Holly R. Chinnery

Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea.

Membrane nanotubes are a recently discovered form of cellular protrusion between two or more cells whose functions include cell communication, environmental sampling, and protein transfer. Although clearly demonstrated in vitro, evidence of the existence of membrane nanotubes in mammalian tissues in vivo has until now been lacking. Confocal microscopy of whole-mount corneas from wild-type, enhanced GFP chimeric mice, and Cx3cr1gfp transgenic mice revealed long (>300 μm) and fine (<0.8 μm diameter) membrane nanotube-like structures on bone marrow-derived MHC class II+ cells in the corneal stroma, some of which formed distinct intercellular bridges between these putative dendritic cells. The frequency of these nanotubes was significantly increased in corneas subjected to trauma and LPS, which suggests that nanotubes have an important role in vivo in cell-cell communication between widely spaced dendritic cells during inflammation. Identification of these novel cellular processes in the mammalian cornea provides the first evidence of membrane nanotubes in vivo.

Toll-like receptors at the ocular surface.

The Toll-like receptor (TLR) family of pathogen recognition molecules has an important role in recognizing microbial pathogens and microbial breakdown products. Activation of TLRs in the corneal epithelium induces CXC chemokine production and recruitment of neutrophils to the corneal stroma. Although essential for pathogen killing, neutrophils can cause extensive tissue damage, leading to visual impairment and blindness. In this review, we examine the role of TLRs in microbial keratitis and in noninfectious corneal inflammation, most commonly associated with contact lens wear. we present recent findings on TLR signaling pathways in the cornea, including MyD88- and TRIF-dependent responses and discuss the role of resident macrophages and dendritic cells. Finally, we examine the potential for targeting the TLR pathway as a potential therapeutic intervention for microbial keratitis and contact lens-associated corneal inflammation.

Novel characterization of monocyte-derived cell populations in the meninges and choroid plexus and their rates of replenishment in bone marrow chimeric mice.

The mouse dura mater, pia mater, and choroid plexus contain resident macrophages and dendritic cells (DCs). These cells participate in immune surveillance, phagocytosis of cellular debris, uptake of antigens from the surrounding cerebrospinal fluid and immune regulation in many pathologic processes. We used Cx3cr1gfp knock-in, CD11c-eYFP transgenic and bone marrow chimeric mice to characterize the phenotype, density and replenishment rate of monocyte-derived cells in the meninges and choroid plexus and to assess the role of the chemokine receptor CX3CR1 on their number and tissue distribution. Iba-1+ major histocompatibility complex (MHC) Class II+ CD169+ CD68+ macrophages and CD11c+ putative DCs were identified in meningeal and choroid plexus whole mounts. Comparison of homozygous and heterozygous Cx3cr1gfp mice did not reveal CX3CR1-dependancy on density, distribution or phenotype of monocyte-derived cells. In turnover studies, wild type lethally irradiated mice were reconstituted with Cx3cr1/gfp-positive bone marrow and were analyzed at 3 days, 1, 2, 4 and 8 weeks after transplantation. There was a rapid replenishment of CX3CR1gfp-positive cells in the dura mater (at 4 weeks) and the choroid plexus was fully reconstituted by 8 weeks. These data provide the foundation for future studies on the role of resident macrophages and DCs in conditions such as meningitis, autoimmune inflammatory disease and in therapies involving irradiation and hematopoietic or stem cell transplantation.

Host Defense at the Ocular Surface

Microbial infections of the cornea frequently cause painful, blinding and debilitating disease that is often difficult to treat and may require corneal transplantation. In addition, sterile corneal infiltrates that are associated with contact lens wear cause pain, visual impairment and photophobia. In this article, we review the role of Toll-Like Receptors (TLR) in bacterial keratitis and sterile corneal infiltrates, and describe the role of MD-2 regulation in LPS responsiveness by corneal epithelial cells. We conclude that both live bacteria and bacterial products activate Toll-Like Receptors in the cornea, which leads to chemokine production and neutrophil recruitment to the corneal stroma. While neutrophils are essential for bacterial killing, they also cause tissue damage that results in loss of corneal clarity. These disparate outcomes, therefore, represent a spectrum of disease severity based on this pathway, and further indicate that targeting the TLR pathway is a feasible approach to treating inflammation caused by live bacteria and microbial products. Further, as the P. aeruginosa type III secretion system (T3SS) also plays a critical role in disease pathogenesis by inducing neutrophil apoptosis and facilitating bacterial growth in the cornea, T3SS exotoxins are additional targets for therapy for P. aeruginosa keratitis.

Turnover of bone marrow-derived cells in the irradiated mouse cornea

In light of an increasing awareness of the presence of bone marrow (BM)‐derived macrophages in the normal cornea and their uncertain role in corneal diseases, it is important that the turnover rate of these resident immune cells be established. The baseline density and distribution of macrophages in the corneal stroma was investigated in Cx3cr1gfp transgenic mice in which all monocyte‐derived cells express enhanced green fluorescent protein (eGFP). To quantify turnover, BM‐derived cells from transgenic eGFP mice were transplanted into whole‐body irradiated wild‐type recipients. Additionally, wild‐type BM‐derived cells were injected into irradiated Cx3cr1+/gfp recipients, creating reverse chimeras. At 2, 4 and 8 weeks post‐reconstitution, the number of eGFP+ cells in each corneal whole mount was calculated using epifluorescence microscopy, immunofluorescence staining and confocal microscopy. The total density of myeloid‐derived cells in the normal Cx3cr1+/gfp cornea was 366 cells/mm2. In BM chimeras 2 weeks post‐reconstitution, 24% of the myeloid‐derived cells had been replenished and were predominantly located in the anterior stroma. By 8 weeks post‐reconstitution 75% of the myeloid‐derived cells had been replaced and these cells were distributed uniformly throughout the stroma. All donor eGFP+ cells expressed low to moderate levels of CD45 and CD11b, with approximately 25% coexpressing major histocompatibility complex class II, a phenotype characteristic of previous descriptions of corneal stromal macrophages. In conclusion, 75% of the myeloid‐derived cells in the mouse corneal stroma are replenished after 8 weeks. These data provide a strong basis for functional investigations of the role of resident stromal macrophages versus non‐haematopoietic cells using BM chimeric mice in models of corneal inflammation.

Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

Retinal Microglia and Uveal Tract Dendritic Cells and Macrophages Are Not CX3CR1 Dependent in Their Recruitment and Distribution in the Young Mouse Eye

PURPOSE The chemokine receptor CX3CR1 is expressed by monocyte-derived dendritic cells (DCs) and macrophages. CX3CR1 mediates leukocyte migration and adhesion in homeostatic and inflammatory conditions. Mice lacking Cx3cr1 have altered distribution and function of DC subpopulations in some tissue microenvironments. The present study compares the distribution of monocyte-derived cells in the normal retina and uveal tract as a prelude to the investigation of the role of CX3CR1 in murine models of ocular disease. METHODS Transgenic mice in which either one (Cx3cr1 gfp/+, heterozygous) or both (Cx3cr1 gfp/gfp, homozygous) copies of the Cx3cr1 gene have been replaced by the enhanced green fluorescent protein (eGFP) reporter gene were used to investigate the role of Cx3cr1 expression on macrophages and DCs in the normal uveal tract and retina. Chimeric mice were used to investigate turnover of these cells in the normal, uninflamed eye. RESULTS Confocal analysis found no significant differences in the density, phenotype or morphology of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice in immunostained iris, ciliary body, or choroidal and retinal wholemounts. Flow cytometry also failed to detect any difference in the density or cell shape of eGFP+ cells between Cx3cr1 gfp/+ and Cx3cr1 gfp/+ mice. Chimeras revealed 73% turnover of monocyte-derived cells in the iris and 63% in the choroid by 6 weeks after transplantation. CONCLUSIONS These data illustrate that homing or migration of DCs and macrophages to the uveal tract and retina in normal young mice is not Cx3cr1 dependent and provide a solid foundation for future studies of monocyte-derived cells and the role of Cx3cr1 in models of ocular disease.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind‐ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell–cell or bridging MNTs and blind‐ended MNTs was greatest between major histocompatibility complex (MHC) class II+ cells during corneal injury or TLR ligand‐mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid‐derived cells in inflamed corneal explants from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that MNTs form de novo at a rate of 15.5 μm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell–cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus‐1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

TLR9 Ligand CpG-ODN Applied to the Injured Mouse Cornea Elicits Retinal Inflammation

During bacterial and viral infections, unmethylated CpG-DNA released by proliferating and dying microbes is recognized by toll-like receptor (TLR) 9 in host cells, initiating innate immune responses. Many corneal infections occur secondary to epithelial breaches and represent a major cause of vision impairment and blindness globally. To mimic this clinical situation, we investigated mechanisms of TLR9 ligand-induced corneal inflammation in mice after epithelial debridement. Application of CpG oligodeoxynucleotides (ODNs) resulted in neutrophil and macrophage infiltration to the cornea and loss of transparency. By 6 hours after CpG-ODN administration, TLR9 mRNA was increased in the cornea and retina. In vivo clinical examination at 24 hours revealed inflammatory infiltrates in the vitreous and retina, which were confirmed ex vivo to be neutrophils and macrophages, along with activated resident microglia. CpG-ODN-induced intraocular inflammation was abrogated in TLR9(-/-) and macrophage-depleted mice. Bone marrow reconstitution of irradiated TLR9(-/-) mice with TLR9(+/+) bone marrow led to restored corneal inflammatory responses to CpG-ODN. Fluorescein isothiocyanate-CpG-ODN rapidly penetrated the cornea and ocular media to reach the retina, where it was present within CD68(+) retinal macrophages and microglia. These data show that topically applied CpG-ODN induces intraocular inflammation owing to TLR9 activation of monocyte-lineage cells. These novel findings indicate that microbial CpG-DNA released during bacterial and/or viral keratitis can cause widespread inflammation within the eye, including the retina.

Regular paperAccumulation of murine subretinal macrophages: effects of age, pigmentation and CX3CR1

Macrophages or activated microglia in the subretinal space are considered a hallmark of some retinal pathologies. We investigated the effects of age, pigmentation and CX(3)CR1 deficiency on the accumulation of macrophages/activated microglia in the outer retina of young and old Cx(3)cr1(gfp/gfp) (CX(3)CR1-deficient) or Cx(3)cr1(gfp/+) mice on either a pigmented (C57BL/6) or albino (BALB/c) background. Quantitative analysis of immunostained retinal-choroidal whole mounts revealed an increase in subretinal macrophage (SRMΦ) numbers in young Cx(3)cr1(gfp/gfp) mice compared with Cx(3)cr1(gfp/+) mice, however the increase was more marked in albino Cx(3)cr1(gfp/gfp) mice. In aged mice, large numbers of SRMΦ/activated microglia replete with autofluorescent debris were noted in both old pigmented Cx(3)cr1(gfp/gfp) and Cx(3)cr1(gfp/+) mice proving this accumulation was not CX(3)CR1-dependent. While CX(3)CR1 deficiency leads to an early onset of SRMΦ accumulation, our data reveal that this change occurs in both aged Cx(3)cr1(gfp/+) and Cx(3)cr1(gfp/gfp) pigmented mice in the absence of marked retinal degeneration and is likely a normal response to aging.