Macrophage activation in obese type 2 diabetes: an enhanced risk for COVID-19 and Acute Respiratory Distress Syndrome?

Objective Hyperactivation of the immune system through obesity and diabetes may enhance infection severity complicated by Acute Respiratory Distress Syndrome (ARDS), the hallmark of severe COVID-19 disease. Objectives: to determine the circulatory biomarkers for macrophage activation at baseline and after serum glucose normalization in obese type 2 diabetes (OT2D) subjects. Methods A case-controlled interventional pilot study in OT2D (n=23) and control subjects (n=23). Subjects underwent hyperinsulinemic clamp normalizing serum glucose. Plasma macrophage-related proteins were determined using Slow Off-rate Modied Aptamer (SOMA)-scan plasma protein measurement at baseline (control and OT2D subjects) and after 1-hour of insulin clamp (OT2D subjects only). Results. Basal M1 macrophage activation was characterized by elevated levels of M1 macrophage-specic surface proteins, CD80 and CD38, and cytokines or chemokines (CXCL1, CXCL5, RANTES) released by activated M1 macrophages. Two potent M1 macrophage activation markers CXCL9 and CXCL10 were decreased in OT2D. Activated M2 macrophages were characterized by elevated levels of plasma CD163, TFGβ-1, MMP7 and MMP9 in OT2D. Conventional mediators of both M1 and M2 macrophage activation markers (IFN-γ, IL-4, IL-13) were not altered. No changes were observed in plasma levels of M1/M2 macrophage activation markers in OT2D in response to acute normalization of glycemia. Conclusion In the basal state, macrophage activation markers are elevated, and these reect the expression of circulatory cytokines, chemokines, growth factors and matrix metalloproteinases in obese individuals with type 2 diabetes, that were not changed by glucose normalisation. These differences may predispose the diabetic individuals to ARDS reecting in increased COVID-19 disease severity.


Introduction
Whether type 2 diabetes (T2D) increases risk for development of acute respiratory distress syndrome (ARDS) has been controversial (1,2). Recently, SARS-CoV-2 infection leading to severe COVID-19 disease has been shown to re ect underlying health conditions (3), diabetes being incontrovertibly associated with poor outcome in patients with COVID-19 (4). Obesity clearly represents a signi cant, independent risk factor, body mass index being positively correlated with risk of ARDS (5). Therefore, the combination of obesity and T2D places patients at high risk for severe infection and poor outcomes.
Acute respiratory distress syndrome (ARDS) results from an excessive and uncontrolled systemic in ammatory response where distinct populations of macrophages, resident alveolar macrophages (AMs), and recruited macrophages from the blood undergo dramatic changes in number and phenotype, playing a causal role in pathogenesis and resolution of ARDS (6). ARDS is the hallmark of severe COVID-19 disease, responsible for the increased mortality associated with SARS-CoV-2 infection particularly in diabetes (4).
Macrophages are a heterogeneous population of innate immune cells that, especially in lung, serve as the rst barriers of defense against extrinsic invaders and airborne particles (7). Macrophages play a pivotal role in in ammatory processes, clearing cellular debris and effecting resolution post-in ammation.
Macrophages undergo polarization in response to the extracellular environment (8,9), including infection, leading to two polarization states: the classically activated phenotype (M1) and the alternatively activated phenotype (M2) (10). M1 macrophages have pro-in ammatory and cytotoxic properties and play a key role in virus clearance. Conversely, M2 (anti-in ammation) cells play a key role in tissue remodeling and matrix deposition post-injury (11).
An increase in tissue macrophages is a common feature of both macro and microvascular diabetic complications, including nephropathy, atherosclerosis, neuropathy, and retinopathy. Hyperglycemia is thought to depress phagocytic ability (16); in vitro studies have shown that high glucose concentrations increased expression of certain M2 polarization markers such as MMP9 and CD169 (17).
Glucose variability has been associated with increased tissue damage through oxidative stress (19) and increased glucose variability was related to more severe ARDS in COVID-19 disease (20). Therefore, we hypothesized that in obese type 2 diabetes (OT2D), even in the basal state, macrophages are activated and thus impaired or aberrant macrophage protein expression is already present in OT2D in comparison to non-diabetic controls, that may increase both infection susceptibility and disease severity, predisposing patients with OT2D to ARDS that is the hallmark of severe COVID-19 disease. To address this question, we measured macrophage markers in plasma of OT2D versus non-diabetic subjects and determined whether alterations in levels of macrophage activation markers in OT2D could be altered by normalizing glucose levels with a hyperinsulinaemic insulin clamp. were on a stable dose of medication (metformin, statin and/or angiotensin-converting enzyme inhibitor/angiotensin receptor blocker) for the preceding three months (21). T2D subjects were excluded if on any anti-glycemic medication other than metformin or with poor glycemic control [HbA1c levels ≥10% (86mmol/mol)]. Control subjects were excluded if diagnosed with type 1 or 2 diabetes or if HbA1c levels >6% (42mmol/mol). The following exclusion criteria were applied for both groups: current smokers, body mass index (BMI) <18 or >50 kg/m 2 , excessive alcohol consumption, renal or liver disease, history or presence of malignant neoplasms within the last 5-years, diagnosis of psychiatric illness, history of pancreatitis or gastrointestinal tract surgery.
Blood processing and biochemical markers measurement: As previously described (21), "blood samples were separated immediately by centrifugation at 2000g for 15-minutes at 4°C, and the aliquots were stored at −80°C, within 30-minutes of blood collection, until batch analysis. High sensitivity C-reactive protein (hsCRP) was measured using Synchron systems CRPH reagent kit (Beckman-Coulter, UK) per manufacturer's protocol. Fasting plasma glucose (FPG) was measured using a Synchron LX 20 analyzer (Beckman-Coulter) per manufacturer's recommended protocol."

SOMA-scan measurements
Plasma protein quanti cation was performed using a Slow Off-rate Modi ed Aptamer (SOMAmer)based protein array, as previously described (version 3.1 of the SomaScan Assay) (22). Brie y, EDTA plasma samples were diluted and the following assay steps were performed in sequence: binding, Catch 1, Cleave, Catch II, elution and quanti cation. Normalization of raw intensities, hybridization, median signal and calibration signal were performed based on the standard samples included on each plate, as previously described (23).

Statistics
No studies detailing changes in macrophage-related proteins in response to hypoglycaemia are available on which to base a power calculation. Sample size for pilot studies has been reviewed by Birkett and Day (24). They concluded a minimum of 20 degrees-of-freedom was required to estimate effect size and variability. Hence, we needed to analyse samples from minimum 20 patients per group. Data trends were visually evaluated; non-parametric tests were applied on non-normal data using the Kolmogorov-Smirnov Test. Comparison between groups was performed using Student's t-test. A p-value of <0.05 was considered statistically signi cant. Statistical analysis was performed using Graphpad Prism (San Diego, CA, USA).

M1 macrophage activation may be induced by Lipopolysaccharide (LPS) in obese subjects with T2D (OT2D):
Classically activated M1 macrophages constitute the rst line of defense against intracellular pathogens and therefore exhibit a high level of phagocytic activity. A signi cant reduction of plasma lipopolysaccharide binding protein (LBP) was found in OT2D versus controls (85311±1453 vs 91747± 3048 RFU of LBP, OT2D vs control, p<0.05) suggesting LPS-mediated activation of M1 macrophages in OT2D ( Figure 1A). Further suggestion of LPS-induced activation of M1 macrophages in OT2D was shown by no change of plasma toll like receptor-4 (TLR4) levels in OT2D (235±11 vs 254±13 RFU of TLR4, OT2D vs control, p=ns) ( Figure 1B) that suggested elevated LPS-mediated endocytosis of TLR-4 in OT2D (25).
Adipose tissue macrophage (ATM) activation in OT2D: Since the T2D subjects in this study were obese, we sought to determine the circulatory markers for activation of adipose tissue macrophages (ATMs) in OT2D. Human ATMs are characterized by their expression of CD163 and we showed in the previous section that the plasma CD163 level was increased in OT2D, suggesting activation of ATMs in OT2D. To determine if those ATMs are derived from circulating monocytes, we measured the monocyte chemoattractant protein-1 (MCP-1, also known as CCL2). Our data indicated that circulatory MCP-1 level did not differ in OT2D compared to control (698±49 vs 760±73 RFU of MCP-1, OT2D vs control, p=ns) ( Figure 4A), suggesting reduced or no migration of monocytederived macrophages in adipose tissue. This observation was supported by the level of macrophage migration inhibitory factor (MIF) which was higher in OT2D (1387±223 vs 1007±29 RFU of MIF-1, OT2D vs control, p<0.05) ( Figure 4B). We further measured the level of netrin-1 to determine if the ATM activation is mediated by a local modulator. Plasma netrin-1 level was higher in OT2D (558±30 vs 477±19 RFU of netrin-1, OT2D vs control, p<0.05) ( Figure 4C). No changes were observed in the plasma levels of adipose tissue macrophages in response to acute normalization of glycemia in OT2D ( Figure   4A-C).

Discussion
This study describes the novel nding of large-scale circulatory macrophage activation markers, showing that both M1 and M2 macrophages are activated in OT2D and were unaffected by acute normalization of glycemia. Our data demonstrate that, in the basal state, classical and alternative activation of macrophages in OT2D were not mediated by conventional mediators (IL-4, IL-13 or IFN-γ); rather, they may be activated by elevated levels of LPS. Increased markers of adipose tissue macrophage activation in OT2D support the model of adipocyte-macrophage interaction and metabolic endotoxemia-induced development of obesity and insulin resistance (27). The circulatory cytokine and chemokine pro les in response to M1 macrophage activation showed a unique pattern in OT2D. Most of the conventional M1 macrophage activation markers (mainly reported in animals) were not elevated in OT2D. However, we found high plasma levels of LPS-induced human M1 surface markers CD80, CD38 and pro-in ammatory chemokines CXCL1, CXCL5 and CCL5, that are released from activated M1 macrophages in OT2D. Our data also revealed that certain subclasses of M2 macrophages that are induced by LPS or TGF-β1 are also activated in OT2D. The elevated levels of M2 activation markers TGF-β1, MMP7 and MMP9, led us to speculate that in certain tissues (e.g. lungs), macrophages are more M2 polarized in the basal state in OT2D. Elevated macrophage activation markers in OT2D were unchanged by acute insulin-induced euglycemia.

LPS may be a mediator of macrophage activation in obese type 2 diabetes
Since our study involved measuring only plasma proteins, we utilized surrogate markers of LPS elevation and its role in M1 macrophage activation in OT2D. We measured the level of plasma LPS-binding protein (LBP) as a determinant of LPS activity in our study subjects and found the LBP level was signi cantly lower in OT2D compared to controls. Previous publications have reported raised levels of LBP in obese subjects (28,29), which is discrepant with our observations. The difference might be a consequence of case selection, as the obese subjects were smokers and consumed alcohol in both of those referenced reports. It is known that, in smokers, LBP levels are raised in bronchoalveolar lavage uid (BALF) (30); this is also the case in heavy drinkers, likely due to injury in icted upon the gastrointestinal barrier by alcohol (31). By contrast, in our study, smoking and alcohol consumption were exclusion criteria. Furthermore, LBP has a dual role in effecting LPS-induced macrophage activation that is concentration dependent; low concentrations of LBP promote LPS-induced activation of mononuclear cells (MNC), while high concentrations inhibit LPS-induced cellular stimulation (32). Moreover, LBP binds to host cells and is internalized, and in the cytoplasm colocalizes with LPS (33). Therefore, decreased LBP levels are re ective of raised LPS levels in the OT2D subjects reported here. The elevated LPS-induced activation of M1 macrophages in OT2D was further con rmed by plasma TLR4 levels that showed no difference between OT2D and controls, suggesting elevated LPS-mediated endocytosis of TLR4 (25).
None of the pro-in ammatory cytokines involved in either M1 or M2 macrophage activation, IFN-γ, IL-4 and IL-13, were increased in OT2D, suggesting that there was no increase of Th1 or Th2-mediated responses in those OT2D subjects. One explanation might be that there were no in ammatory reactions triggered by an invading parasite or allergen in those OT2D cases, as there was no change in plasma CRP in OT2D (Supplementary table 1). M1 macrophage activation in obese type 2 diabetes Elevated CD38 and CD80 levels were found in OT2D, suggesting that LPS-induced M1 macrophage polarization is induced in OT2D. Macrophage activation studies suggest CD38 and CD80 are the markers that best characterize LPS-induced human M1-like macrophage activation (26,34), and CD80 has been reported as a co-stimulatory signaling molecule in alveolar macrophages (35). However, pro-in ammatory cytokines and chemokines released by LPS-TLR4 interaction from M1 macrophage such as MFG-E8 expression were unchanged, suggesting that less mobilization of neutrophils occurs in response to LPSinduced M1 macrophage activation in OT2D.
Two chemokines, IL-8 and RANTES (CCL5), are released from LPS-induced M1 polarized macrophages (26). We did not nd any differences in basal IL-8 level in OT2D; however, plasma RANTES level was signi cantly (~2-fold) higher in OT2D, suggesting RANTES is a unique M1 macrophage activation marker for obese individuals with T2D. This is consistent with a previous report where RANTES (CCL5) was associated with LPS-induced M1 macrophage polarization (36). Moreover, a transcriptional analysis of human alveolar macrophages that were polarized ex vivo using interferon-γ (IFN-γ), revealed the association of RANTES with M1 polarization (37). Therefore, our data suggests lung alveolar macrophages as a possible tissue source of RANTES in OT2D.
Two potent chemokines, CXCL9 and CXCL10 that are markers of IFN-γ-stimulated classical M1 macrophages, were signi cantly reduced in our study subjects. This data is paradoxical to a previous report where LPS-induced elevation of serum CXCL9 and CXCL10 was reported in an LPS-induced ARDS model (38); however, neutralization of IFN-γ showed a marked decrease of plasma CXCL9 and CXCL10 levels (39), suggesting a relationship between stable IFN-γ levels and reduced levels of CXCL9 or CXCL10 in OT2D.

M2 macrophage activation
Our data indicated that M2 macrophages were activated, though there was no change in the basal level of plasma IL-4/IL-3; we therefore hypothesized that M2 macrophages in OT2D are activated either by LPS (M2b) or TGF-β (M2c). Consistent with our hypothesis, we found a signi cant elevation of basal TGF-β1 in OT2D, suggesting M2c is activated in OT2D. M2 macrophage activation was further con rmed by CD163 (Hemoglobin-Haptoglobin Scavenger Receptor) expression that was higher in OT2D. CD163 is an M2 macrophage receptor, and its expression is ampli ed by IL-10, IL-6, M-CSF and glucocorticoids, while TNF-α, IFN-γ, LPS and TGF-β reduce its expression (40). Our data, therefore, suggests a paradoxical effect of cytokines to stimulate the soluble CD163 level in OT2D; elevated circulating CD163 has been reported in obesity and T2D (41).
The level of its ligand CD200 was downregulated in OT2D, though CD200R1, a surface marker of M2 macrophages, did not alter. Lung alveolar macrophages express high levels of CD200R at the basal condition and are upregulated during viral infection. Binding of CD200 (which is expressed on the luminal aspect of the airway epithelium) with CD200R imparts a unidirectional negative signal for resolution of in ammation to the lung alveolar macrophages (42). Therefore, it is likely that an impaired resolution system after lung in ammation is present in OT2D. Two important M2 macrophage receptor markers, dendritic cell speci c ICAM-3 grabbing nonintegrin (DC-SIGN, also known as CD209) and CD36, were signi cantly lower in OT2D. CD209 expression is enhanced by IL-4 but its effect is diminished by IFN-γ and TGF-β (43). So, it is likely that elevated levels of TGF-β1 might play a role in the reduction of CD209 levels that have been related to hyperglycemia.
Two matrix metalloproteinases (MMPs), MMP7 and MMP9, were elevated in OT2D and transcriptional analyses revealed that both are expressed in human monocyte derived M2c macrophages (44) or human alveolar M2 macrophages (45), suggesting the possible activation of human alveolar M2 macrophages in OT2D. LPS induces the expression of both MMP7 and MMP9 in monocyte-derived macrophages (46); therefore, our data strongly suggests that the circulating matrix metalloproteinases MMP7 and MMP9 represent macrophage activation markers in response to LPS stimulus in OT2D. Elevated M2 macrophage markers (especially, TGF-β1, MMP7 and MMP9) indicate that lung alveolar macrophages are activated. In COVID19, the pathogenesis of severe acute respiratory distress syndrome (ARDS) includes pulmonary brosis and edema. The major cellular sources of TGF-β in pulmonary brosis have been shown to be alveolar macrophages and metaplastic type II alveolar epithelial cells (47). Elevated TGF-β1 in OT2D may predispose alveoli in a pre-brotic condition following SARS-CoV-2 infection and may be activated by MMP9 (48). MMP7 has also been reported as a potential peripheral blood biomarker of idiopathic pulmonary brosis (49). Therefore, it is likely that, in OT2D, the lung epithelial barrier integrity is destabilized in response to the broproliferative activity of elevated TGF-β1, MMP7 or MMP9.
We normalized the prevailing hyperglycemia in OT2D subjects using an insulin clamp to investigate whether normalization of glycemia normalized the elevated macrophage markers. Acute normalization, however, did not have any effect upon basal macrophage activation in OT2D. This data is consistent with the LANCET trial, where treatment with insulin versus placebo or metformin did not reduce in ammatory biomarker levels despite improving glucose control (50). However, increased glucose variability has been associated with increased tissue damage in diabetes, perhaps through oxidative stress mechanisms (19) and was associated with more severe ARDS in SARS-CoV-2 infection (20).
Strengths of this study include inclusion of type 2 diabetic subjects with relatively short duration of disease who were relatively treatment naïve; the results may, however, be generalizable to other T2D cohorts. The major limitation of this study is the small numbers in each cohort; with a larger population, even greater differences in macrophage-related protein concentrations may have been discerned. Further limitations are that normalization of blood glucose was a single event and experimental glucose uctuations clamped over a timecourse would have been more robust, and that plasma levels of these proteins may not be re ective of tissue concentrations.