Hyperglycemia Induces Inflammatory Response

Hyperglycemia Induces Inflammatory Response: History

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Subjects: Immunology | Endocrinology & Metabolism

Contributor: Laura Matuschik

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 controls inflammation by the internalization and degradation of hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

diabetes mellitus
hyperglycemia
inflammation
macrophages
CD163
scavenger receptor
hemoglobin-haptoglobin complexes

1. Introduction

Diabetes mellitus is a heterogeneous group of metabolic disorders sharing the common ground of chronic hyperglycemia that induces micro- and macrovascular complications [1,2]. Large clinical trials, i.e., the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS), found that the duration and degree of hyperglycemia correlates with the extent of microvascular complications [3,4]. Hyperglycemia can cause vascular complications by direct and indirect mechanisms, by the activation of detrimental inflammatory pathways in endothelial cells or by activation of immune cells, primarily of myeloid origin [5,6,7,8]. It was suggested that subclinical chronic systemic inflammation creates conditions for vascular damage [9,10,11]. Chronic inflammation is characterized by only moderate elevation of inflammatory cytokines, e.g., TNFα, IL-1β, and IL-6, induced by exogenous or endogenous factors [12]. The major source of inflammatory cytokines in chronic inflammation are macrophages. We have demonstrated previously that human macrophages respond to hyperglycemia by elevated production of predominantly proinflammatory cytokines [6,13]. Hyperglycemia can induce an inflammatory program in innate immune cells on the epigenetic level, as we showed for the enhanced presence of activating histone marks on the promoters of S100A9 and S100A12 genes, responsible for vascular inflammation in diabetes [7,14,15,16]. However, macrophages control inflammation not only by the release of proinflammatory factors, but also by their complex scavenging activity, which can be silent and tolerogenic, or can provoke additional inflammation. The effect of hyperglycemia on this essential function of monocytes and macrophages is unexplored.

CD163 is a scavenger receptor expressed on circulating monocytes and on tissue macrophages in different pathologies [17,18,19,20]. CD163 controls inflammation by the internalization and degradation of hemoglobin-haptoglobin (Hb-Hp) complexes [21]. Expression of CD163 is controlled by pro- and anti-inflammatory factors, where anti-inflammatory agents, such as glucocorticoids and IL-10, stimulate CD163 expression, and proinflammatory cytokines, such as IFNγ and TNFα, suppress CD163 expression [22,23,24,25,26]. The anti-inflammatory cytokine IL-4 had no effect on CD163 protein expression [23,27].

The most comprehensively characterized function of CD163 is its homeostatic role in the scavenging of Hb-Hp complexes formed as a result of intravascular hemolysis. This mechanism physiologically occurs for 10–20% of erythrocytes and increases considerably in pathological conditions, e.g., hemoglobinopathies or inflammation [21,28]. The formation of Hb-Hp complexes protects the endothelium and kidneys from the toxicity of free hemoglobin by preventing renal deposition of free hemoglobin resulting in parenchymal and vascular damage [28,29].

Hb-Hp complexes bind with high affinity to membrane-bound CD163 of both circulating monocytes and tissue macrophages, leading to their internalization and degradation via the cytoprotective and anti-inflammatory heme oxygenase-1 [21,30,31]. This anti-inflammatory capacity is decreased in diabetes mellitus, as CD163 mRNA expression in peripheral blood mononuclear cells (PBMCs) is significantly suppressed in newly diagnosed diabetic patients [32]. Moreover, the percentage of macrophages in atherosclerotic plaques and PBMC expressing CD163 was significantly reduced in diabetic patients compared to non-diabetic individuals [28,33]. Haptoglobin, the protein binding to free extracorpuscular hemoglobin, exists in two known allelic variants: Hp1 and Hp2, leading to three possible phenotypes: Hp1-1, Hp1-2, and Hp2-2 [34]. Haptoglobin serum levels differ considerably from 0.3–3.0 mg/mL between healthy individuals but stay reasonably constant for one individual, and are saturated when 500–1500 mg/L free hemoglobin is present in serum [34,35]. During inflammation, haptoglobin, which is a hepatocyte-produced acute-phase protein, is induced 2–5-fold by the acute-phase mediators IL-1 and IL-6 and can be released locally from storage granules by active neutrophils [36,37]. The clearance and antioxidant capacity of individuals expressing Hp1-1 has been observed to be superior to Hp2-2 individuals [38,39]. Longitudinal prospective studies have demonstrated that the Hp2-2 phenotype is an independent risk factor for the development of cardiovascular disease and increased susceptibility to vascular complications in diabetic individuals in comparison with the homozygous Hp1-1 variant [33,34,40]. As a possible mechanism for the proneness to vascular complications, it has been demonstrated that CD163 is downregulated in macrophages in atherosclerotic plaques of diabetic patients with the Hp2-2 genotype, indicating a compromised hemoglobin clearing capability [33].

2. Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes

In order to sustainably treat the skyrocketing number of patients affected by microvascular complications of diabetes mellitus, it is crucial to broaden our understanding of the immunological mechanisms leading to a derogated control of vascular damage [5,42].

