Natterin an aerolysin-like fish toxin drives IL-1ß-dependent neutrophilic inflammation mediated by caspase-1 and caspase-11 activated by the inflammasome sensor NLRP6.

Natterin is an aerolysin-like pore-forming toxin responsible for the toxic effects of the venom of the medically significant fish Thalassophryne nattereri. Using a combination of pharmacologic and genetic loss-of-function approaches we conduct a systematic investigation of the regulatory mechanisms that control Natterin-induced neutrophilic inflammation in the peritonitis model. Our data confirmed the capacity of Natterin to induce a strong and sustained neutrophilic inflammation leading to systemic inflammatory lung infiltration and revealed overlapping regulatory paths in its control. We found that Natterin induced the extracellular release of mature IL-1ß and the sustained production of IL-33 by bronchial epithelial cells. We confirmed the dependence of both ST2/IL-33 and IL-17A/IL-17RA signaling on the local and systemic neutrophils migration, as well as the crucial role of IL-1α, caspase-1 and caspase-11 for neutrophilic inflammation. The inflammation triggered by Natterin was a gasdermin-D-dependent inflammasome process, despite the cells did not die by pyroptosis. Finally, neutrophilic inflammation was mediated by non-canonical NLRP6 and NLRC4 adaptors through ASC interaction, independent of NLRP3. Our data highlight that the inflammatory process dependent on non-canonical inflammasome activation can be a target for pharmacological intervention in accidents by T. nattereri, which does not have adequate specific therapy.

Anaphylaxis induced by Thalassophryne nattereri venom in mice is an IgE/IgG1-mediated, IL-4-dependent phenomenon.

We hypothesized that beyond the Thalassophryne nattereri venoms ability to induce in mice a strong specific-Th2 response with high levels of specific IgE/IgG1, it would be able to trigger anaphylaxis in sensitized individuals. To investigate whether the venom is capable of inducing an allergic reaction in mice and characterize soluble and cellular mediators involved in this process, BALB/c female mice were sensitized intraperitoneally with decreasing-dose of venom at weekly intervals for 4 weeks and challenged by intraperitoneal, oral or epicutaneous routes with venom 2 weeks later. Our data show that sensitized-mice challenged by all routes showed intense symptoms of anaphylaxis, dependent on the anaphylactic IgG1 and IgE antibodies and mast cells. The late-phase reaction developed after initial symptoms was characterized by the influx of eosinophils, dependent on IL-5, IL-17A and eotaxin produced by Th2 cells in inflamed lungs and skin draining lymph-nodes. Using C57BL/6 deficient mice we demonstrated that IL-4 KO mice failed to develop anaphylactic symptoms or local Th2 inflammation, producing low levels of IgG1 and increased levels of IgG2a. Together our results demonstrated that the venom of T. nattereri has allergenic proteins that can trigger an allergic process, a phenomenon IgE-IgG1 dependent, IL-4-mediated and negatively regulated by IFN-γ.

Stingray venom activates IL-33 producing cardiomyocytes, but not mast cell, to promote acute neutrophil-mediated injury.

One of the hallmarks of acute inflammation is neutrophil infiltration of tissues. We investigated molecular mechanisms implicated in acute neutrophilic inflammation induced by the venom of a freshwater stingray (Potamotrygon cf. henlei) in mice. Ray venom induced early mobilization of neutrophil in the microvasculature of cremaster mice and infiltration of the peritoneal cavity 2 hours after injury, in a dose-response manner. IL-1ß, IL-6, TNF-α, and KC were produced. The neutrophilic infiltration did not occur in mice with ST2 receptor and MyD88 adapters neutralized, or in those with PI3K and p38 MAPK signaling blocked. Drastic reduction of neutrophil infiltration to peritoneal cavities was observed in ST2-/-, TLR2/TLR4-/-, MyD88-/-, TRIF-/- and IL-17A-/- mice, and a partial reduction was observed in IL-18R-/- mice. Mast cell Kit W(sh)/W(sh)-, AHR-, NLRP3-, ICE-, IL-1ß-, P2RX7-, CD39-, IL-17RA-, and TBX21 KO mice retain the ability to induce neutrophilia in peritoneal cavity after ray venom injection. IL-6 and TNF-α alone were insufficient for promote neutrophilia in the absence of ST2 signaling. Finally, abundant production of IL-33 by cardiomyocytes was observed. These results refine our understanding of the importance of the IL-33/ST2 axis and IL-33-producing cardiomyocytes in the early acute neutrophilia induced by freshwater stingray venoms.

