Macrophages are key immune effectors that shape host immunity and drive the initiation and progression of numerous diseases [1,2], including infections, autoimmune disorders, tumors, allergies, and metabolic diseases [[3], [4], [5]]. Upon detecting endogenous or exogenous danger signals, pattern-recognition receptors (PRRs) sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [6]. This recognition triggers an immune response, initiates recruitment of immune cells, and stimulates the release of inflammatory cytokines. Moderate inflammation in tissues and organs promotes repair, whereas excessive inflammation exacerbates tissue damage [7].

USP14 is a deubiquitinase that interacts with substrate proteins, promotes their deubiquitination, sustains their activity, and regulates cell fate [8]. It plays a pivotal role in tumors, metabolic disorders, and immune-related diseases [9]. In immune contexts, USP14 impacts inflammatory pathways and macrophage polarization: it binds and deubiquitinates TRAF6, preserving its activity and enhancing pro-inflammatory responses, and its inhibition promotes autophagy and dampens NF-κB signaling in sepsis models [10,11]. However, whether USP14 governs macrophage responses through additional effectors remains unclear.

Nemo-like kinase (NLK) is a serine/threonine mitogen-activated protein (MAP) kinase that acts as a negative regulator of the Wnt signaling pathway and is critical for maintaining immune homeostasis by directing cell migration, development, and differentiation [12]. NLK suppresses the transcriptional activity of the β-catenin–LEF/TCF complex through direct phosphorylation of TCF4 [13]. It also counters canonical Wnt/β-catenin signaling by activating a TAK1-dependent noncanonical Wnt cascade along with TAK1-binding protein 2 (TAB2) [14]. NLK dampens NF-κB signaling by modulating the IKK complex and preventing p65 nuclear translocation [15]. Despite these roles, potential crosstalk between USP14 and NLK in macrophages has not been defined.

We hypothesized that USP14 intersects with NLK-dependent pathways to shape macrophage inflammatory responses by influencing Wnt–NF-κB crosstalk. To investigate this, we overexpressed and silenced USP14 in RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs). This study delineates how USP14 modulates macrophage activity during inflammatory responses and elucidates the underlying mechanisms, offering insights that could facilitate the development of new therapeutics for inflammation-related diseases.