Philip P. Stashenko, DMD, PhD

The Stashenko laboratory focuses on the role of the immune system in regulating bone mass in oral infections, including periapical and periodontal diseases. These infections elicit innate and adaptive immune responses that protect the host against more widespread infection, but do so at the cost of localized tissue and bone destruction. Bone loss in these and other conditions, including arthritis and osteoporosis, is mediated by osteoclasts, cells that are specialized for the degradation of bone. The interrelationships between the immune response and bone are the subject of the emerging field of osteoimmunology.

Our work seeks to elucidate the molecular mechanisms of osteoclast function, and to use these molecules as diagnostic markers and/or targets for new drugs that prevent unwanted bone loss. Related studies are examining the role of the nervous system, including neurotransmitters such as serotonin and neuropeptides, in modulating bone loss. In parallel, we are investigating how infection-stimulated bone resorption is regulated by cytokines and immune cells in the complex in vivo milieu. A goal of this work is to develop novel immunomodulatory approaches that enhance protective anti-bacterial responses, while concomitantly minimizing tissue destruction.

Molecular mechanisms of osteoclast development and function

Differentiated cells such as osteoclasts express thousands of genes, the vast majority of which are involved in basic cellular processes. However, a subset of genes has specific or highly selective expression, and hence defines the cell's phenotype. Using differential cDNA screening and gene array approaches, we, in collaboration with the Li and Battaglino laboratories, have characterized genes that are expressed predominantly in osteoclasts, and are furthermore critical to the bone resorbing function of this cell type.

These genes include cathepsin K, a cysteine proteinase that is mutated in the human syndrome pycnodysostosis ('Toulouse-Lautrec syndrome'); the G protein modulators RGS10A and B; ATP6i, a unique proton pump subunit that is deficient in about half of the cases of human malignant osteopetrosis ('marble bones'); and the newly discovered NHA2, a novel proton-cation exchanger that is localized to mitochondria and is mutated in the autosomal dominant form of osteopetrosis. Other important genes, not previously known to be expressed in osteoclasts, have also been identified in these studies, including proteases (gelatinase B), chemokines (MIP-1γ), cell-adhesion molecules (ALCAM), transcription factors (Brn3), the serotonin transporter (5-HTT) and serotonin receptors. Additional novel osteoclast-specific genes are under investigation.

The function of a given osteoclast gene in bone resorption is analyzed using a combination of methods, including RNA interference, antibody neutralization and specific chemical inhibitors. Findings from in vitro models are confirmed in vivo, using specific inhibitors or transgenic, knockout and conditional knockout mice. When critical osteoclast genes such as ATP6i are deleted, animals have a profound lack of bone resorption, resulting in bone malformations, stunted growth, and a failure of tooth eruption. Such proteins have great potential as targets for therapeutic agents that interfere with pathological bone resorption.

Recently, together with the Battaglino laboratory, we demonstrated important functions for the serotonin system as well as other neurotransmitters, in regulating osteoclasts and osteoblasts. These and other observations clearly show that the nervous system, including the brain, spinal cord, and peripheral nerves, plays a central role in the maintenance of a healthy, intact skeleton. The importance of these neuroskeletal interactions is perhaps best illustrated in cases of spinal cord injury, in which severe osteoporosis develops very rapidly – ten times faster than occurs with bed rest or microgravity. Understanding the molecular mechanisms that underlie these interactions is of great potential importance in preventing bone loss.

Immunomodulation of infection-stimulated bone resorption

Microbial pathogens have evolved mechanisms that allow them to best evade the immune response and cause disease within the host. We have shown that periodontal and periapical infections with the pathogen Porphyromonas gingivalis (Pg) stimulate a proinflammatory T helper 1 (Th1) response, including the expression of IL-12 and IFNγ, which results in extensive alveolar bone destruction. In contrast Th2-type responses, particularly the anti-inflammatory cytokine IL-10, significantly reduce Pg induced bone loss.

This new paradigm – that it is critical to 'counter' the emergence of the disease-promoting T helper phenotype – is being studied in collaboration with the Campos-Neto laboratory. We have utilized T cell expression cloning to identify antigens of Pg that are immunodominant and elicit Th1 responses, similar to the intact microorganism. When animals are pre-immunized against recombinant Pg antigens using regimens that elicit strong Th1 responses, bone loss is dramatically increased, whereas Th2-inducing regimens reduce bone loss. We are also investigating the role of other T cell subsets, including Treg and Th17 cells, in modulating inflammatory bone loss. Taken together, these studies suggest that new immunotherapeutic strategies can be devised for the prevention of bone loss in chronic inflammatory diseases.

Selected Publications

Leshem O, Kashino SS, Goncalves RB, Suzuki N, Onodera M, Fujimura A, Sasaki H, Stashenko P, Campos-Neto A.  (2008) Th1 biased response to a novel Porphyromonas gingivalis protein aggravates bone resorption caused by this oral pathogen. Microbes Infect. 10(6):664-672.

Sasaki H, Suzuki N, Kent R jr, Kawashima N, Takeda J, Stashenko P.  (2008) T Cell response mediated by myeloid cell-derived IL-12 is responsible for Porphyromonas gingivalis-induced periodontitis in IL-10-deficient mice. J. Immunol.
180(9):6193-6198.

Lesham O, Kashino S, Gonçalves RB, Suzuki N, Onodera M, Fujimura A, Sasaki H, Stashenko P, Campos-Neto A. (2008) Th1 biased response to a novel Porphyromonas gingivalis protein aggravates bone resorption caused by this oral pathogen. Microbes Infect. (in press).

Morse LR, Teng Y, Newton K, Du Y, Liao W, Kohler T, Mueller R, Graves D, Stashenko P, Battaglino RA. (2008) Spinal cord injury causes rapid osteoclastic resorption and growth plate abnormalities in growing rats. Osteoporosis Int. (in press).

Battaglino RA, Pham L, Morse LR, Vokes M, Sharma A, Odgren PR, Yang M, Sasaki H, Stashenko P. (2008) NHA-oc/NHA2: A mitochondrial cation-proton antiporter selectively expressed in osteoclasts. Bone 42(1):180-192.

Pham L, Purcell P, Morse LR, Stashenko P, Battaglino RA. (2007) Expression analysis of nha-oc/NHA2: a novel osteoclast-specific gene. Gene Expr. Patterns 7(8):846-851.

Battaglino RA, Vokes M, Schulze-Spate U, Sharma A, Graves D, Kohler T, Muller R, Yoganathan S, Stashenko P. (2007) Fluoxetine treatment increases trabecular bone formation in mice. J. Cell. Biochem. 100(6):1387-1394.

Schulze-Spate U, Battaglino RA, Fu J, Sharma A, Vokes M, Stashenko P. (2007) Brn3 transcription factors control terminal osteoclastogenesis. J. Cell. Biochem. 102(1):1-12.

Ernst CW, Lee JE, Nakanishi T, Karimbux NY, Rezende TM, Stashenko P, Seki M, Taubman MA, Kawai T. (2007) Diminished forkhead box P3/CD25 double-postive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease. Clin. Exp. Immuno. 148(2):271-280.

Stashenko P, Goncalves RB, Lipkin B, Ficarelli A, Sasaki H, Campos-Neto A. (2007) Th1 immune response promotes severe bone resorption caused by Porphyromonas gingivalis. Am. J. Pathol. 170(1):203-213.

Keles A, Grunes B, DiFuria C, Gagari E, Srinivasan V, Darendeliler MA, Muller R, Kent R Jr, Stashenko P. (2007) Inhibition of tooth movement by osteoprotegerin vs. pamidronate under conditions of constant orthodontic force. Eur. J. Oral Sci. 115(2):131-136.

Yang S, Chen W, Stashenko P, Li Y-P. (2007) Specificity of RGS10A as a key component in the RANKL signaling mechanism for osteoclast differentiation. J. Cell. Sci. 120:3362-3371.

Soukos NS, Chen PS, Morris JT, Ruggiero K, Abernethy AD, Som S, Foschi F, Doucette S, Luschke-Bammann L, Fontana CR, Doukas AG, Stashenko P. (2006) Photodynamic therapy for endodontic disinfection. J. Endod. 32(10):979–984.

Goncalves R, Lesham O, Bernard K, Webb JR, Stashenko P, Campos-Neto A. (2006) T-cell expression cloning of Porphyromonas gingivalis genes coding for T helper biased (Th1/Th2) immune response during infection. Infect. Immun. 74:3958–3966.

Staff

Post-Doctoral Trainees

Emad AlShwaimi, D.D.S.
Otis Chong, D.D.S.
Leslie Morse, D.O.
Matt Nealon, D.D.S.
Hessam Rahimi, D.D.S., Ph.D.

Visiting Scholar

Ahmet Keles, D.D.S., D.M.Sc.

Research Assistant

Lan Pham, B.S.

Student

Rayanne Lee, B.S.