Salmonella Transiently Reside in Luminal Neutrophils in the Inflamed Gut

Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model.Upon S. Tm(wt) infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This "fast cycling" through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut. In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut

<i>S.</i> Tm strains and plasmids.

<p><i>S.</i> Tm strains and plasmids.</p

Luminal neutophils are viable and actively engulf <i>S.</i> Tm.

<p>Streptomycin-treated mice LysM^gfp +/− mice were infected with <i>S.</i> Tm^wt and sacrificed 24h post infection. The cecum was explanted and infected <i>ex vivo</i> with <i>yfp</i> expressing <i>S.</i> Tm^avir, stained with DAPI (DNA; blue) and FM 4–64 (epithelial membranes; red), mounted on an organ holder and analyzed by confocal real time microscopy of <i>S.</i> Tm and GFP⁺ neutrophils. <b>A.</b> Representative confocal image stack showing <i>yfp</i> expressing <i>S.</i> Tm^avir within a GFP⁺ neutrophil. <b>B.</b> Representative confocal images showing live GFP⁺ neutrophils associated with <i>S.</i> Tm^avir. Scale bars: 10µm.</p

Transmigrating neutrophils engulf <i>S.</i> Tm in the gut lumen.

<p><b>A.</b> Model for the interaction of transmigrating PMNs with luminal <i>S.</i> Tm. <i>S.</i> Tm enrich at the epithelial surface via flagella-mediated chemotaxis. PMNs exiting from the mucosa would engulf first those bacteria close to the epithelial border before migrating further into the center of the intestinal lumen. <b>B.</b> Streptomycin-treated mice (n = 3–5) were co-infected with <i>S.</i> Tm^wt and GFP-labeled <i>S.</i> Tm mutant strains carrying pM979 for constitituve <i>gfp</i>-expression (<i>S.</i> Tm^wt<i>; S.</i> Tm^avir; <i>S.</i> Tm<i>^fliGHI</i>; <i>S.</i> Tm<i>^cheY</i>; <i>S.</i> Tm^{avir <i>fliGHI</i>}). 20h post infection, the mice were sacrificed and cecum prepared for immunofluorescence staining of extracellular <i>S.</i> Tm with α-<i>Salmonella</i>-O-antigen group B serum as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034812#s4" target="_blank">materials and methods</a> section. The fraction of intracellular GFP⁺<i>S.</i> Tm was determined as (GFP⁺ LPS⁻/ GFP⁺ LPS⁺) × 100%.</p

<i>Salmonella</i> contained within luminal neutrophils experience iron limitation.

<p>4 groups of streptomycin-treated mice (n = 3) were infected with <i>S.</i> Tm^wt (red; inflammation induced) or <i>S.</i> Tm^avir (blue; no inflammation induced) containing the GFP-reporter plasmids p<i>iroBCDE</i>-<i>gfp</i> (<b>A, C</b>) or p<i>sodB</i>-<i>gfp</i> (<b>B, D</b>), respectively. 20 h post infection, the mice were sacrificed and cecum prepared for immunofluorescence staining of <i>S.</i> Tm with α-<i>Salmonella</i>-O-antigen group B serum as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034812#s4" target="_blank">materials and methods</a> section. The tissue was either not permeabilized (w/o triton) or permeabilized with 0,1% triton prior to staining (+ triton). All bacteria positively stained with the α-<i>Salmonella</i> LPS antiserum were included for analysis and <i>S.</i> Tm GFP-fluorescence intensity (grey levels; y-axis) was determined by automated image analysis (<b>A, B</b>). Representative images of <i>S.</i> Tm^wt or <i>S.</i> Tm^avir in the cecal lumen expressing p<i>iroBCDE</i>-<i>gfp</i> (<b>C</b>) or p<i>sodB</i>-<i>gfp</i> (<b>D</b>) (green). The section was permeabilized with triton-x-100 and stained with α-<i>Salmonella</i> LPS antiserum (red) and phalloidin (blue). Scale bar: 10 µm.</p

<i>S.</i> Tm is associated with CD18⁺ neutrophils in the gut lumen.

<p><b>A.</b> H&E stained section of cecum tissue obtained from streptomycin-treated C57Bl/6 mouse infected with <i>S.</i> Tm^wt for 20 h. Polymorphonuclear cells are detected in the mucosa as well as in the gut lumen (inset). Scale bar: 100 µm; inset: 20 µm; L =  lumen. <b>B.C.</b> Immunofluorescence staining of gut lumina of <i>S.</i> Tm infected mice. Mice (n = 4) were treated with streptomycin and infected with <i>S.</i> Tm^wt pM979 for 20 h. Tissue was fixed and stained with an α-CD18 antibody. Confocal images were obtained and the fraction of CD18⁺ cells of all nucleated cells in the gut lumen was determined (3 independent images/mouse). Scale bar: 10 µm (<b>B</b>). GFP⁺<i>S.</i> Tm co-localize with CD18⁺ cells in the gut lumen Scale bar: 10 µm. GFP (green), CD18 (red) and DNA/nuclei (Sytox-green, grey) (<b>C</b>).</p

Effect of immunomodulatory medication on regional gray matter loss in relapsing–remitting multiple sclerosis — a longitudinal MRI study

Prevention of global gray matter (GM) volume changes in multiple sclerosis (MS) are an objective in clinical trials, but the effect of immunomodulatory medication on regional GM atrophy progression is unclear. MRIs from 86 patients with relapsing-remitting MS (RRMS) followed up for 24 months were analyzed using voxel-based morphometry. An analysis of covariance model (cluster threshold, corrected p<0.05) was used to compare GM volumes between baseline and follow-up while stratified by immunomodulatory medication (IM): Interferone INF-beta-1a (n=34), INF-beta-1b (n=16), glatiramer acetate (GA) (n=15), and no-immunomodulatory treatment (n=21). In the INF-beta-1a/1b group (n=50), significant GM volume reductions were observed during follow-up in fronto-temporal, cingulate and cerebellar cortical brain regions, without significant differences between the INF-beta-1a and INF-beta-1b patients. In the GA group and in unmedicated patients, no significant regional GM volume reductions were observed. In contrast to GA, INF-beta-1a/1b treatment was associated with GM volume reductions in hippocampal/parahippocampal and anterior cingulate cortex. This is the first longitudinal study investigating the effects of IMs on GM in RRMS. Results suggest differences in the dynamics of regional GM volume atrophy in differentially treated or untreated RRMS patients

<i>Salmonella</i> induce expression of the SPI-2 TTSS in neutrophils in the inflamed intestine.

<p>2 groups of streptomycin-treated mice (n = 3) were co-infected with <i>S.</i> Tm^avir containing the GFP-reporter plasmid p<i>ssaG</i>-<i>gfp</i> and either <i>S.</i> Tm^wt (red; inflammation induced) or <i>S.</i> Tm^avir (blue; no inflammation induced). 20h post infection, the mice were sacrificed and cecum prepared for immunofluorescence staining of <i>S.</i> Tm with α-<i>Salmonella</i>-O-antigen group B serum and anti-CD18 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034812#s4" target="_blank">materials and methods</a> section. The tissue was not treated with triton. <b>A</b>. Representative images of intra- and extracellular <i>S.</i> Tm^avir p<i>ssaG</i>-<i>gfp</i> in the cecal lumen. Scale bar: 20µm and 5 µm. α-<i>Salmonella</i> LPS antiserum (blue), CD18 (red) and GFP (green). <b>B.</b> Quantitative image analysis of % <i>gfp</i>-expressing of all LPS⁺<i>S.</i> Tm in the inflamed and non-inflamed gut. In the inflamed gut, the % of extracellular (GFP⁺ LPS⁺) and intracellular (GFP⁺ LPS⁻ and CD18-associated) was determined as well. Bars show the median.</p

Luminal <i>S.</i> Tm are gentamicin-protected.

<p>Gentamicin-protection assay of gut luminal <i>S.</i> Tm. Streptomycin-treated mice (n = 9) were infected with <i>S.</i> Tm^wt. 20h post infection, the mice were sacrificed and cecum contents collected in PBS and split into 4 fractions (untreated; treated with triton-X-100 (0.1%) for 30 minutes; treated with gentamicin (400 µg /ml) for 30 minutes; or treated with triton-X-100 (0.1%) and gentamicin (400 µg/ml) for 30 minutes). Samples were diluted and plated on McConkey agar plates (100 µg/ml streptomycin), allowing specific detection of <i>S.</i> Tm. Bars show mean and StD of <i>S.</i> Tm^wt CFU/ml; xxx  =  p < 0.0001.</p

Spatiotemporal Distribution Pattern of White Matter Lesion Volumes and Their Association With Regional Grey Matter Volume Reductions in Relapsing-Remitting Multiple Sclerosis

The association of white matter (WM) lesions and grey matter (GM) atrophy is a feature in relapsing-remitting multiple sclerosis (RRMS). The spatiotemporal distribution pattern of WM lesions, their relations to regional GM changes and the underlying dynamics are unclear. Here we combined parametric and non-parametric voxel-based morphometry (VBM) to clarify these issues. MRI data from RRMS patients with progressive (PLV, n = 45) and non-progressive WM lesion volumes (NPLV, n = 44) followed up for 12 months were analysed. Cross-sectionally, the spatial WM lesion distribution was compared using lesion probability maps (LPMs). Longitudinally, WM lesions and GM volumes were studied using FSL-VBM and SPM5-VBM, respectively. WM lesions clustered around the lateral ventricles and in the centrum semiovale with a more widespread pattern in the PLV than in the NPLV group. The maximum local probabilities were similar in both groups and higher for T2 lesions (PLV: 27%, NPLV: 25%) than for T1 lesions (PLV: 15%, NPLV 14%). Significant WM lesion changes accompanied by cortical GM volume reductions occurred in the corpus callosum and optic radiations (P = 0.01 corrected), and more liberally tested (uncorrected P &lt; 0.01) in the inferior fronto-occipital and longitudinal fasciculi, and corona radiata in the PLV group. Not any WM or GM changes were found in the NPLV group. In the PLV group, WM lesion distribution and development in fibres, was associated with regional GM volume loss. The different spatiotemporal distribution patterns of patients with progressive compared to patients with non-progressive WM lesions suggest differences in the dynamics of pathogenesis