NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection

Abstract

Background Neospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes.Methods Neospora caninum-infected wild-type (WT) macrophages and Nlrp3 −/− macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed.ResultsMany NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage.ConclusionsThe NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2197-2) contains supplementary material, which is available to authorized users.

NLRP3 Inflammasome Participates in Host Response to Neospora caninum Infection

“…Our previous study showed that N. caninum could induce IL-1β release and caspase-1 activation in murine peritoneal macrophages ( 15 ). To determine if N. caninum also activates the inflammasome in BMDMs, unstimulated and LPS pre-treated BMDMs were infected with N. caninum (parasite:cell MOI = 1 or 3) for 3 h. BMDMs with culture medium alone did not produce measurable levels of IL-1β (p17) or active caspase-1 (p20).…”

“…However, glyburide did not greatly impair the production of IL-6 (Figure 1 J). Our previous study has shown that glyburide could slightly inhibit the replication of N. caninum in Vero cells but not affect its abilities of invasion and viability ( 15 ). These data indicate that K + efflux participates in the signals that activate NLRP3 inflammasome in N. caninum -infected BMDMs.…”

“…Understanding of the immune responses against N. caninum is limited. We have previously demonstrated that the NLRP3 inflammasome can be activated in N. caninum infection in vitro ( 15 ). In this study, we focused on the role of the NLRP3 inflammasome in mediating the immune response against N. caninum at the acute stage of infection.…”

“…Precipitated supernatants and BMDMs extracts were analyzed by immunoblot as described previously ( 15 ). The primary antibodies used were anti-mouse IL-1β (AF-401, R&D, Minneapolis, MN, USA), anti-mouse caspase-1 (p20) (AG-20B-0042, Adipogen, Liestal, Switzerland), anti-ASC (AG-25B-0006-C100, Adipogen, Liestal, Switzerland), anti-NLRP3 (AG-20B-0014, Adipogen, Liestal, Switzerland), and anti-mouse β-actin (Proteintech, Wuhan, China).…”

“…The host innate immune system plays an important role in recognizing microbes and shaping an appropriate adaptive immune response during the early stage of the infection ( 10 ). N. caninum can activate different pattern-recognition receptors (PRRs) of innate immune cells after infecting the host, such as TLR2 ( 11 ), TLR3 ( 12 ), TLR11 ( 13 , 14 ), NOD2 ( 10 ), Dectin-1 ( 9 ), and NLRP3 ( 15 ), and these PRRs consequently induce the host immune response against this parasite.…”