Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice

Sangmi S Park; Michelle Mai; Magdalena Ploszaj; Huchong Cai; Lucas McGarvey; Christian Mueller; Itsaso Garcia-Arcos; Patrick Geraghty

doi:10.1371/journal.pone.0291948

. 2023 Oct 11;18(10):e0291948. doi: 10.1371/journal.pone.0291948

Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice

Sangmi S Park ¹, Michelle Mai ², Magdalena Ploszaj ², Huchong Cai ², Lucas McGarvey ², Christian Mueller ^3,⁴, Itsaso Garcia-Arcos ^1,², Patrick Geraghty ^1,^2,^*

Editor: Doa’a G F Al-u’datt⁵

PMCID: PMC10566687 PMID: 37819895

Abstract

Type 1 diabetes (T1D) is a metabolic disease characterized by hyperglycemia and can affect multiple organs, leading to life-threatening complications. Increased prevalence of pulmonary disease is observed in T1D patients, and diabetes is a leading cause of comorbidity in several lung pathologies. A deficiency of alpha-1 antitrypsin (AAT) can lead to the development of emphysema. Decreased AAT plasma concentrations and anti-protease activity are documented in T1D patients. The objective of this study was to determine whether T1D exacerbates the progression of lung damage in AAT deficiency. First, pulmonary function testing (PFT) and histopathological changes in the lungs of C57BL/6J streptozotocin (STZ)-induced T1D mice were investigated 3 and 6 months after the onset of hyperglycemia. PFT demonstrated a restrictive pulmonary pattern in the lungs of STZ-injected mice, along with upregulation of mRNA expression of pro-fibrotic markers Acta2, Ccn2, and Fn1. Increased collagen deposition was observed 6 months after the onset of hyperglycemia. To study the effect of T1D on the progression of lung damage in AAT deficiency background, C57BL/6J AAT knockout (KO) mice were used. Control and STZ-challenged AAT KO mice did not show significant changes in lung function 3 months after the onset of hyperglycemia. However, histological examination of the lung demonstrated increased collagen accumulation and alveolar space enlargement in STZ-induced AAT KO mice. AAT pretreatment on TGF-β-stimulated primary lung fibroblasts reduced mRNA expression of pro-fibrotic markers ACTA2, CCN2, and FN1. Induction of T1D in AAT deficiency leads to a combined pulmonary fibrosis and emphysema (CPFE) phenotype in male mice.

Introduction

Type 1 diabetes (T1D) is a metabolic disorder characterized by insulin deficiency and subsequent hyperglycemia. Chronic hyperglycemia resulting from T1D can have systemic effects, damaging various organs and leading to numerous complications that contribute to morbidity and mortality of the disease [1]. Different studies show impairment in the lung of T1D patients such as increased airway resistance [2], in addition to decreased lung volumes [3, 4], pulmonary elastic recoil [5, 6], and diffusing capacity of the lung for carbon monoxide (DLCO) [7]. T1D is associated with an increased risk of several respiratory diseases such as emphysema, chronic obstructive pulmonary disease (COPD), asthma, and chronic bronchitis [8]. Diabetes is also a common comorbidity in COPD and idiopathic pulmonary fibrosis (IPF) [9, 10]. TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in the lung [11] and lung tissue histopathological changes with increased inflammatory cell infiltration and thickening of the alveolar septa [12] are reported in animal models of T1D.

Emphysema and loss of the alveolar respiratory surface can occur with alpha-1 antitrypsin (AAT) deficiency. AAT is an anti-protease that is predominantly produced by the liver and secreted into the bloodstream [13]. AAT is well-known for its protective role in the lungs as it neutralizes proteolytic damage of the connective tissue components of the lung by proteases, such as neutrophil elastase [14]. Inherited mutations in the gene coding AAT, SERPINA1, cause AAT deficiency, in which misfolding of AAT proteins triggers their accumulation in the liver and subsequently decreased AAT concentration in the blood and lungs. This can lead to the destruction of alveolar walls in the lungs and the development of emphysema. AAT is also known to play a role in heterogeneous signaling processes not necessarily linked to its anti-protease capacity, including activating phosphatases [15], and inhibiting caspase activity [16]. In the context of infectious disease, AAT has anti-HIV [17] and rhinovirus properties [18], regulates neutrophil activation and degranulation [19, 20], is involved in dendritic cell maturation and promotes regulatory T cell (Treg) differentiation [21]. In the context of inflammatory processes, AAT can increase IL-10 and IL-1Ra release [22], minimize epithelial barrier damage, lower nitric oxide production [23], and regulate IL-8-mediated neutrophil chemotaxis [24]. Finally, in a profibrotic context, AAT decreases renal fibrosis by inhibiting TGFβ-induced epithelial-mesenchymal transition (EMT) [25].

Serum concentration and activity of AAT are lower in T1D patients and are associated with hyperglycemia and the duration of diabetes [26, 27]. AAT with augmentation therapy in young subjects prevents T1D development, prolongs islet allograft survival [28], increases insulin release capacity [29], and inhibits pancreatic B-cell apoptosis [30]. In vivo, studies using T1D mouse models showed that administration of AAT prolonged islet graft survival and inhibited β-cell apoptosis [28, 30]. Additionally, AAT gene therapy prevented the development of T1D and inhibited insulitis [31, 32]. In vitro, AAT increased insulin secretion and protected β-cells from apoptosis in rat pancreatic islets [29]. Despite the suggested role of AAT in the pathogenesis of T1D, the effect of T1D on AAT deficiency is unknown.

To better understand T1D-induced pulmonary complications, the effect of hyperglycemia in the lung was studied by examining the functional and histopathological changes at two-time points following the onset of hyperglycemia using the streptozotocin (STZ)-induced mouse model of T1D. The effect of T1D on the progression of lung damage in AAT deficiency was also investigated using STZ-induced AAT knockout (KO) mice 3 months after the onset of hyperglycemia. These findings show that STZ induces fibrotic changes in the lung and that it leads to accelerated development of combined pulmonary fibrosis and emphysema (CPFE) in the absence of AAT.

Methods

Ethics statement

This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Institutional Animal Care and Use Committee (IACUC) guidelines and the research was conducted according to the principles of the World Medical Association Declaration of Helsinki. SUNY Downstate Health Sciences University’s IACUC approved the protocol (protocol number 15–10482). Animals were monitored several times daily, and any mice exhibiting signs of distress were euthanized. Distress was defined as animals showing one or several of the following characteristics; ruffled fur, arched back, respiratory distress (e.g., gasping), weight loss greater than 20%, and any notable unusual behavior. Investigators consulted with veterinarians if animals appeared distressed. All animals were anesthetized by intraperitoneal (IP) injection of a mixture of ketamine and xylazine. As animals were sedated and paralyzed during pulmonary function testing, euthanized was confirmed by cervical dislocation and no detection of a pulse, followed by vital organ collection.

Animal models

8–13 weeks old male C57BL/6J mice were fasted for 4 hours and intraperitoneally injected with STZ (50 mg/kg) dissolved in citrate buffer for 5 consecutive days to induce T1D. Control mice were injected with citrate buffer for 5 consecutive days. 14 days after the injection, fasting blood glucose was measured and mice that had blood glucose ≥ 250 mg/dl were considered diabetic. Pulmonary function testing (PFT) was performed on the mice 3 and 6 months after the induction of T1D, at which point the mice were euthanized.

AAT KO mice were previously generated by knocking out Serpina1a-e in C57BL/6J mice, using CRISPR-Cas9 as described in Borel et al. [33]. This is a whole-body AAT knockout animal. 8–13 weeks old male AAT KO mice were injected with STZ or citrate buffer as described above. Mice will be designated as follows throughout the paper for simplification: control mice (vehicle-injected mice), STZ mice (STZ-injected mice), AAT KO (vehicle-injected Serpina1a-e knockout mice), and AAT KO STZ (STZ-injected Serpina1a-e knockout mice).

Blood glucose and HbA1c tests

Mice were fasted for 6 hours before measuring blood glucose. Fasting blood glucose levels were measured via tail vein venipuncture using the AlphaTRAK blood glucose monitoring system. To measure glycated hemoglobin (HbA1c), mice were fasted for 4 hours and blood was collected via submandibular bleeding. HbA1c was measured using the DCA Vantage Analyzer (Siemens Diagnostics) using whole blood.

Glucose tolerance testing was performed on mice by fasting them for 4 hours before testing. Glucose solution (250 mg/ml) was injected intraperitoneally at a dose of 2.5 g/kg body weight (i.e., 250 μl injection volume for a 20 g animal). Blood glucose was measured via tail vein venipuncture using the glucometer at 0, 15, 30, 60, 90, and 120 minutes after glucose injection.

Pulmonary function test

Mice were fasted for 4 hours before the test. Mice were anesthetized by intraperitoneal injection of ketamine/xylazine hydrochloride solution (100/10 mg/kg; Millipore Sigma). Mice were tracheostomized and connected to the ventilator via endotracheal cannula to the FlexiVent System (SCIREQ) for PFTs. Mice were paralyzed with pancuronium bromide (1 mg/kg) by intramuscular injection after initiating mechanical ventilation. PFTs were performed, as previously described [34]. The Flexivent software (FlexiWare, Version 7.6, Service Pack 5) was used to calculate respiratory system resistance, pressure-volume loops, compliance, elastance, Newtonian resistance (RN), tissue dampening (G), and tissue elastance (H), as per established the protocol [34] and outlined by the manufacturer. Forced expiratory measurements were also performed to calculate the forced expired volume of 0.1 seconds (FEV_0.1), forced vital capacity (FVC), peak expiratory flow (PEF), and forced expiratory flow at 50% of FVC (FEF₅₀).

Histology

After euthanasia via cervical dislocation, bronchoalveolar lavage (BAL) was collected and the lungs were perfused with 10% formalin for pressure fixation, as previously described [35]. Lung tissues were paraffin-embedded, sectioned, and stained with Masson’s trichrome (Abcam) as recommended by the manufacturer. Images of lung sections were taken at 20x magnification and collagen deposition was measured in the whole upper and lower lobes of the stained lung sections (approximately 100–150 images per lobe; 200–250 images per sample) using the Orbit Image Analysis software (https://www.orbit.bio). The same images were also scored for fibrosis using the modified Ashcroft score as outlined by Hubner et al [36]. Mean linear intercept (MLI) was quantified as an index of airspace size in the upper and lower lobes of stained lung sections to assess morphological changes in lung parenchyma associated with the presence of emphysema, as described by Crowley et al. [37].

Quantitative real-time PCR

Lungs were homogenized in TRI Reagent with 1.0 mm diameter zirconia beads (Biospec Products) for 30 seconds using bead beater disruption (Minibeadbeater-16, BioSpec Products). RNA was extracted as described in the manufacturer’s protocol (Direct-zol RNA miniprep kit, Zymo Research). First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) at 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, followed by a cooling step at 4°C. qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems) with the following thermocycling program: [95°C for 5 min, 95°C for 15 sec, 55°C for 1 min] x 40 times, followed by 65°C to 95°C (0.5°C increment) for 5 sec (C1000 Touch Thermal Cycler, Bio Rad). Hprt1 was used as the housekeeping gene. During RNA isolate, RNA was treated with DNase using the Direct-zol RNA miniprep kit from Zymo Research. RNA (without reverse transcriptase treatment) was tested as a genomic contamination control. 1 ug of total RNA was used for the first strand cDNA template synthesis to generate 20 μl of cDNA. cDNA was dilution by a factor of 4 and 1 μl was used in a 10 μl RT-PCR reaction. Primer sequences are provided in S1 Table.

AAT concentration in plasma

An AAT ELISA assay was performed on mouse plasma as outlined in the manufacturer’s protocol (Mouse Alpha 1-Antitrypsin ELISA kit, ICL Lab). Plasma samples were diluted (1:80,000). Data were presented as AAT concentration in mg/mL plasma. All AAT and wild-type mice were screened for plasma AAT levels to confirm that they were knockouts (see S1 Fig for an example of screening data).

Cell culture

Primary adult human lung fibroblasts were purchased from Lonza. Fibroblasts were cultured in Fibroblast Growth Medium supplemented with 0.5 mL recombinant human insulin, 0.5 mL hFGF-B, 0.5 mL GA-1000 (Gentamicin and Amphotericin B), 10 mL FBS and antibiotics (10,000 units/mL penicillin and 10,000 μg/mL streptomycin). Fibroblast cultures were maintained in collagen I (4 μg/mL)-coated wells in a humidified atmosphere with 5% CO₂ at 37°C. Fibroblast cultures were tested in passages four to seven. Fibroblasts were cultured for 2 hours in serum-free media before supplementation with active plasma purified AAT (MyBioSource, Inc.) and 5 ng/ml recombinant TGFβ (R&D Systems). RNA was extracted from fibroblast for qRT-PCR. Primer sequences are provided in S1 Table.

Statistical analyses

The majority of the data are expressed as dot plots with the means ± S.E.M. highlighted. A comparison of groups was performed by Student’s t-test (two-tailed). Experiments with more than 2 groups were analyzed by 2-way ANOVA with Bonferroni posttests analysis. p values for significance were set at 0.05 All analyses were performed using GraphPad Prism Software (Version 9).

Results

Low-dose STZ injections induce a restrictive pulmonary pattern

STZ mice had a 2-fold increase in fasting blood glucose and an approximately 1.5-fold increase in HbA1c when compared with their controls (Fig 1A). Hyperglycemia was sustained in STZ mice throughout the study period (6 months since the onset of hyperglycemia). Consistently, the glucose clearance rate was decreased in STZ mice, with significantly higher fasting blood glucose levels at all time points compared to vehicle control mice after the glucose challenge (S2A Fig). These data confirmed that STZ injections successfully induced a sustained T1D-like phenotype, and we proceeded to analyze pulmonary parameters.

