Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero-oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA-binding activity. Deletion studies demonstrated that a heme-binding region of Bach1 is confined within its C-terminal region that possesses four dipeptide cysteine-proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA-binding activity of Bach1 as a MafK hetero-oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MARES:

Lipopolysaccharide (LPS), derived from Gram-negative bacteria, is a major cause of acute lung injury and respiratory distress syndrome. Pulmonary surfactant is secreted as a complex mixture of lipids and proteins onto the alveolar surface of the lung. Surfactant phospholipids are essential in reducing surface tension at the air-liquid interface and preventing alveolar collapse at the end of the respiratory cycle. In the present study, we determined that palmitoyl-oleoyl-phosphatidylglycerol and phosphatidylinositol, which are minor components of pulmonary surfactant, and synthetic dimyristoylphosphatidylglycerol regulated the inflammatory response of alveolar macrophages. The anionic lipids significantly inhibited LPS-induced nitric oxide and tumor necrosis factor-␣ production from rat and human alveolar macrophages and a U937 cell line by reducing the LPS-elicited phosphorylation of multiple intracellular protein kinases. The anionic lipids were also effective at attenuating inflammation when administered intratracheally to mice challenged with LPS. Binding studies revealed high affinity interactions between the palmitoyl-oleoylphosphatidylglycerol and the Toll-like receptor 4-interacting proteins CD14 and MD-2. Our data clearly identify important antiinflammatory properties of the minor surfactant phospholipids at the environmental interface of the lung.

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The purpose of the current study was to examine the binding of pulmonary surfactant protein A (SP-A) to TLR4 and MD-2, which are critical signaling receptors for lipopolysaccharides ( In innate immune systems, toll-like receptors (TLRs) 2 are implicated in recognition and signaling of pathogen-associated molecular patterns (1). Stimulation of different TLRs induces distinct patterns of gene expression, which leads to the activation of innate immunity and instructs the development of antigen-specific acquired immunity (2). Among the TLR family, TLR4 plays a critical role in recognition and signaling of bacterial lipopolysaccharide (LPS) (3). TLR4 requires accessory protein MD-2 for an efficient response to LPS (4). We have recently demonstrated the direct interaction between MD-2 and extracellular TLR4 domain (5, 6). MD-2 binds LPS (7), but LPS has been demonstrated to be cross-linked with TLR4 and MD-2 only when coexpressed with CD14 (8), suggesting that LPS is in close proximity to the receptor complex.The lung is constantly challenged by inhaled pathogens, pollutants, and particles that are present in the environment. Pulmonary surfactant, a mixture of lipids and proteins that serves to reduce the surface tension of the alveoli, is involved in the innate immune system of the lung. Recent studies demonstrate that the most abundant component of surfactant protein, surfactant protein A (SP-A), plays important roles in pathogen clearance and inflammatory responses (9 -12). SP-A belongs to the collectin subgroup of the C-type lectin superfamily along with surfactant protein D (SP-D) and mannose-binding lectin. The primary structure of SP-A subunits are composed of a short amino-terminal segment, a collagen-like sequence characterized by Gly-X-Y repeats with an interruption near the midpoint of the domain, a neck domain, and a carbohydrate recognition domain (CRD) (13). Trimeric association occurs by the folding of collagenous domains into triple helices (14) and coiled-coil bundling of ␣-helices in the neck (15). Fully assembled SP-A is a bouquet-like octadecamer consisting of six trimeric subunits that are stabilized by the amino-terminal sequences and disulfide bonds (16).Recent studies from this and other laboratories have demonstrated that SP-A modulates inflammation by interacting with cell surface receptors including CD14 (17), TLR2 (18, 19), signal-inhibitory regulatory protein ␣, and calreticulin/CD91 (20). Although it has been suggested that SP-A activates cellular responses dependent on TLR4 (21), the interactions of SP-A * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan and by Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.

TLRs have been implicated in recognition of pathogen-associated molecular patterns. TLR4 is a signaling receptor for LPS, but requires MD-2 to respond efficiently to LPS. The purposes of this study were to examine the interactions of the extracellular TLR4 domain with MD-2 and LPS. We generated soluble forms of rTLR4 (sTLR4) and TLR2 (sTLR2) lacking the putative intracellular and transmembrane domains. sTLR4 consisted of Glu24-Lys631. MD-2 bound to sTLR4, but not to sTLR2 or soluble CD14. BIAcore analysis demonstrated the direct binding of sTLR4 to MD-2 with a dissociation constant of KD = 6.29 × 10−8 M. LPS-conjugated beads precipitated MD-2, but not sTLR4. However, LPS beads coprecipitated sTLR4 and MD-2 when both proteins were coincubated. The addition of sTLR4 to the medium containing the MD-2 protein significantly attenuated LPS-induced NF-κB activation and IL-8 secretion in wild-type TLR4-expressing cells. These results indicate that the extracellular TLR4 domain-MD-2 complex is capable of binding LPS, and that the extracellular TLR4 domain consisting of Glu24-Lys631 enables MD-2 binding and LPS recognition to TLR4. In addition, the use of sTLR4 may lead to a new therapeutic strategy for dampening endotoxin-induced inflammation.

Vpr, a HIV-1 accessory protein, was believed to be present in the plasma of HIV-1-positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients' viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL-6 was identified as a responsible factor. The induction of IL-6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr-induced IL-6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr-induced formation of OxPC, the phosphorylation of C/EBP-beta, a substrate of MAPK, and IL-6 production. As rIL-6 reactivated viral replication in latently infected cells, our data indicate that rVpr-induced oxidative stress triggers cell-based innate immune responses and reactivates viral production in latently infected cells via IL-6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti-AIDS therapeutics targeting Vpr.

Pulmonary surfactant protein D (SP-D) 3 is a member of the collectin protein family that also includes surfactant protein A (SP-A) and mannose binding lectin (1, 2). The structure of the collectins is characterized by four domains consisting of: 1) an N terminus involved in interchain disulfide bonding, 2) a collagen-like domain, 3) a coiled-coil neck domain, and 4) a carbohydrate recognition domain (CRD) (3). SP-A and mannose binding lectin contain collagenous domains consisting of 23 and 19 repeating Gly-X-Y triplets, respectively, with an interruption at the middle of the collagenous sequence (4, 5). In contrast, SP-D possesses a longer collagenous tail composed of 59 Gly-X-Y repeats without an interruption (6). These differences cause distinct oligomeric organization, with SP-A and mannose binding lectin exhibiting bouquet-like structures consisting of either six or four trimeric subunits (7) and SP-D exhibiting cruciform structures composed of four trimeric subunits (8).Lipopolysaccharide (LPS) is a principal component of the outer membrane of Gram-negative bacteria that activates macrophages and induces a variety of inflammatory mediators, including TNF-␣, IL-1, IL-6, IL-8, and interferon (9). LPS composed of O-antigen, core oligosaccharide, and lipid A is named smooth LPS, and LPS lacking O-antigen and a part of the core oligosaccharides is named rough LPS (10). Toll-like receptor 4 (TLR4) plays a critical role in recognition and signaling by LPS (11, 12). MD-2 binds directly to LPS and is required for TLR4-mediated signaling induced by LPS (13,14). Structural examination of the TLR4-MD-2 complex revealed that MD-2 binds to the concave surface of the N-terminal and central domains of TLR4 (15). A study with recombinant soluble forms of the extracellular TLR4 domain (sTLR4) and MD-2 (sMD-2) from our laboratory indicates the importance of the N-terminal region of TLR4 in MD-2 binding (16).Engineered genetic defects in the pulmonary collectins of mice have revealed their important functions in protecting the lung from microbial infections and inflammation. SP-D-null mice infected with group B Streptococcus or Haemophilus influenza by intra-tracheal instillation show increased inflammation and inflammatory cell recruitment in the lung (17). Increased pulmonary inflammation in LPS (Escherichia coli O55:B5, smooth serotype)-instilled SP-D Ϫ/Ϫ mice and wildtype mice was decreased by intratracheal administration of SP-D and pulmonary surfactant (18). Intratracheal recombinant SP-D prevents endotoxin shock in the newborn preterm