Resumo: O objetivo do trabalho foi avaliar o efeito da endotoxina associada ao formocresol em tecidos periapicais de dentes de macaco, por meio de avaliação histológica. Foram utilizados 36 pré-molares de 3 macacos, que após a abertura coronária e biopulpectomia, foram preenchidos com diferentes medicamentos: Grupo I (12 dentes): mistura de endotoxina/formocresol; Grupo II (15 dentes): endotoxina e curativo de formocresol; Grupo III (controle - 9 dentes): endotoxina. Decorridos 55 dias, os animais foram sacrificados, as peças removidas e submetidas a processamento histológico. Os resultados demonstraram que a associação endotoxina e formocresol, sendo ela na forma de mistura (grupo I) ou por introdução de endotoxina no canal e curativo de formocresol (grupo II) ocasionaram desenvolvimento de inflamação periapical severa, enquanto que a endotoxina sozinha (grupo III) provocou reação periapical moderada. De acordo com a análise estatística não houve diferença nos resultados entre o grupo I e grupo II. E foi observada diferença estatísticamente significante entre os grupos experimentais (grupo I e II) e o grupo controle (grupo III). / Abstract: The aim of this study was to evaluate the effect of endotoxin associated with formocresol on the periapical tissues from monkeys teeth, by using histological evaluation. Thirty-six premolar teeth from 3 monkeys have been used, and after the coronary opening and pulpectomy, they were filled with different medicaments: Group I (12 teeth): endotoxin associated with formocresol; Group II (15 teeth): endotoxin and cotton pullet of formocresol; Group III (control - 9 teeth): endotoxin. After 55 days, the animals were sacrificed and the teeth removed and then submitted to histological processing. The results obtained shown severe inflammation on periapical tissue on group I and on group II, and group III shown moderate inflammation on periapical tissue. / Orientador: Lizeti Toledo de Oliveira Ramalho / Coorientador: Elisa Maria Aparecida Giro / Banca: Denise Madalena Palomaria Spolidorio / Banca: Lourdes Aparecida Martins dos Santos Pinto / Banca: Maria Cristina Borsatto / Banca: Luis José Floriam / Doutor

A própolis é uma substância resinosa, cujas atividades antibacteriana, antiinflamatória, antiviral, fungicida, imuno estimulante, cicatrizante e de anestésico local tem sido valorizadas no uso clínico. O lipopolissacarídeo (LPS) é reconhecidamente uma endotoxina e pode induzir processos inflamatórios. Os objetivos deste trabalho foram: a) analisar in vitro as seguintes propriedades do extrato de própolis verde: 1) perfil físico químico 2) Concentração Inibitória Mínima (CIM) frente à endotoxina da bactéria gram negativa Escherichia coli; e 3) sua atividade imunorregulatória sobre leucócitos de baço de ratos; b) analisar &Prime;in vivo&Prime; a ação do Extrato Etanólico de Própolis (EEP) 10% e 90% e do Extrato de Própolis Puro (EPP), em alvéolos dentais contaminados ou não com lipopolissacarídeo (LPS) bacteriano. Para o estudo in vivo 35 ratos foram submetidos às exodontias dos primeiros molares superiores direito e esquerdo, os quais, imediatamente tiveram o alvéolo dental direito contaminado com 0,1µL de lipopolissacarídeo (LPS) (100µg/kg) e o esquerdo sem tal contaminação. Os grupos com (n=7) para cada tratamento, após 2 semanas: GI- Controle Negativo (CN) - sem tratamento; GII- Tratados com Extrato de Própolis puro (EPP) GIII- Tratados com pasta de própolis a 90% (P90); GIV- Tratados com pasta de própolis a 10% (P10); e, GV- Tratado com veículo das pastas (SB) foram analisados. Os alvéolos foram removidos, desmineralizados, processados pela técnica histológica de rotina, submetidos a secções sistematizadas a 6 µm de espessura e corados em H.E. O volume de osso formado, foi avaliado por contagem de pontos, usando um Sistema Teste sobreposto as imagens capturadas com auxílio de uma câmera acoplada a um microscópio. Observada a normalidade dos dados, procedeu-se o teste ANOVA fatorial e Tukey-Kramer test (p<0,05). In vitro determinou-se a propriedade da própolis, sua CIM e atividade imunorregulatória. In vivo os alvéolos dentais contaminados com lipopolissacarídeo bacteriano e tratados com a própolis verde apresentou maior área de osso neoformado, quando comparado aos demais grupos experimentais. O alvéolo não contaminado e tratado com própolis pura mostrou maior área de fibras colágenas. / Propolis is a resinous substance, whose antibacterial, antiinflammatory, antiviral, antifungal, immune stimulant, and local anesthetic wound healing properties has been considered for clinical practice. The lipopolysaccharide (LPS) is recognized as an endotoxin and can induce inflammatory processes. Our objectives were: a) to analyze in vitro the following properties of green propolis extract: 1) physicochemical profile of green propolis sample, 2) Minimum Inhibitory Concentration (MIC) against endotoxin from gram negative Escherichia coli and 3) its immunoregulatory activity using leukocytes from the spleen of mice,. b) analyze in vivo, the action of propolis ethanol extract (EEP) 10% and 90% and Pure Propolis Extract (EPP) in dental alveoli or not contaminated with lipopolysaccharide (LPS) bacterial. For in vivo study, 35 rats were subjected to extractions of maxillary first molars, right and left, which immediately had the right dental socket contaminated with 0.1 µL of lipopolysaccharide (LPS) (100µg/kg) and left without such contamination. They were divided into groups (n = 7) for each sample after 2 weeks, according to the treatment in the right and left alveoli: GI-Negative Control (NC) - no treatment, GII-Treated Pure Propolis Extract (EPP) GIII-Treated folder with propolis 90% (P90), GIV-Treated folder with propolis 10% (P10) and GV-Treated folder with vehicle (SB). The alveoli were removed, demineralized, processed by routine histologic technique, submitted to systematic sections (6 microns) of thick and stained with HE for histological analysis to assess the new bone tissue volume by point counting method, using a Test system on images captured with the aid of a digital camera attached to a microscope. Observed data normality, we proceeded to ANOVA and Tukey-Kramer tests (p <0.05). In vitro, it was determined the propolis properties, CIM and immunoregulatory activity. In vivo, the alveoli contamined with bacterian lipopolysaccharide and treated with green propolis induced higher bone formation when compared to other groups. The non contamined alveoli and treated with pure propolis showed more quantity of collagen fibers.

Introdução: Processar seguramente os dispositivos cirúrgicos reutilizáveis é um cuidado indireto de Enfermagem do Centro de Material e Esterilização. Muitos desses dispositivos apresentam conformação complexa que dificulta a limpeza, deixando dúvidas quanto ao alcance da esterilidade, ausência de biofilmes e endotoxinas. Entre os produtos utilizados em cirurgia para extração de catarata por facoemulsificação, as vias de irrigação e aspiração são materiais com alto risco de ocasionar endoftalmite ou síndrome tóxica do segmento anterior ocular no próximo usuário. Feitas de silicone, possuem lúmen de diâmetro com 1 a 2,5 mm e comprimento de 1,85 a 2,00 m, configurando-se em material de difícil processamento. Seus fabricantes não apresentam protocolos validados para seu processamento, o que constitui um grave problema de segurança ao paciente. Objetivo: Validar um Procedimento Operacional Padrão (POP) para processamento das vias de irrigação e aspiração do kit de facoemulsificação. Método: Esta pesquisa caracterizou-se, como um estudo laboratorial, utilizando contaminação-desafio. O POP foi elaborado com base no referencial teórico científico atual e na legislação pertinente. Como corpos de prova foram usados tubos de silicone, reproduzindo as dimensões do lúmen e do comprimento das vias de irrigação e aspiração. A análise foi feita por meio da espectroscopia de infravermelho que validou a equivalência da matéria-prima dos corpos de prova ao dispositivo original. Como contaminação-desafio, foi utilizada uma suspensão de Pseudomonas aeruginosa - ATCC 27853 106 UFC/mL acrescida de 20% de albumina humana em meio de cultura enriquecido de Tryptic Soy Broth (TSB) com solução salina balanceada. Para a contaminação dos corpos de prova, foram injetados 3 mL do contaminante desafio no lúmen dos corpos de prova de 1 mm e 7mL nos de 2 mm de diâmetro, e deixados de 1 a 17 horas. Em seguida, estes foram submetidos ao POP proposto. Como desfecho intermediário, foi avaliada a eficácia da limpeza (n=30) com teste de bioluminescência (Clean-Trace ATP water test, 3M®). A eficácia da