the lysosome is a dynamic organelle that operates at an acidic ph and contains numerous enzymes that are critical for cellular degradation pathways .
uptake of extracellular material reaches the lysosome via the endocytic pathway , whereas intracellular cargo is delivered to the lysosome via autophagy .
the lysosome can also play a role in secretion , membrane repair , and cell clearance through the process of lysosomal exocytosis [ 2 , 3 ] .
recently , the importance of lysosomes in cell signaling pathways and nutrient sensing has also become apparent [ 4 , 5 ] .
importantly , the regulation of lysosome structure and function is cell type dependent and is regulated by environmental stimuli .
macrophages are cells of the innate immune system that are important for organ homeostasis , inflammation , host defense , and tissue repair . recently , there has been a renewed interest in macrophage lysosome biology .
the importance of macrophage autophagy in several clinically relevant diseases has helped to fuel this renaissance [ 710 ] . moreover
, it has also come to light that lysosomal pathways activate il-1 release via the inflammasome in several important human diseases including atherosclerosis , gout , and alzheimer 's disease [ 1113 ] . also , of relevance ,
lipid overload and obesity can also induce  lysosomal reprogramming  in adipose tissue macrophages , which may contribute to the metabolic complications of nutrient excess .
together , these and many other studies indicate that additional cellular and molecular studies of lysosome function in macrophages will be critical to understand the role of this organelle in inflammatory diseases
. ex vivo analysis of primary macrophages will be important for mechanistic cell biology experiments investigating lysosome function in phagocytic cells .
the most common sources of primary macrophages include bone marrow - derived macrophages ( bmdms ) and thioglycollate- ( tg- ) elicited peritoneal macrophages ( pmacs ) .
although bmdms and pmacs are derived from very distinct environments they are often used interchangeably to model macrophage biology .
pmacs are monocyte derived cells that are typically isolated from the peritoneal cavity 35 days after tg administration .
thus , pmacs are actively involved in the process of inflammation resolution , which includes the uptake of dead cells and/or debris through efferocytosis or phagocytosis , respectively . in contrast , bmdms are derived from a myeloid precursor cell functionally nave and would be expected to have fewer demands on their endosomal system .
based on this , we hypothesized that the lysosomes would be phenotypically and functionally distinct in these subtypes of primary macrophages . in the current study , we investigated lysosome content , morphology , and function in pmacs and bmdms .
our data demonstrate that pmacs have a larger lysosome volume , increased cathepsin activity , and enhanced expression of several lysosomal genes and proteins .
moreover , using the example of the lipotoxic inflammasome , we provide evidence that these differences in the lysosomal compartment can influence macrophage inflammatory responses .
together , our results argue that the interpretation of data involving lysosome - dependent processes in primary macrophages must take the source of the cells into consideration .
the cd107a ( lamp-1 ) pe conjugated antibody was from ebiosciences ( cat#12 - 1071 ) .
ultrapure - bovine serum albumin ( bsa ) was from lampire and was tested for tlr ligand contamination prior to use .
peritoneal macrophages ( pmacs ) were isolated from c57bl/6 mice 4 days after intraperitoneal injection of 3.85% thioglycollate and plated at a density of 0.9 - 1  10  cells / ml in dmem containing 10% inactivated fetal serum ( ifs ) , 50  u / ml penicillin g sodium , and 50  u / ml streptomycin sulfate ( pen - strep ) , 2  mm l - glutamine , and sodium pyruvate .
bone marrow - derived macrophages ( bmdms ) were prepared by harvesting bone marrow from the femurs and tibias of 812-week - old c57bl/6 mice .
the cells were seeded in 10  cm dishes and differentiated for 6 days in dmem media as above supplemented with 10% supernatant from cmg14.12 cells as a source of m - csf . on day 6 , bmdms were plated at 1  10  cells / ml in media containing 5% supernatant from cmg14.12 cells .
stimulations were performed on the day after harvest in media containing 5% cmg1.12 media . for flow cytometry experiments , macrophages were cultured on low adherence plates ( greiner bio - one ) to facilitate cell harvest .
cells were removed from the plate by washing with pbs followed by 10 minutes with cell stripper ( gibco ) and then 10 minutes with edta / trypsin ( sigma ) .
growth medium was supplemented with palmitate and bsa at a 2  :  1 molar ratio as described previously   and bsa - supplemented media were used as control . for cell stimulations , pbs or lps ( 50  ng / ml ) were added to bsa or free fatty acid containing media .
flow cytometry was performed on a bd biosciences facscalibur machine and the data was analyzed using flowjo software .
wild type ( wt ) c57bl/6 mice were obtained from oriental bioscience and maintained in our mouse colony .
the atg5flox x lysm - cre on the c57bl/6 background were kindly provided by skip virgin ( washington university ) .
mice were maintained in a pathogen - free facility on a standard chow diet ad libitum ( 6% fat ) .
all animal experiments were conducted in strict accordance with nih guidelines for humane treatment of animals and were reviewed by the animal studies committee of washington university school of medicine .
total cellular rna was isolated using qiagen rneasy columns and reversely transcribed using a high capacity cdna reverse transcription kit ( applied biosystems ) .
real - time qrt - pcr was performed using sybr green reagent ( applied biosystems ) on an abi 7500 fast thermocycler .
relative gene expression was determined using the delta - delta ct method normalized to 36b4 expression .
mouse primers sequences were as follows ( all 5-3 ) : 36b4 ( forward - atc cct gac gca ccg ccg tga , reverse - tgc atc tgc ttg gag ccc acg tt ) ; ctsb ( forward - gat caa gga cca cca cat cc , reverse - ctt agg agt gca cgg gag ag ) ; ctsd ( forward - gac agc tcc ccg tgg tag ta , reverse - caa cag aag ctg gtg gac aa ) ; ctsk ( forward - tgc cgt ggc gtt ata cat ac , reverse - cgg cta tat gac cac tgc ct ) ; lamp1 ( forward - tct tca gtg tgc agg tcc ag , reverse - atg agg acg atg agg accag ) ; lamp2a ( forward - cca aat tgg gat cct aac cta , reverse - tgg tca agc agt gtt tat taa ttc c ) ; lamp2b ( forward - ggt gct ggt ctt tca ggc ttg att , reverse - acc acc caa tct aag agc agg act ) : atp6v1h ( forward - aga cag cca gca aca cac tg , reverse - tca cag aaa ctt cgt ggc ag ) ; p62 ( forward - gct gcc cta tac cca cat ct , reverse - cgc ctt cat ccg aga aac ) ; lc3 ( forward - cgt cct gga caa gac caa gt , reverse - att gct gtc ccg aat gtctc ) .
total cellular protein was isolated by lysing cells in 150  mm  nacl , 10  mm  tris ( ph 8) , triton x-100 1% , and 1x protease complete .
proteins were separated on a tgx gradient gel ( 420% ; biorad ) and transferred to a nitrocellulose membrane .
western blotting for cathepsin d and actin was performed using 40  g of total cellular protein .
after the indicated stimulations , pmacs were removed from the plate as described above and fixed in 4% pfa for 15 minutes in the dark at room temperature .
after fixation , cells were permeabilized ( pbs + 0.1% triton x ) for 15 minutes in the dark and washed with facs buffer + 0.1%
triton x. cell were treated with fc receptor block ( bd pharmigen ) for 5 minutes on ice followed by incubation with cd107a ( lamp1 ) pe antibody for 30 minutes in the dark on ice .
after the indicated stimulations , cells were removed from the plate as described above and then stained with 500  nm lysotracker red in tissue culture media for 15 minutes at 37c . after staining ,
cells were washed three times with pbs , harvested as described above , and analyzed by flow cytometry .
for fluorescent dextran experiments , macrophages were incubated with 500  g / ml tetramethylrhodamine- ( tmr- ) dextran for 2  h in regular media .
lysotracker red or tmr - dextran stained cells were fixed for 10 minutes in 4% paraformaldehyde followed by nuclear staining with hoechst 33342 dye .
the cover slips were mounted on glass slides and imaged using a zeiss confocal microscope .
after the indicated stimulations , pmacs were removed from low adherence plates as described above .
the fluorescently labeled cathepsin b substrate was reconstituted per manufacturer 's instructions and diluted in dmem+10% ifs .
macrophages were incubated in 150  l of staining solution for 45 minutes at 37c under 5% co2 with gentle mixing every 10 minutes . after the incubation
, cells were washed 2 times in 1  ml of pbs and analyzed by flow cytometry ( fl2 ) .
il-1 was quantified using a duoset elisa kit ( r&d systems ) according to the manufacturer 's instructions .
groups were compared by paired student 's t - test . a value of p  0.05 was considered significant .
to investigate differences in the lysosome compartment of pmacs compared to bmdms , we quantified lysosome volume in these two sets of primary macrophages using the lysosomotropic dye lysotracker red coupled with flow cytometric analysis . using this approach ,
lysosome content was significantly increased in pmacs relative to bmdms ( figures 1(a ) and 1(b ) ) . in line with this finding ,
pmacs also had significantly higher levels of the lysosomal membrane protein lamp1 compared to bmdm ( figures 1(c ) and 1(d ) ) .
to assess lysosomal protease function , we analyzed the activity of cathepsin b using the fluorogenic substrate magic red .
as shown in figures 1(e ) and 1(f ) , cathepsin b activity was increased in pmacs when compared to bmdms .
lysosomes have several distinct morphologic appearances that can vary based on cell type and activation status including spheroid , ovoid , or tubular .
in addition , the size of lysosomes can vary dramatically . to visualize lysosomes in pmacs and bmdms , we utilized two complementary immunofluorescent approaches : ( 1 ) lysotracker red staining and ( 2 ) tetramethylrhodamine- ( tmr- ) dextran staining .
lysotracker red freely crosses cell membranes and is concentrated in acidic organelles whereas tmr - dextran is taken up via endocytosis and reaches lysosomes in ~30  min .
in addition , the morphology was more frequently ovoid and tubular in pmacs and spherical in bmdms ( figures 2(a)2(d ) ) .
notably , pmac lysosome morphology is similar to that described from adipose tissue macrophages during high fat feeding .
stimulation of bmdms or pmacs with lps did not lead to a significant change in lysosome morphology ( data not shown ) .
emerging data indicates that lysosome biogenesis and function are regulated in part at the transcriptional level .
therefore , we compared the expression of several lysosome and autophagy related genes in bmdms and pmacs .
consistent with the flow cytometry and imaging data , the expression of genes encoding cathepsin proteases , lysosomal membrane proteins , and the vacuolar atpase was increased in pmacs relative to bmdms ( figure 3(a ) ) .
in addition , the autophagy related genes lc3 and p62 were also expressed at a higher level in pmacs ( figure 3(a ) ) .
thus , mrna expression of lysosome and autophagy related genes is increased in pmacs , which is likely related to the expanded lysosome compartment observed in these primary macrophages . at the protein level ,
pro - cathepsin d protein content was increased in pmacs relative to bmdms ( figures 3(b ) and 3(c ) ) .
similar to the flow cytometry data , lamp1 protein content trended towards an increase in pmacs compared to bmdms ( figures 3(d ) and 3(e ) ) .
interestingly , the molecular weight of lamp1 was significantly greater in bmdms suggesting a higher level of glycosylation , a finding which could further affect lysosome function and stability in these two types of primary macrophages ( figure 3(d ) ) . to investigate whether the observed differences in lysosome phenotype modulate macrophage inflammatory responses , we explored the lipotoxic inflammasome as a model system .
we , and others , have shown that the combination of lps and the saturated fatty acid ( sfa ) palmitate triggers the release of il-1 through an nlrp3 and caspase 1-dependent mechanism [ 2022 ] . in prior studies using this system
, it has been shown that il-1 release from pmacs is dependent on lysosome damage and cathepsin proteases , whereas the role of lysosomes in bmdms is less clear . to directly compare pmacs and bmdms using this system , we simultaneously stimulated both cell types with lps in the presence of increasing concentrations of palmitate and the release of il-1 was quantified .
both pmacs and bmdms secreted il-1 in a dose dependent fashion ; however , pmacs consistently secreted 510 times more cytokines than bmdms despite using a similar number of cells ( figures 4(a ) and 4(b ) ) .
importantly , fa uptake was similar between the pmacs and bmdms ( data not shown ) .
consistent with prior studies , il-1 release from pmacs was almost completely prevented by the cathepsin b inhibitor , cao74 , or lysosomal acidification inhibitor , bafilomycin ( baf ) , in palmitate - lps treated pmacs ( figure 4(c ) ) .
this was not the case in bmdms where il-1 release was unaffected by cao74 and actually increased with baf ( figure 4(d ) ) .
thus , lysosome inhibitors differentially influence lipotoxic inflammasome activation in pmacs compared to bmdms , an observation that may account for some of the discrepancies between prior publications [ 2022 ] .
more importantly , these findings argue that the baseline differences in lysosome content and function between pmacs and bmdms can alter the response of these cells to inflammatory stimuli .
lysosomes are also important for the degradation of autophagic cargo , which can also modulate the inflammasome response .
therefore , using atg5 ko cells , we tested the impact of autophagy deficiency on activation of the lipotoxic inflammasome in pmacs versus bmdms .
as can be seen in figures 4(e ) and 4(f ) , the phenotypes observed using autophagy deficient cells were different in these primary macrophage systems . in pmacs , atg5 ko cells had a modest increase in il-1 release after lps treatment ( figure 4(e ) inset ) , but il-1 secretion following palmitate - lps stimulation was reduced compared to wt cells ( figure 4(e ) ) . in distinction ,
atg5 ko bmdms released more il-1 in response to both lps and palmitate - lps treatment ( figure 4(f ) ) .
this was not related to differences in atg5 protein expression , which was similar between the two types of macrophages ( figure 4(g ) ) . instead
, the development of lysosome dysfunction in pmacs treated with palmitate - lps may explain why genetic autophagy deficiency does not further enhance inflammasome activation .
in contrast , atg5 ko bmdms phenocopy cells treated with baf , which also inhibits autophagic flux .
these findings argue that genetic or pharmacologic inhibition of autophagy in bmdms unmasks an important suppressive role for this degradative process on lipotoxic inflammasome activation .
further investigation will be necessary to evaluate the mechanisms that account for these differential phenotypes .
interestingly , recent evidence suggests that autophagy can increase lysosome activity , which could be particularly important for bmdms as they have less active lysosomes at baseline [ 25 , 26 ] .
taken together , this data suggests that pmacs and bmdms respond to lipotoxic stimulation through fundamentally different mechanisms , and at least part of these differences is related to distinct lysosome phenotypes .
the findings presented in this paper illustrate several significant differences between lysosomes in pmacs and bmdms . specifically ,
the  activated  lysosome compartment in pmacs may reflect the signals they receive in the inflammatory setting of peritonitis .
in contrast , bmdms are nave macrophages that have not encountered  activation  signals . using the example of the lipotoxic inflammasome
, we also provide evidence that the distinct lysosome phenotypes observed in bmdms and pmacs can influence macrophage inflammatory function . thus , when conducting experiments of lysosome function with primary macrophages , it is critical to account for baseline lysosome phenotype to ensure appropriate interpretation of data .
we would argue that the choice of bmdm versus pmac for experimentation should be tailored to the specific research question , and a low threshold should exist for comparing multiple macrophage populations .
in addition , further comparative analysis of lysosomes between pmacs , bmdms , and other in vivo isolated macrophages will be necessary to shed additional light on the dynamic regulation of lysosomes in these important leukocytes .
 lysosomes play a critical role in the degradation of both extracellular and intracellular material . 
 these dynamic organelles also contribute to nutrient sensing and cell signaling pathways . 
 macrophages represent a heterogeneous group of phagocytic cells that contribute to tissue homeostasis and inflammation . 
 recently , there has been a renewed interest in understanding the role of macrophage autophagy and lysosome function in health and disease . 
 thioglycollate - elicited peritoneal and bone marrow - derived macrophages are commonly used ex vivo systems to study primary macrophage function . in this study 
 , we reveal dramatic baseline differences in the lysosome morphology and function between these macrophage populations and provide evidence that these differences can be functionally relevant . 
 our results provide important insights into the diversity of lysosomes in primary macrophages and illustrate the importance of accounting for this in data interpretation . 