Background Respiratory syncytial virus (RSV) is a major health care burden with a particularly high worldwide morbidity and mortality rate among infants. Data suggest that severe RSV-associated ...illness is in part caused by immunopathology associated with a robust type 2 response. Objective We sought to determine the capacity of RSV infection to stimulate group 2 innate lymphoid cells (ILC2s) and the associated mechanism in a murine model. Methods Wild-type (WT) BALB/c, thymic stromal lymphopoietin receptor (TSLPR) knockout (KO), or WT mice receiving an anti-TSLP neutralizing antibody were infected with the RSV strain 01/2-20. During the first 4 to 6 days of infection, lungs were collected for evaluation of viral load, protein concentration, airway mucus, airway reactivity, or ILC2 numbers. Results were confirmed with 2 additional RSV clinical isolates, 12/11-19 and 12/12-6, with known human pathogenic potential. Results RSV induced a 3-fold increase in the number of IL-13–producing ILC2s at day 4 after infection, with a concurrent increase in total lung IL-13 levels. Both thymic stromal lymphopoietin (TSLP) and IL-33 levels were increased 12 hours after infection. TSLPR KO mice did not mount an IL-13–producing ILC2 response to RSV infection. Additionally, neutralization of TSLP significantly attenuated the RSV-induced IL-13–producing ILC2 response. TSLPR KO mice displayed reduced lung IL-13 protein levels, decreased airway mucus and reactivity, attenuated weight loss, and similar viral loads as WT mice. Both 12/11-19 and 12/12-6 similarly induced IL-13–producing ILC2s through a TSLP-dependent mechanism. Conclusion These data demonstrate that multiple pathogenic strains of RSV induce IL-13–producing ILC2 proliferation and activation through a TSLP-dependent mechanism in a murine model and suggest the potential therapeutic targeting of TSLP during severe RSV infection.
Background
The epithelial cell‐derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL‐33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, ...TSLP and IL‐33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown.
Methods
BALB/c WT and TSLP receptor‐deficient (TSLPR−/−) mice were challenged intranasally with Alternaria extract (Alt‐Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre‐administered with rTSLP or vehicle, TSLPR−/− mice, and IL‐33 receptor‐deficient (ST2−/−) mice were challenged intranasally with Alt‐Ext or vehicle once or twice to evaluate IL‐33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL‐33, rTSLP + rIL‐33, or vehicle.
Results
Thymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL‐5 and IL‐13 following Alt‐Ext‐challenge compared to WT mice. Further, eosinophilia, protein level of lung IL‐4, IL‐5, and IL‐13, and airway mucus score were also significantly decreased in TSLPR−/− mice compared to WT mice. Endogenous and exogenous TSLP increased Alt‐Ext‐induced IL‐33 release into BALF, and ST2 deficiency decreased Alt‐Ext‐induced TSLP expression in the lung. Further, rTSLP and rIL‐33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro.
Conclusion
Thymic stromal lymphopoietin and IL‐33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt‐Ext‐challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.
We found that TSLPR deficiency significantly decrease the number of lung ILC2, airway eosinophils, and mucus production in mouse challenged with Alternaria extract. TSLP‐TSLPR and IL‐33‐ST2 signaling augments each other's protein release and expression in the lung epithelial cells after aeroallergen challenge. TSLP and IL‐33 signaling increases each other's receptor expression on lung ILC2, and the IL‐33‐augmented TSLPR expression enhances pSTAT5 in the ILC2.
The stimulator of interferon genes (STING) pathway plays an important role in the immune surveillance of cancer and, accordingly, agonists of STING signaling have recently emerged as promising ...therapeutics for remodeling of the immunosuppressive tumor microenvironment (TME) and enhancing response rates to immune checkpoint inhibitors. 2′3′-cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) is the endogenous ligand for STING, but is rapidly metabolized and poorly membrane permeable, restricting its use to intratumoral administration. Nanoencapsulation has been shown to allow for systemic administration of cGAMP and other cyclic dinucleotides (CDN), but little is known about how nanocarriers affect important pharmacological properties that impact the efficacy and safety of CDNs. Using STING-activating nanoparticles (STING-NPs) – a polymersome platform designed to enhance cGAMP delivery – we investigate the pharmacokinetic (PK)-pharmacodynamic (PD) relationships that underlie the ability of intravenously (i.v.) administered STING-NPs to induce STING activation and inhibit tumor growth. First, we demonstrate that nanoencapsulation improves the half-life of encapsulated cGAMP by 40-fold, allowing for sufficient accumulation of cGAMP in tumors and activation of the STING pathway in the TME as assessed by western blot analysis and gene expression profiling. Nanoparticle delivery also changes the biodistribution profile, resulting in increased cGAMP accumulation and STING activation in the liver and spleen, which we identify as dose limiting organs. As a consequence of STING activation in tumors, i.v. administered STING-NPs reprogram the TME towards a more immunogenic antitumor milieu, characterized by an influx of >20-fold more CD4+ and CD8+ T-cells. Consequently, STING-NPs increased response rates to αPD-L1 antibodies, resulting in significant improvements in median survival time in a B16-F10 melanoma model. Additionally, we confirmed STING-NP monotherapy in an additional melanoma (YUMM1.7) and breast adenocarcinoma (E0771) models leading to >50% and 80% reduction in tumor burden, respectively, and significant increases in median survival time. Collectively, this work provides an examination of the PK-PD relationship governing STING activation upon systemic delivery using STING-NPs, providing insight for future optimization for nanoparticle-based STING agonists and other immunomodulating nanomedicines.
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•STING-NPs are polymersomes designed to increase cytosolic delivery of cyclic dinucleotide STING agonists.•Nanoparticle encapsulation of 2′,3′-cGAMP in STING-NPs extends drug half-life and exposure.•Hepatic and splenic toxicities are dose limiting.•Intravenous administration of STING-NPs remodels the tumor immune microenvironment.•Systemic STING-NPs improve survival in models of melanoma and breast cancer.
Decreased expression of the Nlrp3 protein is associated with susceptibility to Crohn's disease. However, the role of Nlrp3 in colitis has not been characterized. Nlrp3 interacts with the adaptor ...protein ASC to activate caspase-1 in inflammasomes, which are protein complexes responsible for the maturation and secretion of interleukin-1β (IL-1β) and IL-18. Here, we showed that mice deficient for Nlrp3 or ASC and caspase-1 were highly susceptible to dextran sodium sulfate (DSS)-induced colitis. Defective inflammasome activation led to loss of epithelial integrity, resulting in systemic dispersion of commensal bacteria, massive leukocyte infiltration, and increased chemokine production in the colon. This process was a consequence of a decrease in IL-18 in mice lacking components of the Nlrp3 inflammasome, resulting in higher mortality rates. Thus, the Nlrp3 inflammasome is critically involved in the maintenance of intestinal homeostasis and protection against colitis.
► The Nlrp3 inflammasome protects against DSS- and TNBS-induced colitis ► Nlrp3 activation in colonic epithelial cells is important for IL-18 production ► IL-18 production by the Nlrp3 inflammasome controls epithelial cell homeostasis
Sex hormones regulate many autoimmune and inflammatory diseases, including asthma. As adults, asthma prevalence is 2-fold greater in women compared to men. The number of group 2 innate lymphoid cells ...(ILC2) is increased in patients with asthma, and we investigate how testosterone attenuates ILC2 function. In patients with moderate to severe asthma, we determine that women have an increased number of circulating ILC2 compared to men. ILC2 from adult female mice have increased IL-2-mediated ILC2 proliferation versus ILC2 from adult male mice, as well as pre-pubescent females and males. Further, 5α-dihydrotestosterone, a hormone downstream of testosterone, decreases lung ILC2 numbers and IL-5 and IL-13 expression from ILC2. In vivo, testosterone attenuated Alternaria-extract-induced IL-5+ and IL-13+ ILC2 numbers and lung eosinophils by intrinsically decreasing lung ILC2 numbers, as well as by decreasing expression of IL-33 and thymic stromal lymphopoietin (TSLP), ILC2-stimulating cytokines. Collectively, these findings provide a foundational understanding of sexual dimorphism in ILC2 function.
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•The number of ILC2 is increased in women with asthma compared to men with asthma•Sex hormones regulate IL-2-dependent ILC2 proliferation and cytokine expression•Testosterone intrinsically and extrinsically attenuates ILC2 airway inflammation
Women have higher asthma prevalence compared to men, and ILC2 are increased in patients with asthma. Cephus et al. show that women with asthma have higher circulating ILC2 numbers compared to men with asthma. Testosterone negatively regulates ILC2 proliferation and cytokine expression, as well as ILC2-mediated allergic airway inflammation.
We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis ...provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus; hippocampal dysgenesis; sterility; and chronic inflammation/infection of lung, middle ear, and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells and suggest that p73 marks these cells for MCC differentiation. In summary, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation, and, like p63, has an essential role in development of tissues.
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•p73 is required for murine MCC differentiation•MCC loss provides a unifying biological defect for phenotypes in p73−/− mice•p73 binds to the genome in proximity to 105 cilia-associated genes, including Foxj1•p73 directly regulates and is required for Foxj1 expression
Using a p73-deficient mouse model, Marshall et al. show that p73 is required for MCC differentiation. ChIP-seq of murine tracheal cells reveals many p73 target genes that regulate MCC differentiation. Lack of expression of key transcriptional regulators of ciliogenesis provides a mechanistic basis for the multiple defects in p73-deficient mice.
Background
Obesity is a risk factor for the development of asthma. However, pharmacologic therapeutic strategies that specifically target obese asthmatics have not been identified. We hypothesize ...that glucagon‐like peptide‐1 receptor agonist (GLP‐1RA) treatment inhibits aeroallergen‐induced early innate airway inflammation in a mouse model of asthma in the setting of obesity.
Methods
SWR (lean) and TALLYHO (obese) mice were challenged intranasally with Alternaria alternata extract (Alt‐Ext) or PBS for 4 consecutive days concurrent with GLP‐1RA or vehicle treatment.
Results
TALLYHO mice had greater Alt‐Ext‐induced airway neutrophilia and lung protein expression of IL‐5, IL‐13, CCL11, CXCL1, and CXCL5, in addition to ICAM‐1 expression on lung epithelial cells compared with SWR mice, and all endpoints were reduced by GLP‐1RA treatment. Alt‐Ext significantly increased BALF IL‐33 in both TALLYHO and SWR mice compared to PBS challenge, but there was no difference in the BALF IL‐33 levels between these two strains. However, TALLYHO, but not SWR, mice had significantly higher airway TSLP in BALF following Alt‐Ext challenge compared to PBS, and BALF TSLP was significantly greater in TALLYHO mice compared to SWR mice following airway Alt‐Ext challenge. GLP‐1RA treatment significantly decreased the Alt‐Ext‐induced TSLP and IL‐33 release in TALLYHO mice. While TSLP or ST2 inhibition with a neutralizing antibody decreased airway eosinophils, they did not reduce airway neutrophils in TALLYHO mice.
Conclusions
These results suggest that GLP‐1RA treatment may be a novel pharmacologic therapeutic strategy for obese persons with asthma by inhibiting aeroallergen‐induced neutrophilia, a feature not seen with either TSLP or ST2 inhibition.
Polygenic obese mice have larger level of Altrernaria extract‐induced TSLP, the number of lung ILC2, neutrophils in the airway, and ICAM‐1 on lung epithelial cells compared with lean mice. GLP‐1RA decreases the Altrernaria extract‐induced IL‐33 and TSLP release, type‐2 inflammation mediated by ILC2, eosinophilia and neutrophilia in the airway, and airway responsiveness in polygenic obese mice. Our findings indicate the potential for a new pharmacological therapeutic strategy to patients with asthma in the setting of obesity.
Abbreviations: ICAM‐1, intercellular adhesion molecule 1; ILC2, group 2 innate lymphoid cells; GLP‐1RA, glucagon‐like peptide‐1 agonist
We previously elucidated the pleotropic role of solute carrier family A1 member 5 (SLC1A5) as the primary transporter of glutamine (Gln), a modulator of cell growth and oxidative stress in non‐small ...cell lung cancer (NSCLC). The aim of our study was to evaluate SLC1A5 as a potential new therapeutic target and candidate biomarker predictive of survival and response to therapy. SLC1A5 targeting was examined in a panel of NSCLC and human bronchial cell lines by RNA interference and by a small molecular inhibitor, gamma‐l‐glutamyl‐p‐nitroanilide (GPNA). The effects of targeting SLC1A5 on cell growth, Gln uptake, ATP level, autophagy and cell death were examined. Inactivation of SLC1A5 genetically or pharmacologically decreased Gln consumption, inhibited cell growth, induced autophagy and apoptosis in a subgroup of NSCLC cell lines that overexpress SLC1A5. Targeting SLC1A5 function decreased tumor growth in NSCLC xenografts. A multivariate Cox proportional hazards analysis indicates that patients with increased SLC1A5 mRNA expression have significantly shorter overall survival (p = 0.01, HR = 1.24, 95% CI: 1.05–1.46), adjusted for age, gender, smoking history and disease stage. In an immunohistochemistry study on 207 NSCLC patients, SLC1A5 protein expression remained highly significant prognostic value in both univariate (p < 0.0001, HR = 1.45, 95% CI: 1.15–1.50) and multivariate analyses (p = 0.04, HR = 1.22, 95% CI: 1.01–1.31). These results position SLC1A5 as a new candidate prognostic biomarker for selective targeting of Gln‐dependent NSCLC.
What's New?
New strategies to overcome lung cancer mortality depend heavily on the discovery of novel therapeutic targets. In non‐small cell lung cancer (NSCLC), a possible target is solute linked carrier family 1A, member 5 (SLC1A5), a major glutamine transporter in NSCLC. This study furthers the promise of SLC1A5 by showing that its expression levels in lung cancer cells can predict cell sensitivity to the inhibitor gamma‐L‐glutamyl‐p‐nitroanilide (GPNA). In NSCLC cell lines, SLC1A5 inactivation led to glutamine starvation and oxidative stress‐mediated autophagy and apoptosis. In NSCLC patients, SLC1A5 expression was associated with poor overall survival.
MicroRNA (miR) are important regulators of gene expression, and aberrant miR expression has been linked to oncogenesis; however, little is understood about their contribution to lung tumorigenesis. ...Here, we determined that miR-31 is overexpressed in human lung adenocarcinoma and this overexpression independently correlates with decreased patient survival. We developed a transgenic mouse model that allows for lung-specific expression of miR-31 to test the oncogenic potential of miR-31 in the lung. Using this model, we observed that miR-31 induction results in lung hyperplasia, followed by adenoma formation and later adenocarcinoma development. Moreover, induced expression of miR-31 in mice cooperated with mutant KRAS to accelerate lung tumorigenesis. We determined that miR-31 regulates lung epithelial cell growth and identified 6 negative regulators of RAS/MAPK signaling as direct targets of miR-31. Our study distinguishes miR-31 as a driver of lung tumorigenesis that promotes mutant KRAS-mediated oncogenesis and reveals that miR-31 directly targets and reduces expression of negative regulators of RAS/MAPK signaling.
The role of respiratory viruses in the transmission of Streptococcus pneumoniae is poorly understood. Key questions, such as which serotypes are most fit for transmission and disease and whether ...influenza virus alters these parameters in a serotype-specific manner, have not been adequately studied. In a novel model of transmission in ferrets, we demonstrated that pneumococcal transmission and disease were enhanced if donors had previously been infected with influenza virus. Bacterial titers in nasal wash, the incidence of mucosal and invasive disease, and the percentage of contacts that were infected all increased. In contact ferrets, viral infection increased their susceptibility to S. pneumoniae acquisition both in terms of the percentage infected and the distance over which they could acquire infection. These influenza-mediated effects on colonization, transmission, and disease were dependent on the pneumococcal strain. Overall, these data argue that the relationship between respiratory viral infections, acquisition of pneumococci, and development of disease in humans needs further study to be better understood.
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