Background

The highly selective, lysosome-dependent catabolic pathway of autophagy is part of normal cellular housekeeping as well as cellular homeostasis. This highly conserved process is responsible for the removal and recycling of damaged cytoplasmic cargo, such as defective organelles or misfolded proteins in the form of aggregates. The core autophagic process involves the formation of double-membrane vesicles, called autophagosomes, that engulf the cytoplasmic cargo and then fuse with the lysosome. After fusion, lysosomal hydrolases are responsible for the degradation (see Figure 1) [1].

Impaired autophagy has been associated with ageing as well as a multitude of different diseases and pathologies, including cancer and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or Huntington’s disease, among others [1,2]. This illustrates the need to counteract impaired autophagy and to explore the possibility of inducing autophagy through treatment options or supplementation with substances. Known autophagy inducers are cellular stress, for example, in the form of caloric restriction or exercise. Further, autophagy is shown to be induced by hypoxia, DNA damage, ER stress, and microbial pathogen infection [3,4]. Heat stress is also a known inducer of autophagy [5], which, in this protocol, can be administered in the form of hyperthermia. This hyperthermia, also known as thermal therapy or heat treatment, uses a specialised heat source in the form of water-filtered infrared-A radiation (wIRA) with a wavelength ranging from 780 to 1,400 nm. For the warming of the whole body, the IRATHERM^®1000M (Von Ardenne Institute of Applied Medical Research GmbH, Dresden, Germany) is used. To heat isolated human primary peripheral blood mononuclear cells (PBMCs), a portable device called IRAcubator with the same heat source is utilised. This is only one example of a treatment method that modulates autophagy, but it demonstrates the need for a reliable and reproducible method. This is particularly important for small, round cells such as PBMCs, which have a high nucleus-to-cytoplasm ratio and for which classic methods of localising and quantifying autophagosomes are ineffective. Prominent methods for autophagy monitoring are transmission electron microscopy (TEM), western blotting, or the use of reporter systems [6]. Besides being very time-consuming and expensive, TEM is rather suited for structural and qualitative analysis as opposed to quantitative analysis. Nevertheless, TEM remains one of the gold standards in autophagy detection, but it is limited to non-living cells. Western blotting requires vast amounts of sample material, making it unsuitable for studies where the sample material is scarce. Further, it is also very time-consuming and expensive. Reporter systems are elegant methods when working with cell lines, but are not feasible when working with primary cells from in vivo treatments.

Other methods, such as CYTO-ID^®, are fast, reliable, and relatively cost-effective, but have severe limitations in small and round cells like PBMCs, as discussed in our previous work [7].

The Guava^® Autophagy Detection kit used in this protocol contains a FITC-labelled anti-LC3 antibody. By optimising the cell number and incubation steps, an ideal staining for PBMCs can be achieved. In addition, the lysosomal inhibitor chloroquine can be used as a positive control. By inhibiting the fusion of the lysosome to the autophagosome, the autophagic process is interrupted, leading to the accumulation of autophagosomes (Figure 1). Therefore, wells that contain chloroquine should show a higher median fluorescent intensity (MFI) if staining has worked properly. For both conditions, additional wells containing chloroquine are prepared as a positive control.

Figure 1. Schematic overview of the autophagy process under different conditions. Under normal conditions, the mature autophagosome fuses with the lysosome, resulting in the degradation of the vesicles (a). In the presence of chloroquine, however, the fusion is inhibited, resulting in accumulation of the autophagosomes (b).

This method proved to be rapid, reliable, and cost-effective. Moreover, it can not only be used to investigate the effects of hyperthermia but can also be adapted to investigate a multitude of treatments and therapies in vivo and ex vivo.