Molecular mechanisms of the induction of IL-12 and its inhibition by IL- 10

Exogenously added IL-10 rapidly inhibited Staphylococcus aureus- or LPS- induced cytokine mRNA expression in human PBMCs and monocytes, with a maximal effect observed when IL-10 was added from 20 h before until 1 h after the addition of the inducers. Nuclear run-on assays revealed that the inhibition of IL-12 p40, IL-12 p35, and TNF-α was at the gene transcriptional level and that the addition of IL-10 to S. aureus- or LPS-treated PBMCs did not affect mRNA stability. The inhibitory activity of IL-10 was abrogated by cycloheximide (CHX), suggesting the involvement of a newly synthesized protein(s). The addition of CHX at 2 h before S. aureus or LPS also inhibited the accumulation of IL-12 p40 mRNA, but did not inhibit IL-12 p35 and TNF-α mRNA. This finding suggests that p40 transcription is regulated through a de novo synthesized protein factor(s), whereas the addition of CHX at 2 h after S. aureus activation caused superinduction of the IL-12 p40, IL-12 p35, and TNF-α genes. These results indicate that in human monocytes, the mechanism(s) of IL-10 suppression of both IL-12 p40 and IL-12 p35 genes is primarily seen at the transcriptional level, and that the induction of the IL-12 p40 and p35 genes have different requirements for de novo protein synthesis.

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-γ responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-γ secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-γ synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-γ. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM. © 2002 Elsevier Science Ltd. All rights reserved.

Avalição de polimorfismos de nucleotídeos simples (SNPs) na região promotora de gene da interleucina 10 em pacientes com Linfoma de Hodgkin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Duração do efeito analgésico e expressão de diferentes metaloproteinases, do colágeno tipo IV e da interleucina-10, em córneas de coelhos tratadas com morfina, após ceratectomia lamelar

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Níveis de mercúrio, prolactina e interleucina 10 em mulheres em idade reprodutiva e puérperas dos municípios de Itaituba e Ananindeua, Pará

Ao mercúrio tem sido atribuída a capacidade de interferir nos sistemas orgânicos imunológico e hormonal, além dos sistemas nervoso e renal frequentemente atingidos por esse agente tóxico. Mulheres em idade fértil ou grávidas constituem um grupo vulnerável a esses efeitos, em relação a si mesmas e seus conceptos. Foi avaliada a exposição ao mercúrio (Hg) e os níveis de prolactina (PRL) e interleucina-10 (IL-10) em 144 mulheres (no pós-parto e cerca de um ano depois) de Itaituba, área sob impacto ambiental do mercúrio e em mulheres de municípios da área metropolitana de Belém, sobretudo Ananindeua, área sem impacto conhecido do mercúrio (156 puérperas e 156 não puérperas). As análises de mercúrio total (Hg-t) em sangue foram feitas por Espectrometria de Absorção Atômica por Vapor Frio. As análises séricas de PRL foram feitas por Ensaio Imunoenzimático com detecção final em fluorescência e as determinações de IL-10 foram realizadas por Ensaio Imunoenzimático de Fase Sólida. Dados demográficos e epidemiológicos foram obtidos através de questionário semi-estruturado. As puérperas de Itaituba apresentaram média de Hg-t, PRL e IL-10 de 13,93 μg/l, 276,20 ng/ml e 39,54 pg/ml, respectivamente. Nas puérperas de Ananindeua as respectivas médias foram 3,67 μg/l, 337,70 ng/ml e 4,90 pg/ml. As mulheres não puérperas de Itaituba apresentaram média de Hg-t de 12,68 μg/l, média de PRL de 30,75 ng/ml e média de IL-10 de 14,20 pg/ml. As médias de Hg-t, PRL e IL-10 das mulheres de Ananindeua foram 2,73 μg/l, 17,07 ng/ml e 1,49 pg/ml, respectivamente. Os níveis de Hg-t, PRL e IL-10 foram maiores em Itaituba (p<0,0001), exceto em relação à PRL das puérperas, maior em Ananindeua. Os níveis semelhantes de Hg-t nas duas avaliações das mulheres de Itaituba (p=0,7056) e a correlação moderada sugerem continuidade da exposição (r=0,4736, p<0,0001). A principal variável preditora dos níveis de mercúrio foi o consumo de peixe nos modelos de regressão múltipla linear e logística. A paridade e os níveis de IL-10 apresentaram associação positiva com a PRL nas puérperas de Itaituba e o peso do recém-nascido e a IL-10, associação positiva com a PRL em puérperas de Ananindeua. A IL-10 apresentou associação negativa com a PRL nas mulheres não puérperas de Itaituba (p=0,0270) e positiva nas mulheres de Ananindeua (p=0,0266). Os níveis de Hg-t estavam associados negativamente com a PRL nas puérperas (p=0,0460) e positivamente com o trabalho em garimpo (p=0,0173) (este também importante para as não puérperas) em Itaituba, segundo os modelos logísticos. A IL-10 esteve associada positivamente à morbidade recente nas puérperas de Itaituba (p=0,0210), negativamente ao consumo de bebida alcoólica (p=0,0178) e positivamente ao trabalho em garimpo nas mulheres não puérperas (p=0,0199). A exposição crônica ao Hg das mulheres de Itaituba, a diferença nos níveis dos fatores imunoendócrinos avaliados em relação às mulheres não expostas e a associação com variáveis epidemiológicas relevantes, sugerem a possibilidade de impactos da exposição no perfil imunoendócrino das mulheres de Itaituba, chamando atenção para a importância da vigilância da saúde dessa população e o possível uso de bioindicadores como a PRL em sua avaliação.

Participação das citocinas Interleucina-12, Interferon-γ, Interleucina-4 e Interleucina-10 e investigação de polimorfismos nos genes do INF-γ (IFNG+874) e da IL-10 (IL10-1082) na malária causada por Plasmodium vivax

A resposta imune na malária é complexa, e os mecanismos de ativação e regulação de linfócitos T efetores e de memória ainda são pouco compreendidos. No presente estudo, determinamos a concentração das citocinas Interferon-γ (IFN-γ), Interleucina-10 (IL-10), Interleucina-4 (IL-4) e Interleucina-12 (IL-12) no soro de indivíduos infectados por Plasmodium vivax, investigamos os polimorfismos no gene do IFN-γ (IFNG+874) e da IL-10 (IL10-1082) e analisamos a associação destes polimorfismos com a concentração das citocinas e com a densidade parasitária. A concentração das citocinas foi determinada por ELISA, e a genotipagem dos polimorfismos IFNG+874 e IL10-1082 foi realizada pelas técnicas de ASO-PCR e PCR-RFLP, respectivamente. Os indivíduos infectados apresentaram níveis séricos de IFN-γ e IL-10 aumentados. A produção de IFN-γ foi maior nos indivíduos primoinfectados, porém não foi associada com a redução da parasitemia. A produção de IL-10 foi alta e associada com altas parasitemias. As citocinas IL-4 e IL-12 não foram detectadas. As freqüências dos genótipos homozigoto mutante AA, heterozigoto AT e selvagem TT do gene do IFN-γ foram 0,51, 0,39 e 0,10, respectivamente. As freqüências dos genótipos homozigoto mutante AA, heterozigoto AG e selvagem GG para IL10 foram 0,49, 0,43 e 0,08, respectivamente. Apenas o polimorfismo do IFN-γ foi associado com níveis reduzidos desta citocina. Na malária causada por P. vivax, houve produção de citocina que caracteriza o perfil Th1 (IFN-γ), com possível participação da IL-10 na imunorregulação.

Associação entre expressão de receptores TLR2 e TLR4 e produção de TNF-alpha e IL-10 por monócitos de gestantes portadoras de pré-eclâmpsia

Preeclampsia (PE) is a pregnancy specific syndrome characterized by a systemic inflammatory response, with higher intensity than that observed in normal pregnancy. Cells of the immune system, such as monocytes and granulocytes are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. The objective of this study was to assess the activation state of monocytes from pregnant women with preeclampsia by endogenous expression of TLR2 e TLR4 receptors and to correlate the expression of TLR2 and TLR4 on monocytes surface of pregnant women with PE with the production of tumor necrosis factor-alpha (TNF- and interleukin-10 (IL-10) by these cells stimulated or not with peptidoglycan (PG) and lipopolysaccharide (LPS), as agonists agents of TLR2 and TLR4, respectively. We evaluated 15 pregnant women with PE, 15 normotensive pregnant women (NT) and 15 non-pregnant (NP). Peripheral blood monocytes were incubates in the presence or absence of LPS or PG. The supernatant obtained after 18h of culture was aspirated and used for TNF- and IL-10 determination by enzyme immunoassay (ELISA). The endogenous expression of TLR2 and TLR4 receptors was evaluated by flow cytometry. Our results showed significant highly concentrations of TNF- and TLR4 expression in monocytes of preeclamptic women when compared with NT and NP. Normal pregnant women presented higher levels of IL-10 in comparison with PE and NP groups. TLR2 expression was similar in the three groups studied. Therefore, our study highlights the important role of TLR4 in PE and the consequent high production of TNF- by monocytes of these patients, as well as the potential mechanism involving low levels of IL-10 in the pathophysiology of the disease. These observations demonstrate the strong link between the pathology of PE and the immune system of these patients

Early IL-10 production is essential for syngeneic graft acceptance

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival. J. Leukoc. Biol. 92: 259-264; 2012.

Exploring the function of IL-10, BTLA and DCs as regulators of immune responses

This thesis focuses on different aspects of immune regulation, both at the cellular and molecular levels. More specifically, this work concentrates on the importance of Interleukin-10, B and T Lymphocyte Attenuator (BTLA), and dendritic cells in respect to immune regulation, with special emphasis on autoimmunity. In this thesis, we show that the cellular source of IL10 production can dramatically influence the outcome of an autoimmune response. We show that T cell-derived IL10 plays an important role in controlling the viability of recently activated T cells, allowing them to become fully functional T effector cells. T cell-specific IL10-deficient mice failed to induce EAE when immunized with MOG peptide. Furthermore, when re-challenged with MOG or other stimuli, these T cells exhibited increased apoptosis rates. Here we report for the first time the generation of a novel mouse model that allows the conditional over-expression of BTLA. We show that BTLA can negatively regulate CD4+ T cells responses, when expressed by the T cells themselves. BTLA over-expression by CD8+ T cells or dendritic cells, however, resulted in enhanced viral clearance. In this study, we show that depletion of DCs, either early on from birth or later in adulthood, does not prevent EAE induction, but instead leads to a lower state of tolerance and stronger immune response. We also show that DCs are responsible for the upregulation of PD-1 on antigen-specific T cells and subsequently induce the formation of Tregs during immune responses.

Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance

We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSIF prevented GVHD in the B6 --> B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSIF (survival, 75% versus 11%, P < .001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent-fashion in vitro. T cells from peg-GCSF-treated IL-10(-/-) donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10(-/-) donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT. (C) 2004 by The American Society of Hematology.

IL-10-producing Regulatory B Cell Development in Human Autoimmune Disease

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of costimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of “regulatory B cells” has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported with interleukin-10 (IL-10) being the most studied. IL-10-producing regulatory B cells predominantly localize within a rare CD1dhiCD5+ B cell subset in mice and the CD24hiCD27+ B cell subset in adult humans. This specific IL-10-producing subset of regulatory B cells have been named “B10 cells” to highlight that the regulatory function of these rare B cells is primarily mediated by IL-10, and to distinguish them from other regulatory B cell subsets that regulate immune responses through different mechanisms. B10 cells have been studies in a variety of animal models with autoimmune disease and clinical settings of human autoimmunity. There are many unsolved questions related to B10 cells including their surface phenotype, their origin and development in vivo, and their role in autoimmunity.

In Chapter 3 of this dissertation, the role of the B cell receptor (BCR) in B10 cell development is highlighted. First, the BCR repertoire of mouse peritoneal cavity B10 cells is examined by single cell sequencing; peritoneal cavity B10 cells have clonally diverse germline BCRs that are predominantly unmutated. Second, mouse B10 cells are shown to have higher frequencies of λ+ BCRs compared to non-B10 cells which may indicate the involvement of BCR light chain editing early in the process of B10 cell development in vivo. Third, human peripheral blood B10 cells are examined and are also found to express higher frequencies of λ chains compared to non-b10 cells. Therefore, B10 cell BCRs are clonally diverse and enriched for unmutated germline sequences and λ light chains.

In Chapter 4 of this dissertation, B10 cells are examined in the healthy developing human across the entire age range of infancy, childhood and adolescence, and in a large cohort of children with autoimmunity. The study of B10 cells in the developing human documents a massive transient expansion during middle childhood when up to 30% of blood B cells were competent to produce IL-10. The surface phenotype of pediatric B10 cells was variable and reflective of overall B cell development. B10 cells down-regulated CD4+ T cell interferon-gamma (IFN-γ) production through IL-10-dependent pathways and IFN-γ inhibited whereas interleukin-21 (IL-21) promoted B cell IL-10 competency in vitro. Children with autoimmunity had a contracted B10 cell compartment, along with increased IFN-γ and decreased IL-21 serum levels compared to age-matched healthy controls. The decreased B10 cell frequencies and numbers in children with autoimmunity may be partially explained by the differential regulation of B10 cell development by IFN-γ and IL-21 and alterations in serum cytokine levels. The age-related changes of the B10 cell compartment during normal human development provide new insights into immune tolerance mechanisms involved in inflammation and autoimmunity.

These studies collectively demonstrate that BCR signals are the most important early determinant of B10 cell development in vivo, that human B10 cells are not a surface phenotype defined developmental B cell subset but a functionally defined regulatory B cell subset that regulates CD4+ T IFN-γ production through IL-10-dependent pathways and that human B10 cell development can be regulated by soluble factors in vivo such as the cytokine milieu. The findings of these studies provide new insights into immune tolerance mechanisms involved in human autoimmunity and the potent effects of IL-21 on human B cell IL-10 competence in vitro open new horizons in the development of autologous B10 cell-based therapies as an approach to treat human autoimmune disease in the future.

Relationship between IL-10 gene -819C/T polymorphism and the risk of inflammatory bowel disease: a meta-analysis.

Background: The -819C/T polymorphism in interleukin 10 (IL-10) gene has been reported to be associated with inflammatory bowel disease (IBD) ,but the previous results are conflicting. Materials and Methods: The present study aimed at investigating the association between this polymorphism and risk of IBD using a meta-analysis.PubMed,Web of Science,EMBASE,google scholar and China National Knowledge Infrastructure (CNKI) databases were systematically searched to identify relevant publications from their inception to April 2016.Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using fixed- or random-effects models. Results: A total of 7 case-control studies containing 1890 patients and 2929 controls were enrolled into this meta-analysis, and our results showed no association between IL-10 gene -819C/T polymorphism and IBD risk(TT vs. CC:OR=0.81,95%CI 0.64- 1.04;CT vs. CC:OR=0.92,95%CI 0.81-1.05; Dominant model: OR=0.90,95%CI 0.80-1.02; Recessive model: OR=0.84,95%CI 0.66-1.06). In a subgroup analysis by nationality, the -819C/T polymorphism was not associated with IBD in both Asians and Caucasians. In the subgroup analysis stratified by IBD type, significant association was found in Crohn’s disease(CD)(CT vs. CC:OR=0.68,95%CI 0.48-0.97). Conclusion: In summary, the present meta-analysis suggests that the IL-10 gene -819C/T polymorphism may be associated with CD risk.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.

Non-major-histocompatibility-complex genetics of ankylosing spondylitis

There is strong evidence from twin and family studies indicating that a substantial proportion of the heritability of susceptibility to ankylosing spondylitis (AS) and its clinical manifestations is encoded by non-major-histocompatibility-complex genes. Efforts to identify these genes have included genomewide linkage studies and candidate gene association studies. One region, the interleukin (IL)-1 gene complex on chromosome 2, has been repeatedly associated with AS in both Caucasians and Asians. It is likely that more than one gene in this complex is involved in AS, with the strongest evidence to date implicating IL-1A. Identifying the genes underlying other linkage regions has been difficult due to the lack of obvious candidates and the low power of most studies to date to identify genes of the small to moderate magnitude that are likely to be involved. The field is moving towards genomewide association analysis, involving much larger datasets of unrelated cases and controls. Early successes using this approach in other diseases indicates that it is likely to identify genes in common diseases like AS, but there remains the risk that the common-variant, common-disease hypothesis will not hold true in AS. Nonetheless, it is appropriate for the field to be cautiously optimistic that the next few years will bring great advances in our understanding of the genetics of this condition.

The enigma of IgE+ B-cell memory in human subjects

Our understanding of the origin and fate of the IgE-switched B cell has been markedly improved by studies in mouse models. The immediate precursor of the IgE-switched B cell is either a relatively naive nonswitched B cell or a mature IgG-switched B cell. These 2 routes are referred to as the direct and indirect pathways, respectively. IgE responses derived from each pathway differ significantly, largely reflecting the difference in time spent in a germinal center and thus time for clonal expansion, somatic hypermutation, affinity maturation, and acquisition of a memory phenotype. The clinical and therapeutic implications for IgE responses in human subjects are still a matter of debate, largely because the immunization procedures used in the animal models are significantly different from classical atopic sensitization to allergens from pollen and mites. On the basis of the limited information available, it seems likely that these atopic IgE responses are characterized by a relatively low IgG/IgE ratio, low B-cell memory, and modest affinity maturation, which fits well with the direct switching pathway. It is still unresolved how the IgE response evolves to cover a wide epitope repertoire involving many epitopes per allergen, as well as many different allergens from a single allergen source. © 2013 American Academy of Allergy, Asthma & Immunology.