title: The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation.
abstract: BACKGROUND
  Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in SFTPC, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.
  
  
  METHODS
  SP-CA116D was expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.
  
  
  RESULTS
  Stable expression of SP-CA116D in MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space.
  
  
  CONCLUSIONS
  We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.
authors.affiliation: Childrens' Hospital of the Ludwig-Maximilians-University, Lindwurmstr, 4, 80337 Munich, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Zarbock
authors.forename: Markus
authors.initials: M
authors.lastname: Woischnik
authors.forename: Christiane
authors.initials: C
authors.lastname: Sparr
authors.forename: Tobias
authors.initials: T
authors.lastname: Thurm
authors.forename: Sunčana
authors.initials: S
authors.lastname: Kern
authors.forename: Eva
authors.initials: E
authors.lastname: Kaltenborn
authors.forename: Andreas
authors.initials: A
authors.lastname: Hector
authors.forename: Dominik
authors.initials: D
authors.lastname: Hartl
authors.forename: Gerhard
authors.initials: G
authors.lastname: Liebisch
authors.forename: Gerd
authors.initials: G
authors.lastname: Schmitz
authors.forename: Matthias
authors.initials: M
authors.lastname: Griese
mesh_terms: D000818:Animals; D001379:Azathioprine / Q000494:pharmacology; D002460:Cell Line; D003520:Cyclophosphamide / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D006886:Hydroxychloroquine / Q000494:pharmacology; D017563:Lung Diseases, Interstitial / Q000235:genetics* / Q000276:immunology; D008775:Methylprednisolone / Q000494:pharmacology; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010743:Phospholipids / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D037721:Pulmonary Surfactant-Associated Protein C / Q000235:genetics* / Q000276:immunology; D011663:Pulmonary Surfactants / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D010743:Phospholipids; D037721:Pulmonary Surfactant-Associated Protein C; D011663:Pulmonary Surfactants; D006886:Hydroxychloroquine; D003520:Cyclophosphamide; D001379:Azathioprine; D008775:Methylprednisolone