A total of 92 patients were enrolled in the study, from which 68 were infected with influenza and 24 had COVID-19. The clinical characteristics of the study participants are summarized in Table 1. Seventy percent of influenza patients were male, with a median age of 48 years. Similarly, COVID-19 affected males (75%) preferentially, with a median age of 50 years. Obesity was more common among influenza patients, while the prevalence of other comorbidities did not differ between both diseases. Cough, dyspnea, fever, myalgia, and arthralgia were the most frequent symptoms of respiratory illness in both participant groups. Interestingly, dyspnea, rhinorrhea, and sputum production were more common during influenza, whereas dry cough and vomit were more frequent among COVID-19 patients (Table 1).
Triage vital signs were similar between groups, except for a higher blood temperature and heart rate in influenza patients. Also, most laboratory parameters routinely tested in emergency departments did not differ between individuals with influenza and COVID-19. The levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and procalcitonin were increased in influenza patients as compared to COVID-19 subjects (Table 1). Similarly, severity of illness scores at admission, including the Sequential Organ Failure Assessment (SOFA), and the Acute Physiology And Chronic Health Evaluation II (APACHE-II), were higher in influenza cases. We should note that despite this, COVID-19 patients showed higher mortality compared to influenza (41% vs. 23%, p=0.1151).
Expression of CXCL17 in the lung of patients with influenza or COVID-19
CXCL17 is constitutively expressed in the respiratory tract and lungs of mice and humans [3, 4]. Under inflammatory conditions, the production of this chemokine is known to be further upregulated [4, 6, 7, 11, 12]. To investigate whether CXCL17 may participate in immunity against influenza A(H1N1) and SARS-CoV-2, we analyzed the tissue expression pattern of CXCL17 in lung autopsy specimens obtained from patients who died of influenza or COVID-19. The histological changes induced in the lungs during influenza were mainly characterized by intra-alveolar inflammatory infiltrates consisting of macrophages and polymorphonuclear cells scattered between areas of edema, hemorrhage, and fibrin deposits (Figure 1a, left upper panel). We also noted that the integrity of alveolar walls and the micro-architecture of the lung were conserved in influenza patients (Supplemental Figure 1). CXCL17 expression was detected mainly within the cytoplasm of infiltrating macrophages, but not in polymorphonuclear cells. The expression of CXCL17 was also detected within alveolar epithelial cells (Figure 1a, left lower panel). Blood vessels and pleura did not express CXCL17 in lung autopsy specimens from influenza patients (data not shown).
Meanwhile, SARS-CoV-2 induced distinctive morphological changes in the infected lung, characterized by an intense inflammatory infiltrate affecting extensive areas of the parenchyma, as well as the thickness of alveolar walls, and partial loss of the histological architecture of the the lung (Supplemental Figure 1 and Figure 1a, right upper panel). These changes are compatible with interstitial pneumonia. As observed in influenza infected lung sections, the expression of CXCL17 in the lungs of deceased COVID-19 patients was also detected within macrophages and alveolar epithelial cells (Figure 1a, right lower panel). CXCL17 was not expressed in endothelial cells of alveolar capillaries, but pleura showed increased CXCL17 expression (data not shown). Collectively, these findings indicate that influenza and SARS-CoV-2 promote the production of CXCL17 in the infected lung.
High serum levels of CXCL17 distinguish influenza from COVID-19
Although CXCL17 is mainly produced at mucosal surfaces, increased serum levels of this chemokine might serve as a readout of active local immune responses. Thus, we addressed whether the CXCL17-mediated responses found in the lungs of influenza- and SARS-CoV-2-infected patients could be also detected in the serum. Our results indicate that CXCL17 levels were significatively elevated in the serum of influenza cases, but not in healthy donors (HD) or COVID-19 subjects. The latter group indeed showed low serum CXCL17 levels, similar to the levels observed in HD (Figure 1b). These findings contrast with the expression of CXCL17 detected in the lung of COVID-19 patients, suggesting that the levels of CXCL17 in serum and its expression in lung tissue specimens do not correlate during the course of the disease. Nonetheles, our lung immunohistochemical analyses only focused on the expression of this chemokine in the late stages of influenza and COVID-19, whereas serum samples were taken within the first day after patients´ hospital admission.
Given that these differences suggested distinctive serum CXCL17 responses during influenza and COVID-19, we next investigated if CXCL17, along with other clinical characteristics, could have certain diagnostic value to discriminate between both viral infections. In an unsupervised clutering analysis, we found that some influenza patients grouped together according to their clinical and laboratory parameters, but another cluster was formed by combined influenza and COVID-19 subjects (Figure 1c). This suggests that the differentiation of the two infections by clinical characteristics would be difficult. Nonetheless, in a random forest analysis, CXCL17 was among the most explicative variables of the viral subtype. Indeed, in a bivariate logistic regression analysis using the variables identified in the random forest algorithm, only CXCL17, along with procalcitonin, showed significant association with influenza (Supplemental Figure 2). LDH and ALP were marginally associated with influenza, whereas platelets showed no correlation with any type of infection. Interestingly, although irrelevant in the random forest algorithm, symptoms such as dyspnea, rhinorrhea, and sputum production were predictors of influenza, whereas dry cough and vomit were associated with COVID-19.
To further estimate the diagnostic value of CXCL17, we performed a ROC curve analysis with the serum levels of CXCL17 of influenza and COVID-19 subjects. We observed that CXCL17 levels could reliably differentiate between influenza and COVID-19, with an AUC of 0.81 (Figure 1e). Using a cut-off value of 841pg/mL, elevated serum levels of this chemokine have a 78.2% sensitivity, 73.5% specificity, 89.2% PPV, 51.3% NPV, and an OR of 8.79 (3.1-26, 95% CI) to distinguish influenza from COVID-19. Together, our results point to the diagnostic use of serum CXCL17 levels in patients with acute respiratory illness to enable distinguishing infection between these viruses.
Dynamics and prognostic value of serum CXCL17 levels in influenza
Next, we evaluated the dynamics of serum CXCL17 levels during influenza. For this purpose, we grouped influenza patients according to the duration of their illness, defined as the period from symptom onset to hospital admission. Interestingly, we found that levels of CXCL17 increased early in influenza patients within the first two days following the onset of symptoms, and levels remained increased during the first two weeks of illness. However, the maximum levels of CXCL17 were observed among influenza patients seeking medical attention three weeks after the onset of symptoms (Figure 2a). In 54 of the 68 influenza patients enrolled in the study, an additional serum sample was obtained seven days (D7) following hospital admission (D0). Although there were no differences in CXCL17 between D0 and D7, most patients showed constant or decreasing chemokine levels (Figure 2b), except for one individual who showed a notable increase in CXCL17 and succumbed to the infection. Overall, the dynamics of CXCL17 levels post-hospital admission were similar in survivors and deceased influenza patients (Figure 2c).
Importantly, serum levels of CXCL17 at D0 were significatively higher in patients who died of influenza as compared to survivors (Figure 2d). Indeed, CXCL17 again was among the variables with the higher importance for influenza-associated mortality in a random forest analysis (Figure 2e), showing an AUC of 0.70 to differentiate both groups in the ROC curve analysis (Figure 2f). Serum levels of CXCL17 above 1,128pg/mL predicted a fatal outcome with a 75% sensitivity and 50% specificity, showing a non-significant OR value for mortality of 3.0 (0.86-9.24, 95% CI). The survival of patients with CXCL17 below such cut-off value was lower at day 14 after admission when compared to individuals with higher levels of this chemokine (Figure 2g, Table 2). Conversely, accumulated survival at days 28 and 60 following hospital admission was lower in influenza patients with serum levels of CXCL17 ≥1,128pg/mL.
Most clinical and laboratory parameters did not differ between the two groups of influenza patients (Supplemental Table 2). Furthermore, none of these clinical characteristics impacted the serum levels of CXCL17 (Figure 3a), and this chemokine was not associated with the severity of ARDS in terms of the PaO2/FiO2 values at admission (Figure 3b). These results together suggest that CXCL17 represents an independent prognostic factor of mortality in influenza. In contrast, no differences in serum CXCL17 levels at admission were observed between survivor- and deceased-COVID-19 patients (Figure 3c).
We also noted that CXCL17 was increased among influenza patients that developed acute kidney injury (AKIN; Figure 3d), and its levels were even higher in individuals requiring renal replacement therapy (Figure 3e). Using the same cut-off value of 1,128pg/mL, elevated serum levels of CXCL17 showed a 73.6% sensitivity, 51% specificity, and a non-significant OR of 2.91 (0.96-8.28, 95% CI) to predict the development of AKIN (Figure 3f). Similarly, increased levels of CXCL17 showed an 87.5% sensitivity, 53.8% specificity, and a significant OR of 8.16 (1.71-38, 95% CI) to predict the need for renal replacement therapy (Figure 3g).
Influenza A(H1N1) pdm09 virus induces the production of CXCL17 in human lung epithelial cells and macrophages
The higher levels of CXCL17 in sera from influenza patients prompted us to investigate the possible cellular sources of CXCL17 during influenza. To this end, we infected human A549 lung alveolar epithelial cells and peripheral blood monocyte-derived macrophages with a clinical isolate of the influenza A(H1N1) pdm09 virus. Interestingly, while both human cell types produced high amounts of CXCL17 at 24 h, 48h, and 72h after the infection, A549 epithelial cells produced lower levels of CXCL17 in response to influenza as compared to macrophages (Figure 3h-i). These findings are cosnsitent with the the expression of CXCL17 in lung macrophages and alveolar epithelial cells in autopsy specimens from influenza patients. This demonstrates that the influenza A(H1N1) pdm09 virus stimulates CXCL17 expression in humans, both in vivo and in vitro.