Characteristics of in vitro infection of human monocytes, by Rickettsia helvetica Microbes and Infection

Eighteen species of rickettsiae are reported to cause infections in humans. One of these is Rickettsia helvetica , which is endemic in European and Asian countries and transmitted by the tick Ixodes ricinus. Besides fever, it has been demonstrated to cause meningitis and is also associated with perimyocarditis. One of the initial targets for rickettsiae after inoculation by ticks is the macrophage/monocyte. How rickettsiae remain in the macrophages/monocytes before establishing their infection in vascular endo- thelial cells remains poorly understood. The main aim of the present study was to investigate the impact on and survival of R. helvetica in a human leukemic monocytic cell line, THP-1. Our results show that R. helvetica survives and propagates in the THP-1 cells. The infection in monocytes was followed for seven days by qPCR and for 30 days by TEM, where invasion of the nucleus was also observed as well as double membrane vacuoles containing rickettsiae, a ﬁ nding suggesting that R. helvetica might induce autophagy at the early stage of infection. Infected monocytes induced TNF- a which may be important in host defence against rickettsial infections and promote cell survival and inhibiting cell death by apoptosis. The present ﬁ ndings illustrate the importance of monocytes to the pathogenesis of rickettsial disease.

Rickettsiae are strictly intracellular bacteria that are louse, tick-, flea or mite-borne. To date, thirty-two species of rickettsiae have been reported and validated of which 18 species have been identified as agents of human disease [1]. The infection may affect many organs and the clinical spectrum range from febrile flu-like illnesses to more severe, even fatal diseases [1,2]. Infections of the central nervous system (CNS), most commonly meningitis, encephalitis and disseminated encephalomyelitis as well as facial palsy or cerebral infarction are known for twelve of the species [3e6]. Reported evidence suggests that the major preferred target cells of rickettsial infection are the monocytes which are the initial targets for rickettsiae in the tick-feeding site [7,8]. Infected monocytes spread the infection further and the vascular endothelial and perivascular cells are invaded by the pathogen causing increased microvascular permeability as a result of rickettsial vasculitis [7,9]. It is important to understand how rickettsiae survive and proliferate in monocytes/macrophages and are potentially disseminated if we are to understand the pathogenesis of rickettsial diseases. Only a few studies have focused on the complex interaction and host cell response between monocyte/macrophages and rickettsia. One shows that Rickettsia akari and Rickettsia typhii may infect and survive within mouse macrophages only causing negligible cytotoxicity [10]. Inhibition of growth or killing of rickettsiae is induced by cytokines in interactions such as interferon-gamma (IFNg) and tumor necrosis factor-a (TNF-a) as well as interleukine-1b (IL-1b). A combination of replication and lysis, the latter more commonly for Rickettsia typhi than for spotted fever rickettsiae, is considered to be the basis for rickettsial pathogenesis allowing rickettsiae to be released and to infect neighbouring cells [11,12]. Rickettsia helvetica is an emerging human pathogen, belonging to the tick-borne spotted fever group of rickettsiae, associated with an eruptive fever, myalgia and perimyocarditis, but also with CNS conditions, such as meningitis. Still its exact pathogenesis remains uncertain [13e16]. The species has been reported from most European countries as well as North Africa, Russia, Asia and Japan [1].
The underlying rationale for the present study was to examine R. helvetica survival and possible influence on human monocytes in an experimental model of an in vitro differentiated human leukemic monocytic cell line, THP-1, which may illustrate implications for R. helvetica as a pathogen.

Rickettsial strain and culture conditions
A previous isolate of R. helvetica from an Ixodes ricinus tick was used and propagated in Vero cells (SigmaeAldrich) plated in T-75 culture flasks as previously described [17]. Uninfected cells were grown in Dulbecco's modified Eagle medium: nutrient mixture F-12 (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum (FBS) (Nordic Biolabs) and 1% penicillinstreptomycin (SigmaeAldrich) in a 37 C incubator. After inoculation with R. helvetica, the cells were subsequently moved to a 32 C incubator in 5% CO 2 in a BSL-2 laboratory. On the 4-5th day post-infection the cells were harvested and assayed for presence of rickettsiae by an indirect fluorescent antibody assay (IFA) using an anti-rickettsial rabbit antiserum and FITC polyclonal swine anti-rabbit IgG immunoglobulin as secondary antibody (F0205, Dako, Agilent Technologies aps). As previously reported rickettsiae were released from the Vero cells by needle disruption or by sonication (6 Â 5 s pulses). The suspension was centrifuged twice at 1000 g 4 C for 10 min to remove cell debris and the supernatant was collected and centrifuged at 17,000 g 4 C for 10 min; the procedure was followed twice. The supernatant was discarded every time and the pellet was resuspended in 1 mL phosphate-buffered saline (PBS) after which time the final pellet was redissolved in 100 mL PBS and the density of rickettsial antigen was estimated using IFA and PCR (calculated to 3000e5000 bacteria/mL) [18].

Culture and inoculation of human monocytes (THP-1)
The human leukemic monocytic cell line, THP-1, (ATCC ® TIB-202™; American type culture collection, Manassas USA) was prepared and expanded in T-75 flasks (Nordic Biolabs) in a 37 C incubator (5% CO 2 ). THP-1 were fed/split using RPMI-1640 media (SigmaeAldrich) supplemented with 10% FBS (Nordic Biolabs) and 1% penicillin-streptomycin (SigmaeAldrich) every other day. To differentiate THP-1, cells were cultured in 24 well plates (250,000 cells per well or 5•10 5 cells/ml) along with 100 ng/ml of phorbol 12-myristate 13-acetate (PMA) (SigmaeAldrich) for 3 h. Subsequently, the medium was replaced with fresh RPMI-1640 and cells were allowed to rest at least 24 h before infection with R. helvetica. Differentiated THP-1 cells were then infected with a given amount of redissolved bacteria pellet used as inoculum (approximately 0.1 bacteria per THP-1 cell). Another 24 well plates of cells were treated in the same way but left uninfected as control. Cells were harvested for immunocytochemistry, PCR and transmission electron microscopy (TEM). The growth medium was not replaced during the cultivation period. The entire content of each well, including medium and cells, was harvested each 24 h interval of infection and for each day, the rickettsia content from the wells were frozen in À70 C and later quantified using qPCR. Experiments were performed in triplicates and repeated twice. To measure cytokine release, the supernatants were collected on 6 consecutive days, starting 24 h after inoculation, and stored immediately in a À20 C freezer. In the electron microscopy study, the cells were cultured for a full month and harvested after 3, 7, 15, 18 and 30 days. The content was stored in buffer containing 2% glutaraldehyde for later examination using TEM.

qPCR
Total cellular DNA was isolated from R. helvetica infected monocytes and uninfected controls. DNA extraction was performed using QIAamp DNA mini kit protocol according to the manufacturer's instructions (Qiagen AB). For quantification of DNA copies, a genus-specific realtime PCR with probe and primers targeting the citrate synthase-encoding gene (gltA) was assayed, as previously described [19]. The harvested cells and bacteria from each well were centrifuged at 17,000 Â g (Lab centrifuge, Sigma) for 20 min to capture all bacteria in both cells and free in culture media, the supernatant was discarded and the pellet was resuspended in 200 mL PBS. In each reaction five ml DNA extracts were used as a template together with 0.25 ml LC Uracil-DNA glycosylase (UNG) (Roche Diagnostics) to minimize the risk of contamination. The reactions were run in a Rotor-Gene 3000 (Qiagen) using Light Cycler® TaqMan® Master (Roche Diagnostics). In each amplification trial, a standard plasmid constructed by cloning the PCR product into a PCR 4-TOPO vector using Invitrogen TOPO® TA Cloning® kit for Sequencing (Thermofisher Scientific) and containing the cloned 74 bp fragment of the gltA gene (nt 1125e1199) was included in 10-fold dilution series containing the R. helvetica gltA gene fragment together with sterile water as a negative control. The experiment was run repeated twice. The standard curve used for quantification of T. helvetica organisms showed linearity in the dilution series between 1.5 and 1.5 Â 10 8 copies (Error 0.0142, Efficiency 1.961, R2 0.997, y ¼ À3.34 Â þ37.68).

ELISA-based cytokine measurements
ELISA was performed to measure the levels of human TNF-a daily of infected and uninfected monocytes, using murine ABTS ELISA kits (Peprotech, Rocky Hill USA) and following manufacturer's protocols. Briefly, ELISA Nunc Maxisorp microplates (Thermofisher Scientific) were coated with diluted capture antibody (1 mg/ml for TNF-a in PBS) overnight at room temperature. Wells were washed 4 times with wash buffer (PBS with 0.05% TWEEN-20 (SigmaeAldrich)), blocked using 1% bovine serum albumin (Nordic Biolabs) in PBS for 1 h at room temperature and washed again. The plates were subsequently incubated with the collected culture medium samples for 2 h at room temperature and then washed. Detection antibody was then applied to the samples for 2 h with another wash step following the incubation. Finally the samples were incubated with an Avidin-HRP conjugate, washed, and exposed to the ABTS liquid substrate. The change in color was monitored after approximately 20 min at 405 nm using a Tecan plate reader (Infinite M200).

