Pathogenesis of Ocular Onchocerciasis

Microfilariae invade both the anterior and the posterior eye. In the latter case, they cause uveitis and chorioretinitis, resulting in loss of vision. In the anterior segment, they are present in the anterior chamber and cornea, where they cause sclerosing keratitis.

Eyes from human cases of onchocerciasis are difficult to obtain and show only the late stages of disease; however, in the cornea these manifest as an infiltrate of monocytic and granulocytic cells in the stroma, often surrounding dead and degenerating worms [22]. More revealing are findings from Onchocerca dermatitis studies, showing microfilariae surrounded by neutrophils, eosinophils or macrophages [23].

Early experimental models of O. volvulus keratitis using guinea pig and murine models demonstrated that prior immunization is essential to develop the corneal opacification and neovascularization characteristic of later stage scle-rosing keratitis, which is consistent with responses found in chronically infected individuals [22, 24, 25]. More recent studies from our group and others showed that keratitis is associated with a predominant CD4+, Th2 response both systemically and in the cornea, that IgE and parasite-specific IgG1 were the predominant isotypes produced, and that the predominant cellular infiltrate is neutrophils [26-28]. The use of B cell-deficient |xMT mice and Fc7R-/— mice revealed that Fc receptors on neutrophils and eosinophils facilitate degranulation of these cells and disruption of corneal clarity [29, 30]. Further studies demonstrated that neutrophil recruitment was mediated by CD31/PECAM-1 and chemokine receptor CXCR2, whereas eosinophil recruitment is dependent on eotaxin, P-selectin and ICAM-1 [27, 31-34].

These findings are consistent with the sequence of events outlined in figure 1: (1) immunization or chronic infection induces a predominant Th2 response, with IL-4 leading to isotype switching to IgE and IgG1, and IL-5 inducing eosinophil differentiation; (2) parasite antigens in the corneal stroma (after microfilaria invasion or injection of parasite antigens) lead to activation of resident cells in the cornea, production of CXC and CC chemokines, elevated expression of adhesion molecules on vascular endothelial cells in the lim-bus, and infiltration of neutrophils and eosinophils to the corneal stroma, and (3) immune complex-mediated cross linking of Fc receptors on neutrophils and eosinophils results in degranulation and release of cationic proteins and other cytotoxic mediators that disrupt normal corneal clarity. In heavily infected individuals, the response to repeated microfilaria invasion over a number of years results in sclerosis and blindness.

Role of Innate Immunity in O. volvulus Keratitis

The role of the innate immune response in Onchocerca keratitis has not been investigated in detail for at least two reasons: firstly, as a model for chronically infected individuals who are presumably sensitized prior to ocular involvement, there would be no example of innate immunity in the eye in the

Lymphoid tissue

Corneal stroma

Lymphoid tissue

Corneal stroma

Corneal Immune Privilege

B cell

Anti-O. volvulus LJ |mmune IgG1 I I ^complexes

B cell

Anti-O. volvulus LJ |mmune IgG1 I I ^complexes

Loss of cornel clarity

Loss of cornel clarity

Fig. 1. Proposed sequence of events in adaptive immune response underlying Onchocerca keratitis.

absence of an adaptive immune response, and secondly, experimental models showed no detectable corneal opacification or neovascularization unless animals were first immunized [24, 25, 28]. However, in vivo confocal microscopy clearly demonstrated a cellular infiltrate in the corneas of unimmunized mice injected intrastromally with parasite antigens. Further, the cellular infiltrate, which was primarily neutrophils, was associated with an increase in corneal thickness and haze [35]. This approach then allowed examination of innate immunity to O. volvulus in the absence of an adaptive immune response.

Using this mouse model of O. volvulus keratitis, we demonstrated that endosymbiotic Wolbachia bacteria are essential for the pathogenesis of O. volvulus keratitis as O. volvulus from individuals depleted of Wolbachia by antibiotic treatment do not induce corneal inflammation [35]. Furthermore, related filarial species containing Wolbachia induce keratitis in contrast to aposymbiotic species lacking Wolbachia [35].

To examine the early host responses to Wolbachia in the cornea, we injected whole microfilariae into the corneal stroma and followed the fate of Wolbachia by immunogold labeling of the major Wolbachia surface protein (WSP) [36]. Figure 2 shows the presence of Wolbachia in the microfilariae in the cornea, with neutrophils in immediate proximity. Figure 3 shows immunogold labeling in neutrophil vacuoles surrounded by primary granules, supporting the notion that Wolbachia are ingested by neutrophils. Furthermore, incubation of neutrophils with Wolbachia stimulates release of TNF-a and CXC chemokines KC/CXCL1 and MIP-2/CXCL2 [36].

Wolbachia and Toll-Like Receptors

Toll-like receptors (TLR) are a family of at least twelve pathogen recognition molecules that respond to microbial products such as lipopolysaccharide (LPS; TLR4), bacterial cell wall components (TLR2), DNA-containing unmethy-lated CG motifs (TLR9) and viral RNA (TLR3, TLR7, and TLR8) [37, 38]. Several reports indicate that Wolbachia activate the innate immune responses via TLR-dependent pathways: (1) Wolbachia activation of macrophages is decreased in C3H/HeJ mice, which have a point mutation in TLR4 that makes it hyporesponsive to LPS [21]; (2) the severity of O. volvulus keratitis was reduced in C3H/HeJ mice [35], and (3) recombinant WSP activates TLR2 and TLR4 [39]. We also show a role for TLR2 in Wolbachia and filarial activation [Gillette-Ferguson et al., submitted]. Although initial reports suggested that Wolbachia have LPS-like activity, sequencing of the Wolbachia revealed no LPS synthase enzymes [40], indicating that TLR2 and TLR4 agonists are more likely to be surface proteins such as WSP and other cell wall components. Our most recent findings show that mice that are deficient in the adaptor molecule myeloid differentiation factor 88 (MyD88), which is common to the signaling pathways of TLR2 and TLR4, do not develop keratitis in response to O. volvulus antigens or to isolated Wolbachia bacteria [41]. Consistent with this observation, isolated neutrophils from MyD88-/- mice are not activated by Wolbachia bacteria or O. volvulus antigens, indicating an essential role for this adaptor molecule at two stages of pathogenesis - production of CXC chemokines by resident cells and neutrophil activation [41].

Taken together, findings from our group and others suggest a role for the innate immune response in Onchocerca keratitis, as shown in figure 4. As TLR2 and TLR4 are expressed in the cornea [42-44], and activation can induce keratitis, we predict that an inflammatory response to Wolbachia is initiated by TLRs on keratocytes, which are likely to be activated after death and degeneration of microfilariae and release of Wolbachia into the confined environment of the corneal stroma. Activated keratocytes can mature into stromal fibroblasts, which produce pro-inflammatory cytokines and CXC chemokines [42, 45], and can induce adhesion molecule expression on vascular endothelial cells [32, 33]. Together, these changes mediate neutrophil recruitment from peripheral, limbal vessels into the avascular corneal stroma and migration through the stromal matrix to the site of microfilaria degradation and release of Wolbachia. A second

Brugia Malayi Wolbachia

Fig. 2. Proximity of neutrophils to Wolbachia in the nematode hypodermis. C57BL/6 mice were injected with microfilariae into the corneal stroma, corneas were removed after 4 or 18 h, and thin sections were immunostained with anti-WSP and visualized with IgG conjugated to 15-nm gold particles. Sections were counterstained with uranyl acetate and lead citrate, and examined by electron microscopy. a, b 4h after injection, WSP was clearly detected inside microfilariae in the corneal stroma (arrows). mf = Microfilariae. c-e 18 h after injection, microfilariae containing Wolbachia were surrounded by neutrophils (PMN). WSP-labeled with gold particles (arrows) are present in the microfilariae adjacent to the neutrophils in either unimmunized (c) or immunized (e) mice. a X4,800. b X8,400. c X5,300. d X 16,000. e X 14,57500 (reprinted with permission [36]).

Fig. 2. Proximity of neutrophils to Wolbachia in the nematode hypodermis. C57BL/6 mice were injected with microfilariae into the corneal stroma, corneas were removed after 4 or 18 h, and thin sections were immunostained with anti-WSP and visualized with IgG conjugated to 15-nm gold particles. Sections were counterstained with uranyl acetate and lead citrate, and examined by electron microscopy. a, b 4h after injection, WSP was clearly detected inside microfilariae in the corneal stroma (arrows). mf = Microfilariae. c-e 18 h after injection, microfilariae containing Wolbachia were surrounded by neutrophils (PMN). WSP-labeled with gold particles (arrows) are present in the microfilariae adjacent to the neutrophils in either unimmunized (c) or immunized (e) mice. a X4,800. b X8,400. c X5,300. d X 16,000. e X 14,57500 (reprinted with permission [36]).

Ocular Onchocerciasis

Fig. 3. Wolbachia in neutrophil vacuoles: immunoelectron microscopy of neutrophils 18 h after injection of microfilariae. Immunogold particles specific for WSP were prominent in neutrophil vacuoles of both immunized (a, b) and unimmunized (c, d) mice. a X11,400. b X45,000. c X24,000. d X67,500 (reprinted with permission [36]).

Fig. 3. Wolbachia in neutrophil vacuoles: immunoelectron microscopy of neutrophils 18 h after injection of microfilariae. Immunogold particles specific for WSP were prominent in neutrophil vacuoles of both immunized (a, b) and unimmunized (c, d) mice. a X11,400. b X45,000. c X24,000. d X67,500 (reprinted with permission [36]).

role for TLR2, TLR4 and MyD88 is therefore ingestion of Wolbachia and activation of neutrophils at this site. As neutrophils express functional TLR2, TLR4 and TLR9 [46], they can produce TNF-a MIP-2 and KC in response to Wolbachia [36], which stimulate further neutrophil infiltration, degranulation and secretion of cytotoxic products such as nitric oxide and myeloperoxidase and oxygen radicals. A cytotoxic effect on keratocytes and corneal endothelial cells will lead to loss of corneal clarity.

Mouse Keratitis Onchocerciasis
Fig. 4. Proposed sequence of events in innate immune responses underlying Onchocerca keratitis. MPO = Myeloperoxidase; NO = nitric oxide.

In chronically infected, untreated individuals, there is also an ongoing adaptive immune response, repeated invasion of microfilariae into the corneal stroma, and consistent worm degeneration and release of Wolbachia. The sustained inflammatory response in the presence of antibody and infiltration of eosinophils and macrophages combine to cause corneal opacification, loss of vision and blindness.

Conclusion

Studies using animal models of river blindness have helped our understanding of the pathogenesis of this disease. Most prominently, they have shown the essential role for endosymbiotic Wolbachia bacteria and the innate immune response in development of keratitis. Future studies will examine the role of TLRs in development of adaptive immunity in this important disease, and may identify targets for immune intervention.

Acknowledgments

This work was supported by NIH grants EY10320, EY11373, and by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation. E.P. is also a recepient of an RPB Senior Investigator Award.

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  • diane
    Is there immunity to river blindness?
    5 years ago

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