Sepsis associated with low vitamin D

Introduction

Theimportance of vitamin D on bone health and calcium homeostasis haslong been understood. However, scientists are beginning to uncoverthat this steroid hormone has important roles in the optimalfunctioning of many organ systems. Vitamin D receptors and25-hydroxyvitamin D-1α-hydroxylase (1α-OHase) have beendiscovered in many extraskeletal tissues and the vitamin D responseelement (VDRE) found in over 900 genes.1 Furthermore, recentepidemiologic and clinical trials have suggested that optimalvitamin D status may be protective against several chronicillnesses, including risk of systemic infection, cardiovasculardisease, lung disease and diabetes.2-6

Highlightingthe importance of the recent discoveries of vitamin D’spleiotropic effects on human health, numerous recent publicationshave explored the definition of vitamin D deficiency and itsprevalence. In 2011, the Institute of Medicine issued a publicationestablishing a serum 25-hydroxyvitamin D (25(OH)D) concentration of20ng/mL as an optimal concentration for skeletal health for the USpopulation.7 In the same year, the Endocrine Societyrecommended a serum 25(OH)D concentration of at least 30 ng/mL foroptimal health benefits.3Using the Endocrine Society’s target,40–100% of US elderly individuals in the community are vitaminD insufficient along with an estimated 1 billion people in theworld.8

Thedeveloping science around vitamin D has begun to reveal potentiallinks between its deficiency and sepsis. The connections betweenthese conditions are still in the early phases of discovery. Thisreview will discuss the various basic and clinical research studiesthat are examining this relationship, covering the data fromexperimental models of infection and sepsis to the clinical data onrespiratory infections and critical illness.

VitaminD and the Immune System

Avital role for vitamin D in the human system was initially indicatedby the discovery of vitamin D receptors (VDR) in nearly all types ofimmune cells, including activated CD4+ and CD8+ T cells, B cells,neutrophils, macrophages and dendritic cells.9 These cells spanthe body’s innate and adaptive immune responses to pathogens.The relationship being uncovered by scientists is complex, yet theunderlying theme seems to be one of modulating the adaptive immuneresponse while promoting innate immunity.10 In regards toadaptive immunity, vitamin D affects the proliferation anddifferentiation of B and T cells and modulates immunoglobulinproduction10 with one study showing suppression of antibodyresponse to pneumococcal antigens in mice.11 While theseeffects may have important roles in autoimmunity, it is not yetclear whether they will have a beneficial role in infection orsepsis. In contrast, vitamin D’s promotional effects on innateimmunity have been more clearly elucidated at this time and will bethe focus in this review.

Theinnate immune system acts to rapidly identify invading organisms andrespond with humoral and cellular defense mechanisms to contain,neutralize and remove offending pathogens. These pathogens areidentified by highly conserved pathogen-associated molecularpatterns (PAMP) that bind to pathogen-recognition receptors (PRR) onimmune cells. The cells that participate in these innate immuneresponses include neutrophils and monocytes as well as epithelialcells that not only provide barrier function but also haveanti-pathogen activity. As discussed below, vitamin D is a keycomponent in several of the pathways of this system.9,12

VitaminD and the Sepsis Cascade

Monocytesplay important roles in the innate immune system as antigenpresenting cells as well as in phagocytosis. Human monocytesrecognize some PAMPs by a family of transmembrane molecules, theToll-Like Receptors (TLRs). TLR4 specifically recognizes and bindsto lipopolysaccharide (LPS), a substance produced by gram-negativebacteria and a potent stimulator of the sepsis inflammatory cascade.Sadeghi et al. demonstrated that human monocytes stimulated with LPSand treated with 1,25-dihydroxyvitamin D (1,25(OH)2D), showeddose-dependent decreases in TLR2 and TLR4 synthesis, with anincrease in CD14, a TLR co-stimulatory molecule.13 They furtherfound that 1,25(OH)2D decreased TNF-α and tissue factor, bothend products of LPS activation and important inflammatory moleculesin sepsis.13 These effects were reversed with the introductionof a VDR antagonist, reinforcing a key role of vitamin D in thissignaling mechanism.13

Furtherstudies have revealed a role for vitamin D in the endothelialresponse to LPS. In sepsis, LPS activates endothelial cells toproduce transcription factor NF-κB, the pro-inflammatorycytokines IL-6 and IL-8, and the chemokine, RANTES. In a study byEquils et al., human endothelial cells treated with 1,25(OH)2D thenstimulated with LPS, showed significant inhibition of thesemolecules when compared with cells only exposed to LPS.14 Thesefindings may suggest that vitamin D acts to modulate thepro-inflammatory endothelial response to LPS.

Overthe past two decades, these intriguing vitamin D-dependent cellularresponses to LPS have also been studied in rat and mouse models ofsepsis. Horiuchi et al. exposed mice simultaneously tointraperitoneal LPS and oral 1,25(OH)2D. Compared with controls,mice that received vitamin D had less expression of the inflammatorymolecule, iTXB2, and a decrease in mortality.15 In 2001,Asakura et al. demonstrated that compared with low-molecular weightheparin, treatment with oral 1,25(OH)2D had equal or improvedeffects on hemostatic parameters and markers of organ dysfunction inrats infused with LPS.16 In 2007, Moller et al. performedplacebo controlled trials of treatment with 1,25(OH)2D in threedifferent rat models of sepsis showing varied results.17 Whilethe different models of sepsis and vitamin D treatments in theseexperiments make them difficult to compare, when combined with thein vitro data they suggest that vitamin D has important modulatoryeffects on the innate immune response to LPS-induced sepsis.

WhileLPS is an important molecule in gram-negative sepsis, vitamin D mayalso have a role in the sepsis cascade induced by fungal organisms.A study by Khoo et al. treated peripheral blood mononuclear cells(PBMC) with 1,25(OH)2D and exposed them to C. albicans.The PBMCs demonstrated significant dose-dependent decreases inproduction of pro-inflammatory cytokines with a decrease inexpression of the PRRs that recognize C. albicans.18

VitaminD and Local Immune Defense

VitaminD’s effects on the innate immune system encompass not only themodulation of the systemic inflammatory response but also the localcontrol of pathogens. In vitro studies have shown that 50,000 -90,000 IU/ml of vitamin D3 inhibited growth or killed strainsofStaphylococcus aureus, Streptococcus pyogenes, Klebsiellapneumoniae, and Escherichia coli.19 Another study oftracheobronchial epithelial cells infected with respiratorysincytial virus showed decreases in inflammatory proteins withoutincreases in viral replication when treated with 1,25(OH)2D.20 Ina model of bovine mastitis, Nelson et al. showed that infiltratingmonocytes from infected mammary tissue increased gene expression ofVDR and 1α-OHase.21 This lends support to a vitaminD-dependent response of the innate immune system that is triggeredby local infection.

Anotherimportant component of the innate immune response is theantimicrobial peptides (AMP). These peptides initiate bacterialkilling by increasing bacterial cell membrane permeability onceinside a phagosome.12 Studies have revealed vitamin D-dependentsteps in this process, demonstrating that the binding of bacterialligands to monocyte PRRs induces gene expression of the VDR and1α-OHase genes.12 In an autocrine fashion, 1α-OHaseactivates 25(OH)D which binds to VDRs and then vitamin D responseelements (VDRE) to induce genes for the AMPs, β defensin 4A andhCAP18.12 The latter molecule is then cleaved to its activeform LL-37.

LL-37in particular has been extensively studied in its role in thevitamin D-dependent pathways of the innate immune system. It isproduced by phagocytic leukocytes, mucosal epithelium andkeratinocytes, and is present in mucosal secretions andplasma.22 LL-37 has shown in vitro microbicidal activityagainst important human pathogens including Pseudomonasaeruginosa, Salmonella typhimurium, Escherichia coli, Listeriamonocytogenes, Staphylococcus epidermidis, Staphylococcus aureus,and vancomycin-resistant entero-cocci.23 In addition todirect microbicidal activity, it has demonstrated disruptionof Pseudomonas aeruginosa biofilms, promotion ofphagocytosis and reactive oxygen species, and chemotaxis of otherimmune cells to sites of infection.22 In one study humanbronchial epithelial cells incubated with 1,25(OH)2D showedincreased production of LL-37 and reduced growth of airwaypathogens, Bordetella bronchioseptica andPseudomonasaeruginosa.24 In another study of human bladder cellschallenged with Escherichia coli, oral vitamin Dpretreatment of subjects increased production of LL-37 andbactericidal activity.25 This effect was neutralized withanti-LL-37 antibodies, highlighting LL-37 as a criticalantibacterial component of the vitamin D mediated immuneresponse.25 LL-37 also appears to be linked to vitamin D statusin humans as a study by Jeng et al. showed a positive correlationbetween plasma concentrations of LL-37 and 25(OH)D among patientsadmitted to the ICU.26

ClinicalResearch on Vitamin D and Infection

Complementingthe basic science research of vitamin D’s effects on theinnate immune system, clinical trials have examined its role in theprevention and control of human infection. The studies havepredominantly studied vitamin D’s role in respiratoryinfections, the most common source of sepsis in the UnitedStates27 and the results have been mixed.

Oneof the early observational studies that pointed toward a connectionbetween vitamin D and respiratory infections was a secondaryanalysis of over 18,000 individuals of the US. National Health andNutrition Examination Survey (NHANES) III. In this study, Ginde etal. found an inverse relationship between serum 25(OH)Dconcentrations and the incidence of upper respiratory infections(URI).28 Vitamin D has since been studied in several clinicaltrials to characterize its role in respiratory infections. In 2010,Sabetta et al. conducted a prospective cohort study showing thatserum 25(OH)D concentrations of 38 ng/mL or greater were associatedwith a 2-fold decrease in the number of upper respiratoryinfections.29 In contrast, Laaksi et al.’s randomizedtrial of vitamin D supplementation (400 IU daily of vitamin D3) didnot show a significant difference in the days absent from work dueto URI, although only about 30% of the intervention group achieved a25(OH)D concentration > 32 ng/mL30. Also of note, this study didshow a significant increase in the number of men remaining healthythroughout the 6-mo study period.30 Another randomized study byLi-Ng et al. revealed that winter month oral vitamin Dsupplementation (2000 IU daily of vitamin D3) did not reduce theincidence, duration or severity of URIs among ambulatory adults.31

Inaddition to these studies on URIs, there have been a number ofstudies examining the relationship between vitamin D and acute lowerrespiratory infections (ALRI) in infants and children. Threecase-control studies of newborns and children admitted with ALRIshowed an association between low vitamin D status and risk ofALRI.32-34 In contrast, two other case-control studies did notreveal an association between vitamin D status and hospitalizationfor ALRI in children, although one of them showed that it wasassociated with admission to the ICU.35,36 In an interventiontrial, Manaseki-Holland et al. showed that a single oral dose of100,000 IU of vitamin D in children admitted for ALRI reduced ratesof recurrence in the subsequent three months, although it did notshorten the duration of the index infection.37Urashima et al.conducted a randomized trial of vitamin D supplementation (1200 IUdaily of vitamin D3) in school aged children during the winter andfound that supplementation significantly decreased the incidence ofinfluenza A infection, especially among children with predisposingrespiratory conditions.38

Thevaried mixed of these studies may be methodological with thedifferent study designs, vitamin D treatments, outcome measures andsample sizes. Rather than nullify the findings from the basicsciences, they help direct future research in this area byindicating that vitamin D’s effects on infection will likelybe small and need large samples sizes and supplementation needs tobe adequate and sustained. In translating this research to sepsis itis important to note that most of the above studies do not focus onspecific infective pathogens. While respiratory infections are themost common cause of sepsis in the US, gram-positive bacteria arethe most common pathogens, followed by gram-negative bacteria with arise in fungi.39 However, the clinical literature on vitamin Dand these organisms is sparse. A case-control study of the NHANES,found an association between low vitamin D and increased nasalcolonization with MRSA.40 Also a cross-sectional study ofveterans with clostridium difficile and staphylococcus aureusinfections, showed more hospitalizations and a 4-fold increase inhospital length of stay among those with serum 25(OH)D < 20ng/mL.41 Despite the limitations of the research on vitamin Dand infection, clinical science is still moving forward in exploringits connections with sepsis.

VitaminD in the Critically Ill

Asvitamin D’s role in the immune system is beginning to bepieced together through its local and systemic effects on immuneresponses to pathogens, there has been an interest in its role incritically ill patients. While this review will focus on theprevalence of vitamin D insufficiency and its connections withsepsis in the critically ill, it is important to note vitamin D’sother potential roles within this population, including patientswith severe hypocalcemia, increased bone turnover in prolongedillness and a possible connection with insulin resistance.42-44

Severalstudies have revealed a high prevalence of vitamin D insufficiencyamong the critically ill, although its association with outcomes hasbeen less clear. A single center prospective observational studyexamined all ICU patients admitted in a spring-summer season andfound serum 25(OH)D concentrations < 24 ng/mL in 79%.45 Springadmission, low albumin and high Simplified Acute Physiology II scorewere all independently associated with low serum 25(OH)Dconcentrations.45 They did not find associations betweenvitamin D, mortality or hospital-acquired infections in the overallgroup or in the septic subgroup.45 McKinney et al. conducted aretrospective study of 136 veterans admitted to the ICU who had aserum 25(OH)D drawn within a month before or after admission to theICU. Ninety-eight percent of the veterans had low serum 25(OH)Dconcentrations.46 The study also demonstrated a significantlyincreased survival rate (69% vs. 44%) among those with serum 25(OH)Dconcentrations greater than 20ng/mL.46 A retrospective study byVenkatram et al. revealed an association between mortality andvitamin D deficiency (25(OH)D < 20 ng/mL) in 437 patients at asingle center ICU.47

Otherstudies have provided more specific data on the relationship betweenvitamin D and septic patients. Jeng et al. showed that vitamin Dinsufficiency was present in 100% of critically ill patients withsepsis, 92% of critically ill patients without sepsis and 66.5% inhealthy controls.26 A prospective study of 66 surgical ICUpatients showed a non-significant trend toward an increased rate ofinfections and sepsis among the vitamin D deficient (< 20ng/mL).48 Braun et al. conducted two retrospective studies onthe same source population investigating this subject. One was aretrospective analysis of 2,399 patients admitted to medical andsurgical ICUs with a 25(OH)D drawn within the year prior toadmission. The data showed a 1.3 and 1.7 fold increase in all-causemortality among vitamin D insufficient and deficient groups (<30ng/mL and < 15ng/mL) respectively and a significant increase inblood culture positivity.49 The other analysis was of 1,325patients with a 25(OH)D drawn 7 d before or after ICU admission andrevealed a significant association between vitamin D deficiency (<15ng/mL) and increased mortality at 30, 90 and 365 d.50 Thevitamin D-mortality association in both analyses was not modified bythe presence of sepsis.49,50 Highlighting the complexity ofconfounding in critically ill and septic patients, a prospectivestudy by Cecchi et al. found a relationship between mortality andvitamin D deficiency among 170 patients with severe sepsis andseptic shock that became insignificant after adjustment.51

Apilot study by Ginde et al. more specifically looked at low vitaminD and its effects on the severity of sepsis. The investigatorsfollowed 81 patients suspected of having an infection in theEmergency Department and examined their severity of illness incomparison to vitamin D status. They found that patients with serum25(OH)D concentrations less than 30ng/mL were more likely atenrollment to have severe sepsis and SOFA (Sepsis-related OrganFailure Assessment) scores ≥ 2 and more likely at 24 h to havesevere sepsis, SOFA scores ≥ 2, APACHE II (Acute Physiology andChronic Health Evaluation II) scores ≥ 25 and dysfunction of twoor more organ systems.52

Overall,the evidence suggests associations between vitamin D depletion andcritical illness-related outcomes with the link to sepsis lessclear. These relationships are likely confounded by many common riskfactors such as age, socio-economic status, obesity, and chronicillness. In regards to the last risk factor, chronically ill anddebilitated individuals likely spend more time indoors withconsequent less sun exposure and more vitamin D deficiency. Theseconfounding associations make it difficult to prove causality incritical-illness outcomes. In addition to these epidemiologicalphenomena, sepsis therapy may also confound this relationship. Onestudy demonstrated this by showing that the hemodilutional effectsof fluid resuscitation can decrease serum 25(OH)D concentration by35%.53 These issues reveal the complexity of research in thisfield and the important confounders that must be accounted for infuture studies.

VitaminD and the Seasonal and Racial Variation in Sepsis

Whileno studies have directly examined the association between theseasonal and geographic variations of serum 25(OH)D concentrationsand the incidence of sepsis, evidence suggests possibleparallels.54 A study surveying the national hospital dischargedatabase from 1979 to 2003 in the USA, showed a significant seasonalvariation of sepsis and severe sepsis with the highest incidences inthe winter and the lowest in the fall, with a parallel variation inthe incidence of the most common cause of sepsis, respiratoryinfections.55 Furthermore, the Northeast USA showed thegreatest seasonal variation in sepsis incidence compared with theSouthern USA which showed the least variation.55 These patternsparallel the annual variations in serum 25(OH)D concentrations, withpeak concentrations in the fall and a nadir after the winter.56 Thisis in part explained by the elliptical orbit of the earth around thesun, changing the solar zenith angle throughout the seasons, whicheffects the amount of UV B radiation reaching the earth’ssurface.57 Interestingly, this seasonal variation of vitamin Dsynthesizing radiation is less pronounced nearer the equator wherethe seasonal variation of sepsis and respiratory infections may belower.57,58 This speculation, however, oversimplifies the othergeophysical factors that affect vitamin D synthesis, including cloudand ozone cover, altitude and surface reflectivity.57

Thereare also human factors that effect vitamin D synthesis including sunprotective behaviors and skin pigmentation, the latter of which mayalso point toward links between vitamin D and racial differences insepsis. Studies have shown that in the US, blacks are nearly twiceas likely to develop sepsis as whites,39 develop moreinfections59 and have higher rates of organ dysfunction withsepsis.60 While vitamin D status was not included in thesestudies, blacks tend to have lower serum 25(OH)D concentrations thanwhites. In a recent review, Grant outlined cross-sectional datashowing serum 25(OH)D concentrations to be 16 ng/mL, 21 ng/mL and 26ng/mL among blacks, Hispanics and whites in the US.61 Thiscould be explained by the fact that increased skin pigmentationleads to less production of vitamin D as melanin absorbs andcompetes for UVB radiation.62 With vitamin D’spleiotropic effects on bodily health, it has been suggested that itmay be a contributing cause for racial disparities across manydiseases61 and its effects on the immune system may provide aconnection with the higher rates of infection and sepsis in blacks.As with the seasonal variation hypothesis, there are no studies thatdirectly examine this relationship, but the speculations warrantfurther investigation.

Conclusion

Themultiple functions of vitamin D in the immune system’sresponse to infection suggest it may be an integral component incombating sepsis. The basic science data point toward vitamin D’srole in the optimal functioning of the innate immune system, in partby producing AMPs such as LL-37; while seeming to temper theinflammatory cascade induced by LPS. The early clinical data on itsrole in preventing and attenuating infections has suggested a linkbut intervention trials have produced mixed results, requiringlarger randomized controlled trials to help define the relationship.Furthermore, clinical data also point toward a role of vitamin D andcritical illness but a direct relationship with sepsis and itsseverity and outcomes is yet to be determined by further research.Some interesting parallel patterns between vitamin D and seasonaland racial variations in sepsis are currently speculative butinteresting questions to be explored. In conclusion, the currentpicture of vitamin D and sepsis is one of a research field early inits course with many important links that provide fertile ground forfurther investigation. Such investigation is warranted as vitamin Dis inexpensive and safe to administer and even incremental benefitsin the outcomes of sepsis may be enacted on a scale to produce asignificant public health impact (Fig. 1, Tables 1, 2and 3).

Figure 1. Morbidityand mortality vitamin D insufficiency and sepsis. Venn diagramreflecting the links between vitamin D’s roles in innateimmune function, clinical infections and sepsis in the criticallyill. The intersections represent the potential increased morbidityand mortality resulting from vitamin D insufficient statespredisposing to and exacerbating sepsis.

Table 1. Basicscience studies of vitamin D and the innate immune response. Summaryof basic science data showing vitamin D’s effects in cell andanimal models of infection and sepsis

1,25D,1,25-dihydroxyvitamin D; 25D, 25-hydroxyvitamin D

Table 2. Clinicalstudies of vitamin D and infections: Summary of observational andexperimental data on vitamin D’s effects in infections

25D,25-hydroxyvitamin D; MRSA, methicillin-resistant Staphylococcusaureus; MSSA, methicillin-sensitive Staphylococcus aureus; RCT,randomized controlled trial

Table 3. Clinicalstudies of vitamin D and sepsis: Summary of observational data onthe links between vitamin D and sepsis among the critically ill

25D,25-hydroxyvitamin D

Disclosureof Potential Conflicts of Interest

Theauthors have no conflicts of interest to declare.

Acknowledgments

Thiswork is supported in part by National Institutes of Health grantsT32 AA013528 (J.A.K. and J.E.H.), K23 AR054334 (V.T.), K24 RR023356(T.R.Z.), R21 HL110044 (G.S.M. and T.R.Z.), U54 RR-024380, P50AA-013757P50, AA013757 (G.S.M.) and the Atlanta Clinical andTranslational Science Institute (UL1 RR025008).

References