• View in gallery
    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).

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    Figure 2—

    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.

  • View in gallery
    Figure 3—

    Mean supernatant IL10 concentrations following exposure of leukocytes to the conditions described in Figure 2. See Figure 2 for remainder of key.

  • View in gallery
    Figure 4—

    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.

  • View in gallery
    Figure 5—

    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.

  • View in gallery
    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.

  • 1. Penna G, Adorini L. 1α,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 2000;164:24052411.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Szymczak I, Pawliczak R. The active metabolite of vitamin D3 as a potential immunomodulator. Scand J Immunol 2016; 83:8391.

  • 3. Altieri B, Muscoqiuri G, Barrea L, et al. Does vitamin D play a role in autoimmune endocrine disorders? A proof of concept. Rev Endocr Metab Disord 2017;18:335346.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Tiosano D, Wildbaum G, Gepstein V, et al. The role of vitamin D receptor in innate and adaptive immunity: a study in hereditary vitamin D-resistant rickets patients. J Clin Endocrinol Metab 2013;98:16851693.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Lin Z, Li W. The roles of vitamin D and its analogs in inflammatory diseases. Curr Top Med Chem 2016;16:12421261.

  • 6. Tokuda N, Levy RB. 1,25-dihydroxyvitamin D3 stimulates phagocytosis but suppresses HLA-DR and CD13 antigen expression in human mononuclear phagocytes. Proc Soc Exp Biol Med 1996;211:244250.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Chandra G, Selvaraj P, Jawahar MS, et al. Effect of vitamin D3 on phagocytic potential of macrophages with live mycobacterium tuberculosis and lymphoproliferative response in pulmonary tuberculosis. J Clin Immunol 2004;24:249257.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Motlagh BM, Ahangaran NA, Froushani SMA. Calcitriol modulates the effects of bone marrow-derived mesenchymal stem cells on macrophage functions. Iran J Basic Med Sci 2015;18:672676.

    • Search Google Scholar
    • Export Citation
  • 9. Verma R, Jung JH, Kim JY. 1,25-dihydroxyvitamin D3 upregulates TLR10 while down-regulating TLR2, 4, and 5 in human monocyte THP-1. J Steroid Biochem Mol Biol 2014;141:16.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Wang H, Zhang Q, Chai Y, et al. 1,25(OH)2D3 downregulates the toll-like receptor 4-mediated inflammatory pathway and ameliorates liver injury in diabetic rats. J Endocrinol Invest 2015;38:10831091.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Fitch N, Becker AB. HayGlass KT. Vitamin D [1,25(OH)2D3] differentially regulates human innate cytokine responses to bacterial versus vital pattern recognition receptor stimuli. J Immunol 2016;196:29652972.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Villaggio B, Soldano S, Cutolo M. 1,25-dihydroxyvitamin D3 downregulates aromatase expression and inflammatory cytokines in human macrophages. Clin Exp Rheumatol 2012;30:934938.

    • Search Google Scholar
    • Export Citation
  • 13. Harishankar M, Afsal K, Banurekha VV, et al. 1,25-dihydroxy vitamin D3 downregulates pro-inflammatory cytokine response in pulmonary tuberculosis. Int Immunopharmacol 2014;23:148152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Neve A, Corrado A, Cantatore FP. Immunomodulatory effects of vitamin D in peripheral blood monocyte-derived macrophages from patients with rheumatoid arthritis. Clin Exp Med 2014;14:275283.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Grubczak K, Lipinska D, Eljaszewicz A, et al. Vitamin D3 treatment decreases frequencies of CD16-positive and TNF-α-secreting monocytes in asthmatic patients. Int Arch Allergy Immunol 2015;166:170176.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Venkatram S, Chilimuri S, Adrish M, et al. Vitamin D deficiency is associated with mortality in the medical intensive care unit. Crit Care Res Pract 2011;15:R292.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Higgins DM, Wischmeyer PE, Queensland KM, et al. Relationship of vitamin D deficiency to clinical outcomes in critically ill patients. JPEN J Parenter Enteral Nutr 2012;36:713720.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Flynn L, Zimmerman LH, McNorton K, et al. Effects of vitamin D deficiency in critically ill surgical patients. Am J Surg 2012;203:379382.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Braun AB, Gibbons FK, Litonjua AA, et al. Low serum 25-hydroxyvitamin D at critical care initiation is associated with increased mortality. Crit Care Med 2012;40:6372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Aygencel G, Turkoglu M, Tuncel AF, et al. Is vitamin D insufficiency associated with mortality of critically ill patients? Crit Care Res Pract 2013;2013:856747

