Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Pittsburgh, PA, USA

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

Anterograde axonal transport – Transport through the cytoplasm of axons from the neuron cell body or soma towards the synapse.

Chemokine – A combined form of the words chemotactic (able to attract cells) and cytokines. Chemokines are a family of low-molecular-weight chemotactic cytokines that attract and activate leukocytes at sites of infection and inflammation. Cytokine – A large and diverse family of intercellular molecular messengers that, like hormones, transmit information from one cell to another. The term is often used to denote polypeptides with immunomodulatory activity.

HSV-1 latency – The retention of a complete viral genome for extended periods without production of infectious virions.

Immunosurveillance – The continuous monitoring function of the immune system whereby it recognizes and reacts against aberrant cells arising within the body.

Regulatory T cells – Specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens.

Retrograde axonal transport – The transport of vesicles from the synaptic region of an axon toward the cell body or soma.

T cells – A population of lymphocytes that derive from bone marrow precursors, mature in the thymus, and play a central role in cell-mediated immunity. Trigeminal ganglia – The ganglia that house the cell bodies of primary afferent neurons innervating the head and neck.

Virion – A mature infectious virus particle existing outside a cell.

Natural History of Herpes Stromal

Keratitis

Although herpes stromal keratitis (HSK) has been studied in a variety of animal models, much of our current knowledge regarding the immunopathogenesis of HSK has derived from murine models due to the plethora of

immunologic reagents available to dissect the immune response in mice. Strain differences in susceptibility to HSK have been noted with A/J>Balb/c>C57BL/6. With most strains of HSV-1, corneal infection requires surface abrasion to facilitate entry of the virus into corneal epithelial cells. An epithelial lesion then forms as a result of virus replication in and destruction of epithelial cells. The lesions are transient, resolving by 4 days post infection (dpi), with eradication of replicating HSV-1 by 7–8 dpi. Healing of the lesions is aided by the rapid turnover of corneal epithelial cells.

Eradication of Replicating HSV-1 from the Corneal Epithelium

Viral clearance appears to be largely a function of the innate immune system, consistent with the rapid kinetics of virus elimination. Evidence suggests a role for neutrophils, natural killer (NK) cells, and gd T cells in the eradication of replicating virus from the cornea. These cells are attracted to and activated within the cornea as a result of the release of cytokines, including cyclooxygenase 2, prostaglandin E2, type 1 interferon, interleukin (IL)-1, IL-6, IL-17, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor (TNF); and chemokines including CXCL10 (IP-10), CXCL9 (MIG), CXCL2 (MIP-2), and CCL3 (MIP-1a). The production of these cytokines and chemokines appears to be initiated by ligation of tolllike receptors (TLRs), especially TLR2 and TLR9, and in response to cytokines including IL-17, IL-6, and interferongamma (IFN-g). The initial neutrophilic infiltrate peaks in HSV-1 infected mouse corneas around 4 dpi, and declines between 6 and 8 dpi, coincident with the peak and decline of viral titers in the mouse cornea.

The adaptive immune system appears to play a less significant role in the initial clearance of HSV-1 from the infected cornea following primary infection. However, it is noteworthy that severe combined immune-deficient (SCID) mice that lack an adaptive immune system do not fully eradicate replicating virus from the cornea. Accordingly, activated CD4 T cells have been detected within the infected cornea by 3 dpi, although their role in inhibiting HSV-1 replication within the cornea has not been established. It is possible, and indeed likely that the failure of SCID mice to eradicate replicating HSV-1 from the cornea during primary infection results from their inability to fully establish and maintain viral latency in the trigeminal

ganglion. Replicating virus in the trigeminal ganglion could then be transported back to and shed into the cornea, perpetuating the corneal infection. The role of T cells in clearing virus from the corneal epithelium following reactivation has not been studied. Antibodies also appear to play an important role in the clearance of HSV-1 from the cornea during both primary and recurrent disease.

HSV-1 Colonization of Sensory Ganglia

During the course of virus replication in epithelia of the cornea, oral, or nasal passages of mice, the virus gains access to the termini of sensory neurons and is transported to neuronal nuclei in trigeminal ganglia. There it replicates briefly and then establishes a latent infection that is characterized by retention of a functional viral genome without production of infectious virions. Evidence suggests that control of acute HSV-1 replication and establishment of latency in trigeminal ganglia is mediated largely by an innate response of macrophages and NK cells mediated by TNFa, IFN-g, and nitric oxide. However, complete eradication of replicating virus requires reinforcement by CD8+ T cells and gd T cells. Latently infected neurons within the ophthalmic branch of trigeminal ganglia then serve as a source of virus for recurrent corneal disease. HSV-specific CD8+ T cells then remain in the trigeminal ganglion in close apposition to infected neurons for the life of the mouse. These CD8+ T cells express a persistent activation phenotype and have been shown to inhibit HSV-1 reactivation from latency both in vivo and in ex vivo ganglionic cultures. A similar association of activated CD8+ T cells with latently infected neurons has also been established in human ganglia. Moreover, psychological stress, which is associated with recurrent herpetic disease in humans, causes a transient compromise of the function of ganglionic CD8+ T cells and HSV-1 reactivation from latency in trigeminal ganglia of mice. These findings suggest that maintenance of HSV-1 latency and prevention of recurrent shedding of virus at mucosal surfaces such as the cornea requires constant immunosurveillance by CD8+ T cells.

Herpes Stromal Keratitis

Following resolution of the epithelial lesion, corneal clarity is reestablished in mice. However, by 7–10 dpi corneal opacity develops accompanied by ingrowth of blood vessels into the normally avascular cornea, and massive leukocytic infiltration. These are manifestations of HSK, which in mice progresses from a non-necrotizing form with little epithelial involvement to severe necrotizing keratitis in which stromal inflammation gives rise to epithelial necrosis. The necrotizing form represents about 7% of human HSK, with 88% exhibiting the non-necrotizing form, and

5% exhibiting a mixture of both forms. It is not clear if the reduced incidence of necrotizing HSK in humans relative to mice is due to medical management of inflammation in human corneas or a reflection of fundamentally different pathogenic mechanisms in most human cases of HSK.

T Cells in HSK

Early studies revealed that HSK failed to develop in athymic nude mice that lacked T cells, but could be reconstituted in these mice by T-cell adoptive transfer. The CD4+ T cell population appears to be the predominant mediator of HSK in most mouse models, with CD8+ T cells tending to reduce disease severity. However, CD8+ T cells can also mediate a milder and more transient form of HSK when mice are deficient in CD4+ T cells or infected with certain strains of HSV-1. Human corneas with HSK contain both CD4+ and CD8+ T cells that are specific for HSV antigens, though the relative contribution of these cells in the progression or resolution of HSK cannot be evaluated. Recently, a population of CD4+ T cells referred to as natural T regulatory cells (nTregs) that co express CD25 and the forkhead/winged-helix transcriptional regulator, FoxP3 were shown to moderate the severity of HSK. The fact that these nTregs maintain the capacity to regulate HSK when expanded in vitro and adoptively transferred into infected mice offers a potentially useful therapeutic modality.

Cytokines and Their Role in HSK

Cytokines provide a means of communication among leukocytes and between leukocytes and parenchymal cells at sites of infection and/or antigen deposition. Under optimal conditions cytokines orchestrate an immunoinflammatory response that eliminates a pathogen with minimal damage to host tissue. In HSK resolution of the immunoinflammatory lesions in the cornea is accompanied by scar tissue formation. This trade-off of strength for clarity is unacceptable in a tissue within the visual axis. Moreover, the fact that in both mice and humans HSK can develop in the absence of replicating virus in the cornea renders HSK a purely immunopathological process. Thus, regulating the production and/or function of the cytokines that orchestrate HSK would reduce tissue damage without compromising protection of the host.

Most studies in the murine models have implicated the Th1 cytokines as playing a primary role in the pathogenesis of HSK. The prototypic Th1 cytokine, IFN-g, aids neutrophil extravasation into the cornea at least in part through upregulation of platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31) on local vasculature. Th1 cells also produce IL-2 that has been shown to directly or indirectly regulate the migration of neutrophils into the central cornea during HSK. The Th1-associated cytokine

IL-12 also contributes to HSK development, presumably through its capacity to regulate IFN-g production. The cytokine IL-6 also plays a central role in orchestrating HSK in part through its ability to induce the neutrophil chemokine MIP-2 (CXCL2).

Recently, the functional repertoire of CD4+ T cells was expanded to include Th17 cells. The IL-17 cytokine family represents the signature cytokines of Th17 cells, and is now implicated in a variety of inflammatory processes and autoimmune diseases. IL-17 is produced in both human and mouse corneas during HSK. Both human and mouse keratocytes express receptors for IL-17, and engagement of this receptor induces their production of neutrophil chemotactic factors as well as GM-CSF. The chemotactic factors attract neutrophils into the cornea while GM-CSF activates them and maintains their viability. Recent studies support a role for IL-17 in neutrophilic infiltration during the early stages of HSK. The requirement for IL-17 is transient and presumably superseded by Th1 cytokines as HSK progresses.

