Background
The metabolic syndrome that may include insulin resistance, NAFLD, chronic low-level inflammation, as well as other serious co-morbidities [
1]. Precipitated mainly by obesity, the metabolic syndrome may eventually lead to type 2 diabetes (T2D) [
1]. Evidence indicate that a number of co-morbid conditions begin to develop even before the onset of impaired glucose tolerance and insulin insensitivity [
2]. One of the co-morbidities that is on the rise globally, in terms of prevalence and incidence, is non-alcoholic fatty liver disease (NAFLD) [
3]. The liver of healthy subjects maintains steady state circulating glucose that quickly responds to dynamic fluctuations of glucose resulting from feeding, fasting, and physical activity. Obesity precipitates glucose intolerance as well as insulin resistance (IR). The theory is that metabolite accumulation resulting from excess nutrient supply are sensed causing a pro-inflammatory response, especially the innate arm. This results in a hepatic inflammatory state that aggravates an already-precarious state of metabolic control [
4]. A number of molecular mechanisms have been identified in the systemic and hepatic inflammatory responses including elevated concentrations of pro-inflammatory immunokines [
4], non-physiologic circulating levels of adipokines [
5], and hepatic accumulation of leukocytes which together with activated Kupffer cells, or independently of them [
6], create a pathologic state of hepatocyte function that contribute to glucose intolerance and IR.
One of the first observations that implicated inflammation in the progression of obesity to the metabolic syndrome and T2D, was the accumulation of activated macrophages inside adipose tissue of obese non-diabetic as well as obese diabetic patients [
7]. These macrophages produce a wide array of pro-inflammatory immunokines that confer IR and impaired glucose uptake [
8]. While macrophages are not the only leukocytes that progressively-accumulate inside hypertrophic adipose, they are the first—together with neutrophils [
9]. Neutrophils comprise the largest population of circulating leukocytes in humans (between 50 and 70% of all circulating leukocytes and together with tissue-resident macrophages are the earliest to be activated and recruited inside an anatomical site that exhibits “damage” and “danger”, including microanatomical regions of hypertrophied adipose, alone or as part of a specific tissue (e.g. skeletal muscle and liver). Activated neutrophils can mobilize tissue-resident leukocytes by producing immunokines, chemokines, as well as by direct contact [
10]. Accumulating data indicate that neutrophils are critical leukocytes in the onset of IR and T2D [
11,
12].
Systemic neutrophil depletion improves fasting blood glucose levels and prevents high fat diet-induced hepatic structural disorders [
13]. Neutrophils are recruited to a site of “damage”/”danger” mainly by the balance of CXCR2 and CXCR4 ligands and the neutrophil cell surface ratio of CXCR2:CXCR4 chemokine receptors [
14]. Mice lacking CXCR2 exhibit impaired neutrophil recruitment and reduced tissue damage in a model of acute pancreatitis suggesting a failure to initiate local inflammation processes [
15]. CXCR2-deficient mice are protected from high fat diet-induced IR and T2D and are characterized by reduced macrophage accumulation in adipose [
16]. One of CXCR2 chemokine ligands, CXCL5, has been shown to be elevated in obese mice and humans, and in mice it was shown to induce IR in a CXCR2-dependent manner [
17]. Another CXCR2 ligand, IL-8, was shown to be elevated in the circulation of obese individuals [
18] and its steady-state circulating levels correlate well with adiposity and insulin resistance [
19].
