HMGB1/TLR4 Signaling Affects Regulatory T Cells in Acute Lung Injury

Figures & data

Table 1 Characteristics of Healthy and ARDS Subjects

	ARDS	Healthy
Male/female^a	33/25	21/15
Median age, yr^b	51 (22–68)	47 (26–70)
Weight (kg)^b	52 (46–78)	56 (51–72)
BMI (kg.cm^−2)^b	28.2 (25–36)	28.6 (26–43)
Survivors/nonsurvivors, n^a	36/22
Servere sepsis/septic shock, n^a	34/24
MODS score^b	7 (6–12)
APACHE II^b	29.3 (19–42)
Median lung score^b	2.8 (2.2–4.1)
SOFA^b	11 (8–15)
SAPS^b	59 (46–68)
Median PaO2/FIO2^b	129 (102–186)
Endotoxin (Eu/mL)^b	2.9 (0.13–14.45)	0.05 (0.001–0.08)
CRP (mg/L)^b	211.2 (28.9–645.3)	1.2 (1.2–2.2)
PCT (ng/mL)^b	2.11 (0.06–22.54)	0.008 (0.004–0.02)
IL–6 (pg/mL)^b	319 (45–5000)	5.6 (0–7)
Underlying disorder	15 post-op
	13 pancreatitis
	10 trauma
	9 obstruction
	7 cancer
	2 diabetes
	1 each cirrhosis, sickle cell crisis
Clinical Infection	32 intra-abdominal
	13 catheter/bloodstream
	9 urinary tract (15)
	4 skin and soft tissue

Notes: ^aData are expressed as number. ^bData are expressed as medians and their interquartile ranges.

Figure 1 Elevated HMGB1 levels and reduced Treg proportions in patients with sepsis-induced ALI. (A) Circulating HMGB1 levels in plasma were measured in patients with sepsis (n=34), septic shock (n=24), and healthy controls (n=36) using an ELISA. **p<0.01. (B) Plasma HMGB1 levels in surviving (n=36) and nonsurviving (n=22) patients with ARDS. *p<0.05 and **p<0.01. (C) Representative two-dimensional scatter diagrams of CD4⁺CD25⁺CD127^low Tregs in the peripheral blood of patients with ARDS and healthy controls were analyzed using flow cytometry. (D) The percentage of CD4⁺CD25⁺CD127^low Tregs in the peripheral blood of patients with ARDS (n=58) and healthy controls (n=36); means±SD, **p<0.01. (E) Correlation between the percentage of CD4⁺CD25⁺CD127^low Tregs and HMGB1 levels in patients with ARDS; R²=0.535, p<0.001. The analyses in this figure were performed using permutation t-tests (A, B, and D) and Pearson’s correlation analyses (E).

Figure 2 TLR4 deletion accelerates lung injury and the local inflammatory response induced by the HMGB1 instillation. (A) Pathological changes in lung tissues from TLR4 mice with different genotypes 48 h after an intratracheal instillation of HMGB1. (B) The morphological changes was quantified by using lung injury scoring system (C) Changes in the W/D weight ratio of the lung tissue 48 h after an intratracheal instillation of HMGB1 in mice with different TLR4 genotypes. (D) TNF-α levels were measured in BAL fluid from the control, WT, and TLR4^-/- groups using an ELISA. (E) Serum TNF-α was measured by ELISA in the control, WT, and TLR4^-/- groups. (F) IL-1β levels were measured in BAL fluid from the control, WT, and TLR4^-/- groups using an ELISA. (G) Serum IL-1β levels were measured in the control, WT, and TLR4^-/- groups using an ELISA, *p<0.05 between 3 groups by using Tukey’s test, ^&p<0.05 between WT and TLR4^-/- group by using t-test.

Table 2 Changes of CTLA-4 and FOXP3 Expression of CD4+CD25+Treg in Different Groups of Mice with ALI (X±s)

Group	CTLA-4	FOXP3
Control	38.602±3.54	52.61±7.69
WT	19.65±1.46*	34.91±4.92*
TLR4^−/-	63.09±5.72*	65.16±4.93*

Note: *p<0.05.

Figure 3 TLR4 reversed the CTLA-4 and FOXP3 downregulation caused by the HMGB1 instillation. (A) Changes in CTLA-4 and FOXP3 expression in CD4⁺CD25⁺ Tregs from different groups of mice with acute lung injury. (B) TGF-β levels in CD4⁺CD25⁺ Tregs from different groups. (C) IL-10 levels in CD4⁺CD25⁺ Tregs from different groups. (D) TGF-β content in CD4⁺CD25⁺ Tregs from mice with different genotypes stimulated with HMGB1. (E) IL-10 content in CD4⁺CD25⁺ Tregs from mice with different genotypes stimulated with HMGB1, *p<0.05 between 3 groups by using Tukey’s test, and ^&p<0.05 between WT and TLR4-/- group by using t-test.

Figure 4 TLR4 deletion reverses HMGB1-induced polarization towards the Th1 phenotype. (A) The CCK-8 method was used to detect the effects of CD4⁺CD25⁺ Tregs on the proliferation of CD4⁺CD25⁻ T cells from different groups. (B) Changes in IL-2 contents in cocultures of CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T cells. (C) IL-4 contents in cocultures of CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T cells. (D) IFN-γ contents in cocultures of CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T cells, *p<0.05 between 3 groups by using Tukey’s test, and ^&p<0.05 between WT and TLR4^-/- group by using t-test.