Regulating the production or blocking the function of the Th1 and Th17 cytokines offer potential therapeutic benefit in preventing the progression of HSK. In addition, the Th1/Th2/Treg cytokine IL-10 has a proven capacity to ameliorate HSK. The general anti-inflammatory properties include inhibition of the production and function of IFN-g.

Antigen-Presenting Cells in HSK

Antigen-presenting cell (APC) refers to the ability of certain cells, including macrophages, dendritic cells, and B lymphocytes to take up, process, and present antigenic peptides to naive CD4+ T cells in the context of major histocompatibility complex class II (MHC class II) and co-stimulatory molecules. The once prevalent view that the normal cornea is devoid of these specialized cells has now been dispelled by several recent histological studies of mouse and human corneas. Dendritic cells (DCs) are present in the basal layer of the corneal epithelium and macrophages are present throughout the corneal stroma, with the density of both cell types gradually diminishing from the peripheral to the central cornea.

During HSV-1 infection of the corneal epithelium, the resident DCs are reinforced by immigrants from the limbus. Studies employing ultraviolet irradiation to diminish DCs or cautery to increase their presence in the cornea prior to HSV-1 infection suggested a role for these cells in directly or indirectly inducing a delayed-type hypersensitivity response in lymphoid organs. Since DCs exhibit transient survival following HSV-1 infection, it is likely that either noninfected corneal DCs acquire viral antigens from corneal epithelial cells for direct presentation to T cells in the lymph nodes, or infected corneal DCs travel to the lymph nodes where they are phagocytosed by

resident DCs and viral antigens are cross-presented to T cells. The DC depletion studies also suggested an important role for corneal DC in presenting HSV-1 antigens to CD4+ T cells upon infiltration of the infected cornea. This is consistent with the observation that CD4+ T cells induce a second and more profound infiltration of DC into the cornea at the time of onset of HSK. It should be noted, however, that the method of DC depletion employed in these early studies was not specific, and the role of DC and DC subpopulations in the efferent and afferent limbs of the CD4+ T cell response in HSK awaits confirmation in studies employing specific depletion strategies now available.

Although many cell types can be induced to express MHC class II antigens, professional APCs are uniquely able to co-express the co-stimulatory molecules that are required for the activation of naive and, in many cases, effector CD4+ T cells. In a mouse model of HSK, blocking the interaction of the ligands B7-1 (CD86) and B7-2 (CD80) on APCs with the co-stimulatory molecule CD28 on CD4+ T cells locally within the cornea inhibited the HSK progression. In contrast, ligation of the inhibitory receptor program death 1 (PD-1) by its ligand B7-H1 (CD274) appears to inhibit HSK progression, though it is not clear if this effect is exerted in the cornea or lymphoid organs. HSK progression was not influenced by blocking the interaction of the APC activating receptor OX40 by its T-cell ligand OX40L (CD154). These studies suggest that local inhibition of co-stimulatory molecule or augmentation of inhibitory molecule signaling in CD4+ Tcells might hold therapeutic potential in the management of HSK.

Chemokine Involvement in HSK

Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes into sites of inflammation. Chemokines not only orchestrate the innate immune response that eradicates replicating HSV-1 from the cornea (see above), but also have a central role in regulating HSK. Defining the role of chemokines in inflammatory processes is complicated by the fact that they are pleiotropic, have overlapping functions, share receptors, and can bind to multiple receptors. Not surprisingly, studies of their involvement in HSK, have in some cases, produced conflicting results. While IP-10 (CXCL10) and MCP-1 (CCL2) appear to be important for directing CD4+ T cells into the cornea, IP-10 might also inhibit neovascularization (see below). The chemokine CCL20, expressed by HSV-1-infected corneal epithelial cells and corneal keratocytes when stimulated by IL-1b and TNFa, appears to be involved in DC infiltration during both epithelial lesions and the secondary infiltration during HSK development. The chemoattractants MIP-2 (CXCL2), MIP-1a (CCL3), and RANTES (CCL5) have all been implicated in guiding neutrophils into the cornea

during HSK. A variety of neutralizing antibodies, and peptide and nonpeptide inhibitors of these chemokines have been developed and might have therapeutic potential in managing HSK, though their successful use will likely be complicated by the redundant nature of the chemokines.