Immunohistochemistry
Infected and uninfected cell cultures, respectively, were fixated by incubating in 4% paraformaldehyde for 10 min, then washed with PBS twice, applied to microscope slide wells, dried and fixed in acetone as previously described [20]. To visualize rickettsiae the cells were stained with rabbit anti-rickettsial serum and FITC polyclonal swine anti-rabbit IgG immunoglobulin as secondary antibody (F0205, Dako, Agilent Technologies). Cell nuclei were stained with DAPI, Vector Laboratories Vectashield mounting medium (VWR, International). When estimating the number of infected cells, the total area of these cells was calculated in ten independent fields of view at 400Â magnification using a measure option for area determination in the microscope's photo system (Lumenera, Infinity Analyze 3-1, Olympus BX60).

Transmission electron microscopy
Harvested R. helvetica-infected THP-1 cells were processed for morphological analysis by fixation in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) supplemented with 0.1 M sucrose. Post fixation was performed in 1% osmium tetroxide dehydration in ethanol and embedded in Agar 100 resin (Agar Scientific). Ultrathin sections were cut and stained using 4% uranyl acetate and Reynolds lead citrate. The cells were inspected in a Tecan G2 transmission electron microscope at 60 kV and the images were prepared by BIOVIS facility.

Statistical analysis
Statistical analyses including regression analysis and t-test were conducted using SAS® proprietary Software 9.4 (TS1M6), licensed to Uppsala university and executed on the X64_10 PRO platform.

Results
The qPCR, immunohistochemical and TEM findings confirm that R. helvetica replicates and survives within the human monocytes, THP-1 cells.

qPCR
The number of rickettsial copies in the cultivation series was separately analysed for each day using PCR (Fig. 1). The curve, representing mean values, demonstrate an initial short-lag phase (day 1) followed by an exponential increase in copies (day 2e4), after which the curve levels out. The recorded replication rate of bacteria was highest on the fourth day of infection, a smaller dip in copy number in the curve was seen on the fifth day before a stationary phase established. The generation time was calculated to be 20.5 h during the first 4 days and 35.7 h until day seven.

ELISA based TNF-a assay
The infected cells production of the cyto-and chemokines is central to the local inflammatory response to rickettsial infection.
Microbial products that activate monocytes lead to the production of proinflammatory cytokines among which TNF-a plays a leading role. We therefore monitored the expression of that cytokine during the first day's post-infection. TNF-a levels increased steadily during the first four-five days of observation after inoculation after which a certain stabilization occurred (Fig. 2AeB). ELISA's were performed in duplicates.

Transmission electron microscopy
TEM revealed rickettsiae in the monocytes "cytoplasma", initially with a smaller number (days 2e3) thereafter with increasing numbers of bacteria up to the period between 7 and 15 days of cultivation, after which time the number was largely unchanged with a tendency towards decreasing numbers. Even after 30 days of culture, the infected monocytes seemed to tolerate the rickettsia well with few signs of cell death (Fig. 3). The ultrastructural morphology of rickettsiae was in accordance with the morphology of Vero-cell-cultured R. helvetica. The majority of the bacteria, some of them about to divide, were found in the cytoplasm, along with host cell organelles such as mitochondria and different type of granules or vesicles that are distinguished from rickettsia in that they do not divide with binary fission, and also lack electron-dense outer membranes. After 7e30 days of cultivation, individual bacteria could also be seen invading the cell nucleus (Fig. 4), a phenomenon that was also illustrated by IFA. We also observed autophagy at the ultrastructural level. The definition of an autophagosome is a doublemembrane-enclosed vacuole containing undigested cytoplasmic contents. During the first three days some double-membrane vacuoles containing rickettsiae could be seen (Fig. 5). The findings suggest that R. helvetica might induce autophagy at the early stage of infection.

Immunohistochemistry
In immunohistochemistry rickettsiae were readily visible inside the cytoplasm of the monocytes already from day 2e3, often single to few organisms (Fig. 6). At this time an estimated 1e2% of cells were infected, whereas approximately 20e30% were infected on 5e6 days of culture and many cells cytoplasm had been loaded with bacteria.

Discussion
By means of PCR, immunohistochemistry and TEM, we have shown that R. helvetica is capable of infecting, surviving and replicating in human monocytes THP-1 cells. The rickettsial load in infected THP-1 cells during a 96 h period is most similar with R. helvetica replication observed previously in Vero cells and the multiplication of Rickettsia slovaca in L929 and Vero cells [17,21]. R. helvetica appears to maintain a low-to-moderate infection load after reaching a plateau phase of growth and more rarely lyse their host cell. Normally, a death phase occurs after the stationary phase. In this study, we did not follow the infection long enough to assess this phase. Earlier results indicate, however, that the phase of DNA degradation occurs later for R. helvetica compared to, for example, R. rickettsii and R. slovaca an assumption that is also consistent with the findings in TEM where there was a tendency towards slowly decreasing numbers and rickettsiae were still seen in the cytoplasm after day 15 [21,22].
It is known from other reports that rickettsiae, after inducing endocytosis and being internalized in the cell, escape from the phagosome by secreting phospholipase D and hemolysin C. In the cytosol, rickettsiae express a surface protein, Sca2, which contributes to the activation of actin polymerization, which propels the rickettsiae through the cytoplasm and through the cell membrane to nearby cells [23,24].
Normally, during infection, microbial products activate monocytes which leads to production of cytokines, which triggers the inflammatory cascade early on. Activated rickettsia infected monocytes are reported capable of producing a number of cytokines as IL-1b, IL-6, IFN-g and TNF-a contributing to the inflammatory process [10,25]. The activation of monocytes upon infection depends on the rickettsial species, for example R. australis induces the release of IL-1b and IL-18 and correspondingly IL-6 and IL-12 are increased upon Rickettsia conorii infection [26] Some rickettsiae such as Orientia tsutsugamushi, R. conorii and Rickettsia parkeri predominantly infect monocytes in vivo. It is therefore proposed that at least some rickettsiae may use monocytes as a vehicle for transport through the body, for example O. tsutsugamushi and possibly R. typhi [9,26].
In the present in vitro study R. helvetica-infected monocytes induced TNF-a production, probably a response to rickettsial LPS [27]. Similar findings have been reported in in vitro infection of human monocyte-derived macrophages with R. conorii which stimulates their production of TNF-a [28]. In the present study higher concentrations of TNF-a was observed until day 4e6 after inoculation. It is consistent with a study of tumor necrosis factor production under conditions of experimental rickettsial infection caused by agents of Astrakhan spotted fever where the concentrations of TNF increased by day 4 of ASF, the peak concentrations were observed on day 6 [29]. In addition to other effects, such as proinflammatory cytokine and mediator of apoptosis, TNF-a has also anti-rickettsial activity in that it restricts the growth of Rickettsia prowazekii in mouse L929 cells and inhibits the growth of R. conorii in human HEp-2 cells [30]. TNF-a synergizes with IFN-g in activating the bactericidal activity by inducing the release of nitric oxide [30,31]. In vivo antigen-presenting cells activated by rickettsial antigens produce high levels of TNF-a and IFN-g, increasing the anti-rickettsial activity of monocytes/macrophages [9]. The suppression of apoptosis by NF-kB is also an important component of TNF-a biology promoting cell survival and inhibiting cell death [32].
The presence and morphology of R. helvetica growing in THP-1 cells was further demonstrated by TEM showing a DNA-filled matrix circumscribed by a plasma membrane and two leaflets. An increasing number of rickettsiae with typical morphology in the cytoplasm was seen during the first days, after which time a stationary phase established and the number stabilized and decreased slightly, similar replication characteristics to what has also been shown previously for R. helvetica in a Vero cell line [17]. Single bacteria were detected in the cell nucleus, using IFA and TEM, from the third day post-infection and thereafter (Figs. 4 and  6). Intracellular replication, and mainly for the typhus group, also the lysis mechanism, are important parts of rickettsial pathogenesis which allow rickettsiae to be released and to infect nearby cells [7,9]. This is consistent with findings from previous studies in which low cytotoxicity and lysis have been shown for peritoneal macrophages infected with R. akari and R. typhii, respectively [10]. Previous studies have also shown that the ability to survive and proliferate in macrophages differs between a non-pathogenic and a pathogenic rickettsia, for example virulent R. conorii survives and proliferate in human macrophage-like cells, while nonvirulent Rickettsia montanensis is rapidly destroyed [8,33]. Prevention of apoptosis allows rickettsiae to multiply and for Rickettsia rickettsia, it has been reported that activation induced by TNF-a, of the    15) were also seen in the nucleus (B) and (C). In C stained with DAPI, representing the same image section as (B). The location in the nucleus is more clearly seen when the finding is assessed with different depths of field. transcription factor, nuclear factor-kappa B, protects the host cell against infection-induced apoptotic death and prolongs infection and thus is critical for host cell survival [34,35]. Single autophagosomes in monocytes were seen on TEM images up to day 7 but not thereafter, suggesting that R. helvetica is unlikely to induce this process or induce a modified autophagy process. Another report has shown that the surface OmpB protein is critical for rickettsial virulence and acts as a protective shield to obstruct and avoid autophagy recognition, thereby revealing a bacterial mechanism to evade antimicrobial autophagy [36]. However, autophagic activity and its markers need to be studied in more detail to allow a correct interpretation of the phenomena associated with R helvetica infection [37].
Our data suggest that R. helvetica infection in monocytes is supported in association with induced elevation of TNF-a. R. helvetica's ability to survive in monocytes and possibly spread further to endothelial cells supports a preliminary assessment of this species as a human pathogen. However, the exact mechanisms of the pathogenesis and the significance of this infection need further study.

Declaration of competing of interest
The authors declare that they have no competing interests.