So far, a number of studies have tried to elucidate the role and regulation of CD163 in pathological conditions, such as diabetes mellitus or inflammation, altogether [32,33,43,44]. In recent studies, mainly samples of already differentiated tissue macrophages [33,45], undifferentiated peripheral blood mononuclear cells [32], or the plasma concentration of the shed receptor, soluble CD163 [46,47], were used. Compared to them, our group used human primary peripheral blood macrophages derived from circulating monocytes.

IFNγ alone is an effective suppressor of CD163 expression on human primary macrophages, thus impairing the scavenging capacity of proinflammatory macrophages. These results, although being observed after a longer duration of cultivation (6 days), correlate with the findings of other studies in which CD163 mRNA and surface expression were decreased on freshly isolated or one-day old peripheral blood monocytes from healthy individuals after IFNγ stimulation [22,26].

Whereas the mRNA expression of CD163 was significantly downregulated in M(IL-4) compared to M0, the surface expression of CD163 was not affected by stimulation with IL-4. In agreement with this observation, Staples et al. described a downregulation of CD163 mRNA expression upon stimulation with IL-4 [48] and both Sulahian et al. and van den Heuvel et al. demonstrated that stimulation with IL-4 did not alter CD163 surface expression compared to M0 [23,27]. Thus, healing macrophages should possess a compensatory mechanism to ensure sufficient levels of surface CD163 to ensure control over the inflammatory response.

Apart from IFNγ- and IL-4-mediated regulation of CD163, hyperglycemia elicited an additional suppression of CD163 mRNA in M(IFNγ) compared to normoglycemia. This finding is in line with the observation that CD163 mRNA expression in PBMCs of diabetic individuals was significantly lower compared to PBMCs of healthy subjects [32]. Additionally, clinical studies not only found that pre-diabetic subjects displayed a significant increase in proinflammatory M(IFNγ), but also showed that diabetic patients had an elevated M1/M2 ratio correlating with a higher prevalence of microangiopathy [42,49].

The decrease of CD163 surface expression in hyperglycemia is congruent with the results of Levy et al., who found that PBMCs acquired from diabetic individuals expressed significantly less CD163 on their cell surface than those from healthy donors [33]. This reduced scavenging capacity might lead to an elevated heme toxicity contributing to endothelial damage and indicating the susceptibility of the diabetic patient to vascular complications due to dysfunctional control of tissue damage [5,50]. The clinically observed heterogeneity of manifestation and onset of vascular diabetic complications correlates with the observed donor-dependent response of macrophages to hyperglycemia. Cytokines, such as IFNγ and IL-4, define the direction of macrophage response, whereas hyperglycemia interferes by enhancing or annulating this cytokine effect.

Suppression of CD163 expression in M(IFNγ) in hyperglycemia raised the question of whether high-glucose conditions additionally have a direct impact on the scavenging function of CD163. Although the two tested variants of CD163′s ligand haptoglobin Hp1-1 and Hp2-2 differ considerably in their molecular structure [34,51], the uptake patterns of Hb-Hp complexes matched CD163 mRNA and surface expression patterns. Remarkably, higher uptake was found for Hb-Hp2-2 complexes compared to Hb-Hp1-1 complexes in all three macrophage subpopulations, correlating with a higher affinity of the Hp2-2 variant for the CD163-binding site located in the scavenger receptor cysteine-rich (SRCR) domain 3 [21,52]. Whether the structure of haptoglobin itself is the crucial factor in the process of Hb-Hp complex internalization is still controversial. An in vitro study performed on monocytes showed that the uptake of Hb-Hp complexes is competitively inhibited by free hemoglobin, thus indicating a common binding site of free and complexed hemoglobin and demonstrating that CD163–hemoglobin interactions are not affected by changes in structure resulting from the binding process of hemoglobin to haptoglobin [53]. This seems to be in contradiction to various other studies describing CD163 as the specific receptor for hemoglobin complexed to haptoglobin but not the free hemoglobin molecule [21,39].

There have not been any studies reporting a qualitative derogation of the CD163 scavenging function. However, the clinically observed proneness to vascular complications in diabetic patients is enhanced by the limited availability—and therefore limited capacity to mitigate vessel damage—of CD163 in proinflammatory conditions [50].

In vitro studies showed the activation of protein-kinase C- and casein-kinase-dependent macrophage pathways by cross-linking of cell surface CD163, triggering the release of proinflammatory cytokines, such as IL-1β and IL-6 [23,54,55]. Moreover, it was found in human atherosclerotic plaques that the exposure to Hb-Hp complexes leads to a particular macrophage phenotype, named M(Hb) or Mhem [56,57]. This phenotype is characterized by an abundant expression of surface CD163, downregulated cytokine production, and the lack of lipid withholding [58,59,60]. As these macrophages are particularly present in areas of hemorrhage and neoangiogenesis, a role in plaque vascularization, microvessel leakage, and inflammation of the surrounding endothelium has been suggested [56], thus questioning the long-established notion that CD163⁺ macrophages are involved in the resolution of inflammation [26,50]. Adding to a possible role of CD163 in proinflammatory macrophage activation, alveolar spaces of severely infected COVID-19 lungs contained a large amount of CD163⁺ macrophages as a sign of altered airway macrophage populations and correlating with diffuse alveolar damage and worse patient outcomes [61]. Moreover, the serum levels of soluble CD163, as a marker of macrophage activation, were enhanced in COVID-19 patients [62,63].

As a possible factor contributing to diverging results, the haptoglobin variants Hp1-1 and Hp2-2 were found to have not only differences in function, but also in the involvement in pathological conditions. For instance, the clearance and antioxidant capacity of Hp1-1 by binding hemoglobin was superior to the clearance of Hp2-2 [38,39]. The release of anti-inflammatory IL-10 in response to the binding of Hb-Hp1-1 complexes to CD163 was increased compared to Hb-Hp2-2 complexes [64]. Regarding the clinical significance of the different haptoglobin variants, it has been shown in longitudinal prospective studies that the Hp2-2 phenotype is an independent risk factor for the development of cardiovascular disease in diabetic individuals in comparison with the homozygous Hp1 variant [34,40]. Additionally, it has been demonstrated that CD163 is downregulated in macrophages of atherosclerotic plaques of diabetic patients with the Hp2-2 genotype, indicating a constrained hemoglobin clearing capacity [33].

To detect the inflammatory response of Hb-Hp scavenging in hyperglycemic conditions, we selected a number of read-out cytokines displaying the complex interaction and different stages of an inflammatory reaction. In healthy individuals, the process of inflammation serves a homeostatic purpose, containing pro- and anti-inflammatory phases [65]. In individuals suffering from type 2 diabetes mellitus, however, the balance is tilted towards the proinflammatory side, manifested by an upregulation of proinflammatory intracellular pathways [66] and an elevation in circulating inflammatory markers, such as C-reactive protein, IL-6, and TNFα [67,68,69].

Hyperglycemia enhanced the proinflammatory response of M(IFNγ) to Hb-Hp complex uptake by stimulating the production of TNFα, IL-1β, IL-6, IL-8, and IL-1RA, supporting the observation that hyperglycemia itself can induce the secretion of proinflammatory cytokines [6].

A statistically significant increase in TNFα secretion, the ‘classical player’ of the acute-phase immune response [70], could only be detected 6 h after the stimulation with Hb-Hp complexes in hyperglycemia, regardless of the present haptoglobin variant. This finding corresponded to the clinical observation of elevated TNFα-levels being detected in newly diagnosed diabetic patients compared to a healthy cohort [71,72].

Elevated IL-6 release might contribute to diabetes progression, as an in vitro study reported the induction of cellular insulin resistance of hepatocytes by IL-6-triggered impairment of insulin receptor signal transduction [74]. Another in vitro study showed that lower concentrations (20 µg/mL) of Hb-Hp1-1 complexes induced the secretion of proinflammatory IL-6 while higher concentrations (100 µg/mL) of Hb-Hp1-1 complexes, however, led to increased CD163 surface expression on monocytes, in terms of a positive anti-inflammatory feedback loop [75].

Hyperglycemia increased the secretion of both IL-1β and its natural inhibitor IL-1RA, confirming our previous data [6]. Increased IL-1RA secretion could be a possible compensation mechanism or negative feedback for the enhanced release of IL-1β, whose serum level was found to be elevated in diabetic patients, contributing to insulin resistance and progression of atherosclerotic lesions in obesity [76]. A compensatory role of IL-1RA might also be an explanation for the controversial observations concerning IL-1RA levels in hyperglycemia: on the one hand, elevated IL-1RA levels could be correlated with insulin resistance [75,77]; on the other hand, IL-1RA was found to diminish markers of inflammation in the blood samples of diabetic patients and enhance beta cell function [78].

In general, Hb-Hp1-1 complex uptake was the strongest stimulus for M(IFNγ) for acute (6 h) cytokine release; however, cytokine secretion was diminished after 24 h. Contrarily, Hb-Hp2-2 complex uptake resulted in the increased secretion of all read-out cytokines after 24 h, indicating a transcriptional upregulation of cytokine production. Hb-Hp2-2 complex uptake is able to create a long-lasting elevated release of proinflammatory cytokines is in line with the numerous clinical observations of Hp2-2 possessing a lesser antioxidative capacity than Hp1-1 [38,39], inducing a lesser anti-inflammatory response than Hp1-1 [64] and even being an independent risk factor for the development of cardiovascular disease in diabetic patients [34,40].

As an element of the pathophysiology of diabetes, these mechanisms promote the low-grade chronic inflammation present in the vascular system of diabetic patients and support the development of vascular complications. This shows that—in addition to a more proinflammatory setting—diabetes mellitus is a disease characterized by less effective anti-inflammatory damage control [5,65].

Further research using monocytes isolated from patients with non-compensated hyperglycemia is needed to validate our findings, and to determine the level of contribution of the Hp-Hb-induced inflammatory monocyte response to systemic inflammation. We also believe that in the future, the effect of currently used medications for diabetic patients should also be analyzed for their ability to minimize Hp-Hb-induced inflammation.

This entry is adapted from the peer-reviewed paper 10.3390/ijms23031385

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