Unlike Th1/Th17 cells, Th2/Th9 cells selectively migrate to the limbus/conjunctiva and initiate an eosinophilic infiltration process.

In this study we compared polarized mouse T-helper (Th) lymphocytes of four populations, sensitized against an ocular antigen, for their patterns of migration and induction of inflammatory processes in recipient mouse eyes expressing the target antigen. Th1, Th2, Th9 and Th17 cells transgenically expressing T-cell receptor (TCR) specific against hen egg lysozyme (HEL) were adoptively transferred to recipient mice expressing HEL in their eyes. Recipient eyes collected 4 or 7 days post injection were analyzed for histopathological changes.

Shedding New Light on the Process of "Licensing" for Pathogenicity by Th Lymphocytes.

Th cells sensitized against autoantigens acquire pathogenicity following two sequential events, namely activation by their target Ag and a process named "licensing." In this study, we analyzed these processes in a transgenic mouse system in which TCR-transgenic Th cells specific to hen egg lysozyme (HEL) are adoptively transferred to recipients and induce inflammation in eyes expressing HEL. Our data show that the notion that the lung is the organ where "licensing" for pathogenicity takes place is based on biased data collected with cells injected i.

Human βA3/A1-crystallin splicing mutation causes cataracts by activating the unfolded protein response and inducing apoptosis in differentiating lens fiber cells.

βγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.

Modulation of V-ATPase by βA3/A1-Crystallin in Retinal Pigment Epithelial Cells.

We have previously demonstrated that βA3/A1-crystallin, a member of the β/γ-crystallin superfamily, is expressed in the astrocytes and retinal pigment epithelial (RPE) cells of the eye. In order to understand the physiological functions of βA3/A1-crystallin in RPE cells, we generated conditional knockout (cKO) mice where Cryba1, the gene encoding βA3/A1-crystallin, is deleted specifically from the RPE using the Cre-loxP system. By utilizing the cKO model, we have shown that this protein is required by RPE cells for proper lysosomal degradation of photoreceptor outer segments (OS) that have been internalized in phagosomes and also for the proper functioning of the autophagy process.

βA3/A1-crystallin is a critical mediator of STAT3 signaling in optic nerve astrocytes.

We have previously reported that in the Nuc1 rat, which has a spontaneous mutation in Cryba1 (the gene encoding βA3/A1-crystallin), astrocytes exhibit decreased Notch signaling, leading to reduced promoter activity for glial fibrillary acidic protein (GFAP). Interestingly, in both Nuc1 astrocytes and in wild type astrocytes following knockdown of Cryba1, vascular endothelial growth factor (VEGF) secretion is decreased. This has led us to explore signaling mediators that could be regulated by βA3/A1-crystallin to modulate both GFAP and VEGF.

Increased Lipocalin-2 in the retinal pigment epithelium of Cryba1 cKO mice is associated with a chronic inflammatory response.

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice.

Phenotypes of Th lineages generated by the commonly used activation with anti-CD3/CD28 antibodies differ from those generated by the physiological activation with the specific antigen.

T-helper (Th) lineages have been generated in vitro by activating CD4 cells with anti-CD3/CD28 antibodies during polarization. Physiologically, however, the generation of Th lineages is by activation with the specific antigen presented by antigen-presenting cells (APC). Here, we used T-cell receptor (TCR)-transgenic mice to compare the phenotypes of Th1, Th9 and Th17 lineages when generated by either one of the two activation modes.

Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling.

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy.

Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin.

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. To study the developmental processes that require intact ubiquitin, we executed the most extensive characterization of the lens proteome to date.

Differential involvement of Th1 and Th17 in pathogenic autoimmune processes triggered by different TLR ligands.

The interaction between TLRs and their cognate ligands triggers both the innate and adaptive immune systems, and thus can play a pivotal role in the defense against pathogen invasion. This work investigates the differentiation of naive CD4 cells into Th1 or Th17 phenotypes in mice treated with different TLR ligands. We use a model system in which naive transgenic cells specific to hen egg lysozyme are adoptively transferred into recipients that express hen egg lysozyme in the lens of the eye.

Impaired endolysosomal function disrupts Notch signalling in optic nerve astrocytes.

