Ashu Sharma

Interleukin-17 regulates expression of the CXC chemokine LIX/CXCL5 in osteoblasts: implications for inflammation and neutrophil recruitment

Interleukin (IL)‐17 is the founding member of an emerging family of inflammatory cytokines whose functions remain poorly defined. IL‐17 has been linked to the pathogenesis of rheumatoid arthritis, and numerous studies implicate this cytokine in inflammation‐induced bone loss. It is clear that a major function of IL‐17 is to amplify the immune response by triggering production of chemokines, cytokines, and cell‐surface markers, ultimately leading to neutrophil chemotaxis and inflammation. As an IL‐17 signaling deficiency in mice causes a dramatic reduction in neutrophil chemotaxis and a consequent increased susceptibility to bacterial infection, it is important to define gene targets involved in IL‐17‐mediated neutrophil trafficking. Here, we demonstrate that IL‐17 and tumor necrosis factor α (TNF‐α) cooperatively induce the lipopolysaccharide‐inducible CXC chemokine (LIX; a.k.a., CXC chemokine ligand 5, Scya5, or murine granulocyte chemotactic protein‐2) in the preosteoblast cell line MC3T3. LIX is induced rapidly at the mRNA and protein levels, likely through the activation of new gene transcription. Conditioned media from MC3T3 cells treated with IL‐17 and/or TNF‐α stimulates neutrophil mobility potently, and LIX is a significant contributing factor to this process. In addition, IL‐17 cooperates with bacterial components involved in periodontal disease to up‐regulate LIX expression. This study is the first demonstration of LIX expression in bone cells and has implications for inflammatory bone diseases such as arthritis and periodontal disease.

Porphyromonas gingivalis Vesicles Enhance Attachment, and the Leucine-Rich Repeat BspA Protein Is Required for Invasion of Epithelial Cells by “Tannerella forsythia”

ABSTRACT The human oral cavity harbors more than 500 species of bacteria. Periodontitis, a bacterially induced inflammatory disease that leads to tooth loss, is believed to result from infection by a select group of gram-negative periodontopathogens that includes Porphyromonas gingivalis, Treponema denticola, and “Tannerella forsythia” (opinion on name change from Tannerella forsythensis pending; formerly Bacteroides forsythus). Epithelial cell invasion by periodontopathogens is considered to be an important virulence mechanism for evasion of the host defense responses. Further, the epithelial cells with invading bacteria also serve as reservoirs important in recurrent infections. The present study was therefore undertaken to address the epithelial cell adherence and invasion properties of T. forsythia and the role of the cell surface-associated protein BspA in these processes. Further, we were interested in determining if P. gingivalis, one of the pathogens frequently found associated in disease, or its outer membrane vesicles (OMVs) could modulate the epithelial cell adherence and invasion abilities of T. forsythia. Here we show that epithelial cell attachment and invasion by T. forsythia are dependent on the BspA protein. In addition, P. gingivalis or its OMVs enhance the attachment and invasion of T. forsythia to epithelial cells. Thus, interactions between these two bacteria may play important roles in virulence by promoting host cell attachment and invasion.

Virulence mechanisms of Tannerella forsythia

The subgingival periodontal pocket of humans harbors more than 500 bacterial species. Periodontitis is a chronic inflammation of the periodontium with multi-factorial etiology. It is initiated due to colonization as subgingival biofilms by a group of gram-negative anaerobes. The disease progresses as a result of the direct effects of bacterial virulence factors on host tissues as well as the self-damaging host responses to the colonizing bacteria (83). While no single species has been implicated as the primary pathogen and the available evidence is consistent with a polymicrobial disease etiology, the red-complex bacteria consisting of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia has been strongly implicated in the onset of periodontitis (83). On the other hand, investigations utilizing the 16S ribotyping have also implicated novel phylotypes in the absence of detectable red-complex bacteria associated with periodontal lesions (44). In support of T. forsythia as being a suspected periodontal pathogen, the organism satisfies the necessary criteria postulated by Socransky and co-workers (82, 83). For instance: (i) its association and increased levels in periodontitis (83), (ii) evidence of host responses to its antigens (2, 81, 94) (iii) its ability to cause disease in animal models (2, 40, 79, 84), and (iv) expression of virulence factors which can potentially contribute to the disease process (described in detail in this review). T. forsythia is an anaerobic gram-negative member of the Cytophaga-Bacteroides family which was initially described as Bacteroides forsythus by Tanner et al. (87) and later reclassified as Tannerella forsythia by Sakamoto et al. (74) based on 16S rRNA phylogenetic analysis. T. forsythia is associated more frequently and/or in higher levels with various forms of the disease, including gingivitis, chronic and aggressive periodontitis, than with health (for review see Ref. (86)). Several studies have also implicated T. forsythia in the progression of clinical attachment loss associated with periodontitis (11, 18, 19, 50, 85). Moreover, a recent study has suggested that T. forsythia infection is more likely to cause periodontitis in overweight women than in normal weight women (7). According to another recent study, overweight or obese individuals have an overgrowth of T. forsythia compared to normal weight individuals, thus subjecting overweight and obese individuals to a higher risk of developing periodontal disease (20). In spite of the overwhelming evidence implicating T. forsythia in pathogenesis, this bacterium remains an understudied organism. This is partly due to the fastidious growth requirement for culturing this bacterium, as well as the fact that genetic manipulations of this organism are difficult to perform (30, 73). Moreover, there are no gene complementation systems currently available for the organism. While T. forsythia is the sole member of the new genus Tannerella, uncultivated oral phylotypes BU045, BU063, 97 and 997 are its closest relatives (95). These phylotypes have long rod-like segmented structure, and though they are frequently found in various periodontal disease-associated plaques, they are present only in low numbers, do not proliferate to high densities and therefore, are considered not relevant to disease pathogenesis (95). Studies in animal models have demonstrated the virulence potential of T. forsythia. For example, T. forsythia caused skin abscesses in rabbits (84) and mice (2, 93) as well as alveolar bone loss in mice (79) and rats (40). These in vivo studies also showed that the pathogenic potential of T. forsythia was enhanced in the presence of other bacteria. For instance, abscess formation in rabbits and in mice was enhanced synergistically when Fusobacterium nucleatum or P. gingivalis were the coinfecting partners of T. forsythia. Similarly, a synergy was observed relative to alveolar bone loss in rats following oral infection with the red-complex consortium, P. gingivalis, T. denticola and T. forsythia (40). These results demonstrate that T. forsythia is a pathogenic organism which might play synergistic roles in inflammation along with other periodontal pathogens. Therefore, in order to fully understand the mechanisms underlying the pathogenesis associated with T. forsythia, it would be important to identify the virulence functions of the organism and determine how these factors are regulated in response to coexisting bacteria and to host-derived factors. It is likely that T. forsythia might influence the physiology and virulence of coexisting periodontal pathogens. For this purpose, physical, chemical, and metabolic interactions are expected to occur, which might further involve bacterial two component sensor-regulator systems. So far, only a few putative virulence factors have been identified in T. forsythia: trypsin-like (17) and PrtH proteases (72), sialidases SiaH (6, 35) and NanH (88), a leucine-rich repeat cell-surface-associated and secreted protein BspA (81), an apoptosis-inducing activity (61), alpha-D-glucosidase and N-acetyl-beta-glucosaminidase (32), a hemagglutinin (59), components of the bacterial S-layer (71, 73), and methylglyoxal production (53).

Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction

ABSTRACT Toll-like receptors (TLRs) are important signal transducers that mediate inflammatory reactions induced by microbes through pattern recognition of virulence molecules such as lipopolysaccharide (LPS) and lipoproteins. We investigated whether proinflammatory cytokine responses induced by certain bacterial protein adhesins may also depend on TLRs. In differentiated THP-1 mononuclear cells stimulated by LPS-free recombinant fimbrillin (rFimA) from Porphyromonas gingivalis, cytokine release was abrogated by monoclonal antibodies (MAbs) to CD14 and TLR4 but not to TLR2. Similar experiments using anti-β2 integrin MAbs suggested that β2 integrins (CD11/CD18) also play a role in cytokine induction by rFimA or native fimbriae. Minor fimbriae (distinct from the fimA-encoded major fimbriae) of P. gingivalis induced proinflammatory cytokine release in a CD14- and TLR2-dependent mode. Cytokine induction by BspA, a leucine-rich repeat protein from Bacteroides forsythus, depended heavily on CD14 and TLR2. We also found that the ability of the streptococcal protein AgI/II to stimulate cytokine release depended partially on CD14 and TLR4, and the AgI/II segment that possibly interacts with these receptors was identified as its N-terminal saliva-binding region. When THP-1 cells were exposed to rFimA for 24 h, surface expression of CD14 and CD18 was decreased and the cells became hyporesponsive to cytokine induction by a second challenge with rFimA. However, tolerance induction was abolished when the THP-1 cells were pretreated with rFimA in the presence of either anti-CD14 MAb or anti-TLR4 MAb. Induction of cross-tolerance between rFimA and LPS correlated with downregulation of the pattern recognition receptors involved. Our data suggest that the CD14-TLR2/4 system is involved in cytokine production and tolerance induction upon interaction with certain proinflammatory bacterial protein adhesins.

Multiple Functions of the Leucine-Rich Repeat Protein LrrA of Treponema denticola

ABSTRACT The gene lrrA, encoding a leucine-rich repeat protein, LrrA, that contains eight consensus tandem repeats of 23 amino acid residues, has been identified in Treponema denticola ATCC 35405. A leucine-rich repeat is a generally useful protein-binding motif, and proteins containing this repeat are typically involved in protein-protein interactions. Southern blot analysis demonstrated that T. denticola ATCC 35405 expresses the lrrA gene, but the gene was not identified in T. denticola ATCC 33520. In order to analyze the functions of LrrA in T. denticola, an lrrA-inactivated mutant of strain ATCC 35405 and an lrrA gene expression transformant of strain ATCC 33520 were constructed. Characterization of the mutant and transformant demonstrated that LrrA is associated with the extracytoplasmic fraction of T. denticola and expresses multifunctional properties. It was demonstrated that the attachment of strain ATCC 35405 to HEp-2 cell cultures and coaggregation with Tannerella forsythensis were attenuated by the lrrA mutation. In addition, an in vitro binding assay demonstrated specific binding of LrrA to a portion of the Tannerella forsythensis leucine-rich repeat protein, BspA, which is mediated by the N-terminal region of LrrA. It was also observed that the lrrA mutation caused a reduction of swarming in T. denticola ATCC 35405 and consequently attenuated tissue penetration. These results suggest that the leucine-rich repeat protein LrrA plays a role in the attachment and penetration of human epithelial cells and coaggregation with Tannerella forsythensis. These properties may play important roles in the virulence of T. denticola.

Tannerella forsythia-induced Alveolar Bone Loss in Mice Involves Leucine-rich-repeat BspA Protein

Tannerella forsythia (formerly Bacteroides forsythus) is one of the periodontal pathogens recently implicated in the development of periodontal disease. The cell-surface-associated, as well as the secreted, leucine-rich-repeat protein (BspA) of this bacterium have been suggested to play roles in bacterial adherence, and also in inflammation, by triggering release of pro-inflammatory cytokines from monocytes and chemokines from osteoblasts, leading to inflammation and bone resorption. In this study, we sought to determine the pathogenic potential of T. forsythia and the in vivo role of the BspA protein in pathogenesis in the mouse model of infection-induced alveolar bone loss. The results showed alveolar bone loss in mice infected with the T. forsythia wild-type strain, whereas the BspA mutant was impaired. In conclusion, evidence is presented in support of T. forsythia as an important organism involved in inducing alveolar bone loss, and the BspA protein is an important virulence factor of this bacterium.

Porphyromonas gingivalis Fimbriae Mediate Coaggregation with Streptococcus oralis through Specific Domains

Fimbriae are major adhesive components on the cell surface of Porphyromonas gingivalis. In this study, we evaluated the role of fimbriae in coaggregation with Streptococcus oralis. Fimbriae purified from P. gingivalis competitively inhibited the coaggregation by 100% at a concentration of 50 ug/mL. On the other hand, the same amount of lipopolysaccharide isolated from P. gingivalis was inhibited by only 25% of the level of the fimbriae. A fimA-inactivated mutant of P. gingivalis failed to show distinct coaggregation activity. Fimbriae added to a solution of various strains of streptococci caused their self-aggregation at a concentration of 10 to 30 ug/mL. The self-aggregation induced by fimbriae was inhibited by \-arginine (20 to 40 mM/L). Iodinated fimbriae reacted with S. oralis cells immobilized on the nitrocellulose membrane, and 100°C heating of the cells diminished the binding abilities. Recombinant fimbrillin (r-Fim, corresponding to whole residues 1 to 337 of native fimbrillin) of P. gingivalis also showed 100% inhibition of the coaggregation. The r-Fim variant (residues 1 to 286) lacking the C-terminal 51 residues was as inhibitory as r-Fim. However, the variant (residues 1 to 265) without the C-terminal 72 residues lost 77% of the inhibitory activity. These findings suggested that residues 266 to 286 contain a domain involved in the coaggregation of P. gingivalis with S. oral is. Inhibition by three polypeptides corresponding to residues 266 to 286, 266 to 337, and 287 to 337 was studied. Peptides 266 to 286 and 266 to 337 inhibited by 96 and 100%, respectively, at a concentration of 1.5 nmol/mL. Peptide 287 to 337 also showed a significant inhibitory effect but to a slightly lesser extent than that of peptide 266 to 286. P. gingivalis fimbriae appear to be involved in coaggregation with streptococci, probably through an adhesive protein molecule(s) of the latter, and the fimbriae possess several domains in the C-terminal residues 266 to 337 for interaction with S. oralis.

Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

ABSTRACT The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for P. gingivalis fimbriae. We hypothesize that adherence of P. gingivalis fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.

A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression

Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth-supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacteriums proteinaceous surface (S)-layer lattice and other glycoproteins. Herein, we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress T-helper (Th)17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen may act to ensure its persistence in the host likely through suppression of Th17 responses. In addition, our data suggest that the bacterium then induces the Toll-like receptor 2–Th2 inflammatory axis that has previously been shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases.

Toll-Like Receptor 2-Mediated Interleukin-8 Expression in Gingival Epithelial Cells by the Tannerella forsythia Leucine-Rich Repeat Protein BspA

ABSTRACT Tannerella forsythia is a gram-negative anaerobe strongly associated with chronic human periodontitis. This bacterium expresses a cell surface-associated and secreted protein, designated BspA, which has been recognized as an important virulence factor. The BspA protein belongs to the leucine-rich repeat (LRR) and bacterial immunoglobulin-like protein families. BspA is, moreover, a multifunctional protein which interacts with a variety of host cells, including monocytes which appear to respond to BspA through Toll-like receptor (TLR) signaling. Since gingival epithelium forms a barrier against periodontal pathogens, this study was undertaken to determine if gingival epithelial cells respond to BspA challenge and if TLRs play any role in BspA recognition. This study was also directed towards identifying the BspA domains responsible for cellular activation. We provide direct evidence for BspA binding to TLR2 and demonstrate that the release of the chemokine interleukin-8 from human gingival epithelial cells by BspA is TLR2 dependent. Furthermore, the LRR domain of BspA is involved in activation of TLR2, while TLR1 serves as a signaling partner. Thus, our findings suggest that BspA is an important modulator of host innate immune responses through activation of TLR2 in cooperation with TLR1.