Inflammasome-Dependent Induction of Adaptive NK Cell Memory.

Monobenzone is a pro-hapten that is exclusively metabolized by melanocytes, thereby haptenizing melanocyte-specific antigens, which results in cytotoxic autoimmunity specifically against pigmented cells. Studying monobenzone in a setting of contact hypersensitivity (CHS), we observed that monobenzone induced a long-lasting, melanocyte-specific immune response that was dependent on NK cells, yet fully intact in the absence of T- and B cells. Consistent with the concept of "memory NK cells," monobenzone-induced NK cells resided in the liver and transfer of these cells conferred melanocyte-specific immunity to naive animals. Monobenzone-exposed skin displayed macrophage infiltration and cutaneous lymph nodes showed an inflammasome-dependent influx of macrophages with a tissue-resident phenotype, coinciding with local NK cell activation. Indeed, macrophage depletion or the absence of the NLRP3 inflammasome, the adaptor protein ASC or interleukin-18 (IL-18) abolished monobenzone CHS, thereby establishing a non-redundant role for the NLRP3 inflammasome as a critical proinflammatory checkpoint in the induction of hapten-dependent memory NK cells.

Extracorporeal photopheresis promotes IL-1ß production.

Extracorporeal photopheresis (ECP) is a widely used clinical cell-based therapy exhibiting efficacy in heterogenous immune-mediated diseases such as cutaneous T cell lymphoma, graft-versus-host disease, and organ allograft rejection. Despite its documented efficacy in cancer immunotherapy, little is known regarding the induction of immunostimulatory mediators by ECP. In this article, we show that ECP promotes marked release of the prototypic immunostimulatory cytokine IL-1ß. ECP primes IL-1ß production and activates IL-1ß maturation and release in the context of caspase-1 activation in monocytes and myeloid dendritic cells. Of interest, IL-1ß maturation by ECP was fully intact in murine cells deficient in caspase-1, suggesting the predominance of an inflammasome-independent pathway for ECP-dependent IL-1ß maturation. Clinically, patient analysis revealed significantly increased IL-1ß production in stimulated leukapheresis concentrates and peripheral blood samples after ECP. Collectively, these results provide evidence for promotion of IL-1ß production by ECP and offer new insight into the immunostimulatory capacity of ECP.

iGLuc: a luciferase-based inflammasome and protease activity reporter.

Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro-interleukin (IL)-1ß-Gaussia luciferase (iGLuc) fusion construct, in which pro-IL-1ß-dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.

Correction: AIM2 Drives Joint Inflammation in a Self-DNA Triggered Model of Chronic Polyarthritis.

[This corrects the article DOI: 10.1371/journal.pone.0131702.].

Sequential administration of a MVA-based MUC1 cancer vaccine and the TLR9 ligand Litenimod (Li28) improves local immune defense against tumors.

TG4010 is an immunotherapeutic vaccine based on Modified Vaccinia virus Ankara (MVA) encoding the human tumor-associated antigen MUC1 and human IL-2. In combination with first-line standard of care chemotherapy in advanced metastatic non-small-cell lung cancer (NSCLC), repeated subcutaneous injection of TG4010 improved progression-free survival in phase 2b clinical trials. In preclinical tumor models, MVATG9931, the research version of TG4010, conferred antigen-specific responses against the weak antigen human MUC1. The combination of a suboptimal dose of MVATG9931 and the type B TLR9 ligand Litenimod (Li28) markedly increased survival in a subcutaneous RMA-MUC1 tumor model compared to the treatment with MVATG9931 or Li28 alone. The requirements for this protection were (i) de novo synthesis of MUC1, (ii) Li28 delivered several hours after MVATG9931 at the same site, (iii) at least two vaccination cycles, and (iv) implantation of MUC1-positive tumor cells in the vicinity to the vaccination site. Subcutaneously injected MVATG9931 allowed transient local gene expression and induced the local accumulation of MCP-1, RANTES, M-CSF, IL-15/IL-15R and IP-10. After repeated injection, CD4+ and CD8+ T lymphocytes, B lymphocytes, NK cells, pDCs, neutrophils, and macrophages accumulated around the injection site, local RANTES levels remained high. Delayed injection of Li28 into this environment, led to further accumulation of macrophages, the secretion of IL-18 and IL-1 beta, and an increase of the percentage of activated CD69+ NK cell. Combination treatment augmented the number of activated CD86+ DCs in the draining lymph nodes and increased the percentage of KLRG1+ CD127-CD8+ T cells at the injection site. In vivo depletion of macrophages around the injection site by Clodronate liposomes reduced local IL-18 levels and diminished survival rates significantly. Thus, sequential administration of MVATG9931 and Li28 improves local innate and adaptive immune defense against tumors, arguing for intratumoral delivery of this peculiar sequential combination therapy.

STING Contributes to Abnormal Bone Formation Induced by Deficiency of DNase II in Mice.

OBJECTIVE: Cytosolic DNA sensors detect microbial DNA and promote type I interferon (IFN) and proinflammatory cytokine production through the adaptor stimulator of IFN genes (STING) to resolve infection. Endogenous DNA also engages the STING pathway, contributing to autoimmune disease. This study sought to identify the role of STING in regulating bone formation and to define the bone phenotype and its pathophysiologic mechanisms in arthritic mice double deficient in DNase II and IFN-α/ß/ω receptor (IFNAR) (DNase II-/- /IFNAR-/- double-knockout [DKO] mice) compared with controls. METHODS: Bone parameters were evaluated by micro-computed tomography and histomorphometry in DKO mice in comparison with mice triple deficient in STING, DNase II, and IFNAR and control mice. Cell culture techniques were employed to determine the parameters of osteoclast and osteoblast differentiation and function. NanoString and Affymetrix array analyses were performed to identify factors promoting ectopic bone formation. RESULTS: Despite the expression of proinflammatory cytokines that would be expected to induce bone loss in the skeleton of DKO mice, the results, paradoxically, demonstrated an accumulation of bone in the long bones and spleens, sites of erythropoiesis and robust DNA accrual. In addition, factors promoting osteoblast recruitment and function were induced. Deficiency of STING significantly inhibited bone accrual. CONCLUSION: These data reveal a novel role for cytosolic DNA sensor pathways in bone in the setting of autoimmune disease. The results demonstrate the requirement of an intact STING pathway for bone formation in this model, a finding that may have relevance to autoimmune diseases in which DNA plays a pathogenic role. Identification of pathways linking innate immunity and bone could reveal novel targets for the treatment of bone abnormalities in human autoimmune diseases.

AIM2 Drives Joint Inflammation in a Self-DNA Triggered Model of Chronic Polyarthritis.

Mice lacking DNase II display a polyarthritis-like disease phenotype that is driven by translocation of self-DNA into the cytoplasm of phagocytic cells, where it is sensed by pattern recognition receptors. While pro-inflammatory gene expression is non-redundantly linked to the presence of STING in these mice, the contribution of the inflammasome pathway has not been explored. To this end, we studied the role of the DNA-sensing inflammasome receptor AIM2 in this self-DNA driven disease model. Arthritis-prone mice lacking AIM2 displayed strongly decreased signs of joint inflammation and associated histopathological findings. This was paralleled with a reduction of caspase-1 activation and pro-inflammatory cytokine production in diseased joints. Interestingly, systemic signs of inflammation that are associated with the lack of DNase II were not dependent on AIM2. Taken together, these data suggest a tissue-specific role for the AIM2 inflammasome as a sensor for endogenous DNA species in the course of a ligand-dependent autoinflammatory condition.

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay.

AIM: To test the methodical and pre-analytical performance of a new multiplex cancer biomarker panel using magnetic beads. METHODS: The MILLIPLEX(®) MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 comprises the tumor markers carcinoembryonic antigen, alpha-fetoprotein, total prostate-specific antigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 125, cytokeratine 19-fragment, ß-human chorionic gonadotropin, human epididymis protein 4, osteopontin, prolactin, the cell death and angiogenesis markers soluble Fas, soluble Fas-ligand, tumor necrosis factor related apoptosis-inducing ligand, vascular endothelial growth factor and the immunological markers interleukin-6 (IL-6), IL-8, tumor necrosis factor-α, transforming growth factor α, fibroblast growth factor-2, macrophage migration inhibitory factor, leptin, hepatocyte growth factor, and stem cell factor. We determined intra- and inter-assay imprecision as well as dilution linearity using quality controls and serum pools. Furthermore, the stability of the 24 biomarkers examined in this panel was ascertained by testing the influence of different storage temperatures and time span before centrifugation. RESULTS: For all markers measured in the synthetic internal quality controls, the intra-assay imprecision ranged between 2.26% and 9.41%, while for 20 of 24 measured markers in the physiological serum pools, it ranged between 1.68% and 12.87%. The inter-assay imprecision ranged between 1.48%-17.12% for 23 biomarkers in synthetic, and between 4.59%-23.88% for 18 biomarkers in physiological quality controls. Here, single markers with very low concentration levels had increased imprecision rates. Dilution linearity was acceptable (70%-130% recovery) for 20 biomarkers. Regarding pre-analytical influencing factors, most markers were stable if blood centrifugation was delayed or if serum was stored for up to 24 h at 4 °C and 25 °C after centrifugation. Comparable results were obtained in serum and plasma for most markers. However, great changes were observed for single markers. CONCLUSION: MILLIPLEX(®) MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 assay is a stable and precise method for detection of most biomarkers included in the kit. However, single markers have to be interpreted with care.

Immunoblotting for active caspase-1.

Immunoblotting for caspase-1 is the gold-standard method of detecting inflammasome activation. In contrast to IL-1ß-based readouts, it can be used in an experimental setup independent of de novo gene expression. Here, we present protocols for the preparation and precipitation of supernatant samples containing activated caspase-1 as well as protocols for polyacrylamide gel electrophoresis (PAGE) and protein immunoblotting.

RIG-I detects triphosphorylated RNA of Listeria monocytogenes during infection in non-immune cells.

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.