TLR2−/− Mice Display Decreased Severity of Giardiasis via Enhanced Proinflammatory Cytokines Production Dependent on AKT Signal Pathway

Abstract

Giardia infection is one of the most common causes of waterborne diarrheal disease in a wide array of mammalian hosts, including humans globally. Although numerous studies have indicated that adaptive immune responses are important for Giardia defense, however, whether the host innate immune system such as TLRs recognizes Giardia remains poorly understood. TLR2 plays a crucial role in pathogen recognition, innate immunity activation, and the eventual pathogen elimination. In this study, we investigated the role of TLR2 as a non-protective inflammatory response on controlling the severity of giardiasis. RT-PCR analysis suggested that TLR2 expression was increased in vitro. We demonstrated that Giardia lamblia-induced cytokines expression by the activation of p38 and ERK pathways via TLR2. Interestingly, the expression of IL-12 p40, TNF-α, and IL-6, but not IFN-γ, was enhanced in TLR2-blocked and TLR2−/− mouse macrophages exposed to G. lamblia trophozoites compared with wild-type (WT) mouse macrophages. Further analysis demonstrated that G. lamblia trophozoites reduced cytokines secretion by activating AKT pathway in WT mouse macrophages. Immunohistochemical staining in G. lamblia cysts infected TLR2−/− and WT mice showed that TLR2 was highly expressed in duodenum in infected WT mice. Also, infected TLR2−/− and AKT-blocked mice showed an increased production of IL-12 p40 and IFN-γ compared with infected WT mice at the early stage during infection. Interestingly, infected TLR2−/− and AKT-blocked mice displayed a decreased parasite burden, an increased weight gain rate, and short parasite persistence. Histological morphometry showed shortened villus length, hyperplastic crypt and decreased ratio of villus height/crypt depth in infected WT mice compared with in infected TLR2−/− and AKT-blocked mice. Together, our results suggested that TLR2 deficiency leads to alleviation of giardiasis and reduction of parasite burden through the promotion of proinflammatory cytokines production. For the first time, our results demonstrated that TLR2 played a negative role in host defense against Giardia.

Conjugated linoleic acid enhances intestinal mucosal innate immunity against parasite Giardia lamblia in a murine model

“…Sin embargo, los procesos de inmunidad innata a Giardia son poco entendidos, y se conoce que participan mastocitos, células dendríticas, macrófagos, células epiteliales y proteínas del complemento, así como la producción de óxido nítrico y péptidos antimicrobianos (López-Romero et al, 2015;Li et al, 2016), e incluso la relación con la microbiota en el ambiente intestinal (Fink and Singer, 2017). G. lamblia puede interaccionar con células dendríticas a través de la activación de la vía TLR4-MyD88-p38 y ERK1/2 MAPKs (Lee et al, 2015), e inducir la maduración de células dendríticas, además, se ha observado que macrófagos peritoneales de ratón pueden reconocer a Giardia mediante el receptor TLR2 (Li et al, 2017). La producción de algunas citocinas como TNF-α e IL-6, se ha descrito son necesarias para el control de Giardia en ratón (Zhou et al, 2003).…”

Extracellular Vesicles Secreted by Neospora caninum Are Recognized by Toll-Like Receptor 2 and Modulate Host Cell Innate Immunity Through the MAPK Signaling Pathway

“…On day 7 after isolation, 3 × 10 6 BMDMs were seeded in 6-well culture plates and incubated for 12 h with 50 µg/ml purified N. caninum EVs or Pam3Cys-Ser-(Lys) 4 (Pam3CSK4) (TLR2/TLR1 Agonist, Invitrogen, Carlsbad, CA, USA, 10 µg/ml) ( 27 ). RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as previously described ( 28 ). All RNA precipitates were dissolved in 20 µl of nuclease-free water, and the RNA concentration and integrity were determined using a NanoDrop 2000 instrument (Thermo Scientific, Waltham, MA, USA).…”

Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2

“…T. vaginalis were cultivated in Diamond’s Typticase-yeast extract-maltose medium with 10% FBS at 37°C, only the late-logarithmic-phase trophozoites were used for the assays. Wild-type (WT) female C57BL/6 mice and TLR2 -/- mice were euthanized with over dose of ether ( Rutkowski et al, 2007 ; Li et al, 2017 ) and soaked in 75% ethanol for 15 min, peritoneal cavity were flushed twice with 10 ml PBS (pH 7.4), then cell suspension were centrifuged at 1,000 g for 10 min, and washed twice with PBS.The primary culture cell viability of macrophages obtained by the lavage fluid was determined by trypan blue method (>99%). 3 × 10 6 cells were plated in a well of 6-well tissue culture plates (JET BIOFIL, China) in 1 ml RPMI 1640 containing 10% FBS, 2 mM L -glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin and incubated overnight at 37°C with 5% CO 2 .…”