Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content

Abstract

Objective The relationship between inflammation, obesity and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). We compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC) and RT-PCR of ATM markers. Methods We evaluated a new software program (AMCounter) to help measure ATM using IHC and compared this to flow cytometry and RT-PCR. Results IHC had good intra-individual reproducibility for total (CD68), pro-inflammatory (CD14) and anti-inflammatory (CD206) ATM. The AMCounter improved inter-reader agreement and was more time efficient. Flow cytometry had acceptable intra-individual reproducibility for the percent of CD68+ cells that were CD14+ or CD206+, but not for ATM/g tissue. ATM/g tissue was much greater using IHC than flow cytometry. The flow cytometry and IHC measures of ATM from the same biopsies were not correlated. There were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14 and CD206 ATM’s per 100 adipocytes. Of interest, were also statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14 and adipose flow cytometry measures of CD68+, CD68+/CD14+ and CD68+/CD206+ ATM’s per g tissue. Conclusions The AMCounter software helps reproducibly and efficiency measures of IHC ATM’s. Flow cytometry, immunohistochemistry and RT-PCR measures of adipose inflammation provide somewhat different information.

Preliminary evidence for reduced adipose tissue inflammation in vegetarians compared with omnivores

“…For the purpose of the current study we included the entire spectrum of vegetarian diets. We chose to study obese omnivores and BMI-matched vegetarians (one vegetarian had a BMI of 28 kg/m 2 ) because our database (derived from our methodology studies [16]) indicate that substantial increases in ATMs occur primarily with BMI’s above 30 kg/m 2 . It would be more difficult to detect a difference between omnivores and vegetarians if the omnivores already have a low ATM burden.…”

“…For immunohistochemistry (IHC), adipose tissue samples (~ 350-400 mg) were processed and the staining procedure was performed as previously described [16]. The adipose tissue sections were visualized by light microscopy using an Olympus BX43 microscope.…”

“…The normality of variables was determined using the Shapiro-Wilk test. IHC data are expressed as number of positive cells per 100 adipocytes, however, because adipocyte size may vary between adipose regions and individuals, we also report ATM relative to tissue mass (per gram tissue) as previously described [16]. Senescent cell data is presented per 100 nuclei.…”

Parallels in Immunometabolic Adipose Tissue Dysfunction with Ageing and Obesity

“…Each method has benefits and drawbacks, which are summarised in Table 1 . For example, it has been shown that immunohistochemistry yields reproducible results, but with flow cytometry, the recovery of macrophages after tissue digestion can be a possible source of variation ( 50 ). However, there is good agreement between flow cytometric analysis of adipose tissue cellular composition and the expression mRNA for key cell surface markers across a range of adiposities in humans (Figure 1 ) ( 35 ).…”

Specific macrophage subsets accumulate in human subcutaneous and omental fat depots during obesity

“…Although studies in mice fed HFD overwhelmingly described the ATM accumulation in the VAT of obese animals, 1,2,4,5,8,17 human data are scarcer and more inconsistent. 10,[31][32][33][34] These discrepancies may be explained by the use of different experimental protocols 35 and by the heterogeneity of the human populations evaluated. After dissecting the ATM subsets present in the obese VAT, we find that the net ATM count was the consequence of CLEC5A + ATM loss together with the increment in the numbers of a CD11c + CLEC5A À ATM subset.…”