Abstract Introduction The enteric nervous system (ENS) is essential for gastrointestinal (GI) function and has recently been implicated in regulating immune activity at mucosal sites. However, its broader contributions to systemic immunity remain unclear. Because the ENS interfaces with extrinsic neural circuits, it is well-positioned to relay information between the gut and peripheral tissues. We hypothesized that enteric neurons detect disturbances in intestinal immune homeostasis and transmit signals that coordinate systemic defenses. Methods To test this, we combined an in vivo infection-challenge paradigm in mice with manipulations of ENS activity and measured downstream immune readouts. We also used ex vivo cultures to assess how enteric neurons respond to microbial cues. Results Our findings indicate that activating the ENS promotes rapid protective responses in distal organs. Upon stimulation, enteric neurons released inflammatory mediators, supporting the mobilization of innate immune cells to both intestinal and peripheral sites. Modulating ENS activity altered host susceptibility to a subsequent systemic challenge. Conclusion Together, these results suggest that the ENS functions as a sensor-effector hub, linking local GI infection to rapid, body-wide immune responses, and highlighting a previously understudied role for the ENS in systemic host defenses. Funding Source n/a Topic Categories Neuroimmunology (NEUR)

Fusobacterium nucleatum is a Gram-negative oncobacterium that is associated with colorectal cancer. The molecular mechanisms utilized by F. nucleatum to promote colorectal tumor development have largely focused on adhesin-mediated binding to the tumor tissue and on the pro-inflammatory capacity of F. nucleatum. However, the exact manner in which F. nucleatum promotes inflammation in the tumor microenvironment and subsequent tumor promotion remains underexplored. Here, we show that both living F. nucleatum and sterile F. nucleatum-conditioned medium promote CXCL8 release from the intestinal adenocarcinoma HT-29 cell line. We determined that the observed pro-inflammatory effect was ALPK1-dependent in both HEK293 and HT-29 cells and that the released F. nucleatum molecule had characteristics that match those of the pro-inflammatory ALPK1 ligand ADP-heptose or related heptose phosphates. In addition, we determined that not only F. nucleatum promoted an ALPK1-dependent pro-inflammatory environment but also other Fusobacterium species such as F. varium, F. necrophorum and F. gonidiaformans generated similar effects, indicating that ADP-heptose or related heptose phosphate secretion is a conserved feature of the Fusobacterium genus. By performing transcriptional analysis of ADP-heptose stimulated HT-29 cells, we found several inflammatory and cancer-related pathways to be differentially regulated, including DNA mismatch repair genes and the immune inhibitory receptor PD-L1. Finally, we show that stimulation of HT-29 cells with F. nucleatum resulted in an ALPK1-dependent upregulation of PD-L1. These results aid in our understanding of the mechanisms by which F. nucleatum can affect tumor development and therapy and pave the way for future therapeutic approaches.

The impact of bacterial members of the microbiota on the development of colorectal cancer (CRC) has become clear in recent years. However, exactly how bacteria contribute to the development of cancer is often still up for debate. The impact of bacteria-derived metabolites, which can influence the development of CRC either in a promoting or inhibiting manner, is undeniable. Here, we discuss the effects of the most well-studied bacteria-derived metabolites associated with CRC, including secondary bile acids, short-chain fatty acids, trimethylamine-N-oxide and indoles. We show that the effects of individual metabolites on CRC development are often nuanced and dose- and location-dependent. In the coming years, the array of metabolites involved in CRC development will undoubtedly increase further, which will emphasize the need to focus on causation and mechanisms and the clearly defined roles of bacterial species within the microbiota.

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.

The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.

The human gastrointestinal tract is inhabited by trillions microbial cells that mostly form a symbiotic relationship with the host. Bacteria have been studied best with regard to the function and effects in the intestinal tract. Bacteria can promote development of colorectal cancer (CRC), but also inflammatory bowel disease (IBD). The focus of recent studies has shifted to the contribution of bacteria-derived metabolites, the intermediate or end products of metabolic reactions, on the intestinal tract. In this work, we aimed to further delineate to contribution of microbiota-derived metabolites on the intestinal diseases IBD and CRC. First, in chapter 2, the role of microbiota-derived metabolites in colorectal cancer is comprehensively assessed. Here, the focus is on the impact of established contributors, including secondary bile acids, short-chain fatty acids, trimethylamine-N-oxide, indoles and colibactin. A loss of epithelial barrier integrity is a hallmark of IBD. Therefore, we utilized a screen in chapter 3 using the released factors by bacteria after a two-day culture, which were added to intestinal organoids. Here, we identified the bacterial species Fusobacterium nucleatum and Fusobacterium varium to release a compound that is toxic to intestinal organoids. Butyrate is a short-chain fatty acid that has been reported to be secreted by Fusobacterium species, which was confirmed to be released by our strains. Lastly, both Fusobacterium-released metabolites and butyrate induced autophagy in intestinal epithelial cells, indicating that this prolonged autophagy could result in toxicity to the organoids. All in all, this chapter identified Fusobacterium species to secrete butyrate which promoted autophagy and organoid cytotoxicity. Fusobacterium nucleatum is not only associated with IBD, but also with CRC. Thus, in chapter 4 we wondered whether Fusobacterium species secrete metabolites that could promote development of colorectal cancer. A pro-inflammatory compound activating ALPK1 signaling was identified to be released from Fusobacterium species, likely being the metabolite ADP-heptose. Lastly, RNA-sequencing suggested that ADP-heptose released by Fusobacterium could promote immune evasion of tumor cells by upregulating the inhibitory receptor PD-L1. Thus, Fusobacterium species secrete ADP-heptose that could promote tumor development by evading the immune system via PD-L1 upregulation. In chapter 5 we identified another ADP-heptose secreting bacterium, Campylobacter jejuni, which can cause severe gastroenteritis in individuals. The pro-inflammatory effect of C. jejuni on epithelial cells was dependent on a functional ADP-heptose synthesis pathway in the bacterium, indicating that indeed specifically ADP-heptose or another upstream metabolite was responsible. Additionally, the ADP-heptose derived from C. jejuni activated a wide set of pro-inflammatory genes, indicating that this metabolite could contribute to the severe inflammation induced by C. jejuni infection. The question remained what the broad transcriptional consequences of ADP-heptose on the intestinal epithelium and immune cells were. Therefore, chapter 6 assessed RNA-sequencing after ADP-heptose stimulation of intestinal epithelium and immune cells compared to a classical bacterial pro-inflammatory compound. It was identified that in addition to an extensive pro-inflammatory transcriptional signature, ADP-heptose also promoted a pro-tumor signature in intestinal epithelial cells. In immune cells, ADP-heptose induced a distinct pro-inflammatory signature, indicating that pro-inflammatory consequences of ADP-heptose could have different consequences in epithelial cells compared to immune cells. The research conducted in this thesis provides insight into the consequences of two bacterial-derived metabolites ADP-heptose and butyrate on especially the intestinal epithelium. This is accomplished using to pathogenic bacterial species F. nucleatum and C. jejuni, which additionally provides insights into pathogenic mechanisms employed by these bacterial species. The results expand our knowledge on microbiota-derived metabolites in intestinal diseases such as inflammatory bowel disease, colorectal cancer and acute gastroenteritis as result of C. jejuni infection. Additionally, the research allows for identification of novel therapeutic approaches to combat these metabolites or bacteria in these diseases.