Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and glucocorticoid (GC) counter-regulator, has emerged as an important modulator of inflammatory responses. However, the molecular mechanisms of MIF counter-regulation of GC still remain incomplete. In present study, we investigated whether MIF mediated counter-regulation of anti-inflammatory effect of GC by affecting Annexin 1 in RAW 264.7 macrophages. We found that stimulation of RAW 264.7 macrophages with lipopolysaccharide (LPS) resulted in down-regulation of Annexin 1, while GC dexamethasone (Dex) or Dex plus LPS led to a significant up-regulation of Annexin 1 expression. RNAi-mediated knockdown of intracellular MIF increased Annexin 1 expression with or without incubation of Dex, while Dex-induced Annexin 1 expression was counter-regulated by the exogenous application of recombinant MIF. Moreover, recombinant MIF counter-regulated, in a dose-dependent manner, inhibition of cytosolic phospholipase A2 alpha (cPLA2α) activation and Prostaglandin E₂ (PGE₂) and Leukotriene B₄ (LTB₄) release by Dex in RAW 264.7 macrophages stimulated with LPS. Endogenous depletion of MIF enhanced the effects of Dex reflecting by more deceased cPLA2α expression and lower PGE₂ and LTB₄ release in RAW 264.7 macrophages. Based on these data, we suggest that MIF counter-regulates Dex-induced Annexin 1 expression, further influencing the activation of cPLA2α and the release of eicosanoids. These findings will add new insights into the mechanisms of MIF counter-regulation of GC.

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The central nervous system (CNS) is considered to be immune privileged implying that non-self antigens which gain access into the CNS are unable to invoke an adaptive immune response. Such shielding of the CNS from the adaptive immune response is aided by the cerebrospinal fluid (CSF)-blood barrier, blood-brain barrier and blood-spinal cord barrier. In addition, the expression of immune regulatory molecules by cells of the CNS down-regulate T-cell function and together with low levels of major histocompatibility (MHC) molecules act to limit immune activation and immune-mediated damage in the CNS. While the idea of immune privilege was first discussed over 70 years ago^1,2 by Sir Peter Medawar who was awarded the Nobel Prize with Sir Frank Macfarlane Burnet in 1960 for the discovery of acquired immune tolerance, it is clear that immune privilege is not absolute since immune reactions do, and indeed must take place to control infections in the CNS.

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The naïve T-cell pool in peripheral lymphoid tissues is fairly stable in terms of number, diversity and functional capabilities in spite of the absence of prominent stimuli. This stability is attributed to continuous tuning of the composition of the T cell pool by various homeostatic signals. Despite extensive research into the link between Stat3 and T cell survival, little is known about how Stat3 regulates homeostasis by maintaining the required naïve T-cell population in peripheral lymphoid organs. We assessed whether the elimination of Stat3 in T cells limits T cell survival. We demonstrated that the proportion and number of single positive (SP) thymocytes as well as T cells in the spleen and lymph node were significantly decreased in the Stat3-deficient group as a result of the enhanced susceptibility of Stat3-deleted T lymphocytes to apoptosis. Importantly, expression of the anti-apoptotic Bcl-2 and Bcl-xL was markedly decreased in Stat3-deleted SP thymocytes and T lymphocytes, suggesting that Stat3 helps to maintain the T-cell pool in the resting condition by promoting the expression of Bcl-2 family genes. These findings suggest the importance of Stat3 in the integration of homeostatic cues for the maintenance and functional tuning of the T cell pool.

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The objective of this study was to determine the systemic effects of chronic SIV infection on plasmacytoid dendritic cells (pDCs). pDCs play a critical role in antiviral immunity, but current data is conflicting on whether pDCs inhibit HIV/SIV replication, or, alternatively, contribute to chronic immune activation and disease. Furthermore, previous pDC studies have been complicated by incomplete descriptions of generalized depletion during HIV/SIV infection, and the effects of infection on pDCs outside peripheral blood remain unclear. In scheduled sacrifice studies of naïve and chronically SIV-infected rhesus macaques we evaluated the distribution and functionality of pDCs in multiple tissues using surface and intracellular polychromatic flow cytometry. As previously observed pDCs were reduced in peripheral blood and spleens, but were also depleted in non-lymphoid organs such as the liver. Interestingly, pDCs accumulated up to 4-fold in jejunum, colon and gut-draining, but not peripheral lymph nodes. Most unexpectedly, SIV infection induced a multifunctional IFN-α, TNF-α, and MIP-1β cytokine secretion phenotype, whereas in normal animals these were generally distinct and separate functions. Herein we show a systemic redistribution of pDCs to gut tissues and gut-draining lymph nodes during chronic SIV infection, coupled to a novel multifunctional cytokine-producing phenotype. While pDC accumulation in the mucosa could aid in virus control, over-production of cytokines from these cells could also contribute to the increased immune activation in the gut mucosa commonly associated with progressive lentivirus infections.

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Opportunistic infections with non-tuberculous mycobacteria such as Mycobacterium avium receive renewed attention due to increased incidence and difficulties in treatment. As for other mycobacterial infections, a still poorly understood collaboration of different immune effector mechanisms is required to confer protective immunity. Here we have characterized the interplay of innate and adaptive immune effector mechanismscontributing to containment in a mouse infection model using virulent Mycobacterium avium strain 104 in C57BL/6 mice.

Mycobacterium avium caused chronic infection in mice as evidenced by sustained organ bacterial load. In the liver, bacteria were contained in granuloma-like structures that could be defined morphologically by expression of the antibacterial innate effector protein Lipocalin 2 in adjoining hepatocytes and infiltrating neutrophils, possibly contributing to containment. Circulatory anti-mycobacterial antibodies steadily increased throughout infection and were primarily of the IgM isotype.

Highest levels of interferon-γ were found in infected liver, spleen and serum of mice approximately two weeks post infection and coincided with halt in organ bacterial growth. In contrast, expression of tumor necrosis factor was surprisingly low in spleen compared to liver. We did not detect interleukin-17 in infected organs or Mycobacterium avium-specific T helper 17 cells, suggesting a minor role for T helper 17 cells in this model. A transient and relative decrease in regulatory T cell numbers was seen in spleens.

This detailed characterization of Mycobacterium avium infection in C57BL/6 mice may provide a basis for future studies aiming at gaining better insight into mechanisms leading to containment of infections with non-tuberculous mycobacteria.

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IL-17A, a pro-inflammatory cytokine acting on neutrophil recruitment, is known to play an important role during M. tuberculosis infection, but the role of IL-17A receptor signaling in immune defense against this intracellular pathogen remains poorly documented. Here we have analyzed this signaling using C57BL/6 mice genetically inactivated in the IL-17 receptor A subunit (IL-17RA^-/-). Although early after infection bacterial growth was controlled to the same extent as in wild type mice, IL-17RA^-/- mice were defective in exerting a long-term control of M. tuberculosis infection as demonstrated by a progressively increasing pulmonary bacterial burden and shortened survival time. Compared to infected wild type mice, IL-17RA^-/- mice showed impaired recruitment of neutrophils to the lungs at the early but not the late stage of infection. Pulmonary TNF-α, IL-6 and particularly IL-10 levels were decreased in the absence of IL-17RA-signaling, while IL-1β was increased. CD4⁺- and γδ- mediated IL-17A production was dramatically increased in IL-17RA^-/- mice (confirming part of their phenotype), whereas production of IFN-γ and expression of the bactericidal enzyme iNOS were not affected. Collectively, our data suggest that early but not late neutrophil recruitment is essential for IL-17A mediated long-term control of M. tuberculosis infection and that a functional IFN-γ response is not sufficient to control M. tuberculosis growth when the IL-17RA pathway is deficient. As treatment of auto-immune diseases with anti-IL-17A antibodies is actually being tested in clinical studies, our data suggest that caution should be taken with respect to possible reactivation of tuberculosis.

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Autologous hematopoietic stem cell transplantation (HSCT) for relapsing-remitting multiple sclerosis is a potentially curative treatment, which can give rise to long-term disease remission. However, the mode of action is not yet fully understood. The aim of the study was to evaluate similarities and differences of the CD4+ T cell populations between HSCT-treated patients (n=12) and healthy controls (n=9). More specifically, we performed phenotyping of memory T cells, Tregs, T helper type 1 (Th1) and T helper type 17 (Th17) cells. Further, T cell reactivity to a tentative antigen: myelin oligodendrocyte glycoprotein was investigated in these patient populations. Patients treated with natalizumab (n=15) were included as a comparative group. White blood cells were analyzed with flow cytometry and T cell culture supernatants were analyzed with magnetic bead panel immunoassays.

HSCT-treated patients had similar levels of Tregs, Th1 and Th17 cells as healthy subjects, whereas natalizumab-treated patients had lower frequencies of Tregs, and higher frequencies of Th1 and Th17 cells. Cells from HSCT-treated patients cultured with overlapping peptides from myelin oligodendrocyte glycoprotein produced more TGF-β1 than natalizumab-treated patients suggestive of a suppressive response. Conversely, T cells from natalizumab-treated patients cultured with those peptides produced more IL-17, IL-1 and IL-10 indicating a Th17 response.

In conclusion, we demonstrate circumstantial evidence for the removal of auto-reactive T cell clones as well as development of tolerance after HSCT. These results parallel the long-term disease remission seen post HSCT.

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A sudden upsurge of fever cases with joint pain was observed in outpatient department, Community Health Centre, Rangat during July-August 2010 in Rangat Middle Andaman, India. The etiological agent responsible for the outbreak was identified as Chikungunya virus (CHIKV), by using reverse transcriptase based polymerase chain reaction and IgM ELISA. The study investigated the association of polymorphisms in the human leucocyte antigen class II genes with susceptibility or protection against CHIKV. 101 patients with clinical features suggestive of CHIKV infection and 104 healthy subjects were included in the study. DNA was extracted and typed for DRB1 and DQB1 alleles. Based on the amino acid sequences of HLA-DQB1 retrieved from IMGT/HLA database, critical amino acid differences in the specific peptide binding pockets of HLA-DQB1 molecules were investigated. The frequencies of DRB1 alleles were not significantly different, whereas lower frequency of HLA-DQB1*03:03 was observed in CHIKV patients compared to control population (P=0.001, Pc = 0.024; OR = 0, 95% CI=0.0-0.331; Peto's odds ratio=0.1317,95% CI=0.0428-0.405). Significantly lower frequency of glutamic acid at 86^th position of peptide binding pocket 1 coding HLA-DQB1 genotypes was observed in CHIKV patients compared to healthy controls (P=0.004, OR =0.307, 95% CI = 0.125-0.707). Computational binding predictions of CD4 epitopes of CHIKV NetMHCII revealed HLA-DQ molecules are known to bind more CHIKV peptides as compared to HLA-DRB1 molecules. The results suggest that HLA-DQB1 alleles and critical amino acid differences in the peptide binding pockets of HLA-DQB1 alleles might have role in influencing infection and pathogenesis of CHIKV.

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CD1d-restricted T (NKT) cells are important for controlling herpesvirus infections. Interestingly, herpes simplex virus (HSV) can down-regulate CD1d-mediated activation of NKT cells. We have previously shown that the Thr322 residue in the cytoplasmic tail of human CD1d is important for its intracellular trafficking and functional expression. We proposed that the phosphorylation of T322 is a signal for CD1d lysosomal targeting and subsequent degradation. In the current study, we generated dual mutants by substituting the T322 and S323 residues of wildtype (WT) CD1d with Ala (non-phosphorylatable) or Asp (mimicking phosphorylation) and ectopically expressed them in human embryonic kidney 293 cells. We found that the surface expression levels of the CD1d mutants was in this order: T322AS323A>WT>T322A>S323A>S323D>T322D>T322DS323D. Our results therefore suggest that mimicking the phosphorylation of both T322 and S323 has a cumulative negative effect on the functional expression of CD1d. As previously reported, we also found that upon an HSV infection, antigen presentation by WT CD1d is reduced and the CD1d molecule is degraded. Interestingly, the T322A/S323A double mutation inhibited CD1d degradation and rescued CD1d-mediated antigen presentation following an HSV-1 infection. This suggests that the T322/S323 dyad may be phosphorylated, which then targets CD1d for lysosomal degradation post-infection as a means of immune evasion, explaining (at least in part) the reduced antigen presentation observed. Thus, our findings strongly suggest that T322 and S323 form a dual residue motif that can regulate the functional expression of CD1d during a viral infection.

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Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by LPS plus IFN-γ or IL-4, respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2 RAPA reduced CXCR4, CD206 and CD209 expression and SCGF-β, CCL18 and CCL13 release. On the other side, in M1 RAPA increased CD86 and CCR7 expression and IL-6, TNF-α and IL-1β1γ release while it reduced CD206 and CD209 expression and IL-10, VEGF and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0.1 mg/kg/day) in 12 patients who were treated for at least 1 month prior to islet transplant. Cytokine release by TLR4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mTOR into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.

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Haptoglobin (Hp), a major acute-phase plasma protein, has been found in arthritic synovial fluid (SF). However, the function and structural modifications of Hp in arthritic SF are unknown. To investigate in vivo generation of modified Hp associated with inflammatory disease, we examined a new Hp isoform in SF from patients with rheumatoid arthritis (RA). Specific Hp fragments of 28 kDa and 15 kDa were identified in SF of patients with RA, and the two polypeptides were presumed to be fragments of the Hp β-chain (43 kDa) produced by cleavage with plasmin. The 15-kDa fragment, which is a C-terminal region of Hp, was observed at higher frequency and levels in RA than in osteoarthritis. Plasmin activity was also higher in SF of RA patients. A recombinant 15-kDa Hp fragment up-regulated interlukin-6 expression in monocytic cells. These findings indicate that C-terminal Hp fragment is generated by plasmin in local inflammatory environments and acts as an inflammatory mediator. They further suggest that a specific Hp fragment might be applied as a novel biomarker for the diagnosis and prognosis of inflammatory diseases such as RA.

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Systemic Lupus Erithematosus (SLE) is an autoimmune disease characterized by multiple alterations affecting the normal function of immune cells, such as lymphocytes, dendritic cells (DCs) and monocytes. Although the understanding of autoimmunity has been significantly increased recently, the breakthrough in effective therapies has been modest, making necessary the development of new therapeutic strategies. Here we propose that a new potential target for therapy is hemeoxygenase-1 (HO-1), an enzyme that catalyzes the degradation of the heme group into biliverdin, carbon monoxide (CO) and Fe⁺². These products exhibit immunosuppressive and anti-inflammatory effects, which can contribute to improving tolerance during organ transplantation. Because HO-1 is highly expressed by immune cells involved in SLE pathogenesis, such as monocytes and DCs, we evaluated whether induction of HO-1 expression or the administration of CO could ameliorate disease in the FcγRIIb KO mouse model for SLE. We found that CO administration decreased the expansion of CD11b⁺ cells, prevented the decline of regulatory T cells (Tregs) and reduced anti-histone antibodies observed in untreated FcγRIIb KO mice. Furthermore, CO-treated animals and HO-1 induction showed less kidney damage as compared to untreated mice. These data suggest that HO-1 modulation and CO administration can ameliorate autoimmunity and prevent the lupus symptoms shown by FcγRIIb KO mice, underscoring HO-1 as a potential new target for autoimmune therapy.

