Ubiquitination of interleukin-1 a is associated with increased pro-inflammatory polarization of murine macrophages deficient in the E3 ligase ITCH

Macrophages play critical roles in homeostasis and inflammation. Macrophage polarization to either a pro-inflamma-tory or anti-inflammatory status is controlled by activating inflammatory signaling pathways. Ubiquitination is a posttranslational modification that regulates these inflammatory signaling pathways. However, the influence of protein ubiquitination on macrophage polarization has not been well studied. We hypothesized that the ubiquitination status of key proteins in inflammatory pathways contributes to macrophage polarization, which is regulated by itchy E3 ubiquitin ligase (ITCH), a negative regulator of inflammation. Using ubiquitin proteomics, we found that ubiquitination profiles are different among polarized murine macrophage subsets. Interestingly, interleukin-1 a a ), an important in li-popolysaccharide-induced pro-inflammatory macrophages, which was enhanced in ITCH-deficient macrophages. The ITCH-deficient macrophages increased levels of the mature form of IL-1 a exhibited pro-inflammatory polarization, and reduced deubiquitination of IL-1 a

Macrophages play critical roles in homeostasis and inflammation. Macrophage polarization to either a pro-inflammatory or anti-inflammatory status is controlled by activating inflammatory signaling pathways. Ubiquitination is a posttranslational modification that regulates these inflammatory signaling pathways. However, the influence of protein ubiquitination on macrophage polarization has not been well studied. We hypothesized that the ubiquitination status of key proteins in inflammatory pathways contributes to macrophage polarization, which is regulated by itchy E3 ubiquitin ligase (ITCH), a negative regulator of inflammation. Using ubiquitin proteomics, we found that ubiquitination profiles are different among polarized murine macrophage subsets. Interestingly, interleukin-1a (IL-1a), an important proinflammatory mediator, was specifically ubiquitinated in lipopolysaccharide-induced pro-inflammatory macrophages, which was enhanced in ITCH-deficient macrophages. The ITCH-deficient macrophages had increased levels of the mature form of IL-1a and exhibited pro-inflammatory polarization, and reduced deubiquitination of IL-1a protein.
Finally, IL-1a neutralization attenuated pro-inflammatory polarization of the ITCH-deficient macrophages. In conclusion, ubiquitination of IL-1a is associated with increased pro-inflammatory polarization of macrophages deficient in the E3 ligase ITCH.
Inflammation is the host's response to exogenous pathogen invasion or endogenous signals such as damaged cells. Inflammation is critical for tissue repair but can also lead to local or systemic tissue damage if unchecked (1). Macrophages are plastic immune cells necessary for maintaining homeostasis and controlling inflammation (2). Once activated, macrophages are polarized into pro-inflammatory or anti-inflammatory macrophages (3,4). Given the critical role of macrophages in inflammation, it is important to understand the regulation of macrophage polarization to intervene in unrestrained inflammation.
Macrophages survey their milieu for abnormal microenvironmental stimuli through pattern-recognition receptors. Pathogens and environmental challenges activate macrophages and trigger inflammatory pathways such as the archetypical nuclear factor-kB (NF-kB) pathway, resulting in the production of key inflam-matory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor (TNF) (2). Cytokines differentially activate macrophages, resulting in macrophage polarization. Pro-inflammatory macrophages are activated by numerous factors, including lipopolysaccharide (LPS) and interferon-g (IFN-g) and characterized by increased inducible nitric oxide synthase (iNOS) expression and nitric oxide production, which play a central role in the pathogen-killing inflammatory response. In contrast, anti-inflammatory macrophages are activated by IL-4 and characterized by increased arginase expression to promote ornithine metabolism, which is crucial for cell proliferation and tissue repair (5).
Previous studies linked macrophage activation with the ubiquitination (ub) status of key proteins in inflammatory signal pathways (6). Lysine-48 ub (Lys-48-ub) of the NF-kB inhibitor (IkB), the final checkpoint of the NFkB pathway, leads to IkB degradation and consequent NF-kB-associated inflammation (7). In contrast, TRAF6, an upstream positive regulator of the NF-kB pathway, is modulated by Lys-63-ub, leading to its conformational change and activation (8). However, a comprehensive analysis of ub profiles of polarized macrophages has not been reported. Whether a unique ub signature is associated with individual macrophage subsets is not known.
Ub status of a protein is controlled by both ub and de-ub enzymes. The ub process is catalyzed sequentially by ubiquitin E1, E2, and E3 ligases whereas de-ub is carried out by de-ub enzymes (DUB). ITCH is a well-characterized negative regulator of inflammation that functions as an E3 ligase as well as an adaptor protein of DUB in addition to catalyzing the final step of protein ub (9). ITCH catalyzes the Lys-48-ub of the proinflammatory AP-1 and JunB transcription factors, marking them for degradation, and consequently inhibits the expression of downstream cytokines in T cells (10). In contrast, ITCH forms complexes with the DUB enzyme A20 in mouse embryonic fibroblasts, resulting in inflammation inhibition (11). We reported that ITCH interacts with DUB enzyme CYLD to reverse the Lys-63-ub of TRAF6 and down-regulate the NF-kB pathway in osteoclast precursors (12). However, the role of ITCH as a ub modulator in polarized macrophages has not been studied. Itch is expressed at low levels at baseline. Itch is an NF-kB target-responsive gene, and with pro-inflammatory stimulation such as LPS, the expression of Itch is elevated to negatively regulate inflammation (9). Thus, exploring genes and proteins affected in Itch2/2 macrophages will improve This article contains supporting information. * For correspondence: Lianping Xing, Lianping_xing@urmc.rochester.edu. our understanding of the negative regulation during sustained inflammation.
IL-1 is an important pro-inflammatory cytokine. IL-1 family cytokines include IL-1a and IL-1b; both bind to the same IL-1 receptor and eventually activates NF-kB signaling to induce inflammation (13). The ub of pro-IL-1b promotes its catalytic processing and subsequent maturation (14). However, how the ub and de-ub regulation of IL-1 affects mac polarization is unknown.
In the current study, we show that quiescent, pro-inflammatory, and anti-inflammatory macrophages have distinct ub profiles and elevated ub of IL-1a in LPS-induced pro-inflammatory macrophages ITCH inhibits inflammatory cytokine IL-1a maturation by promoting the de-ub of pro-IL-1a, thereby negatively regulating LPS-macs pro-inflammatory polarization.
Our data indicate that the ub status of key mediators affects macrophage pro-inflammatory polarization, which is regulated by ITCH. Our study provides rationale for modulating protein ub of key regulators in macrophages as an approach to interfere with inflammatory diseases.

Results
The expressional levels of total ubiquitinated proteins are similar in pro-inflammatory and anti-inflammatory macrophages To test whether protein ub is altered during macrophage polarization, we treated WT BMMs with LPS to induce proinflammatory macrophages or IL-4 to induce anti-inflammatory macrophages using the protocol that we have described recently (15,16) in which iNOS was used as the marker for proinflammatory macrophages and CD206 was used as marker for anti-inflammatory macrophages, and PBS-treated cells were used as control. We assessed the expressional levels of total ubiquitinated proteins by Western blot analysis. We validated our cell model by assessing the expression of surface markers and effector genes expression in these cells (Fig. 1, A and B). Consistent with the literature (17), LPS-treated cells (LPSmacs) were stained positively for iNOS whereas IL-4-treated cells (IL-4-macs) were stained positively for CD206, surface markers for pro-inflammatory and anti-inflammatory macrophages, respectively (Fig. 1A). LPS-macrophages had a proinflammatory gene expression profile with ;500to 1000-foldincreases in IL-1b, NOS2, and TNF mRNA levels compared with PBS-macs, whereas IL-4-macs had an anti-inflammatory gene expression profile with a similar -fold increase of IL10 and PPARg mRNA expression (Fig. 1B). Surprisingly, the expressional levels of total ubiquitinated proteins were similar in all three types of macrophages (Fig. 1C), suggesting that changes in ub of specific proteins, rather than the total amount of ubiquitinated proteins, may be associated with macrophage polarization.

Key proteins of pro-inflammatory polarization are differentially ubiquitinated in LPS-induced macrophages
To identify key regulatory proteins in macrophage polarization, we assessed the ub profiles of LPS-, IL-4-, and PBS-macs ( Fig. 2A) using the ubiquitin proteomics approach (18). As expected, ub profiles were different among three macrophage subsets; 30, 32, and 14 proteins were differentially ubiquitinated in PBS-, LPS-, and IL-4-macs, respectively ( Fig. 2B and Table 1 detailed the ubiquitin proteomics findings in Table S1 and PRIDE dataset identifier PXD018743). Among 32 ubiquitinated proteins in pro-inflammatory LPS-macs, iNOS and IL-1a were the fifth and eighth highest ubiquitinated proteins (Fig. 2C). Both iNOS and IL-1 proteins undergo posttranslational modification by ub (19,20). However, how ub of iNOS and IL-1a affects macrophage polarization has not been studied.

ITCH limits pro-inflammatory phenotype and affects the ub status of IL-1a in macrophages
Previous studies reported that the ubiquitin E3 ligase ITCH limits inflammatory responses by negatively regulating LPS- Figure 1. The expressional levels of total ubiquitinated proteins are similar in pro-inflammatory and anti-inflammatory macrophages. BMMs from WT C57BL/6J mice were treated with LPS (100 ng/ml) or IL-4 (100 ng/ ml) for 24 h to induce pro-inflammatory and anti-inflammatory macrophages, respectively. PBS-treated cells were used as controls. A, representative images show that immunofluorescence-stained cells for iNOS (taken at 103 magnification, scale bar = 50 mM), a marker for pro-inflammatory macrophages or CD206, a marker for anti-inflammatory macrophages. n = 3 repeats. B, relative expression of effector genes of pro-or anti-inflammatory macrophages by qPCR. Values are mean 6 S.D. of three wells. -Fold changes of genes were calculated by normalized to actin expression level and then to PBS-macs. Data were analyzed by one-way ANOVA followed by a Tukey test. *, p , 0.05. C, expression of total ubiquitinated proteins was assessed by Western blot analysis using an anti-ubiquitin antibody. n = 2 repeats.
Itch inhibits macrophage polarization via IL-1a de-ub induced NF-kB activation (11,12). However, whether ITCH affects ub of iNOS or IL-1a as a molecular mechanism for its anti-inflammatory effect has not been studied. To explore if ITCH mediated the ub of iNOS and IL-1a in macrophages, we utilized ITCH-deficient cells. Compared with WT LPS-macs, Itch2/2 LPS-macs had 2-fold increased iNOS signal intensity by immunofluorescence staining (Fig. 3, A and B). Expression of NOS2 and IL1b mRNA significantly increased 5.44-fold in Itch2/2 LPS-macs compared with 1.36-fold in WT LPS-macs (Fig. 3C). These data suggest that Itch2/2 macrophages are predisposed to pro-inflammatory polarization. To determine whether ITCH negatively regulates pro-inflammatory polarization by mediating ub of IL-1a or iNOS in macrophages, we assessed the ub status of iNOS and IL-1a in macrophages from Itch2/2 mice Ubiquitinated iNOS levels were comparable in Itch2/2 and WT LPS-macs (4.1 6 0.9 in Itch2/2 versus 3.72 6 0.74 in WT, -fold increase of LPS-macs over WT PBS-macs) (Fig. 4A). However, ubiquitinated IL-1a levels were ;2-fold higher in Itch2/2 compared with WT cells (9.27 6 1.16 in Itch2/2 versus 4.36 6 1.05 in WT, -fold increase of LPS-macs over WT PBS-macs) (Fig. 4B), indicating that the ub status of IL-1a, but not iNOS, may be regulated by ITCH.

ITCH promotes the de-ub of IL-1a in pro-inflammatory macrophages
Elevated ubiquitinated IL-1a in Itch2/2 macrophages (Fig.  4) suggested that ITCH functioned as an adaptor protein for DUB enzymes to remove ubiquitin from IL-1a. To test whether ITCH affected the de-ub of IL-1a, we stimulated macrophages with LPS for 8 h and assessed the levels of ubiquitinated IL-1a 8 h and 16 h after removing LPS from the culture (Fig. 5A). We found that ubiquitinated IL-1a proteins were higher in Itch2/2 macrophages compared with WT macrophages after LPS removal (-fold increase over PBS: 3.76 6 1.00 in Itch2/2 versus 1. 24 5D). In contrast, the expression of IL-1a mRNA was not significantly different between Itch2/2 and WT macrophages after removing LPS (Fig. 5E), suggesting that higher levels of ubiquitinated IL-1a in Itch2/2 macrophages after LPS removal were not because of transcriptional regulation. Because the levels of ubiquitinated IL-1a were higher after LPS removal in macrophage-deficient ITCH, we suspected that ITCH may affect the de-ub process via its adaptor function, i.e. ITCH works with DUB enzymes to reduce IL-1a ubiquitination. To test this possibility, we treated LPS-macs 8 h after LPS removal (Fig. 5B, lanes 3 and 7) with a DUB inhibitor WP1130. . Thus, Itch regulates the ub status of IL-1a by promoting its de-ub process, which can be blocked by ITCH depletion or the DUB inhibition.

Mature form of IL-1a increases in pro-inflammatory macrophage-deficient ITCH
We then studied how reduced IL-1a de-ub contributes to increased pro-inflammatory phenotype in Itch2/2 LPS-macs because of the positive correlation between ubiquitinated IL-1a and inflammation (Figs. 3 and 4). We hypothesized that ub promoted the processing of pro-IL-1a, resulting in its maturation. We found that mature form of IL-1a increased ;2-fold in Itch2/2 LPS-macs compared with WT cells (-fold increase of LPS-macs over WT PBS-macs: 13.33 6 2.57 in Itch2/2 versus 7.98 6 0.039 in WT cells) (Fig. 6A) measured by Western blot analysis based on the different molecular weights of pro-and mature IL-1a. Consistently, secreted IL-1a in the supernatant of Itch2/2 LPS-macs was ;2.5-fold higher than WT (-fold increase of LPS-macs over WT PBS-macs: 62.70 6 4.20 in Itch2/2 versus 24.75 6 2.25 in WT cells) (Fig. 6B) measured using ELISA that measures soluble IL-1a acquired upon maturation. Because the maturation of pro-IL-1a is chiefly catalyzed by granzyme B (21), it is possible that ITCH depletion increases mature IL-1a by increasing the enzymatic activity of granzyme B. To test this, we assessed granzyme B activity using specific substrate Ac-IETD-AFC. Although granzyme B activity increased after LPS stimulation in both WT and Itch2/2 LPS- . Elevated ubiquitinated IL-1a in Itch2/2 pro-inflammatory macrophages. BMMs from Itch2/2 mice or WT littermate controls were treated as in Fig. 3. Whole-cell lysates were immunoprecipitated with anti-iNOS or anti-IL-1a antibodies and IP complexes were blotted with an antiubiquitin antibody. iNOS and IL-1a protein expression in the input whole lysates were used as controls. The intensity of the ubiquitinated iNOS or IL-1a was quantified with ImageJ as average intensity density 3 the area of the smeared band. The levels of ubiquitinated proteins were normalized to actin and then to PBS-treated WT samples. A, ubiquitinated iNOS was increased in LPS-macs, but is similar between WT and Itch2/2 LPS-macs. B, ubiquitinated IL-1a was increased in LPS-macs and was further increased in Itch2/2 LPSmacs. Data were analyzed with two-way ANOVA followed by Sidak's post hoc test. Values are mean 6 S.D. of n = 3. *, p , 0.05 LPS-macs versus PBS-macs; #, p , 0.05 Itch2/2 cells versus WT cells (only the statistics of the comparison between Itch2/2 versus WT with the same treatment are shown).  (Fig. 6C), suggesting that Itch's regulation of IL-1a maturation was independent of granzyme B. To further support that ITCH depletion promotes IL-1a maturation, we knocked down ITCH in WT macrophages using siRNA approach. Compared with control siRNAtransfected cells, cells that were transfected with siRNA Itch had 60% reduction in ITCH protein expression (-fold increase of ITCH in LPS-macs over control PBS-macs: 0.84 6 0.04 in Itch siRNA versus 1.40 6 0.20 in control siRNA) (Fig. 6D). Most importantly, Itch knockdown macrophages showed ;1.6-fold increase of mature IL-1a than control macrophages, a similar increase in Itch2/2 cells over WT cells (-fold increase of LPS-macs over control PBS-macs: 9.78 6 1.15 in Itch siRNA versus 6.21 6 1.19 in control siRNA) (Fig. 6D). These data further indicate that ITCH inhibits IL-1a maturation by decreasing the maturation process of ubiquitinated IL-1a.

IL-1a neutralizing antibody partially inhibits proinflammatory polarization of Itch2/2 macrophages
To further support the argument that IL-1a maturation contributes to susceptibility of Itch2/2 macrophage to proinflammatory polarization, we examined whether IL-1a neutralizing antibodies (IL-1a-Ab) can rescue the increased proinflammatory phenotype in Itch2/2 LPS-macs by immunoflu-orescence staining for iNOS intensity and qPCR analyzing for the expression of NOS2. IL-1a-Ab treatment slightly, about 35%, decreased iNOS intensity in WT cells (-fold increase of LPS-over PBS-macs: 3.  . Increased mature form of IL-1a in Itch2/2 pro-inflammatory macrophages. BMMs from Itch2/2 mice or WT littermate controls were treated as in Fig. 3. A, pro form and mature form of IL-1a are distinguished using molecular weight in Western blot analysis (pro form: 31 kDa, activated form 17 kDa). Pro form IL-1a increased after LPS stimulation, with no difference between WT and Itch2/2 macrophages. Mature form IL-1a was higher in Itch2/2 LPS-macs compared with WT. B, secreted IL-1a in the supernatant was assessed with a ELISA kit, showing higher mature IL-1a in Itch2/2 LPS-macs. C, granzyme B activity was assessed with specific fluorescent substrate acetyl-IETD-AFC (Ac-IETD-AFC). Production of AFC was monitored in a spectrofluorimeter and normalized to WT PBStreated macrophages. D, si-Itch was used to knock down Itch in macrophages. ITCH protein level was assessed to evaluate the effectiveness of the knockdown. Pro form and mature form of IL-1a are distinguished using molecular weight in Western blot analysis. Densitometry analysis for Itch, pro and mature form of IL-1a. Data were analyzed with two-way ANOVA followed by Sidak's post hoc test. Values are mean 6 S.D. of n = 3. *, p , 0.05 LPS-macs versus PBS-macs; #, p , 0.05 Itch2/2 cells versus WT cells (only the statistics of the comparison between Itch2/2 versus WT with the same treatment are shown).

Discussion
Recent studies demonstrated the importance of mac polarization in various pathological conditions, which are controlled by intricate signaling pathway crosstalk. Using ubiquitin pro-teomics, we showed that the ub profiles are distinct in polarized macrophages. We also found that the ub status of IL-1a is associated with its maturation, which is mediated by ubiquitin E3 ligase ITCH. ITCH depletion in macrophages resulted in elevated ubiquitinated IL-1a, decreased IL-1a de-ub, increased mature IL-1a, and more severe pro-inflammatory polarization (Fig. 8). This implies that modulating ub of key proteins may be a viable approach to manipulate macrophage phenotype and interfere with macrophage-mediated diseases.
Previous studies linked protein ub with macrophage polarization. E3 ligases TRAF6 (22), Pellino1 (23), and Praja2 (24) catalyze the Lys-63-ub to activate positive regulators of NF-kB and JunB pathways in macrophages to promote pro-inflammatory polarization. In contrast, E3 ligase Cbl-b inhibits mac proinflammatory polarization by catalyzing the ub of Toll-like receptor 4 for its degradation (25). In this study, we demonstrate that ITCH regulates mac polarization by modulating the ub of pro-inflammatory cytokine IL-1a. These findings suggest that the ub status of specific proteins in macrophages may result in opposite outcomes on their polarization, e.g. negative regulators of inflammation are ubiquitinated for degradation, whereas positive regulators are ubiquitinated for activation.
Using ub proteomics, we reported for the first time that ub profiles of PBS-, LPS-, or IL-4-treated macrophages are different. Specific ubiquitinated proteins in PBS-macs and IL-4macs involved in a wide range of housekeeping proteins such as RNA transcription (26,27), posttranslational modification (28)(29)(30), mitochondria function (31,32), glutamate metabolism (33), intracellular trafficking (34,35), and so on. More interestingly, cellular structural proteins including actin-related protein 10 are specifically ubiquitinated in IL-4-macs, which might be related to the shift to elongated shape during anti-inflammatory macrophage polarization (36). In contrast, among the top eight specifically ubiquitinated proteins identified in LPS-macs (Fig. 2C, and Table 1), seven are known regulators of inflammation, including IL-1a and iNOS. Suppressor of cytokine signaling 2 (SOCS3) is a negative regulator of cytokine signaling by functioning as a part of a multi-subunit E3 ligase complex (37). The substrates of SOCS3 include important inflammatory regulators such as TRAF6, Janus kinase, and insulin receptor substrate 1/2 (37,38). Interferon-induced GTP-binding protein Mx1/Mx2, C-type lectin, and serum  amyloid A3 protein are critical for host anti-viral (39), anti-bacteria (40), and acute inflammatory (41) responses, respectively. However, the effects of ub on the function of these inflammatory regulators during mac polarization are unclear, which warrants future investigation.
We demonstrated that the ub of IL-1a is important for its processing to the mature form, which raises three questions that warrant further investigations. First, although we showed that ITCH indirectly facilitated IL-1a maturation, we did not explore what proteases and cellular compartments are involved in ubiquitinated IL-1a cleavage. Multiple proteases, including neutrophil elastase, granzyme B, cathepsin G, and proteinase-3, have been reported to cleave pro-IL-1a at different amino acid residues (21). Thus, whether ITCH also affects the level or activity of IL-1a processing proteases needs to be explored in the future. Second, the location of IL-1a proteolytic processing can be intracellular or extracellular, depending on the protease (13). We showed that increased secreted soluble IL-1a in the supernatant of Itch2/2 LPS-macs is correlated with more severe inflammation, which can be partially reversed with IL-1a neutralization, suggesting that IL-1a is processed intracellularly and secreted as a mature form. However, it is also possible that ubiquitinated IL-1a is secreted to the extracellular space and exposed to the proteases in the interstitial space. Third, the effects of IL-1a maturation are unclear. Unlike IL-1b, another member in the IL-1 cytokine family whose biological function depends on its catalytic processing and maturation, both pro-IL-1a and mature IL-1a bind to IL1R with comparable kinetics in vitro (42, 43), suggesting that the maturation might not be necessary for IL-1a bioactivity. However, Afonina et al. (21) reported that the bioactivity of mature IL-1a is several times higher than the pro form in vivo. Watanabe and Kobayashi (44) also reported that catalytic processing of IL-1a to mature form promotes the secretion of IL-1a. Increased in vivo bioactivity of mature IL-1a may be related to the solubility of mature IL-1a, which facilitates its dissemination to exert systemic effects, compared with the membrane-bound pro-IL-1a. Furthermore, pro-IL-1a is subjected to intracellular proteasomal degradation (19), which might be protected from the maturation process. We showed that increased secreted IL-1a is correlated with increased inflammation in Itch2/2 macrophages and can be partially reversed by an IL-1a neutralizing Ab, supporting the argument that maturation of IL-1a increases its bioactivity.
We found that ITCH regulates IL-1a ub by promoting its deub. De-ub is a process to remove ubiquitin from proteins and other molecules, which is carried out by DUB. Recent studies demonstrate the important role of DUBs in controlling inflammation. DUB A20 (11) and CYLD (12) negatively regulate NF-kB signaling pathway in macrophages by removing the Lys-63ub on TRAF6. Mice deficient for A20 or CYLD develop chronic inflammatory phenotype. ITCH is a ubiquitin E3 ligase. ITCH catalyzes the Lys-48-ub of the pro-inflammatory AP-1 and JunB transcription factors, marking them for degradation, and consequently inhibits the expression of downstream cytokines in T cells (10). In contrast, ITCH forms complexes with DUB enzymes A20 (11) or CYLD (12) to reverse the Lys-63-ub of TRAF6 and down-regulate the NF-kB pathway in macrophages. Our findings of the involvement of ITCH in IL-1a de-ub and maturation reveal a new molecular mechanism for inhibitory role of ITCH in inflammation.
Pro-inflammatory stimuli culminate in the activation of a few key signaling pathways (e.g. NF-kB), resulting in similar transcriptional changes in macrophages. However, different levels or combinations of stimuli lead to a complex continuum spectrum of phenotypes instead of the simplified M1-M2 paradigm, and markers for polarized macrophage populations are not well-defined (2,45). In the current study, we used iNOS as an in vitro marker for pro-inflammatory macrophages. It is known that iNOS which, in addition to its bactericidal functions, mediates various inflammatory diseases such as posttraumatic osteoarthritis (15). However, iNOS alone is not a reliable marker for mac polarization in vivo. Therefore, we recognize the limitation of using iNOS as a single marker for LPS macrophages. Characterizing specific panels of markers of polarized macrophage population in future in vivo studies is necessary.

Conclusion
We showed that the ub of specific proteins in inflammatory pathways alter macrophage polarization. Specifically, the de-ub of IL-1a regulated by ITCH inhibits pro-inflammatory macrophage polarization by blocking pro-IL-1a maturation. Based on our findings, it is beneficial to modulate the ub of specific proteins to alter the macrophage phenotype as a treatment for inflammatory diseases.

Animals and cell cultures
Itch2/2 mice (3-6 months, male and female) were generated previously on a C57BL/6J background and were genotyped by PCR analysis (12), and WT littermates were used as controls. Bone marrow (BM) cells were flushed from femurs and tibias and cultured with conditioned medium (1:50 dilution) from a macrophage colony-stimulating factor-producing cell line (46) for 3 days in a-MEM with 10% FBS (Gibco 26140-079) to generate BM macrophages (BMMs). BMMs were treated with LPS (Sigma-Aldrich, L4391; 100 ng/ml or 500 ng/ml) (LPS-macs) or IL-4 (R&D Systems, 404-ML-010; 100 ng/ml) (IL-4-macs) for further experiments as described below. PBS-treated WT macrophages (WT PBS-macs) were used as controls. All animal use in this study has been approved by the Animal Care and Use Committee at the University of Rochester.

Quantitative Real-Time PCR
Total RNA was extracted using TRIzol reagent (Invitrogen). cDNAs were synthesized using the iSCRIPT cDNA Synthesis Kit (Bio-Rad, 1708891). Quantitative real-time PCR (qPCR) amplifications were performed in the iCycler (Bio-Rad) real-time PCR machine using the iQ SYBR Green Supermix (Bio-Rad, 1808882) according to the manufacturer's instructions. Actin was amplified on the same plates and used to normalize the data. Each sample was prepared in triplicate, and each experiment was repeated at least three times. -Fold changes of genes of interest were calculated by normalizing to PBS-treated WT macrophages as 1. The sequences of primer pairs for IL1b, NOS2, TNF, IL10, PPAR, IL1a, and actin mRNAs are shown in Table 2.

Immunofluorescence staining
Cells cultured in 96-well plates were fixed with 10% formalin for immunofluorescence staining. Briefly, cells were blocked with 5% normal donkey serum (Jackson ImmunoResearch Laboratories, 017-000-121, diluted in PBS with 0.1% Triton X) for 30 min at room temperature. Cells were incubated with primary antibodies iNOS (Santa Biotechnology, sc-650, 1:200) or CD206 (R&D Systems, AF2535, 1:100) at 4°C overnight, followed by secondary antibodies goat anti-rabbit Alexa Fluor 488 (Abcam, ab150077, 1:500) and rabbit anti-goat Alexa Fluor 568 (Thermo Fisher, A21085, 1:400) at room temperature for 1 h, and finally stained with 49,6-diamidino-2-phenylindole (DAPI, 1 mg/ml) for 10 min. Antibodies were diluted in 1% normal donkey serum (diluted in PBS with 0.1% Triton X). Cells were imaged with an Olympus 1 3 71 microscope at 203 magnification. To quantify iNOS1 cells, ImageJ was used to calculate the intensity density of F4/801 staining. For the quantification of iNOS staining in the 96-well, one image was taken for each well under 103 magnification was imported to the ImageJ software, with a total of three wells for each group. Thresholds were determined for positive and negative staining and maintained the same throughout analysis. Percentage of iNOS1 area was measured using the "Area Fraction" modality.

LC-MS (LC-MS/MS) for ubiquitinated proteins
Primary BMMs pooled from five C57Bl/6 mice were cultured to induce PBS-, LPS-, or IL-4-macs as described above and were collected for ubiquitin proteomics following a published protocol (18). In brief, cells were lysed in freshly prepared urea lysis buffer. Protein concentration was measured using the Pierce BCA protein assay kit (Thermo Fisher, cat. no. 23225). Disulfide bonds were reduced with 2 mM DTT for 1 h at room temperature. Cysteine residues were carbamidomethylated by adding 10 mM iodoacetamide for 30 min incubation at room temperature in the dark. Proteins were precipitated using the chloroform/methanol precipitation method, followed by resuspension in 1 M urea, 50 mM HEPES. Enzymatic digestion was performed using trypsin (Pierce) at a 1:50 enzyme:protein ratio overnight at 37°C. The reaction was quenched in the morning by the addition of formic acid (Pierce) to 1%. The peptide solution was centrifuged for 5 min at 10,000 3 g to remove precipitates. Peptides were desalted using a 130-mg C18 SepPak cartridge (Waters) and then dried down in a Centrivap Concentrator (Labconco).
For immunoaffinity purification of K-e-GG peptides, lyophilized peptides were reconstituted, and ubiquitin-modified peptides were enriched with PTMScan ubiquitin remnant motif (K-e-GG) kit (Cell Signaling Technology, cat. no. 5562). In brief, reconstituted peptides were incubated with K-e-GG-specific antibody cross-inked Protein-A agarose beads, and eluted with IAP elution buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate and 50 mM NaCl, 0.15% (v/v) TFA). Then peptides were desalted with a homemade C18 column.
For LC-MS/MS, K-e-GG-enriched samples were reconstituted in 20 ml TFA (0.1%, JT Baker) and 6 ml (30%) was loaded onto a homemade 30-cm C18 column, with 1.8 mM beads (Sepax) using an Easy nLC-1000 (Thermo Scientific). The mobile phases were 0.1% formic acid in water (JT Baker) and 0.1% formic acid in acetonitrile (JT Baker). The gradient began at 3% B, went to 8% over 5 min, to 30% over 68 min, then to 70% B in 4 min and was held there for 4 min. The gradient was returned to initial conditions in 3 min and the column was allowed to reequilibrate for 10 min. The flow rate was kept constant at 300 nl/min. The peptides were detected using a Q Exactive Plus mass spectrometer (Thermo Scientific), using a data-dependent top 10 method. A full scan from 400-1400 m/z was collected at 70,000 resolution with a maximum ion injection time of 50 ms, and an AGC setting of 1 3 10 6 . The MS2 spectra were collected at 17,500 resolution using a normalized collision energy of 27, an isolation width of 1.5 Da, a maximum ion injection time of 200 ms, and an AGC setting of 5 3 10 4 . Dynamic exclusion was enabled and set at 25 s, with a repeat count of 1.

MS data analysis
MS data were searched using the Mascot search engine (Matrix Science) within the Proteome Discoverer software platform, version 2.2 (Thermo Fisher), using the SwissProt Mus musculus database. Percolator was used as the false discovery rate calculator, filtering out peptides that had q-values greater than 0.01. To semi-quantify the protein relative abundance in multiple samples, the peak area of the extracted ion chromatograms for each peptide precursor in the full scan was calculated using the Minora Feature Detector node within Proteome Discoverer, which also used retention time alignment and precursor mass to link peptides across runs, which reduces the level of missing values. The abundance of an individual protein was calculated as the sum of its peak areas for all peptides derived from that protein. The relative concentration of each protein was determined by comparing the total MS intensities of all identified peptides from that protein in one sample versus those from other samples. Raw dataset of MS was uploaded to PRIDE (identifier PXD018743). Proteins with an abundance above 1 3 10 7 and at least two identified unique peptides that were specifically ubiquitinated in each condition were identified in Table  2. A list of all ubiquitinated proteins with peptide sequences with diglycine tags was included in Table S1.

Immunoprecipitation
Cells were lysed in ub-lysis buffer and subjected to immunoprecipitation (IP) as described before (12). Briefly, 500 mg of proteins were diluted in 100 ml ub-lysis buffer and mixed with 1 mg of antibodies (iNOS and IL-1a), incubated for 1 h at 4°C, and then incubated with prewashed EZview Red Protein A/G Affinity Gel beads (Millipore Sigma, P6486/E3403) overnight at 4°C. The bound antigens were eluted from the beads by boiling in the eluting buffer for 5 min. Eluted samples were fractionated by 8% SDS-PAGE gel and incubated with anti-ubiquitin antibody (Santa Cruz Biotechnology, sc-8017, 1:200).

Deubiquitination assay
BMMs were induced with LPS (500 ng/ml) or PBS control for 8 h. Then cells were washed with PBS and treated with 1 mM N-ethylmaleimide and harvested 8 h or 16 h after removing LPS. Cells were harvested for IP and RT-qPCR as described above.

ELISA
Secreted IL-1a was measured with ELISA. Briefly, secreted IL-1a in 50 ml of freshly collected supernatant was assessed using the mouse IL-1a/IL-1F1 quantikine ELISA kit (R&D System, MLA00) following the manufacturer's instructions.

Granzyme B activity
Whole cell lysate was harvested for granzyme B and caspase activity measurement. Briefly, 100 mg of whole cell lysate was added to caspase assay buffer (Enzo, BML-Kl111-0020) containing 50 mM of fluorogenic Ac-IETD-AFC fluorogenic peptide substrates (Enzo, ALX-260-110-M005) and incubated at 37°C for 1 h. Production of AFC was monitored in a spectro-fluorimeter with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. The results were presented as the -fold change versus the WT PBS-macs.

siRNA transfection to deplete Itch in WT macrophages
Murine Itch siRNA (4390771 assay ID s68414) and BLOCK-iT Alexa Fluor red fluorescent control siRNA (Thermo Fisher, 14750100) were purchased from Thermo Fisher Silencer Select website. The siRNA transfection was performed following the manufacturer's instructions. In brief, BMMs from WT mice at 60% confluence were transfected with 10 mM Itch siRNA and control siRNA diluted in lipofectamine RNAiMAX Reagent (Thermo Fisher, 13778030) for 2 days. Cells were then treated with LPS to induce LPS-macs. Whole cell lysates were harvested for Western blot analysis. The transfection efficacy in cells treated with control siRNA was 87.96% 6 6.29%. The Itch knock-down was determined by Western blotting.

Statistical analysis
Statistical analyses were performed using Prism7 software. Data are presented as mean 6 S.D. One-way analysis of variance followed by Tukey post hoc test was used to compare PBS-, LPS-, and IL-4-treated WT macrophages. Two-way analysis of variance followed by Sidak's post hoc test was used to compare WT or Itch2/2 macrophages with different treatments. p-values less than or equal to 0.05 were considered significant.

Data availability
The MS dataset is uploaded to PRIDE, accessible at PXD018743.