Basic Research—Biology
Expression of Heat Shock Proteins in Periapical Granulomas Steven C. Goodman, DDS,* Ariadne Letra, DDS, MS, PhD,* Samuel Dorn, DDS,* Ana Claudia Araujo-Pires, DDS, MS,† Andreia Espindola Vieira, DDS, MS,† Letıcia Chaves de Souza, DDS, MS,* Mamatha Yadlapati, BDS, MDS,* Gustavo Pompermaier Garlet, DDS, MS, PhD,† and Renato Menezes Silva, DDS, MS, PhD* Abstract Introduction: Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). HSPs protect cells under adverse conditions such as infection, inflammation, and disease. We hypothesize that endodontic infection might result in an imbalance in the expression of heat shock genes, accounting for different clinical outcomes in periapical lesions. Methods: We analyzed the expression of 44 HSPs genes using a pathway-specific real-time polymerase chain reaction array in 93 human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively. Observed variations in the expression of HSP genes were also analyzed based on the classification of periapical granulomas as active or inactive. In addition, U937 cells were differentiated into macrophages, infected with different concentrations of purified Escherichia coli lipopolysaccharide (LPS), and used as templates for the HSP gene array. Protein expression was assessed by immunohistochemistry. Results: The expression of HSP genes was significantly increased in granulomas compared with healthy periodontal ligament (P < .00001). Among the 44 HSP genes, DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in both granulomas and LPS-treated macrophages. DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions, whereas HSPA4 expression was higher in inactive lesions (P < .005). Higher concentrations of LPS led to increased HSP expression in macrophages (P < .0001). Immunocytochemistry confirmed the expression and colocalization of HSPB1 and HSPA6 proteins in the cytoplasm of LPS-infected macrophages. Conclusions: The observed differential expression patterns of HSPs in periapical granulomas and LPS-infected macrophages suggest that HSP genes and proteins are involved in periapical lesion development and may account for different clinical outcomes. Understanding the role of the heat shock response might provide addi-
tional insights into the process of periapical lesion development. (J Endod 2014;-:1–7)
Key Words Apical periodontitis, gene expression, heat shock protein, macrophages, protein expression
T
he most primitive mechanism of cellular protection involves the expression of a polypeptide family called heat shock proteins (HSPs). Although the description of HSPs as ‘‘stress proteins’’ was generated based on initial descriptions, individual HSPs fulfill different biological functions (1). Some HSPs are present in unstressed cells and play important roles in the folding and translocation of polypeptides across the cell membrane (1). However, HSPs are characteristically induced by stress signals such as elevated temperature, reduced oxygen supply, infectious agents, and inflammatory mediators (2). Therefore, HSPs also exert a protective role against harmful environmental conditions and pathogens (1). A brief overview of the role of HSPs is presented in Figure 1. According to their molecular weight, HSPs are subdivided into the following groups: HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), HSPD (HSP60), and HSPB (HSP27) (3). HSPs are especially effective in triggering the innate immune response by activating macrophages and macrophage-like cells (4). HSPs can also increase the cellular response to lipopolysaccharide (LPS) to stimulate the production of prototypic proinflammatory cytokines such as tumor necrosis factor alpha (TNF-a) (4). Macrophages are key players in innate immunity, respond rapidly to danger signals generated from inflamed sites, and have 3 major functions once activated: antigen presentation, phagocytosis, and immunomodulation through the production of various cytokines and growth factors (5). In the context of periapical lesions, macrophages apparently have a key role in lesion expansion and subsequent bone resorption (6, 7). Macrophage activation will elicit a prominent proinflammatory action mediated by lymphocyte-recruiting chemokines, the production of tissue-degrading enzymes, and osteoclastogenic effects (8). However, the differential activation of macrophages that may stimulate the production of diverse intrinsic HSPs can induce the development of tolerizing phenotypes, thus suppressing cellular immune reactions (4). Therefore, considering that HSPs can exert active roles on the modulation of the immune response (9), especially on determining the functional heterogeneity of macrophages (10), we hypothesized that the differential expression of HSPs can be associated with the outcome of periapical lesions and that macrophages may be considered a source of HSPs in the periapical lesion microenvironment.
From the *Department of Endodontics, School of Dentistry, University of Texas Health Science Center at Houston, Houston, Texas; and †Osteoimmunology Lab, Department of Biological Sciences, School of Dentistry of Bauru, University of S~ao Paulo, S~ao Paulo, Brazil. Address requests for reprints to Dr Renato Menezes Silva, Department of Endodontics, University of Texas School of Dentistry at Houston, 7500 Cambridge Street, Suite 6411, Houston, TX 77054. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.10.021
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Figure 1. A schematic representation of the various roles of HSPs. A, HSPs function to repair protein damage caused by oxidative stress (ie, fever and infection). B, HSPs bind to antigens and present them to immunoregulatory cells via CD91 and toll-like receptors. Both mechanisms will contribute to cytokine release and T-cell activation.
Materials and Methods Subjects and Samples This study was approved by the Committee for Protection of Human Subjects at the University of Texas Health Science Center at Houston, Houston, TX. Subjects were patients (age, 15–57 years; average = 38.2 years) presenting with periapical lesions characterized radiographically as rarefaction lesions with the disappearance of the periodontal ligament space and discontinuity of the lamina dura; patients were referred for endodontic surgery when teeth failed to heal after conventional root canal treatment. Patients with medical conditions requiring the use of systemic modifiers of bone metabolism or other assisted drug therapy (ie, systemic antibiotics, anti-inflammatory, or hormonal therapy) during the last 6 months before the initiation of the study, patients with pre-existing conditions such as periodontal disease, and pregnant or lactating women were excluded from the study. Periapical lesion samples were collected and divided into 2 roughly similar fragments and stored in formalin and RNALater (Ambion, Austin, TX) solutions. Samples stored in formalin were submitted to routine histologic processing (formalin-fixed paraffin-embedded tissues) and sectioned for histopathologic analyses. Only cases of periapical granulomas, represented by the presence of capillaries, inflammatory cells, fibroblasts, collagen, and macrophages and without the presence of an epithelial lining, were selected for the study. Periapical cysts in which cavities were further developed and lined by stratified squamous epithelium were excluded. A total of 93 periapical granulomas were selected for the study. Healthy periodontal ligament tissue samples (n = 24) obtained from premolars extracted for orthodontic purposes (patients aged 17–23 years) were stored in RNALater solution (Invitrogen, Carlsbad, CA) and used as control specimens. Only 1 sample per individual was used in the study, either as a case or control sample. Cell Culture U937 cells (ATCC, Manassas, VA), a human monocyte cell line, were cultured in RPMI-1640 Medium (ATCC) supplemented with 2
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10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and Penicillin Streptomycin (VWR, Radnor, PA). The cells were incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2. For experiments, 2.5 105 cells/well were seeded and subjected to phorbol-12myristate-13-acetate (VWR) treatment at a final concentration of 100 nmol/L for 72 hours for differentiation into macrophages. The cells were then washed with phosphate-buffered saline (PBS) and incubated in normal growth medium without phorbol-12-myristate-13-acetate for 24 hours before treatment with purified Escherichia coli LPS (Sigma, St Louis, MO). LPS was used at a final concentration of 1 mg/mL and 10 mg/mL for 24 hours. By the end of the process, cell density increased up to 80% of confluence.
Gene Expression Analyses Total RNA was extracted from the tissue samples using TRIZOL reagent (Life Technologies, Grand Island, NY) and from the cells using TaqMan Cells-to-CT Control Kit (Life Technologies) following manufacturer’s instructions. Next, the RNA pellet was dried under a vacuum and resuspended in 50 mL diethyl pyrocarbonate–treated water. The integrity of RNA samples was checked by analyzing 1 mg total RNA on a 1.2% (w/v) denaturing formaldehyde-agarose gel. After RNA extraction, complementary DNA was synthesized using 3 mg RNA through a reverse-transcription reaction using SuperScript III Reverse Trancriptase (Invitrogen). Sample pools of the 93 human periapical granulomas (cases) and the 24 human healthy periodontal ligament tissue samples (controls) were obtained and used for posterior analyses. We investigated the messenger RNA (mRNA) expression of 44 HSP genes in a pool of the periapical granuloma samples and controls and in the macrophages with and without LPS treatment (1 mg/mL and 10 mg/mL) using a TaqMan Human Heat Shock Proteins Array (Life Technologies, Foster City, CA) in a quantitative real-time reverse-transcription polymerase chain reaction. 18S, GAPDH, HPRT1, and GUSB were used as internal control genes for normalization as provided in the reaction arrays. We chose a pooled sample analysis because of the elevated cost of the arrays. We also investigated the mRNA expression of receptor JOE — Volume -, Number -, - 2014
Basic Research—Biology nuclear factor kappa ligand (RANKL) and osteoprotegerin (OPG) in the individual periapical granuloma samples in order to classify them as active/progressive or inactive/stable as previously described (11). In brief, lesions showing a higher RANKL to OPG ratio (RANKL > OPG) were classified as active/progressive, whereas lesions with a lower RANKL to OPG ratio (RANKL < OPG) were classified as inactive/stable (11). Thereafter, lesions characterized as active and inactive were used as templates for the analyses of HSP expression as described previously. Finally, we investigated the mRNA expression of the proinflammatory cytokines interleukin-1 beta (IL1-B), interleukin-6 (IL-6), RANKL, and TNF-a in the individual active periapical granuloma samples (n = 43) using gene-specific primers and SYBR Green chemistry (Life Technologies) in a quantitative real-time reverse transcription polymerase chain reaction. Beta-actin and 18S were used as an endogenous control gene for normalization. Primer sequences are available on request. All gene expression experiments were performed in triplicates and repeated once.
Data Analyses The mean cycle threshold (Ct) values from triplicate measurements of the target gene with normalization to the internal controls genes were calculated using the delta-delta Ct method (12). Array data were analyzed using DataAssist v.3.01 and Expression Suite v.1.0 software (Life Technologies). Statistical analyses included analysis of variance followed by Bonferroni correction in GraphPad Prism 5.0 (GraphPad Inc, San Diego, CA). A P value #.05 was considered statistically significant. Linear regression analysis was used to test the correlations between the levels of HSPs and proinflammatory cytokine expression in active periapical granulomas represented by P values and r2. Immunocytochemistry and Immunohistochemistry Macrophage cells were fixed for 15 minutes in 4% formaldehyde in PBS. Cells were incubated with anti-Hsp27 (HSPB1) mouse monoclonal antibody (Abcam, Cambridge, MA) and anti-HSPA6 polyclonal rabbit antibody (Abcam) at a final dilution of 1:100 followed by staining with secondary antibodies (Alexa Fluor 488 Goat Anti-Mouse IgG and Alexa Fluor 594 Donkey Anti-Rabbit IgG [Invitrogen]) at a final concentration of 1:1,000. Lastly, cells were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) for nuclear staining. Images were captured using a Leica TCS SP5 laser confocal microscope (Leica Microsystems, Buffalo Grove, IL). Periapical granuloma tissue sections (n = 5) were used for immunohistochemical localization of HSPB1 and HSPA6. Sections were deparaffinized in xylene and rehydrated in ethanol. Antigenic retrieval was performed in citrate buffer (pH = 6.0) at 96 C for 20 minutes. Sections were incubated overnight at 4 C with anti-Hsp27 (HSPB1) and anti-HSPA6 primary antibodies and incubated in secondary antibodies for 1 hour at room temperature. Expression was detected using 30 -diaminobenzidine chromogen and counterstaining with Harris hematoxylin solution (Sigma, St Louis, MO). Negative control sections received PBS to replace primary antibodies. Positive control sections were used as suggested by the antibody manufacturers.
Results Gene Expression Analysis The expression of all 44 HSP genes was significantly increased in the pool of periapical granulomas compared with the pool of healthy periodontal ligaments (P < .00001). DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in the periapical granuJOE — Volume -, Number -, - 2014
lomas (Fig. 2A). Similarly, the expression of HSP genes was significantly higher in LPS-treated macrophages in comparison with macrophages without LPS treatment (P < .0001), with DNAJC3, HSPA4, HSPA6, and HSPB1 being the most highly expressed genes, as seen with the periapical granulomas, and also HSF2BP and HSP90B (Fig. 2C). From the 93 human periapical samples, 43 were classified as active/progressive and 50 as inactive/stable (data not shown). Analyses based on lesion activity status showed an increased expression of DNAJC3, HSPA6/HSPA7, and HSPB1 genes in active lesions (P < .001), whereas HSPA4 expression was higher in inactive lesions (P < .005) (Fig. 2B). Moreover, in active lesions, DNAJC3, HSPA6/HSPA7, and HSPB1 showed a positive correlation with IL-1b mRNA levels (P < .003). DNAJC3 and HSPB1 were also positively correlated with RANKL and TNF-a mRNA levels (P < .0001), whereas HSPA6/HSPA7 and DNAJC3 presented a positive correlation with IL-6 mRNA levels (P < .04), reinforcing their role as immune response activators (Fig. 3).
Protein Expression Analysis Immunocytochemistry analysis of LPS-infected macrophages showed a positive expression of HSPB1 and HSPA6 proteins and their colocalization in cell cytoplasm (Fig. 4A and B). Immunohistochemistry analyses of periapical granuloma tissue sections also showed positive staining of HSPB1 and HSPA6 dispersedly throughout the tissue and also in epithelial cells in the periphery (Fig. 4C and D).
Discussion In this study, we hypothesized that HSPs could play a role in the development process of apical periodontitis and that macrophages could be a source of HSPs in this process. HSPs have numerous functions, including the facilitation of protein folding and antigen-presenting properties to the immune system (13, 14). Furthermore, extracellular HSPs can stimulate the release of TNF-a; IL-1b, -6 and -12; nitric oxide; and chemokines by monocytes/macrophages (15), which can contribute to the development and/or expansion of periapical lesions. To test our hypothesis, we compared the expression of 44 HSP genes in a pool of human periapical granulomas with that in a pool of healthy periodontal ligament tissue samples and in macrophages with and without LPS treatment. HSP genes are subdivided into different gene families based on molecular weight as follows: HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), HSPD (HSP60), and HSPB (HSP27) (3). In the context of our findings for individual gene expression, members of the HSP27 (HSPB1), HSP40 (HSPA6), HSP70 (DNAJC3), and HSP110 (HSPA4) gene families were significantly overexpressed in the periapical granulomas when compared with controls, particularly in the active lesions, as well as in LPS-treated cells. HSF2B and HSP90B, both members of the HSP27 gene family, were also highly expressed in the LPS-treated macrophages. HSPA4, a member of the HSP70 family, was the most highly expressed gene in the inactive granulomas. These findings suggest that HSPs may have modulatory roles during periapical lesion development and that different heat shock genes/proteins may have distinct roles in this process. Further investigation is warranted to assess the role of each HSP in lesion progression or remission. Macrophages are key cell players in the development process of apical periodontitis; they appear to be a source of HSPs in this process. Intriguingly, basal expression levels of HSP genes were detected in the macrophages regardless of LPS treatment although HSP expression was considerably higher in the LPS-treated group and with increased LPS concentration. Our results in the macrophages were consistent with the results obtained with the periapical granulomas, further implicating a role for HSPs in the dynamics of periapical lesions. Furthermore, in
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Figure 2. The expression of HSP genes in periapical granulomas and macrophages with and without LPS treatment. (A) The expression of all 44 HSP genes was significantly increased in the periapical granuloma pool compared with healthy periodontal ligaments (P < .00001). (B) DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions (RANKL > OPG), whereas HSPA4 was highly expressed in inactive lesions (OPG > RANKL) (P < .005). Results show mean Ct values from triplicate measurements of the target gene with normalization to the internal controls. Relative changes in gene expression were calculated using the delta delta Ct method (12). (C) The expression of HSP genes was significantly increased in LPS-treated macrophages in comparison with untreated cells (P < .0001). A higher LPS concentration resulted in increased HSP gene expression; DNAJC3, HSBP1, HSF2BP, HSP90B, HSPA4, and HSPA6/HSPA expression was significantly higher in macrophages treated with 10 mg/mL LPS (P < .001).
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Figure 3. Correlations between the expression of HSPs and the mediators of lesion progression in active periapical granulomas. Total RNA was extracted from individual periapical granulomas (n = 43), and DNAJC3, HSPA6, HSPB1, RANKL, TNF-a, IL-1B and IL-6 mRNA expression was measured by quantitative real-time polymerase chain reaction. Linear regression analysis was used to test the correlations between the levels of HSPs and mediators of lesion progression expression in active periapical granulomas (represented by P values and r2).
this cell-based model, the observed increase in the HSP expression level subsequent to the increase in LPS concentration allowed us to correlate that a higher level of cell stress triggers the release of more HSPs, which then function as endogenous alert signals to the host immune system through their cytokine-like function (9). After stress exposure, a family of heat shock transcription factors rapidly induces the expression of cytoprotective HSPs including the HSP70 molecular chaperone (16). The binding of newly synthesized polypeptides to HSP chaperones and the subsequent release of folded proteins are regulated by continuous cycles of adenosine triphosphate (ATP) hydrolysis and the exchange of ATP for adenosine diphosphate. The HSP70 chaperone represents the major protein folding machinery in the eukaryotic cytosol, and it complexes with 2 cochaperones, HSP40 and HSP110. HSP40 stimulates ATP hydrolysis, whereas HSP110 acts as a nucleotide exchange factor that accelerates adenosine diphosphate dissociation from HSP70 (17). All HSPs investigated in the present study were significantly more expressed in the periapical granulomas and LPS-stimulated macrophages when compared with controls. HSP70, JOE — Volume -, Number -, - 2014
which was more expressed in active lesions, functions to catalyze the proper folding of nascent proteins and, under stressful conditions, refolds the damaged proteins or targets unrepairable proteins for degradation (18). HSP70 has also been suggested as essential for inducible nitric oxide synthase induction in macrophages (19). The activation of macrophages by endotoxins such as LPS, or cytokines, results in the production of inducible nitric oxide synthase and generates copious amounts of nitric oxide (a cytotoxic weapon generated by macrophages), presumably to help kill or inhibit the growth of invading microorganisms (20). In the periapical environment, continuous LPS stimulation can cause accumulation of the damaged protein contributing to cell apoptosis. High levels of HSP70, as presented by the increased expression of DNAJC3 in this study, may suggest an attempt to promote repair and avoid cell apoptosis. The HSPA4 gene was highly expressed in the inactive lesions. HSPA4 is a member of the HSP 110 family (HSP110) that acts as a nucleotide exchange factor of HSP70 chaperones with a special role in protein quality control, maintaining proper protein folding and
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Figure 4. The localization of HSPB1 and HSPA6 proteins in LPS-treated macrophages and human periapical granulomas. Immunocytochemistry analyses showing a positive expression of HSPB1 (green) and HSPA6 (red) in macrophages treated with (A) 1 ug/mL LPS and (B) 10 mg/mL. Cell nuclei are stained blue (DAPI). Note that HSPB1 and HSPA6 colocalize in the cell cytoplasm (1000 magnification). (C and D) Immunohistochemistry analysis of (C) HSPB1 and (D) HSPA6 proteins in 2 human periapical granuloma samples. Note that both HSPB1 and HSPA6 are expressed throughout the tissue and also in the epithelial layers (20 magnification).
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Basic Research—Biology homeostasis (17). From our findings, we may argue that HSPA4 may play a role in the regulatory machinery that re-establishes protein homeostasis to counteract protein-unfolding stress (21). The HSP27 family plays a part in the regulation of epithelial cell growth and differentiation, wound healing, apoptosis, and cell protection against inflammatory cytotoxic mediators (22). The expression of members of the HSP27 family was also up-regulated in active lesions and LPS-treated macrophages. This protein was previously reported to be expressed in the epithelium of periapical lesions (23). HSP27 may play several roles in periapical lesions that include contributing to the migration of epithelial cell rests and to an increased resistance to necrotic and apoptotic cell death (23). Moreover, increased levels of HSP27 in macrophages inhibit the activation of caspase-3, an essential caspase for monocyte apoptosis (3). The presence of stimulatory signals triggers monocyte survival by inhibiting the apoptotic pathway, thus contributing to the maintenance of the inflammatory response (24). HSPs have been linked to the therapy of both cancer and inflammatory diseases, approaches that use contrasting immune properties of these proteins. It would appear that HSP family members HSP60 and HSP70, whether from external sources or induced locally during inflammation, can be processed by antigen-presenting cells and that HSP-derived epitopes activate regulatory T-cells to suppress inflammatory disease (25). Interestingly, in the present study, members of the HSP70 family were highly expressed in both periapical granulomas and LPS-treated macrophages. Furthermore, correlation analyses reinforced a role for different HSPs as immune response activators, with DNAJC3, HSPA6/HSPA7, and HSPB1 being positively correlated with IL-1b mRNA levels in active lesions, and DNAJC3 and HSPB1 also showing a positive correlation with RANKL and TNF-a mRNA levels. A positive correlation between HSPA6/HSPA7 and DNAJC3 with IL-6 mRNA levels provides additional evidence for the critical role of HSPs in triggering the host immune defense against pathogens. In summary, our findings indicate that HSP27, HSP40, and HSP70 gene families play a crucial role in the maintenance of infection and subsequent tissue destruction in apical periodontitis, whereas HSP110 appears to have a role in re-establishing homeostasis in the periapical environment. These results may provide insights into a better understanding of the immune system and allow for the identification of unique markers to improve the diagnosis and treatment of apical periodontitis. Additional mechanistic studies in animal models may help elucidate the specific role for each HSP in apical periodontitis.
Acknowledgments The authors thank the individuals that agreed to participate in this study and Pasha Goodman for invaluable help in the critical review of the manuscript. Supported in part by UTSD startup funds to R.M.S., AAE Foundation to S.C.G., Fundac¸ao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) to G.P.G, and Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nivel Superior (CAPES), BEX 1099/12-4, Brazil to L.C.S.
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The authors deny any conflicts of interest related to this study.
References 1. De Maio A. Heat shock proteins: facts, thoughts, and dreams. Shock 1999;11:1–12. 2. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998;12:3788–96. 3. Voss OH, Batra S, Kolattukudy SJ, et al. Binding of caspase-3 prodomain to heat shock protein 27 regulates monocyte apoptosis by inhibiting caspase-3 proteolytic activation. J Biol Chem 2007;282:25088–99. 4. Van Noort JM, Bsibsi M, Nacken P, et al. The link between small heat shock proteins and the immune system. Int J Biochem Cell Biol 2012;44:1670–9. 5. Fujiwara N, Kobayashi K. Macrophages in inflammation. Curr Drug Targets Inflamm Allergy 2005;4:281–6. 6. Wang CY, Tani-Ishii N, Stashenko P. Bone-resorptive cytokine gene expression in periapical lesions in the rat. Oral Microbiol Immunol 1997;12:65–71. 7. Suzuki N, Okiji T, Suda H. Enhanced expression of activation-associated molecules on macrophages of heterogeneous populations in expanding periapical lesions in rat molars. Arch Oral Biol 1999;44:67–79. 8. Garlet GP. Destructive and protective roles of cytokines in periodontitis: a reappraisal from host defense and tissue destruction viewpoints. J Dent Res 2010; 89:1349–63. 9. Lee CT, Repasky EA. Opposing roles for heat and heat shock proteins in macrophage functions during inflammation: a function of cell activation state? Front Immunol 2012;3:140. 10. Dijkstra CD, Damoiseaux JG. Macrophage heterogeneity established by immunocytochemistry. Prog Histochem Cytochem 1993;27:1–65. 11. Menezes R, Garlet TP, Letra A, et al. Differential patterns of receptor activator of nuclear factor kappa B ligand/osteoprotegerin expression in human periapical granulomas: possible association with progressive or stable nature of the lesions. J Endod 2008;34:932–8. 12. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–8. 13. Csermely P, Schnaider T, Soti C, et al. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998;79:129–68. 14. Srivastava PK, Menoret A, Basu S, et al. Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world. Immunity 1998;8:657–65. 15. Vega VL, Rodriguez-Silva M, Frey T, et al. Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membraneassociated form that activates macrophages. J Immunol 2008;180:4299–307. 16. Anckar J, Sistonen L. Regulation of HSF1 function in the heat stress response: implications in aging and disease. Annu Rev Biochem 2011;80:1089–115. 17. Polier S, Dragovic Z, Hartl FU, et al. Structural basis for the cooperation of Hsp70 and Hsp110 chaperones in protein folding. Cell 2008;133:1068–79. 18. Vabulas RM, Raychaudhuri S, Hayer-Hartl M, et al. Protein folding in the cytoplasm and the heat shock response. Cold Spring Harb Perspect Biol 2010;2:a004390. 19. Zhang L, Liu Q, Yuan X, et al. Requirement of heat shock protein 70 for inducible nitric oxide synthase induction. Cell Signal 2013;25:1310–7. 20. Petruson K, Stalfors J, Jacobsson KE, et al. Nitric oxide production in the sphenoidal sinus by the inducible and constitutive isozymes of nitric oxide synthase. Rhinology 2005;43:18–23. 21. Rampelt H, Kirstein-Miles J, Nillegoda NB, et al. Metazoan Hsp70 machines use Hsp110 to power protein disaggregation. EMBO J 2012;31:4221–35. 22. Welsh MJ, Gaestel M. Small heat-shock protein family: function in health and disease. Ann N Y Acad Sci 1998;851:28–35. 23. Leonardi R, Villari L, Caltabiano M, et al. Heat shock protein 27 expression in the epithelium of periapical lesions. J Endod 2001;27:89–92. 24. Goyal A, Wang Y, Graham MM, et al. Monocyte survival factors induce Akt activation and suppress caspase-3. Am J Respir Cell Mol Biol 2002;26:224–30. 25. Calderwood SK, Stevenson MA, Murshid A. Heat shock proteins, autoimmunity, and cancer treatment. Autoimmune Dis 2012;2012:486069.
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Expression of Heat Shock Proteins in Periapical Granulomas Steven C. Goodman, DDS,* Ariadne Letra, DDS, MS, PhD,* Samuel Dorn, DDS,* Ana Claudia Araujo-Pires, DDS, MS,† Andreia Espindola Vieira, DDS, MS,† Letıcia Chaves de Souza, DDS, MS,* Mamatha Yadlapati, BDS, MDS,* Gustavo Pompermaier Garlet, DDS, MS, PhD,† and Renato Menezes Silva, DDS, MS, PhD* Abstract Introduction: Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). HSPs protect cells under adverse conditions such as infection, inflammation, and disease. We hypothesize that endodontic infection might result in an imbalance in the expression of heat shock genes, accounting for different clinical outcomes in periapical lesions. Methods: We analyzed the expression of 44 HSPs genes using a pathway-specific real-time polymerase chain reaction array in 93 human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively. Observed variations in the expression of HSP genes were also analyzed based on the classification of periapical granulomas as active or inactive. In addition, U937 cells were differentiated into macrophages, infected with different concentrations of purified Escherichia coli lipopolysaccharide (LPS), and used as templates for the HSP gene array. Protein expression was assessed by immunohistochemistry. Results: The expression of HSP genes was significantly increased in granulomas compared with healthy periodontal ligament (P < .00001). Among the 44 HSP genes, DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in both granulomas and LPS-treated macrophages. DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions, whereas HSPA4 expression was higher in inactive lesions (P < .005). Higher concentrations of LPS led to increased HSP expression in macrophages (P < .0001). Immunocytochemistry confirmed the expression and colocalization of HSPB1 and HSPA6 proteins in the cytoplasm of LPS-infected macrophages. Conclusions: The observed differential expression patterns of HSPs in periapical granulomas and LPS-infected macrophages suggest that HSP genes and proteins are involved in periapical lesion development and may account for different clinical outcomes. Understanding the role of the heat shock response might provide addi-
tional insights into the process of periapical lesion development. (J Endod 2014;-:1–7)
Key Words Apical periodontitis, gene expression, heat shock protein, macrophages, protein expression
T
he most primitive mechanism of cellular protection involves the expression of a polypeptide family called heat shock proteins (HSPs). Although the description of HSPs as ‘‘stress proteins’’ was generated based on initial descriptions, individual HSPs fulfill different biological functions (1). Some HSPs are present in unstressed cells and play important roles in the folding and translocation of polypeptides across the cell membrane (1). However, HSPs are characteristically induced by stress signals such as elevated temperature, reduced oxygen supply, infectious agents, and inflammatory mediators (2). Therefore, HSPs also exert a protective role against harmful environmental conditions and pathogens (1). A brief overview of the role of HSPs is presented in Figure 1. According to their molecular weight, HSPs are subdivided into the following groups: HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), HSPD (HSP60), and HSPB (HSP27) (3). HSPs are especially effective in triggering the innate immune response by activating macrophages and macrophage-like cells (4). HSPs can also increase the cellular response to lipopolysaccharide (LPS) to stimulate the production of prototypic proinflammatory cytokines such as tumor necrosis factor alpha (TNF-a) (4). Macrophages are key players in innate immunity, respond rapidly to danger signals generated from inflamed sites, and have 3 major functions once activated: antigen presentation, phagocytosis, and immunomodulation through the production of various cytokines and growth factors (5). In the context of periapical lesions, macrophages apparently have a key role in lesion expansion and subsequent bone resorption (6, 7). Macrophage activation will elicit a prominent proinflammatory action mediated by lymphocyte-recruiting chemokines, the production of tissue-degrading enzymes, and osteoclastogenic effects (8). However, the differential activation of macrophages that may stimulate the production of diverse intrinsic HSPs can induce the development of tolerizing phenotypes, thus suppressing cellular immune reactions (4). Therefore, considering that HSPs can exert active roles on the modulation of the immune response (9), especially on determining the functional heterogeneity of macrophages (10), we hypothesized that the differential expression of HSPs can be associated with the outcome of periapical lesions and that macrophages may be considered a source of HSPs in the periapical lesion microenvironment.
From the *Department of Endodontics, School of Dentistry, University of Texas Health Science Center at Houston, Houston, Texas; and †Osteoimmunology Lab, Department of Biological Sciences, School of Dentistry of Bauru, University of S~ao Paulo, S~ao Paulo, Brazil. Address requests for reprints to Dr Renato Menezes Silva, Department of Endodontics, University of Texas School of Dentistry at Houston, 7500 Cambridge Street, Suite 6411, Houston, TX 77054. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.10.021
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Figure 1. A schematic representation of the various roles of HSPs. A, HSPs function to repair protein damage caused by oxidative stress (ie, fever and infection). B, HSPs bind to antigens and present them to immunoregulatory cells via CD91 and toll-like receptors. Both mechanisms will contribute to cytokine release and T-cell activation.
Materials and Methods Subjects and Samples This study was approved by the Committee for Protection of Human Subjects at the University of Texas Health Science Center at Houston, Houston, TX. Subjects were patients (age, 15–57 years; average = 38.2 years) presenting with periapical lesions characterized radiographically as rarefaction lesions with the disappearance of the periodontal ligament space and discontinuity of the lamina dura; patients were referred for endodontic surgery when teeth failed to heal after conventional root canal treatment. Patients with medical conditions requiring the use of systemic modifiers of bone metabolism or other assisted drug therapy (ie, systemic antibiotics, anti-inflammatory, or hormonal therapy) during the last 6 months before the initiation of the study, patients with pre-existing conditions such as periodontal disease, and pregnant or lactating women were excluded from the study. Periapical lesion samples were collected and divided into 2 roughly similar fragments and stored in formalin and RNALater (Ambion, Austin, TX) solutions. Samples stored in formalin were submitted to routine histologic processing (formalin-fixed paraffin-embedded tissues) and sectioned for histopathologic analyses. Only cases of periapical granulomas, represented by the presence of capillaries, inflammatory cells, fibroblasts, collagen, and macrophages and without the presence of an epithelial lining, were selected for the study. Periapical cysts in which cavities were further developed and lined by stratified squamous epithelium were excluded. A total of 93 periapical granulomas were selected for the study. Healthy periodontal ligament tissue samples (n = 24) obtained from premolars extracted for orthodontic purposes (patients aged 17–23 years) were stored in RNALater solution (Invitrogen, Carlsbad, CA) and used as control specimens. Only 1 sample per individual was used in the study, either as a case or control sample. Cell Culture U937 cells (ATCC, Manassas, VA), a human monocyte cell line, were cultured in RPMI-1640 Medium (ATCC) supplemented with 2
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10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and Penicillin Streptomycin (VWR, Radnor, PA). The cells were incubated at 37 C in a humidified atmosphere of 95% air and 5% CO2. For experiments, 2.5 105 cells/well were seeded and subjected to phorbol-12myristate-13-acetate (VWR) treatment at a final concentration of 100 nmol/L for 72 hours for differentiation into macrophages. The cells were then washed with phosphate-buffered saline (PBS) and incubated in normal growth medium without phorbol-12-myristate-13-acetate for 24 hours before treatment with purified Escherichia coli LPS (Sigma, St Louis, MO). LPS was used at a final concentration of 1 mg/mL and 10 mg/mL for 24 hours. By the end of the process, cell density increased up to 80% of confluence.
Gene Expression Analyses Total RNA was extracted from the tissue samples using TRIZOL reagent (Life Technologies, Grand Island, NY) and from the cells using TaqMan Cells-to-CT Control Kit (Life Technologies) following manufacturer’s instructions. Next, the RNA pellet was dried under a vacuum and resuspended in 50 mL diethyl pyrocarbonate–treated water. The integrity of RNA samples was checked by analyzing 1 mg total RNA on a 1.2% (w/v) denaturing formaldehyde-agarose gel. After RNA extraction, complementary DNA was synthesized using 3 mg RNA through a reverse-transcription reaction using SuperScript III Reverse Trancriptase (Invitrogen). Sample pools of the 93 human periapical granulomas (cases) and the 24 human healthy periodontal ligament tissue samples (controls) were obtained and used for posterior analyses. We investigated the messenger RNA (mRNA) expression of 44 HSP genes in a pool of the periapical granuloma samples and controls and in the macrophages with and without LPS treatment (1 mg/mL and 10 mg/mL) using a TaqMan Human Heat Shock Proteins Array (Life Technologies, Foster City, CA) in a quantitative real-time reverse-transcription polymerase chain reaction. 18S, GAPDH, HPRT1, and GUSB were used as internal control genes for normalization as provided in the reaction arrays. We chose a pooled sample analysis because of the elevated cost of the arrays. We also investigated the mRNA expression of receptor JOE — Volume -, Number -, - 2014
Basic Research—Biology nuclear factor kappa ligand (RANKL) and osteoprotegerin (OPG) in the individual periapical granuloma samples in order to classify them as active/progressive or inactive/stable as previously described (11). In brief, lesions showing a higher RANKL to OPG ratio (RANKL > OPG) were classified as active/progressive, whereas lesions with a lower RANKL to OPG ratio (RANKL < OPG) were classified as inactive/stable (11). Thereafter, lesions characterized as active and inactive were used as templates for the analyses of HSP expression as described previously. Finally, we investigated the mRNA expression of the proinflammatory cytokines interleukin-1 beta (IL1-B), interleukin-6 (IL-6), RANKL, and TNF-a in the individual active periapical granuloma samples (n = 43) using gene-specific primers and SYBR Green chemistry (Life Technologies) in a quantitative real-time reverse transcription polymerase chain reaction. Beta-actin and 18S were used as an endogenous control gene for normalization. Primer sequences are available on request. All gene expression experiments were performed in triplicates and repeated once.
Data Analyses The mean cycle threshold (Ct) values from triplicate measurements of the target gene with normalization to the internal controls genes were calculated using the delta-delta Ct method (12). Array data were analyzed using DataAssist v.3.01 and Expression Suite v.1.0 software (Life Technologies). Statistical analyses included analysis of variance followed by Bonferroni correction in GraphPad Prism 5.0 (GraphPad Inc, San Diego, CA). A P value #.05 was considered statistically significant. Linear regression analysis was used to test the correlations between the levels of HSPs and proinflammatory cytokine expression in active periapical granulomas represented by P values and r2. Immunocytochemistry and Immunohistochemistry Macrophage cells were fixed for 15 minutes in 4% formaldehyde in PBS. Cells were incubated with anti-Hsp27 (HSPB1) mouse monoclonal antibody (Abcam, Cambridge, MA) and anti-HSPA6 polyclonal rabbit antibody (Abcam) at a final dilution of 1:100 followed by staining with secondary antibodies (Alexa Fluor 488 Goat Anti-Mouse IgG and Alexa Fluor 594 Donkey Anti-Rabbit IgG [Invitrogen]) at a final concentration of 1:1,000. Lastly, cells were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen) for nuclear staining. Images were captured using a Leica TCS SP5 laser confocal microscope (Leica Microsystems, Buffalo Grove, IL). Periapical granuloma tissue sections (n = 5) were used for immunohistochemical localization of HSPB1 and HSPA6. Sections were deparaffinized in xylene and rehydrated in ethanol. Antigenic retrieval was performed in citrate buffer (pH = 6.0) at 96 C for 20 minutes. Sections were incubated overnight at 4 C with anti-Hsp27 (HSPB1) and anti-HSPA6 primary antibodies and incubated in secondary antibodies for 1 hour at room temperature. Expression was detected using 30 -diaminobenzidine chromogen and counterstaining with Harris hematoxylin solution (Sigma, St Louis, MO). Negative control sections received PBS to replace primary antibodies. Positive control sections were used as suggested by the antibody manufacturers.
Results Gene Expression Analysis The expression of all 44 HSP genes was significantly increased in the pool of periapical granulomas compared with the pool of healthy periodontal ligaments (P < .00001). DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in the periapical granuJOE — Volume -, Number -, - 2014
lomas (Fig. 2A). Similarly, the expression of HSP genes was significantly higher in LPS-treated macrophages in comparison with macrophages without LPS treatment (P < .0001), with DNAJC3, HSPA4, HSPA6, and HSPB1 being the most highly expressed genes, as seen with the periapical granulomas, and also HSF2BP and HSP90B (Fig. 2C). From the 93 human periapical samples, 43 were classified as active/progressive and 50 as inactive/stable (data not shown). Analyses based on lesion activity status showed an increased expression of DNAJC3, HSPA6/HSPA7, and HSPB1 genes in active lesions (P < .001), whereas HSPA4 expression was higher in inactive lesions (P < .005) (Fig. 2B). Moreover, in active lesions, DNAJC3, HSPA6/HSPA7, and HSPB1 showed a positive correlation with IL-1b mRNA levels (P < .003). DNAJC3 and HSPB1 were also positively correlated with RANKL and TNF-a mRNA levels (P < .0001), whereas HSPA6/HSPA7 and DNAJC3 presented a positive correlation with IL-6 mRNA levels (P < .04), reinforcing their role as immune response activators (Fig. 3).
Protein Expression Analysis Immunocytochemistry analysis of LPS-infected macrophages showed a positive expression of HSPB1 and HSPA6 proteins and their colocalization in cell cytoplasm (Fig. 4A and B). Immunohistochemistry analyses of periapical granuloma tissue sections also showed positive staining of HSPB1 and HSPA6 dispersedly throughout the tissue and also in epithelial cells in the periphery (Fig. 4C and D).
Discussion In this study, we hypothesized that HSPs could play a role in the development process of apical periodontitis and that macrophages could be a source of HSPs in this process. HSPs have numerous functions, including the facilitation of protein folding and antigen-presenting properties to the immune system (13, 14). Furthermore, extracellular HSPs can stimulate the release of TNF-a; IL-1b, -6 and -12; nitric oxide; and chemokines by monocytes/macrophages (15), which can contribute to the development and/or expansion of periapical lesions. To test our hypothesis, we compared the expression of 44 HSP genes in a pool of human periapical granulomas with that in a pool of healthy periodontal ligament tissue samples and in macrophages with and without LPS treatment. HSP genes are subdivided into different gene families based on molecular weight as follows: HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), DNAJ (HSP40), HSPD (HSP60), and HSPB (HSP27) (3). In the context of our findings for individual gene expression, members of the HSP27 (HSPB1), HSP40 (HSPA6), HSP70 (DNAJC3), and HSP110 (HSPA4) gene families were significantly overexpressed in the periapical granulomas when compared with controls, particularly in the active lesions, as well as in LPS-treated cells. HSF2B and HSP90B, both members of the HSP27 gene family, were also highly expressed in the LPS-treated macrophages. HSPA4, a member of the HSP70 family, was the most highly expressed gene in the inactive granulomas. These findings suggest that HSPs may have modulatory roles during periapical lesion development and that different heat shock genes/proteins may have distinct roles in this process. Further investigation is warranted to assess the role of each HSP in lesion progression or remission. Macrophages are key cell players in the development process of apical periodontitis; they appear to be a source of HSPs in this process. Intriguingly, basal expression levels of HSP genes were detected in the macrophages regardless of LPS treatment although HSP expression was considerably higher in the LPS-treated group and with increased LPS concentration. Our results in the macrophages were consistent with the results obtained with the periapical granulomas, further implicating a role for HSPs in the dynamics of periapical lesions. Furthermore, in
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Figure 2. The expression of HSP genes in periapical granulomas and macrophages with and without LPS treatment. (A) The expression of all 44 HSP genes was significantly increased in the periapical granuloma pool compared with healthy periodontal ligaments (P < .00001). (B) DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions (RANKL > OPG), whereas HSPA4 was highly expressed in inactive lesions (OPG > RANKL) (P < .005). Results show mean Ct values from triplicate measurements of the target gene with normalization to the internal controls. Relative changes in gene expression were calculated using the delta delta Ct method (12). (C) The expression of HSP genes was significantly increased in LPS-treated macrophages in comparison with untreated cells (P < .0001). A higher LPS concentration resulted in increased HSP gene expression; DNAJC3, HSBP1, HSF2BP, HSP90B, HSPA4, and HSPA6/HSPA expression was significantly higher in macrophages treated with 10 mg/mL LPS (P < .001).
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Figure 3. Correlations between the expression of HSPs and the mediators of lesion progression in active periapical granulomas. Total RNA was extracted from individual periapical granulomas (n = 43), and DNAJC3, HSPA6, HSPB1, RANKL, TNF-a, IL-1B and IL-6 mRNA expression was measured by quantitative real-time polymerase chain reaction. Linear regression analysis was used to test the correlations between the levels of HSPs and mediators of lesion progression expression in active periapical granulomas (represented by P values and r2).
this cell-based model, the observed increase in the HSP expression level subsequent to the increase in LPS concentration allowed us to correlate that a higher level of cell stress triggers the release of more HSPs, which then function as endogenous alert signals to the host immune system through their cytokine-like function (9). After stress exposure, a family of heat shock transcription factors rapidly induces the expression of cytoprotective HSPs including the HSP70 molecular chaperone (16). The binding of newly synthesized polypeptides to HSP chaperones and the subsequent release of folded proteins are regulated by continuous cycles of adenosine triphosphate (ATP) hydrolysis and the exchange of ATP for adenosine diphosphate. The HSP70 chaperone represents the major protein folding machinery in the eukaryotic cytosol, and it complexes with 2 cochaperones, HSP40 and HSP110. HSP40 stimulates ATP hydrolysis, whereas HSP110 acts as a nucleotide exchange factor that accelerates adenosine diphosphate dissociation from HSP70 (17). All HSPs investigated in the present study were significantly more expressed in the periapical granulomas and LPS-stimulated macrophages when compared with controls. HSP70, JOE — Volume -, Number -, - 2014
which was more expressed in active lesions, functions to catalyze the proper folding of nascent proteins and, under stressful conditions, refolds the damaged proteins or targets unrepairable proteins for degradation (18). HSP70 has also been suggested as essential for inducible nitric oxide synthase induction in macrophages (19). The activation of macrophages by endotoxins such as LPS, or cytokines, results in the production of inducible nitric oxide synthase and generates copious amounts of nitric oxide (a cytotoxic weapon generated by macrophages), presumably to help kill or inhibit the growth of invading microorganisms (20). In the periapical environment, continuous LPS stimulation can cause accumulation of the damaged protein contributing to cell apoptosis. High levels of HSP70, as presented by the increased expression of DNAJC3 in this study, may suggest an attempt to promote repair and avoid cell apoptosis. The HSPA4 gene was highly expressed in the inactive lesions. HSPA4 is a member of the HSP 110 family (HSP110) that acts as a nucleotide exchange factor of HSP70 chaperones with a special role in protein quality control, maintaining proper protein folding and
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Figure 4. The localization of HSPB1 and HSPA6 proteins in LPS-treated macrophages and human periapical granulomas. Immunocytochemistry analyses showing a positive expression of HSPB1 (green) and HSPA6 (red) in macrophages treated with (A) 1 ug/mL LPS and (B) 10 mg/mL. Cell nuclei are stained blue (DAPI). Note that HSPB1 and HSPA6 colocalize in the cell cytoplasm (1000 magnification). (C and D) Immunohistochemistry analysis of (C) HSPB1 and (D) HSPA6 proteins in 2 human periapical granuloma samples. Note that both HSPB1 and HSPA6 are expressed throughout the tissue and also in the epithelial layers (20 magnification).
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Basic Research—Biology homeostasis (17). From our findings, we may argue that HSPA4 may play a role in the regulatory machinery that re-establishes protein homeostasis to counteract protein-unfolding stress (21). The HSP27 family plays a part in the regulation of epithelial cell growth and differentiation, wound healing, apoptosis, and cell protection against inflammatory cytotoxic mediators (22). The expression of members of the HSP27 family was also up-regulated in active lesions and LPS-treated macrophages. This protein was previously reported to be expressed in the epithelium of periapical lesions (23). HSP27 may play several roles in periapical lesions that include contributing to the migration of epithelial cell rests and to an increased resistance to necrotic and apoptotic cell death (23). Moreover, increased levels of HSP27 in macrophages inhibit the activation of caspase-3, an essential caspase for monocyte apoptosis (3). The presence of stimulatory signals triggers monocyte survival by inhibiting the apoptotic pathway, thus contributing to the maintenance of the inflammatory response (24). HSPs have been linked to the therapy of both cancer and inflammatory diseases, approaches that use contrasting immune properties of these proteins. It would appear that HSP family members HSP60 and HSP70, whether from external sources or induced locally during inflammation, can be processed by antigen-presenting cells and that HSP-derived epitopes activate regulatory T-cells to suppress inflammatory disease (25). Interestingly, in the present study, members of the HSP70 family were highly expressed in both periapical granulomas and LPS-treated macrophages. Furthermore, correlation analyses reinforced a role for different HSPs as immune response activators, with DNAJC3, HSPA6/HSPA7, and HSPB1 being positively correlated with IL-1b mRNA levels in active lesions, and DNAJC3 and HSPB1 also showing a positive correlation with RANKL and TNF-a mRNA levels. A positive correlation between HSPA6/HSPA7 and DNAJC3 with IL-6 mRNA levels provides additional evidence for the critical role of HSPs in triggering the host immune defense against pathogens. In summary, our findings indicate that HSP27, HSP40, and HSP70 gene families play a crucial role in the maintenance of infection and subsequent tissue destruction in apical periodontitis, whereas HSP110 appears to have a role in re-establishing homeostasis in the periapical environment. These results may provide insights into a better understanding of the immune system and allow for the identification of unique markers to improve the diagnosis and treatment of apical periodontitis. Additional mechanistic studies in animal models may help elucidate the specific role for each HSP in apical periodontitis.
Acknowledgments The authors thank the individuals that agreed to participate in this study and Pasha Goodman for invaluable help in the critical review of the manuscript. Supported in part by UTSD startup funds to R.M.S., AAE Foundation to S.C.G., Fundac¸ao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) to G.P.G, and Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nivel Superior (CAPES), BEX 1099/12-4, Brazil to L.C.S.
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The authors deny any conflicts of interest related to this study.
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