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IntroductionLevilactobacillus brevis CRL 2013, a plant-derived lactic acid bacterium (LAB) with immunomodulatory properties, has emerged as an efficient producer of γ-aminobutyric acid (GABA). Notably, not all LAB possess the ability to produce GABA, highlighting the importance of specific genetic and environmental conditions for GABA synthesis. This study aimed to elucidate the intriguing GABA-producing machinery of L. brevis CRL 2013 and support its potential for safe application through comprehensive genome analysis.MethodsA comprehensive genome analysis of L. brevis CRL 2013 was performed to identify the presence of antibiotic resistance genes, virulence markers, and genes associated with the glutamate decarboxylase system, which is essential for GABA biosynthesis. Then, an optimized chemically defined culture medium (CDM) was supplemented with monosodium glutamate (MSG) and yeast extract (YE) to analyze their influence on GABA production. Proteomic and transcriptional analyses were conducted to assess changes in protein and gene expression related to GABA production.ResultsThe absence of antibiotic resistance genes and virulence markers in the genome of L. brevis CRL 2013 supports its safety for potential probiotic applications. Genes encoding the glutamate decarboxylase system, including two gad genes (gadA and gadB) and the glutamate antiporter gene (gadC), were identified. The gadB gene is located adjacent to gadC, while gadA resides separately on the chromosome. The transcriptional regulator gadR was found upstream of gadC, with transcriptional analyses demonstrating cotranscription of gadR with gadC. Although MSG supplementation alone did not activate GABA synthesis, the addition of YE significantly enhanced GABA production in the optimized CDM containing glutamate. Proteomic analysis revealed minimal differences between MSG-supplemented and non-supplemented CDM cultures, whereas YE supplementation resulted in significant proteomic changes, including upregulation of GadB. Transcriptional analysis confirmed increased expression of gadB and gadR upon YE supplementation, supporting its role in activating GABA production.ConclusionThese findings provide valuable insights into the influence of nutrient composition on GABA production. Furthermore, they unveil the potential of L. brevis CRL 2013 as a safe, nonpathogenic strain with valuable biotechnological traits which can be further leveraged for its probiotic potential in the food industry.

The emergence and spread of antibiotic resistance threat forced to explore alternative strategies for improving the resistance to pathogens in livestock production. Probiotic lactic acid bacteria represent an alternative for this objective. In this study, seven Lactiplantibacillus plantarum strains from porcine colostrum and milk were isolated, identified and characterized in terms of their abilities to modulate immunity in porcine intestinal epithelial (PIE) cells. Then, two potential immunoregulatory strains were studied in terms of their ability to utilize and grow in wakame (Undaria pinnafida). Isolates were identified by 16S rRNA gene and evaluated by studying their interaction with PIE cells. The expressions of peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain (NODs), host defense peptides (pBD), and type I interferons (IFNs) were evaluated by RT-qPCR. The strain 4M4417 showed a remarkable capacity to differentially regulate the expression of PGRP1, PGRP3, NOD1, NOD2, and pBD1 in PIE cells. On the other hand, the strain 4M4326 was the most efficient to improve the expression of IFN-α and IFN-β in PIE cells challenged with poly (I:C). Both L. plantarum 4M4326 and 4M4417 were characterized in terms of their ability to utilize wakame. Results demonstrated that both strains efficiently grew in wakame-based broth. Our results suggest that L. planatrum 4M4326 and 4M4417 are interesting candidates to develop immunomodulatory feeds based on wakame utilization. These new immunosynbiotic feeds could help to reduce severity of intestinal infections and improve immune health status in pigs.

Early child maltreatment, such as child abuse and neglect, is well known to affect the development of social skills. However, the mechanisms by which such an adverse environment interrupts the development of social skills remain unelucidated. Identifying the period and brain regions that are susceptible to adverse environments can lead to appropriate developmental care later in life. We recently reported an excitatory/inhibitory imbalance and low activity during social behavior in the medial prefrontal cortex (mPFC) of the maternal separation (MS) animal model of early life neglect after maturation. Based on these results, in the present study, we investigated how MS disturbs factors related to excitatory and inhibitory neurons in the mPFC until the critical period of mPFC development. Additionally, we evaluated whether the effects of MS could be recovered in an enriched environment after MS exposure. Rat pups were separated from their dams on postnatal days (PDs) 2–20 (twice daily, 3 h each) and compared with the mother-reared control (MRC) group. Gene expression analysis revealed that various factors related to excitatory and inhibitory neurons were transiently disturbed in the mPFC during MS. A similar tendency was found in the sensory cortex; however, decreased parvalbumin (PV) expression persisted until PD 35 only in the mPFC. Moreover, the number of PV+ interneurons decreased in the ventromedial prefrontal cortex (vmPFC) on PD 35 in the MS group. Additionally, perineural net formation surrounding PV+ interneurons, which is an indicator of maturity and critical period closure, was unchanged, indicating that the decreased PV+ interneurons were not simply attributable to developmental delay. This reduction of PV+ interneurons improved to the level observed in the MRC group by the enriched environment from PD 21 after the MS period. These results suggest that an early adverse environment disturbs the development of the mPFC but that these abnormalities allow room for recovery depending on the subsequent environment. Considering that PV+ interneurons in the mPFC play an important role in social skills such as empathy, an early rearing environment is likely a very important factor in the subsequent acquisition of social skills.

BackgroundThe neural system plays a critical role in controlling gut immunity, and the gut microbiota contributes to this process. However, the roles and mechanisms of gut-brain-microbiota interactions remain unclear. To address this issue, we employed Drosophila as a model organism. We have previously shown that NP3253 neurons, which are connected to the brain and gut, are essential for resistance to oral bacterial infections. Here, we aimed to investigate the role of NP3253 neurons in the regulation of gut immunity.MethodsWe performed RNA-seq analysis of the adult Drosophila gut after genetically inactivating the NP3253 neurons. Flies were reared under oral bacterial infection and normal feeding conditions. In addition, we prepared samples under germ-free conditions to evaluate the role of the microbiota in gut gene expression. We knocked down the genes regulated by NP3253 neurons and examined their susceptibility to oral bacterial infections.ResultsWe found that immune-related gene expression was upregulated in NP3253 neuron-inactivated flies compared to the control. However, this upregulation was abolished in axenic flies, suggesting that the immune response was abnormally activated by the microbiota in NP3253 neuron-inactivated flies. In addition, redox-related gene expression was downregulated in NP3253 neuron-inactivated flies, and this downregulation was also observed in axenic flies. Certain redox-related genes were required for resistance to oral bacterial infections, suggesting that NP3253 neurons regulate the redox responses for gut immunity in a microbiota-independent manner.ConclusionThese results show that NP3253 neurons regulate the appropriate gene expression patterns in the gut and contribute to maintain homeostasis during oral infections.

ObjectiveThe aim of the study is to discuss the efficacy of live vs. remote cadaver surgical training (CST) for minimally invasive surgery (MIS).MethodsA cohort of 30 interns in their first and second years of training were divided into three groups: live observers (n = 12), live participants (n = 6), and remote observers: (n = 12). The interns had the opportunity to either observe or actively participate in two different surgical procedures, namely, laparoscopic lower anterior resection, performed by a colorectal surgical team, and laparoscopic fundoplication, performed by a pediatric surgical team. The procedures were conducted either at a base center or at a remote center affiliated with the institute. Some of the interns interacted directly with the surgical teams at the base center, and others interacted indirectly with the surgical teams from the remote center. All interns were administered questionnaires before and after completion of the CST in order to assess their understanding of various aspects related to the operating room layout/instruments (called “design”), accessing the surgical field (called “field”), understanding of anatomic relations (called “anatomy”), their skill of dissection (called “dissection”), ability to resolve procedural/technical problems (called “troubleshooting”), and their skill in planning surgery (called “planning”) according to their confidence to operate using the following scale: 1 = not confident to operate independently; 4 = confident to operate with a more senior trainee; 7 = confident to operate with a peer; and 10 = confident to operate with a less experienced trainee. A p

AimsEarly postoperative outcome (EPO) was compared between fully laparoscopic Duhamel-Z (F-Dz) and laparoscopy-assisted Duhamel-Z (A-Dz) anastomoses performed for total colonic aganglionosis (TCA).MethodsEPO was assessed quarterly for the first year after F-Dz/A-Dz using a continence evaluation score (CES) based on stool frequency (motions/day) and stool consistency (0 = liquid, 1 = soft, 2 = formed), presence of anal erosion (0 = severe, 1 = moderate, 2 = mild), and incidence of enterocolitis.Surgical technique involved taking the ileostomy down, dissecting the colon laparoscopically, and preparing the pull-through ileum through the stoma wound. In F-Dz (n = 3), a working port (SILS trocar) was inserted, and laparoscopic retrorectal dissection with forceps used to create a retrorectal tunnel from the peritoneal reflection extending downward as narrow as possible along the posterior wall of the rectum to prevent lateral nerve injury and preserve vascularity. After completing the tunnel, the ileum was pulled-through from an incision on the anorectal line and a Z-shaped ileorectal side-to-side anastomosis performed without a blind pouch. In A-Dz (n = 11), the retrorectal pull-through route was created through a Pfannenstiel incision using blunt manual (finger) dissection along the anterior surface of the sacrum.ResultsSubject backgrounds were similar. Mean quarterly data were: frequency (F-Dz: 4.67, 4.67, 4.67, 3.33) vs. (A-Dz: 7.27, 7.09, 6.18, 5.36) p

In vitro culture models that precisely mirror the porcine respiratory epithelium are needed to gain insight into how pathogens and host interact. In this study, a new porcine bronchial epithelial cell line, designated as PBE cells, was established from the respiratory tract of a neonatal pig. PBE cells assumed a cobblestone-epithelial like morphology with close contacts between the cells when they reached confluence. The PBE cell line was characterized in terms of its expression of pattern recognition receptors (PRRs) and its ability to respond to the activation of the Toll-like receptor 3 (TLR3) and TLR4 signaling pathways, which are key PRRs involved in the defense of the respiratory epithelium against pathogens. PBE cells stimulated with poly(I:C) were able to up-regulate the expression of IFN-β, IFN-λ1 (IL-29), IFN-λ3 (IL-28B), the antiviral factors Mx1, OAS1, and PKR, as well as the viral PRRs RIG-1 and MDA5. The expression kinetics studies of immune factors in PBE cells allow us to speculate that this cell line can be a useful in vitro tool to investigate treatments that help to potentiate antiviral immunity in the respiratory epithelium of the porcine host. In addition, poly(I:C) and LPS treatments increased the expression of the inflammatory cytokines TNF-α, IL-6, IL-8, and MCP-1/CCL2 and differentially modulated the expression of negative regulators of the TLR signaling pathways. Then, PBE cells may also allow the evaluation of treatments that can regulate TLR3- and TLR4-mediated inflammatory injury in the porcine airway, thereby protecting the host against harmful overresponses.

BackgroundThe mortality rate due to COVID-19 in kidney transplant recipients (KTRs) is 16.8 to 32%. Vaccination against COVID-19 is expected to contribute to the prevention of infection, severe disease, and mortality; however, it has been reported that the humoral response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine in KTRs is poor. Vaccination strategies against COVID-19 vary from country to country, and in Japan, the third dose is given 6 months after the second dose. Few studies have evaluated long-term humoral responses after the second dose of SARS-CoV-2 mRNA vaccine. In addition, the superiority of BNT162b2 vaccine and mRNA-1,273 vaccine in KTRs regarding humoral response is controversial.MethodsNinety-four KTRs were administered a second dose of the BNT162b2 or mRNA-1,273 vaccines, and anti-spike (anti-S) and anti-nucleocapsid (anti-N) SARS-CoV-2 antibody levels were measured 5 months (149.2 ± 45.5 days) later. The cutoff value of anti-S antibodies was defined ≥50 AU/ml and 1.4 Index for anti-N antibodies. The primary outcome was the rate of seropositivity, and factors associated with an appropriate humoral response were assessed by univariate and multivariate analysis.ResultsOf 94 KTRs, only 45 (47.9%) patients were positive for anti-S antibodies. The median anti-S SARS-CoV-2 IgG antibody titers was 35.3 (Interquartile range 3.8 to 159.7). Anti-N SARS-CoV-2 IgG antibodies in all patients were

ObjectiveHereditary transthyretin amyloidosis (ATTRv) encompasses different phenotypes among various genotypes. The analysis of the natural history and risk factors of faster progression in different genotypes would refine the treatment strategy.MethodsThe clinical manifestations of ATTRv from A97S (p.A117S) of Taiwanese and late-onset V30M (p.V50M) of Japanese were compared. An autopsy study of A97S was performed.ResultsThere existed three unique features in the A97S cohort compared to the V30M cohort: (1) dysphagia, (2) carpal tunnel syndrome (CTS), and (3) onset age. First, dysphagia was common in A97S (53.4%) but not in V30M and served as a contributor to fast disease progression. All phases of swallowing were affected. In the autopsy pathology, there were extensive amyloid deposits in the viscera and nerves of the tongue, larynx, and esophagus. In A97S, 45 patients (43.3%) had a history of CTS before the onset of length-dependent symptoms by 3 years. The amyloid deposition was more prominent in the median nerve than that in the transverse carpal ligament. The onset age at different stages was younger in the A97S cohort than the V30M cohort by 4–5 years.ConclusionThese phenotypic characteristics together with autopsy pathology in A97S are distinct from V30M. Early dysphagia in A97S correlated with fast progression. In A97S, median neuropathy leading to CTS might be in a continuous spectrum of ATTRv course rather than an independent disease entity. Such observations may serve as a foundation to explore and analyze unique phenotypes among various genotypes.

Abstract Systemic sclerosis (SSc) is a multi‐system autoimmune disease. Anti‐neutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against enzymes found within primary granules of neutrophils and lysosomes in monocytes. Although up to 12% of SSc patients have ANCA, only a minority of these patients develop an overlap syndrome with ANCA‐associated vasculitis. We summarize previous reports on SSc patients with ANCA‐associated neuropathy. In all the reported cases, the SSc diagnosis preceded the ANCA‐associated neuropathy diagnosis. Seven of the eight patients with limited cutaneous SSc had interstitial lung disease (ILD). Thus, patients with ANCA‐associated neuropathy in lSSc may be prone to complication with ILD.

Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-β expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-β, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-β, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets.

Potential probiotic or immunobiotic effects of lactic acid bacteria (LAB) isolated from the milk of the South American camelid llama (Lama glama) have not been reported in published studies. The aim of the present work was to isolate beneficial LAB from llama milk that can be used as potential probiotics active against bacterial pathogens. LAB strains were isolated from llama milk samples. In vitro functional characterization of the strains was performed by evaluating the resistance against gastrointestinal conditions and inhibition of the pathogen growth. Additionally, the adhesive and immunomodulatory properties of the strains were assessed. The functional studies were complemented with a comparative genomic evaluation and in vivo studies in mice. Ligilactobacillus salivarius TUCO-L2 showed enhanced probiotic/immunobiotic potential compared to that of other tested strains. The TUCO-L2 strain was resistant to pH and high bile salt concentrations and demonstrated antimicrobial activity against Gram-negative intestinal pathogens and adhesion to mucins and epithelial cells. L. salivarius TUCO-L2 modulated the innate immune response triggered by Toll-like receptor (TLR)-4 activation in intestinal epithelial cells. This effect involved differential regulation of the expression of inflammatory cytokines and chemokines mediated by the modulation of the negative regulators of the TLR signaling pathway. Moreover, the TUCO-L2 strain enhanced the resistance of mice to Salmonella infection. This is the first report on the isolation and characterization of a potential probiotic/immunobiotic strain from llama milk. The in vitro, in vivo, and in silico investigation performed in this study reveals several research directions that are needed to characterize the TUCO-L2 strain in detail to position this strain as a probiotic or immunobiotic that can be used against infections in humans or animals, including llama.

Immunobiotics have emerged as a promising intervention to alleviate intestinal damage in inflammatory bowel disease (IBD). However, the beneficial properties of immunobiotics are strain dependent and, therefore, each strain has to be evaluated in order to demonstrate its potential application in IBD. Our previous in vitro and in vivo studies demonstrated that Lactobacillus jensenii TL2937 attenuates gut acute inflammatory response triggered by Toll-like receptor 4 activation. However, its effect on colitis has not been evaluated before. In this work, we studied whether the TL2937 strain was able to protect against the development of colitis in a dextran sodium sulfate (DSS)-induced mouse model and we delved into the mechanisms of action by evaluating the effect of the immunobiotic bacteria on the transcriptomic response of DSS-challenged intestinal epithelial cells. L. jensenii TL2937 was administered to adult BALB/c mice before the induction of colitis by the administration of DSS. Colitis and the associated inflammatory response were evaluated for 14 days. Mice fed with L. jensenii TL2937 had lower disease activity index and alterations of colon length when compared to control mice. Reduced myeloperoxidase activity, lower production of pro-inflammatory (TNF-α, IL-1, CXCL1, MCP-1, IL-15, and IL-17), and higher levels of immunoregulatory (IL-10 and IL-27) cytokines were found in the colon of TL2937-treated mice. In addition, the treatment of porcine intestinal epithelial (PIE) cells with L. jensenii TL2937 before the challenge with DSS differentially regulated the activation of the JNK pathway, leading to an increase in epithelial cell integrity and to a differential immunotranscriptomic response. TL2937-treated PIE cells had a significant reduction in the expression of inflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6, IL-15), chemokines (CCL2, CCL4, CCL8, CXCL4, CXCL5, CXCL9, CXCL10), adhesion molecules (SELE, SELL, EPCAM), and other immune factors (NCF1, NCF2, NOS2, SAA2) when compared to control cells after the challenge with DSS. The findings of this work indicate that (a) L. jensenii TL2937 is able to alleviate DSS-induced colitis suggesting a potential novel application for this immunobiotic strain, (b) the modulation of the transcriptomic response of intestinal epithelial cells would play a key role in the beneficial effects of the TL2937 strain on colitis, and (c) the in vitro PIE cell immunoassay system could be of value for the screening and selection of new immunobiotic strains for their application in IBD.

Background: Recurrence of IgA nephropathy (IgAN) in the transplanted kidney is associated with graft survival, but no specific treatment is available. Tonsillectomy (TE) reportedly arrests the progression of IgAN in the native kidney. Thus, we conducted a single-center retrospective cohort study to evaluate the effect of TE prior to IgAN recurrence.Methods: Of the 36 patients with biopsy-proven IgAN who underwent kidney transplantation, 27 were included in this study. Nine patients underwent TE at 1 year after kidney transplantation (group 1), and the remaining 18 did not undergo TE (group 2).Results: The rate of histological IgAN recurrence was significantly lower in group 1 than in group 2 (11.1 vs. 55.6%, log-rank p = 0.046). In addition, half of the recurrent patients in group 2 exhibited active lesions, compared to none in group 1. Serum Gd-IgA1 levels decreased after TE in group 1, whereas they remained stable or increased slightly in group 2. In the recurrent cases, IgA and Gd-IgA1 were found in the germinal center in addition to the mantle zone of tonsils. Finally, mesangial IgA and Gd-IgA1 immunoreactivity was reduced after TE in some cases.Conclusion: Our data suggest that TE at 1 year after kidney transplantation might be associated with the reduced rate of histological IgAN recurrence. TE arrested or reduced serum Gd-IgA1 and mesangial Gd-IgA1 immunoreactivity. Therefore, we generated a hypothesis that serum Gd-IgA1 derived from the tonsils may play a pivotal role in the pathogenesis of IgAN. Based on these findings, we need to conduct verification in a prospective randomized controlled trial.

The oral administration of Lacticaseibacillus rhamnosus CRL1505 differentially modulates the respiratory innate antiviral immune response triggered by Toll-like receptor 3 (TLR3) activation in infant mice, improving the resistance to Respiratory Syncytial Virus (RSV) infection. In this work, by using macrophages depletion experiments and a detailed study of their production of cytokines and antiviral factors we clearly demonstrated the key role of this immune cell population in the improvement of both viral elimination and the protection against lung tissue damage induced by the CRL1505 strain. Orally administered L. rhamnosus CRL1505 activated alveolar macrophages and enhanced their ability to produce type I interferons (IFNs) and IFN-γ in response to RSV infection. Moreover, an increased expression of IFNAR1, Mx2, OAS1, OAS2, RNAseL, and IFITM3 was observed in alveolar macrophages after the oral treatment with L. rhamnosus CRL1505, which was consistent with the enhanced RSV clearance. The depletion of alveolar macrophages by the time of L. rhamnosus CRL1505 administration abolished the ability of infant mice to produce increased levels of IL-10 in response to RSV infection. However, no improvement in IL-10 production was observed when primary cultures of alveolar macrophages obtained from CRL1505-treated mice were analyzed. Of note, alveolar macrophages from the CRL1505 group had an increased production of IL-6 and IL-27 suggesting that these cells may play an important role in limiting inflammation and protecting lung function during RSV infection, by increasing the maturation and activation of Treg cells and their subsequent production of IL-10. In addition, we provided evidence of the important role of CD4+ cells and IFN-γ in the activation of alveolar macrophages highlighting a putative pathway through which the intestinal and respiratory mucosa are communicated under the influence of L. rhamnosus CRL1505.

Fucoidan represents fucose-rich sulfated polysaccharides derived from brown seaweeds, which exerts various biological activities applicable for functional foods and therapeutic agents. The objective of the present study was to investigate in vivo effects of fucoidan extracted from Okinawa mozuku (Cladosiphon okamuranus), common edible seaweed in Japan, on immune responses and microbiota composition in zebrafish. We treated larvae and adult zebrafish with Okinawa mozuku (OM) fucoidan by immersion (100 and 500 μg/mL, 3 days) and by feeding (3 weeks), respectively. The effect of OM fucoidan on immune responses in zebrafish larvae was evaluated by live imaging of neutrophils and macrophages as well as quantitative polymerase chain reaction of pro- and anti-inflammatory cytokine genes. Whole microbiota of zebrafish larvae and intestinal microbiota of adult zebrafish treated with OM fucoidan were analyzed by Illumina MiSeq pair-end sequencing of the V3–V4 region of 16S rRNA genes. Fucoidan treatment only slightly affected the composition of the larvae microbiota and the number of neutrophils and macrophages, while pro- and anti-inflammatory cytokine gene expression levels were upregulated in the larvae treated with 500 μg/mL OM fucoidan. In contrast, feeding of OM fucoidan clearly altered the intestinal microbiota composition of adult zebrafish, which was characterized by the emergence and predominance of multiple bacterial operational taxonomic units (OTUs) affiliated with Rhizobiaceae and Comamonadaceae at the expense of E. coli-related Enterobacteriaceae, the dominant OTUs throughout the studied samples. These changes were accompanied by decreased expression levels of pro-inflammatory cytokine il1b in the intestines of the adult zebrafish. Our current study provides the first insights into in vivo modulatory effects of fucoidan on microbiota and immune responses of unchallenged zebrafish, which underscores the potential of fucoidan to play a modulatory role in the diet–microbiota–host interplay.

