Koichi Kubota. Department of Microbiology, Kitasato University School of Medicine

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1 Communication Kitasato Med J 2017; 47: Suppression of the cytopathic effect of herpes simplex virus by the functional T cell hybridoma B6HO3: a subset of innate lymphocytes may have versatile functions for combating early microbial infection Koichi Kubota Department of Microbiology, Kitasato University School of Medicine In a previous study, we found that functional T cell hybridoma B6HO3 cells respond to cell death in bacterial-infected macrophages with the production of IFN-γ that activates the bactericidal function of macrophages. In the present study, we show that B6HO3 cells also have the ability to respond to herpes simplex virus type 1 infection in J774 macrophage cells and Hepa1-6 cells by suppressing the cytopathic effect of this virus. The functional T cell hybridoma system has been reported to preserve the effector functions of parental lymphocytes, and the parental cell of the B6HO3 hybridoma belongs to innate-like T cells. The findings presented here, therefore, lead us to the hypothesis that a subset of innate lymphocytes may be equipped with versatile functions for combating both bacterial and viral infections. Key words: arginase, arginine, innate immunity, type 1 interferon Abbreviations: CPE, cytopathic effect; HSV, herpes simplex virus; NO, nitric oxide; L-NMMA, N G -monomethyl-l-arginine; L-NAME, N G -nitro-l-arginine methyl ester Findings and Discussion W e previously established a functional T cell hybridoma cell line termed B6HO3 from growtharrested hybrids between YACUT lymphoma cells and C3H/He anti-dba mixed lymphocyte culture cells. 1 This hybridoma responded to cell death in bacterial-infected macrophages with the production of IFN-γ, and this property was considered to reflect the effector function of an innate-like T cell, the parental T cell of this hybridoma. 2,3 In the present study, we investigated whether or not cell death caused by viral infection also induces B6HO3 cells to produce IFN-γ. To this end, J774 mouse macrophage cells were infected with herpes simplex virus type 1 (HSV-1) and cocultured with B6HO3 cells for 48 hours; then, the culture supernatants were examined for IFN-γ production. Unlike the results obtained with bacterial infection, 1 IFN-γ was not detected in the culture supernatants (data not shown). During the course of this study, we observed that, while all of the HSV-1-infected J774 cells underwent cell death in the absence of B6HO3 cells, many J774 cells remained alive in the coculture wells, suggesting that B6HO3 cells Received 17 November 2016, accepted 19 December 2016 Correspondence to: Koichi Kubota, Department of Microbiology, Kitasato University School of Medicine Kitasato, Minami-ku, Sagamihara, Kanagawa , Japan koichi-tn@jcom.home.ne.jp 87 inhibited HSV-1 replication. To further examine this suppressive effect of B6HO3 cells on HSV-1 replication, J774 cells were inoculated with HSV-1 and cocultured with B6HO3 cells in a Transwell to separate both cells with a permeable membrane. As shown in Figure 1B, HSV-1 caused cytopathic effect (CPE) on J774 cells when HSV-1- inoculated J774 cells were cultured alone for 72 hours, but when HSV-1-inoculated J774 cells were cocultured for 72 hours with B6HO3 cells in a Transwell, the CPE of HSV-1 was not observed (Figure 1C). This result indicated that B6HO3 cells produced some soluble factor that suppressed the CPE of HSV-1. However, because the suppression of the CPE did not occur when HSV-1- inoculated cells were cultured for 72 hours together with culture supernatants obtained from 48-hour cocultivation of HSV-1-inoculated J774 cells with B6HO3 cells (Figure 1D), the inhibitory factor in the culture supernatants seemed to be quite unstable. Furthermore, the number of J774 cells in the coculture was much lower than that of control J774 cells that were cultured alone (Figure 1A and 1C), suggesting that the growth of J774 cells was also suppressed in the presence of B6HO3 cells. HSV-1

2 Kubota K. itself has the ability to inhibit cell cycle progression. 4 However, it is unlikely that the growth inhibition of J774 cells was attributable to viral infection, because only a subpopulation of the J774 cells was infected with HSV- 1 due to low multiplicity of infection. Thus, the putative CPE-inhibitory factor produced by B6HO3 cells seemed to also have cell growth-inhibitory activity. This was also supported by the fact that the proliferation of B6HO3 cells in the upper well of the Transwell decreased in the coculture (data not shown). The same results were obtained with the CPE of HSV- 1 on mouse hepatoma Hepa1-6 cells (Figure 2B). Confluent Hepa1-6 cells were inoculated with HSV-1 and they were separately cocultured with B6HO3 cells for 72 hours in a Transwell. Control Hepa1-6 cells are shown in Figure 2A. B6HO3 cells suppressed the CPE of HSV-1 (Figure 2C), but culture supernatants obtained from 48-hour cocultivation of B6HO3 cells with HSV- 1-infected Hepa1-6 cells did not have suppressive activity (Figure 2D). Furthermore, the B6HO3-mediated suppression of the CPE of HSV-1 still occurred even when B6HO3 cells in the upper well were removed from the Transwell at 24 hours after coculture (data not shown). This result indicated that B6HO3 cells responding to HSV-1-infected cells completed the inhibition of HSV-1 replication and propagation within the first 24 hours of coculture. Nitric oxide (NO), which has been shown to inhibit HSV-1 replication and cell proliferation, 5-8 is a candidate for the unstable factor produced by B6HO3 cells. Therefore, we next examined the involvement of NO in the B6HO3-mediated suppression of the CPE of HSV-1 by adding the arginine analogs, N G -monomethyl-larginine (L-NMMA) and N G -nitro-l-arginine methyl ester (L-NAME) as inhibitors of NO synthase 9 to the coculture of HSV-1-inoculated Hepa1-6 cells with B6HO3 cells in a Transwell (Figure 3). Figure 3D and E show that neither inhibitor reversed the suppressive activity of B6HO3 cells, Figure 1. B6HO3 cells suppress CPE caused by HSV-1 in J774 macrophage cells. J774 cells ( /well) were plated in a bottom well of a 24-well Transwell plate and cultured for 16 hours. After culture medium was removed, 0.5 ml of HSV-1 in PBS ( TCID50/ml HSV-1) was added to the well, and the plate was incubated at room temperature for 1 hour. Then, PBS was removed and 1 ml of culture medium was added to the well. B6HO3 cells ( ) in 0.5ml of culture medium were added to an upper well of the Transwell. Subsequently, the plate was incubated in a CO2 incubator at 37 for 72 hours (C). (A), Control J774 cells without HSV-1 and B6HO3 cells. (B), HSV-1-inoculated J774 cells without B6HO3 cells. (D), HSV-1- inoculated J774 cells cultured in the mixture of 0.5 ml of fresh culture medium and 0.5 ml of culture supernatants (sup) obtained from 48- hour cocultivation of HSV-infected J774 cells with B6HO3 cells. Cells were observed under a phase contrast microscope at 72 hours after HSV-1 inoculation (100 ). Scale bar = 50μm 88

3 Multifunction of T cell hybridoma B6HO3 indicating that NO is not involved in this phenomenon. Noninvolvement of NO was also confirmed by the fact that nitrite formation was not detected with Griess reagent 10 in the culture supernatants from those cocultures (data not shown). Interestingly, addition of excess L-arginine to the coculture completely reversed the suppressive activity of B6HO3 cells (Figure 3F). Thus, these results unexpectedly revealed that L-arginine concentrations in culture medium play a crucial role in the B6HO3- mediated suppression of the CPE of HSV-1. L-Arginine has long been known to be required for the replication of DNA viruses like HSV and for cell proliferation. 15 It has been reported that activated macrophages produce arginase, thereby depleting arginine in culture medium and causing the suppression of HSV-1 replication when cocultured with HSV-1- infected cells. 16,17 Accordingly, instead of envisaging an inhibitory soluble factor that is directly interfering with HSV-1 replication, one may well hypothesize that B6HO3 cells promptly produce arginase in response to HSV-1- infected cells, thereby consuming arginine in culture medium, resulting in suppression of the CPE of HSV-1. Since arginase is not an unstable enzyme, it is necessary to explain why the coculture supernatants do not have the CPE-suppressive activity. For this, one can further suppose that arginine consumption in culture medium takes place without B6HO3 cells secreting arginase into culture medium; i.e., B6HO3 cells may produce cytosolic arginase and cationic amino acid transporter simultaneously, resulting in rapid reduction of extracellular levels of L- arginine; 18,19 alternatively, B6HO3 cells may produce membrane-bound arginase. 20,21 It is well established that myeloid cells such as macrophages produce arginase upon induction by a variety of stimuli including T-helper type- 2 cytokines, thus being associated with a various aspects of inflammation and immune suppression. 19,22,23 However, there is a dearth of information regarding whether lymphocytes produce arginase or not. 19,24 Recently it has been reported that type 2 innate lymphocytes have the ability to produce arginase, 25 which lends support to our hypothesis. Verification of our hypothesis awaits further investigation of the precise mechanism underlying the B6HO3-mediated suppression of the CPE of HSV-1. Our previous studies showed that B6HO3 hybridoma Figure 2. B6HO3 cells suppress CPE caused by HSV-1 in Hepa1-6 cells. Hepa1-6 cells ( /well) were plated in a 24-well Transwell plate and cultured for 16 hours. The procedure of coculture with B6HO3 was the same as described in Figure 1. (A), Control Hepa1-6 cells. (B), HSV-1-inoculated Hepa1-6 cells without B6H03 cells. (C), HSV-1-inoculated Hepa1-6 cells cocultured with B6HO3 cells. (D), HSV-1-inoculated Hepa1-6 cells cultured in the mixture of 0.5 ml of fresh culture medium and 0.5 ml of culture supernatants (sup) obtained from 48-hour cocultivation of HSV-1-infected Hepa1-6 cells with B6HO3 cells. Cells were observed under a phase contrast microscope at 72 hours after HSV-1 inoculation (100 ). Scale bar = 50μm 89

4 Kubota K. cells produce IFN-γ in response to interleukin-18 released to the interface of the cell conjugates between B6HO3 cells and dying bacterial-infected macrophages. 1,2 B6HO3 hybridoma cells were thought to exhibit the phenotype of an innate-like T lymphocyte, the parental cell of this hybridoma. 3 This notion led us to the findings that subsets of innate lymphocytes respond to dying bacterial-infected macrophages with the production of innate IFN-γ, which plays a crucial role in the bactericidal activation of macrophages, and that this IFN-γ production pathway plays an important role in the early innate defense against bacterial infection. 26,27 The present study shows that, in addition to the ability to respond to bacterial infection, B6HO3 cells can respond to HSV-1 infection and suppress the CPE of HSV-1 without producing IFN-γ. Thus, innate-like T lymphocytes that became the parental T cell of this hybridoma appear to be capable of dealing with both viral infection and bacterial infection through different mechanisms. Further detailed analyses of the phenomenon reported here will pave the way for our Figure 3. Effects of inhibitors of nitric oxide synthase and arginine on the CPE-suppressive activity of B6HO3 The procedure for coculture of HSV-1-inoculated Hepa1-6 cells with B6HO3 cells was the same as that in Figure 2. (A), Control Hepa1-6 cells without HSV-1 inoculation. (B), HSV-1-inoculated Hepa1-6 cells. (C), HSV-1-inoculated Hepa1-6 cells cocultured with B6HO3 cells. (D), The same coculture as in (C) in the presence of L-NMMA (N G -monomethyl-l-arginine) (1 mm). (E), The same coculture as in (C) in the presence of L-NAME (N G -nitro-l-arginine methyl ester) (1 mm). (F), The same coculture as in (C) in the presence of arginine (10 mm). Cells were observed under a phase contrast microscope at 72 hours after HSV-1 inoculation (100 ). Scale bar = 50μm 90

