Assessing Autophagy in Mouse Models and Patients with Systemic Autoimmune Diseases

Abstract

Autophagy is a tightly regulated mechanism that allows cells to renew themselves through the lysosomal degradation of proteins, which are misfolded or produced in excess, and of damaged organelles. In the context of immunity, recent research has specially attempted to clarify its roles in infection, inflammation and autoimmunity. Autophagy has emerged as a spotlight in several molecular pathways and trafficking events that participate to innate and adaptive immunity. Deregulation of autophagy has been associated to several autoimmune diseases, in particular to systemic lupus erythematosus. Nowadays, however, experimental data on the implication of autophagy in animal models of autoimmunity or patients remain limited. In our investigations, we use Murphy Roths Large (MRL)/lymphoproliferation (lpr) lupus-prone mice as a mouse model for lupus and secondary Sjögren’s syndrome, and, herein, we describe methods applied routinely to analyze different autophagic pathways in different lymphoid organs and tissues (spleen, lymph nodes, salivary glands). We also depict some techniques used to analyze autophagy in lupus patient’s blood samples. These methods can be adapted to the analysis of autophagy in other mouse models of autoinflammatory diseases. The understanding of autophagy implication in autoimmune diseases could prove to be very useful for developing novel immunomodulatory strategies. Our attention should be focused on the fact that autophagy processes are interconnected and that distinct pathways can be independently hyper-activated or downregulated in distinct organs and tissues of the same individual.

Lysosomes as a therapeutic target

“…Fluorescence measurement (flow cytometry or fluorescence microscopy) of cellular staining of acidotropic dyes, such as LysoTracker dyes 92,215 Simple to use but is not quantitative as stated by the manufacturer; can be adapted to clinical trial settings Western blot and fluorescence imaging of lysosomal markers such as L AMP1, L AMP2 etc. 216,217 Simple but does not provide information on subcell populations 89 ; can be adapted to clinical trial settings Electron microscopy 218 Provides morphological information but laborious and semiquantitative…”

“…lysosomal function is essentially linked with autophagy activity as autophagy is a lysosomal-dependent degradation pathway. Thus, a series of methods routinely applied for assessing macroautophagy in mouse models and patients with autoimmune diseases is summarized 89 . To ascertain the extent of autophagy defects, a combination of techniques, such as western blot and flow cytometry, measurement of autophagy makers, fluorescent imaging and electron microscopy, in the presence and absence of lysosomal protease inhibitors, is recommended.…”

“…a The presentation of the successive sections follows the text, namely , LSDs, autoimmune diseases and neurodegenerative diseases. Lysosomes are abnormal in splenic B cells from Fas deficient Murphy Roths Large (MRL)/lpr mice, a mouse model of lupus, compared with B cells from healthy CBA/J mice 89 . TFEB expression was increased, indicating an enhanced biogenesis of lysosomes, and the lysosomal volume was raised.…”

Methods in renal research: Measurement of autophagic flux in the renal cortex ex vivo

“…Autophagy is emerging as a fundamental concept in human health, aging and age‐related diseases. This has been evident across several pathological settings including neurological diseases such as Parkinson’s disease and Alzheimer’s disease; atherosclerosis and other cardiovascular diseases; and immunological diseases such as mycobacterium tuberculosis and Sjögren’s syndrome . Similarly, autophagy has been implicated in several manifestations of kidney disease, both acute and chronic kidney diseases such as diabetic nephropathy .…”

“…This has been evident across several pathological settings including neurological diseases such as Parkinson's disease and Alzheimer's disease 1,2 ; atherosclerosis and other cardiovascular diseases 3,4 ; and immunological diseases such as mycobacterium tuberculosis and Sjögren's syndrome. 4,5 Similarly, autophagy has been implicated in several manifestations of kidney disease, both acute [6][7][8][9][10] and chronic kidney diseases such as diabetic nephropathy. 11,12 The significance of autophagy and the role it plays in the development of renal pathology has risen to prominence in recent years, but few have addressed changes in autophagic flux in vivo.…”