Heather W. Stout-Delgado

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.

Aging Promotes Neutrophil-Induced Mortality by Augmenting IL-17 Production during Viral Infection

Morbidity and mortality associated with viral infections increase with age, although the underlying mechanisms are unclear. Here, we investigated whether aging alters inflammatory responses during systemic viral infection and thereby contributes to virus-induced death. We found that infection of aged mice with systemic herpes viruses led to rapid increases in serum IL-17, neutrophil activation, and mortality due to hepatocyte necrosis. In contrast, all young mice survived infection, displaying weaker IL-17 induction and neutrophil activation. Natural killer T (NKT) cells isolated from the livers of aged mice produced more IL-17 than did young cells, and adoptively transferred aged NKT cells induced liver injury in young mice impaired in viral control. Importantly, IL-17 neutralization or neutrophil depletion during viral infection reduced liver damage and prevented death of aged mice. These results demonstrate that, during systemic viral infection, aging alters the host-pathogen interaction to overproduce IL-17, contributing to liver injury and death.

NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages

Altered metabolism has been implicated in the pathogenesis of inflammatory diseases. NADPH oxidase 4 (NOX4), a source of cellular superoxide anions, has multiple biological functions that may be of importance in inflammation and in the pathogenesis of human metabolic diseases, including diabetes. However, the mechanisms by which NOX4-dependent metabolic regulation affect the innate immune response remain unclear. Here we show that deficiency of NOX4 resulted in reduced expression of carnitine palmitoyltransferase 1A (CPT1A), which is a key mitochondrial enzyme in the fatty acid oxidation (FAO) pathway. The reduced FAO resulted in less activation of the nucleotide-binding domain, leucine-rich-repeat-containing receptor (NLR), pyrin-domain-containing 3 (NLRP3) inflammasome in human and mouse macrophages. In contrast, NOX4 deficiency did not inhibit the activation of the NLR family, CARD-domain-containing 4 (NLRC4), the NLRP1 or the absent in melanoma 2 (AIM2) inflammasomes. We also found that inhibition of FAO by etomoxir treatment suppressed NLRP3 inflammasome activation. Furthermore, Nox4-deficient mice showed substantial reduction in caspase-1 activation and in interleukin (IL)-1β and IL-18 production, and there was improved survival in a mouse model of NLRP3-mediated Streptococcus pneumoniae infection. The pharmacologic inhibition of NOX4 by either GKT137831, which is currently in phase 2 clinical trials, or VAS-2870 attenuated NLRP3 inflammasome activation. Our results suggest that NOX4-mediated FAO promotes NLRP3 inflammasome activation.

Impaired NLRP3 Inflammasome Function in Elderly Mice during Influenza Infection Is Rescued by Treatment with Nigericin

The NLRP3 inflammasome is activated in the lung during influenza viral infection; however, the impact of aging on inflammasome function during influenza infection has not been examined. In this study, we show that elderly mice infected with a mouse-adapted strain of influenza produced lower levels of IL-1β during in vitro and in vivo infection. Dendritic cells from elderly mice exhibited decreased expression of ASC, NLRP3, and capase-1 but increased expression of pro–IL-1β, pro–IL-18, and pro–IL-33 compared with dendritic cells from young infected mice. Treatment with nigericin during influenza infection augmented IL-1β production, increased caspase-1 activity, and decreased morbidity and mortality in elderly mice. Our study demonstrates for the first time, to our knowledge, that during influenza viral infection, elderly mice have impaired NLRP3 inflammasome activity and that treatment with nigericin rescues NLRP3 activation in elderly hosts.

Nanoparticle delivery of mycophenolic acid upregulates PD-L1 on dendritic cells to prolong murine allograft survival.

Conventional immunosuppressive drug delivery requires high systemic drug levels to provide therapeutic benefit, but frequently results in toxic side effects. Novel drug delivery methods, such as FDA‐approved poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (NPs), are promising drug delivery platforms to reduce drug doses and minimize toxicity. Using murine models of skin transplantation, we investigated whether PLGA NPs would effectively deliver mycophenolic acid (MPA), a common clinical immunosuppressant, and avoid the toxicity of conventional drug delivery. We found that intermittent treatment with NPs encapsulated with MPA (NP‐MPA) resulted in a significant extension of allograft survival than intermittent conventional MPA treatment even though the concentration of MPA within NP‐MPA was a 1000‐fold lower than conventional drug. Importantly, recipients who were administered NP‐MPA intermittently avoided drug toxicity, whereas those treated with daily conventional drug manifested cytopenias. Dendritic cells (DCs) endocytosed NP‐MPA to upregulate programmed death ligand‐1 (PD‐L1) and displayed a decreased ability to prime alloreactive T cells. Importantly, the ability of NP‐MPA to promote allograft survival was partly PD‐L1 dependent. Collectively, this study indicates that NPs are potent drug delivery tools that extend allograft survival without drug toxicity.

The role of serpinb9/serine protease inhibitor 6 in preventing granzyme B–dependent hepatotoxicity†

Virally infected hepatocytes are resistant to cytotoxic lymphocyte killing by perforin‐dependent and granzyme‐dependent effector mechanisms. The present studies were designed to examine the role of serine protease inhibitor 6 (SPI‐6) in limiting granzyme B–dependent cytotoxic effector mechanisms in the liver. SPI‐6–specific small interfering RNA (siRNA) administration to C57Bl/6J (B6) mice elicited transient alanine aminotransferase (ALT) elevations that were not observed in either granzyme B–deficient B6 (B6.gzmb−/−) or natural killer (NK) cell–depleted B6 mice. When SPI‐6 expression was abolished by siRNA administration at the time of infection with a recombinant, replication‐deficient adenovirus [E1‐deleted adenovirus encoding β‐galactosidase (AdCMV‐LacZ)], earlier and dramatically increased, and earlier ALT elevations were observed in wild‐type B6 but not in B6.gzmb−/− or NK cell–depleted mice. When a 3‐fold higher dose of AdCMV‐LacZ was administered to B6 mice, the coadministration of SPI‐6 siRNA resulted in the early onset of lethal, acute liver failure. Of note, the accelerated clearance of AdCMV‐LacZ was observed in recipients of SPI‐6 siRNA. Conclusion: These results indicate that the regulated expression of SPI‐6 in hepatocytes during viral infection or following noninfectious causes of liver injury protects hepatocytes against excessively vigorous granzyme B–dependent killing but may also delay immune clearance of virally infected hepatocytes. (HEPATOLOGY 2007.)

Age-Dependent Susceptibility to Pulmonary Fibrosis Is Associated with NLRP3 Inflammasome Activation

Aging has been implicated in the development of pulmonary fibrosis, which has seen a sharp increase in incidence in those older than 50 years. Recent studies demonstrate a role for the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome and its regulated cytokines in experimental lung fibrosis. In this study, we tested the hypothesis that age-related NLRP3 inflammasome activation is an important predisposing factor in the development of pulmonary fibrosis. Briefly, young and aged wild-type and NLRP3(-/-) mice were subjected to bleomycin-induced lung injury. Pulmonary fibrosis was determined by histology and hydroxyproline accumulation. Bone marrow and alveolar macrophages were isolated from these mice. NLRP3 inflammasome activation was assessed by co-immunoprecipitation experiments. IL-1β and IL-18 production was measured by ELISA. The current study demonstrated that aged wild-type mice developed more lung fibrosis and exhibited increased morbidity and mortality after bleomycin-induced lung injury, when compared with young mice. Bleomycin-exposed aged NLRP3(-/-) mice had reduced fibrosis compared with their wild-type age-matched counterparts. Bone marrow-derived and alveolar macrophages from aged mice displayed higher levels of NLRP3 inflammasome activation and caspase-1-dependent IL-1β and IL-18 production, which was associated with altered mitochondrial function and increased production of reactive oxygen species. Our study demonstrated that age-dependent increases in alveolar macrophage mitochondrial reactive oxygen species production and NLRP3 inflammasome activation contribute to the development of experimental fibrosis.

Efficacy of a vaccine that links viral epitopes to flagellin in protecting aged mice from influenza viral infection.

Influenza vaccines are less effective in older people than younger people. This impaired ability to protect older people from influenza viral lung infection has important implications as older people suffer a higher morbidity and mortality from influenza viral lung infection than younger people. Therefore, the development of novel effective vaccines that induce protection from influenza viral infections in older people are urgently needed. We had previously shown that direct linking the TLR5 activator, flagellin, to viral peptides induces effective immunity to viral antigens in young mice and people, respectively. In this study, we tested the efficacy of this vaccine platform with the hemagglutinin peptide of the influenza A strain virus (vaccine denoted as STF2.HA1-2) in protecting aged mice from subsequent influenza viral lung infection. We found that a 3.0 μg dose of the vaccine was effective in reducing mortality and increasing clinical well-being during influenza viral lung infection in aged mice. However, this effect was inferior to the response induced in young mice. Defects in the adaptive immune system but not the innate immune system were associated with this reduced effectiveness of the vaccine with aging. Our results indicate that the STF2.HA1-2 vaccine is effective in protecting aged hosts from influenza lung infection, although defects in the adaptive immune system with aging may limit the effectiveness of this vaccine in older people.

Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses

It is well established that granzymes A and B play a role in CTL killing of target cells by the perforin-dependent granule exocytosis pathway. The functions of multiple additional granzymes expressed in CTL are less well defined. In the present studies, CTL generated from mice deficient in dipeptidyl peptidase 1 (DPP1) were used to investigate the contribution of granzyme C to CTL killing of allogeneic target cells. DPP1 is required for activation of granzymes A and B by proteolytic removal of their N-terminal dipeptide prodomains while a significant portion of granzyme C is processed normally in the absence of DPP1. Cytotoxicity of DPP1−/− CTL generated in early (5-day) MLC in vitro and in peritoneal exudate cells 5 days after initial allogeneic sensitization in vivo was significantly impaired compared with wild-type CTL. Following 3 days of restimulation with fresh allogeneic stimulators however, cytotoxicity of these DPP1−/− effector cells was comparable to that of wild-type CTL. Killing mediated by DPP1−/− CTL following restimulation was rapid, perforin dependent, Fas independent and associated with early mitochondrial injury, phosphatidyl serine externalization, and DNA degradation, implicating a granzyme-dependent apoptotic pathway. The increased cytotoxicity of DPP1−/− CTL following restimulation coincided with increased expression of granzyme C. Moreover, small interfering RNA inhibition of granzyme C expression during restimulation significantly decreased cytotoxicity of DPP1−/− but not wild-type CTL. These results indicate that during late primary alloimmune responses, granzyme C can support CTL-mediated killing by the granule exocytosis pathway in the absence of functional granzymes A or B.

Intrahepatic Lymphocyte Expression of Dipeptidyl Peptidase I-Processed Granzyme B and Perforin Induces Hepatocyte Expression of Serine Proteinase Inhibitor 6 (Serpinb9/SPI-6)

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-α treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-α stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-α-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.