esterilização (n=30) foi avaliada por meio da inoculação direta dos corpos de prova em TSB, e a detecção das endotoxinas (n=30), utilizando kit cinético-turbidimétrico. Os experimentos foram acompanhados por grupos controles positivo (n=3) e negativo (n=3). Resultados: a média de ATP obtida após a aplicação do POP de limpeza, expressa em RLUs, foi de 5,25 RLUs para os corpos de prova de 1 mm de diâmetro e 4,40 RLUs para os de 2 mm. A redução da quantidade média de ATP em RLUs foi de 98,5% a 99,98% respectivamente, para os tempos de contato com o contaminante desafio de 1 e 17h. Quanto à quantidade da endotoxina, esta variou de <0,01 UE/mL a 0,0192 UE/mL. Não houve recuperação do micro-organismo teste em nenhuma das amostras do grupo pesquisado. Os resultados dos controles positivo e negativo foram satisfatórios. Conclusão: Os resultados demonstraram que o POP proposto assegura o processamento das vias de irrigação e aspiração do kit de facoemulsificação. Esta conclusão não deve ser extrapolada para os reusos consecutivos autorizados pelos fabricantes, sem a realização de novos testes até o número máximo de reusos permitidos, justificado pelo fato da superfície dos lumens poderem ser alteradas a cada processamento. / Introduction: Reprocessing safely reusable surgical devices is an indirect nursing care in the Material and Sterilization Center. Many of these devices have complex geometry forms which difficult the cleaning process, leaving doubts about the scope of sterility and absence of biofilms and endotoxin. Among the products used in surgery for cataract extraction by phacoemulsification, the tubings of phacoemulsification machine are the materials with a high risk of causing endophthalmitis or toxic anterior segment syndrome in the next patient. Made of silicon, have a lumen diameter of 1 to 2.5 mm and a length of 1.85 to 2.00 m, and configuring itself in difficult materials to reprocessing. The manufacturers of these materials dont present validated protocols for reprocessing, which is a serious problem to the safety of the patient. Objective: To validate a standard operating procedure for reprocessing of the tubings of phacoemulsification machine. Method: This research was characterize as a laboratory study using test soil. The standard protocol was based on current scientific theoretical reference and relevant legislation. Silicone tubes reproducing lumen dimensions and the length of the original tubings of phacoemulsification machine were used as specimens. The analysis by infrared spectroscopy validated the equivalence of the primal material of the specimens to the original device. It was a test soil with Pseudomonas aeruginosa (ATCC 27853) 106 UFC/mL plus 20% human albumin in culture medium supplemented Tryptic Soy Broth (TSB) with balanced salt solution. To contamination of the samples were injected 3 to 7 mL of the test soil in the lumen and left for 1 and 17 hours. Then, these were submitted to the proposed standard protocol. As an intermediate outcome, the cleaning efficiency (n=30) was evaluated with bioluminescence test (Clean-Trace ATP water test, 3M®). The sterilization efficacy (n=30) was evaluated by direct inoculation specimens of silicone tubes in TSB, and the detection of endotoxin (n=30) using kinetic turbidimetric assay. The experiments were accompanied by positive control group (n=3) and negative (n=3). Results: The average ATP obtained after application of standard protocol cleaning, expressed in RLUs, was 5.25 RLUs for the specimens of silicone tubes of 1 mm in diameter and 4.40 RLUs to them 2 mm in diameter. The decrease in the average measure of ATP in RLUs was 98.5% to 99.98%, respectively for the periods of contact with the contaminant of 1h and 17h. As amount of endotoxin that ranged of <0.01 EU/mL to 0.0192 EU/mL. There wasnt recovery of the test microorganism in the samples of the group studied. The results of positive and negative controls were satisfactory. Conclusion: The results demonstrate that proposed standard protocol ensures the reprocessing of tubings of phacoemulsification machine. This conclusion should not be extrapolated to consecutive reuses authorized by the manufacturers without retest until the maximum number of allowed reuses justified by the fact that surface of the lumens can be changed at each reprocessing.

As células dendríticas (DCs) são células apresentadoras de antígeno suscetíveis a muitos sinais de ativação, os quais induzem diferentes padrões de ativação e resposta de linfócitos T. Neste trabalho, estudamos os efeitos de dois sinais de perigo, a febre e o LPS, sobre o fenótipo e função de DCs. A exposição de DCs ao calor não afetou a expressão de CD80 e CD86, capacidade endocítica ou produção de citocinas, características que foram afetadas pelo LPS. Entretanto, DCs expostas ao calor apresentaram uma maior atividade aloestimuladora e maior expressão de CD40. Quando DCs ativadas com LPS foram também estimuladas pelo calor, nenhuma alteração fenotípica na superfície celular foi notada, mas as DCs induziram maior produção de IFN-<font face=\"symbol\">g por linfócitos T alogenêicos e favoreceram a proliferação de linfócitos T CD8+. Esses dados indicam que a febre pode favorecer uma resposta celular, através de sua ação sobre DCs ativadas, com provável participação do CD40. Além de seu significado fisiológico, esse fenômeno pode ter aplicação em estratégias imunoterapêuticas. / Dendritic cells (DCs) are the main antigen presenting cells and susceptible to many activation signals that will induce different patterns of DC activation and of T cell responses. In this work we studied the effects of two danger signals, fever and LPS, on DC phenotype and function. Exposure of immature monocyte-derived dendritic cells to heat did not affect CD80 and CD86 expression, their endocytic ability, or their cytokine production, characteristics that were affected by LPS. However, heat-exposed DCs presented a higher allo-stimulatory activity and enhanced CD40 expression. When LPS activated DCs were also stimulated by heat, no cell surface phenotypic change was noted but these cells induced a higher IFN-<font face=\"symbol\">g secretion by allogeneic T lymphocytes and favored the proliferation of CD8+ cells. These data indicate that fever may cause a bias toward cellular responses, through its action on activated DCs, probably through CD40. Besides its physiological meaning, this phenomenon may have applications in immunotherapeutic strategies.

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T08:58:10Z (GMT). No. of bitstreams: 1 Marinho_ArianeCassiaSalustiano_M.pdf: 2625506 bytes, checksum: 4b9c6de7f98fb06e145d2b86f601d06f (MD5) Previous issue date: 2013 / Resumo: Lipopolisacarídeos (LPS) - endotoxinas são capazes de estimular células a produzirem citocinas pró-inflamatórias envolvidas na destruição tecidual periapical. Os objetivos do presente estudo foram: 1) Verificar e quantificar endotoxinas em canais radiculares infectados de dentes com periodontite apical crônica e sua relação com sinais e sintomas clínicos de origem endodôntica; 2) Avaliar a efetividade do preparo químico-mecânico (PQM) com hipoclorito de sódio (NaOCl) 2,5%; clorexidina gel (CLX) 2% e soro fisiológico (SS-controle) na eliminação de endotoxinas; 3) Avaliar o potencial inflamatório do conteúdo endodôntico, antes (C1) e após a instrumentação do canal radicular (C2) com NaOCl 2,5%, CLX 2% ou soro fisiológico e após uso de EDTA 17% (C3) em cultura de células de macrófagos quanto à produção de citocinas pró-inflamatórias - IL-1?, TNF-?. Amostras foram coletadas de 30 canais radiculares com necrose pulpar e presença de lesão periapical em C1, C2 e C3 utilizando cones de papel estéreis/apirogênicos. Endotoxina foi detectada em 100% dos canais radiculares estudados, representada pela mediana de 18,70 EU/mL. Dentes com presença de dor à percussão e exsudação intracanal foram relacionados com níveis elevados de endotoxina (p<0,05). Após o PQM, significativa redução de endotoxina foi obtida nos canais radiculares: NaOCl 2,5% + EDTA17% (99,75%), CLX gel 2% + EDTA 17% (98,27%), SS+ EDTA 17% (98,71%) (p<0,05). IL1- ? e TNF-? foram produzidos por macrófagos estimulados pelo conteúdo endodôntico (C1>C2>C3). Foi possível concluir que 1) Endotoxinas estavam presentes em todos os casos investigados, apresentando níveis mais elevados nos dentes com dor à percussão e exsudato intracanal. 