Marco Túlio R. Gomes

Plant cysteine proteinases: Evaluation of the pharmacological activity

Cysteine proteinases are involved in virtually every aspect of plant physiology and development. They play a role in development, senescence, programmed cell death, storage and mobilization of germinal proteins, and in response to various types of environmental stress. In this review, we focus on a group of plant defensive enzymes occurring in germinal tissue of Caricaceae. These enzymes elicit a protective response in the unripe fruit after physical stress. We propose that these enzymes follow a strategy similar to mammalian serine proteinases involved in blood clotting and wound healing. We show evidence for the pharmacological role of plant cysteine proteinases in mammalian wound healing, immunomodulation, digestive conditions, and neoplastic alterations.

Critical Role of ASC Inflammasomes and Bacterial Type IV Secretion System in Caspase-1 Activation and Host Innate Resistance to Brucella abortus Infection

Pathogens are detected by innate immune receptors that, upon activation, orchestrate an appropriate immune response. Recent studies revealed the intracellular signaling cascades involved in the TLR-initiated immune response to Brucella abortus infection. However, no report has elucidated the role of inflammasome receptors in Brucella recognition. Therefore, we decided to investigate the function of NLRC4, NLRP3, and AIM2 in sensing Brucella. In this study, we showed that NLRC4 is not required to induce caspase-1 activation and further secretion of IL-1β by B. abortus in macrophages. In contrast, we determined that AIM2, which senses Brucella DNA, and NLRP3 are partially required for caspase-1 activation and IL-1β secretion. Additionally, mitochondrial reactive oxygen species induced by Brucella were implicated in IL-1β production. Furthermore, AIM2, NLRP3, ASC, and caspase-1 knockout mice were more susceptible to B. abortus infection than were wild-type animals, suggesting that multiple ASC-dependent inflammasomes contribute to host protection against infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than pyroptosis, because we observed augmented bacterial burden in IL-1R and IL-18 knockout mice. Finally, we determined that bacterial type IV secretion system VirB and live, but not heat-killed, Brucella are required for full inflammasome activation in macrophages during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes that collectively orchestrate a robust caspase-1 activation and proinflammatory response.

Toll-Like Receptor 6 Plays an Important Role in Host Innate Resistance to Brucella abortus Infection in Mice

ABSTRACT Brucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-κB signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.

The role of innate immune signals in immunity to Brucella abortus

Innate immunity serves as the first line of defense against infectious agents such as intracellular bacteria. The innate immune platform includes Toll-like receptors (TLRs), retinoid acid-inducible gene-I-like receptors and other cytosolic nucleic acid sensors, nucleotide-binding and oligomerization domain-like receptors, adaptors, kinases and other signaling molecules that are required to elicit effective responses against different pathogens. Our research group has been using the Gram-negative bacteria Brucella abortus as a model of pathogen. We have demonstrated that B. abortus triggers MAPK and NF-κB signaling pathways in macrophages in a MyD88 and IRAK-4-dependent manner. Furthermore, we claimed that so far TLR9 is the most important single TLR during Brucella infection. The identification of host receptors that recognize pathogen-derived nucleic acids has revealed an essential role for nucleic acid sensing in the triggering of immunity to intracellular pathogens. Besides TLRs, herein we describe recent advances in NOD1, NOD2, and type I IFN receptors in innate immune pathways during B. abortus infection.

Interleukin-1 Receptor-Associated Kinase 4 Is Essential for Initial Host Control of Brucella abortus Infection

ABSTRACT Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Recent studies have revealed that Toll-like receptor (TLR)-initiated immune response to Brucella spp. depends on myeloid differentiation factor 88 (MyD88) signaling. Therefore, we decided to study the role of the interleukin-1 receptor-associated kinase 4 (IRAK-4) in host innate immune response against B. abortus. After Brucella infection, it was shown that the number of CFU in IRAK-4−/− mice was high compared to that in IRAK-4+/− animals only at 1 week postinfection. At 3 and 6 weeks postinfection, IRAK-4−/− mice were able to control the infection similarly to heterozygous animals. Furthermore, the type 1 cytokine profile was evaluated. IRAK-4−/− mice showed lower production of systemic interleukin-12 (IL-12) and gamma interferon (IFN-γ). Additionally, a reduced percentage of CD4+ and CD8+ T cells expressing IFN-γ was observed compared to IRAK-4+/−. Further, the production of IL-12 and tumor necrosis factor alpha (TNF-α) by macrophages and dendritic cells from IRAK-4−/− mice was abolished at 24 h after stimulation with B. abortus. To investigate the role of IRAK-4 in mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, macrophages were stimulated with B. abortus, and the signaling components were analyzed by protein phosphorylation. Extracellular signal-regulated kinase 1 (ERK1) and ERK2 and p38 as well as p65 NF-κB phosphorylation was profoundly impaired in IRAK-4−/− and MyD88−/− macrophages activated by Brucella. In summary, the results shown in this study demonstrated that IRAK-4 is critical to trigger the initial immune response against B. abortus but not at later phases of infection.

Plant Proteinases and Inhibitors: An Overview of Biological Function and Pharmacological Activity

Proteinases play a fundamental metabolic role during the life cycle in the plant kingdom. By interacting with endogenous or exogenous inhibitors, the proteolytic activity is modulated to meet metabolic requirements. By probing proteolytic enzymes with their inhibitors, it is possible to identify novel functions unrelated to their proteolytic activity. A group of plant proteolytic enzymes stands as a line of defence against environmental changes as their activation is triggered following various types of stress. On the other hand, plants also contain proteinase inhibitors as countermeasures for their protection against insects and pests. Both proteinases and inhibitors emerge as useful tools to combat human diseases. This review focuses on the biochemical characterization of plant proteinases, their inhibitors, the pharmacological potential of proteinases and inhibitors, and new putative emerging functions of proteolytically inhibited proteinases.

5-Lipoxygenase Negatively Regulates Th1 Response during Brucella abortus Infection in Mice

ABSTRACT Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B4 and lipoxin A4 in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen.

The role of NLRP3 and AIM2 in inflammasome activation during Brucella abortus infection

The innate immune system is essential for the detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1β secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1β upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1β by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response.

Toll-like receptor 6 senses Mycobacterium avium and is required for efficient control of mycobacterial infection.

Mycobacterium avium has been reported to signal through both Toll‐like receptor (TLR2) and TLR9. To investigate the role of TLR6 in innate immune responses to M. avium, TLR6, MyD88, TLR2, and TLR2/6 KO mice were infected with this pathogen. Bacterial burdens were higher in the lungs and livers of infected TLR6, TLR2, TLR2/6, and MyD88 KO mice compared with those in C57BL/6 mice, which indicates that TLR6 is required for the efficient control of M. avium infection. However, TLR6 KO spleen cells presented with normal M. avium induced IFN‐γ responses as measured by ELISA and flow cytometry. In contrast, the production of IFN‐γ in lung tissue was diminished in all studied KO mice. Furthermore, only MyD88 deficiency reduced granuloma areas in mouse livers. Moreover, we determined that TLR6 plays an important role in controlling bacterial growth within macrophages and in the production of TNF‐α, IL‐12, and IL‐6 by M. avium infected DCs. Finally, the lack of TLR6 reduced activation of MAPKs and NF‐κB in DCs. In summary, TLR6 is required for full resistance to M. avium and for the activation of DCs to produce proinflammatory cytokines.

Determination of sphingomyelinase-D activity of Loxosceles venoms in sphingomyelin/cholesterol liposomes containing horseradish peroxidase

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.