David C. Briggs

The Inflammation-associated Protein TSG-6 Cross-links Hyaluronan via Hyaluronan-induced TSG-6 Oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).

The Angiogenic Inhibitor Long Pentraxin PTX3 Forms an Asymmetric Octamer with Two Binding Sites for FGF2

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.

Inter-α-Inhibitor Impairs TSG-6-Induced Hyaluronan Cross-Linking

Background: Inflammation/ovulation-associated protein TSG-6 performs multiple functions in hyaluronan (HA)-rich extracellular matrices. Results: Inter-α-inhibitor (IαI) affects HA-TSG-6 interactions and enhancement of cell adhesion while promoting covalent complex formation between IαI heavy chains and HA. Conclusion: IαI dictates TSG-6 activity and remodels HA matrix properties. Significance: These results provide novel insights into the regulation of HA-protein interactions and assembly of biologically important extracellular matrices. Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.

Incorporation of Pentraxin 3 into Hyaluronan Matrices Is Tightly Regulated and Promotes Matrix Cross-linking

Background: The proteins pentraxin 3 (PTX3) and TNF-stimulated gene-6 (TSG-6) and the proteoglycan inter-α-inhibitor (IαI) are known to be involved in the stabilization of hyaluronan (HA)-rich extracellular matrices. Results: PTX3 incorporation into HA matrices is tightly regulated. Conclusion: PTX3, TSG-6, and IαI are sufficient to cross-link HA matrices. Significance: The results provide mechanistic insights into the regulation of HA-protein interactions. Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking.

A Refined Model for the TSG-6 LINK Module in Complex with Hyaluronan: use of Defined Oligosaccharides to Probe Structure and Function.

Background: The polysaccharide hyaluronan is organized through interactions with the protein TSG-6 during inflammation and ovulation. Results: NMR spectroscopy on TSG-6 in the presence of defined sugars provided restraints that allowed modeling of a refined hyaluronan/TSG-6 complex. Conclusion: TSG-6 binding causes bending of hyaluronan that explains its condensation of this polysaccharide. Significance: This provides novel structural insights into protein-hyaluronan interactions. Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was 13C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

Background: TSG-6 (TNF-stimulated gene-6)-dependent transfer of heavy chains from inter-α-inhibitor onto hyaluronan is critical for ovulation. Results: A calcium ion and chelating glutamate within TSG-6 mediate formation of the covalent heavy chain-TSG-6 intermediate. Conclusion: TSG-6 transferase activity rather than hyaluronan binding drives cumulus expansion. Significance: The role of metal ions in hyaluronan-heavy chain formation has been determined. The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.

Homodimerization of the lymph vessel endothelial receptor LYVE-1 through a redox-labile disulfide is critical for hyaluronan binding in lymphatic endothelium

The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an ∼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an “open scissors” conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.

A bug in CUB's clothing: similarity between clostridial CBMs and complement CUBs

The CUB domain* is an all β-sheet structural motif found predominantly in eukaryotic extracellular proteins, including complement C1 components, urchin epidermal growth factor (EGF)-related proteins, bone morphogenetic protein-1, and other immune and inflammation-associated proteins. The primary function of CUB domains is to mediate protein–protein or protein–carbohydrate interactions (http://smart.embl-heidelberg.de/smart/do_annotation.pl?DOMAIN=CUB). In a recent analysis of CUB domains, we have observed that the structures [1] of a new carbohydrate-binding module (CBM) family, CBM51 [formerly known as novel putative carbohydrate-binding molecule (NPCBM)], have a striking structural similarity to the metal-ion-binding subfamily of eukaryotic CUBs (Figure 1a).

The role of complement factor H-like and factor H-related proteins in age-related macular degeneration

T protein 18 kDa (TSPO) is a protein located on the outer membrane of mitochondria. TSPO is expressed mainly in glial cells in the central nervous system and its expression level is highly up-regulated during neuronal injury and neuroinflammation. We previously reported that TSPO acts as a negative regulator of microglia activation. However, it is not known yet whether TSPO is involved in inflammasome signaling. Inflammasomes are multiprotein complexes for caspase-1 activation and IL-1β processing and implicated in many inflammatory diseases. In this talk, I present the evidence that the prototypical TSPO ligand, Ro5-4864 (Ro5) inhibits NLRP3 inflammasome signaling in THP-1 cells using the canonical LPSprimed/ATP-induced NLRP3 inflammasome model. Ro5 efficiently blocked release of IL-1β and caspase-1, and reduced ASC speck formation and NLRP3 translocation to mitochondria. Ro5 also attenuated LPS/ATP-induced perturbation of mitochondrial function by preventing generation of mitochondrial superoxide and depolarization of mitochondrial membrane potential. These results indicate that the immune modulatory effect of the TSPO ligand Ro5 is through inhibition of NLRP3 inflammasome signaling, suggesting Ro5 as a promising agent with efficacy for NLRP3 inflammasome-associated diseases.Kawasaki disease is an acute systemic vasculitis of unknown cause that affects mainly infants and children. Coronary artery lesions (CAL) are one of the most important complications of this disease. An appropriate therapy during acute phase of Kawasaki disease to prevent large CAL has not been established. Recent studies disclosed that aspirin and flurbiprofen appeared to have a negative impact on the suppressive effects of initial intravenous immunoglobulin (IVIG) therapy on CAL development in the acute phase of Kawasaki disease and that an initial single IVIG therapy with delayed administration of anti-inflammatory drugs might be useful for prevention of large CAL. Furthermore, recent study disclosed that variable factors including IVIG resistance, responsiveness, and relapse of disease were associated with CAL complications and that an initial single IVIG therapy may be useful for the prevention of large CAL caused by different factors of Kawasaki disease.Methods: Eighty patients with MS, their ages ranged between 26-71 years, were subjected to complete clinical and neurological history and examination to confirm the diagnosis. All cases were under their regular treatment they were divided into two main groups, Group I received honey, pollen, royal jelly and propolis and were treated with apiacupuncture 3 times weekly, for 12 months, in addition to their medical treatment, while group II remains on their ordinary medical treatment only. IFN-γ, interleukin (IL) 1β, IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNFα) were detected. Apiacupuncture was done by bee stings for regulating the immune system.B cells are the dominant population of B cells in the pleural and peritoneal cavities. They are a significant source of serum antibody, and they make a dominant contribution to low-affinity IgM antibodies that are present in serum of unimmunized mice. B1 cells in the mouse are thought to be derived from precursors in fetal liver rather than from adult bone marrow. They are believed to maintain their cell numbers in adult mice by longevity and homeostatic proliferation. Unlike conventional B cells (B2 cells) and despite the importance of B1 cells in protection from infections and their association with autoimmunity, the mechanism of B1 cell proliferation and function remained poorly understood. Mysm1 is a histone de-ubiquitinase and has been shown to play an essential role in hematopoiesis and lymphocyte development. Our previous study has demonstrated that in Mysm1 deficient mice, B2 cell development is blocked and B2 cell number is significantly lower compared with their counterpart. In this study we found that, in contrast to the dramatically decreased level of B2 cells, the percentage of B1 cells in the spleen and peritoneal cavity of Mysm1 deficient mice was increased compared with that in wild type mice. Mechanistic study has demonstrated that miR150 expression is compromised in B1 cells from Mysm1 deficient mice. Mysm1 controls the transcription of miR150 through regulating the chromatin state of miR150 locus. What’s more, forced expression of miR150 in B1 cells from Mysm1 mice can partly rescue the phenotype. Overall, our study, for the first time, reveals the important role of histone H2A de-ubiquitinase in B1 cell proliferation and development.A (the term comes from the Latin apis, which means “bee.”), or bee therapy, is the use of honey bee products for therapeutic purposes. Bee venom, bee pollen, raw honey, royal jelly, and propolis are products from bees that are generally considered to have medicinal effects. Now days the apitherapy has great interest of biologist, medical doctors and scientist due to their biological and phytochemical as well as pharmacological activities, so the aim of this review was to through more light on the role of bee products and their influence on cytokines.A rhinitis (AR) is characterized by nasal mucosal inflammation resulting from immunoglobulin E (IgE) mediated hypersensitivity reaction. Allergen exposures stimulate infiltration of inflammatory cells within the nasal mucosa, including basophils, eosinophils, mast cells and mononuclear cells. These inflammatory cells release several allergic mediators, such as histamine, cysteinyl leukotrienes and prostaglandins, which sustain the inflammatory reaction and produce characteristic nasal symptoms of sneezing, itching, rhinorrhea and nasal congestion. Bee venom (BV) consists of various biologically active amines, peptides and nonpeptide components and has radio protective, anti-mutagenic, anti-inflammatory, anti-nociceptive and anticancer activities. Two main components of BV, melittin and adolapin, have anti-inflammatory activity that involve inhibition of cycloxygease-2 and phospholipase-A expression and decrease levels of tumour necrosis factor-α interleukin (IL)1, IL-6 and nitric oxide. The anti-allergic activity is associated with marked inhibition of OVA-induced tracheal contraction and histamine release from lung tissue. The mast-cell degranulating peptide binds to the mast cell receptors and inhibits the binding of IgE and production of histamine. BV also inhibits the release of inflammatory mediators similar to non-steroidal anti-inflammatory drugs.There has been a rapid grown in the field of tumor immunobiology in recent years as a result of recent successes in cancer immunotherapies, and it is clear that immune cells play many sometimes conflicting roles in the tumor microenvironment. However, obtaining phenotypic information about the various immune cells in and around the tumor has been a challenge. Existing methods can deliver phenotypic information on homogenous samples (e.g., flow cytometry or PCR) or morphologic information in single stain IHC. We present here a methodology for delivering quantitative per-cell marker expression and phenotyping, analogous to that obtained from flow cytometry, but from cells imaged in situ in FFPE tissue sections. This methodology combines: the sequential multi-marker labeling of up to 6 antigens using antibodies all of the same species; automated multispectral imaging to remove problematic FFPE tissue autofluorescence and correct cross-talk between fluorescent channels; and an automated analysis that can quantitate the per-cell marker expression, determine the cellular phenotype, count these cells separately in the tumor compartment and in the stroma and provide high-resolution images of their distributions. We will show a 6-plex assay in breast cancer showing the application of the multiplexed staining, per-cell quantitation and cellular phenotyping in FFPE tissue sections, as well as methods to explore the spatial distributions of the phenotyped cells in and around the tumor.I vitro, the effect of mesenchymal stromal cell (MSC) on immune suppression has been well studied. However, in vivo, questions regarding MSC optimal therapeutic strategy, homing, survival, and timing for immune suppression are still elusive. To address these questions for systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS) and Sjogren’s syndrome, a MLR/MpJ-Faslpr/J (Faslpr) mice model was employed. Multipotency of bone marrow derived MSC (BM-MSC) was determined by phenotypic analysis and multipotential differentiation assay. Fourto six-month-old Faslpr mice showed onset of autoimmune disease with lymphoproliferation. Optimal infusion dose was determined by serial dilution. Homing and survival of MSC were monitored by fluorescent microscopy. Infusion frequency was deduced from immunomodulatory kinetics study. BM-MSC with passage number less than 10 could properly maintain multi-lineage differentiation capacity and stem cell phenotypes. Eight rounds administration of 10×106 cells/kg BM-MSC improved mouse survival from 62.5% to 92.9% and reversed lymphoproliferation to normal levels at day-21 post-transplantation (PT) (p=0.189). BM-MSC mainly and eventually homed to immune organs at four months PT. Immunomodulatory kinetics showed that the optimal immunosuppression period of BM-MSC occurred from day-7 to day-21 PT in immune organs, lung, and kidney; hence, optimal infusion frequency was deduced as 21 days. In conclusion, an appropriate therapeutic strategy in a pre-clinical autoimmune disease mouse model was established with a defined MSC source as well as optimal infusion dose, frequency, and administration number. It will help to standardize cell preparation, characterization and administration protocol, and minimize the outcome discrepancies between different centers worldwide.M is a worldwide health problem leading to the death of millions of people. The disease is induced by different species of protozoa parasites from the genus Plasmodium. In humans, Plasmodium falciparum is the most dangerous species responsible for severe disease. Despite all efforts to establish the pathogenesis of malaria, it is far from being fully understood. In addition, resistance to existing drugs has developed in several strains and the development of new effective compounds to fight these parasites is a major issue. Recent discoveries indicate the potential role of the renin-angiotensin system (RAS) in malaria infection. Angiotensin receptors have not been described in the parasite genome, however several reports in the literature suggest a direct effect of angiotensin-derived peptides on different aspects of the host-parasite interaction. Here, we highlight new findings on the involvement of the RAS in parasite development and in the regulation of the host immune response in an attempt to expand our knowledge of the pathogenesis of this disease.F anemia (FA) is a genetic disorder of genomic instability, with main clinical symptoms involving congenital abnormalities, infertility, bone marrow failure and a predisposition to the development of several types of cancer, especially acute myelogenous leukemia (AML) and head and neck carcinomas. Homozygous or bi-allelic mutations in one of 16 genes corresponding to distinct proteins prevent the repair of DNA damage caused by inter-strand cross-linking (ICL) agents and maintenance of genomic integrity during the replication process. The pathogenesis of FA affects cellular processes essential for the homeostasis of the organism, such as differentiation, proliferation and apoptosis, among other less evident, as the immune system that seems to reflect a primary defect of the disease. Peripheral cytokines such as TNF-α, INF-γ, IL-10 and IL-1β may be increased in the blood of these patients, while lower levels IL-6 are observed. However, patients with advanced bone marrow failure seem to show a different profile, with increased levels of TGF-β and IL-6. The absolute number of total lymphocytes is reduced, more specifically the populations of B, T CD8+ and NK cells, as well as their activities. Changes are observed in the proportions normally seen among NK cell subpopulations, suggesting defects in the differentiation of these cells. Clinically, these changes are reflected in an increased susceptibility to various pathogens. Here, we will describe aspects of the diversity, plasticity and microenvironment of the immune system and how genomic instability can affect these mechanisms in the immune system of patients with FA.N oxide synthases are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule, having essential roles in many biological processes including the control of blood pressure, regulation of neuronal activity and immune responses. NOS3 (endothelial NOS or e NOS) and NOS2 (inducible NOS or I NOS) have been appreciated as mediators of inflammatory processes. However, considerably less is known about the role of NOS1 (neuronal NOS or n NOS) in inflammation. We have uncovered an important role for this enzyme in regulating TLR4 signaling. We demonstrate that in contrast to the enhanced susceptibility of NOS2 -/and NOS3 -/mice to LPS, NOS1 -/mice are, in fact, more resistant to LPS-induced lethality and tissue injury. We demonstrate that the loss of NOS1 attenuates TLR4stimulated cytokine production and NF-κB activity in vivo and in vitro. Macrophages from NOS1 -/animals demonstrate an LPS-induced decrease in protein levels of the p65 subunit of NF-κB. This decrease in p65 protein correlates with an increase in protein levels of suppressor of cytokine signaling-1 (SOCS1) and increased physical association between SOCS1 and p65. On studying the mechanism of NOS1-regulation of inflammation we found that an early pulse of NOS1-derived NO was required to stabilize p65 in the nucleus of macrophages via the inhibitory S-nitrosation of suppressor of Cytokine Signaling-1 (SOCS1). NOS1-derived NO through nitrosation of Cys147 and Cys179 on SOCS1 permits p65-mediated pro-inflammatory gene transcription and is essential for the mechanism of inflammation. Taken together, our results demonstrate that NOS1 is a fundamental early regulator of gene transcription of the inflammatory response thereby heavily impacting the course, type and duration of the inflammatory process.