Immunological Comparisons of Aged Horses with vs Immunological Comparisons of Aged Horses with vs. without Pituitary Pars Intermedia Dysfunction: Cell-Mediated Immunity PPID Non-PPID Melissa Siard PhD Candidate Gluck Equine Research Center University Of Kentucky

Outline Introduction PPID Immunity of the Aged Horse Materials and Methods Intracellular staining for cytokines Lymphocyte Proliferation Results & Conclusions Summary & Future Research Outline

Equine demographics—Why we care about the aged population Many old horses still being used for recreation, competition, and breeding Horses ≥15 years - 29% of the equine population in the UK (Ireland et al. 2011) Horses ≥20 years – 8-15% of equine population in the US (USDA 2006; Rich 1989) 15-30% of aged equids have PPID (McFarlane et al 2011)

What Is PPID? Pituitary pars intermedia dysfunction Endocrinopathy thought to be caused by dopaminergic degeneration of the pars intermedia Occurs primarily in aged horses PPID

Symptoms Hirsutism/Hypertrichosis Polydipsia/polyuria Immunosuppression & Opportunistic Infections Muscle atrophy Laminitis Hyperhidrosis Abnormal Fat Distribution Behavioral abnormalities Reproductive Infertility Neurologic Impairment McFarlane 2011 Vet. Clin. Equine

How do we diagnose PPID? Elevated basal ACTH Elevated ACTH response to TRH (10 & 30 mins) Elevated cortisol in response to DST Seasonally these levels are much higher in all horses in the fall, but particularly in PPID horses TRH Test: EDTA plasma collected pre, 10 min, & 30 min post TRH injection (1mg/mL saline/horse; i.v.) Dex Suppresion Test: serum collected pre & 19-20 hr post dex injection (0.04 mg/kg; i.m.) Beech et al. 2007 JAVMA

Effect of PPID on immune Function Symptom: Immunosuppression & Opportunistic Infections ACTH has yielded mixed results as to whether it is immunoinhibitory or immunostimulatory. Glucocorticoids like cortisol have been shown to have immunoinhibitory effects on T cells. Effect of PPID on immune function… Ashwell et al. 2000. Annual Review of Immunology 18; 309–345. *Conflicting Data for In Vitro Studies (Johnson, EW, et al. 2005)- Inhibit antibody responses, INFgamma responses and reduce lymphocyte profliferation Enhance memory cytotoxic T-cell responses *Limited Data for In Vivo Studies (Ohya, T, et al. 2009)- Low dose ACTH is first line treatment for West Syndrome Reduced lymphocyte counts, no change in IgG levels Suggested to have reduced immune responses to vaccination + =

Aging Immunologically Inflamm-aging Chronic, low-grade inflammation that occurs systemically with aging Immunosenescence Add stuff from 3 Reduced immune responses to vaccination Decrease in antibody responses to influenza Decrease in CMI responses to influenza Reduced lymphocyte proliferation

Hypothesis Objective: To determine whether PPID affects cell- mediated immune response, specifically lymphocyte proliferation and production of inflammatory cytokines. Hypothesis: PPID impacts immune function of horses to a greater extent than would be expected by aging alone.

Methods Cells thawed, plated, & stimulated with PMA/ionomycin (4 hrs) IFN-g & TNF-a intracellular stained & flow cytometry performed PBMC isolated from heparinized tubes n=8 Aged, non-PPID horses n=8 PPID horses Cells thawed, stained with CFSE, plated, & stimulated with concanavalin A (96 hrs) Flow cytometry performed to determine lymphocyte proliferation Heparin blood tube

Results: Lymphocyte Production of IFNg

Results: Lymphocyte Production of TNFa

Results: Lymphocyte Proliferation P=0.081 for PPID Status overall; P = 0.162 for Con A 5 uL

Results overview No differences in CMI response Lymphocyte proliferation Lymphocyte production of pro-inflammatory cytokines IFNg and TNFa

Conclusions No significant differences in cell-mediated immune responses between PPID vs. non-PPID, aged horse

Future research To further determine if there are differences immunologically aside from those previously examined between PPID vs. aged, non-PPID horses. To determine what mechanistically drives the occurrence of PPID—What causes the adrenergic neurodegeneration of the pituitary pars intermedia? Oxidative stress? Infectious disease? Nutritional deficiencies? Inflammation?

Thank you The authors have no conflicts of interest. Contact: Dr. Amanda Adams at amanda.adams@uky.edu