Serum interleukin-17 predicts severity and prognosis in patients with community acquired pneumonia: a prospective cohort study

Background Some studies previously demonstrated that interleukin-17 (IL-17) involves in pulmonary diseases progression. Nevertheless, the role of IL-17 in community-acquired pneumonia (CAP) remains unknown. This study aims to examine the correlations between serum IL-17 with the severity and prognosis in CAP patients through a prospective cohort study. Methods All 239 CAP patients were recruited. Serum IL-17 was detected by enzyme-linked immunosorbent assay (ELISA). The CAP severity was evaluated through CAP severity scores, including CURB-65, CRB-65, PSI, SMART-COP, CURXO and APACHE II. Results Serum IL-17 was gradually increased consistent with the severity of CAP. Correlative analysis suggested that serum IL-17 was associated with clinical physiologic indicators among CAP patients. Logistic regression indicated that serum IL-17 was positively related to CAP severity scores. Additionally, the prognostic outcomes were tracked among CAP patients. The levels of IL-17 on admission were significantly increased in CAP patients with ICU admission, mechanical ventilation, vasoactive agent, death and longer hospitalization days. Logistic regression analyses revealed serum higher IL-17 on admission elevated the risks of vasoactive agent usage and longer hospital stays in CAP patients. The cut-off concentrations of serum IL-17 for death, ICU admission, mechanical ventilation and ≥ 14 hospital stays were 86.80 ng/mL, 84.92 ng/mL, 84.92 ng/mL and 60.29 ng/mL respectively. Conclusions Serum IL-17 on admission is positively associated with the severity and poor prognosis among CAP patients, revealing that IL-17 may implicate in the pathological process of CAP. Therefore, serum IL-17 may become an effective biomarker for diagnosis, prognosis and therapy for CAP patients.


Introduction
Community-acquired pneumonia is a lung infection as a result of a wide variety of microorganisms, mainly including bacterial pathogens (such as Streptococcus pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus, et al.) and viral (such as human rhinovirus, influenza, parainfluenza virus, respiratory syncytial virus, et al.) [1]. CAP always causes immune system disorders, local and systemic higher inflammatory responses in patients [2]. CAP is consistently a leading cause of respiratory illness around the world [3,4]. It disproportionately affects adults older than 60 years old or children younger than five years old. CAP has resulted in over 60,000 deaths annually and more than 10 billion dollars health care costs in United States [4][5][6][7]. Patients with mild infections are less likely to seek medical attention and diagnosis. Therefore, the true incidence of CAP may be underestimated [8]. If the diagnosis and treatment were not conducted timely and properly, the rate of fatality will rise sharply [9]. Although validated pneumonia severity scores can guide the decision between outpatient and inpatient therapy, appropriate indicators as biomarkers for CAP may further assist with risk stratification. Therefore, a typical biomarker is imminently explored to improve early clinical diagnosis and decrease the complications. As we know, early detection of CAP is critical in improving prognosis for CAP patients.
Interleukin-17 (IL-17) is primarily secreted by T helper 17 cells (Th17), monocyte and eosinophilia [10,11]. Increasing data have proved that IL-17 can recruit neutrophils, activate T cells, stimulate macrophages and epitheliums. Finally, IL-17 produces a range of proinflammatory cytokines and induces inflammatory reaction in the bodies [12,13]. Past works have indicated that IL-17 is highly expressed in rheumatoid arthritis and ulcerative colitis [14,15]. IL-17 also has been shown to play central roles in the process of several lung diseases, including lung fibrosis, emphysema, acute lung injury and pulmonary hypertension [16][17][18][19]. Moreover, in vitro research has confirmed that IL-17 overexpression or recombinant IL-17 administration elevates chemokines and evokes inflammatory reaction of lung [20,21].
At present, IL-17 is considered to an attractive target for inflammatory responses in the bodies. However, the physiological function of IL-17 was not obvious in CAP patients at present. The associations of IL-17 with severity and clinical outcomes of CAP patients were unclear. We speculate that IL-17 takes part in the pathophysiology process of CAP. Consequently, the study aims to explore the associations of serum IL-17 with the severity and prognosis in CAP patients through a prospective cohort study.

Study design and subjects
All CAP patients were enrolled from August 2019 to April 2021 in the Department of Respiratory and Critical Care Medicine of Second Affiliated Hospital. At first, all 288 patients were recruited and agreed to take participate in this research. Twenty-eight patients with incomplete information, 12 lost cases and 9 withdrawing cases were excluded from this research. Finally, 239 CAP patients were enrolled in the present research. All CAP patients were eligible for this research. After obtaining informed consent from the patients, we gathered relevant demographic characteristics and clinical information. At the onset of CAP, CAP patients always showed specific symptoms and signs, primarily including fever, cough, expectoration, and even purulent sputum; with or without chest pain; chest tightness and dyspnea in CAP patients with affected multiple pulmonary lobes. All selected 239 CAP patients in this research must meet certain diagnostic standard: In accord with the clinical manifestations of pneumonia and consistent with chest CT scans, mainly including (1) chest radiograph suggesting either a new patchy infiltrate, leaf or segment consolidation, ground glass opacity or interstitial change; (2) at least one of the following signs: the presence of cough, sputum production and dyspnoea; core body temperature higher than 38.0 °C; auscultatory findings of abnormal breath sounds and rales; or the counts of white blood cell more than 10 × 10 9 L or less than 4 × 10 9 L; (3) occurred in the community, rather than in a hospital [22]. The inclusion criteria were as follows: pneumonia was occurred in the community or within a definite incubation period after hospitalization; these candidates were not admitted to the hospital for the last three months; not pregnant; no other pulmonary diseases, such as pulmonary malignant tumor, pulmonary tuberculosis or immunodeficiency [23,24]; no antibiotic treatment or intervention before hospitalization. Peripheral blood samples were collected and anticoagulated with EDTA within 24 h after hospitalization. Blood routine examination and biochemical detection were performed in all CAP patients. Then, the severity of pneumonia was evaluated using well-recognized CAP severity scores, including Pneumonia Severity Index (PSI), CURB-65, CRB-65, SMART-COP and Acute Physiology and Chronic Health Evaluation II (APACHE II). Moreover, CAP patients were divided into mild patients and severe patients through CURXO score. Severe CAP is a group of patients who requires treatment in the intensive care unit (ICU). Our research has obtained approval from Ethics Committee of Second Affiliated Hospital of Anhui Medical University (YX2021-085). Furthermore, written agreement consent from eligible patients or the patient's next of kin were obtained.

Enzyme-linked immunosorbent assay (ELISA)
Before any treatment, peripheral blood samples were collected and centrifuged. Then samples were stocked in the − 87 °C ultracold refrigerator until use. IL-17 ELISA kits (CSB-E12819h) were purchased from Cusabio, Wuhan, China (https:// www. cusab io. com/). IL-6 ELISA kits (JYM1942Hu) were obtained from Wuhan Colorful Gene Biological Technology Co. The concentrations of serum inflammatory cytokines were detected following the instructions closely [24,25].

Statistical analysis
All statistical analyses were conducted using SPSS 19.0 version software. Demographic characteristics and laboratory parameters were shown as mean ± standard error (SEM) or median with interquartile ranges. Categorical variables were expressed with frequencies and percentages. The correlations between serum IL-17 and clinical characteristics were evaluated using Pearson correlation analysis among CAP patients. The association between serum IL-17 with CAP severity scores and prognostic outcomes were accessed using logistic regression analysis. There were no significant differences (P > 0.05).

Demographics characteristics and clinical information
In this study, 239 CAP patients were recruited. Demographic characteristics and laboratory test results were collected and generalized. The average age was 61.09 years old among CAP patients (Table 1). Among them, female patients accounted for 40.17%. The proportion of smoking in CAP patients was 17.57%. Besides, the comorbidities of CAP patients were analyzed. As shown in Table 1, 64 (26.78%) patients were with hypertension, 22 (9.21%) patients were with diabetes mellitus, 20 (8.37%) patients were with cerebral infarction, 11 (4.60%) patients were with coronary heart disease, 19 (7.95%) patients were with bronchitis and 78 (32.64%) patients were with other diseases. Blood cell content indicated that the count of white blood cell (WBC) was 8.26 × 10 9 /L, the count of neutrophil was 7.79 × 10 9 /L, the count of lymphocyte was 2.00 × 10 9 /L (Table 1). Meanwhile, liver function, renal function and myocardial function were tested among CAP patients. In addition, the median hospital stay is 10.00 days among CAP patients. During hospitalization, 71 cases admitted to the ICU, median hospital stays were 3.21 days, 66 cases were conducted with mechanical ventilation, 34 cases were used with vasoactive agent and 22 cases were died among CAP patients (Table 1). According to CURXO score, CAP patients who met the following two or more criterions were regraded as severe cases: confusion; urea nitrogen > 30 mg/L; respiratory rate > 30/min; abnormal shadows in multilobar or bilateral lungs; PaO 2 < 54 or PaO 2/ FiO 2 < 250 mmHg. Severe CAP patients accounted for 27.62% in CAP patients. The mean of CURB-65, CRB-65, PSI, SMART-COP and APACHE II was 1.31, 1.07, 78.72, 1.97 and 7.95, respectively.

The level of serum IL-17 in CAP patients
The concentration of serum IL-17 was measured and compared among various groups of CAP patients. The severity degrees of pneumonia were assessed by using CAP scoring system, such as CURXO, PSI, CURB-65, CRB-65, SMART-COP and APACHE II. Serum IL-17 was decreased in 0 score than those in the groups of 1-2 and ≥ 3 scores based on CRB-65 score (Fig. 1A). In accordance with SMART-COP score, serum IL-17 was lower in the group of 0-2 and 3-4 scores than those in the group of 7-8 score (Fig. 1B). IL-17 was higher in the group of 5-6 score than these in the group of 0-2 score (Fig. 1B). According to APACHE II score, IL-17 was highest in the group of > 10 score than other groups (Fig. 1C).
The level of IL-17 was elevated in the group of 6 ~ 10 score compared with the group of < 4 score (Fig. 1C). Based on CURB-65 score, IL-17 was gradually increased in line with CURB-65 score (Fig. 1D). Additionally, IL-17 was lower in severe group than these in mild group (CURXO score) (Fig. 1E). Based on PSI score, serum IL-17 was gradually elevated with PSI score elevation (Fig. 1F).

Associations of serum IL-17 with CAP severity scores in CAP patients
The concentration of serum IL-17 was divided into lower than 75% quartile and higher than 75% quartile. Then, the associations were accessed between serum IL-17 and CAP severity scores through logistic regression analysis in CAP patients. Univariable logistic regression analysis found that serum IL-17 was positively associated with 3-4 score (OR = 9.16; 95% CI 3. 16 Table 3).

The levels of serum IL-17 in CAP patients with different prognostic outcomes
Serum IL-17 was compared among CAP patients with different prognostic outcomes. As shown in Fig. 2A-C, the levels of serum IL-17 on admission were increased in patients with mechanical ventilation, vasoactive agent and ICU admission. Moreover, the levels of serum IL-17 on admission were elevated in patients with ≥ 14 hospital stays compared with patients with ≤ 8 hospital stays and from 8 to 14 hospital stays in CAP patients (Fig. 2D). In addition, serum IL-17 was further compared between alive and dead patients on admission. We found that serum IL-17 levels on admission were remarkably increased in the dead cases (Fig. 2E).

The association between serum IL-17 and the prognosis in CAP patients
Univariate and multivariate logistical regression was performed to investigate the association between serum  (Table 4).

ROC curves and cut-off concentration of serum IL-17 for prognosis
The predictive capacities of IL-17 for prognostic outcomes were accessed using receiver operating characteristic (ROC) area under the curve (AUC) among CAP patients. As shown in Fig. 3A    The cut-off concentration of IL-17 for ≥ 14 hospital stays was 60.29 ng/mL (Fig. 3D).

Discussion
This study strengthens the evidence of associations between serum IL-17 with the severity and prognosis among CAP patients. The research mainly found that: (1) Serum IL-17 on admission was gradually risen in parallel with CAP severity scores; (2) IL-17 on admission was closely correlated with many clinicopathological features among CAP patients; (3) IL-17 on admission was positively correlated with CAP severity scores in CAP patients; (4) Serum higher IL-17 on admission elevated the risk of ICU admission, mechanical ventilation, death and longer length of hospitalization among CAP patients during hospitalization. IL-17, one of robust pro-inflammatory cytokines, which takes part in the airway inflammation, is a central factor in the excessive activation of the body's defense system and the excessive inflammatory response [26]. The past studies demonstrated that IL-17 plays key roles in the progression of several lung diseases, including lung fibrosis, emphysema, acute lung injury and pulmonary hypertension [16][17][18][19]. Serum IL-17 is increased in patients with acute respiratory distress syndrome and participates in the occurrence of pneumonia [27,28]. Nevertheless, the role of IL-17 in CAP was ill-defined so far. Therefore, we tested the levels of serum IL-17 among CAP patients with different severity scores. In the current research, we discovered that the level of IL-17 was gradually increased in parallel with the CAP severity scores among CAP patients. In addition, logistic and linear regression analysis indicated that IL-17 was positively associated with CAP severity scores among CAP patients. These findings indicated that IL-17 is positively correlated with the severity of CAP patients. The previous studies from our laboratory have demonstrated that blood routine indices are obviously changed and the count of lymphocyte is dramatically reduced in COVID-19 patients [29][30][31]. Therefore, the associations of serum IL-17 and blood routine indices were evaluated among CAP patients in this cohort study. There were remarkable positive correlations between IL-17 with WBC and neutrophil, inverse correlations between IL-17 with lymphocyte and eosinophil in CAP patients. Moreover, our studies found that multiple organ dysfunction syndromes are always associated with the process of CAP [32][33][34]. IL-17 was positively correlated with uric acid, urea nitrogen, ALT, AST, CKMB and LDH among CAP patients. It is widely known that the occurrence of CAP is due to the invasion of pathogens, inflammatory cytokines and toxic metabolite [35]. Exorbitant inflammation evokes a serious of expanded cascade reaction, leading to systemic inflammation and multiple organ dysfunction syndromes [9,36]. Other research groups have revealed that cytokine storm is observed in COVID-19 patients and inflammation suppression alleviated the severity of COVID-19 [37,38]. In our present research, IL-17 was also shown to be prominently and positively related with inflammatory cytokines in CAP patients. These findings revealed that serum IL-17 may involve in the process of CAP and may be regarded as a biomarker for diagnosis and therapy for CAP patients.
Existing literatures have suggested that IL-17 is involved in the pathophysiology process and associated with the prognosis in pulmonary diseases. A research based on Chinese population found that IL-17 gene polymorphism is correlated with the risk and prognosis among patients with acute respiratory distress syndrome (ARDS) [39]. Several researches revealed that higher circulatory IL-17 is associated with the poor prognosis in patients with glioma and hepatocellular carcinoma [40,41]. So, the association of IL-17 and the prognosis was estimated in CAP patients. Our results reflected that IL-17 on admission was elevated in CAP patients with ICU admission, mechanical ventilation, vasoactive agent, death and longer hospital stays during hospitalization. Logistic regression analysis revealed that serum higher IL-17 on admission was positively associated with the adverse prognostic outcomes among CAP patients. These results showed that the level of serum IL-17 on admission may predict the prognostic outcomes of CAP patients.
Limitations of the research were small sample size and single-center study, a larger sample size and multicenter study is needed to perform in the future. Additionally, due to the limitation of cohort study, only descriptive and correlational analyses were conducted. Further proofs based on basic experiments are needed to confirm this conclusion and mechanism. Besides, IL-17 was just measured in serum. In the next step, IL-17 would be detected in the bronchoalveolar lavage fluid and lung tissues of CAP patients.

Conclusions
This research mainly demonstrated the correlations between serum IL-17 with the severity and prognostic outcomes of CAP patients based on a prospective cohort study. We found that IL-17 is gradually increased in parallel with the severity scores of CAP. Serum IL-17 is positively correlated with the severity and poor prognosis among CAP patients. The findings of this study indicated that IL-17 may be implicated in the pathophysiology processes of CAP. Therefore, it is likely that serum IL-17 may become an effective biomarker of diagnosis, prognosis and therapy for CAP patients in the future clinical works.