IGF1R deficiency attenuates acute inflammatory response in a bleomycin-induced lung injury mouse model

IGF1R (Insulin-like Growth Factor 1 Receptor) is a tyrosine kinase with pleiotropic cellular functions. IGF activity maintains human lung homeostasis and is implicated in pulmonary diseases such as cancer, ARDS, COPD, asthma and fibrosis. Here we report that lung transcriptome analysis in mice with a postnatally-induced Igf1r gene deletion showed differentially expressed genes with potentially protective roles related to epigenetics, redox and oxidative stress. After bleomycin-induced lung injury, IGF1R-deficient mice demonstrated improved survival within a week. Three days post injury, IGF1R-deficient lungs displayed changes in expression of IGF system-related genes and reduced vascular fragility and permeability. Mutant lungs presented reduced inflamed area, down-regulation of pro-inflammatory markers and up-regulation of resolution indicators. Decreased inflammatory cell presence in BALF was reflected in diminished lung infiltration mainly affecting neutrophils, also corroborated by reduced neutrophil numbers in bone marrow, as well as reduced lymphocyte and alveolar macrophage counts. Additionally, increased SFTPC expression together with hindered HIF1A expression and augmented levels of Gpx8 indicate that IGF1R deficiency protects against alveolar damage. These findings identify IGF1R as an important player in murine acute lung inflammation, suggesting that targeting IGF1R may counteract the inflammatory component of many lung diseases.

IGF1R is highly relevant in the murine lung, displaying the highest activation levels of any organ upon challenge with IGF1 17 . Additionally, epithelial-specific Igf1r deficient mice showed disturbed airway epithelial differentiation after naphthalene-induced club cell injury 18 , and mice with compromised IGF1R signalling displayed oxidative stress resistance 19,20 . Moreover, ablation of the macrophage IGF1-IGF1R axis inhibits the NLRP3 inflammasome, a protein complex that is activated in response to BLM-induced acute lung injury, which indicates that IGF1R plays an important role in initiation of the inflammatory process 21,22 . On this basis, we aimed to study the implications of IGF1R on the inflammatory process that occurs during BLM-induced acute lung injury. For this purpose we used the recently characterized Igf1r conditional mutant mice UBC-CreERT2; Igf1r fl/fl (CreERT2) 23 . In this study, lung transcriptome analysis of CreERT2 mice showed differential expression of genes that could serve a protective role in the lung, and it was also demonstrated that IGF1R deficiency confers resistance to BLM-mediated acute lung injury by counteracting the pulmonary inflammatory response. These results contribute toward a better understanding of the importance of IGF1R as a potential target for future therapeutic approaches in lung diseases with an inflammatory component.

Results
Postnatal IGF1R deficiency in CreERT2 mice causes a general inhibition of differentially expressed genes in the prepubertal lung. To study the effect of IGF1R deficiency in the postnatal mouse lung, CreERT2 mice were treated with tamoxifen at four weeks of age to induce Igf1r gene deletion 23 . Quantitative real-time PCR (qRT-PCR) and Western blot analyses on lung extracts of eight-week-old CreERT2 tamoxifen-treated mice verified efficient depletion of IGF1R expression at the RNA and protein levels (81% and 82%, respectively), when compared to their control littermates (Igf1r fl/fl ) (Fig. 1a,b). To determine the impact of Igf1r deficiency on global lung RNA gene expression, RNA-Seq was performed. After bioinformatics analyses comparing CreERT2 vs. Igf1r fl/fl lung mRNA expression profiles, significant changes in gene expression were found (data submitted to Gene Expression Omnibus, accession number GSE88908). Establishing a False Discovery Rate (FDR) <0.1, 65 differentially expressed genes were identified. 18 genes were up-regulated (28%)  Table S1). The most significantly affected biological functions based on GO and Keyword annotations, as well as published reports, are shown in Fig. 1d, and genes assigned to an extended list of these functions are displayed in Supplementary Table S2. Interestingly, the majority of genes in all categories were down-regulated. Most of them fall into three major categories: development/growth (Mki67 and Rps7, up-regulated; Notch3, Foxo1, Epas1 and Tgfbr3, down-regulated), transcriptional regulation and epigenetics (Top2a, Hist1h1d and Hist1h2bb, up-regulated; Crebbp, Ep300, Polr2a, Zbtb34, Zbtb16, Zfp518b and Zfhx3, down-regulated). Additional relevant biological functions of these genes include inflammation and immune response, followed by protumor function, hypoxia/redox and oxidative stress, as well as cell adhesion and migration (Supplementary Table S2). The top ten differentially expressed genes and their major biological functions are listed in Table 1. As expected, Igf1r is the most down-regulated gene, followed by Srrm2 (involved in pre-mRNA splicing), Nr1d2 (transcriptional regulator) and Epas1 (endothelial barrier integrity). The most up-regulated genes are Gpx8 and Cyp1a1, both implicated in anti-oxidative stress, followed by Gnptg (lysosome transport), Ppp1r2-ps4 (pseudogene) and Saa3 and Spon2, both involved in the immune response.
IGF1R deficiency improves mouse survival and alters IGF system gene expression in early stages after BLM-mediated pulmonary injury. To further analyze how IGF1R affects lung homeostasis, CreERT2 mice were treated with BLM to induce lung damage at six weeks of age (D0), and their survival was followed until D21 (Fig. 2a,b). The percentage of survivors after BLM challenge was significantly higher in CreERT2 mice (79%) than in Igf1r fl/fl mice (33%), without gender differences. Interestingly, mortality predominantly affected mice within the first week of treatment, beginning at D3 (Fig. 2b). qRT-PCR and Western blot analyses on lung extracts at D3 verified IGF1R reduced mRNA (88%) and protein (84%) levels in CreERT2 mice (Fig. 2c,d). In our search for possible compensatory effects on IGF system gene expression, we determined the mRNA levels of Igf1, Igfbp3, Igfbp5 and Insr, in addition to the IGF/Ins transcription factor-signaling mediator Foxo1, by qRT-PCR. Igf1 levels were found to be significantly diminished in CreERT2 lungs but conversely, Igfbp3, Igfbp5, Insr and Foxo1 levels were increased (Fig. 2e).

IGF1R depletion protects against lung vascular fragility and permeability, and reduces inflammatory cell presence in BALF after BLM treatment. Since BLM causes an acute increase in total cells
and protein concentration in bronchioalveolar lavage fluid (BALF) 6 , BALFs from saline-(SAL) and BLM-treated mice from both genotypes were analyzed at D3 (Fig. 3a). To evaluate lung vascular fragility and permeability, the presence of erythrocytes and the protein concentration in BALF were quantified. Interestingly, the increased erythrocyte presence (10-fold) found in BALF of Igf1r fl/fl from BLM compared to SAL-treated mice, was not as pronounced (4-fold) in CreERT2 mice. It is important to mention that erythrocyte counts from SAL-treated CreERT2 mice were significantly reduced (3-fold) with respect to Igf1r fl/fl , and were even more accentuated (6-fold) after BLM challenge (Fig. 3b). Only Igf1r fl/fl BALF protein levels were found to be increased (2-fold) when comparing SAL-to BLM-treated mice, whereas in CreERT2 mice, protein levels remained unchanged after BLM treatment (Fig. 3c). In parallel, total and differential cell counts for neutrophils, macrophages and lymphocytes were severely attenuated in BALF from BLM-challenged CreERT2 lungs with respect to their SAL-treated controls (Fig. 3d). Additionally, differential cell counts were also calculated as a proportion with respect to total absolute cell numbers, and expressed as percentages (Supplementary Table S3). Although BLM-treated Igf1r fl/fl mice demonstrated a significant increment in BALF total cells (3-fold) compared to SAL-treated mice, CreERT2 mice did not show such an increase. Differential cell counts for neutrophils, macrophages and lymphocytes in BLM-challenged Igf1r fl/fl mice exhibited the same marked increase. Furthermore, total and differential cell counts in BALF of IGF1R-deficient mice showed a severe attenuation with respect to Igf1r fl/fl (Fig. 3d).

IGF1R deficiency reduces proliferation and attenuates acute lung inflammation and bone marrow neutrophilopoiesis after BLM-challenge.
Inflamed lung areas were measured at D3, and were found to be markedly reduced in BLM-treated Cre-ERT2 lungs (7-fold) (Fig. 4a,b). To verify and further investigate the mechanism by which IGF1R deficiency blocks inflammatory cell recruitment to the lung, mRNA expression analysis of different inflammatory markers was performed. Pro-inflammatory cytokines Tnf, Il1b and Il6 were found to be significantly down-regulated in CreERT2 lungs (Fig. 4c), and the reduction of TNF protein levels was confirmed by ELISA (Fig. 4d). Conversely, mRNA expression levels of the injury resolution-phase markers Csf1, Il13 and Cd209a were significantly increased (Fig. 4e). Despite clearly increased Il13 mRNA levels, protein levels were found to be only slightly increased (Fig. 4f). Cell proliferation evaluated in lung perivascular areas and in the alveolar parenchyma by Ki67 immuno-staining on D3 was found to be substantially reduced in CreERT2 mice (3-fold in both cases) (Fig. 5a,b). As an indirect measure of inflammatory cell presence in the lung, mRNA levels of neutrophil chemotaxis (Cxcl1), neutrophil (Ly6g) and macrophage (Marco and Adgre1) markers were determined. Cxcl1 levels were greatly reduced in IGF1R-deficient lungs, and Ly6g, Marco and Adgre1markers also showed a significant reduction (Fig. 6a). Neutrophilic infiltration found in perivascular areas was 44% and 7% of total infiltrates for Igf1r fl/fl and CreERT2 BLM-challenged lungs, respectively ( Numbers within graphic bars indicate the number of mice analyzed and data are expressed as mean ± SEM. *p < 0.05; **p < 0.01 (Mann-Whitney U test). BLM, bleomycin.
( Fig. 6b, middle panel). In view of the reduced presence of BALF lymphocytes in mutant mice, infiltration into the lung was also assessed by immunostaining for CD3. Accordingly, lymphocyte counts were also diminished (45%) in CreERT2 lungs (Fig. 6b, bottom panel). To verify reduced neutrophil infiltration in IGF1R-deficient mice, total and neutrophil counts were performed in bone marrow cytospins obtained from mice of both genotypes after BLM treatment. As expected, total cells and neutrophils were found to be diminished in CreERT2 mice with respect to Igf1r fl/fl BLM-challenged lungs (2-and 5-fold, respectively) ( Fig. 7a,b).

IGF1R deficiency reduces alveolar damage and HIF1A expression in BLM-challenged lungs.
To determine the effect of IGF1R depletion on alveolar damage after BLM challenge, alveolar epithelial cell type-specific markers were quantified by qRT-PCR on D3. Transcript levels of alveolar epithelial cell type 1 (Aqp5) and 2 (Sftpc) markers were found to be significantly increased in CreERT2 BLM-challenged lungs. However, IGF1R-deficient lungs demonstrated significantly decreased levels of the hypoxia-inducible factor 1 subunit alfa (Hif1a) (Fig. 8a). Since the RNA-seq indicated that the anti-oxidative stress marker Gpx8 was the most up-regulated gene in CreERT2 unchallenged lungs, its mRNA expression was assayed in BLM-treated lungs, and also found to be increased in IGF1R-deficient mice with respect to controls (Fig. 8a). SFTPC and HIF1A expression determined by immunohistochemistry verified mRNA expression levels (Fig. 8b). In accordance, the number of SFTPC + cells was significantly increased (1.6-fold) (Fig. 8c), and conversely, HIF1A relative fluorescence intensity was found to be diminished (1.8-fold) in IGF1R-deficient lungs (Fig. 8d).

