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

Glossary

Biofilm – The microbial secretion of an extracellular matrix surrounding the organisms.

Conidia – These are fungal spores.

Matrix metalloproteinases (MMPs) – The proteases that participate in tissue remodeling, wound healing, and inflammation.

Multipurpose solution (MPS) – The contact lens care products that are used to disinfect daily-wear contact lenses.

Toll-like receptor (TLR) – A family of surface and endosomal receptors that recognize microbial products. TLR signaling induces production of proinflammatory and chemotactic cytokines and antimicrobial peptides.

Contact-Lens-Associated Fungal

Keratitis

In June 2006, the Centers for Disease Control and Prevention (CDC) Fusarium investigation team (Chang and colleagues) reported 318 cases of Fusarium keratitis, with 164 confirmed cases in 33 states and one US territory, although smaller outbreaks were reported in Singapore, Hong Kong, and France. The age group was between 12 and 83, with a median age of 41; 94% wore soft contact lenses; and keratoplasty was needed for 34%. (Examples of contact-lens-associated Fusarium keratitis are shown by Alfonso and colleagues.) The CDC study demonstrated a clear relation to the use of Bausch and Lomb Renu with MoistureLock multipurpose solution (MPS), and the number of cases of Fusarium keratitis dropped shortly after withdrawal of this product. As unopened bottles were sterile, and Fusarium can be readily isolated from sink and shower drains, the CDC report concluded that the source of infection was in the patients’ homes. However, although the report implies that poor lens care habits were involved, it became clear that Fusarium clinical isolates were more resistant to disinfectants in the lens care solution than CDC strains that were used for comparison. Moreover, resistance was related to the ability of the microorganism’s capacity to form biofilm (see below). Reports from several regions of

the USA, including Florida and San Francisco, described cases of contact-lens-associated Fusarium keratitis demonstrating severe corneal opacification and descemetocele formation (hernia of Descemet’s membrane), most of which required keratoplasty. The CDC also reported that the number of cases of Fusarium keratitis dropped after Renu with Moisture Lock was voluntarily withdrawn from the market. However, several cases have been reported subsequently that were not due to this lens care product and were most likely due to increased awareness of Fusarium, although the causes were not always apparent.

Biofilm Formation in Contact-Lens-

Associated Keratitis

Biofilm is defined as microbial secretion of an extracellular matrix surrounding the organisms. Biofilm formation allows the organisms to resist antibiotics (20–1000 times more resistant than planktonic forms), and to host immune responses. The CDC report on the contact-lens-associated outbreak of Fusarium keratitis also suggested that biofilm formation contributes to the resistance phenotype, as bacterial biofilm can form on contact lenses and lens cases. Bacterial biofilms can be generated rapidly on contact lenses and may therefore contribute to the pathogenesis of keratitis and endophthalmitis. Imamura and co-workers showed that Fusarium forms a biofilm on silicone hydrogel contact lenses; furthermore, the architecture, thickness, and composition of the biofilm differ according to the contact lens type. It is likely that the conidia germinate on the contact lens surface, and favorable conditions allow biofilm development. Once the biofilm is formed, the organisms are more resistant to antifungal agents, including those in multipurpose lens care solutions. Consistent with this notion, the Fusarium strain used to test lens care solutions did not form a biofilm and was more sensitive to lens care solutions (Figure 1).

Keratitis Caused by Candida

Candida species are the most common pathogenic yeast associated with keratitis. Candida albicans is part of the normal commensal flora; however, these organisms can cause opportunistic corneal infections in immunosuppressed

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A B C

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Figure 1 Fusarium biofilm formed on different soft contact lenses. (a) Fusarium conidia were incubated with each contact lens for 2 h, after which time the lenses were removed and incubated a further 48 h. Biofilms were formed by FSSC 1-b isolate MRL8609 on soft contact lenses, and their gross morphologies were imaged using a digital camera. All lenses tested supported biofilm formation by strain MRL8609. (b) The Fusarium FSSC 1-b strain MRL8609 was allowed to form mature biofilms on Etafilcon A silicon hydrogel contact lenses and then was stained with ConA and FUN1 dyes to show extracellular matrix (red) and live organisms (green). Stained lens-containing biofilms were analyzed by confocal scanning laser microscopy. Etafilcon A (A), galyfilcon A (B), lotrafilcon A (C), balafilcon

