Evaluation of systemic inflammatory response and lung injury induced by Crotalus durissus cascavella venom

This study investigated the systemic inflammatory response and mechanism of pulmonary lesions induced by Crotalus durissus cascavella venom in murine in the state of Bahia. In order to investigate T helper Th1, Th2 and Th17 lymphocyte profiles, we measured interleukin (IL) -2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF) and interferon gamma (IFN-γ) levels in the peritoneal fluid and macerated lungs of mice and histopathological alterations at the specific time windows of 1h, 3h, 6h, 12h, 24h and 48h after inoculation with Crotalus durissus cascavella venom. The data demonstrated an increase of acute-phase cytokines (IL-6 and TNF) in the first hours after inoculation, with a subsequent increase in IL-10 and IL-4, suggesting immune response modulation for the Th2 profile. The histopathological analysis showed significant morphological alterations, compatible with acute pulmonary lesions, with polymorphonuclear leukocyte (PMN) infiltration, intra-alveolar edema, congestion, hemorrhage and atelectasis. These findings advance our understanding of the dynamics of envenomation and contribute to improve clinical management and antiophidic therapy for individuals exposed to venom.


Introduction
Accidents involving snakes have been considered a neglected disease by the World Health Organization (WHO) since 2007. It has been recognized as a public health issue with approximately 2.5 million envenomation accidents worldwide, leading to 125,000 deaths and many victims with serious permanent sequelae [1]. Although the physiopathology of these accidents is a complex event, it is known that inflammatory mediators play an important role in the a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Throughout the experiment, careful manipulation of the animals was performed only when necessary in a noise free environment. Concerned parties ensured the highest level of comfort possible and animals welfare, minimal suffering and euthanasia with brevity. We have avoided analgesics and anesthetics as they may interfere both in cytokines production and costimulatory signals required for antigen presentation and T lymphocyte differentiation. The research team, under the permanent supervision of the veterinarian, monitored animals throughout the entire experiment. Observations were conducted every hour to evaluate clinical behavior, level of activity, posture, temperament, locomotion, water and food intake. Signs of respiratory distress, pain or abnormal behavior have been evaluated and recorded. Animals were euthanized immediately at protocol predeterminated times (1h, 3h, 6h, 12h, 24h and 48 hours) with an overdose with ketamine (100mg/Kg) and xylazine (10mg/kg) with immediate extraction of peritoneal fluid and lung tissue. No animals were found dead during the experiment period and all mice were euthanized at the predetermined times.

Ethics statement
The study was conducted in accordance with the ethical principles for animal research adopted by the Brazilian Society of Animal Science and the National Brazilian Legislation n˚.11.794/08 and was approved by the State University of Feira de Santana Animal Ethics Committee (CEUA-UEFS-protocol number 006/2018). The research team received obligatory training in animal welfare, handling and sacrifice technics.

Inflammatory response in mice induced by Crotalus durissus cascavella venom
The experiments were performed a total of 72 mice subdivided into control group (n = 5) and experimental group (n = 7) for each determined time. The experimental group received the venom challenge dose of 50μg/kg [3], by the intraperitoneal (i.p.) route, diluted in 500μL of sterile saline solution (NaCl 0.9%) [3]. The control group received 500μL of sterile saline solution (NaCl 0.9%). Both groups were monitored during the entire experiment (1, 3, 6, 12, 24 and 48 hours). The animals were euthanized with an overdose with ketamine (100mg/Kg) and xylazine (10mg/kg) to obtain the peritoneal fluid and lung.

Obtaining the peritoneal fluid
In both groups, 2mL of phosphate saline solution (Phosphate Buffered Saline-PBS) was injected into the peritoneal cavity to obtain the peritoneal fluid. Then, the animals had their abdomens massaged to wash the entire cavity. The peritoneal fluid was extracted using a syringe, centrifuged at 3000rpm at 4˚C, for 20 minutes [33] and the supernatant separated and stored at -20˚C for subsequent cytokine dosages.

Lung tissue disintegration and histopathologic analysis
The inferior lobe of the right lung was removed and stored in 1mL of PBS solution, then, macerated and centrifuged at 3000 rpm at 4˚C, for 20 minutes [33]. Supernatant was separated and stored at 20˚C for subsequent cytokine dosage. The left lung and the superior lobe of the right lung were initially fixed in 10% buffered formalin for a maximum of 48 hours. Fragments underwent ethanol and xylene dehydration and were diaphanized and cut to a width of 4μm and subsequently stained in a hematoxylin-eosin solution (HE) and Masson's trichrome. The cuts were examined, and images captured by Olympus BX 51 microscope with a coupled digital camera (DP25) and digitalized on cellSens software. Morphometric analysis was performed on five randomly selected microscopic fields on lung parenchymal slides in Swiss mice at different exposure times (1h, 3h, 6h, 12h, 24h, 48h). The number of inflammatory cells was counted in mm 2 with a 20x eyepiece and a 20x objective, in an area of 60 mm 2 . Collagen deposition was quantified by counting the areas marked by Masson's trichrome in mm 2 in five randomly selected microscopic fields on the lung parenchyma slides at 6h, 12h, 24h exposure times. All data obtained were analyzed using ImageJ software (USA). Statistical results were evaluated by the nonparametric Mann-Whitney Test with significance of p<0,05. The graphs were generated by GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA).

Cytokine measurements using Cytometric Bead Array (CBA)
Cytokine concentrations in the peritoneal fluid supernatant and macerated lungs were determined using a BD™ CBA Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, USA) with a FACSCalibur flow cytometer (San Francisco, BD Biosciences). Cytometric Bead Array analysis allowed the simultaneous detection of cytokines, TNF, IL-6, IL-4, IL-10, IFNγ, IL-17A and IL-2, and was performed in accordance with the manufacturer´s instructions. In brief, the Th1/ Th2/Th17 cytokine standards were prepared using a vial of lyophilized Mouse and Assay Diluent using the serial dilutions technique. Capture beads were added into each tube containing samples and standards and then incubated for 2 hours at room temperature in the absence of light. The samples were washed with 1 mL buffer at 200g for 5 minutes and resuspended in 150μL wash buffer. Data acquisition was performed using FCAP Array v2.0 software (Soft Flow, Hungary).

Statistical analysis
The data distribution was evaluated using the Kolmogorov-Smirnov test. To compare the time intervals evaluated, normal distribution data were analyzed using one-way analysis of variance (ANOVA) and Tukey´s test. The Kruskall-Wallis test followed by Dunn's Multiple Comparison Test was used in case of skewed distribution. The T student test was employed to compare experiment and control groups, and non-parametric distributions through the Mann-Whitney test. Results were expressed as mean (standard deviation-SD) and median (interquartile range) and values of p<0.05 were considered to be statistically significant. Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad, San Diego, CA, EUA).

Clinical manifestations induced by Crotalus durissus cascavella venom in mice
Experimental animals presented agitated behavior, pruritus and wound licking. One hour after inoculation, subjects showed patterns of nesting behavior, prostration, progressive lethargy and tremors (Fig 1A, 1B and 1C). Peak respiratory discomfort occurs between 6 and 48 hours, characterized by tachypnea with intense abdominal contractions and thoracic effort (Fig 1D and 1E). Animals in the control group showed no clinical deterioration throughout the experimental period.

Determining Th1/Th2/Th17 cytokine levels in the peritoneal fluid and macerated lung
Analysis of the peritoneal fluid and macerated lung indicated that Crotalus durissus cascavella venom induced varied levels of Th1/Th2/Th17 cytokines and regulatory response at all intervals analyzed. Significant differences between the experimental and control groups were found in TNF (Fig 2A), IL-6 ( Fig 2B) and IL-4 ( Fig 2C) concentrations in the peritoneal fluid. The experimental group presented significantly higher TNF (#p<0.05) and IL-6 (#p<0.05) 1 hour after inoculation. IL-6 levels of the experimental group continued to be higher than the control group at the 3-hour temporal window (#p<0.05). There was an increase in IL-4 concentration at the 24-hour time (#p<0.05).
Analyzing the kinetics of cytokine production in the peritoneal fluid of the experimental group, TNF reaches highest levels over the first hour with subsequent gradual decline ( � p<0.05 e �� p<0.01). IL-6 also presented a statistically significant difference in the experimental group ( � p<0.05 e �� p<0.01), with a peak in the first hour and a subsequent progressive decline over the other time windows. IL-4 production remained similar in the first time windows (1, 3 and 6h), dropping off at 12 hours, with elevated late levels at 24 hours (#p<0.05). Levels of IFNγ, IL-17A and IL-2 have not presented a statistically significant difference.
Among cytokines in the macerated lungs (Fig 3), both IL-4, at 3 hour and IL-10, at the 3 and 12 hour times, demonstrated significant difference between the experimental and the control groups (#p<0.05). The remaining analyzed cytokines have not showed significant  differences. During follow up period, only IFN-γ presented a statistically significant profile between the 3 and 48 hour time windows ( � p<0.05), peaking at 3 hours and declining to its lowest level at 48 hours.

Histopathology of lung damage induced by Crotalus durissus cascavella venom
There was no physiopathological alteration of the pulmonary parenchyma in control group ( Fig 4A). Nevertheless, Crotalus durissus cascavella venom induced early recruitment of Each point represents the mean-SD (A and C) and median-IQR (B, D, E, F and G) of animals per group. � p< 0.05 and �� p<0.01 in relation to the venom treatment times and #p<0.05 when compared to the control group.
At six hours, a decrease in the activity of inflammatory response is observed, despite an increase in vascular congestion, alveolar septa thickening and emphysematous areas (Fig 4D). Between 12 and 48 hours, there were perceptible inflammatory infiltrates in the pulmonary parenchyma, hemorrhagic focuses, vascular congestion and bronchial muscle distension ( Fig  4E).

Discussion
Physiopathogenic mechanisms that lead to pulmonary damage induced by Crotalus durissus venom are still not fully understood, especially inflammatory response and cytokine production [2,28,30,31]. The present study observed a systemic inflammatory response induced by Crotalus durissus cascavella venom associated with aggressive acute pulmonary injury characterized by peribronchial inflammatory infiltrate and altered vascular permeability with patchy hemorrhagic foci.
In response to acute antigenic stimulus induced by toxins, a modulated Th1, Th2 or Th17 immune response occurs [24,25]. The systemic action of toxins triggers an inflammatory response and enhance production of several immunological mediators that activates recruitment, proliferation and differentiation of leukocytes [29,34].
An early increase of IL-6 and TNF levels in the peritoneal fluid and macerated lungs suggests that pro-inflammatory cytokines play a key role in acute inflammation [29]. Previous studies have reported a similar inflammatory profile in the first hours after inoculation of C. d. terrificus venom with subsequent late immunomodulation. These characteristics can be related to crotoxin activity in inhibiting neutrophil chemotaxis and modulating adaptive immune response [17,21,[35][36][37].
Snakebite envenoming induces high levels of IL-6, TNF and IL-1β cytokines and may provoke fever, lethargic, vasodilation and a variety of other symptoms resulting from edema induction, T and B cell activation, and leukocyte recruitment [2,38,39]. It is plausible that high levels of these cytokines are directly related to aggressive clinical manifestations, such as prostration, lethargy and huddling.
Increased cytokines levels during the acute stage have been described by several authors in accidents involving venomous animals [4,33,[40][41][42]. Studies involving scorpion venom demonstrated a positive physiopathological correlation between cytokine concentrations and severity of clinical manifestations [43][44][45][46] Systemic toxicity is markedly affected by cytokines release. Increase IL-10 levels in peritoneal fluid at 12 hour and at 3h and 12h in macerated lungs may play an important role in immune response modulation, favoring a shift from a Th1 towards a Th2 cytokine. The increase in IL-4 in macerated lungs at 3h and at 24h in peritoneal fluid strengthened this hypothesis. Similar data were observed with C. d. terrificus venom [29]. It was also shown that Tityus serrulatus scorpion venom induced an increase in IL-10 and a decrease in IL-6 and TNF [46].
Anaphylaxis is a severe acute allergic manifestation with potentially fatal clinical repercussions, usually prompted by IgE mediated hypersensitivity. The levels of IL-4 become particularly elevated and induce an increase susceptibility to vasoactive mediators [48]. Previous studies observed anaphylactic response to snake venom characterized by rising serum IgE levels. These observations suggest that increase levels of IL-4 and IL-6 may be related to the anaphylactic response in the experimental group. Further research is required to provide evidence of this anaphylactic pathway due to their potential implications for the clinical management of patients bitten by Crotalus durissus cascavella.
The low levels of IL-2 found in the macerated lungs and peritoneal fluid indicated an inhibitory effect on lymphoproliferation, as observed in studies in vitro with C. d. terrificus and C. d. Evaluation of Systemic Inflammatory Response And Lung Injury Induced By Crotalus durissus cascavella Venom collilineatus venom, and with C. d. terrificus crotoxin [17,49]. However, short half-life and low circulation of IL-2 [50] may reduce detection levels of this cytokine.
Decline of IFNγ in macerated lungs and the absence of significant levels of IL-17 may be related to IL-4 production. This cytokine plays a pivotal role decreasing the production of Th1 and Th17 cells. However, the increase in IFNγ in the first hours (3h) in macerated lungs, despite the presence of IL-4 and IL-10, may lead to systemic disorders such disseminated vascular coagulopathy [3]. Furthermore, metalloproteinases can activate prothrombin and coagulation factor X, which inhibit platelet aggregation, and lead to apoptotic activity and hemostatic changes [51].
Despite its severity, few studies have examined pulmonary alterations in snakebite accidents [28, [52][53][54]. In crotalic accidents, crotoxin blocks the neuromuscular transmission that contributes to the development of paralysis, muscular respiratory insufficiency and acute respiratory distress [11,28]. The respiratory abnormalities related in this study are similar to the severe cases of respiratory paralysis reported in accidents caused by snakes in the Elapidae family [55,56], and Vipera palaestine [57].
Hemorrhage focuses, congestion, atelectasis and emphysema in the pulmonary parenchyma within 3 hours of exposure demonstrate an early physiopathological pathway of pulmonary damage and impaired respiratory mechanics, leading to acute lung injury (ALI). Similar alterations were observed with Crotalus durissus cascavella and Tityus e Androctonus venom [31, [58][59][60][61]. Respiratory damage have also been attributed to Bothrops jararaca venom, either by hemorrhage due metalloproteinases or by the action of PLA2 which can lead to pulmonary inflammation [62]. These data emphasize that snakebite victims presenting respiratory alterations require intensive monitoring.
Massive PMN cells proliferation in the pulmonary parenchyma, present up to the 48 hour time window, is probably linked to an increase in IL-17 levels [30,31]. Collagen deposition in the pulmonary parenchyma observed at 6, 12 and 24 hours can be related to mechanisms of repair and remodeling following an aggressive inflammatory response [63,64].
In summary, these findings advance our understanding of the pathophysiologic events and acute lung injury (ALI) induced by Crotalus durissus cascavella venom. High levels of acute phase cytokines detected in the peritoneal fluid and macerated lungs induced a systemic inflammatory response. There is a positive correlation between severity of clinical manifestations and pro-inflammatory cytokines levels (IL-6, TNF). Nevertheless, enhanced IL-4 production indicates a dynamic switch from Th1 to Th2 response and may be related to anaphylaxis triggered by venom components. Public policies should to be designed to prevent and improve patient outcomes in snake accidents.