Human cystic fibrosis monocyte derived macrophages display no defect in acidification of phagolysosomes when measured by optical nanosensors

J Cyst Fibros. 2020 Mar;19(2):203-210. doi: 10.1016/j.jcf.2019.09.003. Epub 2019 Sep 6.

Abstract

Background: Defective macrophage phagolysosomal acidification is implicated in numerous lung diseases including Cystic Fibrosis (CF) and may contribute to defective pathogen killing. Conflicting reports relating to phagolysosomal pH in CF macrophages have been published, in part related to the use of pH-sensitive fluorescent probes where potential inadequacies in experimental design can be a contributing factor (e.g. employing probes with incorrect pKa for the cellular compartment of interest). We developed a reliable method to quantify macrophage phagolysosomal pH using surface-enhanced Raman spectroscopy-based nanosensors.

Methods: Monocyte-derived macrophages from CF and healthy control participants were incubated with nanosensors. Live cell imaging identified phagocytosed nanosensors, and surface-enhanced Raman spectroscopy was performed using para-mercaptobenzoic acid functionalised gold nanoparticles which produce Raman spectra that change predictably with their environmental pH. Conventional fluorescence spectroscopy was carried out in comparison. Nanosensor localisation to phagolysosomes was confirmed by transmission electron microscopy.

Results: Nanosensors were actively phagocytosed by macrophages into phagolysosomes and acidification occurred rapidly and remained stable for at least 60 min. There was no difference in phagolysosomal pH between healthy control and CF macrophages (5.41 ± 0.11 vs. 5.41 ± 0.20, p > .9999), further confirmed by inhibiting Cystic Fibrosis Transmembrane Conductance Regulator in healthy control monocyte-derived macrophages.

Conclusions: Optical nanosensors accurately measure macrophage phagolysosomal pH and demonstrate no phagolysosomal acidification defect in human CF monocyte-derived macrophages. Further studies using alveolar macrophages could extend the impact of our findings. Nanosensors represent a novel and precise means to measure organelle functions with widespread potential for the study and monitoring of several lung diseases.

Keywords: Cystic fibrosis*/pathophysiology; Myeloid cells; Nanotechnology; Phagocytosis; SERS; pH.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Biochemical Phenomena
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Cystic Fibrosis* / pathology
  • Cystic Fibrosis* / physiopathology
  • Female
  • Fluorescent Dyes
  • Humans
  • Hydrogen-Ion Concentration
  • Macrophages, Alveolar* / chemistry
  • Macrophages, Alveolar* / physiology
  • Male
  • Metal Nanoparticles
  • Nanotechnology / instrumentation
  • Nanotechnology / methods
  • Phagocytosis
  • Phagosomes* / chemistry
  • Phagosomes* / microbiology
  • Spectrum Analysis, Raman* / instrumentation
  • Spectrum Analysis, Raman* / methods

Substances

  • CFTR protein, human
  • Fluorescent Dyes
  • Cystic Fibrosis Transmembrane Conductance Regulator