Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome

Abstract

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.

Keywords: acute respiratory distress syndrome; alveolar type II cell; influenza; lipidomics; liponucleotide; mouse; phospholipid; surfactant.

Fig. 1.

Influenza A/WSN/33 virus infection significantly alters levels of many surfactant lipid metabolites in mouse alveolar type II (ATII) cells. A: numbers of C57BL/6 mouse ATII cell metabolites significantly (P < 0.05) increased at 2 and/or 6 days after intranasal inoculation with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) relative to mock-infected controls at the same time points [2 and 6 days postinoculation (2 and 6 dpi)] (n = 48 total). B: numbers of ATII cell metabolites significantly decreased at 2 and/or 6 dpi relative to mock-infected controls at the same time points (n = 19 total). A total of 77 metabolites were analyzed. n = 6 per group, except mock-infected mice at 6 dpi (n = 5).

Fig. 2.

Influenza A virus infection decreases ATII cell levels of many major surfactant phospholipids. A–F: effect of mock infection or influenza A virus infection on ATII cell levels of 1,2-dipalmitoyl phosphatidylcholine [DPPC (16:0/16:0)], 1-palmitoyl,2-palmitoleoyl phosphatidylcholine [PPOPC (16:0/16:1)], 1-palmitoyl,2-linoleoyl phosphatidylcholine [PLPC (16:0/18:2)], 1,2-dipalmitoyl phosphatidylglycerol [DPPG (16:0/16:0)], 1-palmitoyl,2-linoleoyl phosphatidylethanolamine [PE (16:0/18:2)], and 1-palmitoyl,2-oleoyl phosphatidylglycerol [POPG (16:0/18:1)]. *P < 0.05, **P < 0.005, #P < 0.001 vs. mock-infected mice at the same time point. §P < 0.001 vs. influenza A virus-infected mice at 2 dpi. n = 6 per group, except mock-infected mice at 6 dpi (n = 5). Data in boxes represent 1st quartile, median, and 3rd quartile for each sample group; whiskers indicate highest and lowest sample values within the group.

Fig. 3.

ATII cells from influenza-infected mice contain increased amounts of several minor surfactant phospholipids and cholesterol. A–F: effect of mock infection or influenza A virus infection on ATII cell levels of 1-stearoyl,2-arachidonoyl phosphatidylserine [PS (18:0/20:4)], 1-palmitoyl,2-linoleoyl phosphatidylinositol [PLPI (16:0/18:2)], 1-stearoyl,2-linoleoyl phosphatidylinositol [SLPI (18:0/18:2)], sphingosine, sphingomyelin, and cholesterol. *P < 0.05, **P < 0.005, #P < 0.001 vs. mock-infected mice at the same time point. †P < 0.05, §P < 0.001 vs. influenza A virus-infected mice at 2 dpi. n = 6 per group, except mock-infected mice at 6 dpi (n = 5). Data in boxes represent 1st quartile, median, and 3rd quartile for each sample group; whiskers indicate highest and lowest sample values within the group.

Fig. 4.

De novo synthesis of DPPC by the CDP-choline pathway. DPPC (16:0/16:0) is synthesized via the CDP-choline (Kennedy) pathway. In this pathway, CTP-phosphocholine cytidylyltransferase-α (CCT-α) makes the liponucleotide cytidine 5′-diphosphate-choline (CDP-choline) from choline phosphate (choline-P) and cytidine 5′-triphosphate (CTP). This reaction, which is rate-limiting for DPPC synthesis, also generates inorganic pyrophosphate (PPi). 1,2-Diacylglycerol [DAG (16:0/16:0)] is synthesized from glycerol-3-phosphate (glycerol-3-P) by sequential acylation. Glycerol-3-P is first palmitoylated at the sn-1 position to form lysophosphatidic acid [LPA (16:0)] by acyl-CoA:glucose-3-P acyltransferase (GPAT). Acyl-CoA:LPA acyltransferase (LPAT) then catalyzes a second palmitoylation of LPA at the sn-2 position to generate phosphatidic acid [PA (16:0/16:0)]. Both reactions also yield CoA (not shown). Dephosphorylation of PA by PA phosphatase (PAP) generates DAG (16:0/16:0). The final reaction of CDP-choline with DAG (16:0/16:0) to yield DPPC (16:0/16:0), glycerol (not shown), and cytidine 5′-monophosphate (CMP; not shown) is catalyzed by choline phosphotransferase (CPT). Enzymes are shown in bold font. Gray circles, phosphate moiety.

Fig. 5.

Availability of CDP-conjugated liponucleotide precursors for de novo DPPC and PE synthesis is reduced in ATII cells isolated from influenza-infected mice. A–F: effect of mock infection or influenza A virus infection on ATII cell levels of glycerol-3-phosphate (glycerol-3-P), 1-oleoyl,3-linoleoyl diacylglycerol [DAG (18:1/18:2)], choline phosphate (choline-P), cytidine 5′-diphosphocholine (CDP-choline), phosphoethanolamine, and cytidine 5′-diphosphoethanolamine (CDP-ethanolamine). *P < 0.05, **P < 0.005, #P < 0.001 vs. mock-infected mice at the same time point. ‡P < 0.005, §P < 0.001 vs. influenza A virus-infected mice at 2 dpi. n = 6 per group, except mock-infected mice at 6 dpi (n = 5). Data in boxes represent 1st quartile, median, and 3rd quartile for each sample group; whiskers indicate highest and lowest sample values within the group.

Fig. 6.

Influenza infection results in abnormal ATII cell lamellar body morphology. A: effect of mock infection for 6 days on ATII cell lamellar body ultrastructure. B: effect of influenza A virus infection on ATII cell lamellar body ultrastructure at 6 dpi. Magnification ×10,000; scale bar = 5 μm. No adjustments of tone or scale were made to captured images.

Fig. 7.

Changes in the ATII cell surfactant lipidome are accompanied by changes in bronchoalveolar lavage fluid (BALF) lipid composition. A–F: effect of mock infection or influenza A virus infection on BALF phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), sphingomyelin (SM), cholesterol, and protein. #P < 0.001 vs. mock-infected mice at the same time point. §P < 0.001 vs. influenza A virus-infected mice at 2 dpi. n = 5 per group for mock-infected mice at 2 and 6 dpi; n = 6 per group for infected mice at 2 and 6 dpi. Data in boxes represent 1st quartile, median, and 3rd quartile for each sample group. Whiskers indicate highest and lowest sample values within the group.