Acute Lung Injury Associated Milieus Regulate the Plasticity of Type I Alveolar Cells <em>via</em> KLF2

Special Article – Acute Lung Injury

Austin J Surg. 2019; 6(1): 1159.

Acute Lung Injury Associated Milieus Regulate the Plasticity of Type I Alveolar Cells via KLF2

Yang X, Sun J, Liu D, Du J, Yang C, Wang H, Wen D, Zeng L* and Jiang J*

State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, China

*Corresponding author: Jian-xin Jiang and Ling Zeng, Research Institute of Surgery, Daping Hospital, Third Military Medical University, China

Received: November 20, 2018; Accepted: January 07, 2019; Published: January 14, 2019

Abstract

Efficient repair and regeneration of alveolar epithelium after acute lung injuries are critical for pulmonary maintenance. Previous studies suggested alveolar type II cells (ATIIs) are progenitors in the adult lung and Alveolar Type I cells (ATIs) are terminally differentiated. However, recent studies develop fresh perspectives, which suggest ATIs may play more roles. Here we show that ATIs exhibit markedly enhanced proliferation capacity and express a set of ATIIs associated genes after treated with damaged lung milieus. These changed characteristics suggest ATIs generate phenotypic plasticity and exhibit dedifferentiated state. We identify KLF2 as a regulator of the module. Knockdown of KLF2 induces gene expression is consistent to those observed in milieu treated cells. These findings demonstrate the unanticipated plasticity of ATIs and suggest the molecular mechanisms controlling ATI-ATII plasticity which deserve more explorations.

Keywords: KLF2; Alveolar cells; ATI; ATII

Introduction

Adult lung consists of two major biologically distinct components including airway compartments and gas-exchanging units. Different component has region-specific stem/progenitor cells, which are critical for repair and regeneration following various injuries to rebuild functional structures. Mature alveoli are covered with two kinds of epithelial cells, type I and type II epithelial cells [1-6]. ATIs are thin cells covering 95-99% of the alveolar surface area, and lie closely with capillaries to facilitate gas diffusion. ATII cells, which cover the rest of the alveolar surface, are cuboidal cells and secrete surfactant proteins. Classical studies suggest ATIs are terminally differentiated, arise from ATIIs during alveolar homeostasis and regeneration process.

However, with advances in technology, fresh perspectives on the plasticity of ATIs are developed [7,8]. When cultured in vitro, ATIs exhibit some plasticity including the capacities to proliferate and express OCT-4, a protein that is involved in establishing and maintaining the undifferentiated pluripotent state. In vivo studies show us that part of ATIs can regenerate ATIIs in the lung and proliferate upon pneumonectomy or oncogenic KRAS expression. Nevertheless, it is still unclear whether ATIs can adopt new phenotypes to participate in regenerative responses? How do ATIs adopt their new phenotypes?

In this study, we focus on the poorly understood plasticity of ATIs in vitro. As alveolar milieus are capable of mobilizing different kinds of cells participating in repair through regulating their characteristics, we use lung extract from ARDS rats to mimic damaged milieu and explore how the plasticity of ATIs changed. To our surprise, ATIs exhibit markedly enhanced proliferation capacity and express a set of ATIIs associated genes after treated with damaged lung extract. These changed characteristics suggested ATIs generate phenotypic plasticity and exhibit dedifferentiated state. Then we explore the regulators of the module. We find KLF2 is contribute to the changed characteristics [9]. Knockdown of KLF2 induces gene expression changes similar to those observed in milieu treated cells. These findings demonstrate the unanticipated plasticity of ATIs and suggest the molecular mechanisms controlling ATI plasticity deserve more explorations, thus further establishing novel therapeutic approaches in treating lung diseases.

Materials and Methods

Animals

All the animals used here were male pathogen-free Sprague- Dawley rats providing by the Third Military Medical University animal center. These animals were housed in pathogen-free facility under a 12:12-h light-dark cycle with controlled room temperature (21-28°C) and humidity, and allowed ad libitum access to food and water. All animal studies were performed in accordance with the local Guide for the Care and Use of Laboratory Animals and were approved by the ethic committee.

