In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor.

In addition to their role in binding antigen, antibodies can regulate immune responses through interacting with Fc receptors (FcRs). In recent years, significant progress has been made in understanding the mechanisms that regulate the activity of IgG antibodies in vivo. In this Review, we discuss recent studies addressing the multifaceted roles of FcRs for IgG (FcgammaRs) in the immune system and how this knowledge could be translated into novel therapeutic strategies to treat human autoimmune, infectious or malignant diseases.

/pdf/fcg-receptors-as-regulators-of-immune-responses-46isonwygz.pdf

Fcγ receptors as regulators of immune responses

PLoS BIOLOGY Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor Jean-Philippe Coppe 1 , Christopher K. Patil 1[ , Francis Rodier 1,2[ , Yu Sun 3 , Denise P. Mun oz 1,2 , Joshua Goldstein 1¤ , Peter S. Nelson 3 , Pierre-Yves Desprez 1,4 , Judith Campisi 1,2* 1 Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America, 2 Buck Institute for Age Research, Novato, California, United States of America, 3 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 4 California Pacific Medical Center Research Institute, San Francisco, California, United States of America Cellular senescence suppresses cancer by arresting cell proliferation, essentially permanently, in response to oncogenic stimuli, including genotoxic stress. We modified the use of antibody arrays to provide a quantitative assessment of factors secreted by senescent cells. We show that human cells induced to senesce by genotoxic stress secrete myriad factors associated with inflammation and malignancy. This senescence-associated secretory phenotype (SASP) developed slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. Remarkably similar SASPs developed in normal fibroblasts, normal epithelial cells, and epithelial tumor cells after genotoxic stress in culture, and in epithelial tumor cells in vivo after treatment of prostate cancer patients with DNA- damaging chemotherapy. In cultured premalignant epithelial cells, SASPs induced an epithelial–mesenchyme transition and invasiveness, hallmarks of malignancy, by a paracrine mechanism that depended largely on the SASP factors interleukin (IL)-6 and IL-8. Strikingly, two manipulations markedly amplified, and accelerated development of, the SASPs: oncogenic RAS expression, which causes genotoxic stress and senescence in normal cells, and functional loss of the p53 tumor suppressor protein. Both loss of p53 and gain of oncogenic RAS also exacerbated the promalignant paracrine activities of the SASPs. Our findings define a central feature of genotoxic stress-induced senescence. Moreover, they suggest a cell-nonautonomous mechanism by which p53 can restrain, and oncogenic RAS can promote, the development of age-related cancer by altering the tissue microenvironment. Citation: Coppe JP, Patil CK, Rodier F, Sun Y, Mun oz DP, et al. (2008) Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor. PLoS Biol 6(12): e301. doi:10.1371/journal.pbio.0060301 Introduction Cancer is a multistep disease in which cells acquire increasingly malignant phenotypes. These phenotypes are acquired in part by somatic mutations, which derange normal controls over cell proliferation (growth), survival, invasion, and other processes important for malignant tumorigenesis [1]. In addition, there is increasing evidence that the tissue microenvironment is an important determinant of whether and how malignancies develop [2,3]. Normal tissue environ- ments tend to suppress malignant phenotypes, whereas abnormal tissue environments such at those caused by inﬂammation can promote cancer progression. Cancer development is restrained by a variety of tumor suppressor genes. Some of these genes permanently arrest the growth of cells at risk for neoplastic transformation, a process termed cellular senescence [4–6]. Two tumor suppressor pathways, controlled by the p53 and p16INK4a/pRB proteins, regulate senescence responses. Both pathways integrate multiple aspects of cellular physiology and direct cell fate towards survival, death, proliferation, or growth arrest, depending on the context [7,8]. Several lines of evidence indicate that cellular senescence is a potent tumor-suppressive mechanism [4,9,10]. Many poten- tially oncogenic stimuli (e.g., dysfunctional telomeres, DNA PLoS Biology | www.plosbiology.org damage, and certain oncogenes) induce senescence [6,11]. Moreover, mutations that dampen the p53 or p16INK4a/pRB pathways confer resistance to senescence and greatly increase cancer risk [12,13]. Most cancers harbor mutations in one or both of these pathways [14,15]. Lastly, in mice and humans, a senescence response to strong mitogenic signals, such as those delivered by certain oncogenes, prevents premalignant lesions from progressing to malignant cancers [16–19]. Academic Editor: Julian Downward, Cancer Research UK, United Kingdom Received June 27, 2008; Accepted October 22, 2008; Published December 2, 2008 Copyright: O 2008 Coppe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: CM, conditioned medium; DDR, DNA damage response; ELISA, enzyme-linked immunosorbent assay; EMT, epithelial–mesenchymal transition; GSE, genetic suppressor element; IL, interleukin; MIT, mitoxantrone; PRE, presenescent; PrEC, normal human prostate epithelial cell; REP, replicative exhaustion; SASP, senescence-associated secretory phenotype; SEN, senescent; shRNA, short hairpin RNA; XRA, X-irradiation * To whom correspondence should be addressed. E-mail: jcampisi@lbl.gov [ These authors contributed equally to this work. ¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America December 2008 | Volume 6 | Issue 12 | e301

Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor

Group A streptococci are model extracellular gram-positive pathogens responsible for pharyngitis, impetigo, rheumatic fever, and acute glomerulonephritis. A resurgence of invasive streptococcal diseases and rheumatic fever has appeared in outbreaks over the past 10 years, with a predominant M1 serotype as well as others identified with the outbreaks. emm (M protein) gene sequencing has changed serotyping, and new virulence genes and new virulence regulatory networks have been defined. The emm gene superfamily has expanded to include antiphagocytic molecules and immunoglobulin-binding proteins with common structural features. At least nine superantigens have been characterized, all of which may contribute to toxic streptococcal syndrome. An emerging theme is the dichotomy between skin and throat strains in their epidemiology and genetic makeup. Eleven adhesins have been reported, and surface plasmin-binding proteins have been defined. The strong resistance of the group A streptococcus to phagocytosis is related to factor H and fibrinogen binding by M protein and to disarming complement component C5a by the C5a peptidase. Molecular mimicry appears to play a role in autoimmune mechanisms involved in rheumatic fever, while nephritis strain-associated proteins may lead to immune-mediated acute glomerulonephritis. Vaccine strategies have focused on recombinant M protein and C5a peptidase vaccines, and mucosal vaccine delivery systems are under investigation.

Pathogenesis of Group A Streptococcal Infections

The precise role of B cells in systemic autoimmunity is incompletely understood. Although B cells are necessary for expression of disease (Chan, O., and M.J. Shlomchik. 1998. J. Immunol. 160:51–59, and Shlomchik, M.J., M.P. Madaio, D. Ni, M. Trounstine, and D. Huszar. 1994. J. Exp. Med. 180:1295–1306), it is unclear whether autoantibody production, antigen presentation, and/or other B cell functions are required for the complete pathologic phenotype. To address this issue, two experimental approaches were used. In the first, the individual contributions of circulating antibodies and B cells were analyzed using MRL/MpJ- Faslpr (MRL/ lpr ) mice that expressed a mutant transgene encoding surface immunoglobulin (Ig), but which did not permit the secretion of circulating Ig. These mice developed nephritis, characterized by cellular infiltration within the kidney, indicating that B cells themselves, without soluble autoantibody production, exert a pathogenic role. The results indicate that, independent of serum autoantibody, functional B cells expressing surface Ig are essential for disease expression, either by serving as antigen-presenting cells for antigen-specific, autoreactive T cells, or by contributing directly to local inflammation.

https://rupress.org/jem/article-pdf/189/10/1639/1119376/98-2243.pdf

A Novel Mouse with B Cells but Lacking Serum Antibody Reveals an Antibody-independent Role for B Cells in Murine Lupus

The primary roles of T cells and B cells in the initiation of systemic autoimmunity are unclear. To investigate the role of B cells, we crossed the "Jh knockout" mutation onto the autoimmune lpr/lpr background. Animals homozygous for both traits were obtained. As expected, these animals lack B cells. These animals also show no signs of autoimmune kidney destruction nor vasculitis, in spite of carrying the lpr/lpr mutation. In contrast, lpr/lpr littermates that had B cells had severe nephritis and vasculitis, as well as autoantibodies. These results demonstrate a primary role for B cells and/or (auto)antibodies in initiating several types of autoimmune-mediated tissue destruction. The implications of this finding for models and therapy of autoimmunity are discussed.

https://rupress.org/jem/article-pdf/180/4/1295/1105727/1295.pdf

The role of B cells in lpr/lpr-induced autoimmunity.

A standard view of B cells in systemic autoimmunity is that they promote lupus by producing autoantibodies (autoAb). However, this view is incomplete because recent studies have revealed that autoimmune disease can be dissociated from autoAb deposition. Furthermore, the spontaneous T-cell activation and organ infiltration in systemic lupus erythematosus patients and animal models are difficult to explain entirely via a direct autoAb-mediated mechanism. In this review, we describe work addressing the B-cell functions of autoantigen presentation and autoAb production in lupus pathogenesis. In the JHD-MRL-Faslpr strain (JHD/lpr), a B-cell-deficient version of the lupus-prone MRL-Faslpr (MRL/lpr) mouse, spontaneous nephritis and dermatitis is abrogated, demonstrating that B cells have a primary role in disease. B cells play a similar role in Fas-intact, lupus-prone MRL mice. To address the role of autoantigen presentation, we analyzed transgenic mice which have B cells that cannot secrete immunoglobulin (mIgM transgenic mice). The restoration of B cells without antibody caused substantial interstitial nephritis and vasculitis although less marked than the intact MRL/lpr controls. To address the role of autoAb, we infused serum from aged MRL/lpr mice into JHD/lpr mice. At most, mild to no nephritis was observed in the infused mice. These results indicate that B cells are promoting autoimmunity in mechanisms other than autoAb secretion, and we describe a model depicting these B-cell roles in the context of other inflammatory events in lupus.

The central and multiple roles of B cells in lupus pathogenesis

The only established role for complement in mediating immunologic renal disease involves elaboration of leukochemotactic factors and neutrophil-dependent glomerular injury. In the passive Heymann nephritis (PHN) model of experimental membranous nephropathy, rats injected with sheep antibody to rat proximal tubular brush border antigen (Fx1A) form subepithelial deposits of sheep IgG and rat complement (C3), and develop heavy proteinuria after 5 d without glomerular inflammatory changes. To study the role of complement in mediating proteinuria in PHN, 16 rats were treated daily with cobra venom factor from before antibody injection to maintain C3 levels at 0.5). Nephritogenic doses of both the noncomplement-fixing F(ab')2 portion and the gamma 2 subclass of anti-Fx1A IgG produced subepithelial deposits of immunoglobulin without C3, but proteinuria did not occur despite glomerular deposition of up to 70 microgram/2 kidneys of gamma 2. However, glomerular deposition of as little as 60 microgram of gamma 1 produced C3 fixation in vivo and heavy proteinuria. No neutrophil exudate could be detected histologically in PHN from the time of antibody injection through development of proteinuria. Proteinuria in five PHN rats depleted of neutrophils to 0.5). These results demonstrate that proteinuria in the PHN model of membranous nephropathy is complement-dependent and strongly suggest a neutrophil-independent mechanism. Thus a new role for the complement system in mediating immunologic glomerular injury is identified.

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A new role for complement in experimental membranous nephropathy in rats.

Idiotypic cross-reactions were evaluated in 60 polynucleotide-binding monoclonal lupus autoantibodies produced by human-human hybridomas that were derived from seven unrelated patients with SLE. Three antiidiotype reagents were prepared by immunization of rabbits or a mouse with monoclonal autoantibodies from two patients. Binding of the three reagents to their corresponding idiotypes was inhibited by one or more polynucleotides, an indication that the antiidiotypes reacted with the variable regions of the autoantibodies. Each antiidiotype appeared to detect a different idiotypic determinant. Of the 60 monoclonal autoantibodies tested, 40 reacted in one or more competitive immunoassays; 15 reacted with one antiidiotype, 10 reacted with two antiidiotypes and 15 reacted with three antiidiotypes. A monoclonal antiidiotype reagent cross-reacted with autoantibodies from six of the seven patients. The idiotypic cross-reactions of immunoglobulins from unrelated patients suggest that the autoantibodies are derived from related families of germ line genes that are expressed by patients with SLE.

https://rupress.org/jem/article-pdf/158/3/718/1093850/718.pdf

Michael P. Madaio

Papers

A Novel Mouse with B Cells but Lacking Serum Antibody Reveals an Antibody-independent Role for B Cells in Murine Lupus

The role of B cells in lpr/lpr-induced autoimmunity.

The central and multiple roles of B cells in lupus pathogenesis

A new role for complement in experimental membranous nephropathy in rats.

Idiotypic cross-reactions of monoclonal human lupus autoantibodies.