Tim4, a macrophage receptor for apoptotic cells, binds polystyrene microplastics via aromatic-aromatic interactions.

Understanding the interface between microplastics and biological systems will provide new insights into the impacts of microplastics on living organisms. When microplastics enter the body, they are engulfed preferentially by phagocytes such as macrophages. However, it is not fully understood how phagocytes recognize microplastics and how microplastics impact phagocyte functions. In this study, we demonstrate that T cell immunoglobulin mucin 4 (Tim4), a macrophage receptor for phosphatidylserine (PtdSer) on apoptotic cells, binds polystyrene (PS) microparticles as well as multi-walled carbon nanotubes (MWCNTs) through the extracellular aromatic cluster, revealing a novel interface between microplastics and biological systems via aromatic-aromatic interactions. Genetic deletion of Tim4 demonstrated that Tim4 is involved in macrophage engulfment of PS microplastics as well as of MWCNTs. While Tim4-mediated engulfment of MWCNTs causes NLRP3-dependent IL-1ß secretion, that of PS microparticles does not. PS microparticles neither induce TNF-α, reactive oxygen species, nor nitric oxide production. These data indicate that PS microparticles are not inflammatory. The PtdSer-binding site of Tim4 contains an aromatic cluster that binds PS, and Tim4-mediated macrophage engulfment of apoptotic cells, a process called efferocytosis, was competitively blocked by PS microparticles. These data suggest that PS microplastics do not directly cause acute inflammation but perturb efferocytosis, raising concerns that chronic exposure to large amounts of PS microplastics may cause chronic inflammation leading to autoimmune diseases.

Carbon nanotube recognition by human Siglec-14 provokes inflammation.

For the design and development of innovative carbon nanotube (CNT)-based tools and applications, an understanding of the molecular interactions between CNTs and biological systems is essential. In this study, a three-dimensional protein-structure-based in silico screen identified the paired immune receptors, sialic acid immunoglobulin-like binding lectin-5 (Siglec-5) and Siglec-14, as CNT-recognizing receptors. Molecular dynamics simulations showed the spatiotemporally stable association of aromatic residues on the extracellular loop of Siglec-5 with CNTs. Siglec-14 mediated spleen tyrosine kinase (Syk)-dependent phagocytosis of multiwalled CNTs and the subsequent secretion of interleukin-1ß from human monocytes. Ectopic in vivo expression of human Siglec-14 on mouse alveolar macrophages resulted in enhanced recognition of multiwalled CNTs and exacerbated pulmonary inflammation. Furthermore, fostamatinib, a Syk inhibitor, blocked Siglec-14-mediated proinflammatory responses. These results indicate that Siglec-14 is a human activating receptor recognizing CNTs and that blockade of Siglec-14 and the Syk pathway may overcome CNT-induced inflammation.

Shaping of T Cell Functions by Trogocytosis.

Trogocytosis is an active process whereby plasma membrane proteins are transferred from one cell to the other cell in a cell-cell contact-dependent manner. Since the discovery of the intercellular transfer of major histocompatibility complex (MHC) molecules in the 1970s, trogocytosis of MHC molecules between various immune cells has been frequently observed. For instance, antigen-presenting cells (APCs) acquire MHC class I (MHCI) from allografts, tumors, and virally infected cells, and these APCs are subsequently able to prime CD8+ T cells without antigen processing via the preformed antigen-MHCI complexes, in a process called cross-dressing. T cells also acquire MHC molecules from APCs or other target cells via the immunological synapse formed at the cell-cell contact area, and this phenomenon impacts T cell activation. Compared with naïve and effector T cells, T regulatory cells have increased trogocytosis activity in order to remove MHC class II and costimulatory molecules from APCs, resulting in the induction of tolerance. Accumulating evidence suggests that trogocytosis shapes T cell functions in cancer, transplantation, and during microbial infections. In this review, we focus on T cell trogocytosis and the related inflammatory diseases.

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

Low-dose mithramycin exerts its anticancer effect via the p53 signaling pathway and synergizes with nutlin-3 in gynecologic cancers.

MDM2 is a direct negative regulator of p53. The p53-independent mdm2-P1 and p53-dependent mdm2-P2 promoters have been recently shown to harbor Sp1 binding sites. Mithramycin, an inhibitor of Sp1 DNA binding, has been used clinically to treat hypercalcemia and some types of neoplastic disorders. In this study, we investigated the mechanisms behind the anticancer effect of mithramycin. In gynecologic cancer cells expressing wild-type p53, mithramycin stabilized p53 and increased the expression of the p53 downstream target genes PUMA and p21, arrested the cell cycle, and induced apoptosis. This activation of the p53 signaling pathway was a specific effect of MTH at concentrations <50 nm. Mithramycin temporally decreased transcription of both the mdm2-P1 and -P2 promoters. This was followed by a subsequent increase of mdm2-P2 promoter activity by activated p53. Up-regulated MDM2 was in its active form, and consequently attenuated p53 activity. Although mithramycin activated p53 and suppressed the growth of human gynecologic cancer cell xenografts in mice, this was accompanied with a secondary up-regulation of MDM2. Combined treatment with mithramycin and nutlin-3, a drug that inhibits MDM2-p53 interaction, overcame a secondary up-regulation of MDM2 and synergistically inhibited cancer cell growth by inducing apoptosis through activation of the p53 signaling pathway. These observations provide a better understanding of the mechanisms of mithramycin activity, and suggest a potential role for combining mithramycin and nutlin-3 as a chemotherapeutic treatment for gynecologic cancers.

Level of reactive oxygen species induced by p21Waf1/CIP1 is critical for the determination of cell fate.

p21(WAF(1)/)(CIP(1)) is a well-known cell cycle regulatory protein which is overexpressed in several cancer cell lines, and known to determine cell fate. We generated three recombinant adenovirus vectors that expressed either the full-length p21 (Ad-p21F), a p21 mutant with a deletion of the C-terminal proliferative cell nuclear antigen (PCNA) binding domain (Ad-p21N), or a p21 mutant with a deletion of the N-terminal cyclin-dependent kinase binding domain (Ad-p21C). We transfected these vectors into five cancer cell lines. Premature senescence was induced in all of the lines only following transfection with Ad-p21N and Ad-p21F. In addition, apoptosis was also induced in LoVo and HCT116 cells that harbored wild-type p53 and the reactive oxygen species (ROS) level was higher than in senescent cells. Finally, the induction of apoptosis was inhibited by using siRNA to downregulate p53. This observation implies that there is a feedback signaling loop involving p21/ROS/p53 in apoptotic responses. It appears to be, at least in part, driven by high levels of p21 protein. Next, we investigated the cell death effect of endogenous p21 protein on cell fate using sodium butyrate (NaB). Treatment with 1 mM NaB or 2 to 5 mM NaB induced senescence or apoptosis, respectively. The level of intracellular ROS in 5 mM NaB treated cells was 2-fold higher, compared with that in 1 mM NaB treated cells. We also demonstrated that DNA damage response signals including ataxia telangiectasia mutated, gammaH2AX, and p38 MAPK were involved in NaB-induced cell death. The magnitude of intracellular ROS levels in response to p21 elicited either senescence or apoptosis in the cancer cell lines.