Increased lytic efficiency of bovine macrophages trained with killed mycobacteria

RESEARCH ARTICLE Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria Ramon A. Juste 1¤ *, Marta Alonso-Hearn 1 , Joseba M. Garrido 1 , Naiara Abendaño 1 , Iker A. Sevilla 1 , Christian Gortazar 2 , Jose ´de la Fuente 2,3 , Lucas Dominguez 4 1Animal Health Department, NEIKER-Tecnalia, Berreaga, 1, 48160 Derio, Bizkaia, Spain, 2SaBio, Instituto de Investigacio ´n en Recursos Cinege ´ticos, IREC (CSIC, UCLM, JCCM), Ronda de Toledo, 13071, Ciudad Real, Spain, 3Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 McElroy Hall, Stillwater, OK, 74078, United States of America, 4Centro de Vigilancia Sanitaria Veterinaria (VISAVET), Universidad Complutense, Avenida Puerta de Hierro, 28040, Madrid, Spain ¤Current address: SERIDA, Carretera de Oviedo, 33300 Villaviciosa, Asturias, Spain *[email protected] Abstract Innate immunity is evolutionarily conserved in multicellular organisms and was considered to lack memory until very recently. One of its more characteristic mechanisms is phagocytosis, the ability of cells to engulf, process and eventually destroy any injuring agent. We report the results of an ex vivo experiment in bovine macrophages in which improved clearance of Mycobacterium bovis (M.bovis) was induced by pre-exposure to a heat killed M. bovis preparation. The effects were independent of humoral and cellular adaptive immune responses and lasted up to six months. Specifically, our results demonstrate the existence of a training effect in the lytic phase of phagocytosis that can be activated by killed mycobacteria, thus suggesting a new mechanism of vaccine protection. These findings are compatible with the recently proposed concept of trained immunity, which was developed to explain the observation that innate immune responses provide unspecific protection against pathogens including other than those that originally triggered the immune response. Introduction Innate immunity is evolutionarily conservedin multicellularorganisms and is the most primitive component of the immune system. It is basedon non-specificmechanisms that, in contrast to the adaptive immune response, were consideredto lack memory until very recently. The traditional paradigmin immunologyis that innate immunity—as opposed to adaptive immunity— lacks of memory-likeproperties.In plants and invertebrates, however, memoryeffectfor innate host defence has been recently recognizedand definedas training to differentiate from specific immune memory. Therefore, the term 'trained immunity' was proposed to describethe PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 1 / 12 a11111 OPEN ACCESS Citation: Juste RA, Alonso-Hearn M, Garrido JM, Abendaño N, Sevilla IA, Gortazar C, et al. (2016) Increased Lytic Efficiency of Bovine Macrophages Trained with Killed Mycobacteria. PLoS ONE 11 (11): e0165607. doi:10.1371/journal. pone.0165607 Editor: Anil Kumar Tyagi, University of Delhi— South Campus, INDIA Received: May 19, 2016 Accepted: October 15, 2016 Published: November 7, 2016 Copyright: ©2016 Juste et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding for these studies was provided by the EU project WildTBVac (Contract #613799) and by grants from the Instituto Nacional de Investigacio ´n y Tencologı ´a Agraria y alimentaria (INIA, RTA2011-00049) and the Ministry of Science (MINECO, AGL2014-56305) and European Funds Regional Development (FEDER). potentiating effectsof an exposureto microbial agents or vaccines on innate immune responses, which would thus mount a more effectiveimmune response against related and sometimeseven unrelated infections.This training leads to increasedcytokineproduction via epigeneticreprogrammingof monocytes[1]. Although the epidemiologicalevidenceof BacilleCalmette-Guérin (BCG)vaccine effectivenessagainst tuberculosisis referred to as proof of the trainedimmunity concept in higheranimals [2], no experimentalevidencehas beenproduced to support the hypothesis that phagocytosismight be playing a critical role in this process. Here, we report an improved clearance of Mycobacterium bovis in bovinemacrophages lasting up to six months and independentof specificimmune responses.To discriminatebetweenthe adaptive and innate immune responses in cattle sensitizedwith a killedM.bovis preparation, monocytederivedmacrophages (MDM) from sensitizedand control cattle were tested ex vivo for phagocyticactivity while monitoring for systemic specificimmune responses. Our results demonstrate the existence of a training effect in the lytic phase of phagocytosisthat can be activated by killedmycobacterial cells. This suggests a new mechanism of immune protection by whichlow-intensity primingenhancesearly phagocyticresponses independentlyof humoral and cellular adaptive immune responses for, at least, six months. This mechanism would support the empirical evidenceof protection induced by killed mycobacterial vaccines [3,4,5].These findings fit well with the recently proposed general concept of trained immunity, which was developed to explain the observationthat innate immune responses provide unspecificprotection against pathogens including other than those that originallytriggeredthe immune response. Materials and Methods Ethics statement Animals usedin thisstudy were submitted only to procedures that according to European (Directive2010/63/EU of the European Parliament and of the Councilof 22 September2010 on the protection of animals usedfor scientificpurposes.Chapter 1, Article1, Section5, paragraphs b and f)and Spanish (Real Decreto53/2013, de 1 de febrero, por el que se establecenlas normas básicas aplicables para la protección de los animales utilizadosen experimentacióny otros fines científicos,incluyendo la docencia,Article2, Section5, Paragraphs b and f)legislation on experimentalanimals are exempt from its application. The animals, belongingto a registered commercial farm supervisedby the local livestock authority (Serviciode Ganadería de la Diputación Foral de Bizkaia) were submitted only to the introductionof a needlein accordance with goodveterinarypractice and were not killedin relationship with this study. Animals and treatment Twenty-five 3to 6-month-old Limousin (6), Pyrenean (8) or crossed (11) cattle originallypurchased from 8 different farms and currently housed in a small commercial feedlotwere intramuscularlyinjectedwith 2 ml of a suspensionof heat-inactivated cells of a localM.bovis isolate (NEIKER MS#1403) in Montanide 50 (SEPPIC, Paris, France) (MdR) or left untreated. Ten animals receiveda fulldose of 10 7 colony forming units (CFU) (FD group), ten a reduced dose of 10 3 CFU (RD group), and 5 animals servedas untreated controls (UC group). All breeds were present in each treatment group. Two animals from the FD group, 3 from the RD group and 2 from the UC group were lost becauseof unrelated disease(1), traumatism (1) or sale (5) before the final test. Humoral immune response: ELISA test On day 0, immediately before treatment, and at 15, 43, 78 and 186 days post-treatment (dpt), blood was obtained from each animal via the tail vessels and collectedin EDTA Vacutainer Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 2 / 12 Competing Interests: The authors have declared that no competing interests exist. tubes (Becton,Dickinsonand Company, Sparks, MD, USA). The blood samples were analysed using an in-house anti-M.bovis ELISA with bovinePPD as the antigen [5]. This assay includes a serumMycobacterium phlei pre-absorption step to reduce non-specificreactivity caused by environmental mycobacteria. The results of this test were expressed as relative optical density (OD) calculatedas raw serumOD divided by the OD value of the positive control. IFN-γrelease assay (IGRA) Immediately before treatment and at 15, 43, 78 and 186 dpt bloodstimulation was performed within the first 8 h after sampling in 24-well culture plates (Becton-Dickinson1, Franklin Lakes,NJ) with either phosphate-buffered saline (PBS), avian purifiedprotein derivative (PPDAV) (CZ Veterinaria1SA, Porriño, Spain) or bovinepurifiedprotein derivative (PPDBOV) (CZ Veterinaria1SA, Porriño, Spain) tuberculin.After incubationfor 16–24 h at 37°C + 5–7% CO 2 , plasma was separated by centrifugationand frozenat -20°C until testing. Subsequently, a commercialIGRA (BovigamTM, Fisher Scientific,Schlieren,Switzerland) was performedin accordance with the manufacturer’s instructions. Intradermal reaction At 46 and 81 dpt, each animal was intradermallyinoculatedat threespots on each side of the neck (ST) with 0.1 ml of standard bovine and avian tuberculin(CZV, SA. Porriño, Spain) or PBS using a Dermojetdevice(AKRA Dermojet,Pau, France). Reactionswere read after 72 h according to the Spanish TB control program regulations. Phagocytosis assay The phagocytosisassays were performedat 78 and 186 dpt to estimate the bacterialload reduction associatedwith each treatment as previously described[6,7]. For the first phagocyticassay, peripheralbloodwas drawn into heparinizedVacutainer tubes, diluted 1:2 in Hanks balanced salt solution (HBSS), layered over 10 ml of Ficoll-Paque gradient (1.084 g/cm 3 ) and centrifuged at 900 x g for 30 min. The cell interphase was collectedand centrifugedat 400 x g for 10 min to remove platelets from peripheralbloodmonocyticcells (PBMCs). For the secondtest, macrophages were magneticallyenrichedfrom PBMCs using magneticactivated cell sorting (MACS) technology(Miltenyi Biotech, BergischGaldbach,Germany). In both cases, bovine monocytes were resuspendedin RPMI-1640 supplemented with 20 mM L-glutamine, 10% heat-inactivated bovineserum,100 U ml -1 penicillinG and 100 mg ml -1 streptomycin sulfate (Lonza) and seededinto 24-well plates at a density of 4 x 10 5 cells ml -1 . After 2 h at 37°C in a humidified5% CO 2 incubator, non-adherent cellswere removed. Adherent cells were incubatedfor 7 days at 37°C to allow differentiationto MDM prior to infection.Differentiated MDMs were inoculated with a single-cell suspension of M.bovis at an MOI (multiplicity of infection;bacteria:cells)of 10:1. After 2 h and 7 days, the supernatant was removed from three wells, and the cells were washed twice with HBSS to remove extracellularbacteria.Infected macrophages were lysed by vigorous pipetting with 0.5 ml of 0.1% Triton X-100 (Sigma-Aldrich)in sterile water for 10 min. Supplemented Mycobacteria Growth indicator tubes (MGIT) (Becton,Dickinson and Company, Sparks, MD) were inoculatedwith 0.1 ml of each initial bacterialsuspension and with the 2 h and 7 d p. i. cell lysates. The tubes were incubatedat 37 ± 2°C for up to 41 days in a BactecMGIT 960 instrument(Becton,Dickinsonand Company). The earliest instrumental indication of positivity(i.e., time to detection[TTD]) for each tube was recorded. The predicted number of bacteriain each positive tube was calculated using previously generated mathematical formulas which relate TTD(in days) to estimatedlog 10 CFUs. Bacterialcell Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 3 / 12 reductionbetween2 h and 7 days p.i. were calculatedby dividingthe estimated log 10 CFUs at day 7 by that at 2 h p.i. Cytokines gene expression At 186 dpt, MDMs purifiedfrom peripheralbloodof treated and untreated animals were infectedex vivo with M.bovis. At 24 h pi, the infectedMDM were washed in 0.5 ml of cold HBSB, mixed with 50 μl of Lysis Solutionand incubatedat room temperature for fiveminutes to allow RNA release into the Lysis solution (PowerSYBR1Green Cells-to-Ctkit (Life Technologies,Carlsbad,CA).DNAse I was addedto the Lysis Solutionto allow genomic DNA degradation at this step. Next, 5 μl of Stop Solution were mixed into the lysate to inactivate the lysis solution so that it would not interfere with the reverse transcription (RT) or polymerase chain reaction (PCR).Cell lysates (10 μl) were then reverse transcribedto synthesize cDNA using2.5 μl of 20 X RT Enzyme Mix, 25 μl of 2X SYBR RT Buffer, and 12.5 μl of Nuclease-free water. The reactionmixtures were incubatedat 37°C for 60 min and thenat 95°Cfor 5 min to inactivatethe RT enzyme.Finally, the synthesized cDNAs were amplified by real-timePCR usingPower SYBR Green PCR Master Mix and PCR primers for the bovineIL1-α,TGF-β, BCL2,TNF-αand C3 genes as previously described[6]. The β-actin gene was used as the endogenouscontrol genein the assays. To determinethe changes in gene expression (Relative quantification, RQ), the following formula was used:RQ = 2-Δ(ΔC T ) were ΔC T is C T (target gene)—C T (β-actin)and Δ(ΔC T ) is ΔC T (experimental)—ΔC T (control). Results were expressed as RQ of transcriptioncompared to thoseof control uninfectedcells. Statistical analysis ELISA and IGRA optical density readings,estimated bacterialcounts, reductions in bacterial load and cytokineexpression estimates were compared using the GLM procedure in the SAS 9.1 software (SAS Institute, Cary, NC) with the Tukey-Kramer multiple-comparison post-test. Correlations betweenvariables to explore their associationwith innate or adaptive responses were examined by the Kendall correlation and principal component analysis with SAS software. In all predictiveanalyses, differenceswere considered statisticallly significantat p<0.05. Results Assessment of humoral and cell-mediated immune responses in cattle treated with a heat killed M bovis preparation For adaptive immunity, three standard tests based on humoral and cell-mediatedimmune responses were conductedto determine:i) antigen-specificantibody production,ii) IFN-γ release by peripheral blood lymphocytes and iii) cell infiltrationinto the skin [8]. None of the treated groups exhibited humoral responses at 0, 15 and 43 dpt) (Fig 1A). However, the group treated with the highestdose of the killedM.bovis preparation displayed a significantincrease in antibody levelsby day 78 that were slightly higherby day 186. The control and low-dose groups on day 186 also exhibited slight increases in antibody levels relative to their minimal readings, but the antibody levels did not differamong these groups. These patterns are consistent with the anamnestic effectof the tuberculinantigens used in the skin test, whichincreases the reactivity of infected anergic animals [9]. The cellular response as measured by IFN-γ release assay (IGRA) by peripheralbloodlymphocytes revealed a substantial specificresponse by 15 dpt in the FD group that was significantlydifferent from that of the control group (Fig 1B). However, both the control and low-dose immune responses converged at a very low level on day 43 when the immune response of the FD group reached a maximum. Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 4 / 12 Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 5 / 12 Regardingthe intradermaltest, at the firsttest only the FDgroup exhibited a significant increase in mean skin thickness,whereas the control and low-doseRD groups exhibited negligibleincreases in skin thickness.By the secondtest, the high-dosegroup remained at a similar reactivitylevel, whereas reactivity remained very low in both the control and RD groups. With the exception of the low-level response of the RD group, these specificimmune response dynamics were consistent with expectations. M.bovis phagocytosis assessment in bovine macrophages purified from blood of treated and untreated cattle At 78 and 186 dpt, MDM purifiedfrom peripheralbloodof treated and untreated animals were infectedex vivo with M.bovis. Bacterialload associatedwith each treatment at 2 h and 7 days p. i. was estimated as previously described[6,7]. In the phagocytosistest, bacterialcell uptake did not differamong the groups (S2 Table). Fig 2 summarises the results of the two phagocytosisassays for average bacterialload reductionat 7 days p. i. The FD group exhibited an average reductionof 83.37%, whereas the RD group displayed a reductionof 88.03%. By contrast, the bacterialload increasedslightly in the control group. Notably, in the phagocytosis assay performedat 78 dpt, the control group exhibited an approximately 50% reductionthat made the reductionsin the treated groups appear less dramatic by comparison over time (Table 1). This response could be a short-term effect caused by intradermal sensitization with tuberculinor it might be related to individual variation. Interestingly, the rate of infection clearance was much higher in this sampling than in the sampling conductedthree months after the last skin test (186 dpt). This smaller but more widespread reductiontogether with the variable reductionsshortly after skin sensitizationor boostingand three months later suggest that phagocyticmemory might not be constant and definitive but vary with time and tend to wane after severalmonths. This effectof thetreatment on the macrophages lytic capability has not beendescribedpreviouslyand may have important implications for explainingnatural and vaccine protection mechanisms during chronic intracellular infections. Assessment of cytokine profiles in MDMs purified from treated and untreated cattle and infected ex vivo with M.bovis At 24 h pi, RNA was extractedfrom M.bovis-infectedMDMs and reverse transcribedto cDNA to measure IL1-α,TGF-β, BCL2,TNF-αand C3 expression levels by using qRT-PCR (S2 Table). The cytokineprofiles of the groups did not differsignificantly, except the C3 levels, which were significantly higherin both treated groups when compared to controls (p = 0.0006; p = 0.0327). Furthermore,C3 expression levels were significantlyhigherin the FD group than Fig 1. Immune dynamics throughout the experiment. (A) ELISA: Specific humoral immune response in relative optical density units in TB ELISA. *Comparison between MdR-FD and Control (p = 0.0007) and MdR-RD (<0.0001) groups at day 78. ** Comparison between MdR-FD and Control (p = 0.0239) and MdR-RD (p<0.0110) groups at day 186. Other days differences are not statistically significant at p<0.0500. (B) IFN: Specific cellular immune response in optical density units in Interferon γrelease assay. * Comparison between MdR-FD and Control (p = 0.0061) and MdR-RD (p = 0.0051) groups at day 15. ** Comparison between MdR-FD and Control (p = 0.0003) and MdR-RD (<0.0110) groups at day 43. *** Comparison between MdR-FD and Control (p = 0.0190) and MdR-RD (p = 0.0580) groups at day 78. **** Comparison between MdR-FD and Control (p = 0.0043) and MdR-RD (p<0.0001) groups at day 186. Other days differences are not statistically significant at p<0.0500. (C) Skin test: Mean group skin test results in mm. *Comparison between Control and MdR-FD (p<0.0001) or MdR-RD (p = 0.9678) and between both MdR groups (p<0.0001) at day 43. ** Comparison between Control and MdR-FD (p<0.0001) or MdR-RD (p = 0.5456) and between both both MdR groups (p<0.0001) at day 78. Control: Untreated control group. MdR-FD: Full-dose group. MdR-RD: Reduced-dose group. doi:10.1371/journal.pone.0165607.g001 Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 6 / 12 the RD group (p = 0.0042). This findingis consistent with the previously reported increase in the expression of C3 in wild boar tissues treated with this inactivated M.bovis-basedimmunogen [5,10]. In addition, the up-regulation of C3 in the lymph nodesand tonsils has beencorrelated with tuberculosisresistance in wild boar [11]. Relationships between immunological variables Principal component analysis revealed two main associationsbetweenthe studied variables (Fig 3). The first component represented the specificimmune response becausehumoral and cellular specificresponse variables tended to cluster on the positive end. The second component represented bacteriallysis becausethe bacterialload at 7 days and load reductionclearly alignedalong this axis. Strikingly, these two variables did not exhibit any correlation with the first component, suggestingthat phagocyticactivity decreases as specificmechanisms become dominant in the immune response. Therefore, phagocytosisbecomesless relevant as more specific mechanisms are triggered.The lack of correlation betweenspecificmechanisms and actual M.bovis clearance suggest that the innate component of the immune response is uncoupled from both specificresponses and bacterialuptake. Discussion Our results demonstrate that sensitizing young animals with an inactivated mycobacterial preparation by the parenteral route can boostthe bacterialkillingefficiencyof infectedmacrophages. This is the first study to demonstrate a memoryeffecton phagocyticlysis in a higher mammal. This phenomenon differs from that reportedin invertebrates, in which the learning effectappears to occuronly at the uptake phase [12]. Becauseno cells other than macrophages were supposed to be present in our MDM infectedcultures given that both variants of selective Fig 2. Average mycobacterial load reduction according to the treatment calculated as the quotient between log 10 CFU at 2 h minus log 10 CFU at 7 days and log 10 CFU at 2 h. The four decimal numbers in the base of the bars are the p values for differences between the control and sensitized groups. The number between MdR-FD and MdR-RD at the end of the bars represent the p value for the difference between sensitized groups. Control: Untreated control group. MdR-FD: Full-dose group. MdR-RD: Reduced-dose group. doi:10.1371/journal.pone.0165607.g002 Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 7 / 12 procedures applied yielded similar results, it could be assumed that the memoryeffect was maintained by macrophages themselves and not by lymphocytes interactingwith them.Additionally, both the lack of correlation with specificcellular and humoral immune responses and the positiveassociationwith the non-specificIFN-γresponse furtherdemonstrate that phagocytosisdoesnot depend on specificimmune responses.Our results confirmthat trainedimmunity is present in organisms other than miceand lower vertebrates at the macrophage level. Most importantly, our results indicate that this effectremains active in young adults for at least six months after exposure.Intriguingly, lower doses of killed-mycobacteriamight be more efficient at inducingtrainedimmune responses than higher doses, in agreement with field observations [3,13,14]. Mycobacteria are powerfulimmunogens,and this property has long been used to stimulate immune responses by adding mycobacteria immunogens to antigens of interest in complete Table 1. Results of the phagocytosis assays. MD78 MD186 Treatment Calf code Uptake 7 days Reduction Uptake 7 days Reduction Time Treatment Time Treatment Full Dose 1656 C 6.0183 0.0000 100% • 83% a • 63% b • 83% c . . . • 84% a • 78% b • 14% c 1657 C . . . . . . 3621 L . . . 3.2502 0.7869 76% 4159 C . . . 3.9288 0.5586 86% 4166 C 5.9311 0.0000 100% 4.5379 0.0000 100% 4168 C 6.4247 6.3932 0% 3.9444 0.7647 81% 4170 C . . . . . . 6314 P 6.4535 0.0000 100% 5.2150 0.6587 87% 6315 P 6.2336 0.0000 100% 3.6865 0.9331 75% 6568 C 6.8783 0.0000 100% 3.7228 0.7680 79% Reduced Dose 0723 L 5.6854 0.0000 100% • 95% a • 88% b • 67% c 4.3161 0.6490 85% • 89% a • 89% b • 16% c 1654 L . . . . . . 1655 P . . . . . . 4160 L . . . 4.5011 0.8700 81% 4163 C 6.5538 1.3163 80% 6.1269 0.6506 89% 4164 L 6.7075 0.0000 100% 5.5988 0.0000 100% 4167 C . . . 5.1879 0.5797 89% 5049 P 6.6816 0.0000 100% 4.5457 0.7110 84% 6310 P 6.4247 0.7983 88% . . . 9681 P 6.7596 0.0000 100% . . . Control 1658 C 6.4247 0.0000 100% • 56% a • 0% b • 50% c . . . • -15% a • 0% b • 0% c 3877 C . . . . . . 4165 L 6.3833 5.4100 15% 4.7049 6.0817 -29% 6312 P 6.3428 6.0872 4% 5.3117 5.9606 -12% 9682 P 6.8916 0.0000 100% 3.6748 3.6748 0% Individual bacterial cell reduction (Time) between 2 h and 7 days p.i. were calculated by dividing the difference between the estimated log 10 CFUs at day 7 and that at 2 h p.i. by the log 10 CFU at 2 h p.i. Group bacterial reduction was calculated as follows a Mean log 10 CFU reduction from uptake to end of culture at 7 days p.i. b Reduction in mean log 10 CFU in the treated group compared with the control group c Proportion of individuals with 100% reduction in the bacterial load in macrophages. C: Crossed; P: Pyrenean; L: Limousin. doi:10.1371/journal.pone.0165607.t001 Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 8 / 12 Freund’s adjuvant [15]. This immunogenicityis consistent with the high stability of immune response-inducinggenes,which is interpreted as an evolutionaryadaptation favouring longterm survivalin organs and subsequent dissemination[16,17]. In addition,mycobacterialvaccines prevent other diseasesnon-specifically[2,18,19]. This study results suggest that the long held principle that tuberculosiskilledvaccinesdo not work might not be trueand that Tuberculosisvaccination couldbe achievedwithout using live bacilli,which posea risk to immunocompromised individuals of any age who submit to BCGtherapy, such as cancer patients. It is veryimportant to defineappropriate immunization protocols that can take advantage of the non-specificand powerfuleffect of killedvaccines on phagocytosis.These vaccination strategies may also provide protection against infectionsor immune dysfunctionsother than M. bovis. Another interesting findingof this study, which unfortunately could not be extended,is that the training effect of macrophage phagocytosispersists for at least several months. In summary, we present evidencethat bovine macrophages can be “trained” to improve their lytic capacity, rather than just particleuptake, likely by epigeneticreprogramming [20, 21]. Our results are consistent with the concept of trained innate immunity recently proposed by Netea et al. [1]. Evidence from this study suggests that efficient priming of the phagocytic Fig 3. Principal component analysis of the relationships between variables. Note that bacterial uptake is negatively correlated with humoral and cellular specific immune responses. The bacterial reduction exhibits no correlation with these specific variables. Bacterial reduction defines the second factor, which is weakly correlated with the non-specific IFN response. IFNBOV: IFN with bovine PPD; IFNAVI: IFN with avian PPD; IFNPBS: IFN blank; IDBOV: intradermal test with bovine PPD; IDAVI: intradermal test with avian PPD; IDPBS: intradermal test blank; CFU2H: M.bovis CFU logarithm at 2 h after culture inoculation; CFU 7D: M.bovis CFU logarithm at 7 days after culture inoculation; MYCRED: M.bovis CFU reduction between 2 h and 7 days after inoculation. doi:10.1371/journal.pone.0165607.g003 Trained Macrophages PLOS ONE | DOI:10.1371/journal.pone.0165607 November 7, 2016 9 / 12