TM4SF19-mediated control of lysosomal activity in macrophages contributes to obesity-induced inflammation and metabolic dysfunction.

Adipose tissue (AT) adapts to overnutrition in a complex process, wherein specialized immune cells remove and replace dysfunctional and stressed adipocytes with new fat cells. Among immune cells recruited to AT, lipid-associated macrophages (LAMs) have emerged as key players in obesity and in diseases involving lipid stress and inflammation. Here, we show that LAMs selectively express transmembrane 4 L six family member 19 (TM4SF19), a lysosomal protein that represses acidification through its interaction with Vacuolar-ATPase. Inactivation of TM4SF19 elevates lysosomal acidification and accelerates the clearance of dying/dead adipocytes in vitro and in vivo. TM4SF19 deletion reduces the LAM accumulation and increases the proportion of restorative macrophages in AT of male mice fed a high-fat diet. Importantly, male mice lacking TM4SF19 adapt to high-fat feeding through adipocyte hyperplasia, rather than hypertrophy. This adaptation significantly improves local and systemic insulin sensitivity, and energy expenditure, offering a potential avenue to combat obesity-related metabolic dysfunction.

CarRS Two-Component System Essential for Polymyxin B Resistance of Vibrio vulnificus Responds to Multiple Host Environmental Signals.

Enteropathogenic bacteria express two-component systems (TCSs) to sense and respond to host environments, developing resistance to host innate immune systems like cationic antimicrobial peptides (CAMPs). Although an opportunistic human pathogen Vibrio vulnificus shows intrinsic resistance to the CAMP-like polymyxin B (PMB), its TCSs responsible for resistance have barely been investigated. Here, a mutant exhibiting a reduced growth rate in the presence of PMB was screened from a random transposon mutant library of V. vulnificus, and response regulator CarR of the CarRS TCS was identified as essential for its PMB resistance. Transcriptome analysis revealed that CarR strongly activates the expression of the eptA, tolCV2, and carRS operons. In particular, the eptA operon plays a major role in developing the CarR-mediated PMB resistance. Phosphorylation of CarR by the sensor kinase CarS is required for the regulation of its downstream genes, leading to the PMB resistance. Nevertheless, CarR directly binds to specific sequences in the upstream regions of the eptA and carRS operons, regardless of its phosphorylation. Notably, the CarRS TCS alters its own activation state by responding to several environmental stresses, including PMB, divalent cations, bile salts, and pH change. Furthermore, CarR modulates the resistance of V. vulnificus to bile salts and acidic pH among the stresses, as well as PMB. Altogether, this study suggests that the CarRS TCS, in responding to multiple host environmental signals, could provide V. vulnificus with the benefit of surviving within the host by enhancing its optimal fitness during infection. IMPORTANCE Enteropathogenic bacteria have evolved multiple TCSs to recognize and appropriately respond to host environments. CAMP is one of the inherent host barriers that the pathogens encounter during the course of infection. In this study, the CarRS TCS of V. vulnificus was found to develop resistance to PMB, a CAMP-like antimicrobial peptide, by directly activating the expression of the eptA operon. Although CarR binds to the upstream regions of the eptA and carRS operons regardless of phosphorylation, phosphorylation of CarR is required for the regulation of the operons, resulting in the PMB resistance. Furthermore, the CarRS TCS determines the resistance of V. vulnificus to bile salts and acidic pH by differentially regulating its own activation state in response to these environmental stresses. Altogether, the CarRS TCS responds to multiple host-related signals, and thus could enhance the survival of V. vulnificus within the host, leading to successful infection.

Nonsteroidal Anti-Inflammatory Drug Conjugated with Gadolinium (III) Complex as an Anti-Inflammatory MRI Agent.

Studies have been actively conducted to ensure that gadolinium-based contrast agents for magnetic resonance imaging (MRI) are accompanied by various biological functions. A new example is the anti-inflammatory theragnostic MRI agent to target inflammatory mediators for imaging diagnosis and to treat inflammatory diseases simultaneously. We designed, synthesized, and characterized a Gd complex of 1,4,7-tris(carboxymethylaza) cyclododecane-10-azaacetylamide (DO3A) conjugated with a nonsteroidal anti-inflammatory drug (NSAID) that exerts the innate therapeutic effect of NSAIDs and is also applicable in MRI diagnostics. Gd-DO3A-fen (0.1 mmol/kg) was intravenously injected into the turpentine oil-induced mouse model, with Gd-DO3A-BT as a control group. In the in vivo MRI experiment, the contrast-to-noise ratio (CNR) was higher and persisted longer than that with Gd-DO3A-BT; specifically, the CNR difference was almost five times at 2 h after injection. Gd-DO3A-fen had a binding affinity (Ka) of 6.68 × 106 M-1 for the COX-2 enzyme, which was 2.1-fold higher than that of fenbufen, the original NSAID. In vivo evaluation of anti-inflammatory activity was performed in two animal models. In the turpentine oil-induced model, the mRNA expression levels of inflammatory parameters such as COX-2, TNF-α, IL-1ß, and IL-6 were reduced, and in the carrageenan-induced edema model, swelling was suppressed by 72% and there was a 2.88-fold inhibition compared with the saline group. Correlation analysis between in vitro, in silico, and in vivo studies revealed that Gd-DO3A-fen acts as an anti-inflammatory theragnostic agent by directly binding to COX-2.

Liver X receptor controls follicular helper T cell differentiation via repression of TCF-1.

Liver X receptor (LXR) is a critical regulator of cholesterol homeostasis that inhibits T cell receptor (TCR)-induced proliferation by altering intracellular sterol metabolism. However, the mechanisms by which LXR regulates helper T cell subset differentiation remain unclear. Here, we demonstrate that LXR is a crucial negative regulator of follicular helper T (Tfh) cells in vivo. Both mixed bone marrow chimera and antigen-specific T cell adoptive cotransfer studies show a specific increase in Tfh cells among LXRß-deficient CD4+ T cell population in response to immunization and lymphocytic choriomeningitis mammarenavirus (LCMV) infection. Mechanistically, LXRß-deficient Tfh cells express augmented levels of T cell factor 1 (TCF-1) but comparable levels of Bcl6, CXCR5, and PD-1 in comparison with those of LXRß-sufficient Tfh cells. Loss of LXRß confers inactivation of GSK3ß induced by either AKT/Extracellular signal-regulated kinase (ERK) activation or Wnt/ß-catenin pathway, leading to elevated TCF-1 expression in CD4+ T cells. Conversely, ligation of LXR represses TCF-1 expression and Tfh cell differentiation in both murine and human CD4+ T cells. LXR agonist significantly diminishes Tfh cells and the levels of antigen-specific IgG upon immunization. These findings unveil a cell-intrinsic regulatory function of LXR in Tfh cell differentiation via the GSK3ß-TCF1 pathway, which may serve as a promising target for pharmacological intervention in Tfh-mediated diseases.

Flavonoid-Conjugated Gadolinium Complexes as Anti-Inflammatory Theranostic Agents.

In this study, we designed, synthesized, and evaluated gadolinium compounds conjugated with flavonoids as potential theranostic agents for the treatment of inflammation. These novel theranostic agents combine a molecular imaging agent and one of three flavonoids (galangin, chrysin, and 7-hydroxyflavone) as anti-inflammatory drugs as a single integrated platform. Using these agents, MR imaging showed contrast enhancement (>10 in CNR) at inflamed sites in an animal inflammation model, and subsequent MR imaging used to monitor the therapeutic efficacy of these integrated agents revealed changes in inflamed regions. The anti-inflammatory effects of these agents were demonstrated both in vitro and in vivo. Furthermore, the antioxidant efficacy of the agents was evaluated by measuring their reactive oxygen species scavenging properties. For example, Gd-galangin at 30 µM showed a three-fold higher ROS scavenging of DPPH. Taken together, our findings provide convincing evidence to indicate that flavonoid-conjugated gadolinium compounds can be used as potentially efficient theranostic agents for the treatment of inflammation.

Mulberry Component Kuwanon C Exerts Potent Therapeutic Efficacy In Vitro against COVID-19 by Blocking the SARS-CoV-2 Spike S1 RBD:ACE2 Receptor Interaction.

There has been an immense effort by global pharmaceutical companies to develop anti-COVID-19 drugs, including small molecule-based RNA replication inhibitors via drug repositioning and antibody-based spike protein blockers related to cell entry by SARS-CoV-2. However, several limitations to their clinical use have emerged in addition to a lack of progress in the development of small molecule-based cell entry inhibitors from natural products. In this study, we tested the effectiveness of kuwanon C (KC), which has mainly been researched using in silico docking simulation and can serve as an effective building block for developing anti-COVID-19 drugs, in blocking the spike S1 RBD:ACE2 receptor interaction. KC is a natural product derived from Morus alba L., commonly known as mulberry, which has known antiviral efficacy. Molecular interaction studies using competitive ELISA and the BLItz system revealed that KC targets both the spike S1 RBD and the ACE2 receptor, successfully disrupting their interaction, as supported by the in silico docking simulation. Furthermore, we established a mechanism of action by observing how KC prevents the infection of SARS-CoV-2 spike pseudotyped virus in ACE2/TPRSS2-overexpressing HEK293T cells. Finally, we demonstrated that KC inhibits clinical isolates of SARS-CoV-2 in Vero cells. Future combinations of small molecule-based cell entry inhibitors, such as KC, with the currently prescribed RNA replication inhibitors are anticipated to significantly enhance the efficacy of COVID-19 therapies.

Complex regulatory networks of virulence factors in Vibrio vulnificus.

The fulminating zoonotic pathogen Vibrio vulnificus is the causative agent of fatal septicemia in humans and fish, raising tremendous economic burdens in healthcare and the aquaculture industry. V. vulnificus exploits various virulence factors, including biofilm-related factors and exotoxins, for its persistence in nature and pathogenesis during infection. Substantial studies have found that the expression of virulence factors is coordinately regulated by numerous transcription factors that recognize the changing environments. Here, we summarize and discuss the recent discoveries of the physiological roles of virulence factors in V. vulnificus and their regulation by transcription factors in response to various environmental signals. This expanded understanding of molecular pathogenesis would provide novel clues to develop an effective antivirulence therapy against V. vulnificus infection.

MBP-11901 Inhibits Tumor Growth of Hepatocellular Carcinoma through Multitargeted Inhibition of Receptor Tyrosine Kinases.

Hepatocellular carcinomas (HCCs) are aggressive tumors with a poor prognosis. Approved first-line treatments include sorafenib, lenvatinib, and a combination of atezolizumab and bevacizumab; however, they do not cure HCC. We investigated MBP-11901 as a drug candidate for HCC. Cell proliferation and cytotoxicity were evaluated using normal and cancer human liver cell lines, while Western blotting and flow cytometry evaluated apoptosis. The anticancer effect of MBP-11901 was verified in vitro through migration, invasion, colony formation, and JC-1 MMP assays. In mouse models, the tumor volume, tumor weight, and bodyweight were measured, and cancer cell proliferation and apoptosis were analyzed. The toxicity of MBP-11901 was investigated through GOT/GPT and histological analyses in the liver and kidney. The signaling mechanism of MBP-11901 was investigated through kinase assays, phosphorylation analysis, and in silico docking simulations. Results. MBP-11901 was effective against various human HCC cell lines, leading to the disappearance of most tumors when administered orally in animal models. This effect was dose-dependent, with no differences in efficacy according to administration intervals. MBP-11901 induced anticancer effects by targeting the signaling mechanisms of FLT3, VEGFR2, c-KIT, and PDGFRß. MBP-11901 is suggested as a novel therapeutic agent for the treatment of advanced or unresectable liver cancer.

Nanovesicle-Mediated Targeted Delivery of Immune Checkpoint Blockades to Potentiate Therapeutic Efficacy and Prevent Side Effects.

Despite the clinically proven efficacies of immune checkpoint blockades, including anti-cytotoxic T lymphocyte-associated protein 4 antibody (αCTLA-4), the low response rate and immune-related adverse events (irAEs) in cancer patients represent major drawbacks of the therapy. These drawbacks of αCTLA-4 therapy are mainly due to the suboptimal activation of tumor-specific cytotoxic T lymphocytes (CTLs) and the systemic nonspecific activation of T cells. To overcome such drawbacks, αCTLA-4 is delivered by dendritic cell-derived nanovesicles presenting tumor antigens (DCNV-TAs) that exclusively interact with tumor-specific T cells, leading to selective activation of tumor-specific CTLs. Compared to conventional αCTLA-4 therapy, treatment with αCTLA-4-conjugated DCNV-TAs significantly inhibits tumor growth and reduces irAEs in syngeneic tumor-bearing mice. This study demonstrates that the spatiotemporal presentation of both αCTLA-4 and tumor antigens enables selective activation of tumor-specific T cells and potentiates the antitumor efficacy of αCTLA-4 without inducing systemic irAEs.

Single sequence phase shifting spectrally resolved interferometry for an in-line thin film thickness measurement using spectral reflectance and phase.

A novel, to the best of our knowledge, thickness measurement scheme based on a phase shifting spectrally resolved interferometry is proposed for a thin film measurement from spectral reflectance and phase in a single sequence. This purpose is realized by introducing a spectral reflectance phase surface (SRPS). The spectral phase, originally existing in the form of an imaginary term, is revealed as a real term with the spectral reflectance due to the proposition of SRPS. Therefore, the reflectance and phase are contained in a single parameter, SRPS, and are measured in a single sequence. The phase shift error compensation is presented to form the accurate SRPS and ensure the performance of the measurement. The thickness of the sample is determined as the measured SRPS, and the theoretical SRPSs are compared by the cross correlation method. The performance of the proposed system is validated by measuring several samples of the SiO2 on a silicon substrate and comparing the results with the commercial ellipsometer.

Single-sequence stable spectroscopic reflectometry using simultaneous measurement of incident light and reflected light: publisher's note.

This publisher's note contains corrections to Appl. Opt.60, 8915 (2021).APOPAI0003-693510.1364/AO.435321.

Single-sequence stable spectroscopic reflectometry using simultaneous measurement of incident light and reflected light.

We proposed a new scheme of spectroscopic reflectometry (SR) that improves the stability with respect to the intensity variations of the incident light and simplifies the measurement procedure. The proposed method enables this by simultaneously measuring the incident light and the reflected light. Using the multi-order retarder, we modulate the incident light intensity into a high-frequency signal at the spectral domain. Even though the spectrometer measures the sum of reflected light and modulated incident light, each signal can be separately obtained using the Fourier transform. The proposed method is verified through the measurement of SiO2 thin film on the Si substrate with various incident light intensities.

Critical regulation of follicular helper T cell differentiation and function by Gα13 signaling.

GPCR-Gα protein-mediated signal transduction contributes to spatiotemporal interactions between immune cells to fine-tune and facilitate the process of inflammation and host protection. Beyond this, however, how Gα proteins contribute to the helper T cell subset differentiation and adaptive response have been underappreciated. Here, we found that Gα13 signaling in T cells plays a crucial role in inducing follicular helper T (Tfh) cell differentiation in vivo. T cell-specific Gα13-deficient mice have diminished Tfh cell responses in a cell-intrinsic manner in response to immunization, lymphocytic choriomeningitis virus infection, and allergen challenges. Moreover, Gα13-deficient Tfh cells express reduced levels of Bcl-6 and CXCR5 and are functionally impaired in their ability to adhere to and stimulate B cells. Mechanistically, Gα13-deficient Tfh cells harbor defective Rho-ROCK2 activation, and Rho agonist treatment recuperates Tfh cell differentiation and expression of Bcl-6 and CXCR5 in Tfh cells of T cell-specific Gα13-deficient mice. Conversely, ROCK inhibitor treatment hampers Tfh cell differentiation in wild-type mice. These findings unveil a crucial regulatory role of Gα13-Rho-ROCK axis in optimal Tfh cell differentiation and function, which might be a promising target for pharmacologic intervention in vaccine development as well as antibody-mediated immune disorders.

Synthesis, Characterization, and Anticancer Activity of Benzothiazole Aniline Derivatives and Their Platinum (II) Complexes as New Chemotherapy Agents.

We describe the synthesis, characterization, molecular modeling, and in vitro anticancer activity of three benzothiazole aniline (BTA) ligands and their corresponding platinum (II) complexes. We designed the compounds based on the selective antitumor properties of BTA, along with three types of metallic centers, aiming to take advantage of the distinctive and synergistic activity of the complexes to develop anticancer agents. The compounds were characterized using nuclear magnetic resonance spectrometry, Fourier transform infrared spectroscopy, mass spectrometry, elemental analysis, and tested for antiproliferative activity against multiple normal and cancerous cell lines. L1, L2, and L1Pt had better cytotoxicity in the liver, breast, lung, prostate, kidney, and brain cells than clinically used cisplatin. Especially, L1 and L1Pt demonstrated selective inhibitory activities against liver cancer cells. Therefore, these compounds can be a promising alternative to the present chemotherapy drugs.

A Nitric Oxide-Responsive Transcriptional Regulator NsrR Cooperates With Lrp and CRP to Tightly Control the hmpA Gene in Vibrio vulnificus.

Nitric oxide (NO) is an important antimicrobial effector produced by the host innate immune system to counteract invading pathogens. To survive and establish a successful infection, a fulminating human pathogen Vibrio vulnificus expresses the hmpA gene encoding an NO dioxygenase in an NO-responsive manner. In this study, we identified an Rrf2-family transcriptional regulator NsrR that is predicted to contain the Fe-S cluster coordinated by three cysteine residues. Transcriptome analysis showed that NsrR controls the expression of multiple genes potentially involved in nitrosative stress responses. Particularly, NsrR acts as a strong repressor of hmpA transcription and relieves the repression of hmpA upon exposure to NO. Notably, nsrR and hmpA are transcribed divergently, and their promoter regions overlap with each other. Molecular biological analyses revealed that NsrR directly binds to this overlapping promoter region, which is alleviated by loss of the Fe-S cluster, leading to the subsequent derepression of hmpA under nitrosative stress. We further found that a leucine-responsive regulatory protein (Lrp) negatively regulates hmpA in an NsrR-dependent manner by directly binding to the promoter region, presumably resulting in a DNA conformation change to support the repression by NsrR. Meanwhile, a cyclic AMP receptor protein (CRP) positively regulates hmpA probably through repression of nsrR and lrp by directly binding to each promoter region in a sequential cascade. Altogether, this collaborative regulation of NsrR along with Lrp and CRP enables an elaborate control of hmpA transcription, contributing to survival under host-derived nitrosative stress and thereby the pathogenesis of V. vulnificus.

Type 17 immunity promotes the exhaustion of CD8+ T cells in cancer.

BACKGROUND: Multiple types of immune cells producing IL-17 are found in the tumor microenvironment. However, their roles in tumor progression and exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) remain unclear. METHODS: To determine the role of type 17 immunity in tumor, we investigated the growth of B16F10 melanoma and the exhaustion of CD8+ TILs in Il17a-/- mice, Il17aCreR26DTA mice, RORγt inhibitor-treated mice, or their respective control mice. Adoptive transfer of tumor-specific IL-17-producing T cells was performed in B16F10-bearing congenic mice. Anti-CD4 or anti-Ly6G antibodies were used to deplete CD4+ T cells or CD11b+Gr-1hi myeloid cells in vivo, respectively. Correlation between type 17 immunity and T cell exhaustion in human cancer was evaluated by interrogating TCGA dataset. RESULTS: Depletion of CD4+ T cells promotes the exhaustion of CD8+ T cells with a concomitant increase in IL-17-producing CD8+ T (Tc17) cells in the tumor. Unlike IFN-γ-producing CD8+ T (Tc1) cells, tumor-infiltrating Tc17 cells exhibit CD103+KLRG1-IL-7Rαhi tissue resident memory-like phenotypes and are poorly cytolytic. Adoptive transfer of IL-17-producing tumor-specific T cells increases, while depletion of IL-17-producing cells decreases, the frequency of PD-1hiTim3+TOX+ terminally exhausted CD8+ T cells in the tumor. Blockade of IL-17 or RORγt pathway inhibits exhaustion of CD8+ T cells and also delays tumor growth in vivo. Consistent with these results, human TCGA analyses reveal a strong positive correlation between type 17 and CD8+ T cell exhaustion signature gene sets in multiple cancers. CONCLUSION: IL-17-producing cells promote terminal exhaustion of CD8+ T cells and tumor progression in vivo, which can be reversed by blockade of IL-17 or RORγt pathway. These findings unveil a novel role for IL-17-producing cells as tumor-promoting cells facilitating CD8+ T cell exhaustion, and propose type 17 immunity as a promising target for cancer immunotherapy.

A critical role for Th17 cell-derived TGF-ß1 in regulating the stability and pathogenicity of autoimmune Th17 cells.

Pathogenic conversion of Th17 cells into multifunctional helper T cells or Th1 cells contributes to the pathogenesis of autoimmune diseases; however, the mechanism regulating the plasticity of Th17 cells remains unclear. Here, we found that Th17 cells expressed latent TGF-ß1 in a manner dependent on autocrine TGF-ß1. By employing IL-17-producing cell-specific Tgfb1 conditional knockout and fate-mapping systems, we demonstrated that TGF-ß1-deficient Th17 cells are relatively susceptible to becoming IFN-γ producers through IL-12Rß2 and IL-27Rα upregulation. TGF-ß1-deficient Th17 cells exacerbated tissue inflammation compared to TGF-ß1-sufficient Th17 cells in adoptive transfer models of experimental autoimmune encephalomyelitis and colitis. Thus, TGF-ß1 production by Th17 cells provides an essential autocrine signal for maintaining the stability and regulating the pathogenicity of Th17 cells in vivo.

Coaxial spectroscopic imaging ellipsometry for volumetric thickness measurement.

We propose spectroscopic imaging ellipsometry that can measure spectral ellipsometric signals in the entire field of view simultaneously without areal scanning or operation of polarization devices. The proposed imaging ellipsometry is configured in a coaxial optical structure so that the high magnification objective lens is applicable and the spatial resolution is highly increased. Without the operation of polarization components and to efficiently obtain the spectral data in the object plane, the ellipsometric parameters are encoded into the high frequency in the spectral domain and are measured by an imaging Michelson interferometer. The volumetric thickness measurement by the proposed method was verified by comparing the thickness results of the SiO2/Si sample that has four different thicknesses with commercial ellipsometer results.

A critical regulation of Th2 cell responses by RORα in allergic asthma.

Allergic asthma is a chronic inflammatory disease of the lung and the airway, which is characterized by aberrant type 2 immune responses to otherwise unharmful aeroallergens. While the central role of Th2 cells and type 2 cytokines in the pathogenesis of allergic asthma is well documented, the regulation and plasticity of Th2 cells remain incompletely understood. By using an animal model of allergic asthma in IL-4-reporter mice, we found that Th2 cells in the lung expressed higher levels of Rora than those in the lymph nodes, and that treatment with an RORα agonist SR1078 resulted in diminished Th2 cell responses in vivo. To determine the T cell-intrinsic role of RORα in allergic asthma in vivo, we established T cell-specific RORα-deficient (Cd4creRoraf/f) mice. Upon intranasal allergen challenges, Cd4creRoraf/f mice exhibited a significantly increased Th2 cells in the lungs and the airway and showed an enhanced eosinophilic inflammation compared to littermate control mice. Studies with Foxp3YFP-creRoraf/f mice and CD8+ T cell depletion showed that the increased Th2 cell responses in the Cd4creRoraf/f mice were independent of Treg cells and CD8+ T cells. Our findings demonstrate a critical regulatory role of RORα in Th2 cells, which suggest that RORα agonists could be effective for the treatment of allergic diseases.