Increased Adipose Tissue Expression of Interferon Regulatory Factor (IRF)-5 in Obesity: Association with Metabolic Inflammation

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  Figure 1. Enhanced adipose interferon regulatory factor-5 (IRF5) expression in obesity correlates with body mass index (BMI), body fat percentage, age, and levels of glycated hemoglobin (HbA1c). Adipose tissue IRF5 gene expression was determined in 53 non-diabetic individuals grouped as six lean, 18 overweight, and 29 obese using real-time RT-PCR. IRF5 protein expression was determined in six lean, eight overweight, and eight obese individuals using immunohistochemistry (IHC) as described in Materials and Methods. The data (mean ± SEM) show that (A) IRF5 adipose gene expression was elevated in overweight (P = 0.05) and obese (P = 0.008) compared with lean individuals. (B) As expected, IRF5 protein expression (arbitrary units) was also higher in obese (P = 0.004) compared with lean subjects; however, the difference between overweight and lean individuals could not attain statistical significance (P = 0.18). Furthermore, IRF5 adipose gene expression associated positively with clinical markers including (C) BMI (r = 0.37, P = 0.008), (D) body fat percentage, age (r = 0.51, P = 0.0004), and (E) HbA1c (r = 0.41, P = 0.02). (F) Adipose IRF gene and protein expression was found to be mutually…

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  Figure 2. Increased interferon regulatory factor-5 (IRF5) protein expression in the adipose tissue. Adipose IRF5 protein expression was determined by immunohistochemistry (IHC) in six lean, eight overweight, and eight obese individuals and by confocal microscopy (CM), five individuals each, as described in Materials and Methods. The representative images obtained from five independent determinations with similar results show elevated adipose IRF5 protein expression in overweight and obese individuals compared with lean: (A) Red arrows in 100× magnification IHC images and (B) yellow arrows in 40× magnification CM images. Scale bar = 50 μM.

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  Figure 3. Increased adipose tumor necrosis factor-α (TNF-α) expression in obesity. Adipose TNF-α gene expression was determined in six lean, 18 overweight, and 27 obese non-diabetic individuals using real-time RT-PCR, while TNF-α protein expression was also determined in five lean, 13 overweight, and 10 obese non-diabetic individuals using immunohistochemistry (IHC) as described in Materials and Methods. The data (mean ± SEM) show that (A) TNF-α adipose gene expression was higher in obese (P = 0.02) compared with lean individuals. (B) TNF-α protein expression (expressed as IHC intensity in arbitrary units) was also elevated both in overweight (P = 0.0009) and obese (P < 0.0001) compared with lean individuals. (C) The representative IHC images obtained from three independent determinations with similar results show increased adipose tissue TNF-α expression (blue arrows, 100× magnification) in overweight and obese individuals compared with lean. Scale bar = 50 μM. (D) IRF5 and TNF-α gene expression correlated positively in the adipose tissue (r = 0.50, P = 0.0004). The asterisks *, ***, and **** represent significance levels of P ≤ 0.05, P < 0.001, and P < 0.0001, respectively.

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  Figure 4. Elevated adipose interleukin-6 (IL-6) expression in obesity. Adipose tissue IL-6 gene expression was determined in five lean, six overweight, and 19 obese non-diabetic individuals using real-time RT-PCR, while IL-6 protein expression was also determined in six lean, 14 overweight, and 10 obese non-diabetic individuals using immunohistochemistry (IHC) as described in Materials and Methods. The data (mean ± SEM) show that (A) IL-6 adipose gene expression was elevated in obese (P = 0.03) compared with lean individuals. (B) IL-6 protein expression (IHC intensity expressed in arbitrary units) was also increased both in overweight (P = 0.0004) and obese (P < 0.0001) as compared with lean individuals. (C) The representative IHC images obtained from three independent determinations with similar results show elevated adipose tissue IL-6 expression (brown arrows, 100× magnification) in overweight and obese as compared with lean individuals. Scale bar = 50 μM. (D) IRF5 and IL-6 gene expression did not correlate in the adipose tissue (r = 0.26, P = 0.07). The asterisks *, ***, and **** represent significance levels of P ≤ 0.05, P < 0.001, and P < 0.0001, respectively.

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  Figure 5. Upregulated adipose tissue chemokine CXC motif ligand-8 (CXCL8) expression in obesity. Adipose CXCL8 gene expression was determined in five lean, 18 overweight, and 27 obese non-diabetic individuals using real-time RT-PCR, while CXCL8 protein expression was determined in six lean, 14 overweight, and 10 obese non-diabetic individuals using immunohistochemistry (IHC) as described in Materials and Methods. The data (mean ± SEM) show that (A) adipose CXCL8 gene expression was upregulated in obese (P = 0.05) compared with lean individuals. (B) CXCL8 protein expression (IHC intensity in arbitrary units) was also higher in obese (P = 0.0001) compared with lean individuals. (C) The representative IHC images obtained from three independent determinations with similar results show upregulated adipose CXCL8 expression (purple arrows, 100× magnification) in overweight and obese as compared with lean individuals. Scale bar = 50 μM. (D) IRF5 and CXCL8 gene expression was associated with each other in the adipose tissue (r = 0.40, P = 0.0004). The asterisks * and *** represent significance levels of P ≤ 0.05 and P < 0.001, respectively.

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  Figure 6. Adipose chemokine CC motif ligand-5 (CCL5) protein expression is elevated in obesity. CCL5 transcripts/messenger RNA (mRNA) expression was assessed in four lean, 18 overweight, and 23 obese non-diabetic subjects using real-time RT-PCR, while CCL5 protein expression was determined in six lean, 14 overweight, and 10 obese non-diabetic subjects using immunohistochemistry (IHC) as described in Materials and Methods. The data (mean ± SEM) show that (A) increase in CCL5 transcripts in overweight and obese compared with lean individuals was non-significant (P > 0.05). (B) CCL5 protein expression (IHC intensity shown in arbitrary units) was increased in obese (P < 0.0001) as compared with lean subjects. (C) The representative IHC images obtained from three independent determinations with similar results show increased CCL5 adipose expression (turquoise arrows, 100× magnification) in overweight and obese as compared with lean individuals. Scale bar = 50 μM. (D) IRF5 and CCL5 gene expression correlated in the adipose tissue (r = 0.40, P = 0.02). The **** asterisks represent significance level of P < 0.0001.

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  Figure 7. Increased adipose chemokine CC motif ligand-2 (CCL2) expression in obesity. CCL2 gene expression was determined in six lean, 18 overweight, and 23 obese non-diabetic individuals using real-time RT-PCR, while CCL2 protein expression was also assessed in six lean, 14 overweight, and 10 obese non-diabetic individuals using immunohistochemistry (IHC) as described in Materials and Methods. The data (mean ± SEM) show that (A) adipose CCL2 mRNA expression was upregulated in obese (P = 0.05) compared with lean subjects. (B) CCL2 protein expression (IHC intensity shown as arbitrary units) was also higher in overweight as well as obese individuals (P < 0.0001) as compared with lean. (C) The representative IHC images obtained from three independent determinations with similar results show elevated CCL2 adipose expression (cyan arrows, 100× magnification) in overweight and obese as compared with lean individuals. Scale bar = 50 μM. (D) There was no association found between IRF5 and CCL2 gene expression in the adipose tissue (r = 0.12, P = 0.42). The asterisks * and **** represent significance levels of P ≤ 0.05 and P < 0.0001, respectively.

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  Figure 8. Adipose interferon regulatory factor-5 (IRF5) gene expression correlates with the circulatory C-reactive protein (CRP) and adiponectin levels. Plasma levels of CRP and adiponectin were measured in 35 individuals including lean, overweight, and obese by ELISA as described in Materials and Methods. The data (mean ± SEM) show that (A) adipose expression of IRF5 correlated positively with CRP levels (r = 0.37, P = 0.03) and (B) negatively with adiponectin levels (r = −0.43, P = 0.009).

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  Figure 9. Increased adipose IRF5 expression concurs with metabolic inflammation. The illustration depicts a proposed model of metabolic inflammation, in support of the data presented, wherein the increase in obesity parallels with IRF5 adipose upregulation (positive associations with BMI, body fat percentage, age, and HbA1c). These changes in the IRF5 expression are in agreement with a wide range of markers of adipose inflammation including inflammatory cytokines/chemokines, TLRs and TLR-associated signaling molecules, inflammatory macrophage markers, as well as systemic immune-metabolic markers i.e., a positive association with circulatory CRP and a negative association with adiponectin levels. The two-way arrows (⇌) represent a mutual association. Taken together, these changes support the IRF5 upregulation as a novel marker for adipose tissue inflammation in obesity.