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-CI73T 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 . 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 , 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-CΔexon4 or SP-CL188Q, 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 . Consequently, I73T still allows for the production of mature SP-C in vivo.
Stable transfection of MLE-12 cells with SP-CWT or SP-CI73T led to the intracellular accumulation of proSP-CI73T processing intermediates which were not found in cells with proSP-CWT, but corresponded well to species in the BAL fluid of patients with this mutation  (Figure 1). The first step in proSP-C processing is a cleavage at the C-terminal end . Using an EGFP-tag fused to the C-terminus of proSP-C showed no difference in processing intermediates of proSP-CWT and proSP-CI73T (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-CWT nor proSP-CI73T 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-CWT intermediates, that were also identified for proSP-CI73T, 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-CWT. 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-CI73T 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 .
Immunofluorescence assay of stably transfected MLE-12 showed that proSP-CI73T 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 . Up to 80% of used lung surfactant is known to be reinternalized by AECII from alveolar space . Although immunofluorescence does not allow the distinction between different EGFP-positive species depicted in Figure 1B, we speculate that the proSP-CI73T 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-CWT, but rarely proSP-CI73T, 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-CWT moving it toward early endosomal vesicles. On the other hand, methylprednisolone showed the capacity to partially correct the mislocalization/routing defect of proSP-CI73T (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-CI73T 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 . 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-CWT-and proSP-CI73T-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 . In the SP-CI73T 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 . 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-CI73T-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-CI73T 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-CI73T 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, ).
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 . 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.