Patients with idiopathic pulmonary fibrosis (IPF) often experience precipitous deteriorations, termed “acute exacerbations” (AE), marked by diffuse alveolitis and altered gas exchange, resulting in a significant loss of lung function or mortality. The missense isoleucine to threonine substitution at position 73 (I73T) in the alveolar type 2 cell-restricted surfactant protein-C (SP-C) gene (SFTPC) has been linked to clinical IPF. To better understand the sequence of events that impact AE-IPF, we leveraged a murine model of inducible SP-CI73T (SP-CI73T/I73TFlp+/−) expression. Following administration of tamoxifen to 8–12-wk-old mice, an upregulation of SftpcI73T initiated a diffuse lung injury marked by increases in bronchoalveolar lavage fluid (BALF) protein and histochemical evidence of CD45+ and CD11b+ cell infiltrates. Flow cytometry of collagenase-digested lung cells revealed a transient, early reduction in SiglecFhiCD11blowCD64hiCD11chi macrophages, countered by the sequential accumulation of SiglecFloCD11b+CD64CD11cCCR2+Ly6C+ immature macrophages (3 d), Ly6G+ neutrophils (7 d), and SiglecFhiCD11bhiCD11clo eosinophils (2 wk). By mRNA analysis, BALF cells demonstrated a time-dependent phenotypic shift from a proinflammatory (3 d) to an anti-inflammatory/profibrotic activation state, along with serial elaboration of monocyte and eosinophil recruitment factors. The i.v. administration of clodronate effectively reduced total BALF cell numbers, CCR2+ immature macrophages, and eosinophil influx while improving survival. In contrast, resident macrophage depletion from the intratracheal delivery of clodronate liposomes enhanced SftpcI73T-induced mortality. These results using SftpcI73T mice provide a detailed ontogeny for AE-IPF driven by alveolar epithelial dysfunction that induces a polycellular inflammation initiated by the early influx of proinflammatory CCR2+Ly6Chi immature macrophages.

This article is featured in In This Issue, p.2515

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease (ILD) characterized by the disruption of distal lung architecture that ultimately leads to scar formation, abnormal gas exchange, and respiratory failure. The IPF lung is marked by the pathognomonic histology of usual interstitial pneumonitis composed of temporally and spatially heterogeneous areas of fibroblast/myofibroblast accumulation, coupled with extracellular matrix deposition, disrupted alveolar architecture, and subpleural honeycombing (13). In part, because of incomplete understanding of its pathogenesis, the current “gold-standard” therapy is based on “antifibrotic” (i.e., pirfenidone, nintedanib) strategies, which at best only slow the deterioration in pulmonary function (4).

Although initial preclinical and translational modeling focused on aberrant mesenchymal expansion in IPF pathogenesis, alveolar type 2 (AT2) epithelial cell dysfunction has re-emerged as a key mechanism driving aberrant lung remodeling through refined cross-talk with all parenchymal populations (5). This notion is bolstered by epidemiological studies and case reports that identified both inherited and sporadic monogenetic mutations in key genes related to telomere length and pulmonary surfactant system proteins (3, 6). Over 60 mutations in the surfactant protein-C (SP-C) gene (SFTPC), an AT2-specific product, have been described in both adult and pediatric ILD patients. Functionally, SP-C is known to intimately interact with surfactant lipids because of its hydrophobic structure, resulting in surface tension modulation in the distal lung. The missense substitution (g.1286T > C), resulting in a change of isoleucine to threonine at position 73 in the SFTPC proprotein (“SP-CI73T”), represents the most common human ILD mutation (7). In vitro studies of epithelial cell lines expressing mutant SP-CI73T offered mechanistic insights into the cellular changes seen in SFTPCI73T patients, which were each marked by organellar disruption, defective proteostasis, and disruption of cell survival pathways (macroautophagy and mitophagy) (8). To link epithelial dysfunction with fibrotic remodeling in vivo, we developed a, to our knowledge, novel preclinical allelic knockin murine model designed to express mutant SftpcI73T in an inducible manner (9), thus allowing temporal characterization of key pathways and cellular constituents that drive the aberrant injury/repair response.

Acute exacerbations (AE) represent severe clinical deteriorations in patients with chronic ILD (AE-ILD or AE-IPF) that are associated with a high mortality and characterized by extensive inflammatory cell infiltrates, hypoxic respiratory failure, and histological evidence of diffuse alveolar damage superimposed upon the existing PF pathology. Loss of pulmonary function and increased fibrogenic burden represent key downstream effects associated with survivors, corroborating the notion that the components of innate and/or adaptive immunity can contribute to aberrant lung remodeling. Although longitudinal data from IPF cohorts are challenging to acquire, analysis of bronchoalveolar lavage fluid (BALF) has shown that elevated levels of chemokines involved in myeloid cell recruitment (MIP-3, CCL22, and CCL17) correlate with poor disease outcomes (1012). These biomarkers have also been observed in several preclinical models of lung fibrosis (13, 14), therefore supporting the notion that peripheral myeloid cells mobilized during inflammation have the potential to persist in the tissue and participate in lung remodeling (1518). Given that traditional approaches to AE-PF such as corticosteroids have unproven efficacy (19), a deeper understanding of the immune cell dynamics driving tissue remodeling are needed to improve efficacy and specificity of current anti-inflammatory strategies.

To address the unmet need for a temporal characterization of the inflammation contributing to lung remodeling, the current studies aim to define the sequence of early events resulting in polycellular inflammation during AE-PF. Leveraging the SftpcI73T mouse model, we assessed the ontogeny of the recruitment of these immune cell subsets in lung tissue and BALF following the induction of mutant SP-CI73T expression. Furthermore, using depletion protocols, we provide evidence for a key role of peripheral Ly6Chi monocytes in the initiation of the injury. Taken together, these studies contribute to a refined understanding of epithelial–immune cell cross-talk in acute episodes of sterile inflammation occurring in PF exacerbations.

Tamoxifen (TAM; nonpharmaceutical grade) was purchased from Sigma-Aldrich (St Louis, MO). Clodronate liposomes (CLOD) were purchased from Encapsula NanoSciences (Brentwood, TN). Giemsa cytological stain was purchased from Sigma-Aldrich. Abs used for immunohistochemical and flow cytometric analysis were as follows: CD45 (1:250; Santa Cruz Biotechnology); eosinophil peroxidase (EPX) (1:150; Santa Cruz Biotechnology); myeloperoxidase (1:150; Novus Biologicals); inducible NO synthase (iNOS) (1:200; Abcam); CD11b (1:1500; Abcam); FIZZ-1 (1:800; Stemcell Technologies); CD64 (1:250; Bioss Antibodies); CCR2 (1:400; Abcam); CCR4 (1:400; Abcam); CX3CR1 (Bioss Antibodies); CD16/32 (eBioscience), CD11b (eFluo450; eBioscience); fixable viability dye (eFluo780; eBioscience); SiglecF (PE-CF594; BD Biosciences); CD45 (PerCP5.5; BioLegend); CD11c (BV705; BioLegend); Ly6G (AF700; BioLegend); Ly6C (BV510; BioLegend); CD64 (PE/Cy7; BioLegend); CD3 (BUV395; BioLegend); CD43 (PE; BioLegend); and CCR2 (AF647; BioLegend). All other reagents were purchased from Thermo Fisher Scientific (Waltham, MA) or Sigma-Aldrich.

TAM-inducible SP-CI73TFlp mice were generated as previously reported (9). Briefly, the hemagglutinin–SP-CI73T−Neo hypomorphic founder line was crossed with a mouse line expressing a FLP recombinase variant (Flp-O) under the control of the mutated estrogen receptor 2 (ER2) knocked into the Rosa26 locus (The Jackson Laboratory, Bar Harbor, ME) to generate the inducible SP-CI73TFlp-O line. TAM treatment of adult SP-CI73T/I73TFlp+/− mice was initiated at 8–12 wk of age. Both male and female animals were used for the studies. Control group mice are represented as pooled data from TAM-treated SP-CI73T/I73TFlp−/− genotypes or vehicle (oil)–treated SP-CI73T/I73TFlp+/−mice. All mice were housed under pathogen-free conditions in an American Association of Laboratory Animal Care–approved barrier facility at the Perelman School of Medicine of the University of Pennsylvania. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania.

Whole lungs were fixed by tracheal instillation of 10% neutral buffer formalin at a constant pressure (25 cm H2O). Following paraffin embedding, 6-μM sections were cut and stained with H&E by the Pathology Core Laboratory of Children’s Hospital of Philadelphia. Immunostaining of deparaffinized tissue sections was performed as previously described (20). Briefly, after Ag retrieval using citrate buffer (10.2 mM sodium citrate [pH 6] for 20 min) and the quenching of endogenous peroxidase with 3% hydrogen peroxide in methanol (30 min), nonspecific binding was blocked with 10% goat or rabbit serum according to primary Ab origin. Appropriate serum/IgG controls, each diluted in blocking buffer, were applied for overnight incubation at 4°C in a humidified chamber. Following incubation with biotinylated secondary antisera (VECTASTAIN Elite ABC Kit; Vector Laboratories, Burlingame, CA) for 30 min (room temperature), staining was visualized using a Peroxidase Substrate Kit DAB (Vector Laboratories) and counterstained with Harris Modified Hematoxylin (Thermo Fisher Scientific).

Invasive measurement of inspiratory capacity and static lung compliance was performed as part of a terminal harvest. Mice were anesthetized with i.p. pentobarbital, tracheas cannulated with an 18-gauge metal stub adapter, and connected to a small-rodent flexiVent ventilator (SCIREQ Scientific Respiratory Equipment, Toronto, Ontario, CA). Following the establishment of standardized basal ventilatory support at a respiratory rate of 150 breaths/min and a tidal volume of 10 ml/kg of body weight, static lung compliance was measured using the PVs-P (27 cm H2O) maneuver defined in the manufacturer’s software.

BALF was collected from mice using five sequential lavages of 1 ml sterile saline and processed for analysis as previously described (9). Briefly, cell pellets obtained by centrifuging BALF samples at 400 × g for 6 min were resuspended in 1 ml of PBS, and total cell counts were determined using a NucleoCounter (New Brunswick Scientific, Edison, NJ). Differential cell counts were determined manually from BALF cytospins stained with modified Giemsa for 20 min to identify macrophages, lymphocytes, eosinophils, and neutrophils. Total protein content of cell-free BALF was determined by the Bradford method with bovine IgG as a standard as described (9).

First-return aliquots of cell-free BALF were analyzed for CX3CL1 levels using a Luminex platform (MilliporeSigma, Burlington, MA) by the Human Immunology Core at the Perelman School of Medicine.

Following BALF collection, lungs were cleared of blood by cardiac perfusion with saline solution, removed from the chest cavity, minced, and transferred into a 50-ml conical tube and incubated (37°C, 30′) in DMEM plus 5% FBS plus 2 mg/ml collagenase D (catalog no. 11088866001; Roche, Indianapolis, IN). Digested lungs were passed through 70-μm nylon mesh to obtain a single-cell suspension, counted, and mixed with ACK Lysis Buffer (Thermo Fisher Scientific) to remove any remaining RBCs. BALF and tissue cell pellet (5 × 105 cells) were resuspended in 100 μl staining buffer (PBS +0.1% sodium azide) and incubated with anti-mouse CD16/32 Ab (Fc block, eBioscience, San Diego, CA) for 10 min at 4°C to block nonspecific binding. This was followed by a 30-min incubation with fluorescently tagged Abs or appropriate isotype controls (0.25–1.5 μg/106 cells) for 30 min (4°C). Cells were then spun and resuspended in staining buffer for viability staining (30 min at 4°C). Cells were fixed in 2% paraformaldehyde and analyzed with an LSRFortessa (BD Biosciences, San Jose, CA) or FACSAria (BD Biosciences) for cell sorting experiments. Immune cells were identified by forward and side scatter, followed by doublet discrimination of CD45+ viable cells. Specific subsets were identified using a gating strategy modified from Misharin et al. (21), as we have published (9), using FlowJo software (FlowJo, Ashland, OR).

RNA, extracted from BALF cell pellets, was purified in RLT buffer using the RNeasy kit (QIAGEN, Valencia, CA) and served as a template for cDNA preparation using the RETROscript Kit (QIAGEN) following the manufacturer’s protocol. Quantitative single-plex PCRs (qPCR) were performed using Taq polymerase and TaqMan qPCR kits (Applied Biosystems/Thermo Fisher Scientific) on a QuantStudio 7 Flex Real-Time PCR System. The commercial primer sets (all from Applied Biosystems/Thermo Fisher Scientific) were as follows: Ccl2 (MCP-1) (catalog no. Mm00441242_m1), Ccl17 (thymus- and activation-regulated chemokine) (catalog no. Mm01244826_g1), Cxcl1 (catalog no. Mm04207460_m1), Il6 (catalog no. Mm00446190_m1), Il5 (catalog no. Mm00439646_m1), Ccl11 (eotaxin) (catalog no. Mm0041238_m1), and 18s (catalog no. Mm03928990_g1). Results are expressed as relative quantities of RNA (versus untreated or wild-type controls) after the normalization of all values to the corresponding 18s RNA content.

Clodronate, encapsulated in liposomes, was used to deplete monocytes/macrophages in blood and lung (22). Control liposomes (CL) contained PBS only. For intratracheal (i.t.) treatment, each animal received 50 μl (5 mg of clodronate per milliliter of total suspension volume) of CLOD or CL. For i.v. administration, mice were restrained so that the tail vein was accessible, and an insulin syringe was used to administer liposomes (150 mg/kg) 2 h after i.p. TAM injections.

All data are presented with dot plots and group mean ± SEM unless otherwise indicated. Statistical analyses were performed with GraphPad InStat (GraphPad Software, San Diego, CA). Student t test was used for paired data; for analyses involving multiple groups, one-way or two-way ANOVA was performed with post hoc testing as indicated, and survival analyses were performed using log-rank (Mantel–Cox) test. In all cases, statistical significance was considered at p 0.05.

Induction of mutant SP-CI73T expression by i.p. TAM administration resulted in significant changes in lung histology, barrier function, and physiology. Whereas minimal histological changes were observed at 3 d postinduction, marked increases in alveolar septal thickening accompanied by epithelial hyperplasia were notable beginning at 7 d (Fig. 1A). In addition, time-dependent increases in alveolar septal disruption, tissue edema, and hemorrhage occurred in association with increased perivascular cellularity. Consistent with prior observations (9), we also found increased BALF total protein at 7 d postinduction, along with reduced total inspiratory capacity and increased tissue stiffness, as measured by quasistatic lung compliance (Table I).

FIGURE 1.

Parenchymal lung injury and alteration in immune cell composition induced by SP-CI73T expression. (A) High-power images of H&E-stained sections (100×) of control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM administration (250 mg/kg). (B) Immunohistochemical staining of lungs stained for CD45/immune cells (left panels), CD11b/migrating cells (center panels), and CD64/mature macrophages (right panels). Original magnification ×200; inset original magnification ×600. Images shown are representative of three to five animals per group.

FIGURE 1.

Parenchymal lung injury and alteration in immune cell composition induced by SP-CI73T expression. (A) High-power images of H&E-stained sections (100×) of control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM administration (250 mg/kg). (B) Immunohistochemical staining of lungs stained for CD45/immune cells (left panels), CD11b/migrating cells (center panels), and CD64/mature macrophages (right panels). Original magnification ×200; inset original magnification ×600. Images shown are representative of three to five animals per group.

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Table I.
Tissue inflammation, injury, and lung mechanics
Protein Content (μg/ml)Cell Count (106)Inspiratory Capacity (ml)Static Compliance (ml/cmH2O)Tissue Damping (G, cmH2O/ml)
CTL (n = 11) 438.2 ± 38.3 0.26 ± 0.02 0.74 ± 0.02 0.067 ± 0.002 4.59 ± 0.15 
SP-CI73T/I73TFlp+/−      
 3 d (n = 8) 455.9 ± 61.6 0.19 ± 0.03 0.66 ± 0.03 0.057 ± 0.003 5.11 ± 0.28 
 7 d (n = 7) 2266.6 ± 422.5* 0.99 ± 0.18* 0.51 ± 0.05* 0.040 ± 0.005* 7.62 ± 0.74* 
 2 wk (n = 14) 2245.2 ± 245.0* 2.66 ± 0.32* 0.46 ± 0.03* 0.031 ± 0.004* 8.84 ± 1.35* 
Protein Content (μg/ml)Cell Count (106)Inspiratory Capacity (ml)Static Compliance (ml/cmH2O)Tissue Damping (G, cmH2O/ml)
CTL (n = 11) 438.2 ± 38.3 0.26 ± 0.02 0.74 ± 0.02 0.067 ± 0.002 4.59 ± 0.15 
SP-CI73T/I73TFlp+/−      
 3 d (n = 8) 455.9 ± 61.6 0.19 ± 0.03 0.66 ± 0.03 0.057 ± 0.003 5.11 ± 0.28 
 7 d (n = 7) 2266.6 ± 422.5* 0.99 ± 0.18* 0.51 ± 0.05* 0.040 ± 0.005* 7.62 ± 0.74* 
 2 wk (n = 14) 2245.2 ± 245.0* 2.66 ± 0.32* 0.46 ± 0.03* 0.031 ± 0.004* 8.84 ± 1.35* 

Summary analysis for BALF protein content, BALF cell counts, and in vivo pulmonary function testing (inspiratory capacity, static compliance, and tissue damping) from control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and TAM-treated (250 mg/kg i.p.) SP-CI73T/I73TFlp+/− mice at 3 and 7 d and 2 wk. Note that control data represents pooled data from 3 d (n = 3), 7 d (n = 4), and 2 wk (n = 4). Data are represented as mean ± SEM.

*

p < 0.05 compared with control SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice by ANOVA using Tukey post hoc test.

These alterations were accompanied by increases in BALF cell counts, highlighting a mounting inflammatory response that peaked at 2 wk after TAM (Table I). This finding was mirrored by immunohistochemistry showing the accumulation of CD45+ cells in the lung tissue (Fig. 1B, left panels) and perivascular clusters of CD11b+ immune cells, suggesting their peripheral origin (Fig. 1B, center panels). Using the Fc receptor CD64 as a marker of macrophage maturity, early time-dependent decreases in fully differentiated macrophages were observed for up to 2 wk (Fig. 1B, right panels).

Examination of BALF cytospins obtained from SP-CI73T mice after TAM treatment revealed dynamic changes in a mixed population of effector cells occurring over the course of 2 wk (Supplemental Fig. 1). To further characterize these cells, BALF and digested lung tissue were each analyzed by flow cytometry using a previously described Ab panel and sorting strategy (9, 21) to identify resident SigF+CD11b alveolar macrophages, Ly6G+ neutrophils, SigF+CD11c eosinophils, CD3+ lymphocytes, and Ly6Chi immature macrophages (Fig. 2). Correct population gating was validated using Giemsa staining of FACS-derived cytospins (Fig. 2, insets).

FIGURE 2.

Flow cytometric analysis of CD45+ populations in BALF and lung tissue. Gating strategy used to identify myeloid subsets in control (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM administration (250 mg/kg). BALF and enzymatically digested tissue immune cells were gated on CD45+ following the exclusion of doublets. Populations were identified based on a modified protocol from Misharin et al. (21) and as previously described (9). Alveolar macrophages were gated-based SigF+CD11b expression and confirmed using CD11c, CD64, neutrophils (CD11b+Ly6G+), eosinophils (SigF+CD11b+CD11c), lymphocytes (CD3+CD11b), and immature macrophages (CD11b+CD64Ly6Chi). Insets represent Giemsa-stained cytospins of FACS-sorted subsets.

FIGURE 2.

Flow cytometric analysis of CD45+ populations in BALF and lung tissue. Gating strategy used to identify myeloid subsets in control (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM administration (250 mg/kg). BALF and enzymatically digested tissue immune cells were gated on CD45+ following the exclusion of doublets. Populations were identified based on a modified protocol from Misharin et al. (21) and as previously described (9). Alveolar macrophages were gated-based SigF+CD11b expression and confirmed using CD11c, CD64, neutrophils (CD11b+Ly6G+), eosinophils (SigF+CD11b+CD11c), lymphocytes (CD3+CD11b), and immature macrophages (CD11b+CD64Ly6Chi). Insets represent Giemsa-stained cytospins of FACS-sorted subsets.

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Using this strategy, we first observed a significant diminution of SigF+CD11c+ CD64+CD11b alveolar macrophages in BALF and lung tissue digests within 3 d of SP-CI73T induction (Fig. 3A, Supplemental Fig. 2A). Whereas the number of these cells returned to control levels by 7 d in the BALF, their relative abundance continued to remain low for up to 2 wk in the lung tissue (Supplemental Fig. 2A). Conversely, BALF and tissue Ly6C+ immature macrophages were increased during this same early inflammatory phase (Fig. 3B, Supplemental Fig. 2A). Previously, we had shown that SP-CI73T expression increased BALF levels of cytokines involved in the recruitment of monocytes and activated macrophages (CCL2, CCL17), which was mirrored by similar changes in cytokine mRNA expression in AT2 cells (9). qPCR analysis of BALF immune cells indicate that these populations also contribute to Ccl2/Mcp1 production (Fig. 3C), whereas Ccl17 mRNA expression remained unchanged 3 d and 2 wk following mutant SP-CI73T induction (Fig. 3D). Immunohistochemical analysis of lung sections also showed increased expression of the CCL17 receptor, CCR4, on macrophages at 3 d and returned to control levels at 2 wk (Fig. 3E). By comparison, progressive increases in numbers of CCR2+ mononuclear cells in the lung were found beginning 3 d postinduction, whereas CCR2 expression was absent on neutrophils and eosinophils (Fig. 3F). Similarly, numbers of monocytes/macrophages expressing the chemokine receptor CX3CR1 were incrementally increased in the lung during the evolution of injury (Fig. 3G), mirrored by commensurate increases in BALF levels of its ligand CX3CL1 (Fig. 3H).

FIGURE 3.

Characterization of lung macrophage and peripheral monocyte dynamics in SP-CI73T mutant mice. (A and B) Flow cytometric analysis and quantification of BALF for (A) absolute numbers of SigF+CD11c+CD11b macrophages and (B) Ly6C+ immature macrophages (absolute numbers left y-axis and relative percentage right y-axis) collected from control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 and 4 wk following i.p. TAM administration (250 mg/kg). (C and D) qPCR analysis of BALF cells collected from control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg) for markers associated with recruitment of (C) monocytes (Mcp1) and (D) monocyte/macrophages (Ccl17). Data are represented as mean ± SEM (n = 3–8). *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice SP-CI73T/I73TER−/− by one-way ANOVA using Tukey post hoc test. (EG) Immunohistochemical staining of lungs stained for (E) CCR4, (F) CCR2, and (G) CX3CR1 at the indicated time points, showing prominent monocyte/macrophage staining (arrowheads). Note peak expression for CCR4 3 d after mutant induction and time-dependent increases in numbers of CCR2+ and CX3CR1+ macrophages. Original magnification ×200; inset original magnification ×600. Representative images are shown (n = 4). (H) CX3CL1 ELISA of BALF from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg). Data are represented as mean ± SEM (n = 6–11). Controls were pooled from controls at all three time points. *p < 0.05 versus control group using one-way ANOVA followed by Tukey post hoc test. #p < 0.05 versus control group for percent of BALF Ly6Chi immature macrophages (reflecting the right axis of Fig. 3B).

FIGURE 3.

Characterization of lung macrophage and peripheral monocyte dynamics in SP-CI73T mutant mice. (A and B) Flow cytometric analysis and quantification of BALF for (A) absolute numbers of SigF+CD11c+CD11b macrophages and (B) Ly6C+ immature macrophages (absolute numbers left y-axis and relative percentage right y-axis) collected from control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 and 4 wk following i.p. TAM administration (250 mg/kg). (C and D) qPCR analysis of BALF cells collected from control mice (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg) for markers associated with recruitment of (C) monocytes (Mcp1) and (D) monocyte/macrophages (Ccl17). Data are represented as mean ± SEM (n = 3–8). *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice SP-CI73T/I73TER−/− by one-way ANOVA using Tukey post hoc test. (EG) Immunohistochemical staining of lungs stained for (E) CCR4, (F) CCR2, and (G) CX3CR1 at the indicated time points, showing prominent monocyte/macrophage staining (arrowheads). Note peak expression for CCR4 3 d after mutant induction and time-dependent increases in numbers of CCR2+ and CX3CR1+ macrophages. Original magnification ×200; inset original magnification ×600. Representative images are shown (n = 4). (H) CX3CL1 ELISA of BALF from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg). Data are represented as mean ± SEM (n = 6–11). Controls were pooled from controls at all three time points. *p < 0.05 versus control group using one-way ANOVA followed by Tukey post hoc test. #p < 0.05 versus control group for percent of BALF Ly6Chi immature macrophages (reflecting the right axis of Fig. 3B).

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Macrophages are known to participate in both acute lung injury and resolution/fibrosis (23). Therefore, we sought to temporally define phenotypic changes that occurred after SP-CI73T–induced injury. To this end, we measured mRNA expression of markers canonically associated with proinflammatory (classically activated [M1]; iNOS [Nos2]; Il6) and anti-inflammatory/profibrotic (alternatively activated [M2]; Arg1, Fizz1) polarization. Because of the low baseline expression of inflammatory genes, we expressed quantitated gene expression based on their δ-cycle threshold (dCt) relative to the reference gene 18s (lower dCt, approximately higher expression). Although M1 gene expression was increased starting at 3 d after TAM administration, the most dynamic changes observed were on M2 macrophage markers, including Arg1 and Fizz1 at later time points (2 wk) (Fig. 4A). Using 2−ΔΔCt method, we also measured Arg1:Nos2 expression ratio, a surrogate for macrophage activation (24, 25), and found a time-dependent shift to an anti-inflammatory state (3 d ratio = 10.0; 7 d ratio = 30.4; 2 wk ratio = 72.7). We further validated these findings at the protein level by staining lung sections and cytospins for iNOS as well as FIZZ1. Consistent with mRNA analysis, there was upregulation of iNOS in BALF macrophages at 3 d, which persisted up to 2 wk (Fig. 4B). Conversely, staining for M2 markers increased steadily through the first 2 wk (Fig. 4C).

FIGURE 4.

Phenotypic shift of BALF cells following SP-CI73T–induced injury. (A) qPCR analysis of BALF cells collected from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg) for markers associated with M1 (Nos2 and Il6) and M2 (Arg1, Fizz1) activation. Data are represented as dCt relative to 18s and shown as mean ± SEM (n = 3–6). *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice SP-CI73T/I73TER−/− by one-way ANOVA, followed by Tukey post hoc test. (B and C) Immunohistochemical and cytospin staining of lungs for (B) iNOS and (C) FIZZ-1. Representative images are shown (200×; n = 3–5) from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg).

FIGURE 4.

Phenotypic shift of BALF cells following SP-CI73T–induced injury. (A) qPCR analysis of BALF cells collected from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg) for markers associated with M1 (Nos2 and Il6) and M2 (Arg1, Fizz1) activation. Data are represented as dCt relative to 18s and shown as mean ± SEM (n = 3–6). *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice SP-CI73T/I73TER−/− by one-way ANOVA, followed by Tukey post hoc test. (B and C) Immunohistochemical and cytospin staining of lungs for (B) iNOS and (C) FIZZ-1. Representative images are shown (200×; n = 3–5) from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg).

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Using the same gating strategy (Fig. 2), analysis of BALF and tissue-derived cells both demonstrated a steady accumulation of Ly6G+ neutrophils, peaking at 7 d (Fig. 5A, Supplemental Fig. 2A), and SigF+CD11c eosinophils, peaking at 2 wk (Fig. 5D, Supplemental Fig. 2A). Consistent with these findings, immunohistochemical staining of tissue sections showed increased numbers of myeloperoxidase-positive neutrophils at 7 d (Fig. 5B) as well as EPX-positive eosinophils 2 wk postinduction (Fig. 5E). Furthermore, consistent with our BALF differential analysis, the number of BALF CD3+ cells also steadily increased, although the relative percentage of these cells remained unchanged in the tissue (Supplemental Fig. 2B).

FIGURE 5.

Characterization of BALF neutrophil and eosinophil recruitment in SP-CI73T mutant mice. Flow cytometric analysis and quantification of BALF (A) Ly6G+ neutrophil and (D) SigF+CD11c eosinophil subsets collected from BALF in control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 and 4 wk following i.p. TAM administration (250 mg/kg). Absolute quantitation was performed by multiplying the relative abundance of each myeloid subset by the BALF cell counts. Data are represented as mean ± SEM (n = 25–51). Immunohistochemical staining of lungs stained for (B) myeloperoxidase and (E) EPX. Original magnification ×200; inset original magnification ×600. Representative images are shown (n = 3–5). qPCR analysis of BALF cells collected from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg) for markers associated with (C) neutrophil (Cxcl1) and (F) eosinophil (Eotaxin/Ccl11 and Il5) recruitment. *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice or SP-CI73T/I73TFlp−/− by one-way ANOVA, followed by Tukey post hoc test.

FIGURE 5.

Characterization of BALF neutrophil and eosinophil recruitment in SP-CI73T mutant mice. Flow cytometric analysis and quantification of BALF (A) Ly6G+ neutrophil and (D) SigF+CD11c eosinophil subsets collected from BALF in control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 and 4 wk following i.p. TAM administration (250 mg/kg). Absolute quantitation was performed by multiplying the relative abundance of each myeloid subset by the BALF cell counts. Data are represented as mean ± SEM (n = 25–51). Immunohistochemical staining of lungs stained for (B) myeloperoxidase and (E) EPX. Original magnification ×200; inset original magnification ×600. Representative images are shown (n = 3–5). qPCR analysis of BALF cells collected from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 d and 2 wk following i.p. TAM administration (250 mg/kg) for markers associated with (C) neutrophil (Cxcl1) and (F) eosinophil (Eotaxin/Ccl11 and Il5) recruitment. *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice or SP-CI73T/I73TFlp−/− by one-way ANOVA, followed by Tukey post hoc test.

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As previous work from our group identified a central role for epithelial cells in the early recruitment of peripheral subsets of these granulocytes through the elaboration of key cytokines (9), we sought to determine whether BALF immune cells could also participate in this process. Interestingly, whereas BALF cells were not a source of Cxcl1 (Fig. 5C), we did observe an increased and persistent expression of Ccl11/Eotaxin and Il5 up to 2 wk after TAM (Fig. 5F).

As a result of this detailed temporal characterization, we identified peripheral Ly6Chi monocytes as the first peripheral subset infiltrating into the tissue through the activation of the MCP-1/CCR2 axis and hypothesized that their participation was crucial in driving lung injury and alveolitis caused by SftpcI73T expression. We next tested whether pharmacological depletion of resident alveolar macrophages or peripheral monocytes with CL and CLOD would alter the course of early and late inflammatory processes. Consistent with a role of resident macrophages in the maintenance of homeostasis, the depletion of alveolar macrophages using i.t. CLOD resulted in a significant increase in mortality within 7 d post-SftpcI73T induction (Fig. 6A). At this time point, although histological analysis of lungs from TAM-induced mice showed relatively mild cellular infiltration, resident macrophage depletion revealed extensive parenchymal remodeling and the appearance of conspicuous pockets of inflammatory cells, resembling bronchoalveolar lymphoid tissue (Fig. 6B). Of note, neither control nor CLOD affected lung architecture. Conversely, i.v. delivery of CLOD, previously shown to alter peripheral monocyte homeostasis, resulted in enhanced survival post-SftpcI73T induction (90% TAM plus CLOD versus 54% TAM at 2 wk) (Fig. 6C). This protective effect on mortality was reflected in the histology with animals receiving i.v. CLOD, demonstrating reduction in inflammatory cells, particularly in the alveolar and perivascular regions and overall attenuation of parenchymal lung damage (Fig. 6D). The blunted inflammatory response was also reflected in diminished numbers of total BALF cells in mice treated at both 7 d and 2 wk after TAM induction (Fig. 6E).

FIGURE 6.

The effect of depletion of alveolar macrophages or peripheral monocytes with clodronate liposomes on lung injury and survival of SP-CI73T mutant mice is dependent upon the route of administration. Kaplan–Meier survival analysis from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and TAM-induced (250 mg/kg; i.p.) SP-CI73T/I73TFlp+/− mice following (A) i.t. (50 μl/mouse given 48 h prior to induction) or (C) i.v. (150 μg/kg given 2 h postinduction) CLOD or CL administration (n = 3 for CL group collected at terminal day; n = 10–32 for all other groups). Mean survival using death or body weight <75% on two consecutive days as endpoints is shown. *p < 0.05 compared with CTL. #p < 0.05 compared with TAM-treated mice by log-sum (Mantel–Cox) rank test. H&E-stained sections (40×) from SP-CI73T cohorts treated with i.t. or i.v. clodronate as indicated. (B) i.t. CL-treated CTL, i.t. CLOD-treated CTL, or SP-CI73T/I73TFlp+/− mice 6 d following TAM induction alone or TAM plus i.t. CLOD as labeled. Representative images are shown (n = 1–5). (D) i.v. CL-treated CTL, CLOD-treated CTL, or SP-CI73T/I73TFlp+/− mice 6 d following TAM induction alone or TAM plus i.v. CLOD. Representative images are shown (n = 4–5). (E) BALF cell counts from SP-CI73T/I73TFlp+/− mice (n = 10–32) harvested 3 and 7 d and 2 wk after TAM induction. A TAM + CLOD group was cotreated with i.v. CLOD (150 μg/kg administered at 2 h after TAM). CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) received i.v. CLOD or CL (150 μg/kg administered at 2 h after TAM or oil injection) as indicated. Data are represented as mean ± SEM (n = 3 for each CL group harvested at terminal day; n = 8–18 for all other groups). *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice; **p < 0.05 compared with TAM-treated SP-CI73T/I73TFlp+/− mice; ***p < 0.05 for SP-CI73T/I73TFlp+/− mice TAM + CLOD compared with SP-CI73T/I73TFlp+/− TAM, all analyzed by one-way ANOVA using Tukey post hoc test.

FIGURE 6.

The effect of depletion of alveolar macrophages or peripheral monocytes with clodronate liposomes on lung injury and survival of SP-CI73T mutant mice is dependent upon the route of administration. Kaplan–Meier survival analysis from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and TAM-induced (250 mg/kg; i.p.) SP-CI73T/I73TFlp+/− mice following (A) i.t. (50 μl/mouse given 48 h prior to induction) or (C) i.v. (150 μg/kg given 2 h postinduction) CLOD or CL administration (n = 3 for CL group collected at terminal day; n = 10–32 for all other groups). Mean survival using death or body weight <75% on two consecutive days as endpoints is shown. *p < 0.05 compared with CTL. #p < 0.05 compared with TAM-treated mice by log-sum (Mantel–Cox) rank test. H&E-stained sections (40×) from SP-CI73T cohorts treated with i.t. or i.v. clodronate as indicated. (B) i.t. CL-treated CTL, i.t. CLOD-treated CTL, or SP-CI73T/I73TFlp+/− mice 6 d following TAM induction alone or TAM plus i.t. CLOD as labeled. Representative images are shown (n = 1–5). (D) i.v. CL-treated CTL, CLOD-treated CTL, or SP-CI73T/I73TFlp+/− mice 6 d following TAM induction alone or TAM plus i.v. CLOD. Representative images are shown (n = 4–5). (E) BALF cell counts from SP-CI73T/I73TFlp+/− mice (n = 10–32) harvested 3 and 7 d and 2 wk after TAM induction. A TAM + CLOD group was cotreated with i.v. CLOD (150 μg/kg administered at 2 h after TAM). CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) received i.v. CLOD or CL (150 μg/kg administered at 2 h after TAM or oil injection) as indicated. Data are represented as mean ± SEM (n = 3 for each CL group harvested at terminal day; n = 8–18 for all other groups). *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice; **p < 0.05 compared with TAM-treated SP-CI73T/I73TFlp+/− mice; ***p < 0.05 for SP-CI73T/I73TFlp+/− mice TAM + CLOD compared with SP-CI73T/I73TFlp+/− TAM, all analyzed by one-way ANOVA using Tukey post hoc test.

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The protective effects of systemic clodronate (i.v. CLOD) administration on the early injury were reflected in alterations in the dynamics of the immune cell composition of BALF and lung tissue. As noted previously, induction of SftpcI73T mutant was associated with early reduction in SigF+CD11b alveolar macrophages paired with increased Ly6Chi immature macrophages in the BALF (Fig. 3). Systemic clodronate had no effects on alveolar macrophage numbers in BALF (Supplemental Fig. 3A) and tissue digests (data not shown). Conversely, i.v. CLOD resulted in reduced immature macrophage numbers in the BALF during the early (3 d) inflammatory phase but not in total Ly6Chi cells in the tissue (Fig. 7A, 7C). Further analysis on Ly6Chi immature macrophage subpopulations revealed that CLOD had significant effects on BALF and tissue burden of proinflammatory Ly6C+CCR2+ immature macrophages at 3 d (Fig. 7B, 7D). Histochemical analysis of lung sections further confirmed the reduction of CCR2+ cells in SP-CI73T–induced mice following CLOD treatment (Fig. 7E). Additional qPCR analysis of recovered BALF immune cells showed that CLOD-treated SftpcI73T mice exhibited no alteration in the gene expression of factors involved in monocyte recruitment (Mcp1) nor macrophage activation (Fizz1, Arg1, Nos2, and Il6) (Supplemental Fig. 4). CLOD depletion also had no effect on neutrophil infiltration (Supplemental Fig. 3B), although there was significant reduction in eosinophils in both BALF and tissue at 2 wk, assessed by flow cytometry analysis of SigF+CD11c cells (Fig. 8A, 8B) as well as immunohistochemistry for EPX (Fig. 8C).

FIGURE 7.

i.v. monocyte depletion with CLOD results in altered monocyte/macrophage composition in SP-CI73T mice. (A and C) Changes in the relative percentage of Ly6Chi immature macrophages in BALF (A) and lung tissue (C) from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM (250 mg/kg) and i.v. (CLOD) (150 μg/kg, 2 h after TAM injection) administration. Data are represented as mean ± SEM (n = 5–12). (B and D) Changes in the relative percentage of Ly6ChiCCR2+ immature macrophages in BALF (B) and tissue (D) from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM (250 mg/kg) and i.v. CLOD (150 μg/kg, 2 h after TAM injection) administration. Data are represented as mean ± SEM (n = 3–8). All analysis was considered significant. (E) Representative immunohistochemical staining for CCR2 expression of lung sections from SP-CI73T mice (four lungs/condition) harvested 14 d after TAM induction with (TAM + CLOD) or without (TAM) clodronate cotreatment as indicated. CLOD represents SP-CWT/WTFlp+/+ CTL cotreated with clodronate alone. *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice by one-way ANOVA using Tukey post hoc test.

FIGURE 7.

i.v. monocyte depletion with CLOD results in altered monocyte/macrophage composition in SP-CI73T mice. (A and C) Changes in the relative percentage of Ly6Chi immature macrophages in BALF (A) and lung tissue (C) from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM (250 mg/kg) and i.v. (CLOD) (150 μg/kg, 2 h after TAM injection) administration. Data are represented as mean ± SEM (n = 5–12). (B and D) Changes in the relative percentage of Ly6ChiCCR2+ immature macrophages in BALF (B) and tissue (D) from CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM (250 mg/kg) and i.v. CLOD (150 μg/kg, 2 h after TAM injection) administration. Data are represented as mean ± SEM (n = 3–8). All analysis was considered significant. (E) Representative immunohistochemical staining for CCR2 expression of lung sections from SP-CI73T mice (four lungs/condition) harvested 14 d after TAM induction with (TAM + CLOD) or without (TAM) clodronate cotreatment as indicated. CLOD represents SP-CWT/WTFlp+/+ CTL cotreated with clodronate alone. *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice by one-way ANOVA using Tukey post hoc test.

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FIGURE 8.

Peripheral monocyte depletion with i.v. CLOD results in altered eosinophil numbers in SP-CI73T mice. (A and B) Changes in BALF and tissue SigFhiCD11b+CD11c eosinophils from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM (250 mg/kg) and i.v. CLOD (150 μg/kg, 2 h after TAM injection) administration. Data are represented as mean ± SEM (n = 3–8). All analysis was considered significant. *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice by one-way ANOVA using Tukey post hoc test. (C) Immunohistochemical analysis of vehicle and clodronate (150 μg/kg)-treated CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 2 wk following i.p. TAM administration (250 mg/kg). Lungs stained for EPX. Representative 40× images from three to five separate animals at each condition are shown.

FIGURE 8.

Peripheral monocyte depletion with i.v. CLOD results in altered eosinophil numbers in SP-CI73T mice. (A and B) Changes in BALF and tissue SigFhiCD11b+CD11c eosinophils from control (CTL) (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 3 and 7 d and 2 wk following i.p. TAM (250 mg/kg) and i.v. CLOD (150 μg/kg, 2 h after TAM injection) administration. Data are represented as mean ± SEM (n = 3–8). All analysis was considered significant. *p < 0.05 compared with CTL SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice by one-way ANOVA using Tukey post hoc test. (C) Immunohistochemical analysis of vehicle and clodronate (150 μg/kg)-treated CTL (TAM-treated SP-CWT/WTFlp+/+ or oil-treated SP-CI73T/I73TFlp+/− mice) and SP-CI73T/I73TFlp+/− mice 2 wk following i.p. TAM administration (250 mg/kg). Lungs stained for EPX. Representative 40× images from three to five separate animals at each condition are shown.

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Mutations of the SFTPC gene have been linked to various forms of diffuse parenchymal lung disease (DPLD) in humans, namely IPF and childhood ILD (chILD) in adults and pediatric patients, respectively. The clinical course of these DPLDs is often punctuated by AE, which are marked by overt spikes in the inflammatory burden to the lung associated with high mortality from superimposed acute lung injury, which often results in a significant reduction of pulmonary function among survivors (26). There is an unmet need for new therapies for IPF/ILD because of incomplete understanding of the pathogenesis of both the underlying fibrosis and AE because prior pharmacological interventions (corticosteroids, cytokine modulation, antifibrotics) have focused on attenuating downstream responses that at best result in delay, rather than prevention, of pulmonary function decline (2729).

Part of this challenge to the development of effective therapies has been the absence of preclinical experimental models that translate to IPF/chILD patients. Our laboratory has recently developed two murine models of spontaneous PF each driven by a distinct functional class of SFTPC mutation that localize within either the BRICHOS (C121G mutant) or “linker” (I73T) domains of the SP-C proprotein (9, 30). Mutant SFTPC BRICHOS isoforms are misfolded and produce extensive AT2 endoplasmic reticulum stress and apoptosis (30). In contrast, non-BRICHOS mutants such as SFTPCI73T mistraffic to the AT2 plasma membrane, accumulate in late endosomes, and generate a different toxic gain of function AT2 cell phenotype marked by an acquired block in macroautophagy and hyperproliferation (8, 9). Importantly, although expression of these two mutants result in divergent molecular, biochemical, and cellular phenotypes, each is characterized by an overlapping lung phenotype, which includes inflammation and extensive tissue remodeling that established a central role for AT2 cells in the initiation of the inflammatory response through the release of a diverse array of factors involved in the recruitment and the activation of immune cell subsets. In this study, the present work extends these prior observations as we now use the SP-CI73T non-BRICHOS model to temporally define and characterize the role of resident (alveolar macrophages) and peripheral (monocytes, neutrophils, and eosinophils) immune cell subsets accumulating in the lung prior to and during the alveolitis, parenchymal injury, lung injury, and early remodeling following the induction of mutant SftpcI73T expression. Furthermore, using clodronate depletion, we establish a detrimental role for proinflammatory Ly6C+ monocytes in injury progression.

Understanding the AT2–immune cell cross-talk coordinating both the recruitment of CD45+ effector cells and systemic inflammation is pivotal to expanding and validating new therapeutic paradigms for IPF and chILD. A large part of our understanding of the mechanisms of acute and chronic inflammatory lung disease is the product of experimental models that rely on exogenous exposures (ozone, LPS, or bleomycin), which can nonspecifically affect a myriad of parenchymal subsets. Conversely, the SP-CI73T model represents a new and clinically relevant approach to characterize endogenous sterile inflammation and fibrogenesis initiated by epithelial cell stress (9, 31). Leveraging this approach, the induction of mutant SftpcI73T expression was associated with an initial tissue inflammatory response (Fig. 1) in association with a transient decrease in SigF+CD11b resident alveolar macrophages (Fig. 2). Alveolar macrophages represent the first line of defense against pathogen exposure and may be important for maintaining homeostasis of the alveolar niche, a response achieved in coordination with the lung epithelium and mesenchyme (32, 33). Because of their extensive phenotypic plasticity through adaption to the surrounding chemical milieu (34, 35), macrophages have been classified into two major activation states: M1 macrophages, essential in the early response to proinflammatory signals (TNF-α, IFN-γ) and M2 macrophages, important in the termination of inflammation as well as in tissue remodeling following Th2 stimulation (IL-10, TGF-β, IL-4, and IL-13) (23). Consistent with this notion, we found sequential M1 (3 d; Nos2, Il6) and M2 (2 wk; Arg1, Fizz1) activation following the induction of mutant SftpcI73T expression (Fig. 4). It remains to be determined whether the change in activation status observed over time is the result of the phenotypic shift of resident macrophages, influx of activated peripheral subsets, or rather a combination of the two, which will be elucidated in future studies.

Under homeostatic conditions, the alveolar macrophage pool self-maintains through proliferation (36), whereas a combination of recruitment and maturation of peripheral monocytes is indispensable to overcome loss-of-resident subsets following injury (37, 38). Bone marrow chimeras and monocyte reporter mice have been necessary to pinpoint and study the phenotypic changes taking place during the shift from monocyte to immature macrophages and, ultimately, to mature alveolar macrophage (15, 39). In accordance with this notion, the initial reduction in BALF and tissue CD11c+CD64+SigF+CD11b alveolar macrophages upon SP-CI73T mutant protein induction was countered by the accumulation of CD11b+Ly6Chi immature macrophages. This immature myeloid subset was previously shown to be recruited through a CCL2/MCP-1 axis (40) and is congruent with our previous findings of increased MCP-1 levels in the BALF within 3 d postinduction of the SP-CI73T mutant (9). Although the BALF levels of CCL2/MCP-1 returned to control values by 7 d, we found sustained Ccl2 transcript levels in epithelial and immune cells, accompanied by the steady accumulation of CD11b+Ly6ChiCCR2+ immature macrophages in the lung for up to 2 wk (Fig. 3B, 3F). Although this could likely generate a more localized gradient for CCL2/MCP-1 not detectable in whole BALF, we cannot exclude the existence of parallel monocyte recruitment signals. Indeed, our results support this notion by showing increased levels of CCL17 protein and CCR4 expression, thus complementing previous cytokine analysis proposing CCL17/CCR4 axis participation in the development of a noncanonical M2/Th2 inflammatory phenotype (9) in clinical IPF (12, 41). Our data also show that whereas both epithelial and immune cells can contribute to persistent CCL2/MCP-1 production (9), AT2 cells were the sole source of Ccl17 expression, thus confirming a central role for the epithelium in the immune response in this model and likely in clinical IPF (10, 12, 13).

