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1.  Fyn-ADAP signaling via Carma1-Bcl10-MAP3K7 signalosome exclusively regulates inflammatory cytokine production in NK cells 
Nature immunology  2013;14(11):10.1038/ni.2708.
Inflammation is a critical component of the immune response. However, acute or chronic inflammation can be highly destructive. Uncontrolled inflammation forms the basis for allergy, asthma, and multiple autoimmune disorders. Here, we identify a signaling pathway that is exclusively responsible for inflammatory cytokine production but not for cytotoxicity. Recognition of H60+ or CD137L+ tumor cells by murine NK cells led to efficient cytotoxicity and inflammatory cytokine production. Both of these effector functions required Lck, Fyn, PI(3)K-p85α, PI(3)K-p110δ, and PLC-γ2. However, the complex of Fyn and the adapter ADAP exclusively regulated inflammatory cytokine production but not cytotoxicity in NK cells. This unique function of ADAP required a Carma1-Bcl10-MAP3K7 signaling axis. Our results identify molecules that can be targeted to regulate inflammation without compromising NK cell cytotoxicity.
PMCID: PMC3855032  PMID: 24036998
2.  Exon 6 variants carried on systemic lupus erythematosus (SLE) risk haplotypes modulate IRF5 function 
Autoimmunity  2010;44(2):82-89.
Interferon response factor 5 (IRF5) regulates innate immune responses to viral infection. IRF5 genetic variants have been shown to be strongly associated with risk for systemic lupus erythematosus (SLE). Functional roles of IRF5 exon 6 structural variants that occur as part of a SLE risk-associated haplotype, including a 30-bp in/del (in/del-10) and a 48-bp splice-site variant (SV-16), have not been established. In this study, we used IRF5 deficient cells overexpressing human IRF5 variants to investigate the roles of exon 6 in/del-10 and SV-16 in regulation of the apoptosis response, nuclear translocation, and ability to transactivate IRF5 responsive cytokines. We found that expression of IRF5 isoforms including either SV-16 or in/del-10 confers ability of IRF5 to impair the apoptotic response and correlates with reduced capacity for IRF5 nuclear translocation in MEFs after a DNA-damaging stimulus treatment. Interestingly, the presence or absence of both SV-16 and in/del-10 results in abrogation of both the anti-apoptotic and enhanced nuclear translocation effects of IRF5 expression. Only cells expressing IRF5 bearing SV-16 show increased IL-6 production upon LPS stimulation. MEFs expressing hIRF5 variants containing in/del-10 showed no significant difference from the control; however, cells carrying hIRF5 lacking both SV-16 and in/del-10 showed reduced IL-6 production. Our overall findings suggest that exon 6 SV-16 is more potent than in/del-10 for IRF5-driven resistance to apoptosis and promotion of cytokine production; however, in/del-10 co-expression can neutralize these effects of SV-16.
PMCID: PMC3104271  PMID: 20695768
SLE; IRF5 variants; exon 6; apoptosis; nuclear translocation
3.  Defective positive selection results in T cell lymphopenia and increased autoimmune diabetes in ADAP deficient BDC2.5-Bl/6 mice 
European journal of immunology  2008;38(4):986-994.
Adhesion and Degranulation Promoting Adapter protein (ADAP), a positive regulator of T cell receptor (TCR) signaling, is required for thymocyte development and T cell homeostasis. To investigate the role of ADAP in a T cell-driven autoimmune response, we generated ADAP-deficient, BDC2.5 TCR transgenic, diabetes-prone (C57Bl/6) mice (BDC/B6). We observed a striking enhancement of diabetes incidence in ADAP-deficient mice, both in animals homozygous for I-Ag7, and in mice carrying one I-Ab allele (BDC/B6g7/b). Increased disease correlates with significantly reduced numbers of pathologic CD4+ T cells in the mice. Consistent with a state of functional lymphopenia in ADAP-deficient BDC/B6g7/b mice, T cells display increased homeostatic proliferation. Transfer of syngeneic lymphocytes or T cells both blocks ADAP-dependent diabetes and relieves exaggerated homeostatic T cell proliferation observed in ADAP-deficient mice. Marked attenuation in cellularity of the CD4+ single-positive (SP) thymocyte compartment in ADAP-deficient BDC/B6g7/b animals suggests a mechanism for induction of the lymphopenia. We conclude that inefficient positive selection in ADAP deficiency results in lymphopenia that leads to enhanced autoimmune diabetes in the BDC/B6g7/b model. Our findings support the notion that ineffective thymic T cell output can be a powerful causative factor in lymphopenia-driven autoimmune diabetes.
PMCID: PMC2829296  PMID: 18383041
ADAP; Autoimmune Diabetes; BDC2.5; Lymphopenia; Thymocyte development
4.  Dissociating hippocampal and basal ganglia contributions to category learning using stimulus novelty and subjective judgments 
NeuroImage  2011;55(4):1739-1753.
We identified factors leading to hippocampal and basal ganglia recruitment during categorization learning. Subjects alternated between blocks of a standard trial and error category learning task and a subjective judgment task. In the subjective judgments task subjects categorized the stimulus and then instead of receiving feedback they indicated the basis of their response using 4 options: Remember: Conscious episodic memory of previous trials. Know-Automatic: Automatic, rapid response accompanied by conscious awareness of category membership. Know-Intuition: A “gut feeling” without fully conscious knowledge of category membership. Guess: Guessing. In addition, new stimuli were introduced throughout the experiment to examine effects of novelty. Categorization overall recruited both the basal ganglia and posterior hippocampus. However, basal ganglia activity was found during Know judgments (both Automatic and Intuition), whereas posterior hippocampus activity was found during Remember judgments. Granger causality mapping indicated interactions between the basal ganglia and hippocampus, with the putamen exerting directed influence on the posterior hippocampus, which in turn exerted directed influence on the posterior caudate nucleus. We also found a region of anterior hippocampus that showed decreased activity relative to baseline during categorization overall, and showed a strong novelty effect. Our results indicate that subjective measures may be effective in dissociating basal ganglia from hippocampal dependent learning, and that the basal ganglia are involved in both conscious and unconscious learning. They also indicate a dissociation within the hippocampus, in which the anterior regions are sensitive to novelty, and the posterior regions are involved in memory based categorization learning.
PMCID: PMC3123902  PMID: 21255655
Classification learning; implicit learning; striatum; medial temporal lobe; salience; functional connectivity
5.  BTI1, an Azoreductase with pH-Dependent Substrate Specificity ▿ †  
Applied and Environmental Microbiology  2011;77(12):4223-4225.
The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.0 for methyl red, methyl orange, and BHQ-10, to pH >8.3 for flame orange.
PMCID: PMC3131635  PMID: 21531830
6.  Dissociating the Contributions of Independent Corticostriatal Systems to Visual Categorization Learning Through the Use of Reinforcement Learning Modeling and Granger Causality Modeling 
NeuroImage  2009;50(2):644-656.
We dissociated the contributions to learning of four corticostriatal “loops” (interacting striatal and cortical regions): motor (putamen and motor cortex), visual (posterior caudate and visual cortex), executive (anterior caudate and prefrontal cortex), and motivational (ventral striatum and ventromedial frontal cortex). Subjects learned to categorize individual repeated images into one of two arbitrary categories via trial and error. We identified (1) Regions sensitive to correct categorization, categorization learning, and feedback valence (2) Regions sensitive to prediction error (violation of feedback expectancy) and reward prediction (expected feedback associated with category response), using reinforcement learning modeling and (3) Directed influences between regions using Granger causality modeling. Each loop showed a unique pattern of sensitivity to each of these factors. Both the motor and visual loops were involved in acquisition of categorization ability: activity during correct categorization increased across learning, and was sensitive to reward prediction. However, the posterior caudate received directed influence from visual cortex, whereas the putamen exerted directed influence on motor cortex. The motivational and executive loops were involved in feedback processing: both regions were sensitive to feedback valence, which interacted with learning across scans. However, the motivational loop activity reflected prediction error, whereas executive loop activity reflected reward prediction, consistent with the executive loop role in integrating reward and action. Granger causality modeling found directed influences between striatal and cortical regions within each loop. Across loops, the motor loop exerted directed influence on the executive loop which is consistent with the role of the executive loop in integrating feedback with stimulus-response history.
PMCID: PMC2824057  PMID: 19969091
7.  Distinct Regulation of Integrin-Dependent T Cell Conjugate Formation and NF-κB activation by the Adapter Protein ADAP1 
Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation promoting adapter protein (ADAP) to control both integrin adhesive function and NF-κB transcription factor activation. We have investigated the molecular basis by which ADAP controls these events in primary murine ADAP-/- T cells. Naïve DO11.10/ADAP-/- T cells show impaired adhesion to OVAp-bearing antigen presenting cells (APCs) that is restored following reconstitution with wild-type ADAP. Mutational analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55, which is otherwise absent from ADAP-/- T cells. Interestingly, forced expression of SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55 are required for optimal LFA-1 function. In addition, the ADAP proline rich domain is required for optimal antigen-induced activation of CD69, CD25, and Bcl-xL, but is not required for assembly of the CARMA1/Bcl10/MALT1 signaling complex and subsequent TCR-dependent NF-κB activity. Our results indicate that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs, in which the ADAP/SKAP55 module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1 mediated NF-κB activation.
PMCID: PMC2593878  PMID: 18802088
8.  PRAM-1 Is Required for Optimal Integrin-Dependent Neutrophil Function 
Molecular and Cellular Biology  2004;24(24):10923-10932.
PML-retinoic acid receptor alpha (RARα) regulated adaptor molecule 1 (PRAM-1) is an intracellular adaptor molecule that is upregulated during the induced granulocytic differentiation of promyelocytic leukemic cells and during normal human myelopoiesis. This report describes the generation of PRAM-1-deficient mice and an analysis of the function of this adaptor in neutrophil differentiation and mature neutrophil function. We demonstrate here that neutrophil differentiation is not impaired in PRAM-1-deficient mice and that PRAM-1-deficient neutrophils function normally following engagement of Fcγ receptors. In contrast, mature PRAM-1-null neutrophils exhibit significant defects in adhesion-dependent reactive oxygen intermediate production and degranulation. Surprisingly, other integrin-dependent responses, such as cell spreading and activation of several signaling pathways, are normal. Together, these findings demonstrate the uncoupling of key integrin-dependent responses in the absence of PRAM-1 and show this adaptor to be critical for select integrin functions in neutrophils.
PMCID: PMC533979  PMID: 15572693

Results 1-8 (8)