We have shown that in vitro, IL-10hiPD-L1hi regulatory B cells (Bregs) directly attenuate HIV-specific CD8+-mediated CTL activity. Bregs also modulate APC and CD4+ T cell function; herein we characterize the Breg compartment in uninfected (HIVNEG), HIV-infected “elite controllers” (HIVEC), ART-treated (HIVART), and viremic (HIVvir), subjects, and in vitro, assess the impact of Bregs on anti-HIV CTL generation and activity after reactivation of HIV latent reservoirs using suberoylanilide hydroxamic acid (SAHA). We find that Bregs from HIVEC and HIVART subjects exhibit comparable IL-10 expression levels significantly higher than HIVNEG subjects, but significantly lower than HIVVIR subjects. Bregs from HIVEC and HIVART subjects exhibit comparable PD-L1 expression, significantly higher than in HIVVIR and HIVNEG subjects. SAHA-treated Breg-depleted PBMC from HIVEC and HIVART subjects, displayed enhanced CD4+ T-cell proliferation, significant upregulation of antigen-presentation molecules, increased frequency of CD107a+ and HIV-specific CD8+ T cells, associated with efficient elimination of infected CD4+ T cells, and reduction in integrated viral DNA. Finally, IL-10-R and PD-1 antibody blockade partially reversed Breg-mediated inhibition of CD4+ T-cell proliferation. Our data suggest that, possibly, via an IL-10 and PD-L1 synergistic mechanism; Bregs likely inhibit APC function and CD4+ T-cell proliferation, leading to anti-HIV CTL attenuation, hindering viral eradication.During HIV infection, IL-10/IL-10 receptor and programmed death-1 (PD-1)/programmed death-1-ligand (PD-L1) interactions have been implicated in the impairment of cytotoxic T lymphocyte (CTL) activity. Despite antiretroviral therapy (ART), attenuated anti-HIV CTL functions present a major hurdle towards curative measures requiring viral eradication. Therefore, deeper understanding of the mechanisms underlying impaired CTL is crucial before HIV viral eradication is viable. The generation of robust CTL activity necessitates interactions between antigen-presenting cells (APC), CD4 EC) often exhibit robust CTL activity CD8 cytotoxic T lymphocyte (CTL) activity is critical in controlling viral replication during HIV infection (reviewed in ART), viral replication is suppressed but the virus persists because early in infection HIV establishes latent reservoirs and upon ART interruption, HIV replication is detected + T cells as was previously hypothesized. In contrast, Shan et al determined that post reactivation of latent reservoirs an efficient CTL response was indispensable to clear infected cells. Since CTL responses are impaired in HIVART subjects, Margolis and Hazuda ART subjects, IL-10 expressing regulatory B cells  attenuate anti-HIV CTL activities in vitro by directly inhibiting the proliferation of antigen-specific cytotoxic CD8+ T cells in a partially IL-10 dependent manner +CD24hiCD38hi Bregs impair CTL activity during chronic Hepatitis B virus infection + T cells and antigen presenting cells (APC) are also critical in generating effective CTL responses + T cell proliferation and APC function In ART-treated HIV-infected subjects ; healthy controls HIVNEG (n = 20), HIVART (n = 20), and viremic HIV-infected subjects (HIVVIR) (n = 17). Breg frequencies were similar between HIVEC and HIVNEG groups, and were lower in the HIVVIR group, . Surprisingly, Breg frequencies were lowest in the HIVART group . By intracellular cytokine staining (VIR subjects were IL-10 positive compared to Bregs from HIVEC (p = 0.0006), HIVART (p<0.0001) and HIVNEG (p<0.0001) subjects. Further, Bregs from HIVEC and HIVART subjects exhibit comparable frequencies of IL-10 positive cells, significantly higher than in HIVNEG subjects  on CD8+ T cells of all subjects investigated  in 80% of the HIVART subjects  subjects studied , we investigated if this leads to enhanced clearance of infected cells. By intracellular cytokine staining (+ T cells in PBMC from HIVEC (p = 0.0021) and HIVART, (p = 0.0236)   .+ T cells EC subjects, Breg depletion led to enhanced DC expression of MHC-I (p = 0.0154) and MHC-II (p = 0.0008) as well as B-cell expression of MHC-II (p = 0.0076) (ART subjects Breg depletion led to enhanced DC expression of MHC-I (p = 0.0034), and MHC-II (p = 0.0076) as well as B-cell expression of MHC-II (p = 0.0331) and proliferation of CD4+ T cells  and proliferating CD4 0.0076) . Similarhi T cells significantly contribute to viral persistence NEG, HIVVIR, HIVAVIR, and HIVEC  and HIVVIR (p = 0.0421). Bregs from HIVART expressed 34% and 38% more PD-L1 than Bregs from HIVNEG and HIVVIR respectively. Interestingly, in HIVEC, HIVART and HIVVIR, compared to other B cell subsets, Bregs expressed significantly higher levels of PD-L1  and IL-2, either cultured with medium alone, co-cultured with non-Breg B cells, with Bregs, or co-cultured with Bregs supplemented either with anti-IL-10 receptor (IL-10R) blocking antibody, a PD-1 blocking antibody or both anti-IL-10R and anti-PD-1 blocking antibodies combined, as described elsewhere + T cells co-cultured with Bregs proliferated significantly less that those co-cultured with non-Bregs  did not robustly inhibit CD4+ T cell proliferation.Regulatory B cells (Bregs) have been attributed divergent phenotypic  as the measure of CTL-competency and determined that following reactivation of latent reservoirs using SAHA, Breg-depletion modulated critical mediators of robust CTL generation including antigen presentation and CD4+CD24hiCD38hi Bregs represent the predominant IL-10 producing B-cell subset, consistent with previous data suggesting a similar role for Bregs during hepatitis B viral infection Taken together our results suggest that during HIV infection CD19NEG) subjects had a median CD4 count of 777 cells/µl (range: 380–1487). HIV-infected viremic (HIVVIR) subjects had a median CD4 count of 495 cells/µl (range: 240–1136) and median viral load of 40909 copies/ml (range: 11023–3140000). Antiretroviral-treated HIV-infected (HIVART) subjects had a median CD4 count of 435 cells/µl (range: 212–1076) and median viral load of 40 copies/ml (range: 0–800). HIV-infected “Elite” controllers (HIVEC) had a median CD4 count of 763 cells/µl (range: 454–1595) and median viral load of 48 copies/ml (range: 20–1226). HIVEC were characterized as HIV-infected subjects capable of maintaining their viral load at <2000 copies/ml without ART as previously described All studies were performed after signed informed written research consent by each study subject. The study was reviewed and approved by the Institutional Review Board of the Rush University Medical Center, University of California—San Francisco (UCSF), and the University of Iowa City VAMC and University of Iowa. HIV-uninfected (HIV+ T cells were identified by binding to MHC-1-APC Dextramer® (Immudex) and HIV-infected CD4+ T cells were identified by binding to KC-57-PE antibody (Beckman Coulter). All samples were acquired on an LRSII  flow cytometer and the data was analyzed using FlowJo software (Tree Star Inc).To determine endogenous intracellular IL-10 production, PBMC were cultured for 48 hours; during the final 5 hours of incubation the cultures were supplemented with Brefeldin A , PMA  and Ionomycin . After incubation the cells were washed, stained for viable cells , surface stained, fixed/permeabilized (Fix/Perm Kit BD Biosciences) and stained for intracellular IL-10 . To determine spontaneous expression of IL-10 by Bregs from HIV-infected individuals and healthy controls, PBMCS were incubated overnight, stimulated for the final 5 hours and stained as described for healthy controls. The following antibodies were used for immunophenotyping of PBMC: CD19-ECD (Beckman Coulter), PD-L1-PE-Cy7 (eBioscience), CD24-PE, CD38-FITC, HLA-DR-PE-Cy7, CD4-Pacific Blue, CD8-APC-H7, Lineage-1-FITC, CD11c- AF-700, HLA-ABC-PE-C7 and CD107a-PE-C5 . HIV-specific CD8+ individuals were stimulated with HIV-peptide (NIH AIDS repository) pool spanning nef, env, gag and pol (2 ug/ml each) or 500 µM SAHA (Sigma). After 96 hours the frequencies of either CD8+CD107a+ (cytotoxic CD8+ T cells), infected CD4+ T cells  and proliferation of CD4+ T cells were determined by flow-cytometry. To determine the effect of PD-L1/PD-1 and IL-10/IL-10R interactions in Breg-mediated inhibition of CD4+ T cell proliferation, VPD450-labeled FACS-purified CD4+ T cells were activated using Anti-Biotin MACSiBead loaded CD2, CD3, and CD28 antibodies  and IL-2 , cultured with medium alone, with Bregs, with Bregs and IL-10R blocking antibody , with Bregs and PD-1 blocking antibody  or with Bregs with both IL-10R and PD-1 blocking antibodies. After 72 hours, proliferation was determined by flow cytometry.Proliferation dye VPD450 (BD Bioscience)-labeled total or Breg-depleted PBMCs from HIV+ T cells was determined by binding to APC-labeled HLA-A2-restricted SL9 (SLYNTVATL) HIV-Gag epitope MHC-I-Dextramer . Cells of HLA-A2 typed HIV+ individuals were washed twice with PBS, and incubated with 10 µl Dextramer for 10 minutes at room temperature, stained with antibodies and analyzed by flow-cytometry.The frequency of antigen-specific CD85′-TCAAGTAGTGTGTGCCCGTCTGTT-3′ and reverse 5′-AGCTCCTCTGGTTTCTCTTTCGCT-3′; and GAPDH mRNA forward 5′-CTTCAACGACCACTTTGT-3′ and reverse 5′-TGGTCCAGGGGTCTTACT-3′. Fold change in RNA expression was calculated by relative quantification using the comparative cycle threshold method. GAPDH expression was used as an endogenous control.RNA was isolated using Qiagen RNeasy Kit, according to the manufacturer's recommendations. Subsequently, cDNA was synthesized using the Quantitect Reverse Transcription kit . Real-time RT-PCR was performed using a Quantitect SYBR Green PCR kit (Qiagen) in a 7900HT Fast Real-Time PCR system . Melting curve analysis was performed to ensure that the primers amplified the desired amplicon and that primer-dimers were absent. Primers used were: Long Terminal Repeats (LTR) mRNA forward Results are expressed as mean ± standard error of the mean (SEM) or as indicated. GraphPad Prism software, version 5.03 was used for all statistical analysis. The statistical significance p value between group parameters was determined using either unpaired or paired tests . The statistical dependence between variables was calculated using the Spearman rank correlation analysis. p values of <0.05 were considered statistically significant.Figure S1Detection of infected CD4+ T cells using the KC-57 antibody. To determine the specificity of the KC-57 antibody, total or Breg-depleted PBMC from an HIVART subject, were supplemented with HIV-peptides  and as a control PBMC were left unstimulated. After 4 days in culture, intracellular staining for KC-57 was performed and the frequency of stained infected cells determined by flow cytometry. Representative dot plots from 3 independent experiments are shown.(TIF)Click here for additional data file.Figure S2Breg B-cells from HIV-infected subjects express high levels of Annexin V and low levels of Bcl-2. To determine the cause of Breg loss in HIV-infected subjects by flow cytometry we assessed the frequency of determined Annexin V positive and intracellular Bcl-2 positive Bregs and non-Bregs (mature and memory B cells) in HIV-infected  and (a) HIVNEG subjects. P values determined by Graphpad Prism software are indicated.(TIF)Click here for additional data file.


Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a high-quality draft genome sequence for Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated from root nodules of Alnus nitida.Members of the actinomycete genus  Frankia consists of filamentous actinobacteria that form nitrogen-fixing symbiotic associations with a wide variety of woody angiosperms termed actinorhizal plants  and include both narrow-host-range and mid-host-range symbionts. Since only two members of cluster I have been sequenced, strains ACN14a and CcI3  Joint Genome Institute (JGI) using a combination of 454-GS-FLX-Titanium (http://www.jgi.doe.gov). The 454 Titanium standard data and the 454 paired-end data were assembled together with Newbler, version 2.3 (The draft genome of Titanium  and IlluTitanium  techniqusion 2.3 , and ression 2.3 , and theFrankia QA3 was resolved to 1 scaffold containing 121 contigs consisting of 7,590,853 bp with a G+C content of 72.6%, 6,493 candidate protein-encoding genes, 46 tRNA genes, and 3 rRNA regions.The high-quality draft genome of Frankia strain QA3 have been deposited in GenBank with accession number AJWA00000000.The whole draft genome sequences of


Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs).in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice.To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production.S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs. Bone marrow mesenchymal stem cells (BMMSCs) are hierarchical postnatal stem/progenitor cells capable of self-renewing and differentiating into osteoblasts, chondrocytes, adipocytes, and neural cells ,2. BMMSCBMMSCs are thought to be derived from the bone marrow stromal compartment, initially appearing as adherent, single colony clusters (colony-forming unit-fibroblasts [CFU-F]), and subsequently proliferating on culture dishes . To datelpr/J mice were purchased from Jackson Laboratory . Female immunocompromised mice (Beige nude/nude XIDIII) were purchased from Harlan . All animal experiments were performed under the institutionally approved protocols for the use of animal research (USC #10874 and 10941).Female C3H/HeJ, C57BL/6J, and C3MRL-FasAnti Oct4, SSEA4, Runx2, OCN, active β catenin and β catenin were purchased from Millipore . Anti alkaline phosphatase (ALP) antibody was purchased from Abcam . Anti Sca-1-PE, CD34-PE, CD34-FITC, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3ε and CD28 antibodies were purchased from BD Bioscience . Anti Foxp3-PE, IL17-PE, and IFNγ-APC antibodies were purchased from eBioscience . Unconjugated anti CD34, CD73, and CD105, NOS2 were purchased from Santa Cruz Biosciences . Anti β actin antibody was purchased from Sigma .6 into 100 mm culture dishes  at 37°C and 5% CO2. Non-adherent cells were removed after two days and attached cells were maintained for 16 days in alpha minimum essential medium  supplemented with 20% fetal bovine serum , 2 mM L-glutamine, 55 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Colony-forming attached cells were passed once for further experimental use.The single suspension of bone marrow derived all nucleated cells (ANCs) from femurs and tibias were seeded at a density of 15 × 10ECM coated dishes were prepared as described previously . Briefly6) were seeded into 100 mm culture dishes and cultured for two days. The culture supernatant with floating cells was collected and centrifuged to obtain putative non-attached BMMSCs. The cells were re-seeded at indicated numbers on ECM-coated dishes. After 2 days, the floating cells in the cultures were removed with PBS and the attached cells on ECM were maintained for an additional 14 days. Colony-forming attached cells were passed once and sub-cultured on regular plastic culture dishes for further experiments. For some stem cell characterization analyses, we collected SSEA4 positive S-BMMSCs using the MACS magnetic separation system  and expanded in the cultures.Bone marrow-derived ANCs . After 16 days, the cultures were washed with PBS and stained with 1% toluidine blue solution in 2% paraformaldehyde (PFA). A cell cluster that had more than 50 cells was counted as a colony under microscopy. The colony number was counted in five independent samples per each experimental group.4 cells/well) was seeded on two-well chamber slides (Nunc) and cultured for two to three days. The cultures were incubated with BrdU solution (1:100) (Invitrogen) for 20 hours, and stained with a BrdU staining kit (Invitrogen). BrdU-positive and total cell numbers were counted in ten images per subject. The BrdU assay was repeated in five independent samples for each experimental group.The proliferation of BMMSCs and S-BMMSCs was performed using the bromodeoxyuridine (BrdU) incorporation assay. Each cell population . The finite population doublings were determined by cumulative addition of total numbers generated from each passage until the cells ceased dividing.A total of 0.5 × 106 cells) were incubated with 1 µg of R-Phycoerythrin (PE). (PE)-conjugated antibodies or isotype-matched control immunoglobulin Gs (IgGs)  at 4°C for 45 minutes. Samples were analyzed by a fluorescence-activated cell sorting (FACS)Calibur flow cytometer (BD Bioscience). For dual color analysis, the cells were treated with PE-conjugated and fluorescein isothiocyanate (FITC)-conjugated antibodies or isotype-matched control IgGs (1 µg each). The cells were analyzed on FACSCalibur (BD Bioscience).BMMSCs or S-BMMSCs (0.2 × 103/well) were fixed with 4% PFA. The samples were incubated with the specific or isotype-matched mouse antibodies (1:200) overnight at 4°C, and treated with Rhodamine-conjugated secondary antibodies . Finally, chamber slides were mounted using Vectashield mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) .The cells subcultured on eight-well chamber slides (Nunc)  ceramic powders  and subcutaneously transplanted into eight-week-old immunocompromised mice. After eight weeks, the transplants were harvested, fixed in 4% PFA and then decalcified with 5% ethylenediaminetetraacetic acid , followed by paraffin embedding. The paraffin sections were stained with H & E and analyzed by an NIH Image-J. The newly-formed mineralized tissue area from five fields was calculated and shown as a percentage to total tissue area.A total of 4.0 × 10BMMSCs and S-BMMSCs were cultured under osteogenic culture conditions containing 2 mM β-glycerophosphate (Sigma), 100 μM L-ascorbic acid 2-phosphate and 10 nM dexamethasone (Sigma). After induction, the cultures were stained with alizarin red or alkaline phosphatase.For adipogenic induction, 500 nM isobutylmethylxanthine, 60 μM indomethacin, 500 nM hydrocortisone, 10 μg/ml insulin (Sigma), 100 nM L-ascorbic acid phosphate were added to the culture medium. After 10 days, the cultured cells were stained with Oil Red-O and positive cells were quantified by using an NIH Image-J. Total RNA was also isolated from cultures after 10 days induction for further experiments.6 cell pellets were cultured under chondrogenic medium containing 15% FBS, 1% ITS (BD), 100 nM dexamethasone, 2 mM pyruvate (SIGMA), and 10 ng/ml transforming growth factor beta 1 (TGFβ1) in (D)MEM (Invitrogen) for threeweeks. Cell pellets were harvested at three weeks post induction, fixed overnight with 4% PFA and then, sections were prepared for staining.For chondrogenic induction, 1 × 10Extraction of total RNA and RT-PCR were performed according to standard procedures. Primer information is described in Additional materials and methods [see Additional file A total of 20 µg of protein was used and SDS-PAGE and western blotting were performed according to standard procedures. Detailed procedures are described in Additional materials and methods [see Additional file 4/dish, STEMCELL Technologies, Vancouver, BC, V5Z 1B3, Canada) under hematopoietic differentiation medium (STEMCELL Technologies) with or without erythropoietin  for seven days. Whole bone marrow cells and linage negative bone marrow cells (Linage-cells) were used as positive controls. The results are representative of five independent experiments.BMMSCs and S-BMMSCs were cultured onto 35 mm low attach culture dishes (2 × 10S-BMMSCs and BMMSCs were treated with 1 mM L-NG-monomethyl-arginine (L-NMMA)  or 0.2 mM 1400 W  to inhibit total nitric oxide synthase (NOS) or inducible nitric oxide synthase (iNOS), respectively.6/well) were cultured on 24-well plates with or without cytokines  and chemicals  at the indicated concentration and days. The supernatant from each culture was collected and nitric oxide concentration measured using a Total Nitric Oxide and Nitrate/Nitrite Parameter Assay kit (R&D Systems) according to the manufacturer's instruction.BMMSCs (0.2 × 106 of S-BMMSCs or BMMSCs was seeded on each lower chamber. Activated spleen cells (1 × 106/chamber), which were pre-stimulated with plate-bound anti CD3ε antibody (3 µg/ml) and soluble anti CD28 antibody (2 µg/ml) for two days, were loaded in the upper chambers. Both chambers were filled with a complete medium containing (D)MEM  with 10% heat-inactivated FBS, 50 µM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate (Sigma), 1% non-essential amino acid , 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. To measure the spleen cells viability, cell counting kit-8  was used. For apoptosis of spleen cells analyses, Annexin V-PE apoptosis detection kits I (BD Bioscience) were used and analyzed on FACSCalibur (BD Bioscience).The transwell system (Corning) was used for co-culture experiments. A total of 0.2 × 10+CD25- T-lymphocytes (1 × 106/well), collected using a CD4+CD25+ Treg isolation kit (Miltenyi Biotec), were pre-stimulated with plate-bound anti CD3ε antibody (3 µg/ml) and soluble anti CD28 antibody (2 µg/ml) for two days. These activated T-lymphocytes were loaded on 0.2 × 106 BMMSCs or S-BMMSCs cultures with recombinant human TFGβ1 (2 ng/ml) (R&D Systems) and recombinant mouse IL2 (2 ng/ml) (R&D Systems). For Th17 induction, recombinant human TFGβ1 (2 ng/ml) and recombinant mouse IL6 (50 ng/ml)  were added. After three days, cells in suspension were collected and stained with anti CD4-PerCP, anti CD8a-FITC, anti CD25-APC antibodies (each 1 µg) for 45 minutes on ice under dark conditions. The cells were then stained with anti Foxp3-PE antibody (1 µg) using a Foxp3 staining buffer kit (eBioscience) for cell fixation and permeabilization. For Th17, cells in suspension were stained with anti CD4-FITC  for 45 minutes on ice under dark conditions followed by intercellular staining with anti-IL 17 antibody  using a Foxp3 staining buffer kit. The cells were analyzed on FACSCalibur.CD4+/CD73+ double sorted cells (0.1 × 106 cells/10 g body weight) were infused into MRL/lpr mice via the tail vein at 10 weeks of age (n = 6 each group). In the control group, MRL/lpr mice received PBS (n = 5). All mice were sacrificed at two weeks post transplantation for further analysis. The protein concentration in urine was measured using a Bio-Rad Protein Assay .Under general anesthesia, C3H/HeJ-derived BMMSCs, S-BMMSCs, L-NMMA pre-treated BMMSCs (1 mM for five days), or CD34Peripheral blood serum samples were collected from mice. Autoantibodies, sRANKL and CTX were analyzed by ELISA using commercially available kits  according to their manufactures' instructions. The results were averaged in each group. The intra-group differences were calculated between the mean values.6) were treated with PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8a, APC-conjugated anti-CD25 antibodies, and stained with R-PE-conjugated anti-Foxp3 antibody using a Foxp3 staining buffer kit (eBioscience). To measure Th17 cells, PBMNCs (1 × 106) were incubated with PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8a, followed by treatment with R-PE-conjugated anti-IL-17 and APC-conjugated anti-IFNγ antibodies using a Foxp3 staining buffer kit. The cells were then analyzed on FACSCalibur .To detect Tregs, peripheral blood mononuclear cells (PBMNCs)  from bone marrow were plated onto regular plastic culture dishes for two days and all non-attached cells were subsequently transplanted into immunocompromised mice subcutaneously using HA/TCP as a carrier. At eight weeks post-transplantation, newly formed bone was identified in the transplants by H & E staining  could capture higher numbers of CFU-Fs when compared to plastic cultures [see Additional file To determine whether a subset of BMMSCs remain in culture suspension, ANCs  and lipoprotein lipase (lpl) , mineralized nodule accumulation under the osteogenic inductive cultures, and bone regeneration when transplanted into immunocompromised mice using HA/TCP as a carrier Figures  and 2B.  Figures  and 2D.  Figures . Collect+ S-BMMSCs co-expressed BMMSC-associated markers CD73 or Octamer-4 (Oct4), as evidenced by flow cytometric analysis  IgG and IgM antibodies  in peripheral blood when compared to BMMSCs  suggesting that mouse S-BMMSCs probably lose their immunomodulation property under long culture expansion.Since the immunomodulation property of MSCs is one of the essential factors for MSC characterization, allogenic S-BMMSC transplantation into MRL/d Figure . Two weed Figure  and redud Figure . It apped Figure . As expe Figures  and 4E a Figures  in the p Figures , S-BMMSC Figures  and 4F. n Figure  but S-BMn Figure . Next, f Figures . In addi/lpr mice [see Additional file Furthermore, we showed that S-BMMSCs were superior to BMMSCs in terms of reducing increased numbers of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts in the distal femur epiphysis of MRLin vitro experimental data suggested that NO production is an essential factor for BMMSC-mediated immunomodulation.Recently, immunomodulatory properties were identified as an important stem cell characteristic of BMMSCs, leading to the utilization of systemic infused BMMSCs to treat a variety of immune diseases -21. Here+CD25+Foxp3+ Tregs is required for immunotolerance [in vitro co-culture system. When naïve-T-cells were co-cultured with S-BMMSCs or regular BMMSCs in the presence of IL-2 and TGF-β1, S-BMMSCs showed a significant up-regulation of Treg levels compared to regular BMMSCs . These data indicate that endogenous S-BMMSCs could be isolated from bone marrow using CD34 and CD73 antibodies double sorting.In order to identify whether there are functional endogenous S-BMMSCs, we used fluorescence activated cell sorting (FACS) to isolate CD34 and CD73 double-positive cells from bone marrow ANCs which resulted in the recovery of 3.77% double-positive cells [see Additional file Additionally, we used the same BMMSC-ECM isolation approach to reveal the existence of human S-BMMSCs (hS-BMMSC) that possess stem cell properties including multipotent differentiation and self-renewal but lack expression of CD34 (data not shown). hS-BMMSCs showed elevated NO and kynurenine production which indicate high indoleamine 2,3-dioxygenase (IDO) activity when compared to regular BMMSCs [see Additional file Adherent BMMSCs are able to proliferate and undergo osteogenic differentiation, providing the first evidence of CFU-F as precursors for osteoblastic lineage . For ove1 (CD29), CD44, Thy-1 (CD90), CD71, vascular cell adhesion molecule-1 (CD106), activated leukocyte cell adhesion molecule (CD166), STRO-1, GD2, and melanoma cell adhesion molecule (CD146) [M (CD11b) and CD14. However, recent studies have implied that mouse BMMSCs might express the hematopoietic surface molecules, CD45 [+ S-BMMSCs for proliferation and differentiation assays in this study. Interestingly, previous experimental evidence appeared to support a notion that HSCs are capable of differentiating into mesenchymal cells [in vivo [Due to the heterogeneity of the BMMSCs, there is no single, unique marker allowing for BMMSC isolation, rather an array of cell molecules are utilized to profile BMMSCs. It is widely accepted that BMMSCs express SH2 CD105), SH3/SH4 CD73), integrin β, SH3/SH43, integr (CD146) ,31,32. Nes, CD45  and CD34es, CD45 . To ensu[in vivo . Thus, iin vitro and failed to rescue lethally-irradiated mice. The mechanism that may contribute to the up-regulated immunomodulatory function was associated with high NO production in S-BMMSCs and a NO-driven high Tregs level [In this study, we have identified a novel subset of S-BMMSCs that failed to form adherent CFU-F in regular culture dishes, but were capable of adhering on mesenchymal stem cell-produced ECM and differentiating into osteoblasts, adipocytes and chondrocytes from both C3H/HeJ and C57BL/6J mice. S-BMMSCs co-expressed the HSC marker CD34 with the MSC markers CD73 and Oct4, excluding the potential of HSC contamination. Furthermore, S-BMMSCs were found to be distinct from HSC because they lacked the ability to differentiate into hematopoietic cell lineages gs level . NO is ags level .This is the reason that S-BMMSCs showed a superior therapeutic effect in treating SLE mice.+ mesenchymal stem cell population including platelet derived growth factor receptor-β (CD140b), human epidermal growth factor 2/ErbB2 (CD340) and frizzled-9 (CD349) [-CD45-CD31-Sca-1+ [ex vivo expansion to enrich cell numbers for tissue regeneration or systemic therapies by plastic adherent assay. In addition to identifying a novel sub-population of BMMSCs that possess enhanced immunomodulatory properties when compared to regular BMMSCs, we showed that CD34+/CD73+ BMMSCs could be isolated directly from whole bone marrow and that CD34+/CD73+ BMMSCs are endogenous S-BMMSCs with higher NO production, and are superior in treating SLE-like mice when compared to regular BMMSCs.One successful approach is to isolate cells that express specific molecules on their cell surfaces using monoclonal antibodies and cell sorting technologies. Enriched populations of BMMSCs have been isolated from human bone marrow aspirates using a STRO-1 monoclonal antibody in conjunction with antibodies against VCAM-1/CD106 , CD146 , low aff (CD349) . Further1-Sca-1+ . Another1-Sca-1+ . All appin vitro, suggesting the presence of non-adherent BMMSCs in primary CFU-F cultures [in vivo for specific functional needs. Added evidence indicated that osteocalcin-positive cells in circulation were able to differentiate into osteoblastic cells when cultured in the presence of TGFβ [Recently, non-adherent bone marrow cells (NA-BMCs) were identified ,45. The cultures , suggest of TGFβ . HoweverA new subset of BMMSCs (S-BMMSCs) which failed to adhere to culture dishes possesses similar stem cell properties as those seen in BMMSCs, including CFU-F, stem cell markers, osto-, adipo-, and chondro-genic differentiation. However, S-BMMSC showed distinct features including expression of CD34 and a superior immunomodulation property through high NO production. These findings suggest that it is feasible to improve immunotherapy by identifying new subset BMMSCs.7AAD: 7aminoactinomycineD; ALP: alkaline phosphatase; ANCs: all nucleated cells; BMMSCs: bone marrow mesenchymal stem cells; BrdU: bromodeoxyuridine; CFU-F: colony forming unit fibroblastic; CTX: C-terminal telopeptides of type I collagen; DAPI: 4', 6-diamidino-2-phenylindole; (D)MEM: (Dulbecco's) modified Eagle's medium; ECM: extracellular cell matrix; ELISA: enzyme-linked immunosorbent assay; EPO: erythropoietin; FACS: fluorescence-activated cell sorting; FBS: fetal bovine serums; FITC: fluorescein isothiocyanate; H & E: hematoxylin and eosin; HA/TCP: hydroxyapatite/tricalcium phosphate; HSC: hematopoietic stem cell; IDO: indoleamine 2,3-dioxygenase; IFNγ: interferon gamma; IgG: immunoglobulin G; IL-1β: interleukin-1 beta; iNOS: inducible NOS; L-NMMA: L-NG-monomethyl-arginine; lpl: lipoprotein lipase; NF-κB: nuclear factor-kappa B; NOS: nitric oxide synthase; PBMNCs: peripheral blood mononuclear cells; PBS: phosphate-buffered saline; PE: phycoerythrin; PFA: paraformaldehyde; pparγ2: peroxisome proliferator-activated receptor gamma 2; RT-PCR: reverse transcriptase polymerase chain reaction; S-BMMSC: BMMSCs in suspension; SLE: systemic lupus erythematosus; SP: spleen; sRANKL: soluble runt-related NF-κB ligand; SSEA: stage-specific embryonic antigen; TGFβ: transforming growth factor beta; Th17: T helper 17 cells; TRAP: tartrate-resistant acid phosphatase; Tregs: regulatory T cells.The authors declare that they have no competing interests.KA and YY: contributions to conception and design of experiments, acquisition of data, analysis and interpretation of data. TY, CC, LT, and YJ: contributions to acquisition of data, analysis and interpretation of data. XC and SG: contributions to drafting the manuscript and revising critically. SS: contributions to conception and design, drafting the manuscript, and giving final approval of the version to be published. All authors have read and approved the manuscript for publication.Figures S1 to S8 and Additional materials and methods. Figure S1. ECM coated dish could capture a greater number of CFU-F. CFU-f number in ECM coated dish compared to regular dish. Figure S2. -CD34-BMMSCs showed similar property with S-BMMSCsCD45. (A) CFU-f number. (B) Flow cytometric analysis. Figure S3. S-BMMSCs extended survival rate of lethal dose of irradiated mice. The life span of irradiated mice. MRL/lpr miceFigure S4. Osteoclast activity in S-BMMSC-treated . (A) Osteoclast number. (B) sRANKL level. (C) CTX level. MRL/lpr miceFigure S5. L-NMMA pre-treated BMMSC transplantation failed to ameliorate disease phenotype of . (A) Anti dsDNA (IgG) level. (B) Anti dsDNA (IgM) level. (C) Urine protein level. (D) Tregs level. (E) Th17 level. (F) Ratio between Tregs/Th17. Figure S6. Inhibition of NO production in BMMSCs. (A) NO level with inhibitors. (B) iNOS level by western blot. Figure S7. Endogenous S-BMMSCs in mice bone marrow. (A) Cell sorting result. (B) CFU-f number. (C) Osteogenic differentiation in vitro. (D) NO level. Figure S8. Human bone marrow contains S-BMMSCs (hS-BMMSCs). (A) NO level. (B) Kynurenine production. (C) Kynurenine production in co-culture system. (D) T cell apoptosis induction by hS-BMMSCs. Additional materials and methods describe about TRAP staining, Histomotry, Rescue lethal dose irradiated mice, and Isolation of CD34+CD73+ double positive cells.Click here for file


Chemokines are involved in multiple aspects of pathogenesis and cellular trafficking in tumorigenesis. In this study, we report that the latest member of the C-X-C-type chemokines, CXCL17 (DMC/VCC-1), recruits immature myeloid-derived cells and enhances early tumor progression.in vitro, but CXCL17-expressing NIH3T3 cells could form vasculature-rich tumors in immunodeficient mice. Our data showed that CXCL17-expressing tumor cells increased immature CD11b+Gr1+ myeloid-derived cells at tumor sites in mice and promoted CD31+ tumor angiogenesis. Extensive chemotactic assays proved that CXCL17-responding cells were CD11b+Gr1highF4/80− cells (∼90%) with a neutrophil-like morphology in vitro. Although CXCL17 expression could not increase the number of CD11b+Gr1+ cells in tumor-burdened SCID mice or promote metastases of low metastatic colon cancer cells, the existence of CXCL17-responding myeloid-derived cells caused a striking enhancement of xenograft tumor formation.CXCL17 was preferentially expressed in some aggressive types of gastrointestinal, breast, and lung cancer cells. CXCL17 expression did not impart NIH3T3 cells with oncogenic potential These results suggest that aberrant expression of CXCL17 in tumor cells recruits immature myeloid-derived cells and promotes tumor progression through angiogenesis. The tumor microenvironment is the result of an extremely complex series of biological events that depend on tumor cell interaction with the responding host cells + monocytes and CD11c+ immature dendritic cells (DCs) from human peripheral blood +Gr1+ myeloid-derived cells at tumor sites and promotes angiogenesis and tumorigenesis. Moreover, some human tumor cells expressed CXCL17, and a xenograft tumor model using SCID mice showed rapid tumor formation with rich microvasculature. Therefore, we propose that the aberrant expression of CXCL17 in tumor cells may play a pivotal role in tumor progression.Chemokines are involved in the growth and progression of many tumor types, although they were previously regarded mainly as indispensable chemotactic cytokines for immunity and inflammation G12V-transformants gave rise to numerous and large colonies. The level of proliferation of mCXCL17-3T3 cells was not altered relative to that of LacZ-expressing NIH3T3 (LacZ-3T3) cells under regular adherent culture conditions (data not shown). However, subcutaneous injection of mCXCL17-3T3 cells into nude mice showed more rapid tumor formation than that produced by LacZ-3T3 cells  cells formed tumors in athymic nude mice T3 cells . The higactivity . Moreovehttp://www.broadinstitute.org/ccle/home). Moreover, preliminary immunohistochemical analyses showed that CXCL17 was positive in some clinical specimens    and S3A.rcinoma) . Thus, f3) (in vivo through an increase in tumor vessels.We further investigated whether CXCL17 expression could induce rapid tumor growth of CXCL17-negative DLD-1 colon cancer cells in SCID mice. Mouse CXCL17-expressing DLD-1 (CXCL17-DLD-1) cells were generated and injected into the subcutaneous space of SCID mice . Strikin3) . These rWhether CXCL17 expression affects the metastatic potential of tumor cells is of immense interest in tumor biology. While representative CXCL17-positive colon cancer cells such as HT-29, KM12, and Colon-205 cells showed hematogenous distant metastases with respect to their own properties see , CXCL17-+Gr-1+ cells had increased in number at tumor sites  are sensitive to CXCL17.Furthermore, in an effort to identify CXCL17-responding cells, FACS analysis was performed using migrated cells by recombinant mCXCL17. Responding cells were predominantly CD11b-positive and Gr-1-positive (93%), but F4/80-negative . Similarng cells . Thus, t+ vasculatures in the CXCL17-SW620 tumor also increased in number in the presence of CXCL17-responding myeloid-derived cells (We further addressed whether CXCL17-responding myeloid-derived cells could promote tumorigenesis of CXCL17-expressing tumor cells. Migrated myeloid-derived cells in the CXCL17-dependent manner were collected from the lower chamber of the chemotaxis assay (4-hr exposure), and these cells together with CXCL17-expressing SW620 cells were injected subcutaneously into SCID mice . In comped cells . Thus, ted cells , VEGF-A in vivo and formed tumors, 2) some of the human cancer cells expressed CXCL17, 3) they recruited immature CD11b+Gr-1+F4/80− myeloid-derived cells, and 4) the recruited myeloid-derived cells by CXCL17 could promote tumor growth in vivo.Herein, we have demonstrated the potential role of chemokine CXCL17 in tumor formation. The remarkable features presented in this study include: 1) CXCL17-producing cells increased angiogenesis While CXCL17 was identified as a potential chemokine to human immature DCs and monocytes We could successfully monitor tumor angiogenesis using Doppler-based high-resolution ultrasonography  without +Gr-1+ myeloid-derived cells at tumor sites and was coupled with enhanced tumorigenicity and increased vasculature in vivo (+) or granulocytic (Ly-6G+), and function as systemic immune suppressors and promoters of tumor angiogenesis et al.in vivo, and tumor growth between CXCL17- and LacZ-expressing Colon26 cells was not segregated. This dichotomy may have resulted from the specific properties derived from the different cell lines. Additionally, it has previously been shown that MDSCs can enter tumors and differentiate into mature macrophages (TAMs) or neutrophils Various types of myeloid cells have been demonstrated to promote tumor progression by direct immune suppression  in vivo . It may Our results and those of other researchers indicate that CXCL17 may have a dual biological role in tumorigenesis. While the migration of immature myeloid cells was induced at lower concentration levels (0.1–0.2 ug/mL), that of endothelial cells needed a high concentration of CXCL17 (1.0–2.5 ug/mL). The latter case resulted from the physiological conditions for a chemokine. Similar endothelial cell activation through CXCL17 was demonstrated by high-efficient adenoviral transduction of CXCL17 cDNA, in which endothelial cells demonstrated increased levels of VEGF-A, basic FGF, and PDGF CXCL17 gene is activated in malignant cells remains unknown, CXCL17 seems to be involved in the complex tumor microenvironment in some human cancers. Further understanding of the properties of CXCL17 expression and homing cells should provide important clues in efforts to elucidate the components of the tumor microenvironment.Chemokines are emerging as key mediators in the recruitment of a number of different cell types to the tumor microenvironment All animal experiments in this study were approved by the Animal Ethics Review Board of Jichi Medical University (#10–132) and Takasaki University of Health and Welfare (#1119), performed in accordance with the institutional guide for laboratory animals, and followed the principles of laboratory animal care formulated by the National Society for Medical Research. All clinical samples were procured and used according to a protocol approved by the Medical Ethics Committee of Gunma University (followed by principles detailed in the Declaration of Helsinki) from the Pathology Archive of Gunma University and its affiliated hospital. All patients provided written informed consent for the use of their tissues, and all patient information associated with this study was obtained in de-identified format.scid/J (NOD/SCID) and Crlj:SHO-PrkdcscidHrhr (CHO) mice (8–12 weeks old) were purchased from Charles River Japan .BALB/c, BALB/c AJcl-nu/nu (BALB/c nude) and C.B-17/Icr-scid/scidJcl (C.B-17 SCID) mice (8–12 weeks old) were purchased from CLEA Japan, Inc. , and NOD C.B-17-Prkdc2 and 95% air at 37°C.NIH3T3 cells and all human cancer cell lines were obtained from the American Type Culture Collection  and the Health Science Research Resources Bank (HSRRB) . Cells were maintained in Dulbecco’s modified Eagle’s medium , RPMI1640 medium (Sigma-Aldrich), McCoy’s 5A medium , or Leibovitz L-15 medium (Sigma-Aldrich), supplemented with 10% heat-inactivated fetal calf serum (FCS) and additional components appropriate for each cell line Anti-mouse CXCL17  antibody was obtained from R&D Systems . Anti-human CXCL17  and anti-CD31 antibodies were purchased from Santa Cruz Biotechnology . Flow cytometric analysis involved the use of phycoerythrin (PE)-conjugated anti-mouse CD11b mAb, fluorescein isothiocyanate (FITC)-conjugated anti-Gr-1 (Ly-6G) mAb, FITC-conjugated anti-mouse F4/80 mAb, and isotype-matched IgG controls, all of which were purchased from eBioscience .Pertussis toxin (PTX) was obtained from Sigma-Aldrich . Recombinant mouse and human CXCL17 (DMC/VCC1), and recombinant mouse CCL2 (MCP-1) were purchased from R&D Systems and PeproTech , respectively.5′-ATG AAG CTT CTA GCC TCT CCC-3′; mCXCL17 anti-sense: 5′-CTA TAA GGG CAG CGC AAA GCT TGC-3′; hCXCL17 sense: 5′-ATG AAA GTT CTA ATC TCT TCC CTC-3′; hCXCL17 anti-sense: 5′-CTA CAA AGG CAG AGC AAA GCT TCT TAG C-3′. The PCR product was then inserted into the TOPO® PCR Cloning vector . Following confirmation of the nucleotide sequence, the EcoRI cDNA fragments were inserted into the pQCXIN retroviral vector  to generate pQCXIN-mCXCL17 and pQCXIN-hCXCL17, respectively. The retroviral expression plasmids for H-RasG12V  have been previously described Total RNA was extracted from cells using Isogen® . Two micrograms of total RNA were used for first-strand synthesis using SuperScript III™ reverse transcriptase . PCR was then performed using ExTaq polymerase . The full-length encoding sequences of mouse CXCL17  and human CXCL17  were amplified from lung-derived cDNA of mice and human MKN-1 cells using the following respective primers: mCXCL17 sense: 5′-ATG ACG AGG GCC TGG AGT GTG-3′; human VEGF-A anti-sense: 5′-CCT ATG TGC TGG CCT TGG TGA G-3′. PCR conditions for each set of primers included initial treatment at 95°C for 2 min, followed by 35 cycles consisting of denaturation at 95°C for 15 sec, annealing at 57°C for 30 sec, and then extension at 72°C for 2 min. PCR products were analyzed using a 1% agarose gel.For expression analysis, the aforementioned and following primers were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense: 5′-GTA TCG TGG AAG GAC TCA TG-3′; and GAPDH anti-sense: 5′-AGT GGG TGT CGC GCT GTT GAA G-3′; human VEGF-A sense: G12V cDNA, and the establishment of firefly  luciferase-expressing cells has been previously described To generate CXCL17-expressing cells, GP2-293 packaging cells (Clontech) were cotransfected with pQCXIN-mCXCL17 and pVSV-G (Clontech), a plasmid encoding the viral envelope glycoprotein (VSV-G) of the vesicular stomatitis virus, using Lipofectamine 2000 (Invitrogen). Supernatants from transfected GP2-293 were incubated with ∼50% confluent cells in the presence of polybrene . Transduced cells were propagated and maintained in medium containing G418 geneticine  or puromycin . Similar retroviral transductions were conducted for hCXCL17, LacZ, and H-Ras4) in DMEM containing 10% FCS were suspended in 0.4% agarose . The cell suspension was then layered onto a solidified 0.53% agarose bottom layer containing 10% FCS-DMEM in a 35-mm plate and incubated at 37°C and 5% CO2 for 14–21 days until evaluation. Each assay was performed in duplicate and repeated three times with similar results.For soft agar colony formation assays, cells (1×105 per well of a 12-well plastic plate in triplicate) were seeded and incubated for the indicated time points, and then adherent cells were counted using a hemocytometer with trypan blue exclusion.For cell proliferation assays, cells . Plasmid DNA was transfected into HT-29 cells and stable CXCL17-knockdown cells were established in medium containing puromycin . Several clones were isolated per target sequence and CXCL17 expression levels were monitored by RT-PCR. Notably, cell proliferation properties did not alter among parental and transfected cells (data not shown).6) were washed with PBS and incubated with mAb for 30 min at 4°C. Following washing with 0.1% FCS-PBS, cells (5×104) were analyzed using FACSCalibur  and FlowJo analysis software .For the flow cytometric analysis, cells  compound . Frozen tissue sections (5 um) were probed with anti-CD31, followed by appropriate Alexa Fluor 594-labelled secondary antibodies . In sections stained with anti-CD31 antibody, CD31-positive tubular structures within the tumor were considered blood vessels. CD31-positive vessels were counted in five random sections.in lieu of the primary antibody, and those not treated with the primary antibody, were utilized as negative controls.Immunohistochemical analyses used xenografts of HT-29 (colon cancer), NCI-H460 , and A549  cell lines. These were obtained from the American Type Culture Collection . Additionally, surgically resected specimens from human lung, breast, and colon cancer  were examined. Tissue samples were fixed with 4% formaldehyde for one day and then embedded in paraffin routinely. Three-um-thick sections were prepared and stained with hematoxylin and eosin (H&E). Adjacent sections were reacted with primary antibody to human CXCL17  overnight at 4°C. For coloration, a commercially available biotin-streptavidin immunoperoxidase kit  and 3,3′-diaminobenzidine were employed, and counterstained lightly with hematoxylin. Prior to immunostaining, specimens required antigen retrieval by autoclaving  in 10 mM citrate buffer (pH 6.0). Specimens incubated with normal rabbit IgG (2 ug/ml) 6) were injected into the subcutaneous space of mice (the hind limb or the abdomen) Cells in exponential growth phase were harvested by trypsinization and washed twice in PBS prior to injection. C.B-17 SCID mice were treated by injection of anti-asialo GM1 Abs  into the peritoneal cavity one day prior to the operation. For the subcutaneous injections, cells (1 × 10In vivo tumor progression at the skin was examined using the Vevo770 high-resolution ultrasound (US) system with contrast mode software . During the US imaging session, the animals were anesthetized with a 2%/98% isoflurane/oxygen mixture, and body temperature was monitored and maintained at 37°C using a warming plate. Coupling gel was applied to the tumor area, and 3-dimensional (3D) mode and power Doppler scout images established a field of view with vascularity present. To enhance vessel imaging, liposomal microbubbles  6 cells/24 well-plate or 5×106 cells/6 well-plate) were placed on top of the 3-um pore size filters in duplicate, whereas 0.2% BSA-containing RPMI1640 with and without chemokines were placed into the lower chamber (exposed at the indicated concentrations). Following 4 hr at 37°C, migrated cells that had fallen to the bottom of the plate were counted using a hemocytometer and their phenotypes were analyzed by a flow cytometer. PTX treatment occurred at a concentration of 100 ng/ml for 2 hr, and heat-inactivation of recombinant CXCL17 was performed at 95°C for 1 min. Two or three independent experiments were performed with similar results.Spleen cells isolated from mice were used for chemotaxis assays. Cells . Differences between groups were considered significant if P<0.05.P values based on the two-sided Student’s Figure S1Cell morphology of allografts derived from CXCL17-expressing NIH3T3 cells. Specimens from allografts were stained using H&E . Transplanted cells are indicated in each panel.(TIF)Click here for additional data file.Figure S2CXCL17 mRNA expression in various human cell lines. (A) Analysis of human CXCL17 mRNA expression in human cancer cell lines using RT-PCR. Upper panel, CXCL17; lower panel, GAPDH as an internal control. Cell line names are indicated on each panel.(TIF)Click here for additional data file.Figure S3Immunohistochemistry for CXCL17 in the xenograft tumor and in surgically resected specimens from human lung adenocarcinoma. (A) Immunohistochemistry for CXCL17 in the xenograft tumor of A549 and NCI-H460 cells. (B) Surgically resected lung cancers were probed with anti-human CXCL17 antibodies. Magnification is x 100. The brown color  indicates positive staining. Left panels, H&E staining; middle panels, staining with normal rabbit IgG; right panels, anti-human CXCL17 IgG.(TIF)Click here for additional data file.Figure S4Enhanced tumor formation in CXCL17-expressing SW620 colon cancer cells. (A) CXCL17-transfected SW620 cells (1×106) were transplanted into the subcutaneous space of C.B-17 SCID mice and tumor volume was measured at the indicated time points by Vevo770. (B) Blood flow analysis using Vevo770 Doppler-based ultrasound imaging analysis in CXCL17-SW620 cells. It is worth noting that tumors of CXCL17-expressing SW620 cells showed increased blood flow signals in the early tumorigenic stage, but not those of control LacZ-SW620 cells. (C) Stronger blood flow signals in CXCL17-SW620 tumors compared to LacZ-SW620 tumors at the equivalent tumor volume (75 mm3).(TIF)Click here for additional data file.Figure S5Metastatic potential in CXCL17-expressing human cancer cell lines. (A) Luciferase-expressing colon cancer cells were injected into the left ventricle of NOD/SCID mice. Metastatic growth was monitored by tumor-derived photons in vivo. Representative images around 30–40 days following tumor injection are shown. It is worth noting that DLD-1, SW620 and HCT-15 cells were less metastasized than HT-29, KM-12 and Colo205 cells. (B) Ex vivo luminescent inspection of metastasized organs after intra-cardiac injection of tumor cells. (C) Luciferase and CXCL17 doubly expressing DLD-1 cells were injected into the portal vein of SCID mice and then examined by in vivo luminescent imaging.(TIF)Click here for additional data file.Figure S6Increase in CXCL17-responding cells in tumor-bearing BALB/c mice. Parental (unmanipulated) Colon26 cells were transplanted subcutaneously into BALB/c mice, and tumor-bearing conditions in BALB/c mice conditions were generated . (A) Spleen cells were isolated from the indicated mice and the number of CD11b+Gr-1+ cells was determined by a flow cytometer. It is interesting to note that CD11b+Gr-1+ cells in BALB/c Tumor increased more than in naïve tumor-free mice. (B) Spleen cells were isolated from the indicated mice and used for the chemotaxis assay using mouse recombinant CXCL17. *, P<0.001 (n = 4). One of two independent experiments with similar results is shown.(TIF)Click here for additional data file.Figure S7Flow cytometric analysis of CXCL17-responding cells. (A) Spleen cells were isolated from SCID mice and used for the chemotaxis assay using human recombinant CXCL17. (B) Human recombinant CXCL17-responding cells were stained with PE-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse F4/80 and FITC-conjugated anti-mouse Gr-1 mAbs, and then analyzed using flow cytometry. Isotypes, staining with PE-conjugated and/or FITC-conjugated isotype-matched control IgGs; Upper chamber, remaining cell population; lower chamber, responding cell population. (C) Differential population of CXCL17-responding cells from CCL2-responding cells. The dotted rectangle represents the major population preferentially responding to the indicated chemokine.(TIF)Click here for additional data file.Figure S8Enhanced migration of HUVECs with CXCL17. (A) RT-PCR analysis of VEGF-A mRNA expression in HUVECs following recombinant hCXCL17 treatment. HUVECs were exposed to CXCL17 at the indicated concentrations for 10 hr. Upper panel, VEGF-A; lower panel, GAPDH as an internal control. (B) Migration assay of HUVECs in the presence of recombinant hCXCL17. Lines represent reference points for the scratch with a pipette tip. One of two independent experiments with similar results is shown.(TIF)Click here for additional data file.Materials and Methods S1(DOC)Click here for additional data file.


Background. The disruption of physiologic vascular smooth muscle cell (VSMC) migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2). Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy. AGFP protein detectable in coprimary culture of infected rat VSMCs confirmed an expression ratio exceeding 90%  via Boyden chamber for 48 hours. To gain mechanistic insight concerning BMP-2-mediated VSMC migration facilitation, microarray analyses were performed to determine global gene expression changes in BMP-2 overexpressing VSMCs. In VSMCs overexpressing BMP-2, 554 genes were downregulated, and 437 genes were upregulated. Not surprisingly, genes involved in the BMP signaling pathway  were among those upregulated during BMP-2 overexpression. Although our gene chip array determined myosin Va was consistently upregulated during BMP-2 overexpression, myosin Vb and Vc were not detected. In addition, expression of smooth muscle alpha-actin, a characterizing marker of the systole phenotype, remained unchanged. The mRNA and protein levels of OPN and MGP, characterizing markers of the diastolic phenotype, were markedly altered Figures .Via RT-PCR, we determined adenoviral-mediated BMP-2 overexpression significantly increased myosin Va mRNA sevenfold compared to control , in a doWe generated three myosin Va-specific siRNA constructs. Compared to control or other generated siRNA constructs, siRNA-construct-3 (henceforth termed siRNA-3) significantly reduced the expression of both myosin Va mRNA  and protμM U0126  analysis revealed the relationship and interaction between BMP-2, Erk1/2, myosin Va, and actin . We inveμM U0126 . U0126 aμM U0126 . Togethe2+) plays a pivotal role in physiological biochemistry, acting as a second messenger in signal transduction pathways, involved with neurotransmitter release, all muscular cell type contraction, and fertilization [2+ in the signaling mechanism between BMP-2 and Erk1/2 by recording intracellular [Ca2+]i oscillations within VSMCs. After loading the Ca+-sensitive dye Fluo-4 AM, we monitored the time-dependent change of [Ca2+]i within a colony's individual cells. 2+ oscillation. Without external Ca2+ supplementation, prolonged BMP-2 administration activated a large transient increase in Ca2+, followed by a burst of Ca2+ spikes. The average amplitude of spontaneous oscillations increased intracellular Ca2+ concentration ([Ca2+]i) (F/F0) plotted as a function of time demonstrates oscillatory changes in [Ca2+]i. Such [Ca2+]i oscillations significantly increased in cells subjected to 250 ng/mL BMP-2  . IndividmL BMP-2 . These d2+ involvement.We have made several important observations in the current study. Firstly, we demonstrated BMP-2 overexpression in VSMCs augments both unicellular and multicellular population motility. Secondly, we demonstrated BMP-2 overexpression increases the expression of myosin Va but not myosin Vb or Vc. Finally, we provide mechanistic evidence of ERK1/2 involvement in the modulation of myosin Va expression by BMP-2, with CaMyosin Va directly assembles actin into antiparallel bundles , 27 loca2+, but previous reports have not revealed any direct relationship between BMP-2 and myosin Va i oscillations in cultured VSMCs, further activated in the presence of BMP-2. The Erk signaling pathway can be stimulated by increased [Ca2+]i oscillations, augmenting Erk1/2 phosphorylation [2+ as a second messenger in this system are ongoing.Spontaneous calcium oscillations occur in cells originating from excitable tissues of muscular, neuronal, and embryonic stem origin –35. Calc2+ , 25. In rylation , 37, sup in vivo depending upon functional demands and can be contractile, proliferative, migratory, and/or synthetic [α. The present study advances the notion BMP-2 may modulate SMC phenotype towards a synthetic state. Further studies are necessary to determine whether Ca2+ oscillations may have phenotypic implications.VSMCs can change phenotypeynthetic , 39. We In summary, our study demonstrates BMP-2 enhances VSMC migration via Erk1/2 signaling activation, which regulates myosin Va expression. Inhibition of BMP-2-induced myosin Va expression may represent a potential future therapeutic strategy attenuating atherosclerosis.


Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These “orphan” cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI + cell expresses a specific immunity protein that binds to its cognate toxin and inactivates it to prevent cell suicide. Here we identify genes for toxic tips that are not attached to the stick protein. Each of these “orphan” tips has toxic activity, which is blocked by its associated immunity protein. Remarkably, the orphan tips of some bacterial species are often found on the stick proteins in other species, suggesting that cells load and deliver different tips. We also report on a system called Rhs, which encodes another predicted stick-like protein that also carries variable tips. We found that the tips of Rhs proteins are toxic and that Rhs systems encode immunity proteins that specifically block toxin activity. CDI and Rhs toxin tip diversity may represent a microbial arms race, driven by the competition for environmental resources.Recent work from our laboratories has shown that many bacteria express contact-dependent growth inhibition (CDI) systems in which stick-like proteins on the cell surface deliver toxic tips into target cells. Over 60 distinct toxic tips have been identified in bacteria, and our data indicate that each CDI Myxococcus xanthusEscherichia coli isolate EC93 Dickeya dadantii 3937 cdi locus also encodes a small CdiI immunity protein immediately downstream of cdiA. CdiI protects EC93 cells from CdiA-mediated growth autoinhibition Many bacteria lead social lives in communities where they cooperate and compete with members of their own species, as well as those of other species Neisseria and Moraxella species to over 5,600 residues for some Dickeya and Pseudomonas strains Dickeya dadantii 3937 and uropathogenic E. coli (UPEC) 536 possess distinct toxic nuclease activities, and that the corresponding CdiI proteins bind to their cognate CdiA-CT and block nuclease activity both in vitro and in vivoCDI systems are widespread amongst α-, β-, and γ-proteobacteria rhs genes were first identified in E. coli by C.W. Hill and colleagues, and were named rearrangement hotspots based on their role in chromosome duplications CdiA proteins share a number of characteristics with the Rhs protein family. The cdiBAI genes, CDI systems often contain fragmentary gene pairs that resemble cdiA-CT/cdiI toxin/immunity modules. The predicted cdiA-CT fragments generally lack translation initiation signals but encode the VENN peptide motif that demarcates the CdiA-CT region in full-length CdiA proteins. These “orphan” CdiA-CT proteins possess growth inhibitory activities, which are specifically neutralized by the corresponding orphan CdiI immunity proteins. Moreover, we show that the orphan cdiA-CT/cdiI region is actively transcribed in E. coli EC93. Although the orphan CdiA-CT does not appear to be synthesized, functional orphan CdiI immunity protein is produced in EC93. We also show that the Rhs systems of D. dadantii 3937 encode toxin/immunity pairs. Rhs-CTs from D. dadantii 3937 inhibit cell growth when expressed in E. coli, and this toxic activity is specifically neutralized by the cognate RhsI protein encoded immediately downstream. These results suggest that Rhs constitutes another class of cell-surface proteins involved in intercellular competition, and that orphan CT/immunity modules may represent a reservoir of toxin/immunity diversity for both CDI and Rhs systems.Here we show that many CDI systems have an unusual genetic organization similar to that described for some Rhs loci cdi locus in E. coli EC93 revealed two short open reading frames (ORFs) immediately downstream of the cdiI immunity gene , cdiA-CTo3PB1(II) and cdiA-CTo4PB1(II)) share significant regions of homology with the upstream cdiAPB1(II) gene. The extent of these homologous regions varies between orphans, but the homology to full-length cdiAPB1(II) is limited to regions upstream of the VENN encoding sequences for cdiA-CTo2PB1(II) and cdiA-CTo3PB1(II) (cdiA-CTo1PB1(II) gene shares no significant identity with the full-length cdiAPB1(II) gene beyond the sequence encoding the VENN peptide (cdiA-CToPB1(II) genes are unrelated to one another, but have homology to cdiA genes and cdiA-CT fragments from other bacteria. The predicted orphan CdiA-CTo1PB1(II) (UniProt B2K3A6) is related to CdiA-CTs from E. coli A0 34/86  and Enterobacter cloacae subsp. cloacae ATCC 13047 , as well as to orphan CdiA-CTs from Dickeya zeae Ech1591  and Citrobacter rodentium ICC168 . Orphan CdiA-CTo2PB1(II) (B2K3A4) is related to the CdiA-CT from Serratia proteamaculans 568 . Orphan CdiA-CTo3PB1(II) (B2K3A2) is related to a CdiA-CT from Erwinia amylovora CFBP1430  and to orphan CdiA-CTs from E. coli EC869  and Neisseria meningitidis MC58 . Finally, orphan CdiA-CTo4PB1(II) (B2K3A0) is related to the CdiA-CT encoded by the adjacent cdiAPB1(II) gene (39% identity over 179 residues), and also to CdiA-CTs from Klebsiella pneumoniae 342  and Dickeya dadantii Ech586 .Examination of CDI regions in other bacteria shows that orphan de motif . These odiI gene . Three oPB1(II) . In cont peptide . DownstrY. pseudotuberculosis PB1/+ and Neisseria meningitidis FAM18, have well-ordered arrays of orphan cdiA-CT gene fragments downstream of cdiBAI, other species have more complex genomic arrangements. Klebsiella variicola At-22 and Erwinia pyrifoliae DSM 12163 both contain cdiA-CT fragments that do not encode VENN and lack associated cdiI genes  to the orphan cdiA-CTo1254 from E. coli strain 254 , the predicted CdiA(I) protein is identical in all fully sequenced Y. pestis strains with the exception of the Microtus 91001 strain. CdiA(I) sequences N-terminal to the VENN motif are essentially identical in Microtus 91001 and other Y. pestis strains, though there is a six amino acid residue deletion within the hemagglutinin repeat region of Microtus 91001. However, following the VENN motif, the CdiA-CT91001(I) of Microtus 91001 diverges from that of other Y. pestis strains  gene  protein from Microtus 91001 contains an orphan CdiA-CT effector domain from the CO92 strain.All sequenced  strains . Pairwis(I) gene , produciY. pseudotuberculosis PB1/+ and IP31758 strains. The main cdiA-CTIP31758(II) and cdiIIP31758(II) sequences of Y. pseudotuberculosis IP31758 are essentially identical to the cdiA-CTo4PB1(II)/cdiIo4PB1(II) orphan module from strain PB1/+  (cdiA-CTo1PB1(II)/cdiIo1PB1(II) module of strain PB1/+ is nearly identical to cdiA-CTo2IP31758(II)/cdiIo2IP31758(II) from IP31758 , and the orphan cdiA-CTo2PB1(II)/cdiIo2PB1(II) from PB1/+ is 98% identical  to a cdiA gene fragment (which lacks the VENN encoding sequence) and its associated cdiI gene in strain IP31758  shared by eotides) . Additio IP31758 . There acdiA-CT/cdiI modules are merely unused, nonfunctional remnants of full-length cdiA/cdiI genes, then there should be no selective pressure to maintain CdiA-CT toxin activity and CdiI immunity function. To determine whether orphan cdiA-CT/cdiI modules encode functional proteins, we characterized the orphan CdiA-CT/CdiI proteins from E. coli EC93. As described above, CdiA-CTo1EC93 and CdiIo1EC93 are very similar to the UPEC 536 CdiA-CTUPEC536/CdiIUPEC536 pair that we have previously characterized o1EC93 and CdiIo1EC93 bind to one another as predicted for a toxin/immunity pair. We introduced a translation initiation signal upstream of the cdiA-CTo1EC93 fragment and co-expressed orphan CdiA-CTo1EC93 with CdiIo1EC93 immunity protein carrying a hexa-histidine (His6) epitope tag at its C-terminus. Ni2+-affinity purification of His6-tagged CdiIo1EC93 under non-denaturing conditions resulted in co-purification of CdiA-CTo1EC93 (data not shown), demonstrating that the two proteins bind each other. Given the similarity between CdiA-CTo1EC93 and CdiA-CTUPEC536, we next tested whether the CdiIUPEC536 and CdiIo1EC93 immunity proteins are able to bind near-cognate CdiA-CTs. His6-tagged CdiI proteins were first separated from their cognate CdiA-CT proteins by Ni2+-affinity chromatography under denaturing conditions. The individual proteins were then refolded and tested for binding to their cognate partners. Refolded CdiA-CTo1EC93 and CdiA-CTUPEC536 were able re-bind to their cognate CdiI proteins  peptide-tagged proteins to the ClpXP protease. We tagged the C-terminus of CdiIo1EC93 with the ssrA(DAS) peptide and co-expressed it with CdiA-CTo1EC93 in E. coli ΔsspB cells. Degradation of tagged CdiIo1EC93 was then initiated by induction of SspB synthesis from a plasmid-borne arabinose-inducible promoter, resulting in growth arrest after approximately 2 hours -tagged CdiIo13937 , confirming that all of these ORFs are present on a single transcript. We next sought to detect the CdiA-CTo1EC93 protein by immunoblot using polyclonal antibodies raised against the homologous CdiA-CTUPEC536 from UPEC 536. Although this antiserum detects CdiA-CTo1EC93 produced from a heterologous expression system in E. coli K-12, we were unable to detect CdiA-CTo1EC93 in whole-cell lysates of EC93 (data not shown).To determine whether orphan nscripts . This andiIEC93 . Additio3 region , indicatcdiA-CTo1EC93 fragment, the EC93 cdiIo1EC93 gene has an initiation codon and a properly spaced Shine-Dalgarno sequence, indicating that the CdiIo1EC93 protein is likely to be synthesized in EC93 cells. To determine if orphan CdiIo1EC93 is indeed expressed, we tested whether EC93 cells are resistant to CDI mediated by the chimeric CdiAEC93-CTo1EC93 protein. Because EC93 cells are CDI+, the chimeric inhibitor strain must itself be immune to EC93-mediated CDI. Therefore, we introduced a cosmid encoding the CdiAEC93-CTo1EC93 chimera into EC93 and used the resulting strain as the inhibitor for these experiments. Wild-type EC93 was not inhibited by EC93 expressing the CdiAEC93-CTo1EC93 chimera, but the EC93 ΔcdiA-CTo1EC93/ΔcdiIo1EC93 strain was inhibited approximately 103-fold (o1EC93 immunity protein was expressed from a plasmid in the EC93 ΔcdiA-CTo1EC93/ΔcdiIo1EC93 cells (o1EC93 immunity protein is produced from the orphan locus in EC93.In contrast to the orphan 103-fold . This gr93 cells . Taken t91001(I) of Y. pestis Microtus strain 91001 (Q74T84) is similar to the C-terminal region of an Rhs/YD-repeat protein from Waddlia chondrophila WSU 86–1044 (D6YTT8)  from Y. pestis Microtus 91001 (27% identity over 48 residues), suggesting this locus encodes a toxin/immunity protein pair. Intriguingly, Rhs-CT sequences are variable and demarcated by a well-conserved peptide motif (PxxxxDPxGL) analogous to the VENN motif in CdiA proteins rhs loci containing numerous “silent cassettes” that resemble CDI orphan modules Y. pseudotuberculosis strains IP31758 and IP32953, the rhs-CT and putative rhsI sequences are completely unrelated to one another, but the surrounding genomic regions are clearly homologous between the strains  is related to the CT region of a putative Rhs repeat protein  from Pseudomonas fluorescens SBW25, and Rhs-CTo23937 (E0SGM2) is related to the CT of a predicted YD-repeat protein  from Dickeya zeae 1591.In the course of our bioinformatic analyses, we found that many CdiA-CT sequences share significant identity with the C-terminal regions of Rhs proteins . For exa(D6YTT8) . The W.  strains . This hohs genes . In addiE. coli rhsA locus may encode a toxin/immunity pair rhs genes from D. dadantii 3937, which contains three full-length rhs genes that we have termed rhsA (Dda3937_01758), rhsB (Dda3937_02773) and rhsC (Dda3937_04312). Each of these rhs genes is closely followed by a small ORF that encodes a possible immunity protein. Additionally, the rhsC locus contains the two orphan rhs-CT gene fragments described above. We first tested RhsB-CT3937 for growth inhibitory function, because this domain contains an HNH endonuclease motif found in other cytotoxic proteins 3937 together with an ssrA(DAS)-tagged version of the putative RhsIB3937 immunity protein in E. coli cells, and found that growth was arrested upon degradation of RhsIB3937-(DAS)  degradation (+sspB cells. Each of the three Rhs-CT/RhsI-(DAS) plasmids were introduced into sspB+ cells expressing individual RhsI proteins, and the transformed cell suspensions plated onto selective media containing L-arabinose to induce Rhs-CT/RhsI-(DAS) synthesis. Transformants carrying the Rhs-CT/RhsI-(DAS) expression plasmid were only obtained if the cells also expressed the cognate RhsI immunity protein  expression construct can be maintained stably in  protein . TherefocdiBAI genes are followed by an array of orphan cdiA-CT/cdiI modules. These arrays can be extensive, with eleven orphan gene pairs in the cdi locus of E. coli EC869. The widespread occurrence of orphan cdiA-CT/cdiI modules in diverse bacterial species argues that these gene pairs confer a selective advantage. Although the majority of orphan cdiA-CT genes lack translation initiation signals, orphan cdiI genes appear to be fully capable of expression. Therefore, bacteria could maintain orphan modules to build a repertoire of immunity genes to protect themselves from neighboring bacteria that express diverse CdiA proteins. Broad range immunity would clearly confer an advantage and our data demonstrate that EC93 does in fact express its orphan CdiI immunity protein. However, if bacteria are collecting immunity genes, then it is unclear why the toxin-encoding cdiA-CT fragments are retained in the process. One model to explain these findings is that orphan modules allow the bacterium to change its toxin/immunity profile by recombination between the highly conserved VENN-encoding sequences. This may have occurred in the cdi locus of Y. pestis Microtus 91001, where a large deletion has loaded an orphan cdiA-CT/cdiI module onto the main cdiA gene. However, such a strategy for generating CdiA-CT diversity could have serious shortcomings. First, although reloading CdiA with a new toxic C-terminal domain would produce a novel weapon, the recombination would also delete cdiI and render the cell susceptible to the original CdiA protein expressed by neighboring bacteria that have not recombined their cdi locus. Second, simple recombination between cdiA and distal orphan cdiA-CT genes would delete all intervening cdiA-CT/cdiI modules. An alternative model is that recombination between cdiA (or rhs) and the orphan regions occurs following tandem duplication of the loci. This mechanism would allow cdiA-CT/cdiI modules to be rearranged without the loss of immunity or genetic diversity, because a copy of the original cdi/rhs system would be present The results presented here show that many CDI systems have a complex genetic organization in which the rhs genes were first described in E. coli, and were originally thought to be rearrangement hot spots because of a recombination event between nearly identical sequences within the rhsA and rhsB genes E. coli RhsA blocks the recovery of stationary phase cells yibA) encoded immediately downstream of rhsAPseudomonas savastanoi pv. savastanoi was found to be associated with bacteriocin activity rhs genes are linked to valine-glycine repeat (Vgr)-encoding genes that are associated with Type VI secretion systems rhs and vgr genes extends to a functional relationship remains to be determined.The results presented here have also revealed a connection between CDI and Rhs systems. The Myxococcus xanthus plays an important role in social motility M. xanthus YD repeat protein has a signal sequence and is presumably exported to the surface. The gene encoding Q1CXS7 (MXAN_6679) is closely followed by a small ORF that is suggestive of an rhsI gene. However, it is not clear that immunity would be required in this signaling pathway. Perhaps this small ORF encodes a peptide that is involved in receiving the signal from the C-terminal region of the YD repeat protein. Recent work on the entomopathogen Pantoea stewartii suggests another function for a class of Rhs-related proteins termed Ucp (for you cannot pass) P. stewartii genome contains seven ucp homologs that encode large proteins  with similar N-terminal regions and variable C-terminal sequences. The Ucp1 protein mediates bacterial aggregation and is a virulence factor required for pathogenicity against the aphid host. It was postulated that Ucp1 and other Ucp proteins function primarily as adhesins and that C-terminal variability is driven by the need to evade host immune responses Although the Rhs systems examined here appear to be growth inhibitory, there are indications that Rhs proteins have other signaling modalities. Youdarian and Hartzell found that an Rhs-related YD repeat protein (Q1CXS7) from Finally, another intriguing example of Rhs signaling has been proposed for teneurins. The teneurin protein family is comprised of four paralogs that are present in all vertebrates www.webact.org) with BLASTN comparisons. The UniProt, GenBank and gene locus accession numbers for each full-length CdiA and Rhs protein discussed in this work are presented in Pairwise sequence comparisons were performed using GCG Gap or pairwise BLAST. Related CdiA-CT, Rhs-CT, CdiI, RhsI and orphan sequences were found using TBLASTN against assembled bacterial genomes, because these regions are often not annotated and thus not represented in the non-redundant protein database. Pairwise comparisons of genomic regions were performed using WebACT  and EC93orph-Spe (ACT AGT GGC ATT AGA TAG CTT ATC TAT TTT TGC5′ - ATC ) (restriction endonuclease sites are underlined), followed by digesting with NcoI and SpeI, and ligation to plasmid pET21S o1EC93 and C-terminally His6-tagged CdiIo1EC93. The EC93 orphan immunity gene was amplified using primers #1658 (5′ - CAA CAA GCA TGC CCC GAC TTT GAG ACC AGA ATA TC) and #1664 (5′ - ATC AGG AGC ATG GTA TAT GAC AAC ATT TAG ATC). The resulting PCR product was digested with SphI and ligated to EcoRV/SphI digested plasmid pBR322 under control of the tet promoter.The orphan cdiA-CT/cdiI modules from D. dadantii 3937 and E. coli EC869 were amplified using 3937orph-Nco (CCA TGG TGG AGA ATA ACT ATC TGA GCA G5′ - AAG ) and 3937orph-Spe (ACT AGT AGG CTG GTA ATC TTC ATA TTC C5′ - TCT ); and EC869orph11-Nco (CCA TGG GCA CAA ACC AGT CTC TGA CCT TCG5′ - ATT ) and EC869orph11-Spe (ACT AGT ACC TTT GCA GCG ACT CAA GGC CAG5′ - TCT ), respectively. The rhs-CT3937/rhsI3937 modules from D. dadantii 3937 were amplified using the following primer pairs: rhsB-Nco (CCA TGG AAA GTA ATT ACG GTT ATG TCC5′ - CAG ) and rhsB-Spe (A CTA GTA ATT TTT CTT GAT TTA TAT TTT ACA AGC5′ -AA); rhs-orph1-Nco (C CAT GGG GTT GGT GGG ATG TCC GC5′ - TC) and rhs-orph1-Spe (ACT AGT GCC ATC AAG GTA TAC AGA AGG5′ - AAA ); and rhs-orph2-Nco (CCA TGG GGC TGG CAG GGG GGC TG5′ - ACC ) and rhs-orph2-Spe (ACT AGT AAC AGC TTT GTA ATA ATC GTG5′ - TTT ). All PCR products were digested with NcoI and SpeI and ligated to pET21S. The D. dadantii rhsI3937 genes were amplified from pET21S constructs using primer pET-Pst (CTG CAG CAG CCA ACT CAG TGG5′ - CGG ) in conjunction with primers: B-NcorhsI (CCA TGG ATA TTG AAA ATG C5′ - TAA ), o1-NcorhsI (CCA TGG ATT CTA GTG ATA AG5′ - TCA ), and o2-NcorhsI (CCA TGG ATG CTG AAC AAT TTG5′ - AAT ). The resulting PCR products were digested with NcoI and PstI and ligated to plasmid pCH450 Orphan DAS-top (5′ - CTA GTG CTG CGA ACG ATG AAA ATT ACT CCG AAA ATT ATG CGG ATG CGT CTT AAT G) and DAS-bot (5′ - GAT CCA TTA AGA CGC ATC CGC ATA ATT TTC GGA GTA ATT TTC ATC GTT CGC AGC A), and ligated to SpeI/BamHI digested plasmid pKAN E. coli hisS gene was cloned into pKAN(DAS) using SacI and SpeI sites. The resulting hisS-(DAS) SacI/BamHI fragment was subcloned into plasmid pTrc99A (Amersham Pharmacia) to generate plasmid pTrc(DAS). All cdiA-CT/cdiI and rhs-CT/rhsI modules were subcloned into pTrc(DAS) using NcoI and SpeI restriction sites. The sspB and sspB(Δ47) genes were amplified using SspB-Nde (5′ - GAG TTA ATC CAT ATG GAT TTG TCA CAG C) in combination with SspBΔ47-Bam (5′ - TGC GGA TCC TTA ATT CAT GAT GCT GGT ATG TTC ATC GTA GGC) and SspB-Bam (5′ - ATA TGA TTG CCA GGA TCC CGC TAT TTT ATT AAG TC), respectively. Both PCR products were digested with NdeI and BamHI and ligated to plasmid pCH410, allowing L-arabinose control of SspB and SspB(Δ47) expression A cassette encoding the ssrA(DAS) peptide tag (AANDENYSENYADAS) was generated from oligonucleotides, cdiA-CTo1EC93/cdiIo1EC93 module from E. coli EC93 was fused to the full-length EC93 cdiAEC93 gene in multiple steps. A fragment of cdiAEC93 (upstream of the VENN encoding sequence) was amplified using primers, #1527 (5′ - GAA CAT CCT GGC ATG AGC G) and #1758 (5′ - CAA GCT CAG CGA GTT ATT CTC AAC CGA GTT CCT ACC TGC CTG). The EC93 orphan module was amplified using primers, #1759 (5′ - CAG GCA GGT AGG AAC TCG GTT GAG AAT AAC TCG CTG AGC TTG) and #1663 (5′ - GGT CTG GTG TCT AAC CTT TGG G). The two products were combined by overlapping-end PCR using primers #1527 and #1663, and the resulting product digested with SphI and AvrII and ligated to plasmid pDAL660Δ1-39 The orphan 6 complexes were overproduced and purified by Ni2+-affinity chromatography as described 2+-nitrilotriacetic acid resin with native elution buffer [20 mM sodium phosphate (pH 7.0) –10 mM β-mercaptoethanol – 250 mM imidazole], followed by dialysis in storage buffer [20 mM sodium phosphate (pH 7.0) – 150 mM NaCl – 10 mM β-mercaptoethanol]. CdiA-CT and CdiI-His6 proteins were separated from one another by Ni2+-affinity chromatography with denaturing buffer [20 mM sodium phosphate (pH 7.0) – 10 mM β-mercaptoethanol – 6 M guanidine-HCl]. Denatured proteins were refolded by dialysis into storage buffer. All purified proteins were quantified by absorbance at 260 nm using the following molar extinction coefficients: CdiA-CTUPEC536, 12,950 M−1 cm−1; CdiIUPEC536, 8,480 M−1 cm−1; CdiA-CTo1EC93, 11,460 M−1 cm−1; and CdiIo1EC93, 11,460 M−1 cm−1. The specificity of CdiA-CT/CdiI-His6 binding interactions was determined by Ni2+-affinity co-purification as described UPEC536 and CdiA-CTo1EC93 proteins was determined as described All CdiA-CT/CdiI-HisE. coli strain CH4180 (×90 ΔsspB) was co-transformed with pTrc(DAS) orphan module constructs and either the SspB or SspB(Δ47) arabinose-inducible expression plasmids. The resulting strains were grown at 37°C with aeration in LB media supplemented with 150 µg/mL ampicillin and 10 µg/mL tetracycline (to maintain plasmids) to mid-log phase and re-diluted into fresh media to an optical density at 600 nm (OD600) of 0.05. After 40 min, SspB or SspB(Δ47) expression was induced by the addition of 0.4% L-arabinose. Cell growth was tracked by measuring the OD600 every 30 min after induction. Cells expressing CdiA-CTo1EC93/CdiIo1EC93-DAS were harvested into an equal volume of ice-cold methanol and RNA extracted as described His probetRNA (5′ - CAC GAC AAC TGG AAT CAC) and 1BAla probetRNA (5′ - TCC TGC GTG AGC AG) as probes.E. coli sspB+ cells expressing RhsIB3937, RhsIo13937 and RhsIo23937 were transformed with plasmids encoding rhs-CT/rhsI(DAS) modules under control of the PBAD promoter E. coli ΔsspB cells were also transformed in the same manner with arabinose-inducible rhs-CT/rhsI(DAS) constructs to confirm that growth inhibition was dependent upon RhsI-DAS degradation.cdiA-CTEC93 region and cdiIEC93 gene were deleted from rifampicin-resistant E. coli strain EC93 (DL3852) using allelic exchange as described cdiA-CTEC93 was amplified using oligonucleotides #1683 (GAG CTC GAA CAT CCT GGC ATG AGC G5′ - CAA CAA ) and #1684 (5′ - CAG CGA GTT ATT CTC AAC AAC AAC TA CGA GTT CCT ACC TGC CTG) (SacI restriction endonuclease site is underlined). Sequence downstream of cdiIEC93 (including the cdiIEC93 stop codon) was amplified using oligonucleotides #1685 (5′ - CAG GCA GGT AGG AAC TCG tag TTG TTG TTG AGA ATA ACT CGC TG) and #1686 (TCT AGA CCC GAC TTT GAG ACC AGA ATA TC5′ - CAA CAA ) (XbaI restriction endonuclease site is underlined). The two PCR products were combined by overlapping-end PCR using primers #1683 and #1686, and the resulting product digested with SacI and XbaI and ligated to suicide vector pRE112 The main cdiA-CToEC93/cdiIoEC93 module was deleted from rifampicin-resistant EC93 in a similar manner. Sequence upstream of cdiA-CTo1EC93 was amplified using oligonucleotides #1714 (GAG CTC GTG AAG GTG GGC TTA CTC AG5′ - CAA CAA ) and #1715 (5′ - CGA CTT TGA GAC CAG AAT ATC TAT TTA CTC AAC AAC AAC TAT TTT CTG TCT AAG) (SacI restriction endonuclease site is underlined). Sequence downstream of cdiIo1EC93 (including the cdiIo1EC93 stop codon) was amplified using oligonucleotides #1716 (5′- CTT AGA CAG AAA ATA GTT GTT GTT GAG TAA ATA GAT ATT CTG GTC TCA AAG TCG) and #1717 (TCT AGA CCC GTA AGT ATG CTT ATC CCA TG5′ - CAA CAA ) (XbaI restriction endonuclease site is underlined). The two PCR products were combined by overlapping-end PCR using primers #1714 and #1717, and the resulting product digested with SacI and XbaI and ligated to suicide vector pRE112 The orphan E. coli strain EPI100 carrying plasmids pWEB-TNC (CDI−), pDAL660Δ1-39 (CdiAEC93), or pDAL879 (CdiAEC93-CTo1EC93 chimera) were grown overnight at 37°C in LB media supplemented 100 µg/mL of ampicillin. Overnight cultures were diluted into fresh medium to OD600 of 0.05, and incubated at 37°C with aeration until the culture reached mid-log phase (OD600≈0.3). The log-phase inhibitor cultures were then mixed with target E. coli cells – CAG18439 pBR322 (no CdiI), pDAL741 (CdiIEC93), or pDAL867 (CdiIo1EC93) – at an inhibitor to target cell ratio of 10∶1. The co-cultures were incubated for 2 hr at 37°C with aeration. Viable target cell counts were determined by serially dilution of the co-cultures into M9 salt solution followed by plating onto LB agar supplemented with 10 µg/mL of tetracycline.o1 expression in EC93, growth competitions were conducted with streptomycin-resistant EC93 (DL6104) carrying pDAL879 (CdiAEC93-CTo1EC93 chimera) as the inhibitor strain. Target strains were rifampicin-resistant EC93 carrying plasmid pBR322 (no CdiI), and rifampicin-resistant EC93 ΔcdiA-CTo1 ΔcdiIo1 cells carrying pBR322 (no CdiI) or pDAL867 (CdiIo1EC93). Growth competitions were conducted as described above except that mid-log phase cells were mixed and co-cultured at an inhibitor to target ratio of 1∶1. Viable target cell counts were determined by serial dilution of the co-cultures into M9 salt solution followed by plating onto LB agar supplemented with 150 µg/mL of rifampicin.To assay CdiIcdiA-CTEC93/cdiIEC93 and cdiA-CTo1EC93/cdiIo1EC93, followed by treatment with RNase-free DNase I (Roche) to remove contaminating chromosomal DNA. RNA (0.5 µg) was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad). A control without reverse transcriptase was also prepared to assess chromosomal DNA contamination. Quantitative PCR was carried out on a Bio-Rad MyiQ single-color real-time PCR detection system using SYBR green supermix. The following primers sets were used for amplification: #1700 (5′ - GGT GAA GGT GGG CTT ACT CA) and #1701 (5′ - TGA TGT GAC AGA GCC AAA GC) for cdiA-CTEC93; #1698 (5′ - TGC TAT GTA CTG TAC TTG GTC) and #1699 (5′ - TAA AGC CTA TGG GAT TCC T) for cdiIEC93; #1647 (5′ - ACT GAC CGC TGA TGA ACT GG) and #1648 (5′ - AGT AGC CGC TTG AAC CTG CAC) for cdiA-CTo1EC93; #1649 (5′ - TGA ACC CAA CAG TCG CTC TTC) and #1650 (5′ - GTC TTC CCC AGC CAG AGG AT) for cdiIo1EC93; #1568 (5′ - TCA CCC CAG TCA TGA ATC AC) and #1569 (5′ - TGC AAC TCG ACT CCA TGA AG) for 16S rRNA. Thermal cycling conditions were: 95°C for 5 min for polymerase activation and collection of experimental well factors and 40 cycles at 95°C for 10 s; 56°C for 30 s and 72°C for 30s followed by a melting curve (55°C to 95°C) to analyze the end product. Data were analyzed using the iQ5 optical system software (Bio-Rad) and exported to Microsoft Excel and Prism 5.0 for further analysis. For each target gene, a standard curve was generated to assess PCR efficiency (E) allowing the expression level (e) to be determined, where (e) = (Etarget)−Ct target/(Eref)−Ct refTotal RNA was isolated from wild-type EC93 and EC93 strains deleted for Figure S1E. coli UPEC 536. A) Pairwise alignment of CdiA-CTo1EC93 and CdiA-CTUPEC536 shows 76% sequence identity. Numbering begins at the Val residue of the conserved VENN motif. B) Alignment of CdiIo1EC93 and CdiIUPEC536 shows 35% sequence identity.The EC93 orphan CdiA-CT/CdiI protein pair is related to the CdiA-CT/CdiI proteins from (TIF)Click here for additional data file.Figure S2cdiA-CT fragments contain varying amounts of cdiA sequence upstream of the region encoding VENN. A) The region II cdi locus from Y. pseudotuberculosis PB1/+. CTo1 to CTo4 designate the orphan cdiA-CToPB1(II) genes numbered according to their position in the locus. Dots above cdiAPB1(II) represent every 500 amino acids of the encoded protein. B) Pairwise alignments of the full-length Y. pseudotuberculosis PB1/+ region II cdiA gene with the linked orphan cdiA-CT sequences. The region where similarity with the full-length cdiAPB1(II) gene begins is shown for each orphan cdiA-CT sequence. Gray shading indicates nucleotide identity, and the numbers correspond to amino acid residues of the full-length CdiAPB1(II) protein. Amino acid sequences are given in one-letter code and asterisks (*) indicate termination codons. C) The nucleotide and predicted amino acid sequences for the VENN-encoding regions are presented. Orphan sequences shaded in gray are identical to that of the full-length cdiAPB1(II) gene.Orphan (TIF)Click here for additional data file.Figure S3Y. pseudotuberculosis PB1/+ cdiA-CT nucleotide sequences. The nucleotide sequences encoding orphan cdiA-CTs from the Y. pseudotuberculosis PB1/+ region II cdi locus were aligned to the full-length cdiAPB1(II) gene. Numbering corresponds to the full-length CdiAPB1(II) protein sequence. Black indicates positions of sequence identity with the full cdiAPB1(II) gene. Sequence identity falls off abruptly after the VENN-encoding region (grey).Multiple sequence alignment of (PDF)Click here for additional data file.Figure S4cdiA-CT fragments. The orphan cdiA-CTo1 of E. coli 254 is related to orphan cdiA-CTo7 of E. coli EC869. The conserved region is shaded gray, and the left and right junctions of the conserved region are presented in detail. Conservation begins at the VENN-encoding regions for cdiA-CTo1254 and cdiA-CTo7EC869 (left junction) and extends through the predicted orphan cdiI genes to the VENN encoding regions of the following orphan cdiA-CT genes (99% sequence identity in 1048 nucleotides). The sequences then diverge immediately after VENN encoding regions cdiA-CTo2254 and cdiA-CTo8EC869.VENN-encoding sequences demarcate orphan (TIF)Click here for additional data file.Figure S5I-CT of Y. pestis Microtus 91001 is unrelated to CdiA-CTs from other Y. pestis strains. Pairwise alignment of CdiA(I) proteins encoded by the region I cdi loci of Y. pestis Microtus 91001 and Y. pestis CO92. Numbers correspond to amino acid residues of the full-length predicted CdiA(I) proteins. Regions of sequence identity are shaded gray and the VENN peptide motif is boxed.The CdiA(TIF)Click here for additional data file.Figure S6cdiA-CT/cdiI modules from D. dadantii 3937 and E. coli EC869 encode functional toxin/immunity pairs. A) Purification of orphan CdiA-CT/CdiI-His6 proteins. CdiA-CTo13937/CdiIo13937-His6 and CdiA-CTo11EC869/CdiIo11EC869-His6 complexes were purified by Ni2+-affinity chromatography under non-denaturing conditions and analyzed by SDS-PAGE. B) Growth curves of E. coli ΔsspB cells expressing orphan CdiA-CT/CdiI-DAS complexes. Degradation of CdiI-DAS proteins was initiated by the addition of L-arabinose to induce SspB synthesis. Control cells express SspB(Δ47), which does not deliver CdiI-DAS proteins to the ClpXP protease. Growth curves with square symbols represent control strains expressing SspB or SspB(Δ47), but not orphan CdiA-CT/CdiI-DAS complexes.Orphan (TIF)Click here for additional data file.Figure S7I-CT of Y. pestis Microtus 91001 is related to an Rhs-CT from Waddlia chondrophila. Pairwise alignment of Y. pestis Microtus CdiA-CT91001(I) (Q74T84) and the C-terminal region of a predicted Rhs/YD-repeat protein from Waddlia chondrophila WSU 86–1044 (D6YTT8). Regions of sequence identity are shaded gray and the PxxxxDPxGL and VENN peptide motifs are boxed.The CdiA(TIF)Click here for additional data file.Figure S8Y. pseudotuberculosis IP31758 and Y. pseudotuberculosis IP32953. Regions of identity are shaded gray. Sequences diverge abruptly after the DPxGL motif (boxed).The C-terminal regions of Rhs proteins are variable. Pairwise alignment of related Rhs proteins from (TIF)Click here for additional data file.Table S1rhs-CT/rhsI orphan modules is indicated for each Rhs system.Homologies between selected CdiA-CT and Rhs-CT sequences. Rhs-CT sequences were identified by BLAST searches using selected CdiA-CTs as the query sequences. The presence of probable (XLS)Click here for additional data file.Table S2Sequence identifiers for CdiA and Rhs proteins. The UniProt, GenBank and locus tag accession numbers are presented for each CdiA and Rhs protein discussed in the text.(XLS)Click here for additional data file.