STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

Dihydroartemisinin is an inhibitor of trained immunity through Akt/mTOR/HIF1α signaling pathway.

Trained immunity is mechanistically defined as the metabolically and epigenetically mediated long-term functional adaptation of the innate immune system, characterized by a heightened response to a secondary stimulation. Given appropriate activation, trained immunity represents an attractive anti-infective therapeutic target. Nevertheless, excessive immune response and subsequent inflammatory cascades may contribute to pathological tissue damage, indicating that the negative impacts of trained immunity appear to be significant. In this study, we show that innate immune responses such as the production of extracellular traps, pro-inflammatory cytokines, and autophagy-related proteins were markedly augmented in trained BMDMs. Furthermore, heat-killed C. albicans priming promotes the activation of the AIM2 inflammasome, and AIM2-/- mice exhibit impaired memory response induced by heat-killed C. albicans. Therefore, we establish that the AIM2 inflammasome is involved in trained immunity and emerges as a promising therapeutic target for potentially deleterious effects. Dihydroartemisinin can inhibit the memory response induced by heat-killed C. albicans through modulation of mTOR signaling and the AIM2 inflammasome. The findings suggest that dihydroartemisinin can reduce the induction of trained immunity by heat-killed C. albicans in C57BL/6 mice. Dihydroartemisinin is one such therapeutic intervention that has the potential to treat of diseases characterized by excessive trained immunity.

Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.

IFI204 protects host defense against Staphylococcus aureus-induced pneumonia by promoting extracellular traps formation.

Interferon-inducible protein 204 (IFI204) is an intracellular DNA receptor that can recognize DNA viruses and intracellular bacteria. Extracellular traps (ETs) have been recognized as an indispensable antimicrobial barrier that play an indispensable role in bacterial, fungal, parasitic, and viral infections. However, how ETs form and the mechanisms by which IFI204 function in Staphylococcus aureus pneumonia are still unclear. Moreover, by in vitro experiments, we proved that IFI204 deficiency decreases the formation of ETs induced by Staphylococcus aureus in a NOX-independent manner. More importantly, Deoxyribonuclease I (DNase I) treatment significantly inhibited the formation of ETs. IFI204 contributed to ETs formation by promoting citrullination of histone H3 and the expression of PAD4 (peptidylarginine deiminase 4). Altogether, these findings highlight the potential importance of IFI204 for host defense against S. aureus USA300-TCH1516 infection.

Stimulator of Interferon Genes (STING) Promotes Staphylococcus aureus-Induced Extracellular Traps Formation via the ROS-ERK Signaling Pathway.

Stimulator of interferon genes (STING) is a cytosolic DNA sensor or directly recognizes bacterial cyclic dinucleotides, which is required for the detection of microbial infection. Extracellular traps (ETs) are known to be part of the antimicrobial defense system. However, the implication of STING in ETs formation during microbial infection remains unknown. Here, we showed that STING contributed to Staphylococcus aureus (S. aureus)-induced ETs formation through the ROS-ERK signaling. STING deficiency exhibited decreased cell-free DNA (cfDNA) level, reduced expression of citrullinated histone H3 (CitH3), and diminished DNA colocalization with CitH3 and myeloperoxidase (MPO). Interestingly, NADPH oxidase-derived reactive oxygen species (ROS) promoted ETs formation, accompanied by increased activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in S. aureus-stimulated bone marrow-derived macrophages (BMDMs). Corresponding to less ROS production, decreased ERK1/2 activation was shown in STING-/- BMDMs after S. aureus infection. Importantly, inhibiting the ROS-ERK signal reduced the ETs formation and the differences disappeared between WT and STING-/- BMDMs after S. aureus infection. Moreover, STING-/- BMDMs exhibited significantly increased levels of extracellular bacteria compared to WT BMDMs regardless of phagocytosis. In addition, such differences disappeared after DNase I treatment. DNase I treatment also facilitated pathogen colonization without affecting the inflammatory cells infiltration and pro-inflammatory factors secretion following pulmonary S. aureus infection. Furthermore, STING-/- mice presented decreased levels of cfDNA and CitH3, along with increased bacterial colonization compared to WT mice. Altogether, these findings highlighted that STING promoted ETs formation via the ROS-ERK signal for host defense against S. aureus infection.

STING Contributes to Host Defense Against Staphylococcus aureus Pneumonia Through Suppressing Necroptosis.

STING (Stimulator of interferon genes) is known as an important adaptor protein or direct sensor in the detection of nucleotide originating from pathogens or the host. The implication of STING during pulmonary microbial infection remains unknown to date. Herein, we showed that STING protected against pulmonary S.aureus infection by suppressing necroptosis. STING deficiency resulted in increased mortality, more bacteria burden in BALF and lungs, severe destruction of lung architecture, and elevated inflammatory cells infiltration and inflammatory cytokines secretion. STING deficiency also had a defect in bacterial clearance, but did not exacerbate pulmonary inflammation during the early stage of infection. Interestingly, TUNEL staining and LDH release assays showed that STING-/- mice had increased cell death than WT mice. We further demonstrated that STING-/- mice had decreased number of macrophages accompanied by increased dead macrophages. Our in vivo and in vitro findings further demonstrated this cell death as necroptosis. The critical role of necroptosis was detected by the fact that MLKL-/- mice exhibited decreased macrophage death and enhanced host defense to S.aureus infection. Importantly, blocking necroptosis activation rescued host defense defect against S.aureus pneumonia in STING-/- mice. Hence, these results reveal an important role of STING in suppressing necroptosis activation to facilitate early pathogen control during pulmonary S.aureus infection.

