Carla Lima
email: carla.lima@butantan.gov.br

Maria Alice

Pimentel Falcao

Aline Ingrid Andrade-Barros

Lidiane Ana Carolina Seni-Silva

Zito Grund

Eniko Balogh

Katia Conceiçao

Valérie Quesniaux

Bernhard Ryffel

Monica Lopes-Ferreira

Maria Alice Pimentel

Lidiane Zito Grund

Valerie F Quesniaux

Natterin an aerolysin-like

Keywords: Natterin, aerolysin, neutrophilia, NLPR6/NLRC4, caspase-11/caspase-1, IL-

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

In the North and Northeast of Brazil, Thalassophryne nattereri is responsible for several accidents amongst fishermen and bathers [1; 2]. According to Fonseca and Lopes-Ferreira [START_REF] Fonseca | Clinical and experimental studies regarding poisoning caused by a fish Thalassophryne nattereri (niquim)[END_REF], the palm of the hands or soles of the feet are the most commonly areas affected. The main symptoms of envenomation including local edema and excruciating pain that develops immediately after the accident and can remain for several days were reproduced in mice [START_REF] Lopes-Ferreira | Thalassophryne nattereri fish venom: biological and biochemical characterization and serum neutralization of its toxic activities[END_REF].

These symptoms are followed by intense necrosis [START_REF] Lopes-Ferreira | Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice[END_REF] characterized by vascular alterations as stasis and presence of thrombi in venules, focal transient constrictions in arterioles and increased vascular permeability with consequent ischemia. The Thalassophryne nattereri venom (VTn) lacks direct pro-coagulant activity, but exerts a strong cytolytic action on platelets and endothelial cells in vitro [START_REF] Lopes-Ferreira | Hemostatic effects induced by Thalassophryne nattereri fish venom: a model of endothelium-mediated blood flow impairment[END_REF]. The effects of VTn on the delayed recruitment of neutrophils [START_REF] Lima | Characterisation of local inflammatory response induced by Thalassophryne nattereri fish venom in a mouse model of tissue injury[END_REF] and its action decreasing collagen fiber content during the healing phase by the activation of matrix metalloproteinases (MMP-2 and MMP-9) [START_REF] Pareja-Santos | Delayed local inflammatory response induced by Thalassophryne nattereri venom is related to extracellular matrix degradation[END_REF] are added to the mechanisms responsible for ischemic and necrotic injury characterized by the lack of anti-inflammatory drug therapy. Indomethacin (a cyclooxygenase inhibitor), dexamethasone (a steroid anti-inflammatory agent), cyproheptadine (an antagonist of serotonin receptors), fentanyl (an opioid analgesic), or L-NAME (an inhibitor of nitric oxide synthase)

did not affect the venom-induced nociceptive and edematogenic responses [START_REF] Lopes-Ferreira | Kininogenase activity of Thalassophryne nattereri fish venom[END_REF].

Based on the fact that VTn induces pain, edema and necrosis dependent on its kininogenase activity [START_REF] Lopes-Ferreira | Kininogenase activity of Thalassophryne nattereri fish venom[END_REF], using combined pharmacological, proteomic, and transcriptomic approaches we isolated into purified fractions of the venom by chromatography and identified in these fractions as well as in the cDNA library of the venom-producing gland the Natterin family - [10; 11], which are responsible for the main toxic effects of the VTn [9; 10; 12].

Natterins 1 and 2 are very close related, presenting 84% of identity; and both orthologs present identity around 40% with Natterins 3 and 4. Natterin P is the shortest among the Natterin family (71 amino acids long), and shows high homology (84% of identity) mainly to the Natterin 4 at the first 55 amino acids residues [START_REF] Magalhães | Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom[END_REF]. Structurally, Natterins 1-4 contain in the N-terminal region two DM9

domains 13] followed in C-terminal by an aerolysin domain into a so-called domain 2

[ important for toxicity [START_REF] Magalhães | Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom[END_REF], membrane penetration [START_REF] Jia | Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein[END_REF], and being able to activate the NLRP3 inflammasome-dependent caspase-1 maturation leading to IL--dependent neutrophilic inflammation [ 17].

for presenting identity with the aerolysin domain in the Cterminal region of Natterin proteins from the venom of the Thalassophryne nattereri,

Plotosus lineatus -

Gadus morhua, Lampetra morii,

- 15
Oyster Crassostrea gigas [START_REF] Unno | Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Mannose-Specific Lectin CGL1 from the Pacific Oyster Crassostrea gigas[END_REF] and Cnidaria Acropora digitifera [START_REF] Gacesa | Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia)[END_REF].

Due to their capacity to form pore resulting in the activation of inflammation, a tissue specific manner expression (gills, followed by expression in head kidney, skin and spleen), and also differently expression during the development and after infections, these natterin-like proteins have been described as defense molecules for the host, promoting protection against pathogens and microorganisms [START_REF] Chen | The poreforming protein Aep1 is an innate immune molecule that prevents zebrafish from bacterial infection[END_REF].

Given that Natterin aerolysin-like pore-forming toxin medically significant fish T. nattereri and the envenomation is characterized by the lack of anti-inflammatory drug therapy, we hypothesized that the inflammatory response triggered by Natterin may be related to its aerolysin domain involved in the activation of the inflammasome complex helping to promote neutrophilic inflammation into peripheral tissues. To test this hypothesis a mouse model of peritonitis was used with a combination of genetic loss-of-function and pharmacologic approaches to investigate the regulatory mechanisms controlling acute neutrophilic inflammation induce by Natterin.

RESULTS

Natterin induces a robust neutrophilic inflammation in peritonitis model

To gain insights into the capacity of Natterin to induce neutrophilic inflammatory response, we undertook in vivo investigations using this isolated family of fish venom toxins in a murine model of peritonitis. First, the dose-response of neutrophil recruitment to peritoneal cavity of wild type (WT) C57BL/6J (BL/6) mice induced by Natterin was shown in Figure 1A. In control-mice (0h), neutrophils (red line) were barely detectable, and resident macrophages were the major myeloid cells in the peritoneal cavity (data not shown). The high amount (x 10 5 ) of neutrophils induced 2h post-injection was dose-dependent for Natterin in the range of 0.1 to 1 g (i.p. injection in 500 L), which remained high at 10 g. The infiltration of neutrophils triggered by all doses of Natterin 2h into peritoneal cavity was not accompanied by a significant increase in the amount of eosinophils at his time point (2h, blue line, Fig. 1B).

In addition, the kinetics of inflammation induced by Natterin was shown in Figure 1B.

Natterin at 1 g promoted a high and sustained infiltration of leukocytes until 6h, decreasing at 24h.

At this time (24h) when neutrophilia decreased the infiltration of eosinophils increased.

These results confirm the capacity of Natterin to induce a strong and sustained neutrophilic inflammation in murine model of peritonitis.

Natterin induces the release of mature IL-in exudate of peritoneal cavity

We start exploring the mechanisms involved in Natterin-induced neutrophilic inflammation by measuring different mediators in supernatants of peritoneal exudates. IL--

[26], but it also plays a key role in promoting cellular senescence, promoting the senescenceassociated secretory phenotype, SASP, a response characterized by secretion of IL-6 and IL-8 [START_REF] Acosta | A complex secretory program orchestrated by the inflammasome controls paracrine senescence[END_REF].

Active IL-1 is inducible and requires posttranslational proteolysis by inflammatory caspases to signal via IL-1R to promote neutrophil recruitment. As shown in Figure 1C, peritonitis induced by Natterin was accompanied by elevated levels of cytokines as IL-1 , IL-6, and TNF-which jointly promotes neutrophil recruitment, as well as the chemokine MCP-1 that regulate the migration and infiltration of macrophages. In contrast, Natterin did not promote the release of KC (CXCL1), chemokine controlling neutrophils recruitment (data not shown). Finally, we observed a production of PGE2, a pro-inflammatory mediator [28] produced in response to Natterin (Fig. 1D).

Our results show that the acute neutrophil recruitment to the site of inflammation induced by Natterin was associated with extracellular release of mature IL-, IL-6, TNF-and PGE2, independent of KC.

Natterin induces IL-33-expressing bronchial epithelial cells

In the search for the mediators involved in neutrophilic infiltration, we also investigated if

Natterin was able to produce IL-33, an alarmin resident constitutively in the nucleus, primarily of epithelial cells, endothelial cells, and fibroblasts that is released when barriers are breached and binds to a heterodimeric receptor comprising ST2 and IL-1 receptor accessory protein -IL-1RAcP [START_REF] Martin | Interleukin 33 is a guardian of barriers and a local alarmin[END_REF].

For this, we chose to use of an elegant strain of mice that constitutively express the il-33 gene built with a fluorescent report, allowing us to localize the IL-33-producing cell subtypes in different tissues after Natterin i.p. injection. We demonstrated the production of IL-33 by bronchial epithelial cells at 2h post-Natterin i.p. injection in IL-33/citrine reporter (citR) (right, Fig. 2A). Twenty-four hours later Natterin induced several-fold higher IL-33 positive cells compared to 2h (Fig. 2B). The histological examination of lung sections of both, WT or citR mice receiving i.p. injection of sterile PBS (control-mice) revealed absence of IL-33-expressing cells (left, Fig. 2A). Also, no detection of IL-33 was associated with vascular structures of the lung sections from control-or Natterin-injected citR mice (data not shown). No detection of IL-33-expressing cells was observed in peritoneal membrane of control-or Natterin-injected citR mice (Fig. 2C).

Natterin drives lung neutrophilic inflammation mediated by IL-33/ST2 engagement

To better define the mechanism by which cytokines are involved in neutrophilic inflammation induced by Natterin, we employed knockout mice (KO) in the genes of IL-17A, IL-17RA, TNF-, and WT BL/6 mice treated with anti-ST2 receptor neutralizing antibody via i.p. injection, a classical route used for the control of pulmonary inflammation in murine models of asthma, chronic obstructive pulmonary disease (COPD) or bronchopulmonary dysplasia [START_REF] Xu | IL-33-induced neutrophil extracellular traps degrade fibronectin in a murine model of bronchopulmonary dysplasia[END_REF][START_REF] Lee | Blockade of IL-33/ST2 ameliorates airway inflammation in a murine model of allergic asthma[END_REF][START_REF] Kearley | Resolution of allergic inflammation and airway hyperreactivity is dependent upon disruption of the T1/ST2-IL-33 pathway[END_REF].

In Figure 3A, we confirmed that the recruitment of neutrophils promoted by Natterin stimulation was completed dependent on ST2 receptor, since a drastic reduction of 97 ± 0.1% was observed in the number of neutrophils recruited to peritoneal cavities of mice that had blocked ST2 receptors by treatment with anti-ST2 neutralizing Abs 30 min before Natterin injection, confirming the importance of ST2/IL-33 signaling on the neutrophils migration. IL-17A/IL-17RA signaling was also decisive for the recruitment of neutrophils (Fig 3B), since the deficiency of both genes in BL/6 mice injected with Natterin resulted in a strongly reduction in the number of neutrophils into peritoneal cavities (reduction of 86 ± 0.1% and 89 ± 2%, respectively). Moreover in Figure 3C we also observed that TNF-KO mice exhibit a partial decrease of 68 ± 2% in neutrophil recruitment to peritoneal cavity 2h post-Natterin injection compared to Natterin-injected WT mice.

The demonstrated production of IL-33 by bronchial epithelial cells at 2h and mainly at 24h post-Natterin i.p. injection (Fig. 2A and Fig. 2B) and the complete dependence of ST2 on neutrophil recruitment (Fig. 3A) were accompanied by intense and late infiltration of MPO-positive neutrophils into airways of mice at 24h (brown color, Fig. 3D) compared to PBS-injected control mice, characterizing a systemic inflammatory syndrome. In contrast, no infiltration of neutrophils was observed into the lungs of mice treated with anti-ST2 receptor neutralizing antibody before Natterin injection (Fig. 3D), implying IL-33/ST2 as a key driver of local (peritoneal cavity) and remote (lung) systemic inflammation induced by Natterin.

For discharged the role of reverse transendothelial migration (rTEM) [START_REF] Nourshargh | Reverse Migration of Neutrophils: Where, When, How, and Why?[END_REF] in the recruitment of neutrophils into the lungs directly from the peritoneal cavity, we treated mice before Natterin injection with selective pharmacological intervention of c-MET by capmatinib [START_REF] Finisguerra | MET is required for the recruitment of anti-tumoural neutrophils[END_REF]. As shown in Figure 3E the previous treatment of Natterin-injected WT mice with a potent and selective c-Met tyrosine kinase inhibitor (HY13404) did not reverse the neutrophilic infiltration into the lungs and mice presented the same amount of MPO-positive neutrophils in the lungs after Natterin injection compared to Natterininjected WT mice, demonstrating that pulmonary infiltration was derived from bone marrow-mature neutrophils and migration through the bloodstream to the inflamed lung tissues. However, we cannot rule out that pulmonary neutrophils came from the pulmonary marginal reservoir. Within the lung alone, this pool constitutes the most prominent reservoir of neutrophils in the systemic circulation ( 40% of total body neutrophils), enabling rapid neutrophil recruitment inside of the tissue following injury and/or infection.

IL-1/IL-1R-dependent neutrophilia requires caspase-1 and caspase-11

To determine the relative contributions of IL-1 and IL-1 to neutrophil recruitment induced by Natterin, we utilize IL-1 / KO or IL-1 KO mice and deficient mice in type I IL-1 receptor (IL-1R KO). We observed that IL-1R KO (73 ± 2%) and IL-1 / KO (82 ± 4%) mice exhibit a significant decrease in neutrophil recruitment to the peritoneal cavity 2h post-injection. IL-1 KO presented an inhibition of 74 ± 2% in the recruitment of neutrophils compared to BL/6 WT Natterin injected-mice, suggesting that another ligand of IL-1R receptor as ILinjury as confirmed in mice that had blocked IL-by treatment with anti-IL-neutralizing specific monoclonal Abs 30 min before Natterin injection (Fig. 4A).

Next we confirmed the role of caspase-1 and caspase-11 in Natterin-neutrophil mobilization (Fig. 4B). First, we found that ICE KO (Casp1 Casp11 129mt/129mt ) mice deficient in both caspase-1 and caspase-11 were not susceptible to neutrophil recruitment induced by Natterin (decrease of 82 ± 0.4%). Moreover, neutrophilic infiltration in peritoneal cavities of mice was completely prevented when caspase-1 was blocked by the inhibitor YVAD-CMK Ac or caspase-11 by wedelolactone (99 ± 0.1%).

We further investigated the dependence of IL-1 and caspase-1 and caspase-11 on the modulation of Natterin-induced lung neutrophilia. We noticed in Figure 4D that neutralization of IL-1by specific antibody or inhibition of caspase-1 and caspase-11 by treatment with YVAD-CMK and wedelolactone respectively completely blocked neutrophil influx into the lungs 24h after Natterin injection into peritoneum.

Our results demonstrate that caspase-1 and caspase-11 were required for processing of pro-IL-

, and together with IL-1 both cytokines control the systemic neutrophilic response.

Natterin promotes neutrophilic infiltration via gasdermin-D-dependent process

Physiological roles for gasdermin-D in both pyroptosis and IL-1 / ILinflammasome signaling have been extensively characterized in macrophages and other mononuclear leukocytes [START_REF] Jorgensen | Pyroptosis triggers pore-induced intracellular traps (PITs) that capture bacteria and lead to their clearance by efferocytosis[END_REF]. Next, we investigated the role of gasdermin-D in the recruitment of neutrophils induced by Natterin and found that like IL-1R1 KO, IL-lizing-mice, and caspase-1/11 KO mice, gasdermin-D gene deficiency mice also displayed an intense decrease in the number of neutrophils (99% ± 0.1%) recruitment to the peritoneal cavity 2h post-injection, compared to Natterininjected WT mice (Fig. 4C).

We tested whether pyroptosis drives the influx of neutrophils induced by Natterin in pretreated mice with glycine, a potent inhibitor of pyroptosis [START_REF] Weinberg | The role of glycine in regulated cell death[END_REF]. In Figure 4C we demonstrated that WT mice treated with the cyto-protective agent glycine demonstrated intense neutrophil infiltration following Natterin injection compared to control-mice.

Together, these data demonstrate that the neutrophilic inflammation triggered by Natterin was a gasdermin-D-dependent inflammasome process, despite the cells did not die by pyroptosis and remained viable and continue to participate in the inflammatory response.

7. NLRP6/NLRC4, but not NLRP3, drives the neutrophilic infiltration induced by Natterin NLRP3, the most extensively studied member of inflammasome family has been implicated in the sensing of a multiplicity of pathogens as bacteria, virus, fungus, parasites, and several aerolysinlike pore-forming toxins from bacteria or viruses [37; 38]. In response to cellular stress the NLRP3 inflammasome activates multimerization of the adaptor molecule ASC (apoptosis-associated specklike protein with a caspase recruitment domain) and pro-caspase-1, resulting in the processing and secretion of the pro-inflammatory cytokine IL-39].

Next we tested the functional relevance of NLRP3 and ASC to Natterin-induced neutrophilia.

In Figure 5A, we observed that the recruitment of neutrophils in ASC deficient mice was drastically decreased (80 ± 0.4%) compared to Natterin-injected WT mice. However in NLRP3 KO or mice treated with the inhibitor of NLRP3-dependent ASC oligomerization, MCC950 [START_REF] Coll | A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases[END_REF], the recruitment of neutrophils remained elevated compared with BL/6 WT Natterin-mice.

In the search for the requirement for another member besides NLRP3 to activate the ASC/caspases complex assembly, we investigated the participation of sensors such as NLRP6 and NLRC4 adaptor, both expressed and active in epithelial cells [START_REF] Duncan | The NLRC4 Inflammasome[END_REF].

Our results in Figure 5B demonstrated that the absence of NLRP6 virtually abolished (98 ± 0.5%) the recruitment of neutrophils into the peritoneal cavities of mice injected with Natterin. In Figure 5C we further found that NLRC4 deficiency in BL/6 mice led to significantly decrease of neutrophil infiltration (62 ± 3%) after injection of Natterin. However, we also confirmed that mitochondrial anti-viral signaling protein (MAVS) a sensor required for recruitment of NLRP3 to the mitochondria and IL-42] was not involved in neutrophilic inflammation induce by Natterin (Fig. 5D).

These results are indicative that IL--1/caspase-11-dependent neutrophilic inflammation was mediated by non-canonical sensors through ASC interaction.

DISCUSSION

Here in this work, our objective was to investigate the regulatory mechanisms controlling acute neutrophilic inflammation induce by Natterin , specifically its ability to induce IL--dependent neutrophilic inflammation mediated by inflammasome activation. For this, using a combination of genetic loss-of-function and pharmacologic approaches we conduct a systematic investigation in mice of the regulatory mechanisms that control Natterin-induced neutrophilic inflammation in the peritonitis model.

Neutrophils are typically the first responders to infection and injury where they help to contain and destroy antigens through a number of mechanisms [START_REF] Kolaczkowska | Neutrophil recruitment and function in health and inflammation[END_REF]. Our data confirm the capacity of Natterin Biologically active cytokine IL--ILthrough active caspase-1, a cysteine protease [START_REF] Howard | IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha[END_REF]. Recent reports show a role for caspase-11 in regulating the activation of a non-canonical inflammasome that promotes cell death as well as IL-1

and IL-1 secretion and neutrophil recruitment during infection response [START_REF] Kayagaki | Non-canonical inflammasome activation targets caspase-11[END_REF].

Through multiple systemic and local effects, ILand immune responses. IL-of multiple pro-inflammatory genes such as IL-6, IL-8, MCP-1 (or CCL2), IL- [START_REF] Dinarello | Overview of the IL-1 family in innate inflammation and acquired immunity[END_REF]. By enhancing the production of the alarmin IL-33 and subsequent release by damaged cells, ILrecruitment and activation to the site of inflammation [47; 48]. IL-33/ST2 axis seems to be involved in Th17 [START_REF] Vocca | IL-33/ST2 axis controls Th2/IL-31 and Th17 immune response in allergic airway diseases[END_REF] response and IL-17A seems to up-regulate IL-33 expression in NHEK, maybe by induction of ERK, p38/MAPK, and JAK/STAT pathways [START_REF] Martin | Interleukin 33 is a guardian of barriers and a local alarmin[END_REF].

Further, the participation of IL-33 in neutrophil infiltration was demonstrated be dependent on IL-33R expression on peritoneal mast cells and on dependent-or independent mast cell release of TNF- [50; 51]. Our data here highlight the view that neutrophils and Th17 cells establish a communication network in chronic inflammatory diseases, once we confirmed that Natterin-induced systemic neutrophilia was totally dependent on the binding of IL-33 to its ST2 receptor with the entire participation of IL-17A/IL-17RA signaling, as well as partially the cytokine TNF-An increasing body of evidence supports the role of pro-inflammatory cytokines like IL-, IL-17 and TNF-in the pathophysiology of neutrophilic diseases similarly to classic monogenic autoinflammatory diseases, suggesting common physiopathological mechanisms [START_REF] Marzano | A Comprehensive Review of Neutrophilic Diseases[END_REF]. Neutrophilic inflammation, Th1 and Th17 cells establish a communication network in the airways of some severe asthmatics, refractory to treatment by inhaled or oral corticosteroids. Also, the association between IL-17 and neutrophilic inflammation is well-documented in other diseases such as psoriasis, where targeting the IL-17R with Brodalumab reduces neutrophil infiltration of the skin with improvement in disease.

Our data confirming the role of IL-33/ST2 as a mechanism underlying the systemic effect of Natterin inducing the recruitment of neutrophils to the inflamed cavity and later to the lung highlight that Natterin is a potent pro-inflammatory molecule and are evidence that a large number of cells may sense and respond to Natterin.

Altogether our data adds another level of complexity to the Natterin-induced neutrophilic inflammatory process. First, we demonstrated that the IL-1/IL-1R-dependent neutrophilic inflammation induced by Natterin was mediated by caspase-1 and caspase-11 activated by the inflammasome sensor NLRP6 in an ASC dependent-manner. And second, we found that the neutrophilic inflammation triggered by Natterin was gasdermin-D-dependent, but was not affected by glycine treatment. In murine macrophages, glycine markedly delays pyroptosis (via an unknown mechanism), but does not inhibit IL-1 release or assembly of N-gasdermin D pores [53; 54], which led us to think that the neutrophilia induced by IL-1-dependent Natterin is mediated by its excretion in the gasdermin-D-dependent process without the requirement of cell lysis by pyroptosis, as recently described by others [START_REF] Batista | Gasdermin-D-dependent ILduring chronic Toxoplasma gondii infection[END_REF][START_REF] Bulek | Epithelial-derived gasdermin D mediates nonlytic IL-57[END_REF][57][START_REF] Diamond | Salmonella typhimurium-induced IL-1 release from primary human monocytes requires NLRP3 and can occur in the absence of pyroptosis[END_REF][START_REF] Zanoni | An endogenous caspase-11 ligand elicits interleukin-1 release from living dendritic cells[END_REF][START_REF] Chen | The neutrophil NLRC4 inflammasome selectively promotes ILpyroptosis during acute Salmonella challenge[END_REF].

Unlike several studies showing that different bacterial [61; 38; 17; 37] aerolysin-like poreforming toxins open pores in the membrane bilayer and trigger NLRP3 inflammasome-dependent caspase-1 activation IL--dependent neutrophilic inflammation, the activation of NLRP6/NLRC4 inflammasome by Natterin seems to be determinant for the for neutrophil recruitment, independently of NLRP3.

