Genes and Immunity (2014) 15, 404–412 & 2014 Macmillan Publishers Limited All rights reserved 1466-4879/14 www.nature.com/gene

ORIGINAL ARTICLE Enhanced LPS-induced peritonitis in mice deficiency of cullin 4B in macrophages

M-H Hung, Y-R Jian, C-C Tsao, S-W Lin and Y-H Chuang

Cullin 4B (CUL4B), a member of the cullin protein family, is a scaffold protein of the CUL4B–RING–E3 ligase complex that ubiquitinates intracellular proteins.CUL4B’s targets include cell cycle-regulated proteins and DNA replication-related molecules. In this study, we generated myeloid-specific Cul4b-deficient mice (Cul4bf/y;LysM-CreKI/KI) to investigate the influence of Cul4b deficiency on innate immunity, especially on the function of macrophages. Our results show that an intraperitoneal injection of lipopolysaccharide (LPS) led to a significant decrease in body weights and increased leukocyte infiltrates with increased chemokines in the peritoneal cavity of Cul4bf/y;LysM-CreKI/KI mice. However, the proinflammatory cytokines, IL-6 and TNF-a did not increase in LPS-injected Cul4bf/y;LysM-CreKI/KI mice. Furthermore, bone marrow-derived macrophages from Cul4bf/y;LysM-CreKI/KI mice secreted higher levels of chemokines but lower levels of TNF-a and IL-6 upon LPS stimulation. Of note, increased proliferation of Cul4b-deficient macrophages was also observed. These results show that myeloid-specific Cul4b deficiency worsens LPS-induced peritonitis. In addition, Cul4b deficiency leads to enhanced DNA replication and proliferation, increased production of chemokines but a decreased production of proinflammatory cytokines of macrophages. Our data highlight a new role of cullin family, CUL4B, in the immune system.

Genes and Immunity (2014) 15, 404–412; doi:10.1038/gene.2014.32; published online 5 June 2014

INTRODUCTION In addition, short hairpin RNA knockdown of Cul4b results Cullins are molecular scaffold proteins in cullin–RING-based E3 in a significant reduction of TNF-a protein and its mRNA ubiquitin ligases (CRLs) with crucial roles in posttranslational in lipopolysaccharide (LPS)-stimulated mouse macrophage ubiquitin modifications of cellular proteins. Cullins are evolutio- RAW264.7 cells.12 These results imply that CUL4B has a role in narily conserved from yeast to mammals. To date, eight the development and function of monocytes/macrophages. mammalian cullins (cullin 1, 2, 3, 4A, 4B, 5, 7 and 9) have been Innate immunity is the first line of defense against invasion by identified.1 Cullin-mediated ubiquitination requires substrate- pathogens. They are not specific to a particular pathogen in the recruiting receptors that are typically joined to the CRL complex way that adaptive immune responses are. Innate immune cells by a linker protein.2 The C-terminal domain of cullin 4B (CUL4B) recognize pathogen-associated molecular patterns of pathogens binds to ROC1 or ROC2 (ring of cullins). This recruits and by their pattern-recognition receptors. They then secrete anti- allosterically activates an E2 enzyme that transfers ubiquitin to microbial peptides, cytokines or chemokines to kill the pathogens the substrate, and assembles this complex into a CUL4B–RING E3 and to recruit more immune cells to the local site.13,14 ubiquitin ligase (CRL4B). The N-terminal domain of CUL4B Macrophages have a critical role as regulators of the immune interacts with DDB1 (damaged DNA binding protein 1), which response in both nonspecific innate immunity and acts as a linker protein linking CRL4B to a family of DWD specific adaptive immunity. They originate from bone marrow (DDB1-binding WD40) proteins that function as substrate stem cells and proliferate and differentiate in response to receptors and recruit different substrates to the CUL4B–ROC1–E2 growth factors, particularly macrophage colony-stimulating factor catalytic core. CRL4B has been shown to cause ubiquitylation and (M-CSF).15 Macrophages migrate through the blood as blood subsequent 26S proteasome-mediated degradation of several monocytes, enter tissues and form tissue macrophages. When proteins.3,4 CUL4B is involved in a diverse array of functions such pathogens invade, macrophages initiate an inflammatory as DNA replication,5 cell cycle control,6,7 histone modification8 and response against the pathogens that include: ingesting the breakdown of steroid–hormone receptors.9 pathogens, secreting cytokines, chemokines and reactive oxygen Several mutations including truncating, splice-site and missense species, presenting antigens and stimulating other immune cells. variants at conserved amino acids in the Cul4b gene on Xq24 were They also conclude the inflammatory response by clearing identified in patients with X-linked intellectual disability syndrome. immune cells and debris.15,16 In addition, macrophages engulf Patients lacking CUL4B exhibit structural or functional abnormal- and kill microorganisms and secrete factors that afford them key ities in multiple systems, notably in the central nervous system, roles in tissue repair and remodeling.15 skeleton and hematopoiesis. Frequent increases in the number of To gain insight into the role of CUL4B in innate immunity, and monocytes in the peripheral blood are observed in patients especially on the function of macrophages, we generated with X-linked intellectual disability syndrome, whereas the myeloid-specific Cul4b-deficient mice (Cul4bf/y;LysM-CreKI/KI) and total white blood cell counts are in the normal range.10,11 examined the acute inflammatory response induced by LPS

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan. Correspondence: Associate Professor Y-H Chuang or Professor S-W Lin, Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, No. 1, Chang-Te Street, Taipei 10016, Taiwan. E-mail: [email protected] or [email protected] Received 28 January 2014; revised 24 April 2014; accepted 24 April 2014; published online 5 June 2014 Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 405

Figure 1. No obvious abnormal in Cul4bf/y;LysM-CreKI/KI mice. Cul4bf/y;LysM-CreKI/KI mice were generated by breeding of Cul4bf/y mice with LysM-CreKI/KI mice. (a, b) PCR analysis of tail genomic DNA from Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice. The PCR product for the Cul4b floxed allele is 494 bp (a) cre recombinase knock-in is 700 bp and wild-type allele is 350 bp (b). (c) Genotyping of bone marrow-derived macrophages and splenocytes from Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were detected by PCR. The PCR products for the Cul4b floxed allele is 494 bp and deleted allele is 569 bp. Mf, macrophage; Lym, lymphocyte. (d) The expression of Cul4b in bone marrow-derived macrophages was examined by RT-PCR. (e) The expression of CUL4B in bone marrow-derived macrophages was examined by western blot. (f) The body length and body weight of Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were measured. (g) The frequencies and absolute number of subpopulations of peripheral white blood cells (WBCs) of Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice. (h) Quantitative analyses were done to determine the number of red blood cells (RBCs), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and red blood cell volume distribution width (RDW) values and the number of platelets (PLT) in Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice. n ¼ 12–13 mice per group. stimulation and the functions of Cul4b-deficient macrophages CUL4B in the innate immune response and the function of were assayed. Our results demonstrate that myeloid-specific Cul4b macrophages. deficiency enhances LPS-induced acute peritonitis by upregulat- ing the recruiting of leukocytes including macrophages, neutro- phils and lymphocytes. In macrophages, Cul4b deficiency leads to RESULTS an enhanced DNA replication and cell proliferation, increased Successful deletion of Cul4b in macrophages production of chemokines and a decreased production of To delete the Cul4b gene in macrophages, we bred Cul4bf/y mice proinflammatory cytokines. Our data highlight a new role for to LysM-Cre knock-in mice expressing Cre recombinase under the

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 404 – 412 Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 406

Figure 2. Increased leukocyte infiltration in LPS-induced peritonitis of Cul4bf/y;LysM-CreKI/KI mice. Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were intraperitoneally injected with 1 mg kg À 1 LPS. Peritoneal cavities were washed at 24, 48 and 72 h, and lavage fluids were collected. (a) The percentage of body weight change of mice was analyzed. (b) The numbers of infiltrating leukocytes in the peritoneum were counted. (c) The numbers of macrophages, granulocytes and lymphocytes. (d) Serum levels of IL-6 and TNF-a were measured at the indicated time points. (e) The levels of IL-6 and TNF-a in the peritoneal lavage fluid were determined by enzyme-linked immunosorbent assay. (f) The infiltrated leukocytes were collected for total RNA isolation at 24 h, and quantitative RT-PCR of IL-6 and TNF-a was performed. n ¼ 4–8 mice per group. *Po0.05; **Po0.01.

control of a lysozyme M promoter. This was done to restrict Cul4b fragments of the predicted 494 bp and Cre recombinase knock-in ablation to myeloid cells and avoid the perinatal death associated of 700 bp in Cul4bf/y;LysM-CreKI/KI mice (Figures 1a and b). We then with whole-animal Cul4b ablation in mice.17,18 Genotyping of tail genotyped the macrophage genomic DNA from Cul4bf/y; genomic DNA by PCR confirmed the Cul4b floxed allele with DNA LysM-CreKI/KI mice to confirm the deletion of the Cul4b gene in

Genes and Immunity (2014) 404 – 412 & 2014 Macmillan Publishers Limited Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 407 (Figure 1g). The numbers of red blood cells and platelets and hemoglobin, hematocrit, mean corpuscular volume, red cell distribution width of Cul4bf/y;LysM-CreKI/KI mice were no different from those of control Cul4bf/y mice (Figure 1h).

