Aerosol-induced brucellosis increases TLR-2 expression and increased complexity in the microanatomy of astroglia in rhesus macaques

Abstract

Brucella melitensis, a bacterial pathogen and agent of epizootic abortion causes multiple pathologies in humans as well as a number of agriculturally important animal species. Clinical human brucellosis manifests as a non-specific, chronic debilitating disease characterized by undulant fever, arthropathies, cardiomyopathies and neurological sequelae. These symptoms can occur acutely for a few weeks or persist for months to years. Within the brain, endothelial and glial cells can be infected leading to downstream activation events including matrix metalloprotease (MMP) and cytokine secretion and Toll-like receptor (TLR) signaling. These events are likely to lead to tissue remodeling, including morphologic changes in neuronal and glial cells, which are linked to neurological complications including depressive behavior, immune activation and memory loss. Our hypothesis was that B. melitensis infection and neurobrucellosis would lead to activation of astrocytes through upregulation of TLR2 and stimulate concurrent changes in the microanatomy. All six animals were infected via inhalation route. TLR2 expression was approximately doubled in white matter astrocytes of infected rhesus macaques. There was also a 50% increase in the number of astrocytes per unit area in subcortical white matter tracts suggesting increased innate immune activation. This coincided with dramatic increases in the length and complexity of the cell arbor of hypertrophic astrocytes in both cortical gray and white matter. Thus, aerosol-induced brucellosis results in dramatically increased innate immune activation of astrocytes in the absence of widespread neuroinflammation.

Keywords: Brucella; astrocyte; gliosis; innate immune activation; toll-like receptor.

Figure 1

Histologic examination of macaque brain tissues of animals infected with B. melitensis. Two of six animals infected with Brucella had evidence of focally mild to moderate nonsupperative encephalitis (A,B). Four of six animals had no histologic evidence of encephalitis (C).

Figure 2

Innate immune activation is coincident with increased astrocyte numbers in white matter. In control macaques, very few GFAP-positive (red) astrocytes express TLR2 (A, green). Arrowheads demonstrate double positive cells. In contrast, more astrocytes express TLR2 following infection with B. melitensis (B). To confirm if this increase was significant, the proportion of double labeled cells was calculated. 3.9% of white matter astrocytes were TLR2 positive (C). This increased to 9.2% (P = 0.0008) with infection. This was coincident with an increased number of astrocytes within the white matter (D). Due to the low expression of GFAP positive cells in cortical gray matter, the percentage of TLR2 and GFAP double-labeled astrocytes could not be evaluated quantitatively. Significance set at ^*p < 0.05, Scale bar = 25 um.

Figure 3

Schematic of astrocytes and parameters measured. Astrocytes were identified using GFAP antibody. Images were imported into Neurolucida and examined in Neurolucida explorer. At left, an astrocyte from a control macaque (A). At right, an astrocyte from a macaque infected with B. melitensis (B). Each concentric ring represents 10 μm.

Figure 4

B. melitensis infection induces astrocyte hypertrophy. The total length of processes of astrocytes from infected macaques were increased in both gray and white matter (A, G). Similarly, the volume of the processes was increased (B, H). This was coincident with a hypertrophied cell body (C, I). It was curious that there were no changes in the number of primary processes (D, J). Therefore, the increased arbor was a result of increased branching (E, K) and thus, tip quantity (F, L) in astrocytes of macaques infected with Brucella. ^*p < 0.05.

Figure 5

Astrocytes have increased complexity following Brucella infection. In both gray and white matter astrocytes there was an increase in the number of intersections from 20 μm onwards (A, B, respectively). This was borne out by the increased number of bifurcations in the astrocyte processes (C, D) leading to increased complexity of the astrocytes.