TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma, and respiratory reactions to COX-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody-secreting cells (ASCs), including plasma cells and plasmablasts, in their polyp tissue; in addition, their nasal polyp (NP) IgE levels are correlated with disease severity and recurrence of nasal polyposis. OBJECTIVE: We sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of NP ASCs from patients with AERD and CRSwNP. METHODS: NP tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. NP cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with or without IL-5 and analyzed by bulk RNA sequencing. RESULTS: Compared with polyp tissue from patients with CRSwNP, polyp tissue from patients with AERD contained significantly more ASCs and had increased ASC expression of IL-5Rα. ASCs from patients with AERD expressed higher protein levels of B-cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. ASCs from patients with AERD also expressed more IL5RA, IGHE, and cell cycle- and proliferation-related transcripts (CCND2, MKI67, CDC25A, and CDC25B) than did ASCs from patients with CRSwNP. Stimulation of plasma cells from patients with AERD with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from patients with CRSwNP induced few transcriptomic changes. CONCLUSION: NP tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation than do ASCs from patients with aspirin-tolerant CRSwNP.

Type 2 inflammation drives an airway basal stem cell program through insulin receptor substrate signaling.

BACKGROUND: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a type 2 (T2) inflammatory disease associated with an increased number of airway basal cells (BCs). Recent studies have identified transcriptionally distinct BCs, but the molecular pathways that support or inhibit human BC proliferation and differentiation are largely unknown. OBJECTIVE: We sought to determine the role of T2 cytokines in regulating airway BCs. METHODS: Single-cell and bulk RNA sequencing of sinus and lung airway epithelial cells was analyzed. Human sinus BCs were stimulated with IL-4 and IL-13 in the presence and absence of inhibitors of IL-4R signaling. Confocal analysis of human sinus tissue and murine airway was performed. Murine BC subsets were sorted for RNA sequencing and functional assays. Fate labeling was performed in a murine model of tracheal injury and regeneration. RESULTS: Two subsets of BCs were found in human and murine respiratory mucosa distinguished by the expression of basal cell adhesion molecule (BCAM). BCAM expression identifies airway stem cells among P63+KRT5+NGFR+ BCs. In the sinonasal mucosa, BCAMhi BCs expressing TSLP, IL33, CCL26, and the canonical BC transcription factor TP63 are increased in patients with CRSwNP. In cultured BCs, IL-4/IL-13 increases the expression of BCAM and TP63 through an insulin receptor substrate-dependent signaling pathway that is increased in CRSwNP. CONCLUSIONS: These findings establish BCAM as a marker of airway stem cells among the BC pool and demonstrate that airway epithelial remodeling in T2 inflammation extends beyond goblet cell metaplasia to the support of a BC stem state poised to perpetuate inflammation.

Reconstituting Spore Cortex Peptidoglycan Biosynthesis Reveals a Deacetylase That Catalyzes Transamidation.

Some bacteria survive in nutrient-poor environments and resist killing by antimicrobials by forming spores. The cortex layer of the peptidoglycan cell wall that surrounds mature spores contains a unique modification, muramic-δ-lactam, that is essential for spore germination and outgrowth. Two proteins, the amidase CwlD and the deacetylase PdaA, are required for muramic-δ-lactam synthesis in cells, but their combined ability to generate muramic-δ-lactam has not been directly demonstrated. Here we report an in vitro reconstitution of cortex peptidoglycan biosynthesis, and we show that CwlD and PdaA together are sufficient for muramic-δ-lactam formation. Our method enables characterization of the individual reaction steps, and we show for the first time that PdaA has transamidase activity, catalyzing both the deacetylation of N-acetylmuramic acid and cyclization of the product to form muramic-δ-lactam. This activity is unique among peptidoglycan deacetylases and is notable because it may involve the direct ligation of a carboxylic acid with a primary amine. Our reconstitution products are nearly identical to the cortex peptidoglycan found in spores, and we expect that they will be useful substrates for future studies of enzymes that act on the spore cortex.

Optimizing cryopreservation of nasal polyp tissue for cellular functional studies and single-cell RNA sequencing.

KEY POINTS: Mast cell numbers were reduced in samples cryopreserved as whole tissue chunks. Thawed epithelial cells had reduced proliferation rates when preserved as dissociated cell suspensions. The right cryopreservation method to choose may depend on the goals and cell-type focus of the project.

Increased epithelial mTORC1 activity in chronic rhinosinusitis with nasal polyps.

Background: The airway epithelium plays a central role in the pathogenesis of chronic respiratory diseases such as asthma and chronic rhinosinusitis with nasal polyps (CRSwNP), but the mechanisms by which airway epithelial cells (EpCs) maintain inflammation are poorly understood. Objective: We hypothesized that transcriptomic assessment of sorted airway EpCs across the spectrum of differentiation would allow us to define mechanisms by which EpCs perpetuate airway inflammation. Methods: Ethmoid sinus EpCs from adult patients with CRS were sorted into 3 subsets, bulk RNA sequenced, and analyzed for differentially expressed genes and pathways. Single cell RNA-seq (scRNA-seq) datasets from eosinophilic and non-eosinophilic CRSwNP and bulk RNA-seq of EpCs from mild/moderate and severe asthma were assessed. Immunofluorescent staining and ex vivo functional analysis of sinus EpCs were used to validate our findings. Results: Analysis within and across purified EpC subsets revealed an enrichment in glycolytic programming in CRSwNP vs CRSsNP. Correlation analysis identified mammalian target of rapamycin complex 1 (mTORC1) as a potential regulator of the glycolytic program and identified EpC expression of cytokines and wound healing genes as potential sequelae. mTORC1 activity was upregulated in CRSwNP, and ex vivo inhibition demonstrated that mTOR is critical for EpC generation of CXCL8, IL-33, and CXCL2. Across patient samples, the degree of glycolytic activity was associated with T2 inflammation in CRSwNP, and with both T2 and non-T2 inflammation in severe asthma. Conclusions: Together, these findings highlight a metabolic axis required to support epithelial generation of cytokines critical to both chronic T2 and non-T2 inflammation in CRSwNP and asthma.

Augmented Reality Microscopy in the Management of Cerebellopontine Lesions and Microvascular Decompression: A Pilot Study.

Objective: To evaluate whether augmented reality microscopy surgical fluorescence technology, already Food and Drug Administration approved for vascular neurosurgery, can aid in lateral skull base surgery during cerebellopontine (CPA) tumor resection and microvascular decompression. Study Design: Pilot prospective uncontrolled observational cohort study. Setting: An academic tertiary care hospital. Patients: Those who underwent retrosigmoid craniotomy for CPA tumor resection or microvascular decompression for hemifacial spasm, trigeminal neuralgia or pulsatile tinnitus. 11 patients were recruited: 4 underwent CPA tumor resection and 7 underwent microvascular decompression. Interventions: Augmented reality microscopy with fluorescence imaging was utilized to visualize vascular flow intraoperatively. A postoperative surgeon questionnaire was administered to assess the intraoperative efficacy of this technology. Main Outcome Measures: Efficacy of technology in aiding with CPA tumor resection and microvascular decompression. Results: For all 7 microvascular decompression cases, surgeons agreed that the technology aided in identifying areas where disease was affecting tissues with no cases of vascular occlusion identified. In 3 of the 4 CPA tumor resection cases, surgeons agreed that the technology identified areas of vascular flow within the CPA and the tumor. Vascular patency of the sigmoid-transverse sinus was also confirmed. No significant adverse effects were noted except 1 instance of severe-to-profound sensorineural hearing loss. Conclusions: Our study shows that the augmented reality fluorescence technology works during lateral skull base surgery as it can confirm intraoperative vascular integrity. Our data also suggest that this technology may improve visualization of ambiguous vasculature and blood flow to diseased tissue.

Quantifying tinnitus suppression in cochlear implantation using tinnitus interval-limited tracking.

OBJECTIVES: Quantify the effects of cochlear implants (CI) on tinnitus suppression in patients with previous cochlear implantation using a novel audiologic sequence: Tinnitus Interval Limited Tracking (TILT). STUDY DESIGN: Prospective cohort study. METHODS: Consecutive patients with tinnitus and previous cochlear implantation for profound hearing loss underwent an audiologic testing sequence called TILT. Patients rated tinnitus severity using the validated Tinnitus Handicap Inventory (THI) as well as a visual analog scale at baseline and in a variety of audiologic scenarios. Changes in tinnitus severity between scenarios allow for the isolation of the effects of masking and electrical stimulation on the reduction of tinnitus. RESULTS: Twenty patients were enrolled, 10 of whom have tinnitus with average THI 30.2 (standard deviation 22.6). Patients had an acute decrease in tinnitus severity when their CIs were turned on, even in the absence of noise in a soundproof booth. This effect reversed once the CIs were turned off. This effect was greater in magnitude than with masking that occurred with the presentation of soft speech. Acute tinnitus severity trended toward improvement with increased level of presented speech. Degree of improvement was not correlated with THI. CONCLUSION: Acute tinnitus suppression in patients using CIs is multifactorial. Masking plays a role; however, it cannot sufficiently account for the totality of symptom improvement experienced by CI patients. Quantifiable tinnitus suppression observed when a CI is turned on, even in the absence of audiologic stimulation, suggests that electrical stimulation is involved in the mechanism of symptom improvement in these patients. LEVEL OF EVIDENCE: 4 Laryngoscope, 130: 2047-2052, 2020.