Abstract
Eosinophils are terminally differentiated granulocytes that have long been considered as destructive cells associated with Th2 type immune responses such as allergic inflammation and helminth infections. Recently, eosinophils have been actively studied as multifunctional leukocytes regulating an array of physiological responses through interaction with other immune cells. In this study, we examined the expression and function of Toll-like receptors (TLRs) in eosinophilic EoL-1 cells and demonstrated the expression of a number of immune mediators in activated EoL-1 cells and their interaction with the macrophage cell line THP-1 upon TLR4 ligand stimulation. EoL-1 cells differentiated with butyrate increased expression of TLR3, TLR4, and TLR7 at mRNA and protein level with flow cytometry analysis. Mature eosinophils derived from human cord blood CD34+ cells were subjected to RNA-sequencing, and showed the expression of a panel of TLR transcripts and TLR4 was the most highly expressed TLR. Among the cognate ligands of TLR3, TLR4, and TLR7, lipopolysaccharide (LPS) or palmitic acid significantly increased mRNA expression of immune mediators in differentiated EoL-1 cells. Notably, Western blot analysis of palmitic acid-treated differentiated EoL-1 cells showed significantly up-regulated expression of Th2 type cytokines and transcription factors driving eosinophil differentiation. To evaluate functional significance of TLR4 ligand-stimulated eosinophils, we added conditioned media (CM) from EoL-1 cells to differentiated THP-1 cells and assessed the expression of M1 macrophage or M2 macrophage-related markers. M1 and M2 macrophage markers were significantly upregulated by CM from LPS and palmitic acid stimulated EoL-1 cells, respectively. In addition, the adipose tissue of obese mice, where eosinophils are decreased due to obesity-induced inflammation, showed significantly decreased frequency of M2 macrophages, despite an increase in the total macrophage numbers. Based on these collective data, we proposed that eosinophils regulate both inflammatory and anti-inflammatory polarization of macrophages through functional changes induced by different TLR4 ligands.
Highlights
Eosinophils are terminally differentiated innate immune cells, classically described as being involved in allergic diseases and helminth parasitic infections (Gleich and Adolphson, 1986; Rothenberg and Hogan, 2006)
To further dissect the molecular mechanisms behind distinctive cytokine production induced by LPS or palmitic acid in differentiated eosinophilic EoL-1 cells (dEoL-1) cells, we examined expression of MAP kinases (MAPK) signaling molecules, which activate in response to TLR4 stimulation (Rao, 2001; Prince et al, 2011), and eosinophil-lineage associated transcription factors
We examined EoL-1 cells and showed a significant increase of TLR3, TLR4, and TLR7 at both mRNA and protein level by butyrate-induced differentiation (Figures 1, 2)
Summary
Eosinophils are terminally differentiated innate immune cells, classically described as being involved in allergic diseases and helminth parasitic infections (Gleich and Adolphson, 1986; Rothenberg and Hogan, 2006). Increasing evidence supports that eosinophils are multifunctional leukocytes regulating various physiologic responses; eosinophils support tissue regeneration (Heredia et al, 2013), promote homeostatic intestinal immune responses (Jung et al, 2015), regulate adipose tissue macrophage polarization and glucose metabolism (Wu et al, 2011; Lee et al, 2018), and mediate the killing of bacteria, viruses, and fungi (Inoue et al, 2005; Dyer et al, 2009; Linch et al, 2009; Qiu et al, 2014). It is necessary to identify the underlying mechanism that supports the multifunctional aspects of eosinophils as immune-modulatory cells. TLR7 is known to be the most prominent TLR in eosinophils, as supported by TLR7-induced eosinophil activation in asthma (Hiraguchi et al, 2011), and activation of neutrophil upon TLR7 ligation in the eosinophilic cell line EoL-1 (Kim et al, 2018). The precise expression profile and functions of TLRs in eosinophils have remained largely unknown, especially in modulating immune responses by interacting with other immune cells
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