Eric T. Clambey

Hypoxia-inducible factor-1 alpha–dependent induction of FoxP3 drives regulatory T-cell abundance and function during inflammatory hypoxia of the mucosa

Recent studies have demonstrated dramatic shifts in metabolic supply-and-demand ratios during inflammation, a process resulting in localized tissue hypoxia within inflammatory lesions (“inflammatory hypoxia”). As part of the adaptive immune response, T cells are recruited to sites of inflammatory hypoxia. Given the profound effects of hypoxia on gene regulation, we hypothesized that T-cell differentiation is controlled by hypoxia. To pursue this hypothesis, we analyzed the transcriptional consequences of ambient hypoxia (1% oxygen) on a broad panel of T-cell differentiation factors. Surprisingly, these studies revealed selective, robust induction of FoxP3, a key transcriptional regulator for regulatory T cells (Tregs). Studies of promoter binding or loss- and gain-of-function implicated hypoxia-inducible factor (HIF)-1α in inducing FoxP3. Similarly, hypoxia enhanced Treg abundance in vitro and in vivo. Finally, Treg-intrinsic HIF-1α was required for optimal Treg function and Hif1a–deficient Tregs failed to control T-cell–mediated colitis. These studies demonstrate that hypoxia is an intrinsic molecular cue that promotes FoxP3 expression, in turn eliciting potent anti-inflammatory mechanisms to limit tissue damage in conditions of reduced oxygen availability.

Disruption of the Murine Gammaherpesvirus 68 M1 Open Reading Frame Leads to Enhanced Reactivation from Latency

ABSTRACT Murine gammaherpesvirus 68 (γHV68, or MHV-68) is a genetically tractable, small animal model for the analysis of gammaherpesvirus pathogenesis. The γHV68 genome is colinear with the genomes of other sequence gammaherpesviruses, containing large blocks of conserved genes interspersed by a number of putative genes without clear homologs in the other gammaherpesviruses. One of these putative unique genes, the M1 open reading frame (ORF), exhibits sequence homology to a poxvirus serine protease inhibitor, SPI-1, as well as to another γHV68 gene, M3, which we have recently shown encodes an abundantly secreted chemokine binding protein. To assess the contribution of the M1 ORF to γHV68 pathogenesis, we have generated a recombinant γHV68 in which the M1 ORF has been disrupted through targeted insertion of alacZ expression cassette (M1.LacZ). Although M1.LacZ replicated normally in tissue culture, it exhibited decreased splenic titers at days 4 and 9 postinfection in both immunocompetent and immunodeficient mice. Despite decreased levels of acute virus replication, M1.LacZ established a latent infection comparable to wild-type (wt) γHV68, but exhibited an approximately fivefold increase in efficiency of reactivation from latency. M1.LacZ also caused severe vasculitis of the great elastic arteries in gamma interferon receptor (IFN-γR)-deficient mice with a frequency comparable to wt γHV68, but did not cause the mortality or splenic pathology observed with wt γHV68 infection of IFN-γR-deficient mice. Restoration of M1 ORF sequences into M1.LacZ (M1 marker rescue, or M1.MR) demonstrated that M1.LacZ phenotypic alterations in growth in vivo and latency were not due to the presence of additional mutations located elsewhere in the M1.LacZ genome. Generation of a second M1 mutant virus containing a deletion at the 5′ end of the M1 ORF (M1Δ511), but lacking the LacZ expression cassette, revealed the same latency phenotype observed with the M1.LacZ mutant. However, M1Δ511 was not attenuated for acute virus replication in the spleen. We conclude that (i) the induction of arteritis in γHV68-infected IFN-γR-deficient mice can occur in the absence of splenic pathology and mortality, (ii) replication during acute infection is not the primary determinant for the establishment of latent infection, and (iii) the M1 ORF, or a closely linked gene, encodes a gene product that functions to suppress virus reactivation.

Equilibrative nucleoside transporter 1 (ENT1) regulates postischemic blood flow during acute kidney injury in mice

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) - a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1-/- mice. Comprehensive examination of adenosine receptor-knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.

Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function.

Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies.

Non‐malignant clonal expansions of CD8+ memory T cells in aged individuals

Summary:  CD8+ T cells provide a major line of defense against intracellular pathogens. Upon encounter with antigen, CD8+ T cells go through three distinct phases involving proliferation, contraction, and differentiation to become eventually long‐lived CD8+ memory T cells. CD8+ memory T cells provide long‐term protection against infection by intracellular pathogens. CD8+ memory T‐cell proliferation and survival are regulated by many factors, including cytokines, and CD8+ memory T cells are stably maintained over a period of months to years. In aged humans and mice, however, there are significant alterations to the CD8+ memory T‐cell compartment with frequent development of monoclonal expansions of CD8+ memory T cells in healthy individuals. Interestingly, CD8+ clonal expansions are not malignant and do not progress to lymphomas, suggesting that these cells must still be under certain constraints. In this review, we discuss our current understanding of factors that contribute to and regulate these CD8+ clonal expansions as well as the impact of CD8+ clonal expansions on immune function of the aged. In addition, we discuss similarities and differences between CD8+ clonal expansions observed in humans and mice, and we postulate that CD8+ clonal expansions represent a spectrum of biological outcomes ranging from antigen‐driven to antigen‐independent phenomena.

Perioperative Organ Injury

Despite the fact that a surgical procedure may have been performed for the appropriate indication and in a technically perfect manner, patients are threatened by perioperative organ injury. For example, stroke, myocardial infarction, acute respiratory distress syndrome, acute kidney injury, or acute gut injury are among the most common causes for morbidity and mortality in surgical patients. In the current review, the authors discuss the pathogenesis of perioperative organ injury, and provide select examples for novel treatment concepts that have emerged over the past decade. Indeed, the authors are of the opinion that research to provide mechanistic insight into acute organ injury and identification of novel therapeutic approaches for the prevention or treatment of perioperative organ injury represent the most important opportunity to improve outcomes of anesthesia and surgery.

Dendritic cell subsets require cis-activation for cytotoxic CD8 T-cell induction

Dendritic cells (DCs) are required for the induction of cytotoxic T cells (CTL). In most tissues, including the lung, the resident DCs fall into two types, respectively expressing the integrin markers, CD103 and CD11b. The current supposition is that DC function is predetermined by lineage, designating the CD103+ DC as the major cross-presenting DC able to induce CTL. Here we show that Poly I:C (TLR3 agonist) or R848 (TLR7 agonist) do not activate all endogenous DCs. CD11b+ DCs can orchestrate a CTL response in vivo in the presence of TLR7 agonist but not TLR3 agonist, whereas CD103+ DCsrequire ligation of TLR3 for this purpose. This selectivity does not extend to antigen cross-presentation for T cell proliferation but is required for induction of cytotoxicity. Thus, we demonstrate that the ability of DCsto induce functional CTLs isspecific to the nature of the pathogen associated molecular pattern (PAMP) encountered by endogenous DC.

CD73+ regulatory T cells contribute to adenosine-mediated resolution of acute lung injury

Acute lung injury (ALI) is characterized by alveolar injury and uncontrolled inflammation. Since most cases of ALI resolve spontaneously, understanding the endogenous mechanisms that promote ALI resolution is important to developing effective therapies. Previous studies have implicated extracellular adenosine signaling in tissue adaptation and wound healing. Therefore, we hypothesized a functional contribution for the endogenous production of adenosine during ALI resolution. As a model, we administered intratracheal LPS and observed peak lung injury at 3 d, with resolution by d 14. Treatment with pegylated adenosine‐deaminase to enhance extracellular adenosine breakdown revealed impaired ALI resolution. Similarly, genetic deletion of cd73, the pacemaker for extracellular adenosine generation, was associated with increased mortality (0% wild‐type and 40% in cd73–/– mice; P<0.05) and failure to resolve ALI adequately. Studies of inflammatory cell trafficking into the lungs during ALI resolution revealed that regulatory T cells (Tregs) express the highest levels of CD73. While Treg numbers in cd73–/– mice were similar to controls, cd73‐deficient Tregs had attenuated immunosuppressive functions. Moreover, adoptive transfer of cd73‐deficient Tregs into Rag–/– mice emulated the observed phenotype in cd73–/– mice, while transfer of wild‐type Tregs was associated with normal ALI resolution. Together, these studies implicate CD73‐dependent adenosine generation in Tregs in promoting ALI resolution.—Ehrentraut, H., Clambey, E. T., McNamee, E. N., Brodsky, K. S., Ehrentraut, S. F., Poth, J. M., Riegel, A. K., Westrich, J. A., Colgan, S. P., Eltzschig, H. K. CD73+ regulatory T cells contribute to adenosine‐mediated resolution of acute lung injury. FASEB J. 27, 2207–2219 (2013). www.fasebj.org

Adora2b adenosine receptor engagement enhances regulatory T cell abundance during endotoxin-induced pulmonary inflammation.

Anti-inflammatory signals play an essential role in constraining the magnitude of an inflammatory response. Extracellular adenosine is a critical tissue-protective factor, limiting the extent of inflammation. Given the potent anti-inflammatory effects of extracellular adenosine, we sought to investigate how extracellular adenosine regulates T cell activation and differentiation. Adenosine receptor activation by a pan adenosine-receptor agonist enhanced the abundance of murine regulatory T cells (Tregs), a cell type critical in constraining inflammation. Gene expression studies in both naïve CD4 T cells and Tregs revealed that these cells expressed multiple adenosine receptors. Based on recent studies implicating the Adora2b in endogenous anti-inflammatory responses during acute inflammation, we used a pharmacologic approach to specifically activate Adora2b. Indeed, these studies revealed robust enhancement of Treg differentiation in wild-type mice, but not in Adora2b −/− T cells. Finally, when we subjected Adora2b-deficient mice to endotoxin-induced pulmonary inflammation, we found that these mice experienced more severe inflammation, characterized by increased cell recruitment and increased fluid leakage into the airways. Notably, Adora2b-deficient mice failed to induce Tregs after endotoxin-induced inflammation and instead had an enhanced recruitment of pro-inflammatory effector T cells. In total, these data indicate that the Adora2b adenosine receptor serves a potent anti-inflammatory role, functioning at least in part through the enhancement of Tregs, to limit inflammation.

CD8 T cell clonal expansions & aging: a heterogeneous phenomenon with a common outcome.

A highly diverse CD8 T cell repertoire is thought to be critical for maintaining appropriate immune defenses against a variety of pathogens. However, in many aged individuals, the diversity of T cell receptors is significantly reduced by the presence of large, monoclonal expansions of CD8 memory T cells. While ongoing research is focused on understanding the molecular alterations in these expansions, one major hurdle to this goal is the apparent heterogeneity of CD8 clonal expansions, which is apparent even in reductionist systems. In this review, we discuss current evidence that CD8 clonal expansions are a heterogeneous phenomenon, and our evolving understanding of what this heterogeneity tells us about CD8 memory T cell homeostasis and how it is altered in aged individuals.