Thank you for believing in me every time I had a hard time believing in myself. You had high expectations and yet were able to be incredibly supportive and optimistic in the most challenging times - a quality that I have admired and for which I want to thank you. And more than anything, thank you for being such a warm and thoughtful person – I can't even count the countless little thoughtful gestures that came naturally to you and that you displayed over the past seven years, but their impact remains.
Thank you for going above and beyond the call of duty to teach me the ropes in graduate school. Roger, I would like to mention here that my interview with you on Skype before I came to Vanderbilt had made me feel very positive about going to graduate school here, and I especially want to thank you for your understanding, support and guidance during my first year in the Interdisciplinary Graduate Program (IGP). Kathy, I want to thank you for your strong support of the VISP program; this amazing program no longer exists and I was among the lucky ones who had the opportunity to be a part of it.
Amanda, you are no longer a part of Vanderbilt, but my gratitude to VISP would be incomplete without expressing my gratitude to you—thank you so much for being so helpful to the 2011 VISP student cohort while they were settling into graduate school and Nashville.
INTRODUCTION
Disturbances in any of these factors can lead to ER stress and activation of the UPR. In lungs of IPF patients, ER stress markers have been primarily identified in type II AECs ( 27 , 33 ). In type II AEC lines, overexpression of mutant SFPTC genes (either exon 4 deleted or L188Q SFTPC) results in ER stress and increased apoptosis.
Together, these data indicate that ER stress in AECs is not sufficient to induce lung fibrosis. Along these lines, induction of ER stress due to expression of mutant SFTPC (exon4 deletion) in A549 cells increased collagen production and secretion (104). Specifically, ER stress-induced JNK activation has been shown to induce M2 polarization in mouse peritoneal macrophages ( 95 ).
Similarly, silica has been shown to induce alveolar macrophage apoptosis via ER stress in vitro (112).
MATERIALS AND METHODS
In addition, double immunofluorescence for TUNEL and pro-SPC was performed, and double-positive cells were counted at 10 HPF in each lung section. To detect in vitro cytotoxicity, the lactate dehydrogenase (LDH) assay (Promega) was performed according to the manufacturer's instructions. Cells were maintained in DMEM (Invitrogen, Life Technologies) with 2 mM L-glutamine, supplemented with FBS (10%) and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin).
Twenty-four hours later, the medium was changed to fresh DMEM with FBS and antibiotics, and the cells were exposed to hypoxia (1.5% O2) using an in vitro hypoxia chamber (Coy Laboratory) or normoxia (21% O2) for an additional 24– . Cells were transfected with a 10-fold multiplicity of infection (MOI) of Lenti HIF under antibiotic-free conditions using SureENTRY™ Transduction Reagent (Qiagen) (5 ug/ml) according to the manufacturer's instructions. Briefly, MLE12 cells were seeded at 40%–50% confluence in 6-well plates in DMEM with FBS but without antibiotics and grown overnight in a standard cell culture incubator.
Cells were transfected with 25 nM Smartpool siRNA using Lipofectamine RNAi Max (Life Technologies) according to the manufacturer's instructions. Twenty-four hours later, the medium was replaced with fresh DMEM (with FBS and antibiotics) and the cells were exposed to hypoxia (1.5% O2) or normoxia (21% O2) for an additional 48 hours. For chemical inhibition of IRE1α and PERK, MLE12 cells were plated at 70% confluency in 6-well plates in DMEM.
After RNA isolation, DNA digestion was performed using the DNA-free DNA Removal Kit (Ambion Thermo Scientific), and cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen) or Superscript VILO Master Mix (Invitrogen) according to the instructions from the manufacturer. The following primer sets were used: XBP1 forward cleaved 5'-GAGTCCGCAGCAGGTG-3' and Twenty-four hours later, MLE12 cells were transfected with Smartpool siRNA for CHOP or control non-targeting siRNA.
CD45+ cells were depleted by passing a CD45-coated single-cell suspension through LD columns on a QuadroMACS separator (Macs Miltenyi Biotech) according to the manufacturer's instructions. EpCAM+ cells were collected by passing the EpCAM-coated single-cell suspension through LS columns on a QuadroMACS separator according to the manufacturer's instructions.
HYPOXIA WORSENS LUNG FIBROSIS THROUGH EXPRESSION OF ER STRESS EFFECTOR
We found that the number of TUNEL+ AECs was significantly lower in CHOP–/– mice treated with repeated bleomycin compared with WT controls ( Fig. 10E ). Notably, no difference in fibrosis was observed between WT and CHOP–/– mice used as controls in these experiments. To assess the effect of CHOP on potential profibrotic pathways, we analyzed gene expression by RNA sequencing of EpCAM+ (EpCAM is also known as CD326) cells isolated from the lungs of WT and CHOP–/– mice after repeated bleomycin treatment (Figure 12).
Among this set of differentially expressed genes, 127 genes were differentially expressed in epithelial cells from WT and CHOP-/- mice after repeated bleomycin (Figure 12A). As shown in Figure 13, no significant differences were detected in the number of immune/inflammatory cells in the lungs of WT and CHOP–/– mice (alveolar macrophages, interstitial macrophages, B or T lymphocytes), with the exception of neutrophils and dendritic cells, which were reduced in the lungs of CHOP–/–. To test whether CHOP mediates the excess pulmonary fibrosis resulting from exposure to hypoxia after bleomycin treatment, we treated WT and CHOP–/– mice with a single i.t.
As shown in Figure 18, A–C , the hypoxia-induced exacerbation of lung fibrosis was completely abrogated in CHOP–/– mice. In lungs harvested 10 days after bleomycin, quantification of pro-SPC and TUNEL double-positive cells showed that AEC apoptosis was increased in WT mice exposed to hypoxia compared with WT mice maintained in normoxia and that this increase in lungs of CHOP- is mitigated. /– mice (Figure 18D). We also investigated whether CHOP–/– mice had differences in lung immune/inflammatory cell populations in the bleomycin followed by hypoxia model.
Similar to our findings at day 21 post-bleomycin, lymphocyte numbers were similar in the lungs of WT and CHOP–/– mice under normoxia and hypoxia. Regarding macrophage polarization, no difference in the pattern of M1/M2 markers was observed between WT and CHOP–/– mice after bleomycin treatment under normoxia or hypoxia (Figure 20C). In pulmonary fibrosis, previous studies on the role of CHOP have yielded conflicting results, with two reports showing that CHOP–/– mice had reduced fibrosis after a single dose of i.t.
Specifically, CHOP deficiency in bleomycin-treated L188Q SFTPC/CHOP–/– mice reduced fibrosis only to the level observed in bleomycin-treated WT mice, and similarly, CHOP–/– mice exposed to hypoxia after a single dose of bleomycin developed the equivalent of lung fibrosis. with that of WT mice treated with a single dose of bleomycin and maintained in normoxia. Next, we evaluated these targets in vivo and found that expression of activator of transcription factor 5 (ATF5), growth arrest and DNA damage inducible α (GADD45A), and BCL2-interacting protein 3 (BNIP3L) were reduced in the lungs of CHOP-/ – mice compared to WT mice after repeated bleomycin injury (Figure 28), thus validating these targets as potential downstream CHOP mediators affecting AEC survival in this model. Expression of CHOP-dependent mediators of apoptosis in the lungs of CHOP-deficient mice treated with repetitive bleomycin.
We found that (a) in addition to the PERK/ATF4 arm, which is generally considered to be dominant in CHOP activation, the IRE1α/XBP1 arm of the UPR is also required for hypoxia-induced upregulation of CHOP in AECs, and (b) CHOP regulates the expression of several by apoptosis of associated genes in AECs after exposure to hypoxia, including GADD45A, ATF5, and BNIP3L, as verified in the lungs of CHOP–/– mice after repeated bleomycin injury.
THE ROLE OF HYPOXIA-INDUCIBLE FACTOR (HIF) IN LUNG FIBROSIS
Next, we evaluated the effect of HIF signaling on the expression of pro-fibrotic mediators in AECs. Together, these studies suggest that hypoxia increases the expression of pro-fibrotic mediators in AECs, at least some of which are regulated through HIF signaling. Epithelial HIF signaling is not essential for mediating the effects of hypoxia on pulmonary fibrosis in the 'bleomycin + hypoxia' model.
To evaluate whether epithelial HIF signaling affects lung fibrosis in mice, we generated mice with lung epithelial cell-specific deletion of HIF1α and HIF2α. We found that despite significant HIF activation (Figure 32), epithelial HIF1/2 deletion did not alter lung fibrosis (Figure 33) in mice treated with bleomycin followed by exposure to hypoxia. Epithelial HIF-deficient mice are not protected from excessive lung fibrosis induced by exposure to hypoxia.
Although epithelial HIF signaling did not protect against lung fibrosis in mice treated with bleomycin followed by exposure to hypoxia, we wondered whether HIF signaling could regulate the expression of pro-fibrotic mediators in this model. In summary, these studies show that epithelial HIF is not essential to mediate the effects of hypoxia on lung fibrosis in the 'bleomycin + hypoxia' model. Epithelial HIF-deficient mice and littermate controls had similar expression of pro-fibrotic mediators following bleomycin treatment by exposure to hypoxia.
Epithelial HIF signaling plays an important role in mediating lung fibrosis after repetitive bleomycin injury. HIF1/2A/A and HIF1/2fl/fl mice were studied using the repetitive bleomycin model and lungs were harvested 2 weeks after the last dose. These studies demonstrate that although epithelial HIF signaling does not influence lung fibrosis after treatment with a single dose of bleomycin or treatment with bleomycin followed by exposure to hypoxia, it appears to play an important role in the progression of fibrosis after repeated bleomycin injury in mice. Hypoxia and/or HIF signaling are involved in regulating the expression of pro-fibrotic mediators, including collagen (122).
In our work, we found that exposure to hypoxia increases the expression of several pro-fibrotic mediators (CCN1, CTGF, collagen1, PDGFB, VEGF and TGF) of which few (PDGFB and VEGF) are regulated by HIF in AECs. However, epithelial targeting of HIF did not affect fibrosis in mice treated with a single dose of bleomycin followed by exposure to normoxia or hypoxia.
CONCLUDING REMARKS AND FUTURE DIRECTIONS
