The inflammatory chemokine Cxcl18b exerts neutrophil-specific chemotaxis via the promiscuous chemokine receptor Cxcr2 in zebrafish

Cxcl18b is a chemokine found in zebrafish and in other piscine and amphibian species. Cxcl18b is a reliable inflammatory marker; however, its function is yet to be elucidated. Here, we found that Cxcl18b is chemotactic towards neutrophils, similarly to Cxcl8a/Interleukin-8, the best characterised neutrophil chemoattractant in humans and teleosts. Like Cxcl8a, Cxcl18b-dependent recruitment required the chemokine receptor Cxcr2, while it was unaffected by depletion of the other two neutrophil receptors cxcr1 and cxcr4b. To visualise cxcl18b induction, we generated a Tg(cxcl18b:eGFP) reporter line. The transgene is induced locally upon bacterial infection with the fish pathogen Mycobacterium marinum, but strikingly is not directly expressed by infected cells. Instead, cxcl18b is induced by non-phagocytic uninfected cells that compose the stroma of the granulomas, typical inflammatory lesions formed upon mycobacterial infections. Together, these results suggest that Cxcl18b might be an important contributor to neutrophil chemotaxis in the inflammatory microenvironment and indicate that the zebrafish model could be explored to further investigate in vivo the biological relevance of different Cxcl8-like chemokine lineages

Rab27a and SMPD2 KD cells release less exosomes and show a loss in directional migration.

<p><b>(A)</b> Exosomes were purified from differentiated control (NS shRNA), Rab27a shRNA (sh1; sh3), or SMPD2 shRNA (sh2; sh4) KD cells after treatment with fMLP (2 nM, 30 min) and analyzed using a bead-based flow cytometry assay with CD63-FITC, CD81-PE, and CD11b-APC conjugated antibodies. Panels show quantitative analysis from three independent experiments as mean ± SD. Evaluation of exosome flow cytometry was done as described in the legend of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s004" target="_blank">S2A Fig</a>. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s006" target="_blank">S4B Fig</a>, for flow cytometry graphs. <b>(B–C)</b> Exosomes were purified from differentiated control and shRNA KD cells stimulated with 2 nM fMLP. The exosomes were lysed and their LTB₄ content measured by EIA. Results from four independent experiments are shown as mean ± SD in pg/ml/10⁸ cells (<b>B</b>) or pg/ml/μg of exosome protein (<b>C</b>). *** and NS indicate <i>p</i> < 0.0001 and <i>p</i> > 0.05, respectively, compared to corresponding control PLB-985 cells. <b>(D)</b> Differentiated control and shRNA KD cells were stimulated with subsaturating (1 nM) or saturating (1 μM) fMLP for 10 min, and the amount of LTB₄ in the supernatant was assessed by EIA. Results from three independent experiments are shown as mean ± SD. The symbols ** and *** indicate <i>p</i> < 0.001 and <i>p</i> < 0.0001, respectively, compared to corresponding NS shRNA controls. (<b>E)</b> EZ-Taxiscan chemotaxis towards 1 nM of control and KD cell lines. Corresponding migration speeds and CI were calculated from four different experiments and represented as mean ± SD. See legend of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.g004" target="_blank">Fig 4E</a> for details. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s017" target="_blank">S8 Movie</a>. (<b>F)</b> Differentiated NSshRNA, Rab27a, or SMPD2 KD cells or PLB-985 cells overexpressing LTB₄R1 were plated on fibronectin-coated plates for 10 min and uniformly stimulated uniformly with 1 nM fMLP. At specific time points, samples were subjected to western analyses using an antibody against phospho and total myosin light chain II (MLCII). Data are representative of three independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s007" target="_blank">S5C Fig</a>, for quantification.</p

Exosomes mediate the paracrine and autocrine effects of LTB₄.

