Fig 2: TLR2 deletion alters Treg cell frequency without affecting other immune populations. FACS analysis of immune cells in tumors (N = 6 per group), blood (N = 5 neuT-TLR2KO and 6 neuT-TLR2WT) and spleens (N = 3 per group) from neuT-TLR2KO and neuT-TLR2WT mice. Graphs show mean ± SEM of the percentage of (a-c) lymphocytes and myeloid cells from among CD45+ cells, (d-f) CD4+ and CD8+ from among CD3+ T cells, (g) M0, M1, M1/M2 and M2 from among total tumor macrophages, (h-j) Tregs from among CD4+ T cells. Each dot represents a mouse. (k) FACS analysis of peripheral Tregs in TLR2KO and TLR2WT mice. Graphs show mean ± SEM of the percentage of Tregs from among CD4+ T cells (N = 8 per group). (l) FACS analysis of TLR2 expression on Tregs and CD4+ CD25- cells from TLR2WT mice. Graphs show mean ± SEM of the percentage of TLR2+ cells and of TLR2 MFI (N = 8 per group). A representative density plot of Treg staining on CD4+ T cells and representative histograms of isotype control Ab or TLR2 staining on Treg and CD4+ CD25- effector T cells are shown. (m) FACS analysis of Tregs from TLR2WT mice upon ex vivo stimulation of blood T lymphocytes with TLR2 activating ligands. Graphs show mean ± SEM of the percentage of Tregs from among CD4+ cells. Each dot represents a mouse (N = 5 per group). Results come from at least 3 independent experiments. Unpaired Student’s t test with Welch’s correction.

Fig 3: Microbial TLR2 agonist cross the blood-brain barrier and induce neurodegeneration after systemic delivery. a Zymosan or lipoteichoic acid were repeatedly injected intraperitoneally to naïve animals or to animals that have been implanted with an ICV pump continuously administering the TLR2 antagonist CU-CPT22. Mice were sacrificed at day 14 to assess neuronal loss. b Following 3 intraperitoneal injections of zymosan or lipoteichoic acid there was significant more loss in cortical neuronal counts in 5xFAD than in wt mice. ICV administration of TLR2 antagonist, CU-CPT22 abolished zymosan-induced neuronal loss. Dashed line represents the baseline NeuN count of untreated wt (blue) and 5xFAD (orange) mice. *Compared with untreated mice. c In wt mice that received intraperitoneal injections of zymosan, followed by intravenous injection of Biocytin-TMR, the BBB tracer was found exclusively within CD31+ blood vessels. d In 5xFAD, zymosan-injected mice Biocytin-TMR crossed the BBB effectively and accumulated in parenchymal brain cells. e–g In 5xFAD mice, pretreated with three intraperitoneal zymosan injections, a fourth injection of fluorescent-labeled zymosan resulted in multiple fluorescent foci in the brain parenchyma (enlarged f inserted in e). Arrowheads show a fluorescent zymosan particle associated with CD31+ blood vessels, arrows show a fluorescent zymosan particle that exited the blood vessel. 3-D confocal images confirmed the exiting of labeled zymosan from CD31+ blood vessels into the brain parenchyma (g)

Fig 4: TLR2KO BC cells display impaired tumorigenicity. (a) Tumor-free survival and (b) mean tumor volume of TLR2WT and TLR2KO mice (N = 6 per group, from 2 independent experiments) injected orthotopically with 1 × 106 WT-874 or KO-M26 cells. (c) Mean tumor volume of BALB/c mice orthotopically injected with 1 × 104 4T1 cells left untreated (N = 5), transfected with scrambled siRNAs (N = 7) or siRNAs to TLR2 (N = 7). (d) Mean tumor volume of NSG mice injected orthotopically with 1 × 106 WT-874, WT-879, WT-880, KO-C26, KO-M26 or KO-E26 cells (N = 6 per group from two independent experiments). (a) Log-rank (Mantel-Cox) of KO-M26 cells injected into TLR2WT and TLR2KO mice vs WT-874 cells injected into TLR2WT mice. (b) two-way ANOVA test.

Fig 5: Zymosan-induced neurotoxicity is mediated by the brain microglia. a Staining of cortical sections from 5xFAD brain showed that TLR2 immune-reactivity co-localized with Iba1+ microglia. There were rare cells with neuron-like morphology that also expressed TLR2. b Experimental time line of minocycline treatment and zymosan injection. c Quantification of cortical neurons showed that minocycline treatment did not affect neuronal counts, but abolished the neurotoxic effect induced by ICV injection of zymosan. Dashed line represents the baseline NeuN count of untreated wt (blue) and 5xFAD (orange) mice. *ANOVA f (3, 20) = 22.093, p < 0.001