Two-dimensional (2D) materials have attracted considerable interest in recent years due to their many fascinating properties. However, close attention to the potential impact of these materials on human health and the environment is of critical importance for the safe and sustainable implementation of 2D materials and 2D material-enabled technologies. We and others have addressed the interactions of graphene-related materials including graphene oxide (GO) with the immune system using both in vitro and in vivo models. In some studies, a head-to-head comparison was made between graphene-related materials and carbon nanotubes, especially multi-walled carbon nanotubes, and in most if not all cases, the graphene-related materials were found to be less hazardous, even though, chemically speaking, the materials are essentially the same. But perhaps we need to look beyond cell viability/cell death (a common endpoint in toxicology). Could graphene or other atomically thin materials modulate the immune system without killing cells? Could other “players” be involved (apart from the 2D materials, and the immune system)? To address these questions, we investigated the impact of GO on the microbiome, as well as the role of the microbiome (and its metabolites) for the impact of GO on the immune system, using zebrafish as our model system.
The gut microbiome (i.e., the collection of bacteria that dwell in our gut, primarily in the large bowel or colon) is sometimes considered as our “forgotten organ”. However, several studies published in recent years have shown that the gut microbiome regulates normal (physiological) responses. Hence, metabolic signals generated by the microbiome are known to modulate the immune system of the host. Naturally, the complex interactions between the microbiome and its host can only be studied in a living organism – but this doesn’t mean that mice are the best model. In fact, there are species differences also when it comes to the structure and function of the gut microbiome. Therefore, a model – such as the zebrafish – can be used to demonstrate a mechanism, but the same results cannot a priori be used to draw conclusions about “risk”.
We exposed adult zebrafish to a “low” dose (50 µg/L) or a “high” dose (500 µg/L) of GO for 7 days. We employed wild-type (WT) as well as aryl hydrocarbon (Ah) receptor-deficient fish, as the AhR is a key “sensor” of foreign substances and microbial metabolites. Then, the fish were sacrificed, and 16S rRNA gene sequencing was performed to address possible changes in the gut microbiome. We found that GO triggered significant changes in the gut microbiome, and these changes were AhR-dependent. Next, we generated germ-free zebrafish embryos (i.e., zebrafish lacking a gut microbiome), and could show that GO triggered AhR activation as well as the upregulation of markers of immune cells in this model. To better understand the impact of GO when combined with microbial metabolites, we performed single-cell RNA sequencing (scRNA-seq) on whole embryos (Figure 1), and on immune cells sorted from zebrafish embryos based on the expression of lck (a common marker of lymphoid cells). Essentially, scRNA-seq is an advanced method that allows for an understanding of the genetic “blueprint” for cellular function at high resolution. Moreover, one can define cell types based on their gene expression. Hence, the colorful “clusters” in Figure 1 reflect distinct cell populations in zebrafish identified using scRNA-seq. We could thus show that GO plus butyric acid (BA) (a short-chain fatty acid that is produced by the gut microbiome) elicited a specific population of immune cells belonging to the “primitive” or innate immune system called innate lymphoid cells or ILCs. Specifically, we identified cells resembling human type 2 ILCs, based on gene expression. However, further studies are needed to functionally characterize these ILC2-like cells.
Figure 1. scRNA-seq analysis of wild-type (WT) germ-free (GF) zebrafish embryos exposed to graphene oxide (GO). (a) 2D projection of tSNE analysis showing the lck+ lymphocytes (cluster 5) in the control. (b) 2D projection of tSNE analysis showing the two separate lck+ clusters in embryos exposed to GO + BA, i.e., lck+ innate lymphoid cell (ILC)-like cells (nitr+rag1-, cluster 5) and lck+ T cells (nitr-rag1+, cluster 15).
To conclude, we could show, using the zebrafish as our model, that GO elicits a so-called type 2 immune response. Type 2 immunity is best known for its protective role against parasitic infections, as well as for its role in allergic diseases such as asthma. Overall, we have provided evidence that GO can influence the crosstalk between the microbiome and the immune system in an AhR-dependent manner. This was possible thanks to a combination of model systems, including adult zebrafish as well as zebrafish embryos. To cut a long story short, we used an array of models to arrive at a simple conclusion: yes, GO can influence the immune system without killing cells. Our findings also served to underscore that the microbiome needs to be factored in if we are to understand the impact of 2D materials, and possibly other nanomaterials, on the host.
Fadeel B, et al. Safety assessment of graphene-based materials: focus on human health and the environment. ACS Nano. 2018;12(11):10582-10620.
Hernández PP, Strzelecka PM, Athanasiadis EI, Hall D, Robalo AF, Collins CM, Boudinot P, Levraud JP, Cvejic A. Single-cell transcriptional analysis reveals ILC-like cells in zebrafish. Sci Immunol. 2018;3(29):eaau5265.
Peng G, Fadeel B. Understanding the bidirectional interactions between two-dimensional materials, microorganisms, and the immune system. Adv Drug Deliv Rev. 2022;188:114422.
Peng G, Sinkko HM, Alenius H, Lozano N, Kostarelos K, Bräutigam L, Fadeel B. Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor. Nat Nanotechnol. 2023;18(1):42-48.
Stockinger B, Shah K, Wincent E. AHR in the intestinal microenvironment: safeguarding barrier function. Nat Rev Gastroenterol Hepatol. 2021;18(8):559-570.
Bengt Fadeel, M.D., Ph.D., is a Full Professor of Medical Inflammation Research at the Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. He served as chairman of the expert panel of the national nanosafety platform, SweNanoSafe (2017-2021), and is currently chairman of the platform. He also serves on the editorial board of several academic journals including Current Opinion in Toxicology, Frontiers in Toxicology, and Toxicological Sciences. He is a member of the health & environment WP of the Graphene Flagship (2013-2023).
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