Lactobacilli and bifidobacteria derived from infant intestines may activate macrophages and lead to different IL-10 secretion

ABSTRACT

In this study, three strains of lactobacilli and bifidobacteria originally isolated from healthy infants, were tested for their abilities to activate RAW264.7 cells. Gene expression and cytokine production of interleukin-10 (IL-10) of RAW264.7 cells were evaluated. The activation of extracellular regulated protein kinases 1/2 (ERK1/2), p38, and nuclear factor-κB (NK-κB) were also assessed. These results suggest lactobacilli and bifidobacteria in infants may promote production of IL-10 in macrophages, conferring a protective effect in hosts suffering from inflammation. Dimerization of TLR2 and MyD88 and subsequent phosphorylation of the key downstream signaling molecules, such as MAPKs and NK-κB, may be one of the key underlying mechanisms of activation of macrophages by these microbes. Bifidobacteria and lactobacilli induced macrophages to secrete IL-10 in a different manner, which may relate to their abilities to activate key signaling pathways mediated by TLR2 and MyD88.

GRAPHICAL ABSTRACT

Lactobacilli and bifidobacteria derived from infant intestines may activate macrophages and lead to different IL-10 secretion

KEYWORDS:

• Lactobacillus
• Bifidobacterium
• macrophages
• interleukin-10
• signal pathway

Inflammatory bowel diseases (IBDs), which include two subtypes: ulcerative colitis (UC) and Crohn’s disease (CD), are the results of inflammatory caused by chronic idiopathic disorders of the gastrointestinal tract [1]. The incidence of IBDs is rapidly increasing worldwide [2], particularly in South America, Eastern Europe, Asia, and Africa [3].Recent research demonstrated that the highest incidence of CD is in Hesse, Germany (322/100000), and the lowest incidence is in São Paulo, Brazil (0.9/100000). UC had the highest incidence (505/100000) in Norway and the lowest incidence in Romania (2.42/100000) [3]. There is still no practical and effective therapy for IBDs, resulting in IBDs becoming a major public health concern.

Increased incidence of IBDs results from conditions with poor hygiene, local culture, and Westernization [4]. Although the etiology of IBDs remains unclear, emerging scientific evidence indicates that dysregulation of the mucosal immune response results from abnormal gut microbiota in genetically susceptible individuals may play a role in the pathogenesis of IBDs [5]. Studies conducted by Martinez [6] and Scanlan [7] demonstrated that the stability and biodiversity of dominant bacteria in the feces of patients with IBDs were significantly lower than those of normal people. Individuals with IBDs have also been shown to have decreased levels of Lactobacillus, Bifidobacterium, Firmicutes, and Bacteroidetes; and increased levels of Proteobacteria [8]. These changes of intestinal flora lead to the imbalance or destruction of intestinal microecology and the activation of innate and adaptive immune systems, which exacerbates the degree of chronic inflammation. In addition, increased secretion of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-12, and IL-1β, has been found to increase intestinal permeability and destroy intestinal barrier integrity, thereby increasing the severity of IBDs [9]. Therefore, maintaining the balance of intestinal microbiota and reducing chronic inflammation are crucial in the control of IBDs.

The current therapies for IBDs include amino salicylic acid treatment, administration of immunosuppressants, hormone therapy, and other biological agents. However, these conventional therapies have been reported to have negative side effects and no significant influences on the disease treatment [10]. There are many emerging therapies for IBDs, such as probiotics, therapies that block TNF or T cells, and therapies that inhibit inflammatory cell migration and adhesion [11]. Probiotics are live microorganisms that confer health benefits to the host mainly by regulating intestinal microecology and immune response. Some specific probiotic strains, mainly belong to Lactobacillus and Bifidobacterium, have been used to treat a variety of gastrointestinal diseases such as IBDs, irritable bowel syndrome (IBS), and gastritis [12]. Mennigen et al. found that the intervention of VSL#3 (consisting of several Bifidobacterium, Lactobacillus, and Streptococcus strains) can increase the secretion of anti-inflammatory factor IL-10, inhibit the production of pro-inflammatory factor IL-12 and IL-1β, thereby relieving IBDs-related symptoms [13]. Lactobacilli and bifidobacteria are the predominant intestinal microbes in early life and play an important role in the development of immune system. We have previously shown that lactobacilli and bifidobacteria derived from infant intestines have strong immunomodulatory function through interactions with macrophages [14,15], which may be one of the underlying mechanisms for their role in the promotion and regulation of immune responses in their hosts. However, these immunomodulatory effects may be strain-specific/dependent, and more scientific evidence should be obtained from well-designed strain-based studies.

IL-10 was initially named cytokine synthesis inhibitory factor (CSIF) [16], and it is primarily produced by mononuclear macrophages, T cells, B cells, Th1/2 cells, and other lymphocytes [17,18]. The major biological functions of IL-10 include inhibition of the proliferation of Th1 and NK cells; inhibition of the production of major proinflammatory cytokines, such as IL-1, IL-6, IL-12, and TNF; inhibition of the production of reactive nitrogen oxides; and stimulation of the activation of B lymphocytes [19]. Many studies have revealed the important role of IL-10 in the etiology of IBDs [20,21]. Mice were found to develop spontaneous chronic enterocolitis when IL-10 or its receptors are genetically disrupted [22]. In addition, IL-10 signaling in macrophages, which maintains the M2-like regulatory phenotype of lamina propria macrophages, is essential for maintaining mucosal immunological tolerance and preventing the development of IBDs [23,24]. A probiotic strain that can activate macrophages to produce IL-10 may have the potential to improve the quality of life of patients with IBDs, shorten the duration of symptoms, and potentially treat the disease.

In this study, lactobacilli and bifidobacteria, originally isolated from healthy infants, were tested for their abilities to activate RAW264.7 cells, a murine macrophage cell line, and their effects of producing anti-inflammatory cytokine IL-10. Lactobacilli and bifidobacteria assayed in this study were found to promote the production of IL-10 and activate the key signaling pathways in macrophages in different degrees, which may confer a potent protective effect in hosts suffering from inflammatory diseases.

Materials and methods

Cell line and culture medium

Murine macrophage cell line RAW264.7 was obtained from Jennio-bio Co., Ltd (Guangzhou, China). These cells were grown in RPMI 1640 medium (Gibco, USA), supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Millipore, USA). The cells were cultured at 37 °C with 5% CO₂. Purified insoluble peptidoglycan (PGN) from Staphylococcus aureus was purchased from Sigma, USA.

Bacterial strains and media

L. paracasei 207–27 (207–27), L. paracasei 212–27 (212–27), L. paracasei 218–27 (218–27), B. longum 199–1 (199–1), B. breve 207–1 (207–1); B. breve 208–18 (208–18) were originally isolated from healthy newborns born in Chengdu, China [25,26]. These strains were selected by pre-studies which was already performed with tolerance against acid and bile-salt, their adhesion to the intestinal mucosa. The results have not published yet. The bacteria were identified using phenotypic and genetic methods and deposited in The Microorganism Collection of the West China School of Public Health, Sichuan University.

Lactobacilli strains were cultured for 48 h at 37 °C in de Man Rogosa Sharpe broth (Land Bridge, China). Bifidobacterium strains were cultured under the same conditions in TOS propionate agar medium (Eiken, Japan). Both lactobacilli and bifidobacteria were collected by centrifugation at 1000 × g for 10 min and were resuspended in phosphate-buffered saline (Hyclone, USA), and the concentration of bacteria was adjusted to 1.0 × 10⁹ CFU/mL.

Co-culture of strains and macrophages

The strains were co-cultured with macrophages, with PGN and serum-free 1640 RPMI medium as a positive and negative control respectively, for 24 h. The supernatants of the RAW264.7 cell culture were then collected by centrifugation at 13,000 rpm for 10 min and stored at −80 °C until use. We also harvested RAW264.7 cells to isolate total RNA. The bacteria were co-cultured with RAW264.7 for 5 min, 1 h, or 12 h, to collect the total protein. Another group was co-cultured for 24 h to collect the cells and the supernatant.

Cell viability assay

RAW264.7 cells were seeded in 96-well plates at a concentration of 1 × 10⁶ cells/mL following the addition of either Lactobacillus or Bifidobacterium. After co-culturing for 24 h, the cell viability was measured using the WST-8 assay (Cell Counting Kit-8, Dojindo). A 10 mL of WST-8 solution was added per well and the absorbance was measured at 450 nm after 2 h of incubation in a humidified incubator.

Expression of IL-10 mRNA in macrophages

5 × 10⁵ RAW264.7 cells were cultured in 24-well culture plates and treated with 1 × 10⁷ cells of one of three strains of Lactobacilli, 207–27, 212–27, and 218–27, or three strains of Bifidobacteria,199–1, 207–1, and 208–18, as well as with PGN (20 ng/mL) or serum-free RPMI 1640 medium. 24 h after treatment, the total RNA of RAW264.7 cells was extracted using the GENOUT^TM TRIzol RNA Isolation Kit (LABGENE, China) and was reverse-transcribed using the iScript^TM cDNA Synthesis Kit (Bio-Rad, USA). Quantitative real-time polymerase chain reaction (qPCR) was performed using SsoFast^TM EvaGreen® Supermix (Bio-Rad) to quantify IL-10 gene expression using the CFX96 Touch^TM (Bio-Rad). The β-actin gene was used as an endogenous control to normalize expression. This assay was carried out using primers for IL-10 (sense 5′-GACCAGCTGGACAACATACT-3′; antisense 5′-GAGGGTCTTCAGCTTCTCAC-3′), and β-actin (sense 5′-GTGGGCCGCTCTAGGCACCAA-3′; antisense 5′-CTCTTTGATGTCACGCACGATTTC-3′). After a hot start, for IL-10, the amplification profile included 1 cycle of 30 s enzyme activation at 98 °C, and 40 cycles of 5 s denaturation at 98 °C and 5 s annealing at 57.7 °C. For β-actin, the profile included 1 cycle of 30 s at 98 °C, and 40 cycles of 5 s at 98 °C and 5 s at 64.5 °C.

IL-10 determination using ELISA

The IL-10 concentration of all cell culture supernatants was detected using quantitative sandwich ELISA (Quantikine® ELISA, R&D, USA). The results from the cytokine analyses are expressed as the average of three independent experiments. Each experiment was performed with three replicates to account for intra-assay variation.

Western blotting

The RAW264.7 cell line was cultured in a 12-well culture plate with 5 × 10⁵ cells per well overnight. Bacterial strains, PGN, RPMI 1640 or gram – negative bacteria cell wall Lipopolysaccharide (LPS, Hyclone, USA) (20 ng/mL) was added to co-culture with the cells for 5 min, 1 h, or 12 h. Cells were then lysed on ice in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, sodium pyrophosphate, β-glycerophosphate, Na₃VO₄, leupeptin).We centrifuged cell lysates at 13,000 × g for 10 min to remove the insoluble debris and stored them at −80°C until use. Total protein concentrations were determined using the bicinchoninic acid assay method (Beyotime, China). Sample Loading Buffer (0.02% bromophenol blue) was added, and samples were boiled at 100°C for 5 min, separated by SDS-PAGE, and then transferred onto a polyvinylidene difluoride membrane (Millipore). After blocking for 1 h with 5% BSA in PBST, blots were incubated with rabbit anti-phospho-p44/p42 MAPK (Cell Signaling Technology, USA), rabbit anti-phospho-p38 (Cell Signaling Technology), rabbit anti-phospho-transcription factor of the nuclear factor κB (Cell Signaling Technology), rabbit anti-MyD88 (Cell Signaling Technology), mouse-anti-p44/p42 MAPK (Santa, USA), mouse-anti-p38 (Santa), and rabbit anti-NF-κB (Cell Signaling Technology) and incubated at 4 °C overnight. Blots were incubated with secondary HRP-conjugated anti-rabbit immunoglobulin-G (IgG) antibody and anti-mouse IgG antibody for 1 h to detect binding. Target protein was visualized by Molecular Image® ChemiDoc^TM XRS+ (Bio-Rad).

