Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies, and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 μM and one site had a weaker affinity with a KD2 of 60.0 μM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 μM histatin 5 attenuated (p < 0.05) 0.02 μM HagB-induced CCL3/MIP-1α, CCL4/MIP-1β, and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

FL 32610, USA) for providing the recombinant HagB that was used in the experiment. I would also like to thank the Department of Periodontics at the University of Iowa College of Dentistry for their support, especially Dr. Kim Brogden for his excellent guidance and mentorship. iv ABSTRACT Histatins, a group of proteins produced by human salivary glands, have a variety of innate immune functions including the ability to: kill oral microorganisms, neutralize toxins, inactivate protease/collagenase activities, inhibit co-aggregation of oral bacteria, and inhibit lipopolysaccharide mediated activities. Hemagglutinin B (HagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, induces a robust cytokine and chemokine response in human myeloid dendritic cells. In this study, I hypothesize that histatin 5 can attenuate a HagB-induced chemokine response. Objectives: To characterize an expanded cytokine and chemokine response induced in human myeloid dendritic cells by HagB, and to determine if prior incubation of HagB with histatin 5 attenuates these responses. Methods: In my first experiment, 0.040 M HagB was mixed with dilutions of histatin 5 and histatin 8 (Sigma, 0.04 to 40.0 M), incubated at 37C for 30 minutes, and added to 2 x 10 4 human myeloid dendritic cells (Lonza,Walkersville, MD). At 24 hours, culture media was removed, and 6 cytokines and chemokines (pg/ml) were determined in cell-free supernatants (Millipore, Billerica, MA) using the Luminex 100 IS instrument (Luminex, Austin, TX). In my second experiment, 0.040 M HagB was mixed with 40.0 M histatin 5 only (e.g., 1:1000), incubated at 37C for 30 minutes, and added to 2 x 10 4 human myeloid dendritic cells. At 0, 1, 2, 4, 8, 16, and 24 hours post-inoculation, culture media was removed, and 26 cytokines and chemokines (pg/ml) were determined in cell-free supernatants. Results: In both