Granzyme B in aging and age-related pathologies.

Aging is a major risk factor for pathologies that manifest later in life. Much attention is devoted towards elucidating how prolonged environmental exposures and inflammation promote biological (accelerated) tissue aging. Granzymes, a family of serine proteases, are increasingly recognized for their emerging roles in biological aging and disease.

The complex web of FasL: cell type-specific roles in affecting and controlling acute graft-vs-host disease.

FasL has divergent roles in both causing graft-vs-host disease and preventing this condition, which depends on the immune cell type that expresses it.

Cytokine induction of HIF-1α during normoxia in A549 human lung carcinoma cells is regulated by STAT1 and JNK signalling pathways.

Hypoxia inducible factor-1ɑ (HIF-1ɑ) is the regulatory subunit of the HIF-1 transcription factor that is a regulator of cell physiological responses to hypoxia. However, the biological function and regulatory mechanisms controlling HIF-1α in normoxia are poorly understood. Here, we first examined the role of HIF-1α in the inflammatory activation of A549 human lung carcinoma cells in normoxia.

Dysbiosis of the Female Murine Gut Microbiome Exacerbates Neutrophil-mediated Vascular Allograft Injury by Affecting Immunoregulation by Acetate.

Background: The gut microbiota affects immune responses that cause organ transplant rejection, but the mechanisms by which this occurs remain poorly understood.

Methods: We have examined, in a murine model, how disruption of the gut microbiota with antibiotics early in life alters this microbial community later in life to affect immune responses that injure vascular allografts.

Results: Analysis of 16S rRNA and whole genome sequencing of the gut microbiota demonstrated that early life disruption of this microbial community with antibiotics caused a reduction in taxa and enzymatic genes involved in the synthesis of acetate, an immunoregulatory metabolite in mice and humans.

Prevention of vascular-allograft rejection by protecting the endothelial glycocalyx with immunosuppressive polymers.

Systemic immunosuppression for the mitigation of immune rejection after organ transplantation causes adverse side effects and constrains the long-term benefits of the transplanted graft. Here we show that protecting the endothelial glycocalyx in vascular allografts via the enzymatic ligation of immunosuppressive glycopolymers under cold-storage conditions attenuates the acute and chronic rejection of the grafts after transplantation in the absence of systemic immunosuppression. In syngeneic and allogeneic mice that received kidney transplants, the steric and immunosuppressive properties of the ligated polymers largely protected the transplanted grafts from ischaemic reperfusion injury, and from immune-cell adhesion and thereby immunocytotoxicity.

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies.

Immunologic Effects of the Microbiota in Organ Transplantation.

The microbiota is a community of microbes that colonizes body surfaces. It has many effects that influence immune activation and regulation. The success of organ transplantation is limited by rejection of grafts by the immune system so it is important to understand how immunologic responses are controlled in this setting.

Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1.

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions.

Disruption of the Gut Microbiota With Antibiotics Exacerbates Acute Vascular Rejection.

Background: The gut microbiota influences many immunological processes but how its disruption affects transplant rejection is poorly understood.

Methods: Interposition grafting of aortic segments was used to examine vascular rejection. The gut microbiota was disrupted in graft recipients using an antibiotic cocktail (ampicillin, vancomycin, metronidazole, neomycin sulfate) in their drinking water.

IL-6 expression is correlated with increased T-cell proliferation and survival in the arterial wall in giant cell arteritis.

Giant cell arteritis (GCA) is the most common vasculitis in adults affecting large and medium-sized arteries. IL-6 and T cell accumulation within the arterial wall contribute to the pathogenesis of GCA, and blockade of IL-6 activity is efficacious in its treatment. We examined the relationship between levels of IL-6 expression and immunological processes that control the expansion of T cells in GCA-positive temporal artery biopsies.

The Sialidase (Kdnase) Contributes to Cell Wall Integrity and Virulence in Amphotericin B-Treated Mice.

is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an -sialidase from that prefers the sialic acid substrate, 2-keto-3-deoxy-D--D--nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase in .

Graft-Derived IL-6 Amplifies Proliferation and Survival of Effector T Cells That Drive Alloimmune-Mediated Vascular Rejection.

Background: IL-6 is an inflammatory cytokine that controls effector T cell responses but the mechanisms by which it controls allogeneic immune responses and vascular rejection that leads to transplant arteriosclerosis (TA) are poorly understood.

Methods: We have examined the mechanism by which IL-6 contributes to the pathogenesis of vascular rejection and TA using a murine aortic interposition model of vascular rejection.

Results: The absence of IL-6 production from artery graft cells reduced the development of vascular rejection and arteriosclerotic thickening.

Immune-mediated vascular injury and dysfunction in transplant arteriosclerosis.

Solid organ transplantation is the only treatment for end-stage organ failure but this life-saving procedure is limited by immune-mediated rejection of most grafts. Blood vessels within transplanted organs are targeted by the immune system and the resultant vascular damage is a main contributor to acute and chronic graft failure. The vasculature is a unique tissue with specific immunological properties.

Bim regulates alloimmune-mediated vascular injury through effects on T-cell activation and death.

Objective: Bim is a proapoptotic Bcl-2 protein known to downregulate immune responses and to also be required for antigen-induced T-cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T-cell responses in a model of vascular rejection.

Positive feedback regulation of human inducible nitric-oxide synthase expression by Ras protein S-nitrosylation.

The production of nitric oxide (NO) by inducible NO synthase (iNOS) regulates many aspects of physiology and pathology. The expression of iNOS needs to be tightly regulated to balance the broad ranging properties of NO. We have investigated the feedback regulation of cytokine-induced iNOS expression by NO in human cells.

Induction of endothelial nitric oxide synthase expression by IL-17 in human vascular endothelial cells: implications for vascular remodeling in transplant vasculopathy.

IL-17 is a signature cytokine of Th17 cells, a recently described subset of effector CD4 T cells implicated in the development of several pathologies. We have examined the role of IL-17 in regulating endothelial NO synthase (eNOS) expression in human vascular endothelial cells (ECs) because of the key role of eNOS in determining the pathological outcome of immune-mediated vascular diseases. In cultured ECs, IL-17 increased expression of eNOS, eNOS phosphorylation at Ser(1177), and NO production.

Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP(S) expression.

The nature of inflammatory signals determines the outcome of T cell responses. However, little is known about how inflammatory cytokines provided to human CD8 T cells during activation affects their susceptibility to post-activation cell death. We have examined and compared the effects of the inflammatory cytokine IL-12, as well as the combination of IL-1, IL-6, and IL-23 (IL-1/6/23) on the susceptibility of primary human CD8 T cells to post-activation cell death.

CD155 on human vascular endothelial cells attenuates the acquisition of effector functions in CD8 T cells.

Objective: CD155 is a cell surface protein that has recently been described to exert immune regulatory functions. We have characterized the expression of CD155 on human vascular endothelial cells (ECs) and examined its role in the regulation of T-cell activation.

Methods And Results: CD155 was expressed on resting human vascular ECs and was upregulated in an interferon-γ (IFNγ)-dependent manner.

IFN-gamma primes intact human coronary arteries and cultured coronary smooth muscle cells to double-stranded RNA- and self-RNA-induced inflammatory responses by upregulating TLR3 and melanoma differentiation-associated gene 5.

Atherosclerosis of native coronary arteries and graft arteriosclerosis in transplanted hearts are characterized by activation of innate and adaptive immune responses. Nucleic acids generated by infections or cell death have been detected within arteriosclerotic lesions, and it is known that microbial and synthetic nucleic acids evoke inflammatory responses in cultured vascular cells. In this study, we report that model RNA, but not DNA, instigated robust cytokine and chemokine production from intact human coronary arteries containing both intrinsic vascular cells and resident/infiltrating leukocytes.

CXCL12 induction of inducible nitric oxide synthase in human CD8 T cells.

Background: We reported previously that inducible nitric oxide synthase (iNOS) expression in graft-infiltrating human T cells that is confined to the bystander population contributes to T- cell-mediated rejection of allograft arteries in a humanized mouse model. Herein we examine whether CXCL12, a chemokine thought to contribute to recruitment of bystander T cells, induces iNOS in human CD8 T cells.

Methods: Human CD8 T cells were treated with CXCL12 and iNOS expression was examined.

Interferon-gamma induces X-linked inhibitor of apoptosis-associated factor-1 and Noxa expression and potentiates human vascular smooth muscle cell apoptosis by STAT3 activation.

Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs.

Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature.

Inducible NO synthase (iNOS) in human T cells is implicated in the pathogenesis of graft arteriosclerosis. Here we analyze the regulation and role of iNOS in human peripheral blood T cells. Allogeneic endothelial cells (EC) or dermal fibroblasts induce iNOS mRNA and protein expression, as well as enzymatic activity in primary human CD8 T cells.

Detection of apoptosis in cardiovascular diseases.

The past decade has seen a surge in research devoted to understanding the role of cell death in the pathogenesis of various forms of cardiovascular disease. In particular, apoptosis has received much attention owing to the tightly regulated biochemical nature of this form of cell death and the realization of potential therapeutic opportunities. The current chapter describes a few of the more widely used protocols for detecting and quantifying apoptosis in cardiovascular tissues.

Granzyme B induces endothelial cell apoptosis and contributes to the development of transplant vascular disease.

Endothelial cell death induced by cytotoxic T cells is a key initiating event in the development of transplant vascular disease (TVD), the leading cause of late solid organ transplant failure. We studied the role of the granzyme B (GrB) pathwaye, which is one of the main mechanisms by which T cells induce apoptosis of allogeneic targets, in the pathogenesis of TVD. Granzyme B, in combination with perforin (pfn), induced apoptosis of cultured endothelial cells.

Granzyme B induces smooth muscle cell apoptosis in the absence of perforin: involvement of extracellular matrix degradation.

Objective: T cell-induced cytotoxicity, of which granzyme B is a key mediator, is believed to contribute to the pathogenesis of inflammatory vascular diseases. In this report, we investigate the mechanism of granzyme B-induced smooth muscle cell (SMC) death.

Methods And Results: The addition of purified granzyme B alone to cultured SMCs caused a significant reduction in cell viability.