Combination of IL-33 with PD-1 blockade augment mILC2s-mediated anti-tumor immunity.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) represent one of the main tissue-specific innate lymphoid cell populations, which are key drivers of cytokine secretion in their occupational niche. However, the precise involvement of ILC2s in cancer immunity and their potential impact on immunotherapeutic approaches remain poorly understood. METHODS: The proportion of ILC2s originating from various tissue sources were quantified through flow cytometry, along with the determination of CD4+ T cell and CD8+ T cell percentages. Flow cytometry was also employed to assess IFN-γ production and programmed cell death protein-1 (PD-1) expression in T cells. Immunohistochemistry was utilized to detect IL-33 expression in tumor tissues, while immunofluorescence was employed to confirm the infiltration of ILC2s in both murine and human tumor tissues. RESULTS: In this study, we provide evidence that intra-tumoral ILC2s in lung adenocarcinoma (LUAD) exist in a quiescent state. However, the activation of intra-tumoral ILC2s is induced by IL-33 specifically in a natural ILC2s (nILC2, ST2+KLRG1-) phenotype. Considering the pivotal role of PD-1 in cancer immunotherapy and its immunoregulatory functions, we investigated the synergistic effects of IL-33 and anti-PD-1 and found that their combination enhances anti-tumor immunity and improves the efficacy of immunotherapy. Moreover, this combination leads to the upregulation of activated mature ILC2s (mILC2, ST2+KLRG1+) phenotype, thereby highlighting the activated ILC2s as a novel enhancer of the immunoregulatory properties of anti-PD-1. CONCLUSIONS: Collectively, these findings underscore the significance of ILC2s and their contribution to the anti-tumor response in the context of cancer immunotherapy. Consequently, the simultaneous targeting of ILC2s and T cells represents a potentially promising and widely applicable strategy for immunotherapeutic interventions.

Novel prognostic signature for lung adenocarcinoma based on immune-related mRNA pairs.

Lung adenocarcinoma (LUAD) is a highly lethal malignant tumor. While the involvement of multiple mRNAs in the progression of LUAD is well established, the potential diagnostic value of immune-related mRNAs (irmRNAs) in LUAD remains largely unexplored. In this study, we utilized RNA-seq, clinical data, and immune-related gene information from LUAD patients to identify differentially expressed immune-related mRNAs (DEirmRNAs) and developed a predictive risk model based on specific DEirmRNA pairs closely linked with patient prognosis. We classified patients into high-risk and low-risk groups and analyzed factors such as survival rate, clinical characteristics, gene enrichment, immune cell infiltration, tumor mutation load, and drug susceptibility. We confirmed the expression levels of these DEirmRNAs in tumor tissues using qRT-PCR assay. Our results showed that the low-risk group had a longer survival time and lower tumor mutation burden (TMB) and microsatellite instability (MSI) compared to the high-risk group. The high-risk group also had a significant reduction in the number of certain immune cells and a lower half-maximum inhibitor concentration (IC50). We identified specific DEirmRNA pairs that were up-regulated or down-regulated in tumor tissues compared to adjacent tissues. Our prognostic risk model based on DEirmRNA pairs could be used to predict the prognosis of LUAD patients and provide reference for better treatment.

Activation of the GPR35 on ILC2 drives immunosuppression to promote lung cancer progression.

Lung cancer is the most common cancer type with poor prognosis. While G protein-coupled receptor 35 (GPR35) is a potent stimulator of tumor growth, group 2 innate lymphoid cells (ILC2) have shown dual effects in tumorigenesis. Intriguingly, inflammation induced GPR35 activation leads to an upregulation in the markers associated with ILC2. Here, we reported that GPR35 knockout mice exhibited a significantly reduced tumor growth and altered immune infiltration in tumors. Furthermore, activating GPR35 in different mouse models promoted tumor development by enhancing the production of IL-5 and IL-13, thereby facilitating the formation of the ILC2-MDSC axis. Moreover, we found that GPR35 was a poor prognostic factor in patients with lung adenocarcinoma. Together, our findings suggest the potential application of targeting GPR35 in cancer immunotherapy.

