Perturbation of ubiquitin homeostasis promotes macrophage oxidative defenses

The innate immune system senses microbial ligands through pattern recognition and triggers downstream signaling cascades to promote inflammation and immune defense mechanisms. Emerging evidence suggests that cells also recognize alterations in host processes induced by infection as triggers. Protein ubiquitination and deubiquitination are post-translational modification processes essential for signaling and maintenance of cellular homeostasis, and infections can cause global alterations in the host ubiquitin proteome. Here we used a chemical biology approach to perturb the cellular ubiquitin proteome as a simplified model to study the impact of ubiquitin homeostasis alteration on macrophage function. Perturbation of ubiquitin homeostasis led to a rapid and transient burst of reactive oxygen species (ROS) that promoted macrophage inflammatory and anti-infective capacity. Moreover, we found that ROS production was dependent on the NOX2 phagocyte NADPH oxidase. Global alteration of the ubiquitin proteome also enhanced proinflammatory cytokine production in mice stimulated with a sub-lethal dose of LPS. Collectively, our findings suggest that major changes in the host ubiquitin landscape may be a potent signal to rapidly deploy innate immune defenses.


Fig. S1
: DUB inh used in this study. A) Structure of the DUB inhibitors DUB inh and DUB inh C6. Structure of the DUB inh -biotin compound. The arrow highlights the key cyano group. B) RAW264.7 whole cell lysates were incubated with DUB inh -Biotin or DCN-biotin before immunoprecipitation using streptavidin-coated beads. The retained proteins were revealed by silver stain. Data are representative of three independent experiments. Full-length gel is shown in Figure S7A.

Fig. S4: DUB inh induces ROS generation independently of UPR signaling.
A) RAW264.7 cells were treated for 0.5h with 3.5 µM DUB inh . Following treatment, cells were washed and subsequently incubated for the indicated period of time in fresh medium. As control, cells were treated with 10 µM thapsigargin or 10 µg/ml of tunicamycin for 4h. Whole cells lysates were used for immunoblotting against CHOP or actin as a loading control. B) RAW264.7 were treated as above. PCR was performed to amplify the Xbp1 mRNA and products were digested with the Pst1 endonuclease. The unspliced (U) form of Xbp1 contains a Pst1 cleavage site, which will generate two smaller fragments compared to the Xbp1 spliced (S) form. The percentage of Xbp1 splicing was calculated by band densitometry as follow: Xbp1(S) / [Xbp1(U)+Xbp1(S)]. Results for A) and B) are from two independent experiments. RAW264.7 cells were incubated for 0.5h with 50 µM 4µ8C C) or 50 µM GSK-PERK D). DUB inh (or equivalent volume of DMSO) was added on top at a final concentration of 3.5 µM for 0.5h. After incubation, the medium was removed, and cells were infected with L. monocytogenes (MOI 1) for 0.5h. Following infection, cells were washed and new medium containing 10 µg/ml of gentamicin was added. Intracellular bacteria were enumerated at 6h p.i. The data represent percent of intracellular L. monocytogenes growth compared to DMSO-only treated cells. Results were obtained from three independent experiments performed in triplicate E) RAW264.7 cells were incubated with 300 µM TUDCA or medium only for 1h. DUB inh (or equivalent volume of DMSO) was added on top at a final concentration of 3.5 µM for 0.5h before staining for ROS detection. The results from four experiments represent the percentage of cells stained for ROS (% ROS + cells). Significant differences were calculated using one-way ANOVA and Tukey's multiple comparison test on the unmodified data (NS, not significant, *** p < 0.001). Full-length immunoblots and gel are shown in Figure S8A and B respectively.

Fig. S5
: DUB inhibition induces ROS generation through NOX2 complex without affecting protein expression levels of gp91, p22 or p67. A) RAW264.7 cells or WT and gp91 phox-/y iBMDM incubated overnight with 100 ng/ml of LPS and INF-g were treated for 0.5h with 3.5 µM DUB inh . Following treatment, cells were washed and subsequently incubated for the indicated period of time in fresh medium. Whole cells lysates were used for immunoblotting against gp91 phox and GAPDH or b-actin as loading controls. Results are from 2 independent experiments. Full-length blots are presented in supplementary figure 8C. B) WT iBMDM incubated overnight with 100 ng/ml of LPS and INF-g were treated for 0.5h with 3.5 µM DUB inh . Following treatment, cells were washed and subsequently incubated for 0.5h. Whole cells lysates were used for immunoblotting against gp91 phox , p22 phox , p67 phox and GAPDH as loading controls. C) pBMDM isolated from WT, Nox -/-(NOX1 KO) or Nox4 -/-(NOX4 KO) mice and incubated overnight with 100 ng/ml of LPS and INF-g were treated for 0.5h with 3.5 µM DUB inh or equivalent volume of DMSO before staining for 0.5h with the ROS dye. Quantification of mean fluorescence intensity (MFI) from 2 independent experiments was calculated using FlowJo software. Full-length immunoblots are shown in Figure S8C and D.  Figure 1A. Lines highlighted by a star represent sample unrelated to this study.  Figure S1C. B) Full length immunoblots for results shown in Figure 1B. C) Full length immunoblots for results shown in Figure 4F.  Figure S4A. B) Full length gel for results shown in Fig. S4B. The top band (arrowhead) represents hybrid product between spliced and unspliced PCR fragments. C) Full length immunoblots for results shown in Figure S5A. D) Full length immunoblots for results shown in Figure S5B. Lines highlighted by a star represent sample unrelated to this study.
Cell viability. RAW264.7 cells were seeded at a density of 3.5 x 10 4 cells per well in a 96-well plate and allowed to adhere overnight. Cells were treated with increasing concentrations of EerI or DBeQ for 7.5h. LDH release assay (Cytotox 96; Promega) was performed according to the manufacturer's instructions.
Xbp1 splicing assay. RNA was extracted using the RNeasy Mini Kit (QIAGEN) from samples treated with 10 µg/ml of tunicamycin for 4h or with 3.5µM DUB inh or equivalent volume of DMSO for the indicated time. cDNA synthesis was performed using 1 µg of RNA and Xbp1 transcripts were amplified using following primers: forward 5´-GAACCAGGAGTTAAGAACACG-3´ and reverse 5´-AGGCAACAGTGTCAGAGTCC-3´. Amplification was done using using 30 cycles at 94 o C for 1 min, 60 o C for 1 min and 72 o C for 1 min. PCR products were further digested with the restriction enzyme PstI and loaded onto a 3% agarose gel to differentiate between the spliced and unspliced forms of Xbp1.

Methods for compound synthesis
All starting monomers were obtained from commercial suppliers and used without further purification. Routine 1 H NMR spectra were recorded at 400 MHz on a Varian 400, Varian 500, or Bruker 400 Avance instrument with chloroform-d or DMSO-d6 as solvent. Chemical shift values are recorded in d units (ppm). Mass spectra were recorded on a Micromass TofSpec-2E Matrix-Assisted, Laser-Desorption, Time-of-Flight Mass Spectrometer in a positive ESI mode (TOFES + ) unless otherwise noted. High resolution mass spectrometry (HRMS) analysis was performed on an Agilent Q-TOF system. Analytical HPLC was performed on an Agilent 1100 series instrument with an Agilent Zorbax Eclipse Plus C18 (4.6 mm × 75 mm, 3.5 μm particle size) column with the gradient 10% acetonitrile/water (1 min), 10−90% acetonitrile/water (6 min), and 90% acetonitrile/water (2 min) flow = 1 mL/min. Thin-layer chromatography (TLC) was performed on silica gel GHLF plates (250 µm) purchased from Analtech. Column chromatography was carried out in the flash mode utilizing silica gel (220−240 mesh) purchased from Silicycle. Extraction solutions were dried over anhydrous sodium sulfate or magnesium sulfate prior to concentration. Combustion analyses were carried out by Robertson Microlit Laboratories, Ledgewood, NZ.