Lipopolysaccharide-Induced CD300b Receptor Binding to Toll-like Receptor 4 Alters Signaling to Drive Cytokine Responses that Enhance Septic Shock.

Receptor CD300b is implicated in regulating the immune response to bacterial infection by an unknown mechanism. Here, we identified CD300b as a lipopolysaccharide (LPS)-binding receptor and determined the mechanism underlying CD300b augmentation of septic shock. In vivo depletion and adoptive transfer studies identified CD300b-expressing macrophages as the key cell type augmenting sepsis. We showed that CD300b, and its adaptor DAP12, associated with Toll-like receptor 4 (TLR4) upon LPS binding, thereby enhancing TLR4-adaptor MyD88- and TRIF-dependent signaling that resulted in an elevated pro-inflammatory cytokine storm. LPS engagement of the CD300b-TLR4 complex led to the recruitment and activation of spleen tyrosine kinase (Syk) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). This resulted in an inhibition of the ERK1/2 protein kinase- and NF-κB transcription factor-mediated signaling pathways, which subsequently led to a reduced interleukin-10 (IL-10) production. Collectively, our data describe a mechanism of TLR4 signaling regulated by CD300b in myeloid cells in response to LPS.

Structure-based developmental toxicity and ASD-phenotypes of bisphenol A analogues in embryonic zebrafish.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that has become more prevalent in recent years. Environmental endocrine disruptor bisphenol A (BPA) has been linked to ASD. BPA analogues (BPs) are structure-modified substitutes widely used as safer alternatives in consumer products, yet few studies have explored the developmental neurotoxicity (DNT) of BPA analogues. In the present study, we used the larval zebrafish model to assess the DNT effects of BPA and its analogues. Our results showed that many BPA analogues are more toxic than BPA in the embryonic zebrafish assay regarding teratogenic effect and mortality, which may partially due to differences in lipophilicity and/or different substitutes of structural function groups such as CF3, benzene, or cyclohexane. At sublethal concentrations, zebrafish embryos exposed to BPA or BPs also displayed reduced prosocial behavior in later larval development, evidenced by increased nearest neighbor distance (NND) and the interindividual distance (IID) in shoaling, which appears to be structurally independent. An in-depth analysis of BPA, bisphenol F (BPF), and bisphenol S (BPS) revealed macrocephaly and ASD-like behavioral deficits resulting from exposures to sublethal concentrations of these chemicals. The ASD-like behavioral deficits were characterized by hyperactivity, increased anxiety-like behavior, and decreased social contact. Mechanistically, accelerated neurogenesis that manifested by increased cell proliferation, the proportion of newborn mature neurons, and the number of neural stem cells in proliferation, as well as upregulated genes related to the K+ channels, may have contributed to the observed ASD-like morphological and behavioral alterations. Our findings indicate that BPF and BPS may also pose significant risks to ASD development in humans and highlight the importance of a comprehensive assessment of DNT effects for all BPA analogues in the future.

CD300f associates with IL-4 receptor α and amplifies IL-4-induced immune cell responses.

IL-4 receptor (R) α, the common receptor chain for IL-4 and IL-13, is a critical component in IL-4- and IL-13-mediated signaling and subsequent effector functions such as those observed in type 2 inflammatory responses. Nonetheless, the existence of intrinsic pathways capable of amplifying IL-4Rα-induced responses remains unknown. In this study, we identified the myeloid-associated Ig receptor CD300f as an IL-4-induced molecule in macrophages. Subsequent analyses demonstrated that CD300f was colocalized and physically associated with IL-4Rα. Using Cd300f(-/-) cells and receptor cross-linking experiments, we established that CD300f amplified IL-4Rα-induced responses by augmenting IL-4/IL-13-induced signaling, mediator release, and priming. Consistently, IL-4- and aeroallergen-treated Cd300f(-/-) mice displayed decreased IgE production, chemokine expression, and inflammatory cell recruitment. Impaired responses in Cd300f(-/-) mice were not due to the inability to generate a proper Th2 response, because IL-4/IL-13 levels were markedly increased in allergen-challenged Cd300f(-/-) mice, a finding that is consistent with decreased cytokine consumption. Finally, CD300f expression was increased in monocytes and eosinophils obtained from allergic rhinitis patients. Collectively, our data highlight a previously unidentified role for CD300f in IL-4Rα-induced immune cell responses. These data provide new insights into the molecular mechanisms governing IL-4Rα-induced responses, and may provide new therapeutic tools to target IL-4 in allergy and asthma.

Chronic co-exposure to low levels of brominated flame retardants and heavy metals induces reproductive toxicity in zebrafish.

Brominated flame retardants (BFRs) and heavy metals (HMs) are two main types of pollutants in electronic waste recycling sites, which are also ubiquitously detectable in environmental media and human tissues. However, the adverse health effects of exposure to the mixture of these types of pollutants are unknown. In this study, we investigated the reproductive toxicity of a mixture of decabromodiphenyl ether (BDE-209), tetrabromobisphenol A, cadmium chloride, and lead acetate (PbAc) at the environmental relevant levels. Zebrafish were waterborne and exposed to chemical mixtures for one generation. The reproductive effects were evaluated for F0 adults and F1 offspring. Chemical residues were also analyzed in the exposed adults and their eggs at the end of exposure. Our findings demonstrated that exposure to the chemical mixture for 150 days had no effect on the survival rate of zebrafish, but it decreased body length and weight in females and increased body weight and condition factor in males. The mixture exposure resulted in a female-biased sex ratio in adults and decreased sperm density and motility in males and egg production in females. For the F1 offspring, decreased fertilization, delayed hatching, and increased malformation were found in all exposure groups. In conclusion, chronic co-exposure to BFRs and HMs at the environmental relevant levels not only affected growth, sex ratio, and sperm quantity/quality and egg production in adults but also reduced the reproductive success in the offspring, implying that multi-pollutants in the environmental media may pose a public health risk to other exposed organisms or human beings.

Chronic PFOS Exposure Disrupts Thyroid Structure and Function in Zebrafish.

Perfluorooctane sulfonic acid (PFOS), as a potential endocrine disrupting chemical, is widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on thyroid in aquatic organisms and the underlying mechanisms are largely unknown. The present study assessed the effect of chronic PFOS exposure on thyroid structure and function using zebrafish model. Zebrafish at 8 h post fertilization (hpf) were exposed to PFOS (250 µg/l) until 120 d post fertilization (dpf). Thyroid hormone (T3 and T4) level, thyroid morphology and thyroid function related gene expression were evaluated in zebrafish at 120 dpf. Our findings demonstrated that chronic PFOS exposure altered thyroid hormone level, thyroid follicular cell structure and thyroid hormone related gene expression, suggesting the validity of zebrafish as an alternative model for PFOS chronic toxicity screening.

Strain-specific innate immune signaling pathways determine malaria parasitemia dynamics and host mortality.

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis.

Mouse IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses.

FcR specific for pentameric IgM (FCMR) is expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity, and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.

Leukocyte-associated Ig-like receptor-1-deficient mice have an altered immune cell phenotype.

Cross-linking of the collagen binding receptor leukocyte-associated Ig-like receptor-1 (LAIR-1) in vitro delivers an inhibitory signal that is able to downregulate activation-mediated signals. To study the in vivo function of LAIR-1, we generated LAIR-1(-/-) mice. They are healthy and fertile and have normal longevity; however, they show certain phenotypic characteristics distinct from wild-type mice, including increased numbers of splenic B, regulatory T, and dendritic cells. As LAIR-1(-/-) mice age, the splenic T cell population shows a higher frequency of activated and memory T cells. Because LAIR-1(+/+) and LAIR-1(-/-) T cells traffic with equal proficiency to peripheral lymphoid organs, this is not likely due to abnormal T lymphocyte trafficking. LAIR-1(-/-) mice have lower serum levels of IgG1 and, in response to T-dependent immunization with trinitrophenyl-OVA, switch less efficiently to Ag specific IgG2a and IgG2b, whereas switching to IgG1 is not affected. Several mouse disease models, including experimental autoimmune encephalitis and colitis, were used to examine the effect of LAIR-1 deficiency, and no differences in the responses of LAIR-1(-/-) and LAIR-1(+/+) mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in regulating immune cells and suggest that any adverse effects of its absence may be balanced in vivo by other inhibitory receptors.

Cutting edge: mouse CD300f (CMRF-35-like molecule-1) recognizes outer membrane-exposed phosphatidylserine and can promote phagocytosis.

Reportedly, CD300f negatively regulates interactions between dendritic and T cells and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines, an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, cosedimentation, and surface plasmon resonance using phospholipid-containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.

LAIR-1 agonism as a therapy for acute myeloid leukemia.

Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.

C-terminal peptides of chemokine-like factor 1 signal through chemokine receptor CCR4 to cross-desensitize the CXCR4.

CKLF1, a human cytokine that is a functional ligand for CCR4, is upregulated in various inflammation and autoimmune diseases. CKLF1 contains at least two secreted forms, the C-terminal peptides C19 and C27. Chemically synthesized C19 and C27 can interact with CCR4 and attenuate allergic inflammation. In this study, we found C19 and C27 could inhibit SDF-1-induced CXCR4-mediated chemotaxis and promote CXCR4 internalization. The inhibitory effect was due to desensitization of CXCR4, which was mediated by CCR4. Further experiments confirmed that CXCR4 desensitization required activation of PI3K/PKC pathway. Altogether our data elucidate the mechanism of C19- and C27-induced CXCR4 desensitization.

Cancer immunotherapy by NC410, a LAIR-2 Fc protein blocking human LAIR-collagen interaction.

Collagens are a primary component of the extracellular matrix and are functional ligands for the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor (LAIR)-1. LAIR-2 is a secreted protein that can act as a decoy receptor by binding collagen with higher affinity than LAIR-1. We propose that collagens promote immune evasion by interacting with LAIR-1 expressed on immune cells, and that LAIR-2 releases LAIR-1-mediated immune suppression. Analysis of public human datasets shows that collagens, LAIR-1 and LAIR-2 have unique and overlapping associations with survival in certain tumors. We designed a dimeric LAIR-2 with a functional IgG1 Fc tail, NC410, and showed that NC410 increases human T cell expansion and effector function in vivo in a mouse xenogeneic-graft versus-host disease model. In humanized mouse tumor models, NC410 reduces tumor growth that is dependent on T cells. Immunohistochemical analysis of human tumors shows that NC410 binds to collagen-rich areas where LAIR-1+ immune cells are localized. Our findings show that NC410 might be a novel strategy for cancer immunotherapy for immune-excluded tumors.

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells.

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

Overexpression of CHMP6 induces cellular oncosis and apoptosis in HeLa cells.

Cell death can proceed via at least two distinct pathways, apoptosis and oncosis. Apoptosis is an energy-dependent process characterized morphologically by cell shrinkage, whereas oncosis is defined as a prelethal pathway leading to cell death associated with cellular swelling, organelle swelling, and increased membrane permeability. In this study, we found that overexpression of chromatin modifying protein 6 (CHMP6) induced cell death by a series of experiments, including morphological observation, intracellular ATP determination, caspase-3 activity, and flow cytometry. Typical morphological characteristics consistent with oncosis were observed by transmission electron microscopy. Simultaneously, we obtained some results that indicated apoptosis, but the anti-apoptotic gene Bcl-xL and caspase family inhibitor Z-VAD-FMK had little effect on CHMP6-induced cell death. These results suggest that CHMP6 overexpression can cause cell death, predominantly via oncosis and to a certain extent via apoptosis, and that CHMP6 might be a novel regulator involved in both oncosis and apoptosis.

Two C-terminal peptides of human CKLF1 interact with the chemokine receptor CCR4.

Human chemokine-like factor 1 (CKLF1) exhibits chemotactic effects on leukocytes. A previous study demonstrated that CKLF1 is a functional ligand for human CC chemokine receptor 4 (CCR4). In this study, N-terminal amino acid sequencing of secreted CKLF1 protein showed that it contains at least two peptides, C27 and C19. To examine whether C27 or C19 play a role via CCR4, C27 and C19 were chemically synthesized and analyzed by chemotaxis, calcium mobilization, and receptor internalization assays in CCR4-tranfected HEK293 cells or Hut78 cells. The chemotaxis assay showed that C27 could induce chemotaxis to CCR4-transfected HEK293 cells or Hut78 cells while C19 had weaker chemotactic activity, especially in Hut78 cells. C27- or C19-induced chemotaxis was abolished by pertussis toxin, suggesting the involvement of a Gi/o pathway. C27- or C19-induced chemotaxis was also inhibited by an antagonist of CCR4 that show good binding potency, excellent chemotaxis inhibitory activity and selectivity toward CCR4, suggesting that their chemotactic activity specifically involved CCR4. The chemotactic response of CCR4-tranfected HEK293 cells to C27 or C19 was markedly inhibited by preincubation with TARC/CCL17. TARC/CCL17 effectively desensitized the calcium mobilization induced by C27 or C19. Similarly, both of C27 or C19 also desensitized the calcium mobilization and chemotaxis of CCR4-tranfected HEK293 cells in response to TARC/CCL17, suggesting that they might interact with a common receptor. Both C27- and C19-induced clear internalization of CCR4-EGFP. These results confirm that the secreted peptides of CKLF1, C27 and C19, have functional activation via CCR4.

Liposome Preparation for the Analysis of Lipid-Receptor Interaction and Efferocytosis.

Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important "eat-me" signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction-mediated signaling events controlling efferocytosis by cells like professional phagocytes. Here, we describe methods to prepare liposomes, perform liposome-based lipid-receptor binding assays, use liposomes to block efferocytosis, and utilize liposome-coated beads as apoptotic cell surrogates for phagocytosis. © 2018 by John Wiley & Sons, Inc.

Developmental and behavioral alterations in zebrafish embryonically exposed to valproic acid (VPA): An aquatic model for autism.

Autism spectrum disorder (ASD) has complex neurodevelopmental impairments and origins that are linked to both genetic and environmental factors. Hence, there is an urgency to establish animal models with ASD-like characteristics to understand the underlying mechanisms of ASD. Prenatal exposure to valproic acid (VPA) produced ASD-like symptoms in humans, rats, and recently zebrafish. The present study investigated the use of VPA exposure to generate an ASD model in zebrafish. Early life stage exposures produced ASD-like phenotypes in the developing brain development and behavioral changes in embryonic and larval zebrafish. Our findings revealed that treating zebrafish embryos with VPA starting at 8h post fertilization (hpf) resulted in significant: increase in the ASD macrocephalic phenotype; hyperactivity of embryo/larvae movement behaviors; and increases of ASD-like larval social behaviors. Further analysis showed increases in cell proliferation, the proportion of mature newborn neurons, and neural stem cell proliferation in the brain region, which may contribute to the brain overgrowth and macrocephaly observed following VPA exposure. Our study demonstrated that VPA exposure generates ASD-like phenotypes and behaviors, indicating that zebrafish is an alternative model to investigate underlying ASD mechanisms.

An alternative splice form of CMTM8 induces apoptosis.

Previous studies have demonstrated that the chemokine-like factor (CKLF)-like MARVEL transmembrane domain containing 8 (CMTM8) protein accelerates the ligand-induced clearance of epidermal growth factor receptor (EGFR) from the cell surface. The absence of EGFR-mediated signaling induces cells to undergo apoptosis via caspase-dependent and -independent pathways. Here we report the cloning and sequencing of an alternative splice form of CMTM8, obtained from a human blood cDNA library, that utilizes apoptotic pathways distinct from CMTM8. The alternative splice variant arises from a deletion of exon 2 that prevents the expression of a full-length MARVEL domain, and cytosolic YXXPhi motifs. Nevertheless, CMTM8-v2 maintains the ability to induce apoptosis via caspase-dependent and -independent pathways to inhibit cell growth and colony formation. CMTM8 and CMTM8-v2 display different expression profiles and distinct subcellular localization patterns, while operating via different mechanisms to induce apoptosis. CMTM8-v2 did not affect EGFR internalization, implying that the MARVEL domain and/or the cytosolic YXXPhi motifs are necessary for CMTM8 to accelerate ligand-induced EGFR internalization.

Dendritic cells expressing immunoreceptor CD300f are critical for controlling chronic gut inflammation.

Proinflammatory cytokine overproduction and excessive cell death, coupled with impaired clearance of apoptotic cells, have been implicated as causes of failure to resolve gut inflammation in inflammatory bowel diseases. Here we have found that dendritic cells expressing the apoptotic cell-recognizing receptor CD300f play a crucial role in regulating gut inflammatory responses in a murine model of colonic inflammation. CD300f-deficient mice failed to resolve dextran sulfate sodium-induced colonic inflammation as a result of defects in dendritic cell function that were associated with abnormal accumulation of apoptotic cells in the gut. CD300f-deficient dendritic cells displayed hyperactive phagocytosis of apoptotic cells, which stimulated excessive TNF-α secretion predominantly from dendritic cells. This, in turn, induced secondary IFN-γ overproduction by colonic T cells, leading to prolonged gut inflammation. Our data highlight a previously unappreciated role for dendritic cells in controlling gut homeostasis and show that CD300f-dependent regulation of apoptotic cell uptake is essential for suppressing overactive dendritic cell-mediated inflammatory responses, thereby controlling the development of chronic gut inflammation.

Emerging role of CD300 receptors in regulating myeloid cell efferocytosis.

Engulfment of apoptotic cells is predominantly executed by phagocytes via the recognition of "eat me" signals like phosphatidylserine (PS). Various PS-specific receptors exist on phagocytes, including Tyro3, Axl, and MerTK receptor tyrosine kinases (TAMs), T-cell immunoglobulin and mucin domain containing 1 and 4 (TIM1/4), and the newly identified CD300 family. The aim of the present auto-commentary is to highlight recent findings regarding the Cd300lf and Cd300lb receptors and their emerging roles in the development of autoimmune disease.