Bacterial Peptidoglycan Fragments Differentially Regulate Innate Immune Signaling.

The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) and N-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors, such as NOD-like receptors. However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide-containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited because of their synthetic accessibility. Inspired by disaccharide-containing compounds produced from the gut microbe Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone-marrow-derived macrophages. The data reveal distinct gene induction patterns for monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one activator-one pathway program, as biologically relevant fragments induce transcriptional programs to different degrees. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.

T cell fate following Salmonella infection is determined by a STING-IRF1 signaling axis in mice.

The innate immune response following infection with entero-invasive bacterial species is triggered upon release of cyclic di-guanylate monophosphate (c-di-GMP) into the host cell cytosol. Bacterial c-di-GMP activates the intracellular Sensor Stimulator of Interferon Genes (STING), encoded by Tmem173 in mice. Here we identify Interferon Regulatory Factor (IRF) 1 as a critical effector of STING-mediated microbial DNA sensing that is responsible for TH17 cell generation in the mucosal immune system. We find that STING activation induces IRF1-dependent transcriptional programs in dendritic cells (DCs) that define T cell fate determination, including induction of Gasdermin D, IL-1 family member cytokines, and enzymes for eicosanoid synthesis. Our results show that IRF1-dependent transcriptional programs in DCs are a prerequisite for antigen-specific TH17 subspecification in response to microbial c-di-GMP and Salmonella typhimurium infection. Our identification of a STING-IRF1 signaling axis for adaptive host defense control will aid further understanding of infectious disease mechanisms.

GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses.

Dendritic cell (DC) activation is a critical step for anti-tumor T cell responses. Certain chemotherapeutics can influence DC function. Here we demonstrate that chemotherapy capable of microtubule destabilization has direct effects on DC function; namely, it induces potent DC maturation and elicits anti-tumor immunity. Guanine nucleotide exchange factor-H1 (GEF-H1) is specifically released upon microtubule destabilization and is required for DC activation. In response to chemotherapy, GEF-H1 drives a distinct cell signaling program in DCs dominated by the c-Jun N-terminal kinase (JNK) pathway and AP-1/ATF transcriptional response for control of innate and adaptive immune responses. Microtubule destabilization, and subsequent GEF-H1 signaling, enhances cross-presentation of tumor antigens to CD8 T cells. In absence of GEF-H1, anti-tumor immunity is hampered. In cancer patients, high expression of the GEF-H1 immune gene signature is associated with prolonged survival. Our study identifies an alternate intracellular axis in DCs induced upon microtubule destabilization in which GEF-H1 promotes protective anti-tumor immunity.

Microbial recognition by GEF-H1 controls IKKε mediated activation of IRF5.

During infection, transcription factor interferon regulatory factor 5 (IRF5) is essential for the control of host defense. Here we show that the microtubule-associated guanine nucleotide exchange factor (GEF)-H1, is required for the phosphorylation of IRF5 by microbial muramyl-dipeptides (MDP), the minimal structural motif of peptidoglycan of both Gram-positive and Gram-negative bacteria. Specifically, GEF-H1 functions in a microtubule based recognition system for microbial peptidoglycans that mediates the activation of IKKε which we identify as a new upstream IKKα/ß and IRF5 kinase. The deletion of GEF-H1 or dominant-negative variants of GEF-H1 prevent activation of IKKε and phosphorylation of IRF5. The GEF-H1-IKKε-IRF5 signaling axis functions independent of NOD-like receptors and is critically required for the recognition of intracellular peptidoglycans and host defenses against Listeria monocytogenes.

Deletion of the putative tumor suppressor gene, G0s2, does not affect progression of Eµ-Myc driven lymphomas in mice.

Several recent reports have suggested that the G0/G1 switch gene 2 (G0S2) is a potential tumor suppressor in leukemia. Here we show that deletion of the G0s2 gene in mouse does not affect the latency of cancer progression in the Eµ-Myc model of lymphoma. Our findings do not rule out the possibility that G0S2 may be playing a role in other forms of leukemia, but clearly show that the commonly used Eµ-Myc transgenic is not the correct model to conduct studies on G0s2.

Inhibition of adipose triglyceride lipase (ATGL) by the putative tumor suppressor G0S2 or a small molecule inhibitor attenuates the growth of cancer cells.

The G0/G1 switch gene 2 (G0S2) is methylated and silenced in a wide range of human cancers. The protein encoded by G0S2 is an endogenous inhibitor of lipid catabolism that directly binds adipose triglyceride lipase (ATGL). ATGL is the rate-limiting step in triglyceride metabolism. Although the G0S2 gene is silenced in cancer, the impact of ATGL in the growth and survival of cancer cells has never been addressed. Here we show that ectopic expression of G0S2 in non-small cell lung carcinomas (NSCL) inhibits triglyceride catabolism and results in lower cell growth. Similarly, knockdown of ATGL increased triglyceride levels, attenuated cell growth and promoted apoptosis. Conversely, knockdown of endogenous G0S2 enhanced the growth and invasiveness of cancer cells. G0S2 is strongly induced in acute promyelocytic leukemia (APL) cells in response to all trans retinoic acid (ATRA) and we show that inhibition of ATGL in these cells by G0S2 is required for efficacy of ATRA treatment. Our data uncover a novel tumor suppressor mechanism by which G0S2 directly inhibits activity of a key intracellular lipase. Our results suggest that elevated ATGL activity may be a general property of many cancer types and potentially represents a novel target for chemotherapy.

Cyclooxygenase-2 inhibitors down-regulate osteopontin and Nr4A2-new therapeutic targets for colorectal cancers.

BACKGROUND & AIMS: Cyclooxygenase-2 inhibitors reduce colon cancer risk by mechanisms that are not fully understood. We performed microarray analysis of adenomas from Apc(Delta14/+) mice to identify genes that respond to these drugs. METHODS: Apc(Delta14/+) mice were given a single daily injection of parecoxib for up to 9 weeks; intestinal tracts of these and control mice were analyzed by microarray analysis, immunohistochemistry, in situ hybridization, and quantitative real-time polymerase chain reaction. Findings were further assessed using Apc(lox/lox)vil-CreER(T2) mice, the CT26 cancer cell line, and human colon tumor samples. RESULTS: Microarray analysis revealed that osteopontin, a marker of colon cancer progression, was down-regulated in polyps from Apc(Delta14/+) mice given parecoxib compared with controls. Apc(Delta14/+) mice given parecoxib had longer survival times and reduced polyp burdens. Osteopontin was quickly down-regulated by parecoxib in intestinal polyps from Apc(Delta14/+) mice, and 2 components of the osteopontin regulatory network-the orphan nuclear receptor NR4A2 and Wnt/beta-catenin signaling-were sequentially repressed. NR4A2 activated the osteopontin promoter in CT26 cells; this effect was blocked by mutation of the NR4A2 binding response element, cotransfection of a dominant-negative form of NR4A2, and small inhibitory RNA against NR4A2. NR4A2 levels were increased throughout tumor progression in Apc(Delta14/+) mice but, unlike osteopontin, did not correlate with tumor stage. NR4A2 levels were reduced in adenomas from patients treated with rofecoxib. CONCLUSIONS: Down-regulation of osteopontin, probably through blockade of NR4A2 and Wnt signaling, is an important component of the antitumor activity of cyclooxygenase-2 inhibitors. These factors might be developed as therapeutic targets for intestinal cancers.

Nitric oxide and indoleamine 2,3-dioxygenase mediate CTLA4Ig-induced survival in heart allografts in rats.

Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin (CTLA4Ig) leads to transplantation tolerance in mice depending on indoleamine 2,3-dioxygenase (IDO). We have shown that CTLA4Ig induces indefinite heart allograft survival in rats and that nitric oxide (NO) was implicated in the in vitro active tolerogenic mechanisms mediated by dendritic cells (DCs). Here we studied the in vivo tolerogenic mechanisms by which CTLA4Ig induces graft survival in rats receiving a cardiac allograft. Treatment of recipients with the IDO inhibitor 1-methyltryptophan (1-MT) did not abrogate the indefinite graft survival observed with CTLA4Ig alone. This was also the case after administration of the inducible nitric oxide synthase inhibitor aminoguanidine when again, indefinite allograft survival was maintained. However, administration of both inhibitors led to acute rejection. We show that IDO and NO are responsible for the impaired capacity of DCs from CTLA4Ig-treated rats to stimulate allogeneic T cells. In conclusion, we show that NO and IDO mediate CTLA4Ig-induced tolerance in rat allograft recipients.

Heme oxygenase-1 inhibits rat and human breast cancer cell proliferation: mutual cross inhibition with indoleamine 2,3-dioxygenase.

Heme oxygenase-1 (HO-1) is the rate limiting enzyme of heme catabolism whereas indoleamine 2,3 dioxygenase (IDO) catabolizes tryptophan through the kynurenine pathway. We analyzed the expression and biological effects of these enzymes in rat and human breast cancer cell lines. We show that rat (NMU and 13762) but not human cells (MCF-7 and T47D) express HO-1. When overexpressed, we found this enzyme to have anti-proliferative and proapoptotic effects by antioxidant mechanisms in these four cell lines. We show that IDO is expressed by rat and human breast cancer cells. IDO inhibition with 1-MT and siRNA leads to diminished proliferation in rat cells. In contrast, HO-1 negative human cell lines increase proliferation upon IDO inhibition. Since we also demonstrate that IDO inhibits the anti-proliferative HO-1, we propose that IDO has opposite effects on proliferation depending on the coexpression or not of HO-1. We also describe that HO-1 inhibits IDO at the post-translational level through heme starvation. In vivo, we show that rat normal breast expresses HO-1 and IDO. In contrast, N-nitrosomethylurea-induced breast adenocarcinomas only express IDO. In conclusion, we show that HO-1/IDO cross-regulation modulates apoptosis and proliferation in rat and human breast cancer cells.