ssGPL does not contribute to the resistance of <i>M</i>. <i>avium</i> to LL-37.

<p>CFU determination of <i>M</i>. <i>avium</i>^ssGPL and <i>M</i>. <i>avium</i>^ΔssGPL serovar 8 after incubation with 0, 10, 25, and 100 μg/ml LL-37 and 20 μg/ml gentamicin. ***p<0.0001. Data are the mean ± SEM of 3 independent experiments.</p

Loss of LL-37 activity after exposure to NTM or NTM-derived lipids.

<p><i>E</i>. <i>coli bioassays</i> were used to evaluate LL-37 activity. (A) LL-37 remaining in NTM (but not <i>Mtb</i>) culture supernatant no longer kills <i>E</i>. <i>coli</i>. (B) <i>E</i>. <i>coli</i> survives in untreated or boiled NTM culture supernatants to which fresh LL-37 was added. (C) <i>E</i>. <i>coli</i> survival following incubation with <i>M</i>. <i>abscessus</i> or <i>M</i>. <i>intracellulare</i> derived cell fractions. CM = cell membrane, CW = cell wall, ICW = insoluble cell wall fraction.</p

<i>E</i>. <i>coli</i> is susceptible to LL-37.

<p>(A) Log₁₀ CFU of <i>E</i>. <i>coli</i> after 4 hours of incubation with 0, 10, or 25 μg/ml of LL-37. **p< 0.001; ***p<0.0001. Data are the mean ± SEM of 6 independent experiments. (B) Images were taken of each serial dilution on LB agar from <i>E</i>. <i>coli</i> cultures incubated for 4 hours in the absence or presence of 25 μg/ml of LL-37.</p

LL-37 demonstrates broad-spectrum antimicrobial activity.

<p>Log₁₀ CFU after 1–8 hours of incubation of a (A) laboratory isolate of <i>Salmonella enteriditis</i> or clinical isolates of (B) <i>Salmonella enteriditis</i> (Uganda) or (C) <i>Salmonella non-typhi</i> (Nairobi) with 0–50 μg/ml of LL-37. *p< 0.01; **p< 0.001; ***p<0.0001. Data are the mean ± SEM of 3–6 independent experiments. (D) <i>Mtb</i> H37Rv were incubated with 10 μg/ml LL-37 and the percent change in CFU calculated after 96 hours incubation. n = 3 independent experiments.</p

nsGPL do not mediate the resistance of <i>M</i>. <i>abscessus</i> to LL-37.

<p>(A-B) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(+) in the presence of the indicated concentrations of native LL-37 (p = 0.69). Data are the mean ± SEM of 4 independent experiments. (B) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(-) in the presence of the indicated concentrations of native LL-37. Data are the mean ± SEM of 4 independent experiments. *p<0.01; **p< 0.001; ***p<0.0001. (C) Thin-layer chromatography demonstrates the presence and absence of GPL in <i>M</i>. <i>abscessus</i>^nsGPL(+) and <i>M</i>. <i>abscessus</i>^nsGPL(-), respectively. (D-E) CFU determination of <i>M</i>. <i>abscessus</i>^nsGPL(+) and <i>M</i>. <i>abscessus</i>^nsGPL(-), respectively in the presence of the indicated concentrations of scrambled LL-37 peptide. Data are the mean ± SEM of 3 independent experiments. *p<0.01; **p< 0.001; ***p<0.0001.</p

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

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

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

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 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