Infection-mediated priming of phagocytes protects against lethal secondary Aspergillus fumigatus challenge

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection

Reducing hypoxia and inflammation during invasive pulmonary aspergillosis by targeting the Interleukin-1 receptor

International audience1 Hypoxia as a result of pulmonary tissue damage due to unresolved inflammation during invasive pulmonary aspergillosis (IPA) is associated with a poor outcome. Aspergillus fumigatus can exploit the hypoxic microenvironment in the lung, but the inflammatory response required for fungal clearance can become severely disregulated as a result of hypoxia. Since severe inflammation can be detrimental to the host, we investigated whether targeting the interleukin IL-1 pathway could reduce inflammation and tissue hypoxia, improving the outcome of IPA. The interplay between hypoxia and inflammation was investigated by in vivo imaging of hypoxia and measurement of cytokines in the lungs in a model of corticosteroid immunocompromised and in Cxcr2 deficient mice. Severe hypoxia was observed following Aspergillus infection in both models and correlated with development of pulmonary inflammation and expression of hypoxia specific transcripts. Treatment with IL-1 receptor antagonist reduced hypoxia and slightly, but significantly reduced mortality in immunosuppressed mice, but was unable to reduce hypoxia in Cxcr2 −/− mice. Our data provides evidence that the inflammatory response during invasive pulmonary aspergillosis, and in particular the IL-1 axis, drives the development of hypoxia. Targeting the inflammatory IL-1 response could be used as a potential immunomodulatory therapy to improve the outcome of aspergillosis. Humans continuously inhale spores of the fungus Aspergillus fumigatus, which is a ubiquitous mould in soil and decaying organic debris. Although rarely causing disease in immunocompetent individuals, A. fumigatus can cause lethal invasive pulmonary aspergillosis (IPA) in immunocompromised patients, with mortality varying between 30% and 90

A sublethal infection primes bone marrow and blood phagocytes to increased Dectin-1 and CXCR2 expression.

<p>To investigate the priming process following the subethal infection, mice were either sacrificed at day 0 (Naïve) or at day 10 following infection with the sublethal dose (Primed). (A) CXCR2 expression on bone marrow and blood neutrophils (GR1^high CD11b⁺ population). Dectin-1 expression levels on bone marrow and blood neutrophils (GR1^high CD11b⁺ population) (B), and macrophages (F4/80⁺CD11b⁺GR-1^- population) (C). (D) Dectin-1 expression levels on BAL neutrophils and macrophages. Representative histograms show level of expression (left panel) and quantification of respective values in graphs (right panel). (E) Dynamic of Dectin-1 level expression on both neutrophils (left panel) and macrophages (right panel) in the BALs in the three infection settings. “SL” or “L” mice were sacrificed at 0 h (naïve), 12 18, 24, 48, 72 and 144 h p.i. and “Re-inf” mice were sacrificed at 0 h (10 days after the SL infection, day of the re-infection), 12, 18,24, 48, 72 and 144 h p.i. Data (mean ± SEM) represent 2 to 3 independent experiment with n = 5 mice per experiment. Statistically significant differences were determined using a Two way ANOVA with Bonferroni post-test (+p<0.05, ++p<0.01; *p<0.05, **p<0.01, ***p<0.001Re-Inf <i>vs</i> SL dose; #p<0.05, ##p<0.005 ###p<0.001 LL dose <i>vs</i> Re-Inf mice).</p

Protection correlates with early production of pro-inflammatory mediators.

<p>Mice were either infected at day 0 with a sublethal (SL) or a lethal (L) concentration of <i>A</i>. <i>fumigatus</i> conidia. Re-infected (Re-Inf) mice were first infected with a SL dose and 10 days later were challenged with the L dose. Mice were sacrificed at 0, 12, 18, 24, 48, 72, and 144 h p.i. The time point 0 h p.i. of the Re-Inf mice corresponds to the day 10 of the SL mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153829#pone.0153829.s001" target="_blank">S1 Fig</a>). The BAL and lungs tissue homogenates were were assessed by ELISA for quantification of (A) IL-1α, andIL-1ββ, (B) G-CSF, TNF α and IL-6, and (C) IL-17. Data (mean ± SEM) represent 2 to 3 independent experiments with n = 5 mice per experiment. Statistically significant differences were determined using a Two way ANOVA with Bonferroni post-test (+p<0.05, ++p<0.01, +++p<0.001 SL vs LL dose; *p<0.05, **p<0.01, ***p<0.001 Re-Inf vs SL dose; #p<0.05, ##p<0.01, ###p<0.001 LL dose vs Re-Inf mice).</p

Phagocytes from protected mice internalize and kill the conidia efficiently.

<p>Mice were either infected at day 0 with a sublethal (SL) or a lethal (L) concentration of <i>A</i>. <i>fumigatus</i> conidia. Re-infected (Re-Inf) mice were first infected with a SL dose and 10 days later were challenged with the L dose. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153829#pone.0153829.s001" target="_blank">S1 Fig</a>). (A) Representative pictures showing phagocytosis of conidia by BAL neutrophils (left panel) and macrophages (right panel) in the three infection settings at 48 h post infection. Mice were infected with FITC (green) labelled conidia, and the non-phagocytosed conidia were stained using an anti-conidia antibody detected by a secondary Texas Red-conjugated antibody (red). DNA was labelled with Hoechst stain (blue). Using fluorescence microscopy, the phagocytosed conidia were detected only in green while the outside FITC and Texas red conidia appear in yellow. Germination was indicated by the arrowheads pointing to non-phagocytozed and germinating conidia and to piercing hyphae (in red). The percentage of phagocytosis was estimated as the ratio of the number of ingested conidia to the total number of conidia bound to 100 phagocytes (B) Percentages of neutrophils (PMNs) and alveolar macrophages (AMs) involved in the phagocytosis in the lethal and re-infected groups at D1 and D2 h p.i. Within each histogram, is represented the percentage of either neutrophils or alveolar macrophages participating to the phagocytosis (C) BALs were collected and analyzed by flow cytometry to evaluate the killing potential. Plots are gated on GR1^high CD11b⁺ neutrophils and show expression level of the ROS (left panel) and MPO (right panel) production. Data (mean ± SEM) represent 2 to 3 independent experiments with 5 mice per experiment. Statistically significant differences were determined using a Two way ANOVA with Bonferroni post-test (+p<0.05, ++p<0.01, +++p<0.001 L dose <i>vs</i> SL dose; *p<0.05, **p<0.01, ***p<0.001 Re-Inf <i>vs</i> SL dose; #p<0.05, ###p<0.001 LL dose <i>vs</i> Re-Inf mice).</p

Mice recovering from sublethal <i>A</i>. <i>fumigatus</i> conidia inoculum are protected in a T-, B-lymphocytes or NK independent manner.

<p>Mice were either infected at day 0 with a sublethal (SL) or a lethal (L) concentration of <i>A</i>. <i>fumigatus</i> conidia. Re-infected (Re-Inf) mice were first infected with a SL dose and 10 days later were challenged with the L dose. The mice survival was followed on a daily basis during 15 days p.i. (A) <i>A</i>. <i>fumigatus-</i>emitted bioluminescence images from the thorax of representative BALB/c mice taken using the IVIS spectrum (Perkin) (B) Quantification (photons per second) of bioluminescence signal from mice chest areas using Living Image software (Perkin). (C) Modulation of the body weight in the three infection settings. (D) Colony forming unit from lungs homogenates. Mice were sacrificed from the “SL” group at day 1 and 10 and lung homogenates were collected and plated to determine the colony forming units (CFU). (E) Survival rate of BALB/c mice in the three infection settings. (F) Representative lung sections from mice at 48 h p.i. Left panels show Hemalun and right panel methenamine silver stained lung sections. Hemalun staining visualizes the inflammatory foci (purple), whereas silver staining visualizes fungal elements (black). (G) Survival rate of C57BL/6 mice in in the three conditions (H) Survival rate RAG^-/-γc^-/-mice in the three conditions. (****p = 0.0001 L dose <i>vs</i> SL dose; ####p<0.0001 L dose <i>vs</i> Re-Inf mice; n = 10 to 15 mice per group).</p