high - density lipoprotein ( hdl ) , known as antiatherosclerotic lipoprotein , is also involved in innate immunity .
for instance , hdl binds to lipopolysaccharides ( lps ) and lipoteichoic acid ( lta ) derived from microorganisms and neutralizes the physiological activity of these molecules [ 2 , 3 ] .
additionally , hdl is known to have a bactericidal activity toward yersinia enterocolitica serotype o:3 ; this activity is mediated by the complement system . 

mycobacterium avium ( m. avium ) is a species of nontuberculous mycobacteria and causes opportunistic infections in immunocompromised hosts .
although the incidence of m. avium infection is increasing worldwide , its pathogenicity remains poorly understood unlike that of m. tuberculosis .
, cholesterol performs an important function in the invasion and survival of mycobacteria inside macrophages [ 711 ] .
cholesterol accumulation in the host cell membrane is observed at the entry point of m. tuberculosis and of other mycobacteria , and cholesterol depletion inhibits the invasion of cells by mycobacteria .
after phagocytosis of mycobacteria , they are enveloped by the cell membrane containing cholesterol - rich domains ; tryptophan aspartate - containing coat protein ( taco ) is recruited to the phagosomes and prevents the fusion of these organelles with lysosomes .
consequently , the engulfed mycobacteria evade degradation by lysosomes and survive inside the host cell .
the invading mycobacteria modulate lipid metabolism in the host cell and promote formation of lipid droplets ( lds ) , which are mainly composed of neutral lipids such as triacylglycerol and cholesteryl ester .
lds physiologically contribute to lipid storage and lipid metabolism and are available to mycobacteria as a carbon source [ 10 , 14 ] .
scavenger receptor type b1 ( sr - b1 ) , a receptor of apolipoprotein a - i ( apoa - i ) present in hdl as a main component , is generally known to perform the function of transferring esterified cholesterol from matured hdl into the cytosol in accordance with the cholesterol gradient .
sr - b1 was also identified as a nonopsonic phagocytic receptor for mycobacteria because suppression of sr - b1 expression attenuates phagocytosis of mycobacteria [ 16 , 17 ] .
we screened various bacteria for binding with hdl and found that m. avium exhibits a stronger interaction with hdl than do other gram - negative bacilli .
the fact that hdl binds to m. avium has never been reported and the effect for innate immunity is unclear .
the aim of the present study was to elucidate the molecular mechanism of hdl 's binding to m. avium and the physiological meaning of this interaction .

 m. avium ( atcc 700737 ) was cultured for 3 weeks in middlebrook 7h9 broth ( difco ) supplemented with the oleic acid - albumin - dextrose complex ( oadc ; becton dickinson ) .
gram - negative bacteria ( isolated at the clinical laboratory , medical hospital of tokyo medical and dental university ) were cultured on trypticase - soy - agar with 5% sheep blood ( becton dickinson ) before use .
the thp-1 cell line was obtained from atcc ( manassas , va ) and maintained at 210  10 cells / ml in the rpmi 1640 medium ( sigma - aldrich ) supplemented with 10% heat - inactivated fetal bovine serum ( invitrogen ) , with a penicillin - streptomycin - l - glutamine solution ( wako ) , and 1x nonessential amino acids ( gibco ) .
the thp-1 cells were induced to differentiate into thp-1 macrophages by phorbol myristate acetate ( pma , sigma - aldrich ) for 72  h. after washing with phosphate - buffered saline ( pbs ) , the thp-1 macrophages were incubated in the serum - free rpmi 1640 supplemented with nutridoma - sp ( roche ) and then infected with m. avium ( multiplicity of infection [ moi ] 20  :  1 ) with or without hdl ( 50  g protein / ml ) for 24  h at 35c in a humidified atmosphere containing 5% co2 . hdl ( 1.0631.210  g / ml )
hdl and apoa - i were dialyzed against pbs and stored at 4c and 20c , respectively , until use .
bacteria ( 3  10 ) were washed with pbs and then incubated with 40  l of normal human serum ( nhs ) , heat - inactivated serum ( his ) , hdl , apoa - i , or pbs for 10  min at 37c . after washing with pbs , the bacteria were mixed with lysis buffer ( 50  mm tris - hcl ph 8.0 , 150  mm nacl , 0.1% sds , and 0.5% sodium deoxycholate ) .
the lysates were then analyzed by electrophoresis on 12.5% sds - polyacrylamide gels , and the separated proteins were transferred onto polyvinylidene fluoride ( pvdf ) membranes ( millipore ) .
