Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis

Abstract

Background: One hallmark of sepsis is the reduced number of lymphocytes, termed lymphopenia, that occurs from decreased lymphocyte proliferation or increased cell death contributing to immune suppression. Histone modification enzymes regulate immunity by their epigenetic and non-epigenetic functions; however, the role of these enzymes in lymphopenia remains elusive.

Methods: We used molecular biological approaches to investigate the high expression and function of a chromatin modulator protein arginine N-methyltransferase 4 (PRMT4)/coactivator-associated arginine methyltransferase 1 in human samples from septic patients and cellular and animal septic models.

Results: We identified that PRMT4 is elevated systemically in septic patients and experimental sepsis. Gram-negative bacteria and their derived endotoxin lipopolysaccharide (LPS) increased PRMT4 in B and T lymphocytes and THP-1 monocytes. Single-cell RNA sequencing results indicate an increase of PRMT4 gene expression in activated T lymphocytes. Augmented PRMT4 is crucial for inducing lymphocyte apoptosis but not monocyte THP-1 cells. Ectopic expression of PRMT4 protein caused substantial lymphocyte death via caspase 3-mediated cell death signalling, and knockout of PRMT4 abolished LPS-mediated lymphocyte death. PRMT4 inhibition with a small molecule compound attenuated lymphocyte death in complementary models of sepsis.

Conclusions: These findings demonstrate a previously uncharacterised role of a key chromatin modulator in lymphocyte survival that may shed light on devising therapeutic modalities to lessen the severity of septic immunosuppression.

Keywords: ARDS; Bacterial Infection; Lymphocyte Biology; Pneumonia; Respiratory Infection.

Figure 1

LPS and Escherichia coli increases PRMT4 protein expression in lymphocytes in vitro, and PRMT4 is increased in experimental septic models. (A–C) Jurkat cells (A), SKW6.4 cells (B) and THP-1 cells (C) were treated with LPS as indicated, and cell lysates were immunoblotted with PRMT4 and β-actin antibodies. Independent experiments, n=3. (D) Jurkat cells were treated with live E. coli as indicated, and cell lysates were immunoblotted with PRMT4 and β-actin antibodies. Densitometry was plotted in the lower panel. Independent experiments, n=3. (E) Lysates of peripheral blood leucocytes from deidentified human samples with or without sepsis were immunoblotting analysed with PRMT4 and β-actin. (F) PRMT4 protein levels were determined by ELISA from blood plasma from septic patients (n=53) and non-septic control patients (n=53). Lines indicate the median and IQR, Mann-Whitney U test, p=0.0004. (G) CLP procedures were subjected to C57BL/6 J mice for 48 hours; mice sera were collected from untreated controls (n=5) and polymicrobial infected mice (n=10) for PRMT4 ELISA analysis. (H, I) Leucocytes isolated from BALF in LPS-treated mouse were immunofluorescent stained with PRMT4 antibody. PRMT4 expression was visualised using confocal microscopy; the nuclei were stained by DAPI (H). Total cells were counted and positively stained granular and agranular cells were presented as percentage (I). A total of 300 granulocytes and 100 agranulocytes were counted. (J) Isolated CD4+ and CD8+ cells from LPS-treated mouse were lysed and immunoblotting analysed with PRMT4 antibody. Independent experiments, n=3. Scale bar=100 µm. *P=0.05–0.01, **P=0.01–0.001, ***P=0.001–0.0001, ****P<0.0001. BALF, bronchoalveolar lavage fluid; CLP, cecal ligation and puncture; DAPI, (4′,6-diamidino-2-phenylindole); LPS, lipopolysaccharide; PRMT4, protein arginine N-methyltransferase 4.

Figure 2

PRMT4 gene expression increases on activation in CD4+ T cells. CD4+ cells were isolated from the spleen of a mouse (strain C57BL/6J). The mixture of naïve, unstimulated T cells and CD4 T cells activated with anti-CD3/CD28 comprising a total of 10 000 cells each were applied to single-cell RNA sequencing. UMAP lots as two dimensional were used to plot the expression of CD4-specific genes CD4 (A) and CD3e (B), naïve T cell-specific genes Sell (C) and IL7r (D), CD4+ cell activation increased PRMT4 (E), IL2 (F), IL2ra (G), as well as CD69 (H) gene expression. PRMT4, protein arginine N-methyltransferase 4; UMAP, uniform manifold approximation and projection.

Figure 3

LPS increases PRMT4 expression and activates caspase 3 in lymphocytes. (A–C) Jurkat cells (A), SKW6.4 cells (B) and THP-1 cells (C) were treated with LPS as indicated. Cell lysates were subjected to immunoblotting for PRMT4, cleaved caspase 3, cleaved caspase 9 and β-actin. The densitometric results were plotted in the lower panels. Independent experiments, n=3. (D, E) Primary mouse splenic lymphocytes (D) and human peripheral blood T cells (E) were treated with LPS as indicated. Cell lysates were analysed by PRMT4, cleaved caspase 3 and β-actin immunoblotting. The plotted data are shown in the lower panels. Independent experiments, n=3. (F) The faecal material from mouse cecum was cultured in an LB plate overnight. Jurkat cells were treated with aforementioned gut-derived live bacteria for 2 hours. Cell lysates were immunoblotting analysed with PRMT4, cleaved caspase 3, cleaved caspase 9 and β-actin. The plotted data are shown in the lower panel. Independent experiments, n=3. *p=0.05–0.01, **p=0.01–0.001, ***p=0.001–0.0002, **** p=0.0001. LPS, lipopolysaccharide; PRMT4, protein arginine N-methyltransferase 4.

Figure 4

Caspase 3 activation is PRMT4 dependent in lymphocytes. (A, B) Overexpression of PRMT4 increased cleaved caspase 3 baseline levels in Jurkat cells (A) and SKW6.4 cells (B). Relative expression of cleaved caspase 3 was plotted in the lower panel. (C) PRMT4 overexpression does not activate caspase 3 in THP-1 cells. (D) Ectopic expression of PRMT4 enhances LPS-induced caspase 3 activation in Jurkat cells. (E) KO of PRMT4 in Jurkat cells with the CRISPR/Cas9 technique. (F) KO of PRMT4 limits LPS-induced caspase 3 activation. (G) Lentiviral expression of PRMT4 enhances LPS-mediated caspase 3 activation and depletion of PRMT4 by lenti-shPRMT4 reduces cleaved caspase 3 in mouse splenic lymphocytes. Independent experiments, n=3. *P=0.05–0.01, **P=0.01–0.001, ***P=0.001–0.0001. KO, knockout; LPS, lipopolysaccharide; neg, negative; PRMT4, protein arginine N-methyltransferase 4.

Figure 5

High protein level of PRMT4 causes lymphocyte death. (A, B) FACS analysis of apoptosis in PRMT4 KO or overexpressed Jurkat cells with or without LPS treatment. Data of (A) were quantitated in (B). (C) Lenti-PRMT4 or shRNA particles were delivered intratracheally into the mouse. Mouse splenic T cells were isolated and treated with LPS for 18 hours, and viable cells were counted. (D) Jurkat cells were treated with LPS and a range of PRMT4 inhibitors as indicated for 3 hours. Cell lysates were analysed for cleaved caspase 3. Relative expression of cleaved caspase 3 in each group is plotted in the lower panel. Independent experiments, n=3. (E) Isolated mouse splenic T cells were treated with LPS and TP064; cleaved caspase 3 was immunoblotting analysed and plotted in the lower panel. Independent experiments, n=3. *P=0.05–0.01, *P=0.01–0.001, ***P=0.001–0.0001. FACS, fluorescence-activated cell sorting; KO, knockout; LPS, lipopolysaccharide; neg, negative; OE, PRMT4 overexpression; PRMT4, protein arginine N-methyltransferase 4; sh, PRMT4 shRNA; Vec, vector.

Figure 6

Inhibition of PRMT4 suppresses splenic lymphocyte death in an LPS challenged mouse model. (A) PRMT4 was knocked down or overexpressed by IT administrated lentiviral constructs for 14 D. LPS or PRMT4 inhibitor were given intratracheally) as indicated for 24 hours (n=8). Spleen tissues were stained with TUNEL. (B) TUNEL-positive cells in spleen tissues were quantitated. (C, D) CD4+ lymphocytes were isolated from splenic tissues in aforementioned PRMT4 knockdown or overexpression experiments (A) and analysed with flow cytometry. CD4 was used as a T-cell marker. Percentage of apoptosis was quantitated in (D) (n=3). (E) Survival studies were conducted in the LPS lung injury model; mice were observed for 48 hours (n=10). (F, G) Two-stage meta-analysis was conducted using two independent sets of murine data using LPS-only group as reference: PRMT4+LPS (F) and TP064+LPS (G). The data of shPRMT4 group are not shown because the HR was not computable. Two independent experiments were conducted (n=26, (10, 16)). Scale bar=100 µm. LPS, lipopolysaccharide; PRMT4, protein arginine N-methyltransferase 4.

Figure 7

Inhibition of PRMT4 suppresses splenic lymphocyte death in a polymicrobial sepsis model. (A) CP was performed in PRMT4 knocked down or overexpressed mice (n=8). TP064 (0.2 µg/mouse) was administrated intravenously in one group for 48 hours. Spleen tissues were stained with TUNEL. (B) TUNEL-positive cells in spleen tissues. (C, D) Isolated splenic CD4+ T cells were analysed by flow cytometry (C). CD4 was used as a T-cell marker. The data from (C) are plotted in (D). (E) Survival studies were conducted in the CLP model, and mice were observed for 5 days (n=16). (F–H) Meta-analysis was conducted among two independent sets of murine data using CLP only as reference group: PRMT4+CLP (F), shPRMT4+CLP (G) and TP064+CLP (H). Two independent experiments were conducted (n=26, (10, 16)). Scale bar=100 µm. CLP, cecal ligation and puncture; PRMT4, protein arginine N-methyltransferase 4.