    • Search Google Scholar
    • Export Citation
  • 21. Amrein K, Zajic P, Schnedl C, et al. Vitamin D status and its association with season, hospital and sepsis mortality in critical illness. Crit Care 2014;18:R47.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Moromizato T, Litonjua AA, Braun AB, et al. Association of low serum 25-hydroxyvitamin D levels and sepsis in the critically ill. Crit Care Med 2014;42:97107.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Kamr AM, Dembek KA, Reed SM, et al. Vitamin D metabolites and their association with calcium, phosphorous, and PTH concentrations, severity of illness, and mortality in hospitalized equine neonates. PLoS One 2015;2010:E0127684.

    • Search Google Scholar
    • Export Citation
  • 24. Holowaychuk MK, Birkenheuer AJ, Li J, et al. Hypocalcemia and hypovitaminosis D in dogs with induced endotoxemia. J Vet Intern Med 2012;26:244251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Jaffey JA, Backus RC, McDaniel KM, et al. Serum vitamin D concentrations in hospitalized critically ill dogs. PLoS One 2018;13:e0194062.

  • 26. Biswas SK, Lopez-Collazo E. Endotoxin tolerance: new mechanisms, molecules and clinical significance. Trends Immunol 2009;30:475487.

  • 27. Cavaillon JM, Adib-Conquy M. Bench-to-bedside review: endotoxin tolerance as a model of leukocyte reprogramming in sepsis. Crit Care 2006;10:233.

    • Search Google Scholar
    • Export Citation
  • 28. Friedrich I, Spillner J, Lu E-X, et al. Induction of endotoxin tolerance improves lung function after warm ischemia in dogs. Am J Physiol Lung Cell Mol Physiol 2003;284:L224L231.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Medvedev AE, Kopydlowski KM, Vogel SN. Inhibition of lipopolysaccharide-induced signal transduction in endotoxintolerized mouse macrophages: dysregulation of cytokine, chemokine, and Toll-like receptor 2 and 4 gene expression. J Immunol 2000;164:55645574.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Sato S, Takeuchi O, Fujita T, et al. A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways. Int Immunol 2002;14:783791.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. del Fresno C, García-Rio F, Gómez-Piña V, et al. Potent phagocytic activity with impaired antigen presentation identifying lipopolysaccharide-tolerant human monocytes: demonstration in isolated monocytes from cystic fibrosis patients. J Immunol 2009;182:64946507.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Deitschel SJ, Kerl ME, Chang CH, et al. Age-associated changes to pathogen-associated molecular pattern-induced inflammatory mediator production in dogs. J Vet Emerg Crit Care (San Antonio) 2010;20:494502.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Fowler BL, Axiak SM, DeClue AE. Blunted pathogen-associated molecular pattern motif induced TNF, IL-6 and IL-10 production from whole blood in dogs with lymphoma. Vet Immunol Immunopathol 2011;144:167171.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Jaffey JA, Amorim J, DeClue AE. Effects of calcitriol on phagocytic function, toll-like receptor 4 expression, and cytokine production of canine leukocytes. Am J Vet Res 2018;79:10641070.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Axiak-Bechtel SM, Tsuruta K, Amorim J, et al. Effects of tramadol and o-desmethyltramadol on canine innate immune system function. Vet Anaesth Analg 2015;42:260268.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Zhang Y, Axiak-Bechtel S, Friedman Cowan C, et al. Evaluation of immunomodulatory effect of recombinant human granulocyte-macrophage colony-stimulating factor on polymorphonuclear cell from dogs with cancer in vitro. Vet Comp Oncol 2017;15:968979.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. DeClue AE, Yu DH, Prochnow S, et al. Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes. Vet J 2014;200:270275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Müller K, Haahr PM, Diamant M, et al. 1,25-dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level. Cytokine 1992;4:506512.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39. Panichi V, De Pietro S, Andreini B, et al. Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium. Kidney Int 1998;54:14631469.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Tan ZX, Chen YH, Xu S, et al. Calcitriol inhibits tumor necrosis factor α and macrophage inflammatory protein-2 during lipopolysaccharide-induced acute lung injury in mice. Steroids 2016;112:8187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. van Dissel JT, van Langevelde P, Westendorp RGL, et al. Anti-inflammatory cytokine profile and mortality in febrile patients. Lancet 1998;351:950953.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Gogos CA, Drosou E, Bassaris HP, et al. Pro- versus anti-inflammatory cytokine profile in patients with severe sepsis: a marker for prognosis and future therapeutic options. J Infect Dis 2000;181:176180.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Niino M, Fukazawa T, Miyazaki Y, et al. Suppression of IL-10 production by calcitriol in patients with multiple sclerosis. J Neuroimmunol 2014;270:8694.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Härter L, Mica L, Stocker R, et al. Increased expression of toll-like receptor-2 and −4 on leukocytes from patients with sepsis. Shock 2004;22:403409.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45. Wittebole X, Coyle SM, Kumar A, et al. Expression of tumour necrosis factor receptor and toll-like receptor 2 and 4 on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating. Clin Exp Immunol 2005;141:99106.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Brandl K, Glück T, Huber C, et al. TLR-4 surface display on human monocytes is increased in septic patients. Eur J Med Res 2005;10:319324.

    • Search Google Scholar
    • Export Citation
  • 47. Scherberich JE, Kellermeyer M, Ried C, et al. 1-α-calcidiol modulates major human monocyte antigens and toll-like receptors TLR2 and TLR4 in vitro. Eur J Med Res 2005;10:179182.

    • Search Google Scholar
    • Export Citation
  • 48. Cohen-Lahav M, Shany S, Tobvin D, et al. Vitamin D decreases NFκB activity by increasing IκBα levels. Nephrol Dial Transplant 2006;21:889897.

  • 49. Cohen-Lahav M, Douvdevani A, Chaimovitz C, et al. The anti-inflammatory activity of 1,25-dihydroxyvitamin D3 in macrophages [retracted in: J Steroid Biochem Mol Biol 2011;127:444]. J Steroid Biochem Mol Biol 2007;103:558562.

    • Search Google Scholar
    • Export Citation
  • 50. Stio M, Martinesi M, Bruni S, et al. The vitamin D analogue TX 527 blocks NF-κB activation in peripheral blood mononuclear cells of patients with Crohn's disease. J Steroid Biochem Mol Biol 2007;103:5160.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51. Peng L, Malloy PJ, Feldman D. Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter. Mol Endocrinol 2004;18:11091119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52. Crescioli C, Ferruzzi P, Caporali A, et al. Inhibition of prostate cell growth by BXL-628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia. Eur J Endocrinol 2004;150:591603.

    • Search Google Scholar
    • Export Citation
  • 53. Sano J, Oguma K, Kano R, et al. High expression of Bcl-xL in delayed apoptosis of canine neutrophils induced by lipopolysaccharide. Res Vet Sci 2005;78:183187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54. Pintado CO, Carracedo J, Rodriguez M, et al. 1α,25-dihydroxyvitamin D3 (calcitriol) induced apoptosis in stimulated T cells through an IL-2 dependent mechanism. Cytokine 1996;8:342345.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55. Sadeghi K, Wessner B, Laggner U, et al. Vitamin D3 downregulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns. Eur J Immunol 2006;36:361370.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 56. Rassnick KM, Muindi JR, Johnson CS, et al. Oral bioavailability of DN101, a concentrated formulation of calcitriol, in tumor-bearing dogs. Cancer Chemother Pharmacol 2011;67:165171.

    • Crossref
    • Search Google Scholar
    • Export Citation

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Effects of calcitriol on apoptosis, toll-like receptor 4 expression, and cytokine production of endotoxin-primed canine leukocytes

Jared A. JaffeyComparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Juliana AmorimComparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Amy E. DeClueComparative Internal Medicine Laboratory, Veterinary Health Center, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Abstract

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs.

SAMPLE Blood samples from 6 healthy adult dogs.

PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10−7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry.

RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes.

CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

Abstract

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs.

SAMPLE Blood samples from 6 healthy adult dogs.

PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10−7M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry.

RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes.

CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

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).

Figure 1—
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.

Figure 2—
Figure 2—

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

Figure 3—
Figure 3—

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.

Figure 4—
Figure 4—

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

Figure 5—
Figure 5—

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).

Figure 6—
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

a.

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.

b.

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.

c.

Sigma-Aldrich, St Louis, Mo.

d.

Thermo Fisher Scientific, Carlsbad, Calif.

e.

Milliplex MAP canine cytokine-chemokine panel, EMD Millipore Corp, Billerica, Mass.

f.

BD Pharmingen, BD Biosciences, San Jose, Calif.

g.

Beckman Coulter Inc, Brea, Calif.

h.

Summit software, version 5.2.0.7477, Dako, Carpinteria, Calif.

i.

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

References

  • 1. Penna G, Adorini L. 1α,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J Immunol 2000;164:24052411.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Szymczak I, Pawliczak R. The active metabolite of vitamin D3 as a potential immunomodulator. Scand J Immunol 2016; 83:8391.

  • 3. Altieri B, Muscoqiuri G, Barrea L, et al. Does vitamin D play a role in autoimmune endocrine disorders? A proof of concept. Rev Endocr Metab Disord 2017;18:335346.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Tiosano D, Wildbaum G, Gepstein V, et al. The role of vitamin D receptor in innate and adaptive immunity: a study in hereditary vitamin D-resistant rickets patients. J Clin Endocrinol Metab 2013;98:16851693.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Lin Z, Li W. The roles of vitamin D and its analogs in inflammatory diseases. Curr Top Med Chem 2016;16:12421261.

  • 6. Tokuda N, Levy RB. 1,25-dihydroxyvitamin D3 stimulates phagocytosis but suppresses HLA-DR and CD13 antigen expression in human mononuclear phagocytes. Proc Soc Exp Biol Med 1996;211:244250.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Chandra G, Selvaraj P, Jawahar MS, et al. Effect of vitamin D3 on phagocytic potential of macrophages with live mycobacterium tuberculosis and lymphoproliferative response in pulmonary tuberculosis. J Clin Immunol 2004;24:249257.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Motlagh BM, Ahangaran NA, Froushani SMA. Calcitriol modulates the effects of bone marrow-derived mesenchymal stem cells on macrophage functions. Iran J Basic Med Sci 2015;18:672676.

    • Search Google Scholar
    • Export Citation
  • 9. Verma R, Jung JH, Kim JY. 1,25-dihydroxyvitamin D3 upregulates TLR10 while down-regulating TLR2, 4, and 5 in human monocyte THP-1. J Steroid Biochem Mol Biol 2014;141:16.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Wang H, Zhang Q, Chai Y, et al. 1,25(OH)2D3 downregulates the toll-like receptor 4-mediated inflammatory pathway and ameliorates liver injury in diabetic rats. J Endocrinol Invest 2015;38:10831091.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Fitch N, Becker AB. HayGlass KT. Vitamin D [1,25(OH)2D3] differentially regulates human innate cytokine responses to bacterial versus vital pattern recognition receptor stimuli. J Immunol 2016;196:29652972.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Villaggio B, Soldano S, Cutolo M. 1,25-dihydroxyvitamin D3 downregulates aromatase expression and inflammatory cytokines in human macrophages. Clin Exp Rheumatol 2012;30:934938.

    • Search Google Scholar
    • Export Citation
  • 13. Harishankar M, Afsal K, Banurekha VV, et al. 1,25-dihydroxy vitamin D3 downregulates pro-inflammatory cytokine response in pulmonary tuberculosis. Int Immunopharmacol 2014;23:148152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Neve A, Corrado A, Cantatore FP. Immunomodulatory effects of vitamin D in peripheral blood monocyte-derived macrophages from patients with rheumatoid arthritis. Clin Exp Med 2014;14:275283.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Grubczak K, Lipinska D, Eljaszewicz A, et al. Vitamin D3 treatment decreases frequencies of CD16-positive and TNF-α-secreting monocytes in asthmatic patients. Int Arch Allergy Immunol 2015;166:170176.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16. Venkatram S, Chilimuri S, Adrish M, et al. Vitamin D deficiency is associated with mortality in the medical intensive care unit. Crit Care Res Pract 2011;15:R292.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Higgins DM, Wischmeyer PE, Queensland KM, et al. Relationship of vitamin D deficiency to clinical outcomes in critically ill patients. JPEN J Parenter Enteral Nutr 2012;36:713720.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18. Flynn L, Zimmerman LH, McNorton K, et al. Effects of vitamin D deficiency in critically ill surgical patients. Am J Surg 2012;203:379382.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19. Braun AB, Gibbons FK, Litonjua AA, et al. Low serum 25-hydroxyvitamin D at critical care initiation is associated with increased mortality. Crit Care Med 2012;40:6372.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20. Aygencel G, Turkoglu M, Tuncel AF, et al. Is vitamin D insufficiency associated with mortality of critically ill patients? Crit Care Res Pract 2013;2013:856747

    • Search Google Scholar
    • Export Citation
  • 21. Amrein K, Zajic P, Schnedl C, et al. Vitamin D status and its association with season, hospital and sepsis mortality in critical illness. Crit Care 2014;18:R47.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Moromizato T, Litonjua AA, Braun AB, et al. Association of low serum 25-hydroxyvitamin D levels and sepsis in the critically ill. Crit Care Med 2014;42:97107.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. Kamr AM, Dembek KA, Reed SM, et al. Vitamin D metabolites and their association with calcium, phosphorous, and PTH concentrations, severity of illness, and mortality in hospitalized equine neonates. PLoS One 2015;2010:E0127684.

    • Search Google Scholar
    • Export Citation
  • 24. Holowaychuk MK, Birkenheuer AJ, Li J, et al. Hypocalcemia and hypovitaminosis D in dogs with induced endotoxemia. J Vet Intern Med 2012;26:244251.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25. Jaffey JA, Backus RC, McDaniel KM, et al. Serum vitamin D concentrations in hospitalized critically ill dogs. PLoS One 2018;13:e0194062.

  • 26. Biswas SK, Lopez-Collazo E. Endotoxin tolerance: new mechanisms, molecules and clinical significance. Trends Immunol 2009;30:475487.

  • 27. Cavaillon JM, Adib-Conquy M. Bench-to-bedside review: endotoxin tolerance as a model of leukocyte reprogramming in sepsis. Crit Care 2006;10:233.

    • Search Google Scholar
    • Export Citation
  • 28. Friedrich I, Spillner J, Lu E-X, et al. Induction of endotoxin tolerance improves lung function after warm ischemia in dogs. Am J Physiol Lung Cell Mol Physiol 2003;284:L224L231.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. Medvedev AE, Kopydlowski KM, Vogel SN. Inhibition of lipopolysaccharide-induced signal transduction in endotoxintolerized mouse macrophages: dysregulation of cytokine, chemokine, and Toll-like receptor 2 and 4 gene expression. J Immunol 2000;164:55645574.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Sato S, Takeuchi O, Fujita T, et al. A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways. Int Immunol 2002;14:783791.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. del Fresno C, García-Rio F, Gómez-Piña V, et al. Potent phagocytic activity with impaired antigen presentation identifying lipopolysaccharide-tolerant human monocytes: demonstration in isolated monocytes from cystic fibrosis patients. J Immunol 2009;182:64946507.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32. Deitschel SJ, Kerl ME, Chang CH, et al. Age-associated changes to pathogen-associated molecular pattern-induced inflammatory mediator production in dogs. J Vet Emerg Crit Care (San Antonio) 2010;20:494502.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33. Fowler BL, Axiak SM, DeClue AE. Blunted pathogen-associated molecular pattern motif induced TNF, IL-6 and IL-10 production from whole blood in dogs with lymphoma. Vet Immunol Immunopathol 2011;144:167171.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Jaffey JA, Amorim J, DeClue AE. Effects of calcitriol on phagocytic function, toll-like receptor 4 expression, and cytokine production of canine leukocytes. Am J Vet Res 2018;79:10641070.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Axiak-Bechtel SM, Tsuruta K, Amorim J, et al. Effects of tramadol and o-desmethyltramadol on canine innate immune system function. Vet Anaesth Analg 2015;42:260268.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 36. Zhang Y, Axiak-Bechtel S, Friedman Cowan C, et al. Evaluation of immunomodulatory effect of recombinant human granulocyte-macrophage colony-stimulating factor on polymorphonuclear cell from dogs with cancer in vitro. Vet Comp Oncol 2017;15:968979.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. DeClue AE, Yu DH, Prochnow S, et al. Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes. Vet J 2014;200:270275.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 38. Müller K, Haahr PM, Diamant M, et al. 1,25-dihydroxyvitamin D3 inhibits cytokine production by human blood monocytes at the post-transcriptional level. Cytokine 1992;4:506512.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39. Panichi V, De Pietro S, Andreini B, et al. Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium. Kidney Int 1998;54:14631469.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Tan ZX, Chen YH, Xu S, et al. Calcitriol inhibits tumor necrosis factor α and macrophage inflammatory protein-2 during lipopolysaccharide-induced acute lung injury in mice. Steroids 2016;112:8187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. van Dissel JT, van Langevelde P, Westendorp RGL, et al. Anti-inflammatory cytokine profile and mortality in febrile patients. Lancet 1998;351:950953.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Gogos CA, Drosou E, Bassaris HP, et al. Pro- versus anti-inflammatory cytokine profile in patients with severe sepsis: a marker for prognosis and future therapeutic options. J Infect Dis 2000;181:176180.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Niino M, Fukazawa T, Miyazaki Y, et al. Suppression of IL-10 production by calcitriol in patients with multiple sclerosis. J Neuroimmunol 2014;270:8694.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Härter L, Mica L, Stocker R, et al. Increased expression of toll-like receptor-2 and −4 on leukocytes from patients with sepsis. Shock 2004;22:403409.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45. Wittebole X, Coyle SM, Kumar A, et al. Expression of tumour necrosis factor receptor and toll-like receptor 2 and 4 on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating. Clin Exp Immunol 2005;141:99106.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Brandl K, Glück T, Huber C, et al. TLR-4 surface display on human monocytes is increased in septic patients. Eur J Med Res 2005;10:319324.

    • Search Google Scholar
    • Export Citation
  • 47. Scherberich JE, Kellermeyer M, Ried C, et al. 1-α-calcidiol modulates major human monocyte antigens and toll-like receptors TLR2 and TLR4 in vitro. Eur J Med Res 2005;10:179182.

    • Search Google Scholar
    • Export Citation
  • 48. Cohen-Lahav M, Shany S, Tobvin D, et al. Vitamin D decreases NFκB activity by increasing IκBα levels. Nephrol Dial Transplant 2006;21:889897.

  • 49. Cohen-Lahav M, Douvdevani A, Chaimovitz C, et al. The anti-inflammatory activity of 1,25-dihydroxyvitamin D3 in macrophages [retracted in: J Steroid Biochem Mol Biol 2011;127:444]. J Steroid Biochem Mol Biol 2007;103:558562.

    • Search Google Scholar
    • Export Citation
  • 50. Stio M, Martinesi M, Bruni S, et al. The vitamin D analogue TX 527 blocks NF-κB activation in peripheral blood mononuclear cells of patients with Crohn's disease. J Steroid Biochem Mol Biol 2007;103:5160.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51. Peng L, Malloy PJ, Feldman D. Identification of a functional vitamin D response element in the human insulin-like growth factor binding protein-3 promoter. Mol Endocrinol 2004;18:11091119.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52. Crescioli C, Ferruzzi P, Caporali A, et al. Inhibition of prostate cell growth by BXL-628, a calcitriol analogue selected for a phase II clinical trial in patients with benign prostate hyperplasia. Eur J Endocrinol 2004;150:591603.

    • Search Google Scholar
    • Export Citation
  • 53. Sano J, Oguma K, Kano R, et al. High expression of Bcl-xL in delayed apoptosis of canine neutrophils induced by lipopolysaccharide. Res Vet Sci 2005;78:183187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54. Pintado CO, Carracedo J, Rodriguez M, et al. 1α,25-dihydroxyvitamin D3 (calcitriol) induced apoptosis in stimulated T cells through an IL-2 dependent mechanism. Cytokine 1996;8:342345.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55. Sadeghi K, Wessner B, Laggner U, et al. Vitamin D3 downregulates monocyte TLR expression and triggers hyporesponsiveness to pathogen-associated molecular patterns. Eur J Immunol 2006;36:361370.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 56. Rassnick KM, Muindi JR, Johnson CS, et al. Oral bioavailability of DN101, a concentrated formulation of calcitriol, in tumor-bearing dogs. Cancer Chemother Pharmacol 2011;67:165171.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

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