Natural Killer Cell Transcript 4 promotes the development of SjÓ§gren's syndrome via activation of Rap1 on B cells.

Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with SjÓ§gren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases.

The herpes simplex virus-1 transactivator infected cell protein-4 drives VEGF-A dependent neovascularization.

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.

Loss of mandibular lymph node integrity is associated with an increase in sensitivity to HSV-1 infection in CD118-deficient mice.

Type I IFNs are potent antiviral cytokines that contribute to the development of the adaptive immune response. To determine the role of type I IFNs in this process in an infectious disease model, mice deficient in the type I IFN receptor (CD118(-/-)) were ocularly infected with HSV-1 and surveyed at times post infection in the nervous system and lymph node for virus and the host immune response. Virus titers were elevated in the trigeminal ganglia and brain stem with virus disseminating rapidly to the draining lymph node of CD118(-/-) mice. T cell and plasmacytoid dendritic cell infiltration into the brain stem was reduced in CD118(-/-) mice following infection, which correlated with a reduction in CXCL10 but not CXCL9 expression. In contrast, CXCL1 and CCL2 levels were up-regulated in the brainstem of CD118(-/-) mice associated with an increase in F4/80(+) macrophages. By day 5 post infection, there was a significant loss in T, NK, and plasmacytoid dendritic cell numbers in the draining lymph nodes associated with an increase in apoptotic/necrotic T cells and an appreciable lack of HSV-specific CD8(+) T cells. The adoptive transfer of HSV-specific TCR transgenic CD8(+) T cells into CD118(-/-) mice at the time of infection modestly reduced viral titers in the nervous system suggesting in addition to the generation of HSV-specific CD8(+) T cells, other type I IFN-activated pathways are instrumental in controlling acute infection.

Lymphoid precursors are directed to produce dendritic cells as a result of TLR9 ligation during herpes infection.

Hematopoietic stem and progenitor cells were previously found to express Toll-like 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. TNFalpha mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNFalpha-/- 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. Common myeloid progenitors are normally a good source of DCs, but this potential was reduced by TLR9 ligation. Thus, alternate differentiation pathways may be used to produce innate effector cells in health and disease.

Dysregulation of CXCR3 signaling due to CXCL10 deficiency impairs the antiviral response to herpes simplex virus 1 infection.

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.

Vav1 is Essential for HIF-1α Activation via a Lysosomal VEGFR1-Mediated Degradation Mechanism in Endothelial Cells.

The vascular response to hypoxia and ischemia is essential for maintaining homeostasis during stressful conditions and is particularly critical for vital organs such as the heart. Hypoxia-inducible factor-1 (HIF-1) is a central regulator of the response to hypoxia by activating transcription of numerous target genes, including vascular endothelial growth factor (VEGF). Here we identify the guanine nucleotide exchange factor (GEF) Vav1, a regulator of the small Rho-GTPase and cell signaling in endothelial cells, as a key vascular regulator of hypoxia. We show that Vav1 is present in the vascular endothelium and is essential for HIF-1 activation under hypoxia. So, we hypothesized that Vav1 could be a key regulator of HIF-1 signaling. In our findings, Vav1 regulates HIF-1α stabilization through the p38/Siah2/PHD3 pathway. In normoxia, Vav1 binds to vascular endothelial growth factor receptor 1 (VEGFR1), which directs Vav1 to lysosomes for degradation. In contrast, hypoxia upregulates Vav1 protein levels by inhibiting lysosomal degradation, which is analogous to HIF-1α regulation by hypoxia: both proteins are constitutively produced and degraded in normoxia allowing for a rapid response when stress occurs. Consequently, hypoxia rapidly stabilizes Vav1, which is required for HIF-1α accumulation. This shows that Vav1 is the key mediator controlling the stabilization of HIF1α in hypoxic conditions. With this finding, we report a novel pathway to stabilize HIF-1, which shows a possible reason why clinical trials targeting HIF-1 has not been effective. Targeting Vav1 can be the new approach to overcome hypoxic tumors.

Oxygen Tension Regulates Lysosomal Activation and Receptor Tyrosine Kinase Degradation.

Oxygen sensing is crucial for adaptation to variable habitats and physiological conditions. Low oxygen tension, or hypoxia, is a common feature of solid tumors, and hypoxic tumors are often more aggressive and resistant to therapy. Here we show that, in cultured mammalian cells, hypoxia suppressed lysosomal acidification/activation and receptor tyrosine kinase (RTK) degradation. Hypoxia down-regulated mTORc1, reducing its ability to activate transcription factor EB (TFEB), a master regulator of V-ATPase, the lysosomal proton pump. Hypoxia prevented epidermal growth factor receptor (EGFR) degradation in tumor tissues, whereas activation of lysosomes enhanced tumor cell response to anti-EGFR treatment. Our results link oxygen tension and lysosomal activity, provide a molecular explanation of the malignant phenotype associated with hypoxic tumors, and suggest activation of lysosomes may provide therapeutic benefit in RTK-targeted cancer therapy.

Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist.

Neutrophilic granule protein (NGP) was previously reported as a granular protein of neutrophils in mouse, but the function has not been known clearly. We found the presence of the possible signal peptide in NGP and validated this protein is circulating in the bloodstream. In our findings, NGP is being modified post-translationally in Golgi apparatus and endoplasmic reticulum, which is a universal character of secretory molecules with a signal peptide. The secreted NGP protein could be detected both in vitro and in vivo. NGP has sequence similarity with an antimicrobial protein cathelicidin, and we observed the aspect of inflammation of NGP. Interestingly, NGP interacts with the complex of LPS and LPS binding protein (LBP). This interaction blocks the binding of the complex of LPS and LBP to TLR4 and the downstream inflammatory signals. Furthermore, the inhibitory function of NGP against the inflammatory effect of LPS could be observed in both in vitro and in vivo. With these findings, we report NGP is a novel secretory protein to mask LPS and inhibit its function.

An increase in herpes simplex virus type 1 in the anterior segment of the eye is linked to a deficiency in NK cell infiltration in mice deficient in CXCR3.

In response to ocular herpes simplex virus type 1 (HSV-1) infection in mice, a rapid induction or increase in the local expression of chemokines, including CXCL10, is found. The present study investigated the role of the receptor for CXCL10, CXCR3, in the host response to corneal HSV-1 infection. Mice deficient in CXCR3 (CXCR3(-/-)) were found to have an increase in infectious virus in the anterior segment of the eye by day 7 postinfection. Coinciding with the increase, selective chemokines, including CCL2, CCL3, CCL5, CXCL9, and CXCL10, were elevated in the anterior segment of the HSV-1-infected CXCR3(-/-) mice. In contrast, there was a time-dependent reduction in the recruitment of natural killer (NK) cells (NK1.1(+)CD3(-)) into the anterior segment of CXCR3(-/-) mice. A reduction in NK cells residing in the anterior segment of mice following antiasialoGM1 antibody treatment resulted in an increase in infectious virus. No other leukocyte populations infiltrating the tissue were modified in the absence of CXCR3. Collectively, the loss of CXCR3 expression specifically reduces NK cell mobilization into the cornea in response to HSV-1.

The role of chemokines during herpes simplex virus-1 infection.

Herpes simplex virus-type 1 is among the most prevalent and successful humans pathogens. Although infection is largely uncomplicated in the immunocompetent human host, HSV-1 infection can cause blinding corneal disease, and individuals with defects in innate or adaptive immunity are susceptible to herpes simplex encephalitis. Chemokines regulate leukocyte trafficking to inflamed tissues and play a crucial role in orchestrating the immune response to HSV-1 infection. In this review we will focus on the pathways that induce chemokine expression during HSV-1 infection and the implications of chemokine signaling on control of viral replication.

Intact TRL 9 and type I interferon signaling pathways are required to augment HSV-1 induced corneal CXCL9 and CXCL10.

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.

CXCL10 expressing hematopoietic-derived cells are requisite in defense against HSV-1 infection in the nervous system of CXCL10 deficient mice.

The chemokine CXCL10 is crucial for the control of viral replication through the regulation of mobilization of antigen-specific T cells to sites of infection. CXCL10 is highly expressed both at sites of inflammation as well as constitutively within lymphoid organs by both bone marrow (BM)-derived and non-BM-derived cells. However, the relative immunologic importance of CXCL10 expressed by these divergent sources relative to HSV-1 infection is unknown. Using mouse chimeras reconstituted with either wild type or CXCL10 deficient mouse BM, we show BM-derived, radiation-sensitive cells from wild type mice were solely responsible for resistance to HSV-1 in the trigeminal ganglia and brain stem. The resistance was not reflected by a deficiency in the recruitment of effector cells to sites of inflammation or expression of chemokines or IFN-gamma and likely results from additional, yet-to-be-determined factors emanating from wild type, BM-derived cells.

VEGF-A expression by HSV-1-infected cells drives corneal lymphangiogenesis.

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.

CD4+ T cell migration into the cornea is reduced in CXCL9 deficient but not CXCL10 deficient mice following herpes simplex virus type 1 infection.

The role of CXCL9 and CXCL10 in the ocular immune response to herpes simplex virus type 1 (HSV-1) infection was investigated using mice deficient in either CXCL9 or CXCL10. CXCL10 but not CXCL9 deficient mice showed an increase in sensitivity to ocular virus infection as measured by an elevation in virus titer recovered in the tear film and corneal tissue. The increase in virus was associated with an increase in the expression of the chemokine CCL2 but no significant change in the infiltration of CD4(+) T cells or NK cells into the corneal stroma. In contrast, a significant reduction in CD4(+) T cell infiltration into the cornea was found in CXCL9 deficient mice following HSV-1 infection consistent with the absence of CXCL9 expression and reduction in expression of other chemokines including CCL3, CCL5, CXCL1, and CXCL10. Collectively, the results suggest a non-redundant role for CXCL9 and CXCL10 in response to ocular HSV-1 infection in terms of controlling virus replication and recruitment of CD4(+) T cells into the cornea.

The lack of RNA-dependent protein kinase enhances susceptibility of mice to genital herpes simplex virus type 2 infection.

Mice deficient in RNA-dependent protein kinase (PKR-/-) or deficient in PKR and a functional 2',5'-oligoadenylate synthetase (OAS) pathway (PKR/RL-/-) are more susceptible to genital herpes simplex virus type 2 (HSV-2) infection than wild-type mice or mice that are deficient only in a functional OAS pathway (RL-/-) as measured by survival over 30 days. The increase in susceptibility correlated with an increase in virus titre recovered from vaginal tissue or brainstem of infected mice during acute infection. There was also an increase in CD45+ cells and CD8+ T cells residing in the central nervous system of HSV-2-infected PKR/RL-/- mice in comparison with RL-/- or wild-type control animals. In contrast, there was a reduction in the HSV-specific CD8+ T cells within the draining lymph node of the PKR/RL-/- mice. Collectively, activation of PKR, but not of OAS, contributes significantly to the local control and spread of HSV-2 following genital infection.