Wendy E. Walker

The circadian clock controls toll-like receptor 9-mediated innate and adaptive immunity

Circadian rhythms refer to biologic processes that oscillate with a period of ~24 hr. These rhythms are sustained by a molecular clock and provide a temporal matrix that ensures the coordination of homeostatic processes with the periodicity of environmental challenges. We demonstrate the circadian molecular clock controls the expression and function of Toll-like receptor 9 (TLR9). In a vaccination model using TLR9 ligand as adjuvant, mice immunized at the time of enhanced TLR9 responsiveness presented weeks later with an improved adaptive immune response. In a TLR9-dependent mouse model of sepsis, we found that disease severity was dependent on the timing of sepsis induction, coinciding with the daily changes in TLR9 expression and function. These findings unveil a direct molecular link between the circadian and innate immune systems with important implications for immunoprophylaxis and immunotherapy.

Absence of Innate MyD88 Signaling Promotes Inducible Allograft Acceptance

Prior experimental strategies to induce transplantation tolerance have focused largely on modifying adaptive immunity. However, less is known concerning the role of innate immune signaling in the induction of transplantation tolerance. Using a highly immunogenic murine skin transplant model that resists transplantation tolerance induction when innate immunity is preserved, we show that absence of MyD88, a key innate Toll like receptor signal adaptor, abrogates this resistance and facilitates inducible allograft acceptance. In our model, absence of MyD88 impairs inflammatory dendritic cell responses that reduce T cell activation. This effect increases T cell susceptibility to suppression mediated by CD4+CD25+ regulatory T cells. Therefore, this study provides evidence that absence of MyD88 promotes inducible allograft acceptance and implies that inhibiting innate immunity may be a potential, clinically relevant strategy to facilitate transplantation tolerance.

Aging Impairs IFN Regulatory Factor 7 Up-Regulation in Plasmacytoid Dendritic Cells during TLR9 Activation

Plasmacytoid dendritic cells (pDCs) are innate sensors that produce IFN-α in response to viral infections. Determining how aging alters the cellular and molecular function of these cells may provide an explanation of increased susceptibility of older people to viral infections. Hence, we examined whether aging critically impairs pDC function during infection with HSV-2, a viral pathogen that activates TLR9. We found that impaired IFN-α production by aged murine pDCs led to impaired viral clearance with aging. Upon TLR9 activation, aged pDCs displayed defective up-regulation of IFN-regulatory factor 7, a key adaptor in the type I IFN pathway, as compared with younger counterparts. Aged pDCs had more oxidative stress, and reducing oxidative stress in aged pDCs partly recovered the age-induced IFN-α defect during TLR9 activation. In sum, aging impairs the type I IFN pathway in pDCs, and this alteration may contribute to the increased susceptibility of older people to certain viral infections.

Dual Signaling of MyD88 and TRIF Is Critical for Maximal TLR4-Induced Dendritic Cell Maturation

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-β (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.

Nurine myeloid dendritic cell‐dependent toll‐like receptor immunity is preserved with aging

The immune response is the result of the interplay between innate and adaptive immunity, yet the impact of aging on this interaction is unclear. Addressing this fundamental question will be critical for the development of effective vaccines for the rapidly rising older subpopulation that manifests increased prevalence of malignancies and infections. Therefore, we undertook the current study to investigate whether aging impairs toll‐like receptor (TLR) function in myeloid dendritic cells and whether this leads to reduced T‐cell priming. Our results demonstrate that innate TLR immune priming function of myeloid bone marrow derived and splenic dendritic cells (DC) is preserved with aging using both allogeneic and infectious murine experimental systems. In contrast, aging impairs in vitro and in vivo intrinsic T‐cell function. Therefore, our results demonstrate that myeloid DCs manifest preserved TLR‐mediated immune responses with aging. However, aging critically impairs intrinsic adaptive T‐cell function.

Immunity’s fourth dimension: approaching the circadian-immune connection

The circadian system ensures the generation and maintenance of self-sustained ~24-h rhythms in physiology that are linked to internal and environmental changes. In mammals, daily variations in light intensity and other cues are integrated by a hypothalamic master clock that conveys circadian information to peripheral molecular clocks that orchestrate physiology. Multiple immune parameters also vary throughout the day and disruption of circadian homeostasis is associated with immune-related disease. Here, we discuss the molecular links between the circadian and immune systems and examine their outputs and disease implications. Understanding the mechanisms that underlie circadian-immune crosstalk may prove valuable for devising novel prophylactic and therapeutic interventions.

Aging Augments IL-17 T cell Alloimmune Responses

As increasing numbers of elderly patients require solid organ transplantation, the need to better understand how aging modifies alloimmune responses increases. Here, we examined whether aged mice exhibit augmented, donor‐specific memory responses prior to transplantation. We found that elevated donor‐specific IL‐17, but not IFN‐γ, responses were observed in aged mice compared to young mice prior to transplantation. Further characterization of the heightened IL‐17 alloimmune response with aging demonstrated that memory CD4+ T cells were required. Reduced IL‐2 alloimmune responses with age contributed to the elevated IL‐17 phenotype in vitro, and treatment with an anti‐IL‐17 antibody delayed the onset of acute allograft rejection. In conclusion, aging leads to augmented, donor‐specific IL‐17 immune responses that are important for the timing of acute allograft rejection in aged recipients. IL‐17 targeting therapies may be useful for averting transplant rejection responses in older transplant recipients.

ELF4 is critical for induction of type I interferon and the host antiviral response

Induction of type I interferon is a central event of innate immunity, essential for host defense. Here we report that the transcription factor ELF4 is induced by type I interferon and upregulates interferon expression in a feed-forward loop. ELF4 deficiency leads to reduced interferon production, resulting in enhanced susceptibility to West Nile virus encephalitis in mice. After viral infection, ELF4 is recruited by STING, interacts with and is activated by the MAVS-TBK1 complex, and translocates into the nucleus to bind interferon promoters. Cooperative binding with ELF4 increases the binding affinity of interferon regulatory factors IRF3 and IRF7, which is mediated by EICE elements. Thus, in addition to identifying a regulator of innate immune signaling, we uncovered a role for EICE elements in interferon transactivation.

Neonatal B Cells Suppress Innate Toll-Like Receptor Immune Responses and Modulate Alloimmunity

It has been known for decades that neonates are susceptible to transplant tolerance, but the immunological mechanisms involved remain to be fully elucidated. Recent evidence indicates that the maturation state of DCs responding to an allograft may have a profound impact on whether immunity or tolerance ensues. Given that TLR activation is a key process leading to DC maturation, we hypothesized that DCs from neonates have defective TLR immune responses. Contrary to our hypothesis, we found that murine neonatal DCs demonstrated enhanced TLR responses in comparison to adult counterparts in vitro. However, we found that neonatal B cells possess unique immunoregulatory functions as they impaired DC responses to TLR activation in an IL-10-dependent fashion. Functionally, we demonstrated that TLR-activated neonatal, but not adult, B cells impaired Th1, but not Th2, T cell alloimmune responses in vitro and in vivo, in models of alloimmune priming and allotransplantation. We conclude that neonatal B cells possess unique immunoregulatory properties that inhibit DC function and modulate alloimmunity in our murine experimental systems.

MKK3 regulates mitochondrial biogenesis and mitophagy in sepsis-induced lung injury.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.