Neutrophil extracellular trap formation linked to idiopathic pulmonary fibrosis severity and survival.

RATIONALE: Neutrophil counts in bronchoalveolar lavage fluid in idiopathic pulmonary fibrosis are associated with worse outcomes; however, the underlying mechanisms are unknown. Neutrophil extracellular trap formation is associated with worse outcomes in several chronic lung diseases however, there is an unknown role in idiopathic pulmonary fibrosis. OBJECTIVES: To determine the relationship between neutrophil extracellular trap formation in the lungs of idiopathic pulmonary fibrosis patients and clinical outcomes. METHODS: In a discovery cohort of 156 patients with idiopathic pulmonary fibrosis, we measured neutrophil extracellular trap markers in bronchoalveolar lavage fluid, including extracellular DNA, DNA-myeloperoxidase complexes, calprotectin, and neutrophil elastase. We assessed the correlation of these markers with baseline pulmonary function and survival. In a subset of 50 patients, label-free quantitative proteomics was performed to identify the source of extracellular DNA. A validation cohort of 52 patients was similarly assessed. MEASUREMENTS AND MAIN RESULTS: Neutrophil extracellular trap markers in the discovery cohort were significantly correlated with worse pulmonary function (p<0.03). Higher levels of these markers predicted worse survival after adjusting for gender, age, and baseline physiologic severity (hazard ratio range: 1.79 - 2.19). Proteomics revealed a significant correlation between extracellular DNA levels and proteins related to neutrophil extracellular trap formation. The validation cohort showed that extracellular DNA levels were associated with reduced pulmonary function (p=0.04) and trend towards worse survival (hazard ratio=2.16). CONCLUSIONS: Neutrophil extracellular trap formation markers were associated with disease severity and worse survival in idiopathic pulmonary fibrosis. These findings suggest neutrophil extracellular trap formation contributes to lung injury and decreased survival and may represent a potential therapeutic target.

Reprogramming of breast tumor-associated macrophages with modulation of arginine metabolism.

HER2+ breast tumors have abundant immune-suppressive cells, including M2-type tumor associated macrophages (TAMs). While TAMs consist of the immune-stimulatory M1-type and immune-suppressive M2-type, M1/M2-TAM ratio is reduced in immune-suppressive tumors, contributing to their immunotherapy refractoriness. M1 vs. M2-TAM formation depends on differential arginine metabolism, where M1-TAMs convert arginine to nitric oxide (NO) and M2-TAMs convert arginine to polyamines (PAs). We hypothesize that such distinct arginine metabolism in M1- vs M2-TAMs is attributed to different availability of BH4 (NO synthase cofactor) and that its replenishment would reprogram M2-TAMs to M1-TAMs. Recently, we reported that sepiapterin (SEP), the endogenous BH4 precursor, elevates the expression of M1-TAM markers within HER2+ tumors. Here, we show that SEP restores BH4 levels in M2-TAMs, which then redirects arginine metabolism to NO synthesis and converts M2-TAMs to M1-TAMs. The reprogrammed TAMs exhibit full-fledged capabilities of antigen presentation and induction of effector T cells to trigger immunogenic cell death of HER2+ cancer cells. This study substantiates the utility of SEP in metabolic shift of HER2+ breast tumor microenvironment as a novel immunotherapeutic strategy.

Changes in Expression of Tumor Suppressor Gene RKIP Impact How Cancers Interact with Their Complex Environment.

Tumor microenvironment (TME) is the immediate environment where cancer cells reside in a tumor. It is composed of multiple cell types and extracellular matrix. Microenvironments can be restrictive or conducive to the progression of cancer cells. Initially, microenvironments are suppressive in nature. Stepwise accumulation of mutations in oncogenes and tumor suppressor genes enables cancer cells to acquire the ability to reshape the microenvironment to advance their growth and metastasis. Among the many genetic events, the loss-of-function mutations in tumor suppressor genes play a pivotal role. In this review, we will discuss the changes in TME and the ramifications on metastasis upon altered expression of tumor metastasis suppressor gene RKIP in breast cancer cells.

Novel 3D Flipwell system that models gut mucosal microenvironment for studying interactions between gut microbiota, epithelia and immunity.

Gut mucosa consists of stratified layers of microbes, semi-permeable mucus, epithelium and stroma abundant in immune cells. Although tightly regulated, interactions between gut commensals and immune cells play indispensable roles in homeostasis and cancer pathogenesis in the body. Thus, there is a critical need to develop a robust model for the gut mucosal microenvironment. Here, we report our novel co-culture utilizing 3D Flipwell system for establishing the stratified layers of discrete mucosal components. This method allows for analyzing synchronous effects of test stimuli on gut bacteria, mucus, epithelium and immune cells, as well as their crosstalks. In the present report, we tested the immuno-stimulatory effects of sepiapterin (SEP, the precursor of the cofactor of nitric oxide synthase (NOS)-BH4) on the gut mucosal community. We previously reported that SEP effectively reprogrammed tumor-associated macrophages and inhibited breast tumor cell growth. In our co-cultures, SEP largely promoted mucus integrity, bacterial binding, and M1-like polarization of macrophages. Conversely, these phenomena were absent in control-treated cultures. Our results demonstrate that this novel co-culture may serve as a robust in vitro system to recapitulate the effects of pharmacological agents on the gut mucosal microenvironment, and could potentially be expanded to test the effects outside the gut.

S-Nitrosylation in Tumor Microenvironment.

S-nitrosylation is a selective and reversible post-translational modification of protein thiols by nitric oxide (NO), which is a bioactive signaling molecule, to exert a variety of effects. These effects include the modulation of protein conformation, activity, stability, and protein-protein interactions. S-nitrosylation plays a central role in propagating NO signals within a cell, tissue, and tissue microenvironment, as the nitrosyl moiety can rapidly be transferred from one protein to another upon contact. This modification has also been reported to confer either tumor-suppressing or tumor-promoting effects and is portrayed as a process involved in every stage of cancer progression. In particular, S-nitrosylation has recently been found as an essential regulator of the tumor microenvironment (TME), the environment around a tumor governing the disease pathogenesis. This review aims to outline the effects of S-nitrosylation on different resident cells in the TME and the diverse outcomes in a context-dependent manner. Furthermore, we will discuss the therapeutic potentials of modulating S-nitrosylation levels in tumors.

Correction of arginine metabolism with sepiapterin-the precursor of nitric oxide synthase cofactor BH4-induces immunostimulatory-shift of breast cancer.

Immunotherapy is a first-line treatment for many tumor types. However, most breast tumors are immuno-suppressive and only modestly respond to immunotherapy. We hypothesized that correcting arginine metabolism might improve the immunogenicity of breast tumors. We tested whether supplementing sepiapterin, the precursor of tetrahydrobiopterin (BH4)-the nitric oxide synthase (NOS) cofactor-redirects arginine metabolism from the pathway synthesizing polyamines to that of synthesizing nitric oxide (NO) and make breast tumors more immunogenic. We showed that sepiapterin elevated NO but lowered polyamine levels in tumor cells, as well as in tumor-associated macrophages (TAMs). This not only suppressed tumor cell proliferation, but also induced the conversion of TAMs from the immuno-suppressive M2-type to immuno-stimulatory M1-type. Furthermore, sepiapterin abrogated the expression of a checkpoint ligand, PD-L1, in tumors in a STAT3-dependent manner. This is the first study which reveals that supplementing sepiapterin normalizes arginine metabolism, improves the immunogenicity and inhibits the growth of breast tumor cells.

S-Nitrosylation: An Emerging Paradigm of Redox Signaling.

Nitric oxide (NO) is a highly reactive molecule, generated through metabolism of L-arginine by NO synthase (NOS). Abnormal NO levels in mammalian cells are associated with multiple human diseases, including cancer. Recent studies have uncovered that the NO signaling is compartmentalized, owing to the localization of NOS and the nature of biochemical reactions of NO, including S-nitrosylation. S-nitrosylation is a selective covalent post-translational modification adding a nitrosyl group to the reactive thiol group of a cysteine to form S-nitrosothiol (SNO), which is a key mechanism in transferring NO-mediated signals. While S-nitrosylation occurs only at select cysteine thiols, such a spatial constraint is partially resolved by transnitrosylation, where the nitrosyl moiety is transferred between two interacting proteins to successively transfer the NO signal to a distant location. As NOS is present in various subcellular locales, a stress could trigger concerted S-nitrosylation and transnitrosylation of a large number of proteins involved in divergent signaling cascades. S-nitrosylation is an emerging paradigm of redox signaling by which cells confer protection against oxidative stress.

Reconstituting Breast Tissue with Organotypic Three-dimensional Co-culture of Epithelial and Stromal Cells in Discontinuous Extracellular Matrices.

Co-culture systems utilizing reconstituted or synthetic extracellular matrix (ECM) and micropatterning techniques have enabled the reconstruction of surface epithelial tissues. This technique has been utilized in the regeneration, disease modeling and drug screening of the surface epithelia, such as the skin and esophagus. On the other hand, the reconstruction of glandular epithelia would require more intricate ECM organizations. Here we describe a protocol for a novel three-dimensional organotypic co-culture system for the reconstruction of mammary glands that utilizes the discontinuous ECM. In this technique, primary mammary fibroblasts first establish a layer of the connective tissue rich in collagen I. Then, mammary epithelial cells form acinar structures, the functional glandular units, within the laminin-rich basement membrane embedded in the connective tissue. This method allows for the regeneration of the in vivo-like architecture of mammary glands and could be utilized for monitoring the real-time response of mammary glands to drug treatment.