Search Results (50)

The generation of memory CD8⁺ T cells is essential for establishing protective T cell immunity against pathogens and cancers. However, the cellular and molecular mechanisms underlying memory CD8⁺ T cell formation remain incompletely understood. Reliance on specific pathogen-free (SPF) models, characterized by restricted microbial exposure, may limit our understanding of physiologically relevant immune memory development. This study reveals that CD1d-restricted NKT cells regulate central memory T cell (TCM) generation exclusively in a microbe-rich (“dirty”) environment. Under non-SPF housing, CD1d⁺/⁻ and Ja18⁺/⁻ mice exhibited enhanced TCM formation compared to NKT-deficient controls (CD1d⁻/⁻/Ja18⁻/⁻), demonstrating that microbial experience is required for NKT-mediated TCM regulation. Mechanistically, CD1d-restricted NKT cells increased IL-15Rα expression on CD4⁺ T cells in CD1d⁺/⁻ mice, potentiating IL-15 trans-presentation and thereby activating the IL-15/pSTAT5/Eomes axis critical for TCM maintenance. Functional validation through adoptive transfer of CFSE-labeled OT-1 memory cells revealed an NKT cell-dependent survival advantage in CD1d⁺/⁻ hosts. This provides direct evidence that microbiota-experienced niches shape immune memory. Collectively, these findings establish CD1d-restricted NKT cells as physiological regulators of TCM generation and suggest their potential utility as vaccine adjuvants to enhance protective immunity. Full article

The composition of the gut microbiome, defined by environmental factors, significantly affects research outcomes, with variations observed across animal facilities. Efforts to standardize led to the definition of the ‘Altered Schaedler flora’ (ASF), comprising eight bacterial groups. Our data highlights the variability of ASF under pathogen contact. Feces from two wild-type strains (C57Bl/6J and Balb/c mice) with and without proven infection was collected in two different animal facilities and analyzed. The data show a significant difference in the quantity (either reduction or increase) of the eight ASF bacterial groups when comparing infected and non-infected mice across different housing areas (SPF-specific pathogen-free, quarantine, and conventional-experimental areas) within a facility, as well as in comparison to another facility. Furthermore, strain-specific differences are also evident, with certain ASF groups showing a reduction in quantity at one facility but an increase at the other, comparing the same housing area. Comparative studies across facilities confirmed the necessity of baseline determination for accurate ASF analysis. Performing ASF analysis, facilitated by in-house qPCR (quantitative polymerase chain reaction) kits, offers prompt and precise microbiome profiling, enhancing experimental accuracy and health monitoring in animal research settings. Full article

Prolonged sleep deprivation impairs brain function and increases the risk of mental health disorders. Cichorium intybus L. Oligo-polysaccharides (JSO), bioactive compounds derived from chicory, belong to the category of food-medicine homologous substances, possess gut microbiota-modulating and anti-inflammatory properties, and serve as a natural prebiotic, having significant research value in food science. This research examined the anxiolytic properties of JSO in a murine model subjected to chronic sleep deprivation (CSD) stress and explored the mechanisms behind this effect, providing experimental evidence for the development of Cichorium intybus L. as a functional food. Specific pathogen-free (SPF) KM male mice were allocated at random into six experimental groups: the control group, the CSD model group, the diazepam (10 mg/kg) group, and the JSO treatment groups at low (50 mg/kg), medium (100 mg/kg), and high (200 mg/kg) doses. Following 3 weeks of CSD, anxiety-like behaviors were assessed using the open field test, elevated plus maze test, light–dark box test, forced swim test, and marble-burying test. To analyze the composition of gut microbiota, 16S rRNA sequencing was employed, while protein expression in the BDNF, PI3K/AKT/mTOR, and NLRP3 inflammasome pathways was detected by Western blot. Behavioral analysis indicated that JSO (at doses of 100 and 200 mg/kg) markedly enhanced both the time allocated to open arms and the number of entries into open arms in the elevated plus maze test (p < 0.05). JSO (at doses of 50 and 200 mg/kg) significantly elevated transitions in the light–dark box test (p < 0.05), all JSO doses drastically cut down marble-burying behavior (p < 0.001, p < 0.01, p < 0.01). The 16S rRNA sequencing indicated that JSO intervention increased Bacteroidetes abundance while reducing Actinobacteria. Western blot analysis demonstrated that JSO significantly downregulated the ratios of p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, BAX/BCL-2, as well as the expression levels of NLRP3, ASC, Caspase-1, and IL-6 proteins (p < 0.05), while upregulating hippocampal BDNF (p < 0.05). These results indicate that JSO ameliorates CSD-induced anxiety-like behaviors by restoring gut microbiota homeostasis, regulating the BDNF-PI3K/AKT/mTOR and BAX/BCL-2 apoptosis pathways, and suppressing NLRP3 inflammasome-mediated neuroinflammation. Full article

