NF-kappaB is a negative regulator of IL-1beta secretion as revealed by genetic and pharmacological inhibition of IKKbeta

Abstract

IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

Figure 1. Increased endotoxin-induced mortaility is associated with elevated circulating IL-1β in Ikkβ^Δmye mice

(A) Survival after endotoxin injection (30 mg/kg, E. coli O111:B4) of Ikkβ^F/F (black) and Ikkβ^Δmye mice (grey), (n = 4–6). (B) IL-1β, (C) TNF-α and (D) IL-6 plasma levels after LPS administration. (E, I) Survival of Ikkβ^F/F (solid black lines) and Ikkβ^Δmye mice (solid grey lines) after LPS administration in the presence of IL-1ra (E) and sTNFRII (I). Dashed lines represent survival without inhibitors. (F, J) IL-1β, (G) TNF-α and (H) IL-6 plasma levels in mice given LPS plus the indicated inhibitors. Dashed line represents respective plasma levels of Ikkβ^Δmye mice without inhibitor. Data are averages of at least 4 animals per time point.

Figure 2. Depletion of either macrophages or neutrophils improves survival in Ikkβ^Δmye mice

(A, D) Survival of Ikkβ^F/F (black lines) and Ikkβ^Δmye mice (grey lines) depleted of macrophages (A) or neutrophils (D). Dashed lines represent survival without depletion. (B, E) IL-1β and (C, F) TNF-α plasma levels after LPS administration to macrophage-depleted (B, C) or neutrophil-depleted (E, F) mice. Dashed line represents respective plasma levels in Ikkβ^Δmye mice without depletion. Data are averages of at least 4 animals per time point.

Figure 3. Ikkβ^Δ mice develop granulocytosis and show massively increased circulating IL-1β after endotoxin exposure

(A) Complete and differential blood counts and spleen weights of Ikkβ^F/F and Ikkβ^Δ mice. Data are average values for 5 mice of each genotype examined two weeks after a single poly-(I:C) injection. (B, C) Blood smears stained with Wright-Giemsa and (D) number of CD11b⁺/Gr-1⁺ cells in spleens of of Ikkβ^F/F and Ikkβ^Δ mice analyzed two weeks after poly-(I:C) injection. (E) Survival after endotoxin injection (20 mg/kg, E. coli O111:B4) of Ikkβ^F/F (black) and Ikkβ^Δ mice (grey), (n = 4–6). (F) IL-1β and (G) TNF-α plasma levels 1, 2 and 6 hrs after LPS administration.

Figure 4. Increased IL-1β release correlates with elevated apoptosis of IKKβ-deficient macrophages and is inhibited by PAI-2

(A) Relative levels of IL-1β and TNF-α mRNA after incubation of Ikkβ^F/F (black bars) and Ikkβ^Δ (grey bars) BMDM with LPS (100 ng/ml). (B) Immunoblot analysis of intracellular pro-IL-1β, pro-TNF-α and cleaved caspase-3 in macrophages after LPS stimulation (100 ng/ml). (C) IL-1β levels in supernatants of Ikkβ^F/F (black bars) and Ikkβ^Δ (grey bars) macrophages after LPS stimulation. (D) Immunoblot analysis of processed IL-1β and TNF-α in supernatants of cultured macrophages. (E) IL-1β in supernatants of LPS stimulated Ikkβ^F/F (black) and Ikkβ^Δ (grey) macrophages in the presence of Ac-YVAD-cmk (100 μM), Z-VAD-fmk (10 μM) and TPCK (10 μM). Data are averages of at least three animals. (F) Loss of IKKβ activity enhances caspase-1 activation. WT or IKKβ-deficient macrophages were pretreated with ML120B (30 μM) or DMSO and either left unstimulated or incubated with LPS (100 ng/ml). After 22 hrs, culture supernatants were collected and analyzed by immunoblotting for secretion of activated caspase-1 (p17). The highest mobility represents uncleaved pro-caspase-1. (G) Reconstitution of IKKβ-deficient macrophages with PAI-2 blocks apoptosis and IL-1β release. Bone marrow of Ikkβ^Δ mice was retrovirally transduced with EGFP-PAI-2. EGFP-positive cells were sorted, differentiated into macrophages and stimulated with LPS (100 ng/ml) for 24 hrs. Apoptosis was determined by annexin V staining and IL-1β levels in supernatants were determined by ELISA.