Porphyrin-containing Gadolinium Complex as a Tumor-targeting Magnetic Resonance Imaging (MRI) Contrast Agent.

OBJECTIVE: Gadolinium diethylenetriaminepentaacetic di[5-(4'-amidophenyl)-10,15,20- tris(4'-sulfonatophenyl) porphyrin trisodium salt] (Gd-DTPA-2APTSPP) was synthesized by the reaction of diethylenetriaminepentaacetic dianhydride with 5-(4'-aminophenyl)-10,15,20-tris(4'-sulfonatophenyl) porphyrin and subsequently chelation with gadolium chloride. METHODS: This gadolinium complex was characterized and its properties in vitro and in vivo were also evaluated. Compared with Gd-DTPA, Gd-DTPA-2APTSPP possessed high relaxivity r1, low cytotoxicity to HeLa cells and high enhanced signal intensities of the VX2 carcinoma in rabbits for a prolonged time. CONCLUSION: Moreover, Gd-DTPA-2APTSPP can distinguish the VX2 carcinoma from the reactive hyperplasia incited by inflammation and normal tissues in rabbits. Therefore, Gd-DTPA-2APTSPP can be taken up selectively by tumors and show the potential as a tumor-targeting MRI contrast agent.

Porphyrin-containing polyaspartamide gadolinium complexes as potential magnetic resonance imaging contrast agents.

Porphyrin-containing polyaspartamide ligands (APTSPP-PHEA-DTPA) were synthesized by the incorporation of diethylenetriaminepentaacetic acid (DTPA) and 5-(4'-aminophenyl)-10,15,20-tris(4'-sulfonatophenyl) porphyrin, trisodium salt (APTSPP) into poly-α,ß-[N-(2-hydroxyethyl)-l-aspartamide] (PHEA). These ligands were further reacted with gadolinium chloride to produce macromolecule-gadolinium complexes (APTSPP-PHEA-DTPA-Gd). Experimental data of (1)H NMR, IR, UV and elemental analysis evidenced the formation of the polyaspartamide ligands and gadolinium complexes. In vitro and in vivo property tests indicated that APTSPP-PHEA-DTPA-Gd possessed noticeably higher relaxation effectiveness, less toxicity to HeLa cells, and significantly higher enhanced signal intensities (SI) of the VX2 carcinoma in rabbits with lower injection dose requirement than that of Gd-DTPA. Moreover, APTSPP-PHEA-DTPA-Gd was found to greatly enhance the contrast of MR images of the VX2 carcinoma, providing prolonged intravascular duration, and distinguished the VX2 carcinoma and normal tissues in rabbits according to MR image signal enhancements. These porphyrin-containing polyaspartamide gadolinium complexes can be used as the candidates of contrast agents for targeted MRI to tumors.

Dynamic enhancement patterns of solitary pulmonary nodules at multi-detector row CT and correlation with vascular endothelial growth factor and microvessel density.

BACKGROUND AND OBJECTIVE: Differential diagnosis of malignant solitary pulmonary nodules (SPNs) from benign ones is difficult based on imaging manifestations. This study was to assess dynamic enhancement patterns of SPNs detected with multi-detector row computed tomography (MDCT), correlate SPN manifestations of MDCT to the expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD), thus to explore the potential value of MDCT imaging in the diagnosis of SPNs. METHODS: Fifty pathologically and one clinically confirmed patients with SPNs (diameter

[Mechanism of inhibition of tumor angiogenesis by Bletilla colloid: an experimental study].

OBJECTIVE: To study the mechanism of inhibition of tumor angiogenesis by Bletilla colloid. METHODS: Human Hep-G2 hepatocellular carcinoma cells were cultured and treated with Bletilla colloid of different concentrations. Pure culture of Hep-G2 cells was used as control and pure culture medium without Hep-G2 cell was used as blank control. The concentration of vascular endothelial growth factor (VEGF) in the supernatant was detected by ELISA. The apoptosis and proliferation of the Hep-G2 cells were examined by flow cytometry. Cells of human endothelial cell line ECV-304 were cultured in the supernatant of culture media of Hep-G2 treated with Bletilla colloid of different concentrations. MTT method was used to observe the growth of the ECV-304 endothelial cells. Eighty rats were made animal model of transplanted Walker-256 hepatoma and then randomly divided into 4 groups of 20 rats 12 - 14 days after to undergo transarterial chemoembolization (TACE) treated with normal saline, 5-fluouracil (5-Fu), iodized poppy seed oil, and 5-Fu-Blettila microsphere respectively. Two weeks after, the rats were killed and the tumors were taken out. Immunohistological staining was conducted to detect the expression and localization of factor VIII, VEGF, and basic fibroblast growth factor (b-FGF). The microvcascular density (MVD) was calculated by counting the factor VIII positive endothelial cells. RESULTS: There was no statistically significant difference in the apoptosis rate, proliferation rate and supernatant VEGF level between the control group and the Bletilla groups. The inhibition rates of ECV-304 endothelial cell growth were 57.6%, 66.7%, 86.4%, 87.5%, and 94.8% in the groups of Bletilla of the concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 micro g/ml respectively in a dose-dependent manner. The MVD of tumor was 59 +/- 34 per field in the 5-Fu-Blatilla group,significantly lower than those in the other 3 groups (F = 5.177, P = 0.003). The expression of VEGF and the expression of b-FGF were not significantly different among the 4 TACE groups. CONCLUSION: Bletilla colloid inhibits angiogenesis after TACE, potentially, through inhibition of the binding of vascular endothelial growth factor to its receptor.