Conjugation of Met1-linked polyubiquitin (Met1-Ub) by the linear ubiquitin chain 
assembly complex (LUBAC) is an important regulatory modification in innate 
immune signaling. So far, only few Met1-Ub substrates have been described, and 
the regulatory mechanisms have remained elusive. We recently identified that the 
ovarian tumor (OTU) family deubiquitinase OTULIN specifically disassembles 
Met1-Ub. Here, we report that OTULIN is critical for limiting Met1-Ub 
accumulation after nucleotide-oligomerization domain-containing protein 2 (NOD2) 
stimulation, and that OTULIN depletion augments signaling downstream of NOD2. 
Affinity purification of Met1-Ub followed by quantitative proteomics uncovered 
RIPK2 as the predominant NOD2-regulated substrate. Accordingly, Met1-Ub on RIPK2 
was largely inhibited by overexpressing OTULIN and was increased by OTULIN 
depletion. Intriguingly, OTULIN-depleted cells spontaneously accumulated Met1-Ub 
on LUBAC components, and NOD2 or TNFR1 stimulation led to extensive Met1-Ub 
accumulation on receptor complex components. We propose that OTULIN restricts 
Met1-Ub formation after immune receptor stimulation to prevent unwarranted 
proinflammatory signaling.