Cutting Edge: ATP13A2 Is an Endolysosomal Regulator of TLR9/7 Activation in Human Plasmacytoid Dendritic Cells.

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.

Mitigating hERG Liability of Toll-Like Receptor 9 and 7 Antagonists through Structure-Based Design.

hERG is considered to be a primary anti-target in the drug development process, as the K+ channel encoded by hERG plays an important role in cardiac re-polarization. It is desirable to address the hERG safety liability during early-stage development to avoid the expenses of validating leads that will eventually fail at a later stage. We have previously reported the development of highly potent quinazoline-based TLR7 and TLR9 antagonists for possible application against autoimmune disease. Initial experimental hERG assessment showed that most of the lead TLR7 and TLR9 antagonists suffer from hERG liability rendering them ineffective for further development. The present study herein describes a coordinated strategy to integrate the understanding from structure-based protein-ligand interaction to develop non- hERG binders with IC50 >30 µM with retention of TLR7/9 antagonism through a single point change in the scaffold. This structure-guided strategy can serve as a prototype for abolishing hERG liability during lead optimization.

The RNase MCPIP3 promotes skin inflammation by orchestrating myeloid cytokine response.

CCCH zinc finger proteins resolve immune responses by degrading the mRNAs of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-6. Here we report that one such family member, monocyte chemotactic protein-induced protein 3 (MCPIP3, also named ZC3H12C or Regnase-3), promotes skin inflammation by simultaneously enhancing TNF in macrophages and repressing IL-6 in plasmacytoid dendritic cells (pDCs). MCPIP3 is positively associated with psoriasis pathogenesis, and highly expressed by macrophages and pDCs. MCPIP3-deficient macrophages produce less TNF and IL-12p40. However, MCPIP3-deficient pDCs secrete significantly more IL-6. This enhanced intradermal IL-6 may alleviate imiquimod-induced skin inflammation. As a result, MCPIP3-deficient mice are protected from imiquimod-induced psoriasiform lesions. Furthermore, early exposure to pDC-derived IL-6 suppresses macrophage-derived TNF and IL-12p40. Mechanistically, MCPIP3 could directly degrade mRNAs of IL-6, Regnase-1, and IκBζ. In turn, Regnase-1 could degrade MCPIP3 mRNAs. Our study identifies a critical post-transcriptional mechanism that synchronizes myeloid cytokine secretion to initiate autoimmune skin inflammation.

TLR9 Polymorphisms Might Contribute to the Ethnicity Bias for EBV-Infected Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is a rare malignancy in most parts of the world, but is endemic in some ethnic groups. The association of NPC with the Epstein-Barr virus (EBV) is firmly established; however, the mechanism is still unclear. TLR9 is well known for its essential role in viral pathogen recognition and activation of innate immunity. Here, we report a set of TLR9 polymorphisms in the TIR-2 domain of the TLR9 protein collected from the EBV-infected NPC samples from northeast Indian populations sharing the aforesaid ethnicity. The occurrence of mutations is significantly high in these samples as we found a p value of <0.0001 at a significance level of 0.05. These might play an important role for the lack of function of TLR9 and thus for the higher occurrence of EBV-mediated NPC in such ethnic groups.

Mechanical Cues for T Cell Activation: Role of Piezo1 Mechanosensors.

T cells are activated in response to the recognition of antigenic peptides on major histocompatibility complex molecules (pMHC) by the T cell receptors (TCR) and induction of downstream signaling. The strength of interaction between specific TCR with pMHC is a key defining factor for optimal T cell activation. But a number of studies have also suggested a crucial involvement of mechanical cues within the immunological synapse. However, a dedicated mechanosensor molecule that senses and transduces the mechanical cues to optimize TCR activation was, until very recently, not known. The putative candidates included the TCR itself as well as the integrins within the immunological synapse. Recently, the Piezo family of proteins was discovered as professional mechanosensors implicated in a number of physiological processes. One of the two Piezo family members, Piezo1, is expressed in human T cells. Recent data suggest that Piezo1 functions as a professional mechanotransducer at the immunological synapse during TCR-pMHC interaction, and thus play a crucial role in human T cell activation. Thus, the domain of T cell activation has gathered a new mechanoregulatory dimension, which should incite further studies for detailed elucidation of the mechanistic underpinnings and its translational implications.

Lactate Induces Pro-tumor Reprogramming in Intratumoral Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells are the most efficient producers of type I interferons, viz. IFNα, in the body and thus have the ability to influence anti-tumor immune responses. But repression of effective intra-tumoral pDC activation is a key immuno-evasion strategy exhibited in tumors-tumor-recruited pDCs are rendered "tolerogenic," characterized by deficiency in IFNα induction and ability to expand regulatory T cells in situ. But the tumor-derived factors that drive this functional reprogramming of intra-tumoral pDCs are not established. In this study we aimed at exploring if intra-tumoral abundance of the oncometabolite lactate influences intra-tumoral pDC function. We found that lactate attenuates IFNα induction by pDCs mediated by intracellular Ca2+ mobilization triggered by cell surface GPR81 receptor as well as directly by cytosolic import of lactate in pDCs through the cell surface monocarboxylate transporters, affecting cellular metabolism needed for effective pDC activation. We also found that lactate enhances tryptophan metabolism and kynurenine production by pDCs which contribute to induction of FoxP3+ CD4+ regulatory T cells, the major immunosuppressive immune cell subset in tumor microenvironment. We validated these mechanisms of lactate-driven pDC reprogramming by looking into tumor recruited pDCs isolated from patients with breast cancers as well as in a preclinical model of breast cancer in mice. Thus, we discovered a hitherto unknown link between intra-tumoral abundance of an oncometabolite resulting from metabolic adaptation in cancer cells and the pro-tumor tolerogenic function of tumor-recruited pDCs, revealing new therapeutic targets for potentiating anti-cancer immune responses.

Activity-guided development of potent and selective toll-like receptor 9 antagonists.

TLR9 is one of the major innate immune receptors expressed in the endosomes of pDCs and B cells in humans. Aberrant TLR9 activation is implicated in several autoimmune and metabolic disorders as well as in sepsis, making this receptor an important therapeutic target, though specific TLR9 antagonists are yet to be available for clinical use. Here we elucidate the importance of specific physiochemical properties through substitution patterns in quinazoline scaffold to achieve potent hTLR9 inhibition at < 50 nM as well as > 600 fold selectivity against hTLR7, another closely related TLR that shares downstream signaling with TLR9 but plays distinct roles in physiology and pathology. Assays were performed using hPBMC and reporter cell lines. Favorable in vitro ADME profile, pharmacokinetics as well as validation in a clinically relevant in vivo TLR9-inhibition efficacy model in mice establish these novel TLR9-antagonists as candidate therapeutic agents in relevant clinical contexts.

KLK5 induces shedding of DPP4 from circulatory Th17 cells in type 2 diabetes.

OBJECTIVE: Increasing plasma levels and activity of dipeptidyl peptidase-4 (DPP4 or CD26) are associated with rapid progression of metabolic syndrome to overt type 2 diabetes mellitus (T2DM). While DPP4 inhibitors are increasingly used as anti-hyperglycemic agents, the reason for the increase in plasma DPP4 activity in T2DM patients remains elusive. METHODS: We looked into the source of plasma DPP4 activity in a cohort of 135 treatment naive nonobese (BMI < 30) T2DM patients. A wide array of ex vivo, in vitro, and in silico methods were employed to study enzyme activity, gene expression, subcellular localization, protease identification, surface expression, and protein-protein interactions. RESULTS: We show that circulating immune cells, particularly CD4+ T cells, served as an important source for the increase in plasma DPP4 activity in T2DM. Moreover, we found kallikrein-related peptidase 5 (KLK5) as the enzyme responsible for cleaving DPP4 from the cell surface by directly interacting with the extracellular loop. Expression and secretion of KLK5 is induced in CD4+ T cells of T2DM patients. In addition, KLK5 shed DPP4 from circulating CD4+ T helper (Th)17 cells and shed it into the plasma of T2DM patients. Similar cleavage and shedding activities were not seen in controls. CONCLUSIONS: Our study provides mechanistic insights into the molecular interaction between KLK5 and DPP4 as well as CD4+ T cell derived KLK5 mediated enzymatic cleavage of DPP4 from cell surface. Thus, our study uncovers a hitherto unknown cellular source and mechanism behind enhanced plasma DPP4 activity in T2DM.

Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.

Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

INTRODUCTION: Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H(2)O(2), which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. MATERIALS AND METHODS: Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. CONCLUSION: NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Dendritic cells and antigen trapping technology--a revolution in vaccine/immunotherapy strategy.

Vaccines based on dendritic cells--the immune system's key responders to foreign invaders--grabbed the spotlight of this decade. Scientists have devised a dozen different ways to make dendritic cell vaccines. They have linked dendritic cells with all kinds of antigens, including peptides derived from gene mutations, tumor/pathogen RNA, viral vectors, and with whole pathogen/tumor lysate. And they are adding cytokines such as granulocyte macrophage colony stimulating factor or interleukin 4 during dendritic cell growth or maturation or at the site of vaccination to try to boost response. We are still learning the best way to generate the dendritic cells, load them with the antigen and send them to the right place in the body, and use of the biological stage of development of dendritic cells that is best suited to stimulate a response. In the present review attempts have been made to present a comprehensive synopsis of the history, development and ramifications of evolving knowledge on dendritic cell biology and the prospects for being developed as a rational immunotherapeutic tool. Further clinical studies are warranted.

Granulocyte-macrophage colony-stimulating factor drives monocytes to CD14low CD83+ DCSIGN- interleukin-10-producing myeloid cells with differential effects on T-cell subsets.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has long been found to have growth-promoting effects on multipotent haematopoietic lineages, specifically granulocytes and macrophages. GM-CSF combined with interleukin-4 (IL-4) drives monocytes to become myeloid dendritic cells (mDCs) in vitro. We report that culturing human monocytes with GM-CSF alone generates myeloid cells (GM-Mono) that have lower expression of CD14 than monocytes and that fail to express DC-SIGN. GM-Monos, however, express CD83 and the transcription factor PU.1, although at a lower level than the conventional mDCs generated in the presence of GM-CSF and IL-4. On stimulation with tumour necrosis factor-alpha, interferon-gamma and anti-CD40 monoclonal antibody, the GM-Monos predominantly produced IL-10 but were less efficient in IL-12 production. In a primary allogeneic mixed lymphocyte reaction, GM-Monos induced hyporesponsiveness and IL-10-biased cytokine production in CD4(+) T cells. In fresh mixed lymphocyte reaction, GM-Monos inhibited conventional mDC-induced allogeneic CD4(+) T-cell proliferation. GM-Mono-induced inhibition of allogeneic CD4(+) T-cell proliferation was partially attributed to IL-10. Interestingly, GM-Monos neither induced hyporesponsiveness in allogeneic CD8(+) T cells nor inhibited conventional mDC-induced allogeneic CD8(+) T-cell proliferation. Taken together, we characterize monocyte-derived CD14(low) CD83(+) cells generated by GM-CSF that can induce tolerance or stimulation of T cells depending on T-cell subsets.