Inside-Out

ACVP at Experimental Biology 2013

For the ninth consecutive year, the American College of Veterinary Pathologists (ACVP) has partnered with the American Society for Investigative Pathology (ASIP) to host a symposium at the annual Experimental Biology meeting. Experimental Biology is a scientific forum addressing advances in the fields of anatomy, physiology, biochemistry, pathology, nutrition, and pharmacology. The meeting is sponsored by 6 societies that are part of the Federation of American Societies for Experimental Biology, including ASIP; ACVP is a guest society. The 2013 meeting was held in Boston, Massachusetts, April 20 through 24. Organized through the ACVP Intersociety Experimental Pathology Committee, the joint ACVP/ASIP session is designed to highlight the shared interests and perspectives of its members. In an annual meeting attended by more than 14 000 scientists, Experimental Biology is also a unique opportunity to enhance visibility of the ACVP and to illustrate the power of a comparative biomedical background in leading advances in medicine and our understanding of pathogenic mechanisms. The 2013 session was led by ACVP Diplomates Dr Michael Oglesbee (chair), The Ohio State University, and Dr Tim LaBranche (co-chair), Pfizer Corporation. The strategy was to design a session that addresses a novel and emerging area of study with potential broad relevance and thus interest. Emphasis was placed on highlighting the importance of a comparative biomedical approach, particularly in the area of animal modeling. ASIP membership has repeatedly expressed an interest in heat shock protein (HSP) biology, and shifting focus from the roles of intracellular HSP to the role of extracellularly released HSP is a novel and emerging area of investigation.

Background

HSPs are molecular chaperones that are primarily known for their intracellular functions. They bind hydrophobic protein domains that are exposed during the process of folding and unfolding or as intermediates in the assembly of multimeric complexes. Binding of HSPs to client proteins thus maintains solubility of the complex, guiding proper folding and/or assembly reactions that are necessary to maintain cellular homeostasis or to reestablish homeostasis following cell stress. The term chaperone reflects the concept that HSPs prevent 2 hydrophobic domains from coming together in an undesirable manner. The term heat shock protein was coined because of their increased expression following hyperthermic treatment. Hyperthermia causes protein unfolding, and HSP induction is part of a cell response to maintain homeostasis, either by facilitating refolding or by directing terminally denatured proteins to sites of proteasomal or lysosomal degradation. HSP induction is thus a defining feature of the unfolded protein response, a form of the cellular stress response, and detection of HSP induction is thus used as an indicator of cellular toxicity. Major families of HSPs are defined by molecular mass, and members of the 70-kDa family are both constitutively expressed and/or highly stress inducible (eg, hsp70), with expression of the highly inducible isoforms serving as sensitive indicators of changing demands for chaperone functions.

Emerging is an increased appreciation of the degree to which HSPs can be released from cells in both physiologic and pathologic states and the diverse roles that these extracellular HSPs may play. Initially, release was focused on that associated with cell death, and the functions attributed to the released HSPs centered on their ability to modulate immune function and to stimulate proinflammatory responses. The view was that necrosis would trigger proinflammatory or immunostimulatory responses through the release of HSPs but that HSP release would not occur in instances of apoptosis, a form of “silent” cell death that is key to organ development and other physiological states. Immune modulation was largely focused on the ability of the extracellular HSP to deliver antigen for cross-presentation by macrophages and other antigen-presenting cells. That capability was the rationale for isolating HSP-antigen complexes from tumor cells in an attempt to induce antitumor immunity. But now we appreciate the fact that HSPs may be released in a variety of physiologic and pathophysiologic states, and the function of the extracellular HSP can be varied. Release is regulated differently in different tissues, and there are species differences in HSP expression. The diverse roles of extracellular HSPs make them an important determinant of host responses in a great variety of disease states. “Inside-Out: Extracellular Roles for Heat Shock Proteins” was designed to illustrate these emerging concepts. Focus was placed on hsp70, with particular emphasis on the central nervous system, this being a useful framework in which to address current advances in our understanding of release mechanisms and extracellular functions.

Extracellular HSP and Neuroimmune Responses

The session began with Dr Oglesbee’s review of HSP biology in general and then a more in-depth discussion of the role that hsp70 plays in mediating antiviral immunity in the infected brain. Studies using canine distemper virus and measles virus (MeV) show that within the cell, hsp70 stimulates virus gene expression. But within neurons, this virus-host relationship results in the extracellular release of hsp70 from viable cells. The extracellular hsp70 may then engage Toll-like receptors (ie, TLR-4) on brain macrophages to induce a robust type 1 interferon response that is essential to developing the adaptive immune responses that drive viral clearance. Mice lack constitutive expression of hsp70, unlike humans and dogs, so that the contribution of hsp70 could be examined by creating stably transfected mouse cell lines and transgenic mice that constitutively express hsp70. Increasing intracellular levels of hsp70 within neurons results in higher levels of extracellular release following infection, which in turn enhances expression of type 1 interferon in brain macrophages and reduces viral neurovirulence. ⁷ This finding supports an adaptive role for fever in response to microbial infection, since fever is a potent inducer of hsp70 in all tissues. MeV infection of neurons is noncytolytic, and the release of hsp70 was shown to be secretory and not associated with cell death. This finding shows that viable neurons do possess mechanisms to alert the immune system to the presence of an intracellular threat, with extracellular hsp70 serving as a danger signal to the presence of viral infection. Studies with vesicular stomatitis virus (VSV) were used to demonstrate the more broad virological relevance of this host-protective mechanism and the ability of hsp70 to inhibit neurovirulence by a more fulminate and cytopathic neuronal infection. Extracellular release of hsp70 from VSV-infected neurons was unanticipated, given that cell death is thought to involve apoptosis, requiring that we revisit programmed cell death in response to viral infection and to distinguish between apoptosis and proinflammatory programmed cell death (pyroptosis).

Two general take-home messages were developed in this first presentation. First, extracellular hsp70 is part of a more general neuroinflammatory response. While adaptive in context of microbial infection, the same release may be triggered by noninfectious tissue injuries, and in this case the response may be maladaptive. Two examples were illustrated, where elevations of hsp70 in cerebrospinal fluid were documented in dogs with spinal cord reperfusion injury and those with steroid-responsive meningitis-arteritis. The second major point applies to use of the mouse in modeling neuroinflammatory responses in general. Mice are unique in that they lack constitutive expression of hsp70, which is characteristic of humans, dogs, and other animals, and hsp70 can mediate potent neuroinflammatory and neuroimmune responses. To what degree do mice model neuroinflammatory diseases of humans, dogs, and other animals when the difference in basal expression of hsp70 is not addressed? ⁹

Dynamics of HSP Release and Function

Dr Stuart Calderwood of the Harvard Medical School followed with a presentation focused on more basic mechanisms of HSP release from cells and the varied functions that extracellular HSPs may have. The context for the first part of the discussion was the age-old question, “How do you release a leaderless protein from a viable cell?” This has been the dilemma long posed by investigators studying the release of interleukin 1 (IL-1) or fibroblast growth factor 1. Dr Calderwood illustrated the similarities in regulation of extracellular release of IL-1 and hsp70. Although not addressed, the discussion set the stage for considering mechanisms by which cells of diverse histogenesis release signals through microvesicular shedding and exosome secretion. One of the most frequently encountered exosomal proteins is hsp70, where the surface location of hsp70 allows it to engage cell-surface TLR-4. Hsp70 is certainly not alone in this regard, and we must consider other immunogenic HSPs such as hsp60 and hsp90. The response of the target cell to these HSPs was then discussed, beginning with HSPs binding to scavenger receptors such as SREC1 (scavenger receptor expressed by endothelial cells 1) and SRA (scavenger receptor A). SREC1 binding is key to the internalization of HSP-antigen complexes that drive cross-presentation of antigen through both MHC I and II pathways. A key concept was developed in which the TLR4 response to HSPs (eg, hsp70) may be dependent on the scavenger receptor that is engaged, and TLR4 interactions may be indirect. ⁸ HSP bound to SREC-1 appears to stimulate TLR4 signaling to enhance the production of proinflammatory cytokines and type 1 interferon. On the other hand, HSP bound to SRA may suppress TLR4 signaling. Many cell types express TLR4 (although these are predominantly macrophages in the brain), and they differ in their expression of scavenger receptors, reminding us that the role of extracellular HSPs may be more dynamic than simply that of a proinflammatory molecule. In fact, that role may change with duration of HSP release, being initially proinflammatory but with the ability to subsequently downregulate its proinflammatory effects with more prolonged increases—the “off switch.”

Extracellular HSP and the Physiologic Stress Response

Dr Monika Fleshner, University of Colorado, Boulder, examined the role of hsp70 as a damage-associated molecular pattern (DAMP) that is released by stress. In this instance, stress was defined at the animal (physiological) level, using tail shock in rats as the model. Tail shock was shown to increase plasma levels of hsp70, and this increase was dependent on α-adrenergic signaling. The source of the hsp70 is unknown, although vascular endothelial cells and gut epithelial cells are the primary sources being considered. The hsp70 is bioactive, enhancing macrophage bacterial killing following subcutaneous inoculation with Escherichia coli. ² Characterization of the plasma hsp70 showed that it was both free and exosome associated, with increases in both being driven by α-adrenergic signaling. That signaling did not increase total exosome levels, but it did dramatically increase the hsp70 content of exosomes. Exosomes isolated from rats exposed to acute stress were effective at decreasing tissue swelling in response to subcutaneous E. coli inoculation, and the effect was more potent than the administration of free hsp70. These results have 2 important general implications. The first points to the therapeutic potential of administering hsp70-containing exosomes in the bacteria- or virus-infected patient. The second is a consideration that applies to experimental manipulation of animal models, and this point stirred a lively discussion. We place much focus on interpreting physiological stress responses in routine toxicity studies, ⁶ yet the role of extracellular HSPs has not been considered either as a stress biomarker or as a factor that may influence other experimental readouts. The consideration is particularly relevant to studies of inflammation and immunity, and we should consider the potentially different influences of acute vs chronic stress on plasma hsp70 levels and its function. Unknown is the influence of increased plasma hsp70 levels induced by acute stress on the cerebrospinal fluid, although previous work by Awad et al ¹ shows that hsp70 release can be regulated independently between these 2 compartments.

Extracellular HSP in Neurodegenerative Disorders

Dr Pamela McLean, Mayo Clinic, Florida, concluded the session with a talk examining a function of hsp70 that has not been considered: the ability of extracellular hsp70 to influence protein aggregates that are part of neurodegenerative disorders. This potential was addressed in a model of Parkinson disease. α-Synuclein aggregation drives the disorder, with soluble oligomers being released from cells to mediate cytotoxicity, possibly through membrane pore-forming potential of the oligomers. Increasing expression levels of hsp70 in a transgenic model showed that extracellular hsp70 diminished oligomerization and therefore toxicity of α-synuclein. ^4,5 The same beneficial effect was demonstrated using pharmacologic inhibitors of hsp90 that are well known for their induction of hsp70. Increased intracellular levels of hsp70 translated into increased extracellular hsp70 release. The released form of α-synuclein and hsp70 is exosomal, ³ with high levels of exosome formation being a characteristic of neurons that may normally play a role in cell-to-cell communication. While highlighting the therapeutic potential of extracellular hsp70 in neurodegenerative disorders, the findings also raise intriguing questions regarding pathogenesis. Although extracellular hsp70 may diminish toxicity of α-synuclein oligomers, does it facilitate cell-to-cell transmission or even transmission between individuals via the olfactory route? And in light of the previous presentations, what are the costs in terms of proinflammatory effects of extracellular hsp70?

Collectively, the session highlighted underappreciated roles of heat shock proteins in diverse conditions, ranging from physiologic responses to stress to inflammatory, immune, and degenerative disorders. Species-specific differences in HSP expression and the effects of acute stressors were shown to have potentially profound implications on the interpretation of experimental results that are based on animal experimentation. These considerations apply to studies of pathogenesis, toxicologic pathology, physiology, and experimental therapeutics. The broad appeal was reflected in a high level of attendance at the session.

ACVP at Experimental Biology 2014

The 2014 Experimental Biology meeting will be held in San Diego, California, April 26 through 30. The ACVP/ASIP session is entitled “Precision Medicine Opportunities in Medical Research: Learning from the Canine Genome Project” and will be chaired by Dr Tim LaBranche, Pfizer Corporation, and Dr. Melissa Schutten, Genentech. Four speakers are participating in the session. Dr Phil Sponenberg, Professor of Pathology and Genetics, Virginia-Maryland Regional College of Veterinary Medicine, will address canine genetics in the context of changes associated with domestication and the correlation with pathological phenotypes. Dr Gustavo Aquirre, Professor of Medical Genetics and Ophthalmology, University of Pennsylvania College of Veterinary Medicine, will discuss the molecular basis of inherited blindness in dogs and humans. Dr Matthew Breen, Professor of Genetics, North Carolina State University College of Veterinary Medicine, will discuss comparative genomic analyses and cytogenetics of canine cancers. And Dr Jaime Modiano, Professor of Comparative Oncology, University of Minnesota College of Veterinary Medicine, will discuss the genetics of canine cancer immunology and immunotherapy. For those wishing to participate in the 2014 Experimental Biology meeting, the abstract submission deadline is November 8, 2013.