<p><b>(A)</b> Cell tracks of neutrophils chemotaxing towards a ~50 pM/μm gradient of fMLP under agarose (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#sec010" target="_blank">Materials and Methods</a>). <b>(i)</b> DMSO-treated cells stained with cytotracker red. <b>(ii)</b> MK886-treated cells stained with cytotracker red. <b>(iii–iv)</b> MK886-treated cells stained with cytotracker red mixed with DMSO-treated cells stained with cytotracker green: <b>(iii)</b> shows the tracks of MK886-treated red cells in the mixture and <b>(iv)</b> shows tracks of DMSO-treated green cells in the mixture. Speed and CI were calculated from the tracks of 40 cells and averaging over six independent movies. The temporal location of cells in the X or Y direction is coded according to the color map shown. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s019" target="_blank">S10 Movie</a>. <b>(B)</b> Cell tracks of neutrophils chemotaxing towards fMLP as described in legends of A. In this case, neutrophils were treated with GW4869. Red cells were tracked in panels i, ii, and iii. In panel iv, DMSO-treated cells stained with cytotracker green were tracked. Speed and CI were calculated from the tracks of 40 cells and averaging over six independent movies. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s015" target="_blank">S6B Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s020" target="_blank">S11 Movie</a>. <b>(C)</b> Graphs depicting the change in the speed of control- (labeled blue) or inhibitor- (labeled red) treated cells as a function of time. The mean average change in the angular deviation (Δα) between two consequent tracks is presented in the bar graph. Data were calculated from the tracks of 40 cells and averaging over six independent movies. *** indicates <i>p</i> < 0.0001 compared to DMSO treated cells.</p

Exosomes Mediate LTB₄ Release during Neutrophil Chemotaxis

<div><p>Leukotriene B₄ (LTB₄) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB₄ and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB₄ receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB₄ release. Our findings establish that the exosomal pool of LTB₄ acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.</p></div

Exosomes activate neutrophils in an LTB₄-dependent manner.

<p><b>(A)</b> Exosomes were purified from differentiated mCherry-5LO or CD63-GFP cells pretreated with either DMSO or MK886 (1 μM, 30 min) and subsequently stimulated with 2 nM fMLP. The exosomes were lysed and their LTB₄ content measured by EIA. Results from four independent experiments are shown as mean ± SD. The symbols ***, **, *, and NS indicate <i>p</i> < 0.0001, <i>p</i> < 0.001, <i>p</i> < 0.01, and <i>p</i> > 0.05, respectively, compared to corresponding DMSO-treated controls. <b>(B)</b> Detection of CD63 on exosomes purified from differentiated mCherry-5LO or CD63-GFP cells that were pretreated with either DMSO or MK886 (1 μM, 30 min) and subsequently stimulated with 2 nM fMLP in a bead-based flow cytometry assay. Relative median fluorescence intensities obtained from four independent experiments are shown as mean ± SD. NS indicates statistical insignificance (<i>p</i> > 0.05) of the number of exosomes derived from MK886-treated cells compared to DMSO controls. <b>(C)</b> Neutrophil adhesion to fibronectin-coated plates was observed before and 15 min after the addition of 10 μg exosomes derived from mCherry-5LO cells. Differential interference contrast images are representative of three independent experiments. <b>(D)</b> LTB₄ (10 nM) or exosomes (50 μg/ml) isolated from mCherry or mCherry-5LO cells was added to the neutrophils for 15 min and pAkt S473 and p44/42 MAPK (Erk1/2) (T202/Y204) were determined by western analysis using specific antibodies. Data are representative of four independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s005" target="_blank">S3A Fig</a> for quantitation. <b>(E)</b> EZ-Taxiscan chemotaxis towards fMLP, LTB₄, exosomes derived from mCherry-5LO expressing cells, or control buffer. The images show paths of individual cells migrating as circles (from red to blue with increasing time) overlaid onto the final frame. Corresponding migration speeds and chemotaxis index (CI) were calculated from three different experiments and represented as mean ± SD. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s014" target="_blank">S5 Movie</a>. <b>(F)</b> Neutrophils were treated with DMSO or LY223982 (10 μM for 30 min) and allowed to migrate towards LTB₄ or exosomes derived from mCherry-5LO expressing cells. Corresponding migration speeds and CI are calculated from 3 different experiments and represented as mean ± SD. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s005" target="_blank">S3B Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s015" target="_blank">S6</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s016" target="_blank">S7</a> Movies. <b>(G)</b> Exosomes derived from mCherry, mCherry-5LO, or CD63-GFP expressing cells were divided into two parts. One part was used to measure LTB₄ levels to be added exogenously to neutrophils (control or LY223982 treated). The other part was added to neutrophils and pAkt (S473) levels were assessed after 15 min incubation. Data are representative of three independent experiments. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s005" target="_blank">S3C Fig</a> for quantification.</p

Exosomes mediate signal relay during neutrophil chemotaxis.

<p>Tracks, speed, and CI of mixtures of cytotracker-labeled cell lines migrating under-agarose towards a ~50 pM/μm gradient of fMLP. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s009" target="_blank">S7 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s021" target="_blank">S12 Movie</a>. <b>(A)</b> Differentiated cells overexpressing the LTB₄R1 (labeled with cytotracker green, shown as blue tracks) in a mixture with NS shRNA cells (labeled with cytotracker red, shown as red tracks) treated with DMSO (left) or CsH (right). Composite graph shows displacement tracks of both types of cells. Speed and CI were calculated from the tracks of 40 cells and averaging over 8 independent movies. <b>(B)</b> Differentiated cells overexpressing the LTB₄R1 (labeled with cytotracker green, shown as blue tracks) treated with CsH in a mixture with NS shRNA cells (labeled with cytotracker red, shown as red tracks) treated with DMSO (left) or MK866 (right). Composite graph shows displacement tracks of both types of cells. Speed and CI were calculated from the tracks of 40 cells and averaging over 4 independent movies. <b>(C)</b> Differentiated cells overexpressing the LTB₄R1 (labeled with cytotracker green, shown as blue tracks) in a mixture with Rab27a sh1 (labeled with cytotracker red, shown as red tracks) or SMPD2 sh2 (right) cells treated with DMSO. Composite graph shows displacement tracks of both types of cells. Speed and CI were calculated from the tracks of 40 cells and averaging over 4 independent movies. The symbols ***, **, *, and NS indicate <i>p</i> < 0.0001, <i>p</i> < 0.001, <i>p</i> < 0.01, and <i>p</i> > 0.05 respectively compared to CsH treated PLB-LTB₄R1 cells.</p

Ultrastructural analysis of the distribution of 5-LO in chemotaxing neutrophils.

<p>Representative immunogold EM images depicting the distribution of 5-LO in various cellular compartments in neutrophil chemotaxing towards fMLP. <b>(A)</b> Immunogold image stained with a normal rabbit IgG. <b>(B–E)</b> Immunogold images stained with a 5-LO-specific antibody. <b>(F)</b> Quantification of 5-LO positive staining in MVB, non-MVB vesicles, and mitochondria. <b>(G–H)</b> Progressive magnification of sections <b>(i through iii)</b> of neutrophils migrating towards fMLP showing formation and release of 5-LO-containing MVBs. <b>(I)</b> Fusion of 5-LO-containing MVB with plasma membrane and release of exosomes in cell synapse. Ne: nuclear envelope, Nu: nucleus, MVB: multivesicular body, ILV: intraluminal vesicles, Ex: exosome, PM: plasma membrane, CS: cell synapse.</p

Neutrophils release exosomes containing LTB₄ and LTB₄-synthesizing enzymes upon fMLP addition.

<p><b>(A)</b> Differentiated mCherry-5LO cells were plated on fibronectin-coated plates, and time-lapse images were captured before and after a uniform stimulation with 10 nM fMLP. Fluorescent images are representative of six independent experiments. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s010" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s011" target="_blank">S2</a> Movies. <b>(B)</b> Differentiated mCherry-5LO cells were allowed to chemotax under agarose towards an fMLP gradient. Time-lapse images were captured 30 min after the addition of fMLP, 700 μm away from the chemoattractant well. Gradient slope ~ 50 pM/μm (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#sec010" target="_blank">Materials and Methods</a>). Fluorescent images are representative of 20 independent experiments. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s012" target="_blank">S3 Movie</a>. <b>(C)</b> Differentiated CD63-GFP/mCherry-5LO coexpressing cells were allowed to chemotax as described in B. Fluorescent images are representative of eight independent experiments. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s003" target="_blank">S1 Fig</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s013" target="_blank">S4 Movie</a>. <b>(D)</b> Negatively stained EM images of pellets obtained from filtration and ultracentrifugation of supernatants of fMLP-stimulated neutrophils (upper panel) and after further purification on a discontinuous iodixanol gradient (lower panel). Vesicle sizes were determined from five vesicles from five fields from three independent experiments. <b>(E)</b> Equal amounts of protein from purified exosomes, crude ultracentrifugation pellets, and total cell lysates (10 μg) were subjected to western analysis using Calnexin, GRP94, CD81, and CD63 specific antibodies. Results are representative of three independent experiments. <b>(F)</b> Detection of CD63 (upper panel) or CD81 (lower panel) on exosomes obtained from fMLP-stimulated or dimethyl sulfoxide (DMSO)-treated neutrophils in a bead-based flow cytometry assay. Relative median fluorescence intensities obtained from three independent experiments are shown as mean ± standard deviation (SD). *** and NS (Not Significant) indicate <i>p</i> < 0.0001 and <i>p</i> > 0.05, respectively, compared to the isotype control. Also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002336#pbio.1002336.s004" target="_blank">S2 Fig</a>. <b>(G)</b> Crude ultracentrifugation pellets were loaded on a discontinuous iodixanol gradient, total protein from each fraction precipitated and subjected to western analysis using antibodies for the exosomal marker HSC70 or LTB₄ synthesizing enzymes. 5% of the total input was analyzed. Results are representative of three independent experiments. <b>(H)</b> Representative immunogold EM images of exosomes from stimulated neutrophils stained with CD63 or 5-LO antibodies. <b>(I)</b> Purified exosomes from fMLP-stimulated neutrophils treated with DMSO or MK886 (1 μM, 30 min) were lysed and their LTB₄ content measured using EIA. Results from four independent experiments are shown as mean ± SD. *** and NS indicate <i>p</i> < 0.0001 and <i>p</i> > 0.05, respectively, compared to the LTB₄ content of exosomes purified from vehicle treated nonstimulated cells.</p

The Antiviral Factor SERINC5 Impairs the Expression of Non-Self-DNA

SERINC5 is a restriction factor that becomes incorporated into nascent retroviral particles, impairing their ability to infect target cells. In turn, retroviruses have evolved countermeasures against SERINC5. For instance, the primate lentiviruses (HIV and SIV) use Nef, Moloney Murine Leukemia Virus (MLV) uses GlycoGag, and Equine Infectious Anemia Virus (EIAV) uses S2 to remove SERINC5 from the plasma membrane, preventing its incorporation into progeny virions. Recent studies have shown that SERINC5 also restricts other viruses, such as Hepatitis B Virus (HBV) and Classical Swine Fever Virus (CSFV), although through a different mechanism, suggesting that SERINC5 can interfere with multiple stages of the virus life cycle. To investigate whether SERINC5 can also impact other steps of the replication cycle of HIV, the effects of SERINC5 on viral transcripts, proteins, and virus progeny size were studied. Here, we report that SERINC5 causes significant defects in HIV gene expression, which impacts virion production. While the underlying mechanism is still unknown, we found that the restriction occurs at the transcriptional level and similarly affects plasmid and non-integrated proviral DNA (ectopic or non-self-DNA). However, SERINC5 causes no defects in the expression of viral RNA, host genes, or proviral DNA that is integrated in the cellular genome. Hence, our findings reveal that SERINC5’s actions in host defense extend beyond blocking virus entry

Loss of tetherin antagonism by Nef impairs SIV replication during acute infection of rhesus macaques.

Most simian immunodeficiency viruses use Nef to counteract the tetherin proteins of their nonhuman primate hosts. Nef also downmodulates cell-surface CD4 and MHC class I (MHC I) molecules and enhances viral infectivity by counteracting SERINC5. We previously demonstrated that tetherin antagonism by SIV Nef is genetically separable from CD4- and MHC I-downmodulation. Here we show that disruption of tetherin antagonism by Nef impairs virus replication during acute SIV infection of rhesus macaques. A combination of mutations was introduced into the SIVmac239 genome resulting in three amino acid substitutions in Nef that impair tetherin antagonism, but not CD3-, CD4- or MHC I-downmodulation. Further characterization of this mutant (SIVmac239AAA) revealed that these changes also result in partial sensitivity to SERINC5. Separate groups of four rhesus macaques were infected with either wild-type SIVmac239 or SIVmac239AAA, and viral RNA loads in plasma and sequence changes in the viral genome were monitored. Viral loads were significantly lower during acute infection in animals infected with SIVmac239AAA than in animals infected with wild-type SIVmac239. Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post-infection that fully restored anti-tetherin activity and partially restored anti-SERINC5 activity. These observations reveal overlap in the residues of SIV Nef required for counteracting tetherin and SERINC5 and selective pressure to overcome these restriction factors in vivo