bluuebunny/crossref_metadata_2025 huggingface dataset:
Mucosal Clearance of Capsule-Expressing Bacteria Requires Both TLR and Nucleotide-Binding Oligomerization Domain 1 Signaling
Authors: Zola Tracey A. , Lysenko Elena S. , Weiser Jeffrey N.
Published: April 2014

Expression of capsular polysaccharide by bacterial pathogens is associated with increased resistance to host clearance mechanisms, in particular by evading opsonization and uptake by professional phagocytes. The potential for rapid progression of disease caused by encapsulated bacteria points to the importance of innate immunity at the mucosal surface where infection is initiated. Using a murine model of nasopharyngeal colonization, host immune components that contribute to the mucosal clearance of capsule-expressing bacteria were investigated. Clearance of encapsulated Haemophilus influenzae (Hi) required both TLR and nucleotide-binding oligomerization domain (NOD) signaling pathways, whereas individual deficiencies in each of these signaling cascades did not affect clearance of nonencapsulated strains. Moreover, clearance of Hi-expressing capsular polysaccharide required the recruitment of neutrophils to the site of infection, and ex vivo phagocytic bacterial killing required expression of the NOD1 signaling pathway. Conversely, redundancies within these innate immune pathways of non-neutrophil cells were sufficient to promote mucosal clearance of nonencapsulated Hi. Our findings reveal a role for NOD1 in protection from encapsulated pathogens. In addition, this study provides an example of a microbial virulence determinant that alters the requirements for host signaling to provide effective protection.
Interaction between RANTES Promoter Variant and CCR5Δ32 Favors Recovery from Hepatitis B
Authors: Thio Chloe L. , Astemborski Jacquie , Thomas Rasmi , Mosbruger Timothy , Witt Mallory D. , Goedert James J. , Hoots Keith , Winkler Cherie , Thomas David L. , Carrington Mary
Published: April 2014

Recovery from acute hepatitis B virus (HBV) infection occurs in 95% of adult-acquired infections. A 32-bp deletion in CCR5 (CCR5Δ32), which encodes for a nonfunctional receptor, increases the likelihood of recovery. Using 181 subjects with persistent HBV infection and 316 who had recovered, we tested the hypothesis that an epistatic interaction between functional polymorphisms in RANTES (a CCR5 ligand) and CCR5 impacts recovery. Specific models designed to assess individual contributions of compound genotypes demonstrated that the only combination associated with recovery from an HBV infection was RANTES −403A with CCR5Δ32 (odds ratio 0.36, p = 0.02). Because the phenotypic consequence of −403A is reported to be higher levels of RANTES, we propose a model in which excess RANTES in combination with low CCR5 favors recovery from an HBV infection, which will require validation through functional testing.
Aging Mice Exhibit a Functional Defect in Mucosal Dendritic Cell Response against an Intracellular Pathogen
Authors: Moretto Magali M. , Lawlor Elizabeth M. , Khan Imtiaz A.
Published: April 2014

Down-regulation of the immune response in aging individuals puts this population at a potential risk against infectious agents. In-depth studies conducted in humans and mouse models have demonstrated that with increasing age, the T cell immune response against pathogens is compromised and response to vaccinations is subdued. In the present study, using a mouse model, we demonstrate that older animals exhibit greater susceptibility to Encephalitozoon cuniculi infection, and their ability to evoke an Ag-specific T cell response at the gut mucosal site is reduced. The dampening of T cell immunity was due to the defective priming by the dendritic cells (DC) isolated from the mucosal tissues of aging animals. When primed with DC from younger mice, T cells from older animals were able to exhibit an optimal Ag-specific response. The functional defect in DC from older mice can be attributed to a large extent to reduced IL-15 message in these cells, which can be reversed by addition of exogenous IL-15 to the cultures. IL-15 treatment led to optimal expression of costimulatory molecules (CD80 and CD86) on the surface of older DC and restored their ability to prime a T cell response against the pathogen. To our knowledge, this is the first report which demonstrates the inability of the DC population from aging animals to prime a robust T cell response against an infectious agent. Moreover, the observation that IL-15 treatment can reverse this defect has far-reaching implications in developing strategies to increase vaccination protocols for aging populations.
Dysregulation of CXCR3 Signaling due to CXCL10 Deficiency Impairs the Antiviral Response to Herpes Simplex Virus 1 Infection
Authors: Wuest Todd R. , Carr Daniel J. J.
Published: April 2014

The chemokine, CXCL10, chemotactic for NK cells, activated T cells, and dendritic cells is highly expressed during viral infections, including HSV-1. The importance of this chemokine to the control of HSV-1 infection was tested using mice deficient in CXCL10 (CXCL10−/−). Following corneal infection, HSV-1 viral titers were elevated in the nervous system of CXCL10−/− mice, which correlated with defects in leukocyte recruitment including dendritic cells, NK cells, and HSV-1-specific CD8+ T cells to the brain stem. In the absence of NK cells and HSV-1-specific CD8+ T cells in wild-type (WT) or CXCL10−/− mice, similar levels of virus were recovered in the nervous system, suggesting these cells are responsible for the observed defects in the control of viral replication in CXCL10−/− mice. Leukocyte mobilization was also compared between WT, CXCL10−/−, and mice deficient in the only known receptor for CXCL10, CXCR3 (CXCR3 −/−). NK cell mobilization was comparably reduced in both CXCL10−/− and CXCR3−/− mice relative to WT animals. However, the reduction in mobilization of HSV-1-specific CD8+ T cells in CXCL10−/− was not observed in CXCR3−/− mice following HSV-1 infection. The defect was not the result of an alternative receptor for CXCL10, as Ag-specific CD8+ T cell recruitment was not reduced in mice which were deficient in both CXCL10 and CXCR3. Thus, CXCL10 deficiency results in reduced mobilization of HSV-1-specific CD8+ T cells as a result of dysregulation of CXCR3 signaling.
Anti-Inflammatory Effects of Sphingosine Kinase Modulation in Inflammatory Arthritis
Authors: Lai Wen-Qi , Irwan Anastasia Windy , Goh Hong Heng , Howe Hwee Siew , Yu David T. , Valle-Oñate Rafael , McInnes Iain B. , Melendez Alirio J. , Leung Bernard P.
Published: April 2014

Sphingosine kinase (SphK) is a key enzyme in the sphingolipid metabolic pathway responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P). SphK/S1P play a critical role in angiogenesis, inflammation, and various pathologic conditions. Recently, S1P1 receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling via S1P1 enhances synoviocyte proliferation, COX-2 expression, and prostaglandin E2 production. Here, we examined the role of SphK/S1P in RA using a potent SphK inhibitor, N,N-dimethylsphingosine (DMS), and a molecular approach against one of its isoenzymes, SphK1. We observed that levels of S1P in the synovial fluid of RA patients were significantly higher than those of osteoarthritis patients. Additionally, DMS significantly reduced the levels of TNF-α, IL-6, IL-1β, MCP-1, and MMP-9 in cell-contact assays using both Jurkat-U937 cells and RA PBMCs. In a murine collagen-induced arthritis model, i.p. administration of DMS significantly inhibited disease severity and reduced articular inflammation and joint destruction. Treatment of DMS also down-regulated serum levels IL-6, TNF-α, IFN-γ, S1P, and IgG1 and IgG2a anti-collagen Ab. Furthermore, DMS-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Moreover, similar reduction in incidence and disease activity was observed in mice treated with SphK1 knock-down via small interfering RNA approach. Together, these results demonstrate SphK modulation may provide a novel approach in treating chronic autoimmune conditions such as RA by inhibiting the release of pro-inflammatory cytokines.
Infiltrated Neutrophils Acquire Novel Chemokine Receptor Expression and Chemokine Responsiveness in Chronic Inflammatory Lung Diseases
Authors: Hartl Dominik , Krauss-Etschmann Susanne , Koller Barbara , Hordijk Peter L. , Kuijpers Taco W. , Hoffmann Florian , Hector Andreas , Eber Ernst , Marcos Veronica , Bittmann Iris , Eickelberg Oliver , Griese Matthias , Roos Dirk
Published: April 2014

Various inflammatory diseases are characterized by tissue infiltration of neutrophils. Chemokines recruit and activate leukocytes, but neutrophils are traditionally known to be restricted in their chemokine receptor (CR) expression repertoire. Neutrophils undergo phenotypic and functional changes under inflammatory conditions, but the mechanisms regulating CR expression of infiltrated neutrophils at sites of chronic inflammation are poorly defined. Here we show that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis highly express CR on their surface that are absent or only marginally expressed on circulating neutrophils, i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4, as measured by flow cytometry, immunohistochemistry, and confocal microscopy. The induction of CR surface expression on infiltrated neutrophils was functionally relevant, because receptor activation by chemokine ligands ex vivo modulated neutrophil effector functions such as respiratory burst activity and bacterial killing. In vitro studies with isolated neutrophils demonstrated that the surface expression of CR was differentially induced in a cytokine-mediated, protein synthesis-dependent manner (CCR1, CCR3), through Toll-like (CXCR3) or NOD2 (CCR5) receptor engagement, through neutrophil apoptosis (CCR5, CXCR4), and/or via mobilization of intracellular CD63+ granules (CXCR3). CR activation on infiltrated neutrophils may represent a key mechanism by which the local inflammatory microenvironment fine-tunes neutrophil effector functions in situ. Since the up-regulation of CR was exclusively found on infiltrated neutrophils at inflammatory sites in situ, the targeting of these G protein-coupled receptors may have the potential to site-specifically target neutrophilic inflammation.
A Novel Targeted Inhibitor of the Alternative Pathway of Complement and Its Therapeutic Application in Ischemia/Reperfusion Injury
Authors: Huang Yuxiang , Qiao Fei , Atkinson Carl , Holers V. Michael , Tomlinson Stephen
Published: April 2014

Bioavailability and therapeutic efficacy of soluble Crry, a mouse inhibitor of all complement activation pathways, is significantly enhanced when linked to a fragment of complement receptor 2 (CR2), a receptor that targets C3 activation products. In this study, we characterize alternative pathway-specific inhibitors consisting of a single or dimeric N-terminal region of mouse factor H (fH; short consensus repeats 1–5) linked to the same CR2 fragment (CR2-fH and CR2-fHfH). Both CR2-fH and CR2-fHfH were highly effective at inhibiting the alternative pathway in vitro and demonstrated a higher specific activity than CR2-Crry. CR2-fH was also more effective than endogenous serum fH in blocking target deposition of C3. Target binding and complement inhibitory activity of CR2-fH/CR2-fHfH was dependent on CR2- and C3-mediated interactions. The alternative pathway of complement plays a role in intestine ischemia/reperfusion injury. However, serum fH fails to provide protection against intestine ischemia/reperfusion injury although it can bind to and provide cell surfaces with protection from complement and is present in plasma at a high concentration. In a mouse model, CR2-fH and CR2-fHfH provided complete protection from local (intestine) and remote (lung) injury. CR2-fH targeted to the site of local injury and greatly reduced levels of tissue C3 deposition. Thus, the targeting mechanism significantly enhances alternative pathway-specific complement inhibitory activity of the N-terminal domain of fH and has the potential to reduce side effects that may be associated with systemic complement blockade. The data further indicate alternative pathway dependence for local and remote injury following intestinal ischemia/reperfusion in a clinically relevant therapeutic paradigm.
In Vivo BLyS/BAFF Neutralization Ameliorates Islet-Directed Autoimmunity in Nonobese Diabetic Mice
Authors: Zekavat Ghazal , Rostami Susan Y. , Badkerhanian Armen , Parsons Ronald F. , Koeberlein Brigitte , Yu Ming , Ward Christopher D. , Migone Thi-Sau , Yu Liping , Eisenbarth George S. , Cancro Michael P. , Naji Ali , Noorchashm Hooman
Published: April 2014

B lymphocytes are required for the pathogenesis of autoimmune diabetes in NOD mice. Previous studies established that a lymphopenic transitional (TR) B cell compartment reduces the competitive constraint on the entry of newly emerging TR B cells into the splenic follicle (FO), thereby disrupting a peripheral negative selection checkpoint in NOD mice. Thus, development of clinically feasible immunotherapeutic approaches for restoration of appropriate negative selection is essential for the prevention of anti-islet autoimmunity. In this study we hypothesized that in vivo neutralization of the B lymphocyte stimulator (BLyS/BAFF) may enhance the stringency of TR→FO selection by increasing TR B cell competition for follicular entry in NOD mice. This study demonstrated that in vivo BLyS neutralization therapy leads to the depletion of follicular and marginal zone B lymphocytes. Long-term in vivo BLyS neutralization caused an increased TR:FO B cell ratio in the periphery indicating a relative resistance to follicular entry. Moreover, in vivo BLyS neutralization: 1) restored negative selection at the TR→FO checkpoint, 2) abrogated serum insulin autoantibodies, 3) reduced the severity of islet inflammation, 4) significantly reduced the incidence of spontaneous diabetes, 5) arrested the terminal stages of islet cell destruction, and 6) disrupted CD4 T cell activation in NOD mice. Overall, this study demonstrates the efficacy of B lymphocyte-directed therapy via in vivo BLyS neutralization for the prevention of autoimmune diabetes.
Differential Expression and Molecular Associations of Syk in Systemic Lupus Erythematosus T Cells
Authors: Krishnan Sandeep , Juang Yuang-Taung , Chowdhury Bhabadeb , Magilavy Abigail , Fisher Carolyn U. , Nguyen Hang , Nambiar Madhusoodana P. , Kyttaris Vasileios , Weinstein Arthur , Bahjat Rena , Pine Polly , Rus Violeta , Tsokos George C.
Published: April 2014

Diminished expression of TCR ζ and reciprocal up-regulation and association of FcRγ with the TCR/CD3 complex is a hallmark of systemic lupus erythematosus (SLE) T cells. In this study we explored whether differential molecular associations of the spleen tyrosine kinase Syk that preferentially binds to FcRγ contribute to pathological amplification of signals downstream of this “rewired TCR” in SLE. We detected higher amounts of Syk expression and activity in SLE compared with normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, phospholipase C-γ1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.
Cutting Edge: Critical Role for Mesothelial Cells in Necrosis-Induced Inflammation through the Recognition of IL-1α Released from Dying Cells
Authors: Eigenbrod Tatjana , Park Jong-Hwan , Harder Jürgen , Iwakura Yoichiro , Núñez Gabriel
Published: April 2014

Endogenous danger signals released from necrotic cells are thought to be sensed by phagocytes leading to secretion of IL-1α and neutrophilic recruitment. However, the mechanisms for IL-1α production and IL-1α-mediated sterile inflammation remain poorly understood. We report here that necrotic cell extracts elicited little secretion of CXCL1 and IL-6 from macrophages but robust production in mesothelial cells. The induction of CXCL1 as well as activation of NF-κB and MAPKs by cytosolic extracts required the presence of IL-1α in the necrotic cell. Conversely, expression of IL-1R and MyD88 but not IL-1α, RICK, TLR2, TLR4, TRIF, or inflammasome components in mesothelial cells was critical for the production of CXCL1. Furthermore, IL-1α was critical to induce the recruitment of neutrophils in the peritoneal cavity via CXCR2. These studies show that IL-1α is a key danger signal released from necrotic cells to trigger CXCL1 secretion and recruitment of neutrophils via IL-1R/MyD88 on neighboring mesothelial cells.
γ-Aminobutyric Acid Transporter 1 Negatively Regulates T Cell-Mediated Immune Responses and Ameliorates Autoimmune Inflammation in the CNS
Authors: Wang Ying , Feng Dechun , Liu Guoxiang , Luo Qingqiong , Xu Yan , Lin Shuting , Fei Jian , Xu Lingyun
Published: April 2014

γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter of the CNS, and GABA transporter 1 (GAT-1) is critical in maintaining a GABA reservoir and associated functions. The wide expression of GAT-1 in the CNS prompted us to explore its role in neuroimmunological disorders. In mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, we found that the expression levels of GAT-1 mRNA and protein in spinal cord were greatly suppressed as compared with those in naive mice and irrelevant Ag-immunized mice. Therefore, we induced EAE in GAT-1−/− mice and found that the disease was significantly aggravated and was accompanied by some nonclassic EAE signs. Mononuclear cells from GAT-1−/− mice with EAE showed much higher Ag-specific proliferative responses. Proinflammatory cytokine production in these mice was also greatly up-regulated. Further studies revealed that GAT-1 deficiency induced vigorous immune responses by enhancing IκB kinase phosphorylation and NF-κB-DNA binding activity, as well as strengthening the T-bet-STAT1 circuit signaling pathway. Finally, we found that GAT-1 was expressed only on activated T cells primed with Ags, but not on B cells or macrophages. These findings indicate that GAT-1 is a critical modulator in T cell-mediated immune responses and in EAE pathogenesis.
Inhibition of Tumor Growth by NK1.1+ Cells and CD8+ T Cells Activated by IL-15 through Receptor β/Common γ Signaling in trans
Authors: Rowley Jesse , Monie Archana , Hung Chien-Fu , Wu T.-C.
Published: April 2014

IL-15 is an important cytokine involved in the survival and function of CD8+ T cells and NK cells. IL-15 can be presented by IL-15Rα (IL-15RA) to bind with the shared IL-2/IL-15Rβ and common γ-chains, which activate signaling pathways on NK cells and CD8+ T cells. In the present study, we characterized the function of trans-presented IL-15 on NK cells and CD8+ T cells using TC-1 tumor cells transduced with a retrovirus encoding IL-15 linked to IL-15RA (IL-15/IL-15RA). We demonstrated that the expression of IL-15/IL-15RA on TC-1 cells led to increased percentages of tumor-infiltrating NK cells, NKT cells, and CD8+ T cells, resulting in the inhibition of tumor growth in challenged mice. Additionally, in vivo Ab depletion experiments demonstrated that NK1.1+ cells and CD8+ T cells were important in this inhibition of tumor growth. Furthermore, this accumulation of immune cells and inhibition of tumor growth was abolished by a single amino acid mutation in the common γ-chain binding site on IL-15. We also observed that IL-15/IL-15RA-transduced TC-1 cells led to the activation of STAT5 in NK and CD8+ T cells in trans, which was abolished in the mutated IL-15/IL-15RA-transduced TC-1 cells. Taken together, our data suggest that common γ-chain binding-dependent activation of the shared IL-15/IL-2Rβ/common γ signaling pathway may play an important role in the activation of NK cells and CD8+ T cells, resulting in IL-15/IL-15RA trans-presentation-mediated inhibition of tumor growth.
A Novel ICOS-Independent, but CD28- and SAP-Dependent, Pathway of T Cell-Dependent, Polysaccharide-Specific Humoral Immunity in Response to IntactStreptococcus pneumoniaeversus Pneumococcal Conjugate Vaccine
Authors: Chen Quanyi , Cannons Jennifer L. , Paton James C. , Akiba Hisaya , Schwartzberg Pamela L. , Snapper Clifford M.
Published: April 2014

Polysaccharide (PS)- and protein-specific murine IgG responses to intact Streptococcus pneumoniae (Pn) are both dependent on CD4+ T cell help, B7-dependent costimulation, and CD40/CD40 ligand interactions. However, the primary PS-specific, relative to protein-specific, IgG response terminates more rapidly, requires a shorter period of T cell help and B7-dependent costimulation, and fails to generate memory. In light of the critical role for ICOS/ICOS ligand interactions in sustaining T cell-dependent Ig responses and promoting germinal center reactions, we hypothesized that this interaction was nonessential for PS-specific IgG responses to Pn. We now demonstrate that ICOS−/−, relative to wild-type, mice elicit a normal PS-specific IgG isotype response to Pn, despite marked inhibition of both the primary and secondary IgG anti-protein (i.e., PspA, PspC, and PsaA) response. A blocking anti-ICOS ligand mAb injected during primary Pn immunization inhibits both the primary anti-protein response and the generation of protein-specific memory, but has no effect when injected during secondary immunization. In contrast to Pn, both PS- and protein-specific IgG responses to a pneumococcal conjugate vaccine are inhibited in ICOS−/− mice. ICOS−/− mice immunized with intact Pn or conjugate exhibit nearly complete abrogation in germinal center formation. Finally, although mice that lack the adaptor molecule SAP (SLAM-associated protein) resemble ICOS−/− mice (and can exhibit decreased ICOS expression), we observe that the PS-specific, as well as protein-specific, IgG responses to both Pn and conjugate are markedly defective in SAP−/− mice. These data define a novel T cell-, SAP-, and B7-dependent, but ICOS-independent, extrafollicular pathway of Ig induction.
Improved Efficacy of a Tolerizing DNA Vaccine for Reversal of Hyperglycemia through Enhancement of Gene Expression and Localization to Intracellular Sites
Authors: Solvason Nanette , Lou Ya-Ping , Peters Wendy , Evans Erica , Martinez Josefina , Ramirez Uriel , Ocampo Andrea , Yun Rui , Ahmad Saira , Liu Edwin , Yu Liping , Eisenbarth George , Leviten Michael , Steinman Lawrence , Garren Hideki
Published: April 2014

Insulin is a major target for the autoimmune-mediated destruction of pancreatic β cells during the pathogenesis of type I diabetes. A plasmid DNA vaccine encoding mouse proinsulin II reduced the incidence of diabetes in a mouse model of type I diabetes when administered to hyperglycemic (therapeutic mode) or normoglycemic (prophylactic mode) NOD mice. Therapeutic administration of proinsulin DNA was accompanied by a rapid decrease in the number of insulin-specific IFN-γ-producing T cells, whereas prophylactic treatment was accompanied by enhanced IFN-γ-secreting cells and a decrease in insulin autoantibodies. Adoptive transfer experiments demonstrated that the protection was not mediated by induction of CD25+/CD4+ T regulatory cells. The efficacy of the DNA vaccine was enhanced by increasing the level of expression of the encoded Ag, more frequent dosing, increasing dose level, and localization of the protein product to the intracellular compartment. The efficacy data presented in this study demonstrate that Ag-specific plasmid DNA therapy is a viable strategy for preventing progression of type I diabetes and defines critical parameters of the dosing regime that influences tolerance induction.
Sialic Acid Residues Are Essential for the Anaphylactic Activity of Murine IgG1 Antibodies
Authors: Silva Sandriana R. , Casabuono Adriana , Jacysyn Jacqueline F. , Favoretto Bruna C. , Fernandes Irene , Macedo Mahasti S. , Couto Alicia S. , Faquim-Mauro Eliana L.
Published: April 2014

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to FcγRIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules.
CCR6 Regulates the Migration of Inflammatory and Regulatory T Cells
Authors: Yamazaki Tomohide , Yang Xuexian O. , Chung Yeonseok , Fukunaga Atsushi , Nurieva Roza , Pappu Bhanu , Martin-Orozco Natalia , Kang Hong Soon , Ma Li , Panopoulos Athanasia D. , Craig Suzanne , Watowich Stephanie S. , Jetten Anton M. , Tian Qiang , Dong Chen
Published: April 2014

Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two T cell subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-β and requires two nuclear receptors, RORα and RORγ. Th17 cells also express the CCR6 ligand CCL20, which is induced synergistically by TGF-β and IL-6, which requires STAT3, RORγ and IL-21. Th17 cells, by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration.
Acute Pulmonary Lipopolysaccharide Tolerance Decreases TNF-α without Reducing Neutrophil Recruitment
Authors: Natarajan Sudha , Kim Jiyoun , Remick Daniel G.
Published: April 2014

Pulmonary LPS exposure plays a key role in exacerbation of lung diseases such as chronic obstructive pulmonary disease and asthma. However, little is known about the effects of repeated LPS exposure in the lung microenvironment. We have developed a novel murine model of pulmonary LPS tolerance induced by intratracheal (i.t.) administration of LPS. First, we show that pulmonary LPS exposure does not induce whole-body refractoriness to systemic LPS, because i.t. administration followed by i.p. administration did not decrease plasma TNF-α. However, a local refractory state can be induced with two i.t. LPS exposures. Pulmonary LPS tolerance was induced by i.t. administration of 100 ng LPS at time 0 and 48 h. Nontolerant mice received PBS at time 0 and LPS at 48 h. Bronchoalveolar lavage levels of TNF-α were significantly attenuated in tolerant mice vs nontolerant mice (1597 pg/ml vs 7261 pg/ml). TNF-α mRNA was significantly reduced in bronchoalveolar lavage cells (5-fold) and lung tissue (10-fold). No reduction was seen in neutrophil numbers in the bronchoalveolar lavage fluid, myeloperoxidase activity, or expression of neutrophil chemoattractants CXCL1 and CXCL2, reflecting the specificity of the response. The reduction in TNF-α was accompanied by a significant increase in soluble receptors, TNF-SRI (159 pg/ml vs 206 pg/ml) and TNF-SRII (1366 pg/m vs 2695 pg/ml). In conclusion, pulmonary LPS tolerance results in a specific reduction in TNF-α expression, while the neutrophilic response is unaffected. This response may be a mechanism to limit tissue damage by reducing TNF-α levels, while still maintaining the antimicrobial capacity of the lung.
Preferential Use of DH Reading Frame 2 Alters B Cell Development and Antigen-Specific Antibody Production
Authors: Schelonka Robert L. , Zemlin Michael , Kobayashi Ryoki , Ippolito Gregory C. , Zhuang Yingxin , Gartland G. Larry , Szalai Alex , Fujihashi Kohtaro , Rajewsky Klaus , Schroeder Harry W.
Published: April 2014

All jawed vertebrates limit use of DH reading frames (RFs) that are enriched for hydrophobic amino acids. In BALB/c mice, DFL16.1 RF2 encodes valine and isoleucine. To test whether increased use of RF2 affects B cell function, we examined B cell development and Ab production in mice with an IgH allele (ΔD-DμFS) limited to use of a single, frameshifted DFL61.1 gene segment. We compared the results of these studies to wild-type mice, as well as those previously obtained in mice limited to use of either a single normal DH or a single inverted DH that forces use of arginine in CDR-H3. All three of the mouse strains limited to a single DH produced fewer immature B cells than wild type. However, whereas mice limited to a single normal DH achieved normal B cell numbers in the periphery, mice forced to preferentially use RF2 had reduced numbers of mature B cells in the spleen and bone marrow, mirroring the pattern previously observed in mice enriched for charged CDR-H3s. There were two exceptions. B cells in the mice using RF2 normally populated the marginal zone and peritoneal cavity, whereas mice using inverted RF1 had increased numbers of marginal zone B cells and decreased numbers of B1a cells. When challenged with several T-dependent or T-independent Ags, Ag-specific Ab titers in the mice forced to use RF2 were altered. These findings indicate that B cell development and Ag-specific Ab production can be heavily influenced by the global amino acid content of the CDR-H3 repertoire.
Induction of HIV Transcription by Nef Involves Lck Activation and Protein Kinase Cθ Raft Recruitment Leading to Activation of ERK1/2 but Not NFκB
Authors: Witte Vanessa , Laffert Bernd , Gintschel Patricia , Krautkrämer Ellen , Blume Katja , Fackler Oliver T. , Baur Andreas S.
Published: April 2014

The Nef protein of HIV-1 is a key promoter of disease progression, owing to its dramatic yet ill-defined impact on viral replication. Previously, we have shown that Nef enhances embryonic ectodermal development Tat-mediated transcription in a manner depending on Lck and the cytoplasmic sequestration of the transcriptional repressor embryonic ectodermal development. In this study, we report that Lck is activated by Nef and targets protein kinase Cθ downstream, leading to the translocation of the kinase into membrane microdomains. Although microdomain-localized protein kinase Cθ is thought to induce the transcription factor NFκB, we unexpectedly failed to correlate Nef-induced signaling events with enhanced NFκB activity. Instead, we observed an increase in ERK MAPK activity. We conclude that Nef-mediated signaling cooperates with Nef-induced derepression and supports HIV transcription through an ERK MAPK-dependent, but NFκB-independent, pathway.
Reassessment of the Role of Mut S Homolog 5 in Ig Class Switch Recombination Shows Lack of Involvement in cis- and trans-Switching
Authors: Guikema Jeroen E. J. , Schrader Carol E. , Leus Niek G. J. , Ucher Anna , Linehan Erin K. , Werling Uwe , Edelmann Winfried , Stavnezer Janet
Published: April 2014

When B cells are activated after immunization or infection, they exchange the gene encoding the Ig H chain C region by class switch recombination (CSR). CSR generally occurs by an intrachromosomal deletional recombination within switch (S) region sequences. However, ∼10% of CSR events occur between chromosome homologs (trans- or interallele CSR), suggesting that the homologous chromosomes are aligned during CSR. Because the Mut S homolog 4 (Msh4) and Msh5 bind to Holliday junctions and are required for homologous recombination during meiosis in germ cells, we hypothesized these proteins might be involved in trans-chromosomal CSR (trans-CSR). Indeed, Msh4-Msh5 has recently been suggested to have a role in CSR. However, we find a large variety of alternative splice variants of Msh5 mRNA in splenic B cells rather than the full-length form found in testis. Most of these mRNAs are unlikely to be stable, suggesting that Msh5 might not be functional. Furthermore, we find that msh5 nullizygous B cells undergo CSR normally, have unaltered levels of trans-CSR, normal levels of DNA breaks in the Sμ region, and normal S-S junctions. We also show that the S-S junctions from cis- and trans-CSR events have similar lengths of junctional microhomology, suggesting trans-CSR occurs by nonhomologous end joining as does intrachromosome (cis)-CSR. From these data, we conclude that Msh5 does not participate in CSR.
Recognition of Toxoplasma gondii by TLR11 Prevents Parasite-Induced Immunopathology
Authors: Yarovinsky Felix , Hieny Sara , Sher Alan
Published: April 2014

TLRs are thought to play a critical role in self/non-self discrimination by sensing microbial infections and initiating both innate and adaptive immunity. In this study, we demonstrate that in the absence of TLR11, a major TLR involved in recognition of Toxoplasma gondii, infection with this protozoan parasite induces an abnormal immunopathological response consisting of pancreatic tissue destruction, fat necrosis, and systemic elevations in inflammatory reactants. We further show that this immunopathology is the result of non-TLR dependent activation of IFN-γ secretion by NK cells in response to the infection. These findings reveal that in addition to triggering host resistance to infection, TLR recognition can be critical for the prevention of pathogen-induced immune destruction of self tissue.
Trehalose Dimycolate Elicits Eosinophilic Skin Hypersensitivity in Mycobacteria-Infected Guinea Pigs
Authors: Otsuka Atsushi , Matsunaga Isamu , Komori Takaya , Tomita Kadusa , Toda Yoshinobu , Manabe Toshiaki , Miyachi Yoshiki , Sugita Masahiko
Published: April 2014

Delayed-type hypersensitivity represents high levels of protein Ag-specific adaptive immunity induced by mycobacterial infection, and can be monitored in the Ag-challenged skin. Besides protein Ags, recent evidence has suggested that a substantial immunity directed against glycolipid Ags is also elicited in response to mycobacterial infection, but skin hypersensitivity to this class of Ags has not been fully assessed. To address this issue directly, glycolipid-specific skin reactions were evaluated in guinea pigs infected with Mycobacterium avium complex (MAC). Significant skin induration was observed in MAC-infected, but not mock-infected, guinea pigs, following intradermal administration of a mixture of MAC-derived glycolipids. Surprisingly, this glycolipid-specific skin response involved up-regulated expression of IL-5 mRNA in situ and marked local infiltration of eosinophils. Challenge experiments with individual glycolipid components detected an outstanding capability for trehalose dimycolate (TDM), but not a structurally related glycolipid, glucose monomycolate, to elicit the skin response. T lymphocytes derived from the spleen of MAC-infected, but not uninfected, guinea pigs specifically responded to TDM in vitro by up-regulating IL-5 transcription, and this response was not blocked by Abs that reacted to the known guinea pig group 1 CD1 proteins. Finally, the eosinophilic skin hypersensitivity to TDM was also elicited in guinea pigs vaccinated with bacillus Calmette-Guerin, which contrasted sharply with the classical delayed-type hypersensitivity response to the purified protein derivative. Therefore, the TDM-elicited eosinophilic response defines a new form of hypersensitivity in mycobacterial infection, which may account for local infiltration of eosinophils often observed at the site of infection.
A Role for Connexin43 in Macrophage Phagocytosis and Host Survival after Bacterial Peritoneal Infection
Authors: Anand Rahul J. , Dai Shipan , Gribar Steven C. , Richardson Ward , Kohler Jeff W. , Hoffman Rosemary A. , Branca Maria F. , Li Jun , Shi Xiao-Hua , Sodhi Chhinder P. , Hackam David J.
Published: April 2014

The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.
West Nile Virus-Specific CD4 T Cells Exhibit Direct Antiviral Cytokine Secretion and Cytotoxicity and Are Sufficient for Antiviral Protection
Authors: Brien James D. , Uhrlaub Jennifer L. , Nikolich-Žugich Janko
Published: April 2014

CD4 T cells have been shown to be necessary for the prevention of encephalitis during West Nile virus (WNV) infection. However, the mechanisms used by Ag-specific CD4 T cells to protect mice from WNV encephalitis remain incompletely understood. Contrary to the belief that CD4 T cells are protective because they merely maintain the CD8 T cell response and improve Ab production, in this study we provide evidence for the direct antiviral activity of CD4 T cells that functions to protect the host from WNV encephalitis. In adoptive transfers, naive CD4 T cells protected a significant number of lethally infected RAG−/− mice, demonstrating the protective effect of CD4 T cells independent of B cells and CD8 T cells. To shed light on the mechanism of this protection, we defined the peptide specificities of the CD4 T cells responding to WNV infection in C57BL/6 (H-2b) mice, and used these peptides to characterize the in vivo function of antiviral CD4 T cells. WNV-specific CD4 T cells produced IFN-γ and IL-2, but also showed potential for in vivo and ex vivo cytotoxicity. Furthermore, peptide vaccination using CD4 epitopes conferred protection against lethal WNV infection in immunocompetent mice. These results demonstrate the role of direct effector function of Ag-specific CD4 T cells in preventing severe WNV disease.
Sequence Variations in 185/333 Messages from the Purple Sea Urchin Suggest Posttranscriptional Modifications to Increase Immune Diversity
Authors: Buckley Katherine M. , Terwilliger David P. , Smith L. Courtney
Published: April 2014

The 185/333 gene family is highly expressed in two subsets of immune cells in the purple sea urchin in response to immune challenges. The genes encode a surprisingly diverse set of transcripts, which is a function of the variable presence or absence of blocks of shared sequences, known as elements that generate element patterns. Diversity is also the result of a significant level of point mutations. Together, variable element patterns and single nucleotide polymorphisms result in many unique transcripts. The 185/333 genes only have two exons, with the variable element patterns encoded entirely within the second exon. The diversity of the gene family may be the result of frequent recombination among the 185/333 genes that generates a mosaic distribution of element sequences among the genes. A comparative analysis of the sequences for the genes and messages from individual sea urchins indicates that these two sequence sets have largely different nucleotide sequences and appear to use different element patterns. Furthermore, the nucleotide substitution patterns between genes and messages reveal a strong bias toward transitions, particularly cytidine to uridine conversions. These data are consistent with cytidine deaminase activity and may represent a novel form of immunological diversification in an invertebrate immune response system.
Adenosine 5′-Monophosphate-Activated Protein Kinase Promotes Macrophage Polarization to an Anti-Inflammatory Functional Phenotype
Authors: Sag Duygu , Carling David , Stout Robert D. , Suttles Jill
Published: April 2014

Herein, we demonstrate a role of AMP-activated protein kinase (AMPK) as a potent counterregulator of inflammatory signaling pathways in macrophages. Stimulation of macrophages with anti-inflammatory cytokines (i.e., IL-10 and TGFβ) resulted in the rapid phosphorylation/activation of AMPK, whereas stimulation of macrophages with a proinflammatory stimulus (LPS) resulted in AMPK dephosphorylation/inactivation. Inhibition of AMPKα expression by RNA interference dramatically increased the mRNA levels of LPS-induced TNF-α, IL-6, and cyclooxygenase-2. Likewise, expression of a dominant negative AMPKα1 in macrophages enhanced TNF-α and IL-6 protein synthesis in response to LPS stimulation, while diminishing the production of IL-10. In contrast, transfection of macrophages with a constitutively active form of AMPKα1 resulted in decreased LPS-induced TNF-α and IL-6 production, and heightened production of IL-10. In addition, we found that AMPK negatively regulated LPS-induced IκB-α degradation and positively regulated Akt activation, accompanied by inhibition of glycogen synthase kinase β and activation of CREB. Thus, AMPK directs signaling pathways in macrophages in a manner that suppresses proinflammatory responses and promotes macrophage polarization to an anti-inflammatory functional phenotype.
CD40 Mediates Retinal Inflammation and Neurovascular Degeneration
Authors: Portillo Jose-Andres C. , Van Grol Jennifer , Zheng Ling , Okenka Genevieve , Gentil Katrin , Garland Alejandra , Carlson Eric C. , Kern Timothy S. , Subauste Carlos S.
Published: April 2014

Retinopathies are major causes of visual impairment. We used a model of ischemic retinopathy to examine the role of CD40 in the pathogenesis of retinal injury. Retinal inflammation, loss of ganglion cells, and capillary degeneration were markedly attenuated in ischemic retinas of CD40−/− mice. Up-regulation of NOS2 and COX2 after retinal ischemia were blunted in CD40−/− mice. NOS2-COX-2 up-regulation in ischemic retinas from wild-type mice was at least in part explained by recruitment of NOS2+COX-2+ leukocytes. Up-regulation of KC/CXCL1 and ICAM-1 also required CD40. Retinal endothelial and Muller cells expressed CD40. Stimulation of these cells through CD40 caused ICAM-1 up-regulation and KC/CXCL1 production. Bone marrow transplant experiments revealed that leukocyte infiltration, ganglion cell loss, and up-regulation of proinflammatory molecules after retinal ischemia were dependent on CD40 expression in the retina and not peripheral blood leukocytes. These studies identified CD40 as a regulator of retinal inflammation and neurovascular degeneration. They support a model in which CD40 stimulation of endothelial and Muller cells triggers adhesion molecule up-regulation and chemokine production, promoting the recruitment of leukocytes that express NOS2/COX-2, molecules linked to neurovascular degeneration.
FOXP3 Induced by CD28/B7 Interaction Regulates CD25 and Anergic Phenotype in Human CD4+CD25− T Lymphocytes
Authors: Scottà Cristiano , Soligo Marzia , Camperio Cristina , Piccolella Enza
Published: April 2014

Among the signals necessary to generate CD4+CD25+FOXP3+ T cells from CD4+CD25−FOXP3− T cells, a pivotal role is played by CD28. However, in humans, it is not known whether CD28 signaling independently of TCR promotes forkhead box protein 3 (FOXP3) expression and regulates CD4+CD25+FOXP3+ T cell functions. To address this issue, starting from our previous experience, we analyzed the unique signals delivered by CD28 following stimulation by its natural ligand B7. Our results show that, in primary CD4+CD25− T cells, CD28 signals independent of TCR-mediated stimulatory pathways are sufficient to induce the transcription of FOXP3 in a small number of CD4+CD25− T cells committed to express FOXP3. These signals are dependent on CD28-derived PI3K/Akt pathways and resistant to cyclosporin A. In addition, we demonstrated that translated FOXP3 was recruited to CD25, Il-2, and Ctla4 target promoters. CD28-mediated FOXP3 expression was transient and correlated with CD25 expression. The presence of FOXP3 in CD28-activated CD4+CD25− T cells correlated with a transient unresponsiveness to antigenic stimuli. The addition of exogenous IL-2 did not influence either FOXP3 or CD25 expression but rescued CD28-activated T cells from apoptosis. Our results, demonstrating that FOXP3 expression driven solely by the CD28/B7 interaction inhibited T cell activation, support the role of CD28 in the regulation of peripheral tolerance and suggest a new mechanism through which it could occur.