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Autotaxin (ATX) is a secreted enzyme that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) and choline. ATX has been implicated in multiple chronic inflammatory diseases, but little is known about its role in the development of inflammatory bowel disease (IBD). Here, we investigated how ATX contributed to intestinal inflammation during colitis. We found that ATX expression levels were upregulated in the intestines of ulcerative colitis (UC) patients in acute state as well as in the intestines of dextran sulfate sodium (DSS)-induced colitis mice, which is likely due to increased infiltration of inflammatory cells including macrophages. Intriguingly, the inhibition of ATX activity led to reduced production of inflammatory cytokines, as well as attenuated colitis. These findings suggest that ATX may display strong pro-inflammatory properties. Supporting this, treatment with recombinant mouse ATX (rmATX) increased the production of inflammatory cytokines and enzymes in mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMDM), whereas silencing ATX by siRNA reduced LPS-stimulated production of pro-inflammatory factors. Notably, we found that the levels of LPA2 (an LPA receptor) were dramatically upregulated in rmATX-treated RAW264.7 cells and DSS-treated mice. Gene silencing of lpa2 in RAW264.7 cells by siRNA led to reduced production of inflammatory cytokines. Moreover, adenovirus-mediated delivery of lpa2 short hairpin RNA into DSS-treated mice ameliorated colitis. Collectively, our research suggests that ATX may exacerbate DSS-induced colitis by activating LPA2 receptor in macrophages and represent a promising target for the treatment of IBD. KEY MESSAGES: Increased ATX expression and secretion in colitic colons are likely due to increased infiltration of inflammatory cells including macrophages. Recombinant ATX promotes, but ATX silencing inhibits, the production of inflammatory cytokines in LPS-stimulated RAW264.7 cells and BMDM. •LPA2 mediates the pro-inflammatory effects of ATX on macrophages. Inhibition of ATX and downregulation of LPA2 ameliorate DSS-induced colitis.

Background Solid transplant patients are susceptible to Pneumocystis jirovecii pneumonia (PJP). While the vast majority of PJP cases occur within the first 6 months after transplantation, very few PJP cases are seen beyond 1 year post-transplantation (late-onset PJP). PJP and coronavirus disease 2019 (COVID-19, caused by infection with SARS-CoV-2) share quite a few common clinical manifestations and imaging findings, making the diagnosis of PJP often underappreciated during the current COVID-19 pandemic. To date, only 1 case of kidney transplantation who developed COVID-19 and late-onset PJP has been reported, but this patient also suffered from many other infections and died from respiratory failure and multiple organ dysfunction syndrome. A successful treatment of kidney patients with COVID-19 and late-onset PJP has not been reported. Case presentation We present a case of a 55-year-old male kidney transplant patient with COVID-19 who also developed late-onset PJP. He received a combined treatment strategy, including specific anti-pneumocystis therapy, symptomatic supportive therapy, adjusted immunosuppressive therapy, and use of antiviral drugs/antibiotics, ending with a favorable outcome. Conclusions This case highlights the importance of prompt and differential diagnosis of PJP in kidney transplant patients with SARS-CoV-2 infection. Further studies are required to clarify if kidney transplant patients with COVID-19 could be prone to develop late-onset PJP and how these patients should be treated.

Background Ulcerative colitis (UC) is a chronic inflammatory condition linked to intestinal microbial dysbiosis, including the expansion of E. coli strains related to extra-intestinal pathogenic E. coli. These “pathobionts” exhibit pathogenic properties, but their potential to promote UC is unclear due to the lack of relevant animal models. Aims We explored the potential to establish a mouse model of GI infection by the UC-associated E. coli strain p19A, as well as characterize the pathogenic features of p19A. Methods We used a representative UC pathobiont strain (p19A), and mice lacking single immunoglobulin and toll-interleukin 1 receptor domain (SIGIRR), a deficiency increasing susceptibility to gut infections. Vancomycin-pretreated Sigirr-/- mice were subsequently gavaged with the control E. coli DH10B (a derivative of commensal strain K-12) or p19A. One day after infection, mice were exposed to 2.5% dextran sodium sulfate (DSS) in their drinking water for another 4 days. Results Strain p19A was found to adhere to the cecal mucosa of Sigirr-/- mice, causing modest inflammation. Moreover, it dramatically worsened DSS-induced colitis. This potentiation was attenuated using a p19A strain lacking α-hemolysin genes, or when we targeted pathobiont adherence using a p19A strain lacking the adhesin FimH, or following treatment with FimH antagonists. Conclusions Thus, UC pathobionts adhere to the intestinal mucosa, and worsen the course of colitis in susceptible hosts in a manner dependent on specific virulence factors, including α-hemolysin and FimH. Funding Agencies CCC, CIHR