Increased leukocyte infiltration in LPS-induced peritonitis of Cul4bf/y;LysM-CreKI/KI mice The immune response to microbial infections must be tightly controlled to guarantee pathogen elimination while preventing tissue damage by an uncontrolled inflammation. Macrophages have been shown to be activated and to secrete a variety of inflammatory cytokines and mediators in response to LPS, an endotoxin derived from Gram-negative bacteria. Intraperitoneal injections of LPS induce peritonitis with an influx of leukocytes and cytokines.15 We analyzed the inflammatory responses of Cul4bf/y;LysM-CreKI/KI mice in mild LPS peritonitis. LPS (1 mg kg À 1) was intraperitoneally injected to Cul4bf/y;LysM-CreKI/KI and Cul4bf/y mice. We found that the body weight of both groups dropped during the first 2 days after the LPS injection. However, unlike Cul4bf/y mice, Cul4bf/y;LysM-CreKI/KI mice were weaker, inactive and kept losing body weight at 72 h after LPS injection (Figure 2a). Peritoneal lavage fluid cells were significantly increased after LPS injection in Cul4bf/y;LysM-CreKI/KI mice compared with Cul4bf/y mice (Figure 2b). At 24 h after LPS injection, the numbers of early- infiltrated granulocytes were significantly higher in Cul4bf/y; LysM-CreKI/KI mice than in Cul4bf/y mice. A significant increase in macrophages and neutrophils were noted in Cul4bf/y;LysM-CreKI/KI Figure 3. Chemokine profile in LPS stimulation of Cul4bf/y and Cul4bf/y; mice 48 h after LPS stimulation. Surprisingly, the number of LysM-CreKI/KI mice. The levels of chemokines in the peritoneal lavage lymphocytes also increased in LPS-injected Cul4bf/y;LysM-CreKI/KI fluid at 4 h after LPS injection of Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice mice (Figure 2c). were determined by a dot blot immunoassay. (a) Representative dot However, there was no difference in the serum levels of TNF-a plots of chemokine profile. (b) List of chemokines in the array. Each and IL-6 in Cul4bf/y;LysM-CreKI/KI and Cul4bf/y mice after LPS chemokine is represented as duplicate spots in the array. RS: injection (Figure 2d). IL-6 production in peritoneal lavage fluid reference spot, used as positive control of this assay. (c) The relative f/y KI/KI f/y KI/KI after LPS injection was not different between Cul4b ;LysM-Cre expression of chemokines in LPS-injected Cul4b ;LysM-Cre mice to f/y Cul4bf/y mice were analyzed. n ¼ 3 independent experiments. and Cul4b mice, whereas TNF-a in peritoneal lavage fluid was undetectable in both groups (Figure 2e). Of note, by using quantitative RT-PCR, we found that TNF-a and IL-6 mRNA f/y macrophages. Bone marrow-derived macrophages from Cul4b ; expression levels in cells collected from peritoneal lavage fluid KI/KI f/y LysM-Cre and Cul4b mice were collected and the macrophage of Cul4bf/y;LysM-CreKI/KI mice were significantly lower than those of genomic DNA was extracted and subjected to PCR analysis. Cul4bf/y mice (Figure 2f), suggesting a reduced TNF-a and IL-6 As shown in Figure 1c, Cre-mediated deletion resulted in the production per infiltrating leukocyte in LPS-injected Cul4bf/y; f/y appearance of a 569-bp PCR fragment in macrophages of Cul4b ; LysM-CreKI/KI mice. LysM-CreKI/KI mice, whereas a 494-bp PCR fragment of the non- f/y deleted gene was seen in macrophages of Cul4b mice. The Increased chemokine production in peritoneal lavage of deletion efficiency was 90–95% in bone marrow-derived macro- LPS-induced peritonitis of Cul4bf/y;LysM-CreKI/KI mice phages. In addition, the 494-bp PCR fragment of the non-deleted To see whether Cul4bf/y;LysM-CreKI/KI mice injected with LPS gene were observed in splenocytes of both Cul4bf/y;LysM-CreKI/KI secrete more chemokines to attract more polymorphonuclear and Cul4bf/y mice, suggesting that the deletion of Cul4b in Cul4bf/y; leukocytes, inflammatory macrophages and lymphocytes to the LysM-CreKI/KI mice was macrophage specific (Figure 1c). We peritoneal cavity, we examined the peritoneal lavage fluid’s then measured CUL4B expression in macrophages of Cul4bf/y; chemokine expression of LPS-injected mice. As shown in Figures LysM-CreKI/KI and Cul4bf/y mice. By reverse transcription PCR 3a and b, CCL6, chemerin, CCL11, IL-16, CXCL10, CCL2, CXCL-1, (RT-PCR) and western blot assay, we found that the expression CXCL5, CCL8, CCL12, CCL9/10, CCL5 and CXCL12 expression levels of cul4b mRNA and protein in bone marrow-derived macrophages were high in LPS-injected mice. CCL21, CXCL13, CCL27, CXCL16, from Cul4bf/y mice was seen but the cul4b mRNA and protein CCL22, CXCL9 and CXCL2 expression levels were low in LPS- expression was almost undetectable in macrophages from injected mice. Increased expression of chemokines was noted Cul4bf/y;LysM-CreKI/KI mice. Quantitative analysis revealed that in Cul4bf/y;LysM-CreKI/KI mice as compared with Cul4bf/y mice 0–6% of CUL4B expression was shown in macrophages from (Figure 3c). Cul4bf/y;LysM-CreKI/KI mice as compared with that from Cul4bf/y mice (Figures 1d and e). Increased proliferation of Cul4B-deficient macrophages To investigate the role of CUL4B in the function of macrophages, f/y KI/KI Physical characteristics of Cul4b ;LysM-Cre mice we cultured bone marrow-derived macrophages, which are a The Cul4bf/y;LysM-CreKI/KI mice had a normal appearance with homogeneous population of primary and quiescent cells, and average body lengths and body weights comparable to those in tested the function of macrophages from Cul4bf/y;LysM-CreKI/KI Cul4bf/y mice (Figure 1f). Moreover, peripheral white blood cell mice. After 7 days of culturing, the cell shape and morphology counts and subpopulations including neutrophils, basophils, of the differentiated macrophages was comparable in Cul4bf/y; eosinophils, lymphocytes and monocytes in Cul4bf/y;LysM-CreKI/KI LysM-CreKI/KI and Cul4bf/y mice (data not shown). However, the mice were unchanged when compared with Cul4bf/y mice total differentiation efficiency was different. The number of

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 404 – 412 Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 408

Figure 4. Increased proliferation in Cul4b-deficient macrophages. (a) Bone marrow cells (1 Â 107) from Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were cultured in 20% L929-conditioned medium for 7 days and bone marrow-derived macrophages were counted. (b, c) Proliferation of macrophages stimulated with/without M-CSF was detected by [3H]-thymidine incorporation assay as shown in counts per minute (c.p.m.) (b) and stimulation index (c). Stimulation index was calculated by dividing the mean c.p.m. of stimulated wells by the mean c.p.m. of nonstimulated wells. n ¼ 4–9 mice per group. *Po0.05; **Po0.01.

macrophages in Cul4bf/y;LysM-CreKI/KI mice significantly increased (TLR4) on the Cul4B-deficient and control macrophages was not as compared with the control mice (Figure 4a). To further different (Figure 6b), suggesting that the decreased cytokine investigate whether Cul4B deficiency led to the increased production of Cul4B-deficient macrophages on LPS stimulation is proliferation of macrophages, we measured the proliferation of not due to the low expression of the LPS receptor. We further Cul4B-deficient macrophages and compared it with that of examined the chemokine production of macrophages stimulated macrophages from Cul4bf/y mice. [3H]-Thymidine incorporation with LPS. LPS-stimulated bone marrow-derived macrophages showed that Cul4B-deficient macrophages had a significantly secreted CCL6, CCL27, CXCL16, IL-6, CXCL10, CCL2, CXCL1, CCL8, higher proliferation rate without any stimulation compared with CCL12, CCL3/CCL4, CCL9/10, CXCL2, CCL5 and CXCL12. CUL4B- that of controls. The increased proliferation rate was also found in deficient macrophages secreted higher levels of CCL6, CCL27, Cul4B-deficient macrophages stimulated with M-CSF, which is a CXCL16, CCL2, CXCL1, CCL8, CCL12, CCL3/CCL4, CCL9/10, CXCL2, cytokine essential for both the proliferation and differentiation of CCL5 and CXCL12 than control macrophages, and same level of monocytes/macrophages (Figure 4b). In fact, Cul4B-deficient CXCL10 as control macrophages (Figures 6c and d). Taken macrophages had an increased-fold (stimulation index) of together, LPS-stimulated CUL4B-deficient macrophages secrete proliferation on stimulation with M-CSF compared with that of more chemokines but less proinflammatory cytokines than control control macrophages (Figure 4c). To confirm whether the increase macrophages. in [3H]-thymidine incorporation observed above was due to DNA synthesis rather than DNA repair, macrophages under M-CSF No difference between the ability of phagocytosis of Cul4B- stimulation were labeled with propidium iodide (PI) and the DNA deficient macrophages as compared with control macrophages was analyzed by flow cytometry. As showed in Figure 5a, an increased percentage of cells in the S phase were seen in Cul4B- The phagocytosis and destruction of pyogenic bacteria by macrophages is an important host defense mechanism used to deficient macrophages in the steady state. In addition, 20 h of 19 serum starvation increased the proportion of cells in the S phase limit infection. We assayed the phagocytic ability of Cul4B- at 12 and 24 h of culturing with or without stimulation (Figures 5b deficient and control macrophages by flow cytometry of and c). green fluorescent protein-bearing Escherichia coli uptake by macrophages. As shown in Figure 7, the phagocytic ability of macrophages from Cul4bf/y mice increased with time from 0.5 to Decreased production of proinflammatory cytokines of 2 h but decreased at 4 h because of the destruction of bacteria by Cul4B-deficient macrophages macrophages. The same result were seen with Cul4B-deficient f/y f/y macrophages, suggesting that macrophages from Cul4b ;LysM- We stimulated bone marrow-derived macrophages from Cul4b ; KI/KI LysMCreKI/KI and Cul4bf/y mice with LPS and detected the TNF-a Cre mice had the same ability to kill bacteria as the controls. and IL-6 production in macrophages. Similar to the results of the in vivo assay, Figure 2f shows that Cul4B-deficient macrophages have significantly lower levels of TNF-a and IL-6 production on DISCUSSION stimulation with LPS compared with that of control macrophages Patients with CUL4B deficiency frequently have increased numbers (Figure 6a). However, the expression level of Toll-like receptor 4 of monocytes in their peripheral blood.10,11 In addition, CUL4B

Genes and Immunity (2014) 404 – 412 & 2014 Macmillan Publishers Limited Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 409 LPS-stimulated Cul4b-deficient macrophages secrete higher levels of chemokines that may attract polymorphonuclear leukocytes, macrophages and different subsets of lymphocytes than control macrophages. In addition, an increased proliferative response of bone marrow-derived macrophages from Cul4bf/y;LysM-CreKI/KI mice was found in vitro. Taken together, our results demonstrate that a Cul4b deficiency enhances LPS-induced peritonitis and that Cul4b regulates cell cycle and cytokine and chemokine production in macrophages. Resident macrophages have a fundamental role as immune sentinels, initiating inflammatory responses, clearing inflammatory debris and restoring homeostatic tissue environments. After recognizing the initial microbial challenge, resident macrophages drive the influx of inflammatory leukocytes, classically neutrophils but also monocytes, as a source of inflammatory macro- phages.20,21 To study the effect of Cul4b deficiency in the immune responses of macrophages to bacterial infection, we used the LPS-induced peritonitis model to mimic a Gram-negative bacterial infection in mice. Our results demonstrate that after the LPS injection, Cul4bf/y;LysM-CreKI/KI mice have an enhanced inflammation, suggesting that Cul4b is involved in the macrophage’s immune response to infection. In fact, many viruses, including members of the paramyxovirus, herpesvirus, lentivirus and hepadnavirus families, are known to disrupt CRL4 ubiquintylation pathway to evade innate cellular antiviral mechanisms and otherwise benefit viral propagation.22 For example, viruses from the paramyxovirus family, including simian virus 5, mumps virus and human parainfluenza virus type 2, use CUL4–DDB1 to degrade signal transducer and activator of transcription proteins that respond to interferon signaling and initiate cellular antiviral responses.23,24 In this study, we found an increase in macrophage infiltration in Cul4bf/y;LysM-CreKI/KI mice injected with LPS and an increased proliferation of Cul4b-deficient macrophages stimulated with M-CSF. Previous studies have shown that macrophages proliferate during newborn development, adulthood and acute inflammation in young adult mice.25–27 During an acute inflammatory response, when substantial numbers of inflammatory macrophages are recruited from the circulation, tissue-resident macrophages survive and then undergo a transient and intense proliferative Figure 5. Increased S phase of cell cycle in Cul4b-deficient burst in situ to repopulate in the tissue.25–27 It is possible that the macrophages. Bone marrow-derived macrophages from Cul4bf/y increased macrophage infiltrate seen in Cul4bf/y;LysM-CreKI/KI mice f/y KI/KI and Cul4b ;LysM-Cre mice were assayed the cell cycle by staining injected with LPS is due to an increased macrophage in situ with PI. (a) Cell cycle distribution of macrophages without proliferation and an increase in peripheral monocyte recruitment synchronization and stimulation. (b) Cell cycle distribution of to the peritoneal cavity by chemoattractants.15 macrophages with synchronization by serum starvation but without In this study, we found that LPS-stimulated macrophages from stimulation was assayed. (c) Cell cycle distribution of macrophages f/y KI/KI with synchronization by serum starvation and stimulation with Cul4b ;LysM-Cre mice secrete less IL-6 and TNF-a as compared M-CSF was assayed. n ¼ 4 mice per group. *Po0.05. with control macrophages. This is consistent with a previously published study demonstrating that short hairpin RNA knock- downs of CUL4B result in a significant reduction of TNF-a protein knockdown macrophages secrete lower level of TNF-a when and mRNA in LPS-stimulated mouse macrophage RAW264.7 stimulated with LPS.12 These results indicate that CUL4B affects cells.12 They also demonstrated that CUL4B acts as a licensing the development, proliferation and function of monocytes/ factor for loading of tristetraprolin (TTP)-containing TNF-a macrophages. In this study, we investigated the role of Cul4b in messenger ribonucleoproteins on to the polysomes and that innate immunity, especially on the function of macrophages from reduced CUL4B levels shunt the TNF-a message from the myeloid-specific Cul4b-deficient mice (Cul4bf/y;LysM-CreKI/KI). polysomes into the mRNA decay pathways.12 TTP is an ARE- Our results demonstrate that Cul4bf/y;LysM-CreKI/KI mice have binding protein of the CCCH zinc finger family that regulates the significantly reduced body weight and increased infiltrated stability of a number of cytokine and chemokine messages immune cells including granulocytes, macrophage and including TNF-a and also GM-CSF, IL-6, IL-10, CCL2, CCL3, CXCL1, lymphocytes with increased chemokine production after an in addition to many other inflammatory mediators.28–31 Hence, intraperitoneal LPS injection. Although we have seen a greater we suppose that the decreased production of both TNF-a and immune cell infiltration, the production of proinflammatory IL-6 in LPS-stimulated Cul4b-deficient macrophages is associated cytokines in the peritoneal lavage fluid did not increase in with TTP regulation. However, in our LPS-stimulated Cul4b- Cul4bf/y;LysM-CreKI/KI mice compared with control Cul4bf/y mice. deficient macrophages, other TTP-regulated chemokines, such as Meanwhile, the in vitro stimulated bone marrow-derived CCL3 and CXCL1, did not decrease but instead increased, macrophages from Cul4bf/y;LysM-CreKI/KI mice with LPS demon- suggesting that additional regulatory mechanism between strate that macrophages from Cul4bf/y;LysM-CreKI/KI mice secrete these cytokines and chemokines operate in Cul4b-deficient less IL-6 and TNF-a compared with control mice. However, macrophages.

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 404 – 412 Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 410

Figure 6. Decreased production of TNF-a and IL-6 but increased production of chemokines in Cul4b-deficient macrophages. (a) Bone marrow- derived macrophages from Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were stimulated with LPS for 4 h and proinflammatory cytokines TNF-a and IL-6 were detected by enzyme-linked immunosorbent assay. n ¼ 8 mice per group. *Po0.05. (b) TLR4 expression on resting bone marrow- derived macrophages from Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were determined by flow cytometry. The quantity of surface molecules per cell is indicated by the mean fluorescence intensity. (c, d) Bone marrow-derived macrophages from Cul4bf/y and Cul4bf/y;LysM-CreKI/KI mice were stimulated with LPS for 4 h and culture supernatants chemokines were determined by a dot blot immunoassay. (c) Representative dot plots of chemokine profile and the list of chemokines in the array. Each chemokine is represented as duplicate spots in the array. RS: reference spot, used as positive control of this assay. (d) The relative expression of chemokine in Cul4b-deficient macrophages was analyzed. n ¼ 3 independent experiments.

Depending on the microenvironment, macrophages can and CXCL16, were increased in M1 macrophages. Therefore, we acquire distinct functional phenotypes, referred to as classically suggest that Cul4b does not contribute to the M1 macrophage activated, proinflammatory macrophages (M1) and alternatively polarization but it is important in the function of M1 macrophages. activated, anti-inflammatory macrophages (M2). In the presence of Patients with CUL4B deficiency have frequent elevated bacterial products such as LPS, macrophages are polarized to the numbers of monocytes in the peripheral blood.10,11 However, M1 phenotype and produce high levels of cytokines TNF-a, IL-6, in our myeloid-specific Cul4b-deficient mice, peripheral blood IL-1b, IL-12, IL-23 and chemokines CXCL9, CXCL10, CCL2, CCL3, monocytes did not increase. In addition, Cul4bf/y;Sox2-Cre mice, CCL4, CXCL8, CXCL16, and increased levels of reactive oxygen a Cul4b-deficient model for the nonsyndromic model of X-linked species that aid in the clearance of invading pathogens.32,33 By intellectual disability syndrome, also does not have increased contrast, M2 macrophages, expressing the chemokines CCL17, peripheral blood monocytes.34 This discrepancy may be due to CCL22 and CCL24, tune inflammatory responses, scavenge debris the natural differences in the frequency of leukocyte subsets and promote angiogenesis, tissue remodeling and repair.33 In our between humans and mice.35 There was relatively low frequency LPS-stimulated Cul4b-deficient macrophages, some cytokines/ (in our data, 0.086–1.490%) of monocytes in the peripheral white chemokines IL-6 and TNF-a were decreased while other blood cells of mice. However, we did find an increased pro- cytokines/chemokines, such as CXCL9, CXCL10, CCL2, CCL3/CCL4 liferation of bone marrow-derived macrophages in Cul4b-deficient

Genes and Immunity (2014) 404 – 412 & 2014 Macmillan Publishers Limited Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 411 Committee (IACUC) of National Taiwan University College of Medicine and College of Public Health.

Complete blood count Peripheral blood was sampled by cheek blood, and a complete blood count was performed on an automated hematology analyzer (Cell-Dyn 3700, Abbott, Abbott Park, IL, USA).

Bone marrow-derived macrophages Bone marrow was isolated from femurs and tibias of Cul4bf/y;LysM-CreKI/KI and Cul4bf/y mice. Bone marrow cells were incubated with 20% L929- conditioned medium for 7 days to allow differentiation and maturation of macrophages. The purity of macrophages stained with anti-Ly6G and anti-F4/80 antibodies and determined by flow cytometry (Ly6G À F4/80 þ ) and cell morphology was X99%. Figure 7. No difference in the phagocytic ability of Cul4b-deficient and control macrophages. Phagocytic ability of macrophages was detected by flow cytometry of green fluorescent protein-bearing LPS-induced peritonitis E. coli uptake by macrophages. Mice were intraperitoneally injected with LPS (1 mg kg À 1, LPS of E. coli serotype 0111:B4, Sigma-Aldrich, St Louis, MO, USA). Sera were collected at various times after LPS administration (0, 4, 24 and 48 h) for detection of mice. Therefore, we suppose that the increased frequency and proinflammatory cytokines IL-6 and TNF-a. At selected time points, mice numbers of monocytes in the peripheral blood of patients with were euthanized and peritoneal cavities were lavaged with 3 ml of CUL4B deficiency are due to an increased proliferation of phosphate-buffered saline containing 0.02% EDTA. Differential cell counts monocytes. Additional studies are needed to confirm this. were performed by counting at least 400 cells on cytocentrifuged Our results showed that Cul4bf/y;LysM-CreKI/KI mice injected with preparations (Cytospin 3, Shandon, Pittsburgh, PA, USA) stained with Liu’s LPS secrete more chemokines, CCL6, chemerin, CCL11, IL-16, stain and then differentiating them by standard morphological criteria. The 1 CXCL10, CCL2, CXCL-1, CXCL5, CCL8, CCL12, CCL9/10, CCL5 and lavage fluids were stored at À 20 C. Cytokines were evaluated by enzyme- linked immunosorbent assay and chemokines were determined by CXCL12, to attract more polymorphonuclear leukocytes, inflam- Dot-blot-based mouse chemokine antibody array (Mouse Chemokine matory macrophages and lymphocytes to the peritoneal cavity. Array Kit) as recommended by the manufacturer (R&D Systems, CCL2, CCL5, CCL6, CCL8, CCL9/10, CCL11, CXCL10 and CXCL12 Minneapolis, MN, USA). Cell pellets were then prepared for RNA extraction have been reported to attract lymphocytes such as CD4 and CD8 T and quantitative reverse transcription RT-PCR analysis as described below. cells, Th1 and Th2 T cells, B cells and NK cells.33,36,37 The significantly increased lymphocytes were found even at 24 h after RNA isolation and quantitative RT-PCR LPS injection in Cul4bf/y;LysM-CreKI/KI mice, suggesting that those To assess the expression of TNF-a and IL-6, total RNA was isolated using recruited lymphocytes might be innate immune cells, such as NK TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) in cells, innate lymphoid cells, gd T cells, NKT cells and B1 cells. accordance with the user’s manual. For RT-PCR, total RNA was reverse However, the antigen presenting ability of cul4b-deficient transcribed into cDNA and PCR was performed on a 7500 Fast Real-Time macrophages to stimulate adaptive T cells needs further study. PCR System (Applied Biosystems, Carlsbad, CA, USA). b-Actin served as This is the first study to investigate the role of the CUL4B in the internal controls and H2O served as a negative control. SYBR Green DNA- immune system. Our study demonstrates that a Cul4b deficiency binding dye (Thermo, Waltham, MA, USA) was used in the amplification results in increased leukocyte recruitment and an aggravated reactions included oligonucleotide primers for TNF-a, IL-6 and b-actin. peritonitis in LPS-injected mice. In addition, CUL4B regulates Fluorescence signals were analyzed during each of 40 cycles (94 1C for 15 s, 1 the cell cycle and cytokine and chemokine production of 60 C for 1 min). TNF-a and IL-6 mRNA expression were normalized to internal controls, b-actin. Primers used in PCR are as follows. TNF-a forward macrophages. However, Cul4b deficiency does not change the 50-CCCCAAAGGGATGAGAAGTTC-30; reverse 50-TGAGGGTCTGGGCCATAG phagocytic ability of macrophages. AA-30. IL-6 forward 50-CTCTGGGAAATCGTGGAAATG-30; reverse 50-AAGTG- CATCATCGTTGTTCATACA -30. b-Actin forward 50-CACAGTGTTGTCTGGTGG TA-30; reverse 50-GACTCATCGTACTCCTGCTT-30. MATERIALS AND METHODS Proliferation assay of macrophages Generation and genotyping of macrophage-specific 4 Cul4b-deficient mice Bone marrow-derived macrophages (5 Â 10 per well) were cultured in 96-well plates in the presence of M-CSF (10 ng ml À 1) for 18 h. 1 mCi of We have generated mice carrying a loxP-flanked Cul4b allele for 3 3 [ H]-thymidine was pulsed for another 6 h of culture. [ H]-Thymidine conditional knockouts previously.34 To delete the Cul4b gene in f/y KI/KI uptake was measured in a beta scintillation counter (Packard Instrument, macrophages, Cul4b mice were bred with LysM-Cre mice in which Meriden, CT, USA). the Cre recombinase is expressed under control of the murine lysozyme M gene regulatory region.38 Routine genotyping was performed by PCR reaction with primers 50-CATCTTTAGC CTCTTGTGCT-30 paired with Cell cycle analysis of macrophages 50-AAAAGCCTACGTTTATGTGC-30 for the floxed allele (494 bp) or paired Bone marrow-derived macrophages were synchronized by serum starva- 0 0 34 with 5 -AGCCTGGTCTACAAAGTTGA-3 for the deleted allele (569 bp). tion for 24 h and stimulated with or without M-CSF (10 ng ml À 1). After 12 The PCR primers for detection of the LysM-Cre knock-in gene were and 24 h, cells were collected, fixed, PI-stained and analyzed for cell cycle 50-CTTGGGCTGCCAGAATTTCTC-30 paired with 50-CCCAGAAAGCCAGATTA phase distribution with fluorescence-activated cell sorting using a CG-30 (700 bp), while paired with 50-TTACAGTCGGCCAGGCTGAC-30 for the FACSCalibur system (Becton Dickinson Biosciences, San Diego, CA, USA). wild-type mice (350 bp). The PCR reaction condition was 94 1C for 2 min; 35 The data were processed to determine cell populations in the G1, S, and cycles of 94 1C for 30 s, 61 1C for 1 min and 72 1C for 1 min; and 72 1C for G2/M phases. 7 min. Male mice (8–12 weeks) were used in this study under specific pathogen-free conditions. Mice were maintained in the Animal Center of the College of Medicine, National Taiwan University. This study was carried Phagocytic ability of macrophages out in strict accordance with the recommendations in the Guide for the Phagocytosis of macrophage were measured using commercial test kits Care and Use of Laboratory Animals of the National Taiwan University. (Phagotest, Glycotope Biotechnology, Heidelberg, Germany). The percen- The protocol was approved by the Institutional Animal Care and Use tage of cells having performed phagocytic ability are analyzed as their

& 2014 Macmillan Publishers Limited Genes and Immunity (2014) 404 – 412 Innate immunity in mice deficiency of cullin 4B in macrophages M-H Hung et al 412 mean fluorescence intensity (number of ingested green fluorescent 15 Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol protein-bearing E. coli). 2005; 5: 953–964. 16 Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K. Development of Statistical analysis monocytes, macrophages, and dendritic cells. Science 2010; 327: 656–661. 17 Jiang B, Zhao W, Yuan J, Qian Y, Sun W, Zou Y et al. Lack of Cul4b, an E3 ubiquitin Results are expressed as the mean±s.e.m. Nonparametric Mann–Whitney ligase component, leads to embryonic lethality and abnormal placental tests were performed using Prism software (GraphPad Software, San Diego, development. PLoS One 2012; 7: e37070. CA, USA) for all statistical analyses. Comparisons giving P-values 0.05 p 18 Liu L, Yin Y, Li Y, Prevedel L, Lacy EH, Ma L et al. Essential role of the CUL4B were considered significant. ubiquitin ligase in extra-embryonic tissue development during mouse embry- ogenesis. Cell Res 2012; 22: 1258–1269. 19 Underhill DM, Goodridge HS. Information processing during phagocytosis. Nat CONFLICT OF INTEREST Rev Immunol 2012; 12: 492–502. The authors declare no conflict of interest. 20 Davies LC, Jenkins SJ, Allen JE, Taylor PR. Tissue-resident macrophages. Nat Immunol 2013; 14: 986–995. 21 Cailhier JF, Partolina M, Vuthoori S, Wu S, Ko K, Watson S et al. Conditional ACKNOWLEDGEMENTS macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation. J Immunol 2005; 174: 2336–2342. This work was supported by grants from the National Science Council, Taiwan 22 Randow F, Lehner PJ. Viral avoidance and exploitation of the ubiquitin system. (NSC99-2320-B-002-060-MY3). We thank Dr Chun-Yu Chen for critical reading the Nat Cell Biol 2009; 11: 527–534. manuscript and technical support. We thank the technical services provided by the 23 Andrejeva J, Poole E, Young DF, Goodbourn S, Randall RE. The p127 subunit ‘Transgenic Mouse Model Core Facility of the National Core Facility Program for (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted Biotechnology, National Science Council’ and the ‘Gene Knockout Mouse Core degradation of STAT1 by the V protein of the paramyxovirus simian virus 5. J Virol Laboratory of National Taiwan University Center of Genomic Medicine’. We thank the 2002; 76: 11379–11386. Taiwan Mouse Clinic (NSC 102-2325-B-001-042), which is funded by the National 24 Ulane CM, Horvath CM. Paramyxoviruses SV5 and HPIV2 assemble STAT protein Research Program for Biopharmaceuticals (NRPB) at the National Science Council ubiquitin ligase complexes from cellular components. 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