Tumor necrosis factor alpha and interleukin-6 facilitate corneal lymphangiogenesis in response to herpes simplex virus 1 infection.

UNLABELLED: Herpes simplex virus 1 (HSV-1) is a common human pathogen of clinical significance due to its association with vision impairment and encephalitis. In a mouse model of ocular neovascularization, we have previously shown that HSV-1 elicits the genesis of lymphatic vessels into the cornea proper through epithelial cell expression of vascular endothelial growth factor A (VEGFA) dependent upon expression of VEGFR2 during acute infection. We hypothesized that other factors may be involved in lymphangiogenesis, with proinflammatory cytokines as the leading candidates. In the absence of infection or inflammation, intrastromal administration of tumor necrosis factor alpha (TNF-α) coupled with VEGFA elicited lymphatic vessel genesis significantly above either factor alone as well as a vehicle control. Consistent with this observation, anti-TNF-α antibody (Ab) blocked HSV-1-mediated corneal lymphangiogenesis within the first 5 days postinfection. However, TNF-α-deficient (TNF-α(-/-)) mice displayed a level of corneal vessel growth similar to that shown by wild-type (WT) controls. To investigate the likely redundant nature of cytokines, PCR array analysis of HSV-1-infected TNF-α(-/-) mice was conducted, and it revealed several factors elevated above those found in HSV-1-infected WT mice, including interleukin-1ß (IL-1ß), platelet-derived growth factor, angiopoietin 2, insulin-like growth factor 2, and IL-6. Subconjunctival administration of neutralizing Ab to IL-6 blocked lymphangiogenesis in TNF-α(-/-) mice. Whereas the cornea levels of IL-6 were significantly reduced, there was no appreciable change in the level of IL-1ß or other proangiogenic factors analyzed. Collectively, the results suggest in addition to VEGFA, TNF-α and IL-6 promote and likely synergize with VEGFA in corneal lymphangiogenesis during acute HSV-1 infection. IMPORTANCE: We have identified at least two proinflammatory cytokines expressed locally that are involved in the genesis of lymphatic vessels in the normally avascular cornea in response to HSV-1 infection. This finding provides the basis to target IL-6 and TNF-α as additional proangiogenic factors in the cornea during the development of herpetic stromal keratitis as a means to alleviate further neovascularization and tissue pathology associated with the host immune response to the pathogen.

HSV-1 targets lymphatic vessels in the eye and draining lymph node of mice leading to edema in the absence of a functional type I interferon response.

Herpes simplex virus type-1 (HSV-1) induces new lymphatic vessel growth (lymphangiogenesis) in the cornea via expression of vascular endothelial growth factor by virally infected epithelial cells. Here, we extend this observation to demonstrate the selective targeting of corneal lymphatics by HSV-1 in the absence of functional type I interferon (IFN) pathway. Specifically, we examined the impact of HSV-1 replication on angiogenesis using type I IFN receptor deficient (CD118(-/-)) mice. HSV-1-induced lymphatic and blood vessel growth into the cornea proper was time-dependent in immunocompetent animals. In contrast, there was an initial robust growth of lymphatic vessels into the cornea of HSV-1-infected CD118(-/-)mice, but such vessels disappeared by day 5 postinfection. The loss was selective as blood vessel integrity remained intact. Magnetic resonance imaging and confocal microscopy analysis of the draining lymph nodes of CD118(-/-) mice revealed extensive edema and loss of lymphatics compared with wild-type mice. In addition to a loss of lymphatic vessels in CD118(-/-) mice, HSV-1 infection resulted in epithelial thinning associated with geographic lesions and edema within the cornea, which is consistent with a loss of lymphatic vasculature. These results underscore the key role functional type I IFN pathway plays in the maintenance of structural integrity within the cornea in addition to the anti-viral characteristics often ascribed to the type I IFN cytokine family.

Comparison of the host immune response to herpes simplex virus 1 (HSV-1) and HSV-2 at two different mucosal sites.

A study was undertaken to compare the host immune responses to herpes simplex virus 1 (HSV-1) and HSV-2 infection by the ocular or genital route in mice. Titers of HSV-2 from tissue samples were elevated regardless of the route of infection. The elevation in titers of HSV-2, including cell infiltration and cytokine/chemokine levels in the central nervous system relative to those found following HSV-1 infection, was correlative with inflammation. These results underscore a dichotomy between the host immune responses to closely related alphaherpesviruses.

