Alveolar epithelial cell growth hormone releasing hormone receptor in alveolar epithelial inflammation

Abstract

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells’ gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-β and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-β, TNF-α, TGFβ-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

Keywords:

• Inflammation
• growth hormone releasing hormone
• MIA-602
• alveolar epithelial cells
• mesenchymal
• post-inflammatory fibrosis

Introduction

Acute lung injury characterized by inflammation and edema occurs in many clinical contexts, and it is sometimes followed by pulmonary fibrosis. Alveolar epithelial type 2 cells (AT2 cells) respond to alveolar injury by proliferating and relining alveolar surfaces denuded of alveolar type 1 epithelial cells (AT1 cells).¹ AT2 cells re-epithelialize the alveolar surface and transdifferentiate into AT1 cells. AT2 cells undergo transition to intermediate epithelial and progenitor-like states.² After diverse injuries, they differentiate into AT1-like cells through a pre-alveolar type 1 transitional state. AT2 cells may also transdifferentiate into metaplastic basal cells, through intermediates near fibroblasts and Wnt-responsive epithelial progenitors.³

Growth hormone releasing hormone receptor (GHRH-R) is present in lung tissue and is expressed in AT2 cells. Several lines of evidence mechanistically link the GHRH-R with inflammation. Inhibition of the GHRH-R using an antagonistic peptide (e.g., MIA-602) reduced inflammation and fibrosis in lungs of mice treated with bleomycin.⁴ GHRH-R antagonist inhibits JAK/STAT signaling and Th17 cell differentiation reducing ocular and neural inflammation.⁵ GHRH-R antagonists also protect the lung vasculature from unfolded protein response, maintain endothelial barrier integrity and have anti-inflammatory effects in sarcoid granulomas.^6–8 Pro-inflammatory JAK/STAT and NF-kB pathways are linked to GHRH-R activation (e.g., by GHRH-R agonist MR-409) and are downregulated by GHRH-R antagonists (e.g., by MIA-602).^9–11

We employed human iAT2 cells to determine mechanisms by which LPS or pro-inflammatory cytokines typically found in lungs could modulate iAT2 cell expression of pro-inflammatory mediators and mesenchymal markers. These cells, derived from human iPSC, provide a uniquely useful model that is reproducible and remains consistent from experiment to experiment.^12,13 While AT2 cells obtained from explanted human lungs are sometimes available, their use may be limited by the availability of tissue, the possible presence of disease and limited proliferation in culture.^14,15

We tested whether exposure to LPS or cytokines led to iAT2 cell expression of pro-inflammatory cytokines. We also investigated the effects exogenous cytokines on the expression of mesenchymal markers by iAT2 cells. We hypothesized that AT2 cells participate actively in inflammation and could also promote fibrosis by producing local pro-inflammatory signals in response to injury. The overall goal of this work has been to determine specific mechanisms by which the GHRH-R in iAT2 cells facilitates inflammation, which could subsequently lead to post-inflammatory pulmonary fibrosis through intercellular signaling.

Methods

Peptides

GHRH antagonist MIA-602 [(PhAc-Ada)⁰, D-Arg^2,28, Fpa₅⁶, Ala⁸, Har^9,29, Tyr(Me)¹⁰, His^11,20, Orn^12,21, Nle²⁷]-hGHRH(1-29)-NH₂ and agonist MR-409 (N-Me-Tyr¹,D-Ala², Phe⁶, Asn⁸, Orn¹², Abu¹⁵, Orn²¹, Nle²⁷, Asp²⁸, Arg²⁹-NH-Me) was synthesized in the laboratory as described.¹⁶

Purification of crude peptides was done on a Beckman Gold HPLC system (Beckman Coulter, Inc., Brea, CA) equipped with a 127 P solvent Module and 166 P UV–vis Detector using an XBridge™ reversed phase column (10 × 150 mm packed with C4 silica gel, 130 Å pore size, 5 µm particle size [Waters, Milford, MA]). Peptides were eluted with 0.1% aqueous TFA and 0.1% TFA in 70% aqueous acetonitrile (MeCN) in a linear gradient.

iAT2 cell model

Human iAT2 cells (SFTPC^tdTomato+) were obtained from the Center for Regenerative Medicine at Boston University School of Medicine.^12,13 Cells were maintained in CK + DCI medium (referred to hereafter as cell culture medium), which comprises GSK-3 inhibitor CHIR9902 (3 μM), recombinant human keratinocyte growth factor (rhKGF) (10 ng/mL) and dexamethasone (50 nM).

Initially, cells were treated with 500 ng/mL LPS (E. coli O55:B5, Sigma) alone or LPS plus 1 or 5 μM receptor antagonist MIA-602 for 48 h. This time point was chosen empirically because the cells remained viable and gene expression increased sufficiently to be readily detected. Each treatment was used in 8 wells/plate. Each experiment was repeated at least three times (separate plates on different days) with similar results.

In subsequent experiments, iAT2 cells were incubated for 48 h with a mixture of pro-inflammatory cytokines like those found in bronchoalveolar lavage fluid (BALF), including TGFβ-1 (300 pg/ml], IL1-β (10 pg/ml), TNF-α (100 pg/ml), IL-8 (1500 pg/ml), MCP1 (700 pg/ml), IL-33 (40 pg/ml), TSLP (100 pg/ml), IL-13 (2500 pg/ml) and IL-4 (160 p/ml).¹⁷ Cytokines were dissolved in medium, and then incubated with iAT2 cells as a “cocktail” for 48 h. Cells remained viable in these conditions, allowing assessment of both gene and protein expression. Each treatment was used in 8 wells/plate. The experiment was repeated at least three times (separate plates) with similar results.

RNA was isolated subsequently for gene expression assays. Cellular lysates were also harvested to obtain protein for western blotting.

Single cell RNA-sequencing

scRNA-seq libraries were prepared per the Single Cell 3′ Reagent Kit User Guide v2 (10× Genomics).¹⁸ We used Cell Ranger version 1.3.1 (10× Genomics) to process raw sequencing data and Cell Ranger R kit version 2.0.0 with Seurat suite version 2.0.0 for downstream analysis. For clustering, principal-component analysis was performed for dimension reduction. Cell Ranger R kit was used for modeling gene expression with negative binomial distribution to identify genes enriched in specific clusters. Benjamini-Hochberg procedure was used for correcting errors of multiple testing.

Cell proliferation assay

Cell proliferation assays were done in cell culture medium, described above, with or without LPS or cytokines. iAT2 cells (1 × 10⁴) were plated in Matrigel-coated 96-wells using cell culture medium. Cells were treated with 1 or 5 µM native GHRH or its analogs, agonist MR-409 or antagonist MIA-602, for four days. In controls, an equal volume of peptide dissolved in buffer (0.5% DMSO in PBS) was added. Viable cells at the end of treatment were quantified using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt)-based chemiluminescent method (Promega).¹⁹ During a 1-h incubation, viable iAT2 cells converted tetrazolium into a formazan product. Absorbance at 490 nm was recorded using a 96-well plate reader. The absorbance was proportional to the number of cells in proliferation assays. Each treatment was used in 8 wells/plate. The experiment was repeated at least three times (separate plates) with similar results.

Western blots

Protein from cell lysates was electrophoresed on 4–12% SDS-PAGE gels and transferred to an Immobilon-P PVDF membrane (Millipore). 20 µg of protein were loaded in each lane. Membranes were blocked with 5% nonfat milk, then incubated with specific primary antibodies overnight at 4 °C. The following primary antibodies were used at appropriate dilutions to probe western blots: SP-B (Santa Cruz Biotech), SP-C (Seven Hills Bioreagents), Cytokeratin 5 (Abcam), N-cadherin (Cell Signaling Technology) Vimentin (Abcam) GAPDH (Santa Cruz Biotech). See Supplementary Data for details. After incubation with secondary antibody, bands were visualized using Clarity Western ECL Substrate (Bio-Rad).

Each western blot was repeated on at least three separate occasions. Representative examples are shown in the figures. For quantification, digital images obtained by C-DiGit Blot Scanner (LI-COR) were analyzed using Image Studio software.

Normal and IPF lung tissue

Normal lung tissue lysates (n = 2) were obtained from Cell Sciences, Newburyport, MA and IPF lung tissues (n = 2) were obtained from the National Heart, Lung and Blood Institute (NHLBI) Biologic Specimen and Data Repository (BioLINCC), Bethesda, MD. De-identified tissue acquisition was approved by the Miami VAHS Institutional Review Board. We used normal lung tissue and lung tissue from patients with IPF only to demonstrate the presence of the GHRH-R in human lungs, as lung tissue from patients with post-inflammatory pulmonary fibrosis is typically not available.

Quantitative RT-PCR

RNA was isolated using RNeasy Mini kits (Qiagen). One µg of total RNA was reverse transcribed into cDNA using iScript cDNA Synthesis kits (Bio-Rad). For quantitative real-time PCR, 0.05–5 ng cDNA and 0.5 µM of each primer in 20 µl of 1× iQ SYBR Green Supermix (Bio-Rad) were amplified using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). All reactions were done in triplicate, and gene expression analysis conducted using CFX Manager software (Bio-Rad). Representative examples are shown.

Immunofluorescence

iAT2 cells were harvested and plated on Matrigel-coated cover glasses. Cells were incubated with primary antibody overnight at 4° C (1:250 dilution in 1× PBS/1% BSA/0.1% Triton X-100). The next day, cells were rinsed with PBS and incubated with secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or tetramethyl rhodamine (TRITC) (1:1000 dilution). After PBS washing, nuclei were stained with DAPI. Slides were mounted with Prolong Gold Antifade Reagent (ThermoFisher). Images were captured with a Nikon Eclipse Ti microscope.

Signaling pathway analysis

iAT2 cells were incubated with the cytokines described above for 48 h. RNA isolated was used in the RT² Profiler PCR Array (Human Signal Transduction PathwayFinder, Qiagen) to explore signaling pathways activated by exposure to pro-inflammatory cytokines and growth factors described above.

Data analysis

All data are reported as means ± standard error of the mean (SEM) or standard deviation (SD) as described. Student’s t test (two-tailed) was used for comparing two independent groups when the distribution was normal, and the Mann-Whitney U test was used when not normal. The Shapiro-Wilk test was used to assess normality. Multiple group comparisons were done by one-way or two-way ANOVA followed by Dunnett’s or Bonferroni’s post-hoc tests for differences between groups.²⁰ Statistical analyses were done using GraphPad Prism (GraphPad Software, Boston, MA).

Results

iAT2 cell phenotype

We used human alveolar type 2 epithelial cells derived from induced pluripotent stem cells (iAT2 cells). We extensively phenotyped the iAT2 cells to confirm their identity and function. Cells were cultured on Matrigel, where they formed small monolayers or spheroidal groups. On immunofluorescent staining, the cells expressed surfactant protein C and surfactant protein B.

Western blot data shown in Figure 1 confirm qualitatively that these cells produce surfactant protein C (SP-C), surfactant protein B (SP-B), lysophosphatidylcholine acyltransferase 1 (LPCAT1) and HTII-280, consistent with their alveolar epithelial lineage.^21,22

Figure 1. Expression of alveolar epithelial cell type 2 markers in iAT2 cells derived from human iPSC. Each specific protein was detected by western blotting. Normal human lung tissue was used as the positive control (Lane 1). iAT2 cells expressed both surfactant proteins B and C, confirming differentiation (Lane 2). LPCAT1, Lysophosphatidylcholine Acyltransferase 1; HTII-280, 280–300 kDa protein specific to apical surface of AT2 cells; SP-B, Surfactant Protein B; SP-C, Surfactant Protein C.

Single cell RNA sequencing (RNA-seq) of iAT2 cells

We completed single cell RNA-seq of iAT2 cells that had been maintained in cell culture medium or treated with the mixture of cytokines for 48 h. Several arbitrarily defined clusters (seven are shown) of iAT2 cells were defined by principal component analysis.

One population (cluster 2 at the displayed resolution) was characterized by expression of typical markers for AT2 cells, including SFTPB, SFTPC, SFTPA1, SLC34A2, CTSH, and NAPSA. iAT2 cells in cell culture medium also expressed HOPX and KRT19. iAT2 cells, however, did not express typical AT1 markers such as AQP3 and GRAMD2A. In these conditions, iAT2 cells showed no or low expression of transdifferentiation-related genes (SNAI1, PDGFA, KRT5, KRT17, ITGB6, TIMP2), Wnt pathway-related genes (WNT5A, MMP7) interleukin signaling genes (IL1A, CXCL6, CXCL8), ECM-remodeling associated genes (ACTA2, COL1A1, TNC, PLAU), and conducting airway epithelial markers (SOX2, MUC5B).^23,24 Incubation with cytokines did not significantly change the clusters defined by PCA. However, changes in gene expression were detected (Figure 2a).

Figure 2. iAT2 cell single cell RNA sequencing. (a) Single cell RNA-seq revealed multiple clusters within the iAT2 cell population. (b) Volcano plot showing the six most prominently down (left) and up (right) regulated genes after cytokine exposure. As examples, Cellular communication network factor 1, SRY-box transcription factor 4, Early growth response 1, KLF transcription factor 6, Fibronectin 1 and Claudin 18 were markedly over expressed. Cell adhesion molecule 6, S100 calcium binding protein P, Ferritin heavy chain 1, Solute carrier family 34 member 2, Chitinase 3 like 2 and Cystatin B were markedly under expressed.

The volcano plot (Figure 2b) further elucidates differential gene expression due to the cytokines. Examples of genes prominently down regulated are shown at left of the plot, and examples of genes prominently up regulated are shown at the right of the plot. Some of the most significantly down regulated genes included CCN1, SOX4, EGR1, KLF6, FN1 and CLDN18. Some of the most significantly upregulated genes included CEACAM6, S100P, FTH1, SLC34A2, SLC6A14 and CHI3L2.

The heat maps (Figure 3) qualitatively demonstrate the effects of cytokine exposure on gene expression, which confirms effects of cytokines causing up regulation and down regulation of multiple genes differentiating the two conditions.

Figure 3. iAT2 cell single cell RNA sequencing. Heat map showing genes highly expressed in clusters. Surfactant proteins B and C were highly expressed in cluster 2, as was HOPX, consistent with the cells’ alveolar epithelial differentiation. Differential gene expression due to cytokines is shown qualitatively by the distribution of up and down regulated genes on the heat maps and by the prominently expressed genes listed in the text columns to the left of each heat map.

Several gene ontology changes due to cytokines were also evident. Qualitative examination of gene ontology showed enrichment of genes related to interferon signaling, GTPase activity, TGF-β receptor signaling among others due to exposure to the cytokines.

Expression of GHRH-R in lung tissue and cells

We qualitatively assessed the expression of GHRH-R in lungs from normal humans and lungs from patients with IPF. As shown in Figure 4, normal human lung tissue (lanes 1 and 2) expressed full length pituitary type GHRH-R (pGHRH-R), while IPF lung tissue (lanes 3 and 4) expressed predominantly SV1, the splice variant of pGHRH-R known to support fibroblast proliferation.²⁵ iAT2 cells (lane 5), normal lung fibroblasts (lane 6) and IPF lung fibroblasts (lane 7) also expressed SV1. These data confirm the presence of pituitary type GHRH receptor or its functional splice variant in normal and diseased human lungs, implying their clinical relevance.

Figure 4. Expression of GHRH-R in lung tissues and iAT2 cells. Western blotting was done to confirm protein expression of the GHRH receptor in lung tissues and iAT2 cells. The full-length pituitary type GHRH-R (pGHRH-R) was predominantly expressed in normal lungs, while the truncated splice variant 1 (SV1) was expressed strongly in lungs from patients with IPF, as well as in cultured iAT2 cells, normal lung fibroblasts and fibroblasts isolated from patients with IPF.

iAT2 cell proliferation is modulated by GHRH-R

We tested the proliferative response of iAT2 cells to GHRH-R activation or inhibition. As shown in Figure 5a, both GHRH and its agonistic analog, MR-409, stimulated proliferation of iAT2 cells in a dose-dependent manner, while MIA-602, an antagonist of GHRH-R, inhibited proliferation (data shown are the percent increase compared to medium alone [100%]). In the presence of pro-inflammatory cytokines, iAT2 cell proliferation was significantly reduced but was partially restored by GHRH and MR-409 (Figure 5b) (data shown are the percent increase compared to the cytokine mix [100%]). These data clearly demonstrate that the GHRH-R is physiologically expressed in lung epithelium and that modulation of the GHRH-R may protect iAT2 cells’ proliferative capacity from damage caused by inflammation.

Figure 5. Effect of GHRH and peptide analogs on proliferation of iAT2 cells. (a) GHRH and its agonist, MR-409, stimulated proliferation of iAT2 cells in CK + DCI medium, while GHRH antagonist MIA-602 significantly decreased proliferation of iAT2 cells, demonstrating a trophic role for the GHRH-R in iAT2 cells. (b) Growth inhibition, induced by cytokines (CK), was partially reversed by native GHRH peptide or its agonist, MR-409. These results indicate that GHRH-R activation protects iAT2 cells from growth inhibition by cytokines. (Data are means ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 and ns, not significantly different; one-way ANOVA and Bonferroni’s correction.).

Effects of pro-inflammatory LPS on iAT2 cell gene expression

To test whether LPS could increase inflammatory and profibrotic gene expression, iAT2 cells were incubated with LPS (500 ng/ml) in cell culture medium. As shown in Figure 6, LPS significantly increased gene expression of IL1-β and TNF-α by iAT2 cells. These increases were effectively inhibited by GHRH-R antagonist MIA-602.

Figure 6. iAT2 cells express pro-inflammatory genes after incubation with LPS. iAT2 cells were incubated with LPS (500 ng/ml) alone or LPS plus 1 or 5 µM MIA-602 for 48 h. Quantitative RT-PCR was used to analyze the expression of IL-1β, TNF-α and TGF-β1 in the iAT2 cells. Compared to non-treated cells, iAT2 cells treated with LPS showed significantly enhanced expression of IL-1β and TNF-α (but not TGF-β1). Incubation with GHRH-R antagonist, MIA-602, at the same time significantly counteracted the effect of LPS. (Data are shown as medians with the upper/lower quartiles in the Box and Whisker plots, LPS vs LPS + MIA-602 ***, p < 0.001, ****, p < 0.0001; ns, not significantly different; One-way ANOVA and Bonferroni’s correction.).

We also assessed if LPS could affect the expression of selected genes associated with epithelial-mesenchymal transition. As shown in Figure 7, LPS significantly increased the expression of ACTA2, CTGF, SERPINE1, FN1, COL1A1, TNC, WNT5A and MMP7, indicating possible mesenchymal reprogramming of iAT2 cells. MIA-602, the GHRH-R antagonist, inhibited these increases in a way that would prevent amplification of pro-fibrotic signaling.

Figure 7. iAT2 cells express mesenchymal genes after incubation with LPS. iAT2 cells were incubated with LPS (500 ng/mL) alone or LPS plus 1 or 5 µM MIA-602 for 48 h. RNA was isolated and used for RT-PCR to detect expression of ACTA2, CTGF, SERPINE1, FN1, COL1A1, TNC, WNT5A and MMP 7 genes by the iAT2 cells. In each case, LPS significantly enhanced expression of genes typical of mesenchymal differentiation, and GHRH-R antagonist, MIA-602, suppressed the LPS-induced mesenchymal gene expression. (Data are shown as medians with the upper/lower quartiles in the Box and Whisker plots, LPS vs LPS + MIA-602 ***, p < 0.001, ****, p < 0.0001; One-way ANOVA and Bonferroni’s correction.).

