Therapeutic targeting of CCR1 attenuates established chronic fungal asthma in mice

Abstract

CC chemokine receptor 1 (CCR1) represents a promising target in chronic airway inflammation and remodeling due to fungus-associated allergic asthma. The present study addressed the therapeutic effect of a nonpeptide CCR1 antagonist, BX-471, in a model of chronic fungal asthma induced by Aspergillus fumigatus conidia. BX-471 treatment of isolated macrophages inhibited CCL22 and TNF-alpha and promoted IL-10 release. BX-471 also increased toll like receptor-9 (TLR9) and decreased TLR2 and TLR6 expression in these cells. When administered daily by intraperitoneal injection, from days 15 to 30 after the initiation of chronic fungal asthma, BX-471 (3, 10, or 30 mg kg(-1)) dose-dependently reduced airway inflammation, hyper-responsiveness, and remodeling at day 30 after conidia challenge. The maximal therapeutic effect was observed at the 10 mg kg(-1) dose. In summary, the therapeutic administration of BX-471 significantly attenuated experimental fungal asthma via its effects on both innate and adaptive immune processes.

Figure 1

Spontaneous CCL3, CCL6, CCL22 (top panel), TNF-α and IL-10 (bottom panel) generation by isolated macrophages from naïve mice. ELISA was used to determine specific chemokine and cytokine levels in cell-free supernatants removed at 24 h after culture. BX-471 (at 10 μM) or the vehicle (3% DMSO) for this CCR1 antagonist was included in these 24-h cultures. Data are mean±s.e.m. of triplicate or quadruplicate samples. ^*P⩽0.05, ^***P⩽0.001 compared with chemokine or cytokine levels detected in culture wells containing vehicle-treated macrophages. ^τP⩽0.05 compared with CCL22 levels detected in vehicle-treated macrophages.

Figure 2

Quantitative TAQMAN PCR analysis of TLR2, TLR6, and TLR9 transcript expression in vehicle- and BX-471-treated macrophages. The fold-increase in TLR transcript expression above transcript levels in untreated mouse macrophages is shown. Data are mean±s.e.m. of triplicate or quadruplicate samples.

Figure 3

Gomori methenamine silver (GMS) staining of whole lung samples from vehicle (a)- and BX-471-treated (3 mg kg⁻¹) (b), 10 mg kg⁻¹ (c) and 30 mg kg⁻¹ (d) groups at day 30 after conidia challenge. A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or BX-471 at one of the three doses indicated above on a daily basis until day 30. Whole lungs samples were processed used routine histological techniques. Mice that received 10 mg kg⁻¹ of BX-471 showed no evidence of fungal material (highlighted by black stained material in mononuclear cells) retention in contrast to the control and other BX-471 treatment groups. Original magnification was × 200.

Figure 4

Leukocyte counts in bronchoalveolar lavage (BAL) samples from vehicle- and BX-471-treated groups at day 30 after A. fumigatus conidia challenge into A. fumigatus-sensitized mice. A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or on a daily basis until day 30. BAL cells were dispersed onto microscope slides using a cytospin, and eosinophils, lymphocytes, neutrophils, and macrophages were differentially stained with Wright–Giesma stain. Values are expressed as mean±s.e.m. ^*P⩽0.05, ^***P⩽0.001 compared with vehicle-treated group.

Figure 5

Hematoxylin and eosin (H/E) staining of whole lung samples from vehicle (a)- and BX-471-treated (3 mg kg⁻¹) (b), 10 mg kg⁻¹ (c) and 30 mg kg⁻¹ (d) groups at day 30 after conidia challenge. A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or BX-471 at one of the three doses indicated above on a daily basis until day 30. Whole lungs samples were processed using routine histological techniques. Mice that received 10 mg kg⁻¹ of BX-471 showed the greatest reduction in peribronchial inflammation compared the control and other BX-471 treatment groups. Original magnification was × 200.

Figure 6

Airway hyper-responsiveness in vehicle- and BX-471-treated groups at day 30 after A. fumigatus conidia challenge into A. fumigatus-sensitized mice (top panel). A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or on a daily basis until day 30. Peak increases in airway resistance or hyper-responsiveness (units=cm H₂O⁻¹ ml⁻¹ s⁻¹) were determined at each time point after the intravenous injection of methacholine. Values are expressed as mean±s.e.m.; n=5/group/time point. ^*P⩽0.05, ^***P⩽0.001 compared with baseline airway responses measured in the appropriate vehicle- or BX-471-treated group. ^τττP⩽0.001 compared with the airway responses evoked by the 420 μg kg⁻¹ methacholine challenge in the vehicle-treated group. The percent inhibition of airway hyper-responsiveness in the BX-471 treatment groups relative to the vehicle control group is shown in the bottom panel.

Figure 7

Whole lung levels of C-C chemokine receptor-1 (CCR1) agonists CCL3 (a), CCL5 (b), and CCL6 (c) from vehicle (a)- and BX-471-treated (3 mg kg⁻¹) (b), 10 mg kg⁻¹ (c), and 30 mg kg⁻¹ (d) groups at day 30 after conidia challenge. A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or BX-471 at one of the three doses indicated above on a daily basis until day 30. CCL3, CCL5, and CCL6 were measured using a specific ELISA as described in the Methods section. Data are expressed as mean±s.e.m.; n=5 group⁻¹. ^*P⩽0.05, ^**P⩽0.01 compared with whole lung chemokine levels in the vehicle-treated group.

Figure 8

Periodic Acid Schiff (PAS) staining of whole lung samples from vehicle (a)- and BX-471-treated (3 mg kg⁻¹) (b), 10 mg kg⁻¹ (c), and 30 mg kg⁻¹ (d) groups at day 30 after conidia challenge. A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or BX-471 at one of the three doses indicated above on a daily basis until day 30. Whole lungs samples were processed using routine histological techniques. Mice that received 10 mg kg⁻¹ of BX-471 showed the greatest reduction in goblet cell metaplasia compared the control and other BX-471 treatment groups. Original magnification was × 200.

Figure 9

Trichrome Masson staining of whole lung samples from vehicle (a)- and BX-471-treated (3 mg kg⁻¹) (b), 10 mg kg⁻¹ (c), and 30 mg kg⁻¹ (d) groups at day 30 after conidia challenge. A. fumigatus-sensitized mice were challenged intratracheally with live A. fumigatus conidia and 15 days later groups of five mice received vehicle alone or BX-471 at one of the three doses indicated above on a daily basis until day 30. Whole lungs samples were processed using routine histological techniques. Mice that received 10 mg kg⁻¹ of BX-471 showed the greatest reduction in peribronchial fibrosis compared the control and other BX-471 treatment groups. Original magnification was × 200.