Increased <i>in vivo</i> M2 macrophage generation in the absence of Btk.

<p>(A–C) WT and <i>Btk^−/−</i> mice were injected i.v. with 5,000 live <i>S.mansoni</i> eggs. (A) The percentage of pulmonary M2 macrophages was evaluated by flow cytometry using F4/80 and CD11b co-staining. (B–C) Induction of M2-associated genes was determined by qPCR. (D) WT and <i>Btk^−/−</i> mice were injected i.p. with approximately 800 ng Chitin. Peritoneal cells were collected by lavage after 48 hours and gene induction of M2-associated genes was determined by qPCR. In all cases peritoneal macrophages were pooled after isolation, treatment groups consisted of 3–4 animals, and experiments were performed in triplicate. Student’s paired <i>t</i> test was performed comparing gene induction following <i>in vivo</i> exposure to <i>S.mansoni</i> eggs or Chitin as indicated in WT and Btk^−/− peritoneal macrophages. Results shown are mean±SD from three independent experiments. * = p≤0.05.</p

Impaired TLR4-mediated induction of peritoneal M1 cells in <i>Btk^−/−</i> mice <i>in vivo.</i>

<p>(A–E) Peritoneal macrophages were harvested from WT or <i>Btk^−/−</i> mice following i.p. LPS injection. (A) Representative plots demonstrate the gating strategies for macrophage discrimination where total peritoneal macrophages were defined following co-staining with CD11b APC-Cy7 and F4/80 PE-Cy7. (B–C) Macrophage subsets were further distinguished as F4/80^intCD11b^hi M1-like macrophages and F4/80^hiCD11b^hi M2-like macrophages (M1 and M2 gates, respectively). The relative percentage of M1 (B) and M2 (C) macrophages within the total macrophage gate were determined for WT or <i>Btk^−/−</i> mice following i.p. LPS injection. (D) Expression of CD86 and MHC Class II was determined by flow cytometry. (E) MHC Class II expression was examined within the defined M1 and M2 gates following co-staining with F4/80 and CD11b. (F) Ly6C was determined by flow cytometry. In all cases data is presented as percent increased expression above background as determined by staining with the relevant isotype control (indicated by markers in panel (D). (G–H) Peritoneal macrophages harvested from WT or <i>Btk^−/−</i> mice were treated <i>ex vivo</i> with LPS (100 ng/ml) for the indicated time course and the induction of M1- (G) and M2- (H) associated genes was determined by real time PCR (qPCR). Peritoneal macrophages were pooled after isolation, with each treatment group consisting of 3–4 animals, and all experiments were performed in triplicate. Student’s paired <i>t</i> test was performed comparing gene induction in Btk^−/− peritoneal macrophages to WT cells at the indicated time points. Results shown are mean±SD from three independent experiments. * = p≤0.05.</p

Btk^−/− macrophages have impaired ability to expand <i>in vitro</i> into M1 cells.

<p>(A–D) Peritoneal macrophages were extracted from WT and <i>Btk^−/−</i> mice and treated <i>ex vivo</i> with an M1 (LPS plus IFN-y) or an M2 (IL-4 plus IL-13) polarizing cocktail for 24 hr and induction of M1 (A–B) and M2- associated (C–D) genes determined by qPCR. In all cases peritoneal macrophages were pooled after isolation, with each treatment group consisting of 3–4 animals, and all experiments were performed in triplicate. Student’s paired <i>t</i> test was performed comparing gene induction in Btk^−/− peritoneal macrophages to WT cells following treatment with polarizing cocktails as indicated. Results shown are mean±SD from three independent experiments. * = p≤0.05.</p

Proposed transcriptional regulation of macrophage polarization in the absence of Btk.

<p>(A) This study has demonstrated that in response to LPS and IFN-γ Btk contributes to M1 polarizing of myeloid cells via promoting the phosphorylation of Akt and subsequently the p65 subunit of NFκB, in addition to enhancing to phosphorylation of STAT1. (B) In the absence of Btk exposure of myeloid cells to LPS and IFN-γ results in the preferential induction of M2 associated genes and preferentially recruitment of M2 cells <i>in vivo</i>. Previous studies in <i>Btk^−/−</i> mice have observed increased levels IL-10 systemically following LPS treatment. IL-10 is known to activate STAT3 and there is some evidence to suggest that STAT3 may also play a role in promoting M2 macrophage polarization. Thus in the absence of Btk, in response to M1 polarizing stimuli increased IL-10 production together with reduced phosphorylation of key signaling intermediaries, combined with activation of p50 the inhibitory subunit of NF-κB could potentially account for the observed preferential skew towards an M2 phenotype. Additionally this study has shown that in response to IL-4 and IL-13 Btk^−/− cells demonstrate an increased capacity to polarize towards an M2 phenotype as a result of enhanced STAT6 phosphorylation and increased SHIP1 expression.</p

The PDGF-BB-SOX7 axis-modulated IL-33 in pericytes and stromal cells promotes metastasis through tumour-associated macrophages

Signalling molecules and pathways that mediate crosstalk between various tumour cellular compartments in cancer metastasis remain largely unknown. We report a mechanism of the interaction between perivascular cells and tumour-associated macrophages (TAMs) in promoting metastasis through the IL-33–ST2-dependent pathway in xenograft mouse models of cancer. IL-33 is the highest upregulated gene through activation of SOX7 transcription factor in PDGF-BB-stimulated pericytes. Gain- and loss-of-function experiments validate that IL-33 promotes metastasis through recruitment of TAMs. Pharmacological inhibition of the IL-33–ST2 signalling by a soluble ST2 significantly inhibits TAMs and metastasis. Genetic deletion of host IL-33 in mice also blocks PDGF-BB-induced TAM recruitment and metastasis. These findings shed light on the role of tumour stroma in promoting metastasis and have therapeutic implications for cancer therapy