Messenger RNA vaccines encoding for the SARS-CoV-2 spike protein have proven effective at reducing the number of severe cases of COVID-19 (1). However, the emergence of new variants of concern (2), limited vaccine distribution (3), and waning immunity (4) remain major problems that highlight the need for more effective therapies against the acute manifestations of COVID-19. Such therapies, in turn, require a deeper understanding of the cellular and molecular mechanisms underlying this disease. Moreover, it is now known that patients with acute COVID-19 present increased blood levels of pro-inflammatory cytokines (e.g., IL-6, IL-8, TNFα) that drive neutrophil mobilization and are associated with poor prognosis in severe cases (5, 6). Although neutrophils may contribute to viral clearance via cytokine release, phagocytosis, and production of extracellular traps (NETs), a growing body of evidence indicates that excessive neutrophil mobilization becomes pathogenic in COVID-19 (7). Along these lines, we and others recently demonstrated that blood neutrophils from patients with COVID-19 are hyperactivated and display enhanced survival and migration (6, 8). In addition, this excessive neutrophil mobilization has been confirmed by the increased markers of NET production found in blood from patients with acute COVID-19 (8, 9).

Until now, IL-26 has emerged as an intriguing mediator of host defense in lung disorders due to its antibacterial and neutrophil-mobilizing properties (10–16). However, it may also possess anti-viral potential; IL-26 was recently shown to bind viral RNA intermediates and inhibit the replication of the Hepatitis C virus, a virus that, just like SARS-CoV-2, contains a positive-sense single-stranded RNA genome (17). In addition, we have previously shown that IL-26 acts via its receptor complex (IL-10R2/IL-20R1) to enhance the IL-8- and fMLP-mediated chemotaxis of neutrophils in vitro (10), as well as the LPS-dependent accumulation of neutrophils in the lungs of mice in vivo (13). Furthermore, we and others have forwarded evidence that IL-26 alone or bound to NETs triggers the production of the neutrophil-related cytokines IL-6, IL-8, and TNFα in unsorted bronchoalveolar lavage (BAL) cells (10) and peripheral blood mononuclear cells (PBMCs) (18). Finally, we have shown that activation of TLR2, TLR3, TLR4, TLR7, and TLR8, all of which mediate SARS-CoV-2 recognition (19–23), elicits the release of IL-26 in human primary bronchial epithelial cells (TLR3, TLR7, and TLR8) (11), primary lung fibroblasts (TLR4) (24), alveolar macrophages (TLR4) (10), blood neutrophils (TLR4) (12), and the alveolar epithelial cell line A549 (TLR2 and TLR4) (12).

Despite its inherent antiviral and neutrophil-mobilizing properties, the involvement of IL-26 in COVID-19 has not been investigated in a conclusive manner. Therefore, in the current pilot study, we quantified the concentration of IL-26 in plasma samples from patients with acute COVID-19 (COVID-19 group), compared with healthy control subjects (Control group), and analyzed its association with the neutrophil-mobilizing cytokines IL-6, IL-8, and TNFα. We then characterized how the concentration of IL-26 associates with different markers of neutrophil activation, migration, survival, and NET production. Finally, we also determined the correlation between the plasma concentration of IL-26 and different markers of tissue damage and inflammation, as well as hematological parameters relevant to COVID-19. In doing so, we obtained strong evidence that the kinocidin IL-26 is increased at the systemic level and associates with several markers of hyperinflammation and tissue damage in acute COVID-19.

Although several previous studies have associated excessive neutrophil mobilization with poor prognosis in patients with acute COVID-19 (6, 8, 30), ours is the first to show that an increase in the plasma concentration of IL-26 correlates with signs of neutrophil mobilization in this disease. From an immunological point-of-view, it is feasible that IL-26 is involved in acute COVID-19, given its dual role as an antiviral and neutrophil-mobilizing mediator of host defense. It is known that the expression of IL-26 occurs constitutively in several immune and structural cells, and that it is enhanced upon TLR stimulation (31). Moreover, the genetic material of SARS-CoV-2 is recognized by TLR3 (21), TLR7 (21, 22), and TLR8 (22), critical receptors that are known to mediate the production of IL-26 in primary human bronchial epithelial cells (11). Furthermore, the SARS-CoV-2 spike protein can bind and activate TLR4 in a way that is comparable to LPS (19, 23). This may prove to be crucial, given that we have previously shown that exposure to LPS elicits a pronounced increase in IL-26 production in the lungs of healthy volunteers in vivo (10), as well as in blood neutrophils, primary human lung fibroblasts, and alveolar macrophages in vitro (10, 12, 24). Finally, the SARS-CoV-2 envelope protein is known to bind and activate TLR2 (20), which has been shown to cause the release of IL-26 in A549 cells, a cell line derived from human alveolar epithelial cells (12).

The relationship between IL-26 and the neutrophil-mobilizing cytokines IL-6, IL-8, and TNFα that we found in the current study is likely to be multifaceted. On the one hand, it is known that similar stimuli (e.g., TLR4 stimulation) can trigger the concomitant production of IL-6, IL-8, IL-26, and TNFα (31, 32). On the other hand, IL-26 alone or bound to NETs can induce the production of IL-8 and TNFα in unsorted BAL cells (10) and IL-6 in PBMCs (18), while inhibiting the production of the same cytokines in primary bronchial epithelial cells (11). Furthermore, it has previously been shown that TNFα together with IL-1β elicit the expression of IL-26 in human primary arterial smooth muscle cells (27), and that IL-26 enhances the IL-8-mediated chemotaxis of neutrophils isolated from human blood (10). Now, in the current study, we found that the enhanced plasma concentration of IL-26 displays a strong correlation with that of IL-8, and a somewhat weaker one with that of TNFα, in the COVID-19 group. Moreover, we found that IL-8 and IL-26 display comparable correlations with CD47, a marker of neutrophil prolonged survival, in the COVID-19 group. Thus, it seems possible that IL-26 is more functionally related to IL-8 than to IL-6 or TNFα.

Interestingly, although it has previously been shown that IL-26 enhances the pro-inflammatory potential of NETs and other forms of extracellular DNA (18, 27), we now failed to prove a correlation between the plasma concentration of IL-26 and the investigated markers of NET formation within the COVID-19 group. This was the case even though IL-8 and TNFα displayed such correlations. Nevertheless, we observed that the plasma concentration of IL-26 displays a trend towards a positive correlation with the concentrations of dsDNA (p = 0.08) and cell-free nucleosomes (p = 0.12) in the COVID-19 group, and it seems feasible that statistical significance could have been reached if the study material had included a larger number of patients in the COVID-19 group. However, we did prove a positive correlation between the plasma concentration of IL-26 and those of both markers of NET formation when the COVID-19 and Control groups were pooled, a finding that suggests a mechanistic link between IL-26 and NET production, possibly representing normal immunology rather than a unique pathological feature. If such an immunological mechanism does exist, it further implicates IL-26 in antimicrobial host defense in a wider sense, given the risk for bacterial infection that normally follows the damage of mucosal surfaces caused by viral infections.

Among the different markers of inflammation and tissue damage analyzed in the COVID-19 group, we proved a positive correlation between the plasma concentration of IL-26 and that of LDH, an established marker of tissue damage (33). Notably, increased serum LDH is known to be associated with severe COVID-19 (34, 35), and a recent study demonstrated that the serum concentration of LDH correlates in a positive manner with the degree of lung injury in patients with acute COVID-19 (34). In addition, we found that the plasma concentration of IL-26 is associated with decreased MCH, a hematological aberration detectable in mild, and further decreased in severe, cases of COVID-19 (36, 37). Taken together, these findings are suggestive of a mechanistic link between IL-26 and severe COVID-19 that deserves further investigation in larger study materials.

It is true that a previous study by Caterino M. et al. (38) failed to detect differences in the serum concentrations of IL-26 among COVID-19 patients with mild, moderate, or severe disease. However, their study did not include healthy control subjects. We think that their uncontrolled approach contributed to the failure to obtain evidence for the involvement of IL-26 in COVID-19. Furthermore, the fact that Caterino M. et al. studied only 27 patients in total, and as few as 6 with severe disease, argues that low statistical power made it impossible to detect differences among sub-groups with varying disease severity. Similarly, the limited size of our current study material, and the fact that it only included 5 patients with mild COVID-19 (hospitalized without supplemental oxygen), can explain why we failed to detect a statistically significant difference in the plasma concentrations of IL-26 between COVID-19 patients with mild or severe disease (p-value > 0.99, not shown), or a direct correlation between the plasma concentration of IL-26 and an assessment of disease severity (i.e., number of hospitalization days per patient). Moreover, we think that methodological aspects played a role in the study by Caterino M. et al., because they measured IL-26 in serum, whose generation requires blood coagulation—a likely confounder for cytokine release—and found that the concentration of IL-26 was below their technical limit of detection in 20 out of 27 patients. These results are in sharp contrast with our assessment of IL-26 in plasma, in which only 2 out of 49 patients in the COVID-19 group had a concentration of IL-26 below the technical detection limit.

In summary, our current pilot study forwards evidence that systemic IL-26 is markedly increased in patients with acute COVID-19, and that it correlates with neutrophil-mobilizing cytokines, a marker of prolonged neutrophil survival, and with markers of tissue damage and hematological alteration, the latter of which are known to signify severe COVID-19. Thus, IL-26 is involved in acute COVID-19, and it seems feasible that this intriguing kinocidin plays an important role in the hyperinflammation associated with acute COVID-19, a possibility that motivates further investigation into the clinical potential of IL-26 as a target for diagnosis, monitoring, and therapy in this deadly disease.

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 the Swedish Ethical Review Authority in Gothenburg (Diary No. 2020-02579). The patients/participants provided their written informed consent to participate in this study.

EIC, SE, SKG, L-OC, and AL designed the outline of the study. AK, ÅK, and L-OC collected the patient material. SE, KP, and MP performed the neutrophil isolation, flow cytometry evaluation, and measurement of markers of NET formation. EIC, SE, KP, and MP measured blood cytokines. EIC and SE analyzed and compiled the collected data and performed all statistical analyses. SKG, L-OC and AL interpreted and supervised the data and statistical analyses. EIC and AL drafted the original manuscript that was read and reviewed by all authors. All authors contributed to the article and approved the submitted version.

This work was financially supported by the Swedish Asthma Allergy Foundation (AL: # F2019-0031), Swedish Research Council (AL: # 2021-01527; L-OC: # 2016-01234) and Region Stockholm (AL: ALF # 2018-0088).

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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