Blood long non‐coding RNA intersectin 1–2 is highly expressed and links with increased Th17 cells, inflammation, multiple organ dysfunction, and mortality risk in sepsis patients

Abstract Background Long non‐coding RNA intersectin 1–2 (lnc‐ITSN1‐2) exacerbates inflammation and promotes T‐helper (Th) cell differentiation, also serves as a biomarker in critical illness diseases. However, its clinical role in sepsis remains obscure. Hence, the study aimed to explore the relationship of lnc‐ITSN1‐2 with Th cells, inflammation, disease severity, multiple organ dysfunction, and mortality risk in sepsis. Methods Peripheral blood mononuclear cells (PBMC) were isolated from 95 sepsis patients and 50 health controls, followed by lnc‐ITSN1‐2 evaluation using RT‐qPCR. PBMC Th1, Th17 cells and their secreted cytokines in serum were detected by flow cytometry and ELISA, respectively. Results Lnc‐ITSN1‐2 in sepsis patients was higher than it in health controls (Z = −7.328, p < 0.001). Lnc‐ITSN1‐2 correlated with increased interferon‐gamma (p = 0.009), Th17 cells (p = 0.022), and interleukin‐17A (p = 0.006), but not Th1 cells (p = 0.169) in sepsis patients. Moreover, lnc‐ITSN1‐2 had a positive connection with C‐reactive protein (p = 0.001), acute pathologic and chronic health evaluation (APACHE) II (p = 0.024), and sequential organ failure assessment (SOFA) scores (p = 0.022). Regarding SOFA subscales, lnc‐ITSN1‐2 linked with elevated respiratory system score (p = 0.005), cardiovascular system score (p = 0.007), and renal system score (p = 0.004) but no other subscales. Besides, lnc‐ITSN1‐2 had an increasing trend, but no statistical difference, in septic deaths compared to survivors (Z = −1.852, p = 0.064). Conclusion Lnc‐ITSN1‐2 reflects sepsis progression and unfavorable prognosis to some extent, which may serve as a potential biomarker to improve the management of sepsis patients.


| INTRODUC TI ON
Sepsis is featured as an uncontrolled host response to infection, which causes approximately 30 million new cases and around 5.3 million deaths every year. [1][2][3][4] Meanwhile, the pathogenesis of sepsis is complex, including systemic inflammatory response, immune disorder, endoplasmic reticulum stress, and other pathophysiological processes, eventually causing multiple organ dysfunction and leading to high mortality. [5][6][7] Apart from the above understanding of the pathogenesis of sepsis, several treatment methods have been established for sepsis patients including antibiotics, hemodynamic management, infection source control, etc.; however, their mortality risk remains high. [8][9][10][11][12] Thus, it is helpful to explore potential biomarkers for sepsis, based on which clinicians could manage patients timely to improve their prognosis.
In the current study, we aimed to evaluate the relationship of lnc-ITSN1-2 with Th1 cells, Th17 cells, inflammation, multiple organ injury, and 28-day mortality in sepsis patients.

| Participants
From January 2019 to February 2021, this study consecutively recruited 95 sepsis patients. The recruitment criteria were set as (i) diagnosis of sepsis in terms of the sepsis-3 criteria (2016 version) 24 ; (b) aged more than 18 years; (c) hospitalized for sepsis treatment within 24 h of symptom onset. The sepsis patients were excluded from the study if they met the following conditions: (i) unwilling to provide peripheral blood (PB) samples for study use; (ii) complicated with autoimmune disease, solid tumor, or hematologic malignancy; (iii) pregnant and lactating woman. In addition, during the same period, this study also recruited 50 healthy participants with matched age and gender to sepsis patients as health controls. The exclusion criteria for sepsis patients were appropriate for health controls, and the health controls who had abnormalities in the physical examination were also ineligible for the study. The Ethics Committee of Zhongshan Hospital Affiliated to Xiamen University approved the study.

| Data collection
Clinical characteristics were collected after admission within 24 h for further analysis, including age, gender, body mass index (BMI), smoke status, drink status, comorbidities, primary infection site, primary organism, C-reactive protein (CRP) level, Acute Physiology and Chronic Health Evaluation II (APACHE II) score, and Sequential Organ Failure Assessment (SOFA) score. Additionally, the sepsis patients were closely followed up until death or up to 28 days, and mortality during 28-day follow-up was recorded.

| RT-qPCR assay
The RT-qPCR assay was performed for determining the lnc-ITSN1-2 in PBMCs. In brief, total RNA was extracted by PureZOL RNA isolation reagent (Bio-Rad); then, was reversely transcribed to cDNA using iScript ™ cDNA Synthesis Kit (with random primer) (Bio-Rad); besides, the qPCR was executed by SYBR ® Green Real-time PCR Master Mix (Toyobo). The primers were designed according to a previous study. 21 Subsequently, the lnc-ITSN1-2 was analyzed using the 2 −ΔΔCt method (GAPDH as an internal control).

| Statistics
Graphics were plotted using GraphPad Prism 7.02 (GraphPad Software Inc.), and statistical analyses were completed using SPSS 24.0 (IBM). Difference of lnc-ITSN1-2 between sepsis patients and health controls was compared by Wilcoxon's rank-sum test.
Association of two variables was analyzed by Spearman's rank correlation test. Correlation of lnc-ITSN1-2 with primary infection site and primary organism was determined by the Kruskal-Wallis H rank-sum test or Wilcoxon's rank-sum test. Comparison of lnc-ITSN1-2, Th1 cells, IFNγ level, Th17 cells and IL-17A level between deaths and survivors were evaluated by Wilcoxon's rank-sum test.
Statistical significance was concluded if a p-value less than 0.05.
Other clinical properties about patients were displayed in Table 1.

| DISCUSS ION
The clinical role of lnc-ITSN1-2 in inflammatory-related or critical ill diseases has drawn wide attention. For instance, a study reports that high lnc-ITSN1-2 has been found in ankylosing spondylitis patients, meanwhile, it relates to increased IL-1β cytokine 25 ; another study displays that lnc-ITSN1-2 of intestinal mucosa and peripheral blood mononuclear cell in IBD patients is higher than that in health controls 18 ; in critical ill diseases, the high expression of lnc-ITSN1-2 has been observed in SAP and AIS patients. 16,22 However, the role of lnc-ITSN1-2 in sepsis needs to be further explored. Thus, our research invited 95 sepsis patients and 50 health controls to compare lnc-ITSN1-2. Our results displayed lnc-ITSN1-2 was upregulated in sepsis patients. The explanations might be that sepsis is characterized by systematic inflammation; meanwhile, lnc-ITSN1-2 mediated inflammation by NOD2/RIP2/nuclear factor-κB (NF-κB) signaling pathway, which could reflect the level of inflammation to some extent 21,26 ; thus, lnc-ITSN1-2 was highly expressed in sepsis patients than it in health controls.
Recently, it is reported that lnc-ITSN1-2 relates to inflammation and disease severity/activity in critical ill diseases. For example, prior research presents that lnc-ITSN1-2 correlates with increased CRP, Ranson's score, APACHE II score in SAP patients 16 ; meanwhile, lnc-ITSN1-2 positively associates with cognitive impairment, CRP, TNFα and IL-17 levels in AIS patients. 22 However, the relationship between lnc-ITSN1-2 and disease severity in sepsis is not fully understood. In our study, lnc-ITSN1-2 was associated with elevated Th17 cells, inflammation, and APACHE II score in sepsis patients. The reason might be that (1) lnc-ITSN1-2 might mediate miR-125a to activate CD4 + T cell, induce Th17 cell differentiation, and then release the pro-inflammatory cytokines 18 ; therefore, lnc-ITSN1-2 correlated with elevated Th17 cells and inflammation; (2) miR-125a is a well-known anti-inflammatory factor, which inhibits inflammation by regulating the Wnt/β-catenin and NF-κB pathways 27 ; thus, lnc-ITSN1-2 might target miR-125a (mentioned above) to promote inflammation, thus causing multiple organ injury, which indirectly increased disease severity in sepsis patients.
Besides, our study presented the relationship between lnc-ITSN1-2 and multiple organ dysfunction in sepsis patients, which showed that the lnc-ITSN1-2 related to raised SOFA score; moreover,

TA B L E 3
Correlation of lnc-ITSN1-2 expression with primary infection site and primary organism it was positively associated with respiratory system, cardiovascular system, and renal system injury. The data might be caused by that (1) high lnc-ITSN1-2 regulated the inflammation (described above), which accelerated multiple organ injury in sepsis patients; (2) lnc-ITSN1-2 might facilitate cell injury and apoptosis, 28 which directly induced the multiple organ injury.
At present, lnc-ITSN1-2 as a prognostic biomarker for critical ill diseases has been illustrated such as AIS and SAP. 16,22 For example, lnc-ITSN1-2 has a connection with worse recurrence-free survival in AIS patients 22 ; regarding SAP, lnc-ITSN1-2 in deaths is higher than that in survivors; meanwhile, it discloses decent predictive value for elevated mortality risk. 16 In the present study, we evaluated the relationship of lnc-ITSN1-2 with prognosis in sepsis patients, which illustrated that lnc-ITSN1-2 presented an increasing trend in deaths.
The possible explanation was listed as follows: (1) lnc-ITSN1-2 had a connection with high inflammation, APACHE II score, SOFA score, which led to high mortality risk; (2) the sample size of this study was relatively small; therefore, lnc-ITSN1-2 only exhibited an increasing trend, but no statistical difference in deaths compared to survivors.
The current study was cross-sectional research, which revealed that lnc-ITSN1-2 might be a potential biomarker of sepsis, while the causality between sepsis and lnc-ITSN1-2 was hard to explore. Some limitations still existed in the current research: (1) the sample size of sepsis patients was only 95, which might affect the power in statistics, thus, subsequent study should involve more sample size to verify our conclusion; (2) the mechanism of lnc-ITSN1-2 facilitating Th17 cell differentiation and multiple organ dysfunction in sepsis was not clear, which should be explored in the following study; (3) the change of lnc-ITSN1-2 in sepsis patients was unclear after recovery, which should be explored in following study; (4) level of lnc-ITSN1-2 in urine and tissue apart from PBMC in sepsis patients could be investigated; (5) the quantification of lnc-ITSN1-2 was relative in the current study, the absolute quantification could be explored in future; (6) the level of lnc-ITSN1-2 in septic shock could be explored in the further study; (7) whether lnc-ITSN1-2 was variated by treatment regimens such as antibiotics, anti-inflammation in sepsis could be explored in future.
In conclusion, lnc-ITSN1-2 is highly expressed and correlates with inflammation, multiple organ dysfunction, and mortality risk in sepsis patients, indicating lnc-ITSN1-2 may serve as a potential biomarker in sepsis patients.

ACK N OWLED G M ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.