2) O preparo químico-mecânico foi eficaz na redução do conteúdo de endotoxinas, independente da substância química auxiliar testada; 3) O potencial inflamatório do conteúdo endodôntico foi demonstrado pela produção de IL1- ? e TNF-?, exercendo maior atividade inflamatória contra macrófagos nas amostras iniciais, quando comparado com as obtidas após o preparo químico-mecânico / Abstract: Lipopolysaccharide (LPS)-endotoxin are able to stimulate cells to produce proinflammatory cytokines involved in the periapical tissue destruction. The aims of this study were: 1) To verify and quantify endotoxin in infected root canals of teeth with apical periodontitis and correlate the levels with clinical signs and symptoms of the primary endodontic infections; 2) To assess the effectiveness of chemo-mechanical preparation (CMP) with 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) and saline solution (SS), as negative control, for the elimination of endotoxins; 3) To evaluate the inflammatory potential of endodontic content before (C1) and after the CMP (C2) with 2.5% NaOCl, 2% CHX or SS and after final rinse with 17% EDTA (C3) in macrophage cell culture regarding the production of proinflammatory cytokines - IL-1 ? and TNF-?- via the enzyme-linked immunosorbent assay (ELISA). Samples were collected from 30 canals with pulpal necrosis and apical periodontitis in C1, C2 and C3 using sterile non-pyrogenic/paper points. Endotoxin was detected in 100% of root canals studied, represented by the median of 18.70 EU/mL. Teeth with tenderness to percussion and intracanal exudate were related to high levels of endotoxin (p< 0.05). After CMP, significant reduction of endotoxin was obtained in root canals instrumented with 2.5% NaOCl + 17% EDTA (99.75%), CHX 2% + 17% EDTA (98.27%) and SS + EDTA 17% (98,71%) (p< 0.05). IL1-? and TNF-? were produced by the macrophages in response to the endodontic content (C1>C2>C3). It was possible to conclude that 1) Endotoxin were present in all cases investigated, showing higher levels in the teeth with tenderness to percussion and intracanal exudate; 2) CMP was effective in reducing the endotoxic content, regardless of the auxiliary chemical substance; 3) The inflammatory potential of endodontic content was demonstrated by the production of IL1-? and TNF-? in all cases. The infectious content present in the root canal in the initial samples exerted greater inflammatory activity against macrophages compared to the residual content after CMP / Mestrado / Endodontia / Mestra em Clínica Odontológica

Inhalation of organic dust, including endotoxin, has been associated with inflammatory response of the pulmonary system. Limited studies have evaluated the work shift effects of endotoxin on respiratory outcomes for workers in the dairy industry, such as spirometry changes. Measurement techniques for exhaled nitric oxide (eNO) have been standardized by the American Thoracic Society (ATS) and used as a biomarker to identify diseases marked with lung inflammation. Dairy parlor workers are known to work long hours in one location with little job variability. The objectives of this study were to quantify exposure concentrations of inhalable dust and endotoxin among dairy parlor workers, evaluate acute cross-shift changes in respiratory status using spirometry, and assess the effectiveness of exhaled nitric oxide for detecting cross-shift bronchial inflammation changes. The cross-sectional study recruited 62 dairy parlor workers from 10 large herd dairy farms across Iowa, Minnesota, Wisconsin, and South Dakota. Data collected before and after the work shifts included spirometry tests, eNO measurements, and pulmonary symptom questionnaires. Personal breathing zone exposure to inhalable dust was assessed during the shift using Button Aerosol Samplers. Gravimetric analysis was used to determine airborne concentrations of inhalable dust and endotoxin concentration was determined using the recombinant factor C assay. Inhalable dust concentrations ranged from 0.09 - 4.95 mg/m3 with a geometric mean of 0.58 mg/m3. Inhalable endotoxin concentrations ranged from 4-1968 EU/m3 with a geometric mean of 117 EU/m3. The study participants pre-shift forced expiratory volume in the first second (FEV1) as a percentage of predicted was an average of 93.4%. Study group cross-shift FEV1 decreased by -1.16%. Six participants with moderate post-shift concentrations of eNO had an average FEV1 cross-shift change of -3.19%. Dairy parlor workers are exposed to concentrations of organic dusts that may adversely impact health. Future studies should test interventions in milking parlors to reduce dust exposure among dairy workers.

Within hours after its initiation, the severe systemic inflammatory response of sepsis shifts to an adaptive anti-inflammatory state with coincident immunosuppression. This anti-inflammatory phenotype is characterized by diminished proinflammatory cytokine gene expression in response to toll-like receptor (TLR) stimulation with bacterial endotoxin/lipopolysaccharide (LPS), also known as endotoxin tolerance/adaptation. Our and other studies have established that gene-specific reprogramming following TLR4 responses independently represses transcription and translation of proinflammatory genes such as tumor necrosis factor alpha (TNFα). We also previously demonstrated that TNFα and interleukin (IL)-6 mRNA translation is repressed in endotoxin-adapted THP-1 human monocytes by an miRNA-based mechanism involving the argonaute family protein argonaute 2 (Ago2). Here, we further define the molecular nature of reprogramming translation by showing that TLR4-induced microRNA-146 promotes a feed-forward loop that modifies the subcellular localization of the RNA-binding protein RBM4 (RNA-binding motif protein 4) and promotes its interaction with Ago2. This interaction results in the assembly of a translation-repressor complex that disrupts TNFα and IL-6 cytokine synthesis in endotoxin-adapted THP-1 monocytes. This novel molecular path prevents the phosphorylation of RBM4 on serine-309 by p38 MAPK (mitogen-activated protein kinase), which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or protein phosphatase inhibition by okadaic acid increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear relocalization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction.

Within hours after its initiation, the severe systemic inflammatory response of sepsis shifts to an adaptive anti-inflammatory state with coincident immunosuppression. This anti-inflammatory phenotype is characterized by diminished proinflammatory cytokine gene expression in response to toll-like receptor (TLR) stimulation with bacterial endotoxin/lipopolysaccharide (LPS), also known as endotoxin tolerance/adaptation. Our and other studies have established that gene-specific reprogramming following TLR4 responses independently represses transcription and translation of proinflammatory genes such as tumor necrosis factor alpha (TNFα). We also previously demonstrated that TNFα and interleukin (IL)-6 mRNA translation is repressed in endotoxin-adapted THP-1 human monocytes by an miRNA-based mechanism involving the argonaute family protein argonaute 2 (Ago2). Here, we further define the molecular nature of reprogramming translation by showing that TLR4-induced microRNA-146 promotes a feed-forward loop that modifies the subcellular localization of the RNA-binding protein RBM4 (RNA-binding motif protein 4) and promotes its interaction with Ago2. This interaction results in the assembly of a translation-repressor complex that disrupts TNFα and IL-6 cytokine synthesis in endotoxin-adapted THP-1 monocytes. This novel molecular path prevents the phosphorylation of RBM4 on serine-309 by p38 MAPK (mitogen-activated protein kinase), which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or protein phosphatase inhibition by okadaic acid increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear relocalization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction.

An anti-inflammatory phenotype with pronounced immunosuppression develops during sepsis, during which time neutrophils and monocytes/macrophages limit their Toll-like receptor 4 responses to bacterial lipopolysaccharide (LPS/endotoxin). We previously reported that during this endotoxin-tolerant state, distinct signaling pathways differentially repress transcription and translation of proinflammatory cytokines such as TNFα and IL-6. Sustained endotoxin tolerance contributes to sepsis mortality. While transcription repression requires chromatin modifications, a translational repressor complex of Argonaute 2 (Ago2) and RNA-binding motif protein 4 (RBM4), which bind the 3′-UTR of TNFα and IL-6 mRNA, limits protein synthesis. Here, we show that Dcp1 supports the assembly of the Ago2 and RBM4 repressor complex into cytoplasmic processing bodies (p-bodies) in endotoxin-tolerant THP-1 human monocytes following stimulation with LPS, resulting in translational repression and limiting protein synthesis. Importantly, this translocation process is reversed by Dcp1 knockdown, which restores TNFα and IL-6 protein levels. We also find this translational repression mechanism in primary macrophages of septic mice. Because p-body formation is a critical step in mRNA translation repression, we conclude that Dcp1 is a major component of the translational repression machinery of endotoxin tolerance and may contribute to sepsis outcome.

Heterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Gαi2 (-/-) or Gαi1/3 (-/-) mice were investigated. LPS- or SA-induced production of TNFα, IL-6, IFNγ, IL-12, IL-17, GM-CSF, MIP-1α, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p < 0.05) in splenocytes harvested from Gαi2(-/-) mice compared with WT mice. The effect of Gαi protein depletion was remarkably isoform specific. In splenocytes from Gαi1/3 (-/-) mice relative to WT mice, SA-induced IL-6, IFNγ, GM-CSF, and IP-10 levels were decreased (59% to 86%, p < 0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Gαi2 (-/-) and Gαi1/3 (-/-) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Gαi2 (-/-) and Gαi1/3 (-/-) mice relative to WT mice. The disparate response of splenocytes from the Gαi2 (-/-) relative to Gαi1/3 (-/-) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that Gi2 and Gi1/3 proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli.