Six to eight weeks old mice were used in this study. C57BL/6 (B6, CD45.2⁺) mice were purchased from the Shanghai Laboratory Animal Center (SLAC, Shanghai, China). CD45.1⁺ mice (B6 background) were obtained from the Jackson Laboratory. Tlr2^−/−, tlr4^−/−, and tlr9^−/− (B6 background) mice were gifts from Dr. Shaobo Su (Sun Yat-sen University, Guangdong, China). All mice were housed in a specific pathogen-free facility.

For establishing the asthma model, mice were immunized at days 0 and 7 i.p., with 100 μg of OVA (grade V; Sigma–Aldrich, Saint Louis, MO, USA) in 100 μL of sterile saline and adsorbed in 50 μL of Imject alum (Thermo Scientific, Waltham, MA, USA). At days 14, 15, and 16, mice were administered i.n., with 100 μg of OVA in 50 μL of sterile saline after anesthetized with sodium pentobarbital (50 μg/g body weight, i.p., Merck, Whitehouse Station, NJ, USA). Mice were harvested and analyzed at day 21. For the experiment of TLR activation, WT mice were injected (i.p., or i.n.) with 2.5–10 μg of Pam3CSK4 (InvivoGen, San Diego, CA, USA) or 25 μg of CpG (Sangon Biotech, Shanghai, China) at indicated time points.

To obtain cells in pulmonary alveoli, we performed bronchoalveolar lavage as described before (20, 21) with slight modifications. The whole lung was perfused through the trachea with 1 mL of phosphate-buffered saline (PBS) containing 5 mM of EDTA for 4 times. The bronchoalveolar lavage fluid (BALF) was collected and cell pellets were pooled. To obtain single-lung-cell suspensions, lungs were minced and digested in RPMI-1640 medium containing 0.1% collagenase I (Sigma–Aldrich) and 5% fetal bovine serum (Gibco) for 60 min at 37°C. After filtration through a 200-gauge steel mesh, the medium was centrifuged and the red blood cells (RBCs) were removed by RBC lysis buffer (BioLegend). The cells were washed and collected for further experiments. For the isolation of peritoneal cells, mice were injected i.p., with 5 mL of sterile saline containing 5 mM of EDTA. The abdomen was rubbed gently for 2 min, and the lavage fluids were collected. Lavage fluids were centrifuged, and cell pellets were collected. Cell numbers were counted by Countstar® (Shanghai Ruiyu Biotech, China).

For analyses of levels of cytokines and antibody in BALF, the lung was perfused through the trachea with 1 mL of PBS containing 5 mM of EDTA. Lavage fluids were centrifuged, and the supernatants were collected. Levels of IL-4, IL-5, and IgE in BALF and serum were measured by using ELISA kits from Dakewe Biotech (Shenzhen, China) according to the manufacturer's instructions.

Flow cytometry analysis were performed as reported before (22) with slight modifications. For surface staining, single cells were incubated with fluorescein-labeled monoclonal antibodies for 30 min at 4°C after blockade of the Fc receptor, then washed twice and analyzed. For IL-13 staining, cells were stimulated with 50 ng/mL of PMA, 1 μg/mL of ionomycin and 10 μg/mL of monensin (all from Sigma–Aldrich) for 4 h at 37°C. After staining of cell-surface markers, cells were fixed and permeabilized with a Foxp3 Staining Buffer Set (BioLegend) according to the manufacturer's protocols and then stained with anti-mouse IL-13 or isotype control overnight at 4°C. Samples were analyzed by BD LSRII or LSRFortessa system (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (Tree Star, Ashland, OR, USA). Antibodies and isotype controls were purchased form BD Biosciences, BioLegend (San Diego, CA, USA), or eBioscience (San Diego, CA, USA) (Supplementary Table 1).

The mice were immunized with 100 μg of OVA in 100 μL of sterile saline and adsorbed in 50 μL of Imject alum. The peritoneal lavage fluids (PLF) were collected, and pMφ, inflammatory monocytes and neutrophils in PLF were purified by FACSAria II cell sorter (BD Biosciences). Purified cells were resuspended in saline and injected i.p., into the recipient mice at indicated time points.

Total RNA was extracted from 2–5 × 10⁵ purified inflammatory monocytes using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's protocols. Then, gene expression was analyzed using SYBR™ Premix Ex Taq kit (TaKaRa Bio, Kusatsu, Japan) and quantified using the ^ΔΔCt method. The primers used in this study were shown as following: GAPDH, 5′-tgcccagacatcatccctg-3′ (Forward) and 5′-tcagatccacgacggacaca-3′(Reverse); IL-6, 5′-taacagataagctggagtc-3′ (Forward) and 5′-taggtttgccgagtaga-3′ (Reverse); TNF-α, 5′-tctcattcctgcttgtggc-3′ (Forward) and 5′-cacttggtggtttgctacg-3′ (Reverse); IL-12, 5′- gcagcgtgggagtgggatgtg-3′(Forward) and 5′-gggcatcgggagtccagtcca-3′ (Reverse); IFN-γ, 5′-gccatcagcaacaacataagc-3′ (Forward) and 5′-gagctcattgaatgcttggc-3′ (Reverse); IL-4, 5′-caacccccagctagttgtca-3′(Forward) and 5′- cgttgctgtgaggacgtttg-3′(Reverse); IL-5, 5′-agcaatgagacgatgaggct-3′(Forward) and 5′- gtacccccacggacagtttg-3′(Reverse); IL-13, 5′- tcttgcttgccttggtggtc-3′(Forward) and 5′- ggggagtctggtcttgtgtg-3′(Reverse). All primers were synthesized by Sangon Biotech.

Lung tissues without lavage were fixed in 10% (v/v) formalin for >24 h and embedded in paraffin. Sections (6 μm) were stained with hematoxylin and eosin. Peribronchial and perivascular inflammation was assessed by a semi-quantitative scoring system as described before (23). The lung inflammation was scored following the scale: 0, normal; 1, few cells; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells two to four cells deep; and 4, a ring of inflammatory cells of more than four cells deep.

All data were expressed as mean ± SEM and analyzed using the Student's two-tailed t-test. A value of P < 0.05 was considered statistically significant.

To find out the cellular and molecular mechanisms of the protective function of TLR signaling in allergic asthma, we established the mouse model of OVA-induced allergic asthma by peritoneal sensitization using OVA/alum and intranasal challenge with OVA (Figures 1A,B), which is the most widely used mouse model of allergic asthma (24). To confirm if TLR signaling had a protective role in our mouse model, we established OVA-induced allergic asthma in tlr2^−/−, tlr4^−/−, and tlr9^−/− mice, and WT mice were used as the control. The results showed that, compared with WT mice, tlr2^−/−, and tlr4^−/− mice exhibited increased infiltration of leukocytes, especially asthma-related eosinophils, in alveoli (Figures 1C,D), and IgE level in serum was increased significantly in tlr2^−/− and tlr9^−/− mice after allergic asthma establishment (Figure 1E). Activation of various TLRs by agonists has been reported to prevent OVA-induced allergic asthma (25–27). To confirm this protective effect of TLR agonists in our mouse model, we treated WT mice intraperitoneally (i.p.) with the TLR2 agonist Pam3CSK4 or TLR9 agonist CpG (Figure 2A). The treatment of TLR agonists could significantly reduce lung injury (Figures 2B,C) and the number of infiltrating leukocytes and eosinophils in alveoli after allergic asthma establishment (Figures 2D,E). Therefore, these results indicated that activation of TLR signaling protected against OVA-induced allergic asthma in our mouse model, which was consistent with previous reports.

To identify which phase (e.g., sensitization or challenge) of OVA-induced allergic asthma could be affected by TLR signaling, we treated WT mice with Pam3CSK4 by peritoneal and intranasal routes, respectively (Figure 3A). We found that only treating mice i.p., with Pam3CSK4 protected against OVA-induced allergic asthma, characterized by reduced lung injury and less inflammatory cells, especially eosinophils, infiltration in alveoli when compared with WT control mice, but treating mice intranasally (i.n.) with Pam3CSK4 had no this protective effect (Figures 3B–E), implying that TLR agonist treatment might mainly influence the sensitization response in the peritoneal cavity. To prove this hypothesis, different from the mode of administration in Figure 3A, we only treated mice i.p., with Pam3CSK4 at the sensitization stage, and the Pam3CSK4 was i.p., injected after OVA/alum sensitization (Figures 3F). The results showed that Pam3CSK4 injection only at sensitization stages showed equivalent protection compare to the mode of administration in Figures 3A,G. Thus, these results indicated that TLR signaling-mediated protective effect mainly affect the sensitization phase of OVA-induced allergic asthma.

To explore which type of inflammatory cells in the peritoneal cavity could be regulated by TLR signaling at the stage of sensitization, we first monitored the dynamic change of inflammatory cells in the peritoneal cavity after OVA/alum sensitization (Supplementary Figure 1 in Supplementary Material). Inflammatory monocytes and neutrophils were recruited and increased in the peritoneal cavity, along with the decrease of pMφ, after OVA/alum sensitization (Figure 4A). Analyses of TLRs expression on cells showed that expression of TLR2 and TLR4 decreased on pMφ (Figure 4B) but increased on recruited inflammatory monocytes and neutrophils over time after OVA/alum sensitization (Figures 4C,D). Meanwhile, there were no changes of TLR2 and TLR4 expressions on monocytes and neutrophils in blood after OVA/alum sensitization (Figures 4E,F), which means that the microenvironment in peritoneal cavity after OVA/alum sensitization promoted the expression of TLR2 and TLR4. Our previous study found that pMφ almost could not be found in the peritoneal cavity 4 hours after OVA/alum sensitization (28). However, we also found that treating mice i.p., with Pam3CSK4, LPS, or CpG 4 h after sensitization also could significantly attenuate OVA-induced allergic asthma (Figures 3A,B). Thus, activation of TLR signaling in inflammatory monocytes and/or neutrophils (but not pMφ) might be able to attenuate OVA-induced allergic asthma.

To further determine which type of cells, inflammatory monocytes and/or neutrophils, play a critical role in OVA-induced allergic asthma, we purified pMφ, inflammatory monocytes, and neutrophils from OVA/alum-sensitized CD45.1 WT mice (Supplementary Figure 2 in Supplementary Material) and then adoptively transferred them to CD45.2 recipient mice, respectively (Figure 5A). All three cells transfer could induce allergic symptoms in recipient mice after OVA challenge even without OVA/alum sensitization, but transfer of inflammatory monocytes could induce more severe asthma in recipient mice than transfer of pMφ or neutrophils (Figures 5B,C). Taken together, these results suggested that activation of TLR signaling in inflammatory monocytes might be the main reason for the protective function of TLR signaling in OVA-induced allergic asthma.

To explore if the recruited inflammatory monocytes could be regulated by TLR signaling and if this effect could affect subsequent asthma disease, we treated the purified inflammatory monocytes with the TLR2 agonist Pam3CSK4 in vitro before transfer into recipient mice (Figure 6A). The result showed that Pam3CSK4-treated inflammatory monocytes induced milder allergic symptoms in recipient mice after OVA challenge when compared with Pam3CSK4-untreated inflammatory monocytes (Figures 6B–D). It has been reported that allergic asthma is an allergen-induced Th2 inflammatory disease, and activation of TLR signaling prevents OVA-induced allergic asthma in mice through promoting Th1 response (26). Consistent with previous reports, recruited inflammatory monocytes significantly up-regulated the expression of Th1-associated cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, after stimulating with TLR2 agonist Pam3CSK4 in vitro (Figure 6E). Furthermore, there was no significant change of the expression of IL-13 and IL-4 after stimulating with TLR2 agonist Pam3CSK4 in vitro, but IL-5 expression was down-regulated after stimulation (Figure 6F). Taken together, these results suggested that activation of TLR signaling in recruited inflammatory monocytes could promote the expression of Th1-associated cytokines to inhibit allergen-induced Th2 response and finally protected mice against OVA-induced allergic asthma.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.