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in a Site-Specific FashionStreptococci Engage TLR13 on Myeloid Cells

Kirschning, Sachin D. Deshmukh and Philipp HennekeGoldmann, Claudia Waskow, Zhijian J. Chen, Carsten J.Gharun, Roland Elling, Patrick Trieu-Cuot, Tobias Julia Kolter, Reinhild Feuerstein, Evelyne Spoeri, Kourosh

http://www.jimmunol.org/content/196/6/2733doi: 10.4049/jimmunol.1501014February 2016;

2016; 196:2733-2741; Prepublished online 12J Immunol 

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The Journal of Immunology

Streptococci Engage TLR13 on Myeloid Cells in aSite-Specific Fashion

Julia Kolter,*,†,1 Reinhild Feuerstein,*,†,1 Evelyne Spoeri,* Kourosh Gharun,*,†

Roland Elling,*,‡ Patrick Trieu-Cuot,x Tobias Goldmann,{ Claudia Waskow,‖

Zhijian J. Chen,# Carsten J. Kirschning,** Sachin D. Deshmukh,*,†† and

Philipp Henneke*,‡‡

Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among

the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predom-

inantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and

response. In this article, we show that TLR13 is the critical mouse macrophage (MF) receptor in the response to group B

Streptococcus, both in bone marrow–derived MFs and in mature tissue MFs, such as those residing in the lamina propria of

the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent

cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and

dermal MFs. Furthermore, a specific role for TLR13 with respect to MF function is supported by the response to staphylococci,

where TLR13 and UNC-93B limit the cytokine response in bone marrow–derived MFs and microglia, but not in dermal MFs. In

summary, TLR13 is a critical and site-specific receptor in the single MF response to b-hemolytic streptococci. The Journal of

Immunology, 2016, 196: 2733–2741.

Toll-like receptors constitute the best characterized familyof pattern recognition receptors (1). Proper TLR functionin resident immune cells, especially tissue macrophages

(MFs), is required to hold potentially virulent bacteria in check atmucocutaneous sites (2–4). Yet, the policing function of MFs,which stabilizes surface niches for colonizing bacteria, and thedangerous hyperinflammatory response of myeloid cells duringbacterial spread result from similar interaction principals betweenbacterial effectors and TLRs. Hence, it is critical that the MF

response is site specific and well adapted to locally prevalentbacterial species and to the likely mode of contact with the bac-

teria. As an example, the presence of cell-free bacterial toxins intissue areas close to the mucosa should have different implicationsfor the TLR-dependent activation of MFs than the contact withwhole bacteria entering the tissue. The subcellular discriminationof bacterial effectors by MFs is particularly well understood forEscherichia coli, where engagement of TLR4/MD2 by LPS at theplasma membrane induces recruitment of the adapter proteins TIRdomain-containing adaptor protein and MyD88, which results inTNF and IL-6 formation. In contrast, engagement of TLR4/MD2in the endosome, which is the LPS–TLR interface when whole E.coli are ingested, uses the adapters TRAM and TRIF and inducestype I IFNs (5). An analogous model for Gram-positive bacteriacomprises engagement of plasma membrane–expressed TLR2heteromers by lipopeptides and activation of endosomal TLRs bybacterial nucleic acids (6–8). We (6, 9) and other investigators (8)showed that MF activation by streptococci heavily depends onMyD88 and on the endoplasmic reticulum protein UNC-93B,which targets TLRs to the endosomal membrane. Yet, all of thewell-characterized endosomal TLRs that have homologs in hu-mans (TLR3, TLR7, TLR8, TLR9) are dispensable in mice. Thebacterial effector that drives the MyD88–UNC-93B pathway wasidentified as ssRNA. In mice, additional TLRs (i.e., TLR11,TLR12, and TLR13) are expressed in the endosomal compart-ment. Among these, TLR11 was first found to have a dedicatedligand: profilin from Toxoplasma gondii (10). Yet, although therole, if any, of TLR11 and TLR12 in the recognition of Gram-positive bacteria remains unknown, TLR13 was found to interactwith RNA from staphylococci and streptococci and to depend onthe methylation status of the bacterial RNA (11–13). However,little is known about the contribution of TLR13 to the inflam-matory program in MFs infected with streptococci or any otherGram-positive bacteria. This is largely due to the fact that the dataon streptococci interacting with cells from TLR13-knockout miceare mostly limited to the response to isolated bacterial RNA,whereas data from in vitro or in vivo infection are sparse (14, 15).

*Center for Chronic Immunodeficiency, Medical Center, University of Freiburg,79106 Freiburg, Germany; †Faculty of Biology, University of Freiburg, 79104 Frei-burg, Germany; ‡Division of Infectious Diseases and Immunology, Department ofMedicine, University of Massachusetts Medical School, Worcester, MA 01655; xIn-stitute Pasteur, Unite de Biologie des Bacteries Pathogenes a Gram-Positif, CNRSERL3526, 75724 Paris Cedex 15, France; {Institute of Neuropathology, MedicalCenter, University of Freiburg, 79106 Freiburg, Germany; ‖Regeneration in Hema-topoiesis and Animal Models of Hematopoiesis, Faculty of Medicine, TechnicalUniversity, 01307 Dresden, Germany; #Southwestern Medical School, Universityof Texas, Dallas, TX 75390; **Institute of Medical Microbiology, Medical Center,University of Essen, 45147 Essen, Germany; ††Center for Sepsis Control and Care,Medical Center, University of Jena, 07747 Jena, Germany; and ‡‡Center for Pediat-rics and Adolescent Medicine, Medical Center, University of Freiburg, 79106 Frei-burg, Germany

1J.K. and R.F. contributed equally to this work.

Received for publication April 30, 2015. Accepted for publication January 14, 2016.

