Identification and Quantitation of Coding Variants and Isoforms of Pulmonary Surfactant Protein A

Pulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC–MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid

Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of <i>Mycobacterium tuberculosis</i> in Human Macrophages

<div><p></p><p>Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to <i>Mycobacterium tuberculosis</i> (<i>MTB</i>). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to <i>MTB</i>. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular <i>MTB</i>. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular <i>MTB</i> in human macrophages via induction of apoptosis and autophagy.</p></div

Inhibition of NFκB activation induces autophagy in THP-1 cells.

<p>(<b>A</b>) Control THP-1 cells (0.1% DMSO) and THP-1 cells subjected to serum starvation, 5 µM BAY, <i>MTB</i> infection, or both <i>MTB</i>+BAY for 24 hrs, followed by immunoblotting of nuclear-free whole cell lysates for LC3 and β-actin. A representative immunoblot of three independent experiments is shown. (<b>B</b>) Human THP-1 cells were transduced with lentivirus-GFP-LC3 and differentiated into macrophages, followed by infection with <i>MTB</i> for 24 hrs in the absence or presence of 5 µM BAY. The cells were fixed and stained with DAPI to visualize the nuclei (blue) and the number of GFP-positive punctae were quantified. <i>Upper panel</i>, representative immunofluorescence images of three independent experiments; <i>lower panel</i>, average number of GFP-LC3 punctae per cell. The data shown represent the mean ± SEM of duplicate wells/condition from three independent experiments. (<b>C</b>) <i>MTB</i>-infected THP-1 cells treated with BAY were incubated with or without 3-MA, an inhibitor of the early phase of the autophagic pathway. After 48 hrs, the cells were lysed and nuclear-free whole cell lysates (20 µg per lane) were separated by SDS-PAGE and immunoblotted for LC3-I, LC3-II and β-actin. The bar graph represents the relative densities of the LC3-II bands normalized for their corresponding β-actin bands for two independent experiments. (<b>D</b>) THP-1 cells were infected with <i>MTB</i> H37Rv alone, <i>MTB</i>+5 µM BAY, or <i>MTB</i>+BAY+6 mM 3-MA for 4 days and cell-associated <i>MTB</i> was quantified. Data shown are mean ± SEM from two independent experiments performed in duplicates. *p<0.05, **p<0.01, ***p<0.001.</p

TNFα and IFNγ levels in MDM and AM infected with <i>MTB</i> H37Rv with or without NFκB inhibition.

<p>Primary human (<b>A</b>) MDM or (<b>B</b>) AM were infected with <i>MTB</i> H37Rv and after 1 hr, 24 hrs, 4 days, or 8 days of infection, supernatants were assayed for TNFα by ELISA and IFNγ by electrochemiluminescence. Data shown are estimated means with standard error bars from linear mixed model fits, based on seven independent experiments (MDM) or nine independent experiments (AM). *p<0.05 and **p<0.01 for cytokine expression in macrophages infected with <i>MTB</i> alone (closed circles) vs. <i>MTB</i>+BAY (closed squares). Control = (open circles) and BAY = (open squares).</p

<i>MTB</i> H37Rv induces NFκB binding to its <i>cis</i>-regulatory element.

<p>(<b>A</b>) THP-1 cells were pre-treated with 0.1% (v/v) DMSO or 5 µM BAY for 1 hr, followed by infection with <i>MTB</i> H37Rv at the indicated times. An EMSA was performed with an oligonucleotide that corresponds to the consensus binding sequence for NFκB. Data shown are representative of three independent experiments. (<b>B</b>) Primary human MDM or (<b>C</b>) AM were pre-incubated with 0.1% DMSO or 5 µM BAY for 1 hr, then infected with <i>MTB</i> for 3 hrs, and followed by an EMSA to assay for NFκB binding. Data shown are representative of two independent experiments for MDM and AM. NS = non-specific band. SS = supershift band.</p

Inhibition of NFκB activation by BAY 11-7082 reduces the viability of intracellular <i>MTB</i> in human macrophages.

<p>(A) THP-1 cells, (B) MDM, or (C) AM were pretreated with 0.1% (v/v) DMSO (open squares) or 5 µM BAY (closed circles) for 1 hr, followed by infection with <i>MTB</i> H37Rv. One hr, 4 days, or 8 days after infection, the cells were lysed and cultured for <i>MTB</i>. (D) Differentiated THP-1 cells were pretreated with 0.1% (v/v) DMSO (open squares) or 5 µM BAY (closed circles) for one 1 hr, followed by infection with <i>MTB</i>-H37Rv-GFP. One hr, 4 days, or 8 days after infection, fluorescent intensity was measured by Cytofluor II microplate fluorometer. Data shown as mean ± SEM. n = 4 for THP-1 cells in (A) and n = 2 for THP-1 cells in (D), n = 7 volunteers for MDM, n = 9 volunteers for AM. *p<0.05, **p<0.01, ***p<0.001.</p

Inhibition of NFκB activation increases apoptosis of infected macrophages.

<p>(<b>A</b>) THP-1 cells were infected with <i>MTB</i> H37Rv for 4 and 8 days with or without BAY, and apoptosis measured by TUNEL. Data for THP-1 cells are the mean ± SEM of four independent experiments. (<b>B</b>) Primary human MDM and (<b>C</b>) AM were infected with <i>MTB</i> with or without BAY, cultured for 4 days, and apoptosis measured by TUNEL. The percentage (%) numbers above the bars indicate the % cells with positive TUNEL stain. Data for MDM and AM are the mean ± SEM of three independent experiments. n.s. = not significant, **p<0.01, ***p<0.001. (<b>D</b>) THP-1 cells were infected with <i>MTB</i>, 5 µM BAY 11-7082, or both. After 48 hrs, nuclear-free whole cell lysates isolated, and western blot performed for cytochrome c. The membranes were also immunoblotted for β-actin. The bar graph above the immunoblot represent the mean relative density measurements for cytochrome c bands normalized for the densities of the corresponding β-actin band. The data shown are representative of two independent experiments. **p<0.01. (<b>E</b>) THP-1 cells were infected with <i>MTB</i> H37Rv-GFP for 1 hr, stained with DAPI, and viewed under both differential interference contrast (DIC) and fluorescent imaging under 630× magnification (panels 1–3). An area of panel 3 was magnified further on the computer screen (panel 4). Data shown are representative of two independent experiments.</p

Diagram of the mechanisms by which NFκB activation promotes the intracellular survival of <i>MTB</i>.

<p>Based on our experimental findings, NFκB activation enhanced the intracellular survival of <i>MTB</i> through inhibition of apoptosis and autophagy in infected macrophages. Since NFκB can also induce the production of its inhibiting molecule IκBα (blue line) and NFκB inhibition of autophagy could potentially prevent degradation of IKK (red line), the ultimate effect of NFκB on survival of intracellular <i>MTB</i> in macrophages is likely a complex process. IKK = IκBα kinase.</p

Inhibition of NFκB activation using a dominant-negative to IκBα.

<p>(<b>A</b>) THP-1 cells were transduced with or without AdV-GFP or AdV-S32/36A-IκBα at a MOI of 30∶1 for 5 hrs and then infected with <i>MTB</i> H37Rv. After 4 days of infection, the cells were lysed and cell-associated <i>MTB</i> quantified. (<b>B</b>) Wildtype THP-1 cells with or without pre-treatment with 5 µM BAY were infected with <i>MTB</i> H37Rv. Other THP-1 cells were transduced with AdV-GFP or AdV-S32/36A-IκBα at a MOI of 30∶1 for 5 hrs and then infected with <i>MTB</i> H37Rv. After 24 hrs of infection, the supernatants were measured for IL-8 by ELISA. Data are means ± SEM of two independent experiments performed in duplicates. *p<0.05, ** p<0.01.</p

Inhibition of caspase-3 activation abrogates the effects of BAY.

<p>(<b>A</b>) THP-1 cells were cultured with 5 µM BAY, <i>MTB</i>, or <i>MTB</i>+BAY with or without 10 µM of the caspase-3 inhibitor z-DEVD-fmk for 48 hrs. After the indicated time, the cells were lysed and activated caspase-3 quantified by ELISA. Data shown are the mean ± SEM of two independent experiments performed in duplicates. *p<0.05, **p<0.01. (<b>B</b>) THP-1 cells were infected with <i>MTB</i> H37Rv alone (open diamonds), <i>MTB</i>+BAY (closed squares), or <i>MTB</i>+BAY+z-DEVD-fmk (semi-closed triangles). One hr, 2 days, and 4 days after infection, THP-1 cells were lysed and cultured for <i>MTB</i>. (<b>C</b>) The same treatment conditions as in (A) were repeated with THP-1 cells for 2 days followed by measurement of apoptosis using TUNEL staining. Data shown are the mean ± SEM of two independent experiments. *p<0.05, **p<0.01 and ***p<0.001.</p