Expression of TLR2, TLR3, TLR4, and TLR7 on pulmonary lymphocytes of Schistosoma japonicum-infected C57BL/6 mice

Abstract

Despite the paramount role of TLRs in the induction of innate immune and inflammatory responses, there is a paucity of studies on the role of TLRs in Schistosoma japonicum infection. Here, we observed obvious infiltration of inflammatory cells in S. japonicum-infected C57BL/6 mouse lungs. Expression and release of IFN-γ, IL-4, and IL-17 were significantly higher in pulmonary lymphocytes from infected mice compared with control mice in response to anti-CD3 plus anti-CD28 mAbs. Higher percentages of TLR2, TLR3, TLR4, and TLR7 were expressed on such lymphocytes, and the TLR agonists PGN, Poly I:C, LPS, and R848 induced a higher level of IFN-γ. However, a higher level of IL-4 was found in the supernatant of pulmonary lymphocytes from infected mice stimulated by these TLR agonists plus CD3 Ab. Only R848 plus anti-CD3 mAb could induce a higher level of IFN-γ in such lymphocytes. TLR expressions were then compared on different pulmonary lymphocytes after infection, including T cells, B cells, NK cells, NKT cells, and γδT cells. The expression levels of TLR3 on T cells, B cells, NK cells, and γδT cells were increased in the lungs after infection. NK cells also expressed higher levels of TLR4 after infection of control mice. Collectively, these findings highlight the potential role of TLR expression in the context of S. japonicum infection.

Keywords: TLRs; cytokines; ligands; lung.

Figure 1.

The histopathological changes in the lung of infected C57BL/6 mice. (a) Sections of the lung of normal mice (left panels) and infected mice (right panels) were examined by H&E staining (×100). The multi-cellular granuloma could be observed in the infected group. (b) The levels of IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-17, and GM-CSF were detected by CBA. The data are representative of six experiments, each with three or four replicates per group (*P < 0.05, **P < 0.01; the error bars indicate SD).

Figure 2.

TLR expressions in lymphocytes isolated from control or infected mouse lung. (a) The accumulation of TLR2, TLR3, TLR4, and TLR7 mRNA was quantified by using qPCR. (b) The expression of TLR2, TLR3, TLR4, and TLR7 protein from control or infected lung were analyzed by flow cytometry. Flow cytometric analysis from one representative experiment. (c) Average percentages of TLRs in normal and infected mice were calculated from FACS data (n = 6) (*P < 0.05, **P < 0.01; the error bars indicate SD).

Figure 3.

TLRs regulate IFN-γ and IL-4 secretion. Single lung cell suspensions of normal and infected mice were prepared and then cultured in the presence of different ligands plus anti-CD3 mAb. The culture supernatants were collected after 72 h of incubation for detection of IFN-γ and IL-4 by ELISA. The data are representative of four experiments, each with three or four replicates per group (*P < 0.05; the error bars indicate SD).

Figure 4.

The percentages and absolute numbers of T cells, B cells, NK cells, and γδT cells. (a) Flow cytometric analysis of CD3, CD19, NK1.1, and γδTCR expression on normal and infected mice lung lymphocytes is shown. Representative FACS plots are shown (n = 6). The numbers represent the percentage of cells in each subset. (b) Average percentages of T cells, B cells, NK cells, NKT cells, and γδT cells were calculated from FACS with the number of lymphocytes counted under microscope. Cell numbers are from different cell subsets (*P < 0.05; the error bars indicate SD).

Figure 5.

TLR expressions in different lymphocytes isolated from infected and uninfected mouse lung. (a) The percentages of TLR2⁺, TLR3⁺, TLR4⁺, and TLR7⁺ expressed on T cells, B cells, NK cells, NKT cells, and γδT cells, respectively. Flow cytometric analysis from one representative experiment. (B) The percentages of different TLRs expressed on T cells, B cells, NK cells, NKT cells, and γδT cell in the lung were calculated. The results represent for ten independent experiments (*P < 0.05, **P < 0.01; the error bars indicate SD).