Blocking toll-like receptor 2

C29 (MedChemExpress, USA) is a TLR2 inhibitor that prevents ligand-induced interaction of TLR2 with MyD88 and blocks MAPKs and NF-κB activation. C29 was added into the culture wells (to a final concentration of 50 ng/mL) for TLR2 blocking for 1 h at 37 °C. The bacterial strains, PGN, or RPMI 1640 was added into each well for an extra 30 min at 37 °C before the western blotting experiment. Another group was incubated for 24 h at 37 °C for RT-PCR and ELISA.

Statistical analysis

Statistical analysis was performed using SPSS 22.0 software. The results are presented as the mean ± standard deviation. The differences among groups were performed using Student’s t-test analyses and Dunnett’s test analyses. Statistical significance was considered P < 0.05.

Results

Growth of RAW264.7 cells after stimulated with lactobacilli and bifidobacteria

After 24 h co-culturing, RAW264.7 cells had no differences in growth between the tested group and non-treated control, and the viability of cells approached 100% (Figure 1).

Figure 1. Effect of lactobacilli and bifidobacteria strain on cells after 24 h. We set the density of RAW264.7 at 1 × 10⁶ and the final concentration of the strains were 10⁷ CFU/mL. There is no significant difference between any two groups

IL-10 mRNA expression by RAW264.7 after stimulation with Lactobacilli and Bifidobacteria

The tested bifidobacteria activated RAW264.7 cells to show stronger IL-10 mRNA expression, while did less the tested lactobacilli (Figure 2). B. breve 208–18 and B. longum 199–1 stimulated RAW264.7 cells to significantly express IL-10 mRNA to a greater extent than PGN (P < 0.05). B. breve 207–1 stimulated RAW264.7 cells to express more IL-10 than the control (P < 0.05). The tested lactobacilli indicated the expression of IL-10 mRNA less than did PGN and no statistically significant differences between them and control (Figure 2).

Figure 2. IL-10 gene relative expression in RAW264.7 by stimulated by lactobacilli and bifidobacteria strains for 24 h. (*) indicate statistically significant differences between experimental group and PGN (20 ng/mL); (#) indicate statistically significant differences between experimental group and serum-free RPMI 1640 medium

IL-10 secretion by RAW264.7 after stimulation with Lactobacilli and Bifidobacteria

Bifidobacteria and lactobacilli induced the secretion of IL-10 from RAW264.7 cells (Figure 3). Both the results were statistically different (P < 0.05, P < 0.05) from the control. The bifidobacteria demonstrated a greater ability to stimulate the secretion of IL-10 than the lactobacilli. The bifidobacteria induced secretion of IL-10 significantly more than PGN treatment (P < 0.05), while lactobacilli resulted in less secretion of IL-10 than PGN treatment (P < 0.05; Figure 3).

Figure 3. Bifidobacteria and lactobacilli can increase the IL-10 secretion by stimulating macrophage RAW264.7. (*) indicate statistically significant differences between experimental group and PGN (20 ng/mL); (#) indicate statistically significant differences between experimental group and serum-free RPMI 1640 medium

Activation of multiple signaling pathways by Lactobacilli and Bifidobacteria

LPS activated the IL-10 secreting signaling pathways, while PGN had a reduced effect as compared to LPS. B. breve 207–1 activated ERK1/2, NF-κB, and p38 (Figure 4(a)). ERK1/2 and p38 were activated maximally when co-cultured for 1 h. However, NF-κB was activated maximally when the early time. L. paracasei 207–27 only activated ERK1/2 (Figure 4(b)).

Figure 4. Bifidobacteria and lactobacilli can activate TLR2-MyD88-mediated multiple signal pathway which may mediated secreting of IL-10.(a)Macrophages stimulated by B. breve 207–1 total protein was tested by western blotting, analyzed using rabbit anti-phospho-p44/p42 MAPK, rabbit anti-phospho-p38, rabbit anti-phospho-Transcription factor of the nuclear factor κB, rabbit anti-MyD88,mouse-anti-p44/p42 MAPK, mouse-anti-p38, rabbit anti-NF-κB (b) Macrophages stimulated by L.paracasei 207–27 total protein was tested by western blotting and analyzed using the same antibody

Secretion of IL-10 via TLR2-MyD88-mediated multiple signal pathway stimulated by bifidobacteria and lactobacilli

TLR2 inhibitor C29 was added into the co-culture system and the secretion of IL-10 and activation of related multiple signaling pathways were also tested (Figures 5-7). After C29 was added, the secretion of IL-10 from RAW264.7 significantly declined, both in gene expression (Figure 5) and secretion (Figure 6) in the presence of the bifidobacteria and lactobacilli. C29 significantly reduced the activation of ERK1/2 and NF-κB signals as measured by western blotting, when stimulated by B. breve 207–1, which it reduces less the activation of these signals stimulating by L. paracasei 207–27 (Figure 7).

Figure 5. After C29 treated and stimulated by B. breve 207–1, L. paracasei 207–27 IL-10 mRNA relative expression was tested. (*) indicated statistically significant differences between two groups

Figure 6. After C29 treated and stimulating by B. breve 207–1, L. paracasei 207–27 IL-10 secretion was measured by ELISA. (*) indicated statistically significant differences between two groups

Figure 7. After C29 treated and stimulating by B. breve 207–1, L. paracasei 207–27, TLR2-MyD88-mediated multiple signal pathway which regulate the secretion of IL-10

Discussion

Macrophages are known to play an essential role in the regulation of host immunity. Macrophages have the ability to present antigens to specific immune cells and take up foreign substances, and to also differentiate into the M1 or M2 phenotype, due to stimulation by a variety of environmental factors. The M1 or M2 phenotype is characterized based on the cytokines that are secreted. The secretion of high levels of the proinflammatory cytokines IL-12 and TNF-α is conventionally considered to be a feature of M1-type of macrophages, which are primarily involved in antibacterial functions and phagocytosis. Secretion of high levels of IL-10 is a feature of M2-type macrophages, which mainly play a role in anti-inflammatory processes and repair of damaged tissues [27]. Recent studies have indicated that enhancement of M2 macrophages may be a beneficial strategy in the management of various physiological disorders and noninfectious diseases, which are characterized by systemic and local inflammation. These include diseases such as obesity, IBS, IBD, diabetes, and allergic diseases [28].

Bifidobacteria and lactobacilli are the predominant microbes in the intestinal microbiota in humans and particularly in infants. Emerging scientific evidence from various studies has indicated that these intestinal microbes may play an important role in the development and maturation of immunity. Lactobacilli and bifidobacteria derived from infant intestines and breast milk are considered to have more specific health-promoting effects on human body, which makes them as ideal conditant for developing new potential probiotics [29,30]. In our previous studies, bifidobacteria derived from healthy infant intestines have been shown to interact with macrophages in different manners; bacterial strains that taxonomically belonging to B. breve, B. bifidum, and B. infantis, also called infant-type bifidobacteria, potentially induced IL-10 to a greater extent than bifidobacteria derived from adults, from the taxonomic group B. adolecentis [15,31]. Recently, lactobacilli, which are known to induce IL-12, a proinflammatory cytokine, have been found to also induce IL-10 [14,28,32]. However, the further studies are still needed to know more interaction between these bacteria and macrophages to use them more effectively.

In this study, each of the tested lactobacilli and bifidobacteria originally isolated from infant intestines significantly activated macrophages to induce IL-10 expression and secretion in a strain-dependent manner. These results agree with our previous studies, in which infant bifidobacterial and lactobacilli stimulated macrophages to secret anti- proinflammatory cytokine IL-10 in vitro, although a different macrophage cell line was used [14,15]. The results from the present study also show that bifidobacteria activated macrophages to produce IL-10 to a greater extent than lactobacilli, even though both strains were derived from infants. This has not been found in our previous studies [14,15] and other similar studies in human primary macrophages, human macrophage cell line or mice dendritic cells [33–35]. Instead, the co-culture of peripheral blood mononuclear cells and Bifidobacterium and Lactobacillus strains, which were originated from the pharmaceutical probiotic VSL#3, a probiotic product known to relieve IBDs, indicated that the cell debris of Bifidobacterium strains induced more IL-10 than Lactobacillus cell debris [36]; but the underlying mechanism is still unclear. We then further explored the differences between two bacteria in activating IL-10 related signaling pathways.

TLR2 signaling is crucial for the induction of IL-10 production by macrophages stimulated by gram-positive bacteria. TLR signaling through MyD88 leads to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). The MAPK cascade composes of three major pathways including extracellular signal-regulated kinases (ERKs), which are comprised of ERK1 and ERK2, and p38 [37]. In the present study, a strain of bifidobacteria (B. breve 207–1) and a strain of lactobacilli (L. paracasei 207–27) with the strongest IL-10 inducing ability among the tested bifidobacteria and the test lactobacilli, were selected to test and compare their ability in activating TLR2-related signaling pathways.

The tested B. breve 207–1 strain led to greater levels of phosphorylation of p38, ERK1/2, and NF-κB than the L. paracasei 207–27 strain. After exposure to the B. breve 207–1 strain, p38, ERK1/2, and NF-κB were highly phosphorylated, although the degree of activation caused by the tested bifidobacteria increased and decreased with time. While L. paracasei 207–27 strain only stimulated the phosphorylation of ERK1/2. These results suggest that L. paracasei 207–27 and B. breve 207–1 could interact with macrophages in a different manner. Recent study by Baik et al. indicated that even subtle alterations in the structural molecules of PGN in gram-positive bacteria can cause different biological functions to the host, possibly by binding to diverse functional proteins in gastrointestinal tract, such as proteins related to cytoskeleton, gene expression, microbial adhesion, and mucosal integrity [38]. In this study, different PGN structures in the cell walls of lactobacilli and bifidobacteria may be one of reasons why they showed different IL-10 inducing abilities and induced the phosphorylation of TLR2-mediated intercellular signaling pathway to different degrees.

MyD88 is the key molecule in TLR signaling pathways. When the extracellular leucine-rich repeats (LRRs) region of TLR binds a ligand, the intracellular toll/IL-1 receptor domain (TIR) of TLR becomes allosteric to recruit the ligand protein family. After combining the c-terminal region of MyD88 with TIR, the amino (N)-terminal death domain (DD) region of MyD88 becomes allosteric to recruit serine/threonine protein kinases, which contain a DD region, causing the phosphorylation of IL-1 R-associated kinases (IRAKs). After the formation of the MyD88- IRAKs- TRAF-6 complex through recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF-6), TGF-β-activated kinase 1/TAK1-binding protein 1 (TAK1/TAB) and the inhibitor of nuclear factor-κB-kinase complex (IKKs) will be activated. If subsequent signal conduction relies on an NF-κB transcription-dependent pathway, TAK1 can activate NF-κB-inducing kinase (NIK). The activation of NIK leads to activation of IKKα and IKKβ, resulting in phosphorylation of IκB. This results in gene expression due to the release of p50 and p65 subunits of NF-κB. However, in the NF-κB transcription non-dependent pathway, TAK1 will first activate mitogen-activated protein kinase kinase 3 (MKK3), phosphorylate p38, MAPKs to allow p50 and p65 of NF-κB to enter the nucleus resulting in gene expression [37,39,40]. The dimerization of TLR2 and MyD88 and the phosphorylation of p38, MAPKs, and NF-κB are crucial to this signaling process.

In this study, C29 was used to prevent dimerization of TLR2 and MyD88 so that the transmission of TLR-2-mediated intercellular downstream signaling could be blocked. In the presence of C29, the phosphorylation of p38, ERK1/2, and NF-κB in macrophages exposed to the bacteria significantly decreased. In addition, mRNA expression and protein levels of IL-10 were also suppressed. These results indicate that lactobacilli and bifidobacteria can stimulate macrophages to secret IL-10 through dimerization of TLR2 and MyD88 and activation of MAPKs and nuclear factor-κB (NF-κB). IL-10 was also slightly detected even in the presence of C29, suggesting that components of the bacteria, such as DNA or RNA, may activate IL-10 secretion in the macrophages using a different underlying mechanism [41]. For example, this activation could be combined with TLR9 to activate the expression of other target genes, resulting in gene expression [42]. Future work is needed to profile the activation of other TLRs and the downstream signaling pathways, as well as to identify the key component that leads to the differences in activation of macrophages between bifidobacteria and lactobacilli.

IL-10 and related biological agents can be used in the treatment of many autoimmune diseases, psoriasis, IBDs, diabetes, and rheumatoid arthritis. Research [43] shows that IL-10 can effectively prevent the secretion of inflammatory mediators, thus improving arthritis. Goudy [44] found that direct intramuscular injection of a recombinant adeno-associated virus (AAV) vector containing mouse IL-10 cDNA was protective against type 1 diabetes in non-obese diabetic mice. Yang [45] transfected the Epstein-Barr virus gene group with AAV vector in an open reading frame, the BCRF-1 gene, which has a high homology with mouse IL-10, to obtain sustained expression of vIL-10 in vivo. This can also significantly reduce the incidence of pancreatitis and prevent the occurrence of diabetes. Its protective effect is related to sustained gene expression and protein production. A clinical trial [46] showed that using IL-10 treatment can improve quality of life in patients with IBD. Use of probiotics has also been shown to enhance anti-inflammatory effects and improve the symptoms associated with IBDs [47–49]. The present study identified that lactobacilli and bifidobacteria isolated from healthy infants may have the potential to be probiotics, which can be used in the treatment of inflammatory diseases. Further studies will be conducted to investigate the anti-inflammatory effects of these bacterial strains in vivo using an IBD mouse model.

Conclusions

In conclusion, lactobacilli and bifidobacteria isolated from intestines of healthy infants in this study can stimulate macrophages to produce IL-10. The underlying mechanism may include the dimerization of TLR2 and MyD88, resulting in the activation of the MAPK cascade and NF-κB. Development of a better understanding of the molecular targets of inflammatory diseases, including investigation of the anti-inflammatory mechanisms of probiotics, may be essential for the development of treatments for IBDs. The present study provides a theoretical basis for the prevention of inflammatory diseases using probiotics.

Author contributions

Fang He, Qiwei Chen, Zihao Luo, Jiehua Chen, Xiaolei Ze and Xuguang Zhang participated in the design of this study. Zihao Luo and Huijing Liang conducted the experiment. Zihao Luo, Huijing Liang and Zhonghua Miao analyzed the experimental data. Fang He, Qiwei Chen, Ming Li, Xi Shen, Huijing Liang and Zihao Luo revised the manuscript.

Acknowledgments

This study was supported by By-Health Nutrition Science Research Fund of Chinese Nutrition Society (CNS). We also appreciate the support of Public health and Preventive Medicine Provincial Experiment Teaching Center at Sichuan University and Food Safety Monitoring and Risk Assessment Key Laboratory of Sichuan Province.

Disclosure statement

No potential conflict of interest was reported by the authors.