Effects of cytokines on iAT2 cell gene expression

We used the cytokines described above to test whether those would affect gene expression of pro-inflammatory and mesenchymal genes.¹⁷ Gene expression of pro-inflammatory mediators including IL1-β, TNF-α and TGF-β1 were significantly elevated after incubation with cytokines. As shown in Figure 8, the cytokines also increased extracellular matrix genes (FN1, COLA1A1, TNC), myofibroblast activation-associated genes (ACTA2, CTGF, SERPINE1, MMP7) and noncanonical Wnt ligand WNT5A.

Figure 8. Cytokines stimulate iAT2 cell expression of pro-inflammatory mediators. iAT2 cells were incubated with cytokines for 48 h. Quantitative RT-PCR revealed significant increases in genes related to extracellular matrix (ECM) deposition and myofibroblast activation. The increase of pro-inflammatory cytokine expression suggested positive feedback, stimulating an inflammatory response by iAT2 cells. (Data are means ± SEM; ****, p < 0.0001; Unpaired student t-test.).

Cytokines promote mesenchymal protein expression in iAT2 cells

We treated iAT2 cells with the cytokine mixture every 48 h. After two weeks, in the absence of cytokines, iAT2 cells appropriately expressed surfactant protein B and N-cadherin. iAT2 cells treated with cytokines decreased expression of surfactant protein B and highly expressed cytokeratin 5, N-cadherin and vimentin (Figure 9). These data suggested that cytokines stimulated iAT2 cells to both undergo basal cell transdifferentiation and become more mesenchymal in character.

Figure 9. iAT2 cells treated with cytokines expressed mesenchymal proteins. iAT2 cells were treated with cytokine cocktail every other day for 2 wk. The loss of AT2 marker SP-B and expression of cytokeratin 5, N-cadherin, and vimentin were consistent with transdifferentiation toward a basal cell or myofibroblast phenotype and expression of mesenchymal markers. The band intensities, after being normalized to GAPDH, indicated a 6.9-fold decrease in SP-B and 1.2-fold increase in N-cadherin. The increases in cytokeratin 5 and vimentin were not calculated because they are barely detectable in the cells not treated with cytokines.

Cytokines activate pro-inflammatory pathways in iAT2 cells

iAT2 cells were treated as above with cytokines for 48 h. RNA was isolated and used in Human Fibrosis RT² Profiler PCR array and Human Signal Transduction PathwayFinder RT² Profiler PCR arrays (Qiagen). In the fibrosis array, the most changed genes were associated with ECM remodeling (EDN1, SERPINE1, PLAU, ITGB6, ITGB8, ITGAV, MMP3 and TIMP2) and pro-fibrotic signaling (CAV1, SMAD3, PDGFA/B, IL1A, SNAI1). Several pathways, including Wnt/B-catenin and NF-kB, which have been shown to have pro-inflammatory effects in a variety of cells, were activated by cytokines in iAT2 cells.^26–28 Based on gene expression (>300% compared to vehicle-treated), the following pathways were activated by cytokines: Wnt signaling (MMP7 [1204%], CCND2 [435%]); NF-kB signaling (BIRC3 [566%], ICAM1 [521%], CCL5 [395%], IFNG [310%], TNF [301%]); and, JAK/STAT signaling (SOCS3 [341%], IRF1 [312%]).

Discussion

Growth hormone, the production of which is regulated by GHRH-R, is required for alveolar epithelial cell homeostasis.^29,30 The GH receptor is expressed in lung fibroblasts, and its expression is substantially decreased in IPF lungs. The GH axis supports AT2 progenitor renewal, limiting severity of lung fibrosis after alveolar denudation. Given the potential importance of GHRH-R signaling in the lung, the objectives of our work were to delineate mechanisms by which the GHRH-R could modulate inflammation and possible subsequent fibrosis through activity in iAT2 cells.

GHRH belongs to a peptide family that includes glucagon, secretin, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). GHRH-R binds GHRH and stimulates the production of GH essential for development and tissue repair. GHRH is secreted primarily by the hypothalamus, but many tissues, including the lungs, produce it locally.²⁹

GHRH exerts multiple effects relevant to lung growth and repair. GHRH-R antagonists inhibit tumor stromal fibrosis in experimental models.^31,32 GHRH-R is expressed in AT2 cells from normal human subjects and patients with IPF. GHRH-R protein is distributed throughout normal mouse lungs and is readily detected on cultured iAT2 epithelial cells (Cui et al., unpublished data). GHRH-R and its physiologically active splice variant, SV1, are differentially expressed in normal human lungs and in lungs from patients with IPF. SV1 expression facilitates fibroblast proliferation in response to exogenous GHRH. GHRH-R agonists increase fibroblast proliferation and accelerate wound healing.³² GHRH also stimulates the expression of α-smooth muscle actin (α-SMA), which confers contractile activity in myofibroblasts. GHRH and similar peptides regulate the proliferation of fibroblasts through the modulation of extracellular signal-regulated kinase (ERK) and PI3/Akt pathways.³¹

We used cultured, human AT2 cells derived from induced pluripotent stem cells to investigate whether these underwent changes in phenotype when subjected to pro-inflammatory LPS or cytokines. iAT2 cells we used expressed epithelial markers SFTPB, SFTPC and HOPX, in addition to KRT17, KRT19, CTGF and FN1 on single cell RNA-sequencing. We therefore used these cells as a previously validated model of AT2 cells that could reveal mechanisms of post-inflammatory lung fibrosis, although this model does not fully recapitulate the lung in vivo, as airway, vascular, mesenchymal and inflammatory cells are not present.

AT2 cells serve normally as progenitors of basal and AT1 cells, which can reline injured epithelial surfaces. AT2 cells in situ coexist and interact with other cells including AT1 cells, alveolar macrophages, fibroblasts and immune cells. AT2 cells may differentiate toward a mesenchymal phenotype, as demonstrated histologically in an LPS mouse lung injury model.¹ After treatment of mice with LPS, the alveolar surface is relined by AT2 cell derived AT1 cells, which limit vascular permeability.

Epithelial cells have been assumed capable of transitioning toward a mesenchymal phenotype.³³ Transition is characterized by loss of epithelial markers including e-cadherin, occludens, claudins and cytokeratins, followed by expression of mesenchymal markers, including N-cadherin, vimentin, fibronectin, integrins and matrix metalloproteinases. Our data confirmed some of these reported phenotypic changes in iAT2 cells after LPS or cytokine exposure, which represent a response to the stress of inflammatory mediators.

Although most surviving patients with acute lung injury undergo resolution of permeability edema and inflammation, some develop a fibrotic lung phenotype. Rather than progressing to resolution of alveolar epithelial injury, myofibroblasts proliferate in the interstitial space impairing gas exchange and resulting in abnormal remodeling, which can cause persistent structural and functional respiratory defects.³⁴

AT2 cell injury produces subsets of epithelial cells with characteristics of basal and AT1 cells.^35,36 Progenitors responsive to Wnt signaling regenerate the alveolar epithelium after inflammation, and blocking Wnt/β-catenin attenuates bleomycin-induced fibrosis.³⁷ AT2 cells may transdifferentiate into pathologic fibroblasts in response to TGF-β1; AT2 cells can also transdifferentiate into metaplastic alveolar KRT5⁺ basal cells through alveolar-basal intermediates.³⁸ Transdifferentiation of AT2 cells toward AT1 cells (HOPX positive) is an important mechanism that may allow repair of the alveolar epithelial surface.^39,40

Expression of basal and mesenchymal characteristics is an important response of AT2 cells to stress. Their ability to produce both inflammatory and fibrotic mediators like TGF-β1 is a novel finding and an important mechanism that could amplify further damage.

We recognize the obvious limitations of these conceptual and experimental approaches. Cultured alveolar AT2 cells were studied in isolation; no macrophages, fibroblasts or other cell types were present. Thus, the AT2 cell response to LPS or cytokines may not exactly mimic that occurring in the lung. Further, we did not extend these studies to an animal model treated with LPS or bleomycin to induce acute lung injury and fibrosis. We focused on gene expression at the RNA level, so we did not report cytokine concentrations in iAT2 cell culture medium or lysates.⁴¹ Importantly, although clearly useful as models, neither LPS nor cytokines exactly mimic inflammation in the lung given its complexity incorporating multiple cell types. The concentrations of LPS used may not exactly reflect those occurring in the lung during sepsis; and the cytokine mixture employed may have unanticipated effects resulting from interactions among cytokines.

AT2 cells are active participants in mechanisms leading to inflammation. The data reinforce the basis for further investigation into signaling pathways that regulate AT2 cell expression of mesenchymal genes. Importantly, they lead to new areas of investigation that could reveal therapeutic targets, including the GHRH-R and downstream intracellular signaling pathways such as JAK/STAT and NF-kB. Finding that AT2 cells can express genes that lead to both inflammatory and fibrotic stimuli is important and relevant to clinical outcomes. Pro-inflammatory mediators released by iAT2 cells may enhance myofibroblast proliferation and foster mesenchymal cell production of collagen.

Authors’ contributions

Cui, T planned and executed experiments, analyzed data, prepared figures, wrote and edited the manuscript. Wangpaichitr, M conducted experiments, analyzed data and assisted with writing the manuscript. Schally, A suggested testing and provided MIA-602 and MR-409, assisted in experimental design and thoroughly edited the manuscript. Griswold, A analyzed RNA-seq data and prepared figures for the manuscript. Vidaurre, I planned and executed experiments, analyzed data, prepared figures and edited the manuscript. Sha, W synthesized and prepared peptides for use in experiments. Jackson, R planned, supervised and assisted in experiments, reviewed experimental and statistical data and drafted the manuscript.

Abbreviations
ACTA2	=	actin alpha 2, Smooth Muscle
Akt	=	protein kinase from thymoma Akt-8 (protein kinase B)
AT1	=	alveolar epithelial cell type 1
AT2	=	alveolar epithelial cell type 2
BALF	=	bronchoalveolar lavage fluid
COL1A1	=	type I collagen
CTGF	=	connective tissue growth factor
DAPI	=	4′,6-diamidino-2-phenylindole
ECM	=	extracellular matrix
EMT	=	epithelial to mesenchymal transition
ERK	=	extracellular signal regulated kinase
FN1	=	fibronectin
GH	=	growth hormone
GHRH	=	growth hormone releasing hormone
GHRH-R	=	growth hormone releasing hormone-receptor
HOPX	=	homeodomain only protein
iAT2	=	induced alveolar type 2 cell
IL1-β	=	interleukin 1-beta
IPF	=	idiopathic pulmonary fibrosis
iPSC	=	inducible pluripotent stem cell
LPCAT1	=	Lysophosphatidylcholine acyltransferase 1
JAK/STAT	=	Janus kinase/signal transducer and activator of transcription
KRT	=	keratin
LPCAT1	=	lysophosphatidylcholine acyltransferase 1
LPS	=	lipopolysaccharide
MAPK	=	mitogen-activated protein kinase
MCP1	=	chemokine ligand 2 or monocyte chemoattractant protein 1
mTOR	=	mechanistic target of rapamycin
MTT	=	3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NAPSA	=	napsin A aspartic peptidase
NF-kB	=	nuclear factor kappa B
PAK1	=	P21 (RAC1) activated kinase 1
PI3K	=	phosphoinositide 3-kinase
SFTPB	=	surfactant protein B
SP-C	=	surfactant protein C
SFTPC	=	surfactant protein C
SMA	=	smooth muscle actin
SV1	=	splice variant 1
TEM	=	transmission electron microscopy
TGF-B	=	transforming growth factor beta
TNC	=	tenascin C
TNF-a	=	tumor necrosis factor alpha
UPR	=	unfolded protein response
Wnt	=	Wingless and Int-1

Acknowledgements

The authors thank Fei Tang, PhD for biostatistical assistance in experimental design and analysis of the data; and, they thank Irving Vidaurre for technical assistance.

Disclosure statement

A.V.S. and R.J. are listed as co-inventors on patents of GHRH analogs, which were assigned to the University of Miami and Department of Veterans Affairs.

Additional information

Funding

This work was supported by the Miami VAHS Research Service, a Distinguished Medical Research Scientist Award (AVS) and Merit Review grant (MW) from the VA Office of Research and Development.