Determination of Microplastics in Omega-3 Oil Supplements.

Microplastics (MPs) are ubiquitous in the environment, posing a threat to ecosystems and causing increasing concerns regarding their impacts on the human body through exposure. However, there has been limited research on the presence of MPs in functional foods, despite them being consumed for health improvement. This study aimed to investigate MP occurrence in various omega-3 oils and oil products in the Korean market and its relation to the source of raw material or manufacture. MPs were investigated in omega-3 capsules and raw oil, sourced from both plant-based (PB) and animal-based (AB) sources. We developed a method of direct filtration with acetone washing for collecting and characterizing MPs larger than 5 µm using micro-Raman spectroscopy. The average number of MPs by mass was found to be 1.2 ± 1.7 MPs/g for PB raw oil, 2.2 ± 1.7 MPs/g for AB raw oil, 3.5 ± 3.9 MPs/g for PB capsule oil, and 10.6 ± 8.9 MPs/g for AB capsule oil. Polypropylene and polyethylene terephthalate were the major MP species (83-95%) found in omega-3 oil. The proportions based on size range remained consistent across all groups, with a trend of being detected at higher rates as the size decreased. The results reveal that the main reason for the MP contamination of omega-3 oil is not the source of raw material but the manufacturing and packaging process.

Identification and characterization of microplastics in nasal irrigation fluids: A preliminary study.

KEY POINTS: Microplastics were identified in nasal irrigations Polypropylenes, which were the main component of the nozzle, were commonly identified Additional studies are needed to understand the biological relevance of microplastics in nasal irrigations.

Microplastics in gastrointestinal tracts of gentoo penguin (Pygoscelis papua) chicks on King George Island, Antarctica.

Microplastics (< 5 mm) have been found in marine ecosystems worldwide, even in Antarctic ecosystems. In this study, the stomach and upper intestines of 14 dead gentoo penguin (Pygoscelis papua) chicks were collected and screened for microplastics on King George Island, a gateway to Antarctic research and tourism. A total of 378 microplastics were identified by Fourier-transform infrared spectroscopy, with 27.0 ± 25.3 microplastics per individual. The detected number of microplastics did not increase with the mass of penguin chicks, suggesting no permanent accumulation of microplastics. However, the concentration of microplastics was much higher (9.1 ± 10.8 microplastics per individual within the size range 100-5000 µm) than the previously reported concentration in the penguin feces, and a greater number of smaller microplastics were found. Marine debris surveys near the breeding colony found various plastic (79.3%) to be the most frequent type of beached debris, suggesting that local sources of marine plastic waste could have contributed to microplastic contamination of penguin chicks being fed by parents that forage in nearby seas. This finding confirms the presence of microplastics in an Antarctic ecosystem and suggests the need for stronger waste management in Antarctica and a standardized scheme of microplastic monitoring in this once-pristine ecosystem.

Detection of microplastic traces in four different types of municipal wastewater treatment plants through FT-IR and TED-GC-MS.

Large amounts of microplastics are discharged into wastewater treatment plants (WWTPs), from where some of them are released into natural waterbodies on account of their not being fully eliminated by WWTPs. To investigate the behavior and emission of microplastics from WWTPs, we selected four WWTPs with different treatment technologies, including anaerobic-anoxic-aerobic (A2O), sequence batch reactor (SBR), media, and membrane bioreactor (MBR). The number of microplastics detected using Fourier transform infrared (FT-IR) spectroscopy ranged from 520 to 1820 particles/L in influent and from 0.56 to 2.34 particles/L in effluent. The microplastic removal efficiencies of four WWTPs were over 99%, indicating that the type of treatment technologies did not significantly affect the removal rate of microplastics. In the unit process for each WWTP, the major stages relating to microplastic removal were the secondary clarifier and tertiary treatment processes. Most microplastics detected were categorized as fragments and fibers, while other types were hardly detected. The size of more than 80% of microplastic particles detected in WWTPs ranged between 20 and 300 µm, indicating that they were significantly smaller than the size threshold defined for microplastics. Therefore, we used thermal extraction-desorption coupled with gas chromatography-mass spectroscopy (TED-GC-MS) to evaluate the microplastic mass content in all four WWTPs, and the results were compared with those of the FT-IR analysis. In this method, only four components, namely polyethylene, polypropylene, polystyrene, and polyethylene terephthalate, were analyzed because of the analysis limitation, and the total microplastic concentration represented the sum of four components concentrations. The influent and effluent microplastic concentrations estimated by TED-GC-MS ranged from not detectable to 160 µg/L and 0.04-1.07 µg/L, respectively, indicating a correlation coefficient of 0.861 (p < 0.05) between the TED-GC-MS and FT-IR results, when compared to the combined abundance of the four microplastic components by FT-IR analysis.

Analysis of microplastics in various foods and assessment of aggregate human exposure via food consumption in korea.

Evidence of microplastics in humans has recently been demonstrated. The primary route of human exposure to microplastics is consumption of contaminated food and water. However, quantitative estimations of exposure to microplastics are limited, which hinders human health risk assessments. In this study, abundances of microplastics were measured in eight food types, comprising 90 products of table salts, soy sauces, fish sauces, salted seafood, seaweed, honey, beer, and beverage. Aggregate human exposure to microplastics via food consumption was assessed based on the number and mass of microplastics, using deterministic calculations and Monte Carlo simulations. The determinations revealed that average adult Koreans likely ingest 1.4 × 10-4 and 3.1 × 10-4 g of microplastics per week, respectively. These results are orders of magnitude smaller than earlier estimates of 0.1-5 g of microplastics per week that likely chose experimental outliers. Therefore, careful selection of literature data and estimation methods is needed to provide more realistic exposure estimations from microplastic counts. This study extends our understanding of MP occurrence in food and provides a more thorough estimate of aggregate microplastic exposure via food consumption.

Comparative profiling and exposure assessment of microplastics in differently sized Manila clams from South Korea by µFTIR and Nile Red staining.

The accumulation of microplastics in marine organisms is an emerging concern. Due to trophic transfer, the safety of seafood is under investigation in view of the potential negative effects of microplastics on human health. In this study, market samples of Manila clams (Ruditapes philippinarum) from South Korea were segregated into two groups of considerably different size (p < 0.05), namely small clams with shell length of 40.69 ± 3.97 mm, and large clams of shell length 51.19 ± 2.86 mm. Comparative profiling of the number, size, shape, and polymer type of microplastics were performed using µFTIR imaging and Nile red staining. Overall, µFTIR detected only 1559 microplastics while 1996 microplastics were counted based on staining from 61 Manila clams (30 small and 31 large), leading to an overestimation of 18 to 75 %. Comparable microplastics concentration, based on µFTIR, were observed at 2.70 ± 1.66 MP/g or 15.64 ± 9.25 MP/individual for the small samples, and 3.65 ± 1.59 MP/g or 41.63 ± 16.90 MP/individual for the large ones (p > 0.05). Particle diameters of 20-100 µm was the most dominant, accounting for 44.6 % and 46.5 % of all microplastics from the small and large groups, respectively. Particles, with a circularity (resemblance to a circle) value between 0.6 and 1.0, were the most prevalent, followed by fragments and fibers. At least 50 % of microplastics from the small and large samples were polystyrene, making it the most abundant polymer type. Despite the substantial difference in the size of the animals, only a weak to moderate correlation was observed between microplastics content and the physical attributes of the clams such as shell length and weight, (soft) tissue weight, and total weight (Spearman's coefficient < 0.5). The estimated intake of microplastics by the Korean population was 1232 MP/person/year via small clams, 1663 MP/person/year via large clams, and 1489 MP/person/year via clams independent of size.

Microplastics in Food: A Review on Analytical Methods and Challenges.

Human exposure to microplastics contained in food has become a significant concern owing to the increasing accumulation of microplastics in the environment. In this paper, we summarize the presence of microplastics in food and the analytical methods used for isolation and identification of microplastics. Although a large number of studies on seafood such as fish and shellfish exist, estimating the overall human exposure to microplastics via food consumption is difficult owing to the lack of studies on other food items. Analytical methods still need to be optimized for appropriate recovery of microplastics in various food matrices, rendering a quantitative comparison of different studies challenging. In addition, microplastics could be added or removed from ingredients during processing or cooking. Thus, research on processed food is crucial to estimate the contribution of food to overall human microplastic consumption and to mitigate this exposure in the future.

CD137 Signaling Regulates Acute Colitis via RALDH2-Expressing CD11b-CD103+ DCs.

CD137, a potent costimulatory receptor for CD8+ T cells, is expressed in various non-T cells, but little is known about its regulatory functions in these cells. In this study, we show that CD137 signaling, specifically in intestinal CD11b-CD103+ dendritic cells (DCs), restricts acute colitis progression. Mechanistically, CD137 engagement activates TAK1 and subsequently stimulates the AMPK-PGC-1α axis to enhance expression of the Aldh1a2 gene encoding the retinoic acid (RA) metabolizing enzyme RALDH2. RA can act on CD11b+CD103- DCs and induce SOCS3 expression, which, in turn, suppresses p38MAPK activation and interleukin-23 (IL-23) production. Administration of RA in DC-specific CD137-/- mice represses IL-23-producing CD11b+CD103- DCs and TH17 cells, indicating that RA is a major inhibitory effector molecule against intestinal CD11b+CD103- DCs. Additionally, the therapeutic effect of the anti-CD137 antibody is abrogated in DC-specific CD137-/- mice. Taken together, our results define a mechanism of paracrine immunoregulation operating between adjacent DC subsets in the intestine.

CCR5-mediated Recruitment of NK Cells to the Kidney Is a Critical Step for Host Defense to Systemic Candida albicans Infection.

C-C chemokine receptor type 5 (CCR5) regulates the trafficking of various immune cells to sites of infection. In this study, we showed that expression of CCR5 and its ligands was rapidly increased in the kidney after systemic Candida albicans infection, and infected CCR5-/- mice exhibited increased mortality and morbidity, indicating that CCR5 contributes to an effective defense mechanism against systemic C. albicans infection. The susceptibility of CCR5-/- mice to C. albicans infection was due to impaired fungal clearance, which in turn resulted in exacerbated renal inflammation and damage. CCR5-mediated recruitment of NK cells to the kidney in response to C. albicans infection was necessary for the anti-microbial activity of neutrophils, the main fungicidal effector cells. Mechanistically, C. albicans induced expression of IL-23 by CD11c+ dendritic cells (DCs). IL-23 in turn augmented the fungicidal activity of neutrophils through GM-CSF production by NK cells. As GM-CSF potentiated production of IL-23 in response to C. albicans, a positive feedback loop formed between NK cells and DCs seemed to function as an amplification point for host defense. Taken together, our results suggest that CCR5-mediated recruitment of NK cells to the site of fungal infection is an important step that underlies innate resistance to systemic C. albicans infection.

Anti-CD137 Suppresses Tumor Growth by Blocking Reverse Signaling by CD137 Ligand.

CD137 (4-1BB) is a T-cell costimulatory molecule, and agonstic CD137 antibodies are currently being evaluated in the clinic as cancer immunotherapy. Recently, it was found that CD137-/- mice or mice injected with agonistic anti-CD137 antibodies exhibit heightened antitumor responses, contrary to expectations based on other knowledge of CD137 function. Here, we report findings related to reverse signaling by CD137 ligand (CD137L) in antigen-presenting dendritic cells (DC) in tumors that address these paradoxical results. Specifically, CD137L suppressed intratumoral differentiation of IL12-producing CD103+ DC and type 1 tumor-associated macrophages (TAM). Differentiation of these cell types is important because they are required to generate IFNγ-producing CD8+ cytotoxic T lymphocytes (Tc1). Notably, CD137L blockade increased levels of IL12 and IFNγ, which promoted intratumoral differentiation of IFNγ-producing Tc1, IL12-producing CD103+ DC, and type 1 TAM within tumors. Our results offer an explanation for the paradoxical effects of CD137 blockade, based on differential immunomodulatory effects of CD137 signaling and reverse signaling in T cells and DC, respectively. Further, they show how CD137L blockade can seed a forward-feedback loop for activation of CD103+ DC/type 1 TAM and Tc1 that can create a self-perpetuating cycle of highly effective immunosurveillance. Cancer Res; 77(21); 5989-6000. ©2017 AACR.

Inhibition of acute lethal pulmonary inflammation by the IDO-AhR pathway.

The lung is a prototypic organ that was evolved to reduce immunopathology during the immune response to potentially hazardous endogenous and exogenous antigens. In this study, we show that donor CD4+ T cells transiently induced expression of indoleamine 2,3-dioxygenase (IDO) in lung parenchyma in an IFN-γ-dependent manner early after allogeneic hematopoietic stem cell transplantation (HSCT). Abrogation of host IDO expression by deletion of the IDO gene or the IFN-γ gene in donor T cells or by FK506 treatment resulted in acute lethal pulmonary inflammation known as idiopathic pneumonia syndrome (IPS). Interestingly, IL-6 strongly induced IDO expression in an IFN-γ-independent manner when deacetylation of STAT3 was inhibited. Accordingly, a histone deacetylase inhibitor (HDACi) could reduce IPS in the state where IFN-γ expression was suppressed by FK506. Finally, l-kynurenine produced by lung epithelial cells and alveolar macrophages during IPS progression suppresses the inflammatory activities of lung epithelial cells and CD4+ T cells through the aryl hydrocarbon receptor pathway. Taken together, our results reveal that IDO is a critical regulator of acute pulmonary inflammation and that regulation of IDO expression by HDACi may be a therapeutic approach for IPS after HSCT.

Intratumorally Establishing Type 2 Innate Lymphoid Cells Blocks Tumor Growth.

A long-standing question in the field of tumor immunotherapy is how Th2 cytokines block tumor growth. Their antitumor effects are particularly prominent when they are secreted continuously in tumors, suggesting that Th2 cytokines may create a tumor microenvironment unfavorable for tumor growth independently of adaptive immunity. In this study, we show that local production of IL-33 establishes a high number of type 2 innate lymphoid cells (ILC2s) with potent antitumor activity. IL-33 promotes secretion of a massive amount of CXCR2 ligands from ILC2s but creates a tumor microenvironment where tumor cells express CXCR2 through a dysfunctional angiogenesis/hypoxia/reactive oxygen species axis. These two signaling events converge to reinforce tumor cell-specific apoptosis through CXCR2. Our results identify a previously unrecognized antitumor therapeutic pathway wherein ILC2s play a central role.

IL-33 Enhances Host Tolerance to Candida albicans Kidney Infections through Induction of IL-13 Production by CD4+ T Cells.

Susceptibility to systemic Candida albicans infection is determined by immune resistance, as well as by the ability to control Candida-induced immunopathologies. We showed previously that exogenous IL-33 can increase resistance to peritoneal C. albicans infection by regulating multiple steps of the neutrophil anti-Candida response. In this study, using a mouse model of systemic candidiasis, we observed that IL-33 administration limited fungal burden and inflammation and increased survival. In kidneys, IL-33 seemed to directly act on neutrophils and CD4(+) T cells: IL-33 administration enhanced fungal clearance by increasing neutrophil phagocytic activity without which Candida proliferation was uncontrollable. In contrast, IL-33 stimulated CD4(+) T cells to produce IL-13, which, in turn, drove the polarization of macrophages toward the M2 type. Furthermore, the absence of IL-13 abolished IL-33-mediated polarization of M2 macrophages and renal functional recovery. In addition, IL-33 and IL-13 acted synergistically to increase M2 macrophage polarization and its phagocytic activity. Overall, this study identifies IL-33 as a cytokine that is able to induce resistance and tolerance and suggests that targeting resistance and tolerance simultaneously with therapeutic IL-33 may benefit patients with systemic candidiasis.

IL-33-induced hematopoietic stem and progenitor cell mobilization depends upon CCR2.

IL-33 has been implicated in the pathogenesis of asthma, atopic allergy, anaphylaxis, and other inflammatory diseases by promoting the production of proinflammatory cytokines and chemokines or Th2 immune responses. In this study, we analyzed the in vivo effect of IL-33 administration. IL-33 markedly promoted myelopoiesis in the bone marrow and myeloid cell emigration. Concomitantly, IL-33 induced hematopoietic stem and progenitor cell (HSPC) mobilization and extramedullary hematopoiesis. HSPC mobilization was mediated mainly through increased levels of CCL7 produced by vascular endothelial cells in response to IL-33. In vivo treatment of IL-33 rapidly induced phosphorylation of ERK, JNK, and p38, and inhibition of these signaling molecules completely blocked the production of CCL7 induced by IL-33. Consistently, inhibitor of CCR2 markedly reduced IL-33-mediated HSPC mobilization in vivo and migration of HSPCs in response to CCL7 in vitro. IL-33-mobilized HSPCs were capable of homing to, and of long-term reconstitution in, the bone marrow of irradiated recipients. Immune cells derived from these recipients had normal antifungal activity. The ability of IL-33 to promote migration of HSPCs and myeloid cells into the periphery and to regulate their antifungal activity represents a previously unrecognized role of IL-33 in innate immunity. These properties of IL-33 have clinical implications in hematopoietic stem cell transplantation.

CD137 in chronic graft-versus-host disease.

Chronic graft-versus-host disease (GVHD) occurs in recipients of allogeneic hematopoietic stem cell transplantation with a high frequency. Preclinical animal chronic GVHD models outlined in this chapter allow for the delineation of events that occur during chronic GVHD development. The DBA/2 → (C56BL/6 × DBA/2)F1 (BDF1) model is characterized by systemic lupus erythematosus (SLE)-like phenotype. The B10.D2 → Balb/c model presents many features of autoimmune scleroderma. The former model is useful in defining how alloreactive donor CD4(+) T cells break B-cell tolerance, whereas the latter model is suitable for dissecting the pathogenesis of organ fibrosis. Our laboratory has demonstrated that injection of a single dose of strong CD137 agonists can prevent or cure chronic GVHD in these two models. In general, these models are particularly suited to screening the immunomodulatory therapeutics.

Interleukin-33: a mediator of inflammation targeting hematopoietic stem and progenitor cells and their progenies.

Inflammation is defined as a physiological response initiated by a variety of conditions that cause insult to the body, such as infection and tissue injury. Inflammation is triggered by specialized receptors in the innate immune system, which recognize microbial components known as pathogen-associated molecular patterns or endogenous signals produced by damaged cells (damage-associated molecular patterns). IL-33 is a cytokine that is released predominantly at the epithelial barrier when it is exposed to pathogens, allergens, or injury-inducing stimuli. IL-33 target cells are various, ranging from hematopoietic stem and progenitor cells (HSPCs) and essentially all types of their progeny to many non-hematopoietic cells. The pleiotropic actions of IL-33 suggest that IL-33 is involved in every phase of the inflammatory process. In this review, we discuss recent advances in the understanding of how IL-33 orchestrates inflammatory responses by regulating HSPCs and innate immune cells.

IL-33 priming regulates multiple steps of the neutrophil-mediated anti-Candida albicans response by modulating TLR and dectin-1 signals.

IL-33 is known to play an important role in Th2 immunity. In this study, we investigated the effect of IL-33 pretreatment on anti-fungal response using an acute Candida albicans peritoneal infection model. IL-33 pretreatment induced a rapid fungal clearance and markedly reduced the C. albicans infection-associated mortality. The priming effect of IL-33 occurred during multiple steps of the neutrophil-mediated anti-fungal response. First, the anti-fungal effect occurred due to the rapid and massive recruitment of neutrophils to the site of infection as a result of the release of CXCR2 chemokines by peritoneal macrophages and by reversal of the TLR-induced reduction of CXCR2 expression in neutrophils during IL-33 priming. Second, conditioning of neutrophils by IL-33 activated the TLR and dectin-1 signaling pathways, leading to the upregulation of complement receptor 3 expression induced by C. albicans. Upregulated CR3 in turn increased the phagocytosis of opsonized C. albicans and resulted in the production of high levels of reactive oxygen species and the subsequent enhanced killing activity of neutrophils. Taken together, our results suggest that IL-33 can regulate the anti-fungal activity of neutrophils by collaborative modulation of the signaling pathways of different classes of innate immune receptors.

Host CD25+CD4+Foxp3+ regulatory T cells primed by anti-CD137 mAbs inhibit graft-versus-host disease.

CD25(+)CD4(+)Foxp3(+) regulatory T cells (Tregs) play a pivotal role in the maintenance of self-tolerance and regulation of immune responses. Previous studies have demonstrated that CD137 signals can promote proliferation and survival of Tregs in vitro. Here, we show that in vivo CD137-induced expansion of Tregs in naive mice was dependent upon IL-2 secreted by memory T cells. Tregs primed by anti-CD137 mAbs had a higher immunosuppressive capacity. Preconditioning with anti-CD137 mAbs significantly inhibited graft-versus-host disease (GVHD) in the C57BL/6 → (C57BL/6 × DBA/2) F1 acute GVHD model. In this disease model, a high proportion of host Tregs remained long-term in the recipient spleen, whereas donor hematopoietic cells replaced other host bone marrow-derived cells. Transient depletion of Tregs before transfer of donor cells completely abrogated the inhibitory effect of anti-CD137 mAbs on GVHD. In addition, adoptive transfer of anti-CD137-primed Tregs ameliorated GVHD. Our results demonstrate that it is possible to enhance the survival and/or the immunosuppressive activity of host Tregs in nonmyeloablative GVHD, and that 1 way of accomplishing this is through the prophylactic use of anti-CD137 mAbs in nonmyeloablative GVHD.

Anti-CD137 mAb Deletes Both Donor CD4 and CD8 T Cells in Acute Graft-versus-host Disease.

We previously demonstrated that in vivo engagement of CD137, a member of TNF receptor superfamily, can delete allorective CD4(+) T cells through the induction of activation-induced cell death (AICD) in chronic graft-versus-host disease (cGVHD) and subsequently reverse established cGVHD. In this study, we further showed that agonistic anti-CD137 mAb was highly effective in triggering AICD of donor CD8(+) T cells as well as donor CD4(+) T cells in the C57BL/6→unirradiated (C57BL/6 × DBA/2)F1 acute GVHD model. Our results suggest that strong allostimulation should facilitate AICD of both alloreactive CD4(+) and CD8(+) T cells induced by CD137 stimulation. Therefore, depletion of pathogenic T cells using agonistic anti-CD137 mAb combined with potent TCR stimulation may be used to block autoimmune or inflammatory diseases mediated by T cells.

A novel method for procuring a large quantity of mature murine eosinophils in vivo.

Eosinophils are rare hematopietic cells that normally constitute only 1~3% of peripheral blood leukocytes. It would be of help for the purpose of research to obtain a large quantity of eosinophils. In this study, we wanted to develop a novel strategy to induce massive expansion of murine eosinophils in vivo, based on the observation showing that treatment of IL-33 induces eosinophilia in mice. We generated an EL-4 lymphoma cell line (herein named EL-4-IL-33) that was engineered to secrete an active form of IL-33. We found that Siglec-F(+) granulocyte numbers increased by 1850-fold in the peritoneal cavity 10 days after inoculation with 1×107 EL-4-IL-33 cells. This number corresponds to 74-fold increase, as compared with the number of Siglec-F(+) granulocytes in mice that received wild-type EL-4 cells. Siglec-F(+) granulocytes expanded by IL-33 had the circular nucleus and expressed eosinophil-specific genes. They also showed some functional characteristics of eosinophils in that they had the ability to respond to IL-5 for survival and eotaxin-1 for chemoattaxis and to produce bioactive eosinophil peroxidase, suggesting that these cells are genuine eosinophils. Our results indicate that sustained secretion of IL-33 by lymphoma cells in the peritoneal cavity is highly effective in increasing peritoneal eosinophil numbers. Therefore, our simple method to obtain eosinphils on a large scale might be of value for eosinophil studies.