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Background Nod-like receptors (NLRs) are critical to innate immune activation and induction of adaptive T cell responses. Yet, their role in autoinflammatory diseases of the central nervous system (CNS) remains incompletely defined. The NLR, Nlrp12, has been reported to both inhibit and promote neuroinflammation in an animal model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), where its T cell-specific role has been investigated. Uveitis resulting from autoimmunity of the neuroretina, an extension of the CNS, involves a breach in immune privilege and entry of T cells into the eye. Here, we examined the contribution of Nlrp12 in a T cell-mediated model of uveitis, experimental autoimmune uveitis (EAU). Methods Mice were immunized with interphotoreceptor retinoid-binding protein peptide 1–20 (IRBP1–20) emulsified in Complete Freund’s adjuvant, CFA. Uveitis was evaluated by clinical and histopathological scoring, and comparisons were made in WT vs. Nlrp12−/− mice, lymphopenic Rag1−/− mice reconstituted with WT vs. Nlrp12−/− CD4+ T cells, or among bone marrow (BM) chimeric mice. Antigen-specific Th-effector responses were evaluated by ELISA and intracellular cytokine staining. Cellular composition of uveitic eyes from WT or Nlrp12−/− mice was compared using flow cytometry. Expression of Nlrp12 and of cytokines/chemokines within the neuroretina was evaluated by immunoblotting and quantitative PCR. Results Nlrp12−/− mice developed exacerbated uveitis characterized by extensive vasculitis, chorioretinal infiltrates and photoreceptor damage. Nlrp12 was dispensable for T cell priming and differentiation of peripheral Th1 or Th17 cells, and uveitis in immunodeficient mice reconstituted with either Nlrp12−/− or WT T cells was similar. Collectively, this ruled out T cells as the source of Nlrp12-mediated protection to EAU. Uveitic Nlrp12−/− eyes had more pronounced myeloid cell accumulation than uveitic WT eyes. Transplantation of Nlrp12−/− BM resulted in increased susceptibility to EAU regardless of host genotype, but interestingly, a non-hematopoietic origin for Nlrp12 function was also observed. Indeed, Nlrp12 was found to be constitutively expressed in the neuroretina, where it suppressed chemokine/cytokine induction. Conclusions Our data identify a combinatorial role for Nlrp12 in dampening autoimmunity of the neuroretina. These findings could provide a pathway for development of therapies for uveitis and potentially other autoinflammatory/autoimmune diseases of the CNS.

We recently discovered a role for Nod2 within T cells in controlling Th17-immunity, such that deletion of Nod2 exacerbated experimental autoimmune arthritis and uveitis. Protection against opportunistic Candida albicans infection relies on intact Th17-responses. Thus, we considered whether the T cell intrinsic Nod2 might control antifungal Th17 responses at the cost of increasing host susceptibility to autoimmunity. Nod2−/− mice infected with 105 (LD50) Candida albicans had increased survival and reduced fungal burden in the kidney within 24–72 h post-infection, suggesting a critical role for Nod2 in C. albicans immunity. Rag−/− mice reconstituted with Nod2fl/fl/CD4-cre CD4+ T cells and infected with C. albicans had decreased fungal burden compared to control CD4-Cre T cell recipients, and IL-17 depletion reverted the phenotype. After 48h infection, Nod2fl/fl/CD4-cre CD4+ T cells in the kidney had increased activation (CD69), increased proportion of T effector cells, and decreased Tregs compared to controls, suggesting endogenous Nod2 negatively regulates fungal-triggered Teff/Th17 cell responses. We next asked how Nod2 might function in SKG mice that are genetically susceptible to arthritis. Whereas Nod2−/− SKG mice developed an exacerbated form of arthritis compared to Nod2+/+ SKG controls, these same mice cleared C. albicans infection better than Nod2+/+ SKG mice. Cumulatively our data indicate a critical role for T cell intrinsic Nod2 in antifungal Th17 immunity. Given these studies we posit that human NOD2 genetic variants may offer enhanced ability to fight fungal infection via a T cell intrinsic mechanism, at the cost of triggering autoimmunity. Supported by grants from VA (CDA-2 IK2BX004523 and Merit I01BX002180) and NIH (R01 EY025250).

