The main physiologic role of vitamin D is regulation of calcium and phosphorous homeostasis and bone metabolism.1–3 However, vitamin D also has a physiologically important role in both the innate and adaptive immune systems.1–3 Vitamin D receptor expression has been detected in most immune cells, and many immune cells produce 1-α hydroxylase, enabling them to synthesize calcitriol.1,2
In humans, calcitriol attenuates inflammation while also enhancing protective antimicrobial functions of leukocytes.2,4,5 More specifically, calcitriol augments phagocytosis,6–8 downregulates TLR4 expression,9–11 and reduces proinflammatory cytokine production by human leukocytes in vitro.12–15 Similarly, calcitriol blunts proinflammatory cytokine production in canine neutrophils and monocytes in vitroa; however, in contrast to its effects on human leukocytes,16 calcitriol reportedly has no significant effect on phagocytosis or TLR4 expression of canine neutrophils and monocytes in vitro.a
Hypovitaminosis D in critically ill humans with sepsis has been associated with an increased risk of death, compared with the risk for those without this deficiency.16–22 Similarly, hypovitaminosis D has been associated with death in critically ill foals with sepsis.23 In dogs, hypovitaminosis D has been identified with experimentally induced24 and naturally occurring25,b sepsis. Although the role of hypovitaminosis D in sepsis and the association between hypovitaminosis D and outcome are poorly understood, the immunomodulatory effects of calcitriol are believed to be a contributing factor.
Sepsis is a complex paradigm defined by its dynamic nature, which is characterized initially by overexuberant inflammation, followed by an anti-inflammatory immunosuppressive state.26 This shift is believed to be the result of genetic reprogramming that involves downregulation of proinflammatory pathways and enhanced expression of anti-inflammatory genes.27 Endotoxin tolerance is a term used to describe the immunocompromised phase of sepsis and results in hyporesponsiveness of polymorphonuclear cells to pathogen-associated molecular pattern motifs.26 This phenomenon has been demonstrated in dogs in vivo, in which endotoxin priming induces a reduction in the capacity of leukocytes to produce TNF following restimulation with LPS.28 Furthermore, several murine and human in vitro studies29–31 have involved endotoxin priming of leukocytes followed by LPS stimulation as models of sepsis and endotoxin tolerance.29–31
The purpose of the study reported here was to evaluate the effects of calcitriol on proinflammatory and anti-inflammatory cytokine production, neutrophil and lymphocyte apoptosis, and neutrophil and monocyte TLR4 expression in naive (unprimed) and endotoxin-primed canine leukocytes to provide a better understanding of the immunomodulatory role of vitamin D in dogs during sepsis. We believed use of both naive and endotoxin-primed leukocytes would allow determination of whether calcitriol exerted different effects on the basis of leukocyte priming status. We hypothesized that calcitriol exposure would reduce leukocyte production of TNF, increase leukocyte production of IL10, increase neutrophil apoptosis while decreasing lymphocyte apoptosis, and decrease neutrophil and monocyte TLR4 expression by naive and endotoxin-primed cells.
Materials and Methods
Animals
The study protocol was approved by the University of Missouri Animal Care and Use Committee (protocol No. 7334). Six healthy neutered adult dogs of various breeds and an age range of 2 to 6 years were used. Dogs were owned by students of the University of Missouri, who provided their consent for inclusion. 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
Calcitriolc was dissolved in 75% ethanolc 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 1 tube containing sodium heparin as anticoagulant. Blood samples were diluted 1:2 with RPMI 1640 culture mediumd containing 200 U of penicillin/mL and 200 mg of streptomycin/mL and then divided into 2 tubes such that each dog served as its own control. Leukocytes in blood-RPMI mixture from 1 tube were primed with LPS from Escherichia coli O127:B8c (endotoxin; final concentration, 100 ng/mL), and PBS solution was added to the unprimed control tube. After 1 hour of leukocyte incubation with LPS or PBS solution, blood-RPMI mixture from each of the endotoxin-primed and unprimed tubes was transferred into each of 2 tubes, and contents of these second sets of tubes were then incubated with calcitriol (final concentration, 2 × 10−7M) or ethanol (13.5 × 10−2M; negative control substance) for 24 hours at 37°C in 5% CO2 in the dark.
Leukocyte cytokine production
After incubation with calcitriol or control, blood-RPMI mixture was transferred to 24-well plates and stimulated with LPS from E coli O127:B8 (final concentration, 100 ng/mL) or control substance (PBS solution), as described elsewhere.32–35 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 TNF and IL10 concentrations were measured in duplicate with a canine cytokine-specific multiplex bead-based assaye as described elsewhere.35 Results were recorded as median fluorescence intensity and cytokine concentration.
TLR4 expression
Leukocyte TLR4 expression was evaluated as described elsewhere.34 Results of flow cytometric analysis were recorded as percentage of neutrophils and monocytes expressing TLR4.
Neutrophil and lymphocyte apoptosis
Neutrophil and lymphocyte apoptosis was assessed with an annexin V–FITC and PI kit.f Red blood cells in 100 μL of calcitriol- and control substance–treated blood samples were lysed with cold distilled water and 10× PBS solution, then incubated with annexin V–FITC and PI for 15 minutes. Binding buffers were added, and then flow cytometric analysis was performed. Samples blocked with recombinant annexin V were used as negative control samples and analyzed immediately.
Flow cytometry
Flow cytometry was performed at the University of Missouri Cell and Immunology Core Facility by use of a flow cytometerg and associated data analysis software.h A minimum of 15,000 events/sample was recorded. For evaluation of TLR4, these events were 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.34 Gated cells were evaluated via creation of a histogram, with unstained cells used to determine the limits by which to interpret phycoerythrin positivity as described elsewhere.36 For assessment of apoptosis, cells were applied to a plot of forward scatter versus side scatter to identify and gate the neutrophils and lymphocyte population on the basis of their size and granularity.34,37 Each cell type was then analyzed for fluorescence, with cells that were positive for FITC identified as apoptotic cells as described elsewhere37 (Figure 1).
Example of graphic results of flow cytometry showing the gating scheme used to detect apoptotic cells. First granulocytes and then lymphocytes (black circles) were identified on a plot of forward scatter versus side scatter (A) and applied to (arrow) FITC versus PI scatterplots, resulting in identification of apoptosis-negative (ie, negative for FITC; B) and apoptotic cells (ie, positive for FITC; C).
Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1071
Statistical analysis
Statistical analysis was performed with commercially available software.i The Shapiro-Wilk test was used to assess the data for normality. Leukocyte cytokine production capacity was compared between cell treatment groups by means of the paired t test. A Kruskal-Wallis 1-way ANOVA on ranks was performed for between-group comparisons of lymphocyte and neutrophil apoptosis as well as neutrophil and monocyte TLR4 expression. One-way ANOVA was performed to detect differences in leukocyte cytokine production between groups, followed by a Tukey post hoc test for between-group comparisons. For all testing, values of P > 0.05 were considered significant.
Results
Confirmation of expected in vitro model effects
Priming of leukocytes with endotoxin (LPS) resulted in significantly greater production of TNF, compared with production by unprimed leukocytes (Figure 2). However, when the effects of subsequent LPS stimulation on endotoxin-primed and unprimed leukocytes were compared, a notable difference was identified. Stimulation of unprimed leukocytes with LPS resulted in significantly more TNF production, compared with production by unstimulated (PBS solution–exposed) cells. Conversely, for primed leukocytes, additional stimulation with LPS did not result in significantly greater TNF production, compared with production by unstimulated endotoxin-primed leukocytes. This suggested that endotoxin tolerance was successfully induced. Similarly, stimulation of unprimed leukocytes with LPS resulted in a significant increase in IL10 production, compared with production by unstimulated unprimed leukocytes (Figure 3), whereas the endotoxin-primed leukocytes stimulated with LPS had no such increase in IL10 production.
Mean supernatant TNF concentrations following exposure of leukocytes in blood samples from 6 dogs to Escherichia coli LPS (primed cells) or PBS solution (unprimed cells), incubation with calcitriol (2 × 10−7M) or control substance (ethanol), and then exposure again to LPS or PBS solution. 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.79.10.1071
Mean supernatant IL10 concentrations following exposure of leukocytes to the conditions described in Figure 2. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1071
Effects of calcitriol on cytokine production by endotoxin-primed and unprimed canine leukocytes
For unprimed leukocytes, calcitriol exposure significantly reduced LPS-stimulated TNF production (Figure 2). A similar effect was detected for endotoxin primed leukocytes. Calcitriol exposure had no significant effect on LPS-stimulated IL10 production by unprimed leukocytes (Figure 3). However, calcitriol exposure significantly increased LPS-induced IL10 production by endotoxin-primed leukocytes.
Neutrophil and lymphocyte apoptosis
As expected, endotoxin priming increased neutrophil survival rates (Figure 4) with no effect on lymphocyte survival rates (Figure 5). Calcitriol exposure had no effect on proportions of neutrophils or lymphocytes with apoptosis regardless of whether leukocytes were primed or unprimed with endotoxin.
Mean percentage of neutrophils with apoptosis following exposure of leukocytes in blood samples from 6 dogs to LPS or PBS solution and incubation with calcitriol (2 × 10−7M) or control substance (ethanol). Notice that the y-axis is discontinuous. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1071
Box-and-whisker plots showing percentages of lymphocytes with apoptosis following exposure to the conditions described in Figure 4. The horizontal line within each box represents the median; the bottom and top boundaries of each box represent the 25th and 75th percentiles, respectively; and the whiskers represent the range of the data, excluding outliers (asterisks). No significant (P = 0.50) difference was identified among the 4 groups. See Figures 2 and 4 for remainder of key.
Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1071
TLR4 expression
Neutrophil and monocyte TLR4 expression was not significantly altered by endotoxin priming. Additionally, calcitriol exposure had no significant effect on neutrophil and monocyte TLR4 expression by endotoxin-primed or unprimed cells (Figure 6).
Box-and-whisker plots showing percentages of neutrophils and monocytes with TLR4 expression following exposure to the conditions described in Figure 4. No significant (P = 0.76) difference was identified among the 4 groups. See Figures 2, 4, and 5 for remainder of key.
Citation: American Journal of Veterinary Research 79, 10; 10.2460/ajvr.79.10.1071
Discussion
In the present study, we evaluated the effects of calcitriol exposure on leukocyte cytokine production, neutrophil and monocyte TLR4 expression, and neutrophil and lymphocyte apoptosis of unprimed and endotoxin-primed leukocytes in canine blood samples. Calcitriol exposure significantly decreased endotoxin-induced leukocyte production of TNF by unprimed and primed cells. Conversely, calcitriol did not significantly affect production of IL10 by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Additionally, calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor did it affect the magnitude of neutrophil or monocyte TLR4 expression by unprimed or endotoxin-primed cells.
The results supported our hypothesis that calcitriol exposure would significantly decrease TNF production by unprimed and endotoxin-primed leukocytes, suggesting that calcitriol was able to down-regulate the proinflammatory behavior of canine leukocytes. These results were in agreement with reported in vitro findings for humans and mice, in which calcitriol blunts TNF production by endotoxin-primed leukocytes.38–40 We believe these findings indicate that calcitriol could represent a novel immunomodulatory treatment for critically ill dogs with sepsis. However, immunologic data from our study should not be directly applied to clinical settings without additional ex vivo evidence of the effects that calcitriol has on leukocyte TNF production capacity in dogs with sepsis.
