Donor-derived regulatory dendritic cell infusion modulates effector CD8+ T cell and NK cell responses after liver transplantation.

Immune cell-based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+ T cells and CD16bright natural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+ DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+ (IFN-γ+) CD4+ and CD8+ T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+ T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+ T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.

Complement C3-Targeted Gene Therapy Restricts Onset and Progression of Neurodegeneration in Chronic Mouse Glaucoma.

Dysregulation of the complement system is implicated in neurodegeneration, including human and animal glaucoma. Optic nerve and retinal damage in glaucoma is preceded by local complement upregulation and activation, but whether targeting this early innate immune response could have therapeutic benefit remains undefined. Because complement signals through three pathways that intersect at complement C3 activation, here we targeted this step to restore complement balance in the glaucomatous retina and to determine its contribution to degeneration onset and/or progression. To achieve this, we combined adeno-associated virus retinal gene therapy with the targeted C3 inhibitor CR2-Crry. We show that intravitreal injection of AAV2.CR2-Crry produced sustained Crry overexpression in the retina and reduced deposition of the activation product complement C3d on retinal ganglion cells and the inner retina of DBA/2J mice. This resulted in neuroprotection of retinal ganglion cell axons and somata despite continued intraocular pressure elevation, suggesting a direct restriction of neurodegeneration onset and progression and significant delay to terminal disease stages. Our study uncovers a damaging effect of complement C3 or downstream complement activation in glaucoma, and it establishes AAV2.CR2-Crry as a viable therapeutic strategy to target pathogenic C3-mediated complement activation in the glaucomatous retina.

Loss of Fractalkine Signaling Exacerbates Axon Transport Dysfunction in a Chronic Model of Glaucoma.

Neurodegeneration in glaucoma results in decline and loss of retinal ganglion cells (RGCs), and is associated with activation of myeloid cells such as microglia and macrophages. The chemokine fractalkine (FKN or Cx3cl1) mediates communication from neurons to myeloid cells. Signaling through its receptor Cx3cr1 has been implicated in multiple neurodegenerative diseases, but the effects on neuronal pathology are variable. Since it is unknown how FKN-mediated crosstalk influences RGC degeneration in glaucoma, we assessed this in a chronic mouse model, DBA/2J. We analyzed a DBA/2J substrain deficient in Cx3cr1, and compared compartmentalized RGC degeneration and myeloid cell responses to those in standard DBA/2J mice. We found that loss of FKN signaling exacerbates axon transport dysfunction, an early event in neurodegeneration, with a significant increase in RGCs with somal accumulation of the axonal protein phosphorylated neurofilament, and reduced retinal expression of genes involved in axon transport, Kif1b, and Atp8a2. There was no change in the loss of Brn3-positive RGCs, and no difference in the extent of damage to the proximal optic nerve, suggesting that the loss of fractalkine signaling primarily affects axon transport. Since Cx3cr1 is specifically expressed in myeloid cells, we assessed changes in retinal microglial number and activation, changes in gene expression, and the extent of macrophage infiltration. We found that loss of fractalkine signaling led to innate immune changes within the retina, including increased infiltration of peripheral macrophages and upregulated nitric oxide synthase-2 (Nos-2) expression in myeloid cells, which contributes to the production of NO and can promote axon transport deficits. In contrast, resident retinal microglia appeared unchanged either in number, morphology, or expression of the myeloid activation marker ionized calcium binding adaptor molecule 1 (Iba1). There was also no significant increase in the proinflammatory gene interleukin 1 beta (Il1ß). We conclude that loss of fractalkine signaling causes a selective worsening of axon transport dysfunction in RGCs, which is linked to enhanced Nos-2 expression in myeloid cells. Our findings suggest that distinct mechanisms may contribute to different aspects of RGC decline in glaucoma, with axonal transport selectively altered after loss of Cx3cr1 in microglia and/or macrophages.

Glial coverage in the optic nerve expands in proportion to optic axon loss in chronic mouse glaucoma.

Within the white matter, axonal loss by neurodegeneration is coupled to glial cell changes in gene expression, structure and function commonly termed gliosis. Recently, we described the highly variable expansion of gliosis alebosco@neuro.utah.edu in degenerative optic nerves from the DBA/2J mouse model of chronic, age-related glaucoma. Here, to estimate and compare the levels of axonal loss with the expansion of glial coverage and axonal degeneration in DBA/2J nerves, we combined semiautomatic axon counts with threshold-based segmentation of total glial/scar areas and degenerative axonal profiles in plastic cross-sections. In nerves ranging from mild to severe degeneration, we found that the progression of axonal dropout is coupled to an increase of gliotic area. We detected a strong correlation between axon loss and the aggregate coverage by glial cells and scar, whereas axon loss did not correlate with the small fraction of degenerating profiles. Nerves with low to medium levels of axon loss displayed moderate glial reactivity, consisting of hypertrophic astrocytes, activated microglia and normal distribution of oligodendrocytes, with minimal reorganization of the tissue architecture. In contrast, nerves with extensive axonal loss showed prevalent rearrangement of the nerve, with loss of axon fascicle territories and enlarged or almost continuous gliotic and scar domains, containing reactive astrocytes, oligodendrocytes and activated microglia. These findings support the value of optic nerve gliotic expansion as a quantitative estimate of optic neuropathy that correlates with axon loss, applicable to grade the severity of optic nerve damage in mouse chronic glaucoma.

Neurodegeneration severity can be predicted from early microglia alterations monitored in vivo in a mouse model of chronic glaucoma.

Microglia serve key homeostatic roles, and respond to neuronal perturbation and decline with a high spatiotemporal resolution. The course of all chronic CNS pathologies is thus paralleled by local microgliosis and microglia activation, which begin at early stages of the disease. However, the possibility of using live monitoring of microglia during early disease progression to predict the severity of neurodegeneration has not been explored. Because the retina allows live tracking of fluorescent microglia in their intact niche, here we investigated their early changes in relation to later optic nerve neurodegeneration. To achieve this, we used the DBA/2J mouse model of inherited glaucoma, which develops progressive retinal ganglion cell degeneration of variable severity during aging, and represents a useful model to study pathogenic mechanisms of retinal ganglion cell decline that are similar to those in human glaucoma. We imaged CX3CR1(+/GFP) microglial cells in vivo at ages ranging from 1 to 5 months by confocal scanning laser ophthalmoscopy (cSLO) and quantified cell density and morphological activation. We detected early microgliosis at the optic nerve head (ONH), where axonopathy first manifests, and could track attenuation of this microgliosis induced by minocycline. We also observed heterogeneous and dynamic patterns of early microglia activation in the retina. When the same animals were aged and analyzed for the severity of optic nerve pathology at 10 months of age, we found a strong correlation with the levels of ONH microgliosis at 3 to 4 months. Our findings indicate that live imaging and monitoring the time course and levels of early retinal microgliosis and microglia activation in glaucoma could serve as indicators of future neurodegeneration severity.

Evaluating changes in matrix-based, recovery-adjusted concentrations in paired data for pesticides in groundwater.

Pesticide concentration data for waters from selected carbonate-rock aquifers in agricultural areas of Pennsylvania were collected in 1993-2009 for occurrence and distribution assessments. A set of 30 wells was visited once in 1993-1995 and again in 2008-2009 to assess concentration changes. The data include censored matched pairs (nondetections of a compound in one or both samples of a pair). A potentially improved approach for assessing concentration changes is presented where (i) concentrations are adjusted with models of matrix-spike recovery and (ii) area-wide temporal change is tested by use of the paired Prentice-Wilcoxon (PPW) statistical test. The PPW results for atrazine, simazine, metolachlor, prometon, and an atrazine degradate, deethylatrazine (DEA), are compared using recovery-adjusted and unadjusted concentrations. Results for adjusted compared with unadjusted concentrations in 2008-2009 compared with 1993-1995 were similar for atrazine and simazine (significant decrease; 95% confidence level) and metolachlor (no change) but differed for DEA (adjusted, decrease; unadjusted, increase) and prometon (adjusted, decrease; unadjusted, no change). The PPW results were different on recovery-adjusted compared with unadjusted concentrations. Not accounting for variability in recovery can mask a true change, misidentify a change when no true change exists, or assign a direction opposite of the true change in concentration that resulted from matrix influences on extraction and laboratory method performance. However, matrix-based models of recovery derived from a laboratory performance dataset from multiple studies for national assessment, as used herein, rather than time- and study-specific recoveries may introduce uncertainty in recovery adjustments for individual samples that should be considered in assessing change.

Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression.

Glucose is a fundamental metabolite, yet how cells sense and respond to changes in extracellular glucose concentration is not completely understood. We recently reported that the MondoA:Mlx dimeric transcription factor directly regulates glycolysis. In this article, we consider whether MondoA:Mlx complexes have a broader role in sensing and responding to glucose status. In their latent state, MondoA:Mlx complexes localize to the outer mitochondrial membrane, yet shuttle between the mitochondria and the nucleus. We show that MondoA:Mlx complexes accumulate in the nucleus in response to glucose and 2-deoxyglucose (2-DG). Furthermore, nuclear localization of MondoA:Mlx depends on the enzymatic activity of hexokinases. These enzymes catalyze conversion of glucose to glucose-6-phosphate (G6P), which is the first step in the glycolytic pathway. Together, these findings suggest that MondoA:Mlx monitors intracellular G6P concentration and translocates to the nucleus when levels of this key metabolite increase. Transcriptional profiling experiments demonstrate that MondoA is required for >75% of the 2-DG-induced transcription signature. We identify thioredoxin-interacting protein (TXNIP) as a direct and glucose-regulated MondoA:Mlx transcriptional target. Furthermore, MondoA:Mlx complexes, via their regulation of TXNIP, are potent negative regulators of glucose uptake. These studies suggest a key role for MondoA:Mlx complexes in the adaptive transcriptional response to changes in extracellular glucose concentration and peripheral glucose uptake.

A C. elegans Myc-like network cooperates with semaphorin and Wnt signaling pathways to control cell migration.

Myc and Mondo proteins are key regulators of cell growth, proliferation, and energy metabolism, yet often overlooked is their vital role in cell migration. Complex networks of protein-protein and protein-DNA interactions control the transcriptional activity of Myc and MondoA confounding their functional analysis in higher eukaryotes. Here we report the identification of the transcriptional activation arm of a simplified Myc-like network in Caenorhabditis elegans. This network comprises an Mlx ortholog, named MXL-2 for Max-like 2, and a protein that has sequence features of both Myc and Mondo proteins, named MML-1 for Myc and Mondo-like 1. MML-1/MXL-2 complexes have a primary function in regulating migration of the ray 1 precursor cells in the male tail. MML-1/MXL-2 complexes control expression of ECM components in the non-migratory epidermis, which we propose contributes to the substratum required for migration of the neighboring ray 1 precursor cells. Furthermore, we show that pro-migratory Wnt/beta-catenin and semaphorin signaling pathways interact genetically with MML-1/MXL-2 to determine ray 1 position. This first functional analysis of the Myc superfamily in C. elegans suggests that MondoA and Myc may have more predominant roles in cell migration than previously appreciated, and their cooperation with other pro-migratory pathways provides a more integrated view of their role in cell migration.

MondoA-Mlx heterodimers are candidate sensors of cellular energy status: mitochondrial localization and direct regulation of glycolysis.

Transcription factors can be sequestered at specific organelles and translocate to the nucleus in response to changes in organellar homeostasis. MondoA is a basic helix-loop-helix leucine zipper transcriptional activator similar to Myc in function. However, unlike Myc, MondoA and its binding partner Mlx localize to the cytoplasm, suggesting tight regulation of their nuclear function. We show here that endogenous MondoA and Mlx associate with mitochondria in primary skeletal muscle cells and erythroblast K562 cells. Interaction between MondoA and the mitochondria is salt and protease sensitive, demonstrating that it associates with the outer mitochondrial membrane by binding a protein partner. Further, endogenous MondoA shuttles between the mitochondria and the nucleus, suggesting that it communicates between these two organelles. When nuclear, MondoA activates transcription of a broad spectrum of metabolic genes, including those for the glycolytic enzymes lactate dehydrogenase A, hexokinase II, and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3. Regulation of these three targets is mediated by direct interaction with CACGTG sites in their promoters. Consistent with its regulation of glycolytic targets, MondoA is both necessary and sufficient for glycolysis. We propose that MondoA communicates information about the intracellular energy state between the mitochondria and the nucleus, resulting in transcriptional activation of glycolytic target genes.