Faculty Bibliography

Current treatment of hemophilia consists of the administration of recombinant clotting factors, such as factor VIII (FVIII). However, patients with severe hemophilia can mount immune responses targeting therapeutically administered FVIII through inhibitory immunoglobulins that limit treatment efficacy. Induction of immune tolerance to FVIII in hemophilia has been extensively studied but remains an unmet need. We found that nondepleting anti-CD4 monoclonal antibodies (mAbs) are effective in inducing long-term tolerance to FVIII in different strains of hemophilic mice. Tolerance induction was facilitated when anti-CD4 mAbs were administered together with FVIII adsorbed in an adjuvant (alum). The observed state of tolerance was antigen specific, with mice remaining immune competent to respond to different antigens. Importantly, we found that following immunization with FVIII, the primed cells remained susceptible to tolerance induction. Studies with Foxp3-deficient and interleukin 10 (IL-10)-deficient mice demonstrated that the underlying tolerance mechanism is Foxp3 independent but requires IL-10. Our data show that an adjuvant, when administered together with a tolerizing agent such as nondepleting anti-CD4, can facilitate the induction of long-term tolerance to recombinant proteins, possibly not only in hemophilia but also in other diseases that are treated with potentially immunogenic therapeutics.

Diabetes is a major risk factor for atherosclerosis. Although atherosclerosis is initiated by deposition of cholesterol-rich lipoproteins in the artery wall, the entry of inflammatory leukocytes into lesions fuels disease progression and impairs resolution. We show that diabetic mice have increased numbers of circulating neutrophils and Ly6-C(hi) monocytes, reflecting hyperglycemia-induced proliferation and expansion of bone marrow myeloid progenitors and release of monocytes into the circulation. Increased neutrophil production of S100A8/S100A9, and its subsequent interaction with the receptor for advanced glycation end products on common myeloid progenitor cells, leads to enhanced myelopoiesis. Treatment of hyperglycemia reduces monocytosis, entry of monocytes into atherosclerotic lesions, and promotes regression. In patients with type 1 diabetes, plasma S100A8/S100A9 levels correlate with leukocyte counts and coronary artery disease. Thus, hyperglycemia drives myelopoiesis and promotes atherogenesis in diabetes.

Dendritic cells (DC) are professional APCs that regulate innate and adaptive immunity. The role of fatty-acid synthesis in DC development and function is uncertain. We found that blockade of fatty-acid synthesis markedly decreases dendropoiesis in the liver and in primary and secondary lymphoid organs in mice. Human DC development from PBMC precursors was also diminished by blockade of fatty-acid synthesis. This was associated with higher rates of apoptosis in precursor cells and increased expression of cleaved caspase-3 and BCL-xL and downregulation of cyclin B1. Further, blockade of fatty-acid synthesis decreased DC expression of MHC class II, ICAM-1, B7-1, and B7-2 but increased their production of selected proinflammatory cytokines including IL-12 and MCP-1. Accordingly, inhibition of fatty-acid synthesis enhanced DC capacity to activate allogeneic as well as Ag-restricted CD4(+) and CD8(+) T cells and induce CTL responses. Further, blockade of fatty-acid synthesis increased DC expression of Notch ligands and enhanced their ability to activate NK cell immune phenotype and IFN-gamma production. Because endoplasmic reticulum (ER) stress can augment the immunogenic function of APC, we postulated that this may account for the higher DC immunogenicity. We found that inhibition of fatty-acid synthesis resulted in elevated expression of numerous markers of ER stress in humans and mice and was associated with increased MAPK and Akt signaling. Further, lowering ER stress by 4-phenylbutyrate mitigated the enhanced immune stimulation associated with fatty-acid synthesis blockade. Our findings elucidate the role of fatty-acid synthesis in DC development and function and have implications to the design of DC vaccines for immunotherapy.

Muscle-specific kinase (MuSK) is essential for each step in neuromuscular synapse formation. Before innervation, MuSK initiates postsynaptic differentiation, priming the muscle for synapse formation. Approaching motor axons recognize the primed, or prepatterned, region of muscle, causing motor axons to stop growing and differentiate into specialized nerve terminals. MuSK controls presynaptic differentiation by causing the clustering of Lrp4, which functions as a direct retrograde signal for presynaptic differentiation. Developing synapses are stabilized by neuronal Agrin, which is released by motor nerve terminals and binds to Lrp4, a member of the low-density lipoprotein receptor family, stimulating further association between Lrp4 and MuSK and increasing MuSK kinase activity. In addition, MuSK phosphorylation is stimulated by an inside-out ligand, docking protein-7 (Dok-7), which is recruited to tyrosine-phosphorylated MuSK and increases MuSK kinase activity. Mutations in MuSK and in genes that function in the MuSK signaling pathway, including Dok-7, cause congenital myasthenia, and autoantibodies to MuSK, Lrp4, and acetylcholine receptors are responsible for myasthenia gravis.

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.