This work was supported by grants from the German Federal Ministry of Educationand Research (Grant 01EO0803 to P.H.) and by the German Research Council(EL790/1-1 to R.E. and HE3127/5-1 and HE3127/12-1 to P.H.).

Address correspondence and reprint requests to Prof. Philipp Henneke, Center forChronic Immunodeficiency, Medical Center, University of Freiburg, BreisacherStrasse 117, 79106 Freiburg, Germany. E-mail address: philipp.henneke@uniklinik-freiburg.de

Abbreviations used in this article: BMDM, bone marrow–derived MF; GBS, group BStreptococcus; hf, heat-fixed; MF, macrophage; PML, polymorphonuclear leukocyte;qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species; WT, wild-type.

Copyright� 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501014

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Traditionally, MFs were regarded as terminally differentiatedstates of invading monocytes. Yet, compelling recent evidenceindicates that the origin of MFs and MF equivalents is highly sitespecific. Certain tissue MF types (e.g., Kupffer cells, peritoneal,lung, splenic red pulp MFs, and microglia), are long-lived, residein their specific tissue in a steady-state, and are not replenished byinfiltrating monocytes (16). These MFs originate from differentsources (e.g., the fetal liver and yolk sac) (17, 18).Intestinal MFs, the largest subset of MFs in the human body,

recently were shown to originate predominantly from CCR2-dependent Ly6Chigh monocytes in adult mice, with a negligiblecontribution of embryonic precursor cells after 8 wk of life (19).Like the intestinal mucosa, the skin is a major habitat for colo-nizing bacteria and an important physical barrier system againstinvading pathogens. Yet, the origin of resident dermal MFs is onlypartially understood. There appears to be a significant contributionof bone marrow– and blood-derived mononuclear cells to thedermal MF pool, yet it is unclear whether a subset of dermal MFsmay self-renew and differentiate at site (20, 21). In contrast,microglia, the tissue MF equivalent of the CNS, originate fromyolk sac progenitors, which seed during early embryogenesis.Without major insults, such as meningitis or radiation damage,microglia are not replenished by cells of the hematopoietic system(17, 22). Accordingly, intestinal MFs, dermal MFs, and microgliarepresent a spectrum with respect to origin and renewal, and theyare the most interesting candidates to be analyzed for the influenceof the MF origin on pathogen sensing.Thus, we analyzed the role of TLR13 in the cytokine response to

streptococci and staphylococci, in primary mouse monocytes, inmicroglia, and in MFs derived from the bone marrow, the intes-tine, and the dermis.Our data indicate that endosomal TLR13 is the main MF re-

ceptor involved in the recognition of streptococci in resident tissueMFs, including microglia. In contrast, TLR2 plays only a sec-ondary role under conditions of immediate physical contact be-tween streptococci and MFs. The polarization of streptococcalsensing toward TLR13 appears to occur during differentiation inthe tissue, because TLR13 is redundant in Ly6Chigh monocytes,which are precursors of intestinal and most dermal MFs. Fur-thermore, the increased role of TLR13 does not directly resultfrom “natural“ contact with the microbiome, because microgliaisolated from the sterile neonatal brain do not substantially differfrom adult intestinal MFs in their TLR13 usage.

Materials and MethodsAnimals and cell lines

All mice were on the C57BL/6J or C57BL/6N genetic background.Mice harboring a UNC-93B-H412R (3d) mutation were kindly pro-vided by Marina Freudenberg (University of Freiburg) (6). TLR132/2

mice were described elsewhere (12). Flt3tdRFP mice were generatedunder specific pathogen–free conditions in the animal facility of theTechnical University Dresden by breeding Flt3Cre BAC-transgenicmice (23) mice with tdRFP reporter mice (24). Mice used for ex-periments were between 6 and 12 wk old and were matched forage and sex. All animal experiments were approved by the Federal Min-istry for Nature, Environment, and Consumer’s protection of the state ofBaden-Wuerttemberg.

Bacterial strains

For infections, the group B Streptococcus (GBS) strain NEM316 (wild-type[WT]) and its isogenic lipoprotein diacylglyceryl transferase–deficientmutant NEM2188 (Dlgt) were grown to mid-log phase in Todd-Hewittbroth (25). Bacterial concentrations were determined with an opticalspectrophotometer (OD600). CFU were counted by serial dilutions on bloodagar plates (COS; bioMerieux). Staphylococcus aureus SA113 and itsisogenic lipoprotein diacylglyceryl transferase–deficient mutant strain

SA113 (Dlgt) were kindly provided by Fritz Goetz (T€ubingen, Germany).For heat-fixation, bacteria were incubated for 30 min at 80˚C with shakingafter adjusting the cell number to 1010 CFU/ml, as previously described(6). For S. aureus, the strain Newman was used unless indicated otherwise.For RNA extractions, GBS was grown as described above, washed in PBS,and lysed in RLT buffer by adding 250–500 mM glass beads with vigorousshaking (50 Hz, 5 min). RNA was extracted according to the supplier’sprotocol (RNeasy Mini Kit; QIAGEN).

Differentiation of bone marrow–derived MFs

Eight- to twelve-week-old donor mice were sacrificed and sprayed with70% ethanol. Then, femurs and tibias were rinsed with sterile PBS, sep-arated, and opened. Bone marrow cells were flushed with a 27-gauge needleand passed through a 40-mm cell strainer. Pelleted cells were resuspendedin RPMI 1640 medium supplemented with 10% FBS, antibiotics (cipro-floxacin, 10 mg/ml), and either GM-CSF (50 ng/ml) or, where indicated,M-CSF (20 ng/ml, PeproTech) and plated in T75 cell culture flasks(Greiner CELLSTAR). Bone marrow–derived MFs (BMDMs) were usedfor experiments after 10–12 d of differentiation. The resulting cells wereCD45highCD11bhighF4/80highCD64high.

