bluuebunny/crossref_metadata_2025 huggingface dataset:
Key Molecular Contacts Promote Recognition of the BAFF Receptor by TNF Receptor-Associated Factor 3: Implications for Intracellular Signaling Regulation
Authors: Ni Chao-Zhou , Oganesyan Gagik , Welsh Kate , Zhu Xiuwen , Reed John C. , Satterthwait Arnold C. , Cheng Genhong , Ely Kathryn R.
Published: April 2014

B cell-activating factor belonging to the TNF family receptor (BAFF-R), a member of the TNFR superfamily, plays a role in autoimmunity after ligation with BAFF ligand (also called TALL-1, BLyS, THANK, or zTNF4). BAFF/BAFF-R interactions are critical for B cell regulation, and signaling from this ligand-receptor complex results in NF-κB activation. Most TNFRs transmit signals intracellularly by recruitment of adaptor proteins called TNFR-associated factors (TRAFs). However, BAFF-R binds only one TRAF adaptor, TRAF3, and this interaction negatively regulates activation of NF-κB. In this study, we report the crystal structure of a 24-residue fragment of the cytoplasmic portion of BAFF-R bound in complex with TRAF3. The recognition motif 162PVPAT166 in BAFF-R is accommodated in the same binding crevice on TRAF3 that binds two related TNFRs, CD40 and LTβR, but is presented in a completely different structural framework. This region of BAFF-R assumes an open conformation with two extended strands opposed at right angles that each make contacts with TRAF3. The recognition motif is located in the N-terminal arm and intermolecular contacts mediate TRAF recognition. In the C-terminal arm, key stabilizing contacts are made, including critical hydrogen bonds with Gln379 in TRAF3 that define the molecular basis for selective binding of BAFF-R solely to this member of the TRAF family. A dynamic conformational adjustment of Tyr377 in TRAF3 occurs forming a new intermolecular contact with BAFF-R that stabilizes the complex. The structure of the complex provides a molecular explanation for binding affinities and selective protein interactions in TNFR-TRAF interactions.
TLR2 Recognizes a Bacterial Lipopeptide through Direct Binding
Authors: Vasselon Thierry , Detmers Patricia A. , Charron Dominique , Haziot Alain
Published: April 2014

The TLRs play an important role in the initiation of cellular innate immune responses to a wide range of bacterial products, including LPS and lipoproteins. Although rapid progress has been made on signaling functions of activated TLRs, the molecular mechanisms that lead to TLR activation are still poorly understood. We report in this study that the extracellular domain of TLR2 interacts directly with synthetic bacterial lipopeptide (sBLP), a potent analog of bacterial lipoproteins. Using fluorescently labeled sBLP complexed to soluble recombinant CD14 (rsCD14), we observed specific binding of sBLP to the surface of cells expressing TLR2 transgenes and to a recombinant soluble form of the TLR2 ectodomain. TLR2-mediated binding of sBLP at the cell surface did not require prior induction of intracellular signals. In addition, using a chimeric TLR2/TLR4 construct, we showed that the leucine-rich region of TLR2 carries the specificity for binding of the agonist and for initiating signaling. Specific binding of fluorescent sBLP to purified sTLR2 required sCD14. However, sCD14 was not part of the complex formed by soluble TLR2 and sBLP. Together, these data provide evidence that TLR2 recognizes sBLP through direct binding.
TLRs Govern Neutrophil Activity in Aspergillosis
Authors: Bellocchio Silvia , Moretti Silvia , Perruccio Katia , Fallarino Francesca , Bozza Silvia , Montagnoli Claudia , Mosci Paolo , Lipford Grayson B. , Pitzurra Lucia , Romani Luigina
Published: April 2014

Polymorphonuclear neutrophils (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of fungal infection. PMNs, however, may act as double-edged swords, as the excessive release of oxidants and proteases may be responsible for injury to organs and fungal sepsis. To identify regulatory mechanisms that may balance PMN-dependent protection and immunopathology in fungal infections, the involvement of different TLR-activation pathways was evaluated on human PMNs exposed to the fungus Aspergillus fumigatus. Recognition of Aspergillus and activation of PMNs occurred through the involvement of distinct members of the TLR family, each likely activating specialized antifungal effector functions. By affecting the balance between fungicidal oxidative and nonoxidative mechanisms, pro- and anti-inflammatory cytokine production, and apoptosis vs necrosis, the different TLRs ultimately impacted on the quality of microbicidal activity and inflammatory pathology. Signaling through TLR2 promoted the fungicidal activity of PMNs through oxidative pathways involving extracellular release of gelatinases and proinflammatory cytokines while TLR4 favored the oxidative pathways through the participation of azurophil, myeloperoxidase-positive, granules and IL-10. This translated in vivo in the occurrence of different patterns of fungal clearance and inflammatory pathology. Both pathways were variably affected by signaling through TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9. The ability of selected individual TLRs to restore antifungal functions in defective PMNs suggests that the coordinated outputs of activation of multiple TLRs may contribute to PMN function in aspergillosis.
IFN Regulatory Factor 3-Dependent Induction of Type I IFNs by Intracellular Bacteria Is Mediated by a TLR- and Nod2-Independent Mechanism
Authors: Stockinger Silvia , Reutterer Benjamin , Schaljo Barbara , Schellack Carola , Brunner Sylvia , Materna Tilo , Yamamoto Masahiro , Akira Shizuo , Taniguchi Tadatsugu , Murray Peter J. , Müller Mathias , Decker Thomas
Published: April 2014

Like viruses, intracellular bacteria stimulate their host cells to produce type I IFNs (IFN-α and IFN-β). In our study, we investigated the signals and molecules relevant for the synthesis of and response to IFN by mouse macrophages infected with Listeria monocytogenes. We report that IFN-β is the critical immediate-early IFN made during infection, because the synthesis of all other type I IFN, expression of a subset of infection-induced genes, and the biological response to type I IFN was lost upon IFN-β deficiency. The induction of IFN-β mRNA and the IFN-β-dependent sensitization of macrophages to bacteria-induced death, in turn, was absolutely dependent upon the presence of the transcription factor IFN regulatory factor 3 (IRF3). IFN-β synthesis and signal transduction occurred in macrophages deficient for TLR or their adaptors MyD88, TRIF, or TRAM. Expression of Nod2, a candidate receptor for intracellular bacteria, increased during infection, but the protein was not required for Listeria-induced signal transduction to the Ifn-β gene. Based on our data, we propose that IRF3 is a convergence point for signals derived from structurally unrelated intracellular pathogens, and that L. monocytogenes stimulates a novel TLR- and Nod2-independent pathway to target IRF3 and the type I IFN genes.
Human Monocytes Infected with Yersinia pestis Express Cell Surface TLR9 and Differentiate into Dendritic Cells
Authors: Saikh Kamal U. , Kissner Teri L. , Sultana Afroz , Ruthel Gordon , Ulrich Robert G.
Published: April 2014

TLR9 recognizes DNA sequences containing hypomethylated CpG motifs and is a component of the innate immune system highly conserved during eukaryotic evolution. Previous reports suggested that the expression of TLR9 is restricted to plasmacytoid dendritic cells and B lymphocytes. Our results indicate that low levels of TLR9 are present on the cell surface of freshly isolated human monocytes, and expression is greatly increased by infection with Yersinia pestis. Enhanced cell surface TLR9 coincided with elevated levels of cytoplasmic TLR9 and recruitment of MyD88. Infected monocytes differentiated into mature dendritic cells, expressed IFN-α, and stimulated proliferative and cytotoxic T cell responses specific to Y. pestis. Furthermore, uninfected B cells and monocytes both increased cell surface TLR9, CD86, and HLA-DR in response to treatment with CpG-containing oligonucleotides, whereas cell surface TLR9 was down-modulated on infected dendritic cells by the addition of agonist oligonucleotide. Our results suggest that increased expression of TLR9 on the surface of infected cells may serve a role as an activation signal to other cells of the immune system.
Innate Imprinting by the Modified Heat-Labile Toxin of Escherichia coli (LTK63) Provides Generic Protection against Lung Infectious Disease
Authors: Williams Andrew Evan , Edwards Lorna , Humphreys Ian Robert , Snelgrove Robert , Rae Aaron , Rappuoli Rino , Hussell Tracy
Published: April 2014

In a healthy individual, the lung contains few lymphoid cells. However, amplified immune responses, as exemplified during lung infection, can cause extensive tissue damage. We have previously demonstrated that one lung infection modulates the immunopathological outcome to a subsequent unrelated pathogen. Mimicking heterologous immunity may provide a means of enhancing both innate and acquired immunity. We now show that prior lung administration of a modified heat-labile toxin from Escherichia coli (LTK63) enhances immunity to respiratory syncytial virus, influenza virus, and the fungus Cryptococcus neoformans. Treatment with LTK63 decreased lung inflammation and tissue damage and improved the ability to resolve the infection. APCs expressing the activation markers MHC class II, CD80, and CD40 increased in number in the lung. LTK63 treatment increased the pathogen-specific IgA response in the nasal mucosa and simultaneously decreased inflammatory cytokine production (IFN-γ and TNF-α) after infection. The number of activated CD8+CD44+ T cells and the respiratory syncytial virus- or influenza-specific CD8-proliferative responses increased, although the total inflammatory infiltrate was reduced. LTK63 treatment matured lung APCs (LTK63 prevented efficient presentation of whole OVA to DO11.10 cells, whereas OVA peptide presentation was unaffected), enhanced immunity in both a Th1 and Th2 environment, was long lasting, and was not pathogen or host strain specific; the protective effects were partially independent of T and B cells. Innate imprinting by toxin-based immunotherapeutics may provide generic protection against infectious disease in the lung, without the need for coadministered pathogen-specific Ag.
Exploiting Type 3 Complement Receptor for TNF-α Suppression, Immune Evasion, and Progressive Pulmonary Fungal Infection
Authors: Brandhorst T. Tristan , Wüthrich Marcel , Finkel-Jimenez Bea , Warner Thomas , Klein Bruce S.
Published: April 2014

TNF-α is crucial in defense against intracellular microbes. Host immune cells use type 3 complement receptors (CR3) to regulate excess TNF-α production during physiological clearance of apoptotic cells. BAD1, a virulence factor of Blastomyces dermatitidis, is displayed on yeast and released during infection. BAD1 binds yeast to macrophages (Mφ) via CR3 and CD14 and suppresses TNF-α, which is required for resistance. We investigated whether blastomyces adhesin 1 (BAD1) exploits host receptors for immune deviation and pathogen survival. Soluble BAD1 rapidly entered Mφ, accumulated intracellularly by 10 min after introduction to cells, and trafficked to early and late endosomes. Inhibition of receptor recycling by monodansyl cadaverine blocked association of BAD1 with Mφ and reversed TNF-α suppression in vitro. Inhibition of BAD1 uptake with cytochalasin D and FcR-redirected delivery of soluble BAD1 as Ag-Ab complexes or of wild-type yeast opsonized with IgG similarly reversed TNF-α suppression. Hence, receptor-mediated entry of BAD1 is requisite in TNF-α suppression, and the route of entry is critical. Binding of soluble BAD1 to Mφ of CR3−/− and CD14−/− mice was reduced to 50 and 33%, respectively, of that in wild-type mice. Mφ of CR3−/− and CD14−/− mice resisted soluble BAD1 TNF-α suppression in vitro, but, in contrast to CR3−/− cells, CD14−/− cells were still subject to suppression mediated by surface BAD1 on wild-type yeast. CR3−/− mice resisted both infection and TNF-α suppression in vivo, in contrast to wild-type and CD14−/− mice. BAD1 of B. dermatitidis thus co-opts normal host cell physiology by exploiting CR3 to subdue TNF-α production and foster pathogen survival.
Helminth Antigens Modulate TLR-Initiated Dendritic Cell Activation
Authors: Kane Colleen M. , Cervi Laura , Sun Jie , McKee Amy S. , Masek Katherine S. , Shapira Sagi , Hunter Christopher A. , Pearce Edward J.
Published: April 2014

There is increasing awareness that helminth infections can ameliorate proinflammatory conditions. In part, this is due to their inherent ability to induce Th2 and, perhaps, regulatory T cell responses. However, recent evidence indicates that helminths also have direct anti-inflammatory effects on innate immune responses. In this study, we address this issue and show that soluble molecules from the eggs of the helminth parasite Schistosoma mansoni (SEA) suppress LPS-induced activation of immature murine dendritic cells, including MHC class II, costimulatory molecule expression, and IL-12 production. SEA-augmented LPS-induced production of IL-10 is in part responsible for the observed reduction in LPS-induced IL-12 production. However, analyses of IL-10−/− DC revealed distinct IL-10-independent suppressive effects of SEA. IL-10-independent mechanisms are evident in the suppression of TLR ligand-induced MAPK and NF-κB signaling pathways. Microarray analyses demonstrate that SEA alone uniquely alters the expression of a small subset of genes that are not up-regulated during conventional TLR-induced DC maturation. In contrast, the effects of SEA on TLR ligand-induced DC activation were striking: when mixed with LPS, SEA significantly affects the expression of >100 LPS-regulated genes. These findings indicate that SEA exerts potent anti-inflammatory effects by directly regulating the ability of DC to respond to TLR ligands.
Reconstitution of Chemotactic Peptide-Induced Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase Activation in Transgenic COS-phox Cells
Authors: He Rong , Nanamori Masakatsu , Sang Hairong , Yin Hong , Dinauer Mary C. , Ye Richard D.
Published: April 2014

A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91phox, p22phox, p67phox, and p47phox, which respond to phorbol ester and arachidonic acid with O⨪2 production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCδ, but not PKCα, -βII, and -ζ, is necessary for the COS-phox cells to respond to fMLP. A role of PKCδ in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCδ translocation and the sensitivity of fMLP-induced O⨪2 production to rottlerin, a PKCδ-selective inhibitor. Optimal reconstitution also requires phospholipase C-β2 and PI3K-γ. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40phox potentiated fMLP-induced O⨪2 production and raised the level of O⨪2 in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O⨪2 production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.
Demonstration of the Involvement of Outer Surface Protein E Coiled Coil Structural Domains and Higher Order Structural Elements in the Binding of Infection-Induced Antibody and the Complement-Regulatory Protein, Factor H
Authors: McDowell John V. , Wolfgang Jill , Senty Lauren , Sundy Christina M. , Noto Michael J. , Marconi Richard T.
Published: April 2014

Factor H (fH) is an important regulator of the alternative complement cascade. Several human pathogens have been shown to bind fH to their surface, a process that facilitates immune evasion or cell to cell interaction. Among the pathogens that bind fH are some Borrelia species associated with Lyme disease and relapsing fever. The fH-binding proteins of the Lyme spirochetes form two classes (I and II). In Borrelia burgdorferi B31MI, class I includes the outer surface protein E (OspE) paralogs, L39, N38, and P38, whereas the class II group includes A68 and additional proteins that have not yet been identified. To identify the OspE determinants involved in fH and OspE-targeting infection-induced Ab (iAb) binding, deletion, random, and site-directed mutagenesis of L39 were performed. Mutations in several different regions of L39 abolished fH and or iAb binding, indicating that separable domains and residues of OspE are required for ligand binding. Some of the mutants that lost the ability to bind fH, iAb, or both had only a single amino acid change. Site-directed mutagenesis of three putative coiled coil motifs of OspE revealed that these higher order structures are required for fH binding but not for iAb binding. The data presented within demonstrate that the binding of fH and iAb to the OspE protein is mediated by higher order structures and protein conformation. These studies advance our understanding of fH binding as a virulence mechanism and facilitate ongoing efforts to use fH-binding proteins in the development of microbial vaccines.
Herpesvirus-Specific CD8 T Cell Immunity in Old Age: Cytomegalovirus Impairs the Response to a Coresident EBV Infection
Authors: Khan Naeem , Hislop Andrew , Gudgeon Nancy , Cobbold Mark , Khanna Rajiv , Nayak Laxman , Rickinson Alan B. , Moss Paul A. H.
Published: April 2014

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.
IL-27 Signaling Compromises Control of Bacterial Growth in Mycobacteria-Infected Mice
Authors: Pearl John E. , Khader Shabaana A. , Solache Alejandra , Gilmartin Leigh , Ghilardi Nico , deSauvage Fred , Cooper Andrea M.
Published: April 2014

Resistance to tuberculosis (TB) is dependent on the induction of Ag-specific CD4 Th1 T cells capable of expressing IFN-γ. Generation of these T cells is dependent upon IL-12p70, yet other cytokines have also been implicated in this process. One such cytokine, IL-27, augments differentiation of naive T cells toward an IFN-γ-producing phenotype by up-regulating the transcription factor T-bet and promoting expression of the IL-12Rβ2 chain allowing T cells to respond to IL-12p70. We show that the components of IL-27 are induced during TB and that the absence of IL-27 signaling results in an altered disease profile. In the absence of the IL-27R, there is reduced bacterial burden and an increased lymphocytic character to the TB granuloma. Although the number of Ag-specific CD4 IFN-γ-producing cells is unaffected by the absence of the IL-27R, there is a significant decrease in the level of mRNA for IFN-γ and T-bet within the lungs of infected IL-27R−/− mice. Ag-specific CD4 T cells in the lungs of IL-27R−/− also produce less IFN-γ protein per cell. The data show that expression of IL-27 during TB is detrimental to the control of bacteria and that although it does not affect the number of cells capable of producing IFN-γ it does reduce the ability of CD4 T cells to produce large amounts of IFN-γ. Because IFN-γ is detrimental to the survival of effector T cells, we hypothesize that the reduced IFN-γ within the IL-27R−/− lung is responsible for the increased accumulation of lymphocytes within the mycobacterial granuloma.
HIV-1-Induced Migration of Monocyte-Derived Dendritic Cells Is Associated with Differential Activation of MAPK Pathways
Authors: Wilflingseder Doris , Müllauer Brigitte , Schramek Herbert , Banki Zoltan , Pruenster Monika , Dierich Manfred P. , Stoiber Heribert
Published: April 2014

From the site of transmission at mucosal surfaces, HIV is thought to be transported by DCs to lymphoid tissues. To initiate migration, HIV needs to activate DCs. This activation, reflected by intra- and extracellular changes in cell phenotype, is investigated in the present study. In two-thirds of the donors, R5- and X4-tropic HIV-1 strains induced partial up-regulation of DC activation markers such as CD83 and CD86. In addition, CCR7 expression was increased. HIV-1 initiated a transient phosphorylation of p44/p42 ERK1/2 in iDCs, whereas p38 MAPK was activated in both iDCs and mDCs. Up-regulation of CD83 and CD86 on DCs was blocked when cells were incubated with specific p38 MAPK inhibitors before HIV-1-addition. CCR7 expression induced by HIV-1 was sufficient to initiate migration of DCs in the presence of secondary lymphoid tissue chemokine (CCL21) and MIP-3β (CCL19). Preincubation of DCs with a p38 MAPK inhibitor blocked CCR7-dependent DC migration. Migrating DCs were able to induce infection of autologous unstimulated PBLs in the Transwell system. These data indicate that HIV-1 triggers a cell-specific signaling machinery, thereby manipulating DCs to migrate along a chemokine gradient, which results in productive infection of nonstimulated CD4+ cells.
The Virulence Function of Streptococcus pneumoniae Surface Protein A Involves Inhibition of Complement Activation and Impairment of Complement Receptor-Mediated Protection
Authors: Ren Bing , McCrory Mark A. , Pass Christina , Bullard Daniel C. , Ballantyne Christie M. , Xu Yuanyuan , Briles David E. , Szalai Alexander J.
Published: April 2014

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2−/−), CR3 (CR3−/−), or CR4 (CR4−/−) were challenged with WU2, a PspA+ capsular serotype 3 pneumococcus, and its PspA− mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD−/−), LFA-1-deficient (LFA-1−/−), and CD18-deficient (CD18−/−) mice. We found that FD−/−, CR3−/−, and CR4−/− mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA− pneumococci were virulent only in FD−/− and CR1/2−/− mice. Normal mouse serum supported more C3 deposition on pneumococci than FD−/− serum, and more iC3b was deposited onto the PspA− than the PspA+ strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.
O-Acetylation of Cryptococcal Capsular Glucuronoxylomannan Is Essential for Interference with Neutrophil Migration
Authors: Ellerbroek Pauline M. , Lefeber Dirk J. , van Veghel Richard , Scharringa Jelle , Brouwer Ellen , Gerwig Gerrit J. , Janbon Guilhem , Hoepelman Andy I. M. , Coenjaerts Frank E. J.
Published: April 2014

The capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans has been shown to interfere with neutrophil migration. Although several receptors have been implied to mediate this process, the structural perspectives are unknown. Here, we assess the contribution of 6-O-acetylation and xylose substitution of the (1→3)-α-d-mannan backbone of GXM, the variable structural features of GXM, to the interference with neutrophil migration. We compare chemically deacetylated GXM and acetyl- or xylose-deficient GXM from genetically modified strains with wild-type GXM in their ability to inhibit the different phases of neutrophil migration. Additionally, we verify the effects of de-O-acetylation on neutrophil migration in vivo. De-O-acetylation caused a dramatic reduction of the inhibitory capacity of GXM in the in vitro assays for neutrophil chemokinesis, rolling on E-selectin and firm adhesion to endothelium. Genetic removal of xylose only marginally reduced the ability of GXM to reduce firm adhesion. In vivo, chemical deacetylation of GXM significantly reduced its ability to interfere with neutrophil recruitment in a model of myocardial ischemia (65% reduction vs a nonsignificant reduction in tissue myeloperoxidase, respectively). Our findings indicate that 6-O-acetylated mannose of GXM is a crucial motive for the inhibition of neutrophil recruitment.
In Vivo and In Vitro Roles of IL-21 in Inflammation
Authors: Pelletier Martin , Bouchard Amélie , Girard Denis
Published: April 2014

IL-21 is a cytokine known to mediate its biological action via the IL-21R, composed of a specific chain, IL-21Rα, and the common γ-chain (CD132). Recent data suggest that IL-21 possesses proinflammatory properties. However, there is no clear evidence that IL-21 induces inflammation in vivo and, curiously, the interaction between IL-21 and neutrophils has never been investigated, despite the fact that these cells express CD132 and respond to other CD132-dependent cytokines involved in inflammatory disorders. Using the murine air pouch model, we found that IL-21 induced inflammation in vivo, based on recruitment of neutrophil and monocyte populations. In contrast to LPS, administration of IL-21 into the air pouch did not significantly increase the concentration of IL-6, CCL5, CCL3, and CXCL2. We demonstrated that HL-60 cells expressed IL-21Rα, which is down-regulated during their differentiation toward neutrophils, and that IL-21Rα is not detected in neutrophils. Concomitant with this, IL-21 induced Erk-1/2 phosphorylation in HL-60 cells, but not in neutrophils. To eliminate the possibility that IL-21 could activate neutrophils even in the absence of IL-21Rα, we demonstrated that IL-21 did not modulate several neutrophil functions. IL-21-induced Erk-1/2 phosphorylation was not associated with proliferation or differentiation of HL-60 toward neutrophils, monocytes, or macrophages. IL-21Rα was detected in human monocytes and monocyte-derived macrophages, but IL-21 increased CXCL8 production only in monocyte-derived macrophages. We conclude that IL-21 is a proinflammatory cytokine, but not a neutrophil agonist. We propose that IL-21 attracts neutrophils indirectly in vivo via a mechanism independent of IL-6, CCL3, CCL5, and CXCL2 production.
Nonredundant Function of Phosphodiesterases 4D and 4B in Neutrophil Recruitment to the Site of Inflammation
Authors: Ariga Miyako , Neitzert Barbara , Nakae Susumu , Mottin Genevieve , Bertrand Claude , Pruniaux Marie Pierre , Jin S.-L. Catherine , Conti Marco
Published: April 2014

Neutrophils have been implicated in the pathogenesis of many inflammatory lung diseases, including chronic obstructive pulmonary disease and asthma. With this study, we investigated how disruption of cAMP signaling impacts the function of neutrophil recruitment to the lung. Four genes code for type 4 phosphodiesterases (PDE4s), enzymes critical for regulation of cAMP levels and cell signaling. Ablation of two of these genes, PDE4B and PDE4D, but not PDE4A, has profound effects on neutrophil function. In a paradigm of mouse lung injury induced by endotoxin inhalation, the number of neutrophils recovered in the bronchoalveolar lavage was markedly decreased in PDE4D−/− and PDE4B−/− mice 4 and 24 h after exposure to LPS. Acute PDE4 inhibition with rolipram had additional inhibitory effects on neutrophil migration in PDE4B−/− and, to a lesser extent, PDE4D−/− mice. This decreased neutrophil recruitment occurred without major changes in chemokine accumulation in bronchoalveolar lavage, suggesting a dysfunction intrinsic to neutrophils. This hypothesis was confirmed by investigating the expression of adhesion molecules on the surface of neutrophils and chemotaxis in vitro. CD18 expression was decreased after ablation of both PDE4B and PDE4D, whereas CD11 expression was not significantly affected. Chemotaxis in response to KC and macrophage inflammatory protein-2 was markedly reduced in PDE4B−/− and PDE4D−/− neutrophils. The effect of PDE4 ablation on chemotaxis was comparable, but not additive, to the effects of acute PDE4 inhibition with rolipram. These data demonstrate that PDE4B and PDE4D play complementary, but not redundant, roles in the control of neutrophil function.
Adenine Nucleotides Inhibit Cytokine Generation by Human Mast Cells through a Gs-Coupled Receptor
Authors: Feng Chunli , Mery Amin G. , Beller Elizabeth M. , Favot Christa , Boyce Joshua A.
Published: April 2014

ATP and ADP activate functionally distinct G protein-coupled purinergic (P2Y) receptors. We determined the expression and function of adenine nucleotide-specific P2Y receptors on cord blood-derived human mast cells (hMCs). Human MCs expressed mRNA encoding the ADP-specific P2Y1, P2Y12, and P2Y13 receptors; the ATP/UTP-specific P2Y2 receptor; and the ATP-selective P2Y11 receptor. ADP (0.05–50 μM) induced calcium flux that was completely blocked by a P2Y1 receptor-selective antagonist and was not cross-desensitized by ATP. Low doses of ADP induced strong phosphorylation of ERK and p38 MAPKs; higher doses stimulated eicosanoid production and exocytosis. Although MAPK phosphorylation was blocked by a combination of P2Y1- and P2Y12-selective antagonists, neither interfered with secretion responses. Unexpectedly, both ADP and ATP inhibited the generation of TNF-α in response to the TLR2 ligand, peptidoglycan, and blocked the production of TNF-α, IL-8, and MIP-1β in response to leukotriene D4. These effects were mimicked by two ATP analogues, adenosine 5′-O-(3-thiotriphosphate) and 2′,3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate (BzATP), but not by adenosine. ADP, ATP, adenosine 5′-O-(3-thiotriphosphate), and 2′,3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate each induced cAMP accumulation, stimulated the phosphorylation of CREB, and up-regulated the expression of inducible cAMP early repressor, a CREB-dependent inhibitor of cytokine transcription. Human MCs thus express several ADP-selective P2Y receptors and at least one Gs-coupled ADP/ATP receptor. Nucleotides could therefore contribute to MC-dependent microvascular leakage in atherosclerosis, tissue injury, and innate immunity while simultaneously limiting the extent of subsequent inflammation by attenuating the generation of inducible cytokines by MCs.
IL-10 Released by Concomitant TLR2 Stimulation Blocks the Induction of a Subset of Th1 Cytokines That Are Specifically Induced by TLR4 or TLR3 in Human Dendritic Cells
Authors: Re Fabio , Strominger Jack L.
Published: April 2014

Recognition of microbial products through TLRs triggers the expression of several cytokines that regulate innate and adaptive immunity. Signaling by various TLRs is not equivalent and leads to differential gene induction. This study analyzed the responses of human dendritic cells (DCs) and PBMCs stimulated with agonists of TLR2, TLR3, TLR4, TLR5, and TLR7, first individually and then in combination. Several cytokines were equally induced by all TLR agonists, but four genes, IFN-β, IFN-γ-inducible protein 10 (IP-10), IL-12p35, and IL-15, showed a very restricted pattern of induction. Thus, each TLR appears to possess a distinctive ability to activate DCs or PBMCs, suggesting that TLR-mediated responses cannot be simply cataloged as resembling either TLR2 (MyD88 dependent) or TLR4 (MyD88 independent) and that other signaling modalities may exist. The analysis of DC and PBMC activation by combinations of TLR agonists revealed that TLR2 agonists are able to block the induction of IP-10, IL-12p35, and IFN-γ, but not IL-15 and IFN-β, by TLR3 and TLR4. TLR2 stimulation led to rapid release of IL-10 that is responsible for inhibition of IP-10 and IL-12p35 induction. Cross-talk between different TLRs may modify the primary responses of TLR to their agonist, adding a further level of complexity to the regulation of innate immunity.
Immunostimulatory DNA Reverses Established Allergen-Induced Airway Remodeling
Authors: Youn Cho Jae , Miller Marina , Baek Kwang Je , Han Ji Won , Nayar Jyothi , Lee Sook Young , McElwain Kirsti , McElwain Shauna , Raz Eyal , Broide David H.
Published: April 2014

To determine whether immunostimulatory sequences of DNA (ISS) can reverse established airway remodeling, mice that had developed airway remodeling following 3 mo of repetitive OVA challenges, were treated with ISS for 1–3 mo. Systemic administration of ISS to mice that had already developed established airway remodeling significantly reduced the degree of airway collagen deposition (assessed by lung collagen content, peribronchial trichrome staining, and immunostaining with anticollagen type III and type V Abs). ISS reduced bronchoalveolar lavage and lung levels of TGF-β1 and reduced the number of TGF-β1-positive eosinophils and TGF-β1-positive mononuclear cells recruited to the airway. In vitro studies demonstrated that ISS inhibited TGF-β1 expression by macrophages (RAW 264.7 cell line and bone marrow-derived macrophages). In addition, ISS significantly reduces lung levels of expression of the chemokine thymus- and activation-regulated chemokine, as well as the number of peribronchial CD4+ lymphocytes that express Th2 cytokines that promote peribronchial fibrosis. Overall, these studies demonstrate that ISS can reverse features of airway collagen deposition by reducing levels of lung TGF-β1, as well as by reducing levels of the chemokine thymus- and activation-regulated chemokine and the numbers of peribronchial CD4+ lymphocytes that drive the ongoing Th2 immune response.
Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-γ
Authors: Fulkerson Patricia C. , Zimmermann Nives , Hassman Lynn M. , Finkelman Fred D. , Rothenberg Marc E.
Published: April 2014

The expression of distinct chemokines within the asthmatic lung suggests that specific regulatory mechanisms may mediate various stages of asthmatic disease. Global transcript expression profiling was used to define the spectrum and kinetics of chemokine involvement in an experimental murine model of asthma. Seventeen chemokines were induced in the lungs of allergen-inoculated mice, as compared with saline-treated mice. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. Seven (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen-induced chemokines were induced early after allergen challenge and remained induced throughout the experimental period. Three chemokines (CXCL2, CCL3, and CCL17) were induced only during the early phase of the inflammatory response after the initial allergen challenge, while seven chemokines (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13) were increased only after a second allergen exposure. Unexpectedly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent, and many of the identified chemokines were overexpressed in STAT6-deficient mice, providing an explanation for the enhanced neutrophilic inflammation seen in these mice. Notably, IFN-γ and STAT1 were shown to contribute to the induction of two STAT6-independent chemokines, CXCL9 and CXCL10. Taken together, these results show that only a select panel of chemokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6; instead, many of the allergen-induced chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coordinate regulation by STAT1, STAT6, and IFN-γ.
Protective and Pathological Roles of Virus-Specific and Bystander CD8+ T Cells in Herpetic Stromal Keratitis
Authors: Banerjee Kaustuv , Biswas Partha Sarathi , Kumaraguru Udayasankar , Schoenberger Stephen P. , Rouse Barry T.
Published: April 2014

Herpetic stromal keratitis (HSK), resulting from corneal HSV-1 infection, represents a T cell-mediated immunopathologic lesion. In T cell transgenic mice on a SCID or RAG knockout background, the T cells mediating lesions are unreactive to viral Ags. In these bystander models, animals develop ocular lesions but are unable to control infection. Transfer of HSV-immune cells into a CD8+ T cell bystander model resulted in clearance of virus from eyes, animals survived, and lesions developed to greater severity. However, the adoptively transferred CD8+ T cells were not evident in lesions, although they were readily detectable in the lymphoid tissues as well as in the peripheral and CNS. Our results indicate that viral-induced tissue damage can be caused by bystander cells, but these fail to control infection. Immune CD8+ T cells trigger clearance of virus from the eye, but this appears to result by the T cells acting at sites distal to the cornea. A case is made that CD8+ T cell control is expressed in the trigeminal ganglion, serving to curtail a source of virus to the cornea.
Fas Death Receptor Signaling Represses Monocyte Numbers and Macrophage Activation In Vivo
Authors: Brown Nathaniel J. , Hutcheson Jack , Bickel Emily , Scatizzi John C. , Albee Lee D. , Haines G. Kenneth , Eslick Joy , Bradley Kathleen , Taricone Elsa , Perlman Harris
Published: April 2014

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1β and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.
Role of CD44 and Hyaluronan in Neutrophil Recruitment
Authors: Khan Adil I. , Kerfoot Steven M. , Heit Bryan , Liu Lixin , Andonegui Graciela , Ruffell Brian , Johnson Pauline , Kubes Paul
Published: April 2014

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44−/− and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44−/− mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44−/− mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44−/− mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44−/− mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.
Induction of IL-8 Release in Lung Cells via Activator Protein-1 by Recombinant Baculovirus Displaying Severe Acute Respiratory Syndrome-Coronavirus Spike Proteins: Identification of Two Functional Regions
Authors: Chang Ya-Jen , Liu Catherine Y.-Y. , Chiang Bor-Luen , Chao Yu-Chan , Chen Ching-Chow
Published: April 2014

The inflammatory response and the intracellular signaling pathway induced by severe acute respiratory syndrome (SARS)-coronavirus (CoV) were studied in lung epithelial cells and fibroblasts. SARS-CoV spike (S) protein-encoding plasmid induced activations of IL-8 promoter and AP-1, but not NF-κB in these cells. Mutation of the AP-1, not the κB site, abolished the SARS-CoV S protein-induced IL-8 promoter activity. IL-8 release was effectively induced by vAtEpGS688, a baculovirus exhibiting the aa 17–688 fragment of S protein, and this induction was attenuated by the angiotensin-converting enzyme 2 Ab. Recombinant baculovirus expressing different deletion and insertion fragments identified the functional region of S protein from aa 324–688 (particularly the N-terminal aa 324–488 and the C-terminal aa 609–688), which is responsible for IL-8 production. Activations of AP-1 DNA-protein binding and MAPKs after vAtEpGS688 transduction were demonstrated, and SARS-CoV S protein-induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades. These results suggested that the S protein of SARS-CoV could induce release of IL-8 in the lung cells via activations of MAPKs and AP-1. The identification of the functional domain for IL-8 release will provide for the drug design on targeting specific sequence domains of S protein responsible for initiating the inflammatory response.
Fas Ligand Is Required for Resolution of Granulomatous Experimental Autoimmune Thyroiditis
Authors: Wei Yongzhong , Chen Kemin , Sharp Gordon C. , Braley-Mullen Helen
Published: April 2014

We previously suggested that CD8+ T cells promoted resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) at least in part through regulation of Fas ligand (FasL) expression on thyroid epithelial cells. To directly evaluate the role of the Fas pathway in G-EAT resolution, Fas- and FasL-deficient mice on the NOD.H-2h4 background were used as recipients of activated G-EAT effector cells. When MTg-primed wild-type (WT) donor splenocytes were activated and transferred to WT recipients, thyroid lesions reached maximal severity on day 20 and resolved on day 50. Fas, FasL, and FLIP were up-regulated, and many apoptotic inflammatory cells were detected in recipient thyroids on day 20. Fas was predominantly expressed by inflammatory cells, and FasL and FLIP were mainly expressed by thyroid epithelial cells. After depletion of CD8+ T cells, G-EAT resolution was delayed, FLIP and FasL were predominantly expressed by inflammatory cells, and few inflammatory cells were apoptotic. When WT donor splenocytes were transferred to gld recipients, disease severity on day 20 was similar to that in WT recipients, but resolution was delayed. As in CD8-depleted WT recipients, there were few apoptotic inflammatory cells, and FLIP and FasL were expressed primarily by inflammatory cells. These results indicated that the expression of functional FasL in recipient mice was critical for G-EAT resolution. WT cells induced minimal disease in lpr recipients. This was presumably because donor cells were eliminated by the increased FasL on lpr recipient cells, because donor cells were not eliminated, and the mice developed G-EAT if lpr recipients were given anti-FasL mAb.
Selective Growth, In Vitro and In Vivo, of Individual T Cell Clones from Tumor-Infiltrating Lymphocytes Obtained from Patients with Melanoma
Authors: Zhou Juhua , Dudley Mark E. , Rosenberg Steven A. , Robbins Paul F.
Published: April 2014

In recent clinical trials in patients with metastatic melanoma, adoptive transfer of tumor-reactive lymphocytes mediated the regression of metastatic tumor deposits. To better understand the role of individual T cell clones in mediating tumor regression, a 5′ RACE technique was used to determine the distribution of TCR β-chain V region sequences expressed in the transferred cells as well as in tumor samples and circulating lymphocytes from melanoma patients following adoptive cell transfer. We found that dominant T cell clones were present in the in vitro-expanded and transferred tumor-infiltrating lymphocyte samples and certain T cell clones including the dominant T cell clones persisted at relatively high levels in the peripheral blood of the patients that demonstrated clinical responses to adoptive immunotherapy. However, these dominant clones were either undetected or present at a very low level in the resected tumor samples used for tumor-infiltrating lymphocyte generation. These data demonstrated that there was selective growth and survival, both in vitro and in vivo, of individual T cell clones from a relatively small number of T cells in the original tumor samples. These results suggest that the persistent T cell clones played an active role in mediating tumor regression and that 5′ RACE analysis may provide an important tool for the analysis of the role of individual T cell clones in mediating tumor regression. A similar analysis may also be useful for monitoring autoimmune responses.