Honors & Awards
Physician Scientist Career Development Award, Dermatology Foundation (2014, 2015, 2016)
Doctor of Philosophy, Stanford University, IMMUN-PHD (2010)
Doctor of Medicine, Stanford University, MED-MD (2010)
Bachelor of Science, University of California Davis, Neuro Physiology & Behavior (2002)
Paul Khavari, Postdoctoral Faculty Sponsor
Genomic analysis of mycosis fungoides and Sézary syndrome identifies recurrent alterations in TNFR2.
2015; 47 (9): 1056-1060
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.
View details for DOI 10.1038/ng.3370
View details for PubMedID 26258847
A LncRNA-MAF:MAFB Transcription Factor Network Regulates Epidermal Differentiation
2015; 32 (6): 693-706
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.
View details for DOI 10.1016/j.devcel.2015.01.028
View details for Web of Science ID 000351841900008
View details for PubMedID 25805135
FceRI expression and dynamics on mast cells.
Methods in molecular biology (Clifton, N.J.)
2015; 1220: 239-255
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.
View details for DOI 10.1007/978-1-4939-1568-2_15
View details for PubMedID 25388255
- Recurrent point mutations in the kinetochore gene KNSTRN in cutaneous squamous cell carcinoma NATURE GENETICS 2014; 46 (10): 1060-1062
Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells.
journal of allergy and clinical immunology
2013; 132 (4): 922-32 e1 16
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.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.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.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.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.
View details for DOI 10.1016/j.jaci.2013.05.004
View details for PubMedID 23810240
Endothelin-1 Augments Na+/H+ Exchange Activity in Murine Pulmonary Arterial Smooth Muscle Cells via Rho Kinase
2012; 7 (9)
Excessive production of endothelin-1 (ET-1), a potent vasoconstrictor, occurs with several forms of pulmonary hypertension. In addition to modulating vasomotor tone, ET-1 can potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. It is well established that changes in cell proliferation and migration in PASMCs are associated with alkalinization of intracellular pH (pH(i)), typically due to activation of Na(+)/H(+) exchange (NHE). In the systemic vasculature, ET-1 increases pH(i), Na(+)/H(+) exchange activity and stimulates cell growth via a mechanism dependent on protein kinase C (PKC). These results, coupled with data describing elevated levels of ET-1 in hypertensive animals/humans, suggest that ET-1 may play an important role in modulating pH(i) and smooth muscle growth in the lung; however, the effect of ET-1 on basal pH(i) and NHE activity has yet to be examined in PASMCs. Thus, we used fluorescent microscopy in transiently (3-5 days) cultured rat PASMCs and the pH-sensitive dye, BCECF-AM, to measure changes in basal pH(i) and NHE activity induced by increasing concentrations of ET-1 (10(-10) to 10(-8) M). We found that application of exogenous ET-1 increased pH(i) and NHE activity in PASMCs and that the ET-1-induced augmentation of NHE was prevented in PASMCs pretreated with an inhibitor of Rho kinase, but not inhibitors of PKC. Moreover, direct activation of PKC had no effect on pH(i) or NHE activity in PASMCs. Our results indicate that ET-1 can modulate pH homeostasis in PASMCs via a signaling pathway that includes Rho kinase and that, in contrast to systemic vascular smooth muscle, activation of PKC does not appear to be an important regulator of PASMC pH(i).
View details for DOI 10.1371/journal.pone.0046303
View details for Web of Science ID 000309973900128
View details for PubMedID 23029469
The Chymase Mouse Mast Cell Protease 4 Degrades TNF, Limits Inflammation, and Promotes Survival in a Model of Sepsis
AMERICAN JOURNAL OF PATHOLOGY
2012; 181 (3): 875-886
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.
View details for DOI 10.1016/j.ajpath.2012.05.013
View details for Web of Science ID 000309251100016
View details for PubMedID 22901752
Rabaptin-5 regulates receptor expression and functional activation in mast cells
2008; 112 (10): 4148-4157
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-5's 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.
View details for DOI 10.1182/blood-2008-04-152660
View details for Web of Science ID 000260691300035
View details for PubMedID 18698003
Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis
2008; 14 (4): 392-398
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.
View details for DOI 10.1038/nm1738
View details for Web of Science ID 000254674100025
View details for PubMedID 18376408
Roles of RabGEF1/Rabex-5 domains in regulating Fc epsilon RI surface expression and Fc epsilon RI-dependent responses in mast cells
2007; 109 (12): 5308-5317
RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (-/-) mast cells and examined Fc epsilon RI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in -/- mast cells, including decreased basal Fc epsilon RI expression, slowed Fc epsilon RI internalization, elevated IgE + Ag-induced degranulation and IL-6 production, and the decreased ability of -/- cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface Fc epsilon RI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen-activated -/- mast cells. By contrast, correction of these -/- phenotypes required a functional Vps9 domain. Thus, Fc epsilon RI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.
View details for DOI 10.1182/blood-2007-01-067363
View details for Web of Science ID 000247360200045
View details for PubMedID 17341663
RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (8): 2659-2664
We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (Fc epsilonRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (-/-) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1-/- BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1-/- BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1-/- BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1-/- BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.
View details for Web of Science ID 000235554900034
View details for PubMedID 16533754
Chronic hypoxia elevates intracellular pH and activates Na+/H+ exchange in pulmonary arterial smooth muscle
AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY
2005; 289 (5): L867-L874
Chronic hypoxia (CH), caused by many lung diseases, results in pulmonary hypertension due, in part, to increased muscularity of small pulmonary vessels. Pulmonary arterial smooth muscle cell (PASMC) proliferation in response to growth factors requires increased intracellular pH (pHi) mediated by activation of Na+/H+ exchange (NHE); however, the effect of CH on PASMC pHi homeostasis is unknown. Thus we measured basal pHi and NHE activity and expression in PASMCs isolated from mice exposed to normoxia or CH (3 wk/10% O2). pHi was measured using the pH-sensitive fluorescent dye BCECF-AM. NHE activity was determined from Na+-dependent recovery from NH4-induced acidosis, and NHE expression was determined by RT-PCR and immunoblot. PASMCs from chronically hypoxic mice exhibited elevated basal pHi and increased NHE activity. NHE1 was the predominate isoform present in mouse PASMCs, and both gene and protein expression of NHE1 was increased following exposure to CH. Our findings indicate that exposure to CH caused increased pHi, NHE activity, and NHE1 expression, changes that may contribute to the development of pulmonary hypertension, in part, via pH-dependent induction of PASMC proliferation.
View details for DOI 10.1152/ajplung.00455.2004
View details for Web of Science ID 000232469800023
View details for PubMedID 15964895