In vitro Culture with Cytokines Provides a Tool to Assess the Effector Functions of ILC2s in Peripheral Blood in Asthma

Figures & data

Table 1 Study Subjects

Characteristics	HC (n=12)	MA (n=11)	P value
Age (y), mean±SEM	40.3±4.6	44.0±5.3	0.9558
Sex, female/male	10/2	5/6	0.0894
Asthma, n	0	11
Ever smoked (%)	0	9.1	0.4783
Current smoker (%)	0	0
Ever had immunotherapy	0	0
FEV1 (% predicted), Mean	95	78	0.0309*
Medications, n
ICS	0	4	0.0373*
LABA	0	3	0.0932
SABD	0	11	<0.001
LTM	0	5	0.0137*
Skin test, positive/subjects tested	0/2	9/10	0.0455*

Abbreviations: HC, healthy control; MA, mild asthma; ICS, Inhaled corticosteroids; LABA, long-acting beta-agonists; SABD, short-acting bronchodilators; LTM, leukotriene modifiers. *P<0.05.

Figure 1 FACS analysis of blood ILC2 numbers. PBMCs from healthy control subjects (Control, n=12) and asthma subjects (Asthma, n=11) were analyzed by FACS. Panel (A) shows the gating strategy for ILC2s. Panels (B and C) show summaries of total numbers of PBMCs per milliliter of blood and proportion of conventional (CD127⁺CRTH2⁺) ILC2s in PBMCs. Panels (D and E) show total numbers of conventional (CD127⁺CRTH2⁺) and nonconventional (CD127⁺CRTH2⁻) ILC2s per milliliter of blood, respectively. Each dot represents 1 subject, and horizontal bars indicate the means. Male and female subjects are represented by open and closed symbols, respectively.

Figure 2 IL-33-induced cytokine production by PBMCs. PBMCs from healthy control subjects (Control) or mild asthma subjects (Asthma) were cultured with media alone, IL-33 alone (50 ng/mL), or IL-33 plus IL-2 (20 U/mL) (A, C, E). The levels of IL-5, IL-13, and IFN-γ in culture supernatants were determined by ELISA. Each dot represents 1 subject, and horizontal bars indicate the means. Alternatively, PBMCs were cultured with the indicated concentration of IL-33 with or without IL-2 (B, D, F). Data are presented as means±SEMs (n=8–12 in each group). *p < 0.05 between the groups indicated by horizontal lines.

Figure 3 IL-25-induced-cytokine production by PBMCs. PBMCs from healthy control subjects (Control) or mild asthma subjects (Asthma) were cultured with media alone, IL-25 alone (50 ng/mL), or IL-25 plus IL-2 (20 U/mL) (A, C, E). The levels of IL-5, IL-13, and IFN-γ in culture supernatants were determined by ELISA. Each dot represents 1 subject, and horizontal bars indicate the means. Alternatively, PBMCs were cultured with the indicated concentration of IL-25 with or without IL-2 (B, D, F). Data are presented as means±SEMs (n=8–12 in each group). *p < 0.05 between the groups indicated by horizontal lines.

Figure 4 IL-12 effects on cytokine production by PBMCs. PBMCs from healthy control subjects (Control, n=4) or mild asthma subjects (Asthma, n=6) were cultured with IL-33 alone (50 ng/mL), IL-33 plus IL-2 (20 U/mL), or a combination of IL-33, IL-2 and IL-12 (5 ng/mL). The levels of IFN-γ (A) and IL-5 (B) in culture supernatants were determined by ELISA. Each dot represents 1 subject, and horizontal bars indicate the means.

Figure 5 FACS analysis of IL-5 production by PBMCs. PBMCs from asthma subjects were cultured with IL-33 plus IL-2 for 4 days and with brefeldin A for the final 3 hours. The cells were then stained for intracellular IL-5 (or with isotype control antibody), lineage markers (CD3, CD11c, CD14, CD19, CD34, CD56, CD94, CD123, and FcεRIα) and CD127. IL-5 production was analyzed by gating on lineage-positive (Lin⁺) cells and the Lin⁻CD127⁺ cell population. Data are from one representative subject from three asthmatic subjects, which showed concordant findings.