Dynamic changes in Mcl-1 expression regulate macrophage viability or commitment to apoptosis during bacterial clearance
Helen M. Marriott, Colin D. Bingle, Robert C. Read, Karen E. Braley, Guido Kroemer, Paul G. Hellewell, Ruth W. Craig, Moira K.B. Whyte, David H. Dockrell
Helen M. Marriott, Colin D. Bingle, Robert C. Read, Karen E. Braley, Guido Kroemer, Paul G. Hellewell, Ruth W. Craig, Moira K.B. Whyte, David H. Dockrell
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Article Infectious disease

Dynamic changes in Mcl-1 expression regulate macrophage viability or commitment to apoptosis during bacterial clearance

Abstract

Macrophages are critical effectors of bacterial clearance and must retain viability, despite exposure to toxic bacterial products, until key antimicrobial functions are performed. Subsequently, host-mediated macrophage apoptosis aids resolution of infection. The ability of macrophages to make this transition from resistance to susceptibility to apoptosis is important for effective host innate immune responses. We investigated the role of Mcl-1, an essential regulator of macrophage lifespan, in this switch from viability to apoptosis, using the model of pneumococcal-associated macrophage apoptosis. Upon exposure to pneumococci, macrophages initially upregulate Mcl-1 protein and maintain viability for up to 14 hours. Subsequently, macrophages reduce expression of full-length Mcl-1 and upregulate a 34-kDa isoform of Mcl-1 corresponding to a novel BH3-only splice variant, Mcl-1Exon-1. Change in expression of Mcl-1 protein is associated with mitochondrial membrane permeabilization, which is characterized by loss of mitochondrial inner transmembrane potential and translocation of cytochrome c and apoptosis-inducing factor. Following pneumococcal infection, macrophages expressing full-length human Mcl-1 as a transgene exhibit a delay in apoptosis and in bacterial killing. Mcl-1 transgenic mice clear pneumococci from the lung less efficiently than nontransgenic mice. Dynamic changes in Mcl-1 expression determine macrophage viability as well as antibacterial host defense.

Authors

Helen M. Marriott, Colin D. Bingle, Robert C. Read, Karen E. Braley, Guido Kroemer, Paul G. Hellewell, Ruth W. Craig, Moira K.B. Whyte, David H. Dockrell

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Figure 3

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Pneumococcal infection of macrophages results in increased expression of...
Pneumococcal infection of macrophages results in increased expression of a proapoptotic Mcl-1 isoform. (A) Western blot of total protein from MDMs at the indicated time points after infection with pneumococci, probed with anti–Mcl-1 and anti-actin antibodies showing the appearance of the band of approximately 34 kDa. The donor is representative of 1 of the series in Figure 1C. The mean levels of nuclear apoptosis at each time point for this donor are indicated. (B) Western blot of total protein from U937 cells or MDMs (3 donors in 3 separate experiments) 20 hours after infection with pneumococci or mock infection and U937 cells transfected with Mcl-1Exon-1 or empty vector probed with anti–Mcl-1 antibody, with in vitro translation of Mcl-1, Mcl-1S/°TM, and Mcl-1Exon-1 as standards. (C) Genomic organization of Mcl-1 and Mcl-1Exon-1. The filled regions represent the coding region of the 2 transcripts. (D) Percentage of nonviable infected or mock-infected U937 cells 16 hours after nucleofection with vectors containing full-length Mcl-1, Mcl-1Exon-1, Bax, or empty vector. Results represent the analysis of 3 separate experiments. P < 0.05, Spn– Mcl-1 vs. Spn– Mcl-1Exon-1; P < 0.05, Spn+ Mcl-1 vs. Spn+ Mcl-1Exon-1; Student’s paired t test. (E) Percentage of EGFP-positive cells demonstrating features of apoptosis after DAPI staining in the same experiments as in D. P < 0.05, Spn– Mcl-1 vs. Spn– Mcl-1Exon-1; P < 0.05, Spn+ Mcl-1 vs. Spn+ Mcl-1Exon-1; P < 0.05, Spn+ empty vector vs. Spn+ Mcl-1; Student’s paired t test.