1. 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

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

3. 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)

4. 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

5. 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

6. 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 & hr post dex injection (0.04 mg/kg; i.m.)
Beech et al JAVMA

7. 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 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 + =

8. 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

9. 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.

10. 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

11. Results: Lymphocyte Production of IFNg

12. Results: Lymphocyte Production of TNFa

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

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

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

16. 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?

17. Thank you
The authors have no conflicts of interest. Contact: Dr. Amanda Adams at