Ecto-5'-nucleotidase CD73 modulates the innate immune response to influenza infection but is not required for development of influenza-induced acute lung injury

Abstract

Extracellular nucleotides and nucleosides are important signaling molecules in the lung. Nucleotide and nucleoside concentrations in alveolar lining fluid are controlled by a complex network of surface ectonucleotidases. Previously, we demonstrated that influenza A/WSN/33 (H1N1) virus resulted in increased levels of the nucleotide ATP and the nucleoside adenosine in bronchoalveolar lavage fluid (BALF) of wild-type (WT) C57BL/6 mice. Influenza-induced acute lung injury (ALI) was highly attenuated in A1-adenosine receptor-knockout mice. Because AMP hydrolysis by the ecto-5'-nucleotidase (CD73) plays a central role in and is rate-limiting for generation of adenosine in the normal lung, we hypothesized that ALI would be attenuated in C57BL/6-congenic CD73-knockout (CD73-KO) mice. Infection-induced hypoxemia, bradycardia, viral replication, and bronchoconstriction were moderately increased in CD73-KO mice relative to WT controls. However, postinfection weight loss, pulmonary edema, and parenchymal dysfunction were not altered. Treatment of WT mice with the CD73 inhibitor 5'-(α,β-methylene) diphosphate (APCP) also had no effect on infection-induced pulmonary edema but modestly attenuated hypoxemia. BALF from CD73-KO and APCP-treated WT mice contained more IL-6 and CXCL-10/IFN-γ-induced protein 10, less CXCL-1/keratinocyte chemoattractant, and fewer neutrophils than BALF from untreated WT controls. BALF from APCP-treated WT mice also contained fewer alveolar macrophages and more transforming growth factor-β than BALF from untreated WT mice. These results indicate that CD73 is not necessary for development of ALI following influenza A virus infection and suggest that tissue-nonspecific alkaline phosphatase may be responsible for increased adenosine generation in the infected lung. However, they do suggest that CD73 has a previously unrecognized immunomodulatory role in influenza.

Keywords: CD73; acute lung injury; adenosine; influenza; mouse.

Fig. 1.

Influenza A/WSN/33 (H1N1) virus infection reduces cd73 gene expression, but not CD73 enzymatic activity, in wild-type (WT) mouse lung. A and B: effect of intranasal infection of WT C57BL/6 mice with H1N1 influenza A/WSN/33 (10,000 plaque-forming units/mouse) on whole lung homogenate ecto-5′-nucleotidase (cd73) gene expression (fold change relative to uninfected mice, n = 3 per time point) and whole lung homogenate CD73 activity (nmol P_i·mg protein⁻¹·min⁻¹, n = 8 per time point). *P < 0.05 vs. uninfected WT mice. Values are means ± SE.

Fig. 2.

Cardiopulmonary dysfunction and viral replication are not significantly altered in influenza-infected C57BL/6-congenic CD73-knockout (CD73-KO) mice. A–C: effect of infection of CD73-KO mice with H1N1 influenza A/WSN/33 virus on body weight (BWT, %change from day 0, n > 15 per mouse strain), carotid arterial O₂ saturation (Sa_O2, n > 15 per mouse strain), and heart rate (beats/min, n > 15 per mouse strain) at 0–6 days postinfection (dpi). D: effect on viral titers in lung homogenates at 6 dpi (n = 8–9 per mouse strain). #P < 0.001 vs. WT mice. Values are means ± SE.

Fig. 3.

CD73-KO mice are not protected from pulmonary edema following influenza infection. Effects of H1N1 influenza A/WSN/33 infection for 6 days on lung water content (wet-to-dry weight ratio) in WT controls and CD73-KO mice (n = 11 per mouse strain). Values are means ± SE.

Fig. 4.

Detrimental effects of influenza infection on pulmonary compliance and resistance are not attenuated in CD73-KO mice. A–D: effects of H1N1 influenza A/WSN/33 infection for 6 days on static lung compliance (C_ST, ml/cmH₂O × 10), dynamic lung compliance (C_DYN, ml/cmH₂O × 10), baseline total lung resistance (R_BASAL, cmH₂O·s·ml⁻¹), and maximal lung resistance following nebulization of 50 mg/ml methacholine (R_MAX, cmH₂O·s·ml⁻¹). #P < 0.001 vs. WT at the same time point. Values are means ± SE (n = 15 per mouse strain).

Fig. 5.

Neutrophil counts and protein levels are reduced in bronchoalveolar lavage fluid (BALF) from influenza A virus-infected CD73-KO mice. A–D: effects of H1N1 influenza A/WSN/33 infection for 6 days on BALF alveolar macrophages (AMs, n = 7–9 per mouse strain), BALF neutrophils (PMNs, n = 7–9 per mouse strain), BALF lymphocytes (Lymphs, n = 7–9 per mouse strain), and BALF protein (μg/ml, n = 13 and 6 for WT and CD73-KO mice, respectively). *P < 0.05; #P < 0.001 vs. WT mice. Values are means ± SE.

Fig. 6.

Treatment of WT mice with the CD73 inhibitor adenosine 5'-(α,β-methylene) diphosphate (APCP) reduces influenza-induced hypoxemia and BALF leukocyte counts. A–F: effects of treatment of H1N1 influenza A/WSN/33-infected mice with 200 μl of saline or 200 μl of saline containing 20 mg/kg APCP by oral gavage (every other day) on body weight (%change from day 0, n > 15 per treatment group) and carotid arterial O₂ saturation (n > 15 per treatment group) at 0–6 dpi and lung water content (wet-to-dry weight ratio, n = 7–9 per treatment group), BALF alveolar macrophages and neutrophils (AMs and PMNs, n = 5–9 per treatment group), BALF adenosine (n = 3–5 per treatment group), and BALF protein (n = 3–5 per treatment group) at 6 dpi. UNTx, untreated C57BL/6 mice; saline-Tx, saline-treated C57BL/6 mice; APCP-Tx, APCP-treated C57BL/6 mice. *P < 0.05 vs. UNTx. Values are means ± SE.

Fig. 7.

Tissue-nonspecific alkaline phosphatase (TNAP) expression and activity is not higher in CD73-KO mouse lung. A–D: effects of H1N1 influenza A/WSN/33 infection for 6 days on whole lung homogenate tnap gene expression (fold change relative to uninfected mice), whole lung homogenate TNAP and GAPDH protein expression by Western blot analysis (no adjustments were made to original images other than conversion from color to gray scale), whole lung homogenate TNAP protein expression by densitometry of Western blot bands (normalized to GAPDH), and whole lung homogenate TNAP enzymatic activity (U/l). Values are means ± SE (n = 3 per group).