The regulation of TNFα production after heat and endotoxin stimulation is dependent on Annexin-A1 and HSP70

Abstract

Febrile temperatures can induce stress responses which protect cells from damage and can reduce inflammation during infections and sepsis. However, the mechanisms behind the protective functions of heat in response to the bacterial endotoxin LPS are unclear. We have recently shown that Annexin-1 (ANXA1)-deficient macrophages exhibited higher TNFα levels after LPS stimulation. Moreover, we have previously reported that ANXA1 can function as a stress protein. Therefore in this study, we determined if ANXA1 is involved in the protective effects of heat on cytokine levels in macrophages after heat and LPS. Exposure of macrophages to 42 °C for 1 h prior to LPS results in an inhibition of TNFα production, which was not evident in ANXA1(-/-) macrophages. We show that this regulation involves primarily MYD88-independent pathways. ANXA1 regulates TNFα mRNA stability after heat and LPS, and this is dependent on endogenous ANXA1 expression and not exogenously secreted factors. Further mechanistic studies revealed the possible involvement of the heat shock protein HSP70 and JNK in the heat and inflammatory stress response regulated by ANXA1. This study shows that ANXA1, an immunomodulatory protein, is critical in the heat stress response induced after heat and endotoxin stimulation.

Fig. 1

Heat pretreatment followed by TLR agonist stimulation inhibits cytokine production. WT- or ANXA1-deficient ^(−/−) macrophages were incubated at either 37 or 42 °C for 1 h prior to stimulation with (a, b, c) LPS (100 ng/ml) or (d, e, f) CPG ODN1826 (1 μM) or (g) Poly(I:C) (10 μg/ml) for 24 h, and the indicated cytokines were measured using ELISA. **p < 0.01 vs LPS 37 °C control. ^# p < 0.01 vs WT 42 ºC

Fig. 2

TRIF is involved in the regulation of TNFα production after heat and LPS. Macrophages from WT, MYD88-deficient mice, or TRIF inactive mutant mice were stimulated with heat and (a) LPS or (b) CPG ODN1826 or (c) Poly(I:C) as described in Figure 1. ^# p < 0.05 vs WT 37 ºC control; *p < 0.05 vs respective 37 °C controls; ns:  not significant

Fig. 3

Endogenous ANXA1 is involved in TNFα production after heat and LPS. a Experimental set up for b where supernatants from WT macrophages incubated at either 37 or 42 °C for 1 h were used to treat WT or ANXA1 ^−/− macrophages. TNFα production was measured by ELISA after 24 h after LPS stimulation. c Experimental set up for d where WT or ANXA1 ^−/− macrophages were stimulated with heat for 1 h and media was replaced. LPS was added for 24 h and TNFα production was measured by ELISA. *p< 0.05, **p < 0.01 vs respective LPS 37 °C control

Fig. 4

ANXA1 regulates TNFα mRNA expression and stability. WT or ANXA1^−/− macrophages were incubated at either 37 or 42 °C for 1 h prior to stimulation with LPS (100 ng/ml). a TNFα mRNA expression was measured using qPCR after the indicated time points. (b,c,d) TNFα mRNA stability was analyzed after actinomycin treatment. Half-life times were calculated using the equation –ln (2) / decay constant

Fig. 5

JNK activation is enhanced, while MKP1 is inhibited in the absence of ANXA1. WT or ANXA1^−/− macrophages were stimulated with LPS for 30 and 60 min or incubated at either 37 or 42 °C for 1 h prior to stimulation with a media or b LPS (100 ng/ml). Cells were lysed for Western blotting for the respective proteins after 30 and 60 min of LPS. Lane numbers are indicated. Fold change was determined using mean gray values of bands divided by their respective total proteins

Fig. 6

JNK functions upstream of HSP70 in regulation of TNFα production after heat and LPS. WT or ANXA1^−/− macrophages were incubated at either 37 or 42 °C for 1 h prior to stimulation with LPS (100 ng/ml). Cells were lysed for a Western blotting for HSP70 after 30 and 60 min of LPS or b real-time PCR for HSP70 mRNA after 30 min of LPS. c Cells were pretreated for 1 h with the HSP70 inhibitor VER155008 prior to heat and LPS treatment. Cells were lysed for Western blotting for the indicated proteins after 30 min of LPS, and d TNFα production was measured by ELISA after 24 h. e Cells were pretreated for 1 h with the JNK inhibitor SP600125 (10 μM) prior to heat and LPS treatment. Cells were lysed for Western blotting for the indicated proteins after 30 min of LPS, and d TNFα production was measured by ELISA after 24 h. *p < 0.05, **p < 0.01 vs respective LPS controls

Fig. 7

Summary of pathways involved. Upon heat treatment and LPS stimulation, a ANXA1 promotes HSP70 expression transcriptionally and translationally. b This leads to increased MKP1 expression, c which in turn inhibits the activation of the MAP kinases JNK, ERK, and p38, d which leads to a reduction in TNFα production. e JNK can also inhibit HSP70 to control its expression. In the absence of ANXA1, f HSP70 expression is reduced, g which leads to lower MKP1 expression, h which in turn increases the activation of the MAP kinases JNK, ERK, and p38. i This results in an increase in TNFα production