Serum sPD1 and sPDL1 as Biomarkers for Evaluating the Immune State of Lung Adenocarcinoma Patients.

A large proportion of cancer patients benefit from immune checkpoint therapy, while few studies focused on the relationship between soluble PD1 (sPD1) and soluble PDL1 (sPDL1) in serum and immune status of patients. ILC2 and M2 were confirmed to be related to immunosuppression in tumor patients. To determine whether sPD1 and sPDL1 are correlated with the ratio of ILC2 and M2 is helpful to explore the possibility of using sPD1 and sPDL1 as tumor molecular markers. Our results showed an immune balance toward ILC2 and M2-like monocytes in patients with LUAD compared with healthy controls. Meanwhile, decreased CD4+T and CD8+T cells, as well as elevated PD1+CD8+T cells, were found in patients with LUAD. The relative mRNA expression levels of ILC2- and M2-characteristic cytokines were also upregulated accompanied by decreased mRNA expression levels of ILC1- and M1-characteristic cytokines in patients with LUAD compared to healthy controls. Moreover, elevated ILC2 frequencies as well as the amount of IL-13 were positively correlated with the amount of sPD1, however, there was no correlation between them and sPDL1. These results suggested that sPD1 and sPDL1 can serve as diagnostic markers to predict the immune state of cancer patients.

Anti-ST2 Nanoparticle Alleviates Lung Inflammation by Targeting ILC2s-CD4+T Response.

BACKGROUND: Asthma has been regarded as an inflammatory disease, and group 2 innate lymphoid cells (ILC2s) are implicated in asthma pathogenesis. However, no strategy is available to block ILC2s function. Efficiency is also limited due to the use of systemic or subcutaneous routes of administration. The purpose of this study was to investigate the effects of nanoparticles targeting suppression of tumorigenicity 2 (ST2), which is the ILC2 receptor, to alleviate lung inflammation in the murine model of asthma. METHODS: The ultra-small SPIO nanoparticles were firstly synthesized, OVA-induced mice were administered by anti-ST2-conjugated nanoparticles. The inflammatory degree of the lung was investigated by H&E. The percentages of ILC2s and CD4+T cells in bronchoalveolar lavage fluid (BALF) and lung tissue were determined by FACS. Th2-cytokine and OVA-IgE levels were detected by real-time PCR and ELISA, respectively. RESULTS: Treatment with anti-ST2-conjugated nanoparticles significantly alleviated airway inflammation, IL-33 and IL-13 levels and the percentage of CD4+T cells. The percentage of ILC2s was increased, whereas the levels of IL-13 and IL-5 expressed by ILC2s were reduced. CONCLUSION: In the present study, we demonstrated that anti-ST2-conjugated nanoparticles can efficiently control lung inflammation in OVA-induced mice by reducing the ability of ILC2s to produce IL-5 and IL-13, thereby reducing CD4+T cells. Our study also demonstrated that the nanoparticle delivery system could improve the performance of anti-ST2, which may be used as a strategic tool to expand the current drug market.

HtrA1 upregulates the expression of ADAMTS-5 in HNPCs via the ERK/NF-κB/JNK signaling pathway.

Intervertebral disc degeneration (IDD) is a form of chronic inflammation and is one of the most common disorders reported to be involved in low back pain (LBP). The pathophysiology of degeneration is not completely understood, but the consensus is that the degradation of extracellular matrix (ECM) proteins in the disc is the leading factor contributing to IDD. High temperature requirement A1 (HtrA1) is serine protease that has been shown to be increased in degenerated intervertebral discs as a result of an increase in the expression of matrix metalloproteinases (MMPs), but no study has focused on the effect of HtrA1 on a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs). In the present study, we successfully isolated human nucleus pulposus cells (HNPCs) from IDD patients who were our research subjects to elaborate on the potential role of HtrA1 in the pathogenesis of IDD. We confirmed that HtrA1 has the potential to induce the expression of ADAMTS-5 in a dose-dependent manner. Consistently, this was mediated by the ERK, NF-κB and JNK pathways. By using inhibitors of these pathways, the increase in ADAMTS-5 could be reduced. Our findings indicated that HtrA1 can induce the expression of ADAMTS-5 in HNPCs via the ERK/NF-κB/JNK signaling pathway, and our study also elucidated the involved induction mechanisms in HNPCs, which may provide new insights for the treatment of IDD.

Cyclic Guanosine Monophosphate (cGMP)-Dependent Protein Kinase II Blocks Epidermal Growth Factor (EGF)/Epidermal Growth Factor Receptor (EGFR)-Induced Biological Effects on Osteosarcoma Cells.

BACKGROUND The present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells. MATERIAL AND METHODS We transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 µM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt. RESULTS Stimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP. CONCLUSIONS Our results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Obesity May Provide Pro-ILC3 Development Inflammatory Environment in Asthmatic Children.

The prevalence of obesity in children has dramatically increased in the last few decades, and obesity has also emerged as an important risk factor for asthma. Innate mechanisms have been shown to be involved in both diseases, particularly through the recently described innate lymphoid cells (ILCs), in which ILC3s have been linked to obesity both in human and in murine models. The aim of this study was to explore whether being overweight in asthmatic children was associated with elevated circulating ILC3 or elevated messenger RNA (mRNA) levels of RORC, IL-17A, and IL-22. Our results showed significantly elevated ILC3 frequencies in overweight asthmatic children compared with nonoverweight controls based on the detection of Lin+CD127+IL-23R+ cells by flow cytometry. Moreover, elevated ILC3 frequencies positively correlated with the mRNA expression of RORC which has been identified as a transcription factor of ILC3s. The relative mRNA expression level of IL-17A was also upregulated in overweight compared to nonoverweight children, as was the relative mRNA level of IL-22. However, there were no correlations between ILC3 frequencies or the expressions of RORC, IL-17A, and IL-22 and asthma severity. These results suggested that childhood obesity is an independent factor that is associated with an elevated frequency of circulating ILC3s and higher expressions of RORC, IL-22, and IL-17A.

HtrA1 up-regulates expression of MMPs via Erk1/2/Rock-dependent pathways.

BACKGROUND: There are few studies that have identified the potential role of a high temperature requirement A1 (HtrA1) in intervertebral disc degeneration (IDD). This study was undertaken to investigate the regulatory role of HtrA1 in the pathogenesis of IDD. MATERIAL AND METHODS: The mRNA levels of HtrA1 and matrix metalloproteinases (MMPs) of human intervertebral disc degeneration tissues were measured by real-time quantitative PCR, and a correlation between the expression level of HtrA1 and MMPs was also investigated. Human nucleus pulposus cells (HNPCs) were challenged with rHtrA1, and expression of MMPs was measured by real-time quantitative PCR, Western blotting, and ELISA. Moreover, to analyze the mechanism by which HtrA1 up-regulates MMPs, ERK1/2/ROCK signaling pathway inhibitors were also used. RESULTS: We found significant increases in mRNA expression of HtrA1 and MMP1, 3, 9, and 13 in IDD tissues compared with control. HtrA1 expression level was associated with the levels of MMP1, 3, and 13. Expression of MMP1, 3, and 13 mRNA and protein were significantly increased in HNPCs treated by rHtrA1. Moreover, administration of the ERK1/2 signaling pathway inhibitor or ROCK signaling pathway inhibitor decreased rHtrA1-induced MMPs production. Therefore, changes in HtrA1 expression could be involved in the pathogenesis of IDD. CONCLUSION: Our findings indicate that HtrA1 can induce increases in MMPs in HNPCs via the ERK1/2/ROCK signaling pathway, thus providing new insights into the role of HtrA1 in the pathogenesis of IDD.

Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging.

BACKGROUND Degrading enzymes play an important role in the process of disc degeneration. The objective of this study was to investigate the correlation between the expression of high temperature requirement serine protease A1 (HtrA1) in the nucleus pulposus and the T2 value of the nucleus pulposus region in magnetic resonance imaging (MRI). MATERIAL AND METHODS Thirty-six patients who had undergone surgical excision of the nucleus pulposus were examined by MRI before surgery. Pfirrmann grading of the target intervertebral disc was performed according to the sagittal T2-weighted imaging, and the T2 value of the target nucleus pulposus was measured according to the median sagittal T2 mapping. The correlation between the Pfirrmann grade and the T2 value was analyzed. The expression of HtrA1 in the nucleus pulposus was analyzed by RT-PCR and Western blot. The correlation between the expression of HtrA1 and the T2 value was analyzed. RESULTS The T2 value of the nucleus pulposus region was 33.11-167.91 ms, with an average of 86.64±38.73 ms. According to Spearman correlation analysis, there was a rank correlation between T2 value and Pfirrmann grade (P<0.0001), and the correlation coefficient (rs)=-0.93617. There was a linear correlation between the mRNA level of HtrA1 and T2 value in nucleus pulposus tissues (a=3.88, b=-0.019, F=112.63, P<0.0001), normalized regression coefficient=-0.88. There was a linear correlation between the expression level of HtrA1 protein and the T2 value in the nucleus pulposus tissues (a=3.30, b=-0.016, F=93.15, P<0.0001) and normalized regression coefficient=-0.86. CONCLUSIONS The expression of HtrA1 was strongly related to the T2 value, suggesting that HtrA1 plays an important role in the pathological process of intervertebral disc degeneration.

Correlation between the Expression of High Temperature Requirement Serine Protease A1 in Nucleus Pulposus Tissue and the Degree of Intervertebral Disc Degeneration.

Objective To investigate the correlation between the expression level of high temperature requirement serine protease A1 (HtrA1) in nucleus pulposus and the degree of intervertebral disc degeneration (Pfirrmann grade).Methods Thirty-six patients who underwent excision of nucleus pulposus were examined by magnetic resonance imaging (MRI) before operation,and the Pfirrmann grading of all patients was performed according to the sagittal T2 weighted MRI.The expression of HtrA1 in nucleus pulposus tissue was detected by reverse transcription polymerase chain reaction and Western blot.The correlation between the expression level of HtrA1 in nucleus pulposus tissue and Pfirrmann grade was analyzed.Results MRI in all 36 patients showed that there were 3 cases of Pfirrmann grade Ⅰ,10 cases of grade Ⅱ,11 cases of grade Ⅲ,7 cases of grade Ⅲ,and 5 cases of grade Ⅴ.The mRNA and protein expressions of HtrA1 in nucleus pulposus tissue increased with the increase of Pfirrmann grade.There were significant differences in the expression level of HtrA1 among different Pfirrmann grade groups (P<0.05) except for the difference between grade Ⅲ and Ⅴ (P>0.05).Spearman rank correlation analysis revealed that there was a rank correlation between expression level of HtrA1 and Pfirrmann grade (P<0.000 1).Conclusion The mRNA and protein expressions of HtrA1 in nucleus pulposus tissue increase with the increase of Pfirrmann grade,indicating HtrA1 is correlated with the degree of intervertebral disc degeneration.

Risk factors of recompression of cemented vertebrae after kyphoplasty for osteoporotic vertebral compression fractures.

PURPOSE: To evaluate the risk factors correlated with loss of cemented vertebral body height after kyphoplasty in patients with osteoporotic vertebral compression fractures. METHODS: Thirty-four consecutive patients with single-level osteoporotic vertebral compression fractures who underwent kyphoplasty in the Affiliated Hospital of Jiangsu University between January 2012 and August 2014 were retrospectively analysed. Eight independent variables (age, gender, body mass index, pre-operative T-score in bone mineral density, the volume of polymethylmethacrylate injected, pre-operatively vertebral body height, the restoration of body height and the distance between polymethylmethacrylate and endplate) were assessed. The recompression of body height was the dependent variable. Multivariate linear regression analyses were used to determine the factors associated with recompression of body height. RESULTS: Multiple linear regression analyses indicated that the recompression of cemented vertebral body height was correlated with the distance between polymethylmethacrylate and endplate (P = 0.008, b' = 0.489). The final multiple linear regression model, which included only the distance between polymethylmethacrylate and endplate, resulted in a formula that accounted for 41.02 % of the recompression of body height. CONCLUSIONS: The distance between polymethylmethacrylate and endplate is an important risk factor of recompression of cemented vertebrae after kyphoplasty for patients with osteoporotic vertebral compression fractures.