Clinical outcomes in a murine model after envenoming by the Amazonian scorpions Tityus strandi and Tityus dinizi.

The Brazilian Amazon is home to a rich fauna of scorpion species of medical importance, some of them still poorly characterized regarding their biological actions and range of clinical symptoms after envenoming. The Amazonian scorpion species Tityus strandi and Tityus dinizi constitute some of the scorpions in this group, with few studies in the literature regarding their systemic repercussions. In the present study, we characterized the clinical, inflammatory, and histopathological manifestations of T. strandi and T. dinizi envenoming in a murine model using Balb/c mice. The results show a robust clinical response based on clinical score, hyperglycemia, leukocytosis, increased cytokines, and histopathological changes in the kidneys and lungs. Tityus strandi envenomed mice presented more prominent clinical manifestations when compared to Tityus dinizi, pointing to the relevance of this species in the medical scenario, with both species inducing hyperglycemia, leukocytosis, increased cytokine production in the peritoneal lavage, increased inflammatory infiltrate in the lungs, and acute tubular necrosis after T. strandi envenoming. The results presented in this research can help to understand the systemic manifestations of scorpion accidents in humans caused by the target species of the study and point out therapeutic strategies in cases of scorpionism in remote regions of the Amazon.

Evaluation of anti-inflammatory activity of kaurenol: Experimental evaluation and mechanistic insights.

BACKGROUND: Kaurenol, a diterpene alcohol found in Copaifera langsdorffii Desf. (known as "copaiba"), is historically used in traditional medicine for inflammatory conditions. OBJECTIVES: This study aims to comprehensively assess the potential anti-inflammatory and antinociceptive properties of kaurenol. METHODS: To this end, the following experiments were conducted to evaluated toxicity: locomotor performance and acute toxicity; nociception: acetic acid-induced writhing and formalin-induced antinociception; and anti-inflammatory activity: carrageenan and dextran-induced paw edema at 10, 20, and 40 mg/kg, and measurement of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in macrophages at 1, 3, and 9 µg/ml. RESULTS: Kaurenol did not show significant locomotor changes, acute toxicity, and central analgesic activity in the first phase of formalin test at dosages tested. Kaurenol showed 53%, 64%, 64%, and 58% of inhibition in the acetic acid-induced writhing, second phase of formalin test, carrageenan and dextran-induced paw edema, respectively. CONCLUSION: The anti-inflammatory activity was associated with the regulation of NO release and probably with the regulation of mediators, such as serotonin and prostaglandin in vascular permeability, as well as by being associated with the regulation of IL-6 and IL-10. Kaurenol display anti-inflammatory activity but has no analgesic activity.

Heterologous expression of Ts8, a neurotoxin from Tityus serrulatus venom, evidences its antifungal activity.

In this study we expressed the Ts8, a neurotoxin from Tityus serrulatus scorpion venom, in Pichia pastoris yeast. We evaluated the peptide expression in different conditions, such as pH, temperature, and addition of casamino acids supplement. Analyses of expressed products by mass spectrometry and Edman degradation showed that rTs8 has sites that allow its cleavage by yeast proteases released into the culture medium. The casamino acids addition was favourable for toxin expression, however, was not sufficient to minimize proteolytic degradation. Functional assays with recombinant toxin fragments and native toxins have demonstrated the release of cytokines such as TNF-α and IL-1ß in some peptides tested. In addition, the toxins were shown to inhibit the Pichia pastoris growth in antifungal test and were not toxic to alveolar macrophages cells at the concentrations analyzed The electrophysiological screening, by voltage clamp technique, showed that the rTs8 fragment with the highest molecular weight inhibited the Kv1.3 channel, whereas the N-terminal fragment had no activity on the ion channels tested.

Vascular involvement in Behçet's disease: the immunopathological process.

Behçet's disease is a rare form of systemic vasculitis that affects small to large vessels. It is characterized by mucocutaneous, pulmonary, cardiovascular, gastrointestinal, and neurological manifestations. Its clinical presentation is quite wide, ranging from milder cases to severe cases, with multisystemic involvement, characteristically with exacerbations and remissions. Its etiopathogenesis is still unclear, although there is evidence of genetic, environmental, and immunological factors, such as the association with the HLA-B51 allele. In conjunction, all of these point to an abnormal immunopathological process, with activation of cells of innate and adaptive immunity, such as NK cells, neutrophils, and T cells, which generate specific response patterns and cytokines capable of generating mediators that can damage and inflame blood vessels, resulting in venous and arterial occlusions and/or aneurysm formation.

Acometimento vascular na doença de Behçet: o processo imunopatológico / Vascular involvement in Behçet's disease: the immunopathological process

Resumo A doença de Behçet constitui uma forma rara de vasculite sistêmica, que acomete de pequenos a grandes vasos. É caracterizada por manifestações mucocutâneas, pulmonares, cardiovasculares, gastrointestinais e neurológicas. Sua apresentação clínica é bastante ampla, variando de casos mais brandos a casos graves, com acometimento multissistêmico, caracteristicamente com exacerbações e remissões. Suas causas ainda são desconhecidas; entretanto, há evidências genéticas, ambientais e imunológicas, como a associação com o alelo HLA-B51. Todas essas, em conjunto, apontam para um processo imunopatológico anormal, com ativação de células da imunidade inata e adaptativa, como as células natural killer, neutrófilos e células T, que geram padrões de respostas e citocinas específicos capazes de gerar mediadores que podem lesionar e inflamar o sistema vascular, resultando em oclusões venosas, arteriais e/ou formação de aneurismas.

Abstract Behçet's disease is a rare form of systemic vasculitis that affects small to large vessels. It is characterized by mucocutaneous, pulmonary, cardiovascular, gastrointestinal, and neurological manifestations. Its clinical presentation is quite wide, ranging from milder cases to severe cases, with multisystemic involvement, characteristically with exacerbations and remissions. Its etiopathogenesis is still unclear, although there is evidence of genetic, environmental, and immunological factors, such as the association with the HLA-B51 allele. In conjunction, all of these point to an abnormal immunopathological process, with activation of cells of innate and adaptive immunity, such as NK cells, neutrophils, and T cells, which generate specific response patterns and cytokines capable of generating mediators that can damage and inflame blood vessels, resulting in venous and arterial occlusions and/or aneurysm formation.

The ethanolic extract of Terminalia argentea Mart. & Zucc. bark reduces the inflammation through the modulation of cytokines and nitric oxide mediated by the downregulation of NF-κB.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia argentea Mart. & Zucc. (Combretaceae), popularly known as "capitão do campo", is native from the Brazilian cerrado, which is used in folk medicine to treat inflammatory diseases. AIM OF THE STUDY: We aimed to investigate the anti-inflammatory effects, toxicity and mechanisms of action regarding the use of the hydroalcoholic extract of T. argentea bark. MATERIALS AND METHODS: Toxicity was determinate in vitro using the macrophage lineage J774.1 without LPS. Cells were treated with 0.5; 2; 8; 32 and 125 µg/mL of the plant extract. Cell viability was assessed by MTT colorimetric assay. The production of nitrite and cytokines was also determined in the supernatants. A NF-κB reporter assay using RAW macrophages was employed to elucidate the impact of the plant extract on the expression of such molecule. In mice, toxicity was assessed by orally given an intermediate to high concentration of the plant extract on a single dose (1000 or 5000 mg/kg) or low and intermediate doses (300 or 1000 mg/kg) twice daily for 14 days. Blood samples were collected for biochemical analysis. The anti-inflammatory activity was assessed using the air-pouch model with or without pre-inoculation with the inflammatory stimuli LPS (0.5 µg/mL), followed by treatment with plant extract at 5, 60 or 300 mg/kg administered in the air pouch (subcutaneous injection). After 4 h, mice were euthanized and the air pouches washed with 2 mL heparinized PBS (10 IU/mL). Then, the local production in the air pouch wash of cytokines, total proteins and leukocytes was assessed. RESULTS: No signals of toxicity were observed either in cells or mice. Regardless the concentration used in vitro, the extract exhibited a significant anti-inflammatory activity, as perceived by the reduction of the inflammatory cytokines IL-1ß, TNF-α and IL-6 and nitrites on cell supernatants. This was concomitant with a downregulation in NF-κB and elevated levels of IL-10. In mice, similar effects were observed, especially when the plant extract was given at 300 mg/kg, inhibiting the release of IL-1ß, TNF-α, IL-6 and proteins, as well as increasing the release of IL-10. CONCLUSIONS: Altogether, our results demonstrated that the hydroalcoholic extract of T. argentea bark has anti-inflammatory activity without inducing toxicity in cells or living animals. This activity seems to be chiefly influenced by a downregulation in NF-κB, inflammatory cytokines and production of nitrite along with augmented concentration of IL-10.

Eicosanoid pathway on host resistance and inflammation during Mycobacterium tuberculosis infection is comprised by LTB4 reduction but not PGE2 increment.

The functions of eicosanoids, a family of potent biologically active lipid mediators, are not restricted to inflammatory responses and they also act as mediators of the pathogenesis process. However, the role of eicosanoids in tuberculosis remains controversial. To investigate the specific role of LTB4 in Mycobacterium tuberculosis (Mtb) infection, we used 5-lipoxygenase-deficient (5-LO-/-) mice and WT (sv129) mice inoculated intranasally with LTB4 (encapsulated in PLGA microspheres). We showed that deficiency of the 5-LO pathway was related to resistance to Mtb infection. LTB4 inoculation increased susceptibility to Mtb in 5-LO-/- mice but not in WT mice, resulting in worsening of lung inflammation and tissue damage. In infected WT mice, most supplementary LTB4 was metabolized to the inactive form 12-oxo-LTB4 in the lung. A high amount of PGE2 was detected during Mtb infection, and pharmacological inhibition of COX-2 induced a significant reduction of bacterial load and an improved innate immune response in the lungs, independently of baseline LTB4 levels. COX-2 inhibition with celecoxib significantly reduced PGE2 levels, enhanced IFN-γ production and NO release, and increased macrophage phagocytosis of Mtb. The results suggest that a balance between PGE2/LTB4 is essential in the pathogenesis process of tuberculosis to prevent severe inflammation. Moreover, optimal levels of PGE2 are required to induce an effective innate response in the early phase of Mtb infection. Thus, pharmacological modulation of eicosanoid production may provide an important host-directed therapy in tuberculosis.

First report on BaltCRP, a cysteine-rich secretory protein (CRISP) from Bothrops alternatus venom: Effects on potassium channels and inflammatory processes.

CRISPs represent a family of cysteine-rich secretory proteins with molecular mass between 20 and 30 kDa and a highly conserved specific pattern of 16 cysteine residues. In this work, we isolated and characterized a novel CRISP from Bothrops alternatus venom, named BaltCRP, also evaluating its effects on different isoforms of potassium channels (Kv1.1; Kv1.2; Kv1.3; Kv1.4; Kv1.5; Kv2.1; Kv10.1 and Shaker) and on inflammatory processes in vivo. This toxin has a molecular mass of 24.4 kDa and pI around 7.8. Electrophysiological experiments using voltage clamp techniques showed that BaltCRP can affect the currents of Kv1.1; Kv1.3; Kv2.1 and Shaker channels. In addition, BaltCRP induced inflammatory responses characterized by an increase of leukocytes in the peritoneal cavity of mice, also stimulating the production of mediators such IL-6, IL-1ß, IL-10, PGE2, PGD2, LTB4 and CysLTs. Altogether, these results demonstrated that BaltCRP can help understand the biological effects evoked by snake venom CRISPs, which could eventually lead to the development of new molecules with therapeutic potential.

Scorpion envenomation and inflammation: Beyond neurotoxic effects.

Scorpion envenomation results in a wide range of clinical manifestations that are mostly attributed to the activation of the autonomic nervous system by venom toxins. In fact, sympathetic and parasympathetic disturbances play important roles during poisoning. However, scorpion venom also induces a complex hyperinflammatory state that occurs parallel to systemic inflammatory response syndrome and acute sepsis. After a scorpion sting, innate immune cells are exposed to the venom molecules, which bind to pattern recognition receptors and activate pro-inflammatory pathways that contribute toward the promotion of severe symptoms, such as pulmonary edema, and eventually lead to death. In this review, we highlight studies that pointed out inflammation as a major pathological facet of scorpion envenomation, so as to provide novel targets to improve therapeutics for scorpionism.

Global proteomic and functional analysis of Crotalus durissus collilineatus individual venom variation and its impact on envenoming.

Individual variations studies are important to understand the snakebite envenoming and to improve the antivenom production and its effectiveness. In this way, the objective of this study was a comparative analysis of intraspecific variation in the venom composition of 22 Crotalus durissus collilineatus specimens through proteomic techniques. Venoms were fractionated by RP-FPLC, and analyzed by SDS-PAGE and mass spectrometry. Although similar, chromatographic and electrophoretic profiles showed significant qualitative and quantitative differences. Some venom components were identified for the very first time in C. d. collilineatus, such as glutathione peroxidase, nerve growth factor, 5'-nucleotidase, angiotensin-converting enzyme, carboxypeptidase, phosphodiesterase, glutaminyl cyclase and phospholipase B. Regarding hyaluronidase activity, 2 venoms did not present detectable enzyme activity in the tested amounts. Additionally, in vivo crotalic envenoming in mice showed that venoms from different specimens resulted in diversified changes of biochemical and immunological parameters, such as creatine kinase and interleukin 6. This study demonstrated significant intraspecific variations in the venom of C. d. collilineatus, which may impact the production and effectiveness of the antivenom therapy. BIOLOGICAL SIGNIFICANCE: This study performed the proteomic and functional analyzes of 22 C. d. collilineatus individual venoms and verified the occurrence of quali and quantitative variations among them. The venoms evaluated caused envenomings with different changes in biochemical and immunological parameters. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness. In addition, this study was able to identify for the first time 8 different proteins in this subspecies venom, increasing knowledge about its composition and showing that it is a source of these proteins with possible biotechnological applications.

Expanding biological activities of Ts19 Frag-II toxin: Insights into IL-17 production.

Tityus serrulatus (Ts) venom is composed of a mixture of toxins presenting diverse biological functions. However, although this venom has been studied over the past three decades, omics analysis revealed that most of its toxins are not identified or their biological activities are unknown. Ts19 Frag-II is included in this group, which function is still uncertain. This study aimed to expand the biological activities of Ts19 Frag-II through in vivo investigation. Our results demonstrates that mice challenged with Ts19 Frag-II presented biochemical alterations, increasing serum levels of urea, ALT and ß-globulin, besides decreasing γ-globulins. Moreover, this toxin was also able to induce immunological alterations, increasing NO, IL-6, TNF-α and IL-17, being considered a proinflammatory toxin. The increase of IL-17 was unprecedented regarding Ts toxins and could be a result of the overall produced-effect of cells of innate immunity cells (neutrophils, monocytes, natural killers and lymphoid tissue inducers - LTis) as well as of adaptive immunity (Th17 cells). This study expanded the biological activities of Ts19 Frag-II, suggesting that this toxin could be contributing to the Ts envenoming through alterations of biochemical parameters as well as triggering the inflammatory response.

Non-disulfide-bridged peptides from Tityus serrulatus venom: Evidence for proline-free ACE-inhibitors.

The present study purifies two T. serrulatus non-disulfide-bridged peptides (NDBPs), named venom peptides 7.2 (RLRSKG) and 8 (KIWRS) and details their synthesis and biological activity, comparing to the synthetic venom peptide 7.1 (RLRSKGKK), previously identified. The synthetic replicate peptides were subjected to a range of biological assays: hemolytic, antifungal, antiviral, electrophysiological, immunological and angiotensin-converting enzyme (ACE) inhibition activities. All venom peptides neither showed to be cytolytic nor demonstrated significant antifungal or antiviral activities. Interestingly, peptides were able to modulate macrophages' responses, increasing IL-6 production. The three venom peptides also demonstrated potential to inhibit ACE in the following order: 7.2>7.1>8. The ACE inhibition activity was unexpected, since peptides that display this function are usually proline-rich peptides. In attempt to understand the origin of such small peptides, we discovered that the isolated peptides 7.2 and 8 are fragments of the same molecule, named Pape peptide precursor. Furthermore, the study discusses that Pape fragments could be originated from a post-splitting mechanism resulting from metalloserrulases and other proteinases cleavage, which can be seen as a clever mechanism used by the scorpion to enlarge its repertoire of venom components. Scorpion venom remains as an interesting source of bioactive proteins and this study advances our knowledge about three NDBPs and their biological activities.

Physicochemical characterization by AFM, FT-IR and DSC and biological assays of a promising antileishmania delivery system loaded with a natural Brazilian product.

The control and treatment of Leishmaniasis, a neglected and infectious disease affecting approximately 12 million people worldwide, are challenging. Leishmania parasites multiply intracellularly within macrophages located in deep skin and in visceral tissues, and the currently employed treatments for this disease are subject to significant drawbacks, such as resistance and toxicity. Thus, the search for new Leishmaniasis treatments is compulsory, and Ocotea duckei Vattimo, a plant-derived product from the biodiverse Brazilian flora, may be a promising new treatment for this disease. In this regard, the aim of this work was to develop and characterize a delivery system based on solid lipid nanoparticles (SLN) that contain the liposoluble lignan fraction (LF) of Ocotea duckei Vattimo, which targets the Leishmania phagolysosome of infected macrophages. LF-loaded SLNs were obtained via the hot microemulsion method, and their physical and chemical properties were comprehensively assessed using PCS, AFM, SEM, FT-IR, DSC, HPLC, kinetic drug release studies, and biological assays. The size of the developed delivery system was 218.85±14.2 nm, its zeta potential was -30 mV and its entrapment efficiency (EE%) was high (the EEs% of YAN [yangambin] and EPI-YAN [epi-yangambin] markers were 94.21±0.40% and 94.20±0.00%, respectively). Microscopy, FT-IR and DSC assays confirmed that the delivery system was nanosized and indicated a core-shell encapsulation model, which corroborated the measured kinetics of drug release. The total in vitro release rates of YAN and EPI-YAN in buffer (with sink conditions attained) were 29.6±8.3% and 34.3±8.9%, respectively, via diffusion through the cellulose acetate membrane of the SLN over a period of 4 h. After 24 h, the release rates of both markers reached approximately 45%, suggesting a sustained pattern of release. Mathematical modeling indicated that both markers, YAN and EPI-YAN, followed matrix diffusion-based release kinetics (Higuchi's model) with an estimated diffusion coefficient (D) of 1.3.10(-6) cm(2)/s. The LF-loaded SLNs were non-toxic to murine macrophages (20-80 µg mL(-1) range) and exerted a prominent anti-leishmanial effect (20 µg mL(-1)). These data suggest this new and well-characterized lipid nanoparticle delivery system safely and effectively kills Leishmania and warrants further clinical investigation.

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice.

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO's role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80(+)/CD80(+) and F4/80(+)/CD86(+) cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection.

Antiedematogenic Evaluation of Copaifera langsdorffii Leaves Hydroethanolic Extract and Its Major Compounds.

Inflammatory disorders affect many people worldwide, and medicinal plants are used to ameliorate these health problems. This paper reports the antiedematogenic and analgesic evaluation of Copaifera langsdorffii Desf. leaves hydroethanolic extract (Cop) and two of its isolated compounds: quercetin-3-O-α-l-rhamnopyranosyl (quercitrin) and kaempferol-3-O-α-l-rhamnopyranosyl (afzelin). For that, the following experimental protocols were undertaken locomotor performance, writhing induced by acetic acid, antinociceptivity induced by formalin, hot plate latency, paw oedema induced by carrageenan and dextran, and cell migration induced by lipopolysaccharide (LPS), as well as the measurement of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10) in macrophages. Neither the extract nor the isolated compounds displayed analgesic activity. The obtained results showed that C. langsdorffii extract possesses antiedematogenic properties acting on peripheral sites, whereas quercitrin and afzelin are not involved. Moreover, these properties are not associated with cell migration inhibition, TNF-α, IL-6, or IL-10 regulation.

PPAR-γ activation by Tityus serrulatus venom regulates lipid body formation and lipid mediator production.

Tityus serrulatus venom (TsV) consists of numerous peptides with different physiological and pharmacological activities. Studies have shown that scorpion venom increases pro-inflammatory cytokine production, contributing to immunological imbalance, multiple organ dysfunction, and patient death. We have previously demonstrated that TsV is a venom-associated molecular pattern (VAMP) recognized by TLRs inducing intense inflammatory reaction through the production of pro-inflammatory cytokines and arachidonic acid-derived lipid mediators prostaglandin (PG)E2 and leukotriene (LT)B4. Lipid bodies (LBs) are potential sites for eicosanoid production by inflammatory cells. Moreover, recent studies have shown that the peroxisome proliferator-activated receptor gamma (PPAR-γ) is implicated in LB formation and acts as an important modulator of lipid metabolism during inflammation. In this study, we used murine macrophages to evaluate whether the LB formation induced by TsV after TLR recognition correlates with lipid mediator generation by macrophages and if it occurs through PPAR-γ activation. We demonstrate that TsV acts through TLR2 and TLR4 stimulation and PPAR-γ activation to induce LB formation and generation of PGE2 and LTB4. Our data also show that PPAR-γ negatively regulates the pro-inflammatory NF-κB transcription factor. Based on these results, we suggest that during envenomation, LBs constitute functional organelles for lipid mediator production through signaling pathways that depend on cell surface and nuclear receptors. These findings point to the inflammatory mechanisms that might also be triggered during human envenomation by TsV.

TLR2, TLR4 and CD14 recognize venom-associated molecular patterns from Tityus serrulatus to induce macrophage-derived inflammatory mediators.

Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense Tityus serrulatus venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.

Ts6 and Ts2 from Tityus serrulatus venom induce inflammation by mechanisms dependent on lipid mediators and cytokine production.

Inflammatory mediators are thought to be involved in the systemic and local immune response induced by the Tityus serrulatus scorpion envenomation. New functional aspects of lipid mediators have recently been described. Here, we examine the unreported role of lipid mediators in cell recruitment to the peritoneal cavity after an injection with Ts2 or Ts6 toxins isolated from the T. serrulatus scorpion venom. In this report, we demonstrate that following a single intraperitoneal (i.p.) injection of Ts2 or Ts6 (250 µg/kg) in mice, there was an induction of leukocytosis with a predominance of neutrophils observed at 4, 24, 48 and 96 h. Moreover, total protein, leukotriene (LT)B(4), prostaglandin (PG)E(2) and pro-inflammatory cytokine levels were increased. We also observed an increase of regulatory cytokines, including interleukin (IL)-10, after the Ts2 injection. Finally, we observed that Ts2 or Ts6 injection in 5-lipoxygenase (LO) deficient mice and in wild type (WT) 129sv mice pre-treated with LTs and PGs inhibitors (MK-886 and celecoxib, respectively) a reduction the influx of leukocytes occurs in comparison to WT. The recruitment of these cells demonstrated a phenotype characteristic of neutrophils, macrophages, CD4 and CD8 lymphocytes expressing GR1+, F4/80+, CD3+/CD4+ and CD3+/CD8+, respectively. In conclusion, our data demonstrate that Ts2 and Ts6 induce inflammation by mechanisms dependent on lipid mediators and cytokine production. Ts2 may play a regulatory role whereas Ts6 exhibits pro-inflammatory activity exclusively.

Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators.

Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 µg/mL) of venom or toxins pre-stimulated or not with LPS (0.5 µg/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-α in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-α production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation; additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases.