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. 2023 Oct 27:14:1271342.
doi: 10.3389/fimmu.2023.1271342. eCollection 2023.

Modulating asthma-COPD overlap responses with IL-17 inhibition

Leandro do Nascimento Camargo  1   2 , Renato Fraga Righetti  1   2 , Francine Maria de Almeida  1 , Tabata Maruyama Dos Santos  1   2 , Silvia Fukuzaki  1 , Nilo Arthur Bezerra Martins  1 , Miguel Cantadori Barbeiro  1 , Beatriz Mangueira Saraiva-Romanholo  1 , Fernanda Degobbi Tenorio Quirino Dos Santos Lopes  1 , Edna Aparecida Leick  1 , Carla Máximo Prado  3 , Iolanda de Fátima Lopes Calvo Tibério  1
Affiliations

Modulating asthma-COPD overlap responses with IL-17 inhibition

Leandro do Nascimento Camargo et al. Front Immunol. .

Abstract

Background: IL-17 is a modulator of the inflammatory response and is implicated in lung remodeling in both asthma and chronic obstructive pulmonary disease (COPD). Well as and probably in patients with asthma-COPD overlap (ACO).

Methods: In this study, we evaluated the response of the airways and alveolar septa to anti-IL-17 treatment in an ACO model. Fifty-six male BALB/c mice were sensitized with ovalbumin (OVA group), received porcine pancreatic elastase (PPE group), or both (ACO group). Mice were then treated with either anti-IL-17 monoclonal antibody or saline. We evaluated hyperresponsiveness, bronchoalveolar lavage fluid (BALF) cell counts, and mean alveolar diameter. We quantified inflammatory, response, extracellular matrix remodeling, oxidative stress markers, and signaling pathway markers.

Results: Anti-IL-17 treatment in the ACO anti-IL-17 group reduced the maximum response of respiratory system Rrs, Ers, Raw, Gtis, this when compared to the ACO group (p<0.05). There was a reduction in the total number of inflammatory cells, neutrophils, and macrophages in the BALF in the ACO anti-IL-17 group compared to the ACO group (p<0.05). There was attenuated dendritic cells, CD4+, CD8+, FOXP3, IL-1β, IL-2, IL-6, IL-13, IL-17, IL-33 in ACO anti-IL-17 group in airway and alveolar septum compared to the ACO group (p<0.05). We observed a reduction of MMP-9, MMP-12, TIMP-1, TGF-β, collagen type I in ACO anti-IL-17 group in airway and alveolar septum compared to the ACO group (p < 0.05). We also observed a reduction of iNOS and 8-iso-PGF2α in the airways and in the alveolar septum was reduced in the ACO anti-IL-17group compared to the ACO group (p < 0.05). Regarding the signaling pathways, NF-kB, ROCK-1, and ROCK-2 in the airway and alveolar septum were attenuated in the ACO anti-IL-17 group when compared to the ACO group (p<0.05).

Conclusions: Our results suggest that inhibiting IL-17 modulates cell-associated cytokine production in lung tissue, extracellular matrix remodeling, and oxidative stress in ACO through the modulation of NF-kB and FOXP3.

Keywords: anti-IL-17; asthma; asthma-COPD overlap (ACO); extracellular matrix remodeling; inflammation; oxidative stress.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
The sensitization protocol was conducted for 28 days. On days 1 and 14, BALB/c mice received intraperitoneal (i.p.) injection of OVA solution. On days 21, 23, 25, and 27, the animals were exposed to 1% OVA aerosol for 30 minutes. The control group received intraperitoneal saline solution and 0.9% saline solution aerosol. PPE instillation was performed intratracheally on day 21. Treatment groups were given neutralizing anti-IL-17 antibody intraperitoneally on days 22, 23, 25, and 27. On day 28, respiratory system mechanics measurements and bronchoalveolar lavage collection were performed.
Figure 2
Figure 2
Description of the primary antibodies used in the immunohistochemical staining of the lung samples.
Figure 3
Figure 3
Results of airway hyperresponsiveness in relation to respiratory mechanics parameters (Rrs, Ers, Raw, Gtis, and Htis). The mean ± standard error of the maximum response after methacholine challenge for all experimental groups. Values are presented as percentages. Resistance (Rrs); Elastance (Ers); Resistance of larger airways (Raw); Resistance of smaller airways or tissues (Gtis) and elastance of lung tissues (Htis). *p < 0.05, compared to the SAL group; +p < 0.05 comparado ao grupo PPE; #p < 0.05 comparado ao grupo OVA &p < 0.05, compared to the OVA, PPE, and ACO groups.
Figure 4
Figure 4
Results of mean alveolar diameter and qualitative analysis for IL-6. (A) Mean ± standard error of LM evaluation for all experimental groups. Values are presented in micrometers. *p < 0.05, compared to the SAL group; &p < 0.05, compared to the PPE group. (B) Photomicrograph panel showing LM in HE staining (C) and IL-6 in the airway. All figures are presented at a magnification of 400× in the OVA and PPE groups, with scale bars = 40 µm.
Figure 5
Figure 5
Results of eosinophils, neutrophils, macrophages, lymphocytes, and total cells in Bronchoalveolar Lavage Fluid (BALF). Mean ± standard error of BALF assessment for all experimental groups. Values are presented in x104 cells/mL. *p < 0.05, compared to the SAL group; &p < 0.05, compared to the OVA, PPE, and ACO groups.
Figure 6
Figure 6
Results of positive cell expression assessed by hematoxylin and eosin (HE) staining for eosinophils and immunohistochemistry for IL-5 in the airways. Mean ± standard error of eosinophil and IL-5 assessments for all experimental groups. Values are presented in cells/104 µm2. *p < 0.05, compared to the SAL group; +p < 0.05 comparado ao grupo PPE; #p < 0.05 comparado ao grupo OVA &p < 0.05, compared to the OVA and ACO groups.
Figure 7
Figure 7
Results of positive cell expression assessed by immunohistochemistry for cell-associated cytokine production in the airway, and levels of IL-6 and IL-17 measured by ELISA. Mean ± standard error of CD4+, CD8+, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17, and IL-33 assessment for all experimental groups. Values are presented in positive cells/104 µm2 and pg/mL for IL-6 and IL-17. *p < 0.05, compared to the SAL group; +p < 0.05 comparado ao grupo PPE; #p < 0.05 comparado ao grupo OVA &p < 0.05, compared to the OVA, PPE, and ACO groups.
Figure 8
Figure 8
Results of positive cell expression assessed by immunohistochemistry for cell-associated cytokine production in the alveolar septa. Mean ± standard error of CD4+, CD8+, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13, IL-17, and IL-33 assessment for all experimental groups. Values are presented in positive cells/104 µm2. *p < 0.05, compared to the SAL group; +p < 0.05 comparado ao grupo PPE; #p < 0.05 comparado ao grupo OVA &p < 0.05, compared to the OVA, PPE, and ACO groups.
Figure 9
Figure 9
Results of positive cell expression assessed by immunohistochemistry for extracellular matrix remodeling in the airway and levels of TGF- β measured by ELISA. Mean ± standard error of MMP-9, MMP-12, TIMP-1, TGF-β and total collagen fiber content, types I, III, and V assessment for all experimental groups. Values are presented in positive cells/104 µm2, % and pg/mL for TGF-β. *p < 0.05, compared to the SAL group; +p < 0.05 comparado ao grupo PPE; #p < 0.05 comparado ao grupo OVA &p < 0.05, compared to the OVA, PPE, and ACO groups.
Figure 10
Figure 10
Results of positive cell expression assessed by immunohistochemistry for signaling pathways in the airway and alveolar septa. Mean ± standard error of dendritic cells, FOXP3, p65-NFκB, ROCK-1 and ROCK-2 assessment for all experimental groups. Values are presented in positive cells/104 µm2 *p < 0.05, compared to the SAL group; +p < 0.05 comparado ao grupo PPE; #p < 0.05 comparado ao grupo OVA &p < 0.05, compared to the OVA, PPE, and ACO groups.
Figure 11
Figure 11
Photomicrograph of immunohistochemical staining of airway walls to detect IL-17, total collagen fiber, iNOS, and NF-κB (400× magnification)/Scale bar = 40 μm.
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