Ocular Onchocerciasis : The Role of Toll-Like Receptor-2 and Interferon-γ

River Blindness is still highly prevalent in subsaharan Africa with millions of people suffering from the infection with Onchocerca volvulus. This filarial worm harbors the endosymbiont Wolbachia which was found to be essential for worm survival. The immune system of chronically infected individuals is constantly stimulated with worm proteins, lipids, carbohydrates and released Wolbachia from dying worms. In this study, I investigated the activation of the adaptive immune system by O. volvulus and Wolbachia in a mouse model of onchocercal keratitis. Toll-like receptors (TLRs) are innate immune receptors that are activated by conserved bacterial and viral products. Lateley, it was also demonstrated that fungal and helminth products can activate selected TLRs. During filarial infection, stimulation occurs through both bacterial and parasitic products, I therefore focused on the role of TLR2 and TLR4 in the pathogenesis of adaptive immune responses and keratitis development. Using a mouse model of onchocercal keratitis, I immunized C57BL/6, TLR2-/- and TLR4-/- mice with O. volvulus protein extract and examined granulocyte migration to the corneal stroma following local injection of this protein extract. I found that TLR2 was required for IFNγ production by splenocytes, CXC chemokine production in the cornea and neutrophil migration to the corneal stroma, but not for IL-5 production or eosinophil migration. IFNγ was shown to increase surface expression of TLR2 on macrophages, priming them for further activation via TLR2 agonists like onchocercal protein extract. The pro-inflammatory cytokines produced by macrophages induced chemokine production by corneal fibroblasts. This is one pathway of IFNγ enhanced neutrophil migration to the corneal stroma. Other parts of this study showed that pro-inflammatory Th1 responses were predominantly induced by the endosymbiont Wolbachia rather than the filarial worm itself, whereas Th2 responses were mainly induced by the worm. One wolbachial protein that has been previously described and acts as immunomodulator is the Wolbachia surface protein. I characterized TLR2-dependent activation of primary macrophages and splenocytes and identified the region 121-240 of the protein as the main immunostimulatory domain

ST2 Deficiency Does Not Impair Type 2 Immune Responses during Chronic Filarial Infection but Leads to an Increased Microfilaremia Due to an Impaired Splenic Microfilarial Clearance

Background Interactions of the Th2 cytokine IL-33 with its receptor ST2 lead to amplified Type 2 immune responses. As Type 2 immune responses are known to mediate protection against helminth infections we hypothesized that the lack of ST2 would lead to an increased susceptibility to filarial infections. Methodology/Principal Finding ST2 deficient and immunocompetent BALB/c mice were infected with the filarial nematode Litomosoides sigmodontis. At different time points after infection mice were analyzed for worm burden and their immune responses were examined within the thoracic cavity, the site of infection, and systemically using spleen cells and plasma. Absence of ST2 led to significantly increased levels of peripheral blood microfilariae, the filarial progeny, whereas L. sigmodontis adult worm burden was not affected. Development of local and systemic Type 2 immune responses were not impaired in ST2 deficient mice after the onset of microfilaremia, but L. sigmodontis infected ST2-ko mice had significantly reduced total numbers of cells within the thoracic cavity and spleen compared to infected immunocompetent controls. Pronounced microfilaremia in ST2-ko mice did not result from an increased microfilariae release by adult female worms, but an impaired splenic clearance of microfilariae. Conclusions/Significance Our findings suggest that the absence of ST2 does not impair the establishment of adult L. sigmodontis worms, but is important for the splenic clearance of microfilariae from peripheral blood. Thus, ST2 interactions may be important for therapies that intend to block the transmission of filarial disease

der Rheinischen‐Friedrich‐Wilhelms‐Universität Bonn

de

The CD40-autophagy pathway is needed for host protection despite IFN-Γ-dependent immunity and CD40 induces autophagy via control of P21 levels.

Autophagy degrades pathogens in vitro. The autophagy gene Atg5 has been reported to be required for IFN-γ-dependent host protection in vivo. However, these protective effects occur independently of autophagosome formation. Thus, the in vivo role of classic autophagy in protection conferred by adaptive immunity and how adaptive immunity triggers autophagy are incompletely understood. Employing biochemical, genetic and morphological studies, we found that CD40 upregulates the autophagy molecule Beclin 1 in microglia and triggers killing of Toxoplasma gondii dependent on the autophagy machinery. Infected CD40(-/-) mice failed to upregulate Beclin 1 in microglia/macrophages in vivo. Autophagy-deficient Beclin 1(+/-) mice, mice with deficiency of the autophagy protein Atg7 targeted to microglia/macrophages as well as CD40(-/-) mice exhibited impaired killing of T. gondii and were susceptible to cerebral and ocular toxoplasmosis. Susceptibility to toxoplasmosis occurred despite upregulation of IFN-γ, TNF-α and NOS2, preservation of IFN-γ-induced microglia/macrophage anti-T. gondii activity and the generation of anti-T. gondii T cell immunity. CD40 upregulated Beclin 1 and triggered killing of T. gondii by decreasing protein levels of p21, a molecule that degrades Beclin 1. These studies identified CD40-p21-Beclin 1 as a pathway by which adaptive immunity stimulates autophagy. In addition, they support that autophagy is a mechanism through which CD40-dependent immunity mediates in vivo protection and that the CD40-autophagic machinery is needed for host resistance despite IFN-γ

Deficiency in ST2 leads to pronounced microfilaremia and increased adult worm length.

<p>A, microfilariae count per 50 μl of peripheral blood of ST2-ko mice and wild type (WT) controls throughout <i>L. sigmodontis</i> infection. B, microfilarial burden in the thoracic cavity 60 days post <i>L. sigmodontis</i> infection. C, percentage of ST2-ko mice and WT controls that develop patent infections. D, embryogram of female worms 60dpi (6 mice per group and two female worms per mouse). E, adult worm burden in ST2-ko mice and WT controls 35, 60 and 100 dpi, (F and G) length of male and female <i>L. sigmodontis</i> worms in WT and ST2-ko mice during infection. A and C show pooled data from three independent experiments with a minimum of 8 mice per group. B shows pooled data from two independent experiments and E-G show representative data of two independent experiments with a minimum of 6 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05.</p

Reduced Th2 cytokine production after in vitro restimulation of thoracic cavity cells of acute, but not chronically <i>L. sigmodontis</i> infected ST2-ko mice.

<p>Isolated cells from the thoracic cavity of individual <i>L. sigmodontis</i> infected wild type (WT) or ST2-ko mice were cultured in vitro with either <i>L. sigmodontis</i> antigen (LsAg, left panel) or ConA (right panel). IL-4 (A,B), IL-5 (C,D), IL-10 (E,F), IFNγ (G,H), IL-33 (I,J) and IL-25 (K,L) within the culture supernatants were measured on days 35, 60 or 100 post infection. Data is representative for two independent experiments with at least 5 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05, **p<0.01.</p

Absence of the ST2 receptor does not change thoracic cavity IL-4, IL-5, IL-13, IL-25, IL-33 and IFNγ levels during <i>L. sigmodontis</i> infection.

<p>Local concentrations of IL-4 (A), IL-5 (B), IL-13 (C), IL-25 (D), IL-33 (E) and IFNγ (F) in the thoracic cavity lavage prior to infection (day 0) and 35, 60, and 100 days post <i>L. sigmodontis</i> infection of wild type (WT) and ST2-ko mice. Data is representative for two independent experiments with at least 5 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test.</p