CXCR2 blockade has been explored as a possible treatment of several neutrophil-driven pathologies [
20]. We surmised that impairing neutrophil accumulation could improve insulin sensitivity, prevent the progression of high fat diet induced diabetes, and prevent the progression towards NAFLD. We used a selective CXCR2 antagonist, AZD5069 [
20] to test our hypothesis. AZD5069 was initially developed to inhibit neutrophil recruitment into, and activation inside, inflamed airway tissue [
20]. Although pre-clinical studies indicated good efficacy in a number of airway inflammatory conditions, outcomes in human trials were not as impressive, even though the study agent was very well-tolerated with few side effects [
21]. CXCR2 ligands are expressed in the pancreas, adipose and liver [
22], suggesting that under potentially-stressful states, their secretion can be expected to recruit and activate neutrophils, which in turn would exacerbate and amplify a low grade inflammatory condition [
23]. In spite of the evidence linking neutrophils and CXCR2 to T2D and possibly T2D-associated NAFLD [
17], to date there are no studies aimed at short-circuiting neutrophil function and neutrophil-driven inflammation independently of macrophages—well known participants in the progression of HFD pathology towards NAFLD/NASH [
24‐
35]—in progressive obesity-driven metabolic dysregulation that results in T2D and associated NAFLD. Herein, we show for the first time, using the selective CXCR2 antagonist AZD5069 in high fat fed mice, improved insulin-induced suppression of hepatic glucose production, decreased hepatic lipid storage, and a significant prevention of progression towards liver pathology reminiscent of NAFLD.
Discussion
“Pre-diabetes” is characterized by impaired glucose tolerance with an insidious onset of IR, even though fasting plasma glucose (FPG) and HbA1c values may well be within a range considered physiological. If treated early, the metabolic syndrome—mostly reflected as clinical T2D onset—can be delayed or prevented [
43]. While metformin is often the first approach, it is neither insufficient on its own to prevent the progression towards T2D nor to attenuate the underlying low grade chronic inflammation which accompanies the progression towards IR in insulin-sensitive tissues as well as the impairment of pancreatic β cell function [
44]. Low-grade systemic inflammation persists during clinical T2D [
45]. Hyperglycemia and hyperlipidemia, characteristic of T2D, further fuel inflammation, adding to the impairment and damage to insulin-producing and insulin-sensitive tissues and organs through the mechanisms that underlie glucose toxicity and lipotoxicity. By intercepting key immune cellular actors driving this inflammation and/or activating those that attenuate or suppress it, we postulated we could improve insulin sensitivity and β cell function. Improvements in these variables can lead to a decreased risk of developing NAFLD and T2D.
Epidemiologic evidence indicates that at least 50% of individuals diagnosed with T2D also exhibit NAFLD/NASH [
46,
47] which, independent of all other T2D comorbid conditions, can accelerate the progression of metabolic and cardiovascular diseases [
47]. In obese individuals, NAFLD/NASH is a co-morbidity that coincides with IR [
48]. There is strong evidence that inflammation driving IR promotes metabolic abnormalities in liver [
49]. Among all leukocyte populations other than macrophages, neutrophils appear to be just as, if not more relevant, in the onset and progression of obesity-driven IR [
12,
50‐
52]. Moreover increasing evidence suggest that neutrophils precipitate NAFLD/NASH in obese T2D patients [
53,
54].
Neutrophils are a leukocyte population that, with macrophages [
24‐
35], are the earliest to accumulate in insulin-sensitive tissues with weight gain [
12,
50,
51]. We therefore tested whether targeting only neutrophil accumulation independently of macrophages—whose role is already well-established in the progression of NAFLD/NASH [
24‐
35]—could improve insulin sensitivity, prevent the progression of HFD-induced IR, and possibly prevent the progression towards a liver pathology that could be prodromal of, and/or precipitate NAFLD/NASH. Despite a mechanistic rationale linking neutrophils and CXCR2 to IR and possibly IR-associated NAFLD [
17], to date, there are no studies aimed at short-circuiting only neutrophil function and only neutrophil-driven inflammation in progressive obesity-driven metabolic dysregulation that results in IR and associated liver pathology precipitating NAFLD/NASH. We chose to use a selective CXCR2 antagonist, AZD5069 [
20] since the pancreas, adipose and liver express CXCR2 ligands [
22] under stress conditions. Hence, such ligands are able to recruit and activate neutrophils, which in turn may amplify a low grade inflammatory condition [
23]. Use of this selective CXCR2 antagonist also allowed us to dissect the effect of neutrophil-selective impairment independently of macrophages on insulin sensitivity and liver pathology under HFD conditions.
The major findings of this study are that AZD5069 formulated HFD that cause causes liver pathology reminiscent of NAFLD with features suggesting NASH, can improve insulin sensitivity, modestly prevent weight gain and reduce fasting glucose in mice fed a HFD. Remarkably, mice treated with AZD5069 exhibited an improvement in liver pathology with reduced lobular inflammation and neutrophil accumulation, decreased hepatocyte ballooning (Additional file
1: Figure S1A) concomitant with improved NAS score. At the mechanistic level, AZD5069 may be beneficial on attenuating the progression of T2D-associated NAFLD/NASH most probably by preventing the activation and minimizing the accumulation of neutrophils into the liver, independently of macrophages, as well as inside the peripheral insulin-sensitive tissues where HFD causes adipose hypertrophy and accumulation. Although our outcomes support a protective role for AZD5060 in the progression of impaired insulin sensitivity and liver pathology, it remains to be determined whether this agent or others in its drug class (CXCR2 antagonists alone) can be effective when treatment is initiated at a later stage of pathology to reverse the progression of hepatic lesions reminiscent of NAFLD/NASH. Other studies in HFD models of NAFLD/NASH have observed improved liver pathology and NAS score under neutrophil-depleting conditions [
13]. Additionally, AZD5069 treatment also reduced liver lipid content and hepatic glucose production, both hallmarks of IR in NALFD/NASH. In this study, we measured adiponectin because of its known anti-inflammatory and insulin-sensitizing properties [
55]. We hypothesized that AZD5069 would reduce neutrophil accumulation in hypertrophic adipose tissue with the benefit of removing restraint on adiponectin production by neutrophil-produced immunokines. However, our data did not support this hypothesis as an increase in adiponectin was not increased in HFD + AZD fed mice compared to those in the HFD arm (Fig.
4B).
It is worth noting that, although neutrophils are expected to be the primary cells targeted by AZD5069, there are other tissues where the agent may be exerting its actions in its overall effects on improvement of insulin sensitivity. For example, previous studies have shown a CXCR2 mediated inhibitory effect on insulin-induced glucose transport in muscle cells mediated by activation of the JAK/STAT pathway and stimulation of the expression of SOCS-2, a known insulin receptor inhibitor [
56]. In addition, scientific evidence highlights IL-8 as a main adipocytokine producing insulin resistance via the inhibition of insulin-induced Akt phosphorylation in adipocytes [
57,
58]. Furthermore, very recent data highlighted a specific role of CXCR2 expressed in adipocytes in the modulation of murine adipocyte response to high glucose concentration and shed light on its role in the regulation of the proinflammatory response and insulin sensitivity [
59]. Thus, CXCR2 antagonists may very well be acting at multiple sites to achieve a global improvement in insulin sensitivity. This furthers strengthens the rationale that these agents can be potentially helpful treatments in insulin resistance-incident obesity and T2D. We extend these observations and suggest that this class of compounds may also be important, through these same mechanisms, to improve T2D-associated liver pathology such as NAFLD/NASH.
HFD-induced, increased hepatic lipogenesis is an important metabolic abnormality underlying the pathogenesis of hepatic steatosis in insulin-resistant livers. NASH is histologically similar to alcohol-induced steatohepatitis, a disease that can progress to cirrhosis and liver failure. Many of the factors implicated in the development of alcoholic steatohepatitis are also known to be associated with NASH. These factors can be grouped into two broad categories: factors causing an increase in oxidative stress and factors promoting expression of proinflammatory cytokines. Briefly, under HFD conditions, triglyceride and free fatty acid formation contributes to peripheral as well as hepatic insulin resistance [
60‐
65]. The mechanisms in liver, previously-identified, include increased free fatty acid oxidation, oxidative stress and free fatty acid-induced upregulation and activation of PPARγ and AMP Kinase. Together, these diet-induced effects can cause hepatocytes to metabolically-shift towards a pro-apoptotic, inflammation-triggering state. Cui et al. [
66] have shown that a CXCL8 analog, referred to as K11R/G31P, designed to be an antagonist of CXCL8 ligands, binding to CXCR1 and CXCR2 receptors, prevented progression of insulin resistance and liver pathology reminiscent of hepatosteatosis, when administered into young
db/db mice. Along with reduced accumulation of neutrophils into liver, these investigators also discovered that antagonist administration was associated with suppression of gluconeogenesis by decreased hepatic glucose-6-phosphatase and phosphoenol pyruvate carboxykinase activities. Additionally, they observed increased levels of phosphorylated Akt in liver [
66]. It remains to be determined if AZD5069, which targets only CXCR2 without any action on macrophages, or other agents in its drug class interfere or modify these biochemical, enzymatic, and biological processes, directly or indirectly, thus strengthing the view that neutrophils are primary leukocytes in driving high fat diet/obesity-associated liver pathology. While CXCR2 antagonism prevents the accumulation of neutrophils inside inflamed liver, its effects on metabolic activity are very likely to contribute, independently of effects on neutrophils, to the effects we observed in this study. The expression of CXCR2 on insulin-responsive muscle and adipose indicates that AZD5069 could be acting on these tissues, concomitant with actions on neutrophils, as CXCR2 activation in muscle and adipose results in impaired insulin-induced glucose transport [
56] and insulin resistance [
57,
58].
