Canine Cytokine Research Articles

Abstract 2837: Conservation of JAK-STAT signaling in canine lymphocytes responding to human IL-6, IL-7, and IL-1 alpha

Abstract Dogs share physiological similarities with humans, positioning them as valuable models for studying the efficacy and safety of drugs and their underlying mechanisms of action. The JAK-STAT pathway, a highly conserved signaling pathway across species, including humans and dogs, presents an opportunity to investigate the therapeutic potential of cytokines that utilize this pathway. In this study, we aimed to evaluate the functional activity of three canine cytokines on canine cells. To assess stimulation of the JAK/STAT pathway, we used a flow cytometry assay that measures phospho-STAT in activated canine or human lymphocytes in whole blood specimens. Testing of commercially available human and canine IL-6 proteins revealed that canine lymphocytes respond poorly to most canine IL-6 products, whereas all recombinant human IL-6 products stimulated STAT phosphorylation in canine lymphocytes, despite sharing low sequence similarity with canine IL-6. In human T cells, both canine IL-6 and human IL-6 stimulated STAT phosphorylation, demonstrating biological activity from the canine IL-6 products. Several JAK inhibitors inhibited cytokine-induced STAT phosphorylation in both canine and human CD4 T-cells, consistent with JAK inhibition, but targeting the IL-6 receptor with tocilizumab only blocked IL-6-induced STAT phosphorylation in human but not canine CD4 T-cells in whole blood. These findings demonstrate the potential of our assay system to assess cross-species cytokine reactivity, determine if JAK/STAT inhibitors used clinically might be useful in treating inflammatory conditions in dogs, and identify novel canine-specific inhibitors. Additionally, we explored the induction of IL-8 by human and canine IL-1 alpha in IL-1 alpha sensitive human cells. Human IL-1 alpha failed to stimulate a canine IL-1 alpha sensitive cell line, suggesting species-specific differences in IL-1 alpha activity. Furthermore, we discovered that canine IL-7 and human IL-7 are species-specific. In summary, this proof-of-concept study highlights the capability to assess the activation of STATs and pathway inhibition by JAK inhibitors in canine whole blood using flow cytometry. We confirmed that human IL-6 stimulates JAK/STAT signaling in canine CD4 T-cells. Our results also revealed significant differences in species cross-reactivity of human IL-6, IL-7, and IL-1 alpha, underscoring the importance of species-specific investigations. Leveraging the similarities and differences between canine and human cytokines targeting the JAK-STAT pathway can enhance drug development efforts and contribute to the advancement of safer and more effective therapies for both humans and pet dogs with similar diseases. This study was funded by NCI Contract No. 75N91019D00024. Citation Format: King Leung Fung, Melissa L. Breiner, James B. Mitchell, Ralph E. Parchment. Conservation of JAK-STAT signaling in canine lymphocytes responding to human IL-6, IL-7, and IL-1 alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2837.

Modulation of In Vitro Macrophage Responses via Primary and Secondary Bile Acids in Dogs

Bile acids (BA) are important metabolites secreted into the intestinal lumen and impacted by luminal microbes and dietary intake. Prior studies in humans and rodents have shown that BAs are immunologically active and that primary and secondary BAs have distinct immune properties. Therefore, the composition of the gut BA pool may influence GI inflammatory responses. The current study investigated the relative immune modulatory properties of primary (cholic acid, CA) and secondary BAs (lithocholic acid, LCA) by assessing their effects on canine macrophage cytokine secretion and BA receptor (TGR5) expression. In addition, RNA sequencing was used to further interrogate how CA and LCA differentially modulated macrophage responses to LPS (lipopolysaccharide). We found that exposure to either CA or LCA influenced LPS-induced cytokine production via macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis.

Canine immune cells express high levels of CB1 and CB2 cannabinoid receptors and cannabinoid-mediated alteration of canine cytokine production is vehicle-dependent

