PWM-Induced B Cell Activation Figure 2. Contribution of TLR ligands to B cell activation. A: Stimulation of human B cells with TLR ligands. CFSE-labelled CD19+ human B cells were stimulated with or without TLR ligands (TLR2 ligand MALP-2, TLR4 ligand LPS (0.1 and 1 mg/ml) or TLR9 ligand CpG ODN 2006 (CpG)) and/or BCR stimuli (5 mg/ml SpA or anti-Ig (aIg)). After 5 days cells were harvested, stained with anti-CD20 and analyzed by flow cytometry. The graphs depict cell survival (percentage of live gated cells; upper right angle) and cell proliferation (CFSE dilution; % proliferating cells of live gated cells (left) where indicated). The experiment shown is representative of n$3 experiments. B–D: Assessment of TLR2 activity in PWM preparations. HEK293 cells were transfected with pTLR2 or lipofectamine alone (Lf). B: non-transfected and pTLR2-transfected HEK293 cells were stained for TLR2 expression with anti-TLR2 mAb or the respective isotype control as indicated. C: HEK293 cells transfected with pTLR2 or Lf only were stimulated with Pam3CSK4 (P3) or PWM (10 mg/ml). After 24 hours cellular supernatants were collected and analyzed for IL-8 secretion. One representative experiment of n$3 experiments is shown. D: HEK293 cells transfected with pTLR2 were pretreated with anti-TLR2 mAb or the isotype control before stimulation with Pam3CSK4 (P3) or PWM (10 mg/ml). IL-8 was quantified in the 24 hour supernatants. E: 16S rDNA PCR. DNA isolation and PCR amplification of bacterial 16S ribosomal DNA from DNA from E. coli (EC), a negative clinical specimen (NC), a positive clinical sample (CS) and the PWM preparation (PW) or the water control (H2O), with an expected PCR fragment size of approximately 900 bp. doi:10.1371/journal.pone.0029806.g002 this lot of PWM (Lot. 0) induced basal IL-8 secretion from non- transfected HEK293 cells. This indicated that the PWM preparation (Lot. 0) contained immune stimulatory components acting independently of TLR2, a result well compatible with conserved PWM responsiveness in TLR2-deficient murine B cells (Fig. 1E). Nevertheless, PWM-induced IL-8 production was almost doubled in the presence of TLR2 expression (Fig. 2C) and, when compared to the isotype control, reduced by approximately 50% by an anti-TLR2 neutralizing monoclonal antibody (mAb) (Fig. 2D). This proved the presence of TLR2-active substances within the PWM preparation. PWM contains bacterial DNA Since TLR9-dependent sensing of bacterial DNA is a potent inductor of B cell proliferation (Fig. 2A) we also analyzed the bacterial DNA content of PWM preparations. To this end, we amplified ribosomal bacterial 16S DNA by PCR. Our results showed that bacterial DNA was detectable in PWM preparation (Lot. 0) (Fig. 2E). Sequence analysis of the 16S rDNA sequence revealed that the sequence corresponds to Propionibacterium acnes with 99% homology. Bacterial DNA might, thus, represent an additional stimulatory PWM component. Neutralization of TLR2 diminishes the proliferative response to PWM in human B cells To assess the relevancy of the TLR2 activity present in the PWM preparations we stimulated human B cells with Pam3CSK4 or PWM in the absence or presence of the TLR2-blocking mAb or the isotype control already used in Fig. 3A. To better visualize TLR2-mediated B cell activity both stimuli were combined with SpA, that we previously identified as an important enhancer of TLR2 recognition in B cells [7]. In contrast to the experiments performed with murine B cells the results revealed an important contribution of TLR2 activity to PWM-induced proliferation of human B cells: the percentage of proliferating B cells was reduced under TLR2-neutralizing conditions in both Pam3CSK4- and PWM-stimulated B cells in the presence and absence of SpA (Fig. 3A). Nevertheless, these data also indicated that other TLR2- independent stimuli are involved in PWM-induced B cell activation. DNAse treatment of PWM reduces the B cell stimulatory capacity of PWM To assess the impact of TLR9 ligands on B cell mitogenicity of PWM preparations we treated PWM Lot. 0 with DNAse and tested these fractions in regards to mitogenic activity on human B cells. Proliferative activity was markedly reduced when compared to B cells stimulated with PWM incubated with reaction buffer but no enzyme (Fig. 3B). By contrast, DNAse treatment of TLR2- active lipopeptide Pam3CSK4 did not alter its stimulatory capacity, nor did addition of heat-inactivated DNAse to the medium affect B cell responsiveness to stimulation (data not shown). We concluded that bacterial DNA contributes to PWM- triggered stimulation of human B cells. PWM enables B cell stimulation with atypical DNA ODN According to a common view DNA ODN lacking the phosphorothioate (PTO) modification and naked bacterial DNA display reduced B cell activity (Fig. 3B) and [20]. Thus, our findings raised the question how the stimulatory effect of the PWM-contained bacterial DNA was conferred. We proposed that the lectin might facilitate bacterial DNA-driven B cell activation. To test this hypothesis we combined PWM with DNA oligodeox- ynucleotides (ODN) that by themselves display comparably low mitogenic activity (Fig. 3B), e.g. a PTO-modified GpC control ODN that lacks the TLR9-stimulatory CpG motif (GpC PTO) and a CpG ODN lacking the PTO modification previously shown to be essential for cellular uptake and activity of CpG ODN [21] (CpG PO). CFSE dilution experiments revealed that these ‘‘less active’’ ODN displayed B cell activity when combined with PWM and synergistically enhanced the mitogenic property of the PWM, independent of prior DNAse treatment. As shown in Fig. 3B the comparably weak proliferative response to PTO-modified GpC ODN was strongly augmented in the presence of PWM; and unmodified phosphodiester CpG ODN gained stimulatory activity and augmented PWM-induced proliferation. Altogether, these data demonstrated that the PWM preparation contains substances that promote the activity of otherwise inactive synthetic DNA ODN and/or facilitate TLR9-mediated B cell activation. Mitogenic activity correlates with presence of TLR ligands in PWM preparations To further assess the impact of TLR-active substances in PWM preparations on PWM-induced B cell activation we compared four different commercially available PWM preparations in regards to their mitogenic potential and TLR stimulation. The results showed that PWM Lot. A, B and C were superior to Lot. D in the induction of human B cell proliferation (Fig. 4A). This increase in stimulatory activity correlated with higher endotoxin content determined by limbulus assay (LAL assay) (Fig. 4B) and with stronger TLR2-activity in the HEK293 system (Fig. 4C). Notably, TLR2 activity lay below the detection threshold when measured in 1 mg/ml PWM, but was detectable at a concentration of 10 mg/ ml. Comparable levels of IL-8 secretion were achieved by stimulation of transfected HEK293 cells with 300 ng/ml of TLR2 ligand Pam3CSK4. Interestingly, when compared to Lot. 0 in these more recently purchased lots of PWM the levels of TLR2 activity were overall lower, IL-8 induction was restricted to TLR2-transfected HEK293 cells and we did not amplify bacterial DNA by PCR. In fact, in Lot. D, which was purchased as ‘‘highly purified’’ PWM, even LPS content and TLR2 activity were negligible. This PWM preparation also failed to induce B cell PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e29806