Altered transcriptional responses in the lungs of aged mice after influenza infection.

BACKGROUND: Influenza causes a serious infection in older individuals who are at the highest risk for mortality from this virus. Changes in the immune system with age are well known. This study used transcriptomic analysis to evaluate how aging specifically affects the functional host response to influenza in the lung. Adult (12-16 weeks) and aged (72-76 weeks) mice were infected with influenza and lungs were processed for RNA analysis. RESULTS: Older mice demonstrated a delayed anti-viral response on the level of transcription compared to adults, similar to the immunologic responses measured in prior work. The transcriptional differences, however, were evident days before observable differences in the protein responses described previously. The transcriptome response to influenza in aged mice was dominated by immunoglobulin genes and B cell markers compared to adult animals, suggesting immune dysregulation. Despite these differences, both groups of mice had highly similar transcriptional responses involving non-immune genes one day after inoculation and T cell genes during resolution. CONCLUSIONS: These results define a delayed and dysregulated immune response in the lungs of aged mice infected with influenza. The findings implicate B cells and immunoglobulins as markers or mechanisms of immune aging. In addition to discovering new therapeutic targets, the findings underscore the value of transcription studies and network analysis to characterize complex biological processes, and serve as a model to analyze the susceptibility of the elderly to infectious agents.

Glycolysis Inhibition Induces Functional and Metabolic Exhaustion of CD4+ T Cells in Type 1 Diabetes.

In Type 1 Diabetes (T1D), CD4+ T cells initiate autoimmune attack of pancreatic islet ß cells. Importantly, bioenergetic programs dictate T cell function, with specific pathways required for progression through the T cell lifecycle. During activation, CD4+ T cells undergo metabolic reprogramming to the less efficient aerobic glycolysis, similarly to highly proliferative cancer cells. In an effort to limit tumor growth in cancer, use of glycolytic inhibitors have been successfully employed in preclinical and clinical studies. This strategy has also been utilized to suppress T cell responses in autoimmune diseases like Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA). However, modulating T cell metabolism in the context of T1D has remained an understudied therapeutic opportunity. In this study, we utilized the small molecule PFK15, a competitive inhibitor of the rate limiting glycolysis enzyme 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 3 (PFKFB3). Our results confirmed PFK15 inhibited glycolysis utilization by diabetogenic CD4+ T cells and reduced T cell responses to ß cell antigen in vitro. In an adoptive transfer model of T1D, PFK15 treatment delayed diabetes onset, with 57% of animals remaining euglycemic at the end of the study period. Protection was due to induction of a hyporesponsive T cell phenotype, characterized by increased and sustained expression of the checkpoint molecules PD-1 and LAG-3 and downstream functional and metabolic exhaustion. Glycolysis inhibition terminally exhausted diabetogenic CD4+ T cells, which was irreversible through restimulation or checkpoint blockade in vitro and in vivo. In sum, our results demonstrate a novel therapeutic strategy to control aberrant T cell responses by exploiting the metabolic reprogramming of these cells during T1D. Moreover, the data presented here highlight a key role for nutrient availability in fueling T cell function and has implications in our understanding of T cell biology in chronic infection, cancer, and autoimmunity.

A Noncanonical Role for Plasminogen Activator Inhibitor Type 1 in Obesity-Induced Diabetes.

Obesity is a major risk factor for type 2 diabetes because of chronic hepatic inflammation and resultant insulin resistance. Hepatocyte growth factor (HGF) is responsible for resetting hepatic homeostasis after injury following activation by urokinase-type plasminogen activator (u-PA; encoded by the PLAU gene). Plasminogen activator inhibitor type-1 (PAI-1; encoded by the SERPINE1 gene), a u-PA inhibitor and antifibrinolytic agent, is often elevated in obesity and is linked to cardiovascular events. We hypothesized that, in addition to its role in preventing fibrinolysis, elevated PAI-1 inhibits HGF's activation by u-PA and the resultant anti-inflammatory and hepatoprotective properties. Wild-type and PAI-1 knockout (KO) mice on a high-fat diet both became significantly heavier than lean controls; however, the obese KO mice demonstrated improved glucose metabolism compared with wild-type mice. Obese KO mice also exhibited an increase in conversion of latent single-chain HGF to active two-chain HGF, coinciding with an increase in the phosphorylation of the HGF receptor (HGFR or MET, encoded by the MET gene), as well as dampened inflammation. These results strongly suggest that, in addition to its other functions, PAI-mediated inhibition of HGF activation prohibits the resolution of inflammation in the context of obesity-induced type 2 diabetes.

Lymphocyte Activation Gene-3 Maintains Mitochondrial and Metabolic Quiescence in Naive CD4+ T Cells.

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics, we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo, LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3-/- CD4+ T cells, solidifying that LAG-3 controls these processes. Lag3-/- CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation, enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally, enhancement of STAT5 activation, as a result of LAG-3 deficiency, contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses.

Reactive Oxygen Species and Their Implications on CD4+ T Cells in Type 1 Diabetes.

SIGNIFICANCE: Previous work has indicated that type 1 diabetes (T1D) pathology is highly driven by reactive oxygen species (ROS). One way in which ROS shape the autoimmune response demonstrated in T1D is by promoting CD4+ T cell activation and differentiation. As CD4+ T cells are a significant contributor to pancreatic ß cell destruction in T1D, understanding how ROS impact their development, activation, and differentiation is critical. Recent Advances: CD4+ T cells themselves generate ROS via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and electron transport chain activity. Moreover, T cells can also be exposed to exogenous ROS generated by other immune cells (e.g., macrophages and dendritic cells) and ß cells. Genetically modified animals and ROS inhibitors have demonstrated that ROS blockade during activation results in CD4+ T cell hyporesponsiveness and reduced diabetes incidence. Critical Issues and Future Directions: Although the majority of studies with regard to T1D and CD4+ T cells have been done to examine the influence of redox on CD4+ T cell activation, this is not the only circumstance in which a T cell can be impacted by redox. ROS and redox have also been shown to play roles in CD4+ T cell-related tolerogenic mechanisms, including thymic selection and regulatory T cell-mediated suppression. However, the effect of these mechanisms with respect to T1D pathogenesis remains elusive. Therefore, pursuing these avenues may provide valuable insight into the global role of ROS and redox in autoreactive CD4+ T cell formation and function.

Treatment with a Catalytic Superoxide Dismutase (SOD) Mimetic Improves Liver Steatosis, Insulin Sensitivity, and Inflammation in Obesity-Induced Type 2 Diabetes.

Oxidative stress and persistent inflammation are exaggerated through chronic over-nutrition and a sedentary lifestyle, resulting in insulin resistance. In type 2 diabetes (T2D), impaired insulin signaling leads to hyperglycemia and long-term complications, including metabolic liver dysfunction, resulting in non-alcoholic fatty liver disease (NAFLD). The manganese metalloporphyrin superoxide dismustase (SOD) mimetic, manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnP), is an oxidoreductase known to scavenge reactive oxygen species (ROS) and decrease pro-inflammatory cytokine production, by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. We hypothesized that targeting oxidative stress-induced inflammation with MnP would assuage liver complications and enhance insulin sensitivity and glucose tolerance in a high-fat diet (HFD)-induced mouse model of T2D. During 12 weeks of feeding, we saw significant improvements in weight, hepatic steatosis, and biomarkers of liver dysfunction with redox modulation by MnP treatment in HFD-fed mice. Additionally, MnP treatment improved insulin sensitivity and glucose tolerance, while reducing serum insulin and leptin levels. We attribute these effects to redox modulation and inhibition of hepatic NF-κB activation, resulting in diminished ROS and pro-inflammatory cytokine production. This study highlights the importance of controlling oxidative stress and secondary inflammation in obesity-mediated insulin resistance and T2D. Our data confirm the role of NF-κB-mediated inflammation in the development of T2D, and demonstrate the efficacy of MnP in preventing the progression to disease by specifically improving liver pathology and hepatic insulin resistance in obesity.

Reactive oxygen species are required for driving efficient and sustained aerobic glycolysis during CD4+ T cell activation.

The immune system is necessary for protecting against various pathogens. However, under certain circumstances, self-reactive immune cells can drive autoimmunity, like that exhibited in type 1 diabetes (T1D). CD4+ T cells are major contributors to the immunopathology in T1D, and in order to drive optimal T cell activation, third signal reactive oxygen species (ROS) must be present. However, the role ROS play in mediating this process remains to be further understood. Recently, cellular metabolic programs have been shown to dictate the function and fate of immune cells, including CD4+ T cells. During activation, CD4+ T cells must transition metabolically from oxidative phosphorylation to aerobic glycolysis to support proliferation and effector function. As ROS are capable of modulating cellular metabolism in other models, we sought to understand if blocking ROS also regulates CD4+ T cell activation and effector function by modulating T cell metabolism. To do so, we utilized an ROS scavenging and potent antioxidant manganese metalloporphyrin (MnP). Our results demonstrate that redox modulation during activation regulates the mTOR/AMPK axis by maintaining AMPK activation, resulting in diminished mTOR activation and reduced transition to aerobic glycolysis in diabetogenic splenocytes. These results correlated with decreased Myc and Glut1 upregulation, reduced glucose uptake, and diminished lactate production. In an adoptive transfer model of T1D, animals treated with MnP demonstrated delayed diabetes progression, concurrent with reduced CD4+ T cell activation. Our results demonstrate that ROS are required for driving and sustaining T cell activation-induced metabolic reprogramming, and further support ROS as a target to minimize aberrant immune responses in autoimmunity.

Splenectomy promotes indirect elimination of intraocular tumors by CD8+ T cells that is associated with IFNγ- and Fas/FasL-dependent activation of intratumoral macrophages.

Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8(+) T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1(-/-) mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mϕ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mϕs limited CD8 T cell-mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mϕs by CD8(+) T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8(+) T cells and Mϕs to limit the immunosurveillance of intraocular tumors.

Mn porphyrin regulation of aerobic glycolysis: implications on the activation of diabetogenic immune cells.

AIMS: The immune system is critical for protection against infections and cancer, but requires scrupulous regulation to limit self-reactivity and autoimmunity. Our group has utilized a manganese porphyrin catalytic antioxidant (MnTE-2-PyP(5+), MnP) as a potential immunoregulatory therapy for type 1 diabetes. MnP has previously been shown to modulate diabetogenic immune responses through decreases in proinflammatory cytokine production from antigen-presenting cells and T cells and to reduce diabetes onset in nonobese diabetic mice. However, it is unclear whether or not MnP treatment can act beyond the reported inflammatory mediators. Therefore, the hypothesis that MnP may be affecting the redox-dependent bioenergetics of diabetogenic splenocytes was investigated. RESULTS: MnP treatment enhanced glucose oxidation, reduced fatty acid oxidation, but only slightly decreased overall oxidative phosphorylation. These alterations occurred because of increased tricarboxylic acid cycle aconitase enzyme efficiency and were not due to changes in mitochondrial abundance. MnP treatment also displayed decreased aerobic glycolysis, which promotes activated immune cell proliferation, as demonstrated by reduced lactate production and glucose transporter 1 (Glut1) levels and inactivation of key signaling molecules, such as mammalian target of rapamycin, c-myc, and glucose-6-phosphate dehydrogenase. INNOVATION: This work highlights the importance of redox signaling by demonstrating that modulation of reactive oxygen species can supplant complex downstream regulation, thus affecting metabolic programming toward aerobic glycolysis. CONCLUSION: MnP treatment promotes metabolic quiescence, impeding diabetogenic autoimmune responses by restricting the metabolic pathways for energy production and affecting anabolic processes necessary for cell proliferation.

Influence of CD8+ T regulatory cells on intraocular tumor development.

The interior of the eye, or uvea, is a site of immune privilege where certain immune responses are attenuated or completely excluded to protect non-regenerating tissues essential for vision. One consequence of this immunoregulation is compromised immune mediated elimination of intraocular tumors. For example, certain murine tumor cell lines which are rejected by host immune responses when transplanted in the skin grow progressively when placed in the anterior chamber (a.c.) of the eye. Progressive ocular tumor growth occurs despite induction of tumor-specific CD8+ T cell responses capable of eliminating a subsequent tumor challenge in the skin or opposite eye. Why these CD8+ T effectors fail to eliminate established ocular tumors is not known. It is well appreciated that growth of tumors in the a.c. induces the generation of immunosuppressive CD8+ T regulatory (Treg) cells. However, the contribution of CD8+ Treg in ocular tumor progression remains unclear. Several studies indicate that these CD8+ Treg target responding CD4+ T cells to inhibit their induction of macrophage-dependent delayed type hypersensitivity (DTH) responses to tumor antigens (Ags). However, induction of tumor-specific CD4+ T cell responses does not assure intraocular tumor elimination. This review is focused on how CD8+ Treg could influence the tumoricidal activity of ocular tumor-specific CD8+ T effector cells.