title: Caspase-3-independent apoptotic pathways contribute to interleukin-32γ-mediated control of Mycobacterium tuberculosis infection in THP-1 cells.
abstract: BACKGROUND
  Macrophages are the primary effector cells responsible for killing Mycobacterium tuberculosis (MTB) through various mechanisms, including apoptosis. However, MTB can evade host immunity to create a favorable environment for intracellular replication. MTB-infected human macrophages produce interleukin-32 (IL-32). IL-32 is a pro-inflammatory cytokine and has several isoforms. We previously found that IL-32γ reduced the burden of MTB in human macrophages, in part, through the induction of caspase-3-dependent apoptosis. However, based on our previous studies, we hypothesized that caspase-3-independent death pathways may also mediate IL-32 control of MTB infection. Herein, we assessed the potential roles of cathepsin-mediated apoptosis, caspase-1-mediated pyroptosis, and apoptosis-inducing factor (AIF) in mediating IL-32γ control of MTB infection in THP-1 cells.
  
  
  RESULTS
  Differentiated human THP-1 macrophages were infected with MTB H37Rv alone or in the presence of specific inhibitors to caspase-1, cathepsin B/D, or cathepsin L for up to four days, after which TUNEL-positive cells were quantified; in addition, MTB was quantified by culture as well as by the percentage of THP-1 cells that were infected with green fluorescent protein (GFP)-labeled MTB as determined by microscopy. AIF expression was inhibited using siRNA technology. Inhibition of cathepsin B/D, cathepsin L, or caspase-1 activity significantly abrogated the IL-32γ-mediated reduction in the number of intracellular MTB and of the percentage of GFP-MTB-infected macrophages. Furthermore, inhibition of caspase-1, cathepsin B/D, or cathepsin L in the absence of exogenous IL-32γ resulted in a trend toward an increased proportion of MTB-infected THP-1 cells. Inhibition of AIF activity in the absence of exogenous IL-32γ also increased intracellular burden of MTB. However, since IL-32γ did not induce AIF and because the relative increases in MTB with inhibition of AIF were similar in the presence or absence of IL-32γ, our results indicate that AIF does not mediate the host-protective effect of IL-32γ against MTB.
  
  
  CONCLUSIONS
  The anti-MTB effects of IL-32γ are mediated through classical caspase-3-dependent apoptosis as well as caspase-3-independent apoptosis.
authors.affiliation: Department of Medicine, Denver Veterans Affairs Medical Center, Denver, CO, USA. BaiX@njhealth.org.|Departments of Medicine and Academic Affairs, National Jewish Health, D509, Neustadt Building, 1400 Jackson Street, Denver, CO, 80206, USA. BaiX@njhealth.org.|Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA. BaiX@njhealth.org.
authors.forename: Xiyuan
authors.initials: X
authors.lastname: Bai
authors.affiliation: Department of Medicine, Denver Veterans Affairs Medical Center, Denver, CO, USA. KinneyW@njhealth.org.|Departments of Medicine and Academic Affairs, National Jewish Health, D509, Neustadt Building, 1400 Jackson Street, Denver, CO, 80206, USA. KinneyW@njhealth.org.|Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA. KinneyW@njhealth.org.
authors.forename: William H
authors.initials: WH
authors.lastname: Kinney
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei andTri-Service General Hospital; National Defense Medical Center, Taipei, Taiwan. soa@mail.ndmctsgh.edu.tw.
authors.forename: Wen-Lin
authors.initials: WL
authors.lastname: Su
authors.affiliation: Department of Medicine, Denver Veterans Affairs Medical Center, Denver, CO, USA. BaiA@njhealth.org.|Departments of Medicine and Academic Affairs, National Jewish Health, D509, Neustadt Building, 1400 Jackson Street, Denver, CO, 80206, USA. BaiA@njhealth.org.
authors.forename: An
authors.initials: A
authors.lastname: Bai
authors.affiliation: Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA. Alida.Ovrutsky@ucdenver.edu.
authors.forename: Alida R
authors.initials: AR
authors.lastname: Ovrutsky
authors.affiliation: Department of Medicine, Denver Veterans Affairs Medical Center, Denver, CO, USA. Jennifer.Honda@ucdenver.edu.|Departments of Medicine and Academic Affairs, National Jewish Health, D509, Neustadt Building, 1400 Jackson Street, Denver, CO, 80206, USA. Jennifer.Honda@ucdenver.edu.|Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA. Jennifer.Honda@ucdenver.edu.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Honda
authors.affiliation: Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands. m.netea@aig.umcn.nl.
authors.forename: Mihai G
authors.initials: MG
authors.lastname: Netea
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA. Marcela.Henao_Tamayo@ColoState.EDU.
authors.forename: Marcela
authors.initials: M
authors.lastname: Henao-Tamayo
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA. D.Ordway@Colostate.edu.
authors.forename: Diane J
authors.initials: DJ
authors.lastname: Ordway
authors.affiliation: Division of Infectious Diseases, University of Colorado Denver Anschutz Medical Campus, Aurora, CO, USA. cdinare333@aol.com.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Dinarello
authors.affiliation: Department of Medicine, Denver Veterans Affairs Medical Center, Denver, CO, USA. ChanE@njhealth.org.|Departments of Medicine and Academic Affairs, National Jewish Health, D509, Neustadt Building, 1400 Jackson Street, Denver, CO, 80206, USA. ChanE@njhealth.org.|Department of Medicine, University of Colorado School of Medicine, Aurora, CO, USA. ChanE@njhealth.org.
authors.forename: Edward D
authors.initials: ED
authors.lastname: Chan
mesh_terms: D017209:Apoptosis; D058491:Bacterial Load; D002460:Cell Line; D003593:Cytoplasm / Q000382:microbiology; D006801:Humans; D007378:Interleukins / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000276:immunology*
chemical_list: C496971:IL32 protein, human; D007378:Interleukins