Minipig Model of Atopic Dermatitis: Assessment of In Vivo and In Vitro Activity of Recombinant Porcine Interleukin-4 and Interleukin-13

Scientific Poster
Minipig Model of Atopic Dermatitis Assessment of In Vivo and In Vitro Activity of Recombinant Porcine Interleukin-4 and Interleukin-13 - SciPos101

Abstract

Atopic Dermatitis is a common skin condition that clinically presents as erythematic, dry, pruritic skin. Minipigs are used frequently for toxicity/safety of dermally applied products, and thus a model of AD in minipigs would be beneficial for pre-clinical efficacy tests of such medications. This study assessed sensitivity of Hanford minipigs to recombinant porcine (rp) IL-4 and IL-13. Peripheral Blood Mononuclear Cells (PBMC) isolated from female Hanford minipigs demonstrated approximately a 4 fold increase in STAT6 phosphorylation when challenged with rpIL-4, but not rpIL-13. When female Hanfords received a single intradermal dose of rpIL-4 or rpIL-13, erythema and edema was not different from vehicle control dose sites. However, repeat intradermal injections for a period of five days did elicit increased erythema and edema in rpIL-4 dose sites relative to vehicle control, but not rpIL-13 dose sites. The peak irritation was observed approximately 5 minutes after dose administration, similar to histamine injections in minipigs. Interestingly, perivascular or dermal lymphocytes were observed in ~25-38% of rpIL-4 and rpIL-13 dose sites, but were not present in the vehicle control sites. Perivascular eosinophils were observed in ~25% of the rpIL-4 dose sites, while not observed in vehicle or rpIL-13 dose sites. This suggests that intradermal injection of rpIL-4 and rpIL-13 may recruit lymphocytes to dermal tissues. These findings that rpIL-4 and rpIL-13 appear to have biological activity in Hanford minipigs, and are good candidates for further exploration in developing a porcine model of Atopic Dermatitis.

Authors

Jones, M.R.¹; Zhong, M.¹; Johnson, S.¹; Brocksmith, D.¹,2; Bouchard G.F.¹,2; Stricker-Krongrad, A.¹ ¹Sinclair Research Center, LLC, Auxvasse, MO, USA; ²Sinclair BioResources, LLC, Auxvasse, MO, USA

Introduction

Atopic Dermatitis (AD) is a common skin condition that clinically presents as erythematic and pruritic skin. While multiple factors contribute to the pathophysiology of the disease, skin-barrier dysfunction [1, 2], and prominent Th2-mediated immune responses are characteristic of the AD. Interleukin-4 (IL-4) and Interleukin-13 (IL-13) are thought to be contributing factors to the pathogenesis of AD [3], and treatments targeting IL-4 and IL-13 mediated pathways have shown to be beneficial in human patients diagnosed with AD [4]. While multiple animal models of AD exist [5, 6], species specific limitations can deter translational efficacy of therapies developed for humans. Miniature swine are used frequently for toxicity/safety of dermally applied products [7], and thus a model of AD in miniature swine would be beneficial for pre-clinical efficacy tests of such medications.

Materials & Methods

In vitro activity of rpIL-4 and rpIL-13 was quantified using an ELISA for pSTAT6, and flow cytometry for pSTAT1, pSTAT3, and pSTAT6. In brief, Peripheral Blood Mononuclear Cells were isolated from female Hanford minipigs, and treated with rpIL-4 or rpIL-13 (IBI Scientific) for a period of 10-30 minutes. Upon completion of cytokine treatment, cell lysates were prepared for ELISA analysis, or fixed in preparation for staining and analysis using flow cytometry.

In vivo activity was assessed in multiple phases. During Phase 1, one animal received a single dose of vehicle (1% porcine serum in 1 X PBS), histamine (1 mg/dose site), rpIL4 (5-20 µg/dose site), rpIL-13 (5-20 µg/dose site), rpIL-4/rpIL-13 combination (2.5- 20/2.5-20 µg dose site) to assess dermal irritation. During Phase 2, animals received daily intradermal injections of vehicle or rpIL-4 (2-6 µg/dose site) for a period of 5 days. During Phase 3, one animal received daily intradermal injections of rpIL-13 (2-6 µg/dose site) for a period of 5 days. Photographs were taken of the dose sites to characterize the reaction at approximately 5, 20, and 60 minutes post dose. Phase 2 and Phase 3 animals were euthanized, and dose sites were collected and preserved in 10% NBF for H&E staining and histopathological analysis.

Results

Peripheral Blood Mononuclear Cells (PBMC) isolated from female Hanford minipigs demonstrated approximately a 4-fold increase in STAT6 phosphorylation when challenged with rpIL-4, but not rpIL-13 when measured by ELISA (Table 1). STAT6 phosphorylation demonstrated a 2 fold increase when treated with rpIL-4 when measured by Flow Cytometry, but STAT1 and STAT3 phosphorylation were not significantly increased in rpIL-4 or rpIL-13 treated cells (Figure 1).

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When female Hanfords received a single intradermal dose of rpIL-4 or rpIL-13, erythema and edema was not different from vehicle control dose sites (Figure 2). However, repeat intradermal injections for a period of five days did elicit increased erythema and edema in rpIL-4 dose sites relative to vehicle control, but not rpIL-13 dose sites (Figures 3, 4). The peak irritation was observed approximately 5 minutes after dose administration, similar to histamine injections in minipigs. Interestingly, perivascular and dermal lymphocytes were observed in ~25-38% of rpIL-4 and rpIL-13 dose sites, but were not present in the vehicle control sites. Perivascular eosinophils were observed in ~25% of the rpIL-4 dose sites, while not observed in vehicle or rpIL-13 dose sites. However, these differences were minimal, and did not demonstrate lesions typical of atopic dermatitis. Representative photographs for histopathology of sites dosed with rpIL-4 and rpIL-13 are shown in Figures 5-10.

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Discussion

This study demonstrated in vitro and in vivo activity of rpIL-4 in Hanford minipigs, while rpIL-13 did not appear to demonstrate the similar effects. These results demonstrate that rpIL-4 can induce signal transduction via STAT-6 phosphorylation in minipig PBMC, and ultimately lead to mild inflammation when injected intradermally. The histopathology suggests that that intradermal injection of rpIL-4 and rpIL-13 may be capable of recruiting lymphocytes to dermal tissues. While 5 days of repeat intradermal injections of either rpIL-4 or rpIL-13 did not result in characteristic lesions associated with atopic dermatitis, histopathology findings demonstrate the potential for biological activity of both cytokines. It may require a longer dosing period, greater dose concentration, or increase in dosing frequency to create a porcine model of atopic dermatitis.

References

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  2. Boguniewicz, M. and D.Y. Leung, Atopic dermatitis: a disease of altered skin barrier and immune dysregulation. Immunol Rev, 2011. 242(1): p. 233-46.
  3. Gittler, J.K., et al., Progressive activation of T(H)2/T(H)22 cytokines and selective epidermal proteins characterizes acute and chronic atopic dermatitis. J Allergy Clin Immunol, 2012. 130(6): p. 1344-54.
  4. Beck, L.A., et al., Dupilumab treatment in adults with moderate-to-severe atopic dermatitis. N Engl J Med, 2014. 371(2): p. 130-9.
  5. Jin, H., et al., Animal models of atopic dermatitis. J Invest Dermatol, 2009. 129(1): p. 31-40.
  6. Avci, P., et al., Animal models of skin disease for drug discovery. Expert Opin Drug Discov, 2013. 8(3): p. 331-55.
  7. Stricker-Krongrad, A., C.R. Shoemake, and G.F. Bouchard, The Miniature Swine as a Model in Experimental and Translational Medicine. Toxicol Pathol, 2016. 44(4): p. 612-23.

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