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic Salmonella gastroenteritis model, Ab-mediated neutralization of IL-22 impaired intestinal epithelial barrier integrity and, consequently, exaggerated expression of proinflammatory cytokines. As disease normally resolved, neutralization of IL-22 caused luminal narrowing of the cecum—a feature reminiscent of fibrotic strictures seen in Crohn disease patients. Corresponding to the exaggerated immunopathology caused by IL-22 suppression, Salmonella burdens in the gut were reduced. This enhanced inflammation and pathogen clearance was associated with alterations in gut microbiome composition, including the overgrowth of Bacteroides acidifaciens. Our findings thus indicate that IL-22 plays a protective role by limiting infection-induced gut immunopathology but can also lead to persistent pathogen colonization.

Mammalian cells express an array of toll-like receptors to detect and respond to microbial pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). These clinically important attaching and effacing (A/E) pathogens infect the apical surface of intestinal epithelial cells, causing inflammation as well as severe diarrheal disease. Because EPEC and EHEC are human-specific, the related murine pathogen Citrobacter rodentium has been widely used to define how hosts defend against A/E pathogens. This study explored the role of TLR9, a receptor that recognises unmethylated CpG dinucleotides present in bacterial DNA, in promoting host defence against C. rodentium. Infected Tlr9-/- mice suffered exaggerated intestinal damage and carried significantly higher (10-100 fold) pathogen burdens in their intestinal tissues as compared with wild type (WT) mice. C. rodentium infection also induced increased antimicrobial responses, as well as hyperactivation of NF-κB signalling in the intestines of Tlr9-/- mice. These changes were associated with accelerated depletion of the intestinal microbiota in Tlr9-/- mice as compared with WT mice. Notably, antibiotic-based depletion of the gut microbiota in WT mice prior to infection increased their susceptibility to the levels seen in Tlr9-/- mice. Our results therefore indicate that TLR9 signalling suppresses intestinal antimicrobial responses, thereby promoting microbiota-mediated colonisation resistance against C. rodentium infection.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic

BACKGROUND: During periods of acute sickness, all animals including humans voluntarily decrease their food intake, a behavior known as “infection-induced anorexia”. Since infection-induced anorexia or fasting is highly conserved amongst mammals, we and others have proposed that it actually promotes host-defense. Several studies have demonstrated that fasting indeed attenuates disease during acute systemic infection or chronic inflammation. AIMS: Since the Gastrointestinal tract is the first organ to be affected by food deprivation, we investigated if fasting alters the course of infection and/or ameliorates intestinal inflammation using a mouse model of S. typhimurium-induced colitis. We furthermore examined if fasting modifies the resident gut microbiota, and whether these modifications are instrumental to alter the course of S. typhimurium infection and colitis. METHODS: S. typhimurium causes severe colitis in streptomycin pre-treated, orally infected mice as early as 12h post infection. C57BL/6 conventional as well as germfree mice were fed or fasted for 24h and orally infected with S. typhimurium, continuing the fast for another 6h to 24h. Additionally, C57BL/6 conventional mice were fed single macronutrients to gauge the effect of different nutrients on the infection. Pathology as well as Salmonella colonization, was assessed by histological and immunofluorescent staining and organ pathogen burden counts; inflammatory gene transcription was measured by qPCR. Microbiome profiling of intestinal contents was performed utilizing 16S rRNA sequencing. RESULTS: Fasting completely abrogated S. typhimurium infection and colitis in conventional C57BL/6 mice, whereas infection still proceeded, albeit in a reduced fashion in germfree mice in concert with minimal signs of inflammation. Microbiome profiles between fed and fasted groups differed greatly in terms of beta diversity with the fasting group showing a consistent increase in the genus Akkermansia. Feeding mice glucose alone was not sufficient to re-establish the infection, but preliminary results indicate that other macronutrients can partially restore Salmonella colonization. CONCLUSIONS: Taken together, our findings show that prolonged fasting dramatically protects the host against S. typhimurium infection, in part by altering the intestinal microbiome and increasing colonization resistance, likely by limiting nutrient availability for the pathogenic bacteria. Additionally our results indicate that food deprivation impairs Salmonella virulence and reduces its ability to promote an inflammatory environment independent of changes in the microbiome. FUNDING AGENCIES: CCC, CIHRNSERC

Enteropathogenic Escherichia coli (EPEC)-induced diarrhea is often associated with disruption of intestinal epithelial tight junctions. Although studies have shown alterations in the expression and localization of bicellular tight junction proteins during EPEC infections, little is known about whether tricellular tight junction proteins (tTJs) are affected. Using Caco-2 cell monolayers, we investigated if EPEC is capable of targeting the tTJ protein tricellulin. Our results demonstrated that at 4 h postinfection, EPEC induced a significant reduction in tricellulin levels, accompanied by a significant loss of transepithelial resistance (TEER) and a corresponding increase in paracellular permeability. Conversely, cells overexpressing tricellulin were highly resistant to EPEC-induced barrier disruption. Confocal microscopy revealed the distribution of tricellulin into the plasma membrane of infected epithelial cells and confirmed the localization of EPEC aggregates in close proximity to tTJs. Moreover, infections with EPEC strains lacking genes encoding specific type III secreted effector proteins demonstrated a crucial role for the effector EspG1 in modulating tricellulin expression. Complementation studies suggest that the EspG-induced depletion of tricellulin is microtubule dependent. Overall, our results show that EPEC-induced epithelial barrier dysfunction is mediated in part by EspG1-induced microtubule-dependent depletion of tricellulin.

BACKGROUND: Attaching and effacing (A/E) pathogens such as Enterohemorrhagic E. coli (EHEC) are important causes of diarrheal disease worldwide. Interestingly, little is known about how these bacteria obtain key nutrients, and whether these nutrients modulate pathogen colonization and gut infection. For example, fucose is an important sugar and bacterial food source cleaved from host glycans found on the intestinal mucosal surface via the actions of commensal microbes expressing fucosidase enzymes. Interestingly, although the mouse A/E pathogen Citrobacter rodentium does not itself express a fucosidase, recent studies have found that it and EHEC use a fucose sensing system to modulate their pathogenicity in vitro. At present, it is unclear what role fucose metabolism may play in A/E pathogen virulence and metabolism in vivo. To address this, we focused on C. rodentium enzyme L-fuculose kinase (encoded by the fucK gene), which plays a key role in the L-fucose metabolic pathway and fucose utilization as a food source. AIMS: We investigated the roles of fucose and the enzyme L-fuculose kinase in controlling C. rodentium pathogenesis. METHODS: Wildtype (WT) and ΔfucK C. rodentium were used for in vitro assays (type three secretion, growth and adhesion assays) and for in vivo infection. C57Bl/6 mice were infected either separately, or with both C. rodentium strains at a dose of 10^8 colony forming units. In some infections, mice were pretreated with streptomycin (20mg 24h pre-infection) or were fed fucose (200ul of 25mM L-fucose 2x/day). Bacterial burdens, pathology score and competitive assay were assessed on day 6 post-infection. RESULTS: In vitro virulence assays identified no significant differences between ΔfucK and WT C. rodentium regarding type three secretion, growth or adherence to cultured epithelial cells. Moreover, both WT and ΔfucK C. rodentium readily colonized the intestines of mice either pretreated (or not), with streptomycin, with pathogen burdens, localisation and histological pathology scores similar between the two strains. In contrast, simultaneous infection by both strains (competitive assay) within the same mice revealed the ΔfucK strain was significantly impaired when competing with WT C. rodentium (no streptomycin). In contrast, ΔfucK was able to equally compete with WT C. rodentium when streptomycin pretreatment was given to deplete commensal bacteria. Feeding fucose to these streptomycin treated mice again reduced the ability of the ΔfucK strain to compete with WT C. rodentium. CONCLUSIONS: These results indicate that the fucK-dependent fucose metabolic pathway promotes but is not essential to C. rodentium pathogenesis. Moreover these findings suggest that commensal microbes play a key role in controlling fucose availability in the gut, and thereby impact A/E pathogen metabolism and virulence. FUNDING AGENCIES: CCC, CIHRNSERC, FRQS

BACKGROUND: Ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory conditions of the gastrointestinal (GI) tract that have been linked to intestinal microbial dysbiosis. While adherent-invasive Escherichia coli are associated with CD, recent studies have identified E. coli of phylogroup B2 as being frequently isolated from UC patients. B2 E. coli isolated from UC patients (such as p19A) harbour extra-intestinal pathogenic E. coli virulence factors including alpha hemolysin genes (hlyI, II). Correspondingly, the p19A strain causes cell death and barrier dysfunction in human epithelial colorectal cell lines, however its role in intestinal immunopathology is unclear because of the lack of a suitable animal model. AIMS: The current study explores the potential to establish a mouse model of GI infection by the UC-associated E. coli strain p19A, as well as defines the mechanisms by which it promotes colitis. METHODS: C57BL/6 and Sigirr (Single Ig IL-1R-related Receptor) deficient mice were orally administered with 5 mg vancomycin 6 hours prior to the oral infection with 10(8) colony forming units (CFU) of P19A. Sigirr(-/-) mice were examined because they show increased susceptibility to infection by enteric bacterial pathogens. Intestinal tissues as well as feces were homogenized and plated to enumerate CFU. An in vivo imaging system visualized the colonization of p19A in the intact intestines. To test the impact of pre-existing p19A colonization on experimental colitis, mice were colonized with DH10B (non-pathogenic E. coli control), p19A wild-type strain or p19A-ΔhlyI, II (a mutant strain lacking hemolysin genes). The next day, mice were given 3% (wt/vol) dextran sulfate sodium (DSS) in their drinking water for 4 days. Mice were monitored daily and euthanized to collect samples for further analysis. RESULTS: Vancomycin pretreatment led to persistent p19A colonization of the intestinal lumen of C57BL/6 mice, based on luciferase based imaging and fecal shedding data. Furthermore, p19A colonized the intestinal mucosal surface of Sigirr(-/-) mice. While p19A infection caused only minimal pathology on its own, it dramatically worsened the course of DSS colitis, in concert with deep penetration of the damaged colonic mucosa. Notably, a p19A strain deficient in hemolysin genes was severely attenuated in its ability to promote DSS colitis in Sigirr(-/-) mice. CONCLUSIONS: Our findings provide evidence that a UC E. coli pathobiont can readily and persistently colonize the intestines of susceptible hosts, and significantly worsen the course of colitis. This model thus facilitates research into the role played by UC associated E. coli pathobionts in the pathogenesis of IBD. FUNDING AGENCIES: CCC, CIHRNSERC, MITACs, and CHILD Foundation

Effectors translocated into the host cell by Salmonella enterica serovar Typhimurium are critical for bacterial virulence. For many effectors, the mechanisms of their interactions with host pathways are not yet understood. We have recently found an interaction between the SPI-2 effector SseL and oxysterol-binding protein (OSBP). We show here that SseL binds the N-terminus of OSBP and that S. Typhimurium infection results in redistribution of OSBP. We furthermore demonstrate that OSBP is required for efficient replication of intracellular S. Typhimurium. This suggests that S. Typhimurium hijacks OSBP-dependent pathways to benefit its intracellular life-style, possibly by SseL- and OSBP-mediated manipulation of host lipid metabolism.

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.

The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenicEscherichia coli(EPEC), enterohemorrhagicE. coli(EHEC), andCitrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by thesepDandsepLmutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals.IMPORTANCEMany bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to cause disease. The secreted proteins perform different functions at various stages during infection and are classified into three substrate categories (T3SS components, translocators, and effectors). They all contain secretion signals at their N termini, but how their secretion hierarchy is determined is poorly understood. Here, we show that the N-terminal secretion signals from different substrate categories all behave the same and do not confer substrate specificity. We further characterize the secretion signals of the translocators and identify a translocator-specific signal, demonstrating that substrate-specific secretion signals are required in regulating T3SS substrate hierarchy.

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)–Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus . This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus . Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB.

The Salmonella effector protein SopB has previously been shown to induce activation of Akt and protect epithelial cells from apoptosis in vitro . To characterize the role of Akt2 in host defense against Salmonella enterica serovar Typhimurium infection, wild-type (WT) mice and mice lacking Akt2 (Akt2 knockout [KO] mice) were infected using a Salmonella acute gastroenteritis model. Infected Akt2 KO mice showed a more pronounced morbidity and mortality associated with higher bacterial loads in the intestines and elevated levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and MCP-1, in the colons at 1 day postinfection compared to those shown in WT mice. Histopathological assessment and immunohistochemical analysis of cecal sections at 1 day postinfection revealed more severe inflammation and higher levels of neutrophil infiltration in the ceca of Akt2 KO mice. Flow cytometry analysis further confirmed an increase in the recruitment of Gr-1 + CD11b + neutrophils and F4/80 + CD11b + macrophages in the intestines of infected Akt2 KO mice. Additionally, enhanced levels of annexin V + and terminal transferase dUTP nick end labeling-positive (TUNEL + ) apoptotic cells in the intestines of infected Akt2 KO mice were also observed, indicating that Akt2 plays an essential role in protection against apoptosis. Finally, the differences in bacterial loads and cecal inflammation in WT and Akt2 KO mice infected with WT Salmonella were abolished when these mice were infected with the sopB deletion mutant, indicating that SopB may play a role in protecting the mice from Salmonella infection through the activation of Akt2. These data demonstrate a definitive phenotypic abnormality in the innate response in mice lacking Akt2, underscoring the important protective role of Akt2 in Salmonella infection.

BACKGROUND: Vasoactive intestinal peptide (VIP), a 28 amino acid neuropeptide, exhibits potent anti-inflammatory effects in autoimmune and inflammatory diseases. Interestingly, recent ex vivo studies suggest that VIP can promote the production of proinflammatory Th17 cells (primarily secrete cytokine IL-17A). While Th17 cells likely aggravate inflammation in autoimmune diseases, they are known to be protective against Citrobacter rodentium induced colitis-a mouse model of colitis. AIMS: The aim of this study was to examine the in vivo significance of VIP-Th17 signaling axis during an enteric pathogen (Citrobacter rodentium) infection. METHODS: Wild type mice, VIP deficient (Vip(-/-) ) mice and VIP treated Vip(-/-) mice were infected with C. rodentium for 10 days. Body weight loss and survival rates were recorded daily. Bacterial counts in the intestinal and systemic sites were determined by an in vivo imaging system and plating method. Mucosal damages were analyzed by Haemotoxylin and Eosin staining. The production of cytokines (such as IL-1β, IL-6, IL-17A, IL-22 and TNF-α) were quantified by quantitative PCR, ELISA and FACS. RESULTS: Vip (-/-) mice infected with C. rodentium showed dramatic body weight loss, decreased survival rates, increased bacterial colonization in intestinal and systemic sites, worsened mucosal damages/inflammatory responses as well as reduced production of Th17 cell responses. Importantly, exogenous treatment of Vip(-/-) mice with recombinant VIP ameliorated C. rodentium-induced colitis, accompanied by restoration of Th17 cell responses. CONCLUSIONS: We provide the first in vivo evidence that VIP promotes Th17 immune responses thereby protecting against C. rodentium induced colitis. FUNDING AGENCIES: CCC

BACKGROUND: Group 3 innate lymphoid cells (ILC3s) are a class of innate lymphocytes that are emerging as key players in regulating intestinal inflammation, immunity and homeostasis. Through the production of the cytokine IL-22, ILC3s can modulate the function of intestinal epithelial cells (IECs), helping to maintain the IEC barrier integrity and promoting host defense against bacterial pathogens (such as Citrobacter rodentium). Recently, a handful studies have shown that ILC3s can communicate with the nervous system in the gut where neurons and immune cells are enriched. It remains largely unexplored how this neuroimmune communication regulates the number and function of ILC3s in the gut. AIMS: To understand how the neuropeptide - vasoactive intestinal peptide (VIP), controls the expansion and function of ILC3s thereby regulating intestinal immunity under baseline and infection conditions. METHODS: Wildtype (WT), Vip(-/-) and Rag1(-/-) mice were used. ILC3s and dendritic cells were isolated from the intestinal lamina propria using percoll gradients. ILC3s migration towards VIP was measured by a chemotaxis assay. The number and function of ILC3s was assessed using FACS. IL-22, RegIII-β, and RegIII-γ production was quantified by qPCR and/or ELISA. WT and Vip(-/-) mice were infected with C. rodentium for 4–10 days, and their susceptibility to infection assessed by measuring body weight loss, bacterial counts, and histological changes. Recombinant VIP, IL-22 or retinoic acid (RA) was IP injected into Vip(-/-) mice, with the mice subsequently analyzed for their susceptibility to C. rodentium infection. ILC3s were also isolated from Rag1(-/-) mice and injected into Vip(-/-) mice by the IV route, followed by infection with C. rodentium. RESULTS: VIP induced ILC3s migration through the receptor VPAC2. VIP also promoted gut homing receptor CCR9 expression on intestinal ILC3s, by enhancing RA production by intestinal dendritic cells. The loss of VIP led to reduced activation and recruitment of intestinal ILC3 cells, less IL-22 production, and abnormal IEC barrier function. Accordingly, Vip(-/-) mice proved highly susceptible to C. rodentium infection, suffering dramatic body weight loss, increased pathogen colonization, and worsened intestinal damage. Notably, recombinant IL-22 adminstration was sufficient to rescue Vip(-/-) mice from C. rodentium infection. Moreover, treatment with RA or VIP, or adoptive transfer of ILC3 cells were all able to increase the intestinal production of IL-22 and protect Vip(-/-) mice from infection. CONCLUSIONS: Our results reveal a novel neuroimmune axis whereby the neuropeptide VIP controls the activation and recruitment of ILC3s in the gut, thereby playing a key role in promoting mucosal defense against intestinal pathogens. FUNDING AGENCIES: CCC, CIHRNSERC