With the increased popularity and societal acceptance of marijuana and cannabidiol (CBD) use in humans, there is an interest in using cannabinoids in veterinary medicine. There have been a few placebo-controlled clinical trials in dogs suggesting that cannabis-containing extracts are beneficial for dogs with inflammatory diseases such as osteoarthritis, and there is growing interest in their immunosuppressive potential for the treatment of immune-mediated diseases. Since cannabinoids exhibit anti-inflammatory and immunosuppressive effects in many species, the purpose of these studies was to examine whether the plant-derived cannabinoids, CBD and Δ9-tetrahydrocannabinol (THC), would also suppress immune function in canine peripheral blood mononuclear cells (PBMCs). Another goal was to characterize expression of the cannabinoid receptors, CB1 and CB2, in canine immune cells. We hypothesized that CBD and THC would suppress stimulated cytokine expression and that both cannabinoid receptors would be expressed in canine immune cells. Surprisingly, cannabinoid suppressive effects in canine PMBCs were quite modest, with the most robust effect occurring at early stimulation times and predominantly by THC. We further showed that cannabinoid-mediated suppression was dog- and vehicle-dependent with CBD and THC delivered in dimethyl sulfoxide (DMSO) producing more immune suppressive effects as compared to ethanol (ETOH). PCR, flow cytometry, and immunohistochemical staining demonstrated that both CB1 and CB2 are expressed in canine immune cells. Together these data show that canine immune cells are sensitive to suppression by cannabinoids, but more detailed studies are needed to further understand the mechanisms and broad effects of these compounds in the dog.

Detection of pro-inflammatory cytokines in healthy canine tears using Canine Cytokine SpikeMix™ mass spectrometry via multiple reaction monitoring.

To evaluate the efficacy of the Canine Cytokine SpikeMix™ and MRM-MS for detecting pro-inflammatory cytokines in canine tears from healthy research Beagles. A complete ophthalmic examination was performed on 15 healthy research Beagles to verify no ophthalmic diseases. Tears were collected OU by placing a Weck-Cel® cellulose spear in the ventral conjunctival fornix for 1 min. The Weck-Cel® spear was placed in a 2.0 mL tube with a centrifuge filter forcing tears to flow through the filter into the bottom of the tube. The tears were analyzed using the Canine Cytokine SpikeMix™ and MRM-MS. Descriptive data from this study was reported as the normalized total peak area (nTPA) and median (range) using data imported from the online MRM-MS Skyline program. The level of 16 pro-inflammatory cytokines was successfully detected in all 15 dogs. The four cytokines with the highest median amounts in the samples were IL-2 = 0.1243 (0.019-6.7289), IL-6 = 0.964 (0.0036-16.9365), TNFα = 0.1644 (0.0096-0.7138), and CSF-2 = 0.4022 (0.1475-2.6208). This study revealed that 16 pro-inflammatory cytokines in canine tears from healthy dogs can be detected with Canine Cytokine SpikeMix™ and MRM-MS.

Fuzapladib in a randomized controlled multicenter masked study in dogs with presumptive acute onset pancreatitis.

Currently, no specific treatment is available for acute onset pancreatitis (AP), and management relies on symptomatic and supportive standard of care (SOC). Fuzapladib is a novel leukocyte function-associated antigen type-1 (LFA-1) activation inhibitor, blocking activation and subsequent adhesion and migration of neutrophils, potentially decreasing the risk of pancreatitis progression and systemic inflammation. Evaluate the safety and clinical response of dogs with AP after 3 days of administration of fuzapladib. Sixty-one client-owned dogs with presumptive AP. Randomized, masked, and placebo controlled multicenter study. Sixty-one dogs with AP were included for safety assessment, whereas 35 evaluable cases (fuzapladib, n = 16; placebo, n = 19) were included for clinical evaluation. Clinical improvement was assessed based on the change in the modified clinical activity index (MCAI) score on Day 3 compared to Day 0. Secondary variables included canine acute pancreatitis clinical severity index (CAPCSI) scores and serum concentrations of canine pancreatic lipase immunoreactivity, cytokines, and C-reactive protein. Fuzapladib was well tolerated by all treated dogs. Mean change in MCAI scores was significantly higher in the fuzapladib-treated (-7.75) than the placebo group (-5.68; P = .02, 95% confidence interval [CI] for the difference, -4.33, -0.35), suggesting clinical improvement in fuzapladib-treated dogs. No significant difference was found in any of the secondary variables between groups. Administration of fuzapladib to dogs was safe, and a favorable response was detected in 2 clinical activity scores. Effects of fuzapladib on survival and duration of hospitalization were not studied.

Pro-inflammatory cytokines in aqueous humor from dogs with anterior uveitis and post-operative ocular hypertension following phacoemulsification, primary glaucoma, and normal healthy eyes.

