Effects of Nano and Microplastics on the Inflammatory Process: In Vitro and In Vivo Studies Systematic Review

Background : Microplastics (MPs) and Nanoplastics (NPs) are plastic fragments that spread in the environment and accumulate in the human body, so they have been becoming a worldwide environmental concern because of their potential human health effects. The aim of this systematic review was to investigate the prospective impact of MPs and NPs on the inflammatory process. Methods : Electronic article search was performed on PubMed, Scopus and Web of Science international databases from 1 Jan 2012 to 31 Dec 2021. Screenings of titles, abstracts and full texts were performed according to the Preferred Reporting Item for Systematic Review and Meta-analyses (PRISMA). The methodological quality of the studies was checked by the Toxicological data Reliability Assessment Tool. Results : Electronic article search identified 125 records, from which 6 in vitro , 11 in vivo and 2 both in vivo and in vitro studies were included. Both in vivo and in vitro studies have showed an increase ofdifferent inflammatory outcomes (Interleukines, Tumor necrosis factor, Chemokines, Interferones, Transcription factors, Growth factors, Oxydoreductase, Proteins and others), thus it seems to confirm the association withthe exposure to microplastics of different types, sizes, exposure times and exposed species. Conclusions : This systematic review seems to support the relationship between the exposure to MPs and the inflammatory processboth in vivo and in vitro . Greater caution is needed about the role of NPs because ofa very small number of studies. Additional high-quality studies are warranted to confirm these results, especially the research should be focused on NPs being lacking literature.


Introduction
Microplastics are the consequence of the high production of plastics and of its unmanaged release in the environment.Extreme solidity at room temperature, excellent electrical, thermal, and acoustic insulation, portability, ease of use thanks to the lightweight and cheapness are the main properties of plastic.These properties made it the most widespread and the most used synthetic material worldwide, causing a global pollution [1][2][3].It was found in soil, sea, air, and also in the Arctic sea ice [4,5].
MPs and NPs size seems to favor their penetration into animal and vegetal tissues and cells besides accumulation in organs [11][12][13], causing alterations in physiological process [3].Some studies highlighted the capable of MPs and NPs to cause toxicity, chronic inflammation and increase risk of neoplasm [7,[14][15][16][17].The in vivo studies show that both MPs and NPs are absorbed and accumulated in the tissues altering the correct functioning of organs and systems [18][19][20].
The human body is continuously exposed to microplastics through digestion, inhalation or dermal contact.Human's ingestion is considered the simplest form of exposure to MPs and NPs [12,13,21]; which could cause an inflammatory response.In vitro test showed that NPs alter the cell membrane surface and trigger the inflammatory process [18].Also, the exposures to MPs through inhalation and dermal contact were identified as triggers of lung and skin pro-inflammatory responses [17,22,23] which can lead to an increased risk of developing cancer [24].
The aim of the present study was to review the in vitro and in vivo studies that evaluated the impacts of exposure disagreement between the two researchers was resolved through consensus session with a third researcher (M.Fe.).
In the screening phase, this systematic review was split into two main sections: (a) The first referring exclusively to in vitro models and, (b) The second referring exclusively to in vivo models, both for the assessment of the inflammatory capacity of NPs and MPs through the evaluation of the inflammatory biomarkers.
We excluded: (i) studies whose method of inflammatory assessment was not clear, or incompletely described or that do not evaluate, or only evaluate the inflammatory of MPs/NPs qualitatively; (ii) studies that do not evaluate inflammatory through methods specific for MPs/NPs; (iii) studies that only report other health effects (e.g., carcinogenic effect); (iv) studies other than in vitro, e.g., in silico; and (v) studies not reporting statistical data.

