CCAAT/enhancer-binding protein α-dependent regulation of granule formation in mast cells by intestinal bacteria.

The antiallergic effects of gut microbiota have been attracting attention in recent years, but the underlying cellular and molecular mechanisms have not yet been fully understood. In this study, we aimed to investigate these mechanisms specifically focusing on mast cells. Mast cells retain intracellular granules containing various inflammatory mediators such as histamine, which are released outside the cells upon IgE and allergen stimulation. We previously reported that increased expression of the transcription factor, CCAAT/enhancer-binding protein α (C/EBPα), suppresses granule formation in mast cells and that Lacticaseibacillus casei JCM1134T (LC) upregulates C/EBPα levels. Here, granule formation in mouse bone marrow-derived mast cells was suppressed in a MyD88-dependent manner after LC treatment due to C/EBPα-dependent downregulation of the genes encoding serglycin (SRGN) and mast cell protease 4 (Mcpt4). Furthermore, C/EBPα expression was regulated by DNA methylation in the 5' region far upstream of the transcription start site. LC suppressed DNA methylation of specific CpG motifs in the 5' region of the C/EBPα gene. These results conclude that specific gut microbial components, such as those from LC, suppress granule formation in mast cells by inhibiting SRGN and Mcpt4 expression via reduced C/EBPα gene methylation.

Lactobacillus casei displaying MCP2α and FlaC delivered by PLA microspheres effectively enhances the immune protection of largemouth bass (Micropterus salmoides) against LMBV infection.

Largemouth bass ranavirus (LMBV) seriously affects the development of largemouth bass (Micropterus salmoides) industry and causes huge economic losses. Oral vaccine can be a promising method for viral disease precaution. In this study, MCP2α was identified as a valuable epitope region superior to MCP and MCP2 of LMBV by neutralizing antibody experiments. Then, recombinant Lactobacillus casei expressing the fusion protein MCP2αC (MCP2α as antigen, C represents flagellin C from Aeromonas hydrophila as adjuvant) on surface was constructed and verified. Further, PLA microsphere vaccine loading recombinant MCP2αC L. casei was prepared. The PLA microspheres vaccine were observed by scanning electron microscopy and showed a smooth, regular spherical surface with a particle size distribution between 100 and 200 µm. Furthermore, we evaluated the tolerance of PLA-MCP2αC vaccine in simulated gastric fluid and simulated intestinal fluid, and the results showed that PLA-MCP2αC can effectively resist the gastrointestinal environment. Moreover, the protective effect of PLA-MCP2αC against LMBV was evaluated after oral immunization and LMBV challenge. The results showed that PLA-MCP2αC effectively up-regulated the activity of serum biochemical enzymes (T-SOD, T-AOC, LZM, complement C3) and induced the mRNA expression of representative immune genes (IL-1ß, TNF-α, IFN-γ, MHC-IIα, Mx, IgM) in spleen and head kidney tissues. The survival rate of largemouth bass vaccinated with PLA-MCP2αC increased from 24 % to 68 %. Meanwhile, PLA-MCP2αC inhibited the LMBV burden in spleen, head kidney and liver tissues and attenuated tissue damage in spleen. These results suggested that PLA-MCP2αC can be used as a candidate oral vaccine against LMBV infection in aquaculture.

The Phagocytosis of Lacticaseibacillus casei and Its Immunomodulatory Properties on Human Monocyte-Derived Dendritic Cells Depend on the Expression of Lc-p75, a Bacterial Peptidoglycan Hydrolase.

The human gut symbiont Lacticaseibacillus (L.) casei (previously Lactobacillus casei) is under intense research due to its wide range of immunomodulatory effects on the human host. Dendritic cells (DCs) are crucial players in the direct and indirect communication with lactobacilli in the gastrointestinal tract. Here, we demonstrate that human monocyte-derived DCs (moDCs) are able to engulf L. casei BL23, in which the intact bacterial cell wall and morphology have a key role. The absence of the bacterial cell-wall-degrading enzyme, Lc-p75, in L. casei cells causes remarkable morphological changes, which have important consequences in the phagocytosis of L. casei by moDCs. Our results showed that the Lc-p75 mutation induced defective internalization and impaired proinflammatory and T-cell-polarizing cytokine secretion by bacteria-exposed moDCs. The T helper (Th) 1 and Th17 cell activating capacity of moDCs induced by the mutant L. casei was consequently reduced. Moreover, inhibition of the phagocytosis of wild-type bacteria showed similar results. Taken together, these data suggested that formation of short bacterial chains helps to exert the potent immunomodulatory properties of L. casei BL23.

