Apolipoprotein B containing lipoproteins contribute to host innate immune pulmonary defense against Staphylococcus aureus

Circulating lipoproteins are increasingly recognized as innate immune effector molecules. Severe hypolipidemia, which may occur with trauma or critical illness, is clinically associated with bacterial pneumonia. Importantly, pneumonia caused by Staphylococcus aureus is a major cause of morbidity and mortality in both hospital- and community-acquired pneumonia cases. Here we report that lipoprotein deficiency impairs early pulmonary innate defense against S. aureus quorum-sensing dependent pathogenesis. Specifically, apoB levels in the lung early post-infection are significantly reduced with lipoprotein deficiency, coinciding with impaired host control of S. aureus agr-signaling and increased agr-dependent morbidity and inflammation. Given that lipoproteins also inhibit LTA- and LPS-mediated inflammation, these results suggest that hypolipidemia may broadly impact post-trauma pneumonia susceptibility to both Gram positive and Gram negative pathogens. Together with previous reports demonstrating that hyperlipidemia also impairs lung innate defense, these results suggest that maintenance of normal serum lipoprotein levels is necessary for optimal host innate defense in the lung

Automated Computational Processing of 3-D MR Images of Mouse Brain for Phenotyping of Living Animals

Magnetic resonance (MR) imaging provides a method to obtain anatomical information from the brain in vivo that is not typically available by optical imaging because of this organ's opacity. MR is nondestructive and obtains deep tissue contrast with 100-µm^3 voxel resolution or better. Manganese-enhanced MRI (MEMRI) may be used to observe axonal transport and localized neural activity in the living rodent and avian brain. Such enhancement enables researchers to investigate differences in functional circuitry or neuronal activity in images of brains of different animals. Moreover, once MR images of a number of animals are aligned into a single matrix, statistical analysis can be done comparing MR intensities between different multi-animal cohorts comprising individuals from different mouse strains or different transgenic animals, or at different time points after an experimental manipulation. Although preprocessing steps for such comparisons (including skull stripping and alignment) are automated for human imaging, no such automated processing has previously been readily available for mouse or other widely used experimental animals, and most investigators use in-house custom processing. This protocol describes a stepwise method to perform such preprocessing for mouse

Alterations of functional circuitry in aging brain and the impact of mutated APP expression

Alzheimer's disease (AD) is a disease of aging that results in cognitive impairment, dementia and death. Pathognomonic features of AD are amyloid plaques composed of proteolytic fragments of the amyloid precursor protein (APP) and neurofibrillary tangles composed of hyperphosphorylated tau protein. One type of familial Alzheimer's disease (FAD) occurs when mutant forms of APP are inherited. Both APP and tau are components of the microtubule-based axonal transport system, which prompts the hypothesis that axonal transport is disrupted in AD, and that such disruption impacts cognitive function. Transgenic mice expressing mutated forms of APP provide preclinical experimental systems to study AD. Here we perform manganese-enhanced magnetic resonance imaging (MEMRI) to study transport from hippocampus to forebrain in four cohorts of living mice: young and old wild-type and transgenic mice expressing a mutant APP with both Swedish and Indiana mutations (APPSwInd). We find that transport is decreased in normal aging and further altered in aged APPSwInd plaque-bearing mice. These findings support the hypothesis that transport deficits are a component of AD pathology and thus may contribute to cognitive deficits

Alterations of functional circuitry in aging brain and the impact of mutated APP expression

Alzheimer's disease (AD) is a disease of aging that results in cognitive impairment, dementia and death. Pathognomonic features of AD are amyloid plaques composed of proteolytic fragments of the amyloid precursor protein (APP) and neurofibrillary tangles composed of hyperphosphorylated tau protein. One type of familial Alzheimer's disease (FAD) occurs when mutant forms of APP are inherited. Both APP and tau are components of the microtubule-based axonal transport system, which prompts the hypothesis that axonal transport is disrupted in AD, and that such disruption impacts cognitive function. Transgenic mice expressing mutated forms of APP provide preclinical experimental systems to study AD. Here we perform manganese-enhanced magnetic resonance imaging (MEMRI) to study transport from hippocampus to forebrain in four cohorts of living mice: young and old wild-type and transgenic mice expressing a mutant APP with both Swedish and Indiana mutations (APPSwInd). We find that transport is decreased in normal aging and further altered in aged APPSwInd plaque-bearing mice. These findings support the hypothesis that transport deficits are a component of AD pathology and thus may contribute to cognitive deficits

Nox2 Modification of LDL Is Essential for Optimal Apolipoprotein B-mediated Control of <em>agr</em> Type III <em>Staphylococcus aureus</em> Quorum-sensing

<div><p><em>Staphylococcus aureus</em> contains an autoinducing quorum-sensing system encoded within the <em>agr</em> operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within <em>agr</em> has generated four <em>agr</em> specific groups, <em>agr</em> I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives <em>agr</em> signaling. Because <em>agr</em> signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize <em>agr</em> signaling. We determined whether two host defense factors that inhibit AIP1-induced <em>agr</em>I signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced <em>agr</em>III signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against <em>agr</em>III signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against <em>S. aureus agr</em>III infection by binding the secreted signaling peptide, AIP3, and preventing expression of the <em>agr</em>-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes <em>agr</em> signaling by all four <em>agr</em> alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against <em>agr</em> signaling. These data reveal a novel role for oxLDL in host defense against <em>S. aureus</em> quorum-sensing signaling.</p> </div

ROS-dependent oxidation of LDL but not of AIP3 mediates antagonism of <i>agr</i>III-dependent quorum sensing.

<p>(A, B) LDL, apoB or oxLDL at equimolar concentrations of apoB were treated as shown with (A) HOCl for 30 min at 37°C, with or without prior addition of the ROS scavenger N-acetylmethionine (NAM), or (B) singlet oxygen (5 min on ice). 10 nM ROS-treated lipoproteins or controls were cultured overnight with <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] plus 50 nM AIP. <i>agr</i>:P3 promoter activation was measured by flow cytometry. (C) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with 50 nM AIP3 plus LDL treated with increasing concentrations of singlet oxygen. (D) Measurement of LDL lipid oxidation before and after exposure to singlet oxygen or HOCl. Commercially available oxLDL and bovine serum albumin are given as controls. Data represent the mean ± SEM. (E) AIP1 and AIP3 were incubated with buffer or HOCl for 30 min at 37°C, treated with NAM to scavenge remaining ROS, and cultured at 50 nM for 2 h with <i>agr</i>I isolate LAC [<i>agr</i>::P3-yfp] or <i>agr</i>III MW2 [<i>agr</i>::P3-yfp], respectively. Data was measured by flow cytometry. Data points represent the mean ± SEM normalized to 100% activation by untreated AIP and statistics are in reference to this control. (F) AIP1 and AIP3 were exposed to singlet oxygen by incubation with rose bengal in the presence or absence of light, prior to culture with <i>agr</i>I isolate LAC [<i>agr</i>::P3-yfp] or <i>agr</i>III MW2 [<i>agr</i>::P3-yfp]. Data points represent the mean ± SEM normalized to 100% activation by untreated AIP and statistics are in reference to this control. (G) AIP1 was combined with buffer or a 10-fold molar excess of ethylthioacetate prior to exposure to singlet oxygen, followed by culture with <i>agr</i>I isolate LAC [<i>agr</i>::P3-yfp]. Data points represent the mean ± SEM of the mean channel fluorescence. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Optimal apolipoprotein B-mediated antagonism of <i>agr</i>III-signaling requires oxidation of LDL via Nox2.

<p>(A) Schematic representation of AIP1 and AIP3. (B) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with broth control or 10 nM LDL, apoB or oxLDL, and <i>agr</i>::P3 activation was measured by flow cytometry. Colony forming units (cfus) were determined by plating on sheep blood agar. (C) Anti-apoB antibody reverses antagonism of <i>agr</i>::P3 promoter activation in MW2. Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with broth control, 10 nM apoB or oxLDL, along with 30 nM control IgG or 30 nM apoB-specific IgG. (D) LDL, ApoB or oxLDL binding to immobilized AIP3 was measured by SPR following lipoprotein incubation with apoB-specific IgG or control IgG. Data were normalized to the mean ± SEM of LDL binding in the absence of antibody. (E) Blood was collected from wild-type or <i>Nox2^−/−</i> mice. After clearing, the serum was heat inactivated and diluted to 10% in TSB for overnight culture with strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp]. <i>agr</i>::P3 promoter activation was measured by flow cytometry. Data reported are the mean ± SEM normalized to broth control. (F) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] cultured 4 h with 100 nM AIP plus 10% sera from <i>Nox2^−/−</i> mice along with 50 nM LDL or 50 nM oxLDL. Data points represent the mean ± SEM normalized to broth control. (G) Immunoblot detection of apoB and oxidized LDL. Control LDL and oxLDL plus serum from wild-type and <i>Nox2^−/−</i> mice were vacuum transferred to nitrocellulose and stained for oxLDL using monoclonal antibody E06 or rabbit polyclonal antibody to apoB. A representative blot is shown. Band intensity was quantified using Carestream Molecular Imaging software (New Haven, Connecticut), and data normalized to oxLDL or wild-type sera with E06/apoB ratios equal to 1. (H) Strain <i>agr</i>III MW2 [<i>agr</i>::P3-yfp] was cultured overnight with exogenous AIP3 (50 nM) and 10 nM of either human LDL, human oxLDL, LDL purified from wild-type mice, LDL purified from <i>Nox2^−/−</i> mice or broth control. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.</p

Reduction of serum apoB levels in <i>Nox2</i> knockout mice but not wild-type mice significantly increases susceptibility to <i>agr</i>III-mediated <i>S. aur</i>eus virulence.

<p>(A) Blood was collected from wild-type or <i>Nox2^−/−</i> mice treated with 4APP or vehicle control. After clearing, the serum was heat inactivated and diluted to 10% in TSB for overnight culture with <i>agr</i>III MW2 [<i>agr</i>::P3-yfp]. <i>agr</i>::P3 promoter activation was measured by flow cytometry. Data reported are the mean ± SEM normalized to broth control at 100%. (B) Air-pouch schematic showing locations of lumen, epidermis, dermis and muscle. (C–E) Air-pouches were generated on the backs of 8 to 12 week old wild-type or <i>Nox2^−/−</i> mice. Where indicated 4APP treated mice were injected i.p. with 100 µl of 5 mg/ml 4APP or vehicle control at 48 h and 24 h prior to infection with MW2 or its <i>agr</i> deletion mutant (Δ<i>agr</i>) at the indicated concentrations. At time zero, air-pouches were injected with early exponential phase MW2. At 28 h post-infection, the following parameters were determined and data reported as the mean ± SEM: (Left to right) Representative confocal images of dermis from indicated pouches stained with TO-PRO-3 (Invitrogen; blue) and anti-<i>S. aureus</i> antibody (green fluorophore) (Scale bar = 20 µm); Quantification of bacterial density in dermis of pouches; Morbidity was scored on a 0–14 point scale and was based on weight loss, appearance, level of dehydration, mobility and responsiveness; Bacterial burden (Log CFU) in spleen. (C) Wild-type mice treated with 4APP or vehicle control. (D) <i>Nox2^−/−</i> mice treated with 4APP or vehicle control. (E) Wild-type and <i>Nox2^−/−</i> mice. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Effect of oxLDL on antagonism of <i>agr</i>III dependent virulence factor production by MRSA and MSSA clinical isolates.

<p>(A–D) Bacteria were cultured overnight in the presence of media control or oxLDL (50 nM). (A) RNAIII and (B) <i>hla</i> transcription relative to 16S rRNA was determined by qRT-PCR (C) Alpha-hemolysin content of culture supernatants was measured by the ability to lyse rRBCs. (D) Supernatants from (C) were also assessed for lipase activity determined by rate of cleavage of the triglyceride substrate tributyrin. Data reported as the mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p