NLRP6 has a wide range of functions in innate immune signaling suppressing inflammation [62; 63] or playing a role as an inflammasome [START_REF] Chen | A functional role for Nlrp6 in intestinal inflammation and tumorigenesis[END_REF][START_REF] Elinav | NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis[END_REF][START_REF] Delanoye-Crespin | Nod-like receptor pyrin domain-containing protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury[END_REF][START_REF] Levy | Microbiota-modulated metabolites shape the intestinal microenvironment by regulating NLRP6 inflammasome signaling[END_REF][START_REF] Wang | Nlrp6 regulates intestinal antiviral innate immunity[END_REF][START_REF] Birchenough | A sentinel goblet cell guards the colonic crypt by triggering Nlrp6-dependent Muc2 secretion[END_REF][START_REF] Hara | The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection[END_REF]. Recently, Leng et al [START_REF] Leng | NLRP6 self-assembles into a linear molecular platform following LPS binding and ATP stimulation[END_REF] provided biochemical and cellular evidences of an alternative intracellular LPS recognition mechanism through NLRP6.

NLRC4 inflammasome acts as an adaptor to recruit and activate caspase-1 and requires NAIP for binding to ligands [START_REF] Li | Cryo-EM structures of ASC and NLRC4 CARD filaments reveal a unified mechanism of nucleation and activation of caspase-1[END_REF][START_REF] Tenthorey | Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes[END_REF][START_REF] Kofoed | Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity[END_REF]. We proposed that the cytosolic Natterin could be recognized by NLRP6

facilitating their interaction with NLRC4 allowing inflammasome assembly through ASC interaction and resulting in caspase-11 activation, corroborating the involvement of NLRP6 in inflammation.

Interestingly, our data extend those previously published by others showing NLRP3 as the most extensively member of inflammasome family implicated in the sensing of several aerolysin-like pore-forming toxins from bacteria or other such as fungal trichothecene mycotoxin [START_REF] Kankkunen | Trichothecene mycotoxins activate NLRP3 inflammasome through a P2X7 receptor and Src tyrosine kinase dependent pathway[END_REF], viral viraporins [START_REF] Ito | Encephalomyocarditis virus viroporin 2B activates NLRP3 inflammasome[END_REF], bee venom melittin [START_REF] Palm | Role of the inflammasome in defense against venoms[END_REF], and the frog aerolysin-like [START_REF] Xiang | Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection[END_REF] and emphasized that the NLRP6/NLRC4-dependent neutrophil-mediated response may be part of an innate immune mechanism underlying aerolysin from fish venoms.

In conclusion, w were able to clarify that the IL--dependent neutrophilic inflammation induced by Natterin is the result of non-canonical activation of the inflammasome complex with the participation of cytosolic NLRP6/NLRC4 sensors. Our data highlight that this 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.

METHODS

Mice

Natterin preparation

The purified 30-45 kDa Natterin proteins from T. nattereri fish venom were prepared with a pool of venom collected in different months of the year in the state of Alagoas. The venom was fractionated by cation exchange chromatography, using the fast protein liquid chromatography system (FPLC -Pharmacia, Uppsala, Sweden) according to Lopes-Ferreira et al [START_REF] Lopes-Ferreira | Kininogenase activity of Thalassophryne nattereri fish venom[END_REF]. Endotoxin content was evaluated (resulting in a total dose < 0.8 pg LPS) with QCL-1000 chromogenic Limulus amoebocyte lysate assay (Bio-

Acute inflammation induced by Natterin and pharmacological treatments

WT or KO mice (n = 3 to 5/group) were intraperitoneally (i.p.) injected with Natterin at 0.1, 1 or 10 g in 500 L. As negative-control mice were injected i.p. with PBS, and as positive-control mice were injected with 10 g of LPS (E. coli 055:B5, Sigma) in 500 L. After 2, 6, and 24h the peritoneum exudates were harvested for total and differential cell count and for processed proteins determination. Independent group of WT mice was pre-treated 30 min before with MCC950 at 100 mg/Kg; anti-ST2 neutralizing antibody at 25 g/mL (16-9333-82 ); anti-IL-(IgG mabg-mIL--5, Invivogen); YVAD-CMK Ac at 1.8 mg/mL, (17-8603-78-6, Invivogen); wed -200648, Santa Cruz); and glycine at 5 mM (00163, Chem-Impex). An independent group of mice was also treated 2 times a day 24h before Natterin injection with capmatinib at 10 mg/Kg (HY13404, INCB28060, MedChenExpress). Groups of mice were injected with an equivalent volume of NaCl, PBS or isotype control (rat IgG, or rat IgG1 or rat IgG2b). Mice were killed after 2 or 24h for analysis.

Peritoneal cell suspension collection

At time points indicated after Natterin injection, mice were killed, and peritoneum exudates were harvested with 2 x 2.5 mL of cold PBS + 10 mM EDTA for cell suspensions that were centrifuged at 1500 rpm for 10 min at 4°C. The supernatants were stored at -20°C; and cell pellets were resuspended in 1 mL of PBS + 0.1% BSA. The total leukocyte count was performed in a 10 squares of Malassez chamber with Turk solution. For differential counts, aliquots containing 100 L of cell suspension were applied on glass slides, subjected to centrifugation at 1000 rpm for 10 min and stained Cytospin with kit Diff-Quick Stain Set and analyzed in an optical microscope with a 40 x objective. For differential cell counts, 300 leukocytes were enumerated and identified as mononuclear cells, eosinophils or polymorphonuclear neutrophils, based on staining and morphologic characteristics using a light microscope Axio Imager A1 (Carl Zeiss, Germany) with an AxioCam ICc1 digital camera (Carl Zeiss).

Cytometric bead array for determination the level of cytokines

Concentrations of peritoneal exudates of IL-6 and TNF-were determined by cytometric bead modification. Briefly, 50 L samples were subjected to analysis in duplicate using the cytometric bead array kit on a FACSCalibur cytometer. Cytokine concentrations were quantified using CellQuestPro and CBA software (Becton Dickinson). The detection limits for IL-6 and for TNF-were 1.4 pg/mL and 0.9 pg/mL, respectively.

Quantification of IL-1 and MCP-1

IL-1 , KC and MCP-1 levels were measured in exudates of peritoneal cavity using a specific two-site sandwich ELISA with OpEIA Kits (BD-Pharmingen, San Diego, CA, USA). Biotinylated monoclonal antibodies binding has been detected using streptavidin-horseradish peroxidase complex -tetramethylbenzidine) substrate solution containing hydrogen peroxide.

Detection limit was 7.8 pg/mL for both IL-1 and MCP-1 and 25 pg/mL for KC.

Eicosanoid assay

Concentrations of PGE2 were measured in the peritoneal exudate lavage fluid collected 2h

after Natterin or PBS injection, by a specific enzymatic immunoassay, using a commercial kit (Cayman Chemicals, MI, USA) aliquots of each sample were incubated with the eicosanoid conjugated with acetyl cholinesterase and the specific rabbit antiserum in 96-well microtitration plates, coated with anti-rabbit IgG mouse monoclonal antibody. After addition of the substrate, the absorbances of the samples were recorded at 412 nm in a microplate reader, and concentration of the eicosanoid was estimated from standard curve.

Immunofluorescence for detection of IL-33

Natterin (1 g) or sterile saline in 500 L were injected i.p. into BL/6 WT or IL-33/citrine reporter (citR) mice. After 2 or 24h mice were anesthetized with a ketamine (50 mg/mL)/xylasine (25 mg/mL) and perfused through the ascending aorta with saline followed by 4% formaldehyde. After perfusion, lung and peritoneal membrane were removed and post fixed. For cryosectioning, fixed organs were transferred to 40% sucrose in phosphate buffer, mounted in OCT compound (Neg-50, immunofluorescence. All of the sections were stained with DAPI (sc-300415) and fluoromount-G (00-4958, eBioscience) was added to the slides prior to mounting with cover slips and imaged with a fluorescence microscope OLYMPUS BX51, objective 10 x, magnification bar 200 m. For each group of mice, four stained lung sections from each mouse were analyzed and IL-33-positive cells were counting using an image processing package FIJI Image J.

Immunohistochemical for detection of MPO in the lungs

Sections harvested from lungs 24h post Natterin injection were deparaffinized, rehydrated, and incubated in 3% H 2 O 2 in PBS for 10 min at room temperature to block endogenous peroxidase activity. The sections were then washed with PBS three times, incubated in 0.1% trypsin (Sigma Chemical) in 0.1% CaCl 2 at 37°C for 10 min, and rinsed with 4M HCl for 15 min. After nonspecific binding blocking the lung sections were incubated for 1h with anti-MPO (sc-271881, Santa Cruz, at 1/500). After incubation, anti-IgM HRP (A8786, Sigma, at 1/5000) was detected by incubating the sections for 5 min with chromogenic substrate for the peroxidase 3, 3'-diaminobenzidine (DAB) (Sigma), providing a brown reaction product. Sections were counterstained with hematoxylin and lungs were imaged with an upright microscope (Axiolab, Carl Zeiss, Oberkochen, Germany) coupled to a photographic camera (AxioCam Icc1, Carl Zeiss, Oberkochen, Germany) using a 10/0.3 longitudinal distance objective/numeric operture and 1.6 optovar (Carl Zeiss, Oberkochen, Germany).

For each group of mice, four stained lung sections from each mouse were analyzed and MPO-positive cells were counting using an image processing package FIJI Image J.

Statistical analysis

All values were expressed as mean ± SEM. Experiments using 3 to 5 mice per group were performed independently two times. Parametric data were evaluated using analysis of variance, followed by the Bonferroni test for multiple comparisons. Non-parametric data were assessed using the Mann-Whitney test. Differences were considered statistically significant at p < 0.001 using

  First revision Manuscript Number: INTIMP-D-20-00258 Lima C et al. HIGHLIGHT Natterin is aerolysin-like Natterin induced a local and systemic neutrophilic inflammation IL-1 /IL--1R1, IL-33/ST2, and IL-17A/IL-17RA signals controlled neutrophilia Neutrophilia required caspase-11 and caspase-1, but not NLRP3 activation Neutrophilia was mediated by a gasdermin-D-dependent process without pyroptosis Neutrophilia was mediated by ASC-dependent sensors NLRP6 and NLRC4

  to induce a strong and sustained neutrophilic inflammation in murine model of peritonitis leading to systemic inflammatory lung infiltration and reveal overlapping regulatory paths in the control of neutrophilia, including the co-participation of the IL-33/ST2, and IL-17A/IL-17RA signals dependent on IL-1 /IL--1R1.

Male or female 7 8

 8 weeks old C57BL/6J wild types (WT) or IL-1 / -, IL-1 -, IL-1R-, ICE-(Casp1 Casp11 129mt/129mt ), NLRP3-, ASC-, IL-17RA-, IL-17A-, NLRP6-, NLRC4-, MAVS-, Gasdermin-D-, and TNF--deficient mice KO, and IL-33/citrine reporter (citR) mice (all on a BL/6 background) were obtained from a colony at Transgenose Institute (Orleans, France). Mice were maintained in sterile microisolators with sterile rodent feed and acidified water, and were housed in positive-pressure air-conditioned units (25°C, 50% relative humidity) on a 12h light/dark cycle. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Brazilian and French College of Animal Experimentation. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Butantan Institute (Permit Number: 9381060819) and of Transgenose Institute (Permit Numbers: 1085 and 1088).

Figure 2 .

 2 Figure 2. Natterin induces IL-33-expressing bronchial epithelial cells. Natterin (1 g) in 500 L of

Figure 3 .

 3 Figure 3. Natterin drives lung neutrophilic inflammation mediated by IL-33/ST2 engagement.

Figure 4 .

 4 Figure 4. IL-1/IL-1R-dependent neutrophilia requires caspase-11/caspase-1 and Gasdermin-D.

Figure 5 .

 5 Figure 5. NLRP6/NLRC4, but not NLRP3 drives the neutrophilic infiltration induced by

  Figure1

  FIGURE TITLES AND LEGENDSFigure1. Natterin induces a strong and sustained neutrophilic inflammation in murine model of peritonitis. Natterin (0.1, 1 and 10 g) in 500 L of PBS was injected i.p. into BL/6 WT mice and as a negative-control mice were injected i.p. with PBS. Two hours later, peritoneal cells were harvested and aliquots were applied on glass slides, subjected to centrifugation and stained with Diff-Quick Stain kit and leukocytes were imaged with a microscope Axio Imager A1 (Carl Zeiss, Germany) for count (A). Natterin (1 g) in 500 L of PBS was injected i.p. into BL/6 WT mice and as a negativecontrol mice were injected i.p. with PBS. After 2, 6, and 24h the peritoneum exudates were harvested and aliquots were applied on glass slides, subjected to centrifugation and stained with Diff-Quick

Stain kit and leukocytes were imaged for count (B). Two hours after Natterin (1 g) injection, the peritoneum exudates were harvested for cytokine determination (C) by cytokine-specific ELISA (IL-1 and MCP-1 or cytometric bead array (IL-6 and TNF-); and PGE2 (D) levels determination by cytokine-specific ELISA. The results represent the mean ± SEM of 3-5 animals/group. *p < 0.001 compared with negative-control group.

ACKNOWLEDGMENTS

This work was supported by the São Paulo Research Foundation -FAPESP (Grants: 2019/10500-7; 2018/17413-0; 2013/07467-1), CNPq, and le Centre National de la Recherche Scientifique (CNRS), by European funding in Region Centre-Val de Loire (FEDER 2016-00110366 BIO-TARGET and EX005756 BIO-TARGET II). Further the support of the animal services (Nathalie Froux, Jeremy Paumier, Caroline Betrand, and Mathilde Favrat). ACKNOWLEDGMENTS This work was supported by the São Paulo Research Foundation -FAPESP (Grants: 2019/10500-7; 2018/17413-0; 2013/07467-1), CNPq, and le Centre National de la Recherche Scientifique (CNRS), by European funding in Region Centre-Val de Loire (FEDER 2016-00110366 BIO-TARGET and EX005756 BIO-TARGET II). Further the support of the animal services (Nathalie Froux, Jeremy Paumier, Caroline Betrand, and Mathilde Favrat).

COMPETING FINANCIAL AND/OR NON-FINANCIAL INTERESTS

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AUTHOR CONTRIBUTIONS

Maria