Host Cell Preference of Toxoplasma gondii Cysts in Murine Brain: A Confocal Study

Abstract

Toxoplasma gondii is a protozoan parasite that is widely prevalent in humans and typically results in a chronic infection characterized by cysts located predominantly in the central nervous system. In immunosuppressed hosts, such as patients with HIV infection, the infection can be reactivated from the cysts in the brain resulting in a severe and potentially fatal encephalitis. Studies suggest that the chronic infection may also have neuropathological and behavioral effects in immune competent hosts. An improved understanding of tissue cyst behavior is of importance for understanding both the reactivation as well as the neurophysiological consequences of chronic infection. In vivo studies have identified neurons as host cells for cysts but in vitro studies have found that astrocytes can also foster development of the cysts. In this study we have addressed the question of which neural cell tissue cysts of T. gondii reside during chronic infection using a mouse model. Mice were infected with Me49 Strain T. gondii and the intracellular localization of the cysts analyzed during the development and establishment of a chronic infection at 1, 2, and 6 months post infection. Brains were fixed, cryosectioned, and stained with FITC-Dolichos biflorans to identify the Toxoplasma cysts and they were labeled with cell specific antibodies to neurons or astrocytes and then analyzed using confocal fluorescence microscopy. Cysts were found to occur almost exclusively in neurons throughout chronic infection. No cysts were identified in astrocytes, using the astrocyte marker, GFAP. Astrocyte interactions with neuronal-cysts, however, were frequently observed.

Figure 1. Confocal microscopy of cysts in murine brain

(A) Cryosection of murine brain labeled with Dolichos biflorans-FITC (DB-FITC) showing the well-defined staining of the cyst wall; (B) field of 5 cysts showing a typical cyst cluster consisting of a large cyst (> 20 μm in diameter) surrounded by 2 smaller cysts (note the 2 small cysts in the close vicinity); (C) Maximal Image Projection of field with 4 cysts double-labeled with stained with DB-FITC (green) and anti-neurofilament heavy chain (NF-H; red); note the close association of the neurofilaments with the cyst wall as shown in the side views of cysts (a and b, arrows; note that a and b are side views of area indicated at the yellow line); (D) Single slice of cyst in C (starred) showing the close opposition of the NF-H with the cyst wall; (E) single slice of a small cyst in a neuron; (F) Maximal Image Projection of field with 2 cysts double-labeled with DB-FITC (green) and anti-GFAP (red); (G and H) single slices of 2 cysts in F showing the absence of GFAP filaments around the cyst wall; note that the GFAP filaments (red) are present outside the cyst wall, but not closely opposed to the cyst wall.