Figure 5. Increased IL-1β release is independent of apoptosis in IKKβ-deficient neutrophils

(A) Levels of IL-1β and TNF-α mRNAs in neutrophils after LPS stimulation were examined by RNase protection. (B) Immunoblot analysis of intracellular pro-IL-1β and pro-TNF-α in neutrophils after LPS stimulation. (C, D) IL-1β and TNF-α secretion by LPS-stimulated neutrophils determined by ELISA. (E) Number of annexin V positive Gr-1⁺ cells determined by flow cytometry. (F) Immunoblot analysis of cleaved caspase-3 in neutrophils after LPS stimulation (100 ng/ml). (G) IL-1β secretion by LPS-stimulated wt and caspase-1^−/− macrophages and neutrophils. (H) Bioassay of supernatants of LPS-stimulated wt and caspase-1^−/− neutrophils using EL4 cells.

Figure 6. Elevated serine protease activity in IKKβ-deficient neutrophils accounts for enhanced IL-1β secretion

(A) Hydrolysis of MeOSuc-AAPV-pNA was used to determine serine protease activity in Ikkβ^F/F (black bars) and Ikkβ^Δ neutrophils (grey bars) treated with LPS and the protease inhibitors SLPI (1μg/ml) or 3, 4-DCIC (10 μM). (B) PR3, serpin B1/MNEI and PAI-2 expression in LPS–stimulated neutrophils determined by immunoblotting. (C) Immunoblot analysis of IL-1β expression in supernatants of neutrophils 16 hrs after LPS stimulation in the presence of Ac-YVAD-cmk (100 μM), MeOSuc-AAPV-cmk (500 μM) or TPCK (10 μM). (D, E) Immunoblot analysis of IL-1β produced in HEK293 cells after incubation with increasing amounts of purified (D) NE and (E) PR3 without or in the presence or AAPV. (F) Bioassay of extracts used in (D) and (E) in EL4 cells. (G–L) Survival of Ikkβ^F/F mice (black lines) and Ikkβ^Δ mice (grey lines) after pretreatment with the serine protease inhibitor MeOSuc-AAPV-cmk (1 mg/mouse) 1 hr before LPS application (G) or Ac-YVAD-cmk (J) and corresponding IL-1β (H, K) and TNF-α (I, J) plasma levels. Dashed lines in (G and J) represent survival without inhibitor and in (H, I) and (K, L) they represent plasma IL-1β and plasma TNF-α levels of Ikkβ^Δmye mice without inhibitor, respectively.

Figure 7. Prolonged pharmacological inhibition of IKKβ enhances IL-1β secretion and endotoxic shock

(A–C) Survival and corresponding IL-1β and TNF-α plasma levels of LPS-challenged wt mice given 300 mg/kg of the IKKβ inhibitor ML120B by oral gavage 1 hr prior to LPS administration (30 mg/kg). (D) Survival and corresponding IL-1β (E) and TNF-α (F) plasma levels in wt mice given 300 mg/kg ML120B by oral gavage twice daily for four days before LPS challenge. (G) Schematic representation of the proposed dual role of IKKβ-dependent NF-κB activation in regulation of IL-1β secretion by macrophages and neutrophils: in neutrophils, which appear to be a rapid but minor (albeit critical) source of IL-1β, IKKβ driven NF-κB positively regulates the transcription of pro-IL-1β mRNA and serine protease inhibitor genes whose products inhibit the activity of PR3, which can process pro-IL-1β. Secretion of biologically active IL-1β by neutrophils acts together with LPS to augment IL-1β secretion by macrophages, which represent the major source of this cytokine. In the macrophage, NF-κB controls pro-IL-1β mRNA synthesis as well as the expression of genes such as PAI-1, Bcl-x_L and ICEBERG whose products inhibit caspase-1 activation.