Of Mice and Men: Proteasome's Role in LPS-Induced Inflammation and Tolerance

Silswal, Neerupma; Reis, Julia; Qureshi, Asaf A.; Papasian, Christopher; Qureshi, Nilofer

doi: 10.1097/SHK.0000000000000743
Basic Science Aspects
Editor's Choice

ABSTRACT: The molecular basis responsible for tolerance following inflammatory response to lipopolysaccharide (LPS) is not well understood. We hypothesized that inflammation/tolerance in monocytes/ macrophages is dependent on the proteases of proteasome. To test our hypothesis, first, we examined the expression of different proteasome subunits in different human and mouse monocytes/macrophages. Secondly, we investigated the effect of proteasome subunits/ proteases on LPS-induced expression of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) during inflammation and tolerance using mouse RAW 264.7 macrophages, THP1 cells, and cluster of differentiation 14 positive (CD14+) human monocytes. We found that RAW 264.7 cells (XYZ), mouse peritoneal resident, thioglycollate-elicited macrophages, primed RAW 264.7 (XYZ, LMP), and human monocytes (LMP) expressed different types of proteasome subunits/activities. Cells containing predominantly either LMP subunits (such as THP-1 and human monocytes), or only X, Y, Z subunits (RAW 264.7 cells not primed) could only induce TNF-α, but not NO, while cells containing all five to six subunits (XYZ, LMP) of the proteasome could induce both mediators in response to LPS. Distinct states of inflammation/tolerance in LPS treated cells, strongly correlated with an upregulation or downregulation of proteasome's subunits (proteases), respectively. Moreover, interferon-γ treatment of tolerant cells caused robust induction of proteasome's subunit expression in mouse macrophages and human monocytes, and cells regained their ability to respond to LPS. These studies are vital for understanding function of proteasome's subunits during inflammation/tolerance in mouse and human cells, and for design of therapeutic strategies for all diseases based on inflammation.

*Department of Basic Medical Science, School of Medicine, and Shock/Trauma Research Center, School of Medicine, Kansas City, Missouri

Department of Pharmacology and Toxicology, School of Pharmacy, University of Missouri Kansas City, Kansas City, Missouri

Address reprint requests to Nilofer Qureshi, PhD, Department of Basic Medical Science, School of Medicine, Shock/Trauma Research Center, University of Missouri Kansas City, 2411 Holmes Street, Kansas City, MO 64108. E-mail: qureshin@umkc.edu.

Received 13 May, 2016

Revised 31 May, 2016

Accepted 25 August, 2016

This study was supported by grants R01-GM 50870 (NQ), R01-GM102631 (NQ), and R01 GM102631S1 (NQ), from National Institutes of Health, NIGMS.

The authors report no conflicts of interest.

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The host's inflammatory response to bacterial infection has both beneficial and detrimental effects. The primary beneficial effect is damage, death, and elimination of invading bacteria, while the detrimental effects are due to collateral damage to tissues attributable to inflammation. If the inflammatory response is localized, tissue damage is relatively limited and can usually be repaired. When inflammation becomes widespread, a systemic inflammatory response syndrome may develop, potentially leading to tolerance and ultimately resulting in decreased intravascular volume and blood pressure, decreased tissue perfusion, organ failure, and death (1, 2). It is not clear whether tolerance is harmful (weak immune system) or beneficial (repair) to the host.

Tolerance is thought to occur as a compensatory measure to reduce inflammation in the host and has been extensively studied over the past 70 years (3–5). When the initial lipopolysaccharide (LPS) treatment tranduces a signal in the cell via interaction with membrane CD14, LPS-binding protein, toll-like receptor 4 (TLR4), and MD2 proteins, it recruits adaptor proteins, that leads to activation of NF-κB, and other transcription factors ultimately resulting in upregulation of gene and protein expression of inflammatory cytokines (6). Following this cascade of events in macrophages, a state of tolerance sets in, such that a second LPS treatment of macrophages fails to induce select proinflammatory cytokines, such as TNF-α, interleukin-12 (IL-12), interferon-β (IFN-β), and interleukin-6 (IL-6) which is dependent on mitogen-activated protein kinases (MAPKs), and NF-κB signaling pathways. On the other hand, nitric oxide (NO) release is differentially regulated in response to LPS and its induction is not affected during tolerance. Several theories have been put forth for tolerance observed in LPS-treated cells; however, the mechanisms by which these processes are developed at the cellular level are still not well understood (3, 4, 6). Foster et al. and El Gazaar et al. have provided evidence that epigenetic changes at level of chromatin are induced in the promoters of affected genes in tolerized cells, leading to altered transcriptional regulation (7, 8). Medvedev et al. (9) showed that recruitment and activation of downstream molecules to the TLR4 receptor complex was inhibited in tolerized macrophages.

