Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2-independent of its chiral state-significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.

TRAF3 has diverse signaling functions, which vary by cell type. Uniquely in B lymphocytes, TRAF3 inhibits homeostatic survival. Highlighting the role of TRAF3 as a tumor suppressor, loss-of-function TRAF3 mutations are associated with human B-cell malignancies, while B-cell-specific deletion of TRAF3 in mice leads to autoimmunity and lymphoma development. The role of TRAF3 in inhibiting noncanonical NF-κB activation, CD40 and BAFF-R signaling to B cells is well documented. In contrast, TRAF3 enhances many T-cell effector functions, through associating with and enhancing signaling by the T-cell receptor (TCR)-CD28 complex. The present study was designed to determine the role of TRAF3 in signaling via the B-cell antigen receptor (BCR). The BCR is crucial for antigen recognition, survival, proliferation, and antibody production, and defects in BCR signaling can promote abnormal survival of malignant B cells. Here, we show that TRAF3 is associated with both CD79B and the BCR-activated kinases Syk and Btk following BCR stimulation. BCR-induced phosphorylation of Syk and additional downstream kinases was increased in TRAF3 −/− B cells, with regulation observed in both follicular and marginal zone B-cell subsets. BCR stimulation of TRAF3 −/− B cells resulted in increased surface expression of MHC-II, CD80, and CD86 molecules. Interestingly, increased survival of TRAF3 −/− primary B cells was resistant to inhibition of Btk, while TRAF3-deficient malignant B-cell lines showed enhanced sensitivity. TRAF3 serves to restrain normal and malignant BCR signaling, with important implications for its role in normal B-cell biology and abnormal survival of malignant B cells.

TRAF3 is an adaptor protein that binds to a variety of receptor types on immune cells and exerts multiple regulatory functions in signaling pathways. TRAF3 functions are cell type and context dependent. B cell-TRAF3 inhibits homeostatic NFκB2 activation and other pro-survival signaling pathways; restraint of survival is a B cell-unique TRAF3 function. Loss of B cell-TRAF3 in vivo contributes to early autoimmunity and later lymphomagenesis. TRAF3 inhibits B cell TLR functions, including cytokine production (IL-6, IL-10, IL-12p40 and TNFα), Ig isotype switching, and antibody production in response to ligands for TLRs 3, 4, 7/8 and 9. A key knowledge gap is identifying the molecular mechanisms by which TRAF3 mediates this regulation. Our working hypothesis is that B cell-TRAF3 regulates the activity, recruitment, and/or post-translational modification of specific TLR signaling proteins. Our recent data show that B cell-TRAF3 forms a heterocomplex with TRAF6, associating with the tyrosine kinase Syk and inhibiting early TLR signals, such as pIRAK1 degradation. In the absence of TRAF3, TRAF6 shows a greater association with several TLR signaling proteins. These results suggest that TRAF3 may inhibit TRAF6 access to the TLR signaling complex, and thus early TLR signaling. Further studies aimed at providing new insights into the molecular mechanisms contributing to the regulation of B cell-TRAF3 in TLR signaling can inform selection and development of more targeted therapies for B cell malignancies and autoimmunity. Supported by VA Merit Review IO1 BX001702 NIH RO1 AI62656 NIH P50 CA97274 NIH T32 Training Grant AI07485

TRAF3 is an adaptor protein that binds to receptors of the TNFR superfamily and exerts multiple regulatory functions in signaling pathways. However, functions are cell type and context dependent. B cell TRAF3 inhibits homeostatic NFκB2 activation, CD40/BAFFR signaling and other pro-survival signaling pathways; restraint of survival is a B cell unique TRAF3 function. Loss of function mutations of human TRAF3 are detected in B cell lymphoma and multiple myeloma. In addition to homeostatic survival, we found that TRAF3 inhibits B cell TLR signaling, a pathway implicated in B cell lymphomagenesis and autoimmunity. Numerous reports describe TLR function in myeloid cells, but specific TLR signaling pathways in B cells are under-studied. B cell TRAF3 inhibits TLR-mediated cytokine and Ab production, and Ig class switching. Our <underline>working hypothesis</underline> is that B cell TRAF3 regulates the expression, recruitment and/or post-translational modification of TLR signaling proteins. Preliminary data show that B cell TRAF3 associates with signaling proteins MyD88 and IRAK1, and the kinase Syk following TLR stimulation. We are currently investigating how this association normally restrains the functions of these proteins in promoting TLR signals to B cells. We expect that information gained will provide new insights into the molecular mechanisms contributing to the regulation of B cells by TRAF3 in TLR signaling, potentially informing selection and development of more targeted therapies for B cell malignancies and autoimmunity.