An integrated multi-omics analysis of identifies distinct molecular characteristics in pulmonary infections of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) can cause severe acute infections, including pneumonia and sepsis, and cause chronic infections, commonly in patients with structural respiratory diseases. However, the molecular and pathophysiological mechanisms of P. aeruginosa respiratory infection are largely unknown. Here, we performed assays for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptomics, and quantitative mass spectrometry-based proteomics and ubiquitin-proteomics in P. aeruginosa-infected lung tissues for multi-omics analysis, while ATAC-seq and transcriptomics were also examined in P. aeruginosa-infected mouse macrophages. To identify the pivotal factors that are involved in host immune defense, we integrated chromatin accessibility and gene expression to investigate molecular changes in P. aeruginosa-infected lung tissues combined with proteomics and ubiquitin-proteomics. Our multi-omics investigation discovered a significant concordance for innate immunological and inflammatory responses following P. aeruginosa infection between hosts and alveolar macrophages. Furthermore, we discovered that multi-omics changes in pioneer factors Stat1 and Stat3 play a crucial role in the immunological regulation of P. aeruginosa infection and that their downstream molecules (e.g., Fas) may be implicated in both immunosuppressive and inflammation-promoting processes. Taken together, these findings indicate that transcription factors and their downstream signaling molecules play a critical role in the mobilization and rebalancing of the host immune response against P. aeruginosa infection and may serve as potential targets for bacterial infections and inflammatory diseases, providing insights and resources for omics analyses.

circFANCA accelerates the malignant process of OSCC by modulating miR-34a/PA28γ signaling.

OBJECTIVES: To investigate the upstream regulatory molecules of proteasomal activator 28γ (PA28γ), and explore its specific regulatory mechanism and potential clinical significance in OSCC. MATERIALS AND METHODS: qPCR was used to examine miR-34a, circFANCA and PSME3 expression. Western blotting was adopted to detect PA28γ expression. Transwell experiments were conducted to evaluate OSCC cell migration and invasion ability. FISH was used to evaluate the subcellular localization of circFANCA and miR-34a, and RNA pull-down verified the interaction between them. The expression of circFANCA and miR-34a in clinical cohorts was assessed by ISH, and the results were subjected to survival analysis using Kaplan-Meier analysis. RESULTS: Here, we proved that miR-34a expression is lower in highly aggressive OSCC tissues and cell lines. Notably, miR-34a can downregulate PA28γ expression and inhibit OSCC invasion and migration. Next, we confirmed that circFANCA promoted OSCC cell metastatic ability by sponging miR-34a. Importantly, interfering with miR-34a rescued the malignant progression of OSCC induced by silencing circFANCA. Finally, clinical data showed lower miR-34a expression and higher circFANCA expression were associated with poor prognosis in OSCC patients. CONCLUSION: The circFANCA/miR-34a/PA28γ axis facilitates the metastasis of OSCC, and circFANCA and miR-34a have potential to serve as prognostic markers for OSCC patients.

Impact of single-cell RNA sequencing on understanding immune regulation.

Single-cell RNA sequencing (scRNA-seq), one of the most powerful technologies, can describe the transcriptomic heterogeneity of single cells and reveal previously unreported cell types or states in complex tissues. With the rapid development of scRNA-seq, it has renewed our view of cellular heterogeneity and its significance for deeply understanding cell development and function. There are a large number of studies applying scRNA-seq to investigate the heterogeneity of immune cells and disease pathogenesis, focusing on differences among every individual cell, which have provided novel inspiration for disease therapy and biological processes. In this review, we describe the development of scRNA-seq and its application in immune-related physiological states, regulatory mechanisms and diseases. In addition, we further discuss the opportunities and challenges of scRNA-seq in immune regulation.

Subinhibitory Cefotaxime and Levofloxacin Concentrations Contribute to Selection of Pseudomonas aeruginosa in Coculture with Staphylococcus aureus.

