Ligand-mediated cytoplasmic retention of the Ah receptor inhibits macrophage-mediated acute inflammatory responses.

The Ah receptor (AHR) has been shown to exhibit both inflammatory and anti-inflammatory activity in a context-specific manner. In vivo macrophage-driven acute inflammation models were utilized here to test whether the selective Ah receptor modulator 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA360) would reduce inflammation. Exposure to SGA360 was capable of significantly inhibiting lipopolysaccharide (LPS)-mediated endotoxic shock in a mouse model, both in terms of lethality and attenuating inflammatory signaling in tissues. Topical exposure to SGA360 was also able to mitigate joint edema in a monosodium urate (MSU) crystal gout mouse model. Inhibition was dependent on the expression of the high-affinity allelic AHR variant in both acute inflammation models. Upon peritoneal MSU crystal exposure SGA360 pretreatment inhibited neutrophil and macrophage migration into the peritoneum. RNA-seq analysis revealed that SGA360 attenuated the expression of numerous inflammatory genes and genes known to be directly regulated by AHR in thioglycolate-elicited primary peritoneal macrophages treated with LPS. In addition, expression of the high-affinity allelic AHR variant in cultured macrophages was necessary for SGA360-mediated repression of inflammatory gene expression. Mechanistic studies revealed that SGA360 failed to induce nuclear translocation of the AHR and actually enhanced cytoplasmic localization. LPS treatment of macrophages enhanced the occupancy of the AHR and p65 to the Ptgs2 promoter, whereas SGA360 attenuated occupancy. AHR ligand activity was detected in peritoneal exudates isolated from MSU-treated mice, thus suggesting that the anti-inflammatory activity of SGA360 is mediated at least in part through AHR antagonism of endogenous agonist activity. These results underscore an important role of the AHR in participating in acute inflammatory signaling and warrants further investigations into possible clinical applications.

ER stress and distinct outputs of the IRE1α RNase control proliferation and senescence in response to oncogenic Ras.

Oncogenic Ras causes proliferation followed by premature senescence in primary cells, an initial barrier to tumor development. The role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in regulating these two cellular outcomes is poorly understood. During ER stress, the inositol requiring enzyme 1α (IRE1α) endoribonuclease (RNase), a key mediator of the UPR, cleaves Xbp1 mRNA to generate a potent transcription factor adaptive toward ER stress. However, IRE1α also promotes cleavage and degradation of ER-localized mRNAs essential for cell death. Here, we show that oncogenic HRas induces ER stress and activation of IRE1α. Reduction of ER stress or Xbp1 splicing using pharmacological, genetic, and RNAi approaches demonstrates that this adaptive response is critical for HRas-induced proliferation. Paradoxically, reduced ER stress or Xbp1 splicing promotes growth arrest and premature senescence through hyperactivation of the IRE1α RNase. Microarray analysis of IRE1α- and XBP1-depleted cells, validation using RNA cleavage assays, and 5' RACE identified the prooncogenic basic helix-loop-helix transcription factor ID1 as an IRE1α RNase target. Further, we demonstrate that Id1 degradation by IRE1α is essential for HRas-induced premature senescence. Together, our studies point to IRE1α as an important node for posttranscriptional regulation of the early Ras phenotype that is dependent on both oncogenic signaling as well as stress signals imparted by the tumor microenvironment and could be an important mechanism driving escape from Ras-induced senescence.

Differentiated State of Initiating Tumor Cells Is Key to Distinctive Immune Responses Seen in H-RasG12V-Induced Squamous Tumors.

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-RasG12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1-/- background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1+/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198-210. ©2017 AACR.

Genetic and pharmacological analysis identifies a physiological role for the AHR in epidermal differentiation.

Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr(-/-) and Ahr(+/+) murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr(-/-) keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr(+/+) keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM) SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression, and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology.

The TGFß1 pathway is required for NFκB dependent gene expression in mouse keratinocytes.

The transforming growth factor-beta 1 (TGFß1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFß1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFß1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFß1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFß1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFß1 induction of NFκB-luciferase. TGFß1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFß1 and this was blocked in NFκB+/- and -/- keratinocytes and by the IκB super-repressor. To test the effects of the TGFß1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFß1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGFß1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFß1.

CD8(+) T cells mediate RAS-induced psoriasis-like skin inflammation through IFN-γ.

The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RAS(V12G) in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses, and increased T helper type 1 (Th1)/Th17 and T cell type 1 (Tc1)/Tc17 skin infiltration. The majority of skin-infiltrating CD8(+) T cells coexpressed IFN-γ and IL-17A. When RAS was expressed on a Rag1-/- background, microabscess formation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation were suppressed. Depletion of CD8(+), but not CD4(+), T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17(+) γδ T cells, and epidermal hyperproliferation to levels similar to a Rag1-/- background. Reconstitution of Rag1-/- inducible RAS mice with purified CD8(+) T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ, but not of IL-17A, in CD8(+) T-cell-reconstituted Rag1-/- mice expressing RAS blocked CD8-mediated skin inflammation, inducible nitric oxide synthase expression, and keratinocyte hyperproliferation. These results show that CD8(+) T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ.

Extended JAK activation and delayed STAT1 dephosphorylation contribute to the distinct signaling profile of CNS neurons exposed to interferon-gamma.

Although interferon-gamma (IFN-γ) plays a critical role in the noncytolytic elimination of many neurotropic viral infections, the signaling response to this cytokine has not been extensively characterized in primary CNS neurons. We previously demonstrated that the IFN-γ response at the signaling and gene expression levels is temporally extended in primary mouse hippocampal neurons, as compared to the transient response of primary mouse embryonic fibroblasts (MEF). We hypothesize that the protracted kinetics of STAT1 phosphorylation in IFN-γ-treated neurons are due to extended receptor activation and/or delayed STAT1 dephosphorylation in the nucleus. Here, we show that in response to IFN-γ, the Janus kinases (JAK1/JAK2) associated with the neuronal IFN-γ receptor complex remain active for an extended period as compared to MEF. Experimental inactivation of JAK1/JAK2 in neurons after IFN-γ treatment did not reverse the extended STAT1 phosphorylation phenotype. These results suggest that the extended kinetics of neuronal IFN-γ signaling are a product of distinct negative feedback mechanisms operating at both the receptor and within the nucleus.