Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii
© The Author(s) 2017
Received: 11 October 2016
Accepted: 12 March 2017
Published: 12 April 2017
Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1β, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host’s immune response.
Coxiella (C.) burnetii is a Gram-negative, obligate intracellular pathogen and causative agent of Q fever, a widely distributed zooanthroponosis . The disease appears as an acute, flu-like and self-limiting illness, or manifests as a chronically progressing infection (e.g., endocarditis, premature delivery in pregnant women). C. burnetii has a broad host spectrum, which includes birds, reptiles, arthropods and domestic and wild mammals. Sources of human infections often are infected sheep, goats or cattle . In livestock, C. burnetii infection is inapparent in most cases . If disease manifests, referred to as Coxiellosis, reproductive disorders such as abortions, stillbirth in goats and sheep or delivery of weak newborns in cattle were observed . The main route of C. burnetii transmission is via inhalation of infected aerosols or dust, especially when contaminated with C. burnetii birth products (placental membranes and fluids) of goat and sheep, but also by feces and milk [4, 5]. An unprecedented large Q-fever outbreak occurred from 2007 to 2011 in the Netherlands, where more than 4000 human cases were notified and approximately 52 000 ruminants were culled as part of the countermeasures taken to control the epidemic .
Main shedding of C. burnetii with about 109 bacteria per gram placenta is observed during parturition in sheep and goat . Coxiella organisms are primarily detected in trophoblast cells in the placentomes [2, 7, 8]. Shedding of bacteria by milk of asymptomatic cattle was observed to persist for several months . Dairy cows seem to be more chronically infected with C. burnetii than small ruminants. Guatteo et al.  could also show that Coxiella shedding was scarce and sporadic in feces of cattle, whereas permanent and sporadic shedding was observed by milk. PCR analysis of bovine bulk tank milk samples detected more than 102 C. burnetii DNA equivalents per milliliter . Muskens et al.  believe that the localization of the pathogen in the bovine udder is the critical factor for a secretion of bacteria into the milk but it is currently unknown which cell types facilitate persistence and replication of C. burnetii in the mammary gland.
The chain of events implicated in C. burnetii transmission between animals of the reservoir species have poorly been studied at the cellular level. Mononuclear phagocytes, e.g., macrophages and monocytes, are considered the major host cells during natural infection . We recently showed that Coxiella organisms invade primary bovine monocyte-derived and alveolar macrophages in vitro and slowly replicate in these cells without significantly activating them . Even though alveolar macrophages probably represent the first target cell for C. burnetii when entering the body, it is highly likely that alveolar epithelial cells also become exposed to and infected by these bacteria in the early stages of the infection as described in rodent models [14, 15]. At the site of entry, the lung epithelium therefore may determine the character of the subsequent immune response in concert with alveolar macrophages. It is also perceivable that epithelial cells at the exit sites are determinative for persistence in and bacterial transmission of C. burnetii from the reservoir host [1, 4].
In general, C. burnetii is able to grow in a number of cell types, like Vero cells or fibroblast cells . All these cell types poorly mirror the natural cell environment to investigate infection processes in domestic animals. Therefore the present study aimed at developing an in vitro cell system deploying bovine epithelial cell lines from lung, placenta, gut and udder tissues. We studied the permissiveness and host cell response of different epithelial cells with two Coxiella strains: a virulent strain (Nine Mile I), expressing full-length lipopolysaccharide (“smooth LPS”), and an avirulent strain (NM phase II). NMII lacks the full O-polysaccharide I chain and sugars located in the LPS I outer core (called “rough LPS”) .
Materials and methods
Epithelial cell culture
Cell lines used in this study
Dulbecco’s Modified Eagle Medium (DMEM, 1.0 g/mL glucose)
Iscove’s Modified Dulbecco’s Medium (IMDM)/Ham’s F12 nutrient mix [1:1]
DMEM (4.5 g/mL glucose)/Ham’s F12 nutrient mix [1:1]
DMEM (4.5 g/mL glucose)/Ham’s F12 nutrient mix [1:1]
Advanced DMEM/Ham’s F12 nutrient mix [1:1] + additional componentsd
Coxiella (C.) burnetii infection and sampling
Cells were infected with C. burnetii strain “Nine Mile phase I RSA 493” (NMI) and “Nine Mile phase II clone 4” (NMII) (phase I-LPS and phase II-LPS expressing variants, respectively) in multi-well cell culture plates at 37 °C and 5% CO2. Both C. burnetii strains were supplied, propagated and purified as previously described .
Cells were cultured in 24-well plates (Corning® Costar®, Sigma) until at least 80% confluence was reached and infected with NMI and NMII with a MOI of 100 as described above. Cell vitality was monitored by MTT and LDH assay (according to manufacturer’s instructions). After 7 days of infection (see above), cells were fixed with 4% formaldehyde for 24 h and stored at 4 °C. Cells were washed with 1× PBS and permeabilized with 100% ice cold methanol for 1 min followed by incubation with 50 nM NH4Cl for 15 min and PBS/FBS solution (1% FBS) for 30–45 min in the dark at room temperature to block unspecific binding sites. Cells were stained for 30 min at room temperature using an Anti-Coxiella antibody [1:5000 in PBS/FCS] (kindly provided by Anja Lührmann, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany) followed by incubation (30 min–1 h) with a fluorochrome-conjugated secondary antibody [1:500 in PBS/FCS] [Anti-rabbit IgG (H + L), F(ab’) 2 Fragment (Alexa Fluor® 594 Conjugate), New England Biolabs, Frankfurt, Germany], counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature followed by washing with 1× PBS. To characterize the replication compartment of C. burnetii, infected cells were labeled with the acidic marker LysoTracker Red DND-99 (Invitrogen, Darmstadt, Germany). After infection, cells were incubated with LysoTracker (1:5000 in test media) for 2 h at 37 °C following fixation as described above. The cells were mounted in DABCO (1,4-diazabicyclo[2.2.2]-octane, 2% in Glycerol). Samples were viewed under a fluorescence microscope [Olympus CK40; camera: Leica DFC420C, software: Leica Application Suite (LAS) Version 3.7.0 (Build: 681)]. Cultures incubated in PBS without primary antibodies served as negative controls.
Transmission electron microscopy (TEM)
Cells were cultured in 6-well plates (Corning® Costar®, Sigma) and infected with infection strategy B3 as described above. The culture supernatant was removed and cells were fixed with 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.2) for 2 h at 4 °C, detached with a cell scraper from the culture plate, collected in a reaction tube and centrifuged to obtain a cell pellet . The cell pellet was embedded in 2% agarose and sectioned to 1 mm3 cubes. Cubes were post fixed in 2% osmium tetroxide and embedded in araldite Cy212. Ultrathin sections (85 nm) were stained with uranyl acetate and lead citrate. They were examined at an accelerating voltage of 80 kV by transmission electron microscopy (Tecnai12, FEI, Eindhoven, Netherlands).
Quantitative real-time PCR for determination of intracellular C. burnetii genome equivalents (GE)
To estimate the number of cell associated bacteria, DNA from the cell containing fraction of the cultures of infection strategy A and B was purified with the Invisorb® DNA Cleanup-Kit (Stratec, Birkenfeld, Germany) according to manufacturer’s instructions. The number of genome equivalents (GE) was monitored by quantitation of the isocitrate dehydrogenase (icd) gene by quantitative real-time PCR (qPCR) . The icd gene is highly conserved within the species C. burnetii and occurs as single copy in the Coxiella genome. Ct values of technical duplicates varied by less than 0.51 and were used to calculate GE considering values obtained with an entrained icd harbouring plasmid standard. Invasion rate and replication efficiency were calculated from four biological replicates (independent cell cultures, tested in two technical replicates each) as described in the legend to Figure 1.
Flow cytometry analysis
Cells (4 × 105 cells/well, 24 well plates) were detached by Trypsin/EDTA solution and transferred to microtiter plates (V-shape; Greiner Bio-One GmbH, Frickenhausen, Germany) and pelletized by centrifugation (400 × g, 4 min and 4 °C). For detection of CR3 [CD11b, MM12A, diluted 1:250 (VMRD, Pullman, WA, USA)] and αVβ3 [CD61, diluted 1:100 (AbD Serotec, Düsseldorf, Germany)], cells were incubated with 50 µL diluted primary antibody for 20 min. After washing (washing buffer: 1× PBS, 0.5% FCS), cells were incubated with secondary antibody anti-mouse IgG1-APC (Southern Biotech, Birmingham, USA) diluted 1:1000 in 1× PBS for 20 min. Finally cells were washed again and analyzed with BD FACSCanto™II (Becton–Dickinson, Heidelberg, Germany). Data was analyzed with BD FACSDIVA™ software (version 6).
Reverse transcription and cytokine-specific real time PCR
Sequences of primers used in this study
F: GCG ATA CTC ACT CTT CTA CCT TCG A
R: TCG TAC CAG GAA ATG AGC TTG AC
F: ACC TGA ACC CAT CAA CGA AAT G
R: TAG GGT CAT CAG CCT CAA ATA ACA
F: CTG AAG CAA AAG ATC GCA GAT CTA
R: CTC GTT TGA AGA CTG CAT CTT CTC
F: TCT TCT CAA GCC TCA AGT AAC AAG T
R: CCA TGA GGG CAT TGG CAT AC
Unless otherwise indicated, Mann–Whitney U test was used to compare two different samples. A p value of ≤0.05 (“a” or “*”) show a statistically significant difference at the 95% confidence level, a p value of ≤0.01 (“b” or “**”) at the 99% confidence level.
Bovine udder epithelial cells exhibited highest permissiveness for C. burnetii propagation
NMI and NMII, both showed a very low invasiveness within the first 24 h after inoculation (lower left graphs in Figures 2A and B; “invasion B”) under the experimental conditions applied. The udder epithelial cell line was only slightly more susceptible to incorporate C. burnetii than the lung and placental cell lines but differences did not reach statistical significance. Interestingly, although numbers markedly varied between biological replicates, intestinal epithelial cells exhibited the highest susceptibility for C. burnetii invasion within the first 24 h after inoculation.
Increases in C. burnetii-specific GE numbers during 7 days of culture following strategy A (upper right graphs in Figures 2A and B), reflect efficacy of C. burnetii replication but may also be influenced by invasion events having occurred later than 24 h. In order to dissect these two phenomena, excess, i.e., not yet cell-bound C. burnetii particles were removed 24 h after inoculation (“strategy B”; lower right graphs in Figures 2A and B). GE numbers were then quantified at day 7 and calculated relative to the numbers found cell-associated after 24 h of culture. It became apparent that udder epithelial cells were most effective in supporting replication of NMI and NMII (“replication B”): while numbers of NMI increased by approx. 16 000-fold from day 1 to day 7, numbers of NMII increased by approx. 330-fold (p = 0.032). Replication efficiency of NMI and NMII in intestinal epithelial cells was not significantly different from lung and maternal placental cells but fetal placental cells exhibited an intermediate support of C. burnetii replication.
Large Coxiella containing vacuoles (CCV) were formed in gut and udder epithelial cells without affecting viability of the cell cultures
Epithelial cells infected with NMI or NMII retained their typical cell morphology after 7 days of infection and displayed no signs of cell death processes, e.g., disrupted cell membrane or fragmented nuclei. These observations could be confirmed by cell vitality assays. Independent of the C. burnetii strain, infection neither affected metabolic activity nor cytoplasmic membrane integrity of any of the epithelial cell lines after 1 and 7 days of inoculation compared to uninfected control cells (data not shown).
CR3 and αvβ3 surface expression does not correlate with epithelial susceptibility to C. burnetii invasion
C. burnetii infection failed to induce a consistent inflammatory response in bovine epithelial cells
The bovine epithelial cell lines utilized in this study were selected as surrogates of certain steps in the infection path C. burnetii follows inside a mammalian host. The lung represents the entry site for the pathogen to establish the infection with alveolar macrophages considered to play a key role . Placenta, gut and udder are known replication sites immediately prior to transmission events . Here we demonstrated that C. burnetii invaded and replicated in bovine epithelial cells from the different organs without destroying the cell integrity or inducing a substantial immune response. Although the C. burnetii strain NMI is considered more “virulent” than NMII as deduced from cell culture and rodent experiments, the invasiveness and replication efficacy in the bovine cell lines was similar. Epithelial cells from different organs differed in the individual kinetics of two steps in the cellular infection process. Udder epithelial cells were most effective in propagating C. burnetii mainly because of particularly supporting replication after bacterial invasion. Intestinal epithelial cells, by contrast, particularly supported bacterial invasion. The comparably higher replication efficacy detected for the latter cells when applying experimental strategy A as compared to strategy B points to ongoing invasion events even after 24 h of incubation which had paralleled bacterial replication. The interaction pattern with C. burnetii of these two cell types represented the extremes displayed by the cell lines under consideration with the suitability of lung, maternal and fetal epithelial cells to act as C. burnetii host cells ranging in-between.
Inoculation of bovine mammary gland epithelial cell cultures yielded the highest amounts of C. burnetii compared to cultures of epithelial cells from lung, gut and placenta after an incubation time of up to 7 days. Previous histological investigations of tissues from infected cattle primarily detected the pathogen in mammary gland epithelial cells (reviewed in ). Tropism for udder tissues in bovines seems to provide the basis for the high numbers of C. burnetii shed by milk in this species [9, 24, 25]. It is considered that consumption of dairy products poses a low risk for Q fever infections in humans  even though Benson et al.  had found that most bulk tank milk samples contained viable Coxiella organisms and human consumption of C. burnetii-containing milk leads to a rise in specific serum antibody titers in absence of clinical disease. A high bacterial load in raw milk was also described by Enright et al. , who observed up to 10 000 infective doses of C. burnetii per mL of milk from infected dairy cows. Similar bacterial load was observed by Schaal  after quantitative analysis of Coxiella containing milk. High numbers of C. burnetii in milk at least contribute to spreading of the agent within cattle herds. Newborn calves fed C. burnetii-containing milk excrete the bacteria in their feces and urine into the environment . Therefore a dairy herd showing no symptoms of Coxiellosis could still be a C. burnetii reservoir for transmission via the tick-independent infection cycle .
Typical C. burnetii containing structures (CCV) were observed in udder and intestinal epithelial cells by transmission electron microscopy. Mature CCV were seen at 7 days pi in udder cells. Findings are comparable to findings in Vero cells [30, 31]. This indicates that both NMI and NMII undergo a complete replication cycle in these udder epithelial cells. The formation of large CCV requires protein secretion by C. burnetii  and depends on the actin cytoskeleton of the infected cells .
Small ruminants, like goats and sheep, shed C. burnetii more frequently via birth products  which contain huge numbers of bacteria and are the main source of environmental contamination and subsequent aerogenic transmission to humans. In ovine placentas, van Moll et al.  found Coxiella organisms in huge amounts in trophoblast cells which were embedded in acutely inflamed tissue. By contrast, bovine placentas generally contain few or moderate numbers of cells staining positive with Coxiella-specific antibodies and with rarely detectable Coxiella-like organisms. Our results of infection experiments with bovine fetal and maternal placental epithelial cell lines are in line with these ex vivo observations and imply that the many tissue-specific properties the placental cell lines have retained also comprise determinants of permissiveness for C. burnetii infection [35, 36].
Numbers of C. burnetii in the feces of infected small ruminants exceed numbers in bovine feces pointing to another possible shedding route at least in sheep and goats . Feces of aborting ewes contain up to 107 GE of Coxiella per gram . Our in vitro study showed that epithelial cells other than mammary gland epithelium also were capable of internalizing C. burnetii but there was little further propagation of bacterial numbers within the cells. In organs implicated in C. burnetii shedding by cattle, these cells apparently represent rather unsuitable target cells which may be the in vitro correlate of the comparably low numbers of C. burnetii detected in the feces and birth fluids of bovines [9, 10].
At the entry site, lung epithelial cells are the first contact of Coxiella when entering the host organism after aerial transmission. It may be argued, that significant replication is not necessary at the entry site but the bacteria just cross the epithelial barrier to reach phagocytosing cells. Calverley et al.  concluded that recruited monocytes play an important role in the infection process because they control the distribution of bacteria from the lung. Additionally, resident alveolar immune cells were shown in a mouse model to possess a high susceptibility to Coxiella infection  and human and bovine alveolar macrophages can be infected with C. burnetii in vitro [13, 39]. There is cumulating evidence, therefore, that lung epithelial cells, being less susceptible to C. burnetii infection (, this study), do not act as replication sites for the bacteria and as such are poorly implicated in C. burnetii transmission between animals and in persistence in different hosts.
Bovine epithelial cells from different tissues varied in their susceptibility to C. burnetii invasion and support of replication with little correlation between the two properties as udder epithelial cells particularly supported C. burnetii replication whereas intestinal epithelial cells displayed an enhanced susceptibility for C. burnetii invasion. Invasion of C. burnetii into cells is reported to be influenced by the biochemical composition of the LPS . In a variety of cells, C. burnetii strains with phase I and phase II LPS exhibit a different uptake kinetic in that a virulent phase I strain attached slower than the avirulent strain because of the mechanisms these organisms utilize to enter the host cells. Coxiella uses specific eukaryotic receptors such as integrins on macrophages and monocytes to adhere and invade . C. burnetii phase I particles bind the leukocyte response integrin (αvβ3), whereas the avirulent C. burnetii additionally deploy complement receptor 3 (CR3) . Bovine udder epithelial cells exhibited an enhanced αvβ3 expression whereas CR3 was essentially absent from the surface of all cell lines studied. Martinez et al. already described the first C. burnetii protein involved in host cell invasion . OmpA is a surface protein of Coxiella that increased the internalization within non-phagocytic cells without necessity of Coxiella-specific receptors. Further investigations are required to assess the role of the adhesin OmpA and αvβ3 for bacterial attachment, invasion and cellular activation in bovine epithelial cells.
Coxiella organisms can activate immune cells in a strain-dependent manner. Especially avirulent strains promote a higher pro-inflammatory cytokine production compared to virulent strains  which may be linked to LPS phase-related differences in attachment of the bacteria . We observed a general failure to induce immune responses which particularly holds for udder epithelial cells independent of the phase-type of the NM variant and the day post-infection. Enterobacterial LPS is a very potent stimulant for immune reactions via pattern recognition receptors (PRRs)  and was included as positive control in our studies. LPS-stimulated udder epithelial cells showed an upregulation of pro-inflammatory cytokines. The failure of C. burnetii to initiate epithelial immune responses does not result from a process actively steered by a metabolically active pathogen, because infection studies with heat-inactivated NMI and NMII yielded similar results (data not shown). Invasive bacteria normally induce a rapid pro-inflammatory cytokine production as part of the defense mechanisms of the host. Different from bovine epithelial cells, attachment to or invasion of C. burnetii into macrophages stimulate a pro-inflammatory immune response to recruit additional immune cells [13, 39]. However, these responses are regulated in a complex manner. Rasmussen et al.  described a delay in cytokine expression in Chlamydia spp.-infected cells and that chlamydial invasion alone did not induce an immune response. In human colon epithelial cells, activation of cytokine response upon bacterial invasion is dependent on a special set of signals . The attachment alone did not sufficiently stimulate the immune system for bacterial clearance. Activation of epithelial cells by Candida albicans, a common epidermal pathogen, is regulated via two phases of signal pathway activation . On the one hand there is a morphological recognition of the fungus via PRRs and on the other hand a second trigger leads to production of cytokines and further immune reactions inside the host cells. The molecular basis for failure of bovine epithelial cells investigated in our study to initiate inflammatory responses remains to be determined. It can be assumed, though, that this property of C. burnetii is instrumental to create a replicative niche in the reservoir host for persistence of the pathogen, similar to what Ben Amara et al.  had suggested for human trophoblasts.
The in vitro cell system established and characterized in this study will be useful to further the understanding of the chain of events during the infection process of Coxiella organisms inside the bovine host in respect to epithelial cells as possible target cells. Bovine udder epithelial cells seem to have the highest permissiveness for C. burnetii and promote bacterial replication without losing the cell vitality. The udder cell line used was initially isolated from a mammary gland and became permanently cultivable by continuous passaging without deploying artificial transformation. The similarity to primary bovine mammary epithelial cells constitutes a big advantage for further investigations. Even though cell lines assessed in our study significantly varied in their permissiveness, the results strongly imply that C. burnetii can make use of epithelial cells beside immune cells as target cells for successful transmission between animals and into the environment as the cells survived the infection for substantial periods of time while helping the pathogen to evade the hosts immune response. For reasons of availability we chose bovine cells and evaluated the invasion and replication of C. burnetii by using two biological variants of a commonly used prototype strain. Cattle are not the main source of human infection but may shed C. burnetii. The high prevalence of C. burnetii genotype ST 20 recently identified in bovine milk in the U.S.  points to host species-specific adaptations within of the species of C. burnetii. After the proof-of-principle provided in this manuscript, studies are now in progress to assess quantitative differences in the interaction of different Coxiella genotypes with bovine epithelial cells.
The authors declare that they have no competing interests.
Design of study and experiments: CM, KS. Generation and characterization of cell lines: PG, PR, NH, CP. Cell infection assays, fluorescence microscopy and real-time PCR: KS, KB. Electron microscopy; ELT. Statistical analysis: KS. Interpretation of data and drafting of the manuscript: KS, KB, ELT, IDJ, CM. All authors read and approved the final manuscript.
The authors would like to thank Anja Müller, Anke Hinsching, Lisa Wirker und Wolfram Maginot (Friedrich-Loeffler-Institut, Institute of Molecular Pathogenesis) for their excellent technical assistance.
The study was financially supported by the European Commission (FP7 programme in the framework of the project “Antigone—ANTIcipating the Global Onset of Novel Epidemics”, Project Number 278976). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
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