Background/Objectives: Medicinal herbs produce valuable substances with therapeutic potential. The chemical structures of those substances are often converted by gut microbiota. Our previous studies showed that several kinds of bioactive molecules are newly generated in fermented medicinal herbal extract with plant-derived lactic acid bacteria (LABs). Methods: The fermented extract of Atractylodes Japonica Rhizoma (AJR), which is designated as “Byakujutsu” in Japan, with a plant-derived LAB strain IJH-SONE68 was prepared and whether the fermented extract could help reduce symptoms of food allergies, especially wheat intolerance, was confirmed using animal model. Results: It has been found that the fermented extract significantly ameliorates the anaphylaxis score (from 3.0 to 1.0, p = 0.003) of gliadin-induced allergic model mice (specific-pathogen-free, BALB/cJ) accompanied with the modulation of serum total immunoglobulin E (IgE) (from 778 to 518 ng/mL, p = 0.006), interferon (IFN)-γ (from 6.6 to 9.5 pg/mL, p < 0.001), and interleukin (IL)-4 (from 32.0 to 9.1 pg/mL, p < 0.001) levels. Conclusions: The fermented AJR extract may modulate the Th1/Th2 cell balance to alleviate the symptoms of gliadin-induced anaphylaxis in mice. The present study supports the view that the fermentation of medicinal herbal extract prepared using LABs may be a useful procedure for producing therapeutic potential compounds to maintain health. Full article

Background: Deoxynivalenol (DON) poses a threat to animal and human health, particularly causing damage to the nervous system. Intestinal flora can regulate the nervous system through the gut–brain axis; however, there is currently a lack of evidence on the effect of changing the intestinal flora on the damage to the nervous system caused by DON. Therefore, this study aims to investigate the effect of gut microbiota ablation on neurotoxicity induced by exposure to deoxynivalenol. Methods: One hundred-twenty (120) specific pathogen-free (SPF) male C57BL/6j mice were randomly divided into four groups (control group, microbiota-uncleaned group + 5 mg/kg/BW DON, microbiota-cleared group, and microbiota-cleared group + 5 mg/kg/BW DON). The open field and Morris behavior tests were used to evaluate behavior changes after DON exposure. After 14 days of treatment, the mice were euthanized and brain tissues were collected for further analysis. Results: The tests showed that DON exposure led to anxiety and decreased learning ability in mice with no gut microbiota ablation. We also observed pathological changes including neuronal shrinkage, degeneration, and cortical edema in the mice with no microbiota ablation after DON exposure. In addition, the protein and mRNA levels of tight junction proteins and anti-inflammatory factors were decreased in the mice with no microbiota ablation after DON exposure compared with mice with ablated microbiota. Conclusions: We concluded that the presence of microbiota plays a key role in the neurotoxicity induced by DON; thus, ablation of the intestinal microbiota can effectively improve brain damage caused by DON. Full article

This study explored the effects of antimicrobiota vaccines on the acquisition of Borrelia and Rickettsia, and on the microbiota composition of Ixodes ricinus ticks. Using a murine model, we investigated the immunological responses to live Staphylococcus epidermidis and multi-antigenic peptide (MAP) vaccines. Immunized mice were infected with either Borrelia afzelii or Rickettsia helvetica, and subsequently infested with pathogen-free I. ricinus nymphs. We monitored the tick feeding behavior, survival rates, and infection levels. Additionally, we employed comprehensive microbiota analyses, including the alpha and beta diversity assessments and microbial co-occurrence network construction. Our results indicate that both live S. epidermidis and MAP vaccines elicited significant antibody responses in mice, with notable bactericidal effects against S. epidermidis. The vaccination altered the feeding patterns and fitness of the ticks, with the Live vaccine group showing a higher weight and faster feeding time. Microbiota analysis revealed significant shifts in the beta diversity between vaccine groups, with distinct microbial networks and taxa abundances observed. Notably, the MAP vaccine group exhibited a more robust and complex network structure, while the Live vaccine group demonstrated resilience to microbial perturbations. However, the effects of antimicrobiota vaccination on Borrelia acquisition appeared taxon-dependent, as inferred from our results and previous findings on microbiota-driven pathogen refractoriness. Staphylococcus-based vaccines altered the microbiota composition but had no effect on B. afzelii infection, and yielded inconclusive results for R. helvetica. In contrast, previous studies suggest that E. coli-based microbiota modulation can induce a pathogen-refractory state, highlighting the importance of both bacterial species and peptide selection in shaping microbiota-driven pathogen susceptibility. However, a direct comparison under identical experimental conditions across multiple taxa is required to confirm this taxon-specific effect. These findings suggest that antimicrobiota vaccination influences tick fitness and microbiota assembly, but its effects on pathogen transmission depend on the bacterial taxon targeted and the selected peptide epitopes. This research provides insights into the need for strategic bacterial taxon selection to enhance vaccine efficacy in controlling tick-borne diseases. Full article

Laboratory mice are instrumental for preclinical research but there are serious concerns that the use of a clean standardized environment for specific-pathogen-free (SPF) mice results in poor bench-to-bedside translation due to their immature immune system. The aim of the present study was to test the importance of the gut microbiota in wild vs. SPF mice for evaluating host immune responses in a house-dust-mite-induced allergic airway inflammation model without the influence of pathogens. The wild mouse microbiome reduced histopathological changes and TNF-α in the lungs and serum when transplanted to microbiota-depleted mice compared to mice transplanted with the microbiome from SPF mice. Moreover, the colonic gene expression of Gata3 was significantly lower in the wild microbiome-associated mice, whereas Muc1 was more highly expressed in both the ileum and colon. Intestinal microbiome and metabolomic analyses revealed distinct profiles associated with the wild-derived microbiome. The wild-mouse microbiome thus partly reduced sensitivity to house-dust-mite-induced allergic airway inflammation compared to the SPF mouse microbiome, and preclinical studies using this model should consider using both ‘dirty’ rewilded and SPF mice for testing new therapeutic compounds due to the significant effects of their respective microbiomes and derived metabolites on host immune responses. Full article

Neuropathic pain (NPP) is a devastating and unbearable painful condition. As prevailing treatment strategies have failed to mitigate its complications, there remains a demand for effective therapies. Electroacupuncture (EA) has proved a potent remedial strategy in NPP management in humans and mammals. However, past studies have investigated the underlying mechanism of the analgesic effects of EA on NPP, focusing primarily on adenosine receptors in peripheral tissues. Herein, we elucidate the role of the adenosine (Adora-3) signaling pathway in mediating pain relief through EA in the central nervous system, which is obscure in the literature and needs exploration. Specific pathogen-free (SPF) male adult mice (C57BL/6 J) were utilized to investigate the effect of EA on adenosine metabolism (CD73, ADA) and its receptor activation (Adora-3), as potential mechanisms to mitigate NPP in the central nervous system. NPP was induced via spared nerve injury (SNI). EA treatment was administered seven times post-SNI surgery, and lumber (L4–L6) spinal cord was collected to determine the molecular expression of mRNA and protein levels. In the spinal cord of mice, following EA application, the expression results revealed that EA upregulated (p < 0.05) Adora-3 and CD73 by inhibiting ADA expression. In addition, EA triggered the release of adenosine (ADO), which modulated the nociceptive responses and enhanced neuronal activation. Meanwhile, the interplay between ADO levels and EA-induced antinociception, using an Adora-3 agonist and antagonist, showed that the Adora-3 agonist IB-MECA significantly increased (p < 0.05) nociceptive thresholds and expression levels. In contrast, the antagonist MRS1523 exacerbated neuropathic pain. Furthermore, an upregulated effect of EA on Adora-3 expression was inferred when the Adora-3 antagonist was administered, and the EA treatment increased the fluorescent intensity of Adora-3 in the spinal cord. Taken together, EA effectively modulates NPP by regulating the Adora-3 signaling pathway under induced pain conditions. These findings enhance our understanding of NPP management and offer potential avenues for innovative therapeutic interventions. Full article

