Kinetics of corneal leukocytes by intravital multiphoton microscopy

Corneal immune privilege is integral in maintaining the clear avascular window to the foreign world. The presence of distinct populations of corneal leukocytes (CLs) in the normal cornea has been firmly established. However, their precise function and kinetics remain, as of yet, unclear. Through intravital multiphoton microscopy (IV-MPM), allowing themeans to accumulate critical spatial and temporal cellular information, we provide details for long-term investigation of CL morphology and kinetics under steady state and following inflammation. Significant alterations in size and morphology of corneal CD11c+ dendritic cells (DCs) were noted following acute sterile inflammation, including cellvolume(4364.4±489.6 vs. 1787.6±111.0mm3,P<0.001)andsphericity (0.82±0.01 vs. 0.42± 0.02,P<0.001) comparedwith steady state. Furthermore, IV-MPManalyses revealed alterations in both theCD11c+DC and major histocompatibility complex class II (MHC)-II+ mature antigen-presenting cell population kinetics during inflammation, including track displacement length (CD11c: 16.57±1.41 vs. 4.64±0.56 μm, P < 0.001;MHC-II: 9.03± 0.37 vs. 4.09±0.39,P<0.001) andvelocity (CD11c: 1.91±0.07μm/minvs. 1.73±0.1302μm/min; MHC-II: 2.97±0.07 vs. 1.62 ± 0.08, P < 0.001) compared with steady state. Our results reveal in vivo evidence of sessile CL populations exhibiting dendritic morphology under steady state and increased velocity of spherical leukocytes following inflammation. IV-MPMrepresents a powerful tool to study leukocytes in corneal diseases in context.Fil: Seyed-Razavi, Yashar. Tufts University; Estados UnidosFil: Lopez, Maria J.. Tufts University; Estados UnidosFil: Mantopoulos, Dimosthenis. Harvard Medical School; Estados UnidosFil: Zheng, Lixin. Harvard Medical School; Estados UnidosFil: Massberg, Steffen. Ludwig Maximilians Universitat; Alemania. Harvard Medical School; Estados UnidosFil: Sendra, Victor German. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina. Tufts University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Harris, Deshea L.. Tufts University; Estados UnidosFil: Hamrah, Pedram. Harvard Medical School; Estados Unido

Corneal macrophages: an investigation of the cell-cell and neuro-immune interactions of local macrophages in homeostatic and injured murine corneas

The cornea is the most anterior portion of the corneoscleral envelope and, together with the conjunctiva, provides a physical and immunological barrier to foreign particles and pathogens. Like other epithelial tissues, sensory nerves densely innervate the corneal epithelium. The innervation serves a reflex function to protect the eyes and to regulate tear film secretion. The cornea also plays a major role in the refraction of light. Accordingly, its transparency is crucial in maintaining vision. An inflammatory response to infection or trauma can, however, cause the oedema of the collagenous stroma, altering the particular arrangement of keratocytes embedded within the collagenous lamellae, thus compromising vision. Despite the existence of resident macrophages and dendritic cells (DCs), distributed throughout the corneal epithelium and stroma, very little is known about their biological role, movement, method of communication over long distances, and interactions with other cell types in the cornea following injury to the epithelium. This project, therefore, looked to investigate cellular communication between resident immune cells within the corneal stroma. The first contribution of this study to the field of ocular immunology was the in vivo characterisation of membrane nanotubes (MNTs) in the murine cornea using live cell imaging. In vitro studies have provided strong evidence that MNTs enable communication and the exchange of proteins and organelles between connected immune cells. Analysis of corneal flatmounts from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that both DCs and macrophages were capable of forming MNTs following abrasion of the corneal epithelium and exposure to pathogenic stimuli (lipopolysaccharide – LPS), in response to a clinically relevant HSV-1 infection, as well as other stress stimuli. Myeloid derived cells in the iris and dura mater were also found to be capable of forming MNTs, suggesting MNTs in vivo are not a phenomenon limited to the cornea. Analysis of live-cell imaging of fresh explanted corneas revealed MNTs to form de novo, extending from the cell soma at a greater rate than those previously reported in cell culture studies. Whilst the Cx3cr1GFP expression was apparent in all MNTs captured, indicating cytoplasmic continuity within the MNT and between connected cells, the narrow diameter of MNTs in the mouse cornea likely precludes the transfer of large cellular organelles. A second contribution of this study to the field of ocular immunology was the identification of a novel sub-population of resident macrophages that intimately associated with peripheral corneal nerves. This sub-population of cells was therefore nominated as “nerve-associated macrophages” (NAMs). Investigations of other ocular and non-ocular connective tissues revealed some interaction between resident immune cells and large βIII-tubulin+ peripheral nerve fibres in the skin, dura, iris and connective tissue of the cremaster muscle. This association was not as distinctive as the peripheral nerve trunks of the cornea, indicating that the lack of a myelin sheath around nerves in the cornea might facilitate a more distinct interaction between immune cells and nerves than in other myelinated nerves. This study also revealed NAMs disassociate from peripheral corneal nerve trunks within two hours of injury to the central corneal epithelium; this disassociation was found to be Cx3cr1-dependent. Application of anaesthetic to a naïve cornea also revealed a decrease in NAM density, suggesting that alterations in the corneal sensory nerves affect NAMs. NAM density returned to baseline 72 hours after corneal injury. These findings may implicate NAMs to possibly be the early responders to corneal damage, and as such, a potential alternative ocular surface defence mechanism. In summary, the findings presented here support a role for NAMs in the peripheral cornea and MNTs following injury to the central corneal epithelium. Furthermore, it highlights that MNTs and NAMs both respond to stress stimuli and likely play an important role in the early stages of the innate immune response in the mammalian cornea. In conclusion, these data provide evidence to refute the notion that resident mononuclear phagocytes in the cornea are immunologically inert, as well as providing direct evidence to suggest that such cells are interconnected with one another and with sensory nerves of the cornea, and may contribute to shaping an adaptive immune response. A collective understanding of the cell biology of resident corneal immune cells, such as the early response of NAM and role of MNTs in corneal infectious disease progression, can ultimately lead to the development of potential therapeutic interventions aimed at regulating and reducing the harmful effects of inflammatory responses in the cornea

Corneal macrophages: an investigation of the cell-cell and neuro-immune interactions of local macrophages in homeostatic and injured murine corneas

