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. 2023 Jan 31;15(1):77-89.
doi: 10.21037/jtd-22-715. Epub 2023 Jan 10.

Allergen-specific immunotherapy with Alutard SQ improves allergic inflammation in house-dust mites-induced allergic asthma rats through inactivation of the HMGB1/TLR4/NF-κB pathway

Affiliations

Allergen-specific immunotherapy with Alutard SQ improves allergic inflammation in house-dust mites-induced allergic asthma rats through inactivation of the HMGB1/TLR4/NF-κB pathway

Yingying Zhai et al. J Thorac Dis. .

Abstract

Background: Allergen-specific immunotherapy (AIT) is the only available safe, effective, and long-term treatment for allergic airway diseases, including allergic asthma. However, the potential molecular mechanism of AIT in ameliorating airway inflammation remains unknown.

Methods: Rats were sensitized and challenged with house dust mite (HDM) and administered with Alutard SQ or/and high mobility group box 1 (HMGB1) inhibitor, ammonium glycyrrhizinate (AMGZ) or HMGB1 lentivirus. The total and differential cell counts in rat bronchoalveolar lavage fluid (BALF) were detected. Hematoxylin and eosin staining (H&E) was performed to examine the pathological lesions in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the expression of inflammatory factors in lungs, BALF, and serum. Quantitative real-time PCR (qRT-PCR) was used to measure the levels of inflammatory factors in the lungs. Western blot assay was used to evaluate the expression of HMGB1, Τoll-like receptor 4 (TLR4), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the lungs.

Results: Consequently, AIT with Alutard SQ attenuated airway inflammation, the total and differential cells in BALF, and expression of Th (T helper)2 related cytokines and transforming growth factor beta 1 (TGF-β1). The regimen also upregulated Th-1-related cytokine expression by inhibiting the HMGB1/TLR4/NF-κB pathway in HDM-induced asthmatic rats. Furthermore, AMGZ, a HMGB1 antagonist, amplified the functions of AIT with Alutard SQ in the asthma rat model. Nevertheless, overexpression of HMGB1 reversed the functions of AIT with Alutard SQ in the asthma rat model.

Conclusions: In summary, this work demonstrates the role of AIT with Alutard SQ, which inhibits the HMGB1/TLR4/NF-κB signaling pathway in allergic asthma management.

Keywords: Asthma; HMGB1/TLR4/NF‐κB pathway; airway inflammation; allergen-specific immunotherapy (AIT) with Alutard SQ; house dust mite.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-22-715/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Alutard SQ ameliorates HDM-induced airway inflammation. (A) The total and differential cell counts in the BALF of the rat from normal, asthma (HDM), asthma + Alutard SQ low dose (HDM + ALD), asthma + Alutard SQ middle dose (HDM + AMD), and asthma + Alutard SQ high dose (HDM + AHD) groups. (B) H&E staining of lung tissue in different groups. Error bars represent + SD. N=6 mice per group, *P<0.05, vs. normal; #P<0.05, vs. HDM. HDM, house dust mite; ALD, Alutard SQ low dose; AMD, Alutard SQ middle dose; AHD, Alutard SQ high dose; BALF, bronchoalveolar lavage fluid; H&E, hematoxylin & eosin; SD, standard deviation.
Figure 2
Figure 2
Alutard SQ attenuates protein expression of IL-4, IL-5, IL-6, IL-13, TGF-β1, total and allergen-specific IgE, and induces IFN-γ protein expression in allergic asthma rat models. ELISA analysis of protein levels of IL-4, IL-5, IL-6, IL-13, IFN-γ, TGF-β1, and total and allergen-specific IgE in the BALF, serum, and lung tissues derived from different groups of rats. Error bars represent + SD. N=6 mice per group, *P<0.05, vs. normal; #P<0.05, vs. HDM. HDM, house dust mite; ALD, Alutard SQ low dose; AMD, Alutard SQ middle dose; AHD, Alutard SQ high dose; IL, interleukin; IFN-γ, interferon gamma; Ig, immunoglobulin; sIg, specific immunoglobulin; TGF-β1, transforming growth factor beta 1; ELISA, enzyme-linked immunosorbent assay.
Figure 3
Figure 3
Alutard SQ attenuates mRNA expression of IL-4, IL-5, IL-6, IL-13, and total IgE, but upregulates the mRNA expression of IFN-γ in allergic asthma rat models. qRT-PCR analysis of the mRNA levels of IL-4, IL-5, IL-6, IL-13, IFN-γ, and total IgE in the lung tissues derived from different groups of rats. Error bars represent + SD. N=6 mice per group, *P<0.05, vs. normal; #P<0.05, vs. HDM. IL, interleukin; HDM, house dust mite; ALD, Alutard SQ low dose; AMD, Alutard SQ middle dose; AHD, Alutard SQ high dose; qRT-PCR, quantitative real-time polymerase chain reaction; Ig, immunoglobulin; IFN-γ, interferon-gamma.
Figure 4
Figure 4
Alutard SQ prevents HDM-induced airway inflammation by suppressing the HMGB1/TLR4/NF-κB signal pathway. (A) Western blot analysis of the protein expression of HMGB1, TLR4, p-p65, and p65 in lung tissues derived from normal, HDM, HDM + ALD, HDM + AMD, and HDM + AHD groups of rats. (B) Western blot analysis of the protein levels of HMGB1, TLR4, p-p65, and p65 in the lung tissues obtained from HDM, HDM + AHD, HDM + AHD + AMGZ (HMGB1 inhibitor ammonium glycyrrhizinate), and HDM + AHD + HMGB1 lentivirus groups of rats. (C) The total and differential cell count in the BALF. (D) H&E staining of the lung tissue. Error bars represent + SD. N=6 mice per group, *P<0.05, vs. normal; #P<0.05, vs. HDM; $P<0.05, vs. HDM+AHD. HDM, house dust mite; ALD, Alutard SQ low dose; AMD, Alutard SQ middle dose; AHD, Alutard SQ high dose; AMGZ, ammonium glycyrrhizinate; HMGB1, high mobility group box 1; TLR4, Τoll-like receptor 4; p-p65, phospho-p65; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; BALF, bronchoalveolar lavage fluid; H&E, hematoxylin & eosin.
Figure 5
Figure 5
Alutard SQ improves HDM-provoked asthmatic inflammation by inactivating the HMGB1/TLR4/NF-κB signal pathway. ELISA analysis of protein expression of IL-4, IL-5, IL-6, IL-13, IFN-γ, TGF-β1, and total and allergen-specific IgE in the BALF, serum, and lung tissues derived from HDM, HDM + AHD, HDM + AHD + AMGZ, and HDM + AHD + HMGB1 lentivirus groups of rats. Error bars represent + SD. N=6 mice per group, *P<0.05, vs. normal; #P<0.05, vs. HDM; $P<0.05, vs. HDM + AHD. IL, interleukin; HDM, house dust mite; AHD, Alutard SQ high dose; AMGZ, ammonium glycyrrhizinate; HMGB1, high mobility group box 1; BALF, bronchoalveolar lavage fluid; IFN-γ, interferon-gamma; Ig, immunoglobulin; sIg, specific immunoglobulin; TGF-β1, transforming growth factor beta 1; TLR4, Toll-like receptor 4; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.
Figure 6
Figure 6
Autard SQ alleviates IL-4, IL-5, IL-6, IL-13, and total IgE mRNA expression, while it induces the mRNA expression of IFN-γ in the HDM-induced asthmatic rat by inactivating the HMGB1/TLR4/NF-κB signal pathway. mRNA levels of IL-4, IL-5, IL-6, IL-13, IFN-γ and total IgE in the lung tissues obtained from different groups of rats. Error bars represent + SD. N=6 mice per group, #P<0.05, vs. HDM; $P<0.05, vs. HDM + AHD. IL, interleukin; Ig, immunoglobulin; HDM, house dust mite; AHD, Alutard SQ high dose; AMGZ, ammonium glycyrrhizinate; HMGB1, high mobility group box 1; IFN-γ, interferon-gamma; TLR4, Toll-like receptor 4; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; SD, standard deviation.

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