Takashi Akazawa

Tumor-Secreted Lactic Acid Promotes IL-23/IL-17 Proinflammatory Pathway

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.

Two isoforms of a nucleotide-sugar pyrophosphatase/phosphodiesterase from barley leaves (Hordeum vulgare L.) are distinct oligomers of HvGLP1, a germin-like protein

Two isoforms of ADPglucose pyrophosphatase/phosphodiesterase (AGPPase) have been characterized using barley leaves (Hordeum vulgare L.). Whilst one of the isoforms, designated as soluble AGPPase1 (SAGPPase1), is soluble in low ionic strength buffers, the other, SAGPPase2, is extractable using cell wall hydrolytic enzymes or high salt concentration solutions, thus indicating that it is adventitiously bound to the cell wall. Both AGPPase isoforms are highly resistant to SDS, this characteristic being utilized to purify them to homogeneity after zymographic detection of AGPPase activity in SDS‐containing gels. N‐terminal and internal amino acid sequencing analyses revealed that both SAGPPase1 and SAGPPase2 are distinct oligomers of the previously designated HvGLP1, which is a member of the ubiquitously distributed group of proteins of unknown function designated as germin‐like proteins (GLPs).

Enhancement of antitumor natural killer cell activation by orally administered Spirulina extract in mice

Oral administration of hot‐water extract of Spirulina, cyanobacterium Spirulina platensis, leads to augmentation of NK cytotoxicity in humans. Here, we applied to syngeneic tumor‐implant mice (C57BL/6 versus B16 melanoma) Spirulina to elucidate the mechanism of raising antitumor NK activation. A B16D8 subcell line barely expressed MHC class I but about 50% expressed Rae‐1, a ligand for NK activation receptor NKG2D. The Rae‐1‐positive population of implant B16 melanoma was effectively eliminated in the tumor mass progressed in mice. This antitumor activity was induced in parallel with IFN‐γ and abolished in mice by treatment with asialoGM‐1 but not CD8β Ab, suggesting the effector is NK cell. NK cell activation occurred in the spleen of wild‐type mice medicated with Spirulina. This Spirulina‐mediated enhanced NK activation was abrogated in MyD88 –/– mice but not in TICAM‐1 –/– mice. The NK activating properties of Spirulina depending on MyD88 were confirmed with in vitro bone marrow‐derived dendritic cells expressing TLR2/4. In D16D8 tumor challenge studies, the antitumor effect of Spirulina was abolished in MyD88 –/– mice. Hence, orally administered Spirulina enhances tumoricidal NK activation through the MyD88 pathway. Spirulina exerted a synergistic antitumor activity with BCG–cell wall skeleton, which is known to activate the MyD88 pathway via TLR2/4 with no NK enhancing activity. Spirulina and BCG–cell wall skeleton synergistically augmented IFN‐γ production and antitumor potential in the B16D8 versus C57BL/6 system. We infer from these results that NK activation by Spirulina has some advantage in combinational use with BCG–cell wall skeleton for developing adjuvant‐based antitumor immunotherapy. (Cancer Sci 2009)

IL-23-dependent and -independent enhancement pathways of IL-17A production by lactic acid

Interleukin-17A (IL-17A) is a cytokine produced by T(h)17 cells that plays an important role in inflammatory and autoimmune diseases and cancer. Stimulation with IL-6, transforming growth factor-β , IL-21, IL-1β and IL-23 is required for differentiation of T(h)17 cells and the production of IL-17A. Recently, we reported that tumor-derived lactic acid enhances the toll-like receptor (TLR) ligand-mediated expression of IL-23, leading to increased IL-17A production. Tumor cells secrete large amounts of lactic acid due to the up-regulation of glycolysis, which is known as the Warburg effect. Even without TLR ligand stimulation, lactic acid enhanced antigen-dependent IL-17A production from splenocytes in an IL-23-dependent manner. Here, we show that macrophages and effector/memory CD4(+) T cells are the primary cell types involved in the ability of lactic acid to boost IL-17A production. Although lactic acid suppressed the proliferation of T(h)1 and T(h)17 cells, T(h)17 cells still secreted large amounts of IL-17A. CD40 ligand-CD40 interactions were involved in the up-regulation of IL-17A by lactic acid through IL-12/23p40 production. A new cytokine containing the IL-12/23p40 subunit, but not IL-23, IL-12 or the IL-12p40 homodimer, is a candidate for involvement in the up-regulation of IL-17A. IL-1β also increased IL-17A expression; however, IL-1β, CARD9 and MyD88 signaling pathways activated by known intrinsic inflammatory mediators were hardly required for the enhanced activity induced by lactic acid. Our results show that lactic acid functions as an intrinsic inflammatory mediator that activates IL-23-dependent and -independent pathways, resulting in the promotion of chronic inflammation in tumor microenvironments.

Two-step colocalization of MORC3 with PML nuclear bodies

Many functional subdomains, including promyelocytic leukemia nuclear bodies (PML NBs), are formed in the mammalian nucleus. Various proteins are constitutively or transiently accumulated in PML NBs in a PML-dependent manner. MORC3 (microrchidia family CW-type zinc-finger 3), also known as NXP2, which consists of GHL-ATPase, a CW-type zinc-finger and coiled-coil domains, is localized in PML NBs, where it recruits and activates p53 to induce cellular senescence. Interestingly, we found that MORC3 can form PML-independent nuclear domains (NDs) in mouse hematopoietic cells and even in Pml-deficient cells. Here, we show that MORC3 colocalizes with PML by a two-step molecular mechanism: the PML-independent formation of MORC3 NDs by the ATPase cycle, and the association of MORC3 with PML via the SUMO1-SUMO-interacting motif (SIM). Similarly to other members of the GHL-ATPase family, MORC3 functions as a ‘molecular clamp’. ATP binding induces conformational changes in MORC3, leading to the formation of MORC3 NDs, and subsequent ATP hydrolysis mediates the diffusion and binding of MORC3 to the nuclear matrix. MORC3 might clamp DNA or nucleosomes in MORC3 NDs via the CW domain. Furthermore, the SUMOylation of MORC3 at five sites was involved in the association of MORC3 with PML, and SUMO1-unmodified MORC3 formed NDs independently of PML.

Tumor immunotherapy using bone marrow-derived dendritic cells overexpressing Toll-like receptor adaptors

Myeloid dendritic cells (mDCs) play an important role in the initiation of immune responses to cancer and infectious diseases. Toll‐like receptors (TLRs) expressed on mDCs recognize microbial products to elicit signals for mDC maturation, including cytokine production, antigen‐presentation and induction of effector cells. TLR agonists work as adjuvants to modulate the function of mDCs. In TLR signaling, MyD88 and TRIF/TICAM‐1 are major TLR adaptor molecules, which when overexpressed are able to transduce downstream signals without TLR stimuli. We successfully introduced the adaptors into mouse bone marrowderived mDCs using lentiviral vectors. Introduction of MyD88 into mDCs in vitro led to the production of IL‐6 and IL‐12p40 while introduction of TICAM‐1 stimulated interferon (IFN)‐alpha production. Expression of TICAM‐1, but not MyD88, in mDCs slightly induced the co‐stimulatory molecule CD86, while significant upregulation of CD86 was observed in response to other TLR stimuli. Both MyD88 and TICAM‐1 augmented allogeneic mixed lymphocyte reaction (MLR). Ex vivo mouse spleen cells pre‐exposed to tumor antigen exhibited antitumor cytotoxicity when incubated with MyD88‐ or TICAM‐1‐expressing mDCs. Using mDC adoptive transfer and a syngeneic mouse tumor implant model, we established an antitumor immunotherapy whereby tumor growth is retarded by adaptor‐manipulated mDCs.

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

Failure of mycoplasma lipoprotein MALP-2 to induce NK cell activation through dendritic cell TLR2

Macrophage-activating lipopeptide 2 (MALP-2), a mycoplasmal diacylated lipopeptide with palmitic acid moiety (Pam2), activates Toll-like receptor (TLR) 2 to induce inflammatory cytokines. TLR2 is known to mature myeloid dendritic cells (mDC) to drive mDC contact-mediated natural killer (NK) cell activation. Here we tested if MALP-2 activates NK cells through stimulation of TLR2 on mDC. Although synthetic MALP-2 with 6 or 14 amino acids (a.a.) stretch (designated as s and f) matured mDC to induce IL-6, IL-12p40 and TNF-α to a similar extent, they far less activated NK cells than Pam2CSK4, a positive control of 6 a.a.-containing diacyl lipopeptide. MALP-2s and f were TLR2/6 agonists and activate the MyD88 pathway similar to Pam2CSK4, but MALP-2s having the CGNNDE sequence acted on mDC TLR2 to barely induce external NK activation. Even the s form, with slightly high induction of IL-6 compared to the f form, barely induced in vivo growth retardation of NK-sensitive implant tumor. Pam2CSK4 and MALP-2 have the common lipid moiety but different peptides, which are crucial for NK cell activation. The results infer that MALP-2 is applicable to a cytokine inducer but not to an adjuvant for antitumor NK immunotherapy.

Innate immune therapy with a Bacillus Calmette-Guérin cell wall skeleton after radical surgery for non-small cell lung cancer: A case-control study

PurposeWe investigated whether adjuvant immunotherapy with Bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) and surgical resection was better than resection, with or without other adjuvant therapy, for patients with non-small cell lung cancer (NSCLC).MethodsThe case group comprised 71 patients who underwent radical surgery for NSCLC, followed by BCG-CWS immunotherapy, with follow-up data available. The case-control study was designed with one control selected for each case-group patient. Each control was matched by pathological stage and year of birth (±5 years). BCG-CWS 200 μg was inoculated intracutaneously in the upper arm four times per week (sensitization phase); then at 4-week intervals (therapeutic phase).ResultsThe case-group patients received 45 ± 22.6 (average ± SD) cycles of BCG-CWS inoculation. Overall 5-year and 10-year survival rates were 71% and 61% for the case-group patients, and 63% and 43% for the control-group patients. The survival rate of the case group was better than that of the control group (not significant; P = 0.114). The same trend was seen in the patients with stage III or N+ NSCLC (not significant; P = 0.114, P = 0.168). There were no life-threatening adverse events.ConclusionsBCG-CWS immunotherapy seemed to improve survival after resection of NSCLC, especially locally advanced NSCLC. Moreover, this immunotherapy did not compromise quality of life during treatment.

Adjuvant engineering for cancer immunotherapy: Development of a synthetic TLR2 ligand with increased cell adhesion

The development of effective immunoadjuvants for tumor immunotherapy is of fundamental importance. The use of Mycobacterium bovis bacillus Calmette‐Guérin cell wall skeleton (BCG‐CWS) in tumor immunotherapy has been examined in various clinical applications. Because BCG‐CWS is a macromolecule that cannot be chemically synthesized, the development of an alternative synthetic molecule is necessary to ensure a constant supply of adjuvant. In the present study, a new adjuvant was designed based on the structure of macrophage‐activating lipopeptide (MALP)‐2, which is a Toll‐like receptor (TLR)‐2 ligand similar to BCG‐CWS. Macrophage‐activating lipopeptide‐2, [S‐(2,3‐bispalmitoyloxypropyl)Cys (P2C) – GNNDESNISFKEK], originally identified in a Mycoplasma species, is a lipopeptide that can be chemically synthesized. A MALP‐2 peptide was substituted with a functional motif, RGDS, creating a novel molecule named P2C‐RGDS. RGDS was selected because its sequence constitutes an integrin‐binding motif and various integrins are expressed in immune cells including dendritic cells (DCs). Thus, this motif adds functionality to the ligand. P2C‐RGDS activated DCs and splenocytes more efficiently than MALP‐2 over short incubation times in vitro, and the RGDS motif contributed to their activation. Furthermore, P2C‐RGDS showed higher activity than MALP‐2 in inducing migration of DCs to draining lymph node, and in inhibiting tumor growth in vivo. This process of designing and developing synthetic adjuvants has been named “adjuvant engineering,” and the evaluation and improvement of P2C‐RGDS constitutes a first step in the development of stronger synthetic adjuvants in the future. (Cancer Sci 2010)