TLR5 activation induces secretory interleukin-1 receptor antagonist (sIL-1Ra) and reduces inflammasome-associated tissue damage

Abstract

Toll-like receptor-5 (TLR5)-mediated detection of flagellin induces nuclear factor (NF)-κB-mediated transcription of host defense gene expression, whereas recognition of intracellular flagellin by interleukin (IL)-1-converting enzyme protease-activation factor (IPAF) results in maturation/secretion of the inflammasome cytokine IL-1β. The potent effects of IL-1β are counter-regulated by secretory IL-1 receptor antagonist (sIL-1Ra). We studied the roles of flagellin receptors in regulating the expression of IL-1β and sIL-1Ra and their subsequent roles in inflammation. Flagellin induced sIL-1Ra in intestinal epithelia and macrophages in a dose- and time-dependent manner, whereas IL-1β was only induced in macrophages. In vivo, flagellin-induced sIL-1Ra, but not IL-1β, was absolutely dependent upon TLR5 expressed on non-hemopioetic cells. Thus, loss of TLR5 increased the IL-1β/sIL-1Ra ratio on flagellin treatment, which correlated with increased inflammatory pathology in response to this product. Furthermore, the flagellin/TLR5 interaction was important for the induction of sIL-1Ra and limiting inflammatory pathology on Salmonella infection. Finally, reduced sIL-1Ra levels in TLR5KO mice correlated with spontaneous colitis. Taken together, we demonstrate that intestinal epithelia, despite not expressing IL-1β, secrete sIL-1Ra in a TLR5-dependent manner suggesting that loss of TLR5 may promote inflammation by increasing IL-1β activity. Thus, optimizing the balance between inflammasome cytokines and their endogenous inhibitors might prove a useful strategy to treat inflammatory disorders.

Figure 1. Secretion of sIL-1Ra by intestinal epithelial cells (IEC)

Confluent human model intestinal epithelia (HT29) were stimulated with indicated doses of flagellin (FliC), EGF or 100 IU/mL of human IFNα, IFNβ or IFNγ. Supernatants were taken after 24h of culture or at indicated time periods for IL-8 or sIL-1Ra analysis by ELISA. (A) Dose-dependent secretion of sIL-1Ra. (B) sIL-1Ra and (C) IL-8 time-dependent secretion after stimulation with 100 ng/mL of FliC (Triangle) or control PBS (Circle). (D) IL-8 and (E) sIL-1Ra secretion after cells were pre-incubated for 30 minutes with DMSO or MG262 in DMSO (10nM) and then stimulated with 100 ng/mL of FliC. (F) Induction of sIL-1Ra by type I and type II interferons. (G) Induction of sIL-1Ra by EGF.* p<0.05

Figure 2. Flagellin deficiency attenuates Salmonella’s ability to induce sIL-1Ra in IEC

Confluent human intestinal epithelial cells (HT29) were exposed to 1×10⁸ CFU/mL of either flagellate S. Typhimurium or its isogenic aflagellate mutant. After 3h of infection, cells were washed 3 times and incubated with medium containing 50 μg/mL of gentamicin for 6h. (A) IL-8 and (B) sIL-1Ra were measured in culture supernatant by ELISA. * p<0.05.

Figure 3. Secretion of sIL-1Ra by murine macrophages

Confluent mouse macrophage cell line J774A.1 were grown in 24-well plate and stimulated with indicated doses of flagellin (FliC), 20ng of LPS or 100 IU/mL of mouse IFNγ. Supernatants were collected after 24h of culture or at indicated time periods for IL-1β or sIL-1Ra analysis by ELISA. (A) sIL-1Ra production by murine macrophages after stimulation with FliC (10μg/mL), mouse IFNγ (100 IU/mL) or LPS (20 ng/mL). (B) Dose-dependent and (C) time-dependent secretion of sIL-1Ra by J774A.1 cells after stimulation with flagellin. (D) IL-1β and (E) sIL-1Ra production in response to FliC (10μg/mL) by resident peritoneal wild-type (WT), TLR5-deficient (T5KO) and IPAF-deficient (IPAFKO) macrophages measured by ELISA. * p<0.05

Figure 4. Reduced induction of IL-1β and sIL-1Ra by flagellin deficient Salmonella in macrophages

Confluent mouse macrophages were grown in 24-well plate (2×10⁵ cells/well) and exposed to 1×10⁸ CFU/mL of either flagellate S. Typhimurium or its isogenic aflagellate mutant. After 1h of infection, cells were washed 3 times and incubated with medium containing 50 μg/mL of gentamicin for 24 h. Supernatants were assayed for (A) TNFα, (B) IL-1β and (C) sIL-1Ra by ELISA. (D) The IL-1β activity was determined by calculating the ratio of IL-1β/sIL-1Ra. * p<0.05

Figure 5. Flagellin induced sIL-1Ra is TLR5-dependent in vivo

Six to eight week old wild-type (WT), TLR5-deficient (T5KO), IPAF-deficient (IPAFKO) or IPAF/TLR5 (DKO) mice (n=5) were administered i.p. 200 μL of PBS or 10 μg FliC/mouse in 200 μL of PBS. After 1h, serum was collected and assayed for (A) sIL-1Ra, (B) KC and (C) IL-6 by ELISA. Colons were cultured for 24h and (D) sIL-1Ra, (E) KC, (F) IL-6 and (G) IL-1β in supernatants were assayed by ELISA. (H) Total RNAs from colon were isolated and Pro-IL-1β mRNA levels were measured by qRT-PCR. (I) IL-1β/sIL-1Ra ratio. The data is representative of 3 independent experiments.* p<0.05

Figure 6. Flagellin induced sIL-1Ra is dependent on non-hemopoietic cells in vivo

Seven to eight week old WT or TLR5KO mice irradiated and generated WT→WT, WT→TLR5KO, TLR5KO→TLR5KO and TLR5KO→WT bone marrow chimeras were generated as described in Methods. After 8 weeks they were challenged with either PBS or flagellin (10 μg/mouse) i.p. and after 1h blood was collected and serum sIL-1Ra levels were analyzed by ELISA. * p<0.05

Figure 7. Flagellin administration induces adverse effects in TLR5-deficient mice

Six to eight week old wild-type (WT) or TLR5-deficient (T5KO) mice were administered either 100 μL of PBS or flagellin (FliC; 25μg/mouse) in 100 μL PBS on alternate days over a period of 20 days. (A) At the end of the experiment, relative change in body weight between the 1^st and the 10^th injection was calculated. (B) Colonic MPO and (C) cecum MPO. (D) H&E stained colon from mice which received FliC. The results are representative of two independent experiments (n=8). * p<0.05

Figure 8. Salmonella flagellin and TLR5 interaction is required for in vivo sIL-1Ra secretion

(A) Eight week old BALB/_CJ mice were pretreated with streptomycin and 24 h later infected orally with flagellate S. Typhimurium or its aflagellate isogenic mutant. After 48 h post infection, mice were bled and serum analysed by ELISA for sIL-1Ra levels. (B) Eight week old wild-type (WT), TLR5KO (T5KO), IPAFKO and IPAF/TLR5 DKO mice were orally infected with flagellate S. Typhimurium or its aflagellate isogenic mutant (1×10⁸ CFU/mouse). Twelve hour post infection, mice were bled and serum sIL-1Ra quantified by ELISA. The results are representative of two independent experiments. * p<0.05

Figure 9. Systemic sIL-1Ra inversely correlates with severity of spontaneous colitis in TLR5-deficient mice

Eight week old wild-type (WT) or TLR5-deficient (T5KO) mice with low serum amyloid A (SAA) levels (mild colitis), with high SAA levels (robust colitis) or with rectal prolapse (severe colitis) were bled and serum sIL-1Ra levels were measured by ELISA. * p<0.05

Figure 10