Epithelial LIF signaling limits apoptosis and lung injury during bacterial pneumonia

Abstract

During bacterial pneumonia, alveolar epithelial cells are critical for maintaining gas exchange and providing antimicrobial as well as pro-immune properties. We previously demonstrated that leukemia inhibitory factor (LIF), an IL-6 family cytokine, is produced by type II alveolar epithelial cells (ATII) and is critical for tissue protection during bacterial pneumonia. However, the target cells and mechanisms of LIF-mediated protection remain unknown. Here, we demonstrate that antibody-induced LIF blockade remodels the lung epithelial transcriptome in association with increased apoptosis. Based on these data, we performed pneumonia studies using a novel mouse model in which LIFR (the unique receptor for LIF) is absent in lung epithelium. Although LIFR is expressed on the surface of epithelial cells, its absence only minimally contributed to tissue protection during pneumonia. Single-cell RNA-sequencing (scRNAseq) was conducted to identify adult murine lung cell types most prominently expressing Lifr, revealing endothelial cells, mesenchymal cells, and ATIIs as major sources of Lifr. Sequencing data indicated that ATII cells were significantly impacted by pneumonia, with additional differences observed in response to LIF neutralization, including but not limited to gene programs related to cell death, injury, and inflammation. Overall, our data suggest that LIF signaling on epithelial cells alters responses in this cell type during pneumonia. However, our results also suggest separate and perhaps more prominent roles of LIFR in other cell types, such as endothelial cells or mesenchymal cells, which provide grounds for future investigation.

INTRODUCTION

Pneumonia is an acute lower respiratory condition most often caused by infectious pathogens. Such conditions can result in severe damage to the alveolar barrier leading to excessive fluid in the lungs (1). Although strategies such as antibiotics and vaccines have well-established benefits, the effectiveness of microbe-targeting approaches in the context of lung infection is confounded by several factors, such as antibiotic resistance, remarkably diverse etiology (2), and a threat of immunopathology that often endures in the absence of detectable pathogens. This demands a better understanding of host pathways dictating pneumonia outcome, including those controlling both antimicrobial defense and tissue protection.

We and others have now shown that leukemia inhibitory factor (LIF), an IL-6 family cytokine, limits immunopathology in the settings of both bacterial and viral pneumonia (3, 4). LIF signals through its unique receptor, LIFR, and the ubiquitously expressed coreceptor, gp130, to activate the JAK/STAT pathway (5), along with others. Activation of alveolar epithelial STAT3 has been shown by our laboratory and others to limit acute lung injury (6–8), and our prior studies indicate LIF is both necessary and sufficient for this process (3, 6). Although we also identified type II alveolar epithelial cells (ATII) as a predominant source of LIF during pneumonia (9), its target cells and protective effects remain unknown.

Type I (ATI) and type II alveolar epithelial cells are integral to the barrier separating the distal lung from the capillaries. ATIs are mainly responsible for gas exchange whereas ATIIs are important for the production of surfactant proteins that both relieve surface tension and carry out antimicrobial functions (10). ATIIs can also self-proliferate or differentiate into ATIs upon injury (11). As a predominant site of injury following lower respiratory tract infections, the alveolar epithelium is crucial for maintaining the alveolar-capillary interface and limiting severe injury such as that inherent to acute respiratory distress syndrome (ARDS). Hence, maintaining epithelial barrier integrity in the initial stages of pneumonia is crucial for both maintaining and repairing the lung parenchyma in the wake of an infectious challenge, but the biological pathways contributing to such protection are poorly defined.

For the present study, we hypothesized that lung epithelium is an important target of LIF during pneumonia. Our results indicate robust effects of LIF on epithelial cells during pneumonia, which are associated with regulation of lung apoptosis. However, our findings also suggest that LIF-induced protection extends beyond that which occurs in the epithelium, such that its biological activity in alternative targets merits important consideration.

MATERIALS AND METHODS

Mice

Mice with homozygous floxed alleles for LIFR (generated from Lifr^{tm1a(EUCOMM)Hmgu}; EUCOMM, #RQ84) were crossed with a mouse containing a tamoxifen-inducible Cre-recombinase transgene knocked into the Nkx2-1 locus (Nkx2-1^{tm1.1(cre/ERT2)Zjh}; Jackson Laboratories, #014552) to create the EpiLIFR^Δ/Δ mouse. C57BL/6 mice were obtained from Jackson Laboratories. SPC-GFP mice were a generous gift from Dr. John K. Heath (12) and have been backcrossed over 15 generations onto a C57BL/6 background at Boston University. For tamoxifen administration, mice were injected intraperitoneally with tamoxifen (Sigma, #T5648-5G) in corn oil (75 mg/kg, 20 mg/mL stock solution) once per day for 5 consecutive days to induce Cre-mediated recombination. All mice were 6–15 wk of age at the time of use, and both male and female mice were used. Mice were housed at Boston University’s animal facilities, and all protocols were consistent with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (13), as approved by the Institutional Animal Care and Use Committee at Boston University.

Pneumonia Models and Recombinant Murine LIF Administration

Mice were anesthetized intraperitoneally with ketamine (50 mg/kg) and xylazine (5 mg/kg) before intratracheal installation of 50 µL saline containing ∼1 × 10⁶ CFU (colony forming units) Escherichia coli (serotype O6:K2:H1; ATCC #19138) into the left bronchus as previously described (3). Where indicated, bacteria were coinstilled with 10 µg of normal goat IgG (R&D Systems, #AB108) or a neutralizing goat polyclonal IgG targeting murine LIF (R&D Systems, #AB449). Antibody specificity was established by the indicated vendors. In separate experiments, 50 ng recombinant mouse LIF protein (R&D Systems, #8878-LF-100/CF) was coinstilled intratracheally with E. coli. At the indicated timepoints, mice were euthanized by isoflurane overdose, and samples were collected for the indicated analyses.

Bacteriology

E. coli which is an important cause of hospital-acquired pneumonia (14–16) has been routinely used by our laboratory and others (3, 6, 17, 18) as a model for acute respiratory infection in mice. The pathogen was grown overnight on 5% sheep’s blood agar plates at 37°C with 5% CO₂ for 15 h before making a solution with sterile saline at an OD₆₀₀ of 0.3, used as an estimate of final concentrations. The actual inoculum was then calculated by plating serial dilutions on 5% sheep’s blood agar plates. Bacteremia was also determined by plating 100 µL of serially diluted blood collected from the inferior vena cava. Total colonies were counted and reported as colonies per milliliter of blood.

Bronchoalveolar Lavage

Bronchoalveolar lavage fluid (BALF) was collected as previously described (6, 19). Briefly, the heart-lung block was removed and tied to a 20-gauge blunted stainless steel catheter via the trachea. Once secured, the lungs were lavaged 10 times with 1 mL of cold phosphate-buffered saline (PBS). Supernatants from the first milliliter of BALF were stored at −80°C after centrifugation (300 g, 5 min, 4°C), and the cell pellets were combined with that of the other 9 mL of BALF to determine total cell counts, which were calculated by the LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems). After cytocentrifugation and Camco staining (Cambridge Diagnostic, #702), differential counts were determined at each timepoint.

Protein and Cytokine Measurements

BALF protein concentrations were measured by using a mouse magnetic Luminex assay (Bio-Rad, #171I50001 and #171I60001) on a Bio-Plex 200 multiplexing analyzer system. The panel included LIF, G-CSF, IL-10, IL-6, TNFa, IL-1b, IL-17, GM-CSF, and KC. BALF albumin was measured via enzyme-linked immunosorbent assay (ELISA; Bethyl Laboratories, #E99-134).

RNA Extraction, RT-PCR, and Gel Electrophoresis

Snap-frozen lungs were homogenized using a Bullet Blender, and total RNA was purified using Qiagen’s RNeasy Mini kit (#74104) as previously described (3). SuperScript IV First-Strand Synthesis System (Invitrogen, #18091050) was used according to the manufacturer’s directions to reverse transcribe RNA into cDNA. GoTaq G2 Green Master Mix (Promega, #M782A) was used to amplify cDNA by PCR using custom primers flanking exon 4 of the LIFR sequence (Fwd: 5' - AAA TTC CAG CTC TTT CAC CTG G -3'; Rev: 5' - AGG TCT GAG GTC CAG TTC CA -3') from Integrated DNA Technologies. Samples were run on a 2% agarose gel with ethidium bromide at 120 V for 50 min.

Immunoblotting

Snap-frozen lungs were homogenized using a Bullet Blender and extracted for protein as previously described (3). Immunoblots were run on NuPage 4%–12% Bis-Tris gels (Thermo Fisher Scientific, #NP0335PK2) and probed for LIFR (Abcam, #202847), pSTAT3 (Cell Signaling Technologies, #9145S), STAT3 (#12640S), and pan-actin (#4968S). Antibody specificity was established by the indicated vendors. Blots were imaged using the LI-COR Odyssey CLx fluorescent imaging system, and subsequent analyses were done using ImageStudioLite software. Full Western blots have been included as Supplemental Fig. S3.

Histology and Immunofluorescence

Lungs were fixed in 4% paraformaldehyde (Ted Pella Inc, #18505) and embedded in paraffin as previously described (3). An in situ cell death detection kit (Sigma Aldrich, #12156792910) was used according to the manufacturer’s directions on 8-µm sections to detect apoptosis. Sections were also counterstained after TUNEL staining using an anti-pro-SPC rabbit antibody (Seven Hills Bioreagents, #WRAB-9337) for type II alveolar epithelium, anti-podoplanin hamster antibody (Invitrogen, #14–5381-85) for type I alveolar epithelium, and anti-CD31 goat antibody (R&D, #AF3628) for endothelial cells. The following secondary antibodies were used: donkey anti-rabbit Alexa Fluor 647 (Invitrogen, #A31573), goat anti-hamster Alexa Fluor 647 (Jackson ImmunoResearch Laboratories, #107–606-142), and donkey anti-goat Alexa Fluor 647 (Invitrogen, #A32849). Tissue slides were permeabilized with 0.2% Triton X-100 (PBS-T) and blocked with 5% donkey serum. The primary antibody was diluted in PBS-T overnight at 4°C. Secondary antibodies were diluted in PBS-T and incubated for 1 h at room temperature. DAPI (Invitrogen, #R37606) was used to stain nuclei. Slides were mounted with ProLong Diamond Antifade Mountant (Invitrogen, #P36970), and images were captured on a Leica DM4 B light microscope with integrated LAS X software for morphometry or a Zeiss Axio Observer fluorescence microscope. Quantification of cell numbers was done by averaging 20 blinded and randomized images per animal. Similarly, hematoxylin and eosin (H&E) staining was performed on fixed and paraffin-embedded lung sections. Lung tissue was fixed overnight, washed with PBS and saline, then dehydrated through increasingly concentrated ethanol washes, followed by xylene clearing and paraffin infiltration. Histology scores were calculated using recommended guidelines from the American Thoracic Society (20).

