Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

Figure 40.3 Mutations in Cryg. Histological sections of cataractous lenses from two different mutant lines are shown. The mutations have been induced by ENU in the Cryga gene (A) or in the

always smaller than in the wild type. With the aid of in situ hybridization techniques with a probe detecting all Cryg transcripts in embryonic sections, a lower extent of Cryg transcripts was detected in the Crygbnop mutants beginning from E13.5. The first morphological abnormality in the mutant lenses was observed as a swelling of lens fibers at E15.5 (Santhiya et al., 1995). A common feature in three Cryg mutants investigated (including Crygbnop) was inhibition of a Mg2+-dependent DNase in the lens. The decrease of DNase activity followed the same directionality (Crygens > Crygbnop > Cryget) as the decrease in the relative content of water-soluble lens protein, which might be used as a rough indicator of the severity of cataractogenesis (Graw and Liebstein, 1993). Although Cryg mutation-mediated mechanisms of cataract formation are not fully understood, it is known that the mutations interfere with the breakdown of the lens fiber cell nuclei during terminal differentiation. The alteration of this process was demonstrated for three mouse Cryg mutants (Crygbnop, Crygeelo, Cryget); in these mutants, it was shown that the mutant γ-crystallins contributed to the formation of amyloid-like fibers in the lens fiber cell nuclei (Sandilands et al., 2002).

Some of the inherited cataracts in humans are also related to mutations in the γ-crystallin-encoding genes. In humans, two of the six CRYG genes on chromosome 2 are pseudo-

Crygd gene (B). Inset in the overview marks the magnification, which is given below. The differences in severity are apparent. Bars = 100 μm. See color plate 38. (From Graw et al., 2004.)

genes, but mutations associated with a clinical phenotype have been found up to now only in CRYGC and CRYGD. Surprisingly, the CRYGD-P23T mutation was observed in five independent families from different continents and reported to be causative for phenotypically diverse cataractous features. In all cases, the wild-type sequence CCAC CCCAA changes to CCACACCAA. Moreover, one of the dominant human CRYGD mutations (W156X) is identical to the dominant mouse Lop12 mutation; the common single base-pair exchange is G→A, which cannot be explained by a slippage mechanism during DNA replication. Consequently, in mouse and human, 18 amino acids are missing at the C-terminus, leading to a dominant phenotype (in contrast to the 16 amino acids that are missing in the recessive mouse Crygs mutation).

Cytoskeletal Proteins There are three major lens cytoskeletal proteins, filensin (CP94 or beaded filament structural protein 1; gene symbol Bfsp1), phakinin (also referred to as CP49 or beaded filament structural protein 2; gene symbol Bfsp2), and vimentin. Since vimentin knockout mutants do not show an ocular phenotype, the two Bfsp genes are important for lens transparency. CP49 and filensin, together with α-crystallins, have been localized at unique cytoskeletal structures within the lens fiber cells known as

beaded filaments. They are considered to be important in facilitating the chaperone activity of α-crystallin assemblies. Mutations in these two genes lead to cataract formation.

Disruption of the Bfsp1 gene reduced levels of filensin’s assembly partner CP49 and prevented the assembly of beaded filaments. These knockouts began to show evidence of light scattering by 2 months and worsened with age. Heterozygous animals exhibited an intermediate phenotype, showing a moderate light scattering at 5 months (Alizadeh et al., 2003).

However, the knockout of the Bfsp2 gene does not lead to cataracts, even if the absence of CP49 causes a subtle loss of optical clarity in the lens (Alizadeh et al., 2002; Sandilands et al., 2003). Moreover, a deletion of the splice-acceptor site in exon 2 of the mouse Bfsp2 results in a splicing of exon 1 to exon 3 and causes a frameshift in the reading frame as well as the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. The phenotype of this mutation is also subtle, as described for the knockout of the entire gene. Since this mutation is present in several mouse strains (129, 101, and CBA), it might interfere with other mutations or targeted deletions, and therefore it might have important implications for lens studies using these strains (Sandilands et al., 2004). Mutations in CP49-encoding gene BFSP2 were shown to be responsible for dominant cataracts in humans (Conley et al., 2000; Jakobs et al., 2000); their phenotypes, however, seem to be variable ranging from congenital nuclear, sutural, or stellate cataracts to juvenile-onset cataracts.

