinki and approved by the ethics committee of Pasteur Institute of Iran (IR.PII.REC.1398.060). All methods were carried out in accordance with relevant guidelines and regulations.

Investigation of the Preventive Efects of Lactobacillus/ Bifdobacterium Mixture.
To study the efect of probiotics before infammation induction, the Lactobacillus/Bifdobacterium mixture was frst added to HT-29 cell line, and after 6 hours, SP-ETEC and SP-Salmonella typhi were added to induce infammation.

Investigation of the Terapeutic Efects of Lactobacillus/ Bifdobacterium Mixture at the Beginning of Infammation.
To show the efcacy of Lactobacillus/Bifdobacterium at the beginning of infammation induction Lactobacillus/Bifdobacterium and sonicated pathogens were added to the HT-29 cell line simultaneously.

Investigation of the Terapeutic Efects of Lactobacillus/ Bifdobacterium Mixture after Infammation Induction.
To study the efect of Lactobacillus/Bifdobacterium mixture after infammation induction, SP-ETEC and SP-Salmonella typhi were frst added to the HT-29 cell line, and Lactobacillus/Bifdobacterium was added after 6 hours to determine the presumed efects. Each treatment was performed in two separate biological replicates and three separate technical replicates.
In the next step, each well was washed twice with PBS to remove the nonadherent bacteria. Tese treatments were performed in duplicate and cell culture was maintained at 37°C and 5% CO 2 for up to 48 hours. Te determination of MOI was performed as previously described [15].

Cytokine Assays.
To measure the production of proinfammatory cytokines, including IL-6 and IL-1β, the cell culture supernatant was centrifuged at 6000 rpm, the sediment was discarded, and the supernatant was collected to determine using an ELISA kit (Karmanian Pars Gene, Iran) according to the manufacturer's protocols.

RT-PCR of Infammatory Signaling Pathway Genes.
Total RNA was extracted according to the manufacturer's instructions (Roche, Germany). Te cDNA template was synthesized with the cDNA synthesis kit (Yekta Tajhiz, Iran) according to the manufacturer's instructions. Te online PrimerBank website (http://pga.mgh.harvard.edu/ primerbank) was used to choose the qPCR primers (Table 1). All the primers were tested using gradient PCR to get an appropriate annealing temperature. Te mRNA quantifcation of studied genes was evaluated with the ABI step one plus detection system (Applied Biosystems, USA co) using SYBR Green master mix (Amplicon Bio, Denmark). All the reactions were performed in duplicate. Te formula RQ � 2 −ΔΔCt was used to get relative gene expression in the comparative CT method [16]. Te appropriate internal control gene, glyceraldehyde 3-phosphate dehydrogenase (gapdh), was selected as a housekeeping gene to normalize the data.

Statistical Analysis.
Graphs and statistical analyses of the data were performed using GraphPad Prism 8 software to compare the variables of the diferent groups. Statistical diferences between diferent groups, including control (C), sonicated pathogen (SP), the frst administration of Lactobacillus/Bifdobacterium and second administration of sonicated pathogen (LBP), the frst administration of sonicated pathogen and the second administration of Lactobacillus/Bifdobacterium (PLB), and the concurrent administration of Lactobacillus/Bifdobacterium and sonicated pathogen (P + LB), were determined using ANOVA. P values < 0.05 were considered statistically signifcant. Results were expressed as standard deviation (SD).

Results
Te efcacy of probiotics in up-or down-regulation of JAK/ STAT and infammatory genes was investigated by comparing probiotic-treated HT-29 cells with negative control cells (unexposed HT-29 cells) and HT-29 cells exposed to the sonicated pathogen as the positive control. It should be said that the concept of treatment phases refers to Lactobacillus/ Bifdobacterium treatments before, after, and during the induction of infammation.