A. Arrows indicate extracellular matrix in the biofilms. Similar results were found for C. albicans (not shown). Reprinted from Imamura, Y., Chandra, J., Mukherjee, P. K., et al. (2008). Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy

52: 171–182.

individuals or following trauma or surgery. In contrast to filamentous fungi in which trauma is the major predisposing condition, Candida is primarily associated with therapeutic contact lenses, steroid use or immunosuppressive disease, and corneal surgery. In these dimorphic organisms, the yeast stage initially infects the cornea, and then germinates to form pseudohyphae in the corneal stroma. Candida produces a number of proteases and phospholipases (particularly phospholipase B) that facilitate their penetration through the cornea and contribute to tissue destruction. Using C. albicans mutants in a murine model of keratitis, Jackson and colleagues showed that C. albicans virulence depends on expression of genes encoding or regulating hyphal formation, but not genes regulating adherence.

Fungal Keratitis Associated with Trauma

Although relatively rare in North America and Europe, filamentous fungi are among the most common causes of microbial keratitis and corneal ulcers in India, China, and Ghana. In the southern USA, Fusarium solani and Fusarium

oxysporum are the most common causes of mycotic keratitis, with Aspergillus species being the second most common cause, including A. fumigatus, A. niger, and A. nidulans.

Corneal trauma is the main predisposing factor, and the incidence of fungal keratitis increases during harvest season, which is consistent with the majority of cases associated with agricultural work, where it affects more males than females of working age. Trauma can be caused by numerous factors, such as airborne soil and plant material. Filamentous fungi are ubiquitous in the environment, especially on plants, and the fungal spores (conidia) can penetrate the corneal epithelium, germinate in the stroma, and if unchecked by the host response or effective antifungal therapy, fungal hyphae will grow in the corneal stroma, penetrate Descemet’s membrane, and invade the anterior chamber.

Role of Matrix Metalloproteinases in Fungal

Keratitis

Matrix metalloproteinases (MMPs) have an important role in tissue remodeling, wound healing, and inflammation. Rohini and co-workers examined human tears from fungal keratitis patients and corneal sections after keratoplasty and detected elevated collagenases MMP-2 and MMP-8, and the MMP-9 gelatinase, which is consistent with the activation and degranulation of infiltrating neutrophils. In addition to microbial killing, which is primarily mediated by oxygen radicals, neutrophils also prevent microbial dissemination by releasing MMPs and causing local tissue damage. Dong and co-workers as well as Mitchell and co-workers showed that MMP-2 and MMP-9 were also elevated in rabbit and mouse models of Fusarium and Candida keratitis, and Lin and colleagues demonstrated a role for MMP-8 in corneal inflammation by mediating breakdown of collagen and release of chemotactic Pro–Gly–Pro peptides, which then facilitate neutrophil migration through the cornea. Although differences between Aspergillus and Fusarium growth in the stroma have been reported by Xie and colleagues, it is not clear at present how this relates to MMP activity, or if there is a difference in protease production by these organisms.

Role of Innate Immunity in Fungal Keratitis

Since the 1960s, it has been apparent that the host immune response regulates fungal growth and the outcome of the infection. In rabbit and murine models of Fusarium and Candida keratitis in which either conidia or yeast is applied topically to the abraded epithelium, or is injected intrastromally, a neutrophil-rich cellular infiltrate into the corneal stroma ultimately clears the infection. However, subverting the host response by systemic

treatment with cyclophosphamide leads to increased hyphal penetration of the corneal stroma, decreased cellular infiltration, (especially neutrophils), and the unchecked growth of hyphae, causing corneal perforation. These results indicate that the host response plays a critical role in restricting fungal growth in the cornea. To characterize the host response to fungal challenge, Candida-infected corneas were processed for microarray analysis, which demonstrated that the proinflammatory cytokines, interleukin-1 (IL-1) and tumor necrosis factoralpha (TNF-a), were upregulated. To characterize the innate immune response, Tarabishy and colleagues injected Fusarium conidia into the corneal stroma of immunocompetent C57BL/6 and mice on the same genetic background in which genes related to the Toll-Like Receptor (TLR) family of pathogen recognition molecules were knocked out. Figure 2 shows that 6 h after intrastromal injection of 10 000 conidia, hyphae were detected in the corneal stroma of C57BL/6 mice and in mice in which the gene for the MyD88 adaptor molecule common to most TLRs and IL1R1 is mutated. Although a cellular infiltrate was detected in the peripheral cornea of C57BL/6 mice, this infiltrate was absent in MyD88–/– mice. Figure 3 shows that whereas C57BL/6 mice rapidly develop corneal opacification associated with a pronounced infiltrate and clear the organisms, MyD88–/– mice had delayed cellular infiltration, and even though neutrophils were recruited to the corneal stroma,

Fungal isolate

Figure 2 Fusarium strain differences in biofilm formation on lotrafilcon A lenses. Biofilms were formed in the absence or presence of DEB (which inhibits biofilm formation) on a lotrafilcon A lens using the FSSC 2-c ATCC 36031 reference isolate or clinical isolate FSSC 1-b (MRL8609), FOSC 3-a (MRL8996). Biofilms were quantified using the XTT metabolic activity assay. Data represent means (+/– SDs) calculated from three separate experiments. Reprinted from Imamura, Y., Chandra, J., Mukherjee, P. K., et al. (2008). Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy 52: 171–182.

they were unable to clear the organisms (Figures 4 and 5). Fusarium hyphae are detected throughout the cornea (Figure 5), which perforated within 4 days. Subsequent experiments showed that IL-1R1 is required for neutrophil recruitment to the cornea, whereas TLR4 is important for fungal killing.

Murine Model of Aspergillus Keratitis

Aspergillus species, the second most common cause of fungal keratitis after Fusarium, are also ubiquitous in the environment, and most people inhale hundreds of conidia daily. Although pulmonary aspergillosis occurs primarily in immunosuppressed individuals, this is not the case in keratitis, where the risk factors are similar to those of Fusarium, that is, the highest incidence is associated with agriculture and trauma. In addition, Aspergillus conidia are smaller than Fusarium conidia, can therefore penetrate deeper into the lungs, and likely also penetrate deeper into the corneal stroma. We generated a strain of Aspergillus fumigatus expressing a red fluorescent protein, and injected conidia into the corneal stroma of C57BL/6 mice. Figure 6 shows that after 24 h, the cornea is opaque. However, Figure 6(b) also shows that the presence of corneal opacities coincides with the presence of Aspergillus. Figure 6(c) shows higher magnification of hyphae in the corneal stroma. Ongoing studies are examining the role of the host response and Aspergillus virulence factors in the pathogenesis of this disease.

MyD88–/– C57BI/6

Figure 3 In vivo confocal microscopy of Fusarium keratitis in C57BL/6 and MyD88–/– corneas. C57BL/6 and MyD88–/– mice were injected intrastromally with 1 104 conidia from a clinical isolate of F. oxysporum. After 6 h, mice were examined by in vivo confocal microscopy (Confoscan). Representative images from the central and peripheral corneal stroma are shown. Note the presence of hyphae in the central corneal stroma of C57BL/6 and MyD88–/– mice (a, c); however, a cellular infiltrate is present in the peripheral cornea of C57BL/6, but not MyD88–/– mice (b, d). Reprinted from Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. Journal of Immunology

181: 593–600.

MyD88–/– C57BL/6

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Clinical score

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3

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C57BL/6 MyD88–/–

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Figure 5 Impaired cellular infiltration in MyD88–/– mice (a, b) Histological analysis in MyD88–/– corneas after PAS staining shows Fusarium hyphae penetrating Descemet’s membrane after 24 h, and growing in the stroma and anterior chamber after 48 h despite the presence of a cellular infiltrate. In contrast, there was an early and pronounced cellular infiltrate in C57BL/6 mice (c, d) comprised mostly of neutrophils. Reprinted from Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. Journal of Immunology 181: 593–600.

