Lymphatic vessel transit seeds cytotoxic resident memory T cells in skin draining lymph nodes.

Lymphatic transport shapes the homeostatic immune repertoire of lymph nodes (LNs). LN-resident memory T cells (TRMs) play an important role in site-specific immune memory, yet how LN TRMs form de novo after viral infection remains unclear. Here, we tracked the anatomical distribution of antiviral CD8+ T cells as they seeded skin and LN TRMs using a model of vaccinia virus-induced skin infection. LN TRMs localized to the draining LNs (dLNs) of infected skin, and their formation depended on the lymphatic egress of effector CD8+ T cells from the skin, already poised for residence. Effector CD8+ T cell transit through skin was required to populate LN TRMs in dLNs, a process reinforced by antigen encounter in skin. Furthermore, LN TRMs were protective against viral rechallenge in the absence of circulating memory T cells. These data suggest that a subset of tissue-infiltrating CD8+ T cells egress from tissues during viral clearance and establish a layer of regional protection in the dLN basin.

Lymphatic vessel transit seeds precursors to cytotoxic resident memory T cells in skin draining lymph nodes.

Resident memory T cells (TRM) provide rapid, localized protection in peripheral tissues to pathogens and cancer. While TRM are also found in lymph nodes (LN), how they develop during primary infection and their functional significance remains largely unknown. Here, we track the anatomical distribution of anti-viral CD8+ T cells as they simultaneously seed skin and LN TRM using a model of skin infection with restricted antigen distribution. We find exquisite localization of LN TRM to the draining LN of infected skin. LN TRM formation depends on lymphatic transport and specifically egress of effector CD8+ T cells that appear poised for residence as early as 12 days post infection. Effector CD8+ T cell transit through skin is necessary and sufficient to populate LN TRM in draining LNs, a process reinforced by antigen encounter in skin. Importantly, we demonstrate that LN TRM are sufficient to provide protection against pathogenic rechallenge. These data support a model whereby a subset of tissue infiltrating CD8+ T cells egress during viral clearance, and establish regional protection in the draining lymphatic basin as a mechanism to prevent pathogen spread.

Infection-induced lymphatic zippering restricts fluid transport and viral dissemination from skin.

Lymphatic vessels are often considered passive conduits that flush antigenic material, pathogens, and cells to draining lymph nodes. Recent evidence, however, suggests that lymphatic vessels actively regulate diverse processes from antigen transport to leukocyte trafficking and dietary lipid absorption. Here we tested the hypothesis that infection-induced changes in lymphatic transport actively contribute to innate host defense. We demonstrate that cutaneous vaccinia virus infection by scarification activates dermal lymphatic capillary junction tightening (zippering) and lymph node lymphangiogenesis, which are associated with reduced fluid transport and cutaneous viral sequestration. Lymphatic-specific deletion of VEGFR2 prevented infection-induced lymphatic capillary zippering, increased fluid flux out of tissue, and allowed lymphatic dissemination of virus. Further, a reduction in dendritic cell migration to lymph nodes in the absence of lymphatic VEGFR2 associated with reduced antiviral CD8+ T cell expansion. These data indicate that VEGFR2-driven lymphatic remodeling is a context-dependent, active mechanism of innate host defense that limits viral dissemination and facilitates protective, antiviral CD8+ T cell responses.

Epithelial Pyroptosis in Host Defense.

Pyroptosis is a lytic form of cell death that is executed by a family of pore-forming proteins called gasdermins (GSDMs). GSDMs are activated upon proteolysis by host proteases including the proinflammatory caspases downstream of inflammasome activation. In myeloid cells, GSDM pore formation serves two primary functions in host defense: the selective release of processed cytokines to initiate inflammatory responses, and cell death, which eliminates a replicative niche of the pathogen. Barrier epithelia also undergo pyroptosis. However, unique mechanisms are required for the removal of pyroptotic epithelial cells to maintain epithelial barrier integrity. In the following review, we discuss the role of epithelial inflammasomes and pyroptosis in host defense against pathogens. We use the well-established role of inflammasomes in intestinal epithelia to highlight principles of epithelial pyroptosis in host defense of barrier tissues, and discuss how these principles might be shared or distinctive across other epithelial sites.

PGD2 and CRTH2 counteract Type 2 cytokine-elicited intestinal epithelial responses during helminth infection.

Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Gait Deficits and Loss of Striatal Tyrosine Hydroxlase/Trk-B are Restored Following 7,8-Dihydroxyflavone Treatment in a Progressive MPTP Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is caused by neurodegeneration of nigrostriatal neurons, resulting in dopamine (DA) stimulated motor deficits. Like brain derived neurotrophic factor (BDNF), 7,8-dihydroxyflavone (DHF) is an agonist of the tropomyosin receptor kinase-B (TrkB) and stimulates the same secondary cascades that promote neuronal growth, survival and differentiation. We used our progressive mouse model of PD by administering increasing doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over 4 weeks (5 days/week), and then treated mice with DHF for 4 weeks after the cessation of the toxin injections (i.e., restoration). Mice treated with DHF recovered motorically, even after MPTP administration. Despite a 75% loss of tyrosine hydroxylase (TH) expression in the dorsolateral (DL) striatum in the MPTP group, mice treated with DHF had a recovery comparable to that found in the respective control. There was no recovery of DA tissue levels within the DL striatum. In both the DL striatum and substantia nigra (SN)/midbrain, phosphorylated TrkB and secondary messengers were significantly increased following DHF compared to the MPTP only group. Expression of the sprouting biomarker, superior cervical ganglion 10 (SCG10), was increased ∼20% in the DL striatum and 66% in the SN/midbrain in mice treated with DHF compared to the MPTP only group. We report that after 4 weeks of progressive MPTP administration, DHF can restore motor deficits and TH within the DL striatum in a TrkB-dependent manner. Our data suggests that DHF may help alleviate motor symptoms of PD and restore the loss of DA terminals within the striatum.

Glatiramer Acetate Reverses Motor Dysfunction and the Decrease in Tyrosine Hydroxylase Levels in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease and there are no effective treatments that either slow or reverse the degeneration of the dopamine (DA) pathway. Using a 4-week progressive MPTP (1-methyl-1,2,3,6-tetrahydropyridine) neurotoxin model of PD, which is characterized by neuroinflammation, loss of nigrostriatal DA, and motor dysfunction, as seen in patients with PD, we tested whether post-MPTP treatment with glatiramer acetate (GA), an immunomodulatory drug, could reverse these changes. GA restored the grip dysfunction and gait abnormalities that were evident in the MPTP treated group. The reversal of the motor dysfunction was attributable to the substantial recovery in tyrosine hydroxylase (TH) protein expression in the striatum. Within the substantia nigra pars compacta, surface cell count analysis showed a slight increase in TH+ cells following GA treatment in the MPTP group, which was not statistically different from the vehicle (VEH) group. This was associated with the recovery of BDNF (brain derived neurotrophic factor) protein levels and a reduction in the microglial marker, IBA1, protein expression within the midbrain. Alpha synuclein (syn-1) levels within the midbrain and striatum were decreased following MPTP, while GA facilitated recovery to VEH levels in the striatum in the MPTP group. Although DA tissue analysis revealed no significant increase in striatal DA or 3,4-Dihydroxyphenylacetic acid levels (DOPAC) in the MPTP group treated with GA, DA turnover (DOPAC/DA) recovered back to VEH levels following GA treatment. GA treatment effectively reversed clinical (motor dysfunction) and pathology (TH, IBA1, BDNF expression) of PD in a murine model.

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Dietary therapy restores glutamatergic input to orexin/hypocretin neurons after traumatic brain injury in mice.

Study Objectives: In previous work, dietary branched-chain amino acid (BCAA) supplementation, precursors to de novo glutamate and γ-aminobutyric acid (GABA) synthesis, restored impaired sleep-wake regulation and orexin neuronal activity following traumatic brain injury (TBI) in mice. TBI was speculated to reduce orexin neuronal activity through decreased regional excitatory (glutamate) and/or increased inhibitory (GABA) input. Therefore, we hypothesized that TBI would decrease synaptic glutamate and/or increase synaptic GABA in nerve terminals contacting orexin neurons, and BCAA supplementation would restore TBI-induced changes in synaptic glutamate and/or GABA. Methods: Brain tissue was processed for orexin pre-embed diaminobenzidine labeling and glutamate or GABA postembed immunogold labeling. The density of glutamate and GABA immunogold within presynaptic nerve terminals contacting orexin-positive lateral hypothalamic neurons was quantified using electron microscopy in three groups of mice (n = 8 per group): Sham/noninjured controls, TBI without BCAA supplementation, and TBI with BCAA supplementation (given for 5 days, 48 hr post-TBI). Glutamate and GABA were also quantified within the cortical penumbral region (layer VIb) adjacent to the TBI lesion. Results: In the hypothalamus and cortex, TBI decreased relative glutamate density in presynaptic terminals making axodendritic contacts. However, BCAA supplementation only restored relative glutamate density within presynaptic terminals contacting orexin-positive hypothalamic neurons. BCAA supplementation did not change relative glutamate density in presynaptic terminals making axosomatic contacts, or relative GABA density in presynaptic terminals making axosomatic or axodendritic contacts, within either the hypothalamus or cortex. Conclusions: These results suggest TBI compromises orexin neuron function via decreased glutamate density and highlight BCAA supplementation as a potential therapy to restore glutamate density to orexin neurons.

Plastic changes at corticostriatal synapses predict improved motor function in a partial lesion model of Parkinson's disease.

In Parkinson's disease, striatal dopamine depletion leads to plastic changes at excitatory corticostriatal and thalamostriatal synapses. The functional consequences of these responses on the expression of behavioral deficits are incompletely understood. In addition, most of the information on striatal synaptic plasticity has been obtained in models with severe striatal dopamine depletion, and less is known regarding changes during early stages of striatal denervation. Using a partial model of nigral cell loss based on intranigral injection of the proteasome inhibitor lactacystin, we demonstrate ultrastructural changes at corticostriatal synapses with a 15% increase in the length and 30% increase in the area of the postsynaptic densities at corticostriatal synapses 1 week following toxin administration. This increase was positively correlated with the performance of lactacystin-lesioned mice on the rotarod task, such that mice with a greater increase in the size of the postsynaptic density performed better on the rotarod task. We therefore propose that lengthening of the postsynaptic density at corticostriatal synapses acts as a compensatory mechanism to maintain motor function under conditions of partial dopamine depletion. The ultrastructure of thalamostriatal synapses remained unchanged following lactacystin administration. Our findings provide novel insights into the mechanisms of synaptic plasticity and behavioral compensation following partial loss of substantia nigra pars compacta neurons, such as those occurring during the early stages of Parkinson's disease.

Cyclosporin promotes neurorestoration and cell replacement therapy in pre-clinical models of Parkinson's disease.

BACKGROUND: The early clinical trials using fetal ventral mesencephalic (VM) allografts in Parkinson's disease (PD) patients have shown efficacy (albeit not in all cases) and have paved the way for further development of cell replacement therapy strategies in PD. The preclinical work that led to these clinical trials used allografts of fetal VM tissue placed into 6-OHDA lesioned rats, while the patients received similar allografts under cover of immunosuppression in an α-synuclein disease state. Thus developing models that more faithfully replicate the clinical scenario would be a useful tool for the translation of such cell-based therapies to the clinic. RESULTS: Here, we show that while providing functional recovery, transplantation of fetal dopamine neurons into the AAV-α-synuclein rat model of PD resulted in smaller-sized grafts as compared to similar grafts placed into the 6-OHDA-lesioned striatum. Additionally, we found that cyclosporin treatment was able to promote the survival of the transplanted cells in this allografted state and surprisingly also provided therapeutic benefit in sham-operated animals. We demonstrated that delayed cyclosporin treatment afforded neurorestoration in three complementary models of PD including the Thy1-α-synuclein transgenic mouse, a novel AAV-α-synuclein mouse model, and the MPTP mouse model. We then explored the mechanisms for this benefit of cyclosporin and found it was mediated by both cell-autonomous mechanisms and non-cell autonomous mechanisms. CONCLUSION: This study provides compelling evidence in favor for the use of immunosuppression in all grafted PD patients receiving cell replacement therapy, regardless of the immunological mismatch between donor and host cells, and also suggests that cyclosporine treatment itself may act as a disease-modifying therapy in all PD patients.

MPTP-induced parkinsonism in mice alters striatal and nigral xCT expression but is unaffected by the genetic loss of xCT.

Nigral cell loss in Parkinson's disease (PD) is associated with disturbed glutathione (GSH) and glutamate levels, leading to oxidative stress and excitotoxicity, respectively. System xc- is a plasma membrane antiporter that couples cystine import (amino acid that can be further used for the synthesis of GSH) with glutamate export to the extracellular environment, and can thus affect both oxidative stress and glutamate excitotoxicity. In the current study, we evaluated the involvement of system xc- in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. Our results indicate that the expression of xCT (the specific subunit of system xc-) undergoes region-specific changes in MPTP-treated mice, with increased expression in the striatum, and decreased expression in the substantia nigra. Furthermore, mice lacking xCT were equally sensitive to the neurotoxic effects of MPTP compared to wild-type (WT) mice, as they demonstrate similar decreases in striatal dopamine content, striatal tyrosine hydroxylase (TH) expression, nigral TH immunopositive neurons and forelimb grip strength, five weeks after commencing MPTP treatment. Altogether, our data indicate that progressive lesioning with MPTP induces striatal and nigral dysregulation of system xc-. However, loss of system xc- does not affect MPTP-induced nigral dopaminergic neurodegeneration and motor impairment in mice.

Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson's disease.

Parkinson's disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of α-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study α-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human α-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic α-synuclein existed primarily in an unbound, soluble pool. In contrast, α-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing α-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for α-synuclein's binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, α-synuclein aggregates are selectively found in presynaptic terminals.

Loss of leucine-rich repeat kinase 2 (LRRK2) in rats leads to progressive abnormal phenotypes in peripheral organs.

The objective of this study was to evaluate the pathology time course of the LRRK2 knockout rat model of Parkinson's disease at 1-, 2-, 4-, 8-, 12-, and 16-months of age. The evaluation consisted of histopathology and ultrastructure examination of selected organs, including the kidneys, lungs, spleen, heart, and liver, as well as hematology, serum, and urine analysis. The LRRK2 knockout rat, starting at 2-months of age, displayed abnormal kidney staining patterns and/or morphologic changes that were associated with higher serum phosphorous, creatinine, cholesterol, and sorbitol dehydrogenase, and lower serum sodium and chloride compared to the LRRK2 wild-type rat. Urinalysis indicated pronounced changes in LRRK2 knockout rats in urine specific gravity, total volume, urine potassium, creatinine, sodium, and chloride that started as early as 1- to 2-months of age. Electron microscopy of 16-month old LRRK2 knockout rats displayed an abnormal kidney, lung, and liver phenotype. In contrast, there were equivocal or no differences in the heart and spleen of LRRK2 wild-type and knockout rats. These findings partially replicate data from a recent study in 4-month old LRRK2 knockout rats and expand the analysis to demonstrate that the renal and possibly lung and liver abnormalities progress with age. The characterization of LRRK2 knockout rats may prove to be extremely valuable in understanding potential safety liabilities of LRRK2 kinase inhibitor therapeutics for treating Parkinson's disease.