The role of irisin on the polarization of monocyte-derived macrophages from pediatric patients with type 2 diabetes mellitus

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The role of irisin on the polarization of monocyte-derived macrophages from pediatric patients with type 2 diabetes

mellitus

A thesis presented as a partial requirement to obtain the academic degree of:

Master of Science in Biomedical Sciences

By:

Adrián Marcelo González Gil

Monterrey, Nuevo León, June 3, 2021

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Dedication

I dedicate this work to my beloved wife Rosario, and my family and friends, whose constant support and love were crucial to help me navigate this challenging time in my life.

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Acknowledgements

I would like to thank everyone who supported me throughout my two-year journey in the master’s program and my prior Social Service in Research year, which helped me develop a passion for biomedical research and endeavor to pursue a career as a physician-scientist.

To my advisors, Dr. Leticia Elizondo Montemayor and Dr. Elena González Castillo, for their invaluable guidance and constant feedback and support. They have helped me grow not only as a physician-scientist, but also as a person. To all members of the Cardiovascular and Metabolomics Research Group, for their valuable feedback during seminars.

To Dr. Eduardo Vázquez, who advised me on the analysis of flow cytometry experiments and shared reagents in times of dire need. To Isabel García, who always kindly helped me troubleshoot when I faced technical difficulties with the equipment in the laboratory.

To my fellow students and colleagues, Eder Luna, Esteban Valdez, Francisco Robles, Paulina Hernández, Monica Velásquez, Abel Gutiérrez, Mario Chapa, Sofía Acosta, Carolina Zertuche, Silvia López, Bernardo Martínez, José Romeo Villarreal Calderón, Alejandro Torres Quintanilla, Marina Peschard, Sofía Huerta, Daniel Roffe, Mariana Navarro, Itzel Treviño, Felipe Salazar, and Bianca Nieblas, who made this challenging time more enjoyable and constantly supported each other. To Eder Luna, for his valuable input and insight, which contributed positively to the outcome of this project. To José Romeo Villarreal Calderon, Alejandro Torres Quintanilla, and Luisa Reyes, who kindly taught me some of the laboratory techniques used in this project.

To my medical school and alma mater, Tecnólogico de Monterrey School of Medicine and Health Sciences (granted a full academic scholarship for my studies), and CONACYT (granted a graduate student maintenance scholarship).

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Abstract

The role of irisin on the polarization of monocyte-derived macrophages from pediatric patients with type 2 diabetes mellitus

Adrian M. Gonzalez-Gil, M.D.

Irisin is a novel peptide hormone released from skeletal muscle following acute bouts of physical exercise. Besides its originally described role on adipose tissue browning, its direct anti- inflammatory properties have been recently described. Specifically, when murine RAW264.7 macrophages were pre-treated with irisin, both gene and protein expression of key proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, decrease following exposure to lipopolysaccharide (LPS), while levels of the anti-inflammatory cytokine IL-10 increase. These changes have been linked to decreased TLR4 protein expression and activity of NF-ΚB. As well, in murine models, irisin has been described to favor an alternatively activated M2 macrophage phenotype, both from a M0 unstimulated state and a M1 classical proinflammatory state. Recently, the importance of an anti-inflammatory microenvironment to maintain the differentiation and function of brown adipose tissue has been emphasized in preclinical models, which could imply that irisin exerts its global metabolic effects indirectly through its action on adipose tissue macrophages, at least partially. Thus, irisin and its downstream pathways could represent a novel therapeutic target in type 2 diabetes mellitus (T2DM), a disease state characterized by a systemic low-grade proinflammatory state and lower circulating levels of irisin compared with healthy controls. In humans, evidence shows that M1 macrophage infiltrates in adipose tissue originate from circulating monocytes, but no studies have described the role of irisin on any type of human immune cell.

Using monocyte-derived macrophages (MDMs) obtained from peripheral blood mononuclear cells (PBMCs) from a previously characterized cross-sample of pediatric T2DM patients (n=15) and matched healthy controls (n=8), we sought to determine differences in MDM immunophenotypes between groups, their response to in vitro treatment with recombinant human irisin, and to correlate patients’ basal monocyte subsets with their clinical and biochemical parameters and MDM phenotypes. In order to standardize the procedures previous to testing cryopreserved samples of T2DM and control pediatric patients, blood was obtained from one healthy adult individual. In this subject, in vitro irisin treatment significantly increased CD163 MFI. T2DM subjects had higher proportions of circulating intermediate monocytes (IMs) relative to healthy controls, which correlated positively with body fat percentage and the inflammatory marker hs-CRP and negatively with HDL-c. MDMs from T2DM subjects had similar polarization profiles compared with controls when exposed to IL-4 and IFN-γ and LPS. However, macrophage polarization capacity, as measured by M1 (CD80) and M2 (CD163, CD200R) marker MFI, was significantly associated with basal monocyte proportions when considering all participants. Upon irisin treatment, CD163 upregulation was no longer observed in MDMs from patient samples, but a trend towards decreased NF-κB activation was noted. Our results provide preliminary evidence in favor of irisin’s anti-inflammatory role in human macrophages but must be replicated in future studies with larger sample sizes. On the other hand, increased IMs in pediatric T2DM might suggest enhanced monocyte migration and differentiation to macrophages in obese white adipose tissue or to vascular atherosclerotic lesions early in disease evolution, which warrants future longitudinal and mechanistic studies.

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1. Problem Statement

1.1. Background

Obesity, defined as a body mass index of >30 kg/m² or>90^th percentile for age and sex in the pediatric population (Prevention, 2016), is considered by many a modern day pandemic. It is associated with increased morbidity and mortality and is a risk factor for cardiometabolic diseases, including the metabolic syndrome (MetS), type 2 diabetes mellitus (T2DM), hypertension (HTN), and ischemic heart disease (IHD) (Hruby & Hu, 2015). T2DM is the most common type of diabetes worldwide, representing more than 90% of all cases of diabetes (DeFronzo et al., 2015). The International Diabetes Federation estimated that in 2013, 382 million adults aged 20-70 had T2DM, and 80% of cases lived in low- and middle-income countries. Its prevalence is expected to increase to 592 million people by 2035. Importantly, about 30% of T2DM cases remain undiagnosed (DeFronzo et al., 2015). In Mexico, prevalence of diabetes has consistently increased in the last decades, with the most recent National Survey of Health and Nutrition (ENSANUT) reporting a 9.4%

prevalence in adults (Hernandez Avila et al., 2016). If not adequately controlled with medical treatment and lifestyle changes, T2DM is associated with early morbidity and mortality due to micro- and macrovascular complications and increased susceptibility to life-threatening infections.

This often poses a challenge in clinical practice, as glycemic control depends largely on the patients’

strict adherence to medical treatment (Polonsky & Henry, 2016).

Of greater importance, incidence of T2DM in children and adolescents has been observed to increase steadily for the last three decades, which goes in hand with the childhood obesity epidemic (Bloomgarden, 2004; Dileepan & Feldt, 2013). In Mexico, there is no data regarding prevalence or incidence of T2DM in children and adolescents, but is estimated to represent approximately 50% of all diabetes cases (Frenk & Marquez, 2010). This proportion is clearly linked to childhood obesity because 34% of children and 36% of adolescents are either overweight or obese in Mexico, making it the country with most childhood obesity worldwide (Romero-Martínez et al., 2017). This is important because the low-grade systemic inflammatory state that accompanies obesity and favors the development of insulin resistance and T2DM, a process in which M2 alternatively activated anti-inflammatory macrophages in white adipose tissue (WAT) are replaced by M1 classically activated proinflammatory macrophages that play a crucial role in this process (Chawla et al., 2011; Donath & Shoelson, 2011; Reilly & Saltiel, 2017), begins and is maintained since early childhood (Aygun et al., 2005; Schwarzenberg & Sinaiko, 2006). T2DM represents a serious public health concern that requires urgent implementation of prevention strategies and novel therapeutic targets to reduce burden of disease and healthcare costs.

T2DM is frequently asymptomatic until late in disease progression when insulin deficiency and uncontrolled hyperglycemia are established, which may result in the classical diabetic syndrome of polydipsia, polyuria, blurred vision, nocturia, fatigue, and rarely, weight loss. However, in most cases, clinical presentation is indolent, and the patient is not aware of the disease due to the slow and gradual development of signs and symptoms. In fact, symptoms are frequently documented

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retrospectively once the diagnosis is established (McCulloch, 2019). T2DM may be clinically silent until micro- and macrovascular complications settle, which include diabetic nephropathy, neuropathy, and retinopathy; acute coronary syndrome, stroke, peripheral artery disease, among others (DeFronzo et al., 2015). Due to the asymptomatic and slowly progressive nature of T2DM, screening strategies have been implemented for high risk groups, as well as preventive measures, such as weight loss and physical activity regimes (Association, 2021b). Diagnostic criteria for diabetes may include any of the following: (1) fasting glucose ≥126 mg/dL, (2) glycated hemoglobin (HbA1c) ≥ 6.5%, (3) random glucose ≥200 mg/dL in the presence of symptoms, or (4) glucose ≥200 mg/dL as measured 2 hours following an oral glucose challenge (Association, 2021a).

Unfortunately, to date, T2DM remains uncurable in most cases. Thus, prevention of complications through strict metabolic control and reduction of cardiovascular risk factors via pharmacologic and lifestyle interventions remain a cornerstone in its management (Association, 2021b, 2021c, 2021d). The mechanisms of some currently used drugs in diabetes are shown in figure 1 (Trevor & Katzung, 2015). Although pharmacologic therapy is effective in reducing glycemia and reduces the rate of microvascular complications (Association, 2021c), there are currently no approved drugs that are specifically directed against the inflammatory component of the disease.

In fact, clinical trials employing agents with known anti-inflammatory properties have been conducted in adult patients with T2DM. Although some results have been promising, there is still no conclusive evidence regarding the clinical utility of any of them (Pollack et al., 2016).

Figure 1: Mechanisms of action of commonly used antidiabetic drugs. The inflammatory component that is in part responsible of insulin resistance in the disease is not directly targeted by currently used medications.

Figure taken from (Trevor & Katzung, 2015). DPP-IV: Dipeptidyl peptidase-4; GLP-1: Glucagon-like peptide-1;

SGLT-2: Sodium/glucose cotransporter 2.

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For decades, it has been recognized that physical exercise exerts systemic anti-inflammatory effects, and that it is useful in preventing the development of obesity, T2DM, and cardiovascular disease (Gleeson et al., 2011). The current consensus is that the efficacy of exercise in the prevention of T2DM is partially due to this anti-inflammatory effect. To date, the molecular mechanisms remain elusive and incompletely understood (Gleeson et al., 2011), but are beginning to be deciphered with the study of myokines, among which irisin is one of the most comprehensively studied (Huh, 2018;

Pedersen & Febbraio, 2012).

Irisin is a peptide hormone cleaved from its precursor, fibronectin type III domain- containing protein 5 (FNDC5), via mechanisms that are likely dependent on ADAM family proteases (Yu et al., 2019). FNDC5 is a transmembrane protein whose expression is regulated by the transcription factor peroxisome proliferator-activated receptor-gamma (PPAR-γ) and its coactivator peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). Irisin was first described by Boström and colleagues as a molecule released from SkM in PGC-1α- overexpressing mice subjected to exercise (Boström et al., 2012). Its originally described function of white adipose tissue (WAT) “browning” involved phenotypic transformation of murine subcutaneous white adipocytes to “brite” adipocytes which were characterized by increased expression of uncoupling protein 1 (UCP1), thermogenesis, and energy expenditure. WAT

“browning” was associated with improved insulin sensitivity and weight loss in mice fed a high-fat diet (Boström et al., 2012). Multiple independent research groups have confirmed that irisin exists and circulates in humans (Perakakis et al., 2017). Since its discovery, the role of irisin in the pathophysiology of obesity-related disorders has gathered great interest from the medical and scientific community, which has resulted in extensive basic and clinical research studies (Polyzos et al., 2018). Among its many proposed beneficial pleiotropic effects (Korta et al., 2019), irisin has been described to possess anti-inflammatory and antioxidant properties in a wide variety of in vitro and animal models (Askari et al., 2018), including macrophages (Dong et al., 2015; Mazur-Bialy, 2017;

Mazur-Bialy et al., 2018; Mazur-Bialy & Pocheć, 2021; Agnieszka Irena Mazur-Bialy et al., 2017; Ye et al., 2019), with some authors even reporting M2 polarization from both an unpolarized state (Dong et al., 2015) and a full-blown M1 state (Ye et al., 2019) . However, there are no published studies that have explored the immunomodulatory effect of irisin on human immune cells. Thus, in the present work, using flow cytometry, we sought to explore whether recombinant human irisin could favor anti-inflammatory M2 polarization in human monocyte-derived macrophages (MDMs) obtained from cryopreserved peripheral blood mononuclear cell (PBMC) samples from a previously established cohort of pediatric T2DM subjects and healthy controls. Our findings might support future research and eventually the use of irisin as a potential anti-inflammatory therapeutic target in context of obesity and type 2 diabetes mellitus (T2DM).

Importantly, three monocyte populations with different phenotype and functions have been described in humans, and the proportions and functionality of these monocyte subsets are known to vary according to the health status of the individual (Kapellos et al., 2019; Wong et al., 2012), which could potentially affect their differentiation and polarization potential in vivo based on

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in vitro evidence (Boyette et al., 2017). Particularly, monocytes from adult subjects with obesity and/or T2DM have been described to show altered proportions in their subsets and to be “primed”

towards a proinflammatory or dysfunctional phenotype in a myriad of studies (de Matos et al., 2019;

Devêvre et al., 2015; Friedrich et al., 2019; Giulietti et al., 2007; Nikiforov et al., 2017; Satoh et al., 2010; Valtierra-Alvarado et al., 2020; Yang et al., 2012), but no studies have described monocyte subsets and function in context of pediatric patients with T2DM. Furthermore, physical exercise has been described to modulate monocyte function towards an anti-inflammatory phenotype (Aw et al., 2018; Gleeson et al., 2011) even in individuals with obesity (de Matos et al., 2019) but the mechanisms remain unknown; irisin, a known mediator of physical activity, could be one of the mechanistic links. Thus, in addition, we sought to determine basal monocyte subsets for both studied groups, and to correlate the monocyte subsets with patients’ clinical and biochemical characteristics, including irisin, as well as the immunophenotype obtained in experiments with MDMs and the response to irisin treatment in vitro.

1.2. Problem statement

1.2.1 Research questions

The present work seeks to answer the following questions that to the best of our knowledge, remain unanswered in the literature:

1. What effect does recombinant human irisin have on human MDMs’ immunophenotype in the healthy state under different polarization conditions?

2. What is the distribution of monocyte subsets in peripheral blood from pediatric T2DM subjects compared to healthy controls, and how is this associated to clinical, biochemical, and inflammatory parameters?

3. If monocyte subset proportions are altered in pediatric T2DM, how does this affect their differentiation potential in vitro?

4. In MDMs obtained from pediatric T2DM patients and healthy controls, what effect does treatment with recombinant human irisin have on their polarization, and is this effect dependent on basal monocyte immunophenotype?

1.2.1 Rationale and hypothesis

Irisin has been shown to possess anti-inflammatory and antioxidant properties in in vitro models using murine macrophage cell lines. On the other hand, it is known that circulating monocytes from adult T2DM patients are primed towards a proinflammatory phenotype. Therefore, an M2 anti-inflammatory phenotype will be favored when monocyte-derived macrophages (MDMs) are treated with human recombinant irisin in vitro. Furthermore, MDMs’ response to irisin will differ between healthy controls and T2DM patients, an effect that will be dependent on the basal proportion of proinflammatory monocyte subpopulations.

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H0: In vitro treatment with irisin is not associated with decreased surface M1 markers or increased M2 markers. Basal monocyte subpopulations between T2DM and controls do not differ. MDMs’ response to irisin is not different between healthy controls and T2DM patients.

H1: In vitro treatment with irisin is associated with decreased surface M1 markers and increased M2 markers. Basal monocyte subpopulations between T2DM and controls are different. MDMs’

response to irisin is different between healthy controls and T2DM patients.

1.3. Objectives

1.3.1. General Objective

To determine if irisin exerts anti-inflammatory effects on MDMs by favoring an M2 phenotype and suppressing M1 polarization, and whether this effect differs between pediatric T2DM patients and healthy controls.

1.3.2. Specific Objectives

1. To determine the ideal culture conditions for M1 and M2 MDM differentiation using monocytes obtained from cryopreserved PBMCs through a negative selection protocol.

2. To determine the biologic activity of recombinant human irisin when used to treat MDMs in vitro as measured by M1 and M2 cell surface markers, as well as inflammatory pathway components, using flow cytometry.

3. To determine basal monocyte subpopulations in peripheral blood mononuclear cells obtained from pediatric T2DM patients and matched healthy controls and correlate them with clinical and biochemical parameters.

4. To determine immunophenotypic differences between MDMs from T2DM pediatric patients and those from healthy controls, as well as the difference in response to in vitro irisin treatment.

5. To correlate patients and controls’ MDM immunophenotypes with the basal proportions of monocyte subsets.

1.4. Justification

T2DM is a global health problem that up to this day remains incurable. In recent decades, a tendency towards increased incidence of the disease in children and adolescents has been noted, which has been intimately associated with the increased prevalence of childhood obesity.

Unfortunately, once the disease has been established, it becomes irreversible in most cases and requires life-long treatment with pharmacologic and lifestyle interventions to prevent micro- and macrovascular complications, which are responsible for a significant fraction of overall morbidity and mortality worldwide, and especially in Mexico. Thus, there is a growing need to generate effective strategies focused on T2DM prevention, as well as novel pharmacologic therapies that delay or inhibit its progression. In the last two decades, the low-grade proinflammatory state that

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accompanies obesity, in which M1 macrophages in WAT play a central role, has been investigated as a crucial component in T2DM disease pathogenesis, and thus, modulation of macrophage immunophenotype in context of metabolic disease has received great attention from the scientific community. In this regard, irisin, a novel hormone-like peptide released by skeletal muscle during acute bouts of exercise, has been shown to exert diverse antidiabetic metabolic effects in preclinical studies, among which a strong anti-inflammatory effect on macrophages, which are direct mediators of the obesity-related inflammatory state, has been reported. However, irisin’s immunomodulatory role on human immune cells has never been explored. Considering well- established differences between murine and human immunobiology and in monocyte subsets, it is often not possible to extrapolate results from studies in mice directly to humans. Thus, studies with cells and tissues of human origin are warranted. If irisin’s anti-inflammatory action was confirmed in human macrophages, this could aid in the elucidation of new pathways for macrophage function modulation in context of human metabolic disease, which could prepare ground for future mechanistic studies. On the other hand, to date, monocyte subsets in pediatric T2DM have not been described in the literature, which could potentially render new insights and hypotheses on inflammatory mechanisms early in the time course of the disease or serve as biomarkers of ongoing vascular damage associated with T2DM.

1.5. Scope

The present work seeks to explore whether in vitro treatment with human recombinant irisin has a direct effect on monocyte-derived macrophage (MDM) immunophenotype, as measured by M1 (CD80) and M2 (CD163 and CD200R) cell surface markers and inflammatory pathway components (TLR-4 and NF-κB) by means of flow cytometry. We recognize the complexity of immune signaling pathways and continuum of macrophage phenotypes in vivo, in which multiple and diverse mechanisms participate (Drareni et al., 2019; Parisi et al., 2018). Herein, we use a simplified in vitro model of the WAT microenvironment and do not assess all the potential effects that irisin could have on MDMs, which are possibly vast considering the available evidence in other types of cells and tissues, as recently reviewed by Rabiee et al (Rabiee et al., 2020).

We sought to identify associations between monocyte subsets and clinical and biochemical parameters in a cohort of pediatric subjects with T2DM and healthy controls. However, as a limitation of a cross-sectional design, causality cannot be established, as we did not perform an intervention or longitudinally followed-up patients in time. Likewise, cryopreserved PBMCs used in this study were altruistically donated and collected for a previous protocol, which imposed certain limitations on study design, as elaborated later.

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2. Theoretical Framework

2.1 Obesity and type 2 diabetes mellitus

2.1.1. T2DM pathogenesis

The pathogenesis of T2DM is complex and multifactorial, with both genetic and environmental factors playing a role. However, it has been proposed that the environmental component is far more important than the genetic one, given that genetic variants associated with the disease have only been described to increase the risk of developing the disease by 10-20%, and it is estimated that up to 90% of all T2DM cases can be prevented with only lifestyle changes, such as a maintaining a healthy diet, having a BMI of less than 25 kg/m², engaging in moderate intensity exercise for at least 30 minutes daily, avoiding tobacco, and consuming alcohol with moderation (DeFronzo et al., 2015). In fact, it is estimated that up to 54% of the general population has at least one risk locus for T2DM (Morris et al., 2012), but most subjects do not develop the disease in the absence of risk factors. In this regard, the strongest risk factor for the development of T2DM has been determined to be overweight or obesity (DeFronzo et al., 2015), which is due, among other mechanisms, to a chronic low-grade systemic proinflammatory state that directly causes insulin resistance. Indeed, increased serum concentrations of inflammatory markers, such as C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), have been strongly associated with increased risk of developing T2DM (Wang et al., 2013). Likewise, increased circulating adiponectin, an adipokine with anti-inflammatory properties that is released by healthy adipocytes in conditions of normal weight, is associated with decreased risk (Li et al., 2009).

The main metabolic defects observed in T2DM are insulin resistance in skeletal muscle (SkM), the liver, and adipose tissue, as well as compromised insulin secretion by pancreatic β-cells (DeFronzo et al., 2015), all of which contribute to hyperglycemia. As well, other determinants of glycemia have been described, which are also altered in T2DM and are collectively known as the

“ominous octet”, as shown in figure 2. β-cell resistance to glucagon-like peptide 1 (GLP-1) contributes to their gradual loss of function, while persistent increased levels of glucagon (due to loss of insulin-induced suppression) and increased sensitivity to glucagon in the liver, contribute to excessive and inappropriate gluconeogenesis in the liver. Insulin resistance in adipocytes triggers an accelerated lipolysis (due to decreased suppression of hormone-sensitive lipase) that leads to increased circulating free fatty acids, which accumulate ectopically in SkM and the liver, where they exacerbate insulin resistance, and in pancreatic β-cells themselves, which results in their dysfunction, as described later. In the kidney, chronic hyperglycemia triggers an upregulation of type 2 sodium-glucose co-transporters (SGLT2), which increases the threshold for glycosuria and favors glucose retention, further favoring hyperglycemia. Furthermore, hypothalamic resistance to the anorexigenic effects of insulin, leptin, GLP-1, amylin, and YY peptide have been described in obesity and T2DM, which facilitates further weight gain and its cardiometabolic consequences. A proinflammatory state that begins early in the state of obesity further contributes to disease progression and exacerbates disease manifestations (DeFronzo et al., 2015).

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©2021 ADRIAN MARCELO GONZALEZ GIL 17 Figure 2: The ominous octet of hyperglycemia in T2DM: In the present work, we emphasize the role of inflammation, specifically macrophages, in T2DM (lower right). Figure taken from: (DeFronzo et al., 2015).

AMPK: AMP-activated protein kinase; DPP4: Dipeptidyl peptidase-4; GLP-1 RA: Glucagon-like peptide-1 receptor agonists; IκB: I-kappa-B; MAPK: Mitogen-activated protein kinase; NF-κB:Nuclear factor kappa-light- chain-enhancer of activated B cells; ROS: Reactive oxygen species; SGLT-2: Sodium/glucose cotransporter 2;

TLR-4: Toll-like receptor 4; TNF: Tumor necrosis factor; TZDs: Thiazolidinediones.

2.1.2. The role of monocytes and macrophages in metainflammation

Adipose tissue is composed of adipocytes, extracellular matrix, and cells of the stromal vascular fraction (SVF). In physiologic conditions, resident macrophages comprise less than 15% of the SVF and are mostly polarized to an M2 anti-inflammatory and insulin-sensitizing phenotype, which is characterized by increased production of interleukin-10 (IL-10) in response to eosinophil- derived IL-4 and IL-13 (Wynn et al., 2013). In contrast, in the obese state, Ly6c^hi monocytes, which are analogous to CD14^hiCD16^- classical monocytes in humans (Guilliams et al., 2018), infiltrate adipose tissue in response to stressed adipocyte-derived monocyte chemoattractant protein-1 (MCP-1; also known as CCL2) and differentiate to macrophages in situ, increasing the proportion of macrophages to 45-60% of the SVF. These macrophages then acquire a proinflammatory M1 classical phenotype in response to local stress signals and are characterized by abundant production of TNF-α, IL-1β, and IL-6 (see figure 3). Consequently, local inflammation and insulin resistance ensue, as discussed in further detail below.

Monocytes are mononuclear cells that originate from myeloid precursors in the bone marrow. In humans, three main subpopulations in peripheral blood have been described, each of which differ in phenotype and function: classical CD14⁺⁺CD16^-, intermediate CD14⁺⁺CD16⁺, and non- classical CD14⁺CD16⁺ monocytes (Kapellos et al., 2019). It is widely accepted that classical

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monocytes are the precursors of macrophages that have infiltrated obese adipose tissue, but intermediate and non-classical monocytes have also been suggested to play a pathogenic role in conditions of metabolic stress (Devêvre et al., 2015; Poitou et al., 2011). Specifically, increased proportions of proinflammatory non-classical (Friedrich et al., 2019) and intermediate monocytes (Friedrich et al., 2019; Jagannathan et al., 2019; Poitou et al., 2011) have been described in adults with T2DM. This is of interest because it has been shown that the latter monocyte subset may express up to 2.5-fold levels of toll-like receptor 4 (TLR4) (Skinner et al., 2005), an observation that has also been confirmed in adults with T2DM (Yang et al., 2012). Importantly, monocytes from patients with obesity, which express higher levels of TLR4 and TLR8 compared with healthy controls, secrete TNF-α and IL-1β more abundantly following in vitro stimulation with lipopolysaccharide (LPS) or ssRNA (Devêvre et al., 2015). As well, monocytes from adults with T2DM also show a tendency towards a proinflammatory phenotype, as evidenced by increased basal expression of proinflammatory cytokines (Giulietti et al., 2007; Satoh et al., 2010), increased TNF-α, IL-1β, IL-6, and IL-8 production following in vitro stimulation with IFN-γ (Giulietti et al., 2007; Nikiforov et al., 2017), and increased IL-6 production after LPS stimulus (Yang et al., 2012) compared with monocytes from healthy controls. These findings may represent an important contributory role of monocytes to the systemic inflammatory state observed in T2DM. In the context of diabetes, it has been hypothesized that this phenomenon could be due to the presence of advanced glycation end products (AGEs), which are found in higher concentrations in the circulation and that activate the immune response via pathways downstream of their receptors (RAGEs), among which the transcription factor nuclear factor kappa-B (NF-κB) plays a central role (Jin et al., 2015).

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©2021 ADRIAN MARCELO GONZALEZ GIL 19 Figure 3: The role of M1 and M2 macrophages in adipose tissue metabolism. Figure taken from (Wynn et al., 2013). AAM: Alternatively activated (M2) macrophage; CAM: Classically activated (M1) macrophage; CCL2:

Chemokine (C-C motif) ligand 2 [MCP-1: Monocyte-chemoattractant protein-1]; CCL5: Chemokine (C-C motif) ligand 5; CCL8: Chemokine (C-C motif) ligand 8; CCR2: C-C chemokine receptor type 2; Eos: Eosinophil; IFN-γ:

Interferon gamma; IL: Interleukin; ILC2: Type 2 innate lymphoid cells; IRF3: Interferon regulatory factor 3; JNK:

c-Jun N-terminal kinase; KLF4: Kruppel-like factor 4; Ly6c: Lymphocyte antigen 6 complex; Mono: Monocyte;

MR: Mineralocorticoid receptor; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; OPN:

Osteopontin; PPAR: Peroxisome proliferator-activated receptor; STAT6: Signal transducer and activator of transcription 6; TNF: Tumor necrosis factor; Treg: Regulatory T cell.

2.1.3. The role of the obesity-related low-grade systemic proinflammatory state in T2DM pathogenesis

Obesity-associated insulin resistance plays a central role in T2DM pathogenesis. Insulin resistance is partially explained by mechanisms linked to the low-grade proinflammatory state that characterizes obesity (Calle & Fernandez, 2012; DeFronzo et al., 2015; Donath & Shoelson, 2011).

As mentioned previously, monocytes and macrophages are key cellular components of metainflammation that play a crucial role in insulin resistance and endothelial dysfunction that accompany obesity (Donath & Shoelson, 2011; Reilly & Saltiel, 2017; Wensveen et al., 2015).

The full sequence of events leading to full-blown WAT inflammation in obesity remains incompletely understood, but comprehensive models have been proposed by (Wensveen et al., 2015) and (Reilly & Saltiel, 2017). Briefly, hypertrophic adipocytes, through pathways that could involve hypoxia, mechanical stress, damage-associated molecular patterns (DAMPs) originating from necrotic adipocytes in the vicinity (e.g., double-stranded DNA and heat shock proteins), gut- derived endotoxemia secondary to impaired gut permeability in obesity, among others, secrete soluble mediators (e.g., MCP-1) that favor the recruitment of monocytes. Subsequently, proinflammatory cytokines secreted by adipocytes favor the differentiation of newly infiltrated monocytes to M1 proinflammatory macrophages, as shown in figure 4 (Reilly & Saltiel, 2017).

Furthermore, increased levels of adipocyte-derived FFAs bind TLR-4 in macrophages in conjunction with fetuin A, a hepatokine that has been shown to be upregulated in conditions of metabolic stress and is correlated with insulin resistance (Meex & Watt, 2017). As adipocytes grow in size and number due to excess nutrients, there is augmented production of leptin and decreased production of adiponectin, which participate in pro- and anti-inflammatory signaling pathways, respectively, further favoring M1 over M2 polarization (Wensveen et al., 2015). Besides monocytes, NK cells and CD8⁺ T-cells have also been shown to infiltrate obese WAT and become activated by stressed adipocytes through contact-dependent mechanisms, which drives production of IFN-γ, the M1- licencing cytokine by excellence. Moreover, M1 macrophages further recruit CD4⁺ T-cells via production of the chemokine CXCL16, which in presence of a proinflammatory microenvironment, are primed to a Th1 phenotype that is characterized by high production of IFN-γ, while CD8⁺ T-cells and B-cells are further recruited in the later stages of the process. Furthermore, the exaggerated production of TNF-α and IL-1β by M1 macrophages itself contributes to activate downstream NF-κB

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and the NLRP3 inflammasome, which results in a positive feedback autocrine loop of enhanced transcription and secretion of proinflammatory cytokines, respectively (Wensveen et al., 2015).

Given that adaptive immune responses are generally considered to cease once the offending antigen is eliminated (Murphy & Weaver, 2017b), it is expected that WAT inflammation continues indefinitely in obesity, as necrotic adipocytes are never fully cleared from WAT by macrophages.

Instead, “crown-like” structures of necrotic adipocytes surrounded by abundant M1 macrophages develop, and their presence correlates with insulin resistance and T2DM in adults (Bigornia et al., 2012) , potentially via mechanisms described next.

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©2021 ADRIAN MARCELO GONZALEZ GIL 21 Figure 4: Proposed origins and sequence of events leading to the obesity-associated low-grade proinflammatory state. Figures taken from (Reilly & Saltiel, 2017) (top) and (Wensveen et al., 2015) (bottom).

See text for details. B: B lymphocyte; CD4: Helper T lymphocyte; CD8: Cytotoxic T lymphocyte; DAMPs:

Damage-associated molecular patterns; ECM: Extracellular matrix; FFA: Free fatty acids; IFN-γ: Interferon gamma; IL: Interleukin; M1: Classically activated macrophage; M2: Alternatively activated macrophage; MCP- 1: Monocyte chemoattractant protein 1; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells;

Nφ: Neutrophil; NK: Natural killer cell; NKp46:Natural cytotoxicity triggering receptor 1; NKp46-L: NKp46 ligand; NLR:NOD-like receptor; NLRP3: NLR family pyrin domain containing 3; PAMP: Pathogen-associated molecular pattern; RhoA-Rock: Ras homolog family member A and Rho-associated protein kinase; TLR: Toll- like receptor; TNF: Tumor necrosis factor.

Overall, M1 macrophages perpetuate the local proinflammatory environment in WAT and eventually, cytokine overproduction results in spill-over to the systemic circulation (Wensveen et al., 2015), which contributes to systemic in addition to local insulin resistance via mechanisms that involve alterations in pathways downstream to the insulin receptor, as shown in figure 5 (DeFronzo et al., 2015). Specifically, proinflammatory cytokines induce insulin resistance by activating kinases such as IκB kinase-β (Iκκ-β), JUN amino-terminal kinase 1 (JNK1), and p38 mitogen-activated protein kinases (MAPKs), which phosphorylate serine residues in insulin-receptor substrate (IRS) proteins (Arkan et al., 2005; de Alvaro et al., 2004). These cytokines also induce insulin resistance by stimulating the production of suppressor of cytokine signaling (SOCS) proteins that result in a signaling blockade downstream to the insulin receptor (Howard & Flier, 2006; Lebrun & Van Obberghen, 2008). Inflammation-induced insulin resistance operates physiologically in context of viral and bacterial infections, which serves to deviate glucose away from peripheral tissues to fuel activated immune cells that rely on glycolysis for their function (Wynn et al., 2013). However, when chronic, insulin resistance becomes pathologic and contributes to metabolic derangements that eventually cause a vicious self-perpetuating cycle that leads to the development of T2DM. In fact, the global effects of hyperglycemia, dyslipidemia, increased circulating FFAs (which exacerbate insulin resistance themselves), and hypertension frequently observed in obesity and T2DM are largely explained by the summed effect of insulin resistance in insulin-responsive tissues (Reilly &

Saltiel, 2017). As well, the inflammatory state accelerates atherogenesis and contributes to macrovascular complications of diabetes, such as stroke and coronary artery disease (Fatkhullina et al., 2016).

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©2021 ADRIAN MARCELO GONZALEZ GIL 22 Figure 5: Proposed mechanisms of insulin resistance in context of obesity. See text for details. Figure taken from (DeFronzo et al., 2015). DAG: Diacylglycerol; ER: Endoplasmic reticulum; FA: Fatty acid; FFA: Free fatty acid; IKK:IκB kinase; IL: Interleukin; IRE1α: Inositol-requiring enzyme 1 alpha; IRS: Insulin receptor substrate;

JNK: JUN amino-terminal kinase; mTOR: Mammalian target of rapamycin; PAI1: Plasminogen activator inhibitor-1; PI3K:Phosphoinositide 3-kinase; PKC: Protein kinase C; RBP4: Retinol-binding protein 4; ROS:

Reactive oxygen species; SOCS: Suppressor of cytokine signaling; TLR4: Toll-like receptor 4; TNF: Tumor necrosis factor; TNFR: Tumor necrosis factor receptor; UPR: Unfolded protein response; XBP1: X-box binding protein 1.

Importantly, inflammation is also involved in β-cell degeneration in the context of T2DM in a pathologic process coined as “insulitis” (Donath & Shoelson, 2011), as evidenced by high local concentrations of IL-1β, cytotoxic T-cell infiltrates, and amyloid deposits in pancreatic islets, which induce dysfunction and apoptosis of β-cells that are already stressed due to gluco- and lipotoxicity (see figure 6). On the other hand, endoplasmic reticulum stress, which is induced by chronically increased demand for insulin, also plays an important role in β-cell loss by activating the unfolded protein response (UPR), which if unresolved, leads to apoptosis (Fonseca et al., 2011). Furthermore, the UPR is also involved directly in insulin resistance in peripheral tissues (DeFronzo et al., 2015).

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©2021 ADRIAN MARCELO GONZALEZ GIL 23 Figure 6: Mechanisms of β-cell stress and dysfunction in context of obesity and T2DM. Among one of the mechanisms, inflammatory mediators have been hypothesized to play an important role in β-cell dysfunction (Donath & Shoelson, 2011; Fonseca et al., 2011). ASC: Apoptosis‑associated speck‑like protein containing a CARD; ATP: Adenosine triphosphate; CCL2: Chemokine (C-C motif) ligand 2 [MCP-1: Monocyte- chemoattractant protein-1]; CXCL8:Chemokine (C-X-C motif) ligand 8 [IL-8]; ER: Endoplasmic reticulum; FFA:

Free fatty acids; IFN-γ: Interferon gamma; IL-1β: Interleukin 1 beta; IL-1R: Interleukin 1 receptor; IL-1RA:

Interleukin 1 receptor antagonist; MyD88: Myeloid differentiation primary‑response protein 88; NF-κB:

Nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3:NLR family pyrin domain containing 3;

TLR: Toll-like receptor; TNF: Tumor necrosis factor; TXNIP: thioredoxin‑interacting protein; TXR: Thioredoxin.

Yet another recognized consequence of macrophage-mediated meta-inflammation is brown and beige adipose tissue (BAT) dysfunction, which is relevant considering the insulin- sensitizing and energy-dissipating properties of these adipose depots (Bartelt & Heeren, 2014).

Indeed, as recently reviewed by Villarroya and colleagues, there is a growing body of evidence supporting the concept that M2 anti-inflammatory macrophages are crucial for BAT activity and that proinflammatory cytokines impair its function (Villarroya et al., 2018). This is in line with the decreased BAT function that has been described in patients with obesity in many studies, as reviewed by Alcala et al. (Alcalá et al., 2019).

The proposed mechanisms through which macrophages modulate BAT function are summarized in figure 7. Briefly, M1 macrophages have been described to reduce local levels of

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norepinephrine through its internalization and catabolism (Pirzgalska et al., 2017; Villarroya et al., 2018), to impair sympathetic nerve ending sprouting in adipose tissue (Wolf et al., 2017), and to hinder beige differentiation through TNF-α and integrin contact-dependent mechanisms (Chung et al., 2017). All of these mechanisms diminish BAT activity, an expected consequence considering that β3 adrenoceptor-mediated signaling is crucial for activation of lipolysis and thermogenesis in BAT (Larabee et al., 2020). In addition, a phenomenon of “catecholamine resistance” secondary to chronic exposure to TNF-α has been described in adipocytes in the context of obesity (Reilly &

Saltiel, 2017) which further adds to BAT function impairment. On the other hand, M2 macrophages could activate BAT by supporting non-inflammatory removal of adipocyte remnants, by secreting natural PPAR-γ ligands that promote beige adipogenesis, such as 9- and 13-hydroxyoctadecadienoic (HODE) acids (Lee et al., 2016), or through direct synthesis of norepinephrine (Nguyen et al., 2011;

Qiu et al., 2014), although the latter mechanism has been challenged and must be explored further (Fischer et al., 2017; Villarroya et al., 2018). Other immune cells, such as eosinophils, ILC2, and iNKT cells have also been hypothesized to play a direct role in BAT function, but may also exert their function indirectly my modulating the phenotype of macrophages in a paracrine fashion through cytokine-dependent mechanisms (Villarroya et al., 2018).

Altogether, restoring BAT function has been viewed as a potential therapeutic target in obesity (Scheele & Nielsen, 2017), and thus, restoring the M2 phenotype in obese adipose tissue depots could indirectly result in increased BAT activity, potentially restoring insulin sensitivity. In this regard, a potential therapeutic target is irisin, whose first-described function to increase WAT browning (Boström et al., 2012) could also be in fact an indirect effect of its recently reported M2- polarizing properties (Dong et al., 2015; Ye et al., 2019), as discussed in the following sections.

Figure 7: Macrophages and other immune cells control BAT function. See text for details. Figure taken from:

(Villarroya et al., 2018). ADM2: Adrenomedullin 2; CCL: CC chemokine ligand; FGF21: Fibroblast growth factor 21; IL: Interleukin; IL-4Rα: IL-4 receptor alpha chain; ILC2: Type 2 innate lymphoid cells; iNKT: Invariant natural killer T cell; MetEnk: Methionine enkephalin; METRNL; Meteorin-like; MAOA: Monoamine oxidase-A; NE:

Norepinephrine; Plex A4: Plexin A4; Sem-6A: Semaphorin-6A; TH: Tyrosine hydroxylase; TNF-α: Tumor necrosis factor alpha; VCAM-1: Vascular cell adhesion molecule 1.

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2.2. Irisin

2.2.1. A pleiotropic molecule?

Irisin has been shown to exert effects in multiple target tissues in preclinical studies, which are summarized in figure 8. Besides its originally described role in adipose tissue browning, there is in vitro and in vivo evidence, mostly in murine models, that irisin may exert other favorable metabolic effects directly or indirectly, acting mainly in adipose tissue, SkM, and the liver (Perakakis et al., 2017), but also on pancreatic β-cells (Zhang et al., 2018) and a wide variety of other cell types, including inflammatory cells (Gonzalez-Gil & Elizondo-Montemayor, 2020; Rabiee et al., 2020), which are the focus of this work. In human primary white adipocytes, irisin has been shown to increase glucose uptake, possibly by increasing expression of GLUT-4 transporters via a mechanism that is independent of insulin (J. Y. Huh et al., 2014), and in 3T3-L1 murine adipocytes, to stimulate lipolysis through a mechanism dependent on hormone-sensitive lipase and to inhibit lipid accumulation by decreasing adipocyte size (Xiong et al., 2015). In SkM, irisin modulates metabolic processes by phosphorylating and activating Adenosine 5'-Monophosphate (AMP)-Activated Protein Kinase (AMPK), which results in increased expression of GLUT-4, HK2, PPARA, and decreased expression of PYGM and PCK1 in irisin-treated human primary myocytes, leading to enhanced glucose and lipid uptake and decreased glycogenolysis and gluconeogenesis (Joo Young Huh et al., 2014). Similar to effects observed in adipose tissue, irisin also stimulates mitochondrial biogenesis and thermogenesis in C2C12 murine skeletal myocytes (Vaughan et al., 2014). In AML12 murine hepatocytes, irisin has been shown to attenuate oxidative stress (Batirel et al., 2014), to promote glycogenesis through activation of glycogen synthase, and to inhibit gluconeogenesis via downregulation of PCK1 and G6PC through the PI3K-AKT-FOXO1 pathway in HepG2 cells (Liu et al., 2015), as well as to decrease lipogenesis and accumulation of intracellular lipids in AML12 cells and murine primary hepatocytes (Park et al., 2015).

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by A.M.G.G. (2021). (b) Proposed mechanisms for irisin’s metabolic effects in skeletal muscle, white adipose tissue, and the liver. Taken from (Perakakis et al., 2017). ACC:Acetyl-CoA carboxylase; ADAM10: A Disintegrin

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©2021 ADRIAN MARCELO GONZALEZ GIL 27 and metalloproteinase domain-containing protein 10; AMPK: AMP-activated protein kinase; Arg-1: Arginase 1; ATP: Adenosine triphosphate; BAT: Brown or beige adipose tissue; cAMP: Cyclic adenosine monophosphate; CD: Cluster of differentiation; CNS: Central nervous system; COX2: Cyclooxygenase 2; ERK:

Extracellular signal-regulated kinase; FABP4: Fatty acid binding protein 4; FAS: Fatty acid synthase; FNDC5:

Fibronectin type III domain-containing protein 5; FOXO1: Forkhead box protein O1; G6PC: Glucose-6- phosphatase; GLUT4: Glucose transporter type 4; GSK3: Glycogen synthase kinase 3; GS: Glycogen synthase;

HK2: Hexokinase 2; HO-1: Heme oxygenase 1; HSL: Hormone-sensitive lipase; IL: Interleukin; MAPK: Mitogen- activated protein kinase; MyD88: Myeloid differentiation primary response 88; NF-κB:Nuclear factor kappa- light-chain-enhancer of activated B cells; Nrf2: Nuclear factor erythroid 2-related factor 2; PCK1:

Phosphoenolpyruvate carboxykinase 1; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PI3K:Phosphoinositide 3-kinase; PKA: Protein kinase A; PPAR:Peroxisome proliferator- activated receptor; PRMT3: Protein arginine N-methyltransferase 3; PYGM: Glycogen phosphorylase, muscle isoform; SkM: Skeletal muscle;SREBP1C: Sterol regulatory element-binding protein 1c; TFAM:Mitochondrial transcription factor A; TLR4: Toll-like receptor 4; TNF: Tumor necrosis factor; UCP: Uncoupling protein; WAT:

White adipose tissue;

2.2.2. Irisin in clinical studies

The physiologic relevance of irisin in humans, and even its existence, has been questioned (Raschke et al., 2013). Indeed, evidence is still limited in human tissues (Maak et al., 2021).

Consequently, although much remains to be explored about the physiology of irisin in humans, based on preclinical observations, irisin or its downstream targets could play a therapeutic role in obesity, the MetS, T2DM, and other metabolic diseases (Polyzos et al., 2018). This has set ground for researchers to study the association between irisin and clinical and paraclinical parameters in a wide range of pathologic contexts in humans. However, clinical studies in obesity, MetS, and T2DM to date have generated controversy in both adults (Polyzos et al., 2018) and children (Elizondo- Montemayor et al., 2018) due to contradictory findings and inconsistencies between results and irisin’s predicted metabolic roles. These discrepancies have been attributed to many factors, including the lack of standardization of commercial ELISA kits used to measure circulating irisin, nonspecific binding of antibodies to other serum proteins, suboptimal study designs, and heterogeneity of analyzed subjects, all of which make findings difficult to interpret (Perakakis et al., 2017). Nevertheless, this path of controversy has already been journeyed by other hormones discovered in previous decades, including leptin, adiponectin, and resistin (Perakakis et al., 2017).

Further well-designed future studies are expected to respond to many unresolved questions and controversies.

2.2.3. The possible anti-inflammatory properties of irisin: Preclinical models

Recently, interest has grown around irisin’s putative immunomodulatory role. There is preclinical evidence that irisin possesses anti-inflammatory and antioxidant properties in a wide variety of tissues, which has led it to be considered a potential therapeutic target in inflammatory diseases (Askari et al., 2018). Studies that target these properties are summarized in table 1. It has

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been demonstrated that irisin decreases production of proinflammatory cytokines and attenuates oxidative stress in murine macrophage and adipocyte cell lines (Mazur-Bialy, 2017; A. I. Mazur-Bialy et al., 2017; Mazur-Bialy et al., 2018; Mazur-Bialy & Pocheć, 2021; Agnieszka Irena Mazur-Bialy et al., 2017) and in murine in vivo models of obesity (Xiong et al., 2018). However, it is currently unclear whether these actions could be translated to humans, as there are currently no reports that have directly determined the role of irisin in human immune cells of any type. Nevertheless, Li et al reported that administration of irisin to cultured fragments of whole human visceral and subcutaneous adipose tissue significantly reduced mRNA expression of key proinflammatory cytokines TNF-α, IL-6, MCP-1, and MIP-1α, while increasing expression of the anti-inflammatory cytokine IL-10 (H. Li et al., 2019). These findings suggest that irisin could act on human adipocytes or cells of the stromal vascular fraction, including macrophages. It is also currently unknown whether irisin, by itself, is sufficient to exert significant anti-inflammatory actions in vivo or if it requires cooperation with Th2-type cytokines, such as IL-4. This would be expected, as the αVβ5 integrin heterodimer, irisin’s newly identified receptor in osteocytes and adipocytes (Kim et al., 2019), has been demonstrated to be upregulated by IL-4 in a PPAR-γ-dependent manner in human PBMCs and to promote M2 macrophage polarization (Yao et al., 2018).

On the molecular level, irisin has been hypothesized to exert its anti-inflammatory effects by modifying signaling pathways involved in the activation of NF-κB (Jiang et al., 2020; Jin et al., 2020; Agnieszka Irena Mazur-Bialy et al., 2017; Yu et al., 2020). Specifically, irisin was shown to decrease protein levels of TLR4 and downstream MyD88, which was associated with decreased NF- κB activity in RAW 264.7 macrophages (Agnieszka Irena Mazur-Bialy et al., 2017). A similar effect has been independently observed in other cell types, including hippocampal neurons in context of a murine ischemia-reperfusion injury model (Yu et al., 2020) and human proximal tubule cells (Jin et al., 2020). The TLR4 signaling pathway is a crucial activator of the innate immune system and an ancient defense mechanism against virulent microorganisms that is conserved in all mammal species. However, TLR4 is not only activated by LPS on the surface of Gram-negative bacteria (Murphy & Weaver, 2017a), but also by other ligands, such as FFAs (e.g. palmitate) that are coupled to fetuin A (Meex & Watt, 2017), as previously discussed. FFAs are elevated in the context of obesity and T2DM, and are also directly involved in the establishment of insulin resistance by inhibiting molecules downstream to the insulin receptor (Shi et al., 2006). Thus, by interfering in the TLR4 pathway, irisin could potentially inhibit or attenuate proinflammatory responses, which could in turn explain the enhanced basal insulin sensitivity in peripheral tissues that is observed in physically active individuals.

Besides modulating the TLR4/NF-κB pathway directly, a complementary explanation for irisin’s reported anti-inflammatory effects involves modulation of endogenous antioxidant defense systems (Deng et al., 2018; Du et al., 2019; Mazur-Bialy et al., 2018; Mazur-Bialy & Pocheć, 2021) and the pleiotropic energy sensor AMPK (Jiang et al., 2020; X. Li et al., 2019; Xiong et al., 2018; Ye et al., 2019). Mazur-Bialy and colleagues recently described the kinetics of nuclear factor (erythroid- derived-2) like 2 (Nrf2)-dependent heme oxygenase-1 (HO-1) and other antioxidant enzyme

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expression in RAW 264.7 macrophages when irisin is applied both 2 hours before and 2 hours after LPS stimulation (Mazur-Bialy & Pocheć, 2021). In rat intestinal epithelial cells (IEC-6) subjected to hypoxia/reoxygenation injury, the anti-inflammatory effects of irisin were abrogated following Nrf2 siRNA transfection (Du et al., 2019), further evidencing that irisin’s anti-inflammatory effects depend, at least partially, on enhanced activity of Nrf2. Considering that Nrf2 and NF-κB pathways are intimately intertwined and are known to engage in molecular crosstalk through complex transcriptional and post-transcriptional mechanisms (Ahmed et al., 2017; Cuadrado et al., 2018;

Lingappan, 2018; Wardyn et al., 2015), it is likely that irisin, through its antioxidant actions, may indirectly influence the inflammatory response in multiple cell types.

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Report Model M1/M2 Mediators TLR

4 My D88

NF- κB

ROS Nrf2 AOE AMPK SIRT1 (X. Li et al.,

2019)

Sepsis-induced lung injury (in vivo &

A549 cells)

- ↓TNF-α, IL- 1β, MCP-1

- - ↓ - - - ↑ ↑

(Du et al., 2019) Intestinal I/R injury (in vivo & IEC-6

cells)

- ↓TNF-α, IL- 1β, IL-6

- - - ↓ ↑ ↑ - -

(Yu et al., 2020) Brain I/R injury (in vivo)

- - ↓ ↓ ↓ - - - - -

(Deng et al., 2018)

AGE-induced endothelial dysfunction (HUVECs)

- ↓IL-1β, IL- 18 (↓NLRP3)

- - - ↓ - - - -

(Jin et al., 2020) Renal injury caused by sepsis (HK-2

cells)

- ↓TNF-α, IL- 1β, IL-6

- - ↓ - - - - -

(Jiang et al., 2020)

Spinal cord injury (in vivo)

- ↓TNF-α, IL- 1β, IL-6;

COX-2, iNOS

- - ↓ ↓ - ↑SOD2 ↑ -

(H. Li et al., 2019)

Whole human visceral/subcutane ous adipose tissue

culture

- ↓TNF-α, IL- 1β, MCP-1, MIP-1α,

↑IL-10

- - - - - - - -

(Mazur-Bialy &

Pocheć, 2021)

RAW 264.7 murine macrophages

- ↓HMGB1 - - - ↓ ↑ ↑HO-

1, ↓ GpX, SOD1,

Cat-9

- -

(Agnieszka Irena Mazur-Bialy et

al., 2017)

RAW 264.7 murine macrophages

- ↓TNF-α, IL- 1β, IL-6,

MCP-1, HMGB1

↓ ↓ ↓ - - - - -

(Ye et al., 2019) RAW 264.7 murine macrophages

↑CD206, ARG-1;

↓CD86

↑ TGF-β,

↓TNF-α

- - - - - - ↑ -

(Dong et al., 2015)

Mouse peritoneal macrophages

↑CD206, CD163,

↓CD86

↓TNF-α, IL- 6, MIP-1α, MIP-1β

- - - - - - ↑ ? -

(Xiong et al., 2018)

FNDC5^-/- mice fed HFD, LPS-treated RAW 264.7 &

peritoneal macrophages

↑CD206, MGL1

↓CD11c, CD68

↓TNF-α, IL- 1β, IL-6, IL-8,

↑IL-10

- - ↓ - - - ↑ -

AGE: Advanced glycation end product; AMPK: AMP-activated protein kinase; AOE: Antioxidant enzymes; Cat-9: Catalase-9; CD: Cluster of differentiation; COX2: Cyclooxygenase-2; Gpx: Glutathione peroxidase; HMGB1:High mobility group box 1; HO-1: Heme oxygenase 1; I/R:

Ischemia-reperfusion; IL: Interleukin; iNOS: Inducible nitric oxide synthase; M1/M2: Macrophage polarization markers; MCP-1: Monocyte chemoattractant protein 1; MGL1: Macrophage galactose-type lectin 1; MIP: Macrophage inflammatory protein; MyD88: Myeloid differentiation primary response 88; Nrf2: Nuclear factor erythroid 2-related factor 2; ROS: Reactive oxygen species; SOD1: Superoxide dismutase 1; SIRT1: Sirtuin 1; TGF-β: Transforming growth factor beta; TLR-4: Toll-like receptor 4; TNF: Tumor necrosis factor;

The role of irisin on the polarization of monocyte-derived macrophages from pediatric patients with type 2 diabetes mellitus

The role of irisin on the polarization of monocyte-derived macrophages from pediatric patients with type 2 diabetes

mellitus

A thesis presented as a partial requirement to obtain the academic degree of:

Master of Science in Biomedical Sciences

By:

Adrián Marcelo González Gil

Monterrey, Nuevo León, June 3, 2021

Dedication

Acknowledgements

Abstract

The role of irisin on the polarization of monocyte-derived macrophages from pediatric patients with type 2 diabetes mellitus

Table of contents

1. Problem Statement

1.1. Background

1.2. Problem statement

1.3. Objectives

1.4. Justification

1.5. Scope

2. Theoretical Framework

2.1 Obesity and type 2 diabetes mellitus

2.2. Irisin