Features of NASH in the progressive liver pathology was observed reliably in our HFD-only treated mice. We are very aware of the limitations of the HFD used in this study in its ability to cause clinically- and molecularly-distinguishable NASH and we acknowledge that other diets can be more “NASHogenic” [
67‐
69]. However, there is an ongoing and vigorous discussion concerning which of these diets can elicit insulin resistance and verifiable NASH concomitantly [
67‐
69]. We therefore chose, in this study, to use a HFD that is generally accepted to concomitantly cause insulin resistance and liver pathology inside a reasonable timeframe. Inflammation and steatosis were consistently seen in liver sections from mice in the control treatment arm. The pathology resembles what is often observed in liver biopsies from humans exhibiting NAFLD/NASH. The most remarkable finding in liver sections from mice treated with AZD5069 was an improved composite NAS score. NAS is a widely-accepted method of evaluating human NAFLD and NASH [
70,
71]. Recently, NAS has been shown to be verifiably-associated with change in fibrosis stage in humans [
72]. In our study, the lower NAS score in liver sections from mice treated with AZD5069 is a consequence of attenuated lobular inflammation, steatosis, and hepatocyte ballooning. In contrast to the composite NAS score, the attenuation in fibrosis in the liver sections of AZD5069-treated mice was moderate without any signs of regression. Nevertheless, the lower density of collagen 1A1, α-SMA, and galectin-3; molecules that reflect the progression of liver fibrosis and macrophage activation [
73], suggests that AZD5069, acting to prevent the activation and accumulation of neutrophils into the liver, can achieve some protective effect against fibrosis. Increased α-SMA and collagen 1a1 levels reflect the activation of hepatic stellate cells and their mobilization into myofibroblast-like cells [
74]. Hence, the effect of AZD5069 acting to prevent the activation and accumulation of neutrophils into the liver, may disallow this conversion, thereby preventing collagen deposition and fibrosis. The unraveling of these potential mechanisms, where neutrophils and a neutrophil-based microenvironmental inflammatory state drive them, compel further study of CXCR2 antagonists alone or in combination with other drugs that act at different stages of the pathophysiology of the disease. For example, medications that decrease body weight and thus % fat in the liver, incretins that act by both decreasing liver fat and improving inflammation, other insulin sensitizers such as pioglitazone or CHS-131 that act as PPARγ agonists and finally with medications that may act as antifibrotic agents.
A limitation of our study, herein, are the known differences between human and rodents in regard of CXCR1/2 expression and function. In humans, IL-8/CXCL8 (and CXCL6, also known as GCP-2) exerts its activity by activating both CXCR1 and CXCR2 [
75‐
77], whereas the other ELR-CXC chemokines selectively bind CXCR2 [
77]. By contrast, up until recently, only one functional ELR-CXC receptor was identified in mice and was characterized as the homologue of human CXCR2 [
78,
79] however, the orthologous murine CXCR1 [
80,
81] was identified and subsequently confirmed to be a functional receptor [
82], specifically activated by murine GCP-2, human GCP-2/CXCL6, and human IL-8/CXCL8. Thus, given possible differences between mice and humans, the data obtained herein using CXCR2 selective inhibition by AZD5069 caution that further studies in humans are warranted and the outcomes in this study should be verified in human T2D patients.
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