Sirtuin 2 regulates neutrophil functions through NAD+ synthesis pathway in virus infection.

Neutrophils play an important role in antiviral immunity, but the underlying mechanisms remain unclear. Here, we found that SIRT2 deficiency inhibited the infiltration of neutrophils, as well as the secretion of inflammatory cytokines and the formation of neutrophil extracellular traps (NETs), ameliorating disease symptoms during acute respiratory virus infection. Mechanistically, SIRT2 deficiency upregulates quinolinic acid (QA)-producing enzyme 3-hydroxyanthranilate oxygenase (3-HAO) and leads to expression of quinolinate phosphoribosyltransferase (QPRT), which promotes the synthesis of QA for NAD+ and limits viral infection when de novo NAD+ synthesis is blocked. Tryptophan-2,3-oxygenase expressed in epithelial cells metabolizes tryptophan to produce kynurenine and 3-hydroxyaminobenzoic acid, which is a source of intracellular QA in neutrophils. Thus, our findings reveal a previously unrecognized QPRT-mediated switch in NAD+ metabolism by exploiting neutrophil-derived QA as an alternative source of replenishing intracellular NAD+ pools induced by SIRT2 to regulate neutrophil functions during virus infection, with implications for future immunotherapy approaches.

The Microbiome Protocols eBook initiative: Building a bridge to microbiome research.

The Microbiome Protocols eBook (MPB) serves as a crucial bridge, filling gaps in microbiome protocols for both wet experiments and data analysis. The first edition, launched in 2020, featured 152 meticulously curated protocols, garnering widespread acclaim. We now extend a sincere invitation to researchers to participate in the upcoming 2nd version of MPB, contributing their valuable protocols to advance microbiome research.

Quantitative and dynamic profiling of human gut core microbiota by real-time PCR.

The human gut microbiota refers to a diverse community of microorganisms that symbiotically exist in the human intestinal system. Altered microbial communities have been linked to many human pathologies. However, there is a lack of rapid and efficient methods to assess gut microbiota signatures in practice. To address this, we established an appraisal system containing 45 quantitative real-time polymerase chain reaction (qPCR) assays targeting gut core microbes with high prevalence and/or abundance in the population. Through comparative genomic analysis, we selected novel species-specific genetic markers and primers for 31 of the 45 core microbes with no previously reported specific primers or whose primers needed improvement in specificity. We comprehensively evaluated the performance of the qPCR assays and demonstrated that they showed good sensitivity, selectivity, and quantitative linearity for each target. The limit of detection ranged from 0.1 to 1.0 pg/µL for the genomic DNA of these targets. We also demonstrated the high consistency (Pearson's r = 0.8688, P < 0.0001) between the qPCR method and metagenomics next-generation sequencing (mNGS) method in analyzing the abundance of selected bacteria in 22 human fecal samples. Moreover, we quantified the dynamic changes (over 8 weeks) of these core microbes in 14 individuals using qPCR, and considerable stability was demonstrated in most participants, albeit with significant individual differences. Overall, this study enables the simple and rapid quantification of 45 core microbes in the human gut, providing a promising tool to understand the role of gut core microbiota in human health and disease. KEY POINTS: • A panel of original qPCR assays was developed to quantify human gut core microbes. • The qPCR assays were evaluated and compared with mNGS using real fecal samples. • This method was used to dynamically profile the gut core microbiota in individuals.

Toxicity assessment of Cucurbita pepo cv Dayangua and its effects on gut microbiota in mice.

BACKGROUND: Cucurbita pepo cv Dayangua (CPD) is an edible plant with diverse pharmacological properties. The current research on CPD has primarily focused on initial investigations of its chemical composition and pharmacological effects, and no comprehensive toxicity assessment has been conducted to date. METHODS: In the present study, the toxicity of CPD was evaluated through both acute and sub-chronic oral toxicity tests in mice. 16S rDNA sequencing was used to analyze the composition of the gut microbiota of mice at different time points to observe the effect of CPD on these microbial communities. RESULTS: In the acute toxicity test, CPD exhibited low toxicity, with a median lethal dose (LD50) > 2000 mg/kg. The sub-chronic toxicity test indicated that CPD administration at doses of 200, 400, and 600 mg/kg did not cause mortality or significant organ damage in mice. Furthermore, analysis of the gut microbiota after gavage administration of CPD at 400 and 600 mg/kg revealed an improved abundance of some beneficial gut bacteria. CONCLUSIONS: In summary, no acute or sub-chronic toxic effects were observed in mice following the oral administration of CPD. CPD did not affect the structure and diversity of the gut microbiota and may contribute to an increase in the number of beneficial gut bacteria.

Lactobacillus delbrueckii subsp. bulgaricus Alleviates Acute Injury in Hypoxic Mice.

Acute mountain sickness (AMS) is a common ailment in high-altitude areas caused by the body's inadequate adaptation to low-pressure, low-oxygen environments, leading to organ edema, oxidative stress, and impaired intestinal barrier function. The gastrointestinal tract, being the first to be affected by ischemia and hypoxia, is highly susceptible to injury. This study investigates the role of Lactobacillus delbrueckii subsp. bulgaricus in alleviating acute hypoxic-induced intestinal and tissue damage from the perspective of daily consumed lactic acid bacteria. An acute hypoxia mouse model was established to evaluate tissue injury, oxidative stress, inflammatory responses, and intestinal barrier function in various groups of mice. The results indicate that strain 4L3 significantly mitigated brain and lung edema caused by hypoxia, improved colonic tissue damage, and effectively increased the content of tight junction proteins in the ileum, reducing ileal permeability and alleviating mechanical barrier damage in the intestines due to acute hypoxia. Additionally, 4L3 helped to rebalance the intestinal microbiota. In summary, this study found that Lactobacillus delbrueckii subsp. bulgaricus strain 4L3 could alleviate acute intestinal damage caused by hypoxia, thereby reducing hypoxic stress. This suggests that probiotic lactic acid bacteria that exert beneficial effects in the intestines may alleviate acute injury under hypoxic conditions in mice, offering new insights for the prevention and treatment of AMS.

SIRT3 Negatively Regulates TFH-Cell Differentiation in Cancer.

Follicular helper T (TFH) cells are essential for inducing germinal center (GC) reactions to mediate humoral adaptive immunity in tumors; however, the mechanisms underlying TFH-cell differentiation remain unclear. In this study, we found that the metabolism sensor sirtuin 3 (SIRT3) is critical for TFH-cell differentiation and GC formation during tumor development and viral infection. SIRT3 deficiency in CD4+ T cells intrinsically enhanced TFH-cell differentiation and GC reactions during tumor development and viral infection. Mechanistically, damaged oxidative phosphorylation (OXPHOS) compensatively triggered the NAD+-glycolysis pathway to provide a cellular energy supply, which was necessary for SIRT3 deficiency-induced TFH-cell differentiation. Blocking NAD+ synthesis-glycolysis signaling or recovering OXPHOS activities reversed the TFH-cell differentiation induced by SIRT3 deficiency. Moreover, the mTOR and hypoxia-inducible factor 1α (HIF1α) signaling axis was found to be responsible for TFH-cell differentiation induced by SIRT3 deficiency. HIF1α directly interacted with and regulated the activity of the transcription factor Bcl6. Thus, our findings identify a cellular energy compensatory mechanism, regulated by the mitochondrial sensor SIRT3, that triggers NAD+-dependent glycolysis during mitochondrial OXPHOS injuries and an mTOR-HIF1α-Bcl6 pathway to reprogram TFH-cell differentiation. These data have implications for future cancer immunotherapy research targeting SIRT3 in T cells.

The adaptor protein ECAP, the corepressor LEUNIG, and the transcription factor BEH3 interact and regulate microsporocyte generation in Arabidopsis.

Histospecification and morphogenesis of anthers during development in Arabidopsis (Arabidopsis thaliana) are well understood. However, the regulatory mechanism of microsporocyte generation at the pre-meiotic stage remains unclear, especially how archesporial cells are specified and differentiate into 2 cell lineages with distinct developmental fates. SPOROCYTELESS (SPL) is a key reproductive gene that is activated during early anther development and remains active. In this study, we demonstrated that the EAR motif-containing adaptor protein (ECAP) interacts with the Gro/Tup1 family corepressor LEUNIG (LUG) and the BES1/BZR1 HOMOLOG3 (BEH3) transcription factor to form a transcription activator complex, epigenetically regulating SPL transcription. SPL participates in microsporocyte generation by modulating the specification of archesporial cells and the archesporial cell-derived differentiation of somatic and reproductive cell layers. This study illustrates the regulation of SPL expression by the ECAP-LUG-BEH3 complex, which is essential for the generation of microsporocytes. Moreover, our findings identified ECAP as a key transcription regulator that can combine with different partners to regulate gene expression in distinct ways, thereby facilitating diverse processes in various aspects of plant development.

Characterization of the follicular fluid microbiota based on culturomics and sequencing analysis.

Introduction. The human oocyte microenvironment is follicular fluid, which is important for follicle growth, ovulation and maturation of the oocyte. The micro-organisms present in follicular fluid could be a predictor of in vitro fertilization outcomes.Hypothesis/Gap Statement. Women with follicular fluid colonized with micro-organisms can be asymptomatic, but the presence of some genera in the follicular fluid correlates with in vitro fertilization.Aim. To confirm the existence of micro-organisms in follicular fluid, and to profile the micro-organisms present in follicular fluid sampled from women undergoing in vitro fertilization with different outcomes.Methodology. Women undergoing in vitro fertilization (n=163) were divided into different subgroups according to their in vitro fertilization outcomes. Their follicular fluid samples were collected, and among them, 157 samples were analysed by 16S rDNA sequencing, and 19 samples were analysed using culturomics.Results. The culturomics results suggested that the 19 follicular fluid samples were not sterile. The isolation rates for Streptococcus, Finegoldia and Peptoniphilus were >50 % in the 19 samples. Linear discriminant analysis effect size analysis showed differential bacteria abundance according to the pregnancy rate, the rate of normal fertilization, the rate of high-quality embryos and the rate of available oocytes. The sequencing results showed that micro-organisms could be detected in all 157 samples. Pseudomonas, Lactobacillus, Comamonas, Streptococcus and Acinetobacter were detected in all of the samples, but with a wide range of relative abundance. Pseudomonas, Lactobacillus, Ralstonia and Vibrio constituted a notable fraction of the microbiota.Conclusions. Follicular fluid is not sterile. Micro-organisms in follicular fluid could be a predictor of in vitro fertilization outcomes.

Deacetylase sirtuin 2 negatively regulates myeloid-derived suppressor cell functions in allograft rejection.

Although myeloid-derived suppressor cells (MDSCs) are critical for allograft survival, their regulatory mechanism remains unclear. Herein, our results showed that metabolism sensor sirtuin 2 (SIRT2) negatively regulates the functions of MDSCs in inducing allogeneic skin graft rejection. Genetic deletion of SIRT2 in myeloid cells (Sirt2Δmye) increased the number of CD11b+Gr1+ MDSCs in bone marrow, spleens, draining lymph nodes, and allografts, inhibited the production of proinflammatory cytokine tumor necrosis factor ɑ, enhanced the production of anti-inflammatory cytokine interleukin 10, and potentiated the suppressive activation of MDSCs in prolonging allograft skin survival. C-X-C motif chemokine receptor 2 is critical for mediating the recruitment and cytokine production of MDSCs induced by SIRT2. Mechanistically, Sirt2Δmye enhanced NAD+ levels, succinate dehydrogenase subunit A (SDHA) activities, and oxidative phosphorylation (OXPHOS) levels in MDSCs after transplantation. Pharmacologically blocking nicotinamide phosphoribosyltransferase effectively reverses the production of cytokines and suppressive activities of MDSC induced by Sirt2Δmye. Blocking OXPHOS with knockdown of SDHA or pharmacological blocking of SDHA significantly restores Sirt2Δmye-mediated stronger MDSC suppressive activity and inflammatory factor productions. Thus, our findings identify a previously unrecognized interplay between NAD+ and SDH-mediated OXPHOS metabolic pathways in regulating MDSC functions induced by the metabolic sensor SIRT2 in allogeneic transplantation.

Amelioration of intestinal barrier function and reduction of blood lead level in adult women with recurrent spontaneous abortion by a novel product of dietary fiber mixture, Holofood.

BACKGROUND: The elevated circulating toxins secondary to the impairment of intestinal barrier integrity commonly elicit a chronic inflammatory response and finally contribute to multiple diseases. These toxins, including bacterial by-products and heavy metals, are the potent risk factors for the development of recurrent spontaneous abortion (RSA). Preclinical evidence suggests that several dietary fibers can restore intestinal barrier function and decrease the accumulation of heavy metals. However, it is uncertain whether treatment with a newly developed blend of dietary fibers product (Holofood) benefits patients with RSA. METHODS: In this trial, we enrolled 70 adult women with RSA, who were randomly assigned into the experiment group and the control group in a 2:1 ratio. Upon the basis of conventional therapy, subjects in the experiment group (n = 48) received 8 weeks oral administration with Holofood three times daily at a dose of 10 g each time. Subjects without Holofood consumption were set as the control (n = 22). Blood samples were collected for the determinations of metabolic parameters, heavy mental lead, and the indices related to intestinal barrier integrity (D-lactate, bacterial endotoxin, and diamine oxidase activity). RESULTS: The reduction amplitude in blood lead from baseline to week 8 was 40.50 ± 54.28 (µg/L) in the experiment group as compared with 13.35 ± 36.81 (µg/L) in the control group (P = 0.037). The decreased level of serum D-lactate from baseline to week 8 was 5.58 ± 6.09 (mg/L) in the experiment group as compared with - 2.38 ± 8.90 (mg/L, P < 0.0001) in the control group. The change in serum DAO activity from baseline to week 8 was 3.26 ± 2.23 (U/L) in the experiment group as compared with - 1.24 ± 2.22 (U/L, P < 0.0001) in the control group. Participants who received Holofood had a greater decline in blood endotoxin from baseline to week 8 than those in the control group. Moreover, by comparing with the self-baseline, Holofood consumption significantly decreased the blood levels of lead, D-lactate, bacterial endotoxin, and DAO activity. CONCLUSION: Our results suggest that Holofood affords a clinically relevant improvements in blood lead level and intestinal barrier dysfunction in patients with RSA.

Hippo Kinase MST1-Mediated Cell Metabolism Reprograms the Homeostasis and Differentiation of Granulocyte Progenitor Cells.

The mechanism of the development of granulocyte progenitor cells into neutrophils under steady-state and pathological conditions remains unclear. In this study, our results showed that with the development of neutrophils from hematopoietic stem cells to mature neutrophils, the expression level of the Hippo kinase MST1 gradually increased. Mst1-specific deficiency in myeloid cells caused neutrophilia, with an expanded granulocytic compartment resulting from a cell-autonomous increase in the number of granulocyte-macrophage progenitors under steady-state conditions and during Listeria monocytogenes infection. Mechanistically, mTOR and HIF1α signaling are required for regulating the balance between glycolysis and succinate dehydrogenase-mediated oxidative phosphorylation, which is crucial for Mst1-/--induced proliferation of granulocyte-monocyte progenitors, lineage-decision factor C/EBPα expression, and granulopoiesis. HIF1α directly regulated C/EBPα promoter activities. Blocking mTOR and HIF1α or adjusting the balance between glycolysis and succinate dehydrogenase-mediated oxidative phosphorylation reversed the granulopoiesis induced by Mst1-/- under steady-state conditions or infection in mice. Thus, our findings identify a previously unrecognized interplay between Hippo kinase MST1 signaling and mTOR-HIF1α metabolic reprogramming in granulocyte progenitor cells that underlies granulopoiesis.

Curtanaerobium respiraculi gen. nov., sp. nov., a novel anaerobic bacterium isolated from human bronchoalveolar lavage fluid.

Two related anaerobic strains, designated as SWB101512T and SWB19611, were isolated from the bronchoalveolar lavage fluid of two lung cancer patients. Cells were Gram-stain-positive, non-motile and non-spore-forming. Growth could be observed at 26-45 °C (optimum, 37 °C), pH 5.0-8.5 (optimum, pH 7.0) and with 0.5-2.0 % (v/w) NaCl (optimum, 1.0%). The 16S rRNA gene sequences of SWB101512T and SWB19611 showed the highest similarities to Denitrobacterium detoxificans DSM 21843T (91.1 and 91.3 %, respectively). The phylogenetic tree based on the 16S rRNA gene sequences and the core genome sequences demonstrated that the two strains clustered together and formed a distinct lineage within the family Eggerthellaceae. The DNA G+C contents of strains SWB101512T and SWB19611 were 62.0 and 61.9 mol%, respectively. The predominant cellular fatty acids of strains SWB101512T and SWB19611 were C16 : 0 DMA (27.8 and 28.8 %, respectively). The respiratory menaquinone in both strains was menaquinone 6 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids, three glycolipids and three unidentified lipids. Based on evidence from phenotypic, chemotaxonomic and genomic analyses, a new genus and species belonging to the family Eggerthellaceae, named Curtanaerobium respiraculi gen. nov., sp. nov. is proposed. The type strain is SWB101512T (=GDMCC 1.2991T=JCM 35330T).

Characterization of Lung and Oral Microbiomes in Lung Cancer Patients Using Culturomics and 16S rRNA Gene Sequencing.

Recently, microbiota dysbiosis in lung cancer has attracted immense attention. Studies on lung microbes are mostly based on sequencing, which has left the potentially functional bacteria with extremely low abundance uncovered. In this study, we characterized and compared the lung and oral cavity microbiotas using culturomics and 16S rRNA gene sequencing. Of the 198 bacteria identified at the species level from bronchoalveolar lavage fluid (BALF) samples, Firmicutes was predominant (39.90%). Twenty bacterial species isolated from BALF samples were present in at least half of the patients and were also highly abundant in oral samples. Of all isolated strains, Streptococcus and Veillonella were highly dominant. The abundance of Prevotella and Veillonella decreased from the oral cavity to the lung, whereas that of Pseudomonas increased. Linear discriminant analysis effect size demonstrated that Prevotella was more abundant in the healthy samples than in the cancerous ones, which is in accordance with the isolation of Prevotella oralis only from the healthy group using culturomics. Moreover, Gemella sanguinis and Streptococcus intermedius were isolated only from the non-small-cell lung cancer (NSCLC) group, and 16S rRNA gene sequencing showed that they were higher in the NSCLC than in the small-cell lung cancer group. Furthermore, while Bacillus and Castellaniella were enriched in lung adenocarcinoma, Brucella was enriched in lung squamous cell carcinoma. Overall, alterations were observed in the microbial community of patients with lung cancer, whose diversity might be site and pathology dependent. Using culturomics and 16S rRNA gene amplicon sequencing, this study has provided insights into pulmonary and oral microbiota alterations in patients with lung cancer. IMPORTANCE The relationship between lung microbiota and cancer has been explored based on DNA sequencing; however, culture-dependent approaches are indispensable for further studies on the lung microbiota. In this study, we applied a comprehensive approach combining culturomics and 16S rRNA gene amplicon sequencing to detect members of the microbiotas in saliva and BALF samples from patients with unilateral lobar masses. We found alterations in the microbial community of patients with lung cancer, whose diversity might be site and pathology dependent. These features may be potential bacterial biomarkers and new targets for lung cancer diagnosis and treatment. In addition, a lung and oral microbial biobank from lung cancer patients was established, which represents a useful resource for studies of host-microbe interactions.

Aryl hydrocarbon receptor nuclear translocator limits the recruitment and function of regulatory neutrophils against colorectal cancer by regulating the gut microbiota.

BACKGROUND: Although the role and mechanism of neutrophils in tumors have been widely studied, the precise effects of aryl hydrocarbon receptor nuclear translocator (ARNT) on neutrophils remain unclear. In this study, we investigated the roles of ARNT in the function of CD11b+Gr1+ neutrophils in colitis-associated colorectal cancer. METHODS: Wild-type (WT), ARNT myeloid-specific deficient mice and a colitis-associated colorectal cancer mouse model were used in this study. The level and functions of CD11b+Gr1+ cells were evaluated by flow cytometry and confocal microscopy. RESULTS: We found that ARNT deficiency drives neutrophils recruitment, neutrophil extracellular trap (NET) development, inflammatory cytokine secretion and suppressive activities when cells enter the periphery from bone marrow upon colorectal tumorigenesis. ARNT deficiency displays similar effects to aryl hydrocarbon receptor (AHR) deficiency in neutrophils. CXCR2 is required for NET development, cytokine production and recruitment of neutrophils but not the suppressive activities induced by Arnt-/- in colorectal cancer. The gut microbiota is essential for functional alterations in Arnt-/- neutrophils to promote colorectal cancer growth. The colorectal cancer effects of Arnt-/- neutrophils were significantly restored by mouse cohousing or antibiotic treatment. Intragastric administration of the feces of Arnt-/- mice phenocopied their colorectal cancer effects. CONCLUSION: Our results defined a new role for the transcription factor ARNT in regulating neutrophils recruitment and function and the gut microbiota with implications for the future combination of gut microbiota and immunotherapy approaches in colorectal cancer.

Safety Evaluation and Probiotic Potency Screening of Akkermansia muciniphila Strains Isolated from Human Feces and Breast Milk.

Akkermansia muciniphila is considered a next-generation probiotic because of its immense potential to regulate disorders. We isolated 31 strains of A. muciniphila from feces or breast milk of healthy people. After genome sequencing, assembly, and analysis, we selected six strains (AM01 to AM06) for further exploration. We first analyzed their general characteristics, including morphological description, growth characteristics, and physiological and biochemical characteristics, and then confirmed their genetic characteristics, including GC content, putative virulence factors, and antibiotic resistance genes. We next investigated the tolerance of these strains to artificial gastric and intestinal fluids and bile salts to evaluate their survival potential in the digestive tract. Drug sensitivity tests were also conducted based on the analysis of the antibiotic resistance genes of these strains. Furthermore, we examined the genetic stability and acute toxicity of two strains (AM02 and AM06) in mice. Finally, the safety of AM06 was evaluated in normal mice and nude mice. AM06 exhibited adaptability to pH changes. Since AM02 and AM03 showed more resistance to antibiotics than AM01 and AM04 to AM06, their potential clinical application may be limited. Both AM02 and AM06 were genetically and phenotypically stable and safe in normal mice, and AM06 was safe in nude mice. Considering all this together, AM06 is a safe A. muciniphila strain and exhibits a great potential for use as a probiotic strain among the isolated strains. IMPORTANCE In this study, we isolated 30 strains of Akkermansia muciniphila from different samples of human feces, and for the first time we isolated an A. muciniphila strain from human breast milk. This isolation verified the existence of microbes in human breast milk, which suggests that A. muciniphila can be vertically propagated from mother to infant and participates in the formation of the early gut microbiome. We then systematically evaluated the potential for use as a probiotic of this A. muciniphila strains according to the FAO/WHO recommendation. We confirmed that the AM06 strain isolated from breast milk has no virulence factors and is genetically stable and nonpathogenic for both normal mice and nude mice. Moreover, its tolerance to pH changes and bile salts indicates its desirable probiotic properties. Thus, we propose that the AM06 strain of A. muciniphila is safe for use as a probiotic candidate.

Parvimonas micra activates the Ras/ERK/c-Fos pathway by upregulating miR-218-5p to promote colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in the world, and a strong relationship exists between CRC and gut microbiota, which affects the occurrence, development, and metastasis of cancer. Bioinformatics-based analyses revealed that the abundance of Parvimonas micra (P. micra) in the feces of patients with cancer is significantly higher than that in healthy people. Therefore, an important relationship may exist between P. micra and CRC. METHODS: We first confirmed that P. micra can promote the proliferation of cell lines through cell experiments and mouse models. Then we selected the signaling pathways and content of exosomes to promote the development of CRC by transcriptomics and microRNA sequencing. Finally, we confirmed that P. micra promoted CRC development through miR-218-5p/Ras/ERK/c-Fos pathway through the in vivo and in vitro experiments. RESULTS: First, it was confirmed by in vitro and in vivo experiments that P. micra can promote the development of CRC. Transcriptome analysis after the coincubation of bacteria and cells revealed that P. micra promoted cell proliferation by activating the Ras/ERK/c-Fos pathway. Furthermore, microRNA sequencing analysis of the cells and exosomes showed that miR-218-5p and protein tyrosine phosphatase receptor R (PTPRR) were the key factors involved in activating the Ras/ERK/c-Fos pathway, and the miR-218-5p inhibitor was used to confirm the role of microRNA in xenograft mice. CONCLUSION: This experiment confirmed that P. micra promoted the development of CRC by upregulating miR-218-5p expression in cells and exosomes, inhibiting PTPRR expression, and ultimately activating the Ras/ERK/c-Fos signaling pathway.

Regulation of follicular T helper cell differentiation in antitumor immunity.

Follicular T helper (Tfh) cells are a subset of CD4+ T cells that play an important role in the formation of germinal centers and the maturation and differentiation of affinity-matured B cells. Recent studies have demonstrated important functions of Tfh cells in tertiary lymphoid structures of tumors, revealing great potential of Tfh cells in tumor immunity. However, Tfh development is incompletely understood. The differentiation of Tfh cells is a complex, multistage process regulated at the DNA, RNA and protein levels. This review just summarizes current research on the molecular mechanisms of Tfh cell differentiation to better understand the role of Tfh cells in antitumor immunity.

The hippo kinase MST1 negatively regulates the differentiation of follicular helper T cells.

Follicular helper T (TFH ) cells are essential for inducing germinal centre (GC) reactions to mediate humoral adaptive immunity and antiviral effects, but the mechanisms of TFH cell differentiation remain unclear. Here, we found that the hippo kinase MST1 is critical for TFH cell differentiation, GC formation, and antibody production under steady-state conditions and viral infection. MST1 deficiency intrinsically enhanced TFH cell differentiation and GC reactions in vivo and in vitro. Mechanistically, mTOR and HIF1α signalling is involved in glucose metabolism and increased glycolysis and decreased OXPHOS, which are critically required for MST1 deficiency-directed TFH cell differentiation. Moreover, upregulated Foxo3 expression is critically responsible for TFH cell differentiation induced by Mst1-/- . Thus, our findings identify a previously unrecognized relationship between hippo kinase MST1 signalling and mTOR-HIF1α-metabolic reprogramming coupled with Foxo3 signalling in reprogramming TFH cell differentiation.

Immunomagnetic-bead enriched culturomics (IMBEC) for isolating pathobionts from feces of colorectal cancer patients.

Culturomics employs various cultivating conditions to obtain different types of bacteria and new species. However, current culturomics lacks a highly efficient method for isolating specific pathobionts. Immunomagnetic bead technology, which uses magnetic beads conjugated with antibodies for capturing the antigen to realize enrichment of the targets, has been employed as an alternative method. In this study, we developed a novel method, immunomagnetic bead-enriched culturomics (IMBEC), in which magnetic bead-conjugated antibodies purified from the fecal samples of patients with colorectal cancer (CRC) were used to enrich and isolate potential pathobionts. A protocol for enriching potential pathobionts via immunomagnetic capture was developed by optimizing the concentrations of coupling reagents, NaCl, and detergent. The efficacy of pathobiont enrichment was compared between antibody-coated magnetic beads (antibody group) and nonconjugated blank magnetic beads (blank group). To determine the proinflammatory potential of isolates from both groups, we investigated their ability to induce cytokine production in THP-1 macrophages. This protocol was employed for isolating bacteria from 10 fecal samples of patients with CRC, which were simultaneously compared with those isolated from the blank group. A total of 209 bacterial species were isolated from both groups, including 173 from the antibody group, 160 from the blank group, and 124 from both groups. Bacteria isolated from the antibody group produced more proinflammatory cytokines than those isolated from the blank group. IMBEC is a promising method for relatively specific isolation of potential pathobionts for a particular disease of interest.

Modulation of tumor-associated macrophages in colitis-associated colorectal cancer.

Intestinal macrophages are the most abundant immune cells in the small and large intestine, which maintain intestinal homeostasis by clearing invading bacteria and dead cells, secreting anti-inflammatory cytokines, and inducing tolerance to symbiotic bacteria and food particles. In addition, as antigen-presenting cells, they also participate in eliciting adaptive immune responses through bridging innate immune responses. After the intestinal homeostasis is disrupted, the damaged or apoptotic intestinal epithelial cells cannot be effectively cleared, and the infection of exogenous pathogens and leakage of endogenous antigens lead to persistent intestinal inflammation. Long-term chronic inflammation is one of the important causes of colitis-associated carcinogenesis (CAC). Tumor microenvironment (TME) is gradually formed around tumor cells, in which tumor associated macrophage (TAMs) is not only the builder, but also regulated by TME. This review just briefly summarized the role of intestinal macrophages under physiological and pathological inflammatory and cancerous conditions, and current therapeutic strategies for intestinal diseases targeting macrophages.