A non-BRICHOS surfactant protein c mutation disrupts epithelial cell function and intercellular signaling

Biophysically active pulmonary surfactant contains a mixture of lipids and hydrophobic surfactant proteins B (SP-B) and C (SP-C). A normal composition and homeostasis of pulmonary surfactant is critical for its surface-tension-reducing properties and gas exchange in the alveolar region of the lung. SP-C is synthesized exclusively by alveolar type II cells (AECII) as a 21 kDa preprotein (proSP-C). ProSP-C is further processed to the 4.2 kDa mature protein through a sequence of proteolytic cleavages before being secreted together with lipids and other surfactant components to the alveolar surface [1–4]. Surfactant secretion is accomplished by fusion of lamellar bodies, AECII-specific organelles for surfactant assembly and storage, with the plasma membrane [5]. SNARE proteins, in particular syntaxin 2 and SNAP-23, are required for regulated surfactant secretion. Both proteins are associated with the plasma membrane and to some degree with lamellar bodies [6, 7]. In parallel to secretion, AECII reinternalize and recycle surfactant components from the alveolar surface by means of endocytosis via clathrin-dependent and clathrin-independent pathways, which include routing to early endosomes and multivesicular bodies [5].

Interstitial lung disease (ILD) is a heterogeneous group of diseases of known and unknown etiology [8–10]. Several histological and clinical subtypes of ILD are linked to the SP-C protein deficiency caused by mutations of the corresponding SFTPC gene [3]. Many SP-C mutations cluster within the preprotein's BRICHOS domain and lead to misfolding of the preprotein, aberrant trafficking and processing [11]. To date, all affected individuals with BRICHOS domain mutations have been heterozygous with no detectable mature SP-C in their lungs, suggesting a dominant-negative effect of the mutant allele (e.g. Δexon4, L188Q) [11, 12]. Moreover, in cell lines expressing BRICHOS domain mutations, proSP-C forms perinuclear aggregates, consistent with the cell's inability to clear aggregated misfolded proteins and a toxic gain-of-function [12, 13]. Various pathologic mechanisms for these mutations causing chronic accumulation of misfolded proSP-C have been proposed, such as induction of endoplasmic reticulum (ER) stress, cytotoxicity, and caspase 3- and caspase 4-mediated apoptosis [14–16]. These factors might contribute to ILD through cell injury and death of AECII. In addition to the BRICHOS domain mutations, a second class of SFTPC mutations has emerged. A heterozygous missense mutation, leading to a substitution of threonine for isoleucine at position 73 of the proSP-C, is the most frequent SFTPC mutation [17, 18]. There is a strong variability in the phenotype of these patients, ranging from asymptomatic to early fatal cases [19]. I73T SP-C (SP-C^I73T) is marked by mistrafficking of the preprotein to the endosomal compartment and by preserved secretion of both mature and aberrant proSP-C and proSP-B forms and their intra-alveolar accumulation [17, 20, 21]. Yet, current knowledge on SP-C^I73T lacks a precise understanding of the proSP-C processing abnormalities, concurrent cell stress response and cytotoxicity, as well as perturbations of the surfactant composition and secretion.

Current treatment of the genetic interstitial lung diseases in children is unfortunately empirical. Corticosteroids are anti-inflammatory and stimulate surfactant protein transcription [22, 23]. Chloroquine and its less toxic derivative hydroxychloroquine [24, 25] are used and believed to act on the lysosomal function, i.e. reduce vesicle fusion, exocytosis and proteolytic degradation [26, 27] or stimulate lamellar body biogenesis [28]. Thus, there is a need to define the cellular mechanism of the currently applied treatments. Potential therapeutic targets are cell chaperones which assist in normal protein folding and are part of the cytoprotective mechanism that keeps control over misfolded proteins either by folding them correctly or by directing them to the degradation pathway [29, 30]. Δexon4 SFTPC mutation and proSP-C^Δexon4 accumulation upregulate the major ER chaperone BiP in an attempt to maintain surfactant biosynthesis in the presence of ER stress [14]. The regulation of other chaperones, like HSP90, HSP70, calreticulin and calnexin, is unknown. Even so, without pharmacological manipulation, such cytoprotective mechanisms may not be sufficient to maintain production of the bioactive surfactant with a normal lipid/protein composition. In addition, AECII, stressed by aberrantly processed proteins, might signal to and activate the surrounding cells, particularly those of immune system, which could contribute to the SP-C-associated disease.

The goal of this study was to investigate the intracellular disturbances and intercellular signaling of AECII affected by SP-C^I73T expression and the ability of the pharmaceuticals commonly used in ILD therapy to modulate some of the cellular mechanisms behind the diseases. We demonstrate the impact of I73T mutation on proSP-C processing, AECII stress tolerance, surfactant lipid composition and activation of the cells of the immune system. In addition, we investigate modulation of the disease cellular mechanisms by pharmaceutical drugs applied in the ILD therapy.

Mutations in the SFTPC gene are a known cause of surfactant deficiency and very variable genetic ILD in children and adults. We investigated the intracellular disturbances and intercellular signaling of MLE-12 cells expressing SP-C^I73T and the ability of pharmaceutical drugs used in ILD therapy to modulate some of the cellular consequences of SP-C deficiency caused by this mutation. MLE-12 cells were chosen as a model system since they contain structures, which resemble lamellar bodies seen in AECII [37]. The presence of lamellar body-like structures in the cells used was confirmed by electron microscopy (data not shown). Here we named the organelles detectable as LAMP3-positive vesicles, lamellar body-like structures.

A potential limitation of the study is that our system corresponds rather to a homozygous than to a heterozygous SFTPC mutation where one WT copy is still present. Although endogenous SP-C is expressed in the MLE-12 cells [38], expression of exogenous SP-C from the CMV promoter present on the plasmid vector is likely higher. However, all known patients with SP-C mutations are heterozygous, expressing one copy of the wild type gene. Thus, the experimental model reflects the in vivo condition. In addition, in contrast to SP-C^Δexon4 or SP-C^L188Q, I73T is a non-BRICHOS domain mutation and does not lead to the conformational disease observed for BRICHOS domain mutations where the mutant allele acts in a dominant-negative way [13]. Consequently, I73T still allows for the production of mature SP-C in vivo[17].

Stable transfection of MLE-12 cells with SP-C^WT or SP-C^I73T led to the intracellular accumulation of proSP-C^I73T processing intermediates which were not found in cells with proSP-C^WT, but corresponded well to species in the BAL fluid of patients with this mutation [17] (Figure 1). The first step in proSP-C processing is a cleavage at the C-terminal end [2]. Using an EGFP-tag fused to the C-terminus of proSP-C showed no difference in processing intermediates of proSP-C^WT and proSP-C^I73T (Figure 1, right). This means that (a) the first cleavage step happening at C-terminus is not influenced by this mutation and (b) the mutation does not interfere with the export from the ER and Golgi, because this cleavage occurs after trafficking through these compartments [2, 3]. In addition, immunofluorescence assays showed neither proSP-C^WT nor proSP-C^I73T retention in the ER compartment (Figure 2), supporting the conclusions made from the immunoblots. To examine the processing following the first C-terminal cleavage, we applied N-terminal protein tags. Dominant proSP-C^WT intermediates, that were also identified for proSP-C^I73T, were the species after the first C-terminal cleavage (Figure 1, bands #2 and #6), and the species before the first N-terminal cleavage (Figure 1, band #8). The primary full-length translation product (Figure 1, bands #1 and #5) was only faintly detectable for proSP-C^WT. Expression of proSP-C in this model is under control of a CMV-promoter, not the native SP-C promoter. It is therefore unlikely that a feedback mechanism is responsible for a higher expression of proSP-C^I73T intermediates. It is more likely that the I73T mutation slows down the processing and/or trafficking of the mutant proSP-C, leading to accumulation of incompletely processed proSP-C. It is not known how this mutation affects the folding of proSP-C, but subtle changes in conformation may also be responsible for the abundance of another processing intermediate, of size ~17 kDa (Figure 1, band #3 and #7). This intermediate can be found in the BAL fluid of patients with the I73T mutation, suggesting that this proSP-C form is being secreted from AECII along with the mature SP-C that is produced by AECII regardless of the presence of the I73T mutation [17].

Immunofluorescence assay of stably transfected MLE-12 showed that proSP-C^I73T colocalized often with EEA1 positive vesicles (Figure 2A), confirming our previous report (12). Early endosomes generally contain material that is taken up by endocytosis and is either recycled or routed for degradation [32]. Up to 80% of used lung surfactant is known to be reinternalized by AECII from alveolar space [5]. Although immunofluorescence does not allow the distinction between different EGFP-positive species depicted in Figure 1B, we speculate that the proSP-C^I73T species in the EEA1 positive compartment might be primarily the additional preprotein species accumulating in the I73T mutant. They are secreted only by the AECII with the I73T mutation and might be reinternalized as well. On the other hand, proSP-C^WT, but rarely proSP-C^I73T, colocalized with syntaxin 2, a SNARE protein involved in the secretion of lung surfactant, found in the plasma membrane and lamellar bodies of AECII (Figure 2B). Interestingly, our data propose the influence of hydroxychloroquine and methylprednisolone on localization and routing of proSP-C^WT moving it toward early endosomal vesicles. On the other hand, methylprednisolone showed the capacity to partially correct the mislocalization/routing defect of proSP-C^I73T (Figure 5).

The expression of mutated proteins frequently results in elevated cell stress. This has been shown for the BRICHOS domain SP-C mutations L188Q and Δexon4 [14, 15]. We found that the constitutive expression of SP-C^I73T moderately increased cell lethality under normal growth conditions (Figure 3A), maybe as a result of the ability of the cellular system to adapt to the presence of stress, as reported in [39]. The additional exogenous stress, imposed in our experiments by exposure to pharmaceuticals used in ILD therapy, might shift this balance out of the tolerable range. Treatment of the cells with azathioprine drug almost doubled the number of dying I73T mutant cells compared to WT. This aggravation was much less pronounced in the presence of methylprednisolone, hydroxychloroquine or cyclophosphamide.

Intracellular stress is in part handled by endogenous chaperones. Still without pharmacological boost, such cytoprotective mechanisms may not always be sufficient to normalize the cell function and maintain production of the bioactive surfactant with a normal lipid/protein composition. We determined the change in expression of the four important chaperones under the influence of the same ILD drugs. We found that the influence of azathioprine on the chaperones was almost the same in proSP-C^WT-and proSP-C^I73T-expressing cells, leaving no protection for additional stress, being a potent stress factor per se (Figure 4A-D). In contrast, hydroxychloroquine treatment led to an 81% increase in HSP90, and 75% increase in calreticulin expression in I73T mutant cells over WT cells (Table 1), thereby possibly protecting the cells against the additional stress and enhancing the ER folding capacity. HSP90 seemed to be targeted by all tested pharmaceuticals, while calnexin levels were refractory to stimulation (Figure 4). Treatment with the four drugs did not change the pattern of the proSP-C processing bands observed in the immunoblots in Figure 1A (data not shown).

The lipid composition of the stable MLE-12 cells was similar to that previously described in human foetal AECII, especially with regard to PC composition [40]. In the SP-C^I73T expressing cells we found a pronounced drop of total cellular PC, whereas LPC was increased (Figure 6A, additional file 1: supplemental Table S1). It is known that PC is degraded to LPC by an intrinsic phospholipase A2-like activity, and that LPC is toxic to various cells [41]. Increased LPC may therefore be a result of increased phospholipase activity due to the presence of mutated SP-C. SP-C dysfunction may also lead to a diminished activity of acyltransferases which reacetylate LPC. LPC is a known inhibitor of the lung surfactant activity and has the ability to penetrate directly into interfacial films to impair lowering of the alveolar surface tension during dynamic compression [42, 43]. Elevated LPC levels in the SP-C^I73T-expressing cells could also explain the heightened sensitivity towards exogenous stress described above. Generation of LPC cannot account for the decrease of PC mass in SP-C^I73T expressing cells, but additional factors, which directly interfere with the synthesis and packaging of PC, must also be responsible. This is in line with the observed grossly altered pattern of the fatty acid species of the different phospholipid classes, including PC in SP-C^I73T cells (Table 2, additional file 1: supplemental Table S1).

AECII secrete the surfactant phospholipids into the alveolar space where it lowers surface tension. Among phospholipids secreted by the I73T mutants PC was again decreased by 27% and LPC was increased by 57%, compatible with a reduced surfactant function [40, 43]. Treatment with methylprednisolone or hydroxychloroquine ameliorated the increase in intracellular and secreted LPC and decrease in secreted PC, but did not completely correct it (Figure 6A, B). The capacity of the treatment with methylprednisolone and hydroxychloroquine to correct the lipid disturbances caused by I73T mutation (Figure 6B) represent one of the mechanisms by which these treatments are empirically helpful in some patients with I73T mutations (own unpublished results, [19]).

Lastly, the index patient with the I73T mutation in our previous study displayed a mild interstitial chronic inflammation and most of the infiltrated leukocytes were CD3+ and CD4+ T-lymphocytes [17]. We found that cells with the I73T mutation released soluble factors into the medium that increase surface expression of CCR2 and CXCR1 on CD4+ lymphocytes and CXCR1 on neutrophiles (Figure 7A-C). When activated, the high affinity IL-8 receptor CXCR1 mediates antibacterial killing capacity [36, 44]. Increases in surface expression levels of CCR2 and CXCR1, respectively, might have the potential to modulate the pulmonary immune response with regard to antibacterial (CXCR1) and profibrotic (CCR2) responses [36, 45]. However, the soluble factors involved in the induction of chemokine receptor expression as well as the functional consequences of this phenomenon remain to be addressed in future studies.

We showed impaired proSP-C processing, altered cellular stress tolerance and unfavorable changes of the surfactant lipid composition in a murine AECII model cell line. Some of the demonstrated cellular aspects behind the disease could be modulated with drugs used in the therapy of ILD patients, thereby giving insight into their potential therapeutic mechanism on a cellular level. We also demonstrated that AECII with I73T mutation could signal to the surrounding cells of the immune system through secretion of soluble factors. Therefore, our study adds to understanding of the effects that SFTPC mutations impose on (pro)SP-C and AECII cell biology and pave the way for a more precise pharmacological targeting in patients with SP-C deficiency.

This paper contains parts of the doctoral thesis of Christiane Sparr. Until 2009 Sunčana Kern published as Sunčana Moslavac.