Tu2023

A Novel Source of Intestinal Damage: The HIV Protease Inhibitor RitonavirWorsens Damage Caused by COX Inhibitors.Andrea Mencarelli, Barbara Renga, Sabrina Cipriani, Claudio D'Amore, Daniela Francisci,Franco Baldelli, Eleonora Distrutti, Annibale Donini, Stefano Fiorucci

Background. The protease inhibitor ritonavir is part of highly active anti-retroviral therapy(HAART) used successfully in the treatment of human immunodeficiency virus (HIV) infec-tion. There is evidence that ritonavir alters intestinal permeability and induce damage tothe small intestine. Because HIV infected patients taking HAART are at high risk of developingcardiovascular complications, there might be a need to use low dose of aspirin (ASA) toprevent inschemic event. Similarly, long term survival exposes HIV infects persons to detri-mental interaction of ritonavir with NSAIDs, since both agents might cause intestinal injury.Aims. To test whether ritonavir worsens intestinal injury caused by NSAIDs and ASA.Methods. C57BL6 mice were treated for 25 days with ritonavir (50 mg/Kg/day per os) andthan for others 5 days with the combination of ritonavir plus ASA (50 mg/Kg/day per os)or naproxen (100 mg/Kg/day per os). In a second set of experiments C57BL6 mice weretreated for 25 days with ritonavir (50 mg/Kg/day per os) alone or in combination with thePGE2 analog misoprostol (100 μg/Kg per os). Results. Ritonavir administration per se causedintestinal damage and its co-administration in combination with naproxen or ASA exacerbatedthe severity of intestinal damage and intestinal inflammation as assessed by measuringhaematocrit, MPO activity, relative mRNA expression of iNOS, MCP-1 and VLA-1 (Figure).All treatments caused reduced mucosal PGE2 . Co-administration of misoprostol, a PGE2analogue, protected against intestinal damage induced by naproxen and ritonavir. Conclu-sions. The protease inhibitor ritonavir causes intestinal damage and its association withNSAIDs or ASA worsens damage caused by the COX-inhibitors. Misoprostol protects fromdamage caused by ritonavir. Further studies are need to clarify whether this observationhas a clinical readout.

Intestinal injury caused by ASA and ritonavir in mice

Tu2024

AIEC Instigates Chronic Colitis by Altering Microbiota Composition and ItsInherent Pro-Inflammatory PotentialBenoit Chassaing, Omry Koren, Frederic A. Carvalho, Ruth E. Ley, Andrew T. Gewirtz

BACKGROUND: Inflammatory bowel diseases (IBD) are characterized by a seemingly aber-rant mucosal immune response to the gut microbiota with disease development dictated byboth genetics and environmental factors. A model exemplifying this notion is our recentdemonstration that colonization of adherent-invasive E. coli (AIEC) during microbiota acqui-sition results in chronic colitis in mice that lacked the flagellin receptor TLR5 (T5KO). Thatsuch colitis persisted after the AIEC was cleared suggests that pathobionts such as AIECmightact as instigators of inflammation possibly resulting in a change inmicrobiota composition thatmight have greater potential to activate pro-inflammatory gene expression. GOAL: Examinethe extent to which transient colonizationwith AIEC lastingly affectedmicrobiota compositionand its innate immune activating ability. METHODS: Germ-free mice (WT and T5KO) wereinoculated with AIEC strain LF82 and placed in standard housing to allow establishmentof a complex microbiota that displaced (i.e. eliminated) AIEC in both strains of mice. Feceswere assayed for the inflammatory marker lipocalin-2, total bacterial load by PCR andmicrobiota composition by 454 sequencing. Innate immune activating potential of fecalmicrobiotas was assayed via TLR4 and TLR5 reporter cells. RESULTS: Transient colonizationby AIEC inWTmice did not alter inflammatory markers, bacterial loads, microbiota composi-tion, nor its pro-inflammatory potential. In contrast, transient colonization of T5KO mice byAIEC resulted in chronic inflammation, associated with alteration in microbiota composition,which correlated with microbiota components exhibiting greater induction of pro-inflamma-tory gene expression in both TLR4 and TLR5 reporter cells. Colonization of T5KO byaflagellate AIEC did not alter the microbiota's innate immune activating ability nor resultin chronic inflammation. CONCLUSION: AIEC, and perhaps other pathobionts, may instigatechronic inflammation in susceptible hosts by altering the gut microbiota composition soas to give it an inherently greater ability to activate innate immunity/pro-inflammatorygene expression.

Tu2025

High-Throughput Deep Sequencing, Cecal Content Transplants and SelectiveColonization Demonstrate That Enteric Dysbiosis Drives the RadiationGastrointestinal Syndrome and Identify Potential Bacterial Targets forRadiation Intestinal InjuryChristopher D. Packey, Nitsan Maharshak, Nida Waheed, Emery Harris, Susan Li, ScottE. Plevy, Ian M. Carroll, Ryan B. Sartor

Background: Radiation intestinal injury limits therapeutic dosing for malignancies. Thecause of initial morbidity/mortality after radiation is the gastrointestinal (GI) syndrome,which has unclear mechanisms and no FDA-approved therapies. Methods: High-throughputsequencing of the 16S rRNA (16S) gene and quantitative real-time PCR were performed onmucosa/lumen samples from the jejunum, cecum, and distal colon in 3 groups of SPF wild-type (WT) C57BL/6 (B6) mice: 1) non-irradiated 2) exposed to 15 Gy and 3) exposed to15 Gy and treated with antibiotics (n=5/group). 16S data was processed through QIIMEpipeline. Cecal contents from non-irradiated ("conventional") and post-irradiation("dysbiotic") donor mice were transferred to germ-free (GF) and SPF WT B6 (n=4/group)

S-907 AGA Abstracts

irradiated recipient mice, or to GF interleukin 10-deficient (IL-10-/-) and WT 129 SvEv(129) recipient mice (n=6-7/group), after which clinical responses were assessed. Probioticsor antibiotics were administered to lethally irradiated mice in separate experiments to assesstheir impact on dysbiosis and GI syndrome. Results: Radiation altered mucosal/luminalenteric microbiota composition (p=0.001) and increased colonic microbial diversity (p=0.0005). 16S taxonomic analysis showed that radiation decreased the intestinal Firmicutesphylum (0.35-0.56 fold change; p,0.0001) and Lactobacillus spp. (p,0.02). Consumptionof the probiotic VSL#3, comprised of several Lactobacillus spp., prevented radiation colitis(n=5 mice/group) and promoted eubiosis. Conversely, radiation increased the intestinalProteobacteria phylum (291.3-3036.1 fold change; p=0.05) and Escherichia genus(p,0.0001). In selective colonization experiments, Escherichia coli NC101 mono-associatedgnotobiotic mice exhibited accelerated morbidity/mortality following radiation compared toEnterococcus faecalis OG1RF mono-associated and GF mice. Ciprofloxacin treatment pre-vented most radiation-induced microbiota alterations and rescued mice from radiation GIsyndrome. In cecal transfer experiments, GF and SPF WT B6 mice receiving a dysbioticmicrobiota from irradiated mice experienced accelerated morbidity/mortality compared toGF and SPF mice inoculated with conventional microbiota following equivalent radiationdoses. IL-10-/- 129 mice colonized with a conventional or dysbiotic microbiota and WT 129mice inoculated with a dysbiotic microbiota lost weight, while WT 129 mice inoculatedwith conventional microbiota gained weight appropriately. Conclusions: Intestinal dysbiosisdrives the radiation GI syndrome. Microbiota constituents may contribute to this syndrome(E. coli) or confer protection (Lactobacillus spp.). Agents that prevent/correct enteric dysbioses,including VSL#3 and ciprofloxacin, are promising prophylaxis/treatment options for radiationintestinal injury and should be studied in clinical trials.

Tu2026

A Murine Model to Assess the Impact of Chronic Campylobacter jejuniColonization on GI HealthMeghan Wymore Brand, Amanda E. Ramer-Tait, Anne-Marie C. Overstreet, AlbertJergens, Orhan Sahin, Qijing Zhang, Michael J. Wannemuehler

Bacterial pathogens such as Campylobacter jejuni may induce self-limiting infections of thegastrointestinal (GI) tract that are influenced by the microbiota in healthy individuals. Thereis little known about the pathogenesis of C. jejuni in inflammatory bowel disease (IBD)patients who develop a dysbiotic microbiota. It has been suggested that the GI communitycomposed of the altered Schaedler flora (ASF, eight cultivable bacterial strains) metabolicallyresembles that of the dysbioticmicrobiota of IBD patients. The ability of C. jejuni to persistentlycolonize a host with a dysbiotic microbiota may deleteriously influence the severity of GIdisease by affecting host immune responses and mucosal gene expression. Previous studieswith C. jejuni in conventional microbiota immunocompetent murine models demonstratelow and irregular colonization rates and lack of disease. The goal of this study was todemonstrate that the pathogenesis of C. jejuni is differentially influenced by the complexityof the microbiota. We hypothesized that C. jejuni would colonize defined microbiota (DM)mice and induce mild mucosal inflammation characterized by ASF antigen-specific immuneresponses and differential mucosal gene expression. The ASF DM model can be used toevaluate antigen-specific immune responses and quantitative microbial changes within thecontext of a stable microbial community. C3H/HeN:TAC DM mice and conventionalmicrobiota C3H/HeN mice were colonized with several strains of C. jejuni. Subsequentmucosal inflammation was induced with 1.5 % dextran sodium sulfate (DSS). Persistenceof colonization, adaptive immune responses, histopathological changes, and host gene expres-sion (e.g., Fut2, B3galt5, Ceacam12, Cyp4b1, and Ugt8a) were evaluated. C. jejuni persistentlycolonized DM mice (107-109 CFU/gram feces) for ≥ 45 days and induced mild mucosalinflammation, while C. jejuni did not stably colonize conventional microbiota C3H/HeNmice. DM mice colonized with C. jejuni developed ASF antigen-specific serum IgG but onlyC. jejuni antigen-responsive CD4+ T cells. Low dose DSS treatment did not enhance diseaseseverity over C. jejuni or DSS alone. Differential colonic gene expression was detected betweencontrol and C. jejuni infected DM mice. These data indicate that mice harboring a lesscomplex microbiota (i.e., dysbiotic) are more susceptible to colonization by C. jejuni versusthose with a conventional microbiota, resulting in the induction of ASF antigen-specificimmune responses. In comparison to previous results, the disease outcome induced by C.jejuni was less severe in comparison to that induced by Helicobacter bilis. In conclusion, thepathogenesis of C. jejuni is differentially influenced by the complexity of the microbiota,and DMmice offer the opportunity to evaluate the impact of a chronic host-provocateur inter-action.

Tu2027

The Chemotherapeutic Agent Doxorubicin Induces Contrasting MicrobialResponses in the Jejunum and Distal Ileum That May Contribute toDifferential Injury PatternsChristopher D. Packey, Kelly Gewain, Ryan B. Sartor, Christopher M. Dekaney

Background: Intestinal dysbiosis has recently been associated with many disease processes,including the inflammatory bowel diseases. We hypothesize that increased injury in mid-jejunum (JEJ) compared to distal ileum (DI) in response to doxorubicin (Dox) may berelated to differences in bacterial composition and shifts. Methods: Eight specific pathogen-free C57BL6/J mice were administered (20 mg/kg i.p.) Dox. At 6 (n=4) and 24 h (n=4) afteradministration, mice were sacrificed and tissue was harvested from JEJ and DI. Healthycontrol mice (n=4) were sacrificed and tissues harvested similarly. DNA was isolated fromall tissue samples and 16S rRNA quantitative real-time PCR was performed using SYBRGreen and universal and bacterial group specific primers. Statistical analyses were performedusing GraphPad Prism software. Results: At 6 h after administration of Dox, total numbersof luminal and mucosal bacteria were decreased in JEJ (7.2*103 from 1.5*105; p=0.05) andDI (3.6*104 from 1.0*105). By 24 h, total bacterial numbers began to normalize. Thepercentage of mucosal bacteria increased at 6 h after Dox administration, both in JEJ (24.3%from 2.2%) and DI (41.3% from 1.3%). By 24 h, mucosal bacteria percentages began tonormalize. There were differences between JEJ and DI regarding alterations in bacterial groupconcentrations induced by Dox. Clostridium coccoides subgroup members were decreased in

AG

AA

bst

ract

s