The global Sigmar1 knockout (Sigmar1^−/−) mice were obtained from the Mutant Mouse Resource Regional Center in a mixed background (C57BL6×129S/SvEv) and backcrossed with C57BL6 for 10 generations (Bernard-Marissal et al., 2015; Abdullah et al., 2018). Both male and female mice of 2 years of age were used for all the experiments to understand the pathophysiological roles of Sigmar1 protein in mice lungs. All mice housed in cages were accommodated in a well-controlled environment with a 12-h light-dark cycle and provided with regular chow diet ad libitum food and water. All animals were cared for and handled according to the Guide for the Care and Use of Laboratory Animals (Council, 2010). The Institutional Animal Care and Use Committee at LSUHS-Shreveport approved all experimental procedures and protocols for using animals. The formalin-fixed human lung tissues were obtained from individuals with no clinical signs of lung diseases during autopsy in collaboration with the Department of Pathology and Translational Pathobiology at LSU Health Sciences Center-Shreveport. Human lung tissues used in this study were deemed non-human research by the local IRB owing to the exclusive use of postmortem samples.

Lung tissues were isolated from deeply anesthetized Sigmar1^−/− and Wt mice and immediately fixed in 10% buffered formalin, processed, and embedded in paraffin as described previously. The formalin-fixed human lung tissues were obtained from a collaborator and a co-author of this manuscript (James Traylor), which were collected from individuals with no clinical sign of lung diseases during autopsy. Paraffin-embedded tissue blocks were cut into 5 µm serial sections and used for different types of histological staining. 5 μm thin sections were deparaffinized, rehydrated, and stained for Hematoxylin and Eosin (H&E) using H&E staining kit (H-3502, Vector Laboratories) following the manufacturer’s protocol. Picrosirius red and Masson’s trichome staining were used to assess the deposition of collagen content in the lung tissue and determine the fibrotic area, respectively (Molnar et al., 2017). All stained tissue sections were imaged using an Olympus BX40 brightfield microscope using ×10 objective lens in a blinded investigator manner. The percentage of collagen content was measured from Picrosirius red staining (stained red), and the fibrotic area was measured from the Masson’s trichrome staining (stained blue) using National Institutes of Health (NIH) ImageJ software v1.6.0. (Bethesda, MD) (Bhuiyan et al., 2016; Abdullah et al., 2018). 10–15 microscopic fields of lung tissues were randomly selected for quantifications and at least six mice were used per genotype, including male and female. Images were quantified in an analyst-blinded fashion.

Paraffin-embedded human and mouse lung tissue sections (5 µm thin) were deparaffinized and rehydrated before staining following the previously described protocol (Abdullah et al., 2020). Briefly, antigen unmasking solution (H-3301, Vector Laboratories, Burlingame, CA) was used to retrieve antigen using a heat-induced manner. Slides were incubated with 0.3% v/v hydrogen peroxide to block endogenous peroxidases, followed by 5% serum (Vector Laboratories, Burlingame, CA) blocking at room temperature. Subsequently, the tissue sections were incubated with primary antibodies overnight at 4°C (Sigmar1 1:100 dilution, 61,994, Cell Signaling Technology, Danvers, MA) and with secondary antibodies at room temperature in a humidified box. The amplified signal was obtained between antigen-antibodies for visualization by using Vectastain Elite Peroxidase Kit (Vector Laboratories, Burlingame, CA) followed by Dako DAB Chromogen system (DAKO, Carpinteria, CA) according to the manufacturer’s protocol. Lung tissue sections were then counterstained with hematoxylin, dehydrated, and mounted using Cytoseal XYL mounting medium (Thermo Fisher Scientific, Waltham, MA). Images were taken using Olympus BX40 brightfield microscope under ×10 objective lens with Olympus cellSens imaging software (Olympus Life Science, Waltham, MA). The DAB-precipitated brown positive areas were considered Sigmar1 positive areas.

Paraffin-embedded 5 µm thin tissue sections from human and mouse lung tissues were stained for immunofluorescence microscopy to determine Sigmar1’s presence and expression in lungs, as previously described (Bhuiyan et al., 2013b; Aishwarya et al., 2022). Briefly, the lung sections were deparaffinized, rehydrated, and antigen-unmasked by boiling at 100°C for 30 min with 10 mmol/L sodium citrate buffer (pH 6.0). A signal enhancer was used for better imaging for 30 min on the tissue sections followed by blocking buffer (1% bovine serum albumin, 0.1% fish skin gelatin in cold water, and 1% tween 20 in PBS) incubation for 1 h at room temperature. The blocked lung tissue sections were then incubated with primary antibody overnight at 4°C (Sigmar1 1:100 dilution, 61,994, Cell Signaling Technology, Danvers, MA) in a humid slide stain box. After 16 h, the tissue sections were washed with PBS and incubated with Alexa Flour conjugated secondary antibody (1:100 dilution) for 1 h 30 min at room temperature in the humid chamber. The stained sections were counterstained with DAPI (Invitrogen, Waltham, MA) for 5 min to stain nuclei and mounted using Vectashield Hardest Antifade mounting media for fluorescence staining (H1400; Vector Laboratories, Burlingame, CA). Sections were left in a dark place to dry for at least 4 hours and then imaged using a Nikon A1R high-resolution confocal microscope with Nikon NIS elements software (v.4.13.04) under ×10 objective lens.

Total proteins were isolated from the lung tissues of Wt and Sigmar1^−/− mice and whole cell tissue lysates were prepared for Western blotting following previously described protocol (Abdullah et al., 2018; Abdullah et al., 2020; Aishwarya et al., 2022). The harvested lung tissues were lysed in an ice-cold lysis buffer called Cell Lytic M (Sigma-Aldrich, St. Louis, MO) supplemented with Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) using a bead homogenizer and subsequent sonication of the homogenized material. The lysed cell materials were centrifuged to precipitate insoluble cell debris, and supernatants with soluble proteins were collected. Protein concentrations of lung tissue lysates were measured using the modified Bradford Reagent (Bio-Rad Laboratories, Hercules, CA). An equal amount of proteins were used to prepare samples using 6x Laemmli’s sample buffer for Western blotting. 20–30 µg of proteins were loaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation according to molecular weights and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA). The PVDF membranes containing proteins were then blocked using 5% non-fat skim milk and incubated with primary antibodies overnight at 4°C on a rocker at a slow speed. The membranes were then washed with 1x TBS-T buffer and incubated with alkaline-phosphatase conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h 30 min at room temperature on the slow rocker. Finally, the antibodies’ probed membranes were developed using ECF substrate (Amersham, Little Chalfont, UK) and imaged on a ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Hercules, CA). The scanned images were viewed in Bio-Rad Image Lab software v.6.0.1 and densitometric analysis was performed in NIH ImageJ software v1.6.0 (Bethesda, MD). Ponceau S protein stain dye (Acros Organics, Geel, Belgium) was used on the transferred PVDF membrane to see the transfer efficiency and could be used as a loading control. The primary antibodies used for immunoblotting in this study include-anti-Sigmar1 (1:1000; 61,994, Cell Signaling Technology, Danvers, MA), anti-pNFkB (1:1000; 3033S, Cell Signaling Technology), NFkB (1:1000; 4764S, Cell Signaling Technology), anti-Fibronectin (1:1000; ab2413, Abcam, Cambridge, UK), anti-pSMAD2 ser245/250/255 (1:1000; 3104S, Cell Signaling Technology), anti-pSMAD2 ser465/467 (1:1000; 3101S, Cell Signaling Technology), anti-SFTP-A (1:1000; PA5-79987, Invitrogen, Waltham, MA), anti-SFTP-B (1:1000; PA5-42000, Invitrogen), anti-SFTP-C (PA5-102493, Invitrogen), anti-SFTP-D (1:1000; BS-1583R, Bioss Antibodies, Woburn, MA), and anti-β-Actin (1:1000; sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA).

Lung tissues isolated from the Wt and Sigmar1^−/− mice were immediately cut into small pieces (1 mm (Su, 1982)), overnight fixed with 3% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer and underwent a post-fixation step using 1% osmium tetroxide (OsO₄). Subsequently, the tissue pieces were counterstained with uranyl acetate and lead salts and embedded in low-viscosity epoxy resins. The resin-embedded tissue blocks were then microscopically cut into thin sections, stained with toluidine blue staining to visualize the ultrastructural organizations of the sections and confirm the tissue integrity, and finally, imaged under JEOL-JEM-1400 transmission electron microscope (JEOL, Peabody, MA) using an advanced microscopy techniques digital camera (Woburn, MA). Male and female lung tissue sections from both Wt and Sigmar1^−/− mice were analyzed in an investigator-blinded manner (N = 1 male and 1 female mice per group).

All in vivo experiments were investigator-blinded and animals were randomly selected for each group/genotype. The unbiased data collection was confirmed using a numerical ear tagging system for the animals used in the experiments. All the imaging studies included alphanumerically labeled paraffin blocks, tissue slides, and microscopic images. The individual mouse and image identifier numbers were cross-referenced and matched to proceed with the analysis. All data are presented in box and whiskers plots, where boxes represent inter-quartile ranges, whiskers represent data distribution, and lines represent medians. GraphPad Prism software version 8 (La Jolla, CA) was used for all the data analyses for statistical significance. The data were first tested for normality distribution and the data sets that passed the normality assumption were analyzed using parametric unpaired Student’s t-test for groups of two. However, data sets that failed to pass the normality assumption test, were analyzed using non-parametric Kruskal–Wallis test. A p-value of equal or less than 0.05 was considered as statistically significant.

Sigmar1 is a multifunctional ubiquitously expressed protein that is widely expressed in mammalian cell types (Aishwarya et al., 2021). Several pharmacological studies using ligand binding assays demonstrated the presence of Sigmar1 in the lung (Seth et al., 1997; Maruo et al., 2000; Langa et al., 2003; Collier et al., 2007; Lever et al., 2012; Lever et al., 2014; Lever et al., 2015). However, Sigmar1 expression in the rodent and human pulmonary system has not been well characterized yet. In the present study, we used paraffin-fixed human (with no clinical record of lung disease) and mouse (Wt) lung tissue sections and performed immunohistochemical (IHC) staining using anti-Sigmar1 antibody. The expression of Sigmar1 is determined as brown precipitates, and the sections were counterstained with hematoxylin (blue) for nuclei. The immunohistochemistry staining showed the presence of Sigmar1 in healthy human lung tissue as well as in Wt mouse lung tissues (Figure 1A). IHC staining showed ubiquitous expression of Sigmar1 in all cell types of the pulmonary system and particularly, increased level of Sigmar1 positive staining was observed in the bronchiolar epithelium. A negative control staining of lung tissue from Sigmar1 global knockout mouse (Sigmar1^−/−) confirmed the specificity of Sigmar1 antibody. Paraffin fixed lung tissue sections were also used for immunofluorescence (IF) staining using anti-Sigmar1 antibody (green), indicating Sigmar1 expression in the human and mouse lung tissues (Figure 1B). Both the IHC and IF staining confirmed the ubiquitous expression of Sigmar1 in the pulmonary systems of human and mouse.

After determining Sigmar1’s expression in the lung tissue, we used Sigmar1 null mouse to determine whether Sigmar1 ablation in mice developed any pathological changes in the lung of the aged mice. Sigmar1^−/− mice were reported to have a normal life cycle but were reported to develop several neurological and cardiac pathological changes (Aishwarya et al., 2021). Therefore, we used the aged mice to characterize the lung pathology in the aged mice. Formalin-fixed paraffin tissue sections were used for hematoxylin and eosin (H&E) staining to observe the structural organizations of the lung tissue. H&E staining showed altered alveolar structures, which is the most critical functional unit of lung for exchanging gas, in the absence of Sigmar1 (Figure 2A). Alteration in alveolar structures include not fully open alveoli, pneumocytes disarray, and lots of nucleus infiltration in absence of Sigmar1 in mice. In addition, Sigmar1^−/− lungs showed evidence of higher extent of immune cells infiltration indicated by more nuclear staining in purple compared to Wt littermate control (Figure 2A). We have also checked the protein expression level of a very common marker of inflammatory respiratory illness, phosphorylated nuclear factor kappa B (NFkB). The protein expression of phospho-NFkB was significantly higher in Sigmar1 deficient lung lysates (Figure 2B). Lung tissues from three mice per group were used for each experiment. All these data suggested that Sigmar1 null mice develop signs of pulmonary inflammation over aging.

We also determined collagen deposition and fibrotic remodeling of lung tissues in aged Sigmar1^−/− and Wt mice using Sirius red and Masson’s trichrome staining of histological lung tissue sections. Sirius red staining showed significantly increased collagen deposition area, and Masson’s trichrome staining showed significantly higher fibrotic area in the Sigmar1^−/− lung sections compared to Wt lung sections (Figures 3A, B). Consistent with this, we also observed an increased expression level of extracellular matrix protein called fibronectin, which is a well-known fibrotic marker, in Sigmar1^−/− lung lysates compared to Wt littermate control (Figures 4A, B). As sustained phosphorylation of SMAD2 protein is reported in TGF-β-induced pulmonary fibrosis (Ard et al., 2019), we also measured the protein level of phosphorylated SMAD2 in the lung cell lysates. Sigmar1^−/− lung lysates showed significantly increased phosphorylated SMAD2 at serine 245/250/255 and serine 465/467 sites compared to Wt lungs (Figures 4A, B) (n = 3 per group for histological staining and Western blot analysis). All these data together suggest that Sigmar1^−/− lungs develop pulmonary fibrotic remodeling over aging.

To further understand the direct role of Sigmar1 in respiratory physiology, we have analyzed the ultrastructural components of lung tissue isolated from Sigmar1^−/− and Wt littermate controls using transmission electron microscopy (TEM). Toluidine blue staining of semithin sections of the lungs demonstrated the structural organizations of the organelles of lungs including bronchioles (B), alveoli (Al), smooth muscle cells (Sm), connective tissue (Ct), type-I alveolar epithelial cells (PI), and type-II alveolar epithelial cells (PII) (Figure 5). The Sm lining around the B in Sigmar1^−/− lungs showed thicker compared to Wt lungs. We performed TEM analysis of these semithin sections for more detailed ultrastructural analysis (Figure 6). The control lungs showed type-II alveolar epithelial cells (PII) with numerous multilamellar bodies (LBs) of different sizes and densely packed lipid lamellae. Interestingly, the transmission electron micrographs of lung tissue sections showed noticeable different structure and pattern of LBs in Sigmar1^−/− lungs compared to control Wt lungs. However, PII cells in Sigmar1^−/− lungs showed a partial loss in lamellae membranous structures in the lamellar bodies (Figure 6). In addition, Sigmar1^−/− lungs also showed accumulation of numerous large endosomes (LE) with scarce amorphous content, elongated mitochondria with comparatively less dense mitochondrial cristae (M), and increased level of collagen (C) deposition (Figure 6).

Pulmonary surfactant is required to lower the alveolar air-water interface to reduce the surface tension. Surfactant is composed of a unique phospholipid called dipalmitoylphosphatidyl-choline, and four associated proteins, including SFTP-A, SFTP-B, SFTP-C, and SFTP-D (Froh et al., 1990). Most of the surfactant components are stored and released by the multilamellar bodies in the type-II alveolar epithelial cells. Since our data showed an altered structural pattern of lamellar bodies in type-II pneumocytes in Sigmar1^−/− mouse lungs, we further investigated if this affected the protein expression levels of the four surfactant proteins. Western blot analysis and densitometric quantification showed the increased protein levels for the SFTP-A, SFTP-B, SFTP-C, and SFTP-D precursor proteins in the aged Sigmar1^−/− mouse lungs compared to age match littermate Wt mouse lungs (Figure 7). Lungs tissues were collected from three mice per group for comparing surfactant proteins levels in lungs.