Partial Least Square Path Modeling: Basic Concepts issues and Application

Abstract

Previous studies in our laboratory have shown a differential activation of the mitogen-activated protein kinases (MAPKs) in primary bone marrow-derived macrophages following infection with pathogenic Myco- bacterium avium compared to the activation following infection with nonpathogenic Mycobacterium smegmatis. Additionally, M. smegmatis-infected macrophages produced significantly elevated levels of tumor necrosis factor alpha (TNF-) compared to the levels produced by M. avium-infected macrophages. The TNF- production was dependent on both p38 and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. However, the macrophage transcription factors downstream of the MAPKs, which were required for TNF- production, remained undefined. In this study we determined that the transcription factor cyclic AMP response element binding protein (CREB) is significantly more activated in M. smegmatis-infected macrophages than in M. avium-infected macrophages. We also found that CREB activation was dependent on p38 and protein kinase A but not on ERK 1/2 or calmodulin kinase II. Moreover, mutating the cAMP-responsive element on the TNF- promoter resulted in significantly diminished promoter activity following M. smegmatis infection but not M. avium infection. The inability of macrophages infected with M. avium to sustain MAPK activation and to produce high levels of TNF- was due, in part, to an increase in serine/threonine phosphatase PP2A activity. Our studies are the first to demonstrate an important role for the transcription factor CREB in TNF- production by mycobacterium-infected macrophages, as well as a role for M. avium’s induction of PP2A phosphatase activity as a mechanism to limit macrophage activation. Mycobacterium avium is an opportunistic pathogen that af- fects people with suppressed immune systems, particularly people with late-stage human immunodeficiency virus or chronic lung diseases. In AIDS patients, M. avium is commonly disseminated and can involve almost any internal organ, espe- cially the liver, spleen, and bone marrow. Moreover, M. avium is responsible for increased morbidity and mortality in human immunodeficiency virus-infected individuals (6). M. avium is a facultative intracellular pathogen that resides within the phagosome of the host macrophage. The macro- phage is the first line of defense against invading microorganisms, and it functions to phagocytose and subsequently destroy these invaders within a phagolysosome. However, following phagocyto- sis, M. avium, like other species in the genus Mycobacterium, including Mycobacterium tuberculosis and Mycobacterium leprae, has been shown to halt the maturation of the phagosome through coordinated blocking of lysosome fusion with the phagosome. Thus, the M. avium-containing phagosome fails to acquire lyso- somal lytic enzymes, nor does it acidify, due to a scarcity of the * Corresponding author. Mailing address: Department of Biology, University of Notre Dame, 130 Galvin Life Science Center, Notre Dame, IN 46556. Phone: (574) 631-3734. Fax: (574) 631-7413. E-mail: schorey.1@nd.edu. † Present address: Benaroya Research Institute at Virginia Mason, Immunology, Seattle, WA 98101. 514 proton ATPase