Dual role for CXCR3 and CCR5 in asthmatic type 1 inflammation

Abstract

Background: Many patients with severe asthma (SA) fail to respond to type 2 inflammation-targeted therapies. We previously identified a cohort of subjects with SA expressing type 1 inflammation manifesting with IFN-γ expression and variable type 2 responses.

Objective: We investigated the role of the chemotactic receptors C-X-C chemokine receptor 3 (CXCR3) and C-C chemokine receptor 5 (CCR5) in establishing type 1 inflammation in SA.

Methods: Bronchoalveolar lavage microarray data from the Severe Asthma Research Program I/II were analyzed for pathway expression and paired with clinical parameters. Wild-type, Cxcr3^-/-, and Ccr5^-/- mice were exposed to a type 1-high SA model with analysis of whole lung gene expression and histology. Wild-type and Cxcr3^-/- mice were treated with a US Food and Drug Administration-approved CCR5 inhibitor (maraviroc) with assessment of airway resistance, inflammatory cell recruitment by flow cytometry, whole lung gene expression, and histology.

Results: A cohort of subjects with increased IFN-γ expression showed higher asthma severity. IFN-γ expression was correlated with CXCR3 and CCR5 expression, but in Cxcr3^-/- and Ccr5^-/- mice type 1 inflammation was preserved in a murine SA model, most likely owing to compensation by the other pathway. Incorporation of maraviroc into the experimental model blunted airway hyperreactivity despite only mild effects on lung inflammation.

Conclusions: IFNG expression in asthmatic airways was strongly correlated with expression of both the chemokine receptors CXCR3 and CCR5. Although these pathways provide redundancy for establishing type 1 lung inflammation, inhibition of the CCL5/CCR5 pathway with maraviroc provided unique benefits in reducing airway hyperreactivity. Targeting this pathway may be a novel approach for improving lung function in individuals with type 1-high asthma.

Keywords: CCL5; CCR5; CXCL10; CXCL9; CXCR3; IFN-γ; maraviroc; severe asthma.

FIG 1:

IFN-γ is elevated in a sub-group of asthma patients with more severe disease. A Microarray data from SARP I/II bronchoalveolar lavage cell pellet RNA (BAL) was analyzed. IFNG expression in BAL is shown across asthma severity groups; while there was no statistical difference in median, a population of asthma patients with significantly elevated IFNG expression compared to healthy controls is noted; these patients were identified by a cutoff of two standard deviations above the healthy control median (y=7.467, dotted line). B After dividing the cohort into IFN-γ high and low, the prevalence of these groups was compared by asthma severity (Fisher’s Exact test). C BAL Cell differentials (% cell content) were compared for lymphocytes and neutrophils between IFN-γ High and Low groups; Mann-Whitney U test. D and E Daily use of oral corticosteroid (OCS) and the presence of frequent exacerbations (≥3 in the prior 12 months) were compared between IFN-γ High and Low Groups (Fisher’s Exact Test)

FIG 2:

There is a strong correlation between CXCR3 and CCR5 in asthma that suggests a dual chemotactic role. A Microarray data from SARP I/II bronchoalveolar lavage cell pellet RNA (BAL) was analyzed. CXCL10 and CCL5 expression in BAL was compared between IFN-γ High and Low groups (Mann-Whitney U test). B Expression of the Type 2 chemokine IL4 was elevated in IFNG high subjects, while no difference was observed in the eosinophilic cytokine IL5 or the Type 3 cytokine IL17A (Mann-Whitney U test). C Both CXCR3 and CCR5 correlate with IFNG suggesting a role in promoting Type 1 inflammation (Spearman Nonparametric Correlation). D There is strong correlation between both CCR5 and CXCR3 suggesting both pathways are upregulated in certain patients (Spearman Nonparametric Correlation).

FIG 3:

Cxcr3^−/− mice have a similar inflammatory profile and phenotype to C57Bl/6 mice in a murine severe asthma model. A Whole lung PCR compared cytokine expression between naïve and severe asthma model (SA) exposed WT and Cxcr3^−/− mice (one-way ANOVA with α<0.05 for each panel, Sidak’s post-hoc test for within treatment comparison shown; data representative of two independent experiments). B H&E staining of lung sections shows similar inflammation between treatment groups in both WT mice and Cxcr3^−/− mice (20x magnification, representative sections) C Whole lung PCR compared expression of the Cxcr3 and Ccr5 genes in WT and Cxcr3^−/− mice (Student’s T-test, data representative of 2 independent experiments).

FIG 4:

Ccr5^−/− mice also have a similar inflammatory profile and phenotype to WT mice in a murine severe asthma model. A Whole lung PCR compares cytokine expression between naïve and severe asthma model (SA) exposed WT mice and Ccr5^−/− mice (One-way ANOVA with α<0.05 is significant for each panel, Sidak’s post-hoc test for within treatment comparison; data are pooled from 2 independent experiments). B H&E staining of lung sections shows similar inflammation between treatment groups in both WT mice and Ccr5^−/− mice (20x magnification, representative sections). C Whole lung PCR compared expression of the Ccr5 and Cxcr3 genes in WT and Ccr5^−/− mice (Student’s T-test, data pooled from 2 independent experiments).

FIG 5:

Dual Receptor inhibition significantly reduces IFN-γ⁺ cell recruitment to the lungs in a severe asthma model. A Assessment of lung T-lymphocyte (CD3⁺CD4⁺) cell populations by ICS in Cxcr3^−/− mice shows no significant difference in T-cell subtype recruitment (Student’s T-test, data representative of 2 independent experiments). B WT mice were treated with maraviroc or no treatment with a non-significant trend to decrease in IFN-γ⁺ and IL-17a⁺ cells noted in maraviroc treated mice with no change in IL-13⁺ cells (Student’s T-test, data pooled from 2 independent experiments). C Cxcr3^−/− mice were treated with maraviroc or no treatment with significant reductions in IFN-γ⁺ and IL-17a⁺ cells with maraviroc treatment and no change in IL-13⁺ cells (Student’s T-test, data pooled from 2 independent experiments).

FIG 6:

Maraviroc effectively inhibits Airway hyperreactivity without modifying lung inflammation. A WT mice (WT, n = 7), Cxcr3^−/− mice (n=5) and Ccr5^−/− mice(n=6) were subjected to the SA model and underwent airway resistance testing with increasing concentrations of methacholine (data pooled from 2 separate experiments, Kruskall-Wallis testing). B WT mice were exposed to the SA model (Severe Asthma, n = 16) and a subset were treated with either maraviroc throughout the model beginning pre-sensitization (Maraviroc Base, n = 8) or post-sensitization but prior to challenges (Maraviroc Treat, n = 8) (data pooled from 4 experiments, Kruskall-Wallis testing with Dunn’s post hoc testing for intergroup comparisons). C WT and Maraviroc treated mice (Maraviroc) completed the full SA model and underwent assessment of whole lung cytokine expression by PCR with no significant differences observed. D Histology from WT and pre-challenge Maraviroc treated mice showed a modest improvement but residual inflammation in the maraviroc group (H&E staining, 20x magnification).