IL-1α Signaling Is Critical for Leukocyte Recruitment after Pulmonary Aspergillus fumigatus Challenge

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine lung

IL-1α Signaling Is Critical for Leukocyte Recruitment after Pulmonary Aspergillus fumigatus Challenge

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine lung

Enhancement of CD8⁺ T cell response by FGK4.5 treatment during VSV infection is dependent on CD70.

<p>C57BL/6 mice were left uninfected, or i.v. infected with 2×10³ cfu LM-ova, or with 2×10⁵ pfu VSV-ova alone, or 2×10⁵ pfu plus 100 µg FGK4.5 treated i.p. one day after infection, or 2×10⁵ pfu VSV-ova plus 100 µg FGK4.5 one day post infection and 300 µg FR70, a blocking CD70 mAb, on days −1, +1, and +3 relative to infection. The magnitude of the Ova/K^b-specific memory CD8⁺ T cell population in the (A) peripheral blood and in the (B) spleen was quantified 60 days post infection. (C) To assess protective immunity, memory mice were re-challenged with 5×10⁵ cfu LM-ova. Three days later bacterial burden was assessed in the spleen and liver. Each experiment contained 4–5 mice per group and data are representative of at least two independent experiments. Statistical significance was determined using a one-way ANOVA with a Bonferroni’s post-test (***p<0.001; **p<0.01; *p<0.05).</p

FGK4.5 treatment enhances CD8⁺ T cell responses induced by VSV infection.

<p>C57BL/6 mice were i.v. infected with 2×10⁵ pfu VSV-ova and treated i.p. with 100 µg FGK4.5, an agonistic CD40 mAb, one day post infection. (A) Sixty days after primary infection the Ova/K^b-specific CD8⁺ memory T cell population in the blood was quantified by Ova/K^b-tetramer staining. (B) To assess whether activating CD40 would induce protective immunity, vaccinated mice were re-challenged with 5×10⁵ cfu LM-ova sixty day after VSV-ova vaccination. Three days later spleens and liver were assessed for bacterial burden. Statistical significance was determined using a one-way ANOVA with a Bonferroni’s post-test (**p<0.01). Data are representative of at least three independent experiments.</p

Protective immunity induced by VSV is short lived.

<p>C57BL/6 mice were i.v infected with 2×10⁵ pfu VSV-ova or 2×10³ cfu LM-ova. (A) 180 days after primary infection mice were re-challenged with 5×10⁵ cfu LM-ova. Three days later livers and spleens (data not shown) were plated to assess bacterial burden. Statistical significance was determined using a one-way ANOVA with a Bonferroni’s post-test (**p<0.01). (B) Naïve C57BL/6 or memory mice infected 30, 60, 90, or 180 days previously with either VSV-ova or LM-ova were challenged with 5×10⁵ CFU of LM-ova. Three days post-challenge bacterial burden was quantified in the spleen and liver. The fold reduction of LM-ova burden at 72 h post-challenge in vaccinated versus naïve animals was calculated from representative time-point out to ∼6 months post-vaccination. This was done by dividing the LM burden in the naïve animals by those found in vaccination animals to get a fold-reduction in LM burden. Data are representative of at least two independent experiments at each time-point.</p

FGK4.5 treatment during VSV vaccination enhances the functional avidity and poly-functionality of secondary effector CD8⁺ T cells.

<p>C57BL/6 mice were i.v. infected with 2×10³ cfu LM-ova, or with 2×10⁵ pfu VSV-ova, or 2×10⁵ pfu VSV-ova plus treated i.p. with 100 µg FGK4.5 one day post infection. Ninety days after primary challenge mice were re-challenged with 5×10⁴ cfu LM-ova. Seven days later the sensitivity to antigen and functionality of the secondary effector CD8⁺ T cell response was assessed by intracellular cytokine staining. Spleen lymphocytes were stimulated with one to 0.00001 µg/mL of the SIINFEKL peptide. Cells were fixed and rendered permeable before staining with antibodies IFNγ, TNFα, and IL-2. We tested (A) the percent IFNγ and (B) the percent IFNγ over the maximum; at all peptide dilutions. Next we analyzed the (C) geometric mean fluorescence intensity (MFI) of the CD8⁺ T-cells for IFNγ, the (B) percent of IFNy expressing cells also expressing TNFα and (E) the IFNγ expressing cells also expressing IL-2. Results shown for 0.001 and 1 µg/mL SIINFEKL only (Figures C, D, E). Statistical significance was determined using a one-way ANOVA with a Bonferroni’s post-test ***p<0.001**p<0.01; *p<0.05). Each group contained 4–5 mice per group.</p

Memory CD8⁺ T cell induced by VSV and LM infection differ in their differentiation status.

<p>C57BL/6 mice were i.v infected with 2×10⁵ pfu VSV-ova or 2×10³ cfu LM-ova. Sixty days post-infection the cell surface phenotype (A) and transcription factor profile (B) of the Ova/K^b-specific CD8⁺ T cell memory populations was assessed by flow cytometry. (A–B) Analysis is on gated specifically on CD11a^high Ova/K^b-specific CD8⁺ T cells and the mean fluorescence intensity (MFI) of each transcription factors BLIMP1, T-bet, and Eomes from individual mice was graphed. Blue line represents LM-ova and orange line represents VSV-ova. (C) To test whether blocking the PD-1 receptor could induce protective immunity mice were treated with 300 µg of RMP1-14, a blocking mAb to PD-1, at the time of re-challenge. Mice were re-challenged with 5×10⁵ cfu LM-ova and three days later the bacterial burden in the liver and spleen was assessed. Statistical significance was determined using an unpaired Student’s t-test (*p<0.05; ***p<0.001). Data are representative of at least two independent experiments.</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

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