The cornea is the most anterior portion of the corneoscleral envelope and, together with the conjunctiva, provides a physical and immunological barrier to foreign particles and pathogens. Like other epithelial tissues, sensory nerves densely innervate the corneal epithelium. The innervation serves a reflex function to protect the eyes and to regulate tear film secretion. The cornea also plays a major role in the refraction of light. Accordingly, its transparency is crucial in maintaining vision. An inflammatory response to infection or trauma can, however, cause the oedema of the collagenous stroma, altering the particular arrangement of keratocytes embedded within the collagenous lamellae, thus compromising vision. Despite the existence of resident macrophages and dendritic cells (DCs), distributed throughout the corneal epithelium and stroma, very little is known about their biological role, movement, method of communication over long distances, and interactions with other cell types in the cornea following injury to the epithelium. This project, therefore, looked to investigate cellular communication between resident immune cells within the corneal stroma. The first contribution of this study to the field of ocular immunology was the in vivo characterisation of membrane nanotubes (MNTs) in the murine cornea using live cell imaging. In vitro studies have provided strong evidence that MNTs enable communication and the exchange of proteins and organelles between connected immune cells. Analysis of corneal flatmounts from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that both DCs and macrophages were capable of forming MNTs following abrasion of the corneal epithelium and exposure to pathogenic stimuli (lipopolysaccharide – LPS), in response to a clinically relevant HSV-1 infection, as well as other stress stimuli. Myeloid derived cells in the iris and dura mater were also found to be capable of forming MNTs, suggesting MNTs in vivo are not a phenomenon limited to the cornea. Analysis of live-cell imaging of fresh explanted corneas revealed MNTs to form de novo, extending from the cell soma at a greater rate than those previously reported in cell culture studies. Whilst the Cx3cr1GFP expression was apparent in all MNTs captured, indicating cytoplasmic continuity within the MNT and between connected cells, the narrow diameter of MNTs in the mouse cornea likely precludes the transfer of large cellular organelles. A second contribution of this study to the field of ocular immunology was the identification of a novel sub-population of resident macrophages that intimately associated with peripheral corneal nerves. This sub-population of cells was therefore nominated as “nerve-associated macrophages” (NAMs). Investigations of other ocular and non-ocular connective tissues revealed some interaction between resident immune cells and large βIII-tubulin+ peripheral nerve fibres in the skin, dura, iris and connective tissue of the cremaster muscle. This association was not as distinctive as the peripheral nerve trunks of the cornea, indicating that the lack of a myelin sheath around nerves in the cornea might facilitate a more distinct interaction between immune cells and nerves than in other myelinated nerves. This study also revealed NAMs disassociate from peripheral corneal nerve trunks within two hours of injury to the central corneal epithelium; this disassociation was found to be Cx3cr1-dependent. Application of anaesthetic to a naïve cornea also revealed a decrease in NAM density, suggesting that alterations in the corneal sensory nerves affect NAMs. NAM density returned to baseline 72 hours after corneal injury. These findings may implicate NAMs to possibly be the early responders to corneal damage, and as such, a potential alternative ocular surface defence mechanism. In summary, the findings presented here support a role for NAMs in the peripheral cornea and MNTs following injury to the central corneal epithelium. Furthermore, it highlights that MNTs and NAMs both respond to stress stimuli and likely play an important role in the early stages of the innate immune response in the mammalian cornea. In conclusion, these data provide evidence to refute the notion that resident mononuclear phagocytes in the cornea are immunologically inert, as well as providing direct evidence to suggest that such cells are interconnected with one another and with sensory nerves of the cornea, and may contribute to shaping an adaptive immune response. A collective understanding of the cell biology of resident corneal immune cells, such as the early response of NAM and role of MNTs in corneal infectious disease progression, can ultimately lead to the development of potential therapeutic interventions aimed at regulating and reducing the harmful effects of inflammatory responses in the cornea

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&ndash;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&thinsp;&mu;m/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell&ndash;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

Melanoblasts Populate the Mouse Choroid Earlier in Development Than Previously Described

Purpose: Human choroidal melanocytes become evident in the last trimester of development, but very little is known about them. To better understand normal and diseased choroidal melanocyte biology we examined their precursors, melanoblasts (MB), in mouse eyes during development, particularly their relation to the developing vasculature and immune cells. Methods: Naïve B6(Cg)-Tyrc-2J/J albino mice were used between embryonic (E) day 15.5 and postnatal (P) day 8, with adult controls. Whole eyes, posterior segments, or dissected choroidal wholemounts were stained with antibodies against tyrosinase-related protein 2, ionized calcium binding adaptor molecule-1 or isolectin B4, and examined by confocal microscopy. Immunoreactive cell numbers in the choroid were quantified with Imaris. One-way ANOVA with Tukey's post hoc test assessed statistical significance. Results: Small numbers of MB were present in the presumptive choroid at E15.5 and E18.5. The density significantly increased between E18.5 (381.4 ± 45.8 cells/mm2) and P0 (695.2 ± 87.1 cells/mm2; P = 0.032). In postnatal eyes MB increased in density and formed multiple layers beneath the choriocapillaris. MB in the periocular mesenchyme preceded the appearance of vascular structures at E15.5. Myeloid cells (Ionized calcium binding adaptor molecule-1-positive) were also present at high densities from this time, and attained adult-equivalent densities by P8 (556.4 ± 73.6 cells/mm2). Conclusions: We demonstrate that choroidal MB and myeloid cells are both present at very early stages of mouse eye development (E15.5). Although MB and vascularization seemed to be unlinked early in choroidal development, they were closely associated at later stages. MB did not migrate into the choroid in waves, nor did they have a consistent relationship with nerves