apoa - i was detected with a goat anti - apoa - i polyclonal antibody ( academy bio - medical company ) followed by a peroxidase- ( pod- ) conjugated rabbit anti - goat igg antibody ( medical & biological laboratories ) . finally , apoa - i was visualized using hydroperoxide and 3,3-diaminobenzidine as the substrate .
this analysis was carried out using a previously described method   with a slight modification .
lipids were extracted from the outer cell wall of m. avium by means of folch 's extraction procedure   and were then spotted onto a nitrocellulose membrane .
the membrane was sequentially incubated with 5% ( w / v ) skim milk and 50  g / ml hdl in 10  mm tris - hcl ( ph 8.0 ) containing 140  mm nacl and 0.1% tween 20 ( tbs - t ) for 1  h and 5  h , respectively , at room temperature .
the membrane was washed with tbs - t and then incubated with a pod - conjugated anti - apoa - i polyclonal antibody ( cosmo bio ) .
the lipids obtained from m. avium were spotted onto three silica gel 60 plates ( merck millipore ) .
the tlc plates were simultaneously developed with chcl3/ch3oh ( 95  :  5 , v / v ) .
after the run was completed , one of the plates was treated with 20% sulfuric acid solution to visualize the separated lipids , and the other 2 were subjected to tlc blot analysis according to a previously described method .
one membrane was incubated with 1% bovine serum albumin ( bsa ) in tbs - t and subsequently incubated with 83  g / ml of apoa - i in tbs - t at 4c overnight .
the membrane was washed with tbs - t and incubated with the pod - conjugated anti - apoa - i polyclonal antibody ( binding site ) .
apoa - i was visualized as described above . to analyze by matrix - assisted laser desorption ionization time - of - flight mass spectrometry ( maldi - tof ms ) ,
the lipid bound to apoa - i was extracted from the other membrane by means of chcl3/ch3oh ( 2  :  1 , v / v ) according to comparison with the position of the apoa - i spot on the membrane visualized by the tlc blotting described above .
live m. avium cells ( 3  10/ml ) were briefly sonicated to disperse clumps and mixed with the middlebrook 7h9 broth supplemented with oadc with or without hdl ( 50  g protein / ml ) .
absorbance of the medium at 530  nm was monitored for 144  h. this assay was carried out according to the previously described method [ 22 , 23 ] .
briefly , m. avium was autoclaved and stained with 0.5  mg / ml fluorescein isothiocyanate ( fitc , dojin laboratories ) in pbs for 30  min .
after exhaustive washing , we resuspended the stained m. avium at 3  10/ml in pbs and then stored the suspension at 80c until use .
thp-1 macrophages ( 10/well ) were cultured with the fitc - conjugated m. avium in the presence of hdl ( 50  g protein / ml ) , apoa - i ( 50  g / ml ) , or bsa ( 50  g / ml ) .
after quenching extracellular fluorescence using trypan blue ( 1.2  mg / dl ) , we fixed the cells with cellfix ( becton dickinson ) and subjected them to flow cytometric analysis on a navios flow cytometer ( beckman coulter ) .
the fitc - conjugated e. coli , which was prepared by the similar method described above , was also analyzed by the phagocytosis assay as the reference .
thp-1 macrophages ( 10/well ) were infected with live m. avium ( moi 20  :  1 ) with or without hdl ( 50  g protein / ml ) for 24  h. the cells engulfing m. avium were washed three times with pbs and further cultured in the absence of m. avium and hdl for 0 , 24 , and 48  h. then , thp-1 macrophages were lysed with pbs containing 0.1% sds to recover the engulfed m. avium .
the lysates diluted 100-fold with pbs were cultured on middlebrook 7h11 agar plates ( difco ) supplemented with oadc .
thp-1 macrophages ( 5  10/well ) were infected with live m. avium ( moi 20  :  1 ) with or without hdl ( 50  g protein / ml ) for 24  h. after being fixed with a 10% formaldehyde solution for 15  min , they are stained with oil red o ( oro ) for 5  min .
the cells were counterstained with mayer 's hematoxylin for 2  min in samples to be examined under a light microscope .
for quantification of lds amount , infected thp-1 macrophages ( 5  10/well ) were stained with only oro as described above .
oro was then extracted in 500  l of 100% isopropyl alcohol , and absorbance was measured at 540  nm as described previously .
all of the values were shown by the mean  standard error of the mean ( sem ) .
cfus , the number of lds , and the others were statistically analyzed by paired student 's t - test , kruskal - wallis test , and multifactorial analysis of variance ( anova ) followed by tukey post hoc tests , respectively .
the protocol of this study was approved by the research ethics committee of tokyo medical and dental university ( decision number 1491 ) .
to screen the bacteria for interaction with hdl , some species of bacteria that were incubated with normal human serum ( nhs ) were analyzed by western blotting with an anti - apoa - i antibody ( figure 1(a ) ) .
the apoa - i signal was well visible in the lysate of m. avium incubated with nhs but only barely visible in samples from other gram - negative bacteria . to confirm the direct interaction between apoa - i and m. avium
, we carried out western blotting of the lysate of m. avium incubated with heat - inactivated serum ( his ) , nhs , isolated hdl , or purified apoa - i ( figure 1(b ) ) .
the apoa - i signal was well visible after incubation not only with nhs but also with the his , hdl , and apoa - i , indicating that hdl bound directly to m. avium via apoa - i .
we hypothesized that apoa - i interacts with the abundant lipids on the m. avium cell wall because of amphipathic -helices present in the apoa - i molecule . to test this hypothesis
, we performed a western blot assay of the lipids extracted from the outer cell wall of m. avium ( figure 2(a ) ) .
apoa - i was detected in the spots of the extracted lipids incubated with hdl and the intensity of signals increased with the amount of spotted lipids ( at a constant hdl concentration ) .
the tlc blot assay was carried out to determine whether apoa - i binds to a specific lipid molecule of m. avium ( figure 2(b ) ) .
several lipid spots on the tlc plate were nonspecifically visualized by a charring reagent ( figure 2(b ) , left ) .
one of those spots visibly interacted with apoa - i by tlc blot analysis ( figure 2(b ) , right ) , indicating that apoa - i formed a complex with this specific lipid of m. avium .
the lipids extracted from the apoa - i - positive spot of the tlc blot were analyzed by maldi - tof ms ( supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4353620 ) .
two prominent peaks at m / z 1243.8 and m / z 1271.8 were observed along with the several peaks with an interval of 28  da , suggesting that the lipid that bound to apoa - i included a variety of fatty acid moieties .
the growth curve analysis of m. avium in the presence or absence of hdl was performed to determine whether hdl directly kills m. avium ( figure 3 ) .
no significant difference was observed in the absorbance of the medium with or without hdl for 6 days .
this finding indicated that hdl itself had no bactericidal activity toward m. avium . to find out whether hdl participates in the nonopsonic phagocytosis of m. avium by thp-1 macrophages
, we used flow cytometry to analyze the effect of hdl on the number of thp-1 macrophages engulfing fitc - conjugated m. avium ( figure 4(a ) ) .
the percentage of fitc - positive cells decreased with the addition of hdl and apoa - i but did not change significantly after addition of bsa .
the percentage of fitc - positive cells which is obtained using fitc - conjugated e. coli indicated a similar result to that of fitc - conjugated m. avium ; however the addition of hdl did not decrease the percentage of fitc - positive cells .
when the number of fitc - positive cells of control samples was set to 100% , hdl and apoa - i decreased the number of fitc - positive cells to 68.7%   1.5% and 83.3%   0.4% , respectively ( figure 4(b ) ) .
bsa did not affect the engulfment of m. avium by thp-1 macrophages ( 101.8%   2.0% ) .
in contrast , fitc - positive cells did not decrease , if anything increased , in the case of fitc - conjugated e. coli .
assay of cfus was performed to assess the number of live m. avium inside thp-1 macrophages by means of m. avium recovered from the lysed thp-1 macrophages ( figure 5(a ) ) .
the results that were expressed in cfu / well were ( 8.5  1.2 )  10 and ( 11.7  0.6 )  10 ( mean  se ) in the presence and the absence of hdl , respectively .
this observation is consistent with the result obtained by the flow cytometric analysis ( figure 4 ) , namely , that hdl attenuated the engulfment of m. avium by thp-1 macrophages . to assess the survival rate of m. avium inside thp-1 macrophages ,
the infected thp-1 macrophages were further cultured in the absence of m. avium and hdl for 24 and 48  h. the number of cfus per well indicated a tendency to decrease in a time - dependent manner ( figure 5(b ) ) .
however , no significant difference was observed in the decreasing profiles between the presence and the absence of hdl , suggesting that the coexistence of hdl could not affect bacterial killing after ingestion by thp-1 macrophage . after the infection of thp-1 macrophages with live m. avium ,
lds inside the cells were stained with oil red o ( oro ) ( figure 6(a ) ) .
the numbers of lds per cell were significantly increased by the coexistence of live m. avium and hdl ( figure 6(b ) ) .
lds amounts were also estimated by the absorbance at 540  nm of the extracted oro ( figure 6(c ) ) .
the existence of hdl or m. avium alone indicated no significant effect in the quantity of oro compared with the control .
however , the coexistence of hdl and m. avium caused an increase in the lds amount .
in this study , we found that hdl binds to m. avium via the interaction between apoa - i ( the main structural component of hdl ) and a specific lipid molecule of m. avium .
although a structure of the specific lipid molecule was analyzed by maldi - tof - ms / ms ( data not shown ) , we failed to identify it among the known lipid molecules of m. avium .
it should be noted that m. avium causes inflammation and increases permeability of blood vessels , increasing the chance of interaction with leaked hdl .
if this specific lipid of m. avium plays a role in host pathogenesis , hdl may neutralize the activity of this lipid , just as in the case of lps and lta
. it could be important to identify the lipid molecule in order to elucidate a participation of hdl in m. avium infection .
we explored the influence of hdl 's binding to m. avium on an innate immunity in experiments with thp-1 macrophages .
hdl does not directly kill m. avium , but the presence of hdl significantly decreases the number of thp-1 macrophages engulfing m. avium , and hdl indicated no effect on bacterial killing after ingestion .
sr - b1 , known as an apoa - i receptor , also plays a role in nonopsonic phagocytosis of mycobacteria [ 16 , 17 ] . according to these observations
, we can hypothesize that hdl competitively inhibits the binding of m. avium to sr - b1 .
in this study , we did not analyze sr - b1 , and further research is needed to assess the interaction between m. avium and hdl , including the participation of sr - b1 . when mycobacteria are recognized by toll - like receptors 2 or 6 ( tlr2/6 ) , lds formation is enhanced by tlr signaling [ 25 , 26 ] .
moreover , some researchers proposed a mechanism behind the enhancement of lds formation during mycobacterial infection : upregulation of a series of scavenger receptors including sr - b1 . in our study , m. avium infection did not apparently upregulate lds formation under serum - free condition ; however , the coexistence of hdl increased the number and the amount of lds in thp-1 macrophages , suggesting that hdl - cholesterol is utilized as a raw material for lds formation .
two mechanisms may explain this observation . according to one mechanism , hdl - cholesterol is internalized together with m. avium via phagocytosis . according to the other ,
although approximately 30% of the engulfment of m. avium by thp-1 macrophages was attenuated by the coexistence of hdl ( figure 4 ) , the hdl - cholesterol influx is enhanced by upregulation of sr - b1 in thp-1 macrophages engulfing m. avium .
consequently , tlr signaling and hdl - cholesterol influx via sr - b1 may promote lds formation in the presence of both m. avium and hdl .
on the basis of the present study , we believe that hdl plays a crucial role in the infection of thp-1 macrophages by m. avium .
identification of the lipid molecule of m. avium that binds to hdl via apoa - i and the analysis of the expression of the scavenger receptor , sr - b1 , in an upcoming project , should shed some light on the pathogenesis of m. avium infections and on the relevant immune response .
hdl affects m. avium infection through the binding between apoa - i , the main component of hdl , and a specific lipid of m. avium .
 high - density lipoprotein ( hdl ) is involved in innate immunity toward various infectious diseases . concerning bacteria , 
 hdl is known to bind to lipopolysaccharide ( lps ) and to neutralize its physiological activity . on the other hand 
 , cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment . 
 however , the pathogenicity of mycobacterium avium ( m. avium ) infection , which tends to increase worldwide , remains poorly studied . 
 here we report that hdl indicated a stronger interaction with m. avium than that with other gram - negative bacteria containing abundant lps . 
 a binding of apolipoprotein ( apo ) a - i , the main protein component of hdl , with a specific lipid of m. avium might participate in this interaction . hdl did not have a direct bactericidal activity toward m. avium but attenuated the engulfment of m. avium by thp-1 macrophages . 
 hdl also did not affect bacterial killing after ingestion of live m. avium by thp-1 macrophage . 
 furthermore , hdl strongly promoted the formation of lipid droplets in m. avium - infected thp-1 macrophages . 
 these observations provide new insights into the relationship between m. avium infection and host lipoproteins , especially hdl . 
 thus , hdl may help m. avium to escape from host innate immunity . 
1. Introduction 
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion