Single-cell RNA sequencing of human eosinophils from nasal polyps reveals eosinophil heterogeneity in chronic rhinosinusitis tissue.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by type 2 inflammation in the US, yet the actual roles of eosinophils in CRSwNP are largely unclear. OBJECTIVE: To reveal the roles and heterogeneity of eosinophils in nasal polyp (NP) tissue by single cell RNA-Sequencing (scRNA-Seq) analysis of NP tissue. METHODS: Sinonasal tissues (NP and control sinus tissue) and patient matched peripheral blood (PB) samples were obtained from 5 control patients and 5 patients with CRSwNP. Eosinophils were enriched prior to processing for scRNA-Seq. The gene expression profiles in eosinophils were determined by the microwell-based scRNA-Seq technology (BD Rhapsody platform). We predicted the overall function of NP eosinophils by gene ontology (GO) enrichment and pathway analyses and confirmed expression of selected genes by flow cytometry. RESULTS: After filtering out contaminating cells, we detected 5,542 eosinophils from control PB, 3,883 eosinophils from CRSwNP PB, 101 eosinophils from control sinus tissues (not included in further analyses) and 9,727 eosinophils from NPs by scRNA-Seq. We found that 204 genes were down-regulated, and 354 genes were up-regulated in NP eosinophils, compared to all PB eosinophils (>1.5-fold, Padj<0.05). Up-regulated genes in NP eosinophils were associated with activation, cytokine-mediated signaling, growth factor activity, NF-κB signaling and anti-apoptotic molecules. NP eosinophils displayed 4 clusters revealing potential heterogeneity of eosinophils in NP tissue. CONCLUSIONS: Elevated eosinophils in NP tissue appear to exist in several subtypes that may play important pathogenic roles in CRSwNP, in part via controlling inflammation and hyperproliferation of other cells.

Beneficial and adverse effects of dam construction in canal tannery wastewater effluent with a high content of chromium in Hazaribagh, Bangladesh.

BACKGROUND: Blockage to divide downstream canals into upstream canals, into which tannery wastewater including a high concentration of trivalent chromium [Cr(III)] is directly discharged, has been constructed in Hazaribagh, a tannery built-up area in Bangladesh. However, there has been no study to verify the environmental significance of blockage construction for water pollution of Cr in nature. METHODS: Consecutive fixed area monitoring for a total of 164 water samples collected outside and inside Hazaribagh from 2014 to 2023 was carried out to clarify the effects of stagnant and flowable canal water in the presence or absence of blockage on Cr(III) and hexavalent Cr [Cr(VI)] concentrations. RESULTS: Since pollution of Cr(III) and Cr(VI) in Buriganga River (outside Hazaribagh) was not serious, this study then focused on their pollution in canal water (inside Hazaribagh) in the nonblockage period, blockage construction period and blockage destruction period. As expected, the mean Cr(III) concentration in downstream canal water samples in the blockage construction period was more than 98% lower than that in the upstream canal water samples in the same period, while the concentrations were comparable in downstream and upstream canal water samples in the nonblockage period and blockage destruction period. Unexpectedly, the mean concentration of Cr(VI) in the upstream canal water samples in the blockage construction period was 38.6-fold and 3.3-fold higher than that in the downstream canal water samples and the Cr(VI) guideline value by the US-EPA, respectively. CONCLUSION: This study demonstrated for the first time not only a merit of decreased Cr(III) pollution but also a demerit of increased Cr(VI) pollution in stagnant water derived from blockage construction in natural environments. This bitter lesson obtained by the enclosure of Cr(III)-polluted water is globally applicable for water pollution of Cr(III), which is used in various industries including the leather industry.

Th2 cell-derived histamine is involved in nasal Th2 infiltration in mice.

OBJECTIVE: Histamine derived from mast cells and basophils plays important roles in inducing allergic symptoms. Although T cells also produce histamine, the involvement of the histamine produced from T cells has remained enigmatic. We sought to reveal the roles of T helper 2 (Th2) cell-derived histamine in nasal allergic disorders. METHODS: The histamine production from Th2 cells was measured by EIA. The mRNA expression of histidine decarboxylase (HDC) was measured by real-time PCR. To investigate the roles of Th2 cell-derived histamine in vivo, we analyzed an antigen-specific Th2 cell transfer mouse model. RESULTS: Th2 cells produced histamine by T cell receptor stimulation, and these properties were specific for Th2 cells, but not Th1 cells and naïve CD4 T cells. The histamine produced from Th2 cells was involved in the infiltrations of Th2 cells in response to antigen exposure. CONCLUSION: These results suggest that Th2 cell-derived histamine play important roles in nasal allergic disorders.

Th2 cells and macrophages cooperatively induce allergic inflammation through histamine signaling.

Histamine, which is mainly produced by mast cells and basophils, participates in various allergic symptoms, and some studies have reported that macrophages also produce histamine. Moreover, recent studies have revealed that macrophages, especially alternatively activated macrophages (M2) induced by T helper 2 (Th2) cytokines, such as interleukin (IL)-4 and IL-13, participate in the pathogenesis of allergic diseases. The major source of Th2 cytokines is antigen-specific Th2 cells. To elucidate the relationship between histamine, macrophages, and Th2 cells in allergic inflammation, we established a macrophage-Th2 cell co-culture model in vitro and an antigen-specific Th2 cell transfer mouse model of rhinitis. In vitro analyses indicated that macrophages produce histamine by interacting with antigen-specific Th2 cells through the antigen. Furthermore, Th2 cells and macrophages cooperatively elicited rhinitis in the mouse model. We determined that histamine induces Th2- and macrophage-elicited sneezing responses through H1 receptor signaling, whereas it induces nasal eosinophil infiltrations through H4 receptor signaling. Collectively, these results indicate a novel histamine production mechanism by macrophages, in which Th2 cells and macrophages cooperatively induce nasal allergic inflammation through histamine signaling.

[Th2 cells and macrophages induce novel type-I-hypersensitivity-like reaction].

In recent decades, many patients have been suffering from allergic rhinitis including Japanese cedar pollinosis, which is becoming a national disease in Japan. There is other upper airway intractable disease, called eosinophilic sinusitis. The elucidation of the pathogenesis of upper airway intractable disease is demanded for the development of novel therapies. Many researches about allergic pathogenesis have focused on IgE-mast cells pathway, however, there are the patients with allergic symptoms induced by non-IgE mediated mechanisms. The patients who show allergic rhinitis-like symptoms, such as sneezing, nasal discharge, and nasal clotting, without allergen-specific IgE, are diagnosed as non-allergic rhinitis. The precise mechanisms of non-allergic rhinitis are totally unclear. We have investigated the non-IgE mediated nasal symptoms, because the elucidation of non-IgE mediated mechanisms might lead to the elucidation of other upper airway intractable disease. We established antigen-specific Th2 cells transfer model and revealed the novel allergic mechanisms induced by Th2 cells, macrophages and endotoxin. Although Th2 cells play important roles in allergic diseases, the main function of Th2 cells are thought to produce Th2 cytokines, such as interleukin (IL)-4, IL-5, IL-13. We revealed the new functions of Th2 cells in allergic diseases. In addition, we found the novel histamine production mechanisms using in vitro macrophages and Th2 cells co-culture model. Both macrophages and Th2 cells produced histamine by the interaction through antigen. Our observations suggested the existence of the novel allergic mechanisms distinct from IgE-mast cells pathway.

Safety profile and immunological response of dual sublingual immunotherapy with house dust mite tablet and Japanese cedar pollen tablet.

BACKGROUND: There have been no studies of dual administration of sublingual immunotherapy (SLIT) tablets for perennial and seasonal allergic rhinitis. This trial (JapicCTI-184014) was conducted to investigate the safety profile and immunological response during dual therapy with SQ house dust mite (HDM) and Japanese cedar pollen (JCP) SLIT tablets. METHODS: This was a multicenter, open-label, randomized trial of 109 Japanese patients with coexisting HDM and JCP allergic rhinitis who had positive tests for HDM- and JCP specific IgE (≥0.7 kU/L). Patients were allocated to receive HDM (N = 54) or JCP (N = 55) SLIT tablets alone for 4 weeks followed by 8 weeks of dual therapy with both SLIT tablets administered within 5 min of each other. Adverse events (AEs), adverse drug reactions (ADRs), and serum IgE and IgG4 specific for HDM (Dermatophagoides farinae, Dermatophagoides pteronyssinus) and JCP were recorded. RESULTS: The percentage of subjects with AEs and ADRs was similar between the two groups and between the two periods of monotherapy and dual therapy. Most AEs and ADRs were mild in severity, and no serious events were observed. The most common ADRs were local events in the oral cavity. Levels of IgE and IgG4 specific for HDM (D. farinae, D. pteronyssinus) and JCP were increased after treatment with HDM and JCP SLIT tablets, respectively. CONCLUSIONS: Dual therapy with both SLIT tablets administered within 5 min after 4 weeks of monotherapy with HDM or JCP tablet was well tolerated and induced the expected immunological responses.

Activation of group 2 innate lymphoid cells exacerbates and confers corticosteroid resistance to mouse nasal type 2 inflammation.

Both Th2 cells and group 2 innate lymphoid cells (ILC2s) contribute to allergic diseases. However, their exact role and relationship in nasal allergic disorders are unclear. In this study, we investigated the cooperation of Th2 cells and ILC2s in a mouse model of nasal allergic disorder. To differentially activate Th2 cells and/or ILC2s in nasal mucosa, mice were intra-nasally administered ovalbumin (OVA) antigen, papain, an ILC2-activator, or both for 2 weeks. Epithelial thickness and number of eosinophils in the nasal mucosa were evaluated at 24 h after the final challenge. Intra-nasal administration of OVA and papain preferentially activated Th2 cells and ILC2s, respectively, in the nose. Both OVA and papain increased the nasal epithelial thickness and number of eosinophils, and their coadministration significantly enhanced the symptoms. Although T-/B-cell-deficient mice showed severely decreased nasal symptoms induced by OVA or OVA-plus-papain, the mice still showed slight papain-induced nasal symptoms. In ILC2-deficient mice, OVA-plus-papain-induced nasal symptoms were suppressed to the same level as OVA-alone. Similarly, IL-33- and ST2-deficient mice showed decreased OVA-plus-papain-induced nasal symptoms. IL-5 induced eosinophilia only, but IL-13 contributed to both nasal epithelial thickening and eosinophilia induced by OVA-plus-papain. Dexamethasone ameliorated OVA-alone-induced nasal epithelial thickening. However, OVA-plus-papain-induced nasal epithelial thickening was only partially controlled by dexamethasone. These results demonstrate that IL-33/ST2-pathway-mediated ILC2 activation exacerbated Th2-cell-induced nasal inflammation by producing IL-13. Although Th2-cell-alone-induced nasal inflammation was controlled by corticosteroid treatment, the activation of ILC2s conferred treatment resistance. Therefore, ILC2s and their activators could be therapeutic targets for treatment-refractory nasal allergic disorders.

Allergen endotoxins induce T-cell-dependent and non-IgE-mediated nasal hypersensitivity in mice.

BACKGROUND: Allergen-mediated cross-linking of IgE on mast cells/basophils is a well-recognized trigger for type 1 allergic diseases such as allergic rhinitis (AR). However, allergens may not be the sole trigger for AR, and several allergic-like reactions are induced by non-IgE-mediated mechanisms. OBJECTIVE: We sought to describe a novel non-IgE-mediated, endotoxin-triggered nasal type-1-hypersensitivity-like reaction in mice. METHODS: To investigate whether endotoxin affects sneezing responses, mice were intraperitoneally immunized with ovalbumin (OVA), then nasally challenged with endotoxin-free or endotoxin-containing OVA. To investigate the role of T cells and mechanisms of the endotoxin-induced response, mice were adoptively transferred with in vitro-differentiated OVA-specific TH2 cells, then nasally challenged with endotoxin-free or endotoxin-containing OVA. RESULTS: Endotoxin-containing, but not endotoxin-free, OVA elicited sneezing responses in mice independent from IgE-mediated signaling. OVA-specific TH2 cell adoptive transfer to mice demonstrated that local activation of antigen-specific TH2 cells was required for the response. The Toll-like receptor 4-myeloid differentiation factor 88 signaling pathway was indispensable for endotoxin-containing OVA-elicited rhinitis. In addition, LPS directly triggered sneezing responses in OVA-specific TH2-transferred and nasally endotoxin-free OVA-primed mice. Although antihistamines suppressed sneezing responses, mast-cell/basophil-depleted mice had normal sneezing responses to endotoxin-containing OVA. Clodronate treatment abrogated endotoxin-containing OVA-elicited rhinitis, suggesting the involvement of monocytes/macrophages in this response. CONCLUSIONS: Antigen-specific nasal activation of CD4+ T cells followed by endotoxin exposure induces mast cell/basophil-independent histamine release in the nose that elicits sneezing responses. Thus, environmental or nasal residential bacteria may exacerbate AR symptoms. In addition, this novel phenomenon might explain currently unknown mechanisms in allergic(-like) disorders.

Murine allergic rhinitis and nasal Th2 activation are mediated via TSLP- and IL-33-signaling pathways.

Thymic stromal lymphopoietin (TSLP) and IL-33 are epithelium-derived proallergic cytokines that contribute to allergic diseases. Although the involvement of TSLP in allergic rhinitis (AR) is suggested, the exact role of TSLP in AR is poorly understood. Furthermore, the relative contribution of TSLP and IL-33 in nasal allergic responses has not been described. In this study, we examined the roles of TSLP and IL-33 in AR by analyzing acute and chronic AR models. Acute AR mice were intraperitoneally immunized with ragweed, then intranasally challenged with ragweed pollen for four consecutive days. Chronic AR mice were nasally administrated ragweed pollen on consecutive days for 3 weeks. In both models, TSLP receptor (TSLPR)-deficient mice showed defective sneezing responses and reduced serum ragweed-specific IgE levels compared with wild-type (WT) mice. Analyses of bone-marrow chimeric mice demonstrated that hematopoietic cells were responsible for defective sneezing in TSLPR-deficient mice. In addition, FcεRI(+)-cell-specific TSLPR-deficient mice showed partial but significant reduction in sneezing responses. Of note, Th2 activation and nasal eosinophilia were comparable between WT and TSLPR-deficient mice. ST2- and IL-33-deficient mice showed defective Th2 activation and nasal eosinophilia to acute, but not chronic, ragweed exposure. TSLPR and ST2 double-deficient mice showed defective Th2 activation and nasal eosinophilia even after chronic ragweed exposure. These results demonstrate that TSLPR signaling is critical for the early phase response of AR by controlling the IgE-mast-cell/basophil pathway. The IL-33/ST2 pathway is central to nasal Th2 activation during acute allergen exposure, but both TSLPR and ST2 contribute to Th2 responses in chronically allergen-exposed mice.