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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4−/− (Ipaf−/−), and caspase-1−/− derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7−/− macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.

A general, combinatorial library method for the rapid identification of high-affinity peptide ligands of protein modular domains is reported. The validity of this method has been demonstrated by determining the sequence specificity of four Src homology 2 (SH2) domains derived from protein tyrosine phosphatase SHP-1 and SHP-2 and inositol phosphatase SHIP. A phosphotyrosyl (pY) peptide library was screened against the SH2 domains, and the beads that carry high-affinity ligands of the SH2 domains were identified and peptides were sequenced by partial Edman degradation and mass spectrometry. The results reveal that the N-terminal SH2 domain of SHP-2 is capable of recognizing four different classes of pY peptides. Binding competition studies suggest that the four classes of pY peptides all bind to the same site on the SH2 domain surface. The C-terminal SH2 domains of SHP-1 and SHP-2 and the SHIP SH2 domain each bind to pY peptides of a single consensus sequence. Database searches using the consensus sequences identified most of the known as well as many potential interacting proteins of SHP-1 and/or SHP-2. Several proteins are found to bind to the SH2 domains of SHP-1 and SHP-2 through a new, nonclassical ITIM motif, (V/I/L)XpY(M/L/F)XP, which corresponds to the class IV peptides selected from the pY library. The combinatorial library method should be generally applicable to other protein domains.

IntroductionHuman natural killer (NK) cells are CD56 ϩ CD3 Ϫ large granular lymphocytes of the innate immune system. 1,2 NK cells participate in early responses against infection or malignant transformation. In addition to their potent cytolytic activity, NK cells have an important immunoregulatory function in that they produce cytokines and chemokines when activated. In particular, NK cells produce IFN-␥, a critical cytokine for the clearance of infectious pathogens and tumor surveillance, 3 in response to a wide variety of stimuli, including both soluble factors and cellular interactions. 4,5 Dendritic cells and monocytes stimulated with bacterial cell wall components release monokines such as IL-12 and IL-18, which synergistically induce rapid and robust production of IFN-␥ by NK cells. 6 NK cells also express the low-affinity receptor for the Fc fragment of immunoglobulin (Ig)G (Fc␥RIIIA, CD16), which is the activating receptor required for triggering antibody dependent cellular cytotoxicity (ADCC) as well as the induction of IFN-␥. 7 IL-12 monokine stimulation in combination with CD16 activation induces a synergistic induction of IFN-␥ in NK cells, but to a lesser extent than does IL-12 and IL-18 costimulation. 8 This observation has recently been shown to have implications in the antibody therapy of breast cancer patients. In fact, the antitumor actions of the anti-HER2 monoclonal antibody trastuzumab are enhanced by IL-12 treatment in vivo, and this effect is dependent on NK cell production of IFN-␥. 9 The regulation of NK cell IFN-␥ production involves positive and negative mediators, such as kinases and phosphatases, as well as transcription factors. 10-14 SHIP1 is a hematopoietic cell specific 5Ј inositol phosphatase. 15 We have previously shown that SHIP1 is expressed differentially in CD56 bright and CD56 dim NK cell subsets, and is negatively modulated by the costimulation of IL-12 and IL-18. 13 SHIP1, by dampening the PI3K pathway, is able to negatively regulate IFN-␥ production by monokines and CD16 stimulation in both human and mouse NK cells. 13,16 MicroRNAs (miRs) are a highly conserved class of small, noncoding RNAs with important regulatory functions in proliferation, differentiation, signal transduction, immune responses, and carcinogenesis. 17 miRs regulate gene expression posttranscriptionally by forming imperfect base pairs with sequences in the 3Ј untranslated region (UTR) of genes. In turn, this prevents protein accumulation by repressing translation or by inducing mRNA degradation. 18 Recently, a general role of miRs in regulation of NK cell activation, survival, and function has been shown using conditional deletion of Dicer or Dgcr8. 19 A specific role of miR-150 in regulating development and maturation of mouse NK cells has also been reported. 20 Further, it has been shown that miR-181 promotes human NK cell development by regulating Notch signaling. 21 26 Of interest, miR-155 is overexpressed in NK-cell lymphoma/leukemia and this correlates with low levels of SHIP1 expression and u...

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.

Francisella tularensis is a gram-negative facultative bacterium that causes the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. In order to help understand the mechanisms by which this occurs, we performed Affymetrix microarray analysis on transcripts from blood monocytes infected with the virulent Type A Schu S4 strain. Results showed that expression of several host response genes were reduced such as those associated with interferon signaling, Toll-like receptor signaling, autophagy and phagocytosis. When compared to microarrays from monocytes infected with the less virulent F. tularensis subsp. novicida, we found qualitative differences and also a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes in the Schu S4 strain. Notably, the PI3K / Akt1 pathway appeared specifically down-regulated following Schu S4 infection and a concomitantly lower cytokine response was observed. This study identifies several new factors potentially important in host cell subversion by the virulent Type A F. tularensis that may serve as novel targets for drug discovery.

The Gram-negative bacterium Francisella novicida infects primarily monocytes/macrophages and is highly virulent in mice. Macrophages respond by producing inflammatory cytokines that confer immunity against the infection. However, the molecular details of host cell response to Francisella infection are poorly understood. In this study, we demonstrate that F. novicida infection of murine macrophages induces the activation of Akt. Inhibition of Akt significantly decreases proinflammatory cytokine production in infected macrophages, whereas production of the anti-inflammatory cytokine IL-10 is enhanced. Analysis of the mechanism of Akt influence on cytokine response demonstrated that Akt promotes NF-κB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with phagosomal escape. Infection with mglA mutants of F. novicida elicited sustained activation of Akt in comparison to cells infected with wild-type F. novicida. Concomitantly, there was significantly higher proinflammatory cytokine production and lower IL-10 production in cells infected with the mglA mutant. Finally, transgenic animals expressing constitutively active Akt displayed a survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida. Together, these observations indicate that Akt promotes proinflammatory cytokine production by F. novicida-infected macrophages through its influence on NF-κB, thereby contributing to immunity against F. novicida infection.

Purpose: Natural killer (NK) cells express an activating Fc receptor (FcgRIIIa) that mediates antibody-dependent cellular cytotoxicity (ADCC) and production of immune modulatory cytokines in response to antibody-coated targets. Cetuximab is a therapeutic monoclonal antibody directed against the HER1 antigen. We hypothesized that the NK cell response to cetuximab-coated tumor cells could be enhanced by the administration of NK cell^stimulatory cytokines. Experimental Design: Human NK cells stimulated with cetuximab-coated tumor cells and interleukin-2 (IL-2), IL-12, or IL-21 were assessed for ADCC and secretion of IFN-g and T cell^recruiting chemokines. IL-21 and cetuximab were given to nude mice bearing HER1-positive xenografts. Results: Stimulation of human NK cells with cetuximab-coated tumor cells and IL-2, IL-12, or IL-21 resulted in 3-fold to 10-fold higher IFN-g production than was observed with either agent alone. NK cell^derived IFN-g significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1a, and RANTES) that could direct the migration of naive and activated Tcells. IL-2, IL-12, and IL-21enhanced NK cell ADCC against tumor cells treated with cetuximab. The combination of cetuximab, trastuzumab (an anti-HER2 monoclonal antibody), and IL-21 mediated greater NK cell cytokine secretion and ADCC than any agent alone. Furthermore, administration of IL-21enhanced the effects of cetuximab in a murine tumor model. Conclusions: These results show that cetuximab-mediated NK cell activity can be significantly enhanced in the presence of NK cell^stimulatory cytokines. These factors, therefore, may be effective adjuvants to administer, in combination with cetuximab, to patients with HER1-positive malignancies.