Introduction
The outcome of microbial infections depends on both the virulence of the infecting organism and the veracity of the host response. The balance of these two entities will determine the severity of disease and the longevity of infection. While in some infections tissue damage is a direct result of cytotoxic activity of microbial products such as exotoxins, the host inflammatory response causes collateral tissue damage associated with antimicrobial activity. Microbial virulence may then be defined as the ability of the organisms to survive in the presence of the host immune response. This chapter will focus on infections of the anterior eye based on exposure to the ocular surface, but will also discuss infections of the posterior eye, including endophthalmitis and ocular toxoplasmosis.
Microbes in the environment
Most of the organisms that cause severe ocular infections are either ubiquitous in the environment or are part of our normal body flora. These microbes are opportunistic pathogens and require breach of the physical barriers of the eye. For example, Pseudomonas aeruginosa and Acanthamoeba are normal freshwater organisms present in ponds, lakes and household water supplies, including showerheads. Similarly, Aspergillus and Fusarium moulds, which are plant saprophytes and pathogens, are ubiquitous in the air we breathe, although spore counts are higher in hot and humid areas of the world, and in agricultural regions, especially during harvest seasons. Aspergillus is among the most widely distributed organisms worldwide and, except in extremely cold environments, most individuals inhale Aspergillus spores on a daily basis. In humid conditions Aspergillus is the cause of the green mould found on bread and other foods, and routine examination of the average bathroom will detect Aspergillus and Fusarium spores, Pseudomonas , Serratia and Acanthamoeba (which feeds on bacteria and fungi). The number of organisms in the environment is generally higher in warmer climates and during the summer in temperate climates.
In addition to the external environment, several organisms are present in the normal body flora of the human conjunctiva (adenovirus), the skin (staphylococci) and the nasopharynx (streptococcus pneumoniae, candida albicans). Most individuals also harbour herpes simplex virus (HSV) and herpes zoster as latent forms in the trigeminal ganglia. HSV is the most common cause of corneal infections in the USA and other industrialized countries and causes resurgent keratitis following viral exit from latency. Toxoplasma gondii is also ubiquitous in the environment and humans are infected following ingestion of cat faecal material or infected meat.
The exceptions to pathogens with near ubiquitous distribution are those that are very restricted geographically or which thrive under conditions of poor hygiene. The bacterium that causes trachoma ( Chlamydia trachomatis ) is the major example of an organism that thrives in unsanitary environments, as it is readily transmitted by flies that breed on human waste. Onchocerca volvulus , the cause of river blindness, and Loa loa (eyeworm) are highly adapted human parasites that require an intermediate insect vector, and are found primarily in Africa. These will be discussed later in this chapter. Table 8-1 lists many of the causes of ocular infection.
| Group/phylum | Genus, species | Major site of infection |
|---|---|---|
| Viruses | Adenovirus | Conjunctiva |
| Herpes simplex | Cornea | |
| Herpes zoster | Cornea | |
| CMV | Retina | |
| Obligate intracellular | Chlamydia | Conjunctiva, cornea (trachoma) |
| Gram-negative bacteria | PA | Cornea |
| Sm | Cornea | |
| Gram-positive bacteria | SP | Conjunctiva, cornea, vitreous |
| Sa | Cornea | |
| Bacillus cereus | Vitreous | |
| Yeast | Candida | Cornea |
| Mould | Fusarium | |
| Aspergillus | ||
| Protozoa | Toxoplasma | Cornea |
| Acanthamoeba | ||
| Microsoporidia | ||
| Helminths | Toxocara canis | Retina |
| Filarial | Onchocerca volvulus (river blindness) | Cornea, retina |
| Loa loa (eye-worm) | Conjunctiva |
* Although a search of case reports will produce a much longer list of organisms, the table includes only the most common causes of ocular infection.
Host defences at the ocular surface
Physical barriers
Blinking is a very effective cleansing mechanism and eyelashes can trap microbes, preventing access to the globe. In addition, the eyelids contain sebaceous glands that secrete lactic acid and fatty acids in a low pH environment, which has a direct inhibitory effect on bacterial replication. Tears also contain antimicrobial compounds including lacritin, lactoferrin, lipocalin lysozyme, β-defensins and other antimicrobial peptides. The tear film also contains secretory immunoglobulin A (IgA), IgG and complement that contribute to protection against bacterial invasion (see Ch. 7 ). Protection is also afforded by neutrophils in the tears, which increase in numbers following sleep when the eyelids are closed.
Epithelium
The corneal epithelium has a glycocalyx on the apical surface composed of large proteoglycan mucins including MUC1, MUC4 and MUC16 (see Ch. 4 ). This mucin layer provides a physical barrier that restricts bacterial adherence to corneal epithelial cells in addition to releasing mucins into the tear film ( Fig. 8-1 ) . Epithelial cells also form tight junctions that are a very effective barrier against subsequent penetration to the corneal stroma; infections therefore generally occur as a consequence of traumatic injury or alteration of the corneal surface microenvironment. Epithelial cells also secrete antimicrobial peptides including β-defensins, cathelicidin (LL-37) and calprotectin (S100A8/A9), and by degradation of cytokeratin-6A in corneal epithelial cells. These keratin-derived antimicrobial peptides (KDAMPs) have a distinct secondary structure and exhibit broad antimicrobial activity.
Resident macrophages and dendritic cells in the cornea
The normal mammalian cornea was generally thought to be devoid of immune cells; however, with the advent of improved immunostaining methods, together with examining whole-mount corneas rather than histological sections, an entire network of macrophages and dendritic cells was revealed. Most bone marrow-derived cells in the normal corneal stroma have characteristics of macrophages, with dendritic cells more prominent in the peripheral limbal region of the cornea and in the basement membrane of the epithelium (Bowman’s membrane). These cells extend pseudopodia (‘periscopes’) through to the apical surface, presumably to detect microbes or and microbial products ( Fig. 8-2A–D ). Nanotubes that appear to connect distant cells can also be detected in the corneal stroma during inflammation ( Fig. 8-2D ).
Pathogen recognition receptors and recruitment of neutrophils
Most nucleated cells are able to recognize and respond to microbial products, although macrophages and dendritic cells are specifically adapted for this purpose. These cells express multiple copies of surface receptors that recognize bacterial and fungal proteins, carbohydrates, lipids and DNA and RNA, and are termed pathogen recognition receptors (PRRs) (see Ch. 7 , p. 379 ). Ligand binding initiates intracellular signalling events that result in production of pro-inflammatory and chemotactic cytokines. These are invariant receptors encoded in the germ line genes, which is in contrast to T and B cells, where receptors are generated following gene rearrangement. Further, whereas mature T and B cells primarily recognize specific peptides (and in some cases well-defined carbohydrates), the invariant receptors associated with innate immunity recognize conserved proteins, lipids and nucleic acids primarily associated with microbes. Though not a focus of the current chapter, these receptors can also recognize endogenous self-antigens, danger-associated molecular patterns (DAMPs), which include silica, uric acid or asbestos crystals, and advanced glycation end products. Among the best characterized receptors are cell surface and endosomal Toll-like receptors, cell surface C-type lectins, and intracellular NOD-like receptors (NLRs).
Toll-like receptors (TLR)
TLR family members are single transmembrane receptors that recognize structurally conserved microbial products; further, activation of these receptors leads to production of pro-inflammatory and chemotactic cytokines that mediate recruitment of neutrophils, macrophages and lymphocytes to the site of infection. As shown in Figure 8-3 , TLRs are located in cholesterol rich regions of the plasma cell membrane (lipid rafts) and in endosomes, and can recognize lipids, proteins or nucleic acids. Lipid-binding TLRs include TLR2, which forms heterodimers with TLR1 or TLR6 to bind lipopeptides, and TLR4/MD-2, which recognize the lipid A moiety of lipopolysaccharide (LPS). TLR5 and TLR11 recognize proteins, and are activated by bacterial flagellin (TLR5) or uropathogenic E. coli or Toxoplasma gondii profilin (TLR11). TLR3, TLR7, TLR8 and TLR9 are located on endosomal membranes and bind viral and bacterial nucleic acids. With the exception of TLR3, all TLRs stimulate the cells through the MyD88 common adaptor molecule, leading to NF-κB translocation to the nucleus and expression of genes encoding pro-inflammatory cytokines and chemotactic cytokines (chemokines). TLR3 and TLR4 activate the TRIF pathway, which induces IRF3 transcription and production of type I interferons that mediate antiviral responses. TLR4 activation involves accessory molecules, including lipopolysaccharide (LPS)-binding protein and CD14, which combine to extract single endotoxin molecules from the outer membrane and form monomeric endotoxin, and MD-2, which is the receptor for the lipid A moiety of LPS. CD14 also chaperones TLR4 from the plasma membrane to endosomes in order to activate the TRIF pathway.
NOD-like receptors
NOD-like receptors (NLRs) comprise an intracellular family of pathogen recognition molecules which also activate the NF-κB complex, leading to expression of pro-inflammatory and chemotactic cytokines. NOD2 has been well characterized and shown to recognize muramyl dipeptide on degraded bacterial cell wall peptidoglycan, and can therefore respond to invading Gram-negative and Gram-positive bacteria ( Box 8-1 ). However, as Gram-positive bacteria have more peptidoglycan in the cell wall, NOD2 activation occurs following infection by staphylococci or streptococci.
Mutations in NOD2 are associated with susceptibility to autoimmune diseases that include Crohn’s disease, which is a common and painful form of inflammatory bowel disease, and Blau’s syndrome, which is manifest by multiple autoimmune disorders, including a severe form of uveitis. Although not completely understood, some of the polymorphisms of NOD2 result in hyperresponsiveness to MDP from otherwise harmless commensal bacteria in the intestine and skin.
NLRs include NLRP3, which recognizes bacterial toxins and crystals, and NLRC4, which recognizes flagellin of Gram-negative bacteria such as Pseudomonas aeruginosa ( Fig. 8-4 ). Once activated, these NLRs form a large, multi-protein complex called an inflammasome, which activates caspase-1 and cleaves IL-1β, IL-18 and IL-33 from the inactive pro-form, to the bioactive, mature form of these cytokines. Prolonged activation of inflammasomes also leads to caspase-1-mediated cell death, termed pyroptosis.
C-type lectins
In addition to bacterial products, host cells recognize fungal cell wall components by activation of C-type lectins on the cell surface ( Fig. 8-5 ). Dectin-1 recognizes β-glucan, whereas Dectin-2 and Dectin-3 recognize α-mannans, and are activated after either clustering of Dectin-1 or heterodimerization of Dectin-2 and Dectin-3, which leads to production of pro-inflammatory and chemotactic cytokines.
Neutrophils
Neutrophils are the first cells to respond to invading microbes. They are the most abundant leucocytes in the blood of normal individuals, comprising ~25% total white cells. Neutrophils in the blood constitutively express receptors for chemokines, specifically CXCR1, which binds IL-8/CXCL8, and CXCR2, which binds CXCL1, CXCL2 and CXCL5 (although murine neutrophils express CXCR1, they do not produce IL-8 and primarily respond to CXCL1, 2 and 5 through CXCR2). Neutrophils also constitutively express adhesion molecules that bind to receptors on vascular endothelial cells ( Box 8-2 ).
CXC chemokines such as IL-8 are directly (and specifically) chemotactic for neutrophils, whereas pro-inflammatory cytokines such as IL-1α, IL-1β and TNF-α can induce increased expression of vascular cell adhesion molecules on capillary endothelial cells in the limbus. Expression of these adhesion molecules has a critical role in tethering, binding and facilitating neutrophil transmigration into the corneal stroma ( Fig. 8.6 ). This is a general mechanism for extravasation of leucocytes such as lymphocytes, where expression of specific adhesion molecules and chemokines on vascular endothelial cells are recognized by specific receptors on different cell types, thereby coordinating the cellular recruitment to the tissue.
Neutrophils (and other leucocytes) are recruited from capillaries (including limbal blood vessels) to infected tissues through a sequence of events in which pro-inflammatory cytokines produced at the site of infection induce expression of adhesion molecules on vascular endothelial cells. Selectins mediate tethering, intracellular adhesion molecules (ICAM-1, -2 and VCAM-1) bind to integrins on the neutrophils, and chemokines stimulate their transmigration across the vascular endothelium ( Fig. 8-6A ). Once in the tissue, migration of neutrophils to the site of infection is also dependent on a chemokine gradient, especially in an avascular tissue such as the cornea, where cells need to migrate from peripheral limbal vessels.
The response of neutrophils to bacteria and yeasts involves phagocytosis, degranulation and neutrophil extracellular trap formation (NETs, Fig. 8-6B ). If they are unable to ingest the much larger fungal hyphae or Acanthamoeba , they can bind to the pathogen surface and release cytotoxic components, including reactive oxygen and nitrogen species, antimicrobial peptides, serine proteases and matrix metalloproteinases. They can also undergo NETosis, releasing these cytotoxic components in the context of a DNA/histone-rich net. Neutrophils also express pathogen recognition molecules and ligand activation stimulates production of pro-inflammatory and chemotactic cytokines that exacerbate neutrophil infiltration and recruit other cells such as macrophages and T cells. The role of macrophages and dendritic cells in the retina and uveal tract is discussed in Chapters 1 and 2 . However, these mediators are also cytotoxic, and can contribute to loss of epithelial cells and keratocytes. Further matrix metalloproteinases can degrade the stromal collagen resulting in visual impairment and corneal scarring.
Adaptive immunity to microbial infection
Although most bacterial infections are extracellular and are dealt with rapidly by innate immunity, intracellular pathogens such as mycobacteria, protozoa and especially viruses survive longer and can induce an adaptive immune response. Thus they stimulate T- and B-cell responses which control infection and regulate the severity of infection. For example, CD4 cells play an important regulatory role in herpes simplex keratitis, and CD8 cells regulate herpes latency in trigeminal ganglia. Also, long-term exposure to airborne fungal spores induces systemic T-cell responses that likely regulate the severity of subsequent corneal infection. Toxoplasmosis and onchocerciasis are examples of chronic infections in which adaptive immunity plays an important role in determining disease severity and outcome. Other factors that affect adaptive immunity and increased susceptibility to microbial infections include immunosuppressive drugs and HIV infection (see also Ch. 7 , p. 437 ).
Ocular infections worldwide
Contact lenses
Contact lenses are a major risk factor for microbial keratitis. Approximately 134 million people worldwide wear contact lenses, and lens wear is the most common risk factor for corneal infections in the industrialized world. Long-term contact lenswear inhibits epithelial cell proliferation and migration and suppresses limbal stem cell production of basal corneal epithelial cells. Soft contact lenses, especially extended wear lenses, alter the microenvironment of the ocular surface by reducing the flow and effectiveness of tears, and trapping microbes at the cell surface. Further, poor hygiene in relation to lens cases and lens care solutions facilitates growth of bacteria and fungi, often forming multi-organism biofilms which are more resistant to antibiotics and lens care solutions. In this outbreak, one Lens care solution was found to be ineffective killing clinical and environmental isolates of Fusarium , resulting in over 300 cases of keratitis in the USA, Europe and Singapore in 2005/2006. Similarly, the increased incidence of Acanthamoeba keratitis in Chicago in 2007 was initially thought to be due to a lens care solution, resulting in withdrawal of the product; however, later findings showed the outbreak was due instead to reduced chlorination of the Chicago river.
Viral infections of the eye
There are several viruses that infect the eye, including adenovirus, which causes epidemic keratoconjunctivitis (serotypes 3, 7, 8 and 19), and pharyngoconjunctival fever (serotypes 1, 2, 3, 5, 7 and 14). Human papillomavirus causes epithelial proliferation resulting in formation of benign papilloma (warts) on the lids and conjunctiva. Herpes zoster ophthalmicus causes an extremely painful corneal infection as well as extensive involvement of the skin in the same dermatome served by the ophthalmic division of the trigeminal nerve (see Ch. 1 , p. 74 ) and occurs following resurgence of latency from the nerve. However, globally, herpes simplex virus 1 is the most common cause of ocular viral infections.
Herpes stromal keratitis
Herpes simplex virus 1 (HSV-1) is among the most common causes of ocular infections worldwide, as evidenced by high seroprevalence rates in industrialized and developing countries. Infection is most often asymptomatic, but oral and genital lesions are common manifestations of infection. However, HSV-1 can also cause herpes stromal keratitis (HSK) and can infect the eyelids, conjunctiva, cornea, uveal tract and retina. As with oral and genital infection, HSK can occur repeatedly and cause progressive corneal scar formation.
Following primary infection of the corneal epithelium, the virus enters corneal neurones and migrates to the trigeminal ganglia, which provides sensory innervation to the cornea (see Ch. 1 , p. 14 ). HSV-1 then enter a latent state where viral DNA is present in neurones but no infectious virus is produced. Following exposure to ultraviolet light, the virus can be reactivated and axonally transported into the corneal epithelium, which is highly innervated. Immune suppression also leads to reactivation, indicating an essential role of the host immune response to maintain latency (discussed below).
Primary infection.
TLR9 expression on corneal epithelial cells is important in the initial activation by HSV-1 infections and can be induced by HSV-1 DNA alone. These cells produce type 1 interferons (IFN-α/β), which inhibit viral replication. Natural killer cells are also recruited to the corneal stroma and produce IFN-γ and tumour necrosis factor α (TNF-α), which activate macrophages. CD4 + Th1, Th17 and T regulatory (Treg) cells also play an important role in limiting the primary response, with the suppressive activity of Tregs balancing the pro-inflammatory and pro-angiogenic activity of Th17 cells.
Angiogenesis and lymphangiogenesis.
Blood and lymph vessel formation of the normally avascular cornea is a characteristic feature of HSK, and is important to initiate an adaptive immune response to the virus as lymphatic vessels transport viral antigens to draining nodes, and blood vessels transport mature HSV-1-specific T cells to the cornea. However, angiogenesis also impairs visual acuity, and neovascularization is tightly regulated by selective production of vascular endothelial cell growth factors and receptor antagonists.
Latency.
In the course of primary corneal infection, the virus enters the axons of sensory neurones and is transported in a retrograde manner to the cell bodies, which are located in the trigeminal ganglia, where viral DNA is inserted into the nucleus. This episomal stage of the virus is maintained by expression of HSV-1 latency-associated transcripts. HSV-1 specific CD8 + T cells are found in close association with infected neurones and can form immunological synapses in which the T-cell receptor and CD8 molecules are in the same proximity as class I receptors on the neurones ( Fig. 8-7 ). Although IFN-γ is important, release of perforin in CD8 cell lytic granules is essential to maintain latency, which occurs without killing the neurones.