Data Collection and Synthesis
The following descriptive and quantitative information was extracted from each of the eligible study for both sections, i.e., in vitro and in vivo studies: authors and year of publication, type and size of MPs/NPs, dose/exposure time, inflammatory biomarkers, animals (in vivo studies)/cell models (in vitro studies), assay(s) and outcomes.Information was summarized and organized in tables and for each table studies can be identified by their listed study details  (First-Author name and year of publications).For in vitro and in vivo studies, we created 10 different tables summarizing for specific outcomes (Interleukine, Tumor necrosis factors, Chemochine, Interferon, Transcriptor factors, Growth factors, Oxidoreductase, Proteins and Others).

Study Quality Appraisal
The methodological quality of the studies has been checked using the Toxicological data Reliability Assessment Tool (ToxR Tool) guidelines for reporting randomized clinical trials for in vitro and in vivo studies [26].In particular, two researchers (E.P., M.P.) performed data selection, extraction and quality assessment independently.Any disagreement between the two researchers was resolved by consensus session with a third researcher (M.F.).

Results
The initial search retrieved a total of 125 studies, from which 55 were excluded because of duplicate records.A total of 70 studies were screened based on the title and abstract, from which 28 were excluded, resulting in 42 fulltext studies that were considered potentially eligible for inclusion.A total of 23 studies were excluded because of carried out using a mixture of contaminants including MPs and lack of statistical data analysis.Finally, 19 studies (6 in vitro, 11 in vivo and 2 both in vivo and in vitro) were included in this review [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45].
The full process of article collection, screening, and eligibility assessment is presented in Fig. 1.
Changes in levels of all investigated outcomes (Interleukines, Tumor necrosis factor, Chemokines, Interferons, Transcription factors, Growth factors, Oxydoreductase, Proteins and others) have been summarised in Tables 2-10.
Below we have summarized the main results for each individual study included in the review by outcomes specifying different type of plastic exposure and study design.
Table 2 (Ref.[27][28][29][30][31][32][36][37][38][39][41][42][43][44][45]) shows an increase in interleukins 6, 8 and 1β.In particular, after exposure to PS 6 studies (three in vitro studies [29,37,42], two in vivo studies [27,32] and one both in vivo and in vitro [39]) showed an increase in IL-6; four studies showed an increase in IL-β1 (one in vitro studies [37] and three in vivo studies [27,30,36]) and two studies showed an increase in IL-8 (one in vitro study [29] and one in vivo study [36]).Only one in vivo study [45], which investigated MPs without specifying their type, showed an increase in IL-4 and IL-5.At the same time, only one in vivo study [31] out of three investigated exposures to PE detecting an increase in IL-8; two in vivo studies [28,31] out of three showed an increase in IL-6 and one in vivo study [31] out of three showed an increase in IL-1β.The remaining studies, that investigated the other types of micro/nanoplastics (PP, NPs, polymers, PS-NH2, PVC) or fibers (LFb), highlighted an increase in IL-1β while no significant increase was detected for other cytokines.Only one in vivo study [28], concerning exposure to PE, showed a decrease in IL-2 and IL-5.Instead, only one in vitro study [38] with exposure to PVC reported a decrease in IL-8 (Table 2).
Table 4 (Ref.[28,32,37,39]) shows the trend of different chemokines following exposure to PP (one study in vitro [37]), PS (three studies respectively one in vitro [37], one study in vivo [32] and one both [39] and PE (one study in vivo [28]).Only one study [37] reported an increase in MIP-1β following exposure to both PP and PS.Whereas only exposure to PS (two studies [32,39]) showed an increase in both CCL11 and CXCL10.Regarding the exposure to PE only one study [28] evaluated both RANTES and IP-10 detecting an increase in the first cytokine and a decrease in the second (Table 4).
In Table 5 (Ref.[30,44,45]) we have reported the trend of INF levels following exposure to MPs (two in vivo studies [44,45]), NPs (one in vivo study [44]) and PS (one in vivo study [30]).Both exposure to MPs and NPs did not change the trend of IFN-y [45] and INFPHI1 [44] levels.Finally, only exposure to PS was associated with an increase in IFNy [30] (Table 5).
Table 7 (Ref.[28,32]) shows the trend of growth factors after exposure to PE (one study in vivo [28]) and PS (one study in vivo [32]).There was an increase in GCSF [28] after exposure to PE whereas exposure to PS did not modify the trend of FGF8 [32] (Table 7).
Table 8 (Ref.[32,35,36,41]) shows the enzymes with antioxidant action investigated by the studies included in the review.In general, exposure to PS (two in vivo studies [32,36], PP (one in vivo study [41], LDPE (one in vivo study [35] and SFB, LFB (one in vivo study [41] showed an increase in all the enzymes investigated.In particular, after exposure to PS the values of CAT [36], GPx [36] were increased, whereas the trend of SOD (two studies [32,36]) was detected in increase in only one [36] of the two studies.Finally, exposure to LDPE [35] reported an increase in CAT, GRd and GST and no change in values for GPx and SOD, whereas the study by Zhao which investigated the exposure to PP, SFb and LFb showed an increase in SOD only following exposure to LFB [41] (Table 8).

Risk of Bias
The results of the quality assessment of the studies are reported in Figs.2,3 and Supplementary Tables 1,  2. In particular, only two in vitro studies didn't report information on the source/origin of the test system [29,39].Only one author did not report the necessary information on test system properties, and on conditions of cultivation and maintenance.Only one study reported the number of replicates [34].Concerning in the vivo studies, only one study didn't report information on the source/origin of the test system [41].Four studies did not give the sex of the test organism [32,35,40,44].One study did not give age or body weight of the test organisms at the start of the study [40].One study did not give information on the housing or feeding conditions in case of repeated dose toxicity studies [39].[39], from table 6 due to none of these outcomes studied.Notes: IRE1α, Inositol-requiring transmembrane kinase endoribonuclease-1α; ATF6, Activating transcription factor 6; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; ERK 1-2, Extracellular signal-regulated kinases 1-2; TLR4, Tool -like receptor 4; AP-1, Activator protein 1; IRF5, Interferon Regulatory Factor 5; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PPAR-γ, Peroxisome proliferator-activated receptotγ; GATA4, GATA-binding protein 4; p-JNK, p-Jun N-terminal kinases; AKT, RAC(Rho family)-alpha serine/threonine-protein kinase; mTOR, mechanistic target of rapamycin; Nrf2-HO1, Nuclear factor erythroid 2-related factor 2 linked to Hemeoxygenase 1; PS, Polystyrene; PE, Polyethylene.* p < 0.05; ** p < 0.01; *** p < 0.001.N.A.,Not applicable.