Flow Cytometry and Cell Sorting

Lungs were enzymatically digested into single cell suspensions and stained as described previously (21, 22). Lungs were digested at 37°C and 100 RPM for 45 min in an elastase solution containing 4.5 U/mL elastase (Worthington Biochemical, #LS002292) and 100 units/mL DNase I (Qiagen, #79256). Cells in suspension were subsequently filtered through 100-, 70-, and 40-µm nylon mesh. After red blood cell lysis (Sigma, #R7757), cells were counted on the LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems) and stained with a combination of the following antibodies: CD45-FITC (Biolegend, #103108), EpCAM-APC (Biolegend, #118214), CD31-PacBlue (Biolegend, #102422), EpCAM-eFluor 450 (Invitrogen, #48–5791-82), Podoplanin-SB600 (Invitrogen, #63–5381-82), CD24-BUV737 (BD Biosciences, #612832), CD104-APC (Biolegend, #123611), LIFR-PE (R&D Systems, #FAB5990P), 7AAD (BD Biosciences, #559925), CD45-PerCP-Cy5.5 (Biolegend, #103132), and TACS Annexin V-FITC Apoptosis Detection kit (R&D Systems, #4830-01-K). Flow cytometry was performed using BD LSRII and fluorescence-activated cell storting (FACS) was performed using BD FACSARIA II at the Boston University Flow Cytometry Core Facility. Data were analyzed using FlowJo (v. 10) software. Unstained cells, single-stained UltraComp eBeads (Invitrogen, #01–2222-42), and fluorescence-minus-one (FMO) controls were used for each experiment.

Microarray

Epithelial cells (7AAD−, CD45−, and EpCAM+) were isolated by FACS 6 h and 24 h after intratracheally being challenged with E. coli in the presence of control IgG or anti-LIF IgG for microarray analysis at the Boston University Microarray and Sequencing Resource Core Facility. Cells were centrifuged and resuspended in RNAprotect immediately after sorting. RNA was isolated from sorted cells using the RNeasy Micro kit, following the manufacturer’s instructions (Qiagen). RNA quality was validated using an Agilent bioanalyzer, and microarrays were performed for each sample using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. Probes on the array were mapped to the mouse genome using a custom CDF file as described (23), which can be found at http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/genomic_curated_CDF.asp. Following data normalization, filtering, and preprocessing, fold changes were calculated between experimental groups. GSEA (v. 2.2.1) (24) was used to identify biological terms, pathways, and processes that were coordinately upregulated or downregulated within each pairwise comparison. All data have been deposited in the NCBI Gene Expression Omnibus (Accession No.: GSE179764; http://www.ncbi.nlm.nih.gov/geo/).

Single-Cell RNA Sequencing

C57BL/6 mice were intratracheally challenged in their left lobes for 24 h with IgG saline, IgG E. coli or anti-LIF E. coli, resulting in a single lung sample from three separate conditions. Left lung lobes were digested at 37°C and 100 rpm for 30 min in a solution containing 9.5 U/mL elastase (Worthington Biochemical, #LS002292), 20 U/mL collagenase I (Worthington Biochemical, #NC9693955), 5 U/mL dispase (Worthington Biochemical, #NC9199795), and 50 U/mL DNase I (Qiagen, #79256). Single-cell suspensions were subsequently processed as described in materials and methods for Flow Cytometry and Cell Sorting for FACS. An equal ratio of live CD45+, CD45-EpCAM+, and CD45-EpCAM− cells were combined for each condition. ScRNAseq libraries were generated at Boston University’s Department of Medicine Single Cell Sequencing Core Facility and sequenced by Boston University’s Microarray and Sequencing Resource Core Facility on an Illumina NextSeq 2000 instrument according to Illumina and 10X Genomics guidelines (Illumina). Basic single cell RNA-seq data analysis was performed by BU’s DOM Single Cell Sequencing Core Facility. Preprocessed data (.rds) was submitted to BioTuring Browser (v. 2.9.23 for Mac OS, developed by BioTuring Inc., San Diego, CA) for visualization and downstream analyses. The differential expression analyses were performed by Venice (BioTuring Inc.). All data have been deposited in the NCBI Gene Expression Omnibus (Accession No.: GSE179855; http://www.ncbi.nlm.nih.gov/geo/).

Cell Culture

The murine alveolar epithelial cell line E10 (25), obtained from Dr. Alvin Malkinson (University of Colorado, Aurora, CO), was cultured in CMRL-1066 medium containing 10% FBS, 2 mM l-glutamine (Glutamax), 100 U/mL penicillin, and 100 µg/mL streptomycin as previously described (21). The murine AT2 cell-derived cell line MLE15 (26), obtained from Dr. Jeffrey A. Whitsett (Cincinnati Children’s Hospital Medical Center, Cincinnati, OH), was cultured in a 1:1 mixture of DMEM and F12 medium supplemented with 2% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 µg/mL insulin, 10 µg/mL transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, and 10 mM HEPES as previously described (21). MLE-12 cells were purchased from American Type Culture Collection and maintained as recommended by the provider. Cells were seeded at 250,000 cells/well in 24-well plates and incubated overnight (37°C, 5% CO₂) until ∼80% confluent. They were then stimulated with 50 ng/mL of recombinant murine LIF (R&D Systems, #8878LF025/CF) for 30 min, and cell lysates were collected for protein extraction, which was then stored at −80°C until use.

Statistics

Statistical analyses were performed using GraphPad Prism (GraphPad, La Jolla, CA). Data are presented as means ± SEM. Comparisons between two groups were performed using a Student’s t test. One-way analysis of variance (ANOVA) was used to analyze data when comparing more than 2 data sets, followed by a Dunnett post hoc test for multiple comparisons. Data were tested for normality (Shapiro–Wilk test) and homoscedasticity (F test) and were log transformed if either condition was not met. Comparisons were considered significant when P < 0.05.

RESULTS

LIF Blockade Increases Cellular Apoptosis during Pneumonia

Previously, we reported exaggerated lung injury during an Escherichia coli-induced pneumonia following antibody-induced LIF blockade (3). The capacity of LIF to activate epithelial STAT3 (3, 6), along with the established importance of epithelial STAT3 for limiting acute lung injury (6–8) suggested LIF as a mediator of epithelial cytoprotection. To comprehensively assess the impact of LIF neutralization on lung epithelium, we performed transcriptional profiling on sorted CD45-/EpCAM+ epithelial cells after 6 and 24 h of intratracheal E. coli in the presence of control IgG or anti-LIF IgG (Fig. 1A). As predicted, there were hundreds of differentially expressed genes (FDR < 0.05) due to pneumonia at both time points, albeit more so with LIF neutralization, suggesting a greater sensitivity to infection under these circumstances. By 6 h, Gene Set Enrichment Analysis (GSEA) revealed that apoptosis-related pathways such as “Positive Regulation of Caspase Activity” and “Caspase Activation” were upregulated based on the transcriptional signatures detected in anti-LIF-treated mice when compared with IgG-treated controls (Fig. 1, B and C). To corroborate these findings, C57BL/6 mice were intratracheally (i.t.) challenged with E. coli for 24 h in the presence of control IgG or anti-LIF IgG and assessed by fluorescence microscopy using TUNEL staining to visualize apoptotic cells (Fig. 2A). Significantly more TUNEL+/DAPI+ cells were detected following LIF blockade. However, TUNEL+ cells could not be specifically attributed to any combination of ATII cells, ATI cells, or endothelial cells when counterstained for pro-SPC, podoplanin, or CD31, respectively (Fig. 2B), perhaps owing to downstream effects in other cell types and/or insufficient precision to confidently identify apoptotic cells with this method. To identify and quantify the apoptotic cells with anti-LIF treatment, flow cytometry using annexin V and propidium iodide (PI) was performed on C57BL/6 mice after 24 h E. coli challenge (Fig. 2C). A slightly higher but insignificant percentage of apoptotic (Annexin V+/PI-) epithelial cells were present in lungs of anti-LIF-treated mice (Fig. 2D). This trend was not observed in CD45+ myeloid cells (data not shown) or CD45−/EpCAM−/CD31+ endothelial cells (Fig. 2E). Thus, while LIF regulates apoptosis during pneumonia, the identity of these apoptotic cells remains unclear.

Figure 1.

Epithelial reprogramming following leukemia inhibitory factor (LIF) blockade. Lung epithelial cells (7AAD−, CD45−, EpCAM+) from C57BL/6 mice were isolated by FACS 6 h and 24 h after i.t. E. coli in the presence of control IgG or anti-LIF IgG for microarray analysis (n = 3). A: Venn diagrams illustrate the number of differentially expressed genes (FDR < 0.05) due to pneumonia. B and C: Gene Set Enrichment Analysis (GSEA) at 6 h after infection revealed categories consistent with apoptosis in anti-LIF treated mice (vs. IgG controls). E. coli, Escherichia coli; i.t., intratracheal.

Figure 2.

Effect of leukemia inhibitory factor (LIF) blockade on lung apoptosis during pneumonia. A and B: C57BL/6 mice were challenged with i.t. E. coli for 24 h in the presence of control IgG or anti-LIF IgG. TUNEL staining (red) was applied to 4% PFA-fixed paraffin-embedded lung tissue sections. DAPI (blue) was used to stain nuclei. Pro-SPC was used to counterstain for ATII cells, podoplanin for ATI cells, and CD31 for endothelial cells (green). TUNEL images were taken at ×40 magnification and quantified by counting and averaging the number of TUNEL+/DAPI+ cells in 20 images/mouse (n = 6–8). Counterstained images were taken at ×63 magnification. C: low cytometry was used to quantify apoptosis (Annexin V+/PI−) in epithelial cells (CD45−/EpCAM+). The gating strategy excluded doublets and used annexin V and propidium iodide to distinguish apoptotic cells. Summarized data are illustrated for epithelial cells (CD45−/EpCAM+) (D) and endothelial cells (CD45−/EpCAM-/CD31+) (n = 11) (E). P value indicated on each graph vs. IgG as determined using a Student’s t test. ATII, type II alveolar epithelial cells; E. coli, Escherichia coli.

LIFR is Expressed on Epithelium and Contributes to Tissue Protection during Pneumonia

