Imiquimod and interferon-alpha augment monocyte-mediated astrocyte secretion of MCP-1, IL-6 and IP-10 in a human co-culture system

Abstract

Toll-like receptor 7 (TLR7)-activation has been implicated as a significant mechanism of neuroinflammation triggered by ssRNA viruses. Infiltration of monocytes into the brain and astrocyte activation occurs during in vivo TLR7-mediated neuroinflammation. The objective here was to determine whether the TLR7 agonist, imiquimod, and interferon-alpha (IFN-α), promote monocyte-mediated astrocyte secretion of pro-inflammatory factors. Using a human primary co-culture system, we demonstrate that monocytes, together with imiquimod and IFN-α, promote astrocyte secretion of MCP-1, IL-6 and IP-10. Furthermore, TLR7-induced monocyte-derived IL-1β is critical for promoting the astrocyte response. Overall, this study provides a potential mechanism for TLR7-mediated neuroinflammation.

Keywords: Astrocyte; Co-culture; IL-1beta; Imiquimod; Interferon-alpha; Monocyte.

Figure 1:. Human primary monocytes induce human U251 and primary astrocytes to produce MCP-1 and IL-6.

Human primary monocytes (N=5 for C-D and N=5 for G-H) were purified from healthy donors and co-cultured with U251 or primary astrocytes (donor 1). Astrocytes were cultured at 2×l0⁵ cells/mL and monocytes were added at various concentrations to establish monocyte:astrocyte ratios. Cells were co-cultured for 24 h, with a Golgi block added 4 h before cell harvest. Intracellular staining and flow cytometry were performed to determine astrocyte production of MCP-1 and IL-6. FSC-A and FSC-H were used to separate the astrocyte population from the monocytes. Panels A-B and E-F are flow cytometry plots of astrocyte production of MCP-1 and IL-6 comparing astrocytes alone (0:1 ratio) to a monocyte to astrocyte ratio of 1:5. For statistical analysis, dataset was first log transformed. Next, a one-way (C and G) or repeated measures (RM) (D and H) analysis of variance (ANOVA) with a Dunnett’s multiple comparisons post-test was performed. * denotes a statistical difference from 0:1 ratio group (p<0.05). Graphs are mean +/− SEM.

Figure 2:. Imiquimod/R837 treatment alone and with IFN-α, enhances monocyte-mediated astrocyte production of MCP-1, IL-6 and IP-10.

(A-F) Human primary monocytes were co-cultured with U251 or primary astrocytes (donor 1) at a ratio of 1:20 (monocyte:astrocyte) and stimulated with IFN-α (50, 100 and 200U/mL) or R837 (0.1, 1 and 10μg/mL). Cells were co-cultured for 24 h, with a Golgi block added 4 h before cell harvest. An aliquot of supernatant was collected prior to the addition of the Golgi block. Intracellular staining and flow cytometry were performed to determine astrocyte production of MCP-1, IL-6 and IP-10. The right panels in A-F are supernatant levels of cytokines in co-culture measured via LEGENDplex™ and/or ELISAmax™. The N values for monocytes isolated from human donors was N=9 and N=3 for left and right panels, respectively, in A, C, E, and N=5 for right and left panels in B, D, F. (G-L) IFN-α (100U/mL) and R837 (1μg/mL) were added alone and in combination to the co-culture and astrocyte production of MCP-1, IL-6 and IP-10 was determined (N=6 for G-I and N=5 for J-L). For A-F, dataset was log transformed and a RM ANOVA with a Dunnett’s multiple comparisons post-test was performed. For G-L, dataset was log transformed and a RM ANOVA with a Tukey’s post-test was performed. For A-F, * denotes a statistical difference from NS of the respective ratio group (p<0.05). Graphs are mean +/− SEM.

Figure 3:. IL-1β is a major factor governing the monocyte-mediated astrocyte response to R837.

(A-C) Human monocytes (N=9) were co-cultured with U251 astrocytes at a ratio of 1:20 (monocyte:astrocyte) in either normal wells (N) or 0.4μm transwells (T). Co-culture was stimulated with IFN-α (100U/mL) or R837 (10μg/mL). Cells were co-cultured for 24 h, with a Golgi block added 4 h before cell harvest. (D) Human monocytes (N=9) were cultured alone at l×l0⁴ cells/mL. Monocytes were either left untreated (NS), treated with IFN-α (100U/mL) or R837 (10μg/mL). Monocytes were cultured for 20 h and supernatants were collected. LEGENDplex™ was used to measure supernatant levels of MCP-1, TNF-α, IL-1β, IFN-γ, IL-6 and IP-10. (E-J) Human primary monocytes (N=6 for E-G and N=5 for H-J) were co-cultured with astrocytes (U251 or primary donor 1) at a ratio of 1:20 and stimulated with IFN-α (100U/mL) or R837 (10μg/mL). Anti-IL-1β was added to the co-culture at a concentration of 1μg/mL. An isotype (1 μg/mL) served as the control. Cells were co-cultured for 24 h, with a Golgi block added 4 h before cell harvest. For A-C, a RM two-way ANOVA with a Sidak’s multiple comparisons post-test was performed. * denotes a statistical difference from the NS group in normal (N) wells (p<0.05) and # denotes a statistical difference from the NS group in transwells (T). $ denotes a statistical difference between normal wells and transwells. For D, a log transformation was performed if there was unequal variance (F test). Then an unpaired t-test was performed to determine significant differences between media and each treatment. A RM ANOVA with a Dunnett’s multiple comparisons post-test was performed to determine differences between NS and either IFNα or R837-treated monocytes. # denotes a statistical difference between media control. * denotes a statistical difference from the NS (p<0.05). For E-J, a paired t-test was performed to determine significant differences between isotype control and anti-IL-1β group. A log transformation was performed for H-J prior to the paired t-test. * denotes a statistical difference from the isotype control (p<0.05). Graphs are mean +/− SEM.

Figure 4:. Replacement of monocytes with recombinant IL-1β in the control, R837 and IFN-α cultures, promotes a similar profile of MCP-1, IL-6 and IP-10 production by astrocytes.

(A-F) Astrocytes (U251 and primary donor 1) were treated with varying concentrations of recombinant IL-1β (0, 0.5, 1, 5, 10, 20, 50 and 100pg/mL). (G-L) Astrocytes were left untreated (NS), treated with IFN-α (100U/mL) or R837 (10μg/mL) alone or in combination with IL-1β (20pg/mL). Cells were incubated for 20 h and a Golgi block was added for 4 h. Intracellular staining and flow cytometry were performed to determine astrocyte production of MCP-1, IL-6 and IP-10. A RM ANOVA with a Dunnett’s multiple comparisons post-test was performed for A-L. * denotes a statistical difference from 0pg/mL for A-F and the respective NS treatment group for G-L. Graphs are mean +/− SEM.

Figure 5:. The intermediate monocyte population positively associates with U251 astrocyte production of IL-6.

Human primary monocytes (N=16) were co-cultured with U251 astrocytes at a ratio of 1:20 and stimulated with IFN-α (100U/mL) or R837 (10μg/mL). Cells were co-cultured for 24 h, with a Golgi blocker added 4 h before cell harvest. Intracellular staining and flow cytometry were performed to determine astrocyte production of MCP-1, IL-6 and IP-10. Extracellular staining of CD14 and CD16 was performed on an aliquot of monocytes prior to co-culture. The percent of each monocyte population was determined. A correlation analysis (Prism 7, GraphPad) was performed between monocyte subsets and U251 astrocyte production of MCP-1, IL-6 or IP-10.