Animal experiments were performed with male adult C57BL/6J mice (8–12-weeks old). Animals were maintained in specific pathogen-free animal facility. LysM^ΔCommd10 and Cx3cr1^ΔCommd10 mice were generated by crossing Lyz2^cre and Cx3cr1^cre (26) mice with Commd10^fl/fl mice, respectively, which were purchased from the EUCOMM consortium (strain EM: 05951) (C57BL/6J background). Experiments with LysM^ΔCommd10 and Cx3cr1^ΔCommd10 mice were performed on mice heterozygous for these genes.

Intestinal biopsies were obtained from 16 terminal ileums and colons of IBD patients with active disease according to pathological assessment of nearby biopsies and from 14 age and sex-matched healthy controls. Biopsies were immediately frozen in liquid nitrogen for further analyses. Peripheral blood (20 ml) was obtained from 15 IBD patients or healthy controls. PBMCs were enriched from whole blood by Ficoll density gradient media (Ficoll-PaqueTM PLUS, GE Healthcare). CD14⁺CD16⁻HLA-DR⁺ cells were sorted using FACS-Aria machine (BD Biosciences) and used immediately for RNA isolation.

Studies with human cells and tissues were carried out in accordance with the recommendations of Tel-Aviv Sourasky Medical Center Helsinki committee. All subjects gave written informed consent in accordance with the Declaration of Helsinki. All procedures involving human subjects were reviewed and approved by the Institutional Review Boards (Tel-Aviv Sourasky Medical Center IRB approval # TLV-0579-10). All mouse studies were carried out in accordance with the recommendations of Tel-Aviv Sourasky Medical Center ethical committee for animal studies. The protocols were approved by the local committee (# 16-6-13).

BMDM were prepared by flushing BM from the femur and tibia and culturing in RPMI medium containing FBS (10%), penicillin (100 IU/ml), streptomycin (100 μg/ml) and macrophage-colony stimulating factor (M-CSF, 20 ng/ml), at 37°C in 5% CO₂. Media was supplemented every 2–3 days. On day 6, 500,000 BMDM were seeded and subjected to LPS stimulation (Sigma-Aldrich, L2880 Lot #025M4040V) (100 ng/ml) for 3 h, or to Escherichia coli (ATCC 25922) at multiplicity of infection (MOI) = 1, with early log phase bacteria. 30 min after infection cells were washed and supplemented with gentamicin (100 ng/ml) to eliminate extracellular bacteria. At indicated times macrophages were washed twice with PBS^−/− (without Ca⁺⁺ and Mg⁺⁺), and collected for western blot analyses. Neutrophils were isolated from BM by magnetic separation using the neutrophil isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany cat# 130-092-332). Neutrophils were enriched to high purity (above 99%) and identified using flow cytometry as CD11b⁺Ly6G⁺ cells. About 2,000,000 neutrophils were seeded in RPMI containing 10% FBS, 1% L-Glutamine and 0.1% penicillin streptomycin, and incubated for 30 min in a 5% CO₂ at 37°C. Non-adherent cells were removed and neutrophils were subjected to LPS (100 ng/ml) for 3 h. ATP (InvivoGen, cat# Tlrl-atp1) was added in the last 30 min of the experiment.

Spleens were subjected to mechanical meshing and filtered through 200 μM wire mesh. The cell sediments were washed and pellet was lysed for erythrocytes by 2 min incubation with ACK buffer composed of 0.15 M NH₄Cl, and 0.01 M KHCO₃, and then washed again with PBS^−/− (without Ca²⁺ and Mg²⁺). Ly6C^hi monocytes were defined as CD45⁺CD11b⁺CD115⁺ Ly6C^hi and sorted to high purity (above 90%) using the SH800 sorter machine (Sony Biotechnology Inc). Cells were subjected to LPS as detailed above for BMDM.

Spleen and thymus were subjected to mechanical meshing into a sterile RPMI containing 10% FBS, 1% L-Glutamine and 0.1% penicillin streptomycin. Erythrocytes were lysed by incubation with ACK buffer for 2 min. Cells were plated and selective adherence to plastic was used to enrich non-adherent lymphocytes.

Mice at the same range of body mass were i.p. injected with LPS, 0.2 mg/mouse, in 100 μl saline. Mice were sacrificed at indicated time points.

Dextran sulfate sodium (DSS, MP Biomedicals, cat# 160110) was administered in drinking water (1.5%) for 4 and 7 days. Colitis severity was assessed using the murine colonoscopy system (IMAGE1 SPIES™ series, Karl Storz, Germany). Quantification of colitis severity was graded as previously described (27). Digitally recorded video files were processed with Windows Movie Maker software (Microsoft).

In the LPS-induced systemic inflammation experiments, some of the mice received an i.p. injection of 400 μl anti-mouse CCR2 mAb (clone MC-21)-conditioned media (30 μg Ab/ml), as previously described. Injections were started at 12 h prior to LPS challenge, and repeated every 24 h until sacrifice. In the DSS-induced colitis experiment, MC-21 was injected every 24 h starting at day 1 of DSS, as previously described (10).

Hepatic non-parenchymal cells were isolated from liver as previously described (4). In brief, mice were perfused with 10 ml of cold PBS^−/− and the liver was excised. Livers were cut into small fragments, incubated with shaking (37°C, 250 rpm for 45 min) with 5 ml digestion buffer [5% FCS, 0.5 mg/ml collagenase VIII (Sigma-Aldrich, Rehovot, Israel, C5138-500MG) in PBS^+/+ (with Ca²⁺ and Mg²⁺)]. Subsequently, livers were subjected to mechanical meshing and filtered through 200 μM wire mesh. This was followed by three cycles of washings with PBS^−/− at 40 g from which the supernatant was taken, omitting the parenchymal cell pellet. The supernatant was then centrifuge (400 g) and cell pellet was lysed for erythrocytes by 2 min incubation with ACK buffer (0.15 M NH₄C and 0.01 M KHCO₃).

Colonic lamina propria phagocytes were isolated as previously described (10). In brief, extra-intestinal fat tissue was carefully removed and colons were then flushed of their luminal content with cold PBS^−/−, opened longitudinally, and cut into 3–5 mm pieces. Epithelial cells and mucus were removed by 40 min incubation with 5 ml HBSS^−/− (without Ca²⁺ and Mg²⁺) containing 5% FBS, 2 mM EDTA, and 1 mM DTT (Sigma) at 37°C shaking at 250 rpm. Colon pieces were then incubated with shaking (37°C, 250 rpm for 45 min) with 5 ml digestion buffer [5% FCS, 1 mg/ml collagenase VIII (Sigma-Aldrich, Rehovot, Israel, C5138-500MG) in PBS^+/+]. The cell suspension was then filtered through 200 μM wire mesh and washed with PBS^−/−.

Total protein from BMDM, neutrophils, lymphocytes and mentioned tissues was extracted in ice cold RIPA buffer (C-9806S, Cell Signaling Tech. Beverly, Massachusetts) containing protease inhibitors (P8340, Sigma Aldrich St. Louis, Missouri). Proteins were detected by immunoblotting using standard techniques. Antibodies that were used: caspase-11 (sc-374615), GAPDH (sc-47724), β-ACTIN (sc-47778), Pol II (sc-9001), GCN5 (sc-20698) from Santa Cruz; caspase-1(AG-20B-0042) from adipogen; anti-COMMD10 antibody (GTX121488) from Genetex, pP65 (3033) and P65 (6956) from Cell Signaling. Blots were incubated with HRP-conjugated secondary antibodies, and subjected to chemiluminescent detection using the MicroChemi imaging system (DNR Bio-Imaging Systems, Israel). P65 subcellular fractionation was performed using NE-PER nuclear/cytoplasmic extraction kit (78835, Thermo scientific, Paisley, UK) per manufacturer's instructions. Equivalent protein amounts were loaded for both nuclear and cytoplasmic fractions.

Human blood CD14⁺ monocytes were isolated using the magnetic monocyte isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany cat# 130-091-153). Cells were cultured in RPMI medium containing FBS (10%), penicillin (100 IU/ml), streptomycin (100 μg/ml) and macrophage-colony stimulating factor (M-CSF, 20 ng/ml), at 37°C in 5% CO₂. Media was supplemented every 2–3 days. On day 6 cells were plated in 6-well plates (~5 × 10⁵ cells/well) for 18–24 h before transfection. Cells were transfected with 200 nM ON-TARGETplus SMARTpool siRNA targeting Commd10 or with non-targeting scrambled siRNA [Thermo Scientific, Dharmacon (Illkirch, France)] using HiPerFect transfectant reagent (Qiagen, Courtaboeuf, France) for 48 h, per manufacturer's instructions. Cells were then stimulated with 100 ng/ml LPS for 1 h.

Total RNA was extracted from tissues with TRIzol® reagent (Invitrogen, United States), and from BMDM or sorted Ly6C^hi monocytes using the RNeasy Mini Kit (QIAGEN, Germany). RNA was reverse transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, California). All PCR reactions were performed with SYBR Green PCR Master Mix kit (Applied Biosystems) and Applied Biosystems 7300 Real-Time PCR machine. Quantification of PCR signals of each sample was performed by the ΔCt method normalized to Gapdh housekeeping gene. For the list of gene specific primers see Table S1.

The following anti-mouse antibodies were used (dilutions are indicated): CD45 (clone 30-F11, 1:100), CD11b (clone M1/70, 1:300), Ly6C (clone HK1.4, 1:300), CD115 (clone AFS98, 1:100), CD64 (clone X54-5/7.1, 1:50), Ly6G (clone 1A8, 1:100), and Tim4 (clone RMT4-54, 1:50), MHCII (clone M4-114.15.2, 1:200), CX₃CR1 (clone SA011F11, 1:100), CD14 (clone M5E2, 1:100), CD16 (clone 3G8, 1:100) all purchased from BioLegend, San Diego, CA, USA. Anti-mouse F4/80 (clone A3-1, 1:50) was purchased from BIORAD. Cells were analyzed with BD FACSCanto™ II (BD Bioscience). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR, United States).

Mouse plasma and supernatants from BMDM, BM neutrophils or sorted Ly6C^hi monocytes were collected and kept in −80°C until assessed. The levels of the cytokines TNFα, IL-1β, and IFNγ were assessed with the DuoSet ELISA kit (R&D Systems). Plasma from mice at 4 h following LPS challenge was subjected to multiplex cytokine array using the mouse cytokine array Q1 (R&D Systems), and analyzed by Q-analyzer software for QAM-CYT-1.

Mice received FITC-conjugated dextran by gavage (Sigma-Aldrich, Cat# FD4, 160 mg/100 g of body mass) 20 h after LPS stimuli. Four hours later, plasma was collected and FITC fluorescence signal was analyzed using the Synergy 2 Multi- Mode Reader (BioTek Instruments Inc).

Tissues were weighed and briefly homogenized (Polytron PT-MR 2100, Kinematica AG) in 1 ml PBS^−/−. Appropriate dilutions were seeded on LB agar plates and incubated at 37°C for 24 h. The number of colonies was counted and CFU per gram-tissue mass was calculated.

Raw microarray data were downloaded from the Immunological Genome Project data Phase 2 (GSE37448 microarray datasets) using Partek GS 6.6 (http://www.partek.com/pgs). Blood Ly6C^hi monocytes (n = 3) and neutrophils (n = 4) were selected. Raw gene expression data were obtained for Commd10. The gene expressions of the adipocyte marker adiponectin (Adipoq) and of the hepatocyte and cholangiocyte marker Cytokeratin18 (Krt18) were also obtained to set background expression levels.

In most experiments, statistical differences between two groups were determined using the unpaired two tailed t-test with GraphPad. Survival data (Figure 2C) were analyzed by Logrank (Mental-cox method) and Gehan-Breslow-Wilcoxon tests using GraphPad, comparing the Commd10^fl/fl and LysM^ΔCommd10 groups. Significance was defined if p-value was <0.05 as following: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

The role of COMMD proteins in the regulation of myeloid cell function has been so far overlooked, in particular with respect to COMMD10, given the embryonic lethality of COMMD10-knockout mice. Therefore, we generated conditional COMMD10 knockout mice (Commd10^fl/fl) that allow specific targeting of COMMD10-deficiency to myeloid cells by crossing them with Lyz2^cre mice. The resulting LysM^ΔCommd10 mice exhibited normal birth rate and life expectancy (data not shown). Efficient COMMD10 protein-deficiency was evident in macrophages generated from the spleen and BM, but not in lymphoid cells extracted from the spleen and thymus (Figure 1A). Both splenic and bone marrow-derived macrophages (BMDM) exhibited specific reduction in Commd10 expression, while the expression of all other Commd genes was intact (Figure 1B). Therefore, the LysM^ΔCommd10 mice provided us with a model to study the immunoregulatory role of COMMD10 in myeloid cells. We assessed the effect of COMMD10-deficiency on LPS-induced innate immune responses in BMDM. LPS-stimulated BMDM from LysM^ΔCommd10 mice showed increased transcription of pro-inflammatory cytokines including TNFα (Tnf), IL-1β (Il1b) and the MX Dynamin Like GTPase 1 (Mx1) (Figure 1C). Supernatants from overnight cultures of LysM^ΔCommd10 BMDM contained higher levels of the secreted cytokines TNFα, IFNγ and IL-1β (Figure 1D). Therefore, COMMD10-deficiecny in Ly6C^hi monocyte-derived BMDM exhibit increased inflammatory cytokine production in response to LPS.

COMMD1, the prototype member of the COMMD family, was initially reported to inhibit NF-κB-mediated transcription (28). Further studies in human embryonic kidney cells genetically manipulated to overexpress distinct COMMD proteins revealed that COMMD10 is also capable of inhibiting TNF-mediated NF-κB activation (21). Yet, the physiological relevance of this finding to immune cells, specifically monocytes, remains elusive. In this respect, LPS-challenged COMMD10-deficient BMDM exhibited intensified and more persistent phosphorylation of P65 (Figure 1E) as well as increased translocation of P65 to the nucleus (Figure 1F). Increased levels of phosphorylated P65 (pP65) and IL-1β secretion were also evident in COMMD10-deficient BMDM infected with the Gram^neg bacteria Escherichia coli (E. coli) (Figures 1G,H,I). To further validate these findings in human CD14^hi monocytes, which are the equivalent of mouse Ly6C^hi monocytes, we utilized siRNA to silence Commd10 gene expression in M-CSF-differentiated CD14^hi monocyte-derived macrophages. Even partial reduction in COMMD10 expression was sufficient to induce greater activation of NF-κB in response to LPS, as manifested by increased nuclear translocation of P65 (Figure 1J). Therefore, these results establish a role for COMMD10 in suppressing NF-κB activation.

We next examined the response of LysM^ΔCommd10 mice to systemic LPS-induced systemic inflammation. Analysis of an array of plasma cytokines at 4 h following LPS challenge revealed higher levels of pro-inflammatory cytokines and chemokines including IFNγ, IL-6, IL-9, CCL5 (RANTES) and IL-1β in the LysM^ΔCommd10 vs. Commd10^fl/fl mice (Figure 2A). More direct analysis of TNFα levels further revealed its elevated expression in the plasma of LysM^ΔCommd10 mice (Figure 2B). This augmented cytokine storm witnessed in the LysM^ΔCommd10 mice may induce tissue damage and impair survival. Indeed, mortality rates of LysM^ΔCommd10 mice were significantly higher over a period of 72 h in comparison with Commd10^fl/fl control mice (p = 0.02; Figure 2C). These results suggest that COMMD10-deficiency induces a hyper-inflammatory state that impairs overall survival following acute endotoxic shock.

Inflammasome activation in macrophages is essential to ensure adequate host defense against invading microbes, yet it must be finely controlled to avoid overt tissue damage (14). While both blood monocytes and macrophages are prime producers of IL-1β, monocytes naturally express higher levels of activated caspase-1 allowing its maturation and release even following a single stimulus of TLR-2 or−4 (18). To determine whether Ly6C^hi monocytes were responsible for the excessive IL-1β production in the serum of LPS-challenged LysM^ΔCommd10 mice (Figure 2A), we induced their ablation by preemptive treatment with the anti-CCR2 MC-21 antibody (10, 29). Indeed, MC-21 treatment specifically depleted Ly6C^hi monocytes tissue infiltrates in the liver (Figure S1A) and colon (Figure S1B) of LPS-challenged mice, but was inert to tissue-resident macrophages or neutrophils in these compartments. In addition, we crossed Commd10^fl/fl with Cx3cr1^cre mice giving rise to Cx3cr1^ΔCommd10 mice. In these mice, cre-driven recombination (and the ensuing COMMD10-deficiency) is efficient in tissue-resident macrophages, significantly less in circulating CX₃CR1^loLy6C^hi monocytes and hardly affects CX₃CR1^neg neutrophils (26). Plasma IL-1β levels were profoundly elevated in LysM^ΔCommd10 vs. Commd10^fl/fl mice at 4 h following LPS challenge (Figure 3A). Strikingly, this elevation was ameliorated following Ly6C^hi monocyte ablation (Figure 3A). Moreover, plasma IL-1β levels were similar in both Commd10^fl/fl and Cx3cr1^ΔCommd10 mice (Figure 3A). These phenomena were mirrored in the transcription of Il1b in the liver (Figure 3B). The activation of caspases-1 and-11 can both drive IL-1β release (15, 16). In this respect, in comparison with Commd10^fl/fl mice, there was significantly higher expression of pro-caspase-11 as well as activation of both caspase-1 and-11 in the liver of LysM^ΔCommd10, but not Cx3cr1^ΔCommd10 mice, at 4 h following LPS challenge (Figures 3C,D). Strikingly, MC-21-mediated Ly6C^hi monocyte ablation completely abolished the activation of these caspases in the liver (Figures 3C,D). In alignment, flow cytometry analysis showed increased infiltration of Ly6C^hi monocytes, defined as CD11b⁺Ly6C^hiLy6G⁻, to the livers of LysM^ΔCommd10 vs. Commd10^fl/fl mice. In contrast, COMMD10-deficiency had no effect on the abundancy of resident CD11b^intF4/80⁺Tim4⁺ KCs or infiltrating CD11b⁺Ly6G⁺Ly6C^lo neutrophils (Figure 3E). Similarly, increased activation of caspases-1 and-11 was observed in LysM^ΔCommd10 spleen (Figure 3F) and colon (Figure 3G), and in both cases, it was ameliorated by MC-21-driven ablation of Ly6C^hi monocytes. Moreover, there was a trend for increased infiltration of Ly6C^hi monocytes to the LysM^ΔCommd10 colon with no clear difference in the abundance of CD11b⁺F4/80⁺CD64⁺ resident lpMF and Ly6G⁺ neutrophils (Figure 3H). To directly delineate the effect of COMMD10-deficiency on inflammasome activation in mature circulating Ly6C^hi monocytes, these cells were sorted to high purity from their splenic reservoir according to their simultaneous expression of CD45, CD11b, CD115 (Csf-1R) and Ly6C (data not shown). In the absence of COMMD10, Ly6C^hi monocytes exhibited a strong trend toward increased transcription of Il1b and significantly increased secretion of IL-1β in response to LPS (Figure 3I). Of note, in contrast to Ly6C^hi monocytes, there were reduced levels of IL-1β production in the supernatants of LPS and adenosine triphosphate (ATP)-stimulated COMMD10-deficient BM neutrophils (Figures S2A,B). Using published data sets available from the ImmGen Consortium database we could further show that Commd10 gene expression was near background levels in blood neutrophils and significantly higher in blood Ly6C^hi monocytes (Figure S3). Taken together, these findings implicate COMMD10 as a negative regulator of inflammasome activity in Ly6C^hi monocytes, but not in tissue-resident macrophages and neutrophils, during LPS-induced systemic inflammation.

Defective intestinal epithelial tight junction (TJ) barrier has been implicated as an important pathogenic factor in sepsis (30). It may lead to bacterial translocation from the intestinal lumen to the mesenteric nodes, liver and systemic circulation, and induce severe complications resulting in high mortality rates. Excess production of inflammatory cytokines, and specifically IL-1β, can damage TJ protein expression and function (31, 32). Therefore, we hypothesized that the increased mortality rates of LPS-challenged LysM^ΔCommd10 mice (Figure 2C) may be the result of impaired intestinal barrier function. Indeed, a significant breach in barrier function was evident in LysM^ΔCommd10 mice at 24 h following LPS challenge, as confirmed by markedly increased intestinal translocation of the orally administered fluorescent probe FITC-dextran (Figure 4A). Ly6C^hi monocyte depletion partially mitigated barrier leakage (Figure 4A). In agreement with the impaired barrier function, expression of colonic tight-junction claudin genes Cldn 1, 2 and 15 was decreased in the colons of LysM^ΔCommd10 mice at 48 h following LPS challenge (Figure 4B). Ly6C^hi monocyte depletion restored their expression to the level present in Commd10^fl/fl colons (Figure 4B). In alignment with their increased mortality (Figure 2A), bacterial counts were elevated in mesenteric lymph nodes (MLN), liver and spleen of LysM^ΔCommd10 mice (Figure 4C). Collectively, these findings mark an important role for COMMD10 in the protection of intestinal barrier function from Ly6C^hi monocyte-driven inflammation during sepsis.

The majority of colonic-resident lpMFs rely on constant replenishment by blood Ly6C^hi monocytes attracted by local tonic low-grade inflammatory stimuli (8, 9, 11, 12). Newly arriving Ly6C^hi blood monocytes are normally conditioned by IL-10 to acquire a non-inflammatory gene-expression profile, while differentiating into CX₃CR1^hi lpMFs (33, 34). However, this conditioning process fails when they enter the inflamed gut, and hence differentiate instead into pro-inflammatory CX₃CR1^lo effector cells that actively promote gut inflammation (10, 13). Interestingly, IL-1β was recently shown to be the key mediator driving intestinal inflammation in mice and patients with IL-10 receptor deficiency (35). Therefore, we next studied the effect of COMMD10-deficiency in myeloid cells on intestinal inflammation in an acute model of dextran sodium sulfate (DSS)-induced colitis. Remarkably, live colonoscopy assessment revealed significantly aggravated colitis in LysM^ΔCommd10 vs. Commd10^fl/fl control mice (Figure 5A; Movie S1). In the colon of Cx3cr1^cre mice, cre-mediated recombination is mainly targeted to resident lpMFs (34). Notably, the colitis score of Cx3cr1^ΔCommd10 mice was similar to that of Commd10^fl/fl control mice (Figure 5A; Movie S1), suggesting that COMMD10 does not play a critical anti-inflammatory role in CX₃CR1^hi lpMFs. The aggravated colitis in LysM^ΔCommd10 mice was also reflected in the reduced length of their colon in comparison with that of Commd10^fl/fl or Cx3cr1^ΔCommd10 (Figure 5B).

Colonic infiltrating Ly6C^hi monocytes become the dominant pro-inflammatory myeloid effector cells at day 4 of DSS-induced colitis (10). Therefore, we next investigated the effect of COMMD10-deficiency on Ly6C^hi monocyte inflammasome activity at this time point. Similarly to their response to LPS-induced systemic inflammation (Figure 3A), LysM^ΔCommd10 mice exhibited a prominent increase in plasma levels of IL-1β, which was completely ameliorated upon MC-21-governed Ly6C^hi monocyte ablation. Further emphasizing that Ly6C^hi monocytes are the main source for the augmented plasma IL-1β production, Cx3cr1^ΔCommd10 mice exhibited a significant reduction in plasma IL-1β, even in comparison to control Commd10^fl/fl mice. Thus, these results exclude a contribution of tissue-resident macrophages to the overall increase of plasma IL-1β observed in the LysM^ΔCommd10 mice (Figure 6A). At day 7, the augmented plasma IL-1β production in LysM^ΔCommd10 mice was still apparent (Figure 6B). Plasma TNFα levels were also elevated in the LysM^ΔCommd10 mice at day 4 and 7 (Figure 6C). We next examined whether generation of Ly6C^hi monocytes and neutrophils in the BM or their egress to the circulation were affected by the specific COMMD10-deficiencies in LysM^ΔCommd10 and Cx3cr1^ΔCommd10 mice. However, flow cytometry analysis of BM and blood of these transgenic mice at day 4 of DSS-induced colitis revealed no clear differences in their levels (Figures 6D,E). Hence, these results suggest that Ly6C^hi monocyte hyper-inflammasome activity, rather than availability in the circulation, is responsible for the increased plasma levels of IL-1β in LysM^ΔCommd10 mice. Surprisingly in the colon, there were significantly reduced fractions of Ly6C^hi monocytes and neutrophils in both LysM^ΔCommd10 and Cx3cr1^ΔCommd10 mice, with no clear difference in the abundance of colonic lpMFs (Figure 6F). This was further corroborated by showing reduced number per tissue mass of colonic Ly6C^hi monocytes, but not of lpMFs, in both LysM^ΔCommd10 and Cx3cr1^ΔCommd10 mice (Figure 6G). In agreement with this, the colonic expression of the genes encoding for the Ly6C^hi monocyte and neutrophil recruitment chemokines Ccl2, Cxcl1, and Cxcl2, respectively, was markedly reduced in both mice (Figure 6H). Finally, LysM^ΔCommd10 colons exhibited reduced local transcription of Il1b gene, which is in alignment with the declined accumulation of Ly6C^hi monocytes (Figure 6H). Therefore, these results suggest that circulating Ly6C^hi monocytes are responsible for the augmented systemic production of IL-1β.

The results above highlight an important negative immunoregulatory role for COMMD10 in tuning Ly6C^hi monocyte-driven inflammation during colitis (Figure 6). Therefore, downregulation of COMMD10 levels may be essential to license their inflammatory behavior. Indeed, in comparison to Ly6C^hi monocytes sorted from steady state splenic reservoirs, there was a profound decrease in the transcription of Commd10 in their Ly6C^hi monocyte counterparts sorted from splenic reservoirs and inflammatory colons at day 4 of DSS-induced colitis (Figure 7A). Furthermore, Commd10 gene expression was also profoundly reduced in the transition of Ly6C^hi monocytes from circulation (splenic reservoir) to colonic tissue in the context of colitis (Figure 7A). A functionally similar pro-inflammatory CD14^hi monocyte population has been identified within inflamed tissues of IBD patients (36, 37). Notably, there was also significant decline in the expression of COMMD10 gene (Figure 7B) and protein (Figure 7C) in circulating CD14^hi monocytes sorted from patients with active IBD vs. healthy subjects. This reduction in COMMD10 expression was further evident in colonic or ileal tissue biopsies extracted from IBD patients vs. healthy subjects at both gene (Figure 7D) and protein (Figure 7E) levels.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.