Inflammatory effect of Bothropstoxin-I from the Bothrops jararacussu venom mediated 1 by NLRP3 inflammasome involves ATP and P2X7 receptor 2

23 24 Muscle tissue damage is one of the local effects described in bothropic envenomations. 25 Bothropstoxin-I (BthTX-I), from B. jararacussu venom, is a K49-phospholipase A 2 that 26 induces a massive muscle tissue injury, and, consequently, local inflammatory reaction. The 27 NLRP3 inflammasome is a sensor that triggers inflammation by activating caspase 1 and 28 releasing IL-1  and/or inducing pyroptotic cell death in response to tissue damage. We, 29 therefore, aimed to address activation of NLRP3 inflammasome by BthTX-I-associated injury 30 and the mechanism involved in this process. Intramuscular injection of BthTX-I results in 31 infiltration of neutrophils and macrophages in gastrocnemius muscle, which is reduced in 32 NLRP3- and Caspase-1-deficient mice. The in vitro IL-1β production induced by BthTX-I- 33 inperitoneal macrophages requires caspase 1/11, ASC and NLRP3 and is dependent of ATP- 34 induced K + efflux and P2X7R. BthTX-I induces a dramatic release of ATP from C2C12 35 myotubes, therefore representing the major mechanism for P2X7R-dependent inflammasome 36 activation in macrophages. A similar result was obtained when human monocyte-derived 37 macrophages were treated with BthTX-I. These findings demonstrated the inflammatory 38 effect of BthTX-I on muscle tissue, pointing out a role for the ATP released by damaged cells 39 for the NLRP3 activation on macrophages, contributing to the understanding of the 40 microenvironment of the tissue damage of the Bothrops envenomation. 41

. 67 Recently, we showed the coordinated cellular infiltration initiated by neutrophils, 68 followed by classical and alternative (M1 and M2) macrophage subsets in injured muscle 69 tissue triggered by BthTX-I injection. Furthermore, we demonstrated a direct correlation 70 between the macrophage subsets that, by distinct cytokines production, exert a crucial role in 71 the inflammation phase and myogenesis process (15). 72 As previously postulated, the innate cells, through distinct receptors (PRRs), such as inflammation (17). However, the innate molecular mechanisms that detect these components 80 and initiate the inflammatory response remain under investigation. 81 Among distinct germ-line-encoded receptors expressed by innate cells, the NLRs can be 82 activated by both PAMPs and DAMPs, leading to the production of cytokines and 83 chemokines mediating the inflammation (16). Several NLRs are able to the assembly of 84 inflammasome that results in the production of IL-1β and IL-18, such as NLRP3, which can 85 be activated not only by microbial molecules, but also by several endogenous or exogenous 86 agents (16, 18). In agreement with this, Palm and Medzhitov (19) showed that the  inflammasome via NLRP3. However, the mechanisms involved in this process is not 93 completely elucidated. Therefore, here we evaluated the role of inflammasome activation and 94 the mechanisms involved in this process. Our results show that the sequential recruitment of 95 neutrophils followed by macrophages triggered by BthTX-I injection at distinct time points is 96 mediated by NLRP3-inflammasome. Furthermore, BthTX-I is able to induce IL-1β 97 production dependent on NLRP3-activation by macrophages. We also verified that BthTX-I    In some experiments, the pan-caspase inhibitor Z-YVA-FMK (50 μM, Sigma-Aldrich) 167 was added to PM after 2 hours of LPS priming; or LPS-primed PM was treated with KCl 168 (50mM) and NaCl (50mM) for 1 hour before BthTX-I stimulation. Moreover, 5x10 6 PM were 169 eventually cultured in the presence of C2C12 "conditioned medium", as described below.

170
To assess the participation of the P2X7 receptor on NLRP3 activation of PM, the cells 171 were incubated with LPS, and, after 90 minutes, it was added the A437980 (10mM, Sigma-  Differentiated C2C12 cells were incubated with BthTX-I (10 μg/mL) for 6 hours, then 181 centrifuged and used for electron microscopy analysis. Supernatants were collected for IL-1, 182 LDH, and ATP measurement, or pooled and used as a "conditioned medium" for PM culture.

BthTX-I induces NLRP3 inflammasome activation in LPS-primed peritoneal macrophages 291
In order to study the mechanism involved in the inflammasome activation by BthTX-I, 292 we evaluated the ability of the toxin to activate the NLRP3 inflammasome in macrophages 293 considering their role on local inflammation triggered by the venom.

294
Therefore, we treated LPS-primed peritoneal macrophages with 10µg/mL BthTX-I for 6 295 hours and measured caspase-1 activation and IL-1 production. ATP (1 mM) was used as a 296 positive control for NLRP3 inflammasome activation.  Figure 2C). 314 In addition, the IL-1 release from peritoneal macrophages of mice deficient of caspase-315 1, NLRP3 or ASC, but not NLRC4 (IPAF), was quite completely abolished ( Figure 2D).    Interestingly, BthTX-I also induced IL-1β production by human macrophages, and it 381 was dependent oncaspase-1 activation since lower IL-1β production was detected in cells 382 culture primed or not with LPS and incubated with the toxin plus caspase-1 inhibitor 383 (parthenolide) ( Figure 4E). Furthermore, blocking of IL-1β production by human-derived 384 macrophages incubated with ATP or BthTX-I was also verified in cultures with high KCl 385 concentration, as shown in figure 4E. These results suggest, therefore, that BthTX-I is able to 386 activate the NLRP3 inflammasome via ATP mediated by P2X7R and involving K + -efflux.  Therefore, considering our data that NLRP3/IL-1 participates in the inflammatory response 481 elicited by the BthTX-I, the interference of this process by using the anakinra, as an 482 interleukin (IL)-1 receptor antagonist (Ra) (50, 51), could be useful in to minimize the 483 exacerbated local reaction triggered by the snakebite envenomation.

484
In summary, we demonstrated that BthTX-I is able to induce inflammasome activation 485 in vitro promoting the production of IL-1β dependent of caspase-1, NLRP3 and ASC in 486 macrophages. In addition, BthTX-I induced intense ATP release by C2C12 myotubes, which 487 as a DAMP, together with the toxin were able to activate NLRP3 via P2X7R in macrophages 488 producing IL-1β. Therefore, our data contribute to a better understanding of the molecular 489 mechanisms of inflammasome activation in response to a toxin that is involved in