Another protein, one associated with the lens cytoskeleton, is the Nhs1 protein, which is affected in the human Nance-Horan syndrome (NHS). Recently, a large insertion between exon 1 and exon 2 of the mouse Nhs1 gene was shown to underlie the X-linked dominant cataract Xcat, which was recovered after parental irradiation. Histological analysis during embryonic development revealed that in the affected embryos, the primary fiber cells are irregularly arranged and show small foci of cellular disintegration; the fibers progressively degenerate. Molecularly, the insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. The presence of Nhs1 exon 1 is critical for localization of the protein to the cytoplasm, whereas proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein (Huang et al., 2006).

Mouse models for metabolic cataracts

Sugar-Induced Cataracts in the Mouse Inborn errors in the galactose pathway and diabetes are known risk factors for the development of cataracts in humans. Sugar is

converted to the corresponding sugar alcohol, which accumulates in the lens and creates osmotic problems, eventually leading to cataract.

For a long time, appropriate mouse models were missing. One hypothesis was that the enzyme aldose reductase (responsible for the conversion of sugar to its alcohol) has a very low activity in the mouse compared to human. Therefore, it was not surprising that the galactokinase (Glk1) knockout in the mouse does not suffer from cataracts at all. However, the introduction of a human aldose reductase transgene into a Glk1-deficient background resulted in cataract formation within the first postnatal day (Ai et al., 2000). This result highlights the importance of aldose reductase in sugar-dependent cataract formation.

Another candidate for cataract formation under diabetic conditions came from investigation of the bifunctional protein DCoH (dimerizing cofactor for HNF1). It acts as an enzyme in intermediary metabolism (gene symbol Pcbd1: pterin 4α-carbinolamine dehydratase) and as a binding partner of the HNF1 family of transcriptional activators. Knockout mutants of Pcbd1 are viable and fertile but display hyperphenylalaninemia and a predisposition to cataracts. Lens opacities were visually detectable in about 20% of the Pcbd1 null mice, if maintained on the outbred CD1 genetic background. The age at onset varied widely, with the earliest detection at 12 days; most of the affected animals had developed cataracts by the age of 24 weeks. The incidence of cataract formation was reduced in the C57BL/6J inbred genetic background (Bayle et al., 2002).

Protein-Bound Carbohydrates and Cataract Besides the free sugars of the intermediate metabolic pathways, sugar residues are present in a variety of glycoproteins. One of the corresponding enzymes is the α(1,3)-galac- tosyltransferase, which catalyzes the addition of galactose in an α(1,3) configuration to particular glycoproteins. Therefore, it is also referred to as glycoprotein galactosyltransferase α1,3 (gene symbol Ggta1); the corresponding gene is mapped to chromosome 2. The transfer of galactose to particular glycoproteins creates a highly immunogenic epitope that is present in all mammals except humans, apes, and Old World monkeys. Ggta1 knockout mice have impaired glucose tolerance and decreased insulin sensitivity, and develop cataracts. A white pinhead opacity was observed in one eye at an average age of 36–37 days and in the second eye 1–2 days later. Rapid progression to full opacities occurred on average within 7–8 days. Early nuclear and posterior cortical changes, as well as fiber folds and swollen sutures, have been observed (see Dahl et al., 2006, and references therein).

Another interesting mouse model for congenital cataracts was characterized by targeting the gene coding for perlecan (Hspg2, mapped to chromosome 4). Perlecan is a large,

multidomain, heparan sulfate proteoglycan found in all basement membranes; besides type IV collagen and laminin, it is a core protein of the lens capsule. Therefore it is not surprising that the homozygous deletion of the exon 3 by gene targeting leads to leakage of cellular material through the lens capsule and degeneration of the lens within 3 weeks after birth. In detail, loss of exon 3 removes the attachment sites for three heparan sulfate side chains composed of linear polysaccharides. It is speculated that this deletion might also change the affinity of perlecan for basic fibroblast growth factor. The cataractogenic potential of deletion within the Hspg2 gene is dramatically enhanced in double knockouts, including both Hspg2 and Col18a1; the Col18a1 knockouts do not have any eye phenotype (Rossi et al., 2003).

