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. 2021;13(6):345-358.
doi: 10.1159/000515738. Epub 2021 May 31.

TLR7 Ligation Inhibits TLR8 Responsiveness in IL-27-Primed Human THP-1 Monocytes and Macrophages

Natalya Odoardi  1 , Olena Kourko  1 , Carlene Petes  1 , Sameh Basta  1 , Katrina Gee  1
Affiliations

TLR7 Ligation Inhibits TLR8 Responsiveness in IL-27-Primed Human THP-1 Monocytes and Macrophages

Natalya Odoardi et al. J Innate Immun. 2021.

Abstract

Regulation of proinflammatory cytokine expression is critical in the face of single-stranded RNA (ssRNA) virus infections. Many viruses, including coronavirus and influenza virus, wreak havoc on the control of cytokine expression, leading to the formation of detrimental cytokine storms. Understanding the regulation and interplay between inflammatory cytokines is critical to the identification of targets involved in controlling the induction of cytokine expression. In this study, we focused on how the antiviral cytokine interleukin-27 (IL-27) regulates signal transduction downstream of Toll-like receptor 7 (TLR7) and TLR8 ligation, which recognize endosomal single-stranded RNA. Given that IL-27 alters bacterial-sensing TLR expression on myeloid cells and can inhibit replication of single-stranded RNA viruses, we investigated whether IL-27 affects expression and function of TLR7 and TLR8. Analysis of IL-27-treated THP-1 monocytic cells and THP-1-derived macrophages revealed changes in mRNA and protein expression of TLR7 and TLR8. Although treatment with IL-27 enhanced TLR7 expression, only TLR8-mediated cytokine secretion was amplified. Furthermore, we demonstrated that imiquimod, a TLR7 agonist, inhibited cytokine and chemokine production induced by a TLR8 agonist, TL8-506. Delineating the immunomodulatory role of IL-27 on TLR7 and TLR8 responses provides insight into how myeloid cell TLR-mediated responses are regulated during virus infection.

Keywords: Inflammation; Interferon; Interleukin-27; Macrophages; Monocytes; Toll-like receptor; Toll-like receptor 7; Toll-like receptor 8.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
TLR7- and TLR8-mediated p38, ERK1/2, and NF-κB activation is differentially modulated in IL-27-pretreated THP-1 and PMA-THP-1 cells. a THP-1 cells and PMA-THP-1 cells were treated with or without IL-27 (50 ng/mL) for 16 h, washed, then stimulated with agonists: IMQ (5 μg/mL), TL8-506 (TL8) (500 ng/mL), R848 (5 μg/mL), or CL075 (2.5 μg/mL) for 15 min. Lysates were run on SDS-PAGE gels for immunoblot analysis and probed for phosphorylated proteins: p-p38, p-p42/44 ERK1/2. Membranes were then stripped and reprobed for corresponding pan protein counterparts as a loading control. b Fold change was calculated by normalizing respective phosphorylated lanes to pan controls and then calculating fold change over medium control. Images are representative of at least 2 independent experiments. c THP-1 XBlue cells and PMA-THP-1 XBlue cells were treated with IL-27 (50 ng/mL) overnight, then stimulated with IMQ (5 μg/mL), TL8 (500 ng/mL), R848 (5 μg/mL), or CL075 (2.5 μg/mL) for 24 h to allow for NF-κB/AP-1-induced SEAP production and secretion. SEAP production was quantified using a colorimetric QUANTI-BlueTM assay measured at 650 nm. Data represent the mean and standard deviation of 3 independent experiments. An ordinary one-way ANOVA with multiple comparisons was used to determine statistical significance between pairs as indicated. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. IMQ, imiquimod; R848, resiquimod; SEAP, secreted embryonic alkaline phosphatase; TLR7, Toll-like receptor 7; TLR8, Toll-like receptor 8; IL-27, interleukin-27. ERK1/2, extracellular signal-regulated kinases 1/2; PMA, phorbol 12-myristate 13-acetate.
Fig. 2
Fig. 2
Treatment with IL-27 results in elevated cytokine and chemokine production in response to TLR8 or TLR7/8 agonists. THP-1 (left column) and PMA-THP-1 (right column) cells were pretreated with IL-27 (50 ng/mL) for 16 h, washed, then stimulated with IMQ (5 μg/mL), TL8 (500 ng/mL), R848 (5 μg/mL), or CL075 (2.5 μg/mL) for 24 h. Cell-free supernatants were collected for detection of TNF-α (a, b), IL-6 (c, d), IL-8 (e, f), or RANTES (g, h) by ELISA. Data represent the mean and SEM of at least 5 replicate experiments. A paired, one-way ANOVA with multiple comparisons and Bonferroni correction was used for statistical analyses between agonists and medium control as indicated by asterisks above bars or between pairs as indicated by asterisks above lines. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. IMQ, imiquimod; R848, resiquimod; TLR7, Toll-like receptor 7; TLR8, Toll-like receptor 8; IL-27, interleukin-27; IL-6, interleukin-6; IL-8, interleukin-8; ELISA, enzyme-linked immunosorbent assay; PMA, phorbol 12-myristate 13-acetate.
Fig. 3
Fig. 3
Stimulation of monocytes and macrophages with TLR7 agonist IMQ results in reduced cytokine production. THP-1 (left) and PMA-THP-1 (right) cells were pretreated with IL-27 (50 ng/mL) for 16 h, washed, then stimulated with either IMQ (5 μg/mL) alone, TL8-506 (TL8) (500 ng/mL) alone, or 500 ng/mL of TL8 with increasing doses of IMQ (2.5–20 μg/mL) for 24 h. Cell-free supernatants were collected for detection of TNF-α (a, b), IL-6 (c, d), IL-8 (e, f), or RANTES (g, h) by ELISA. Data represent the mean and SEM of at least 3 replicate experiments. A paired, one-way ANOVA with multiple comparisons was used for statistical analyses between co-stimulated samples against TL8 alone (black bars) as indicated by asterisks above bars, in IL-27 pretreated co-stimulated samples versus IL-27 pretreated TL8-stimulated samples (gray bars) as indicated by asterisks above lines, or between pairs as indicated by asterisks above lines. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. IMQ, imiquimod; R848, resiquimod; TLR7, Toll-like receptor 7; TLR8, Toll-like receptor 8; IL-27, interleukin-27; IL-6, interleukin-6; IL-8, interleukin-8; ELISA, enzyme-linked immunosorbent assay; PMA, phorbol 12-myristate 13-acetate.
Fig. 4
Fig. 4
IL-27 enhances both TLR7 and TLR8 mRNA levels, but differentially modulates protein expression. a THP-1 cells and PMA-THP-1 cells were treated with or without IL-27 (50 ng/mL) for 4 h and qPCR was performed to examine changes in TLR7 (left) or TLR8 (right) mRNA expression. b THP-1 and PMA-THP-1 cells were stimulated with IL-27 (50 ng/mL) for 16 h then fixed, permeabilized, then stained for intracellular TLR7 or TLR8 expression in comparison to medium cultured cells. Unstained cells (auto; shaded histogram) are included for negative controls. Histograms displayed are representative of 5 independent experiments with at least 14 replicates total. c Fold change was calculated between IL-27-treated samples and medium controls. Each point represents a replicate value within an independent experiment. Data were analyzed using students t tests for statistical analyses between pairs as indicated. ns, not significant; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. TLR7, Toll-like receptor 7; TLR8, Toll-like receptor 8; IL-27, interleukin-27; qPCR, quantitative real-time polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate.
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