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E. coli can be divided into three major subgroups depending on their pathogenic traits: nonpathogenic (commensals), pathogenic causing intestinal infections, and extraintestinal pathogenic E. coli (ExPEC). The unique ability of ExPEC strains to colonize different sites and to cause a heterogeneous group of diseases is due in part to the versatile genome of E. coli, which are able to remodel their genetic repertoire by acquiring and losing virulence attributes. ExPEC has a considerable impact on public health in Minnesota and the US, but still represents an under-appreciated health threat when compared to intestinal pathogenic E. coli. Remarkably, the annual number of reported cases due to ExPEC-induced disease is more than 100 fold higher than annual cases due to E. coli O157:H7. Food animals serve as a reservoir for ExPEC in humans, and raw chicken has been implicated as a major source of ExPEC transmission. Of further significance is that increased antibiotic resistance of ExPEC and the lack of broadly acting vaccines are leaving fewer treatment options. Our study focuses on bolstering innate immunity during E. coli infection, which offers a very rapid and broad response. Neutrophils (also referred to as polymorphonuclear cells or granulocytes) are the most abundant leukocyte population in the blood of humans, and due to their microbicidal products and rapid recruitment into sites of infection, they play a critical role in early bacterial clearance. Our findings indicate that a disintegrin and metalloprotease-17 (ADAM17) expressed by leukocytes functions as an important gatekeeper of neutrophil infiltration into sites of bacterial infection. ADAM17 is a membrane-associated metalloprotease that regulates the density of various cell surface receptors, as well as the release of soluble pro-inflammatory factors, a process referred to as ectodomain shedding. ADAM17 is constitutively expressed by leukocytes and its enzymatic activity is very rapidly induced upon cell activation. We have generated conditional ADAM17 gene knockout mice with an ADAM17 deficiency in all leukocytes. These mice demonstrate greatly impaired shedding of ADAM17 substrates when compared to control mice. We have begun examining the role of ADAM17 on the early host response using an E. coli abdominal infection model. Of interest is that peritoneal E. coli levels and its hematogenous spread were markedly reduced in ADAM17-null mice when compared to control mice. A rapid influx of neutrophils is necessary to reduce bacterial spread, and an examination of peritoneal neutrophil infiltration revealed this occurred in an accelerated manner in conditional
ADAM17 knockout mice. Here we will further investigate the impact of leukocyte ADAM17 on the early host response during E. coli abdominal infection.
<p>NON-TECHNICAL SUMMARY: <br/>Extraintestinal pathogenic Escherichia coli (ExPEC) is part of the normal bowel flora, but possess genes that allow them to infect any extra-intestinal location, including the bloodstream, cerebral spinal fluid, surgical wounds, and abdominal sites in humans. In Minnesota, ExPEC strains are believed to be important zoonotic and foodborne pathogens, and in the United States the medical-economical ramifications from ExPEC infections amount to billions of dollars in costs per annum. Of further significance is that increased antibiotic resistance of ExPEC and the lack of broadly acting vaccines are leaving fewer treatment options. Our long-term goal is to identify new therapeutic targets that will bolster the host response to ExPEC pathogens. A key component of the host response to infection by E. coli is robust neutrophil recruitment. ADAM17 is a
metalloprotease expressed by neutrophils and its proteolytic activity is rapidly induced upon their activation, which regulates the density of cell surface adhesion molecules and the release of various soluble pro-inflammatory factors. In our preliminary studies, gene-targeting ADAM17 in leukocytes resulted in accelerated neutrophil recruitment and enhanced bacterial clearance in a mouse model of abdominal E. coli infection. The central hypothesis of our proposal is that ADAM17 over-activation in leukocytes impairs neutrophil effector activities and promotes bacterial infection. The overall objective of our study is to determine the mechanisms by which ADAM17 exerts itself on the host response during E. coli infection. The impact of our findings is that they will significantly advance our understanding of the mechanisms of action of ADAM17 during E. coli infection and indicate the
therapeutic potential of this immune modulator.
<p>APPROACH: <br/>For our proposed studies, age-matched conditional ADAM17 knockout mice and control mice will be intraperitoneally injected with serial 10-fold dilutions of E. coli 0111:B4, a bacterial strain used by others for abdominal infection in mice, and monitored for 7 days. An LD50 for each cohort will be determined using a modified method of Reed and Muench. We will also examine bacterial dissemination and end organ damage. For instance, conditional ADAM17 knockout and control mice will be i.p. infected with E. coli and all mice will be euthanized at 6, 12, 24, 48 and 72 hours post-infection. At each time point, we will collect blood, peritoneal lavage fluid, and liver and lung samples. A portion of the liver and lung tissue will be homogenized in cold PBS and bacterial counts determined by serial dilutions using standard microbiologic methods. A portion of the
liver and lung will also be embedded in paraffin by standard procedures for histologic examination. Tissue sections will be stained with the anti-neutrophil marker Gr-1 as well as hematoxylin and eosin, and then processed for light microscopy to quantify neutrophil infiltration and tissue damage. The Comparative Pathology Shared Resource core facility at the University of Minnesota, which is located one floor up from the PI's lab, will assist in the tissue preparation and pathology scoring, done in a blinded fashion. There is a large body of evidence demonstrating an overwhelming release of pro-inflammatory cytokines during the onset of E. coli infection, and therefore we will also assess the local and systemic cytokine/chemokine response by conditional ADAM17 knockout and control mice i.p. injected with E. coli. The levels of a multitude of cytokines and chemokines in the collected
peritoneal lavage fluid and peripheral blood will be quantified by a multiplex ELISA. We currently use a Cytometric Bead Array (Becton Dickinson) analyzed with a BD FACSCanto and Bio-Plex kits (Bio-Rad Laboratories) analyzed on a Bio-Plex 200 instrument. These instruments are located in the Cellular and Protein Analysis Core Lab that Dr. Walcheck oversees. Using the Bio-Plex instrument, for instance, we can simultaneously quantify ~ 40 cytokines and chemokines. This broad array of factors will provide an extensive assessment of the inflammatory state in conditional ADAM17 knockout and control mice. Animal numbers are based on power analysis calculations. The criterion for significance has been set at 0.05. The proposed sample sizes will have power of ? 80% to yield a statistically significant result. Individual experiments are designed to use a minimum number of animals and data
will be pooled from repeated experiments to increase the sample size. If the overall ANOVA is found to be significant, then individual comparisons will be made between the groups of mice. Statistical comparison techniques will be utilized to maintain an overall significance level of 5%.
<p>PROGRESS: 2012/10 TO 2013/06<br/>Target Audience: University of Minnesota faculty and students, particularly in theCollege of Veterinary Medicine and the Academic Health Center. The research and medical communities to inform and extend knowledge related to the underlying mechanisms of immunosupression by excessive inflammation duringExPECinfection.Target audiences also include the people of the state of Minnesota and theUnited States asExPEC has a considerable impact on public health. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Project funding suported two postdoctoral associates. Drs.Hemant Kumar, Ph.D (0.8 FTE) and Nooshin Bahaie, MD, PhD (0.5 FTE). Drs. Kumar and Bahaie providedessential experience in lab management, molecular and cellular biology, mouse colony maintenance, and
infectious disease. Both contributed significantly to the data generated. Currently, Dr. Kumar is continueing his postdoctoral training in my lab. Dr.Bahaie has entered a MD residiency program at the Scripps Research Institute. How have the results been disseminated to communities of interest? Publications are in preparation. Data has also been presented at the University of Minnesota, including at the Inflammation Group monthly meetings and in the College of Veterinary Medicine seminar series. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported