The novel methyltransferase SETD4 regulates TLR agonist-induced expression of cytokines through methylation of lysine 4 at histone 3 in macrophages Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Yuyun Zhong, Ping Ye, Zhuzhong Mei, Sui Huang, Mengyi Huang, Yue Li, Shixian Niu, Shuqi Zhao, Junwei Cai, Juan Wang, Hequn Zou, Yong Jiang, Jinghua Liu Abstract The production of inflammatory cytokines is closely related to pathogen-associated molecular pattern (PAMP)-triggered activation of the Toll-like receptor (TLR), intracellular signal transduction pathways such as MAPK and NF-κB, and histone modifications. Histone methylation, a type of histone modifications, is mainly accomplished by a class of SET family proteins containing highly conserved SET domains. In the present study, we found that SET domain-containing protein 4 (SETD4) regulated inflammatory cytokines in response to TLR agonists. LPS stimulation led to the enhanced SETD4 expression, while the increased IL-6 and TNF-α release from LPS-stimulated RAW264.7 cells was attenuated by depletion of SETD4 using RNA interference. The results were further confirmed in BMDMs and pMφ isolated from SETD4-deficient mice where SETD4−/− macrophages treated with LPS, BLP or Poly(I:C) showed down-regulated IL-6 and TNF-α mRNA and protein levels when compared with SETD4+/+ macrophages. Moreover, the mRNA levels of all NF-κB-dependent genes including IL-1β, IL-10, NFKBA, DUSP1, CCL2, CCL5, and CXCL10 in SETD4−/− macrophages were substantially reduced. To further clarify the regulatory mechanism(s) by which SETD4 modulates inflammatory cytokines, we examined the effect of SETD4 on the activation of MAPK and NF-κB signalling pathways, and found that knockout of SETD4 had no effect on phosphorylation of p38, ERK, JNK, p65, and IκBα. Notably, SETD4 translocated quickly from the cytosol to the nucleus upon LPS stimulation, suggesting that SETD4 may exert its regulatory function downstream of the MAPK and NF-κB pathways. To characterize this, we performed an in vitro HMTase assay to measure histone methyltransferase (HMTase) activity of SETD4. H3K4me1 and H3K4me2 levels were enhanced dramatically with the supplementation of SETD4, whereas both H3K4me1 and H3K4me2 were strongly attenuated in SETD4−/− BMDMs. Moreover, the LPS-stimulated recruitment of H3K4me1 and H3K4me2 at both TNF-α and IL-6 promoters was severely impaired in SETD4−/− BMDMs. Collectively, these results demonstrate that SETD4 positively regulates IL-6 and TNF-α expression in TLR agonist-stimulated macrophages by directly activating H3K4 methylation.

EMCHD2019 - Abstracts Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s):

Isolation of cypress gibberellin-regulated protein: Analysis of its structural features and IgE binding competition with homologous allergens Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Lisa Tuppo, Claudia Alessandri, Ivana Giangrieco, Michela Ciancamerla, Claudia Rafaiani, Maurizio Tamburrini, Maria Antonietta Ciardiello, Adriano Mari Abstract The presence in cypress pollen of an important allergen, belonging to the gibberellin-regulated protein (GRP) family, has been suggested for many years. However, it has never been isolated and sometimes the homologous peach allergen, Pru p 7, has been used as a surrogate to perform immunological investigations. The aim of this study has been the isolation and molecular characterization of the GRP contained in the Cupressus sempervirens pollen. This protein, named Cypmaclein, has been purified from the natural source using conventional biochemical methods consisting in different chromatographic separations. Cypmaclein has been identified by direct protein sequencing of the N-terminal region and of internal fragments of the molecule. In SDS-PAGE, its apparent molecular mass is slightly higher than that of Pru p 7. Nevertheless, the mass spectrometry experiments reveal that the exact molecular mass of Cypmaclein (6821.88 Da) is very close to that of Pru p 7 (6909.90 Da). Two regions of Cypmaclein have been sequenced providing 50% of its primary structure. A high overall sequence identity of Cypmaclein with all the analyzed GRP has been observed, although in the N-terminal region the high identity is limited to the homolog of Cryptomeria japonica. In circular dichroism experiments Cypmaclein produced a spectrum overlapping that of Pru p 7. However, the comparative analysis of Cypmaclein, Pru p 7 and Pun g 7 IgE reactivity revealed a behavior that was not completely overlapping, thus suggesting that the IgE epitopes are only partially shared. In single point highest inhibition achievable assays performed with the FABER test, Cypmaclein efficiently competed with the allergenic peach and pomegranate GRP in the binding of specific IgE of patients sensitized to Pru p 7. In conclusion, the natural cypress pollen GRP has been isolated for the first time, its structural features have been investigated and its cross-reactivity with Pru p 7 and Pun g 7 has been demonstrated. This protein is now available for further investigations aimed at understanding its clinical relevance in the allergy to cypress pollen. In addition, the prevalence of sensitization directly to Cypmaclein, and not limited to the homologs, can be defined.

Anti-human CD63 monoclonal antibody COS3A upregulates monocyte-induced IL-10 excretion leading to diminution of CD3-mediated T cell response Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Siriwan Wansook, Kodchakorn Mahasongkram, Nuttaphol Chruekamlow, Supansa Pata, Watchara Kasinrerk, Panida Khunkaewla Abstract Human CD63 has been reported to play a role either as an inhibitor or as a co-stimulator of T- cell responses, although the mechanism of this is unclear. In this study, an anti-human CD63 monoclonal antibody (mAb) COS3A was used to monitor the role of CD63 in T-cell activation. MAb COS3A could inhibit CD3-mediated T-cell proliferation and CD25 expression in peripheral blood mononuclear cells (PBMCs), used as a study model, but the suppressive effect was not observed when purified T-cells were used instead of PBMCs. The inhibitory phenomenon was associated with downregulation of IL-2 and IFN-γ by T-cells, but upregulation of IL-10 by monocytes. Neutralizing IL-10 with anti-IL-10 mAb improved the T-cell response, indicating the role of IL-10 in T-cell suppression. In this study, monocytes were demonstrated to play a role in impeding T-cell activation by the anti-CD63 mAb COS3A. This is the first evidence that anti-CD63 mAb induces IL-10 secretion by monocytes, which later play a role in T-cell hypo-responsiveness.

Protection to immune system of mice by N-acetyl tryptophan glucoside (NATG) against gamma radiation induced immune suppression Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Poonam Malhotra, Ashutosh K Gupta, Darshana Singh, Saurabh Mishra, Shravan K Singh, Raj Kumar Abstract Immune system is a critical modulator of radiation-induced biological effects. In this study, we have assessed protective potential of N-acetyl tryptophan glucoside (NATG) pre-treatment in bone marrow of gamma radiation challenged mice. Isolated bone marrow cells were analysed for cell cycle progression by flow cytometry, while various pro-/anti-inflammatory cytokine profiles were performed by ELISA method. Overall radioprotective ability of NATG in ensuring protection against gamma radiation-induced damage was assessed by evaluating whole body survival analysis and haematological studies on 9 Gy irradiated mice with/without NATG pre-treatment. Results exhibited pre-treatment with 150 mg/kg b.wt oral administration of NATG as most effective against 9 Gy radiation exposure. Moreover, NATG showed non-interfering effect on cell cycle progression in pre-treated irradiated mice group when compared to radiation alone group. In addition, cytokine expression analysis indicated significant (p > 0.05) elevation in levels of IFN-γ, IL-2, IL-12, IL-13 and IL-17 in NATG pre-treated irradiated mice in comparison to radiation alone group. On the contrary, NATG pre-treatment was observed to alleviate levels of TNF-α and IL-10 significantly (p < 0.05) in radiated group as compared to only irradiated mice group. Furthermore, NATG pre-treatment to 9 Gy radiation exposed mice aided in restoring their haematological parameters in terms of haemoglobin counts, RBC counts, WBC counts, hematocrit levels, platelets and granulocyte levels in comparison to irradiated alone mice, thus enhancing their immune system and contributing towards a better survival against gamma radiation-induced deleterious effects. Conclusively, this study highlights the potential of NATG as a prospective radiation countermeasure agent against ionizing radiation-induced assaults to the immune system.

Cucurbitacin E ameliorates lipopolysaccharide-evoked injury, inflammation and MUC5AC expression in bronchial epithelial cells by restraining the HMGB1-TLR4-NF-κB signaling Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Jin Shang, Weihua Liu, Chunyan Yin, Haiping Chu, Meizhen Zhang Abstract Asthma is a chronic inflammatory disorder of airway affecting people from childhood to old age, and is characterized by airway epithelial dysfunction. Cucurbitacin E (CuE), a tetracyclic triterpene isolated from Cucurbitaceae plants, has been recently proved to exert anti-inflammation and immunology regulation activities. Nevertheless, its roles in asthma remains poorly defined. In the current study, CuE had little cytotoxicity on cell viability of human bronchial epithelial cell line BEAS-2B. Moreover, lipopolysaccharide (LPS) exposure inhibited cell viability and induced cell apoptosis, which was reversed following CuE pretreatment. Additionally, CuE administration suppressed LPS-induced inflammatory cytokine production, including TNF-α, IL-6, and IL-8. Simultaneously, supplementation with CuE decreased the transcripts and releases of mucin 5AC (MUC5AC) in LPS-treated BEAS-2B cells. Intriguingly, CuE inhibited LPS-evoked activation of the high-mobility group box1 (HMGB1)-TLR4-NF-κB signaling by reducing the expression of HMGB1, TLR4 and p-p65 NF-κB. Notably, restoring this pathway by elevating HMGB1 expression largely offset the protective function of CuE against LPS-triggered cell injury, inflammatory response and MUC5AC expression. Consequently, these findings highlight that CuE can ameliorate human bronchial epithelial cell insult and inflammation under LPS-simulated asthmatic conditions by blocking the HMGB1-TLR4-NF-κB signaling, thereby supporting its usefulness as a promising therapeutic agent against asthma.

The histamine-4 receptor antagonist JNJ7777120 prevents immune abnormalities by inhibiting RORγt/T-bet transcription factor signaling pathways in BTBR T+ Itpr3tf/J mice exposed to gamma rays Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Sheikh F. Ahmad, Ahmed Nadeem, Mushtaq A. Ansari, Saleh A. Bakheet, Haneen A. Al-Mazroua, Mohammad R. Khan, Abdullah F. Alasmari, Wael A. Alanazi, Homood M. As Sobeai, Sabry M. Attia Abstract Autism is a neurodevelopmental disorder characterized by deficits and qualitative impairments in communication and implicit skill learning. Its prevalence is higher than previous estimates, and treatments have limited efficacy and are costly. Here, we assessed the therapeutic potential of JNJ77777120 (JNJ), a histamine-4 receptor (H4R) antagonist, using BTBR T+ Itpr3tf/J (BTBR) mice, a confirmed model of autism, and C57BL/6J (C57) mice, a commonly chosen reference strain. We first examined the effects of JNJ treatment on BTBR mice exposed to gamma-rays (irradiation-exposed) using a three-chambered apparatus. We further investigated the possible molecular mechanisms through which JNJ administration modulates IL-17A-, RORγT-, IL-22-, T-bet-, STAT3-, ICOS-, and Foxp3-producing CD8+ T cells in the spleens of irradiation-exposed BTBR mice. The effects of JNJ administration on the mRNA and protein expression of IL-17A, RORγT, IL-22, T-bet, STAT-3, pSTAT3, IL-10, and Foxp3 in brain tissue were also explored. Results showed that JNJ treatment with irradiation exposure increased social interactions in BTBR mice compared to that in irradiation-exposed BTBR mice. Additionally, JNJ-treated and irradiation-exposed BTBR mice exhibited decreases in IL-17A-, RORγT-, IL-22-, T-bet-, and STAT3-producing CD8+ T cells and increases in ICOS- and Foxp3-producing CD8+ T cells. Moreover, JNJ treatment and irradiation exposure in BTBR mice regulated the mRNA and protein expression levels of IL-17A, RORγT, IL-22, T-bet, STAT3, pSTAT-3, IL-10, and Foxp3 in the brain tissue. These results suggest that JNJ is useful for the treatment of autism, as this H4R antagonist could block inflammatory cytokine production and transcription factor signaling.

Expression and presentation of immune-related membrane proteins of fish by a cell surface display platform using insect cells Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Satoshi Tasumi, Keisuke Kobayashi, Shihori Takanashi, Shuichi Asakawa, Osamu Nakamura, Kiyoshi Kikuchi, Yuzuru Suzuki Abstract Cell surface display is a useful platform to examine the interactions between two proteins of interest, such as immune receptors and ligands. This technique is also useful for studies on the immune receptors of lower vertebrates and invertebrates. However, in many cases, the commonly used cell culture temperature is relatively high for proteins from such organisms. Since insect cells can be cultured at lower temperatures than many other cells, and since they are equipped with “quality control” system, which is advantageous for the presentation of properly folded proteins, we anticipated that the insect cell surface display system could be more suitable for that type of research. In the present study, multiple cloning site of the commercially available expression vector pIB/V5-His was modified, and whether this vector could be useful to present fish immune-related membrane proteins was investigated. Using this plasmid, fugu’s CD8α and CC chemokine receptor 7 could be presented on the cell surface. The clones of the lamprey variable lymphocyte receptors obtained previously by the yeast surface display (YSD) system as hen’s egg lysozyme (HEL) binders also could be presented on the cell surface and bound to HEL. These results suggest that functional immune-related membrane proteins can be presented on the insect cell surface, indicating that this system is useful for immunological studies on exothermal animals.

Three-dimensional structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Akihiro Nishiguchi, Nobutaka Numoto, Nobutoshi Ito, Takachika Azuma, Masayuki Oda Abstract Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of VH and DH gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.

Giardia excretory-secretory proteins modulate the enzymatic activities of mast cell chymase and tryptase Publication date: October 2019 Source: Molecular Immunology, Volume 114 Author(s): Zhiqiang Li, Dimitra Peirasmaki, Staffan Svärd, Magnus Åbrink Abstract Background Mast cells are involved in the host immune response controlling infection with the non-invasive intestinal protozoan parasite Giardia intestinalis. Experimental infections in rodents with G. intestinalis showed increased intestinal expression of mucosal and connective mast cell specific proteases suggesting that both mucosal and connective tissue mast cells are recruited and activated during infection. During infection Giardia excretory-secretory proteins (ESPs) with immunomodulatory capacity are released. However, studies investigating potential interactions between Giardia ESPs and the connective tissue mast cell specific serine proteases, i.e. human chymase and mouse mast cell protease (mMCP)-4 and, human and mouse tryptase (mMCP-6) remain scarce. Results We first investigated if soluble Giardia proteins (sGPs), which over-lap extensively in protein content with ESP fractions, from the isolates GS, WB and H3, could induce mast cell activation. sGPs induced a minor activation of bone marrow derived mucosal-like mast cells, as indicated by increased IL-6 secretion and no degranulation. Furthermore, sGPs were highly resistant to degradation by human tryptase while human chymase degraded a 65 kDa sGP and, wild-type mouse ear tissue extracts degraded several protein bands in the 10 to 75 kDa range. In striking contrast, sGPs and ESPs were found to increase the enzymatic activity of human and mouse tryptase and to reduce the activity of human and mouse chymase. Conclusion Our finding suggests that Giardia ssp. via enhancement or reduction of mast cell protease activity may modulate mast cell-driven intestinal immune responses. ESP-mediated modulation of the mast cell specific proteases may also increase degradation of tight junctions, which may be beneficial for Giardia ssp. during infection.