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.

LCCL peptide cleavage after noise exposure exacerbates hearing loss and is associated with the monocyte infiltration in the cochlea.

Acoustic trauma induces an inflammatory response in the cochlea, resulting in debilitating hearing function. Clinically, amelioration of inflammation substantially prevents noise-induced hearing loss. The Limulus factor C, Cochlin, and Lgl1 (LCCL) peptide plays an important role in innate immunity during bacteria-induced inflammation in the cochlea. We aimed to investigate the LCCL-induced innate immune response to noise exposure and its impact on hearing function. METHODS: We used Coch (encodes cochlin harboring LCCL peptide) knock-out and p.G88E knock-in mice to compare the immune responses before and after noise exposure. We explored their hearing function and hair cell degeneration. Moreover, we investigated distinct characteristics of immune responses upon noise exposure using flow cytometry and RNA sequencing. RESULTS: One day after noise exposure, the LCCL peptide cleaved from cochlin increased over time in the perilymph space. Both Coch-/- and CochG88E/G88E mutant mice revealed more preserved hearing following acoustic trauma compared to wild-type mice. The outer hair cells were more preserved in Coch-/- than in wild-type mice upon noise exposure. The RNA sequencing data demonstrated significantly upregulated cell migration gene ontology in wild-type mice than in Coch-/- mice following noise exposure, indicating that the infiltration of immune cells was dependent on cochlin. Notably, infiltrated monocytes from blood (C11b+/Ly6G-/Ly6C+) were remarkably higher in wild-type mice than in Coch-/- mice at 1 day after noise exposure. CONCLUSIONS: Noise-induced hearing loss was attributed to over-stimulated cochlin, and led to the cleavage and secretion of LCCL peptide in the cochlea. The LCCL peptide recruited more monocytes from the blood vessels upon noise stimulation, thus highlighting a novel therapeutic target for noise-induced hearing loss.

Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu.

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1ß secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1ß rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1ß-secreting cells in DAMP-rich inflammatory region.

Mitofusin-2 Promotes the Epithelial-Mesenchymal Transition-Induced Cervical Cancer Progression.

High expression of mitofusin-2 (MFN2), a mitochondrial fusion protein, has been frequently associated with poor prognosis of patients with cervical cancer. Here, we aimed to identify the function of MFN2 in cervical cancer to understand its influence on disease prognosis. To this end, from cervical adenocarcinoma, we performed an MTT assay and quantitative RT-PCR (qRT-PCR) analysis to assess the effects of MFN2 on the proliferation and of HeLa cells. Then, colony-formation ability and tumorigenesis were evaluated using a tumor xenograft mouse model. The migration ability related to MFN2 was also measured using a wound healing assay. Consequently, epithelial-mesenchymal transition (EMT) of MFN2-knockdowned HeLa cells originating from adenocarcinoma. markers related to MFN2 were assessed by qRT-PCR. Clinical data were analyzed using cBioPortal and The Cancer Genome Atlas. We found that MFN2 knockdown reduced the proliferation, colony formation ability, migration, and in vivo tumorigenesis of HeLa cells. Primarily, migration of MFN2-knockdowned HeLa cells decreased through the suppression of EMT. Thus, we concluded that MFN2 facilitates cancer progression and in vivo tumorigenesis in HeLa cells. These findings suggest that MFN2 could be a novel target to regulate the EMT program and tumorigenic potential in HeLa cells and might serve as a therapeutic target for cervical cancer. Taken together, this study is expected to contribute to the treatment of patients with cervical cancer.

Endogenous DEL-1 restrains melanoma lung metastasis by limiting myeloid cell-associated lung inflammation.

Distant metastasis represents the primary cause of cancer-associated death. Pulmonary metastasis is most frequently seen in many cancers, largely driven by lung inflammation. Components from primary tumor or recruited leukocytes are known to facilitate metastasis formation. However, contribution of target site-specific host factor to metastasis is poorly understood. Here, we show that developmental endothelial locus-1 (DEL-1), an anti-inflammatory factor abundant in the lung and down-regulated by inflammatory insults, protects from melanoma lung metastasis independently of primary tumor development and systemic immunosurveillance. DEL-1 deficiency is associated with gene profiles that favor metastatic progression with inflammation and defective immunosurveillance. Mechanistically, DEL-1 deficiency primarily influences Ly6G+ neutrophil accumulation in lung metastatic niche, leading to IL-17A up-regulation from γδ T cells and reduced antimetastatic NK cells. In support, neutrophil depletion or recombinant DEL-1 treatment profoundly reverses these effects. Thus, our results identify DEL-1 as a previously unrecognized link between tumor-induced inflammation and pulmonary metastasis.

3D Imaging of the Transparent Mycobacterium tuberculosis-Infected Lung Verifies the Localization of Innate Immune Cells With Granuloma.

Using a novel tissue-clearing method, we aimed to visualize the three-dimensional (3D) distribution of immune cells within Mycobacterium tuberculosis (Mtb)-infected mice lungs. Ethyl cinnamate-based tissue clearing of Mtb-infected mice lungs was performed to obtain transparent lung samples, which were then imaged using a light sheet fluorescence microscope. Using the 3D images, we performed quantitative analysis of the immune cell population within multiple granulomas. In addition, to compare the data from the tissue clearing method, we performed histopathological and immunofluorescence analyses, and flow cytometry. We then created 3D images of the Mtb-infected lung that successfully demonstrated the distribution of blood vessels, immune cells, and granulomas. Since the immune cells within a granuloma could be separately selected and counted, the immune cell population within a specific lesion could be quantified. In addition, macroscopic analysis, e.g., the size or shape of a granuloma, as well as microscopic analysis could be performed as intact lung samples were used. The use of the tissue clearing method in infected lungs could be a novel modality for understanding the role of the immune system in the pathogenesis of tuberculosis.

In vivo monitoring of dynamic interaction between neutrophil and human umbilical cord blood-derived mesenchymal stem cell in mouse liver during sepsis.

BACKGROUND: Sepsis is a global inflammatory disease that causes death. It has been reported that mesenchymal stem cell (MSC) treatment can attenuate inflammatory and septic symptoms. In this study, we investigated how interactions between neutrophils and human umbilical cord blood (hUCB)-MSCs in the liver of septic mice are involved in mitigating sepsis that is mediated by MSCs. Accordingly, we aimed to determine whether hUCB-MSC application could be an appropriate treatment for sepsis. METHODS: To induce septic condition, lipopolysaccharide (LPS) was intraperitoneally (i.p.) injected into mice 24 h after the intravenous (i.v.) injection of saline or hUCB-MSCs. To determine the effect of hUCB-MSCs on the immune response during sepsis, histologic analysis, immunoassays, and two-photon intravital imaging were performed 6 h post-LPS injection. For the survival study, mice were monitored for 6 days after LPS injection. RESULTS: The injection (i.v.) of hUCB-MSCs alleviated the severity of LPS-induced sepsis by increasing IL-10 levels (p < 0.001) and decreasing mortality (p < 0.05) in septic mice. In addition, this significantly reduced the recruitment of neutrophils (p < 0.001) to the liver. In hUCB-MSC-treated condition, we also observed several distinct patterns of dynamic interactions between neutrophils and hUCB-MSCs in the inflamed mouse liver, as well as vigorous interactions between hepatic stellate cells (HSCs or ito cells) and hUCB-MSCs. Interestingly, hUCB-MSCs that originated from humans were not recognized as foreign in the mouse body and consequently did not cause graft rejection. CONCLUSIONS: These distinct interaction patterns between innate immune cells and hUCB-MSCs demonstrated that hUCB-MSCs have beneficial effects against LPS-induced sepsis through associations with neutrophils. In addition, the immunomodulatory properties of hUCB-MSCs might enable immune evasion in the host. Taken together, our results suggest the prospects of hUCB-MSCs as a therapeutic tool to inhibit inflammation and alleviate pathological immune responses such as sepsis.

Particulate matter-induced senescence of skin keratinocytes involves oxidative stress-dependent epigenetic modifications.

Ambient air particulate matter (PM) induces senescence in human skin cells. However, the underlying mechanisms remain largely unknown. We investigated how epigenetic regulatory mechanisms participate in cellular senescence induced by PM with a diameter <2.5 (PM2.5) in human keratinocytes and mouse skin tissues. PM2.5-treated cells exhibited characteristics of cellular senescence. PM2.5 induced a decrease in DNA methyltransferase (DNMT) expression and an increase in DNA demethylase (ten-eleven translocation; TET) expression, leading to hypomethylation of the p16INK4A promoter region. In addition, PM2.5 led to a decrease in polycomb EZH2 histone methyltransferase expression, whereas the expression of the epigenetic transcriptional activator MLL1 increased. Furthermore, binding of DNMT1, DNMT3B, and EZH2 to the promoter region of p16INK4A decreased in PM2.5-treated keratinocytes, whereas TET1 and MLL1 binding increased, leading to decreased histone H3 lysine 27 trimethylation (H3K27Me3) and increased H3K4Me3 in the promoter of p16INK4A. PM2.5-induced senescence involved aryl hydrocarbon receptor (AhR)-induced reactive oxygen species (ROS) production. ROS scavenging dampened PM2.5-induced cellular senescence through regulation of DNA and histone methylation. Altogether, our work shows that skin senescence induced by environmental PM2.5 occurs through ROS-dependent the epigenetic modification of senescence-associated gene expression. Our findings provide information for the design of preventive and therapeutic strategies against skin senescence, particularly in light of the increasing problem of PM2.5 exposure due to air pollution.

Real-Time Monitoring of Cancer Cells in Live Mouse Bone Marrow.

Disseminated tumor cells in the bone marrow environment are the main cause of systemic metastasis after curative treatment for major solid tumors. However, the detailed biological processes of tumor biology in bone marrow have not been well defined in a real-time manner, because of a lack of a proper in vivo experimental model thereof. In this study, we established intravital imaging models of the bone marrow environment to enable real-time observation of cancer cells in the bone marrow. Using these novel imaging models of intact bone marrow and transplanted bone marrow of mice, respectively, via two-photon microscopy, we could first successfully track and analyze both the distribution and the phenotype of cancer cells in bone marrow of live mouse. Therefore, these novel in vivo imaging models for the bone marrow would provide a valuable tool to identify the biologic processes of cancer cells in a real-time manner in a live animal model.

Mitofusin-2 Expression Is Implicated in Cervical Cancer Pathogenesis.

BACKGROUND/AIM: It remains unclear whether mitofusin-2 (MFN2) functions as a tumour suppressor or oncogene in cancer progression. In this study we, therefore, aimed to investigate the effect of MFN2 on the pathogenesis of cervical cancer. MATERIALS AND METHODS: MFN2 expression was detected in seven healthy cervical, 64 cervical intraepithelial neoplasia (CIN), and 120 cervical squamous cell carcinoma (SCC) tissues by immunohistochemistry. Moreover, biological function of MFN2 in cervical cancer was investigated in vitro. RESULTS: MFN2 levels exhibited a tendency to gradually increase from healthy cervical tissue to CIN to SCC. Moreover, MFN2 expression was significantly associated with higher T-stage (p=0.008) and lymph node metastasis (p<0.001). The proliferative, migratory, and invasive abilities of MFN2-knockdown cells were significantly lower (p<0.001, p<0.001, and p<0.001, respectively) than control cells. CONCLUSION: MFN2 may be involved in cervical cancer pathogenesis as an oncogene and might serve as a biomarker of cervical SCC.

TAGLN2 polymerizes G-actin in a low ionic state but blocks Arp2/3-nucleated actin branching in physiological conditions.

