Differential TLR7-mediated cytokine expression by R848 in M-CSF- versus GM-CSF-derived macrophages after LCMV infection

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) play an important role in macrophage (MФ) development by influencing their differentiation and polarization. Our goal was to explore the difference between M-CSF- and GM-CSF-derived bone marrow MФ responsiveness to TLR7-mediated signalling pathways that influence cytokine production early after infection in a model of acute virus infection. To do so, we examined cytokine production and TLR7-mediated signalling at 1 h post-lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) infection. We found that R848-induced cytokine expression was enhanced in these cells, with GM-CSF cells exhibiting higher proinflammatory cytokine expression and M-CSF cells exhibiting higher anti-inflammatory cytokine expression. However, R848-mediated signalling molecule activation was diminished in LCMV-infected M-CSF and GM-CSF macrophages. Interestingly, we observed that TLR7 expression was maintained during LCMV infection of M-CSF and GM-CSF cells. Moreover, TLR7 expression was significantly higher in M-CSF cells compared to GM-CSF cells. Taken together, our data demonstrate that although LCMV restrains early TLR7-mediated signalling, it primes differentiated MФ to enhance expression of their respective cytokine profiles and maintains levels of TLR7 expression early after infection.

Keywords: GM-CSF; M-CSF; R848; TLR7; cytokines; macrophages.

Fig. 1.

Phenotyping of bone marrow-derived MФ cultured in M-CSF versus GM-CSF. Cells were differentiated with either M-CSF or GM-CSF for 7 days in RPMI with 10 % FBS. On day 7 cells were harvested and co-stained with anti-F4/80 and anti-CD11b antibodies and analysed via flow cytometry. Data shown are representative of three independent biological replicates.

Fig. 2.

LCMV nucleoprotein expression in infected BMDMs (M-CSF and GM-CSF). In vitro LCMV-NP expression in M-CSF- or GM-CSF-induced cells after infection with LCMV. M-CSF (a) and GM-CSF (b) cells were infected at an m.o.i. of 3 for 24 h. Uninfected cells were used in the same labelling protocol to indicate the negative controls (white histogram) and were used to gate on positive NP stained cells (light grey). These data are a representative of three independent biological replicates.

Fig. 3.

Infection with LCMV causes differential cytokine production in M-CSF- and GM-CSF-derived MФ. Murine bone marrow-derived MФ cultured in M-CSF or GM-CSF for 7 days were exposed to media conditions, mock infection, or LCMV infection for 1, 3, or 6 h before the supernatants were harvested. Supernatants were harvested for cytokine analysis by ELISA: TNF-α (a), IL-6 (b), IL-12/23p40 (c) and IL-10 (d). Data are representative of at least three independent biological replicates and are presented as the mean±sd of three technical replicate wells. Statistical significance for differences in cytokine expression between mock- and LCMV-infected cells was calculated using a two-way ANOVA with Tukey’s multiple comparisons test. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Fig. 4.

GM-CSF MФ exhibit enhanced proinflammatory cytokine expression in response to LCMV infection and R848 stimulation. GM-CSF MФ and M-CSF MФ were either infected with LCMV (m.o.i.=3) or mock and then stimulated with R848 (1 µg ml⁻¹) for 1 h, 3 h and 6 h. GM-CSF and M-CSF cell-free supernatants were collected for the measurement of TNF-α (a), IL-6 (b), IL-12/23p40 (c), IL-23 (d), IL-12p70 (e) and IL-10 (f) by ELISA. Data presented are the mean±sd of three independent biological replicates, each with three technical replicate wells. Statistical significance for differences in cytokine expression between R848 treated and untreated cells was calculated using Tukey’s multiple comparisons test. To determine statistical significance between mock- and LCMV-infected cells, Bonferroni’s multiple comparisons were performed. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

Fig. 5.

Phosphorylation of p38, p42/44 and NF-kB is decreased in LCMV-infected M-CSF and GM-CSF MФ in response to R848. Murine bone marrow-derived MФ were differentiated in M-CSF- or GM-CSF-enriched media for 7 days before infection. MФ were infected with mock or LCMV for 1 h and then exposed to R848 (5 µg ml⁻¹) for 15 min. (a) Cells were then harvested and lysed for immunoblot for phosphorylated (p)-p38, p-42/44 and p-NF-κBp65. Blots were stripped and reproved with pan p38, p42/44 and NF-kB antibodies. Immunoblots shown are representative of at least three independent biological replicates. (b) Densitometry analysis was performed on three independent biological replicates and is graphed as the mean MFI±sd of the fold change compared to mock medium (MOCK MED) controls for M-CSF and GM-CSF. Statistical significance for differences in phosphorylation levels between R848-treated and untreated cells was calculated using Tukey’s multiple comparisons test. **P≤0.01, ***P≤0.001, ****P≤0.0001.

Fig. 6.

Flow cytometry analyses of TLR7 expression in M-CSF MФ and GM-CSF MФ. M-CSF (a) and GM-CSF MФ (b) cells were fixed and permeabilized prior to intracellular staining for TLR7 (shaded grey histogram). The geometric mean fluorescence intensity (MFI) of TLR7 expression is indicated in the top right corner of each histogram. Isotype controls are indicated by white histograms. Data shown are representative of three independent biological replicates. The average MFI±sd (c) was calculated from three biological replicates. Statistical significance for differences in TLR7 expression between R848-treated and untreated cells was calculated using Tukey’s multiple comparisons test. To determine statistical significance between mock- and LCMV-infected cells, Bonferroni’s multiple comparisons were performed. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.