Progress and Challenges in the Use of MAP1LC3 as a Legitimate Marker for Measuring Dynamic Autophagy In Vivo

Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy in tissue samples and in vivo via a protein, which centrally intervenes in the autophagy pathway, the microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3), which is the most widely used marker and the first identified to associate with autophagosomal structures. These approaches are presented and discussed in terms of pros and cons. Some recommendations are provided to improve the reliability of the interpretation of results.


Introduction
Autophagy (from the ancient Greek "αὐτóϕαγoς", meaning "self-eating") is a critical component of cell homeostasis. This pivotal process contributes to the cellular quality control and energetic regulation of tissues via the lysosomal processing and recycling of vital cellular components [1]. Three main forms of autophagy are classically described that are macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA), but in fact, there are many more forms and subtypes of each (as the different types of microautophagy, for example), working concomitantly in the cell to control its viability. Some of these processes are highly selective, some others are less, but all share a certain degree of selectivity to mediate the degradation of specific targets [2][3][4][5][6]. Table 1. Examples of autophagic and lysosomal activators and inhibitors (non-exhaustive list) 1 .
Discovered in our team, a 21-mer phosphopeptide called P140, which primarily targets CMA in vitro and ex vivo and corrects altered macroautophagy occurring in inflammatory pathological contexts, showed its therapeutic effectiveness both in animal models of autoimmunity [26][27][28][29][30] and in patients with lupus [31,32]. Given to lupus mice with active disease, P140 reduced the overexpression level of lysosomal-associated membrane protein (LAMP)2A and HSPA8, two key players of CMA [33][34][35]. In vitro, P140 inhibited CMA in a cell line that stably expressed a CMA reporter [34,35]. In NOD.H-2h4 mice that develop a primary Sjögren's-like syndrome, P140 rescued sick mice from some autophagy defects presumably associated with the ULK1 complex, and significantly reduced the formation of tertiary lymphoid structures in salivary glands, a hallmark characteristic of this disease in patients [30]. The expression of BECN1, which was raised in diseased NOD.H-2h4 mice, a feature that could explain, at least in part, the clinical and biological beneficial effect of P140 in this mouse model.
Another peptide, the cell-penetrating peptide Tat-BECN1, rather acts as an activator of autophagy [36]. It was found to exert protection against viral and bacterial infection both in vitro and in vivo, and some effectiveness in other pathological conditions such as heart failure in response to cardiac hypertrophy, for example [4]. The activity of some rationally-designed analogs of Tat-BECN1 peptide, like Tat-D11 (Novus Biologicals), prove significantly more potent than the original sequence in different assays and indications. A peptide named humanin initially identified from surviving neurons in patients with Alzheimer's disease (AD), was described as a direct enhancer of CMA that acts by increasing substrate binding and translocation into lysosomes [37]. It interacts with HSP90 and stabilizes the binding of this chaperone to CMA cargos as they bind to the lysosomal Cells 2020, 9, 1321 4 of 19 membrane. It was shown that humanin exerts cardioprotective and neuroprotective properties in diseases such as AD, cardiovascular diseases, stroke, myocardial infarction, diabetes, and cancer. These peptides and other molecules that target specific autophagy elements represent potential therapeutics that might be successfully applied to treat patients affected by pathologies for which current drugs are clearly insufficient with severe secondary effects, or to individuals who are refractory to the existing medications.
When developing new chemical strategies of intervention that target autophagy, an important step is to demonstrate that in vivo, and not only in vitro or ex vivo, the molecule is effective on the expected autophagic target that has been identified in the upstream investigations. Indeed, in vivo, it may well be that the molecule induces its effect as thought, i.e., directly on the expected autophagy component but it might also act via an autophagy-independent mechanism or on an alternative metabolic system that in fine impact autophagy. It is effectively well known that numerous autophagy-related markers are also involved in other metabolic pathways. It is the case of BECN1, HSPA8, MTOR, and many others [4,23]. Yet, measuring autophagy in vivo or even in tissue samples (biopsies) collected from patients, for example, remains a challenge. Assessment of autophagic flux in whole organisms is even more challenging. Specific assays are limited and often remain non validated given the variety of tissues and organisms that are studied. The aim of this review is to gather useful information compiled from the existing literature and generated by our own experience, to analyze the validity of proposed methods and to discuss their potential and limitations. An emphasis is placed on microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3) that is central in the autophagy process and especially in macroautophagy. Present at low and variable levels of expression in a normal situation, its distribution is widespread, for example in the bone marrow, brain, heart, placenta, thyroid, bladder, and several other organs and tissues.

The Microtubule-Associated Protein 1A/1B-Light Chain 3 (MAP1LC3)
Together with sequestosome (SQSTM1)/p62, MAP1LC3 is the most widely used marker that is followed experimentally to evaluate the extent of autophagy in cells. MAP1LC3 is a member of the highly conserved ATG8 protein family. It was initially described in the yeast (S. cerevisiae) and later identified in mammals [38,39]. MAP1LC3 belongs to a large family of seven members composed of MAP1LC3A, 3B, 3B2, 3C, and the γ-aminobutyric acid (GABA)-receptor-associated proteins (GABARAP, GABARAPL1, GABARAPL2) proteins. The NMR structure of MAP1LC3B (residues 1-120) has been determined ( Figure 1) [40]. The members of this protein family share a common ubiquitin-like (Ubl) structures present at the C-terminus and two α-helices at their N-terminus, which are likely involved in protein-protein and lipid-protein interactions and post-translational modifications that occur during the autophagy process [41][42][43][44]. In the cytosol, newly synthesized MAP1LC3B is cleaved by the autophagy-related 4B cysteine peptidase (ATG4B) at the C-terminal glycine residues 120 leading to MAP1LC3-I ( Figure 1). Phosphatidylethanolamine (PE)-conjugation of MAP1LC3-I resulting in the lipidated MAP1LC3-II form requires ATG7 and ATG3, as well as the ATG12-ATG5-ATG16L1 complex [45][46][47]. Formation of the latter involves conjugation of ATG12 to ATG5, in a process mediated by ATG7 and ATG10, and further binding of the ATG12-ATG5 conjugate to an ATG16L1 dimer. In this process, ATG7 plays a central role as this E1-like activating enzyme enables both the lipidation of ATG8 proteins (MAP1LC3) and conjugation of ATG12 to ATG5. It is the only enzyme common to both conjugation pathways. At the isolation membrane and autophagosomes, MAP1LC3-II displays significant functions in the autophagy process, such as elongation, sealing isolation membranes, recognizing cargos, etc. [47]. The Ubl structures encompass β-strands with hydrophobic pockets, which are implicated in protein interactions. MAPL1LC3 is also present in the nucleus of a variety of cell types. In response to starvation, nuclear MAP1LC3 is deacetylated and trafficked out of the nucleus into the cytoplasm where it exerts its functions in autophagy. The protein is also enriched in nucleoli where it binds nuclear and nucleolar constituents, such as microtubule-associated protein 1B, tubulin, and ribosomal proteins. MAP1LC3 proteins interact with many cofactors and ligands. All interacting proteins display a common ubiquitin-binding domain (UBD) and a short hydrophobic LC3-interacting region (LIR) motif that contains an N-terminal sequence W (tryptophan)xxL (leucine). The W residue is surrounded by other aromatic residues, tyrosine (Y) and phenylalanine (F), acidic residues, and sometimes serine (S) and threonine (T) residues to compose a consensus W/F/YXXL/I/V motif. These residues interact with the basic residues present in the Ubl domain of MAP1LC3 via electrostatic bridges ( Figure 1). LIR motif-containing proteins encompass two hydrophobic residues, which are accommodated into the MAP1LC3 binding pockets [48]. The LIR motifs are post-translationally regulated and contribute to the different functions displayed by MAP1LC3 proteins, including selective autophagy process [48][49][50][51].
MAP1LC3 plays a key role in the autophagosome biogenesis machinery from the isolation membrane to the lysosomal stage. The recruitment of MAP1LC3 and other ATG proteins triggers vesicle expansion in a concerted manner and intervenes in the initial steps of membrane curvature. During this complex sequential process, MAP1LC3 is finely regulated by diverse post-translational modifications, which favor or on the contrary alter its activity. Thus, phosphorylation of MAP1LC3B at Thr 50 by serine/threonine-protein kinases STK3/STK4 is essential for the autophagosomelysosomal fusion process [52]. Phosphorylation at residue Ser 12 by phosphokinase A (PKA) keeps MAP1LC3B unavailable for conjugation to PE and inhibits the autophagy process [53]. This modification is essential for phagophore expansion; its failure leads to defects in autophagosome formation. During stress or starvation conditions, PKA activity is inhibited and MAP1LC3B remains available for conjugation. On the other hand, acetylation regulates the MAP1LC3B translocation from the nucleus to the cytosol. The reason for the presence of LC3B in the nucleus remains an unanswered question. Acetylation at residues Lys 49 and Lys 51 makes the MAP1LC3B protein confined to the nucleus and not available for cytosolic ligases [54]. Of note, Lys 49 and Lys 51 form ionic interactions with ligands, especially with the LIR motifs. In stress conditions, especially starvation, deacetylation of MAP1LC3B by sirtuin 1 results in the availability of MAP1LC3B for ligases [54]. MAP1LC3 proteins interact with many cofactors and ligands. All interacting proteins display a common ubiquitin-binding domain (UBD) and a short hydrophobic LC3-interacting region (LIR) motif that contains an N-terminal sequence W (tryptophan)xxL (leucine). The W residue is surrounded by other aromatic residues, tyrosine (Y) and phenylalanine (F), acidic residues, and sometimes serine (S) and threonine (T) residues to compose a consensus W/F/YXXL/I/V motif. These residues interact with the basic residues present in the Ubl domain of MAP1LC3 via electrostatic bridges ( Figure 1). LIR motif-containing proteins encompass two hydrophobic residues, which are accommodated into the MAP1LC3 binding pockets [48]. The LIR motifs are post-translationally regulated and contribute to the different functions displayed by MAP1LC3 proteins, including selective autophagy process [48][49][50][51].
MAP1LC3 plays a key role in the autophagosome biogenesis machinery from the isolation membrane to the lysosomal stage. The recruitment of MAP1LC3 and other ATG proteins triggers vesicle expansion in a concerted manner and intervenes in the initial steps of membrane curvature. During this complex sequential process, MAP1LC3 is finely regulated by diverse post-translational modifications, which favor or on the contrary alter its activity. Thus, phosphorylation of MAP1LC3B at Thr 50 by serine/threonine-protein kinases STK3/STK4 is essential for the autophagosome-lysosomal fusion process [52]. Phosphorylation at residue Ser 12 by phosphokinase A (PKA) keeps MAP1LC3B unavailable for conjugation to PE and inhibits the autophagy process [53]. This modification is essential for phagophore expansion; its failure leads to defects in autophagosome formation. During stress or starvation conditions, PKA activity is inhibited and MAP1LC3B remains available for conjugation. On the other hand, acetylation regulates the MAP1LC3B translocation from the nucleus to the cytosol. The reason for the presence of LC3B in the nucleus remains an unanswered question. Acetylation at residues Lys 49 and Lys 51 makes the MAP1LC3B protein confined to the nucleus and not available for cytosolic ligases [54]. Of note, Lys 49 and Lys 51 form ionic interactions with ligands, especially with the LIR motifs. In stress conditions, especially starvation, deacetylation of MAP1LC3B by sirtuin 1 results in the availability of MAP1LC3B for ligases [54].

Current MAP1LC3-Based Methods Designed for Autophagy Research
MAP1LC3-based assays are widely used in current autophagy research [22,[55][56][57][58]. In general, they offer the possibility to monitor the cellular autophagic activity in a routine manner using conventional laboratory equipment. A number of advantageous criteria, such as sensitivity, performance, specimen throughput, volume requirements, the limit of detection, ease of execution, instrument workspace, and the costs of equipment and disposal, are met. Commonly, the detection of processed MAP1LC3B-II by Western blot (Figure 2) or fluorescence studies, together with electron microscopy for autophagosome formation (Figure 3), is sufficient to qualify autophagy in cells.

Current MAP1LC3-Based Methods Designed for Autophagy Research
MAP1LC3-based assays are widely used in current autophagy research [22,[55][56][57][58]. In general, they offer the possibility to monitor the cellular autophagic activity in a routine manner using conventional laboratory equipment. A number of advantageous criteria, such as sensitivity, performance, specimen throughput, volume requirements, the limit of detection, ease of execution, instrument workspace, and the costs of equipment and disposal, are met. Commonly, the detection of processed MAP1LC3B-II by Western blot (Figure 2) or fluorescence studies, together with electron microscopy for autophagosome formation (Figure 3), is sufficient to qualify autophagy in cells. Without the use of Baf A1, it would be unpredictable to assess whether the compound is increasing the autophagosome synthesis or inhibiting the autophagosome degradation. Modified from [59]. See abbreviations in the abbreviations section.  Without the use of Baf A1, it would be unpredictable to assess whether the compound is increasing the autophagosome synthesis or inhibiting the autophagosome degradation. Modified from [59]. See abbreviations in the abbreviations section.

Current MAP1LC3-Based Methods Designed for Autophagy Research
MAP1LC3-based assays are widely used in current autophagy research [22,[55][56][57][58]. In general, they offer the possibility to monitor the cellular autophagic activity in a routine manner using conventional laboratory equipment. A number of advantageous criteria, such as sensitivity, performance, specimen throughput, volume requirements, the limit of detection, ease of execution, instrument workspace, and the costs of equipment and disposal, are met. Commonly, the detection of processed MAP1LC3B-II by Western blot (Figure 2) or fluorescence studies, together with electron microscopy for autophagosome formation (Figure 3), is sufficient to qualify autophagy in cells. Without the use of Baf A1, it would be unpredictable to assess whether the compound is increasing the autophagosome synthesis or inhibiting the autophagosome degradation. Modified from [59]. See abbreviations in the abbreviations section.  These basic methods, applied to primary cells and cell lines, are often adequate for the first series of screening tests, but they may appear insufficient to further develop knowledge for therapeutic purposes. Indeed, they suffer each from intrinsic limitations that can introduce errors in the data analyses and survey results (Table 2). Furthermore, and most importantly, they often apply to cells and not to tissues and organs and are routinely performed in vitro/ex vivo and not in vivo. Table 2. MAP1LC31-based standard assay for assessing autophagy and their limitations 1 .

Techniques Special Attention, Limitations and Pitfalls
Western blot assay ( Figure 2) • Examining the expression of MAP1LC3-I and -II in the presence and absence of protease inhibitors is an absolute requirement 2.  [56]. Quantification can also be made manually by a trained and blinded observer.

•
Discrimination of true autophagosomes devoid of MAP1LC3 aggregates, which are formed due to the aggregate prone proteins and autophagy-independent manner can be difficult.

•
In GFP-or mRFP-GFP-MAP1LC3 constructs, labelling may not give absolute results, especially if the pH of lysosomes is altered in pathological situations (as in lupus, for example, in which the mean lysosomal pH is raised [35]). • Use of samples with or without inhibitors should be maintained for the better comparison (except for a few probes, e.g., GFP-MAP1LC3-RFP-MAP1LC3∆G).

•
Assays based on the red fluorescent protein Keima cannot be used with fixed cells because the assay completely relies on lysosomal acidity [71].

Flow cytometry
• Detects the different forms of endogenous MAP1LC3 (incl. MAP1LC3-I, MAP1LC3-II) proteins without any discrimination. • Improved speed and statistical power when determining autophagic flux using tandem MAP1LC3 fusion proteins.
Multispectral imaging flow cytometry • Homogenous, mix-and-read assay that takes advantage of the required proximity of the donor and acceptor species for the generation of signal [77].

•
No direct information obtained on lysosomal degradation. • Time consuming. • Technical errors, e.g., poor-fixation, sometimes leads to over or under looking observations [78]. • Conventional methods, but not advanced electron microscopy methods, are not suitable to determine the volume and size of the inner cell compartments, due to the thin sections [60].

Long-lived protein degradation
• Proteasome inhibitors should be used to specify the action of autophagy.

•
Labelling efficiency is always a question, e.g., special culture media, without methionine, is required in non-radioactive labelling [79].

Techniques Special Attention, Limitations and Pitfalls
LDH sequestration assay • Lysosomal inhibitors always need to be used to measure autophagic flux [80].

Dextran sequestration assay
• Loading fluorescently labelled dextran into cells is a delicate operation [81,82]. 1 A few other conventional methods and/or nonspecific methods, such as isotope release from long-lived protein degradation (radiolabeling long-lived proteins with radioactive amino acid residues, such as [ 14 C]-leucine, [ 3 H]-leucine, [ 14 C]-valine, or [ 35 S]-methionine) and LDH assay (using LDH as a cargo) are not reviewed here as they are not routinely used. 2 We are well aware that determining the ATG protein levels or the number of autophagosomes alone does not provide the overall estimation of autophagic activity since the process is very dynamic, and MAP1LC3-II can quickly degrade within the lysosomes. In fact, the two opposite scenarios, namely induction of autophagy or blockade in the downstream steps of autophagy leading to defective degradation, can result in an increased number of autophagosomes. Therefore, it is necessary to evaluate the autophagic flux by measuring the level of expression of MAP1LC3-I and -II in the presence of lysosomal inhibitors such as bafilomycin A1 or others. See abbreviations in the abbreviations section.
In general, these methods have the capability for a high sample throughput and are used to sift large numbers of such samples for further application. As indicated, however, they also have some disadvantages and intrinsic limitations. The results that are generated to support possible changes in autophagy processes have also to be distinguished from effects induced by the many other forms of cell death [22,83].

MAP1LC3-Based Methods Designed for Studying Autophagy in Tissues
Several methods have been described to detect MAP1LC3 directly in fresh and fixed tissues collected from patients and experimental animal models. They are listed in Table 3 with their respective advantages and limitations (see also the Box 1). Table 3. MAP1LC3-based methods to measure autophagy in biopsies.

Immunohistochemistry
• High throughput analysis of MAP1LC3 localization in tissue arrays.

•
Availability of fixed tissues in the clinic. • Co-localization with additional autophagy-related proteins can be analyzed.
• Availability of MAP1LC3 isoform specific antibodies with sufficient sensitivity for FFPE tissue sections. • Quantifying MAP1LC3 punctae needs experienced pathologist.
• A lot of tissue is needed to extract enough protein.

•
Requires protein extraction from a cell mixture.

•
Isolation of pure cell populations from the tissues would be needed to analyze cell-specific levels of MAP1LC3 expression.

•
No information on MAP1LC3 localization.
In-situ hybridization [85] • Highly specific for MAP1LC3 isoforms. • Allows to assess MAP1LC3 isoform expression levels in different cell types.
• MAP1LC3 mRNA expression is not a "marker" of autophagy activity per se. Specific considerations that apply when organs/tissue are studied -The autophagic process (type, regulation, intensity) can differ from cell to cell, organ to organ/tissue in the same individual. -Many fluorescence methods used in autophagy monitoring require specialized fixation methods, fresh tissue preparation, and rapid visualization. Most of the classical methods are not applicable in the case of the brain.
Examples are highlighted in Figure 4 using mouse colonic tissues, and human lung cancer tissues and cell lines.
A variety of suppliers provide adequate antibodies that should be evaluated and finely calibrated in each cell/tissue and methodological condition to gain an optimal signal/background ratio. A non-exhaustive list is given in Table 4.
As indicated, although MAP1LC3-II has been widely used as a marker to detect autophagy in various tissue samples from patients (biopsies or extraction of tissues at autopsy) or animal models, these assays cannot provide information on the dynamics of autophagy process and flux.
• Allows to assess MAP1LC3 isoform expression levels in different cell types.
• MAP1LC3 mRNA expression is not a "marker" of autophagy activity per se.
• One needs to assume that MAP1LC3 mRNA levels correlate with protein expression.
See abbreviations in the abbreviations section. MAP1LC3A/B immunohistochemistry staining of FFPE-H1299 NSCLC cells that were either left untreated (Ctrl) or treated with everolimus, a selective inhibitor of MTOR activity used to stimulate autophagy. Detailed instructions on how the quantification of LC3B dot formation is done can be found in [84,86,87]. See abbreviations in the abbreviations section.
A variety of suppliers provide adequate antibodies that should be evaluated and finely calibrated in each cell/tissue and methodological condition to gain an optimal signal/background ratio. A nonexhaustive list is given in Table 4. Detecting co-localization of key autophagy proteins or post-translational modifications associated with an active autophagy state might represent new approaches to better describe autophagic activity in tissue. In addition, the quality of the tissue materials from patients and animals, e.g., the time from removing the specimen to tissue fixation, is key to adequately assess autophagy. Clinical studies sometimes rely on quantifying MAP1LC3 staining intensity rather than analyzing MAP1LC3 dot formation to evaluate autophagic activity in archived patient samples. This is clearly less conclusive and should be avoided. Analyzing MAP1LC3 punctae formation, although not measuring the autophagic flux per se, better represents increased autophagic activity ( Figure 4B,C) [87]. Improved and new techniques are critical to enabling autophagy flux analyses in the human tissues. Table 4. Non-exhaustive list of MAP1LC3 antibodies.

MAP1LC3-Based Methods Designed for Autophagy In Vivo
A number of approaches have been described to detect MAP1LC3 in living experimental animals. They are listed in Table 5 with respective performances and merits.
Despite the availability of various approaches for analyzing the autophagy in vivo in experimental models, and their potential use to screen therapeutic molecules and study the fundamental process of autophagy, their translation into the clinic remains limited. Further research is necessary to develop suitable probes that are safer and can be used in humans to analyze autophagy in vivo.

Method Advantages Limitations
Transgenic mice expressing GFP-MAP1LC3 and fluorescence microscopy Allow the formation of autophagosomes to be studied. Used to study macroautophagy and mitophagy.
Do not permit the formation of autolysosome to be studied as GFP loses its fluorescence at acidic pH in lysosomes. Other transgenic mice are required [88] No measurement of autophagic flux. Cells possess auto-fluorescent punctate structures such as lipofuscin that is detectable in the green spectra. Always compare to non-transgenic control littermates [66]. Measure the basal (low) and induced autophagy flux in embryos and tissues of zebrafish and mice [70]. 1 SBI-0206965 (Adooq Bioscience), a potent and selective inhibitor of ULK1, can be used to inhibit autophagy in vivo; is given intraperitoneally into mice at 2 mg/kg body weight in DMSO, once per day for 7 days [89]; other autophagy blockers can be used in vivo as control, e.g., CQ/HCQ, NH 4 Cl, bafilomycin A1 (in certain strict conditions), colchicine, vinblastine, and the inhibitor of lysosomal enzymes leupeptide [66]. 2 Measurement of chaperone-mediated autophagy (CMA) activity in vivo has been described [90]. See abbreviations in the abbreviations section.

Conclusions
This short survey clearly identifies the crucial need for developing more performant assays permitting to screen autophagy regulator molecules in vivo. In view of the lack of suitable techniques to analyze autophagy flux in vivo in humans, the best possible solution would be to investigate autophagy flux by using cells derived from the affected organs/tissues of the patients with aberrations in the autophagy. Alternatively, induced pluripotent stem cell technology (3D tissue cultures or organoids) could be exploited in order to reprogram human somatic cells to obtain disease-relevant cell types for the investigations. Genome-wide studies have reported several disease-associated loci and genes that affect autophagy [4,91]. Though ex vivo, these cells provide an opportunity to investigate the role of autophagy in the pathogenesis of diseases and to get a clue on if therapeutic molecules that are deemed beneficial in the animal models could be used in the patients.
It is also important that MAP1LC3-based analyses need to be complemented with lysosomal function to confirm the intact autophagy process. Though MAP1LC3 analyses indicate the autophagy process, the functional intactness of lysosomes is critical to complete the process via degradation of macromolecules and damaged cellular components. In fact, several muscular disorders such as X-linked myopathy with excessive autophagy, inclusion body myositis (IBM), IBM-associated with Paget's disease of the bone, frontotemporal dementia, and amyotrophic lateral sclerosis are associated with reduced proteolytic activity of lysosomes through enhanced autophagosome biogenesis was observed [92,93]. This feature has also been observed in lupus settings (Wang and Muller, unpublished data).
We deliberately focused this article on MAP1LC3-based analyses and macroautophagy. We listed the different ways of MAP1LC3 quantification in different settings and the arguments pro and against these measurements (see also Box 1 for recommendation). We emphasized here the importance of complementing MAP1LC3 autophagy measurements with nonMAP1LC3-based techniques. Other markers should be systematically followed such as SQSTM1, BECLIN-1, WIPI-1, ATG5/12, ATG14, ATG16L1, VPS34, LAMP2A, and others if possible, to support the results obtained with MAP1LC3. It is also important to distinguish the noncanonical autophagy process known as LC3-associated phagocytosis (LAP) from the canonical autophagy process. LAP and canonical autophagy processes do not require hierarchical intervention of all of the ATG proteins [94]. Both pathways do not play the same functions in cellular homeostasis and physiology, and therefore differentiating both processes can be highly relevant [95]. Recent studies on monocyte/macrophages revealed that LAP has a significant role in phenotype differentiation, particularly on the anti-inflammatory phenotype [96]. In addition to its role in regulating autoimmune response [97], LAP-mediated protective response against hepatic and systemic inflammation are also documented [98].
As reviewed in this article, biochemical assays, electron microscopy, and light microscopy are useful in assessing the autophagic process, however, these assays are cell type-, context-, and time-dependent. Immunologists or cell biologists who have equipped the laboratory with many techniques, should consider using as many markers possible and also, at least, two independent techniques to confirm the autophagic process. Autophagic flux analyses are especially recommended. Despite advancements in the methods to measure autophagy, there is still a lack of completeness, which should be answered with future developments. Novel methods, both in vitro and especially in vivo, to detect changes in the autophagy process in living tissues, are particularly awaited. They are decisive to discover and develop new, more effective drugs that target the autophagy machinery for patient care.  Conflicts of Interest: S.M. is co-inventor of CNRS-ImmuPharma patents on P140 peptide; has received research funds (paid to institution). The other authors declare no conflicts of interest. All declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Glossary
Amphisomes are single-membrane compartment issue from the fusion of autophagosomes and endosomes Autolysosomes are single-membered acidic vesicle formed by the fusion of autophagosome and lysosome Autophagosomes are well discriminated double-membrane vesicles in which intracellular material (incl. organelles and fragments of organelles) are sequestered and delivered to the lysosome for degradation Endosomes are single-membrane compartment formed by the process of endocytosis

Lysosomes
are digestive organelles of the cell, which bear an acidic environment (pH 4.5 to 5.0) to degrade material via different processes

v-ATPases
are membrane-associated proton pumps, which maintain the acidic environment in organelles, such as the endosomes, lysosomes, trans-Golgi network, and secretory granules.