Investigational compound Oclacitinib (Fig. 1) was synthesized in the laboratories of Zoetis (Kalamazoo, MI, USA).

Janus kinase enzyme activity assays and kinase selectivity panels

Recombinant human active kinase domains for JAK1 (amino acids 852–1142; NP_002218), JAK2 (amino acids 808–1132; NP_004963), JAK3 (amino acids 781–1124; NP_000206), and TYK2 (amino acids 870–1187; NP_003322) were used in isolated enzyme assays using Caliper microfluidics technology to determine potency of oclacitinib against the JAK family members, as previously described (Meyer et al., 2010). Sequence homology to the analogous sequences in the canine JAK enzymes are 98, 98, 100, and 90%, respectively (Figure S1). Invitrogen kinase panel testing (Life Technologies, Grand Island, NY, USA) was performed to determine potency of oclacitinib toward 38 different non-JAK kinases using their SelectScreenTM Kinase Profiling Services (Madison, WI, USA). Oclacitinib was evaluated at a concentration of 1 lM. Kinasespecific assay conditions and data analyses are described on their Web site http://www.lifetechnologies.com/us/en/home/ products-and-services/services/custom-services/screening-andprofiling-services/selectscreen-profiling-service/selectscreen-kinaseprofiling-service.html This service utilizes the Z’-Lyte technology for all kinase screening. All tests were run in duplicate.

Fig. 1. Chemical structure of oclacitinib. Oclacitinib is a cyclohexylamino pyrrolopryimidine.

JAK enzymes are 98, 98, 100, and 90%, respectively (Figure S1). Invitrogen kinase panel testing (Life Technologies, Grand Island, NY, USA) was performed to determine potency of oclacitinib toward 38 different non-JAK kinases using their SelectScreenTM Kinase Profiling Services (Madison, WI, USA). Oclacitinib was evaluated at a concentration of 1 lM. Kinasespecific assay conditions and data analyses are described on their Web site http://www.lifetechnologies.com/us/en/home/ products-and-services/services/custom-services/screening-andprofiling-services/selectscreen-profiling-service/selectscreen-kinaseprofiling-service.html This service utilizes the Z’-Lyte technology for all kinase screening. All tests were run in duplicate.

Interleukin-2 cytokine function

Canine whole blood was collected in heparin sulfate tubes from beagle dogs as well as dogs from mixed breeds. Whole blood (20 lL) was plated in 96-well plates (Corning Costar, Tewksbury, MA, USA) with 180 lL of DMEM complete medium (Dulbecco’s modified Eagle medium; 10% heat-inactivated fetal bovine serum; 100 U/mL penicillin; 100 lg/mL streptomycin; Gibco Life Technologies, Grand Island, NY, USA) containing vehicle control or oclacitinib (0.001–10 lM), concanavalin A (ConA; 1 lg/mL; Sigma-Aldrich, St. Louis, MO, USA), and canine interleukin-2 (IL-2; 50 ng/mL; R&D Systems, Minneapolis, MN, USA). Plates were incubated at 37 °C for 48 h. Tritiated thymidine, 0.4 lCi per well (Perkin Elmer, Waltham, MA, USA), was added for 20 additional hours. Plates were frozen and then thawed, washed, and filtered using a Brandel MLR-96 cell harvester (Gaithersburg, MD, USA) and prewet filter mats (Perkin Elmer). Filters were dried and placed into filter sample bags (Perkin Elmer) with 10 mL of scintillant (Perkin Elmer). Sealed filters were counted on an LKB Wallac 1205 Betaplate liquid scintillation counter (Pharmacia, Uppsala, Sweden). Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.

Interleukin-4 cytokine function

The CellSensor STAT6-bla RA-1 human B-cell line from Invitrogen (Life Technologies, Grand Island, NY, USA) was used. Cells were propagated in Opti-MEM Reduced Serum Medium containing 5% heat-inactivated fetal bovine serum, 100 lM nonessential amino acids, 1 mM sodium pyruvate, 1% penicillin/streptomycin, and 556 ng/mL of CD40 Ligand (Invitrogen, Life Technologies) overnight at 37 °C, 5% CO2. Cells were then plated in 384-well assay plates, black-wall, clear bottom (Costar Corning, Corning, NY, USA) at a density of 20 000 cells per well. Oclacitinib (0.0000381–10 lM) or vehicle control was added to cells for 2 h. Ten nanograms per milliliter hIL-4 (Invitrogen) was then added to cell cultures for 5 h. Activation of the STAT6-beta-lactamase reporter gene by IL-4 was determined by detecting beta-lactamase activity with the LiveBLAzerTM-FRET B/G substrate (CCF-4 AM; Invitrogen) for 2 h. Fluorescence emission values at 460 and 530 nm were obtained using a fluorescent plate reader. The 460/ 530 nm ratios were expressed as percent control, and dose– response data were analyzed using a 4-parameter logistic equation.

Interleukin-6 cytokine function

The CellSensor STAT3-bla HEK293T human epithelial cell line from Invitrogen was used. Cells were plated into 384-well assay plates, black-wall, clear bottom (Costar Corning) at a density of 1.875 X 10 raised to the 5th power, cells per well in DMEM high glucose medium containing 5% FBS (Gibco Life Technologies) and incubated at 37 °C, 5% CO². Oclacitinib (0.0000954–25 lM) or vehicle control was added to cells for 1 h. Twenty nanograms per milliliter hIL-6 (Invitrogen) was then added to cell cultures for 5 h. Activation of the STAT3-beta-lactamase reporter gene by IL-6 was determined by detecting beta-lactamase activity with the LiveBLAzerTM-FRET B/G substrate (CCF-4 AM; Invitrogen). Fluorescence emission values at 460 and 530 nm were obtained using a fluorescent plate reader. The 460/530 nm ratios were expressed as percent control, and dose–response data were analyzed using a 4-parameter logistic equation.

Interleukin-13 cytokine function

The HT-29 human colonic epithelial cell line from American Type Culture Collection (ATCC, Manassas, VA, USA) was used. Cells were propagated in McCoy’s 5A medium containing 10% FBS, 50 U/mL penicillin, 50 lg/mL streptomycin, and 2 mM L-glutamine (Gibco Life Techologies). Cells were trypsinized from flasks, washed in fresh medium, and resuspended in 96- well assay plates (Costar Corning) at a density of 3x10 raised to the 5th power cells per well. Oclacitinib (0.0015–30 lM)) or vehicle control was added to cells while on ice for 30 min. One nanogram per milliliter hIL-13 (R&D Systems) was then added. Cells were incubated in a 37 °C water bath for 30 min then fixed in 1.75% formaldehyde in PBS, washed in PBS containing 0.5% BSA, and incubated overnight at -20 °C in absolute methanol. Fixed cells were stained with PE-labeled antibody to human pSTAT6 (pY641; BD Biosciences, San Jose, CA, USA). Samples were analyzed with an FACSCalibur equipped with a plate-based autosampler (BD Biosciences) and analyzed using FlowJo software, version 7.6.1 (Tree Star Inc., Ashland, OR, USA). Data were expressed as mean fluorescence and then expressed as percent control. Dose–response data were then analyzed using a 4-parameter logistic equation.

Interleukin-31 cytokine function

The canine monocytic cell line DH82 (ATCC) was plated at a density of 100 000 cells per well in 96-well plates and grown overnight in MEM medium (Gibco Life Technologies) containing 15% FBS (Gibco Life Technologies) and 10 ng/mL canine IFNc (R&D Systems). After 24 h, growth medium was replaced with serum-free MEM for 2 h. Test compound or vehicle control was then added for 1 h to generate an 11-point titration curve (ranging from 0.0001 to 10 lM). Cells were treated with 1 lg/mL of canine IL-31 (in-house generated) for 5 min. Cytokine treatment was terminated by removing medium and adding AlphaScreen SureFireTM lysis buffer (Perkin Elmer). Activation of pSTAT3 was detected using Perkin Elmer AlphaScreen SureFireTM STAT3 p-Y705 kit. Data were expressed as mean Alpha Screen Signal units and then expressed as percent control. Dose–response data were then analyzed using a 4- parameter logistic equation.

