The NLRP3 inflammasome is activated by nanoparticles through ATP, ADP and adenosine.

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO2) and titanium dioxide (nano-TiO2) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO2 or nano-TiO2 caused a significant increase in P2Y1, P2Y2, A2A and/or A2B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1ß secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2A or A2B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1ß secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.

ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation.

Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1ß (IL-1ß) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1ß production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.

NLRP3 inflammasome is required in murine asthma in the absence of aluminum adjuvant.

BACKGROUND: Inflammasome activation with the production of IL-1ß received substantial attention recently in inflammatory diseases. However, the role of inflammasome in the pathogenesis of asthma is not clear. Using an adjuvant-free model of allergic lung inflammation induced by ovalbumin (OVA), we investigated the role of NLRP3 inflammasome and related it to IL-1R1 signaling pathway. METHODS: Allergic lung inflammation induced by OVA was evaluated in vivo in mice deficient in NLRP3 inflammasome, IL-1R1, IL-1ß or IL-1α. Eosinophil recruitment, Th2 cytokine, and chemokine levels were determined in bronchoalveolar lavage fluid, lung homogenates, and mediastinal lymph node cells ex vivo. RESULTS: Allergic airway inflammation depends on NLRP3 inflammasome activation. Dendritic cell recruitment into lymph nodes, Th2 lymphocyte activation in the lung and secretion of Th2 cytokines and chemokines are reduced in the absence of NLRP3. Absence of NLRP3 and IL-1ß is associated with reduced expression of other proinflammatory cytokines such as IL-5, IL-13, IL-33, and thymic stromal lymphopoietin. Furthermore, the critical role of IL-1R1 signaling in allergic inflammation is confirmed in IL-1R1-, IL-1ß-, and IL-1α-deficient mice. CONCLUSION: NLRP3 inflammasome activation leading to IL-1 production is critical for the induction of a Th2 inflammatory allergic response.

DNA vaccination of macaques with several different Nef sequences induces multispecific T cell responses.

CD8(+) T lymphocytes play a key role in controlling viremia during primary human immunodeficiency virus-1 and in maintaining disease-free infection. It has recently been shown that DNA immunization of rhesus monkeys can elicit strong, long-lived antigen-specific cytotoxic T lymphocyte (CTL) responses. In previous work, it was shown that macaque CTL responses to lipopeptide vaccination were directed against a limited number of epitopes. In the present study, we used the DNA immunization approach to enlarge T cell responses to several epitopes and to multiple isolates. We immunized macaques with a mixture of six plasmids reflecting the variability of Nef epitopic regions in the simian immunodeficiency virus (SIV) mac251 primary isolate. The Nef genes from viruses included in the SIVmac251 primary isolate were sequenced and the six selected sequences were individually subcloned into the pCI vector, under cytomegalovirus enhancer/promoter control, and injected into macaques. We show that DNA immunization with Nef sequences induced interferon-gamma (IFN-gamma) secreting cell responses directed against several regions of Nef. Reacting T cell lines were expanded in vitro and multispecific CTL responses mapping the 96-138 Nef region were analyzed. Several peptides recognized by CTL were identified and studies using peptides reflecting the variability of Nef indicated that all of the Nef variants were recognized in the 96-138 region. Moreover, CTL responses were directed against an immunodominant epitope located in a functional region within the Nef protein that is essential for viral replication. This work shows that our approach of DNA immunization with several sequences induced multispecific T cell responses recognizing variants included in the SIVmac251 primary isolate.

Specific vaccine therapy in chronic hepatitis B: induction of T cell proliferative responses specific for envelope antigens.

In a pilot study, it was established that specific therapy by standard anti-hepatitis B virus (HBV) vaccination may be effective in reducing HBV replication and canceling the immune tolerance to hepatitis B surface antigen (HBsAg) particles in about 50% of persons with chronic active HBV replication. In the present study, the vaccine-induced immune responses were analyzed during an ongoing controlled multicenter vaccine trial. Vaccination elicited peripheral blood mononuclear cell proliferative responses specific for envelope antigen in 7 of 27 subjects given HBsAg. The responses induced by the vaccines were mediated by CD4+ T lymphocytes, and at least three different epitopes were recognized. HBV-specific CD4+ T lymphocytes produced high levels of interferon-gamma [corrected] and belonged to a T helper 1 subset. Reduction of serum HBV DNA in some of these persons suggests that induction of CD4+ T cell responses could be important in controlling viremia during vaccine therapy of chronic HBV carriers.

Immunotherapy of chronic hepatitis B by anti HBV vaccine.

Vaccine therapy is now used in various infectious diseases. The hepatitis B virus (HBV) leads to chronic infection in around 5% of patients with a high risk of chronic active hepatitis which may result in cirrhosis and hepatocellular carcinoma. The partial efficacy of antiviral therapies (40% of sustained inhibition of HBV replication), their cost, their possible side effects and the immune-mediated pathology of HBV infection explain the need of new immune therapies in treating HBV infection. Experimental and clinical evidences suggest the usefulness of vaccine therapy in HBV chronic infection. In a pilot and opened study, forty-six consecutive chronic HBsAg carriers with chronic hepatitis and detectable serum HBV DNA were given 3 standard injections of the GenHevac B vaccine at one month interval. Six months after the first injection, 12 patients (26.1%) had undetectable HBV DNA while 8 others showed significant decrease (more than 50%) in HBV DNA titers. Six of these 12 responders received a standard course of alpha-Interferon (5 MU thrice weekly subcutaneously for 4 months) and all six had still undetectable HBV replication at the end of follow-up. Among the 34 non responders to vaccine, 20 were given alpha-interferon and 2 the monophosphate derivate of Vidarabine: 12 of these 22 patients stopped HBV replication and in all 12, vaccine therapy had induced a significant decrease of HBV replication before the antiviral treatment with a decrease of mean serum HBV DNA from 392 pg/ml before to 217 pg/ml after vaccine therapy. In an ongoing controlled study, using the same vaccine schedule, serum HBV DNA disappeared more frequently after 6 months, in patients who were given a preS2/S vaccine (7/35) than in patients who received a S vaccine (1/21) or no vaccine (1/32). In responders to vaccine, an induction of specific proliferative responses was observed and this may contribute to the potential efficacy of anti-HBV vaccine therapy. No side-effect or vaccine-induced escape-mutants occurred during the follow-up. In summary, serum HBV DNA disappeared in 28 of the 46 patients (60.9%) who were given vaccine therapy, with (64.2%) or without (55.6%) Interferon. These results are not different at 6 months and at the end of follow-up from those of 43 HBsAg chronic carriers who were given only an antiviral treatment. Active immune therapy against HBV appears efficient and less expensive than antiviral therapies in stopping HBV replication. Such results need to be confirmed by the completed results of our controlled, randomized trial which is now conducted in our unit.

HLA-dependent variations in human immunodeficiency virus Nef protein alter peptide/HLA binding.

In human immunodeficiency virus (HIV) infection, sequence variations within defined cytotoxic T lymphocyte (CTL) epitopes may lead to escape from CTL recognition. In a previous report, we have shown that the variable central region of HIV Nef protein (amino acids 73-144) that contains potential CTL epitopes, can escape the CTL response. We suggested that this non recognition occurs through a variety of mechanisms. In particular, we provided evidence that HIV Nef sequences recovered from HLA-A11-expressing individuals have alterations in the major anchor residues essential for binding of the two Nef epitopes (amino acids 73-82 and 84-92) to the HLA-A11 molecule. Here, we investigate in more detail whether variations in autologous Nef sequences affect HLA binding, leading to CTL escape. Potential epitopes were sought by testing Nef peptides containing the HLA-A11-specific motif or related motifs. We confirmed that only the two previously described epitopes identified in cytolysis tests have optimal reactivity with the HLA-A11 molecule. We then sequenced several viral variants from donors that do not express the HLA-A11 molecule and compared the variability of these epitopes with those obtained from HLA-A11-expressing individuals. One substitution (Leu85) found in the sequences isolated from both populations increase the reactivity of the HLA-A11-restricted epitope 84-92, and might explain the difference in immunogenicity observed between the two HLA-A11-restricted epitopes from HLA-A11+ individuals. In addition, selective variations were only detected in virus isolated from HLA-A11-expressing individuals. Furthermore, examination of the association of variant peptides with the HLA-A11 molecule demonstrated that a single substitution within the minimal epitope could not always completely abrogate HLA binding, suggesting that multiple alterations within a particular epitope may accumulate during disease progression, allowing the virus to escape CTL recognition.

Identification of multirestricted immunodominant regions recognized by cytolytic T lymphocytes in the human immunodeficiency virus type 1 Nef protein.

Peripheral blood mononuclear cells from a large number of human immunodeficiency virus (HIV)-seropositive donors were used to analyze the CD8+ T-cell response to each part of the Nef protein of HIV-1/LAI. This report identifies an immunodominant region (amino acids 73 to 144) in the Nef protein that was recognized by 97% of the NEF responder donors. This peptide sequence was dissected into four epitopic regions (amino acids 73 to 82, 83 to 97, 113 to 128, and 126 to 144), each of which was recognized under different HLA class I restrictions. Short overlapping peptides were used to sensitive the target cells for cytolysis and so to determine if these epitopic regions were multirestricted. Each region was found to contain several epitopes recognized with different HLA molecules. Thus, the central region of the Nef protein, a regulatory protein expressed early in HIV-infected cells, is rich in epitopic sequences which are found to be similar in many infected individuals and which can be recognized in association with at least ten HLA class I molecules. Their implications for the vaccination of humans with peptide sequences are discussed.

Impaired cytotoxic T lymphocyte recognition due to genetic variations in the main immunogenic region of the human immunodeficiency virus 1 NEF protein.

Human immunodeficiency virus (HIV) induces strong responses from human histocompatibility leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL). In a previous report we identified an immunodominant region (amino acids 73-144) in the NEF protein that was recognized by CD8+ class I-restricted CTL of most asymptomatic individuals. Analysis of the 73-144 region by peptide sensitization, experiments using overlapping peptides corresponding to the LAI isolate identified the peptide sequences located between residues 73 and 82 or 84 and 92 and the peptide sequence between residues 134 and 144 as cognate peptides for HLA-A11- and HLA-B18-restricted epitopes, respectively. This report describes the variable demonstrable reactivities of CTL obtained from HLA-A11 or HLA-B18 seropositive, asymptomatic patients who all had a response to the virus NEF protein, but who did not always recognize appropriate cognate peptides. The high mutation rate of HIV probably facilitates the selection of mutants that can avoid the cellular immune response. We therefore analyzed the variability of these epitopes restricted by HLA-A11 and HLA-B18. We sequenced several viral isolates from HLA-A11 and HLA-B18 donors who recognized certain HLA-peptide complexes and from those who did not. A CTL sensitization assay was used to show that some mutations led to a great reduction in CTL activity in vitro. This might be due to failure of the mutated epitope to bind major histocompatibility complex class I molecule. A simple assay was used to detect peptides that promoted the assembly of class I molecules. Some of these mutations at major anchor positions prevented HLA-A11/peptide binding, and consequently impaired recognition of the HLA-peptide complex by the T cell receptor.

Diversity of the V3 region of HIV in Paris, France.

OBJECTIVE: To carry out, within France, a large-scale molecular epidemiological investigation on the principal neutralizing determinant of HIV-1, located in the third variable region (V3) of the envelope protein. Such investigations are of the utmost importance in the identification and monitoring of the distribution and spread of different viral strains internationally. DESIGN: Using polymerase chain reaction (PCR), we examined the genetic variation of the V3 region sequences of 28 HIV-infected patients from Paris, France. RESULTS: Comparison of the Parisian V3 loop sequences with other published data indicates that the range of diversity in France is included within that of a large group that contains sequences from North America, the rest of Europe, Japan, India and Africa. Variability appears to be lower in the V3 loop than in its flanking regions. Five out of the six putative N-linked glycosylation sites show preferential alterations to charged amino acids. We report two motifs at the tip of the loop that have not been described previously. CONCLUSIONS: The structural homogeneity and the wide geographic representation of the major V3 group suggests that a common strategy could be applied to a large proportion of isolates in the development of a broad-spectrum HIV vaccine.

Functional bioassays for B cell growth factors using polyclonally activated murine spleen B cells.

In this study, we have set up and optimized three distinct T cell-independent polyclonal B cell activation assay systems using highly T cell-depleted murine spleen B cells which were either preactivated in vitro with lipopolysaccharide (LPS) or costimulated with anti-IgM antibodies (anti-mu) or dextran sulfate (DxS). Using these assays, we have investigated the effects of recombinant human or murine interleukins, as well as those of a partially purified T cell-derived factor, designated BSF-LPS. Our results show that none of the interleukins, alone or in combination, is able to maintain growth of the LPS-preactivated B cell blasts, even at the highest concentrations tested, whereas the addition of our BSF-LPS preparation to the cultures significantly increases DNA synthesis. As expected, recombinant murine IL-4 (r mu IL-4) causes a substantial proliferation of anti-mu costimulated B cells. Such anti-mu costimulated B cells also respond, to a lower degree, to recombinant human IL-1 alpha (r hu IL-1 alpha), and do not significantly proliferate upon addition of r mu IL-2, r mu IL-5 or BSF-LPS. On the other hand, r mu IL-5 stimulates very efficiently DxS-costimulated B cells proliferation, whereas r hu IL-1 alpha only exerts a marginal effect; r mu IL-2, r mu IL-4 or BSF-LPS did not maintain growth of DxS-costimulated B cells. We believe that such a thorough investigation of the particular behaviour of activated B cell subpopulations towards various lymphokines provides the background for setting up a valuable bioassay method to differentiate the various interleukins acting on B cells through parallel use of the three distinct T cell-independent polyclonal B cell activation assay systems.