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Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1 β . This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1–RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-d-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

Latency-reversing agents (LRAs) have been tested in HIV-1–infected individuals in the hopes of activating the latent viral reservoir and contributing to efforts to eradicate the virus. Robert F. Siliciano and his colleagues now report that most individual LRAs fail to reactivate latent virus in cells from infected individuals and that in vitro models of latency do not adequately reflect the ability of these agents to induce latent virus ex vivo . HIV-1 persists in a latent reservoir despite antiretroviral therapy (ART) 1 , 2 , 3 , 4 , 5 . This reservoir is the major barrier to HIV-1 eradication 6 , 7 . Current approaches to purging the latent reservoir involve pharmacologic induction of HIV-1 transcription and subsequent killing of infected cells by cytolytic T lymphocytes (CTLs) or viral cytopathic effects 8 , 9 , 10 . Agents that reverse latency without activating T cells have been identified using in vitro models of latency. However, their effects on latently infected cells from infected individuals remain largely unknown. Using a new ex vivo assay, we demonstrate that none of the latency-reversing agents (LRAs) tested induced outgrowth of HIV-1 from the latent reservoir of patients on ART. Using a quantitative reverse transcription PCR assay specific for all HIV-1 mRNAs, we demonstrate that LRAs that do not cause T cell activation do not induce substantial increases in intracellular HIV-1 mRNA in patient cells; only the protein kinase C agonist bryostatin-1 caused significant increases. These findings demonstrate that current in vitro models do not fully recapitulate mechanisms governing HIV-1 latency in vivo . Further, our data indicate that non-activating LRAs are unlikely to drive the elimination of the latent reservoir in vivo when administered individually.

Neutrophil extracellular traps (NET) formation is part of the neutrophil response to infections, but excessive or inappropriate NETosis may trigger the production of autoantibodies and cause organ damage in autoimmune disorders. Spontaneously netting neutrophils are not frequent and induction of NET in vitro by selected stimuli is necessary to investigate their structure. In the present work, the protein composition and post-translational modifications of NET produced under different stimuli have been studied by means of proteomic analysis. Neutrophils from healthy donors were stimulated by PMA, A23187, Escherichia coli LPS or untreated; after three hours, cells were washed, treated with DNase and supernatants collected for mass spectrometry. Data were analyzed by unsupervised hierarchical clustering analyses. We identified proteins contained in NETs of any source or exclusive of one stimulus: LPS-induced and spontaneous NET diverge in protein composition, while PMA- and A23187-induced NET appear more similar. Among the post-translational modifications we examined, methionine sulfoxidation is frequent especially in PMA- and LPS-induced NETs. Myeloperoxidase is the protein more extensively modified. Thus, proteomic analysis indicates that NETs induced by different stimuli are heterogeneous in terms of both protein composition and post-translational modifications, suggesting that NET induced in different conditions may have different biological effects.

Angiogenic sprouting, the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from within the cellular microenvironment, such as biochemical gradients and perfusion. However, many of these cues are missing in current in vitro models of angiogenic sprouting. We here describe an in vitro platform that integrates both perfusion and the generation of stable biomolecular gradients and demonstrate its potential to study more physiologically relevant angiogenic sprouting and microvascular stabilization. The platform consists of an array of 40 individually addressable microfluidic units that enable the culture of perfused microvessels against a three-dimensional collagen-1 matrix. Upon the introduction of a gradient of pro-angiogenic factors, the endothelial cells differentiated into tip cells that invaded the matrix. Continuous exposure resulted in continuous migration and the formation of lumen by stalk cells. A combination of vascular endothelial growth factor-165 (VEGF-165), phorbol 12-myristate 13-acetate (PMA), and sphingosine-1-phosphate (S1P) was the most optimal cocktail to trigger robust, directional angiogenesis with S1P being crucial for guidance and repetitive sprout formation. Prolonged exposure forces the angiogenic sprouts to anastomose through the collagen to the other channel. This resulted in remodeling of the angiogenic sprouts within the collagen: angiogenic sprouts that anastomosed with the other perfusion channel remained stable, while those who did not retracted and degraded. Furthermore, perfusion with 150 kDa FITC-Dextran revealed that while the angiogenic sprouts were initially leaky, once they fully crossed the collagen lane they became leak tight. This demonstrates that once anastomosis occurred, the sprouts matured and suggests that perfusion can act as an important survival and stabilization factor for the angiogenic microvessels. The robustness of this platform in combination with the possibility to include a more physiological relevant three-dimensional microenvironment makes our platform uniquely suited to study angiogenesis in vitro.

THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages.

Breast cancer (BC) is a common female malignancy, worldwide. BC death is predominantly caused by lung metastasis. According to previous studies, Dihydrotanshinone I (DHT), a bioactive compound in Salvia miltiorrhiza Bunge (S. miltiorrhiza), has inhibitory effects on numerous cancers. Here, we investigated the anti-metastatic effect of DHT on BC, where DHT more strongly inhibited the growth of BC cells (MDA-MB-231, 4T1, MCF-7, and SKBR-3) than breast epithelial cells (MCF-10a). Additionally, DHT repressed the wound healing, invasion, and migration activities of 4T1 cells. In the 4T1 spontaneous metastasis model, DHT (20 mg/kg) blocked metastasis progression and distribution in the lung tissue by 74.9%. DHT reversed the formation of neutrophil extracellular traps (NETs) induced by phorbol 12-myristate 13-acetate, as well as ameliorated NETs-induced metastasis. Furthermore, it inhibited Ly6G+Mpo+ neutrophils infiltration and H3Cit expression in the lung tissues. RNA sequencing, western blot, and bioinformatical analysis indicated that TIMP1 could modulate DHT acting on lung metastasis inhibition. The study demonstrated a novel suppression mechanism of DHT on NETs formation to inhibit BC metastasis.

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

Synovitis, a common clinical symptom for osteoarthritis (OA) patients, is highly related to OA pathological progression and pain manifestation. The activated synovial macrophages have been demonstrated to play an important role in synovitis, but the mechanisms about macrophage activation are still not clear. In this study, we found that the exosome-like vesicles from osteoarthritic chondrocytes could be a new biological factor to stimulate inflammasome activation and increase mature IL-1β production in macrophages. The degraded cartilage explants produced more exosome-like vesicles than the nondegraded ones, while the exosome-like vesicles from chondrocytes could enter into joint synovium tissue and macrophages. Moreover, the exosome-like vesicles from osteoarthritic chondrocytes enhanced the production of mature IL-1β in macrophages. These vesicles could inhibit ATG4B expression via miR-449a-5p, leading to inhibition of autophagy in LPS-primed macrophages. The decreased autophagy promoted the production of mitoROS, which further enhanced the inflammasome activation and subsequent IL-1β processing. Ultimately, the increase of mature IL-1β may aggravate synovial inflammation and promote the progression of OA disease. Our study provides a new perspective to understand the activation of synovial macrophages and synovitis in OA patients, which may be beneficial for therapeutic intervention in synovitis-related OA patients.