Differential Contribution of Ca2+-Dependent Mechanisms to Hyperexcitability in Layer V Neurons of the Medial Entorhinal Cortex

Temporal lobe epilepsy is characterized by recurrent seizures in one or both temporal lobes of the brain; some in vitro models show that epileptiform discharges initiate in entorhinal layer V neurons and then spread into other areas of the temporal lobe. We previously found that, in the presence of GABAA receptor antagonists, stimulation of afferent fibers, terminating both at proximal and distal dendritic locations, initiated hyperexcitable bursts in layer V medial entorhinal neurons. We investigated the differential contribution of Ca2+-dependent mechanisms to the plateaus underlying these bursts at proximal and distal synapses. We found that the NMDA glutamatergic antagonist D,L-2-amino-5-phosphonovaleric acid (APV; 50 μM) reduced both the area and duration of the bursts at both proximal and distal synapses by about half. The L-type Ca2+ channel blocker nimodipine (10 μM) and the R- and T-type Ca2+ channel blocker NiCl2 (200 μM) decreased the area of the bursts to a lesser extent; none of these effects appeared to be location-dependent. Remarkably, the perfusion of flufenamic acid (FFA; 100 μM), to block Ca2+-activated non-selective cation currents (ICAN) mediated by transient receptor potential (TRP) channels, had a location-dependent effect, by abolishing burst firing and switching the suprathreshold response to a single action potential (AP) for proximal stimulation, but only minimally affecting the bursts evoked by distal stimulation. A similar outcome was found when FFA was pressure-applied locally around the proximal dendrite of the recorded neurons and in the presence of a selective blocker of melastatin TRP (TRPM) channels, 9-phenanthrol (100 μM), whereas a selective blocker of canonical TRP (TRPC) channels, SKF 96365, did not affect the bursts. These results indicate that different mechanisms might contribute to the initiation of hyperexcitability in layer V neurons at proximal and distal synapses and could shed light on the initiation of epileptiform activity in the entorhinal cortex

CCR5 Is Essential for NK Cell Trafficking and Host Survival following Toxoplasma gondii Infection

The host response to intracellular pathogens requires the coordinated action of both the innate and acquired immune systems. Chemokines play a critical role in the trafficking of immune cells and transitioning an innate immune response into an acquired response. We analyzed the host response of mice deficient in the chemokine receptor CCR5 following infection with the intracellular protozoan parasite Toxoplasma gondii. We found that CCR5 controls recruitment of natural killer (NK) cells into infected tissues. Without this influx of NK cells, tissues from CCR5-deficient (CCR5(−/−)) mice were less able to generate an inflammatory response, had decreased chemokine and interferon γ production, and had higher parasite burden. As a result, CCR5(−/−) mice were more susceptible to infection with T. gondii but were less susceptible to the immune-mediated tissue injury seen in certain inbred strains. Adoptive transfer of CCR5(+/+) NK cells into CCR5(−/−) mice restored their ability to survive lethal T. gondii infection and demonstrated that CCR5 is required for NK cell homing into infected liver and spleen. This study establishes CCR5 as a critical receptor guiding NK cell trafficking in host defense

NK Cells Help To Induce CD8(+)-T-Cell Immunity against Toxoplasma gondii in the Absence of CD4(+) T Cells

CD8(+) T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4(+) T-cell help was needed for launching in vivo CD8(+) T-cell activity against these pathogens and tumors. However, recently CD4(+) T-cell-independent CD8 responses during several microbial infections including those with Toxoplasma gondii have been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4(+) T cells, T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8(+) T-cell immunity. Depletion of NK cells inhibited the generation of CD8(+) T-cell immunity in CD4(−/−) mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8(+) T-cell immunity against T. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8(+) T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4(+) T cells, NK cells can play an important role in induction of primary CD8(+) T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection

Interleukin-17/Interleukin-17 Receptor-Mediated Signaling Is Important for Generation of an Optimal Polymorphonuclear Response against Toxoplasma gondii Infection

We investigated the role of interleukin-17 (IL-17)/IL-17 receptor (IL-17R)-mediated signaling in the protective immunity against Toxoplasma gondii. IL-17R(−/−) mice developed a normal adaptive immunity against the parasite. However, increased mortality in the knockout animals can be attributed to a defect in the migration of polymorphonuclear leukocytes to infected sites during early infection

Expression of CCR5 and CXCR3 Chemokine Ligands and IFNγ in <i>T. gondii</i>-Infected Mice

<p>Quantitative PCR was performed on total RNA isolated from spleen, lung, liver, and small intestine of wild-type control and CCR5^−/− C57BL/6 mice (female, 5–6 wk old, three or four mice per group) infected orally with 15–50 cysts of 76K strain of <i>T. gondii</i>. Tissues were harvested from uninfected and at days 3, 5, and 7 PI. Data are displayed as copies of cytokine or chemokine per copies of GAPDH, with wild type in black bars and CCR5^−/− in hatched bars ± standard error of the mean. Statistics were performed using Student's t-test. Sets of symbols: * indicates comparisons with uninfected wild type, ^ indicates comparisons between days 5 and 7 PI; and # indicates comparisons between wild type and CCR5^−/−. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ^ <i>p</i> < 0.05, ^ ^ <i>p</i> < 0.01, ^ ^ ^ <i>p</i> < 0.001, # <i>p</i> < 0.05, ## <i>p</i> < 0.01, ### <i>p</i> < 0.001.</p

Survival, Histopathology, and Parasite Load of CCR5^−/− and CCR5^+/+ Mice in a C57BL/6x129 Background following Infection with Different Doses of <i>T. gondii</i>

<div><p>(A) Survival. Female CCR5^−/− mice (5–8 wk old) and age-matched CCR5^−/− wild-type littermate controls, both in a C57BL/6x129 background, were challenged perorally with the cysts of the 76K strain of <i>T. gondii</i>. The animals were monitored for survival on daily basis. There were six animals per group and the experiment was performed three times with similar results. All CCR5^+/+ mice survived at the three doses of cysts tested.</p><p>(B) Histopathology. Photomicrographs of hematoxylin and eosin-stained liver and ileum of small intestine isolated from infected CCR5^+/+ mice and CCR5^−/− mice at day 14 PI. Wild-type liver: Moderate fatty change is seen in hepatocytes, with small foci of mixed polymorphonuclear and mononuclear infiltration. Bar, 10 μm. CCR5^−/− liver: Severe fatty changes are seen in hepatocytes with mononuclear and polymorphonuclear inflammatory nodules. Bar, 10 μm. Wild-type small intestine: The superficial mucosa is missing in some places (arrow) and no parasite multiplication is evident. Bar, 100 μm. CCR5^−/− small intestine. The superficial mucosa is largely missing and a mixed inflammatory infiltrate including polymorphonuclear cells is evident within the lamina propria and superficial mucosa. Bar, 100 μm. Inset: Higher magnification with arrow pointing to tachyzoites in lamina propria of CCR5^−/− mice. Bar, 75 μm.</p><p>(C) Level of parasite DNA<i>.</i> Groups of CCR5^−/− mice and wild-type littermate controls both on a C57BL/6x129 background were infected perorally with 20 cysts of <i>T. gondii</i>. At day 14 PI, small intestine, liver, spleen, lung, and brain from both CCR5^−/− and wild-type mice (three mice per group) were collected and the parasite load in the tissues was determined by competitive PCR. The experiment was performed twice with similar results.</p></div