Dietary Supplementation with Lactobacilli Improves Emergency Granulopoiesis in Protein-Malnourished Mice and Enhances Respiratory Innate Immune Response

This work studied the effect of protein malnutrition on the hemato-immune response to the respiratory challenge with Streptococcus pneumoniae and evaluated whether the dietary recovery with a probiotic strain has a beneficial effect in that response. Three important conclusions can be inferred from the results presented in this work: a) protein-malnutrition significantly impairs the emergency myelopoiesis induced by the generation of the innate immune response against pneumococcal infection; b) repletion of malnourished mice with treatments including nasally or orally administered Lactobacillus rhamnosus CRL1505 are able to significantly accelerate the recovery of granulopoiesis and improve innate immunity and; c) the immunological mechanisms involved in the protective effect of immunobiotics vary according to the route of administration. The study demonstrated that dietary recovery of malnourished mice with oral or nasal administration of L. rhamnosus CRL1505 improves emergency granulopoiesis and that CXCR4/CXCR12 signaling would be involved in this effect. Then, the results summarized here are a starting point for future research and open up broad prospects for future applications of probiotics in the recovery of immunocompromised malnourished hosts.     

Stimulation of macrophages by immunobiotic Lactobacillus strains: influence beyond the intestinal tract

Lactobacillus rhamnosus CRL1505 (Lr1505), L. rhamnosus CRL1506 (Lr1506) and L. casei CRL431 (Lc431) are able to stimulate intestinal immunity, but only Lr1505 and Lc431 are able to stimulate immunity in the respiratory tract. With the aim of advancing the understanding of the immunological mechanisms involved in stimulation of distant mucosal sites, this study evaluated the effects of orally administered probiotics on the functions of alveolar and peritoneal macrophages. Compared to a control group, these three lactobacilli were able to significantly increase phagocytic and microbicidal activities of peritoneal macrophages. After intraperitoneal challenge with pathogenic Candida albicans, mice treated with immunobiotics had significantly lower pathogen counts in infected organs. Moreover, lactobacilli‐treated mice had a stronger immune response against C. albicans. On the other hand, only Lc1505 and Lc431 were able to improve activity of and cytokine production by alveolar macrophages. Only in these two groups was there better resistance to respiratory challenge with C. albicans, which correlated with improved respiratory immune response. The results of this study suggest that consumption of some probiotic strains could be useful for improving resistance to infections in sites distant from the gut by increasing the activity of macrophages at those sites.     

Inorganic polyphosphate from the immunobiotic Lactobacillus rhamnosus CRL1505 prevents inflammatory response in the respiratory tract

Lactobacillus (L.) rhamnosus CRL1505 accumulates inorganic polyphosphate (polyP) in its cytoplasm in response to environmental stress. The aim of this study was to evaluate the potential effects of polyP from the immunobiotic CRL1505 on an acute respiratory inflammation murine animal model induced by lipopolysaccharide (LPS).First, the presence of polyP granules in the cytoplasm of CRL1505 strain was evidenced by specific staining. Then, it was demonstrated in the intracellular extracts (ICE) of CRL1505 that polyP chain length is greater than 45 phosphate residues. In addition, the functionality of the genes involved in the polyP metabolism (ppk, ppx1 and ppx2) was corroborated by RT-PCR. Finally, the possible effect of the ICE of CRL1505 strain containing polyP and a synthetic polyP was evaluated in vivo using a murine model of acute lung inflammation. It was observed that the level of cytokines pro-inflammatory (IL-17, IL-6, IL-2, IL-4, INF-γ) in serum was normalized in mice treated with ICE, which would indicate that polyP prevents the local inflammatory response in the respiratory tract. The potential application of ICE from L. rhamnosus CRL1505 as a novel bioproduct for the treatment of respiratory diseases is one of the projections of this work.     

Influence of orally administered Lactobacillus GG on respiratory immune response in a murine model of diet‐induced obesity

Mice with diet‐induced obesity were fed with Lactobacillus rhamnosus GG (LGG) suspended in saline or saline alone (control mice). Pulmonary mRNA expression of IFN‐γ; IFN‐α receptor 1; CD247 antigen; killer cell lectin‐like receptor subfamily K, member 1; TNF‐α; IL‐12 receptor β1 and IL‐2 receptor β, and the proportion of Lactobacillales in feces were significantly greater in the LGG group than in the control mice (P < 0.05 and P < 0.01, respectively). These results suggest that LGG alters the respiratory immunity of obese subjects through having a potent impact on intestinal immunity.     

Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH

Aims: To evaluate the efficacy of a novel microencapsulation procedure using whey protein and pectin to improve the survival rate of Lactobacillus rhamnosus CRL 1505 to low pH and bile. Methods and Results: Lactobacillus rhamnosus CRL 1505 was encapsulated by ionotropic gelation using pectin (PE) and pectin–whey protein (PE–WP). Both types of beads (MC_PE/WP and MC_PE–WP/WP) were covered with a layer of whey protein by complex coacervation. The noncapsulated lactobacilli were not sensitive to bile salts but to acid. Both microparticles protected Lact. rhamnosus CRL 1505 at pH 2·0, but only MC_PE/WP was effective at pH 1·2. Conclusions: The combination of ionotropic gelation and complex coacervation techniques is efficient to obtain microcapsules of pectin covered with whey proteins. The MC_PE/WP beads were more stable than the MC_PE–WP/WP beads in simulated gastric conditions, thus offering better protection to Lact. rhamnosus CRL 1505 at low pH. Significance and Impact of the Study: Pectin beads with a whey protein layer (MC_PE/WP) could be used as probiotic carrier in functional foods of low pH (e.g. apple juice), thus protecting Lact. rhamnosus CRL 1505 against the stressful conditions of the gastric tract.     

Intermediate chains of exopolysaccharides from Lactobacillus rhamnosus RW‐9595M increase IL‐10 production by macrophages

Aims: To evaluate the immunosuppressive properties of the exopolysaccharide (EPS) from high‐EPS producer Lactobacillus rhamnosus RW‐9595M on inflammatory cytokines produced by macrophages. Methods and Results: The conditioned media (CM) were produced by macrophages treated with parental Lact. rhamnosus ATCC 9595 and its isogenic variant, the high‐EPS producer Lact. rhamnosus RW‐9595M, and the levels of TNF‐α, IL‐6, IL‐10 and IL‐12 were evaluated. Results revealed that CM from parental Lact. rhamnosus induced higher levels of TNF‐α, IL‐6 and IL‐12 but inhibited IL‐10 production, whereas its mucous variant induced low or no TNF‐α and IL‐6. Addition of purified EPS to macrophages treated with parental Lact. rhamnosus decreased the inflammatory cytokines and inhibited the metabolic activity of lymphocytes. The intermediate polysaccharide chains (16–30 units) produced by time‐controlled hydrolysis of EPS increased the IL‐10 produced by macrophages. Conclusions: Polysaccharide chains of EPS induced immunosuppression by the production of macrophagic anti‐inflammatory IL‐10. Significance and impact of the Study: These results indicate that the EPS from Lact. rhamnosus RW‐9595M may be useful as a new immunosuppressive product in dairy food.     

Valsartan independent of AT1 receptor inhibits tissue factor,TLR‐2 and ‐4 expression by regulation of Egr‐1 through activation of AMPK in diabetic conditions

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)‐1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT₁R) can reduce tissue factor (TF) and toll‐like receptor (TLR)‐2 and ‐4 by regulating Egr‐1 in THP‐1 cells and aorta in streptozotocin‐induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr‐1, TF, TLR‐2 and ‐4 which were significantly reduced by valsartan. HG increased Egr‐1 expression by activation of PKC and ERK1/2 in THP‐1 cells. Valsartan increased AMPK phosphorylation in a concentration and time‐dependent manner via activation of LKB1. Valsartan inhibited Egr‐1 without activation of PKC or ERK1/2. The reduced expression of Egr‐1 by valsartan was reversed by either silencing Egr‐1, or compound C, or DN‐AMPK‐transfected cells. Valsartan inhibited binding of NF‐κB and Egr‐1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF‐α, IL‐6 and IL‐1β) production and NF‐κB activity in HG‐activated THP‐1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT₁R in THP‐1 cells or CHO cells, which were devoid of AT₁R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr‐1, TLR‐2, ‐4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin‐induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr‐1 regulation.     