Construction and immune efficacy of recombinant Lactobacillus casei strains expressing Malt from Aeromonas veronii.

Aeromonas veronii is an important zoonotic pathogen that causes significant economic losses in the aquaculture industry. The use of probiotics in aquaculture is a practical alternative to antibiotics to promote animal health and aid in disease prevention. In the present study, we aimed to construct a recombinant Lactobacillus casei(surface-displayed or secretory) strain containing Malt from A. veronii TH0426 and assess its potential as an oral vaccine. A 1314-bp Malt gene fragment was successfully amplified and cloned into a prokaryotic protein expression system. Protein expression in resulting recombinant strains Lc-MCS-Malt (surface-displayed) and Lc-pPG-Malt (secretory) was then verified by Western blotting and indirect immunofluorescence. A single band was observed on the Western blots, with the molecular weight of the corresponding protein shown to be 48 kDa. Furthermore, a fluorescent signal for Lc-MCS-Malt was observed by fluorescence microscopy. At 0, 7, 16, 25, and 34 days post-immunization, tissue and blood samples were collected from common carp orally administered with the recombinant L. casei strains for immune-related index analyses. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-1ß, TNF-α, and IFN-γ genes in the group immunized with recombinant L. casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Results also showed that recombinant L. casei could stimulate the mucosa through colonization of the intestine, resulting in increased transcription of IL-10, IL-1ß, TNF-α, and IFN-γ. Immunity and colonization assays also showed that after 34 days of fasting, recombinant L. casei were still present in the intestines of the immunized fish. Common carp that received Lc-MCS-Malt(53.3%) and Lc-pPG-Malt (46.7%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our findings suggested that recombinant L. casei can adequately protect fish and improve immunity, providing a theoretical basis for the future development of an oral Lactobacillus vaccine for use in aquaculture.

Immunogenicity evaluation of recombinant Lactobacillus casei W56 expressing bovine viral diarrhea virus E2 protein in conjunction with cholera toxin B subunit as an adjuvant.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored. RESULTS: Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer's patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals. CONCLUSIONS: The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.

Effects of recombinant Lactobacillus casei on growth performance, immune response and disease resistance in crucian carp, Carassius auratus.

In the present study, we constructed two recombinant Lactobacillus casei (L. casei) Lc-pPG-1-AcrV (surface-displayed) and Lc-pPG-2-AcrV (secretory) constitutively expressing AcrV protein of Aeromonas veronii (A. veronii). Expression of recombinant AcrV protein was verified by western blot and immunofluorescence technique. Compared with PBS group, the final weight (FW), weight gain (WG) and specific growth rate (SGR) of fish fed Lc-pPG-1-AcrV, Lc-pPG-2-AcrV and Lc-pPG diets after 56 days observed significantly increase (p < 0.05), while the feed conversion ratio (FCR) showed a significantly decrease (p < 0.05). The recombinant L. casei strains were orally administrated to crucian carp, and significant increased (p < 0.05) the immunoglobulin M (IgM), elevated the acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM) and superoxide dismutase (SOD) activity in serum. Moreover, leukocytes phagocytosis percentage and index of the recombinant L. casei were both enhanced. The results demonstrated that the recombinant L. casei could elicit systemic immune responses and increase the serum immunological index. The Interleukin-10 (IL-10), Interleukin-1ß (IL-1ß), interferon-γ (IFN-γ) and Tumor Necrosis Factor-α (TNF-α) levels in liver, spleen, kidney and intestine have up regulated significantly in tissues (p < 0.05), suggesting that the recombinant L. casei has the ability to induce expression of cytokines and enhance the innate immune response. Higher survival rates were exhibited that crucian carp immunized with Lc-pPG-1-AcrV (67.5%) and Lc-pPG-2-AcrV (52.5%) after challenge with A. veronii. In conclusion, these two recombinant L. casei vaccine were effective in improving crucian carp growth, immunity response and disease resistance. The recombinant L. casei strains may be a promising candidate for the development of an oral vaccine against A. veronii.

Oral immunization with recombinant Lactobacillus casei expressing flaB confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio.

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-ß, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora.

Expression of the alpha toxin of Clostridium perfringens in Lactobacillus casei genome and evaluation of its immune effects in mice.

We previously developed a stable and marker-free Lactobacillus casei strain (PPαT Δupp) that contained a chromosomally integrated expression cassette (PPαT) that enabled the surface expression of the Clostridium perfringens alpha toxin. To measure immune responses against the alpha toxin, specific-pathogen-free BALB/c mice were inoculated with L. casei PPαT Δupp by oral gavage. Then, specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM). The results showed that alpha toxin-specific IgA and IgG antibodies and cytokines were markedly increased following immunization. Natural alpha toxin challenge and neutralization tests were performed. The results showed that immunized mice can fully resist 1.5 minimum lethal doses of toxin. These results indicated that the immunized mice can produce not only humoral immunity, but also cellular immunity. These results provide a new pathway for the development of a safe, effective, and food-grade vaccine.

Oral immunization with recombinant Lactobacillus casei expressing OmpAI confers protection against Aeromonas veronii challenge in common carp, Cyprinus carpio.

Aeromonas veronii is a gram-negative pathogen capable of infecting both fish and mammals, including humans, and natural infection in fish results in irreparable damage to the aquaculture industry. Lactic acid bacteria (LAB) have a number of properties that make them attractive candidates as delivery vehicles for presentation to the mucosa sites of compounds with pharmaceutical interest, in particular vaccines. In this study, we generated two recombinant Lactobacillus casei (surface-displayed or secretory) expressing the OmpAI of A.veronii and evaluated the effect on immune responses in fish model. A 1022 bp gene fragment of the 42 kDa OmpAI antigen of A.veronii was cloned into pPG-1 (surface-displayed) and pPG-2 (secretory) and electrotransformed into Lactobacillus casei CC16. The recombinant plasmid in L.casei could be stably inherited over 50 generations, and production of OmpAI protein had slight limited effects on cells growth. Treatment of common carp with the recombinant vaccine candidate stimulated high serum or skin mucus specific antibody titers and induced a higher lysozyme, ACP, SOD activity, while fish fed with Lc-pPG or PBS had no detectable immobilizing immune responses. Expression of IL-10, IL-ß, IFN-γ, TNF-α genes in the group immunized with recombinant L.casei were significantly (P < 0.05) up regulated as compared with control groups, indicating that inflammatory response and cell immune response were triggered. Further, viable recombinant L.casei strains were directly delivered and survive throughout the intestinal tract, the recombinant OmpAI was also detected in intestine mucosal. The results showed that common carp received Lc-pPG1-OmpAI (66.7%) and Lc-pPG2-OmpAI (50.0%) had higher survival rates compared with the controls after challenge with A.veronii, indicating that Lc-pPG1-OmpAI and Lc-pPG2-OmpAI had beneficial effects on immune response and enhanced disease resistance of common carp against A.veronii infection. Our study here demonstrates, for the first time, the ability of recombinant L.casei as oral vaccine against A.veronii infection in carps. The combination of OmpAI delivery and LAB approach may be a promising mucosal therapeutic agent for treating and controlling A.veronii.

Effects of Lactobacillus casei and Enterococcus faecalis on growth performance, immune function and gut microbiota of suckling piglets.

This study was carried out to investigate the effects of orally administrated Lactobacillus casei and Enterococcus faecalis on performance, immune function and gut microbiota of suckling piglets. Neonatal piglets (n = 120) were randomly assigned to 4 groups, with 30 suckling piglets in each group. The piglets were from 15 litters, one male and one female piglet were selected for each group in each litter. The Control group was administrated with normal saline, the other groups with L. casei or E. faecalis or a combination of L. casei and E. faecalis at a ratio of 3:1. Each piglet was orally administrated with 1, 2, 3 and 4 ml probiotics or normal saline at the age of 1, 7, 14 and 21 d, respectively. The piglets were weaned at the age of 21 d. The results showed that compared with the Control group, the average daily gain of piglets administrated with probiotics was significantly increased, and the diarrhoea rate and mortality were significantly decreased (p < 0.05). After supplementation of the combined probiotics, the protease activity in stomach, duodenum and colon was increased and in all supplemented groups, the immunoglobulin A concentration in plasma was significantly higher (p < 0.05). The combined probiotics significantly increased villus length and the expression level of transforming growth factor-ß in the jejunum (p < 0.05) but decreased the expression level of the jejunal tumour necrosis factor-α (p < 0.05). In addition, probiotics could regulate gut microbiota and increase microbial similarity coefficients for keeping piglet gut microbiota stable.

Chemical characterization and immunomodulatory properties of polysaccharides isolated from probiotic Lactobacillus casei LOCK 0919.

The Lactobacillus casei strain, LOCK 0919, is intended for the dietary management of food allergies and atopic dermatitis (LATOPIC® BIOMED). The use of a probiotic to modulate immune responses is an interesting strategy for solving imbalance problems of gut microflora that may lead to various disorders. However, the exact bacterial signaling mechanisms underlying such modulations are still far from being understood. Here, we investigated variations in the chemical compositions and immunomodulatory properties of the polysaccharides (PS), L919/A and L919/B, which are produced by L. casei LOCK 0919. By virtue of their chemical features, such PS can modulate the immune responses to third-party antigens. Our results revealed that L919/A and L919/B could both modulate the immune response to Lactobacillus planatarum WCFS1, but only L919/B could alter the response of THP-1 cells (in terms of tumor necrosis factor alpha production) to L. planatarum WCFS1 and Escherichia coli Nissle 1917. The comprehensive immunochemical characterization is crucial for the understanding of the biological function as well as of the bacteria-host and bacteria-bacteria cross-talk.

Immune Response of Healthy Adults to the Ingested Probiotic Lactobacillus casei Shirota.

Daily ingestion of a probiotic drink containing Lactobacillus casei Shirota (LcS; 1.3 × 1010 live cells) by healthy adults for (1) 4-week LcS, (2) 6-week discontinuation of LcS and (3) a final 4 weeks of LcS was investigated. There was a significant increase in expression of the T cell activation marker CD3+ CD69+ in ex vivo unstimulated blood cells at weeks 10 and 14, and there was a significant increase in the NK cell marker CD3+ CD16/56+ in ex vivo unstimulated blood cells at weeks 4, 10 and 14. Expression of the NK cell activation marker CD16/56+ CD69+ in ex vivo unstimulated blood cells was 62% higher at week 10 and 74% higher at week 14. Intracellular staining of IL-4 in ex vivo unstimulated and PMA-/ionomycin-stimulated CD3+ ß7+ integrin blood cells was significantly lower at weeks 10 and 14. Intracellular staining of IL-12 in ex vivo unstimulated and LPS-stimulated CD14+ blood cells was significantly lower at weeks 4, 10 and 14. Intracellular staining of TNF-α in LPS-stimulated CD14+ blood cells was significantly lower at weeks 4, 10 and 14. Mucosal salivary IFN-γ, IgA1 and IgA2 concentrations were significantly higher at week 14, but LcS did not affect systemic circulating influenza A-specific IgA or IgG and tetanus-specific IgG antibody levels. In addition to the decrease in CD3+ ß7+ integrin cell IL-4 and a reduced CD14+ cell pro-inflammatory cytokine profile, at week 14 increased expression of activation markers on circulating T cells and NK cells and higher mucosal salivary IgA1 and IgA2 concentration indicated a secondary boosting effect of LcS.

A phase trial of the oral Lactobacillus casei vaccine polarizes Th2 cell immunity against transmissible gastroenteritis coronavirus infection.

