IL-33 Enhances IFNγ and TNFα Production by Human MAIT Cells: A New Pro-Th1 Effect of IL-33.

Mucosal-associated invariant T (MAIT) cells represent a distinct T cell population restricted by the MHC-class-I-related molecule, MR1, which recognizes microbial-derived vitamin B2 (riboflavin) metabolites. Their abundance in humans, together with their ability to promptly produce distinct cytokines including interferon γ (IFNγ) and tumor necrosis factor α (TNFα), are consistent with regulatory functions in innate as well as adaptive immunity. Here, we tested whether the alarmin interleukin 33 (IL-33), which is secreted following inflammation or cell damage, could activate human MAIT cells. We found that MAIT cells stimulated with IL-33 produced high levels of IFNγ, TNFα and Granzyme B (GrzB). The action of IL-33 required IL-12 but was independent of T cell receptor (TCR) cross-linking. MAIT cells expressed the IL-33 receptor ST2 (suppression of tumorigenicity 2) and upregulated Tbet (T-box expressed in T cells) in response to IL-12 or IL-33. Electronically sorted MAIT cells also upregulated the expression of CCL3 (Chemokine C-C motif ligand 3), CD40L (CD40 Ligand), CSF-1 (Colony Stimulating Factor 1), LTA (Lymphotoxin-alpha) and IL-2RA (IL-2 receptor alpha chain) mRNAs in response to IL-33 plus IL-12. In conclusion, IL-33 combined with IL-12 can directly target MAIT cells to induce their activation and cytokine production. This novel mechanism of IL-33 activation provides insight into the mode of action by which human MAIT cells can promote inflammatory responses in a TCR-independent manner.

Cefoxitin treatment of MRSA leads to a shift in the IL-12/IL-23 production pattern in dendritic cells by a mechanism involving changes in the MAPK signaling.

Methicillin resistant Staphylococcus aureus (MRSA) constitute a serious health care problem worldwide. This study addresses the effect of ß-lactam treatment on the ability of clinically relevant MRSA strains to induce IL-12 and IL-23. MRSA strains induced a dose-dependent IL-12 response in murine bone-marrow-derived dendritic cells that was dependent on endocytosis and acidic degradation. Facilitated induction of IL-12 (but not of IL-23) called for activation of the MAP kinase JNK, and was suppressed by p38. Compromised peptidoglycan structure in cefoxitin-treated bacteria - as denoted by increased sensitivity to mutanolysin -caused a shift from IL-12 towards IL-23. Moreover, cefoxitin treatment of MRSA led to a p38 MAPK-dependent early up-regulation of Dual Specificity Phosphatase (DUSP)-1. Compared to common MRSA, characteristics associated with a persister phenotype increased intracellular survival and upon cefoxitin treatment, the peptidoglycan was not equally compromised and the cytokine induction still required phagosomal acidification. Together, these data demonstrate that ß-lactam treatment changes the MRSA-induced IL-12/IL-23 pattern determined by the activation of JNK and p38. We suggest that accelerated endosomal degradation of the peptidoglycan in cefoxitin-treated MRSA leads to an early expression of DUSP-1 and accordingly, a reduction in the IL-12/IL-23 ratio in dendritic cells. This may influence the clearance of S. aureus.

Frontline Science: CD40 signaling restricts RNA virus replication in Mϕs, leading to rapid innate immune control of acute virus infection.

Many acute viral infections target tissue Mϕs, yet the mechanisms of Mϕ-mediated control of viruses are poorly understood. Here, we report that CD40 expressed by peritoneal Mϕs restricts early infection of a broad range of RNA viruses. Loss of CD40 expression enhanced virus replication as early as 12-24 h of infection and, conversely, stimulation of CD40 signaling with an agonistic Ab blocked infection. With peritoneal cell populations infected with the filovirus, wild-type (WT) Ebola virus (EBOV), or a BSL2 model virus, recombinant vesicular stomatitis virus encoding Ebola virus glycoprotein (rVSV/EBOV GP), we examined the mechanism conferring protection. Here, we demonstrate that restricted virus replication in Mϕs required CD154/CD40 interactions that stimulated IL-12 production through TRAF6-dependent signaling. In turn, IL-12 production resulted in IFN-γ production, which induced proinflammatory polarization of Mϕs, protecting the cells from infection. These CD40-dependent events protected mice against virus challenge. CD40-/- mice were exquisitely sensitive to intraperitoneal challenge with a dose of rVSV/EBOV GP that was sublethal to CD40+/+ mice, exhibiting viremia within 12 h of infection and rapidly succumbing to infection. This study identifies a previously unappreciated role for Mϕ-intrinsic CD40 signaling in controlling acute virus infection.

Expression of leukotriene B4 receptor 1 defines functionally distinct DCs that control allergic skin inflammation.

Leukotriene B4 (LTB4) receptor 1 (BLT1) is a chemotactic G protein-coupled receptor expressed by leukocytes, such as granulocytes, macrophages, and activated T cells. Although there is growing evidence that BLT1 plays crucial roles in immune responses, its role in dendritic cells remains largely unknown. Here, we identified novel DC subsets defined by the expression of BLT1, namely, BLT1hi and BLT1lo DCs. We also found that BLT1hi and BLT1lo DCs differentially migrated toward LTB4 and CCL21, a lymph node-homing chemoattractant, respectively. By generating LTB4-producing enzyme LTA4H knockout mice and CD11c promoter-driven Cre recombinase-expressing BLT1 conditional knockout (BLT1 cKO) mice, we showed that the migration of BLT1hi DCs exacerbated allergic contact dermatitis. Comprehensive transcriptome analysis revealed that BLT1hi DCs preferentially induced Th1 differentiation by upregulating IL-12p35 expression, whereas BLT1lo DCs accelerated T cell proliferation by producing IL-2. Collectively, the data reveal an unexpected role for BLT1 as a novel DC subset marker and provide novel insights into the role of the LTB4-BLT1 axis in the spatiotemporal regulation of distinct DC subsets.

A simplified method to produce mRNAs and functional proteins from synthetic double-stranded DNA templates.

We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning.

Hizikia fusiforme extract enhances dendritic cell maturation in vitro and in vivo.

Dendritic cells (DCs) are play critical roles in the priming and regulation of immune responses. DCs rapidly process and convey these antigens to prime antigen-specific T cells. Therefore, regulation of DCs functions is important for immunity and immunotherapies. Immune adjuvants for DCs activation are needed to improve the efficacy of vaccines against tumors and many infectious diseases. Therefore, we demonstrate that H. fusiformis extract can regulate DCs maturation and activation. H. fusiformis extract induced costimulatory molecules (CD 80 and CD86), antigen-presenting molecules (major histocompatibility complex (MHC) I and II), CCR7 expression, and interleukin (IL)-12 production in DCs. These effects are associated with upregulation of mitogen-activated protein kinase (MAPK) signaling pathway. In addition, H. fusiformis extract induces costimulatory molecules on splenic DCs and activated CD8+ T cells in vivo. Taken together, these findings suggest that H. fusiformis extract may be a potential efficient immune therapeutic compound in DCs-mediated immunotherapies. ABBREVIATIONS: CTL: cytotoxic T lymphocytes; DCs: dendritic cells; ERK: extracellular signal-regulated kinases; IL: interleukini; JNK: c-Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; MHC: major histocompatibility complex.

Synergistic phage-surfactant combination clears IgE-promoted Staphylococcus aureus aggregation in vitro and enhances the effect in vivo.

Currently, topical antibiotic treatment is a major strategy for decolonisation of Staphylococcus aureus, although it may result in antibiotic resistance or recolonisation of the organism. Recently, application of bacteriophages in the treatment of S. aureus infection has attracted attention. However, a single administration of bacteriophages did not effectively decolonise S. aureus in our first trial in vivo. Using a bacteriophage (pSa-3) and surfactant combination in vitro, we showed an increased (>8%) adsorption rate of the bacteriophage on the host. Moreover, the combination increased the eradication of immunoglobulin E (IgE)-stimulated aggregation, as the surfactant promoted the dissociation of S. aureus aggregates by decreasing the size by 75% and 50% in the absence and presence of IgE, respectively. Furthermore, the combined treatment significantly decolonised the pathogen with an efficacy double that of the phage-only treatment, and decreased the expression of pro-inflammatory cytokine genes (IL-1ß, IL-12 and IFNγ) for 5 days in the second in vivo trial. These results suggest that the bacteriophage-surfactant combination could act as an alternative to antibiotics for S. aureus decolonisation in patients with dermatitis.

BTLA-Expressing Dendritic Cells in Patients With Tuberculosis Exhibit Reduced Production of IL-12/IFN-α and Increased Production of IL-4 and TGF-ß, Favoring Th2 and Foxp3+ Treg Polarization.

Little is known about how tuberculosis (TB) impairs dendritic cell (DC) function and anti-TB immune responses. We previously showed that the B and T lymphocyte attenuator (BTLA), an immune inhibitory receptor, is involved in TB pathogenesis. Here, we examined whether BTLA expression in TB affects phenotypic and functional aspects of DCs. Active TB patients exhibited higher expression of BTLA in myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs) subsets compared with healthy controls (HCs). BTLA expression was similarly high in untreated TB, TB relapse, and sputum-bacillus positive TB, but anti-TB therapy reduced TB-driven increases in frequencies of BTLA+ DCs. BTLA+ DCs in active TB showed decreased expression of the DC maturation marker CD83, with an increased expression of CCR7 in mDCs. BTLA+ DCs in active TB displayed a decreased ability to express HLA-DR and to uptake foreign antigen, with a reduced expression of the co-stimulatory molecule CD80, but not CD86. Functionally, BTLA+ DCs in active TB showed a decreased production of IL-12 and IFN-α as well as a reduced ability to stimulate allogeneic T-cell proliferative responses. BTLA+ mDCs produced larger amounts of IL-4 and TGF-ß than BTLA- mDCs in both HCs and APT patients. BTLA+ DCs from active TB patients showed a reduced ability to stimulate Mtb antigen-driven Th17 and Th22 polarizations as compared to those from HCs. Conversely, these BTLA+ DCs more readily promoted the differentiation of T regulatory cells (Treg) and Th2 than those from HCs. These findings suggest that TB-driven BTLA expression in DCs impairs the expression of functional DC surrogate markers and suppress the ability of DCs to induce anti-TB Th17 and Th22 response while promoting Th2 and Foxp3+ Tregs.

TLR9 Sensing of Self-DNA Controls Cell-Mediated Immunity to Listeria Infection via Rapid Conversion of Conventional CD4+ T Cells to Treg.

CD4+ T lymphocytes are crucial for controlling a range of innate and adaptive immune effectors. For CD8+ cytotoxic T lymphocyte (CTL) responses, CD4+ T cells can function as helpers (TH) to amplify magnitude and functionality or as regulatory cells (Treg) capable of profound inhibition. It is unclear what determines differentiation to these phenotypes and whether pathogens provoke alternate programs. We find that, depending on the size of initial dose, Listeria infection drives CD4+ T cells to act as TH or induces rapid polyclonal conversion to immunosuppressive Treg. Conversion to Treg depends on the TLR9 and IL-12 pathways elicited by CD8α+ dendritic cell (DC) sensing of danger-associated neutrophil self-DNA. These findings resolve long-standing questions regarding the conditional requirement for TH amongst pathogens and reveal a remarkable degree of plasticity in the function of CD4+ T cells, which can be quickly converted to Tregin vivo by infection-mediated immune modulation.

Myeloid Cell-Targeted Nanocarriers Efficiently Inhibit Cellular Inhibitor of Apoptosis for Cancer Immunotherapy.

Immune-checkpoint blockers can promote sustained clinical responses in a subset of cancer patients. Recent research has shown that a subpopulation of tumor-infiltrating dendritic cells functions as gatekeepers, sensitizing tumors to anti-PD-1 treatment via production of interleukin-12 (IL-12). Hypothesizing that myeloid cell-targeted nanomaterials could be used to deliver small-molecule IL-12 inducers, we performed high-content image-based screening to identify the most efficacious small-molecule compounds. Using one lead candidate, LCL161, we created a myeloid-targeted nanoformulation that induced IL-12 production in intratumoral myeloid cells in vivo, slowed tumor growth as a monotherapy, and had no significant systemic toxicity. These results pave the way for developing combination immunotherapeutics by harnessing IL-12 production for immunostimulation.

Rescue, Purification, and Characterization of a Recombinant HSV Expressing a Transgenic Protein.

In the previous chapter, we describe the engineering of a HSV-BAC genome by galK recombineering. Here we describe the procedures to reconstitute, or regenerate, the replicating recombinant virus, and the methods to purify it and characterize it for the correct expression of the transgene. We present the example of R-115, a recombinant expressing murine interleukin 12 (mIL12) from the US1-US2 intergenic region. A specific method for the production of highly purified virions by iodixanol gradient, suitable for in vivo applications, is also detailed.

Mannan Enhances IL-12 Production by Increasing Bacterial Uptake and Endosomal Degradation in L. acidophilus and S. aureus Stimulated Dendritic Cells.

The mannose receptor (MR) is a C-type lectin involved in endocytosis and with a poorly defined ability to modulate cellular activation. We investigated the effect of mannan treatment prior to stimulation of murine bone marrow-derived dendritic cells with the Gram-positive bacteria Lactobacillus acidophilus NCFM (L. acidophilus) on the induction of Interleukin (IL)-12. Mannan enhanced the IL-12 production induced by L. acidophilus in a dose dependent manner (up to 230% enhancement). Additionally, mannan-enhanced IL-12 induction was also demonstrated with another Gram-positive bacteria, Staphylococcus aureus (S. aureus), while an IL-12 reducing effect was seen on Escherichia coli stimulated cells. Furthermore, the expression of Interferon ß (Ifnb) was increased in cells treated with mannan prior to stimulation with L. acidophilus. The addition of mannan but not of bacteria led to endocytosis of MR, while addition of mannan prior to L. acidophilus or S. aureus resulted in increased endocytosis of bacteria, a faster killing of endocytosed bacteria, and increased reactive oxygen species production. Expression of signaling lymphocytic activation molecule (SLAMF)1 shown previously to be involved in the facilitation of endosomal degradation was upregulated by mannan but not by L. acidophilus and S. aureus. The IL-12 enhancement by mannan but not the IL-12 induced by the bacteria was abrogated by addition of inhibitors of clathrin coated pits (chlorpromazine and monodansylcadaverine). Furthermore, the addition of acid sphingomyelinase, a facilitator of ceramide raft formation, prior to addition of L. acidophilus enhanced the IL-12 production and the endocytosis of bacteria. In summary, our results show that mannan increases the IL-12 production induced by some Gram-positive bacteria through MR-endocytosis, which increases bacterial endocytosis and endosomal killing. The differential effect of MR activation on the IL-12 production induced by Gram-positive and Gram-negative bacteria may influence the immune response toward allergens and other glycoproteins.

ß-Catenin stabilization in NOD dendritic cells increases IL-12 production and subsequent induction of IFN-γ-producing T cells.

Dendritic cells (DC) from diabetes-prone NOD mice and patients with type 1 diabetes (T1D) produce excess IL-12 that drives development of ß-cell-destroying IFN-γ-producing T cells. The molecular mechanisms that control IL-12 production in T1D are unclear. In this study, we report that ß-catenin, a multifunctional protein involved in inflammation, is dramatically increased in DC from NOD mice. We further investigated the mechanisms leading to accumulation of ß-catenin in NOD DC and its role in the inflammatory pathogenic responses associated with T1D. Hyperphosphorylation of ß-catenin at a stabilizing residue, serine 552, mediated by activation of Akt, appears to lead to ß-catenin accumulation in NOD DC. Elevated ß-catenin in DC correlated with IL-12 production and induction of IFN-γ-producing CD4 cells. On the one hand, knockdown/inhibition of ß-catenin significantly reduced NOD DC production of IL-12 and their ability to induce IFN-γ-producing CD4 cells. On the other hand, overexpression of ß-catenin in control DC resulted in increased IL-12 production and induction of IFN-γ-production in T cells. Additionally, we found that ß-catenin inhibitors decreased NF-κB activation in NOD DC and IFN-γ production by NOD T cells in vivo. These data strongly suggest that accumulation of ß-catenin in DC from NOD mice drives IL-12 production, and consequently, development of pathogenic IFN-γ-producing T cells. Targeting the defect responsible for ß-catenin accumulation and subsequent overproduction of pro-inflammatory cytokines by NOD DC could be an effective therapeutic strategy for the prevention and/or treatment of T1D.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from naïve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells.

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

Exogenous intrapleural injection of interleukin-27 may improves outcome and prognosis in patients with tuberculous pleural effusion.

We hypothesize that exogenous intrapleural injection of interleukin-27 may improve outcome and prognosis in patients with tuberculous pleural effusion (TPE). Studies have found that the balance of Th1/Th2 determines the development trend of TPE. High concentrations of IFN-γ and TNF-α in pleural effusion are associated with pleural adhesion in patients with TPE. Interleukin-27 is a member of the IL-12 family, and IL-27 has a dual regulatory effect on Th1 immunity. On one hand, IL-27 can promote the initial CD4+ T cell proliferation by inducing the expression of T-bet, IL-12Rß2 and ICAM-1 in the initial CD4+ T cells, and also promote its differentiation into Th1 cells and IFN-γ production in the early infection. On the other hand, in the case of high Th1 polarization, IL-27 induced STAT3 phosphorylation and inhibited TNF and IL-12 production in activated peritoneal macrophages, indicating a novel feedback mechanism by which IL-27 can modulate excessive inflammation, thereby preventing damage to the body caused by excessive immune response. Studies haves confirmed that after stimulation of antigen by mononuclear cells in TPE, the Th1 and Th2 cell subsets and Th1/Th2 ratio markedly increase, and the increase of Th1 is more obvious than that of Th2. Therefore, compared to patients with TPE in the high-level IL-27 group, we hypothesized that pleural effusion is absorbed more slowly, pleural thickening is more obvious, pleural adhesions are more extensive, and the incidence of thoracic collapse is higher in the low-level IL-27 group under the same conditions of anti-tuberculosis treatment. However, exogenous intrapleural injection of IL-27 may induce Stat3 phosphorylation and inhibit TNF and IL-12 production, finally reduces the secretion of IFN-γ and TNF-α. This negative regulation inhibits the excessive inflammatory reaction caused by tuberculosis infection, reduces pleural adhesion, pleural thickening and local pleural tissue damage, thereby improving the prognosis of patients.

Immunoregulatory Functions of the IL-12 Family of Cytokines in Antiviral Systems.

Members of the interleukin 12 (IL-12) family have been known to be inflammatory factors since their discovery. The IL-12 family consists of IL-12, IL-23, IL-27, IL-35, and a new member, IL-39, which has recently been identified and has not yet been studied extensively. Current literature has described the mechanisms of immunity of these cytokines and potential uses for therapy and medical cures. IL-12 was found first and is effective in combatting a wide range of naturally occurring viral infections through the upregulation of various cytokines to clear the infected cells. IL-23 has an essential function in immune networks, can induce IL-17 production, and can antagonize inhibition from IL-12 in the presence of T helper (Th) 17 cells, resulting in type II IFN (IFN-γ) regulation. IL-27 has a competitive relationship to IL-35 because they both include the same subunit, the Epstein-Barr virus-induced gene3 (EBi3). This review provides a simple introduction to the IL-12 family and focuses on their functions relevant to their actions to counteract viral infections.

IL12 and IFNγ secretion by donor mononuclear cells in response to host antigens may predict acute GVHD after HSCT.

One persistent problem of allogeneic hematopoietic stem cell transplantation (HSCT) is acute graft versus host disease (GVHD). The role of cytokines in the pathogenesis of GVHD has been acknowledged. We aimed, in the current study, to investigate the possibility of prediction of acute GVHD through investigating the pattern of interleukin 12 (IL12) and interferon gamma (IFNγ) production of both patients' origin and donors' origin. A total of 45 patients, receiving allogeneic peripheral blood (PB) stem cells from an identical sibling, were included in the study. Patients' plasma was collected after conditioning, during aplastic phase (representing patients' origin) and after engraftment (representing donors' origin). In addition an aliquot from the graft was used as responders in mixed lymphocyte culture (MLC) for 3 days with patients' mitomycin-treated mononuclear cells as stimulators. Culture supernatant was used for detection of IL12 and IFNγ of donors' origin. Fourteen patients developed acute GVHD. In culture supernatant, IL12 was detectable in 7/14 cases with and in none of 31 cases without acute GVHD (p= <0.001). The corresponding figures for IFNγ were 10/14 and 3/31 with significantly higher IFNγ level in cases with than in cases without acute GVHD (p = 0.001). At engraftment the corresponding figures were 7/14 and 5/31 for IL12 and 11/14 and 7/31 for IFNγ with significantly higher cytokine levels in cases with acute GVHD (p = 0.008 and p = 0.001 respectively). At a cutoff of 0.89 pg/ml, IL12 in culture supernatant may predict acute GVHD with absolute specificity of 100% and a sensitivity of 50%. In conclusion, IL12 and IFNγ of donors' origin not of patients' origin may predict the occurrence of acute GVHD. The MLC model may allow prediction of acute GVHD upfront before conditioning of the patient or mobilization of the donor.

Co-delivery of immunomodulators in biodegradable nanoparticles improves therapeutic efficacy of cancer vaccines.

To improve the efficacy of cancer vaccines we aimed to modulate the suppressive tumor microenvironment. In this study, the potential of intratumoral immune modulation with poly (I:C), Resiquimod (R848) and CCL20 (MIP3α) was explored. Biodegradable polymeric nanoparticles were used as delivery vehicles for slow and sustained release of these drugs in the tumor area and were combined with specific immunotherapy based on therapeutic peptide vaccination in two aggressive murine carcinoma and lymphoma tumor models. Whereas nanoparticle delivery of poly (I:C) or R848 improved therapeutic efficacy, the combination with MIP3α remarkably potentiated the cancer vaccine antitumor effects. The long-term survival increased to 75-100% and the progression free survival nearly doubled on mice with established large carcinoma tumors. The potent adjuvant effects were associated with lymphoid and myeloid population alterations in the tumor and tumor-draining lymph node. In addition to a significant influx of macrophages into the tumor, the phenotype of the suppressor tumor-associated macrophages shifted towards an acute inflammatory phenotype in the tumor-draining lymph node. Overall, these data show that therapeutic cancer vaccines can be potentiated by the combined nanoparticle mediated co-delivery of poly (I:C), R848 and MIP3α, which indicates that a more favorable milieu for cancer fighting immune cells is created for T cells induced by therapeutic cancer vaccines.

Association between genotypes of rs34436714 of NLRP12 and serum tumor necrosis factor-alpha in inflammatory bowel disease: A case-control study.

We aimed to investigate the impact of the single nucleotide polymorphisms of rs34436714 of the NOD-like receptor protein 12 gene on the production of tumor necrosis factor-alpha (TNFα) in patients with inflammatory bowel disease (IBD)In a matched case-control study 90 patients with IBD, 56 with Crohn disease (CD) and 34 with ulcerative colitis, were genotyped and compared to 98 healthy comparators matched for age and gender. Expression level of TNFα, interleukin (IL)-6, IL-12, and soluble triggering receptor expressed on myeloid cells were measured in patients' sera. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated for TNFα production.Serum TNFα was greater among carriers of GT/TT genotypes than GG genotypes of rs34436714. Stimulated TNFα production was also higher in carriers of GT/TT genotypes. The frequency of CD with fistulizing behavior and with CD involving the small intestine was greater among carriers of GT/TT genotypes than of the GG genotype. Distribution of the GG, GT, and TT genotypes of rs34436714 were in Hardy-Weinberg equilibrium in both groups. The genotype distribution was the same in both groups.Carriage of minor frequency alleles of rs34436714 was accompanied by greater circulating levels of TNFα and by greater capacity for stimulated TNFα production by PBMCs. These alleles had an impact on the phenotype of patients with CD.

IL12 Abrogates Calcineurin-Dependent Immune Evasion during Leukemia Progression.

Exploitation of the immune system has emerged as an important therapeutic strategy for acute lymphoblastic leukemia (ALL). However, the mechanisms of immune evasion during leukemia progression remain poorly understood. We sought to understand the role of calcineurin in ALL and observed that depletion of calcineurin B (CnB) in leukemia cells dramatically prolongs survival in immune-competent but not immune-deficient recipients. Immune-competent recipients were protected from challenge with leukemia if they were first immunized with CnB-deficient leukemia, suggesting robust adaptive immunity. In the bone marrow (BM), recipients of CnB-deficient leukemia harbored expanded T-cell populations as compared with controls. Gene expression analyses of leukemia cells extracted from the BM identified Cn-dependent significant changes in the expression of immunoregulatory genes. Increased secretion of IL12 from CnB-deficient leukemia cells was sufficient to induce T-cell activation ex vivo, an effect that was abolished when IL12 was neutralized. Strikingly, recombinant IL12 prolonged survival of mice challenged with highly aggressive B-ALL. Moreover, gene expression analyses from children with ALL showed that patients with higher expression of either IL12A or IL12B exhibited prolonged survival. These data suggest that leukemia cells are dependent upon calcineurin for immune evasion by restricting the regulation of proinflammatory genes, particularly IL12. SIGNIFICANCE: This report implicates calcineurin as an intracellular signaling molecule responsible for immune evasion during leukemia progression and raises the prospect of re-examining IL12 as a therapeutic in leukemia.