Post-treatment vascular leakage and inflammatory responses around brain cysts in porcine neurocysticercosis

Abstract

Cysticidal treatment of neurocysticercosis, an infection of humans and pig brains with Taenia solium, results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations. Treatment-induced pericystic inflammation and its association with blood brain barrier (BBB) dysfunction, as determined by Evans blue (EB) extravasation, was studied in infected untreated and anthelmintic-treated pigs. We compared the magnitude and extent of the pericystic inflammation, presence of EB-stained capsules, the level of damage to the parasite, expression of genes for proinflammatory and regulatory cytokines, chemokines, and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained (blue) and non stained (clear) cysts. Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment. The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours. Expression levels of the proinflammatory genes for IL-6, IFN-γ, TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues, and were generally highest at 120 hr. Additionally, expression of some markers of immunoregulatory activity (IL-10, IL-2Rα) were decreased in EB-stained capsules. An increase in other markers for regulatory T cells (CTLA4, FoxP3) was found, as well as significant increases in expression of two metalloproteases, MMP1 and MMP2 at 48 hr and 120 hr post-treatment. We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response, largely limited to pericystic tissues with compromised vascular integrity. Because treatment induced inflammation occurs in porcine NCC similar to that in human cases, this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation.

Fig 1. Clear and blue-stained capsules (EB capsules) on the surface and cut surfaces of brains from infected pigs.

Photographs of the surface and coronal sections of representative brains from an untreated pig (A and B) and a pig treated with praziquantel 48 h previously (C and D), showing cysts with clear (white arrowheads) and blue capsules (blue arrowheads) visible within the parenchyma and on the surface.

Fig 2. EB capsules show increased inflammation and cyst wall damage compared to clear capsules.

Cysts were harvested from the brains of infected untreated pigs (n = 3; see Methods) or pigs treated with PZQ 48h (n = 4) or 120h (n = 4) earlier. For each cyst, scores reflecting the degree and extent of the inflammatory cell infiltrates (A) and cyst wall damage (B) at 48h (PZQ 48h) and 120h (PZQ 120h) after PZQ treatment (see Methods and S1 Fig.) are shown (open squares refer to clear capsules and closed squares, to EB capsules, bar = mean). Statistical significance in comparison between groups is represented by asterisks (*: p<0.05; **: p<0.01; ***: p<0.005).

Fig 3. Upregulation of proinflammatory genes in EB capsules compared to clear capsules.

Expression levels of RNA in six types of brain tissue samples, uninfected brain tissue distant from cysts (no cysts), clear pericystic brain tissue in untreated pigs (UT Clear) and at 48h post treatment (PZQ 48 Clear) and EB capsules in untreated pigs (UT Blue) and at 48h (PZQ 48 h Blue) and 120h post treatment (PZQ 120 h Blue) were quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays. Gene expression levels are depicted as fold increase of studied RNA over reference (housekeeping) RNA (18S rRNA), TNF-α (A; number [n] of samples for the indicated types of capsules are shown below each bar) and IL-6 (B). The bars represent means and CI₉₅ for the group. Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.

Fig 4. Upregulation of both Th1 and Th2 markers, IFN-γ and IL-13, in blue capsules following PZQ treatment.

Expression levels of RNA in brain tissues and EB-stained and clear capsules for IFN-γ (A; number [n] of samples for the indicated types of capsules are shown below each bar), and IL-13 (B). In each graph, the bars represent means and CI₉₅ for the group. Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.

Fig 5. Increased expression of CD-25 and IL-10 in EB capsules following PZQ treatment.

Expression levels of RNA in brain tissue samples from EB-stained and clear capsules were quantified (See Methods). Shown are expression levels, depicted as fold increase over the RNA levels for the reference (housekeeping) gene (18S rRNA), encoding four representative regulatory T cells (Treg) phenotyping and functional markers: CD25 (A; number [n] of samples for the indicated types of capsules are shown below each bar), FoxP3 (B), IL-10 (C) and CTLA4 (D). Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.

Fig 6. Increased expression of fibrosis/granuloma promoting genes in EB and clear capsules.

Methods and tissues examined are identical to those analyzed in Figs. 3–5. Shown are four genes representative of fibrosis and granuloma promoting molecules: MMP1 (A; number [n] of samples for the indicated types of capsules are shown below each bar; n = 7, 6, 3, 6, 6, respectively), MMP9 (B), TIMP1 (C) and TIMP2 (D). Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.