COVID-19 is a recently emerged viral disease caused by a betacoronavirus, SARS-CoV-2. The viral infection has spread globally in a short span of time affecting 196 million people and causing approximately 4.2 million deaths as of 29^th July 2021 (John Hopkins University Coronavirus tracker).

Majority of the infected individuals exhibit either no or mild symptoms and about 15% develop severe pneumonia with approximately 6% progressing to acute respiratory distress syndrome (ARDS) and multiorgan failure (Guan et al., 2020). Inflammatory cytokines, IL-6, IL-10 and TNF-alpha have been identified as independent predictors of disease severity and clinical outcome (Del Valle et al., 2020; Dhar et al., 2021). Even, spike protein of SARS-CoV-2 is reported to induce inflammatory cytokines in macrophages (Khan et al., 2021). One of the hypotheses for progression to serious disease complications is imbalanced host immune response with ineffective early innate immunity followed by impaired adaptive immune responses and hyper-inflammation (Blanco-Melo et al., 2020; Tay et al., 2020).

Several studies reported alterations in both innate and adaptive arms of the host immunity against SARS-CoV-2 infection in COVID-19 patients (Diao et al., 2020; Zhang et al., 2020; Zheng et al., 2020; Munje et al., 2021). During airway infections, neutrophils are the first line of defense. In response to viral infection, neutrophils migrate to the site of infection and initiate the antiviral defense mechanism. Recent studies have reported accumulation of neutrophils in severe COVID-19 patients compared to non-severe patients (Huang et al., 2020; Kuri-Cervantes et al., 2020; Liao et al., 2020; Tan et al., 2020; Wang D. et al., 2020). In a COVID-19 patient with fatal outcome, large number of neutrophils were found in autopsied lung infiltrates (Barnes et al., 2020). Moreover, increased neutrophil to lymphocyte ratio in peripheral blood of severe COVID-19 patients (Zhang et al., 2020) suggest that neutrophil activation may modulate immune response after SARS-CoV-2 infection. Upon neutrophil activation or death, alarmins including alpha-defensins, S100A8/A9 heterodimers (calprotectin) and myeloperoxidase (MPO) proteins are released extracellularly in circulation (Faurschou and Borregaard, 2003; Tardif et al., 2015). Till date, COVID-19 pathogenesis has focused on macrophages, T and B cells. It has been suggested that neutrophilia is associated with disease severity in COVID-19. However, it is still unknown whether neutrophils drive the inflammation or act as bystanders. In this study, we aimed to quantitate the circulatory levels of neutrophil’s secretory proteins in different clinical presentations of COVID-19 patients.

Table 1 summarizes the demographic features and hematological/biochemical parameters among the COVID-19 patients enrolled in this study. Of the 109 patients studied, 23 were viral RNA positive asymptomatic contacts of COVID-19 index cases while 63 patients were symptomatic with mild (n=32) or severe (n=31) disease. Among 31 patients suffering from severe disease, 19 (61.3%) required mechanical ventilation for oxygen support and 11 (35.5%) succumbed to the infection.

This study provides comparative, circulatory expression profiles of DEFA1, S100A8/A9 and MPO in COVID-19 patients with different clinical presentations. Though SARS-CoV-2 infection involves neutrophil activation, a marked difference was noted in the association of these markers with disease severity. Of note, raised DEFA1 and MPO levels characterized symptomatic infections while elevated levels of calprotectin marked SARS-CoV-2 infections irrespective of clinical presentation ( Figure 1 ). Further, MPO and calprotectin levels did not differ among patients with mild or severe disease. An important observation was a significant rise in DEFA1 levels during the first week post-disease onset in severe than non-severe patients ( Figure 2A ). Odds-ratio analysis showed that DEFA1 could act as potential biomarker in predicting disease severity (OR=11.34). Of note, at 314 ng/mL DEFA1 levels, accurate prediction of disease severity in differentiating mild from severe disease patients could be determined with 68% sensitivity and 91% specificity ( Figure 3G ). This significant observation with small numbers need to be extended to a larger cohort with prospective follow-up studies.

Single cell RNA sequencing analyses showed the increased presence of dysfunctional neutrophils expressing S100A8, S100A9, DEFA3 and DEFA4 genes in the PBMCs of severe COVID-19 patients suggesting association of the phenotypic alterations in neutrophils with disease severity (Schulte-Schrepping et al., 2020). Very recently, increased serum DEFA1 levels were reported in COVID-19 patients suffering from acute respiratory distress syndrome (Kerget et al., 2021). Other clinical presentations were not studied. Contrary to the biomarker role of alpha-defensins, antiviral role of DEFA1 was established in in-vitro wherein HNP1 inhibited the viral infection at the step of viral entry by disrupting the fusion of the SARS-CoV-2 virus with host membrane (Xu et al., 2021). Similarly, antiviral activity of intestinal alpha-defensin, HD-5 released from Paneth cells and of β-defensin, hBD-2 was shown to be due to blocking of the entry of spike expressing pseudovirus into ACE2 expressing cells (Wang C. et al., 2020; Zhang et al., 2021). At the site of infection, neutrophils can exert their antiviral activities by increasing the expression of peptides such as, defensins and promote degranulation. However, excessive release of defensins and neutrophil elastase led to cell death of bronchial epithelial cells thereby increasing permeability of blood vessels and causing acute lung injury (Grommes and Soehnlein, 2011). The mechanism(s) by which defensins contribute to disease severity during SARS-CoV-2 infection are not yet known.

Elevated calprotectin levels in the blood of COVID-19 patients with severe disease are consistent with a study from Michigan, USA (Shi et al., 2020). In addition, we report that elevation of calprotectin was a function of SARS-CoV-2 infection and was not related to disease severity. The observation of higher expression of S100A8 and S100A9 genes in the PBMCs of the patients with severe disease but not in patients with mild disease is noteworthy (Silvin et al., 2020). The discrepancy between gene expression and protein levels (this study) could be due to differences in sampling timings, increase only at transcription level or transient rise in the circulatory proteins missed by our study. In children suffering with multisystem inflammatory syndrome (MIS-C) after SARS-CoV-2 infection, rise in S100 protein levels was similar to other inflammatory disorder such, as Kawasaki disease (Rodriguez-Smith et al., 2021). Disease severity was associated with increased, circulatory S100B protein levels as well (Aceti et al., 2020).

So far, several multi-omics studies revealed that genes involved in neutrophil function and activation are highly upregulated and neutrophil degranulation is the most prominent defense pathway in SARS-CoV-2 infected patients (Akgun et al., 2020; Schulte-Schrepping et al., 2020; Bankar et al., 2021). Elevated levels of MPO in nasopharyngeal samples of COVID-19 patients were identified by proteomic analysis (Akgun et al., 2020). Our observations of increased expression of alpha defensins and calprotectin in severe patients on the 1^st day of hospitalization are in line with previous proteomic profiling study (Meizlish et al., 2021). Of note, fatal outcome was associated with a further rise in the expression of DEFA1 and calprotectin levels in the patients with severe disease ( Figure 3E ) and 91% required mechanical ventilation. These results confirm the findings of Shi et al. (2020). Taken together, it seems that neutrophil activation signature can predict mortality in COVID-19 patients. However, there was a distinct possibility of the role of secondary infections in modulating the expression of three neutrophil secretory proteins in severe COVID-19 patients. Importantly, similar to the reports of higher proportion of secondary infection in patients from China (Zhou et al., 2020), Italy (De Santis et al., 2021), France (Roger et al., 2021) and UK (Hughes et al., 2020), 61% of severe patients from our study suffered from secondary infection. The observations of raised levels of these proteins much before the appearance of secondary infection ( Figures 3B–D ) provide important evidence for the association of higher expression levels of DEFA1/Calprotectin/MPO levels with COVID-19 severity. Moreover, the significant rise in DEFA1 levels in severe patients in comparison of patients with mild disease was independent of any confounding factors associated with disease severity (p=0.02). In our study, 53% of patients undergoing early intubation (0-3 days after hospitalization) developed secondary infection. This is in line with findings that mechanically ventilated COVID-19 patients are at high risk of developing secondary infection (Risa et al., 2021).

Multiple models have predicted neutrophils, lymphocytes, C-reactive protein (a biomarker for inflammation), and D-dimer (a measure of blood clotting) as markers for disease severity in SARS-CoV-2 infection (Zhou et al., 2020). Our findings of increased neutrophil-lymphocyte ratio along with raised D-dimer and CRP levels in the patients with severe disease confirm earlier observations (Chen et al., 2020; Liu et al., 2020; Yao et al., 2020; Zhou et al., 2020). A positive correlation was obtained between increased neutrophil-lymphocyte ratio with increased levels of DEFA1 in severe patients. A weak positive correlation between CRP and calprotectin levels was also observed in our study, probably due to small sample size. It is pertinent to note here that calprotectin was found to be superior than CRP in identifying patients requiring mechanical ventilation at any point during their hospitalization (Shi et al., 2020). Circulatory calprotectin was identified as an independent variable influencing disease severity (Silvin et al., 2020). Recognizing potential role of alpha defensins and calprotectin in augmenting inflammatory response, a clinical trial using an adjunctive therapy, cytosorb, is underway. This formulation acts by absorbing a broad spectrum of cytokines, DAMPs and PAMPs to reduce their circulatory levels and ameliorate immunopathology (Tay et al., 2020).

In summary, our data reveals that neutrophil activation as assessed by levels of circulatory alpha defensins and calprotectin varies with disease severity. DEFA1 seems to be a potential biomarker of severity, needing further evaluation. Association of raised DEFA1 and calprotectin with poor clinical outcome raises possibility of crucial roles of these molecules in the COVID-19 pathogenesis. Given the urgent need for effective treatment of COVID-19 patients, efficacy of anti-neutrophil therapies to combat neutrophil activation needs to be undertaken on priority.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Institutional Ethics Committee of Bharati Hospital & Research Centre at Bharati Vidyapeeth Deemed University (IEC/2020/25). Informed written consent was obtained from each subject before participating in this study. This study was conducted in accordance to the ethical standards of the Helsinki Declaration of 1975, as revised in 2013. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Conceptualization, study design and supervision: VA. Methodology: SS and SC. Patient’s recruitment and clinical management: PJ, PD, and SP. Data analysis and interpretation: SS and SP. Writing of the original manuscript: SS and SC. Review and editing of the manuscript: VA and AM. All authors contributed to the manuscript and approved the submitted version.

This work was partially supported by Indian Council for Medical Research, ICMR. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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