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.

The renin-angiotensin system promotes arrhythmogenic substrates and lethal arrhythmias in mice with non-ischaemic cardiomyopathy.

AIMS: The progression of pathological left ventricular remodelling leads to cardiac dysfunction and contributes to the occurrence of malignant arrhythmias and sudden cardiac death. The underlying molecular mechanisms remain unclear, however. Our aim was to examine the role of the renin-angiotensin system (RAS) in the mechanism underlying arrhythmogenic cardiac remodelling using a transgenic mouse expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). This mouse model exhibits progressive cardiac dysfunction leading to lethal arrhythmias. METHODS AND RESULTS: Subcutaneous administration of aliskiren, a direct renin inhibitor, significantly suppressed the progression of pathological cardiac remodelling and improved survival among dnNRSF-Tg mice while reducing arrhythmogenicity. Genetic deletion of the angiotensin type 1a receptor (AT1aR) similarly suppressed cardiac remodelling and sudden death. In optical mapping analyses, spontaneous ventricular tachycardia (VT) and fibrillation (VF) initiated by breakthrough-type excitations originating from focal activation sites and maintained by functional re-entry were observed in dnNRSF-Tg hearts. Under constant pacing, dnNRSF-Tg hearts exhibited markedly slowed conduction velocity, which likely contributes to the arrhythmogenic substrate. Aliskiren treatment increased conduction velocity and reduced the incidence of sustained VT. These effects were associated with suppression of cardiac fibrosis and restoration of connexin 43 expression in dnNRSF-Tg ventricles. CONCLUSION: Renin inhibition or genetic deletion of AT1aR suppresses pathological cardiac remodelling that leads to the generation of substrates maintaining VT/VF and reduces the occurrence of sudden death in dnNRSF-Tg mice. These findings demonstrate the significant contribution of RAS activation to the progression of arrhythmogenic substrates.

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.

Inhibition of N-type Ca2+ channels ameliorates an imbalance in cardiac autonomic nerve activity and prevents lethal arrhythmias in mice with heart failure.

AIMS: Dysregulation of autonomic nervous system activity can trigger ventricular arrhythmias and sudden death in patients with heart failure. N-type Ca(2+) channels (NCCs) play an important role in sympathetic nervous system activation by regulating the calcium entry that triggers release of neurotransmitters from peripheral sympathetic nerve terminals. We have investigated the ability of NCC blockade to prevent lethal arrhythmias associated with heart failure. METHODS AND RESULTS: We compared the effects of cilnidipine, a dual N- and L-type Ca(2+) channel blocker, with those of nitrendipine, a selective L-type Ca(2+) channel blocker, in transgenic mice expressing a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). In this mouse model of dilated cardiomyopathy leading to sudden arrhythmic death, cardiac structure and function did not significantly differ among the control, cilnidipine, and nitrendipine groups. However, cilnidipine dramatically reduced arrhythmias in dnNRSF-Tg mice, significantly improving their survival rate and correcting the imbalance between cardiac sympathetic and parasympathetic nervous system activity. A ß-blocker, bisoprolol, showed similar effects in these mice. Genetic titration of NCCs, achieved by crossing dnNRSF-Tg mice with mice lacking CACNA1B, which encodes the α1 subunit of NCCs, improved the survival rate. With restoration of cardiac autonomic balance, dnNRSF-Tg;CACNA1B(+/-) mice showed fewer malignant arrhythmias than dnNRSF-Tg;CACNA1B(+/+) mice. CONCLUSIONS: Both pharmacological blockade of NCCs and their genetic titration improved cardiac autonomic balance and prevented lethal arrhythmias in a mouse model of dilated cardiomyopathy and sudden arrhythmic death. Our findings suggest that NCC blockade is a potentially useful approach to preventing sudden death in patients with heart failure.

Increased expression of HCN channels in the ventricular myocardium contributes to enhanced arrhythmicity in mouse failing hearts.

BACKGROUND: The efficacy of pharmacological interventions to prevent sudden arrhythmic death in patients with chronic heart failure remains limited. Evidence now suggests increased ventricular expression of hyperpolarization-activated cation (HCN) channels in hypertrophied and failing hearts contributes to their arrythmicity. Still, the role of induced HCN channel expression in the enhanced arrhythmicity associated with heart failure and the capacity of HCN channel blockade to prevent lethal arrhythmias remains undetermined. METHODS AND RESULTS: We examined the effects of ivabradine, a specific HCN channel blocker, on survival and arrhythmicity in transgenic mice (dnNRSF-Tg) expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor, a useful mouse model of dilated cardiomyopathy leading to sudden death. Ivabradine (7 mg/kg per day orally) significantly reduced ventricular tachyarrhythmias and improved survival among dnNRSF-Tg mice while having no significant effect on heart rate or cardiac structure or function. Ivabradine most likely prevented the increase in automaticity otherwise seen in dnNRSF-Tg ventricular myocytes. Moreover, cardiac-specific overexpression of HCN2 in mice (HCN2-Tg) made hearts highly susceptible to arrhythmias induced by chronic ß-adrenergic stimulation. Indeed, ventricular myocytes isolated from HCN2-Tg mice were highly susceptible to ß-adrenergic stimulation-induced abnormal automaticity, which was inhibited by ivabradine. CONCLUSIONS: HCN channel blockade by ivabradine reduces lethal arrhythmias associated with dilated cardiomyopathy in mice. Conversely, cardiac-specific overexpression of HCN2 channels increases arrhythmogenicity of ß-adrenergic stimulation. Our findings demonstrate the contribution of HCN channels to the increased arrhythmicity seen in failing hearts and suggest HCN channel blockade is a potentially useful approach to preventing sudden death in patients with heart failure.

Reciprocal expression of MRTF-A and myocardin is crucial for pathological vascular remodelling in mice.

Myocardin-related transcription factor (MRTF)-A is a Rho signalling-responsive co-activator of serum response factor (SRF). Here, we show that induction of MRTF-A expression is key to pathological vascular remodelling. MRTF-A expression was significantly higher in the wire-injured femoral arteries of wild-type mice and in the atherosclerotic aortic tissues of ApoE(-/-) mice than in healthy control tissues, whereas myocardin expression was significantly lower. Both neointima formation in wire-injured femoral arteries in MRTF-A knockout (Mkl1(-/-)) mice and atherosclerotic lesions in Mkl1(-/-); ApoE(-/-) mice were significantly attenuated. Expression of vinculin, matrix metallopeptidase 9 (MMP-9) and integrin ß1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in Mkl1(-/-) mice than in wild-type mice. In cultured vascular smooth muscle cells (VSMCs), knocking down MRTF-A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF-A expression in dedifferentiated VSMCs was the downregulation of microRNA-1. Moreover, the MRTF-A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF-A could thus be a novel therapeutic target for the treatment of vascular diseases.

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.

Change of positive selection pressure on HIV-1 envelope gene inferred by early and recent samples.

HIV-1 infection has been on the rise in Japan recently, and the main transmission route has changed from blood transmission in the 1980s to homo- and/or hetero-sexual transmission in the 2000s. The lack of early viral samples with clinical information made it difficult to investigate the possible virological changes over time. In this study, we sequenced 142 full-length env genes collected from 16 Japanese subjects infected with HIV-1 in the 1980s and in the 2000s. We examined the diversity change in sequences and potential adaptive evolution of the virus to the host population. We used a codon-based likelihood method under the branch-site and clade models to detect positive selection operating on the virus. The clade model was extended to account for different positive selection pressures in different viral populations. The result showed that the selection pressure was weaker in the 2000s than in the 1980s, indicating that it might have become easier for the HIV to infect a new host and to develop into AIDS now than 20 years ago and that the HIV may be becoming more virulent in the Japanese population. The study provides useful information on the surveillance of HIV infection and highlights the utility of the extended clade models in analysis of virus populations which may be under different selection pressures.

Within-host co-evolution of Gag P453L and protease D30N/N88D demonstrates virological advantage in a highly protease inhibitor-exposed HIV-1 case.

To better understand the mechanism of HIV group-specific antigen (Gag) and protease (PR) co-evolution in drug-resistance acquisition, we analyzed a drug-resistance case by both bioinformatics and virological methods. We especially considered the quality of sequence data and analytical accuracy by introducing single-genome sequencing (SGS) and Spidermonkey/Bayesian graphical models (BGM) analysis, respectively. We analyzed 129 HIV-1 Gag-PR linkage sequences obtained from 8 time points, and the resulting sequences were applied to the Spidermonkey co-evolution analysis program, which identified ten mutation pairs as significantly co-evolving. Among these, we focused on associations between Gag-P453L, the P5' position of the p1/p6 cleavage-site mutation, and PR-D30N/N88D nelfinavir-resistant mutations, and attempted to clarify their virological significance in vitro by constructing recombinant clones. The results showed that P453L(Gag) has the potential to improve replication capacity and the Gag processing efficiency of viruses with D30N(PR)/N88D(PR) but has little effect on nelfinavir susceptibility. Homology modeling analysis suggested that hydrogen bonds between the 30th PR residue and the R452Gag are disturbed by the D30N(PR) mutation, but the impaired interaction is compensated by P453L(Gag) generating new hydrophobic interactions. Furthermore, database analysis indicated that the P453L(Gag)/D30N(PR)/N88D(PR) association was not specific only to our clinical case, but was common among AIDS patients.

Advanced dynamic monitoring of cellular status using label-free and non-invasive cell-based sensing technology for the prediction of anticancer drug efficacy.

Quantitative evaluation of anticancer drug efficacy using in vitro cell-based assays is useful for cancer patients, particularly those who show unconventional cancer development. Nevertheless, conventional chemosensitivity testing often requires widely used labeling agents and time-consuming laboratory procedures that provide low reliability. Label-free non-invasive cell-based assays are desired for dynamic monitoring of cellular status. This critical review first describes conventional chemosensitivity testing and then advanced label-free cell-based technology used to screen anticancer drugs through dynamic monitoring of cellular status, focusing on dosage and the use of drug-resistant cancer cells. Results from label-free cell-based approaches are compared with those of conventional chemosensitivity testing. The cellular statuses, addressed in terms of respective mechanisms and disadvantages, are extracellular fluxes of proton (H(+)), O(2), and anticancer drugs, cell morphology changes, cell-environment interaction, and mitochondrial membrane potential. Finally, a cell-based systems outlook is presented. This paper represents a step toward efficient and accurate initial screening of anticancer drugs and development of compounds and their combined use to achieve pharmacodynamic and pharmacokinetic interactions, and chemotherapy evaluation of particular anticancer drugs for individual patients.

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.

Development of signaling echo method for cell-based quantitative efficacy evaluation of anti-cancer drugs in apoptosis without drug presence using high-precision surface plasmon resonance sensing.

We propose a rapid and label-free quantitative evaluation of anti-cancer drug efficacy in apoptosis using live cancer cells cultured on a sensor chip. The resultant custom-made, high-precision surface plasmon resonance (SPR) sensor monitors the inner mitochondrial membrane's potential change (DeltaPsi(m)). For trans-membrane anti-cancer drugs to be effective, continuous potential changes arising from the crossing of the membrane by the drug of interest prevent DeltaPsi(m) monitoring. Herein, we report on a novel signaling echo method that avoids this disturbance; the cancer cells are incubated with a specific anti-cancer drug, with subsequent removal of the drug before SPR measurements. The cell reaction without any drug was monitored as the differential SPR angle rate of change for 10 min from 30 min after a sensor chip was set on a prism. The cell reaction after 60 min pre-incubation with a drug was significantly related to the conventional cell viability after 48 h (P <0.001).

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.

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain.

During coevolution with the host, HIV-1 developed the ability to hijack the cellular ubiquitin/proteasome degradation pathway to counteract the antiviral activity of APOBEC3G (A3G), a host cytidine deaminase that can block HIV-1 replication. Abrogation of A3G function involves the HIV-1 Vif protein, which binds A3G and serves as an adapter molecule to recruit A3G to a Cullin5-based E3 ubiquitin ligase complex. Structure-guided mutagenesis of A3G focused on the 14 most surface-exposed Lys residues allowed us to identify four Lys residues (Lys-297, 301, 303, and 334) that are required for Vif-mediated A3G ubiquitination and degradation. Substitution of Arg for these residues confers Vif resistance and restores A3G's antiviral activity in the presence of Vif. In our model, the critical four Lys residues cluster at the C terminus, opposite to the known N-terminal Vif-interaction region in the protein. Thus, spatial constraints imposed by the E3 ligase complex may be an important determinant in Vif-dependent A3G ubiquitination.

Inferring within-patient HIV-1 evolutionary dynamics under anti-HIV therapy using serial virus samples with vSPA.

BACKGROUND: Analysis of within-patient HIV evolution under anti-HIV therapy is crucial to a better understanding the possible mechanisms of HIV drug-resistance acquisition. The high evolutionary rate of HIV allows us to trace its evolutionary process in real time by analyzing virus samples serially collected from the same patient. However, such studies are still uncommon due to the lack of powerful computational methods designed for serial virus samples. In this study, we develop a computational method, vSPA (viral Sequential Pathway Analysis), which groups viral sequences from the same sampling time into clusters and traces the evolution between clusters over sampling times. The method makes use of information of different sampling times and traces the evolution of important amino acid mutations. Second, a permutation test at the codon level is conducted to determine the threshold of the correlation coefficient for clustering viral quasispecies. We applied vSPA to four large data sets of HIV-1 protease and reverse transcriptase genes serially collected from two AIDS patients undergoing anti-HIV therapy over several years. RESULTS: The results show that vSPA can trace within-patient HIV evolution by detecting many amino acid changes, including important drug-resistant mutations, and by classifying different viral quasispecies coexisting during different periods of the therapy. CONCLUSION: Given that many new anti-HIV drugs will be available in the near future, vSPA may be useful for quickly providing information on the acquisition of HIV drug-resistant mutations by monitoring the within-patient HIV evolution under anti-HIV therapy as a computational approach.

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.