Differential susceptibility of transgenic mice expressing human surfactant protein B genetic variants to Pseudomonas aeruginosa induced pneumonia

Abstract

Surfactant protein B (SP-B) is essential for lung function. Previous studies have indicated that a SP-B 1580C/T polymorphism (SNP rs1130866) was associated with lung diseases including pneumonia. The SNP causes an altered N-linked glycosylation modification at Asn129 of proSP-B, e.g. the C allele with this glycosylation site but not in the T allele. This study aimed to generate humanized SP-B transgenic mice carrying either SP-B C or T allele without a mouse SP-B background and then examine functional susceptibility to bacterial pneumonia in vivo. A total of 18 transgenic mouse founders were generated by the DNA microinjection method. These founders were back-crossed with SP-B KO mice to eliminate mouse SP-B background. Four founder lines expressing similar SP-B levels to human lung were chosen for further investigation. After intratracheal infection with 50μl of P. aeruginosa solution (1×107 CFU/mouse) or saline in SP-B-C, SP-B-T mice the mice were sacrificed 24 hours post-infection and tissues were harvested. Analysis of surfactant activity revealed differential susceptibility between SP-B-C and SP-B-T mice to bacterial infection, e.g. higher minimum surface tension in infected SP-B-C versus infected SP-B-T mice. These results demonstrate for the first time that human SP-B C allele is more susceptible to bacterial pneumonia than SP-B T allele in vivo.

Surfactant protein D attenuates acute lung and kidney injuries in pneumonia-induced sepsis through modulating apoptosis, inflammation and NF-κB signaling

“…The hSP-D cDNA fragment was cloned into the basic 3.7-hSP-C/SV40 vector and the recombinant construct was confirmed by DNA sequencing. The processes of recombinant DNA were performed based on the standard methods of molecular cloning 58 , 59 . The 5.3-kb DNA fragment was microinjected into fertilized oocytes from WT C57BL/6 mice.…”

“…Therefore, all experiments in this study were performed through intratracheal instillation of bioluminescent P . aeruginosa Xen5 at a dose of 1 × 10 6 CFU/50 μl/mouse to induce bacterial pneumonia 59 , 60 . Briefly, mice were anesthetized using intraperitoneal ketamine/xylazine (90 mg/kg ketamine, 10 mg/kg xylazine) injection.…”

“…Bacterial solution was intratracheally inoculated into the lung of mice in pneumonia groups and 50 μl of sterile saline in sham group. At 48 h post infection, mice were anesthetized to obtain blood samples, and then sacrificed to harvest BALF, lungs and kidneys for further analyses 59 .…”

Up‐regulation of SFTPB expression and attenuation of acute lung injury by pulmonary epithelial cell‐specific NAMPT knockdown

“…A variant polymorphism of the SFTPB gene has been associated with ARDS (26,27). Ge et al (28) demonstrated that in SFTPB humanized mice, the human SFTPB C allele of the 1580C/T polymorphism (single nucleotide polymorphism rs1130866) was more susceptible to bacterial pneumonia than the SFTPB T allele in vivo. Pneumonia is a frequent cause of ARDS.…”

The effect of diet-induced serum hypercholesterolemia on the surfactant system and the development of lung injury

“…Samples were incubated at 37 °C for at least 1 h prior to assessing the surface tension reducing properties using a constrained sessile drop surfactometer (CSD) as previously reported [27]. Briefly, the system involved a pedestal, 3 mm in diameter with a 1 mm pinhole, placed within a chamber incubated at 37 °C.…”