Unsaturated Fatty Acids Promote the Phagocytosis
of P.aeruginosa and R.equi by RAW264.7Macrophages
Stephanie Adolph •Herbert Fuhrmann •
Julia Schumann
Received:19July 2012/Accepted:8August 2012/Published online:18August 2012ÓSpringer Science+Business Media,LLC 2012
Abstract In the present study,using the murine monocyte/macrophage cell line RAW264.7as a model system,we analyzed the phagocytosis rate and the bactericidal capacity of polyunsaturated fatty acids (PUFA)-enriched macrophages against Pseudomonas aeruginosa and Rhodococcus equi .The P.aeruginosa strain ATCC 10145,the virulent R.equi strain ATCC 33701,and the non-virulent R.equi strain ATCC 6939were examined.Flow cytometric detection of intracellular microorganisms in combination with viability assays were used to determine the impact of PUFA on the number of engulfed,surviving as well as replicating bacteria.Macro-phage enrichment with PUFA resulted in an increase of the internalization rate of the microorganisms by the immune cells.Moreover,an impeding
action of the unsaturated fatty acids on the intracellular survival rates of the virulent strains P.aeruginosa ATCC 10145and R.equi ATCC 33701could be observed.The n-3fatty acid docosahexaenoic acid (DHA)as well as the n-6fatty acid arachidonic acid (AA)showed the most pronounced effects.Taken together,our data support the idea of supplementing PUFA to immunocompromised indi-viduals as well as to people suffering from chronic infections with P.aeruginosa or R.equi to improve macrophage phag-ocytic and microbicidal activity.
Introduction
Phagocytosis,a major effector function of macrophages,is of vital importance in a host’s immune defense.The multi-
step process encompasses particle recognition followed by binding,regulated ingestion,and finally the destruction of internalized particles.The ability of macrophages to bind,engulf,and kill bacteria defines the bactericidal activity of the immune cells [7].
The microbicidal properties of macrophages are of par-ticular relevance with regard to facultative intracellular pathogens,such as Pseudomonas aeruginosa or Rhodo-coccus equi ,which are described to survive or even to replicate within the phagocytes [12,19,21,22].Espe-cially immunocom
promised individuals are endangered to develop serious illness due to infection with P.aeruginosa or R.equi [12,19,21,22].To persist within the host’s macrophages,the pathogens have evolved distinct mecha-nisms of survival.P.aeruginosa is a nosocomial pathogen,which infects the pulmonary tract,the urinary tract,the ear,burns,and wounds [21].The disease agent exhibits a mul-titude of enzymatic and mutational mechanisms of resis-tance,and is known for its abilities to form biofilms and to synchronize gene expression by the intercellular commu-nication mechanism,quorum sensing [19,21].Moreover,the quorum sensing molecules released by P.aeruginosa are reported to actively modulate defense reactions of host immune cells [11].R.equi ,the causative agent of pyogra-nulomatous pneumonia,interferes with the normal pathway of phagosome maturation [12,22].Phagosomes containing virulent R.equi are described to be only mildly acidic (pH 6.5)and to be characterized by the absence of the proton-pumping vacuolar ATPase [12,22].Of note,there are non-virulent R.equi strains as well.Non-virulent R.equi lack the vap plasmids,which are thought to be essential for impeding phagosome maturation and,thus,virulence [8,20,22].Nevertheless,even non-virulent R.equi strains are described to survive,but not to replicate,within macrophages for several hours due to the natural resistance of R.equi against
S.Adolph ÁH.Fuhrmann ÁJ.Schumann (&)
Faculty of Veterinary Medicine,Institute of Physiological Chemistry,University of Leipzig,An den Tierkliniken 1,04103Leipzig,Germany
e-mail:julia.schumann@vmf.uni-leipzig.de
modulateCurr Microbiol (2012)65:649–655DOI 10.1007/s00284-012-0207-3
low pH as well as distinct radical intermediates of macro-phage respiratory burst[5,8,20,22].
Phagocytosis is primarily a membrane-associated pro-cess.At this,the extension of cell-membrane pseudopodia and the encircling of particles are enabled by an active remodeling of the membrane and the clustering of partic-ular membrane receptors[9,16].The structure of the plasma membrane and thefluidity of the lipid bilayer, therefore,are likely to play an important role for the phagocytic activity of a macrophage.Both,structure and fluidity,strongly depend on the fatty acid composition of the membrane phospholipids.Accordingly,a plasma membrane enrichment with polyunsaturated fatty acids (PUFA)is long discussed as a promising approach to improve the phagocytic activity of macrophages[2,3,16]. Macrophage membrane lipid composition,in fact,can be modulated depending on the availability of particular fatty acids as shown by us and others[2,3,16,17].Moreover, several in vitro studies report the enrichment of macro-phages with PUFA to result in an increase in the phago-
cytic activity of the cells[3,6,16].The degree of unsaturation of the plasma membrane and the ratio of saturated to unsaturated fatty acids were found to correlate with the phagocytosis rates of macrophages[3,16]. However,these results are limited to the phagocytosis of zymosan particles or of killed,non-virulent bacteria.Data concerning the impact of PUFA enrichment of macro-phages on the phagocytic activity of the cells with respect to viable and infectious pathogens such as P.aeruginosa or R.equi are lacking.Hence,in the present study,using the murine monocyte/macrophage cell line RAW264.7as a model system,we analyzed the phagocytosis rate and the bactericidal capacity of PUFA-enriched macrophages against these two disease agents.To obtain a comprehen-sive insight,besides the P.aeruginosa strain ATCC10145, both the virulent R.equi strain ATCC33701and the non-virulent R.equi strain ATCC6939were examined.The counts of engulfed,surviving as well as replicating bacteria were determined by means offlow cytometric detection of intracellular microorganisms in combination with viability assays(CFU assay,MTT assay).This study for thefirst time provides an informative basis as to whether a PUFA supplementation of macrophages could be of benefit to improve the phagocytic and the microbicidal activity of the immune cells in case of infection with pathogenic bacteria. Materials and Methods
Materials
All chemicals and reagents were obtained from Sigma-Aldrich(Taufkirchen,Germany)unless noted otherwise.Cell cultureflasks were purchased from Greiner Bio-One (Frickenhausen,Germany).HEPES(25mmol/L)-buffered RPMI1640culture medium containing300mg/L L-glu-tamine and fetal bovine serum gold were acquired from PAA Laboratories GmbH(Co¨lbe,Germany).
Cell Culture
The permanent mouse monocyte/macrophage cell line RAW264.7(ATCC number TIB-71)was used.The RAW264.7cells were cultured in RPMI1640medium supplemented with4.5g/L glucose and5%FCS(basic medium).The fatty acids alpha-linolenic acid(LNA; C18:3n3),eicosapentaenoic acid(EPA;C20:5n3),docosa-hexaenoic acid(DHA;C22:6n3),linoleic acid(LA; C18:2n6),or arachidonic acid(AA;C20:4n6)(all Biotrend, Ko¨ln,Germany)were included in the culture medium at 15l mol/L using ethanol as a vehicle(0.2%v/vfinal ethanol concentration).Cells were incubated in75cm2cell culture flasks at a density of19106cells/mL for72h at37°C and 5%CO2in a humidified atmosphere.
Fluorescein labeling of Bacteria
Log-phase cultures of the bacterial strains P.aeruginosa ATCC10145,R.equi ATCC33701(virulent),an
d R.equi ATCC6939(non-virulent)were centrifuged at10,000rpm for5min and washed twice in phosphate-buffered saline (PBS).The microorganisms were resuspended at concen-trations of19108cells/mL(determined by turbidity measurement)in PBS containing0.2mg/mL offluorescein isothiocyanate and incubated for30min in the dark at 37°C.Labeled bacteria were separated from freefluores-cent dye by three washing steps using PBS.The viability of the microorganisms was verified by growing the bacteria on tryptic soy agar plates.
Flow Cytometry
RAW264.7were seeded in six-well cell culture plates at a density of  3.39105cells/mL in the media described above.Co-cultivation of macrophages withfluorescein-labeled P.aeruginosa ATCC10145,R.equi ATCC33701, or R.equi ATCC6939was performed for45min in a bacterium/cell ratio of10:1.At the end of the incubation period,samples were set on ice to stop ongoing phagocy-tosis,washed three times with PBS,andfixed with1% formaldehyde in PBS.Fluorescence signals of non-inges-ted bacteria were quenched with1.0mg/mL trypan blue. The quenching solution was added to the samples imme-diately prior toflow cytometric analysis.Cells were ana-lyzed on a FacsCalibur by means of Cellquest Pro3.1 software(all Becton–Dickinson,Heidelberg,Germany).
The phagocytosis rate represents the fraction of RAW264.7 with one or more ingested bacteria(fluorescent cells) within the total population.The analysis was performed in triplicates in three independent experiments for each combination of fatty acid supplement and bacterial strain.
CFU Assay
RAW264.7were seeded in96-well cell culture plates at a density of  2.59104cells/mL in the media described above.Replicate cultures were overlaid with bacterial sus-pension adjusted to yield a bacterium/cell ratio of2:1for P.aeruginosa ATCC10145or3:1for R.equi ATCC33701 as well as for R.equi ATCC6939.The cells were co-cul-tivated for30min followed by two washing steps with PBS to remove extracellular bacteria.Infected RAW264.7were either lysed with PBS containing0.5%Triton X-100or refed with PUFA-enriched media.Further cultivation was performed for0.5/3h before lysis for P.aeruginosa or rather0.5/24h before lyses for the both R.equi strains.The cell lysates were diluted serially,and100l L of each dilu-tion was plated on tryptic soy agar plates.Microcolonies were counted after24h(P.aeruginosa)or48h(R.equi strains)of incubation at37°C.The numbers of colony-forming units(CFU)per milliliter were calculated by multiplying the number of colonies by the dilution factor and the volume of the total sample in proportion to the 100l l sample taken for setting the plate count.The analysis was performed in six independent experiments for each combination of fatty acid
supplement and bacterial strain. MTT Assay
RAW264.7were seeded,infected,and lysed as described for the CFU assay.Cell lysates containing internalized bacteria were cultured for24h(P.aeruginosa)or48h(R.equi strains) at37°C.3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT)was dissolved in PBS at5mg/mL.10l L of the MTT solution was added to40l L of the cell lysates in the final24h of incubation.Afterward,the converted dye was solubilized by an addition of50l L of acidic isopropanol (pH4.7).Absorbance was read on a Wallac Victor21420 multiplate reader at590nm and analysis done by means of WorkOut1.5software(all Perkin Elmer,Rodgau,Germany). The data were quantified by means of a bacteria standard curve set up for each bacterial strain tested.The analysis was per-formed in triplicates in six independent experiments for each combination of fatty acid supplement and bacterial strain. Statistical Analysis
The data are shown as mean±standard deviation(SD). One-way analysis of variance followed by Students t test was used to identify significant differences between means. The statistical analysis was carried out by means of GraphPad Prism4(GaphPad Software,La Jolla,USA).In all cases,P\0.05was assumed to indicate significant differences.
Results and Discussion
Phagocytosis Rates
The phagocytosis of the pathogens by the macrophages is the priority action of the innate immune cells.The effective internalization of disease agents is a precondition for pre-venting bacteria dissemination throughout the body.The phagocytosis rate,therefore,is of crucial importance in the initial immune defense against pathogens.
Unsupplemented Macrophages
The phagocytosis rates of RAW264.7macrophages were assessed by means offlow cytometry.For P.aeruginosa, the phagocytosis rate of unsupplemented macrophages was found to be very low in comparison to the phagocytosis rates observed for the R.equi strains tested(Fig.1). Moreover,a difference in the phagocytosis rates of the non-virulent R.equi ATCC6939and the virulent R.equi ATCC 33701could be seen(Fig.1).The number of unsupple-mented RAW264.7with internalized non-virulent R.equi ATCC6939was about1.5times higher than the number of unsupplemented macrophages with internalized virulent R.equi ATCC33701(Fig.1).
The data underline the impact of the type of bacteria on the macrophage-mediated phagocytosis processes.The target cells of R.equi are macrophages[12,22].Thus, R.equi,unlike P.aeruginosa,does
not exhibit mechanisms to prevent phagocytosis,which results in elevated phago-cytosis rates.Furthermore,the virulence of the microor-ganisms seems to be of importance.It is assumed that the surface structure of a bacterium decisively influences the phagocytosis rate of macrophages[10].The known dif-ferences in the surface structures of virulent and non-virulent R.equi[22]therefore may provide the basis for the differing phagocytosis rates observed.
PUFA-Supplemented Macrophages
Enrichment of RAW264.7with PUFA from both the n-3 and the n-6families resulted in a marked increase in phagocytosis rates(Fig.1).Regardless of the bacterial strain tested due to fatty acid supplementation,a boost in the number of macrophages with internalized microor-ganisms could be observed(Fig.1).For P.aeruginosa,the
phagocytosis rate was enhanced up to 5.6times compared to the unsupplemented macrophages (Fig.1).Regarding the R.equi strains tested,the rise in the phagocytosis rate due to the PUFA enrichment of macrophages was up to 2.7times for the non-virulent R.equi ATCC 6939and up to 3.5times for the virulent R.equi ATCC 33701(Fig.1).
The promoting effect of unsaturated fatty acids on the internalization of zymosan particles or heat-kill
ed microor-ganisms by macrophages is well documented [2,3,6,9,16].However,we show here for the first time that PUFA increase the phagocytosis rates of macrophages during co-cultivation of the immune cells with viable bacteria.Our data provide evidence that the PUFA raise the phagocytic activity of macrophages against different types of bacteria,which vary in their surface structure.It is discussed that the boost in the macrophage phagocytosis rates in the context of a fatty acid supplementation may be due the modulation of the physical character of the macrophage membrane [2,3,9,16].The alteration of membrane fluidity seems to be of particular importance [2,3,9,16].In fact,data from our group and others [2,3,16,17]demonstrate PUFA to be readily incor-porated into the macrophage plasma membrane.Supple-mentation of macrophages with unsaturated fatty acids is known to result in the modification of the membrane orga-nization,membrane trafficking as well as signal transduction mechanisms [13],which is likely to mediate the increase in phagocytic activity.More recently,PUFA were reported to induce the differentiation of macrophages into the anti-inflammatory M2phenotype [14].M2macrophages are characterized by higher phagocytosis rates compared to
the
Fig.1Phagocytosis rates of RAW264.7macrophages cultured in basic medium or in medium supplemented with 15l mol/L LNA,EPA,DHA,LA,or AA after 45min of co-cultivation with fluorescein-labeled,viable bacteria (P.aeruginosa ATCC 10145,R.equi ATCC 33701,R.equi ATCC 6939).a Representative flow cytometric dot plots for the co-cultivation with R.equi ATCC 33701.b Histogram of flow cytometric data.Data are mean ±SD (N =3,n =3).Means denoted by different letters are significantly different
pro-inflammatory M1phenotype[18].Thus,the enrichment of macrophages with unsaturated fatty acids may promote the phagocytic activity of the immune cells in two inde-pendent ways:(i)an increase in membranefluidity and(ii)a receptor-mediated switch in macrophage polarization.
Survival Rates
The internalization of a pathogen in a plasma membrane-derived phagosome is linked with the maturation of the phagosome to a phagolysosome.Bacteria,which are not able to interfere with the process of phagolysosome formation, are degraded by the combined action of a low pH,hydrolytic enzymes as well as reactive oxygen and nitrogen interme-diates inside the phagolysosome.However,
pathogens,such as P.aeruginosa and R.equi,have evolved mechanisms to survive,allowing them to remodel the phagosome and to rest in the antimicrobial environment[5,8,12,19–22]. Unsupplemented Macrophages
The survival rates of P.aeruginosa as well as R.equi inside the RAW264.7were assessed both by the MTT and the CFU assays with comparable results.For P.aeruginosa, after30min of internalization by the control macrophages, a reduction in the number of viable bacteria by about20% could be seen(Fig.2).Nevertheless,after3h of internal-ization,there was an increase in the number of viable bacteria by2.5times compared to the initial number of phagocytosed bacteria(Fig.2).For R.equi,the survival rates in unsupplemented macrophages were found to depend on the virulence of the strain tested.The number of the non-virulent R.equi ATCC6939dropped as early as after30min of internalization by about65%,and stayed almost constant within a period of24h(Fig.2).In con-trast,the number of the virulent R.equi ATCC33701 decreased by only7%30min after internalization,and then rebounded within24h,thereby slightly exceeding the initial number of phagocytosed bacteria(Fig.2).
The survival rates observed are in accordance with the literature.Both virulent strains,P.aeruginosa as well as R.equi ATCC33701,are described to replicate within macrophages[12,19,21,22].The bacterial growth can be divided into two parts:(i)an initial lag phase and(ii)a propagation phase[8].Th
e differences in the rate of mul-tiplication observed between P.aeruginosa and R.equi ATCC33701are due to the pathogen-specific doubling times and do not result from a differentiated action of the RAW264.7macrophages.Non-virulent R.equi strains, lacking the vapA plasmid,are described to survive,but do not replicate inside the macrophages[5,8,20,22].Con-sistently,we observed a bacteriostatic effect of the mac-rophages on the non-virulent R.equi ATCC6939.The functions of the vapA plasmid-encoded proteins have yet not been fully explained[22].It is,however,speculated that these proteins prevent the acidification of the phago-some.Although able to survive at a low pH[20],the acidic milieu is likely to counteract the propagation of R.equi ATCC6939.
PUFA-Supplemented Macrophages
The supplementation of RAW264.7with fatty acids from both the n-3and the n-6families resulted in reduced sur-vival rates of the virulent bacteria tested(Fig.2).The PUFA enrichment of the macrophages was found to enhance the killing of the pathogens within thefirst30min after internalization(Fig.2).Furthermore,an impeding action of the unsaturated fatty acids on bacteria replication inside the macrophages could be observed(Fig.2).The PUFA effects were particularly apparent for R.equi ATCC 33701.Due to PUFA supplementation,the drop in the number of viable R.equi ATCC33701was up to nine times more pronounced(Fig.2).In addition,except for LNA,even24h
after internalization,the number of viable R.equi ATCC33701was beneath the initial number of phagocytosed bacteria(Fig.2).Regarding P.aeruginosa, 30min after internalization,the number of viable bacteria was up to1.4times lower for PUFA-supplemented mac-rophages compared to unsupplemented cells(Fig.2).The replication rates of P.aeruginosa within the RAW264.7 were decreased from2.5times(without PUFA supple-mentation)to  1.7times(with AA supplementation) (Fig.2).Nevertheless,the enrichment of the culture med-ium with fatty acids did not result in any difference in the survival rates of the non-virulent R.equi6939in com-parison to unsupplemented macrophages(Fig.2).
To our knowledge,it is thefirst time that unsaturated fatty acids were shown to impact the survival of the virulent bac-teria P.aeruginosa and R.equi ATCC33701within macro-phages.So far,a modulatory effect of PUFA on intracellular pathogen survival has been solely described for Staphylo-coccus aureus and Escherichia coli[4,23].Here too,an increase in the intracellular killing of the pathogens was observed[4,23].Moreover,we could show the macrophage enrichment with PUFA to result in a slowdown of bacterial growth.Although not sufficient for bacterial clearance,the unsaturated fatty acids support the host’s immune defense against P.aeruginosa and R.equi ATCC33701.It is of par-ticular importance to note that fatty acids from both the n-3 and the n-6families mediate the effects de
scribed.For R.equi ATCC33701,the n-3PUFA DHA was found to act most effectively;for P.aeruginosa,the n-6PUFA AA showed the most pronounced impact.Thus,our data indicate that mainly the carbon chain length and the number of double bonds and to a lesser extent the fatty acid family are critical for the potency

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