Biomedicines | Free Full-Text | Elevated NET, Calprotectin, and Neopterin Levels Discriminate between Disease Activity in COVID-19, as Evidenced by Need for Hospitalization among Patients in Northern Italy

Coronavirus disease 2019 (COVID-19) is initially characterized by flu-like symptoms, which can be associated with pneumonia and multi-organ damage. The final clinical outcome varies greatly. While some patients may only experience a mild or moderate upper airway illness that does not require hospitalization, others may develop severe disease and life-threatening acute respiratory distress syndrome (ARDS). Older age and the presence of co-morbidities, such as diabetes mellitus, obesity, and immunosuppression, have been associated with more aggressive COVID-19 and higher mortality [1,2,3,4].

There is evidence that the more aggressive disease is largely caused by a hyperinflammatory and prothrombotic response (immunothrombosis) triggered by the SARS-CoV-2 virus and mediated by the host’s deregulated immune response [5,6]. Besides the increased production of proinflammatory cytokines, additional mediators of innate immunity, such as complement activation, are involved. In particular, the high levels of circulating complement activation products, together with systemic proinflammatory cytokines, are crucial in causing endothelial perturbation. Moreover, the tissue deposition of complement components may contribute to the induction of a pro-adhesive and thrombophilic endothelial phenotype as an amplification thrombo-inflammatory loop ultimately responsible for the thrombophilic state in COVID-19 [1].

Studies have shown that the hyperproduction of proinflammatory cytokines, the release of damage-associated molecular patterns during tissue injury, and an increased occurrence of thrombotic events are all associated with the involvement of neutrophils [7]. Specifically, the increased formation of low-density neutrophils and the generation of neutrophil extracellular traps (NETs) play significant roles in the immunopathology of the disease. They are closely correlated with its severity and poor prognosis [8,9,10]. An intense systemic inflammation with high levels of proinflammatory cytokines and the associated endothelial perturbation characterize SARS-CoV-2-infected patients who require hospitalization because of a more severe disease [11,12].

Calprotectin, formerly L1 protein, is the main cytosolic protein in neutrophils and monocytes [13]. Elevated levels of extracellular calprotectin are not only found in autoimmune diseases [14,15] but have also been detected in severe COVID-19, suggesting that neutrophils are involved in inflammation and respiratory exacerbation in COVID-19 [16]. Calprotectin is a major component of NETs, which are usually involved in host defense for the destruction of invading pathogens [17]. Increased NET levels have been found in hospitalized COVID-19 patients [10], where they can obstruct capillary circulation [18]. In severe COVID-19, NETs are considered to be a driver of endothelial damage for the ensuing immune thrombosis [19,20]. However, the formation of NET is counteracted by DNase, which is an enzyme that destroys NETs via the degradation of their DNA content [21]. In contrast to the previous and current findings in COVID-19 patients, we only detected moderately increased levels of calprotectin among the other parameters (NETs, neopterin, and syndecan-1) tested in COVID-19 convalescent blood donors in Oslo [22].

Additional studies have found that calprotectin and NET levels are raised in vaccine-induced thrombocytopenia and thrombosis (VITT) after vaccination with ChAdOx1 nCoV-19 [23,24,25]. Moreover, recently, we reported increased DNase levels in the ChAdOx1 nCoV-19-vaccinated individuals, as measured via a novel DNase test, which correlated with their previously measured NET values [26].

Neopterin is an anti-oxidant agent produced by mononuclear phagocytes upon stimulation with IFNγ, which is produced by T cells and NK cells when viruses, cancer cells, and other proinflammatory danger signals activate them [27]. IFN-related pattern recognition receptors (PRRs) are activated by pathogen-associated (PAMPs) and damage-associated (DAMPs) molecular patterns in the mucosa [28]. Previously, neopterin has been used as an unspecific screening test for virus infections in Austrian blood banks [29,30]. Elevated neopterin levels have also been found during SARS-CoV-2 infection and at higher levels in severe than mild disease [31].

The complement activation products C5a and sC5b-9 have been shown to increase rapidly in COVID-19 patients [32,33,34,35] and are predictors for the hospitalization of COVID-19 patients in Northern Italy [11]. Also, a range of other proinflammatory cytokines, growth factors, and hematological parameters increased during COVID-19, more so in severely affected patients during the first pandemic wave [11].

We confirmed, in our cohort, that older age is associated with a higher risk of hospitalization due to more severe disease, as previously reported [2], and we looked at additional biomarkers.

The increase in H3-NET levels in hospitalized versus non-hospitalized patients is consistent with the widely accepted involvement of neutrophil in COVID-19. The complement system is activated in COVID-19, and complement deposits can be found in damaged tissues such as the lungs, which have a chemotactic effect on neutrophils and ultimately favor NETosis [11,32,33,34,35,41]. We also showed increased levels of IL-8 in sera from the same cohort of patients in Northern Italy [11]. IL-8 may, in turn, cooperate in recruiting neutrophils to the inflammatory site, and there is evidence that high receptor-saturating IL-8 concentrations promote NETosis [42]. The resulting enhanced NETosis is associated with thrombosis, which leads to further complications at the center of the inflammatory insult [19,20].

Hence, the increase in calprotectin, mainly derived from neutrophils, is unsurprising in our COVID-19 cohort, in whom secondary pneumonia gives an influx of neutrophils to the lungs. It has been shown that calprotectin, which is a heterodimer of S100A8 (MRP-8) and S100A9 (MRP-14), is deposited on the endothelium of venules in inflamed tissue and also promotes the extravasation of leukocytes [38].

Neopterin is an old test for emerging infections [29,30], and it has also been documented for SARS-CoV-2 infection [31]. There is evidence that higher levels of neopterin in severe COVID-19 may be used as a predictive marker for disease severity [43,44]. This is confirmed here by the increased levels of neopterin and the correlation with other inflammatory markers in our COVID-19 cohort. Moreover, we find that neopterin concentrations can discriminate between hospitalized and non-hospitalized patients, similar to calprotectin and NET values. Previously, this has especially been shown for the complement activation products C5a and sC5b-9 and other proinflammatory markers [11].

Since heparin is known to be involved in NET formation/degradation and most COVID-19 hospitalized patients were treated with heparin (Fragmin), such patients could not be included when measuring NET levels [38,39]. Moreover, calprotectin cannot be measured accurately in patients treated with heparin because the S100A9 (MRP-14) dimer part of calprotectin has affinity and interacts with heparin [38]. Therefore, heparin-treated patients were excluded from the current paper.

The correlation between NET and NLR levels should be discussed because NLR is a parameter for physiological stress and particularly important with regard to lung affection and pneumonia during SARS-CoV-2 infection, where NETosis plays a critical role. The negative correlation between NETs and platelet count may indicate that neutrophil activation contributes to platelet activation, consumption, and/or involvement in the formation of leuco-aggregates [45].

There is a general agreement that SARS-CoV-2 plays a direct role in tissue damage, but the intense immune response further contributes to the clinical manifestations [5]. The relationships of NETs, calprotectin, and neopterin with COVID-19 hospitalization and, simultaneously, with the biomarkers of the innate and adaptive immune responses potentially responsible for tissue damage are consistent with this view.