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Characterization of immune cells in psoriatic adipose tissue.

Rose S, Stansky E, Dagur PK, Samsel L, Weiner E, Jahanshad A, Doveikis J, Naik HB, Playford MP, McCoy JP, Mehta NN - J Transl Med (2014)

Bottom Line: Rarely have these populations been verified with confirmatory methodologies or functional studies.Further, both CD16+CD56(Lo) and CD16-CD56(Hi) NK cells were found to correlate inversely with body mass index.The relationship between the predominant CD16+CD56(Lo) NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Adipose tissue normally contains immune cells that regulate adipocyte function and contribute to metabolic disorders including obesity and diabetes mellitus. Psoriasis is associated with increased risk for metabolic disease, which may in part be due to adipose dysfunction, which has not been investigated in psoriasis. There is currently no standardized method for immunophenotyping human adipose tissue. In prior studies, characteristic phenotypic markers of immune cell populations identified in animal models or in other human tissues have been applied in a similar manner to human adipose tissue. Rarely have these populations been verified with confirmatory methodologies or functional studies. Thus, we performed a comprehensive phenotypic and functional analysis of immune cell populations in psoriatic adipose tissue.

Methods: Conventional and imaging flow cytometry were used to define immune cell populations in biopsy specimens of psoriatic adipose tissue (n = 30) including T cells, B cells, NK cells, NKT cells, neutrophils, and macrophages. Relationships between adipose immune cell types and body mass index were determined using Spearman regression analysis, and multivariate linear regression analysis was performed to adjust for cardiometabolic disease risk factors.

Results: These analyses revealed a wide range of cell surface receptors on adipose tissue macrophages, which may serve a dual purpose in immunity and metabolism. Further, both CD16+CD56(Lo) and CD16-CD56(Hi) NK cells were found to correlate inversely with body mass index. The relationship between the predominant CD16+CD56(Lo) NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use.

Conclusions: Together, these studies enhance our understanding of adipose immune cell phenotype and function, and demonstrate that examination of adipose tissue may provide greater insight into cardiometabolic pathophysiology in psoriasis.

No MeSH data available.


Related in: MedlinePlus

Flow cytometric analysis of surface receptor expression and phagocytic function in adipose tissue macrophages. (A) ATM populations were examined for expression of TLR2, TLR4, CD11c, CD274, ABCA1, CD163, SR-B1, LOX-1, MSR1, CD36, RAGE, and CX3CR1. CD14+HLADRII-CD206- (green histograms), CD14+HLADRII+CD206- (blue histograms), and CD14+HLADRII+CD206+ (red histograms) cells are presented compared to FMO control staining for each marker (orange histograms). (B) Mean fluorescence intensity values for each surface marker depicted in (A) were compared in CD14+HLADRII-CD206-, CD14+HLADRII+CD206-, and CD14+HLADRII+CD206+ ATM. Kruskall-Wallis testing with post-hoc Dunn’s multiple comparison testing was performed to determine whether surface macrophage marker expression was statistically different among the different subsets. p values < 0.05 were considered to be statistically significant. (C) The pHrodo system was utilized to demonstrate phagocytosis of opsonized bioparticles by enriched CD14+ monocytes (Monocytes, left panel, positive control), positively selected CD14+ ATM (Enriched ATM, center panel), and whole adipose tissue, gated on CD14+ ATM (Whole ATM, right panel) by flow cytometry. Green dots represent bioparticles incubated with antibody staining cocktail and no cells (acellular negative control). Red dots represent monocytes or ATM incubated with bioparticles at 4°C (cellular negative control). Blue dots represent monocytes or ATM incubated with bioparticles at 37°C. Enriched ATM and Whole ATM were further gated into 3 subpopulations based on HLADRII and CD206 expression. Phagocytosis of opsonized bioparticles is depicted for each subpopulation of Enriched ATM and Whole ATM and is reported as percentages of viable cells containing bioparticles.
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Fig4: Flow cytometric analysis of surface receptor expression and phagocytic function in adipose tissue macrophages. (A) ATM populations were examined for expression of TLR2, TLR4, CD11c, CD274, ABCA1, CD163, SR-B1, LOX-1, MSR1, CD36, RAGE, and CX3CR1. CD14+HLADRII-CD206- (green histograms), CD14+HLADRII+CD206- (blue histograms), and CD14+HLADRII+CD206+ (red histograms) cells are presented compared to FMO control staining for each marker (orange histograms). (B) Mean fluorescence intensity values for each surface marker depicted in (A) were compared in CD14+HLADRII-CD206-, CD14+HLADRII+CD206-, and CD14+HLADRII+CD206+ ATM. Kruskall-Wallis testing with post-hoc Dunn’s multiple comparison testing was performed to determine whether surface macrophage marker expression was statistically different among the different subsets. p values < 0.05 were considered to be statistically significant. (C) The pHrodo system was utilized to demonstrate phagocytosis of opsonized bioparticles by enriched CD14+ monocytes (Monocytes, left panel, positive control), positively selected CD14+ ATM (Enriched ATM, center panel), and whole adipose tissue, gated on CD14+ ATM (Whole ATM, right panel) by flow cytometry. Green dots represent bioparticles incubated with antibody staining cocktail and no cells (acellular negative control). Red dots represent monocytes or ATM incubated with bioparticles at 4°C (cellular negative control). Blue dots represent monocytes or ATM incubated with bioparticles at 37°C. Enriched ATM and Whole ATM were further gated into 3 subpopulations based on HLADRII and CD206 expression. Phagocytosis of opsonized bioparticles is depicted for each subpopulation of Enriched ATM and Whole ATM and is reported as percentages of viable cells containing bioparticles.

Mentions: Conventional flow cytometry demonstrated various immune cell subsets in psoriatic adipose tissue using classic phenotypic markers. The ability of these markers to correctly identify adipose immune cells was confirmed by imaging flow cytometry. This technology combines features of brightfield microscopy, flow cytometry, and immunofluorescence microscopy in a single instrument, thus allowing for high-resolution characterization of cell populations in tissues [35]. Imaging flow cytometry verified ATM by positive CD14 staining, abundant cytoplasm, and round to U-shaped nuclei consistent with macrophage morphology (Figure 3, panels A-C). Three subsets of ATM were present based on HLADRII and CD206 expression (Figure 3), and ATM rarely expressed CD16 (mean 1.47 ± 1.77%, Additional file 6: Figure S3). Conventional flow cytometry was then performed to uncover ATM markers that may link metabolism and immunity in psoriasis (Figure 4). ATM expressed a wide array of surface receptors with roles in microbial pattern recognition (TLR2, TLR4), immune suppression (CD274), cell adhesion (CD11c), cholesterol trafficking (ABCA1), chemokine binding (CX3CR1), and macromolecule clearance (CD163, SR-B1, LOX-1, MSR1, RAGE, and CD36) (Figure 4A). Mean fluorescence intensity (MFI) values for these receptors were strikingly different among the macrophage subpopulations (Figure 4B). TLR2, CD11c, MSR1, and HLADRII expression were highest in HLADRII+CD206+, intermediate in HLADRII+CD206-, and lowest in HLADRII-CD206- ATM (Figure 4B). LOX-1 expression was lowest in the HLADRII+CD206- population, CD36 expression was lowest in the HLADRII+CD206+ subset, and RAGE expression was highest in HLADRII-CD206-, intermediate in HLADRII+CD206-, and lowest in HLADRII+CD206+ cells (Figure 4B). ATM phagocytic function was also assessed by flow cytometry using the pHrodo system, which utilizes bioparticles that fluoresce only upon acidification within phagocyte endosomes [36]. Phagocytosis was equivalent among the 3 ATM subpopulations (Figure 4C).Figure 3


Characterization of immune cells in psoriatic adipose tissue.

Rose S, Stansky E, Dagur PK, Samsel L, Weiner E, Jahanshad A, Doveikis J, Naik HB, Playford MP, McCoy JP, Mehta NN - J Transl Med (2014)

Flow cytometric analysis of surface receptor expression and phagocytic function in adipose tissue macrophages. (A) ATM populations were examined for expression of TLR2, TLR4, CD11c, CD274, ABCA1, CD163, SR-B1, LOX-1, MSR1, CD36, RAGE, and CX3CR1. CD14+HLADRII-CD206- (green histograms), CD14+HLADRII+CD206- (blue histograms), and CD14+HLADRII+CD206+ (red histograms) cells are presented compared to FMO control staining for each marker (orange histograms). (B) Mean fluorescence intensity values for each surface marker depicted in (A) were compared in CD14+HLADRII-CD206-, CD14+HLADRII+CD206-, and CD14+HLADRII+CD206+ ATM. Kruskall-Wallis testing with post-hoc Dunn’s multiple comparison testing was performed to determine whether surface macrophage marker expression was statistically different among the different subsets. p values < 0.05 were considered to be statistically significant. (C) The pHrodo system was utilized to demonstrate phagocytosis of opsonized bioparticles by enriched CD14+ monocytes (Monocytes, left panel, positive control), positively selected CD14+ ATM (Enriched ATM, center panel), and whole adipose tissue, gated on CD14+ ATM (Whole ATM, right panel) by flow cytometry. Green dots represent bioparticles incubated with antibody staining cocktail and no cells (acellular negative control). Red dots represent monocytes or ATM incubated with bioparticles at 4°C (cellular negative control). Blue dots represent monocytes or ATM incubated with bioparticles at 37°C. Enriched ATM and Whole ATM were further gated into 3 subpopulations based on HLADRII and CD206 expression. Phagocytosis of opsonized bioparticles is depicted for each subpopulation of Enriched ATM and Whole ATM and is reported as percentages of viable cells containing bioparticles.
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Related In: Results  -  Collection

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Fig4: Flow cytometric analysis of surface receptor expression and phagocytic function in adipose tissue macrophages. (A) ATM populations were examined for expression of TLR2, TLR4, CD11c, CD274, ABCA1, CD163, SR-B1, LOX-1, MSR1, CD36, RAGE, and CX3CR1. CD14+HLADRII-CD206- (green histograms), CD14+HLADRII+CD206- (blue histograms), and CD14+HLADRII+CD206+ (red histograms) cells are presented compared to FMO control staining for each marker (orange histograms). (B) Mean fluorescence intensity values for each surface marker depicted in (A) were compared in CD14+HLADRII-CD206-, CD14+HLADRII+CD206-, and CD14+HLADRII+CD206+ ATM. Kruskall-Wallis testing with post-hoc Dunn’s multiple comparison testing was performed to determine whether surface macrophage marker expression was statistically different among the different subsets. p values < 0.05 were considered to be statistically significant. (C) The pHrodo system was utilized to demonstrate phagocytosis of opsonized bioparticles by enriched CD14+ monocytes (Monocytes, left panel, positive control), positively selected CD14+ ATM (Enriched ATM, center panel), and whole adipose tissue, gated on CD14+ ATM (Whole ATM, right panel) by flow cytometry. Green dots represent bioparticles incubated with antibody staining cocktail and no cells (acellular negative control). Red dots represent monocytes or ATM incubated with bioparticles at 4°C (cellular negative control). Blue dots represent monocytes or ATM incubated with bioparticles at 37°C. Enriched ATM and Whole ATM were further gated into 3 subpopulations based on HLADRII and CD206 expression. Phagocytosis of opsonized bioparticles is depicted for each subpopulation of Enriched ATM and Whole ATM and is reported as percentages of viable cells containing bioparticles.
Mentions: Conventional flow cytometry demonstrated various immune cell subsets in psoriatic adipose tissue using classic phenotypic markers. The ability of these markers to correctly identify adipose immune cells was confirmed by imaging flow cytometry. This technology combines features of brightfield microscopy, flow cytometry, and immunofluorescence microscopy in a single instrument, thus allowing for high-resolution characterization of cell populations in tissues [35]. Imaging flow cytometry verified ATM by positive CD14 staining, abundant cytoplasm, and round to U-shaped nuclei consistent with macrophage morphology (Figure 3, panels A-C). Three subsets of ATM were present based on HLADRII and CD206 expression (Figure 3), and ATM rarely expressed CD16 (mean 1.47 ± 1.77%, Additional file 6: Figure S3). Conventional flow cytometry was then performed to uncover ATM markers that may link metabolism and immunity in psoriasis (Figure 4). ATM expressed a wide array of surface receptors with roles in microbial pattern recognition (TLR2, TLR4), immune suppression (CD274), cell adhesion (CD11c), cholesterol trafficking (ABCA1), chemokine binding (CX3CR1), and macromolecule clearance (CD163, SR-B1, LOX-1, MSR1, RAGE, and CD36) (Figure 4A). Mean fluorescence intensity (MFI) values for these receptors were strikingly different among the macrophage subpopulations (Figure 4B). TLR2, CD11c, MSR1, and HLADRII expression were highest in HLADRII+CD206+, intermediate in HLADRII+CD206-, and lowest in HLADRII-CD206- ATM (Figure 4B). LOX-1 expression was lowest in the HLADRII+CD206- population, CD36 expression was lowest in the HLADRII+CD206+ subset, and RAGE expression was highest in HLADRII-CD206-, intermediate in HLADRII+CD206-, and lowest in HLADRII+CD206+ cells (Figure 4B). ATM phagocytic function was also assessed by flow cytometry using the pHrodo system, which utilizes bioparticles that fluoresce only upon acidification within phagocyte endosomes [36]. Phagocytosis was equivalent among the 3 ATM subpopulations (Figure 4C).Figure 3

Bottom Line: Rarely have these populations been verified with confirmatory methodologies or functional studies.Further, both CD16+CD56(Lo) and CD16-CD56(Hi) NK cells were found to correlate inversely with body mass index.The relationship between the predominant CD16+CD56(Lo) NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Adipose tissue normally contains immune cells that regulate adipocyte function and contribute to metabolic disorders including obesity and diabetes mellitus. Psoriasis is associated with increased risk for metabolic disease, which may in part be due to adipose dysfunction, which has not been investigated in psoriasis. There is currently no standardized method for immunophenotyping human adipose tissue. In prior studies, characteristic phenotypic markers of immune cell populations identified in animal models or in other human tissues have been applied in a similar manner to human adipose tissue. Rarely have these populations been verified with confirmatory methodologies or functional studies. Thus, we performed a comprehensive phenotypic and functional analysis of immune cell populations in psoriatic adipose tissue.

Methods: Conventional and imaging flow cytometry were used to define immune cell populations in biopsy specimens of psoriatic adipose tissue (n = 30) including T cells, B cells, NK cells, NKT cells, neutrophils, and macrophages. Relationships between adipose immune cell types and body mass index were determined using Spearman regression analysis, and multivariate linear regression analysis was performed to adjust for cardiometabolic disease risk factors.

Results: These analyses revealed a wide range of cell surface receptors on adipose tissue macrophages, which may serve a dual purpose in immunity and metabolism. Further, both CD16+CD56(Lo) and CD16-CD56(Hi) NK cells were found to correlate inversely with body mass index. The relationship between the predominant CD16+CD56(Lo) NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use.

Conclusions: Together, these studies enhance our understanding of adipose immune cell phenotype and function, and demonstrate that examination of adipose tissue may provide greater insight into cardiometabolic pathophysiology in psoriasis.

No MeSH data available.


Related in: MedlinePlus