<i>Il1r1</i>-deficient mice are highly susceptible to <i>Aspergillus fumigatus</i> infection.

<p>Age-matched C57BL/6 or <i>Il1r1</i>-deficient mice were infected i.t. with 5×10⁷ CEA10 conidia and at indicated time-points, mice were euthanized, BALF collected, and lungs saved for histological analysis. <b>(A)</b> Formalin-fixed lungs were paraffin embedded, sectioned, and stained with H&E (top) or GMS (bottom) for analysis by microscopy. Representative lung sections from C57BL/6 and <i>Il1r1</i>-deficient mice infected with CEA10 for 48 h are shown using either the 4× (left) or 20× (right) objectives. <b>(B)</b><i>A. fumigatus</i> germination rates were assessed over the first 72 h of infection by microscopically counting both the number of conidia and number of germlings in GMS-stained section. <b>(C)</b> Survival of C57BL/6, <i>Pycard</i>^−/−, and <i>Il1r1^−/−</i> mice challenged i.t. with 1.5×10⁷<i>A. fumigatus</i> (CEA10) conidia was then monitored for survival over the first 96 h (Mantel-Cox log-rank test, p = 0.0002). Data are representative of 2 independent experiments at each time point consisting of at least 5 mice per group. <b>(D)</b> Total macrophage (left panel) and neutrophil (right panel) recruitment in the BALF was measured at 12, 24, and 48 h post-infection. Data are representative of at least 2 independent experiments at each time point consisting of 3–5 mice per group. Bar graphs show the group mean ± one SEM. Statistically significant differences were determined using Student’s t-test (*p < 0.05; **p < 0.01).</p

C57BL/6 mice treated with IL-1α neutralizing antibody were more susceptible to <i>Aspergillus fumigatus</i> infection.

<p>C57BL/6 mice treated with isotype control antibody or IL-1α neutralizing antibody were infected i.t. with 5×10⁷ CEA10 conidia. At the indicated time points mice were euthanized, BALF collected and lungs saved for histological analysis. <b>(A)</b> Formalin-fixed lungs were paraffin embedded, sectioned and stained with H&E (top) or GMS (bottom) for analysis by microscopy. Representative lung sections from C57BL/6 mice treated with isotype control antibody (left) or with anti-IL-1α antibody (right) and infected with CEA10 for 48 h are shown using either the 4× (left) or 20× (right) objectives. <b>(B)</b><i>A. fumigatus</i> germination rates at 48 h after challenge was determined by microscopically counting both the number of conidia and number of germlings in GMS-stained section. <b>(C)</b> Total macrophage (left panel) and neutrophil (right panel) recruitment in the BALF was measured at 24 and 48 h post-infection via cytospins. Data are representative of two independent experiments consisting of 4–5 mice per group. Bar graphs show the group mean ± one SEM. Statistically significant differences were determined using a Student’s t-test (*p < 0.05, ***p < 0.001).</p

CCR2⁺ monocyte regulate early IL-1α and CXCL1 expression.

<p>C57BL/6 or CCR2-depleter mice were treated i.p. with 250 ng of DT 24 h prior to challenge with 5×10⁷ Af293 conidia. <b>(A)</b> Naïve C57BL/6 or CCR2-depleter mice or C57BL/6 or CCR2-depleter mice challenged eight hours prior were euthanized and the BALF and lung tissue collected for flow cytometric analysis to assess depletion of target cells by DT. Plots are gated on CD45⁺ CD11b⁺ cells and show Ly6c and Ly6g staining, which identify the CCR2⁺ monocytes and neutrophils, respectively. <b>(B)</b> IL-1α and <b>(C)</b> CXCL1 protein levels in the lung parenchyma at 8 h post-challenge with 5×10⁷ conidia of <i>A. fumigatus</i> strain Af293 were measured using ELISA assays. Bar graphs show the group means ± one SEM. <b>(D)</b> Eight hours post-challenge with 5×10⁷ conidia of <i>A. fumigatus</i> strain Af293, neutrophils in the BALF were enumerated. Data are representative <b>(B-C)</b> or pooled <b>(D)</b> from two independent experiments consisting of 4 mice per group. Each symbol represents an individual mouse and the line represents the group mean. Statistically significant differences were determined using a one-way ANOVA with Bonferroni’s post-test compared C57BL/6 mice (*p < 0.05, **p < 0.01).</p

IL-1α signaling enhances expression of leukocyte recruiting chemokines.

<p>C57BL/6 mice treated with either isotype control antibody or IL-1α neutralizing antibody, <i>Il1r1</i>-deficient and <i>Pycard</i>-deficient mice were infected with 5×10⁷ CEA10 conidia and at 24 hours post-infection, mice were euthanized, BALF collected, and lung tissue homogenized. Cytokine and chemokine levels in the lung homogenates were measured using 12-plex multiplex Luminex assay, similar trends were observed in BALF. Data are representative of two independent experiments consisting of 4–5 mice per group. Bar graphs show the group mean ± one SEM. Statistically significant differences were determined using a Kruskal-Wallis one-way ANOVA with Dunn’s post-test (*p < 0.05, **p < 0.01).</p

Treatment of <i>Il1r1</i>-deficient mice with CXCL1 partially increases resistance to <i>Aspergillus fumigatus</i> infection.

<p>C57BL/6 mice and <i>Il1r1</i>-deficient mice were challenged i.t. with 5×10⁷ CEA10 conidia. Three hours post-challenge mice were given 0.5 μg CXCL1 i.t. or PBS alone. Twenty-four hours post-infection, mice were euthanized, BALF collected, and lungs saved for histological analysis. <b>(A)</b> Formalin-fixed lungs were paraffin embedded, sectioned and stained with H&E (top) or GMS (bottom) for analysis by microscopy. Representative lung sections from <i>Il1r1</i>-deficient mice challenged with CEA10 for 48 h and treated with either PBS or CXCL1 are shown using either the 4× (left) or 20× (right) objectives. <b>(B)</b><i>A. fumigatus</i> germination rates were assessed at 48 h of infection by microscopically counting both the number of conidia and number of germlings in GMS-stained section. Number of conidia and number of germlings were counted for each GMS-stained section to quantify the percent germination. <b>(C)</b> Macrophage and neutrophil recruitment in <i>Il1r1</i>-deficient mice 24 h post-challenge infected with <i>A. fumigatus</i> treated with PBS or CXCL1 given i.t. was determined via cytospins.<b>(D)</b> Bone marrow neutrophils from C57BL/6 and <i>Il1r1</i>-deficient mice were incubated with CEA10 germlings <i>in vitro</i> at a 10:1 ratio in normoxia for 2 h. The XTT assay was used to determine percent fungal damage. <b>(E)</b> Lung damage and <b>(F)</b> leakage were assessed by measuring LDH and albumin, respectively. Data is representative of at least two independent experiments consisting of three to five mice per group, except for the bone marrow neutrophil anti-hyphal XTT assay which is a single experiment which consisted of pooled bone marrow neutrophils from three mice done in triplicate. Each symbol represents an individual mouse or replicate and the line represents the group mean. Statistically significant differences were determined using a one-way ANOVA with Bonferroni’s post-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant).</p

C57BL/6 mice show differential expression of IL-1α and IL-1ß after <i>A. fumigatus</i> infection.

<p>Mice were infected i.t. with 5×10⁷ CEA10 conidia and at indicated time-points, mice were euthanized, bronchoalveolar lavage fluid (BALF) collected, and lung tissue homogenized. IL-1β <b>(A)</b>, IL-18 <b>(B)</b>, IL-1α <b>(C)</b>, and IL-1Ra <b>(D)</b> levels in lung homogenate (IL-1α) and BALF (IL-1β, IL-18, and IL-1Ra) were measured using ProcartaPlex Mouse Cytokine & Chemokine 36-plex Immunoassay or ELISA. Data are representative of four mice per time point and two independent experiments. Each dot represents the mean ± one SEM.</p