Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Abstract

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines.

Inhibitory effect of BMAP-28 on Leptospiral Lipopolysaccharide-Induced TLR2-Dependent Immune Response in Bovine Cells

“…We found that there was rapid up-regulation of proinflammatory cytokines at low concentrations of L-LPS stimulation in BFF_NCC1 cells. In previous studies, LPS from Leptospira species had a specific immune response, which was recognized by TLR2 in human monocytes, TLR2 in pig fibroblasts, and both TLR2 and TLR4 in mouse macrophages (10, 20). To elucidate whether TLR2 plays an important role in the activation response of L-LPS in bovine cells, in the present study, BFF_NCC1 cells were used as a cell model based on certain characteristics (high expression level of TLR2 and lack of TLR4 mRNA expression).…”

Interaction between Leptospiral Lipopolysaccharide and Toll-like Receptor 2 in Pig Fibroblast Cell Line, and Inhibitory Effect of Antibody against Leptospiral Lipopolysaccharide on Interaction

“…Total RNA was isolated from the cells using the NucleoSpin RNA II kit (Takara Bio, Japan) according to manufacturer’s protocol, and reverse transcription was performed using PrimeScript RT reagent kit (Takara Bio, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) reactions were performed on a Thermal Cycler Dice Real-time PCR System II (Takara Bio, Japan) as same procedure as previous experiment ( Guo et al, 2014 ), using SYBR green I mixture (Takara Bio, Japan) and the following primers: swine TLR2 (forward: 5′-GCATGAAGATGATGTGGGCC-3′, reverse: 5′-TAGGAGTCCTGCTCACTGTA-3′), TLR4 (forward: 5′-TGGAACAGGTATCCCAGAGG-3′, reverse: 5′-CAGAATCCTGAGGGAGTGGA-3′), interleukin (IL)-6 (forward: 5′-TGGATAAGCTGCAGTCACAG-3′, reverse: 5′-ATTATCCGAATGGCCCTCAG-3′), IL-8 (forward: 5′-GCTCTC TGTGAGGCTGCAGTT-3′, reverse: 5′-TTTATGCACTGG CATCGAAGTT-3′) and swine β-actin (forward: 5′-CATCACCATCGGCAACGA -3′, reverse: 5′-GCGTAGAGGTCCTTGCGGATGT-3′) serves as an internal control. Each sample was prepared in triplicate and the average value was used as the expression value.…”

“…However, the knowledge of TLRs function in pig innate immunity during Leptospira infection is not clear yet. Our previous study showed that L-LPS is capable of inducing the activation of TLR2 rather than TLR4 in pig fibroblasts (PEFs_NCC1), and causing the expression of proinflammatory cytokines ( Guo et al, 2014 ). The immune response of L-LPS was weak in PEFs_NCC1 cell line; therefore we try to use another pig cell line to elicit pig innate immunity response of L-LPS sufficiently.…”

Phagocyte Escape of Leptospira: The Role of TLRs and NLRs

“…The most striking feature of innate recognition of leptospires is their potent lipoprotein-induced TLR2 activation ( 112 , 114 , 122 – 124 ). It was first shown that LipL32, the major lipoprotein of leptospires, is a TLR2 agonist in human macrophages and hamster ovary cells ( 114 ) and in mouse renal tubular proximal cells ( 125 ).…”

“…We also determined that the recognition of leptospires occurred through dimers of TLR2 and TLR1 ( 115 ) specific for triacylated lipoproteins that are present in leptospires ( 111 ). More recently, it was shown that leptospires also stimulate pig and bovine fibroblasts through TLR2 ( 122 , 123 ). The abundant lipoproteins present in the membranes of leptospires easily explain their potent TLR2 activity, at least in vitro .…”