Ruminants exhibit stronger tolerance to gossypol, an anti-nutritional factor, compared to monogastric animals. We transplanted Hu sheep rumen microbiota into male mice to investigate the role of rumen microbiota in animal gossypol tolerance. Thirty specific-pathogen-free (SPF) male C57BL/6 mice were randomly divided into three groups: normal diet (CK group), gossypol diet (FG group), and rumen microbiota transplantation (FMT group, gossypol diet). The pathological changes in the liver and small intestine of the mice, the organ coefficient, and sperm parameters were analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the blood and lactate dihydrogen-X (LDH-X) levels in the testicular tissue were also measured. The results showed that body weight, feed intake, sperm concentration, sperm motility, and LDH-X levels in the FMT group increased (p < 0.05) compared with the FG group, while the enzyme activities of ALT, AST, and AST/ALT decreased (p < 0.05). In the FMT group, the injury to liver cells was alleviated, the structure of the small intestine was intact, and the villus height and the ratio of villus height to crypt depth (V/C) were higher than those in the FG group (p < 0.05). And there were no differences in various organ coefficients and sperm deformity rates among the three groups (p > 0.05), but compared with the FG group, mice in the FMT group showed tendencies closer to those in the CK group. Rumen microbiota transplantation relieved the reproductive toxicity and liver damage induced by gossypol in male mice and improved the tolerance of recipient animals to gossypol. Additionally, rumen microbes improved the intestinal structural integrity of recipients. Full article

Iron deficiency is the number one nutritional problem worldwide. Iron uptake is regulated at the intestine and is highly influenced by the gut microbiome. Blood from the intestines drains directly into the liver, informing iron status and gut microbiota status. Changes in either iron or the microbiome are tightly correlated with the development of metabolic dysfunction-associated steatotic liver disease (MASLD). To investigate the underlying mechanisms of the development of MASLD that connect altered iron metabolism and gut microbiota, we compared specific pathogen free (SPF) or germ-free (GF) mice, fed a normal or low-iron diet. SPF mice on a low-iron diet showed reduced serum triglycerides and MASLD. In contrast, GF low-iron diet-fed mice showed increased serum triglycerides and did not develop hepatic steatosis. SPF mice showed significant changes in liver lipid metabolism and increased insulin resistance that was dependent upon the presence of the gut microbiota. We report that total body loss of mitochondrial iron importer Mitoferrin2 (Mfrn2⁻/⁻) exacerbated the development of MASLD on a low-iron diet with significant lipid metabolism alterations. Our study demonstrates a clear contribution of the gut microbiome, dietary iron, and Mfrn2 in the development of MASLD and metabolic syndrome. Full article

The normal growth and development of skeletal muscle is essential for the health of the body. The regulation of skeletal muscle by intestinal microorganisms and their metabolites has been continuously demonstrated. Acetate is the predominant short-chain fatty acids synthesized by gut microbiota through the fermentation of dietary fiber; however, the underlying molecular mechanisms governing the interaction between acetate and skeletal muscle during the rapid growth stage remains to be further elucidated. Herein, specific pathogen-free (SPF) mice, germ-free (GF) mice, and germ-free mice supplemented with sodium acetate (GS) were used to evaluate the effects of acetate on the skeletal muscle growth and development of young mice with gut microbiota deficiency. We found that the concentration of serum acetate, body mass gain, succinate dehydrogenase activity, and expression of the myogenesis maker gene of skeletal muscle in the GS group were higher than those in the GF group, following sodium acetate supplementation. Furthermore, the transcriptome analysis revealed that acetate activated the biological processes that regulate skeletal muscle growth and development in the GF group, which are otherwise inhibited due to a gut microbiota deficiency. The in vitro experiment showed that acetate up-regulated Gm16062 to promote skeletal muscle cell differentiation. Overall, our findings proved that acetate promotes skeletal muscle growth and development in young mice via increasing Gm16062 expression. Full article

