Necroptosis and Caspase-2-Mediated Apoptosis of Astrocytes and Neurons, but Not Microglia, of Rat Hippocampus and Parenchyma Caused by Angiostrongylus cantonensis Infection.

Hongli Zhou,Yue Hu,Zhiyue Lv,Paron Dekumyoy,Minyu Zhou,Zhe Chen,Ping Huang,Yubin Ma,Yanin Limpanont,Yixin Cheng

doi:10.3389/fmicb.2019.03126

Abstract

Infection with the roundworm Angiostrongylus cantonensis is the main cause of eosinophilic meningitis worldwide. The underlying molecular basis of the various pathological outcomes in permissive and non-permissive hosts infected with A. cantonensis remains poorly defined. In the present study, the histology of neurological disorders in the central nervous system (CNS) of infected rats was assessed by using hematoxylin and eosin staining. Quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot and immunofluorescence (IF) were used in evolutions of the transcription and translation levels of the apoptosis-, necroptosis-, autophagy-, and pyroptosis-related genes. The distribution of apoptotic and necroptotic cells in the rat hippocampus and parenchyma was further detected using flow cytometry, and the features of the ultrastructure of the cells were examined by transmission electron microscopy (TEM). The inflammatory response upon CNS infection with A. cantonensis evolved, as characterized by the accumulation of a small number of inflammatory cells under the thickened meninges, which peaked at 21 days post-infection (dpi) and returned to normal by 35 dpi. The transcription levels and translation of caspase-2, caspase-8, RIP1 and RIP3 increased significantly at 21 and 28 dpi but decreased sharply at 35 dpi compared to those in the normal control group. However, the changes in the expression of caspase-1, caspase-3, caspase-11, Beclin-1 and LC3B were not obvious, suggesting that apoptosis and necroptosis but not autophagy or pyroptosis occurred in the brains of infected animals at 21 and 28 dpi. The results of RT-qPCR, western blot analysis, IF, flow cytometry and TEM further illustrated that necroptosis and caspase-2-mediated apoptosis occurred in astrocytes and neurons but not in microglia in the parenchyma and hippocampus of infected animals. This study provides the first evidence that neuronal and astrocytic necroptosis and caspase-2-mediated apoptosis are induced by A. cantonensis infection in the parenchymal and hippocampal regions of rats at 21 and 28 dpi but these processes are negligible at 35 dpi. These findings enhance our understanding of the pathogenesis of A. cantonensis infection and provide new insights into therapeutic approaches targeting the occurrence of cell death in astrocytes and neurons in infected patients.

Highlights

The rat lungworm Angiostrongylus cantonensis, a food-borne zoonotic parasite, has remained the most common causative agent of neuro-angiostrongyliasis characterized by eosinophilic meningitis or eosinophilic meningoencephalitis worldwide since its first description in 1933 (Chen, 1935; Hu et al, 2018)
To investigate the mRNA levels of molecules related to cell death, we first determined the levels of RIP3 and caspase-2 in the epencephalon, brainstem, and whole brain of the animals from the normal control and experimental groups (Figure 2)
The results show that the rats infected with A. cantonensis exhibited significantly elevated mRNA levels of RIP3 and caspase-2 at 14, 21, and 28 dpi but decreased levels at 35 dpi in the hippocampus and parenchyma, whereas there were no obvious differences in the levels of LC3B, Beclin, caspase-1, IL-1β or caspase-11, suggesting that necroptosis and apoptosis but not autophagy or pyroptosis occurred in the brains of the infected rats

Summary

Introduction

The rat lungworm Angiostrongylus cantonensis, a food-borne zoonotic parasite, has remained the most common causative agent of neuro-angiostrongyliasis characterized by eosinophilic meningitis or eosinophilic meningoencephalitis worldwide since its first description in 1933 (Chen, 1935; Hu et al, 2018). A. cantonensis is the most common infectious cause of eosinophilic meningitis in Southeast Asia and the Pacific Basin (Dunn et al, 2019) and distinct pathological outcomes of rats and mice with A. cantonensis infection have been observed over the past several decades (Courdurier et al, 1964; Wallace and Rosen, 1969; Uchida et al, 1984), the pathogenicity and pathophysiology of neuro-angiostrongyliasis remain unclear (Morassutti and GraeffTeixeira, 2012). Determining the molecular events that occur in the rat brain will explain the higher tolerance and better adaptation to A. cantonensis of permissive hosts than of non-permissive hosts, and help to better elucidate the pathogenetic mechanisms of A. cantonensis

Methods

Results

Conclusion