Scott L. Townley

Discovery of circulating microRNAs associated with human prostate cancer using a mouse model of disease

Circulating microRNAs (miRNAs) are emerging as useful non‐invasive markers of disease. The objective of this study was to use a mouse model of prostate cancer as a tool to discover serum miRNAs that could be assessed in a clinical setting. Global miRNA profiling identified 46 miRNAs at significantly altered levels (p ≤ 0.05) in the serum of TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice with advanced prostate cancer compared to healthy controls. A subset of these miRNAs with known human homologues were validated in an independent cohort of mice and then measured in serum from men with metastatic castration‐resistant prostate cancer (mCRPC; n = 25) or healthy men (n = 25). Four miRNAs altered in mice, mmu‐miR‐141, mmu‐miR‐298, mmu‐miR‐346 and mmu‐miR‐375, were also found to be at differential levels in the serum of men with mCRPC. Three of these (hsa‐miR‐141, hsa‐miR‐298 and hsa‐miR‐375) were upregulated in prostate tumors compared with normal prostate tissue, suggesting that they are released into the blood as disease progresses. Moreover, the intra‐tumoral expression of hsa‐miR‐141 and hsa‐miR‐375 were predictors of biochemical relapse after surgery. This study is the first to demonstrate that specific serum miRNAs are common between human prostate cancer and a mouse model of the disease, highlighting the potential of such models for the discovery of novel biomarkers.

Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response

Chemokines are essential for homeostasis and activation of the immune system. The chemokine ligand/receptor pairing CCL20/CCR6 is interesting because these molecules display characteristics of both homeostatic and activation functions. These dual characteristics suggest a role for CCR6 in the priming and effector phases of the immune response. However, while CCR6 has been implicated in the effector phase in several models, a role in the priming phase is less clear. Herein we analyze the role of CCR6 in these two important arms of the immune response during experimental autoimmune encephalomyelitis (EAE). Both CCR6 and its chemokine ligand CCL20 were up-regulated in the draining lymph nodes and spinal cord during EAE, and CCR6 was up-regulated on CD4+ T cells that had divided following induction of EAE. The functional role of this expression was demonstrated by impaired development of EAE in gene-targeted CCR6-deficient mice and in mice treated either with a neutralizing anti-CCR6 Ab or with a novel receptor antagonist. Inhibition of EAE was due to reduced priming of autoreactive CD4+ T cells probably as a result of impaired late-stage influx of dendritic cells into draining lymph nodes. This was accompanied by reduced egress of activated lymphocytes from the lymph nodes. These results demonstrate a novel role for CCR6 in the mechanism of autoreactive lymphocyte priming and emigration to the efferent lymphatics.

Antagonism of the chemokine receptors CXCR3 and CXCR4 reduces the pathology of experimental autoimmune encephalomyelitis.

Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11(4–79), was obtained. CXCL11(4–79) strongly inhibited the migration of activated mouse T cells in response to all three high‐affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12(P2G2), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4+ T cells in the CNS. Further investigation demonstrated that CXCL12(P2G2) inhibited the sensitization phase, whereas CXCL11(4–79) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.

PI3Kδ drives the pathogenesis of experimental autoimmune encephalomyelitis by inhibiting effector T cell apoptosis and promoting Th17 differentiation

The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.

Mast cells, neuropeptides, histamine, and prostaglandins in UV-induced systemic immunosuppression.

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.

