Can Lacticaseibacillus rhamnosus CRL1505 postbiotic improve emergency myelopoiesis in immunocompromised mice?

We evaluated whether viable and non-viable Lacticaseibacillus rhamnosus CRL1505 (Lr05V or Lr05NV, respectively) was able to improve emergency myelopoiesis induced by Streptococcus pneumoniae (Sp) infection. Adult Swiss-mice were orally treated with Lr05V or Lr05NV during five consecutive days. The Lr05V and Lr05NV groups and untreated control group received an intraperitoneal dose of cyclophosphamide (Cy-150 mg/kg). Then, the mice were nasally challenged with Sp (107 UFC/mice) on day 3 post-Cy injection. After the pneumococcal challenge, the innate and myelopoietic responses were evaluated. The control group showed a high susceptibility to pneumococcal infection, an impaired innate immune response and a decrease of hematopoietic stem cells (HSCs: Lin-Sca-1+c-Kit+), and myeloid multipotent precursors (MMPs: Gr-1+Ly6G+Ly6C-) in bone marrow (BM). However, lactobacilli treatments were able to significantly increase blood neutrophils and peroxidase-positive cells, while improving cytokine production and phagocytic activity of alveolar macrophages. This, in turn, led to an early Sp lung clearance compared to the control group. Furthermore, Lr05V was more effective than Lr05NV to increase growth factors in BM, which allowed an early HSCs and MMPs recovery with respect to the control group. Both Lr05V and Lr05NV were able to improve BM emergency myelopiesis and protection against respiratory pathogens in mice undergoing chemotherapy.

Lacticaseibacillus rhamnosus CRL1505 Peptidoglycan Modulates the Inflammation-Coagulation Response Triggered by Poly(I:C) in the Respiratory Tract.

Lacticaseibacillus rhamnosus CRL1505 beneficially modulates the inflammation-coagulation response during respiratory viral infections. This study evaluated the capacity of the peptidoglycan obtained from the CRL1505 strain (PG-Lr1505) to modulate the immuno-coagulative response triggered by the viral pathogen-associated molecular pattern poly(I:C) in the respiratory tract. Adult BALB/c mice were nasally treated with PG-Lr1505 for two days. Treated and untreated control mice were then nasally challenged with poly(I:C). Mice received three doses of poly(I:C) with a 24 h rest period between each administration. The immuno-coagulative response was studied after the last administration of poly(I:C). The challenge with poly(I:C) significantly increased blood and respiratory pro-inflammatory mediators, decreased prothrombin activity (PT), and increased von Willebrand factor (vWF) levels in plasma. Furthermore, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and thrombomodulin (TM) expressions were increased in the lungs. PG-Lr1505-treated mice showed significant modulation of hemostatic parameters in plasma (PT in %, Control = 71.3 ± 3.8, PG-Lr1505 = 94.0 ± 4.0, p < 0.01) and lungs. Moreover, PG-Lr1505-treated mice demonstrated reduced TF in F4/80 cells from lungs, higher pro-inflammatory mediators, and increased IL-10 compared to poly(I:C) control mice (IL-10 in pg/mL, Control = 379.1 ± 12.1, PG-Lr1505 = 483.9 ± 11.3, p < 0.0001). These changes induced by PG-Lr1505 correlated with a significant reduction in lung tissue damage. Complementary in vitro studies using Raw 264.7 cells confirmed the beneficial effect of PG-Lr1505 on poly(I:C)-induced inflammation, since increased IL-10 expression, as well as reduced damage, production of inflammatory mediators, and hemostatic parameter expressions were observed. In addition, protease-activated receptor-1 (PAR1) activation in lungs and Raw 264.7 cells was observed after TLR3 stimulation, which was differentially modulated by PG-Lr1505. The peptidoglycan from L. rhamnosus CRL1505 is able to regulate inflammation, the procoagulant state, and PAR1 activation in mice and macrophages in the context of the activation of TLR3 signaling pathways, contributing to a beneficial modulation of inflammation-hemostasis crosstalk.

Topical Administration of Lactiplantibacillus plantarum Accelerates the Healing of Chronic Diabetic Foot Ulcers through Modifications of Infection, Angiogenesis, Macrophage Phenotype and Neutrophil Response.

This work aimed to evaluate the adjuvant treatment to surgical debridement using topical applications of Lactiplantibacillus plantarum ATCC 10241 cultures in complicated diabetic foot ulcers as compared to diabetic foot ulcers receiving surgical wound debridement. A randomised controlled trial was performed involving 22 outpatients with complicated diabetic foot ulcers that either received surgical debridement (SuDe, n = 12) or surgical debridement plus topical applications of L. plantarum cultures (SuDe + Lp, n = 10) every week during a 12 week treatment period. Compared to patients receiving SuDe, patients treated with SuDe + Lp exhibited significantly increased fibroplasia and angiogenesis, as determined by Masson's trichrome staining and the study of CD34 cells, α-smooth muscle actin to semi-quantify vascular area, number of vessels and endothelial cells. In addition, a promotion of the polarisation of macrophages from M1 (CD68) to M2 (CD163) phenotype was observed in SuDe + Lp patients with remarkable differences in the tissue localisation. Bacterial counts were significantly diminished in the SuDe + Lp group compared to the SuDe group. Ex vivo assays, using polymorphonuclears isolated from peripheral blood of patients with diabetes and healthy individuals and challenged with Staphylococcus aureus demonstrated that the addition of L. plantarum supernatants significantly improved the phagocytosis of these cells. L. plantarum-secreted components increased the neutrophils bactericidal activity and regulated the netosis induced by S. aureus. At day 49, the average wound area reduction with SuDe + Lp was 73.5% compared with 45.8% for SuDe (p < 0.05). More patients progressed to closure with SuDe + Lp compared with SuDe treatment, indicating the ability of L. plantarum to accelerate the healing. At day 60, 60% of patients treated with SuDe + Lp achieved 100% of wound area reduction compared with 40% for SuDe. We propose that SuDe + Lp could be an effective adjuvant to surgical debridement when SuDe is not satisfactory for patients with complicated diabetic foot ulcers. The treatment is cheap and easy to apply and the product is easy to obtain.

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.

The Role of Immunobiotics and Postbiotics in the Recovery of Immune Cell Populations From Respiratory Mucosa of Malnourished Hosts: Effect on the Resistance Against Respiratory Infections.

Malnutrition is associated with a state of secondary immunodeficiency, which is characterized by a worsening of the immune response against infectious agents. Despite important advances in vaccines and antibiotic therapies, the respiratory infections are among the leading causes of increased morbidity and mortality, especially in immunosuppressed hosts. In this review, we examine the interactions between immunobiotics-postbiotics and the immune cell populations of the respiratory mucosa. In addition, we discuss how this cross talk affects the maintenance of a normal generation of immune cells, that is crucial for the establishment of protective innate and adaptive immune responses. Particular attention will be given to the alterations in the development of phagocytic cells, T and B lymphocytes in bone marrow, spleen and thymus in immunosuppression state by protein deprivation. Furthermore, we describe our research that demonstrated that the effectiveness of immunobiotics nasal administration in accelerating the recovery of the respiratory immune response in malnourished hosts. Finally, we propose the peptidoglycan from the immunobiotic Lactobacillus rhamnosus CRL1505 as the key cellular component for the effects on mucosal immunity, which are unique and cannot be extrapolated to other L. rhamnosus or probiotic strains. In this way, we provide the scientific bases for its application as a mucosal adjuvant in health plans, mainly aimed to improve the immune response of immunocompromised hosts. The search for safe vaccine adjuvants that increase their effectiveness at the mucosal level is a problem of great scientific relevance today.

Characterization of Weissella viridescens UCO-SMC3 as a Potential Probiotic for the Skin: Its Beneficial Role in the Pathogenesis of Acne Vulgaris.

Previously, we isolated lactic acid bacteria from the slime of the garden snail Helix aspersa Müller and selected Weissella viridescens UCO-SMC3 because of its ability to inhibit in vitro the growth of the skin-associated pathogen Cutibacterium acnes. The present study aimed to characterize the antimicrobial and immunomodulatory properties of W. viridescens UCO-SMC3 and to demonstrate its beneficial effect in the treatment of acne vulgaris. Our in vitro studies showed that the UCO-SMC3 strain resists adverse gastrointestinal conditions, inhibits the growth of clinical isolates of C. acnes, and reduces the adhesion of the pathogen to keratinocytes. Furthermore, in vivo studies in a mice model of C. acnes infection demonstrated that W. viridescens UCO-SMC3 beneficially modulates the immune response against the skin pathogen. Both the oral and topical administration of the UCO-SCM3 strain was capable of reducing the replication of C. acnes in skin lesions and beneficially modulating the inflammatory response. Of note, orally administered W. viridescens UCO-SMC3 induced more remarkable changes in the immune response to C. acnes than the topical treatment. However, the topical administration of W. viridescens UCO-SMC3 was more efficient than the oral treatment to reduce pathogen bacterial loads in the skin, and effects probably related to its ability to inhibit and antagonize the adhesion of C. acnes. Furthermore, a pilot study in acne volunteers demonstrated the capacity of a facial cream containing the UCO-SMC3 strain to reduce acne lesions. The results presented here encourage further mechanistic and clinical investigations to characterize W. viridescens UCO-SMC3 as a probiotic for acne vulgaris treatment.

Lactobacillus rhamnosus postbiotic-induced immunomodulation as safer alternative to the use of live bacteria.

Many attempts have been made to search for safer immunomodulatory agents that enhance the immune response and reduce the number and severity of infections in at-risk populations. The use of postbiotics, non-viable microbial cells or cell fractions that confer a health benefit to the consumer, represents a safe and attractive way to modulate and enhance the immune function in order to improve human health. Therefore, the aim of this work is to evaluate the immunoregulatory effect of Lactobacillus rhamnosus CRL1505 postbiotics in a complex culture system using human intestinal epithelial cells (IECs) and dendritic cells (DCs) differentiated from peripheral blood mononuclear cells. First, we demonstrated that L. rhamnosus CRL1505 differentially modulate human IECs and DCs after the challenge with the TLR4 agonist LPS. The CRL1505 strain down-regulated CD40, CD80 and CD86 expression in DCs, and increased their production of TNF-α, IL-1ß, IL-6 and IL-10. Interestingly, the non-viable strain was able to modulate the immune response of both types of human cells. Then, we showed that cell wall (CW1505) and peptidoglycan (PG1505) from L. rhamnosus CRL1505 modulated TLR4-triggered immune response in IECs and DCs. Of interest, CW1505 showed a strong stimulatory effect while the PG1505 presented immune characteristics that were more similar to viable and non-viable CRL1505. To date, several molecules of immunobiotics were identified, that can be connected to specific host-responses. We hereby demonstrated that peptidoglycan of L. rhamnosus CRL1505 is a key molecule for the immunobiotic properties of this strain in human IECs and DCs. Likewise, the result of these studies could provide predictive tools for the in vivo efficacy of postbiotics and the scientific basis for their future applications in immunocompromised patients.

Improvement of Myelopoiesis in Cyclophosphamide-Immunosuppressed Mice by Oral Administration of Viable or Non-Viable Lactobacillus Strains.

Myelosuppression is the major dose-limiting toxicity of cancer chemotherapy. There have been many attempts to find new strategies that reduce myelosuppression. The dietary supplementation with lactic acid bacteria (LAB) improved respiratory innate immune response and the resistance against respiratory pathogens in immunosupressed hosts. Although LAB viability is an important factor in achieving optimal protective effects, non-viable LAB are capable of stimulating immunity. In this work, we studied the ability of oral preventive administration of viable and non-viable Lactobacillus rhamnosus CRL1505 or L. plantarum CRL1506 (Lr05, Lr05NV, Lp06V or Lp06NV, respectively) to minimize myelosuppressive and immunosuppressive effects derived from chemotherapy. Cyclophosphamide (Cy) impaired steady-state myelopoiesis in lactobacilli-treated and untreated control mice. Lr05V, Lr05NV and Lp06V treatments were the most effective to induce the early recovery of bone marrow (BM) tissue architecture, leukocytes, myeloid, pool mitotic and post-mitotic, peroxidase positive, and Gr-1Low/High cells in BM. We selected the CRL1505 strain for being the one capable of maintaining its myelopoiesis-enhancing properties in its non-viable form. Although the CRL1505 treatments do not modify the Cy ability to induce apoptosis, both increased the incorporation of BrdU in BM cells. Consequently, Lr05NV and Lr05V treatments were able to promote early recovery of LSK cells (Lin-Sca-1+c-Kit+ cells), multipotent progenitors (Lin-Sca-1+c-Kit+CD34+ cells), and myeloid cells (Gr-1+Ly6G+Ly6C- cells) with respect to the untreated Cy control. In addition, these treatments were able to increase the frequency of IL17A-producing innate lymphoid cells in the intestinal lamina propria (IL-17A+RORγt+CD4-NKp46+ cells) after Cy injection. These results were correlated with an increase in the IL-17A serum levels, a GM-CSF high expression and a CXCL12 lower expression in BM. Therefore, both Lr05V and Lr05NV treatments are able to activate beneficially the IL-17A/GM-CSF axis and accelerate the recovery of Cy-induced immunosuppression by increasing BM myeloid precursors. We demonstrated for the first time the beneficial effect of CRL1505 strain on myelopoiesis affected by a chemotherapeutic drug. Furthermore, Lr05NV could be a good and safe resource for reducing chemotherapy-induced leukopenia. The results are a starting point for future research and open up broad prospects for future applications of the immunobiotics.

The Role of Alveolar Macrophages in the Improved Protection against Respiratory Syncytial Virus and Pneumococcal Superinfection Induced by the Peptidoglycan of Lactobacillus rhamnosus CRL1505.

The nasal priming with nonviable Lactobacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulates the respiratory innate immune response in infant mice, improving their resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, it was found that NV1505 or PG1505 significantly enhance the numbers of CD11c+SiglecF+ alveolar macrophages (AMs) producing interferon (IFN)-ß. In this work, we aimed to further advance in the characterization of the beneficial effects of NV1505 and PG1505 in the context of a respiratory superinfection by evaluating whether their immunomodulatory properties are dependent on AM functions. Macrophage depletion experiments and a detailed study of their production of cytokines and antiviral factors clearly demonstrated the key role of this immune cell population in the improvement of both the reduction of pathogens loads and the protection against lung tissue damage induced by the immunobiotic CRL1505 strain. Studies at basal conditions during primary RSV or S. pneumoniae infections, as well as during secondary pneumococcal pneumonia, brought the following five notable findings regarding the immunomodulatory effects of NV1505 and PG1505: (a) AMs play a key role in the beneficial modulation of the respiratory innate immune response and protection against RSV infection, (b) AMs are necessary for improved protection against primary and secondary pneumococcal pneumonia, (c) the generation of activated/trained AMs would be essential for the enhanced protection against respiratory pathogens, (d) other immune and nonimmune cell populations in the respiratory tract may contribute to the protection against bacterial and viral infections, and (e) the immunomodulatory properties of NV1505 and PG1505 are strain-specific. These findings significantly improve our knowledge about the immunological mechanisms involved in the modulation of respiratory immunity induced by beneficial microbes.

Immunomodulatory Properties of Bacterium-Like Particles Obtained From Immunobiotic Lactobacilli: Prospects for Their Use as Mucosal Adjuvants.

Non-viable lactic acid bacteria (LAB) have been proposed as antigen delivery platforms called bacterium-like particles (BLPs). Most studies have been performed with Lactococcus lactis-derived BLPs where multiple antigens were attached to the peptidoglycan surface and used to successfully induce specific immune responses. It is well-established that the immunomodulatory properties of LAB are strain dependent and therefore, the BLPs derived from each individual strain could have different adjuvant capacities. In this work, we obtained BLPs from immunomodulatory (immunobiotics) and non-immunomodulatory Lactobacillus rhamnosus and Lactobacillus plantarum strains and comparatively evaluated their ability to improve the intestinal and systemic immune responses elicited by an attenuated rotavirus vaccine. Results demonstrated that orally administered BLPs from non-immunomodulatory strains did not induce significant changes in the immune response triggered by rotavirus vaccine in mice. On the contrary, BLPs derived from immunobiotic lactobacilli were able to improve the levels of anti-rotavirus intestinal IgA and serum IgG, the numbers of CD24+B220+ B and CD4+ T cells in Peyer's patches and spleen as well as the production of IFN-γ by immune cells. Interestingly, among immunobiotics-derived BLPs, those obtained from L. rhamnosus CRL1505 and L. rhamnosus IBL027 enhanced more efficiently the intestinal and systemic humoral immune responses when compared to BLPs from other immunobiotic bacteria. The findings of this work indicate that it is necessary to perform an appropriate selection of BLPs in order to find those with the most efficient adjuvant properties. We propose the term Immunobiotic-like particles (IBLPs) for the BLPs derived from CRL1505 and IBL027 strains that are an excellent alternative for the development of mucosal vaccines.

Nasal Priming with Lactobacillus rhamnosus CRL1505 Stimulates Mononuclear Phagocytes of Immunocompromised Malnourished Mice: Improvement of Respiratory Immune Response.

The effect of Lactobacillus rhamnosus CRL1505 (Lr) on macrophages (Ma) and dendritic cells (DC) in the orchestration of anti-pneumococcal immunity was studied using malnutrition and pneumococcal infection mouse models. Monocytes (Mo), Ma, and DC in two groups of malnourished mice fed with balanced diet (BCD) were studied through flow cytometry; one group was nasally administered with Lr (BCD+Lr group), and the other group was not (BCD group). Well-nourished (WNC) and malnourished (MNC) mice were used as controls.Malnutrition affected the number of respiratory and splenic mononuclear phagocytes. The BCD+Lr treatment, unlike BCD, was able to increase and normalize lung Mo and Ma. The BCD+Lr mice were also able to upregulate the expression of the activation marker MHC II in lung DC and to improve this population showing a more significant effect on CD11b+ DC subpopulation. At post-infection, lung Mo values were higher in BCD+Lr mice than in BCD mice and similar to those obtained in WNC group. Although both repletion treatments showed similar values of lung Ma post-infection, the Ma activation state in BCD+Lr mice was higher than that in BCD mice. Furthermore, BCD+Lr treatment was able to normalize the number and activation of splenic Ma and DC after the challenge.Lr administration stimulates respiratory and systemic mononuclear phagocytes. Stimulation of Ma and DC populations would increase the microbicide activity and improve the adaptive immunity through its antigen-presenting capacity. Thus, Lr contributes to improved outcomes of pneumococcal infection in immunocompromised hosts.

Evaluation of the Immunomodulatory Activities of the Probiotic Strain Lactobacillus fermentum UCO-979C.

Lactobacillus fermentum UCO-979C, a strain isolated from a human stomach, was previously characterized by its potential probiotic properties. The UCO-979C strain displayed the ability to beneficially regulate the innate immune response triggered by Helicobacter pylori infection in human gastric epithelial cells. In this work, we conducted further in vitro studies in intestinal epithelial cells (IECs) and in vivo experiments in mice in order to characterize the potential immunomodulatory effects of L. fermentum UCO-979C on the intestinal mucosa. Results demonstrated that the UCO-979C strain is capable to differentially modulate the immune response of IECs triggered by Toll-like receptor 4 (TLR4) activation through the modulation of TLR negative regulators' expression. In addition, we demonstrated for the first time that L. fermentum UCO-979C is able to exert its immunomodulatory effect in the intestinal mucosa in vivo. The feeding of mice with L. fermentum UCO-979C significantly increased the production of intestinal IFN-γ, stimulated intestinal and peritoneal macrophages and increased the number of Peyer's patches CD4+ T cells. In addition, L. fermentum UCO-979C augmented intestinal IL-6, reduced the number of immature B220+CD24high B cells from Peyer's patches, enhanced the number of mature B B220+CD24low cells, and significantly increased intestinal IgA content. The results of this work revealed that L. fermentum UCO-979C has several characteristics making it an excellent candidate for the development of immunobiotic functional foods aimed to differentially regulate immune responses against gastric and intestinal pathogens.

Nasal immunization with recombinant chimeric pneumococcal protein and cell wall from immunobiotic bacteria improve resistance of infant mice to Streptococcus pneumoniae infection.

Respiratory tract infections and invasive disease caused by Streptococcus pneumoniae in high-risk groups are a major global health problem. Available human vaccines have reduced immunogenicity and low immunological memory in these populations, as well as high cost as a public health strategy in poor communities. In addition, no single pneumococcal protein antigen has been able to elicit protection comparable to that achieved using protein-polysaccharide conjugate vaccines. In this context, chimeric pneumococcal proteins raise as potential good vaccine candidates because of their simplicity of production and reduced cost. The aim of this work was to study whether the nasal immunization of infant mice with the recombinant chimeric pneumococcal protein (PSFP) was able to improve resistance to S. pneumoniae, and whether the immunomodulatory strain Lactobacillus rhamnosus CRL1505 or its cell wall (CW1505) could be used as effective mucosal adjuvants. Our results showed that the nasal immunization with PSPF improved pneumococcal-specific IgA and IgG levels in broncho-alveolar lavage (BAL), reduced lung bacterial counts, and avoided dissemination of pneumococci into the blood. Of interest, immunization with PSPF elicited cross-protective immunity against different pneumococcal serotypes. It was also observed that the nasal immunization of infant mice with PSPF+CW1505 significantly increased the production of pneumococcal-specific IgA and IgG in BAL, as well as IgM and IgG in serum when compared with PSPF alone. PSPF+CW1505 immunization also improved the reduction of pneumococcal lung colonization and its dissemination in to the bloodstream when compared to PSPF alone. Our results suggest that immunization with PSPF together with the cell wall of the immunomodulatory strain L. rhamnosus CRL1505 as a mucosal adjuvant could be an interesting alternative to improve protection against pneumococcal infection in children.

Are the immunomodulatory properties of Lactobacillus rhamnosus CRL1505 peptidoglycan common for all Lactobacilli during respiratory infection in malnourished mice?

Previously, we reported that Lactobacillus rhamnosus CRL1505 peptidoglycan (PG05) improves the innate immune response in immunocompromised-malnourished mice after Streptococcus pneumoniae infection. This study extends those previous findings by demonstrating that the dietary recovery of malnourished mice with nasal administration of PG05 improves not only the innate immune response but the respiratory and systemic adaptive humoral response as well. PG05 enhanced the Th2 response, the recovery of B cells, and the concentration and opsonophagocytic activity of anti-pneumococcal antibodies. In addition, by performing comparative studies with the peptidoglycans from lactobacilli of the same species (L. rhamnosus CRL534) or with similar immunomodulatory properties (L. plantarum CRL1506), we demonstrated here that PG05 has unique immunomodulatory properties that cannot be extended to peptidoglycans from other probiotic strains. However, the knowledge of the molecular characteristics of PG05 is indispensable to understand immunomodulatory abilities of L. rhamnosus CRL1505.

Peptidoglycan from Immunobiotic Lactobacillus rhamnosus Improves Resistance of Infant Mice to Respiratory Syncytial Viral Infection and Secondary Pneumococcal Pneumonia.

Several research works have demonstrated that beneficial microbes with the capacity to modulate the mucosal immune system (immunobiotics) are an interesting alternative to improve the outcome of bacterial and viral respiratory infections. Among the immunobiotic strains with the capacity to beneficially modulate respiratory immunity, Lactobacillus rhamnosus CRL1505 has outstanding properties. Although we have significantly advanced in demonstrating the capacity of L. rhamnosus CRL1505 to improve resistance against respiratory infections as well as in the cellular and molecular mechanisms involved in its beneficial activities, the potential protective ability of this strain or its immunomodulatory cellular fractions in the context of a secondary bacterial pneumonia has not been addressed before. In this work, we demonstrated that the nasal priming with non-viable L. rhamnosus CRL1505 or its purified peptidoglycan differentially modulated the respiratory innate antiviral immune response triggered by toll-like receptor 3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that peptidoglycan from L. rhamnosus CRL1505 significantly improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-ß+ alveolar macrophages with the consequent increases of IFN-γ, IL-10, and IFN-ß in the respiratory tract. Our results also showed that the increase of these three cytokines is necessary to achieve protection against respiratory superinfection since each of them are involved in different aspect of the secondary pneumococcal pneumonia that have to be controlled in order to reduce the severity of the infectious disease: lung pneumococcal colonization, bacteremia, and inflammatory-mediated lung tissue injury.

Inorganic salts and intracellular polyphosphate inclusions play a role in the thermotolerance of the immunobiotic Lactobacillus rhamnosus CRL 1505.

In this work, the thermotolerance of Lactobacillus rhamnosus CRL1505, an immunobiotic strain, was studied as a way to improve the tolerance of the strain to industrial processes involving heat stress. The strain displayed a high intrinsic thermotolerance (55°C, 20 min); however, after 5 min at 60°C in phosphate buffer a two log units decrease in cell viability was observed. Different heat shock media were tested to improve the cell survival. Best results were obtained in the mediumcontaining inorganic salts (KH2PO4, Na2HPO4, MnSO4, and MgSO4) likely as using 10% skim milk. Flow cytometry analysis evinced 25.0% live cells and a large number of injured cells (59.7%) in the inorganic salts medium after heat stress. The morphological changes caused by temperature were visualized by transmission electronic microscopy (TEM). In addition, TEM observations revealed the presence of polyphosphate (polyP) granules in the cells under no-stress conditions. A DAPI-based fluorescence technique, adjusted to Gram-positive bacteria for the first time, was used to determine intracellular polyP levels. Results obtained suggest that the high initial polyP content in L. rhamnosus CRL 1505 together with the presence of inorganic salts in the heat shock medium improve the tolerance of the cells to heat shock. To our knowledge, this is the first report giving evidence of the relationship between polyP and inorganic salts in thermotolerance of lactic acid bacteria.

The Role of Microbiota and Immunobiotics in Granulopoiesis of Immunocompromised Hosts.

The number of granulocytes is maintained by a regulated balance between granulopoiesis in the bone marrow and clearance and destruction in peripheral tissues. Granulopoiesis plays a fundamental role in the innate immune response. Therefore, factors affecting the normal granulopoiesis lead to alterations in innate defenses and reduce the resistance against infections. In this study, we give a description on recent advances regarding the molecular and cellular events that regulate steady-state and emergency granulopoiesis, which are crucial processes for the generation of protective innate immune responses. Particular attention will be given to emergency granulopoiesis alterations in immunosuppression states caused by malnutrition and chemotherapy. The role of microbiota in maintaining a steady-state granulopoiesis and the immunological mechanisms involved are also discussed. Moreover, we describe the findings of our laboratory demonstrating that the dietary supplementation with immunobiotics is an interesting alternative to improve steady-state and emergency granulopoiesis, the respiratory innate immune response, and the resistance against respiratory pathogens in immunocompromised hosts.

Transcriptomic Analysis of the Innate Antiviral Immune Response in Porcine Intestinal Epithelial Cells: Influence of Immunobiotic Lactobacilli.

Lactobacillus rhamnosus CRL1505 and Lactobacillus plantarum CRL1506 are immunobiotic strains able to increase protection against viral intestinal infections as demonstrated in animal models and humans. To gain insight into the host-immunobiotic interaction, the transcriptomic response of porcine intestinal epithelial (PIE) cells to the challenge with viral molecular associated pattern poly(I:C) and the changes in the transcriptomic profile induced by the immunobiotics strains CRL1505 and CRL1506 were investigated in this work. By using microarray technology and reverse transcription PCR, we obtained a global overview of the immune genes involved in the innate antiviral immune response in PIE cells. Stimulation of PIE cells with poly(I:C) significantly increased the expression of IFN-α and IFN-ß, several interferon-stimulated genes, cytokines, chemokines, adhesion molecules, and genes involved in prostaglandin biosynthesis. It was also determined that lactobacilli differently modulated immune gene expression in poly(I:C)-challenged PIE cells. Most notable changes were found in antiviral factors (IFN-α, IFN-ß, NPLR3, OAS1, OASL, MX2, and RNASEL) and cytokines/chemokines (IL-1ß, IL-6, CCL4, CCL5, and CXCL10) that were significantly increased in lactobacilli-treated PIE cells. Immunobiotics reduced the expression of IL-15 and RAE1 genes that mediate poly(I:C) inflammatory damage. In addition, lactobacilli treatments increased the expression PLA2G4A, PTGES, and PTGS2 that are involved in prostaglandin E2 biosynthesis. L. rhamnosus CRL1505 and L. plantarum CRL1506 showed quantitative and qualitative differences in their capacities to modulate the innate antiviral immune response in PIE cells, which would explain the higher capacity of the CRL1505 strain when compared to CRL1506 to protect against viral infection and inflammatory damage in vivo. These results provided valuable information for the deeper understanding of the host-immunobiotic interaction and their effect on antiviral immunity. The comprehensive transcriptomic analyses successfully identified a group of genes (IFN-ß, RIG1, RNASEL, MX2, A20, IL27, CXCL5, CCL4, PTGES, and PTGER4), which can be used as prospective biomarkers for the screening of new antiviral immunobiotics in PIE cells and for the development of novel functional food and feeds, which may help to prevent viral infections.

Immunobiotic Lactobacillus strains reduce small intestinal injury induced by intraepithelial lymphocytes after Toll-like receptor 3 activation.

OBJECTIVE: Intestinal intraepithelial lymphocytes (IELs) play critical roles in disrupting epithelial homeostasis after Toll-like receptor (TLR)-3 activation with genomic rotavirus dsRNA or the synthetic dsRNA analog poly(I:C). The capacity of immunobiotic Lactobacillus rhamnosus CRL1505 (Lr1505) or Lactobacillus plantarum CRL1506 (Lp1506) to beneficially modulate IELs response after TLR3 activation was investigated in vivo using a mice model. RESULTS: Intraperitoneal administration of poly(I:C) induced inflammatory-mediated intestinal tissue damage through the increase of inflammatory cells (CD3(+)NK1.1(+), CD3(+)CD8αα(+), CD8αα(+)NKG2D(+)) and pro-inflammatory mediators (TNF-α, IL-1ß, IFN-γ, IL-15, RAE1, IL-8). Increased expression of intestinal TLR3, MDA5, and RIG-I was also observed after poly(I:C) challenge. Treatment with Lr1505 or Lp1506 prior to TLR3 activation significantly reduced the levels of TNF-α, IL-15, RAE1, and increased serum and intestinal IL-10. Moreover, CD3(+)NK1.1(+), CD3(+)CD8αα(+), and CD8αα(+)NKG2D(+) cells were lower in lactobacilli-treated mice when compared to controls. The immunomodulatory capacities of lactobacilli allowed a significant reduction of intestinal tissue damage. CONCLUSIONS: This work demonstrates the reduction of TLR3-mediated intestinal tissue injury by immunobiotic lactobacilli through the modulation of intraepithelial lymphocytes response. It is a step forward in the understanding of the cellular mechanisms involved in the antiviral capabilities of immunobiotic strains.

Draft Genome Sequence of Lactobacillus plantarum CRL1506, an Immunomodulatory Strain Isolated from Goat Milk.

This report describes a draft genome sequence of Lactobacillus plantarum CRL1506, a probiotic strain with immunomodulatory properties isolated from goat milk. The reads generated by a whole-genome shotgun (WGS) strategy on an Illumina MiSeq sequencer were assembled into contigs with a total size of 3,228,096 bp. The draft genome sequence of L. plantarum CRL1506 will be useful for further studies of specific genetic features of this strain and for understanding the mechanisms of its immunobiotic properties.