The Aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNFα, and IFNγ cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNFα- and IFNγ-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes.

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Åberg CH, Kelk P, Johansson A (2015). Aggregatibacter actinomycetemcomitans: virulence of its leukotoxin and association with aggressive periodontitis. Virulence 6: 188-195.
Argueta JGM, Shiota S, Yamaguchi N, Masuhiro Y, Hanazawa S (2006). Induction of Porphyromonas gingivalis GroEL signaling via binding to Toll-like receptors 2 and 4. Oral Microbiol Immunol 21: 245-251.
Boesze-Battaglia K, Brown A, Walker L, Besack D, Zekavat A, Wrenn S, Krummenacher C, Shenker BJ (2009). Cytolethal distending toxin-induced cell cycle arrest of lymphocytes is dependent upon recognition and binding to cholesterol. J Biol Chem 17: 10650-10658.
Boesze-Battaglia K, Walker LP, Zekavat A, Dlakić M, Scuron MD, Nygren P, Shenker BJ (2015). The Aggregatibacter actinomycetemcomitans cytolethal distending toxin active subunit CdtB contains a cholesterol recognition sequence required for toxin binding and subunit internalization. Infect Immun 83: 4042-4055.
Böyum A (1968). Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97: 77-89.
Bulut Y, Michelsen KS, Hayrapetian L, Naiki Y, Spallek R, Singh M, Arditi M (2005). Mycobacterium tuberculosis heat shock proteins use diverse Toll-like receptor pathways to activate pro-inflammatory signals. J Biol Chem 280: 20961-20967.
Goulhen F, Hafezi A, Uitto VJ, Hinode D, Nakamura R, Grenier D, Mayrand D (1998). Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillusactinomycetemcomitans. Infect Immun 66: 5307-5313.
Haubek D, Johansson A (2014). Pathogenicity of the highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans and its geographic dissemination and role in aggressive periodontitis. J Oral Microbiol 14: 6.
Herbert BA, Novince CM, Kirkwood KL (2016). Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis. Mol Oral Microbiol 31: 207-227.
Kato S, Nakashima K, Nagasawa T, Abiko Y, Furuichi Y (2013). Involvement of Toll-like receptor 2 in apoptosis of Aggregatibacter actinomycetemcomitans-infected THP-1 cells. J Microbiol Immunol Infect 46: 164-170.
Kim TH, Shin SJ, Park YM, Jung ID, Ryu SW, Kim DJ, Park JH, Park JH (2015). Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells. Cytokine 71: 139-144.
Kirby AC, Meghji S, Nair SP, White P, Reddi K, Nishihara T, Nakashima K, Willis AC, Sim R, Wilson M et al. (1995). The potent bone resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL. J Clin Invest 96: 1185-1194.
Koga T, Kusuzaki T, Asakawa H, Senpuku H, Nishihara T, Noguchi T (1993). The 64 kilodalton GroEL-like protein of Actinobacillusactinomycetemcomitans. J Periodontal Res 28: 475-477.
Korostoff J, Wang JF, Kieba I, Miller M, Shenker BJ, Lally ET (1998). Actinobacillus actinomycetemcomitans leukotoxin induces apoptosis in HL-60 cells. Infect Immun 66: 4474-4483.
Lally ET, Kieba IR, Sato A, Green CL, Rosenbloom J, Korostoff J, Wang JF, Shenker BJ, Ortlepp S, Robinson MK et al. (1997). RTX toxins recognize a beta2 integrin on the surface of human target cells. J Biol Chem 272: 30463-30469.
Mangan DF, Taichman NS, Lally ET, Wahl SM (1991). Lethal effects of Actinobacillus actinomycetemcomitans leukotoxin on human T lymphocytes. Infect Immun 59: 3267-3272.
Mayer MP, Bueno LC, Hansen EJ, DiRienzo JM (1999). Identification of a cytolethal distending toxin gene locus and features of a virulence-associated region in Actinobacillus actinomycetemcomitans. Infect Immun 67: 1227-1237.
Nalbant A, Chen C, Wang Y, Zadeh HH (2003). Induction of T-cell apoptosis by Actinobacillus actinomycetemcomitans mutants with deletion of ltxA and cdtABC genes: possible activity of GroEL-like molecule. Oral Microbiol Immunol 18: 339-349.
Nishida E, Hara Y, Kaneko T, Ikeda Y, Ukai T, Kato I. (2001). Bone resorption and local interleukin-1α and interleukin-1β synthesis induced by Actinobacillus actinomycetemcomitansand Porphyromonas gingivalis lipopolysaccharide. J Periodontal Res36: 1-8.
Paju S, Goulhen F, Asikainen S, Grenier D, Mayrand D, Uitto V (2000). Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation. FEMS Microbiol Lett 182: 231-235.
Park SR, Kim DJ, Han SH, Kang MJ, Lee JY, Jeong YJ, Lee SJ, Kim TH, Ahn SG, Yoon JH et al. (2014). Diverse Toll-like receptors mediate cytokine production by Fusobacterium nucleatumand Aggregatibacter actinomycetemcomitans in macrophages. Infect Immun 82: 1914-1920.
Patil C, Rossa C Jr, Kirkwood KL (2006). Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activatedprotein kinase pathways in periodontal ligament fibroblasts. Oral Microbiol Immunol 21: 392-398.
Res PC, Thole JE, de Vries RR (1991) Heat shock proteins in immunopathology. Curr Opin Immunol 3: 924-929.
Saygılı T, Akıncılar SC, Akgul B, Nalbant A (2012). Aggregatibacter actinomycetemcomitans GroEL protein promotes conversion ofhuman CD4+ T cells into IFNγ IL10 producing Tbet+ Th1 cells.PLoS ONE 7: e49252.
Sfakianakis A, Barr CE, Kreutzer D (2001). Mechanisms of Actinobacillus actinomycetemcomitans-induced expression of interleukin-8 in gingival epithelial cells. J Periodontol 72: 1413-1419.
Shenker BJ, Hoffmaster RH, McKay TL, Demuth DR (2000). Expression of the cytolethal distending toxin (Cdt) operon in Actinobacillus actinomycetemcomitans: evidence that the CdtB protein is responsible for G2 arrest of the cell cycle in human T-cells. J Immunol165: 2612-2618.
Shenker BJ, Walker LP, Zekavat A, Dlakić M, Boesze-Battaglia K (2014). Blockade of the PI-3K signalling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro-inflammatory cytokines. Cell Microbiol 16: 1391-1404.
Slots J, Ting M (1999). Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in human periodontal disease: occurrence and treatment. Periodontol 2000 20: 82-121.
Tabeta K, Yoshie H, Yamazaki K (2001). Characterization of serum antibody to Actinobacillus actinomycetemcomitans GroEL-like protein in periodontitis patients and healthy subjects. Oral Microbial Immunol 16: 290-295.