Proteomic signature reveals modulation of human macrophage polarization and functions under differing environmental oxygen conditions: Oxygen tension modulates macrophage polarization

International audienceMacrophages are innate immune cells which can react to a large number of environmental stimuli thanks to a high degree of plasticity. These cells are involved in a variety of tissue functions in homeostasis, and they play essential roles in pathological contexts. Macrophages’ activation state, which determines their functional orientation, is strongly influenced by the cellular environment. A large body of macrophage literature is devoted to better defining polarizations from a molecular viewpoint. It is now accepted that a multidimensional model of polarization is needed to grasp the broad phenotype repertoire controlled by environmental signals. The study presented here aimed, among other goals, to provide a molecular signature of various polarizations in human macrophages at the protein level so as to better define the different macrophage activation states. To study the proteome in human monocyte-derived macrophages as a function of their polarization state, we used a label-free quantification approach on in-gel fractionated and LysC/Trypsin digested proteins. In total, 5102 proteins were identified and quantified for all polarization states. New polarization-specific markers were identified and validated. Because oxygen tension is an important environmental parameter in tissues, we explored how environmental oxygen tension, at either atmospheric composition (18.6% O2) or “tissue normoxia” (3% O2), affected our classification of macrophage polarization. The comparative results revealed new polarization-specific makers which suggest that environmental oxygen levels should be taken into account when characterizing macrophage activation states. The proteomic screen revealed various polarization-specific proteins and oxygen sensors in human macrophages. One example is arachidonate 15-lipoxygenase (ALOX15), an IL4/IL13 polarization-specific protein, which was upregulated under low oxygen conditions and is associated with an increase in the rate of phagocytosis of apoptotic cells. These results illustrate the need to consider physicochemical parameters like oxygen level when studying macrophage polarization, so as to correctly assess their functions in tissue.&nbsp

Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6.

International audienceThe most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed

Unpacking Pandora's Box: Innovative Techniques for Effectively Counseling Asylum Applicants Suffering From Post-Traumatic Stress Disorder

46 p. ; This article was previously published in v.4 no.2 of the Hastings Race and Poverty Law Journal.Many asylum seekers are denied relief in the United States because the asylum officer or immigration judge evaluating the case does not believe the asylum applicant is telling the truth. The applicant may appear not to be credible because his story of persecution lacks sufficient detail and is inconsistent. A victim of severe and often prolonged trauma may develop post-traumatic stress disorder (PTSD), which profoundly affects his ability to tell a consistent and detailed story of past persecution. Thus, an asylum seeker suffering from PTSD as a result of traumatic experiences, desperately in need of a safe haven, may be denied asylum due to the symptoms of his affliction. This article is timely in light of recent changes in immigration law that have considerably raised both evidentiary requirements and the standard for obtaining asylum. These changes create an asylum process that poses significant obstacles for asylum seekers and dramatically reduces their chances of being granted asylum. This article proposes practical methods for use throughout the lawyer-client relationship in order to help an asylum seeker credibly tell his story of past persecution. This article incorporates recent scientific research on PTSD and its affect on memory in recommending the counseling techniques in this article. These techniques may assist an asylum seeker in consistently recalling the details of past persecution that establish a well-founded fear of returning to his home country

Virulence of single and double <i>GRA</i> knockout strains in CD1 mice.

<p>One hundred tachyzoites of the parental (Δ<i>ku80</i>) or knockout (Δ<i>gra)</i> strains were injected intraperitoneally (i.p.) into 8 week old female CD1 mice and survival was followed for 30 days (n = 8 for Δ<i>gra3</i>, Δ<i>gra4</i>, Δ<i>gra7</i>, Δ<i>gra9</i>, Δ<i>gra3</i>Δ<i>gra5</i> and Δ<i>gra3</i>Δ<i>gra7</i>, n = 4 for Δ<i>gra2</i>, Δ<i>gra5</i>, Δ<i>gra6</i>, Δ<i>gra8</i>, Δ<i>gra2</i>Δ<i>gra4</i>, Δ<i>gra2</i>Δ<i>gra6</i>, and Δ<i>gra4</i>Δ<i>gra6</i>). A) Survival of mice infected with parasites containing single <i>GRA</i> knockouts for IVN-localized GRA proteins. B) Survival of mice infected with parasites containing single <i>GRA</i> knockouts for PVM-localized GRA proteins. C) Survival of mice infected with parasites containing double <i>GRA</i> knockouts. Significance was determined by a Student’s t-test comparing the knockout strain with the parental.</p

Intracellular replication rate of single Δ<i>gra</i> knockout parasite strains.

<p>Intracellular replication rate of single Δ<i>gra</i> knockout parasite strains.</p

Indirect immunofluorescence assay verifies deletion of GRA proteins.

<p>IFA validation of the deletion of GRA2-9. HFFs were infected overnight with Δ<i>gra</i> knockout strains or with the parental strain. Infected cells were fixed, permeabilized with 0.002% saponin and incubated with the appropriate primary antibodies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159306#sec002" target="_blank">Material and Methods</a>), followed by Alexa 488-coupled goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies. After labeling both the host- and parasite nuclei were revealed with Hoechst 33342; coverslips were mounted in Mowiol and observed by epifluorescence. To distinguish the shape of ∆gra mutants’ vacuoles in the absence of fluorescent signal, we chose to artificially increase their exposure time, which may lead to some artificial background noise. Scales: 5 μm.</p

PV formation by Δ<i>gra2-9</i> single knockout strains in normal and lipid-free media.

<p>HFFs were pre-equilibrated in lipid-free D10 media (lfD10), then infected overnight with a single Δ<i>gra</i> knockout parasite strain or the Δ<i>ku80</i> parental strain. All parasite strains were passaged 3 times in lfD10 medium prior to infecting HFFs. Infected cells were fixed, permeabilized with 0.002% saponin, incubated with rabbit serum anti-GRA6 (all strains but Δ<i>gra6</i>) or with rabbit serum anti-GRA4 (Δ<i>gra6</i>) and with mAb anti-SAG1, visualized with goat anti-rabbit IgG coupled to Alexa 488 and goat anti-mouse IgG coupled to Alexa 594, mounted in Mowiol and observed by epifluorescence. To distinguish the shape of ∆gra mutants’ vacuoles in the absence of fluorescent signal, we chose to artificially increase their exposure time, which may lead to some background noise. Scales: 5 μm.</p

Strains used or developed in this study.

<p>Strains used or developed in this study.</p

Strategy for removal of the <i>HXGPRT</i> selectable marker from a <i>GRA</i> gene locus.

<p>A) Removal of the <i>HXGPRT</i> selectable marker from RHΔ<i>ku80</i>Δ<i>gra</i>::<i>HXGPRT</i>. This strategy is representative of each of the <i>HXGPRT</i> removal attempts. Following transfection with an <i>HXGPRT</i>-excised plasmid, clones lacking <i>HXGPRT</i> but containing the 5’ and 3’ flanking regions of the original <i>GRA</i> locus were generated by double-crossover homologous recombination, using 6TX selection. B) Validation of the <i>HXGPRT</i> removal from the <i>gra4</i> gene locus. The parental strain exhibits a PCR5 amplicon of ~ 2,300 bp, corresponding to a DNA fragment that includes the <i>HXGPRT</i> coding sequence. Clones 1–12 exhibit a PCR5 amplicon of ~ 1,100 bp, which corresponds to the removal of <i>HXGPRT</i>. M: DNA size ladder; C: control parental strain.</p

Construction and validation of the single <i>GRA2-9</i> knockout strains.

<p>A) Strategy for disruption of a <i>GRA</i> gene locus by double crossover homologous recombination in type I RHΔ<i>ku80</i>Δ<i>hxgprt</i> using a plasmid, pΔGRA, engineered to contain the 5’ and 3’ target flanks surrounding the <i>HXGPRT</i> selectable marker. Successful recombination events were selected by mycophenolic acid plus xanthine (MPA+X). This strategy is representative of each of the gene knockout attempts. B) Validation of the Δ<i>gra4</i> targeted gene deletion by PCR. The control parental strain is positive for PCR 1 (~ 380 bp) and PCR 2 (~ 670 bp) but negative for PCR 3 (~ 1,100 bp) and PCR4 (~ 1,200 bp). Clones 1–12 exhibit the banding pattern of a targeted Δ<i>gra4</i> gene knockout,. M: DNA size ladder. C) Western blot validation of the deletion of GRA2-9. Cell lysates from the equivalent of 2 x 10⁷ parasites were loaded per lane, separated on a 13% SDS-PAGE (non-reduced conditions), probed with primary antibodies (indicated on the side of the gels), then with goat anti-mouse IgG or goat anti-rabbit IgG, both coupled to peroxidase and visualized by chemiluminescence.</p