Cholesterol Metabolism and Cataract One of the “old” syndromic, dominant mouse cataract mutants is the X-linked bare patches (Bpa). Whereas hemizygous males die before birth, heterozygous females have patches of bare skin. Lens cortical “frost figure” opacities are present. Molecular analysis showed that mutations in the gene Nsdhl (encoding an NAD(P)H steroid dehydrogenase-like protein) are responsible for the phenotype in two independent Bpa and three independent striated (Str) alleles (Liu et al., 1999). At the time it was published, it was the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. In total, 10 alleles are reported (three spontaneous, two chemically induced, and five irradiation induced). It is also the first cataract phenotype shown to be related to the cholesterol pathway.

Mouse models for senile cataracts

The Emory mouse is a well-characterized genetic model for age-onset cataract. Emory mice develop cataracts at 5–6 months (early cataract strain) or 6–8 months (late cataract strain). Emory mouse cataracts increase in severity with age and first develop in the anterior superficial cortex region of the lens. They eventually progress into the anterior deep cortex region and ultimately result in complete lens opacification. Emory mouse cataracts are also associated with changes in numerous biochemical parameters and gene expression levels in the lens (see Sheets et al., 2002, and references therein). The first unpublished results from our laboratory concerning the mapping of the underlying mutation strongly suggest that the late Emory cataract (gene symbol Em) is a complex genetic disease that results from mutations in several genes, which must interact to produce this particular type of cataract.

Another genetic mouse model for senile cataracts is the senescence-accelerated mouse (SAM), which was identified at Kyoto University in 1970 on an AKR/J background strain. There are eight senescence-prone (SAM-P) strains, which

are characterized by an earlier onset and more rapid advancement of senescence resulting from a significantly shorter life span. Cataracts have been found in the SAM-P/1 and SAM-P/9 strains. The earliest change was the appearance of a ripple mark body at about 3 months of age. The number of rippled rings increased with age. These changes later induced refractive distortion of retinal vessels. Wholemount flat preparations of the epithelium showed that the number of cells was markedly decreased in advanced stages of cataract. In the late stages of life, the lens cortex became liquefied and developed into a mature cataract (Nishimoto et al., 1993). The mode of inheritance and the linkage to a particular chromosome still remain to be investigated.

Recently, the phenotypic characterization of a conditional knockout of the murine Nbn gene (encoding nibrin) was reported. The Nbn gene is the mouse homologue for the human Nijmegen breakage syndrome gene. All Nbn-defi- cient lenses develop cataracts at an early age due to altered lens fiber cell differentiation, including disruption of normal lens epithelial and fiber cell architecture and incomplete denucleation of fiber cells. In addition, Nbn-deficient lenses show dysregulated transcription of various crystalline genes. These features implicate a function of Nbn in terminal differentiation of the lens fiber cells and cataractogenesis (Yang et al., 2006). The encoded protein nibrin has a role in DNA double-strand-break sensing in response to DNA damage and repair. Since defects in DNA damage repair are frequently associated with premature aging processes, the mutation in the Nbn gene might be a first hint at the participation of the corresponding repair and cell cycle control proteins in the formation of age-related cataracts.

Open questions and conclusions

Database searches indicate additional mouse mutants exhibiting diverse forms of cataract that have not yet been characterized with respect to their mutations. One of them is Tim (translocation-induced circling mutation), which is associated with a reciprocal chromosomal translocation between chromosomes 4 and 17. Affected mice develop an anterior subcapsular cataract that appears after birth and is progressive and accompanied by abnormal head tossing and circling behavior. The most likely explanation for the phenotype is that the translocation breakpoint disrupted a gene or its regulation; this breakpoint remains to be determined (Smith et al., 1999). Other mutants include the following (details and references are available on the Web site of the Jackson Laboratory [www.informatics.jax.org]):

• The mutation vacuolated lens (vl) is mapped to mouse chromosome 1 and leads to opaque white lenses. Additionally, the mutants are characterized by a white belly spot and spina bifida. Small lens vacuoles are present at birth.

