Lymphangiogenesis plays a pivotal role in diverse pathological conditions. Here, we demonstrate that a carbohydrate-binding protein, galectin-8, promotes pathological lymphangiogenesis. Galectin-8 is markedly upregulated in inflamed human and mouse corneas, and galectin-8 inhibitors reduce inflammatory lymphangiogenesis. In the mouse model of corneal allogeneic transplantation, galectin-8-induced lymphangiogenesis is associated with an increased rate of corneal graft rejection. Further, in the murine model of herpes simplex virus keratitis, corneal pathology and lymphangiogenesis are ameliorated in Lgals8−/− mice. Mechanistically, VEGF-C-induced lymphangiogenesis is significantly reduced in the Lgals8−/− and Pdpn−/− mice; likewise, galectin-8-induced lymphangiogenesis is reduced in Pdpn−/− mice. Interestingly, knockdown of VEGFR-3 does not affect galectin-8-mediated lymphatic endothelial cell (LEC) sprouting. Instead, inhibiting integrins α1β1 and α5β1 curtails both galectin-8- and VEGF-C-mediated LEC sprouting. Together, this study uncovers a unique molecular mechanism of lymphangiogenesis in which galectin-8-dependent crosstalk among VEGF-C, podoplanin and integrin pathways plays a key role.

Background:The ability of autotransporter (AT) to translocate polypeptides with multiple disulfide bonds is controversial. Results: Surface display of functional chymotrypsin (4 S-S) and M18 scFv (2 S-S) was quantitatively characterized. Conclusion: Surface display of functional recombinant protein with multiple disulfide bonds can be achieved using AT system. Significance: Displaying recombinant proteins with disulfide bonds enhances utility of ATs.

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 the means 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 cell volume (4364.4 ± 489.6 vs. 1787.6 ± 111.0 μm, P < 0.001) and sphericity (0.82 ± 0.01 vs. 0.42 ± 0.02, P < 0.001) compared with steady state. Furthermore, IV-MPM analyses revealed alterations in both the CD11c 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) and velocity (CD11c: 1.91 ± 0.07 μm/min vs. 1.73 ± 0.1302 μm/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-MPM represents a powerful tool to study leukocytes in corneal diseases in context.-Seyed-Razavi, Y., Lopez, M. J., Mantopoulos, D., Zheng, L., Massberg, S., Sendra, V. G., Harris, D. L., Hamrah, P. Kinetics of corneal leukocytes by intravital multiphoton microscopy.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.

SUMMARY The presence and potential functions of resident plasmacytoid dendritic cells (pDCs) in peripheral tissues is unclear. We report that pDCs constitutively populate naïve corneas and are increased during sterile injuries or acute herpes simplex virus 1 (HSV-1) keratitis. Their local depletion leads to severe clinical disease, nerve loss, viral dissemination to the trigeminal ganglion and draining lymph nodes, and mortality, while their local adoptive transfer limits disease. pDCs are the main source of HSV-1-induced IFN-α in the corneal stroma through TLR9, and they prevent re-programming of regulatory T cells (Tregs) to effector ex-Tregs. Clinical signs of infection are observed in pDC-depleted corneas, but not in pDC-sufficient corneas, following low-dose HSV-1 inoculation, suggesting their critical role in corneal antiviral immunity. Our findings demonstrate a vital role for corneal pDCs in the control of local viral infections.

The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögren's syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n = 20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 106 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren's syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.

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The isolation of human monoclonal antibodies (hmAb) has emerged as a versatile platform in a wide variety of contexts ranging from vaccinology to therapeutics. In particular, the presence of high titers of circulating auto-antibodies is implicated in the pathology and outcome of autoimmune diseases. Therefore, the molecular characterization of these hmAb provides an avenue to understanding the pathogenesis of autoimmune diseases. Additionally, the phenotype of the auto-reactive B cells may have direct relevance for therapeutic intervention. In this report, we describe a high-throughput single-cell assay, microengraving, for the screening, characterization and isolation of anti-citrullinated protein antibodies (ACPA) from peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients. Stimulated B cells are profiled at the single-cell level in a large array of sub-nanoliter nanowells (~105), assessing both the phenotype of the cells and their ability to secrete cyclic-citrullinated peptide (CCP)-specific antibodies. Single B cells secreting ACPA are retrieved by automated micromanipulation, and amplification of the immunoglobulin (Ig) heavy and light chains is performed prior to recombinant expression. The methodology offers a simple, rapid and low-cost platform for isolation of auto-reactive antibodies from low numbers of input cells and can be easily adapted for isolation and characterization of auto-reactive antibodies in other autoimmune diseases.