type 2 diabetes ( t2d ) is characterized by hyperglycemia and dyslipidemia resulting from a combination of insulin resistance in the skeletal muscle , liver , and adipose tissues , and impaired insulin secretion from the pancreatic islets , which is putatively due to impaired immune response .
patients with t2d exhibit increased susceptibility to infection , impaired wound healing , and other inflammatory or degenerative manifestations .
it is the macrophage system dysfunction that leads to the major alterations among these innate immune abnormalities [ 3 , 4 ] .
macrophage system including circulating monocytes , tissue macrophages , and dendritic cells is characterized by a high level of plasticity and widespread tissue distribution .
it was observed that phagocytes in type 2 diabetic patients were in a heightened state of oxidative stress accompanied by impaired bactericidal activity and chemotaxis [ 57 ] .
furthermore , the receptor functions , levels of cytokines expression , and release of the macrophages derived from the peripheral blood of t2d patients all changed dramatically [ 810 ] .
monocytes derived from bone marrow remain in the blood circulation system for 1 - 2 days before they migrate into the peripheral tissues , where they differentiate into fully mature resident macrophages . in innate immunity ,
resident macrophage population is composed of various cells , including macrophages in the peritoneal cavity , which is thought fully developed and adaptable to their local microenvironment with a wide diversity in terms of their function and phenotype [ 12 , 13 ] .
the evidence that macrophages could display different activation states with distinct properties provided us with clues to understand diverse functions of macrophages .
two polarized states in activated macrophages have been defined , the pro - inflammatory , classically activated state m1 induced by type i cytokines ( e.g. , tnf ) or bacterial products ( e.g. , lps ) and the anti - inflammatory , alternatively activated phenotype m2 induced by il-4 , il-10 , or glucocorticoid hormones [ 15 , 16 ] .
these different phenotypes of macrophages were expressed in such a manner that macrophages display a balanced , integrated pattern of functions [ 14 , 17 ] .
most of the current understanding of macrophage biology came from analyses of cells drawn from the mouse peritoneal cavity , which became widely used as a macrophage source as early as the 1960s . a recent study reported marked difference between resident and induced macrophages , such as the efficacy of killing pathogenic microorganisms and antigen presentation .
furthermore , studies about alteration of macrophage characteristics in diabetes were performed in streptozotocin- ( stz- ) induced type 1 diabetic mouse model , which was accompanied by immunological dysfunction .
it is hard to determine whether the dysfunctions of macrophages in these diabetes hosts were solely caused by diabetes itself or just by the host immunological dysfunction . to the best of our knowledge ,
little is known about the polarized states of rpms . to provide evidence showing the effects of dm with long duration on cell numbers , morphology , and innate immunity functions of macrophages in vivo
, we used resident peritoneal macrophages ( rpms ) from 5-month - old db / db mice obtained by abdominal cavity lavage to study the changes .
the result may help us to understand the susceptibility of diabetic patients to infectious diseases and other complications in the late stage of diabetes .
  ethylenediaminetetraacetic acid ( edta ) , n - formyl - l - methionyl - l - leucyl - l - phenylalanine ( fmlp ) , paraformaldehyde , modified wright - giemsa stain solution , and fluorescent red , carboxylate - modified polystyrenelatex beads were purchased from sigma - aldrich ( st .
rpmi-1640 , fetal bovine serum ( fbs ) , penicillin / streptomycin , and phosphate buffered saline ( pbs ) were obtained from gibco brl ( grant island , ny , usa ) .
bovine serum albumin ( bsa ) was ordered in bd pharmingen ( san diego , ca , usa ) .
eight - to - twelve - week - old male db / db ( c57bl/6j - lepr / lepr ) mice and littermate controls ( c57bl/6j background ) were fed chow ( labdiet 5001 ) and bought from the model animal research center of nanjing university ( nanjing , china ) .
all mice were kept in a specific pathogen - free facility and given free access to food and water .
the experiment was performed strictly in accordance with the institutional guidelines for the care and use of laboratory animals of shanghai jiao tong university .
approximately 6  ml cold serum - free rpmi-1640 medium was injected intraperitoneally after mice were killed under ether anesthesia . after a gentle massage ,
peritoneal fluid containing cells was harvested and centrifuged at 700  g for 5  min at 4c .
the pellet was resuspended with 5  ml of rpmi-1640 containing 10% fcs , 100  units / ml penicillin , and 100  g / ml streptomycin and cultured at 37c in a 35  mm dish ( corning , ny , usa ) for one hour , and only attached macrophages were used for the following experiments .
adherent cells were collected with 5  mm edta ( sigma - aldrich ) in ice - cold pbs ( gibco brl ) .
the cell viability was more than 90% , and more than 90% of adherent cells were f4/80 macrophages , as reported previously [ 22 , 23 ] .
rpms from db / db or control mice ( 1  10 ) were cultured in 24-well plates containing 12  mm sterilized glass slides per well for 2  h. for microscopic studies , the adherent cells were stained by modified wright - giemsa stain ( sigma - aldrich ) according to manufacturer 's protocol .
the glass slides were rinsed briefly in running deionized water ( ph7.2 ) and dried in air thoroughly before capturing images by a microscope ( olympus bx51 , olympus optical , tokyo , japan ) .
rpms from db / db or c57bl/6j mice were collected then cultured on 24-well plates containing 12  mm glass slides at a concentration of 2  10 macrophages per well for 2  h. the adherent macrophages were fixed in 4% paraformaldehyde ( sigma - aldrich ) for 20  min then incubated in pbs with 0.5% bsa at 4c for 30  min in order to avoid nonspecific binding to fcr .
primary antibodies against f4/80 ( emr1 : r&d systems , usa ) and cy3-labeled second antibodies ( beyotime institute of biotechnology , shanghai , china ) were sequentially incubated with macrophages for overnight at 4c and 2  h at room temperature , respectively .
after dapi ( invitrogen , san diego , ca , usa ) staining , cells were analyzed under laser confocal scanning microscopy ( lsm510 , zeiss , germany ) .
at least 5  10 rpms were incubated with 2.4  g2 ( bd biosciences ) for 10  min at room temperature then with primary antibodies or the matching control isotypes for 30  min at 4c . thereafter
, the cells were rinsed twice and resuspended in pharmingen stain buffer ( bd biosciences ) for assay by facscalibur flow cytometer ( becton dickinson , franklin lakes , nj ) .
the data were analyzed with cell quest software ( becton dickinson , mountain , ca , usa ) . for three - color staining ,
rpms were stained with phycoerythrin - cy5 ( pe - cy5- ) labeled anti - f4/80 monoclonal antibodies ( mabs ) ( ebiosciences , san diego , ca , usa ) , phycoerythrin ( pe- ) labeled anti - cd40 ( ebiosciences ) mabs , or fluorescein isothiocyanate ( fitc- ) labeled anti - cd206 mabs ( biolegend , san diego , ca , usa ) for 30  min at 4c .
rpms ( 4  10 ) were seeded in a 6-well plate then incubated with fluorescent red , carboxylate - modified polystyrenelatex beads ( 8  10 beads ) for 2  h at 37c , while the control were incubated at 4c .
the cells were washed with pbs before harvest and then resuspended in pbs containing 5% bsa .
the phagocytic activity of rpms was determined by measuring phagocytic rate and mean fluorescence intensity ( mfi ) with facscalibur flow cytometer ( becton and dickinson ) .
the adherence capacity of rpms was evaluated by the adherence index ( ai ) , capacity of adherent to plastic surfaces .
briefly , 500  l rpmi-1640 complete medium containing 7.5  10 rpms was placed in each eppendorf tube , incubated at 37c with 5% co2 in a shaking bath for 10 , 30 , or 60  min , respectively .
ai was calculated according to the equation : ai = 100  ( number of macrophages ( ml  supernatant)/number of macrophages ( ml  original  sample))100 .
chemotaxis experiment was carried out in 6.5  mm polycarbonate membrane inserts ( costar , cambridge , ma , usa ) with a 5  m pore size .
briefly , 100  l rpms suspension ( 1.3  10/well ) was deposited in the insert and 600  l rpmi-1640 containing fmlp ( sigma - aldrich ) ( 0.1  mmol ) was added into the lower compartment to induce chemotaxis for 2  h .
after the nonmigrated cells were removed from the polycarbonate membrane upper side , the migrated cells which were on the lower side were fixed and stained by crystal violet staining solution ( beyotime institute of biotechnology ) . the chemotactic index ( ci )
is presented as the total number of migrated cells of 16 random selected microscopic fields ( at 400 magnification ) .
student 's unpaired t - test for comparison of means was used to compare means of each group .
to determine the long effect of t2d on cell numbers and cellular morphology of rpms in db / db mice , we identified resident macrophages by detecting the surface antigens f4/80 firstly .
the purity of mouse f4/80 rpms was routinely determined by fcm before each assay , which was more than 90% ( figure 1(a ) ) . as shown in figure 1(b ) , the total cell numbers of f4/80 rpms in each db / db mouse were far greater than that of control ( p < 0.01 , n = 6 ) . however , after the data were standardized with body weight , the result of rpm numbers reversed ( figure 1(c ) ) .
the morphology of rpms was determined by wright - giemsa stain and a two - photon laser scanning . as shown in figures 1(e ) and 1(g ) ,
freshly isolated f4/80 rpms from db / db mice were nearly round shape , while that from control mice displayed spindle - shaped or irregular morphology ( figures 1(d ) and 1(f ) ) .
thus , a significant alteration of cell numbers and morphology of f4/80 rpms occurred in the mice with a long duration of dm .
the nonspecific phagocytic capacity of f4/80 rpms against fluorescence - modified polystyrenelatex beads was assessed by using fcm and shown in figure 2 . as compared with mice in the control group ( figure 2(b ) ) , cell - related fluorescence intensity corresponding to the peak of the histograms moved to the left in db / db mice , which indicated that fewer fluorescent beads were ingested by macrophages ( figure 2(d ) ) .
both the phagocytic rate ( figure 2(e ) , p < 0.001 , n = 4 ) and mfi of rpms from db / db mice were much lower than those of control group ( figure 2(f ) , p < 0.001 , n = 4 ) . to further determine other functional changes like adhesion and chemotaxis in macrophage from mice with a long duration of diabetes , we investigated the chemotaxis and adhering capacity of rpms .
it was showed in figures 3(b ) and 3(d ) that rpms from control or db / db mice migrated to the lower side of the filter after exposure to 0.1  mmol fmlp for 2  h by crystal violet staining .
the values of ci of rpms in diabetic mice were lower than those of control without significant difference ( figure 3(e ) , p > 0.05 , n = 4 ) .
the adhering capacity of rpms was assayed at selected time points ( 10  min , 30  min , and 60  min ) , respectively , after macrophages were placed in each eppendorf tube for a shaking bath as described in section 2 . as shown in figure 3(f ) , the values of ai of rpms from db / db mice were all significantly lower than those of the controls ( p < 0.01 , n = 4 ) . the expression of cd40 on macrophages after activation is critically related to the efficient promotion of adaptive immunity by macrophages .
thus , we detected cd40 and cd206 expressions on rpms from db / db or control mice by fcm to study the polarized state as described in section 2 .
as shown in figure 4(c ) , the percentage of m2 ( f4/80/cd40/cd206 ) macrophages ( p <
0.01 , n = 4 ) of rpms in db / db mice was significantly higher than that of control mice , and the percentage of m1 ( f4/80/cd40/cd206 ) macrophages of rpms in db / db mice reduced accordingly .
in the present study , we explored the pathogenesis of diabetes mellitus ( dm ) with the classical type 2 diabetic mouse model .
it was demonstrated in our study that long duration of t2d significantly altered cell numbers , morphology , phenotypes , and functions of resident peritoneal macrophages .
the severe complications of diabetes including impaired wound healing and accelerated atherosclerosis may increase patient 's susceptibility to infection and make the infected tissue difficult to be repaired . it was well accepted that the dysfunction of the macrophage system may contribute to these complications .
macrophages play important roles in innate immunity , which help host to defense microbial invaders through producing various cytokines and phagocytosis .   in our study , it was found that the cell numbers of f4/80 rpms in each 5-month - old db / db mouse increased obviously , which was contradicted with a previous study performed in stz - induced type 1 diabetic ( t1d ) mice without standardization of mouse 's body weight .
ma et al . presumed that the reduction of f4/80 rpms in t1d mice was probably related with shortened life span of macrophages and/or the blocking differentiation of macrophages from monocytes or bone marrow cells ( bmcs ) .
another preliminary study in vitro also showed that only a low level of macrophages was generated in nod bmcs after being stimulated with gm - csf .
interestingly , when the number of f4/80 rpms with mouse 's body weight was standardized , the result was reversed .
as db / db mice are larger while stz - induced t1d mice are much smaller than control , the number of f4/80 rpms from each mouse may be positively correlated with animal body weight .
nevertheless , as it is well accepted that diabetes and obesity lead to a chronic low - grade inflammation , diabetes - associated inflammation is correlated with elevated macrophage cell numbers .
previous report about the percentage of bone marrow - derived cells expressing the macrophage marker f4/80 ( f4/80 ) in perigonadal , perirenal , mesenteric , and subcutaneous adipose tissue by immunohistochemical analysis revealed that it was significantly and positively correlated with both adipocyte size and body mass .
however , the specific mechanism of different macrophage distribution between adipose tissue and abdominal cavity is still unclear . in the present study
, we demonstrated for the first time that the number of rpms from each mouse was positively correlated with animal body weight .
furthermore , we were the first to find that the rpms from db / db mice were nearly round in shape as compared with those of control mice with spindle - shaped or irregular morphology .
it is not surprising that macrophage heterogeneity presents in different organs or tissues , as well as even in a single organ .
recent studies showed that macrophage phenotypic and functional heterogeneity might be related to a wide diversity of factors , including different precursor cells , different differentiation stages , different microenvironments , or even different stimulatory factors .
the response of f4/80 peritoneal macrophages from stz - induced diabetic mouse at 4 months and control mouse to ifn- and lps was also different , which demonstrated that macrophages from diabetic mice produced much less tnf- and il-6 than those of control mice through akt - mtor and erk pathways .
hence , this morphologic heterogeneity in rpms between db / db and control mice may hint at the discrepancy of cell physiological functions ; the potential mechanism might be related with the alteration of microenvironments , levels of blood glucose , dyslipidemia , and advanced glycation end products .
macrophages can engulf foreign pathogens , microorganisms and dead cells , which are essential steps of cellular immunity and a part of innate immunity .
it was found that phagocytic capacity of f4/80 rpms from 5-month - old diabetic mice was lower than that of control with method of detecting the amount of phagocytosis fluorescence - modified polystyrenelatex beads by fcm .
the decreased nonspecific phagocytosis ability of macrophages in diabetic hosts was consistent with several reports about the diabetic host with increased susceptibility to microorganism infection [ 33 , 34 ] .
furthermore , remarkably impaired adhesion ability of rpms was also observed in db / db mice .
attenuated adhesion ability in rpms from db / db mice may also be responsible for the dysfunction immunity though this test could not completely mimic the process of macrophages adhesion to endothelial cells in vivo as a first step in the inflammation reaction . however , the mechanismsof macrophages from diabetic hosts with inhibited adhesion capacity need further study .
lastly , the chemotaxis vitality of rpms from db / db mice was also found to be impaired although no significant difference between the two groups was identified . because the strong relationship between the adipose tissue macrophage content and indicators of adiposity in diabetic mouse provides a mechanism for the increased adipose tissue production of proinflammatory molecules and acute phase proteins , such as tnf- , il-6 , and mcp-1 . and increased adipocyte volume and number in diabetic mice are also positively correlated with leptin production , while negatively correlated with production of adiponectin [ 36 , 37 ] . as it is well accepted that huge array of environmental factors ( including cytokines , chemokines , pattern recognition receptors , and hormones ) differentially regulates macrophage response , in our study the decreased nonspecific phagocytosis , adhesion , and chemotaxis ability of rpms in diabetic hosts may be related to the abnormal microenvironment in db / db mouse . on the other hand ,
these findings support the possibility that interrupted wound healing , and complications associated with inflammatory in dm patients , at least in part , is due to the dysfunction of macrophages in phagocytosis , adhesion , and chemotaxis . during enhanced recruitment in response to disease states ,
inflammatory monocytes are recruited in response to cytokine cues and undergo differentiation into two broad but distinct subsets of macrophages that are categorized as either classically activated ( m1 ) or alternatively activated ( m2 ) .
the expression of costimulatory molecules such as cd80 , cd86 , cd40 , and cd54 on macrophages after activation is critically related to the efficient promotion of adaptive immunity by macrophages .
cd206 expression is a specific marker for il-4-induced m2 macrophages , which was used by c. sun as the m2 marker in a flow cytometry analysis .
hence , we selected the markers cd40 and cd206 as m1 and m2 macrophages , respectively , for our study .
our result showed the proportion of anti - inflammatory m2-type rpms was doubled in db / db group compared with the control group .
this result was similar with another study in vitro , which showed that markers on peritoneal macrophages isolated from stz - induced diabetes mice at 4 months after activation were significantly lower compared to control mice as determined by the percentage of positive cells and mfi of cd86 and cd54 expression ; whereas arginase activity , another important marker for alternatively activated macrophages , was significantly higher in peritoneal macrophages isolated from stz - induced diabetes mice than those of control mice when stimulated with il-4 .
macrophages not only get involved in the etiology of dm and its chronic complications but also defense pathogens , and their proper activated state determines the result of the host response to pathogen aggression .
the m1 program of macrophages is usually associated with protection during acute infectious diseases and thought to damage neighboring tissues ; m2 macrophages having immunosuppressive and tissue - repairing functions play critical roles in the resolution of harmful inflammation by producing anti - inflammatory mediators [ 40 , 41 ] .
therefore , an increased proportion of m2 rpms in the db / db mice may be related with the chronic low - grade inflammation in abdominal cavity .
however , db / db mouse is a genetically deficient mouse , and il-4 signaling in db / db macrophages was alleviated compared with db/ or macrophages from control mice , which indicated that the impaired il-4 pathway in macrophages from the db / db mouse model of t2d may induce a state of il-4 resistance and finally disrupt il-4-dependent m2 phenotype macrophage yield .
our study clearly showed that an increased percentage of m2 type rpms accompanied with remarkable innate immunity dysfunction occurred in type 2 diabetic mice in vivo with a long duration .
this study may help us to understand the susceptibility of diabetic patients to infectious diseases and other complications in the patients of diabetes .
however , further investigation is needed to clarify the mechanisms involved and possible methods to ameliorate this alteration .
 type 2 diabetes ( t2d ) is associated with perturbed innate immunity . 
 macrophages , bridging innate immunity and metabolic disturbances , play important roles in controlling immune homeostasis . however , the effect of long - term diabetic milieu ( dm ) on the functions and phenotypes of macrophages is still not clear . in this study , we used resident peritoneal macrophages ( rpms ) from 5-month - old db / db mice to investigate the changes of macrophages . 
 it was found that rpms in db / db mice significantly reduced phagocytosis and adhesion capacity . 
 after standardization with body weight , the number of f4/80 +  rpms markedly reduced in db / db mice , and , furthermore , the macrophages skewed to m2-polarizated macrophages . 
 the results of morphology found that the rpms shape of db / db mice was nearly round , but the rpms shape of control mice was spindle - shaped and irregular . in this study , we found the cell numbers , morphology , and innate immunity functions of rpms in 5-month - old type 2 diabetic mice ( db / db mice ) obtained by abdominal cavity lavage were significantly altered . 
 importantly , we also found the remarkably increased m2-rpms in diabetic mice for the first time . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion