BACKGROUND: Pseurotins, a family of secondary metabolites of different fungi characterized by an unusual spirocyclic furanone-lactam core, are suggested to have different biological activities including the modulation of immune response. PURPOSE: Complex characterization of the effects of pseurotin D on human lymphocyte activation in order to understand the potential of pseurotin to modulate immune response in humans. METHODS: CD4+ and CD8+ T cells and CD19+ B cells isolated from human blood were activated by various activators simultaneously with pseurotin D treatment. The effects of pseurotin were tested on the basis of changes in cell viability, apoptosis, activation of signal transducers and activators of transcription (STAT) signaling pathways, production of tumor necrosis factor (TNF)-α by T cells, expression of activation markers CD69 and CD25 on T cells and Human Leukocyte Antigen-DR isotype (HLA-DR) on B cells, and the differentiation markers CD20, CD27, CD38, and immunoglobulin (Ig) D on B cells. RESULTS: Pseurotin D significantly inhibited the activation of both CD4+ and CD8+ human T cells complemented by the inhibition of TNF-α production without significant acute toxic effects. The Pseurotin D-mediated inhibition of T-cell activation was accompanied by the induction of the apoptosis of T cells. This corresponded with the inhibited phosphorylation of STAT3 and STAT5. In human B cells, pseurotin D did not significantly inhibit their activation; however, it affected their differentiation. CONCLUSIONS: Our results advance the current mechanistic understanding of the pseurotin-induced inhibition of lymphocytes and suggest pseurotins as new attractive chemotypes for future research in the context of immune-modulatory drugs.

Department of Experimental Biology Faculty of Science Masaryk University 625 00 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences 612 65 Brno Czech Republic

International Clinical Research Center St Anne's University Hospital 656 91 Brno Czech Republic

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Varga J., Frisvad J.C., Samson R.A. Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Stud. Mycol. 2011;69:57–80. doi: 10.3114/sim.2011.69.05. PubMed DOI PMC

Maiya S., Grundmann A., Li X., Li S.M., Turner G. Identification of a hybrid PKS/NRPS required for pseurotin A biosynthesis in the human pathogen Aspergillus fumigatus. Chembiochem. 2007;8:1736–1743. doi: 10.1002/cbic.200700202. PubMed DOI

Tsunematsu Y., Fukutomi M., Saruwatari T., Noguchi H., Hotta K., Tang Y., Watanabe K. Elucidation of pseurotin biosynthetic pathway points to trans-acting C-methyltransferase: Generation of chemical diversity. Angew. Chem. Int. Ed. Engl. 2014;53:8475–8479. doi: 10.1002/anie.201404804. PubMed DOI PMC

Ishikawa M., Ninomiya T. Chemical modification of pseurotin A: One-pot synthesis of synerazol and pseurotin E and determination of absolute stereochemistry of pseurotin E. J. Antibiot. 2008;61:692–695. doi: 10.1038/ja.2008.99. PubMed DOI

Bloch P., Tamm C., Bollinger P., Petcher T.J., Weber H.P. Pseurotin, a new metabolite of Pseudeurotium ovalis Stolk having an unusual hetero-spirocyclic system. Helv. Chim. Acta. 1976;59:133–137. doi: 10.1002/hlca.19760590114. PubMed DOI

Jo D., Han S. Total syntheses of spirocyclic PKS-NRPS-based fungal metabolites. Chem. Commun. 2018;54:6750–6758. doi: 10.1039/C8CC02315H. PubMed DOI

Fisch K.M. Biosynthesis of natural products by microbial iterative hybrid PKS-NRPS. RSC Adv. 2013;3:18228–18247. doi: 10.1039/c3ra42661k. DOI

Ando O., Satake H., Nakajima M., Sato A., Nakamura T., Kinoshita T., Furuya K., Haneishi T. Synerazol, a New Antifungal Antibiotic. J. Antibiot. 1991;44:382–389. doi: 10.7164/antibiotics.44.382. PubMed DOI

Komagata D., Fujita S., Yamashita N., Saito S., Morino T. Novel neuritogenic activities of pseurotin A and penicillic acid. J. Antibiot. 1996;49:958–959. doi: 10.7164/antibiotics.49.958. PubMed DOI

Asami Y., Kakeya H., Komi Y., Kojima S., Nishikawa K., Beebe K., Neckers L., Osada H. Azaspirene, a fungal product, inhibits angiogenesis by blocking Raf-1 activation. Cancer Sci. 2008;99:1853–1858. doi: 10.1111/j.1349-7006.2008.00890.x. PubMed DOI PMC

