The level of H2 O2 -type oxidative stress regulates virulence of Theileria-transformed leukocytes

International audienceTheileria annulata infects predominantly macro-phages, and to a lesser extent B cells, and causes a widespread disease of cattle called tropical theileriosis. Disease-causing infected macro-phages are aggressively invasive, but this viru-lence trait can be attenuated by long-term culture. Attenuated macrophages are used as live vaccines against tropical theileriosis and via their charac-terization one gains insights into what host cell trait is altered concomitant with loss of virulence. We established that sporozoite infection of mono-cytes rapidly induces hif1-α transcription and that constitutive induction of HIF-1α in transformed leukocytes is parasite-dependent. In both infected macrophages and B cells induction of HIF-1α acti-vates transcription of its target genes that drive host cells to perform Warburg-like glycolysis. We propose that Theileria-infected leukocytes main-tain a HIF-1α-driven transcriptional programme typical of Warburg glycolysis in order to reduce as much as possible host cell H 2O2 type oxidative stress. However, in attenuated macrophages H2O2 production increases and HIF-1α levels conse-quently remained high, even though adhesion and aggressive invasiveness diminished. This indicates that Theileria infection generates a host leukocytes hypoxic response that if not properly controlled leads to loss of virulence

Un vaccin recombinant contre la theileriose tropicale des bovins à theileria annaluta

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Role of c-Jun-target genes in infected macrophage adhesion to fibronectin.

<p><b>A</b>, Infected macrophage adhesion is downregulated in the vaccine line (V-Delta 169) compared to virulent (V) Jed 4. RT-qPCR determined levels of genes (<i>cd69</i>, <i>icam-1</i>, <i>cd49</i> and <i>itgb5</i>) potentially involved in mediating adhesion. <b>B</b>, Virulent Jed 4 binds at high density to fibronectin compared to the V-Delta 169 vaccine line; 70% of fibronectin binding capacity is lost upon ablation of c-Jun.</p

Suppression of c-Jun leads to down-regulation of AP-1 activity.

<p><b>A</b>, Flag-tagged dominant-negative mutant of c-Jun, lacking the first 169 amino acid transactivation domain (Flag-Δ169) was introduced into <i>T. annulata</i>-infected macrophages. <b>B</b>, Suppression of c-Jun reduces AP-1 activity in <i>T. annulata</i>-infected macrophages, as measured by 3XTRE-driven luciferase activity (TRE-luc). The activity of AP-1 is 20 times lower in the vaccine line (V-Delta 169) compared to the control line (V). <b>C</b>, Western blot analysis of the expression of two different AP-1 family members in vaccine and control lines showing that expression of c-Jun is decreased and ATF2 increased in vaccine line. In spite of an increase in ATF2 protein the level of phospho-ATF2 (p-ATF2) is markedly reduced. Protein amounts were compared to actin and decreases in p-ATF2 and c-Jun quantified using Image J (C1 and C2).</p

Dissemination of Jed 4 macrophages in Rag2/γC mice.

<p><b>A (Virulent line) & B (Engineered attenuated line)</b>, The Ts4 micro-satellite locus was amplified from DNA extracted from the lungs (tracks 1–7), kidneys (tracks 8–11) and spleens (tracks 12–18) of 18 mice injected with virulent Jed 4 macrophages. Apart from a Ts4 specific band in the positive control no parasite DNA was amplified from extracts of lungs (tracks 1–8), kidneys (tracks 9–11) and spleens (tracks 12–18) of 18 mice injected with V-Delta 169 macrophages. (+) = Positive control DNA, (−) = negative control water, MW = molecular-weight size marker (200 bp ladder).</p

Infection of six cynomolgus macaques with SIVmac251 and establishment of chronic infection.

<p>(<b>A</b>) vRNA load in plasma. (<b>B</b>) Evolution of total CD4 T cell numbers in the blood over time (percentage of baseline). (<b>C</b>) vRNA in RB. (<b>D</b>) vRNA in PLNs. ULD = under the limit of detection. P values are given for non-parametric Wilcoxon rank sum test and was considered significant if p < 0.05.</p

SIV infection induces ISG expression during both phases of infection in PLNs.

<p>(<b>A</b>) Venn diagram showing the number of ISGs expressed at D9, M3, or both time points. (<b>B</b>) Comparison of mean ISG expression FC at D9 and M3 p.i. for the 51 ISGs expressed at both time points. (<b>C</b>) Venn diagram of ISGs expressed at D9 p.i., splitting them into those only inducible by type I IFN (Type I), only inducible by type II IFN (Type II), and those inducible by both IFN types (left) and comparison of their mean induction levels (right). (<b>D</b>) Venn diagram of ISGs expressed at M3 p.i., splitting them into those only inducible by type I IFN, those only inducible by type II IFN, and those inducible by both IFN types (left) and comparison of their induction levels (right). (<b>E</b>) Venn diagram of ISGs expressed at D9 p.i., splitting them into those displaying at least one ISRE, one GAS, or both sequence motifs in their promoters (left) and comparison of their induction levels (right). (<b>F</b>) Venn diagram of ISGs expressed at M3 p.i., splitting them into those displaying at least one ISRE, one GAS, or both in their promoters (left) and comparison of their induction levels (right). (<b>G</b>) Functional enrichment of type I-, type II-, and type I&II ISG subsets. The p value was calculated by the Fisher test with the Benjamini-Hochberg correction for multiple comparisons. The -log(p-value) is given for the most significant processes. In C, D, E, and F, the p value is given for Welch's unequal variances <i>t</i>-test, considering p < 0.05 to be significant.</p

Evolution of the ISG-associated functional signature during infection.

<p>Functional enrichment of ISGs differentially expressed at either (<b>A</b>) D9 p.i., (<b>B</b>) M3 p.i., or (<b>C</b>) both time points (D9&M3), was performed using IPA. The right-tailed Fisher Exact Test with the Benjamini-Hochberg correction for multiple tests was used to identify processes showing statistically significant over-representation of focus ISGs. Over-represented functional or pathway processes have more focus genes than expected by chance and the–log(p-values) of the most significant functions are plotted in the radar plot.</p

Heat-map signatures of differentially expressed ISGs.

<p>The differentially expressed genes of the ISG cluster with the most significant functions are represented as heat-maps. The color intensity shown at each time point is related to the FC in RNA expression: grey for not differentially expressed (not DEG), yellow for FC values between 1 and 2, orange for FC values between 2 and 5, and red for FC values above 5.</p

Transcriptome profiling reveals inter-tissue differences and the evolution of differential gene expression post-infection.

<p>Two critical time points, day 9 (D9) and month 3 (M3) p.i., were compared to baseline (before viral infection) (<b>A</b>) Number of genes with significant differential expression relative to baseline in RBs, PLNs, and PBMCs. A two-sample t-tests (p < 0.05) was used to identify differentially expressed genes (<b>B</b>) Multidimensional scaling (MDS) representation showing the segregation of biological samples based on the altered expression of the 1,638 genes with FC > 2. (<b>C</b>) Venn diagram showing the distribution of differentially expressed genes over time in RBs, PLNs, and PBMCs. (<b>D</b>) Evolution of RNA expression of interferons and interferon receptors in RBs, PLNs, and PBMCs at D9 and M3 p.i. Heatmap representing the FC from baseline for the six SIVmac251 infected macaques.</p