Granulocyte/macrophage colony-stimulating factor cytokine function

The Cell Sensor irf-1-bla TF-1 human erythroleukemia cell line from Invitrogen was used. Cells were plated in 96-well plates (Costar Corning) at a density of 2x10 raised to the 6th power cells per well in Opti-MEM Reduced Serum Medium (Invitrogen) containing 5% heat-inactivated fetal bovine serum, 100 lM nonessential amino acids, 1 mM sodium pyruvate, 1% penicillin/streptomycin (all from Gibco Life Technologies) and incubated at 37 °C, 5% CO2. Oclacitinib (0.00061–10 lM) or vehicle control was added to cell cultures for 2 h. Cells were then stimulated with 10 ng/mL GM-CSF (Invitrogen) for 5 h. Activation of the STAT5-beta-lactamase reporter gene by GM-CSF-1 was determined by detecting beta-lactamase activity with the LiveBLAzerTM-FRET B/G substrate (CCF-4 AM; Invitrogen) using a fluorescent plate reader. Fluorescence emission values at 460 and 530 nm were obtained using a fluorescent plate reader. The 460/530 nm ratios were expressed as percent control, and dose–response data were analyzed using a 4-parameter logistic equation.

Erythropoietin cytokine function

Canine bone marrow cells were isolated under sterile conditions from the humerus of beagle dogs and placed in warmed DMEM medium (Gibco Life Technologies). The sample was filtered through a stainless steel wire mesh (Sigma-Aldrich) and gently ground with a glass pestle. The suspension was then passed through a 100-lm cell strainer and washed three times in warmed Alsever’s solution (Sigma-Aldrich). The final pellet was resuspended in 20 mL of DMEM complete medium. Cell suspensions were plated in 96-well plates (Costar Corning) at a density of 2x10 raised to the 5th power cells per well in DMEM complete medium containing oclacitinib (0.001–10 lM) or vehicle control, and erythropoietin (0.2 U per well Erythropoietin; R&D Systems). Plates were incubated at 37 °C for 48 h. Tritiated thymidine, 0.4 lCi per well (Perkin Elmer), was added for 20 additional hours. Plates were frozen and then thawed, washed, and filtered using a Brandel MLR-96 cell harvester and prewet filter mats (Perkin Elmer). Filters were dried at 60 °C for 1 h and placed into filter sample bags (Perkin Elmer) with 10 mL of scintillant (Perkin Elmer). Sealed filters were counted on an LKB Wallac 1205 Betaplate liquid scintillation counter. Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.

Interleukin-12 cytokine function,/p>

Human whole blood was collected in heparin sulfate tubes. Whole blood (75 uL per well) was plated in 384-well plates (Costar Corning) containing vehicle control or oclacitinib (0.000143–37.5 um). Treated whole blood was then stimulated with mouse anti-human CD3 (1 ug/mL; BD Biosciences), mouse anti-human CD28 (10 ng/mL; BD Biosciences), and recombinant human uL-12 (10 ng/mL; R&D Systems) overnight at 37 °C, 5% CO2. Whole blood was spun to separate plasma, and 20 uL of plasma was collected. Interferon gamma levels were quantitated in plasma using Meso IFN-gamma 384-well plate kit and a Meso Scale Discovery SECTOR Imager 6000 (Rockville, MD, USA) following the manufacturer’s instructions. Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.

Interleukin-23 cytokine function

Human whole blood was collected in heparin sulfate tubes. Whole blood (70 uL) was plated in 96-well, deep-well, V-bottom plates (Corning) and incubated at 37 °C for 30 min. Vehicle control or oclacitinib (0.000143–37.5 lM) was added to plates and incubated at 37 °C for 30 min. IL-23 (100 ng/mL; R&D Systems) was then added to plates and incubated at 37 °C for 15 min. Reactions were stopped by the addition of 1x Phosflow lysis/fix buffer (BD Biosciences). Plates were incubated at RT for 15 min. Cells were washed in PBS buffer containing 1% heat-inactivated fetal bovine serum (Gibco Life Technologies) and 0.01% sodium azide and permeablized in 90% ice cold methanol for 30 min. Cells washed and stained with AlexaFluor 647-conjugated anti-pSTAT3 antibody (BD Biosciences) diluted 1:150. Plates were incubated at 4 °C overnight. Samples were transferred to 96-well U-bottom plates (Costar Corning) for flow cytometric analysis. Flow cytometric analysis was performed using a FACSCalibur flow cytometer equipped with the BD High Throughput sampler using CellQuest software (BD Biosciences). Thirty thousand cellular events were collected per sample, and data were analyzed using FlowJo software (Tree star). Lymphocyte populations showing pSTAT3 staining were enumerated across dose groups. Data were expressed as percent control, and dose– response data were then analyzed using a 4-parameter logistic equation.

RESULTS

Inhibitory activity of oclacitinib against JAK family members was determined in isolated enzyme systems. Oclacitinib inhibited JAK1, JAK2, JAK3, and TYK2 by 50% at concentrations (IC50’s) of 10, 18, 99, and 84 nM, respectively (Table 1). Oclacitinib was most potent against the JAK1 enzyme, showing a 1.8-fold selectivity for JAK1 vs. JAK2 and 9.9-fold selectivity toward JAK1 vs. JAK3 (Table 1). Oclacitinib did not significantly inhibit a panel of 38 other kinases when tested at a concentration of 1 lM (Table 2).

Effects of oclacitinib on the function of cytokines involved in inflammation (IL-2 and IL-6), allergy (IL-4 and IL-13), pruritus (IL-31) as well as hematopoiesis (erythropoietin and GM-CSF), and innate immune cell responses (IL-12 and IL-23) were specifically evaluated. Oclacitinib was able to inhibit IL-2, IL-4, IL-6, IL-13, and IL-31 with IC50’s ranging from 36 to 249 nM. Oclacitinib was much less potent against erythropoietin and GM-CSF, with IC50’s above 1000 nM for both of these cytokines. Oclacitinib did not significantly inhibit IL-12 or IL-23 function (IC50’s > 3000 nM) (Table 3).

DISCUSSION

Janus kinase inhibitors are being developed by numerous pharmaceutical companies for the treatment of a variety of inflammatory, autoimmune, and hematologic disorders in humans. The selectivity profile toward JAK family members varies between these different inhibitors, and desired profiles are influenced in part by the disease indication of interest (Menet et al., 2013). A variety of cytokines implicated in allergy require activation of a cytokine receptor that utilizes JAK1 as one of the obligate JAK partners (Fig. 2). Therefore, a JAK1 selective inhibitor may be useful for the treatment of allergic conditions in dogs. In this study, we characterized the in vitro activity of the novel JAK inhibitor oclacitinib and showed it preferentially inhibited JAK1-dependent cytokine receptor complexes involvedin inflammation and pruritus, common clinical signs in canine allergic skin disease.

Table 1. Inhibitory activity of oclacitinib against Janus kinase (JAK) family members in isolated enzyme systems

Table 2.

In isolated enzyme systems, oclacitinib was shown to inhibit JAK family members, most effectively JAK1, while not significantly inhibiting a variety of other non-JAK kinases, suggesting oclacitinib is a selective JAK inhibitor. Exploration of oclacitinib’s selectivity pattern in cell model systems showed that oclacitinib preferentially inhibited pro-allergic, pro-inflammatory, and pruritogenic cytokines that bind receptors utilizing the JAK1 enzyme as one of the JAK partners to mediate intracellular signaling. For example, oclacitinib inhibited IL-2 and IL-4 that bind receptor complexes that engage both JAK1 and JAK3 enzymes to trigger signal transduction cascades. IL-6, IL-13, and IL-31 were also inhibited by oclacitinib. These cytokines bind receptor complexes that activate JAK1 and JAK2 enzymes and possibly TYK2 under some circumstances (Schindler & Plumlee, 2008) (Fig. 2). Oclacitinib inhibited these JAK1-dependent cytokines with IC50’s ranging from 36 to 249 nM. The JAK1-dependent cytokines evaluated in this study play a role in many aspects of allergy. IL-4 is a cytokine produced by Th2 cells, mast cells, and eosinophils. It functions to regulate immunoglobulin class switching in B cells and enhances the expression of FceRI, the high affinity receptor that binds allergen-specific IgE on a variety of immune cells. IL-4 can also induce proliferation, survival, and/or chemotaxis of many cell types such as B cells, mast cells, and eosinophils, some of the key players in allergy (Izuhara et al., 2002). IL-13 is also produced by Th2 cells, and receptors for this cytokine have been found on epithelial cells and macrophages. Its function has been studied more from the perspective of allergic airway disease, so the understanding of its role in allergic skin disease is limited (Wills-Karp & Finkelman, 2008). IL-6 is a cytokine produced by a variety of cell types including T cells and macrophages and plays a key role in activating the innate immune system (e.g., neutrophil migration to inflamed sites) and promoting B-cell differentiation and antibody production (Mihara et al., 2012). IL-2 regulates T-cell proliferation and can induce the production of T-cell cytokines such as IFN-c. Both IL-2 and IFN-c transcripts have been found to be upregulated in the skin of atopic dogs (Nuttall et al., 2002), suggesting that IL-2 can contribute to cellular and molecular changes that drive inflammation in the skin. Finally, IL-31 is a recently identified cytokine that can induce scratching behavior in dogs when injected into the animal and has also been found in dogs with naturally occurring AD (Gonzales et al., 2013), suggesting IL- 31 may be an important cytokine driving pruritic responses in dogs. The ability to inhibit JAK1-dependent cytokines discussed above is a desirable trait to have for a treatment of allergic skin disease where chronic inflammation and pruritus are important clinical signs to control in the animal.

Table 3. Inhibitory activity of oclacitinib in cell model systems

Oclacitinib appears to have specificity toward cytokines involved in allergy, inflammation, and pruritus but shows reduced activity toward cytokines involved in hematopoiesis (GM-CSF and erythropoietin; IC50’s > 1000 nM). Many hematopoietic cytokines bind homodimeric receptors that do not recruit JAK1 but instead recruit JAK2/JAK2 pairs for signal transduction and induction of proliferation of bone marrow cells (Schindler & Plumlee, 2008) (Fig. 2). Although oclacitinib appeared to be potent against JAK2 in isolated enzyme assays, potency toward cytokines that engage receptor complexes utilizing JAK2/JAK2 pairings was not significant in cell-based assays. The isolated enzyme reactions are contrived systems where the enzymes are artificially activated, and truncated versions of the enzyme are used in tests instead of full-length enzymes in their natural state, associated with their appropriate receptor within a cell, and activated by their natural ligand. Homology between the human and canine JAK2 amino acid sequences 808–1132, the sequence of the truncated JAK2 protein used in isolated enzyme reactions, is 98%, whereas homology between the human and canine full-length JAK2 protein is 94%. Although not significantly different, it is possible that we could not engineer the JAK2 enzyme to completely mimic its natural state within the cell, which could cause data generated from enzyme assays to be dramatically different from cell-based data. A similar disconnect in potency between the IC50 generated in JAK2 isolated enzyme assays and JAK2- dependent cell-based assays has also been noted with tofacitinib where the IC50 against JAK2 in the enzyme assay was reported to be 4.1 nM, but the IC50 was 1377 nM in wholeblood assays evaluating the activity of GM-CSF on human monocytic cells (Meyer et al., 2010). As oclacitinib was less potent at inhibiting the function of cytokine receptor complexes utilizing JAK2/JAK2 pairings compared with the cytokines that could activate receptors that engaged JAK1/JAK3, JAK1/JAK2, or JAK1/JAK2/TYK2 pairings, these data suggest inhibition of JAK1 is driving the selectivity pattern of oclacitinib in cells.

Oclacitinib showed no activity toward the function of IL-12 and IL-23 (IC50’s > 3000 nM) in human cell-based assays. IL- 12 and IL-23 bind receptors that recruit JAK2 and TYK2 reconfirming minimal or no activity toward cytokine-receptor complexes that are not dependent on the JAK1 enzyme (Fig. 2). These cytokines are produced by antigen-presenting cells in response to microbial pathogens such as viruses, bacteria, and parasites and play a key role in engaging the adaptive immune system. Their activity is essential in preventing serious infections and inducing appropriate responses to fight off foreign invaders (Vignali & Kuchroo, 2012). Expanding our panel of cytokine testing to other JAK-dependent cytokines involved in similar innate immune cell activities such as type I interferons (IFN b/x/t) and type II IFN-y will be important to better understand oclacitinib’s effects on cytokines involved in innate immune cell responses.

As some of our cell model systems used to test the potency and selectivity of oclacitinib were human cell model systems, particularly those cytokine-receptor complexes that utilized the JAK2/TYK2 pairings (e.g., IL-12 and IL-23 functional cellbased tests), it is important to keep in mind the homology between the human and canine JAK sequences. The sequence homology between human and canine full-length JAK1, JAK2, JAK3, and TYK2 proteins are 98%, 94%, 100%, and 83%, respectively (Figure S1). Oclacitinib did not inhibit IL-12 and IL-23 function in human cell-based assays; however, these cytokines activate receptors that utilize JAK2 and TYK2 pairings. Human TYK2 is only 83% homologous to canine TYK2, which is much lower than the other JAK family members. Therefore, it is important to continue investigations of oclacitinib potency and selectivity in canine cell model systems to determine whether this initially observed selectivity pattern toward JAK1-dependent cytokines will hold true in canine receptor systems, particularly those dependent on JAK2/TYK2 pairings.

Fig. 2. Cytokine receptor families that utilize Janus kinase (JAK) enzymes for signaling. Cytokines are small proteins secreted by cells that can produce a variety of responses such as growth, development, differentiation, and activation of immune cells as well as nonimmune cell types. Cytokines exert these biological effects by binding to receptors on the surface of cells. Several cytokine receptors rely on the association and activation of JAK enzymes on the cytoplasmic portion of the receptor in order to transmit signals to the nucleus and induce necessary changes within the cell. Cytokine receptors can be groups according to the types of JAKs that are recruited to the receptor complexes. Many cytokines involved in allergy, inflammation, and pruritus bind receptor complexes that utilize JAK1. For example, IL-2 and IL-4 will bind receptor complexes that recruit JAK1 and JAK3. IL-6 and IL-13 bind receptors that engage JAK1, JAK2, and TYK2, and IL-31 will engage receptors that activate JAK1 and JAK2. In contrast, several cytokines involved in hematopoiesis (GM-CSF, erythropoietin) or innate immune cell defenses (IL-12 and IL-23) activate receptors dependent on JAK2/JAK2 or JAK2/TYK2 pairings.

Oclacitinib (APOQUEL) was recently approved for the control/treatment of pruritus associated with allergic dermatitis and the control or treatment of AD in dogs at least 12 months of age in the US and EU. In the present study, oclacitinib was shown to inhibit JAK1-dependent cytokines involved in allergy, inflammation, and pruritus, suggesting that these are the mechanisms by which oclacitinib is effectively treating allergic skin diseases in dogs. We recently disclosed the results of a placebo-controlled field trial evaluating the efficacy of oclacitinib in client-owned dogs with allergic dermatitis (Cosgrove et al., 2013). Oclacitinib produced a significant reduction in mean Owner Pruritus Visual Analog Scale (VAS) scores in these dogs compared with placebo-treated animals as early as Day 1 and on all other evaluation days of the study (Days 1–7; P < 0.0001). The Veterinary Dermatitis VAS scores were also significantly reduced on Day 7 (P < 0.0001) in the oclacitinibtreated animals compared with placebo. Therefore, inhibition of JAK1-dependent cytokines involved in allergy, inflammation, and pruritus is an effective and novel way to treat clinical signs associated with canine allergic skin disease.

SOURCES OF FUNDING

This study was self-funded.

CONFLICT OF INTEREST

Zoetis employees are Zoetis Inc. shareholders.

ACKNOWLEDGMENTS

The authors would like to acknowledge Tom Wilson for informatics support.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Figure S1. Sequence homology between human and canine JAK family members.

REFERENCES

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