Multiple functional therapeutic effects of TnP: A small stable synthetic peptide derived from fish venom in a mouse model of multiple sclerosis.

The pathological condition of multiple sclerosis (MS) relies on innate and adaptive immunity. New types of agents that beneficially modify the course of MS, stopping the progression and repairing the damage appear promising. Here, we studied TnP, a small stable synthetic peptide derived from fish venom in the control of inflammation and demyelination in experimental autoimmune encephalomyelitis as prophylactic treatment. TnP decreased the number of the perivascular infiltrates in spinal cord, and the activity of MMP-9 by F4/80+ macrophages were decreased after different regimen treatments. TnP reduces in the central nervous system the infiltration of IFN-γ-producing Th1 and IL-17A-producing Th17 cells. Also, treatment with therapeutic TnP promotes the emergence of functional Treg in the central nervous system entirely dependent on IL-10. Therapeutic TnP treatment accelerates the remyelination process in a cuprizone model of demyelination. These findings support the beneficial effects of TnP and provides a new therapeutic opportunity for the treatment of MS.

Neutrophils releasing IL-17A into NETs are essential to plasma cell differentiation in inflamed tissue dependent on IL-1R.

Interleukin (IL) 17A in chronic inflammation is also produced by innate immune cells as neutrophils. Mice with chronic humoral response induced by venom of Thalassophryne nattereri (VTn) proved to be a good tool for evaluating the impact of IL-17A on the development of long-lived plasma cells in the inflamed peritoneal cavity. Here, we report that VTn induces IL-17A production by neutrophils accumulating in the peritoneal cavity and triggers the extrusion of IL-17A along with neutrophil extracellular traps (NETs). Neutrophil depletion reduced the number of IL17A-producing cells in VTn-immunized mice and blocked the differentiation of long-lived plasma cells. Specific antibody production and survival of long-lived plasma cells was ablated in VTn-immunized mice deficient in CD4, while CD28 signaling had the opposite effect on differentiation of long-lived plasma cells. Further, maturation of long-lived plasma cells in inflamed peritoneal cavity was IL-1R1 and COX-2 dependent. Finally, when both the Raf-MEK-ERK pathway and the IL-17A or IL-1R1 activities were blocked, neutrophils were unable to promote the differentiation of memory B cells into long-lived plasma cells, confirming the essential role of neutrophils and IL-17A along with NETs in an IL-1/IL-1R-dependent manner as the novel helping partner for plasma cell differentiation in chronically inflamed tissues.

Thalassophryne nattereri fish venom: from the envenoming to the understanding of the immune system.

Thalassophryne nattereri (niquim) is a venomous fish found off North and Northeast coast of Brazil, where it is known by the severity of the accidents involving humans. This review article is divided into four topics. The first one provides a brief description of the animal biology and its distribution off Brazilian coastal waters, the venom apparatus, signs and symptoms observed in envenomated humans and also describes envenomation in mice. The second topic describes the use of modern genetic approach and mass spectrometry for identification of highly expressed genes in its venom glands and the sequence of major toxins. The third chapter offers a detailed study of tissue injury induced by the venom and reveals the role of toxins that impair inflammation reduction. Finally, the fourth section expands the understanding of many extrinsic and intrinsic essential factors in maintaining survival of memory B cell compartment. Our results demonstrate the wide possibilities for research in the area of toxinology, also the necessity of interconnection among biochemistry, pharmacology and immunology areas for the expansion of knowledge and for generation of innovation.

Airborne pollutant ROFA enhances the allergic airway inflammation through direct modulation of dendritic cells in an uptake-dependent mechanism.

Studies suggest that airborne pollutants are important cofactors in the exacerbation of lung diseases. The role of DC on the exacerbation of lung inflammation induced by particulate matter pollutants is unclear. We evaluated the effects of residual oil fly ash (ROFA) on the phenotype and function of bone marrow-derived dendritic cells (BMDCs) in vitro and lung dendritic cells (DCs) in vivo, and the subsequent T-cell response. In a model of asthma, exposure to ROFA exacerbated pulmonary inflammation, which was attributed to the increase of eosinophils, IL-5- and IFN-γ-producing T cells, and goblet cells as well as decreased number of Treg and pDC. However, the ROFA showed no ability to modulate the production of anaphylactic IgE. In vitro studies showed that ROFA directly induced the maturation of DCs up-regulating the expression of co-stimulatory molecules and cytokines and MMP production in an uptake-dependent and oxidative stress-dependent manner. Furthermore, ROFA-pulsed BMDC transferred to allergic mice exacerbated eosinophilic inflammation as well as promoted increased epithelial and goblet cells changes. Thus, pollutants may constitute an important and risk factor in the exacerbation of asthma with inhibition of the negative regulatory signals in the lung, with enhanced mDC activation that sustains the recruitment of effector T lymphocytes and eosinophil.

Thalassophryne nattereri fish venom: from the envenoming to the understanding of the immune system

Thalassophryne nattereri (niquim) is a venomous fish found off North and Northeast coast of Brazil, where it is known by the severity of the accidents involving humans. This review article is divided into four topics. The first one provides a brief description of the animal biology and its distribution off Brazilian coastal waters, the venom apparatus, signs and symptoms observed in envenomated humans and also describes envenomation in mice. The second topic describes the use of modern genetic approach and mass spectrometry for identification of highly expressed genes in its venom glands and the sequence of major toxins. The third chapter offers a detailed study of tissue injury induced by the venom and reveals the role of toxins that impair inflammation reduction. Finally, the fourth section expands the understanding of many extrinsic and intrinsic essential factors in maintaining survival of memory B cell compartment. Our results demonstrate the wide possibilities for research in the area of toxinology, also the necessity of interconnection among biochemistry, pharmacology and immunology areas for the expansion of knowledge and for generation of innovation.

Thalassophryne nattereri fish venom: from the envenoming to the understanding of the immune system

Thalassophryne nattereri (niquim) is a venomous fish found off North and Northeast coast of Brazil, where it is known by the severity of the accidents involving humans. This review article is divided into four topics. The first one provides a brief description of the animal biology and its distribution off Brazilian coastal waters, the venom apparatus, signs and symptoms observed in envenomated humans and also describes envenomation in mice. The second topic describes the use of modern genetic approach and mass spectrometry for identification of highly expressed genes in its venom glands and the sequence of major toxins. The third chapter offers a detailed study of tissue injury induced by the venom and reveals the role of toxins that impair inflammation reduction. Finally, the fourth section expands the understanding of many extrinsic and intrinsic essential factors in maintaining survival of memory B cell compartment. Our results demonstrate the wide possibilities for research in the area of toxinology, also the necessity of interconnection among biochemistry, pharmacology and immunology areas for the expansion of knowledge and for generation of innovation.

The hierarchical process of differentiation of long-lived antibody-secreting cells is dependent on integrated signals derived from antigen and IL-17A.

Switched CD19-positive memory B cells purified from mice with chronic immune response against Thalassophrynenattereri venom proteins were cultured with venom or cytokines. Our results confirm the existence of a hierarchic process of differentiation: activated memory B cells progressively acquire increasing levels of CD138 and decreasing levels of CD45R/B220 to finally arrive at ASC with B220(neg) phenotype, which are IgG1-secreting cells. Only Bmem from peritoneal cavity or bone marrow of VTn immunized mice presented the capacity to generate ASC functionally active. IL-17A or IL-21/IL-23/IL-33 improves the ability of venom to induce intracellular IgG of peritoneal derived-ASC. Cognate stimulation with venom and IL-17A is sufficient to down-regulate the expression of CD45R/B220. BAFF-R is up-regulated in splenic or medullar derived-ASC stimulated by venom, CpG or cytokines. Only splenic derived-ASC up-regulate Bcl-2 expression after CpG or the combination of IL-21/IL-23/IL-33 stimulation. Finally, the activation of ASC for IgG1 secretion is triggered by venom proteins in peritoneal cavity and by IL-17A in medullar niche. These results show the importance of the integration of signals downstream of BCR and IL17-A receptors in modulating ASC differentiation, focusing in the microenvironment niche of their generation.

TLR2, TLR4 and the MyD88 signaling are crucial for the in vivo generation and the longevity of long-lived antibody-secreting cells.

This study was undertaken to gain better insights into the role of TLRs and MyD88 in the development and differentiation of memory B cells, especially of ASC, during the Th2 polarized memory response induced by Natterins. Our in vivo findings demonstrated that the anaphylactic IgG1 production is dependent on TLR2 and MyD88 signaling, and that TLR4 acts as adjuvant accelerating the synthesis of high affinity-IgE. Also, TLR4 (MyD88-independent) modulated the migration of innate-like B cells (B1a and B2) out of the peritoneal cavity, and the emigration from the spleen of B1b and B2 cells. TLR4 (MyD88-independent) modulated the emigration from the spleen of Bmem as well as ASC B220(pos). TLR2 triggered to the egress from the peritoneum of Bmem (MyD88-dependent) and ASC B220(pos) (MyD88-independent). We showed that TLR4 regulates the degree of expansion of Bmem in the peritoneum (MyD88-dependent) and in BM (MyD88-independent) as well as of ASC B220(neg) in the spleen (MyD88-independent). TLR2 regulated the intensity of the expansion of Bmem (MyD88-independent) and ASC B220(pos) (MyD88-dependent) in BM. Finally, TLR4 signals sustained the longevity of ASC B220(pos) (MyD88-independent) and ASC B220(neg) into the peritoneum (MyD88-dependent) and TLR2 MyD88-dependent signaling supported the persistence of B2 cells in BM, Bmem in the spleen and ASC B220(neg) in peritoneum and BM. Terminally differentiated ASC B220(neg) required the cooperation of both signals through TLR2/TLR4 via MyD88 for longevity in peritoneum, whereas Bmem required only TLR2/MyD88 to stay in spleen, and ASC B220(pos) rested in peritoneum dependent on TLR4 signaling. Our data sustain that earlier events on memory B cells differentiation induced in secondary immune response against Natterins, after secondary lymph organs influx and egress, may be the key to determining peripheral localization of innate-like B cells and memory B cells as ASC B220(pos) and ASC B220(neg).

The longevity of Th2 humoral response induced by proteases natterins requires the participation of long-lasting innate-like B cells and plasma cells in spleen.

The generation of long-lived antibody-secreting cells (ASC) and memory B cells are critical events for an effective vaccine and the choice of adjuvant can influence these processes. Various cellular and molecular mechanism involved in the protease action that determine Th2 responses have been identified. However, direct or indirect actions in the regulation of the induction, survival and longevity of ASC in differential compartments remain largely unknown. We investigated whether the proteolytic activity of proteins are determinant for the modulation of the memory immune response in mice, promoting the differentiation of memory B cells to terminally differentiated end stage cells. Here, we show that the proteolytic activity of Natterins, from the venom of Thalassophryne nattereri Brazilian fish, besides inducing a Th2 response with plasmatic titers of high-affinity antigen-specific IgE over extended periods is sufficient for the generation of signals that contribute to the formation of a survival niche in the spleen, essential for the longevity of the main subtype of ASC with B220(neg) phenotype.

Role of interplay between IL-4 and IFN-γ in the in regulating M1 macrophage polarization induced by Nattectin.

Recently our group described that Nattectin, a C-type lectin of the venom of Thalassophryne nattereri shows a potent pro-inflammatory capacity. Here, we demonstrated that Nattectin is able to induce M1 macrophage marker iNOS, and up-regulate the expression of MHC class II, CD80, CD86 and CD40 molecules. The increase in MHC class II and CD49a integrin expression with MMP-9 production and endocytic capacity depend on lectin function of Nattectin. Moreover, the polarization of peritoneal and bone marrow-derived macrophages induced by Nattectin to M1 profile is dependent on Th1 cytokines (IL-12 and IFN-γ), and negatively regulated by Th2 cytokines (IL-4, IL-10 and IL-13). Also we reveal that IL-4 play a dual role in this polarization: a regular action of IL-4 was seen in the negative regulation of the CD40 expression, but an unexpected positive regulation was seen in the expression of CCR7 and MHC class II. Finally, our in vivo studies showed that the influx of neutrophils and small peritoneal macrophage--F4/80(low)MHCII(hi) induced by Nattectin is totally dependent on IL-4 and IFN-γ cytokines. Furthermore, the induction of IL-6 release is negatively regulated by IL-4 and positively regulated by IL-12 and IFN-γ. Together, the results allowed us to expand the knowledge about the regulation of macrophage activation, as well as confirmed the ability of Nattectin, a fish C-type lectin, as an important immunomodulatory agent.

IL-5 and IL-17A are critical for the chronic IgE response and differentiation of long-lived antibody-secreting cells in inflamed tissues.

Prolonged survival of long-lived antibody-secreting cells in the BM has been implicated as a key component of long-term humoral immunity. The current study was designed to uncover the extrinsic signals required for the generation and maintenance of ASC in several niches (peritoneum, spleen and bone-marrow). Our results show that protein mixture of the Thalassophryne nattereri venom induced a chronic Th2 humoral response that is characterized by splenic hyperplasia with GC formation and venom retention by follicular DCs. Retention of B1a in the BM were observed. In the late phase (120d) of chronic venom-response the largest pool of ASC into the peritoneal cavity consisted of B220(neg)CD43(high) phenotype; the largest pool of ASC into spleen was constituted by B220 positive cells (B220(high) and B220(low)), whereas the largest pool of ASC into in the BM was constituted by the B220(high)CD43(low) phenotype; and finally, terminally differentiated cells (B220(neg)CD43(high)) were only maintained in the inflamed peritoneal cavity in late phase. After 120d a sustained production of cytokines (KC, IL-5, TNF-α, IL-6, IL-17A and IL-23) and leukocytes recruitment (eosinophils, mast cells, and neutrophils) were induced. IL-5- and IL-17A-producing CD4+ CD44+ CD40L+ Ly6C+ effector memory T cells were also observed in peritoneal cavity. Finally, treatment of venom-mice with anti-IL-5- and anti-IL17A-neutralizing mAbs abolished the synthesis of specific IgE, without modifying the splenic hyperplasia or GC formation. In addition, IL-5 and IL-17A negatively regulated the expansion of B1a in peritoneal cavity and BM, and promoted the differentiation of these cells in spleen. And more, IL-5 and IL-17A are sufficient for the generation of ASC B220(neg) in the peritoneal cavity and negatively regulate the number of ASC B220(pos), confirming that the hierarchical process of ASC differentiation triggered by venom needs the signal derived from IL-5 and IL-17A.

Nattectin a fish C-type lectin drives Th1 responses in vivo: licenses macrophages to differentiate into cells exhibiting typical DC function.

