Teresa A. Doggett

Noncanonical autophagy promotes the visual cycle.

Phagocytosis and degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is fundamental to vision. Autophagy is also responsible for bulk degradation of cellular components, but its role in POS degradation is not well understood. We report that the morning burst of RPE phagocytosis coincided with the enzymatic conversion of autophagy protein LC3 to its lipidated form. LC3 associated with single-membrane phagosomes containing engulfed POS in an Atg5-dependent manner that required Beclin1, but not the autophagy preinitiation complex. The importance of this process was verified in mice with Atg5-deficient RPE cells that showed evidence of disrupted lysosomal processing. These mice also exhibited decreased photoreceptor responses to light stimuli and decreased chromophore levels that were restored with exogenous retinoid supplementation. These results establish that the interplay of phagocytosis and autophagy within the RPE is required for both POS degradation and the maintenance of retinoid levels to support vision.

The snake venom protein botrocetin acts as a biological brace to promote dysfunctional platelet aggregation

Botrocetin is a snake venom protein that enhances the affinity of the A1 domain of plasma von Willebrand factor (vWF) for the platelet receptor glycoprotein Ibα (GPIbα), an event that contributes to bleeding and host death. Here we describe a kinetic and crystallographic analysis of this interaction that reveals a novel mechanism of affinity enhancement. Using high-temporal-resolution microscopy, we show that botrocetin decreases the GPIbα off-rate two-fold in both human and mouse complexes without affecting the on-rate. The key to this behavior is that, upon binding of GPIbα to vWF-A1, botrocetin prebound to vWF-A1 makes no contacts initially with GPIbα, but subsequently slides around the A1 surface to form a new interface. This two-step mechanism and flexible coupling may prevent adverse alterations in on-rate of GPIbα for vWF-A1, and permit adaptation to structural differences in GPIbα and vWF in several prey species.

The plasticity of regulatory T cell function.

Regulatory T cells (Tregs) can suppress a wide variety of cell types, in diverse organ sites and inflammatory conditions. Whereas Tregs possess multiple suppressive mechanisms, the number required for maximal function is unclear. Furthermore, whether any interrelationship or cross-regulatory mechanisms exist to orchestrate and control their utilization is unknown. In this study, we assessed the functional capacity of Tregs lacking the ability to secrete both IL-10 and IL-35, which individually are required for maximal Treg activity. Surprisingly, IL-10/IL-35 double-deficient Tregs were fully functional in vitro and in vivo. Loss of IL-10 and IL-35 was compensated for by a concurrent increase in cathepsin E (Ctse) expression, enhanced TRAIL (Tnfsf10) expression, and soluble TRAIL release, rendering IL-10/IL-35 double-deficient Tregs functionally dependent on TRAIL in vitro and in vivo. Lastly, whereas C57BL/6 Tregs are normally IL-10/IL-35 dependent, BALB/c Tregs, which express high levels of cathepsin E and enhanced TRAIL expression, are partially TRAIL dependent by default. These data reveal that cross-regulatory pathways exist that control the utilization of suppressive mechanisms, thereby providing Treg functional plasticity.

Autophagy supports survival and phototransduction protein levels in rod photoreceptors

Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod cell loss is the primary pathologic event.

An Immunogenic Peptide in the A-box of HMGB1 Protein Reverses Apoptosis-induced Tolerance through RAGE Receptor

Background: The role of caspase-1 in regulating the immunogenic properties of HMGB1 has not been previously reported. Results: We have mapped a peptide in the A-box of HMGB1 that reverses tolerance through RAGE. Conclusion: Inflammasome signaling regulates the immunogenic activity of HMGB1. Significance: Immunogenic peptides within the HMGB1 A-box may be exploited to reverse immune tolerance in sepsis patients. Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

IL-18 Immunotherapy for Neovascular AMD: Tolerability and Efficacy in Nonhuman Primates.

PURPOSE Age-related macular degeneration is the most common form of central retinal blindness in the elderly. Of the two end stages of disease, neovascular AMD-although the minority form-is the most severe. Current therapies are highly successful at controlling progression of neovascular lesions; however, a significant number of patients remain refractory to treatment and the development of alternative and additive therapies to anti-VEGFs is essential. METHODS In order to address the translational potential of interleukin (IL)-18 for use in neovascular AMD, we initiated a nonhuman primate tolerability and efficacy study for the use of intravitreally (IVT) administered clinical grade human IL-18 (SB-485232). Cynomolgus monkeys were injected IVT with increasing doses of human IL-18 (two each at 1000, 3000, and 10,000 ng per eye). In tandem, 21 monkeys were administered nine laser burns in each eye prior to receiving IL-18 as an IVT injection at a range of doses. Fundus fluorescein angiography (FFA) was performed on days 8, 15, and 22 post injection and the development of neovascular lesions was assessed. RESULTS We show intravitreal, mature, recombinant human IL-18 is safe and can reduce choroidal neovascular lesion development in cynomolgus monkeys. CONCLUSIONS Based on our data comparing human IL-18 to current anti-VEGF-based therapy, clinical deployment of IL-18 for neovascular AMD has the potential to lead to a new adjuvant immunotherapy-based treatment for this severe form of central blindness.

Strategies for the Use of Live Recombinant Avirulent Bacterial Vaccines for Mucosal Immunization

Publisher Summary Live recombinant avirulent microorganisms can be used as antigen delivery vehicles to especially induce mucosal immune responses following immunization of individuals by oral, intranasal, and intravaginal routes. There are a number of issues of importance in considering the use of live microorganisms for this purpose. One is whether the vaccine induces just a localized mucosal immune response or a generalized response with production of secretory IgA in all secretory glands, and from mucosal tissues throughout the body. Bacteria in at least eight genera have been used to achieve immune responses at mucosal sites. It is likely that others will be evaluated in the future. Lactobacillus fermentum has been used to express foreign antigens, but since these organisms seem not to colonize except in the colon, which may not be the best inductive site for a mucosal immune response. Repetitive oral immunizations of recombinant L. fermentum are required to achieve adequate immune responses. Streptococcus gordonii has been genetically engineered to express antigens. Vibrio cholerae and certain strains of Escherichia coli represent microorganisms with the ability to adhere to specific mucosal tissues, but without the ability to survive following uptake into M cells and entry into phagocytic cells in the gut-associated lymphoid tissue (GALT).

Autophagy supports color vision

Cones comprise only a small portion of the photoreceptors in mammalian retinas. However, cones are vital for color vision and visual perception, and their loss severely diminishes the quality of life for patients with retinal degenerative diseases. Cones function in bright light and have higher demand for energy than rods; yet, the mechanisms that support the energy requirements of cones are poorly understood. One such pathway that potentially could sustain cones under basal and stress conditions is macroautophagy. We addressed the role of macroautophagy in cones by examining how the genetic block of this pathway affects the structural integrity, survival, and function of these neurons. We found that macroautophagy was not detectable in cones under normal conditions but was readily observed following 24 h of fasting. Consistent with this, starvation induced phosphorylation of AMPK specifically in cones indicating cellular starvation. Inhibiting macroautophagy in cones by deleting the essential macroautophagy gene Atg5 led to reduced cone function following starvation suggesting that cones are sensitive to systemic changes in nutrients and activate macroautophagy to maintain their function. ATG5-deficiency rendered cones susceptible to light-induced damage and caused accumulation of damaged mitochondria in the inner segments, shortening of the outer segments, and degeneration of all cone types, revealing the importance of mitophagy in supporting cone metabolic needs. Our results demonstrate that macroautophagy supports the function and long-term survival of cones providing for their unique metabolic requirements and resistance to stress. Targeting macroautophagy has the potential to preserve cone-mediated vision during retinal degenerative diseases.

Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GPIb/spl alpha/-vWF tether bond

The ability of platelets to tether to and translocate on injured vascular endothelium relies on the interaction between the platelet glycoprotein receptor Ib alpha (GPIb/spl alpha/) and the A1 domain of von Willebrand factor (vWF-A1). We now report that the GPIb/spl alpha/-vWF-A1 tether bond displays similar kinetic attributes as the selectins including: 1) the requirement for a critical level of flow to initiate adhesion, 2) short-lived tethering events at sites of vascular injury in vivo, and 3) a fast intrinsic dissociation rate constant, k/sub off//sup 0/ (3.45 /spl plusmn/ 0.37 s/sup -1/). Values for k/sub off/, also varied exponentially (4.2 /spl plusmn/ 0.8 s/sup -1/ to 7.3 /spl plusmn/ 0.4 s/sup -1/) as a function of the force applied to the bond (from 36 to 217pN). The biological importance of rapid bond dissociation is demonstrated by kinetic characterization of the naturally occurring A1 domain mutation, I546V that results in spontaneous binding of plasma vWF to circulating platelets in flowing blood. This mutation resulted in a loss of the shear threshold phenomenon, a /spl sim/6-fold reduction in k/sub off/, but no significant alteration in the ability of the tether bond to resist shear-induced forces. Thus, flow dependent adhesion and rapid and force-dependent kinetic properties are the predominant features of the GPIb/spl alpha/-vWF-A1 tether bond.

Expression of the rfa, LPS Biosynthesis Promoter in Salmonella typhimurium During Invasion of Intestinal Epithelial Cells

Salmonella regulates transcription of many of its genes in response to environmental conditions encountered inside or outside the eukaryotic cells it infects. In this paper, we examined Salmonella typhimurium gene expression within epithelial cells, by using bacterial luciferase as a reporter. We focused on gene expression controlled by Salmonella rfa promoter, using lac promoter as a control. We observed down regulation for both promoters during the initial 2 h of invasion. The decreased levels of luciferase activity appeared to be due to metabolic changes, since we observed similar results with tissue culture medium alone. Gene expression stabilized to a new steady state for the Salmonella rfa promoter, while a lac promoter activity steadily decreased. Bacterial luciferase activity was a good indicator of intracellular numbers and allowed us to detect as few as 1000 bacterial cells/infected monolayer. Both promoters were not dependent on host protein synthesis for expression.