Tenascin-C inactivation impacts lung structure and function beyond lung development

Tenascin-C (TNC) is an extracellular matrix protein expressed at high levels during lung organogenesis. Later, TNC is only transiently de novo expressed to orchestrate tissue repair in pathological situations. We previously showed that TNC inactivation affects lung development and thus evaluated here the implications on lung function in newborn/adult mice. Respiratory function parameters were measured in anesthetized and mechanically ventilated wild-type (WT) and TNC-deficient mice at 5 (P5) and 90 (P90) days of age under basal conditions, as well as following high tidal volume (HTV) ventilation. At P5, TNC-deficient mice showed an increased static compliance (Cst) and inspiratory capacity (IC) relative to WT at baseline and throughout HTV. At P90, however, Cst and IC were only elevated at baseline. Control non-ventilated newborn and adult TNC-deficient mice showed similar lung morphology, but less alpha smooth muscle actin (α-SMA) around small airways. SMA + cells were decreased by 50% in adult TNC-deficient lungs and collagen layer thickened around small airways. Increased surfactant protein C (SP-C) and altered TGFβ and TLR4 signaling pathways were also detected. Thus, TNC inactivation-related defects during organogenesis led to persisting functional impairment in adulthood. This might be of interest in the context of pulmonary diseases with thickened airway smooth muscle layer or ventilation heterogeneity, like asthma and COPD.

Tenascin-C (TNC) is a large hexameric protein and the founding member of the tenascin family of extracellular matrix (ECM) proteins 1,2 . TNC binds to cell surface receptors, ECM proteins or, more recently discovered, soluble factors or pathogens to regulate cell adhesion, migration, proliferation and differentiation 1, [3][4][5] . TNC is a complex protein with post-transcriptional (alternative splicing) and post-translational (glycosylation, citrullination, proteolytic processing) regulations giving rise to numerous isoforms having sometimes opposite effects on cell behavior, moreover in a cell-dependent manner 5,6 . Its expression is highly regulated in time and space, with a strong expression during specific morphogenetic events throughout embryogenesis and organogenesis especially in the lung 1,2,5,7 . In the growing lung, it is expressed at the epithelial-mesenchymal interface at the tips of developing airways during branching morphogenesis, and throughout lung mesenchyme during alveolarization 8,9 . In adult tissues, there is very little or no TNC expression, except in small foci of tissues subjected to high tensile stress or in certain stem cell niches 1, 8 . Otherwise, TNC is de novo re-expressed only transiently in pathological situations like inflammation or tissue damage, or permanently in diseases like cancer or rheumatoid arthritis 1, 10,11 .
TNC is highly expressed in many chronic lung diseases like chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), respiratory distress syndrome (RDS) and asthma, where it is even considered as a marker of severity [12][13][14][15][16] . In the 1990's, two research groups generated TNC-deficient mice: Saga et al. 17 and Forsberg et al. 18 . Both animal models showed that TNC inactivation was not lethal and did not induce any reproductive alteration. Studies in TNC knockout (KO) mice showed that lung disease phenotype was largely reduced in the absence of TNC in acute lung injury 19 , in selective epithelial lung injury 20 or in ovalbumin-induced asthma 21 .
