Severe acute respiratory syndrome (SARS) was first identified in Guangdong Province in China (28). Over the ensuing 9 months, more than 8,000 cases were identified throughout the world, with a ϳ10% case fatality rate. A novel coronavirus, SARS coronavirus (SARS-CoV), was identified as the causative agent (6,17,29,32). Initial investigations indicated that the virus spread to humans from infected exotic animals such as Himalayan palm civets (Paguma larvata) and Chinese ferret badgers (Melogale moschata) (12); more recent work has suggested that the natural reservoirs for the virus are wild bat populations in China (19,24). Although SARS has not recurred in human populations to a significant extent since 2003, the potential severity of such a recurrence has spurred interest in developing an animal model for the human disease.SARS-CoV infects and replicates in mice, ferrets, hamsters, and several species of nonhuman primates (cynomolgus and rhesus macaques, African green monkeys, and common marmosets) (reviewed in reference 37). However, none of these animals develop a clinical disease that is reproducible and equivalent in severity to that observed in SARS patients. A mouse model would be useful for answering many questions about SARS pathogenesis and for testing vaccine efficacy, in part because reagents for the study of the immune response are widely available. However, other than aged or immunocompromised (STAT1 Ϫ/Ϫ ) mice (37), these animals do not develop significant clinical disease, and lethality has not been demonstrated in any murine model of SARS. With the goal of developing a more robust murine model, we generated transgenic (Tg) mice in which expression of hACE2 (human angiotensin-converting enzyme 2, the primary host cell receptor for SARS-CoV [23]) was targeted to epithelial cells. While human ACE2 and murine ACE2 (mACE2) molecules are very homologous, mACE2 does not support SARS-CoV binding as efficiently as hACE2 (22). Here we show that the transgenic expression of hACE2 in epithelia converts a mild SARS-CoV infection into a rapidly fatal disease. MATERIALS AND METHODSMice. All animal studies were approved by the University of Iowa and the Veterans Administration Institutional Animal Care and Use committees. Mice transgenic for expression of hACE2 (K18-hACE2 mice) were generated as follows (see Fig. 1A). The hACE2 coding sequence was PCR amplified from IMAGE consortium clone ID 5243048 (ATCC, Manassas, VA) and cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). The lacZ coding sequence in the previously described pK18mTElacZ-K18i6x7pA construct (16) (a kind gift from Jim Hu, Hospital for Sick Children, Toronto, Canada) was then replaced by the hACE2 coding sequence to create pK18-hACE2. 5Ј of the hACE2 coding sequence, this plasmid contains 2.5 kb of upstream genomic sequence, the promoter, and the first intron (with a mutation in the 3Ј splice acceptor site to reduce exon skipping) of the human cytokeratin 18 (K18) gene as well as a translational enhancer sequence from alfalfa mosaic vi...

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) proteinpseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

(25D 3 ) 3 (2). The second step is conventionally known to take place in the kidneys, but increasing number of tissues have been found to express 1␣-hydroxylase (Cyp27B1), the enzyme responsible for the final and rate-limiting step in the synthesis of the active 1,25-dihydroxyvitamin D 3 (1,25D 3 ) (3, 4). Expression of 1␣-hydroxylase has been reported in epithelial cells of the skin (keratinocytes) (5, 6), intestine (7, 8), breast (9), and prostate (10) and in cells of the immune system including macrophages (11-13), monocytes VDR modulates the expression of a long list of genes in a celland tissue-specific manner. A number of these genes play a role in immunity. The first evidence suggesting that vitamin D has important immunomodulatory effects comes from epidemiological data showing that individuals with low 25D 3 levels are more susceptible to Mycbacterium tuberculosis infection and often have a more severe course of disease (20,21). Furthermore, several casecontrol studies have found an association between VDR polymorphisms and susceptibility to tuberculosis (22)(23)(24). A recent translational study indicates that the reason for these findings is insufficient levels of the antimicrobial protein, cathelicidin, in individuals with low vitamin D levels. In this study, activation of TLR2/1 by a mycobacterial ligand triggered up-regulation of cathelicidin mRNA and increased killing of mycobacteria by macrophages in the presence of vitamin D (25). A subsequent study in skin epithelial cells (keratinocytes) showed that skin injury or infection (TLR2/6 ligand) leads to activation of vitamin D and upregulation of at least two vitamin D-dependent genes, the cathelicidin antimicrobial peptide and the TLR coreceptor, CD14, involved in innate immunity (6). The genes encoding for cathelicidin (26 -28) and CD14 (29) have VDREs within their promoters and are positively regulated by vitamin D. Both are primarily expressed in myeloid cells but have also been found in respiratory

verting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1␤ and TNF␣ acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface. a disintegrin and metalloprotease 17; severe acute respiratory syndrome; coronavirus SEVERE ACUTE RESPIRATORY SYNDROME (SARS) emerged as a regional and global health threat in 2002-2003 resulting in ϳ8,000 cases and 800 deaths. The causative agent was identified as a novel human coronavirus Refs. 8,33,51). Studies (13,47) of patients with SARS demonstrated that the respiratory tract is a major site of SARS-CoV infection and disease-associated morbidity. In 2003, angiotensin-converting enzyme 2 (ACE2) was discovered as a receptor for the virus (39). ACE2 is expressed in vascular endothelia, lung, renal and cardiovascular tissues, testes, and epithelia of the small intestine (9,17,18). It is a terminal carboxypeptidase that cleaves a single residue from ANG II, generating ANG-(1-7) (10, 32, 60). In addition, ACE2 cleaves the terminal residues from several other bioactive peptides, including neurotensin, dynorphin A (1-13), apelin-13, and des-Arg bradykinin (9, 60). As a type I transmembrane protein, ACE2 is comprised of a short cytoplasmic domain, a transmembrane domain, and a large ectodomain (57). A region of the ACE2 ectodomain that includes the first ␣-helix and lysine 353 and proximal residues of the N-terminus of ␤-sheet 5 interacts with high affinity with the receptor-binding domain of the SARS-CoV S glycoprotein (40).Previ...

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.

STAT (signal transducer and activator of transcription) proteins combine with cytokine receptors and receptor-associated kinases in distinct protein/protein interactions that are critical for STAT-dependent signal transduction events, but the nature of any subsequent STAT interactions with DNA-binding proteins in the nucleus is less certain. Based on assays of DNA/protein binding and activity of transfected reporter plasmids, we determined that occupation of contiguous DNA-binding sites for Stat1 (the first member of the STAT family) and the transcriptional activator Sp1 are both required for full activation of the intercellular adhesion molecule-1 gene by interferon-␥. Thus, Stat1 binding to DNA cannot by itself be equated with biologic actions of Stat1. In co-immunoprecipitation experiments, we also obtained evidence of direct and selective Stat1/Sp1 interaction (in primary culture cells without overexpression), further indicating that Stat1/Sp1 synergy confers an element of specificity in the pathway leading to cytokine-activated transcription and cytokine-dependent immunity and inflammation. STAT1 proteins act as critical intermediates in cytokine-dependent gene activation based on their dual capacities for signal transduction (at the cell surface) and activation of transcription (in the nucleus) (1). Signal transduction depends on programmed assembly of cytokine receptors, receptor-associated JAK kinases, and in some cases serine kinases, that recruit and activate specific STAT proteins (2-4). Phosphorylated/activated STATs then dimerize, translocate to the nucleus, and direct transcription of specific target genes. For example, the first member of the STAT family (designated Stat1␣) undergoes tyrosine 701 and serine 727 phosphorylation in response to IFN-␥ (5). This activation step is triggered by IFN-␥-dependent oligomerization of the IFN-␥ receptor and consequent cross-phosphorylation of receptor-associated Jak1 and Jak2 kinases and the receptor ␣-chain (6). Receptor phosphorylation enables ␣-chain recruitment of Stat1 via its SH2 domain. Stat1 then undergoes phosphorylation and release from the receptor as a homodimer that can translocate to the nucleus and bind to a specific DNA element (7,8). Thus, distinct protein/protein interactions are critical for Stat1-dependent signal transduction events at the IFN-␥ receptor, but the nature of Stat1 interactions with other proteins (especially other transcription factors) in the nucleus is less certain. In the present report, we take advantage of a primary cell culture model with selective IFN-␥ responsiveness of the intercellular adhesion molecule-1 (ICAM-1) gene (9, 10) in order to study the basis for Stat1-dependent transcription. The results offer the first evidence that Stat1-mediated transactivation depends on synergistic interaction with another transcriptional activator (Sp1). EXPERIMENTAL PROCEDURESMaterials-Recombinant human IFN-␥ was from Genentech (San Francisco, CA); unlabeled dATP and dGTP were from Boehringer Mannheim; [␣-32 P]dCTP was from DuPo...

Cytokine effects on immunity and inflammation often depend on the transcription factors termed signal transducers and activators of transcription (STATs), so STAT signaling pathways are candidates for influencing inflammatory disease. We reasoned that selective IFN responsiveness of the first STAT family member (Stat1) and Stat1-dependent immune-response genes such as intercellular adhesion molecule-1 (ICAM-1), IFN regulatory factor-1 (IRF-1), and Stat1 itself in airway epithelial cells provides a basis for detecting cytokine signaling abnormalities in inflammatory airway disease. On the basis of nuclear localization and phosphorylation, we found that epithelial Stat1 (but not other control transcription factors) was invariably activated in asthmatic compared with normal control or chronic bronchitis subjects. Furthermore, epithelial levels of activated Stat1 correlated with levels of expression for epithelial ICAM-1, IRF-1, and Stat1, and in turn, ICAM-1 levels correlated with T-cell accumulation in tissue. However, only low levels of IFN-γ or IFN-γ-producing cells were detected in airway tissue in all subjects. The results therefore provide initial evidence linking abnormal behavior of STAT pathways for cytokine signaling to the development of an inflammatory disease. In that context, the results also change the current scheme for asthma pathogenesis to one that must include a localized gain in transcriptional signal ordinarily used for a T helper 1-type cytokine (IFN-γ) in combination with allergydriven overproduction of T helper 2-type cytokines.

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