A fundamental bimodal role for neuropeptide Y1 receptor in the immune system

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY) in the central nervous system is a major regulator of numerous physiological functions, including stress. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five Y receptors (Y1, Y2, Y4, Y5, and y6). Using Y1-deficient (Y1−/−) mice, we showed that Y1−/− T cells are hyperresponsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signaling through Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, Y1−/− mice were resistant to T helper type 1 (Th1) cell–mediated inflammatory responses and showed reduced levels of the Th1 cell–promoting cytokine interleukin 12 and reduced interferon γ production. This defect was due to functionally impaired antigen-presenting cells (APCs), and consequently, Y1−/− mice had reduced numbers of effector T cells. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. Our findings uncover a sophisticated molecular mechanism regulating immune cell functions that can lead to stress-induced immunosuppression

The Y1 receptor for NPY: a novel regulator of immune cell function

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- Tcells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell–mediated inflammatory responses and showed reduced levels of the Th1 cell–promoting cytokine interleukin 12 and reduced interferon γ production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicatedthat this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system

HBEC take up fluorescently labelled antigen via actin-dependent mechanisms and form conjugates with T cells.

<p>Flow cytometry histograms depicting level of uptake of FITC-OVA (<i>A</i>) and Lucifer yellow (<i>C</i>) by HBEC at 37°C (blue line) vs background uptake at 4°C (red line). Data are representative of three independent experiments. Inhibition of FITC-OVA (<i>B</i>) and Lucifer yellow (<i>D</i>) uptake by HBEC cells pre-incubated with 10 mM Cytochalasin D (CCD). <i>C</i>, Flow cytometry histogram depicting level of uptake of Lucifer yellow by HBEC at 37°C (blue line) vs background uptake at 4°C (red line). Data are representative of three independent experiments Percentage increase in mean fluorescence intensity (MFI) is calculated as follows: (MFI following uptake at 37°C/MFI following uptake at 4°C)×100. Data are pooled from three independent experiments (n = 3 per experiment) and are expressed as mean +/− SD. ** and *** indicates statistically significant differences between control and CCD treatment as assessed by Student t test (p, 0.001, p<0.001 respectively). Representative flow cytometry plots indicating the levels of conjugation between HBEC and CD4⁺<i>(E)</i> and CD8⁺<i>(F)</i> cells. HBEC were labeled with PKH67 and isolated T cells labeled with PKH26 and equal numbers of cells were co-cultured for 30 min prior to flow cytometric analysis.</p

Parasitaemia and survival curves of <i>Plasmodium berghei</i>.

<p>PbK-infection at the non-encephalitogenic dose was used to model NCM (closed triangle), PbK-infection at the encephalitogenic dose (PbK^1/2, open triangle) and PbA-infection to model CM (closed square) (n = 10). (<b>A</b>) Parasitaemia and (<b>B</b>) survival curves during the course of the infections. No difference in percentage parasitaemia on day 7 p.i by the Mann Whitney test: PbK (closed triangle) vs PbK^1/2 (open triangle) p = 0.6286; PbK^1/2 (open triangle) vs PbA (closed square) p = 1; PbK (closed triangle) vs PbA (closed square) p = 0.5333. Note, parasitaemia values for PbA infected mice after day 7 are not shown, since none were surviving. Parasitaemia values for PbK and PbK^1/2 after day 7 p.i diverge. Parasitaemia is shown as mean ± SD. CBA mice (n = 10, per group) infected with PbK^1/2 or PbA parasites develop CM, with a cumulative incidence of 70% and 100% respectively. Comparison of survival curves PbK (closed triangle) vs PbK^1/2 (open triangle): Log-rank (Mantel-Cox) Test p<0.001 and Gehan-Breslow-Wilcoxon Test p<0.002. PbK^1/2 (open triangle) vs PbA (closed square): Log-rank (Mantel-Cox) Test p<0.001 and Gehan-Breslow-Wilcoxon Test p<0.001. PbK (closed triangle) vs PbA^1/2 (closed square): Log-rank (Mantel-Cox) Test p<0.001 and Gehan-Breslow-Wilcoxon Test p<0.001.</p

Detection, clearance, and tissue distribution of MP following adoptive transfer.

<p>(<b>A</b>) Clearance of transferred MP within circulation following adoptive transfer. The presence of transferred PKH67-labeled MP was detected via flow cytometry in the blood of recipient mice. MP were detectable immediately following intravenous injection; healthy recipients (circles) cleared the MP quicker than PbA infected recipients (squares). Data are mean ± SEM from <i>n</i> = 3 per group. (<b>B</b>) Microparticles localised in brain microvessels of CM⁺ recipient mice. Smears were prepared from healthy and CM⁺ mice, recipients of PKH67-labelled MP (green) purified from healthy or CM⁺ donor mice (<i>n = </i>3). MP were activated <i>in vitro</i> with calcium ionophore (A23187), a known vesiculating agonist, labelled, intravenously transferred and allowed to circulate for 1 h. Brain smears were fixed and counter-stained with Texas Red Lectin and DAPI to identify vessels (red) and nuclei (blue), respectively. MP from CM⁺ donors can only be detected in the microvessels of CM⁺ recipient mice (arrows). Imaged on Olympus IX71. Insert: MP from CM⁺ donor lodged in CM⁺ brain vessel, imaged using oil immersion ×60 on Olympus FluoView FV 1000 confocal microscope. Arrows indicate MP lining the endothelium amongst other cells and trapped in birfurcation of vessels. (<b>C</b>) Summary of MP localisation in cerebral vessels. MP from CM⁺ donors can only be detected in the microvessels of CM⁺ recipient mice, as indicated by green tick.</p

Cell-specific MP in plasma during the course of <i>Plasmodium berghei</i> infection of CBA mice.

<p>PbK (closed triangle), PbK^1/2 (open triangle) or PbA (closed square) (<b>A</b>) PMP/µL plasma; (<b>B</b>) EMP/µL plasma; (<b>C</b>) RMP/µL plasma; (<b>D</b>) MMP/µL plasma; (<b>E</b>) LMP/µL plasma. Data shown from <i>n = 10</i> PbK- (closed triangle) and <i>n = </i>5 PbK^1/2 infected (open triangle) mice on day 0, 2, 4, 6, 7, 14 and 18. Data shown from PbA infected mice (closed square) <i>n = </i>11 day 0, <i>n = </i>10 day 2, <i>n = </i>10 day 4, <i>n = </i>12 day 6, <i>n = 6</i> day 7, <i>n = 5</i> day 8 and represented as mean ± SEM *p<0.05, **p<0.001, ***p<0.0001. Inconsistencies in sample group size are due to the fragile health status of mice during the period of morbidity.</p

Expression of markers relevant to antigen presentation and T cell activation on HBEC.

<p>Histograms represent flow cytometry results from unstimulated and cytokine stimulated HBEC cells 18 h following stimulation. HBEC were stimulated with either 10 ng/ml TNF (blue line), 50 ng/ml IFNg (green line), or 10 ng/ml TNF+50 ng/ml IFNg (orange line) and compared to unstimulated cells (red line). Cells were stained with mAbs against CD54 (ICAM-1), Endoglin (CD105), MHC II (HLA-DR), ICOSL (CD275), CD40, CD80 and CD86 as per manufacturers instructions. Data are representative of four independent experiments.</p

Transferred EMP generated <i>in vitro</i> induce CM-like pathology in healthy mice.

<p>(<b>A</b>) Detection of PKH-67 labelled EMP in the blood of recipient mice following transfer. Purified EMP (400×10³/mouse) from TNF stimulated and resting mouse brain microvascular endothelial cell line B3 harvested <i>in vitro</i>, were transferred into PbA infected and healthy recipient mice. Clearance of fluorescently labeled EMP from the blood was monitored by flow cytometry. Latex beads (400×10³/mouse, control) can be detected in constant circulation up to 30 minutes post transfer. Data represent the mean from <i>n</i> = 3. Closed circle/solid line indicates beads transferred into healthy mice, open circle/interrupted line indicates beads transferred into infected mice, closed square/solid line indicates NS- EMP transferred into healthy mice, closed triangle/solid line indicates NS-EMP transferred into infected mice, open square/interrupted line indicates TNF-EMP transferred into healthy mice and open triangle/interrupted line indicates TNF-EMP transferred into infected mice. (<b>B</b>) Detection of CD54 and CD106 on <i>in vitro</i> generated MP derived from mouse brain microvascular endothelial cells. Graph shows the expression of CD54 (white bar) and CD106 (black bar) on resting and TNF-stimulated EMP. Data are mean ± SD, *p<0.05. The numbers of CD54⁺ and CD106⁺ EMP in the supernatant increased following TNF-stimulation (mean ± SEM MP/µL; CD54: 9.850±1.064 to 38.050±9.925; 74.11% CD54⁺ shift CD106: 86.183±9.981 to 133.250±19.860; 35.32% CD106⁺ shift). (<b>C</b>) Representative haematoxylin-eosin brain and lung sections from healthy and PbA infected mice treated with PBS or EMP (<i>n = </i>3) on day 6 p.i. Morphometric analysis reveals that EMPs induce significant pathology in healthy brain and lung. In the brain, the arrows heads indicate areas of engorged vessels and haemorrhage. In the lung, multifocal lesions, abnormal cellularity in the alveolar septa, plasma in alveoli and haemorrhage. Treatment with EMP in healthy mice induces haemorrhage in the brain (white arrow head) and increased cellularity (white arrow head) and atelectasis (white arrow) in the lung of recipient mice. Such pathology is not observed in the mice treated with PBS or beads alone. Scale bar (bottom left) indicates 40 µm. (<b>D</b>) Effects of <i>in vitro</i> generated EMP on healthy and PbA infected brain and lung in the following treated groups: PBS (white bar), beads (light grey), NS-EMP (dark grey) and TNF-EMP (black). Representative Haematoxylin-Eosin images (<i>n = </i>3) per group were scored for histopathological signs (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003839#ppat-1003839-t003" target="_blank">Table 3</a>).</p