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

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 immunofluoresence 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 (~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

MyD88-Deficient Mice Exhibit Decreased Parasite-Induced Immune Responses but Reduced Disease Severity in a Murine Model of Neurocysticercosis▿

The symptomatic phase of neurocysticercosis (NCC), a parasitic disease of the central nervous system (CNS) in humans, is characterized by inflammatory responses leading to neuropathology and, in some cases, death. In an animal model of NCC in which mice were intracranially inoculated with the parasite Mesocestoides corti, the infection in mice lacking the myeloid differentiation primary response gene 88 (MyD88−/−) resulted in decreased disease severity and improved survival compared with that in wild-type (WT) mice. The CNS of MyD88−/− mice was more quiescent, with decreased microgliosis and tissue damage. These mice exhibited substantially reduced primary and secondary microglial nodule formations and lacked severe astrogliotic reactions, which were seen in WT mice. Significantly reduced numbers of CD11b+ myeloid cells, αβ T cells, γδ T cells, and B cells were present in the brains of MyD88−/− mice in comparison with those of WT mice. This decrease in cellular infiltration correlated with a decrease in blood-brain barrier permeability, as measured by reduced fibrinogen extravasation. Comparisons of cytokine expression indicated a significant decrease in the CNS levels of several inflammatory mediators, such as tumor necrosis factor alpha, gamma interferon, CCL2, and interleukin-6, during the course of infection in MyD88−/− mice. Collectively, these findings suggest that MyD88 plays a prominent role in the development of the hyperinflammatory response, which in turn contributes to neuropathology and disease severity in NCC

Ly6C(high) monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1(GFP/+) mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C(low) monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C(high) and Ly6C(low) monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C(high) monocytes, but that these cells may transition through a Ly6C(low) state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C(high) monocytes via help from CD4⁺ T cells

CX₃CR1-GFP⁺ cells upregulate PD-L2 as they extravasate and accumulate in liver tissue.

<p>(<b>A and B</b>) Representative contour plots and graphs displaying PD-L2 expression on liver leukocytes isolated from uninfected or infected <i>CX₃CR1^gfp/+</i> mice gated on live, single, lineage (CD3, B220, DX5 and Siglec F) negative, SSC^low cells. n = 4 mice/group. (<b>C</b>) qRT-PCR analysis of Relmα expression. (<b>D</b>) Representative contour plots show blood/tissue partitioning of GFP⁺ liver leukocytes from CX₃CR1^GFP/+ mice using <i>in vivo</i> CD45 staining. Plots display cells gated on single, live, lineage negative cells. (<b>E</b>) The proportion of CD45⁺ (white) or CD45⁻ (black) cells gated on live single, lineage⁻, GFP⁺ cells. (<b>F</b>) Contour plots display blood/tissue partitioning of PDL2⁺, GFP⁺ cells gated on single, live, lin-, CX₃CR1-GFP⁺ cells. (<b>G</b>) Proportions of GFP⁺PDL2⁺ cells that are CD45⁺ (white) or CD45⁻ (black) as in (<b>E</b>). (<b>H</b>). qRT-PCR analysis of YM1 expression in liver leukocytes sorted from 8 week infected mice using <i>in vivo</i> CD45 staining to isolate CD45−(tissue) GFP+ granuloma macrophages from CD45+ (blood) GFP+, Ly6C^high and Ly6C^low monocytes. CD11b-GFP− cells from infected mice were used as a negative control. Dots represent individual mice from one of two experiments. Statistical significance was determined by One-way ANOVA with Dunnett's test. ** = p<0.001, *** = p<0.0001. Results are representative of at least two experiments that include n = 3–4 mice/group.</p

GFP⁺Ly6C^low monocytes exhibit patrolling behavior, while GFP⁺Ly6C^high cells transit rapidly through the sinusoids.

<p>(<b>A</b>) High magnification snapshot showing tracks of GFP+ crawling cells (white) and GFP+ cells that move rapidly through the sinusoids (yellow) in a steady state uninfected liver. Scale bar = 50 µm. (<b>B</b>) Representative tracks of crawling monocytes in steady state uninfected livers. (<b>C and E</b>) Intravital confocal microscopy of an uninfected <i>Cx₃cr1^gfp/+</i> mouse showing GFP+ (green) monocytes that are either Ly6C^high (<b>C</b>) or Ly6C^low (<b>E</b>) after intravenous anti-Ly6C Ab staining (red). (<b>D and F</b>) Time-lapse images from an uninfected <i>Cx₃cr1^gfp/+</i> mouse injected with anti-Ly6C Ab showing two GFP+ Ly6C^high monocytes (<b>D</b>) and two GFP+ Ly6C^low monocytes migrating through the sinusoids (<b>F</b>). GFP is shown in green, anti-Ly6C in red, and nuclei in blue. Snapshots were taken in single z planes. Scale bars = 20 µm. Tracks are shown in white.</p

CX₃CR1-GFP⁺ monocytes accumulate in the liver when <i>S. mansoni</i> eggs appear without proliferation.

<p>(<b>A–C</b>) Representative flow cytometry plots and graphs display the proportion of GFP⁺ Ly6C^high and Ly6C^low monocytes from CX₃CR1^GFP/+ mice gated on live, single, lineage (CD3, B220, DX5 and Siglec F) negative, SSC^low cells. (<b>D–E</b>) qRT-PCR analysis of CCR2 and eGFP expression in the liver. (<b>F</b>) Representative contour plots show <i>in situ</i> EdU labeling of liver leukocytes gated on live, single, lineage negative, CD11b⁺ cells. Results are representative of two experiments that included n = 3–4 mice/group. Statistical significance was determined by One-way ANOVA with Dunnett's test. * = p<0.05, ** = p<0.001, *** = p<0.0001.</p

The presence of <i>S. mansoni</i> eggs alters the patrolling behavior of CX₃CR1-GFP⁺ monocytes in the liver sinusoids.

<p>(<b>A–C</b>) Maximum projections of Z stacks from the livers of (<b>A</b>) uninfected <i>Cx₃cr1^gfp/+</i> mice and <i>S. mansoni-</i>infected <i>Cx₃cr1^gfp/+</i> mice with an egg encapsulated in a granuloma (<b>B</b>), or with an egg in the blood vessels (<b>C</b>). Sinusoidal vessels (dark areas) and tissue architecture are visualized by nuclear staining (blue) and auto-fluorescence in the red channel. Parasite eggs are auto-fluorescent and can be seen in red. White tracks showing the paths of individual motile CX₃CR1-GFP⁺ cells (green) are overlayed onto images. Scale bars = 50 µm (<b>D</b>) Log-transformed mean speed (µm/min), (<b>E</b>) track duration of motile GFP⁺ cells, (<b>F</b>) arrest coefficient (fraction of time cell crawls <2 µm/min) and (<b>G</b>) confinement ratio (Displacement/track length). Motility data is pooled from 3 mice for uninfected mice (n = 68 cells), 5 mice for fully developed granulomas (n = 182 cells). Data for exposed eggs in the vasculature is pooled from 6 mice (n = 143). Scale bars = 100 µm. * = p<0.05, ** = p<0.001, *** = p<0.0001.</p