A paucity of information is available for any species regarding the in vitro effects of calcitriol on IL10 production by unprimed or endotoxin-primed leukocytes. This information would be important for assessing the potential value of calcitriol as a novel immunomodulatory treatment for critically ill individuals with sepsis, independent of species. In humans with sepsis, nonsurvivors have a significantly greater plasma IL10-to-TNF concentration ratio than survivors at hospital admission or after 48 hours of hospitalization.41,42 This anti-inflammatory response not only correlates with severity of disease but also represents a pragmatic marker of sepsis-induced immunosuppression.42
We found that calcitriol had no effect on LPS-stimulated production of IL10 by unprimed canine leukocytes. However, calcitriol exposure significantly increased the IL10 production capacity of endotoxin-primed leukocytes. These results differ from reported findings for humans, for whom calcitriol has no effect on IL10 production by endotoxin-primed peripheral blood mononuclear cells in vitro.43 One possible explanation for the conflicting results is that the present study focused on the IL10 production of all leukocytes in blood samples rather than on specific leukocyte cell lines. This global approach was pursued to more closely mimic the effect calcitriol has following endotoxin exposure in vivo. Additional research investigating the effects that calcitriol has on IL10 production in specific cell lines in dogs is needed to better define the therapeutic potential of calcitriol for dogs with sepsis.
Regulation of the innate immune response is a delicate balance that, when disrupted, can result in deleterious inflammation and immunotolerance. The imbalance of the innate immune response in humans with sepsis is multifactorial; however, endotoxin-induced upregulation of leukocyte TLR4 expression is believed to contribute to this phenomenon.44–46 In the study reported here, priming of leukocytes in canine blood samples with endotoxin had no significant effect on neutrophil and monocyte TLR4 expression. These results corresponded with previously reported findings for dogs,36 but were different from the effect endotoxin has on human leukocyte TLR4 expression both in vivo and in vitro (ie, an increase in TLR4 expression).44–46 Furthermore, incubation of unprimed or endotoxin-primed leukocytes in canine blood samples with calcitriol had no significant effect on neutrophil and monocyte TLR4 expression. In humans, incubation of endotoxin-primed blood samples with calcitriol has no effect on peripheral blood monocyte TLR4 expression.47
The shift from a proinflammatory leukocyte state to an anti-inflammatory state in the absence of TLR4 downregulation suggested involvement of a postreceptor mechanism. Calcitriol and its analogs directly block basal and cytokine-stimulated NFκB activity by increasing expression of IκBα, the NFκB inhibitory protein, by macrophages and peripheral blood mononuclear cells.48–51 Moreover, calcitriol indirectly inhibits NFκB signaling by upregulating the expression of insulin-like growth factor-binding protein 3 and clusterin that interfere with NFκB activation.51,52 Additional research into the effects of calcitriol on the NFκB inflammatory pathway is needed to establish this relationship in dogs. However, the results reported here provided mechanistic rationale that calcitriol might represent a novel treatment for dogs with sepsis by dampening inflammation without compromising leukocyte TLR4 expression.
As expected, a significant decrease in the degree of neutrophil apoptosis was identified in the present study following leukocyte priming with endotoxin. Delayed apoptosis of canine neutrophils induced by endotoxin in vitro is believed to be associated with an inducible increase in B-cell lymphoma extralarge protein expression.53 An increase in neutrophil viability during sepsis can fortify microbial killing but can also potentiate severe tissue or organ damage. In contrast with our hypothesis, calcitriol had no effect on the degree of neutrophil apoptosis in blood samples containing endotoxin-primed leukocytes. This indicated that calcitriol was able to modulate inflammatory cytokine production without affecting neutrophil viability, regardless of whether cells were unprimed or endotoxin primed. It follows that calcitriol may be able to reduce the harmful effects associated with proinflammatory cytokine production without compromising the protective potential of viable neutrophils in dogs with sepsis.
Incubation of unprimed or endotoxin-primed leukocytes in canine blood samples with calcitriol had no significant effect on lymphocyte apoptosis. In humans, calcitriol causes a concentration-dependent decrease in viability of phytohemagglutinin-stimulated T lymphocytes in vitro.54 The proposed mechanism by which calcitriol reduces T lymphocyte viability includes blockage in the G1 phase of T cell cycle and reduction in IL2 secretion.54 Our findings might have indicated that calcitriol has minimal effect on the apoptotic pathway of basal and activated lymphocytes in dogs in vitro. Alternatively, this difference in findings between species may have resulted from the mitogen used for stimulation as well as the incubation time. Additional research involving various lymphocyte activators and incubation times would be needed to explore this possibility.
One limitation of the present study was that evaluation of TLR4 expression in neutrophils and monocytes was performed concurrently. This was done to investigate the effect that calcitriol had on integral components of the innate immune response, providing a more accurate replication of endotoxin exposure in vivo. Additional studies involving neutrophil- and monocyte-specific monoclonal antibodies are needed to evaluate the effect that calcitriol has on canine endotoxin-primed neutrophils and monocytes independently, in vitro. Another limitation was that TNF and IL10 were used as sole markers of pro- and anti-inflammatory immune responses, respectively. This focused approach allowed a general interpretation of the immunomodulatory impact that calcitriol had on the cytokine production capacity of LPS-stimulated, endotoxin-primed leukocytes. However, additional studies involving a broad panel of both proinflammatory and anti-inflammatory cytokines in blood samples containing pathogen-associated molecular pattern motif–primed and unprimed leukocytes are needed to garner a thorough understanding of the immunologic role of calcitriol in dogs.
Another limitation was that we used only 1 calcitriol concentration (2 × 10−7M) and incubation time (24 hours) to evaluate neutrophil and lymphocyte apoptosis. Little information is available regarding the effect that calcitriol has on the viability of neutrophils and lymphocytes in any species. Perhaps calcitriol has a concentration- and time-dependent effect on viability of endotoxin-primed neutrophils and lymphocytes. Additional research involving several calcitriol concentrations and incubation times may provide information integral to understanding whether the potential therapeutic benefits of calcitriol in dogs with sepsis may be time dependent. Similarly, neutrophil and monocyte TLR expression was evaluated with the use of only 1 calcitriol concentration and incubation time. The rationale for this approach was based on our previous worka as well as extrapolation from in vitro studies in humans.9,55 Moreover, the calcitriol concentration that we used is attainable in dogs following oral and IV administration56 and is therefore clinically relevant.
We believe that the results reported here high-light several important aspects of the canine innate immune response to endotoxin, endotoxin tolerance, and the role of calcitriol in modulating the aforementioned responses in vitro. The stimulation of unprimed leukocytes with LPS resulted in a significant increase in leukocyte TNF and IL10 production capacity, whereas this response was not observed for endotoxin-primed leukocytes when stimulated with LPS, confirming the presence of endotoxin tolerance in dogs in vitro. Furthermore, endotoxin priming had no significant effect on neutrophil and monocyte TLR4 expression or lymphocyte viability, but did reduce the amount of neutrophil apoptosis. Additionally, calcitriol induced an anti-inflammatory status in unprimed and endotoxin-primed leukocytes without compromising neutrophil and monocyte TLR4 expression or altering neutrophil and lymphocyte viability in vitro. However, the effects of calcitriol appeared to vary on the basis of leukocyte priming status. This variation may have represented a specific inflammatory pathway augmented by calcitriol in vitro. Overall, we believe our findings provide a pathomechanistic rationale for future research into the effects of calcitriol in critically ill dogs with sepsis. However, additional research is needed before immunologic data can be applied from healthy dogs to dogs with endotoxin-primed leukocytes (ie, sepsis) in clinical settings.
Acknowledgments
Supported by a University of Missouri College of Veterinary Medicine Clinician Scientist Grant.
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 |
| NFκB | Nuclear factor κB |
| PI | Propidium iodide |
| TLR | Toll-like receptor |
| TNF | Tumor necrosis factor |
Footnotes
Jaffey J, Amorim J, DeClue AE. In vitro effects of vitamin D on phagocytosis, TLR4, and cytokine production (abstr), in Proceedings. Am Coll Vet Intern Med Forum 2017;775.
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.
Sigma-Aldrich, St Louis, Mo.
Thermo Fisher Scientific, Carlsbad, Calif.
Milliplex MAP canine cytokine-chemokine panel, EMD Millipore Corp, Billerica, Mass.
BD Pharmingen, BD Biosciences, San Jose, Calif.
Beckman Coulter Inc, Brea, Calif.
Summit software, version 5.2.0.7477, Dako, Carpinteria, Calif.
SigmaPlot, version 13, Systat Software Inc, San Jose, Calif.
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