Induction of Anti-inflammatory Cytokines in CD11b- and Gr-1- Positive Cells after Neuropathogenic Coronaviral Infection

Mu-3 virus (Mu-3), a neuropathogenic strain of the mouse hepatitis virus, induces the apoptosis of pyramidal neurons in CA2 and CA3 subregions of the hippocampus at 4 days post-inoculation (dpi), without showing destructive changes or viral invasion there at 3 dpi. Since it has been considered that the apoptotic lesions occur through the indirect effects of infection, local expression of cytokines was examined in the brain in our previous study, revealing that the anti-inflammatory cytokine IL-10 is produced by pyramidal neurons of CA2 and CA3 after infection. However, infection of primary brain culture failed to elevate IL-10 production. Therefore, in order to examine whether Mu-3 infection elevates IL-10 production, CD11b-expressing Peritoneal Exudate Cells (PECs) were used, which showed an increased level of IL-10 production in the supernatant of PEC culture after infection. The finding that IL-10-producing cells expressed Lewis X (Lex) supports our previous hypothesis that Lex expression is involved in the immunosuppressive state induced by viral infection. In addition, in combination with an experiment involving ex vivo infection, it was indicated that Mu-3 elicits M2 macrophages or Gr-1+CD11b+ Myeloid-Derived Suppressor Cells (MDSCs) that produce IL-10 and TGF-β to circumvent the host immune response after infection.


Introduction
The Mutant virus Mu-3, isolated from the srr7 virus (srr7) [1,2], causes the apoptosis of pyramidal neurons in CA2 and CA3 subregions of the hippocampus after infection [3]. Apoptosis of the pyramidal neurons in the hippocampus is observed after infection with various kinds of viruses, as introduced in our previous report [3]. It has been considered that the apoptotic lesions after infection are not caused by the direct infection of neurons, but induced through indirect effects [3][4][5], triggered by immune responses that involve elevated levels of proinflammatory cytokines detected in the course of diseases. However, our study using the homogenized tissue or frozen sections obtained from Mu-3-infected mice failed to detect the production of proinflammatory cytokines in CA2 and CA3 subregions of the hippocampus [6]. Thereafter, we examined the involvement of antiinflammatory cytokines, and found that IL-10 is expressed in pyramidal neurons in CA2 and CA3 subregions of the hippocampus [6]. TGF-β, which is associated with Theiler's Murine Encephalomyelitis Virus (TMEV) infection [4], was also detected in the hippocampus, although the expression level was low, and the expression was not limited to CA2 and CA3 subregions, but was also observed in a CA1 subregion. The involvement of anti-inflammatory cytokines in the development of the lesions observed in the hippocampus after infection with Mu-3 corresponds to our previous findings that Mu-3, srr7, and its maternal clone, JHM virus strain (JHMV) cl-2 virus (cl-2), induce an immunosuppressive state in the infected mice, which have a shrunk spleen with reduced cell population and the neuropathology showing very low-level inflammatory cell infiltration in the brain parenchyma in spite of a wide area of brain damage [2,7,8]. The leukocytes derived from the infected mice show unresponsiveness to the stimulation by Lipopolysaccharide (LPS) [9,10], mimicking the state in Endotoxin Tolerance (ET), or LPS tolerance, where a population of CD11b mid Gr-1 mid suppressor Monocytes/Macrophages (Mo/Mas) plays a central role, secreting the anti-inflammatory cytokines IL-10 and TGF-β [11].
Although we observed IL-10 expression in pyramidal neurons in the hippocampus, we could not detect the induction of anti-inflammatory cytokines using brain tissue cultures after infection with MU-3, probably because of different population of neurons in the pyramidal layers in vivo and tissue cultures [6]. Therefore, in order to examine whether Mu-3 is capable of triggering the production of antiinflammatory cytokines after infection, we used PECs, which are known to produce anti-inflammatory cytokines under the ET condition, and express the Lewis X (Le x ) carbohydrate structure after infection with srr7, accompanied by a shift of the Gr1 + CD11b + cell population [12]. In this report, we showed that IL-10 is produced in the PECs after infection with JHMV for the first time in the world.

Viruses and animals
A neuropathogenic MHV strain, Mu-3, was used in this study. This virus was propagated and titrated in DBT cells, as previously described [3]. Specific pathogen-free inbred BALB/c mice purchased from Charles River (Tokyo, Japan) were housed in a specific pathogen-free animal facility, and maintained according to the guidelines with respect for the Helsinki Declaration set by the ethical committee of our university. For the experiment, mice were transferred to a Biosafety Level 3 (BSL-3) laboratory after obtaining permission from the committee of our university. Six-seven-week-old mice were used for all experiments. Mice were inoculated with 1 × 10 2 Plaque-Forming Unit (PFU)/50 μL of Mu-3 virus into the right frontal lobe under deep anesthesia using ethyl ether (SHOWA-ETHER, Tokyo, Japan).

Preparation of peritoneal exudate cells
Thioglycolate Medium (TM) (BD Diagnostics, Loveton Circle Sparks, MD, USA) was prepared as previously described [12]. Four days after the injection of 2 ml of TM into the peritoneal cavity, Peritoneal Exudate Cells (PECs) were collected. To obtain adherent PECs, the cells were seeded at 2 × 10 5 per well in eight-well glassbottomed chamber slides (Nalge Nunc International), and cultured as previously described [12]. After overnight culture, the adherent cells were inoculated with 1.0 × 10 1 PFU (plaque-forming units) of Mu-3 virus per well and incubated for the specified time period.

Immunofluorescence
Fixed cultured cells on slide-glasses were processed for immunostaining using the relevant antibodies (Table 1), followed by nuclear counterstaining using Hoechst 33342 (Invitrogen, Carlsbad, Calif., USA), as previously described [12]. Some cells were stained with hematoxylin (Sigma) to observe nuclear structures. Stained cell cultures were mounted with gold antifade reagent (Invitrogen) and examined using a confocal laser scanning microscope (Leica Microsystems, Knowlhill, UK) or fluorescence microscope (KEYENCE, Osaka, Japan).

Flow cytometry
PECs infected with 1.3 × 10 5 PFU of Mu-3 virus in vitro in suspension culture [12], or infected with 1.0 × 10 5 PFU of Mu-3 virus in vivo in the peritoneal cavity for 24 h, were collected and processed for cytometry using a FACSCalibur (BD Biosciences) after staining with fluorochrome-conjugated antibodies (Table 1) as previously described [12]. Appropriate isotype controls were used in all cases. Data were analysed using FlowJo software (Tree Star, Ashland, OR, USA).  Table 1: Antibodies and reagents used in this study.

The production of cytokines detected by in vitro assay
Because an examination using primary brain cultures failed to detect the production of anti-inflammatory cytokines [6], the in vitro inducibility of cytokines by Mu-3 infection was studied using PECs, which produce measurable IL-10 under the ET state [13], in order to examine whether Mu-3 infection is able to trigger the production of anti-inflammatory cytokines. At 24 h post inoculation (hpi), the infection spread to all areas of the culture accompanied by the detachment of many cells from the bottom, showing variable levels of cytokines, usually low-levels, in the culture supernatant (data not shown). Therefore, the levels of cytokine production were examined, comparing with those of mock infection (Mock), during eight to 16 hours after in vitro inoculation with Mu-3. The production of proinflammatory cytokines was variable. A slight increase in the level was observed in IL-6 production, and a marked one was detected in that of TNF-α and MCP-1. The IFN-β production was completely suppressed after infection (Figure 1), as previously reported [14]. In contrast, the elevated levels of anti-inflammatory cytokines were detected after infection. In the supernatant of PEC culture, the level of IL-10 increased after in vitro inoculation with Mu-3, reaching its peak at 12 hpi, and declined afterwards (Figure 1). The production of TGF-β showed more rapid elevation after infection than that of IL-10. The levels of TGF-β reached their peak at 8 hpi. Immunofluorescent studies using adherent PECs revealed that, as reported previously [12], PECs expressed CD11b and Gr-1 (Figure 2a). The shape of nuclei was that of classical macrophages, that is small and round or rod-shaped (Figures 2a and 2b). The morphology of PECs did not show marked changes after in vitro infection, except for syncytium formation, and this is markedly different from those infected in vivo as described below. Although, in the pictures, the numbers of the cells expressing the markers look to have increased after infection, this is due to stained syncytial cells, and the numbers did not show marked differences after infection when marker-expressing cells were counted (data not shown), as we previously reported [12]. PECs infected in vivo in the peritoneal cavity were collected by spinoculation at 24 hpi (V (24 hpi)) (b). PECs on the slide glasses were processed to detect CD11b, Gr-1 or nucleus using Hoechst 33342 (H). In order to observe the nuclear structure, some were stained by Hematoxylin (He).
Next, we examined the production of anti-inflammatory cytokines in the cultured adherent PECs. IL-10 was produced by Gr-1-or CD11b-positive cells, especially those near syncytia (Figures 3A and  3B). Many CD11b-positive (CD11b + ) cells near the syncytia have been reported to extend their foot processes to connect syncytia [12]. Cells near syncytia and those already involved in the syncytia, but located at the periphery showed the marked expression of IL-10 ( Figure 3B). However, in the area with the marked expression of viral antigens, IL-10 was not colocalized ( Figure 3D). In the mock-infected culture, IL-10 expression was not prominent. Colocalization of Le x and IL-10 was detected at the periphery of syncytia as well as in solitary cells near the syncytia, supporting our previous hypothesis that Le x expression is involved in the syncytium formation and immunosuppressive state induced by viral infection. The Le x expression was prominent in mock infected cells (data not shown) as we previously reported [12]. In order to compare the sensitivity to detect cytokines between the multiplex bead-based immunoassay and immunostaining, IFN-β, whose production was totally inhibited after infection by the former (Figure  1), was immunostained, and the latter was proven to be more sensitive than the other (Figures 3A-3F), as we detected IFN-β in the in vivo assay, with immunostaining showing IFN-β-producing cells both near and at a distant from infected sites deep in the brain parenchyma during the early phase of infection, but not in the infected cells [15].

Mo/Mas detected by ex vitro assay
In order to assess in vivo responses to infection, we performed an ex vivo assay. Three days after the injection of Thioglycollate Medium (TM), Mu-3 was injected into the peritoneal cavity. The PECs collected at 24 hpi were seeded in glass-bottomed chamber slides. However, almost no cells adhered to the bottom (data not shown). Therefore, we employed cytospin procedure [2]. The morphology of PECs with in vivo infection markedly differed from that of those with in vitro infection, with ring-shaped nuclei in more than 80% of the PECs (Figure 2b). The ring-shaped nucleus is a hallmark of differentiated Gr-1 + CD11b + Myeloid-Derived Suppressor Cells (MDSCs) (16). The flow cytometric analysis revealed that only a small population of PECs in suspension culture shifted to the CD11b mid Gra-1 mid population after in vitro infection (Figure 4a), which corresponds to the observation in the adherent PECs (Figure 2a), and our previous report [12]. However, a large CD11b + Gra-1 + population appeared after in vivo infection (Figure 4b). The inflow of suppressor cells, namely differentiated Gr-1 + CD11b + MDSCs, into the peritoneal cavity should have occurred after in vivo infection.

Discussion
The studies on the immunological pathogenesis of MHV including neuropathogenic JHMV strains have been mainly focused on a robust innate immune response in the fulminant and acute inflammation induced by virulent viral strains, or T cell-mediated immune responses in chronic inflammation induced by attenuated types of viruses in animal models for studying human demyelinating diseases [16]. Therefore, the interests have been mainly focused on immunostimulating proinflammatory cytokines, and we are the first to report that one of the JHMV strains induces the production of antiinflammatory cytokines after infection. There are two types of inflammatory responses caused by viral infection. One is a marked one [16], and the other is a very mild one, typically observed in the neuropathogenic retroviral infection [17]. The viruses cl-2 [18], its attenuated mutant virus srr7 [19], and Mu-3 [3] derived from srr7 as a mutant clone, cause the latter type of response after infection, showing the neuropathologies with very mild inflammation in the brain parenchyma in spite of marked brain injury. A marked inflammation is observed only in the meninges during the early phase of infection with the viruses. The population of exudate cells in the meninges are rich in Mo/Mas, which are the initial target of infection [3,7,8].
The finding, presented in this report that Mo/Mas produce antiinflammatory cytokines after infection, only partially explains the neuropathology with mild inflammation in the brain parenchyma, because the production of IL-10 was observed in the pyramidal neurons in the CA2 and CA3 subregions of the hippocampus and not in the CD11b-positive Mo/Mas [6] in the area where brain injury occurs without prominent inflammation [3]. However, we could prove that Mu-3 infection triggered the production of anti-inflammatory cytokines, which explains the low response to LPS stimulation after infection with srr7, mimicking the state of ET [9,10].
ET is a classic form of protective mechanisms whereby cells receiving low doses of endotoxin, enter a transient state of hyporesponsiveness to the next endotoxin challenges [11]. Activated macrophages that play a key role in ET have been recognized as M2 macrophages, an immunosuppressive subset of macrophages, which appear in both the blood stream [20] and lymphoid organs [21], as well as in local inflammatory sites as exuded cells, such as PECs [13]. The term M2-like Gr-1 + CD11b + myeloid suppressor cell is also employed to identify immunosuppressive macrophages that appear in ET [11]. Gr-1 + CD11b + myeloid suppressor cells, or Gr-1 + CD11b + Myeloid-Derived Suppressor Cells (MDSCs), the name of which is often used to identify suppressor cells that appear around tumors, comprise a heterogeneous population [22], and are not a distinctly different population of M2 macrophages, as the term M2-like Gr1 + CD11b + myeloid suppressor cell indicates. For example, M2 macrophages among MDSCs can be differentiated into the M1 type [23]. Both M2 macrophages and MDSCs mediate immunosuppression by secreting IL-10, which is used as one of the markers to identify M2 macrophages [24].
Thus, we also showed that Mu-3 infection induced the appearance of MDSCs or M2 macrophages, which were Gr-1-, CD11b-, and IL-10positive cells with ring-shaped nucleus. The observation by an immunostaining and a flow cytometry that the population shifted to those immunosuppressive Mo/Mas, which consisted of only a small population among PECs after in vitro infection, explains the slight increase in the levels of some proinflammatory cytokines after infection. In contrast, the production of IFN-β measured in the culture supernatant was totally abolished, which has been considered to occur through direct interference in the cascades to produce IFN-β by coronaviral infection [14]. However, at eight hpi, less than 0.1% of the PECs should be infected, because they were inoculated at a Multiplicity of Infection (M.O.I.) of 0.5 × 10 -4 . The influence of anti-inflammatory cytokines should be considered to explain the down-regulation of IFNβ production after MHV infection. In contrast to in vitro infection, the in vivo infection of PECs induced a large population of Gr-1 + CD11b + cells among PECs, which should have occurred after the migration of Gr-1 + CD11b + myeloid cells into the peritoneal cavity [13]. This finding indicates that in vivo infection causes overwhelming immunosuppressive state throughout the body accompanied by accelerated anti-inflammatory cytokine production compared with that observed on the in vitro infection.
The appearance of IL-10-and Le x -double positive cells supported our previous study, using gene manipulated mice that do not express Le x , revealing that Le x expression is involved in the immunosuppressive state caused by infection with srr7 [9]. Many complex sugar structures including LPS have tolerogenic properties [25]. Furthermore, their binding partners, glycan-binding proteins or lectins play as varied roles in immunological responses. Le x is a natural ligand of dendritic cell-specific intercellular-adhesion-moleculegrabbing non-integrin (DC-SIGN), a C-type lectin-like receptor. DC-SIGN initiates a Raf-1-indipendent pathway after interaction with fucose, which is located at the end of the Le x carbohydrate structure, resulting in the induction of anti-inflammatory cytokines [26].
Our series of studies has shown that highly neurovirulent viruses invade and spread in the brain using the host reactions triggered by infection, which includes not only immune responses and induction of Le x expression, but also the production of unique extracellular matrices, which form an Astrocytic Reticular Network (ARN) after infection [15], and are used as a pathway for the viruses to enter into the brain parenchyma from the meninges, the initial site of infection [10,27].