Objective The prevalence of drug-resistant Mycobacterium tuberculosis (Mtb) strains is exacerbating the global burden of tuberculosis (TB), highlighting the urgent need for new treatment strategies for TB. Methods The recombinant adenovirus vaccine expressing cyclic di-adenosine monophosphate (c-di-AMP) phosphodiesterase B (CnpB) (rAd-CnpB), was administered to normal mice via mucosal immunization, either alone or in combination with drug therapy, to treat Mtb respiratory infections in mice.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of antibodies in serum and bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was performed to assess the transcription levels of cytokines interferon γ(IFN-γ) and interleukin 10(IL-10) in mouse lungs. Flow cytometry was used to determine the proportions of CD4⁺ and CD8⁺ T cell subsets in the lungs and spleens. ELISA was employed to measure the levels of cytokines IFN-γ, IL-2, IL-10, inflammatory factors IL-6, and tumor necrosis factor α (TNF-α) secreted by spleen cells following antigen stimulation. The bacteria loads in the lungs and spleens of Mtb-infected mice were enumerated by plate counting methods. Resluts Intranasal immunization with rAd-CnpB induced high titers of IgG in mouse serum and the production of IgG and IgA in BALF, along with alterations in T lymphocyte subsets in the lungs and spleens. Administration of rAd-CnpB, either alone or in combination with drugs, to Mtb-infected mice significantly increased serum IgG levels as well as IgA and IgG levels in BALF. rAd-CnpB immunization promoted the secretion of CnpB-specific cytokines and inflammatory factors by splenocytes in Mtb-infected mice. However, rAd-CnpB immunotherapy, either alone or combined with drugs, did not significantly affect the bacterial loads in the lungs and spleens of mice with Mtb respiratory infections. Conclusion Mucosal immunization with rAd-CnpB induced significant mucosal, humoral and cellular immune responses in mice, and significantly enhanced CnpB-specific cellular immune responses in Mtb-infected mice.
Animals ; Adenoviridae/immunology* ; Mycobacterium tuberculosis/genetics* ; Mice ; Female ; Phosphoric Diester Hydrolases/genetics* ; Tuberculosis Vaccines/administration & dosage* ; Tuberculosis/prevention & control* ; Mice, Inbred BALB C ; Cytokines ; Lung/microbiology* ; Immunization ; Bronchoalveolar Lavage Fluid/immunology* ; Immunity, Mucosal

Endosomes are characterized by the presence of various phosphoinositides that are essential for defining the membrane properties. However, the interplay between endosomal phosphoinositides metabolism and innate immunity is yet to be fully understood. Here, our findings highlight the evolutionary continuity of RAB-10/Rab10's involvement in regulating innate immunity. Upon infection of Caenorhabditis elegans with Pseudomonas aeruginosa, an increase in RAB-10 activity was observed in the intestine. Conversely, when RAB-10 was absent, the intestinal diacylglycerols (DAGs) decreased, and the animal's response to the pathogen was impaired. Further research revealed that UNC-16/JIP3 acts as an RAB-10 effector, facilitating the recruitment of phospholipase EGL-8 to endosomes. This leads to a decrease in endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and an elevation of DAGs, as well as the activation of the PMK-1/p38 MAPK innate immune pathway. It is noteworthy that the dimerization of UNC-16 is a prerequisite for its interaction with RAB-10(GTP) and the recruitment of EGL-8. Moreover, we ascertained that the rise in RAB-10 activity, due to infection, was attributed to the augmented expression of LET-413/Erbin, and the nuclear receptor NHR-25/NR5A1/2 was determined to be indispensable for this increase. Hence, this study illuminates the significance of endosomal PI(4,5)P2 catabolism in boosting innate immunity and outlines an NHR-25-mediated mechanism for pathogen detection in intestinal epithelia.
Animals ; Caenorhabditis elegans/genetics* ; Endosomes/immunology* ; Caenorhabditis elegans Proteins/immunology* ; Phosphatidylinositol 4,5-Diphosphate/immunology* ; Immunity, Innate ; Pseudomonas aeruginosa/immunology* ; rab GTP-Binding Proteins/genetics* ; Diglycerides/metabolism*

OBJECTIVE: To observe the effects of moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4) on immune function and intestinal flora in healthy rats, thereby investigating the underlying mechanism of moxibustion on immune function. METHODS: Twenty 8-week-old SD rats were randomly divided into a young blank group and a young moxibustion group, with 10 rats in each group. Similarly, twenty 8-month-old SD rats were randomly divided into a middle-aged blank group and a middle-aged moxibustion group, with 10 rats in each group. The rats in the two moxibustion groups received moxibustion at "Mingmen" (GV 4) and "Guanyuan" (CV 4), 15 min per session, once daily, five times a week, for a total of four months. The rats in the two blank groups were fed under normal conditions. After the intervention, thymus and spleen indexes were calculated; the morphology of thymus and spleen tissues was observed using HE staining; the flow cytometry was used to detect the expression of CD and CD T lymphocytes and the CD/CD ratio was calculated; ELISA was used to measure the serum levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-17 (IL-17); 16S rRNA high-throughput sequencing was used to analyze the intestinal flora. Additionally, the correlation between the relative abundance of intestinal flora and serum levels of TNF-α, IFN-γ, IL-6, IL-10 and IL-17 was analyzed. RESULTS: Compared with the young blank group, the young moxibustion group exhibited an increase in the cortical area of thymus tissue with tighter lymphocyte arrangement; compared with the middle-aged blank group, the middle-aged moxibustion group showed an increase in thymus index (P<0.05) and an increase in the cortical area of thymus tissue. There were no significant differences in spleen index between the 2 moxibustion groups and the 2 blank groups (P>0.05). There were no significant differences in the expression of CD, CD, and CD/CD ratio between the 2 moxibustion groups and the corresponding blank groups (P>0.05). Compared with the young blank group, the young moxibustion group had elevated IL-6 level (P<0.05); compared with the middle-aged blank group, the middle-aged moxibustion group had decreased IL-10 and IL-17 levels (P<0.05). Compared with the young blank group, the young moxibustion group exhibited increased Sobs index, Ace index, and Chao index (P<0.01, P<0.05), as well as increased relative abundance of Spirochaetota, Treponema, Turicibacter, Rikenellaceae_RC9_gut_group (P<0.05), and decreased relative abundance of Dubosiella (P<0.05). Compared with the middle-aged blank group, the middle-aged moxibustion group had increased relative abundance of Spirochaetota, Treponema, norank_f_Peptococcaceae (P<0.05), and decreased relative abundance of Proteobacteria, Allobaculum, and Faecalibaculum (P<0.05). Correlation analysis revealed that relative abundance of Eubacterium_xylanophilum_group and unclassified _f_Lachnospiraceae was negatively correlated with serum TNF-α level (r=-0.39, P=0.03; r=-0.24, P=0.04), while relative abundance of norank_f_norank_o_Clostridia_UCG-014 and Lactobacillus was positively correlated with serum TNF-α level (r=0.37, P=0.04; r=0.43, P=0.02). The relative abundance of Roseburia and Monoglobus was negatively correlated with serum IFN-γ level (r=-0.40, P=0.02; r=-0.44, P=0.01), while relative abundance of Lactobacillus was positively correlated with serum IL-10 level (r=0.43, P=0.02). CONCLUSION Moxibustion could improve immune function in healthy rats, and its mechanism may be related to the regulation of relative abundance of intestinal flora.
Rats ; Animals ; Moxibustion ; Rats, Sprague-Dawley ; Interleukin-10/genetics* ; Interleukin-17 ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/genetics* ; Gastrointestinal Microbiome ; RNA, Ribosomal, 16S ; Interferon-gamma ; Immunity

This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type Ⅰ interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.
Animals ; Swine ; African Swine Fever Virus/metabolism* ; Membrane Proteins/metabolism* ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction/genetics*

DNA sensor, a kind of pattern recognition receptor (PRR), is widely expressed in innate immune cells. It activates the inflammatory signaling pathways and triggers an innate immune response by recognizing the pathogens or DNA in abnormal host cells. DNA-dependent activator of IFN-regulatory factors (DAI) is the first cytoplasmic DNA receptor discovered, which plays an important role in regulating the innate immune responses characterized by induction of interferon and programmed cell death. The article summarizes the molecular characteristics of DAI, its downstream signaling pathways, and its role and mechanism in anti-infective immunity, tumor immunity and inflammatory diseases. It also makes a preliminary exploration of the correlation between DAI and transplantation immunology, and provides a new target for the therapy of various immune diseases.
DNA/metabolism* ; Receptors, Pattern Recognition ; Immunity, Innate ; Signal Transduction/genetics* ; DNA-Binding Proteins/genetics*

