Essential Role of the Innate Immune Adaptor RIP2 in the Response to Otitis Media

Abstract

Intracellular nucleotide binding and oligomerization domain (NOD) and Toll-like (TLR) receptors have emerged as pivotal sensors of infection. Both Nod1 and Nod2 contain a caspase activation and recruitment domain (CARD) that interacts with the adaptor protein RIP2 (receptor-interaction protein-2). This leads to ubiquitination of RIP2 and in turn to the activation of NFκB and MAPK transcription factors, to command the host defensive response against pathogenic infections. RIP2 is also activated by TLRs 2 and 4, although the mechanism of this activation is less. The role of RIP2 in otitis media (OM) pathogenesis has yet to be examined. Herein, we used in vivo animal models including C57BL/6 wild-type (WT) and RIP2−/− knockout mice inoculated in the middle ear (ME) with non-typeable Haemophilus influenzae (NTHi), a common human OM pathogen, to evaluate the expression of RIP2 and its signaling genes at the cellular level to determine the role of RIP2 in OM pathogenesis and recovery. The Nod1, Nod2, and Ripk2 genes are minimally expressed in the normal ME. However, they are strongly upregulated during acute OM, as are many genes related to RIP2 signaling. However, while signaling genes were expressed by various ME cell types, only mucosal epithelial and stromal cells expressed the NODs, RIP2, and signaling genes required for the activation of the host defensive response. Whereas WT mice clear ME bacteria and recover from OM within 5 days after infection, RIP2-deficient mice show persistent ME bacterial carriage and inflammation to at least 15 days. This includes significantly prolonged mucosal hyperplasia and ME leukocytic infiltration. Recruitment of macrophages is also delayed in comparison to WT mice. Thus, RIP2 is required to elicit a robust innate immune response that promotes bacterial clearance and increases host innate resistance. The results also identify the structural cells of the ME mucosa, as opposed to leukocytes, as the primary sites of NOD/RIP2 activity in the infected ME.

Immunomodulatory Response of the Middle Ear Epithelial Cells in Otitis Media

“…OM leads to increased gene expression of PRR and effector molecules (34). Furthermore, it has been shown in numerous animal models that disorders of innate immune system, e.g., absence of PRRs such as TLR-2 or NLR or disruptions in the signal transduction chains via MyD88 or RIP2, lead to bacterial persistence and the resulting chronification of OM (10,15,26,35,36).…”

Expression of Truncated Products at the 5′-Terminal Region of RIPK2 and Evolutive Aspects that Support Their Biological Importance

“…After activation by bacterial peptidoglycans, NOD1 and NOD2 associate with RIP-2 via the CARD–CARD interaction and promote the expression of immune response and inflammatory genes through the nuclear factor kappa B (NF-κB) signaling ( Inohara and Nunez 2003 ; Topal and Gyrd-Hansen 2021 ). NOD signaling may also cooperate with Toll-like receptors (TLRs) in detecting bacteria ( Kobayashi, et al 2002 ), but there is no consensus on the mechanisms involved in the activation of RIP-2 by TLRs (reviewed by ( Kurabi, et al 2022 )).…”