TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation.

Autophagy is a fundamental biological process of the eukaryotic cell contributing to diverse cellular and physiological functions including cell-autonomous defense against intracellular pathogens. Here, we screened the Rab family of membrane trafficking regulators for effects on autophagic elimination of Mycobacterium tuberculosis var. bovis BCG and found that Rab8b and its downstream interacting partner, innate immunity regulator TBK-1, are required for autophagic elimination of mycobacteria in macrophages. TBK-1 was necessary for autophagic maturation. TBK-1 coordinated assembly and function of the autophagic machinery and phosphorylated the autophagic adaptor p62 (sequestosome 1) on Ser-403, a residue essential for its role in autophagic clearance. A key proinflammatory cytokine, IL-1ß, induced autophagy leading to autophagic killing of mycobacteria in macrophages, and this IL-1ß activity was dependent on TBK-1. Thus, TBK-1 is a key regulator of immunological autophagy and is responsible for the maturation of autophagosomes into lytic bactericidal organelles.

Brightfield multiplex immunohistochemistry with multispectral imaging.

Brightfield microscopy is the preferred method of pathologists for diagnosing solid tumors, utilizing common staining techniques such as hematoxylin and eosin staining and immunohistochemistry (IHC). However, as our understanding of the complex tumor microenvironment grows, there is increasing demand for multiplexed biomarker detection. Currently, multiplexed IHC assays are almost exclusively based on immunofluorescence because brightfield techniques are limited by the broad spectral absorption of chromogens and a reliance on conventional 3-channel color cameras. In this work, we overcome these limitations by combining new chromogens possessing narrow absorbance bands with matched illumination channels and monochrome imaging. Multiplex IHC was performed using four or five covalently deposited chromogens and hematoxylin nuclear stain to preserve morphological context and detail. Brightfield illumination was provided with a tungsten lamp/filter wheel combination or filtered light emitting diodes to provide up to 12 illumination wavelengths. In addition, an automated rapid imaging system was developed, using a synchronized 12-LED illuminator, that could capture images at all wavelengths in under 1 s. In one example, a four-biomarker multiplex assay was designed and used to distinguish regions of adenocarcinoma and squamous cell carcinoma in non-small cell lung cancer. The technology was also validated with a five-biomarker assay in prostate cancer. Spectrally unmixed images of each biomarker demonstrated concordant expression patterns with DAB single stain on serial sections, indicating faithful identification of each biomarker. In each assay, all chromogens were well resolved by spectral unmixing to remove spectral crosstalk. While further characterization and refinement of the assay, and improvements in automation and user interface are necessary for pathologist acceptance, this approach to multiplex IHC and multispectral imaging has the potential to accelerate adoption of multiplexing by combining the medical value of high-order multiplexing with the speed, pathologist familiarity, and broadly established clinical utility of brightfield microscopy.

Chromogenic immunohistochemical quadruplex provides accurate diagnostic differentiation of non-small cell lung cancer.

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Delivery of cytosolic components by autophagic adaptor protein p62 endows autophagosomes with unique antimicrobial properties.

Autophagy allows cells to self-digest portions of their own cytoplasm for a multitude of physiological purposes, including innate and adaptive immunity functions. In one of its innate immunity manifestations, autophagy, is known to contribute to the killing of intracellular microbes, including Mycobacterium tuberculosis, although the molecular mechanisms have been unclear. Here, we delineated sequential steps of the autophagic pathway necessary to control intracellular M. tuberculosis and found that in addition to autophagy initiation and maturation, an accessory autophagy-targeting molecule p62 (A170 or SQSTM1) was required for mycobactericidal activity. The p62 adaptor protein delivered specific ribosomal and bulk ubiquitinated cytosolic proteins to autolysosomes where they were proteolytically converted into products capable of killing M. tuberculosis. Thus, p62 brings cytosolic proteins to autolysosomes where they are processed from innocuous precursors into neo-antimicrobial peptides, explaining in part the unique bactericidal properties of autophagic organelles.

Covalently deposited dyes: a new chromogen paradigm that facilitates analysis of multiple biomarkers in situ.

Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.

In Situ Detection of Protein Complexes and Modifications by Chemical Ligation Proximity Assay.

Protein function is often regulated by protein-protein interactions and post-translational modifications. Detection of these important biological phenomena in fixed biological samples could serve as an invaluable tool in biomedical research, drug development, as well as clinical cancer diagnostics and prognostics. We report here a novel methodology which utilizes unique antibody bioconjugates capable of forming proximity induced chemical ligation to enable in situ detection of proximal targets in fixed biological samples. Using this new methodology, we demonstrate in situ visualization of various protein heterodimers/complexes and post-translational modifications such as phosphorylation and ubiquitination. This new method offers high specificity, sensitivity, flexibility, and ease of use. In addition, the assay preserves critical contextual and heterogeneity information on biomarkers in clinically relevant samples.

Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy.

The majority of studies on autophagy, a cytoplasmic homeostasis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.

Higher order Rab programming in phagolysosome biogenesis.

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.

A novel 5x multiplex immunohistochemical staining reveals PSMA as a helpful marker in prostate cancer with low p504s expression.

The ability to combine multiple immunohistochemical (IHC) markers within a single tissue section facilitates the evaluation and detection of co-expressions, while saving tissue. A newly developed 5x multiplex (MPX) IHC staining of five different IHC markers (Basal cell cocktail (34ßE12 + p63), p504s (SP116), ERG (EPR3864), Ki-67 (30-9), PSMA (EP192)) was applied on whole sections of n = 37 radical prostatectomies (RPE) including normal and cancerous tissue. Four different colors including brown, magenta, yellow and teal coded for different stainings, whereas magenta was used twice for nuclear Ki-67 and cytosolic / membranous PSMA. The staining of multiplex IHC was compared to single stains of ERG, PSMA and p504s. The proper staining of the basal cell cocktail and Ki-67 could be assessed by internal positive controls in the multiplex staining. The proportion of PSMA and p504s expression revealed a significant correlation between multiplex and single stains (p < 0.01) as well as a concordant staining pattern for ERG (n = 14 prostate cancers were identified ERG positive with both methods). Our proof of concept study demonstrates a robust staining pattern of all five different antibodies with this newly developed 5x MPX IHC. This approach facilitates the recognition of prostate cancer, in particular by adding PSMA in cases with low p504s expression.

Th1-Th2 polarisation and autophagy in the control of intracellular mycobacteria by macrophages.

Autophagy is a major intracellular pathway for the lysosomal degradation of long-lived cytoplasmic macromolecules and damaged or surplus organelles. More recently, autophagy has also been linked with innate and adaptive immune responses against intracellular pathogens, including Mycobacterium tuberculosis, which can survive within macrophages by blocking fusion of the phagosome with lysosomes. Induction of autophagy by the Th1 cytokine IFN-gamma enables infected macrophages to overcome this phagosome maturation block and inhibit the intracellular survival of mycobacteria. Conversely, the Th2 cytokines IL-4 and IL-13 inhibit autophagy in murine and human macrophages. We discuss how differential modulation of autophagy by Th1 and Th2 cytokines may represent an important feature of the host response to mycobacteria.

A Method to Reuse Archived H&E Stained Histology Slides for a Multiplex Protein Biomarker Analysis.

Archived Hematoxylin and Eosin (H&E) stained pathology slides are routinely stored to index formalin-fixed paraffin-embedded (FFPE) sample tissue blocks. FFPE blocks are clinically annotated human tumor specimens that can be valuable in studies decades after the tissue is collected. If stored properly, they have the potential to yield a valuable number of serial sectioned slides for diagnostic or research purposes. However, some retrospective studies are limited in scope because the tissue samples have been depleted or not enough material is available in stored blocks for serial sections. The goal of these studies was to determine if archived H&E-stained slides can be directly reutilized by optimizing methods to de-stain and then re-stain the H&E stained slides to allow the detection of several biomarkers of interest using a conjugated antibody with chromogen multiplex immunohistochemistry procedure. This simple but innovative procedure, combined with image analysis techniques, demonstrates the ability to perform precise detection of relevant markers correlated to disease progression in initially identified tumor regions in tissue. This may add clinical value in retaining H&E slides for further use.

Autophagic proteolysis of long-lived proteins in nonliver cells.

Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes with lysosomes, the engulfed cargo is degraded. The proteolysis of longlived proteins by macroautophagy is a standard, specific measure of autophagic degradation and represents an end-point assay for the pathway. The assay is based on a pulse-chase approach, whereby cellular proteins are radiolabeled by an isotopically marked amino acid, the short-lived, rapidly turned over, proteins are allowed to be degraded during a long chase period, and then the remaining, stable radiolabeled proteins are subjected to autophagic degradation. The classical application of this method has been in hepatocytes, but the recent growth of interest in autophagy has necessitated adaptation of this method in nonliver cells. Here we describe a protocol to quantify autophagic degradation of longlived proteins in macrophages. This chapter details the method of analyzing autophagic proteolysis in RAW264.7 mouse macrophages.

The Mycobacterium tuberculosis phagosome.

Tuberculosis is currently the most devastating human bacterial disease, causing millions of deaths annually and infecting an overwhelming percentage of the global population. Its success as a scourge lies in the ability of Mycobacterium tuberculosis to prevent normal phagolysosome biogenesis, essential to the destruction of invading microorganisms, inside macrophages. Recent work has identified host GTPases involved in the block of normal phagolysosome biogenesis during mycobacterial infection and has provided a set of methods, in particular efficient macrophage transfection, which will prove essential in examining the role of host effectors in this process.

Phosphoinositides in phagolysosome and autophagosome biogenesis.

Interconversions of phosphoinositides play a pivotal role during phagocytosis and at the subsequent stages of phagosomal maturation into the phagolysosome. Several model systems have been used to study the role of phosphoinositides in phagosomal membrane remodelling. These include phagosomes formed by inanimate objects such as latex beads, or pathogenic bacteria, e.g. Mycobacterium tuberculosis. The latter category provides naturally occurring tools to dissect membrane trafficking processes governing phagolysosome biogenesis. M. tuberculosis persists in infected macrophages by blocking Rab conversion and affecting Rab effectors. One of the major Rab effectors involved in this process is the type III phosphatidylinositol 3-kinase hVPS34. The lipid kinase hVPS34 and its enzymatic product PtdIns3P are critical for the default pathway of phagosomal maturation into phagolysosomes. Mycobacteria block PtdIns3P production and thus arrest phagosomal maturation. PtdIns3P is also critical for the process of autophagy, recently recognized as an effector of innate immunity defenses. Induction of autophagy by pharmacological, physiological, or immunological means, overcomes mycobacterial phagosome maturation block in a PtdIns3P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3P and PtdIns3P-dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host-pathogen interactions, and innate and adaptive immunity defense mechanisms meet.

Bacterial luciferase is naturally destabilized in Mycobacterium tuberculosis and can be used to monitor changes in gene expression.

Reporter systems efficient at monitoring temporal gene expression in slow-growing mycobacteria would significantly aid the characterization of gene expression in specific environments. Bacterial luciferase is a reporter that has not been widely used to study gene expression in mycobacteria. This report describes the determination of the degradation of bacterial luciferase in Mycobacterium tuberculosis H37Ra and its utility as a reporter of temporal gene expression in this slow-growing mycobacterium. The inducible/repressible alanine dehydrogenase promoter of M. tuberculosis H37Rv was used to track the decay kinetics of Vibrio harveyi luciferase in both mid-log phase and stationary phase grown M. tuberculosis H37Ra, which proved to be highly similar during both phases of growth. The luciferase reporter was then used to detect changes in expression from the heat-shock promoter, phsp60, of M. bovis BCG during M. tuberculosis H37Ra growth in culture. Quantitative real-time PCR analysis of groEL2, the hsp60 homologue in M. tuberculosis, displayed a similar pattern of expression to phsp60-driven luciferase. These results strongly suggest that the luciferase reporter can be used to monitor temporal changes in gene expression in M. tuberculosis and may serve as a novel system to examine gene expression under specific conditions.

Comparison of methods in the detection of ALK and ROS1 rearrangements in lung cancer.

INTRODUCTION: The use of targeted therapies toward specific oncogenic driver mutations has become a critical factor in the treatment of patients with lung cancer. It is therefore essential to utilize tests with high performance characteristics. Fluorescence in situ hybridization (FISH) is the standard method for detecting anaplastic lymphoma kinase (ALK) and ROS1 rearrangements in non-small-cell lung cancer but the utility of other methods such as immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) is unclear. METHODS: Three hundred and sixty-two lung cancer patients were tested with FISH, CISH, and IHC using three ALK antibodies (ALK1, 5A4, D5F3) and one ROS1 antibody in the detection of ALK and ROS1 rearrangements. RESULTS: There was a 97.4% concordance (298 of 306) between FISH and CISH for detection of ALK rearrangements. The ROS1 rearrangement status had a 97% (291 of 300) concordance between CISH and FISH. ALK protein expression was observed in 6 of 341 samples with the ALK1 and 5A4 antibodies and 5 of 341 samples with D5F3. All three antibodies stained each of the ALK FISH-positive samples (100% sensitivity). ROS1 protein expression was observed in 2 of 322 samples. One of three samples with a ROS1 rearrangement by FISH showed ROS1 protein expression (33.3% sensitivity). CONCLUSION: Our findings show good correlation between FISH versus CISH in the detection of ALK and ROS1 rearrangements. FISH versus IHC showed good correlation in the detection of ALK rearrangements but showed weak correlation in the detection of ROS1 rearrangements. These results suggest CISH and IHC could be complimentary detection methods to FISH in the detection of ALK and ROS1 rearrangements.

Rapid two-temperature formalin fixation.

Formalin fixation is a mainstay of modern histopathologic analysis, yet the practice is poorly standardized and a significant potential source of preanalytical errors. Concerns of workflow and turnaround time drive interest in developing shorter fixation protocols, but rapid protocols can lead to poor histomorphology or inadequate downstream assay results. Additionally, assays such as immunohistochemistry for phosphorylated epitopes have historically been challenging in the context of formalin-fixed tissue, indicating that there may be room for improvement in this process that is fundamental to the practice of anatomic pathology. With these issues in mind, we studied basic formalin biochemistry to develop a novel formalin fixation protocol that involves a pre-incubation in subambient temperature formalin prior to a brief exposure to heated formalin. This new protocol is more rapid than standard protocols yet preserves histomorphology and yields tissue that is compatible with an expanded set of downstream clinical and research assays, including immunohistochemistry for phosphorylated epitopes.

Lysosomal positioning coordinates cellular nutrient responses.

mTOR (mammalian target of rapamycin) signalling and macroautophagy (henceforth autophagy) regulate numerous pathological and physiological processes, including cellular responses to altered nutrient levels. However, the mechanisms regulating mTOR and autophagy remain incompletely understood. Lysosomes are dynamic intracellular organelles intimately involved both in the activation of mTOR complex 1 (mTORC1) signalling and in degrading autophagic substrates. Here we report that lysosomal positioning coordinates anabolic and catabolic responses with changes in nutrient availability by orchestrating early plasma-membrane signalling events, mTORC1 signalling and autophagy. Activation of mTORC1 by nutrients correlates with its presence on peripheral lysosomes that are physically close to the upstream signalling modules, whereas starvation causes perinuclear clustering of lysosomes, driven by changes in intracellular pH. Lysosomal positioning regulates mTORC1 signalling, which in turn influences autophagosome formation. Lysosome positioning also influences autophagosome-lysosome fusion rates, and thus controls autophagic flux by acting at both the initiation and termination stages of the process. Our findings provide a physiological role for the dynamic state of lysosomal positioning in cells as a coordinator of mTORC1 signalling with autophagic flux.

Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria.

IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease.

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages.

Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.