Viral respiratory infections are of major importance because of their capacity to cause of a high degree of morbidity and mortality in high-risk populations, and to rapidly spread between countries. Perhaps the best example of this global threat is the infectious disease caused by the new SARS-CoV-2 virus, which has infected more than 4 million people worldwide, causing the death of 287,000 persons according to the WHO's situation report on May 13, 2020. The availability of therapeutic tools that would be used massively to prevent or mitigate the detrimental effects of emerging respiratory viruses on human health is therefore mandatory. In this regard, research from the last decade has reported the impact of the intestinal microbiota on the respiratory immunity. It was conclusively demonstrated how the variations in the intestinal microbiota affect the responses of respiratory epithelial cells and antigen presenting cells against respiratory virus attack. Moreover, the selection of specific microbial strains (immunobiotics) with the ability to modulate immunity in distal mucosal sites made possible the generation of nutritional interventions to strengthen respiratory antiviral defenses. In this article, the most important characteristics of the limited information available regarding the immune response against SARS-CoV-2 virus are revised briefly. In addition, this review summarizes the knowledge on the cellular and molecular mechanisms involved in the improvement of respiratory antiviral defenses by beneficial immunobiotic microorganisms such as Lactobacillus rhamnosus CRL1505. The ability of beneficial microorganisms to enhance type I interferons and antiviral factors in the respiratory tract, stimulate Th1 response and antibodies production, and regulate inflammation and coagulation activation during the course of viral infections reducing tissue damage and preserving lung functionally, clearly indicate the potential of immunobiotics to favorably influence the immune response against SARS-CoV-2 virus.

It was reported that exopolysaccharides (EPSs) from lactobacilli are able to differentially modulate mucosal antiviral immunity. Although research has described the ability of EPSs derived from Streptococcus thermophilus to modulate the mucosal immune system, their impact on antiviral immunity was less explored. In this work, we investigated the capacity of the EPS-producing S. thermophilus ST538 to modulate the innate antiviral immune response triggered by the activation of the Toll-like receptor 3 (TLR3) in porcine intestinal epitheliocytes (PIE cells). Moreover, in order to study the immunomodulatory potential of S. thermophilus ST538 EPS, we successfully developed two mutant strains through the knockout of the epsB or epsC genes. High-performance liquid chromatography and scanning electron microscopy studies demonstrated that the wild type (WT) strain produced as high as 595 μg/ml of EPS in the skim milk medium, while none of the mutant strains (S. thermophilus ΔepsB and ΔepsC) were able to produce EPS. Studies in PIE cells demonstrated that the EPS of S. thermophilus ST538 is able to significantly improve the expression of interferon β (IFN-β), interleukin 6 (IL-6), and C-X-C motif chemokine 10 (CXCL10) in response to TLR3 stimulation. The role of EPS in the modulation of antiviral immune response in PIE cells was confirmed by comparative studies of cell free culture supernatants and fermented skim milks obtained from S. thermophilus ΔepsB and ΔepsC. These results suggest that S. thermophilus ST538 could be used as an immunobiotic strain for the development of new immunologically functional foods, which might contribute to improve resistance against viral infections.

Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNβ, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-β and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells.

Studies have demonstrated that lipoteichoic acid (LTA) is involved in the immunomodulatory properties of some immunobiotic lactobacilli. The aim of this work was to evaluate whether LTA contributes to the capacity of Lactobacillus plantarum CRL1506 in modulating the intestinal innate antiviral immune response. A D-alanyl-lipoteichoic acid biosynthesis protein (dltD) knockout CRL1506 strain (L. plantarumΔdltD) was obtained, and its ability to modulate Toll-like receptor (TLR)-3-mediated immune response was evaluated in vitro in porcine intestinal epithelial (PIE) cells and in vivo in Balb/c mice. Wild-type (WT) CRL1506 (L. plantarum WT) was used as positive control. The challenge of PIE cells with the TLR3 agonist poly(I:C) significantly increased interferon (IFN)-β, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 expressions. PIE cells pretreated with L. plantarumΔdltD or L. plantarum WT showed higher levels of IFN-β while only L. plantarum WT significantly reduced the expression of IL-6 and MCP-1 when compared with poly(I:C)-treated control cells. The oral administration of L. plantarum WT to mice prior the intraperitoneal injection of poly(I:C) significantly increased IFN-β and IL-10 and reduced intraepithelial lymphocytes (CD3+NK1.1+CD8αα+) and pro-inflammatory mediators (TNF-α, IL-6, and IL-15) in the intestinal mucosa. Similar to the WT strain, L. plantarumΔdltD-treated mice showed enhanced levels of IFN-β after poly(I:C) challenge. However, treatment of mice with L. plantarumΔdltD was not able to increase IL-10 or reduce CD3+NK1.1+CD8αα+ cells, TNF-α, IL-6, or IL-15 in the intestine. These results indicate that LTA would be a key molecule in the anti-inflammatory effect induced by the CRL1506 strain in the context of TLR3-mediated inflammation.

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.

Plant viruses are thought to be essentially harmful to the lives of their cultivated crop hosts. In most cases studied, the interaction between viruses and cultivated crop plants negatively affects host morphology and physiology, thereby resulting in disease. Native wild/non-cultivated plants are often latently infected with viruses without any clear symptoms. Although seemingly non-harmful, these viruses pose a threat to cultivated crops because they can be transmitted by vectors and cause disease. Reports are accumulating on infections with latent plant viruses that do not cause disease but rather seem to be beneficial to the lives of wild host plants. In a few cases, viral latency involves the integration of full-length genome copies into the host genome that, in response to environmental stress or during certain developmental stages of host plants, can become activated to generate and replicate episomal copies, a transition from latency to reactivation and causation of disease development. The interaction between viruses and host plants may also lead to the integration of partial-length segments of viral DNA genomes or copy DNA of viral RNA genome sequences into the host genome. Transcripts derived from such integrated viral elements (EVEs) may be beneficial to host plants, for example, by conferring levels of virus resistance and/or causing persistence/latency of viral infections. Studies on viral latency in wild host plants might help us to understand and elucidate the underlying mechanisms of latency and provide insights into the raison d’être for viruses in the lives of plants.

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.

Adipocytes are the most important cell type in adipose tissue playing key roles in immunometabolism. We previously reported that nine members of the Toll-like receptor (TLR) family are expressed in an originally established porcine intramuscular pre-adipocyte (PPI) cell line. However, the ability of TLR ligands to modulate immunometabolic transcriptome modifications in porcine adipocytes has not been elucidated. Herein, we characterized the global transcriptome modifications in porcine intramuscular mature adipocytes (pMA), differentiated from PPI, following stimulation with Pam3csk4, Poly(I:C) or LPS which are ligands for TLR2, TLR3, and TLR4, respectively. Analysis of microarray data identified 530 (218 up, 312 down), 520 (245 up, 275 down), and 525 (239 up, 286 down) differentially expressed genes (DEGs) in pMA following the stimulation with Pam3csk4, Poly(I:C), and LPS, respectively. Gene ontology classification revealed that DEGs are involved in several biological processes including those belonging to immune response and lipid metabolism pathways. Functionally annotated genes were organized into two groups for downstream analysis: immune response related genes (cytokines, chemokines, complement factors, adhesion molecules, and signal transduction), and genes involved with metabolic and endocrine functions (hormones and receptors, growth factors, and lipid biosynthesis). Differential expression analysis revealed that EGR1, NOTCH1, NOS2, TNFAIP3, TRAF3IP1, INSR, CXCR4, PPARA, MAPK10, and C3 are the top 10 commonly altered genes of TLRs induced transcriptional modification of pMA. However, the protein-protein interaction network of DEGs identified EPOR, C3, STAR, CCL2, and SAA2 as the major hub genes, which were also exhibited higher centrality estimates in the Gene-Transcription factor interaction network. Our results provide new insights of transcriptome modifications associated with TLRs activation in porcine adipocytes and identified key regulatory genes that could be used as biomarkers for the evaluation of treatments having immunomodularoty and/or metabolic functional beneficial effects in porcine adipocytes.

Modern animal and crop production practices are associated with the regular use of antimicrobials, potentially increasing selection pressure on bacteria to become resistant. Alternative approaches are needed in order to satisfy the demands of the growing human population without the indiscriminate use of antimicrobials. Researchers have brought a different perspective to solve this problem and have emphasized the exploitation of animal- and plant-associated microorganisms that are beneficial to their hosts through the modulation of the innate immune system. There is increasing evidence that plants and animals employ microbial perception and defense pathways that closely resemble each other. Formation of pattern recognition receptor (PRR) complexes involving leucine-rich repeat (LRR)-containing proteins, mitogen-activated protein kinase (MAPK)-mediated activation of immune response genes, and subsequent production of antimicrobial products and reactive oxygen species (ROS) and nitric oxide (NO) to improve defenses against pathogens, add to the list of similarities between both systems. Recent pioneering work has identified that animal and plant cells use similar receptors for sensing beneficial commensal microbes that are important for the maintenance of the host's health. Here, we reviewed the current knowledge about the molecular mechanisms involved in the recognition of pathogenic and commensal microbes by the innate immune systems of animal and plants highlighting their differences and similarities. In addition, we discuss the idea of using beneficial microbes to modulate animal and plant immune systems in order to improve the resistance to infections and reduce the use of antimicrobial compounds.

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-β, Myxovirus resistance gene A (MxA) and RNaseL.

The scientific community has reported several cases of microbes that exhibit elevated rates of antibiotic resistance in different regions of the planet. Due to this emergence of antimicrobial resistant microorganisms, the use of antibiotics as promoters of livestock animals’ growth is being banned in most countries around the world. One of the challenges of agricultural immunology therefore is to find alternatives by modulating the immune system of animals in drug-independent safe food production systems. In this regard, in an effort to supplant antibiotics from bovine feeds, several alternatives were proposed including the use of immunomodulatory probiotics (immunobiotics). The purpose of this review is to provide an update of the status of the modulation of intestinal antiviral innate immunity of the bovine host by immunobiotics, and the beneficial impact of immunobiotics on viral infections, focused on intestinal epithelial cells (IECs). The results of our group, which demonstrate the capacity of immunobiotic strains to beneficially modulate Toll-like receptor 3-triggered immune responses in bovine IECs and improve the resistance to viral infections, are highlighted. This review provides comprehensive information on the innate immune response of bovine IECs against virus, which can be further investigated for the development of strategies aimed to improve defenses in the bovine host.

Corynebacterium pseudodiphtheriticum is a Gram-positive bacterium found as a member of the normal microbiota of the upper respiratory tract. It was suggested that C. pseudodiphtheriticum may be potentially used as a next-generation probiotic for nasal application, although no deep studies were performed in this regard. We hypothesized that human isolate C. pseudodiphtheriticum strain 090104 is able to modulate the respiratory innate immune response and beneficially influence the resistance to viral and bacterial infections. Therefore, in the present study we investigated how the exposure of infant mice to nasal priming with viable or non-viable C. pseudodiphtheriticum 090104 influences the respiratory innate immune response triggered by Toll-like receptor (TLR)-3 activation, the susceptibility to primary Respiratory Synsytial Virus (RSV) infection, and the resistance to secondary Streptococcus pneumoniae pneumonia. We demonstrated that the nasal priming with viable C. pseudodiphtheriticum 090104 differentially modulated TLR3-mediated innate antiviral immune response in the respiratory tract of infant mice, improving their resistance to primary RSV infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that viable C. pseudodiphtheriticum improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-β+ alveolar macrophages. Of interest, non-viable bacteria did not have the same protective effect, suggesting that C. pseudodiphtheriticum colonization is needed for achieving its protective effect. In conclusion, we present evidence that nasal application of viable C. pseudodiphtheriticum could be thought as an alternative to boost defenses against RSV and secondary pneumococcal pneumonia, which should be further studied and validated in clinical trials. Due to the absence of a long-lasting immunity, re-infection with RSV throughout life is common. Thus, a possible perspective use could be a seasonal application of a nasal probiotic spray to boost respiratory innate immunity in immunocompetent subjects.

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-β+ alveolar macrophages with the consequent increases of IFN-γ, IL-10, and IFN-β 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.

The mucosal tissues of the gastrointestinal tract are the main portal entry of pathogens such as rotavirus (RVs), which is a leading cause of death due to diarrhea among young children across the globe and a major cause of severe acute intestinal infection in livestock animals. The interactions between intestinal epithelial cells (IECs) and immune cells with RVs have been studied for several years, and now it is known that the innate immune responses triggered by this virus can have both beneficial and detrimental effects for the host. It was demonstrated that natural RVs infection in infants and experimental challenges in mice result in the intestinal activation of pattern recognition receptors (PRRs) like Toll-like receptor 3 (TLR3) and striking secretion of pro-inflammatory mediators that can lead to increased local tissue damage and immunopathology. Therefore, modulating desregulated intestinal immune responses triggered by PRRs activation are a significant promise for reducing the burden of RVs diseases. The ability of immunoregulatory probiotic microorganisms (immunobiotics) to protect against intestinal infections such as those caused by RVs, are among the oldest effects studied for these important group of beneficial microbes. In this review, we provide an update of the current status on the modulation of intestinal antiviral innate immunity by immunobiotics, and their beneficial impact on RVs infection. In addition, we describe the research of our group that demonstrated the capacity of immunobiotic strains to beneficially modulated TLR3-triggered immune response in IECs, reduce the disruption of intestinal homeostasis caused by intraepithelial lymphocytes, and improve the resistance to RVs infections.