Susan E. Murray

Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor-2

The actions of corticotropin-releasing hormone (Crh), a mediator of endocrine and behavioural responses to stress, and the related hormone urocortin (Ucn) are coordinated by two receptors, Crhr1 (encoded by Crhr) and Crhr2 (refs 4,5). These receptors may exhibit distinct functions due to unique tissue distribution and pharmacology. Crhr-null mice have defined central functions for Crhr1 in anxiety and neuroendocrine stress responses. Here we generate Crhr2−/− mice and show that Crhr2 supplies regulatory features to the hypothalamic-pituitary-adrenal axis (HPA) stress response. Although initiation of the stress response appears to be normal, Crhr2−/− mice show early termination of adrenocorticotropic hormone (Acth) release, suggesting that Crhr2 is involved in maintaining HPA drive. Crhr2 also appears to modify the recovery phase of the HPA response, as corticosterone levels remain elevated 90 minutes after stress in Crhr2−/− mice. In addition, stress-coping behaviours associated with dearousal are reduced in Crhr2–/– mice. We also demonstrate that Crhr2 is essential for sustained feeding suppression (hypophagia) induced by Ucn. Feeding is initially suppressed in Crhr2−/− mice following Ucn, but Crhr2−/− mice recover more rapidly and completely than do wild-type mice. In addition to central nervous system effects, we found that, in contrast to wild-type mice, Crhr2−/− mice fail to show the enhanced cardiac performance or reduced blood pressure associated with systemic Ucn, suggesting that Crhr2 mediates these peripheral haemodynamic effects. Moreover, Crhr2−/− mice have elevated basal blood pressure, demonstrating that Crhr2 participates in cardiovascular homeostasis. Our results identify specific responses in the brain and periphery that involve Crhr2.

Animal models of CRH excess and CRH receptor deficiency display altered adaptations to stress.

This review highlights new information gained from studies using recently developed animal models that harbor specific alterations in corticotropin-releasing hormone (CRH) pathways. We discuss features of a transgenic mouse model of chronic CRH overexpression and two mouse models that lack either CRH receptor type 1 (CRH-R1) or type 2 (CRH-R2). Together these models provide new insights into the role of CRH pathways in promoting stability through adaptive changes, a process known as allostasis.

NF-κB–inducing kinase plays an essential T cell–intrinsic role in graft-versus-host disease and lethal autoimmunity in mice

NF-κB–inducing kinase (NIK) is an essential upstream kinase in noncanonical NF-κB signaling. NIK-dependent NF-κB activation downstream of several TNF receptor family members mediates lymphoid organ development and B cell homeostasis. Peripheral T cell populations are normal in the absence of NIK, but the role of NIK during in vivo T cell responses to antigen has been obscured by other developmental defects in NIK-deficient mice. Here, we have identified a T cell–intrinsic requirement for NIK in graft-versus-host disease (GVHD), wherein NIK-deficient mouse T cells transferred into MHC class II mismatched recipients failed to cause GVHD. Although NIK was not necessary for antigen receptor signaling, it was absolutely required for costimulation through the TNF receptor family member OX40 (also known as CD134). When we conditionally overexpressed NIK in T cells, mice suffered rapid and fatal autoimmunity characterized by hyperactive effector T cells and poorly suppressive Foxp3(+) Tregs. Together, these data illuminate a critical T cell–intrinsic role for NIK during immune responses and suggest that its tight regulation is critical for avoiding autoimmunity.

A Genetic Model of Stress Displays Decreased Lymphocytes and Impaired Antibody Responses Without Altered Susceptibility to Streptococcus pneumoniae

Stress pathways affect immune function, the most notable of these pathways being activation of the hypothalamic-pituitary-adrenal (HPA) axis. Although HPA activation has generally been relegated to an immunosuppressive role, recent evidence suggests that stress and HPA activation can be immunoenhancing in certain situations. To investigate specific effects of stress on immune function, we used a genetic model of chronic stress wherein transgenic mice overexpress corticotropin-releasing hormone (CRH), a primary mediator of the stress response. In these mice, CRH is overproduced in the brain, leading to chronic activation of the HPA axis. We found that CRH-transgenic mice have decreased leukocyte numbers in lymphoid compartments, with preferential loss of B lymphocytes. They also exhibit decreased Ab production and impaired isotype switching in response to immunization with a thymus-dependent Ag, phosphocholine-keyhole limpet hemocyanin. Despite these deficits, immunization protected CRH-transgenic and wild-type mice equally well against lethal challenge with Streptococcus pneumoniae, an encapsulated Gram-positive bacterium known to require Ab-mediated opsonization for clearance. While IgG responses are severely depressed in these mice, IgM titers are only modestly decreased. This fairly robust IgM response may be sufficient to protect against S. pneumoniae. Additionally, while total leukocyte numbers are decreased in these mice, neutrophil numbers are increased. This increase in number of neutrophils may compensate for the depressed IgG response, allowing adequate host defense during chronic stress.

OX40-Mediated Differentiation to Effector Function Requires IL-2 Receptor Signaling but Not CD28, CD40, IL-12Rβ2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-γ. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-γ in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-γ secretion.

IL-7 enhances Ag-specific human T cell response by increasing expression of IL-2R α and γ chains

Abstract Interleukin-7 has demonstrated potent enhancing effects on the growth and differentiation of several immature cell types, including thymocytes, and on survival of resting and antigen activated T cells. In this study, we evaluated the effects of IL-7 on post-thymic antigen-specific T cells from human blood. IL-7 was found to enhance proliferation responses and IFN-γ secretion of myelin or recall Ag-specific Th1 cells through the selective up-regulation of the IL-2Rα and γ but not β chains in both an Ag-dependent and Ag-independent manner, but did not affect monocytes, B cells, or NK cells. These functions of IL-7 enhanced the detection of Th1 but not Th2 cell frequency by >2.5 fold, and promoted selection of Ag-specific Th1 cells by the limiting dilution method. Moreover, IL-7 pretreatment conferred increased resistance of CD4+ T cells to CD8+ cell lysis. These studies demonstrate that IL-7 promotes the growth and survival of circulating Ag-specific human Th1 cells through a mechanism that probably involves the γc common receptor for IL-2 family members that includes IL-7.

A Cell-Intrinsic Requirement for NF-κB–Inducing Kinase in CD4 and CD8 T Cell Memory

NF-κB–inducing kinase [(NIK), MAP3K14] is an essential kinase linking a subset of TNFR family members to the noncanonical NF-κB pathway. To assess the cell-intrinsic role of NIK in murine T cell function, we generated mixed bone marrow chimeras using bone marrow from NIK knockout (KO) and wild-type (WT) donor mice and infected the chimeras with lymphocytic choriomeningitis virus (LCMV). The chimeras possess an apparently normal immune system, including a mixture of NIK KO and WT T cells, and the virus was cleared normally. Comparison of the NIK KO and WT CD4 and CD8 T cell responses at 8 d post infection revealed modest but significant differences in the acute response. In both CD4 and CD8 compartments, relatively fewer activated (CD44hi) NIK KO T cells were present, but within the CD44hi population, a comparable percentage of the activated cells produced IFN-γ in response to ex vivo stimulation with antigenic LCMV peptides, although IL-7R expression was reduced in the NIK KO CD8 T cells. Assessment of the LCMV-specific memory at 65 d post infection revealed many more LCMV-specific WT memory T cells than NIK KO memory T cells in both the CD4 and the CD8 compartments, although the small number of surviving NIK KO memory T cells responded to secondary challenge with virus. These results demonstrate a cell-intrinsic requirement for NIK in the generation and/or maintenance of memory T cells in response to acute viral infection.

Cell-Intrinsic Role for NF-kappa B-Inducing Kinase in Peripheral Maintenance but Not Thymic Development of Foxp3 + Regulatory T Cells in Mice

NF-κB inducing kinase (NIK, MAP3K14) is a key signaling molecule in non-canonical NF-κB activation, and NIK deficient mice have been instrumental in deciphering the immunologic role of this pathway. Global ablation of NIK prevents lymph node development, impairs thymic stromal development, and drastically reduces B cells. Despite altered thymic selection, T cell numbers are near normal in NIK deficient mice. The exception is CD4+ regulatory T cells (Tregs), which are reduced in the thymus and periphery. Defects in thymic stroma are known to contribute to impaired Treg generation, but whether NIK also plays a cell intrinsic role in Tregs is unknown. Here, we compared intact mice with single and mixed BM chimeric mice to assess the intrinsic role of NIK in Treg generation and maintenance. We found that while NIK expression in stromal cells suffices for normal thymic Treg development, NIK is required cell-intrinsically to maintain peripheral Tregs. In addition, we unexpectedly discovered a cell-intrinsic role for NIK in memory phenotype conventional T cells that is masked in intact mice, but revealed in BM chimeras. These results demonstrate a novel role for NIK in peripheral regulatory and memory phenotype T cell homeostasis.

Overproduction of corticotropin-releasing hormone blocks germinal center formation: role of corticosterone and impaired follicular dendritic cell networks

Corticotropin-releasing hormone (CRH) is a central mediator in the response to stress, coordinating behavioral, autonomic and neuroendocrine activation. CRH overproduction is implicated in several affective disorders, including major depression, panic-anxiety disorder and anorexia--diseases also associated with altered immune function. We investigated the link between CRH overdrive and immune function using CRH transgenic mice. Following immunization, CRH transgenic mice fail to form germinal centers; chronic glucocorticoid administration recapitulates this effect in wild-type mice. Regulation of germinal centers by glucocorticoids appears to be mediated, in part, through effects on follicular dendritic cells (FDC), providing a novel mechanism by which CRH dysregulation may significantly impair humoral immune responses.

Fibroblast-adapted human CMV vaccines elicit predominantly conventional CD8 T cell responses in humans

Cytomegalovirus (CMV)-based vaccines have shown remarkable efficacy in the rhesus macaque model of acquired immune deficiency syndrome, enabling 50% of vaccinated monkeys to clear a subsequent virulent simian immunodeficiency virus challenge. The protective vaccine elicited unconventional CD8 T cell responses that were entirely restricted by MHC II or the nonclassical MHC I molecule, MHC-E. These unconventional responses were only elicited by a fibroblast-adapted rhesus CMV vector with limited tissue tropism; a repaired vector with normal tropism elicited conventional responses. Testing whether these unusual protective CD8 T responses could be elicited in humans requires vaccinating human subjects with a fibroblast-adapted mutant of human CMV (HCMV). In this study, we describe the CD8 T cell responses of human subjects vaccinated with two fibroblast-adapted HCMV vaccines. Most responses were identified as conventional classically MHC I restricted, and we found no evidence for MHC II or HLA-E restriction. These results indicate that fibroblast adaptation alone is unlikely to explain the unconventional responses observed in macaques.