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The critical roles of kinase AKT in tumor cell proliferation, apoptosis and protein synthesis have been widely recognized. But AKT also plays an important role in the immune modulation. Recent studies have confirmed that kinase AKT can regulate the innate immune cell (neutrophil, macrophage and dendritic cell) development and functions. Studies have showed that different isoforms of kinase AKT have different effects in regulating immunity related diseases mainly through the mTOR dependent or independent pathway. The purpose of this review is just that illustrate the immune modulaton of kinase AKT on the innate immune cell development, survival and function.

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The current study sought to delineate the gene expression profile of the host response in the caecum and colon during acute infection with C. difficile in a mouse model of infection, and to investigate the nature of the unfolded protein response in this process. The infected mice displayed a significant up-regulation in the expression of chemokines (Cxcl1, Cxcl2 and Ccl2), numerous pro-inflammatory cytokines (Ifng, Il1b, Il6, and Il17f), as well as Il22 and a number of anti-microbial peptides (Defa1, Defa28, Defb1, Slpi and Reg3g) at the site(s) of infection. This was accompanied by a significant influx of neutrophils, dendritic cells, cells of the monocyte/macrophage lineage and all major subsets of lymphocytes to these site(s). However, CD4 T cells of the untreated and C. difficile-infected mice expressed similar levels of CD69 and CD25. Neither tissue had up-regulated levels of Tbx21, Gata3 or Rorc. The caeca and colons of the infected mice showed a significant increase in eIF2α phosphorylation, but neither the splicing of Xbp1 nor the up-regulation of endoplasmic reticulum chaperones, casting doubt on the full-fledged induction of the unfolded protein response by C. difficile. They also displayed significantly higher phosphorylation of AKT and STAT3, an indication of pro-survival signalling. These data underscore the local, innate, pro-inflammatory nature of the response to C. difficile and highlight eIF2α phosphorylation and the IL-22-pSTAT3-RegIIIγ axis as two of the pathways that could be utilised to contain and counteract the damage inflicted on the intestinal epithelium.

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Th2 cells are critical to host defense against helminth infection and the pathogenesis of allergic diseases. The differentiation of Th2 cells from naïve CD4 T cells is controlled by intricate transcriptional mechanisms. At the precursor stage of naïve CD4 T cells, transcriptional mechanisms maintain the potential and in the meantime prevent spontaneous differentiation to Th2 fate. In addition, intrachromosomal interactions important for coordinated expression of Th2 cytokines pre-exist in naïve CD4 T cells. Upon TCR engagement, naïve CD4 T cells are induced by polarizing signals of the IL-4/Stat6 and Jagged/Notch pathways to up regulate the expression of GATA-3. Once up regulated, GATA-3 drives Th2 and suppresses Th1 differentiation in a cell autonomous fashion. During the differentiation, the Th2 cytokine locus, as well as the IFN-gamma locus, undergoes chromatin remodeling and epigenetic modifications that contribute to the somatic memory of Th2 cytokine gene expression pattern. Once differentiated, Th2 effector cells promptly produce Th2 cytokines upon TCR stimulation, which is regulated by concerted actions of GATA-3, TCR signaling, enhancers and the Th2 locus control region. This review provides a detailed account of the transcriptional regulatory events at these different stages of Th2 differentiation.

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Natural Killer (NK) cell mediated contact sensitivity recently was described in mice. Here, we confirm NK cell mediated contact sensitivity (CS) in SCID and RAG1^-/- mice but not in SCID-_beige mice, which have nonfunctional NK cells that lack NK cell granules. NK cell mediated CS was transferred by liver mononuclear cells and the DX5⁺ fraction of liver cells, confirming that NK cells mediate contact sensitivity in the absence of T and B cells. Participation of NKT cells and B-1 cells was ruled out using Jα18^-/- and JH^-/- mice respectively. Remarkably, NK cell mediated CS was observed just 1-hr after immunization and was detectable as early as 30 min. post challenge. Further, we examined cytokine requirements for NK cell mediated CS, and found that liver mononuclear cells from IL-12^-/-, IFN-γ^-/- and IFN-αR^-/- donors fail to transfer NK cell mediated CS to naïve hosts.

Our studies clearly show that DNFB sensitized NK cells mediate very rapid, Ag-specific cell-mediated immunity, with features of both innate and acquired immune responses.

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Inflammatory Bowel Disease (IBD) is characterized by damage to the gut mucosa and systemic inflammation. We sought to evaluate the role of chronic inflammation on circulating T cell activation in human subjects with Crohn's Disease and ulcerative colitis. We studied 54 IBD patients and 28 healthy controls. T cell activation and cycling were assessed in whole blood samples by flow cytometry. Levels of lipopolysaccharide (LPS) were measured in serum by limulus amebocyte lysate assay, and plasma levels of inflammatory markers and LPS-binding proteins by ELISA. The proportions of circulating CD4+ and CD8+ T lymphocytes in cycle (Ki67+) are increased in IBD patients when compared to these proportions in controls. CD8+ T cells from IBD patients are also enriched for cells that expressed CD38 and HLA-DR, and proportions of these cells are related to plasma levels of IL-6 and CRP in these patients. Intracellular interleukin-2 and interferon-γ levels were elevated in resting and polyclonally activated CD4+ and CD8+ T cells in IBD patients when compared to levels from healthy controls. Surprisingly, we did not find increased levels of LPS in the serum of IBD patients. We did, however, find a signature of recent microbial translocation, as levels of LPS-binding protein (LBP) are increased in the plasma of IBD patients compared to plasma levels in healthy controls; LBP levels are also directly related to proportions of CD38 HLA-DR expressing CD4+ and CD8+ T cells. Local damage to the GI tract in IBD may result in systemic inflammation and T cell activation.

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Pathogenic New World hantaviruses cause severe disease in humans characterized by a vascular leak syndrome, leading to pulmonary edema and respiratory distress with case fatality rates approaching 40%. Hantaviruses infect microvascular endothelial cells without conspicuous cytopathic effects, indicating that destruction of the endothelium is not a mechanism of disease. In humans, high levels of inflammatory cytokines are present in the lungs of patients that succumb to infection. This, along with other observations, suggests that disease has an immunopathogenic component. Currently the only animal model available to study hantavirus disease is the Syrian hamster, where infection with Andes virus (ANDV), the primary agent of disease in South America, results in disease that closely mimics that seen in humans. Conversely, inoculation of hamsters with a passaged Sin Nombre virus (SNV), the virus responsible for most cases of disease in North America, results in persistent infection with high levels of viral replication. We found that ANDV elicited a stronger innate immune response, whereas SNV elicited a more robust adaptive response in the lung. Additionally, ANDV infection resulted in significant changes in the blood lymphocyte populations. To determine whether the adaptive immune response influences infection outcome, we depleted hamsters of CD4⁺ and CD8⁺ T cells prior to infection with hantaviruses. Depletion resulted in inhibition of virus-specific antibody responses, although the pathogenesis and replication of these viruses were unaltered. These data show that neither hantavirus replication, nor pathogenesis caused by these viruses, is influenced by the adaptive immune response in the Syrian hamster.

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Indomethacin is a cyclooxygenase inhibitor, and shows therapeutic potential for various eosinophilic skin diseases, particularly eosinophilic pustular folliculitis. One of the unique characteristics of indomethacin is that, unlike other non-steroidal anti-inflammatory drugs, it is a potent agonist of chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for prostaglandin (PG)D2. This study investigated the pharmacological actions of indomethacin on eosinophil migration to clarify the actual mechanisms underlying the therapeutic effects of indomethacin on eosinophilic pustular folliculitis. Eosinophils exhibited chemokinetic and chemotactic responses to both PGD2 and indomethacin through CRTH2 receptors. Pretreatment of eosinophils with indomethacin greatly inhibited eosinophil migration to PGD2 and, to a much lesser extent, to eotaxin (CCL11); these effects could be mediated by homologous and heterologous desensitization of eosinophil CRTH2 and CCR3, respectively, by agonistic effects of indomethacin on CRTH2. Indomethacin also cancelled a priming effect of Δ¹²-PGJ2, a plasma metabolite of PGD2, on eosinophil chemotaxis to eotaxin. Indomethacin down-modulated cell surface expression of both CRTH2 and CCR3. Hair follicle epithelium and epidermal keratinocytes around eosinophilic pustules together with the eccrine apparatus of palmoplantar lesions of eosinophilic pustular folliculitis were immunohistochemically positive for lipocalin-type PGD synthase. Indomethacin may exert therapeutic effects against eosinophilic skin diseases in which PGD2-CRTH2 signals play major roles by reducing eosinophil responses to PGD2.

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IL-33 and its receptor ST2 are over-expressed in clinical colitis tissue. However, the significance of these observations is at present unknown. Significantly, we demonstrate here that IL33 and ST2 are the primary early genes induced in the inflamed colon of BALB/c mice following dextran sulphate sodium (DSS)-induced experimental ulcerative colitis. Accordingly diarrhoea and DSS induced colon inflammation were impaired in ST2^-/- BALB/c mice and exacerbated in WT mice by treatment with exogenous recombinant IL-33, associated respectively with reduced and enhanced expression of chemokines (CXCL9 and CXCL10), inflammatory (IL-4, IL-13, IL-1, IL-6, IL-17), and angiogenic (VEGF) cytokines in vivo. The exacerbation effect of treatment with recombinant IL-33 on DSS-induced acute colitis was abolished in IL-4^-/- BALB/c mice. Thus, IL-33 signalling via ST2, by inducing an IL-4-dependent immune response may be a major pathogenic factor in the exacerbation of ulcerative colitis.

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Anti-Gal is the most abundant natural antibody in humans constituting ~1% of immunoglobulins. Anti-Gal is naturally produced also in apes and Old World monkeys. The ligand of anti-Gal is a carbohydrate antigen called the “α-gal epitope” with the structure Galα1-3Galβ1-4GlcNAc-R. The α-gal epitope is present as a major carbohydrate antigen in nonprimate mammals, prosimians and New World monkeys. Anti-Gal can contribute to several immunological pathogeneses. Anti-Gal IgE produced in some individuals causes allergies to meat and to the therapeutic monoclonal antibody cetuximab, all presenting α-gal epitopes. Aberrant expression of the α-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves’ Disease. α-Gal epitopes produced by Trypanosoma cruzi interact with anti-Gal and induce “autoimmune like” inflammatory reactions in Chagas Disease. Anti-Gal IgM and IgG further mediate rejection of xenografts expressing α-gal epitopes. Because of its abundance, anti-Gal may be exploited for various clinical uses. It increases immunogenicity of microbial vaccines (e.g., flu vaccine) presenting α-gal epitopes by targeting them for effective uptake by APC. Tumor lesions are converted into vaccines against autologous tumor associated antigens by intratumoral injection of α-gal glycolipids which insert into tumor cell membranes. Anti-Gal binding to α-gal epitopes on tumor cells targets them for uptake by APC. Accelerated wound healing is achieved by application of α-gal nanoparticles which bind anti-Gal, activate complement, recruit and activate macrophages that induce tissue regeneration. This therapy may be of further significance in regeneration of internally injured tissues such as ischemic myocardium and injured nerves.

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Swine leukocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4⁺T cells. A human dominant-negative mutant class II transactivator (CIITA-DN) transgene under a CAG promoter with an endothelium-specific Tie2 enhancer was constructed. CIITA-DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA-DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4⁺T cell response to cells from CIITA-DN and wild-type (WT) pigs was compared. Lymphocyte subset numbers and T cell function in CIITA-DN pigs were compared with WT pigs. The expression of SLA class II on antigen-presenting cells from CIITA-DN pigs was significantly reduced (40-50% reduction compared with WT; P<0.01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P<0.01). The human CD4⁺T cell response to CIITA-DN pAECs was significantly weaker than to WT pAECs (60-80% suppression; P<0.01). Although there was a significantly lower frequency of CD4⁺ cells in the PBMCs from CIITA-DN (20%) than from WT (30%) pigs (p<0.01), T cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA-DN pigs should be partially protected from the human cellular immune response.

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Activation of CD4+ T cells helps establish and maintain immune responses. During infection with Lymphocytic Choriomeningitis Virus (LCMV) clone 13, the CD4+ T cells responses are lost. In this study, we were interested in the nature of the CD4+ T cell responses following infection with LCMV clone 13. To pursue this question, we infected C57BL/6 mice with LCMV clone 13. We used a GP66-80 MHC Class II tetramer to determine whether or not the CD4+ T cells were present following infection with LCMV clone 13. We determined that the cells were present and antigen specific, but not functional. We attributed their dysfunction to the presence of CD4+ T cell inhibitory ligands. We further stained for presence of CD4+ T cell inhibitory ligands. We found that the during chronic infection the number of CD4+ T cells expressing PD-1 and CD160 were greater over the time course study than the other CD4+ T cell inhibitory ligands. These data show that using CD4+ T cell inhibitory ligands as a reagent for characterization can help aid in understanding the complex immune responses associated with persistent infections.

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It has been reported that the initiation of HAART is associated with the development of reversal reaction (RR) in co-infected HIV / leprosy patients. Nevertheless, the impact of HIV and HAART therapy on the cellular immune response to M. leprae (ML) remains unknown. In the present study, we observed that ex vivo PBMCs of both RR and RR/HIV patients presented increased percentages of activated CD4⁺T cells when compared with the HC group. The frequency of CD8⁺CD38⁺ cells increased in the PBMCs of RR / HIV but not in RR patients when compared with the HC group. RR and RR/HIV skin lesion cells presented similar percentages of activated CD4⁺ cells, but the numbers of activated CD8⁺ cells were higher in RR/HIV in comparison to the RR group. The frequency of IFN-γ-producing cells was high in response to ML regardless of HIV co-infection. In ML-stimulated cells, there was an increase in TCM CD4⁺ T cell frequencies in the RR and RR/HIV groups, but an increase in TCM CD8⁺ T cell frequency was only observed in the RR/HIV group. ML increased granzyme B⁺ TEM CD8⁺ T cell frequencies in the RR/HIV PBMCs, but not in the HC and RR groups. Our data suggest that the increased expression of TEM CD8⁺ T cells together with greater perforin/granzyme B production could be an additional mechanism leading to the advent of RR in co-infected patients. Moreoever, this increased expression may explain the severity of RR occurring in these patients.

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Approaches to evaluate T-cell responses to Epstein-Barr virus (EBV) include the enzyme-linked immunospot (ELISPOT), which quantify cells capable of immediate IFN-γ secretion upon antigen stimulation. However, evaluation of expandable EBV-specific memory T cells in an ELISPOT formant has not been described previously. We quantified EBV-specific T-cell precursors with high proliferative capacity by using a peptide-based cultured IFN-γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15-mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant (SOT) recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV-specific T-cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared to standard ELISPOT responses. T-cell responses quantified by cultured ELISPOT were mainly mediated by CD4⁺ T cells and a marked pattern of immunodominance to latent-phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV-specific T-cell responses were altered in SOT recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung-transplanted patient with EBV-associated diseases. Analysis of T-cell responses to EBV by ELISPOT assays might provide new insights in the pathogenesis of EBV-related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.

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Schistosomiasis japonica is a severe tropical disease caused by the parasitic worm Schistosoma japonicum. Among the most serious pathological effects of S. japonicum infection are hepatic lesions (cirrhosis and fibrosis) and portal hypertension. Interleukin-17 (IL-17) is a pro-inflammatory cytokine involved in the pathogenesis of many inflammatory and infectious conditions, including schistosomiasis. We infected C57BL/6 mice with Schistosoma japonicum and isolated lymphocytes from the liver to identify cell subsets with high IL-17 expression and release using flow cytometry and ELISA. Expression and release of IL-17 was significantly higher in hepatic lymphocytes from infected mice compared to control mice in response to both non-specific stimulation with anti-CD3 mAb+/anti-CD28 mAb and phorbol 12-myristate 13-acetate (PMA) plus ionomycin. We then compared IL-17 expression in three hepatic T cell subsets, Th, NKT, and γδT, to determine the major source of IL-17 during infection. IL-17 was induced in all three subsets by PMA + ionomycin, but γδT lymphocytes exhibited the largest increase in expression. We then established a mouse model to further investigate the role of IL-17 in granulomatous and fibrosing inflammation against parasite eggs. Reducing IL-17 activity using anti-IL-17A antibodies decreased infiltration of inflammatory cells and collagen deposition in infected C57BL/6 mouse liver. The serum levels of soluble egg antigen (SEA)-specific IgGs were enhanced by anti-IL-17A mAb blockade, suggesting that IL-17 normally serves to suppress this humoral response. These findings suggest that γδT cells are the most IL-17-producing cells and that IL-17 contributes to granulomatous inflammatory and fibrosing reactions in S. japonicum-infected C57BL/6 mouse liver.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Heat Shock Proteins (HSP) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen presenting cells (APC), including dendritic cells (DC). Targeting is achieved through binding of HSP to distinct cell surface receptors and is followed by antigen internalisation, processing and presentation. An improved understanding of the interaction of HSP with DC has driven the development of numerous HSP-containing vaccines, designed to deliver antigens directly to DC. Studies in mice have shown that for cancers, such vaccines generate impressive immune responses and protection from tumour challenge. However, translation to human use, as for many experimental immunotherapies, has been slow partly because of the need to perform trials in patients with advanced cancers where demonstration of efficacy is challenging. Recently, the properties of HSP have been utilised for development of prophylactic vaccines against infectious diseases including tuberculosis, and meningitis. These HSP-based vaccines, in the form of pathogen-derived HSP antigen complexes, or recombinant HSP combined with selected antigens in vitro, offer an innovative approach against challenging diseases where broad antigen coverage is critical.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Alzheimer's disease (AD) is the most common form of dementia, with prevalence progressively increasing with aging. Pathological hallmarks of the disease include accumulation of amyloid-beta (Aβ) peptides and neurofibrillary tangles in the brain associated with glial activation and synpatotoxicity. In addition, AD involves peripheral and brain-endogenous inflammatory processes that appear to enhance disease progression. More than a decade ago a new therapeutic paradigm has emerged for AD, namely the activation of the adaptive immune system directly against the self-peptide Aβ, aiming at lowering its accumulation in the brain. This was the first time that a brain peptide was used to vaccinate human subjects in a manner similar to classic viral or bacterial vaccines. The vaccination approach took several turns, from initially active to passive and then back to modified active vaccines. As the two first approaches to date failed to show sufficient efficacy, the latter is presently being evaluated in ongoing clinical trials. The present review summarizes the immunogenic characteristics of Aβ in human and mice and discusses past, present and future Aβ-based immunotherapeutic approaches for AD. We emphasize potential pathogenic and beneficial roles of CD4 T cells in light of the pathogenesis and the general decline in T-cell responsiveness evident in the disease.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Impaired local cellular immunity contributes to the pathogenesis of persistent high-risk human papillomavirus (HR-HPV) infection and related cervical intraepithelial neoplasia (CIN), but the underlying molecular mechanisms remain unclear. Recently, the PD-1/PD-L1 (CD279/CD274) pathway was demonstrated to play a critical role in attenuating T-cell responses and promoting T-cell tolerance during chronic virus infections. In this study, we examined the expression of PD-1 and PD-L1 on cervical T cells and dendritic cells (DCs), respectively, from 40 women who were HR-HPV–negative (-) or HR-HPV–positive (+) with CIN grades 0, I and II-III. We also measured IFN-γ, IL-12 and IL-10 levels in cervical exudates. The most common HPV type was HPV 16, followed by HPV 18, 33, 51 and 58. PD-1 and PD-L1 expression on cervical T cells and DCs, respectively, was associated with HR-HPV positivity and increased in parallel with increasing CIN grade. The opposite pattern was observed for CD80 and CD86 expression on DCs, which decreased in HR-HPV (+) patients in parallel with increasing CIN grade. Similarly, reduced levels of the Th1-type cytokines IFN-γ and IL-12 and increased levels of the Th2-type cytokine IL-10 in cervical exudates correlated with HR-HPV positivity and CIN grade. Our results suggest that upregulation of the inhibitory PD-1/PD-L1 pathway may negatively regulate cervical cell-mediated immunity to HPV and contribute to the progression of HR-HPV–related CIN. These results may aid in the development of PD-1/PD-L1 pathway-based strategies for immunotherapy of HR-HPV–related CIN.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

ADP-ribosylation is the addition of one or more (up to some hundreds) ADP-ribose moieties to acceptor proteins. There are two major families of enzymes that catalyze this reaction: extracellular ADP-ribosyl-transferases (ARTs), which are bound to the cell membrane by a GPI anchor or are secreted, and poly(ADP-ribose)-polymerases (PARPs), which are present in the cell nucleus and/or cytoplasm. Recent findings revealed a wide immunological role for ADP-ribosylating enzymes. ARTs, by sensing extracellular NAD concentration, can act as danger detectors. PARP-1, the prototypical representative of the PARP family, known to protect cells from genomic instability, is involved in the development of inflammatory responses and several forms of cell death. PARP-1 also plays a role in adaptive immunity by modulating the ability of dendritic cells to stimulate T cells or by directly affecting differentiation and functions of T and B cells. Both PARP-1 and PARP-14 (CoaSt6) KO mice were described to display reduced Th2 cell differentiation and allergic responses. Our recent findings showed that PARP-1 is involved in the differentiation of Foxp3⁺ regulatory T cells, suggesting a role for PARP-1 in tolerance induction. Also ARTs regulate Treg cell homeostasis by promoting Treg apoptosis during inflammatory responses. PARP inhibitors ameliorates immune-mediated diseases in several experimental models, including rheumatoid arthritis, colitis, experimental autoimmune encephalomyelitis, and allergy. All together these findings show that ADP-ribosylating enzymes, in particular PARP-1, play a pivotal role in the regulation of immune responses and may represent a good target for new therapeutic approaches in immune-mediated diseases.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Innate lymphoid cells (ILCs) is the collective term for a group of related innate lymphocytes, including NK cells and the more recently appreciated non-NK ILCs, which all lack rearranged antigen-receptors such as those expressed by T and B cells. Similar to NK cells, the newly discovered ILCs depend on the transcription factor Id2 and the common γ-chain of the IL-2 receptor for development. However, in contrast to NK cells, non-NK ILCs also require IL-7. In addition to the cytotoxic functions of NK cells, assuring protection against tumor development and viruses, new data indicate that ILCs contribute to a wide range of homeostatic and pathophysiological conditions in various organs via specialized cytokine-production capabilities. Here we summarize current knowledge on ILCs with a particular emphasis on their tissue-specific effector functions, in the gut, liver, lungs and uterus. When possible, we try to highlight the role these cells play in humans.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumor necrosis factor-alpha (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. TNF-α exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR-1 and sTNFR-2). The signaling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated M. bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1^-/- mice at day 3 after BCG infection. These results suggest a potential role for TNFR1 in mycobacteria induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain dependent manner.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Inflammatory bowel disease (IBD) is characterized by dysregulated immune responses in inflamed intestine, with dominance of IL-17-producing cells and deficiency of Treg cells. The aim of this study was to investigate the effect and mechanisms of sirolimus, an inhibitor of the mammalian target of rapamycin (mTOR), on immune responses in a murine model of Crohn's disease (CD). Murine colitis was induced by intrarectal administration with 2,4,6-trinitrobenzene sulfonic acid (TNBS) at day 0. Mice were then i.p.-treated with sirolimus daily for three days. The gross and histological appearances of the colon and the numbers, phenotype and cytokine production of lymphocytes were compared with these characteristics in a control group. Sirolimus treatment significantly decreased all macroscopic, microscopic and histopathologic parameters of colitis analyzed. The therapeutic effects of sirolimus were associated with a down-regulation of proinflammatory cytokines TNF-α, IL-6 and IL-17A. Intriguingly, sirolimus administration resulted in a prominent up-regulation of regulatory cytokine TGF-β. Supporting the hypothesis that sirolimus directly affects functional activity of CD4⁺CD25⁺ Treg cells, we observed a remarkable enhancement of FoxP3 expression in colon tissues and isolated CD4⁺ T cells of sirolimus-treated mice. Simultaneously, sirolimus treatment led to a significant reduction in the number of CD4⁺IL-17A⁺ T cells in the MLN cells as well as IL-17A production in MLN cells. Therefore, sirolimus may offer a promising new therapeutic strategy for the treatment of IBD.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Microbial infections are a major cause of infant mortality worldwide as a result of impaired immune defenses in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin (IL)-27 is a heterodimeric cytokine of the IL-12 family that is produced primarily by antigen presenting cells and is immunosuppressive toward a variety of immune cell types. Here we show that IL-27 gene expression is elevated in cord blood-derived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration in which elevated IL-27 gene expression may impact immune responses in mice. Age-dependent analysis of IL-27 gene expression indicated that levels of IL-27 remain significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine-stained splenocytes further confirmed these results. IL-27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal-maternal interface since we demonstrate dose-responsive gene expression to progesterone in macrophages. Neutralization of IL-27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL-27 during incubation with the Mycobacterium bovis BCG vaccine augmented the level of interferon (IFN)-γ elicited from allogeneic CD4⁺ T lymphocytes. This suggests that blocking IL-27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Dendritic cells (DC) are the main immune mediators inducing primary immune responses. DC generated from Monocytes (MoDC) are a model system to study the biology of DC in vitro, as they represent inflammatory DC in vivo. Previous studies on the generation of MoDC in horses indicated that there was no distinct difference between immature and mature DC and that the expression profile was distinctly different from humans, where CD206 is expressed on immature MoDC while CD83 is expressed on mature MoDC. Here we describe the kinetics of equine MoDC differentiation and activation analysing both phenotypic and functional characteristics. Blood monocytes (Mo) were first differentiated with equine granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) generating immature DC (iMoDC). These cells were further activated with a cocktail of cytokines including interferon-gamma (IFNγ) but not CD40L to obtain mature DC (mMoDC). In order to determine the expression of a broad range of markers for which no monoclonal antibodies were available to analyse the protein expression, microarray and qPCR analysis were performed to carry out gene expression analysis. This study demonstrates that equine iMoDC and mMoDC can be distinguished both phenotypically and functionally but the expression pattern of some markers including CD206 and CD83 is dissimilar to the human system.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins – OmpS1 and OmpS2 – that are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunising mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor (TLR) 4 agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of TNF and IL-6, and OmpS2 was also able to induce IL-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunisation of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T-lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunised with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus (A(H1N1)pdm09). Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

In addition to archetypal cognitive defects, Down Syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess altered T cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a 3-4 fold decrease in the number and proportion of immature, double negative (DN) thymocyte progenitors. In addition, there were two-fold less double-positive (DP) and CD4 single-positive (SP) thymocytes in Ts65Dn thymii. Reflecting this deficient thymic function, there were decreased naïve T cells in the spleen and polyclonal stimulation of peripheral T cells exhibited a marked reduction in proliferation suggesting a senescent phenotype. In contrast B cell progenitors were unchanged in the bone marrow of Ts65Dn mice, but in the spleen, there were decreased transitional and follicular B cells and these cells proliferated less upon antigen receptor stimulus but not in response to LPS. As a potential mechanism for diminished thymic function, immature thymocyte populations expressed diminished levels of the cytokine receptor IL-7Rα, which was associated with decreased proliferation and increased apoptosis. Increased oxidative stress and inhibition of the Notch pathway were identified as possible mediators of decreased IL-7Rα expression in Ts65Dn mice. The data suggest that immature thymocyte defects underlie immune dysfunction in DS and that increased oxidative stress and reduced cytokine signaling may alter lymphocyte development in Ts65Dn mice.

© 2013 The Authors. Immunology © 2013 Blackwell Publishing Ltd

A critical component of vaccine design is to generate and maintain antigen-specific memory lymphocytes of sufficient quantity and quality to give the host life-long protection against re-infection. Therefore, it is important to understand how memory T cells acquire the ability for self-renewal while retaining a potential for heightened recall of effector functions. During acute viral infection or following vaccination, antigen-specific T cells undergo extensive phenotypic and functional changes during differentiation to the effector and memory phases of the immune response. The changes in cell phenotype that accompany memory T-cell differentiation are predominantly mediated through acquired transcriptional regulatory mechanisms, in part achieved through epigenetic modifications of DNA and histones. Here we review our current understanding of epigenetic mechanisms regulating the off-on-off expression of CD8 and CD4 T-cell effector molecules at naive, effector and memory stages of differentiation, respectively, and how covalent modifications to the genome may serve as a mechanism to preserve ‘poised’ transcriptional states in homeostatically dividing memory cells. We discuss the potential of such mechanisms to control genes that undergo on-off-on patterns of expression including homing and pro-survival genes, and the implications on the development of effector-memory and central-memory T-cell differentiation. Lastly, we review recent studies demonstrating epigenetic modifications as a mechanism for the progressive loss of transcriptional adaptation in antigen-specific T cells that undergo sustained high levels of T-cell receptor signalling.

T cells are exquisitely poised to respond rapidly to pathogens and have proved an instructive model for exploring the regulation of inducible genes. Individual genes respond to antigenic stimulation in different ways, and it has become clear that the interplay between transcription factors and the chromatin platform of individual genes governs these responses. Our understanding of the complexity of the chromatin platform and the epigenetic mechanisms that contribute to transcriptional control has expanded dramatically in recent years. These mechanisms include the presence/absence of histone modification marks, which form an epigenetic signature to mark active or inactive genes. These signatures are dynamically added or removed by epigenetic enzymes, comprising an array of histone-modifying enzymes, including the more recently recognized chromatin-associated signalling kinases. In addition, chromatin-remodelling complexes physically alter the chromatin structure to regulate chromatin accessibility to transcriptional regulatory factors. The advent of genome-wide technologies has enabled characterization of the chromatin landscape of T cells in terms of histone occupancy, histone modification patterns and transcription factor association with specific genomic regulatory regions, generating a picture of the T-cell epigenome. Here, we discuss the multi-layered regulation of inducible gene expression in the immune system, focusing on the interplay between transcription factors, and the T-cell epigenome, including the role played by chromatin remodellers and epigenetic enzymes. We will also use IL2, a key inducible cytokine gene in T cells, as an example of how the different layers of epigenetic mechanisms regulate immune responsive genes during T-cell activation.

Naive T cells differentiate and become distinct subsets in response to changes in the cytokine milieu. Such specialization arises through a complex and dynamic utilization of cis-regulatory enhancer elements. In this brief essay, we review recent findings on the relative contributions of sensors of the cytokine milieu, especially the signal transducer and activator of transcription family transcription factors, ‘master regulators’, and other transcription factors in the enhancer architecture of T cells. These findings provide new insights into how signal transduction impinges upon the genome.

Mature naive CD4 T-cells possess the potential for an array of highly specialized functions, from inflammatory to potently suppressive. This potential is encoded in regulatory DNA elements and is fulfilled through modification of chromatin and selective activation by the collaborative function of diverse transcription factors in response to environmental cues. The mechanisms and strategies employed by transcription factors for the programming of CD4 T-cell subsets will be discussed. In particular, the focus will be on co-operative activity of environmental response factors in the initial activation of regulatory DNA elements and chromatin alteration, and the subsequent role of ‘master regulator’ transcription factors in defining the fidelity and environmental responsiveness of different CD4 T-cell subsets.

We identified CD8⁺ CD122⁺ regulatory T cells (CD8⁺ CD122⁺ Treg cells) and reported their importance in maintaining immune homeostasis. The absence of CD8⁺ CD122⁺ Treg cells has been shown to lead to severe systemic autoimmunity in several mouse models, including inflammatory bowel diseases and experimental autoimmune encephalomyelitis. The T-cell receptors (TCRs) expressed on CD8⁺ CD122⁺ Treg cells recognize the target cells to be regulated. To aid in the identification of the target antigen(s) recognized by TCRs of CD8⁺ CD122⁺ Treg cells, we compared the TCR diversity of CD8⁺ CD122⁺ T cells with that of conventional, naive T cells in mice. We analysed the use of TCR-Vβ in the interleukin 10-producing population of CD8⁺ CD122⁺ T cells marked by high levels of CD49d expression, and found the significantly increased use of Vβ13 in these cells. Immunoscope analysis of the complementarity-determining region 3 (CDR3) of the TCR β-chain revealed remarkable skewing in a pair of Vβ regions, suggesting the existence of clonally expanded cells in CD8⁺ CD122⁺ T cells. Clonal expansion in Vβ13⁺ cells was confirmed by determining the DNA sequences of the CDR3s. The characteristic TCR found in this study is an important building block for further studies to identify the target antigen recognized by CD8⁺ CD122⁺ Treg cells.

Fli-1 belongs to the Ets transcription factor family and is expressed in haematopoietic cells, including most of the cells that are active in immunity. The mononuclear phagocytes, i.e. monocytes, macrophages and dendritic cells, originate in haematopoietic stem cells and play an important role in immunity. To assess the role of Fli-1 in mononuclear phagocyte development in vivo, we generated mice that express a truncated Fli-1 protein, lacking the C-terminal transcriptional activation domain (Fli-1^ΔCTA). Fli-1^ΔCTA/ΔCTA mice had significantly increased populations of haematopoietic stem cells and common dendritic cell precursors in bone marrow compared with wild-type littermates. Significantly increased classical dendritic cells, plasmacytoid dendritic cells, and macrophage populations were found in spleens from Fli-1^{∆CTA/∆CTA} mice compared with wild-type littermates. Fli-1^ΔCTA/ΔCTA mice also had increased pre-classical dendritic cell and monocyte populations in peripheral blood mononuclear cells. Furthermore, bone marrow reconstitution studies demonstrated that expression of Fli-1 in both haematopoietic cells and stromal cells affected mononuclear phagocyte development in mice. Expression of Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic growth factor, in multipotent progenitors was statistically significantly increased from Fli-1^{∆CTA/∆CTA} mice compared with wild-type littermates. Fli-1 protein binds directly to the promoter region of the Flt3L gene. Hence, Fli-1 plays an important role in the mononuclear phagocyte development, and the C-terminal transcriptional activation domain of Fli-1 negatively modulates mononuclear phagocyte development.

Complement factor H (Cfh) is a key regulator of the complement cascade and protects C57BL/6 mice from immune complex-mediated complement-dependent glomerulonephritis. In chronic serum sickness (CSS) there are increased deposits of immune complexes in the glomeruli with inflammation and a scarring phenotype. As cucurmin is an effective anti-inflammatory agent and reduces complement activation, we hypothesized that it should alleviate renal disease in this setting. To determine the effectiveness of curcumin, an apoferritin-induced CSS model in Cfh-deficient (Cfh^−/−) mice was used. Curcumin treatment (30 mg/kg) given every day in parallel with apoferritin reduced glomerulonephritis and enhanced kidney function (blood urea nitrogen, 45·4 ± 7·5 versus 35·6 ± 5·1; albuminuria, 50·1 ± 7·1 versus 15·7 ± 7·1; glomerulonephritis, 2·62 + 0·25 versus 2 + 0·3, P < 0·05). In line with reduced IgG deposits in mice with CSS given curcumin, C9 deposits were reduced indicating reduced complement activation. Mice treated with curcumin had a significant reduction in the number of splenic CD19⁺ B cells and the ratio of CD19 : CD3 cells (P < 0·05) with no change in the T-cell population. Myeloperoxidase assay showed reduced macrophages in the kidney. However, a significant reduction in the M2 subset of splenic macrophages by apoferritin was prevented by curcumin, suggesting a protective function. Curcumin treatment reduced mRNA expression of inflammatory proteins monocyte chemoattractant protein-1 and transforming growth factor-β and matrix proteins, fibronectin, laminin and collagen. Our results clearly illustrate that curcumin reduces glomerulosclerosis, improves kidney function and could serve as a therapeutic agent during serum sickness.

In monoclonal gammopathies (MG) and multiple myeloma (MM), normal natural cytotoxicity receptors (NCR) expression (NCR1/NKp46, NCR2/NKp44, NCR3/NKp30) is observed in natural killer (NK) cells. Nonetheless, except in plasma cell leukemia, few tumor plasmocytes are present in PB, while NK studies have been performed on peripheral blood (PB). For this reason we focused our attention on NK from bone marrow (BM). Our study demonstrates that the down-regulation of NCR3/NKp30 is only detectable in NK from BM but not in PB, and shows a drastic decrease of both NKG2D and CD244/2B4/p38 expression in NK from BM in comparison with PB. In conclusion, our data more precisely describe the mechanism of immune escape of MG/MM from innate immunity since we show a drastic down regulation of 3 major activating NK receptors (NCR3/NKp30, NKG2D and CD244/2B4/p38) at the site of tumor, i.e BM, that was undetectable in PB. Further studies regarding immune regulatory drugs in MG/MM will imperiously require the assessment of immune cell status not only in PB but also in BM to obtain more relevant data regarding anti-tumor efficacy.

HIV-specific T cells that produce interferon-γ (IFN-γ) are present in the genital tract of HIV-infected women although these do not provide protection against genital HIV shedding. Because polyfunctional HIV-specific T cells have been implicated in better HIV control than those with a single function, this study aimed to investigate whether polyfunctional T cells were present at the female genital mucosa. Cervical cytobrush-derived T cells were obtained from chronically HIV-infected women and compared with blood. CD3⁺ T cells from both compartments were expanded with Dynal anti-CD3/CD28 expander beads for 14 days and flow cytometry was used to evaluate four T-cell functions (CD107a, IFN-γ, tumour necrosis factor-α and macrophage inflammatory protein-1β) from 16 women. The majority of Gag-specific T-cell responses in the female genital tract were monofunctional, although low frequencies of HIV Gag-specific polyfunctional CD8⁺ T cells were detected at the cervix in 81·3% (13/16) of women. The ability of CD8⁺ T cells at both the cervix and in blood to express CD107a and to exhibit polyfunctional responses (two or more functions) following Gag stimulation was inversely associated with plasma viral load and positively associated with blood CD4 counts, suggesting that clinical status impacted on the functionality of HIV-specific T cells at the mucosa, in a similar way to blood. HIV Gag-specific cervical T cells were largely monofunctional. Polyfunctional T cells were detected at the cervix in women with high blood CD4 count and low plasma viral load but these did not protect from HIV genital shedding.

We have previously demonstrated that the anti-inflammatory prostaglandin 15-deoxy-Δ 12,14-prostaglandin J₂ (15dPGJ₂) delays inflammation-induced preterm labour in the mouse and improves pup survival through the inhibition of nuclear factor-κB (NF-κB) by a mechanism yet to be elucidated. 15dPGJ₂ is an agonist of the second prostaglandin D₂ receptor, chemoattractant receptor homologous to the T helper 2 cell (CRTH2). In human T helper cells CRTH2 agonists induce the production of the anti-inflammatory interleukins IL-10 and IL-4. We hypothesized that CRTH2 is involved in the protective effect of 15dPGJ₂ in inflammation-induced preterm labour in the murine model. We therefore studied the effects of a specific small molecule CRTH2 agonist on preterm labour and pup survival. An intrauterine injection of lipopolysaccharide (LPS) was administered to CD1 mice at embryonic day 16, ± CRTH2 agonist/vehicle controls. Mice were killed at 4.5 hr to assess fetal wellbeing and to harvest myometrium and pup brain for analysis of NF-κB, and T helper type 1/2 interleukins. To examine the effects of the CRTH2 agonist on LPS-induced preterm labour, mice were allowed to labour spontaneously. Direct effects of the CRTH2 agonist on uterine contractility were examined ex vivo on contracting myometrial strips. The CRTH2 agonist increased fetal survival from 20 to 100% in LPS-treated mice, and inhibited circular muscle contractility ex vivo. However, it augmented LPS-induced labour and significantly increased myometrial NF-κB, IL-1β, KC-GRO, interferon-γ and tumour necrosis factor-α. This suggests that the action of 15dPGJ₂ is not via CRTH2 and therefore small molecule CRTH2 agonists are not likely to be beneficial for the prevention of inflammation-induced preterm labour.

Human metapneumovirus (hMPV) is the second most common cause of acute lower respiratory tract infections in children, causing a significant public health burden worldwide. Given that hMPV can repeatedly infect the host without major antigenic changes, it has been suggested that hMPV may have evolved molecular mechanisms to impair host adaptive immunity and, more specifically, T-cell memory. Recent studies have shown that hMPV can interfere with superantigen-induced T-cell activation by infecting conventional dendritic cells (DCs). Here, we show that hMPV infects mouse DCs in a restricted manner and induces moderate maturation. Nonetheless, hMPV-infected DCs are rendered inefficient at activating naive antigen-specific CD4⁺ T cells (OT-II), which not only display reduced proliferation, but also show a marked reduction in surface activation markers and interleukin-2 secretion. Decreased T-cell activation was not mediated by interference with DC–T-cell immunological synapse formation as recently described for the human respiratory syncytial virus (hRSV), but rather by soluble factors secreted by hMPV-infected DCs. These data suggest that although hMPV infection is restricted within DCs, it is sufficient to interfere with their capacity to activate naive T cells. Altogether, by interfering with DC function and productive priming of antigen-inexperienced T cells, hMPV could impair the generation of long-term immunity.

The IMiDs^® immunomodulatory compounds lenalidomide and pomalidomide are agents with anti-inflammatory, immunomodulatory and anti-cancer activity. An excellent success rate has been shown for multiple myeloma in phase I/II clinical trials leading to Food and Drug Administration approval of lenalidomide. One mechanism by which these drugs could enhance anti-tumour immunity may be through enhanced dendritic cell (DC) function. Thalidomide, a compound structurally related to lenalidomide and pomalidomide, is known to enhance DC function, and we have investigated whether its analogues, pomalidomide and lenalidomide, also have functional effects on DCs. We used mouse bone marrow-derived DCs treated with 5 or 10 μm pomalidomide, or lenalidomide from day 1 of culture. Treatment with IMiD^® immunomodulatory compounds increased expression of Class I (H2-Kb), CD86, and pomalidomide also increased Class II (I-Ab) expression in bone marrow-derived DCs, as measured by flow cytometry. Fluorescent bead uptake was increased by up to 45% when DCs were treated with 5 or 10 μm pomalidomide or lenalidomide compared with non-treated DCs. Antigen presentation assays using DCs primed with ovalbumin, and syngeneic T cells from transgenic OTI and OTII mice (containing MHC restricted, ovalbumin-specific, T cells) showed that both pomalidomide and lenalidomide effectively increased CD8⁺ T-cell cross-priming (by up to 47%) and that pomalidomide alone was effective in increasing CD4⁺ T-cell priming (by 30%). Our observations suggest that pomalidomide and lenalidomide enhance tumour antigen uptake by DCs with an increased efficacy of antigen presentation, indicating a possible use of these drugs in DC vaccine therapies.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are bioactive n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in fish oil that exert immunosuppressive effects. A significant amount of literature shows that n-3 LCPUFAs suppress dendritic cell (DC) function in vitro; however, few studies have determined if the effects are emulated at the animal level. In this study, we first focused on the functional consequences of 5% (weight/weight) fish oil on splenic CD11c⁺ DCs. Administration of n-3 LCPUFAs, modelling human pharmacological intake (2% of total kcal from EPA,1·3% from DHA), to C57BL/6 mice for 3 weeks reduced DC surface expression of CD80 by 14% and tumour necrosis factor-α secretion by 29% upon lipopolysaccharide stimulation relative to a control diet. The n-3 LCPUFAs also significantly decreased CD11c⁺ surface expression and phagocytosis by 12% compared with the control diet. Antigen presentation studies revealed a 22% decrease in CD69 surface expression on transgenic CD4⁺ T lymphocytes activated by DCs from mice fed fish oil. We then determined if the functional changes were mechanistically associated with changes in lipid microdomain clustering or plasma membrane microviscosity with n-3 LCPUFAs, as reported for B and T lymphocytes. Fish oil administration to mice did not influence cholera-toxin induced lipid microdomain clustering or microviscosity, even though EPA and DHA levels were significantly elevated relative to the control diet. Overall, our data show that n-3 LCPUFAs exert immunosuppressive effects on DCs, validating in vitro studies. The results also show that DC microdomain clustering and microviscosity were not changed by the n-3 LCPUFA intervention used in this study.

n-Butyrate deriving from bacterial fermentation in the mammalian intestine is a key determinant in gastrointestinal homeostasis. We examined the effects of this short-chain fatty acid and Toll-like receptor 2 (TLR) and TLR4 engagement on inflammatory/immunity-associated genes, cyclo-oxygenases (COXs), prostaglandins (PGs) and leukotrienes (LTs) in human monocytes. Before RNA isolation, freshly isolated human monocytes were co-incubated for different time-points with 1 mm n-butyrate alone or in combination with bacterial stimuli. Based on a knowledge-driven approach, a signature of 180 immunity/inflammation-associated genes was picked and real-time PCR analysis was performed. Pathway analysis was carried out using a web-based database analysing program. Based on these gene expression studies the findings were evaluated at the protein/mediator level by Western blot analysis, FACS and ELISA. Following co-incubation with n-butyrate and lipopolysaccharide, key enzymes of the eicosanoid pathway, like PTGS2 (COX-2), TXS, ALOX5, LTA4H and LTC4S, were significantly up-regulated compared with stimulation with lipopolysaccharide alone. Furthermore, release of the lipid mediators PGE₂, 15d-PGJ_2, LTB₄ and thromboxane B₂ was increased by n-butyrate. Regarding signalling, n-butyrate had no additional effect on mitogen-activated protein kinase and interfered differently with early and late phases of nuclear factor-κB signalling. Our results suggest that among many other mediators of eicosanoid signalling n-butyrate massively induces PGE₂ production by increasing the expression of PTGS2 (COX-2) in monocytes following TLR4 and TLR2 activation and induces secretion of LTB₄ and thromboxane B₂. This underscores the role of n-butyrate as a crucial mediator of gut-specific immunity.