Toll-Like Receptor 3–TRIF Pathway Activation by Neospora caninum RNA Enhances Infection Control in Mice

Abstract

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)-TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3 Ϫ/Ϫ and TRIF Ϫ/Ϫ mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-␥), and nitric oxide (NO). Unlike those of T. gondii, N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-␣ and IFN-␤ in the presence of the parasite. Furthermore, TRIF Ϫ/Ϫ infected macrophages produced lower levels of IL-12p40, while exogenous IFN-␣ replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease.

Immediate Interferon Gamma Induction Determines Murine Host Compatibility Differences between Toxoplasma gondii and Neospora caninum

“…Despite this discrepancy, other aspects of those studies (51,56) are consistent with our results. For one, our data clearly show increased susceptibility to N. caninum in MyD88 knockout mice based on higher parasite burdens at the later stages of acute infection ( Fig.…”

“…due to the higher effective inoculum (10 6 trypan blue-negative parasites versus 10 6 total tachyzoites in the present study). Differences in inocula and parasite strains may also contribute to the differences between the present study and another showing that TLR3 knockout mice were more susceptible to N. caninum than were WT mice (in this case after infection with 10 7 tachyzoites [56]). In our study, we observed small differences in parasite burdens and IFN-␥ levels at 4 hpi and no impact on parasite control in TLR3 Ϫ/Ϫ mice, as N. caninum infections were controlled within the same time frame as in WT mice.…”

“…For our studies, we did not normalize input parasites by dye exclusion, but based on plaque assays for these studies, we typically observe ϳ10 to 20% viability of input parasites. Therefore, it is likely that our "effective" dose is significantly lower than 10 6 tachyzoites, and this is why we did not observe any mortality while others did (including a more recent study showing 100% mortality in MyD88 Ϫ/Ϫ mice infected with 10 7 total N. caninum tachyzoites [56]).…”

“…Previous work in vitro has shown that N. caninum infection induced type I interferon responses that are dependent on TLR3 signaling (55) in human cells. Additionally, TLR3 has been shown to be important for mouse resistance to N. caninum (although with a different N. caninum strain and at a 10-fold-higher dose than those used here [56]). To investigate the possibility that type I interferons might also be produced during the immediate host response to N. caninum, we tested serum samples from our in vivo experiments for detectable IFN-␣ and IFN-␤ during the first 48 h after both T. gondii and N. caninum infections in BALB/c mice.…”

Interplay Between Reactive Oxygen Species and the Inflammasome Are Crucial for Restriction of Neospora caninum Replication

“…MyD88-dependent TLR signaling has been reported to play a key role in parasite recognition and induction of suitable immune response against this parasite (Mineo et al, 2009(Mineo et al, , 2010Beiting et al, 2014;Gurung and Kanneganti, 2016). However, activation of MyD88-independent TLR pathways, such as the TRIFdependent TLR3 signaling, are also important for activation of the immune response against N. caninum, as potent inducers of type I interferons (Beiting et al, 2014;Miranda et al, 2019). In parallel, NLRs have also emerged as important components of the innate immune system, due to its ability to recognize and eliminate intracellular parasites (Zamboni and Lima-Junior, 2015;Coutermarsh-Ott et al, 2016).…”