Reagents

shRNA kits of Tert, Perk and Atf4, antibodies (Abs) of TERT (Clone#: C-12, Cat#: sc-377,511), ATF4 (B-3, sc-390,063), CD19 (B-1, sc-390,244, fluorochrome: AF488), MHC II (7–1 H): sc-13,556, AF546), CD11c (G-3, sc-398,725, AF594), CD14 (H-4, sc-515,785; 61D3, sc-52,457, AF648), CD16 (YFC 120.5, sc-58,962, AF700), CD163 (ED2, sc-58,965, AF790), CD141 (D-3, sc-13,164, AF488), CD103 (OX62, sc-53,085, AF546), CD172a (C-7, sc-376,884, AF594) and F4/80 (D-11, sc-365,340, AF648) were purchased from Santa Cruz Biotech (Santa Cruz, CA). Abs of PERK (ab229912), p-PERK (phospho T982; ab192591), p-ATF4 (phospho S245; ab28830) were purchased from abcam (Cambridge, MA). CMIP Ab was purchased from MyBioSource (MBS9603486; San Diego, CA). Ab of XCR1-PE, ELISA kits of EPX, tryptase, Mcpt1, OVA-specific IgE, IL-4, IL-5, IL-10, and IL-13 were purchased from Dakewe BioMart (Shenzhen, China). IL-10 neutralizing Ab (JES5-2A5) was bought from eBioscience (San Diego, CA). Reagents and materials for RT-qPCR, Western blotting and IP were purchased from Invitrogen (Carlsbad, CA).

Ethics statement

The study protocol was approved by the human ethics committee and the animal ethics committee at our institution. A written informed consent was obtained from each human subject. All animal experiments were conducted in accordance to the ARRIVE guidelines.

Human subjects

Patients with perennial allergic rhinitis (AR) were enrolled into this study. The diagnosis of AR was carried out by our experienced physicians based on the routine procedures established in our department, which can be found elsewhere [29, 30]. Briefly, the inclusive criterion of AR was that patients had perennial AR for more than two years, positive skin prick test results, and positive serum specific IgE. Subjects with any of the following conditions were excluded, which were severe organ diseases, cancers, autoimmune diseases, and those under treatment with immune suppressive agents for any reason. In addition, healthy control (HC) subjects were also enrolled, who had no allergic disease history, negative skin prick test results, and negative serum sIgE. The human ethics committee at our institution approved the human experimental protocol (approve #2022-0036). A written informed consent was obtained from each human subject. The demographic data are presented in Table 1.

Table 1 Demographic data of human subjectsCollection of nasal secretions (NS)

A piece of degreasing cotton (about 1 g) was gently placed in the middle nasal meatus. It was taken out 5 min later. NS was squeezed out from the cotton, and analyzed in other experiments.

Total nasal symptom score (TNSS)

TNSS was recorded from each human subject following reported method [31]. TNSS includes three parts: 1, nasal obstruction (scored 0, 1, 2, 3), nasal itching/sneezing (scored 0, 1, 2, 3), secretion/nasal discharge (scored 0, 1, 2, 3). Human subjects were asked to record TNSS daily for one week prior to the enrollment.

Real-time quantitative RT-qPCR (RT-qPCR)

RNAs were extracted from cells harvested from relevant experiments. The first strand of cDNA was synthesized with the RNA samples using a reverse transcription kit following the manufacturer’s instructions. In the presence of relevant primers (Table S1 in supplemental materials), the samples were amplified in a qPCR device (Bio Rad CFX96) using the SYBR Green Master Mix. The results were calculated with the 2-∆∆Ct method with the reference gene β-actin, and presented as relative expression (RE).

Preparation of mouse airway mononuclear cells (AMCs)

The lungs were excised right after the sacrifice, cut into small pieces, incubated with collagenase IV (0.5 mg/ml) for 20 min at 37 °C with mild agitation. Single cells were filtered through a cell strainer (70 μm first, then 40 μm). AMCs were isolated from the single cells by the Percoll gradient density centrifugation.

Establishment of an AR mouse model

Following the established procedures [17], mice were treated with the ovalbumin (OVA)-aluminum hydroxide (alum) protocol as illustrated in Fig. S4 in the supplemental materials. The animal experimental protocols were approved by the Animal Ethics Committee of Shenzhen University (Approve#2022-008).

Assessment of the AR responses in mice

The AR responses in mice include the AR-like clinical symptoms (nasal itch and sneezing), the amounts of allergic mediators [eosinophil peroxidase (EPX) and mouse mast cell protease-1 (Mcpt1)], and Th2 cytokines (IL-4, IL-5, and IL-13) in the nasal lavage fluid (NLF), and increase in the amounts of specific IgE (sIgE). The AR response was evaluated following the established procedures of our laboratory that also can be found in our previous reports [17].

Collection of NLF

The trachea was opened in the neck right after the sacrifice. A syringe needle was inserted into the trachea towards the nasal direction. A saline solution (1 ml per mouse) was injected into the upper respiratory tract and recovered from the nostrils.

Colocalization of CMIP and ubiquitin

The PVDF membrane of the IP experiment for detecting the CMIP-TERT complex was treated with peel-re-staining procedures and stained with ubiquitin Ab following the Western blotting procedures.

RNA interference (RNAi)

DCs were transfected with the shRNA kits (Santa Cruz Biotech) of Perk, or Atf4, or Tert, or control reagents following the manufacturer’s instruction. The effects of RNAi were checked in the cells by Western blotting 2 days after the transfection.

Over expression of TERT in DCs

Naïve DCs were transfected with Tert-expression plasmids (labeled with His, provided by the San Gong Biotech, Shanghai, China) or control plasmids following the manufacturer’s instruction. The recombinant TERT in DCs were checked by Western blotting two days later.

Preparation of RNA samples from peripheral DCs

Blood samples were collected from each human subject through the ulnar vein puncture. Peripheral blood mononuclear cells (PBMCs) were isolated by the gradient density Percoll centrifugation. DCs were purified from the PBMCs by flow cytometry using CD11c as a surface marker. RNAs were extracted from DCs with the TRIzol reagents.

RNA sequencing (RNAseq)

RNA samples were extracted from DCs and subjected to RNAseq analysis. The procedures of RNAseq were carried out by the professional staff of the biotech company (BGI, Shenzhen, China). The data were analyzed by the technical staff of the company. The results of differentially expressed genes (DEGs) are presented by volcano plots and heatmaps. The data of RNAseq were verified by conventional RT-qPCR.

Western blotting

Proteins were extracted from cells collected from relevant experiments, separate by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), and transferred onto a PVDF (polyvinylidene difluoride) membrane. After blocking with 5% skim milk for 30 min, the membrane was incubated with the primary Abs (diluted to 200 ng/ml; the types of Abs are indicated in figures and legends) overnight, followed by incubating with HRP (horseradish peroxidase) conjugated second Abs (diluted to 20 ng/ml) for 2 h. Washing with TBST (Tris-buffered saline containing 0.05% Tween 20) three times was performed after each incubation. Enhanced chemiluminescence was utilized to develop the immunoblots on the membrane and were photographed using an imaging device (UVP, Cambridge, UK). Immunoblots were subjected to densitometry using ImageJ software when necessary.

Flow cytometry (FCM)

Single cells were prepared from relevant experiments. In the surface staining, cells were incubated with Abs of interest (Ab types are detailed in figures and legends; diluted to 0.5 µg/ml) or isotype IgG for 30 min at 4 °C. The cells were washed with FCM buffer (phosphate buffered saline, PBS, containing 2% bovine serum albumin, BSA) three times, and analyzed with a flow cytometer (BD FACSCanto II). In the intracellular staining, cells were fixed with 1% paraformaldehyde (containing 0.05% Triton X-100 to increase the permeability of the membrane) for 1 h. The cells were then processed with the same procedures as the surface staining. The data were analyzed with Flowjo software (TreeStar Inc., Ashland, OR). The data obtained from the isotype IgG staining were used as the gating references.

Mice

Male C57/BL6 mice (6–8 weeks old) were purchased from the Guangdong Experimental Animal Center (Foshan, China). Tertf/f and Itgax-Cre mice were purchased from Jackson Laboratory (Bar Harbor, Main). A mouse strain carrying DCs deficient of Tert gene expression (the Tertf/fItgax-Cre mice) was generated in-house by crossing Tertf/f mice with Itgax-Cre mice. Tertf/fItgax-Cre mice were used in experiments after 5 generations. The gene knocking out system was activated by feeding Tertf/fItgax-Cre mice with tamoxifen (Sigma Aldrich; 20 mg/mouse in 0.3 ml corn oil) daily for 5 consecutive days. Mice were maintained in a specific pathogen-free facility at Shenzhen University with free access to food and water.

Cell culture

The cells were cultured in a RPMI1640 medium supplemented by 2 mM of L-glutamine, 10% fetal calf serum, 0.1% streptomycin and 100 U/ml penicillin. Cell viability exceeded 99% based on the Trypan blue exclusion test.

Enzyme-linked immunosorbent assay (ELISA)

The amounts of serum specific IgE (sIgE) and cytokines in the serum or culture supernatant were evaluated by ELISA using commercial reagent kits based on the recommended protocols.

Immunoprecipitation (IP)

Proteins were extracted from cells harvested from relevant experiments and precleared by incubating with protein G agarose beads for 2 h. Supernatant was collected and incubated with Abs (1 µg/ml) of interest or isotype IgG overnight. The immune complexes were precipitated by incubating with protein G agarose beads for 2 h. The beads were collected by centrifugation at 5,000 g for 10 min. Proteins were eluted from the beads, and subjected to the analysis of Western blotting.

Statistics

The difference between two groups was determined by the Student’s t-test, or Mann Whitney test. ANOVA followed by Dunnett’s test or Bonferroni test was performed for the data of more than two groups. Pearson correlation coefficient assay or Spearman correlation coefficient assay was conducted to determine the correlation between groups.