Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jan 31;38(1):26-32.
doi: 10.14348/molcells.2015.2136. Epub 2014 Dec 15.

The Early Induction of Suppressor of Cytokine Signaling 1 and the Downregulation of Toll-Like Receptors 7 and 9 Induce Tolerance in Costimulated Macrophages

Affiliations
Free PMC article

The Early Induction of Suppressor of Cytokine Signaling 1 and the Downregulation of Toll-Like Receptors 7 and 9 Induce Tolerance in Costimulated Macrophages

Hyo-Ji Lee et al. Mol Cells. .
Free PMC article

Abstract

Toll-like receptors (TLR) 7 and 9 transduce a cellular signal through the MyD88-dependent pathway and induce the production of inflammatory mediators against microbial nucleotide components. The repeated stimulation of TLR4 leads to endotoxin tolerance, but the molecular mechanisms of tolerance induced through the costimulation of individual TLR has not yet been established, although endosomal TLRs share signaling pathways with TLR4. In the present study, mouse macrophages were simultaneously stimulated with the TLR7 agonist, gardiquimod (GDQ), and the TLR9 agonist, CpG ODN 1826, to examine the mechanism and effector functions of macrophage tolerance. Compared with individual stimulation, the costimulation of both TLRs reduced the secretion of TNF-α and IL-6 through the delayed activation of the NF-κB pathway; notably, IL-10 remained unchanged in costimulated macrophages. This tolerance reflected the early induction of suppressor of cytokine signaling-1 (SOCS-1), according to the detection of elevated TNF-α secretion and restored NF-κB signaling in response to the siRNA-mediated abrogation of SOCS-1 signaling. In addition, the restimulation of each TLRs using the same ligand significantly reduced the expression of both TLRs in endosomes. These findings revealed that the costimulation of TLR7 and TLR9 induced macrophage tolerance via SOCS-1, and the restimulation of each receptor or both TLR7 and TLR9 downregulated TLR expression through a negative feedback mechanisms that protects the host from excessive inflammatory responses. Moreover, the insufficient and impaired immune response in chronic viral infection might also reflect the repeated and simultaneous stimulation of those endosomal TLRs.

Keywords: NF-κB; SOCS-1; TLRs; inflammation; macrophage; tolerance.

Figures

Fig. 1.
Fig. 1.
Costimulation and/or restimulation of TLR7 and TLR9 decreased the levels of proinflammatory cytokine production in mouse macrophages. (A) Mouse macrophages were stimulated with either an agonist for TLR7 (GDQ) or TLR9 (ODN 1826) or both (GDQ + ODN1826) overnight (1st), and the production of TNF-α in the supernatant was measured using ELISA. LPS stimulation was introduced as a positive control. □ Raw264.7 macrophage cell line, and ▪ BMDMs from Balb/c mice. (B) After the first stimulation, the cells were washed with PBS and incubated with culture media overnight. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd), and the production of TNF-α was measured using ELISA. The levels of IL-6 (C) and IL-10 (D) were measured in RAW264.7 cells under the same conditions. Significant differences were indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and n.s., not significant (P < 0.05).
Fig. 2.
Fig. 2.
Delayed activation of NF-κB signaling in cells simultaneously stimulated with both agonists for TLR7 and TLR9. (A) RAW264.7 cells were stimulated with either an agonist for TLR7 or TLR9 or both. The cell lysates were extracted, and the changes in expression levels of TLR signaling downstream molecules were assessed using Western blot analysis. Bar graphs indicate the results of the relative densitometry compared with β-actin protein. (B) Nuclear translocation of p65 was identified within cytoplasmic (CE) and nuclear fractions (NE) by Western blotting against p65. (C) RAW264.7 cells were transfected with the NF-κB-luc reporter gene. The transfected cells were treated with either an agonist for TLR7 and TLR9 or both for 4 h (1st), followed by washing with PBS and incubation in culture media for 12 h. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd), and the NF-κB promoter activity was measured through luciferase assay. Significant differences were indicated as **P < 0.01, ***P < 0.001, and n.s., not significant (P < 0.05).
Fig. 3.
Fig. 3.
Upregulation of SOCS1 after TLR7 and TLR9 costimulation. (A) RAW264.7 cells were stimulated with either an agonist for TLR7 or TLR9 or both for the indicated time. The protein expression in the total lysates was analyzed by Western blotting using antibodies against A20, IRAK-M, SOCS-1 and β-actin. (B) The mRNA expression levels of A20, SOCS-3, SOCS-1, IRAK-M and β-actin were determined through RT-PCR. (C) RAW264.7 cells were transfected with siRNA targeting SOCS-1 mRNA or scrambled siRNA and stimulated with either an agonist for TLR7 or TLR9 or both overnight (1st). After stimulation, the cells were washed with PBS and incubated with culture media overnight. Subsequently, the cells were restimulated with TLR agonists for 1 h (2nd) and the production of TNF-α was measured through ELISA.
Fig. 4.
Fig. 4.
Recovered NF-κB signaling in the costimulated cells when SOCS-1 was abolished (A) RAW264.7 cells were transfected with siRNA targeting SOCS-1 mRNA or scrambled siRNA and stimulated with either an agonist for TLR7 or TLR9 or both for indicated time points. The whole cell lysates were extracted, and Western blot analysis showed the cellular levels of IRAK-1, IKK-β, IκBα and SOCS-1. α-tubulin was used as a loading control. (B) RAW264.7 cells were stimulated with each agonist for TLR7 and TLR9 or both for indicated time points, and then nuclear extracts were immunoprecipitated with anti-phospho-NF-κB p65 or anti-SOCS-1, followed by western blot analysis.
Fig. 5.
Fig. 5.
The costimulation and/or restimulation of TLR7 and TLR9 decrease the expression of TLR7 and TLR9. (A) RAW264.7 cells were stimulated with either an agonist for TLR7 or TLR9 or both overnight (1st), subsequently the cells were washed with PBS and incubated with culture media overnight. After incubation, the cells were restimulated with TLR agonists for 1 h (2nd) and the expression of TLR7 and TLR9 was measured using RT-PCR. (B) The cells were incubated with either an agonist for TLR7 and TLR9 or both for 4 or 16 h, and the expression of endosomal TLR7 and TLR9 was measured using flow cytometry. The data are presented as the mean fluorescence intensity (MFI) (C).

Similar articles

See all similar articles

Cited by 11 articles

See all "Cited by" articles

References

    1. Akira S. Innate immunity to pathogens: diversity in receptors for microbial recognition. Immunol. Rev. 2009;227:5–8. - PubMed
    1. Chang Z.L. Important aspects of Toll-like receptors, ligands and their signaling pathways. Inflamm. Res. 2010;59:791–808. - PubMed
    1. Chang J.J., Lacas A., Lindsay R.J., Doyle E.H., Axten K.L., Pereyra F., Rosenberg E.S., Walker B.D., Allen T.M., Altfeld M. Differential regulation of toll-like receptor pathways in acute and chronic HIV-1 infection. Aids. 2012;26:533–541. - PMC - PubMed
    1. Chen W.H., Toapanta F.R., Shirey K.A., Zhang L., Giannelou A., Page C., Frieman M.B., Vogel S.N., Cross A.S. Potential role for alternatively activated macrophages in the secondary bacterial infection during recovery from influenza. Immunol. Lett. 2012;141:227–234. - PMC - PubMed
    1. Cross A., Asher L., Seguin M., Yuan L., Kelly N., Hammack C., Sadoff J., Gemski P., Jr. The importance of a lipopolysaccharide-initiated, cytokine-mediated host defense mechanism in mice against extraintestinally invasive Escherichia coli. J. Clin. Invest. 1995;96:676–686. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback