Quantification of potential alterations in pulmonary innate immunity and hyperresponsiveness during respiratory syncytial virus infection

Source: IOWA STATE UNIVERSITY submitted to

QUANTIFICATION OF POTENTIAL ALTERATIONS IN PULMONARY INNATE IMMUNITY AND HYPERRESPONSIVENESS DURING RESPIRATORY SYNCYTIAL VIRUS INFECTION

PROJECT DIRECTOR: Ackermann, M. R. Williamson, M.

PERFORMING ORGANIZATION
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
AMES,IA 50011

NON TECHNICAL SUMMARY: Respiratory syncytial viruses (RSV) are common respiratory pathogens of cattle and sheep. Current vaccines are not fully effective and reinfections can occur. The long-term consequence of RSV infections is poorly understood but evidence suggests that infected young individuals are predisposed to secondary microbial infections and allergic conditions. Project findings may lead to ways to enhance antimicrobial peptide production and, thereby, reduce the severity of the initial RSV infection, and the incidence of reinfections.

OBJECTIVES: Respiratory syncytial viruses (RSV) are common respiratory pathogens of cattle and sheep. The pneumonia caused by bovine RSV is often severe and complicated by secondary infections. Current vaccines are not fully effective, reinfections are not uncommon, and pneumonia in neonates caused by these viruses increases the incidence of other respiratory problems (secondary infections, allergic disease [asthma]) later in life. The ability of RSV to: 1) breech the innate immune system to establish infection and predispose the lung the secondary pathogens, and 2) increase lung hypersensitivity post-RSV pneumonia are poorly understood. Our overall hypothesis is that: RSV infection of the lung permanently alters the innate immune system and neuroregulatory elements of inflammation (substance P-mast cell axis) and thereby predisposes the lung to secondary infections and hyperresponsiveness. Studies outlined in this proposal will quantify changes in: 1) antimicrobial peptides production by lung epithelial cells, which are increasingly recognized important elements of the innate immune system, and 2) the substance P-mast cell axis, a key component of the pulmonary hypersensitivity pathway, during development, progression and resolution of RSV pneumonia in an ovine model. The hypothesis will be tested with the following three specific aims: 1) Define the alterations of the neuroregulatory elements of inflammation (substance P-mast cell axis) during the development, progression and resolution of RSV pneumonia in an ovine RSV-infection model. 2) Determine the extent to which antimicrobial peptide production (a component of the innate immune system) is altered during the development, progression and resolution of RSV pneumonia in an ovine RSV-infection model. 3) Determine the extent to which previous RSV infection enhances the severity of secondary bacterial infections.

APPROACH: Twenty-four lambs of either sex and mixed breed, ranging from 2 to 3 months of age, and free of clinical signs of illness and fever, will be randomly assigned into six groups. They will be sedated and inoculated intrabronchially (right and left cranial lobes) by fiberoptic bronchoscope with an inoculum that we have previously determined will induce RSV pneumonia. Control lambs will be sedated and inoculated similarly with PFS. Following the inoculations, the effects of xylazine sedation will be reversed with telozolidine. The lambs will be obtained from Iowa State University (ISU), Laboratory Animal Resources. The animals will be maintained in isolation rooms in accordance with the methods approved by the American Association of Laboratory Animal Care. One hour prior to sacrificing lambs, each animal will receive intravenous injections of bromodeoxyuridine (BrDU). BrDU is incorporated in the DNA of dividing cells and will be used as a marker to quantify proliferation of hyperplastic bronchiolar cells (which occur in RSV pneumonia and may reflect immature cells lacking antimicrobial peptide) and mast cells. Four lambs from each group (including PFS-inoculated controls) will be necropsied at days 3 (initiation of RSV pneumonia), 21 (progression of infection into chronicity), and 90 (resolved lesions) post-inoculation. Animals will be euthanized by intravenous pentobarbital overdose. Lesions are easily recognized grossly at all time points (days 3, 21, 90) post-inoculation and are roughly 10-20 centimeters in width, involving from 25-40% of the lung lobe centered around the initial site of inoculation. Another group of lambs will be depleted of substance P fibers by subcutaneous injections of capsaicin using a protocol developed in our laboratory. Control lambs receiving PFS lack gross and histologic lesions and tissue will be collected from same region as infected animals. Structural changes in bronchioles and alveoli as well as substance P-mast cell, and NK-1 and antimicrobial peptide production will be quantified in lung tissue collected from the lambs in each inoculation group and compared between treatment groups. For specific aim 3, groups of substance P competent and depleted lambs will be inoculated and assessed for clinical signs (respiration rate, temperature, appetance). Ninety days after the initial RSV or PFS inoculation, the lambs will receive a second inoculation of M. haemolytica or PFS. The lambs will be necropsied 3 days after the second inoculation and lung tissue will be assessed for the antimicrobial peptide production, substance P-mast cell and NK-1 production as above, and also the extent of lesion severity.

CRIS NUMBER: 0188110 SUBFILE: CRIS
PROJECT NUMBER: IOWV-411-23-0015 SPONSOR AGENCY: CSREES
PROJECT TYPE: ANIMAL HEALTH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2000 TERMINATION DATE: Sep 30, 2003

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION

Knowledge Area (KA)	Subject (S)	Science (F)	Objective (G)	Percent
311	3610	1160	4.2	100%

CLASSIFICATION HEADINGS
KA311 - Animal Diseases
S3610 - Sheep, live animal
F1160 - Pathology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks

RESEARCH EFFORT CATEGORIES

BASIC	(N/A)%
APPLIED	100%
DEVELOPMENTAL	(N/A)%

KEYWORDS: sheep; respiratory syncytial virus; virus diseases (animals); vaccines; anti microbial agents; peptides; mast cells; defensins; respiratory diseases; animal diseases; tissue analysis; immunity; infection; hypersensitivity; animal pathology; asthma; immunology; inflammation; epithelial cells; disease severity; bacterial diseases (animals)

PROGRESS: Oct 1, 2000 TO Sep 30, 2003
Substance P contributes to the physiologic homeostasis of pulmonary airways and vasculature. During pneumonia, alterations in substance P production and receptor expression can influence bronchoconstriction and vascular perfusion. The distribution of substance P fibers, mast cells and substance P receptor (neurokinin-1 receptor [NK-1R]) in lungs of normal sheep and sheep with acute (1 day), subacute (15 days) and chronic (45 days) bronchopneumonia caused by Mannheimia haemolytica was determined by immunohistochemistry for three rabbit polyclonal antibodies generated to the cytosolic C-terminal portion of NK-1 R (residues 393-407). There were significant differences between antibodies in distribution of NK-1R immunoreactivity and with age, but not between treatments. Substance P fibers were decreased and mast cells were increased. The work suggests that: 1.) antibodies to the same NK-1R cytosolic region can vary in their specificity and affinity, 2.) NK-1R is widely distributed in ovine lung, 3.) the expression of NK-1R decreases with age, 4.) the density of NK-1R does not change during progression of bacterial (M.haemolytica) bronchopneumonia, and 5) infection increases mast cells and decreases substance P fibers. A model of paramyxoviral infection in lambs was then developed to assess the effect of viral infection on substance P fibers, receptors and mast cells. Parainfluenza virus-3 (PI-3V) is a common respiratory pathogen of cattle and sheep. Bovine viral diarrhea virus (BVDV) is a common bovine pathogen that may enhance respiratory disease. The purpose of the study was to: 1) compare acute, subacute and chronic pulmonary lesions in PI-3/BVDV versus PI-3 inoculated lambs, 2) determine cellular distribution of PI-3 and BVDV antigens within the lung by immunohistochemistry, and 3) assess expression of beta-defensin during viral pneumonia. Two groups of neonatal lambs were inoculated intranasally and intratracheally with PI-3/BVDV, PI-3 alone, or sterile media. Both infected lambs and controls were bled and euthanized 3, 6, and 17 days post-inoculation (p.i.), and the respiratory tract tissues were collected. The histopathological lesions were similar in both groups of animals. By immunohistochemistry, PI-3 viral antigen at 3 days p.i. was abundant and present within the cytoplasm of the bronchiolar epithelial cells and to a lesser extent in the macrophages and type II pneumocytes, and very rarely in the bronchial epithelial cells. On day 6 p.i., the amount of viral antigen varied among the animals from small amounts in the areas with most severe lesions to complete absence. Viral antigen was not detectable by immunohistochemistry on day 17 p.i. In conclusion, the simultaneous inoculation of PI-3 and BVDV viruses in the lung of neonatal lambs does not enhance lesion severity, or persistence, and distribution of PI-3 antigen. These tissues will be assessed for substance P receptor to determine if viral pneumonia alters expression.

IMPACT: 2000-10-01 TO 2003-09-30 Hypersensitivity reactions in lung are health problems for domestic animals, humans, and other species. Substance P and it's receptor are key elements in the hypersensitivity reacion and paramyxoviral infection can predispose to hypersensitivity. The work determines the extent of substance P receptor changes during pneumonia and may lead to enhanced prevention/therapeutic strategies.

PUBLICATION INFORMATION: 2000-10-01 TO 2003-09-30
Ramirez-Romero R, Brogden KA, Gallup JM, Sonea IM, Ackermann MR: Mast cell density and substance P-like immunoreactivity during the initiation and progression of lung lesions in ovine Mannheimia (Pasteurella) haemolytica pneumonia. Microbial Pathogenesis 30(6):325-35, 2001.

PROJECT CONTACT INFORMATION

NAME:	Ackermann, M. R.
PHONE:	515-294-3647
FAX:	515-294-5423