In lung development, we previously showed that TNC inactivation induced a defect in branching morphogenesis (early lung development) 22 and alveolarization (late lung development) 23 . In early lung development, absence of TNC reduced the number of branches of the developing airways, and in late development, it delayed Basal pulmonary function is persistently altered in tnc Ko mice. To determine the impact of TNC inactivation on the respiratory function, we measured basal respiratory function in anesthetized and mechanically ventilated newborn (P5) and adult (P90) WT and TNC KO mice (Fig. 3). At P5 and at P90, there were obvious changes in the shape of the PV curves between TNC KO and WT animals (Fig. 3e,j). Relative to WT, static compliance (Cst), an index of the distensibility of the respiratory system, and parameter A, an estimate of the inspiratory capacity (IC), were both significantly higher in TNC KO animals under basal conditions (Fig. 3a,f,b,g). There was no statistical difference in parameter K, a measure of the overall curvature of deflation limb of the PV curves, nor in hysteresis (area), estimating atelectasis existing before the PV loop (Fig. 3c,h,d,i). We also calculated the work of breathing (WOB), which is an indicator of increased effort to breathe, from the Figure 1. TNC isoforms in newborn and adult WT mice. Lung total RNA (lanes 1-2; two WT samples) was amplified by RT-qPCR with two sets of primers surrounding the alternatively spliced fnIII domains of TNC gene: primers 5f-6r, which immediately border the alternatively spliced region, and primers 5s-6as, located at the 5' end of fnIII domain 5 and at 3' end of fnIII domain 6. Panel a: drawing of the mouse TNC gene, with the localization of the primers pairs. Panel b,c: RT-qPCR from newborn lungs; Panel d,e: RT-qPCR from adult lungs; Panel b,d: RT-qPCR with primers 5s-6as; Panel c,e: RT-qCR with primers 5f-6r. Each fnIII domain is 273 nucleotides long. RT-qPCRs are run on 2% agarose gels with 100 bp DNA ladder as size marker. Isoforms are indicated by arrows on the right side of the gel. area under the inflation limb of the PV curves (Fig. 3e,j). WOB at time 0 was similar in the newborn TNC KO and WT mice at P5 (1.84 ± 0.16 vs 1.86 ± 0.16 J/L, respectively) as well as in the adult mice at P90 (1.72 ± 0.13 vs 1.58 ± 0.31 J/L, respectively, for TNC KO and WT animals). Since TNC expression was observed in tissues exposed to tension, we then investigated TNC KO and WT mice under a mechanical stress induced by a high tidal volume (HTV) profile. Newborn and adult mice were both subjected during one hour to a HTV protocol and respiratory function parameters were recovered every 15 minutes (Fig. 4). Following HTV, differences in the shape of the PV curves between WT and TNC KO animals were only apparent in the newborn mice (Fig. 4e,j). Like what was observed under baseline conditions, static compliance and IC both remained elevated in the TNC KO newborn mice relative to WT during the whole HTV ventilation challenge (Fig. 4a,b). Parameter K was identical in both genotypes during the whole experiment (Fig. 4c) and hysteresis tended to be higher in the newborn TNC KO animals throughout the whole HTV challenge (p ≤ 0.1 at each time point; statistically significant only at the end of the ventilation) (Fig. 4d). In the adult mice, similar PV curves were obtained in the TNC KO and WT mice following 60 minutes of HTV (Fig. 4j), and, consistently, both genotypes showed superimposed values for all related parameters ( Fig. 4f-i). In order to test the hypothesis of a change in surfactant production, surfactant protein B and C (SP-B and SP-C) protein expression were evaluated by Western blot. SP-C, but not SP-B, protein expression was slightly increased in adult TNC KO lung samples ( Fig. 4k-m). collagen staining is increased around small airways in adult tnc Ko lungs. To evaluate whether certain structures or structural proteins were modified concurrently, histological stainings were performed on P5 lung sections. Hematoxylin-eosin (H&E) staining of lung sections indicated a similar morphology in both TNC KO and WT newborn lungs (Fig. 5a,b). However, when comparing ventilated animals to controls of the same age, we observed a strong thickening and a cellular infiltration of the inter-saccular septa in ventilated animals (both WT and TNC KO) suggesting an inflammatory reaction in response to the HTV ventilation (Fig. 5c,d).
Resorcin-fuchsin staining of elastin fibers did not highlight any difference in elastin distribution between TNC KO and WT lungs (Fig. 5e,f). Collagen fibers staining with Masson trichrome showed a faint collagen staining which was not different in TNC KO compared to WT lung (Fig. 5g,h).
The same histological stainings as for newborn (P5) lungs were performed on P90 lung sections. We did not find any morphological differences between TNC KO and WT lungs with hematoxylin-eosin staining (Fig. 6a,b). Elastin staining and protein expression were similar in both TNC KO and WT lungs (Fig. 6c,d,j,k). However, especially around small airways and at the tips of the developing septa, but also to a lesser degree around large airways and arterial blood vessels (Fig. 7a,b). Nevertheless, whole lung α-SMA protein expression was identical in both genotypes (Fig. 7g,i). At P90, α-SMA immunostaining was largely weaker in TNC KO lung around small airways and to a lesser extend around larger airways and arterial blood vessels ( Fig. 7c-f). Moreover, flow cytometry experiments showed a 50% decrease (p = 0.087) in the percentage of SMA + cells in TNC KO lungs (Fig. 7o), with a similar percentage of decrease in both genotypes, in SMA + desmin+ cells (smooth muscle cells) and SMA + vimentin+ cells (myofibroblasts) (Fig. 7p,q). Interestingly, the percentage of proliferating cells (ki67 +) of both populations was even lower in TNC KO samples, compared to the same population in WT (Fig. 7r,s). Whole lung α-SMA protein expression was, however, overall slightly increased in TNC KO animals ( Fig. 7h,j). In order to test other major proteins of smooth muscle actin fibers, whole lung protein expression of smooth muscle myosin heavy chain (MYH11) and phosphorylated form of smooth muscle myosin light chain (PMLC2) was checked in parallel. Expression of both proteins were the same in both adult TNC KO and WT animals ( Fig. 7k-n).

tnc-related tGfβ and TLR4 pathways are upregulated in adult TNC KO lungs. Expression of
transforming growth factor beta (TGFβ) pathway components was evaluated by RT-qPCR in P90 lungs to see if the principal TNC-related pathway was affected in parallel to lung function in TNC KO lungs. TGFβ1 and TGFβ receptor 1 (TGFβR1), -R2 and -R3 mRNA expression were largely increased in TNC KO lungs, whereas TGFβ2 and −3 were unchanged (Fig. 8a). Smad2 and Smad3 mRNAs were also largely increased in TNC KO lungs, but the expression of their phosphorylated protein was only slightly increased, as seen by Western blot (Fig. 8b-e). TNC was also shown to be a ligand for Toll-like receptor 4 (TLR4) and activate the myeloid differentiation primary response 88 (Myd88)-dependent branch of the TLR4 pathway 24,25 . We looked at the expression of two intermediates of this pathway. TLR4 and MyD88 mRNA expression were also highly up-regulated in TNC KO lungs (Fig. 8f).

Discussion
Based on the previously described critical role of TNC in lung development, we hereby explored how TNC-deficiency affected lung function in newborn mice and whether this had repercussions persisting in adulthood. The aim of this study was thus to evaluate homozygous TNC KO versus WT mice at two different time points: newborn (P5) and adult age (P90 (3 months)). Animals were tested for their lung function under basal conditions and after a mechanical stress challenge in order to highlight functional alterations that may otherwise be silent. www.nature.com/scientificreports www.nature.com/scientificreports/ It is well known that different domains compose TNC protein and control its interaction with numerous binding partners 4,26 . Mouse TNC gene possesses 8 constant and 6 alternatively spliced fnIII domains, each corresponding to an exon 2,6 . Our results confirmed that whereas mRNAs of short and long isoforms of TNC were found in newborn lungs, only short isoforms were detected in adult lungs. This is in agreement with the collection of studies made in different species and recently published as a review 6 . Short TNC isoforms promote cell attachment, cell differentiation and the formation of focal adhesions, whereas long TNC isoforms promote cell migration and are expressed when morphogenesis is very active, like in embryogenesis or cancer growth 6,8 . At the protein level, we also confirmed that TNC was highly expressed in the parenchyma but not in the conducting airways in newborn WT lung. However, in adult lungs, TNC protein was undetectable, in accordance with previously published reports 27 .
Functional evaluation of the newborn TNC KO and WT mice showed that static compliance and inspiratory capacity were elevated in the TNC KO animals under basal conditions, indicating a higher respiratory system distensibility than their WT counterpart. This difference persisted during one hour of mechanical stress. Interestingly, no alteration was observed in the general lung morphology of TNC KO animals relative to WT. As assessed, static compliance reflects the intrinsic elastic properties of the respiratory system including surface tension. Two ECM proteins are mainly responsible for elastic properties of the lung: elastin and collagen. Collagen represents 50% of lung ECM and is principally composed of fibrillar collagen (collagen I and III). Elastin represents 18% of lung ECM and is made essentially of crosslinked elastin surrounded by fibrillin microfibrils 28 . No difference in elastin or collagen staining was observed between TNC KO and WT newborn lungs. Surface tension depends on the size of airspaces and the amount and distribution of surfactant. We did not detect any morphological difference between TNC KO and WT lungs in hematoxylin-eosin staining, but morphometric measurements done by Mund and Schittny in TNC KO lungs showed the existence of a larger lung volume at P2-21, accompanied by a smaller length of the free septal edge (length of alveolar entrance rings), a reduced formation (anlage) of new alveolar septa, and a reduced alveolar surface area at P7 (as compared to WT); this result  www.nature.com/scientificreports www.nature.com/scientificreports/ was indicative of a delay in classical alveolarization, which was further shown to be caught up during continued alveolarization 23 . Regarding surfactant, no implication of TNC in surfactant metabolism has been shown so far and surfactant protein C (SP-C) immunostaining did not reveal any difference between TNC KO and WT lung sections (data not shown).
Alpha-SMA is a marker of airway smooth muscle (ASM) cells, vascular smooth muscle cells and myofibroblasts in the lung. ASM is the contractile element responsible for bronchoconstriction, airway luminal diameter and airway resistance. ASM also produces ECM proteins, pro-and anti-inflammatory mediators and growth factors necessary to influence the proliferation, migration and apoptosis of other resident cells like epithelial cells, fibroblasts, or ASM cells themselves [29][30][31] . In newborn TNC KO mice, we observed a weaker α-SMA staining around small airways and at the tips of the developing septa, and to a lesser extent around large airways and blood vessels. It is not clear whether ASM cell layer is thinner or α-SMA is less expressed in ASM cells in TNC KO animals. However, this could potentially be associated with a more heterogeneous ventilation in the TNC KO mice as a result of more easily collapsible small airways. This might also provide an explanation to the fact that compliance remained elevated in the newborn mice following the mechanical stress challenge as the weaker α-SMA staining found at the tips of the free septa (most probably myofibroblasts), representing a reduced α-SMA content in the alveolar entrance rings, would also be expected to result in a decreased tissue resistance and more www.nature.com/scientificreports www.nature.com/scientificreports/ airway instability during HTV. Overall, the differences in morphometric measurements showing an emphysematous profile of TNC KO lungs compared to WT lungs 23 , and the reduced branching observed in TNC KO lungs explants during early lung development 22 could explain the increased static compliance and inspiratory capacity observed in newborn TNC-deficient mice.
Adult TNC KO mice, like the newborn ones, also displayed under basal conditions a higher static compliance and inspiratory capacity than their age-matched WT controls, indicating a persistent functional impairment following TNC inactivation. However, under HTV ventilation, both genotypes behaved in a similar manner suggesting that despite its underlying functional impairment, the adult TNC KO lungs were able to cope during the mechanical stress challenge. Adult mice have been previously shown to be more susceptible to HTV mechanical ventilation than newborn mice 32 . Therefore, when newborn mice were ventilated with a tidal volume of 40 ml/kg, adult mice were ventilated with 25 ml/kg for a similar challenge. No striking differences in lung morphology or elastin staining/protein expression were either observed between adult TNC KO and WT mice. Morphometric measurements done previously by Mund and Schittny also showed a similar lung volume, length of the free septal edge, formation (anlage) of new septa and alveolar surface area in both genotypes at 3 months of age (P86) 23 . No difference in SP-C immunostaining was either observed in TNC KO versus WT lung sections (data not shown), but a slight increase in SP-C, but not SP-B, protein expression, speaking for an enhanced efficacy of surfactant, was observed in whole lung lysates from adult TNC KO mice relative to WT. On the other hand, there was clearly a weaker α-SMA immunostaining around small airways and to a lesser extend around large airways and blood vessels as in the newborn mice and, interestingly, collagen staining revealed a thickened subepithelial collagen layer around small airways in TNC KO lungs. These differences in collagen and surfactant might have protected the adult TNC KO lungs during HTV, which is susceptible to induce lung injury as a result of repetitive overdistention and derecruitment. These differences might therefore explain the observed unchanged static compliance in TNC-deficient mice at P90 relative to WT and the lack of a similar effect at P5. Finally, the slight increase in SP-C could also have contributed in the increase in static compliance and inspiratory capacity seen under basal conditions in the adult TNC KO mice.
In adult lungs, flow cytometry experiments confirmed the large decrease in SMA + cells observed in TNC KO lungs by immunohistochemistry. Both smooth muscle cells (SMC) and myofibroblasts cell numbers were www.nature.com/scientificreports www.nature.com/scientificreports/ decreased. Moreover, the proliferating fractions of these two populations were also lower in TNC KO compared to WT lungs, pointing to a general decrease in SMA + cells proliferation in the absence of TNC. SMA + cells originate from mesenchymal cells during branching morphogenesis (peribronchiolar stage) for smooth muscle cells, and during alveolar stage for myofibroblasts. During branching morphogenesis, pluripotent mesenchymal cells present at the distal end of each growing branch migrate in a distal to proximal direction along the branches. Under the combined action of factors from mesodermal and epithelial origin acting in a cooperative way, they give rise to pools of SMC progenitors and then differentiated SMCs. These factors are principally: fibroblast growth factor 10/9 (FGF10/9), Wnts, sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), platelet-derived growth factor receptor (PDGFR), but also vascular endothelial growth factor (VEGF) or TGFβ 33,34 . Wnt secreted by the adjacent developing epithelium is necessary for expansion and differentiation of SMC progenitors, and it was shown to act through a Wnt/TNC/PDGFR pathway 35 . This could indicate that absence of TNC impairs SMC progenitors pool formation, and therefore, explains the lower number of SMA + cells in TNC KO adult lungs.
At the molecular level though, whole lung α-SMA, but also myosin heavy and light (phosphorylated form) chain protein expression was either slightly upregulated or unchanged in adult TNC KO lungs. It is possible that the discrepancy between α-SMA immunohistochemistry/flow cytometry and Western blot results comes from the monomer versus polymer status of α-SMA protein. Indeed, actin monomers polymerize in actin filaments (with troponin and tropomyosin), and complexes with myosin filaments to form smooth muscle fibers. Alpha-SMA monomers could be detected by Western blot (denaturing gels), but not be uncovered by immunohistochemistry or flow cytometry. Or it could simply be that each SMA + cell expresses higher levels of SMA in TNC KO than in WT lungs. SMA gene expression is under the control of many factors among which TNC, through the TLR4/Myd88 pathway. Indeed, TNC was shown to induce the synthesis of inflammatory markers through the Myeloid differentiation primary response 88 (MyD88)-dependent signaling pathway of TLR4 24,25 . But TNC also induces α-SMA, collagen I and TGFβ expression, as well as myofibroblast differentiation/recruitment, through TLR4 receptor, while in return, TGFβ induces TNC through the TGFβ pathway 13,[36][37][38][39][40][41][42][43][44] . In our experiments, TGFβ pathway intermediates were not down-regulated in TNC KO lungs, but rather up-regulated, maybe as a compensatory mechanism. mRNA expression of TGFβ1 and of all three TGFβ receptors were increased, while TGFb2 and −3 mRNA expression were unchanged. This is in accordance with the fact that TGFβ1 is expressed both during embryogenesis and adulthood, whereas TGFβ2 and −3 are abundantly expressed essentially in developing organs 45  Increased TNC expression was reported in lungs of patients with asthma and chronic lung inflammation 10,47,48 . Increased TNC expression was also found in lungs of COPD patients, where an obstructive component also occurs in form of chronic bronchitis 15  showed that TNC KO lungs were protected from fibrosis, with less SMA + myofibroblasts in the lung, and TNC inactivation prevented constitutively active TGFβ to induce the differentiation of fibroblasts into myofibroblasts 19 . ASM hypertrophy is largely implicated in diseases like asthma, but also bronchitis and COPD, where thickening of ASM and airway remodeling lead to a decrease in bronchial diameter or airway obstruction 50,51 . The effect of down-regulation of α-SMA + cells following TNC inactivation could therefore constitute an interesting approach in the prevention or treatment of obstructive pulmonary diseases with thickened ASM layer or heterogeneous ventilation. Indeed, the same Wnt/TNC/PDGFR pathway regulating SMC formation and differentiation during lung development was recently shown to recur in airway remodeling in chronic asthma 52,53 .
Altogether, the results hereby presented demonstrate that TNC deficiency induces defects in lung developmental processes having functional implications that persist in adulthood. TNC being no more expressed at this stage, the functional alteration of adult TNC KO mice probably originated from lung development alterations, but subsisted in adulthood in part due to persistent architectural/structural modifications (SMA + cells, collagen) and impaired gene expression (TGFβ and TLR4 pathways). Silencing of TNC expression might be an interesting approach for the prevention of ASM thickening in patients with functional and structural airway obstruction or ventilation heterogeneity, like in asthma or COPD.

Animals. All experiments were approved by the animal ethic commissions of the Federal Food Safety and
Veterinary Office, and the Veterinary Service of the Canton Bern and performed in accordance with the Swiss Federal Act in Animal protection guidelines and regulations. TNC KO mice 18 were bred on a 129SV background. Postnatal day 5 (P5) and 3 months old (P90) TNC KO and WT animals were anesthetized with a mixture of Midazolam (5 mg/kg BW), Fentanyl (0.05 mg/kg BW) and Medetomidin (0.5 mg/kg BW), and then intratracheally intubated and connected to a computer-controlled piston ventilator (flexiVent, SCIREQ Scientific Respiratory Equipment Inc., Montreal, Canada) for mechanical ventilation and for the construction of pressure-driven pressure-volume (PV) curves. The animals were first ventilated with a standard mouse ventilation profile (respiratory rate: 150/min; tidal volume: 10 ml/kg; PEEP: 3cmH 2 O) and PV curves were constructed in a step-wise manner under closed-chest conditions (basal conditions). Then, mechanical ventilation was switched to a high amplitude ventilation mode (HTV) and the subjects were ventilated for 1 h with room air at a respiratory rate of 60/min, an Scientific RepoRtS | (2020) 10:5118 | https://doi.org/10.1038/s41598-020-61919-x www.nature.com/scientificreports www.nature.com/scientificreports/ increased tidal volume (25 ml/kg for the adults and 40 ml/kg for the newborns) and a PEEP of 0cmH 2 O. During that time, PV curves were recorded every 15 minutes, while the animals were kept on a heating pad with rectal temperature being maintained at 35-37 °C and heart rate monitored by ECG. The Salazar-Knowles equation 54 was fitted to the pressure and volume signals at each plateau of the deflation limb of the PV curves directly in the flexiVent operating system (flexiWare version 7.2) and the following parameters were automatically obtained after each measurement: Static compliance (Cst): describing the distensibility of the respiratory system (lungs and chest walls) at 5 cmH 2 O, parameter A: an estimate of the inspiratory capacity and parameter K: the exponential function capturing the curvature of the PV curves. In addition, the area between the inflation and deflation limbs of the PV curve (hysteresis) was calculated, as well as the work-of-breathing (WOB) from the area under the inflation limb, normalized to the maximal volume.
At the end of an experiment, the animal was sacrificed with an overdose of the anesthetic mixture. The left lung lobe was ligated and snap-frozen for RNA/protein extraction. The right lobes were inflated with freshly prepared paraformaldehyde 4% at a pressure of 20 cmH 2 O, fixed and stored for histological analyses. Pressure was maintained during fixation for at least 2 hours at 4 °C, to prevent the lung from recoiling. Non-ventilated age-matched animals were used as controls.