Unlocking bat immunology: establishment of pteropus alecto bone marrow-derived dendritic cells and macrophages

ABSTRACT Bats carry and shed many emerging infectious disease agents including Ebola virus and SARS-like Coronaviruses, yet they rarely display clinical symptoms of infection. Bat epithelial

or fibroblast cell lines were previously established to study the bat immune response against viral infection. However, the lack of professional immune cells such as dendritic cells (DC)

and macrophages has greatly limited the significance of current investigations. Using _Pteropus alecto (P. alecto_) GM-CSF plus IL4, FLT3L and CSF-1, we successfully generated bat bone

marrow-derived DC and macrophages. Cells with the phenotype, morphology and functional features of monocyte-derived DC, _bona fide_ DC or macrophages were obtained in GM-CSF/IL4, FLT3L or

CSF-1 cultures, respectively. The successful generation of the first bat bone marrow-derived immune cells paves the way to unlocking the immune mechanisms that confer host resilience to

pathogens in bats. SIMILAR CONTENT BEING VIEWED BY OTHERS DECIPHERING BAT INFLUENZA H18N11 INFECTION DYNAMICS IN MALE JAMAICAN FRUIT BATS ON A SINGLE-CELL LEVEL Article Open access 27 May

2024 DIFFERENCES IN ACUTE PHASE RESPONSE TO BACTERIAL, FUNGAL AND VIRAL ANTIGENS IN GREATER MOUSE-EARED BATS (_MYOTIS MYOTIS_) Article Open access 10 September 2022 THE ROLE OF PLASMACYTOID

DENDRITIC CELLS (PDCS) IN IMMUNITY DURING VIRAL INFECTIONS AND BEYOND Article Open access 22 May 2024 INTRODUCTION Bats serve as reservoir hosts for viruses that are related to many deadly

emerging diseases in humans including Nipah virus, Hendra virus, SARS-like Coronavirus and Ebola virus1,2,3,4,5. Interestingly, bats carrying these viruses, which are pathogenic in humans

and other mammals, show no clinical signs of diseases under natural or experimental infection conditions3,6,7,8,9. This unique ability may reflect an unknown interaction between these bat

viruses and the bat immune system as a result of extensive co-evolution over a long period of time10. Development of bat cell lines is essential for studying the bat immune system,

particularly viral-host interaction under _in vitro_ conditions. To this end, various non-immune bat cell lines, originating from either insectivorous or fruit-bats and covering multiple

species, had been constructed including _Pteropus, Eidolon, Hypsignathus, Rousettus, Epomops, Myotis_ and _Tadarida_ bats11,12,13,14. These cell lines, either primary or immortalized,

supported bat viral infection studies and basic host responses. In contrast to the rapid clearance or reduction of bat viruses evident in _in vivo_ experiments, viral replication does not

appear to be significantly different to other hosts. However, when comparing the multiple _in vitro_ studies in these bat cell lines14,15,16, bat viruses exhibited subversion of the bat

immune system11,16. These observations prompted us to ask whether these bat cells serve as an appropriate model for studying the bat immune response. Dendritic cells (DC) are professional

antigen-presenting cells that initiate and regulate the pathogen-specific adaptive immune responses and are central to the development of immunologic memory and tolerance17,18,19, whereas

macrophages are critical effector cells and regulators of inflammation and the innate immune responses20,21. Possibly equipped with all the major innate immune recognition receptors, they

can secrete cytokines, interferons and pro-inflammatory factors to activate and recruit immune cells to the site of infection upon recognition of pathogens18,19,20,21,22,23. Understanding

how bat DC and macrophages respond to viruses is critical for studying bat antiviral immunology. However, while several reports characterize non-hematopoietic bat cell lines11,12,13,14,

there is currently no report of successful culture or isolation of bat DC or macrophages. In this study, we characterize the first bat bone marrow-derived DC and macrophages. We utilised

overexpressed _Pteropus alecto (P. alecto_) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), or FMS-like tyrosine kinase 3 ligand (FLT3L) to generate bone

marrow-derived DC, while colony-stimulating factor 1 (CSF-1, M-CSF) allowed the generation of bone marrow-derived macrophages by adapting previously established protocols24,25,26. The

successful generation of these bat immune cells will fill this urgently needed technological gap and greatly facilitate our understanding of any bat-specific immune mechanisms contributing

to their resistance to viral diseases. RESULTS/DISCUSSION PHENOTYPIC AND MORPHOLOGICAL CHARACTERIZATION OF _P. ALECTO_ BM-DERIVED DENDRITIC CELLS AND MACROPHAGES We hypothesised that

similarly to human and mouse bone marrow (BM)-derived mononuclear cells (MNC), bat BM-derived MNC would differentiate into macrophages in the presence of CSF-1, into _bona fide_ dendritic

cells (DC) in the presence of FLT3L, and into monocyte-derived DC in the presence of GM-CSF + IL-424,25,26,27. Based on sequences obtained from the _P. alecto_ genome28, we produced

recombinant _P. alecto_ CSF-1, GM-CSF, IL-4-GFP fusion proteins and a fusion protein comprising the functional unit of _P. alecto_ FMS-like tyrosine kinase 3 ligand (FLT3L) called

vaccibodies (Supplementary Fig. S1a,b). _P. alecto_ FLT3L vaccibodies were initially produced to detect FLT3L-expressing cells among primary bat MNC but it showed good functional activity

and, thus, was used in this study. In order to characterise BM-derived MNC by flow cytometry, we first validated that antibodies directed against human or mouse membrane molecules allowed to

detect membrane molecules with a similar cellular expression pattern in _P. alecto_ bat (Fig. 1a and Supplementary Fig. S1c). Antibodies previously described as cross-species reactive and

targeting membrane proteins that showed good conservation between _P. alecto_ human and mouse were used (Supplementary Fig. S1a). We also used an anti-CD3 intracellular domain (highly

conserved across-species) and a commercial anti-bat IgG (Martìnez Gòmez _et al_., _Scientific Reports_, in press). Among live CD44+ lung MNC, CD11b+ cells could be split into MHC-IIlo/− and

MHC-II+ cells while putative CD3+IgG− T cells and CD3−IgG+ B cells could be detected among CD11b− cells (Supplementary Fig. S1c). This analysis showed that both CD11b+MHC-II− (putative

monocyte/macrophages) and CD11b+MHC-II+ (putative DC) expressed high levels of another myeloid cell marker, CD172a (SIRPα), but did not express the B (IgG) and T (CD3) cell lineage markers

(Fig. 1a). Using these antibodies, we next analysed _P. alecto_ BM-derived MNC cultured for 6 days (D6) with _P. alecto_ FLT3L vaccibodies, GM-CSF+IL-4 (GM/IL-4) or CSF-1 and compared them

to the BM cells at Day 0 (D0, _ex vivo_) by flow cytometry (Fig. 1b–d, see Supplementary Fig. S1d for the total gating strategies). At D6, adherent cells from the three different culture

conditions showed an increased size (FSC-A) and granulosity (SSC-A) as compared to _ex vivo_ BM cells (Fig. 1b), suggesting _in vitro_ acquired activation in our culture conditions. It is

important to note that most cells were adherent in these three culture conditions, while this was not the case when cultured in the absence of any of these growth factors. While 53.5% of _ex

vivo_ BM cells (D0) expressed the myeloid marker CD11b, 65.7% and 69.9% of D6 FLT3L- and GM/IL-4- cultured cells expressed CD11b, respectively. When cells were cultured 6 days in the

presence of CSF-1, the proportion of CD11b+ cells reached 84.8% (Fig. 1b,c). Cells were also analysed for expression of CD172a (SIRPα) and for MHC-II, with this latter molecule being

expressed at an intermediate level by immature BM-derived dendritic cells (BM-DC) and at a high level by mature BM-DC obtained from both human or mouse BM29. From the five bats tested, among

CD44+CD11b+ cells, a well-defined population of CD172a+MHC-IIhi cells was observed only when cells were cultured 6 days with the pan-DC growth factor FLT3L (Fig. 1b,d). A population of

CD172a+/loMHC-IIint cells could also be detected in both D6 FLT3L and GM/IL-4– cultured BM-derived cells, and only in low proportion in CSF-1-cultured BM-derived cells (Fig. 1b,d). Giemsa

staining (Fig. 1e and Supplementary Fig. S1e) and confocal microscopy (DiC/CD11b staining, Fig. 1f) of D6 cultured cells showed that some FLT3L-treated bat BM-DC (FLT3L-DC) had the typical

dendritic morphology of _bona fide_ DC. Cells with such dendritic morphology were not observed in the other culture conditions (Fig. 1e, f and Supplementary Fig. S1e). GM-CSF/IL-4 cultures

(GM/IL4-DC) gave rise to large, granulous cells with a rough membrane resembling monocyte-derived DC while CSF-1 cultures (CSF-1-MΦ) gave rise to cells with a classical large, foamy

macrophage morphology (Fig. 1e,f and Supplementary Fig. S1e). T-CELL ALLOSTIMULATORY AND PHAGOCYTIC CAPACITIES OF _P. ALECTO_ BM-DERIVED DC AND MACROPHAGES We next evaluated functional

capacities of D6 cultured _P. alecto_ BM-derived cells. A major functional specialization of DC is their strong T-cell allostimulatory capacity. To date, the only antibody capable of

staining for bat T cells is the intracellular anti-CD3 (Fig. 1a), thus preventing us from purifying live bat T cells. Therefore, D6-cultured BM cells (10,000 cells) were co-cultured for

another 6 days with 100,000 CFSE-labelled total allogenic lung (n = 4) or spleen (n = 2) MNC (containing T cells, see Fig. 1a; Fig. 2a–c, see Supplementary Fig. S2a for total gating

strategy). As controls, lung or spleen MNC were cultured either alone or in the presence of PaKiT03 cells (_P. alecto_ kidney cell line). Large (FSC-Ahi) “blastic” cells, a classical feature

of activated T cells, could be detected only when lung MNC were co-cultured with BM-derived cells, particularly with FLT3L- and GM/IL-4- cultured BM-DC, and to a lower extent with

CSF-1-cultured BM-macrophages (Fig. 2a). Such blastic cells were completely absent from the lung or spleen MNC cultured alone, and only very few blastic cells could be detected in lung MNC

cultured with PaKiT03 cells (Fig. 2a). The greatest cellular viability was obtained in the presence of FLT3L-DC and GM/IL4-DC (80% of live cells among total cells) with more than 3,000

viable cells detected by flow cytometry (Fig. 2b). These non-adherent blastic cells were mostly negative for CD11b (Supplementary Fig. S2), and were more numerous and had a lower granulosity

than the BM-derived cells initially placed into culture. Furthermore, blastic cells were positive for CFSE (Fig. 2c and Supplementary Fig. S2a), which indicates that they were mostly

derived from the allogenic lung MNC and that they did not correspond to the BM-derived DC or macrophages. While only a small proportion of lung MNC proliferated (CFSElo) when cultured alone

(8.3%), a strong proliferation was observed when BM-derived cells were present, particularly in the presence of FLT3L-DC and GM-CSF/IL4 cultures (84.0% and 83.05% of proliferation,

respectively), and to a lesser extent in the presence of CSF-1-MΦ (70.2%) (Fig. 2c and Supplementary Fig. S2). While both the viability and number of cells recovered when cultures were done

in the presence of PaKIT03 cells were low (Fig. 2a), these later were able to induce some proliferation (45.8%), but to a much lower level as compared to BM-derived DC and macrophages (Fig.

2c). Thus, this stronger allostimulatory capacity of FLT3L-DC is in line with the presence of MHC-IIhi (putative “mature”) cells in the BM-derived cells (see Fig. 1a,c), while GM/IL4-DC,

which comprises a majority of MHC-IIint (putative “immature”) cells, showed a slightly lower allostimulatory capacity. We next evaluated the phagocytic capacity of primary lung (_ex vivo_)

and BM-derived MNC (Fig. 3). We first confirmed that primary lung CD11b+ MNC were phagocytes since they were capable of phagocytising fluorescent polystyrene beads while CD11b− (lymphoid)

cells were incapable to do so (Fig. 3a, see Supplementary Fig. S3a for the total gating strategy and Supplementary Fig. S3b for the no-bead control). Among CD11b+CD172a+ cells, this function

was maximal in MHC-IIint putative macrophages, followed by MHC-II− putative monocytes and finally, by MHC-IIhi putative cDC (Fig. 3a). Similarly to primary CD11b+ Lung MNC, BM-derived DC

and macrophages were also phagocytic (Fig. 3b). Contrary to primary lung MNC, the greatest phagocytosis was observed in MHC-IIhi cells. As a control, PaKiT03 cells were analysed in parallel

and did not show any phagocytic activity (Supplementary Fig. S3c). To confirm that the BM-derived cells did engulf the beads, CSF-1-MΦ cultured in the presence of fluorescent polystyrene

beads were analysed using the Anmis Imagestream platform. More than 10,000 cells were analysed for each condition. When CSF-1-MΦ were cultured with beads (0.5–1 μm) at 4 °C, very few cells

(2.2%) engulfed at least one bead, while 7.5% did so when culture was carried out at 37 °C (Fig. 3c). As a control, PaKiT03 cells were cultured the same way and minimal cells (1.1%) could

uptake a fluorescent bead (Supplementary Fig. S3d). CYTOKINE AND INTERFERON RESPONSES OF _P. ALECTO_ BM-DERIVED DC AND MACROPHAGES A major effector function of DC and macrophages is to

secrete pro-inflammatory cytokines, anti-viral type I and type III interferons (IFN) as well as upregulating costimulatory molecules upon activation23. We examined the expression of mRNA

coding for cytokines and costimulatory molecules in bat BM-derived DC and macrophages upon stimulation with Toll-like receptor 3 (TLR3) ligand (poly I:C) and TLR7/8 ligand CL097 (Fig. 4).

Tumour necrosis factor alpha (TNFα), a central orchestrator of inflammation, showed the strongest induction in macrophages following TLR7/8 triggering (Fig. 4a). We next evaluated the

expression of the different chains of the heterodimeric cytokines, IL-12 and IL-23. These cytokines stimulate IFNγ production and cytotoxicity by T cells and NK cells, and also participate

in the polarization of naïve T cells into Th1 or Th17 cells, respectively. Bioactive IL-12p70 and IL-23 are heterodimers sharing the IL-12/IL-23p40 chain that is associated with the specific

IL-12p35 and IL-23p19 chains, respectively30,31,32. IL-12/IL-23p40 transcripts were strongly induced in both FLT3L-DC and macrophages by CL097 and to a lesser extent in GM/IL4-DC. IL-23p19

was mostly induced in macrophages, while IL-12p35 was only strongly induced in GM/IL-4-DC. We next evaluated the expression of genes involved in type-I and type-III anti-viral IFN production

(Fig. 4b). IRF7 is a transcription factor that activates the transcription of genes coding for Type-I IFN such as IFNβ and Type-III IFN such as IFNλ2. These IFNs can then stimulate the

expression of IFN-stimulated anti-viral genes, including MX Dynamin-like GTPase I (MX1). Following TLR3 stimulation, MX1 transcript levels were increased in the three types of BM-derived

myeloid cells while macrophages showed the strongest induction of IRF7 and IFNβ. Interestingly, FLT3L-DC showed the greatest capacity to produce IFNλ2. Poly I:C triggers IFN-III production

only by _bona fide_ cross-presenting mouse CD8α+ and human CD141+ conventional DC1 (cDC1)33. Such strong IFNλ2 production by FLT3L-generated, bat BM-derived DC indicates that they share this

functional specialisation with mouse and human cDC. This observation might also indicate that the strong IFNλ2 transcript expression previously observed in bat primary splenocytes

stimulated with poly I:C might have originated from primary _bona fide_ cDC34. Costimulatory molecules (CD40, CD80 and CD83) showed variable induction levels following TLR3- (poly I:C) or

TLR7/8- (CL097) triggering (Fig. 4c). TLR3-triggering induced a low increase in CD40 mRNA expression in GM/IL4-DC and macrophages only, while increase in CD80 and CD83 mRNA expression levels

were only observed in macrophages. Following TLR7/8-triggering, FLT3L-DC showed the greatest increase in both CD80 and CD83 expression, while CD40 was induced also at low levels only in

GM/IL4-DC and macrophages. This further indicates variation in the degree of maturation and functionality between each generated cell subtype that may be utilised for various functional

assays. CONCLUSION Here we present the first documented methodology for the production of monocyte-derived DC, _bona fide_ DC and macrophages from bat bone marrow. We utilised the

established method of recombinant GM-CSF/IL-4, FLT3L or CSF-1 proteins as commonly used on mouse bone marrow and applied this strategy to a wild animal species. Once these proteins are

cloned from the endogenous species and expressed we believe this method could be utilised in any wild species of animal. While simplistic in nature this achievement allows investigation of

viral zoonotic experiments in phenotypically functional immune cells, investigation of immune cell function and interaction between different immune cells. These resources were previously

limited due to constant need of catching wild bats and the lack of research-oriented bat colonies for fresh materials. We show that such immune cells can be generated in large enough

quantities for functional studies, transcriptional profiling, analysis of surface markers and even antigen presentation studies. This will facilitate further research into potential

mechanisms behind bat-borne zoonotic infection “spill-over” from bats to humans or livestock. It will also open new avenues in understanding specific immune mechanisms that bats have

established to reduce or eliminate clinical diseases while concurrently living with various pathogens. METHODS BATS SAMPLE COLLECTION AND CELL ISOLATION All animal processing work was

conducted in accordance with approved guidelines, methods and permits from the Queensland Animal Science Precinct and the CSIRO Australian Animal Health Laboratory. All experimental

protocols were approved by the Duke-NUS Medical School. Capture and processing of adult bats (_Pteropus alecto_) was conducted as described previously12. To harvest bone marrow (BM) cells,

ribs from the bats were collected and muscle tissues were stripped off the bones before rinsing them in ethanol briefly and then in RPMI 1640 (Gibco) with 10% (v/v) fetal bovine serum FBS.

Caps at both ends of the ribs were then snipped off and RPMI media was used to flush out the BM through the ribs using a needle and syringe. Harvested BM cells were subsequently washed with

PBS, re-suspended in RPMI 1640 with 10% (v/v) (FBS) and 7% (v/v) DMSO, and stored in liquid nitrogen. Bat lung cells were prepared as described previously35. GENERATION OF RECOMBINANT _P.

ALECTO_ GM-CSF, IL-4 AND CSF-1 _P. alecto_ CSF2 (coding for CSF-2 also called GM-CSF), IL-4 and (coding for CSF-1 also called M-CSF) genes have been annotated previously28. The sequences

were sent for gBlock synthesis (IDT) and then cloned into the pcDNA 6.2/EmGFP TOPO vector (Thermo Fisher Scientific) for expression in bat cells. Primers used to subclone _P. alecto_ CSF1

and CSF2 genes from the gene block are shown in Supplementary Table S1. Of note, the CSF1 gene was human codon optimised during synthesis. Endotoxin-free maxipreps of plasmids were prepared

using kits (Omega) and transfected into 5 × 106 PakiT03 cells12 using Lipofectamine 3000 (Thermo Fisher Scientific; 10 μg/10 cm dish) according to the manufacturer’s standard protocol. Six

hours later the media containing transfection reagent was replaced. After 48 h, media containing cytokines was collected and filtered using a 0.8 μm filter before storing at −80 °C until

required. GENERATION OF _P. ALECTO_ FLT3L VACCIBODIES Construction of the vaccibody gene construct has been previously described36. _P. alecto_ FLT3L (acc.nr. ELK18536 aa 22–276) was

synthesised by Genscript and cloned into the vaccibody gene construct using BsmI and BsiWI. mWasabi was cloned into the construct using SfiI, as previously described36. To express the

vaccibodies, the gene constructs were transiently transfected into HEK293T cells grown in 5-layer tissue culture flasks (Falcon Multi-Flask) using Lipofectamine 2000 (Invitrogen).

Vaccibodies were purified by harvesting supernatants and applied them onto a Sepharose 4 Fast Flow column (GE Healthcare) conjugated with an antibody recognizing the CH3 domain of the

vaccibody (clone HP6017), as previously described37. Bound vaccibodies were eluted with 0.1 M Glycin-HCI pH 2.7, dialysed twice in PBS, concentrated using a 50-Kd cutoff Vivaspin column

(Sartorius Stedim Biotech), aliquoted, and stored at −80 °C until use. 6 DAY-CULTURES OF BM PRIMARY MONONUCLEAR CELLS TO GENERATE DENDRITIC CELLS AND MACROPHAGES Frozen _P. alecto_ bone

marrow cells were thawed in 37 °C water bath for 30 s. Each vial of cells (1 ml) were immediately diluted with 9 ml of fresh RPMI 1640 containing 10% (v/v) FBS and spun down at 1300 rpm for

5 min. Cells were resuspended in 10 ml fresh RPMI 1640 containing 10% (v/v) FBS and incubated for 1 h at 37 °C. Three million cells were cultured in 3 ml RPMI 1640 with 10% (v/v) FBS in each

well of 6-well plates. Cells were either supplied with 600 ng/ml FLT3L, 10% GM-CSF + 10% IL-4 supernatant (v/v) or 10% CSF-1 supernatant (v/v) to generate FLT3L-DC, GM/IL-4-DC or CSF-1-MΦ,

respectively. At day 3 (D3), half of the media was replaced with fresh media containing 20% GM-CSF + 20% IL-4 (v/v), 1200 ng/ml FLT3L or 20% CSF-1 (v/v). At day 6 (D6), adherent cells were

dislodged using ice-cold PBS containing 2 mM EDTA at room temperature. Cells were then spun down and resuspended for either flow cytometry staining, confocal staining, phagocytosis assays,

mixed lymphocyte reaction or cultured at cell density of 1–2 × 105 cells/well in 24-well plates for qPCR assays. FLOW CYTOMETRY STAINING The following antibodies (Ab) were used for FACS

analyses: goat polyclonal anti-bat IgG (Novus Biologicals, NB7237) detected using an anti-goat IgG secondary antibody (TermoFisher), mouse IgG1 anti-human CD3-FITC (clone CD3-12, Abcam),

chicken IgY anti-CADM1 (Necl2, SynCAM1, clone 3E1, MBL) detected using a donkey F(ab)’2 anti-chicken IgY-AlexaFluor647 (Jackson Immunoresearch), rat IgG2b anti-CD44-APC/eFluor780 (clone IM7,

eBioscience), rat IgG2b anti-CD11b-Brilliant Violet711 (ITGAM, clone M1/70, BD Biosciences), mouse IgG1 anti-CD172a (SIRPα, clone DH59B, KingFischer Biotech Inc) detected using a polyclonal

goat Ig anti-mouse IgG1-PE/Cy7 (batch Poly4053, Biolegend), and rat IgG2a anti-mouse MHCII-FITC (I-A/I-E, clone 2G9, BD Biosciences). Cells (1–5.106 cells/tube) were washed and incubated

with Live/Dead blue dye (30 min, 4 °C; Invitrogen/Life Technologies) in PBS. Then, 5% heat-inactivated FBS was added (15 min, 4 °C; Sigma Aldrich). Cells were labelled with antibodies, then

washed and stained with secondary reagents. For CD3 intracellular staining, following secondary reagents staining, cells were fixed, permeabilized and stained with the anti-CD3 antibody

using the BD Cytofix/Cytoperm kit (BD Biosciences) following manufacturer’s instructions. The samples were acquired using a FACS FORTESSA (BD Biosciences). Analyses were carried out using

FlowJo V10 (Tree Star Inc). CYTOSPIN AND GIEMSA STAINING Cytospins were prepared from 6-day cultured BM-derived cells stained with Hema 3 system according to manufacturer’s protocol (Fisher

Diagnostics). Cells were then stained as described previously35. CONFOCAL MICROSCOPY _P. alecto_ bone marrow cells were seeded in 6-well plates with 1.5 glass coverslips inside during

differentiation. At day 6, coverslips with cells attached were moved to 24-well plate where cells were fixed in 4% paraformaldehyde/0.37% Gluteraldehyde in TBS at room temperature for 40 

minutes. After removal of fixative, cells were washed three times with TBS, followed by blocking with 5% BSA in TBS for 30 minutes. Cells were stained with anti-CD11b-Brilliant Violet711

(mentioned above, 1:200 in TBS) and Phalloidin 647 (Molecular Probes, Thermo Scientific) for 1 h. Coverslips were mounted in mowiol 4.88 in the dark overnight at room temperature. Single

plane images with differential contrast (DiC) were captured on a Leica SP8 (STED3x) machine with 405/white light lasers. MIXED LYMPHOCYTE REACTION Allogenic _P_. a_lecto_ lung mononuclear

cells (MNC) were labelled with 0.2 μM carboxyfluorescein succinimidyl ester (CFSE) (Thermo Scientific) for 10 min at 37 °C. 10,000 6 day-culture BM-derived DC or macrophages were co-cultured

with 100,000 CFSE labelled lung MNC in the presence of 75 UI/ml of recombinant human IL-2 (R&D Systems) for 6 days in AIM-V medium (Thermo Scientific) supplemented with 10% human

AB-serum (Sigma Aldrich). As controls, lung MNC were cultured 6 days alone or in the presence of 75 UI/ml of recombinant human IL-2. On day 6, cells were analysed by flow cytometry.

PHAGOCYTOSIS ASSAY Approximately 1–2 × 105 macrophage cells/tube (6-day culture BM with CSF-1) were placed in 500 μl of RPMI 1640 medium with 2% FBS and pre-chilled in an ice:water slurry

for 5–10 minutes. Fluorescent microspheres beads (0.50–0.99 μm) (Bang Laboratories) were added at one bead per cell to the suspension in a microfuge tube and incubated either on ice or at 37

 °C for 1 h. Cells were then centrifuged briefly (1,300 rpm, 4 °C for 5 min) and washed twice in 1 ml cold PBS before being resuspended in 25 μl of FACS buffer (PBS, 2% FBS, 2 mM EDTA) and

analysed on the Amnis ImageStream®X Mk II imaging flow cytometer. Samples were acquired at x60 magnification and the percentage of positive/negative cells were quantified with the inbuilt

IDEAS software. STIMULATIONS AND QUANTITATIVE REAL TIME PCR (QPCR) Differentiated BM-derived DC or macrophages were dislodged using cold PBS + 2 mM EDTA at day 7. Cells were placed in a

24-well plate at 1–2 × 105 cells/well and cultured with cytokines-free RPMI medium containing 10% FBS. Cells were then treated with poly I:C (Invivogen) or CL097 (Invivogen) at a final

concentration of 1 μg/ml for 6 h before collection in RLT buffer (QIAGEN) for further RNA extraction. RNA was purified using the RNeasy® Mini Kit (QIAGEN) with on-column DNaseI digestion

step using the RNase-Free DNase Set (QIAGEN). cDNA was subsequently synthesized using QuantiTect® Reverse Transcription Kit (QIAGEN). qPCR was performed in triplicates to determine

transcription levels of various genes coding for TNFα, IL23-p19, IL12-p35, IL12-p40, IFNβ, IFNλ2, IRF7 and Mx1. Reactions were setup using the SensiFAST™ SYBR No-ROX Kit (Bioline) and assays

were run on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) under the following cycling condition: 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s and 57 °C for 30 s, and

ending with a melt profile analysis. Relative expression of the targeted gene was determined by REST formula, relative to the housekeeping gene SNRDP338,39. qPCR primers for IFNβ, IFNλ2, MX1

and IRF7 have been mentioned previously34,40. All primers used in this study are listed in Table S1. STATISTICAL ANALYSIS Paired t-test was used. Differences were defined as statistically

significant when p < 0.05. All these tests were performed using GraphPad Prism 6. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Zhou, P. _et al_. Unlocking bat immunology:

establishment of _Pteropus alecto_ bone marrow-derived dendritic cells and macrophages. _Sci. Rep._ 6, 38597; doi: 10.1038/srep38597 (2016). PUBLISHER'S NOTE: Springer Nature remains

neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES * Calisher, C. H., Childs, J. E., Field, H. E., Holmes, K. V. & Schountz, T.

Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev 19, 531–545, doi: 10.1128/CMR.00017-06 (2006). Article  PubMed  PubMed Central  Google Scholar  * Chua, K. B. et al.

Isolation of Nipah virus from Malaysian Island flying-foxes. Microbes Infect 4, 145–151 (2002). Article  PubMed  Google Scholar  * Ge, X. Y. et al. Isolation and characterization of a bat

SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535–538, doi: 10.1038/nature12711 (2013). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Halpin, K., Young, P.

L., Field, H. E. & Mackenzie, J. S. Isolation of Hendra virus from pteropid bats: a natural reservoir of Hendra virus. J Gen Virol 81, 1927–1932, doi: 10.1099/0022-1317-81-8-1927 (2000).

Article  CAS  PubMed  Google Scholar  * Leroy, E. M. et al. Fruit bats as reservoirs of Ebola virus. Nature 438, 575–576, doi: 10.1038/438575a (2005). Article  ADS  CAS  PubMed  Google

Scholar  * Menachery, V. D. et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat Med 21, 1508–1513, doi: 10.1038/nm.3985 (2015). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Middleton, D. J. et al. Experimental Nipah virus infection in pteropid bats (Pteropus poliocephalus). J Comp Pathol 136, 266–272, doi:

10.1016/j.jcpa.2007.03.002 (2007). Article  CAS  PubMed  Google Scholar  * Setien, A. A. et al. Experimental rabies infection and oral vaccination in vampire bats (Desmodus rotundus).

Vaccine 16, 1122–1126 (1998). Article  CAS  PubMed  Google Scholar  * Swanepoel, R. et al. Experimental inoculation of plants and animals with Ebola virus. Emerg Infect Dis 2, 321–325, doi:

10.3201/eid0204.960407 (1996). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang, L. F., Walker, P. J. & Poon, L. L. Mass extinctions, biodiversity and mitochondrial function:

are bats ‘special’ as reservoirs for emerging viruses? Curr Opin Virol 1, 649–657, doi: 10.1016/j.coviro.2011.10.013 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Biesold,

S. E. et al. Type I interferon reaction to viral infection in interferon-competent, immortalized cell lines from the African fruit bat Eidolon helvum. PLoS One 6, e28131, doi:

10.1371/journal.pone.0028131 (2011). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Crameri, G. et al. Establishment, immortalisation and characterisation of pteropid bat cell

lines. PLoS One 4, e8266, doi: 10.1371/journal.pone.0008266 (2009). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Jordan, I., Horn, D., Oehmke, S., Leendertz, F. H. &

Sandig, V. Cell lines from the Egyptian fruit bat are permissive for modified vaccinia Ankara. Virus Res 145, 54–62, doi: 10.1016/j.virusres.2009.06.007 (2009). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Kuhl, A. et al. Comparative analysis of Ebola virus glycoprotein interactions with human and bat cells. J Infect Dis 204 Suppl 3, S840–849, doi:

10.1093/infdis/jir306 (2011). Article  CAS  PubMed  Google Scholar  * Kruger, N. et al. Surface glycoproteins of an African henipavirus induce syncytium formation in a cell line derived from

an African fruit bat, Hypsignathus monstrosus. J Virol 87, 13889–13891, doi: 10.1128/JVI.02458-13 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Virtue, E. R., Marsh, G.

A., Baker, M. L. & Wang, L. F. Interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines. PLoS One 6, e22488, doi:

10.1371/journal.pone.0022488 (2011). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Banchereau, J. et al. Immunobiology of dendritic cells. Annu Rev Immunol 18, 767–811, doi:

10.1146/annurev.immunol.18.1.767 (2000). Article  CAS  PubMed  Google Scholar  * Dutertre, C. A., Wang, L. F. & Ginhoux, F. Aligning bona fide dendritic cell populations across species.

Cell Immunol 291, 3–10, doi: 10.1016/j.cellimm.2014.08.006 (2014). Article  CAS  PubMed  Google Scholar  * Schlitzer, A., McGovern, N. & Ginhoux, F. Dendritic cells and monocyte-derived

cells: Two complementary and integrated functional systems. Semin Cell Dev Biol 41, 9–22, doi: 10.1016/j.semcdb.2015.03.011 (2015). Article  CAS  PubMed  Google Scholar  * Ginhoux, F. &

Jung, S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat Rev Immunol 14, 392–404, doi: 10.1038/nri3671 (2014). Article  CAS  PubMed  Google Scholar  * Ginhoux,

F., Schultze, J. L., Murray, P. J., Ochando, J. & Biswas, S. K. New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat Immunol 17, 34–40,

doi: 10.1038/ni.3324 (2015). Article  CAS  Google Scholar  * Varol, C., Mildner, A. & Jung, S. Macrophages: development and tissue specialization. Annu Rev Immunol 33, 643–675, doi:

10.1146/annurev-immunol-032414-112220 (2015). Article  CAS  PubMed  Google Scholar  * Yan, Z. Q. & Hansson, G. K. Innate immunity, macrophage activation, and atherosclerosis. Immunol Rev

219, 187–203, doi: 10.1111/j.1600-065X.2007.00554.x (2007). Article  CAS  PubMed  Google Scholar  * Akagawa, K. S., Kamoshita, K. & Tokunaga, T. Effects of granulocyte-macrophage

colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages. J Immunol 141, 3383–3390 (1988). CAS  PubMed  Google

Scholar  * Inaba, K. et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176,

1693–1702 (1992). Article  CAS  PubMed  Google Scholar  * Maraskovsky, E. et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple

dendritic cell subpopulations identified. J Exp Med 184, 1953–1962 (1996). Article  CAS  PubMed  Google Scholar  * Mildner, A., Yona, S. & Jung, S. A close encounter of the third kind:

monocyte-derived cells. Adv Immunol 120, 69–103, doi: 10.1016/B978-0-12-417028-5.00003-X (2013). Article  CAS  PubMed  Google Scholar  * Zhang, G. et al. Comparative analysis of bat genomes

provides insight into the evolution of flight and immunity. Science 339, 456–460, doi: 10.1126/science.1230835 (2013). Article  ADS  CAS  PubMed  Google Scholar  * Villadangos, J. A.,

Schnorrer, P. & Wilson, N. S. Control of MHC class II antigen presentation in dendritic cells: a balance between creative and destructive forces. Immunol Rev 207, 191–205, doi: doi:

10.1111/j.0105-2896.2005.00317.x (2005). Article  CAS  PubMed  Google Scholar  * Harrington, L. E. et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from

the T helper type 1 and 2 lineages. Nat Immunol 6, 1123–1132, doi: 10.1038/ni1254 (2005). Article  CAS  PubMed  Google Scholar  * Oppmann, B. et al. Novel p19 protein engages IL-12p40 to

form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715–725 (2000). Article  CAS  PubMed  Google Scholar  * Trinchieri, G. Interleukin-12:

a proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 13, 251–276, doi:

10.1146/annurev.iy.13.040195.001343 (1995). Article  CAS  PubMed  Google Scholar  * Lauterbach, H. et al. Mouse CD8alpha+ DCs and human BDCA3+ DCs are major producers of IFN-lambda in

response to poly IC. J Exp Med 207, 2703–2717, doi: 10.1084/jem.20092720 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhou, P. et al. Type III IFNs in pteropid bats:

differential expression patterns provide evidence for distinct roles in antiviral immunity. J Immunol 186, 3138–3147, doi: 10.4049/jimmunol.1003115 (2011). Article  CAS  PubMed  Google

Scholar  * Schlitzer, A. et al. IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 38, 970–983, doi:

10.1016/j.immuni.2013.04.011 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Oynebraten, I., Lovas, T. O., Thompson, K. & Bogen, B. Generation of antibody-producing

hybridomas following one single immunization with a targeted DNA vaccine. Scand J Immunol 75, 379–388, doi: 10.1111/j.1365-3083.2011.02639.x (2012). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Fossum, E. et al. Vaccine molecules targeting Xcr1 on cross-presenting DCs induce protective CD8+ T-cell responses against influenza virus. Eur J Immunol 45, 624–635, doi:

10.1002/eji.201445080 (2015). Article  CAS  PubMed  Google Scholar  * Eisenberg, E. & Levanon, E. Y. Human housekeeping genes, revisited. Trends Genet 29, 569–574, doi:

10.1016/j.tig.2013.05.010 (2013). Article  CAS  PubMed  Google Scholar  * Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45

(2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhou, P. et al. IRF7 in the Australian black flying fox, Pteropus alecto: evidence for a unique expression pattern and

functional conservation. PLoS One 9, e103875, doi: 10.1371/journal.pone.0103875 (2014). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We

thank Charlene Foong Shu Fen from the SingHealth Flow Cytometry Core Platform. We thank Antonio Bertoletti and Anthony T. Tan for the use of and help with the Amnis Imagestream. We thank

members of the CSIRO Australian Animal Health Laboratory team led by Gary Crameri and the Queensland Animal Science Precinct team led by Hume Field for their advice and help in catching and

handling bats. This work was supported by grant NRF2012NRF-CRP001–056 from the National Research Foundation in Singapore (F.G. and L-F.W.), by the National Research Foundation Singapore

under its cooperative basic research grant new investigator grant (NMRC/BNIG/2026/2014 for C-A.D and NMRC/BNIG/2040/2015 for A.T.I) and administered by the Singapore Ministry of Health’s

National Medical Research Council, The Research Council of Norway (grant: 220642/H10, E.F.) and the Jebsen Foundation (B.B.). AUTHOR INFORMATION Author notes * Zhou Peng, Chionh Yok Teng,

Irving Aaron T, Dutertre Charles-Antoine and Wang Lin-Fa contributed equally to this work. AUTHORS AND AFFILIATIONS * Programme in Emerging Infectious Disease, Duke-NUS Medical School, 8

College Road, Singapore, 169857, Singapore Peng Zhou, Yok Teng Chionh, Sergio Erdal Irac, Matae Ahn, Justin Han Jia Ng, Florent Ginhoux, Aaron T Irving, Charles-Antoine Dutertre & Lin-Fa

Wang * K.G. Jebsen Center for Research on Influenza Vaccines, Oslo University Hospital, University of Oslo, Oslo, 0027, Norway Even Fossum & Bjarne Bogen * Center for Immune Regulation,

Institute of Immunology, Oslo University Hospital Rikshospitalet, University of Oslo, Oslo, 0424, Norway Bjarne Bogen * Singapore Immunology Network (SIgN), Agency for Science, Technology

and Research (A*STAR), 8A Biomedical Grove, IMMUNOS Building #3-4, BIOPOLIS, Singapore, 138648, Singapore Florent Ginhoux & Charles-Antoine Dutertre Authors * Peng Zhou View author

publications You can also search for this author inPubMed Google Scholar * Yok Teng Chionh View author publications You can also search for this author inPubMed Google Scholar * Sergio Erdal

Irac View author publications You can also search for this author inPubMed Google Scholar * Matae Ahn View author publications You can also search for this author inPubMed Google Scholar *

Justin Han Jia Ng View author publications You can also search for this author inPubMed Google Scholar * Even Fossum View author publications You can also search for this author inPubMed 

Google Scholar * Bjarne Bogen View author publications You can also search for this author inPubMed Google Scholar * Florent Ginhoux View author publications You can also search for this

author inPubMed Google Scholar * Aaron T Irving View author publications You can also search for this author inPubMed Google Scholar * Charles-Antoine Dutertre View author publications You

can also search for this author inPubMed Google Scholar * Lin-Fa Wang View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceptualization:

P.Z., Y.T.C., A.T.I., C.A.D. and L.-F.W.; Experiments: P.Z., Y.T.C., M.A., S.I.E., J.H.J.N., E.F., G.C., A.T.I., C.A.D.; Data analysis: P.Z., Y.T.C., M.A., A.T.I. and C.A.D.; Key reagent:

E.F. and B.B.; Resources: F.G., and L.-F.W.; Writing: P.Z., Y.T.C., A.T.I., F.G., C.A.D. and L.-F.W. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial

interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY FIGURES RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0 International License. The images or

other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the

Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit

http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Zhou, P., Chionh, Y., Irac, S. _et al._ Unlocking bat immunology: establishment of

_Pteropus alecto_ bone marrow-derived dendritic cells and macrophages. _Sci Rep_ 6, 38597 (2016). https://doi.org/10.1038/srep38597 Download citation * Received: 11 March 2016 * Accepted: 10

November 2016 * Published: 09 December 2016 * DOI: https://doi.org/10.1038/srep38597 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get

shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative