Significance Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of sickle cell disease, malaria, and sepsis, but the mechanisms by which heme operates are not completely understood. Here we show that heme induces IL-1β processing through the activation of the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages. Our results suggest that among NLRP3 activators, heme has common as well as unique requirements to trigger inflammasome activation. In vivo, hemolysis and heme cause inflammasome activation. Importantly, macrophages, inflammasome components, and IL-1R contribute to hemolysis-induced lethality. These results highlight the potential of understanding the molecular mechanisms by which heme is sensed by innate immune receptors as a way to identify new therapeutic strategies to treat the pathological consequences of hemolytic diseases.

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Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4 ؊/؊ or to Myd88 ؊/؊ macrophages restored hemeinduced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1 ؊/؊ ) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS. (Blood. 2012;119(10): 2368-2375) IntroductionThe term programmed cell death was used for many years as a synonym of apoptosis, whereas necrosis in the opposite extreme was considered an abrupt and uncontrolled type of cell death. However, recent evidence clearly shows that several nonapoptotic cell death modes including autophagy, pyroptosis, and necrosis also involve elaborate molecular circuitry. 1,2 This scenario was originally revealed in a study showing that depending on the cell type, tumor necrosis factor (TNF) could trigger different cellular fates including survival, apoptosis, and necrosis. 3 On blockage of protein synthesis or NF-B, activation of death cytokine receptors of the TNF superfamily triggers caspase-dependent apoptosis, whereas simultaneous inhibition of caspase reorients the cell death to necrosis. [4][5][6][7] Receptor-interacting protein 1 (RIP1, also known as RIPK1) regulates survival and cell death fates. Mice deficient in Rip1 present extensive apoptosis, dying early after birth. The increased sensitivity to TNF-mediated cell death in Rip1 Ϫ/Ϫ cells correlates with a failure to activate NF-B. 8 Recent work shows that necrotic cell death is highly regulated by the RIP1 and RIP3 kinases (also known as RIPK3). 6,7,9-11 Programmed necrosis can be initiated by several stimuli including DNA damage, oxidative stress, infection, and activation of pattern recognition receptors. 1,2,[12][13][14][15][16][17] Intra or extra vascular hemolysis, rhabdomyolysis, and extensive cell damage cause the release of large quantities of hemeproteins. The oxidation of some hemeproteins including hemoglobin and myoglobin can release the heme moiety promoting further oxidation an...

Excessive release of proinflammatory cytokines by innate immune cells is an important component of the pathogenic basis of malaria. Proinflammatory cytokines are a direct output of Toll-like receptor (TLR) activation during microbial infection. Thus, interference with TLR function is likely to render a better clinical outcome by preventing their aberrant activation and the excessive release of inflammatory mediators. Herein, we describe the protective effect and mechanism of action of E6446, a synthetic antagonist of nucleic acid-sensing TLRs, on experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA. We show that in vitro, low doses of E6446 specifically inhibited the activation of human and mouse TLR9. Tenfold higher concentrations of this compound also inhibited the human TLR8 response to single-stranded RNA. In vivo, therapy with E6446 diminished the activation of TLR9 and prevented the exacerbated cytokine response observed during acute Plasmodium infection. Furthermore, severe signs of ECM, such as limb paralysis, brain vascular leak, and death, were all prevented by oral treatment with E6446. Hence, we provide evidence that supports the involvement of nucleic acid-sensing TLRs in malaria pathogenesis and that interference with the activation of these receptors is a promising strategy to prevent deleterious inflammatory responses that mediate pathogenesis and severity of malaria.immunotherapy | innate immunity | nucleic acid recognition | inflammation

High levels of circulating immunocomplexes (ICs) are found in patients with either infectious or sterile inflammation. We report that patients with either Plasmodium falciparum or Plasmodium vivax malaria have increased levels of circulating anti-DNA antibodies and ICs containing parasite DNA. Upon stimulation with malaria-induced ICs, monocytes express an NF-κB transcriptional signature. The main source of IC-induced proinflammatory cytokines (i.e., tumor necrosis factor alpha [TNF-α] and interleukin-1β [IL-1β])in peripheral blood mononuclear cells from acute malaria patients was found to be a CD14+ CD16 (FcγRIIIA)+ CD64 (FcγRI)high CD32 (FcγRIIB)low monocyte subset. Monocytes from convalescent patients were predominantly of the classical phenotype (CD14+ CD16−) that produces high levels of IL-10 and lower levels of TNF-α and IL-1β in response to ICs. Finally, we report a novel role for the proinflammatory activity of ICs by demonstrating their ability to induce inflammasome assembly and caspase-1 activation in human monocytes. These findings illuminate our understanding of the pathogenic role of ICs and monocyte subsets and may be relevant for future development of immunity-based interventions with broad applications to systemic inflammatory diseases.

Chronic hepatitis C virus (HCV) infection has also been associated with the development of several extrahepatic alterations, including thrombocytopenia, and a variety of pathogenic mechanisms are reported to be implicated in this hematological abnormality. Different studies have succeeded in detecting HCV in platelets with discrepant results. Moreover, most of the studies on HCV-associated thrombocytopenia have failed to provide data concerning the infecting genotype, a factor with prognostic implication in chronically HCV-infected patients. To determine whether thrombocytopenia is an extrahepatic alteration dependent on particular HCV genotypes, and to assess the relationship between thrombocytopenia and detection of HCV-RNA (positive strand) in platelets from patients with chronic HCV infection, 106 anti-HCV+/HCV-RNA+ patients (57 thrombocytopenic and 49 non-thrombocytopenic) were prospectively studied. The infecting genotype was analyzed from sera by using direct nucleotide sequencing of the polymerase chain reaction (PCR) products from core region. Genotypes 1a, 1b, and 3a were more prevalent in our patients, and no association between these genotypes and thrombocytopenia was observed ( p=0.891). HCV-RNA was detected in platelets by reverse transcriptase (RT)-nested PCR in the 5' non-coding region with a higher frequency (60%) in thrombocytopenic patients than in non-thrombocytopenic subjects (35%, p=0.017), suggesting that HCV is directly involved in the process that, at least in part, leads to thrombocytopenia.

One sentence summary: Lipooligosaccharide sialic acid, a key virulence factor for gonococci, can be targeted by novel immunotherapeutics to overcome multidrug-resistant gonorrhea. Editor: Alison Criss ABSTRACTGonorrhea has become resistant to most conventional antimicrobials used in clinical practice. The global spread of multidrug-resistant isolates of Neisseria gonorrhoeae could lead to an era of untreatable gonorrhea. New therapeutic modalities with novel mechanisms of action that do not lend themselves to the development of resistance are urgently needed. Gonococcal lipooligosaccharide (LOS) sialylation is critical for complement resistance and for establishing infection in humans and experimental mouse models. Here we describe two immunotherapeutic approaches that target LOS sialic acid: (i) a fusion protein that comprises the region in the complement inhibitor factor H (FH) that binds to sialylated gonococci and IgG Fc (FH/Fc fusion protein) and (ii) analogs of sialic acid that are incorporated into LOS but fail to protect the bacterium against killing. Both molecules showed efficacy in the mouse vaginal colonization model of gonorrhea and may represent promising immunotherapeutic approaches to target multidrug-resistant isolates. Disabling key gonococcal virulence mechanisms is an effective therapeutic strategy because the reduction of virulence is likely to be accompanied by a loss of fitness, rapid elimination by host immunity and consequently, decreased transmission.

RESUmoEstudo de natureza descritivo-reflexiva, objetivando apresentar as características do pesquisador que utiliza o método Teoria Fundamentada nos Dados e delinear reflexões sobre o desenvolvimento das aptidões do pesquisador para tornar-se um Teórico Fundamentado. A discussão teóri-ca foi embasada nos referenciais dessa metodologia e apoiada na literatura. O artigo apresenta as principais demandas de estudos qualitativos com a Grounded Theory e importantes comportamentos, atitudes e características desenvolvidas pelos pesquisadores. Constata-se que a aprendizagem acerca da Teoria Fundamentada nos Dados vai além da habilidade para operacionalizar um conjunto de procedimentos e técnicas. Implica, também, em desafios de transformação na postura como pesquisador e em novas formas de pensar e pesquisar, reunindo conhecimentos a partir dos dados para formar uma teoria. AbSTRAcTThis descriptive-reflexive study was performed with the objective to present the characteristics of researchers who use the Grounded Theory method, and outline the development of aptitudes for the researcher to become a Grounded Theoretician. The theoretical discussion was based on the frameworks of this methodology and supported by the literature. The article presents the main demands of qualitative studies using Grounded Theory, and important behaviors, attitudes and characteristics developed by the researchers. It is concluded that learning about Grounded Theory involves more than operationalizing a group of procedures and techniques. It also involves facing challenges to change one's attitude as a researcher and develop new ways of thinking and researching, gathering knowledge based on data to form a theory. dEScRiPToRSNursing Nursing research Nursing methodology research Qualitative research RESUmEnEstudio de naturaleza descriptivo-reflexiva que objetiva presentar las características del investigador que utiliza el método Teoría Fundamentada en los Datos y delinear reflexiones sobre el desarrollo de las aptitudes del investigador para transformarse en un Teórico Fundamentado. La discusión teórica se basó en los referenciales de esa metodología y obtuvo apoyo en la literatura. El artículo presenta las principales demandas de estudios cualitativos con la Grounded Theory e importantes comportamientos, actitudes, características desarrolladas por los investigadores. Se constata que el aprendizaje de la Teoría Fundamentada en los Datos va más allá de la habilidad para poner en operatividad un conjunto de procedimientos y técnicas. Implica también desafíos de transformación en la postura como investigador y nuevas formas de pensar e investigar, reuniendo conocimientos a partir de los datos para expresar una teoría. dEScRiPToRESEnfermería Investigación en enfermería Investigación metodológica en enfermería Investigación cualitativa