To investigate whether STZ leads to functional changes in the lungs, we conducted PFT 3 months and 6 months after the onset of hyperglycemia. Forced expiratory volume (FEV_0.1) and forced vital capacity (FVC) were both decreased by approximately 15% (p = 0.0031 and 0.0044) in STZ mice after 3 months, and no difference was observed in FEV/FVC ratios (Fig 1B), which indicates a restrictive lung function pattern. In line with this, the Pressure-Volume (PV) loop shifted downwards and to the right in STZ mice. Inspiratory capacity (IC) and compliance decreased by 14% and 25% (p = 0.0019 and 0.0009), respectively, whereas tissue damping (G) and elastance (H) increased by 1.5-fold and 1.2-fold (p = 0.0211 and 0.0063) in STZ mice, respectively (Fig 1B).

These functional changes were chronic and persisted 6 months after the onset of hyperglycemia (Fig 1C); FEV_0.1 and FVC were still decreased by 18% (p = 0.0001 and 0.0005) in STZ mice, and FEV_0.1/FVC ratio was similar between STZ and control (Fig 1C). PV loops remained shifted down and to the right, and IC and lung compliance were decreased by 18.9% and 25.7% (p < 0.0001 and p = 0.0002), respectively in STZ mice. G and H were increased by 1.5-fold and 1.3-fold (p = 0.0034 and 0.0009), respectively in STZ mice compared to the control mice. These data were consistent with those observed after only 3 months of hyperglycemia. There was no worsening of the pulmonary conditions despite STZ mice showing arrested body weight gain at 3 months of age (S2B Fig).

STZ mice exhibit fibrotic traits in the lung

To further investigate the functional changes in the lungs of STZ mice, we quantified collagen staining in Masson’s trichrome-stained lung tissues. No collagen accumulation was observed in the wild-type mice 3 months after the onset of hyperglycemia as assessed by histological means by 2 separate methods (Fig 2A). However, mRNA expression of fibrotic markers Ccn2 and Fn1 was significantly increased in lung homogenates from STZ mice. By 6 months after the onset of hyperglycemia, STZ mice showed visible accumulation of collagen in the upper (3.5-fold; p = 0.001) and lower lobes (4.3-fold; p < 0.0001) of the lung, along with a significant increase in mRNA expression of Acta2 and Ccn2 (Fig 2B). The modified Ashcroft scoring method also confirmed elevated fibrosis in STZ mice at 6 months post STZ injections (Fig 2B). These results demonstrate the upregulation of fibrotic markers in the lungs of STZ mice as early as 3 months after the onset of hyperglycemia, which precedes collagen accumulation detectable by light microscopy by 6 months. Mean linear intercepts analysis was performed and demonstrated no emphysema development in the wild-type STZ mice at 3- or 6-months post STZ injections (S3 Fig).

Fig 2 — Representative images of the lung sections stained with Masson’s trichrome were taken at 20x magnification. Blue staining representing collagen was quantified in the upper and lower lobes of the lung sections. Lung fibrosis was also scored using the modified Ashcroft method at each time point. *Acta2*, *Ccn2*, and *Fn1* mRNA expressions in the lung were measured by qRT-PCR. (A) 3 months after the onset of hyperglycemia and (B) 6 months after the onset of hyperglycemia. Data were analyzed by unpaired t-test. N = 5 animals per group. *p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.

Plasma AAT concentration is decreased in STZ mice and hyperglycemia is exacerbated in AAT-deficient STZ mice

T1D is associated with decreased expression of AAT, and the significance of this decrease is not understood. AAT deficiency is a risk factor for the onset of emphysema [26, 27]. Consistent with the observations in humans, plasma protein levels of AAT in STZ mice were decreased by 1.5-fold compared to the control mice (p = 0.0079) (Fig 3A) 6 months after the onset of hyperglycemia. To model the effect of T1D on the lung in AAT deficiency, we injected STZ into Serpina1a-e knockout (AAT KO) mice. Fasting blood glucose and HbA1c measured at several time points after the onset of hyperglycemia demonstrated that STZ AAT KO mice exhibited higher fasting blood glucose compared to STZ mice throughout the 3 months study period (Fig 3C).

Fig 3 — (A) Plasma concentration of AAT was measured in control and STZ mice. (B) Schematic showing the timeline of the experiment using vehicle and STZ-injected WT and AAT KO mice. This image was created with BioRender.com. (C) Fasting blood glucose and glycated hemoglobin (HbA1c) were measured in mice every 4 weeks. Data were analyzed by unpaired t-test and two-way ANOVA using Tukey’s post hoc tests. N = 5 to 9 animals per group. *p≤0.05; **p≤0.01; ****p≤0.0001.

STZ AAT KO mice exhibit no significant functional changes in the lung

3 months after the validation of hyperglycemia in STZ AAT KO mice, PFTs were performed, and the results were compared to those from STZ and vehicle control mice (Fig 4). The physiology phenotype appeared to be mild and did not reach significance. AAT KO mice did not show significant differences in FEV/FVC ratios, FEV_0.1, and FVC compared to the control mice (Fig 4A), and their PV loop shifted only slightly upwards and towards the left compared to the control mice (Fig 4B). Consistently, IC, compliance, G, and H were unaffected (Fig 4C).

Fig 4 — PFT was performed in mice 3 months after the onset of hyperglycemia. (A) FEV0.1 and FVC measurements (B) Pressure-volume curve (AUC = Area under the curve) (C) Inspiratory capacity, compliance, tissue damping, and tissue elastance. Data were analyzed by two-way ANOVA using Tukey’s post hoc tests. N = 5 to 7 animals per group. *p≤0.05; **p≤0.01.

AAT KO mice are expected to develop emphysema starting at around 35 weeks of age [33]. Since PFT was performed in the mice at 20–25 weeks of age, it can be postulated that AAT KO mice have not yet developed emphysema. Interestingly, while all of the PFT parameters in STZ AAT KO mice changed in the same direction as STZ mice, they did not reach significance (Fig 4A and 4C). PV loops of STZ AAT KO mice shifted slightly downwards and to the right compared to the AAT KO mice but this shift was not significant (Fig 4B). Overall, no obvious functional changes were observed in AAT KO compared to the control mice and STZ did not seem to affect pulmonary function in AAT KO mice based on PFT results alone.

STZ-induced AAT KO mice exhibit fibrotic and emphysematous histological changes in the lung

Since STZ injections were associated with a fibrotic phenotype in WT mice, we examined Masson’s trichrome-stained lung sections from the AAT-deficient mouse models. STZ AAT KO mice exhibited higher collagen accumulation than AAT KO mice (~ 2-fold) in both the upper and lower lobes of the lung (p < 0.0001 and p = 0.0033). Fibrosis scoring with the modified Ashcroft method further confirmed elevated fibrosis in the STZ AAT KO mice (Fig 5B). When compared with STZ mice, STZ AAT KO mice showed upregulated Acta2 mRNA expression in the lung (p = 0.0019) (Fig 5A and 5B). These observations suggest that the STZ injections caused a more severe increase in collagen deposition and Acta2 gene expression in the AAT deficiency model.

Fig 5 — (A) Representative images of the lung sections stained with Masson’s trichrome were taken at 20x magnification. (B) Blue staining representing collagen was quantified in the upper and lower lobes of the lung sections. *Acta2*, *Ccn2*, and *Fn1* mRNA expressions in the lung were measured by qRT-PCR. (C) Airspace enlargements were assessed by MLI measurements in the upper and lower lobes of the lung. Data were analyzed by two-way ANOVA using Tukey’s post hoc tests. N = 5 animals per group. *≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.

Since AAT KO mice are expected to develop emphysema, MLI were analyzed in the lung sections. These 20–25 weeks-old AAT KO mice had not yet developed emphysema in either the upper or lower lobe of the lung (Fig 5C). In contrast, STZ AAT KO mice exhibited emphysema in the upper lobe of the lung with a significant increase in airway space enlargement compared to AAT KO mice. Taken together, these data suggest that the combination of AAT deficiency and T1D accelerates the progression of emphysema and induces concomitant fibrosis, leading to the development of CPFE.

Altered TGFβ signaling observed in STZ animals in vivo and AAT can counter TGFβ-mediated fibroblast signaling in vitro

To explore inflammation responses in our mouse models, qRT-PCR and Luminex assays were undertaken. Luminex assays were performed on the plasma and BALF of wild-type animals 6 months after the onset of hyperglycemia. Plasma IL6 was decreased and CXCL5 was elevated in STZ mice at 6 months, while no changes in BALF CXCL1, CCL2, IL6, CCL20, CXCL5, and RAGE were observed (S4 Fig). TGF-β gene expression was elevated in the 3-month STZ animal group but not at the 6-month time point (Fig 6A) or in the AAT KO mice in both time points (Fig 6B).

Fig 6 — qRT-PCR was formed to determine (A) TGF gene expression changes in STZ mice at 3- and 6-months post STZ injects and for TGF and CXCL5 gene expression changes in wild-type and AAT KO mice 3-months post STZ injections. (C) qRT-PCR was performed for *CCN2*, *ACTA2*, and *FN1* changes in primary human lung fibroblasts exposed to recombinant TGF-β in the presence or absence of AAT. Data were analyzed by two-way ANOVA using Tukey’s post hoc tests. *≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001.

To determine if AAT counteracted TGF-β responses, primary human lung fibroblasts were exposed to recombinant TGF-β in the presence or absence of AAT. AAT countered TGF-β-induced expression of CCN2, ACTA2, and FN1 (Fig 6C), suggesting direct anti-fibrotic effects of AAT in fibroblasts.

Discussion

In this study, our data show that the STZ mouse model exhibits a pattern of restrictive pulmonary defect, as indicated by decreased FEV and FVC along with decreased inspiratory capacity and compliance. These functional changes were accompanied by increased expression of fibrotic markers Acta2 and Ccn2 as well as accumulation of collagen in the lungs, which is consistent with other studies that suggested that T1D leads to fibrotic development in the lung [11, 12, 38]. In contrast to the changes in pulmonary function and mRNA expression of fibrotic markers in STZ mice at both 3 and 6 months after the onset of hyperglycemia, collagen staining in the lung was increased in STZ mice only at the 6-month time point. This suggests that active tissue repair and ECM deposition [39, 40] start as early as 3 months after the onset of diabetes and becomes detectable by histology at the 6-month time point.

When combined with the AAT-deficient mouse model, STZ accelerated the development of emphysema and led to the progression of CPFE. Emphysema and pulmonary fibrosis exhibit some opposite physiologic effects. Emphysema is characterized by decreased lung elastic recoil and increased lung compliance and volume, and pulmonary fibrosis is characterized by increased lung elastic recoil and decreased lung compliance and volume. Therefore, PFT alone was insufficient to diagnose CPFE in STZ AAT KO mice [41]. However, the histology data demonstrated increased collagen accumulation in the lung of STZ-induced AAT KO mice compared to STZ mice and AAT KO control mice, suggesting that 3 months after the onset of hyperglycemia was insufficient to induce changes in the extracellular matrix (ECM) in wildtype mice but AAT deficiency accelerated this histological change, which is observed only at 6 months in STZ mice. Increased emphysema was also detected in STZ AAT KO mice compared to the control groups, which suggests that STZ accelerated the progression of emphysema. STZ AAT KO mice developed more severe hyperglycemia compared to STZ mice that express AAT, consistent with studies that show the protective role of AAT in the pathogenesis of T1D [30–32]. We also determined that elevated TGF-β expression is observed in the lungs of STZ mice and that AAT counters TGF-β signaling in vitro using human primary lung fibroblast cells.

Potential explanations for accelerated lung damage observed in AAT KO STZ mice include the ability of AAT to modulate inflammatory and immune responses, which is compromised in T1D models. Administration of AAT was shown to prolong islet graft survival and inhibit β-cell apoptosis [28, 30], prevent the development of T1D by preventing cell-mediated autoimmunity, and inhibit insulitis in a genetic T1D mouse model [31, 32]. AAT has anti-inflammatory properties that can suppress TNF-α and MMP-12 secretion and increase cAMP-mediated secretion of IL-10 [42–44]. TNF-α can mediate immune responses observed in the pathogenesis of T1D and inhibiting it prevented the development of T1D in nonobese diabetic (NOD) mice [45]. MMP-12 is a negative regulator of glucose metabolism and is also implicated in the development of emphysema [46, 47]. The immunosuppressive effects of IL-10 can modulate the progression of inflammatory autoimmune diseases, including T1D and COPD [48–50]. Neutrophils isolated from human T1D subjects or mice are primed to produce neutrophil extracellular traps (NETs) [51]. Neutrophils from AAT-deficient subjects have increased neutrophil responses [19, 24, 52] and AAT could counter elevated neutrophil activation, NETs formation, and degranulation. In our study, we detected elevated CXCL5 concentrations in the plasma of STZ mice and increased gene expression of Cxcl5 in the lungs of AAT KO mice. CXCL5 is an important chemoattractant for leukocyte recruitment in mice as they lack IL8 [53]. A recent study demonstrated that monocyte-derived macrophages from ZZ AAT deficient subjects expressed higher levels of CXCL5, in addition to CXCL1 and CXCL8 [54]. AAT is also known to play a role in endothelial [55] and smooth muscle cell [56] immune responses. AAT deficiency can result in vasculitis development with elevated inflammation observed in the blood vessels [57]. In larger central arteries, the balance between elastin and collagen is important in arterial stiffness [58]. Outside of its anti-inflammatory and anti-protease functions, little is known about whether AAT influences the micro- and macrovascular of the pulmonary vasculature. Patients with AAT deficient-related COPD do have increased aortic stiffness that could lead to an increased risk of cardiovascular disease [59]. This may represent a novel area for investigation considering the impact of hyperglycemia on fibrosis and the exaggerated phenotype we observe in the AAT KO animals following STZ injections.

The role of AAT as a regulator of epithelial-mesenchymal transition (EMT) may explain the accelerated development of fibrotic phenotype in AAT KO STZ mouse lungs in our study. AAT can inhibit the Wnt canonical pathway [60], which is involved in the pathogenesis of diabetic nephropathy [61]. AAT treatment can also inhibit renal fibrosis by inhibiting TGF-β-mediated EMT [25]. Our in vitro work suggests that AAT could counter TGF-β-mediated signaling. However, we were not able to detect altered TGF-β gene expression in the AAT KO STZ animals. This may be due to only sampling at the 3-month time point. Additional sampling at other time points may be beneficial to further elucidate the mechanism for the fibrotic profile observed in this study.

One of the limitations of this study is that our AAT deficiency model is a knockout mouse, different from AAT deficiency in humans caused by mutations in the SERPINA1 gene and intrahepatic accumulation of misfolded AAT. Null mutations of the SERPINA1 gene are reported in humans but are very rare [62]. Second, several studies suggest that female mice are resistant to STZ-induced diabetes, therefore we focused on utilizing male mice [63]. This current study can only account for the results observed in male animals. Confirmation of our findings will require testing in female animals. The T1D literature suggests that female mice are more resistant to streptozotocin than male animals [64–68]. A recent study demonstrated that the resistance of female mice to STZ-induced diabetes can be overcome by increasing the dose of STZ [69]. Therefore, it may be possible to undertake a female study with this modified dosing. We also started our experiments when animals were between 8–13 weeks, which may not exactly match the age for onset of T1D in humans. Additionally, the maturational rate of mice does not linearly correlate with humans and some suggest that maturation occurs in mice 150 times faster during the first month of life and 45 times faster over the next five months [70]. Mature adult mice range in age from 3–6 months, which is predicted to be equivalent to humans ranging from 20–30 years. Therefore, 8–13-week-old mice used in our study could be deemed to be close to 15–20 years old in human aging.

Furthermore, the impact of hyperglycemia was previously observed to reduce serum AAT concentration and activity in human T1D subjects [27], but AAT levels typically increase with inflammation and are viewed as an acute-phase reactant [71]. In T1D, there are higher levels of systemic inflammation. Therefore, the reduced AAT levels observed in our model are consistent with human AAT levels in T1D. Other cells and tissues produce AAT but the majority comes from the liver. Perhaps hyperglycemia has some long-term effects on liver function that could contribute to lower AAT levels. Equally, it is unclear how hyperglycemia impacts the activity of AAT. These are areas of interest for future studies. We cannot rule out the possibility that STZ itself could induce liver damage and reduce AAT production. The exact mechanism for the development of CPFE in the AAT KO animals remains unknown, but this will be an important area of future investigation. AAT’s regulation of TGF-β signaling may be a critical event but further work is required to elucidate the exact mechanism. Other investigators have expressed the human non-mutated AAT gene, by a recombinant adeno-associated virus, in nonobese diabetic (NOD) mice and observed significantly reduced insulitis and prevented the development of hyperglycemia [31]. It would be of interest to either express a mutated version of human AAT or administer AAT to the AAT KO mice and observe glucose levels and pulmonary function following STZ-induced hyperglycemia. Finally, we only tested the AAT KO mice 3 months after STZ injections. Additional time points should be explored to determine the acute and long-term impact of AAT deficiency and hyperglycemia on disease severity and earlier inflammation or TGFβ signaling.

Altogether, our study shows that induction of T1D by STZ in mice is associated with the development of pulmonary fibrosis and that the combination of STZ injection and AAT KO accelerates the progression of pulmonary fibrosis as well as emphysema.

Supporting information

S1 Fig. Plasma AAT concentration for the screening of AAT KO mice.

(A) Plasma concentration of AAT was measured in control and STZ mice using a commercially available ELISA. The dotted lines denote the limit of detection of the assay. The data are expressed as dot plots with the means ± S.E.M.

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S2 Fig. Measurement of body weight changes and glucose tolerance testing in mice.

(A) Mice were challenged with glucose (2 g/kg, i.p.), and fasting blood glucose was measured at 0, 15, 30, 60, 90 and 120 min after the challenge. Glucose challenge was performed at 2 months and 5 months since STZ or citrate buffer injections. (B) Body weights of the mice were measured weekly and % body weight change was calculated. (A-B) The bar graphs show the area under the curve (AUC) of curves. Data were analyzed by unpaired t-test. N = 8–18. *p≤0.05, **p≤0.01.

(TIF)

Click here for additional data file.^{(2.4MB, tif)}

S3 Fig. Measurement of mean linear intercept for the assessment of emphysematous changes in the lungs of STZ mice.

Mean free distance in the airspace was assessed by mean linear intercept measurements in the upper and lower lobes of the lung in control or STZ mice exposed to RA or CS for 6 months. Data were analyzed by two-way ANOVA using Tukey’s post hoc tests. N = 5/group.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S4 Fig. Inflammatory changes in the plasma and BAL of STZ mice.

Protein concentrations of CXCL1, CCL2, IL-6, CCL20, CXCL5, and RAGE were measured by Luminex multiplex assay in the A) plasma and B) BAL samples from control and STZ mice. Data were analyzed by Student’s t-test. *p≤0.05; **p≤0.01; ***p≤0.001.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S1 Table. Primer sequences used for qRT-PCR.

(DOCX)

Click here for additional data file.^{(13.6KB, docx)}

Acknowledgments

The authors would like to thank the Pulmonary Division of SUNY Downstate Health Sciences University for its support.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was funded by grants from the Alpha 1 Foundation award numbers 493373 and 614218 (P.G), from the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Numbers R56HL148774 and R01HL148774 (I. G. A.), and NIH Grants (R01-NS088689, R01-DK098252, and R24-OD018259) and the Alpha-1 Foundation (C.M.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health or the Alpha-1 Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PONE-D-23-20311Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient micePLOS ONE

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Reviewer #1: The study by Park SS et al is an interesting study investigating comorbidity of Lung disease with experimental diabetes in KO AAT mice model. The concept of the study is very interesting as it address an important issue. However, there are few issues that need to be addressed

a) The scoring of fibrosis needs to be done using the modified Ashrofts score for histology slides and to measure total collagen content of the lung

b) TGFb is one of the most important profibrotic mediator and should be measured

c) possible mechanism should be discussed in details

d) what is the effect on blood sugar level when AAT is administered to the STZ AATKO mice ?

Reviewer #2: The study aimed to investigate the effect of Type 1 diabetes (T1D) on the progression of lung damage in mice with Alpha-1 antitrypsin (AAT) deficiency. The study induced T1D in C57BL/6J background mice using streptozotocin (STZ) and measured pulmonary functions after 3 and 6 months of hyperglycemia. The study also utilized STZ-challenged AAT knockout mice to test the hypothesis. The results suggest that the induction of T1D in AAT deficiency leads to a combined pulmonary fibrosis and emphysema (CPFE) phenotype in male mice. However, the study has some concerns that require addressing by the authors.

1. Introduction:

a. In recent studies, diabetes has been shown to affect the pro-inflammatory environments and proliferative properties of cells. Additionally, it also influences the micro- and macrovascular of the pulmonary vasculature. It is not clear if T1D also affects similarly during the progression of AAT.

2. Methodology:

a. Why were only male mice used in the study?

b. Provide information on the route of STZ administration in mice.

c. How did the authors confirm the AAT KO in mice? Also, it is not clear if the AAT KO was lung-specific or global.

d. Provide the volume of glucose solution injected in mice.

e. Provide details; how much RNA was used for RT-PCR, and how was the genomic contamination confirmed?

f. The format of supplementary figures and information is not accessible.

g. Please explain how the forced expiration volume and forced vital capacity were measured in mice.

3. Results and discussion:

a. Did authors also consider measuring lung resistance and compliance?

b. In Figure 2B, the collagen percentage increased in the lung section of STZ-injected mice. Does it mean the changes in pulmonary function might be associated with airway remodeling mediated due to collagen depositions?

c. Figure 3B, what exactly does the vehicle (40 mg/kg) mean to be here? Also, the dose of STZ mentioned is 40 mg/kg which does not align with the methodology section (50 mg/kg).

Reviewer #3: It is an important study to understand how antitrypsin deficiency contribute pulmonary fibrosis and emphysema in Type 1 Diabetes.

A few things need to be addressed by the authors such as, importantly,

- The introduction and discussion of this study are very brief, authors need to add more background studies and discuss about it in relation to their current study and data, also adding more references in the article.

And also,

- Does the increased levels of collagen in STZ AAT KO mice result in any functional changes? Did the authors confirm it by pulmonary function tests in the later stages? Or how long it does take to appear any lung function abnormalities after the initiation of the collagen formation in these KO mice?

- Is there any specific reason why AAT go down in T1D patients? Or is the diabetic conditions itself causing AAT down regulation in T1D patients?. Such questions need to be discussed elaborately in the manuscript.

- Authors have a Supplementary Figure 2 , but its mention in the manuscript is missing.

- In page 10, line 225-228, Authors mentioned Figure 3B in a Figure 4.?

- In Page 2, line 28-29, the sentence need to be reconstructed.

- In Page 3, line 57-60 , the sentence need to be reconstructed (may be split in to 2 sentences) .

- In Page 9, line 209-210 , the sentence is incomplete , needs reconstruction.

Reviewer #4: This is an interesting study examining the potential role of ATT in STZ diabetes-induced pulmonary consequences. The study concludes that ATT deficiency accelerates pulmonary fibrosis and emphysema compared to WT, when exposed to STZ (starts at 3 months rather than 6 months). The discussion speculates that this effect of ATT may stem from its role regulating EMT transition and WNT signaling and its anti-inflammatory effects. The detrimental effect of knocking out ATT on STZ-induced lung pathology, may also stem from ATT prominent role in the regulation of neutrophil activation and degranulation (review in Janciauskiene et al., 2018), critical in the modulation of inflammation.

Figure 2A,B shows as much increase in collagen staining at 3 months than at 6 months therefore doesn’t reflect the quantification graph in 2A, that shows no difference between Crl and STZ at 3 months.

All the data shown for ATTKO mice reflects the 3 months post hyperglycemia time point. Is there any data at 6 month? Are the fibrosis and PFTs worse at 6 months compared to 3 months? It would be interesting to know, given that the severity of the outcomes is significantly increased in the WT at 6 months.

There are some discrepancies that should be discussed in the mRNA induction of fibrotic markers. Lines 253-255 state that the data shows more pronounced increase in profibrotic markers for the STZ-treated ATTKO compared to the STZ-treated WT, however the following points need to be clarified:

Figure 2A shows no significant change in Acta 2 at 3 months and a significant increase at 6 months (ctl vs STZ) Figure 5B shows only the 3 months data and a significant increase in ATT-KO with or without STZ compared to Ctrl mice. However, is there a significant change between ATTKO ctl vs ATTKO STZ? Is that more pronounced at 6 months? As it is presented, it shows that Acta2 doesn’t change when ATTKO mice are treated with STZ.

Figure 2A also shows significant increases in Ccn2 and Fn1 mRNA in WT STZ diabetic mice at 3 and 6 months but Figure 5B shows a significant increase only in Ccn2 in WT Ctrl vs STZ but not Fn1 as shown in 2A. There are also no significant differences in Ccn2 or Fn1 in the ATTKO mice with or without STZ.

Reviewer #5: This is a very interesting article.

It was known at low doses (usually given as multiple exposure), STZ induce immune and inflammatory response as autoimmune diabetes.

The authors talk about type 1 diabetes, the study was started on adult mice (8-13 weeks).These are older mice, not young ones.Type 1 diabetes is diabetes in young people, usually

The authors should explain this circumstance.

**********

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Reviewer #1: No

Reviewer #2: Yes: Nilesh Sudhakar Ambhore

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

**********

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PLoS One. 2023 Oct 11;18(10):e0291948. doi: 10.1371/journal.pone.0291948.r002

Author response to Decision Letter 0

21 Aug 2023

Journal requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

Response: We have made these changes.

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

Response: We have updated this section.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: We have updated these sections.

Response: We have included a full ethics statement in the resubmitted version.

5. We are unable to open your Supporting Information file [Supplemental Fig 1 and Supplemental Fig 2]. Please kindly revise as necessary and re-upload.

Response: We submitted the supplemental figures as .EPS files and it appears that they were not added to the version given to reviewers. We now include 4 supplemental figures in this resubmission.

Reviewer #1:

The study by Park SS et al is an interesting study investigating comorbidity of Lung disease with experimental diabetes in KO AAT mice model. The concept of the study is very interesting as it address an important issue. However, there are few issues that need to be addressed

a) The scoring of fibrosis needs to be done using the modified Ashrofts score for histology slides and to measure total collagen content of the lung

Response: We have now performed analysis with the modified Ashcroft score method. Please see Figures 2 and 5B. The data are consistent with the software image analysis.

b) TGFb is one of the most important profibrotic mediator and should be measured

Response: We have performed real-time TGFβ PCR on samples from our 4 groups at the 3-month timeframe and compared the wild type animals at 3- and 6-months post STZ. TGFβ mRNA levels were elevated only in the 3-month wild-type STZ mice. TGFβ was back to baseline in 6-month wild-type STZ mice. Perhaps TGFβ is elevated at an earlier time point in the AAT KO. Please see Figure 6A and where we discuss this on lines 331-351, 378-380, 412-416, and 445-447.

We have also treated human primary lung fibroblasts with recombinant TGFβ in the presence or absence of AAT protein and examined TGFβ-induced ACTA2, CCN2, and FN1 gene expression. AAT reduced TGFβ induction of these fibrosis markers. Please see Figure 6B.

c) possible mechanism should be discussed in details

Response: We have broadened our discussion on possible mechanisms. We have also discussed our new data. Please see lines 390-407, and 412-416.

d) what is the effect on blood sugar level when AAT is administered to the STZ AATKO mice ?

Response: Due to the resubmission deadline, we were unable to perform this experiment. We have outlined this approach in possible future areas for investigation and also outlined previously studies administering AAT to wild type mice and its impact on glucose levels. Please see lines 446-451.

Reviewer #2:

1. Introduction:

Response: We added data on several inflammation markers we investigated in 6-month STZ wild-type animals. We observed a significant increase in CXCL5 levels in the plasma of the STZ mice (Supplemental Figure 4). We do observe increased CXCL5 gene expression in the AAT KO mice but this is not enhanced by STZ at the 3-month time point (Figure 6B). Additional time points or analysis of other tissues other than lung may be needed to study the significance of elevated CXCL5. We have included this in our limitation section in the discussion. Please see lines 451-453

We have also outlined this point in the discussion as to whether AAT influences the micro- and macrovascular of the pulmonary vasculature. Please see lines 398-407.

2. Methodology:

a. Why were only male mice used in the study?

Response: Female mice have been found to be resistant to streptozotocin (Bell et al, Endocrinology. 1994; Le May et al, Proceedings of the National Academy of Sciences of the United States of America. 2006; Leiter et al, Proc Natl Acad Sci U S A 1982; Deeds et al, Lab Anim 2011; Rossini et al, Endocrinology 1978). A recent study demonstrated that the resistance of female mice to STZ-induced diabetes can be overcome by increasing the dose of STZ (Saadane et al, PLoS One 2020). We chose to use the established male model to first determine if the lower dose of STZ-induced diabetes would impact the lungs. We have stated this in the discussion on lines 423-426.

b. Provide information on the route of STZ administration in mice.

Response: Mice were intraperitoneally injected with STZ (50 mg/kg) dissolved in citrate buffer for 5 consecutive days. This is now stated on lines 136-137.

c. How did the authors confirm the AAT KO in mice? Also, it is not clear if the AAT KO was lung-specific or global.

Response: We performed ELISA on the plasma of the AAT KO to confirm depletion of AAT. Dr. Mueller’s lab created this line and it is well characterized in his manuscript, Borel et al, Proc Natl Acad Sci U S A. 2018. We have included the screening ELISA data in Supplemental Figure 1. This mouse line is a whole-body knockout.

d. Provide the volume of glucose solution injected in mice.

Response: Glucose solution (250 mg/ml) was injected intraperitoneally at a dose of 2.5 g/kg body weight. Therefore, a 20 g mouse would receive 50 mg of glucose, which would equate to a volume of 250 μl of the 250 mg/ml stock to obtain a 2.5 g/kg. This volume was based on the animal weight. Please see line 136-137.

e. Provide details; how much RNA was used for RT-PCR, and how was the genomic contamination confirmed?

Response: RNA was treated with DNase during the isolation method using the Direct-zol RNA miniprep kit from Zymo Research. RNA (without reverse transcriptase treatment) was tested as a genomic contamination control. 1 ug of total RNA was used for the first strand cDNA template synthesis to generate 20 μl of cDNA. cDNA was dilution by a factor of 4 and 1 μl was used in a 10 μl RT-PCR reaction. Please see lines 173-177.

f. The format of supplementary figures and information is not accessible.

Response: We apologize for the supplemental files not being provided. We did upload them but were not aware that they were not accessible to the reviewers.

g. Please explain how the forced expiration volume and forced vital capacity were measured in mice.

Response: We have added this information. Please see lines 145-150.

3. Results and discussion:

a. Did authors also consider measuring lung resistance and compliance?

Response: Lung resistance and compliance were measure and are shown in Figures 1 and 4. Tissue damping is a measure of tissue resistance and reflects the energy dissipation in the alveoli.

Response: Yes, we believe that increased collagen likely contributes to the changes in pulmonary function.

c. Figure 3B, what exactly does the vehicle (40 mg/kg) mean to be here? Also, the dose of STZ mentioned is 40 mg/kg which does not align with the methodology section (50 mg/kg).

Response: Thank you for highlighting the error. Yes, 50 mg/kg is the dose of the STZ. We have made this correction in the vehicle group labelling.

Reviewer #3:

It is an important study to understand how antitrypsin deficiency contribute pulmonary fibrosis and emphysema in Type 1 Diabetes.

A few things need to be addressed by the authors such as, importantly,

Response: We have added more information to the introduction and discussion as advised. Please see lines 71-80, 390-407, and 412-416.

Response: We only performed function changes and collagen quantification in AAT KO at the 3-month timepoint post STZ. At 3 months, we do observe collagen changes and function changes in the AAT KO. It would be of interest to test later time point. We have noted this in the limitation section of the discussion. Please see lines 451-453.

Response: This is an area for discussion. A previous paper demonstrated that hyperglycaemia can impaired serum AAT concentration and activity (Sandler et al, Diabetes Res Clin Pract. 1988). Typically, AAT is viewed as a marker for inflammation. In T1D, there are higher levels of systemic inflammation. Therefore, the reduced AAT levels observed in our model is consistent with human AAT levels in T1D. The liver is the major site for AAT production. Other cells and tissues produce AAT but the majority comes from the liver. Perhaps hyperglycemia has some long-term effects on liver function that could contribute to lower AAT levels. Equally, it is unclear how hyperglycemia impacts the activity of AAT. These are areas of interest for future studies. Finally, we cannot rule out that STZ itself could impact on liver damage. We have discussed this on lines 434-443.

- Authors have a Supplementary Figure 2 , but its mention in the manuscript is missing.

Response: We apologize for the supplemental files not being provided. We did upload them but were not aware that they were not accessible to the reviewers.

- In page 10, line 225-228, Authors mentioned Figure 3B in a Figure 4.?

Response: Thank you. We have now corrected this typo.

- In Page 2, line 28-29, the sentence need to be reconstructed.

Response: We have modified this sentence

- In Page 3, line 57-60 , the sentence need to be reconstructed (may be split in to 2 sentences) .

Response: We have modified this sentence

- In Page 9, line 209-210 , the sentence is incomplete , needs reconstruction.

Response: We have modified this sentence

Reviewer #4:

This is an interesting study examining the potential role of ATT in STZ diabetes-induced pulmonary consequences. The study concludes that ATT deficiency accelerates pulmonary fibrosis and emphysema compared to WT, when exposed to STZ (starts at 3 months rather than 6 months). The discussion speculates that this effect of ATT may stem from its role regulating EMT transition and WNT signaling and its anti-inflammatory effects. The detrimental effect of knocking out ATT on STZ-induced lung pathology, may also stem from ATT prominent role in the regulation of neutrophil activation and degranulation (review in Janciauskiene et al., 2018), critical in the modulation of inflammation.

Response: We agree with the reviewer and have broaden the scope of possible mechanisms that could contribute to this phenotype. The review article from Janciauskiene et al 2018 is a good summary of the literature on AAT regulation on neutrophil responses. We now show elevated levels of CXCL5, a neutrophil chemoattractant, in AAT KO mice and in wild-type STZ injected animals. Please see lines 71-80, 390-407, and 412-416.

Response: In Figure 2A the y-axis scale for the percentage of collagen ranges from 0-0.08 %. In figure 2B the y-axis scale ranges from 0-0.25 %. At 3 months the percentage of collagen in the STZ groups are approximately 0.06 % but are 0.17 % at 6 months. Therefore, there is a large difference in collagen from 3 to 6 months. We have adjusted the scale in these figures to be the same range for 3- and 6-month animals.

Does the reviewer mean that the images look similar in terms of collagen? This is a representative image but the graphs represent many images per animal.

Response: We have not performed AAT KO experiments at the 6-months post STZ. Since we observed significant changes at 3 months, we focused only on this time point. We also expected that the severity of both AAT deficiency and STZ injections might make the animals likely not to survive beyond the 6 months. We have added a sentence in a limitation/future studies section. Please see lines 451-453.

Response: We agree with the reviewer and modified this sentence

Response: We do not have data on AAT KO mice with and without STZ at the 6-month time point. For Figure 2, we performed Student t-test. While for Figure 5, we performed two-way ANOVA using Tukey’s post hoc tests. There are no significant changes between AAT KO control vs AAT KO STZ for Acta2 gene expression.

Response: We agree with the reviewer that not all of these fibrotic markers are enhanced in the AAT KO mice at 3-months post STZ. Additional players likely exist that play a significant role in AAT deficiency associated lung fibrosis in STZ injected mice. Equally, with only sampling at one time point, we may have missed some critical changes that could have occurred earlier. We have discussed this on lines 390-407, 412-416, and 451-453.

Reviewer #5:

It was known at low doses (usually given as multiple exposure), STZ induce immune and inflammatory response as autoimmune diabetes.

The authors talk about type 1 diabetes, the study was started on adult mice (8-13 weeks).These are older mice, not young ones.Type 1 diabetes is diabetes in young people, usually

The authors should explain this circumstance.

Response: This is an area for debate and many articles discuss possible comparative age ranges between mice and humans. Overall, the maturational rate of mice does not linearly correlate with humans. It is suggested to occur 150 times faster during the first month of life and 45 times faster over the next five months (Flurkey et al 2007). Mature adult mice range in age from 3 - 6 months, which is equivalent for humans ranging from 20 - 30 years. Therefore, 8–12-week-old mice would be deemed to be somewhere close to 15-20 years old in human aging. Also, the standard approach to induced hyperglycemia in C57BL/6J mice is to inject them with STZ between 8-12 weeks old (Jackson labs and others report this). We chose this time range based on established protocols. Please see lines 426-433.

Flurkey, Currer, and Harrison, 2007. 'The mouse in biomedical research.' in James G. Fox (ed.), American College of Laboratory Animal Medicine series (Elsevier, AP: Amsterdam; Boston).

Attachment

Submitted filename: Response to reviewers - Final.docx

Click here for additional data file.^{(217.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0291948.r003

Decision Letter 1

Doa'a G F Al-u'datt

10 Sep 2023

Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice

PONE-D-23-20311R1

Dear Dr. Geraghty,

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: The manuscript has been improved from the first version. The authors have addressed my previous comments satisfactorily, I have no further major changes to suggest.

Reviewer #5: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #5: No

**********

PLoS One. doi: 10.1371/journal.pone.0291948.r004

Acceptance letter

Doa'a G F Al-u'datt

2 Oct 2023

PONE-D-23-20311R1

Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice

Dear Dr. Geraghty:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Plasma AAT concentration for the screening of AAT KO mice.

(TIF)

Click here for additional data file.^{(535.1KB, tif)}

S2 Fig. Measurement of body weight changes and glucose tolerance testing in mice.

(TIF)

Click here for additional data file.^{(2.4MB, tif)}

S3 Fig. Measurement of mean linear intercept for the assessment of emphysematous changes in the lungs of STZ mice.

(TIF)

Click here for additional data file.^{(1.2MB, tif)}

S4 Fig. Inflammatory changes in the plasma and BAL of STZ mice.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S1 Table. Primer sequences used for qRT-PCR.

(DOCX)

Click here for additional data file.^{(13.6KB, docx)}

Attachment

Submitted filename: Response to reviewers - Final.docx

Click here for additional data file.^{(217.7KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

[pone.0291948.ref001] 1.Mauricio D, Alonso N, Gratacos M. Chronic Diabetes Complications: The Need to Move beyond Classical Concepts. Trends Endocrinol Metab. 2020;31(4):287–95. Epub 2020/02/09. doi: 10.1016/j.tem.2020.01.007 . [DOI] [PubMed] [Google Scholar]

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PERMALINK

Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice

Sangmi S Park

Michelle Mai

Magdalena Ploszaj

Huchong Cai

Lucas McGarvey

Christian Mueller

Itsaso Garcia-Arcos

Patrick Geraghty

Roles

Abstract

Introduction

Methods

Ethics statement

Animal models

Blood glucose and HbA1c tests

Pulmonary function test

Histology

Quantitative real-time PCR

AAT concentration in plasma

Cell culture

Statistical analyses

Results

Low-dose STZ injections induce a restrictive pulmonary pattern

Fig 1. Fasting blood glucose and pulmonary function tests of the vehicle and STZ-injected mice.

STZ mice exhibit fibrotic traits in the lung

Fig 2. Quantification of collagen and fibrotic markers in the lung of the vehicle and STZ-injected mice.

Plasma AAT concentration is decreased in STZ mice and hyperglycemia is exacerbated in AAT-deficient STZ mice

Fig 3. Plasma AAT concentration and fasting blood glucose of vehicle and STZ-injected WT or AAT KO mice.

STZ AAT KO mice exhibit no significant functional changes in the lung

Fig 4. PFT in the vehicle and STZ-injected WT or AAT KO mice.

STZ-induced AAT KO mice exhibit fibrotic and emphysematous histological changes in the lung

Fig 5. Quantification of collagen and fibrotic markers in the lung of the vehicle and STZ-injected WT or AAT KO mice.

Altered TGFβ signaling observed in STZ animals in vivo and AAT can counter TGFβ-mediated fibroblast signaling in vitro

Fig 6. Altered TGFβ signaling observed in STZ animals in vivo and AAT can counter TGFβ-mediated fibroblast signaling in vitro.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Doa'a G F Al-u'datt

Roles

Author response to Decision Letter 0

Decision Letter 1

Doa'a G F Al-u'datt

Roles

Acceptance letter

Doa'a G F Al-u'datt

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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