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. 2019 May 28;10(37):3451-3461.
doi: 10.18632/oncotarget.26919.

Endotoxin tolerance modulates TREG and TH17 lymphocytes protecting septic mice

Mariana M C Andrade  1 , Suely S K Ariga  1 , Denise F Barbeiro  1 , Hermes V Barbeiro  1 , Rosangela N Pimentel  1 , Ricardo C Petroni  1 , Francisco G Soriano  1
Affiliations

Endotoxin tolerance modulates TREG and TH17 lymphocytes protecting septic mice

Mariana M C Andrade et al. Oncotarget. .

Abstract

Background: Tolerance induces a regulated immune response to infection. We hypothesized that tolerance induction modulated profile of T regulatory cell (Treg) and T lymphocyte 17 (Th17) cells and is related cytokine released in septic animals. Methods: Male black C57/6 mice received subcutaneous (s.c.) injections of lipopolysaccharide (LPS) (1 mg/kg) for 5 days, on day 8th was made cecal ligation and puncture (CLP). Blood and spleen tissue were collected for cell analysis and cytokines measurements. Results: Cytokines (interleukin 2 (IL-2), interleukin (IL-6), transforming growth factor β (TGF-β) and interferon γ (INF-γ)) related to Treg and Th17 stimulation were elevated in the spleen of tolerant animals compared to sham. Treg and Th17 lymphocytes showed an increased amount in blood (Treg: 920 ± 84 cells vs. 1946 ± 65 cells, sham vs. tolerant; Th17:38321± 1954 cells vs. 43526 ± 7623 cells, sham vs. tolerant) and spleen (Treg: 5947 ± 273 cells vs. 16521 ± 486 cells, sham vs. tolerant; Th17: 26543 ± 2944 cells vs. 64567 ± 5523 cells, sham vs. tolerant). Treg (135±23 cells) and Th17 (1590 ± 256 cells) cells were reduced in blood of septic animals compared to sham, while CLP tolerant animals presented an increasing number of these cells. Lymphocyte Th17IL6+ were elevated in tolerant and CLP tolerant animals in the blood compared to sham. Conclusion: LPS tolerance was associated with increasing population of Treg and Th17. LPS tolerance reduces the hyper inflammatory response with immunoregulation exerted by Treg and Th17 cells protecting from septic damage.

Keywords: Th17; Treg; lymphocytes; sepsis; tolerance.

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Conflict of interest statement

CONFLICTS OF INTEREST No conflicts of interest, financial or otherwise, are declared by the author(s). The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Lipopolysaccharide-induced tolerance in mice.
C57/6 mice were treated daily with 1 mg/kg of lipopolysaccharide (LPS; tolerant group, n = 30) or saline placebo (naïve group, n = 30) for 5 d. Experiment 1- spleen were collected from animals sacrifeied every day, 4 houras after LPS induction dose. Experiment 2- On day 8, animals were submitted to CLP or SHAM surgery, and samples were collecte after 4 houras.
Figure 2
Figure 2. Mortality following CLP (b) was 80% in naïve mice after 72 h, 40% in tolerant mice after 72 h.
Data from of 30 animals per group. *p<0.05 for a difference between tolerant and naïve groups.
Figure 3
Figure 3. Splenic inflammatory cytokines and growth cytokines during LPS tolerance development.
L-2 (A), IL-4 (B), TGF-β (C), IL12 (D), INFγ (E), IL-6 (F), TNFα (G), IL1ß (H), and IL-10 (I) interleukin levels were determined in basal group (BASAL 0), and 4 hours after each LPS injection, in days 1, 2, 3, 4, 5 and 8 of tolerance induction. Animals per period n=8, data are presented as mean ± SD. (*) p<0.05 compared to BASAL group (n=6 for each group); (§) p<0.05 compared to day 1; (#) p< 0.05 compared to days 3, 4, 5 and 8.
Figure 4
Figure 4. Splenic Th17 cytokines during LPS tolerance development.
IL-17f (A), IL-17a (B), IL-22 (C) and IL-23 (D) interleukin levels were determined in basal group (BASAL), and 4 hours after each LPS injection, in days 1, 2, 3, 4, 5 and 8 of tolerance induction. Animals per period n=8, data are presented as mean ± SD. (*) p<0.05 compared to BASAL group (n=8 for each group); (§) p<0.05 compared to day 1; (#) p< 0.05 compared to days 3, 4, 5 and 7.
Figure 5
Figure 5. LPS tolerance effect on CD4, Treg and Th17 in septic animals.
CD4, Treg and Th17 was quantified in SHAM, SHAM Tolerant, CLP and CLP Tolerant groups. The quantification was performed by flow cytometry in spleen cells and blood cells. CD4 quantification in blood (A) and spleen (B); CD4+CD25+FOXP3+ (Treg cells) amount of cells in spleen (C) and blood (D). Quantification of CD4+IL17+ IL-6 (E) spleen and (F) in blood, and Th17-IL6/Treg ratio (G) in spleen and (H) in blood in. Animals per group n=8, data are presented as mean ± SD. (*) p<0.05 compared SHAM group; (#) p<0.05 compared SHAM TOL group; (§) p<0,05 compared to CLP group.
Figure 6
Figure 6. The LPS tolerance effect on splenic pro-, anti-inflammatory and growth cytokines in septic animals.
IL1ß (A), INFγ (B), TNFα (C), IL-2 (D), IL-4 (E), IL-5 (F), IL-6 (G), GM-CSF (H), IL-10 (I) interleukin levels were determined in SHAM, SHAM Tolerant, CLP and CLP tolerant group. Animals per group n=8, data are presented as mean ± SD. (*) p<0.05 compared to SHAM group; (#) p<0.05 compared to SHAM TOL group; (§) p<0,05 compared to CLP group.
Figure 7
Figure 7. The LPS tolerance effect on splenic Th17 cytokines in septic animals.
IL-17a (A), IL-17f (B), IL-17e (C), IL-21 (D), IL-22 (E) and IL-23 (F) interleukin levels were determined in SHAM, SHAM Tolerant, CLP and CLP tolerant group. Animals per group n=8, data are presented as mean ± SD. (*) p<0.05 compared to SHAM group; (#) p<0.05 compared to SHAM TOL group; (§) p<0,05 compared to CLP group.
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