Short communication: Probiotic induction of interleukin-10 and interleukin-12 production by macrophages is modulated by co-stimulation with microbial components.

Probiotic lactobacilli stimulate macrophages and dendritic cells to secrete cytokines and thereby regulate the immune responses of the host. The balance of the IL-10 and IL-12 production induced by a probiotic is crucial for determining the direction of the immune response. In the present study, we examined the ability of microbial components to modify IL-10 and IL-12 production induced by a popular probiotic strain, Lactobacillus casei strain Shirota (LcS), which itself predominantly induces IL-12 production. Microbial ligands for toll-like receptor (TLR)3 and TLR5 further enhanced the IL-12 induction by LcS, whereas ligands for TLR2, TLR4, TLR7, and TLR9 converted the cytokine production pattern from IL-12 predominant to IL-10 predominant. These results indicate that the probiotic induction of IL-10 and IL-12 production can be flexibly modified by co-stimulation with microbial components. This could explain the variety of immunomodulatory functions (immunoactivation or anti-inflammation) exerted by this probiotic strain.

High-affinity monoclonal IgA regulates gut microbiota and prevents colitis in mice.

Immunoglobulin A (IgA) is the main antibody isotype secreted into the intestinal lumen. IgA plays a critical role in the defence against pathogens and in the maintenance of intestinal homeostasis. However, how secreted IgA regulates gut microbiota is not completely understood. In this study, we isolated monoclonal IgA antibodies from the small intestine of healthy mouse. As a candidate for an efficient gut microbiota modulator, we selected a W27 IgA, which binds to multiple bacteria, but not beneficial ones such as Lactobacillus casei. W27 could suppress the cell growth of Escherichia coli but not L. casei in vitro, indicating an ability to improve the intestinal environment. Indeed W27 oral treatment could modulate gut microbiota composition and have a therapeutic effect on both lymphoproliferative disease and colitis models in mice. Thus, W27 IgA oral treatment is a potential remedy for inflammatory bowel disease, acting through restoration of host-microbial symbiosis.

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor-deficient mice.

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor-deficient (pIgR(-/-) ) mice. The pIgR(-/-) mice exhibited the accumulation of CD8αß(+) T-cell receptor (TCR)-αß(+) IELs (CD8αß(+) αß-IELs) after weaning, but no increase of CD8αß(+) γδ-IELs was detected in pIgR(-/-) TCR-ß(-/-) mice compared with pIgR(+/+) TCR-ß(-/-) mice. When 5-bromo-2'-deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU-labelled cells in SI-IELs was not different between pIgR(+/+) mice and pIgR(-/-) mice. However, the proportion of BrdU-labelled CD8αß(+) -IELs became higher in pIgR(-/-) mice than pIgR(+/+) mice 10 days after discontinuing BrdU-labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR(+/+) TCR-ß(-/-) mice and pIgR(-/-) TCR-ß(-/-) mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αß(+) αß-IELs increased much more in the SI of pIgR(-/-) TCR-ß(-/-) mice than pIgR(+/+) TCR-ß(-/-) mice 8 weeks after the transfer. αß-IELs from pIgR(-/-) mice could produce more interferon-γ and interleukin-17 than those of pIgR(+/+) mice, and intestinal permeability tended to increase in the SI of pIgR(-/-) mice with aging. Taken together, these results indicate that activated CD8αß(+) αß-IELs preferentially accumulate in pIgR(-/-) mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.

Autocrine DNA fragmentation of intra-epithelial lymphocytes (IELs) in mouse small intestine.

Intraepithelial lymphocytes (IELs) are present in the intestinal epithelium. Mechanisms of IELs for the protection of villi from foreign antigens and from infections by micro-organisms have not been sufficiently explained. Although more than 70% of mouse duodenal and jejunal IELs bear γδTCR (γδIELs), the functions of γδIELs are little investigated. We stimulate γδIELs by anti-CD3 monoclonal antibody (mAb) injection. The mAb activates γδIELs to release Granzyme B (GrB) into the spaces surrounding the γδIELs and intestinal villous epithelial cells (IECs). Released GrB induces DNA fragmentation in IECs independently of Perforin (Pfn). IECs immediately repair their fragmented DNA. Activated IELs reduce their cell size, remain for some time in the epithelium after the activation and are ultimately eliminated without leaving the site. We focus our attention on the response of IELs to the released GrB present in the gap surrounding IELs, after activation, in order to examine whether the released GrB has a similar effect on IELs to that observed on IECs in our previous studies. DNA fragmentation is also induced in IELs together with the repair of fragmented DNA thereafter. The time-kinetics of both events were found to be identical to those observed in IECs. DNA fragmentation in IELs is Pfn-independent. Here, we present Pfn-independent "autocrine DNA fragmentation" in IELs and the repair of fragmented DNA in IELs and discuss their biological significance. Autocrine DNA fragmentation has never been reported to date in vivo.

Activation of intra-epithelial lymphocytes; their morphology, marker expression and ultimate fate.

Intraepithelial lymphocytes (IELs) have been considered to play a key role in the defense system of the small intestine. Its mechanism has not been made sufficiently clear. Studies on IELs have been extremely limited to functions of αß T-cell receptor (αßTCR) IELs (αß-IELs). Since, in the mouse duodenum and jejunum, γδ-IELs consist 75 % of IELs, it thus would be inappropriate to argue the mechanism without extensive discussions over the functions of γδ-IELs. In previous studies, we found that the anti-CD3 monoclonal antibody (mAb) injection induced DNA fragmentation in intestinal epithelial cells (IECs) and DNA repair immediately after, that these responses were reproduced by anti-γδTCR mAb not by anti-αßTCR mAb and that the DNA fragmentation was induced by Granzyme B secreted by IELs, totally independent of Perforin. To further explore the functions of IELs in situ, we undertook experiments exclusively focused on IELs, on their changes and ultimate fate after the stimulation in mouse in vivo system. The current study demonstrated that the injected anti-CD3 mAb bound to CD3 on IELs, that the mAb activated γδ-IELs, leading to their degranulation, that changes occurred irreversibly in IELs and finally that activated IELs died in situ. γδ-IELs could be considered to respond to various stimulations most likely without the need of accessory cells ("always ready for rapid response"), to die in situ ("disposable") and thus to respond to the stimulation only once ("a one-shot responder"). These characteristics of γδ-IELs are important to further elucidate the functions of γδ-IELs in the intestinal defense system.

Lactobacillus casei Shirota enhances the preventive efficacy of soymilk in chemically induced breast cancer.

Soy foods are known to be effective for breast cancer prevention. The habitual consumption of soy isoflavones in combination with the probiotic Lactobacillus casei Shirota (LcS) was shown to decrease the risk of breast cancer occurrence in our previous population-based case-controlled study among Japanese women. The present study aimed to elucidate the cooperative prevention mechanism of soymilk and LcS using an animal carcinogenic model. Female Sprague-Dawley rats received a high-fat, AIN-76A diet containing soymilk, LcS, both soymilk and LcS, or none and were orally exposed to 2-amino-1-methyl-6-penylimidazo[4,5-b]pyridine at a dose of 85 mg/kg bodyweight eight times for 2 weeks. The development of palpable mammary tumors was monitored for 17 weeks. Tumor tissues were immunohistochemically examined for estrogen receptor (ER)-α, Ki-67 and CD34. Compared with the control group, the incidence and multiplicity of mammary tumors were reduced by soymilk alone and soymilk in combination with LcS, while tumor volume was decreased by LcS alone and LcS in combination with soymilk. An immunohistochemical analysis revealed that soymilk in combination with LcS more effectively reduced the numbers of ER-α-positive and Ki-67-positive cells in tumors than soymilk alone and that both soymilk and LcS inhibited tumor angiogenesis. These results demonstrated that soymilk prevents the development of mammary tumors and that LcS suppresses tumor growth, potentially enhancing the preventive efficacy of soymilk. The habitual consumption of LcS in combination with soymilk might be a beneficial dietary style for breast cancer prevention.

Involvement of parasympathetic pelvic efferent pathway in psychological stress-induced defecation.

AIM: To investigate the role of the pelvic nerve pathway in stress-induced acceleration of colorectal transit and defecation in rats. METHODS: Surgical transection of rectal nerves (rectal branches of the pelvic nerve), vagotomy (Vag) or adrenalectomy (Adx) were performed bilaterally in rats. Number of fecal pellet output of these rats was measured during 1-h water avoidance stress (WAS). To evaluate the colonic transit, rats were given phenol red through the catheter indwelled in the proximal colon and subjected to WAS. After WAS session, entire colon and rectum were isolated and distribution of phenol red was measured. Distal colonic and rectal transit was evaluated using glass bead. Rats were inserted the glass bead into the distal colon and evacuation rate of the bead was measured. Neural activation was assessed by immunohistochemical staining of c-Fos and PGP9.5 in colonic whole-mount preparations of longitudinal muscle myenteric plexus (LMMP). RESULTS: In the sham-operated rats (sham op), WAS significantly increased defecation and accelerated colorectal transit with marked elevation of plasma corticosterone level. Compared with sham-operated rats, increase in the excretion of fecal pellets during WAS was significantly reduced by rectal nerve transection (RNT) (sham op: 6.9 ± 0.8 vs RNT: 4.3 ± 0.6, P < 0.05) or Vag (sham op: 6.4 ± 0.8 vs Vag: 3.7 ± 1.1, P < 0.05), although corticosterone level remained elevated. Adx-rats significantly increased the defecation despite the lower corticosterone level. Distribution pattern of phenol red showed RNT inhibited distal colonic and rectal transit accelerated by WAS, while Vag inhibited proximal colonic transit. Suppression of distal colonic and rectal transit by RNT was further confirmed by the bead evacuation rate (sham op: 80.0% vs RNT: 53.8%). WAS significantly increased the number of c-Fos-immunoreactive neural cells in the LMMP of the proximal and distal colon, whereas c-Fos expression was decreased by RNT in the distal colon (sham op: 9.0 ± 2.0 vs RNT: 4.4 ± 1.0, P < 0.05) and decreased by Vag in the proximal colon. CONCLUSION: Pelvic nerve conveys WAS stimuli from the brain to the distal colon, and directly activate the myenteric neurons, followed by the increase of its motility.

Granzyme B-dependent and perforin-independent DNA fragmentation in intestinal epithelial cells induced by anti-CD3 mAb-activated intra-epithelial lymphocytes.

We previously found that an i.p. injection of anti-CD3 monoclonal antibody (mAb) into mice caused DNA fragmentation in the intestinal villous epithelial cells (IVECs) of the duodenum and the jejunum. In this study, in order to elucidate the mechanism of DNA fragmentation in IVECs, we searched for the inducer(s) of DNA fragmentation by using immunohistochemistry. The release of cytoplasmic granules from intraepithelial lymphocytes (IELs) and the formation of large gaps between IELs and IVECs were observed electron microscopically after antibody administration. The presence and distribution pattern of Granzyme B (GrB), a serine protease in cytolytic granules present in cytotoxic T lymphocytes and natural killer cells and considered to be the responsible molecule for DNA fragmentation in target cells, was examined in detail in intestinal villi by immunohistology. GrB was detected in cytoplasmic granules in nearly all IELs. The time-kinetics of granule release from IELs after mAb injection coincided not only with that of the extracellular diffusion of GrB, but also with that of DNA fragmentation in IVECs. On the other hand, perforin (Pfn), assumed to cooperate with GrB in DNA fragmentation, could not be detected in IELs, and its release was not confirmed after the anti-CD3 mAb injection. Anti-CD3 mAb injection also induced DNA fragmentation in IVECs in Pfn-knockout mice. These results support the notion that DNA fragmentation in IVECs by the stimulated IELs in the present study is induced by a mechanism involving GrB, but independent of Pfn.

Flexible cytokine production by macrophages and T cells in response to probiotic bacteria: a possible mechanism by which probiotics exert multifunctional immune regulatory activities.

Probiotics have been reported to be efficacious against cancers, infections, allergies, inflammatory bowel diseases and autoimmune diseases, and it is important to explain how such multifunctional activities are realized. Lactobacillus casei Shirota (LcS) is one of these multifunctional probiotics, and its ability to augment the host immune system has been extensively examined. We have shown that the cell wall structure of this probiotic strain is responsible for potently inducing IL-12 production. In addition, we have recently found that LcS differentially controls the inflammatory cytokine responses of macrophages and T cells in either Peyer's patches or the spleen. Other studies revealed that LcS-induced IL-12 production by macrophages is modified when other bacteria or their cell components are simultaneously present. These findings can provide a theoretical basis for understanding the multifunctional activities of specific probiotics.

Peptidoglycan from lactobacilli inhibits interleukin-12 production by macrophages induced by Lactobacillus casei through Toll-like receptor 2-dependent and independent mechanisms.

We previously showed that Lactobacillus strains having a rigid cell wall resistant to intracellular digestion can stimulate macrophages to induce large a quantity of interleukin-12 (IL-12). In this study, we examined the influence of lactobacilli and bacterial cell wall components on IL-12 production by macrophages that was induced by Lactobacillus casei, which has a rigid cell wall. Easily digestible lactobacilli such as Lactobacillus johnsonii and Lactobacillus plantarum or their intact cell walls (ICWs) weakly or very weakly induced IL-12 production by macrophages, and inhibitedL. casei-induced IL-12 production. While the ICW of L. casei was resistant to intracellular digestion and did not inhibit L. casei-induced IL-12 production, its polysaccharide-depleted ICW, i.e. intact peptidoglycan, was sensitive to intracellular digestion and inhibited L. casei-induced IL-12 production. Furthermore, the peptidoglycans of L. johnsonii, L. plantarum and Staphylococcus aureus also inhibited L. casei-induced IL-12 production. Peptidoglycans from lactobacilli suppressed L. casei-induced expression of IL-12p40 but not IL-12p35 mRNA. Inhibition of IL-12 production by peptidoglycan was mitigated in Toll-like receptor 2 (TLR2)-deficient macrophages compared with the inhibition in wild-type macrophages. A derivative of the minimal structural unit of peptidoglycan (6-O-stearoyl-muramyl dipeptide) recognized by nucleotide-binding oligomerization domain 2 (NOD2) could also suppress L. casei-induced IL-12 production. These findings demonstrate that easily digestible bacteria and peptidoglycan suppress IL-12 production through pattern recognition receptors such as TLR2 and NOD2. IL-12 production in the gut may be negatively regulated by the simultaneous inhibitory actions of various resident bacteria that are susceptible to intracellular digestion.

Curriculum vitae of intestinal intraepithelial T cells: their developmental and behavioral characteristics.

The alimentary tract has an epithelial layer, consisting mainly of intestinal epithelial cells (IECs), that is exposed to the exterior world through the intestinal lumen. The IEC layer contains many intestinal intraepithelial T cells (IELs), and the total number of IELs constitutes the largest population in the peripheral T-cell pool. Virtually all gammadelta-IELs and many alphabeta-IELs in the mouse small intestine are known to express CD8 alpha alpha homodimers. A wide range of evidence that supports extrathymic development of these CD8 alpha alpha(+) IELs has been collected. In addition, while several studies identified cells with precursor T-cell phenotypes within the gut epithelium, how these precursors, which are dispersed along the length of the intestine, develop into gammadelta-IELs and/or alphabeta-IELs has not been clarified. The identification of lymphoid cell aggregations named 'cryptopatches' (CPs) in the intestinal crypt lamina propria of mice as sites rich in T-cell precursors in 1996 by our research group, however, provided evidence for a central site, whereby precursor IELs could give rise to T-cell receptor-bearing IELs. In this review, we discuss the development of IELs in the intestinal mucosa and examine the possibility that CPs serve as a production site of extrathymic IELs.

Essential roles of monocytes in stimulating human peripheral blood mononuclear cells with Lactobacillus casei to produce cytokines and augment natural killer cell activity.

We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-alpha, and IL-10. IFN-gamma production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-gamma effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-gamma through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.

Prenatal blockage of lymphotoxin beta receptor and TNF receptor p55 signaling cascade resulted in the acceleration of tissue genesis for isolated lymphoid follicles in the large intestine.

Signaling by lymphotoxin (LT) and TNF is essential for the organogenesis of secondary lymphoid tissues in systemic and mucosal compartments. In this study, we demonstrated that the progeny of mice treated with fusion protein of LTbetaR and IgGFc (LTbetaR-Ig) or LTbetaR-Ig plus TNFR55-Ig (double Ig) showed significantly increased numbers of isolated lymphoid follicles (ILF) in the large intestine. Interestingly, double Ig treatment accelerated the maturation of large intestinal ILF. Three-week-old progeny of double Ig-treated mice showed increased numbers of ILF in the large intestine, but not in the small intestine. Furthermore, alteration of intestinal microflora by feeding of antibiotic water did not affect the increased numbers of ILF in the large intestine of double Ig-treated mice. Most interestingly, mice that developed numerous ILF also had increased levels of activation-induced cytidine deaminase expression and numbers of IgA-expressing cells in the lamina propria of the large intestine. Taken together, these results suggest that ILF formation in the large intestine is accelerated by blockage of LTbetaR and TNFR55 signals in utero, and ILF, like colonic patches, might play a role in the induction of IgA response in the large intestine.

Modulation of natural killer cell activity by supplementation of fermented milk containing Lactobacillus casei in habitual smokers.

BACKGROUND: Habitual smoking significantly reduces natural killer (NK) cell activity. To clarify whether the intake of fermented milk containing lactic acid bacteria restores NK cell activity in habitual smokers, we conducted a placebo-controlled double-blind test. METHODS: Ninety-nine subjects with smoking habits were randomly divided into two groups and daily for 3 weeks were given fermented milk containing Lactobacillus casei or placebo. NK cell activity in the peripheral blood mononuclear cells (PBMNC) was determined before and after the intake. Average number of cigarettes smoked and number of cigarettes that the subjects smoked before giving blood after getting up on the inspection day were asked, and these data were used to adjust the influence of smoking on NK cell activity. RESULTS: NK cell activity in individuals was inversely correlated to numbers of cigarettes smoked. Averaged NK cell activity adjusted by the numbers of cigarettes was significantly higher in individuals drinking fermented milk containing L. casei than those drinking a placebo. However, the proportion of NK cells was not different between individuals drinking either fermented milk containing L. casei or the placebo. CONCLUSIONS: Intake of fermented milk containing lactic acid bacteria was considered effective for restoring the NK cell activity of habitual smokers.

Modulation of Immune Functions by Foods.

Evidence is rapidly accumulating as to the beneficial effects of foods. However, it is not always clear whether the information is based on data evaluated impartially in a scientific fashion. Human research into whether foods modulate immune functions in either intervention studies or randomized controlled trials can be classified into three categories according to the physical state of subjects enrolled for investigation: (i) studies examining the effect of foods in healthy individuals; (ii) studies analyzing the effect of foods on patients with hypersensitivity; and (iii) studies checking the effect of foods on immunocompromized subjects, including patients who had undergone surgical resection of cancer and newborns. The systematization of reported studies has made it reasonable to conclude that foods are able to modulate immune functions manifesting as either innate immunity (phagocytic activity, NK cell activity) or acquired immunity (T cell response, antibody production). Moreover, improvement of immune functions by foods can normalize the physical state of allergic patients or cancer patients, and may reduce the risk of diseases in healthy individuals. Therefore, it is valuable to assess the immune-modulating abilities of foods by measuring at least one parameter of either innate or acquired immunity.

Homeostatic regulation of intestinal villous epithelia by B lymphocytes.

The epithelial cell of the small intestine is one of the most rapidly regenerating cells in the body. However, the cellular mechanism and biological significance underlying this rapid regeneration remain elusive. In this study we examined the intestinal epithelia of mutant mice that lack B and/or T cells and those of normal littermates. The absence of B cells in Ig mu-chain mutant mice or B and T cells in recombination-activating gene (RAG)-2(-/-) as well as SCID mutant mice was associated with a marked acceleration of epithelial cell turnover and an up-regulation of the expression of MHC class II molecules. No such effects were observed in T cell-deficient TCR-delta and -beta double-mutant mice. As far as the goblet cells of villous epithelium are concerned, absolute numbers of them remained the same among these mutant mice that have no B and/or T cells. Alymphoplasia (aly/aly) mutant mice that lacked Peyer's patches and Ig-producing cells in the lamina propria, but harbored a large number of intestinal mucosal T cells, also displayed a significant acceleration of epithelial cell turnover and, to some extent, up-regulated expression of MHC class II molecules. Notably, the accelerated epithelial cell turnover was not observed and returned to normalcy in the Ig mu-chain mutant mice that had been given antibiotic-containing water. These findings indicate that B cells down-regulate the generation and differentiation of intestinal epithelial cells in the normal wild-type condition and suggest that enteric microorganisms are implicated in the accelerated generation of epithelial cells in mice that have no B cells.

Inhibition of B16 melanoma experimental metastasis by interferon-gamma through direct inhibition of cell proliferation and activation of antitumour host mechanisms.

Interferon-gamma (IFN-gamma) has pleiotropic activities other than its antivirus action, including cell growth inhibition, natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activation, and angiogenesis inhibitory activity, and these activities are supposed to be involved in its antitumour activity. However, it has not been completely elucidated which activity is mainly involved in the tumour suppression in vivo. In this study, we analysed inhibitory mechanisms of endogenous IFN-gamma against B16 melanoma experimental metastasis. After intravenous injection of tumour cells, tumour deposits in the lungs and liver were increased and life span was shorter in IFN-gamma(-/-) mice, indicating important roles for IFN-gamma in antitumour mechanisms. Interestingly, tumour deposits were not increased in IFN-gamma receptor (R)(-/-) mice. Furthermore, only low levels of cell-mediated immunity against the tumour and activation of NK cells were observed, indicating that antimetastatic effects of IFN-gamma is not mediated by host cells. The survival period of B16 melanoma-bearing IFN-gamma R(-/-) mice was, however, shorter than wild-type mice. These observations suggest that IFN-gamma prevents B16 melanoma experimental metastasis by directly inhibiting the cell growth, although antitumour host functions may also be involved in a later phase.