Parotid glands have a dysregulated immune response following radiation therapy.

Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data suggest that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous data indicate that post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors.

Diet composition influences the effect of high fat diets on bone in growing male mice.

The effect of diet-induced obesity on bone in rodents is variable, with bone mass increases, decreases, and no impact reported. The goal of this study was to evaluate whether the composition of obesogenic diet may influence bone independent of its effect on body weight. As proof-of-principle, we used a mouse model to compare the skeletal effects of a commonly used high fat 'Western' diet and a modified high fat diet. The modified high fat diet included ground English walnut and was isocaloric for macronutrients, but differed in fatty acid composition and contained nutrients (e.g. polyphenols) not present in the standard 'Western' diet. Eight-week-old mice were randomized into 1 of 3 dietary treatments (n = 8/group): (1) low fat control diet (LF; 10 % kcal fat); (2) high fat 'Western' diet (HF; 46 % kcal fat as soybean oil and lard); or (3) modified high fat diet supplemented with ground walnuts (HF + walnut; 46 % kcal fat as soybean oil, lard, and walnut) and maintained on their respective diets for 9 weeks. Bone response in femur was then evaluated using dual energy x-ray absorptiometry, microcomputed tomography, and histomorphometry. Consumption of both obesogenic diets resulted in increased weight gain but differed in impact on bone and bone marrow adiposity in distal femur metaphysis. Mice consuming the high fat 'Western' diet exhibited a tendency for lower cancellous bone volume fraction and connectivity density, and had lower osteoblast-lined bone perimeter (an index of bone formation) and higher bone marrow adiposity than low fat controls. Mice fed the modified high fat diet did not differ from mice fed control (low fat) diet in cancellous bone microarchitecture, or osteoblast-lined bone perimeter, and exhibited lower bone marrow adiposity compared to mice fed the 'Western' diet. This proof-of-principal study demonstrates that two obesogenic diets, similar in macronutrient distribution and induction of weight gain, can have different effects on cancellous bone in distal femur metaphysis. Because the composition of the diets used to induce obesity in rodents does not recapitulate a common human diet, our finding challenges the translatability of rodent studies evaluating the impact of diet-induced obesity on bone.

Multiagent Intratumoral Immunotherapy Can Be Effective in A20 Lymphoma Clearance and Generation of Systemic T Cell Immunity.

The use of immunotherapies has shown promise against selective human cancers. Identifying novel combinations of innate and adaptive immune cell-activating agents that can work synergistically to suppress tumor growth and provide additional protection against resistance or recurrence is critical. The A20 murine lymphoma model was used to evaluate the effect of various combination immunotherapies administered intratumorally. We show that single-modality treatment with Poly(I:C) or GM-CSF-secreting allogeneic cells only modestly controls tumor growth, whereas when given together there is an improved benefit, with 50% of animals clearing tumors and surviving long-term. Neither heat nor irradiation of GM-CSF-secreting cells enhanced the response over use of live cells. The use of a TIM-3 inhibitory antibody and an OX40 agonist in combination with Poly(I:C) allowed for improved tumor control, with 90% of animals clearing tumors with or without a combination of GM-CSF-secreting cells. Across all treatment groups, mice rejecting their primary A20 tumors were immune to subsequent challenge with A20, and this longstanding immunity was T-cell dependent. The results herein support the use of combinations of innate and adaptive immune activating agents for immunotherapy against lymphoma and should be investigated in other cancer types.

Partially replacing cyclophosphamide with bendamustine in combination with cyclosporine A improves survival and reduces xenogeneic graft-versus-host-disease.

Introduction: The use of allogeneic hematopoietic cell transplantation (allo-HCT) for treating hematological disorders is increasing, but the development of graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality. The use of post-transplant cyclophosphamide (CY) has significantly improved outcomes following allo-HCT, but complications of viral reactivation due to delayed immune reconstitution or relapse remain. Other laboratories are evaluating the potential benefit of lowering the dose of CY given post-transplant, whereas our laboratory has been focusing on whether partially replacing CY with another DNA alkylating agent, bendamustine (BEN) may be advantageous in improving outcomes with allo-HCT. Methods: Here, we utilized a xenogeneic GvHD (xGvHD) model in which immunodeficient NSG mice are infused with human peripheral blood mononuclear cells (PBMCs). Results: We show that a lower dose of CY (25 mg/kg) given on days +3 and +4 or CY (75 mg/kg) given on only day +3 post-PBMC infusion is not sufficient for improving survival from xGvHD, but can be improved with the addition of BEN (15 mg/kg) on day +4 to day +3 CY (75 mg/kg). CY/BEN treated mice when combined with cyclosporine A (CSA) (10mg/kg daily from days +5 to +18 and thrice weekly thereafter), had improved outcomes over CY/CY +CSA treated mice. Infiltration of GvHD target organs was reduced in both CY/CY and CY/BEN treatment groups versus those receiving no treatment. CY/CY +CSA mice exhibited more severe xGvHD at day 10, marked by decreased serum albumin and increased intestinal permeability. CY/BEN treated mice had reductions in naïve, effector memory and Th17 polarized T cells. RNAseq analysis of splenocytes isolated from CY/CY and CY/BEN treated animals revealed increased gene set enrichment in multiple KEGG pathways related to cell migration, proliferation/differentiation, and inflammatory pathways, among others with CY/BEN treatment. Conclusion: Together, we illustrate that the use of CY/BEN is safe and shows similar control of xGvHD to CY/CY, but when combined with CSA, survival with CY/BEN is significantly prolonged compared to CY/CY.

Indomethacin Treatment Post-irradiation Improves Mouse Parotid Salivary Gland Function via Modulation of Prostaglandin E2 Signaling.

Annually, >600,000 new cases of head and neck cancer (HNC) are diagnosed worldwide with primary treatment being surgery and radiotherapy. During ionizing radiation (IR) treatment of HNC, healthy salivary glands are collaterally damaged, leading to loss of function that severely diminishes the quality of life for patients due to increased health complications, including oral infections and sores, cavities, and malnutrition, among others. Therapies for salivary hypofunction are ineffective and largely palliative, indicating a need for further research to uncover effective approaches to prevent or restore loss of salivary gland function following radiotherapy. Previous work in our lab implicated prostaglandin E2 (PGE2) as an inflammatory mediator whose release from radiation-exposed cells promotes salivary gland damage and loss of function. Deletion of the P2X7 purinergic receptor for extracellular ATP reduces PGE2 secretion in irradiated primary parotid gland cells, and salivary gland function is enhanced in irradiated P2X7R-/- mice compared to wild-type mice. However, the role of PGE2 signaling in irradiated salivary glands is unclear and understanding the mechanism of PGE2 action is a goal of this study. Results show that treatment of irradiated mice with the non-steroidal anti-inflammatory drug (NSAID) indomethacin, which reduces PGE2 production via inhibition of cyclooxygenase-1 (COX-1), improves salivary gland function compared to irradiated vehicle-treated mice. To define the signaling pathway whereby PGE2 induces salivary gland dysfunction, primary parotid gland cells treated with PGE2 have increased c-Jun N-terminal Kinase (JNK) activation and cell proliferation and reduced amylase levels and store-operated calcium entry (SOCE). The in vivo effects of blocking PGE2 production were also examined and irradiated mice receiving indomethacin injections have reduced JNK activity at 8 days post-irradiation and reduced proliferation and increased amylase levels at day 30, as compared to irradiated mice without indomethacin. Combined, these data suggest a mechanism whereby irradiation-induced PGE2 signaling to JNK blocks critical steps in saliva secretion manifested by a decrease in the quality (diminished amylase) and quantity (loss of calcium channel activity) of saliva, that can be restored with indomethacin. These findings encourage further attempts evaluating indomethacin as a viable therapeutic option to prevent damage to salivary glands caused by irradiation of HNC in humans.

The complex role of prostaglandin E2-EP receptor signaling in wound healing.

Prostaglandins are critical lipid mediators involved in the wound healing response, with prostaglandin E2 (PGE2) being the most complex and exhibiting the most diverse physiological outputs. PGE2 signals via four G protein-coupled receptors, termed EP-receptors 1-4 that induce distinct signaling pathways upon activation and lead to an array of different outputs. Recent studies examining the role of PGE2 and EP receptor signaling in wound healing following various forms of tissue damage are discussed in this review.

Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions.

Salivary glands sustain collateral damage following radiotherapy (RT) to treat cancers of the head and neck, leading to complications, including mucositis, xerostomia and hyposalivation. Despite salivary gland-sparing techniques and modified dosing strategies, long-term hypofunction remains a significant problem. Current therapeutic interventions provide temporary symptom relief, but do not address irreversible glandular damage. In this review, we summarize the current understanding of mechanisms involved in RT-induced hyposalivation and provide a framework for future mechanistic studies. One glaring gap in published studies investigating RT-induced mechanisms of salivary gland dysfunction concerns the effect of irradiation on adjacent non-irradiated tissue via paracrine, autocrine and direct cell-cell interactions, coined the bystander effect in other models of RT-induced damage. We hypothesize that purinergic receptor signaling involving P2 nucleotide receptors may play a key role in mediating the bystander effect. We also discuss promising new therapeutic approaches to prevent salivary gland damage due to RT.

P2X7 receptor deletion suppresses γ-radiation-induced hyposalivation.

Head and neck cancer treatments typically involve a combination of surgery and radiotherapy, often leading to collateral damage to nearby tissues causing unwanted side effects. Radiation damage to salivary glands frequently leads to irreversible dysfunction by poorly understood mechanisms. The P2X7 receptor (P2X7R) is a ligand-gated ion channel activated by extracellular ATP released from damaged cells as "danger signals." P2X7R activation initiates apoptosis and is involved in numerous inflammatory disorders. In this study, we utilized P2X7R knockout (P2X7R-/-) mice to determine the role of the receptor in radiation-induced salivary gland damage. Results indicate a dose-dependent increase in γ-radiation-induced ATP release from primary parotid gland cells of wild-type but not P2X7R-/- mice. Despite these differences, apoptosis levels are similar in parotid glands of wild-type and P2X7R-/- mice 24-72 h after radiation. However, γ-radiation caused elevated prostaglandin E2 (PGE2) release from primary parotid cells of wild-type but not P2X7R-/- mice. To attempt to uncover the mechanism underlying differential PGE2 release, we evaluated the expression and activities of cyclooxygenase and PGE synthase isoforms. There were no consistent trends in these mediators following radiation that could explain the reduction in PGE2 release in P2X7R-/- mice. Irradiated P2X7R-/- mice have stimulated salivary flow rates similar to unirradiated controls, whereas irradiated wild-type mice have significantly decreased salivary flow rates compared with unirradiated controls. Notably, treatment with the P2X7R antagonist A438079 preserves stimulated salivary flow rates in wild-type mice following γ-radiation. These data suggest that P2X7R antagonism is a promising approach for preventing γ-radiation-induced hyposalivation.