Circulating microRNAs predict biochemical recurrence in prostate cancer patients

Background:Circulating microRNAs (miRNAs) are emerging as promising biomarkers for prostate cancer. Here, we investigated the potential of these molecules to assist in prognosis and treatment decision-making.Methods:MicroRNAs in the serum of patients who had experienced rapid biochemical recurrence (BCR) (n=8) or no recurrence (n=8) following radical prostatectomy (RP) were profiled using high-throughput qRT-PCR. Recurrence-associated miRNAs were subsequently quantitated by qRT-PCR in a validation cohort comprised of 70 patients with Gleason 7 cancers treated by RP, 31 of whom had undergone disease progression following surgery. The expression of recurrence-associated miRNAs was also examined in tumour tissue cohorts.Results:Three miRNAs – miR-141, miR-146b-3p and miR-194 – were elevated in patients who subsequently experienced BCR in the screening study. MiR-146b-3p and miR-194 were also associated with disease progression in the validation cohort, as determined by log-rank tests and Cox proportional hazards regression. Multivariate analysis revealed that miR-146b-3p possessed prognostic information beyond standard clinicopathological parameters. Analysis of tissue cohorts revealed that miR-194 was robustly expressed in the prostate, elevated in metastases, and its expression in primary tumours was associated with a poor prognosis.Conclusion:Our study suggests that circulating miRNAs, measured at the time of RP, could be combined with current prognostic tools to predict future disease progression in men with intermediate risk prostate cancers.

Tea tree oil reduces histamine-induced oedema in murine ears

Abstract. Objective: To examine the effect of topically applied tea tree oil (TTO) on histamine-induced oedema in the ears of mice.¶Methods and results: For BALB/c mice, 10 μ undiluted TTO applied immediately after, but not 30 min before intradermal injection of 600 μg histamine in 10 μl, significantly suppressed oedema development. TTO applied after histamine injection also suppressed histamine-induced oedema in C57/BL6 mice. TTO applied immediately after intradermal injection of compound 48/80 (200 μg in 10 μl saline) also significantly reduced ear swelling. TTO suppressed histamine-induced oedema to the same extent in capsaicin-treated (neuropeptide-depleted) and control mice which suggests that TTO does not inhibit histamine-induced oedema by regulating the activity of peripheral sensory neurons. Terpinen-4-ol, the major water-soluble component of TTO, was equivalent in potency to TTO in the suppression of histamine-induced ear swelling.¶Conclusion: Topical application of TTO, and in particular terpinen-4-ol, may be effective in controlling histamine-induced oedema often associated with Type I allergic immediate hypersensitivities.

A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer

MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial–mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal–epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.

Primary Defect in UVB-Induced Systemic Immunomodulation Does Not Relate to Immature or Functionally Impaired APCs in Regional Lymph Nodes

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m2) to the shaved dorsum of BALB/c mice, CD11c+/FITC+ cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE2 were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c+ lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA323–339) to CD4+ cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-γ levels were increased in the cultures containing CD11c+ cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c+ APCs that directly or by production of soluble mediators (IL-12, PGE2) affect cellular responses in the nodes of UVB-irradiated mice.

Control of Salmonella dissemination in vivo by macrophage inflammatory protein (MIP)-3α/CCL20

While chemokines are clearly important in the generation of protective immunity, the role of individual chemokines in the control of bacterial infection is still poorly understood. In this study, we investigated the role of macrophage inflammatory protein (MIP)-3α/CCL20, a chemokine that attracts activated T and B lymphocytes and immature dendritic cells, in host responses to bacterial infection. CCL20 production was induced in subcutaneous tissue in the BALB/c mouse in response to Salmonella enteritidis, Staphylococcus aureus and zymosan, with S. enteritidis being the most potent. S. enteritidis induced CCL20 production in the spleen following either oral administration or injection into the peritoneal cavity. In contrast, no increase was observed in the Peyers patches. In this model, following intraperitoneal injection, dose-dependent colonization of the spleen and Peyers patches by S. enteritidis, expression of IFNγ and IL-4, and production of antibodies against the S. enteritidis surface antigen SefA were observed. Prior treatment with neutralizing antibodies against CCL20 enhanced bacterial dissemination to the spleen and Peyers patches and strongly biased the IFNγ/IL-4 ratio towards a type 2 profile in the spleen, while the humoral response was unaffected. In contrast, treatment with neutralizing anti-MIP-1α/CCL3 antibodies enhanced the bacterial burden in the Peyers patches but not in the spleen, had no significant effect on the cytokine ratio, but significantly inhibited anti-SefA production. Together, these results demonstrate an important role for CCL20 in the control of bacterial infection and more specifically in the regulation of cell-mediated immunity against intracellular bacteria such as S. enteritidis.