Author, year
Particles type Outcomes GCSF FGF8

Discussion
The in vitro [29,37,38,42,43]and in vivo [27,28,36]studies included in this review seem to confirm an association between the increase in pro-inflammatory interleukins Il-6, IL-8 and IL-1b and the exposure to microplastics of different types, sizes, exposure times and exposed species, whereas the interpretation of the results, relating  to the other interleukins investigated, requires more caution because there are only few heterogeneous studies(42 in vitro, 28 and 45 in vivo) in literature to date.In particular, it is already known that these pro-inflammatory interleukins take part in the acute inflammatory response [46], whereas IL-6 acts as an anti-inflammatory myokine too [47].Furthermore, the results of the studies seem to confirm that the persistence of acute inflammation can become chronic up to result in a further systemic inflammatory action, inducing COPD (Chronic Obstructive Pulmonary Disease), asthma [48] and Inflammatory Bowel Diseases (IBD) [31,38].This process could be caused by the reduction of TEER and by  the expression of the ZO-1 protein, this leads to a loss of epithelial integrity of the barrier cells [29,39,49].This reduction in the integrity of the barrier cells has been confirmed for microplastics of 3 µm size, through vesicles of the plasma membrane the microplastics of 60 nm size can be internalized [50].
The proinflammatory action of TNF-α following exposure to PS, in a size and concentration dependent manner, seems to be confirmed both in vitro [39,42] and in vivo studies [27,30,42].It is already considered as an immune mediator for cell adhesion, migration, angiogenesis and apopto-sis; therefore, its up regulation is a potential indicator of an immune response and inflammation [51].However, it must be pointed out that the studies in the literature have only investigated TNF-α and always in a heterogeneous way in terms of type of plastic, size, species exposed, dose and exposure time that do not allow us a generalization of the results [27,30,[36][37][38][39][42][43][44][45].
Levels related to the chemokine MIP1β, both following exposure to PP and PS, would appear to increase but it is related to a single in vitro study [37], no one in vivo study was found in literature.This result would seem interesting because it is one of the major factors produced by macrophages and monocytes after exposure to bacterial toxins or proinflammatory cytokines [52,53].On the other hand, it is not possible to hypothesize the same result for the other chemokines investigated as they have been included in heterogeneous studies for type of plastic, size, species exposed, dose and exposure time [28,32,37,39].
A few in vivo studies quantifying the levels of IFNPHI-1, IFN-γ and growth factors (GCSF and FGF8) did not allow us to draw conclusions also because they tested exposure to MPs without specifying the type.Only 1 in vivo study claims to have tested the PS [30].The study of IFNPHI-1 and IFN-γ, also considered as cytokines, would be of interest due to their crucial role both in innate and acquired immunity but also as activators of macrophages which is involved in the inflammatory process [54].In addition, GCSF is a glycoprotein that stimulates the bone marrow to produce granulocytes responsible for the acute phase of inflammation whereas FGF8 is responsible for fibroblasts growth that can cause chronic inflammation [55].
Finally, as regards the other outcomes investigated, not classifiable in the aforementioned categories, a potential association emerged between exposure to microplastics of different type, size and exposure time and ROS both in vitro [29,33,37] and in vivo studies [30,35,40,41,45].Although ROS are known to cause chronic oxidative stress including inflammation, alteration of permeability and histopathological damage [56][57][58], it would underline that their formation may also depend on the surface of microplastics.This is supported by the fact that the experiment carried out with NAC-coated (N-acetylcysteine) nanoparticles reduce toxi-city and oxidants, subsequently reducing the toxic effect on THP-1 macrophages [37].
The studies included in the review have various limitations regarding, for example, the use of different animal and cell models, size and type of particles investigated, doses and exposure time or conditions, quantified outcomes and tests used for their quantification.Moreover, it should be noted that most of the authors summarize the results through graphs in which is difficult to obtain numerical data comparable to each other and to estimate the quality.Furthermore, none of the in vivo studies included in the review exposed male and female mice to microplastics at the same time, this may be a limitation as the influence of sex has already been demonstrated for pollutants exposure as for example to metals [59,60].Another limitation of the studies concerns the lack of studies on NPs, in fact, only 2 studies included in this review investigated NPs.Moreover, only one study took into consideration the limits related to methodology [43].In particular, it has been discussed the difficulties of the particles to translocate across the epithelium to the basolateral side in the membrane of 12-well insert, whereas only one study focused on methods to detect MPs in tissues are not appropriate [39].Surprisingly six authors showed no limitation in their studies [30,32,34,37,41,44].
Finally, we have included only English language articles in this review, and it was not possible to compare the results with those of other reviews as to the authors' knowledge there are no reviews like this one in the literature to date.Although the limitations, the results of this review may be useful for the organization of future studies.In particular, they provide information on potential outcomes that could help confirm the hypothesis of an association between exposure to various types of microplastics and the inflammatory process.

Conclusions
In conclusion, this review seems to support the association between the MPs exposure and the inflammation response both in vivo and in vitro.Conversely greater caution is needed regarding the role of NPs due to the very small number of studies in literature.Additional high-quality studies are warranted to confirm these results, especially the research should be focused on NPs being lacking literature.