Search

The phagolysosome is an antimicrobial and degradative organelle that plays key roles in macrophage-mediated inflammatory and homeostatic functions. Whereas mature phagolysosomes are known to sequester and degrade their contents into basic nutrients, they were not previously assigned an active role in amplifying inflammation. We have described a novel macrophage process in which partially digested immunostimulatory PAMPs are released extracellularly from the mature phagolysosome via discrete events we term eructophagy. Eructophagy is induced by proinflammatory stimuli, negatively regulated by IL4 and MTOR, and is dependent on key autophagy proteins, including fusion machinery of degradative and secretory autophagy. We propose that macrophages use eructophagy to release processed PAMPs/DAMPs to amplify local inflammation.

Recognition of pathogen-or-damage-associated molecular patterns is critical to inflammation. However, most pathogen-or-damage-associated molecular patterns exist within intact microbes/cells and are typically part of non-diffusible, stable macromolecules that are not optimally immunostimulatory or available for immune detection. Partial digestion of microbes/cells following phagocytosis potentially generates new diffusible pathogen-or-damage-associated molecular patterns, however, our current understanding of phagosomal biology would have these molecules sequestered and destroyed within phagolysosomes. Here, we show the controlled release of partially-digested, soluble material from phagolysosomes of macrophages through transient, iterative fusion-fission events between mature phagolysosomes and the plasma membrane, a process we term eructophagy. Eructophagy is most active in proinflammatory macrophages and further induced by toll like receptor engagement. Eructophagy is mediated by genes encoding proteins required for autophagy and can activate vicinal cells by release of phagolysosomally-processed, partially-digested pathogen associated molecular patterns. We propose that eructophagy allows macrophages to amplify local inflammation through the processing and dissemination of pathogen-or-damage-associated molecular patterns.

This institutional self-study investigated the use of text-matching software (TMS) to prevent plagiarism by students in a Canadian university that did not have an institutional license for TMS at the time of the study. Assignments from a graduate-level engineering course were analyzed using iThenticate®. During the initial phase of the study, similarity scores from the first student assignments (N = 132) were collected to determine a baseline level of textual similarity. Students were then offered an educational intervention workshop on academic integrity. Another set of similarity scores from consenting participants’ second assignments (n = 106) were then collected, and a statistically significant assignment effect (p

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3−/− mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target., Interferon (IFN) is an important component of antiviral immunity, but can also be exploited by bacteria for immune evasion. Here the authors show that Listeria monocytogenes (Lm) induces type I IFN to suppress the degradation of Lm virulence proteins, ActA and LLO, and promote Lm infection in an IFITM3-dependent manner, thereby hinting at a potential target for antimicrobial therapy.

Macrophages are an important component of the innate immune response. Priming and activation of macrophages is stimulated by cytokines (i.e IFNγ). However, growth hormone (GH) can also stimulate macrophage activation. Based on these observations, the goal of this work was to 1) to compare the transcriptome profile of macrophages activated in vitro with GH and IFNγ, and 2) to assess the impact of GH on key macrophage functional properties like reactive oxygen species (ROS) production and phagosomal proteolysis. To assess the global transcriptional and functional impact of GH on macrophage programming, bone marrow derived macrophages were treated with GH or IFNγ. Our data strongly support a potential link between GH, which wanes with age, and impaired macrophage function. The notable overlap of GH with IFNγ-induced pathways involved in innate immune sensing of pathogens and antimicrobial responses argue for an important role for GH in macrophage priming and maturation. By using functional assays that report on biochemical activities within the lumen of phagosomes, we have also shown that GH alters physiologically relevant processes such as ROS production and proteolysis. These changes could have far reaching impacts on antimicrobial capacity, signaling, and antigen presentation.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.

Historically an enzyme of the immune system, the lysosomal reductase GILT serves a unique homeostatic role in bone resorption., The extracellular bone resorbing lacuna of the osteoclast shares many characteristics with the degradative lysosome of antigen-presenting cells. γ-Interferon–inducible lysosomal thiol reductase (GILT) enhances antigen processing within lysosomes through direct reduction of antigen disulfides and maintenance of cysteine protease activity. In this study, we found the osteoclastogenic cytokine RANKL drove expression of GILT in osteoclast precursors in a STAT1-dependent manner, resulting in high levels of GILT in mature osteoclasts, which could be further augmented by γ-interferon. GILT colocalized with the collagen-degrading cysteine protease, cathepsin K, suggesting a role for GILT inside the osteoclastic resorption lacuna. GILT-deficient osteoclasts had reduced bone-resorbing capacity, resulting in impaired bone turnover and an osteopetrotic phenotype in GILT-deficient mice. We demonstrated that GILT could directly reduce the noncollagenous bone matrix protein SPARC, and additionally, enhance collagen degradation by cathepsin K. Together, this work describes a previously unidentified, non-immunological role for GILT in osteoclast-mediated bone resorption.

Cathepsin X/Z/P is cysteine cathepsin with unique carboxypeptidase activity. Its expression is associated with cancer and neurodegenerative diseases, although its roles during normal physiology are still poorly understood. Advances in our understanding of its function have been hindered by a lack of available tools that can specifically measure the proteolytic activity of cathepsin X. We present a series of activity-based probes that incorporate a sulfoxonium ylide warhead, which exhibit improved specificity for cathepsin X compared to previously reported probes. We apply these probes to detect cathepsin X activity in cell and tissue lysates, in live cells and in vivo, and to localize active cathepsin X in mouse tissues by microscopy. Finally, we utilize an improved method to generate chloromethylketones, necessary intermediates for synthesis of acyloxymethylketones probes, by way of sulfoxonium ylide intermediates. In conclusion, the probes presented in this study will be valuable for investigating cathepsin X pathophysiology.

Respiratory silicosis is a preventable occupational disease that develops secondary to the aspiration of crystalline silicon dioxide (silica) into the lungs, activation of the NLRP3 inflammasome, and IL-1β production. Cathepsin Z has been associated with the development of inflammation and IL-1β production; however, the mechanism of how cathepsin Z leads to IL-1β production is unknown. Here, the requirement for cathepsin Z in silicosis was determined using wildtype mice and mice deficient in cathepsin Z. The activation of the NLRP3 inflammasome in macrophages was studied using wildtype and cathepsin Z-deficient bone marrow-derived murine dendritic cells and the human monocytic cell line THP-1. Cells were activated with silica, and IL-1β release was determined using enzyme-linked immunosorbent assay or IL-1β bioassays. The relative contribution of the active domain or integrin-binding domain of cathepsin Z was studied using recombinant cathepsin Z constructs and the α5 integrin neutralizing antibody. We report that the lysosomal cysteine protease cathepsin Z potentiates the development of inflammation associated with respiratory silicosis by augmenting NLRP3 inflammasome-derived IL-1β expression in response to silica. Secreted cathepsin Z functions non-proteolytically via the internal integrin-binding domain to impact caspase-1 activation and the production of active IL-1β through integrin α5 without affecting transcription levels of NLRP3 inflammasome components. This work reveals a regulatory pathway for the NLRP3 inflammasome that occurs in an outside-in fashion and provides a link between extracellular cathepsin Z and inflammation. Furthermore, it reveals a level of NLRP3 inflammasome regulation that has previously only been found downstream of extracellular pathogens.

In step with the worldwide trend for highereducational institutes to establish areas of research emphasis,the accumulation of resources in key areas has become commonpractice in veterinary facultie...

The synthetic supercooling drug, icilin, and its primary receptor target, the cation channel transient receptor potential (TRP) melastatin-8 (TRPM8), have been described as potent negative regulators of inflammation in the colon. The aim of this study was to determine whether the anti-inflammatory action of icilin could potentially be used to treat autoimmune neuroinflammatory disorders, such as multiple sclerosis (MS). During experimental autoimmune encephalomyelitis (EAE)-a CD4+ T cell-driven murine model of MS-we found that both wild-type (WT) and TRPM8-deficient EAE mice were protected from disease progression during icilin treatment, as evidenced by delays in clinical onset and reductions in neuroinflammation. In vitro, icilin potently inhibited the proliferation of murine and human CD4+ T cells, with the peripheral expansion of autoantigen-restricted T cells similarly diminished by the administration of icilin in mice. Attenuation of both TRPM8-/- and TRP ankyrin-1-/- T-cell proliferation by icilin was consistent with the WT phenotype, which suggests a mechanism that is independent of these channels. In addition, icilin treatment altered the expressional profile of activated CD4+ T cells to one that was indicative of restricted effector function and limited neuroinflammatory potential. These findings identify a potent anti-inflammatory role for icilin in lymphocyte-mediated neuroinflammation and highlight clear pleiotropic effects of the compound beyond classic TRP channel activation.-Ewanchuk, B. W., Allan, E. R. O., Warren, A. L., Ramachandran, R., Yates, R. M. The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response.

Environmental and hormonal factors are implicated in dysimmunity in multiple sclerosis. We investigated whether bisphenol-A, a prominent contaminant with endocrine-disrupting capabilities, altered susceptibility in an inflammatory model of multiple sclerosis. We found that gestational, but not adult, exposure to bisphenol-A increased the development of experimental autoimmune encephalomyelitis in adulthood in male, but not female, mice when a suboptimal disease-inducing immunization was used. Gestational bisphenol-A in male mice primed macrophages in adulthood and raised granulocyte-colony stimulating factor and neutrophil counts/activity postsuboptimal immunization. Neutralizing granulocyte-colony stimulating factor blocked susceptibility to disease in bisphenol-A mice. Early life exposure to bisphenol-A may represent an environmental consideration in multiple sclerosis.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus that grows in macrophages and causes acute pneumonia in pigs. PRRSV causes devastating losses to the porcine industry. However, due to its high antigenic variability and poorly understood immunopathogenesis, there is currently no effective vaccine or treatment to control PRRSV infection. The common occurrence of PRRSV infection with bacterial infections as well as its inflammatory-driven pathobiology raises the question of the value of antibiotics with immunomodulating properties for the treatment of the disease it causes. The macrolide antibiotic Tulathromycin (TUL) has been found to exhibit potent anti-inflammatory and immunomodulating properties in cattle and pigs. The aim of this study was to characterize the anti-viral and immunomodulating properties of TUL in PRRSV-infected porcine macrophages. Our findings indicate that blood monocyte-derived macrophages are readily infected by PRRSV and can be used as an effective cellular model to study PRRSV pathogenesis. TUL did not change intracellular or extracellular viral titers, not did it alter viral receptors (CD163 and CD169) expression on porcine macrophages. In contrast, TUL exhibited potent immunomodulating properties, which therefore occurred in the absence of any direct antiviral effects against PRRSV. TUL had an additive effect with PRRSV on the induction of macrophage apoptosis, and inhibited virus-induced necrosis. TUL significantly attenuated PRRSV-induced macrophage pro-inflammatory signaling (CXCL-8 and mitochondrial ROS production) and prevented PRRSV inhibition of non-opsonized and opsonized phagocytic function. Together, these data demonstrate that TUL inhibits PRRSV-induced inflammatory responses in porcine macrophages and protects against the phagocytic impairment caused by the virus. Research in live pigs is warranted to assess the potential clinical benefits of this antibiotic in the context of virally induced inflammation and tissue injury.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC’s antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense, ssRNA virus of the genus Arterivirus, is a devastating disease of swine worldwide. Key early targets of PRRSV infection in pigs include professional phagocytes in the lung, such as alveolar and interstitial macrophages and dendritic cells, the dysfunction of which is believed to be responsible for much of the associated mortality. In order to study the effect of virus infection on phagocyte function, the development of a robust, reproducible model would be advantageous. Given the limitations of current models, we set out to develop a porcine bone marrow-derived macrophage (PBMMΦ) cell model to study phagosomal maturation and function during PRRSV infection. Derivation of PBMMΦs from marrow using cultured L929 fibroblast supernatant produced a homogeneous population of cells that exhibited macrophage-like morphology and proficiency in Fc-receptor-mediated phagocytosis and phagosomal maturation. PBMMΦs were permissive to PRRSV infection, resulting in a productive infection that peaked at 24 h. Assessment of the effect of PRRSV infection on the properties of phagosomal maturation in PBMMΦs revealed a significant decrease in phagosomal proteolysis and lowered production of reactive oxygen species, but no change in PBMMΦ viability, phagocytosis or the ability of phagosomes to acidify. In this study, we present a new model to investigate PRRSV infection of phagocytes, which demonstrates a significant effect on phagosomal maturation with the associated implications on proper macrophage function. This model can also be used to study the effect on the phagosomal microenvironment of infection by other viruses targeting porcine macrophages.

Although endosomes, lysosomes, and phagosomes require a reductive environment for the optimal activity of disulfide reductases and other thiol-dependent enzymes, how these reductive environments are established and maintained remain unknown. Our goal in this study was to begin to elucidate the redox control systems responsible for maintaining redox-sensitive enzymatic activities in the phagolysosome of murine macrophages. Through the use of specific inhibitors and genetic knockdown of known redox enzymes, we identified redox pathways that influence phagosomal disulfide reduction. In particular, known inhibitors of the NADPH-dependent selenoprotein, thioredoxin reductase, were shown to inhibit phagosomal disulfide reduction and phagosomal proteolysis. This was supported by the observation that conditional deletion of the selenocysteine tRNA in macrophages decreased phagosomal disulfide reduction capacity. In addition, pharmacologic inhibition of the pentose phosphate pathway decreased rates of disulfide reduction and proteolysis in the phagosome, implicating NADPH as a source of phagosomal reductive energy. Finally, by analyzing the effect of extracellular redox couples, such as cysteine:cystine on thiol-dependent phagosomal processes, we demonstrated that the extracellular space can additionally supply the phagosome with reductive energy. Collectively, these data demonstrate that defined cytosolic reductive pathways act in concert with the uptake of cysteine from the extracellular space to support thiol-dependent chemistries in the phagosome.

In addition to debris clearance and antimicrobial function, versatile organelles known as phagosomes play an essential role in the processing of exogenous antigen in antigen presenting cells. While there has been much attention on human leukocyte antigen haplotypes in the determination of antigenic peptide repertoires, the lumenal biochemistries within phagosomes and endosomes are emerging as equally-important determinants of peptide epitope composition and immunodominance. Recently, the lumenal redox microenvironment within these degradative compartments has been shown to impact two key antigenic processing chemistries: proteolysis by lysosomal cysteine proteases and disulfide reduction of protein antigens. Through manipulation of the balance between oxidative and reductive capacities in the phagosome-principally by modulating NADPH oxidase (NOX2) and γ-interferon-inducible lysosomal thiol reductase (GILT) activities-studies have demonstrated changes to antigen processing patterns leading to modified repertoires of antigenic peptides available for presentation, and subsequently, altered disease progression in T cell-driven autoimmunity. This review focuses on the mechanisms and consequences of redox-mediated phagosomal antigen processing, and the potential downstream implications to tolerance and autoimmunity.

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.

The chemistries within phagosomes of APCs mediate microbial destruction as well as generate peptides for presentation on MHC class II. The antimicrobial effector NADPH oxidase (NOX2), which generates superoxide within maturing phagosomes, has also been shown to regulate activities of cysteine cathepsins through modulation of the lumenal redox potential. Using real-time analyses of lumenal microenvironmental parameters, in conjunction with hydrolysis pattern assessment of phagocytosed proteins, we demonstrated that NOX2 activity not only affects levels of phagosomal proteolysis as previously shown, but also the pattern of proteolytic digestion. Additionally, it was found that NOX2 deficiency adversely affected the ability of bone marrow–derived macrophages, but not dendritic cells, to process and present the I-Ab–immunodominant peptide of the autoantigen myelin oligodendrocyte glycoprotein (MOG). Computational and experimental analyses indicated that the I-Ab binding region of the immunodominant peptide of MOG is susceptible to cleavage by the NOX2-controlled cysteine cathepsins L and S in a redox-dependent manner. Consistent with these findings, I-Ab mice that were deficient in the p47phox or gp91phox subunits of NOX2 were partially protected from MOG-induced experimental autoimmune encephalomyelitis and displayed compromised reactivation of MOG-specific CD4+ T cells in the CNS, despite eliciting a normal primary CD4+ T cell response to the inoculated MOG Ag. Taken together, this study demonstrates that the redox microenvironment within the phagosomes of APCs is a determinant in MHC class II repertoire production in a cell-specific and Ag-specific manner, which can ultimately impact susceptibility to CD4+ T cell–driven autoimmune disease processes.

Kubes et al. show that methicillin-resistant Staphylococcus aureus (MRSA) survive and proliferate inside Kupffer cells. Intracellular MRSA is resistant to neutrophil-killing and antibiotics treatment and, when released into the circulation, can infect other organs., Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC’s antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

Macrophages fulfill most of their microbicidal duties in their phagosomes following uptake of microbes. However, some microbes, such as Mycobacterium tuberculosis, have evolved mechanisms to subvert the normal maturation process of their phagocytic compartment to limit the hostility of this environment. The experimental analysis of this process and its subsequent impact on bacterial fitness is technically demanding and has required the development of a broad range of readouts to correlate function and outcome. In this chapter we detail two technically divergent platforms to measure the environment within the phagosomal compartment that contains Mtb in the short term, and more long-term readouts of bacterial fitness and Mtb's reaction to host-derived stresses. The readouts are all fluorescence-based and are adaptable to measurement by a range of platforms, including spectrofluorometry, confocal microscopy, and flow cytometry.

The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated. In this chapter, we detail phagosome-specific, fluorescence-based assays that measure disulfide reduction and the production of reactive oxygen species in live phagocytes such as macrophages and dendritic cells, in real time.

The biochemical processes within the phagosomes of macrophages and dendritic cells are essential to immunity and homeostasis. Measurable properties of the phagosomal lumen include: assessment of various hydrolytic activities, reduction and oxidation events, pH, ion concentrations, and electrochemical gradients. These often-interdependent phagosomal features are commonly evaluated individually, hindering the analysis of the biochemical relationship between these factors within the same phagosome. In addition, the ability of phagosomes within the same cell to behave autonomously is becoming more evident, thus highlighting the need for a technique capable of multiplex analyses of phagosomal lumenal chemistries at the single phagosome level. In this chapter, we outline an approach that is capable of simultaneously measuring multiple phagosomal parameters of individual phagosomes by utilizing specifically designed experimental particles with multiple reporter fluors, in combination with real-time fluorometric measurement via automated microscopy. Subsequent analysis using high-content image analysis software enables each phagosomal parameter to be evaluated in a quantitative or semi-quantitative manner. This approach facilitates investigation of the complex relationship between phagosomal properties in a population of macrophages in real time, at the level of individual phagosomes.

The biochemical processes within the phagosomes of macrophages and dendritic cells are essential to immunity and homeostasis. Measurable properties of the phagosomal lumen include: assessment of various hydrolytic activities, reduction and oxidation events, pH, ion concentrations, and electrochemical gradients. These often-interdependent phagosomal features are commonly evaluated individually, hindering the analysis of the biochemical relationship between these factors within the same phagosome. In addition, the ability of phagosomes within the same cell to behave autonomously is becoming more evident, thus highlighting the need for a technique capable of multiplex analyses of phagosomal lumenal chemistries at the single phagosome level. In this chapter, we outline an approach that is capable of simultaneously measuring multiple phagosomal parameters of individual phagosomes by utilizing specifically designed experimental particles with multiple reporter fluors, in combination with real-time fluorometric measurement via automated microscopy. Subsequent analysis using high-content image analysis software enables each phagosomal parameter to be evaluated in a quantitative or semi-quantitative manner. This approach facilitates investigation of the complex relationship between phagosomal properties in a population of macrophages in real time, at the level of individual phagosomes.

The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated. In this chapter, we detail phagosome-specific, fluorescence-based assays that measure disulfide reduction and the production of reactive oxygen species in live phagocytes such as macrophages and dendritic cells, in real time.

Background Resident macrophages in OA synovial tissue contribute to synovitis through pro-inflammatory mediators driving cartilage loss. What remains unknown is how these macrophages interact with synovial mesenchymal progenitor cells (sMPCs) in the joint. sMPCs have the potential to undergo chondrogenesis, but for yet unknown reasons, this ability is decreased in OA patients. In this study, we sought to identify if alteration of macrophage activity regulates the chondrogenic capacity of sMPCs. Methods An explant model was developed using human synovium obtained from normal individuals and OA patients. These explants were subjected to macrophage depletion and/or cytokine stimulation in order to regulate/deplete the residing macrophage population. Supernatant was collected following a 12-day treatment phase and subjected to inflammatory secretome analysis. sMPCs from the explants were subsequently placed under 21-day chondrogenic differentiation and levels of type II collagen (Col2a), Aggrecan (Acan), and Sox9 gene expression was quantified. Results Inflammatory secretome analysis from OA patients revealed the presence of pro-inflammatory analytes following pro- and anti-inflammatory cytokine stimulation and/or macrophage depletion. Additionally, chondrogenic differentiation of sMPCs was heterogeneously impacted across all OA patients following pro-/anti-inflammatory cytokine stimulation and/or macrophage depletion. Conclusion Tissue resident synovial macrophages can regulate the chondrogenic differentiation of sMPCs after cytokine stimulation in a patient specific manner. The secretion profile of OA synovium was also responsive to cytokine stimulation and/or macrophage depletion as observed by the largely pro-inflammatory milieu upregulated following cytokine stimulation. Electronic supplementary material The online version of this article (doi:10.1186/s12950-016-0120-9) contains supplementary material, which is available to authorized users.

The level of proteolysis within phagosomes of dendritic cells (DCs) is thought to be tightly regulated, as it directly impacts the cell's efficiency to process antigen. Activity of the antimicrobial effector NADPH oxidase (NOX2) has been shown to reduce levels of proteolysis within phagosomes of both macrophages and DCs. However, the proposed mechanisms underlying these observations in these two myeloid cell lineages are dissimilar. Using real-time analysis of lumenal microenvironmental parameters within phagosomes in live bone marrow-derived DCs, we show that the levels of phagosomal proteolysis are diminished in the presence of NOX2 activity, but in contrast to previous reports, the acidification of the phagosome is largely unaffected. As found in macrophages, we show that NOX2 controls phagosomal proteolysis in DCs through redox modulation of local cysteine cathepsins. Aspartic cathepsins were unaffected by redox conditions, indicating that NOX2 skews the relative protease activities in these antigen processing compartments. The ability of DC phagosomes to reduce disulphides was also compromised by NOX2 activity, implicating this oxidase in the control of an additional antigen processing chemistry of DCs.

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies.

Second mitochondrial activator of caspase (Smac)-mimetic compounds and oncolytic viruses were developed to kill cancer cells directly. However, Smac-mimetic compound and oncolytic virus therapies also modulate host immune responses in ways we hypothesized would complement one another in promoting anticancer T-cell immunity. We show that Smac-mimetic compound and oncolytic virus therapies synergize in driving CD8

Macrophages play a central role in tissue homeostasis and the immune system. Their primary function is to internalize cellular debris and microorganisms for degradation within their phagosomes. In this context, their capacity to process and sequester lipids such as triacylglycerides and cholesteryl esters makes them key players in circulatory diseases, such as atheroclerosis. To discover new inhibitors of lipolytic processing within the phagosomal system of the macrophage, we have developed a novel, cell-based assay suitable for high-throughput screening. We employed particles carrying a fluorogenic triglyceride substrate and a calibration fluor to screen for inhibitors of phagosomal lipolysis. A panel of secondary assays were employed to discriminate between lipase inhibitors and compounds that perturbed general phagosomal trafficking events. This process enabled us to identify a new structural class of pyrazole-methanone compounds that directly inhibit lysosomal and lipoprotein lipase activity.

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-κB-dependent genes, type I IFNs, and caspase-dependent IL-1β maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1β maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population-based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G (IgG)-stimulated reactive oxygen species (ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C (PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR-mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG-opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR-triggered, PKC/Syk-dependent local assembly and activation of NOX2.

Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

The phagosome is key to most macrophage functions. It is the site of degradation of particulate material, of bacterial killing and the generation of peptides for antigen presentation. Despite its role at the fulcrum of the innate and acquired immune systems, little is known about the physiology of this organelle in activated macrophages. In this study, we utilize fluorometric techniques to characterize functional alterations in the lumenal environment of the maturing phagosome following stimulation of macrophages with interferon-gamma and/or lipopolysaccharide. In addition to modulating the kinetics of phagosomal acidification, activation results in a phagosome with diminished hydrolytic activities that varies markedly with the activation status of the cell. Differential levels of proteolytic, lipolytic and beta-galactosidase activities were observed in the phagosome but not in the total lysosomal extract, indicating selective delivery of enzymes to the developing phagosome. Despite the suppression of hydrolytic activities observed in early phagosomes, late phagosomes exhibit an enhanced and protracted accumulation of lysosomal cargo. The data are consistent with limiting proteolysis in the early phagosome to maximize epitope generation and antigen presentation while sequestering the degradative capacity in the late phagolysosome.

The cysteine cathepsins B, S, and L are functionally linked to antigen processing, and hence to autoimmune disorders such as multiple sclerosis. Stemming from several studies that demonstrate that mice can be protected from experimental autoimmune encephalomyelitis (EAE) through the pharmacologic inhibition of cysteine cathepsins, it has been suggested that targeting these enzymes in multiple sclerosis may be of therapeutic benefit. Utilizing mice deficient in cysteine cathepsins both individually and in combination, we found that the myelin-associated antigen myelin oligodendrocyte glycoprotein (MOG) was efficiently processed and presented by macrophages to CD4+ T cells in the individual absence of cathepsin B, S or L. Similarly, mice deficient in cathepsin B or S were susceptible to MOG-induced EAE and displayed clinical progression and immune infiltration into the CNS, similar to their wild-type counterparts. Owing to a previously described CD4+ T cell deficiency in mice deficient in cathepsin L, such mice were protected from EAE. When multiple cysteine cathepsins were simultaneously inhibited via genetic deletion of both cathepsins B and S, or by a cathepsin inhibitor (LHVS), MHC-II surface expression, MOG antigen presentation and EAE were attenuated or prevented. This study demonstrates the functional redundancy between cathepsin B, S and L in EAE, and suggests that the inhibition of multiple cysteine cathepsins may be needed to modulate autoimmune disorders such as multiple sclerosis.

Targeting inflammatory function of autoreactive T lymphocytes defines a key facet of therapeutics for autoimmune diseases such as multiple sclerosis (MS). The super-cooling drug icilin has recently been shown to limit systemic inflammation through activation of the TRPM8 calcium channel in murine models of colitis. We thus aimed to define an anti-inflammatory mechanism for the drug candidate icilin and TRPM8 in T cell-mediated adaptive neuroinflammation. Using experimental autoimmune encephalomyelitis (EAE), a CD4+ T cell-driven model of MS, we assayed the influence of icilin on CD4+ T cell activation, proliferation, and effector function. Significant reductions in clinical disease were observed in WT EAE mice receiving icilin treatment, coinciding with fewer infiltrating leukocytes in the central nervous system. Unexpectedly, TRPM8−/− mice treated with icilin also displayed protection from EAE, showing similar reductions in disease score and leukocyte infiltration. Both WT and TRPM8−/− mice receiving icilin treatment showed significant delays in initial disease onset, attributed in vitro to reduced T cell proliferation via icilin-mediated G1 cell cycle arrest. A library of chemically and structurally related icilin analogs was screened to identify novel drug candidates targeting autoimmune T cell function. Amongst the screened drug analogs, a small fraction potently attenuated T cell proliferation in vitro. These results suggest that TRPM8-independent ablation of T cell function drives the anti-inflammatory properties of icilin during EAE. Further, novel drug candidates structurally related to icilin represent a promising class of molecules for limiting T cell responses in MS and other autoinflammatory diseases.

Although it is known that lysosomal cysteine cathepsins require a reducing environment for optimal activity, it is not firmly established how these enzymes are maintained in their reduced-active state in the acidic and occasionally oxidative environment within phagosomes and lysosomes. γ-Interferon-inducible lysosomal thiol reductase (GILT) has been the only enzyme described in the endosomes, lysosomes, and phagosomes with the potential to catalyze the reduction of cysteine cathepsins. Our goal in the current study was to assess the effect of GILT on major phagosomal functions with an emphasis on proteolytic efficiency in murine bone marrow-derived macrophages. Assessment of phagosomal disulfide reduction upon internalization of IgG-opsonized experimental particles confirmed a major role for GILT in phagosomal disulfide reduction in both resting and interferon-γ-activated macrophages. Furthermore we observed a decrease in early phagosomal proteolytic efficiency in GILT-deficient macrophages, specifically in the absence of an NADPH oxidase-mediated respiratory burst. This deficiency was more prominent in IL-4-activated macrophages that inherently possess lower levels of NADPH oxidase activity. Finally, we provide evidence that GILT is required for optimal activity of the lysosomal cysteine protease, cathepsin S. In summary, our results suggest a role for GILT in maintaining cysteine cathepsin proteolytic efficiency in phagosomes, particularly in the absence of high NADPH oxidase activity, which is characteristic of alternatively activated macrophages.

Giardia duodenalis (syn. G. intestinalis , G. lamblia ) infections are a leading cause of waterborne diarrheal disease that can also result in the development of postinfectious functional gastrointestinal disorders via mechanisms that remain unclear. Parasite numbers exceed 10 6 trophozoites per centimeter of gut at the height of an infection. Yet the intestinal mucosa of G. duodenalis -infected individuals is devoid of signs of overt inflammation. G. duodenalis infections can also occur concurrently with infections with other proinflammatory gastrointestinal pathogens. Little is known of whether and how this parasite can attenuate host inflammatory responses induced by other proinflammatory stimuli, such as a gastrointestinal pathogen. Identifying hitherto-unrecognized parasitic immunomodulatory pathways, the present studies demonstrated that G. duodenalis trophozoites attenuate secretion of the potent neutrophil chemoattractant interleukin-8 (CXCL8); these effects were observed in human small intestinal mucosal tissues and from intestinal epithelial monolayers, activated through administration of proinflammatory interleukin-1β or Salmonella enterica serovar Typhimurium. This attenuation is caused by the secretion of G. duodenalis cathepsin B cysteine proteases that degrade CXCL8 posttranscriptionally. Furthermore, the degradation of CXCL8 via G. duodenalis cathepsin B cysteine proteases attenuates CXCL8-induced chemotaxis of human neutrophils. Taken together, these data demonstrate for the first time that G. duodenalis trophozoite cathepsins are capable of attenuating a component of their host's proinflammatory response induced by a separate proinflammatory stimulus.

New therapeutic approaches for gastrointestinal inflammatory disorders are sorely needed. Children infected with the protozoan parasite Giardia duodenalis exhibit attenuated gut inflammatory respon...

Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enable these activities to be rigorously quantified. This unit contains descriptions of several of these assays assessed by different methods of quantitation, including a fluorescence resonance emission transfer (FRET) assay for phagosome/lysosome fusion measured by spectrofluorometry, a fluorogenic assay for the superoxide burst measured by flow cytometry, and a fluorogenic assay for bulk proteolysis measured by confocal microscopy. These assays illustrate both the range of parameters that can be quantified and the flexibility of instrumentation that can be exploited for their quantitation. Curr. Protoc. Immunol. 102:14.34.1-14.34.14. © 2013 by John Wiley & Sons, Inc. Keywords: macrophage; phagosome; phagocytosis; phagosome maturation