PWM-Induced B Cell Activation Figure 5. Contribution of the lectin component. A: Human B cell proliferation in response to PWM (Lot. B and C) in the presence or absence of N,N-di-acetylchitobiose or N,N,N-tri-acetylchitotriose was assessed by 3H-thymidine incorporation given in counts per minute (cpm). The mean values 6 SEM from one representative experiment performed in triplicates of n = 2 is shown. B: Human CD19+ B cells were left unstimulated or stimulated with 0.25 mM GpC PTO ODN (GC) and/or 10 mg/ml of PWM, 5 mg/ml SpA or 10 mg/ml anti-Ig (aIg) for 72 hours. Proliferation was quantified by 3H- thymidine incorporation. The diagram shows the means from n = 4 experiments 6 SEM. The values obtained were normalized to GC = 1 (7106280 cpm = mean 6 SEM). C: Human CD19+ B cells were stained with CFSE and stimulated with highly purified PWM (Lot. D) or Lycopersicon esculentum lectin (LEA) with or without GpC PTO ODN (GC). Proliferation was quantified by CFSE dilution on day 4. The percentage of proliferating cells is provided in each dot plot. The results from two independent donors of n = 4 are shown. doi:10.1371/journal.pone.0029806.g005 PWM-extracted DNA was used as a template (Fig. 2E). Similarly, TLR2-specific ligands (Fig. 2C and 3A) are most likely derived from bacterial lipoproteins, considering that, to date, there is no evidence for plant-derived TLR2 ligands. Although we did not follow this up, it is very likely that the PWM preparations contain low concentrations of other bacterial molecules such as peptido- glycan or lipoteichoic acids. It is well-known that specific bacterial species preferentially interact with plants. Rhizobium spp. for example are plant symbionts while others including most Agrobacterium spp. and Burkholderia spp. are phytopathogens [24]. These genus have a Gram negative cell wall and, thus, carry LPS among other specific microbial molecules that confer bacterial recognition by mammalian pattern recognition receptors. However, despite the attractiveness of the hypothesis, our findings provided no evidence for plant-associated pathogens as a source for TLR ligands and we could not exclude contamination of PWM during the purification procedure: sequencing of the 16S rDNA PCR product in Lot. 0 revealed the presence of DNA from Propionibacterium acnes, a typical inhabitant of human skin containing CG-rich TLR9-stimulatory genomic DNA [25]. Taking into consideration that different classes of bacterial molecules are detectable in the PWM preparation, we propose that the endotoxin content in PWM preparations (Fig. 4B and [10]) could originate from inaccuracies in the chemical purifica- tion procedure as well as from plant-derived bacteria. Neverthe- less, regardless of its origin LPS will contribute to the stimulation of TLR4-carrying immune cells such as human monocytes (Fig. 1B) and murine B cells (Fig. 1E). However - in contrast to monocytes and murine B cells - human B cells lack expression of the lipopolysaccharide sensors CD14 and TLR4 [12,18]. Correlating with receptor expression human B cells fail to respond to LPS despite the presence or absence of a BCR stimulus (Fig. 2A). As a consequence, LPS is not an important PWM component conferring mitogenic activity on human B cells although it may act on other leukocyte subsets. Since polymyxin B is a cationic detergent antagonization of PWM-induced B cell activation does not necessarily need to be limited to LPS, but could also involve bacterial lipoproteins or DNA. Nevertheless, PWM-derived LPS may very well contribute to murine B cell stimulation as was recently reported for a polysaccharide isolated from Acanthopanax senticosus that activated murine B cells and macrophages in a TLR4-dependent manner [4] and a polysaccharide purified from Acanthopanax koreanum that displayed TLR2- and TLR4-dependent B cell stimulatory activity [3]. In contrast to TLR4 stimulation, human peripheral blood B cells respond to TLR2 and TLR9 specific challenge and the response is increased in the presence of BCR stimulation (with SpA or anti-Ig) that was previously found to enable TLR2 activity and to augment the TLR9 response in human B cells (Fig. 2A and [7,17,22]). In contrast to TLR2 ligands PWM displays mitogenic activity in the absence of BCR stimulation (Fig. 1A). This indicates that PWM contains stimulatory agents that either circumvent the requirement for BCR ligation or directly engage the BCR. This effect could be attributed to the lectin compound that binds to cell surface N-acetylglucosamine-containing glyco- proteins [8,26]. Lectin-mediated engagement of rather ill-defined surface receptors may, thus, enable TLR2 activity and/or enhance the activity of other TLRs such as the DNA sensor TLR9 [7,17,22]. Surprisingly, the combination of PWM with SpA failed to increase proliferation although it was recently suggested that combined stimulation of PWM and SAC, a suspension of S. aureus cells with high SpA content, provides a stronger and more reliable stimulus in human PBMC [5]. However, the fact that our experiments were carried out using purified human B cells, e.g. in the absence of antigen presenting cells, may account for this difference. Notably, in our experimental setting a slight decrease in the percentage of proliferating B cells observed when SpA was used together with PWM (Fig. 1A) could indicate that SpA and PWM compete for the same B cell binding sites. Again, we can only speculate whether this could involve a subgroup of B cell receptors such as the VH3+ BCR targeted by SpA [27]. However, this observation was supported by the finding that BCR stimulation with anti-Ig markedly reduced the duration of proliferation and terminal differentiation in B cells stimulated with PWM (Fig. 1A). This provides the notion that BCR stimulation might interfere with the cellular binding of Phytolacca americana lectin. Taking into consideration that only traces of bacterial DNA and lipoproteins are present in the PWM preparations it is evident that high concentrations of PWM (1–10 mg/ml) are required for B cell stimulation. Moreover, our data suggest that the poke weed lectin enables B cell stimulation by low concentrations of bacterial DNA that by themselves would be ineffective. They further highlight that non-CpG DNA sequences and non-PTO modified ODN display immune stimulatory activity in the presence of PWM (Fig. 3B and 5). Interestingly, similar observations were made when B cell stimulation with non-CpG ODN occurred together with a BCR antigen or when ODN lacking B cell activity were targeted to the BCR [28,29]: B cell ODN sequence requirements were found to be less stringent in the presence of BCR activation, and uptake of ODN improved. In the present context, we propose that similarly to a BCR signal PWM sensitizes B cells for pathogen-associated molecular patterns (PAMPs). It can only be speculated whether the positive charge of the Phytolacca americana-derived lectin might enable complexation with negatively charged bacterial mole- cules such as DNA [30]. Similarly to PTO-modification of synthetic ODN or formation of autoimmune complexes as in [28] these complexes could protect DNA from degradation and promote cellular uptake of bacterial DNA. Alternatively, lectin- mediated binding to carbohydrates on cell surface receptors facilitates uptake of these substances and/or cellular activation. Endocytotic uptake, in turn, could provide access to TLR9 and induces intracellular redistribution of TLR9 to the autophago- somes [31,32]. PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29806