Bacterial species in the polymicrobial community evolve interspecific interaction relationships to adapt to the survival stresses imposed by neighbors or environmental cues. Pseudomonas aeruginosa and Staphylococcus aureus are two common bacterial pathogens frequently coisolated from patients with burns and respiratory disease. Whether the application of commonly used antibiotics influences the interaction dynamics of the two species still remains largely unexplored. By performing a series of on-plate competition assays and RNA sequencing-based transcriptional profiling, we showed that the presence of the cephalosporin antibiotic cefotaxime or the quinolone antibiotic levofloxacin at subinhibitory concentration contributes to selecting P. aeruginosa from the coculture with S. aureus by modulating the quorum-sensing (QS) system of P. aeruginosa. Specifically, a subinhibitory concentration of cefotaxime promotes the growth suppression of S. aureus by P. aeruginosa in coculture. This process may be related to the increased production of the antistaphylococcal molecule pyocyanin and the expression of lasR, which is the central regulatory gene of the P. aeruginosa QS hierarchy. On the other hand, subinhibitory concentrations of levofloxacin decrease the competitive advantage of P. aeruginosa over S. aureus by inhibiting the growth and the las QS system of P. aeruginosa. However, pqs signaling of P. aeruginosa can be activated instead to overcome S. aureus. Therefore, this study contributes to understanding the interaction dynamics of P. aeruginosa and S. aureus during antibiotic treatment and provides an important basis for studying the pathogenesis of polymicrobial infections. IMPORTANCE Increasing evidence has demonstrated the polymicrobial characteristics of most chronic infections, and the frequent communications among bacterial pathogens result in many difficulties for clinical therapy. Exploring bacterial interspecific interaction during antibiotic treatment is an emerging endeavor that may facilitate the understanding of polymicrobial infections and the optimization of clinical therapies. Here, we investigated the interaction of cocultured P. aeruginosa and S. aureus with the intervention of commonly used antibiotics in clinic. We found that the application of subinhibitory concentrations of cefotaxime and levofloxacin can select P. aeruginosa in coculture with S. aureus by modulating P. aeruginosa QS regulation to enhance the production of antistaphylococcal metabolites in different ways. This study emphasizes the role of the QS system in the interaction of P. aeruginosa with other bacterial species and provides an explanation for the persistence and enrichment of P. aeruginosa in patients after antibiotic treatment and a reference for further clinical therapy.

Updates on mechanistic insights and targeting of tumour metastasis.

Malignant tumours are one of the major diseases that seriously endanger human health. The characteristics of their invasion and metastasis are one of the main causes of death in cancer patients, and these features cannot be separated from the participation of various molecules-related cells living in the tumour microenvironment and specific structures. Tumour invasion can approximately be divided into several specific steps according to the movement of tumour cells. In each step, there are different actions in the tumour microenvironment that mediate the interactions among substances. Researchers are attempting to clarify every mechanism of the tumour dissemination. However, there is still a long way to the final determination. Here, we review these interactions in tumour invasion and metastasis at the structural, molecular and cellular levels. We also discuss the ongoing studies and the promise of targeting metastasis in tumour therapy.

Exo-circRNAs: a new paradigm for anticancer therapy.

CircRNAs, as new members of long noncoding RNAs, have been the focus of recent investigation. CircRNAs feature a closed continuous loop structure without 5'-3' polarity or a poly A tail. Many studies have reported the potential application of circRNAs in the clinic as new biomarkers and therapeutic targets in different diseases, especially for cancer. Additionally, the exosomes are important vehicles in cell-to-cell communication. And exo-circRNAs are circRNAs in exosomes which can be detected to provide additional evidence for conventional diagnostic methods and can be applied to suppress the malignant progress in cancer. In this review, we describe the biogenesis, characteristics, and functions of circRNAs and exosomes. Specifically, we present a comprehensive update of the promising role of exo-circRNAs in anticancer therapy.

DNA Repair Interacts with Autophagy To Regulate Inflammatory Responses to Pulmonary Hyperoxia.

Oxygen is supplied as a supportive treatment for patients suffering from acute respiratory distress syndrome. Unfortunately, high oxygen concentration increases reactive oxygen species generation, which causes DNA damage and ultimately cell death in the lung. Although 8-oxoguanine-DNA glycosylase (OGG-1) is involved in repairing hyperoxia-mediated DNA damage, the underlying molecular mechanism remains elusive. In this study, we report that ogg-1-deficient mice exhibited a significant increase of proinflammatory cytokines (TNF-α, IL-6, and IFN-γ) in the lung after being exposed to 95% oxygen. In addition, we found that ogg-1 deficiency downregulated (macro)autophagy when exposed to hyperoxia both in vitro and in vivo, which was evident by decreased conversion of LC3-I to LC3-II, reduced LC3 punctate staining, and lower Atg7 expression compared with controls. Using a chromatin immunoprecipitation assay, we found that OGG-1 associated with the promoter of Atg7, suggesting a role for OGG1 in regulation of Atg7 activity. Knocking down OGG-1 decreased the luciferase reporter activity of Atg7. Further, inflammatory cytokine levels in murine lung epithelial cell line cells were downregulated following autophagy induction by starvation and rapamycin treatment, and upregulated when autophagy was blocked using 3-methyladenine and chloroquine. atg7 knockout mice and Atg7 small interfering RNA-treated cells exhibited elevated levels of phospho-NF-κB and intensified inflammatory cytokines, suggesting that Atg7 impacts inflammatory responses to hyperoxia. These findings demonstrate that OGG-1 negatively regulates inflammatory cytokine release by coordinating molecular interaction with the autophagic pathway in hyperoxia-induced lung injury.

Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis.

Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.

Atg7 enhances host defense against infection via downregulation of superoxide but upregulation of nitric oxide.

Pseudomonas aeruginosa is an opportunistic bacterium that can cause serious infection in immunocompromised individuals. Although autophagy may augment immune responses against P. aeruginosa infection in macrophages, the critical components and their role of autophagy in host defense are largely unknown. In this study, we show that P. aeruginosa infection-induced autophagy activates JAK2/STAT1α and increases NO production. Knocking down Atg7 resulted in increased IFN-γ release, excessive reactive oxygen species, and increased Src homology-2 domain-containing phosphatase 2 activity, which led to lowered phosphorylation of JAK2/STAT1α and subdued expression of NO synthase 2 (NOS2). In addition, we demonstrated the physiological relevance of dysregulated NO under Atg7 deficiency as atg7(-/-) mice were more susceptible to P. aeruginosa infection with increased mortality and severe lung injury than wild-type mice. Furthermore, P. aeruginosa-infected atg7(-/-) mice exhibited increased oxidation but decreased bacterial clearance in the lung and other organs compared with wild-type mice. Mechanistically, atg7 deficiency suppressed NOS2 activity by downmodulating JAK2/STAT1α, leading to decreased NO both in vitro and in vivo. Taken together, these findings revealed that the JAK2/STAT1α/NOS2 dysfunction leads to dysregulated immune responses and worsened disease phenotypes.

Ammonia drives dendritic cells into dysfunction.

Ammonia levels are often elevated in patients with cirrhosis or tumors. Patients with these diseases are immunocompromised. In this study, we investigated the effects of ammonia on a member of the immune cell family, the dendritic cells (DCs). Our results demonstrated that ammonia diminished cell count, phagocytosis, and lymphocyte stimulation of DCs. Ammonia also induced DC swelling, excessive reactive oxygen species production, and mitochondrial damage, which may constitute the underlying mechanism of ammonia-induced DC dysfunction. In ammonium chloride (NH4Cl)-loaded mice, DCs exhibited lowered phagocytosis and a weakened immune response to the chicken OVA vaccine. DCs from patients with cirrhosis or ammonia-treated healthy human blood both exhibited diminished phagocytosis. Moreover, tumor cell conditioned medium drove DCs into dysfunction, which could be reversed by ammonia elimination. In a murine colon carcinoma model, we found that ammonia could regulate tumor growth involving DCs and their related immune response. These findings reveal that ammonia could drive DCs into dysfunction, which contributes to the immunocompromised state of patients with cirrhosis or tumors.

IL-37 ameliorates the inflammatory process in psoriasis by suppressing proinflammatory cytokine production.

IL-37 is a potent inhibitor of innate immunity by shifting the cytokine equilibrium away from excessive inflammation. Psoriasis is thought to be initiated by abnormal interactions between the cutaneous keratinocytes and systemic immune cells, triggering keratinocyte hyperproliferation. In the current study, we assessed IL-37 in two well-known psoriasis models: a human keratinocyte cell line (HaCaT) and the keratin 14 VEGF-A-transgenic mouse model. First, we used the HaCaT cell line, which was transiently transfected with an overexpressing IL-37 vector, and tested the effect of IL-37 on these cells using a mixture of five proinflammatory cytokines. IL-37 was effective in suppressing the production of CXCL8, IL-6, and S100A7, which were highly upregulated by the mixture of five proinflammatory cytokines. Keratin 14 VEGF-A-transgenic mice were treated with plasmid coding human IL-37 sequence-formulated cationic liposomes, and we observed potent immunosuppressive effects over the 18-d period. In this model, we observed reduced systemic IL-10 levels, local IFN-γ gene transcripts, as well as mild mast cell infiltration into the psoriatic lesions of the mice. Immunohistochemical analysis indicated that IL-37 was expressed by effector memory T cells, as well as macrophages, in human psoriatic plaques. In conclusion, our studies strongly indicate that IL-37 plays a potent immunosuppressive role in the pathogenesis of both experimental psoriasis models in vitro and in vivo by downregulating proinflammatory cytokines. Importantly, our findings highlight new therapeutic strategies that can be designed to use this immunosuppressive anti-inflammatory cytokine in psoriasis and other inflammatory cutaneous diseases.

Inhibition of p-IκBα Ubiquitylation by Autophagy-Related Gene 7 to Regulate Inflammatory Responses to Bacterial Infection.

BACKGROUND: Klebsiella pneumoniae causes serious infections and healthcare burdens in humans. We have previously reported that the deficiency of autophagy-related gene (Atg) 7 in macrophages (murine alveolar macrophage cell line [MH-S]) induced irregular host immunity against K. pneumoniae and worsened pathologic effects in the lung. In the current study, we investigated the molecular mechanism by which Atg7 influenced K. pneumoniae-induced inflammatory responses. METHODS: Expression levels of Atg7, ubiquitin (Ub), and tumor necrosis factor (TNF) α and phosphorylation of IκBα (p-IκBα) were determined with immunoblotting. Ubiquitylation of p-IκBα was determined with immunoprecipitation. RESULTS: We noted an interaction between Atg7 and p-IκBα, which was decreased in MH-S after K. pneumoniae infection, whereas the interaction between Ub and p-IκBα was increased. Knock-down of Atg7 with small interfering RNA increased p-IκBα ubiquitylation, promoted nuclear factor κB translocation into the nucleus, and increased the production of TNF-α. Moreover, knock-down of Ub with lentivirus-short hairpin RNA Ub particles decreased binding of p-IκBα to Ub and inhibited TNF-α expression in the primary alveolar macrophages and lung tissue of atg7-knockout mice on K. pneumoniae infection. CONCLUSIONS: Loss of Atg7 switched binding of p-IκBα from Atg7 to Ub, resulting in increased ubiquitylation of p-IκBα and intensified inflammatory responses against K. pneumoniae. Our findings not only reveal a regulatory role of Atg7 in ubiquitylation of p-IκBα but also indicate potential therapeutic targets for K. pneumoniae control.

Lyn regulates inflammatory responses in Klebsiella pneumoniae infection via the p38/NF-κB pathway.

Klebsiella pneumoniae (Kp) is one of the most common pathogens in nosocomial infections and is becoming increasingly multidrug resistant. However, the underlying molecular pathogenesis of this bacterium remains elusive, limiting the therapeutic options. Understanding the mechanism of its pathogenesis may facilitate the development of anti-bacterial therapeutics. Here, we show that Lyn, a pleiotropic Src tyrosine kinase, is involved in host defense against Kp by regulating phagocytosis process and simultaneously downregulating inflammatory responses. Using acute infection mouse models, we observed that lyn(-/-) mice were more susceptible to Kp with increased mortality and severe lung injury compared with WT mice. Kp infected-lyn(-/-) mice exhibited elevated inflammatory cytokines (IL-6 and TNF-α), and increased superoxide in the lung and other organs. In addition, the phosphorylation of p38 and NF-κB p65 subunit increased markedly in response to Kp infection in lyn(-/-) mice. We also demonstrated that the translocation of p65 from cytoplasm to nuclei increased in cultured murine lung epithelial cells by Lyn siRNA knockdown. Furthermore, lipid rafts clustered with activated Lyn and accumulated in the site of Kp invasion. Taken together, these findings revealed that Lyn may participate in host defense against Kp infection through the negative modulation of inflammatory cytokines.

Atg7 deficiency impairs host defense against Klebsiella pneumoniae by impacting bacterial clearance, survival and inflammatory responses in mice.

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium that can cause serious infections in humans. Autophagy-related gene 7 (Atg7) has been implicated in certain bacterial infections; however, the role of Atg7 in macrophage-mediated immunity against Kp infection has not been elucidated. Here we showed that Atg7 expression was significantly increased in murine alveolar macrophages (MH-S) upon Kp infection, indicating that Atg7 participated in host defense. Knocking down Atg7 with small-interfering RNA increased bacterial burdens in MH-S cells. Using cell biology assays and whole animal imaging analysis, we found that compared with wild-type mice atg7 knockout (KO) mice exhibited increased susceptibility to Kp infection, with decreased survival rates, decreased bacterial clearance, and intensified lung injury. Moreover, Kp infection induced excessive proinflammatory cytokines and superoxide in the lung of atg7 KO mice. Similarly, silencing Atg7 in MH-S cells markedly increased expression levels of proinflammatory cytokines. Collectively, these findings reveal that Atg7 offers critical resistance to Kp infection by modulating both systemic and local production of proinflammatory cytokines.

FIP200 is involved in murine pseudomonas infection by regulating HMGB1 intracellular translocation.

BACKGROUND: FIP200, a critical autophagy initiating protein, can participate in numerous cellular functions including cancer development; however, its functional role in P. aeruginosa infection of alveolar macrophages is unknown. METHODS: To investigate the role of FIP200 in host defense, we transfected murine alveolar macrophage MH-S cells with FIP200 siRNA. Having confirmed that FIP200 knockdown inhibited PAO1-induced autophagosme formation, we sought to characterize the underlying signaling pathways by immunoblotting. Further, we used fip200 KO mice to study the effects of fip200 deficiency on HMGB1 translocation. RESULTS: We showed that Pseudomonas PAO1 strain infection facilitated autophagosome formation, whereas knockdown of FIP200 inhibited autophagosome formation and HMGB1 expression in MH-S cells. Silencing FIP200 impaired the translocation of HMGB1 to cytosol of MH-S cells and almost abolished acetylation of HMGB1 during PAO1 infection. In contrast, FIP200 overexpression facilitated the cytosol translocation of HMGB1 from nuclei and increased acetylation of HMGB1 in PAO1-infected MH-S cells. Importantly, expression and acetylation of HMGB1 were also significantly down-regulated in fip200 KO mice following PAO1 infection. CONCLUSIONS: Collectively, these findings elucidate that FIP200 may regulate expression and translocation of HMGB1 during PAO1 infection, which may indicate novel therapeutic targets to control pulmonary infection.

Lung microbiome: new insights into the pathogenesis of respiratory diseases.

The lungs were long thought to be sterile until technical advances uncovered the presence of the lung microbial community. The microbiome of healthy lungs is mainly derived from the upper respiratory tract (URT) microbiome but also has its own characteristic flora. The selection mechanisms in the lung, including clearance by coughing, pulmonary macrophages, the oscillation of respiratory cilia, and bacterial inhibition by alveolar surfactant, keep the microbiome transient and mobile, which is different from the microbiome in other organs. The pulmonary bacteriome has been intensively studied recently, but relatively little research has focused on the mycobiome and virome. This up-to-date review retrospectively summarizes the lung microbiome's history, composition, and function. We focus on the interaction of the lung microbiome with the oropharynx and gut microbiome and emphasize the role it plays in the innate and adaptive immune responses. More importantly, we focus on multiple respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), fibrosis, bronchiectasis, and pneumonia. The impact of the lung microbiome on coronavirus disease 2019 (COVID-19) and lung cancer has also been comprehensively studied. Furthermore, by summarizing the therapeutic potential of the lung microbiome in lung diseases and examining the shortcomings of the field, we propose an outlook of the direction of lung microbiome research.

m6A-dependent mature miR-151-5p accelerates the malignant process of HNSCC by targeting LYPD3.

miRNA has emerged as a crucial regulator in various of pathological and physiological processes, yet its precise mechanism of action the detailed mechanism of their action in Head and neck squamous cell carcinoma (HNSCC) remains incompletely understood. This study sheds light on the role of mi-151-5p, revealing its significantly elevated expression in tumor cells, which notably enhances the invasion and migration of HNSCC cells. This effect is achieved through directly targeting LY6/PLAUR Domain Containing 3 (LYPD3) by miR-151-5p, involving complementary binding to the 3'-untranslated regions (3'-UTR) in the mRNA of LYPD3. Consequently, this interaction accelerates the metastasis of HNSCC. Notably, clinical observations indicate a correlation between high expression of miR-151-5p and low levels of LYPD3 in clinical settings are correlated with poor prognosis of HNSCC patients. Furthermore, our investigation demonstrates that glycosylation of LYPD3 modulates its subcellular localization and reinforces its role in suppressing HNSCC metastasis. Additionally, we uncover a potential regulatory mechanism involving the facilitation of miR-151-5p maturation and accumulation through N6-methyladenosine (m6A) modification. This process is orchestrated by methyltransferase-like 3 (METTL3) and mediated by a newly identified reader, heterogeneous nuclear ribonucleoprotein U (hnRNP U). These findings collectively underscore the significance of the METTL3/miR-151-5p/LYPD3 axis serves as a prominent driver in the malignant progression of HNSCC.

PA28γ induces dendritic cell maturation and activates T-cell immune responses in oral lichen planus.

Oral lichen planus (OLP) is a common chronic inflammatory disease of the oral mucosa, the mechanism of its inflammatory progression has not yet been fully elucidated. PA28γ plays a significant role in a variety of immune-related diseases. However, the exact role of PA28γ in the pathogenesis of OLP remains unclear. Here, we demonstrated that PA28γ is overexpressed in epithelial cells and inflammatory cells of OLP tissues but has no significant relationship with OLP subtypes. Functionally, keratinocytes with high PA28γ expression could induce dendritic cell (DC) maturation and promote the T-cell differentiation into Th1 cells in response to the immune response. In addition, we found that a high level of PA28γ expression is associated with high numbers of infiltrating mature DCs and activated T-cells in OLP tissues. Mechanistically, keratinocytes with high PA28γ expression could promote the secretion of C-C motif chemokine (CCL)5, blocking CCL5 or/and its receptor CD44 could inhibit the induction of T-cell differentiation by keratinocytes with high PA28γ expression. In conclusion, we reveal that keratinocytes with high expression of PA28γ in OLP can induce DC maturation and promote T-cell differentiation through the CCL5-CD44 pathway, providing previously unidentified mechanistic insights into the mechanism of inflammatory progression in OLP.