Igarashi Y., Yabuta Y., Sekine A., Fujii K., Harada K., Oikawa T., Sato M., Furumai T., Oki T. Directed biosynthesis of fluorinated pseurotin A, synerazol and gliotoxin. J. Antibiot. 2004;57:748–754. doi: 10.7164/antibiotics.57.748. PubMed DOI

Asami Y., Kakeya H., Onose R., Yoshida A., Matsuzaki H., Osada H. Azaspirene: A novel angiogenesis inhibitor containing a 1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione skeleton produced by the fungus Neosartotya sp. Organ. Lett. 2002;4:2845–2848. doi: 10.1021/ol020104+. PubMed DOI

Anjum K., Bi H., Chai W., Lian X.Y., Zhang Z. Antiglioma pseurotin A from marine Bacillus sp. FS8D regulating tumour metabolic enzymes. Nat. Prod. Res. 2017;32:1–4. doi: 10.1080/14786419.2017.1343329. PubMed DOI

Vasicek O., Rubanova D., Chytkova B., Kubala L. Natural pseurotins inhibit proliferation and inflammatory responses through the inactivation of STAT signaling pathways in macrophages. Food Chem. Toxicol. 2020;141:111348. doi: 10.1016/j.fct.2020.111348. PubMed DOI

Vasicek O., Fedr R., Skoroplyas S., Chalupa D., Sklenar M., Tharra P.R., Svenda J., Kubala L. Natural pseurotins and analogs thereof inhibit activation of B-cells and differentiation into the plasma cells. Phytomedicine. 2020;69:153194. doi: 10.1016/j.phymed.2020.153194. PubMed DOI

Ishikawa M., Ninomiya T., Akabane H., Kushida N., Tsujiuchi G., Ohyama M., Gomi S., Shito K., Murata T. Pseurotin A and its analogues as inhibitors of immunoglobulin E [correction of immunoglobuline E] production. Bioorg. Med. Chem. Lett. 2009;19:1457–1460. doi: 10.1016/j.bmcl.2009.01.029. PubMed DOI

Gould H.J., Sutton B.J. IgE in allergy and asthma today. Nat. Rev. Immunol. 2008;8:205–217. doi: 10.1038/nri2273. PubMed DOI

Bluml S., McKeever K., Ettinger R., Smolen J., Herbst R. B-cell targeted therapeutics in clinical development. Arthritis Res. Ther. 2013;15(Suppl. 1):S4. doi: 10.1186/ar3906. PubMed DOI PMC

U.S. Department of Health and Human Services Centers for Disease Control and Prevention [(accessed on 3 December 2020)];Summary Health Statistics for U.S. Adults: National Health Interview Survey. 2017 Available online: https://www.cdc.gov/nchs/nhis/shs.htm.

Rabb H. The T cell as a bridge between innate and adaptive immune systems: Implications for the kidney. Kidney Int. 2002;61:1935–1946. doi: 10.1046/j.1523-1755.2002.00378.x. PubMed DOI

Li L., Yee C., Beavo J.A. CD3- and CD28-dependent induction of PDE7 required for T cell activation. Science. 1999;283:848–851. doi: 10.1126/science.283.5403.848. PubMed DOI

Hasbold J., Hong J.S., Kehry M.R., Hodgkin P.D. Integrating signals from IFN-gamma and IL-4 by B cells: Positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a. J. Immunol. 1999;163:4175–4181. PubMed

Avery D.T., Ma C.S., Bryant V.L., Santner-Nanan B., Nanan R., Wong M., Fulcher D.A., Cook M.C., Tangye S.G. STAT3 is required for IL-21-induced secretion of IgE from human naive B cells. Blood. 2008;112:1784–1793. doi: 10.1182/blood-2008-02-142745. PubMed DOI

Ding B.B., Yu J.J., Yu R.Y., Mendez L.M., Shaknovich R., Zhang Y., Cattoretti G., Ye B.H. Constitutively activated STAT3 promotes cell proliferation and survival in the activated B-cell subtype of diffuse large B-cell lymphomas. Blood. 2008;111:1515–1523. doi: 10.1182/blood-2007-04-087734. PubMed DOI PMC

Ul-Haq Z., Naz S., Mesaik M.A. Interleukin-4 receptor signaling and its binding mechanism: A therapeutic insight from inhibitors tool box. Cytokine Growth Factor. Rev. 2016;32:3–15. doi: 10.1016/j.cytogfr.2016.04.002. PubMed DOI

Flynn M.J., Hartley J.A. The emerging role of anti-CD25 directed therapies as both immune modulators and targeted agents in cancer. Br. J. Haematol. 2017;179:20–35. doi: 10.1111/bjh.14770. PubMed DOI

Cibrian D., Sanchez-Madrid F. CD69: From activation marker to metabolic gatekeeper. Eur. J. Immunol. 2017;47:946–953. doi: 10.1002/eji.201646837. PubMed DOI PMC

Kimura M.Y., Hayashizaki K., Tokoyoda K., Takamura S., Motohashi S., Nakayama T. Crucial role for CD69 in allergic inflammatory responses: CD69-Myl9 system in the pathogenesis of airway inflammation. Immunol. Rev. 2017;278:87–100. doi: 10.1111/imr.12559. PubMed DOI

Reddy M., Eirikis E., Davis C., Davis H.M., Prabhakar U. Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: An in vitro model to monitor cellular immune function. J. Immunol. Methods. 2004;293:127–142. doi: 10.1016/j.jim.2004.07.006. PubMed DOI

Marchingo J.M., Kan A., Sutherland R.M., Duffy K.R., Wellard C.J., Belz G.T., Lew A.M., Dowling M.R., Heinzel S., Hodgkin P.D. T cell signaling. Antigen affinity, costimulation, and cytokine inputs sum linearly to amplify T cell expansion. Science. 2014;346:1123–1127. doi: 10.1126/science.1260044. PubMed DOI

Zhu J., Paul W.E. CD4 T cells: Fates, functions, and faults. Blood. 2008;112:1557–1569. doi: 10.1182/blood-2008-05-078154. PubMed DOI PMC

Geha R.S., Jabara H.H., Brodeur S.R. The regulation of immunoglobulin E class-switch recombination. Nat. Rev. Immunol. 2003;3:721–732. doi: 10.1038/nri1181. PubMed DOI

Chen T., Zhang B., Lin Y., Pan J., Zhao X.X., Ren S.X. Protective Effects of Prunasin A against the Differentiation of Osteoclasts and Destruction of Cartilage via the Receptor Activator of Nuclear Factor-Kappa-Beta Ligand/Mitogen-Activated Protein Kinase/Osteoprotegerin Pathway in a Rat Model of Arthritis. Pharmacology. 2019;104:216–225. doi: 10.1159/000502537. PubMed DOI

Moriggl R., Topham D.J., Teglund S., Sexl V., McKay C., Wang D., Hoffmeyer A., van Deursen J., Sangster M.Y., Bunting K.D., et al. Stat5 is required for IL-2-induced cell cycle progression of peripheral T cells. Immunity. 1999;10:249–259. doi: 10.1016/S1074-7613(00)80025-4. PubMed DOI

Rajasingh J., Raikwar H.P., Muthian G., Johnson C., Bright J.J. Curcumin induces growth-arrest and apoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cell leukemia. Biochem. Biophys. Res. Commun. 2006;340:359–368. doi: 10.1016/j.bbrc.2005.12.014. PubMed DOI

Chen K., Qiu P.C., Yuan Y., Zheng L., He J.B., Wang C., Guo Q., Kenny J., Liu Q., Zhao J.M., et al. Pseurotin A Inhibits Osteoclastogenesis and Prevents Ovariectomized-Induced Bone Loss by Suppressing Reactive Oxygen Species. Theranostics. 2019;9:1634–1650. doi: 10.7150/thno.30206. PubMed DOI PMC

Kienzler A.K., Rizzi M., Reith M., Nutt S.L., Eibel H. Inhibition of human B-cell development into plasmablasts by histone deacetylase inhibitor valproic acid. J. Allergy Clin. Immunol. 2013;131:1695–1699. doi: 10.1016/j.jaci.2013.01.018. PubMed DOI

Abdelwahed K.S., Siddique A., Mohyeldin M.M., Qusa M.H., Goda A.A., Singh S.S., Ayoub N.M., King J.A., Jois S.D., El Sayed K.A. Pseurotin A as a novel suppressor of hormone dependent breast cancer progression and recurrence by inhibiting PCSK9 secretion and interaction with LDL receptor. Pharmacol. Res. 2020;158:104847. doi: 10.1016/j.phrs.2020.104847. PubMed DOI PMC

Georgiev Y.N., Paulsen B.S., Kiyohara H., Ciz M., Ognyanov M.H., Vasicek O., Rise F., Denev P.N., Lojek A., Batsalova T.G., et al. Tilia tomentosa pectins exhibit dual mode of action on phagocytes as beta-glucuronic acid monomers are abundant in their rhamnogalacturonans I. Carbohydr. Polym. 2017;175:178–191. doi: 10.1016/j.carbpol.2017.07.073. PubMed DOI

Vasicek O., Lojek A., Ciz M. Serotonin and its metabolites reduce oxidative stress in murine RAW264.7 macrophages and prevent inflammation. J. Physiol. Biochem. 2020;76:49–60. doi: 10.1007/s13105-019-00714-3. PubMed DOI

Pseurotin D Induces Apoptosis through Targeting Redox Sensitive Pathways in Human Lymphoid Leukemia Cells