IL-23 is a proinflammatory cytokine consisting of a p19 subunit and a p40 subunit that is shared with IL-12. IL-23 is overexpressed in and around tumor tissues, where it induces local inflammation and promotes tumor development. Many tumor cells produce large amounts of lactic acid by altering their glucose metabolism. In this study, we show that lactic acid secreted by tumor cells enhances the transcription of IL-23p19 and IL-23 production in monocytes/macrophages and in tumor-infiltrating immune cells that are stimulated with TLR2 and 4 ligands. DNA elements responsible for this enhancing activity of lactic acid were detected in a 2.7-kb 5′-flanking region of the human IL-23p19 gene. The effect of lactic acid was strictly regulated by extracellular pH. Furthermore, by inducing IL-23 overproduction, lactic acid facilitated the Ag-dependent secretion of proinflammatory cytokine IL-17 but not IFN-γ by TLR ligand-stimulated mouse splenocytes. Interestingly, this effect was observed even in the absence of TLR ligand stimulation. These results suggest that rather than just being a terminal metabolite, lactic acid is a proinflammatory mediator that is secreted by tumor cells to activate the IL-23/IL-17 proinflammatory pathway but not the Th1 pathway. Targeting the lactic acid-induced proinflammatory response may be a useful approach for treating cancer.

Smoldering inflammation often increases the risk of progression for malignant tumors and simultaneously matures myeloid dendritic cells (mDCs) for cell-mediated immunity. PolyI:C, a dsRNA analog, is reported to induce inflammation and potent antitumor immune responses via the Toll-like receptor 3/Toll-IL-1 receptor domain-containing adaptor molecule 1 (TICAM-1) and melanoma differentiation-associated protein 5/IFN-β promoter stimulator 1 (IPS-1) pathways in mDCs to drive activation of natural killer cells and cytotoxic T lymphocytes. Here, we found that i.p. or s.c. injection of polyI:C to Lewis lung carcinoma tumor-implant mice resulted in tumor regression by converting tumor-supporting macrophages (Mfs) to tumor suppressors. F4/80− Mfs infiltrating the tumor respond to polyI:C to rapidly produce inflammatory cytokines and thereafter accelerate M1 polarization. TNF-α was increased within 1 h in both tumor and serum upon polyI:C injection into tumorbearing mice, followed by tumor hemorrhagic necrosis and growth suppression. These tumor responses were abolished in TNF-α −/− mice. Furthermore, F4/80 + Mfs in tumors extracted from polyI:Cinjected mice sustained Lewis lung carcinoma cytotoxic activity, and this activity was partly abrogated by anti-TNF-α Ab. Genes for supporting M1 polarization were subsequently up-regulated in the tumor-infiltrating Mfs. These responses were completely abrogated in TICAM-1 −/− mice, and unaffected in myeloid differentiation factor 88 −/− and IPS-1 −/− mice. Thus, the TICAM-1 pathway is not only important to mature mDCs for cross-priming and natural killer cell activation in the induction of tumor immunity, but also critically engaged in tumor suppression by converting tumor-supporting Mfs to those with tumoricidal properties.Toll-like receptor | tumor-associated macrophages | TRIF

Ligand stimulation of the Toll-like receptors (TLRs) triggers innate immune response, cytokine production and cellular immune activation in dendritic cells. However, most TLR ligands are microbial constituents, which cause inflammation and toxicity. Toxic response could be reduced for secure immunotherapy through the use of chemically synthesized ligands with defined functions. Here we create an RNA ligand for TLR3 with no ability to activate the RIG-I/MDA5 pathway. This TLR3 ligand is a chimeric molecule consisting of phosphorothioate ODN-guided dsRNA (sODN-dsRNA), which elicits far less cytokine production than poly(I:C) in vitro and in vivo. The activation of TLR3/TICAM-1 pathway by sODN-dsRNA effectively induces natural killer and cytotoxic T cells in tumour-loaded mice, thereby establishing antitumour immunity. Systemic cytokinemia does not occur following subcutaneous or even intraperitoneal administration of sODN-dsRNA, indicating that TICAM-1 signalling with minute local cytokines sufficiently activate dendritic cells to prime tumoricidal effectors in vivo.

Somatic mutation of PIGA in hematopoietic stem cells causes deficiency of glycosyl phosphatidylinositol-anchored proteins in paroxysmal nocturnal hemoglobinuria (PNH) that underlies the intravascular hemolysis but does not account for expansion of the PNH clone. Immune mechanisms may mediate clonal selection but appear insufficient to account for the clonal dominance necessary for PNH to become clinically apparent. Herein, we report 2 patients with PNH whose PIGAmutant cells had a concurrent, acquired rearrangement of chromosome 12. In both cases, der(12) had a break within the 3 untranslated region of HMGA2, the architectural transcription factor gene deregulated in many benign mesenchymal tumors, that caused ectopic expression of HMGA2 in the bone marrow. These observations suggest that aberrant HMGA2 expression, in concert with mutant PIGA, accounts for clonal hematopoiesis in these 2 patients and suggest the concept of PNH as a benign tumor of the bone marrow. ( IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a consequence of nonmalignant clonal expansion of hematopoietic stem cells with somatic mutation of PIGA. 1 Mutant PIGA 2 explains the deficiency of glycosyl phosphatidylinositol-anchored proteins (GPI-APs) that underlies the intravascular hemolysis of PNH. 3 However, PIGAmutant stem cells have no intrinsic proliferative advantage, 4,5 suggesting a 2-step model of pathogenesis.Step 1 of this model, clonal selection, 6,7 is envisioned as a conditional survival advantage that depends on deficiency of 1 or more GPI-APs. The close association of PNH with aplastic anemia, suggests that the selection pressure is immune mediated. 6,7 But, although 60% to 70% of patients with aplastic anemia have small, subclinical populations of GPI-AP Ϫ hematopoietic cells at diagnosis, 8 only 10% to 15% subsequently develop clinically apparent PNH. 9 In the remainder, GPI-AP Ϫ cells persist subclinically or disappear, 8 suggesting that mutant PIGA (and the consequent deficiency of GPI-APs) is necessary for clonal selection but is insufficient to account for the clonal expansion required for clinical manifestations of PNH to become apparent.Clonal expansion, step 2 of the PNH pathogenesis model, is envisioned as a consequence of clonal evolution in which a second somatic mutation bestows on the PIGA-mutant stem cell a proliferative advantage. 10 Herein, we present evidence supporting this 2-step model by showing a concurrent, acquired genetic abnormality in the PIGAmutant cells of 2 patients that establishes a novel mechanism for the nonmalignant clonal hematopoieis characteristic of PNH. Patients, materials, and methods PatientsInformed consent was obtained from patients J20 and US1 according to protocols approved by the Institutional Review Boards of Osaka University Hospital (Osaka, Japan) and the University of Utah School of Medicine (Salt Lake City, UT), respectively. Hybrid cell linesMonocytes derived from J20 or US1 were fused with the hypoxanthine phosphoribosyltransferase-negative mouse myeloma cell line, P...

Aerolysin of the Gram‐negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The α‐toxin of Gram‐positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)‐anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI‐anchored proteins, but nevertheless bound aerolysin poorly and was 10‐fold less sensitive than wild‐type cells. A cDNA of N‐acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N‐glycan is involved in the binding. Removal of mannoses by α‐mannosidase II was important for the binding of aerolysin. In contrast, the α‐toxin killed GnTI‐deficient and wild‐type CHO cells equally, indicating that its binding to GPI‐anchored proteins is independent of N‐glycan. Because SL bound to wild‐type but not to GnTI‐deficient cells, and because a hybrid toxin consisting of SL and the α‐toxin killed wild‐type cells 10‐fold more efficiently than GnTI‐ deficient cells, SL with its binding site for N‐glycan contributes to the high binding affinity of aerolysin.