Children with mutations in the nucleotide binding domain of NOD2 cause the monogenetic inflammatory disease Blau Syndrome, which is characterized by dermatitis, arthritis and uveitis. NOD2 is a well-known microbial sensor important in anti-bacterial immunity. However, the mechanism by which mutated NOD2 causes non-infectious inflammation in Blau is unknown. We recently reported a novel function of Nod2 as a central regulator of T cell homeostasis and as a negative regulator of autoreactive Th17 responses in experimental autoimmune uveitis (EAU) and arthritis. Here, we examined the effect of Blau mutations on T cell function and EAU using Blau knock-in mice carrying the most common Blau-mutation R314Q (corresponding to R334Q in humans) and Blau patient cells. We found that CD4+ T cells from Blau patients and Blau mice, produced increased IL-17 but decreased IFNγ following in vitro TCR-activation compared to control patients and WT mice, respectively. Blau mice developed worsened EAU compared to WT controls, which was accompanied by increased autoreactive (retina-specific) Th17 cells within the eye. Lymphocyte-deplete Rag1−/− mice reconstituted with Blau CD4+ T cells developed worse EAU than WT T cell recipients, indicating that CD4+ T cells expressing Blau-specific Nod2 mutations are sufficient to cause exacerbated EAU. Cumulatively, these data reveal a previously unconsidered role for Th17 cells in Blau Syndrome, and provide new T cell-targeted therapeutic avenues to treat this disease. Supported by grants from VA (CDA-2 IK2BX004523 and Merit I01BX002180) and NIH (R01 EY025250).

Caspase recruitment domain-containing protein 9 (Card9) promotes anti-fungal immunity following C-type lectin receptor Dectin-1 activation by b-glucans. CARD9 has also been implicated in the pathogenesis of T cell-mediated inflammatory diseases including rheumatoid arthritis and ankylosing spondylitis, but how Card9 controls non-infectious inflammation is poorly understood. Using genetically susceptible SKG mice that develop b-glucan (zymosan)-induced, Th17-mediated autoimmune arthritis and spondylitis we found that Card9-deficiency (Card9−/− SKG mice) conferred complete protection from disease. Card9 promoted arthritis via a Dectin-1-mediated mechanism, as the Dectin-1-specific ligand (curdlan) induced disease and bacterial/fungal ligands specific for Dectin-2 or Mincle did not. At 8 weeks post-zymosan Th17 cells and neutrophils were dramatically reduced in Card9−/− SKG mouse joints, indicating an interplay between Card9, neutrophils and Th17 cells in promoting arthritis. Importantly, pre-arthritic Card9−/− SKG mice had reduced neutrophils in lavage fluid 4 hours post-zymosan compared to SKG mice, but no changes in dendritic cells or macrophages. Lavage fluid neutrophils from Card9−/− SKG mice had similar Dectin-1 expression to SKG mice, yet decreased activation (retention of CD62L) and degranulation of primary (CD63) and secondary (CD66b). Akin to Card9−/− SKG mice, neutrophil-deplete SKG mice had decreased Th17 cells and delayed arthritis onset, supporting a pathogenic function for neutrophils in induction of arthritis. These data suggest that Card9 functions downstream of Dectin-1 to promote pathogenic neutrophil responses necessary to induce Th17-mediated arthritis and spondylitis.

Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) is a pattern recognition receptor known to promote anti-bacterial immunity yet its function in fungal disease is less understood. Here we sought to investigate the role of Nod2 in regulating fungal infection. Our results demonstrate that Nod2−/− mice infected with 105 (LD50) Candida albicans had increased survival and reduced kidney pathology compared to WT mice, indicating an important role for Nod2-deficiency in protection against infection. In corroboration, Nod2−/− mice had increased fungal clearance in the kidney within 24–72 h post-infection. T cell-deficient Rag−/− or Nod2−/− Rag−/− mice infected with C. albicans had similar fungal burdens, indicating a cellular role for Nod2 outside of innate and non-hematopoietic cells. In support, Nod2−/− mouse kidneys had increased frequencies of CD4+ T cells 48h post-infection, indicating a potential protective function for Nod2−/− T cells. To evaluate a putative T cell-intrinsic function of Nod2 we infected Rag1−/− mice reconstituted with CD4+ T cells from conditional knockout mice deficient in Nod2 in CD4+ T cells (Nod2fl/fl/CD4-cre) or control mice (CD4-cre). Mice harboring CD4+ T cells from Nod2fl/fl/CD4-cre mice had decreased fungal burden within 48 h and increased Th17 cells in the kidneys akin to the global Nod2−/− mice. Protection conferred by Nod2-deficient CD4+ T cells was abrogated by neutralization of IL-17, indicating the functional importance of the Nod2/IL-17 response in CD4+ T cells in C. albicans pathogenesis. Cumulatively, our data suggest that in opposition to the known protective role of Nod2 in bacterial infections, T cell-intrinsic Nod2 promotes fungal infection.

Background NOD2 is the genetic cause of Blau syndrome, an autoinflammatory disease that manifests as coincident uveitis and arthritis. Since dysregulation of IL-1 signalling is considered a pathogenic mechanism in a number of related autoinflammatory conditions, we examined the extent to which unimpeded interleukin (IL)-1 signalling influences NOD2-dependent inflammation of the eye versus the joint. Methods Mice deficient for IL-1R antagonist (IL-1Ra) were administered the NOD2 agonist muramyl dipeptide (MDP) by systemic (intraperitoneal) or local (intraocular and/or intra-articular) injections. NOD2-deficient mice received an intraocular injection of recombinant IL-1β. Uveitis was evaluated by intravital videomicroscopy and histopathology, and arthritis was assessed by near-infrared imaging and histopathology. Ocular levels of IL-1α, IL-1β and IL-1Ra were quantified by enzyme-linked immunosorbent assay. Results IL-1Ra deficiency did not render mice more responsive to systemic exposure of MDP. Despite the increased production of IL-1R agonists IL-1α and IL-1β in response to intraocular injection of MDP, deficiency in IL-1Ra did not predispose mice to MDP-triggered uveitis, albeit intravascular cell rolling and adherence were exacerbated. NOD2 expression was dispensable for the potential of IL-1 to elicit uveitis. However, we find that IL-1Ra does play an important protective role in arthritis induced locally by MDP injection in the joint. Conclusions Our findings highlight the complexity of NOD2 activation and IL-1 signalling effects that can be compounded by local environmental factors of the target organ. These observations may impact how we understand the molecular mechanisms by which NOD2 influences inflammation of the eye versus joint, and consequently, treatment options for uveitis versus arthritis.

Purpose: Here we investigate the role of donor endothelium on allograft rejection in a lamellar keratoplasty (LK) model using grafts with or without donor endothelium. Methods: Corneal buttons of donor C57BL/6 mice (2.0 mm) were transplanted to lamellar recipient beds (1.5 mm) in BALB/c mice. Two variations of the LK procedure were performed: (1) standard LK (SLK) (n = 13) without donor endothelium and (2) modified LK (MLK) (n = 14) with retained donor endothelium. The graft status was assessed by slit lamp biomicroscopy and scored for stromal opacity, corneal edema, neovascularization, and anterior chamber reaction up to 46 days post-transplantation. Corneas were also observed histologically. Results: The presence of a grafted corneal endothelium promoted graft rejection; 92.9% (13/14) of grafts were rejected in MLK after an average of 8.3 days, while 69.2% (9/13) of grafts were rejected in SLK on average 10.8 days after transplantation. The former’s stromal opacity was significantly greater at all time points after day 14 except for day 21 (p = 0.77) and day 32 (p = 0.25). Corneal edema was significantly greater in the former at all time points after day 10 except for day 21 (p = 0.16). Neovascularization was significantly greater in the former at all time points after day 10 except for day 25 (p = 0.22). Conclusion: Variations of this model of LK may be useful for studies of immunological mechanisms in corneal transplantation. The donor corneal endothelium may serve as a target of the immune response which promotes inflammation, neovascularization, and graft rejection.

Nod2 is a pattern recognition receptor known for its role in antimicrobial immunity. Intriguingly, NOD2 appears to have a role outside of infection, as a single point mutation in this molecule leads to 100% incidence of an inflammatory arthritis called Blau Syndrome. Here we investigate the role of Nod2 in regulation of arthritogenic CD4+ T cells in SKG mice. SKG mice have a mutation in ZAP-70 that results in altered thymic selection and spontaneous production of arthritogenic T cells. Nod2−/− SKG mice have exacerbated arthritis and increased numbers of CD4+IL-17+ (Th17) cells that co-produce TNF and GM-CSF compared to SKG mice. Additionally, Nod2−/− SKG mice have augmented levels of IL-17A in their joint synovial fluid. Exacerbated disease in Nod2−/− SKG mice is independent of sex or SKG-mediated dysbiosis as shown by co-housing studies, but dependent on the skg-mutation. Naïve (non-arthritic) Nod2−/− SKG mice have similar numbers of conventional CD4+ T cells with unaltered Treg frequency or function compared to SKG mice. However, CD4+ T cells isolated from naïve Nod2−/− SKG mice produce increased levels of pro-inflammatory cytokines (IL-17, IFNγ) in response to TCR-dependent or -independent stimulus. Furthermore, transfer of CD4+ T cells isolated from naïve Nod2−/−SKG mice into lymphopenic (Nude) recipients results in worsened arthritis compared to an analogous transfer of SKG-CD4+ T cells. These data suggest a previously unappreciated function of Nod2 as a negative regulator of the proinflammatory capacity and pathogenesis of autoreactive CD4+ T cells in SKG mice. Understanding how Nod2 participates in immune tolerance mechanisms will contribute to our knowledge of the pathophysiology of autoimmunity and arthritis.

This review paper examines and evaluates urine-sampling methodologies in infants and young children, to determine which methods are suitable for use in large biomonitoring surveys or studies of environmental chemicals in children younger than 6 years. Methods for non-toilet-trained children include the use of urine bags, collection pads (e.g., cotton or gauze inserts), disposable diapers, cotton diapers, and the clean catch method. In toilet-trained children, collection methods include use of a commode insert pan as well as specimen collection cups. The advantages and disadvantages of these various methods need to be evaluated with respect to the target population, timing and frequency of collection, minimum sample volume required, method of urine extraction, potential for contamination of the sample, stability of the analyte of interest, and burden on participants and research team. Collection methods must not introduce contamination or affect the integrity of the sample, should be logistically practical, and should minimize discomfort experienced by the child. Although collection of urine samples from children who are not toilet-trained is more challenging than collection from older toilet-trained children, the vulnerability of younger children to the exposure to and health effects of environmental chemicals makes finding suitable methods a priority.

Pseudomonas aeruginosa is known to invade epithelial cells during infection and in vitro. However, little is known of bacterial or epithelial factors modulating P. aeruginosa intracellular survival or replication after invasion, except that it requires a complete lipopolysaccharide core. In this study, real-time video microscopy revealed that invasive P. aeruginosa isolates induced the formation of membrane blebs in multiple epithelial cell types and that these were then exploited for intracellular replication and rapid real-time motility. Further studies revealed that the type three secretion system (T3SS) of P. aeruginosa was required for blebbing. Mutants lacking either the entire T3SS or specific T3SS components were instead localized to intracellular perinuclear vacuoles. Most T3SS mutants that trafficked to perinuclear vacuoles gradually lost intracellular viability, and vacuoles containing those bacteria were labeled by the late endosomal marker lysosome-associated marker protein 3 (LAMP-3). Interestingly, mutants deficient only in the T3SS translocon structure survived and replicated within the vacuoles that did not label with LAMP-3. Taken together, these data suggest two novel roles of the P. aeruginosa T3SS in enabling bacterial intracellular survival: translocon-dependent formation of membrane blebs, which form a host cell niche for bacterial growth and motility, and effector-dependent bacterial survival and replication within intracellular perinuclear vacuoles.

The immune system is governed by dynamic events involving in part direct intercellular interactions between an immune cell and other cells or the cell's environment. Owing to its unique optical characteristics, the eye offers remarkable opportunities for the analysis of the immune system by intravital microscopy. In this review, we present a brief overview of the current state of knowledge of leukocyte trafficking in each of three anatomically distinct and medically important regions of the eye (cornea, iris, retina) as determined by the application of intravital microscopy to animal models of disease. Additionally, we discuss the use of ocular imaging in patients and volunteers. Finally, we examine the future prospects for this field in terms of its potential for impacting our understanding of fundamental immunological phenomena.

We have previously shown that ExoU, a type III secreted cytotoxin of Pseudomonas aeruginosa, causes acute cytotoxicity towards corneal epithelial cells in vitro, and contributes to corneal disease pathology and ocular colonization in vivo. Subsequently, we reported that ExoU represses phagocyte infiltration of infected corneas in vivo. ExoU has patatin-like phospholipase activity that is required for cytotoxic activity in vitro (mammalian cell injury and death) and for disease in a murine model of pneumonia. We hypothesized that the phospholipase activity was required for ExoU-mediated corneal disease and ocular colonization. Using the murine scarification model, corneal disease pathology was examined after inoculation with approximately 10(6)cfu of a P. aeruginosa effector mutant (PA103DeltaexoUexoT::Tc) complemented with either exoU (pUCPexoU), phospholipase-inactive exoU (pUCPexoUD344A) or a plasmid control (pUCP18). Eyes were photographed and disease severity scored at 24 and 48h post-infection. Viable bacteria colonizing infected eyes were quantified at 6 and 48h. Complementation with exoU caused significantly more pathology (increased disease severity scores) and enabled bacteria to better colonize (by approximately 1000-fold) at 48h as compared to phospholipase-inactive exoU which did not differ from plasmid control. Surprisingly, exoU did not contribute to early (6h) colonization. In-vitro assays confirmed that the phospholipase domain of exoU was required for cytotoxicity towards human corneal epithelial cells. Taken together these data show that the phospholipase activity of the P. aeruginosa cytotoxin, ExoU, plays a role in the pathogenesis of corneal infection via mechanism(s) occurring after initial colonization of a susceptible cornea.

Rapidly growing populations and migration to urban areas in developing countries has resulted in a vital need for the establishment of centralized water systems to disseminate potable water to residents. Protected source water and modern, well-maintained drinking water treatment plants can provide water adequate for human consumption. However, ageing, stressed or poorly maintained distribution systems can cause the quality of piped drinking water to deteriorate below acceptable levels and pose serious health risks. This review will outline distribution system deficiencies in developing countries caused by: the failure to disinfect water or maintain a proper disinfection residual; low pipeline water pressure; intermittent service; excessive network leakages; corrosion of parts; inadequate sewage disposal; and inequitable pricing and usage of water. Through improved research, monitoring and surveillance, increased understanding of distribution system deficiencies may focus limited resources on key areas in an effort to improve public health and decrease global disease burden.

During corneal infection, cytotoxic Pseudomonas aeruginosa strains remain mostly extracellular, while invasive strains can enter corneal cells and replicate within them. We tested the hypothesis that ofloxacin, which easily penetrates host cell membranes, would be more effective than the less cell-permeable antibiotic tobramycin, for treatment of corneal infection by an invasive P. aeruginosa strain.A murine model of P. aeruginosa keratitis was used to compare the response to ofloxacin, tobramycin, prednisolone acetate, and non-preserved saline treatment, as well as combination antibiotic-corticosteroid therapy for infection caused by a cytotoxic strain (6206) and an invasive strain (PAO1). Treatment involved hourly eye drop administration for 12 hours.As expected, tobramycin was less effective at eradicating viable bacteria from corneas infected with the invasive strain. Despite rapid sterilization of corneas in other antibiotic treated groups, disease progression occurred during the 12 hour treatment period. Both antibiotics hastened disease resolution over the next 7 days for infections caused by either strain. Corticosteroid use during the 12 hour treatment period was of little added benefit.Differences between invasive and cytotoxic strain infections in their early response to the different therapeutic regimens did not translate to notable differences after 7 days, but the effects of antibiotics in halting disease progression were delayed for both strain types. These results suggest that successful management might be improved by addressing factors contributing to disease progression during sterilization of the cornea by antibiotics.

NOD2 is the genetic cause of Blau syndrome, an autoinflammatory disease that manifests as coincident uveitis and arthritis. Since dysregulation of IL-1 signalling is considered a pathogenic mechanism in a number of related autoinflammatory conditions, we examined the extent to which unimpeded interleukin (IL)-1 signalling influences NOD2-dependent inflammation of the eye versus the joint.Mice deficient for IL-1R antagonist (IL-1Ra) were administered the NOD2 agonist muramyl dipeptide (MDP) by systemic (intraperitoneal) or local (intraocular and/or intra-articular) injections. NOD2-deficient mice received an intraocular injection of recombinant IL-1β. Uveitis was evaluated by intravital videomicroscopy and histopathology, and arthritis was assessed by near-infrared imaging and histopathology. Ocular levels of IL-1α, IL-1β and IL-1Ra were quantified by enzyme-linked immunosorbent assay.IL-1Ra deficiency did not render mice more responsive to systemic exposure of MDP. Despite the increased production of IL-1R agonists IL-1α and IL-1β in response to intraocular injection of MDP, deficiency in IL-1Ra did not predispose mice to MDP-triggered uveitis, albeit intravascular cell rolling and adherence were exacerbated. NOD2 expression was dispensable for the potential of IL-1 to elicit uveitis. However, we find that IL-1Ra does play an important protective role in arthritis induced locally by MDP injection in the joint.Our findings highlight the complexity of NOD2 activation and IL-1 signalling effects that can be compounded by local environmental factors of the target organ. These observations may impact how we understand the molecular mechanisms by which NOD2 influences inflammation of the eye versus joint, and consequently, treatment options for uveitis versus arthritis.

