Eon J. Rios

Genomic analysis of mycosis fungoides and Sézary syndrome identifies recurrent alterations in TNFR2

Mycosis fungoides and Sézary syndrome comprise the majority of cutaneous T cell lymphomas (CTCLs), disorders notable for their clinical heterogeneity that can present in skin or peripheral blood. Effective treatment options for CTCL are limited, and the genetic basis of these T cell lymphomas remains incompletely characterized. Here we report recurrent point mutations and genomic gains of TNFRSF1B, encoding the tumor necrosis factor receptor TNFR2, in 18% of patients with mycosis fungoides and Sézary syndrome. Expression of the recurrent TNFR2 Thr377Ile mutant in T cells leads to enhanced non-canonical NF-κB signaling that is sensitive to the proteasome inhibitor bortezomib. Using an integrative genomic approach, we additionally discovered a recurrent CTLA4-CD28 fusion, as well as mutations in downstream signaling mediators of these receptors.

Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis.

Sepsis is a complex, incompletely understood and often fatal disorder, typically accompanied by hypotension, that is considered to represent a dysregulated host response to infection. Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension. We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe cecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, show reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell–dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.

Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma

Here we report the discovery of recurrent mutations concentrated at an ultraviolet signature hotspot in KNSTRN, which encodes a kinetochore protein, in 19% of cutaneous squamous cell carcinomas (SCCs). Cancer-associated KNSTRN mutations, most notably those encoding p.Ser24Phe, disrupt chromatid cohesion in normal cells, occur in SCC precursors, correlate with increased aneuploidy in primary tumors and enhance tumorigenesis in vivo. These findings suggest a role for KNSTRN mutagenesis in SCC development.

A LncRNA-MAF:MAFB Transcription Factor Network Regulates Epidermal Differentiation

Progenitor differentiation requires remodeling of genomic expression; however, in many tissues, such as epidermis, the spectrum of remodeled genes and the transcription factors (TFs) that control them are not fully defined. We performed kinetic transcriptome analysis during regeneration of differentiated epidermis and identified gene sets enriched in progenitors (594 genes), in early (159 genes), and in late differentiation (387 genes). Module mapping of 1,046 TFs identified MAF and MAFB as necessary and sufficient for progenitor differentiation. MAF:MAFB regulated 393 genes altered in this setting. Integrative analysis identified ANCR and TINCR lncRNAs as essential upstream MAF:MAFB regulators. ChIP-seq analysis demonstrated MAF:MAFB binding to known epidermal differentiation TF genes whose expression they controlled, including GRHL3, ZNF750, KLF4, and PRDM1. Each of these TFs rescued expression of specific MAF:MAFB target gene subsets in the setting of MAF:MAFB loss, indicating they act downstream of MAF:MAFB. A lncRNA-TF network is thus essential for epidermal differentiation.

The Chymase Mouse Mast Cell Protease 4 Degrades TNF, Limits Inflammation, and Promotes Survival in a Model of Sepsis

Mouse mast cell protease 4 (mMCP-4), the mouse counterpart of human mast cell chymase, is thought to have proinflammatory effects in innate or adaptive immune responses associated with mast cell activation. However, human chymase can degrade the proinflammatory cytokine TNF, a mediator that can be produced by mast cells and many other cell types. We found that mMCP-4 can reduce levels of mouse mast cell-derived TNF in vitro through degradation of transmembrane and soluble TNF. We assessed the effects of interactions between mMCP-4 and TNF in vivo by analyzing the features of a classic model of polymicrobial sepsis, cecal ligation and puncture (CLP), in C57BL/6J-mMCP-4-deficient mice versus C57BL/6J wild-type mice, and in C57BL/6J-Kit(W-sh/W-sh) mice containing adoptively transferred mast cells that were either wild type or lacked mMCP-4, TNF, or both mediators. The mMCP-4-deficient mice exhibited increased levels of intraperitoneal TNF, higher numbers of peritoneal neutrophils, and increased acute kidney injury after CLP, and also had significantly higher mortality after this procedure. Our findings support the conclusion that mMCP-4 can enhance survival after CLP at least in part by limiting detrimental effects of TNF, and suggest that mast cell chymase may represent an important negative regulator of TNF in vivo.

Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells

BACKGROUND Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. OBJECTIVES We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. METHODS C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti-2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl-human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. RESULTS Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. CONCLUSIONS Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro.

Rabaptin-5 regulates receptor expression and functional activation in mast cells

Rab5 is a small GTPase that regulates early endocytic events and is activated by RabGEF1/Rabex-5. Rabaptin-5, a Rab5 interacting protein, was identified as a protein critical for potentiating RabGEF1/Rabex-5s activation of Rab5. Using Rabaptin-5 shRNA knockdown, we show that Rabaptin-5 is dispensable for Rab5-dependent processes in intact mast cells, including high affinity IgE receptor (FcepsilonRI) internalization and endosome fusion. However, Rabaptin-5 deficiency markedly diminished expression of FcepsilonRI and beta1 integrin on the mast cell surface by diminishing receptor surface stability. This in turn reduced the ability of mast cells to bind IgE and significantly diminished both mast cell sensitivity to antigen (Ag)-induced mediator release and Ag-induced mast cell adhesion and migration. These findings show that, although dispensable for canonical Rab5 processes in mast cells, Rabaptin-5 importantly contributes to mast cell IgE-dependent immunologic function by enhancing mast cell receptor surface stability.

FcεRI Expression and Dynamics on Mast Cells

Mast cells are key effector and immunoregulatory cells in IgE-associated immune responses, including allergic disorders. IgE antibodies bind to the high-affinity IgE receptor, FcεRI, expressed on the surface of mast cells; antigen-induced cross-linking of FcεRI-bound IgE molecules activates the mast cell to release an array of proinflammatory and immunomodulatory mediators. Because mast cells often respond to very low levels of antigen in vivo, the level of FcεRI expressed on the surface of these cells is an important factor in determining the responsiveness of these cells to antigen. FcεRI surface expression is regulated by a number of processes, including FcεRI stabilization, FcεRI recycling, and antigen-induced internalization. Although members of the Rab family of small GTPases and the ubiquitin ligase, Cbl, have recently emerged as major regulators of many of the membrane trafficking events that govern FcεRI expression levels, the mechanisms and intracellular pathways that regulate FcεRI trafficking remain poorly defined. This chapter outlines a number of flow cytometry-based assays that can be used to investigate cell surface FcεRI expression and dynamics (stabilization, recycling, and internalization) on bone marrow-derived mast cells (BMCMCs), the most commonly used model system for studying mast cells in vitro. Given the importance of FcεRI levels to mast cell responsiveness and function, the characterization of FcεRI expression and dynamics on different mast cell populations is critical when trying to compare IgE-dependent processes between different mast cell populations.