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ORIGINAL ARTICLE Table of Contents  
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Impact of conjugated linoleic acid on obesity and its association with macrophage recruitment: Experimental and immunohistochemical study


1 Department of Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt
2 Department of Physiology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt; Department is Physiology, King Abdulaziz University, Jeddah, Saudi Arabia
3 Department of Physiology, Faculty of Medicine, Menoufia University, Shebeen El-Kom, Egypt

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Date of Submission23-Mar-2022
Date of Decision09-Jul-2022
Date of Acceptance18-Jul-2022
Date of Web Publication14-Nov-2022
 

  Abstract 


Background: Conjugated linoleic acid (CLA) has been shown in humans and animals to have anti-adipose effects. The current study aims to assess the prophylactic and therapeutic impact of CLA and its effect on recruited macrophage type using immunohistochemistry against CD68 and CD 163. Materials and Methods: Forty adult male albino rats of local strain were included in the study and divided into control, CLA-supplemented, obese, CLA-prophylactic obese, and CLA-treated obese groups. Biopsies from visceral fat of the investigated groups were obtained and assessed for histopathological changes and immunohistochemical staining for CD68 and CD163. Results: Obese group showed hypertrophied adipocytes and infiltration by inflammatory cells compared to other groups. The obese group showed a marked increase in the CD68 positivity compared with that in the control and CLA-supplemented groups. CLA-prophylactic and CLA-treated groups showed mild immune reaction with a significant decrease in CD68 positivity compared to the obese group. The obese group showed a significant decrease in the CD163 positivity compared with that in the control and CLA-supplemented groups. Conclusions: Adipose tissue in obese is characterized by inflammation with more M1 than M2 macrophages. CLA could direct the recruited macrophages toward the anti-inflammatory subtype (M2) which encourages its beneficial effects in prophylaxis from obesity.

Keywords: Adipose tissue, CD163, CD68, obesity, rats


How to cite this URL:
Abdou AG, Bendary MA, Abdou SE, Amer GS. Impact of conjugated linoleic acid on obesity and its association with macrophage recruitment: Experimental and immunohistochemical study. J Microsc Ultrastruct [Epub ahead of print] [cited 2022 Nov 30]. Available from: https://www.jmau.org/preprintarticle.asp?id=361120





  Introduction Top


Obesity affects more than half a billion people worldwide, in 2018, 600 million were obese and 1.9 billion were overweight.[2] Between 1990 and 2011, the prevalence of obesity in Egypt grew from 4% to 7%, respectively, and it is anticipated to increase.[3]

This high prevalence of obesity represents a serious worldwide public health problem include coronary heart disease, insulin resistance, heart failure, gall bladder disease, hepatic steatosis, and sleep apnea. Type 2 diabetes, hypertension, arthritis, and other morbidities are all increased by obesity.[4]

Inflammatory cells have been found to be attracted to adipose tissue, including lymphocytes, neutrophils, and macrophages. There are two types of macrophages, M1 (proinflammatory) and M2 (anti-inflammatory). The pattern of macrophage infiltration differs between lean and obese subjects.[5]

Conjugated linoleic acid (CLA), a polyunsaturated fatty acid, is abundant in dairy products and ruminant meats.[6] CLA was proposed to have anti-adipose properties in humans and animals.[7] Previous studies have been purposed that CLA could induce apoptosis in fatty tissue and to inhibit adipose cell proliferation and differentiation.[8]

The current study's goal is to assess CLA's prophylactic and therapeutic effects on obesity, as well as its impact on recruited macrophage types using immunohistochemistry against CD68 (M1 macrophages) and CD 163 (M2 macrophages).


  Materials and Methods Top


Forty adult male albino Sprague Dawley rats weighed (120–160 g each) were included in this work. Rats were purchased from the Ophthalmology Institute's animal house. Rats were kept in a standard laboratory condition with a natural light-dark cycle of 12/12 h. Wire mesh cages were used to keep rats (8rats per cage with dimensions 80 cm × cm 40 × 30 cm) at room temperature (22°C ± 3°C) with food and drink readily available. The rats were left to accommodate the laboratory and animal house circumstances for 1 week before the start of the experiment.

The ethical guidelines established by the review board committee of the Faculty of Medicine, Menoufia University approved the experimental protocol (phy 203/2019) the protocol was approved at March, 2019. The experiment followed international guidelines for the use and care of laboratory animals according to declaration of Helsinki. In this study, the rats were divided into five groups at random, each with eight rats.

Group 1 (control group)

The rats in this group were supplemented with a standard rat chow diet and 0.5 ml olive oil by oral gavage, for 3 months, once per day.

Group 2 (conjugated linoleic acid -supplemented group)

In this group, normal rats were supplemented with 1.5% CLA dissolved in olive oil (equivalent to approximately 1500 mg/kg BW/day) by oral gavage, once daily for 3 months.[9]

Group 3 (obese group)

In this group, obesity was produced in rats by feeding them a high-fat diet (HFD) for 3 months.[10]

Group 4 (conjugated linoleic acid -prophylactic obese group)

The rats of this group were supplemented with HFD concomitant with 1.5% CLA by oral gavage, once daily for 3 months.

Group 5 (conjugated linoleic acid -treated obese group)

In this group, the rats were rendered obese by their supplementation with HFD for 3 months, thereafter they were supplemented once daily with 1.5% CLA by oral gavage for 3 months.

Preparation of high-fat diet

HFD was prepared following the American Institute of Nutrition (AIN). Rats were fed based on the AIN-93M. The HFD is a semi-purified diet formula with 20 g of fat/100 g of diet (19 g of butter oil and 1 g of soybean oil to provide essential fatty acids) compared to the standard commercial rat chow that provides 12.62 kJ/g of diet. This HFD provided 19.34 kJ/g diet, of which fat provides 7.74 kJ (40% fat per weight of diet).[11]

Histopathological study

Specimen from visceral (mesenteric) fat of each rat was taken and fixed in 10% formalin solution, then sent to Pathology Department for processing in the form of dehydration in ethanol solutions and clearing in xylene. The specimens then were impregnated in paraffin and finally, paraffin blocks were prepared. From each block, serial 5 μsections were cut, one to be stained with hematoxylin and eosin for assessment of pathological alterations and the other sections were cut on charged slides for immunohistochemical detection of CD 68 and CD 163.

Immunohistochemistry study

The primary antibodies were ready to use mouse monoclonal antibodies raised against CD 68 (Clone KP, Cat. #AP-9006, lab vision, USA) and CD 163 (clone 10D6, lab vision, USA).

Steps of de-paraffinization and rehydration were applied on the slides, then antigen retrieval in citrate buffer saline (pH 6) for 20 min, followed by cooling at room temperature. Incubation with 6% H2O2 in methanol was done for inhibition of endogenous peroxidase activity. The primary antibodies were incubated on the slides in humidity chamber overnight at room temperature. DAB and Mayer's hematoxylin were used as a brown chromogen substrate and as a counterstain, respectively. Negative control was prepared by replacing the primary antibodies with phosphate buffer saline. Reactive lymph node was used as a positive tissue control for CD68 and CD163. Cytoplasmic or granular brown staining was required to assign positive CD68 and CD 163 expression in any number of cells.

Statistical analysis

The “Statistical Package for the Social Science” (SPSS) version 22 application was used to statistically analyze the data (SPSS Inc., Chicago, Illinois, USA). The qualitative data were analyzed using Fisher's exact and Chi-square tests. P ≤ 0.05 was used as the significant level.


  Results Top


Histopathological changes in different groups

Control and CLA-supplemented groups showed normal architecture of the adipocytes. The obese group showed hypertrophied adipocytes surrounded by inflammatory cells. Nearly normal adipocytes in the CLA-prophylactic obese group and CLA-treated obese group were observed [Figure 1].
Figure 1: Normal adipocytes in control (a) and CLA supplemented (b) groups. Relative hypertrophied adipocytes and immune cell infiltration in adipocytes of obese, (c and d) Adipose tissue of CLA-prophylactic obese and treated groups showing nearly normal adipocytes, (e and f). (H and E, ×200 in b, d, e, f and × 400 in a and c), CLA: Conjugated linoleic acid

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CD68 expression in different groups

CD68 expression appeared as granular brown staining in between the visceral adipocytes in different studied groups. CD68 positive expression was identified in two rats (25%) in the control group, one rat (12.5%) in the CLA-supplemented group, seven rats (87.5%) in the obese group, two rats (25%) in the CLA-prophylactic group, and two rats (25%) in the CLA-treated group [Figure 2]. CD68 positivity in the obese group increased significantly when compared with control, CLA-supplemented, prophylactic, and -treated groups (P = 0.04, 0.01, 0.04, and 0.03, respectively) [Table 1].
Figure 2: M1 macrophages highlighted by CD68 immunostaining in control (a), CLA supplemented (b), obese (c), CLA-prophylactic (d), and CLA-treated groups (e). negative CD68 expression in CLA-treated groups (f). (Immunohistochemical staining × 400), CLA: Conjugated linoleic acid

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Table 1: Immuohistochemical results of CD68 (M1) positivity in between the visceral adipocytes in all studied groups of rats

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CD163 expression in different groups>

CD163 expression appeared as granular brown staining in between the visceral adipocytes in different studied groups. Six rats (75%) in the control group, seven rats (87.5%) in the CLA-supplemented group, one rat (12.5%) in the obese group, seven rats (87.5%) in the CLA-prophylactic group, and one rat (12.5%) in the CLA-treated group showed CD163 positive expression [Figure 3]. The obese group showed a significant decrease in the CD163 positivity compared with that in control, CLA-supplemented, and CLA-prophylactic groups (P = 0.04, 0.01, and 0.01, respectively). On the other hand, the CLA-treated group did not differ from that of the obese group (P = 0.44) [Table 2].
Figure 3: M2 macrophages highlighted by CD163 immunostaining in control (a), CLA supplemented (b), obese (c), CLA-prophylactic (d), and CLA-treated groups (e), Negative CD163 expression in CLA-treated group (f). (Immunohistochemical staining × 400), CLA: Conjugated linoleic acid

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Table 2: Immuohistochemical results of CD163 (M2) positivity in between the visceral adipocytes in all studied groups of rats

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  Discussion Top


The current study demonstrated hypertrophied adipocytes and inflammatory cellular infiltrates in the obese group agreeing with other studies[1],[12] who showed that in obesity, the adipose tissue expands either by enlargement of the fat cells (hypertrophy) or increase in their number (hyperplasia). Preadipocytes transform into mature adipocytes, whereas adipocytes store triglycerides and grow large.

In both obese and nonobese people, the number of fat cells is defined during childhood and adolescence and remains stable throughout maturity.[13] In adulthood obesity, there is adipocyte hypertrophy rather than hyperplasia. However, some investigators suggested adipocyte hyperplasia in adulthood, where they found that obese adults have enlarged lower more than upper body subcutaneous fat through hyperplasia in response to overeating.[14]

On the other hand, CLA-prophylactic and CLA-treated obese groups displayed nearly normal adipocytes and decrease the infiltration of adipose tissue with the inflammatory cells. These results agreed with others[15],[16] who found that CLA decreased the size of fat cells in human white adipose tissue.

In the present work, the infiltration of adipose tissue with inflammatory cells in the obese group was consistent with Kintscher et al.[5] who reported that T-cells and neutrophils infiltrate the adipose tissue before macrophages infiltration. The presence of macrophages in fatty tissue was consistent with Wellen and Hotamisligil's findings,[17] who found a 4–5-fold increase in fatty tissue macrophages in overweight people compared to lean people. Many years ago, it was established that obesity causes macrophage migration into fatty tissue. Xu et al. 2003,[18] were pioneers in discovering many macrophage markers in adipose tissue of leptin-deficient (Lep ob/ob), leptin receptor-deficient (LepR db/db), and diet-induced obese (DIO) mice compared with the lean controls. In the visceral adipose tissues of lean people, macrophages make up about 10%–15% of the stromal vascular cells, however, in obese humans and obese mice, their numbers increased to 40%–50%.[19] These bone marrow-derived adipose tissue macrophages (ATMs) are produced from the circulating monocyte progenitors that pass the vascular endothelium to mature into macrophages in the fatty tissue.[19],[20]

The process by which macrophages infiltrate fatty tissue is considered to be a response to a “stress signals” arising from the hypertrophic adipocytes.[19] In obesity, the lipid-engorged hypertrophic adipocytes have endoplasmic reticulum and mitochondrial stress responses that enhance macrophage infiltration into the fatty tissue.[21] In addition, macrophage infiltration of fatty tissue is caused by many other factors mostly related to adipose tissue hypertrophy such as the fatty acid flux within the adipose tissue, resulting from more stored triglycerides, which leads to macrophage recruitment. Toll-like receptor 4 has been shown to be a receptor for saturated fatty acids and to play a role in the production of inflammatory cytokines by fluxed fatty acids.[22] In addition, the high circulating level of leptin in obese acts as a chemoattractant for macrophages in the adipose tissue.[23] Finally, lower vascular density associated with obesity, as well as hypoxia and adipocyte death, may affect macrophage recruitment.[24]

Inside the fatty tissue, when monocytes are stimulated, they develop into either typically activated macrophages (M1) or alternatively activated macrophages (M2).[20]

In the lean fatty tissue, the major populations of ATMs are M2 macrophages. T helper type 2 (Th2) cytokines such interleukin (IL)-4, IL-10, and IL-13, as well as immune complexes and glucocorticoids, polarize recruited monocytes toward activated M2 macrophages. M2 macrophages secrete anti-inflammatory cytokines like IL-10 and IL-1 receptor antagonists. As a result, M2 macrophages are called anti-inflammatory macrophages and are thought to protect the adipocytes from inflammation in the lean state.[20]

The expression of CD163 and CD180 on the surface of M2 macrophages is one of their distinguishing characteristics.[20],[25] M2 macrophages are incorporated in remodeling, repair, and insulin sensitivity maintenance by producing arginase-1, IL-10, and IL-1Ra.[1]

In contrast, in obesity, the inflammatory cytokine interferon T helper 1 and lipopolysaccharide polarize recruited monocytes toward classically activated M1 macrophages. Tumor necrosis factor (TNF), IL-6, IL-1, IL-12, and MCP-1 are pro-inflammatory cytokines secreted by M1 cells. Therefore, they are usually termed pro-inflammatory macrophages phenotype.[26],[27] Surface expression of CD68, CD40, CD64, CD11c and CD11 are shown in M1 macrophages.[28]

The current study found that obese rats' adipose tissue expressed more CD68 and less CD163 than the control group, as well as a substantial increase in M2 macrophages in the CLA-supplemented and CLA-prophylactic groups was observed compared to the obese group. These results were consistent with those of Xu et al.,[18] who discovered that fatty tissue has a greater level of M1 macrophage markers of genetically obese (Lep ob/ob and LepR db/db) and DIO mice as compared to lean controls. Furthermore, Yu et al.[29] discovered that the macrophage marker CD68 gene expression was enhanced in the obese mice's visceral fat. Moreover, Lumeng et al. (2007)[26] found that HFD consumption shifts murine ATM cytokine expression patterns from M2 like to M1 like. In addition, Odegaard et al., in 2007,[30] discovered that shifts in ATM state from M2 polarized to M1 pro-inflammatory were associated with fatty tissue inflammation in DIO. This shift of ATMs may be explained by the obesity-induced switch of macrophage polarization from M2 macrophages which more resident in lean adipose tissue to M1 macrophages which more abundant in obese adipose tissue.[25]

Furthermore, the obesity-induced ablation of peroxisome proliferator-activated receptor (PPAR), which is necessary for the maturation of M2 macrophages, could explain this ATMs switch. Adipocytes produce the Th2 cytokine IL-13, which promotes macrophage PPAR expression via a signal transducer and activators of the transcription-6 binding site. Inhibition of PPAR prevents macrophages from transforming into the M2 phenotype, resulting in inflammation.[31]


  Conclusion Top


Finally, in this study, CD163 (M2) was highly expressed and CD68 (M1) was minimally expressed in the adipose tissue of CLA-prophylactic obese rats. These obtained results were concomitant with the results of Fiona et al., in 2007[32] who found that CD68 mRNA, ATMs infiltration, and TNF-α decreased in adipose tissue following CLA supplementation. According to these data, CLA could polarize recruited macrophages toward anti-inflammatory M2 macrophages.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Asmaa Gaber Abdou,
Department of Pathology, Faculty of Medicine, Menoufia University, Shebeen El-Kom
Egypt
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_25_22



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