Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
THIOREDOXIN IN NEUTROPHIL-AIRWAY EPITHELIAL INTERACTIONS
Sponsoring Institution
Cooperating Schools of Veterinary Medicine
Project Status
COMPLETE
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0209378
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Sep 10, 2004
Project End Date
Jul 31, 2014
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Pathology, Microbiology & Immunology
Non Technical Summary
Neutrophils play a beneficial role by removing oxidant-injured airway epithelial cells and enhancing subsequent epithelial repair after ambient ozone exposure. The long-term research goals are to investigate mechanisms of pulmonary inflammation and repair with special emphasis on the interaction between neutrophils and airway epithelial cells.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113840104070%
3113840109010%
3113840115010%
3113840116010%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1160 - Pathology; 1150 - Toxicology;
Goals / Objectives
The primary objective of this research is to determine the importance of thioredoxin in neutrophil-airway epithelial cell interactions after oxidant injury. The central hypothesis is that neutrophil-dependent removal of oxidant-injured airway epithelial cells requires thioredoxin production by the injured cells that then enhances neutrophil co-localization, removal of injured cells and subsequent epithelial repair.
Project Methods
The objectives of this proposal will be addressed in four parts. First, the investigators will determine the molecular interaction between thioredoxin and the cell membrane of oxidant-injured airway epithelial cells. Secondly, they will investigate the importance of thioredoxin in neutrophil co-localization to airway epithelial cells, their removal after injury and enhancing subsequent epithelial repair. Immortalized human bronchial epithelial cells will be stably transfected with a thioredoxin expression construct and the investigators will quantify neutrophil co-localization to these cells, neutrophil-dependent removal of oxidant injured cells and epithelial repair using BrdU. To assess the importance of thioredoxin structure in these functions, the investigators will repeat the experiments using monolayers transfected with thioredoxin expression constructs mutated in key cysteine amino acids. Third, they will show thioredoxin is chemotactic for mouse neutrophils. This will lead to the fourth aim of developing a transgenic mouse over-expressing wild type and mutant thioredoxin in airway epithelium and demonstrating the role of thioredoxin in neutrophil emigration and removal of injured cells in vivo.

Progress 10/01/13 to 07/31/14

Outputs
Target Audience:A progress report was submitted to the funding agency. Changes/Problems:A progress report was submitted to the funding agency. What opportunities for training and professional development has the project provided?A progress report was submitted to the funding agency. How have the results been disseminated to communities of interest?A progress report was submitted to the funding agency. What do you plan to do during the next reporting period to accomplish the goals?A progress report was submitted to the funding agency.

Impacts
What was accomplished under these goals? A progress report was submitted to the funding agency.

Publications


    Progress 09/10/04 to 07/31/14

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Final Report Submitted to the funding Agency

    Publications


      Progress 10/01/12 to 09/30/13

      Outputs
      Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

      Impacts
      What was accomplished under these goals? This proposal was submitted to a different sponsor and progress reports were submitted to the appropriate agency.

      Publications


        Progress 10/01/11 to 09/30/12

        Outputs
        Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

        Impacts
        What was accomplished under these goals? This proposal was submitted to a different sponsor and progress reports were submitted to the appropriate agency.

        Publications


          Progress 10/01/10 to 09/30/11

          Outputs
          Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

          Impacts
          What was accomplished under these goals? This project was submitted to a different sponsor and progress reports were submitted to the appropriate agency.

          Publications


            Progress 01/01/10 to 09/30/10

            Outputs
            Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

            Impacts
            What was accomplished under these goals? This proposal was submitted by a different sponsor. Progress Reports were submitted to the appropriate agency.

            Publications


              Progress 01/01/09 to 12/31/09

              Outputs
              OUTPUTS: We are continuing to investigate oxidant injury and repair of airway epithelial cells. We were unsuccessful in generating a transgenic thioredoxin mouse. We have investigated the role of neuropeptides, substance P and CGRP, in airway epithelial injury and repair after ozone exposure. PARTICIPANTS: Karen Oslund, Reen Wu, Dallas Hyde, Ed Schelegle. Training for Karen Oslund: Course on Stereology University of Seattle Sept. 2009. Workshop on chromatin immunoprecipitation. UC Davis Oct. 2009 TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

              Impacts
              We have shown that the neuropeptides, substance P and CGRP, contribute to airway epithelial cell injury and repair. Rats that were treated with a receptor antagonist to either NK-1R, the substance P receptor, or a CGRP receptor antagonist, demonstrated attenuated airway epithelial injury and repair after ozone exposure. Additionally, injured airway epithelial cells demonstrated cytoplasmic localization of Nur77, a mediator of non-apoptotic programmed cell death, suggesting this cell death pathway maybe important in ozone induced injury.

              Publications

              • Oslund KL, Hyde DM, Putney LF, Alfaro MF, Walby WF, Tyler NK, Schelegle ES. Activation of Calcitonin Gene-Related Peptide Receptor during Ozone Inhalation Contributes to Epithelial Injury and Repair. 2009. Journal of Toxicological Pathology. 37(6):805-813.


              Progress 01/01/08 to 12/31/08

              Outputs
              OUTPUTS: The continuing development of this useful transgenic mouse and characterization of the line will be a priority for next year. This work is directly related to specific aim #3 in which will use these mice to study neutrophil recruitment and co-localization in the airway epithelium after ozone exposure. These results are significant because we are advancing towards our ultimate goal of generating a transgenic mouse overexpressing thioreodixn exclusively in the airway epithelium. When created, this mouse will be a valuable to tool to further understand the role of thioredoxin not only in these studies but in diverse models of inflammation and injury involving the lungs. we have completed a study investigating the role of thioredoxin in cytotoxicity after smoke injury. This work has been submitted for publication to the Journal of American Physiology: Lung Cell and Molecular Biology. PARTICIPANTS: Karen Oslund PI Reen Wu Mentor Dallas Hyde Advisor TARGET AUDIENCES: No Target Audiences information reported. PROJECT MODIFICATIONS: No Project Modifications information reported.

              Impacts
              Our preliminary results from the in vitro neutrophil experiments found that neutrophils remove significantly more thioredoxin over-expressing cells compared to wildtypes. Additionally, this result was not affected by neutrophil number i.e. more neutrophils did not result in significantly different results. We are in the process of analyzing additional data from this experiment. These results are significant because it expands the findings from our earlier work that neutrophils preferentially co-localize with airway epithelial cells over-expressing thioredoxin. This work continues to demonstrate the role of thioredoxin in targeting and removing airway epithelial cells expressing thioredoxin. Additionally we have found that thioredoxin is important in protecting subconfluent cultures of airway epithelial cells from smoke induced injury. Low density cultures of several airway epithelial cell types (primary human, monkey and a cell line HBE1) are more susceptible to cytotoxicity induced by cigarette smoke compared to confluent cultures. However, if we over-express thioredoxin in subconfluent cultures, the cells are protected from smoke injury. We feel that this is an important model for airway epithelial cells undergoing regeneration after an injury when they are in a subconfluent state. We hope to investigate this phenomenon in vivo.

              Publications

              • No publications reported this period


              Progress 01/01/07 to 12/31/07

              Outputs
              OUTPUTS: Specific Aims: The specific aims for this project have not changed. This past year a major focus of this project has been breeding and development of a double transgenic mouse. We have successfully isolated and performed a pronuclear injection of a established a line of mice containing a tetracycline inducible plasmid with wildtype thioredoxin. Additionally, these mice have been successfully crossed with CCSP tetracycline regulator mice provided by a researcher in our laboratory. We are in the process of administering doxycycline (a tetracycline derivative in the drinking water of some of the double transgenic mice to determine if thioredoxin will be selectively overexpressed in the airway epithelium. The continuing development of this useful transgenic mouse and characterization of the line will be a priority for next year. This work is directly related to specific aim #3 in which will use these mice to study neutrophil recruitment and co-localization in the airway epithelium after ozone exposure. These results are significant because we are advancing towards our ultimate goal of generating a transgenic mouse overexpressing thioreodixn exclusively in the airway epithelium. When created, this mouse will be a valuable to tool to further understand the role of thioredoxin not only in these studies but in diverse models of inflammation and injury involving the lungs. Secondly, we continuing to investigate neutrophil mediated removal of thioredoxin overexpressing airway epithelial cells in vitro related to specific aim #2. We are in the process of completing a study investigating whether quiescent and activated neutrophils preferentially remove airway epithelial cells over-expressing thioredoxin. Finally, we have completed a study investigating the role of thioredoxin in cytotoxicity after smoke injury. This work has been submitted for publication to the Journal of American Physiology: Lung Cell and Molecular Biology. PARTICIPANTS: Karen Oslund PI Reen Wu Mentor Dallas Hyde Advisor

              Impacts
              Our preliminary results from the in vitro neutrophil experiments found that neutrophils remove significantly more thioredoxin over-expressing cells compared to wildtypes. Additionally, this result was not affected by neutrophil number i.e. more neutrophils did not result in significantly different results. We are in the process of analyzing additional data from this experiment. These results are significant because it expands the findings from our earlier work that neutrophils preferentially co-localize with airway epithelial cells over-expressing thioredoxin. This work continues to demonstrate the role of thioredoxin in targeting and removing airway epithelial cells expressing thioredoxin. Additionally we have found that thioredoxin is important in protecting subconfluent cultures of airway epithelial cells from smoke induced injury. Low density cultures of several airway epithelial cell types (primary human, monkey and a cell line HBE1) are more susceptible to cytotoxicity induced by cigarette smoke compared to confluent cultures. However, if we over-express thioredoxin in subconfluent cultures, the cells are protected from smoke injury. We feel that this is an important model for airway epithelial cells undergoing regeneration after an injury when they are in a subconfluent state. We hope to investigate this phenomenon in vivo.

              Publications

              • No publications reported this period