Loss of interleukin-12 modifies the pro-inflammatory response but does not prevent duct obstruction in experimental biliary atresia

BACKGROUND: Livers of infants with biliary atresia and of neonatal mice infected with rotavirus (RRV) have increased expression of interferon-gamma (IFNγ) and interleukin (IL)-12. While the expression of IFNγ regulates the obstruction of extrahepatic bile ducts by lymphocytes, the role of IL-12 in the pathogenesis of biliary obstruction is unknown. Based on the role of IL-12 as a key proinflammatory cytokine, we hypothesized that loss of IL-12 prevents the obstruction of extrahepatic bile ducts. METHODS: IL12-knockout (IL-12KO) and wild type mice were injected with RRV or saline at day 1 of age and monitored for the development of symptoms. The cellular and molecular phenotypes were determined at days 3, 7, and 14 by real-time PCR and flow cytometry. RESULTS: RRV infection of IL-12KO mice resulted in growth failure, jaundice/acholic stools, and decreased survival similar to wild-type mice. IL-12KO mice had a remarkable neutrophil-rich portal inflammation and epithelial sloughing of extrahepatic bile ducts. Loss of IL-12 decreased but did not abolish the hepatic expression of IFNγ, displayed a remarkable increase in expression of TNFα, IFNα, IFNβ and decreased expression of IL-4 and IL-5. CONCLUSION: Loss of IL-12 did not modify the progression of bile duct obstruction in experimental biliary atresia. However, the inflammatory response was predominantly neutrophil-based and displayed a Th1 response in the absence of IL-12

Thymus‐derived regulatory T cells restrain pro‐inflammatory Th1 responses by downregulating CD

The severity and intensity of autoimmune disease in Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) patients and in scurfy mice emphasizes the critical role played by thymus-derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here we demonstrate that tTregs selectively inhibit CD27/CD70-dependent Th1 priming, while leaving the IL-12-dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN-γ-secreting CD4+ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27-dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responsesin pressSCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Immunodominant PstS1 antigen of mycobacterium tuberculosis is a potent biological response modifier for the treatment of bladder cancer

BACKGROUND: Bacillus Calmette Guérin (BCG)-immunotherapy has a well-documented and successful clinical history in the treatment of bladder cancer. However, regularly observed side effects, a certain degree of nonresponders and restriction to superficial cancers remain a major obstacle. Therefore, alternative treatment strategies are intensively being explored. We report a novel approach of using a well defined immunostimulatory component of Mycobacterium tuberculosis for the treatment of bladder cancer. The phosphate transport protein PstS1 which represents the phosphate binding component of a mycobacterial phosphate uptake system is known to be a potent immunostimulatory antigen of M. tuberculosis. This preclinical study was designed to test the potential of recombinant PstS1 to serve as a non-viable and defined immunotherapeutic agent for intravesical bladder cancer therapy. METHODS: Mononuclear cells (PBMCs) were isolated from human peripheral blood and stimulated with PstS1 for seven days. The activation of PBMCs was determined by chromium release assay, IFN-γ ELISA and measurement of lymphocyte proliferation. The potential of PstS1 to activate monocyte-derived human dendritic cells (DC) was determined by flow cytometric analysis of the marker molecules CD83 and CD86 as well as the release of the cytokines TNF-α and IL-12. Survival of presensitized and intravesically treated, tumor-bearing mice was analyzed by Kaplan-Meier curve and log rank test. Local and systemic immune response in PstS1-immunotherapy was investigated by anti-PstS1-specific ELISA, splenocyte proliferation assay and immunohistochemistry. RESULTS: Our in vitro experiments showed that PstS1 is able to stimulate cytotoxicity, IFN-γ release and proliferation of PBMCs. Further investigations showed the potential of PstS1 to activate monocyte-derived human dendritic cells (DC). In vivo studies in an orthotopic murine bladder cancer model demonstrated the therapeutic potential of intravesically applied PstS1. Immunohistochemical analysis and splenocyte restimulation assay revealed that local and systemic immune responses were triggered by intravesical PstS1-immunotherapy. CONCLUSION: Our results demonstrate profound in vitro activation of human immune cells by recombinant PstS1. In addition, intravesical PstS1 immunotherapy induced strong local and systemic immune responses together with substantial anti-tumor activity in a preclinical mouse model. Thus, we have identified recombinant PstS1 antigen as a potent immunotherapeutic drug for cancer therapy

The Cyclophilin-Binding Agent Sanglifehrin A Is a Dendritic Cell Chemokine and Migration Inhibitor

Sanglifehrin A (SFA) is a cyclophilin-binding immunosuppressant but the immunobiology of action is poorly understood. We and others have reported that SFA inhibits IL-12 production and antigen uptake in dendritic cells (DC) and exhibits lower activity against lymphocytes. Here we show that SFA suppresses DC chemokine production and migration. Gene expression analysis and subsequent protein level confirmation revealed that SFA suppressed CCL5, CCL17, CCL19, CXCL9 and CXCL10 expression in human monocyte-derived DC (moDC). A systems biology analysis, Onto Express, confirmed that SFA interferes with chemokine-chemokine receptor gene expression with the highest impact. Direct comparison with the related agent cyclosporine A (CsA) and dexamethasone indicated that SFA uniquely suppresses moDC chemokine expression. Competitive experiments with a 100-fold molar excess of CsA and with N-Methyl-Val-4-cyclosporin, representing a nonimmunosuppressive derivative of CsA indicated chemokine suppression through a cyclophilin-A independent pathway. Functional assays confirmed reduced migration of CD4+ Tcells and moDCs to supernatant of SFA-exposed moDCs. Vice versa, SFA-exposed moDC exhibited reduced migration against CCL19. Moreover, SFA suppressed expression of the ectoenzyme CD38 that was reported to regulate DC migration and cytokine production. These results identify SFA as a DC chemokine and migration inhibitor and provide novel insight into the immunobiology of SFA

Murine Gammaherpesvirus-68 Inhibits Antigen Presentation by Dendritic Cells

Dendritic cells (DCs) play a central role in initiating adaptive immunity. Murine gammaherpesvirus-68 (MHV-68), like many persistent viruses, infects DCs during normal host colonization. It therefore provides a means to understanding what host and viral genes contribute to this aspect of pathogenesis. The infected DC phenotype is likely to depend on whether viral gene expression is lytic or latent and whether antigen presentation is maintained. For MHV-68, neither parameter has been well defined. Here we show that MHV-68 infects immature but not mature bone marrow-derived DCs. Infection was predominantly latent and these DCs showed no obvious defect in antigen presentation. Lytically infected DCs were very different. These down-regulated CD86 and MHC class I expression and presented a viral epitope poorly to CD8+ T cells. Antigen presentation improved markedly when the MHV-68 K3 gene was disrupted, indicating that K3 fulfils an important function in infected DCs. MHV-68 infects only a small fraction of the DCs present in lymphoid tissue, so K3 expression is unlikely to compromise significantly global CD8+ T cell priming. Instead it probably helps to maintain lytic gene expression in DCs once CD8+ T cell priming has occurred

Potentiating Effects of MPL on DSPC Bearing Cationic Liposomes Promote Recombinant GP63 Vaccine Efficacy: High Immunogenicity and Protection

Visceral leishmaniasis (VL), a vector-transmitted disease caused by Leishmania donovani, is potentially fatal if left untreated. Vaccination against VL has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine is pressing for the control of the disease. Earlier, we observed protective efficacy using leishmanial antigen (Ag) in the presence of either cationic liposomes or monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) against experimental VL through the intraperitoneal (i.p.) route of administration in the mouse model. However, this route of immunization is not adequate for human use. For this work, we developed vaccine formulations combining cationic liposomes with MPL-TDM using recombinant GP63 (rGP63) as protein Ag through the clinically relevant subcutaneous (s.c.) route. Two s.c. injections with rGP63 in association with cationic liposomes and MPL-TDM showed enhanced immune responses that further resulted in high protective levels against VL in the mouse model. This validates the combined use of MPL-TDM as an immunopotentiator and liposomes as a suitable vaccine delivery system

Interleukin-12p40 Modulates Human Metapneumovirus-Induced Pulmonary Disease in an Acute Mouse Model of Infection

The mechanisms that regulate the host immune response induced by human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, are largely unknown. Cytokines play an important role in modulating inflammatory responses during viral infections. IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV. In this work, we demonstrated that in mice deficient in IL-12p40, hMPV infection induced an exacerbated pulmonary inflammatory response and mucus production, altered cytokine response, and decreased lung function. However, hMPV infection in these mice does not have an effect on viral replication. These results identify an important regulatory role of IL-12p40 in hMPV infection

Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells

<div><p>Background</p><p><i>Leishmania major</i> infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of <i>Leishmania</i> parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.</p><p>Methodology/Principal Findings</p><p>Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating <i>L</i>. <i>major</i> Friedlin V1 mutants defective in LPG alone, (FV1 <i>lpg1-</i>), or generally deficient for all PGs, (FV1 <i>lpg2-</i>). Infection with metacyclic, infective stage, <i>L</i>. <i>major</i> or purified LPG induced high levels of <i>IL12B</i> subunit gene transcripts in hDCs, which was abrogated with FV1 <i>lpg1-</i> infections. In contrast, hDC infections with FV1 <i>lpg2-</i> displayed increased <i>IL12B</i> expression, suggesting other PG-related/<i>LPG2</i> dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 <i>lpg1-</i>, FV1 <i>lpg2-</i> infections revealed that FV1 <i>lpg1-</i> mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.</p><p>Conclusions/Significance</p><p>These data suggest that <i>L</i>. <i>major</i> LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring <i>Leishmania</i> surface glycoconjugates that result in modulation of host cellular IL12.</p></div

NO2 inhalation induces maturation of pulmonary CD11c+ cells that promote antigenspecific CD4+ T cell polarization

<p>Abstract</p> <p>Background</p> <p>Nitrogen dioxide (NO₂) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. NO₂is also produced endogenously in the lung during acute inflammatory responses. NO₂can function as an adjuvant, allowing for allergic sensitization to an innocuous inhaled antigen and the generation of an antigen-specific Th2 immune response manifesting in an allergic asthma phenotype. As CD11c⁺antigen presenting cells are considered critical for naïve T cell activation, we investigated the role of CD11c⁺cells in NO₂-promoted allergic sensitization.</p> <p>Methods</p> <p>We systemically depleted CD11c⁺cells from transgenic mice expressing a simian diphtheria toxin (DT) receptor under of control of the CD11c promoter by administration of DT. Mice were then exposed to 15 ppm NO₂followed by aerosolized ovalbumin to promote allergic sensitization to ovalbumin and were studied after subsequent inhaled ovalbumin challenges for manifestation of allergic airway disease. In addition, pulmonary CD11c⁺cells from wildtype mice were studied after exposure to NO₂and ovalbumin for cellular phenotype by flow cytometry and <it>in vitro </it>cytokine production.</p> <p>Results</p> <p>Transient depletion of CD11c⁺cells during sensitization attenuated airway eosinophilia during allergen challenge and reduced Th2 and Th17 cytokine production. Lung CD11c⁺cells from wildtype mice exhibited a significant increase in MHCII, CD40, and OX40L expression 2 hours following NO₂exposure. By 48 hours, CD11c⁺MHCII⁺DCs within the mediastinal lymph node (MLN) expressed maturation markers, including CD80, CD86, and OX40L. CD11c⁺CD11b^-and CD11c⁺CD11b⁺pulmonary cells exposed to NO₂<it>in vivo </it>increased uptake of antigen 2 hours post exposure, with increased ova-Alexa 647⁺CD11c⁺MHCII⁺DCs present in MLN from NO₂-exposed mice by 48 hours. Co-cultures of ova-specific CD4⁺T cells from naïve mice and CD11c⁺pulmonary cells from NO₂-exposed mice produced IL-1, IL-12p70, and IL-6 <it>in vitro </it>and augmented antigen-induced IL-5 production.</p> <p>Conclusions</p> <p>CD11c⁺cells are critical for NO₂-promoted allergic sensitization. NO₂exposure causes pulmonary CD11c⁺cells to acquire a phenotype capable of increased antigen uptake, migration to the draining lymph node, expression of MHCII and co-stimulatory molecules required to activate naïve T cells, and secretion of polarizing cytokines to shape a Th2/Th17 response.</p