The inflammasome adaptor ASC contributes to multiple innate immune processes in the resolution of otitis media

Abstract

This study was designed to understand the contribution of the inflammasome and IL-1β activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1β and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc(-/-)-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1β maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc(-/-) mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.

Keywords: IL-1β; NTHi; cytokine; inflammation; macrophage; middle ear.

Figure 1

A schematic representation of inflammasome activation and signaling in NTHi-mediated OM. TLR signaling in response to pathogen molecules triggers the initial synthesis of the pro-forms of the inflammatory cytokines IL-1β and IL-18. Pathogen moieties and other danger signals can also trigger the activation of inflammasomes based on various innate immune receptors (NLRP-3, NLRC-4/IPAF, RIG-1 and AIM-2). However, the adaptor molecule ASC mediates assembly of most inflammasomes. The inflammasome then recruits and activates caspase-1, which, in turn, cleaves the pro-forms of IL-1β and IL-18, releasing them in their mature/active forms. As IL-1β is prominent in OM,^, inflammasomes seem likely to play a role in inflammatory signaling in the ME during NTHi-induced ME infection in our mouse model. The best-characterized inflammasome is based on NLRP-3, which can be activated by various bacterial molecules and danger signals. However, there are other mechanisms of NLRP-3 activation, including the P2RX7 ATP receptor, ROS formation during oxidative stress, and glucose-mediated release of TXNIP from TRX. The NLRC-4/IPAF inflammasome is activated via its upstream receptors NAIP-2 and NAIP-5, while the AIM-2 and RIG-1 inflammasomes act as sensors for cytosolic bacterial, viral and host dsDNA and RNA, respectively.

Figure 2

Assessment of changes in the gene expression of the different inflammasomes and their related components in MEM after NTHi infection. Mining gene chip microarray data showed significant and early time-dependent increases in transcripts of the inflammasome targets IL-1β and IL-18 after NTHi infection (A). This is accompanied by up-regulation of various inflammasome receptor mRNAs (B), including NLRP-3, RIG-1, AIM-2 and NLRP-4. Up-regulation of the mRNA for the adaptor molecule ASC and various caspases during NTHi induced OM (C) provides the remaining substrates for the inflammasome. The transcripts of various molecules known to activate the different inflammasomes are also increased in (D) and (E), as are those of inflammasome regulators (F). Data are represented numerically in Supplementary Table S1.

Figure 3

Impaired IL-1β processing in Asc^−/− mice. Immunoblotting of mouse MEM tissue 12 h after NTHi infection confirmed the NTHi-dependent induction of pro-IL-1β expression at the protein level, as well as its processing into its shorter, mature, active form. Pro-IL-1β was induced at similar levels in WT and Asc^−/− mice. However, lack of ASC protein resulted in severely reduced pro-IL-1β processing into mature IL-1β in Asc^−/− mice.

Figure 4

Lack of the Asc gene results in enhanced mucosal hyperplasia. (A) Quantitative comparison of mucosal hyperplasia and (B) histology (hematoxylin and eosin) staining of WTand Asc-deficient mice during the course of OM. In Asc^−/− mice mucosal thickness was significantly greater in the first few days after NTHi infection, until full recovery was reached by d 10. *Significantly different than WT mice (P < 0.05), n = 6 ears.

Figure 5

Lack of the Asc gene alters leukocyte recruitment to the ME. (A) Area of the ME lumen occupied by inflammatory cells was used to quantify leukocyte infiltration of the ME cavity (n = 6 ears). The accumulation of ME leukocytes was delayed in Asc^−/− mice. The ME infiltrate was examined at 400 × to identify the neutrophils and macrophages. The recruitment of neutrophils (B) was also delayed, and the number of macrophages (C) was significantly decreased in Asc^−/− mice. HPF: high-power field. *Significantly different from WT mice (P < 0.05), n = 6 ears.

Figure 6

Assessment of macrophages phagocytosis and bacterial clearance. Phagocytosis and killing of NTHi bacteria was assessed in peritoneal macrophages from WT and Asc mice by quantifying the colony counts remaining after loading with NTHi and then 1 h (phagocytosis) or 3 h (killing) incubation with gentamicin to destroy all extracellular bacteria. Bars represent bacteria recovered after lysis of the macrophages. Asc^−/− macrophages exhibited less phagocytic uptake of NTHi (1 h), but once inside the cell a similar proportion of bacteria were killed (3 h). Experiments were performed in triplicate and expressed as mean ± SEM. *Significantly different than WT mice (P < 0.05).

Figure 7

ME expression of genes related to Th17 cell differentiation. There is evidence that caspase-1 activation can play a role in tissue proliferation, mediated by Th17 cytokines. Others have noted these genes are down-regulated in caspase-1 knockout mice in stomach mucosa. Gene array data indicated that mRNA up-regulation of Th17 cytokines and receptors possibly play an early role in the innate immune response of the ME. This provided evidence that these genes may play a role in stimulating mucosal hyperplasia in the ME, and would be hypothesized to be reduced in mice unable to activate caspase-1, as in the case of the Asc^−/− knockout mice used in this study.