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.

The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic.

Previously, we demonstrated that the non-viable strain Lacticaseibacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulated the respiratory innate antiviral immune response triggered by Toll-like receptor (TLR)-3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In this work, we evaluated the effect of other non-viable L. rhamnosus strains and their peptidoglycans on the respiratory immune response and their impact on primary and secondary respiratory infections. In addition, the duration of the protective effect induced by NV1505 and PG1505 as well as their ability to protect against different Streptococcus pneumoniae serotypes were evaluated. Our results showed that among the five selected L. rhamnosus strains (CRL1505, CRL498, CRL576, UCO25A and IBL027), NV1505 and NVIBL027 improved the protection against viral and pneumococcal infections by modulating the respiratory immune response. Of note, only the PG1505 presented immunomodulatory activities when compared with the other purified peptidoglycans. Studies on alveolar macrophages showed that NV1505 and PG1505 differentially modulated the expression of IL-6, IFN-γ, IFN-ß, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1ß, TNF-α, CCL2, CXCL2, CXCL10 and IL-27 in response to pneumococcal challenge. Furthermore, we demonstrated that NV1505 and PG1505 treatments protected mice against secondary pneumococcal pneumonia produced by different serotypes of S. pneumoniae until 30 days after stimulation with poly(I:C). This work advances the characterization of the protective effect of NV1505 and PG1505 by demonstrating that they increase resistance against the pneumococcal serotypes 3, 6B, 14 and 19F, with an effect that lasts up to 30 days after the primary viral inflammation. The results also confirm that the immunomodulatory properties of NV1505 and PG1505 are unique and are not shared by other members of this species, and suggest the existence of a capacity to stimulate trained immunity in alveolar macrophages.

Lactobacillus casei CRL431 modulates hemostatic activation induced by protein malnourishment and pneumococcal respiratory infection.

Previously, we demonstrated that Lactobacillus casei CRL431, a well-known immunomodulatory bacterium, beneficially regulates coagulation activation, fibrin formation in lung, and the pro-inflammatory state induced by protein malnourishment and pneumococcal infection. In this study, we deepen in the understanding of the mechanisms involved in the immunoregulatory activity of L. casei CRL431 during a nutritional repletion process by evaluating (a) platelet and endothelial activation, (b) tissue factor (TF) expression, and (c) protease-activated receptor (PAR) activation in an experimental bacterial respiratory infection model in malnourished mice. Our findings demonstrate for the first time that the repletion diet supplemented with L. casei CRL431 was effective to normalize platelet counts in blood, modulate platelet activation and their recruitment into the lung, and regulate local and systemic TF expression and endothelial activation, which were affected by malnourishment. Streptococcus pneumoniae challenge induced local and systemic increase of platelet counts, PARs activation, P-selectin and TF expression, as well as endothelial activation in both well-nourished and malnourished mice. Malnourished animals evidenced the highest alterations of the parameters evaluated while the mice fed with the probiotic bacterium had similar behavior to normal controls but with lower PAR activation in lung. These results demonstrate that supplementation of repletion diet with L. casei CRL431 is effective to modulate alterations induced by malnourishment and pneumococcal infection, restraining coagulation activation, the inflammatory process, and lung damage. These observations contribute to set the basis for the application of probiotic functional foods to modulate the inflammation-hemostasis interactions altered by malnourishment or bacterial respiratory infections. KEY POINTS: • Pneumococcal infection increases pro-coagulant state induced by protein malnourishment. • Repletion with L. casei CRL431 modulates platelet, TF, and endothelial activation. • L. casei CRL431 improves immune-coagulative response in protein malnourishment.

A Novel In-House Enzyme-Linked Immunosorbent Assay for Genotype 3 Hepatitis E Virus Reveals High Seroprevalence in Blood Donors in Northern Argentina.

The Hepatitis E virus (HEV) is an emergent virus that causes acute hepatitis in immunocompetent hosts and chronic hepatitis in immunocompromised hosts. In Latin America, the main circulating genotype HEV-3 is usually of zoonotic origin. Diagnosis and seroprevalence studies mainly rely on the detection of specific antibodies. There are scarce data on the seroprevalence of HEV infection in Latin America mainly due to the lack of awareness of HEV circulation. Furthermore, in some countries, like Argentina, HEV testing is not included in routine assays. In order to provide tools to deepen the knowledge on HEV epidemiology in South America, we designed a new in-house ELISA based on the native recombinant protein ORF2 aa112-608 and demonstrated its potential for detecting anti-HEV immunoglobulin G (IgG) in human serum samples. The following conditions were determined: an optimal antigen concentration of 0.25 µg/ml, a serum dilution of 1:80, gelatin as a blocking agent, and a secondary antibody dilution of 1:2000. A relative sensitivity of 93.33% (95% CI: 77.9-99.2%) and a relative specificity of 99.4% (95% CI: 96.7-100%) were determined using a panel of previously characterized sera and a gold standard (HEV IgG ELISA, DIA.PRO, Italy). Further, we obtained a very good agreement (κ index = 0.94, 95% CI: 0.87-1.00) with the gold standard. We screened 813 blood donor samples with this newly developed ELISA and found a seroprevalence of 9.23% (95% confidence interval, 7.33-11.43%). We show for the first time evidence of past HEV infection in Tucuman, the most populated city in northern Argentina. We expect that this study will raise the interest of health decision makers who should intercede to include indirect testing of HEV in regular diagnostic protocols. In conclusion, the in-house ELISA developed in this work shows a very good agreement with an already licensed commercial HEV IgG ELISA (DIA.PRO, ITALY), which can be used as an epidemiologic tool for HEV surveillance.

Non-viable immunobiotic Lactobacillus rhamnosus CRL1505 and its peptidoglycan improve systemic and respiratory innate immune response during recovery of immunocompromised-malnourished mice.

The effect of non-viable Lactobacillus rhamnosus CRL1505 and its cell wall and peptidoglycan on respiratory immunity in malnourished mice was studied. Weaned mice were malnourished with a protein-free diet for 21d and received BCD during 7d (BCD) or BCD with nasal non-viable L. rhamnosus CRL1505 (BCD+UV) or its cell wall (BCD+CW) or peptidoglycan (BCD+PG) supplementation during last 2d of the treatment. Malnourished mice without treatment (MNC) and well-nourished mice (WNC) were used as controls. Mice were infected nasally with Streptococcus pneumoniae after treatments. Resistance against pneumococci was reduced in MNC mice. Repletion with BCD reduced lung and blood bacterial cell counts when compared to MNC mice but the counts did not reach the levels of the WNC group. However, when malnourished mice received BCD+UV, BCD+CW or BCD+PG, pneumococci was not detected in lung or blood samples. Pneumococcal infection increased the levels of TNF-α, IL-1ß, IL-6, and IL-10 in the respiratory tract, however the values were lower in MNC than in WNC mice. BCD+UV and BCD+PG groups showed values of phagocytes, IL-1ß and IL-6 that were similar to WNC mice, while TNF-α was significantly higher in those groups when compared to WNC mice. Moreover, BCD+UV and BCD+PG treatments improved levels of respiratory IL-10, reaching values that were superior to those observed in WNC mice. The work demonstrates for the first time that non-viable probiotic bacteria or their cellular fractions could be an interesting alternative as mucosal immunomodulators, especially in immunocompromised hosts in which the use of live bacteria might be dangerous.

Probiotic Lactobacillus strains protect against myelosuppression and immunosuppression in cyclophosphamide-treated mice.

This work evaluated the capacity of two probiotic strains, Lactobacillus casei CRL431 and Lactobacillus rhamnosus CRL1506, to protect against myelosuppression and immunosuppression in cyclophosphamide (Cy)-treated mice. Changes in mature granulocytes and progenitor cells in bone marrow (BM) and blood were studied. In addition, the ability of probiotics to accelerate the recovery of the immune response against the opportunistic pathogen Candida albicans was evaluated. We demonstrated for the first time that the preventive treatment with immunomodulatory lactobacilli such as L. casei CRL431 or L. rhamnosus CRL1506 was able to increase immature myeloid progenitors in the BM, allowing an early recovery of myeloid cells after Cy administration. Probiotic lactobacilli were also capable to induce an early recovery of neutrophils in blood, improve phagocytic cells recruitment to infectious sites and increase the resistance against the opportunistic pathogen C. albicans. Although deeper studies regarding the cellular and molecular mechanisms of probiotic actions are needed, these findings support the idea that strains like CRL431 and CRL1506 may accelerate the recovery of Cy-caused immunosuppression by immunopotentiating myeloid cells. Then, probiotic lactobacilli have the potential to be used as alternatives for lessening chemotherapy-induced immunosuppression in cancer patients.

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.

Immunobiotic lactobacilli reduce viral-associated pulmonary damage through the modulation of inflammation-coagulation interactions.

The exacerbated disease due to immune- and coagulative-mediated pulmonary injury during acute respiratory viruses infection results in severe morbidity and mortality. Identifying novel approaches to modulate virus-induced inflammation-coagulation interactions could be important alternatives for treating acute respiratory viruses infections. In this study we investigated the effect of the probiotic strain Lactobacillus rhamnosus CRL1505 on lung TLR3-mediated inflammation, and its ability to modulate inflammation-coagulation interaction during respiratory viral infection. Our findings reveal for the first time that a probiotic bacterium is able to influence lung immune-coagulative reaction triggered by TLR3 activation, by modulating the production of proinflammatory and anti-inflammatory cytokines as well as expression of tissue factor and thrombomodulin in the lung. We also demonstrated that the preventive treatment with the probiotic bacteria beneficially modulates the fine tune balance between clearing respiratory viruses (respiratory syncytial virus and influenza virus) and controlling immune-coagulative responses in the lung, allowing normal lung function to be maintained in the face of a viral attack. Our data also pinpoint a crucial role for IL-10 in the immune protection induced by L. rhamnosus CRL1505 during respiratory viral infections. These observations might be helpful to propose new preventive or therapeutic approaches to better control virus-inflammatory lung damage using probiotic functional foods.

Developmental expression and role of Kinesin Eg5 during Xenopus laevis embryogenesis.

BACKGROUND: The neural crest is a transient multipotent migratory cell population unique to vertebrates. These cells undergo an epithelial-to-mesenchymal transition and migrate extensively through the embryo. They differentiate into numerous diverse derivatives including the peripheral nervous system, melanocytes,and craniofacial cartilages. The development of the neural crest is mediated by complex interactions of multiple signals and transcription factors. The kinesin Eg5 is a plus end-directed microtubule-based motor protein that is essential for bipolar spindle formation during mitosis and meiosis, axon growth, and mammal embryonic development. RESULTS: We analyzed in detail the expression pattern of eg5 and established that it is expressed at the prospective neural fold, in the premigratory and migratory neural crest. Functional analysis revealed that in Xenopus, early embryogenesis eg5 function is required during neural crest induction, specification, and maintenance. eg5 is also required during neural crest migration and for derivatives formation. Moreover, we demonstrated a hierarchical relationship with the Indian Hedgehog signaling pathway. CONCLUSIONS: Our results show that eg5 is essential for the specification and maintenance of neural crest progenitors during Xenopus early embryogenesis rather than cell proliferation and survival.

Orally administered Lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(I:C).

BACKGROUND: Some studies have shown that probiotics, including Lactobacillus rhamnosus CRL1505, had the potential to beneficially modulate the outcome of certain bacterial and viral respiratory infections. However, these studies did not determine the mechanism(s) by which probiotics contribute to host defense against respiratory viruses. RESULTS: In this work we demonstrated that orally administered Lactobacillus rhamnosus CRL1505 (Lr1505) was able to increase the levels of IFN-γ, IL-10 and IL-6 in the respiratory tract and the number of lung CD3(+)CD4(+)IFN-γ(+) T cells. To mimic the pro-inflammatory and physiopathological consecuences of RNA viral infections in the lung, we used an experimental model of lung inflammation based on the administration of the artificial viral pathogen-associated molecular pattern poly(I:C). Nasal administration of poly(I:C) to mice induced a marked impairment of lung function that was accompanied by the production of pro-inflammatory mediators and inflammatory cell recruitment into the airways. The preventive administration of Lr1505 reduced lung injuries and the production of TNF-α, IL-6, IL-8 and MCP-1 in the respiratory tract after the challenge with poly(I:C). Moreover, Lr1505 induced a significant increase in lung and serum IL-10. We also observed that Lr1505 was able to increase respiratory IFN-γ levels and the number of lung CD3(+)CD4(+)IFN-γ(+) T cells after poly(I:C) challenge. Moreover, higher numbers of both CD103(+) and CD11b(high) dendritic cells and increased expression of MHC-II, IL-12 and IFN-γ in these cell populations were found in lungs of Lr1505-treated mice. Therefore, Lr1505 treatment would beneficially regulate the balance between pro-inflammatory mediators and IL-10, allowing an effective inflammatory response against infection and avoiding tissue damage. CONCLUSIONS: Results showed that Lr1505 would induce a mobilization of cells from intestine and changes in cytokine profile that would be able to beneficially modulate the respiratory mucosal immunity. Although deeper studies are needed using challenges with respiratory viruses, the results in this study suggest that Lr1505, a potent inducer of antiviral cytokines, may be useful as a prophylactic agent to control respiratory virus infection.

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.