Based on the LIF blockade findings, we hypothesized that epithelial cells are both a source (9) and a direct target of LIF during pneumonia. It is well-established that LIF signals through its unique receptor, LIFR, which heterodimerizes with the common IL-6 family coreceptor, gp130, to activate the JAK/STAT pathway, particularly STAT3 (5). To confirm whether alveolar epithelial cells express LIFR, we initially examined three different alveolar epithelial cell lines: E10 (ATI-like), MLE15 (ATII-like), and MLE12 (ATI and II-like) (3, 21, 26). All three cell lines expressed LIFR protein by immunoblot and responded to 30 min of recombinant murine LIF (rmLIF) stimulation as measured by Y705-pSTAT3 activity (Fig. 3, A and B). To extend this observation in vivo, we generated an epithelial-specific (27, 28) inducible LIFR floxed mouse expressing Cre-recombinase under the transcriptional control of the Nkx2-1 promoter targeting all lung epithelial cells (hereby abbreviated as EpiLIFR^Δ/Δ). As part of the validation process, we confirmed that EpiLIFR^Δ/Δ mice express a nonfunctional mutant form of LIFR at the whole lung RNA level (Supplemental Fig. S1), and significantly less LIFR protein was detected on sorted EpiLIFR^Δ/Δ epithelial cells as determined by immunoblot and flow cytometry when compared with wild-type (floxed LIFR, Cre-negative) counterparts (Fig. 3, C and D). When EpiLIFR^Δ/Δ mice were challenged with 24 h intratracheal E. coli following tamoxifen administration, H&E staining indicated that EpiLIFR^Δ/Δ mice had significantly higher lung injury scores [as measured by the recommended American Thoracic Society guidelines (20)] compared with wild-type counterparts (Fig. 3, E and F). However, other metrics including total protein accumulation in BALF as a measure of alveolar edema [1.409 ± 0.3682 (EpiLIFR^Δ/Δ) vs. 1.062 ± 0.3308 (WT)] and average number of TUNEL+/DAPI+ cells per field [3.950 ± 0.2958 (EpiLIFR vs. 3.231 ± 0.2215 (WT)] did not reach statistical significance. To look more closely at LIFR protein distribution in lung epithelium, flow cytometry was used to measure LIFR MFI in ATII (CD45-/EpCAM+/SPC+), ATI (CD45-/EpCAM+/podoplanin+), ciliated (CD45-/EpCAM+/CD104+/CD24^hi), and club (CD45-/EpCAM+/CD104+/CD24^med) cells in mice with surfactant protein C promoter-driven green fluorescent protein (SPC-GFP) (Fig. 3G), which had been previously characterized by our laboratory and others (9, 12, 29). LIFR surface expression was detected across all epithelial subsets analyzed with relative expression appearing to be somewhat higher in alveolar epithelial cells, perhaps indicating that LIFR-related signaling preferentially occurs in the distal airways. Overall, these findings suggest that epithelial LIFR contributes to tissue protection during bacterial pneumonia, although LIFR on other cell types may still be involved.

Figure 3.

Influence of epithelial leukemia inhibitory factor receptor (LIFR) during pneumonia. A: LIFR protein was visualized by immunoblot in lysates from E10, MLE15, and MLE12 (alveolar epithelial cell lines) cells. B: Y705 phosphorylated STAT3 immunoreactivity was measured 30 min after stimulation of the indicated cell lines with recombinant murine LIF stimulation (rmLIF). Each lane is a technical replicate. C: sorted epithelial cells (CD45−/EpCAM+) were pooled from 4 wild-type (WT) and 4 EpiLIFR^Δ/Δ mice to visualize LIFR by immunoblot. D: LIFR median fluorescence intensity (MFI) was measured by flow cytometry for epithelial cells (CD45−/EpCAM+) in single-cell suspensions prepared from the lungs of WT and EpiLIFR^Δ/Δ mice (n = 7). E: EpiLIFR^Δ/Δ mice and WT counterparts were intratracheally challenged with E. coli for 24 h, and left lobes were fixed and paraffin embedded for H&E. F: 20 images per mouse were scored to assess lung injury (n = 5–7). G: LIFR MFI (normalized to FMO) on various lung epithelial cell types was measured by flow cytometry using female SPC-GFP mice (n = 3). Significance versus ATII was determined by one-way ANOVA followed by a Dunnett post hoc test for multiple comparisons. Histograms show LIFR MFI graphed against LIFR FMO for each cell type. P value indicated on each graph vs. WT as determined using a Student’s t test except for Fig. 3G. ATII, type II alveolar epithelial cells; E. coli, Escherichia coli; FMO, fluorescence-minus-one; H&E, hematoxylin and eosin; SPC-GFP, surfactant protein C promoter-driven green fluorescent protein.

Exogenous LIF Delivery Only Modestly Impacts Pneumonia-Induced Injury

Past and present results indicating that LIF is necessary for tissue protection during pneumonia (3, 4) prompted us to determine whether exogenous rmLIF is sufficient to limit immunopathology in this setting. Interestingly, despite the importance of endogenous LIF, co-instillation of rmLIF with E. coli failed to elicit protection compared with control groups as measured by BALF albumin (Fig. 4A). There were also no differences observed in neutrophil recruitment, bacteremia, or BALF cytokines other than LIF itself at 24 h post-infection (Fig. 4, B–D). RmLIF was also administered intravenously 4 h after intratracheal inoculation, resulting in no differences in BALF total protein between the treatment groups at the 24-h timepoint (data not shown). However, we observed a subtle but significant decrease in lung injury score by histology following rmLIF treatment compared with controls (Fig. 4, E and F), indicating that this strategy has a modest protective benefit. Overall, however, these findings suggest that endogenous LIF induction most likely achieves the maximum protective effects afforded by this cytokine.

Figure 4.

Effects of exogenous recombinant murine LIF (rmLIF) administration during pneumonia. A: 50 ng rmLIF was coinstilled with E. coli (1 × 10⁶ CFU) in C57BL/6 mice for 6 and 24 h. Bronchoalveolar lavage fluid (BALF) albumin was measured for each time point by ELISA (n = 5–8). Following 24 h of infection, BALF was collected for determination of neutrophils (PMNs) and alveolar macrophages (AMs) (B) or cytokine concentrations (C) as measured by Luminex (n = 6–8). D: bacteremia was determined following 24 h of infection (n = 4 or 5). E and F: left lobes from female C57BL/6 mice were fixed and paraffin embedded for H&E. 20 images per mouse were scored to assess lung injury (n = 4). P value indicated on each graph vs. control as determined using multiple t tests or Student’s t test. E. coli, Escherichia coli; LIF, leukemia inhibitory factor; H&E, hematoxylin and eosin

ATII, Endothelial, and Mesenchymal Cells Are Prominent Sources of LIFR in the Lungs

Although consistent with our initial hypothesis, the results observed in EpiLIFR^Δ/Δ mice suggest roles for LIFR signaling in other non-epithelial cell types. To address this possibility, single-cell RNA-sequencing (scRNAseq) was performed on C57BL/6 mice to determine which adult murine lung cells express Lifr, as well as how such cells respond during pneumonia. Mice were again intratracheally challenged for 24 h with control IgG + saline, control IgG + E. coli, or anti-LIF IgG + E. coli. Infected lobes were collected and digested into single-cell suspensions for FACS. An equal ratio of live CD45+, CD45−/EpCAM+, and CD45−/EpCAM− cells were combined for each condition and subsequently pooled for single-cell sequencing. Unsupervised analysis by Seurat yielded 21 distinct clusters, representing the lung cellular landscape with and without pneumonia (Fig. 5A). We found that Lifr was most highly expressed in endothelial, mesenchymal, and ATII cells (Fig. 5, B and C), building upon our initial hypothesis of ATII cells as a potential target of LIF signaling. This observation, particularly in nonpneumonic mice, is consistent with other publicly available scRNAseq datasets (30, 31). As expected, there was a significant effect of pneumonia on various cell types, with a particularly prominent shift in the transcriptional profiles of ATII cells (Supplemental Fig. S2 and Supplemental Tables S1–S6). Curiously, it is known that ambient RNA contamination from epithelial cells such as Sftpc can be present in nonepithelial cells in certain contexts, as we suspect is the case in Supplemental Table S2 (32). To further investigate our three cellular groups of interest, single-cell analytical tool BioTuring Browser (33) was used to subplot ATII cells, endothelial cells, and mesenchymal cells (Fig. 5D), illustrating effects of pneumonia as well as LIF blockade. Differential expression analyses between anti-LIF E. coli and IgG E. coli were conducted for each cell type. For ATII cells, there were 2,666 differentially expressed genes (FDR < 0.05), 2,509 of which were downregulated as a result of anti-LIF treatment. The upregulated genes were most significantly associated with the “p53 Pathway” and “Apoptosis” while the downregulated genes were most significantly associated with “Unfolded Protein Response” and “DNA Repair” as determined by Enrichr (Table 1) (34–36). Similar observations were made for endothelial cells (Table 2) and mesenchymal cells (Table 3) as well.

Figure 5.

Single-cell RNA-seq for lung cells following pneumonia with or without LIF blockade. Female C57BL/6 mice were intratracheally challenged for 24 h with saline or E. coli (1 × 10⁶ CFU) with or without LIF neutralization (n = 1/group). Left lobes were collected and digested into single-cell suspensions for FACS. Equal ratios of CD45+, CD45-EpCAM+, and CD45-EpCAM− cells were pooled from a mouse undergoing each condition. Using the ×10 Genomics Chromium System, single cells were captured, and bar-coded cDNA libraries were consolidated for RNA-seq performed on the Illumina NextSeq 2000 System. A: unsupervised clustering by Seurat yielded 21 distinct clusters, representing the lung cellular landscape with and without pneumonia, in the presence of control IgG or anti-LIF. B and C: Lifr is illustrated across lung cell populations, with prominent expression in endothelial cells, fibroblasts/pericytes/stromal cells, and ATII cells as further indicated in violin plots. D: ATII, endothelial, and mesenchymal cells were subplotted using BioTuring Browser. All plots were generated by BioTuring Browser. ATII, type II alveolar epithelial cells; E. coli, Escherichia coli; LIF, leukemia inhibitory factor.

Table 1.

Differential expression analyses were conducted on anti-LIF vs. IgG E. coli for ATII cells by BioTuring Browser

Pathway Name	P Value
Upregulated genes
p53 pathway	0.001037
Apoptosis	0.001724
Interferon γ response	0.005034
Xenobiotic metabolism	0.005034
Pperoxisome	0.009158
IL-2/STAT5 signaling	0.02053
Estrogen response late	0.02093
Fatty acid metabolism	0.0361
Interferon α response	0.04097
Myogenesis	0.07301
Downregulated genes
Myc targets V1	1.74E-87
mTORC1 signaling	3.07E-33
Oxidative phosphorylation	4.56E-27
Myc targets V2	1.04E-17
Unfolded protein response	1.05E-17
E2F targets	1.17E-17
Adipogenesis	1.39E-12
DNA repair	2.07E-12
Protein secretion	2.04E-11
Fatty acid metabolism	7.59E-10

All of the upregulated and downregulated genes were analyzed by Enrichr, and the top 10 pathways associated with these gene changes based on Molecular Signature Database 2020 are listed by P value. ATII, type II alveolar epithelial cells; E. coli, Escherichia coli; LIF, leukemia inhibitory factor.

Table 2.

Differential expression analyses were conducted on anti-LIF vs. IgG E. coli for endothelial cells by BioTuring Browser

Pathway Name	P Value
Upregulated genes
TNF-α Signaling via NF-κB	1.08E-16
Hypoxia	2.53E-09
p53 pathway	5.24E-08
Adipogenesis	0.00018
Reactive oxygen species pathway	0.00748
UV response up	0.00081
Estrogen response early	0.00194
Cholesterol homeostasis	0.01648
Apoptosis	0.00894
Notch signaling	0.08147
Downregulated genes
Myc targets V1	4.40E-59
Oxidative phosphorylation	2.80E-30
Unfolded protein response	2.15E-11
Interferon γ response	3.60E-10
mTORC1 signaling	1.01E-09
Myc targets V2	1.95E-08
Adipogenesis	5.04E-08
Protein secretion	5.11E-08
Interferon α response	6.90E-08
PI3K/AKT/mTOR signaling	6.31E-07

All of the upregulated and downregulated genes were analyzed by Enrichr, and the top 10 pathways associated with these gene changes based on Molecular Signature Database 2020 are listed by P value. ATII, type II alveolar epithelial cells; E. coli, Escherichia coli; LIF, leukemia inhibitory factor.

Table 3.

Differential expression analyses were conducted on anti-LIF vs. IgG E. coli for mesenchymal cells by BioTuring Browser

Pathway Name	P -Value
Upregulated genes
Glycolysis	0.0137
mTORC1 signaling	0.0137
Cholesterol homeostasis	0.06457
IL-6/JAK/STAT3 signaling	0.0755
Pperoxisome	0.08961
PI3K/AKT/mTOR signaling	0.09044
Unfolded protein response	0.097
Spermatogenesis	0.1148
Coagulation	0.1172
UV response up	0.1331
Downregulated genes
Coagulation	0.00051
Epithelial mesenchymal transition	0.00151
TGF-β signaling	0.00175
Xenobiotic Metabolism	0.02193
TNF-α signaling via NF-κB	0.02193
Hypoxia	0.02193
heme metabolism	0.02193
Complement	0.02193
Inflammatory response	0.02193
IL-6/JAK/STAT3 signaling	0.09545

All of the upregulated and downregulated genes were analyzed by Enrichr, and the top 10 pathways associated with these gene changes based on Molecular Signature Database 2020 are listed by P value. ATII, type II alveolar epithelial cells; E. coli, Escherichia coli; LIF, leukemia inhibitory factor.

DISCUSSION

We previously showed that LIF contributes to lung tissue protection during bacterial pneumonia, which has since been corroborated in a model of viral pneumonia (3, 4). We also discovered that ATII cells are the predominant source of LIF production during pneumonia (9). However, where and how LIF signaling elicits lung protection is unknown. The results of this study, to our knowledge, are the first to report cellular targets of LIF, along with corresponding LIF-dependent effects. Our findings demonstrate that epithelial LIFR signaling contributes to tissue protection during pneumonia, corresponding with LIF-dependent regulation of pulmonary apoptosis. While pharmacological administration of exogenous rmLIF did not generate significant protection during bacterial pneumonia, the role of host-derived LIF in lung tissue protection remains important. ScRNAseq also supported LIF-driven effects in ATII cells, including those downstream of STAT3, whereas results from this analysis also implicate endothelial and mesenchymal cells as additional targets of LIF.

To date, LIF biology has primarily been studied in the contexts of stem cell biology, reproduction, and various cancers (27, 37–39). Recently, the importance of LIF-STAT signaling in intestinal epithelium has come to light, in which intestinal epithelial cells were shown to secrete LIF in a mouse colitis model, leading to STAT3 activation and the promotion of repair mechanisms (40). This study followed a separate publication that had established the importance of gp130—the common coreceptor of all IL-6 family cytokines—to intestinal epithelial proliferation in the context of inflammatory bowel diseases (41). In contrast, little is known about the role of epithelial LIF in the context of pneumonia or lung injury. It has been observed that LIF is detected in BALF of patients with ARDS, but the role it plays in disease pathogenesis remains unclear (42). LIF has also been detected in epithelial cells and mesenchymal cells of the upper airways in human lung tissues and cultured cells in response to proinflammatory stimuli, while LIFR was consistently expressed at baseline regardless of further stimulation (43). This prior report suggested that LIF may be involved in the regulation of airway inflammation, perhaps involving the neuroimmune pathways of asthma (44). However, our current study is the first to test epithelial LIF signaling in connection with apoptosis and identifying distal lung cells that express Lifr mRNA in adult mice, indicating potential targets of LIF signaling during pneumonia.

Alveolar epithelial cells, endothelial cells, and mesenchymal cells are the main components of the alveolar-capillary barrier—the primary site of gas exchange, fluid regulation, and immune infiltration during acute lung injury (45). Correspondingly, our results reveal these three cell types as the most prominent sites of Lifr mRNA expression, suggesting that LIF activity may aid the coordination of cellular homeostasis at this interface. ATII cells, specifically, are competent immune cells that are capable of sending and receiving immunomodulatory signals such as cytokines and chemokines (1, 6, 10). Indeed, the immune capacity of epithelial cells is supported by our current findings, which illustrate a substantial shift in cellular transcriptomics in response to pneumonia. Simultaneously, the endothelium is the first barrier to vascular fluid and inflammatory cells that are recruited to the injured lung. Loss of endothelial barrier integrity leads to an accumulation of edema in the lungs, characteristic of pneumonia (45–47). Our scRNAseq data indicated that Lifr expression on endothelial cells was higher than on any other cell type. LIF signaling in endothelium may directly and more significantly contribute to tissue protection during pneumonia as compared with epithelial LIF based on the outcomes observed in EpiLIFR^Δ/Δ mice, which exhibited histological evidence of injury without detectable differences in alveolar edema. It should be noted that incomplete deletion of LIFR in these mice or other LIFR ligands may have contributed to this phenotype. Although the system has its limitations, we and others have reported that the NKX2.1-CreER is highly efficient (29). Furthermore, an alternative LIFR ligand such as oncostatin M (OSM) is extremely unlikely to cause our observed phenotype because its affinity for LIFR is much lower than that of its own receptor OSMR (48), and the consequences of OSM blockade, as reported by our group previously (17), are different from those following LIF manipulation. In a similar vein, it is also entirely possible that cells neighboring the epithelium (e.g., endothelial and/or mesenchymal cells) are on the receiving end of LIF, and that such responses indirectly impact epithelial cells in a manner that extends beyond any signals directly engaged by epithelial LIFR. Future investigations in our laboratory will consider the influence of LIFR on pulmonary endothelial cells and perhaps others during pneumonia. To our knowledge, our scRNAseq data are also the first to explore the effects of E. coli pneumonia on the entire murine lung landscape. Most notably, ATII cells cluster differently in response to pneumonia while immune cells such as alveolar macrophages are less represented in pneumonic conditions and neutrophils increase in response to pneumonia. These findings not only support our hypothesis that ATII cells are significantly involved in the response to infection but also suggest that other gene changes outside of the LIF/STAT3 pathway may also be involved.

