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. 2004 Jan;78(2):576-84.
doi: 10.1128/jvi.78.2.576-584.2004.

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus

Dalia Burzyn  1 , John C Rassa, David Kim, Irene Nepomnaschy, Susan R Ross, Isabel Piazzon
Affiliations

Toll-like receptor 4-dependent activation of dendritic cells by a retrovirus

Dalia Burzyn et al. J Virol. 2004 Jan.

Abstract

Mouse mammary tumor virus (MMTV) is a milk-borne retrovirus that exploits the adaptive immune system. It has recently been shown that MMTV activates B cells via Toll-like receptor 4 (TLR4), a molecule involved in innate immune responses. Here, we show that direct virus binding to TLR4 induced maturation of bone marrow-derived dendritic cells and up-regulated expression of the MMTV entry receptor (CD71) on these cells. In vivo, MMTV increased the number of dendritic cells in neonatal Peyer's patches and their expression of CD71; both these effects were dependent on TLR4. Thus, retroviral signaling through TLRs plays a critical role in dendritic-cell participation during infection.

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Figures

FIG. 1.
FIG. 1.
Expression of costimulatory molecules on BMDCs after MMTV stimulation. BMDCs were incubated for 18 h with MMTV(LA), MMTV plus anti-MMTV antibody, boiled MMTV, or AT-2-treated MMTV. Expression of CD40 (A, C, and D) or CD80 (B) on CD11c+ cells was analyzed by FACS. The dashed lines represent isotype antibody control. The histograms depict representative results. The experiment was performed three times with similar results.
FIG. 2.
FIG. 2.
Proinflammatory cytokine and chemokine production in MMTV-stimulated BMDCs. Cells (106/100 μl) were incubated with LPS (100 ng/ml) or MMTV. Where indicated, LPS and MMTV were boiled (1 h) or pretreated with UV (30 min) or anti-MMTV antibody (Ab). (A) After 18 h, the concentration of TNF-α, IL-6, or IL-12 in the supernatant was measured by ELISA. The data are presented as means plus SD. One experiment out of three is shown. *, P < 0.001 compared to the control group (unstimulated BMDCs). (B) After 2 h, cytokine and chemokine mRNA levels were measured by RPA, using multiprobe sets. One experiment out of three is shown.
FIG. 3.
FIG. 3.
TNF-α production in LPS-tolerized BMDCs. BMDCs from BALB/cJ mice were tolerized for 20 h with LPS (10 ng/ml) and then stimulated with LPS (100 ng/ml) or MMTV for 18 h. The TNF-α concentration in the supernatant was measured by ELISA (A), and total RNA was analyzed by RT-PCR using TNF-α-specific primers (B). The data are representative of three separate experiments with similar results. The error bars indicate SD.
FIG. 4.
FIG. 4.
CD40 and CD80 expression on MMTV-induced BMDCs from C3H/HeN and C3H/HeJ mice. BMDCs were incubated for 18 h with MMTV. Expression of CD40 (A) and CD80 (B) on CD11c+ cells was analyzed by FACS. The data represent the mean fluorescence intensity (MFI) (mean ± SD; n = 3). The experiment was performed three times with similar results.
FIG. 5.
FIG. 5.
Cytokine production in MMTV-induced BMDCs from C3H/HeN, C3H/HeJ, BALB/cJ, and C.C3H Tlr4lps-d (BALB/cJLPS-d) mice. BMDCs (106/100 μl) were incubated with MMTV for 18 h. The concentration of TNF-α (A and D), IL-6 (B), or IL-12 (C) in the supernatant was measured by ELISA. The data represent the means ± SD (n = 3). One experiment out of three is shown. *, P < 0.001 compared to the control group (unstimulated BMDCs).
FIG. 6.
FIG. 6.
Chemokine and cytokine mRNA levels in MMTV-induced BMDCs from C3H/HeN and C3H/HeJ mice. Cells (106/100 μl) were incubated with MMTV or LPS. After 2 h, chemokine (A and C) and cytokine (B and D) mRNA levels were measured by RPA, using multiprobe sets. (A and B) Autoradiographs of RPA gels. −, unstimulated. (C and D) Quantitation of relative expression of chemokine and cytokine mRNAs, as described in Materials and Methods.
FIG. 7.
FIG. 7.
Expression of the MMTV receptor on BMDCs and infection with MMTV. (A to D) BMDCs derived from C3H/HeN (A), C3H/HeJ (B), TLR2−/− (C) and TLR4Lps-d/TLR4Lps-d TLR2−/− (D) mice were incubated for 18 h with MMTV. Expression of CD71+/CD11c+ cells was analyzed by FACS. The histograms depict representative results. Dashed lines represent isotype antibody; solid lines, unstimulated BMDCs; bold lines, MMTV. (E) Data are presented as the increase (n-fold) in the mean fluorescence intensity (MFI) (plus SD) compared to unstimulated cells for each mouse strain. The experiment was performed three times with similar results. *, P < 0.01 (C3H/HeN versus C3H/HeJ); #, P < 0.01 (TLR2−/− versus TLR4Lps-d/TLR4Lps-d TLR2−/−). (F and G) BMDCs were infected with MMTV as described in Materials and Methods. After overnight incubation, DNA was extracted and PCR was performed to look for integrated viral DNA in the cellular genome. The data are representative of three separate experiments with similar results.
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