Ellen J Lee1,2 and Holly L Rosenzweig1,2,3* 1Department of Ophthalmology, Oregon Health & Science University, Portland, OR, USA 2VA Medical Center, Portland, OR, USA 3Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, OR, USA *Corresponding author: Holly L. Rosenzweig, PhD, VA Medical Center, 3710 SW US, Veterans Hospital Rd. Bldg 103, Room E-221A, Mail stop: VA RD E-mail: rosenzwh@ohsu.edu

Rheumatoid arthritis remains a major public health concern, estimated to affect 1.5 million adults in the U.S. However, the etiology and underlying immunological mechanisms of this disease remain largely speculative. SKG mice harbor a spontaneous mutation in ZAP-70 and develop arthritogenic T cells, leading to chronic, self-sustained joint destruction. Arthritis in SKG mice replicates a current theoretical paradigm that environmental factors, including microbes, activate preexisting autoreactive T cells in genetically predisposed individuals to cause disease. Currently, the mechanisms by which innate immune receptors augment T cell activation as well as disease onset and progression are not understood. Here we sought to characterize the role of the intracellular pattern recognition molecule, NOD2, in disease pathology. Using NIR-spectroscopy and clinical scoring methods we determined that SKG mice lacking NOD2 develop markedly worse arthritis when triggered by fungal associated molecules, including curdlan and zymosan, as compared to SKG/NOD2+/+ mice. Intracellular cytokine staining showed no difference between Th17 and Th1 cytokine production by CD4+ and CD8+ T cell splenocytes between genotypes. Importantly, neutralization of IL-17A reduced disease severity in SKG/NOD2−/− mice to levels of disease exhibited by SKG/NOD2+/+, suggesting IL-17A as the prominent worsening factor in SKG/NOD2−/− mice. Our data suggest that NOD2 serves a protective role against autoimmune arthritis, in part due to regulation of IL-17A. Thus, understanding how microbial recognition pathways like NOD2 factor into the induction and pathology of arthritis is key to the development of effective a therapy.

Purpose. Dendritic cells (DCs) are antigen-presenting cells vital for initiating immune responses. In this study the authors examined the in vivo migratory capability of resident corneal DCs to various stimuli. Methods. The authors used mice expressing enhanced yellow fluorescent protein (eYFP) under control of the CD11c promoter to visualize corneal DCs. To assess the distribution and mobility of DCs, normal corneas were imaged in vivo and ex vivo with fluorescence microscopy. Intravital microscopy was used to examine the responses of resident central and peripheral corneal DCs to silver nitrate injury, lipopolysaccharide, microspheres, and tumor necrosis factor (TNF-alpha). In some experiments, TNF-alpha injection was used to first induce centripetal migration of DCs to the central cornea, which was subsequently reinjected with microspheres. Results. In normal corneas, DCs were sparsely distributed centrally and were denser in the periphery, with epithelial-level DCs extending into the epithelium. Videomicroscopy showed that though cell processes were in continuous movement, cells generally did not migrate. Within the first 6 hours after stimulation, neither central nor peripheral corneal DCs exhibited significant lateral migration, but central corneal DCs assumed extreme morphologic changes. An increased number of DCs in the TNF-alpha-stimulated central cornea were responsive to subsequent microsphere injection by adopting a migratory behavior, but not with increased speed. Conclusions. In vivo imaging reveals minimal lateral migration of corneal DCs after various stimuli. In contrast, DCs within the central cornea after initial TNF-alpha injection are more likely to respond to a secondary insult with lateral migration.

Purpose: In contrast to penetrating keratoplasty (PK), the donor cornea in lamellar keratoplasty (LK) remains separated from the host aqueous humor. There is debate about relative merits of each approach, but experimental comparisons have never been performed in animal models. Therefore, the authors developed a murine LK model. Methods: For allogeneic PK and LK surgeries, corneas of C57BL/6 mice were transplanted to BALB/c mice, assessed by slit lamp, and scored for opacity, edema, and neovascularization up to 46 d post-transplantation. Additional PK or LK surgeries were performed for histological assessment. Results: Graft rejection rate was less in LK vs. PK (69.2 vs. 100%), as was neovascularization (84.6 vs. 100%). In LK, inflammatory cells infiltrated primarily the button; in PK, heavier infiltration was observed throughout the cornea. Conclusions: This study demonstrates the feasibility of LK in mice and presents data suggesting that the inflammatory response in LK differs from that in PK.

Purpose The scarified cornea keratitis model was modified to study Pseudomonas aeruginosa infection of healing corneal epithelium. The new model was then used to study the role of ExsA, a transcriptional activator of P. aeruginosa, in bacterial penetration through injured and healing corneal epithelia. Methods Scratch-injured corneas of C57BL/6 mice were allowed to heal for 0, 6, 9, or 12 hours before inoculation with a cytotoxic (6206) or invasive (PAO1) P. aeruginosa strain. Disease progression was monitored for 14 days. The integrity of the healing epithelium was studied in uninfected eyes by fluorescein staining and by histologic examination. In other experiments, the effect of bacterial exsA mutation was studied after 0, 6, or 12 hours of healing. Three hours after infection, these eyes were used to quantify early bacterial colonization levels by viable counts, or they were sectioned to study bacterial penetration through the epithelium by microscopy. Results Corneas remained susceptible to infection 6 but not 12 hours after scratch injury. By 6 hours, the previously exposed stroma was already completely covered by several layers of epithelial cells. Fluorescein staining unexpectedly occurred even after 12 hours of healing time, showing that resistance to infection preceded full restoration of epithelial barrier function. Mutation of exsA reduced both bacterial colonization levels and penetration through the epithelium 3 hours after bacterial inoculation, but only in the 6-hour healing situation, and only for the cytotoxic strain (PA103). Mutation of exsA in the invasive strain (PAO1) had no effect on 3-hour colonization or penetration levels under any circumstances. Conclusions The 6-hour healing infection model showed a role for ExsA in early interactions with the corneal epithelium that was not detectable with the conventional (0-hour) scratch model. Comparison of the 6- and 12-hour healing models, which showed that factors additional to barrier function contribute to defense against infection, could be used to gain new insights into corneal defense mechanisms, and the methods used by bacteria to circumvent them.

PURPOSE The exoenzyme S regulatory protein ExsA regulates a type III secretion system in Pseudomonas aeruginosa. In vitro, cytotoxic strains use this system to secrete exotoxin (Exo)U and ExoT causing cytotoxicity and inhibiting their phagocytosis by epithelial cells. Invasive P. aeruginosa secrete ExoT and ExoS, but exsA mutation has little impact on their short-term interactions with epithelia. In the present study, the contribution of these ExsA-regulated proteins toward corneal infections in vivo was investigated. METHODS After anesthesia, the left cornea of C57BL/6 mice was scratch injured and then inoculated with cytotoxic (PA103) or invasive (PAK) P. aeruginosa or with isogenic mutants in exsA-related genes. Inocula of 10(3) to 10(6) bacteria/5 micro L were used, and at least five animals were assigned to each experimental group. Corneal disease was quantified at regular intervals for 14 days in masked fashion with two different scoring systems. RESULTS For the cytotoxic strain, mutation of either exoU or exoT alone had little effect on virulence, whereas simultaneous mutation of both exoT and exoU or of exsA resulted in a significantly reduced capacity to cause corneal disease. Complementation of the double exoUexoT mutant with exoU alone restored bacterial colonization levels (>3-log increase) and disease severity to wild-type levels. Complementation with exoT alone increased colonization ( approximately 3-log increase) and increased virulence to almost the same levels as wild-type or exoU-complemented infections. Virulence of the invasive strain was not reduced by mutation of exsA or of genes encoding the ExsA-regulated secreted proteins. CONCLUSIONS ExsA contributed to corneal virulence of only cytotoxic P. aeruginosa, with contributions made by both ExoU and ExoT to bacterial survival and disease severity. This differs from cytotoxic P. aeruginosa virulence in the lung, which is ExoU-dependent.