Pro‐inflammatory capacity of classically activated monocytes relates positively to muscle mass and strength

In mice, monocytes that exhibit a pro‐inflammatory profile enter muscle tissue after muscle injury and are crucial for clearance of necrotic tissue and stimulation of muscle progenitor cell proliferation and differentiation. The aim of this study was to test if pro‐inflammatory capacity of classically activated (M1) monocytes relates to muscle mass and strength in humans. This study included 191 male and 195 female subjects (mean age 64.2 years (SD 6.4) and 61.9 ± 6.4, respectively) of the Leiden Longevity Study. Pro‐inflammatory capacity of M1 monocytes was assessed by ex vivo stimulation of whole blood with Toll‐like receptor (TLR) 4 agonist lipopolysaccharide (LPS) and TLR‐2/1 agonist tripalmitoyl‐S‐glycerylcysteine (Pam₃Cys‐SK₄), both M1 phenotype activators. Cytokines that stimulate M1 monocyte response (IFN‐γ and GM‐CSF) as well as cytokines that are secreted by M1 monocytes (IL‐6, TNF‐α, IL‐12, and IL‐1β) were measured. Analyses were adjusted for age, height, and body fat mass. Upon stimulation with LPS, the cytokine production capacity of INF‐γ, GM‐CSF, and TNF‐α was significantly positively associated with lean body mass, appendicular lean mass and handgrip strength in men, but not in women. Upon stimulation with Pam₃Cys‐SK₄, IL‐6; TNF‐α; and Il‐1β were significantly positively associated with lean body mass and appendicular lean in women, but not in men. Taken together, this study shows that higher pro‐inflammatory capacity of M1 monocytes upon stimulation is associated with muscle characteristics and sex dependent.     

Inhibition of cytokine response to TLR stimulation and alleviation of collagen‐induced arthritis in mice by Schistosoma japonicum peptide SJMHE1

Helminth‐derived products have recently been shown to prevent the development of inflammatory diseases in mouse models. However, most identified immunomodulators from helminthes are mixtures or macromolecules with potentially immunogenic side effects. We previously identified an immunomodulatory peptide called SJMHE1 from the HSP60 protein of Schistosoma japonicum. In this study, we assessed the ability of SJMHE1 to affect murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated by toll‐like receptor (TLR) ligands in vitro and its treatment effect on mice with collagen‐induced arthritis (CIA). We show that SJMHE1 not only modulates the cytokine production of murine macrophage (MΦ) and dendritic cell but also affects cytokine production upon coculturing with allogeneic CD4⁺ T cell. SJMHE1 potently inhibits the cytokine response to TLR ligands lipopolysaccharide (LPS), CpG oligodeoxynucleotides (CpG) or resiquimod (R848) from mouse splenocytes, and human PBMCs stimulated by LPS. Furthermore, SJMHE1 suppressed clinical signs of CIA in mice and blocked joint erosion progression. This effect was mediated by downregulation of key cytokines involved in the pathogenesis of CIA, such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐17, and IL‐22 and up‐regulation of the inhibitory cytokine IL‐10, Tgf‐β1 mRNA, and CD4⁺CD25⁺Foxp3⁺ Tregs. This study provides new evidence that the peptide from S. japonicum, which is the ‘safe’ selective generation of small molecule peptide that has evolved during host–parasite interactions, is of great value in the search for novel anti‐inflammatory agents and therapeutic targets for autoimmune diseases.     

The effects of a vegetable‐derived probiotic lactic acid bacterium on the immune response

The objective of this study was to investigate the probiotic properties of the fermented vegetable derived lactic acid bacterium, L. plantarum. L. plantarum 10hk2 showed antibacterial activity against pathogenic bacteria and immunomodulating effects on murine macrophage cell lines. RAW 264.7 cells stimulated with viable cells of this probiotic strain increased the amounts of pro‐inflammatory mediators such as IL‐1β, IL‐6 and TNF‐α, as well as the anti‐inflammatory mediator, IL‐10. ICR mice fed with viable cells of L. plantarum 10hk2 had reduced numbers of enteric Salmonella and Shigella species in comparison to controls from 2 weeks after supplementation, and this effect was observed for up to 4 weeks. The findings of this study suggest that this specific lactic acid bacterial strain, which is derived from vegetable fermentation, holds great promise for use in probiotics and as a food additive since it can reduce the number of some pathogenic bacteria through production of lactic acids.     

Consumption of probiotic Lactobacillus rhamnosus (MTCC: 5897) containing fermented milk plays a key role in development of the immune system in newborn mice during the suckling–weaning transition

Early infancy, the period when offspring rely not only on their own immunity to combat food‐borne antigens but also acquire immunity through maternal sources (via transplacental routes and breast milk), is critical for immune system development Hence the present study was designed to evaluate the effect on offspring of administration of probiotic‐containing fermented milk (PFM) either to mothers during the suckling period or to their offspring after weaning either separately or sequentially. PFM‐fed mice showed enhanced leukocyte functionality in offspring as evidenced by significantly (P < 0.05) increased release of lysosomal enzymes (β‐galactosidase, β‐glucuronidase) in peritoneal fluid and nitric oxide production in culture supernatants of activated macrophages. Further, remarkably reduced levels (P < 0.01) of inflammatory markers (TNF‐α, monocyte chemotactic protein‐1) and allergic antibodies (total and milk specific IgE) were observed in offspring where PFM was fed either to them or to their mothers. However, considerably increased levels (P < 0.05) of SIgA were found in the guts of control and experimental groups animals irrespective of their exposure to PFM. Restoration of Th1/Th2 homeostasis further confirmed the useful effects of PFM supplementation by shifting the cytokine profile (IL‐4, IFN‐γ and IL‐10) with increased IFN‐γ/IL‐4 and reduced IgE/Ig2Ga ratios. Hence, it is logical to conclude that administration of Lactobacillus rhamnosus‐containing (MTCC:5897) fermented milk to mothers during the suckling period and to their offspring after weaning has beneficial effects on the development of newborns immune systems; this effect appears to be more pronounced when mothers are fed with it.     

Effect of riboflavin‐producing bacteria against chemically induced colitis in mice

Aim

To assess the anti‐inflammatory effect associated with individual probiotic suspensions of riboflavin‐producing lactic acid bacteria (LAB) in a colitis murine model.

Methods and Results

Mice intrarectally inoculated with trinitrobenzene sulfonic acid (TNBS) were orally administered with individual suspensions of riboflavin‐producing strains: Lactobacillus (Lact.) plantarum CRL2130, Lact. paracasei CRL76, Lact. bulgaricus CRL871 and Streptococcus thermophilus CRL803; and a nonriboflavin‐producing strain or commercial riboflavin. The extent of colonic damage and inflammation and microbial translocation to liver were evaluated. iNOs enzyme was analysed in the intestinal tissues and cytokine concentrations in the intestinal fluids. Animals given either one of the four riboflavin‐producing strains showed lower macroscopic and histologic damage scores, lower microbial translocation to liver, significant decreases of iNOs+ cells in their large intestines and decreased proinflammatory cytokines, compared with mice without treatment. The administration of pure riboflavin showed similar benefits. Lact. paracasei CRL76 accompanied its anti‐inflammatory effect with increased IL‐10 levels demonstrating other beneficial properties in addition to the vitamin production.

Conclusion

Administration of riboflavin‐producing strains prevented the intestinal damage induced by TNBS in mice.

Significance and Impact of the Study

Riboflavin‐producing phenotype in LAB represents a potent tool to select them for preventing/treating IBD.     

Pathogenesis and association of Mycoplasma pneumoniae infection with cardiac and hepatic damage

The aim of this study was to investigate the pathogenesis of Mycoplasma pneumoniae (MP) infection and its association with cardiac and hepatic damage. Between March 2013 and March 2014, 59 children with MP pneumonia (MPP) and 30 healthy children were enrolled. Serum titers of TLR4, T cell immunoglobulin and mucin‐domain (TIM) 3, IL‐10, TNF‐α, and IFN‐γ were measured both in children with MPP and healthy children. Additionally, MP‐specific antibody titer and creatine kinase‐MB (CK‐MB), and alanine transaminase (ALT) titers were measured in patients with MPP. There were significant differences between the MPP patients and healthy controls in titers of TIM1 (P < 0.01), TLR2 (P = 0.028), TLR4 (P = 0.019), IL‐10 (P < 0.01), TNF‐α (P < 0.01) and IFN‐γ (P < 0.01); however, no significant difference was found in TIM3 titers (P = 0.8181). TIM1 was correlated with CK‐MB (P = 0.025), whereas both TIM1 and TLR2 titers were correlated with MP‐specific antibody titers (P < 0.001; P = 0.003, respectively). Additionally, there were correlations between ALT, TIM3, and TLR2 titers (P = 0.025; P = 0.037, respectively). The titers of TIM1 were significantly higher in patients with cardiac damage (P = 0.007) than in those without it, whereas the titers of TLR2 were significantly higher in patients with hepatic damage (P = 0.026) than in those without it. TLR2, TLR4 and TIM1 may be involved in the process of MP infection. Additionally, TLR2, TLR4, TIM1 and TIM3 may play particular roles in the pathogenesis of MPP‐associated cardiac and hepatic damage.     

Macrophage-adipocyte interaction: marked interleukin-6 production by lipopolysaccharide

Objective: Recent studies suggested macrophages were integrated in adipose tissues, interacting with adipocytes, thereby exacerbating inflammatory responses. Persistent low‐grade infection by gram‐negative bacteria appears to promote atherogenesis. We hypothesized a ligand for toll‐like receptor 4 (TLR4), bacterial lipopolysaccharide (LPS), would further exaggerate macrophage‐adipocyte interaction. Research Methods and Procedures: RAW264.7 macrophage cell line and differentiated 3T3‐L1 preadipocytes were co‐cultured using transwell system. As a control, each cell was cultured independently. After incubation of the cells with or without Escherichia coli LPS, tumor necrosis factor (TNF)‐α and interleukin (IL)‐6 production was evaluated. Results: Co‐culture of macrophages and adipocytes with low concentration of Escherichia coli LPS (1 ng/mL) markedly up‐regulated IL‐6 production (nearly 100‐fold higher than that of adipocyte culture alone, p < 0.01), whereas TNF‐α production was not significantly influenced. This increase was partially inhibited by anti‐TNF‐α neutralizing antibody. Recombinant TNF‐α and LPS synergistically up‐regulated IL‐6 production in adipocytes. However, this increase did not reach the level of production observed in co‐cultures stimulated with LPS. Discussion: A ligand for TLR‐4 stimulates macrophages to produce TNF‐α. TNF‐α, thus produced, cooperatively up‐regulates IL‐6 production with other soluble factors secreted either from adipocytes or macrophages in these cells. Markedly up‐regulated IL‐6 would greatly influence the pathophysiology of diabetes and its vascular complications.     

Suppression of Inflammatory Cytokine Production by ar‐Turmerone Isolated from Curcuma phaeocaulis

Rhizomes of Curcuma phaeocaulis Valeton (Zingiberaceae) have traditionally been used for controlling inflammatory conditions. Numerous studies have aimed to isolate and characterize the bioactive constituents of C. phaeocaulis. It has been reported that its anti‐inflammatory properties are a result of cyclooxygenase‐2 inhibition; however, its effect on the T‐cell function remains to be elucidated. In this study, four known sesquiterpenoids, viz., ar‐turmerone (TM), germacrone (GM), (+)‐(4S,5S)‐germacrone‐4,5‐epoxide (GE), and curzerenone (CZ), were isolated from C. phaeocaulis rhizomes and evaluated for their effects on the CD4⁺ T‐cell function. While GM, GE, and CZ had no effect on the activation of splenic T cells or CD4⁺ T cells, TM suppressed the interferon (IFN)‐γ production, without affecting the interleukin (IL)‐4 expression. TM also decreased the expression of IL‐2 in CD4⁺ T cells, but did not change their cell‐division rates upon stimulation. These results suggest that TM, a major constituent of C. phaeocaulis rhizomes selectively exerts potent anti‐inflammatory effects via suppression of the inflammatory cytokines IFN‐γ and IL‐2.