Discussion
This is the first report of the functional implication of IGF1R in acute lung inflammation using a BLM mouse model. First, we analyzed the lung transcriptome in recently reported IGF1R-deficient mice (CreERT2) 23 identifying differentially expressed genes with potentially protective roles. After BLM challenge, CreERT2 mice showed resistance to BLM-mediated acute lung injury by counteracting lung inflammation and alveolar damage.  Table S1) were found to be up-regulated in CreERT2 lungs. In this regard, it is widely known that H1 histones participate in chromatin condensation therefore repressing gene expression 25,26 . Additionally, the histone acetyltransferases Crebbp and Ep300, both transcriptional co-activators, were found down-regulated in IGF1R-deficient lungs. In an inflammatory context, CREBBP and EP300 were reported to activate NF-κB-mediated pro-inflammatory gene expression in response to oxidative stress [26][27][28] . Thus, increased H1 histone together with lower acetyltransferase expression would result in a more condensed chromatin state, less accessible to transcription factors. Furthermore, the lower expression observed for mitochondrial respiratory chain complexes I (mt-Nd4, mt-Nd5 and mt-Nd6) and III (mt-Cytb) genes (Supplementary Table S1) could result in decreased electron transport chain activity and consequently, in a reduction of reactive oxygen species production, since these complexes were reported to govern the response to hypoxia 29,30 . In parallel, Gpx8 and Cyp1a1, both involved in alleviating oxidative stress and inflammation 31,32 , were the two most up-regulated genes. Overall, these results indicate that IGF1R deficiency could potentially be associated with a higher capacity to endure oxidant-induced injury.
After BLM treatment CreERT2 mice showed improved survival. Similar results were observed in acute lung injury mouse models with compromised IGF1R activity 20,33 . Moreover, IGF1R-deficient lungs showed increased Igfbp3, Igfbp5, Insr and Foxo1 levels after BLM challenge, possibly due to compensatory effects in response to IGF1R deficiency, as reported 18,34 . Specifically, IGFBP3 and IGFBP5 have shown protective properties in the mouse lung [35][36][37] . Remarkably, CreERT2 mice showed decreased total proteins in BALF, an indicator of reduced vascular permeability. This finding together with diminished erythrocyte counts are in accordance with the lower vascular extravasation reported in hypomorphic IGF1R-deficient mice 20 . Furthermore, the decreased presence of different inflammatory cell types in CreERT2 lungs was reflected in BALF cell counts, and supported by reduced proliferation in perivascular and alveolar areas. Diminished Cxcl1 and Ly6g mRNA levels were verified by reduced neutrophilic infiltration into CreERT2 lungs. Considering that normal neutrophil bone marrow counts in mice are around 36.9% 38 , BLM clearly induced bone marrow neutrophilopoiesis in Igf1r fl/fl (54.25%) mice, unlike in IGF1R-deficient mice (25.44%). Similarly, pharmacological IGF1R blocking was recently reported to decrease the number of peripheral white blood cells 17,39 . Altogether, these results demonstrate that the lack of IGF1R efficiently counteracts the acute BLM-induced neutrophilia, a major inflammatory player in this model.
Concerning TNF and Il1b, CreERT2 lungs showed decreased expression of these cytokines, the most relevant in the lung during the early phase of BLM response 10,40 . Accordingly, PREX1, an IGF1R signalling activator, has been shown to have a pro-inflammatory role after BLM treatment, and Prex1-deficient mice mirrored the pro-inflammatory profile shown by our IGF1R-deficient mice at D3 41 . In addition, activation of the IGF1R/PI3K/ AKT/mTOR signalling pathway was reported to promote lung injury and repair 42,43 , and IGF1R signaling promotes TNF-induced activation of NF-kB, a major pathway involved in inflammation 44 . Likewise, IGF1R plays an important role in initiation of the inflammatory process, as ablation of the macrophage IGF1/IGF1R signaling axis in mice inhibits the NLRP3 inflammasome, a protein complex triggered in the lung upon BLM-induced damage 21,22 . During inflammation, while M1 macrophages contribute to tissue injury after excessive production of pro-inflammatory mediators (e.g., TNF and IL1B), M2 macrophages lead to resolution of inflammation and tissue repair upon anti-inflammatory cytokine activation (e.g., IL13 and CSF1) 9,45 . In this regard, both diminished expression of Tnf, Il1b and Il6 as well as elevated levels of Csf1, Il13 and Cd209a found in CreERT2 lungs would promote a pulmonary environment enriched in M2 macrophages. Noteworthy, IL13 was reported to protect against acute hyperoxic lung injury and Cd209a expression was found to be increased in the resolution-phase macrophages after peritonitis induction in mice 46,47 . Altogether, these data support the idea that IGF1R deficiency would facilitate dampening of innate/adaptive immunity and resolution of inflammation.
Following BLM-induced lung injury we found increased expression of the alveolar markers Aqp5 and SFTPC in CreERT2 lungs. Accordingly, AQP5 expression was reported to be decreased in BLM-challenged lungs 48 , and its reduced levels were shown to contribute to abnormal fluid fluxes during pulmonary inflammation in mice 49 . In addition, SFTPC-deficient mice had increased mouse mortality, neutrophilic inflammation, and alveolar damage following BLM treatment 50 . In line with our results, Sfptc mRNA levels were also found to be increased in CreERT2 lungs after allergic airway inflammation 51 . Thus, it appears that IGF1R deficiency confers a protective role against alveolar damage.
As a master transcriptional regulator of the adaptive response to hypoxia, HIF1A uses CREBBP and EP300 as transcriptional co-activators. Thus, decreased Crebbp and Ep300 transcriptional levels in non-challenged CreERT2 lungs could contribute to HIF1A reduced expression after BLM challenge. In accordance, alveolar type  Although we demonstrate a significant reduction of IGF1R expression in CreERT2 lungs, TMX-mediated IGF1R deletion may occur with different degrees of mosaicism in different cell types. Thus, IGF1R generalized deletion cannot be used to deduce in which cells IGF1R signaling is crucial for promoting acute lung inflammation. Furthermore, the variability of intratracheal administration of BLM and the effect of the genetic background on phenotypic variations should also be considered as constraints to this report.
In summary, we have shown that IGF1R deficiency in mice plays a key role in decreasing transcriptional activity, and confers protection against alveolar damage and pulmonary inflammation. Notably, our findings may contribute to understanding the importance of IGF1R as a potential target for future therapeutic approaches in respiratory diseases with persistent damage and inflammation.

Methods
Ethics Statement. All experiments and animal procedures were carried out following the guidelines laid down by the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were revised and approved by the CIBIR Bioethics Committee (refs 03/12 and 13/12). All animals were bred and maintained under specific pathogen-free (SPF) conditions in laminar flow caging at the CIBIR animal facility.
Generation of Igf1r-deficient mice, establishment of the BLM-induced acute lung injury murine model and survival rate. UBC-Cre-ERT2; Igf1r fl/fl double transgenic mice were in a C57BL/6 enriched (at least six generation backcrosses to C57BL/6 strain) mixed genetic background. For experimental purposes, UBC-Cre-ERT2; Igf1r fl/fl double transgenic mice were crossed with Igf1r fl/fl mice to directly generate descendants in equal proportions in the same litter, and Igf1r fl/fl and UBC-Cre-ERT2; Igf1r fl/fl littermates were respectively used as experimental controls and mutants. Tamoxifen (TMX) was administered daily for five consecutive days to four-week-old mice of both genotypes to induce a postnatal Igf1r gene conditional deletion in UBC-Cre-ERT2; Igf1r fl/fl mice, as previously described 23 . Two-month-old and six-week-old tamoxifen-treated UBC-CreERT2; Igf1r fl/fl (CreERT2) and Igf1r fl/fl mice were used for RNAseq analysis, and BLM treatment to induce lung injury, respectively. Six-week-old mice (equal sex proportions) of both genotypes were intra-tracheally instilled with either a single dose of 2.5 µl/g body weight of BLM sulfate (5 U/kg) (EMD Millipore, Billerica, MA) in saline (2 U/ ml) or saline (SAL) at D0, under a ketamine-xylazine anesthetic combination in saline (100:10 mg/kg respectively; 10 μl/g). Animals were monitored for 21 days to determine survival rates of both UBC-CreERT2; Igf1r fl/fl and Igf1r fl/fl mice after BLM challenge. Those animals that reached the human endpoint, as specified by the CIBIR Bioethics Committee protocol ref. 13/12, were also considered to be dead animals at each time point. The human endpoint criteria were applied when there was severe involvement of one of the specific (body weight, respiratory pattern and bleeding), or moderate involvement of two or more of the general (appearance, natural behavior) or specific parameters occurred, according to our expert veterinary evaluation. For acute lung injury studies, animals were sacrificed and tissues were collected at D3 (Fig. 2a).
Tissue and BALF collection. Before tissue collection, animals were euthanized by intraperitoneal injection of 10 μl/g of a ketamine-xylazine anesthetic combination in saline (300:30 mg/kg respectively). Three different sets of mice were used for BALF, mRNA/Western blot/histology and ELISA, respectively. A first set was used to obtain BALF; lungs from saline or BLM-treated mice were lavaged twice with 0.8 ml cold PBS. From the second set, left lungs were inflated with formalin fixative, post-fixed by immersion in formalin for 8-10 h, embedded in paraffin and cut into 3 µm sections for histopathological evaluation or immunohistochemistry; and right lobes were separated and snap frozen in liquid nitrogen for qRT-PCR and Western blot. Left lungs from a third set of animals were harvested for ELISA analysis.
Quantification of BALF. Total cell number was counted and expressed as cells/ml BALF and differential cell counts were performed on May-Grünwald/Giemsa (Sigma-Aldrich, St. Louis, MO) stained cytospins from 4 animals per condition, blind counting a minimum of 300 cells per slide. Cells were determined to be macrophages, lymphocytes and neutrophils using standard morphology criteria. The average number of red blood cells per high-power field was obtained by evaluating 5 different fields on BALF cytospin preparations. Total protein concentration in BALF supernatants was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA).

Histopathological analyses and immunostaining. Quantification of inflammation was determined
in H&E stained sections and expressed as the percentage of inflamed lung area to total section surface. Five BLM-treated left lungs per genotype were used. Inflamed lung areas were defined as darker H&E stained foci, where inflammatory cells accumulate massively, and delimitated manually using the Fiji open-source image processing software package v1.48r (https://fiji.sc).
Immunohistological detection of proliferating cells was performed as described 23 . Ki67 positive cells were counted using 5 or 10 fields per section per animal in perivascular and alveolar areas, respectively. The Ki67-labeling index was calculated as the number of Ki67 positive cells compared to the total cell number. Other histological and immunohistochemical quantifications were performed using 5 BLM-treated animals per genotype evaluating 5 different fields per lung. Determination of neutrophil and lymphocyte infiltration grades were assessed in lung perivascular areas on H&E and CD3 (ab5690 1:200, Abcam, Cambridge, UK) stained lung sections, and expressed as the number of neutrophils or lymphocytes to total cell infiltrates. Quantification of macrophages and alveolar type 2 cells was assessed in alveolar areas immunostained with anti-F4/80 (Clone MCA497GA 1:100, Bio-Rad, Langford, UK), and anti-SFTPC (AB3786, EMD Millipore, Billerica, MA) antibodies respectively, and expressed per unit area. Macrophage diameters were measured and volumes were extrapolated using the sphere volume formula. HIF1A expression was determined using the HIF1A antibody (ab2185 1:100, Abcam, Cambridge, UK) and expressed as relative fluorescence units (RFU) as previously described 18 . HIF1A relative fluorescence was evaluated using the Fiji software package.
Femoral bone marrow isolation. Bone marrow (BM) isolation was performed using 5 BLMtreated animals per genotype (1 femur per animal). After dissection, the femoral heads were incised and femurs were positioned in bottom perforated 0.5 ml tubes placed inside 1.5 ml tubes. After centrifugation at 10000 × g for 15 seconds, BM was suspended in 500 µl PBS and centrifuged at 300 × g for 5 min at 4 °C. Following aspiration of the supernatant, BM pellets were resuspended in 500 µl of ACK lysing buffer (Thermo Fisher Scientific, Waltham, MA) and after 10 minutes of incubation, 1 ml of PBS was added. Total cell numbers were counted and expressed as cells/ml BM and neutrophil counts were performed on May-Grünwald/Giemsa (Sigma-Aldrich, St. Louis, MO) stained cytospins and 5 different fields per slide were blind counted.

RNA isolation, reverse transcription and qRT-PCR.
Inferior lung lobes taken from eight-week-old mice or on D3 post-bleomycin were homogenized in TRIzol Reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA from D3 post-bleomycin-treated mice was reverse transcribed to cDNA using SuperScript II First-Strand Synthesis System (Invitrogen, Carlsbad, CA) as per the manufacturer's specifications. cDNA samples were amplified by qRT-PCR in triplicate on a 7300 Real Time PCR instrument (Applied Biosystems, Foster City, CA), for each primer pair assayed (Supplementary  Table S4). Results were normalized using the 18S rRNA gene as the endogenous control.
Lung transcriptome analysis. RNAseq analysis was performed on eight-week-old tamoxifen-treated UBC-CreERT2; Igf1r fl/fl and Igf1r fl/fl lungs (n = 3 per genotype). Approximately, 1 μg of total RNA from each sample was submitted to the CIBIR Genomics Core Facility for sequencing. Briefly, after verifying RNA quality in an Experion Bioanalyzer (BioRad), TruSeq total RNA libraries were generated according to the manufacturer's instructions (Illumina Inc). The libraries were sequenced in a Genome Analyzer IIx (Illumina Inc) to generate 150 single-end reads. Mus musculus GRCm38.71 (FASTA) from the Ensemble database was used as the reference genome. After removing adapter sequences with the Cutadapt software 53 , mapping to the reference genome was performed with TopHat2 (version 2.5) 54 . Gene expression quantification, normalization, and statistical analyses were performed with SeqSolve (Integromics). Expression data were normalized by calculating the fragments per kilobase of exon per million fragments mapped (FPKM) reads for each gene. Igf1r-transcriptionally regulated genes involved in biological processes were classified according to GO and Keyword annotations. Additionally, a PubMed search, and the Genecards and OMIM tools were used to help assign biological functions.
Western blot analysis. Superior lung lobes were solubilized in a 10 mM Tris/HCl (pH 7.4) buffer containing 0.1% sodium dodecyl sulfate, a protease inhibitor mixture, and DNase (Promega, Fitchburg, WI). Samples were separated in NuPAGE Novex 4-12% Bis-Tris Gel (Invitrogen, Carlsbad, CA) and transferred to a polyvinylidene difluoride membrane (EMD Millipore). Membranes were incubated with primary antibodies for IGF1R (#3027 Cell Signalling Inc., Danvers, MA) and beta-Actin (ab6276 Abcam, Cambridge, UK) at 1:1000 and 1:30000 dilutions respectively, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (DAKO, Agilent technologies, Santa Clara, CA) for IGF1R and beta-Actin respectively, at a 3:10 dilution. Signals were detected using ECL Western Blot Substrate (Thermo Fisher Scientific, Waltham, MA) and Hyperfilm ECL (GE Healthcare, Little Chalfont, UK). Films were scanned and signals in the linear range were quantified using Image J and normalized to beta-Actin levels.
ELISAS. TNF and IL13 levels were determined in homogenized tissue lysates using left lungs with the help of mouse TNF-alpha Quantikine and IL-13 Duoset ELISA Kits (R&D Systems, Minneapolis, MN), according to the manufacturer's guidelines.
Statistics. Statistical analyses were performed using SPSS Statistics Software v21 for Windows. Differences between genotypes were evaluated for significance using the non-parametric Mann-Whitney U test or the Dunn Sidak test for multiple comparisons. Results are shown as mean values ± standard error of the mean (SEM). For all analyses, a p value < 0.05 was considered statistically significant.