Considerable efforts are currently focused on the biology of DC in view of their possible clinical use as adjuvant for the generation of antigen-specific immunity and lifelong immunologic memory or for the treatment of tumors. We assessed the role of Nattectin a C-type lectin identified in the Thalassophryne nattereri fish venom in DC maturation. Nattectin induced a significant neutrophilic recruitment into peritoneal cavity of mice, followed by macrophages, with lipidic mediators and IL-12 p70 synthesis. Macrophages derived from 7day-Nattectin mice were CD11c+CD11b(low)Ly6(high)F4/80R(high) and express high levels of MHC class II and CD80 molecules. Culture of peritoneal exudates derived macrophages from 7day Nattectin-mice and immature BMDCs with Nattectin markedly increased the surface expression of CD40, CD80, CD86, and MHC class II in a dose-dependent manner, and the production of MMP-2 and MMP-9 distributed in nucleus and cytoplasm of cells, that was associated with strong activity in the culture supernatant. Nattectin treated DCs secreted IL-12 p70 and IL-10. The Nattectin-treated BMDC or macrophage-derived DCs were highly efficient at Ag capture. The specific immune response elicited by Nattectin was characterized by the production of specific antibodies IgG1 and mainly IgG2a with IL-10 and IFN-γ synthesis by splenic cells. These results enable us to address that Nattectin induces the recruitment of Ly6C(high) monocytes into the peritoneum, which exhibit a pro-inflammatory profile, where they differentiate into proliferating F4/80R(high) macrophages. Macrophage-derived DCs mature in the presence of the cytokine milieu generated against Nattectin, exhibiting T cell co-stimulatory molecule expression and induced a Th1 polarized response.

Analysis of the inflammatory reaction induced by the catfish (Cathorops spixii) venoms.

Cathorops spixii is one of the most abundant venomous fish of the southeastern coast of the State of São Paulo, and consequently causes a great part of the accidents seen there. The accidents affect mainly fishermen, swimmers and tourists and are characterized by punctiform or wide wounds, erythema, edema, pain, sudoresis, indisposition, fever, nausea, vomiting and secondary infection. The objective of this work was to characterize the inflammatory response induced in mice by both venoms (mucus and sting) of the catfish C. spixii. Our results demonstrated that both venoms induced a great number of rolling and adherent leukocytes in the post-capillary venules of cremaster muscle of mice, and an increase in the vascular permeability in peritoneal cavity. Mucus induced the recruitment of neutrophils immediately after injection followed later by macrophage infiltration. In contrast, the cellular infiltration elicited by sting venom was rapidly resolved. The peritonitis reaction provoked by venoms was characterized by cytokine (IL-6), chemokines (MCP-1 and KC) or lipid mediator (LTB4) production in the peritoneal cavity. The macrophages from 7-day mucus venom-induced exudates upon in vitro mucus venom stimulation, expressed CD11c x MHC class II and release bioactive IL-12p70. On the other hand, sting venom-elicited peritoneal macrophages lost the ability to differentiate into dendritic cells, following re-stimulation in vitro with sting venom, they do not express CD11c, nor do they exhibit sufficient levels of MHC class II. In conclusion, both types of venoms (mucus or sting) promote inflammatory reaction with different profiles, and the inflammatory reaction induced by the first was characterized by antigen persistence in peritoneal cavity that allowed the activation of phagocytic cells with capacity of antigenic presentation.

Experimental immunization with Thalassophryne nattereri fish venom: striking IL-5 production and impaired of B220+ cells.

Murine experimental model have been useful to understanding the toxic as well as the pharmacological properties of the Thalassophryne nattereri venom. However, the specific immune response to T. nattereri venom in mice is yet unclear. Our results showed that the venom elicited in BALB/c mice high levels of specific IgG1 and total IgE isotype with high affinity, accompanied by a striking IL-5 production, what point out to a Th2-like response. Meanwhile, the production of IFN-gamma by lymphocytes pool expanded upon mitogen stimulus, suggests that the venom was also able to activate Th1 clones. Elevated number of antigen-presenting cells expressing CD11c or CD11b from day 4 to 6 supported ongoing antigen presentation process in the primary response and efficient T-cell expansion (increase of CD4(+) cells). In contrast, decreased B220 expression was observed, suggesting that the formation of memory long lived cell compartment. In conclusion, T. natterri venom stimulates an association of cytokine of both Th1 and Th2 profile, with a notable IL-5 production and specific IgG1 and total IgE isotypes secretion. Furthermore, our finding showed that T. natterri venom can affect the B cell fate and induce a memory antibody response through the secretion of protective IgG subclasses. Further studies with the venom protein toxins may provide clues to molecular mechanism regulating proliferation and differentiation of antibody-secreting cells in our model. A better understating of how T. natterri venom can modulate immune response could be useful in therapeutic strategies.