Astrocytes migrate from the optic nerve into the inner retina, forming a template upon which retinal vessels develop. In the Nuc1 rat, mutation in the gene encoding βA3/A1-crystallin disrupts both Notch signalling in astrocytes and formation of the astrocyte template. Here we show that loss of βA3/A1-crystallin in astrocytes does not impede Notch ligand binding or extracellular cleavages.

Suppression of neuroinflammation by astrocytic dopamine D2 receptors via αB-crystallin.

Chronic neuroinflammation is a common feature of the ageing brain and some neurodegenerative disorders. However, the molecular and cellular mechanisms underlying the regulation of innate immunity in the central nervous system remain elusive. Here we show that the astrocytic dopamine D2 receptor (DRD2) modulates innate immunity through αB-crystallin (CRYAB), which is known to suppress neuroinflammation.

αB-Crystallin regulates expansion of CD11b⁺Gr-1⁺ immature myeloid cells during tumor progression.

The molecular chaperone αB-crystallin has emerged as a target for cancer therapy due to its expression in human tumors and its role in regulating tumor angiogenesis. αB-crystallin also reduces neuroinflammation, but its role in other inflammatory conditions has not been investigated. Here, we examined whether αB-crystallin regulates inflammation associated with tumors and ischemia.

Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment.

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL.

The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.

Purpose: We noted an unexpected inheritance pattern of lesions in several strains of gene-manipulated mice with ocular phenotypes. The lesions, which appeared at various stages of backcross to C57BL/6, bore resemblance to the rd8 retinal degeneration phenotype. We set out to examine the prevalence of this mutation in induced mutant mouse lines, vendor C57BL/6 mice and in widely used embryonic stem cells.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored.

Identification of the HSPB4/TLR2/NF-κB axis in macrophage as a therapeutic target for sterile inflammation of the cornea.

Sterile inflammation underlies many diseases of the cornea including serious chemical burns and the common dry eye syndrome. In search for therapeutic targets for corneal inflammation, we defined the kinetics of neutrophil infiltration in a model of sterile injury to the cornea and identified molecular and cellular mechanisms triggering inflammatory responses. Neutrophil infiltration occurred in two phases: a small initial phase (Phase I) that began within 15 min after injury, and a larger second phase (Phase II) that peaked at 24-48 h.

αA- and αB-crystallins interact with caspase-3 and Bax to guard mouse lens development.

The small heat shock protein, α-crystallin, exists in two isoforms, αA and αB, and displays strong ability against stress-induced apoptosis. Regarding their functional mechanisms, we and others have demonstrated that they are able to regulate members in both caspase and Bcl-2 families. In addition, we have also shown that αA and αB may display differential anti-apoptotic mechanisms under certain stress conditions.

Overexpression of human γC-crystallin 5 bp duplication disrupts lens morphology in transgenic mice.

Purpose: To delineate the molecular mechanisms underlying autosomal dominant congenital cataracts caused by a 5 bp duplication in human CRYGC.

Methods: c.119_123dup (CRYGC5bpd) and wild-type human γC-crystallin (CRYGC) were expressed in transgenic mouse lenses by the chicken βB1-crystallin promoter.

Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

Background: The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.

Antigen-specific Th9 cells exhibit uniqueness in their kinetics of cytokine production and short retention at the inflammatory site.

Recently reported lines of Th9 cells, producing IL-9 and IL-10, were generated by polarization with IL-4 and TGF-β and activation with Abs against CD3 and CD28. In this paper, we analyzed features of Th9 lines similarly polarized but activated by the "natural mode" (i.e.

alphaB-crystallin regulation of angiogenesis by modulation of VEGF.

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected.

Unlike Th1, Th17 cells mediate sustained autoimmune inflammation and are highly resistant to restimulation-induced cell death.

Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly.

A suppressive oligodeoxynucleotide inhibits ocular inflammation.

Synthetic oligodeoxynucleotides (ODN) expressing 'suppressive' TTAGGG motifs down-regulate a variety of proinflammatory and T helper type 1 (Th1)-mediated pathological immune responses. The ability of the archetypal suppressive ODN A151 to inhibit ocular inflammation was examined in two murine models: experimental autoimmune uveitis, induced by immunization with a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP) and adoptively transferred ocular inflammation, induced by transferring Th1 cells specific to hen egg lysozyme (HEL) into recipient mice that express HEL in their eyes. A151 treatment suppressed the inflammation in both models.

VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.

VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival.