Early Phase Of Immune Response Research Articles

Transcription factor MafB-mediated inhibition of type I interferons in plasmacytoid dendritic cells.

Type I IFNs (IFN-α and IFN-β), immunomodulatory cytokines secreted from activated plasmacytoid dendritic cells (pDCs), contribute to the innate defense against pathogenic infections and the pathogenesis of the autoimmune disease psoriasis vulgaris. A previous study has shown that an E26 transformation-specific (Ets) family transcription factor Spi-B can transactivate the type I IFN promoter in synergy with IFN regulatory factor (IRF)-7 and is required for type I IFN production in pDCs. However, the mechanism of negative regulation of type I IFNs by pDCs remains unknown. In this study, we report that a basic leucine zipper (bZip) transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB) suppresses the induction of type I IFNs in pDCs. The elevated expression of MafB inhibited the transactivation of type I IFN genes in a dose-dependent manner. At the molecular level, MafB interacted with the Ets domain of Spi-B and interfered with IRF-7-Spi-B complexation. Decreased MafB mRNA expression and degradation of MafB protein in the early phase of immune responses led to the enhancement of type I IFNs in pDCs. In vivo studies indicated that MafB is involved in resistance against imiquimod-induced psoriasis-like skin inflammation. Overall, these findings demonstrate that MafB acts as a negative regulator of type I IFN induction in pDCs and plays an important role in maintaining immune homeostasis.

Cytotoxicity of natural killer cells on canine mammary carcinoma cells

Natural killer (NK) cells play have a crucial role in the early phase of immune responses against various pathogens. We compared characteristics of canine NK cells against two canine mammary carcinoma cell lines, REM134 and CF41.Mg. REM134 showed higher expression of progesterone receptor, proliferative cell nuclear antigen, Ki67, multiple drug resistance, Bmi-1, c-myc, E-cadherin, and human epidermal growth factor receptor type-2 than that of CF41.Mg. For specific expansion and activation of NK cells, we isolated CD5 negative cells from canine peripheral blood mononuclear cells and co-cultured K562 cells in the presence of interleukin (IL)-2, IL-15, and IL-21 for 21 days. As a result, we found that expression markers of activated NK cells such as NKp30, NKp44, NKp46, NKG2D, CD244, perforin, granzyme B, and tumor necrosis factor alpha were highly upregulated. In addition, we found there was upregulated production of interferon gamma of activated NK cells against target cells such as REM134 and CF41.Mg. Specifically, we observed that cytotoxicity of NK cells against target cells was more sensitively reacted to CF41.Mg than REM134. Based on the results of this study, we recommend the development of an experimental application of CF41Mg, which has not been reported in canine mammary carcinoma research. Keywords: canine, mammary carcinoma, natural killer cells, interferon gamma, cytotoxicity

Characterization of cellular and humoral immune responses to alkaline phosphatase from fertile hydatid cysts in the human peripheral blood.

Hydatid cyst, the larval stage of the tapeworm Echinococcus granulosus and a causative agent of cystic echinococcosis, possesses a vast number of antigenic peptides that are constantly presented in the host immune system during infection. Here, we sought to provide more information about the cellular/humoral components engaged in the peripheral immune reactions to the fertile-cyst-derived Echinococcus alkaline phosphatase (E.ALP) in human hosts. Lymphoproliferative and cytokine responses after recall of E.ALP suggested the presence of specific immune reactions against the antigen. Interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 had the highest fold increase over the spontaneous levels in response to hydatid crude antigen (HCA). Recall of E.ALP, as well as its encounter, boosted IFN-γ, TNF-α, IL-2, and IL-6 responses in peripheral blood mononuclear cells cultures (PBMCs). The HCA-driven levels of all the cytokines in the culture supernatants of normal PBMCs were higher than those measured after E.ALP encounter. Immunoglobulin G (IgG)-profile in response to HCA showed the dominance of specific IgG1, IgG2, and IgG4 antibodies, but relatively lower affinity of IgG3 to this antigen. IgG1 and IgG3 were the only isotypes detected in serum responses to E.ALP. Our findings suggested that E.ALP contributes to the early phase of immune responses to the parasite, likely by induction of proinflammatory profiles and clonal expansion of high-affinity IgG1- and IgG3-secreting plasma cells, suggesting the value of E.ALP as a candidate to develop novel therapeutic and immunization strategies.

Activation and selective IL-17 response of human Vγ9Vδ2 T lymphocytes by TLR-activated plasmacytoid dendritic cells.

Vγ9Vδ2 T cells and plasmacytoid dendritic cells (pDCs) are two distinct cell types of innate immunity that participate in early phases of immune response. We investigated whether a close functional relationship exists between these two cell populations using an in vitro co-culture in a human system.pDCs that had been activated by IL-3 and the TLR9 ligand CpG induced substantial activation of Vγ9Vδ2 T cells upon co-culture, which was cell-to-cell contact dependent, as demonstrated in transwell experiments, but that did not involve any of the costimulatory molecules potentially expressed by pDCs or Vγ9V2 T cells, such as ICOS-L, OX40 and CD40L. Activated pDCs selectively induced IL-17, but not IFN-γ, responses of Vγ9Vδ2T cells, which was dominant over the antigen-induced response, and this was associated with the expansion of memory (both central and effector memory) subsets of Vγ9Vδ2 T cells.Overall, our results provide a further piece of information on the complex relationship between these two populations of cells with innate immunity features during inflammatory responses.

The Immunology of CD1- and MR1-Restricted T Cells.

CD1- and MHC-related molecule-1 (MR1)-restricted T lymphocytes recognize nonpeptidic antigens, such as lipids and small metabolites, and account for a major fraction of circulating and tissue-resident T cells. They represent a readily activated, long-lasting population of effector cells and contribute to the early phases of immune response, orchestrating the function of other cells. This review addresses the main aspects of their immunological functions, including antigen and T cell receptor repertoires, mechanisms of nonpeptidic antigen presentation, and the current evidence for their participation in human and experimental diseases.

Structural and expression studies of interferon regulatory factor 8 in Japanese flounder, Paralichthys olivaceus

Interferon regulatory factor 8 (IRF8) plays a role in both innate and adaptive systems in mammals. In this study, the gene and promoter sequences of Japanese flounder, Paralichthys olivaceus, (Po) IRF8 were cloned, and its expression in response to polyinosinic:polycytidylic acid (poly I:C) and lymphocystis disease virus (LCDV) challenges was studied in vivo. The PoIRF8 gene spans over 3.3 kb with a structure of 9 exon–8 intron and encodes 420 amino acids. The putative protein shows the highest sequence identity (69.5–89.0%) to fish IRF8 and possesses a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) of vertebrate IRF8. Phylogenetic analysis classified PoIRF8 into the cluster of fish IRF8 within vertebrate IRF8 group of IRF4 subfamily. A number of transcription factor binding sites were identified in the 2348-bp 5′ flanking region of PoIRF8 gene, including those of transcription factors for type Ⅰ and type Ⅱ interferon (IFN) inducible genes and genes regulating the development and function of lymphomyeloid cells in mammals. The PoIRF8 transcripts were expressed in all examined tissues of healthy flounders, with higher levels observed in the immune relevant tissues. They were up-regulated by both poly I:C and LCDV treatments in the spleen, head kidney, gills and muscle in an early phase of immune responses, with initiation and peak time points of induction prior to type Ⅰ IFN and Mx. Relative to LCDV, the induction by poly I:C was quicker in all four tissues. These results indicate an involvement of PoIRF8 in the host's antiviral responses and a functional conservation of IRF8 between fish and mammals.

Porcine CD8αdim/-NKp46high NK cells are in a highly activated state.

Natural Killer (NK) cells play a crucial role in the early phase of immune responses against various pathogens. In swine so far only little information about this lymphocyte population exists. Phenotypical analyses with newly developed monoclonal antibodies (mAbs) against porcine NKp46 recently revealed that in blood NKp46- and NKp46+ cells with NK phenotype exist with comparable cytotoxic properties. In spleen a third NKp46-defined population with NK phenotype was observed that was characterised by a low to negative CD8α and increased NKp46 expression. In the current study it is shown that this NKp46high phenotype was correlated with an increased expression of CD16 and CD27 compared to the CD8α+NKp46- and NKp46+ NK-cell subsets in spleen and blood. Additionally NKp46high NK cells expressed elevated levels of the chemokine receptor CXCR3 on mRNA level. Functional analyses revealed that splenic NKp46high NK cells produced much higher levels of Interferon-γ and Tumor Necrosis Factor-α upon stimulation with cytokines or phorbol-12-myristate-13-acetate/Ionomycin compared to the other two subsets. Furthermore, cross-linking of NKp46 by NKp46-specific mAbs led to a superior CD107a expression in the NKp46high NK cells, thus indicating a higher cytolytic capacity of this subset. Therefore porcine splenic NKp46high NK cells represent a highly activated subset of NK cells and may play a profound role in the immune surveillance of this organ.

Bcl6 expression specifies the T follicular helper cell program in vivo

T follicular helper cells (Tfh cells) play a pivotal role in germinal center reactions, which require B cell lymphoma 6 (Bcl6) transcription factor. To analyze their relationships with other effector T cell lineages and their stability in vivo, we developed and analyzed a new Bcl6 reporter mouse alone or together with other lineage reporter systems. Assisted with genome-wide transcriptome analysis, we show substantial plasticity of T cell differentiation in the early phase of immune response. At this stage, CXCR5 appears to be expressed in a Bcl6-independent manner. Once Bcl6 is highly expressed, Tfh cells can persist in vivo and some of them develop into memory cells. Together, our results indicate Bcl6 as a bona fide marker for Tfh polarized program.

Intrathymic programming of effector fates in three molecularly distinct γδ T cell subtypes

γδ T cells function in the early phase of immune responses. Although innate γδ T cells have primarily been studied as one homogenous population, they can be functionally classified into effector subsets based on the production of signature cytokines, analogous to adaptive T helper subsets. Unlike adaptive T cells, however, γδ T effector function correlates with genomically encoded TCR chains, suggesting that clonal TCR selection is not the primary determinant of γδ effector differentiation. A high resolution transcriptome analysis of all emergent γδ thymocyte subsets segregated based on TCRγ/δ chain usage indicates the existence of three separate subtypes of γδ effectors in the thymus. The immature γδ subsets are distinguished by unique transcription factor modules that program effector function.

Thermal burn injury induced myeloperoxidase changes in murine obesity model

Inflammatory response due to thermal burn injury was assessed in murine obesity model. Multimodal non invasive imaging of the myeloperoxidase activity was determined for the purpose. Myeloperoxidase (MPO), an inflammatory heme protein is present in myeloid cells‐ neutrophils, microglia, and macrophages, and utilizes hydrogen peroxide to generate reactive oxygen species. We earlier demonstrated using multimodal fluorescence imaging, infiltration of the peritoneal macrophages at the site of burn injury. Here, we determined the changes in MPO activity as a response to burn injury using C57BL/6NTac mice placed under high fat or regular diet. The in vivo MPO activity was detected by whole body luminescence imaging 15 minutes after i.p injection of luminol. luminol a redox‐sensitive compound emits blue luminescence upon exposure to oxidizing agents, and established to have unique specificity to MPO in vivo. We show significant increase in MPO activity as a result of neutrophil activation at the burn injury. Robust activation, deduced from the MPO activity, was observed in the lymph nodes of the murine model that went through the high‐fat diet regime. These real‐time monitoring methodologies of MPO activity detection by in vivo imaging will greatly enhance the understanding of the early phase of immune response to a burn injury and aid in development of better treatments.

High Expression of Fas Ligand on Cord Blood Dendritic Cells: A Possible Immunoregulatory Mechanism After Cord Blood Transplantation

Background Allogeneic cord blood transplantation is associated with less severe graft-versus-host disease (GVHD). Dendritic cells (DCs), as the most potent antigen-presenting cells of the immune system, play a central role in the development of GVHD. Because apoptosis induction is one of the known mechanisms that DCs use to regulate T-cell responses, we studied the immunostimulatory and apoptosis induction capacities of cord blood dendritic cells (CBDCs) and peripheral blood dendritic cells (PBDCs) to evaluate the mechanisms underlying the lower incidence of GVHD after cord blood transplantation. Presence of apoptosis-related markers Fas, Fas ligand (FasL), and CD40 and costimulatory molecules, along with the proportion of myeloid and lymphoid DCs subsets, were also measured on CBDCs and PBDCs. Methods Fresh CBDCs and PBDCs were isolated from cord and peripheral mononuclear cells as lineage-negative cells by using monoclonal antibodies against CD3, CD11b, CD14, CD16, CD19, CD56, CD34, and CD66b. DCs were cocultured with allogeneic T cells, and the effect of CBDCs and PBDCs on T-cell apoptosis and proliferation were determined through flow cytometric analysis and 3H-thymidine incorporation. Results Our findings showed that CBDCs markedly augment apoptosis of CD3 + T-cells. FasL expression on CBDCs was significantly higher than on PBDCs. However, there was no difference between Fas expression on CBDCs and PBDCs. Moreover, CBDCs were poor stimulators of allogenic T cells in mixed leukocyte reaction compared with adult peripheral blood DCs. They also displayed decreased expression of HLA-DR and CD86 molecules. The ratio of lymphoid DCs (CD11c −, CD123 +) to myeloid DCs (CD11c +, CD123 −) was also significantly higher in CBDCs compared with PBDCs. Conclusions It seems that less severe GVHD after cord blood transplantation is due not only to a higher degree of immaturity of CBDCs, but also to delivery of apoptotic signals to the host T cells that recognize allo-MHC molecules on CBDCs in the early phase of immune response.

The up-regulation of large yellow croaker secretory IgM heavy chain at early phase of immune response

An immunoglobulin M (IgM) heavy-chain gene homologue was isolated from the spleen cDNA library of the large yellow croaker Pseudosciaena crocea (LycIgH). The complete cDNA of LycIgH is 1,987 nucleotides long, encoding a protein of 585 amino acids with a putative molecular weight of 64.5 kDa. The deduced LycIgH possesses a typical secretory IgM heavy chain organization with a variable region (V(H)) connected to four constant regions (C(H1-4)) by a diversity segment (D(H)) and a joining segment (J(H)). Tissue expression profile analysis showed that LycIgH was constitutively expressed in gills, intestine, liver, kidney, heart, spleen, muscle, and blood, while at a higher level in spleen, kidney and intestine. Upon stimulation with poly (I: C), the LycIgH transcripts were quickly increased in spleen and kidney at 12 h post induction (with 5.87- and 5.48-fold mRNA increases, respectively), followed by a recovery to normal level at 24 h. The LycIgH transcripts in spleen and kidney induced by inactivated bacterial vaccine reached their peak levels at 48 h (14.53-fold) and 12 h (3.70-fold), respectively. These results indicated the up-regulation of LycIgH expression in spleen and kidney by poly (I: C) or bacterial vaccine occurred at the early phase of induction and was differentially modulated in the two tissues by different stimulations.

Partial and Ineffective Activation of Vγ9Vδ2 T Cells by Mycobacterium tuberculosis-Infected Dendritic Cells

Gammadelta T cells and dendritic cells (DCs) participate in early phases of immune response against Mycobacterium tuberculosis. We investigated whether a close functional relationship exists between these two cell populations using an in vitro coculture in a human system. Vgamma9Vdelta2 T cells induce full maturation of M. tuberculosis-infected immature DCs, as demonstrated by upregulation of the costimulatory CD80, CD86, CD40, and HLA-DR molecules on infected DCs after 24 h of coculture. Reciprocally, infected DCs induced substantial activation of Vgamma9Vdelta2 T cells upon coculture, which was cell-to-cell contact and TCR dependent, as demonstrated in transwell experiments. However, infected DCs selectively induced proliferative, but not cytokine or cytolytic, responses of Vgamma9Vdelta2 T cells, and this was associated with the expansion of phenotypically immature, central memory-type Vgamma9Vdelta2 T cells. Importantly, expansion of central memory Vgamma9Vdelta2 T cells and reduction of the pool of Vgamma9Vdelta2 T cells with immediate effector functions (effector memory and terminally differentiated cells) were also detected in vivo in the peripheral blood of patients with active tuberculosis, which reversed after antimycobacterial therapy. M. tuberculosis-infected DCs produced many different cytokines, but not IL-15, and addition of IL-15 to cocultures of infected DCs and Vgamma9Vdelta2 T cells caused efficient differentiation of these latter with generation of effector memory and terminally differentiated cells, which were capable of reducing the viability of intracellular M. tuberculosis. Overall, this study provides a further piece of information on the complex relationship between important players of innate immunity during mycobacterial infection.

Natural Killer (NK) Cells from Killers to Regulators: Distinct Features Between Peripheral Blood and Decidual NK Cells

Natural killer (NK) cells are a key component of innate immunity, particularly crucial during the early phase of immune responses against certain viruses, parasites, and microbial pathogens. The role of NK cell during pregnancy has been vividly discussed over the past years and it is now becoming increasingly clear that NK cells control pregnancy maintenance at several levels. In normal pregnancy, it appears that they provide benefit by properly secreting cytokines, chemokines and angiogenic factors rather than functioning as cytotoxic effector cells. However, as they are endowed with all the cytolytic weapons, they promptly become capable of attacking fetal and maternal tissues during infection and inflammation.

Dendritic Cells as Arbiters of Peritoneal Immune Responses

During the past few years, there has been a substantial increase in the understanding of innate immunity. Dendritic cells are emerging as key players in the orchestration of this early phase of immune responses, with a role that will translate into the subsequent type of adaptive immune response against infection. Here we provide an overview of dendritic cell differentiation and function, with particular emphasis on those features unique to the immune defense of the peritoneal cavity and in the context of peritoneal dialysis-associated immune responses. The reader is referred to the primary references included in the accompanying list for specific details in this fascinating field.

Lyme Arthritis Synovial γδ T Cells Instruct Dendritic Cells via Fas Ligand

gammadelta T cells participate in the innate immune response to a variety of infectious microorganisms. They also link to the adaptive immune response through their induction of maturation of dendritic cells (DC) during the early phase of an immune response when the frequency of Ag-specific T cells is very low. We observe that in the presence of Borrelia burgdorferi, synovial Vdelta1 T cells from Lyme arthritis synovial fluid potently induce maturation of DC, including production of IL-12, and increased surface expression of CD40 and CD86. The activated DC are then able to stimulate the Vdelta1 T cells to up-regulate CD25. Both of these processes are initiated primarily by Fas stimulation rather than CD40 activation of DC via high expression of Fas ligand by the Vdelta1 T cells. DC are resistant to Fas-induced death due to expression of high levels of the Fas inhibitor c-FLIP. This effect serves to divert Fas-mediated signals from the caspase cascade to the ERK MAPK and NF-kappaB pathways. The findings affirm the importance of the interaction of certain T cell populations with DC during the early phases of the innate immune response. They also underscore the view that as levels of c-FLIP increase, Fas signaling can be diverted from induction of apoptosis to pathways leading to cell effector function.

Reciprocal stimulation of gammadelta T cells and dendritic cells during the anti-mycobacterial immune response.

Gammadelta T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vgamma1 T cells from Tcrb(-/- )mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN-gamma secretion and cytotoxic activity. This activation process was due to a non-cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC-derived IL-12. Reciprocally, Vgamma1 T cells provided a key cytokine, IFN-gamma, which increased IL-12 production by BCG-infected DC. Moreover, exposure of BCG-infected DC to Vgamma1 T cells conditioned the former to prime a significantly stronger anti-mycobacterial CD8 T cell response. Consequently, stimulation of gammadelta T cells and their non-cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine-induced CD8 T cell immunity.

Lysine 58 and Histidine 66 at the C-terminal α-Helix of Monocyte Chemoattractant Protein-1 Are Essential for Glycosaminoglycan Binding

Monocytes rolling on the endothelial cell layer interact with monocyte chemoattractant protein-1 (MCP-1) that is tethered to the proteoglycans on the luminal side of the endothelial cells and consequently initiate adhesion of monocytes in the early phase of immune response. The amino acid residues in MCP-1 involved in tethering to the proteoglycans have not been elucidated. MCP-1 showed binding to [3H]heparin with a KD of 1.5 microM. We substituted lysine or histidine residues at the C-terminal end of MCP-1 with alanine residues and tested these mutants for their ability to bind heparin, heparan sulfate, hyaluronic acid, and chondroitin sulfate-C. Substitution of Lys-58 or His-66 drastically reduced glycosaminoglycan binding. Substitution of Lys-56 or deletion of the five amino acid residues at the C terminus, including Lys-75, did not alter the heparin binding ability, suggesting that the other lysine residues at the C terminus are not involved in glycosaminoglycan binding. MCP-1 and its mutants did not bind hyaluronic acid as strongly as the other subunits of the GAGs. Substitution of Lys-58 or His-66 by alanine that prevented glycosaminoglycan binding did not affect Ca2+ influx, receptor binding, or chemotactic activity elicited by the chemokine on monocytic THP-1 cells. Therefore, we conclude that the Lys-58 and His-66 residues in the C-terminal alpha-helix of MCP-1 are essential for glycosaminoglycan binding and probably for the binding to the endothelial surface proteoglycans.

ICAM-1 induction on hepatocytes as a marker for immune activation of acute liver allograft rejection.

Intercellular adhesion molecule-1 (ICAM-1) induction on hepatocytes was investigated in relation to acute liver allograft rejection, CMV infection, and systemic bacterial infections. Twenty-four liver transplant recipients underwent an episode of acute rejection, 13 developed a symptomatic clinical CMV infection, and 7 had bacterial sepsis. Seven recipients without rejection or infection complications were used as controls. All rejection episodes monitored by frequent FNABs were reversible, and lymphocyte and lymphoid blast-dominated with a with peak of inflammation (7.2 +/- 3.9 corrected increment units [CIU]). The rejections were treated with high-dose steroids, and the inflammation subsided within one week. ICAM-1 was demonstrated from fine needle aspiration biopsy (FNAB) preparations by a monoclonal antibody and immunoperoxidase staining. ICAM-1 was not detected on the hepatocytes immediately after transplantation or in control patients, but was always seen during rejection. ICAM-1 appeared 1-5 days before the onset of inflammation in FNAB. The intensity of ICAM-1 expression increased toward the peak of inflammation and subsided together with inflammation. During CMV infection a mild immune activation was seen in FNAB (peak 2.5 +/- 0.8 CIU) and in blood. An intense ICAM-1 induction also preceded the immune activation caused by CMV, and subsided slowly with successful antiviral treatment. In addition, a slight ICAM-1 induction on the hepatocytes was recorded during bacterial sepsis. ICAM-1 induction on hepatocytes appears to be linked with an early phase of immune response, and it even precedes the lymphoid activation of rejection. However, several infections, such as CMV and bacterial infections, raise an immune response and may also induce ICAM-1. In conclusion, ICAM-1 induction on hepatocytes can be considered an early, though unspecific, marker for acute liver allograft rejection.