Isolation and culture of ATIs

Rat ATIs were isolated and purified using a modification of published methods. Briefly, male SD rat (ag. 4-6-week-old; wt. 100- 160 g) lungs were dissected, perfused, lavaged, and then instilled with dispase (10 U/ml; Corning) for 50 min at 37°C. The resulting lungs were minced, filtered, washed, and incubated in rat IgG-coated Petri dishes in DMEM for 30 min at 37°C. Unattached cells were collected, resuspended in DMEM with 5% FBS, and incubated in dishes for 20 min at 37°C to loosen non-epithelial cells to the extent possible. Unattached cells were collected again, incubated with T1a antibody (5 mg/ml; sigma) in DMEM with 1% FBS for 45 min at 4°C on a rotator, washed and incubated with anti-rabbit IgG microbeads for 15 min at 4°C. The suspensions were washed and selected for ATIs. The purity of fresh isolated T1a-positive cells (AEC1s) was 86.6 ± 1.62%.

Freshly isolated ATIs were seeded at a density of 3×104 cells/ cm2 in high-glucose DMEM supplemented with 10% FBS, 100 units (mg)/ml of penicillin and streptomycin, and maintained in a 5% CO2 humidified 37°C incubator. Culture medium was refreshed every other day.

Acute lung injury animal model

Acute lung injury model was induced through Hemorrhagic Shock/Resuscitation (HR) combined with transtracheal injection of lipopolysaccharide (Escherichia coli serotype O55: B5, Difco Laboratories, Detroit, MI) as previously described. In short, 250±20 g male rats were anesthetization and shaved off the hair on interfeminium. Left femoral artery was carefully isolated, ligated and inserted a catheter to monitor of Mean Arterial Pressure (MAP), blood sampling, and resuscitation. Hemorrhagic shock was triggered by sucking blood, leading to the MAP to 40 mm Hg within 10 min. This blood pressure was maintained by further pumping blood for 1.5 hours. After that, rats were resuscitated with pumped blood plus equal volume of Ringer’s solution, and injected transtracheally 250 ul LPS (4.5 mg/kg in sterilized saline). The sham-operated animals were subjected to surgical procedures and injection of 250 ul sterilized saline without hemorrhagic shock/resuscitation and LPS challenge. All the works were operated cautiously to reduce infectious diseases or other undesirable outcomes.

Preparation of lung extract from normal and injured lungs

Rats were sacrificed at days 1, 3, 5 and 7 after transtracheal injection, respectively. Whole Lung was excised, perfused, segmented. Left lobes were fixed in 4% paraformaldehyde, dehydrated in ethanol, embedded in paraffin. Right lobes were transferred to the mortar containing liquid nitrogen and finely powdered. Then tissue powder was dissolved in DMEM, centrifuged, filtered, aliquoted and frozen at -80°C.

KLF2 silencing by small interfering RNA transfection in ATI cells

ATIs were transfected with validated small interfering RNA (siRNA) duplex oligonucleotides (Genepharma, Shanghai, China) by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as the the transfection reagent according to the manufacturer’s instructions. In brief, ATI cells were grown routinely in DMEM with 10% fetal bovine serum to about 50% confluence, and then treated with 100 nM KLF2 siRNA, or a non-specific duplex siRNA as negative control using lipofectamine. Transfected cells were grown at 37°C for 4h, followed by incubation with fresh culture medium. After 24h or 48h, transfected cells were examined with phasecontrast microscopy, and used in further assays or RNA/protein extraction. The sequences for KLF2 siRNAs and control siRNA used here were: KLF2-rat-104 (sense: 5'-G C A C G G A U G A G G A C C U A A A T T-3', antisense: 5'-U U U A G G U C C U C A U C C G U G C T T-3'); the control siRNA 5 (sense: 5'-U U C U C C G A A C G U G U C A C G U T T-3', anti-sense: 5'-A C G U G A C A C G U U C G G A G A A T T-3').

CCK-8 assay

To monitor cell growth after the treatment of different factors, ATI cell proliferation was quantified by Cell Counting Kit-8 (CCK- 8) (Beyotime, Hangzhou, China) according to the manufacturer’s instructions. Briefly, ATI cells were seeded in each 96-well plate, treated with indicated factors, and further incubated for 24 h or 48 h. Fresh medium containing 10% CCK-8 reagent was added to each well and incubated for 2 h at 37°C. OD450nm value in each well was determined by a microplate reader.

Immunofluorescence staining

Paraffin-embedded lung sections and cultured cells were used for staining. Samples were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked with 10% serum (same host as secondary antibody) in PBS 1 h at room temperature. Cells were stained with antibodies against T1a (SC-1666906; Santa Cruz; 1:200), AQP5 (SC-9890; Santa Cruz; 1:200), Cav-1 ( ) as well as proSPC (AB3786; Millipore; 1:1000), KLF2 (BS-2772R; Bioss; 1:200), vimentin (AB8978; Abcam; 1:200), Oct 4 (11263-1-AP; Proteintech; 1:100) and PCNA (AB29; Abcam; 1:1000). Samples were incubated with primary antibody at 4°C overnight followed by the appropriate donkey antimouse, donkey anti-rabbit, or donkey anti-goat secondary antibodies (Invitrogen) protected from light, at room temperature for 1 h. Negative control slides were only exposed to secondary antibodies (data not shown). Nuclei were visualized with DAPI (D8417; Sigma; 0.5 ug/ml).

RNA extraction and quantitative PCR analysis

RNA was isolated from lung tissues or cultured cell preparations using TRIzol (Invitrogen, CA, USA) according to the manufacturer’s instructions. Reverse transcription reactions were performed with Prime Script RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China). Gene expressions were determined by quantitative real-time PCR using

SYBR Green Real-Time PCR Master Mix (TOYOBO, OSAKA, Japan). Rat β-actin was used as as internal control. Primer sequences were as follows:

Rat β-actin was used as as internal control. Primer sequences were as follows: Rat β-actin was used as as internal control. Primer sequences were as follows: rat T1a sense: 5'-C A G T G T T G C T C T G G G T T T T G G-3', antisense: 5'- -3'; rat AQP5 sense: 5'-G C T T T G G A A T C A G G C A G A A T G-3', antisense: 5'-A G A C C T G G G T T C A C C A T G T C A-3'; rat proSPC sense: 5'- -3', antisense: 5'-A G C A A G C T G T C G T C C C T T T C-3'; rat Nkx2.1 sense: 5'-A C G T G A G C A A G A A C A T G G C C-3', antisense: 5'-G G T G G T T C T G G A A C C A G A T C-3'; rat KLF2 sense: 5'-C T C A G C G A G C C T A T C T T G C C-3', antisense: 5'-C A C G C T G T T T A G G T C C T C A T C C-3'; rat Snx5 sense: 5'-G C C C G G T T A A A A A G C A A A G A T G T-3', antisense: 5'-G C A T G C T T T A T T T C C A G T T C A G A C A-3'; rat β-actin sense: 5'-A G A G A A G C T G T G C T A T G T T G C C C T-3', antisense: 5'-ACCGCTCATTGCCGATAGTGATGA-3'. Data were processed using 2-ΔΔCT method.

Protein extraction and Western blot analysis

Cultured or transfected cells were lysed in RIPA buffer containing 1% PMSF (P0013, Beyotime). Protein concentration was determined by the BCA protein assay kit (P0012, Beyotime). The protein was loaded onto a SDS-PAGE minigel and transferred onto PVDF membrane. After probed with diluted rabbit antibody at 4°C overnight, the blots were subsequently incubated with HRPconjugated secondary antibody (1:5000). Signals were visualized using ECL Substrates (Millipore, MA, USA). GAPDH was used as an endogenous protein for normalization.

Statistical analyses

All data are expressed as mean ± standard deviation of at least three independent experiments. Differences in measured variables between treated groups were assessed by using the Student’s t-tests or one-way ANOVA. P values less than 0.05 are regarded as significant.

Results

The milieus of damaged lung promote ATIs proliferation

ATIs isolated after 1-2 days were rounded and small with the tendency to group together to form small aggregations. During the following culture, cells spread out, flattened, proliferated, and formed a continuous epithelial monolayer. For the cell identification, cells 4 days after isolation were fixed and immunofluorescence were performed (Figure 1A-1C). By counting nine multiple fields (512um×512um) under microscopy, 89.0±2.1 % of total cells were T1a positive (ATIs specific marker). In accordance with previous studies, our ATIs were proSPC positive in a peri-nuclear distribution, which was very different from the punctate cytoplasmic patterns in ATIIs. To detect the proliferation capability, ATIs were plated at a density of 2 *104/well and observed for 18 days. ATIs became motile at day 4, and proliferated until cells reached confluence. The growth curve was showed in (Figure 1D).