BackgroundThe aim of this study was to evaluate the levels of pro-inflammatory cytokines in aqueous humor (AH) from dogs with anterior uveitis and post-operative ocular hypertension (POH) following phacoemulsification, in AH from dogs with primary glaucoma, and in normal healthy eyes with no signs of anterior uveitis or other ocular diseases.MethodsAn exploratory study including 21 samples of AH collected from 15 dogs; post-phacoemulsification with anterior uveitis and POH (‘POH group’, n = 10 samples), primary glaucoma (‘glaucoma group’, n = 6 samples), and normal (‘normal group’, n = 5 samples). Target mass spectrometry via multiple reaction monitoring (MRM-MS) with the Canine Cytokine SpikeMix™ as internal standard was used to measure the pro-inflammatory cytokine levels.ResultsThe MRM-MS method measured 15 pro-inflammatory cytokines. Tumor-necrosis-factor-alpha (TNFα) and interleukin-18 (IL-18) levels in AH were different between all three groups (glaucoma>POH>normal) (p = .05, p = .02, respectively). Additionally, IL-6 was higher in the ‘POH group’ compared to the ‘glaucoma group’ (p = .04) and IL-4 was higher in the ‘POH group’ compared to the ‘normal group’ (p = .04). Intraocular pressure (IOP) was positively associated with increased AH levels of IL-18 (Spearman correlation = .64, p = .03).ConclusionsMRM-MS using the Canine Cytokine SpikeMix™ as an internal standard was established as a method to detect pro-inflammatory cytokine levels in canine AH. The study demonstrated increased levels of IL-4, IL-6, IL-18, and TNFα in AH from canines with POH following phacoemulsification. Primary glaucomatous eyes had the highest levels of IL-18 and TNFα which may indicate that inflammation plays a role in the pathogenesis of primary glaucoma in dogs.

Canine Cytokines Profile in an Endemic Region of L. infantum: Related Factors.

Canine leishmaniosis is caused by infection with parasite Leishmania infantum, which are transmitted by sandflies Phlebotomus. Canine leishmaniosis is an endemic disease in the Mediterranean region. The immune response could vary between hosts and determines the severity of the disease and clinical features. The aim of this study was to analyze the serum levels of cytokines TNF-α, IFN-γ, IL-2, IL-6, and IL-8, which are related to the activation of Th1 or Th2 immune responses in dogs living in the L. infantum endemic region. Moreover, we intend to relate and correlate these levels with different factors, such as sex, age, diet, lifestyle, and breed. Epidemiological data and serum were recovered for seventy-eight dogs, and serum levels of cytokines described previously were analyzed by using the ELISA method. The results showed differences in serum levels of IFN-γ, IL-2, and IL-8 between breeds. The lifestyle also affected serum levels of IL-2. The main conclusion of this study is that Ibizan hounds and crossbred dogs have a serological profile of cytokines that seems to indicate certain protections against infection by L. infantum compared to boxer and purebred breeds.

Kinetics of the inflammatory response during experimental Babesia rossi infection of beagle dogs

Babesia rossi causes severe morbidity and mortality in dogs in sub-Saharan Africa, and the complications associated with this disease are likely caused by an unfocused, excessive inflammatory response. During this experimental B. rossi study we investigated inflammatory marker and cytokine kinetics during infection and after treatment. We aimed to determine whether infectious dose and treatment would influence the progression of the inflammatory response and clinical disease. Six healthy male beagle dogs formed the study population, one was used to raise the infectious inoculum, three were administered a high B. rossi infectious dose (HD group) and two a low infectious dose (LD group). Clinical examination, complete blood count (CBC) and C-reactive protein (CRP) were determined daily. Cytokines were quantified on stored plasma collected during the study, using a canine specific cytokine magnetic bead panel (Milliplex©). The experiment was terminated and treatment administered when predetermined experimental or humane endpoints were reached. Parasitemia occurred on day 1 and 3 in the HD and LD groups respectively. The rate of increase in parasitemia in the HD group was significantly faster than that seen in the LD group. Significant differences were found in heart rate, blood pressure, interferon gamma (INFγ), keratinocyte chemoattractant (KC), INFγ-induced protein 10 (IP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP1), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), IL-6, IL-7, IL-8, IL-10 IL-15, IL-18, CRP, neutrophils and monocytes between groups at multiple time points during the course of the infection. Our findings suggest that the initiation of inflammation occurs before the onset of clinical disease in B. rossi infection and infectious dose influences the onset of the inflammatory response. Treatment enhances the inflammatory response in the immediate post-treatment period which may contribute to disease associated complications. Finally, we found that there is an imbalance in pro/anti-inflammatory cytokine concentrations during infection which may promote parasite replication.

The Increase in Circulating Levels of Pro-Inflammatory Chemokines, Cytokines, and Complement C5 in Canines with Impaired Kidney Function.

Chronic low-grade inflammation is a key contributor to the progression of kidney disease. The release of cytokines and other pro-inflammatory proteins may further contribute to detrimental kidney health by increasing interstitial edema and renal fibrosis. The aim of the present study was to investigate the inflammatory markers in canines who developed renal disease naturally and were diagnosed with renal disease either during life or following necropsy, as assessed by a veterinarian. RNA was isolated from canine blood obtained at necropsy and stored as bioarchived samples from ten canines with renal disease (9.6–14.7 yr) and ten controls (10.1–14.8 yr). At the time of death, the mean blood creatinine concentration and BUN were elevated in dogs with renal disease compared to control (both p < 0.01). Samples were assessed for changes in gene expression using the Canine cytokine RT2 Profiler PCR Array for inflammation. There was a significant increase in C-C Motif Chemokine Ligand 16 (CCL16), C-X-C Motif Chemokine Ligand 5 (CXCL5), Interleukin 16 (IL-16), and Complement Component 5 (C5) (all p < 0.05 vs. con). In addition, there was also a statistically non-significant increase in 49 genes and a down-regulation in 35 genes from a panel of total 84 genes. Pro-inflammatory genes including CCL16, CXCL5, IL-16, and C5 can all contribute to renal inflammation and fibrosis through different signaling pathways and may lead to a progressive impairment of kidney function. Blockade of their activation may be important in ameliorating the initiation and/or the progression of renal disease.

Multiplex Cytokine Analyses in Ear Canals of Dogs Suggest Involvement of IL-8 Chemokine in Atopic Otitis and Otodectic Mange-Preliminary Results.

Simple SummaryAtopic dermatitis is a form of allergy of genetic origin accompanied by itching, during which the animal more easily develops allergies to environmental factors, usually pollen and dust mites. Among the symptoms, otitis is common. The mechanisms of this disease are still not fully understood. In this study, we aimed to demonstrate the detection ability of inflammatory markers (cytokines and chemokines) in the ear canals of atopic dogs suffering from otitis externa compared to healthy dogs and to dogs with parasitic otitis (inflammatory but not allergic otitis). Therefore, we non-invasively sampled the surface of the ear canals of atopic dogs and compared the amounts of certain cytokines and chemokines with those in similar samples taken from ears of healthy dogs and ears with spontaneous ear mite infestation. It appears that concentrations of IL-8 are significantly higher in atopic ears than in healthy ears. Nevertheless, this difference does not appear to be atopic-specific, since the amount of interleukin-8 (IL-8) also increased in ears infested with mites. Further investigations with a larger number of dogs are now required to confirm these results and possibly to find other biomarkers involved in the pathogenesis of canine atopic otitis.Cutaneous cytokines and chemokines are involved in the pathogenesis of human and canine atopic dermatitis. The aim of the present study was to discriminate cytokine expression in the ear canals of atopic dogs with otitis, dogs with non-allergic inflammatory otitis (otodectic mange) and healthy non-atopic dogs. The ear canals of nine atopic dogs suffering from non-infected otitis externa (n = 14 ears), 10 healthy dogs suffering from otodectic mange (n = 20 ears) and 21 healthy controls (39 ears) were swabbed. The concentrations of a panel of 13 cytokines and chemokines on the aural surface were measured by multiplex analyses (Milliplex Canine Cytokine Panel). In addition, Canine Atopic Dermatitis Extent and Severity Index (CADESI)-04 and Otitis Index Score (OTIS3) scores were used to evaluate the overall status of the dogs. The concentration of IL-8 was significantly higher in the ears of atopic dogs and dogs with otodectic mange compared to those of healthy dogs. Significant increases in the levels of IL-10 were also overexpressed in atopic otitis but at lower rates. The concentrations of interleukin(IL)-8 were positively correlated with the OTIS3 hyperplasia score in atopic dogs. Taken together, these results suggest that IL-8 is overexpressed in atopic otitis and otodectic mange and that levels correlate with the otitis severity in atopic dogs.

Automated blood sampling in canine telemetry model: Enhanced assessment of immune liabilities

IntroductionThis manuscript presents a successful integration of multi-timepoint biomarker blood sampling (e.g., cytokines) in a conscious dog cardiovascular study using automated blood sampling via vascular access ports in telemetry instrumented dogs. In addition to determining plasma exposure of the test compound, the assessment of biomarkers of interest allows for more comprehensive preclinical evaluation on a traditional conscious dog cardiovascular (CV) telemetry study especially for immunology and immune-oncology molecules. This model system provides a rapid and efficient means to quickly gain understanding of potential effects on key cardiovascular parameters in large species that are commonly used for preclinical safety evaluations while collecting multiple blood samples for drug and cytokine analysis. MethodsMale beagle dogs were chronically implanted with telemetry devices (PhysioTel™ model D70-PCTP) and vascular access ports (SPMID-GRIDAC-5NC). BASi Culex-L automated blood sampling (ABS) (Bioanalytical Systems, Inc) system was used to collect blood samples at multiple time points for cytokine analysis. Four beagles received low-dose lipopolysaccharide solution (LPS) (0.1 and 0.5 μg/mL). The following cytokines were measured by Milliplex® map Canine Cytokine Magnetic Bead Panel: Interleukin (IL) 2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, TNF-α, MCP-1, KC-like, GM-CSF, IFN gamma, and IP10. ResultsLow dose LPS administration induced a pronounced dose-dependent, transient release of key inflammatory cytokines (IL-2, IL-6, IL-10, TNF-α, MCP-1, and KC-like). Cytokine responses were similar to other canine and human endotoxin models. LPS administration led to an increase in body temperature, heart rate, and mean arterial pressure, as well as a decrease in QTcV interval. ConclusionSuccessful incorporation of cytokine analysis in telemetry instrumented dogs with vascular access ports allows for translational PK/PD modeling of both efficacy and safety of compounds in the immunology as well as the immune-oncology therapeutic areas designed to modulate the immune system. Remote collection of blood samples simultaneously with CV endpoints is a significant enhancement for assessment of biomarkers that are sensitive to animal handling and excitement associated with room disturbances which are obligatory with manual blood collection. Furthermore, implementing this approach has also refined our animal welfare procedure by reducing the handling during a study and thereby reducing stress (positive refinement 3R impact).

Serum Concentration of Inflammatory Cytokines in Dogs with Suspected Acute Pancreatitis.

Acute pancreatitis is an acute inflammatory process in the pancreas that is common in dogs. This study was designed to compare cytokines between healthy dogs and dogs with suspected acute pancreatitis. For the canine cytokine antibody array, three healthy dogs and three dogs with suspected acute pancreatitis were included. Interleukin (IL)-2, IL-6, IL-10, GM-CSF, and TNF-α were not detected in either group based on the results. Conversely, IL-8 (p = 0.035), Monocyte Chemoattractant Protein-1 (MCP)-1 (p = 0.0138), Receptor for Advanced Glycation Endproducts (RAGE) (p = 0.0079), and stem cell factor (SCF) (p = 0.034) were significantly increased in dogs with suspected acute pancreatitis. However, vascular endothelial growth factor (VEGF) (p = 0.6971) did not differ significantly between groups. For the canine serum Enzyme-Linked Immunosorbent Assay (ELISA), eight healthy dogs and eight dogs with suspected acute pancreatitis were included. ELISA revealed that IL-8 (p < 0.0001), MCP-1 (p < 0.0001), RAGE (p = 0.006), and SCF (p = 0.0002) were all significantly upregulated in the experimental group. We confirmed multiple patterns of cytokines in suspected acute pancreatitis of dogs via canine cytokine antibody array using a small quantity of serum. After this procedure, we reevaluated the cytokines, which were significantly increased in dogs with suspected acute pancreatitis, by ELISA, with more samples. Through this study, we confirmed that MCP-1, RAGE, and SCF were newly suggested factors in dogs with suspected acute pancreatitis.

Pluripotency and immunomodulatory signatures of canine induced pluripotent stem cell-derived mesenchymal stromal cells are similar to harvested mesenchymal stromal cells

With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCs harvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC). A combination of deep sequencing and quantitative RT-PCR of the ciMSC transcriptome confirmed that ciMSCs express more genes in common with cBM-MSCs and cAT-MSCs than with the ciPSCs from which they were derived. Both ciMSCs and harvested cMSCs express a range of pluripotency factors in common with the ciPSCs including NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. CXCR-4, which is essential for the homing of MSCs to sites of inflammation, is also detectable in ciMSCs, cAT- and cBM-MSCs, but not ciPSCs. ciMSCs constitutively express the immunomodulatory factors iNOS, GAL-9, TGF-β1, PTGER-2α and VEGF, and the pro-inflammatory mediators COX-2, IL-1β and IL-8. When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCs upregulated their expression of IDO, iNOS, GAL-9, HGF, TGF-β1, PTGER-2α, VEGF, COX-2, IL-1β and IL-8. When co-cultured with mitogen-stimulated lymphocytes, ciMSCs downregulated their expression of iNOS, HGF, TGF-β1 and PTGER-2α, while increasing their expression of COX-2, IDO and IL-1β. Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into their potential use for the management of canine immune-mediated and inflammatory disorders.

Climate-associated drivers of plasma cytokines and contaminant concentrations in Beaufort Sea polar bears (Ursus maritimus)

Assessing polar bear (Ursus maritimus) immune function in relation to environmental stressors, including habitat change, nutritional stress, pathogen prevalence, and pollution, has been identified as critical for improved understanding of the species' health. The objectives of this study were two-fold: 1) to assess the role of climate-associated factors (habitat use, body condition) in explaining the plasma concentrations of contaminants in southern Beaufort Sea (SB) polar bears, and 2) to investigate how climate-associated factors, contaminant concentrations, and pathogen sero-prevalence influence the plasma concentrations of immune-signaling proteins called cytokines. A commercially available multiplex canine cytokine panel was validated for the quantification of five pro- and anti-inflammatory cytokines in polar bear plasma: tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-8, IL-10, and interferon gamma-induced protein 10 (IP-10). This panel was then used to measure cytokine concentrations in 49 SB polar bears sampled in the springs of 2013 and 2014. Mean ∑PCBs (plasma), ∑OCs (plasma), and THg (hair) were 13.01 ± 1.52 ng g−1 w.w. (range: 0.17–52.63), 19.46 ± 1.17 ng g−1 w.w. (range: 6.63–45.82), and 0.49 μg g−1 d.w. (range: 0.99–15.18), respectively. Top models explaining variation in concentrations of plasma PCBs, plasma OC pesticides, and hair THg in SB polar bears included body mass index and/or habitat use (onshore versus offshore), with higher contaminant concentrations in leaner and/or offshore bears. Plasma cytokine concentrations were influenced most strongly by plasma PCBs and age, with little to no influence found for plasma OCs or hair THg concentrations, habitat use, or pathogen sero-prevalence. The lack of association between cytokines and these latter variables is likely due to a temporal disconnect between measured endpoints. The change of polar bear habitat use, feeding ecology, and body condition with ongoing climate warming is affecting exposure to contaminants and pathogens, with potential adverse consequences on a well-balanced immune system.

Combined in vitro IL-12 and IL-15 stimulation promotes cellular immune response in dogs with visceral leishmaniasis.

Domestic dogs are the main reservoir of Leishmania infantum, a causative agent of visceral leishmaniasis (VL). The number of human disease cases is associated with the rate of canine infection. Currently available drugs are not efficient at treating canine leishmaniasis (CanL) and months after the treatment most dogs show disease relapse, therefore the development of new drugs or new therapeutic strategies should be sought. In CanL, dogs lack the ability to mount a specific cellular immune response suitable for combating the parasite and manipulation of cytokine signaling pathway has the potential to form part of effective immunotherapeutic methods. In this study, recombinant canine cytokines (rcaIL-12, rcaIL-2, rcaIL-15 and rcaIL-7) and soluble receptor IL-10R1 (rcasIL-10R1), with antagonistic activity, were evaluated for the first time in combination (rcaIL-12/rcaIL-2, rcaIL-12/rcaIL-15, rcaIL-12/rcasIL-10R1, rcaIL-15/rcaIL-7) or alone (rcasIL-10R1) to evaluate their immunomodulatory capacity in peripheral blood mononuclear cells (PBMCs) from dogs with leishmaniasis. All the combinations of recombinant proteins tested were shown to improve lymphoproliferative response. Further, the combinations rcaIL-12/rcaIL-2 and rcaIL-12/rcaIL-15 promoted a decrease in programmed cell death protein 1 (PD-1) expression in lymphocytes. These same combinations of cytokines and rcaIL-12/rcasIL-10R1 induced IFN-γ and TNF-α production in PBMCs. Furthermore, the combination IL-12/IL-15 led to an increased in T-bet expression in lymphocytes. These findings are encouraging and indicate the use of rcaIL-12 and rcaIL-15 in future in vivo studies aimed at achieving polarization of cellular immune responses in dogs with leishmaniasis, which may contribute to the development of an effective treatment against CanL.