Figure 6 Aspergillus fumigatus in murine cornea. Aspergillus fumigatus was transfected with a plasmid expressing m-cherry. Conidia were injected into the corneal stroma of C57BL/6 mice, and after 24 h, corneas were examined by light (a) and fluorescence (b) microscopy. (c) Whole mount cornea examined by confocal microscopy.

require a combination of human and animal studies to determine the course of events leading to fungal killing and resolution of infection. Human disease correlates are particularly difficult to study; however, Bochud and co-workers showed that polymorphisms in TLR4 are associated with susceptibility to systemic aspergillosis; therefore, it is possible that TLR4 also mediates susceptibility to Aspergillus keratitis.

Conclusions

The pathogenesis of fungal keratitis depends on the balance between the host response and expression of fungal virulence factors. Although some mediators of innate immunity and fungal virulence factors have been identified, it will

Acknowledgments

Studies presented in this article were supported by NIH grant EY18362 (EP) and EY11373 (EP), by DE017486-01A1 (MAG) and R01DE 13932 (MAG), and by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.

See also: Contact Lenses; Corneal Epithelium: Response to Infection; Innate Immune System and the Eye.

Further Reading

Alfonso, E. C., Cantu-Dibildox, J., Munir, W. M., et al. (2006). Insurgence of Fusarium keratitis associated with contact lens wear.

Archives of Ophthalmology 124: 941–947.

Bharathi, M. J., Ramakrishnan, R., Meenakshi, R., et al. (2007). Microbial keratitis in South India: Influence of risk factors, climate, and geographical variation. Ophthalmic Epidemiology 14: 61–69.

Bochud, P. Y., Chien, J. W., Marr, K. A., et al. (2008). Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation.

New England Journal of Medicine 359: 1766–1777.

Chang, D. C., Grant, G. B., O’Donnell, K., et al. (2006). Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. Journal of the American Medical Association 296: 953–963.

Dong, X., Shi, W., Zeng, Q., and Xie, L. (2005). Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea. Current Eye Research 30: 613–620.

Grant, G. B., Fridkin, S., Chang, D. C., and Park, B. J. (2007). Postrecall surveillance following a multistate Fusarium keratitis outbreak, 2004 through 2006. Journal of the American Medical Association 298:

2867–2868.

Imamura, Y., Chandra, J., Mukherjee, P. K., et al. (2008). Fusarium and Candida albicans biofilms on soft contact lenses: Model development, influence of lens type, and susceptibility to lens care solutions. Antimicrobial Agents and Chemotherapy 52: 171–182.

Jackson, B. E., Wilhelmus, K. R., and Mitchell, B. M. (2007). Genetically regulated filamentation contributes to Candida albicans virulence during corneal infection. Microbial Pathogenesis 42: 88–93.

Mitchell, B. M., Wu, T. G., Chong, E. M., Pate, J. C., and Wilhelmus, K. R. (2007). Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis. Cornea 26: 589–593.

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

Rohini, G., Murugeswari, P., Prajna, N. V., Lalitha, P., and Muthukkaruppan, V. (2007). Matrix metalloproteinases (MMP-8, MMP-9) and the tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in patients with fungal keratitis. Cornea 26: 207–211.

Tarabishy, A. B., Aldabagh, B., Sun, Y., et al. (2008). MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2.

Journal of Immunology 181: 593–600.

Wu, T. G., Keasler, V. V., Mitchell, B. M., and Wilhelmus, K. R. (2004). Immunosuppression affects the severity of experimental Fusarium solani keratitis. Journal of Infectious Diseases 190: 192–198.

Xie, L., Zhai, H., Shi, W., et al. (2008). Hyphal growth patterns and recurrence of fungal keratitis after lamellar keratoplasty.

Ophthalmology 115: 983–987.

Yuan, X., Mitchell, B. M., and Wilhelmus, K. R. (2008). Gene profiling and signaling pathways of Candida albicans keratitis. Molecular Vision 14: 1792–1798.

Glossary

Conjunctiva-associated lymphoid tissue (CALT) – It is the physiological protective mucosal immune tissue of the conjunctiva. It consists of lymphoid cells and accessory cells inside the mucosal tissue and can be divided into the epithelial and underlying connective tissue (lamina propria) compartments. It is arranged as a diffuse lymphoid effector tissue along the whole extension of the conjunctiva and has interspersed organized lymphoid follicles for afferent antigen uptake and effector cell generation.

Dendric cells (DCs) – They are a special class of professional antigen-presenting cells (APC, together with macrophages and B-cells). They take up external antigens, degrade them into small fragments (epitopes), present them on MHC-class-II to T-helper cells, and hence, induce immune reactions. Depending on their maturation status which is influenced by the presence of inflammatory signals, they modulate between the inductions of tolerance versus inflammation. They also link the unspecific innate to the induced specific immune system and are hence key modulators of the immune reaction.

Eye-associated lymphoid tissue (EALT) – This tissue summarizes all the lymphoid tissues of the extended mucosal ocular surface, that is, of ocular surface proper (conjunctiva and cornea) along with its mucosal adnexa (the lacrimal-drainage- associated lymphoid tissue, LDALT, and the lymphoid cells inside the lacrimal gland). EALT is in line with the mucosal immune system in other parts of the body (e.g., gut-associated lymphoid tissue (GALT) in the gut and bronchus-associated lymphoid tissue (BALT) in the airways).

High endothelial venules (HEVs) – Specialized postcapillary venules that have an endothelium of bright roundish cells compared to the ordinary flat dense ones. They are located in lymphoid tissues and have tissue-specific adhesion molecules (vascular addressins) on the cell surface that specifically interact with homing receptors on circulating lymphocytes in order to maintain a regulated immigration of lymphocytes into the tissue.

Human leukocyte antigen (HLA) – A system of the major histocompatibility complex () MHC)

in humans. It contains of a number of genes and their respective encoded proteins (that can act as antigens). The term HLA is frequently used to describe immunological self and nonself in the context of transplant rejection.

Intercellular adhesion molecule 1 (ICAM-1) –

An adhesion molecule (CD54 according to the immunological cluster of differentiation, CD, nomenclature) mainly on vascular endothelial cells which is upregulated in inflammation and promotes the increased immigration of leukocytes, that carry corresponding integrin receptors, into the tissue.

Membraneous cells (M-cells) – Also called microfolded cells, they are a special type of cells in the modified epithelium overlying organized lymphoid follicles, the so-called follicle-associated epithelium (FAE). Their name refers to the fact that they have a different, usually smooth, surface ultrastructure compared to the ordinary epithelial cells. They form cellular pockets populated by groups of leukocytes which are separated from the lumen by a thin luminal cytoplasmic sheet. M-cells actively transcytose luminal antigens for uptake by the leukocytes and their subsequent presentation to and activation of T- and B-cells in order to generate antigen-specific effector cells.

Major histocompatibility complex (MHC) – It is differentiated mainly into class-I and class-II. Their encoded proteins on the surface of cells perform the presentation of protein antigen fragments (epitopes) to immune cells. MHC-class-I is found on all nucleated cells and presents antigens produced inside the cell (either own or viral proteins after infection) to cytotoxic CD8 lymphocytes and natural killer cells. MHC-class-II, in contrast, occurs physiologically only on specialized antigenpresenting cells and presents foreign antigens to the CD4 receptor of T-helper cells. In inflammation, it can be upregulated by other cells.

Lipopolysaccharide (LPS) – A component of the outer cell membrane of the wall of Gram-negative bacteria that acts as an endotoxine. The presence of LPS, that is detected for example, by toll-like receptors, signals the pathogenic nature of antigens to the immune system and elicits a strong inflammatory reaction.

Tolerance – Immune tolerance is a status in which the immune system is in a state of nonreactivity

to an antigen in order to prevent inflammatory tissue-destructive reactions. Tolerance is actively generated and directed not only against the bodies’ own cellular self-antigens but also against nonpathogenic external antigens. If tolerance fails, autoimmunological disease or allergy may occur. Tolerance is the default mode of the mucosal immune system, including CALT and EALT, in order to preserve tissue integrity.

Conjunctival Morphology and Function Are Closely Interacting for Immune Surveillance

Epithelial Defense Mechanisms

Epithelial morphology and function

The conjunctiva is a moist mucous organ that consists of a surface epithelium and an underlying loose connective tissue (lamina propria), separated by the epithelial basement membrane. The epithelium of the human conjunctiva has, in contrast to small rodents (e.g., rat and mouse), a stratified nonsquamous morphology and consists of two to three cell layers of cubical cells in most parts. It becomes multilayered and assumes prismatic morphology at the fornix whereas it tends to become squamous toward the limbus (Figure 1(a) and 1(b)). Interspersed mucus-secreting goblet cells occur inside the epithelium as single cells or in small groups as well as intraepithelial

lymphocytes (IELs) that reside mainly in the basal layers (Figure 2(b) and Figure 3(a)) as well as dendritic cells (DCs), that have long narrow extensions, for uptake of antigens from the surface. The conjunctival epithelial surface is covered by small cytoplasmic protrusions (microvilli and microplicae) with a well-developed surface coat of filamentous projections (glycocalyx) that form a meshwork (Figure 1(c) and Figure 2(a)).

Epithelial Immune Surveillance Takes Care of

Environmental Antigens

Physical and physicochemical barriers keep antigens outside

The structure of the conjunctival epithelium already contributes to basic protective mechanisms which can be considered as part of the innate defense. Epithelial cells are mechanically connected by desmosomes and have an apical belt of intercellular junctions including tight junctions that seal the intercellular space and limit the passive para-cellular leakage of antigens in and out of the tissue (Figure 2(a)). This physical cellular barrier is supplemented by the physicochemical barrier of the epithelial mucins, that consist of cell membrane-spanning mucins (glycocalyx) produced by the ordinary epithelial cells and of soluble mucins secreted by the goblet cells which mix with the aqueous phase. Together they form a layer in the range of few micrometers thickness, that is, a sticky gel to which microbes adhere and can hence be cleared by the constant renewal of the preocular tear film. Soluble protective factors, including secretory immunoglobin A (SIgA), are fixed to the mucin layer in order to make it an almost impenetrable and lethal barrier to antigens and in particular to microbes.

The mechanical washing effect of the tear film wipes away antigens and detritus

The tear film is an important functional component of the ocular surface mucosal protection system. Apart from providing the necessary moisture, the constant flow of tears over the ocular surface and in particular, over the cornea, together with the wiping effect of the lid margin with every blink, provides a constant mechanical washing. This discharges antigens and removes dust and cell detritus. Other parts of the ocular surface along the retropalpebral tear film are not so rapidly cleared so that antigens can stay in longer contact with the epithelium. Therefore, the tear film contains a large number of antimicrobial factors that contribute more specifically to the innate immune defense.

Epithelial innate immune defense factors

The innate immune system uses pattern-related receptors (PRRs) that mainly detect conserved pathogen-associated molecular patterns (PAMPs) but also host antigens from destroyed cells. It reacts via effectors, which consist of soluble antimicrobial proteins and peptides (AMPs)

which bind to the microbial cell wall in order to destroy it or which interfere with the microbial metabolism. The innate immune system also employs production of soluble mediators, such as inflammatory cytokines and chemotactic cytokines (chemokines) that functionally couple the innate and adaptive immune answer.

PRRs on epithelial cells provide an external alarm system

As soon as microbial antigens have breached the physicochemical barrier, they get in touch with epithelial PRRs (Figure 2(a), the most prominent of which is presently the diverse family of toll-like receptors (TLRs). Binding

a nuclear transcription factor, nuclear factor kappa B (NFkB), and results in production of inflammatory cytokines such as interleukin 6 (IL-6), interferon gamma (IFN-g), or tumor necrosis factor alpha (TNF-a). Subsequently, these induce the production of chemokines, adhesion molecules, and inducible AMP. Altogether this represents an inflammatory cascade with activation, first

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of the epithelial cells and later also of lamina propria leukocytes and vascular endothelial cells. It induces leukocyte recruitment into the epithelium and their immigration from the blood stream into the tissue.

The normal conjunctival epithelium expresses a number of different TLRs, similar to the cornea. TLR1, 2, 3, 5, and 6 were found in all conjunctival and limbal epithelial cell samples, TLR4 and 9 only inconstantly, but not TLR7, 8, and 10. TLR2 may only occur upon stimulation by IFN-g and bacterial cell wall extract, for example, in patients with ocular allergy. This results in upregulation of inflammatory markers, such as the intercellular adhesion molecule 1 (ICAM-1), human leukocyte antigen (HLA), TNF-a, and IL-8, in a dose-dependent manner. Bacterial-specific TLRs are of interest in ocular allergy because colonization by bacteria is common there. The activation of TLRs represents an important co-factor in ocular allergy and their blockade can significantly inhibit release of inflammatory mediators which may turn out as a promising new therapy option for ocular allergy.

The conjunctival epithelium secretes diverse AMPs

The spectrum of epithelial derived AMPs is distinct for cornea and conjunctiva but overlapping. Conjunctival epithelium produces not only the human b-defensins (hBD)-1,2,3 and further AMPs such as liver-expressed antimicrobial peptides (LEAPs) 1 and 2 and cathelicidin (LL-37) but also macrophage inflammatory protein 3alpha (MIP-3a) and thymosin beta 4 (Tb-4). Some of the AMPs are constitutively produced, whereas others are inducible. hBD-2 is induced by inflammatory cytokines in ocular surface inflammation and by presence of bacterial LPS, while hBD3 is induced by infection and LL-37 by epithelial wounding. Conjunctival AMPs such as LL-37 are active against bacterial (Pseudomonas aeruginosa, Staphylococcus aureus,

Staphylococcus epidermidis) and viral (Herpes simplex virus 1, adenovirus) infection. They can act as multifunctional factors in wound healing and signaling pathways. Interestingly, the antimicrobial activity of some of these AMPs (e.g., hBD-1, hBD-2, and Tbeta-4) is almost completely inhibited in the presence of tear fluid. This may indicate that not all epithelial AMPs are produced in order to act as tear film factors but rather play a major role for local protection inside the conjunctival epithelium itself. Apart from AMPs, there is a plethora of other protective proteins. AMPs continue downstream in the lacrimal drainage system.

Conventional antibacterial factors are surprisingly versatile defense tools

Even the ‘old fashioned’ established antimicrobial proteins in the tear film, such as lysozyme and lactoferrin, have surprising newly detected functions. Apart from being bactericidal, either by lysis of components in the Gram-positive bacterial cell wall (lysozyme) or by interfering with their iron metabolism (lactoferrin), they are also antifungal and antiviral. Through the absorption of the strongly inflammatory bacterial endotoxin lipopolysaccharide (LPS), which is a surface molecule on Gram-negative bacteria, they also act anti-inflammatory. They have further anti-inflammatory functions by their influence on antigenpresenting cells (APCs) and hence appear as key elements in host defense that link innate and adaptive immunity.

Conjunctival Lamina Propria: Morphology and Function of the Diffuse Mucosal Immune System

Diffusely arranged lymphoid and innate cells contribute to conjunctival immune surveillance

The lamina propria contains bone-marrow-derived cells and vessels of different types. Apart from capillaries and lymph vessels, specialized high endothelial venules (HEVs) occur. Vessels serve for the supply with nutrition and discharge of metabolites, for hormonal regulation of the tissue, and also for the migration of immune cells. Lymphocytes, together with accessory leukocyte populations (macrophages, granulocytes, mast cells, and DCs), form a diffuse lymphoid tissue (Figure 3(a)) which is regarded mainly as an effector site of CALT although antigen uptake via DCs can also occur here to a certain extent. The diffuse lymphoid tissue is located in the vast majority of the surface, except for the solitary lymphoid follicles. The thickness of this cell layer depends on the location along the conjunctiva, shows a certain topographical variation, and is frequently only one to two cells wide, which may be a reason why these cells have often been overlooked in the past. It also shows a certain interindividual variation that may depend on the immune status of the person. IEL also functionally belong to the diffuse effector cells (Figure 3(b)).

Different subtypes of lymphocytes occur in the conjunctiva

Diffuse conjunctival lymphocytes are mainly CD3þ T-cells (Figure 3(c)) (whereas CD20þ B-cells are largely restricted to the solitary lymphoid follicles). They are activated (CD45Roþ, CD25þ) and express the human