The intestinal epithelial barrier can prevent the invasion of pathogenic microorganisms and food antigens to maintain a consistent intestinal homeostasis. However, an imbalance in this barrier can result in various diseases, such as inflammatory bowel disease, malnutrition, and metabolic disease. Thus, the aim of this study was to select probiotic strains with epithelial barrier-enhancing ability in cell-based model and further investigate them for their improving effects on colitis mouse and weaned piglet models. The results showed that selected specific cell-free fermentation supernatants (CFSs) from Ligilactobacillus salivarius P1, Lactobacillus gasseri P12, and Limosilactobacillus reuteri G7 promoted intestinal epithelial cell growth and proliferation, strengthening the intestinal barrier in an intestinal epithelial cell line Caco-2 model. Further, the administration of CFSs of L. salivarius P1, L. gasseri P12, and L. reuteri G7 were found to ameliorate DSS-induced colitis in mice. Additionally, spray-dried powders of CFS from the three strains were examined in a weaned piglet model, only CFS powder of L. reuteri G7 could ameliorate the feed/gain ratio and serum levels of D-lactate and endotoxin. In conclusion, a new potential probiotic strain, L. reuteri G7, was selected and showed ameliorating effects in both colitis mouse and weaned piglet models. Full article

Background: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. Methods: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. Results: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). Conclusions: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle. Full article

Gnotobiotic murine models are important to understand microbiota–host interactions. Despite the role of bacteriophages as drivers for microbiome structure and function, there is no information about the structure and function of the gut virome in gnotobiotic models and the link between bacterial and bacteriophage/prophage diversity. We studied the virome of gnotobiotic murine Oligo-MM12 (12 bacterial species) and reduced Altered Schaedler Flora (ASF, three bacterial species). As reference, the virome of Specific Pathogen-Free (SPF) mice was investigated. A metagenomic approach was used to assess prophages and bacteriophages in the guts of 6-week-old female mice. We identified a positive correlation between bacteria diversity, and bacteriophages and prophages. Caudoviricetes (82.4%) were the most prominent class of phages in all samples with differing relative abundance. However, the host specificity of bacteriophages belonging to class Caudoviricetes differed depending on model bacterial diversity. We further studied the role of bacteriophages in horizontal gene transfer and microbial adaptation to the host’s environment. Analysis of mobile genetic elements showed the contribution of bacteriophages to the adaptation of bacterial amino acid metabolism. Overall, our results implicate virome “dark matter” and interactions with the host system as factors for microbial community structure and function which determine host health. Taking the importance of the virome in the microbiome diversity and horizontal gene transfer, reductions in the virome might be an important factor driving losses of microbial biodiversity and the subsequent dysbiosis of the gut microbiome. Full article

Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of nosocomial infections associated with a high mortality rate and represents a serious threat to human health and the increasing frequency of antimicrobial resistance. Cancer patients are more vulnerable to invasive infection due to ulcerative lesions in mucosal surfaces and immune suppression secondary to chemotherapy. In our in vitro study, we observed that probiotics have the potential to yield beneficial effects on intestinal epithelial cells infected with P. aeruginosa. Additionally, probiotics were found to confer advantageous effects on the innate immunity of mice suffering from Salmonella-induced colitis. As a result, we sought to investigate the impact of probiotics on gut-derived P. aeruginosa sepsis induced by chemotherapy. Following chemotherapy, gut-derived P. aeruginosa sepsis was induced in female C57BL/6 mice aged 6–8 weeks, which were raised under specific-pathogen-free (SPF) conditions in an animal center. Prior to the induction of the sepsis model, the mice were administered 1 × 10⁸ colony-forming units (CFU) of the probiotics, namely Lactobacillus rhamnosus GG (LGG) and Bifidobacterium longum (BL) via oral gavage. We observed that LGG or BL amplified the inflammatory mRNA expression in mice undergoing chemotherapy and suffering from gut-derived P. aeruginosa sepsis. This led to a heightened severity of colitis, as indicated by histological examination. Meanwhile, there was a notable decrease in the expression of antimicrobial peptide mRNA along with reduced levels of zonulin and claudin-2 protein staining within mucosal tissue. These alterations facilitated the translocation of bacteria to the liver, spleen, and bloodstream. To our astonishment, the introduction of probiotics exacerbated gut-derived P. aeruginosa sepsis in mice undergoing chemotherapy. Conclusively, we must be prudent when using probiotics in mice receiving chemotherapy complicated with gut-derived P. aeruginosa sepsis. Full article