Isolation of tissue-resident MFs

Intestinal MFs were extracted from the colon and the small intestine.Briefly, intestines were cleaned of mesentery and opened longitudinally,and fecal contents were rinsed off. Epithelial cells were dissociated byshaking twice for 15 min at 37˚C in 2 mM EDTA and 10 mM HEPES inHBSS (colon) or RPMI 1640 (small intestine). Remaining tissue waswashed, minced, and digested three times for 15 min at 37˚C with 0.3 mg/mlCollagenase IV (Worthington), 5 U/ml Dispase (Corning), and 0.5 mg/mlDNase I (Roche) in HBSS/RPMI 1640 supplemented with 2% FCS. Cellsuspensions were passed through a 70-mm strainer. For dermal MFs,mouse ears were subjected to enzymatic digestion by Dispase (1 mg/ml;STEMCELL Technologies), Collagenase II (2 mg/ml; PAA), and DNase I(0.8 mg/ml; Roche) in PBS for 2 h at 1400 rpm with shaking at 37˚C. Afterdigestion, the samples were filtered with a 40-mm cell strainer. Microgliawere isolated as described (26). Briefly, brains of neonatal mice werecollected, and the meninges were removed. Tissue was dissociated bypipetting and centrifugation, and the cell suspension was plated in cellculture flasks coated with 5 mg/ml poly-L-lysine. After 10 d with repeatedmedium exchange, cells were stimulated with 10 ng/ml M-CSF (Pepro-Tech). Three days later, microglia were harvested by orbital shaking for 3 hat 37˚C and 130 rpm. Harvesting was repeated after 7 d using the sameprocedure.

Flow cytometry staining

For flow cytometry, cells were washed with staining buffer (2 mM EDTAand 2% FCS in PBS), and 106 cells were stained for 30 min at 4˚C in 100 mlvolume. Subsequently, cells were washed and analyzed with a 10-colorflow cytometer (Gallios) and Kaluza software (version 1.2; both fromBeckman Coulter). The following anti-mouse Abs were used: CD45–eFluor 450, CD11b–PE-Cy7, MHC class II–eFluor 450 (eBioscience),Ly6G-FITC, Ly6C–PerCP-Cy5.5, CD11c-allophycocyanin (BD Pharmin-gen), CD64–PerCP-Cy5.5 (BioLegend), and F4/80-allophycocyanin (AbDSerotec).

Stimulation of cells

MFs were seeded into 96-well plates in triplicates (8 3 104 cells/well).The following day, stimulants were added in 100 ml cell culture medium.As controls, 100 ng/ml LPS (Sigma) and 5 mg/ml R848 (InvivoGen) wereused. The TLR13 ligand SA19 (RNA oligoribonucleotide with the se-quence 59-GGA CGG AAA GAC CCC GUG G-39, 0.6 mg) and GBS RNA(100 ng/well) were transfected into cells using LyoVec (InvivoGen). Fortransfections, nucleic acids were mixed with LyoVec, according to thesupplier’s instructions, incubated for 20 min at room temperature, andadded directly to the wells. Heat-fixed (hf) bacteria were used at a con-centration of 5 3 107/ml, unless indicated otherwise.

ELISA

TNF-a (R&D Systems), IL-6, and IL-1b (both from BD Biosciences) werequantified using ELISA kits, according to the manufacturer’s instructions.

In vitro blood cell stimulation and intracellular TNF-a staining

Mouse blood was collected from the retrobulbar venous plexus of anes-thetized mice. After RBC lysis (13 RBC Lysis Buffer Solution; eBio-science), leukocytes were washed with PBS and stained with the indicated

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Abs or plated for stimulation. For stimulation, leukocytes were washedtwice and resuspended in RPMI 1640 medium supplemented with 10%FBS with antibiotics (ciprofloxacin, 10 mg/ml). After plating (5 3 105

cells/well, 24-well plate), cells were treated with BD GolgiPlug ProteinTransport Inhibitor and stimulated for 5 h at 37˚C with the indicatedstimulants. For inhibition of phagocytosis, Cytochalasin D (final concen-tration) was added to the culture 60 min before stimulation with GBS orLPS. After harvesting and cell surface staining, cells were fixed, per-meabilized (Cytofix/Cytoperm Kit; BD Biosciences), and stained for in-tracellular TNF-a (anti-mouse TNF-a allophycocyanin; BD Biosciences).TNF-a–producing cell populations were determined by flow cytometricanalysis. Blood inflammatory monocytes were characterized as CD45high

CD11bhighLy6GlowLy6Chigh. Mouse regulatory monocytes were charac-terized as CD45highCD11bhighLy6GlowLy6Clow. Polymorphonuclearleukocytes (PML) were characterized as CD45highCD11bhighLy6Ghigh. ForRNA extractions, blood monocytes were purified from RBC-lysed bloodby immunomagnetic negative selection with the EasySep Mouse MonocyteEnrichment Kit (STEMCELL Technologies), according to the manufac-turer’s instructions.

Ex vivo stimulation of sorted cells, RNA preparation, andquantitative RT-PCR

Cells were sorted by FACS (MoFlo Astrios), resuspended in RPMI 1640medium with 10% FBS plus antibiotics (ciprofloxacin, 10 mg/ml), andplated in 48-well plates (3 3 104 cells/well). Adherent cells were washedonce and stimulated with medium containing the indicated stimulants for2 h at 37˚C. Total RNA was extracted using the RNeasy Micro Kit,according to the instruction manual (QIAGEN). Quantitative RT-PCR(qRT-PCR) was performed as described previously (9). For TLR expres-sion levels, cells were sorted directly into RLT lysis buffer. The followingmouse primer sequences were used (59–39): TNF-a: TCG TAG CAA ACCACC AAG TG (fw), CCT TGT CCC TTG AAG AGA ACC (rev); IL-10:CCC TTT GCTATG GTG TCC TT (fw), TGG TTT CTC TTC CCA AGACC (rev); GAPDH: ACT CCA CTC ACG GCA AAT TC (fw), TCT CCATGG TGG TGA AGA CA (rev); TLR1: TCA AGC ATT TGG ACC TCTCCT (fw), TTG TAC CCG AGA ACC GCT CA (rev); TLR2: TTT GCTGGG CTG ACT TCT CT (fw), AAA TCT CCA GCA GGA AAG CA(rev); TLR4: TTC CTT CTT CAA CCA AGA ACATAG ATC (fw), TTGTTT CAA TTT CAC ACC TGG ATA A (rev); TLR6: CCA AGA ACAAAA GCC CTG AG (fw), TGT TTT GCA ACC GAT TGT GT (rev);TLR7: GGA AAT TGC CCT CGA TGT TA (fw), CAA AAA TTT GGCCTC CTC AA (rev); TLR9: GCT GAA GCT GGA CCT GTC TC (fw),CAG GTT GGG TAG GAA GGA CA (rev); TLR13: ATG TGA AGACCG TGC CTT TG (fw), GGC GGC AGA GAA AAT CCT AC (rev);UNC-93B: CAC CCT TAC TTA CGG CGT CTA (fw), CAT GTT GCCATA CTT CAC CTC T (rev); and MyD88: TCC GGC AAC TAG AACAGA CAG ACT (fw), GCG GCG ACA CCT TTT CTC AAT (rev).

Infection of BMDMs

For infections with living bacteria, BMDMswere seeded into 96-well plates(8 3 104/well). The following day, medium was changed to antibiotic-freeRPMI 1640 medium. Bacteria were grown to an OD600 of 0.4, washedtwice with PBS, and added to the wells. Multiplicity of infection wasverified by dilution series on blood agar. Plates were centrifuged for 10min at 1500 rpm to synchronize infections, and supernatants were col-lected after 8 h (IL-1b) or 24 h (TNF-a). For the 24-h time points, gen-tamicin (250 mg/ml) was added to the culture 2 h postinfection. LPS (100ng/ml) plus 5 mM ATP (InvivoGen), the latter of which was added 20 minbefore the end of the experiment, served as a control for inflammasomeactivation.

Measurement of reactive oxygen species

For reactive oxygen species (ROS) quantification, 1 3 106 BMDMs wereseeded into white multi-well plates. The following day, medium wasreplaced by 50 ml fresh medium. After 2 h, 40 mg/ml lucigenin (Invi-trogen) was added and incubated for 5 min in the dark at room tempera-ture. Stimulants were added in 50 ml to their appropriate finalconcentrations, and fluorescence was analyzed in a MicroLumat Plusreader (Berthold).

Skin infection model

Mouse skin was infected as previously described (3). Briefly, bacteria weregrown to mid-log phase, washed with PBS twice, and resuspended in PBSat ∼3 3 108 CFU/ml. A total of 10 ml suspension was injected intrader-mally into the ear pinna of anesthetized mice (i.p. ketamine/xylazine).Mice were analyzed after 24 h. For CFU counts, ears were homogenized

with beads in 1 ml PBS using a TissueLyser (QIAGEN), and serial dilu-tions were plated on blood agar plates and incubated overnight at 37˚C.

Statistical analysis

Comparative data were analyzed by the two-tailed Student t test or one-wayANOVA, followed by the Bonferroni post hoc test for multiple groupcomparison. Data are presented as mean 6 SEM; p values #0.05 wereconsidered significant. Statistical analysis was performed with GraphPadPrism 6.

ResultsTLR13 is essential for the recognition of streptococci andstaphylococci in BMDMs

BMDMs are widely used workhorses in studies on cellular innateimmunity to bacteria. It was shown that recognition of ssRNA fromGram-positive bacteria is essential for the cytokine response inBMDMs and that endosomal TLRs are the key sensors of RNA inMFs (6, 11). This model was based on the role of the endoplasmicreticulum protein UNC-93B, which acts like a chaperone forendosomal TLRs by allowing for their trafficking and localization.A single point mutation in UNC-93B (H412R, “3d”) abrogatesnucleic acid sensing by TLR7 and TLR9, as well as localization ofthese TLRs to endosomes (27, 28). In concordance, the potent IL-6 response of WT mouse BMDMs to GBS was abrogated whenBMDMs were derived from UNC-93B–deficient (3d) mice(Fig. 1A). Endosomal TLR3, TLR7, TLR8, and TLR9 werereported not to be involved in the recognition of GBS ssRNA (6).Accordingly, we analyzed BMDMs deficient in endosomalTLR13, which was shown to be essential for the sensing of RNA(11), using the 23S rRNA segment SA19 as TLR13 ligand control.We found that TLR132/2 BMDMs were strongly impaired in thecytokine response to GBS (Fig. 1B), which was in accordancewith data on GBS strains masked from TLR13 recognition (14).Moreover, UNC-93B and TLR13 appeared to serve as nonre-dundant signaling components in the same pathway. Notably, theessential roles of UNC-93B and TLR13 were not dependent on theMF polarization status, because GM-CSF– and M-CSF–derivedBMDMs from the respective knockout mice were equally im-paired in their cytokine response (data not shown). Because thecytokine response of MFs to GBS is strongly dependent onphagocytosis (29), we wondered whether TLR13 modulates GBSuptake. However, TLR13-deficient BMDMs showed a normalphagocytic capacity compared with WT BMDMs (data notshown). The role of TLR13 in the cytokine response to S. aureuswas less pronounced than observed for GBS and was only visiblefor low bacterial concentrations (Fig. 1C). The production of ROSfollowing stimulation with GBS and S. aureus was independentof TLR13, because WT and TLR132/2 BMDMs were indistin-guishable in this respect over the entire range of bacterial con-centrations (Fig. 1D). These findings suggested that endosomalTLR13 is particularly important for the cytokine formation inBMDMs in response to Gram-positive bacteria. Moreover, thequantitative contribution of TLR13 differed between bacterialspecies (GBS . staphylococci).Next, we determined how the recognition of GBS lipoproteins by

TLR2 contributes to the cytokine formation in MFs in concert withthe newly established role of TLR13. We used GBS with a dele-tion of the lipoprotein transferase gene lgt. In this strain, the pu-tative TLR2-activating diacylated lipoproteins are not formed,thus TLR2 is not activated (25). In WT BMDMs, lgt-deficient andthe parental WT GBS (strain NEM316) did not differ in cytokineinduction. In contrast, lgt-deficient GBS was even less potentthan WT GBS in UNC-93B–deficient BMDMs (Fig. 1E). Theseresults indicate that the residual immune response to GBS in MFs

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deficient in endosomal TLR signaling is attributable to TLR2 and iscompletely abolished if both TLRs are missing. Because hf bacteriarevealed a notable phenotype under the outlined conditions, we nextchallenged BMDMs with live GBS harvested at mid-logarithmicgrowth phase (Fig. 1F). During infection with live GBS for 24 h,the cytokine response in TLR13-deficient BMDMs was stronglyreduced compared with control cells. In addition, the response tolgt-deficient GBS was further reduced in TLR132/2 BMDMs,whereas lgt-deficient and WT GBS were indistinguishable in WTBMDMs. Furthermore, the production of IL-1b, which requireshemolytic activity by live GBS (30), was strongly dependent onendosomal TLR sensing via UNC-93B (Fig. 1G). Additional deletionof TLR2-activating lipoprotein transferase resulted in complete lossof IL-1b secretion, indicating that TLR2 and TLR13 wereequally important for sensing of live GBS (Fig. 1G).

Endosomal TLRs and TLR2 are dispensable for the recognitionof GBS by monocytes and PML

We next analyzed the TLR-dependent cytokine response to GBS inmonocytes derived from mouse blood. We stimulated primaryblood leukocytes fromWT mice with GBS or, as control, with LPSand analyzed TNF-a production by intracellular staining andFACS. PML (CD45highCD11bhighLy6GhighLy6Cint), patrollingmonocytes (CD45highCD11bhighLy6GlowLy6Clow), and inflamma-tory monocytes (CD45highCD11bhighLy6GlowLy6Chigh) all showed

a robust GBS-induced TNF-a response (Fig. 2A). Surprisingly, theTNF-a response was independent of TLR13 and TLR2 in PMLand Ly6Chigh inflammatory monocytes, because WTand TLR132/2

cells were indistinguishable when challenged with WT or with lgt-deficient GBS (Fig. 2B). Moreover, endosomal TLR signaling wasaltogether dispensable for GBS sensing by myeloid blood cells, asshown by stimulation of UNC-93B–deficient cells (Fig. 2C).These results were confirmed by analyzing TNF-a RNA levels inmonocytes purified from murine blood and stimulated in vitro for2 h (Fig. 2D). Although lack of TLR13 did not affect cytokineformation in monocytes, the receptor was generally functional inthese cells, as verified by stimulation with the TLR13-specificligand SA19 (Fig. 2D). These data indicate that myeloid cellscirculating in the blood are activated by GBS, without substantialinvolvement of endosomal TLRs or TLR2. Because phagocytosisof GBS is required for TLR13 recognition, it seemed possible thatpoor phagocytosis might explain the poor engagement of endo-somal TLRs. However,.80% of monocytes were fluorescent afterincubation with FITC-labeled GBS for 1 h, indicating strongphagocytosis (data not shown). Moreover, when phagocytosis wasinhibited by Cytochalasin D prior to stimulation, the inflammatoryresponse of monocytes to GBS was abrogated (Fig. 2E). In con-trast, the LPS response was only modestly affected, which is inaccordance with published data (31). Therefore, phagocytosis isrequired for the GBS-induced cytokine response in monocytes,

FIGURE 1. TLR13 is essential for the recognition of Gram-positive bacteria in BMDMs. BMDMs from WT and UNC-93B (3d) (A) or TLR132/2 (B and C)

mice were stimulated with LPS (100 ng/ml), R848 (5 mg/ml), SA19 (6 mg/ml in LyoVec transfection medium), hf GBS [53 107/ml (A) and 106/ml, 107/ml, 108/ml

(B)], and hf S. aureus [106/ml, 107/ml, 108/ml (C)]. (D) BMDMs from WTand TLR132/2mice were stimulated with hf GBS or hf S. aureus (106/ml, 107/ml, 108/ml),

and maximum ROS production based on relative light units (RLU) was analyzed within 2.5 h. (E) BMDMs fromWTand UNC-93B (3d) mice were stimulated with

hf WTor lgt-deficient GBS (53 107/ml), and supernatant IL-6 and TNF-a levels were analyzed after 24 h. (F) M-CSF–derived BMDMs from WTand TLR132/2

mice were infected with WTor lgt-deficient GBS (multiplicity of infection [MOI] = 10) for 2 h and then 250 mg/ml gentamicin was added to the culture. Induction

of TNF-a response was measured after 24 h by ELISA. (G) BMDMs from WT and UNC-93B (3d) mice were stimulated with LPS (100 ng/ml), WT GBS, or

lgt-deficient GBS (MOI = 1), and supernatant IL-1b levels were analyzed after 8 h. ATP (5 mM) was added to the LPS control 30 min before the end of the

stimulation. All data are mean 6 SEM of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, two-tailed unpaired t test. ns, not significant.

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but endosomal TLR signaling can be compensated for via otherpathways.Collectively, these data suggest that the use of TLR13 in myeloid

cells by streptococci is specific for the site of residence or origin.

Monocyte-derived intestinal MFs require TLR13 for GBSsensing

In view of the difference between in vitro–differentiated BMDMsand blood monocytes, the role of TLR13 in tissue-resident MFswas most interesting. Depending on the tissue, MFs originatefrom the yolk sac/fetal liver and proliferate in situ, or they arederived from hematopoiesis with constant replenishment by bloodmonocytes. MFs from the colon of adult mice largely originatefrom blood monocytes (19). Moreover, the colon is the naturalcolonization site of GBS, starting early in life (2). Hence, un-derstanding GBS recognition in intestinal MFs is important in thecontext of host resistance against GBS. Accordingly, we sortedMHCIIhighCD11bhighF4/80high colonic MFs from 8–12-wk-oldmice (Fig. 3A). By this age, intestinal MFs derive largely fromhematopoiesis, as shown by their RFP expression in a fate-mapping analysis of Flt3cretdRFP reporter mice (Fig. 3B). Be-cause Flt3 is expressed in hematopoietic stem cells, RFP+ cellsderive from blood monocytes and not the yolk sac (23). We then

challenged the sorted MFs with GBS ex vivo, followed byintracellular staining or qRT-PCR for TNF-a. To allow directcomparison, we used the same functional assay and time frames asfor blood monocytes. Notably, in colonic MFs deficient in eitherUNC-93B or TLR13, the TNF-a response to GBS or lgt-deficientGBS was strongly reduced (Fig. 3C, 3D). Nucleotide sensing byTLR13/UNC-93B, as a key mechanism in GBS recognition, isconserved throughout the mouse intestine, as shown in MFs fromthe small intestine (Fig. 3E). Therefore, monocyte-derived MFswere functionally distinct from blood monocytes, their directprecursors (19). Instead, intestinal MFs sense GBS in a similarfashion as in vitro–differentiated BMDMs.

Dermal MFs require endosomal TLR13 for sensing of GBS

GBS is a well-established cause of soft tissue and bone infections,both in infants and adults (32, 33). In the dermis, an importantbarrier against soft tissue infections caused by streptococci, resi-dent dermal MFs constitute the main immune cell population(34). We showed recently that dermal MFs are essential for therecognition of skin-invading S. aureus. Signaling via the TLRadaptor protein MyD88 mediates the response (3). During infec-tion, the majority of dermal MFs is replenished by blood-derivedLy6Chigh monocytes. To test whether dermal MFs are functionally

FIGURE 2. Endosomal TLRs and TLR2 are dispensable for the recognition of GBS by monocytes and PML. (A) RBC-lysed blood cells from WT mice

were stimulated with LPS (100 ng/ml) or hf GBS (5 3 107/ml) and stained for CD45, CD11b, Ly6G, and Ly6C. Cells were fixed, permeabilized, and

stained for TNF-a intracellularly. TNF-a–producing cell populations were determined by flow cytometry after 5 h. RBC-lysed blood cells from WT

and TLR132/2 (B) and UNC-93B (3d) (C) mice were stimulated with LPS (100 ng/ml), hf WT GBS (5 3 107/ml), and hf lgt-deficient GBS (5 3 107/ml),

and TNF-a–producing PML and Ly6Chigh monocytes were determined as described. (D) Monocytes purified by immunomagnetic negative selection were

stimulated with 100 ng/ml LPS or 108/ml hf GBS or transfected with SA19. After 2 h, cells were lysed in RLT buffer, and cytokine expression levels were

quantified by qRT-PCR. Data represent the mean fold change of TNF-a gene expression levels normalized to GAPDH compared with unstimulated

controls. (E) RBC-lysed blood cells were pretreated with DMSO or 10 mM cytochalasin D for 1 h, followed by incubation with 100 ng/ml LPS or 108/ml hf

GBS for 5 h. Intracellular TNF-a production was measured by flow cytometry. Data are mean 6 SEM of three independent experiments. *p , 0.05, **p ,0.01, ***p , 0.001, ****p , 0.0001, two-tailed unpaired t test. ns, not significant.

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similar to BMDMs and intestinal MFs, or whether they mimicmonocytes, we sorted CD45highCD11bhighCD11clowCD64high

MFs from WT, UNC-93B–deficient, and TLR132/2 mice andstimulated them with WT and lgt-deficient GBS (Fig. 4). UNC-93B–deficient and TLR132/2 dermal MFs were impaired in theirTNF-a response to GBS. This response was further compromisedwhen lipoprotein-deficient GBS was used (Fig. 4B, 4C). This in-dicates that the proinflammatory response to GBS in dermal MFsrelies on TLR13 and TLR2.

Self-renewing microglia are particularly dependent onendosomal nucleotide sensing for GBS recognition

Microglia are regarded as the equivalent of tissue MFs in the CNS.Yet, in many ways, such as function and surface marker expression,microglia differ from tissue-resident MFs. In addition, microgliaderive exclusively from erythromyeloid progenitors in the yolk sacand self-renew during their long lifespan (35). Because GBS is themost important cause of meningoencephalitis in newborn infantsand is the third most frequent cause of bacterial meningoencepha-litis in many parts of the world, the molecular mechanisms

underlying the microglia response to GBS are highly important.Accordingly, we prepared microglia from the brain of newbornmice (P2) and stimulated the cells with GBS or RNA isolated fromGBS. We found that the potent TNF-a response to whole bacteria orRNA in microglia is almost completely abrogated in the absence ofthe TLR13 chaperone UNC-93B (Fig. 5A). The residual, albeit verysmall, response in UNC-93B–deficient microglia was dependent onTLR2 activation by GBS. The dependence of microglia on UNC-93B in bacterial recognition was not restricted to GBS, becausewhole S. aureus preparations barely induced TNF-a in UNC-93B–deficient microglia. TNF-a induction was further reduced inmicroglia, when the absence of staphylococcal nucleic acid rec-ognition was combined with the absence of TLR2 engagement byS. aureus (Fig. 5B). To rule out that the differences between mono-cytes and different MF subsets are the result of different expressionlevels of relevant TLRs or adaptors, we analyzed their expression byqRT-PCR. Compared with MFs, monocytes expressed similar orhigher levels of TLR2 and its coreceptors TLR1 and TLR6, as well asTLR4, endosomal TLR7, TLR9, and TLR13, and the adaptor pro-teins UNC-93B and MyD88 (Fig. 5C).

UNC-93B (3d) mice incompletely control dissemination ofGBS to the blood stream

As a result of the strong phenotype of TLR13 deficiency observedin MFs in vitro, we were interested in the role of endosomal TLR

FIGURE 3. Monocyte-derived intestinal MFs require TLR13 for GBS

sensing. (A) For FACS, intestinal mouse MFs were characterized as

CD11bhighMHCIIhighF4/80high. (B) Intestinal MFs of Flt3cretdRFP mice

were analyzed after 7 and 19 wk of age, and RFP expression was deter-

mined by flow cytometry. (C) Intestinal MFs from WT and UNC-93B (3d)

mice were stimulated with LPS (100 ng/ml), R848 (5 mg/ml), and hf WT

and lgt-deficient GBS (5 3 107/ml) and stained extracellularly for MHC

class II, CD11b, and F4/80 and intracellularly for TNF-a. TNF-a–pro-

ducing cells were detected by flow cytometry. Small intestinal cells (D)

and sorted colonic MFs (E) from WTand TLR132/2 mice were stimulated

with LPS (100 ng/ml) and hf WT and lgt-deficient GBS (5 3 107/ml).

TNF-a production was analyzed by qRT-PCR [colon, (D)] relative to

Gapdh or intracellular TNF-a staining [small intestine, (E)]. All data are

mean 6 SEM of three independent experiments. *p , 0.05, **p , 0.01,

***p , 0.001, two-tailed Student t test.

FIGURE 4. Dermal MFs sense GBS via endosomal TLR13. (A) Dermal

mouse MFs of digested skin were characterized as CD45highCD11bhigh

CD11clowCD64high. FACS-sorted dermal MFs from WT and UNC-93B

(3d) mice (B) or TLR132/2 mice (C) were stimulated with LPS (100 ng/ml),

R848 (5 mg/ml), and hf WT and lgt-deficient GBS (5 3 107/ml) for 2 h;

total RNA was prepared, and qRT-PCR for TNF-a was performed. Values

are expressed relative to Gapdh. All data are mean 6 SEM of three in-

dependent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, two-tailed

unpaired t test. ns, not significant.

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sensing in the pathogenesis of GBS. Because of the phenotypicdiscrepancy between monocytes and MFs, we chose a model thatwas shown to rely on MF presence in staphylococcal infection.Intradermal injection in the ear pinna of WT mice results inneutrophil recruitment and resolution of infection that are de-pendent on the presence of dermal MFs (3). This model is rele-vant for GBS, because local infections of skin and soft tissue oftenprecede neonatal GBS sepsis (32). To verify the importance ofTLR signaling, we used MyD882/2 mice, which are severelyimpaired in the sensing of surface and endosomal TLRs. At 24 hpostinfection, WT mice showed a strong increase in neutrophilnumbers in blood and skin (Fig. 6A, 6C), whereas no bacteria wasdetected in peripheral blood. In contrast, MyD882/2 mice dis-played significantly lower peripheral neutrophil counts, and al-most all mice suffered from bacteremia (Fig. 6B). No significantdifference in the local inflammatory response was noted betweenWT and UNC-93B (3d) mice; however, in 4 of 10 UNC-93B (3d)mice, bacteria were detected in the peripheral blood, which wasaccompanied by a tendency toward lower neutrophil counts in

blood and skin. Collectively, these data point out that TLR sens-ing, including endosomal TLR pathways, is important for a properimmune response to GBS in the skin to recruit effector cells andprevent dissemination of bacteria into the blood stream.

DiscussionPreviously, we found that MFs recognize streptococci throughendosomal sensing of bacterial RNA in strict dependency onMyD88 and UNC-93B (6). We now provide evidence that TLR13is the endosomal receptor for streptococci in differentiated mouseMFs. It seems noteworthy that nucleic acid sensing, as the criticalbottleneck in cytokine induction by streptococci, is shared be-tween MFs derived from diverse tissues, including the brain,which are of different origin and reside in very distinct microen-vironments. TLR2 as an extracellular receptor for lipoproteinscannot compensate for the loss of RNA sensing. However, whenTLR13 or UNC-93B are nonfunctional, TLR2 sensing becomesdetectable under some circumstances, because TLR13-deficientMFs are further impaired in their inflammatory response to lgt-deficient GBS. A previous study on a GBS mutant with acquirednucleic acid methylation interfering with TLR13 recognition (14)suggested that live GBS do not engage TLR13 as an essentialreceptor in MFs. In contrast, we show in this study that TLR13 iscrucial for the recognition of live and fixed bacteria by MFs.Whether strain specific effects beyond methylase activity areimportant for the role of TLR13 in GBS recognition cannot beconcluded with certainty at this stage.Notably, we showed previously that sensing of staphylococci by

resident dermal MFs is independent of TLR2 and endosomalTLRs (3). Accordingly, GBS, but not S. aureus, essentially en-

FIGURE 5. Microglia require endosomal nucleotide sensing for GBS

recognition. (A) In vitro–differentiated microglia from neonatal WT and

UNC-93B (3d) mice were stimulated with LPS (100 ng/ml), R848 (5 mg/ml),

GBS RNA (10 ng/ml in LyoVec transfection medium), and hf WT and lgt-

deficient GBS (107/ml and 108/ml), and supernatant TNF-a levels were

analyzed after 24 h. (B) In vitro–differentiated microglia from WT and

UNC-93B (3d) mice were stimulated with LPS (100 ng/ml), R848 (5 mg/ml),

hf WT S. aureus (strain SA113, 107/ml), hf lgt-deficient S. aureus (Dlgt;

strain SA113, 107/ml), and SA19 (6 mg/ml in LyoVec transfection me-

dium). Supernatant TNF-a levels were analyzed by ELISA after 24 h. Data

are mean6 SEM of six pooled mice in three independent experiments. (C)

Relative expression levels of TLRs and adaptors in microglia, BMDMs,

and sorted blood monocytes were determined by qRT-PCR relative to

Gapdh. Data are mean 6 SEM of at least three mice. *p , 0.05, **p ,0.01, two-tailed unpaired t test.

FIGURE 6. MyD88, but not endosomal TLRs, is essential for host re-

sistance and inflammation control in dermal GBS infection. (A–D) GBS

NEM316 (3 3 106) was injected intradermally in both ears of WT,

MyD882/2, and UNC-93B–deficient mice. Twenty-four hours postinfec-

tion, mice were sacrificed, and blood was analyzed for neutrophil (A) and

CFU counts (B). (C) One ear/mouse was analyzed by flow cytometry for

recruited neutrophils (Ly6-G+Ly6-Cint). (D) The second ear was homogenized

and plated for CFU counts. Data points shown are from three independent

experiments with four mice/group for WT and UNC-93B (3d) mice and for

two or three mice per group for MyD882/2 mice. *p , 0.05, **p , 0.01,

***p , 0.001, one-way ANOVA, Bonferroni multiple-comparison test. ns,

not significant.

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gages similar receptors in primary dermal and intestinal MFs, aswell as in in vitro–differentiated BMDMs. This indicates a ratherelaborate interaction of MFs with bacteria that depends on thebacterial species and the MF origin.The soft tissue GBS infection model analyzed in this study did

not reveal a substantial role for nucleic acid sensing in bacterialresistance and inflammation control, although we observed a trendtoward increased bacteremia in UNC-93B–deficient mice. Incontrast, MyD882/2 mice were severely impaired in bacterialclearance and neutrophil recruitment in the same model. It seemsplausible that the high invasiveness of GBS overwhelms cellularimmunity exerted by dermal MFs, thereby forcing early interac-tion with circulating myeloid cells. The latter respond, as wedemonstrated in this article, to GBS in a TLR13- and UNC-93B–independent fashion and may mask the TLR13/UNC-93B–re-stricted response of dermal MFs.Several questions remain about the role of TLR13 in strepto-

coccal pathogenesis. First, streptococci are highly adapted to thehuman species. They are pathobionts that thrive as part of themicrobiome in normal children and adults, yet they carry a sub-stantial disease burden as causes of sepsis and meningitis. Second,TLR13 is a mouse-specific receptor without a homolog in humans.In contrast, mice lack functional TLR8, which is a well-establishedreceptor of RNA in humans (36). With respect to TLR repertoireand function, humans are similar to cattle, which are widely ac-cepted as the original host of GBS (37). It remains to be estab-lished whether TLR13 equips mice with specific resistance tobacteria or whether it takes over the role of TLR8, which wasrecently identified as a sensor for bacterial RNA in human mye-loid cells (38–40). The latter seems likely, because mononuclearcells from mice and humans use the TLR signaling partnersIRAK4 and MyD88 for the optimal response to streptococci andstaphylococci (41). Finally, spatial organization of signal activa-tion by streptococci is conserved between mice and humans, asindicated by the role of the endosome as the privileged signalingsite in MFs processing streptococci (42). Therefore, conservedmechanisms in innate cellular immunity appear to be present inmice and humans, although the specific TLRs involved are obvi-ously distinct.At the same time, these mechanisms show a considerable degree

of variability, even in highly related cell types from one species. Itseems intriguing that monocytes as circulating MF progenitors donot require TLR13 or UNC-93B and, hence, endosomal TLRsignaling altogether, for a potent cytokine response to streptococciand staphylococci. In line with this data, a recent study of patientsdeficient in MyD88/IRAK4 demonstrated a residual responsive-ness of blood cells to whole heat-killed bacteria, in contrast to thecomplete loss of response to purified bacterial TLR ligands (43).In this context, the discrete role of MyD88 in circulating myeloidcell subsets in response to GBS remains to be established. TLR13is expressed in mouse monocytes and PML yet its function ap-pears to be by-passed by other unidentified recognition systemsfor GBS. Because GBS typically causes blood-borne disease onceit becomes invasive, the TLR13/UNC-93B–independent mode ofinteraction between GBS and monocytes and PML is likely ofimportance in the infection biology of this bacterium. It is con-ceivable that tissue MFs, which reside in close vicinity to mucosalsurfaces potentially colonized with streptococci (e.g., dermal andlamina propria MFs), might profit from sampling the metabolicactivity of bacteria via their RNA. In contrast, robust and highlyredundant recognition systems in circulating leukocytes are re-quired to prevent any blood-borne bacteria from escaping unde-tected. Yet, it remains unclear how monocytes switch to the“TLR13 mode” or “endosomal TLR mode” once they have left the

blood stream and differentiate into MFs. It is well appreciated thatMF differentiation and functional polarization are highly complexprocesses, underlying a network of transcriptional regulationprocesses (44). Hence, further research into site-specific immunityagainst bacteria is important to clarify why MFs as diverse asthose residing in the intestinal wall and in the brain show the sameactivation pattern by bacterial RNA.The identification of TLR13 as a MF-specific streptococcal

receptor offers the perspective for a better understanding of cell-autonomous events triggered by streptococci. These may betransferred to the human system, allowing for the development ofimproved adjunctive sepsis therapy.

AcknowledgmentsWe thankMarina Freudenberg for the provision of knockout mice and Bern-

hard Kremer, Anita Imm, and Jan Bodinek-Wersing for outstanding tech-

nical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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