Diverse impact of a probiotic strain, Lacticaseibacillus paracasei Shirota, on peripheral mononuclear phagocytic cells in healthy Japanese office workers: a randomized, double-blind, controlled trial.

Mononuclear phagocytic cells (MPCs) are classified into monocytes (Mos)/macrophages and dendritic cells (DCs) based on their functions. Cells of MPCs lineage act as immune modulators by affecting effector cells, such as NK cells, T cells, and B cells. This study aimed to investigate the effects of Lacticaseibacillus paracasei strain Shirota (LcS) ingestion on peripheral MPCs, particularly on their expression of functional cell-surface molecules enhanced in healthy adults. Thus, twelve healthy office workers consumed a fermented milk drink containing 1.0 × 1011 cfu of LcS (LcS-FM) or a control unfermented milk drink (CM) once a day for 6 weeks. Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples, and immune cells and functional cell-surface molecules were analyzed. We observed remarkable differences in the expression of HLAABC, MICA, CD40, and GPR43 in plasmacytoid DCs (pDCs) between the LcS-FM and CM groups, whereas no difference was found in CD86 or HLADR expression. The LcS-FM group exhibited higher CD40 expression in both conventional DCs (cDCs) and Mos, especially in type 2 conventional DCs (cDC2s) and classical monocytes (cMos); higher percentages of cMos, intermediate monocytes (iMos), and nonclassical monocytes; and higher numbers of cMos and iMos in PBMCs than the CM group. LcS ingestion increased the expression of HLAABC, MICA, CD40, and GPR43 in pDCs and CD40 in cDCs and Mos, particularly cDC2s and cMos. These results suggest that LcS modulates the function of MPCs that may lead to the regulation of immune effector functions in healthy adults.

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.

Microbiota of the Small Intestine Is Selectively Engulfed by Phagocytes of the Lamina Propria and Peyer's Patches.

Phagocytes such as dendritic cells and macrophages, which are distributed in the small intestinal mucosa, play a crucial role in maintaining mucosal homeostasis by sampling the luminal gut microbiota. However, there is limited information regarding microbial uptake in a steady state. We investigated the composition of murine gut microbiota that is engulfed by phagocytes of specific subsets in the small intestinal lamina propria (SILP) and Peyer's patches (PP). Analysis of bacterial 16S rRNA gene amplicon sequences revealed that: 1) all the phagocyte subsets in the SILP primarily engulfed Lactobacillus (the most abundant microbe in the small intestine), whereas CD11bhi and CD11bhiCD11chi cell subsets in PP mostly engulfed segmented filamentous bacteria (indigenous bacteria in rodents that are reported to adhere to intestinal epithelial cells); and 2) among the Lactobacillus species engulfed by the SILP cell subsets, L. murinus was engulfed more frequently than L. taiwanensis, although both these Lactobacillus species were abundant in the small intestine under physiological conditions. These results suggest that small intestinal microbiota is selectively engulfed by phagocytes that localize in the adjacent intestinal mucosa in a steady state. These observations may provide insight into the crucial role of phagocytes in immune surveillance of the small intestinal mucosa.

Beneficial Effects of Citrus Juice Fermented with Lactobacillus plantarum YIT 0132 on Japanese Cedar Pollinosis.

Recently, the prevalence of allergies in Japan has been increasing. Certain types of fruit juice and lactic acid bacteria are known to alleviate allergic symptoms. Therefore, we examined whether citrus juice fermented by a specific lactic acid bacteria can improve the symptoms of Japanese cedar pollinosis (JCPsis). Lactobacillus plantarum YIT 0132 (LP0132) was selected based on its high proliferative activity in citrus juice and anti-inflammatory interleukin-10-inducing activity. Dietary administration of heat-killed LP0132 cells or citrus juice fermented with LP0132 was found to significantly suppress nasal rubbing in a JCPsis mouse model, indicating relief of allergy symptoms. To evaluate the effects of LP0132-fermented citrus juice on pollinosis symptoms and quality of life (QOL) in humans with JCPsis, a single-blind, placebo-controlled, parallel-group clinical trial was conducted. The participants were 42 adults with JCPsis. They ingested 100 mL of sterilized LP0132-fermented citrus juice (active group) or unfermented citrus juice (placebo group) once daily for 8 weeks. Immediately after the pollen peak when allergy symptoms and QOL loss were most severe, itchy eyes, itchy skin, and QOL loss by JCPsis were alleviated in the active group compared with the placebo group. At 10 weeks after starting the intervention, increased the levels of blood eosinophils were significantly suppressed in the active group compared with the placebo group. We conclude that continuous ingestion of citrus juice fermented with LP0132 may help alleviate the allergy symptoms and impaired QOL caused by JCPsis.

Probiotic upregulation of peripheral IL-17 responses does not exacerbate neurological symptoms in experimental autoimmune encephalomyelitis mouse models.

CONTEXT: It is of great importance to evaluate the safety of probiotics in dysregulated immune conditions, as probiotics can possibly modulate immune functions in the host. OBJECTIVE: We tried to confirm the safety of using Lactobacillus casei strain Shirota (LcS) to help prevent autoimmunity in the central nervous system. METHODS: We used two chronic experimental autoimmune encephalomyelitis (EAE) models, a relapse and remission type EAE model in SJL/J mice and a durable type model in C57BL/6 mice. LcS was administered from 1 week before antigen sensitization until the end of the experiments, and neurological symptoms and histopathological changes of the spinal cord were observed. Immunological parameters were also examined in the SJL/J mouse model. RESULTS: LcS administration did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model but instead tended to improve neurological symptoms in the SJL/J mouse EAE model. LcS administration transiently upregulated IL-17 production by antigen-stimulated lymphocytes of draining lymph nodes 7 days after sensitization. Enhanced production of IL-10 and an increase in the percentage of CD4(+)CD25(+) T regulatory cells were also observed at the same sites. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12, but this expression was not enhanced by LcS administration. CONCLUSION: These results demonstrate that LcS does not exacerbate, but instead may improve EAE depending on the immunization conditions, and that IL-17 responses at peripheral sites may not always result in a worsening of autoimmune diseases.

Well-controlled proinflammatory cytokine responses of Peyer's patch cells to probiotic Lactobacillus casei.

SUMMARY: In order to clarify the probiotic features of immunomodulation, cytokine production by murine spleen and Peyer's patch (PP) cells was examined in response to probiotic and pathogenic bacteria. In spleen cells, probiotic Lactobacillus casei induced interleukin (IL)-12 production by CD11b(+) cells more strongly than pathogenic Gram-positive and Gram-negative bacteria and effectively promoted the development of T helper (Th) type 1 cells followed by high levels of secretion of interferon (IFN)-gamma. Although the levels of IL-12 secreted by PP cells in response to L. casei were lower in comparison with spleen cells, Th1 cells developed as a result of this low-level induction of IL-12. However, IFN-gamma secretion by the L. casei-induced Th1 cells stimulated with a specific antigen was down-regulated in PP cells. Development of IL-17-producing Th17 cells was efficiently induced in PP cells by antigen stimulation. Lactobacillus casei slightly, but significantly, inhibited the antigen-induced secretion of IL-17 without a decrease in the proportion of Th17 cells. No bacteria tested induced the development of IL-10-producing, transforming growth factor-beta-producing or Foxp3-expressing regulatory T cells, thus suggesting that certain probiotics might regulate proinflammatory responses through as yet unidentified mechanisms in PP cells. These data show probiotic L. casei to have considerable potential to induce IL-12 production and promote Th1 cell development, but the secretion of proinflammatory cytokines such as IL-12 and IL-17 may be well controlled in PP cells.

Bacterial teichoic acids reverse predominant IL-12 production induced by certain lactobacillus strains into predominant IL-10 production via TLR2-dependent ERK activation in macrophages.

The cytokine response of macrophages to probiotic lactobacilli varies between strains, and the balance of IL-10/IL-12 production is crucial for determination of the direction of the immune response. To clarify the mechanism whereby Lactobacillus strains differentially induce production of IL-10 and IL-12, we examined the potential relationship between cytokine production and MAPK activation. In mouse peritoneal macrophages, Lactobacillus plantarum potently induced IL-10 but weakly induced IL-12 production, whereas L. casei potently induced IL-12 but weakly induced IL-10 production. Kinetic analysis of the activation of ERK, p38, and JNK showed that L. plantarum induced a more rapid and intense activation of MAPKs, especially of ERK, than L. casei. A selective blockade of ERK activation induced by L. plantarum resulted in a decrease in IL-10 production and a simultaneous increase in IL-12 production. Interestingly, when macrophages were stimulated with a combination of L. plantarum and L. casei, IL-10 production was induced synergistically. We identified cell wall teichoic acid and lipoteichoic acid as key factors for triggering the synergistic induction of IL-10 production, although these teichoic acids alone only weakly induced IL-10 production. The effect of these teichoic acids on IL-10 production was mediated by TLR2-dependent ERK activation. Our data demonstrate that activation of the ERK pathway is critical for determination of the balance of the IL-10/IL-12 response of macrophages to lactobacilli and that predominant IL-12 production induced by certain lactobacilli such as L. casei can be converted into predominant IL-10 production when stimulated in the presence of teichoic acids.

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.

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.