Transcriptomic and Proteomic Changes Driving Pulmonary Fibrosis Resolution in Young and Old Mice.

Bleomycin-induced pulmonary fibrosis in mice mimics major hallmarks of idiopathic pulmonary fibrosis. Yet in this model, it spontaneously resolves over time. We studied molecular mechanisms of fibrosis resolution and lung repair, focusing on transcriptional and proteomic signatures and the effect of aging. Old mice showed incomplete and delayed lung function recovery 8 weeks after bleomycin instillation. This shift in structural and functional repair in old bleomycin-treated mice was reflected in a temporal shift in gene and protein expression. We reveal gene signatures and signaling pathways that underpin the lung repair process. Importantly, the downregulation of WNT, BMP, and TGFß antagonists Frzb, Sfrp1, Dkk2, Grem1, Fst, Fstl1, and Inhba correlated with lung function improvement. Those genes constitute a network with functions in stem cell pathways, wound, and pulmonary healing. We suggest that insufficient and delayed downregulation of those antagonists during fibrosis resolution in old mice explains the impaired regenerative outcome. Together, we identified signaling pathway molecules with relevance to lung regeneration that should be tested in-depth experimentally as potential therapeutic targets for pulmonary fibrosis.

Transcriptomic and proteomic profiling of young and old mice in the bleomycin model reveals high similarity.

The most common preclinical, in vivo model to study lung fibrosis is the bleomycin-induced lung fibrosis model in 2- to 3-mo-old mice. Although this model resembles key aspects of idiopathic pulmonary fibrosis (IPF), there are limitations in its predictability for the human disease. One of the main differences is the juvenile age of animals that are commonly used in experiments, resembling humans of around 20 yr. Because IPF patients are usually older than 60 yr, aging appears to play an important role in the pathogenesis of lung fibrosis. Therefore, we compared young (3 months) and old mice (21 months) 21 days after intratracheal bleomycin instillation. Analyzing lung transcriptomics (mRNAs and miRNAs) and proteomics, we found most pathways to be similarly regulated in young and old mice. However, old mice show imbalanced protein homeostasis as well as an increased inflammatory state in the fibrotic phase compared to young mice. Comparisons with published human transcriptomic data sets (GSE47460, GSE32537, and GSE24206) revealed that the gene signature of old animals correlates significantly better with IPF patients, and it also turned human healthy individuals better into "IPF patients" using an approach based on predictive disease modeling. Both young and old animals show similar molecular hallmarks of IPF in the bleomycin-induced lung fibrosis model, although old mice more closely resemble several features associated with IPF in comparison to young animals.

Mapping the metabolomic and lipidomic changes in the bleomycin model of pulmonary fibrosis in young and aged mice.

Alterations in metabolic pathways were recently recognized as potential underlying drivers of idiopathic pulmonary fibrosis (IPF), translating into novel therapeutic targets. However, knowledge of metabolic and lipid regulation in fibrotic lungs is limited. To comprehensively characterize metabolic perturbations in the bleomycin mouse model of IPF, we analyzed the metabolome and lipidome by mass spectrometry. We identified increased tissue turnover and repair, evident by enhanced breakdown of proteins, nucleic acids and lipids and extracellular matrix turnover. Energy production was upregulated, including glycolysis, the tricarboxylic acid cycle, glutaminolysis, lactate production and fatty acid oxidation. Higher eicosanoid synthesis indicated inflammatory processes. Because the risk of IPF increases with age, we investigated how age influences metabolomic and lipidomic changes in the bleomycin-induced pulmonary fibrosis model. Surprisingly, except for cytidine, we did not detect any significantly differential metabolites or lipids between old and young bleomycin-treated lungs. Together, we identified metabolomic and lipidomic changes in fibrosis that reflect higher energy demand, proliferation, tissue remodeling, collagen deposition and inflammation, which might serve to improve diagnostic and therapeutic options for fibrotic lung diseases in the future.

RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma.

BACKGROUND: RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. METHODS: IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. RESULTS: We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. CONCLUSION: These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Automatization and improvement of µCT analysis for murine lung disease models using a deep learning approach.

BACKGROUND: One of the main diagnostic tools for lung diseases in humans is computed tomography (CT). A miniaturized version, micro-CT (µCT) is utilized to examine small rodents including mice. However, fully automated threshold-based segmentation and subsequent quantification of severely damaged lungs requires visual inspection and manual correction. METHODS: Here we demonstrate the use of densitometry on regions of interest (ROI) in automatically detected portions of the lung, thus avoiding the need for lung segmentation. Utilizing deep learning approaches, the middle part of the lung is found in a µCT-stack and a ROI is placed in the left and the right lobe. RESULTS: The intensity values within the ROIs of the µCT images were collected and subsequently used for the calculation of different lung-related parameters, such as mean lung attenuation (MLA), mode, full width at half maximum (FWHM), and skewness. For validation, the densitometric approach was correlated with histological readouts (Ashcroft Score, Mean Linear Intercept). CONCLUSION: We here show an automated tool that allows rapid and in-depth analysis of µCT scans of different murine models of lung disease.

FKBP51 modulates steroid sensitivity and NFκB signalling: A novel anti-inflammatory drug target.

Steroid refractory inflammation is an unmet medical need in the management of inflammatory diseases. Thus, mechanisms, improving steroid sensitivity and simultaneously decreasing inflammation have potential therapeutic utility. The FK506-binding protein 51 (FKBP51) is reported to influence steroid sensitivity in mental disorders. Moreover, biochemical data highlight a connection between FKBP51 and the IKK complex. The aim of this study was to elucidate whether FKBP51 inhibition had utility in modulating steroid resistant inflammation by increasing the sensitivity of the glucocorticoid receptor (GR) signalling and simultaneously inhibiting NFκB-driven inflammation. We have demonstrated that FKBP51 silencing in a bronchial epithelial cell line resulted in a 10-fold increased potency for dexamethasone towards IL1beta-induced IL6 and IL8, whilst FKBP51 over-expression of FKBP51 reduced significantly the prednisolone sensitivity in a murine HDM-driven pulmonary inflammation model. Immunoprecipitation experiments with anti-FKBP51 antibodies, confirmed the presence of FKBP51 in a complex comprising Hsp90, GR and members of the IKK family. FKBP51 silencing reduced NFκB (p50/p65) nucleus translocation, resulting in reduced ICAM expression, cytokine and chemokine secretion. In conclusion, we demonstrate that FKBP51 has the potential to control inflammation in steroid insensitive patients in a steroid-dependent and independent manner and thus may be worthy of further study as a drug target.

microRNA cluster 106a~363 is involved in T helper 17 cell differentiation.

T-helper cell type 17 (Th17) mediated inflammation is associated with various diseases including autoimmune encephalitis, inflammatory bowel disease and lung diseases such as chronic obstructive pulmonary disease and asthma. Differentiation into distinct T helper subtypes needs to be tightly regulated to ensure an immunological balance. As microRNAs (miRNAs) are critical regulators of signalling pathways, we aimed to identify specific miRNAs implicated in controlling Th17 differentiation. We were able to create a regulatory network model of murine T helper cell differentiation by combining Affymetrix mRNA and miRNA arrays and in silico analysis. In this model, the miR-212~132 and miR-182~183 clusters were significantly up-regulated upon Th17 differentiation, whereas the entire miR-106~363 cluster was down-regulated and predicted to target well-known Th17 cell differentiation pathways. In vitro transfection of miR-18b, miR-106a and miR-363-3p into primary murine Cd4+ lymphocytes decreased expression of retinoid-related orphan receptor c (Rorc), Rora, Il17a and Il17f, and abolished secretion of Th17-mediated interleukin-17a (Il17a). Moreover, we demonstrated target site-specific regulation of the Th17 transcription factors Rora and nuclear factor of activated T cells (Nfat) 5 by miR-18b, miR-106a and miR-363-3p through luciferase reporter assays. Here, we provide evidence that miRNAs are involved in controlling the differentiation and function of T helper cells, offering useful tools to study and modify Th17-mediated inflammation.

Histone deacetylase 6 (HDAC6) plays a crucial role in p38MAPK-dependent induction of heme oxygenase-1 (HO-1) in response to proteasome inhibition.

The proteasome is responsible for the degradation of polyubiquitinated proteins. Inhibition of the proteasome leads to an accumulation of polyubiquitinated proteins and thus to an impairment of the cellular protein homeostasis. To prevent cellular damage on proteasome inhibition there is an up-regulation of several heat shock proteins (Hsps), including Hsp27, Hsp70, and heme oxygenase-1 (HO-1). It was demonstrated that the induction of classical Hsps, such as Hsp27 and Hsp70, is dependent on a HDAC6-dependent mechanism which releases HSF-1 and induces the expression of newly synthesized Hsps. In this study we demonstrate that the up-regulation of HO-1 on proteasome inhibition is mediated by p38MAPK and Nrf-2. Interestingly we found additional evidence, proving the involvement of HDAC6 in the up-regulation of HO-1. By using RNAi technologies against HDAC6 we demonstrate that there is a lack of the expected induction of HO-1, Nrf-2, and phosphorylated p38 (pp38) after proteasome inhibition. Furthermore, we can show that p38 is acetylated in unstressed cells and is a good substrate for HDAC6-mediated deacetylation. Therefore, we propose that on proteasome inhibition HDAC6 deacetylates p38, allowing the subsequent phosphorylation of p38 and resultant activation of NRF-2. NRF-2 enters the nucleus and functions as a transcription factor for HO-1.

Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress.

After oxidative stress, proteins that are oxidatively modified are degraded by the 20S proteasome. However, several studies have documented an enhanced ubiquitination of yet unknown proteins. Because ubiquitination is a prerequisite for degradation by the 26S proteasome in an ATP-dependent manner this raises the question whether these proteins are also oxidized and, if not, what proteins need to be ubiquitinated and degraded after oxidative conditions. By determination of oxidized and ubiquitinated proteins we demonstrate here that most oxidized proteins are not preferentially ubiquitinated. However, we were able to confirm an increase in ubiquitinated proteins 16 h after oxidative stress. Therefore, we isolated ubiquitinated proteins from hydrogen peroxide-treated cells, as well as from control cells and cells treated with lactacystin, an irreversible proteasome inhibitor, and identified some of these proteins by MALDI tandem mass spectrometry. As a result we obtained 24 different proteins that can be categorized into the following groups: chaperones, energy metabolism, cytoskeleton/intermediate filaments, and protein translation/ribosome biogenesis. The special set of identified, ubiquitinated proteins confirms the thesis that ubiquitination upon oxidative stress is not a random process to degrade the mass of oxidized proteins, but concerns a special group of functional proteins.

Interactions of the proteasomal system with chaperones: protein triage and protein quality control.

Unfolded, misfolded, or modified proteins are able to induce proteotoxic cell stress. To prevent proteotoxic stress, it is crucial to have a functional protein quality control system, especially in the cytosol and in the endoplasmic reticulum where proteins are newly synthesized. The leading actors in this protein quality control system are the ubiquitin-proteasomal system and the huge family of heat shock proteins and chaperones. Both systems interact with each other, influencing the decision of whether a protein becomes (re)folded or degraded. Especially upon cellular stress, such as heat shock or oxidative stress, heat shock proteins are drastically upregulated, supporting, and regulating proteasomal degradation of defect proteins. Failure of one of the systems can be compensated partially by the upregulation of the other. Nevertheless, prolonged failure of the proteasome or chaperones results in protein aggregation and cellular dysfunction.

Protein oxidative modification in the aging organism and the role of the ubiquitin proteasomal system.

Living in an oxygen containing environment is automatically connected to oxidative stress. Beside lipids and nucleic acids, especially proteins are very susceptible for oxidative modifications. These oxidative modifications comprise alterations of single amino acids, like the formation of protein carbonyls and methionine sulfoxide, or the aggregation of whole proteins. Due to the ongoing accumulation of protein aggregates during the aging process, the cellular protein quality control system becomes more and more overwhelmed. One essential element of the protein quality control machinery is the ubiquitin proteasomal system which plays therefore a crucial part in the aging process, too. Ubiquitination of proteins is a three step mechanism to tag proteins with a polyubiquitin chain for the proteasome. The proteasome is a regulated, barrel-shaped multi-enzyme complex which is responsible for the degradation of proteins. Although there is no drastic loss of all proteasomal subunits during the aging process, there is a functional decline of the proteasome activity in aging organisms. Impairment of the ubiquitin proteasome system leads to increasing protein aggregation and cellular death. A lot of age related diseases are closely connected to an inhibition of the proteasome and the formation of large protein aggregates. Especially skin aging, atherosclerosis, age-dependent macula degeneration, cataract formation and several neurodegenerative diseases are directly connected to the decline of proteasome function. This review outlines the connections between aging, oxidative stress and protein oxidation, as well as the influence on the ubiquitin proteasomal system and several associated diseases.

Proteins bearing oxidation-induced carbonyl groups are not preferentially ubiquitinated.

A substantial part of soluble, oxidized proteins are degraded by the proteasome. However, it is still under debate whether these oxidized proteins are degraded by the 26S proteasome in an ubiquitin-dependent way or in an ubiquitin-independent way by the 20S proteasome. Therefore, we treated cells with H(2)O(2) and UV-A irradiation and detected protein carbonyls and ubiquitination by immunoblotting. Separation of ubiquitinated proteins from non-ubiquitinated reveals that most oxidized proteins are not ubiquitinated.

Combination of PDT and inhibitor treatment affects melanoma cells and spares keratinocytes.

Photodynamic therapy (PDT) is a potential tool in cancer treatment. Today this therapy is established among others for the treatment of nonmelanoma skin cancer. However, the more dangerous skin cancer--the melanoma--still has to be removed by surgery. Therefore, we investigated the effects of PDT and additional administration of heme oxygenase I (HO-I) and poly(ADP-ribose) polymerase (PARP) inhibitors on the treatment of melanoma cells in comparison to nonmalignant keratinocytes. Therefore, cocultures were established with WM451LU melanoma cells and HaCaT keratinocytes. In the coculture some 65% melanoma cells and 35% HaCaT cells were present before PDT, whereas after PDT the proportion was 41% melanoma cells and 59% HaCaT cells. Combination of both inhibitors improves these results to only 16% melanoma cells and 84% HaCaT cells. PDT is, therefore, a potent skin cancer treatment, which might also be interesting for melanoma treatment. The cytotoxic effects of PDT are largely mediated by ROS. Addition of HO-I and PARP inhibitors could improve the efficiency of photodynamic treatment.