Background: IL-13 is a key T helper cell (Th2) cytokine acts as an important mediator in nasal epithelial remodeling such as goblet cell hyperplasia and loss or dysfunction of ciliated cell. IL-13 contributes to airway remodeling by altering differentiation of airway basal/progenitor cells fate away from ciliated cells toward to goblet cells. The Centriolar coiled coil protein 110 (Cp110) is a critical regulator of cilia formation and is well known to suppress ciliogenesis, but its supportive role in ciliogenesis is not well understood Methods: We investigated IL-13 induced cilia loss in human nasal epithelial cells (hNECs) differentiation process using a pseudo-stratified epithelium in air-liquid interface (ALI) culture to determine how IL-13 alters ciliogenesis Results: We have confirmed that IL-13 reduced ciliated cells with impaired ciliary function by reducing of ciliary beating frequency and increased proportion of goblet cells on hNECs differentiation process in ALI. Immunofluorescence image of untreated hNECs showed that CP110 was a dotted pattern located in the nucleus of the cells at early stages (Day 0, 3 and 7) of ALI. From 14 to 21 day, Cp110 was found uniformly distributed at the base of ciliated cells and beating cilia can be found at the same time with positive staining of Fork head Box J1 (Foxj1) and βIV-tubulin. However, after IL-13 treatment, nuclear Cp110 was apparent and cilia were not found in most areas of lower ciliated cell densities at late stage of hNECs differentiation (Day 14 and 21). The mRNA levels of Cp110 and Foxj1 were decreased in the presence of IL-13 Conclusions: IL-13 may interfere Cp110 localizing to cilia–forming basal bodies to inhibit ciliated cell differentiation
Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group.
ABSTRACT Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8 + cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (T H 1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional T H 1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8 + T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.
During influenza pneumonia, the alveolar epithelial cells of the lungs are targeted by the influenza virus. The distal airway stem cells (DASCs) and proliferating alveolar type II (AT2) cells are reported to be putative lung repair cells. However, their relative spatial and temporal distribution is still unknown during influenza-induced acute lung injury. Here, we investigated the distribution of these cells, and concurrently performed global proteomic analysis of the infected lungs to elucidate and link the cellular and molecular events during influenza pneumonia recovery. BALB/c mice were infected with a sub-lethal dose of influenza H1N1 virus. From 5 to 25 days post-infection (dpi), mouse lungs were subjected to histopathologic and immunofluorescence analysis to probe for global distribution of lung repair cells (using P63 and KRT5 markers for DASCs; SPC and PCNA markers for AT2 cells). At 7 and 15 dpi, infected mouse lungs were also subjected to protein mass spectrometry for relative protein quantification. DASCs appeared only in the damaged area of the lung from 7 dpi onwards, reaching a peak at 21 dpi, and persisted until 25 dpi. However, no differentiation of DASCs to AT2 cells was observed by 25 dpi. In contrast, AT2 cells began proliferating from 7 dpi to replenish their population, especially within the boundary area between damaged and undamaged areas of the infected lungs. Mass spectrometry and gene ontology analysis revealed prominent innate immune responses at 7 dpi, which shifted towards adaptive immune responses by 15 dpi. Hence, proliferating AT2 cells but not DASCs contribute to AT2 cell regeneration following transition from innate to adaptive immune responses during the early phase of recovery from influenza pneumonia up to 25 dpi.
The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.
Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NACHT, LRR, and PYD domains-containing protein 1 (NLRP1) is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131 This cleavage triggers N-glycine-mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and interleukin-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.
Upper airway inflammatory diseases are associated with abnormal expression of nasal epithelial forkhead-box J1 (FOXJ1) which regulates motile cilia formation. We sought to investigate whether aberrant FOXJ1 localizations correlate with the disease severity and the co-existence of allergic rhinitis (AR) or asthma in patients with nasal polyps (NPs).We elucidated localization patterns of FOXJ1 by performing immunofluorescence assays in nasal specimens and cytospin samples from controls and patients with NPs. We also assayed mRNA expression levels of FOXJ1 by using quantitative real-time polymerase chain reaction. Four localization patterns [normal (N), intermediate (I), mislocalization (M), and absence (A)] were defined. A semi-quantitative scoring system was applied for demonstrating FOXJ1 localization in five areas per paraffin section, with individual sections being scored between 0 and 2.FOXJ1 localization score was significantly higher in samples from NPs than in controls (P < 0.001). Elevated FOXJ1 localization scores and down-regulation of FOXJ1 mRNA levels were observed in NPs with co-existing AR or asthma (all P < 0.05). Moreover, FOXJ1 localization scores positively correlated with Lund-Mackay score (r = 0.362, P = 0.007). Of primary cytospin samples, the mean percentage of patients with FOXJ1 localization patterns N, I, M and A was 15.0%, 3.3%, 53.3% and 28.3% in NPs, and 82.5%, 5.0%, 5.0% and 7.5% in controls, respectively (P < 0.001).Aberrant localization of FOXJ1 correlates with the severity and co-existence of AR or asthma in patients with NPs, and might be a novel target for assessment and intervention in NPs.
Ratko Djukanović has consulted and presented at symposia organised by TEVA, Novartis, GlaxoSmithKline and AstraZeneca and has shares in and consults for Synairgen; Dr Asa Wheelock report remuneration from AstraZenica and Harvard Medical School for speaking engagements on SNF-clustering in COPD.; Charles Auffray reports grants from Innovative Medicine Initiative; Kian Fan Chung has received honoraria for participating in Advisory Board meetings of the pharmaceutical industry regarding treatments for asthma and chronic obstructive pulmonary disease and has also been remunerated for speaking engagements; Ian Adcock has received grants from Advisory Board meetings with pharmaceutical companies GSK, A-Z, Novartis, Boehringer Ingelheim and Vectura, and grants on asthma and COPD from Pfizer, GSK, MRC, EU, BI and IMI; Peter Sterk reports grants from IMI Innovative Medicines Initiative, during the conduct of the study; Matthew Loza and Frederic Baribaud are Employees and Shareholders of Janssen Research and Development, a Johnson and Johnson company; John Riley and Ana R Sousa are employees of GSK; the rest of the authors have nothing to disclose. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
In vitro and in vivo research based on cell lines and animals are likely to be insufficient in elucidating authentic biological and physiological phenomena mimicking human systems, especially for generating pre-clinical data on targets and biomarkers. There is an obvious need for a model that can further bridge the gap in translating pre-clinical findings into clinical applications. We have previously generated a model of in vitro differentiated human nasal epithelial cells (hNECs) which elucidated the nasal-initiated repertoire of immune responses against respiratory viruses such as influenza A virus and rhinovirus. To assess their clinical utility, we performed a microarray analysis of influenza virus-infected hNECs to elucidate nasal epithelial-initiated responses. This was followed by a metagenomic analysis which revealed transcriptomic changes comparable with clinical influenza datasets. The primary target of influenza infection was observed to be the initiator of innate and adaptive immune genes, leaning toward type-1 inflammatory activation. In addition, the model also elucidated a down-regulation of metabolic processes specific to the nasal epithelium, and not present in other models. Furthermore, the hNEC model detected all 11 gene signatures unique to influenza infection identified from a previous study, thus supporting the utility of nasal-based diagnosis in clinical settings. In conclusion, this study highlights that hNECs can serve as a model for nasal-based clinical translational studies and diagnosis to unravel nasal epithelial responses to influenza in the population, and as a means to identify novel molecular diagnostic markers of severity.
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