Serum amyloid A promotes emphysema by triggering the reciprocal activation of neutrophils and ILC3s

Dear Editor, Chronic obstructive pulmonary disease (COPD) is a group of irreversible lung diseases that include emphysema and chronic bronchiolitis.1 Many studies have shown that COPD is due to adaptive immune responses, particularly CD8 and CD4 T cells2,3; however, the role(s) that innate immune cells such as innate lymphoid cells (ILCs) and granulocytes play in the pathogenesis of emphysema and COPD is largely unknown. Here, we show that neutrophils, and particularly type 3 innate lymphoid cells (ILC3s), play a key role in the development of COPD with the emphysema phenotype. To explore the roles of ILCs in COPD pathogenesis, we induced the chronic phase of emphysema by treating lipopolysaccharide (LPS) and porcine pancreatic elastase (PPE) in the Rag1–/– (which lack mature B and T cells) and wildtype (WT) mice for 4 weeks (Figures 1A and S1A). This treatment induced emphysema-like changes in the lung (Figures 1B and S1B). Since previous studies suggest that emphysema associates with elevated circulating serum amyloid A (SAA) levels,4,5 we examined the mRNA expression of SAA and found that Saa3 was particularly increased in the emphysematous lung but not in the liver (Figure 1C). Induction of SAA was also confirmed by flow cytometry (Figures 1D,E and S1C,D). Moreover, the level of hSAA1, whose amino acid sequence is most similar to that of mouse SAA3,6 is higher in the sputum of emphysematous patients than in the sputum of non-emphysematous COPD patients (Table S1 and Figure 1F). Monocytes and dendritic cells were the main cellular sources of SAA in the emphysema-induced lungs (Figures 1G,H and S1E,F). Under the same condition, both interferon (IFN)-γ and interleukin (IL)-17AILCs are significantly increased (Figures 1I,J and S1G,H). The same trend for increasing SAA and ILC3s was observed in the acute model of emphysema (Figure S2A–E). Consistent with this, the frequency of ILC3s in the sputum of emphysematous COPD patients was higher than that of non-emphysematous patients (Figure 1K).

Dear Editor, Chronic obstructive pulmonary disease (COPD) is a group of irreversible lung diseases that include emphysema and chronic bronchiolitis. 1 Many studies have shown that COPD is due to adaptive immune responses, particularly CD8 + and CD4 + T cells 2,3 ; however, the role(s) that innate immune cells such as innate lymphoid cells (ILCs) and granulocytes play in the pathogenesis of emphysema and COPD is largely unknown. Here, we show that neutrophils, and particularly type 3 innate lymphoid cells (ILC3s), play a key role in the development of COPD with the emphysema phenotype.
To explore the roles of ILCs in COPD pathogenesis, we induced the chronic phase of emphysema by treating lipopolysaccharide (LPS) and porcine pancreatic elastase (PPE) in the Rag1 -/-(which lack mature B and T cells) and wildtype (WT) mice for 4 weeks ( Figures 1A and S1A). This treatment induced emphysema-like changes in the lung ( Figures 1B and S1B). Since previous studies suggest that emphysema associates with elevated circulating serum amyloid A (SAA) levels, 4,5 we examined the mRNA expression of SAA and found that Saa3 was particularly increased in the emphysematous lung but not in the liver ( Figure 1C). Induction of SAA was also confirmed by flow cytometry (Figures 1D,E and S1C,D). Moreover, the level of hSAA1, whose amino acid sequence is most similar to that of mouse SAA3, 6 is higher in the sputum of emphysematous patients than in the sputum of non-emphysematous COPD patients (Table S1 and Figure 1F). Monocytes and dendritic cells were the main cellular sources of SAA in the emphysema-induced lungs (Figures 1G,H and S1E,F). Under the same condition, both interferon (IFN)-γ + and interleukin (IL)-17A + ILCs are significantly increased (Figures 1I,J and S1G,H). The same trend for increasing SAA and ILC3s was observed in the acute model of emphysema ( Figure S2A-E). Consistent with this, the frequency of ILC3s in the sputum of emphysematous COPD patients was higher than that of non-emphysematous patients (Figure 1K).
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Next, we asked whether SAA itself could provoke emphysema and increase ILC3s. Even a single dose of rhSAA1 caused acute inflammation and emphysema in the lung (Figure 2A,B). Remarkably, the administration of rhSAA1 specifically increased the actively proliferating IL-17A + ILC3s ( Figure 2C-E). In contrast, there were minimal changes in the T H cells ( Figure S3A,B). To test whether SAA directly promoted ILC3 expansion, we treated naïve ILCs with rhSAA1 in vitro and found that rhSAA1 did slightly increase the number of IL-17A + cells ( Figure 2F,G). Since the increase in ILC3s by in vivo rhSAA1 treatment was much higher ( Figure 2D), factors other than SAA may induce the proliferation of ILC3s. To identify those factors, we compared the expression of innate cytokines that can stimulate ILCs and found that only Il1b expression was elevated after rhSAA1 administration ( Figures 2H and  S4A). The increase in IL-1β by rhSAA1 was also confirmed by flow cytometry ( Figure 2I,J), and IL-1β-producing cells were mostly neutrophils (Figures 2K,L and S4B). When naïve neutrophils derived from bone marrow were stimulated with rhSAA1 in vitro, the expression of Il1b (Figure 2M) and of receptors that are known to recognise SAA, namely, formyl peptide receptor 2 (Fpr2), Toll-like receptor 2 (Tlr2) and Tlr4 7 was increased ( Figure S4C). However, this upregulation was not observed in ILCs ( Figure S4D). Moreover, the frequency of neutrophils in the sputum was higher in COPD patients with emphysema ( Figure 2N). In the sputum, neutrophils and IL-1β show a positive correlation, and IL-1β was elevated in emphysematous COPD patients ( Figure 2O,P).
To understand how ILC3s induced the emphysema, we first measured the gene expression of matrix metalloproteinases (MMPs) -1, 2, 8, 9 and 12, which play essential roles in the development of emphysema. 8 Only Mmp12 expression was significantly increased in the lungs from LPS/PPE-treated Rag1 -/mice ( Figure 4A) and reduced by ILC depletion ( Figure 4B). Although alveolar macrophages expressed the highest MMP12 levels, they did not change with emphysema. Instead, neutrophils significantly increased the expression of MMP12 in the emphysema condition ( Figure 4C,D). Therefore, we hypothesised that ILC3s induce neutrophils to express MMP12 in the emphysematous lung. To test this, we co-cultured naïve neutrophils with activated ILC3s and measured the expression of MMP12 ( Figure 4E). MMP12 expression in the neutrophils was low but elevated when neutrophils were co-cultured with activated ILC3s (Figures 4F and  S5A-B). This was also confirmed by flow cytometry, and IL-17 blockade abrogated the MMP12 expression in the neutrophils ( Figure 4G,H). Thus, ILC3s induce emphysema by promoting the MMP12 secretion of neutrophils. Finally, we asked whether this SAA-neutrophil-ILC3 axis is also involved in the pathogenesis of the emphysema phenotype in COPD patients. SAA1, neutrophils and ILC3s were increased in the induced sputum from emphysematous COPD patients and were positively correlated with each other ( Figure 4I-K). Moreover, lung function was decreased significantly as the lung neutrophils and ILC3s increased ( Figure 4L-N). Together, these data suggest that the SAA-neutrophil-ILC3 axis is also applicable to COPD patients with emphysema.
In conclusion, although the role of ILC3s in lung disease has been underestimated due to the rarity, the current study suggested that lung-resident ILC3s may be critical regulators of emphysema and perhaps also acute exacerbation of chronic obstructive pulmonary diseases. The present study showed that ILC3s and neutrophils reciprocally interact to induce emphysema, but ILC3s may be the key emphysema-inducing cells: This is shown by the fact that (1) ILC3 depletion improved the emphysema even in the presence of neutrophils, and (2) ILC3 depletion in emphysema-induced mice eliminated the ability of neutrophils to produce MMP12. Therefore, ILC3-neutrophils could be applied to COPD patients with emphysema through IL-17A-based immunotherapy. To this end, how ILC3s are specifically targeted to modulate local immune responses requires further exploration.

F U N D I N G I N F O R M AT I O N
This study was supported by grants from the Korea Healthcare Technology R&D Project of the Ministry of Health and Welfare, Korea (HI15C3083).

C O N F L I C T O F I N T E R E S T
Authors declare that they have no competing interests.