Lactobacillus/Bifdobacterium Could Generally Decrease the Expression Level of STAT Genes.
Gene expression data from STAT are shown in Figure 1. Te expression level increased after treatment with sonicated pathogens (p < 0.05) compared to negative controls.
Te comparative analysis of STAT1 gene expression showed that the overall trend was downward in most treatment phases, especially compared to the sonicated pathogens after 48 hours of treatment (SP48). Lactobacillus/ Bifdobacterium treatment after infammation induction in the 24 hours of treatment (PLB24) had almost the strongest reduction efect (p < 0.05). Treatment with our probiotic mixture before infammation induction (LBP48) had the least reduction efect. It could be said that the probiotics could have a better reducing efect on the post-treatment status.
In STAT2, the results were similar to those in STAT1. Again, Lactobacillus/Bifdobacterium treatment had the strongest reduction efect after infammation induction in the 24 hours of treatment (PLB24) (p < 0.001). Lactobacillus/ Bifdobacterium treatment with concomitant infammation induction (P + LB48) also had a remarkable reduction efect (p < 0.05). Again, using our probiotic mixture before infammation induction (LBP24 and LBP48) had the least reduction efect. Terefore, the post-treatment had the better reduction efect.
In STAT3, results showed that using Lactobacillus/Bifdobacterium before infammation induction (LBP24 and LBP48) could increase the expression level (p < 0.001). Again, using Lactobacillus/Bifdobacterium after infammation induction (PLB24) had the greatest reduction efect (p < 0.001) and using probiotic mixture before infammation induction has the least reduction efects.
Te comparative analysis of STAT4 gene expression showed that the general trend was downward, especially in the frst 24 hours of treatment. Lactobacillus/Bifdobacterium was able to signifcantly decrease the expression level in all three treatment phases (p < 0.05).
For STAT5, the addition of Lactobacillus/Bifdobacterium before and after infammation induction (LBP24 and PLB24) had signifcant efects on reducing expression levels compared to the positive control in the frst 24 hours of treatment (SP24) (p < 0.01). It should be noted that using Lactobacillus/Bifdobacterium after infammation induction (PLB24 and PLB48) had a reducing efect at both time orders. It has been fully shown that the use of probiotic strains in post-treatment had a better reduction efect.
Te comparative analysis of STAT6 gene expression showed that the general trend was downward in most treatment phases. All treatments with Lactobacillus/Bifdobacterium were able to decrease the expression level, except for P + LB48. Te addition of Lactobacillus/Bifdobacterium before and after infammation induction at both time points (LBP24 and 48, PLB24 and 48) had a reducing efect. Te use  (P) represents pathogen; PLB represents frst pathogen and then Lactobacillus/Bifdobacterium; LBP represents frst Lactobacillus/Bifdobacterium and then sonicated pathogen; P + LB represents sonicated pathogen and then Lactobacillus/Bifdobacterium added simultaneously. Data were considered statistically signifcant when p < 0.05 ( * p < 0.05, * * p < 0.001). Letter (a) indicates the relatedness between C24 and C48 with other treatments, letter (b) shows the relatedness between sp24 and other treatments, and letter (c) shows the relatedness between sp48 with other treatments. Te relatedness between other treatments is shown with brackets.
of Lactobacillus/Bifdobacterium after infammation induction in the frst 24 hours of treatment (PLB24) reduced the expression level to almost zero.
Regarding the expression of STAT genes, it can be said that post-treatment with probiotic strains had the best reduction efects on all six STAT genes. Tis efect could show the suitable therapeutic property of probiotics, which make these strains suitable as complementary treatments.

Signifcant Reduction Efects Could Be Seen via Lactobacillus/Bifdobacterium Treatments on JAK Genes.
Expression data from JAK are shown in Figure 2. Te comparative analysis of JAK genes expression between positive and negative controls showed that the sonicated pathogens could signifcantly increase gene expression, especially after 48 hours of treatment (p < 0.001). A downward trend in gene expression was observed for all JAK genes, including JAK1, JAK2, JAK3, and TYK2.
In JAK1, Lactobacillus/Bifdobacterium treatment had the strongest reduction efect after infammation induction in the 24 hours of treatment (PLB24) (p < 0.001). For JAK2, almost all treatments were able to down-regulate the expression level, except for LBP24. All treatments could have reduction efects in the expression level of JAK3. In TYK2, the same result (the reduction efect) could be seen in all treatments. It should be noted that the expression level in PLB48 decreased to zero (see Figure 2(d)). As with STAT genes, post-treatment with our probiotic strains could reduce the expression of JAK genes (especially in JAK1 and TYK2).  (P) represents pathogen; PLB represents frst pathogen and then Lactobacillus/Bifdobacterium; LBP represents frst Lactobacillus/Bifdobacterium and then sonicated pathogen; P + LB represents sonicated pathogen and then Lactobacillus/Bifdobacterium added simultaneously. Data were considered statistically signifcant when p < 0.05 ( * p < 0.05, * * p < 0.001). Letter (a) indicates the relatedness between C24 and C48 with other treatments, letter (b) shows the relatedness between sp24 and other treatments, and letter (c) shows the relatedness between sp48 with other treatments. Te relatedness between other treatments is shown with brackets. treatment phases, could decrease the expression level to near zero (see Figure 3(b)). Although, as can be seen in Figure 3, all treatments were able to reduce the expression level of infammatory genes, a closer look at the magnitude of the reduction shows that post-treatment had a greater impact on the reduction (especially for the genes NEMO, TIRAP, and RIP). Again, the expression of infammatory genes could show the complementary therapeutic efect of our probiotic strains.

Te Results of Cytokine Production.
Te results of proinfammatory cytokine production could be seen in Figure 4. Cytokine production was signifcantly higher after treatment with SP. However, Lactobacillus/Bifdobacterium treatment signifcantly decreased cytokine production before, after, and during infammation. No signifcant diference was seen between the diferent treatment phases.

