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Mo1684 The Role of Commensal Microbes in Mucosal Defense of the Neonatal Gut
AGA Abstracts
its binding to FPR1. Conclusion: These findings show that synthetic gliadin peptides induce neutrophil migration in an FPR1-dependent manner. The present study emphasizes an emerging concept that PT-gliadin is interpreted and handled by the host mucosa as a danger signal similar to that provided by fMet-Leu-Phe, likewise exerting direct and robust chemoattractant effects on neutrophils. These observations provide new insight into our understanding of how gliadin triggers inflammatory and autoimmune responses. Mo1682 Krüpple-Like Factor 5 Is Required for Normal Intestinal Homeostasis As a Transcription Factor Controlling Proliferation Kristin N. Bell, Noah F. Shroyer Background and Aims: In the adult intestine, all proliferative stem and progenitor cells are located in the crypts of Lieberkühn. Within the intestinal crypts expression of the transcription factor Klf5 is confined to proliferating cells. Homeostatic maintenance of these stem and progenitor cell populations is essential to support the rapid turnover of the intestinal epithelium. Therefore, we hypothesize that Klf5 is required for cellular proliferation to maintain intestinal homeostasis. Methods: To examine the requirement of Klf5, inducible recombination of the Klf5-floxed allele was driven by Villin-creER in mice. Tamoxifen was administered for two consecutive days prior to sacrifice on day three, five, seven, or twenty-eight. Sufficiency of Klf5 was examined utilizing a gain-of-function, doxycycline inducible system (Villin-cre; Rosa26-rtta-ires-GFP; TRE-Klf5 mice) for two and a half days prior to sacrifice. Tissue was analyzed using conventional immunohistochemistry, western blot analysis, and in-situ hybridization. Three-dimensional enteroids derived from jejunal crypts were used for in vitro analysis. Results: Loss of Klf5 resulted in decreased proliferation and increased apoptosis early after recombination. In contrast, tissue analyzed at a later time point showed normal proliferation and lack of crypt cell death. Complementary results were found in vitro as jejunal crypts isolated three days after in vivo tamoxifen administration formed enteroids that survived for nine days in culture; however they failed to produce budding crypts unlike their wildtype counterparts. In contrast, overexpression of Klf5 disrupted adult mouse intestinal morphology within two days while prolonged expression resulted in death. Induction of Klf5 was sufficient to increase the proliferative crypt zone, induce villus blunting, and decrease villus cell differentiation; however, no effect was seen on stem cell numbers. Conclusions: Data gathered from loss-of-function studies suggest a role for Klf5 in crypt proliferation. Furthermore, its importance in epithelial maintenance and cellular turnover is potentially crucial as indicated by increased apoptosis in the crypt zone early after loss of this transcription factor. Together with data showing lack of cell death and normal enteroid formation at later time points suggest that selection against the loss of Klf5 occurs early after recombination. It remains to be determined weather this loss of Klf5 is maintained or replaced by some other mechanism. Increased Klf5 is sufficient to induce hyperproliferative, elongated crypts further defining its role in promoting intestinal crypt proliferation. Collectively, these data highlight a role for Klf5 in epithelial proliferation; further studies are needed to distinguish cell autonomous effects in the transient progenitor cell population versus intestinal stem cells.
Transepithelial neutrophil migration Mo1684 The Role of Commensal Microbes in Mucosal Defense of the Neonatal Gut Emily G. Blosser, Casey T. Weaver, David A. Randolph PURPOSE: Late-onset bacterial sepsis (LOS) is a leading cause of morbidity and mortality among preterm infants worldwide. Infections with Gram-negative bacteria, such as Klebsiella pneumoniae, occurring when bacteria translocate across premature gut epithelium into the bloodstream, can be particularly severe. Preterm infants often undergo intense antibiotic regimens to combat high risk of infection. Paradoxically, prolonged empiric antibiotic therapy is associated with increased risk of intestinal infection and death after adjustment for covariates. Commensal microbes in the mature gut are believed to confer protection from bacterial translocation by competing with pathogens for space and resources, and by inducing development of gut immune defenses. The purpose of this study was to determine how reduced gut microbial diversity affects the risk of LOS and to elucidate the kinetics of colonization. METHODS: We have developed a physiologic mouse model of neonatal LOS in which C57BJ/6 pups, inoculated with K. pneumoniae via oral gavage, become septic as the pathogen translocates across the gut mucosa in an age-dependent manner. To test the effects of reduced gut microbial diversity in this model, pregnant dams were placed on oral vancomycin and/ or gentamicin until pups were 4 days old. Pups were challenged on day 5 with 107 CFU K. pneumoniae. Germ-free and untreated pups served as controls. Real time PCR and in vivo bioluminescent imaging of K. pneumoniae were used to assess disease. High throughput 16S ribosomal RNA sequencing of Naive intestinal samples was used to identify colony founder families present on day 5 of life. RESULTS: Colony diversity and size were significantly reduced in antibiotic-treated pups, which resulted in increased risk of LOS (p<0.01 vs. untreated controls). Vancomycin treatment preserved microbial diversity and survival to a greater extent than gentamicin (p<0.01). Bioluminescent imaging and PCR detection of K. pneumoniae in stool indicated delayed clearance in pups with limited flora (p<0.01). High throughput sequencing of Naive neonatal intestinal samples identified three principal bacterial founder genera - Lactobacillus, Pasteurella, and Streptococcus - the relative abundance of which were significantly altered depending on the presence and type of antibiotic therapy. CONCLUSIONS: The microbiota is important for protection against Gram-negative sepsis. Although diversity of colonizing organisms is limited early in life, antibiotic treatment alters colonization by reducing both the number and diversity of microbes present in the gut. Gram-negative antibiotics may be more detrimental to gut immune development than Grampositive antibiotics, though both significantly alter the abundance of founding species of the microbiota. More studies are needed to determine the role of colony founders in gut immune induction.
Mo1683 Imaging Mucosal Surfaces With Micro-Optical Coherence Tomography Mark E. Kusek, Kengyeh Chu, Linbo Liu, Wayne G. Shreffler, Guillermo J. Tearney, Bryan Hurley Paramount to effective host defense is the mobilization of neutrophils from the vasculature through extracellular matrix and across epithelial barriers in response to pathogens capable of infecting various mucosal surfaces. Although this influx of inflammatory cells is necessary for resolution of infection, it also brings deleterious consequences by mediating nonspecific tissue damage. An in vitro model of intestinal epithelial cells grown on permeable Transwell filters has been previously developed and is widely employed to elucidate key features of host-pathogen interactions as well as neutrophil trans-epithelial migration. Leveraging a novel imaging tool called micro-Optical Coherence Tomography (μOCT), we sought to explore key pathological events occurring at mucosal surfaces modeled using the in vitro Transwell system. The imaging technique, μOCT, enables visualization using near-infrared light of living biological tissues at a cellular level without need for sample fixing or staining. μOCT also supports 3D reconstruction of imaging targets as well as the ability to observe changes occurring within a fixed area of the target over a defined period of time. Results indicate that μOCT is capable of revealing clearly resolvable images of the polarized epithelial monolayer grown on the Transwell filter. Further, treatment of the monolayer with EDTA results in a loss in polarity and barrier integrity that is perceivable upon imaging with μOCT. The dynamic process of neutrophil trans-epithelial migration in response to a gradient of the chemo-attractants, fMLP and LTB4, is observed using μOCT, whereby neutrophils are distinguishable from epithelial cells and migrate in clusters across the epithelial barrier. This imaging technique also has the capability to quantify the number of neutrophils that migrate through the transwell to a chemotactic gradient, making it a valuable tool in examining migration of granulocytes in more detail than established techniques. Investigation of mucosal surfaces with μOCT provides a new visual perspective that will likely lead to novel insight unveiling key mechanistic features of the complex interactions between inflammatory cells, epithelial cells, and pathogens. A better understanding of these mechanisms facilitated by further exploration using μOCT will assist in the development of novel therapeutics to reduce the detrimental effects of infection with pathogens and other diseases that involve mucosal inflammation.
Mo1685 Opposite NOS2-Mediated Roles for Epidermal Growth Factor and Indomethacin in Immature Human Intestinal Inflammation Emanuela Ferretti, Eric Tremblay, David Grynspan, Marcos Bettolli, Karolina M. Burghardt, Corentin Babakissa, Emile Levy, Ernest G. Seidman, Daniel Menard, JeanFrancois Beaulieu Background and Aim: Nitric oxide (NO) is a ubiquitous signaling molecule that is involved in several important physiological and pathological processes in the gastrointestinal system. NO synthase 2 (NOS2) is most widely implicated in the processes of epithelial cell injury/ apoptosis and host immune defense. However, the specific role of NOS2-mediated NO in the inflammation of the immature human intestinal mucosa remains unclear. We have previously reported that EGF attenuates the inflammatory response in the mid-gestation human small intestine using serum-free organ culture (Physiol Genomics 2012, 44(4):26880). Recently, we also reported that the non-steroidal anti-inflammatory drug, indomethacin (INDO), exerts multiple detrimental metabolic effects on the immature human intestine (Genomics 2013, 101(3):171-7). In this study we investigated the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 itself as well as its downstream metabolic pathways. Methods: Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1μM INDO for 48 h using Illumina whole genome microarrays. Ingenuity Pathway Analysis (IPA) software identified several NOS2-related genes and their expression patterns were confirmed by qPCR. Results: By comparing gene expression profiles induced by EGF to that of INDO in the immature ileum, IPA identified three canonical pathways where NOS2 is known to play an important role: "free radical scavenging", "glycolysis/ gluconeogenesis" and "leukocyte extravasation". For instance, we noted that gene expression of several antioxidant enzymes such as DUOX2, GPX2, SOD2 and especially NOS2 were
S-635
AGA Abstracts
its binding to FPR1. Conclusion: These findings show that synthetic gliadin peptides induce neutrophil migration in an FPR1-dependent manner. The present study emphasizes an emerging concept that PT-gliadin is interpreted and handled by the host mucosa as a danger signal similar to that provided by fMet-Leu-Phe, likewise exerting direct and robust chemoattractant effects on neutrophils. These observations provide new insight into our understanding of how gliadin triggers inflammatory and autoimmune responses. Mo1682 Krüpple-Like Factor 5 Is Required for Normal Intestinal Homeostasis As a Transcription Factor Controlling Proliferation Kristin N. Bell, Noah F. Shroyer Background and Aims: In the adult intestine, all proliferative stem and progenitor cells are located in the crypts of Lieberkühn. Within the intestinal crypts expression of the transcription factor Klf5 is confined to proliferating cells. Homeostatic maintenance of these stem and progenitor cell populations is essential to support the rapid turnover of the intestinal epithelium. Therefore, we hypothesize that Klf5 is required for cellular proliferation to maintain intestinal homeostasis. Methods: To examine the requirement of Klf5, inducible recombination of the Klf5-floxed allele was driven by Villin-creER in mice. Tamoxifen was administered for two consecutive days prior to sacrifice on day three, five, seven, or twenty-eight. Sufficiency of Klf5 was examined utilizing a gain-of-function, doxycycline inducible system (Villin-cre; Rosa26-rtta-ires-GFP; TRE-Klf5 mice) for two and a half days prior to sacrifice. Tissue was analyzed using conventional immunohistochemistry, western blot analysis, and in-situ hybridization. Three-dimensional enteroids derived from jejunal crypts were used for in vitro analysis. Results: Loss of Klf5 resulted in decreased proliferation and increased apoptosis early after recombination. In contrast, tissue analyzed at a later time point showed normal proliferation and lack of crypt cell death. Complementary results were found in vitro as jejunal crypts isolated three days after in vivo tamoxifen administration formed enteroids that survived for nine days in culture; however they failed to produce budding crypts unlike their wildtype counterparts. In contrast, overexpression of Klf5 disrupted adult mouse intestinal morphology within two days while prolonged expression resulted in death. Induction of Klf5 was sufficient to increase the proliferative crypt zone, induce villus blunting, and decrease villus cell differentiation; however, no effect was seen on stem cell numbers. Conclusions: Data gathered from loss-of-function studies suggest a role for Klf5 in crypt proliferation. Furthermore, its importance in epithelial maintenance and cellular turnover is potentially crucial as indicated by increased apoptosis in the crypt zone early after loss of this transcription factor. Together with data showing lack of cell death and normal enteroid formation at later time points suggest that selection against the loss of Klf5 occurs early after recombination. It remains to be determined weather this loss of Klf5 is maintained or replaced by some other mechanism. Increased Klf5 is sufficient to induce hyperproliferative, elongated crypts further defining its role in promoting intestinal crypt proliferation. Collectively, these data highlight a role for Klf5 in epithelial proliferation; further studies are needed to distinguish cell autonomous effects in the transient progenitor cell population versus intestinal stem cells.
Transepithelial neutrophil migration Mo1684 The Role of Commensal Microbes in Mucosal Defense of the Neonatal Gut Emily G. Blosser, Casey T. Weaver, David A. Randolph PURPOSE: Late-onset bacterial sepsis (LOS) is a leading cause of morbidity and mortality among preterm infants worldwide. Infections with Gram-negative bacteria, such as Klebsiella pneumoniae, occurring when bacteria translocate across premature gut epithelium into the bloodstream, can be particularly severe. Preterm infants often undergo intense antibiotic regimens to combat high risk of infection. Paradoxically, prolonged empiric antibiotic therapy is associated with increased risk of intestinal infection and death after adjustment for covariates. Commensal microbes in the mature gut are believed to confer protection from bacterial translocation by competing with pathogens for space and resources, and by inducing development of gut immune defenses. The purpose of this study was to determine how reduced gut microbial diversity affects the risk of LOS and to elucidate the kinetics of colonization. METHODS: We have developed a physiologic mouse model of neonatal LOS in which C57BJ/6 pups, inoculated with K. pneumoniae via oral gavage, become septic as the pathogen translocates across the gut mucosa in an age-dependent manner. To test the effects of reduced gut microbial diversity in this model, pregnant dams were placed on oral vancomycin and/ or gentamicin until pups were 4 days old. Pups were challenged on day 5 with 107 CFU K. pneumoniae. Germ-free and untreated pups served as controls. Real time PCR and in vivo bioluminescent imaging of K. pneumoniae were used to assess disease. High throughput 16S ribosomal RNA sequencing of Naive intestinal samples was used to identify colony founder families present on day 5 of life. RESULTS: Colony diversity and size were significantly reduced in antibiotic-treated pups, which resulted in increased risk of LOS (p<0.01 vs. untreated controls). Vancomycin treatment preserved microbial diversity and survival to a greater extent than gentamicin (p<0.01). Bioluminescent imaging and PCR detection of K. pneumoniae in stool indicated delayed clearance in pups with limited flora (p<0.01). High throughput sequencing of Naive neonatal intestinal samples identified three principal bacterial founder genera - Lactobacillus, Pasteurella, and Streptococcus - the relative abundance of which were significantly altered depending on the presence and type of antibiotic therapy. CONCLUSIONS: The microbiota is important for protection against Gram-negative sepsis. Although diversity of colonizing organisms is limited early in life, antibiotic treatment alters colonization by reducing both the number and diversity of microbes present in the gut. Gram-negative antibiotics may be more detrimental to gut immune development than Grampositive antibiotics, though both significantly alter the abundance of founding species of the microbiota. More studies are needed to determine the role of colony founders in gut immune induction.
Mo1683 Imaging Mucosal Surfaces With Micro-Optical Coherence Tomography Mark E. Kusek, Kengyeh Chu, Linbo Liu, Wayne G. Shreffler, Guillermo J. Tearney, Bryan Hurley Paramount to effective host defense is the mobilization of neutrophils from the vasculature through extracellular matrix and across epithelial barriers in response to pathogens capable of infecting various mucosal surfaces. Although this influx of inflammatory cells is necessary for resolution of infection, it also brings deleterious consequences by mediating nonspecific tissue damage. An in vitro model of intestinal epithelial cells grown on permeable Transwell filters has been previously developed and is widely employed to elucidate key features of host-pathogen interactions as well as neutrophil trans-epithelial migration. Leveraging a novel imaging tool called micro-Optical Coherence Tomography (μOCT), we sought to explore key pathological events occurring at mucosal surfaces modeled using the in vitro Transwell system. The imaging technique, μOCT, enables visualization using near-infrared light of living biological tissues at a cellular level without need for sample fixing or staining. μOCT also supports 3D reconstruction of imaging targets as well as the ability to observe changes occurring within a fixed area of the target over a defined period of time. Results indicate that μOCT is capable of revealing clearly resolvable images of the polarized epithelial monolayer grown on the Transwell filter. Further, treatment of the monolayer with EDTA results in a loss in polarity and barrier integrity that is perceivable upon imaging with μOCT. The dynamic process of neutrophil trans-epithelial migration in response to a gradient of the chemo-attractants, fMLP and LTB4, is observed using μOCT, whereby neutrophils are distinguishable from epithelial cells and migrate in clusters across the epithelial barrier. This imaging technique also has the capability to quantify the number of neutrophils that migrate through the transwell to a chemotactic gradient, making it a valuable tool in examining migration of granulocytes in more detail than established techniques. Investigation of mucosal surfaces with μOCT provides a new visual perspective that will likely lead to novel insight unveiling key mechanistic features of the complex interactions between inflammatory cells, epithelial cells, and pathogens. A better understanding of these mechanisms facilitated by further exploration using μOCT will assist in the development of novel therapeutics to reduce the detrimental effects of infection with pathogens and other diseases that involve mucosal inflammation.
Mo1685 Opposite NOS2-Mediated Roles for Epidermal Growth Factor and Indomethacin in Immature Human Intestinal Inflammation Emanuela Ferretti, Eric Tremblay, David Grynspan, Marcos Bettolli, Karolina M. Burghardt, Corentin Babakissa, Emile Levy, Ernest G. Seidman, Daniel Menard, JeanFrancois Beaulieu Background and Aim: Nitric oxide (NO) is a ubiquitous signaling molecule that is involved in several important physiological and pathological processes in the gastrointestinal system. NO synthase 2 (NOS2) is most widely implicated in the processes of epithelial cell injury/ apoptosis and host immune defense. However, the specific role of NOS2-mediated NO in the inflammation of the immature human intestinal mucosa remains unclear. We have previously reported that EGF attenuates the inflammatory response in the mid-gestation human small intestine using serum-free organ culture (Physiol Genomics 2012, 44(4):26880). Recently, we also reported that the non-steroidal anti-inflammatory drug, indomethacin (INDO), exerts multiple detrimental metabolic effects on the immature human intestine (Genomics 2013, 101(3):171-7). In this study we investigated the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 itself as well as its downstream metabolic pathways. Methods: Gene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1μM INDO for 48 h using Illumina whole genome microarrays. Ingenuity Pathway Analysis (IPA) software identified several NOS2-related genes and their expression patterns were confirmed by qPCR. Results: By comparing gene expression profiles induced by EGF to that of INDO in the immature ileum, IPA identified three canonical pathways where NOS2 is known to play an important role: "free radical scavenging", "glycolysis/ gluconeogenesis" and "leukocyte extravasation". For instance, we noted that gene expression of several antioxidant enzymes such as DUOX2, GPX2, SOD2 and especially NOS2 were
S-635
AGA Abstracts