5 Multifunction of T cell hybridoma B6HO3 functional-hybridoma-based prediction that a subset of innate lymphocytes is equipped with versatile functions for combating both bacterial and viral infections. Conflict of interests The author has no conflicts of interest to declare. References 1. Kubota K. A novel functional T cell hybrioma recognizes macrophage cell death induced by bacteria: a possible role for innate lymphocytes in bacterial infection. J Immunol 2006; 176: Kubota K, Kadoya Y. IL-18 provided in dying bacterial-infected macrophages induces IFN-γ production in functional T-cell hybridoma B6HO3 through cell conjugates. Innate immun 2014; 20: Kubota K, Iwabuchi K. Phenotypic changes in growth-arrested T cell hybrids: a possible avenue to produce functional T cell hybridoma. Front Immunol 2014; 5: Ehmann GL, McLean TI, Bachenheimer SL. Herpes simplex virus type 1 infection imposes a G1/S block in asynchronously growing cells and prevents G1 entry in quiescent cells. Virology 2000; 267: Ellermann-Eriksen S. Macrophages and cytokines in the early defence against herpes simplex virus. Virol J 2005; 2: Isobe K, Nakashima I. Nitric Oxide production from a macrophage cell line: interaction with autologous and allogeneic lymphocytes. J Cell Biochem 1993; 53: Croen KD. Evidence for antiviral effect of nitric oxide: Inhibition of herpes simplex virus type 1 replication. J Clin Invest 1993; 91: Karupiah G, Harris N. Inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites. J Exp Med 1995; 181: Rees DD, Palmer RMJ, Schultz R, et al. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 1990; 101: Green LC, Wagner DA, Glogowski J, et al. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982; 126: Becker Y, Olshevsky U, Levitt J. The role of arginine in the replication of herpes simplex virus. J Gen Viol 1967; 1: Inglis VB. Requirement of arginine for the replication of herpes virus. J Gen Virol 1968; 3: Winters AL, Consigli RA. Effects of arginine deprivation on polyoma virus infection of mouse embryo cultures. J Gen Virol 1971; 10: Archard LC, Williamson JD. The effect of arginine deprivation on the replication of vaccinia virus. J Gen Virol 1971; 12: Bach SJ, Simon-Reuss I. Arginase, an antimitotic agent in tissue culture. Biochim Biophys Acta 1953; 11: Wildy P, Gell PGH, Rhodes J, et al. Inhibition of herpes simplex virus multiplication by activated macrophages: a role for arginase? Infect Immun 1982; 37: Bonina L, Nash AA, Arena A, et al. T cellmacrophage interaction in arginase-mediated resistance to herpes simplex virus. Virus Res 1984; 1: Rodriguez PC, Zea AH, DeSalvo J, et al. L-arginine consumption by macrophages modulates the expression of CD3ζ chain in T lymphocytes. J Immuol 2003; 171: Popovic PJ, Zeh HJ 3rd, Ochoa JB. Arginine and immunity. J Nutr 2007; 137 (6 Suppl 2): 1681S-6S. 20. Terayama H, Koji T, Kontani M, et al. Arginase as an inhibitory principle in liver plasma membranes arresting the growth of various mammalian cells in vitro. Biochim Biophys Acta 1982; 720: Fuentes JM, Campo ML, Soler G. Physico-chemical properties of hepatocyte plasma-membrane-bound arginase. Arch Int Physiol Biochim Biophys 1991; 99: Munder M. Arginase: an emerging key player in the mammalian immune system. Br J Pharmacol 2009; 158: Burrack KS, Morrison TE. The role of myeloid cell activation and arginine metabolism in the pathogenesis of virus-induced diseases. Front Immunol 2014; 5: Yu H, Yoo PK, Aguirre CC, et al. Widespread expression of arginase 1 in mouse tissues: Biochemical and physiological implications. J Histochem Cytochem 2003; 51: Bando JK, Nussbaum JC, Liang H-E, et al. Type 2 innate lymphoid cells constitutively express arginase- 1 in the naïve and inflamed lung. J Leukoc Biol 2013; 94: Kubota K. Innate IFN-γ production by subsets of natural killer cells, natural killer T cells and γδ T cells in response to dying bacterial-infected macrophages. Scan J Immunol 2010; 71: Kubota K, Kadoya Y. Innate IFN-γ-producing cells in the spleen of mice early after Listeria monocytogenes infection: importance of microenvironment of the cells involved in the production of innate IFN-γ. Front Immunol 2011; 2: