Mouse modeling of lung function and host resistance in space flight and ground-based flight models

Stephen K. Chapes, PhD

Division of Biology

Space grant awarded and undergraduate fellowship to Ms. Allison Row to work on a NASA funded project (NAG2-1274) in Dr. Chapes' laboratory. Ms. Row learned how to perform flow cytometric analyses on cells being prepared and cultured in simulated space flight setups. She learned to harvest cells and culture them as would be done in the NASAS project.

Space grant supported the research of Marci Hart who was working on NASAS supported research (NCC5-168) to develop a mouse model to study lung function and host resistance in space flight and ground-based space-flight models.

The B10xC2D mouse is characterized by the lack of funciton of three genes, including MHCII, Slc11a1, and Tlr4. The loss of function of these genes make the mice more susceptible to opportunistic lung infections. Because of this natural susceptibility, these mice make novel and ideal tools with which to test the increased susceptibilty to pneumonia and to test therapies to alleviate infection. Moreover, because lung function and environmental air safety are key NASA concerns, there was a need to develop a model that would allow for the assessment of increased susceptibility to lung infections or pathology. Because human studies would be deemed too invasive and potentially unethical, the development of a mouse model was an ideal substitute for these assessments. Ms. Hart's studies characterized the development of pneumonia in B10xC2D mice and demonstrated the feasibility of adoptive transfer therapy to prevent pneumonia.

Ms. Hart's first study examined cell trafficking patterns, graft longevity, and celluar in vivo changes of an established macrophage cell line (C2D) after adoptive transfer to TLR4LPS-del mice, in order to reconstitute immune responses. Adoptively transfered C2D macrophages trafficked in vivo to sites populated by host macrophages and were detected in the spleen, lungs, and lymph nodes significantly earlier than TLR4-expressing bone marrow cells. C2D macrophages were present after several months in vivo and transcribed cytokines and chemokines in respone to the in vivo microenvironment. CXCLI (KC) and TNFa transcription levels of C2D macrophages were different in vivo compared to cells maintained exclusively in vitro. C2D macrophages restored the sensitivity of B10 mice to LPS. These responses of C2D macrophages to the host environment occured without causing significant pathology. Therefore, C2D cells were discovered as unique tools for reconstitution of imared host immunity becaues of their ability to survive in vivo.

Ms. Hart's second study investigates the importance of TLR4-positive macrophages in early recognition and clearance of a pulmonatory bacterial infection. Toll-like receptor 4 (TLR4) is a trans-membrane receptor that is the primary recognition molecule for lipopolysaccharide (LPS) of Gram-negative bacteria. The TLR4LPS-del mouse strains C57BL10/ScN (B10) and STOCK-Abbtml-TLR4LPS-del-Slc11a1s (B10xC2D) are susceptible to pulmonary infections and develop pneumonia when naturally or experimentally infected by the opportunistic bacterium Pasteurella pneumotropica. Since these animals have the TLR4LPS-delgenotype, we hypothesized that reconstitution of these mice with TLR4-positive macrophages would provide resistance to this bacterium. To test this hypothesis, we adoptively transferred a cultured macrophage cell line (C2D macrophages) to B10 and B10xC2D mice by intraperitoneal injection. We compared the performance of C2D macrophages to whole bone marrow cells from C2D mice. We show that C2D macrophages induce earlier transcription of TNFa and the chemokines JE and MIP-2 in the lungs of B10 and B10xC2D mice. In addition, C2D macrophages alter the course of inflamation following experimental Pasteurella challenge. C2D macrophages protect B10xC2D mice which lack CD4+T cells. These data indicate that macrophages are critical cells in pulmonary immunity and that macrophages alone can provide host resistance to P.pneumotropica. This study provided insight into the importance of TLR4-positive macrophages in early recognition and clearance of a pulmonary bacterial infection.

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