bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous and Prostatic Steroid Binding Protein Peptide-Induced Autoimmune Prostatitis in the Nonobese Diabetic Mouse
Authors: Penna Giuseppe , Amuchastegui Susana , Cossetti Chiara , Aquilano Francesca , Mariani Roberto , Giarratana Nadia , De Carli Elena , Fibbi Benedetta , Adorini Luciano
Published: April 2014

Chronic nonbacterial prostatitis is a poorly defined syndrome of putative autoimmune origin. To further understand its pathogenesis, we have analyzed autoimmune prostatitis in the NOD mouse, a strain genetically prone to develop different organ-specific autoimmune diseases. Spontaneous development of autoimmune prostatitis in the NOD male, defined by lymphomonuclear cell infiltration in the prostate gland, is well-established by ∼20 wk of age and is stably maintained afterward. Disease development is indistinguishable in NOD and NOR mice, but is markedly delayed in IFN-γ-deficient NOD mice. A T cell response to the prostate-specific autoantigen prostatic steroid-binding protein (PSBP) can be detected in NOD males before development of prostate infiltration, indicating lack of tolerance to this self Ag. The intraprostatic inflammatory infiltrate is characterized by Th1-type CD4+ T cells, which are able to transfer autoimmune prostatitis into NOD.SCID recipients. We characterize here experimental autoimmune prostatitis, detected by intraprostatic infiltrate and PSBP-specific T cell responses, induced in 6- to 8-wk-old NOD males by immunization with synthetic peptides corresponding to the C1 subunit of PSBP. Three PSBP peptides induce in NOD mice vigorous T and B cell responses, paralleled by a marked lymphomononuclear cell infiltration in the prostate. Two of these peptides, PSBP21–40 and PSBP61–80, correspond to immunodominant self epitopes naturally processed in NOD mice after immunization with PSBP, whereas peptide PSBP91–111 represents a cryptic epitope. These model systems address pathogenetic mechanisms in autoimmune prostatitis and will facilitate testing and mechanistic analysis of therapeutic approaches in this condition.
Sphingosine 1-Phosphate-Mediated Trafficking of Pathogenic Th2 and Mast Cells for the Control of Food Allergy
Authors: Kurashima Yosuke , Kunisawa Jun , Higuchi Morio , Gohda Masashi , Ishikawa Izumi , Takayama Naoko , Shimizu Miki , Kiyono Hiroshi
Published: April 2014

Sphingosine 1-phosphate (S1P) has been proposed as a regulator of lymphocyte trafficking, but its role in mucosa-associated diseases, such as in food allergies, remains to be elucidated. To examine the role of S1P in allergic diseases in the intestine, we used a Th2 cell-mediated Ag-specific allergic diarrhea model and demonstrated that type 1 S1P receptor (S1P1) expression was preferentially associated with pathogenic CD4+ T cells for the development of allergic reactions. Consistent with this demonstration, treatment with FTY720, a modulator of the S1P1, prevented allergic diarrhea by inhibiting the migration of systemically primed pathogenic CD4+ T cells induced by oral challenge with allergen into the large intestine. In addition, FTY720 hampered mast cell infiltration into the large intestine, whereas eosinophil infiltration into the large intestine and total and allergen-specific serum IgE production were comparable between mock- and FTY720-treated groups. These results suggest that modulation of the S1P-mediated pathway to inhibit the migration of pathogenic CD4+ T cells and mast cells into the large intestine could be a novel strategy for preventing allergic diarrhea.
Adult Human Fibroblasts Are Potent Immunoregulatory Cells and Functionally Equivalent to Mesenchymal Stem Cells
Authors: Haniffa Muzlifah A. , Wang Xiao-Nong , Holtick Udo , Rae Michelle , Isaacs John D. , Dickinson Anne M. , Hilkens Catharien M. U. , Collin Matthew P.
Published: April 2014

Bone marrow mesenchymal stem cells (MSC) have potent immunosuppressive properties and have been advocated for therapeutic use in humans. The nature of their suppressive capacity is poorly understood but is said to be a primitive stem cell function. Demonstration that adult stromal cells such as fibroblasts (Fb) can modulate T cells would have important implications for immunoregulation and cellular therapy. In this report, we show that dermal Fb inhibit allogeneic T cell activation by autologously derived cutaneous APCs and other stimulators. Fb mediate suppression through soluble factors, but this is critically dependent on IFN-γ from activated T cells. IFN-γ induces IDO in Fb, and accelerated tryptophan metabolism is at least partly responsible for suppression of T cell proliferation. T cell suppression is reversible, and transient exposure to Fb during activation reprograms T cells, increasing IL-4 and IL-10 secretion upon restimulation. Increased Th2 polarization by stromal cells is associated with amelioration of pathological changes in a human model of graft-vs-host disease. Dermal Fb are highly clonogenic in vitro, suggesting that Fb-mediated immunosuppression is not due to outgrowth of rare MSC, although dermal Fb remain difficult to distinguish from MSC by phenotype or transdifferentiation capacity. These results suggest that immunosuppression is a general property of stromal cells and that dermal Fb may provide an alternative and accessible source of cellular therapy.
LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse
Authors: Graf Beth , Bushnell Timothy , Miller Jim
Published: April 2014

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.
Detection of KIR3DS1 on the Cell Surface of Peripheral Blood NK Cells Facilitates Identification of a Novel Null Allele and Assessment of KIR3DS1 Expression during HIV-1 Infection
Authors: Pascal Véronique , Yamada Eriko , Martin Maureen P. , Alter Galit , Altfeld Marcus , Metcalf Julia A. , Baseler Michael W. , Adelsberger Joseph W. , Carrington Mary , Anderson Stephen K. , McVicar Daniel W.
Published: April 2014

KIR3DL1 is a highly polymorphic killer cell Ig-like receptor gene with at least 23 alleles described, including its activating counterpart, KIR3DS1. Recently, the KIR3DS1 allele has been shown to slow progression to AIDS in individuals expressing HLA-Bw4 with isoleucine at position 80. However, due to the lack of a specific Ab, KIR3DS1 expression and function is not well characterized. In this study, we demonstrate KIR3DS1 expression on a substantial subset of peripheral natural killer cells through its recognition by the mAb Z27. The fidelity of this detection method was confirmed by analysis of KIR3DS1 transfectants and the identification of a novel KIR3DS1 null allele. Interestingly, KIR3DS1 is also expressed by a small proportion of CD56+ T cells. We show that ligation of KIR3DS1 by Z27 leads to NK cell IFN-γ production and degranulation as assessed by expression of CD107a. Furthermore, we document the persistence of KIR3DS1+ NK cells in HIV-1 viremic patients. The high frequency of KIR3DS1 expression, along with its ability to activate NK cells, and its maintenance during HIV-1 viremia are consistent with the epidemiological data suggesting a critical role for this receptor in controlling HIV-1 pathogenesis.
Modulatory Effects of 1,25-Dihydroxyvitamin D3 on Human B Cell Differentiation
Authors: Chen Sheng , Sims Gary P. , Chen Xiao Xiang , Gu Yue Ying , Chen Shunle , Lipsky Peter E.
Published: April 2014

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)2D3 levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)2D3 on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)2D3, although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1α-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)2D3 and/or activation. Importantly, 1,25(OH)2D3 up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)2D3 may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.
Functional Role for IκBNS in T Cell Cytokine Regulation As Revealed by Targeted Gene Disruption
Authors: Touma Maki , Antonini Valeria , Kumar Manoj , Osborn Stephanie L. , Bobenchik April M. , Keskin Derin B. , Connolly John E. , Grusby Michael J. , Reinherz Ellis L. , Clayton Linda K.
Published: April 2014

Triggering of the TCR by cognate peptide/MHC ligands induces expression of IκBNS, a member of the IκB family of NF-κB inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of IκBNS in TCR triggering, we created a targeted disruption of the IκBNS gene. Surprisingly, mice lacking IκBNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, IκBNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-γ than wild-type cells. Transfection analysis demonstrates that IκBNS and c-Rel individually increase IL-2 promoter activity. The effect of IκBNS on the IL-2 promoter, unlike c-Rel, is dependent on the NF-κB rather than the CD28RE site; mutation of the NF-κB site extinguishes the induction of transcription by IκBNS in transfectants and prevents association of IκBNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-γ-linked transcripts in IκBNS knockout T cells. Collectively, our findings demonstrate that IκBNS regulates production of IL-2 and other cytokines induced via “strong” TCR ligation.
Neonatal B Cells Suppress Innate Toll-Like Receptor Immune Responses and Modulate Alloimmunity
Authors: Walker Wendy E. , Goldstein Daniel R.
Published: April 2014

It has been known for decades that neonates are susceptible to transplant tolerance, but the immunological mechanisms involved remain to be fully elucidated. Recent evidence indicates that the maturation state of DCs responding to an allograft may have a profound impact on whether immunity or tolerance ensues. Given that TLR activation is a key process leading to DC maturation, we hypothesized that DCs from neonates have defective TLR immune responses. Contrary to our hypothesis, we found that murine neonatal DCs demonstrated enhanced TLR responses in comparison to adult counterparts in vitro. However, we found that neonatal B cells possess unique immunoregulatory functions as they impaired DC responses to TLR activation in an IL-10-dependent fashion. Functionally, we demonstrated that TLR-activated neonatal, but not adult, B cells impaired Th1, but not Th2, T cell alloimmune responses in vitro and in vivo, in models of alloimmune priming and allotransplantation. We conclude that neonatal B cells possess unique immunoregulatory properties that inhibit DC function and modulate alloimmunity in our murine experimental systems.
Coordinated Involvement of Mast Cells and T Cells in Allergic Mucosal Inflammation: Critical Role of the CC Chemokine Ligand 1:CCR8 Axis
Authors: Gonzalo Jose-Angel , Qiu Yubin , Lora Jose M. , Al-Garawi Amal , Villeval Jean-Luc , Boyce Joshua A. , Martinez-A Carlos , Marquez Gabriel , Goya Iñigo , Hamid Qutayba , Fraser Christopher C. , Picarella Dominic , Cote-Sierra Javier , Hodge Martin R. , Gutierrez-Ramos Jose-Carlos , Kolbeck Roland , Coyle Anthony J.
Published: April 2014

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by ∼70% of CD4+ T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4+ T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4+ effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.
NF-κB p50/p65 Affects the Frequency of Ly49 Gene Expression by NK Cells
Authors: Pascal Véronique , Nathan Neera R. , Claudio Estefania , Siebenlist Ulrich , Anderson Stephen K.
Published: April 2014

In mice, acquisition of Ly49 receptors characterizes one of the developmental stages of NK cells. We previously described a novel Ly49 promoter, Pro1, involved in Ly49 gene regulation in immature NK cells. Pro1 transcriptional activity requires a NF-κB binding site; however, only NF-κB/p50 binding to this element was observed. Cotransfection of NF-κB/p65 with Ly49g Pro1 in LNK cells induced a decrease in the transcriptional activity of the core promoter. Moreover, decreasing NF-κB/p65 protein expression by RNA interference increases Pro1 transcriptional activity. A high rate of NF-κB/p65 degradation in LNK cells correlates with Pro1 activity, since treatment with the proteasome inhibitor MG132 increased levels of NF-κB/p65 protein and decreased Pro1 activity. In addition, analysis of the Ly49 repertoire in NF-κB/p50 null mice reveals a decrease in the proportion of NK cells expressing a given Ly49 molecule. The defect in Ly49 expression is observed in the bone marrow and the spleen with a similar altered pattern of developmental stages in each tissue. The frequency of Ly49 expression in NF-κB/p52 null mice is slightly increased, indicating the specific role of NF-κB/p50 in Ly49 gene activation. These results suggest that NF-κB p50/p65 plays a major role in the initiation of Ly49 gene expression in NK cells.
The Related Adaptors, Adaptor in Lymphocytes of Unknown Function X and Rlk/Itk-Binding Protein, Have Nonredundant Functions in Lymphocytes
Authors: Perchonock Claire E. , Pajerowski Anthony G. , Nguyen Chau , Shapiro Michael J. , Shapiro Virginia Smith
Published: April 2014

Adaptors play a critical role in regulating signaling pathways that control lymphocyte development and activation. Adaptor in lymphocytes of unknown function X (ALX) and Rlk/Itk-binding protein (RIBP) are adaptors related by structure and sequence, coexpressed in T cells. Mice deficient for each adaptor demonstrated that ALX and RIBP, respectively, negatively and positively regulate T cell activation in response to TCR/CD28 stimulation. However, these results did not preclude that they may function redundantly in other cell populations, or in response to other stimuli. Therefore, to understand the relationship between these related adaptors, ALX/RIBP-deficient mice were generated. We demonstrate that although ALX and RIBP are expressed throughout T cell development, T cell development occurs normally in these mice. Using the H-Y TCR transgenic model, positive and negative selection were found to proceed unimpeded in the absence of ALX and RIBP. We demonstrate that RIBP is also expressed in B cells; however, RIBP- and ALX/RIBP-deficient mice had normal B cell development, and responded equivalently to wild type in response to IgM, CD40, B cell-activating factor/B lymphocyte stimulator, CpG, and LPS. Interestingly, T cells deficient in both ALX and RIBP behaved similarly to those deficient in ALX alone during T cell activation in response to TCR/CD28, exhibiting increased IL-2 production, CD25 expression, and proliferation, thus showing that ALX deficiency masked the effect of RIBP deficiency. ALX/RIBP-deficient T cells did not have any alterations in either activation-induced cell death or Th1/2 polarization. Therefore, we did not find any functional redundancy or synergy during lymphocyte development, selection, activation, or survival in ALX/RIBP-deficient mice, demonstrating that these molecules function independently.
Despite Inhibition of Nuclear Localization of NF-κB p65, c-Rel, and RelB, 17-β Estradiol Up-Regulates NF-κB Signaling in Mouse Splenocytes: The Potential Role of Bcl-3
Authors: Dai Rujuan , Phillips Rebecca A. , Ahmed S. Ansar
Published: April 2014

NF-κB plays a major role in regulating the immune system. Therefore, alterations in NF-κB activity have profound effects on many immunopathologies, including inflammation, autoimmunity, and lymphoid neoplasia. We investigated the effects of estrogen (17β-estradiol) on NF-κB in C57BL/6 mice since estrogen is a natural immunomodulator and we have recently reported that estrogen up-regulates several NF-κB-regulated proteins (inducible NO synthase, IFN-γ, and MCP-1). We found that in vivo estrogen treatment had differential effects on NF-κB family members. Estrogen profoundly blocked the nuclear translocation of p65, c-Rel, and Rel-B, partially blocked p52, but permitted translocation of p50. Despite blockade of both the classical (p65/p50) and alternative (RelB/p52) NF-κB activation pathways, estrogen induced constitutive NF-κB activity and increased the levels of cytokines regulated by NF-κB (IL-1α, IL-1β, IL-10, and IFN-γ). Studies involving a NF-κB inhibitor confirmed a positive regulatory role of NF-κB on these cytokines. Remarkably, estrogen selectively induced B cell lymphoma 3 (Bcl-3), which is known to associate with p50 to confer transactivation capabilities, thereby providing a potential link between observed p50 DNA-binding activity and estrogen up-regulation of NF-κB transcriptional activity. Chromatin immunoprecipitation assays confirmed that Bcl-3 bound to the promoter of the NF-κB-regulated inducible NO synthase gene in cells from estrogen-treated mice. Estrogen appeared to act at the posttranscriptional level to up-regulate Bcl-3 because mRNA levels in splenocytes from placebo- and estrogen-treated mice were comparable. The novel findings of differential regulation of NF-κB proteins by estrogen provide fresh insight into potential mechanisms by which estrogen can regulate NF-κB-dependent immunological events.
The p47 GTPases Iigp2 and Irgb10 Regulate Innate Immunity and Inflammation to Murine Chlamydia psittaci Infection
Authors: Miyairi Isao , Tatireddigari Venkat R. R. Arva , Mahdi Olaimatu S. , Rose Lorne A. , Belland Robert J. , Lu Lu , Williams Robert W. , Byrne Gerald I.
Published: April 2014

C57BL/6J mice were 105-fold more resistant to Chlamydia psittaci infection than DBA/2J mice by LD100 determinations. Linkage analysis using BXD recombinant inbred strains revealed a single effector locus at a 1.5-Mbp region on chromosome 11 encoding a cluster of three p47 GTPases (Irgb10, Igtp, and Iigp2). Western blots of infected tissue showed that Irgb10 was elevated in resistant mice and one of the two possible Iigp2 protein isoforms was preferentially expressed in susceptible mice. The BXD39 strain, susceptible at Irgb10 and resistant at Iigp2, had an intermediate phenotype implicating the nonredundant role of these p47 GTPases. C57BL/6J and DBA/2J exhibited a difference in IFN-γ-dependent chlamydial control, which was reversible by Iigp2 small interfering RNA knockdown. Microarrays of infected peritoneal lavage revealed >10-fold up-regulation of neutrophil-recruiting chemokines in susceptible mice and >100-fold increase in macrophage differentiation genes in resistant mice, indicating that the susceptibility pattern involves the stimulation of different inflammatory cell-recruiting pathways. Massive neutrophil recruitment was seen in susceptible mice by histology and flow cytometry, and neutrophil chemokine receptor (CXCR2) knockout mice on a susceptible background survived a lethal challenge, confirming that neutrophil recruitment was required for susceptibility. Congenic Igtp knockout mice also susceptible at Irgb10 and Iigp2 on a resistant background recruited neutrophils and succumbed to infection. We conclude that Irgb10 and Iigp2 act together to confer differential susceptibility against murine chlamydial infection. Data indicate that these p47 GTPases have cell-autonomous effects that result in vastly different inflammatory stimulations, leading to either recovery or death.
The Intrinsic Apoptotic Pathway Is Required for Lipopolysaccharide-Induced Lung Endothelial Cell Death
Authors: Wang Helena L. , Akinci I. Ozkan , Baker Christina M. , Urich Daniela , Bellmeyer Amy , Jain Manu , Chandel Navdeep S. , Mutlu Gökhan M. , Budinger G. R. Scott
Published: April 2014

LPS has been implicated in the pathogenesis of endothelial cell death associated with Gram-negative bacterial sepsis. The binding of LPS to the TLR-4 on the surface of endothelial cells initiates the formation of a death-inducing signaling complex at the cell surface. The subsequent signaling pathways that result in apoptotic cell death remain unclear and may differ among endothelial cells in different organs. We sought to determine whether LPS and cycloheximide-induced cell death in human lung microvascular endothelial cells (HmVECs) was dependent upon activation of the intrinsic apoptotic pathway and the generation of reactive oxygen species. We found that cells overexpressing the anti-apoptotic protein Bcl-XL were resistant to LPS and cycloheximide-induced death and that the proapoptotic Bcl-2 protein Bid was cleaved following treatment with LPS. The importance of Bid was confirmed by protection of Bid-deficient (bid−/−) mice from LPS-induced lung injury. Neither HmVECs treated with the combined superoxide dismutase/catalase mimetic EUK-134 nor HmVECs depleted of mitochondrial DNA (ρ0 cells) were protected against LPS and cycloheximide-induced death. We conclude that LPS and cycloheximide-induced death in HmVECs requires the intrinsic cell death pathway, but not the generation of reactive oxygen species.
Streptococcus suis Serotype 2, an Important Swine and Human Pathogen, Induces Strong Systemic and Cerebral Inflammatory Responses in a Mouse Model of Infection
Authors: Domínguez-Punaro María C. , Segura Mariela , Plante Marie-Michèle , Lacouture Sonia , Rivest Serge , Gottschalk Marcelo
Published: April 2014

Streptococcus suis, an important swine and human pathogen, causes septic shock and meningitis. The pathogenesis of both systemic and CNS infections caused by S. suis is poorly understood. A hematogenous model of infection in CD1 mice was developed to study the systemic release of cytokines during the septic shock phase and the proinflammatory events in the CNS associated with this pathogen. Using a liquid array system, high levels of systemic TNF-α, IL-6, IL-12, IFN-γ, CCL2, CXCL1, and CCL5 were observed 24 h after infection and might be responsible for the sudden death of 20% of animals. Infected mice that survived the early sepsis later developed clinical signs of meningitis and exhibited lesions in the meninges and in numerous regions of the brain, such as the cortex, hippocampus, thalamus, hypothalamus, and corpus callosum. Bacterial Ags were found in association with microglia residing only in the affected zones. In situ hybridization combined with immunocytochemistry showed transcriptional activation of TLR2 and TLR3 as well as CD14, NF-κB, IL-1β, CCL2, and TNF-α, mainly in myeloid cells located in affected cerebral structures. Early transcriptional activation of TLR2, CD14, and inflammatory cytokines in the choroid plexus and cells lining the brain endothelium suggests that these structures are potential entry sites for the bacteria into the CNS. Our data indicate an important role of the inflammatory response in the pathogenesis of S. suis infection in mice. This experimental model may be useful for studying the mechanisms underlying sepsis and meningitis during bacterial infection.
Inhalation of Toluene Diisocyanate Vapor Induces Allergic Rhinitis in Mice
Authors: Johnson Victor J. , Yucesoy Berran , Reynolds Jeff S. , Fluharty Kara , Wang Wei , Richardson Diana , Luster Michael I.
Published: April 2014

Diisocyanates are the leading cause of occupational asthma, and epidemiological evidence suggests that occupational rhinitis is a comorbid and preceding condition in patients who develop asthma. The goal of the present studies was to develop and characterize a murine model of toluene diisocyanate (TDI)-induced rhinitis. Female C57BL/6 mice were exposed to workplace-relevant concentrations of TDI vapor via inhalation for 4 h/day for 12 days with or without a 2-wk rest period and TDI challenge. Mice exposed 12 consecutive weekdays to 50 parts per billion TDI vapor showed elevated total serum IgE and increased TDI-specific IgG titers. Breathing rates were decreased corresponding with increased inspiratory time. TDI exposure elevated IL-4, IL-5, IL-13, and IFN-γ mRNA expression in the nasal mucosa, suggesting a mixed Th1/Th2 immune response. Expressions of mRNA for proinflammatory cytokines and adhesion molecules were also up-regulated. These cytokine changes corresponded with a marked influx of inflammatory cells into the nasal mucosa, eosinophils being the predominant cell type. Removal from exposure for 2 wk resulted in reduced Ab production, cytokine mRNA expression, and cellular inflammation. Subsequent challenge with 50 parts per billion TDI vapor resulted in robust up-regulation of Ab production, cytokine gene expression, as well as eosinophilic inflammation in the nasal mucosa. There were no associated changes in the lung. The present model shows that TDI inhalation induces immune-mediated allergic rhinitis, displaying the major features observed in human disease. Future studies will use this model to define disease mechanisms and examine the temporal/dose relationship between TDI-induced rhinitis and asthma.
Dominant-Negative Inhibitors of Soluble TNF Attenuate Experimental Arthritis without Suppressing Innate Immunity to Infection
Authors: Zalevsky Jonathan , Secher Thomas , Ezhevsky Sergei A. , Janot Laure , Steed Paul M. , O’Brien Christopher , Eivazi Araz , Kung James , Nguyen Duc-Hanh T. , Doberstein Stephen K. , Erard François , Ryffel Bernhard , Szymkowski David E.
Published: April 2014

TNF is a pleiotropic cytokine required for normal development and function of the immune system; however, TNF overexpression also induces inflammation and is associated with autoimmune diseases. TNF exists as both a soluble and a transmembrane protein. Genetic studies in mice have suggested that inflammation in disease models involves soluble TNF (solTNF) and that maintenance of innate immune function involves transmembrane TNF (tmTNF). These findings imply that selective pharmacologic inhibition of solTNF may be anti-inflammatory and yet preserve innate immunity to infection. To address this hypothesis, we now describe dominant-negative inhibitors of TNF (DN-TNFs) as a new class of biologics that selectively inhibits solTNF. DN-TNFs blocked solTNF activity in human and mouse cells, a human blood cytokine release assay, and two mouse arthritis models. In contrast, DN-TNFs neither inhibited the activity of human or mouse tmTNF nor suppressed innate immunity to Listeria infection in mice. These results establish DN-TNFs as the first selective inhibitors of solTNF, demonstrate that inflammation in mouse arthritis models is primarily driven by solTNF, and suggest that the maintenance of tmTNF activity may improve the therapeutic index of future anti-inflammatory agents.
Adenosine Deamination Sustains Dendritic Cell Activation in Inflammation
Authors: Desrosiers Melanie D. , Cembrola Katherine M. , Fakir Michael J. , Stephens Leslie A. , Jama Fatimina M. , Shameli Afshin , Mehal Wajahat Z. , Santamaria Pere , Shi Yan
Published: April 2014

Adenosine is a suppressive agent that protects the host from excessive tissue injury associated with strong inflammation. In tissue stress, higher levels of adenosine signal through adenosine receptors to exert strong anti-inflammatory effects, and thus protect host cells. Existing evidence also suggests that elevated adenosine potently down-regulates the activation of lymphocytes during inflammation. This notion, however, is in contrast with another basic observation that the immune system is highly activated precisely under the same circumstances against pathogens. In this study, we show that inflammatory responses of dendritic cells (DCs) are highly sensitive to adenosine suppression. However, they intrinsically carry high adenosine deaminase activity, which in turn degrades and removes adenosine from the surroundings, cutting off DCs from the suppression. This regulatory mechanism is important in DC responses to pathogen-associated molecular patterns and their activation of T cells. Our findings suggest a mechanism that DCs maintain their hyperreactive state in inflammation despite the general state of suppression, and reveal a regulatory role of adenosine deaminase in DC innate immune responses.
Forkhead Box P3 Regulates TLR10 Expression in Human T Regulatory Cells
Authors: Bell Michael P. , Svingen Phyllis A. , Rahman Meher K. , Xiong Yuning , Faubion William A.
Published: April 2014

Although functionally relevant TLRs can be expressed on human T regulatory (Treg) cells, little is known about the transcriptional control of their expression. We hypothesized that the transcription factor forkhead box P3 (FOXP3) regulates the expression of TLR family members in human Treg cells. Using primary human T cells and a reporter assay in Jurkat T cell lines, we dissected the regulation of TLR10, a TLR highly expressed in human Treg cells. We determined that TLR10 was expressed in human Treg cells through quantitative PCR, Western blotting, and flow cytometry. DNA binding of FOXP3 to a suspected cis-regulatory region in proximity to the transcription start site of TLR10 was established through EMSA and chromatin immunoprecipitation. Transcriptional control of TLR10 by FOXP3 was determined through luciferase reporter assays in Jurkat T cell lines. Relevance of FOXP3 to TLR10 gene transcription in primary T cells was established through the transfection of primary CD4+CD25−FOXP3− T cells with a FOXP3 expression vector, which resulted in prompt production of TLR10 mRNA. Enhanced expression of TLR10 protein in primary Treg cells was induced in a calcium-dependent fashion through TCR activation. The suspected promotional cooperation between FOXP3 and NF-AT was established in the abolition of the luciferase signal upon transfection of a mutant FOXP3 devoid of NF-AT-binding activity. These results suggest that human Treg cells express TLR10, and this expression is regulated through a cooperative complex of FOXP3 and NF-AT.
Multiple Chemokine Receptors, Including CCR6 and CXCR3, Regulate Antigen-Induced T Cell Homing to the Human Asthmatic Airway
Authors: Thomas Seddon Y. , Banerji Aleena , Medoff Benjamin D. , Lilly Craig M. , Luster Andrew D.
Published: April 2014

Human allergic asthma is a chronic inflammatory disease of the airways thought to be driven by allergen-specific Th2 cells, which are recruited into the lung in response to inhaled allergen. To identify chemoattractant receptors that control this homing pattern, we used endobronchial segmental allergen challenge in human atopic asthmatics to define the pattern of chemoattractant receptor expression on recruited T cells as well as the numbers of recruited CD1d-restricted NKT cells and levels of chemokines in the bronchoalveolar (BAL) fluid. CD1d-restricted NKT cells comprised only a small minority of BAL T cells before or after Ag challenge. BAL T cells were enriched in their expression of specific chemoattractant receptors compared with peripheral blood T cells prechallenge, including CCR5, CCR6, CXCR3, CXCR4, and BLT1. Surprisingly, following segmental allergen challenge, no chemoattractant receptor was specifically increased. However, CCR6 and CXCR3, which were expressed on virtually all CD4+ BAL T cells prechallenge, were markedly decreased on all recruited BAL T cells following Ag challenge, suggesting that these receptors were internalized following encounter with ligand in the airway. Our data therefore suggests a role for CCR6 and CXCR3, in conjunction with other chemoattractant receptors, in the recruitment of inflammatory T cells into the BAL during the allergic asthmatic response.
Targeted Deletion of MKK4 Gene Potentiates TNF-Induced Apoptosis through the Down-Regulation of NF-κB Activation and NF-κB-Regulated Antiapoptotic Gene Products
Authors: Sethi Gautam , Ahn Kwang Seok , Xia Dianren , Kurie Jonathan M. , Aggarwal Bharat B.
Published: April 2014

MAPK kinase 4 (MKK4) is a dual-specificity kinase that activates both JNK and p38 MAPK. However, the mechanism by which MKK4 regulates TNF-induced apoptosis is not fully understood. Therefore, we used fibroblasts derived from MKK4 gene-deleted (MKK4-KO) mice to determine the role of this kinase in TNF signaling. We found that when compared with the wild-type cells, deletion of MKK4 gene enhanced TNF-induced apoptosis, and this correlated with down-regulation of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, XIAP, Bcl-2, Bcl-xL, and cFLIP) gene products, all regulated by NF-κB. Indeed we found that TNF-induced NF-κB activation was abrogated in MKK4 gene-deleted cells, as determined by DNA binding. Further investigation revealed that TNF-induced IκBα kinase activation, IκBα phosphorylation, IκBα degradation, and p65 nuclear translocation were all suppressed in MKK4-KO cells. NF-κB reporter assay revealed that NF-κB activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IκBα kinase was modulated in gene-deleted cells. Overall, our results indicate that MKK4 plays a central role in TNF-induced apoptosis through the regulation of NF-κB-regulated gene products.
Identification of Purα as a New Hypoxia Response Factor Responsible for Coordinated Induction of the β2 Integrin Family
Authors: Kong Tianqing , Scully Melanie , Shelley C. Simon , Colgan Sean P.
Published: April 2014

Central to the process of inflammation are hypoxic conditions that lead to the binding of circulating leukocytes to the endothelium. We have previously shown that such binding is mediated by monocytes being able to directly sense hypoxic conditions and respond by inducing their surface expression of the β2 integrin family of adhesion molecules. In this study, we show that coordinated induction of the β2 integrins during direct hypoxia-sensing occurs through transcriptional activation of each of the genes by which they are encoded. Certain of the molecular mechanisms that mediate this activation in transcription are dependent upon hypoxia-inducible factor-1 (HIF-1), whereas others are HIF-1 independent. In search of these HIF-1-independent mechanisms, we identified Purα as a new hypoxia-response factor. Binding of Purα to the HIF-1-independent β2 integrin promoters is induced by hypoxia and mutagenesis of these Purα-binding sites almost completely abolishes the ability of the promoters to respond to hypoxic conditions. Additional studies using siRNA directed against Purα also revealed a loss in the hypoxic response of the β2 integrin promoters. Taken together, our findings demonstrate that hypoxia induces a coordinated up-regulation in β2 integrin expression that is dependent upon transcriptional mechanisms mediated by HIF-1 and Purα.
Extracellular Acidosis Triggers the Maturation of Human Dendritic Cells and the Production of IL-12
Authors: Martínez Diego , Vermeulen Mónica , von Euw Erika , Sabatté Juan , Maggíni Julian , Ceballos Ana , Trevani Analía , Nahmod Karen , Salamone Gabriela , Barrio Marcela , Giordano Mirta , Amigorena Sebastian , Geffner Jorge
Published: April 2014

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-γ, but not IL-4, by Ag-specific CD4+ T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.
Targeting the Primary Tumor to Generate CTL for the Effective Eradication of Spontaneous Metastases
Authors: Yu Ping , Lee Youjin , Wang Yang , Liu Xiaojuan , Auh Sogyong , Gajewski Thomas F. , Schreiber Hans , You Zhaoyang , Kaynor Campbell , Wang Xinzhong , Fu Yang-Xin
Published: April 2014

Metastatic disease is the major cause of morbidity and mortality in cancer. Although surgery, chemotherapy, or radiation can often control primary tumor growth, successful eradication of disseminated metastases remains rare. We have now tested whether direct targeting tumor tissues to generate antitumor immune response before surgical excision produces sufficient CTL against micrometastases. One unsolved problem is whether such response allows coming CTL to be educated and then exit the tumor site. Another unsolved problem is whether these CTL can then patrol and effectively eliminate spontaneously metastasized tumor cells in the periphery. In this study, we have shown that adenovirus-expressing TNFSF14 [LIGHT (name derived from homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes); Ad-LIGHT] inoculated directly into primary 4T1 tumor, a highly aggressive, spontaneously metastasizing mammary carcinoma, followed by surgical removal of the primary tumor can eradicate established and disseminated metastatic tumor cells in the peripheral tissues. Furthermore, we clearly show with a fibrosarcoma model Ag104Ld that local treatment can generate plenty of tumor-specific CTL that exit the primary tumor and infiltrate distal tumors to completely eradicate distal tumors. Therefore, targeting the primary tumor with Ad-LIGHT before surgical excision is a new strategy to elicit better immune response for the eradication of spontaneous metastases.
Programmed Death 1 Expression on HIV-Specific CD4+ T Cells Is Driven by Viral Replication and Associated with T Cell Dysfunction
Authors: D’Souza Michelle , Fontenot Andrew P. , Mack Doug G. , Lozupone Catherine , Dillon Stephanie , Meditz Amie , Wilson Cara C. , Connick Elizabeth , Palmer Brent E.
Published: April 2014

Functional impairment of HIV-specific CD4+ T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4+ and CD8+ T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4+ T cells. To investigate the role of PD-1 in HIV-associated CD4+ T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-γ-producing HIV-specific CD4+ T cells compared with total or CMV-specific CD4+ T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4+ T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4+ T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4+ T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4+ T cells.
Human CD8+ T Cell Memory Generation in Puumala Hantavirus Infection Occurs after the Acute Phase and Is Associated with Boosting of EBV-Specific CD8+ Memory T Cells
Authors: Tuuminen Tamara , Kekäläinen Eliisa , Mäkelä Satu , Ala-Houhala Ilpo , Ennis Francis A. , Hedman Klaus , Mustonen Jukka , Vaheri Antti , Arstila T. Petteri
Published: April 2014

The induction and maintenance of T cell memory is incompletely understood, especially in humans. We have studied the T cell response and the generation of memory during acute infection by the Puumala virus (PUUV), a hantavirus endemic to Europe. It causes a self-limiting infection with no viral persistence, manifesting as hemorrhagic fever with renal syndrome. HLA tetramer staining of PBMC showed that the CD8+ T cell response peaked at the onset of the clinical disease and decreased within the next 3 wk. Expression of activation markers on the tetramer-positive T cells was also highest during the acute phase, suggesting that the peak population consisted largely of effector cells. Despite the presence of tetramer-positive T cells expressing cytoplasmic IFN-γ, PUUV-specific cells producing IFN-γ in vitro were rare during the acute phase. Their frequency, as well as the expression of IL-7Rα mRNA and surface protein, increased during a follow-up period of 6 wk and probably reflected the induction of memory T cells. Simultaneously with the PUUV-specific response, we also noted in seven of nine patients an increase in EBV-specific T cells and the transient presence of EBV DNA in three patients, indicative of viral reactivation. Our results show that in a natural human infection CD8+ memory T cells are rare during the peak response, gradually emerging during the first weeks of convalescence. They also suggest that the boosting of unrelated memory T cells may be a common occurrence in human viral infections, which may have significant implications for the homeostasis of the memory T cell compartment.
Cutting Edge: Vitamin D-Mediated Human Antimicrobial Activity against Mycobacterium tuberculosis Is Dependent on the Induction of Cathelicidin
Authors: Liu Philip T. , Stenger Steffen , Tang Dominic H. , Modlin Robert L.
Published: April 2014

Host defense against intracellular pathogens depends upon innate and adaptive antimicrobial effector pathways. TLR2/1-activation of monocytes leads to the vitamin D-dependent production of cathelicidin and, at the same time, an antimicrobial activity against intracellular Mycobacterium tuberculosis. To determine whether induction of cathelicidin was required for the vitamin D-triggered antimicrobial activity, the human monocytic cell line THP-1 was infected with M. tuberculosis H37Ra and then activated with the active vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25D3). 1,25D3 stimulation resulted in antimicrobial activity against intracellular M. tuberculosis and expression of cathelicidin mRNA and protein. Using small interfering RNA (siRNA) specific for cathelicidin, 1,25D3-induced cathelicidin mRNA and protein expressions were efficiently knocked down, whereas a nonspecific siRNA control had little effect. Finally, 1,25D3-induced antimicrobial activity was completely inhibited in the presence of siRNA against cathelicidin, instead leading to enhanced intracellular growth of mycobacteria. These data demonstrate that cathelicidin is required for the 1,25D3-triggered antimicrobial activity against intracellular M. tuberculosis.
A Pegylated Derivative of α-Galactosylceramide Exhibits Improved Biological Properties
Authors: Ebensen Thomas , Link Claudia , Riese Peggy , Schulze Kai , Morr Michael , Guzmán Carlos A.
Published: April 2014

The glycolipid α-galactosylceramide (αGalCer) has immunomodulatory properties, which have been exploited to combat cancer, chronic inflammatory diseases, and infections. However, its poor solubility makes αGalCer a suboptimal compound for in vivo applications. In this study, a pegylated derivative of αGalCer is characterized, which exhibits improved physical and biological properties. The new compound, αGalCerMPEG, is water-soluble and retains the specificity for the CD1d receptor of αGalCer. The in vitro stimulatory properties on immune cells (e.g., dendritic cells and splenocytes) are maintained intact, even when tested at a 33-fold lower concentration of the active moiety than αGalCer. NK cells isolated from mice treated with αGalCerMPEG also had stronger cytotoxic activity on YAC-1 cells than those obtained from animals receiving either αGalCer or CpG. Intranasal immunization studies performed in mice showed that αGalCerMPEG exerts stronger adjuvant activities than the parental compound αGalCer when tested at 0.35 vs 11.7 nM/dose. Coadministration of β-galactosidase with αGalCerMPEG resulted not only in high titers of Ag-specific Abs in serum (i.e., 1:512,000), but also in the stimulation of stronger Th2 and secretory IgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, and vagina). The new synthetic derivative αGalCerMPEG represents a promising tool for the development of immune interventions against infectious and noninfectious diseases.