PD-L1-expressing dendritic cells contribute to viral resistance during acute HSV-1 infection.

The inhibitory receptor, Programmed Death 1 (PD-1), and its ligands (PD-L1/PD-L2) are thought to play a role in immune surveillance during chronic viral infection. The contribution of the receptor/ligand pair during an acute infection is less understood. To determine the role of PD-L1 and PD-L2 during acute ocular herpes simplex virus type 1 (HSV-1) infection, HSV-1-infected mice administered neutralizing antibody to PD-L1 or PD-L2 were assessed for viral burden and host cellular immune responses. Virus titers were elevated in cornea and trigeminal ganglia (TG) of anti-PD-L1-treated mice which corresponded with a reduced number of CD80-expressing dendritic cells, PD-L1⁺ dendritic cells, and HSV-1-specific CD8⁺ T cells within the draining (mandibular) lymph node (MLN). In contrast, anti-PD-L2 treatment had no effect on viral replication or changes in the MLN population. Notably, analysis of CD11c-enriched MLN cells from anti-PD-L1-treated mice revealed impaired functional capabilities. These studies indicate PD-L1-expressing dendritic cells are important for antiviral defense during acute HSV-1 infection.

Autoregulatory characteristics of a Bacillus anthracis serine/threonine kinase.

BA-Stk1 is a serine/threonine kinase (STK) expressed by Bacillus anthracis. In previous studies, we found that BA-Stk1 activity is modulated through dephosphorylation by a partner phosphatase, BA-Stp1. In this study, we identified critical phosphorylation regions of BA-Stk1 and determined the contributions of these phosphodomains to autophosphorylation and substrate phosphorylation. The data indicate that BA-Stk1 undergoes trans-autophosphorylation within a regulatory domain, referred to as the activation loop, which carries eight putative regulatory serine and threonine residues. We identified activation loop mutants that impacted kinase activity in three different manners: regulation of autophosphorylation (T162), regulation of substrate phosphorylation (T159 and S169), and regulation of overall kinase activity (T163). Tandem mass spectrometry (MS/MS) analysis of the phosphorylation profile of each mutant revealed a second site of phosphorylation on the kinase that was influenced by the phosphorylation status of the activation loop. This second region of the kinase contained a single phosphorylation residue, S214. Previous work has shown S214 to be necessary for downstream substrate phosphorylation, and we have shown that this residue is subject to dephosphorylation by BA-Stp1. These findings indicate a connection between the phosphorylation status of the activation loop and phosphorylation of S214, and this suggests a previously undescribed model for how a bacterial STK shifts from a state of autophosphorylation to targeting downstream substrates.

CXCL1-deficient mice are highly sensitive to pseudomonas aeruginosa but not herpes simplex virus type 1 corneal infection.

PURPOSE: To determine the role of the chemokine CXCL1 on leukocyte recruitment, cytokine production and host resistance during HSV-1 and Pseudomonas aeruginosa infection. METHODS: Viral titer and bacterial load were compared following infection of wild-type (WT) and CXCL1(-/-) mice. Corneal leukocyte recruitment was determined using flow cytometry. Cytokine levels were assessed by luminex-based suspension arrays. Hematoxylin and eosin (H&E) staining, confocal microscopy, and optical coherence tomography (OCT) were used to visualize leukocyte recruitment and corneal thickening. RESULTS: HSV-1-infected WT and CXCL1(-/-) mice possessed similar viral titers in the cornea during late acute infection. Flow cytometry analysis detected similar leukocyte levels in the cornea following infection as well. By comparison, there was a significant increase in P. aeruginosa recovered from CXCL1(-/-) corneas as compared with WT mice. Imaging analysis and histochemical staining revealed impaired leukocyte recruitment to the central cornea and earlier corneal thickening in CXCL1(-/-) mice. IFN-γ, CCL2, and CCL5 protein levels were similar between WT and CXCL1(-/-) corneas following HSV-1 or P. aeruginosa infection. However, CXCL2 levels were significantly reduced in the CXCL1(-/-) corneas following either infection. CONCLUSIONS: The absence of CXCL1 and CXCL2 expression significantly impairs the ability of the host to control P. aeruginosa replication through the recruitment of leukocytes to the central cornea. In contrast, CXCL1, CXCL2, and the cells they recruit, are not required for HSV-1 clearance during acute infection.