C57BL/6J WT, IFN-γ^−/− (B6.129S7-Ifng^tm1Ts/J), IL-10^−/− (B6.129P2-Il10^tm1Cgn/J), and CRISPR/Cas9 knock-in (B6J.129 (Cg)-Gt (ROSA) 26Sortm1.1^{(CAG-cas9*,-EGFP) Fezh}/J: WT^EGFP) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). T-bet^−/− (Tbox21) mice were provided by Dr. Laurie Glimcher (Department of Immunology and Infection Diseases, Harvard School of Public Health). All mice were housed in cages maintained under pathogen-free conditions with air conditioning, a 12 h light and 12 h dark cycle and were provided with food and water ad libitum. The study was approved by the Kyung Hee University animal care and use committee. All of the experiments were performed in accordance with the approved animal protocols and guidelines established by Kyung Hee University [KHUASP (SE)-12-015]. All experiments used females that were 7–9 weeks old.

This study was performed using previously described experimental procedures, which expose mice to mainstream of CS (17, 18). The mice were exposed to the smoke from five consecutive non-filtered cigarettes (Reference 3R4F without filter; University of Kentucky, Lexington, KY, USA) four times a day, 6 days per week, using a smoking apparatus described by previously (18). Female C57BL/6J WT, T-bet^−/−, IFN-γ^−/−, and WT^EGFP mice were exposed to CS for 4 weeks, and CD4⁺ and CD8⁺ depletion, immune cell profiling in lamina propria (LP), hemoccult test and A4B7⁺ cell analysis studies were performed under 2 week CS exposure. All mice were sacrificed 24 h after the last CS exposure.

The ascending part of the colon was homogenized with a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and centrifuged at 10,000 × g for 15 min at 4°C. The colon homogenates were assayed for Th1, Th2, and Th17 using a CBA (BD Sciences, San Diego, CA, USA) using a BD FACSCalibur flow cytometer (BD Sciences).

The bronchoalveolar lavage (BAL) fluid was immediately collected after the mice were sacrificed. The lungs were lavaged and pooled three times with 1 ml of ice-cold PBS. The obtained BAL fluid was centrifuged at 3,000 × g for 10 min at 4°C, and the supernatants (BAL fluid) stored at −80°C. The Th1 cytokines, IFN-γ and TNF-α, were measured using a quantitative sandwich ELISA kit (IFN-γ, and TNF-α; BD Sciences) according to the manufacturer’s protocol. The optical density was measured at 450 nm using a microplate reader (SoftMax Pro software; Sunnyvale, CA, USA). The optical densities obtained for IFN-γ and TNF-α were each divided by the total protein concentrations of the respective BAL fluid samples for standardization purposes. The total protein concentrations were determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.

Total RNA was prepared from frozen colon tissue homogenates with an easy-BLUE™ RNA extraction kit (iNtRON Biotech., Sungnam, Republic of Korea). The cDNA synthesis was carried out for at 42°C and 5 min at 94°C using a cDNA synthesis kit (Bioneer Corporation., Daejeon, Republic of Korea). qRT-PCR for IFN-γ and TNF-α was performed with a SYBR Green I master mix using a Lightcycler^® 480 system (Roche, Basel, Switzerland) as previously described in Jung et al. (19). The IFN-γ, TNF-α, and β-actin genes were amplified using the following primers: IFN-γ: forward (F), 5′-TCA AGT GCG ATA GAT GTG GAA GAA-3′ and reverse (R), 5′-TGG CTC TGC AGG ATT TTC ATG-3′, TNF-α: F, 5′-CAT CTT CTC AAA ATT CGA GTG ACA A-3′ and R, 5′-TGG GAG TAG ACA AGG TAC AAC CC-3′, and β-actin: F, 5′-AGA GGG AAA TCG GTG AC-3′ and R, 5′-CAA TAG TGA CCT GGC GCT-3′. IFN-γ and TNF-α expressions were normalized to β-actin expression (20).

To perform BAL fluid collection, the mice were sacrificed and a tracheal cannula was slowly inserted. Three times via the tracheal cannula, 1 ml of ice-cold PBS was delivered and recovered by gentle manual aspiration. The collected BAL fluid was centrifuged at 3,000 × g for 10 min at 4°C, and the cell pellet was washed and finally resuspended in 1 ml of PBS. First, the total viable cells in the resulting pellet were counted using a trypan blue stain. To count the differential cells (neutrophils, macrophages, and lymphocytes), BAL fluid cells were adhered to glass slides using Cytospin (Sandon, Waltham, MA, USA) with Diff-Quick staining (Life Technologies., Auckland, New Zealand). The stained BAL cell slides were mounted with Canada balsam (Showa Chemical Co. Ltd., Tokyo, Japan). The BAL cells were counted under a light microscope as we previously reported (19, 21). The result was indicated as the cell number × 10⁴.

The mesenteric lymph nodes (MLNs) were disrupted over a wire mesh screen. The colonic LP was isolated into a single-cell suspension as previously described in Bosurgi et al. (22). The lung was dissociated into a single-cell suspension using a mouse lung dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with the gentle MACS™ dissociator, according to the manufacturer’s protocol. The red blood cells were lysed in BD Pharm Lyse™ lysing solution (BD Sciences). The single cells were stimulated in RPMI 1640 supplemented with 10% fetal bovine serum, 50 UI/ml penicillin, and 50 µg/ml streptomycin (Hyclone, Logan, UT, USA) for 5 h with 50 ng/ml PMA/1 μg/ml Ionomycin (Sigma-Aldrich, St. Louis, MO, USA), respectively, in the presence of 0.66 µl/ml BD Golgistop™ protein transport inhibitor (BD Sciences). Intracellular IFN-γ and surface marker CD4 were assessed using a Mouse Th1/Th2/Th17 Phenotyping Kit (BD Sciences) following the manufacturer’s instructions. The stimulated cells were incubated with the following antibodies: CD4-FITC, IFN-γ-PE, and IL-17A-APC (e-Bioscience, San Diego, CA, USA). To examine production of IFN-γ in CD4, CD8, or NK cells, splenocytes were stimulated for 5 h with PMA, Ionomycin, and BD Golgistop™. The cells fixed and stained with cell surface marker CD4-PE, CD8-APC, or NK1.1-FITC (e-Bioscience). Then, intracellular IFN-γ was stained. To elucidate the effect of CS exposure on immune cell population in colonic LP, single cells from colonic LP were stained with the following antibodies: B220-PE, CD4-FITC, CD4-APC, CD8-APC, CD11b-APC, CD25-PE, CD45-FITC, F480-FITC, and Gr1-PE (e-Bioscience). To examine an expression of A4B7, single cells from blood and spleen were stained with CD4-APC and A4B7-PE (e-Bioscience) antibodies. All of the sample data were acquired by a FACSCalibur flow cytometer using Cell Quest Pro software (BD Sciences) and generated in graphical and tabular formats using FlowJo software (Tree Star Inc., Ashland, OR, USA).

mAbs were used for the in vivo depletion of CD4⁺ and CD8⁺ T-cell subsets. The C57BL/6J WT mice were injected once with 500 µg i.p. antimouse CD4 Abs (clone 2.43), CD8 Abs (clone GK1.5), or rat IgG (Sigma-Aldrich) every 3 days from days 3 to 11, in 3-day intervals. The efficacy of CD4⁺ T-cell and/or CD8⁺ T-cell depletion was analyzed using a FACSCalibur flow cytometer.

CD4⁺CD25⁻ T cells were highly purified from the lung draining lymph nodes of WT and IFN-γ^−/− mice using the CD4⁺CD25⁺ T cells Treg isolation kit and magnetic bead separation (Miltenyi Biotec). The purity of the isolated cells was analyzed by flow cytometry prior to AT (>88% of the CD4⁺ cells were CD25⁻). The isolated CD4⁺CD25⁻ T cells (5 × 10⁵/mouse) were suspended in 200 µl PBS and injected via i.v. into IL-10^−/− mice. After 14 days of the AT, the mice were sacrificed.

The mice were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml). The lungs and colons were removed from the perfused mice, fixed with 10% neutral-buffered formalin (IMEB Inc., San Marcos, CA, USA), and embedded in paraffin. A section of the lung (4-µm thick) and transverse colons (7-µm thick) from each mouse were stained with hematoxylin and eosin and visualized on an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with a DP71 digital camera (Olympus). A total histological score of the lung was calculated for each mouse as described previously (18). Briefly, the lung sections were scored from 0 to 5 by readers according to the following criteria: 0 = normal; 1 = very mild; 2 = mild; 3 = moderate; 4 = marked; 5 = severe inflammation. The lung sections were examined under 200× magnifications to quantify the mean alveolar airspace (MAA). MAA was a quantitative assessment of lung structure that was determined by dividing the sum of the alveolar airspace areas divided by the number of identified alveoli using Image Pro-Plus 5.1 software (Media Cybernetics, Inc., Silver Spring, MD, USA). The colon sections were assigned a score from 0 to 3 as described previously (23). The histological analyses were performed in a blinded fashion.

All of the values are presented as the mean ± SEM. The statistical significance (p < 0.05 for all analyses) was assessed using two-tailed Student’s t-test for single comparisons or by two-way ANOVA with repeated measures using Prism 5.01 software (Graphpad Software Inc., San Diego, CA, USA).

First, we wanted to examine whether CS exposure could induce Crohn’s disease-like illness. Therefore, we exposed the mice to CS as described in a previous study (18) and evaluated the impact of CS exposure in the bowel. The colon length was significantly shorter (air group: 77.1 ± 2.3 mm vs. CS group: 56.8 ± 0.8 mm, Figure 1A) and the body weight was markedly reduced (Figure 1B) by CS exposure after 4 weeks of CS exposure. Histologic analysis confirmed that CS exposure significantly increased the infiltration of inflammatory cells in the submucosa, epithelial cell hyperplasia, and mucus thickness in the ascending colon (Figures 1E,F). The immune cell subset analysis in the LP demonstrated that the neutrophils and macrophages were predominantly increased upon CS exposure (Figure S1A in Supplementary Material). In addition, the fecal occult blood test showed that CS exposure for 2 weeks was enough to cause bleeding (Figure S1B in Supplementary Material). These results indicated that CS exposure could cause colitis. Next, we wanted to know whether CS exposure-induced colitis resembles Crohn’s disease (Th1/Th17-dominant colitis) or ulcerative colitis (Th2 dominant colitis). We evaluated cytokine production in inflamed regions in the bowel and found that the production of Th1 cytokines such as IFN-γ and TNF-α was highly increased (37.4- and 3.1-fold higher, respectively), while the production of none of the Th2/17 cytokines, including IL-4 and IL-17A, was increased, indicating that CS exposure-induced colitis resembles Crohn’s disease (Figures 1C,D). To confirm the Th1/17 responses in CS exposure-induced colitis, we analyzed the intracellular expression of IFN-γ and IL-17A on CD4⁺ T cells in MLNs and colonic LP. The results demonstrated that CS exposure significantly increased Th1 but not Th17 in both MLNs and colonic LP, suggesting that CS exposure specifically induces Th1-mediated bowel inflammation (Figures 1G–J).

We have repeatedly shown that CS exposure develops lung inflammation in mice (18, 24). As expected, the mice with CS exposure-induced colitis also showed signs of lung inflammation. A massive recruitment of inflammatory cells was observed in the entire lung, especially around the bronchial and peribronchial layers in the CS-exposed mice (Figure 2C). Likewise, alveolar wall destruction was also observed in their lung tissue, which resulted in enlarged airspaces (Figures 2E,F). Infiltrated immune cells reflecting pathological changes in lung parenchyma (25) were also markedly increased in the CS exposure group compared to the air exposure group (Figure 2E). We attempted to determine the involvement of Th1 and Th17 responses in lung inflammation induced by CS exposure because the Th1 response, but not the Th17 response, was observed in colonic inflammation as triggered by the same stimuli. Interestingly, the results showed that CS exposure activated both Th1 and Th17 responses in the lung, unlike in the bowel (Figures 2A,B,D).

As IFN-γ and IL-17A are secreted both in CD4⁺ T cells and CD8⁺ T cells (26), we wanted to know which T-cell subtype mediated the CS exposure-induced inflammation in the colon and lung. When mice were exposed to CS, a significantly increased IFN-γ⁺CD4⁺ T-cell population was observed in the MLNs, colonic LP, and lung, but there was no significant increase in the IFN-γ⁺CD8⁺ T-cell population (Figure 3A). In addition, IFN-γ producing in CD4⁺ T cells in splenocytes was significantly increased upon CS exposure, not in CD8⁺ T and NK cells (Figure S1C in Supplementary Material). In the case of IL-17A⁺-secreting T cells, there was a significant increase in IL-17A⁺CD4⁺ T cells in the lung but not in the colon. There was no difference in IL-17A⁺CD8⁺ T cells in the lung and colon (Figure 3A). To identify specific T-cell subsets involved in CS exposure-induced colitis, we depleted CD4⁺ T cells or CD8⁺ T cells prior to CS exposure (Figure 3B). CD4⁺ T cell depletion neutralized the effect of CS exposure on colonic length. It also strongly blocked the increase in IFN-γ and TNF-α expression in the lung and the immune cell infiltration into the lung by CS exposure. In the colon, however, CD4⁺ T-cell depletion could not completely inhibit the increased expression of IFN-γ and TNF-α (Figures 3C–F). Although CD8⁺ T-cell depletion also affected colon length and the concentrations of IFN-γ and TNF-α expression in colon, the effect of CD4⁺ T-cell depletion was superior to that of CD8⁺ T-cell depletion in both the colon and lung. These results indicate that CD4⁺ T cells are the major cell population that mediates CS exposure-induced inflammation in the colon and lung, although both CD4⁺/8⁺ T cell subsets are involved.

T-bet is a Th1-specific transcription factor that regulates the production of IFN-γ in CD4⁺ T cells (27). As we observed the relevance of IFN-γ and the role of CD4⁺ T cells for CS-induced inflammation above, we exposed T-bet-deficient mice to CS for 4 weeks to assess the role of the Th1 response in CS exposure-induced colitis and lung inflammation. As shown in Figures 4A,B, T-bet^−/+ mice showed a significant reduction in both the colon length and body weight following CS exposure; however, T-bet^−/− mice exhibited significant protection against CS exposure-induced changes in colon length and body weight. Additionally, T-bet^−/− mice showed unaltered expression of IFN-γ and TNF-α upon CS exposure, while T-bet^−/+ mice showed significantly increased expression of IFN-γ and TNF-α following CS exposure in both colon and lung (Figures 4C,F). In T-bet^−/− mice, CS exposure also blocked the effect on infiltrated immune cell numbers and inflammatory cytokine concentrations (Figures 4D–F). These results indicate more clearly that CS-induced inflammatory responses are influenced by Th1 responses.

To confirm whether CS-induced inflammatory responses are IFN-γ dependent in the colon, we exposed IFN-γ-deficient mice to CS. The CS exposure caused a noticeable decline in the colon length and the body weight in IFN-γ^−/+ mice. Meanwhile, the CS-exposed IFN-γ^−/− mice demonstrated a significantly improved colon length compared to the CS-exposed IFN-γ^−/+ mice (Figure 5A). Protection from body weight loss by IFN-γ deficiency was observed from day 22 (Figure 5B). CS exposure caused the IFN-γ^−/+ mice to express highly increased concentrations of IFN-γ and TNF-α in the colon and lung, while no IFN-γ was detected in IFN-γ^−/− mice, as expected (Figures 5C,D). IFN-γ deficiency significantly inhibited immune cell infiltrations into the lung by CS exposure (Figure 5E). Interestingly, IFN-γ deficiency decreased CS exposure-induced secretion of TNF-α in the colon and also protected the lung from inflammation by CS exposure (Figures 5D–F).

To confirm the role of IFN-γ in the induction of colitis via CS-stimulated CD4⁺ T cells, we performed an AT of lung draining lymph node CD4⁺CD25⁻ T cells from WT or IFN-γ^−/− mice to colitis-prone IL-10^−/− mice (Figure 6A) (28). Fourteen days later, after the AT of isolated lung draining lymph node CD4⁺CD25⁻ T cells from WT mice, body weight loss was observed in IL-10^−/− mice (Figure 6C), but the same cells adoptively transferred from IFN-γ^−/− mice did not cause body weight loss (Figure 6F). The AT of WT cells shortened colon length (Figure 6B) and increased IFN-γ and TNF-α expression (Figure 6D); however, the AT of IFN-γ-deficient cells did not cause those changes (Figures 6E,G), indicating that IFN-γ producing CS-exposed CD4⁺ T cells play a substantial role in inducing colitis. We also examined an expression of gut homing integrin, A4B7, in CD4⁺ T cells (29). When we check it in the blood and spleen, CS exposure increased the expression of A4B7 in CD4⁺ T cells (Figure S1D in Supplementary Material).

To follow the migration of CS-stimulated CD4⁺ T cells into colon, we performed an AT of lung draining lymph node CD4⁺CD25⁻ T cells from WT^EGFP to IL-10^−/− mice. The IL-10^−/− mice which received the AT of CS-stimulated lung draining lymph CD4⁺CD25⁻ T cells observed a significant increase the population of CD4⁺ T cells in MLNs and colon compared to the Air group (Figures 7A,E). However, the other tissues including lung, lung draining lymph, and spleen did not show increase in CD4⁺ population (Figures 7B–D). Altogether, these data demonstrated that CS stimulates CD4⁺CD25⁻ T cells migration from lung draining lymph node to MLNs and colon in IL-10^−/− mice.