Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases

AbstractThe ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.

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Interaction of Antibiotics with Innate Host Defense Factors against Salmonella enterica Serotype Newport

mSphere ◽

10.1128/msphere.00410-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 15

Author(s):

George Sakoulas ◽

Monika Kumaraswamy ◽

Armin Kousha ◽

Victor Nizet

Keyword(s):

Innate Immunity ◽

Immune System ◽

Innate Immune System ◽

Salmonella Enterica ◽

Clinical Performance ◽

Innate Immune ◽

Content Type ◽

Antimicrobial Assay

ABSTRACT It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo. This study examines the pharmacodynamics of antimicrobials that are used to treat Salmonella with each other and with key components of the innate immune system. Antimicrobial synergy was assessed using time-kill and checkerboard assays. Antimicrobial interactions with innate immunity were studied by employing cathelicidin LL-37, whole-blood, and neutrophil killing assays. Ceftriaxone and ciprofloxacin were found to be synergistic in vitro against Salmonella enterica serotype Newport. Ceftriaxone, ciprofloxacin, and azithromycin each demonstrated synergy with the human cathelicidin defense peptide LL-37 in killing Salmonella. Exposure of Salmonella to sub-MICs of ceftriaxone resulted in enhanced susceptibility to LL-37, whole blood, and neutrophil killing. The activity of antibiotics in vivo against Salmonella may be underestimated in bacteriologic media lacking components of innate immunity. The pharmacodynamic interactions of antibiotics used to treat Salmonella with each other and with components of innate immunity warrant further study in light of recent findings showing in vivo selection of antimicrobial resistance by single agents in this pathogen. IMPORTANCE It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo.

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Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection

Infection and Immunity ◽

10.1128/iai.00820-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 405-412 ◽

Cited By ~ 25

Author(s):

Sasha J. Rose ◽

Luiz E. Bermudez

Keyword(s):

Immune System ◽

Mycobacterium Avium ◽

Innate Immune System ◽

Innate Immune ◽

Mononuclear Phagocytes ◽

Bacterial Killing ◽

Content Type ◽

Tnf Α

ABSTRACTMycobacterium aviumsubsp.hominissuisis an opportunistic human pathogen that has been shown to form biofilmin vitroandin vivo. Biofilm formationin vivoappears to be associated with infections in the respiratory tract of the host. The reasoning behind howM. aviumsubsp.hominissuisbiofilm is allowed to establish and persist without being cleared by the innate immune system is currently unknown. To identify the mechanism responsible for this, we developed anin vitromodel using THP-1 human mononuclear phagocytes cocultured with establishedM. aviumsubsp.hominissuisbiofilm and surveyed various aspects of the interaction, including phagocyte stimulation and response, bacterial killing, and apoptosis.M. aviumsubsp.hominissuisbiofilm triggered robust tumor necrosis factor alpha (TNF-α) release from THP-1 cells as well as superoxide and nitric oxide production. Surprisingly, the hyperstimulated phagocytes did not effectively eliminate the cells of the biofilm, even when prestimulated with gamma interferon (IFN-γ) or TNF-α or cocultured with natural killer cells (which have been shown to induce anti-M. aviumsubsp.hominissuisactivity when added to THP-1 cells infected with planktonicM. aviumsubsp.hominissuis). Time-lapse microscopy and the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay determined that contact with theM. aviumsubsp.hominissuisbiofilm led to early, widespread onset of apoptosis, which is not seen until much later in planktonicM. aviumsubsp.hominissuisinfection. Blocking TNF-α or TNF-R1 during interaction with the biofilm significantly reduced THP-1 apoptosis but did not lead to elimination ofM. aviumsubsp.hominissuis. Our data collectively indicate thatM. aviumsubsp.hominissuisbiofilm induces TNF-α-driven hyperstimulation and apoptosis of surveilling phagocytes, which prevents clearance of the biofilm by cells of the innate immune system and allows the biofilm-associated infection to persist.

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Redundant Catalases Detoxify Phagocyte Reactive Oxygen and Facilitate Histoplasma capsulatum Pathogenesis

Infection and Immunity ◽

10.1128/iai.00173-13 ◽

2013 ◽

Vol 81 (7) ◽

pp. 2334-2346 ◽

Cited By ~ 35

Author(s):

Eric D. Holbrook ◽

Katherine A. Smolnycki ◽

Brian H. Youseff ◽

Chad A. Rappleye

Keyword(s):

Immune System ◽

Innate Immune System ◽

Histoplasma Capsulatum ◽

Reactive Oxygen ◽

Innate Immune ◽

Respiratory Pathogen ◽

Human Neutrophils ◽

Content Type

ABSTRACTHistoplasma capsulatumis a respiratory pathogen that infects phagocytic cells. The mechanisms allowingHistoplasmato overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part ofHistoplasma's ability to survive during infection. To probe the contribution ofHistoplasmacatalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protectedHistoplasmafrom peroxide challengein vitroand from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defensesin vitro, CatB was dispensable for lung infection and extrapulmonary disseminationin vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival ofHistoplasmayeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuatedHistoplasmavirulencein vivo. These results demonstrate thatHistoplasma's dual catalases comprise a system that enablesHistoplasmato efficiently overcome the reactive oxygen produced by the innate immune system.

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What Are the Molecular Mechanisms by Which Functional Bacterial Amyloids Influence Amyloid Beta Deposition and Neuroinflammation in Neurodegenerative Disorders?

International Journal of Molecular Sciences ◽

10.3390/ijms21051652 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1652 ◽

Cited By ~ 3

Author(s):

Robert P. Friedland ◽

Joseph D. McMillan ◽

Zimple Kurlawala

Keyword(s):

Amyloid Beta ◽

Neurodegenerative Disorders ◽

Innate Immune System ◽

Molecular Mechanisms ◽

Innate Immune ◽

Know How ◽

Unique Distribution ◽

The Brain

Despite the enormous literature documenting the importance of amyloid beta (Ab) protein in Alzheimer's disease, we do not know how Ab aggregation is initiated and why it has its unique distribution in the brain. In vivo and in vitro evidence has been developed to suggest that functional microbial amyloid proteins produced in the gut may cross-seed Ab aggregation and prime the innate immune system to have an enhanced and pathogenic response to neuronal amyloids. In this commentary, we summarize the molecular mechanisms by which the microbiota may initiate and sustain the pathogenic processes of neurodegeneration in aging.

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Flagellar Motility Is a Key Determinant of the Magnitude of the Inflammasome Response to Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.00054-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2043-2052 ◽

Cited By ~ 40

Author(s):

Yash R. Patankar ◽

Rustin R. Lovewell ◽

Matthew E. Poynter ◽

Jeevan Jyot ◽

Barbara I. Kazmierczak ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune System ◽

Innate Immune System ◽

Innate Immune ◽

Bacterial Motility ◽

Inflammasome Activation ◽

Flagellar Motility ◽

Content Type

ABSTRACTWe previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronicPseudomonas aeruginosainfections, enables bacteria to evade association and ingestion ofP. aeruginosaby phagocytes bothin vitroandin vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation byP. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotileP. aeruginosa. NonmotileP. aeruginosaelicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cellsin vitro. Importantly, nonmotileP. aeruginosaalso elicits reduced IL-1β levelsin vivoin comparison to those elicited by wild-typeP. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

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216 Anti-tumor activity of iosH2 by blocking LILRB2 receptor signalling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0216 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A235-A235

Author(s):

Osiris Marroquin Belaunzaran ◽

Anahita Rafiei ◽

Anil Kumar ◽

Julia Kolibaba ◽

Lorenz Vogt ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Innate Immune System ◽

Cell Activation ◽

Innate Immune ◽

High Affinity ◽

Anti Tumor Activity

BackgroundThe human leukocyte immunoglobulin-like receptor family B (LILR B) acts as check point blockade of the innate immune system by inhibiting leukocyte activation through SHP phosphatase recruitment. Some of the physiological ligands include classical HLA class I molecules, including beta-2-microglobulin (B2M) free open conformers (OC). Natural HLA-OC expression is known from autoimmune disease leading to immune activation by pleiotropic effects since they bind to LILRB and KIR family members reducing Treg and MDSC numbers and increased effector T-cell and NK-cell activation, respectively. We have generated an IgG4-HLA-57 open conformer (OC) molecule (iosH2) with high affinity for LILRB molecules and demonstrate its anti-cancer activity in vitro and in vivo.Methods iosH2 was produced by transient gene expression in CHO cells and purified by standard chromatography. Affinity of iosH2 binding was quantified by ELISA and SPR analysis. HLA-G mediated signaling and competition was assessed using functional cell lines. Effect of iosH2 on activation of SHP1/2 was assessed using Western Blot. Functional assays including in vitro polarization and phagocytosis potential of primary macrophages was assessed by flow cytometry in the presence of iosH2 or isotype control. Effect of iosH2 on T cell activation was evaluated in co-cultures of cancer and T cells. Mouse models were used to assess in vivo activity.Results iosH2 binds to LILRB2 with high affinity and blocks the activation of HLA-G. In addition, iosH2 blocks receptor-mediated activation of SHP1/2. iosH2 promotes a shift from M2 to M1 macrophages with enhanced tumor cell phagocytosis in vitro. iosH2 enhances activation and killing potential of T cells in cancer cells and T cells co-culture assay. iosH2 exerts therapeutic efficacy in mouse transgenic (melanoma) and different syngeneic tumor models (e.g. pancreatic, colon and breast cancer) as monotherapy. Moreover, it acts synergistically in vivo with PD1 blocking antibodies achieving long-term tumor control. Ex vivo tumor sample analysis demonstrates a significant reduction of MDSC and Tregs and a shift towards an activated inflammatory M1 macrophage phenotype. Loss of MDSC functionality was paralleled by enhanced CD8+ T cell expansion and activity.Conclusions iosH2 binds to LILRB2 with high affinity, restores immune cell function in vitro and demonstrates anti-tumor activity in different in vivo mouse models. In addition, it acts synergistically in vivo with PD1. iosH2 is a first-in-class OC therapeutic with robust anti-tumor activity by promoting key components of the innate immune system. Clinical development is under way and phase I trial in preparation.

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Eosinophils and Macrophages within the Th2-Induced Granuloma: Balancing Killing and Healing in a Tight Space

Infection and Immunity ◽

10.1128/iai.00127-19 ◽

2019 ◽

Vol 87 (10) ◽

Cited By ~ 10

Author(s):

Anupama Ariyaratne ◽

Constance A. M. Finney

Keyword(s):

Immune Cells ◽

Host Response ◽

Inflammatory Diseases ◽

Innate Immune ◽

Mouse Strains ◽

Dual Function ◽

Underlying Mechanisms ◽

Parasitic Worms

ABSTRACTGranuloma formation is a key host immune response generated to confine invading pathogens and limit extensive host damage. It consists of an accumulation of host immune cells around a pathogen. This host response has been extensively studied in the context of inflammatory diseases. However, there is much less known about Th2-type granulomas generated in response to parasitic worms. Based onin vitrodata, innate immune cells within the granuloma are thought to immobilize and kill parasites but also act to repair damaged tissue. Understanding this dual function is key. The two billion people and many livestock/wild animals infected with helminths demonstrate that granulomas are not effective at clearing infection. However, the lack of high mortality highlights their importance in ensuring that parasite migration/tissue damage is restricted and wound healing is effective. In this review, we define two key cellular players (macrophages and eosinophils) and their associated molecular players involved in Th2 granuloma function. To date, the underlying mechanisms remain poorly understood, which is in part due to a lack of conclusive studies. Most have been performedin vitrorather thanin vivo, using cells that have not been obtained from granulomas. Experiments using genetically modified mouse strains and/or antibody/chemical-mediated cell depletion have also generated conflicting results depending on the model. We discuss the caveats of previous studies and the new tools available that will help fill the gaps in our knowledge and allow a better understanding of the balance between immune killing and healing.

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Prokaryotic ribosomal RNA stimulates zebrafish embryonic innate immune system

BMC Research Notes ◽

10.1186/s13104-019-4878-8 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Abhishikta Basu ◽

Maki Yoshihama ◽

Tamayo Uechi ◽

Naoya Kenmochi

Keyword(s):

Immune System ◽

Innate Immune System ◽

18S Rrna Gene ◽

Positive Control ◽

Innate Immune ◽

Rrna Gene ◽

Immune Recognition ◽

E Coli

Abstract Objectives Cell-culture studies reported that prokaryotic RNA molecules among the various microbe-associated molecular patterns (MAMPs) were uniquely present in live bacteria and were categorized as viability-associated MAMPs. They also reported that specific nucleotide modifications are instrumental in the discrimination between self and nonself RNAs. The aim of this study was to characterize the in vivo immune induction potential of prokaryotic and eukaryotic ribosomal RNAs (rRNAs) using zebrafish embryos as novel whole animal model system. Additionally, we aimed to test the possible role of rRNA modifications in immune recognition. Results We used three immune markers to evaluate the induction potential of prokaryotic rRNA derived from Escherichia coli and eukaryotic rRNAs from chicken (nonself) and zebrafish (self). Lipopolysaccharide (LPS) of Pseudomonas aeruginosa served as a positive control. E. coli rRNA had an induction potential equivalent to that of LPS. The zebrafish innate immune system could discriminate between self and nonself rRNAs. Between the nonself rRNAs, E. coli rRNA was more immunogenic than chicken rRNA. The in vitro transcript of zebrafish 18S rRNA gene without the nucleotide modifications was not recognized by its own immune system. Our data suggested that prokaryotic rRNA is immunostimulatory in vivo and could be useful as an adjuvant.

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Myeloid 12/15-LOX regulates B cell numbers and innate immune antibody levels in vivo

Wellcome Open Research ◽

10.12688/wellcomeopenres.10308.1 ◽

2017 ◽

Vol 2 ◽

pp. 1 ◽

Cited By ~ 3

Author(s):

Sarah N. Lauder ◽

Victoria J Tyrrell ◽

Keith Allen-Redpath ◽

Maceler Aldrovandi ◽

David Gray ◽

...

Keyword(s):

B Cell ◽

Cell Function ◽

Inflammatory Diseases ◽

Innate Immune ◽

Cell Phenotype ◽

Cell Numbers ◽

Antibody Levels ◽

Immune Antibody

Background: The myeloid enzyme 12/15-lipoxygenase (LOX), which generates bioactive oxidized lipids, has been implicated in numerous inflammatory diseases, with several studies demonstrating an improvement in pathology in mice lacking the enzyme. However, the ability of 12/15-LOX to directly regulate B cell function has not been studied.Methods: The influence of 12/15-LOX on B cell phenotype and function, and IgM generation, was compared using wildtype (WT) and 12/15-LOX (Alox15-/-) deficient mice. The proliferative and functional capacity of splenic CD19+B cells was measuredin vitroin response to various toll-like receptor agonists.Results: WT andAlox15-/-displayed comparable responses. Howeverin vivo, splenic B cell numbers were significantly elevated inAlox15-/-mice with a corresponding elevation in titres of total IgM in lung, gut and serum, and lower serum IgM directed against the 12/15-LOX product, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (HETE-PE).Discussion: Myeloid 12/15-LOX can regulate B cell numbers and innate immune antibody levelsin vivo, potentially contributing to its ability to regulate inflammatory disease. Furthermore, the alterations seen in 12/15-LOX deficiency likely result from changes in the equilibrium of the immune system that develop from birth. Further studies in disease models are warranted to elucidate the contribution of 12/15-LOX mediated alterations in B cell numbers and innate immune antibody generation to driving inflammationin vivo.

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Thawed Mesenchymal Stem Cell Product Shows Comparable Immunomodulatory Potency to Cultured Cells In Vitro and in Polymicrobial Septic Animals

Scientific Reports ◽

10.1038/s41598-019-54462-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Yuan Tan ◽

Mahmoud Salkhordeh ◽

Jia-Pey Wang ◽

Andrea McRae ◽

Luciana Souza-Moreira ◽

...

Keyword(s):

Cultured Cells ◽

Inflammatory Diseases ◽

Endothelial Permeability ◽

Innate Immune ◽

Activated T Cells ◽

Inflammatory Conditions ◽

Significant Difference ◽

Stem Cell Product

AbstractMesenchymal stem cells (MSCs) have been shown to exert immunomodulatory effects in both acute and chronic diseases. In acute inflammatory conditions like sepsis, cell therapy must be administered within hours of diagnosis, requiring “off-the-shelf” cryopreserved allogeneic cell products. However, their immunomodulatory potency, particularly in abilities to modulate innate immune cells, has not been well documented. Herein we compared the stabilities and functionalities of cultured versus thawed, donor-matched MSCs in modulating immune responses in vitro and in vivo. Cultured and thawed MSCs exhibited similar surface marker profiles and viabilities at 0 hr; however, thawed MSCs exhibited higher levels of apoptotic cells beyond 4 hrs. In vitro potency assays showed no significant difference between the abilities of both MSCs (donor-matched) to suppress proliferation of activated T cells, enhance phagocytosis of monocytes, and restore endothelial permeability after injury. Most importantly, in animals with polymicrobial sepsis, both MSCs significantly improved the phagocytic ability of peritoneal lavage cells, and reduced plasma levels of lactate and selected inflammatory cytokines without significant difference between groups. These results show comparable in vitro and in vivo immunomodulatory efficacy of thawed and fresh MSC products, providing further evidence for the utility of a cryopreserved MSC product for acute inflammatory diseases.

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