• View in gallery

    Example of graphic results of flow cytometry showing the gating scheme used to detect phagocytosis. Live leukocytes were first separated from artifact via creation of a propidium iodide histogram (A), then applied to a plot of forward scatter versus side scatter (B). Phagocytic cells (granulocytes and monocytes combined) were identified and applied to an FITC histogram, resulting in identification of phagocytosis-negative (C) and phagocytosis-positive (D) cells.

  • View in gallery

    Example of graphic results of flow cytometry showing the gating scheme used to detect TLR4 expression. First, cells (granulocytes and monocytes combined) were identified on a plot of forward scatter versus side scatter (A) and applied to phycoerythrin scatterplots, resulting in identification of TLR4-negative (B) and TLR4-positive (C) cells.

  • View in gallery

    Mean supernatant TNF concentrations following incubation of blood samples from 8 dogs with calcitriol (10−7M) or control substance (ethanol) and subsequent exposure to LPS, LTA, or MDP. Two replicates were performed per dog. Vertical bars represent SD, and the ends of each horizontal bar indicate the treatments to which provided P value pertains.

  • View in gallery

    Lipopolysaccharide-stimulated TNF concentrations following incubation of blood samples from 3 dogs (represented by different symbols) with calcitriol at various concentrations or control substance (ethanol). Provided P values represent comparisons of mean values for all 3 dogs. See Figure 3 for remainder of key.

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Effects of calcitriol on phagocytic function, toll-like receptor 4 expression, and cytokine production of canine leukocytes

Jared A. Jaffey DVM, MS1, Juliana Amorim DVM2, and Amy E. DeClue DVM, MS3
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  • 1 Comparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 2 Comparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
  • | 3 Comparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

Abstract

OBJECTIVE To determine the in vitro effects of calcitriol on indicators of immune system function in blood samples collected from healthy dogs.

SAMPLE Blood samples from 8 healthy adult dogs.

PROCEDURES Blood samples were incubated with calcitriol (10−7M) or control substance for 24 hours. Afterward, lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate (MDP)-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) were measured with a canine-specific multiplex assay. Phagocytosis of opsonized Escherichia coli and leukocyte expression of constitutive toll-like receptor 4 (TLR4) were evaluated via flow cytometry. Blood samples from 3 dogs were used to create a concentration-response curve to evaluate whether the observed cytokine modulation was concentration dependent.

RESULTS Incubation of canine blood samples with calcitriol resulted in significant decreases in LPS-, LTA-, and MDP-stimulated leukocyte production of TNF but not IL10. Blunting of TNF production was concentration dependent. Leukocyte calcitriol exposure had no significant effect on phagocytosis and TLR4 expression.

CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in canine leukocytes exposed to LPS, LTA, and MDP in vitro, without altering phagocytosis or TLR4 expression. Thus, calcitriol could represent a novel candidate immunomodulatory treatment for dogs.

Abstract

OBJECTIVE To determine the in vitro effects of calcitriol on indicators of immune system function in blood samples collected from healthy dogs.

SAMPLE Blood samples from 8 healthy adult dogs.

PROCEDURES Blood samples were incubated with calcitriol (10−7M) or control substance for 24 hours. Afterward, lipopolysaccharide (LPS)-, lipoteichoic acid (LTA)-, and N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate (MDP)-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) were measured with a canine-specific multiplex assay. Phagocytosis of opsonized Escherichia coli and leukocyte expression of constitutive toll-like receptor 4 (TLR4) were evaluated via flow cytometry. Blood samples from 3 dogs were used to create a concentration-response curve to evaluate whether the observed cytokine modulation was concentration dependent.

RESULTS Incubation of canine blood samples with calcitriol resulted in significant decreases in LPS-, LTA-, and MDP-stimulated leukocyte production of TNF but not IL10. Blunting of TNF production was concentration dependent. Leukocyte calcitriol exposure had no significant effect on phagocytosis and TLR4 expression.

CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in canine leukocytes exposed to LPS, LTA, and MDP in vitro, without altering phagocytosis or TLR4 expression. Thus, calcitriol could represent a novel candidate immunomodulatory treatment for dogs.

A side from its classic role in calcium homeostasis and bone metabolism, vitamin D is a clinically important regulator of both innate and adaptive immune responses.1–3 Vitamin D receptors have been identified in almost all immune cells of the innate and adaptive immune systems, including B lymphocytes, CD4+ and CD8+ T lymphocytes, dendritic cells, macrophages, and neutrophils.2,4,5 In humans, calcitriol attenuates inflammation while also enhancing protective functions such as phagocytosis and antimicrobial-peptide production.2,5,6 In vitro, calcitriol augments phagocytosis,7–9 downregulates TLR4 expression,10–12 and reduces proinflammatory cytokine production by human leukocytes.13–16

Vitamin D deficiency in humans has been linked to several inflammatory or autoimmune diseases, including rheumatoid arthritis,15 inflammatory bowel disease,17 multiple sclerosis,18 asthma,19 type 1 diabetes mellitus,20 and systemic lupus erythematosus.21 Likewise, hypovitaminosis D in dogs has been associated with gastrointestinal disease,22–25 renal failure,26 cardiac disease,27,28 and sepsis.29,30,a Although vitamin D plays an integral role in numerous disease processes, a paucity of information exists regarding the immunomodulatory effects of calcitriol in dogs.

The purposes of the study reported here were to determine whether calcitriol exposure would alter phagocytosis and TLR4 expression by neutrophils and monocytes and to assess its effect on PAMP-stimulated leukocyte production of TNF and IL10. We hypothesized that calcitriol exposure would reduce LPS-, LTA-, and MDP-stimulated leukocyte production of TNF and increase leukocyte production of IL10. Furthermore, we hypothesized that calcitriol exposure would enhance phagocytosis and reduce TLR4 expression by neutrophils and monocytes.

Materials and Methods

Animals

The study protocol was approved by the University of Missouri Animal Care and Use Committee (protocol No. 7334). Eight healthy dogs (5 neutered males and 3 spayed females) owned by faculty members, residents, and students of the University of Missouri were included in the study, with owner consent. Dogs included 4 mixed-breed dogs and 1 each of Australian Cattle Dog, Greyhound, Australian Shepherd, and Labrador Retriever. Ages ranged from 2 to 6 years. The health status of each dog was confirmed by physical examination. The dogs had received no medications or vaccinations for ≥ 1 month before enrollment, except for preventive parasite treatments.

Calcitriol

Calcitriolb was dissolved in 75% ethanolb to make a stock solution containing 24 nmol of calcitriol/mL. The stock solution was then sealed and stored light protected at 4°C.

Blood sample collection and processing

A blood sample (6 mL) was collected from each dog via jugular venipuncture (22-gauge needles) into tubes containing sodium heparin as anticoagulant. Samples were diluted 1:2 with RPMI 1640 culture mediumc containing 200 U of penicillin/mL and 200 mg of streptomycin/mL. Blood-RPMI mixture was incubated with calcitriol (final concentration, 10−7M) or ethanol (6.8 × 10−2M; negative control substance) for 24 hours at 37°C in 5% CO2 in the dark.

For creation of a concentration-response curve to determine whether LPS-stimulated leukocyte production of TNF was concentration dependent, blood samples were used from 3 of the included dogs that had been arbitrarily selected. Blood samples for this purpose were diluted 1:2 with RPMI 1640 culture medium and incubated with calcitriol at various concentrations (10−7, 10−8, 10−9, and 10−10M) or control substance for 24 hours at 37°C in 5% CO2 in the dark.

Leukocyte cytokine production

Following incubation with calcitriol or control substance, blood-RPMI mixture was transferred to 24-well plates and stimulated with LPS from Escherichia coli O127:B8a (final concentration, 100 ng/mL), LTA from Streptococcus faecalisb (final concentration, 1 μg/mL), MDPb (final concentration, 25 μg/mL), or control substance (PBS solution), as described elsewhere.31,32 Plates were incubated for 24 hours at 37°C in 5% CO2 in the dark.

After incubation, plates were centrifuged (400 × g for 7 minutes at room temperature [21°C]). The supernatant was collected and frozen at −80°C for batch analysis. For analysis, supernatant samples were first thawed, and then amounts of TNF and IL10 were measured with a canine cytokine-specific multiplex bead-based assay.d This assay has been validated for use in dogs.33 Each sample was analyzed in duplicate with appropriate control substances and associated data analysis softwaree to determine the median fluorescence intensity and cytokine concentration. The limit of detection of this assay for each cytokine was 48.8 ng/mL, the intra-assay coefficient of variation was < 5%, and the interassay coefficient of variation was < 15%.

Phagocytosis of E coli

Neutrophil and monocyte phagocytic function tests were performed with commercially available test kitsf that have been validated for dogs.34,35 For tests of phagocytic function, 100 μL of each calcitriol-and control substance-treated blood sample was incubated with FITC-labeled opsonized E coli for 10 minutes in a 37°C water bath to stimulate phagocytosis by WBCs; negative control samples were those incubated without bacteria. Samples were placed on ice to extinguish phagocytosis, and quenching solution was added to allow discrimination between attachment and internalization of bacteria by quenching FITC fluorescence of surface-bound bacteria. Samples were washed, RBCs were lysed, and leukocytes were fixed with a lysing solution that contained diethylene glycol and formaldehyde. Propidium iodide was added to stain DNA, allowing exclusion of aggregation artifacts of bacteria or dead cells during flow cytometric analysis.

TLR4 expression

Phycoerythrin-conjugated anti-human CD284g (TLR4) antibody was incubated with 100 μL of calcitriol- and control substance-treated blood samples for 30 minutes in the dark on ice, as described elsewhere.36 Matched phycoerythrin-conjugated mouse IgG1 isotype control was used as negative control samples of the antibody. Cells were then washed twice with flow cytometry buffer and centrifuged at 400 × g for 5 minutes. Red blood cells were lysed by use of ammonia chloride potassium lysis buffer solution (8.26 g of NH4Cl, 1.0 g of KHCO3, and 0.037 g of Na2EDTA in 1.0 L of deionized distilled H2O; pH, 7.2) and then washed. Finally, cells were resuspended in 400 μL of flow cytometry buffer and analyzed immediately via flow cytometry. Results were recorded as percentage of neutrophils and monocytes expressing TLR4.

Flow cytometry

Flow cytometry was performed at the University of Missouri Cell and Immunology Core Facility with a flow cytometerh and associated data analysis software.i A minimum of 15,000 events/sample was recorded. These events were then applied to a plot of forward scatter versus side scatter to identify and gate the neutrophil and monocyte populations concurrently on the basis of their size and granularity. For assessment of phagocytosis, neutrophils and monocytes were then evaluated by creation of a histogram, with unstained cells used to determine the limits of which to interpret FITC positivity as described elsewhere37 (Figure 1). Percentages of neutrophils and monocytes that had phagocytosed E coli were recorded for assessment of phagocytic function. For evaluation of TLR4 expression, neutrophils and monocytes were evaluated by creation of a histogram, with unstained cells used to determine the limits by which to interpret phycoerythrin positivity as described elsewhere36 (Figure 2).

Figure 1—
Figure 1—

Example of graphic results of flow cytometry showing the gating scheme used to detect phagocytosis. Live leukocytes were first separated from artifact via creation of a propidium iodide histogram (A), then applied to a plot of forward scatter versus side scatter (B). Phagocytic cells (granulocytes and monocytes combined) were identified and applied to an FITC histogram, resulting in identification of phagocytosis-negative (C) and phagocytosis-positive (D) cells.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1064

Figure 2—
Figure 2—

Example of graphic results of flow cytometry showing the gating scheme used to detect TLR4 expression. First, cells (granulocytes and monocytes combined) were identified on a plot of forward scatter versus side scatter (A) and applied to phycoerythrin scatterplots, resulting in identification of TLR4-negative (B) and TLR4-positive (C) cells.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1064

Statistical analysis

Statistical analysis was performed with the aid of commercially available software.j The Shapiro-Wilk test was used to assess the data for normality. Data were compared between cell treatment groups by means of the paired t test or 1-way repeated-measures ANOVA, and the post hoc Fisher least significant difference multiple comparisons procedure was performed as indicated. A P value < 0.05 was considered significant.

Results

Cytokine responses

Incubation of blood samples from 8 dogs with calcitriol significantly reduced TNF production by leukocytes following stimulation with LPS, LTA, and MDP (Figure 3). Conversely, incubation of blood samples yielded no increase in leukocyte IL10 production following stimulation with LPS (mean ± SD concentrations: calcitriol, 791 ± 386 pg/mL; control substance, 551 ± 323 pg/mL; P = 0.11), LTA (calcitriol, 595.7 ± 308.8 pg/mL; control substance, 407.9 ± 169.9 pg/mL; P = 0.08), or MDP (calcitriol, 499.3 ± 270.4 pg/mL; control substance, 383.0 ± 183.8 pg/mL; P = 0.12).

Figure 3—
Figure 3—

Mean supernatant TNF concentrations following incubation of blood samples from 8 dogs with calcitriol (10−7M) or control substance (ethanol) and subsequent exposure to LPS, LTA, or MDP. Two replicates were performed per dog. Vertical bars represent SD, and the ends of each horizontal bar indicate the treatments to which provided P value pertains.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1064

Compared with control values, a significant, concentration-dependent decrease in LPS-stimulated leukocyte TNF production was identified when blood samples from 3 dogs were incubated with calcitriol at concentrations ranging from 10−7 to 10−9M (Figure 4). Additionally, a significant decrease from values for 10−10M calcitriol was identified for leukocyte TNF production at calcitriol concentrations from 10−9 to 10−7M.

