Turning up the heat : inflammasome activation by fungal pathogens

Since its first description in 2002 [1], the inflammasome has been implicated in the mechanisms underlying a growing number of infectious, autoimmune, and metabolic diseases [2]. Regarding infectious processes, several studies have shown the involvement of this critical component of innate immunity in the outcome of infection with nearly every class of microbe, including fungi [3]. Innate immunity is the frontline of defense against infection and relies on the ability of its main players (phagocytes and epithelial barriers) to detect conserved components of microbes or pathogen-associated molecular patterns (PAMPs). In fungi, the carbohydrate polymers of the cell wall, such as chitin, β-glucan, and mannan are the major PAMPs recognized by the host’s innate immune cells; this recognition occurs via germline-encoded receptors termed pattern recognition receptors (PRRs) [4]. In addition to PAMPs, endogenous molecules associated with damaged host cells, or damage-associated molecular patterns (DAMPs), are released during tissue injury and activate PRRs. This innate detection system includes the Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and AIM2-like receptors (ALRs). Although the main fungal- recognition PRRs (CLRs and TLRs) are bound to the cytoplasmic membrane of innate immune cells [4], fungal sensing by PRRs located in the cytosol, such as the NLRs and ALRs, is becoming increasingly evident. A number of NLRs and ALRs can assemble into the inflammasome, a multiprotein complex consisted of PRRs such as NLRP3 (NLR family, pyrin domain-containing 3), NLRC4, or AIM2, adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD), and procaspase-1 [3]. Upon formation of the complex, procaspase-1 is cleaved into an active cysteine protease, which further cleaves the proinflammatory cytokines IL-1β and IL-18 into their mature forms, followed by unconventional secretion. IL-1β and IL-18 mediate several innate antimicrobial responses and are critical to direct adaptive Th17/Th1 cellular responses [5]. In addition, inflammasome activation causes pyroptosis, a lytic inflammatory form of cell death [2,5]

Effects of bromopride on expression of metalloproteinases and interleukins in left colonic anastomoses : an experimental study

Anastomotic dehiscence is the most severe complication of colorectal surgery. Metalloproteinases (MMPs) and interleukins (ILs) can be used to analyze the healing process of anastomosis. To evaluate the effects of bromopride on MMP and cytokine gene expression in left colonic anastomoses in rats with or without induced abdominal sepsis, 80 rats were divided into two groups for euthanasia on the third or seventh postoperative day (POD). They were then divided into subgroups of 20 rats for sepsis induction or not, and then into subgroups of 10 rats for administration of bromopride or saline. Left colonic anastomosis was performed and abdominal sepsis was induced by cecal ligation and puncture. A colonic segment containing the anastomosis was removed for analysis of gene expression of MMP-1α, MMP-8, MMP-13, IL-β, IL-6, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). On the third POD, bromopride was associated with increased MMP-1α, MMP-13, IL-6, IFN-γ, and IL-10 gene expression. On the seventh POD, all MMP transcripts became negatively modulated and all IL transcripts became positively modulated. In the presence of sepsis, bromopride administration increased MMP-8 and IFN-γ gene expression and decreased MMP-1, TNF-α, IL-6, and IL-10 gene expression on the third POD. On the seventh POD, we observed increased expression of MMP-13 and all cytokines, except for TNF-α. In conclusion, bromopride interferes with MMP and IL gene expression during anastomotic healing. Further studies are needed to correlate these changes with the healing process

NLRP3 inflammasome activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1b that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1b in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL- 1 receptor, we demonstrated that IL-1b signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus

Extracellular Paracoccidioides brasiliensis phospholipase B involvement in alveolar macrophage interaction

Background: Phospholipase B (PLB) has been reported to be one of the virulence factors for human pathogenic fungi and has also been described as necessary for the early events in infection. Based on these data, we investigated the role of PLB in virulence and modulation of the alveolar pulmonary immune response during infection using an in-vitro model of host-pathogen interaction, i.e. Paracoccidioides brasiliensis yeast cells infecting alveolar macrophage (MH-S) cells. Results: The effect of PLB was analyzed using the specific inhibitor alexidine dihydrochloride (0.25 μM), and pulmonary surfactant (100 μg mL-1), during 6 hours of co-cultivation of P. brasiliensis and MH-S cells. Alexidine dihydrochloride inhibited PLB activity by 66% and significantly decreased the adhesion and internalization of yeast cells by MH-S cells. Genes involved in phagocytosis (trl2, cd14) and the inflammatory response (nfkb, tnf-α, il-1β) were down-regulated in the presence of this PLB inhibitor. In contrast, PLB activity and internalization of yeast cells significantly increased in the presence of pulmonary surfactant; under this condition, genes such as clec2 and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of interleukin-10 (IL-10) and increased the levels of IL-12 and tumor necrosis factor-α (TNF-α). In addition, gene expression analysis of plb1, sod3 and icl1 suggests that P. brasiliensis gene re-programming is effective in facilitating adaptation to this inhospitable environment, which mimics the lung-environment interaction. Conclusion: P. brasiliensis PLB activity is involved in the process of adhesion and internalization of yeast cells at the MH-S cell surface and may enhance virulence and subsequent down-regulation of macrophage activation

The role of T cell subsets and cytokines in the regulation of intracellular bacterial infection

Cellular immune responses are a critical part of the host's defense against intracellular bacterial infections. Immunity to Brucella abortus crucially depends on antigen-specific T cell-mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. T lymphocytes that proliferate in response to B. abortus were characterized for phenotype and cytokine activity. Human, murine, and bovine T lymphocytes exhibited a type 1 cytokine profile, suggesting an analogous immune response in these different hosts. In vivo protection afforded by a particular cell type is dependent on the antigen presented and the mechanism of antigen presentation. Studies using MHC class I and class II knockout mice infected with B. abortus have demonstrated that protective immunity to brucellosis is especially dependent on CD8+ T cells. To target MHC class I presentation we transfected ex vivo a murine macrophage cell line with B. abortus genes and adoptively transferred them to BALB/c mice. These transgenic macrophage clones induced partial protection in mice against experimental brucellosis. Knowing the cells required for protection, vaccines can be designed to activate the protective T cell subset. Lastly, as a new strategy for priming a specific class I-restricted T cell response in vivo, we used genetic immunization by particle bombardment-mediated gene transfer

Análise da força tênsil na cicatrização da parede abdominal de ratos tratados com infliximabe

OBJETIVO: Avaliar os efeitos do infliximabe, anticorpo monoclonal quimérico humano-murino, sobre a força tênsil da ferida operatória abdominal. MÉTODOS: Sessenta ratos, linhagem Wistar, machos, sadios, com peso corporal inicial entre 215 e 390 g e 60 e 90 dias de vida foram distribuídos aleatoriamente em dois grupos, E (Experimental) e C (Controle) com 30 animais cada. Os animais do grupo E receberam por via subcutânea, dose única, de 5mg/Kg de infliximabe, via subcutânea e os animais do grupo C receberam, volume equivalente, de solução de NaCl a 0,9%, via subcutânea. Depois de 48h os animais de ambos os grupos foram submetidos à incisão mediana na parede abdominal com 4 cm de extensão incluindo todos os planos que foram reconstituídos com sutura contínua músculo aponeurótica e pele, separadamente, com fio de nylon 5.0. A seguir os animais grupo E foram separados por sorteio simples em três subgrupos denominados E3, E7 e E14 com dez animais e os do grupo C em C3, C7 e C14 e foram submetidos, respectivamente, à reoperação e eutanásia no terceiro, sétimo e 14º dia pós-operatório. A parede abdominal anterior, ressecada dos animais durante a reoperação, foi cortada, com lamina de bisturi nº 15, perpendicularmente à ferida operatória. Cada espécime, em forma de fita, com 6 cm por 2 cm, foi preso pela extremidade de modo que a linha de sutura ficasse eqüidistante dos pontos de fixação do dinamômetro e realizado o teste de resistência tensil. O dinamômetro, aferido a cada série de medidas, foi calibrado para aplicar velocidade do teste de ruptura de 25 mm/min e o valor de ruptura foi expresso em N (Newtons) Antes da eutanásia a veia cava abdominal foi identificada e puncionada para retirada de sangue para dosagem de TNF-α. RESULTADOS: A média da força tensil encontrada para os animais dos subgrupos E3, E7, E14, C3, C7 e C14 foram, respectivamente, 16,03; 18,69; 27,01; 28,40; 27,22; 29,15 e 24,30 N. Nos resultados dos testes de múltiplas comparações foram encontradas diferenças significantes (p< 0,05) entre os subgrupos E3 e E7 quando comparados com C3, C7e C14. CONCLUSÃO: O infliximabe interferiu na cicatrização da ferida da parede abdominal com diminuição da força de ruptura na fase inflamatória e proliferativa.PURPOSE: To evaluate the effects of infliximab, a murine/human chimeric monoclonal antibody, on the tensile strength of abdominal wall surgical wounds. METHODS: Sixty Wistar healthy male rats with initial body weight between 215 and 390 g and 60 and 90 days of age were randomly assigned into two groups, E (Experimental) and C (Control) with 30 animals each. Group E animals received a single subcutaneous dose of 5mg/Kg of infliximab, and Group C animals received equivalent subcutaneous volume of a solution of 0.9% NaCl. After 48h, animals from both groups were submitted to a 4 cm median incision in the abdominal wall, including all layers that had been reconstituted with continuous suture of the aponeurotic muscle and skin, with 5.0 nylon thread. Then, Group E animals were separated by simple allotment into three subgroups named E3, E7 and E14 with ten animals each, and those from group C into C3, C7, C14 and were submitted, respectively, the reoperation and euthanasia at the third, seventh and fourteenth postoperative day. The anterior abdominal wall, which was resected during reoperation, was cut with No 15 scalpel lamina perpendicularly to the surgical wound. Each specimen, in the form of a 6 cm x 2 cm strip, was fixed by the extremity so that the suture line was equidistant from the fixation points of the dynamometer, in order to undergo the tensile strength test. The dynamometer, which was gauged for each series of measures, was calibrated to apply velocity to the 25 mm/min rupture test; the rupture value was expressed in N (Newton). Prior to euthanasia, the abdominal vena cava was identified and punctured in order to collect blood for TNF-α dosage. RESULTS: The mean tensile strength found for animals from subgroups E3, E7, E14, C3, C7, C14 were, respectively, 16.03, 18.69, 27.01, 28.40, 27.22, 29.15 and 24.30 N. In the results of the multiple comparisons tests, significant differences (p<0.05) was found between subgroups E3 and E7 compared with C3, C7 and C14. CONCLUSION: The infliximab interfered in the healing of the abdominal wall wound decreasing the rupture strength in the inflammatory and proliferative phases

Effects of metoclopramide on the expression of metalloproteinases and interleukins in left colonic anastomoses : an experimental study

PURPOSE: To evaluate the effects of metoclopramide on metalloproteinases (MMP) and interleukins (IL) gene expression in colonic anastomoses in rats. METHODS: Eighty rats were divided into two groups for euthanasia on the 3rd or 7th postoperative day (POD), then into two subgroups for sepsis induction or not, and then into subgroups to receive either metoclopramide or saline solution. Left colonic anastomosis were performed and then analyzed. RESULTS : On the 3rd POD, metoclopramide was associated with increased expression of MMP-1a, MMP-13, and TNF-α. On the 7th POD, the transcripts of all MMPs, TNF-α, IL-1β, IFN-γ, and IL-10 of the treated animals became negatively modulated. In the presence of sepsis, metoclopramide did not change MMPs and decreased IL-6, IL-1β, IFN-γ and IL-10 gene expression on the 3rd POD. On the 7th POD, increased expression of all MMPs, IFN-γ and IL-10 and negative modulated TNF-α and IL-6 gene expression. CONCLUSION: Administration of metoclopramide increased metalloproteinases and interleukins gene expression on the 3rd postoperative day and negatively modulated them on the 7th POD. In the presence of abdominal sepsis, metoclopramide did not change MMPs and decreased ILs gene expression on the 3rd POD. On the 7th POD, the drug increased expression of all MMPs

Alloxan-induced diabetes delays repair in a rat model of closed tibial fracture

A closed fracture was performed on the left tibia of 3-month-old Wistar rats weighing 250 to 350 g that were either healthy (N = 24) or made diabetic with alloxan (N = 24) to investigate the effect of alloxan-induced diabetes on the course of bone fracture healing. Histomorphometric analysis of the fracture site was performed at 7, 14, 25, and 35 days. After 7 days, diabetic rats had significantly less cartilage (P = 0.045) and greater fibrous connective (P = 0.006) tissue formation at the fracture site compared to controls. In contrast, marked callus formation was seen in diabetic rats with significant osteogenesis (P = 0.011, P = 0.010, P = 0.010, respectively, for 14, 25, and 35 days) and chondrogenesis (P = 0.028, P = 0.033, P = 0.019) compared to controls. Radiographic analysis revealed a displaced fracture with poor bone fragment alignment and delayed consolidation at these times in the diabetic group. The levels of alkaline phosphatase were significantly higher in diabetic rats at 25 days (P = 0.009). These results suggest that the initial excessive formation of fibrous connective tissue associated with delay in chondrogenesis and osteogenesis may not provide suitable stability of the fractured site, contributing to the inappropriate alignment of fragments and an increase in the volume of callus in later stages of repair. The resulting displaced fracture in diabetic rats requires long periods for remodeling and complete bone consolidation

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

Background: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts