Cellular mechanisms of fatal respiratory insufficiency in arboviral encephalitis

描述（由申请方提供）：总体目标是确定虫媒病毒性脑炎致死性呼吸功能不全的细胞机制。呼吸功能不全是虫媒病毒性脑炎的严重后果，更具体地说是西尼罗河神经系统疾病，并可导致预后不良。我们已经使用啮齿动物模型来确定神经缺陷引起呼吸窘迫，并且是西尼罗河病毒（WNV）和其他病毒性脑炎死亡的主要病理生理机制。这些发现代表了关于这些病毒如何引起呼吸衰竭和死亡的突破，但是引起这些病理反应的细胞机制是未知的，并且是本申请的主题。待检验的假设是趋化因子CXCR 4/CXCL 12轴延迟病毒特异性T淋巴细胞早期进入含有神经元控制的延髓腹外侧（VLM）实质。此外，我们推测，这种延迟导致更高的病毒载量，和升高的抑制性生长抑素（SST）神经肽或其受体，导致致命的呼吸衰竭。这一假设是基于三个最近的发现。1)我们发现神经性呼吸功能不全是感染WNV和其他虫媒病毒的啮齿动物死亡的主要病理生理机制，这可能与人类患者的致命病毒性脑炎有关。2)值得注意的是，WNV免疫染色的神经元与VLM中的SST染色的神经元紧密共定位（96%），这意味着WNV和/或炎症诱导SST。特别是在前B？tzinger复合体（一个对呼吸节律发生至关重要的区域）内，有许多神经元被SST严重染色。这在WN神经系统疾病中可能具有生物学意义，因为VLM中的SST是呼吸的有效负调节剂。SST神经肽在呼吸的产生和控制中起作用的证据是基于外源性给予VLM SST引起的严重的负性呼吸效应，以及给予SST受体拮抗剂恢复呼吸功能。3)CXCR 4/CXCL 12轴延迟WNV特异性T淋巴细胞从血管周围空间进入脑的非特定区域的实质。不幸的是，致细胞病变的嗜神经病毒，如WNV，需要病毒特异性CD 8 + T细胞渗透到实质中以清除病毒。这种延迟导致病毒载量增加，导致炎症反应增加和WNV感染小鼠的存活率降低。因此，保护性T细胞特异性地延迟进入VLM的作用可能会在免疫病理条件下异常增加SST抑制肽，并导致虫媒病毒诱导的呼吸衰竭。为检验这一假设而设计的具体目标是：目标1。确定WNV特异性T细胞通过CXCR 4/CXCL 12轴的延迟进入是否通过增加SST和VLM实质中的炎性细胞而导致致命性呼吸功能不全。我们将通过与无呼吸功能不全的WNV感染小鼠相比，定量具有呼吸功能不全的WNV感染小鼠的VLM中的炎性细胞和SST来实现这一点。此外，将评价CXCR 4拮抗剂减轻VLM中这些病理事件的能力。目标2.确定SST或其受体的表达增加是否与WNV诱导的呼吸功能不全有关。这将通过确定立体定向注射到VLM中的SST受体拮抗剂是否可以减轻病毒抑制呼吸功能的作用来实现。

DESCRIPTION (provided by applicant): The overall goal is to identify the cellular mechanism of fatal respiratory insufficiency in arboviral encephalitides. Respiratory insufficiency is a serius outcome of arboviral encephalitis, and more specifically West Nile neurological disease, and can result in a poor prognosis. We have used rodent models to establish that neurological deficits cause the respiratory distress and are the primary pathophysiological mechanism of death for West Nile virus (WNV) and other viral encephalitides. These findings represent a breakthrough on how these viruses cause respiratory failure and death, but the cellular mechanism(s) causing these pathological responses are unknown, and are the subject of this application. The hypothesis to be tested is that the chemokine CXCR4/CXCL12 axis delays early entry of viral specific T lymphocytes into the parenchyma of the ventrolateral medulla (VLM) containing respiratory-control neurons. Additionally, we hypothesize that this delay results in higher virus load, and elevated inhibitory somatostatin (SST) neuropeptide or its receptor that contribute to lethal respiratory failure. This hypothesis is based on three recent findings. 1) Our finding that neurological respiratory insufficiency is a primary pathophysiologica mechanism of death in rodents infected with WNV and other arboviruses, which might be relevant to fatal viral encephalitides of human patients. 2) Remarkably, WNV envelope-immunostained neurons are tightly co-localized (96%) with SST-stained neurons in the VLM, which implies that WNV and/or inflammation are inducing SST. Specifically within the pre-B¿tzinger complex, an area critical for respiratory rhythmogenesis, there are many neurons heavily stained for SST. This may be biologically significant in WN neurological disease, because SST in the VLM is a potent negative regulator of breathing. Evidence that SST neuropeptide plays a role in the generation and control of respiration is based on profound negative respiratory effects caused by exogenously administered SST to the VLM, and a restoration of respiratory function by administration of antagonists to SST receptors. 3) CXCR4/CXCL12 axis delays entry of WNV-specific T lymphocytes from the perivascular spaces into the parenchyma of non-specified areas of the brain. Unfortunately, cytopathic neurotropic viruses, such as WNV, require penetration of viral specific CD8+ T cells into the parenchyma for clearance of the virus. This delay results in an increased viral load leading to increased inflammatory responses and decreased survival of WNV-infected mice. Consequently, the effect of delayed entry of protective T cells specifically in the VLM may aberrantly increase the SST inhibitory peptide under immune-pathological conditions and contribute to arboviral-induced respiratory failure. The specific aims designed to test the hypothesis are: Aim 1. Determine if delayed entry of WNV- specific T cells via the CXCR4/CXCL12 axis contributes to fatal respiratory insufficiency by increasing SST, and inflammatory cells specifically in the parenchyma of the VLM. We will accomplish this by quantifying inflammatory cells and SST in the VLM of WNV-infected mice with respiratory insufficiency, as compared to WNV-infected mice without respiratory insufficiency. Additionally, an antagonist of CXCR4 will be evaluated for its ability to mitigate these pathological events in the VLM. Aim 2. Determine if increased expression of SST or its receptor(s) is involved with WNV-induced respiratory insufficiency. This will be accomplished by determining if an antagonist to the SST receptor stereotaxically injected into the VLM can mitigate the effects of the virus to suppress respiratory functions.