Transmissible gastroenteritis coronavirus (TGEV) is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. TGEV is an enteropathogenic coronavirus that causes highly fatal acute diarrhoea in newborn pigs. An oral Lactobacillus casei (L. casei) vaccine against anti-transmissible gastroenteritis virus developed in our laboratory was used to study mucosal immune responses. In this L. casei vaccine, repetitive peptides expressed by L. casei (specifically the MDP and tuftsin fusion protein (MT)) were repeated 20 times and the D antigenic site of the TGEV spike (S) protein was repeated 6 times. Immunization with recombinant Lactobacillus is crucial for investigations of the effect of immunization, such as the first immunization time and dose. The first immunization is more important than the last immunization in the series. The recombinant Lactobacillus elicited specific systemic and mucosal immune responses. Recombinant L. casei had a strong potentiating effect on the cellular immunity induced by the oral L. casei vaccine. However, during TGEV infection, the systemic and local immune responses switched from Th1 to Th2-based immune responses. The systemic humoral immune response was stronger than the cellular immune response after TGEV infection. We found that the recombinant Lactobacillus stimulated IL-17 expression in both the systemic and mucosal immune responses against TGEV infection. Furthermore, the Lactobacillus vaccine stimulated an anti-TGEV infection Th17 pathway. The histopathological examination showed tremendous potential for recombinant Lactobacillus to enable rapid and effective treatment for TGEV with an intestinal tropism in piglets. The TGEV immune protection was primarily dependent on mucosal immunity.

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus.

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus casei ATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L. casei ATCC334 grown in either biofilm or planktonic cultures. TNF-α production was suppressed and the NF-κB pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF-α were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect.

An extensively hydrolysed casein-based formula for infants with cows' milk protein allergy: tolerance/hypo-allergenicity and growth catch-up.

Children with cows' milk protein allergy (CMPA) are at risk of insufficient length and weight gain, and the nutritional efficacy of hypo-allergenic formulas should be carefully assessed. In 2008, a trial assessed the impact of probiotic supplementation of an extensively hydrolysed casein-based formula (eHCF) on acquisition of tolerance in 119 infants with CMPA. First analysis of the study results showed that the studied formula allowed improvement of food-related symptoms. The scoring of atopic dermatitis (SCORAD) index was assessed at randomisation and after 6 months of feeding. A post hoc analysis was performed using WHO growth software's nutritional survey module (WHO Anthro version 3.2.2). All infants who were fed the study formula tolerated it well. The SCORAD index significantly improved from randomisation to 6 months of feeding with the study formula. Anthropometric data indicated a significant improvement in the weight-for-age, length-for-age and weight-for-length z scores, as well as in the restoration of normal BMI. The probiotic supplementation did not show any impact on these parameters. The present data showed that this eHCF was clinically tolerated and significantly improved the SCORAD index and growth indices.

Probiotic Bifidobacterium breve induces IL-10-producing Tr1 cells in the colon.

Specific intestinal microbiota has been shown to induce Foxp3(+) regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103(+) dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103(+) DCs from Il10(-/-), Tlr2(-/-), and Myd88(-/-) mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103(+) DCs failed to induce IL-10 production from co-cultured Il27ra(-/-) T cells. B. breve treatment of Tlr2(-/-) mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103(+) DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4(+) T cells from wild-type mice, but not Il10(-/-) mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties.

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full-term, breast-fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy-protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL-12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll-like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized.

Up-regulation of MDP and tuftsin gene expression in Th1 and Th17 cells as an adjuvant for an oral Lactobacillus casei vaccine against anti-transmissible gastroenteritis virus.

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.

Immune response in nasopharynx, lung, and blood elicited by experimental nasal pneumococcal vaccines containing live or heat-killed lactobacilli as mucosal adjuvants.

This work analyzes the humoral and cellular immune responses induced by live (LcV) and heat-killed (LcM) Lactobacillus casei associated with the pneumococcal antigen (P-Ag) at the nasopharynx level, considering nasal-associated lymphoid tissue (NALT) as the primary inductive site of the mucosal immune system, and lung and blood as effector sites. Levels of P-Ag IgA and IgG antibodies, main types of B and T cells, and cytokines in mucosal and systemic compartments were evaluated. The results showed that both LcM+P-Ag and LcV+P-Ag vaccines effectively induced IgA and IgG anti-P-Ag Abs in the upper and lower respiratory tract and plasma. These results correlated with increased IgA+ cells in NALT and lung that was induced by the experimental vaccines. Moreover, numbers of IgG+ cells increased in the blood. Profiles of inflammatory and regulatory cytokines were evaluated and their possible implications for the defense against pneumococci was assessed. Considering the overall results, the potential mechanisms of immune stimulation induced by LcM and LcV used as adjuvants are discussed. LcV and LcM showed similar effects on the immune system. Strain viability is not crucial for the stimulation of the antigen-specific immune response, and LcM is a convenient and effective mucosal adjuvant.