Figure 4—
Figure 4—

Lipopolysaccharide-stimulated TNF concentrations following incubation of blood samples from 3 dogs (represented by different symbols) with calcitriol at various concentrations or control substance (ethanol). Provided P values represent comparisons of mean values for all 3 dogs. See Figure 3 for remainder of key.

Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1064

Phagocytosis

No significant (P = 0.25) difference was identified in the percentage of neutrophils and monocytes with phagocytosed E coli between blood samples (n = 8) incubated with calcitriol (mean ± SD, 72.8 ± 7.9%) or control substance (74.1 ± 7.6%).

TLR4 expression

Data from 2 dogs were excluded from the analysis of TLR4 expression because of technical difficulties. No significant (P = 0.31) difference in the percentage of neutrophils and monocytes with TLR4 expression was identified between blood samples (n = 6) incubated with calcitriol (mean ± SD, 21.6 ± 7.8%) and control substance (20.9 ± 7.7%).

Discussion

In the study reported here, we evaluated the effects of calcitriol exposure on PAMP-stimulated leukocyte cytokine production, neutrophil and monocyte phagocytosis, and TLR4 expression in blood samples from dogs. Leukocyte production of TNF following stimulation with LPS, LTA, and MDP was significantly reduced when blood samples had been incubated with calcitriol rather than control substance. However, stimulated leukocyte production of IL10 was not significantly increased by calcitriol exposure. Furthermore, incubation of blood samples with calcitriol resulted in no significant increase in neutrophil and monocyte phagocytosis or decrease in TLR4 expression.

These findings suggested that calcitriol attenuated PAMP-stimulated leukocyte production of TNF in healthy dogs in vitro in a concentration-dependent manner. Similarly, in humans, calcitriol impairs leukocyte secretion of TNF following stimulation with LPS, LTA, phorbol myristate acetate, and ionomycin in a concentration-dependent fashion in vitro.38–40 Likewise, vitamin D supplementation significantly reduces circulating concentrations of proinflammatory cytokines in critically ill humans.41

Incubation of canine blood samples with calcitriol in the present study had no effect on neutrophil and monocyte TLR4 expression, suggesting that alterations in TNF production may be attributable to postreceptor signaling or protein production, as has been postulated for humans. Calcitriol and its analogs directly inhibit NFκB activation through an increase in the expression of IκBa, the inhibitory protein in human peripheral blood mononuclear cells and macrophages, and may reduce nuclear translocation of NFκB via subunit p65.42–44 Furthermore, calcitriol indirectly inhibits NFκB signaling by upregulating the expression of insulin-like growth factor-binding protein 3 and clusterin that interfere with NFκB activation.45,46 Research is needed regarding the effects of calcitriol on the NFκB inflammatory pathway to establish this relationship in dogs. In addition, the small number of dogs in the present study may have resulted in insufficient statistical power to identify a difference in granulocyte and monocyte TLR4 expression if such a difference truly existed. Therefore, we could not rule out the possibility that the observed decrease in leukocyte TNF production in dogs was not associated with a decrease in TLR4 expression.

Calcitriol exposure had no effect on PAMP-stimulated leukocyte IL10 production in the present study. The immunomodulatory effect that calcitriol has on PAMP-stimulated leukocyte IL10 production differs greatly within and across numerous species.47–52 Incubation of blood samples and peripheral blood mononuclear cells with PAMP reportedly results in a significant increase in peripheral blood mononuclear cell IL10 production in healthy humans in vitro.47 Interestingly, other research in humans has shown that incubation with calcitriol results in a significant reduction in PAMP-stimulated peripheral blood mononuclear IL10 production.12,50 This dichotomy may be influenced by differences in the cell type investigated, calcitriol concentrations, cell culture incubation times, pattern-recognition receptor ligands, and population heterogeneity. The lack of a significant increase or decrease in PAMP-stimulated IL10 production by canine leukocytes in the present study may therefore have been attributable to the impact of similar variables. Alternatively, our study may have lacked the power to detect a difference had one existed. Larger studies involving different concentrations of calcitriol, immune cell types, and cell culture incubation times are needed to better characterize the immunomodulatory effect that calcitriol has in healthy dogs in vitro.

In contrast to human monocytes and macrophages, for which incubation with calcitriol results in enhanced phagocytosis,7–9,53 phagocytic behavior of canine neutrophils and monocytes was not altered by calcitriol exposure in the present study. We had chosen a 10−7M concentration of calcitriol because it consistently results in a significant increase in human monocyte and macrophage phagocytosis (and decreases human leukocyte TLR4 expression) in vitro.7–10,40,53 It is possible that differences exist in species sensitivity to calcitriol and that the concentration of calcitriol needed to enhance phagocytosis in dogs is different from that needed in humans. This is the situation in postpartum dairy cattle, in which a concentration of approximately 10−4M was needed to increase neutrophil phagocytosis.54 Additional research is warranted into the effect of higher concentrations of calcitriol on leukocyte phagocytosis in dogs. Alternatively, evaluation of phagocytic behavior by neutrophils and monocytes concurrently in the present study could have masked an effect of calcitriol had one existed for either cell type individually.

The present study had several limitations that could be addressed in future studies. One limitation was the evaluation of phagocytosis and TLR4 expression in neutrophils and monocytes concurrently. This was done to assess the effect that calcitriol had on integral constituents of the innate immune response (neutrophils and monocytes), more closely mimicking the response in vivo. Additional research involving dual surface markers is needed to evaluate the effect that calcitriol has in vitro on canine neutrophils and monocytes independently. A second limitation was that phagocytosis and TLR4 expression in neutrophils and monocytes were evaluated at a single calcitriol concentration (10−7M); however, this concentration is attainable in dogs in vivo following oral and IV administration,55 and so we believed it would be clinically relevant. A third limitation was that TNF and IL10 were used as surrogate markers of proinflammatory and anti-inflammatory immune responses, respectively. This approach yielded a general understanding of the immunomodulatory effect calcitriol had on a PAMP-stimulated inflammatory microenvironment. However, additional research involving a broad panel of both proinflammatory and anti-inflammatory cytokines is needed to garner a thorough understanding of the immunologic role of calcitriol in dogs. Lastly, the small number of included dogs may have resulted in limited statistical power to detect differences in granulocyte and monocyte TLR4 expression if such differences truly existed. Therefore, we could not rule out the possibility that the decrease in leukocyte TNF production was not associated with a decrease in TLR4 expression.

The study reported here showed that the downregulation of PAMP-stimulated leukocyte production of TNF was concentration dependent and may not interfere with neutrophil or monocyte phagocytosis or TLR4 expression in dogs in vitro. We believe these findings provide novel information about the immunomodulatory role of calcitriol in dogs and, in doing so, a pathophysiologic rationale for future studies.

Acknowledgments

Supported by a University of Missouri College of Veterinary Medicine Clinician Scientist Grant and a Phi Zeta grant.

Presented in abstract form at the American College of Veterinary Internal Medicine Forum, National Harbor, Md, June 2017.

The authors thank Matt Haight and Savannah Smith for technical support and Kate Anderson for literature review assistance.

ABBREVIATIONS

FITC

Fluorescein isothiocyanate

IL10

Interleukin-10

LPS

Lipopolysaccharide

LTA

Lipoteichoic acid

MDP

N-acetylmuramyl-l-alanyl-d-isoglutamine hydrate

NFκB

Nuclear factor κB

PAMP

Pathogen-associated molecular pattern motif

TLR

Toll-like receptor

TNF

Tumor necrosis factor

Footnotes

a.

Carver A, Koenigshof A. Evaluation of vitamin D, calcitriol, and ionized calcium levels in dogs with sepsis (abstr). J Vet Emerg Crit Care 2016;26(suppl 1):S4.

b.

Sigma-Aldrich, St Louis, Mo.

c.

Thermo Fisher Scientific, Carlsbad, Calif.

d.

Milliplex MAP canine cytokine/chemokine magnetic bead panel, EMD Millipore Corp, Billerica, Mass.

e.

Milliplex Analyst, version 5.1, EMD Millipore Corp, Billerica, Mass.

f.

Phagotest, Orpegen Pharma, Heidelberg, Germany.

g.

HTA125 clone, eBioscience Inc, San Diego, Calif.

h.

CyAn ADP analyzer, Beckman Coulter Inc, Brea, Calif.

i.

Summit, version 5.2.0.7477, Dako, Carpinteria, Calif.

j.

SigmaPlot, version 13, Systat Software Inc, San Jose, Calif.

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Contributor Notes

Address correspondence to Dr. DeClue (decluea@missouri.edu).