Discussion
IBD as a chronic infammatory disease has diferent phases of relapses and remissions. Patients usually present with various symptoms, including diarrhea, abdominal pain, and bloody stools, as well as the production of infammatory cytokines [17]. As mentioned above, abnormal responses of the innate and adaptive immune systems are one of the causative processes involved in IBD. Te inhibition of Treg function is a process that could occur in the pathogenesis of IBD. Treg cells are associated with anti-infammatory cytokines such as IL-10 and therefore have an inhibitory efect on other T cells including T1 and T17. Inhibition of Tregs could lead to overproduction of pro-infammatory cytokines. Te other process that may be involved in the pathogenesis of IBD is the activation of the NF-kB pathway, which is associated with IL-17, and the production of proinfammatory cytokines, including IL-6 [18]. JAK/STAT is another signaling pathway associated with pro- infammatory and anti-infammatory cytokines and therefore may play a crucial role in the pathogenesis of IBD [19]. Te use of an agent, such as probiotics, that could afect these signaling pathways might be suitable to treat or prevent the symptoms of IBD. As mentioned above, the combination of probiotics has better efects on health status compared to individual strains [6]. Terefore, we aimed to use probiotics (Lactobacillus/Bifdobacterium) before, after, and during infammation induction and evaluate the molecular efects of these strains to determine whether probiotics have antiinfammatory efects as preventive and/or therapeutic agents.
Te results of our previous in vivo study showed antiinfammatory efects of probiotics [12]. In the present study, we performed in vitro molecular studies to investigate the exact mechanisms of the anti-infammatory efect of probiotics. Te cytokine assay results were in complete agreement with the previous in vivo results. Te use of Lactobacillus/Bifdobacterium before, after, and during infammation induction was able to signifcantly decrease the production of IL-6 and IL-1β. Molecular study was also consistent with these phenotypic results. Te results of JAK/ STAT and NF-kB were remarkable. Te general trend of all genes examined in the present study after Lactobacillus/ Bifdobacterium treatment was downward. As mentioned above, the association of the JAK/STATs and NF-kB with various cytokines makes these pathways a suitable target for reducing infammation. According to Flamant et al., some pro-infammatory cytokines, including IL-6, IL-9, and IL-12, correlate with severity and disease activity in patients with UC and CD, and STAT1/STAT5, JAK1, JAK3, and TYK2 are associated with these infammatory cytokines. Terefore, any agent that targets the activation of this pathway and inhibits it could be suitable for the treatment of IBD [20].
Zundler et al. have also shown that STATs may play diferent roles in the development of IBD. For example, they demonstrated that upregulation of STAT1 is a primary defect that leads to the initiation of infammation in patients with IBD. Tey also showed that the deletion of some STATs, including STAT4, may protect patients from colitis progression. In addition, the activation of STAT6 has been shown to lead to a reduction in the induction of Tregs, and thus it plays a critical role in the development of colitis in mice [21]. Similar fndings have been reported about JAKs. Leonard et al. have been reported that JAKs are associated with various infammatory cytokines; therefore, several JAK inhibitors are suitable agents to control and treat IBD [22]. On the other hand, targeting NF-kB is also a suitable way to control IBD, as colitis is associated with the excessive activation of the NF-kB pathway [23]. Many of the therapeutic agents used in IBD, including sulfasalazine, infiximab, and anti-TNF-α, have inhibitory efects on the NF-kB pathway [24].

Conclusion
As mentioned above, there are several treatment options, including monotherapy with chemical drugs, the use of immunomodulators, or surgical treatments for severe forms of the disease [25]. Te present study demonstrated the benefcial properties of probiotics in controlling infammation in both phenotypic and molecular assays. Although phenotypic tests and molecular analysis could show the anti-infammatory efects of probiotic strains in all treatment phases, the results of molecular studies showed that the use of Lactobacillus/Bifdobacterium seems to have a stronger reduction efect after infammation induction, as it was able to reduce the expression level towards zero in (P) represents pathogen; PLB represents frst pathogen and then Lactobacillus/Bifdobacterium; LBP represents frst Lactobacillus/Bifdobacterium and then sonicated pathogen; P + LB represents sonicated pathogen and then Lactobacillus/Bifdobacterium were added simultaneously. Data were considered statistically signifcant when p < 0.05 ( * p < 0.05, * * p < 0.001). Letter (a) indicates the relatedness between C24 and C48 with other treatments, letter (b) shows the relatedness between sp24 and other treatments, and letter (c) shows the relatedness between sp48 with other treatments. Te relatedness between other treatments is shown with brackets. some genes, including STAT6, TYK2, and TIRAP. Indeed, using eight diferent probiotic strains might be a cause of such a reduction efect. Terefore, the use of probiotics to control and manage symptoms in patients with IBD, especially those in the relapsing phase and active stage of the disease, would be crucial to improve patients' lifestyle. It should be noted that our study has some potential limitations. Te use of protein assays such as western blotting (immunoblotting) and thorough evaluation of antiinfammatory cytokines along with JAK/STAT inhibitors/ agonists need to be thoroughly explored.

Data Availability
Te datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
Te experimental protocols were established following the Declaration of Helsinki and approved by the ethics committee of Pasteur Institute of Iran (IR.PII.REC.1398.060). All methods were carried out in accordance with relevant guidelines and regulations.