Protective roles of endogenous LIF have now been established during pneumonia (3, 4) as well as endotoxic shock (49) establishing a precedent for leveraging exogenous LIF as a treatment platform. For instance, administration (50) or overexpression (51) of LIF confers protection in response to LPS or hyperoxia, respectively, and several studies now suggest LIF as a potential therapeutic in the setting of neurodegenerative disease (52, 53). We previously reported that intrapulmonary delivery of rmLIF is sufficient to yield a substantial STAT3 response in lung tissue, including the epithelium (3). In the current study, however, rmLIF had only a modest effect on pneumonia outcome, with significant decreases in injury observed histologically but not by other measures such as alveolar edema or cytokine induction, both of which were markedly exaggerated in our prior studies of LIF blockade (3). These results suggest that endogenous LIF levels are sufficient to confer the maximum protection afforded by LIF in response to respiratory infection. We acknowledge, however, that these results must be interpreted with caution, given the possibility of missed effects due to our selected dose or timing regimen.

When considering the impact of pharmacological LIF blockade on all epithelial cells, transcriptomics revealed a scenario in which cells were more reactive to pneumonia (based on elevated transcriptional differences), including gene signatures suggestive of apoptosis and cell death. Yet, these transcriptional events did not equate to detectable differences in the number of annexin V+ apoptotic epithelial cells recovered from anti-LIF-treated mice. While this could be related to technical constraints involved in the recovery of intact epithelial cells from pneumonic lungs, it also suggests that the stress-associated gene alterations in epithelial cells following LIF blockade were insufficient to yield cell death under the experimental conditions tested. Indeed, this is further evidenced by exaggerated TUNEL+ cells that did not distinctly colocalize with ATII, ATI, or endothelial cells. The latter finding again highlights regulation of apoptosis as a potential mechanism underlying LIF-mediated tissue protection but suggests that this effect may be more complex, involving both direct and indirect consequences of LIF signaling. Our own and other’s data consistently indicate the capacity of LIF to activate STAT3 via phosphorylation of Y705 both in vitro and in vivo (3, 6), and this has been shown to mediate both pro- and antiapoptotic signaling. For example, STAT3 modulates apoptosis-related signals such as Fas, Bcl-2, Bcl-xL, and NF-κB (54–56), and failure to initiate this response in lung epithelium reproducibly promotes lung injury (6–8, 18, 54). Furthermore, based on reports of LIF eliciting YAP activity in intestinal epithelium and various cancers (40, 57), the Hippo pathway, which has known homeostatic effects on lung epithelium, may also be involved downstream of LIF during pneumonia. Meanwhile, endothelial STAT3 has also been shown to limit lung injury (58), again supporting this as a candidate liaison for LIF-driven protection. Depending on the cell type or the downstream pathways that are activated, LIF and LIFR have been shown to have opposing effects, suggesting that this cytokine and its receptor are likely context-specific involving multiple cell types and various functional roles. For example, LIF neutralization seems to downregulate “PI3K/AKT/mTOR signaling” in endothelial cells but upregulate it in mesenchymal cells. However, we also observed shared pathways such as upregulated “p53 pathway” and “apoptosis” between epithelial and endothelial cells as a result of LIF blockade, suggesting potential mechanisms behind LIF-mediated tissue protection that require further study.

To our knowledge, our findings are the first to identify cellular sources of Lifr mRNA with and without pneumonia and are also the first to demonstrate epithelial-specific consequences of LIF signaling in this context. The mechanisms downstream of LIF, STAT3, and/or other LIF-driven signaling intermediates are yet to be determined, but we anticipate that future studies involving alternative cell types may lead to novel clinical interventions for patients with or at risk for bacterial pneumonia.

DATA AVAILABILITY

Data will be made available upon reasonable request. Source data are available at NCBI Gene Expression Omnibus (accession #: GSE179855; GSE179764; http://www.ncbi.nlm.nih.gov/geo/).

SUPPLEMENTAL DATA

Supplemental Figs. S1–S3 and Supplemental Tables S1–S6: https://doi.org/10.6084/m9.figshare.15078915.

GRANTS

The study was supported by National Institutes of Health (NIH) Grants R01-HL111449, R01-GM120060, R01-HL124392, R35-HL135756, R01-AI115053, R33-HL137081, and T32-HL007035.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.N., M.R.J., D.N.K., J.P.M., X.V., and K.E.T. conceived and designed research; E.N., E.A., L.A.B., F.T.K., A.T.S., A.M.M., and K.E.T. performed experiments; E.N., E.A., L.A.B., A.T.S., and K.E.T. analyzed data; E.N., A.T.S., M.R.J., D.N.K., J.P.M., X.V., K.E.T., and L.J.Q. interpreted results of experiments; E.N. prepared figures; E.N. drafted manuscript; E.N., E.A., L.A.B., C.V.O., F.T.K., A.T.S., A.M.M., M.R.J., D.N.K., J.P.M., X.V., K.E.T., and L.J.Q. edited and revised manuscript; E.N. and L.J.Q. approved final version of manuscript.