The effects of conjugated linoleic acid supplementation on inflammatory cytokines and adipokines in adults: A GRADE-assessed systematic review and dose–response meta-analysis

Background and aims Many studies have investigated the effect of conjugated linoleic acid (CLA) supplementation on inflammatory cytokines and adipokines. However, the results of these studies are not consistent. Therefore, this systematic review and meta-analysis were designed to comprehensively evaluate the effect of CLA supplementation on inflammatory cytokines and adipokines. Methods Randomized controlled trials (RCTs) examining the effects of CLA supplementation on C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), adiponectin, and leptin, published up to March 2022, were identified through PubMed, SCOPUS, and ISI Web of Science databases. A random-effects model was used to calculate weighted mean differences (WMDs) with 95% confidence intervals (CI) for 42 studies that included 1,109 participants. Results Findings from 42 studies with 58 arms indicated that CLA supplementation significantly decreased IL-6 and TNF-α levels and also slightly increased CRP levels. However, adiponectin and leptin levels did not change after CLA supplementation. A subgroup analysis found that CLA supplementation reduced adiponectin and leptin in women. Conclusion Our results demonstrated that CLA supplementation increased CRP levels and decreased TNF-α and IL-6 levels. Therefore, it seems that CLA can have both proinflammatory and anti-inflammatory roles. Systematic review registration https://www.crd.york.ac.uk/prospero/, identifier (CRD42022331110).


Introduction
Inflammation is a protective reaction by an organism in response to injury, irritation, or infection that eliminates harmful stimuli and initiates the healing process (1). However, uncontrolled or unresolved inflammation can lead to tissue damage as well as the development of chronic inflammatory diseases, including type 2 diabetes, cardiovascular disease, cancer, etc. (2,3). Therefore, the control and prevention of chronic inflammation should be considered in order to prevent some chronic diseases and even improve health.
Weight loss strategies such as dietary interventions and physical activity are commonly used as nonpharmacological interventions to combat and/or prevent proinflammatory conditions (4,5). Despite the considerable benefits of pharmacological therapies, these may exert undesirable side effects and may not be tolerated by some individuals. On the other hand, they may cause drug resistance (reduction of efficiency) and even toxicity (6). Several studies have suggested that dietary strategies, such as certain supplements, can modulate inflammation. For example, a clinical trial has reported that magnesium and vitamin E co-supplementation led to a significant decrease in C-reactive protein (CRP) and a significant increase in total antioxidant capacity levels (7)(8)(9). Another study has suggested that Lglutamine supplementation in the early period of COVID-19 infection may reduce inflammatory responses and boost the immune system (10). Therefore, it is practical to find nutraceuticals and natural compounds with anti-inflammatory effects that may serve as alternative therapies to pharmacological interventions.
Conjugated linoleic acid (CLA) is a collective term for geometric isomers of linoleic acid (C18:2, n-6). This polyunsaturated fatty acid has two double bonds separated by a methylene group. This conjugation of the double bond is generally in positions 9 and 11 or 10 and 12 and may be a cis or trans configuration. Depending on the position and geometry of the double bonds, several isomers of CLA have been identified, such as 9cis,11trans-CLA and 10trans,12cis-CLA (11,12). Most of the beneficial properties of CLA are elicited by these two main isomers. For example, 10trans,12cis-CLA is involved in catabolic processes such as lipolysis and fat oxidation, whereas 9cis,11trans-CLA seems to be the active anabolic agent. In addition, based on the evidence, 10trans,12cis-CLA is thought to be anticarcinogenic, antiobesity, and antidiabetic, whereas 9cis,11trans-CLA is mainly anti-inflammatory (13).
CLA is formed via biohydrogenation by bacterial enzymes known as catalyst, present in the intestine microbiota of ruminant mammals; hence, CLA is mainly found in their flesh and milk (14). Nonetheless, it is present in foods only in finite amounts; therefore, commercialized CLA supplements have been provided to offer potential benefits, such as reduction of body weight and total fat mass, anticancer effects including reducing tumor growth (15,16), reducing insulin resistance, lipid disorders, and oxidative stress, improving liver function in nonalcoholic fatty liver (17), and immunomodulatory effects (18)(19)(20).
A large number of studies have investigated the effect of CLA supplementation on inflammatory cytokines and adipokines. However, the results of these studies are contradictory. For instance, some studies have reported that CLA supplementation had no significant effect on leptin levels (21, 22), whereas others found a significant decrease in serum leptin during CLA supplementation (23, 24). CLA supplementation has even been shown to significantly increase serum leptin levels (25). Also, some studies have reported that CLA supplements lead to a decrease in adiponectin levels (26), while another study has reported that CLA supplements have little or no effect on adiponectin levels (27). However, Sneddon et al. (28) have reported that CLA supplementation was associated with a significant increase in serum adiponectin levels (12%) in young obese subjects. Furthermore, while one study has found that CLA supplementation may increase CRP and tumor necrosis factor-alpha (TNF-a) and marginally decrease interleukin-6 (IL-6), indicating a proinflammatory state (29,30), a recent review article suggests that CLA may have an antiinflammatory effect by reducing inflammatory mediators such as cytokines, particularly IL-6, TNF-a, IFN-g, and IL-1b (30). Therefore, due to the inconsistencies in previous studies, the present study sought to update previous meta-analyses in light of the plethora of new studies. Thus, the current meta-analysis sought to investigate the effects of CLA on inflammatory cytokines and adipokines in adults.

Method and materials
This systematic review has been conducted according to the PRISMA statement (31). The present study was registered at PROSPERO CRD42022331110.

Search strategy
We conducted a comprehensive literature search in scientific databases such as PubMed/Medline, Scopus, Web of Science, EMBASE, the Cochrane databases, and Google Scholar to find relevant prospective studies on the effects of CLA supplementation on inflammatory cytokines, adiponectin, and leptin published up to March 2022. There were no restrictions on the length of time or language of publications. The Participant, Intervention, Comparison/ Control, Outcome (PICOS) search framework was used (32), which stands for population (both healthy and unhealthy adults), intervention (CLA supplementation), comparison (placebo/control group), outcome (changes in CRP, IL-6, TNF, adiponectin, and leptin), and research design (parallel and crossover randomized clinical trials).
The combination of MESH and non-MESH terms were used for the search, as follows: ("conjugated linoleic acid" OR "conjugated fatty acid" OR "boric acid" OR "rumenic acid" OR "CLA") AND (intervention OR "intervention study" OR "intervention studies" OR "controlled trial" OR randomized OR random OR randomly OR placebo OR "randomized controlled trial" OR "randomized clinical trial" OR RCT OR blinded OR "double blind" OR "double blinded" OR trial OR "clinical trial" OR trials OR "Pragmatic Clinical Trial" OR "Cross-Over Studies" OR "Cross-Over" OR "cross-over study" OR parallel OR "parallel study" OR "parallel trial").

Study selection
By reading the titles, abstracts, and full text of the papers as needed, two researchers independently (FS and OA) chose the appropriate articles. The effects of CLA supplementation on inflammatory cytokines and adipokines variables in adults with different health statuses in all human randomized clinical trials) RCTs(. We incorporated studies that satisfied the following criteria: (1) RCTs (parallel or crossover); (2) used oral intake of CLA; (3) examined the effects of CLA supplementation on CRP, IL-6, TNFa, adiponectin, and leptin; (4) had an intervention duration of at least 2 weeks (RCTs with two or more eligible arms were considered as separate studies); (5) were performed on adults (≥18 years old); (6) provided means and standard deviations (SDs) for all outcome variables, or any other effect sizes from which the calculation of mean and SD was possible. The searches were limited to human studies with no language restrictions. Animal studies, reviews, in vitro research, research on kids and teenagers, grey literature, conference abstracts, opinion pieces, books, and RCTs without a placebo or control group were excluded. Studies that used CLA in combination with vitamins or minerals were also excluded.
In the present study, we searched for studies that assessed the effects of CLA supplementation on all inflammatory cytokines. After screening and finding eligible studies, we found that most studies evaluated CRP, IL-6, and TNF. In addition, a limited number of studies have evaluated other inflammatory factors such as IL-1 and IL-8. Therefore, we included studies that evaluated the effects of CLA on CRP, IL-6, and TNF.

Data extraction
After quickly skimming the titles, abstracts, and full text to choose the most pertinent research following a separate review of each qualifying RCT, the following data were gleaned by two independent researchers (OA and FS). Name of the first author, country of origin, year of publication, type of clinical trial, participant characteristics (mean age, body mass index (BMI), sex), randomization, blinding, sample size, number of participants in the intervention and control groups, type and dosage of supplemented CLA, duration of the study, and related data were extracted for additional measurements. The CLA dosages were converted to milligrams per day, whether they were given in grams per day or another unit.

Quality assessment
To rate the quality of the studies, the Cochrane Collaboration technique was utilized (33). Two researchers (SR and GS) independently assessed the methods, and any disagreements between their assessments were resolved through discussion. Each study was evaluated for any bias, including those caused by randomized sequence generation, allocation concealment, participant and staff blindness, outcome assessor blindness, insufficient outcome data, selective reporting, and other biases. Thus, three categories were established: high risk of bias (general risk of bias > 2 high risks), low risk of bias (general risk of bias < 2 high risks), and undetermined risk of bias (general risk of bias = 2 high risk).

Statistical analysis
Stata 11.0 was used to conduct the statistical analysis (Stata Corp, College Station, TX, USA). p-values <0.05 were deemed statistically significant for all two-tailed tests. The pooled weighted mean difference (WMD) was calculated using a random-effects model to take into account any existing heterogeneity (due to the different intervention doses, duration, participant health, sample sizes, and length of intervention) developed by Der Simonian and Laird (34). We computed the mean differences in CRP, IL-6, TNF-a, adiponectin, and leptin between the CLA supplementation and control groups from the preintervention to the postintervention. Formula: SD = square root [(SD at baseline) 2 + (SD at end of study) 2 (2rSD at baseline, SD at end of study)] was used to get the SD of the mean difference (35). For RTCs that only provided standard error of the mean (SEM) information, SD was determined using the following formula: Standard Error of Mean (SEM): SD =, where n is the number of subjects in each group (36). After visual inspection of forest plots or Cochrane's Q test, heterogeneity was evaluated using the I square (I 2 ) statistic (p < 0.05 and I 2 > 40%), with I 2 > 40% considered clinically significant (37). The baseline CRP, IL-6, TNF-a, adiponectin, and leptin levels were analyzed, while the intervention duration (< 12 weeks, ≥ 12 weeks) and dosage of CLA (≥ 3 and < 3 g/ day) were based on the median values of the included studies and health status (healthy: people without any disease; unhealthy: people suffering from a disease such as atherosclerosis, rheumatoid arthritis, diabetes, etc.). Other subgroup analyses were performed according to gender (man, woman), baseline BMI (normal (18.5-24.9 kg/m²), overweight (25-29.9 kg/m²), and obese (>30 kg/m²)) and isomer's type (CLA-c9t11, CLA-t10c12, both). Studies examining the effect of CLA supplementation on inflammatory cytokines and adipokines used the Begg's test and the funnel plot test to evaluate publication bias (38). We used the leave-one-out approach to do a sensitivity analysis to identify how many inferences were dependent on a single sample to examine the influence of each study on the pooled effect size (39). The possible linear association between CLA (mg/day) dose and duration on outcome variables was evaluated using meta-regression, whereas a nonlinear dose-response analysis was used to assess the effect of CLA supplementation dose and duration (40,41).

Result
The flow of study selection The flow chart presented in Figure 1 describes the selection process and the references retrieved from the database.
A total of 8,351 studies were identified in the first step of the literature search of electronic databases, PubMed (n = 3,582), ISI Web of Science (n = 1,991), Scopus (n = 2,310), and the Cochrane library (n = 468). After excluding duplicated (n = 2,419) and irrelevant studies based on titles and abstracts (n = 2,804), 104 potentially relevant articles were considered for full-text review. Out of 104 studies, 62 did not have the desired data. Finally, 42 studies (11,12,16,18,19,27,28, were included in the present meta-analysis, and their characteristics are illustrated in Table 1. The risk of bias assessment is summarized in Table 2.

Publication bias
Although the visual inspection of funnel plots showed slight asymmetries for all of the outcome variables, no significant publication bias was detected according to Begg's test for most of the variables, such as CRP (p = 0.189), IL-6 (p = 0.441), adiponectin (p = 0.198), and leptin (p = 0.050). Publication bias was considerable just for TNF-a (p = 0.042) (Figures 3A-E).

Nonlinear dose-response analysis
The present study conducted a nonlinear dose-response regression to analyze the dose-response relationship between CLA supplementation and inflammatory cytokines and adipokines. We did not find a significant nonlinear relationship between dose (g/day) and changes in CRP [(coefficients = −0.059; p nonlinearity = 0.353)] ( Figure 4A

Meta-regression analysis
Meta-regression analyses were performed to assess whether inflammatory cytokines and adipokines were affected by CLA supplementation doses and intervention duration. We found no significant linear association between intervention dose (g/day) and changes in CRP [(coefficients = 0.279; p linearity = 0.304)] ( Figure 6A Figure 7E).

Sensitivity analysis
The sensitivity analysis demonstrated that no study had a significant impact on the overall findings. Although the overall result of studies reporting data on TNF-a was sensitive to the study by Song  Nonlinear dose-response analysis on the effects of the CLA dosage (mg/day) on (A) CRP (mg/L), (B) IL-6 (pg/ml), (C) TNF-a (ng/ml), (D) adiponectin (µg/ ml), and (E) leptin (ng/ml). Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from random-effects analysis. WMD, weighted mean difference; CI, confidence interval; CLA, conjugated linoleic acid; CRP, C-reactive protein; IL-6, interleukin 6; TNF-a, tumor necrosis factor-alpha.
(16) (WMD = 0.249; 95% CI, −0.016, 0.515). It was shown that the exclusion of every individual study by the sensitivity analysis could not change the direction of the correlation but eliminated the significant effect of CLA on CRP. Table 4 displays the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) profile of CLA supplementation on inflammatory cytokine and adipokine variables together with the certainty in outcomes. For CRP, because of serious limitations in inconsistency, the quality of evidence was moderate. In the case of IL-6, because of very serious limitations in inconsistency and for TNF-a because of very serious limitations in inconsistency ad serious limitations for publication bias, the quality of evidence was low. For both outcomes, including leptin and adiponectin, because of very serious limitations in inconsistency and serious limitations in imprecision, the quality of evidence was very low.

Certainty assessment
Using the GRADE methodology, which was previously outlined, the total degree of evidence certainty across the studies was evaluated and summarized (77).

Discussion
In this review, an analysis of pooling 42 studies indicated that CLA supplementation increased CRP concentrations, decreased IL-6 and TNF-a values, and had no effect on adiponectin and leptin levels. Findings of subgroup analyses showed that CLA consumption increased CRP in subjects with normal CRP levels (less than 3 mg/ dl) and those who were healthy or consumed CLA in doses of ≥3 g/ day. Taking CLA decreased IL-6 in male individuals and unhealthy subjects if the trial duration was less than 12 weeks and when a mixture of two isomers was used. TNF-a was reduced by CLA consumption in studies with an intervention dose of <3 g/day, male Nonlinear dose-response analysis on the effects of the duration of the intervention (week) of CLA on (A) CRP (mg/L), (B) IL-6 (pg/ml), (C) TNF-a (ng/ml), (D) adiponectin (µg/ml), and (E) leptin (ng/ml). Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from random-effects analysis. WMD, weighted mean difference; CI, confidence interval; CLA, conjugated linoleic acid; CRP, C-reactive protein; IL-6, interleukin 6; TNF-a, tumor necrosis factor-alpha.
subjects, healthy persons, or those having normal baseline BMI, and when a mixture of two isomers was used. However, in subgroup analyses based on isomer type, 10trans12cis-CLA isomer significantly increased TNF-a. Supplementation with CLA reduced adiponectin in women. It also decreased leptin in women and unhealthy adults but increased in those with normal baseline BMI. Moreover, the results of the meta-regression analysis showed that there was no significant linear association between intervention doses and duration of supplementation with inflammatory cytokines and adipokines.
The majority of studies included in this meta-analysis used supplements consisting of mixed amounts of CLA isomers. Therefore, since CLA isomers have shown different effects on inflammation (30, 78), it is suggested that this issue be considered in research. It is also important to consider the contents of the placebo used in studies. Some included RCTs have used vegetable oils as a placebo, such as safflower and olive oils. One study has indicated the reduced effect of safflower oil on CRP levels compared to CLA (79). Another study has elucidated the anti-inflammatory effect of olive oils through the prevention of lipooxygenase and enzymes that synthesize leukotriene (80). In other words, prescribing these vegetable oils as a placebo might misinterpret results.
Diet and physical activity can play an important role in inflammatory conditions (81). It is better to consider the effects of these two factors in the result of the present meta-analysis. Although, in some studies, the confounding variables of physical activity and diet were controlled, in some trials, these factors were not considered.
Diet is an important regulatory factor in the immune response. Malnutrition leads to suppression of the immune system, whereas overnutrition leads to immune activation. Some foods have antiinflammatory effects, and there are still controversies about others (81). For example, several studies have reported a positive association between the glycemic index/glycemic load of dietary carbohydrates and inflammatory cytokines (81). Also, a high-fat diet causes excessive body fat accumulation and impairs the immune system. On the other hand, it has been reported that trans and saturated fatty acids are significantly associated with the inflammatory state (82). Random-effects meta-regression plots of the association between dose of CLA (mg/day) and weighted mean difference of (A) CRP (mg/L), (B) IL-6 (pg/ ml), (C) TNF-a (ng/ml), (D) adiponectin (µg/ml), and (E) leptin (ng/ml). Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from random-effects analysis. WMD, weighted mean difference; CI, confidence interval; CRP, C-reactive protein; IL-6, interleukin 6; TNF-a, tumor necrosis factor-alpha. Random-effects meta-regression plots of the association between duration of intervention of CLA and weighted mean difference of (A) CRP (mg/L), (B) IL-6 (pg/ml), (C) TNF-a (ng/ml), (D) adiponectin (µg/ml), and (E) leptin (ng/ml). Horizontal lines represent 95% CIs. Diamonds represent pooled estimates from random-effects analysis. WMD, weighted mean difference; CI, confidence interval; CRP, C-reactive protein; IL-6, interleukin 6; TNF-a, tumor necrosis factor-alpha. Although ruminant trans fatty acids have different effects than industrial trans fatty acids, several studies show that industrial trans fatty acids promote inflammation, whereas ruminant trans fatty acids have been reported to be harmless or even beneficial to health, as well as also having anti-inflammatory properties (83). In relation to physical activity, it has been reported that although exercise produces a short-term inflammatory response, regular exercise in the long term has an "anti-inflammatory" effect (84). One study reported that sedentary subjects had higher levels of inflammatory factors such as IL-6 and TNF-a compared to subjects with higher physical activity (85).
Other confounding factors that can affect inflammatory markers are sleep duration and sleep quality. Recently, studies have reported a relationship between longer sleep duration (>8 h) and increased levels of inflammatory markers such as CRP and IL-6 (86, 87). In addition, a study on healthy subjects reported that higher IL-6 levels were associated with lower sleep quality (88).
Here, we showed CLA consumption increased CRP concentrations, decreased IL-6 and TNF-a, and did not change adiponectin and leptin levels. There are some meta-analyses that have investigated the effect of CLA on the abovementioned markers. For example, Derakhshandeh-Rishehri et al. (2019) (89), working on 15 RCTs, revealed that long-term (24 weeks) CLA intake significantly increased CRP levels (89). A meta-analysis conducted by Haghighatdoost et al. (28) showed a significant increase in CRP (n = 8) and TNF-a levels (n = 8) along with a slight decrease in blood IL-6 (n = 9) following CLA supplementation (29). Another meta-analysis (2018) conducted on 19 RCTs demonstrated that CLA intake had a small but not significant decline in plasma leptin. However, it significantly decreased leptin in obese individuals, with trials lasting for less than 24 weeks (21). A study carried out by Mazidi et al. (89) on 14 RCTs indicated that treatment with CLA had no effect on IL-6 but significantly reduced serum TNF-a and adiponectin, besides enhancing serum CRP concentrations (90). By combining previous and current results, it can be acknowledged that except for the effect of CLA in increasing CRP levels, there is no consensus on other markers (i.e., IL-6, TNF-a, adiponectin, and leptin). Moreover, it is noteworthy to state that exercise, dietary intake, sleep duration, and sleep quality can alter the effects of CLA. Therefore, further welldesigned studies may clarify the role of CLA on the abovementioned markers.
In the present study, the gender-dependent impact of CLA has also been shown in the reduction of IL-6, TNF-a, adiponectin, and leptin. Based on evidence, CLA can induce testosterone biosynthesis (91), and subsequently, testosterone is able to decrease IL-6 expression (92). It has also been stated that circulating CLA concentrations are greater in women than in men (93). Therefore, the aforementioned evidence can justify the different effects seen between men (IL-6 and TNF-a reduction) and women (adiponectin and leptin reduction) after taking CLA.
The effect of CLA on inflammatory responses has generated inconsistent results. This may be due to isomers and tissue-specific responses of CLA. Moreover, across the included RCTs, administration doses of CLA varied from 1.6 to 10.8 mg/day, and the duration of the treatment differed from 4 to 104 weeks. Health characteristics, inflammation status, and even gut microbiome composition of the recruited individuals were also different (78). These variations in the studies can influence the results and interpretations.
For example, in relation to isomer and tissue-specific response, 9cis,11trans-CLA isomer exerts its anti-inflammatory effect by activating the peroxisome proliferator-activated receptor-g (PPARg)-dependent pathway and ultimately reducing the production of proinflammatory cytokines such as TNF-a and IL-6 (30). However, the 10trans,12cis-CLA isomer has been reported to have a proinflammatory effect in adipose tissue, in contrast to its effects on innate immunity and vascular cells. In addition, in adipose tissue, activation of PPARg is contrary to the physiological effects of 10trans,12cis-CLA in vitro and in vivo (78). This isomer downregulates PPAR-g gene expression and increases TNF-a, IL-6, and CRP production in adipose tissue (78, 94). It seems that the reason for this inconsistency is cytokines secreted from adipocytes, which in turn alter PPARg expression and activity in the fat cell. Therefore, inconclusive results regarding the anti-inflammatory properties of CLA are likely due to the subtle proinflammatory effects of the 10trans,12cis-CLA isomers in specific tissues (30, 78).
Overall, it appears that CLA can elicit both anti-inflammatory and proinflammatory features.
Some proposed mechanisms for the effects of CLA isomers on inflammation include the following: (1) Modulation of eicosanoid signaling. This means that CLA reduces the production of inflammatory eicosanoids derived from arachidonic acid (AA) through the inhibition of several steps in the AA cascade. (2) Modulating the transcription of some genes, such as the PPAR gene, plays an important role in the regulation of inflammation. Also, it suppresses the expression of the inducible NO synthase (iNOS) gene, which leads to a decrease in IL-6 production. (3) Inhibition of NF-kB activity, which is a transcription factor involved in cytokine gene expression, cellular adhesion, cell cycle activation, apoptosis, and carcinogenesis and is integrally related to inflammatory responses (78, 95).
This meta-analysis has some limitations that should be addressed. Most of the included studies had a relatively small sample size, which can cause an overestimation of the results. Observation of publication bias for TNF-a findings suggests overestimation of CLA efficiency on TNF-a. Moreover, TNF-a, IL-6, and CRP results were sensitive to some studies.
In conclusion, it is suggested that CLA supplementation can have both proinflammatory and anti-inflammatory roles. It can enhance CRP concentrations while reducing TNF-a and IL-6 levels. Furthermore, CLA is able to decrease adiponectin and leptin in women. It can also decrease leptin in unhealthy adults and increase it in subjects with a normal baseline BMI. Finally, in order to improve the quality of studies, future clinical trials are encouraged to carefully consider CLA-isomer-specific regulation of inflammatory markers and take notice of the contents of the placebo used in their control groups. It is also important to keep in mind the gender-dependent impact of CLA.