Therapeutic Potential of Combining IL-6 and TNF Blockade in a Mouse Model of Allergic Asthma

Abstract

Combined anti-cytokine therapy is a promising therapeutic approach for uncontrolled steroid-resistant asthma. In this regard, simultaneous blockade of IL-4 and IL-13 signaling by Dupilumab (anti-IL-4Ra monoclonal antibody) was recently approved for severe eosinophilic asthma. However, no therapeutic options for neutrophilic asthma are currently available. Recent advances in our understanding of asthma pathogenesis suggest that both IL-6 and TNF may represent potential targets for treatment of severe neutrophilic asthma. Nevertheless, the efficacy of simultaneous pharmacological inhibition of TNF and IL-6 in asthma was not yet studied. To evaluate the potency of combined cytokine inhibition, we simultaneously administrated IL-6 and TNF inhibitors to BALB/c mice with HDM-induced asthma. Combined IL-6/TNF inhibition, but not individual blockade of these two cytokines, led to complex anti-inflammatory effects including reduced Th2-induced eosinophilia and less prominent Th17/Th1-mediated neutrophilic infiltrate in the airways. Taken together, our results provide evidence for therapeutic potential of combined IL-6/TNF inhibition in severe steroid-resistant asthma.

Keywords: HDM-induced asthma; Th17/Th1-induced neutrophilia; Th2-induced eosinophilia; anti-cytokine therapy.

Figure 1

Combined pharmacological inhibition of TNF and IL-6 in acute HDM-induced asthma. (A) Scheme of the experiment. Acute asthma was induced in 6–8-week old BALB/c mice by daily i.n. administration of 20 µg HDM extract per mouse for six days with the sensitization of 5 µg HDM one week prior to the main course (red arrows). Anti-IL-6 antibody (MP5-20F3) (5 µg/g of body weight), anti-TNF inhibitor (Etanercept) (10 µg/g of body weight), and saline as a control were administered i.p. every 48 h for 13 days according to the scheme (blue arrows). Prior to i.n. administration of HDM, mice were anesthetized by 3% isoflurane delivered with oxygen. BALF was collected for analysis 48 h after the last challenge. Frequencies (%) of eosinophils (Siglec-F⁺ CD11c⁻), neutrophils (Ly6G⁺ CD11b⁺) (B) and alveolar macrophages (SiglecF⁺ CD11c⁺) (C) gated on CD45⁺ live cells in the BALF were assessed by flow cytometry. (D) Total cell numbers in the BALF. (E) IgE production (ng/mL) in the BALF was determined by ELISA on day 14. Each point on a diagram represents a single mouse (5–12 mice in each group); mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; #—**** p < 0.0001 (naïve vs. HDM + saline), *** p < 0.001 (naïve vs. HDM + aIL-6), ** p < 0.01 (naïve vs. HDM + aTNF); * p < 0.05 (naïve vs. HDM + aIL-6/aTNF) (one-way ANOVA test was used). HDM, house dust mite; BALF, bronchoalveolar lavage fluid.

Figure 2

Th2- and Th1-cell inflammatory responses in the lungs of HDM-treated mice with simultaneous administration of IL-6 and TNF inhibitors. (A) Frequency (%) of Th2-cells (IL-13⁺) among TCRb⁺ CD4⁺ cells in the lungs. (B) Protein levels of Th2-associated cytokines (pg/mL) in the BALF were measured by multiplex analysis on day 14. (C) Frequency (%) of Th1-cells (TNF⁺ IFNγ⁺) gated on TCRb⁺ CD4⁺ live cells in the lungs. (D) IFNγ production (pg/mL) in the BALF was determined by multiplex analysis on day 14. (E) Frequency (%) of Treg cells (FoxP3⁺ HELIOS⁺) in the lungs and lymph nodes (LN). Data represent mean ± SD, 4–12 mice per group with each point representing a single mouse. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (one-way ANOVA test was used).

Figure 3

Th17-cell response in HDM-treated mice with combined anti-TNF/IL-6 treatment. Representative FACS plots (A) and frequency (%) (B) of IL-17A⁺ RORγt⁺ cells gated on TCRb⁺ CD4⁺ live cells in the lungs of control mice and mice with anti-cytokine treatment. (C) Relative expression of Il17a gene normalized to Actb by quantitative RT-PCR analysis in the lungs 48 h after last HDM challenge. Each point in a diagram represents a single mouse (5–12 mice in each group); mean ± SD. One-way ANOVA test revealed: * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

Assessment of lung histology in mice with severe HDM-induced asthma undergoing combined administration of IL-6 and TNF inhibitors. Simultaneous pharmacological inhibition of TNF and IL-6 led to diminished lung tissue remodeling in severe HDM-induced asthma. (A) Scheme of severe asthma model; 6–8-week-old BALB/c mice were sensitized i.t. with HDM extract on days 0 and 4 (100 μg) (red arrows). On days 14–17, mice were challenged i.t. with HDM (100 μg) (red arrows). Prior to i.t. administration of the allergen, mice were anesthetized by i.p. injection of the Zoletil/Xyla cocktail for anesthesia. Anti-IL-6 (MP5-20F3) (5 μg/g of body weight), anti-TNF (XT3.11) (10 μg/g of body weight) antibodies, and saline as a control were administered intraperitoneally prior to each immunization every 48 h for 17 days (blue arrows); 48 h after the last challenge lungs were perfused by cardiac puncture using 0.9% NaCl and fixed in 4% PFA for further histological assessment of lung tissue. (B) Representative PAS-stained lung tissue sections (original magnification: 50×, scale bar: 500 μm) in control mice with saline and mice injected with anti-TNF and/or anti-IL-6 antibodies (i.p.). (C) Lung inflammation and (D) PAS-positive cell score. (E) Relative expression of Tgfb1 gene normalized to Actb by quantitative RT-PCR analysis in the lungs 48 h after last HDM challenge. Data represent mean ± SD, 5–6 mice per group. * p < 0.05 (one-way ANOVA test was used).

Figure 5

Combined anti-IL-6/TNF administration induces a complex anti-inflammatory effect in the context of severe asthma. Inhibition of IL-6 demonstrated decreased frequency of neutrophils, whereas TNF ablation predominantly suppressed eosinophilic infiltrate into the airways. Moreover, administration of IL-6 neutralizing antibodies prevented the accumulation of Th17- and Th1- cells in the airways, caused by pharmacological TNF ablation. Th2-associated eosinophilia is inhibited under TNF blockade and was not observed under anti-IL-6 administration. On the contrary, combined pharmacological inhibition of IL-6 and TNF had a complex effect consisting of reduction in both Th2-associated eosinophilia and Th1/Th17-mediated neutrophilia and prevention of tissue remodeling in the airways. Therefore, combined anti-IL-6/TNF therapy may be beneficial in inhibiting the key inflammatory responses in allergic airway inflammation and preventing the side effects of monotherapy.