The inflammatory cytokine effect of Pam3CSK4 TLR2 agonist alone or in combination with Leishmania infantum antigen on ex-vivo whole blood from sick and resistant dogs

BackgroundA wide spectrum of clinical manifestations and immune responses exist in canine L. infantum infection. Ibizan hounds are more “resistant” to disease than other dog breeds. Recognition of pathogen-associated molecule patterns by toll like receptors (TLRs) rapidly triggers a variety of anti-microbial immune responses through the induction of pro-inflammatory cytokines such as TNF-α and IL-6 which may play an important role in controlling Leishmania infection. The main objective of this study was to investigate and compare the effect of a TLR2 agonist (TLR2a) alone or in combination with L. infantum antigen (LSA) on ex vivo whole blood cytokine production from healthy seronegative IFN-γ non-producer dogs from an area of low in canine leishmaniosis endemicity (n = 11); sick seropositive dogs with low production of IFN-γ (n = 17) and healthy seronegative or low positive Ibizan hounds with a predominant IFN-γ production (n = 21) from a highly endemic area. Whole blood was stimulated with medium alone (Ø), LSA, concanavalin A, TLR2 (Pam3CSK4) receptor agonist (Ø + TLR2a) and TLR2a and LSA (LSA + TLR2a) for 48 h. Supernatants were harvested for measurement of canine TNF-α and IL-6 cytokines by ELISA.ResultsA significant increase of TNF-α was found in the supernatants of stimulated blood from all groups (Ø + TLR2a and LSA + TLR2a) when compared with medium alone. A similar pattern was observed for IL-6. Interestingly, a significant increase of TNF-α production was only observed when stimulation with LSA + TLR2a was compared with TLR2a alone in Ibizan hounds. A significant increase of TNF-α production was observed with stimulation of LSA + TLR2a when compared with LSA in all groups. Significantly higher concentrations of TNF-α and IL-6 were detected in Ibizan hounds, especially for the Ø + TLR2a and LSA + TLR2a treatments compared with other groups.ConclusionsThis study demonstrated that TLR2a alone enhances the production of the inflammatory cytokines TNF-α and IL-6 in sick, “resistant” and healthy non-infected dogs. In addition, a combination of LSA+TLR2a promoted a synergistic pro-inflammatory effect with TNF-α in Ibizan hounds but not in seropositive sick dogs and seronegative healthy dogs. These findings might suggest the importance of Pam3CSK4 as a possible immunomodulator for CanL.

Cross-reactivity of commercially available anti-human monoclonal antibodies with canine cytokines: establishment of a reliable panel to detect the functional profile of peripheral blood lymphocytes by intracytoplasmic staining.

BackgroundThe process for obtaining monoclonal antibodies against a specific antigen is very laborious, involves sophisticated technologies and it is not available in most research laboratories. Considering that most cytokines remain partially conserved among species during evolution, the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine. In this context, the amino acid sequence from human and canine cytokines have demonstrated 49–96 % homology, suggesting high probability of cross-reactivity amongst monoclonal antibodies. For this, 17 commercially available anti-human monoclonal antibodies [IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-10, IL-12, IL-13, IL-17A, IFN-γ (#1, #2), TNF-α (#1, #2) and TGF-β], were evaluated in vitro for intracellular cytokine detection in a stimulated canine blood culture by flow cytometry and confocal microscopy. Lymphocytes from peripheral blood of healthy and two unhealthy dogs were analyzed.ResultsEleven anti-human mAbs [IL-1α, IL-4, IL-5, IL-6, IL-8 (#1, #2), IL-12, IL-17A, TNF-α (#1, #2) and TGF-β] cross-reacted against canine intracellular cytokines. The specificity of the assays was not affected after Fc-blocking. Three anti-human cytokine mAbs [IL-4, IL-8 (#2) and TGF-β] when evaluated by confocal microscopy also cross-reacted with intracellular canine cytokines. The identification of human mAbs that cross-reacted with canine cytokines may support their use as immunological biomarkers in veterinary medicine studies.ConclusionThe identification of these 11 anti-human cytokine mAbs that cross-reacted with canine cytokines will be useful immunological biomarkers for pathological conditions by flow cytometry and fluorescence microscopy in dogs.

Validation of a commercial canine assay kit to measure pinniped cytokines

The present study was conducted to assess and validate the cross-reactivity of commercially available multiplex human and canine cytokine kits coupled with the Bio-Plex 200 platform to measure cytokines in three pinniped species, harbor seals (Phoca vitulina), gray seals (Halichoerus grypus), and harp seals (Pagophilus groenlandicus). Cytokines are important small proteins that help direct a proper immune response to pathogens. The human cytokine kit allowed the detection of cytokines in the supernatant of mitogen-stimulated human peripheral blood mononuclear cells, but not in the three pinniped species studied, with the exception of TNFα and GM-CSF. In contrast, the canine cytokine kit appeared to cross-react with the majority of cytokines in the three pinniped species tested, including the pro-inflammatory cytokine TNFα, the Th1 cytokine INFγ, and the Th2 cytokine IL-10, an anti-inflammatory cytokine. In addition, the pro-inflammatory cytokines IL-6 and IL-8 were also measured in all pinniped species. Overall, the Bio-Plex 200 platform and the canine multiplex cytokine kit allowed the successful measurement of potentially clinically important pinniped cytokines. This additional tool may provide veterinarians with additional information to detect sub-clinical signs of inflammation or evidence for immune response, which may not be revealed during regular medical evaluation, e.g. physical examination, hematology, and serum chemistry.

Allergen-induced production of IL-31 by canine Th2 cells and identification of immune, skin, and neuronal target cells

The canine cytokine IL-31 induces pruritus in dogs and can be detected in dogs with atopic dermatitis; however very little is understood around its interactions with specific canine cells. We hypothesize that IL-31 is involved in the progression of allergic skin disease by coordinating the interaction between the immune system with skin and neuronal systems. The goal of the following work was to identify cells that produce IL-31 as well as cells that may respond to this cytokine. Peripheral blood mononuclear cells (PBMCs) were collected from naïve and house dust mite (HDM) allergen-sensitized beagle dogs and used for ex vivo characterization of cytokine production assessed using ELISpot and quantitative immunoassay. Sensitization to HDM allergen induced a T-helper type 2 (Th2) cell phenotype characterized by an increase in the production of IL-4 protein. Interestingly, repeated allergen challenge over time also resulted in an increase in IFN-γ. Further evaluation showed that co-stimulation of Th2 polarized cells with antigen and the bacterial component Staphylococcus enterotoxin B (SEB) produced higher levels of IL-31 compared to either stimulant alone. Production of IL-31 when PBMCs were stimulated by T cell mitogens suggests T cells as a source of IL-31. Quantitative real-time PCR was utilized to determine expression of the IL-31 receptor alpha chain in canine cell lines and tissue. Canine monocytic cells, keratinocytes, and dorsal root ganglia were shown to express the IL-31 receptor alpha chain mRNA. In a multifaceted disease such as canine atopic dermatitis, the combination of Th2 polarization and microbial presence may lead to IL-31 mediated effects driving inflammation and pruritus by immune cells, keratinocytes, and direct neuronal stimulation.

In vitro effects of CpG oligodeoxynucleotides delivered by gelatin nanoparticles on canine peripheral blood mononuclear cells of atopic and healthy dogs – a pilot study

Cytosine-phosphate-guanine (CpG) oligodeoxynucleotides offer a novel promising immunotherapeutic approach for atopic dermatitis (AD) both in humans and animals. Gelatin nanoparticles (GNP) enhance and prolong CpG-associated immunomodulatory effects and minimize adverse effects both in vitro and in vivo. Information about the effects of this combination in dogs is lacking. The aim of this study was to evaluate immunological effects of CpG coupled to GNP on canine peripheral blood mononuclear cells (PBMCs) in vitro. Eight dogs with AD, diagnosed by standard criteria and with a concurrent immediate hypersensitivity to house dust mites were included. Control samples were taken from eight healthy, age-matched control dogs without history or evidence of cutaneous or systemic illness. Peripheral blood mononuclear cells of healthy and allergic dogs were incubated with CpG-GNP and the uptake of CpG-GNP was demonstrated using confocal laser scanning microscopy. Cell culture supernatant concentrations of interferon gamma (IFN-γ), interleukin (IL)-4, IL-6 and IL-10 were measured by Canine Cytokine Milliplex. No significant changes in IFN-γ and IL-4 were found when comparing PBMCs incubated with CpG and CpG-GNP with the negative controls in atopic and healthy dogs. Interleukin-6 was not detected in any of the groups. However, a statistically significant increase in IL-10 concentration was found after 24h stimulation with CpG-GNP compared with CpG alone both in atopic and healthy dogs. As IL-10 is considered an immunosuppressive cytokine playing a key role in peripheral tolerance; the reported CpG-GNP formulation could be a new approach in allergy treatment.