Fig. 2 | Communications Biology

Fig. 2

From: Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells

Fig. 2

Wnt5a induces the production of pro-inflammatory cytokines in mouse myeloid cells. a Murine RAW264.7 macrophages, or primary (1°) WT C57Bl/6, MyD88-/-, or Tlr4-/- BMM, were treated with rWnt5a (6 h). Cytokine levels were determined by CBA. (n = 4 and n = 3). Dunnett’s test was used for multiple comparisons. b Wnt5a signalling in WT C57Bl/6, MyD88−/−, and Tlr4−/− mice. Relative protein levels of MCP-1 (CCL2) or TNFα from the experiment shown (a) in different mouse strains are shown. (n = 3). Dunnett’s test was used for multiple comparisons. c Wnt5a induces Tnf, but not Ccl2 (MCP-1) mRNA transcription as shown by using the transcriptional blocker actinomycin D (ActD) and RAW264.7 cells. The protein levels were determined using CBA. (n = 3) Dunnett’s test (black annotation) and Holm-Sidak´s test (grey annotations) was used for multiple comparisons. d HA-Wnt5a-induced activation of the mouse Il10 promoter in human THP-1 cells. LPS (18 h) as positive control. (n = 6). Dunnett’s test was used for multiple comparisons. e Wnt5a-induced IL-10:TNFα protein level ratio in mouse and human cells. (n = 3 and n = 5). Data were analysed by paired t-test. f Wnt5a-induced activation of an AP-1 reporter in mouse RAW264.7 but inconsistently in human THP-1 cells. (n = 8) Data were analysed by paired t-test. g Wnt5a signalling in human myeloid cells is inhibited by ectopic expression of dominant negative (DN)-hTLR2 and DN-hTLR4. DN-TLR2, DN-TLR4 or DN-TLR2/DN-TLR4 plasmids, using THP-1 cells and HA-Wnt5a or Ctrl vector. The protein levels were determined using CBA. The ratio rWnt5a-treated/Ctrl-treated for each pair is shown. (n = 3) Holm-Sidak´s test was used for multiple comparisons. h Wnt5a slightly affects MyD88-independent IRF and NFκB signalling. Overexpression of pcDNA3-HA-Wnt5a (48 h) in THP1-DualTM MyD-KO cells. (n = 4) Data were analysed by paired t-test. i Wnt5a signalling in human primary monocytes is blocked by PMB. Relative levels of IL-10, TNFα, IL-6 (CBA), and IL-8 (ELISA) stimulated with rWnt5a (6 h), with or without PMB treatment, are shown. Error bars indicate SEM (n = 4). Mann–Whitney U test was used for each bar comparison to Ctrl. Error bars indicate SEM; *p < 0.05, **p < 0.01, and ***p < 0.001. Data were analysed by ANOVA or t-test as indicated for each panel

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