DataSheet_1_Psychological stress creates an immune suppressive environment in the lung that increases susceptibility of aged mice to Mycobacterium tuberculosis infection.docx

Age is a major risk factor for chronic infections, including tuberculosis (TB). Elderly TB patients also suffer from elevated levels of psychological stress. It is not clear how psychological stress impacts immune response to Mycobacterium tuberculosis (M.tb). In this study, we used social disruption stress (SDR) to investigate effects of psychological stress in young and old mice. Unexpectedly, we found that SDR suppresses lung inflammation in old mice as evidenced by lower pro-inflammatory cytokine levels in bronchial lavage fluid and decreased cytokine mRNA expression by alveolar macrophages. To investigate effects of stress on M.tb infection, mice were subjected to SDR and then infected with M.tb. As previously reported, old mice were better at controlling infection at 30 days than young mice. This control was transient as CFUs at 60 days were higher in old control mice compared to young mice. Consistently, SDR significantly increased M.tb growth at 60 days in old mice compared to young mice. In addition, SDR in old mice resulted in accumulation of IL-10 mRNA and decreased IFN-γ mRNA at 60 days. Also, confocal microscopy of lung sections from old SDR mice showed increased number of CD4 T cells which express LAG3 and CD49b, markers of IL-10 secreting regulatory T cells. Further, we also demonstrated that CD4 T cells from old SDR mice express IL-10. Thus, we conclude that psychological stress in old mice prior to infection, increases differentiation of IL-10 secreting T cells, which over time results in loss of control of the infection.</p

Design of mannosylated oral amphotericin B nanoformulation: efficacy and safety in visceral leishmaniasis

<p>The aim of this study was to evaluate mannose-anchored thiolated chitosan (MTC) based nanocarriers (NCs) for enhanced permeability, improved oral bioavailability and anti-parasitic potential of amphotericin B (AmB). Transgenic <i>Leishmania donovani</i> parasites expressing red fluorescent protein DsRed2 and imaging-flow cytometry was used to investigate parasitic burdens inside bone marrow-derived macrophages <i>ex vivo.</i> Cytokine estimation revealed that MTC nanocarriers activated the macrophages to impart an explicit immune response by higher production of TNF-α and IL-12 as compared to control. Cells treated with MTC NCs showed a significantly higher magnitude of nitrite and propidium iodide (PI) fluorescence intensity in contrast to cells treated with AmB. Concerning to apparent permeability coefficient (<i>P</i>_app) results, the MTC NCs formulation displayed more specific permeation across the Caco-2 cell monolayer as compared to AmB. The half-life of MTC NCs was about 3.3-fold persistent than oral AmB used as positive control. Also, t oral bioavailability of AmB was increased to 6.4-fold for MTC NCs compared to AmB for positive control. Acute oral evaluation indicated that MTC NCs were significantly less toxic compared to the AmB. Based on these findings, MTC NCs seems to be promising for significant oral absorption and improved oral bioavailability of AmB in leishmaniasis chemotherapy.</p

Pseudomonas aeruginosa pulmonary infection results in S100A8/A9-dependent cardiac dysfunction.

Pseudomonas aeruginosa (P.a.) infection accounts for nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that P.a. infection of the lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and mortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth

Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant <i>L. tarentolae</i> Vaccine against Experimental Murine Visceral Leishmaniasis

<div><p>Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans <i>Leishmania (L.) tarentolae</i> species as a live vaccine vector to deliver specific <i>Leishmania</i> antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against <i>L. infantum</i> infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant <i>L. tarentolae</i> expressing the <i>L. donovani</i> A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant <i>L. tarentolae</i> protects BALB/c mice against <i>L. infantum</i> challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant <i>L. tarentolae</i> as a safe live vaccine candidate against VL.</p> </div

Analysis of the specific humoral response induced in vaccinated and control mice before and after challenge.

<p>Mice were bled after two vaccinations before challenge and 5 weeks after challenge. Sera were obtained from individual mice from each group and pooled (n = 15). G1 to G5 groups are as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002174#pntd-0002174-g002" target="_blank">Figure 2</a>. Before challenge sera were tested for anti rA2-rCPA-rCPB (A) and F/T <i>L. tarentolae</i> A2-CPA-CPB^-CTE-EGFP (B) and after challenge sera were tested for anti F/T <i>L. infantum</i> (C) antibodies by an isotype-specific ELISA. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test (<i>p</i><0.05 denoted as *, <i>p</i><0.01 denoted as **, <i>p</i><0.001 denoted as *** and n.s. denoted as non significant).</p

Liver and splenic parasite burden in all groups following immunization and infectious challenge with <i>L. infantum</i>.

<p>The parasite number in the liver (A, B) or spleen (D, E) was evaluated by Limiting Dilution Assay (LDA) at 2, 4, 8 and 12 weeks post-infection. The parasite burden in the liver was compared between groups G1 and G3, G4 (controls) (A) and between G2 and G3, G5 (controls) (B). Areas contained under the curves (AUC) obtained in A and B were compared between groups G1, G2 and their related controls groups G4 and G5, respectively (C). The parasite burden in spleen was compared between groups G1 and G3, G4 (controls) (D) and between G2 and G3, G5 (controls) (E). Areas contained under the curves (AUC) obtained in D and E were compared between groups G1, G2 and their related controls groups G4, G5, respectively (F). The number of two independent repeats is shown here as mean±S.E. of measures obtained from 4 mice of each group (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002174#s2" target="_blank">Materials and methods</a> for more detail about the groups). G1 [vaccinated with DNA A2-CPA-CPB^-CTE-cSLN (prime) and Live <i>L. tarentolae</i>-A2-CPA-CPB^-CTE (boost)]; G2 [vaccinated with Live <i>L. tarentolae</i>-A2-CPA-CPB^-CTE (prime) and Live <i>L. tarentolae</i>-A2-CPA-CPB^-CTE (boost)]; G3 (control PBS); G4 [(DNA vector alone (prime) and Live <i>L. tarentolae</i> wild type (boost)], and G5 [Live <i>L. tarentolae</i> wild type (prime) and Live <i>L. tarentolae</i> wild type (boost)]. Only significant differences are shown in the graphs. The asterisk sign (*) indicates the significant difference between G1 and G2 with their respective controls G4 and G5, respectively and the plus sign (+) indicates the significant difference between G1 and G2 with G3 control at the indicated time points as determined by Student's test (<i>p</i><0.05 denoted as *, <i>p</i><0.01 denoted as **and <i>p</i><0.001 denoted as ***).</p

Expression of the A2-CPA-CPB^-CTE-EGFP tri-fusion gene by <i>L.tarentolae</i>.

<p>(A) Expression of EGFP by recombinant <i>L. tarentolae</i>-EGFP promastigotes (left) and <i>L. tarentolae</i>-A2-CPA-CPB^-CTE-EGFP promastigotes (right) before and after glinting of fluorescence. (B) Percentage of the EGFP positive population in <i>L. tarentolae</i> transfected with either pLEXSY-EGFP (left) or pLEXSY-A2-CPA-CPB^-CTE-EGFP (clone #5, right) as determined by flow cytometry. (C) Western blot analysis for evaluating expression of the A2-CPA-CPB^-CTE-EGFP fusion protein. A 102.56 kDa band corresponding to the A2-CPA-CPB^-CTE-EGFP protein was detected in the recombinant <i>L. tar</i>-A2-CPA-CPB^-CTE-EGFP by western blotting using an anti-CPB antibody. No band was seen in lane 1 representing a negative control (<i>L. tarentolae</i> wild type).</p

Cytokine production by splenocytes in vaccinated and control mice.

<p>IFN-γ (panel A), IL-10 (panel B), IFNγ/IL-10 ratio (panel C), IL-2 (panel D) and nitrite (NO₂) (panel E) production after stimulation with rA2-rCPA-rCPB (column 1), F/T <i>L. tarentolae</i> A2-CPA-CPB^-CTE-EGFP (column 2) and F/T <i>L. infantum</i> WT (column 3) as recall antigens. G1 to G5 groups are as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002174#pntd-0002174-g002" target="_blank">Figure 2</a>. Each bar represents a mean±S.E. in pg/ml (for all cytokines) or in µm for nitrite. The number of independent repeats was two and all tests were done in triplicates (number of mice per group/time point n = 3) and the results are pooled and shown as mean±S.E. of measures obtained from 6 mice in different groups. The asterisk indicates the significant difference between values at the indicated time points as determined by Student's test (<i>p</i><0.05 denoted as *, <i>p</i><0.01 denoted as **, <i>p</i><0.001 denoted as *** and n.s. denoted as non significant).</p