TAGLN is an actin-binding protein family that comprises three isoforms with theorized roles in smooth muscle differentiation, tumour development, lymphocyte activation, and brain chemistry. However, their fundamental characteristics in regulation of the actin-based cytoskeleton are not fully understood. Here we show that TAGLN2 (including TAGLN1 and TAGLN3) extensively nucleates G-actin polymerization under low-salt conditions, where polymerization would be completely suppressed. The calponin homology domain and actin-binding loop are essential to mechanically connect two adjacent G-actins, thereby mediating multimeric interactions. However, TAGLN2 blocked the Arp2/3 complex binding to actin filaments under physiological salt conditions, thereby inhibiting branched actin nucleation. In HeLa and T cells, TAGLN2 enhanced filopodium-like membrane protrusion. Collectively, the dual functional nature of TAGLN2-G-actin polymerization and Arp2/3 complex inhibition-may account for the mechanisms of filopodia development at the edge of Arp2/3-rich lamellipodia in various cell types.

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

Live Imaging of Influenza Infection of the Trachea Reveals Dynamic Regulation of CD8+ T Cell Motility by Antigen.

During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.

Sepsis lethality via exacerbated tissue infiltration and TLR-induced cytokine production by neutrophils is integrin α3ß1-dependent.

Integrin-mediated migration of neutrophils to infected tissue sites is vital for pathogen clearance and therefore host survival. Although ß2 integrins have been shown to mediate neutrophil transendothelial migration during systemic and local inflammation, relatively little information is available regarding neutrophil migration in sepsis beyond the endothelial cell layer. In this study, we report that integrin α3ß1 (VLA-3; CD49c/CD29) is dramatically upregulated on neutrophils isolated from both human septic patients and in mouse models of sepsis. Compared with the α3ß1 (low) granulocytes, α3ß1 (high) cells from septic animals displayed hyperinflammatory phenotypes. Administration of a α3ß1 blocking peptide and conditional deletion of α3 in granulocytes significantly reduced the number of extravasating neutrophils and improved survival in septic mice. In addition, expression of α3ß1 on neutrophils was associated with Toll-like receptor-induced inflammatory responses and cytokine productions. Thus, our results show that α3ß1 is a novel marker of tissue homing and hyperresponsive neutrophil subtypes in sepsis, and blocking of α3ß1 may represent a new therapeutic approach in sepsis treatment.

Optogenetic control of chemokine receptor signal and T-cell migration.

Adoptive cell transfer of ex vivo-generated immune-promoting or tolerogenic T cells to either enhance immunity or promote tolerance in patients has been used with some success. However, effective trafficking of the transferred cells to the target tissue sites is the main barrier to achieving successful clinical outcomes. Here we developed a strategy for optically controlling T-cell trafficking using a photoactivatable (PA) chemokine receptor. Photoactivatable-chemokine C-X-C motif receptor 4 (PA-CXCR4) transmitted intracellular CXCR4 signals in response to 505-nm light. Localized activation of PA-CXCR4 induced T-cell polarization and directional migration (phototaxis) both in vitro and in vivo. Directing light onto the melanoma was sufficient to recruit PA-CXCR4-expressing tumor-targeting cytotoxic T cells and improved the efficacy of adoptive T-cell transfer immunotherapy, with a significant reduction in tumor growth in mice. These findings suggest that the use of photoactivatable chemokine receptors allows remotely controlled leukocyte trafficking with outstanding spatial resolution in tissues and may be feasible in other cell transfer therapies.

Opposing roles for RhoH GTPase during T-cell migration and activation.

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients ("go" signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition ("stop" signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4(+) T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals.

Monitoring integrin activation by fluorescence resonance energy transfer.

Aberrant integrin activation is associated with several immune pathologies. In leukocyte adhesion deficiency (LAD), the absence or inability of ß(2) integrins to undergo affinity upregulation contributes to recurrent infectious episodes and impaired wound healing, while excessive integrin activity leads to an exaggerated inflammatory response with associated tissue damage. Therefore, integrin activation is an attractive target for immunotherapies, and monitoring the effect of agents on integrin activation is necessary during preclinical drug development. The activation of integrins involves the structural rearrangement of both the extracellular and cytoplasmic domains. Here, we describe methods for monitoring integrin conformational activation using fluorescence resonance energy transfer (FRET).

Outside-in signal transmission by conformational changes in integrin Mac-1.

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the alpha(M) and beta(2) subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.