BACKGROUND: Lung cancer has a high incidence and mortality rate, but the treatment of lung cancer still lacks low toxicity and efficient anti-tumor drugs. Polysaccharide from radix tetrastigme has development value in anti-tumor treatment methods. This study was to observe the effect of polysaccharide from radix tetrastigme on immune response of Lewis lung cancer mice and explore its molecular mechanism. METHODS: Lewis lung cancer mouse models were established and randomly grouped. The spleen polypeptide group was intragastric with 50 mg/kg spleen polypeptide, and the radix tetrastigme polysaccharide low, medium and high dose groups were intragastric with 62.5, 125 and 250 mg/kg radix tetrastigme polysaccharide, respectively, and the model group and the control group were intragastric with equivolume normal saline. Tumor formation and metastasis were compared. Haematoxylin-eosin (HE) staining was used to observe the pathological changes of tumor cells. Macrophage phagocytosis, apoptosis, M1/M2 polarization, T cell subsets and cytokine levels in peripheral blood were detected by flow cytometry. The proliferation activity of macrophages was detected by methyl thiazolyldiphenyl tetrazolium (MTT) assay. Dendritic cell (DC) antigen presenting function was detected by chlorophenol red-β-D-galactopyranoside (CPRG) method. Tumor tissue differentiation antigen cluster 47 (CD47) mRNA and protein expression and macrophage signal regulatory protein α (SIRRP α) expression were detected by real time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). RESULTS: The tumor inhibition rates and anti-metastasis rates in the 3-dose radix tetrastigme polysaccharide group and the spleen polypeptide group were higher than those in the model group, and the pathological injury of tumor tissue were severer, and the positive rate of phagocytosis of ink by macrophages and the efficiency of phagocytosis of tumor cells were increased; the apoptosis rate of macrophages was decreased; the proliferation activity of macrophages, polarization ratio of macrophages to M1 type, DC antigen presenting ability, CD4+, CD4+/CD8+ levels were increased; the level of serum tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and the expression of tumor tissue CD47, macrophage SH2-containing protein tyrosine phosphatase 1 (SHP-1), SH2-containing protein tyrosine phosphatase 2 (SHP-2), and phosphorylation signal regulatory protein α (p-SIRPα) were decreased, and the differences were statistically significant (P<0.05). There were no significant differences in the above indexes between low-dose radix tetrastigme polysaccharide group and spleen polypeptide group (P>0.05), and the effects of radix tetrastigme polysaccharide were dose-dependent. CONCLUSIONS Radix tetrastigme polysaccharide can inhibit tumor growth, metastasis and immune response in Lewis lung cancer mice, and its mechanism may be related to inhibiting SIRP/CD47 signaling pathway.
Mice ; Animals ; CD47 Antigen/genetics* ; Lung Neoplasms/drug therapy* ; Cytokines/genetics* ; Polysaccharides/pharmacology* ; Immunity ; Protein Tyrosine Phosphatases

Autophagy is a highly conserved mechanism for material degradation and recycling in eukaryote cells, and plays important roles in growth, development, stress tolerance and immune responses. ATG10 plays a key role in autophagosome formation. To understand the function of ATG10 in soybean, two homologous GmATG10 genes, namely GmATG10a and GmATG10b, were silenced simultaneously by bean pod mottle virus (BPMV) induced gene silencing. The carbon starvation induced by dark treatment and Western blotting analysis of GmATG8 accumulation level indicated that concurrent silencing GmATG10a/10b resulted in the impairment of autophagy in soybean; disease resistance and kinase assays demonstrated that GmATG10a/10b participated in the immune responses by negatively regulating the activation of GmMPK3/6, indicating that GmATG10a/10b plays a negative regulatory role in immune response in soybean.
Soybeans/genetics* ; Immunity

Bacillus cereus is a common foodborne pathogen. Accidently eating food contaminated by B. cereus will cause vomiting or diarrhea, and even death in severe cases. In the present study, a B. cereus strain was isolated from spoiled rice by streak culture. The pathogenicity and drug resistance of the isolated strain were analyzed by drug sensitivity test and PCR amplification of virulence-associated gene respectively. Cultures of the purified strain were injected intraperitoneally into mice to examine their effects on intestinal immunity-associated factors and gut microbial communities, to provide references for the pathogenic mechanism and medication guidance of these spoilage microorganisms. The results showed that the isolated B. cereus strain was sensitive to norfloxacin, nitrofurantoin, tetracycline, minocycline, ciprofloxacin, spectinomycin, clindamycin, erythrocin, clarithromycin, chloramphenicol, levofloxacin, and vancomycin, but resistant to bactrim, oxacillin and penicillin G. The strain carries seven virulence-associated genes including hblA, hblC, hblD, nheA, nheB, nheC and entFM, which are involved in diarrhea-causing toxins production. After infecting mice, the isolated B. cereus strain was found to cause diarrhea in mice, and the expression levels of immunoglobulins and inflammatory factors in the intestinal mucosae of the challenged mice were significantly up-regulated. Gut microbiome analysis showed that the composition of gut microbial community in mice changed after infection with B. cereus. The abundance of the uncultured_bacterium_f_Muribaculaceae in Bacteroidetes, which is a marker of body health, was significantly decreased. On the other hand, the abundance of uncultured_bacterium_f_Enterobacteriaceae, which is an opportunistic pathogen in Proteobacteria and a marker of dysbacteriosis, was significantly increased and was significantly positively correlated with the concentrations of IgM and IgG. These results showed that the pathogenic B. cereus carrying diarrhea type virulence-associated gene can activate the immune system by altering the composition of gut microbiota upon infection.
Animals ; Mice ; Bacillus cereus/metabolism* ; Food Microbiology ; Immunity, Mucosal ; Diarrhea ; Microbiota ; Enterotoxins/genetics*

Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.
Animals ; Mice ; Rabbits ; Mycobacterium tuberculosis/genetics* ; Tuberculosis ; Antigens, Bacterial ; Cytokines ; Immunity, Cellular

We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics* ; Animals ; DNA ; Immunity, Innate ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine