HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

Innate immune sensing of viral infection results in type I interferon (IFN) production and inflammasome activation. Type I IFNs, primarily IFN-α and IFN-β, are produced by all cell types upon virus infection and promote an antiviral state in surrounding cells by inducing the expression of IFN-stimulated genes. Type I IFN production is mediated by Toll-like receptor (TLR) 3 in HCV infected hepatocytes. Type I IFNs are also produced by plasmacytoid dendritic cells (pDC) after sensing of HIV and HCV through TLR7 in the absence of productive pDC infection. Inflammasomes are multi-protein cytosolic complexes that integrate several pathogen-triggered signaling cascades ultimately leading to caspase-1 activation and generation pro-inflammatory cytokines including interleukin (IL)-18 and IL-1β. Here, we demonstrate that HIV and HCV activate the inflammasome, but not Type I IFN production, in monocytes and macrophages in an infection-independent process that requires clathrin-mediated endocytosis and recognition of the virus by distinct endosomal TLRs. Knockdown of each endosomal TLR in primary monocytes by RNA interference reveals that inflammasome activation in these cells results from HIV sensing by TLR8 and HCV recognition by TLR7. Despite its critical role in type I IFN production by pDCs stimulated with HIV, TLR7 is not required for inflammasome activation by HIV. Similarly, HCV activation of the inflammasome in monocytes does not require TLR3 or its downstream signaling adaptor TICAM-1, while this pathway leads to type I IFN in infected hepatocytes. Monocytes and macrophages do not produce type I IFN upon TLR8 or TLR7 sensing of HIV or HCV, respectively. These findings reveal a novel infection-independent mechanism for chronic viral induction of key anti-viral programs and demonstrate distinct TLR utilization by different cell types for activation of the type I IFN vs. inflammasome pathways of inflammation.     

Role of type I interferons during macrophage activation by lipopolysaccharide

Activation of macrophages by bacterial lipopolysaccharide (LPS) is accompanied by the secretion of type I interferons (IFNs) which can act in an autocrine manner. We examined the role of type I IFNs in macrophage responses to LPS using bone marrow-derived macrophages (BMM) from IFNAR1-/- mice, which lack a component of the type I IFN receptor and do not respond to type I IFNs. We found that, unlike wild-type (WT) BMM, LPS-treated IFNAR1-/- cells failed to produce nitric oxide (NO), or express inducible NO synthase (iNOS), indicating that type I IFNs are essential for all LPS-stimulated NO production in BMM. Exogenously added type II IFN (IFNgamma) rescued these responses in LPS-treated IFNAR1-/- BMM. In contrast to effects on NO, type I IFNs negatively regulated respiratory burst activity in LPS-primed BMM. We also found that while type I IFNs mediated the anti-proliferative effects of lower concentrations of LPS, at higher concentrations LPS acted in a type I IFNs-independent manner. Finally, we report that type I IFNs are a survival factor for BMM. Despite this, the ability of LPS to also prevent apoptosis in BMM was independent of type I IFNs. These findings highlight the diverse roles of type I IFNs in mediating LPS-stimulated macrophage responses.     

Constitutively Active Inflammasome in Human Melanoma Cells Mediating Autoinflammation via Caspase-1 Processing and Secretion of Interleukin-1��

Interleukin-1β (IL-1β) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1β contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1β precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1β via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1β; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1β from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.     

IFN-alpha priming results in a gain of proinflammatory function by IL-10: implications for systemic lupus erythematosus pathogenesis

Interleukin-10 is a predominantly anti-inflammatory cytokine that inhibits macrophage and dendritic cell function, but can acquire proinflammatory activity during immune responses. We investigated whether type I IFNs, which are elevated during infections and in autoimmune diseases, modulate the activity of IL-10. Priming of primary human macrophages with low concentrations of IFN-alpha diminished the ability of IL-10 to suppress TNF-alpha production. IFN-alpha conferred a proinflammatory gain of function on IL-10, leading to IL-10 activation of expression of IFN-gamma-inducible, STAT1-dependent genes such as IFN regulatory factor 1, IFN-gamma-inducible protein-10 (CXCL10), and monokine induced by IFN-gamma (CXCL9). IFN-alpha priming resulted in greatly enhanced STAT1 activation in response to IL-10, and STAT1 was required for IL-10 activation of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma expression in IFN-alpha-primed cells. In control, unprimed cells, IL-10 activation of STAT1 was suppressed by constitutive activity of protein kinase C and Src homology 2 domain-containing phosphatase 1. These results demonstrate that type I IFNs regulate the balance between IL-10 anti- and proinflammatory activity, and provide insight into molecular mechanisms that regulate IL-10 function. Gain of IL-10 proinflammatory functions may contribute to its pathogenic role in autoimmune diseases characterized by elevated type I IFN levels, such as systemic lupus erythematosus.     

Soluble Interleukin-15 Complexes Are Generated In Vivo by Type I Interferon Dependent and Independent Pathways

Interleukin (IL)-15 associates with IL-15Rα on the cell surface where it can be cleaved into soluble cytokine/receptor complexes that have the potential to stimulate CD8 T cells and NK cells. Unfortunately, little is known about the in vivo production of soluble IL-15Rα/IL-15 complexes (sIL-15 complexes), particularly regarding the circumstances that induce them and the mechanisms responsible. The main objective of this study was to elucidate the signals leading to the generation of sIL-15 complexes. In this study, we show that sIL-15 complexes are increased in the serum of mice in response to Interferon (IFN)-α. In bone marrow derived dendritic cells (BMDC), IFN-α increased the activity of ADAM17, a metalloproteinase implicated in cleaving IL-15 complexes from the cell surface. Moreover, knocking out ADAM17 in BMDCs prevented the ability of IFN-α to induce sIL-15 complexes demonstrating ADAM17 as a critical protease mediating cleavage of IL-15 complexes in response to type I IFNs. Type I IFN signaling was required for generating sIL-15 complexes as in vivo induction of sIL-15 complexes by Poly I:C stimulation or total body irradiation (TBI) was impaired in IFNAR-/- mice. Interestingly, serum sIL-15 complexes were also induced in mice infected with Vesicular stomatitis virus (VSV) or mice treated with agonistic CD40 antibodies; however, sIL-15 complexes were still induced in IFNAR-/- mice after VSV infection or CD40 stimulation indicating pathways other than type I IFNs induce sIL-15 complexes. Overall, this study has shown that type I IFNs, VSV infection, and CD40 stimulation induce sIL-15 complexes suggesting the generation of sIL-15 complexes is a common event associated with immune activation. These findings reveal an unrealized mechanism for enhanced immune responses occurring during infection, vaccination, inflammation, and autoimmunity.     

Immunostimulatory properties of human dendritic cells generated using IFN-β associated either with IL-3 or GM-CSF

Despite limited clinical efficacy in large trials, dendritic cells (DC)-based immunization has yielded impressive responses in some patients. Key questions remain to be solved in order to optimize this therapeutic vaccine. Among them, the nature of the DC type used and its state of maturation are pivotal. Besides myeloid DC which are mostly used in clinical trials, a new DC type has been recently described resulting from the differentiation of monocytes in the presence of type I IFNs. In the present study, we analyze the features of type I IFNs DC generated in the presence of either IL-3 (IL-3-DC) or GM-CSF (GM-CSF-DC) and compare their capacity to respond to poly(I:C) and to subsequently trigger T-cell activation. The two DC types disclose a similar immunophenotype characterized by high levels of chemokines receptors, co-stimulatory and HLA molecules expression. After poly(I:C) maturation, both DC types display a marked upregulation of CD80, CD83 and CD86 and the same pattern of gene expression. In addition, poly(I:C) stimulated them to secrete IFN- and IL-12p70. Both DC types elicit potent allogeneic reactions. Priming of autologous T cells by IL-3-DC or GM-CSF-DC pulsed with an HLA-A2 restricted melan-A derived peptide, lead to the expansion of peptide specific CTL secreting high amounts of IFN-.We conclude that poly(I:C) matured IL-3-DC and GM-CSF-DC share similar phenotype and functional properties including the capacity to prime tumor-associated antigen specific CTL.N. Mazouz and O. Detournay equally contributed to this work.     

Anthrax Lethal Factor Cleavage of Nlrp1 Is Required for Activation of the Inflammasome

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.     

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI^−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI^−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI^−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.     

Type I IFN modulates innate and specific antiviral immunity

IFNs protect from virus infection by inducing an antiviral state and by modulating the immune response. Using mice deficient in multiple aspects of IFN signaling, we found that type I and type II IFN play distinct although complementing roles in the resolution of influenza viral disease. Both types of IFN influenced the profile of cytokines produced by T lymphocytes, with a significant bias toward Th2 differentiation occurring in the absence of responsiveness to either IFN. However, although a Th1 bias produced through inhibition of Th2 differentiation by IFN-gamma was not required to resolve infection, loss of type I IFN responsiveness led to exacerbated disease pathology characterized by granulocytic pulmonary inflammatory infiltrates. Responsiveness to type I IFN did not influence the generation of virus-specific cytotoxic lymphocytes or the rate of viral clearance, but induction of IL-10 and IL-15 in infected lungs through a type I IFN-dependent pathway correlated with a protective response to virus. Combined loss of both IFN pathways led to a severely polarized proinflammatory immune response and exacerbated disease. These results reveal an unexpected role for type I IFN in coordinating the host response to viral infection and controlling inflammation in the absence of a direct effect on virus replication.     

Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases

Caspases not only play an essential role during apoptotic cell death, but a subfamily of them-the inflammatory caspases-are associated with immune responses to microbial pathogens. Activation of inflammatory caspases, such as caspase-1 and caspase-5, occurs upon assembly of an intracellular complex, designated the inflammasome. This results in the cleavage and activation of the proinflammatory cytokines IL-1beta and IL-18. Mutations in one of the scaffold proteins of the inflammasome, NALP3/Cryopyrin, are associated with autoinflammatory disorders underscoring the importance of regulating inflammatory caspase activation.     

Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation.

Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human brains. Microglia, as the resident phagocyte, is a strong candidate for the clearance of apoptotic lymphocytes. Apoptosis was induced in cultured autologous thymocytes and in myelin basic protein (MBP)-specific, encephalitogenic T cells from Lewis rats by the addition of 0.1 microg/ml methylprednisolone. The amount of phagocytosis of apoptotic cells was assessed using an in vitro phagocytosis assay. Supernatants were collected to measure microglial cytokine secretion. The state of immune activation in microglia was investigated by a T cell proliferation assay and by flow cytometric analysis of microglial surface expression of immune molecules. Microglia ingested specifically apoptotic cells (apoptotic thymocytes as well as MBP-specific T cells) in contrast to nonapoptotic control cells (p < 0.0001). Subsequent secretion of the proinflammatory cytokines TNF-alpha and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-beta was not affected. Furthermore, ingestion of apoptotic cells led to increased microglial MHC class II expression without concomitant increase in MHC class I, costimulatory molecules, and ICAM expression. The Ag-specific activation of MBP-specific T cells in cocultures with microglia that had ingested apoptotic cells was significantly less than that of identical T cells that interacted with nonphagocytosing microglia. Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of apoptotic cells. The uptake of apoptotic cells by microglia is tolerogenic and results in a reduced proinflammatory cytokine production and a reduced activation of encephalitogenic T cells. This might help to restrict an autoimmune inflammation and minimize damage in the inflamed brain.     

pMGF505-7R determines pathogenicity of African swine fever virus infection by inhibiting IL-1β and type I IFN production

Inflammatory factors and type I interferons (IFNs) are key components of host antiviral innate immune responses, which can be released from the pathogen-infected macrophages. African swine fever virus (ASFV) has developed various strategies to evade host antiviral innate immune responses, including alteration of inflammatory responses and IFNs production. However, the molecular mechanism underlying inhibition of inflammatory responses and IFNs production by ASFV-encoded proteins has not been fully understood. Here we report that ASFV infection only induced low levels of IL-1β and type I IFNs in porcine alveolar macrophages (PAMs), even in the presence of strong inducers such as LPS and poly(dA:dT). Through further exploration, we found that several members of the multigene family 360 (MGF360) and MGF505 strongly inhibited IL-1β maturation and IFN-β promoter activation. Among them, pMGF505-7R had the strongest inhibitory effect. To verify the function of pMGF505-7R in vivo, a recombinant ASFV with deletion of the MGF505-7R gene (ASFV-Δ7R) was constructed and assessed. As we expected, ASFV-Δ7R infection induced higher levels of IL-1β and IFN-β compared with its parental ASFV HLJ/18 strain. ASFV infection-induced IL-1β production was then found to be dependent on TLRs/NF-κB signaling pathway and NLRP3 inflammasome. Furthermore, we demonstrated that pMGF505-7R interacted with IKKα in the IKK complex to inhibit NF-κB activation and bound to NLRP3 to inhibit inflammasome formation, leading to decreased IL-1β production. Moreover, we found that pMGF505-7R interacted with and inhibited the nuclear translocation of IRF3 to block type I IFN production. Importantly, the virulence of ASFV-Δ7R is reduced in piglets compared with its parental ASFV HLJ/18 strain, which may due to induction of higher IL-1β and type I IFN production in vivo. Our findings provide a new clue to understand the functions of ASFV-encoded pMGF505-7R and its role in viral infection-induced pathogenesis, which might help design antiviral agents or live attenuated vaccines to control ASF.     

The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance

The emergence of chronic inflammation during obesity in the absence of overt infection or well-defined autoimmune processes is a puzzling phenomenon. The Nod-like receptor (NLR) family of innate immune cell sensors, such as the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (Nlrp3, but also known as Nalp3 or cryopyrin) inflammasome are implicated in recognizing certain nonmicrobial originated 'danger signals' leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) and IL-18 secretion. We show that calorie restriction and exercise-mediated weight loss in obese individuals with type 2 diabetes is associated with a reduction in adipose tissue expression of Nlrp3 as well as with decreased inflammation and improved insulin sensitivity. We further found that the Nlrp3 inflammasome senses lipotoxicity-associated increases in intracellular ceramide to induce caspase-1 cleavage in macrophages and adipose tissue. Ablation of Nlrp3 in mice prevents obesity-induced inflammasome activation in fat depots and liver as well as enhances insulin signaling. Furthermore, elimination of Nlrp3 in obese mice reduces IL-18 and adipose tissue interferon-γ (IFN-γ) expression, increases naive T cell numbers and reduces effector T cell numbers in adipose tissue. Collectively, these data establish that the Nlrp3 inflammasome senses obesity-associated danger signals and contributes to obesity-induced inflammation and insulin resistance.     

Cutting Edge: A dual role for type I IFNs during polyinosinic-polycytidylic acid-induced NK cell activation

NK cells are cytotoxic lymphocytes that are most efficient at fulfilling their functions after a phase of priming provided by cytokines and/or accessory cells. Although type I IFNs are known to be important in this process, it remains unclear whether they act directly on NK cells or indirectly on accessory cells. We used adoptive transfer experiments and mixed bone marrow chimeras to dissect the requirement for type I IFN signaling in response to the dsRNA analog polyinosinic-polycytidylic acid. We demonstrate that optimal NK cell priming requires type I IFNs to signal on both NK cells and accessory cells. In the absence of IL-15, the residual NK cell activation was strictly dependent on cell-intrinsic IFNAR signaling in NK cells. Our results suggest that type I IFNs produced following viral infection simultaneously target accessory cells for IL-15 transpresentation and NK cells themselves and that these two pathways cooperate for NK cell priming.     

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.     

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.