Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Direct visualization of avian influenza H5N1 hemagglutinin precursor and its conformational change by high-speed atomic force microscopy.

BACKGROUND: Hemagglutinin (HA) of influenza A is one of the key virulence factors that mediates the release of viral components in host cells. HA is initially synthesized as a trimeric precursor (HA0) and then it is cleaved by proteases to become a functional HA. Low pH induces irreversible conformational changes in both HA0 and HA but only HA is fusion compatible. Here, we used high-speed atomic force microscopy (HS-AFM) to record conformational changes in HA0 trimers (H5N1) from neutral to acidic conditions at a millisecond scale. METHODS: Purified HA0 protein was diluted with either neutral Tris-HCl (pH 7.4) or acetic acid-titrated Tris-HCl (pH 5.0) and then loaded onto bare mica. Neutral or acidic Tris-HCl was used as the scanning buffer. HS-AFM movies were recorded and processed using Image J software. RESULTS: The conformation of HA0neutral visualized using HS-AFM was comparable to the HA trimer structures depicted in the PDB data and the AFM simulator. HA0 underwent rapid conformational changes under low pH condition. The circularity and area of HA0acid were significantly higher than in HA0neutral. In contrast, the height of HA0acid was significantly lower than in HA0neutral. CONCLUSIONS: We have captured real-time images of the native HA0 trimer structure under physiological conditions using HS-AFM. By analyzing the images, we confirm that HA0 trimer is sensitive to acidic conditions. GENERAL SIGNIFICANCE: The dynamic nature of the HA structure, particularly in the host endosome, is essential for H5N1 infectivity. Understanding this acidic behavior is imperative for designing therapeutic strategies against H5N1. This article reports a sophisticated new tool for studying the spatiotemporal dynamics of the HA precursor protein.

Nucleoporin Nup58 localizes to centrosomes and mid-bodies during mitosis.

BACKGROUND: Nuclear pore complexes (NPCs) act as nano-turnstiles within nuclear membranes between the cytoplasm and nucleus of mammalian cells. NPC proteins, called nucleoporins (Nups), mediate trafficking of proteins and RNA into and out of the nucleus, and are involved in a variety of mitotic processes. We previously reported that Nup62 localizes to the centrosome and mitotic spindle during mitosis, and plays a role in centrosome homeostasis. However, whether Nup58, a Nup62 subcomplex protein, also localizes to spindle poles is unknown. RESULT: Herein, we show that Nup58 localizes to the nuclear rim during interphase, and to mitotic spindles, centrosomes, and midbodies during mitosis. Our confocal microscopy, live-cell imaging, and stimulated emission depletion nanoscopy results also demonstrated that Nup58 localized to the centrosomes during metaphase and relocalized to midbodies during abscission. Depletion of Nup58 resulted in centrosomal abnormalities and delayed abscission. CONCLUSION: Nup58 localized at the centrosomes and mitotic spindle during metaphase and relocalized at midbodies during abscission. This study highlights the important role of Nup58 in mitosis.

Targeting Nucleoporin POM121-Importin ß Axis in Prostate Cancer.

Nuclear pores are nanomachines acting as gatekeepers of molecular transport across nuclear membranes. In a recent issue of Cell, Rodriguez-Bravo et al. (2018) demonstrates that nuclear pores promote aggressive prostate cancer by snowballing POM121-importin ß-driven nuclear import. Treatment with Importazole, an importin ß inhibitor, impeded tumor aggressiveness, presenting a potential therapeutic strategy.

Implication of Highly Cytotoxic Natural Killer Cells for Esophageal Squamous Cell Carcinoma Treatment.

Esophageal squamous cell carcinoma (ESCC) is an aggressive upper gastrointestinal cancer and effective treatments are limited. Previous studies reported that natural killer (NK) cells expanded by coculturing with K562-mb15-41BBL feeder cells, a genetically modified K562 leukemia cell line that expresses membrane-bound interleukin (IL)-15 and 41BBL ligand, were highly proliferative and highly cytotoxic. Here, we investigated the potential of expanded NK cells for ESCC treatment. We analyzed both genetic and surface expression levels of NKG2D ligands (NKG2DLs) in ESCC using publicly available microarray data sets and ESCC cell lines. The cytotoxicity of resting and of IL-2-activated NK cells against ESCC cell lines was compared with that of expanded NK cells. We then also investigated the effect of epithelial mesenchymal transition (EMT) inducers, GSK3ß inhibitor and epidermal growth factor, on NKG2DLs expressions. As a result, MICA and MICB were significantly overexpressed in ESCC compared with adjacent normal tissues and surface NKG2DLs were expressed in ESCC cell lines. Expanded NK cells were much potent than IL-2-activated and resting NK cells against ESCC cell lines. Blocking of NKG2D with anti-NKG2D monoclonal antibody dampened expanded NK cell cytotoxicity, suggesting that the NKG2DLs-NKG2D interaction is crucial for NK cells to eliminate ESCC cells. EMT inducers concurrently induced EMT and NKG2DLs expression in ESCC cell lines rendering transitioned cells more sensitive to expanded NK cells. In conclusion, expanded NK cells were highly cytotoxic against NKG2DLs-expressing ESCC cells, particularly the EMT phenotype. These results provide a strong rationale for clinical use of these NK cells in ESCC patients.

Accumulation of CD11c+CD163+ Adipose Tissue Macrophages through Upregulation of Intracellular 11ß-HSD1 in Human Obesity.

Adipose tissue (AT) macrophages (ATMs) are key players for regulation of AT homeostasis and obesity-related metabolic disorders. However, the phenotypes of human ATMs and regulatory mechanisms of their polarization have not been clearly described. In this study, we investigated human ATMs in both abdominal visceral AT and s.c. AT and proposed an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1)-glucocorticoid receptor regulatory axis that might dictate M1/M2 polarization in ATMs. The accumulation of CD11c+CD163+ ATMs in both visceral AT and s.c. AT of obese individuals was confirmed at the cellular level and was found to be clearly correlated with body mass index and production of reactive oxygen species. Using our in vitro system where human peripheral blood monocytes (hPBMs) were cocultured with Simpson-Golabi-Behmel syndrome adipocytes, M1/M2 polarization was found to be dependent on 11ß-HSD1, an intracellular glucocorticoid reactivating enzyme. Exposure of hPBMs to cortisol-induced expression of CD163 and RU-486, a glucocorticoid receptor antagonist, significantly abrogated CD163 expression through coculture of mature adipocytes with hPBMs. Moreover, 11ß-HSD1 was expressed in crown ATMs in obese AT. Importantly, conditioned medium from coculture of adipocytes with hPBMs enhanced proliferation of human breast cancer MCF7 and MDA-MB-231 cells. In summary, the phenotypic switch of ATMs from M2 to mixed M1/M2 phenotype occurred through differentiation of adipocytes in obese individuals, and upregulation of intracellular 11ß-HSD1 might play a role in the process.

Total muscle-sparing uniportal video-assisted thoracoscopic surgery lobectomy.

BACKGROUND: Conventional video-assisted thoracoscopic lobectomy uses multiple incisions, including an access incision and several port incisions. This series aims to evaluate the technical feasibility and early results of uniportal video-assisted thoracoscopic surgery (UVATS) lobectomy using a small, total muscle-sparing incision. METHODS: We performed the first UVATS lobectomy in June 2009, and 38 major resections were attempted using this approach until September 2011. A single, small, muscle-sparing incision was made without rib spreading. True anatomic hilar dissection, individual vascular and bronchial ligation, and mediastinal lymph node dissection were performed under thoracoscopic visualization on a monitor. RESULTS: Thirty-two patients (84%) had malignant diseases, and 6 patients (16%) had benign diseases. Of the primary lung cancers, 85% were in stage I. Of the 38 attempted major resections, 32 UVATS lobectomies were successfully completed and 6 were converted to open thoracotomy. The early outcomes of successful UVATS lobectomy were analyzed (32 patients); 97% had no postoperative complications. There were no deaths. Mean pain score was 0.4 on postoperative day 1 and decreased to 0 by 1 week. Ninety-seven percent of patients received only oral analgesia postoperatively. Eight percent of patients experienced mild intercostal neuralgia not requiring treatment. No patients complained of shoulder dysfunction. The median duration of returning to full normal activities was 7 postoperative days. CONCLUSIONS: Total muscle-sparing UVATS lobectomy is technically feasible with low morbidity and mortality rates. Patients had minimal postoperative pain and narcotic use; and good functional outcomes with no shoulder dysfunction and early return to full normal activities.

Massive pulmonary tuberculosis cavity misdiagnosed as pneumothorax.

Massive pulmonary cavity is a rare and serious complication of chronic reactivation tuberculosis. A 38-year-old gentleman had a history of tuberculosis treatment noncompliance 2 years ago. His presenting symptoms were cough, fever, and left-sided pleuritic chest discomfort for 2 months. Chest radiographs showed extensive lung destruction associated with large thick-walled cavities and severe fibrosis of the residual lung. In the emergency department, this was initially misdiagnosed as a large pneumothorax and a chest tube was inserted. Subsequently, this was misdiagnosed again as bronchopleural fistula when brisk air leak was seen. The chest tube did not lead to any radiological or clinical improvement and was removed without incident. This case demonstrates that massive pulmonary cavity can easily be misdiagnosed and tube thoracostomy is unnecessary. Although this condition was previously reported to be associated with a high mortality rate, our patient survived as a result of accurate diagnosis and prompt antituberculosis therapy.