Saad Y. Salim

Air pollution effects on the gut microbiota: a link between exposure and inflammatory disease.

Global incidence rates for inflammatory bowel disease (IBD) have gradually risen over the past 20 years. Genome-wide association studies (GWAS) have identified over 160 genetic loci associated with IBD; however, inherited factors only account for a partial contribution to the disease risk. We have recently shown that urban airborne particulate matter (PM) ingested via contaminated food can alter gut microbiome and immune function under normal and inflammatory conditions. In this addendum, we will discuss how PM can modify the gut microbial form and function, provide evidence on changes seen in intestinal barrier, and suggest a working hypothesis of how pollutants affect the gastrointestinal tract. The significance of the work presented could lead to identifying airborne pollutants as potential risk factors and thus provide better patient care management.

Patients with Inflammatory Bowel Disease Exhibit Dysregulated Responses to Microbial DNA

Background A critical role for the gut epithelium lies in its ability to discriminate between pathogens and commensals and respond appropriately. Dysfunctional interactions between microbes and epithelia are believed to have a role in inflammatory bowel disease (IBD). In this study, we analyzed microbiota and gene expression in IBD patients and examined responses of mucosal biopsies to bacterial DNA. Methods Biopsies were taken from non-inflamed areas of the colon in healthy controls (HC) and Crohns disease (CD) and ulcerative colitis (UC) patients in remission. Biopsies were snap-frozen or cultured with DNA from Lactobacillus plantarum (LP) or Salmonella dublin (SD). Gene expression was analyzed under basal conditions and in response to DNA. Gene networks were analyzed using Ingenuity Pathways software. Mucosal-associated microbiota was analyzed using terminal restriction fragment length polymorphism. Frequency of single nucleotide polymorphisms in NOD2 and TLR9 was assessed. Results Patients with IBD had altered microbiota, enhanced expression of inflammatory genes, and increased correlations between specific gene expression and microbes. Principle component analysis showed CD and UC patients to cluster independently from healthy controls in both gene expression and microbial analysis. DNA from LP stimulated anti-inflammatory pathways in controls and UC patients, but induced an upregulation of IL17A in CD patients. There were no differences in SNP frequencies of TLR9 or NOD2 in the groups. Conclusions Patients with Crohns disease exhibit altered responses to bacterial DNA. These findings suggest that the gut response to bacterial DNA may depend not only on the specific type of bacterial DNA, but also on the host.

Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

ABSTRACT Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes.

I-FABP as Biomarker for the Early Diagnosis of Acute Mesenteric Ischemia and Resultant Lung Injury

Acute mesenteric ischemia (AMI) is a life-threatening condition that can result in multiple organ injury and death. A timely diagnosis and treatment would have a significant impact on the morbidity and mortality in high-risk patient population. The purpose of this study was to investigate if intestinal fatty acid binding protein (I-FABP) and α-defensins can be used as biomarkers for early AMI and resultant lung injury. C57BL/6 mice were subjected to intestinal ischemia by occlusion of the superior mesenteric artery. A time course of intestinal ischemia from 0.5 to 3 h was performed and followed by reperfusion for 2 h. Additional mice were treated with N-acetyl-cysteine (NAC) at 300 mg/kg given intraperitoneally prior to reperfusion. AMI resulted in severe intestinal injury characterized by neutrophil infiltrate, myeloperoxidase (MPO) levels, cytokine/chemokine levels, and tissue histopathology. Pathologic signs of ischemia were evident at 1 h, and by 3 h of ischemia, the full thickness of the intestine mucosa had areas of coagulative necrosis. It was noted that the levels of α-defensins in intestinal tissue peaked at 1 h and I-FABP in plasma peaked at 3 h after AMI. Intestinal ischemia also resulted in lung injury in a time-dependent manner. Pretreatment with NAC decreased the levels of intestinal α-defensins and plasma I-FABP, as well as lung MPO and cytokines. In summary, the concentrations of intestinal α-defensins and plasma I-FABP predicted intestinal ischemia prior to pathological evidence of ischemia and I-FABP directly correlated with resultant lung injury. The antioxidant NAC reduced intestinal and lung injury induced by AMI, suggesting a role for oxidants in the mechanism for distant organ injury. I-FABP and α-defensins are promising biomarkers, and may guide the treatment with antioxidant in early intestinal and distal organ injury.

Exposure to Ingested Airborne Pollutant Particulate Matter Increases Mucosal Exposure to Bacteria and Induces Early Onset of Inflammation in Neonatal IL-10–Deficient Mice

Background:Epidemiological associations between early-life air pollution exposure and increased risk of inflammatory bowel diseases have been shown. Our aim was to determine if exposure to airborne particulate matter (PM10) during the neonatal period would alter colitis in the interleukin (IL)-10−/− mouse model. Methods:IL-10−/− pregnant dams and pups were fed chow ± PM10 (9 &mgr;g/g) and pups were studied at 10, 14, and 20 weeks. Twenty-week-old mice were given 2% dextran sodium sulfate. Metagenomic analysis of stool was performed. Bacterial translocation was assessed by serum lipopolysaccharide and culturing bacteria from mesenteric lymph nodes and spleen. Cytokine expression was measured in gut homogenates using the MesoScale discovery platform. PM10 was applied to CMT93 cells ± J744 macrophages, and resistance and cytokine secretion were assessed. THP-1 macrophages were incubated with Escherichia coli HB101 ± PM10 for assessment of uptake and killing. Results:PM10 exposure increased colonic proinflammatory cytokines and bacterial translocation into mesenteric lymph nodes, whereas IL-17A levels were reduced in PM10-fed 10-week-old mice. Bifidobacterium was decreased in mice fed PM10, whereas serum lipopolysaccharide was increased. PM10 interfered with phagocytosis and killing in THP-1 cells. In coculture, PM10 increased tumor necrosis factor &agr; and fluorescein isothiocyanate–dextran flux. After dextran sodium sulfate treatment, PM10-fed mice responded with increased colonic tumor necrosis factor &agr; and IL-1&bgr; and a larger percentage of PM10-fed mice had live bacteria in the mesenteric lymph nodes. Conclusions:Our data suggest that early exposure to pollution particulates can result in an earlier onset of intestinal disease in genetically susceptible hosts and can alter responses to gut injury in later life.

VSL#3 probiotics provide protection against acute intestinal ischaemia/reperfusion injury

Acute intestinal ischaemia/reperfusion injury (AII/R) is an adaptive physiologic response during critical illness, involving mesenteric vasoconstriction and hypoperfusion. Prevention of AII/R in high risk patient populations would have a significant impact on morbidity and mortality. The purpose of this study was to investigate the protective effects of VSL#3 probiotic treatment in a murine model of AII/R. Adult 129/SvEv mice were subjected to an experimental AII/R model using superior mesenteric artery occlusion. Animals were pre-treated with either three days or two weeks of VSL#3 probiotics. Local tissue injury markers were assessed by levels of myeloperoxidase and activation of nuclear factor kappa B (NFкB). Systemic and local cytokines, including interleukin (IL)-1β, IL- 10, TNFα, and interferon gamma were measured by ELISA and multiplex fluorescent detection. VSL#3 probiotics reduced local tissue inflammation and injury due to AII/R. A two-week course of VSL#3 was more effective than a shorter three-day course. The reduction in local inflammation from the two-week course of VSL#3 is correlated to a significant reduction in levels of active IL-1β, and tissue levels of myeloperoxidase. Levels of active NFкB were significantly elevated in the vehicle-fed AII/R mice, corroborating with tissue inflammation, which were attenuated by VSL#3 administrations. VSL#3 did not cause any systemic inflammation or lung injury. VSL#3 probiotics are effective in reducing local tissue injury from AII/R by down-regulating pro-inflammatory mediators and immune cell recruitment. This study highlights a potential role for VSL#3 in management of patients at high risk for AII/R.

Metabolomic profiling to characterize acute intestinal ischemia/reperfusion injury

Sepsis and septic shock are the leading causes of death in critically ill patients. Acute intestinal ischemia/reperfusion (AII/R) is an adaptive response to shock. The high mortality rate from AII/R is due to the severity of the disease and, more importantly, the failure of timely diagnosis. The objective of this investigation is to use nuclear magnetic resonance (NMR) analysis to characterize urine metabolomic profile of AII/R injury in a mouse model. Animals were exposed to sham, early (30 min) or late (60 min) acute intestinal ischemia by complete occlusion of the superior mesenteric artery, followed by 2 hrs of reperfusion. Urine was collected and analyzed by NMR spectroscopy. Urinary metabolite concentrations demonstrated that different profiles could be delineated based on the duration of the intestinal ischemia. Metabolites such as allantoin, creatinine, proline, and methylamine could be predictive of AII/R injury. Lactate, currently used for clinical diagnosis, was found not to significantly contribute to the classification model for either early or late ischemia. This study demonstrates that patterns of changes in urinary metabolites are effective at distinguishing AII/R progression in an animal model. This is a proof-of-concept study to further support examination of metabolites in the clinical diagnosis of intestinal ischemia reperfusion injury in patients. The discovery of a fingerprint metabolite profile of AII/R will be a major advancement in the diagnosis, treatment, and prevention of systemic injury in critically ill patients.

Swimming in a tsunami of change

We read the recent article on ‘‘Faith-based medical education’’ by Whitehead and Kuper (2017) with much interest. We applaud them for publicly questioning the accepted ‘‘doctrine’’ of competence-based medical education (CBME) by asking the community to consider whether ‘‘the emperor has no clothes’’. We agree that it is dangerous for any community to blindly accept significant change mandated ‘‘from the top’’ as ‘‘gospel’’ without good reason, and we agree that it is important for members of our community to be able to ask critical questions of authority without fear. Further, this article made us ponder on several issues pertaining to change management in medical education, for instance:

Non-Invasive Point-of-Care Device to Diagnose Acute Mesenteric Ischemia

Inadequate blood supply to the intestine can lead to acute mesenteric ischemia (AMI), with a mortality rate ranging from 60% to 90%. This high mortality rate is partially due to late detection and the lack of efficient early diagnostic tests. There is an urgent need for a point-of-care tool for immediate bedside diagnosis. Here we present for the first time a rapid and non-invasive electrochemical biosensor device based on non-faradic impedance spectroscopy to detect intestinal fatty-acid binding protein (I-FABP) as an indication of AMI. The electrochemical biosensors consist of gold interdigitated electrodes that were fabricated using photolithographic techniques on top of silicon dioxide substrates. The electrode surfaces were functionalized with an I-FABP capture antibody (CAnB) to entice the target protein, while gold nanoparticles (GNPs) functionalized with detection antibodies (DAnB-GNPs) were utilized as a novel mechanism to enhance the detection signal. Quantification of the I-FABP concentration in the medium depended on its attachment to CAnB and DAnB-GNPs in a sandwich manner, where the latter boosts the impedance signal through its binding to the I-FABP. This non-invasive non-faradic electric biosensor device demonstrates the potential for bench-to-bedside translation with the goal of decreasing morbidity and mortality from AMI.

Tu1847 Dysregulated Responses to Microbial DNA are Associated With Altered Microflora in Patients With Crohn's Disease

G A A b st ra ct s spheroid that exposes the basolateral membrane of IEC with preserving the cell formation and structure of crypt. Moreover, we have originally generated pIEC from colon. We therefore aim to assess the reaction to flagellin of pIEC via TLR5 on basolateral membrane in small intestine and colon by the alternation of Notch signaling. Methods: pIECs were generated from mouse small intestine and colon by Sato method and original method, respectively. The localization of TLR5 in pIEC was analyzed by immunofluorescence. Flagellin was added to the medium for either 3 hours or 7 days to stimulate TLR5 on basal membrane at the outside of spheroid. Comprehensive genes induced by flagellin were detected by microarray analysis. Notch signaling was suppressed by gamma secretase inhibitor (GSI). Results: pIECs of small intestine and colon were cultured as spheroid shape with keeping cell formation. TLR5 in pIECs was specifically localized on the basal membrane at the outside of the spheroid. Flagellin did not change the cell formation and the phenotypic gene expression of pIECs. However, microarray analysis showed that various genes including cytokines, chemokines and reactive oxygen species were induced by flagellin. Candidate genes were confirmed by quantitative RT-PCR. pIEC generated from MyD88 deficient mice did not react to Flagellin, suggesting that the response to Flagellin is mediated by TLR5 signaling via MyD88. Treatment with GSI inhibited Notch signaling of pIEC, resulting in not only the increase of secretory lineages but also the decrease of absorptive cells and stem cells. Interestingly, Flagellin inducible genes were up-regulated by Notch signal inhibition of pIEC generated from small intestine but not from colon. Conclusion: We have for the first time investigated the natural reaction of IECs by flagellin without the effect of the stromal tissue. IECs play various roles in the invasion of bacteria as a unit at the front line of mucosal defense. Moreover, Notch signaling inhibition in pIECs causes the overreaction to flagellin, suggesting that Notch signaling acceleration of IECs in Crohns disease patients might weaken flagellin response.