bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells
Authors: Juckem Laura K. , Boehme Karl W. , Feire Adam L. , Compton Teresa
Published: April 2014

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (αβ IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-κB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with β1 integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.
Apoptotic Cells Protect Mice against Lipopolysaccharide-Induced Shock
Authors: Ren Yi , Xie Yi , Jiang Guoping , Fan Jianqing , Yeung Joseph , Li Wen , Tam Paul K. H. , Savill John
Published: April 2014

LPS is a main causative agent of septic shock. There is a lack of effective therapies. In vitro studies have shown that uptake of apoptotic cells actively inhibits the secretion by activated macrophages (Mφ) of proinflammatory mediators such as TNF-α and that such uptake increases the antiinflammatory and immunosuppressive cytokine TGF-β. We therefore investigated the protective effect of apoptotic cells against LPS-induced endotoxic shock in mice. The current report is the first study to demonstrate that administration of apoptotic cells can protect mice from LPS-induced death, even when apoptotic cells were administered 24 h after LPS challenge. The beneficial effects of administration of apoptotic cells included 1) reduced circulating proinflammatory cytokines, 2) suppression of polymorphonuclear neutrophil infiltration in target organs, and 3) decreased serum LPS levels. LPS can quickly bind to apoptotic cells and these LPS-coated apoptotic cells can be recognized and cleared by Mφ in a CD14/thrombospondin/vitronectin receptor-dependent manner, accompanied with suppression of TNF-α and enhancement of IL-10 expression by LPS-activated Mφ. Apoptotic cells may therefore have therapeutic potential for the treatment of septic shock.
Molecular Characterization of Helicobacter pylori VacA Induction of IL-8 in U937 Cells Reveals a Prominent Role for p38MAPK in Activating Transcription Factor-2, cAMP Response Element Binding Protein, and NF-κB Activation
Authors: Hisatsune Junzo , Nakayama Masaaki , Isomoto Hajime , Kurazono Hisao , Mukaida Naofumi , Mukhopadhyay Asish K. , Azuma Takeshi , Yamaoka Yoshio , Sap Jan , Yamasaki Eiki , Yahiro Kinnosuke , Moss Joel , Hirayama Toshiya
Published: April 2014

Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IκBα ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca2+ entry (SKF96365), and intracellular Ca2+ channels (dantrolene) blocked VacA-induced IL-8 production. Furthermore, an intracellular Ca2+ chelator (BAPTA-AM), which inhibited VacA-activated p38 MAPK, caused a dose-dependent reduction in VacA-induced IL-8 secretion by U937 cells, implying a role for intracellular Ca2+ in mediating activation of MAPK and the canonical NF-κB pathway. VacA stimulated translocation of NF-κBp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which is a p38MAPK substrate and binds to the AP-1 site of the IL-8 promoter, inhibited VacA-induced IL-8 production. VacA activated an IL-8 promoter containing an NF-IL-6 site, but not a mutated AP-1 or NF-κB site, suggesting direct involvement of the ATF-2/CREB binding region or NF-κB-binding regions in VacA-induced IL-8 promoter activation. Thus, in U937 cells, VacA directly increases IL-8 production by activation of the p38 MAPK via intracellular Ca2+ release, leading to activation of the transcription factors, ATF-2, CREB, and NF-κB.
Phosphatidylinositol 3-Kinase and Xanthine Oxidase Regulate Nitric Oxide and Reactive Oxygen Species Productions by Apoptotic Lymphocyte Microparticles in Endothelial Cells
Authors: Mostefai H. Ahmed , Agouni Abdelali , Carusio Nunzia , Mastronardi M. Letizia , Heymes Christophe , Henrion Daniel , Andriantsitohaina Ramaroson , Martinez M. Carmen
Published: April 2014

Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. We have previously shown that MPs from apoptotic T cells induce endothelial dysfunction, but the mechanisms implicated are not completely elucidated. In this study, we dissect the pathways involved in endothelial cells with respect to both NO and reactive oxygen species (ROS). Incubation of endothelial cells with MPs decreased NO production that was associated with overexpression and phosphorylation of endothelial NO synthase (eNOS). Also, MPs enhanced expression of caveolin-1 and decreased its phosphorylation. Microparticles enhanced ROS by a mechanism sensitive to xanthine oxidase and P-IκBα inhibitors. PI3K inhibition reduced the effects of MPs on eNOS, but not on caveolin-1, whereas it enhanced the effects of MPs on ROS production. Microparticles stimulated ERK1/2 phosphorylation via a PI3K-depedent mechanism. Inhibition of MEK reversed eNOS phosphorylation but had no effect on ROS production induced by MPs. In vivo injection of MPs in mice impaired endothelial function. In summary, MPs activate pathways related to NO and ROS productions through PI3K, xanthine oxidase, and NF-κB pathways. These data underscore the pleiotropic effects of MPs on NO and ROS, leading to an increase oxidative stress that may account for the deleterious effects of MPs on endothelial function.
Complement Receptors 1 and 2 Influence the Immune Environment in a B Cell Receptor-Independent Manner
Authors: Jacobson Amanda C. , Weis Janis J. , Weis John H.
Published: April 2014

The CD21/35 proteins are complement receptors implicated in controlling and interpreting activation states of the innate and acquired immune responses. One defect of CD21/35−/− animals is depressed production of Ag-specific IgG3 which we show is evident in vivo but not in vitro. Gene expression profiles obtained from naive wild-type and CD21/35−/− splenocytes demonstrated enhanced expression of inflammatory mediators from CD11b+ splenocytes in the CD21/35−/− animals. Splenocyte populations between wild-type and CD21/35−/− mice were similar except for a moderate increase in GR1lowCD31+ immature myeloid cells. Furthermore, depletion of neutrophils and other GR1-expressing cells alleviates elevated inflammatory gene expression in the CD21/35−/− spleen. Complement activation also plays a key role in the differential gene expression observed in the CD21/35-deficient mouse as depletion of C3 or inhibition of C3a receptor signaling within the animal returned inflammatory gene expression within the spleen to wild-type levels. Finally, C3 depletion before immunization allowed for the enhanced production of Ag-specific IgG3 production in the CD21/35−/− mouse compared with mock-depleted animals. These data suggest that the overall environment of the CD21/35−/− spleen is quite different from that of the wild-type animal perhaps due to altered complement convertase activity. This difference may be responsible for a number of the phenotypes ascribed to the deficiency of CD21/35 proteins on B cells and follicular dendritic cells.
Destructive Arthritis in the Absence of Both FcγRI and FcγRIII
Authors: Boross Peter , van Lent Peter L. , Martin-Ramirez Javier , van der Kaa Jos , Mulder Melissa H. C. M. , Claassens Jill W. C. , van den Berg Wim B. , Arandhara Victoria L. , Verbeek J. Sjef
Published: April 2014

Fc receptors for IgG (FcγR) have been implicated in the development of arthritis. However, the precise contribution of the individual FcγR to joint pathology is unclear. In this study, the role of the different FcγR was assessed both in an active and in a passive mouse model of arthritis by analyzing disease development in double and triple knockout (KO) offspring from crosses of FcγRI KO, FcγRIII KO, FcγRI/III double KO, or FcR γ-chain KO with the FcγRII KO on C57BL6 background, which is susceptible for collagen-induced arthritis (CIA). In the active CIA model, onset was significantly delayed in the absence of FcγRIII, whereas incidence and maximum severity were significantly decreased in FcγRI/II/III triple KO but not in FcγRII/III double KO and FcγRI/II double KO mice as compared with FcγRII KO animals. Remarkably, fully destructive CIA developed in FcγRI/II/III triple KO mice. In contrast, FcR γ/FcγRII double KO mice were resistant to CIA. These findings were confirmed with the passive KRN serum-induced arthritis model. These results indicate that all activating FcγR play a role in the development of arthritis, mainly in the downstream effector phase. FcγRIII is critically required for early arthritis onset, and FcγRI can substantially contribute to arthritis pathology. Importantly, FcγRI and FcγRIII were together dispensable for the development of destructive arthritis but the FcR γ-chain was not, suggesting a role for another FcR γ-chain associated receptor, most likely FcγRIV. In addition, FcγRII plays a negative regulatory role in both the central and effector phase of arthritis.
Development of Either Split Tolerance or Robust Tolerance along with Humoral Tolerance to Donor and Third-Party Alloantigens in Nonmyeloablative Mixed Chimeras
Authors: Chan William F. N. , Razavy Haide , Luo Bin , Shapiro A. M. James , Anderson Colin C.
Published: April 2014

Hematopoietic chimerism is considered to generate robust allogeneic tolerance; however, tissue rejection by chimeras can occur. This “split tolerance” can result from immunity toward tissue-specific Ags not expressed by hematopoietic cells. Known to occur in chimeric recipients of skin grafts, it has not often been reported for other donor tissues. Because chimerism is viewed as a potential approach to induce islet transplantation tolerance, we generated mixed bone marrow chimerism in the tolerance-resistant NOD mouse and tested for split tolerance. An unusual multilevel split tolerance developed in NOD chimeras, but not chimeric B6 controls. NOD chimeras demonstrated persistent T cell chimerism but rejected other donor hematopoietic cells, including B cells. NOD chimeras also showed partial donor alloreactivity. Furthermore, NOD chimeras were split tolerant to donor skin transplants and even donor islet transplants, unlike control B6 chimeras. Surprisingly, islet rejection was not a result of autoimmunity, since NOD chimeras did not reject syngeneic islets. Split tolerance was linked to non-MHC genes of the NOD genetic background and was manifested recessively in F1 studies. Also, NOD chimeras but not B6 chimeras could generate serum alloantibodies, although at greatly reduced levels compared with nonchimeric controls. Surprisingly, the alloantibody response was sufficiently cross-reactive that chimerism-induced humoral tolerance extended to third-party cells. These data identify split tolerance, generated by a tolerance-resistant genetic background, as an important new limitation to the chimerism approach. In contrast, the possibility of humoral tolerance to multiple donors is potentially beneficial.
1α,25-Dihydroxyvitamin D3 Potentiates the Beneficial Effects of Allergen Immunotherapy in a Mouse Model of Allergic Asthma: Role for IL-10 and TGF-β
Authors: Taher Yousef A. , van Esch Betty C. A. M. , Hofman Gerard A. , Henricks Paul A. J. , van Oosterhout Antoon J. M.
Published: April 2014

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a potent inhibitor of NF-κB expression, can prevent the maturation of dendritic cells in vitro leading to tolerogenic dendritic cells with increased potential to induce regulatory T cells. Herein, we investigated whether the combination of allergen immunotherapy with 1,25(OH)2D3 potentiates the suppressive effects of immunotherapy and whether the immunoregulatory cytokines IL-10 and TGF-β are involved in the effector phase. OVA-sensitized and challenged BALB/c mice displayed airway hyperresponsiveness (AHR) and increased serum OVA-specific IgE levels, bronchoalveolar lavage eosinophilia, and Th2 cytokine levels. In this model, the dose response of allergen immunotherapy 10 days before OVA inhalation challenge shows strong suppression of asthma manifestations at 1 mg of OVA, but partial suppression of bronchoalveolar lavage eosinophilia, IgE up-regulation, and no reduction of AHR at 100 μg. Interestingly, coadministration of 10 ng of 1,25(OH)2D3 with 100 μg of OVA immunotherapy significantly inhibited AHR and potentiated the reduction of serum OVA-specific IgE levels, airway eosinophilia, and Th2-related cytokines concomitant with increased IL-10 levels in lung tissues and TGF-β and OVA-specific IgA levels in serum. Similar effects on suboptimal immunotherapy were observed by inhibition of the NF-κB pathway using the selective IκB kinase 2 inhibitor PS-1145. The suppressive effects of this combined immunotherapy were partially reversed by treatment with mAb to either IL-10R or TGF-β before OVA inhalation challenge but completely abrogated when both Abs were given. These data demonstrate that 1,25(OH)2D3 potentiates the efficacy of immunotherapy and that the regulatory cytokines IL-10 and TGF-β play a crucial role in the effector phase of this mouse model.
Differential Contributions of APC Subsets to T Cell Activation in Nonobese Diabetic Mice
Authors: Marleau Annette M. , Summers Kelly L. , Singh Bhagirath
Published: April 2014

Despite the pivotal role of dendritic cells (DC) in shaping immunity, little is known about their functionality in type 1 diabetes. Moreover, due to the paucity of DC in vivo, functional studies have relied largely upon in vitro-expanded cells to elucidate type 1 diabetes-associated functional abnormalities. In this study, we provide a comprehensive analysis of the functional capabilities of in vivo-derived DC subsets from NOD mice by comparing DC to other NOD APC types and to DC from autoimmune-resistant strains. NOD DC closely resemble those from nonautoimmune strains with respect to costimulation and cytokine production. The exception is the CD8α+CD11b−DC subset which is numerically reduced in NOD spleens, but not in the pancreatic lymph nodes, while DC from both tissues produce little IL-12 in this strain. This defect results in unusual deferral toward macrophage-derived IL-12 in NOD mice; NOD macrophages produce aberrantly high IL-12 levels that can overcompensate for the DC defect in Th1 polarization. APC subset use for autoantigen presentation also differs in NOD mice. NOD B cells overshadow DC at activating islet-reactive T cells, whereas DC and B cells in NOD-resistant mice are functionally comparable. Differential involvement of APC subsets in T cell activation and tolerance induction may prove to be a crucial factor in the selection and expansion of autoreactive T cells.
Polarization of Primary Human Monocytes by IFN-γ Induces Chromatin Changes and Recruits RNA Pol II to the TNF-α Promoter
Authors: Garrett Stacey , Dietzmann-Maurer Kelly , Song Li , Sullivan Kathleen E.
Published: April 2014

Monocyte polarization by IFN-γ or IL-4 drives a complex series of cellular responses leading to increased intracellular killing (IFN-γ) or enhanced healing (IL-4) among other functional responses. We studied the effect of IL-4 and IFN-γ polarization on histone modifications at the TNF-α locus in human primary monocytes. IFN-γ polarization markedly increased the expression of TNF-α, whereas IL-4 treatment decreased the expression. We found that IFN-γ alone increased histone H4 acetylation at the TNF-α promoter. The effect of IFN-γ on TNF-α expression was durable upon cytokine washout and even repolarization with IL-4. Concordantly, IFN-γ-mediated H4 acetylation was also durable. IFN-γ recruited activating transcription factor-2 via p38 to the TNF-α promoter, but inhibition of p38 had minimal effect on H4 acetylation. In a novel finding, we found that IFN-γ recruited RNA Pol II to the human TNF-α promoter via ERK signaling, but did so without initiating transcription, leading to a poised condition. These studies provide an important perspective on monocyte polarization. Polarization by IFN-γ has a durable effect on TNF-α expression, and histone acetylation may provide a mechanism for persistence of the effect.
The Apoptotic Pathway Contributing to the Deletion of Naive CD8 T Cells during the Induction of Peripheral Tolerance to a Cross-Presented Self-Antigen
Authors: Redmond William L. , Wei Cheng-Hong , Kreuwel Huub T. C. , Sherman Linda A.
Published: April 2014

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet β-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim−/− clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.
MyD88-Dependent Pathway in T Cells Directly Modulates the Expansion of Colitogenic CD4+ T Cells in Chronic Colitis
Authors: Tomita Takayuki , Kanai Takanori , Fujii Toshimitsu , Nemoto Yasuhiro , Okamoto Ryuichi , Tsuchiya Kiichiro , Totsuka Teruji , Sakamoto Naoya , Akira Shizuo , Watanabe Mamoru
Published: April 2014

TLRs that mediate the recognition of pathogen-associated molecular patterns are widely expressed on/in cells of the innate immune system. However, recent findings demonstrate that certain TLRs are also expressed in conventional TCRαβ+ T cells that are critically involved in the acquired immune system, suggesting that TLR ligands can directly modulate T cell function in addition to various innate immune cells. In this study, we report that in a murine model of chronic colitis induced in RAG-2−/− mice by adoptive transfer of CD4+CD45RBhigh T cells, both CD4+CD45RBhigh donor cells and the expanding colitogenic lamina propria CD4+CD44high memory cells expresses a wide variety of TLRs along with MyD88, a key adaptor molecule required for signal transduction through TLRs. Although RAG-2−/− mice transferred with MyD88−/−CD4+CD45RBhigh cells developed colitis, the severity was reduced with the delayed kinetics of clinical course, and the expansion of colitogenic CD4+ T cells was significantly impaired as compared with control mice transferred with MyD88+/+CD4+CD45RBhigh cells. When RAG-2−/− mice were transferred with the same number of MyD88+/+ (Ly5.1+) and MyD88−/− (Ly5.2+) CD4+CD45RBhigh cells, MyD88−/−CD4+ T cells showed significantly lower proliferative responses assessed by in vivo CFSE division assay, and also lower expression of antiapoptotic Bcl-2/Bcl-xL molecules and less production of IFN-γ and IL-17, compared with the paired MyD88+/+CD4+ T cells. Collectively, the MyD88-dependent pathway that controls TLR signaling in T cells may directly promote the proliferation and survival of colitogenic CD4+ T cells to sustain chronic colitis.
Formation of IL-7Rαhigh and IL-7Rαlow CD8 T Cells during Infection Is Regulated by the Opposing Functions of GABPα and Gfi-1
Authors: Chandele Anmol , Joshi Nikhil S. , Zhu Jinfang , Paul William E. , Leonard Warren J. , Kaech Susan M.
Published: April 2014

IL-7 is essential for the survival of naive and memory T cells, and IL-7 receptor α-chain (IL-7Rα) expression is dynamically regulated in activated CD8 T cells during acute viral and bacterial infections. Most virus-specific CD8 T cells become IL-7Rαlow and are relatively short-lived, but some escape IL-7Rα repression (referred to as IL-7Rαhigh memory precursor effector cells) and preferentially enter the memory CD8 T cell pool. How antiviral effector CD8 T cells regulate IL-7Rα expression in an “on and off” fashion remains to be characterized. During lymphocytic choriomeningitis virus infection, we found that opposing actions of the transcription factors GABPα (GA binding protein α) and Gfi-1 (growth factor independence 1) control IL-7Rα expression in effector CD8 T cells. Specifically, GABPα was required for IL-7Rα expression in memory precursor effector cells, and this correlated with hyperacetylation of the Il7ra promoter. In contrast, Gfi-1 was required for stable IL-7Rα repression in effector CD8 T cells and acted by antagonizing GABPα binding and recruiting histone deacetylase 1, which deacetylated the Il7ra promoter. Thus, Il7ra promoter acetylation and activity was dependent on the reciprocal binding of GABPα and Gfi-1, and these data provide a biochemical mechanism for the generation of stable IL-7Rαhigh and IL-7Rαlow states in virus-specific effector CD8 T cells.
Effector Memory αβ T Lymphocytes Can Express FcγRIIIa and Mediate Antibody-Dependent Cellular Cytotoxicity
Authors: Clémenceau Béatrice , Vivien Régine , Berthomé Mathilde , Robillard Nelly , Garand Richard , Gallot Géraldine , Vollant Solène , Vié Henri
Published: April 2014

Human memory T cells are comprised of distinct populations with different homing potential and effector functions: central memory T cells that mount recall responses to Ags in secondary lymphoid organs, and effector memory T cells that confer immediate protection in peripheral tissues. In the present study we demonstrate that a proportion of effector memory T cells express FcγRIIIa (CD16), are perforin positive, and directly mediate Ab-dependent cytotoxicity ex vivo. This particular αβ T lymphocyte subset has the morphology of large granular lymphocytes, increases proportionately in vivo during reactive lymphocytosis, and can be detected in vitro among EBV-specific T lymphocytes after stimulation with EBV Ags. Consequently, during a normal immune response, amplification of these effector memory T lymphocytes that are capable of Ab-dependent cytotoxicity may have beneficial or harmful consequences depending on the presence of pathogen- or tissue-specific Abs, respectively.
Sphingosine 1-Phosphate Regulates the Egress of IgA Plasmablasts from Peyer’s Patches for Intestinal IgA Responses
Authors: Gohda Masashi , Kunisawa Jun , Miura Fumi , Kagiyama Yuki , Kurashima Yosuke , Higuchi Morio , Ishikawa Izumi , Ogahara Ikuko , Kiyono Hiroshi
Published: April 2014

It is well established that Peyer’s patches (PPs) are sites for the differentiation of IgA plasma cell precursors, but molecular and cellular mechanisms in their trafficking remain to be elucidated. In this study, we show that alterations in type 1 sphingosine 1-phosphate (S1P) receptor expression during B cell differentiation in the PPs control the emigration of IgA plasma cell precursors. Type 1 S1P receptor expression decreased during the differentiation of IgM+B220+ B cells to IgA+B220+ B cells, but recovered on IgA+B220− plasmablasts for their emigration from the PPs. Thus, IgA+B220− plasmablasts migrated in response to S1P in vitro. Additionally, IgA+ plasmablasts selectively accumulated in lymphatic regions of PPs when S1P-mediated signaling was disrupted by FTY720 treatment. This accumulation of IgA+ plasmablasts in the PPs led to their reduction in the intestinal lamina propria and simultaneous impairment of Ag-specific intestinal IgA production against orally administered Ag. These findings suggest that S1P regulates the retention and emigration of PP B cells and plays key roles in the induction of intestinal IgA production.
Inter-Strain Tissue-Infiltrating T Cell Responses to Minor Histocompatibility Antigens Involved in Graft-Versus-Host Disease as Determined by Vβ Spectratype Analysis
Authors: Zilberberg Jenny , McElhaugh Danielle , Gichuru Loise N. , Korngold Robert , Friedman Thea M.
Published: April 2014

Lethal graft-vs-host disease (GVHD) can be induced between MHC-matched murine strains expressing multiple minor histocompatibility Ag differences. In the B6–>BALB.B model, both CD4+ and CD8+ donor T cells can mediate lethal GVHD, whereas in the B6–>CXB-2 model, only CD8+ T cells are lethal. TCR Vβ CDR3-size spectratyping was previously used to analyze CD8+ and CD4+ T cell responses in lethally irradiated BALB.B and CXB-2 recipients, which showed significant overlap in the reacting repertoires. However, CD4+ T cells exhibited unique skewing of the Vβ2 and 11 families in only BALB.B recipients. These Vβ family reactivities were confirmed by immunohistochemical staining of lingual epithelial infiltrates, and by positive and negative selection Vβ family transfer experiments for GVHD induction in BALB.B recipients. We have now extended these studies to examine the T cell repertoire responses involved in target tissue damage. Infiltrating B6 host-presensitized CD8+ and CD4+ T cells were isolated 8–10 days post-transplant from the spleens, intestines and livers of CXB-2 and BALB.B transplant recipients. For both T cell subsets, the results indicated overlapping tissue skewings between the recipients, also between the tissues sampled within the respective recipients as well as tissue specific responses unique to both the BALB.B and CXB-2 infiltrates. Most notably, the CD4+ Vβ 11+ family was skewed in the intestines of BALB.B but not CXB-2 recipients. Taken together, these data suggest that there are likely to be target tissue-related anti-multiple minor histocompatibility Ag-specific responses in each of the strain recipients, which may also differ from those found in peripheral lymphoid organs.
The Lymphotoxin Pathway Regulates Aire-Independent Expression of Ectopic Genes and Chemokines in Thymic Stromal Cells
Authors: Seach Natalie , Ueno Tomoo , Fletcher Anne L. , Lowen Tamara , Mattesich Monika , Engwerda Christian R. , Scott Hamish S. , Ware Carl F. , Chidgey Ann P. , Gray Daniel H. D. , Boyd Richard L.
Published: April 2014

Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTβR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTβR expression was found in all TEC subsets, but the highest levels were detected in MTS-15+ thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTβR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEClow), as well as maintenance of MTS-15+ fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta−/− and Ltbr−/− mice. Thus, we propose a broader role for LTα1β2-LTβR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEClow for efficient induction of central tolerance.
Release from Regulatory T Cell-Mediated Suppression during the Onset of Tissue-Specific Autoimmunity Is Associated with Elevated IL-21
Authors: Clough Louise E. , Wang Chun Jing , Schmidt Emily M. , Booth George , Hou Tie Zheng , Ryan Gemma A. , Walker Lucy S. K.
Published: April 2014

The activity of regulatory T cells (Treg) is widely accepted to play a central role in preventing pathogenic immune responses against self-Ags. However, it is not clear why such regulation breaks down during the onset of autoimmunity. We have studied self-Ag-specific Treg during the induction of spontaneous diabetes. Our data reveal a shift in the balance between regulatory and pathogenic islet-reactive T cells in the pancreas-draining lymph nodes during disease onset. Treg function was not compromised during disease initiation, but instead conventional T cells showed reduced susceptibility to Treg-mediated suppression. Release from Treg suppression was associated with elevated levels of IL-21 in vivo, and provision of this cytokine abrogated Treg suppression in vitro and in vivo. These data suggest that immunological protection of a peripheral tissue by Treg can be subverted by IL-21, suggesting new strategies for intervention in autoimmunity.
The Adjuvants Aluminum Hydroxide and MF59 Induce Monocyte and Granulocyte Chemoattractants and Enhance Monocyte Differentiation toward Dendritic Cells
Authors: Seubert Anja , Monaci Elisabetta , Pizza Mariagrazia , O’Hagan Derek T. , Wack Andreas
Published: April 2014

Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.
Nonoverlapping Expression of IL10, IL12p40, and IFNγ mRNA in the Marginal Zone and T Cell Zone of the Spleen after Antigenic Stimulation
Authors: Kalies Kathrin , König Peter , Zhang Yong-Ming , Deierling Maria , Barthelmann Julia , Stamm Claudia , Westermann Jürgen
Published: April 2014

The differentiation of CD4+ T cells is regulated by cytokines locally within the compartments of secondary lymphoid organs during adaptive immune responses. Quantitative data about the expression of cytokine mRNAs within the T and B cell zones of lymphoid organs are lacking. In this study, we assessed the expression of multiple cytokine genes within the lymphoid compartments of the spleen of rats after two types of stimulation. First, the spleen was stimulated directly by a blood-derived Ag. Second, the spleen was stimulated indirectly by incoming lymphocytes that had been activated and released during a proceeding immune response at a distant tissue site. Using laser microdissection, we show that the expression of cytokine mRNAs was compartment specific, transient, and preceded cell proliferation after the direct antigenic stimulation. Surprisingly, the indirect stimulation by incoming activated lymphocytes induced similar cytokines in the T cell zone. However, the nonoverlapping expression was lost and IL10 appeared as the major cytokine in all compartments. Thus, tracking two types of immune activation without disturbing the integrity of structures reveals distinct and overlapping events in the compartments of the spleen. This information adds a new dimension to the understanding of immune responses in vivo.
A Conserved IFN-α Receptor Tyrosine Motif Directs the Biological Response to Type I IFNs
Authors: Zhao Wenli , Lee Carolyn , Piganis Rebecca , Plumlee Courtney , de Weerd Nicole , Hertzog Paul J. , Schindler Christian
Published: April 2014

Mammalian type I IFNs (IFN-Is) mediate their potent biological activities through an evolutionarily conserved IFN-α receptor (IFNAR), consisting of IFNAR1 and IFNAR2. These two chains direct the rapid activation of two founding members of the STAT family of transcription factors, STAT1 and STAT2. To understand how IFN-Is direct the recruitment and activation of STATs, a series of mutant murine IFNAR1 and IFNAR2 receptors were generated and evaluated in IFNAR1 and IFNAR2 knockout cells. These studies reveal that a single conserved IFNAR2 tyrosine, Y510, plays a critical role in directing the IFN-I-dependent activation of STAT1 and STAT2, both in murine fibroblasts and macrophages. A second IFNAR2 tyrosine, Y335, plays a more minor role. Likewise, Y510 > Y335 play a critical role in the induction of genes and antiviral activity traditionally associated with IFN-Is.
Immunoproteomic Identification and Serological Responses to Novel Chlamydia pneumoniae Antigens That Are Associated with Persistent C. pneumoniae Infections
Authors: Bunk Sebastian , Susnea Iuliana , Rupp Jan , Summersgill James T. , Maass Matthias , Stegmann Werner , Schrattenholz André , Wendel Albrecht , Przybylski Michael , Hermann Corinna
Published: April 2014

The controversial discussion about the role of Chlamydia pneumoniae in atherosclerosis cannot be solved without a reliable diagnosis that allows discrimination between past and persistent infections. Using a proteomic approach and immunoblotting with human sera, we identified 31 major C. pneumoniae Ags originating from 27 different C. pneumoniae proteins. More than half of the proteins represent Chlamydia Ags not described previously. Using a comparative analysis of spot reactivity Pmp6, OMP2, GroEL, DnaK, RpoA, EF-Tu, as well as CpB0704 and CpB0837, were found to be immunodominant. The comparison of Ab-response patterns of sera from subjects with and without evidence for persisting C. pneumoniae, determined by multiple PCR analysis of PBMC and vasculatory samples, resulted in differential reactivity for 12 proteins, which is not reflected by reactivity of the sera in the microimmunofluorescence test, the current gold standard for serodiagnosis. Although reactivity of sera from PCR-positive donors was increased toward RpoA, MOMP, YscC, Pmp10, PorB, Pmp21, GroEL, and Cpaf, the reactivity toward YscL, Rho, LCrE, and CpB0837 was decreased, reflecting the altered protein expression of persisting C. pneumoniae in vitro. Our data provide the first evidence of a unique Ab-response pattern associated with persistent C. pneumoniae infections, which is a prerequisite for the serological determination of persistently infected patients.
The Long Isoform of Cellular FLIP Is Essential for T Lymphocyte Proliferation through an NF-κB-Independent Pathway
Authors: Zhang Nu , Hopkins Kaycie , He You-Wen
Published: April 2014

Although the long isoform of cellular FLIP (c-FLIPL) has been implicated in TCR-mediated signaling, its role in T cell proliferation remains controversial. Some studies have demonstrated that overexpression of c-FLIPL promotes T cell proliferation and NF-κB activation, whereas others have reported that c-FLIPL overexpression has no effect or even inhibits T cell proliferation. To establish the role of c-FLIPL in T lymphocyte proliferation, we have generated a conditional knockout mouse strain specifically lacking c-FLIPL in T lymphocytes. c-FLIPL−/− mice exhibit severely impaired effector T cell development after Listeria monocytogenes infection in vivo and c-FLIPL-deficient T cells display defective TCR-mediated proliferation in vitro. However, c-FLIPL−/− T cells exhibit normal NF-κB activity upon TCR stimulation. These results demonstrate that c-FLIPL is essential for T lymphocyte proliferation through an NF-κB-independent pathway.
Comparison of Proteins Expressed on Secretory Vesicle Membranes and Plasma Membranes of Human Neutrophils
Authors: Uriarte Silvia M. , Powell David W. , Luerman Gregory C. , Merchant Michael L. , Cummins Timothy D. , Jog Neelakshi R. , Ward Richard A. , McLeish Kenneth R.
Published: April 2014

Secretory vesicles are neutrophil intracellular storage granules formed by endocytosis. Understanding the functional consequences of secretory vesicle exocytosis requires knowledge of their membrane proteins. The current study was designed to use proteomic technologies to develop a more complete catalog of secretory vesicle membrane proteins and to compare the proteomes of secretory vesicle and plasma membranes. A total of 1118 proteins were identified, 573 (51%) were present only in plasma membrane-enriched fractions, 418 (37%) only in secretory vesicle-enriched membrane fractions, and 127 (11%) in both fractions. Gene Ontology categorized 373 of these proteins as integral membrane proteins. Proteins typically associated with other intracellular organelles, including nuclei, mitochondria, and ribosomes, were identified in both membrane fractions. Ingenuity Pathway Knowledge Base analysis determined that the majority of canonical and functional pathways were significantly associated with proteins from both plasma membrane-enriched and secretory vesicle-enriched fractions. There were, however, some canonical signaling pathways that involved proteins only from plasma membranes or secretory vesicles. In conclusion, a number of proteins were identified that may elucidate mechanisms and functional consequences of secretory vesicle exocytosis. The small number of common proteins suggests that the hypothesis that secretory vesicles are formed from plasma membranes by endocytosis requires more critical evaluation.
Pneumocystis Infection Enhances Antibody-Mediated Resistance to a Subsequent Influenza Infection
Authors: Wiley James A. , Harmsen Allen G.
Published: April 2014

In contrast to the detrimental outcomes most often associated with the resolution of coinfections, the model presented here involving a localized Pneumocystis infection of the lung, followed 2 wk later by an influenza virus infection, results in a significant beneficial outcome for the host. In the week following the influenza infection, immunocompetent coinfected animals exhibited an accelerated rate of virus clearance, an accelerated appearance of higher influenza-specific neutralizing Ab titers in their serum and bronchoalveolar lavage fluid (BALF), significantly reduced inflammatory cytokine levels in their BALF, and reduced levels of morbidity relative to animals infected only with influenza virus. The beneficial outcome observed in coinfected immunocompetent animals was dependent on the ongoing resolution of a viable Pneumocystis infection. No differences in viral clearance were detected between coinfected and influenza-only-infected μMT mice or likewise for SCID mice. The accelerated anti-influenza response did not appear to be associated with influenza-specific CD8 T cell-mediated responses or NK cell responses in the lung. Rather, the increased rate of viral clearance was due to the enhancement of the influenza-specific Ab response, which in turn was transiently dependent upon the resolution of the ongoing Pneumocystis infection.
Molecular Mechanisms of Cytokine and Chemokine Release from Eosinophils Activated by IL-17A, IL-17F, and IL-23: Implication for Th17 Lymphocytes-Mediated Allergic Inflammation
Authors: Cheung Phyllis F. Y. , Wong Chun K. , Lam Christopher W. K.
Published: April 2014

IL-17A and IL-17F are members of the IL-17 family that play crucial roles in allergic inflammation. Recent studies reported that IL-17A and IL-17F production from a distinct Th lymphocyte subset, Th17, was specifically induced by IL-23, which was produced by dendritic cells and macrophages in response to microbial stimuli. The IL-23-IL-17 axis might therefore provide a link between infections and allergic diseases. In the present study, we investigated the effects of IL-17A, IL-17F, and IL-23, alone or in combination, on cytokine and chemokine release from eosinophils and the underlying intracellular mechanisms. Human eosinophils were found to constitutively express receptors for IL-17A, IL-17F, and IL-23 at the protein level. IL-17A, IL-17F, and IL-23 could induce the release of chemokines GRO-α/CXCL1, IL-8/CXCL8, and MIP-1β/CCL4 from eosinophils, while IL-17F and IL-23 could also increase the production of proinflammatory cytokines IL-1β and IL-6. Synergistic effects were observed in the combined treatment of IL-17F and IL-23 on the release of proinflammatory cytokines, and the effects were dose-dependently enhanced by IL-23, but not IL-17F. Further investigations showed that IL-17A, IL-17F, and IL-23 differentially activated the ERK, p38 MAPK, and NF-κB pathways. Moreover, inhibition of these pathways using selective inhibitors could significantly abolish the chemokine release induced by IL-17A, IL-17F, and IL-23 and the synergistic increases on IL-1β and IL-6 production mediated by combined treatment of IL-17F and IL-23. Taken together, our findings provide insight for the Th17 lymphocyte-mediated activation of eosinophils via differential intracellular signaling cascades in allergic inflammation.
Kruppel-Like Factor 4 Is Essential for Inflammatory Monocyte Differentiation In Vivo
Authors: Alder Jonathan K. , Georgantas Robert W. , Hildreth Richard L. , Kaplan Ian M. , Morisot Sebastien , Yu Xiaobing , McDevitt Michael , Civin Curt I.
Published: April 2014

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4−/− fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4+/+ cells in competitive repopulation assays. However, hematopoietic “KLF4−/− chimeras” generated by transplantation of KLF4−/− fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115+Gr1+) monocytes, and had reduced numbers of resident (CD115+Gr1−) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4−/− chimeras, bone marrow monocytic cells from KLF4−/− chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.
Mechanism of IL-1β-Induced Increase in Intestinal Epithelial Tight Junction Permeability
Authors: Al-Sadi Rana , Ye Dongmei , Dokladny Karol , Ma Thomas Y.
Published: April 2014

The IL-1β-induced increase in intestinal epithelial tight junction (TJ) permeability has been postulated to be an important mechanism contributing to intestinal inflammation of Crohn’s disease and other inflammatory conditions of the gut. The intracellular and molecular mechanisms that mediate the IL-1β-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to elucidate the mechanisms that mediate the IL-1β-induced increase in intestinal TJ permeability. Specifically, the role of myosin L chain kinase (MLCK) was investigated. IL-1β caused a progressive increase in MLCK protein expression. The time course of IL-1β-induced increase in MLCK level correlated linearly with increase in Caco-2 TJ permeability. Inhibition of the IL-1β-induced increase in MLCK protein expression prevented the increase in Caco-2 TJ permeability. Inhibition of the IL-1β-induced increase in MLCK activity also prevented the increase in Caco-2 TJ permeability. Additionally, knock-down of MLCK protein expression by small interference RNA prevented the IL-1β-induced increase in Caco-2 TJ permeability. The IL-1β-induced increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression. The IL-1β-induced increase in MLCK mRNA transcription and subsequent increase in MLCK protein expression and Caco-2 TJ permeability was mediated by activation of NF-κB. In conclusion, our data indicate that the IL-1β increase in Caco-2 TJ permeability was mediated by an increase in MLCK expression and activity. Our findings also indicate that the IL-1β-induced increase in MLCK protein expression and Caco-2 TJ permeability was mediated by an NF-κB-dependent increase in MLCK gene transcription.