Introduction

Community-acquired pneumonia (CAP), a prevalent infectious disease affecting individuals across all age groups, particularly children, the elderly, and immunocompromised individuals, is noteworthy1. To date, CAP remains a significant contributor to morbidity and mortality in human beings2. Despite ongoing endeavors to lessen the symptoms of severe pneumonia, the mortality rate for patients admitted to the intensive care unit (ICU) continues to be alarmingly high3. Annually, one million patients with CAP require hospitalization, and 60,000 cases die of this disease in the United States4. The impact of CAP on public health is substantial, not only due to its significant morbidity and mortality rates, but also it imposes a considerable burden on healthcare resources5. In addition, the clinical presentation of patients with CAP sometimes fails to align with the severity of the condition3. Early identification and timely intervention of high-risk patients are imperative in mitigating the progression to severe pneumonia and reducing mortality rates.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a recently discovered endoplasmic reticulum stress-induced neurotrophic factor that mitigates cerebral ischemia/reperfusion injury by attenuating the inflammatory response, and has beneficial effects in multiple diseases6. As a crucial cellular self-protective factor, MANF plays a pivotal role in mounting responses to diverse stressors, encompassing endoplasmic reticulum stress, oxidative stress, and neuronal damage7,8. It is known that the most classical role of MANF consists of functioning as a neurotrophic factor, thereby exerting direct neuroprotective effects within the nervous system, and exhibiting cellular protective properties in animal models of non-neurological disease, such as retinal injury, diabetes, liver injury, myocardial infarction, nephrotic syndrome, and others7.

The previous study has revealed that recombinant murine MANF alleviates sepsis-associated with lung injury through repressing endoplasmic reticulum stress9. In addition, an animal experiment found that MANF lessens lipopolysaccharide-induced acute lung injury by attenuating the activation of macrophages10. Endoplasmic reticulum stress is obviously activating in Pseudomonas aeruginosa pneumonia11. Therefore, we thought that MANF may implicated in the course of CAP. However, the precise role of MANF in patients with CAP remains elusive. Further investigation is warranted to elucidate the potential association between serum MANF level with CAP. The objective of this study was to investigate the correlation between serum MANF and CAP, aiming to enable early prediction of CAP severity progression and provide novel insights for CAP treatment.

Materials and methods

Study design and participant’s enrollment

A total of 319 patients confirmed with CAP were enrolled in this prospective cohort study, all of whom were admitted to the Second Affiliated Hospital of Anhui Medical University between November 2021 and December 2022. According to the diagnostic criteria of CAP, eligible patients were recruited in the study. The demographic characteristics and clinical data were gathered. All patients diagnosed with CAP must be at least 18 years, and perform nucleic acid and chest X-ray test prior to hospitalization to rule out COVID-19. The specific exclusion criteria and inclusion criteria were described in detail in our former studies12,13,14. At the same time, all patients should conduct comprehensive clinical observation and monitor to ensure the accuracy and reliability of the study results. For the further evaluating the severity of CAP, severity scores including CURB-65, PSI, SMART-COP, and APACHE II scores were conducted. Moreover, in order to compare the level of serum MANF, age and sex matched healthy volunteers were enrolled. Fasting peripheral blood were collected15. All experimental protocols were approved by Ethics Committee of Second Affiliated Hospital of Anhui Medical University (YJ-YX2021-147). All methods were carried out in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects and/or their legal guardians.

Enzyme-linked immunosorbent assay (ELISA)

Morning fasting blood samples were collected from patients with CAP. Centrifugation of blood specimens were carried out. Then, serum samples were collected and store in an ultra-low temperature refrigerator. The level serum MANF was detected via ELISA in accordance with the previous study16. Human MANF ELISA kits (JYM2692Hu) were provided by Wuhan ColorfulGene Biological Technology Co.

Statistical analysis

All statistical analysis was performed in IBM SPSS Statistics version 22. The quantitative variables were expressed by mean or median. Due to the non-normality, the values of serum MANF level were log-transformed. The categorical variables were shown as frequencies. The differences of baseline characteristics were compared by One-way ANOVA, non-parametric test, or χ2 test depending on the distributed characteristics of raw data. The relationships of serum MANF with baseline characteristics were estimated by Spearman rank correlation analysis. The correlations of serum MANF with severity scores were estimated by linear and logistic regression analyses. The relationship between serum MANF and prognostic outcomes were analyzed via χ2 test and logistic regression analyses. The predictive powers of serum MANF for clinical outcomes were analyzed by receiver operating characteristic (ROC). P-values <0.05 were considered statistically significant.

