Faculty Bibliography

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33-mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization-associated gene profile. Furthermore, miR-33-mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid-producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33-depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4+ T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3+ Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

One of the cardinal features of neural development and adult plasticity is the contribution of activity-dependent signaling pathways. However, the interrelationships between different activity-dependent genes are not well understood. The immediate early gene neuronal-activity-regulated pentraxin (NPTX2 or Narp) encodes a protein that has been associated with excitatory synaptogenesis, AMPA receptor aggregation, and the onset of critical periods. Here, we show that Narp is a direct transcriptional target of brain-derived neurotrophic factor (BDNF), another highly regulated activity-dependent gene involved in synaptic plasticity. Unexpectedly, Narp is bidirectionally regulated by BDNF. Acute BDNF withdrawal results in downregulation of Narp, whereas transcription of Narp is greatly enhanced by BDNF. Furthermore, our results show that BDNF directly regulates Narp to mediate glutamatergic transmission and mossy fiber plasticity. Hence, Narp serves as a significant epistatic target of BDNF to regulate synaptic plasticity during periods of dynamic activity.

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

Neither breakpoints (BPs) nor epidemiological cutoff values (ECVs) have been established for Candida spp. with anidulafungin, caspofungin, and micafungin when using the Sensititre YeastOne (SYO) broth dilution colorimetric method. In addition, reference caspofungin MICs have so far proven to be unreliable. Candida species wild-type (WT) MIC distributions (for microorganisms in a species/drug combination with no detectable phenotypic resistance) were established for 6,007 Candida albicans, 186 C. dubliniensis, 3,188 C. glabrata complex, 119 C. guilliermondii, 493 C. krusei, 205 C. lusitaniae, 3,136 C. parapsilosis complex, and 1,016 C. tropicalis isolates. SYO MIC data gathered from 38 laboratories in Australia, Canada, Europe, Mexico, New Zealand, South Africa, and the United States were pooled to statistically define SYO ECVs. ECVs for anidulafungin, caspofungin, and micafungin encompassing >/=97.5% of the statistically modeled population were, respectively, 0.12, 0.25, and 0.06 mug/ml for C. albicans, 0.12, 0.25, and 0.03 mug/ml for C. glabrata complex, 4, 2, and 4 mug/ml for C. parapsilosis complex, 0.5, 0.25, and 0.06 mug/ml for C. tropicalis, 0.25, 1, and 0.25 mug/ml for C. krusei, 0.25, 1, and 0.12 mug/ml for C. lusitaniae, 4, 2, and 2 mug/ml for C. guilliermondii, and 0.25, 0.25, and 0.12 mug/ml for C. dubliniensis. Species-specific SYO ECVs for anidulafungin, caspofungin, and micafungin correctly classified 72 (88.9%), 74 (91.4%), 76 (93.8%), respectively, of 81 Candida isolates with identified fks mutations. SYO ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin, micafungin, and especially caspofungin, since testing the susceptibilities of Candida spp. to caspofungin by reference methodologies is not recommended.

Background: Data on TDF use during pregnancy for preventing mother-to-child transmission (MTCT) of hepatitis B virus (HBV) are scarce. Methods: Hepatitis B E antigen (HBeAg)-positive mothers with HBV DNA levels >200,000 IU/mL were randomized 1:1 to receive either TDF from gestation week 30-32 to postpartum week 4 or no treatment, and were followed-up until postpartum week 28. All infants received immunoprophylaxis. The primary measurement was the MTCT rate, while endpoints included TDF safety, maternal HBV DNA reduction at delivery, and HBeAg or hepatitis B s antigen loss/seroconversion at postpartum week 28. Results: Among the 200 mothers enrolled in 5 regions of the country, 180 completed the study. At postpartum week 28, the MTCT rate was significantly lower in infants from TDF-treated mothers when compared to those from non-treated mothers, both on per-protocol analysis (0% vs. 6.82%, P = 0.013) and intention-to-treat analysis (5.16% vs. 18.0%, P = 0.007). The safety profile was similar between groups, with no difference in birth defect rates (2.11% with TDF exposure vs. 1.14% without exposure, P = 1.00). HBV DNA levels decreased to <200,000 IU/mL in 68% (66/97) of TDFtreated mothers before delivery compared to 2.0% (2/100) of non-treated mothers (P < 0.001). The HBV serologic outcome did not differ between groups. Conclusions: TDF therapy in late pregnancy for highly viremic mothers effectively reduced MTCT. The treatment was well tolerated, and no safety concerns were identified. TDF therapy should be strongly considered for mothers whose HBV DNA levels exceeded 200,000 IU/mL and started at gestation week 30-32. (Table Presented)

Introduction: Autoimmune hepatitis (AIH) is a T cell-mediated inflammatory process which frequently necessitates liver transplantation. Mincle is a novel pro-inflammatory pattern recognition receptor which is critical in the immune response to mycobacteria, but has not been implicated in liver disease. Methods: C57BL/6 or Mincle-/- mice were treated with Concanavalin- A (ConA) to induce AIH. Alternatively, Mincle inhibitor (6G5), Syk inhibitor (Piceatannol), Hif-1alpha inhibitor (LW6), or C/ EBPbeta inhibitor (Genistein) were employed. Survival, AST, ALT, serum cytokines, liver inflammatory infiltrate and transcription factors were assessed. Results: Mincle was highly expressed on hepatic innate inflammatory cells. Furthermore, Mincle ligand (SAP130) and Mincle signaling intermediates (including p-Syk) were increased in murine and human AIH. Most significantly, Mincle deletion or blockade protected against AIH. Mincle-/- mice had decreased mortality, transaminase levels, and CD45+ hepatic influx as compared to WT mice (Table). p-Syk inhibition also attenuated ConA injury. Mechanistically, we found that Mincle blockade decreased NF-kappabeta related signaling intermediates, including Hif-1alpha, and C/EBPbeta, which is critical for normal macrophage inflammatory responses, specifically nitric oxide production. Accordingly, Mincle-/- macrophages had impaired activation of CD4+ T cells, and Hif-1alpha and C/ EBPbeta blockade reduced the severity of AIH. Conclusions: Our work is the first to show that Mincle is a critical mediator of the hepatotoxic inflammatory response in AIH. Mincle signaling mediates hepatic injury through upregulation of Hif-1alpha and C/ EBPbeta transcription factors leading to robust macrophage-mediated NO production. Our research suggests that targeting Mincle may have efficacy in the treatment of AIH. (Table Presented)

Neurotrophins and glucocorticoids are robust synaptic modifiers, and deregulation of their activities is a risk factor for developing stress-related disorders. Low levels of brain-derived neurotrophic factor (BDNF) increase the desensitization of glucocorticoid receptors (GR) and vulnerability to stress, whereas higher levels of BDNF facilitate GR-mediated signaling and the response to antidepressants. However, the molecular mechanism underlying neurotrophic-priming of GR function is poorly understood. Here we provide evidence that activation of a TrkB-MAPK pathway, when paired with the deactivation of a GR-protein phosphatase 5 pathway, resulted in sustained GR phosphorylation at BDNF-sensitive sites that is essential for the transcription of neuronal plasticity genes. Genetic strategies that disrupted GR phosphorylation or TrkB signaling in vivo impaired the neuroplasticity to chronic stress and the effects of the antidepressant fluoxetine. Our findings reveal that the coordinated actions of BDNF and glucocorticoids promote neuronal plasticity and that disruption in either pathway could set the stage for the development of stress-induced psychiatric diseases.

The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1exon8). We previously reported that PS1 L271V increased amyloid beta (Abeta) 42/40 ratios, while PS1exon8 reduced Abeta42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1exon8 did not rescue Abeta generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1exon8 is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1exon8 in vivo by crossing PS1exon8 transgenics with either PS1-null or Dutch APPE693Q mice. As a control, we crossed APPE693Q with mice expressing a deletion in an adjacent exon (PS1exon9). PS1exon8 did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1exon8 interacts with nicastrin, participating in the gamma-secretase complex formation. These data support that catalytically inactive PS1exon8 is generated physiologically and participates in protein-protein interactions.