Inhibition of Carbonic Anhydrases Enhance the Recovery from Acute Experimental Colitis by Controlling Epithelial Regeneration Emiko Mizognchi, Atsushi Mizognchi, Atul K. Bhan, Daniel K. Podolsky
Background & Aims: Colonic crypt elongation occurs during both chronic colitis and in the recovery phase of acute colitis. The impact of these alterations on epithelial cell function is not fully defined yet. Methods: DNA microarray analyses of colonic epithelial cells (CEC) of WT and TCRet KO mice were performed, and the results were confirmed by RT-PCR and immunohistocbemistry (IHC). The function of selected gene-encoding proteins was examined by administration of specific inhibitors directed toward products identified by expression analysis in WT mice given 4% DSS to induce colitis (DSS-mice). Results: DNA microarray analysis and RT-PCR revealed that expression of detoxification-associated genes, especially carbonic anhydrase (CAR)-IV and phenol sulfotransferase (PST)-I, was markedly downregufated in TCRc~ KO mice compared to WT mice. Of note, CAR-IV mRNA expression was markedly downregnlated in the CEC of TCRa KO mice compared to IL-10 KO mice, CD45RBhi cell transferred model and DSS-mice In contrast, PST-1 mRNA was significantly decreased in all the examined chronic colitis models but not the DSS-mice. IHC showed that CAR protein expressed in the surface epithelium rather than crypts in WT mice but not in TCR,~ KO mice. The intrarectal administration of valproic acid (inhibitor of CAR-I, II and IV) into WT mice significantly enhanced the recovery from DSS-induced acute colitis. In contrast, niflumic acid (inhibitor of PST-I) administration failed to suppress the DSS- colitis. Histologically, markedly increased number Of BrdU + CEC after Day 6 was obseived in the DSS treated mice administrated with valproic acid compared to those with PBS. However the number of BrdU + CEC were markedly decreased and less number of mononu- clear cell infiltration was found in lamina propria on Day 12 in valproic acid administrated mice. Conclusions: The expression of CAR-IV may significantly modulate the development of colitis; it was downregulated in CEC of Th2-mediated but not Th 1 and chemically induced colitis. The in vivo neutralization of CAR activity may provide a therapeutic strategy for active (acute) inflammation.
Paneth Cells In the Terminal Ileum Are the Main Site Of Local NOD2 Gene Expression Sanjay G. Lala, Annabel Bromfield, Sok Hot, Caroline Osborne, Olagunju Ognnbiyi, Satish Keshav
Background and aims: Terminal ileitis is the major form of Crohn's disease associated with mutations in the NOD2 gene. The NOD2 protein interacts with bacterial lipopolysaccharide (LPS) intracellularly, inducing activation of nuclear factor KB (NF-KB). Previous studies show that the gene is mainly expressed by circulating blood monocytes. However, monocytes efficiently detect LPS through cell-surface receptors such as tofi-fike receptor 4 (TLR4), and the mechanisms by which NOD2 mutations specifically cause terminal ileitis, and not other manifestations of Crohn's disease, are unknown. Paneth cells, which are most numerous in the terminal ileum, are critically important in enteric antibacterial defense, and respond to LPS through undefined pathways. We therefore determined if these specialized cells expressed the NOD2 gene, and compared this expression with that in monocytes and macrophages. Methods: In situ hybridization and immunohistochemistry were used to localize NOD2 expression and to positively identify the various cell types. Laser capture microdissection (LCM), and calcium chelation followed by mechanical disruption were used to isolate crypts and villi. Quantitative real-time RT-PCR was used to measure gene expression in isolated cells and LCM specimens. Results: In Crohn's ileitis, NOD2 RNA and protein were expressed by monocytes, but not mature macrophages in the lamina propria or within granulomas. NOD2 expression was most readily detected in Paneth cells in normal and Crohn's disease affected terminal ileum. NOD2 mRNA levels were enriched in isolated crypt compared to villous-epithelial cells, and NOD2 expression was mainly detected in LCM-acquired Paneth cells but not villous epithelial cells. This enrichment in crypt cells paralleled the enrichment for human defensins 5 and 6, which are exclusively expressed by Paneth ceils Paneth ceils in Crohn's disease-affected terminal ileum also strongly expressed tumor necrosis factor
(TNFa) mRNA, further confirming their potential participation in local inflammation. Conclusions: Paneth ceils strongly and specifically express NOD2 in the terminal ileum, and could therefore play an important and hitherto unrecognized role in the development of NOD2-associated Crohn's disease.
Enhanced Adaptive Immune Response to E. coli Flagellin in 1BD Jan-Michael Klapproth, Daniel A. Moore 111, Shanthi V. Sitaraman, Andrew T. Gewirtz
Background: Ffagelfin, the primary component of bacterial flagella, is released by many microbes including commensal E. coli strains. As such, flagellin is abundant in the intestinal lumen. While lumenal flagellin is without affect, flagellin that reaches the basolateral surface of the epithelium promotes recruitment of innate immune cells via activating TLR5-mediated pro-inflammatory gene expression. We have recently shown that, in a mouse model, flagellin placed beyond the epithelium has the potential to activate the adaptive immune system by serving as a T-cell adjuvant. The ~ of this study was to investigate whether flagellin is actually seen by the human adaptive immune system and, if so, whether persons with inflammatory bowel disease (IBD) have a difference in their level of adaptive immune response to this bacterial protein. Methods: Serum was prepared from healthy donors or IBD patients of the Emory Clinic (n>20 per group). Serum IgG titers were measured via ELISA to either flagelfin or LPS purified from the commensal gut E. coli strain F-18 BSA served as a negative control. The specificity of the antibody response to flagellin vs. other bacterial proteins was assessed by using the serum to immunoblot lysates from E. coli F-18 or an aflagellate strain. Results: Serum from all tested donors exhibited detectable serum lgG titer against E. coli flagellin indicating that this protein is indeed attaining access to the adaptive immune system. Most donors had detectable titers to E. coli LPS with the average titer being more than lO-fold lower than that observed to an equal mass of flagellin. No
donors exhibited a significant IgG titer to the control protein BSA. Persons with IBD had substantially elevated lgG titers to flagellin (5-10 fold) compared to healthy control subjects In contrast, we did not observe a significant difference in IgG titers to LPS lmmunoblotting whole bacterial lysates with these human sera indicated that while IBD patients have antibody titers particularly elevated for flageflni, they also exhibit increased titers to E coli proteins in general perhaps, in part, due to fiagellin's adjuvant ability. Conclusions: Bacterial flagellm is a major target of the adaptive immune system in health and especially in inflammatory bowel disease. In light of its adjuvant ability, the flagellin that reaches the adaptive immune system may also be able to promote potentially harmful immune responses to host proteins and thus may play a role in the pathogenesis of IBD. In addition, serum titer to flagellin may be a useful marker of IBD.
Lymphostatin/Efa-1 Inhibits Multiple Pathways of Lymphocyte Activation Shu Y. Gao, Jan-Michael A. Klapproth
Background: Lymphostatin/EHEC Factor of Adherence-1 (LS/Efa-1)is a toxin expressed in Gram negative bacteria capable of causing attaching/effacing lesions (A/E). LS/Efa-1 has been identified in Emeropathogenic- (EPEC), Enterohemorrhagic-, Rabbit Diarrhea E. cofi and C. rodentium and leads to profound suppression of IL-2, -4, and IFN-g expression in mitogen-activated T cell cultures. Goal: To determine the intracellular activation pathway(s) affected by LS/Efa-1 in defined T lymphocyte cultures. Methods: Jurkat E6-1 and D1.1 T cells were pre-incuhated with 50mug/ml lysates per 1X10mio ceils for two hours. Lysates were generated from a cosmid done expressing LS/Efa- 1 and a mutant strain with an insertion mutation that lost inhibitory activity. Following pre-stimulation, T cells were stimulated with anti-CD3 (20X10mio beads/ml, Dynabeads) plus anti-CD28 (25mug/ml) for up to 20 minutes. Activation was terminated at different times and T cell lysates analyzed by Western blot with specific antibodies for phosphorylated tyrosine, NF-AT1, NF-kB p65, I-kB, Erk- 1/-2, and c-jun. Results: Phosphoryfation of Erk-1/-2 was transient and decreased 10-fold in T lymphocytes exposed to LS/Efa-I in comparison to T lymphocytes exposed to lysates from the mutant strain and Erk-1/-2 cytoplasmic protein concentrations remained unchanged Even in the presence of l-kB phosphoryfation, NF-kB p65 was not translocated, and like NF-AT1, remained in the cytoplasm of T cells exposed to LS/Efa-1. Panems of tyrosine phosphorylation did not differ in the presence or absence of LSFEfa-1. In addition, LS/Efa- 1 did not suppress activation of c-jun. Experiments performed with D1.1 cells revealed identical results in the phosphorylation pattern as observed for E6-1 cells. Summary: kS/ Era-1 suppresses activation of multiple pathways the proteins NF-AT1, NF-kB p65 and Erk- 1/-2. Despite I-kB phosphorylation, NF-kB p65 nuclear translocation did not occur in the presence of LS/Efa-1 Conclusion: These findings suggest that LS/Efa-1 plays a role in the response to enteric refections with ME Gram negative bacteria by suppressing activation of the adaptive immune
Role of Salmonella Effector Proteins SipB and SipC In Altered Barrier and Transport Properties of Human Intestinal Epithelium Mbert K. Park, Lone S. Bertelsen, Donald G. Guiney, Kim E. Barrett
Background: We have previously shown that an invA mutant of Salmonella, while deficient in invading intestinal epithelial cells, was capable of inducing similar signaling pathways and chloride secretory responses as wild type Salmonella. To investigate this further we have used sipB and sipC deficient mutants to examine the specific effect of invasion protein translocation on signaling events, chloride secretory responses, and transepithelial resistance (TER) during Salmonella-infection of intestinal epithelial ceils. Methods: Monolayers of Ts+ cells were infected apically (MOI 25) with wild type, sipB, or sipC mutants of Salmonella typhimurium SL1344. Protein phosphoryfation was verified by Western blot analysis, and chloride secretion and TER by electrophysiology. Results: TER decreased significantly over 3 hours in ceils infected with wild type Salmonella (from 870_+40 to 157+2 D,*cm 2, p