Glennice N. Bowen

Respiratory Syncytial Virus Activates Innate Immunity through Toll-Like Receptor 2

ABSTRACT Respiratory syncytial virus (RSV) is a common cause of infection that is associated with a range of respiratory illnesses, from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children <1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected to contribute to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs, including TLR2, TLR6, TLR3, TLR4, and TLR7, that can interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting tumor necrosis factor alpha, interleukin-6, CCL2 (monocyte chemoattractant protein 1), and CCL5 (RANTES). As previously noted, TLR4 also contributes to cytokine activation (L. M. Haynes, D. D. Moore, E. A. Kurt-Jones, R. W. Finberg, L. J. Anderson, and R. A. Tripp, J. Virol. 75:10730-10737, 2001, and E. A. Kurt-Jones, L. Popova, L. Kwinn, L. M. Haynes, L. P. Jones, R. A. Tripp, E. E. Walsh, M. W. Freeman, D. T. Golenbock, L. J. Anderson, and R. W. Finberg, Nat. Immunol. 1:398-401, 2000). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation.

Toll-like receptor-mediated activation of neutrophils by influenza A virus.

Influenza virus infection of the respiratory tract is characterized by a neutrophil infiltrate accompanied by inflammatory cytokine and chemokine production. We and others have reported that Toll-like receptor (TLR) proteins are present on human neutrophils and that granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment enhances IL-8 (CXCL8) secretion in response to stimulation with TLR ligands. We demonstrate that influenza virus can induce IL-8 and other inflammatory cytokines from GM-CSF-primed human neutrophils. Using heat inactivation of influenza virus, we show that viral entry but not replication is required for cytokine induction. Furthermore, endosomal acidification and viral uncoating are necessary. Finally, using single-cell analysis of intracellular cytokine accumulation in neutrophils from knockout mice, we prove that TLR7 is essential for influenza viral recognition and inflammatory cytokine production by murine neutrophils. These studies demonstrate neutrophil activation by influenza virus and highlight the importance of TLR7 and TLR8 in that response.

Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity

Toll-like receptors (TLRs) are critically involved in the innate immune response to bacterial, viral and fungal pathogens. We have studied human peripheral blood mononuclear cells, murine embryonic fibroblasts (MEFs) and a panel of human cell lines, including HEK, HeLa, AGS, ECV304 and U373 cells, for expression of TLR-specific mRNAs and for TLR-ligand dependent cytokine secretion. Peripheral blood cells expressed multiple TLRs; however, many studies have shown that blood contains multiple, heterogeneous cell populations with distinct patterns of TLR expression. Cell lines had variable expression of TLRs, and in most cases lacked TLR2 and TLR8 expression and only weakly expressed mRNAs for TLR5, TLR7 and TLR9. In contrast, MEFs expressed high levels of mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. MEFs were highly responsive to TLR-ligand activation and secreted high levels of both IL-6 and MCP-1 in response to TLR ligands. MEFs from mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrated profound defects in their IL-6 response to their specific ligands, consistent with studies of macrophages and tissues from adult knockout animals. MEF cultures are homogenous and amenable to biochemical analysis and should allow rigorous studies of the contribution of individual TLRs to the innate immune response.

Role of Specific Innate Immune Responses in Herpes Simplex Virus Infection of the Central Nervous System

ABSTRACT Herpes simplex virus 1 (HSV-1) causes a spectrum of disease, including herpes labialis, herpes keratitis, and herpes encephalitis, which can be lethal. Viral recognition by pattern recognition receptors plays a central role in cytokine production and in the generation of antiviral immunity. The relative contributions of different Toll-like receptors (TLRs) in the innate immune response during central nervous system infection with HSV-1 have not been fully characterized. In this study, we investigate the roles of TLR2, TLR9, UNC93B1, and the type I interferon (IFN) receptor in a murine model of HSV-1 encephalitis. TLR2 is responsible for detrimental inflammatory cytokine production following intracranial infection with HSV-1, and the absence of TLR2 expression leads to increased survival in mice. We prove that inflammatory cytokine production by microglial cells, astrocytes, neutrophils, and monocytes is mediated predominantly by TLR2. We also demonstrate that type I IFNs are absolutely required for survival following intracranial HSV-1 infection, as mice lacking the type I IFN receptor succumb rapidly following infection and have high levels of HSV in the brain. However, the absence of TLR9 does not impact survival, type I IFN levels, or viral replication in the brain following infection. The absence of UNC93B1 leads to a survival disadvantage but does not impact viral replication or type I IFN levels in the brain in HSV-1-infected mice. These results illustrate the complex but important roles that innate immune receptors play in host responses to HSV-1 during infection of the central nervous system.

Trefoil Family Factor 2 Is Expressed in Murine Gastric and Immune Cells and Controls both Gastrointestinal Inflammation and Systemic Immune Responses

ABSTRACT Trefoil family factor 2 (TFF2), also known as spasmolytic peptide, is a low-molecular-weight protein that is upregulated in gastric tissues infected with Helicobacter or having other inflammatory conditions, but a precise function is yet to be elucidated. The role of TFF2 in the development of gastritis, colitis, and inflammatory cytokine responses was examined both in vivo and in vitro using wild-type and TFF2 knockout mice. TFF2 knockout and wild-type mice were infected with Helicobacter felis (H. felis) to induce gastritis. Colitis was induced in TFF2 knockout and wild-type mice by administering dextran sodium sulfate (DSS) in drinking water. Histopathology, clinical disease (colitis), and antibody levels (H. felis) were examined. TFF2 expression in tissues was determined by reverse transcriptase PCR, and the inflammatory and proliferative responses of TFF2-expressing macrophages and spleen cells were examined by cytokine enzyme-linked immunosorbent assay, thymidine incorporation, and gene array studies. TFF2 knockout mice have increased susceptibility to H. felis-induced gastritis, with enhanced gastric inflammation. They were also more susceptible to DSS-induced colitis, with prolonged colonic hemorrhage and persistent weight loss. Remarkably, TFF2 expression was not limited to the gastrointestinal tract, as suggested in previous studies, but was also present in macrophages and lymphocytes. The inflammatory and proliferative responses of these immune cell types were dysregulated in TFF2 knockout mice. TFF2−/− cells were hyperresponsive to interleukin 1 beta stimulation but showed normal responses to lipopolysaccharide, suggesting a specific role for TFF2 in interleukin 1 receptor but not Toll-like receptor 4 signaling via their Toll-interleukin 1 resistance domains. TFF2−/− lymphocytes also produced higher levels of interleukin 2 than wild-type cells. Thus, TFF2 was expressed in the gastrointestinal cells and in immune cells and was a negative regulator of gastrointestinal inflammation and immune cell cytokine responses. Our studies suggest that TFF2 not only controls gastrointestinal repair but also regulates mononuclear cell inflammatory responses.

Mutations in bone marrow-derived stromal stem cells unmask latent malignancy

Neoplastic epithelia may remain dormant and clinically unapparent in human patients for decades. Multiple risk factors including mutations in tumor cells or the stromal cells may affect the switch from dormancy to malignancy. Gene mutations, including p53 mutations, within the stroma of tumors are associated with a worse clinical prognosis; however, it is not known if these stromal mutations can promote tumors in genetically at-risk tissue. To address this question, Apc(Min/+) and Apc(Min/+) Rag2(-/-) mice, which have a predilection to mammary carcinoma (as well as wild-type (wt) mice), received mesenchymal stem cells (MSC) with mutant p53 (p53MSC) transferred via tail vein injection. In the wt mouse, p53MSC circulated in the periphery and homed to the marrow cavity where they could be recovered up to a year later without apparent effect on the health of the mouse. No mammary tumors were found. However, in mice carrying the Apc(Min/+) mutation, p53MSC homed to mammary tissue and significantly increased the incidence of mammary carcinoma. Tumor necrosis factor (TNF)-alpha-dependent factors elaborated from mesenchymal cells converted quiescent epithelia into clinically apparent disease. The increased cancer phenotype was completely preventable with neutralization of TNF-alpha or by transfer of CD4(+) regulatory T cells from immune competent donors, demonstrating that immune competency to regulate inflammation was sufficient to maintain neoplastic dormancy even in the presence of oncogenic epithelial and stromal mutations. The significant synergy between host immunity and mesenchymal cells identified here may restructure treatments to restore an anticancer microenvironment.

Discovery of a novel TLR2 signaling inhibitor with anti-viral activity

Blockade of Toll-like receptor (TLR)-mediated inflammatory responses represents a new approach in the development of anti-inflammation therapeutics. In the present study, we have screened for TLR2-mediated inflammation inhibitors from small molecule compound libraries using a sensitive cell line stably expressing TLR2, CD14, and an NF-kappaB-driven-luciferase reporter gene. Lymphocytic choriomeningitis virus (LCMV) was used as a virus model. This arenavirus activates a TLR2/CD14-dependent NF-kappaB signaling pathway. We have identified 10 potential anti-inflammatory compounds out of 101,306 compounds. We further evaluated 1 of these positive compounds, E567. We demonstrated that compound E567 efficiently inhibits both LCMV and Herpes simplex virus 1 (HSV-1) induced cytokine responses in both human and mouse cell cultures. We also demonstrated that E567 inhibits cytokine responses in the mouse. Remarkably, E567 is also capable of inhibiting LCMV replication in mice. This is a new model for developing drugs for use in treating viral illnesses.

T-bet Knockout Prevents Helicobacter felis-Induced Gastric Cancer

Helicobacter infection is the primary risk factor for gastric cancer, with the cytokine environment within the gastric mucosa the strongest predictor of disease risk. Elevated TNF-α, IL-1β, and low IL-10 are associated with the highest risk. In this study, we used C57BL/6 mice to identify T-bet as a central regulator of the cytokine environment during Helicobacter felis infection. We infected male and female C57BL/6 and C57BL/6-T-bet knockout (KO) liter mates with H. felis and examined the bacterial colonization, immune response, and mucosal damage at varying time points. T-bet KO mice maintained infection for 15 mo at similar levels to wild-type mice. Infection and immune response did not differ between male and female mice. Despite sustained infection, T-bet KO mice respond with a blunted Th1 response associated with preservation of parietal and chief cells and protection from the development of gastric cancer. Unexpectedly, T-bet KO mice develop a gastric environment that would not be expected based on the phenotype of T-bet KO CD4 cells alone. T-bet KO mice respond to H. felis infection with a markedly blunted IL-1β and TNF-α and elevated IL-10 levels. Activity of this one master regulator modulates the expression of the key gastric mucosal cytokines associated with gastric cancer and may be a target for therapy to restore immune balance clinically in patients at risk for gastric cancer.