Fig 2: The increased phagocytosis observed in Tlr4−/− macrophages is dependent on TLR3 signalling.a Wildtype and b Tlr4−/− iBMDMs were treated with chemical inhibitors of TLR2, TLR3 and TLR9 for 1 h, then infected with non-opsonised C. neoformans. Data is pooled from three independent experiments (n = 9 per condition). c Wildtype and Tlr4−/− iBMDMs were treated with a TLR3 inhibitor then infected with C. neoformans opsonised with the anti-capsular 18B7 antibody (n = 3 per condition). d, e Tlr4−/− macrophages were pre-treated with optimal and suboptimal concentrations of TLR3 inhibitor and ox-LDL, individually and in combination (n = 3 per condition). Phagocytosis was quantified as the number of internalised cryptococci within 100 macrophages. Data is representative of two independent experiments and is shown as mean ± SEM; statistical significance was evaluated using (a, b, d, e) one-way ANOVA or (c) two-way ANOVA followed by Tukey’s post-hoc test. P-values are shown above each graph. f Wildtype and Tlr4−/− iBMDMs were treated with 0.025% DMSO (control) or 10 μM TLR3 inhibitor for 1 h. MSR1 (also known as CD204) expression was measured using anti-mouse CD204-PE monoclonal antibody. PE-labelled rat IgG2a κ was used as an isotype control. Samples were run through a flow cytometer and analysed in the FlowJo software. Percentages refer to the percentage of MSR1-positive cells. Data is representative of two independent experiments. Source data are provided as a Source Data file.

Fig 3: MyD88 and TRIF are required for nonopsonic uptake of C. neoformans without affecting MSR1 expression.a Wildtype and b Tlr4−/− macrophages were treated with inhibitors of MyD88, IKKβ (a kinase downstream of MyD88 that is necessary for NF-κB activation), TRIF, and TBK1 (a kinase downstream of TRIF that phosphorylates and activates IRF3) (n = 6 per condition). Following pre-treatment with the various inhibitors, cells were infected with non-opsonised C. neoformans. Data are pooled from two independent experiments. c Immortalised BMDMs from wildtype, Tlr4−/−, MyD88−/− and Trif−/− macrophages were infected with non-opsonised C. neoformans (n = 3). Data is representative of three independent experiments. Phagocytosis was quantified as the number of internalised cryptococcus within 100 macrophages. Data is mean ± SEM and analysed using one-way ANOVA followed by Tukey’s post-hoc test. P-values are shown above each graph. d Baseline surface expression of Macrophage Scavenger Receptor 1 (MSR1) (also known as CD204) was measured in wildtype, Tlr4−/−, MyD88−/− and Trif−/− macrophages using anti-mouse CD204-PE antibody. PE-labelled rat IgG2a κ was used as an isotype control. Receptor expression was measured using flow cytometry and analysed using the FlowJo software. Data is representative of two independent experiments. Percentages refer to the percentage of MSR1-positive cells. Source data are provided as a Source Data file.

Fig 4: Proposed model of MSR1-meidated non-opsonic phagocytosis.TLR4 modulates the surface expression of MSR1 through a yet-to-be-identified mechanism, such that loss of TLR4 signalling drives increased surface expression of MSR1. This may be through modulation of MSR1 gene expression or modulation of intracellular MSR1 reservoir trafficking to the plasma membrane. MSR1 is then responsible for the direct binding and internalisation of C. neoformans. MSR1-mediated uptake may rely on the formation of a signalling complex with FcγRII/III. The ITAM domain of FcγRs enables SYK and PI3K activation which then activates Rac GTPases that drive actin polymerisation and phagocytosis. At the same time, TLR3, MyD88 and TRIF may also serve as coreceptors or adaptor proteins leading to the activation of ERK1/2 and p38 which will also drive actin remodelling and pathogen uptake. Figure created with BioRender.com.

Fig 5: Increased uptake in Tlr4−/− macrophages is dependent on FcγRs, SYK, PI3K, ERK1/2 and p38, but not JNK.Tlr4−/− macrophages were pre-treated with inhibitors of a FcγRs (n = 3 per condition), b SYK and PI3K (control, n = 6; treatment, n = 3), and c Mitogen Activate Protein Kinases (MAPKs) (n = 3 per condition) for 1 h, then infected with non-opsonised C. neoformans. The number of internalised fungi per 100 macrophages was quantified from images from a fluorescence microscope. Figures are representative of three independent experiments. Data shown as mean ± SEM; a one-way ANOVA with Tukey’s post-hoc test was performed to evaluate statistical significance. P-values are shown above each graph. d Wildtype and Tlr4−/− iBMDMs were exposed to MAPK inhibitors for 24 h, then cell surface expression of MSR1 was detected with using anti-mouse CD204-PE antibody. PE-labelled rat IgG2a κ was used as an isotype control. Receptor expression was measured using flow cytometry and analysed using the FlowJo software. Data is representative of two independent experiments. Percentages refer to the percentage of MSR1-positive cells. Source data are provided as a Source Data file.