Ubiquitin regulatory X (UBX) domain-containing protein 6 (UBXN6) is an essential cofactor for the activity of the valosin-containing protein p97, an adenosine triphosphatase associated with

diverse cellular activities. Nonetheless, its role in cells of the innate immune system remains largely unexplored. In this study, we report that UBXN6 is upregulated in humans with sepsis

and may serve as a pivotal regulator of inflammatory responses via the activation of autophagy. Notably, the upregulation of UBXN6 in sepsis patients was negatively correlated with

inflammatory gene profiles but positively correlated with the expression of Forkhead box O3, an autophagy-driving transcription factor. Compared with those of control mice, the macrophages

branched-chain amino acids. These metabolic shifts amplify mammalian target of rapamycin pathway signaling, in turn reducing the nuclear translocation of the transcription factor EB and

impairing lysosomal biogenesis. Together, these data reveal that UBXN6 serves as an activator of autophagy and regulates inflammation to maintain immune system suppression during human

sepsis.

The innate immune system serves as a primary defense mechanism that detects and responds to pathogen- or damage-associated molecular patterns during infection and inflammation. Monocytes and

macrophages, as principal cells in the innate immune system, can elicit detrimental inflammatory responses when their function becomes aberrant [1,2,3]. A delicate balance between

inflammation and innate immunity is crucial for effectively orchestrating protective immune responses against various pathogenic invasions [2, 3]. Understanding the mechanisms by which

monocytes/macrophages regulate inflammation and innate immunity is crucial for the development of novel therapeutics to combat infections and manage inflammatory diseases such as sepsis, a

life-threatening systemic inflammatory condition. Despite the importance of this knowledge, the mechanisms underlying the delicate balance between inflammation and innate immunity remain

incompletely understood.

Proteostasis is a dynamic and vital biological process for maintaining protein homeostasis and involves several pathways, such as autophagy, endoplasmic reticulum-associated protein

degradation (ERAD), and proteasomal degradation [4]. Autophagy, a lysosomal catabolic pathway that degrades large protein aggregates and damaged organelles, plays a crucial role in the

regulation of immune and inflammatory responses, thereby partially controlling the pathological processes associated with inflammation [5, 6]. The ERAD pathway is highly conserved in

evolutionary terms and degrades misfolded or unfolded proteins of mammalian cells to maintain protein homeostasis [7, 8]. Valosin-containing protein (VCP)/p97 is a significant member of the

ATPases associated with diverse cellular activities (AAA+) superfamily that facilitates the conversion of chemicals into mechanical energy in many organisms [9,10,11]. The p97 is an abundant

cytosolic protein in mammalian cells that regulates autophagy, ERAD, gene expression, and organelle biogenesis, thereby maintaining cellular homeostasis [10, 12, 13].

Several cofactors ( > 40), including “ubiquitin regulatory X” (UBX) domain-containing proteins, interact with p97 and participate in its functional regulation by facilitating substrate

recruitment, leading to the formation of various p97-cofactor complexes [10, 13]. UBXN6 is a novel p97 cofactor that associates with the p97 complex via both peptide:N-glycanase and

ubiquitin-associated or UBX-containing proteins (PUB) [14, 15] and UBX domains [16]. A recent study revealed that UBXN6 contains p97-remodeling motifs that drive AAA+ remodeling and ring

opening, thereby regulating p97 ATPase activity [17]. Both p97 and UBXN6 preserve lysosomal homeostasis by facilitating the clearance of damaged lysosomes and activating lysophagy

[18,19,20]. In addition, both UBXN6 and p97 control the trafficking of ubiquitylated caveolin-1 within the endocytic pathway [21]. Moreover, p97 activity is essential for the production of

the autophagy-inducing lipid phosphatidylinositol-3-phosphate and for autophagosome biogenesis [22]. A recent study demonstrated that p97, UBXN6, and ANKRD13A act cooperatively to target the

parasitophorous vacuole, thereby restricting Toxoplasma gondii infection in interferon-stimulated human endothelial cells [23]. Nonetheless, the specific involvement of the p97 cofactor

UBXN6 in the regulation of innate immune and inflammatory responses and its clinical relevance remain largely unexplored.

Here, we revealed significantly upregulated levels of UBXN6 in the peripheral blood mononuclear cells (PBMCs) of septic patients. Single-cell RNA sequencing (scRNA-seq) analysis revealed

that UBXN6 was predominantly expressed in human primary monocytes/macrophages. In sepsis patients, UBXN6 levels were negatively correlated with inflammatory gene profiles but positively

correlated with the levels of Forkhead box O3 (FOXO3) and several autophagy/mitophagy-related genes. Next, we established myeloid-specific UBXN6-deficient mice, which exhibited increased

susceptibility to systemic inflammation. Mechanistically, myeloid UBXN6 plays a pivotal role in inducing both autophagy and ERAD by regulating mitochondrial and cellular oxidative stress,

such as reactive oxygen species (ROS), thus influencing inflammatory responses in macrophages. Furthermore, myeloid UBXN6 deficiency shifted immunometabolic remodeling toward aerobic

glycolysis and increased the levels of branched-chain amino acids (BCAAs), thus increasing mammalian target of rapamycin (mTOR) pathway activation and limiting both the nuclear translocation

of transcription factor EB (TFEB) and lysosomal biogenesis. Thus, myeloid UBXN6 is an essential activator of autophagy and controller of inflammation. Our data also highlight the clinical

relevance of UBXN6 in terms of human sepsis, particularly in the context of immunosuppression.

To examine the overall changes in gene expression in sepsis patients, we used deep RNA-seq to perform transcriptome analyses of PBMCs from patients with a poor prognosis (SP), patients who

had recovered (SR), and healthy controls (HC). In total, 1.44 billion raw reads were generated from 8 HCs and 12 sepsis patients (8 SRs and 4 SPs) and trimmed to remove adapter and

low-quality sequences. On average, 68.1 million clean reads were produced per sample and mapped to the reference human genome (Supplementary Table 1). From the resulting alignments, we

identified differentially expressed genes (DEGs) between the SP, SR, and HC groups by performing comparative transcriptome analysis. After controlling for multiple comparisons and a false

discovery rate (FDR) of 5%, we acquired 1,769, 1,840, and 266 DEGs via comparisons between the SR and HC, SP and HC, and SP and SR data (Fig. 1A). To identify potential PBMC biomarkers of

SP, we investigated 604 (484 + 120) DEGs shared by HCs and SPs while excluding those shared by SRs and HCs. Among these DEGs, 356 and 248 DEGs were commonly upregulated or downregulated,

respectively, in SP patients (Fig. 1B). The downregulated DEGs were significantly enriched in immune system-related biological processes, including the cell surface receptor signaling

pathway (GO:0007166), positive regulation of interferon-γ production (GO:0032729), T-cell activation (GO:0008009), Th17 cell differentiation (hsa04659), and cytokine–cytokine receptor

interaction (hsa04060) (Supplementary Fig. 1A, B), suggesting that SP patients exhibited immunosuppressive profiles.

Compared with healthy controls, sepsis patients presented upregulated expression levels of UBXN6. A Diagram illustrating the number of DEGs identified by comparing HCs to SRs, HCs to SPs,

and SRs to SPs. The yellow region highlights DEGs whose expression varies between HCs and SPs but not between HCs and SRs. B Graph depicting the fold changes in 604 genes from the yellow

area of (A), with red indicating upregulated DEGs and blue indicating downregulated DEGs in SP. C Heatmap representing 91 ATG genes with differential expression. Hierarchical clustering of

DEGs was performed on the basis of the Euclidean distance of relative expression. D Expression of UBXN6 in human PBMCs from HCs, SRs, and SPs in our cohort. *q value