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ObjectiveOur goals were to evaluate the antitumour efficacy of Lactobacillus rhamnosus GG (LGG) in combination with immune checkpoint blockade (ICB) immunotherapies on tumour growth and to investigate the underlying mechanisms.DesignWe used murine models of colorectal cancer and melanoma to evaluate whether oral administration of LGG improves the efficacy of ICB therapies. We performed the whole genome shotgun metagenome sequencing of intestinal contents and RNA sequencing of dendritic cells (DCs). In a series of in vitro and in vivo experiments, we further defined the immunological and molecular mechanisms of LGG-mediated antitumour immunity.ResultsWe demonstrate that oral administration of live LGG augmented the antitumour activity of anti-programmed cell death 1 (PD-1) immunotherapy by increasing tumour-infiltrating DCs and T cells. Moreover, the combination treatment shifted the gut microbial community towards enrichment in Lactobacillus murinus and Bacteroides uniformis, that are known to increase DC activation and CD8+tumour recruitment. Mechanistically, treatment with live LGG alone or in combination with anti-PD-1 antibody triggered type I interferon (IFN) production in DCs, enhancing the cross-priming of antitumour CD8+ T cells. In DCs, cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) was required for IFN-β induction in response to LGG, as evidenced by the significant decrease in IFN-β levels in cGAS or STING-deficient DCs. LGG induces IFN-β production via the cGAS/STING/TANK binding kinase 1/interferon regulatory factor 7 axis in DCs.ConclusionOur findings have offered valuable insight into the molecular mechanisms of live LGG-mediated antitumour immunity and establish an empirical basis for developing oral administration of live LGG as a combination agent with ICB for cancer therapies.

Cirrhotic portal hypertension is characterised by development of the decompensating events of ascites, encephalopathy, portal hypertensive bleeding and hepatorenal syndrome, which arise in a setting of cirrhosis-associated immune dysfunction (CAID) and define morbidity and prognosis. CAID describes the dichotomous observations that systemic immune cells are primed and display an inflammatory phenotype, while failing to mount robust responses to pathogen challenge. Bacterial infections including spontaneous bacterial peritonitis are common complications of advanced chronic liver disease and can precipitate variceal haemorrhage, hepatorenal syndrome and acute-on-chronic liver failure; they frequently arise from gut-derived organisms and are closely linked with dysbiosis of the commensal intestinal microbiota in advanced chronic liver disease.Here, we review the links between cirrhotic dysbiosis, intestinal barrier dysfunction and deficits of host-microbiome compartmentalisation and mucosal immune homoeostasis that occur in settings of advanced chronic liver disease. We discuss established and emerging therapeutic strategies targeted at restoring intestinal eubiosis, augmenting gut barrier function and ameliorating the mucosal and systemic immune deficits that characterise and define the course of decompensated cirrhosis.

ObjectiveGut microbiota have been linked to inflammatory bowel disease (IBD) and colorectal cancer (CRC). Akkermansia muciniphila (A. muciniphila) is a gram-negative anaerobic bacterium that is selectively decreased in the faecal microbiota of patients with IBD, but its causative role and molecular mechanism in blunting colitis-associated colorectal cancer (CAC) remain inconclusive. This study investigates how A. muciniphila engages the immune response in CAC.DesignMice were given dextran sulfate sodium to induce colitis, followed by azoxymethane to establish CAC with or without pasteurised A. muciniphila or a specific outer membrane protein (Amuc_1100) treatment. Faeces from mice and patients with IBD or CRC were collected for 16S rRNA sequencing. The effects of A. muciniphila or Amuc_1100 on the immune response in acute colitis and CAC were investigated.Results A. muciniphila was significantly reduced in patients with IBD and mice with colitis or CAC. A. muciniphila or Amuc_1100 could improve colitis, with a reduction in infiltrating macrophages and CD8+ cytotoxic T lymphocytes (CTLs) in the colon. Their treatment also decreased CD16/32+ macrophages in the spleen and mesenteric lymph nodes (MLN) of colitis mice. Amuc_1100 elevated PD-1+ CTLs in the spleen. Moreover, A. muciniphila and Amuc_1100 blunted tumourigenesis by expanding CTLs in the colon and MLN. Remarkably, they activated CTLs in the MLN, as indicated by TNF-α induction and PD-1downregulation. Amuc_1100 could stimulate and activate CTLs from splenocytes in CT26 cell conditioned medium.ConclusionsThese data indicate that pasteurised A. muciniphila or Amuc_1100 can blunt colitis and CAC through the modulation of CTLs.

ObjectiveThe protein post-translational modification (PTM) in host cells can be rewritten by bacterial enzymes and represents an unprecedented mechanism in the communication between intestinal flora and the host. Although Akkermansia muciniphila has been widely investigated as a probiotic and blunts colitis-associated tumourigenesis in mice, there is little understanding regarding whether A. muciniphila is involved in the PTM of colorectal cancer (CRC). This study investigates whether and how A. muciniphila engages in the PTM of host CRC.DesignThe secreting extracellular vesicles from A. muciniphila and purified Amuc_2172 were used for different tumourigenesis mice models. Amuc_2172-induced immune activity of CD8+ cytotoxic T lymphocytes (CTLs) were evaluated in vitro and in vivo. The acetyltransferase activity and downstream target genes of Amuc_2172 were investigated.ResultsAmuc_2172, a general control non-derepressible 5-related acetyltransferase of A. muciniphila, was accessible to colorectal cells by macropinocytosis and functioned as an acetyltransferase of Lys14 on histone H3 (H3K14ac). Elevated H3K14ac on Hspa1a loci promoted the transcription and secretion of heat-shock protein 70 (HSP70) in cancer cells. High level of HSP70 promoted the immune activity of CTLs in vitro and in vivo. Moreover, bioengineered nanoparticles provided a safe and reliable drug delivery strategy of Amuc_2172 for CRC treatment in an allograft mice model.ConclusionAmuc_2172 reprogrammed tumour microenvironment by inducing HSP70 secretion and promoting CTL-related immune response in the process of tumourigenesis.

ObjectiveIntestinal microbiota is implicated in the pathogenesis of autoimmune type 1 diabetes in humans and in non-obese diabetic (NOD) mice, but evidence on its causality and on the role of individual microbiota members is limited. We investigated if different diabetes incidence in two NOD colonies was due to microbiota differences and aimed to identify individual microbiota members with potential significance.DesignWe profiled intestinal microbiota between two NOD mouse colonies showing high or low diabetes incidence by 16S ribosomal RNA gene sequencing and colonised the high-incidence colony with the microbiota of the low-incidence colony. Based on unaltered incidence, we identified a few taxa which were not effectively transferred and thereafter, transferred experimentally one of these to test its potential significance.ResultsAlthough the high-incidence colony adopted most microbial taxa present in the low-incidence colony, diabetes incidence remained unaltered. Among the few taxa which were not transferred, Akkermansia muciniphila was identified. As A. muciniphila abundancy is inversely correlated to the risk of developing type 1 diabetes-related autoantibodies, we transferred A. muciniphila experimentally to the high-incidence colony. A. muciniphila transfer promoted mucus production and increased expression of antimicrobial peptide Reg3γ, outcompeted Ruminococcus torques from the microbiota, lowered serum endotoxin levels and islet toll-like receptor expression, promoted regulatory immunity and delayed diabetes development.ConclusionTransfer of the whole microbiota may not reduce diabetes incidence despite a major change in gut microbiota, but single symbionts such as A. muciniphila with beneficial metabolic and immune signalling effects may reduce diabetes incidence when administered as a probiotic.

BackgroundHelicobacter pylori infection is the most prevalent bacterial infection worldwide. Attempts to develop a vaccine have not been successful, partly due to the absence of well-defined immune correlates of protection. The inflammatory response to H. pylori infection is characterised by the recruitment of T cells expressing markers of tissue-resident memory T (TRM) cells to the gastric mucosa. However, the function of TRM cells in gastric tissue during H. pylori reinfection remained poorly understood.ObjectiveWe aimed to investigate the induction, development and function of gastric TRM cells during primary and secondary H. pylori infection.DesignWe characterised gastric H. pylori–specific TRM cells in mice and humans by flow cytometry, immunohistochemistry, immunofluorescence, ChipCytometry staining and single-cell RNA sequencing. The function of gastric TRM cells was established in H. pylori eradication and reinfection experiments as well as by targeted depletion of Hobit+ TRM cells and neutrophils in mice.ResultsExpression of the transcription factor Hobit governs the induction and development of gastric TRM cells, which largely depend on the presence of the H. pylori virulence factor Cytotoxin-associated gene A. H. pylori-specific CD4+ and CD8+ TRM cells resided long-term in the stomach and conferred complete protection from reinfection with the help of neutrophils. Gastric CD8+ TRM cells exhibited varying Hobit expression levels and clustered into distinct subgroups based on distinct transcriptomic and cytokine profiles, suggesting functional specialisation.ConclusionThese findings establish gastric TRM cells as bona fide correlates of protection against H. pylori, highlighting their potential for future prophylactic and therapeutic strategies.

ObjectiveAntibiotic use is associated with an increased risk of developing multiple inflammatory disorders, which in turn are linked to alterations in the intestinal microbiota. How these alterations in the intestinal microbiota translate into an increased risk for inflammatory responses is largely unknown. Here we investigated whether and how antibiotics promote inflammation via the translocation of live native gut commensal bacteria.DesignOral antibiotics were given to wildtype and induced mutant mouse strains, and the effects on bacterial translocation, inflammatory responses and the susceptibility to colitis were evaluated. The sources of the bacteria and the pathways required for bacterial translocation were evaluated using induced mutant mouse strains, 16s rRNA sequencing to characterise the microbial communities, and in vivo and ex vivo imaging techniques.ResultsOral antibiotics induced the translocation of live native commensal bacteria across the colonic epithelium, promoting inflammatory responses, and predisposing to increased disease in response to coincident injury. Bacterial translocation resulted from decreased microbial signals delivered to colonic goblet cells (GCs), was associated with the formation of colonic GC-associated antigen passages, was abolished when GCs were depleted and required CX3CR1+ dendritic cells. Bacterial translocation occurred following a single dose of most antibiotics tested, and the predisposition for increased inflammation was only associated with antibiotics inducing bacterial translocation.ConclusionsThese findings reveal an unexpected outcome of antibiotic therapy and suggest that bacterial translocation as a result of alterations in the intestinal microflora may provide a link between increasing antibiotic use and the increased incidence of inflammatory disorders.

The central nervous system interacts dynamically with the immune system to modulate inflammation through humoral and neural pathways. Recently, in animal models of sepsis, the vagus nerve (VN) has been proposed to play a crucial role in the regulation of the immune response, also referred to as the cholinergic anti-inflammatory pathway. The VN, through release of acetylcholine, dampens immune cell activation by interacting with α-7 nicotinic acetylcholine receptors. Recent evidence suggests that the vagal innervation of the gastrointestinal tract also plays a major role controlling intestinal immune activation. Indeed, VN electrical stimulation potently reduces intestinal inflammation restoring intestinal homeostasis, whereas vagotomy has the reverse effect. In this review, we will discuss the current understanding concerning the mechanisms and effects involved in the cholinergic anti-inflammatory pathway in the gastrointestinal tract. Deeper investigation on this counter-regulatory neuroimmune mechanism will provide new insights in the cross-talk between the nervous and immune system leading to the identification of new therapeutic targets to treat intestinal immune disease.

Objective The authors tested whether the anti-interleukin (IL)-17A monoclonal antibody secukinumab was safe and effective for the treatment of active Crohn's disease. Design In a double-blind, randomised, placebo-controlled proof-of-concept study, 59 patients with moderate to severe Crohn's disease (Crohn's Disease Activity Index (CDAI) ≥220 to ≤450) were assigned in a 2:1 ratio to 2×10 mg/kg intravenous secukinumab or placebo. The primary end point, addressed by Bayesian statistics augmented with historical placebo information, was the probability that secukinumab reduces the CDAI by ≥50 points more than placebo at week 6. Ancillary analyses explored associations of 35 candidate genetic polymorphisms and faecal calprotectin response. Results 59 patients (39 secukinumab, 20 placebo, mean baseline CDAI 307 and 301, respectively) were recruited. 18/59 (31%) patients discontinued prematurely (12/39 (31%) secukinumab, 6/20 (30%) placebo), 10/59 (17%) due to insufficient therapeutic effect (8/39 (21%) secukinumab, 2/20 (10%) placebo). Fourteen serious adverse events occurred in 10 patients (seven secukinumab, three placebo); 20 infections, including four local fungal infections, were seen on secukinumab versus none on placebo. Primary end point analysis estimated <0.1% probability (∆CDAI (SD) =33.9 (19.7), 95% credible interval −4.9 to 72.9) that secukinumab reduces CDAI by ≥50 points more than placebo. Secondary area under the curve analysis (weeks 4–10) showed a significant difference (mean ΔCDAI=49; 95% CI (2 to 96), p=0.043) in favour of placebo. Post hoc subgroup analysis showed that unfavourable responses on secukinumab were driven by patients with elevated inflammatory markers (CRP≥10 mg/l and/or faecal calprotectin≥200 ng/ml; mean ΔCDAI=62; 95% CI (−1 to 125), p=0.054 in favour of placebo). Absence of the minor allele of tumour necrosis factor-like ligand 1A was strongly associated with lack of response measured by baseline-adjusted changes in calprotectin at week 6 (p=0.00035 Bonferroni-corrected). Conclusions Blockade of IL-17A was ineffective and higher rates of adverse events were noted compared with placebo. Clinical trial registration This trial was registered at ClinicalTrial.gov with the number NCT01009281.