•The mutant blind-sterile (bs) is characterized by bilateral nuclear cataracts, microphthalmia, and glossy coats. The cataracts are detectable at E16. Females are fertile, but males are sterile. The mutation was mapped to mouse chromosome 2.

•The Tcm mutation (total cataract with microphthalmia), a cataract with iris dysplasia and coloboma, and the Ccw mutation, cataract and curly whiskers, are localized to mouse chromosome 4.

•The nuclear posterior polar opacity (Npp) maps to chromosome 5.

•Cat5 (previously To2), a total opacity, is located close to the centromer on chromosome 10.

•Two alleles of Cat3 (Cat3vl, vacuolated lens; Cat3vao, cata-

ract with anterior opacity) arose independently in the F1 generation after paternal γ-irradiation and map to the central

region of chromosome 10.

•The so-called rupture of lens cataract (rlc) was mapped to chromosome 14; a similar form, lr2 (lens rupture 2), was mapped to a close position. The opacity in the rlc/rlc mice becomes apparent at 35–60 days of age; there are no developmental changes reported.

•Finally, a form of cataract that forms postnatally without observed developmental alterations is the Nakano cataract (nct). The mutation was mapped to chromosome 16.

A more detailed analysis of these mutants should allow a more precise description of the mechanisms leading to cataracts. The list of already characterized mutants and the list of not yet characterized mutants (which is still increasing, owing to ongoing mutagenesis screens and improved phenotyping strategies) underline the power of this particular genetic system.

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The pathogenesis of ocular infectious disease is determined by the virulence of the microorganism, the host immune response, and the anatomical features of the site of infection. In the eye, the cornea is unique anatomically, as it must remain optically clear and avascular, yet able to respond rapidly to microbial insult, protecting itself and the interior structures of the eye, such as the retina, from sight-threatening microbially initiated damage. Understanding innate and acquired immune response mechanisms during viral, bacterial, fungal, and parasitic eye infections remains fundamental to the rational design of therapeutic strategies to eliminate the microorganisms, control inflammatory responses, and minimize the action of their virulence factors, thus preventing permanent structural damage to the cornea and interior ocular structures, which would render them optically dysfunctional.

Numerous animal models have provided much information about immune system responses to infection. Among them, the mouse is often regarded as a model of choice. Mice are susceptible to a similar range of microbial infections as humans, and marked differences between inbred strains can be exploited to analyze the genetic basis of infections. In addition, the genetic tools that are available for use in the laboratory mouse, and new techniques to monitor the expression of genes (e.g., bacterial) in vivo, make the mouse the principal experimental animal model for studying mechanisms of infection and immunity (Buer and Balling, 2003).

Herpetic infections of the cornea

Herpetic viral infection of the cornea caused by herpes simplex virus (HSV) is a leading nontraumatic cause of blindness, despite the availability of both immunosuppressive and antiviral drugs (O’Brien and Hazlett, 1996). Acute infection of the corneal surface is a manifestation of virusinduced cytolysis. The corneal stroma may also be involved as a result of recurring infections associated with reactivation from latency. Stromal inflammation reflects an immunopathological process that often leads to corneal scarring, neovascularization, permanent endothelial dysfunction, and vision impairment. HSV also may be causative in dermatitis, blepharitis, conjunctivitis, iridiocyclitis, and retinitis.

The mouse model has greatly furthered our understanding of the role of the immune response in tissue damage. Mice are experimentally infected with certain strains of HSV and develop keratitis. Wounding of the cornea is usually required, and not all mouse strains are susceptible. Herpes stromal keratitis (HSK) in the mouse is a model of disciform nonnecrotizing keratitis in which the inflammation may be dominated eventually by lymphocytes and other mononuclear cells (O’Brien and Hazlett, 1996). HSK is T cell mediated, and in BALB/c mice infected with HSV-1, tissuedestructive inflammation in the cornea (HSK) and other periocular lesions develop about 7 days after viral infection. CD4+ T cells and T helper 1 (Th1)-type cytokines contribute both to the immunopathology in the cornea and to the eradication of viral replication in the skin. Studies in CD4+ T cell–deficient mice showed that these cells preferentially mediate HSK, but in their absence, a high infectious dose of HSV-1 can induce histologically similar but transient HSK that is mediated by CD8+ T cells (Lepisto et al., 2006). Disruption of CD40/154 signaling does not affect the initial expansion of CD4+ T cells in the draining lymph nodes but dramatically reduces the persistence and Th1 polarization of these cells. It was concluded that CD154 signaling is required during the inductive but not the effector phase of the Th1 immune response in the infected cornea, and therefore local disruption of this signaling pathway would not likely be a useful therapy for HSK (Xu et al., 2004). In contrast, others have used mice to show that CD86, a member of the costimulatory family of molecules, is important in the development of cytotoxic T cells and in reducing viral replication in the eyes of HSV-1-infected mice (Osorio et al., 2005). In mice, low delayed-type hypersensitivity (DTH) responses were found to be associated with less severe disease, while high DTH responsive mice exhibited worse disease. IL-10 knockout mice also had worsened disease, while mice given recombinant (r) IL-10 protein by ocular and intraperitoneal routes had less severe lesions (Keadle and Stuart, 2005).

Recurrent HSV-1 usually results from reactivation of latent virus in sensory neurons, followed by transmission to peripheral sites (Liu et al., 2000). The mechanism of this has been extensively studied in mouse models. It was shown that CD8+ T cells that are present in the trigeminal ganglion at

the time of its excision can maintain HSV-1 in a latent state in sensory neurons in ex vivo trigeminal ganglion cultures (Liu et al., 2000). The use of T cell transgenic mice with severe combined immunodeficiency syndrome (SCID) or mice on a recombinase-activating gene–deficient background lacking both T and B lymphocytes also suggests that CD8+ T cell control is expressed in the trigeminal ganglion, serving to curtail the source of the virus to the cornea (Banerjee et al., 2004).

The pathogenesis of corneal scarring and vascularization in HSK is uncertain but appears to reflect a complex interaction of various cytokines, chemokines, and growth factors brought in by inflammatory cells or produced locally in the cornea in response to HSV-1 infection. Evidence suggests that HSV-1 infection disrupts the normal equilibrium between angiogenic and antiangiogenic stimuli, leading to vascularization of the normally avascular cornea. Thrombospondin 1 and 2, matrix proteins involved in wound healing, are potent antiangiogenic factors and may be among the critical players involved. It has been suggested that elucidating their role in corneal scarring and vascularization may lead to improved therapies for HSK (Kaye and Choudhary, 2006).

In this regard, matrix metalloproteinase-9 (MMP-9), a type IV collagenase, has been shown to contribute to corneal neovascularization in the mouse corneal stroma infected with HSV. Neutrophils (PMNs), which invade the cornea soon after infection, were considered a likely source of the matrix-degrading enzyme, since using a PMN-specific monoclonal antibody (mAb) diminished MMP-9 expression, as well as the extent of angiogenesis. MMP-9 knockout mice also had diminished disease, while use of tissue inhibitor of metalloproteinase-1 (TIMP-1), a specific MMP-9 inhibitor, reduced disease in wild-type mice. These data suggest that targeting MMP-9 for inhibition may improve therapy for HSK (Lee et al., 2002). Rapid improvement of HSV-1- induced keratitis also was noted after amniotic membrane transplantation onto the cornea. It was postulated that this was caused by reduced expression of MMP-8 and -9 and increased expression of TIMP-1 (Heiligenhaus et al., 2005). Others have shown that in the virus-infected cornea, inter- leukin-6 (IL-6) promotes corneal infection by acting in an autocrine-paracrine fashion to induce resident corneal cells to make macrophage inhibitory protein-2 (MIP-2) and macrophage inflammatory protein-1α (MIP-1α/CCL3), which in turn recruit PMNs to the virus infection site in the murine cornea (Fenton et al., 2002). Use of a transgenic mouse that overexpresses the IL-1 receptor antagonist (IL-1ra) protein revealed that these mice were markedly resistant to HSK, compared with IL-1ra knockout and C57BL/6 WT control mice. Resistance was the consequence of reduced expression of molecules such as IL-6 and MIP-2, as well as vascular endothelial growth factor (VEGF) production, normally

upregulated directly or indirectly by IL-1. These mice also had a marked reduction in angiogenesis, an essential step in HSK pathogenesis (Biswas et al., 2004a). Mice receiving IL-1ra protein also had diminished disease severity (Biswas et al., 2004b). A better understanding of the role of VEGF in induction of angiogenesis and in the pathogenesis of HSK was derived from experiments in which mice were injected with the VEGF inhibitor mFlt(1-3)-immune globulin G. These mice had diminished angiogenesis, and the severity of lesions after HSV infection was less (Zheng et al., 2001). Administration of a cyclooxygenase 2 (COX-2) selective inhibitor also was tested and was found to decrease PMN infiltration into the HSV-infected cornea and diminished corneal VEGF levels, likely accounting for the reduced angiogenic response noted in these mice (Biswas et al., 2005). In another study, application of plasmid DNA encoding IL18 to the cornea of mice before HSV-1 ocular infection also reduced angiogenesis and diminished HSK lesions (Kim et al., 2005). Systemic injection of VEGF pathway-specific silencing (si) RNAs before infection was another confirmation of the role of VEGF in disease, as knocking down the gene for VEGF inhibited ocular angiogenesis induced by HSV (Kim et al., 2004). The absence of the chemokine receptor CCR5, a shared receptor for the beta chemokines CCL3 and CCL5, appeared to play a role in regulating leukocyte trafficking and control of virus burden, but was not critical to prevent mortality after corneal HSV-1 infection (Carr et al., 2006).

Findings from mouse studies have also suggested that HSK may be the result of either molecular mimicry or bystander activation phenomenon (Wickham and Carr, 2004). Molecular mimicry has been difficult to prove and is based on the tenet that causative viruses express epitopes that cross-react with a host protein and that the initial immune response to viruses carries over to include antihost reactivity. Bystander activation represents a complex that could include the release of normally sequestered antigens from damaged cells that have become immunogenic, alteration of host protein structure, and the subversion of host cells, causing pro-inflammatory mediator production or the synthesis of abnormal products such as autoantibodies (Wickham and Carr, 2004).

Bacterial infections and keratitis

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that rapidly induces bacterial keratitis, a disease often associated with extended-wear contact lens use (O’Brien and Hazlett, 1996). The host innate immune response to this pathogen includes local PMN recruitment, which is essential to control bacterial replication, bacterial spread, and host survival. Nonetheless, PMN persistence in the cornea is also associated with destructive pathology, including stromal

scarring and perforation, potentially requiring corneal transplantation (Steuhl et al., 1987; Hazlett, 2004). PMN infiltration into inflamed tissue is largely controlled by local production of inflammatory mediators. In the mouse, two members of the C-X-C family of chemokines, MIP-2 (functional homologue of human IL-8) and KC, are potent chemoattractants and activators of PMN. In corneal infections, MIP-2 was shown to be the major chemokine that attracts PMNs into the P. aeruginosa-infected cornea, and the persistence of PMNs in the cornea of susceptible (cornea perforates) C57BL/6 versus resistant BALB/c (no corneal perforation) mice was found to correlate with higher MIP-2 chemokine levels (both mRNA and protein) (Kernacki et al., 2000). IL-1, produced by macrophages, monocytes, and resident corneal cells (Niederkorn et al., 1989; Dinarello, 1996), also influences PMN infiltration into tissues (Dinarello and Wolff, 1993). When tested, levels of IL-1α and IL-1β (mRNA and protein) were elevated in the infected cornea of C57BL/6 (susceptible) over BALB/c (resistant) mice. Neutralization of IL-1β in infected C57BL/6 mice (Rudner et al., 2000) reduced disease severity, as evidenced by a reduction in PMNs in the cornea (MPO assay), a decreased bacterial load, and decreased levels of MIP-2 at both the mRNA and protein levels. The use of caspase-1 inhibitor treatment in C57BL/6 mice confirmed these data, even when inhibitor treatment was initiated after disease onset. In addition, improvement was augmented when the caspase- 1 inhibitor was given after infection together with the antibiotic ciprofloxacin (Thakur et al., 2004). A live attenuated P. aeruginosa vaccine has also been tested and been found to elicit outer membrane protein-specific active and passive protection against corneal infection (Zaidi et al., 2006).

The role of T cells in P. aeruginosa corneal infection was first studied in inbred C57BL/6 WT and CD8+ T-deficient, β2-microglobulin knockout mice (on the C57BL/6 background) (Kwon and Hazlett, 1997). Corneas of both groups of mice perforated by 7 days post infection and the histopathology was similar, with infiltration of PMNs within 24 hours post infection. In contrast, corneas of wild-type mice antibody depleted of CD4+ T cells and infected with P. aeruginosa did not perforate at 7 days post infection, versus mice depleted of CD8+ T cells or treated with an irrelevant antibody. Antibody neutralization of IFN-γ before infecting C57BL/6 mice also prevented corneal perforation and was associated with a lower DTH response when compared with C57BL/6 mice similarly treated with an irrelevant antibody. These data support that a CD4+ T cell (Th1)–dominant response following P. aeruginosa infection is associated with genetic susceptibility and corneal perforation in C57BL/6 mice (Kwon and Hazlett, 1997) and provided the first evidence that CD4+ T cells are important in the development of keratitis. In addition, the use of gene array studies confirmed a Th1 versus Th2 bias of C57BL/6 versus BALB/c

mice to infection with Pseudomonas (Huang and Hazlett, 2003). Other studies investigated whether IL-12 (IL-12 p40) was associated with IFN-γ production and the susceptibility response of C57BL/6 mice after P. aeruginosa challenge. IL12 p40 knockout mice (C57BL/6 background) versus wildtype mice were tested to examine disease progression in the endogenous absence of the cytokine. When tested, both groups of mice were susceptible to corneal challenge with P. aeruginosa, with corneal perforation observed 5–7 days post infection. Semiquantitative RT-PCR and ELISA analyses confirmed that IL-12 p40 message and protein levels were elevated after infection in the wild-type mice over the expected absence of IL-12 p40 in the knockout mouse cornea. Immunostaining for IL-12 in wild-type C57BL/6 mice revealed that stromal PMNs were at least one source of the cytokine (Hazlett et al., 2002).

The role of IL-18 and IFN-γ production in the resistance response of the predominantly Th2 responding BALB/c mouse was also tested. Semiquantitative RT-PCR detected IFN-γ mRNA expression levels in the cornea of infected mice at 1–7 days post infection. Cytokine levels were significantly upregulated when compared with control, uninfected normal mouse corneas (Huang et al., 2002). With RT-PCR, IL-18 mRNA expression was detected constitutively in the normal, uninfected cornea, but levels were significantly elevated 1–7 days post infection. To test whether IL-18 regulated IFN-γ production, BALB/c mice were injected with an anti-IL-18 mAb. Treatment decreased corneal IFN-γ mRNA (Huang et al., 2002) levels, and both bacterial load and disease severity increased when compared with IgG-injected control mice. These data provide evidence that IL-18 is critical to the resistance response of BALB/c mice by induction of IFN-γ and that IFN-γ is required for bacterial killing or stasis in the cornea (Hazlett, 2002; Hazlett et al., 2005). Another study showed that the killing effect of IFN-γ was indirect, through regulation of nitric oxide levels (McClellan et al., 2006).

Further study of the resistance response in BALB/c mice examined the role of the pro-inflammatory neuropeptide, substance P (SP), in IFN-γ production. Natural killer cells were required to produce IFN-γ; the cells expressed the neurokinin-1 receptor (a major SP receptor) and directly regulated IFN-γ production through interaction with this receptor (Lighvani et al., 2005), suggesting a unique link between the nervous system and the development of innate immunity in the cornea.

Staphylococcus aureus is a gram-positive organism that is seen in patients with epithelial defect, especially those on prolonged corticosteroid treatment (O’Brien and Hazlett, 1996). The disease progresses gradually and induces stromal opacity. A mouse model of the disease has been developed (Girgis et al., 2003) that should be usable in a large range of studies that were not feasible in a rabbit keratitis model,