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

Enantiomer-Specific Binding of Ruthenium(II) Molecular Wires by the Amine Oxidase of Arthrobacter globiformis

The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire−AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active (“off-Cu”) conformation, and the side chain of the so-called “gate” residue Tyr296 is consistently in the “gate-open” conformation. Among the wires tested, the most stable complex is produced when the wire has a −(CH_2)_4− linker. In this complex, the Ru(II)(phen)(bpy)_2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Λ and Δ isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases

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

Host-Derived Leukotriene B4 Is Critical for Resistance against Invasive Pulmonary Aspergillosis

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how immune competent hosts maintain control of fungal infections while constantly being exposed to fungi is rapidly emerging. It is known that timely neutrophil recruitment to and activation in the lungs is critical to the host defense against development of invasive pulmonary aspergillosis, but the inflammatory sequelae necessary remains to be fully defined. Here, we show that 5-Lipoxygenase (5-LO) and Leukotriene B4 (LTB4) are critical for leukocyte recruitment and resistance to pulmonary A. fumigatus challenge in a fungal-strain-dependent manner. 5-LO activity was needed in radiosensitive cells for an optimal anti-fungal response and in vivo LTB4 production was at least partially dependent on myeloid-derived hypoxia inducible factor-1α. Overall, this study reveals a role for host-derived leukotriene synthesis in innate immunity to A. fumigatus

<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