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. 2008 Dec 12:5:53.
doi: 10.1186/1742-2094-5-53.

Expression and distribution of Toll-like receptors 11-13 in the brain during murine neurocysticercosis

Bibhuti B Mishra  1 , Uma Mahesh Gundra, Judy M Teale
Affiliations

Expression and distribution of Toll-like receptors 11-13 in the brain during murine neurocysticercosis

Bibhuti B Mishra et al. J Neuroinflammation. .

Abstract

The functions of Toll-like receptors (TLRs) 11-13 in central nervous system (CNS) infections are currently unknown. Using a murine model of neurocysticercosis, we investigated the expression and distribution of TLRs 11-13 by using both gene specific real-time PCR analysis and in situ immunofluorescence microscopy in both control and neurocysticercosis brains. In the mock infected brain, mRNAs of TLRs 11-13 were constitutively expressed. Parasite infection caused an increase of both mRNAs and protein levels of all three TLRs by several fold. All three TLR proteins were present in both CNS and immune cell types. Among them TLR13 was expressed the most in terms of number of positive cells and brain areas expressing it, followed by TLR11 and TLR12 respectively. Among the nervous tissue cells, TLRs 11-13 protein levels appeared highest in neurons. However, TLR13 expression was also present in ependymal cells, endothelial cells of pial blood vessels, and astrocytes. In contrast, infiltrating CD11b and CD11c positive myeloid cells predominantly produced TLR11 protein, particularly early during infection at 1 wk post infection (approximately 50% cells). TLRs 12 and 13 proteins were present on approximately 5% of infiltrating immune cells. The infiltrating cells positive for TLRs 11-13 were mostly of myeloid origin, CD11b+ cells. This report provides a comprehensive analysis of the expression of TLRs 11-13 in normal and parasite infected mouse brains and suggests a role for them in CNS infections.

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Figures

Figure 1
Figure 1
M. corti infection elevates mRNA expression of TLRs 11–13 in brain. Total RNA was isolated from infected and mock infected control mouse brains at 1 wk, 3 wk, and 6 wk p.i. using Trizol reagent. Isolated RNA was reversed transcribed to cDNA by using random primers. Levels of TLR specific RNA and housekeeping gene 18S in these samples were measured by Real Time PCR analysis as described below using SYBR green as the detection dye. TLR specific mRNA levels were normalized to the mRNA level of the housekeeping gene 18S in the same sample and expressed in arbitrary units. The normalized values corresponding to the mRNA expression for each TLR gene are shown in the y-axis and represent the mean of three independent experiments. The data obtained were compared using a Student's t-test.
Figure 2
Figure 2
Expression of TLR11 in brains of mock infected control and NCC mice analyzed by immunofluorescence microscopy. Balb/c mice were infected intracranially with M. corti and sacrificed at various times p.i. Brain crysections were analyzed for expression of TLR11 and cell specific markers using fluorochrome conjugated antibodies. Nuclear staining DAPI is blue. (A) Mock infected control stained with TLR11 specific antibody (400×). (B) M. corti infection at 1 wk showing TLR11 positive staining in neurons (400×). (C) M. corti infection at 3 wk when maximum TLR11 expression was observed (400×). (D) M. corti infection at 6 wk showing TLR11 positive staining (400×). (E) Mock infected control mice stained with TLR11 and neuron specific antibody (Neun) (400×). The insert e' represents a 2× magnification of a selected area depicting TLR11 expression (Red) on neurons. (F) M. corti infection at 3 wk showing TLR11 positive neurons, merged image (yellow/orange; 400×). (G) Mock control mice stained with anti-TLR11 and anti-CD11b antibody (400×). (H) M. corti infection at 1 wk showing TLR11 and CD11b double positive cells, merged image (yellow/orange; 400×). Insert h' is a 2× magnification of a selected area to better illustrate TLR11 and CD11b double positive staining (I) The relative levels of TLR11 expression in mock and M. corti infected animals was calculated as described in experimental procedures.
Figure 3
Figure 3
Expression of TLR12 in brains of mock infected control and NCC mice analyzed by immunofluorescence microscopy. Balb/c mice were infected intracranially with M. corti and sacrificed at various times p.i. Brain cryosections were analyzed for expression of TLR12 and cell specific markers using fluorochrome conjugated antibodies. Nuclear staining DAPI is blue. (A) Mock infected control stained with TLR12 specific antibody (400×). (B) M. corti infection at 1 wk showing TLR12 positive staining (400×). (C) M. corti infection at 3 wk when maximum TLR12 expression was observed (400×). (D) M. corti infection at 6 wk showing TLR12 positive staining (400×). (E) Mock control mice stained with TLR12 and neuron specific antibody (Neun) (400×). The insert e' represents a 2× magnification of a selected area depicting TLR12 expression (Red) on neurons. (F) M. corti infection at 3 wk showing TLR12 positive neurons, merged image (yellow/orange; 400×). (G) The relative levels of TLR12 expression in mock and M. corti infected animals was calculated as described in experimental procedures.
Figure 4
Figure 4
Expression of TLR13 in brains of mock infected control and NCC mice analyzed by immunofluorescence microscopy. Balb/c mice were infected intracranially with M. corti and sacrificed at various times p.i. Brain cryosections were analyzed for expression of TLR13 and cell specific markers using fluorochrome conjugated antibodies. Nuclear staining DAPI is blue. (A) Mock infected control stained with TLR13 specific antibody (400×). (B) M. corti infection at 1 wk showing TLR13 positive staining in neurons (400×). (C) M. corti infection at 3 wk when maximum TLR13 expression was observed (400×). (D) M. corti infection at 6 wk showing TLR13 positive staining (400×). (E) Mock control mice stained with TLR13 and neuron specific antibody (Neun) (400×). The insert e' represents a 2× magnification of a selected area depicting TLR13 expression (Red) on neurons. (F) M. corti infection at 3 wk showing TLR13 positive neurons, merged image (yellow/orange; 400×). (G) M. corti infection at 3 wk showing TLR13 positive astrocytes and its processes, merged image (yellow/orange; 400×). The arrow points to the pial vessel expressing TLR13. (H) The relative levels of TLR13 expression in mock and M. corti infected animals was calculated as described in experimental procedures.
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