Aberrant blood MALT1 and its relevance with multiple organic dysfunctions, T helper cells, inflammation, and mortality risk of sepsis patients

Abstract Background MALT1 is linked with multiple organic dysfunctions, inflammatory storm, and T helper (Th) cell differentiation. Herein, the current study aimed to investigate the correlation of peripheral blood mononuclear cell (PBMC) MALT1 with Th1 cells, Th17 cells, and prognosis of sepsis patients. Methods In general, 78 sepsis patients and 40 health controls (HCs) were enrolled. MALT1 expression was detected in PBMCs from all subjects by RT‐qPCR. Besides, Th1 and Th17 cells were measured in PBMCs from sepsis patients by flow cytometry; interleukin 17A (IL‐17A) and interferon gamma (IFN‐γ) were determined in serum from sepsis patients by ELISA. Results MALT1 expression was higher in sepsis patients than HCs (p < 0.001). MALT1 expression was positively correlated with Th17 cells (r s = 0.291, p = 0.038) and IL‐17A (r s = 0.383, p = 0.001), but not with Th1 cells (r s = 0.204, p = 0.151) or IFN‐γ (r s = 0.175, p = 0.125) in sepsis patients. MALT1 expression was positively correlated with APACHE II score (r s = 0.275, p = 0.015), C‐reactive protein (CRP) (r s = 0.257, p = 0.023), and sequential organ failure assessment (SOFA) score (r s = 0.306, p = 0.006) (MALT1 expression was positively correlated with SOFA respiratory system score (r s = 0.348, p = 0.002), and SOFA liver score (r s = 0.260, p = 0.021), but not with SOFA scores in nervous system, cardio vascular system, coagulation, and renal system (all p > 0.05)). MALT1 expression (p = 0.010), Th1 cells (p = 0.010), Th17 cells (p = 0.038), and IL‐17A (p = 0.012), except for IFN‐γ (p = 0.102), elevated in sepsis deaths compared with sepsis survivors. Conclusion PBMC MALT1 is highly expressed in sepsis patients with its overexpression associated with multiple organic dysfunctions, elevated Th17 cells, and increased mortality risk.


| INTRODUC TI ON
Sepsis continues to be a global health problem with in-hospital mortality ranging from 15.6% to 30%. [1][2][3][4][5] Generally, sepsis is featured by severe, potentially fatal, multiple organic dysfunctions caused by dysregulated host response to infection; meanwhile, it is usually accompanied by inflammatory storm. 6 Over the decades, though progressions have been made in the prevention and treatment of sepsis, its incidence still increases unexpectedly; meanwhile, its mortality rate remains unsatisfying. 5,[7][8][9][10] Considering the high mortality of sepsis, it would be valuable to find novel biomarkers for indicating disease severity and further realizing better management for sepsis patients.
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is an intracellular signaling protein, with its coding gene located on chromosome 18 q21. 11 Interestingly, previous studies illuminate that MALT1 is associated with dysregulated immune response to infection and multiple organic dysfunctions (including kidney injury, lung injury, and liver injury). [12][13][14][15][16][17] For instance, one study discloses that MALT1 regulates immune response in human primary T cells and macrophages via inactivating NEDD4-binding protein 1 (N4BP1) and subsequently facilitates the viral reactivation of immunodeficiency virus (HIV)- 1. 12 Beyond that, MALT1 overexpression could result in kidney injury via activating nuclear factor κB (NF-κB) signaling. 15 Additionally, it is illustrated that MALT1 enhances the differentiation of CD4 + T cells into Th1 and Th17 cells, meanwhile the differentiation of CD4 + T cells into Th1 and Th17, which are important in the pathology of sepsis. [18][19][20][21] However, the clinical relevance of MALT1 with sepsis, which is one of the most serious infection-related diseases, still remains elusive.
Herein, the current study aimed to investigate the correlation of MALT1 with Th1 cells, Th17 cells, and prognosis in sepsis patients.

| Subjects
This study was a prospective cohort study. Between January 2019 and February 2021, this study serially enrolled 78 sepsis patients.
The enrollment criteria for sepsis patients were as follows: (i) confirmed as sepsis in accordance with the sepsis-3 criteria 22 ; (ii) over 18 years old; (iii) hospitalized for sepsis treatment within 24h of symptom onset; and (iv) willing to provide peripheral blood (PB) samples. The exclusion conditions for sepsis patients were set as: (i) concomitant with autoimmune disease, hematological disease or solid tumor; and (ii) pregnant or gestational patient. Besides, from January 2019 to February 2021, the study also included 40 healthy subjects without abnormities in medical examinations as health controls (HCs), who were willing to provide PB samples for study use, and the exclusion conditions for sepsis patients were also suitable for health controls. The study protocol was approved by Ethics Committee.

| Data collection
Demographics, disease characteristics, and biochemical indexes were collected for study analysis. Besides, Acute Physiology and Chronic Health Evaluation II (APACHE II) score and Sequential Organ Failure Assessment (SOFA) score were also obtained to evaluate the disease severity of sepsis patients. In addition, the 28-day follow-up information of sepsis patients was also collected, and mortality during follow-up was calculated.

| Sample preparation
For sepsis patients, PB samples were obtained after admission; then, peripheral blood mononuclear cells (PBMCs) were isolated by gradient density centrifugation using Ficoll PM400 (Cytiva), and serum samples were isolated by centrifuge at 3500 revolutions per minute for 10 mins. For HCs, PB samples were obtained after enrollment, and then PBMCs were separated.

| Sample assessment
PBMCs of all sepsis patients as well as all HCs were used to de-

| RT-qPCR assay
MALT1 expression in PBMCs was detected with RT-qPCR. In detail, the total RNA from PBMC was extracted using PureZOL RNA isolation reagent (Bio-Rad); complementary DNA was synthesized by iScript™ cDNA Synthesis Kit (with random primer) (Bio-Rad); qPCR was performed by SYBR ® Green Realtime PCR Master Mix (Toyobo).
Besides, the current primers took reference from a previous study. 21 Additionally, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference; meanwhile, 2 −ΔΔCt method was applied to calculate the MALT1 relative expression.

| Statistical analysis
Graphics and statistical analyses were completed using GraphPad

| Clinical characteristics of sepsis patients
Among the enrolled 78 sepsis patients, the age was 56.6 ± 11.4 years with 49 (62.8%) males (Table 1). Additionally, the body mass index

| Comparison of PBMC MALT1 expression between sepsis patients and HCs
The median PBMC MALT1 expression in sepsis patients and HCs   APACHE II score, mean ± SD 12.6 ± 6.1 SOFA score, mean ± SD 5.4 ± 2.5 Respiratory system 1.3 ± 0.6 Nervous system 0.6 ± 0.6 Cardio vascular system 0.7 ± 0.6  (Table S1). Additionally, in terms of these indexes in discriminating sepsis deaths from sepsis survivors, PBMC MALT1 ( Figure 4A) disclosed a similar value as Th1 cells, IFNγ, Th17 cells, and IL-17A ( Figure 4B) did, while these values were numerically inferior to APACHE II score and SOFA score ( Figure 4C).

| DISCUSS ION
Recently, abnormally elevated MALT1 has been studied in a series of  Several previous studies disclose that MALT1 leads to T cell activation and differentiation into Th1 and Th17 cells in a series of infection-associated diseases including colitis and encephalitis. 21,25,26 In the current study, PBMC MALT1 expression was positively associated with Th17 cells, IL-17A level in sepsis patients, but not with Th1 cells or IFNγ level. A possible explanation could be that: MALT1 facilitates the differentiation of CD4 + T cells into Th17 cells via activating NF-κB signaling; therefore, MALT1 overexpression is linked with elevated Th17 cells and its secreted IL-17A level. 25,27 Currently, SOFA scoring system is the main index for evaluating multiple organic dysfunctions in sepsis. 28 However, previous studies never evaluate the correlation of MALT1 expression with this index in sepsis. Then, the present study disclosed that PBMC MALT1 expression was positively linked with general SOFA score, SOFA respiratory system score, and SOFA liver score in sepsis patients.

1) MALT1 might contribute to systemic inflammatory responses at-
tributable to the infection in sepsis, which would probably lead to a significant lung injury; thus, MALT1 overexpression is correlated with unfavorable SOFA respiratory system score in sepsis patients; besides, MALT1 recruitment leads to caspase 1 activation and pyroptotic death of invariant natural killer T cells, which correspondingly lead to liver injury; thus, MALT1 overexpression is correlated with unfavorable SOFA respiratory system score and SOFA liver score in sepsis patients. [15][16][17] 2) MALT1 expression is positively linked with the recruitment of Th17 cells and IL-17A, which indicates sepsis severity. [29][30][31][32] Correspondingly, MALT1 overexpression might be correlated with sepsis severity in sepsis patients.
In the past decades, the mortality rate of sepsis still remains unsatisfying. 7 Collectively, PBMC MALT1 is highly expressed in sepsis patients with its overexpression associated with multiple organic dysfunctions, elevated Th17 cells, and increased mortality risk, which implies that it could forecast the prognosis of sepsis patients.

ACK N OWLED G EM ENTS
None.

CO N FLI C T S 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
Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.