As monocytes were the first peripheral population recruited in the lung following SP-CI73T induction (Fig. 3), we asked whether the disruption of this process could alter injury progression. i.t. and i.v. CLOD represent widely used experimental methodologies to reduce monocyte/macrophage numbers in the compartment of interest (42). Although its mechanism of action is not targeted to specific macrophage subsets, neither route of administration has been shown to affect interstitial cells (43, 44). Perhaps one of the most interesting findings to emerge from this work is represented by the dichotomous effects of clodronate, depending upon the route of administration. Notable was the observation that resident alveolar macrophage depletion resulted in significantly decreased survival (Fig. 6A), which was accompanied by histological evidence of severe inflammation within 6 d of SP-CI73T induction (Fig. 6B). At higher power, we noted the presence of clusters of neutrophils and lymphocytes. The morphology of these lesions is consistent with a possible acquisition of induced bronchoalveolar lymphoid tissue; however, its definitive identification will require microdissection and flow cytometric characterization of these regions to properly define such structures as published by Harmsen’s group (45). Regardless, the current data intriguingly suggest a pivotal role for resident alveolar macrophages in lung immune homeostasis and in containing the inflammatory burst that develops in response to mutant SP-CI73T induction in AT2 cells (Fig 6B).

Conversely, we found that when systemically administered, clodronate effectively blunted the accumulation of CCR2+Ly6C+ immature macrophages during acute injury while also significantly improving overall survival and modulating inflammatory endpoints, such as total BALF cell numbers, CCR2+ cell influx, and the expansion of eosinophils (Figs. 6, 7). These findings suggest that recruitment of this blood-borne subset plays a deleterious role early in SP-CI73T lung injury, a notion consistent with other experimental models of acute and chronic injury in the lung and elsewhere (15, 46, 47). In addition, our data are consistent with findings indicating a proinflammatory and proinjurious function of peripheral monocytes due, at least in part, to their distinct ontogeny from that of resident macrophages (48). This notion is further supported by experimental models of bleomycin-induced injury showing significant contributions of monocyte-derived alveolar macrophages to the aberrant injury and remodeling (15).

In this model, we observed a consistent and well-defined dynamic accumulation of eosinophils in tissue and BALF after TAM-induced SftpcI73T induction (Fig. 5D, Supplemental Fig. 2A). An exhaustive literature describes the role of eosinophils in parasitic infection, allergic disease, and asthma (49, 50). Complementary, clinical evidence has clearly established eosinophil presence in several subtypes of DPLD, including IPF (18, 51, 52) and in AE (53); however, their exact functions are currently unknown. Eosinophils have been shown to promote parenchymal destruction via the release of highly toxic granules containing hypobromite, superoxide, and peroxide; yet, they are also the source of hematopoietic and growth factors involved in tissue remodeling/fibrosis (e.g., TGF-β) (54, 55). Using this model, we previously showed that AT2 epithelial cells represent a source of cytokines associated with eosinophil recruitment (CCL11) and maintenance (IL-5) upon induction of SP-CI73T (9). In the current studies, we show by qPCR that BALF immune cells also participate in eosinophil mobilization, albeit at a later time point (2 wk), suggesting they may act to amplify ongoing inflammatory cell recruitment. Notably, as Ly6C+ monocytes have been shown to recruit eosinophils in a CCL11-dependent manner (56), our observations that peripheral monocyte depletion results in reduced eosinophil numbers in the lung after SP-CI73T mutant induction further support the notion that these cells could be important in injury progression.

Through analysis of both tissue and BALF, we demonstrated that these compartments are comparable when characterizing mobile effector cell subsets and for which BALF analysis in patients with a variety of ILDs has often been used to assess disease activity. However, because of the complexity of AE, analysis of tissue digests offers additional information of interstitial populations that may contribute to injury initiation and progression. Although our data has focused on the role of myeloid immune cell populations, it is likely that effector cells of lymphoid origin, such as regulatory T cells or the recently characterized lineage-negative innate lymphoid cells, could also play a role. In support to this notion, regulatory T cells have been previously shown to participate in both AE of lung disease and aberrant lung remodeling (57, 58), whereas innate lymphoid cells, despite their elusive nature, have been heavily linked with pulmonary disease states enriched in noncanonical Th2 factors (i.e., IL-5, IL-6, and IL-13) (59, 60). Based on our findings of conspicuous levels of these cytokines after SP-CI73T mutant induction, it is intriguing to propose that this regulatory subset could play a role in the dynamic inflammatory events associated with this preclinical model.

In summary, using a novel model of endogenous, sterile lung injury, the current study demonstrates that expression of a mutant Sftpc protein in the pulmonary epithelium of mice recapitulates many features of AE of DPLD in human patients. In addition to characterizing the ontogeny of the immune cell response to AT2 cell stress, these results offer important insight into the role of peripheral proinflammatory monocytes in the initiation and amplification/progression of inflammatory events in ILD pathogenesis. The complexity of the effector cell cascades observed during SP-CI73T–induced AE supports the notion that a multitarget therapeutic approach will be required to make significant inroads into the treatment of IPF and chILD.

We thank Drs. Debra Laskin and Andrew Gow for valuable input. M.F.B. is an Albert M. Rose Established Investigator of the Pulmonary Fibrosis Foundation.

This work was supported by U.S. Department of Veterans Affairs Merit Review 1I01BX001176 (M.F.B.), National Institutes of Health (NIH) R01 HL119436 (M.F.B.), NIH P30 ES013508 (M.F.B.), and NIH HL129150 (S.M.); J.K. was supported by NIH 2T32 HL007586.

The online version of this article contains supplemental material.

Abbreviations used in this article:

AE

acute exacerbation

AT2

alveolar type 2

BALF

bronchoalveolar lavage fluid

chILD

childhood ILD

CL

control liposome

CLOD

clodronate liposome

dCt

δ-cycle threshold

DPLD

diffuse parenchymal lung disease

EPX

eosinophil peroxidase

ILD

interstitial lung disease

iNOS

inducible NO synthase

IPF

idiopathic PF

i.t.

intratracheal

M1

classically activated

M2

alternatively activated

PF

pulmonary fibrosis

qPCR

quantitative single-plex PCR

SP-C

surfactant protein-C

SP-CI73T

isoleucine to threonine at position 73 in the SFTPC proprotein

TAM

tamoxifen.

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The authors have no financial conflicts of interest.

Supplementary data