Innate Receptors Research Articles

Differential gene expression profile analysis of RIG-I- versus type I IFN-stimulated primary human nasal epithelial cells and protection from influenza virus replication

Abstract Nasal mucosa is a key barrier against upper airway infection including influenza. Trained innate antiviral immunity has the potential to broadly protect the host from a variety of respiratory viruses. Although exposure of cells to type I IFN confers some degree of protection, direct stimulation of innate immune sensors may induce a more effective and longer lasting antiviral immunity. In the present study we analysed the gene expression profile and functional consequence of direct activation of RIG-I by its specific ligand 5′-triphosphate RNA (3pRNA) in primary human nasal epithelial cells, and compared these effects to stimulation of other innate receptors and stimulation by type I IFN. Gene expression analysis was carried out by Lexogen QuantSeq 3′mRNA sequencing. For functional analysis, epithelial cells were exposed to H1N1 influenza virus strain A/PR/8/34. Viral replication was analysed by qPCR. Gene set enrichment analysis revealed upregulation of pathways related to antiviral immune response and type I Interferon signaling. Activation of RIG-I in epithelial cells induced a broad panel of antiviral defense mechanisms which extends beyond those stimulated by recombinant type I IFN. RIG-I-stimulated cells were completely protected from influenza viral replication, while viability of the cells was not reduced. Recombinant IFN-b resulted in lower protection against viral replication compared to 3pRNA, demonstrating that RIG-I induces additional functional antiviral activities not induced by exogenous type I IFN acting alone. Thus, our findings support the concept that stimulation of RIG in epithelial cells is a promising strategy to protect cells from viruses including emerging viral infections of the respiratory tract.

Mechanism of Influenza Virus Induced Inflammatory Cell Death

Abstract RNA viruses and host cell death responses are co-evolved which prompt the fate of immune responses. Inflammatory cell death is a form of programmed signaling cascade which lead to the loss of cell integrity and secretion of leaderless pro-inflammatory cytokines. Host innate immune system exerts inflammatory cell death to curtail virus replication and initiate protective inflammatory and adaptive responses. This also causes host tissue damage if uncontrolled. Elevated inflammatory responses and lung damage are primary causes for morbidity and mortality during pathogenic influenza infections. However, little is known about key mechanisms regulating influenza A virus (IAV) induced cell death and inflammation. We identified Z-DNA Binding Protein 1 (ZBP1) as a sensor of IAV infection to trigger inflammatory cell death in in vitro and in vivo. ZBP1 induced inflammatory cell death is specific to nuclear replicating IAV but not the other RNA viruses those replicate in the cytosol. We found that the ZBP1 translocate to the nucleus prior to the cell death execution. Unlike other innate receptors, ZBP1 recognizes unique patterns of RNA in ribonucleoprotein complexes (vRNPs) of IAV. This nucleate RHIM domain driven oligomeric RIPK3 signaling scaffolds to facilitate RIPK3-Caspase-8 dependent inflammatory cell death (both necroptosis and pyroptosis). Our biochemical studies identified RIG-I dependent activation, ubiquitination of ZBP1 and loss of mitochondrial integrity precedes inflammatory cell death which facilitate the assembly of cell death signaling complexes proximal to the cellular membrane. Overall, our work demonstrates the mechanism of sensing of nuclear replicating IAV vRNPs to trigger programmed inflammatory cell death.

IL-9-secreting tissue-resident memory CD4+ T cells contribute to allergic airway recall responses

Abstract Asthma is a chronic intermittent inflammatory lung disease for which there is no cure. Allergen-specific CD4 T helper (Th) cells that secrete cytokines including IL-4, IL-13, and IL-9 mediate asthma pathogenesis. In a six week chronic model of allergic airway disease, we were able to detect a robust IL-9+CD4+ T cell population in the lungs. Interestingly, the in vivo derived IL-9+CD4+ T cells had a multi-cytokine profile including the production of IL-5, IL-13, and IL-10. IL-9-reporter-positive cells demonstrated high gene expression levels of innate cytokine receptors Il1rl1, Il17rb, and Crlf2 and the transcription factor Gata3. IL-9+CD4+ T cells were found to be tissue-resident through intravenous labeling and had CD4+ tissue-resident memory (Trm) cell markers CD69 and CD11a. To determine the Trm phenotype of lung resident IL-9+ T cells, we developed a memory model using a five week period of rest after six weeks of chronic challenge, before a recall allergen challenge. In the memory model, IL-9+ T cells maintained tissue residency and demonstrated increased cell frequency and IL-9 production, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. These observations further suggest that IL-9 from Trm cell populations plays a novel role in allergen recall responses and is a potential therapeutic target for patients suffering from chronic intermittent asthma.

Toll-like Receptor 2/IL-1β signaling is required for sensing and responding to a commensal bacterium at the ocular surface

Abstract Conjunctiva-associated lymphoid tissue (CALT) resembles a conventional mucosal immune tissue, and harbors a variety of immune cells. CALT is exposed to diverse microorganisms that come in contact with the ocular surface. Previously, we demonstrated that colonization of the ocular mucosa with the commensal bacterium, Corynebacterium mastitidis (C. mast), results in increased resistance of the ocular surface to infectious fungal and bacterial pathogens. Further, we showed that this effect is due to IL-17 produced by Vγ4+γδ T cells that respond to C. mast in an IL-1-dependent fashion. In this study, we show that the toll-like receptor 2 (TLR2) and IL-1β are important for IL-17 production, activation and proliferation of Vg4+ γδ T cells. Using in vivo and in vitro approaches, we show that cells of TLR2 KO mice have reduced production of IL-1β in response to C. mast. These mice also fail to recruit Vg4+γδ T cells and neutrophils to the conjunctiva. We interpret these data to mean that TLR2 is required for sensing this commensal on the conjunctiva and its draining lymph nodes to activate the inflammasome and consequent IL-1 production, which in turn drives the IL-17 response. Our data highlight the importance and the role of the innate receptor TLR2 in the generation of the ocular mucosal immune response and in maintenance of the homeostatic γδ T cell response at the ocular surface.

NOD-like receptors: major players (and targets) in the interface between innate immunity and cancer.

Innate immunity comprises several inflammation-related modulatory pathways which receive signals from an array of membrane-bound and cytoplasmic pattern recognition receptors (PRRs). The NLRs (NACHT (NAIP (neuronal apoptosis inhibitor protein), C2TA (MHC class 2 transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein) and Leucine-Rich Repeat (LRR) domain containing proteins) relate to a large family of cytosolic innate receptors, involved in detection of intracellular pathogens and endogenous byproducts of tissue injury. These receptors may recognize pathogen-associated molecular patterns (PAMPs) and/or danger-associated molecular patterns (DAMPs), activating host responses against pathogen infection and cellular stress. NLR-driven downstream signals trigger a number of signaling circuitries, which may either initiate the formation of inflammasomes and/or activate nuclear factor κB (NF-κB), stress kinases, interferon response factors (IRFs), inflammatory caspases and autophagy. Disruption of those signals may lead to a number of pro-inflammatory conditions, eventually promoting the onset of human malignancies. In this review, we describe the structures and functions of the most well-defined NLR proteins and highlight their association and biological impact on a diverse number of cancers.

CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis

Direct bacterial recognition by innate receptors is crucial for bacterial clearance. Here, we show that the IgA receptor CD89 is a major innate receptor that directly binds bacteria independently of its cognate ligands IgA and c-reactive protein (CRP). This binding is only partially inhibited by serum IgA and induces bacterial phagocytosis by CD11c+ dendritic cells and monocytes and/or macrophages, suggesting a physiological role in innate host defense. Blood phagocytes from common variable immunodeficiency patients bind, internalize, and kill bacteria in a CD89-dependent manner, confirming the IgA independence of this mechanism. Invivo, CD89 transgenic mice are protected in two different models of sepsis: a model of pneumonia and the cecal ligation and puncture (CLP) polymicrobial model of infection. These data identify CD89 as a first-line innate receptor for bacterial clearance before adaptive responses can be mounted. Fc receptors may emerge as a class of innate receptors for various bacteria with pleiotropic roles.

Toll-Like Receptor 7 Enhances Rabies Virus-Induced Humoral Immunity by Facilitating the Formation of Germinal Centers.

Rabies virus (RABV) causes fatal encephalitis in mammals and poses a public health threat in many parts of the world. Vaccination remains the most effective means for prevention and control of rabies. Studies focusing on the mechanism of RABV immunogenicity are necessary for improvement of rabies vaccines. Toll-like receptor 7 (TLR7), an innate receptor sensing single-stranded viral RNA, is important for the induction of innate and adaptive immunity. Our studies revealed that the absence of TLR7 led to a lower antibody production in mice immunized with RABV. It is further found that TLR7 deficiency affected the recruitment of germinal center (GC) B cells and led to lessened GCs formation. Consistently, there were less plasma cells (PCs) and antibody secreting cells (ASC) in TLR7−/− mice than those in wild type (WT) mice, resulting in impaired production of RABV-neutralizing antibodies (VNA). TLR7 deficiency also impaired the generation of memory B cells (MBCs) and the induction of secondary immune responses. Moreover, TLR7 deficiency down-regulated the induction of some cytokines/chemokines, especially IFN-γ, resulting in a Th2-biased antibody production. Overall, our results suggest that TLR7 facilitates the induction of the humoral immunity in response to RABV.

IL-10 Producing B Cells Dampen Protective T Cell Response and Allow Chlamydia muridarum Infection of the Male Genital Tract.

A significant proportion of individuals develop chronic, persistent and recurrent genital tract infections with Chlamydia trachomatis, which has been attributed to the numerous strategies that the bacterium uses to subvert host immune responses. Animal chlamydia models have demonstrated that protective immune response is mediated by CD4+ Th1 cytokine responses. Herein, we demonstrate that early after infecting the male genital tract, C. muridarum triggers the production of IL-10 by splenic and lymph node cells. In addition, C. muridarum triggers IL-6 and TNFα secretion. Data obtained from in vitro and in vivo experiments revealed B cells as the major IL-10 contributors. Indeed, purified B cells produced high amounts of IL-10 and also exhibited enhanced expression of inhibitory molecules such as CD39, PD-L1 and PD1 after C. muridarum stimulation. In vitro experiments performed with sorted cell subsets revealed that Marginal Zone B cells were the main IL-10 producers. In vitro and in vivo studies using TLR-deficient mice indicated that TLR4 signaling pathway was essential for IL-10 production. In addition, in vivo treatments to neutralize IL-10 or deplete B cells indicated that IL-10 and B cells played a significant role in delaying bacterial clearance ability. Moreover, the latter was confirmed by adoptive cell transfer experiments in which the absence of IL-10-producing B cells conferred the host a greater capability to induce Th1 responses and clear the infection. Interestingly, NOD mice, which were the least efficient in clearing the infection, presented much more Marginal Zone B counts and also enhanced TLR4 expression on Marginal Zone B cells when compared to B6 and BALB/c mice. Besides, treatment with antibodies that selectively deplete Marginal Zone B cells rendered mice more capable of inducing enhanced IFNγ responses and clearing the infection. Our findings suggest that B cells play a detrimental role in C. muridarum infection and that activation by innate receptors like TLR4 and IL-10 production by these cells could be used by Chlamydia spp. as a strategy to modulate the immune response establishing chronic infections in susceptible hosts.

Viral DNA Binding to NLRC3, an Inhibitory Nucleic Acid Sensor, Unleashes STING, a Cyclic Dinucleotide Receptor that Activates Type I Interferon

Immune suppression is a crucial component of immunoregulation and a subgroup of nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing proteins (NLRs) attenuate innate immunity. How this inhibitory function is controlled is unknown. A key question is whether microbial ligands can regulate this inhibition. NLRC3 is a negative regulator that attenuates type I interferon (IFN-I) response by sequestering and attenuating stimulator of interferon genes (STING) activation. Here, we report that NLRC3 binds viral DNA and other nucleic acids through its LRR domain. DNA binding to NLRC3 increases its ATPase activity, and ATP-binding by NLRC3 diminishes its interaction with STING, thus licensing an IFN-I response. This work uncovers a mechanism wherein viral nucleic acid binding releases an inhibitory innate receptor from its target.

Breaking down chronic inflammatory diseases: the role of biglycan in promoting a switch between inflammation and autophagy.

It is well established that biglycan, a small leucine-rich proteoglycan, acts as an extracellular matrix-derived danger signal in its soluble form. By binding to innate immunity Toll-like receptors (TLR) 2 and 4, biglycan initiates and perpetuates the inflammatory response. Previous work has conveyed that biglycan's role in inflammation extends far beyond its function as a canonical danger signal. It has been shown that biglycan acts in an anti-inflammatory capacity, wherein it tightly regulates the inflammatory response. In this review, we will discuss a paradigm shift to our understanding of biglycan signaling in inflammation. Mounting evidence suggests that the selective interactions between biglycan, TLRs, and their adapter proteins critically regulate downstream signaling and disease outcome. Biglycan can act as a high-affinity ligand for TLR coreceptors CD14 and CD44, further providing an additional layer of complexity. We propose a novel concept, that biglycan steers signaling toward inflammation by interacting with CD14, whereas it can trigger autophagy by binding to CD44. Thus, biglycan, and perhaps others soluble proteoglycans, could function as molecular switches which could either propagate the signaling of chronic inflammation or promote the resolution of inflammatory processes. Obviously, these new functions have broad implications in the regulation of various inflammatory diseases and could provide the basis for developing novel therapeutic regimens that would selectively target the interactions between biglycan, TLRs, coreceptors, and adapter molecules.

Abstract P2-06-23: New biomarkers for adjuvant bisphosphonate use in triple-negative breast cancer

Abstract We first showed that triple-negative breast cancer (TNBC) patients with low Toll-like receptor 9 (TLR9) expression have significantly worse prognosis than patients with high TLR9 expression. TLR9 is an innate immunity DNA receptor which is also expressed in several cancers, including TNBC. Later, we showed that low-TLR9 cells and tumors are more sensitive to amino-bisphosphonates, especially to zoledronate. Bisphosphonates (BP) inhibit osteoclasts and thereby effectively prevent bone fractures in osteoporosis and in cancer-induced bone disease. BPs also have direct anticancer effects. Interestingly, adjuvant BP use is associated with significantly improved progression-free and overall survival, but only among post-menopausal breast cancer patients. The molecular mechanisms behind this observation is unclear. CD73 is a 70 kDa glycophosphatidylinositol-anchored cell protein, which converts adenosine monophosphate to adenosine and organic phosphate. High CD73 expression has been shown to have cancer cell invasion-promoting properties. On the other hand, CD73 is associated with poor prognosis in TNBC. Interestingly, TLR9 and CD73 have been shown to have inverse relationship in T cells and during colonic inflammation. Currently, we study the relationship of CD73 and TLR9 in vitroand in vivo upon BP treatment. Our preliminary results show that TLR9 and CD73 have an inverse relationship also in TNBC cells. We use both nitrogen-containing (zoledronate and alendronate) and pyrophosphate-like (clodronate) BPs together with doxorubicin to study TNBC cell responses. We have seen that zoledronate and alendronate, but not clodronate, inhibited 4T1 murine triple-negative mammary tumor cell proliferation in a dose-dependent manner. Also, high concentrations of zoledronate and alendronate significantly inhibited 4T1 cell migration. Additionally, CD73 shRNA cells are more sensitive to zoledronate and alendronate, showing morphological changes and disruption in actin organization. Our results suggest that CD73 expression may affect treatment responses to BPs in TNBC. Further, low-CD73 tumors could benefit more of BP therapy, compared with high-CD73 tumors. Citation Format: Sandholm JA, Petruk N, Selander KS, Tuomela JM. New biomarkers for adjuvant bisphosphonate use in triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-23.

Ficolins and the Recognition of Pathogenic Microorganisms: An Overview of the Innate Immune Response and Contribution of Single Nucleotide Polymorphisms.

Ficolins are innate pattern recognition receptors (PRR) and play integral roles within the innate immune response to numerous pathogens throughout the circulation, as well as within organs. Pathogens are primarily removed by direct opsonisation following the recognition of cell surface carbohydrates and other immunostimulatory molecules or via the activation of the lectin complement pathway, which results in the deposition of C3b and the recruitment of phagocytes. In recent years, there have been a number of studies implicating ficolins in the recognition and removal of numerous bacterial, viral, fungal, and parasitic pathogens. Moreover, there has been expanding evidence highlighting that mutations within these key immune proteins, or the possession of particular haplotypes, enhance susceptibility to colonization by pathogens and dysfunctional immune responses. This review will therefore encompass previous knowledge on the role of ficolins in the recognition of bacterial and viral pathogens, while acknowledging the recent advances in the immune response to fungal and parasitic infections. Additionally, we will explore the various genetic susceptibility factors that predispose individuals to infection.

Abstract A050: Intratumoral delivery of a novel STING agonist synergizes with checkpoint blockade to regress multifocal pancreatic cancer

Abstract Immunosuppressive myeloid populations including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are abundant within pancreatic adenocarcinoma (PDAC) tumors and play critical roles in constraining cytotoxic T-cell function in the tumor microenvironment. We hypothesized that intratumoral engagement of innate pathogen recognition receptors such as Stimulator of Interferon Genes (STING) could induce proinflammatory polarization of the myeloid stroma and liberate the antitumor T-cell response to regress refractory PDAC tumors in the presence of checkpoint blockade.We developed and characterized a novel cyclic dinucleotide (CDN) STING agonist IACS-8803, and found that 8803 activates downstream STING signaling in both human (THP-1) and murine (J774) myeloid reporter cells with over 10-fold greater potency than ML-RR-S2-CDA, the first-in-class CDN currently undergoing clinical evaluation (NCT02675439, NCT03172936). Intratumoral delivery of 8803 into subcutaneous B16 melanoma and PDAC tumors additionally revealed a greater capacity to induce tumor regression relative to ML-RR-S2-CDA. In order to evaluate the specific effects of 8803 on the phenotype and function of suppressive myeloid populations, we generated in vitro polarized human M2 macrophages and murine bone marrow-derived MDSC. Upon exposure to 8803, we observe downregulation of M2 markers CD163, LAP/TGF-β, and Arginase on human M2 macrophages, concomitant with upregulation of M1 markers CD86, CD80, and IL-6. Additionally, 8803-stimulated murine MDSC exhibit reduced T-cell suppressive capacity compared to unstimulated MDSC. In these studies, we consistently observe that the magnitude of phenotypic and functional repolarization by 8803 is superior to that of ML-RR-S2-CDA as well as other known CDN, 2’3’-cGAMP and c-di-GMP. Therefore, we describe IACS-8803 as a novel, highly potent STING agonist with the capacity to induce inflammatory repolarization in suppressive myeloid cells of both human and murine origin. We next investigated the capacity for intratumoral delivery of IACS-8803 to sensitize murine pancreatic cancer to checkpoint blockade and to mobilize systemic immunity against disseminated lesions. We utilized mT4-2D, a novel pancreatic cancer cell line from Kras+/LSL-G12D Tp53+/LSL-R172H Pdx1-Cre tumor organoids. We isolateda single cell clone of mT4-2D with reduced in vivo growth kinetics (termed mT4-LS), as well as a clone that maintains the aggressive nature of the parental line (termed mT4-LA). Mice bearing 10-day established orthotopic and subcutaneous mT4-LS tumors received standard regimens of αCTLA-4, αPD-1, or combined αCTLA-4/αPD-1 in the presence or absence of 8803 CDN injected into the orthotopic pancreatic tumor. We find single-agent treatment with 8803, αCTLA-4, αPD-1, or αCTLA-4/αPD-1 can cure 40-60% of mice of both orthotopic and subcutaneous tumors in this system; however, combining 8803 with checkpoint blockade completely eradicates both injected and distal mT4-LS tumors in all mice. We replicated these studies using the highly aggressive and refractory mT4-LA model, and found that combination therapy with intra-pancreatic 8803 and systemic αCTLA-4/αPD-1 significantly extends survival compared to 8803 or αCTLA-4/αPD-1 alone (p=0.001, p=0.0086, respectively). Analysis of treated mT4-LA tumors by flow cytometry reveals that combination therapy enhances the cytotoxic potential of CD8 T-cells at both injected and uninjected lesions, and promotes dendritic cell proliferation within the pancreatic milieu. These studies provide a preclinical rationale for pursuing the use of STING-activating CDN as a localized approach to sensitize refractory PDAC tumors to checkpoint blockade immunotherapy. Citation Format: Casey R. Ager, Maria E. Di Francesco, Philip Jones, Michael A. Curran. Intratumoral delivery of a novel STING agonist synergizes with checkpoint blockade to regress multifocal pancreatic cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A050.

RNA Modifications Modulate Activation of Innate Toll-Like Receptors.

Self/foreign discrimination by the innate immune system depends on receptors that identify molecular patterns as associated to pathogens. Among others, this group includes endosomal Toll-like receptors, among which Toll-like receptors (TLR) 3, 7, 8, and 13 recognize and discriminate mammalian from microbial, potentially pathogen-associated, RNA. One of the discriminatory principles is the recognition of endogenous RNA modifications. Previous work has identified a couple of RNA modifications that impede activation of TLR signaling when incorporated in synthetic RNA molecules. Of note, work that is more recent has now shown that RNA modifications in their naturally occurring context can have immune-modulatory functions: Gm, a naturally occurring ribose-methylation within tRNA resulted in a lack of TLR7 stimulation and within a defined sequence context acted as antagonist. Additional RNA modifications with immune-modulatory functions have now been identified and recent work also indicates that RNA modifications within the context of whole prokaryotic or eukaryotic cells are indeed used for immune-modulation. This review will discuss new findings and developments in the field of immune-modulatory RNA modifications.

Human double negative T cells target lung cancer via ligand-dependent mechanisms that can be enhanced by IL-15

BackgroundThe advents of novel immunotherapies have revolutionized the treatment of cancer. Adoptive cellular therapies using chimeric antigen receptor T (CAR-T) cells have achieved remarkable clinical responses in B cell leukemia and lymphoma but the effect on solid tumors including lung cancer is limited. Here we present data on the therapeutic potential of allogeneic CD3+CD4−CD8− double negative T (DNT) cells as a new cellular therapy for the treatment of lung cancer and underlying mechanisms.MethodsDNTs were enriched and expanded ex vivo from healthy donors and phenotyped by flow cytometry. Functionally, their cytotoxicity was determined against primary and established non-small-cell lung cancer (NSCLC) cell lines in vitro or through in vivo adoptive transfer into xenograft models. Mechanistic analysis was performed using blocking antibodies against various cell surface and soluble markers. Furthermore, the role of IL-15 on DNT function was determined.ResultsWe demonstrated that ex vivo expanded DNTs can effectively lyse various human NSCLC cells in vitro and inhibit tumor growth in xenograft models. Expanded DNTs have a cytotoxic phenotype, as they express NKp30, NKG2D, DNAM-1, membrane TRAIL (mTRAIL), perforin and granzyme B, and secrete IFNγ and soluble TRAIL (sTRAIL). DNT-mediated cytotoxicity was dependent on a combination of tumor-expressed ligands for NKG2D, DNAM-1, NKp30 and/or receptors for TRAIL, which differ among different NSCLC cell lines. Furthermore, stimulation of DNTs with IL-15 increased expression of effector molecules on DNTs, their TRAIL production and cytotoxicity against NSCLC in vitro and in vivo.ConclusionHealthy donor-derived DNTs can target NSCLC in vitro and in vivo. DNTs recognize tumors via innate receptors which can be up-regulated by IL-15. DNTs have the potential to be used as a novel adoptive cell therapy for lung cancer either alone or in combination with IL-15.

Discovery of a New Natural Product and a Deactivation of a Quorum Sensing System by Culturing a "Producer" Bacterium With a Heat-Killed "Inducer" Culture.

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of “inducer” microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS.

Oleoylethanolamide, Neuroinflammation, and Alcohol Abuse.

Neuroinflammation is a complex process involved in the physiopathology of many central nervous system diseases, including addiction. Alcohol abuse is characterized by induction of peripheral inflammation and neuroinflammation, which hallmark is the activation of innate immunity toll-like receptors 4 (TLR4). In the last years, lipid transmitters have generated attention as modulators of parts of the addictive process. Specifically, the bioactive lipid oleoylethanolamide (OEA), which is an endogenous acylethanolamide, has shown a beneficial profile for alcohol abuse. Preclinical studies have shown that OEA is a potent anti-inflammatory and antioxidant compound that exerts neuroprotective effects in alcohol abuse. Exogenous administration of OEA blocks the alcohol-induced TLR4-mediated pro-inflammatory cascade, reducing the release of proinflammatory cytokines and chemokines, oxidative and nitrosative stress, and ultimately, preventing the neural damage in frontal cortex of rodents. The mechanisms of action of OEA are discussed in this review, including a protective action in the intestinal barrier. Additionally, OEA blocks cue-induced reinstatement of alcohol-seeking behavior and reduces the severity of withdrawal symptoms in animals, together with the modulation of alcohol-induced depression-like behavior and other negative motivational states associated with the abstinence, such as the anhedonia. Finally, exposure to alcohol induces OEA release in blood and brain of rodents. Clinical evidences will be highlighted, including the OEA release and the correlation of plasma OEA levels with TLR4-dependent peripheral inflammatory markers in alcohol abusers. In base of these evidences we hypothesize that the endogenous release of OEA could be a homeostatic signal to counteract the toxic action of alcohol and we propose the exploration of OEA-based pharmacotherapies to treat alcohol-use disorders.

Functional Programming of Innate Immune Cells in Response to Bordetella pertussis Infection and Vaccination.

Despite widespread vaccination, B. pertussis remains one of the least controlled vaccine-preventable diseases. Although it is well known that acellular and whole cell pertussis vaccines induce distinct immune functionalities in memory cells, much less is known about the role of innate immunity in this process. In this review, we provide an overview of the known differences and similarities in innate receptors, innate immune cells and inflammatory signalling pathways induced by the pertussis vaccines either licensed or in development and compare this to primary infection with B. pertussis. Despite the crucial role of innate immunity in driving memory responses to B. pertussis, it is clear that a significant knowledge gap remains in our understanding of the early innate immune response to vaccination and infection. Such knowledge is essential to develop the next generation of pertussis vaccines with improved host defense against B. pertussis.

Autophagy and Viral Infection.

Autophagy is an intracellular recycling process that maintains cellular homeostasis by orchestrating immunity upon viral infection. Following viral infection, autophagy is often initiated to curtail infection by delivering viral particles for lysosomal degradation and further integrating with innate pattern recognition receptor signaling to induce interferon (IFN)-mediated viral clearance. However, some viruses have evolved anti-autophagy strategies to escape host immunity and to promote viral replication. In this chapter, we illustrate how autophagy prevents viral infection to generate an optimal anti-viral milieu, and then concentrate on how viruses subvert and hijack the autophagic process to evade immunosurveillance, thereby facilitating viral replication and pathogenesis. Understanding the interplays between autophagy and viral infection is anticipated to guide the development of effective anti-viral therapeutics to fight against infectious diseases.

The regulatory capacity of B cells directs the aggressiveness of CLL

ABSTRACTChronic lymphocytic leukemia (CLL) is associated with abnormal T-cell responses responsible for defective anti-tumor activities. Intriguingly, CLL B cells share phenotypical characteristics with regulatory B (Breg) cells suggesting that they might negatively control the T-cell activation and immune responses. We elaborated an in vitro co-culture system with T cells to evaluate the Breg capacities of CLL B cells following innate Toll-like receptor 9 (TLR9) engagement. We demonstrated that B cells from half of the patients exhibited regulatory capacities, whilst B cells from the remaining patients were unable to develop a Breg function. The T cell sensitivities of all patients were normal suggesting that defective Breg activities were due to intrinsic CLL B cell deficiencies. Thus, TLR-dedicated gene assays highlighted differential signature of the TLR9 negative regulation pathway between the two groups of patients. Furthermore, correlations of the doubling time of lymphocytosis, the time to first treatment, the mutational status of IgVH and the Breg functions indicate that patients with efficient Breg activities have more aggressive CLL than patients with defective Breg cells. Our in vitro observations may open new approaches for adjusting therapeutic strategies targeting the Breg along with the evolution of the disease.