Results

Demographic information and clinical characteristics

The study included 319 patients diagnosed with CAP. According to the tertile of serum MANF level, CAP patients were divided into three groups: Tertile 1 (T1) group, Log MANF lower than 3.15; T2 group, Log MANF from 3.15 to 3.33; T3 group, Log MANF higher than 3.33. As illustrated in Table 1, there was no significant differences of age, body mass index, the numbers of complications, the counts of white blood cell (WBC), neutrophil, lymphocyte, monocyte, eosinophil, and basophil among three groups. Moreover, the etiology was analyzed. There was no difference of streptococcus pneumonae, legionella pneumophila, respiratory virus, or others in CAP patients with different tertiles of serum MANF (Table 1). In addition, the number of male patients were increased across with serum MANF among CAP patients. In addition, the obvious differences of creatinine, D-dimer, C-reactive protein (CRP), and interleukin-6 (IL-6) were observed among three subgroups (Table 1).

Table 1 Demographic information and clinical characteristics.
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Level of serum MANF in CAP patients

The levels of serum MANF in CAP patients and control group were quantified using ELISA. As shown in Supplemental Fig. 1, the level of serum MANF was obviously higher in CAP patients than those in healthy participants. Moreover, the level of serum MANF was gradually elevated in cases in accordance with CURB-65 score (Fig. 1A). In addition, serum MANF was compared in CAP cases with different PSI score. The results indicated that serum MANF was highest in V grade among CAP cases (Fig. 1B). Additionally, the content of serum MANF was elevated in >10 scores grade compared with < 4 scores and 4 ~ 6 scores of APACHE II (Fig. 1C). Lastly, the level of serum MANF was compared in cases with different SMART-COP score. We found that the concentration of serum MANF was higher in 5 ~ 6 and 7 ~ 8 scores than other classifications (Fig. 1D).

Fig. 1
figure 1

The level of serum MANF in CAP patients with different severity. The level of serum MANF was detected using ELISA. (A-D) The differences of serum MANF was compared in CAP patients with different scoring criteria. (A) The level of serum MANF in CAP cases of different CURB-65 scores. (B) The level of serum MANF in CAP cases of different PSI scores. (C) The level of serum MANF in CAP cases of different APACHE II scores. (D) The level of serum MANF in CAP cases of different SMART-COP scores. *P < 0.05, **P < 0.01.

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Associations between serum MANF and scoring criteria

The relationships of serum MANF with different scoring criteria were evaluated by multivariate linear regression analyses. The results hinted that each 1-unit increase of Log MANF was associated with the elevations of 1.050 score in CURB-65, 45.969 score in PSI, 2.602 score in SMART-COP, and 8.260 score in APACHE II, respectively (Table 2). In addition, CAP patients were allocated into three subgroups in terms of the tertiles of serum MANF. Multivariate logistic regression analyses revealed that the odds ratio (OR) was 3.731 (95%CI: 1.279, 10.880) of PSI, and 3.041 (95%CI: 1.104, 8.379) of APACHE II in T3 group compared with T1 group (Table 2).

Table 2 Associations between serum MANF and CAP severity scores.
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Associations between serum MANF and prognostic outcomes

The level of serum MANF on admission was compared in CAP cases with different clinical outcomes. Although there was no difference of serum MANF in CAP cases with hospitalization duration (Fig. 2D), the level of serum MANF was dramatically higher in cases who underwent mechanical ventilation, death, ICU admission during hospitalization (Fig. 2A-C). Besides, the links of serum MANF with different prognostic outcomes were determined. Chi-square test found that the relative risks (RRs) of mechanical ventilation, ICU admission, and 30-day mortality were sensibly elevated in line with the increased serum MANF among CAP cases (Table 3). The potential confounding factors were controlled. Multivariate logistic regression analysis indicated that there were positive relationships between serum MANF and the poorly clinical outcomes among CAP cases during hospitalization (Table 3).

Fig. 2
figure 2

The level of serum MANF in CAP patients with different prognosis. The level of serum MANF was detected using ELISA. (A-D) The differences of serum MANF was compared in CAP patients with different prognosis. (A) The level of serum MANF in CAP cases with and without mechanical ventilation. (B) The level of serum MANF in CAP cases with and without ICU admission. (C) The level of serum MANF in CAP cases with and without death. (D) The level of serum MANF in CAP cases with different hospital length. **P < 0.01.

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Table 3 Associations between log MANF and prognostic outcomes.
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Predictive powers of serum MANF for poorly prognostic outcomes

The predictive powers of serum MANF for different clinical outcomes were estimated by ROC curve. The area under the curve (AUC) for different prognosis were as follows: Mechanical ventilation, 0.83 (95%CI: 0.75, 0.90); ICU admission, 0.83 (95%CI: 0.77, 0.89); 30-Day mortality, 0.84 (95%CI: 0.71, 0.96) (Fig. 3). The cutoff values of serum MANF for different prognosis were shown below: Mechanical ventilation, 3230.6 pg/mL; ICU admission, 4200.5 pg/mL; 30-day mortality, 5105.9 pg/mL (Fig. 3).

Fig. 3
figure 3

The predictive capacities of serum MANF for different prognosis. (A-C) The predictive capacities of serum MANF for different prognostic outcomes were analyzed by ROC curve. (A) The predictive power for mechanical ventilation. (B) The predictive power for ICU admission. (C) The predictive power for death.

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Discussion

The current cohort study predominantly analyzed the relationship of serum MANF with severity and prognosis. Our research primarily found that: Serum MANF level on admission was evidently elevated in severe CAP patients; The level of serum on admission was significantly and positively related with scoring criteria; The level of serum on admission was substantially higher in patients who underwent the poor prognosis during hospitalization; The level of serum MANF on admission was positively correlated with the risk of poor prognosis during hospitalization. The above results hinted that MANF may participate in the disease process of CAP.

Recent studies have found that SARS-CoV-2 infection can activate host immune response and induce endoplasmic reticulum stress in patients with coronavirus disease 201917,18. Not only that, endoplasmic reticulum stress is implicated in the pathological process of pneumonia19,20. In addition, in vitro experiment suggested that Chlamydia pneumoniae infection evokes endoplasmic reticulum stress in murine adipocytes21. CAP is one of inflammatory diseases which mainly incurred by gram-positive bacterium Streptococcus pneumoniae and viruses22,23. Therefore, we guessed that the infection of bacterium or viruses may evoke endoplasmic reticulum stress in lung tissues of CAP patients, and the expression of MANF was changed in the progression of CAP. Then, the level of serum MANF was determined. The results found that the level of serum MANF on admission was increased in severe pneumonia patients. Not only that, the content of serum MANF was progressively increased in line with the scoring criteria of CAP. Linear and logistic regression analyses further showed that there was a positive correlation between serum MANF level and the scoring criteria of CAP. These results hinted that the level of serum MANF has a positive correlation with the severity of CAP.

There is increasing evidence have found that the expression level of MANF is closely related to the prognosis in several diseases. Recent research indicated that the protein and mRNA expression levels of MANF are evidently elevated and positively associated with higher risk of tumor recurrence in patients with hepatocellular carcinoma24. In addition, a prospective observational study found that increased serum MANF concentration can predict the risks of early neurologic deterioration and 90-day bad prognosis in acute intracerebral hemorrhage patients25. Moreover, higher serum MANF content on admission elevates the risk of poor prognosis in patients with severe traumatic brain injury26. Consequently, the relation between serum MANF concentration and the prognosis was explored among CAP patients. The results shown that the concentration of serum MANF was clearly risen in patients who underwent death, mechanical ventilation, ICU admission within 30 days of hospitalization. Logistic regression analysis verified serum MANF on admission was positively related to the frequencies of mechanical ventilation, ICU admission, and death within 30 days of hospitalization. In addition, the predictive capacities of serum MANF level on admission for poor prognostic outcomes were estimated by ROC curve. The data manifested that the level of serum MANF on admission can effectively predict the risks of 30-day mortality, mechanical ventilation, ICU admission during hospitalization. Therefore, the above results demonstrated that the level of serum MANF on admission is positively correlated with poor prognosis in CAP patients.

This perspective cohort study innovatively discovered the relationship of serum MANF with CAP. However, there were several flaws in this research. First, all enrolled participants were from one hospital. These findings should be further vindicated in another cohort. Second, the number of enrolled patients was rather small, a larger sample size is required and proved the results. Third, this study was an epidemiological investigation on the basis of clinical patients, the specific mechanism of MANF elevation in serum and originated cells of MANF production were unclear. In vivo and in vitro experiment can illuminate the physiological processes of MANF increase in CAP patients. Fourth, the level of serum was only detected at hospitalization. However, more times blood drawn for non-medical purpose were prohibited in hospital. Thus, the change of serum MANF was not unclear among CAP cases from the onset of pneumonia to its resolution.

Conclusion

In summary, this evidence demonstrated that serum MANF is positively associated with scoring criteria and poor prognosis in CAP patients. Serum MANF may be of considerable clinical value and can be regarded as a diagnostic and prognostic biomarker in CAP. Of course, more rigorous and uniform studies are required to further analyze the relationship of serum MANF level with CAP, and to explore the practical significances of assisting clinical decisions.