Genome editing of PAR2 through targeted delivery of CRISPR-Cas9 system for alleviating acute lung inflammation via ERK/NLRP3/IL-1β and NO/iNOS signalling

Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases,immune cell infiltration and protease-driven tissue damages.It is an urgent need to explore potential drug strategies for mitigating lung inflammation.Protease-activated receptor 2(PAR2)as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction.However,it has been rarely reported about the role of PAR2 in lung inflammation.This study applied CRISPR-Cas9 system encoding Cas9 and sg RNA(p Cas9-PAR2)for PAR2 knockout and fabricated an anionic human serum albuminbased nanoparticles to deliver p Cas9-PAR2 with superior inflammation-targeting efficiency and stability(TAP/p Cas9-PAR2).TAP/p Cas9-PAR2 robustly facilitated p Cas9-PAR2 to enter and transfect inflammatory cells,eliciting precise gene editing of PAR2 in vitro and in vivo.Importantly,PAR2 deficiency by TAP/p Cas9-PAR2 effectively and safely promoted macrophage polarization,suppressed proinflammatory cytokine releases and alleviated acute lung inflammation,uncovering a novel value of PAR2.It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/p Cas9-PAR2was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/i NOS signalling.Therefore,this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.

Regulatory factors of ILC2 are therapeutic targets for lung inflammation

Type 2 innate lymphoid cells(ILC2s)are an important class of innate immune cells that play a key role in regulating immune responses,maintaining tissue homeostasis,and participating in immune responses induced by inflammatory diseases.In lung inflammation,ILC2s drive the inflammatory response by secreting type 2 cytokines,and have a significant role in tissue repair and the maintenance of barrier function by secreting IL‐9 and antimicrobial peptides.ILC2s activation and function are affected by various regulatory factors,including epithelial‐derived alarmins such as IL‐25,IL‐33,and thymic stromal lymphopoietin,neurotransmitters,metabolites and hormones.These regulatory factors affect the development and activation of ILC2s through signaling pathways under different pathological conditions.An in‐depth study of regulatory factors is expected to provide new targets and strategies for the treatment of lung inflammation.

HTR2A agonists play a therapeutic role by restricting ILC2 activation in papain-induced lung inflammation

Group 2 innate lymphoid cells(ILC2s)are a category of heterogeneous cells that produce the cytokines IL-5 and IL-13,which mediate the type 2 immune response.However,specific drug targets on lung ILC2s have rarely been reported.Previous studies have shown that type 2 cytokines,such as IL-5 and IL-13,are related to depression.Here,we demonstrated the negative correlation between the depression-associated monoamine neurotransmitter serotonin and secretion of the cytokines IL-5 and IL-13 by ILC2s in individuals with depression.Interestingly,serotonin ameliorates papain-induced lung inflammation by suppressing ILC2 activation.Our data showed that the serotonin receptor HTR2A was highly expressed on ILC2s from mouse lungs and human PBMCs.Furthermore,an HTR2A selective agonist(DOI)impaired ILC2 activation and alleviated the type 2 immune response in vivo and in vitro.Mice with ILC2-specific depletion of HTR2A(Il5^(cre/+)·Htr2a^(flox/flox)mice)abolished the DOI-mediated inhibition of ILC2s in a papain-induced mouse model of inflammation.In conclusion,serotonin and DOI could restrict the type 2 lung immune response,indicating a potential treatment strategy for type 2 lung inflammation by targeting HTR2A on ST2+ILC2s.

Increasing stiffness promotes pulmonary retention of ligand-directed dexamethasone-loaded nanoparticle for enhanced acute lung inflammation therapy

Inhaled nanoparticles(NPs)need to penetrate the bronchial mucosa to deliver drug payloads deeply in the lung for amplified local therapy.However,the bronchial mucociliary barrier eliminates NPs rapidly,which considerably limits their mucosal penetration.In this study,we find that surface ligand modification and stiffness adjustment of NPs contribute to the significantly enhanced bronchial mucosal absorption and pulmonary retention of inhaled drugs.We utilize neonatal Fc receptor ligand(FcBP)to modify the rationally designed low stiffness NPs(Soft-NP)and high stiffness NPs(Stiff-NP)to target bronchial mucosa.In an acute lung inflammation rat model,after intranasal administration with dexamethasone-loaded NPs,Stiff-NP endowed with FcBP displays superior therapeutic effects.The in vitro data demonstrate that the promotion effect of FcBP to bronchial mucosal absorption of Stiff-NP dominates over Soft-NP.This could be attributed to the higher affinity between ligand-receptor when incorporating FcBP on the Stiff-NP surface.Meanwhile,high stiffness modulates more actin filaments aggregation to mediate endocytosis,along with strengthened Ca2+signal to enhance exocytosis.Conclusively,we highlight that FcBP-modified NPs with higher stiffness would be a potential pulmonary drug delivery system.

TRIM65 E3 ligase targets VCAM-1 degradation to limit LPS-induced lung inflammation

Although the adhesion molecules-mediated leukocyte adherence and infiltration into tissues is an important step of inflammation,the post-translational regulation of these proteins on the endothelial cells is poorly understood.Here,we report that TRIM65,an ubiquitin E3 iigase of tripartite protein family,selectively targets vascular cell adhesion molecule 1(VCAM-1)and promotes its ubiq-uitination and degradation,by which it critically controls the duration and magnitude of sepsis-induced pulmonary inflammation.TRIM65 is constitutively expressed in human vascular endothelial cells.During TNFa-induced endothelial activation,the protein levels ofTRIM65 and VCAM-1 are inversely correlated.Expression of wild-type TRIM65,but not expression of aTRIM65 mutant that lacks E3 ubiquitin ligase function in endothelial cells,promotes VCAM-1 ubiquitination and degradation,whereas small interference RNA-mediated knockdown of TRIM65 attenuates VCAM-1 protein degradation.Further experiments show that TRIM65 directly interacts with VCAM-1 protein and directs its polyubiquitination,by which TRIM65 controls monocyte adherence and infiltration into tissues during inflammation.Importantly,TRIM65-deficient mice are more sensitive to lipopolysaccharide-induced death,due to sustained and severe pulmonary inflammation.Taken together,our studies suggest that TRIM65-mediated degradation of VCAM-1 represents a potential mechanism that controls the duration and magnitude of infiammation.

The prognostic significance of ALI,PLR,and Ki-67 expression in stageⅢ–Ⅳinoperable non-small cell lung cancer

Objective The aim of the study was to investigate and compare the prognostic value of advanced inflammatory index,platelet/lymphocyte ratio(PLR),and Ki-67 expression in stageⅢ–Ⅳinoperable non-small cell lung cancer(NSCLC)before treatment.Methods The clinical data of 98 inoperable patients with stageⅢ–ⅣNSCLC in our hospital(Fifth Department of Oncology,Hebei General Hospital,Shijiazhuang,China)before treatment were retrospectively analyzed,and advanced lung cancer inflammation index(ALI)was calculated using body mass index(BMI)×serum albumin(ALB)÷neutrophil/lymphocyte ratio(NLR).he optimal cutoff values of ALI and PLR for predicting prognosis is determined.Chi-square test was used to analyze the relationship between patients and clinical characteristics.Kaplan-Meier method was used to calculate the total survival of patients,and log-rank test was used for comparison.Independent prognostic factors were assessed by univariate and multivariate analyses.Spearman correlation was used to analyze the relationship among ALI,PLR,and Ki-67.Results In our study of the 98 cases,the survival time of the patients with ALI<18 was significantly lower than that of patients with ALI>18(P<0.001),with a median survival time of 10 months and 25 months,respectively.The survival time of patients with a PLR<185 was significantly higher than that of patients with a PLR>185(median survival time was 27 months vs.10 months,P<0.001).The higher the Ki-67 expression,the shorter the survival time(P<0.005).The combined ALI and PLR detection results indicated that the survival time of patients with high ALI and low PLR was significantly longer than that of patients with low ALI and high PLR(P<0.001).Univariate analysis showed that smoking history,degree of differentiation,KPS score,Ki-67 expression,ALI value,and PLR affected the prognosis of patients.Multivariate analysis showed that KPS score,ALI value,and Ki-67 expression were independent prognostic factors.Conclusion ALI,PLR,and Ki-67 expression are important predictors of stage III-IV inoperable NSCLC.In terms of the prognostic value,ALI seems to have the best ability to predict patient survival.In addition,the combined detection of ALI and PLR levels before treatment seems to be more helpful in improving our prediction of patient prognosis.Moreover,it is expected to play a role in future clinical applications.

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1^(-/-) mice infected with influenza virus(H1N1)

Respirovirus infection can cause viral pneumonia and acute lung injury （ALl）. The interleukin-1 （IL-1） family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo. IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation. We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 （IL-1R1） deficient mice. Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6, TNF-α, G-CSF, KC, and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1^-/- mice in comparison with that of wild type infected mice. The adaptive immune response against the H1N1 virus in IL-1 R1^-/- mice was impaired with downregulated anti-viral Thl cell, CD8＋ cell, and antibody functions, which contributes to attenuated viral clearance. Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1^-/- mice compared with that in WT infected mice. Moreover, the infected IL-1R1^--/ mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung. Together, these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury, particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.

Pandemic and modern medicine:Time to recognize and correct previous misconceptions

A sudden increase in the number of viral pneumonias with the onset of the COVID-19 pandemic revealed defects in the provision of medical care for acute pneumonia in the lungs.Localization of the inflammatory process and its functional consequences indicate the identity of all forms of acute pneumonia,regardless of the etiology,and the need to develop treatment principles based on the pathogenesis of the disease.

Tectorigenin inhibits the inflammation of LPS-induced acute lung injury in mice

AIM: In a previous study, the anti-inflammatory effects of tectorigenin were disclosed. In this study, the anti-inflammatory effects of tectorigenin on acute lung injury using a lipopolysaccharide(LPS)-induced acute lung injury(ALI) mouse model were investigated. METHOD: The cell-count in the bronchoalveolar lavage fluid(BALF) was measured. The animal lung edema degree was evaluated by the wet/dry weight(W/D) ratio. The superoxidase dismutase(SOD) activity and myeloperoxidase(MPO) activity was assayed using SOD and MPO kits, respectively. The levels of inflammatory mediators, including tumor necrosis factor-α(TNF-α), IL-1β, and IL-6 were assayed using an enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed through HE staining. The inflammatory signal pathway related protein nuclear factor NF-κB p65 mR NA expression was measured by real-time PCR, and the protein level of NF-κB p65 was measured using Western blotting analysis. RESULTS: The data showed that treatment with the tectorigenin markedly attenuated the inflammatory cell numbers in the BALF, decreased nuclear factor NF-κB p65 mR NA level and protein level in the lungs, and improved SOD activity and inhibited MPO activity. Histological studies showed that tectorigenin substantially inhibited LPS-induced neutrophils in lung tissue compared with the model group. CONCLUSION: The results indicated that tectorigenin had a protective effect on LPS-induced ALI in mice.

NLRP3 inflammasome activation determines the fibrogenic potential of PM_(2.5) air pollution particles in the lung

Airborne fine particulate matter(PM_(2.5))is known to cause respiratory inflammation such as chronic obstructive pulmonary disease and lung fibrosis.NLRP3 inflammasome activation has been implicated in these diseases;however,due to the complexity in PM_(2.5)compositions,it is difficult to differentiate the roles of the components in triggering this pathway.We collected eight real-life PM_(2.5)samples for a comparative analysis of their effects on NLRP3 inflammasome activation and lung fibrosis.In vitro assays showed that although the PM_(2.5)particles did not induce significant cytotoxicity at the dose range of 12.5to 100μg/m L,they induced potent TNF-αand IL-1βproduction in PMA differentiated THP-1 human macrophages and TGF-β1 production in BEAS-2B human bronchial epithelial cells.At the dose of 100μg/m L,PM_(2.5)induced NLRP3 inflammasome activation by inducing lysosomal damage and cathepsin B release,leading to IL-1βproduction.This was confirmed by using NLRP3-and ASC-deficient cells as well as a cathepsin B inhibitor,ca-074 ME.Administration of PM_(2.5)via oropharyngeal aspiration at 2 mg/kg induced significant TGF-β1 production in the bronchoalveolar lavage fluid and collagen deposition in the lung at 21days post-exposure,suggesting PM_(2.5)has the potential to induce pulmonary fibrosis.The ranking of in vitro IL-1βproduction correlates well with the in vivo total cell count,TGF-β1 production,and collagen deposition.In summary,we demonstrate that the PM_(2.5)is capable of inducing NLRP3 inflammasome activation,which triggers a series of cellular responses in the lung to induce fibrosis.

Identification of two migratory colon ILC2 populations differentially expressing IL-17A and IL-5/IL-13

Group 2 innate lymphoid cells(ILC2s)play important tissue resident roles in anti-parasite immunity,allergic immune response,tissue homeostasis,and tumor immunity.ILC2s are considered tissue resident cells with little proliferation at steady state.Recent studies have shown that a subset of small intestinal ILC2s could leave their residing tissues,circulate and migrate to different organs,including lung,liver,mesenteric LN and spleen,upon activation.However,it remains unknown whether other ILC populations with migratory behavior exist.In this study,we find two major colon ILC2 populations with potential to migrate to the lung in response to IL-25 stimulation.One subset expresses IL-17A and resembles inflammatory ILC2s(iILC2s)but lacks CD27 expression,whereas the other expresses CD27 but not IL-17A.In addition,the IL-17A^(+)ILC2s express lower levels of CD127,CD25,and ST2 than CD27^(+)ILC2s,which express higher levels of IL-5 and IL-13.Surprisingly,we found that both colon ILC2 populations still maintained their colonic features of preferential expression of IL-17A and CD27,IL-5/IL-13,respectively.Together,our study identifies two migratory colon ILC2 subsets with unique surface markers and cytokine profiles which are critical in regulating lung and colon immunity and homeostasis.

SOCS3 dictates the transition of divergent time-phased events in granulocyte TNF-a signaling

Tumor-necrosis factor-a （TNF-a）-driven nuclear factor-KB （NF-KB） activation and apoptosis are opposing pathways; the growing recognition of these conflicting roles of TNF-a is perplexing. Here, we show that inflammation and apoptosis are time-phased events following TNF-a signaling and that emergence of suppressor of cytokine signaling 3 （SOCS3） expression limits the ongoing NF-KB activation and promotes apoptosis; further, we suggest an altered view of how inflammatory diseases are initiated and sustained. In vitro, TNF-a （50 ng/ml） induced granulocyte SOCS3 protein, inhibited nuclear accumulation of the p65NF-KB subunit and enhanced apoptosis, as shown by DNA laddering, annexin V positivity, and overexpression of caspase-3 and Bax in the late phase, whereas the early phase was marked by NF-KB activation. Conversely, SOCS3 knockdown by small interfering RNA （siRNA） inhibited granulocyte apoptosis and enhanced nuclear accumulation of p65 and 5＇ lipooxygenase expression in the late phase of TNF-a signaling. As apoptosis is associated with SOCS3 abundance, we suggest that these divergent TNF-a-driven events are time-phased, interconnected, opposing control mechanisms and one of the central features through which the immune system resolves pulmonary inflammation. Dysregulation may initiate mucosal inflammation, thus changing the landscape of asthma theraov.