HSDM Theses and Dissertations

Introduction: Secreted Semaphorin-3F (SEMA3F) is a chemorepulsive protein in Neuropilin-2 (NRP2)-expressing cells. It guides nervous and circulatory system patterning during embryogenesis. SEMA3F is frequently deleted in human small cell lung cancer and other cancer types including head and neck squamous cell carcinoma, as such it is a postulated as a potential tumor suppressor gene. Recent data from our group has identified NRP2 in CD4+ T cells. Aims: Our goal is to investigate the role of endogenous Sema3F and NRP2 in oral carcinogenesis, tumor progression, and tumor immunity. Methods: Different stages of oral carcinogenesis were evaluated for NRP2 expression. Sema3F expression was evaluated in OSCC cell lines and compared to normal mucosa. Transgenic mice with Sema3F deletion in adult keratinocytes were used to analyze cancer initiation and progression before or after exposure to carcinogen compared to controls. Tongues of Nrp2-global knockout (KO) or wildtype mice were injected with syngeneic oral cancer xenografts and tumors were evaluated for CD4+ and CD8+ T cell infiltration. Antigen-induced hypersensitivity reactions in Sema3F-KO, K14-Sema3F-iKO, Nrp2-KO, and CD4-Nrp2-KO mice were used to evaluate for ear swelling and CD4+ T cell infiltration in experimental mice compared to controls. Results: Nrp2 expression was absent in normal oral and skin epithelium but was upregulated during the late stages of dysplasia in both human and mouse carcinogenesis. Conversely, Sema3F was present in normal mouse tongue epithelium but decreased in OSCC cell lines. When Sema3F was deleted prior to exposure to carcinogen, the majority of control mice (Sema3F-intact) (25 out of 30) developed carcinoma in situ (CIS) or invasive oral squamous cell carcinoma (OSCC), while only 17% of K14-Sema3F-KO mice (4 out of 23) developed CIS, with none progressing to OSCC. When Sema3F knockout was induced post-carcinogen exposure, mice that lacked Sema3F in their epithelium exhibited more infiltrative tumors compared to controls. Oral cancer grafts displayed increased infiltration of CD4+ and CD8+ lymphocytes in Nrp2-/- mice compared to Nrp2+/+ mice. Mice lacking epithelial Sema3F or Nrp2-expressing T cells exhibited prolonged inflammation, tissue swelling, and CD4+ T cell infiltration, whereas control mice resolved quickly. Conclusion: Sema3F is not a tumor suppressor in OSCC, primarily because normal oral epithelium lacks Nrp2 expression and Sema3F cannot function in an autocrine fashion to inhibit carcinogenesis. Furthermore, mice that lacked Sema3F in their epithelium exhibited reduced carcinogenesis. Cancerous and inflamed tissues lacking Sema3F in the epithelium or Nrp2 on CD4+ T cells exhibited increased immune surveillance and T lymphocyte recruitment abrogating cancer initiation and preventing oral cancer progression. We conclude that the SEMA3F/NRP2 pathway suppresses host immunity and provides new potential targets for immunotherapy in oral cancer.

Introduction: Dental pulp tissue is densely innervated and it has unique immunological characteristics. Upon injury such as deep caries, exposing the pulp, a strong neuronal and immune response occurs which modulates pain and the tissue damage. Our knowledge on the dynamics of initial innate immune response, whether this response is modulated by sensory afferents of the pulp, and how this innate immune response alters tissue damage and/or pain outcome is limited. Materials and Methods: Dental pulp exposure in mice was used as the pulpitis model. To investigate the innate immune response, pulp tissue was collected from the permanent molars at different time points and flow cytometry was performed. To investigate tissue damage, H&E staining was performed. Immunohistochemistry and in-situ hybridization were performed to capture spatial changes of innate immune cells, sensory afferents, and bacteria invasion, respectively. Finally, mechanical pain sensitivity was captured by facial Von Frey stimulation and spontaneous pain was captured using the Mouse Grimace Scale. Data were analyzed using two-way ANOVA, t-tests, or repeated measure of ANOVA with appropriate multiple comparison tests. Results: We found that neutrophils constituted 70-90% of immune cell populations up to day 7. We also found that the neuropeptide CGRP, released from sensory afferents, contributed to the recruitment of myeloid cells (p=0.017) while also increasing spontaneous pain at day 1 (p=0.02). Moreover, when we depleted neutrophils and monocytes, we found that there was more tissue necrosis at day 6 (p=0.002) and bacteria had penetrated deeper parts of the tissue, whereas mechanical pain was lower compared to control group (p=0.03). Conclusions: Neutrophils and monocytes are crucial to protect dental pulp tissue from further bacterial penetration and tissue damage while also contributing to mechanical pain sensitivity. We also found that CGRP modulates innate immune responses either by causing vasodilation or by directly mediating neutrophil recruitment while contributing to spontaneous pain behavior during pulpitis.

Objectives: The aim of this project is to assess expression of specialized pro-resolving lipid mediators (SPMs) and inflammation resolution receptors on oral neutrophils (oPMNs) in oral lichen planus (OLP) compared to healthy controls. FFAR2 binds short chain fatty acids, FFAR4 binds long chain fatty acids, ERV1 binds Resolvin E1, and ALX binds Lipoxin A4. We hypothesize that in OLP, there is decreased oPMN surface expression of inflammation resolution receptors and decreased levels of SPMs, reflecting a failure of the distinct process of inflammation resolution, and hence progression from acute to chronic inflammation in the disease pathogenesis. SPMs activate the inflammation resolution pathways and are potential therapeutic options, especially for steroid refractory OLP. Methods: OLP cases and healthy controls were recruited from the oral medicine clinic at Brigham and Women’s Hospital and the Harvard School of Dental Medicine. Oral rinse samples were collected for oPMN isolation and lipidomics analysis using liquid chromatography-tandem mass spectrometry. oPMNs were labeled and analyzed by flow cytometry. The number/percentage of cells positive for inflammation resolution receptors, quantities of receptors per cell, and levels of lipid mediators of inflammation were compared using two-sample t-tests. Principle component analysis (PCA) was performed to identify clustering trends in lipid profiles and exploratory biomarker analysis was conducted to identify potential salivary biomarkers for OLP. Results: A total of 27 healthy controls and 17 OLP cases were included. Cases had almost double the number of mean total oPMNs (14x10^3, p.01) and viable (CD16+) oPMNs (8x10^3, p=0.01). The mean percentage of ERV1+ oPMNs was higher in cases than controls (84% vs. 67%, p.01). The mean fluorescent intensity (MFI) for receptors in cases vs controls were: 63x10^3 vs. 90x10^3 for ERV1 (p.01), 83x10^3 vs. 66x10^3 for FFAR2 (p=0.02), 53x10^3 vs. 36x10^3 for FFAR4 (p=0.02), and 604x10^3 vs. 690x10^3 for ALX (p=0.45). Overall, pro-inflammatory lipid mediators were upregulated while pro-resolving lipid mediators were downregulated in OLP cases compared to controls. PCA identified that the lipid mediator profiles were generally different between the two groups by the distinct clustering of samples. Prostaglandin F2 alpha (PGF2α) and 20-hydroxy leukotriene B4 (LTB4) had significantly higher levels in cases and showed good discriminatory potential as biomarkers for OLP. Conclusion: In OLP, there were more ERV1+ oPMNs, but less expression of ERV1. There is variation in the lipid mediator profiles between cases and controls, with downregulation of pro-resolution mediators in OLP. These findings possibly indicate a failure of inflammation resolution in OLP, and potentially explain acute/symptomatic exacerbations of this chronic condition. Further research is required to support translational studies using topical preparations of SPMs in the management of OLP.

Background and Objectives: Sleep plays a vital role in maintaining health and well-being. Poor sleep contributes to several adverse health outcomes due to hormonal and metabolic disorders that alters molecular processes that drive cellular immune activation and induce the secretion of immunometabolic biomarkers. Most studies use blood samples for their investigations, in this study we tested both salivary and serum samples to study the effect of sleep variables on immunometabolic biomarkers. There is established evidence on the relationship between poor sleep behavior and inflammatory and metabolic biomarkers. In this study, our aims were to determine the relationship between sleep timing (bedtime), sleep debt and social jetlag with levels of nine salivary and serum immunometabolic biomarkers (C-Reactive Protein (CRP), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleuken-10 (IL-10), Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein-1 (MCP-1), adiponectin, leptin, and insulin). Materials and Methodology: Data were collected from 352 adolescents, 16-18 years old and enrolled in Kuwait’s public high schools. Nine biomarkers were measured in saliva and serum supernatants using multiplex magnetic bead panels on a Luminex 200™ system. For statistical analyses, we conducted mixed effect linear regression models to account for school variable as a random effect to study the association between biomarkers as our outcome variables and sleep variables as our exposure variables, controlling for confounders (age, sex, medical history, nap, screen time, negative dietary habits). We also investigated the role of Nap, BMI and Waist circumference (WC) in these associations. Results: After controlling for confounding variables in all our models we found that (bedtime) sleep timing was statistically significantly associated with an overall elevation for salivary IL-6 and serum (CRP, IL-6, IL-10, MCP-1 and insulin) and reduction in serum IL-8 biomarkers. BMI and WC played a major rule in attenuating some of these associations as confounding variables, for example, leptin biomarker with bedtime. In addition, we displayed a mediation effect of BMI for the associations between bedtime and serum (CRP, IL-6, and insulin). Adolescents who exhibited catch-up sleep on free days (weekends) showed a statistically significant positive association with salivary IL-6 and serum (CRP and IL-6) and negative association with serum IL-8 biomarkers. For students with social jetlag (SJL), only salivary biomarkers (IL-6, VEGF and leptin) showed a statistically significant reduction with increase in SJL. Conclusion: This change of biomarker levels and directions with sleep timing behaviors support the hypothesis that late bedtime and disrupted circadian rhythm promote local and systemic inflammation that can trigger chronic metabolic diseases. Our findings highlight the importance of late bedtime as a strong predictor of oral and systemic inflammation, indexed by salivary and serum biomarkers.