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Consequences of telomere dysfunction in fibroblasts, club and basal cells for lung fibrosis development

TRF1 is an essential component of the telomeric protective complex or shelterin. We previously showed that dysfunctional telomeres in alveolar type II (ATII) cells lead to interstitial lung fibrosis. Here, we study the lung pathologies upon telomere dysfunction in fibroblasts, club and basal cells. TRF1 deficiency in lung fibroblasts, club and basal cells induced telomeric damage, proliferative defects, cell cycle arrest and apoptosis. While Trf1 deletion in fibroblasts does not spontaneously lead to lung pathologies, upon bleomycin challenge exacerbates lung fibrosis. Unlike in females, Trf1 deletion in club and basal cells from male mice resulted in lung inflammation and airway remodeling. Here, we show that depletion of TRF1 in fibroblasts, Club and basal cells does not lead to interstitial lung fibrosis, underscoring ATII cells as the relevant cell type for the origin of interstitial fibrosis. Our findings contribute to a better understanding of proper telomere protection in lung tissue homeostasis.


In vivo measurement of lung function
The mice were anesthetized by intraperitoneal injection of 10 μl/g of a ketamine-medetomidine anesthetic combination in saline (75:1 mg/kg respectively). A MiniVent (Harvard Apparatus, Holliston, MA, USA) was connected to the plethysmograph and the tracheal cannula for animal ventilation at 10 ml/kg of tidal volume and 150 breaths per minute. Data were measured by 2 pressure transducers that detect pressure variations in the chamber (flow) and in the tracheal cannula (pressure).

Sample collection and processing
Animals were euthanized by intraperitoneal injection of 10 μl/g of a ketamine-xylazine anesthetic combination in saline (100:10 mg/kg respectively) after lung function assessment.
Serum was obtained by centrifugation at 3000 xg for 10 min at 4 °C and stored at -80°C. On the other hand, bronchoalveolar lavage fluid (BALF) was centrifuged at 10000 rpm for 5 min at 4 °C and the supernatants were stored at -80 °C to subsequently assess total protein concentration in BALF using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Hereafter, the BALF pellets were resuspended in 500 μl of ACK Lysing Buffer (Thermo Fisher Scientific) and centrifuged at 10000 rpm for 5 min at 4 °C after 10 min of incubation. The supernatants were discarded and 500 µl of PBS 1X were added to the pellet to prepare the BALF cytospin preparations by centrifugation of the slides at 1500 rpm for 5 min.

Quantification of BALF
Total cell number was counted and expressed as cells/ml of BALF, and differential cell counts were performed on May-Grünwald Giemsa (Sigma-Aldrich)-stained cytospins, counting a minimum of 300 cells per slide.
Determination of differential cell counts was performed using standard morphology criteria.

Histopathological analyses and immunostaining
Fiji open source image processing software package v1.48r (http://fiji.sc) was used for the quantification of collagen (Sirius Red) and Vimentin, SMA, E-cadherin and Collagen I positive areas (percentage of DAB), as well as to assess smooth muscle thickness (SMA) and epithelium length measurements. Quantifications in lung sections were performed in 4 different fields or bronchi, respectively in a random way.

Statistics
Statistical analyses were accomplished using SPSS Statistics Software v21 for Windows (IBM, Armonk, NY, USA). For all analyses, a p value<0.05 was considered statistically significant. Results are shown as mean values ± standard error of the mean (SEM). For all analyses, a P value < 0.05 was considered statistically significant.