Angiogenesis in HSV-1 Stromal Keratitis

Neovascularization of the normally avascular cornea appears to be a requisite step in the development of severe HSK in mice. Both vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 have an important role in the neovascularization of corneas with HSK. Production of these factors is induced by IL-1, IL-6, and MIP-2. Corneal cell production of soluble VEGF receptors prevents vascularization of the cornea during steady state. Clearly, the capacity of the soluble VEGF receptor to neutralize VEGF and prevent its binding to VEGF receptors on the limbal vasculariture is overwhelmed by the amount of VEGF produced in HSV-1 infected corneas. Neovascularization provides enhanced corneal access of blood-borne leukocytes that mediate HSK, and blocking neovascularization effectively prevents HSK progression. Based on the established roles for vascularization and VEGF in mouse models of HSK, and the prominent presence of corneal vessels in necrotizing keratitis in humans, therapeutic approaches geared toward blocking vascularization hold great promise in the management of HSK.

Models of HSK Pathogenesis

Despite extensive advances in our understanding of the pathogenesis of HSK in mice, one of the most basic questions (viz., what stimulates the CD4+ T cells that mediate HSK) remains unanswered. Three models of CD4+ T-cell activation in HSV-1 infected corneas have been advanced and supported by experimental data. These include: (1) bystander activation by the cytokine milieu present in the cornea, (2) autoimmunity to corneal tissue resulting from molecular mimicry by viral proteins, and (3) virus-specific activation.

The concept that bystander activation of CD4+ T cells mediated by the cytokine milieu within the infected cornea is sufficient to induce HSK was proposed based on findings in mice whose T cells all express transgenic T- cell receptors (TCRs) specific for ovalbumin (ova). When the corneas of these mice were infected with HSV-1, they developed HSK that was mediated by ova-specific CD4+ T cells. However, these mice failed to control HSV-1 replication in the cornea, and succumbed to infection early during HSK development. Indeed, when HSV-1 replication was controlled in the corneas of these with kinetics similar to that of immunologically normal mice,

the mice failed to develop HSK. Thus, in immunologically normal mice (and presumably people) bystander activation of CD4+ T cells might contribute to, but is not itself sufficient for, the development of HSK.

Other studies incorporating BALB/c mice that are congenic for the IgH locus concluded that HSK develops as a result of an autoaggressive attack of CD4+ T cells on corneal tissue. This autoimmune response was induced by a peptide contained in the viral UL6 protein that mimicked a sequence in a corneal protein and in the Ig heavy chain of one of the congenic strains. The BALB/c strain that expressed the mimicked peptide in its Ig heavy chains did not respond to the peptide due to clonal deletion, and were highly resistant to HSK. In contrast, the congenic strain that failed to express the UL6-mimicked peptide in its Ig heavy chain generated a response to UL6 that crossreacted with a corneal protein. The authors concluded from these findings that HSK is an autoimmune disease arising from antigenic mimicry by the HSV-1 UL6 protein. Unfortunately, these intriguing findings were not reproduced in subsequent studies by another group, and no UL-6-specific or cornea-specific CD4+ T cells have been isolated from mouse or human corneas with HSK. Thus, the involvement of autoimmunity in HSK remains uncertain.

A third possibility is that reactivity of HSV-specific CD4+ T cells to HSV antigens within the cornea triggers HSK. This hypothesis is consistent with the fact that HSV-1-specific CD4+ T cells have been isolated from both human and murine corneas at varying stages of keratitis. Further support for this concept came from a study in which partial tolerization of CD4+ T cells to HSV-1 antigens was associated with reduced severity of HSK. However, no studies to date have established a clear role for HSV-1-specific CD4+ T cells in directly mediating HSK. Based on the available data, it is likely that the initiation of HSK results from stimulation of HSVspecific CD4+ T cells in the cornea, while bystander activation might contribute to the progression and chronicity of the inflammation.

Epidemiology and Pathogenesis of

Human HSK

Clinically, ocular HSV-1 infection remains a significant cause of visual impairment worldwide. In developed nations, there are approximately 8.4–13.2 new ocular HSV-1 infections per 100 000 person-years with an overall incidence, including recurrences, of 20.7–31.5 episodes per 100 000 person-years. Perhaps the most serious manifestation of ocular HSV-1 infection is the potentially blinding HSK, which accounts for only 2% of initial HSV-1 ocular presentations but represents 20–61% of recurrent disease.

Multiple forms of HSK exist and no classification system is universally accepted (here we use a classification system described by Liesegang). HSK commonly presents as a non-necrotizing or immune form, but can also present as the rarer but more serious necrotizing form. Based on limited data, it appears that both the necrotizing and immune presentations of human HSK share the features of neovascularization and leukocytic infiltration seen in the mouse model. Since HSK in the mouse progresses from disease resembling immune HSK to disease resembling necrotizing HSK, it is reasonable to propose that the two forms represent a continuum rather than distinct pathologies. However, there are important differences between the human and mouse HSK, including the fact that HSK rarely leads to corneal perforation in humans, while perforation is a frequent occurrence in the mouse model. Moreover, human HSK is often associated with replicating HSV-1 in the cornea and overlying epithelial lesions, whereas replicating HSV-1 is typically eradicated from the mouse cornea prior to the onset of HSK.

Management of Human HSV Stromal Keratitis and Implications on Pathogenesis

Management of HSK is complicated by the fact that the cells that are responsible for eliminating the virus also contribute to immunopathology in the cornea. Treating the inflammation alone can significantly exacerbate viral replication. In contrast, treating with antivirals alone can lead to uncontrolled inflammatory damage to the cornea. The current standard of care for both non-necrotizing and necrotizing HSV stromal keratitis includes topical corticosteroids and topical antivirals. Corticosteroids are used to diminish the immunopathological component of stromal disease, while the antivirals prevent further viral replication. However, corticosteroids applied to the ocular surface can lead to adverse side effects including development of cataracts or glaucoma. A less well-studied treatment option for patients suffering from HSV stromal keratitis is topical cyclosporin A (CsA) in addition to topical antivirals. Several small noncontrolled trials in humans with HSK suggested that treatment with CsA may be beneficial in cases resistant to corticosteroids. Additionally, studies using murine models of HSK revealed that CsA effectively reduced stromal inflammation and haze in a dose-dependent manner. Similar to corticosteroids, CsA also functions to dampen the immune system. CsA blocks transcriptional activation

of CD4+ T-cell pathways necessary for production of inflammatory cytokines, such as IL-2 and IFN-g. Furthermore, CsA inhibits corneal neovascularization in murine models of HSK.

Successful medical treatment of human HSK has important implications concerning the theories of HSK pathogenesis. CsA functions to inhibit TCR-induced calcineurin signaling that leads to production of inflammatory cytokines, such as IL-2. Therefore, effective treatment of HSK with CsA implicates TCR signaling, favoring CD4+ T-cell activation by viral or autoantigens, but not by cytokines.

Studies in mice emphasize an important role for neovascularization in HSK progression. Mouse studies also implicate VEGF as a critical regulator of vascularization associated with HSK. The current availability of therapeutic agents such as Avastin and Leucentis that inhibit vascularization by blocking VEGF offers the potential for exciting new treatment alternatives.

Acknowledgments

The authors would like to thank Kira Lathrop, MAMS, for technical assistance within this article.

See also: Adaptive Immune System and the Eye: T CellMediated Immunity; Antigen-Presenting Cells in the Eye and Ocular Surface; Avascularity of the Cornea; Corneal Epithelium: Response to Infection; Immunopathogenesis of Pseudomonas Keratitis.

Further Reading

Biswas, P. S. and Rouse, B. T. (2005). Early events in HSV keratitis – setting the stage for a blinding disease. Microbes and Infection 7: 799–810.

Lepisto, A. J., Frank, G. M., and Hendricks, R. L. (2007). How herpes simplex virus type 1 rescinds corneal privilege. In: Niederkorn, J. Y. and Kaplan, H. J. (eds.) Immune Response and the Eye, vol. 92, pp. 203–212. Karger: Basel.

Liesegang, T. J. (1999). Classification of herpes simplex virus keratitis and anterior uveitis. Cornea 18: 127–146.

Metcalf, J. F., Hamilton, D. S., and Reichert, R. W. (1979). Herpetic keratitis in athymic (nude) mice. Infection and Immunity 26: 1164–1171.

Sheridan, B. S., Knickelbein, J. E., and Hendricks, R. L. (2007). CD8 T cells and latent herpes simplex virus type 1: Keeping the peace in sensory ganglia. Expert Opinion on Biological Therapy 7: 1323–1331.

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

Microfilariae – First stage (L1) larvae of filarial nematodes including Onchocera volvulus, the causative agent of river blindness.

TLR (toll-like receptor) – A family of surface and endosomal receptors that are expressed on mammalian cells, including the cornea. These receptors recognize microbial products and transmit cell signals that culminate in the elaboration of chemokines and cytokines.

Wolbachia – Obligate intracellular bacteria that exist as endosymbionts in filarial nematodes including

Onchocerca volvulus.

Onchocerciasis (river blindness) remains endemic in a number of sub-Saharan African countries and has foci in Yemen and in Latin America. Most recent (2006) estimates indicate that there are 37 million individuals infected with Onchocerca volvulus. The life cycle of all filarial nematodes includes transmission through insect vectors, with Simulium blackflies transmitting O. volvulus. First stage larvae (microfilariae, L1) are ingested during a blood meal and migrate through the insect gut, thorax and into the salivary gland having undergone two molts to the third-stage larvae (L3). Infective L3 enter the mammalian host during a second insect blood meal, where they develop to L4 stage and then adult males and females. Adult male and female worms live for over 10 years in subcutaneous tissues, producing millions of microfilariae over their lifespan. Microfilariae can survive for over 1 year in the skin, and can enter the anterior and porterior segments of the eye. While alive, microfilariae appear to cause minimal damage, and individuals can be very heavily infected; however, when the larvae die either by natural attrition or following chemotherapy, the host immune response causes acute and chronic tissue damage, with severe onchodermatitis in the skin, visual impairment, and blindness. Figure 1 shows examples of sclerosing keratitis and of cellular infiltration and vascularization in the corneal stroma of an infected individual in west Africa.

The Role of Endosymbiotic Wolbachia

Bacteria in Onchocerciasis

Intracytoplasmic Rickettsia-like bacteria were first described in filarial nematodes in 1977, and later identified

as Wolbachia pipientis. Although 75% of insect species and a number of crustaceans harbor Wolbachia as endosymbionts, filarial nematodes are the only group of parasitic worms that are infected, most likely because they are the only nematode group with an obligate insect host. In filarial nematodes, Wolbachia are detected with in cells in the hypodermis and uterus, in immature microfilariae in the uterus, and in mature microfilariae in the skin and cornea. The bacteria are most numerous in the mammalian host compared with the insect stage, and appear to have an essential, though poorly understood, role in nematode embryogenesis. Antibiotic (Doxycycline) treatment of filaria-infected individuals effectively sterilizes the adult females, reducing overall microfilaria numbers in the skin and blocking disease transmission.

Role of Wolbachia in Pathogenesis – Evidence from Infected Individuals

The role of Wolbachia in the pathogenesis of filarial disease has been implicated from observations made after antifilarial therapy. Elevated Wolbachia DNA and even intact Wolbachia are detected in the blood, and are associated with the proinflammatory cytokines seen in patients with post-treatment side effects such as fever, edema, and headache. Using quantitative PCR for Wolbachia surface protein (WSP) gene, which is present as a single copy per organism, the number of bacteria per worm was found to be similar in all insect stages of B. malayi. However, within 7 days in the mammalian host, bacteria numbers increased 600-fold and showed a high ratio of Wolbachia/nematode DNA in L4 larvae, indicating rapid bacterial replication within the worms. This Wolbachia load is maintained in adult males, but increases in females during embryogenesis. Wolbachia are therefore a target for antibiotic treatment, and patients given a course of doxycyline in addition to annual ivermectin treatment have reduced systemic microfilariae, and significantly fewer adult worm number in subcutaneous nodules. Wolbachia appear to mediate recruitment of neutrophils, as the number of neutrophils in nodules from doxycycline-treated individuals is greatly reduced compared with untreated individuals. An additional line of evidence for a role for Wolbachia in the pathogenesis of onchocerciasis relates to earlier studies showing that two strains of O. volvulus that differ in virulence exist in West Africa based on DNA probes using a noncoding repeat sequence, and the strain

shown to cause more severe ocular disease has significantly higher Wolbachia loads compared with the second, less virulent strain, indicating a correlation between virulence and Wolbachia in ocular onchocerciasis.

Taken together, these findings strongly support the notion of an important role for Wolbachia in the proinflammatory response and pathogenesis of onchocerciasis.

The Role of Wolbachia in the Pathogenesis of ocular onchocerciasis – Lessons from the Murine Model of

O. volvulus/Wolbachia Keratitis

Microfilariae invade both the anterior and the posterior segments of the eye. In the latter case, they cause uveitis and chorioretinitis, resulting in loss of vision; therefore, although Onchocerca keratitis is more frequent and more readily detectable, corneal transplants are not conducted as the patients generally also have posterior segment disease. Because eyes from human cases of onchocerciasis are not available, the host response to Onchocerca has been examined in the skin of infected individuals. As such, Onchocerca infected individuals show microfilariae in the dermis surrounded by neutrophils, eosinophils, or macrophages. The likely explanation is that neutrophils surround recently dead and degenerating worms in the skin, whereas macrophages and eosinophils migrate to the site at later

time points. Our findings in a murine model of Onchocerca keratitis shows that neutrophils surround microfilariae in the cornea within 24 h and immunogold labeling of the major Wolbachia surface protein shows neutrophils in close proximity to Wolbachia (Figure 2). Using this mouse model of O. volvulus keratitis in which filaria/Wolbachia extracts are injected into the corneal stroma, St. Andre and colleagues demonstrated that endosymbiotic Wolbachia bacteria are essential for the pathogenesis of O. volvulus keratitis as: (1) O. volvulus from individuals depleted of Wolbachia by antibiotic treatment do not induce corneal inflammation; (2) related filarial species containing Wolbachia induce keratitis in contrast to filarial species lacking Wolbachia; and (3) isolated Wolbachia induce neutrophil recruitment to the corneal stroma.

Wolbachia and TLRs in the Cornea

TLRs are surface and endosomal receptors that are expressed in the cornea and respond to microbial products such as lipopolysaccharide (TLR4) and lipoproteins (TLR2). TLR2 forms heterodimers with TLR1 or TLR6 to initiate signaling through adaptor molecules and induce nuclear factor kappa B (NFkB) translocation to the nucleus, and results in production of pro-inflammatory and chemotactic cytokines. Our findings using gene knock-out mice clearly demonstrate that either O. volvulus extracts

PMN

PMN

PMN

mf

mf D

(e)

Figure 2 Presence of endosymbiotic Wolbachia bacteria in microfilaria and adult female worms. C57BL/6 mice were injected into the corneal stroma with microfilariae, corneas were removed after 4 h or 18 h and thin sections were immunostained with anti-Wolbachia Surface Protein (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) 4 h after injection. WSP was clearly detected inside microfilariae in the corneal stroma (arrows). Mf: microfilariae. (c, d) 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. Magnifications:

(a) 4800; (b) 8400; (c) 5300; (d) 16 000. Reprinted with permission from Gillette-Ferguson, I., Hise, A. G., McGarry, H. F., et al. (2004). Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness). Infection and Immunity 72: 5687. (e) Longitudinal section of adult female Brugia malayi immunostained with anti-WSP showing WSP positive microfilariae in the uterus (magnification 400). Reprinted from Science; photomicrograph by Amy Hise.

containing Wolbachia, or isolated Wolbachia bacteria selectively activate TLR2 and TLR6, and adaptor molecules MyD88 and Mal. Figure 3 shows that corneal inflammation (neutrophil infiltration and increased corneal haze) is entirely dependent on activating TLR2. Moreover, studies using chimeric mice have shown that TLR2 expressed on bone-marrow-derived cells have an important role in provoking corneal inflammation.

Taken together, findings from our group and others indicate that Wolbachia induces TLR2 activation in resident macrophages in the corneal stroma, and produce proinflammatory cytokines and CXC chemokines, which mediate neutrophil recruitment from peripheral, limbal vessels into the corneal stroma. Neutrophil responses to Wolbachia are also dependent on TLR2/MyD88, which mediate cytokine production by these cells, and may contribute to degranulation and secretion of reactive oxygen species and matrix metalloproteinases, resulting in cell death and loss of corneal clarity.

In chronically infected, untreated individuals, there is also an ongoing adaptive immune response, due to repeated invasion of microfilariae into the corneal stroma, and prolonged worm degeneration and release of Wolbachia. Infiltrating eosinophils and macrophages also contribute to tissue damage, manifesting as corneal opacification, loss of vision, and blindness. We found that a third role for the TLR2 is filaria/Wolbachia activation of dendritic cells and T-cell production of IFN-g but not IL-4 or IL-5. IFN-g also has an indirect role in enhancing pro-inflammatory and chemotactic cytokine production, thereby increasing neutrophil recruitment to the corneal stroma (Figure 4). Together, these findings demonstrate that TLR2 governs the host response to Wolbachia at several levels, including systemic and corneal responses, and may be a target for blocking corneal inflammation.

Identification of a Wolbachia TLR2/TLR6

Ligand

The TLR2/TLR6 heterodimer is activated by diacylated lipoproteins. To identify possible Wolbachia lipoproteins that activate TLR2/TLR6, Taylor and colleagues searched the lipoprotein databases, and identified Brugia malayi Wolbachia peptidoglycan-associated lipoprotein PAL (wBmPAL). Synthetic diacylated wBmPAL was shown to selectively induce IFN-g production, to induce systemic TNF-a in a murine model of lymphatic filariasis, and to induce corneal inflammation in a TLR2/TLR6–dependent manner. These data indicate that the interaction between these (and likely other) lipoproteins and TLR2/6 in the cornea is essential for the development of O. volvulus keratitis.

Conclusion

River blindness appears to be mostly under control due to mass distribution of ivermectin (Mectizan), which kills microfilariae and has led to reduced prevalence of disease. Recent studies targeting Wolbachia for antibiotic treatment now demonstrate that doxycycline can also be used to treat infected individuals. This is not only a paradigm shift in treating filarial-infected individuals, but given the risk for ivermectin resistance, also provides a critical second approach to treatment, although this is still at the clinical trial stage. The Bill and Melinda Gates Foundation recently provided funds to develop novel antibiotics as an adjunct treatment for river blindness and lymphatic filariasis, and are described in the anti- Wolbachia website. Overall, the increased understanding

of the pathogenesis of this disease and targeting Wolbachia in particular bring these devastating diseases closer to being controlled.

Acknowledgments

We gratefully acknowledge the contribution of our colleagues in these studies, including Illona Gillette-Ferguson, Amy Hise, Achim Hoerauf and Mark Taylor. This work was supported by NIH grants EY10320 and EY11373, by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.

See also: Innate Immune System and the Eye; Pathogenesis of Fungal Keratitis.

(a)

Cells/5 μm section

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Further Reading

Boatin, B. A. and Richards, F. O., Jr. (2006). Control of onchocerciasis.

Advances in Parasitology 61: 349–394.

Brattig, N. W. (2004). Pathogenesis and host responses in human onchocerciasis: Impact of Onchocerca filariae and Wolbachia endobacteria. Microbes and Infection/Institut Pasteur 6: 113–128.

Daehnel, K., Gillette-Ferguson, I., Hise, A. G., et al. (2007). Filaria/ Wolbachia activation of dendritic cells and development of Th1associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness). Parasite Immunology 29: 455–465.

Gentil, K. and Pearlman, E. (2009). IFN-g and IL-1R1 regulate neutrophil recruitment to the corneal stroma in a murine model of Onchocerca volvulus keratitis (river blindness). Infection and Immunity 77(4):

1606–1612.

Gillette-Ferguson, I., Daehnel, K., Hise, A. G., et al. (2007). Toll-like receptor 2 regulates CXC chemokine production and neutrophil recruitment to the cornea in Onchocerca volvulus/Wolbachiainduced keratitis. Infection and Immunity 75: 5908–5915.

Gillette-Ferguson, I., Hise, A. G., McGarry, H. F., et al. (2004). Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness). Infection and Immunity 72: 5687–5692.

Higazi, T. B., Filiano, A., Katholi, C. R., et al. (2005). Wolbachia endosymbiont levels in severe and mild strains of Onchocerca volvulus. Molecular and Biochemical Parasitology 141: 109–112.

Hise, A. G., Daehnel, K., Gillette-Ferguson, I., et al. (2007). Innate immune responses to endosymbiotic Wolbachia bacteria in Brugia

malayi and Onchocerca volvulus are dependent on TLR2, TLR6, MyD88, and Mal, but not TLR4, TRIF, or TRAM. Journal of Immunology 178: 1068–1076.

Hoerauf, A. (2008). Filariasis: New drugs and new opportunities for lymphatic filariasis and onchocerciasis. Current Opinion in Infectious Diseases 21: 673–681.

Hoerauf, A., Mand, S., Adjei, O., Fleischer, B., and Buttner, D. W. (2001). Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet 357: 1415–1416.

Pearlman, E., Johnson, A., Adhikary, G., et al. (2008). Toll-like receptors at the ocular surface. Ocular Surface 6: 108–116.

Saint Andre, A., Blackwell, N. M., Hall, L. R., et al. (2002). The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295: 1892–1895.

Taylor, M. J., Bandi, C., and Hoerauf, A. (2005). Wolbachia bacterial endosymbionts of filarial nematodes. Advances in Parasitology 60: 245–284.

Turner, J., Langley, R. S., Johnston, K. L., et al. (2009). Filarial Wolbachia lipoprotein stimulates innate and adaptive inflammatory responses through TLR2 and TLR6 and induce disease manifestations of lymphatic filariasis and river blindness. Journal of Biological Chemistry 284: 22364–22378.

Relevant Websites

http://www.a-wol.net – A-WOL – Anti-Wolbachia Consortium.