Faculty Bibliography

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.

The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium.

In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport and acid-base balance; mice knocked-out for ClC-2 are blind and sterile. Previous work has suggested that CLC-2 is sorted basolaterally by TIFS812LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modelling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI623LL and QVVA635LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI623LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelial-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting ClC-2 through interactions of ESMI623LL with a highly conserved pocket in their gamma1-sigma1A hemicomplex.

During obesity, macrophages (Mo) accumulate in the visceral adipose tissue (VAT), giving rise to a state of chronic, low-grade inflammation that promotes insulin resistance and type 2 diabetes. We hypothesized that activation of HIF-1alpha in highly-metabolically active Mo sustains inflammation in obese VAT and promotes metabolic dysfunction. We show that hypoxic Mo, like M1-polarized Mo, shift their metabolism from oxidative phosphorylation to glycolysis, leading to the accumulation of HIF-1alpha-stabilizing intermediates. Extracellular flux analysis showed that treating Mo with the hypoxia mimetic CoCl2 or the saturated fatty acid palmitate reduced cellular oxygen consumption and increased the rate of extracellular acidification indicative of enhanced glycolysis. Metabolites known to accumulate during persistent glycolysis, such as succinate and lactic acid, activated HIF-1alpha in Mo and promoted inflammatory gene expression. To test the role of Mo HIF-1alpha in promoting VAT inflammation and metabolic dysfunction in obesity, we fed wild type or Mo-specific HIF-1alpha knock-out mice a high-fat diet (60% kcal fat) for 20 weeks. Notably, the ablation of HIF-1alpha in Mo reduced VAT inflammation as indicated by the reduced accumulation of F4/80+ cells and decreased expression of inflammatory cytokines (TNFalpha, IL-6, MCP-1). In addition, adipose tissue Mo isolated from Mo-HIF-1alpha knock-out mice showed increased expression of markers characteristic of M2 reparative Mo (Ym1, Fizz1, Aldh2) and reduced expression of M1 inflammatory Mo markers (Ccl2, Il1b), compared to Mo from WT mice. Furthermore, Mo-HIF-1alpha knock-out mice showed improved glucose homeostasis and insulin sensitivity, and reduced plasma insulin and free fatty acid levels compared to WT mice. Together, these data indicate that activation of HIF-1alpha in VAT Mo during obesity promotes tissue inflammation and insulin resistance

BACKGROUND: The surgical implantation of materials and devices has dramatically increased over the past decade. This trend is expected to continue with the broadening application of biomaterials and rapid expansion of aging populations. One major factor that limits the potential of implantable materials and devices is the foreign body response, an immunologic reaction characterized by chronic inflammation, foreign body giant cell formation, and fibrotic capsule formation. METHODS: The English literature on the foreign body response to implanted materials and devices is reviewed. RESULTS: Fibrotic encapsulation can cause device malfunction and dramatically limit the function of an implanted medical device or material. Basic science studies suggest a role for immune and inflammatory pathways at the implant-host interface that drive the foreign body response. Current strategies that aim to modulate the host response and improve construct biocompatibility appear promising. CONCLUSIONS: This review article summarizes recent basic science, preclinical, and clinicopathologic studies examining the mechanisms driving the foreign body response, with particular focus on breast implants and synthetic meshes. Understanding these molecular and cellular mechanisms will be critical for achieving the full potential of implanted biomaterials to restore human tissues and organs.

Bone metabolism is a vital process that involves resorption by osteoclasts and formation by osteoblasts, which is closely regulated by immune cells. The neuronal guidance protein Netrin-1 regulates immune cell migration and inflammatory reactions, but its role in bone metabolism is unknown. During osteoclast differentiation, osteoclast precursors increase expression of Netrin-1 and its receptor Unc5b. Netrin-1 binds, in an autocrine and paracrine manner, to Unc5b to promote osteoclast differentiation in vitro, and absence of Netrin-1 or antibody-mediated blockade of Netrin-1 or Unc5b prevents osteoclast differentiation of both murine and human precursors. We confirmed the functional relationship of Netrin-1 in osteoclast differentiation in vivo using Netrin-1-deficient (Ntn1(-/-) ) or wild-type (WT) bone marrow transplanted mice. Notably, Ntn1(-/-) chimeras have markedly diminished osteoclasts, as well as increased cortical and trabecular bone density and volume compared with WT mice. Mechanistic studies revealed that Netrin-1 regulates osteoclast differentiation by altering cytoskeletal assembly. Netrin-1 increases regulator of Rho-GEF subfamily (LARG) and repulsive guidance molecule (RGMa) association with Unc5b, which increases expression and activation of cytoskeletal regulators RhoA and focal adhesion kinase (FAK). Netrin-1 and its receptor Unc5b likely play a role in fusion of osteoclast precursors because Netrin-1 and DC-STAMP are tightly linked. These results identify Netrin-1 as a key regulator of osteoclast differentiation that may be a new target for bone therapies. (c) 2015 American Society for Bone and Mineral Research.