Filifactor alocis is a recently recognized periodontal pathogen; however, little is known regarding its interactions with the immune system. As the first-responder phagocytic cells, neutrophils are recruited in large numbers to the periodontal pocket, where they play a crucial role in the innate defense of the periodontium. Thus, in order to colonize, successful periodontal pathogens must devise means to interfere with neutrophil chemotaxis and activation. In this study, we assessed major neutrophil functions, including degranulation and cell migration, associated with the p38 mitogen-activated protein kinase (MAPK) signaling pathway upon challenge with F. alocis. Under conditions lacking a chemotactic gradient, F. alocischallenged neutrophils had increased migration compared to uninfected cells, indicating that F. alocis increases chemokinesis in human neutrophils. In addition, neutrophil chemotaxis induced by interleukin-8 was significantly enhanced when cells were challenged with F. alocis, compared to noninfected cells. Similar to live bacteria, heat-killed F. alocis induced both random and directed migration of human neutrophils. The interaction of F. alocis with Toll-like receptor 2 induced granule exocytosis along with a transient ERK1/2 and sustained p38 MAPK activation. Moreover, F. alocis-induced secretory vesicle and specific granule exocytosis were p38 MAPK dependent. Blocking neutrophil degranulation with TAT-SNAP23 fusion protein significantly reduced the chemotactic and random migration induced by F. alocis. Therefore, we propose that induction of random migration by F. alocis will prolong neutrophil traffic time in the gingival tissue, and subsequent degranulation will contribute to tissue damage.

dRecent microbiome studies have implicated a role for Filifactor alocis in periodontal disease. In this study, we investigated the colonization and survival properties of F. alocis in a mouse subcutaneous chamber model of infection and characterized host innate immune responses. An infection of 10 9 F. alocis successfully colonized all chambers; however, the infection was cleared after 72 h. F. alocis elicited a local inflammatory response with neutrophils recruited into the chambers at 2 h postinfection along with an increase in levels of the proinflammatory cytokines interleukin 1␤ (IL-1␤), IL-6, and tumor necrosis factor (TNF). F. alocis also induced apoptosis in chamber epithelial cells and neutrophils. Consistent with resolution of infection, neutrophil numbers and cytokine levels returned to baseline by 72 h. Fluorescent in situ hybridization (FISH) and quantitative PCR demonstrated that F. alocis exited the chambers and spread to the spleen, liver, lung, and kidney. Massive neutrophil infiltration was observed in the spleen and lungs, and the recruited neutrophils were in close proximity to the infecting bacteria. Significant epithelial injury was observed in the kidneys. Infection of all tissues was resolved after 7 days. This first in vivo study of the pathogenicity of F. alocis shows that in the chamber model the organism can establish a proinflammatory, proapoptotic local infection which is rapidly resolved by the host concordant with neutrophil influx. Moreover, F. alocis can spread to, and transiently infect, remote tissues where neutrophils can also be recruited.

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H₂O₂ production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.

Filifactor alocis is a newly appreciated pathogen in periodontal diseases. Neutrophils are the predominant innate immune cell in the gingival crevice. In this study, we examined modulation of human neutrophil antimicrobial functions by F. alocis. Both non-opsonised and serum-opsonised F. alocis were engulfed by neutrophils but were not efficiently eliminated. Challenge of neutrophils with either non-opsonised or serum-opsonised F. alocis induced a minimal intracellular as well as extracellular respiratory burst response compared to opsonised Staphylococcus aureus and fMLF, respectively. However, pretreatment or simultaneous challenge of neutrophils with F. alocis did not affect the subsequent oxidative response to a particulate stimulus, suggesting that the inability to trigger the respiratory response was only localised to F. alocis phagosomes. In addition, although neutrophils engulfed live or heat-killed F. alocis with the same efficiency, heat-killed F. alocis elicited a higher intracellular respiratory burst response compared to viable organisms, along with decreased surface expression of CD35, a marker of secretory vesicles. F. alocis phagosomes remained immature by delayed and reduced recruitment of specific and azurophil granules, respectively. These results suggest that F. alocis withstands neutrophil antimicrobial responses by preventing intracellular ROS production, along with specific and azurophil granule recruitment to the bacterial phagosome.

Encephalitogenic T cells not only cause experimental autoimmune encephalomyelitis (EAE), but they induce resistance against subsequent induction of the disease as well. The T cell receptor (TcR) of encephalitogenic T cells is believed to contribute to their vaccinating activity. Findings in support of this assumption include the apparent restricted use of particular TcR elements, such as V beta 8.2. However, results from other laboratories including ours do not support this idea. We previously showed that rat T cells reactive against the conserved encephalitogenic epitope of myelin basic protein [MBP (87-99)] use the TcR in a heterogeneous fashion (Sun, D. et al., Eur. J. Immunol. 1992. 22: 591). Here we show, in Lewis rats, that the TcR beta chain usage of T cells specific for the dominant MBP (68-88) epitope is not restricted to V beta 8.2. Not only did such cells rely on diverse V beta chains, but some non-V beta 8-bearing cells were highly encephalitogenic. We also show that antigen-presenting cells (APC) play an important role in determining the TcR usage of MBP-specific T cells. Stimulation of MBP (68-88)-specific T cell lines by cloned APC derived from different sources resulted in selection of encephalitogenic T cells bearing different TcR beta chains.

In the present study, we have isolated and characterized five myelin basic protein (MBP)-reactive T cell lines directly from the brains of Lewis rats during the early paralytic phase of experimental autoimmune encephalomyelitis (EAE). Each T cell line responded to the dominant encephalitogenic epitope spanning residues 68-88, and did not react against the conserved encephalitogenic epitope [MBP(87-99)] or the nonencephalitogenic MBP epitope [MBP(50-69)]. We determined the T cell receptor (TcR) beta chain usage by polymerase chain reaction, DNA sequencing analysis and by generation of MBP-reactive hybridomas from one of the T cell lines (BT74). The results revealed that brain-infiltrating, MBP-reactive T cells freshly isolated early in the course of the disease exhibit TcR diversity.

We previously demonstrated that a significant proportion of interphotoreceptor retinoid-binding protein (IRBP)-specific uveitogenic T cells in the C57BL/6 mouse and Lewis rat express CD8. The aims of this study were to determine whether some of the IRBP-specific T cells in the B10RIII mouse also express CD8 and whether CD8 and CD4 IRBP-specific T cells in the B10RIII mouse recognize a different or the same antigenic epitope. Our results show that autoreactive CD8 T cells were abundant in B10RIII mice immunized with the uveitogenic peptide IRBP161-180. Using multimers of recombinant H-2D r molecules, we also showed that the binding of the H-2D r fusion protein to IRBP161-180-expanded CD8 T cells was dependent on the peptide complexed with the recombinant molecules. The use of a panel of truncated peptides showed that the truncated 10-mer peptide, IRBP168-177, retained the ability to bind to, and stimulate, IRBP161-180-specific CD8 T cells after complexing with a dimeric MHC class I (H-2D r ) molecule. Finally, adoptive transfer of IRBP161-180-specific T cells stimulated with IRBP168-177 consistently induced mild, but significant, EAU in naïve B10RIII mice.

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