Development of Spontaneous Airway Changes Consistent with Human Asthma in Mice Lacking T-bet Susetta Finotto, Markus F. Neurath, Jonathan N. Glickman, Shixin Qin, Hans A. Lehr, Francis H. Y. Green, Kate Ackerman, Kathleen Haley, Peter R. Galle, Susanne J. Szabo, Jeffrey M. Drazen, George T. De Sanctis, and Laurie H. Glimcher

Supplementary Material

A: Confirmatory measurement of AHR in mice using conventional lung mechanics.
The findings of AHR in unanesthetized T-bet deficient mice, assessed by the non-invasive Penh technique, were confirmed by direct measurement of pulmonary resistance. In this method mice are anesthetized and instrumented for measurement of trans-pulmonary pressure and the respiratory airflow (12). In this assay full dose-response curves for the effects of intravenous methacholine on pulmonary resistance are created; comparisons are made among groups of curves.

Groups of mice (eight mice per group) were sensitized with OVA intraperitoneally and sham challenged with PBS aerosol (OVA/PBS) as previously described (13). Mice lacking the T-bet protein exhibit a dose-related increase in RL compared to control littermate mice. Differences were evaluated for statistical significance (* =P<0.05) by Student's two tailed t test for independent events. Data are given as mean values +/- SEM (wt versus T-bet (-/-): p=0.038 at 100mg/kg; p=0.044 at 330mg/kg; p= 0.0198 at 100mg/kg; wt versus T-bet (+/-): p= 0.037 at 100mg/kg; p=0.014435 at 330mg/kg; p=0.039 at 1000 mg/kg).

Medium version | Full size version

---

B: Peribronchial and perivascular infiltration of eosinophils and lymphocytes.
Quantification of the lymphocytes and eosinophils in the airways of the T-bet KO mice. Lung sections were scored by two pathologists masked as to genotype and treatment. Each characteristic was assigned a from 0 ( no inflammation ) to 4 (maximum infiltrate of eosinophils or lymphocytes).

Eosinophils/ Lymphocytes	WT (+/+)	T-bet (-/-)
Venules	0	3
Bronchi	0	3
Alveoli	0	1
Interstitium	0	2

---

Supplemental Figure 2.
C: Cytokines in BALF from wt and T-bet deficient mice.
We examined whether the structural changes in the airway of T-bet deficient mice were associated with a difference in the patterns of cytokine expression between wt and T-bet deficient mice.

Increased TGF-beta₁ (A), and TNFa (B) in BALF of T-bet näive deficient mice. TGF-beta1 and TNFa were measured by ELISA in BALF of unsensitized mice while IL-4 (C) was measured in BALF of mice sensitized with OVA and challenged with PBS aerosol as described above. Results are reported as mean +/- SEM of a minimum of three mice per group. For all three cytokines the T-bet (-/-) values were significantly (P< 0.05) greater than wt values.

IL-13 in BALF was measured in näive wt and T-bet (-/-) mice. In wt mice the levels were 77.0+/- 46.4 pg/ml and in T-bet (-/-) mice levels were 183+/- 1.41 pg/ml. The levels in T-bet(-/-) mice were significantly (P=0.05) higher than in wt mice.

Medium version | Full size version

---

D. Effect of allergen sensitization and challenge on lung mechanics.
AHR assessed as the concentration of methacholine required to double pulmonary resistance, termed the ED₂₀₀R_L, was determined in wt and T-bet deficient mice sensitized to OVA and receiving OVA or PBS aerosol challenge. In this assay a lower numerical value of the ED₂₀₀R_L denotes enhanced airway responsiveness. The wt animals were less responsive to methacholine than the T-bet deficient animals regardless of their challenge status.

Genotype	0VA/PBS	OVA/OVA
	Log ED 200 RL (mg/ml) methacholine
Wild Type (+/+)	2.46(0.13	2.17±0.06*
T-bet (+/-)	1.87(0.07	1.96±0.08
T-bet (-/-)	1.77(0.092	1.94±0.11
*Significantly less than OVA/PBS, P=0.04

---

E: IL-5 and Eosinophils in bronchoalveolar lavage fluid (BALF)
Bronchoalveolar lavage fluid from wild type and T-bet deficient mice was assayed for IL- 5 using a capture and detection antibody from Rand D Systems. Mice were systemically and locally (by aerosol inhalation) sensitized to ovalbumin as previously described (13) and challenged by exposure to an aerosol of 6% ovalbumin (OVA/OVA) or phosphate buffered saline (PBS) (OVA/PBS). Twenty-four hours after the final challenge, mice were anesthetized and bronchoalveolar lavage performed; concentrations of IL-5 were assayed as noted. The only statistically significant differences occurred between OVA/OVA wt and T-bet(+/-) (p=0.03) and T-bet(-/-) (p=0.05).

Although eosinophils were prominent in histological sections of naïve T-bet deficient mice, no eosinophils were detected in the BALF from T-bet (+/+), T-bet (+/-) or T-bet (-/-) mice sensitized to OVA and challenged with PBS (OVA/PBS). In contrast among mice sensitized to OVA and challenged with OVA (OVA/OVA) the percentage of eosinophils in BALF was :T-bet (+/+), T-bet (+/-) and T-bet (-/-) mice was 82.84+/- 2.79, 79.20+/- 1.9, 84.64+/- 2.69 respectively. Thus there was no effect of T-bet on the recovery of eosinophils from BALF.

Medium version | Full size version

---

F. Immunostaining of mouse airway sections for T-bet and smooth muscle actin.
Frozen sections were fixed in 2% PFA followed by pre-incubation with 2% normal horse serum in PBS/0.5%BSA/0.2% Saponin (blocking solution) for 45 minutes at room temperature. The slides were then incubated overnight at 4°C with the monoclonal anti T-bet antibody (5ug/ml) in PBS/0.5% BSA/0.2% Saponin. Slides were then incubated with biotinylated horse anti-mouse IgG (1:200) followed by streptavidin-conjugated-Cy3 complex (1:1000 in PBS). For detection of alpha smooth-muscle actin sections were incubated overnight with anti-alpha smooth muscle actin-FITC conjugated antibody (1:250 in blocking solution) (Sigma, St. Louis, MI) followed by incubation with Dapi-associated mounting medium (Vector Laboratories Inc., Burlingame, CA )