Signaling Subunit Research Articles

Effective recognition of multiple anions by an azobenzene--Al$^{\mathbf{3+}}$ system

A ready-to-use multifunctional coordinative system, 2,2'-dihydroxyazobenzene --aluminum (III) (DHAB--Al$^{3+})$, was created to recognize several anions effectively through three channels, namely colorimetric detection, UV-Vis spectroscopy, and fluorescence spectroscopy. Under naked eye visualization, the H$_{2}$PO$_{4}^{-}$ can be readily distinguished from the other anions by the DHAB--Al$^{3+}$ system through a change in color from reddish-orange to light yellow. H$_{2}$PO$_{4}^{-}$ was qualitatively detected by using UV-Vis and fluorescence spectra. Differentiating SO$_{3}^{2-}$, HPO$_{4}^{2-}$, and HCO$_{3}^{-}$ anions colorimetrically is difficult; thus, CO$_{3}^{2-}$, SO$_{3}^{2-}$, HPO$_{4}^{2-}$, and HCO$_{3}^{-}$ were discriminated by adopting distinct changes in the UV-Vis spectra. CO$_{3}^{2-}$, HPO$_{4}^{2-}$, and HCO$_{3}^{-}$ were accurately quantified using fluorescence spectra. The DHAB--Al$^{3+}$ system responded to H$_{2}$PO$_{4}^{-}$ and CO$_{3}^{2-}$ via a sensing mechanism based on a displacement approach. By contrast, the system responded to SO$_{3}^{2-}$, HPO$_{4}^{2-}$, and HCO$_{3}^{-}$ based on a binding site--signaling subunit approach. The DHAB--Al$^{3+}$ system can determine the concentration ranges of H$_{2}$PO$_{4}^{-}$ with the naked eye and quantify CO$_{3}^{2-}$, SO$_{3}^{2-}$, HPO$_{4}^{2-}$, and HCO$_{3}^{-}$ through UV-Vis spectra.

Fluorogen-activating proteins provide tunable labeling densities for tracking FcεRI independent of IgE.

Crosslinking of IgE bound FcεRI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators that stimulate the allergic response. Here, we present and characterize the use of fluorogen-activating proteins (FAPs) for single particle tracking of FcεRI to investigate how receptor mobility is influenced after IgE-induced changes in mast cell behavior. FAPs are genetically encoded tags that bind a fluorogen dye and increase its brightness upon binding up to 20,000-fold. We demonstrate that, by titrating fluorogen concentration, labeling densities from ensemble to single particle can be achieved, independent of expression level and without the need for wash steps or photobleaching. The FcεRI γ-subunit fused to a FAP (FAP-γ) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of FcεRI at the single molecule level. We show that the FcεRI γ-subunit dynamics are controlled by the IgE-binding α-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering FcεRI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track FcεRI relative to dynamin–GFP and find that immobilized receptors readily correlate with locations of dynamin recruitment only under conditions that promote rapid endocytosis. These studies demonstrate the usefulness of the FAP system for single molecule studies and have provided new insights into the relationship among FcεRI structure, activity, and mobility.

Peripheral nerve regeneration and NGF-dependent neurite outgrowth of adult sensory neurons converge on STAT3 phosphorylation downstream of neuropoietic cytokine receptor gp130.

After nerve injury, adult sensory neurons can regenerate peripheral axons and reconnect with their target tissue. Initiation of outgrowth, as well as elongation of neurites over long distances, depends on the signaling of receptors for neurotrophic growth factors. Here, we investigated the importance of gp130, the signaling subunit of neuropoietic cytokine receptors in peripheral nerve regeneration. After sciatic nerve crush, functional recovery in vivo was retarded in SNS-gp130(-/-) mice, which specifically lack gp130 in sensory neurons. Correspondingly, a significantly reduced number of free nerve endings was detected in glabrous skin from SNS-gp130(-/-) compared with control mice after nerve crush. Neurite outgrowth and STAT3 activation in vitro were severely reduced in cultures in gp130-deficient cultured neurons. Surprisingly, in neurons obtained from SNS-gp130(-/-) mice the increase in neurite length was reduced not only in response to neuropoietic cytokine ligands of gp130 but also to nerve growth factor (NGF), which does not bind to gp130-containing receptors. Neurite outgrowth in the absence of neurotrophic factors was partially rescued in gp130-deficient neurons by leptin, which activates STAT3 downstream of leptic receptor and independent of gp130. The neurite outgrowth response of gp130-deficient neurons to NGF was fully restored in the presence of leptin. Based on these findings, gp130 signaling via STAT3 activation is suggested not only to be an important regulator of peripheral nerve regeneration in vitro and in vivo, but as determining factor for the growth promoting action of NGF in adult sensory neurons.

17: Structural and functional analysis of the GM-CSF:GM-CSF receptor alpha chain binary complex provide new insights into signalling of the GM-CSF ternary complex

Interleukin (IL)-3, granulocyte–macrophage colony stimulating factor (GM-CSF) and IL-5 are important cytokines that control production and function of myeloid cells and dendritic cells. Over-expression of these cytokines or their receptors can lead to chronic inflammatory diseases and myeloid leukaemias. These cytokines signal through heterodimeric specific receptors consisting of cytokine-specific α chains, and a signalling subunit β c, shared by all three receptors. We have determined the 3D crystal structure of the binary GM-CSF:GM-CSF receptor α chain (GMR α ) complex to 2.8 A resolution, which reveals for the first time all three extracellular domains of the GMR α receptor chain. The structure shows a striking resemblance to the related IL-3R α , IL-5R α and IL-13R α subunits; however, the differing positions of the N-terminal domains suggest that this domain may play a cytokine-specific role in cell signalling. We have used mutagenesis, ligand binding and functional studies to examine residues in GMR α and GM-CSF that define the binding interface, and identified residues involved in higher order complex assembly. Furthermore, the complete structure of the GMR α receptor chain from the binary complex was used to improve the electron density maps of the partial GMR α in the previously published ternary complex, elucidating additional GM-CSF:GMR α interactions and the position of the N-terminal domain of GMR α in the ternary complex. A structural comparison of the GM-CSF binary and ternary complexes revealed numerous major, as well as subtle, conformational changes in cytokine and both receptor chains. These studies enable us to identify differences and similarities in the way GM-CSF, IL-3 and IL-5 interact with their receptors and facilitate different types of cell signaling events.

A Probe for the Selective and Parts‐per‐Billion‐Level Detection of Copper(II) and Mercury(II) using a Micellar Medium and Its Utility in Cell Imaging

AbstractA novel colorimetric probe 1 based on the picolyl moiety has been designed and synthesized. Probe 1 is composed of a pyrene and a bispicolyl amine (BPA) unit, in which the BPA moiety acts as a binding unit and the binding phenomenon is sensed from the changes in the signaling subunit. The probe detects Cu2+ specifically in water and both Cu2+ and Hg2+ efficiently in neutral Brij‐58 micellar media. The probe shows a color change visible to the naked eye upon addition of metal ions. Notably, in a micellar medium, probe 1 can detect both the Cu2+ and Hg2+ ions even at parts‐per‐billion levels. Furthermore, the probe shows ratiometric detection of both the metal ions making the sensing quantitative. The two metal ions could be discriminated both visibly under a UV lamp and with the use of fluorescence spectroscopy. The probe could be also used in biological cell lines for the detection of both Hg2+ and Cu2+ ions.

Selective fluorescent sensor for mercury(II) ion based on an easy to prepare double naphthalene Schiff base

A fluorescent mercury sensor allowing for cheap, sensitive, and highly selective detection of Hg2+ is described. This sensor combines two naphthalene groups as signaling subunits and the presence of imine group which was oxidized to amide with DMSO and Hg2+ confers the coordination capacity required to coordinate Hg2+ ion. This double-naphthalene Schiff base chemosensor (G, Scheme 2) is readily soluble in DMSO and the solution give colorless fluorescent signal, when sensor G reacts with Hg2+ the solution give blue fluorescent signal. The MS, IR and 1H NMR data suggest that the presence of Hg2+ leads to the formation of OHg2+N, which is then converted to a structural change, the CN of G was oxidized to OCNH of G1 (Scheme 1). The test strip based on G was fabricated, which could act as a convenient and efficient Hg2+ test kit. The event is useful for fluorescent detection of Hg2+ at the micromolar level with a detection limit of 5.595×10−8M in DMSO medium.

Fluorescent organic nanoparticles as chemosensor for nanomolar detection of Cs+ in aqueous medium

A new sensor 1 bearing mixed imine and amide linkages is synthesized with simple condensation reaction of diode amine with catechol carbaldehyde. The amide linkages may define the receptor pseudo cavity as these linkages are generally considered to provide rigidity to receptor design. The imine linkages are expected to be responsible for providing binding site as well as fluorescent signaling sub-unit. The 1 is evaluated for their sensor activities in a diverse range of solvents and it has shown the selective binding affinity for Cs+. In order to evaluate the sensor activity in aqueous medium, fluorescent organic nanoparticles (FONs) of receptor 1 are formed using re-precipitation method. FONs of 1 has shown AIE phenomenon in aqueous medium with enhancement in fluorescence intensity at λmax = 412 nm. Fluorescence emission profiles of these FONs have shown some changes in low and high pH range; however it remains stable in pH range 4–9, thus making them appropriate to use in environmental/biological samples. To the best of our knowledge, 1is representing the first example of FONs as chemosensor for nanomolar detection of Cs+ in aqueous media.

Structure of the Chicken CD3ϵδ/γ Heterodimer and Its Assembly with the αβT Cell Receptor

In mammals, the αβT cell receptor (TCR) signaling complex is composed of a TCRαβ heterodimer that is noncovalently coupled to three dimeric signaling molecules, CD3εδ, CD3εγ, and CD3ζζ. The nature of the TCR signaling complex and subunit arrangement in different species remains unclear however. Here we present a structural and biochemical analysis of the more primitive ancestral form of the TCR signaling complex found in chickens. In contrast to mammals, chickens do not express separate CD3δ and CD3γ chains but instead encode a single hybrid chain, termed CD3δ/γ, that is capable of pairing with CD3ε. The NMR structure of the chicken CD3εδ/γ heterodimer revealed a unique dimer interface that results in a heterodimer with considerable deviation from the distinct side-by-side architecture found in human and murine CD3εδ and CD3εγ. The chicken CD3εδ/γ heterodimer also contains a unique molecular surface, with the vast majority of surface-exposed, nonconserved residues being clustered to a single face of the heterodimer. Using an in vitro biochemical assay, we demonstrate that CD3εδ/γ can assemble with both chicken TCRα and TCRβ via conserved polar transmembrane sites. Moreover, analogous to the human TCR signaling complex, the presence of two copies of CD3εδ/γ is required for ζζ assembly. These data provide insight into the evolution of this critical receptor signaling apparatus.

T-Cell Receptor-CD3 Signaling Complex Extracellular Interactions Characterized by Genetic Incorporation of Unnatural Amino Acid Photo-Crosslinkers

T-cells play a central role in the adaptive immune response to invading pathogens and cancer while largely avoiding self-reactivity. The interaction of T-cell receptors (TCRs) displayed on T-cells with peptide-major histocompatibility complexes (pMHCs) on the surface of antigen presenting cells leads to signaling through the TCR-CD3 signaling complexes, which result in the initiation of cellular immune responses. However, how molecular and structural events in TCR-recognition translate through TCR-CD3 complex and generate differences in intracellular signaling is still not clear. Previous studies have shown that the extracellular TCR-CD3 subunits interactions are important for T-cell responses to TCR engagement. The objective of this research is to understand structural organization and specific interactions among TCR and CD3 subunits, and to provide insights into how structural features and mechanisms initiating TCR-triggering determine the outcome of immune responses. To monitor interactions among TCR-CD3 subunits, we developed a system to site-specifically incorporate an unnatural amino acid photo-crosslinker p-Azido-L-phenylalanine (pAzpa) or p-benzoyl-L-phenylalanine (pBpa) into TCR expressed on cell surface. Subsequently, after activation of TCR with pMHC, photo-crosslinked TCR-CD3 complexes were immunoprecipitated and analyzed by Western Blotting. Our analysis showed that specific residues in the TCR DE, CC, FG loops and G strand play critical roles in TCR-CD3 subunit interactions. Moreover, cross-linked high molecular weight complexes suggested TCR-TCR dimerization during the signaling process. Together our results 1) reveal how TCR-CD3 signaling subunits are positioned together to perform their function, 2) suggest how the antigen specific recognition information gets transferred from outside to the signaling molecules inside cell to determine immune responses, 3) provide potential targets for modulating outcome of immune response.

IL-6 signalling in patients with acute ST-elevation myocardial infarction

Cytokines of the IL-6 family have been related to infarct size and prognosis in patients with myocardial infarction. The aims of the present study were to elucidate possible associations between myocardial necrosis and left ventricular impairment and members of the IL-6 transsignalling system including soluble (s) IL-6R and (s) glycoprotein 130 (sgp130) in patients with ST-elevation myocardial infarction (STEMI) treated with primary PCI.In blood samples from 1028 STEMI patients, collected in-hosptial, we found significant correlations between peak TnT and IL-6 and CRP (p < 0.001, all) and between IL-6 and CRP and LV ejection fraction and NT-proBNP (p < 0.001, all). On the contrary, no significant associations were found between peak TnT and sgp130 or sIL-6R. Furthermore sgp130 was significantly elevated in diabetic patients and also associated with the glucometabolic state.In conclusion, circulating levels of IL-6 and CRP, but not the soluble forms of the receptor (sIL-6R) or the receptor signalling subunit (sgp130) were associated with the extent of myocardial necrosis. The biological importance of the IL-6/gp130-mediated signalling pathways in patients with acute myocardial infarction and dysglycemia should be further elucidated.

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice

AbstractNeutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin–dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti–P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation.Key Points

SOCS-1 Mediates Ubiquitylation and Degradation of GM-CSF Receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and the related cytokines interleukin (IL)-3 and IL-5 regulate the production and functional activation of hematopoietic cells. GM-CSF acts on monocytes/macrophages and granulocytes, and several chronic inflammatory diseases and a number of haematological malignancies such as Juvenile myelomonocytic leukaemia (JMML) are associated with deregulated GM-CSF receptor (GMR) signaling. The downregulation of GMR downstream signaling is mediated in part by the clearance of activated GMR via the proteasome, which is dependent on the ubiquitylation of βc signaling subunit of GMR via an unknown E3 ubiquitin ligase. Here, we show that suppressor of cytokine signaling 1 (SOCS-1), best known for its ability to promote ubiquitin-mediated degradation of the non-receptor tyrosine kinase Janus kinase 2 (JAK2), also targets GMRβc for ubiquitin-mediated degradation and attenuates GM-CSF-induced downstream signaling.

Construction of unnatural heterodimeric receptors based on IL‐2 and IL‐6 receptor subunits

Cells have various receptors on their surface for responding to extracellular signals that involve intercellular communication. Although the mechanism of signal transduction by such wild-type receptors has been studied intensively, there has been minimal effort in investigating whether such receptors could signal when unnaturally coupled. In this study, we investigated whether unnatural receptor pairs comprising interleukin-2 (IL-2) and interleukin-6 (IL-6) receptor subunits could transduce a signal through forced dimerization. We replaced the extracellular domain of IL-2R and IL-6R signaling subunits (IL-2Rβ, IL-2Rγ, and gp130) with the FK506-binding protein (FKBP) or the FKBP12-rapamycin binding (FRB) domain, the protein pair known to be heterodimerized by rapamycin. When expressed in a hematopoietic cell line, unnatural heterodimers (IL-2Rβ-gp130 and IL-2Rγ-gp130 pairs) successfully transduced a signal. While the IL-2Rγ-gp130 pair maximally mimicked gp130 signaling, the IL-2Rβ-gp130 pair gave weaker gp130 signaling and no significant induction of IL-2Rβ signaling, indicating a high potential of the IL-2Rγ chain in terms of activating the coupled partners. This is the first report demonstrating that heterodimeric combinations of IL-2R and IL-6R subunits are functional for signaling. Further extension of this approach may attain a creative design of artificial receptor pairs that have distinct signaling properties when compared with natural receptors.

Gene Deficiency in Activating Fcγ Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.

Theoretical Elucidation of the Origin for Assembly of the DAP12 Dimer with Only One NKG2C in the Lipid Membrane

In this work, we have investigated in details the origin of the assembly of the DAP12 dimer with only one NKG2C in the activating immunoreceptor complex from thew two aspects of electronic properties and dynamic structures by performing density functional theory (DFT) calculations and molecular dynamics (MD) simulations. In the DFT calculations, we studied the aggregation ability of the NKG2C(TM) with the DAP12(TM) dimer and the DAP12(TM)-DAP12(TM)-NKG2C(TM) complex by analyzing the electrostatic potentials and frontier molecular orbitals (FMOs), and in the MD simulations we mainly investigated the dynamic structures of the DAP12(TM)-DAP12(TM)-NKG2C(TM) complex and its mutants, as well as the tetramer complex consisting of two DAP12(TM) and two NKG2C(TM) helixes without any restriction. Through the studies of the electrostatic potential, the FMOs, and the dynamic structures, we have provided reasonable explanations to some extent for the experimental observation that only one NKG2C can associate with the DAP12 homodimer. The present theoretical results are expected to give valuable information for further studying the assembly between receptors and signaling subunits.

Cell-to-Cell Variability Analysis Dissects the Plasticity of Signaling of Common γ Chain Cytokines in T Cells

Natural variability in the abundance of signaling regulators can lead to divergence in cell fate, even within genetically identical cells that share a common differentiation state. We introduce cell-to-cell variability analysis (CCVA), an experimental and computational methodology that quantifies the correlation between variability in signaling regulator abundance and variation in the sensitivity of cells to stimuli. With CCVA, we investigated the unexpected effects of the interleukin 2 (IL-2) receptor α chain (IL-2Rα) on the sensitivity of primary mouse T lymphocytes to cytokines that signal through receptors that have the common γ chain (γ(c)). Our work showed that increased IL-2Rα abundance decreased the concentration of IL-2 required for a half-maximal activation (EC(50)) of the downstream effector signal transducer and activator of transcription 5 (STAT5), but reduced the responsiveness to IL-7 or IL-15, without affecting the EC(50) values of other γ(c) cytokines. To investigate the mechanism of the effect of IL-2Rα on γ(c) cytokine signaling, we introduced a Bayesian-inference computational framework that models the formation of receptor signaling complexes with data from previous biophysical measurements. With this framework, we found that a model in which IL-2Rα drives γ(c) depletion through the assembly of functional IL-2R complexes was consistent with both the CCVA data and experimental measurements. The combination of CCVA and computational modeling produced quantitative understanding of the crosstalk between γ(c) cytokine receptor signaling in T lymphocytes.

The BAFF Receptor Transduces Survival Signals by Co-opting the B Cell Receptor Signaling Pathway

SummaryFollicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This “tonic” BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors.

Gp130 activation is regulated by D2–D3 interdomain connectivity

Activation of the IL-6 (interleukin 6) receptor subunit gp130 (glycoprotein 130) has been linked to the formation of complexes with IL-6 and the IL-6 receptor, as well as to gp130 dimerization. However, it has been shown that gp130 is present as a pre-formed dimer, indicating that its activation is not solely dependent on dimerization. Therefore the detailed mechanism of gp130 activation still remains to be deciphered. Recently, deletion mutations of gp130 have been found in inflammatory hepatocellular adenoma. The mutations clustered around one IL-6-binding epitope of gp130 and resulted in a ligand-independent constitutively active gp130. We therefore hypothesized that conformational changes of this particular IL-6-binding epitope precedes gp130 activation. Using a rational structure-based approach we identified for the first time amino acids critical for gp130 activation. We can show that gp130 D2-D3 interdomain connectivity by hydrophobic residues stabilizes inactive gp130 conformation. Conformational destabilization of the EF loop present in domain D2 and disruption of D2-D3 hydrophobic interactions resulted in ligand-independent gp130 activation. Furthermore we show that the N-terminal amino acid residues of domain D1 participate in the activation of the gp130 deletion mutants. Taken together we present novel insights into the molecular basis of the activation of a cytokine receptor signalling subunit.

Colorimetric Hg2+ Sensing in Water: From Molecules toward Low-Cost Solid Devices

A new colorimetric molecular sensor allowing for cheap, fast, sensitive, and highly selective naked-eye detection of Hg(2+) in water is described. This molecule combines a 1,8-diaminoanthraquinone signaling subunit and phosphonic acid esters that confer the water solubility to the dye (R = H). A ready-to-use colorimetric solid sensor was obtained by incorporating an amphiphilic analog (R = OC(12)H(25)) exhibiting similar binding properties and optical responses in an agarose film.

Central inhibition of interleukin-6 trans-signaling during peripheral infection reduced neuroinflammation and sickness in aged mice.

During systemic infection, inflammatory cytokines such as interleukin (IL)-6 are produced in excess in the brain of aged mice and induce severe behavioral deficits. However, no studies have examined how pro-inflammatory IL-6 trans-signaling is involved in the exaggerated production of IL-6 in the aged brain, nor the extent to which IL-6 trans-signaling affects other markers of neuroinflammation, adhesion molecules, and behavior. Therefore, this study investigated in aged mice the presence of IL-6 signaling subunits in microglia; the central effects of soluble gp130 (sgp130)-a natural inhibitor of the IL-6 trans-signaling pathway-on IL-6 production in microglia; and the effects of sgp130 given intracerebroventricularly (ICV) on neuroinflammation and sickness behavior caused by i.p. injection of lipopolysaccharide (LPS). Here we show that microglia isolated from aged mice have higher expression of IL-6 receptor (IL-6R) compared to microglia from adults; and the level of mRNA for ADAM17, the enzyme responsible for shedding membrane-bound IL-6R in trans-signaling, is higher in the hippocampus of aged mice compared to adults. Additionally, we show in aged mice that peripheral LPS challenge elicits a hyperactive IL-6 response in microglia, and selective blockade of trans-signaling by ICV injection of sgp130 mitigates this. The sgp130-associated inhibition of IL-6 was paralleled by amelioration of exaggerated and protracted sickness behavior in aged mice. Taken together, the results show that microglia are important regulators of the IL-6 trans-signaling response in the aged brain and sgp130 exerts an anti-inflammatory effect by inhibiting the pro-inflammatory arm of IL-6 signaling.