Inflammatory lymphangiogenesis plays a crucial role in the development of inflammation and transplant rejection. The mechanisms of inflammatory lymphangiogenesis during bacterial infection, toll-like receptor ligand administration, and wound healing are well characterized and depend on ligands for the vascular endothelial grow factor receptor (VEGFR) 3 that are produced by infiltrating macrophages. But inflammatory lymphangiogenesis in nonlymphoid tissues during chronic viral infection is unstudied. Herpes simplex virus 1 (HSV-1) infection of the cornea is a leading cause of blindness and depends on aberrant host immune responses to antigen within the normally immunologically privileged cornea. We report that corneal HSV-1 infection drives lymphangiogenesis and that corneal lymphatics persist past the resolution of infection. The mechanism of HSV-1–induced lymphangiogenesis was distinct from the described mechanisms of inflammatory lymphangiogenesis. HSV-1–elicited lymphangiogenesis was strictly dependent on VEGF-A/VEGFR-2 signaling but not on VEGFR-3 ligands. Macrophages played no role in the induction of lymphangiogenesis and were not a detectable source of VEGF-A. Rather, using VEGF-A reporter transgenic mice, we have identified infected epithelial cells as the primary source of VEGF-A during HSV-1 infection. Our results indicate that HSV-1 directly induces vascularization of the cornea through up-regulation of VEGF-A expression.

Hematopoietic stem and progenitor cells were previously found to express Tolllike receptors (TLRs), suggesting that bacterial/viral products may influence blood cell formation. We now show that common lymphoid progenitors (CLPs) from mice with active HSV-1 infection are biased to dendritic cell (DC) differentiation, and the phenomenon is largely TLR9 dependent. Similarly, CLPs from mice treated with the TLR9 ligand CpG ODN had little ability to generate CD19 ؉ B lineage cells and had augmented competence to generate DCs. TNF␣ mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNF␣ ؊/؊ mice treated with CpG ODN. Increased numbers of DCs with a lymphoid past were identified in Ig gene recombination substrate reporter mice treated with CpG ODN. TLR9 is highly expressed on lymphoid progenitors, and culture studies revealed that those receptors, rather than inflammatory cytokines, accounted for the production of several types of functional DCs. IntroductionHematopoietic stem cells (HSCs) give rise to progenitors with potential to produce blood cell types with remarkably stable characteristics. Although this process is tightly controlled, recent findings suggest that hematopoiesis is dynamic and also responsive to environmental factors. 1 The loss of differentiation options is gradual, and T lymphocytes, natural killer (NK) cells, and dendritic cells (DCs) can each be made from multiple progenitors under experimental circumstances. 1,2 Indeed, apparently similar DCs arise from distinct myeloid or lymphoid progenitors. 3 This new perspective raises the possibility that choices are made between multiple pathways to replenish effectors of the immune system. Thus, it is important to learn what normal and disease conditions favor particular differentiation routes.Several major categories of DCs have been found in murine bone marrow (BM). Conventional DCs (cDCs) are competent to present antigens, whereas plasmacytoid dendritic cells (pDCs) are potent producers of type I interferon. 3 The pDCs are divisible into 2 subtypes (pDC1 and pDC2) on the basis of RAG-1 expression and patterns of cytokine production. 4 Under experimental conditions, DCs are produced from stem cells, as well as lymphoid and myeloid progenitors. [3][4][5] Flk-2/flt-3 ligand and the associated Stat3 signaling pathway are important for DC differentiation; consequently, efficient progenitors bear the Flk-2/flt-3 receptor. 3 In our experience, the highest yields of pDCs are obtained from the primitive Lin Ϫ c-Kit hi Sca-1 ϩ (LSK) fraction of murine BM. 4 Two recent reports identified a Lin Ϫ Flt3 ϩ c-Kit lo -CD115 ϩ pro-DC population capable of generating pDCs and at least 2 categories of DCs. 6,7 However, greater yields of DCs were produced from more primitive progenitors, and some of those are already restricted to particular DC pathways. 7 Common lymphoid progenitors (CLPs) represent the main pathway to B lineage cells and include most progenitors dest...

The chemokine, CXCL10, chemotactic for NK cells, activated T cells, and dendritic cells is highly expressed during viral infections, including HSV-1. The importance of this chemokine to the control of HSV-1 infection was tested using mice deficient in CXCL10 (CXCL10−/−). Following corneal infection, HSV-1 viral titers were elevated in the nervous system of CXCL10−/− mice, which correlated with defects in leukocyte recruitment including dendritic cells, NK cells, and HSV-1-specific CD8+ T cells to the brain stem. In the absence of NK cells and HSV-1-specific CD8+ T cells in wild-type (WT) or CXCL10−/− mice, similar levels of virus were recovered in the nervous system, suggesting these cells are responsible for the observed defects in the control of viral replication in CXCL10−/− mice. Leukocyte mobilization was also compared between WT, CXCL10−/−, and mice deficient in the only known receptor for CXCL10, CXCR3 (CXCR3 −/−). NK cell mobilization was comparably reduced in both CXCL10−/− and CXCR3−/− mice relative to WT animals. However, the reduction in mobilization of HSV-1-specific CD8+ T cells in CXCL10−/− was not observed in CXCR3−/− mice following HSV-1 infection. The defect was not the result of an alternative receptor for CXCL10, as Ag-specific CD8+ T cell recruitment was not reduced in mice which were deficient in both CXCL10 and CXCR3. Thus, CXCL10 deficiency results in reduced mobilization of HSV-1-specific CD8+ T cells as a result of dysregulation of CXCR3 signaling.

Herpes simplex virus-1 (HSV-1) causes lifelong infection affecting between 50 and 90% of the global population. In addition to causing dermal lesions, HSV-1 is a leading cause of blindness resulting from recurrent corneal infection. Corneal disease is characterized by loss of corneal immunologic privilege and extensive neovascularization driven by vascular endothelial growth factor-A (VEGF-A). In the current study, we identify HSV-1 infected cells as the dominant source of VEGF-A during acute infection, and VEGF-A transcription did not require TLR signaling or MAP kinase activation. Rather than being an innate response to the pathogen, VEGF-A transcription was directly activated by the HSV-1 encoded immediate early transcription factor, ICP4. ICP4 bound the proximal human VEGF-A promoter and was sufficient to promote transcription. Transcriptional activation also required cis GC-box elements common to the VEGF-A promoter and HSV-1 early genes. Our results suggest that the neovascularization characteristic of ocular HSV-1 disease is a direct result of HSV-1's major transcriptional regulator, ICP4, and similarities between the VEGF-A promoter and those of HSV-1 early genes.

Herpes simplex virus type 1 ocular infection elicits a potent inflammatory response including the production of the chemokines, CXCL9 and CXCL10, in mice. Since HSV-1 nucleic acid is recognized by pattern receptors including Toll-like receptor (TLR) 9, we tested the hypothesis that TLR9 is necessary for the early augmentation of CXCL10 following HSV-1 infection. Similar to wild type controls, TLR9 deficient mice constitutively expressed CXCL10 in the cornea. Following infection or stimulation with the deoxycytidylate-phosphate-deoxyguanylate (CpG) motif, CXCL10 levels were significantly elevated in the cornea of wild type but not TLR9 or type I interferon receptor deficient mice. The reduced CXCL10 response in the cornea of TLR deficient mice was correlative with an increase in virus shedding and a reduction in neutrophil infiltration. This is the first report that shows enhanced CXCL10 expression following neurotropic viral replication requires both intact TLR 9 and type I interferon signaling pathways.