Background: Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterised by myofibroblast proliferation and abnormal extracellular matrix accumulation in the lungs. Transforming growth factor (TGF)-β1 initiates key profibrotic signalling involving the SMAD pathway and the small heat shock protein B5 (HSPB5). Tripartite motif-containing 33 (TRIM33) has been reported to negatively regulate TGF-β/SMAD signalling, but its role in fibrogenesis remains unknown. The objective of this study was to elucidate the role of TRIM33 in IPF.Methods: TRIM33 expression was assessed in the lungs of IPF patients and rodent fibrosis models. Bone marrow-derived macrophages (BMDM), primary lung fibroblasts and 3D lung tissue slices were isolated fromTrim33-floxed mice and cultured with TGF-β1 or bleomycin (BLM).Trim33expression was then suppressed by adenovirus Cre recombinase (AdCre). Pulmonary fibrosis was evaluated in haematopoietic-specificTrim33knockout mice and inTrim33-floxed mice that received AdCre and BLM intratracheally.Results: TRIM33 was overexpressed in alveolar macrophages and fibroblasts in IPF patients and rodent fibrotic lungs.Trim33inhibition in BMDM increased TGF-β1 secretion upon BLM treatment. Haematopoietic-specificTrim33knockout sensitised mice to BLM-induced fibrosis. In primary lung fibroblasts and 3D lung tissue slices,Trim33deficiency increased expression of genes downstream of TGF-β1. In mice, AdCre-Trim33inhibition worsened BLM-induced fibrosis.In vitro, HSPB5 was able to bind directly to TRIM33, thereby diminishing its protein level and TRIM33/SMAD4 interaction.Conclusion: Our results demonstrate a key role of TRIM33 as a negative regulator of lung fibrosis. Since TRIM33 directly associates with HSPB5, which impairs its activity, inhibitors of TRIM33/HSPB5 interaction may be of interest in the treatment of IPF.

The E2F transcription factor 1 is subtly regulated along the cell cycle progression and in response to DNA damage by post-translational modifications. Here, we demonstrated that the E3-ubiquitin ligase cellular inhibitor of apoptosis 1 (cIAP1) increases E2F1 K63-poly-ubiquitination on the lysine residue 161/164 cluster, which is associated with the transcriptional factor stability and activity. Mutation of these lysine residues completely abrogates the binding of E2F1 to CCNE, TP73 and APAF1 promoters, thus inhibiting transcriptional activation of these genes and E2F1-mediated cell proliferation control. Importantly, E2F1 stabilization in response to etoposide-induced DNA damage or during the S phase of cell cycle, as revealed by cyclin A silencing, is associated with K63-poly-ubiquitinylation of E2F1 on lysine 161/164 residues and involves cIAP1. Our results reveal an additional level of regulation of the stability and the activity of E2F1 by a non-degradative K63-poly-ubiquitination and uncover a novel function for the E3-ubiquitin ligase cIAP1.

Idiopathic and toxic pulmonary fibrosis are severe diseases starting classically in the subpleural area of the lung. It has recently been suggested that pleural mesothelial cells acquire a myofibroblast phenotype under fibrotic conditions induced by TGF-β1 or bleomycin. The importance and role of inflammation in fibrogenesis are still controversial. In this work, we explored the role of IL-1β/caspase-1 signaling in bleomycin lung toxicity and in pleural mesothelial cell transformation. C57BL/6 mice were intravenously injected with either bleomycin or nigericin or NaCl as control. In vitro, the Met5A cell line was used as a model of human pleural mesothelial cells. Intravenous injections of bleomycin induced lung fibrosis with histologically-proven peripheral distribution, collagen accumulation in the pleural and subpleural area, and overexpression of markers of myofibroblast transformation of pleural cells which migrated into the lung. These events were associated with an inflammatory process with an increase in neutrophil recruitment in pleural lavage fluid and increased caspase-1 activity. TGF-β1 was also overexpressed in pleural lavage fluid and was produced by pleural cells following intravenous bleomycin. In this model, local pleural inhibition of IL-1β with the IL-1β inhibitor anakinra diminished TGF-β1 and collagen accumulation. In vitro, caspase-1 inhibition interfered with Met5A cell transformation into the myofibroblast-like phenotype induced by bleomycin or TGF-β1. Moreover, nigericin, a caspase-1 activator, triggered transformation of Met5A cells and its intra-pleural delivery induced fibrogenesis in mice. We demonstrated, after intravenous bleomycin injection in mice, the role of the pleura and highlighted the key role of IL-1β/caspase-1 axis in this fibrogenesis process.

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by extracellular-matrix (ECM) accumulation in the lungs. Transforming-growth-factor (TGF)-ß1 is a key profibrotic growth factor involving Smad pathway. Tripartite motif containing 33 (TRIM33) has previously been reported to exert a negative control on Smad signaling but its role in fibrogenesis remains unknown. Methods: TRIM33 expression was assessed in IPF. Bleomycin (BLM) or NaCl were injected in conditional hematopoietic-specific TRIM33 knock-out mice. In vitro, Bone Marrow-derived Macrophage (BMDM) and primary lung fibroblasts were depleted for TRIM33 and treated with TGF-ß1. In vitro we characterized the relationship between TRIM33 and HSPB5. Results: TRIM33 was overexpressed in the lung of IPF patients compared to controls. Similar results were observed in fibrososis rodent model. Interestingly, TRIM33 inhibition in BMDM led to a 3-fold increase of TGF-ß1 secretion (ELISA) upon BLM treatment. We therefore used conditional hematopoietic-specific TRIM33 knock-out mice and demonstrated that these mice were more sensitive to BLM compared to WT mice with 1) a significant increase in collagen deposition (Sircol assay), 2) an increase in EMT marker mRNA expression 3) a significant increase (X2) of TGF-ß1 level in BALF. TRIM33 deficient lung fibroblasts were more sensitive to TGF-ß1 treatment with a significantly increased mRNA expression of EMT marker. In vitro, we demonstrated that HSPB5 was able 1) to bind to TRIM33 (BLI) 2) to impair with TRIM33 activity. Conclusion: Our results demonstrate a key role of TRIM33 in lung fibrosis. Inhibitors of TRIM33 and HSPB5 may be of interest for the treatment of IPF

La fibrose pulmonaire idiopathique (FPI) est une maladie dévastatrice caractérisée par l’accumulation de matrice extracellulaire dans les poumons. Le transforming-growth-factor (TGF)-β1 est une cytokine pro-fibrosante qui active la voie des Smads. Nous avons déjà montré que la protéine de choc thermique HSPB5 a un rôle activateur sur cette voie dans la fibrogenèse. TRIpartite Motif-containing 33 (TRIM33) est un régulateur négatif de la voie TGF-β1/Smads mais son rôle dans la fibrogenèse est inconnu. Nous avons étudié, le rôle de TRIM33 dans la FPI dans des modèles in vivo et ex-vivo, ainsi que le potentiel thérapeutique d’inhibiteurs chimiques d’HSPB5 dans des modèles de fibrose pulmonaire.Nous avons montré que TRIM33 est surexprimé dans les foyers fibroblastiques (myofibroblastes) ainsi que dans les macrophages alvéolaires de patients FPI. L’inhibition de cette protéine aggrave la différenciation mésenchymateuse, in vitro et in vivo, mais augmente également la sécrétion de TGF-β1 par les macrophages. En parallèle, nous avons mis en évidence l’efficacité de plusieurs inhibiteurs spécifiques de HSPB5, dans des modèles cellulaires de fibrose. Ces inhibiteurs ont été capables de diminuer l’augmentation des marqueurs de la différenciation mésanchymateuse induite dans le TGF-β1. Un de ces inhibiteurs s’est déjà révélé efficace pour traiter la fibrose pulmonaire induite par la bléomycine chez la souris.En conclusion, ce travail de thèse a permis de mettre en évidence le rôle de TRIM33 dans le développement de la FPI ainsi que l’efficacité d'inhibiteurs spécifiques de HSPB5 qui permettront peut-être un jour le traitement de la fibrose pulmonaire idiopathique.