bluuebunny/crossref_metadata_2025 huggingface dataset:
CTL-Associated Antigen-4 Ligation Induces Rapid T Cell Polarization That Depends on Phosphatidylinositol 3-Kinase, Vav-1, Cdc42, and Myosin Light Chain Kinase
Authors: Wei Bin , da Rocha Dias Silvy , Wang Hongyan , Rudd Christopher E.
Published: April 2014

CTLA-4 can negatively regulate cytokine production and proliferation, increase motility, and override the TCR-induced stop-signal needed for stable T cell-APC conjugation. Despite this, little is known regarding whether CTLA-4 can alter T cell morphology and the nature of the signaling events that could account for this event. In this study, we demonstrate that anti-CTLA-4 and CD3/CTLA-4 induce rapid T cell polarization (i.e., within 15–30 min) with increases in lamellipodia, filopodia, and uropod formation. This was observed with anti-CTLA-4 and CD80-Ig ligation of CTLA-4, but not with anti-CD3 alone, or anti-CD3/CD28 coligation. Polarization required PI3K, the guanine nucleotide exchange factor Vav1, the GTP-binding protein Cdc42, as well as myosin L chain kinase. By contrast, a key downstream target of PI3K, protein kinase B, as well as Rho kinase and RhoA, were not needed. Our results demonstrate that CTLA-4 is a potent activator T cell polarization needed for motility, and this process involves specific set of signaling proteins that might contribute to coreceptor regulation of T cell function.
Cutting Edge: T Cells Respond to Lipopolysaccharide Innately via TLR4 Signaling
Authors: Zanin-Zhorov Alexandra , Tal-Lapidot Guy , Cahalon Liora , Cohen-Sfady Michal , Pevsner-Fischer Meirav , Lider Ofer , Cohen Irun R.
Published: April 2014

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1α (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.
Localization and Characterization of the Mannose-Binding Lectin (MBL)-Associated-Serine Protease-2 Binding Site in Rat Ficolin-A: Equivalent Binding Sites within the Collagenous Domains of MBLs and Ficolins
Authors: Girija Umakhanth Venkatraman , Dodds Alister W. , Roscher Silke , Reid Kenneth B. M. , Wallis Russell
Published: April 2014

Ficolins and mannose-binding lectins (MBLs) are the first components of the lectin branch of the complement system. They comprise N-terminal collagen-like domains and C-terminal pathogen-recognition domains (fibrinogen-like domains in ficolins and C-type carbohydrate-recognition domains in MBLs), which target surface-exposed N-acetyl groups or mannose-like sugars on microbial cell walls. Binding leads to activation of MBL-associated serine protease-2 (MASP-2) to initiate complement activation and pathogen neutralization. Recent studies have shown that MASP-2 binds to a short segment of the collagen-like domain of MBL. However, the interaction between ficolins and MASP-2 is relatively poorly understood. In this study, we show that the MASP-2 binding site on rat ficolin-A is also located within the collagen-like domain and encompasses a conserved motif that is present in both MBLs and ficolins. Characterization of this motif using site-directed mutagenesis reveals that a lysine residue in the X position of the Gly-X-Y collagen repeat, Lys56 in ficolin-A, which is present in all ficolins and MBLs known to activate complement, is essential for MASP-2 binding. Adjacent residues also make important contributions to binding as well as to MASP activation probably by stabilizing the local collagen helix. Equivalent binding sites and comparable activation kinetics of MASP-2 suggest that complement activation by ficolins and MBLs proceeds by analogous mechanisms.
Identification of a Novel Lipid Raft-Targeting Motif in Src Homology 2-Containing Phosphatase 1
Authors: Sankarshanan Mohan , Ma Zhong , Iype Tessy , Lorenz Ulrike
Published: April 2014

The tyrosine phosphatase Src homology 2-containing phosphatase 1 (SHP-1) is a key negative regulator of TCR-mediated signaling. Previous studies have shown that in T cells a fraction of SHP-1 constitutively localizes to membrane microdomains, commonly referred to as lipid rafts. Although this localization of SHP-1 is required for its functional regulation of T cell activation events, how SHP-1 is targeted to the lipid rafts was unclear. In this study, we identify a novel, six-amino acid, lipid raft-targeting motif within the C terminus of SHP-1 based on several biochemical and functional observations. First, mutations of this motif in the context of full-length SHP-1 result in the loss of lipid raft localization of SHP-1. Second, this motif alone restores raft localization when fused to a mutant of SHP-1 (SHP-1 ΔC) that fails to localize to rafts. Third, a peptide encompassing the 6-mer motif directly binds to phospholipids whereas a mutation of this motif abolishes lipid binding. Fourth, whereas full-length SHP-1 potently inhibits TCR-induced tyrosine phosphorylation of specific proteins, expression of a SHP-1-carrying mutation within the 6-mer motif does not. Additionally, although SHP-1 ΔC was functionally inactive, the addition of the 6-mer motif restored its functionality in inhibiting TCR-induced tyrosine phosphorylation. Finally, this 6-mer mediated targeting of SHP-1 lipid rafts was essential for the function of this phosphatase in regulating IL-2 production downstream of TCR. Taken together, these data define a novel 6-mer motif within SHP-1 that is necessary and sufficient for lipid raft localization and for the function of SHP-1 as a negative regulator of TCR signaling.
Pseudomonas aeruginosa Delays Kupffer Cell Death via Stabilization of the X-Chromosome-Linked Inhibitor of Apoptosis Protein
Authors: Ashare Alix , Monick Martha M. , Nymon Amanda B. , Morrison John M. , Noble Matthew , Powers Linda S. , Yarovinsky Timur O. , Yahr Timothy L. , Hunninghake Gary W.
Published: April 2014

Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-α. We hypothesized that early during infection, Kupffer cells are protected against TNF-α-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-α, or both. At early time points, TNF-α induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-α-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-α-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-α-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-α-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-α-induced Kupffer cell death via stabilization of XIAP.
Molecular Characterization of the Interaction between Porins of Neisseria gonorrhoeae and C4b-Binding Protein
Authors: Jarva Hanna , Ngampasutadol Jutamas , Ram Sanjay , Rice Peter A. , Villoutreix Bruno O. , Blom Anna M.
Published: April 2014

Neisseria gonorrhoeae, the causative agent of gonorrhea, is a natural infection only in humans. The resistance of N. gonorrhoeae to normal human serum killing correlates with porin (Por)-mediated binding to the complement inhibitor, C4b-binding protein (C4BP). The entire binding site for both porin molecules resides within complement control protein domain 1 (CCP1) of C4BP. Only human and chimpanzee C4BPs bind to Por1B-bearing gonococci, whereas only human C4BP binds to Por1A strains. We have now used these species-specific differences in C4BP binding to gonococci to map the porin binding sites on CCP1 of C4BP. A comparison between human and chimpanzee or rhesus C4BP CCP1 revealed differences at 4 and 12 amino acid positions, respectively. These amino acids were targeted in the construction of 13 recombinant human mutant C4BPs. Overall, amino acids T43, T45, and K24 individually and A12, M14, R22, and L34 together were important for binding to Por1A strains. Altering D15 (found in man) to N15 (found in rhesus) introduced a glycosylation site that blocked binding to Por1A gonococci. C4BP binding to Por1B strains required K24 and was partially shielded by additional glycosylation in the D15N mutant. Only those recombinant mutant C4BPs that bound to bacteria rescued them from 100% killing by rhesus serum, thereby providing a functional correlate for the binding studies and highlighting C4BP function in gonococcal serum resistance.
Inhibited Aortic Aneurysm Formation in BLT1-Deficient Mice
Authors: Ahluwalia Neil , Lin Alexander Y. , Tager Andrew M. , Pruitt Ivy E. , Anderson Thomas J. T. , Kristo Fjoralba , Shen Dongxiao , Cruz Anna R. , Aikawa Masanori , Luster Andrew D. , Gerszten Robert E.
Published: April 2014

Leukotriene B4 is a proinflammatory lipid mediator generated by the enzymes 5-lipoxygenase and leukotriene A4 hydrolase. Leukotriene B4 signals primarily through its high-affinity G protein-coupled receptor, BLT1, which is highly expressed on specific leukocyte subsets. Recent genetic studies in humans as well as knockout studies in mice have implicated the leukotriene synthesis pathway in several vascular pathologies. In this study, we tested the hypothesis that BLT1 is necessary for abdominal aortic aneurysm (AAA) formation, a major complication of atherosclerotic vascular disease. Chow-fed Apoe−/− and Apoe−/−/Blt1−/− mice were treated with a 4-wk infusion of angiotensin II (1000 ng/min/kg) beginning at 20 wk of age, in a well-established murine AAA model. We found a reduced incidence of AAA formation as well as concordant reductions in the maximum suprarenal/infrarenal diameter and total suprarenal/infrarenal area in the angiotensin II-treated Apoe−/−/Blt1−/− mice as compared with the Apoe−/− controls. Diminished AAA formation in BLT1-deficient mice was associated with significant reductions in mononuclear cell chemoattractants and leukocyte accumulation in the vessel wall, as well as striking reductions in the production of matrix metalloproteinases-2 and -9. Thus, we have shown that BLT1 contributes to the frequency and size of abdominal aortic aneurysms in mice and that BLT1 deletion in turn inhibits proinflammatory circuits and enzymes that modulate vessel wall integrity. These findings extend the role of BLT1 to a critical complication of vascular disease and underscore its potential as a target for intervention in modulating multiple pathologies related to atherosclerosis.
Differences in Expression, Affinity, and Function of Soluble (s)IL-4Rα and sIL-13Rα2 Suggest Opposite Effects on Allergic Responses
Authors: Khodoun Marat , Lewis Christina , Yang Jun-Qi , Orekov Tatyana , Potter Crystal , Wynn Thomas , Mentink-Kane Margaret , Khurana Hershey Gurjit K. , Wills-Karp Marsha , Finkelman Fred D.
Published: April 2014

IL-4 and IL-13 are each bound by soluble receptors (sRs) that block their activity. Both of these sRs (sIL-4Rα and sIL-13Rα2) are present in low nanogram per milliliter concentrations in the serum from unstimulated mice, but differences in affinity and half-life suggest differences in function. Serum IL-4/sIL-4Rα complexes rapidly dissociate, releasing active IL-4, whereas sIL-13Rα2 and IL-13 form a stable complex that has a considerably longer half-life than uncomplexed IL-13, sIL-13Rα2, IL-4, or sIL-4Rα. Approximately 25% of sIL-13Rα2 in serum is complexed to IL-13; this percentage and the absolute quantity of sIL-13Rα2 in serum increase considerably during a Th2 response. sIL-13Rα2 gene expression is up-regulated by both IL-4 and IL-13; the effect of IL-4 is totally IL-4Rα-dependent while the effect of IL-13 is partially IL-4Rα-independent. Inhalation of an IL-13/sIL-13Rα2 complex does not affect the expression of IL-13-inducible genes but increases the expression of two genes, Vnn1 and Pira-1, whose products activate APCs and promote neutrophilic inflammation. These observations suggest that sIL-4Rα predominantly sustains, increases, and diffuses the effects of IL-4, whereas sIL-13Rα2 limits the direct effects of IL-13 to the site of IL-13 production and forms a stable complex with IL-13 that may modify the quality and intensity of an allergic inflammatory response.
Select Plant Tannins Induce IL-2Rα Up-Regulation and Augment Cell Division in γδ T Cells
Authors: Holderness Jeff , Jackiw Larissa , Kimmel Emily , Kerns Hannah , Radke Miranda , Hedges Jodi F. , Petrie Charles , McCurley Patrick , Glee Pati M. , Palecanda Aiyappa , Jutila Mark A.
Published: April 2014

γδ T cells are innate immune cells that participate in host responses against many pathogens and cancers. Recently, phosphoantigen-based drugs, capable of expanding γδ T cells in vivo, entered clinical trials with the goal of enhancing innate immune system functions. Potential shortcomings of these drugs include the induction of nonresponsiveness upon repeated use and the expansion of only the Vδ2 subset of human γδ T cells. Vδ1 T cells, the major tissue subset, are unaffected by phosphoantigen agonists. Using FACS-based assays, we screened primary bovine cells for novel γδ T cell agonists with activities not encompassed by the current treatments in an effort to realize the full therapeutic potential of γδ T cells. We identified γδ T cell agonists derived from the condensed tannin fractions of Uncaria tomentosa (Cat’s Claw) and Malus domestica (apple). Based on superior potency, the apple extract was selected for detailed analyses on human cells. The apple extract was a potent agonist for both human Vδ1 and Vδ2 T cells and NK cells. Additionally, the extract greatly enhanced phosphoantigen-induced γδ T cell expansion. Our analyses suggest that a tannin-based drug may complement the phosphoantigen-based drugs, thereby enhancing the therapeutic potential of γδ T cells.
IL-1α and IL-1β Are Endogenous Mediators Linking Cell Injury to the Adaptive Alloimmune Response
Authors: Rao Deepak A. , Tracey Kevin J. , Pober Jordan S.
Published: April 2014

Preoperative or perioperative ischemic injury of allografts predisposes to graft arteriosclerosis, the major cause of late graft failure. We hypothesize that injured tissues release mediators that increase the production of pathogenic cytokines by alloreactive T cells. We find that freeze-thaw lysates of human endothelial cells (EC) increase both IFN-γ and IL-17 production by human CD4+ T cells activated by HLA-DR+ allogeneic EC. Immunoadsorption of high-mobility group box 1 protein (HMGB1) reduces this activity in the lysates by about one-third, and recombinant HMGB1 increases T cell cytokine production. HMGB1 acts by inducing IL-1β secretion from contaminating monocytes via TLR4 and CD14. Upon removal of contaminating monocytes, the remaining stimulatory activity of EC lysates is largely attributable to IL-1α. Recombinant IL-1 directly augments IFN-γ and IL-17 production by activated memory CD4+ T cells, which express IL-1R1. Furthermore, IL-1 increases the frequency of alloreactive memory CD4+ T cells that produce IL-17, but not those that produce IFN-γ, in secondary cultures. Our results suggest that IL-1, released by injured EC or by HMGB1-stimulated monocytes, is a key link between injury and enhanced alloimmunity, offering a new therapeutic target for preventing late graft failure.
Intrahepatic Lymphocyte Expression of Dipeptidyl Peptidase I-Processed Granzyme B and Perforin Induces Hepatocyte Expression of Serine Proteinase Inhibitor 6 (Serpinb9/SPI-6)
Authors: Stout-Delgado Heather W. , Getachew Yonas , Miller Bonnie C. , Thiele Dwain L.
Published: April 2014

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-α treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-α stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-α-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.
Molecular Mimics Can Induce Novel Self Peptide-Reactive CD4+ T Cell Clonotypes in Autoimmune Disease
Authors: Ercolini Anne M. , Miller Stephen D.
Published: April 2014

It has been postulated that infectious agents may precipitate autoimmune disease when T cell responses raised against the pathogen cross-react with self-peptides, a phenomenon known as molecular mimicry. However, there are very little data available characterizing the similarity between the repertoire of the cross-reactive self-specific T cell population compared with the pathogen-specific T cell repertoire. In this study, we use immunoscope analysis to identify the T cell populations induced upon priming SJL/J mice with a pathogen-derived mimic of the immunodominant encephalitogenic myelin peptide PLP139–151, which is contained within the protease IV protein of Haemophilus influenzae (HAE574–586). We describe an IFN-γ-producing Vβ19+ T cell population in HAE574–586-primed mice that appears to be the “public clonotype” as it expanded in response to peptide in all mice tested. Critically this Vβ19+ T cell population is not expanded in mice primed with the self-peptide PLP139–151, indicating that mimics can induce the expansion of new self-reactive populations not initially present in the periphery of a host. This is the first description of the use of immunoscope analysis to characterize the cross-reactive anti-self T cell response induced by a molecular mimic.
Strength of Stimulus and Clonal Competition Impact the Rate of Memory CD8 T Cell Differentiation
Authors: Sarkar Surojit , Teichgräber Volker , Kalia Vandana , Polley Antonio , Masopust David , Harrington Laurie E. , Ahmed Rafi , Wherry E. John
Published: April 2014

The developmental pathways of long-lived memory CD8 T cells and the lineage relationship between memory T cell subsets remain controversial. Although some studies indicate the two major memory T cell subsets, central memory T (TCM) and effector memory T (TEM), are related lineages, others suggest that these subsets arise and are maintained independently of one another. In this study, we have investigated this issue and examined the differentiation of memory CD8 T cell subsets by tracking the lineage relationships of both endogenous and TCR transgenic CD8 T cell responses after acute infection. Our data indicate that TCR transgenic as well as nontransgenic TEM differentiate into TCM in the absence of Ag. Moreover, the rate of memory CD8 T cell differentiation from TEM into the self-renewing and long-lived pool of TCM is influenced by signals received during priming, including Ag levels, clonal competition, and/or the duration of infection. Although some TEM appear to not progress to TCM, the vast majority of TCM are derived from TEM. Thus, long-lasting, Ag-independent CD8 T cell memory results from progressive differentiation of memory CD8 T cells, and the rate of memory T cell differentiation is governed by events occurring early during T cell priming.
Duration of Antigen Expression In Vivo following DNA Immunization Modifies the Magnitude, Contraction, and Secondary Responses of CD8+ T Lymphocytes
Authors: Hovav Avi-Hai , Panas Michael W. , Rahman Shaila , Sircar Piya , Gillard Geoffrey , Cayabyab Mark J. , Letvin Norman L.
Published: April 2014

The duration of Ag expression in vivo has been reported to have a minimal impact on both the magnitude and kinetics of contraction of a pathogen-induced CD8+ T cell response. In this study, we controlled the duration of Ag expression by excising the ear pinnae following intradermal ear pinnae DNA immunization. This resulted in decreased magnitude, accelerated contraction and differentiation, and surprisingly greater secondary CD8+ T cell responses. Furthermore, we found delayed and prolonged Ag presentation in the immunized mice; however, this presentation was considerably decreased when the depot Ag was eliminated. These findings suggest that the magnitude and the contraction phase of the CD8+ T cell response following intradermal DNA immunization is regulated by the duration rather than the initial exposure to Ag.
LIGHT Is Critical for IL-12 Production by Dendritic Cells, Optimal CD4+ Th1 Cell Response, and Resistance to Leishmania major
Authors: Xu Guilian , Liu Dong , Okwor Ifeoma , Wang Yang , Korner Heinrich , Kung Sam K. P. , Fu Yang-Xin , Uzonna Jude E.
Published: April 2014

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4+ Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT−/− mice were severely impaired in IL-12p40 production following IFN-γ and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT β receptor (LTβR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-γ production in vivo. In an infection model, injection of HVEM-Ig or LTβR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-γ production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTβR-Ig was also reproduced in LIGHT−/− → RAG1−/− chimeric mice. In contrast, L. major-infected LTβ−/− mice do not develop acute disease, suggesting that the effect of LTβR-Ig is not due to blockade of membrane LT (LTα1β2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-γ-producing CD4+ Th1 cells and its blockade results in severe susceptibility to Leishmania major.
Impaired Macrophage Function Underscores Susceptibility to Salmonella in Mice Lacking Irgm1 (LRG-47)
Authors: Henry Stanley C. , Daniell Xiaojou , Indaram Maanasa , Whitesides John F. , Sempowski Gregory D. , Howell David , Oliver Tim , Taylor Gregory A.
Published: April 2014

IRG proteins, or immunity-related GTPases (also known as p47 GTPases), are a group of IFN-regulated proteins that are highly expressed in response to infection. The proteins localize to intracellular membranes including vacuoles that contain pathogens in infected macrophages and other host cells. Current data indicate that the IRG protein Irgm1 (LRG-47) is critical for resistance to intracellular bacteria. This function is thought to be a consequence of regulating the survival of vacuolar bacteria in host cells. In the current work, the role of Irgm1 in controlling resistance to Salmonella typhimurium was explored to further define the mechanism through which the protein regulates host resistance. Irgm1-deficient mice displayed increased susceptibility to this bacterium that was reflected in increased bacterial loads in spleen and liver and decreased maturation of S. typhimurium granulomas. The mice also displayed an inability to concentrate macrophages at sites of bacterial deposition. In vitro, the ability of Irgm1-deficient macrophages to suppress intracellular growth of S. typhimurium was impaired. Furthermore, adhesion and motility of Irgm1-deficient macrophages after activation with IFN-γ was markedly decreased. Altered adhesion/motility of those cells was accompanied by changes in cell morphology, density of adhesion-associated proteins, and actin staining. Together, these data suggest that in addition to regulating the maturation of pathogen-containing vacuoles, Irgm1 plays a key role in regulating the adhesion and motility of activated macrophages.
HIV Impairs TNF-α Mediated Macrophage Apoptotic Response to Mycobacterium tuberculosis
Authors: Patel Naimish R. , Zhu Jinping , Tachado Souvenir D. , Zhang Jianmin , Wan Zhi , Saukkonen Jussi , Koziel Henry
Published: April 2014

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV+ persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-α. In contrast, apoptosis of differentiated HIV+ human U1 macrophages (HIV+ U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-α release, whereas apoptosis and TNF-α release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-α release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-κB nuclear translocation were reduced in HIV+ U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV+ persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-α release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV+ persons.
Src Tyrosine Kinases Mediate Activations of NF-κB and Integrin Signal during Lipopolysaccharide-Induced Acute Lung Injury
Authors: Lee Hui S. , Moon Changsuk , Lee Hye W. , Park Eun-Mi , Cho Min-Sun , Kang Jihee L.
Published: April 2014

Src tyrosine kinases (TKs) are signaling proteins involved in cell signaling pathways toward cytoskeletal, membrane and nuclear targets. In the present study, using a selective Src TK inhibitor, PP1, we investigated the roles of Src TKs in the key pulmonary responses, NF-κB activation, and integrin signaling during acute lung injury in BALB/C mice intratracheally treated with LPS. LPS resulted in c-Src phosphorylation in lung tissue and the phospho-c-Src was predominantly localized in recruited neutrophils and alveolar macrophages. PP1 inhibited LPS-induced increases in total protein content in bronchoalveolar lavage fluid, neutrophil recruitment, and increases in the production or activity of TNF-α and matrix metalloproteinase-9. PP1 also blocked LPS-induced NF-κB activation, and phosphorylation and degradation of IκB-α. The inhibition of NF-κB activation by PP1 correlated with a depression of LPS-induced integrin signaling, which included increases in the phosphorylations of integrin β3, and of the focal adhesion kinase (FAK) family members, FAK and Pyk2, in lung tissue, and reductions in the fibrinogen-binding activity of alveolar macrophages. Moreover, treatment with anti-αv, anti-β3, or Arg-Gly-Asp-Ser (RGDS), inhibited LPS-induced NF-κB activation. Taken together, our findings suggest that Src TKs play a critical role in LPS-induced activations of NF-κB and integrin (αvβ3) signaling during acute lung injury. Therefore, Src TK inhibition may provide a potential means of ameliorating inflammatory cascade-associated lung injury.
Involvement of Vitronectin in Lipopolysaccaride-Induced Acute Lung Injury
Authors: Tsuruta Yuko , Park Young-Jun , Siegal Gene P. , Liu Gang , Abraham Edward
Published: April 2014

Vitronectin is present in large concentrations in serum and participates in regulation of humoral responses, including coagulation, fibrinolysis, and complement activation. Because alterations in coagulation and fibrinolysis are common in acute lung injury, we examined the role of vitronectin in LPS-induced pulmonary inflammation. Vitronectin concentrations were significantly increased in the lungs after LPS administration. Neutrophil numbers and proinflammatory cytokine levels, including IL-1β, MIP-2, KC, and IL-6, were significantly reduced in bronchoalveolar lavage fluid from vitronectin-deficient (vitronectin−/−) mice, as compared with vitronectin+/+ mice, after LPS exposure. Similarly, LPS induced increases in lung edema, myeloperoxidase-concentrations, and pulmonary proinflammatory cytokine concentrations were significantly lower in vitronectin−/− mice. Vitronectin−/− neutrophils demonstrated decreased KC-induced chemotaxis as compared with neutrophils from vitronectin+/+ mice, and incubation of vitronectin+/+ neutrophils with vitronectin was associated with increased chemotaxis. Vitronectin−/− neutrophils consistently produced more TNF-α, MIP-2, and IL-1β after LPS exposure than did vitronectin+/+ neutrophils and also showed greater degradation of IκB-α and increased LPS-induced nuclear accumulation of NF-κB compared with vitronectin+/+ neutrophils. These findings provide a novel vitronectin-dependent mechanism contributing to the development of acute lung injury.
IFN-γ Produced by Human Papilloma Virus-18 E6-Specific CD4+ T Cells Predicts the Clinical Outcome after Surgery in Patients with High-Grade Cervical Lesions
Authors: Seresini Samantha , Origoni Massimo , Lillo Flavia , Caputo Luigi , Paganoni Anna Maria , Vantini Simone , Longhi Renato , Taccagni Gianluca , Ferrari Augusto , Doglioni Claudio , Secchi Piercesare , Protti Maria Pia
Published: April 2014

Cervical neoplastic lesions are associated with infection by high-risk human papilloma viruses (HPVs). HPV-16 and HPV-18 are the most common genotypes. It has been proposed that development of HPV-16-positive cervical lesions is associated with impaired CD4+ T cell immunity against early Ags. The aim of the study was to evaluate whether this impairment also applies to HPV-18. We investigated the presence and the quality of anti-HPV-18 E6 CD4+ T cell responses in the blood of 37 consecutive patients with high-grade cervical lesions, 25 normal donors, and 20 cord bloods. The immune infiltrate in the cervical lesions was also evaluated. The characteristics of the responses were correlated to the clinical outcome. We found that one or more HPV-18 E6 peptides, containing naturally processed epitopes, were able to induce a response in 40–50% of the patients, depending on the effector function tested. Importantly, these percentages rose to 80–100% when HPV-18-positive patients were considered. HPV-18 E6-specific CD4+ T cells produced mixed Th1/Th2 responses and statistical analysis of the cytokines produced revealed that the amount of IFN-γ released could predict infection persistence and/or disease relapse after surgery. Finally, we found that a higher number of infiltrating CD4+ and T-bet+ T cells in the lesions correlated with a favorable clinical outcome. Our results strongly suggest a relevant role for CD4+ T cells in the control of the HPV-18 compared with HPV-16 infections in patients with high-grade cervical lesions and identify an immunologic parameter potentially useful for patients’ stratification.
Invertebrate Immune Systems–Specific, Quasi-Specific, or Nonspecific?
Authors: Rowley Andrew F. , Powell Adam
Published: April 2014

Until recently, it was widely accepted that invertebrates fail to show a high degree of specificity and memory in their immune strategies. Recent reports have challenged this view such that our understanding of the capabilities of the invertebrate immune systems needs to be reassessed. This account critically reviews the available evidence that suggests the existence of a high degree of memory and specificity in some invertebrates and seeks mechanistic explanations of such observations. It is postulated that elevated levels of phagocytosis may be a partial explanation for this phenomenon.
CTLA-4 · FasL Induces Early Apoptosis of Activated T Cells by Interfering with Anti-Apoptotic Signals
Authors: Orbach Ariel , Rachmilewitz Jacob , Parnas Miram , Huang Jui-Han , Tykocinski Mark L. , Dranitzki-Elhalel Michael
Published: April 2014

The fusion protein CTLA-4 · FasL, a paradigmatic “trans signal converter protein”, can attach to APC surfaces and in effect convert B7-activating costimulator signals into inhibitory Fas receptor-generated signals. The present study investigates CTLA-4 · FasL’s mechanism of action. A combination of p27kip and proliferating cell nuclear Ag Western blot and propidium iodide flow cytometric analysis showed no CTLA-4 · FasL effect on cell cycle entry and progression, pointing away from the kind of classical anergy associated with CTLA-4 · Ig. Significantly, CTLA-4 · FasL elicited apoptosis (as detected by annexin-V/propidium iodide costaining) as early as 24 h after T cell activation, suggesting that some coordinate signaling might be capacitating the Fas receptor. Significantly, CTLA-4 · FasL, but not CTLA-4 · Ig, anti-Fas mAb, or the two in combination, abrogated the usual increase in expression of the anti-apototic protein, cFLIP. Furthermore, activation of caspases 8 and 3 were not affected by CTLA-4 · FasL. These findings suggest a model for CTLA-4 · FasL action wherein there is coordinate triggering of a death receptor and suppression of a proapoptotic protein.
Ex Vivo Expansion of CD4+CD25+FoxP3+ T Regulatory Cells Based on Synergy between IL-2 and 4-1BB Signaling
Authors: Elpek Kutlu G. , Yolcu Esma S. , Franke Deanna D. H. , Lacelle Chantale , Schabowsky Rich-Henry , Shirwan Haval
Published: April 2014

Naturally occurring CD4+CD25+FoxP3+ T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-β, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4+CD25− T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.
Ikaros Enforces the Costimulatory Requirement for IL2 Gene Expression and Is Required for Anergy Induction in CD4+ T Lymphocytes
Authors: Thomas Rajan M. , Chunder Neelanjana , Chen Chunxia , Umetsu Sarah E. , Winandy Susan , Wells Andrew D.
Published: April 2014

T cell activation results in dynamic remodeling of the chromatin at the IL2 promoter and induction of IL2 gene transcription. These processes are each dependent upon CD28 costimulation, but the molecular basis for this requirement is not clear. The IL2 promoter contains consensus-binding elements for Ikaros, a lymphocyte-specific zinc-finger DNA-binding protein that can regulate gene expression by recruiting chromatin-remodeling complexes. We find that native Ikaros in CD4+ T cells exhibits sequence-specific binding to these elements in vitro, and interacts with the endogenous IL2 promoter in vivo, in a manner dependent upon its DNA-binding domain. This binding has important consequences on the regulation of the IL2 gene, because CD4+ T cells with reduced Ikaros DNA-binding activity no longer require signals from the TCR or CD28 for histone acetylation at the endogenous IL2 promoter, and no longer require CD28 costimulation for expression of the IL2 gene. Furthermore, CD4+ T cells with reduced Ikaros activity are resistant to clonal anergy induced by TCR ligation in the absence of either CD28 or IL-2R signals. These results establish Ikaros as a transcriptional repressor of the IL2 gene that functions through modulation of chromatin structure and has an obligate role in the induction of anergy.
NKp30 Ligation Induces Rapid Activation of the Canonical NF-κB Pathway in NK Cells
Authors: Pandey Rahul , DeStephan Christine M. , Madge Lisa A. , May Michael J. , Orange Jordan S.
Published: April 2014

Studies of patients with congenital immunodeficiency due to mutation of the NF-κB essential modulator (NEMO) gene have demonstrated that NEMO integrity is required for NK cell cytotoxicity. Thus, we have studied the physiology of NF-κB activation in NK cells during the cytolytic program. In resting ex vivo human NK cells or cell lines, IκB was degraded after 10 min exposure to PMA and ionomycin, or TNF and was maximally degraded by 30 min. Ligation of several NK cell activation receptors including NKp30 induced a similar response and was blocked by pretreatment with the proteosome inhibitor MG132. There was no short-term effect on p100 processing, the signature of noncanonical NF-κB activation. NK cell IκB degradation corresponded to increases in nuclear NF-κB as detected by EMSA. Supershift of stimulated NK cells and fluorescence microscopy of individual NK cells in cytolytic conjugates demonstrated that the p65/p50 heterodimer was the primary NF-κB used. NF-κB function was evaluated in NK92 cells transduced with a κB GFP reporter, and their conjugation with K562 cells or ligation of NKp30 ligation resulted in rapid GFP accumulation. The latter was prevented by the Syk inhibitor piceatannol. Thus, NK cell activation signaling specifically induces transcriptional activation and synthesis of new NF-κB dependent proteins during the initiation of cytotoxicity.
Differential Development of Murine Dendritic Cells by GM-CSF versus Flt3 Ligand Has Implications for Inflammation and Trafficking
Authors: Xu Yuekang , Zhan Yifan , Lew Andrew M. , Naik Shalin H. , Kershaw Michael H.
Published: April 2014

To gain ample numbers of dendritic cells (DCs) for investigation, or for immunotherapy, the culture of DC precursors from bone marrow in either GM-CSF and IL-4 (GM/IL4-DCs) or Flt3L (FL-DCs) has often been used. Despite their common use, the relationship of these culture-derived DCs to those in vivo, and their relative potential for use in immunotherapy, needs further elucidation. In this study we found that in contrast to FL-DCs, highly purified GM/IL4-DCs were larger and more granular, surface Mac-3+, and were comprised of two populations (CD24lowCD11bhigh and CD24highCD11blow). Functionally, although comparable in T cell activation, GM/IL4-DCs produced more inflammatory mediators including TNF-α, IL-10, CCL-2, and NO than FL-DCs upon TLR ligation. However, FL-DCs migrated more efficiently to draining lymph nodes after s.c. injection and produced a different profile of cytokines to GM/IL4-DCs. Developmentally, unlike GM/IL4-DCs, FL-DCs cannot be differentiated from CD11bhighLy6ChighLy6G− monocytes. Collectively, these data suggest that the GM/IL4-DCs are the equivalents of the TNF-α and inducible NO synthase producing DCs in vivo that emerge after inflammation whereas FL-DCs better represent the steady-state resident DCs. The differences between GM/IL4-DCs and FL-DCs have serious implications for DC-based immunotherapeutic strategies.
Soluble CD14 Discriminates Slight Structural Differences between Lipid As That Lead to Distinct Host Cell Activation
Authors: Asai Yasuyuki , Makimura Yutaka , Kawabata Atsushi , Ogawa Tomohiko
Published: April 2014

Soluble CD14 (sCD14) in serum is known to sensitize host cells to LPS. In the present study, the contributions of sCD14 and LPS-binding protein to a lipid A moiety from LPS preparations of periodontopathogenic Fusobacterium nucleatum sp. nucleatum were compared with that of Escherichia coli-type synthetic lipid A (compound 506). F. nucleatum lipid A was identified to be a hexa-acylated fatty acid composed of tetradecanoate (C14) and hexadecanoate (C16), similar to dodecanoate (C12) and C14 in compound 506. The two lipid A specimens exhibited nearly the same reactivity in Limulus amoebocyte lysate assays, though F. nucleatum lipid A showed a weaker lethal toxicity. Both lipid A specimens showed nearly the same activities toward host cells in the absence of FBS, though compound 506 exhibited much stronger activity in the presence of FBS, sCD14, or sCD14 together with LPS-binding protein. Furthermore, native PAGE/Western immunoblot assays demonstrated that F. nucleatum lipid A had a weaker binding to sCD14 as compared with compound 506. These results suggest that sCD14 is able to discriminate the slight structural differences between these lipid As, which causes their distinct host cell activation activities.
Differential Requirements by CD4+ and CD8+ T Cells for Soluble and Membrane TNF in Control of Francisella tularensis Live Vaccine Strain Intramacrophage Growth
Authors: Cowley Siobhán C. , Sedgwick Jonathon D. , Elkins Karen L.
Published: April 2014

During primary infection with intracellular bacteria, the membrane-associated form of TNF provides some TNF functions, but the relative contributions during memory responses are not well-characterized. In this study, we determined the role of T cell-derived secreted and membrane-bound TNF (memTNF) during adaptive immunity to Francisella tularensis live vaccine strain (LVS). Although transgenic mice expressing only the memTNF were more susceptible to primary LVS infection than wild-type (WT) mice, LVS-immune WT and memTNF mice both survived maximal lethal secondary Francisella challenge. Generation of CD44high memory T cells and clearance of bacteria were similar, although more IFN-γ and IL-12(p40) were produced by memTNF mice. To examine T cell function, we used an in vitro tissue coculture system that measures control of LVS intramacrophage growth by LVS-immune WT and memTNF-T cells. LVS-immune CD4+ and CD8+ T cells isolated from WT and memTNF mice exhibited comparable control of LVS growth in either normal or TNF-α knockout macrophages. Although the magnitude of CD4+ T cell-induced macrophage NO production clearly depended on TNF, control of LVS growth by both CD4+ and CD8+ T cells did not correlate with levels of nitrite. Importantly, intramacrophage LVS growth control by CD8+ T cells, but not CD4+ T cells, was almost entirely dependent on T cell-expressed TNF, and required stimulation through macrophage TNFRs. Collectively, these data demonstrate that T cell-expressed memTNF is necessary and sufficient for memory T cell responses to this intracellular pathogen, and is particularly important for intramacrophage control of bacterial growth by CD8+ T cells.
Secretory IgA Mediates Bacterial Translocation to Dendritic Cells in Mouse Peyer’s Patches with Restriction to Mucosal Compartment
Authors: Kadaoui Khalil A. , Corthésy Blaise
Published: April 2014

In addition to fulfilling its function of immune exclusion at mucosal surfaces, secretory IgA (SIgA) Ab exhibits the striking feature to adhere selectively to M cells in the mouse and human intestinal Peyer’s patches (PPs). Subsequent uptake drives the SIgA Ab to dendritic cells (DCs), which become partially activated. Using freshly isolated mouse DCs, we found that the interaction with SIgA was tissue and DC subtype dependent. Only DCs isolated from PPs and mesenteric lymph nodes interacted with the Ab. CD11c+CD11b+ DCs internalized SIgA, while CD11c+CD19+ DCs only bound SIgA on their surface, and no interaction occurred with CD11c+CD8α+ DCs. We next examined whether SIgA could deliver a sizeable cargo to PP DCs in vivo by administering SIgA-Shigella flexneri immune complexes into a mouse ligated intestinal loop containing a PP. We found that such immune complexes entered the PPs and were internalized by subepithelial dome PP DCs, in contrast to S. flexneri alone that did not penetrate the intestinal epithelium in mice. Dissemination of intraepithelial S. flexneri delivered as immune complexes was limited to PPs and mesenteric lymph nodes. We propose that preexisting SIgA Abs associated with microbes contribute to mucosal defense by eliciting responses that prevent overreaction while maintaining productive immunity.