Vladimir V. Kravchenko

CD14 Is a pattern recognition receptor

Septic shock caused by a diverse group of bacterial pathogens is a serious human disease. Recognition of bacterial envelope constituents is one mechanism used by mammalian cells to initiate responses leading to bacterial killing or, unfortunately, responses that also cause fatal septic shock. Here we show that CD14 plays a key role in initiating cell activation by a group of bacterial envelope components from Gram-negative and Gram-positive microorganisms, as well as mycobacteria. We propose that CD14 is a receptor used by mammalian cells to recognize and signal responses to a diverse array of bacterial constituents. This finding defines the molecular basis for innate microbial immunity; implicit in these findings are new possibilities for therapeutics.

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

BMK1/ERK5 regulates serum-induced early gene expression through transcription factor MEF2C.

Big MAP kinase 1 (BMK1), also known as ERK5, is a mitogen‐activated protein (MAP) kinase member whose biological role is largely undefined. We have shown previously that the activity of BMK1 in rat smooth muscle cells is up‐regulated by oxidants. Here, we describe a constitutively active form of the MAP kinase kinase, MEK5(D), which selectively activates BMK1 but not other MAP kinases in vivo. Through utilization of MEK5(D), we have determined that a member of the MEF2 transcription factor family, MEF2C, is a protein substrate of BMK1. BMK1 dramatically enhances the transactivation activity of MEF2C by phosphorylating a serine residue at amino acid position 387 in this transcription factor. Serum is also a potent stimulator of BMK1‐induced MEF2C phosphorylation, since a dominant‐negative form of BMK1 specifically inhibits serum‐induced activation of MEF2C. One consequence of MEF2C activation is increased transcription of the c‐jun gene. Taken together, these results strongly suggest that in some cell types the MEK5/BMK1 MAP kinase signaling pathway regulates serum‐induced early gene expression through the transcription factor MEF2C.

Regulation of the MEF2 family of transcription factors by p38

ABSTRACT Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

Regulation of an essential innate immune response by the p50 subunit of NF-kappaB.

Recognition of bacterial endotoxin (LPS) elicits multiple host responses, including activation of cells of the innate immune system. LPS exposure occurs repeatedly during septicemia, making strict regulation of gene expression necessary. Such regulation might prevent, for example, the continuous production of proinflammatory cytokines such as tumor necrosis factor (TNF), which could lead to severe vascular collapse. Tolerance to LPS is characterized by a diminished production of TNF during prolonged exposure to LPS, and is therefore likely to represent an essential control mechanism during sepsis. In the present study, which uses mice with genetic deletions of the proteins of NF-kappaB complex, we provide data demonstrating that increased expression of the p50 subunit of NF-kappaB directly results in the downregulation of LPS-induced TNF production. This contention is supported by the following observations: (1) tolerance to LPS is not induced in macrophages from p50-/- mice; (2) long-term pretreatment with LPS does not block synthesis of TNF mRNA in p50-/- macrophages (in contrast to wild-type macrophages); (3) ectopic overexpression of p50 reduces transcriptional activation of the murine TNF promoter; and (4) analysis of the four kappaB sites from the murine TNF promoter demonstrates that binding of p50 homodimers to the positively acting kappaB3 element is associated with development of the LPS-tolerant phenotype. Thus, p50 expression plays a key role in the development of LPS tolerance.

Modulation of Gene Expression via Disruption of NF-κB Signaling by a Bacterial Small Molecule

The control of innate immune responses through activation of the nuclear transcription factor NF-κB is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-κB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-κB–responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients.

MDP‐induced interleukin‐1β processing requires Nod2 and CIAS1/NALP3

Nucleotide‐binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold‐induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine‐rich repeat, and pyrin domain‐containing protein 3 (NALP3) has been suggested to be sufficient for MDP‐dependent release of mature IL‐1β, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP‐induced IL‐1β release requires Nod2 and CIAS1/NALP3 as well as receptor‐interacting protein‐2 (Rip2), apoptosis‐associated speck‐like protein containing a caspase activation and recruitment domain (ASC), and caspase‐1. In contrast, MDP‐dependent IL‐6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL‐1β production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.

NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1β (IL-1β) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways.

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Here we have shown that p38 and eIF2α phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.

MOLECULAR-CLONING OF HUMAN P38 MAP KINASE

Mitogen-activated protein (MAP) kinases are intracellular serine/threonine kinases activated by dual phosphorylation of adjacent threonine (T) and tyrosine (Y). A diverse number of extracellular signals induce activation of MAP kinases. Here we describe the cloning of a cDNA encoding human p38 MAP kinase (p38). The amino acid sequence of human p38 is 99.4% identical to mouse p38 [Han et al. (1994) Science 265, 808-11]. Like murine p38, the dual phosphorylation site of human p38 MAP kinase is characterized by a TGY sequence. Previous studies have described activation of p38 following exposure to products of microbial pathogens, physical-chemical stimuli and cytokines. The highly conserved nature of p38 suggests the importance of its function in regulating cellular responses.