Estrogen receptor α and aryl hydrocarbon receptor independent growth inhibitory effects of aminoflavone in breast cancer cells.

BACKGROUND: Numerous studies have implicated the aryl hydrocarbon receptor (AhR) as a potential therapeutic target for several human diseases, including estrogen receptor alpha (ERα) positive breast cancer. Aminoflavone (AF), an activator of AhR signaling, is currently undergoing clinical evaluation for the treatment of solid tumors. Of particular interest is the potential treatment of triple negative breast cancers (TNBC), which are typically more aggressive and characterized by poorer outcomes. Here, we examined AF's effects on two TNBC cell lines and the role of AhR signaling in AF sensitivity in these model cell lines. METHODS: AF sensitivity in MDA-MB-468 and Cal51 was examined using cell counting assays to determine growth inhibition (GI50) values. Luciferase assays and qPCR of AhR target genes cytochrome P450 (CYP) 1A1 and 1B1 were used to confirm AF-mediated AhR signaling. The requirement of endogenous levels of AhR and AhR signaling for AF sensitivity was examined in MDA-MB-468 and Cal51 cells stably harboring inducible shRNA for AhR. The mechanism of AF-mediated growth inhibition was explored using flow cytometry for markers of DNA damage and apoptosis, cell cycle analysis, and ß-galactosidase staining for senescence. Luciferase data was analyzed using Student's T test. Three-parameter nonlinear regression was performed for cell counting assays. RESULTS: Here, we report that ERα-negative TNBC cell lines MDA-MB-468 and Cal51 are sensitive to AF. Further, we presented evidence suggesting that neither endogenous AhR expression levels nor downstream induction of AhR target genes CYP1A1 and CYP1B1 is required for AF-mediated growth inhibition in these cells. Between these two ERα negative cell lines, we showed that the mechanism of AF action differs slightly. Low dose AF mediated DNA damage, S-phase arrest and apoptosis in MDA-MB-468 cells, while it resulted in DNA damage, S-phase arrest and cellular senescence in Cal51 cells. CONCLUSIONS: Overall, this work provides evidence against the simplified view of AF sensitivity, and suggests that AF could mediate growth inhibitory effects in ERα-positive and negative breast cancer cells, as well as cells with impaired AhR expression and signaling. While AF could have therapeutic effects on broader subtypes of breast cancer, the mechanism of cytotoxicity is complex, and likely, cell line- and tumor-specific.

Fungi subvert vaccine T cell priming at the respiratory mucosa by preventing chemokine-induced influx of inflammatory monocytes.

Vaccinologists strive to harness immunity at mucosal sites of pathogen entry. We studied respiratory delivery of an attenuated vaccine against Blastomyces dermatitidis. We created a T cell receptor transgenic mouse responsive to vaccine yeast and found that mucosal vaccination led to poor T cell activation in the draining nodes and differentiation in the lung. Mucosal vaccination subverted lung T cell priming by inducing matrix metalloproteinase 2 (MMP2), which impaired the action of the chemokine CCL7 on egress of CCR2(+) Ly6C(hi) inflammatory monocytes from the bone marrow and their recruitment to the lung. Studies in Mmp2(-/-) mice, or treatment with MMP inhibitor or rCCL7, restored recruitment of Ly6C(hi) monocytes to the lung and CD4(+) T cell priming. Mucosal vaccination against fungi and perhaps other respiratory pathogens may require manipulation of host MMPs in order to alter chemokine signals needed to recruit Ly6C(hi) monocytes and prime T cells at the respiratory mucosa.

Limited model antigen expression by transgenic fungi induces disparate fates during differentiation of adoptively transferred T cell receptor transgenic CD4+ T cells: robust activation and proliferation with weak effector function during recall.

CD4(+) T cells are the key players of vaccine resistance to fungi. The generation of effective T cell-based vaccines requires an understanding of how to induce and maintain CD4(+) T cells and memory. The kinetics of fungal antigen (Ag)-specific CD4(+) T cell memory development has not been studied due to the lack of any known protective epitopes and clonally restricted T cell subsets with complementary T cell receptors (TCRs). Here, we investigated the expansion and function of CD4(+) T cell memory after vaccination with transgenic (Tg) Blastomyces dermatitidis yeasts that display a model Ag, Eα-mCherry (Eα-mCh). We report that Tg yeast led to Eα display on Ag-presenting cells and induced robust activation, proliferation, and expansion of adoptively transferred TEa cells in an Ag-specific manner. Despite robust priming by Eα-mCh yeast, antifungal TEa cells recruited and produced cytokines weakly during a recall response to the lung. The addition of exogenous Eα-red fluorescent protein (RFP) to the Eα-mCh yeast boosted the number of cytokine-producing TEa cells that migrated to the lung. Thus, model epitope expression on yeast enables the interrogation of Ag presentation to CD4(+) T cells and primes Ag-specific T cell activation, proliferation, and expansion. However, the limited availability of model Ag expressed by Tg fungi during T cell priming blunts the downstream generation of effector and memory T cells.

Vaccine-induced protection against 3 systemic mycoses endemic to North America requires Th17 cells in mice.

Worldwide rates of systemic fungal infections, including three of the major pathogens responsible for such infections in North America (Coccidioides posadasii, Histoplasma capsulatum, and Blastomyces dermatitidis), have soared recently, spurring interest in developing vaccines. The development of Th1 cells is believed to be crucial for protective immunity against pathogenic fungi, whereas the role of Th17 cells is vigorously debated. In models of primary fungal infection, some studies have shown that Th17 cells mediate resistance, while others have shown that they promote disease pathology. Here, we have shown that Th1 immunity is dispensable and that fungus-specific Th17 cells are sufficient for vaccine-induced protection against lethal pulmonary infection with B. dermatitidis in mice. Further, vaccine-induced Th17 cells were necessary and sufficient to protect against the three major systemic mycoses in North America. Mechanistically, Th17 cells engendered protection by recruiting and activating neutrophils and macrophages to the alveolar space, while the induction of Th17 cells and acquisition of vaccine immunity unexpectedly required the adapter molecule Myd88 but not the fungal pathogen recognition receptor Dectin-1. These data suggest that human vaccines against systemic fungal infections should be designed to induce Th17 cells if they are to be effective.

Dynamic interplay among monocyte-derived, dermal, and resident lymph node dendritic cells during the generation of vaccine immunity to fungi.

Early innate events that enable priming of antifungal CD4 T cells are poorly understood. We engineered an attenuated fungal vaccine with a model epitope, EalphaRFP, to track vaccine immunity to Blastomyces dermatitidis during yeast recognition, antigen presentation, and priming of naive T cells. After subcutaneous injection of the vaccine, monocyte-derived inflammatory dendritic cells (DCs) are the earliest and largest population that associates with yeast, carrying them into the draining lymph nodes. Despite marked association with yeast, these DCs fail to display surface peptide:MHC complexes or prime naive T cells. Instead, the ability to display antigen and prime CD4 T cells resides with lymph node-resident DCs after antigen transfer from immigrant DCs and with skin migratory DCs. Our work reveals the dynamic interplay among distinct DC subsets that prime naive CD4 T cells after yeast are injected in the skin and discloses the cellular elements underlying vaccine-induced immunity to fungi.