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. 2023 Jan 27:14:1111123.
doi: 10.3389/fimmu.2023.1111123. eCollection 2023.

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels

Johanna Samulin Erdem  1 , Táňa Závodná  2 , Torunn K Ervik  1 , Øivind Skare  1 , Tomáš Hron  3 , Kristine H Anmarkrud  1 , Anna Kuśnierczyk  4   5 , Julia Catalán  6   7 , Dag G Ellingsen  1 , Jan Topinka  2 , Shan Zienolddiny-Narui  1
Affiliations

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels

Johanna Samulin Erdem et al. Front Immunol. .

Abstract

Introduction: Inhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model.

Methods: MH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications.

Results: Our data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure.

Conclusion: Together, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis.

Keywords: epigenetic; fibrosis; inflammation; macrophage; miRNA; nanomaterials; polarization.

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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
Characterization of nanomaterials. Representative SEM images and length measurements in µm of (A) CNC, (B) CNF, (C) NM-400, and (D) NM-401.
Figure 2
Figure 2
Cellular uptake and effects on membrane permeability at 24h post-exposure. Uptake of nanomaterials was investigated by TEM or immuno-TEM in M1 macrophages. Representative images of (A) Control, (B) CNC, (C) CNF, (D) NM-400, and (E) NM-401-exposed cells. C1: 0.15 μg/cm2 and C2: 2.7 μg/cm2. The experiment was repeated twice. Black arrows indicate endosomal structures with nanomaterials. White arrows indicate fibers in cell cytoplasm. L indicates lysosomal structures. (F) Membrane leakage as measured by medium lactate dehydrogenase (LDH) release following NM-401 exposure. Data indicate mean ± SD, (n=3-5), *p<0.05, **p<0.01.
Figure 3
Figure 3
Effects of nanomaterial exposure on the expression of common macrophage polarization markers. Changes in gene expression were assessed by qPCR following exposure to CNC, CNF, NM-400 and NM-401 in (A) M1 and (B) M2 macrophages. C1: 0.15 μg/cm2 and C2: 2.7 μg/cm2. Expression was related to the mean expression in unexposed control cells which was set to 1. Data represent mean ± SE, (n=5), *p<0.05, ***p<0.001.
Figure 4
Figure 4
Effects on the secretion of cytokines and chemokines following exposure to CNC, CNF, NM-400 and NM-401 (high C2 dose). (A) Venn diagram illustrates the temporal differences in affected cytokines and chemokines in exposed M1 and M2 cells. (B) The combined inflammatory potential of nanomaterial exposure was illustrated by the p−values (−log10) of treatment effects for the deregulated proteins. The cutoff line indicates the significance level corrected for multiple testing. Filled symbols indicate values above the cutoff line where the exposure significantly affected the total secretion of cytokines in the specified macrophage subclass. (C) Cytokines and chemokines with significant changes in their secretion in M1 cells and M2 cells at 4 and 24h of exposure compared to controls. Data indicate mean ± CI, (n=4), *p<0.05, **p<0.01, ***p<0.001.
Figure 5
Figure 5
Effects on the expression of genes regulating epigenetic modifications. Changes in gene expression were assessed by qPCR following exposure to CNC, CNF, NM-400 and NM-401. C1: 0.15 μg/cm2 and C2: 2.7 μg/cm2. (A) Heatmap of the mean fold changes in regulated genes following 24h of nanomaterial exposure in M0, M1 and M2 macrophages. (B) Venn diagram illustrates commonly regulated genes in M1 and M2 cells after NM-400 and NM-401 exposure. (C) Genes significantly regulated following exposure to NM-400. (D) Genes significantly regulated following exposure to NM-401. Data represent mean ± SD, (n=5), p<0.05.
Figure 6
Figure 6
CNF and NM-401 exposure induced changes in miRNA expression. (A) Volcano plot of differentially expressed known miRNAs in CNF-exposed M1 cells. (B) Volcano plot of differentially expressed known miRNAs in NM-401-exposed M2 cells. Y-axes show negative decadic logarithm of uncorrected p-values (-log10 P), x-axes show the binary logarithm of fold changes (log2 fold change). Log2 fold change cutoff = 1, and FDR cutoff = 0.1 are indicated. (C) Clustering heat map of differentially expressed miRNAs -log10(CPM). (D) Venn diagrams of differentially expressed miRNAs and (E) their predicted target genes. PANTHER analyzes of the target genes of differentially expressed miRNA in (F) CNF- and (G) NM-401-exposed macrophages, (n=5-6).
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Grants and funding

This work was supported by the National Institute of Occupational Health, Oslo, Norway (Grant number: 201600262). Mass spectrometry-based analyses were performed by the Proteomics and Modomics Experimental Core (PROMEC), Norwegian University of Science and Technology (NTNU) and The Central Norway Regional Health Authority. This facility is a member of the National Network of Advanced Proteomics Infrastructure (NAPI), which is funded by the Research Council of Norway INFRASTRUKTUR-program (project number: 295910). The authors also acknowledge the assistance provided by the Research Infrastructures NanoEnviCz (Project No. LM2015073), supported by the Ministry of Education, Youth, and Sports of the Czech Republic and the project Pro-NanoEnviCz (Reg. No. CZ.02.1.01/0.0/0.0/16_013/0001821) supported by the Ministry of Education, Youth, and Sports of the Czech Republic and the European Union—European Structural and Investments Funds in the frame of Operational Program Research Development and Education.

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