Our data indicate that human B cells responding to antigen plus T cell help, as mimicked by anti-Igμ+α-CD40, have increased potential to up regulate surface TLR4, but the final levels of TLR4 expression and perhaps function is highly dependent on the cytokine milieu. The positive correlation between bioactive IL-4 (as measured by the surrogate eosinophilia) and the percentage of circulating TLR4-positive B cells in type 2 diabetes patients (p≤0.05, data not shown) suggests an unappreciated link between altered cytokine levels and expression/function of TLR4, that together may promote or regulate systemic inflammation in such individuals (McDonnell et al., in press; Jagannathan et al., 2009
; Jagannathan et al., 2010
). The data therefore significantly extend a previous demonstration that IL-4 up regulates B cell TLR4 (Mita et al., 2002
) by linking elevated B cell TLR4 expression, thus mechanisms of B cell TLR4 activation, with inflammatory disease. Whether the cytokine milieu in patients also plays roles in establishing the unexpected pro-inflammatory functions of B cell TLR4 is under investigation.
Thus far, we have demonstrated that B cell TLR4 decreases TLR2-mediated IL-10 secretion (Jagannathan et al., 2009
). B cell TLR4 engagement also decreases B cell IL-8 secretion in samples from CD patients, but B cell TLR4 responds to E. coli LPS by increasing IL-8 secretion by B cells from patients with ulcerative colitis (McDonnell et al., in press). These data indicate that B cell TLR4 up-regulation imparts unexpected disease-associated functions that likely affect the overall inflammatory milieu in affected individuals. Regardless, the data highlight the pitfalls of characterizing diseases such as CD, type 2 diabetes, and periodontal disease as IL-2-dominated (Th1) or IL-4-dominated (Th2) disease. Circulating cells from these patients have signatures of both IL-4 activity (elevated B cell TLR4 activation: , Shin et al., 2009
; McDonnell et al., in press) and either increased IL-2 activity (elevated TLR4 activation in cultured monocytes, Dasu et al., 2010
), or balanced IL-2/IL-4 activity (no change in blood monocyte TLR4 expression, (Jagannathan et al., 2010
) defined herein.
The presence of an elevated percentage of TLR4-positive cells in the naïve B cell population of inflammatory disease patients (McDonnell et al., in press; Jagannathan et al., 2009
) cannot be explained by the strong stimulatory conditions used in this study: memory, but not naïve B cells would have been exposed to such stimuli in vivo. However, published work has shown that peripheral B cell survival is absolutely dependent on low-level “tonic” signaling through the B cell receptor (Bannish et al., 2001
; Lam et al., 1997
) and that even naïve B cells have low-level activation of the Igμ signaling pathway (Fuentes-Panana et al., 2006
). It is possible that Igμ-mediated tonic signaling, along with the elevated levels of soluble and/or cell-associated CD40, TLR ligands and cytokines present in inflammatory disease patients (Al-Attas et al., 2009
; Cani et al., 2007
; Creely et al., 2007
; Jinchuan et al., 2004
; Liu et al., 1999
; Varo et al., 2003
), are sufficient to induce B cell TLR4 activation in vivo, even in the absence of the activation history that characterizes the memory compartment.
Multiple mechanisms could explain differential TLR4 regulation in B cells and monocytes. Previous demonstrations that the TLR4 promoter is packaged into an equivalently active chromatin structure in monocytes and B cells from healthy or inflammatory disease patients (Jagannathan et al., 2009
; Jagannathan et al., 2010
) discount the likelihood that chromatin accessibility plays a critical role in differential TLR4 regulation highlighted by our IL-2/IL-4 experiments. We instead speculated that different concentrations of key TLR4 activating factors (PU.1 and IRFs; (Pedchenko et al., 2005
; Rehli et al., 2000
) in B cells and monocytes explained differential regulation of TLR4 surface levels in the two cell types. However, the ChIP data do not support this possibility: PU. 1 and IRFs associate with the TLR4 promoter approximately equivalently in B cells and monocytes despite higher protein levels of PU.1 and lower protein levels of IRFs in monocytes compared to B cells (DeKoter and Singh, 2000
; Marecki et al., 1999
; Nikolajczyk et al., 1997
). Instead, the agreement between the ChIP and mRNA data, which both lack concordance with the surface staining data, indicates that post-transcriptional regulation plays a critical role in determining TLR4 surface expression on B cells. Post-transcriptional TLR4 regulation has not been attributed to monocytes by either our data or by published studies. However, differences between B cell and monocyte TLR4 regulation raised herein suggest more rigorous approaches are required to formally discount the possibility of additional layers of TLR4 regulation in monocytes. Regardless, different mechanisms of TLR4 regulation in B cells and monocytes are consistent with the finding that TLR4 is up regulated on B cells, but not monocytes or neutrophils, in fresh ex vivo samples from type 2 diabetes patients vs. non-diabetic donors (Jagannathan et al., 2010
). We predict that further studies aimed at understanding post-transcriptional mechanisms of TLR4 regulation in immune system cells may shed light on the role these cells and receptors play in inflammatory disease, and identify new targets for next-generation anti-inflammatory treatments.