For more than a decade, our group has been studying the role of proteasomes in inflammatory responses induced by LPS and other microbial products in mouse cells (6, 10–15). Proteasomes are cytoplasmic organelles that contain three well-characterized proteolytic activities, chymotrypsin-like activity (CT-like, X and LMP7, cleaves at non-polar residues such as tyrosine), postacidic activity (PA, Y, LMP2 cleaves at acidic residues such as aspartic and glutamic acid), and trypsin-like activity (T-like Z and LMP10, cleaves at basic residues such as arginine and lysine residues) attributable to proteasome subunits X (β5), Y (β1), and Z (β2), respectively (16–18). In response to LPS, X, Y, and Z subunits of proteasomes are replaced by low-molecular mass peptides (LMP) 7, β5i), LMP2(β1i), and LMP10 (β2i) in newly synthesized proteasomes in RAW 264.7 cell line. Proteasomes thus transformed contain immunosubunits (11, 14, 19) and this change causes cells to switch their proteolytic activities from predominantly PA activity (Y) to CT-like and T-like activities (LMP7/LMP10) in differentiated cells. This switching in proteolytic activities was thought to lead to enhanced degradation of peptides used for antigen presentation, but now it appears that it might also be important for degradation of regulatory proteins involved in LPS-induced signaling pathways (14).

Our previous studies of inflammatory processes, using mouse macrophages, defined a pivotal role for six proteasome protease subunits X, Y, Z, LMP2, LMP7, and LMP10 in inflammatory processes. We were the first to determine that magnitude of NO and cytokine (e.g., TNF-α, interleukin-1 (IL-1), IL-6) responses to LPS and other agonists could be significantly down-regulated by using proteasome inhibitors, or by altering protease subunit composition of immunoproteasomes in wild-type and knockout mice (11–15, 19). We also showed that multiple LPS-induced pathways are proteasome-dependent, and lactacystin, a well-known proteasome inhibitor that inhibits CT-like activity, markedly suppressed LPS induced inflammatory responses in thioglycollate-elicited mouse macrophages. Moreover, LPS-induced signaling pathways activated by TLR4, Myeloid differentiation factor 88, (MyD88), and primarily TIR-domain-containing adapter-inducing interferon-β (TRIF/TRAM) were altered in LMP7 and LMP10 subunit-deficient macrophages (obtained from knockout mice) such that TNF-α was not affected, but NO, IL-1, IL-12, and IL-6 (TRIF/TRAM pathway) were not induced robustly with LPS; but IFN-γ treatment prior to LPS reversed this defect and these cells behaved normally with respect to NO induction. Collectively, we have demonstrated that LPS induces changes in immunoproteasome composition, especially the CT-like and T-like activities of LMP7 and LMP10 respectively, are critical for inducing proinflammatory (via TRIF/TRAM pathway) responses by mouse macrophages. However, all five to six subunits of proteasomes may be required for optimal cytokine and NO induction by cells in response to LPS.

Our primary objective of the current investigation was to determine proteasome subunits that are regulated during inflammation and tolerance in various human and mouse cells by LPS. We hypothesized that LPS-induced alterations in proteasome subunit composition/levels contributes to development of inflammation/tolerance in cells, and IFN-γ reverses the effects of tolerance by upregulating LMP subunits of proteasome in all cell types. Therefore, to test our hypothesis, we determined contribution of proteasomal subunits/proteases on LPS-induced TNF-α, cytokines, and NO during inflammation and tolerance in RAW 264.7 cells, THP1 cells, and CD14+ monocytes. We have established that all six proteasome protease subunits can be upregulated/downregulated in response to LPS, depending on cell type and both XYZ and LMPs subunits are required for optimal NO induction. Moreover, IFN-γ and LPS reverse the immune paralysis during tolerance and upregulate XYZ and/or LMP subunits of the proteasome.

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Highly purified, deep rough chemotype LPS (Re-LPS) from E. coli D31m4 was prepared, as described by Qureshi et al. (20). For tissue culture studies, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640, heat-inactivated low-endotoxin fetal bovine serum (FBS), and gentamicin were all purchased from Lonza (Walkersville, Md). Human AB serum was purchased from Fisher Scientific (Waltham, Mass). Thioglycollate was purchased from Sigma Aldrich (St. Louis, Mo) and RNeasy mini kit from QIAGEN sciences (Germantown, Md). Proteasome-Glo cell-based luminescent assays for assaying chymotrypsin-like, postacidic, and trypsin-like activities were purchased from Promega (Madison, Wis).

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Animals and cell culture

Six-week-old control female mice (C57BL/6, C3H/FeJ, wild-type [WT], BALB/c, C3H/HeJ, C3H/HeN) were purchased from Charles Rivers Laboratory (Wilmington, Mass). Resident and thioglycollate (TG)-elicited peritoneal macrophages were prepared from mice (4, 13, 14, 19). All studies were conducted following approval from Animal Care and Use Committee at the University of Missouri-Kansas City (IACUC). Mice were injected with saline or 1.5 mL of sterile 4% TG (Difco, Detroit, Mich), in the lower part of the abdomen and after 4 days peritoneal cells were harvested. First, mice were sacrificed using the cervical dislocation method. The mouse was laid on its back on a towel paper, 75% ethanol was sprayed on the whole body. The abdominal skin was held up with a pair of forceps and a small hole was cut out in the middle. Ten milliliters of DMEM medium (without serum) was injected to flush out the peritoneal cavity. After harvesting, the cells were spun down at 1,000 rpm for 10 min, and resuspended with 10 mL DMEM (supplemented with10% serum). The yield of cells usually varied from 1 to 1.5 × 107 TG-elicited peritoneal macrophages/mouse using this technique.

The murine RAW 264.7 macrophage and human monocytic THP-1 cell lines were purchased from ATCC. RAW 264.7 and THP-1 cells were grown using DMEM and RPMI, respectively, supplemented with 10% fetal bovine serum and gentamicin (10 μg/mL) in a humidified incubator with 5% CO2 at 37°C. Human mononuclear fractions of heparinized whole blood were obtained from volunteers recruited in the protocols approved by the University of Missouri, Kansas City Institutional Review Board (IRB). Mononuclear cells were separated from blood by mixing 50 μL of monocyte enrichment cocktail and 25 μL of granulocyte depletion cocktail per milliliter of blood. After incubation for 20 min at room temperature, sample was diluted with equal volume of PBS+2% FBS and 1 mM EDTA. Diluted sample was layered on top of the Ficoll-Paque PLUS and centrifuged for 20 min at 1,200 × g with the brakeoff. The enriched cells were removed from the plasma interface, washed and counted. Monocyte purity was >96% by differential count. Purified human blood monocytes were suspended in a 6-well plate (2 × 106 cells/well) using complete media (RPMI 1640 with human AB serum). After 24 h, cells were washed and tolerance treatment was performed. Cells were pelleted and stored at −20oC after treatment.

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Detection of cell viability

Viability of peritoneal macrophages treated with LPS was determined by a quantitative colorimetric assay with 3-(4,5)-dimethylthiozol-2, 5-diphenyltetrazolium bromide (MTT) as described previously (11, 12). No cell death was observed during the experiments.

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Measurement of TNF-α and NO

The levels of TNF-α in cell culture supernatants were determined by mouse and human TNF-α Quantikine ELISA kit (R&D System, Minneapolis, Minn) according to the manufacturer's instructions. The lower limit of detection for TNF-α using this method is about 5 pg/mL. The presence of NO in the supernatants was assayed using the Griess reagent kit (Sigma-Aldrich, St. Louis, Mo).

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Western blot analysis

Cellular proteins were extracted from cells using a lysis buffer (Sigma) and their concentrations were measured with Pierce BCA protein assay kits (Thermo Scientific, Waltham, Mass). Western blots were used to measure the relative proportions of the proteins of various cells and cell lines. Each well of the gel was loaded with 20 to 40 μg of protein and samples were electrophoresed at a constant 150 V in 1 × Tris glycine buffer for 50 min. Proteins in the gels were transferred onto the Immobilon Transfer Membranes (IPVH 15150; Millipore, Bedford, Mass) using the wet transfer cell. Blots were incubated with the indicated antibodies, and bands were visualized with an enhanced chemiluminescence detection kit (Pierce) as described previously (14, 21). Anti-proteasome antibodies X (catalogue no. AP-121), Y (AP-122), and LMP7 (AP-119) were purchased from Boston Biochem; and Z (catalogue no. SC-54735), LMP7 (K15, AP-SC2020), LMP2 (SC28809), LMP10 (SC-2004), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-2030) were purchased from Santa Cruz (Dallas, Tex). We have used these antibodies in our prior publications and they detect bands that correspond to the correct molecular weight (14). Secondary antibodies were purchased from Santa Cruz.

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Tolerance experiments and quantitative real-time polymerase chain reaction (qRT-PCR)

THP-1 cells, CD14+ monocytes, and RAW 264.7 (1 × 106 cells/well) cells were treated in a 6-well plate as follows: THP-1 cells were first differentiated with phorbol 12-myristate 13-acetate (PMA) (10 ng/mL) for 24 h, before primary treatment with medium (M) or LPS (L10 ng/mL) for 24 h, followed by washing and replacement with M or L (10 ng/mL), for an additional 4 or 24 h. The four combinations are represented as MM, ML, LM, and LL. The first LPS treatment renders cells tolerant to second LPS treatment. In some experiments, tolerized (LM) cells were washed with medium, treated with IFN-γ (50 units/mL) for 4 h (LM+IFNγ), washed, and then followed by another 4 h treatment with LPS (LM+IFNγ+LPS). After the indicated treatment, all cells were washed with PBS and total RNA was extracted by using RNeasy mini kit (Qiagen) as per manufacturer's instructions. Real-time quantitative reverse transcription PCR (qRT-PCR) was performed by using TaqMan RNA-to-Ct one step kit. Primers-probes for all six proteasome subunits of human and mouse (X, Y, Z, LMP7, LMP2, and LMP10) and one-step qRT-PCR kit were obtained from Life Technologies (Foster City, Calif). All reactions were performed in triplicate using an equal amount of mRNA per reaction. Reverse-transcriptase step involved incubation at 48°C for 15 min. The PCR cycling conditions included an initial denaturation of 95° C for 10 min, followed by 40 cycles of 95° C for 15 s, and 60° C for 60 s. qRT-PCR assays were completed using a step-one plus real-time PCR system. Finally, gene expression from cell cultures was normalized (2−ΔΔCT analysis) to GAPDH.

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Proteasome cell-based activity assays

Figure. No caption a...

RAW 264.7 and PMA-differentiated THP-1 cells (5 × 104 cells/well) were treated in a 96-well plate for MM, ML, LM, and LL conditions, as described above for gene expression analysis. The Proteasome-Glo luminescent assays that individually measure the chymotrypsin-like, trypsin-like, and postacidic protease activities associated with proteasome complex were carried out according to manufacturer's protocol, as previously described (14).

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Monocyte/macrophage heterogeneity and development can be correlated to differences in proteasome structure

Since murine cells and human cells have been shown to differ, with respect to their transcriptional responses to LPS (22, 23), we evaluated the proteasome subunits present in various murine and human cells and cell lines, to select cells with distinct proteasome structures. Proteasome subunits in select human or mononuclear cells and murine macrophages are compared in Figure 1A–C and Table 1. We established, through Western analysis, that newly purchased THP-1 cells (human monocytic cell line) expressed LMP2 and LMP10, but did not express LMP7 proteasome subunits (Fig. 1A, lane 1). In contrast, PMA treated, THP-1 cells (Fig. 1A, lane 2), expressed LMP7, but this PMA induced LMP7 expression could be reversed by treatment with LPS (200 ng for 48 h; Fig. 1A, lane 3). There was no change in the expression of LMP2 in the presence or absence of PMA or LPS (Fig. 1A, lanes 1, 2, and 3) while the expression of LMP10 was low in PMA treated cells (Fig. 1A, lane 1) and was not detectable after treatment with PMA or LPS (Fig. 1A, lanes 2 and 3). Further, THP-1 cells grown in media (with glucose) for several weeks, without additional stimulation, began expressing LMP7 (Fig. 1B) and, in fact, contained proteasomes with immuno-subunits LMP2, LMP7, and LMP10, but no detectable X, Y, or Z subunits. In contrast, RAW 264.7 cells expressed mainly X, Y, and Z proteasome subunits, as shown in Figure 1B, Table 1. Although we detected a band that migrated just above the LMP2 band in Western blot of RAW 264.7 cell lysates, our extensive experience suggests that this could be due to either pro-LMP2, mutated LMP2, or some impurity that reacts with anti-LMP2 antibody. CD14+ human monocytes recovered from human blood were found to contain subunits LMP2 and LMP7, but not LMP10, X, Y, and Z (Fig. 1C). These results suggest that there is heterogeneity with respect to proteasome structures in RAW 264.7 macrophages and human monocytes. RAW 264.7 cells expressed all six subunits in response to LPS as published earlier (13), In contrast, THP1 cells expressed only inducible LMP7, 2, and 10 subunits and human monocytes expressed predominantly LMP7 and LMP2.

We sought to compare subunits and protease activities of proteasomes isolated from resident versus TG-elicited peritoneal macrophages from several different inbred strains of mice (there was a 5–10 fold increase in the number of macrophages collected after TG-elicitation, as compared with resident macrophages), to determine whether this heterogeneity of cell type with respect to proteasome structure could contribute to heterogeneity of results described in the literature (Fig. 2). We compared resident versus TG-elicited macrophages from BALB/c (BC), C57BL/6 (B6) mice (wild-type), C3H/HeN (wild-type), and C3H/HeJ mice (24). As demonstrated by Western analysis, most of the macrophages obtained from various mouse strains showed heterogeneity with respect to proteasome subunits. BC resident macrophages expressed predominantly X, Y, and LMP2 subunits, with undetectable levels of LMP7, and barely detectable LMP10. Subunit Z was not present in significant amounts. After TG-elicitation, levels of X and Y subunits decreased (BC-TG), and LMP2 and LMP10 appeared to increase, but there was no detectable increase in expression of LMP7, as shown in Fig. 2, Table 1. B6 resident macrophages lacked X and Z subunits as compared with RAW 264.7 macrophages, and expressed predominantly Y, LMP2, and LMP10; LMP7 was barely detectable. TG-elicited B6 macrophages differed from resident B6 macrophages by expressing LMP7 much more prominently. Interestingly, resident macrophages from C3H/HeJ mice (LPS non-responders) showed lower levels of LMP7 and LMP10 than C3H/HeN (wild-type) mice. Moreover, upon TG treatment of C3H/HeJ mice, X and Y subunit expression was down-regulated and there were slight elevations of LMP7 and LMP10 expression. TG-elicited C3H/HeN macrophages induced slight reductions and increases in expression of Y and LMP7, respectively. There is a dramatic shift for macrophages obtained from B6 mice, C3H/HeJ and C3H/HeN mice, and a minor shift toward increased LMP7 expression for BC macrophage that corresponds to a change in specificity of the proteasome's protease activities from postacidic to chymotrypsin-like and trypsin-like activities (data not presented). These mouse macrophages are very active in inducing LPS-induced TNF-α and NO. However, at times, when TG treatment causes an increase in X, Y, and Z subunits, a decrease in TNF-α and NO induction as compared with resident cells was observed (data not presented). This suggests that some mouse peritoneal macrophages that contain at least five to six proteasome subunits including LMP7 are very active in inducing NO and cytokines in response to LPS. Cells are clearly heterogeneous with respect to proteasome subunits and cells were selected based on differential proteasome structures.

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Proteasome's proteases are upregulated during inflammation and downregulated during tolerance in RAW 264.7, THP-1, and CD14+ monocytes. Reprogramming of tolerant cells leads to down-regulation of proteasome subunits

Our goal was to test the hypothesis that initial interaction of LPS with host macrophages reprograms proteasome's subunit structure and enzymatic activities, leading to LPS-induced pro-inflammatory responses, followed by tolerance. Human blood CD14+ monocytes, RAW 264.7 cells, and PMA-differentiated THP-1 cells were placed in various experimental groups designated as follows: MM, ML, LM, and LL, as described under the Methods section.

As shown in Figure 3A and B, the MM group served as a control, and ML (first LPS treatment, mimicking an initial inflammatory response) showed 81.3 and 44.8-fold upregulation in levels of TNFα protein and 31.3 and 27.8-fold increased mRNA (δCT values) after 4 h, in THP-1 and RAW 264.7 cells, respectively, as expected. However, when cells were pretreated with LPS for 24 h, washed, and then treated with medium (LM) or LPS a second time (LL, representing LPS induced tolerance), TNFα protein expression, and mRNA levels were reduced to only 0.77 fold (RAW 264.7) and 0.69 fold (THP-1), compared with ML group. Thus, initial LPS treatment induced a state of tolerance, demonstrated by reduced levels of expression of TNFα protein and mRNA on re-exposure to LPS, in both cell lines. Next, we examined whether tolerant state in LM cells could be reversed by treatment with IFN-γ. In order to test this, tolerant cells (LM) were pretreated with IFN-γ for 4 h followed by exposure to medium (LM+IFN-γ) or LPS (LM+IFN-γ+LPS) for another 4 h. Our data demonstrated that IFN-γ+LPS treatment completely reversed the state of tolerance; and there was a concomitant 2.85 fold (RAW 264.7) and 17.8 fold (THP1) upregulation of TNF-α protein; and 7.23 fold (RAW 264.7) and 4.81 fold (THP1) increase in gene expression, respectively. (Fig. 3, A and B), In contrast, there was no effect on NO production in tolerant RAW 264.7 cells (Fig. 3C). Besides, THP-1 cells and human monocytes (data not presented) did not produce NO in response to LPS, under any of the conditions tested (Fig. 3C).

In order to determine whether the results of experiments described above, for LPS tolerance, were associated with changes in various protease subunits, we performed identical experiments with respect to LPS or media exposure, and IFN-γ rescue, but measured expression of proteasome subunits (Fig. 4, A–D). In RAW 264.7 cells treated with media initially (i.e., no induction of LPS tolerance), followed by 4 or 24 h of exposure to LPS (ML), there was a substantial increase in production of mRNA for all proteasome subunits; most robust changes were observed for X, Y, Z and LMP subunits after 4 h of LPS exposure (Fig. 4, A and B), but less upregulation at 24 h (Fig. 4, C and D). There was a significant downregulation in expression of all subunits during LM and LL treatments. Treatment of tolerized cells with IFN-γ alone did not rescue, but IFN-γ and LPS, significantly upregulated expression of all LMP and X subunits (Fig. 4, A–D).

In contrast to RAW 264.7 cells, PMA-treated THP1 cells showed no significant effect on X, Y, and Z subunit expression in any of treatments (data not shown), but there was significant upregulation of all three LMP subunits at 4 h after LPS exposure (P <0.05) (Fig. 4E). Moreover, first LPS treatment resulted in significantly lower levels of mRNA for LMP7 and LMP2 in tolerized (LM), compared with non-tolerized cells (ML) (Fig. 4E), and after 24 h LPS treatment, all three LMP subunits were downregulated in tolerized THP1 cells (LM and LL; P <0.05) (Fig. 4F). Interestingly, IFN-γ treatment alone or IFN-γ and LPS was able to upregulate two or three LMP subunits in THP1 cells that were re-exposed to LPS for 4 and/or 24 h (Fig. 4, E and F).

In order to determine whether the results of experiments described above, for LPS tolerance, were associated with changes in various protease activities of the proteasome, we performed identical experiments to those described above with respect to LPS or media exposure, and IFN-γ rescue, but measured chymotrypsin-like, postacidic, and trypsin-like activities of proteasome (Fig. 5, A–C). Compared with media controls (MM), mainly chymotrypsin-like and trypsin-like activities were most significantly affected and upregulated by LPS treatment (ML) in both RAW and THP-1 cells (P <0.05) (Fig. 5, A and C). RAW cells (XYZ) had lower chymotrypsin-like and trypsin-like activities, as compared with THP-1 cells, because the latter cells contained LMP proteasome subunits. In contrast, RAW cells showed a higher postacidic activity (due to Y). Tolerance treatment of THP1 cells with LPS for 24 h (LM) resulted in significantly decreased chymotrypsin-like and trypsin-like activities compared with M, while decrease in postacidic activity was observed only in RAW cells (Fig. 5, A–C), but second LPS treatment for 4 h, in both RAW and THP1 cells (LL) significantly decreased chymotrypsin-like and trypsin-like activities. As observed with proteasome subunits, IFN-γ treatment followed by 4 h LPS exposure significantly increased chymotrypsin-like and trypsin-like activities in both cell lines; while only postacidic activity was revived only in RAW cells (P <0.05) (Fig. 5, A–C).

Furthermore, we carried out similar tolerance experiments with CD14+ human monocytes where MM group served as a control, and ML (first LPS treatment, mimicking an initial inflammatory response) showed a robust upregulation in levels of TNFα protein and mRNA after 4 h (435.5 fold increase in TNFα protein and 70.5 fold increase in TNFα mRNA δCT values) (P <0.05) as expected (Fig. 6, A and B). TNFα (protein and mRNA) expression levels were significantly reduced as compared with the ML group in tolerized cells LM and LL (P <0.05). Thus, initial LPS treatment induced a state of tolerance, demonstrated by reduced levels of TNFα mRNA on re-exposure to LPS in human monocytes. Similarly, when tolerized (LM) human monocytes were treated with IFN-γ and LPS, there was a concomitant increase in TNF-α protein and gene expression (P <0.05) (Fig. 6, A and B) ⋅ However, human monocytes (data not presented) did not produce detectable NO or inducible nitric oxide synthase (iNOS) in response to LPS, under any of the conditions tested, and LPS-induced tolerance on CD14+ human monocytes had no effect on mRNA of X, Y, and Z proteasome subunits (Fig. 6C). There was a significant increase in LMP subunit expression after 4 h LPS exposure (ML) especially (P <0.05) (Fig. 6D). Compared with non-tolerized cells (ML), LPS tolerized human monocytes (LL) differed markedly in that they failed to upregulate LMP subunit expression after re-exposure to LPS for 4 h, as observed with other cell lines. This failure to upregulate LMP subunit expression in CD14+ monocytes was partially rescued by IFN-γ treatment alone (P <0.05) (Fig. 6D). After IFN-γ and LPS treatment of tolerant cells (LM), there was a significant increase in LMP7, LMP2, and LMP10 gene expression (P <0.05) (Fig. 6D). Collectively, all subsets of cells that contained either X, Y, Z, or LMP subunits or both; induced TNF-α in response to LPS and developed tolerance and these cells could be rescued by IFN-γ or IFN-γ and LPS treatments in human monocytes.

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We have established first that human monocyte/mouse macrophage heterogeneity and development can be strongly correlated to differences in proteasome structure/function (Fig. 7). Subunits X, Y, LMP7, and LMP2 were present in mouse macrophages, while subunits Z and LMP10 were not observed in significant levels in all cells described above. Simplistically, cells can now be classified into three major subtypes those containing either X, Y, Z; X, Y, Z, and LMP; or LMP subunits. Moreover, priming with thioglycollate (in vivo), PMA, or LPS reprograms subunits in primed cells from X, Y, Z, to LMPs in newly synthesized proteasomes of peritoneal murine macrophages, which consequently leads to changes in specificities of proteasome's proteases from postacidic to chymotrypsin-like and trypsin-like activities, thus affecting cytokine expression and NO levels. We selected cells containing: X, Y, Z subunits (unprimed RAW 264.7 cells); X, Y, Z, and LMP subunits (primed RAW 264.7 cells), or LMP subunits (human CD14+ monocytes, THP1 cells) for our studies. These cells differed in TNF-α and NO responses induced by LPS. While primed RAW 264.7 cells and murine macrophages containing X, Y, Z and LMP proteasome subunits induced robust levels of TNF-α and NO in response to LPS. Cells that contain only LMP subunits such as THP-1 and human monocytes, or X, Y, Z subunits (RAW 264.7 cells, not primed) induced TNF-α, but not, NO in response to LPS. Thus, suggesting that a combination of constitutive X, Y, Z and inducible subunits LMP in cells are important for efficient LPS-induced NO.

Secondly, we have identified for the first time that proteasome's proteases are upregulated (regardless of cell type) during inflammation (ML) and downregulated during tolerance in RAW 264.7, THP-1, and CD14+ monocytes (LM, LL). Treatment of RAW 264.7 macrophages with LPS (ML) results in induction of relatively high levels of gene expression of all X, Y, Z and LMP proteasome subunit genes that correlates with high TNF-α expression in cells. Then after second LPS treatment, there is downregulation of all proteases and TNF-α levels because cells become tolerant. Treatment with IFN-γ followed by LPS reverses their tolerant phenotype and cells are activated and new proteasome subunits are biosynthesized. In contrast, THP-1 and CD14+ human monocytes contained predominantly LMP subunits as compared with RAW 264.7 cells. Human CD14+ monocytes and THP1 cells do not perform all functions of macrophage and are not capable of inducing NO in response to LPS, but rather induced robust levels of TNF-α. Our data also revealed an upregulation of only LMP subunits, but not XYZ in response to LPS (cytokine induction), and this was followed by downregulation during LPS-induced tolerant human monocytes. This tolerance could be reversed by treatment with IFN-γ or IFN-γ+LPS which resulted in robust upregulation of proteasome's subunits/activities in monocytes. Thus, CD14+monocytes differed in their proteasome structures and function, as compared with mouse peritoneal macrophages.

Mechanistically, tolerance has been previously attributed to several proteins that block signaling, including TNF-α, interleukin-10 (IL-10), transforming growth factor-β (TGF-β), interleukin-1 receptor associated kinase (IRAK) M, (negative regulator of Toll signaling), IRAK1, IRAK-4, iNOS, suppressor of cytokine synthesis (SOCS) 1, SOCS3, (SH2-containingInositol 5′-Phosphatase) SHIP, and at the nuclear level, increase in p50/p50 homodimers, activation of peroxisome proliferator-activated receptor (PPAR)-γ, hypoxia-inducible factor (HIF)-1, and complex mechanism involving histone modification (acetylation, phosphorylation, and methylation) generating a histone code that produces docking sites for proteins regulating chromatin structure and gene transcription (25, 26). This topic on tolerance mechanisms has been recently reviewed by others and us (5, 27–31). Our model (Fig. 7) is consistent with most previous mechanisms of tolerance, because transcription/degradation of all cytokines and signaling proteins mentioned above is proteasome-dependent (12). Importantly, LPS-induced tolerance has been previously shown to be reversed in cells by IFNγ (32), and we suggest that this could be due to introduction of new proteasome LMP subunits in cells, which are primed and induce increased levels of cytokine expression in response to LPS.

Much of recent research on mechanisms of LPS-induced response has been centered on canonical pathway followed by activation of TLR4. Ubiquitin-proteasome pathway plays a pivotal role in LPS-induced signaling and can be envisioned as an on/off switch for inflammation/tolerance (6, 11). The cascade of signaling events that follow via MyD88 or TRIF/TRAM as the adapter proteins leads to the activation of NF-κB, via degradation of ubiquitinated and phosphorylated IκB by the proteasome. Similarly, we have shown that phosphorylation of ERK-1, JNK-1, and p38 (MAPK) is also proteasome dependent (10). Our data suggest that initial interaction of LPS with host macrophages induces the postacidic activity in RAW 264.7 cells, and then chymotrypsin-like, and trypsin-like activities in cells, initiating a process of proteasome-mediated degradation of signaling mediators such as TLR4, IRAK-1, IRAKM, phosphorylated interferon regulatory factor 3, TNF receptor associated factor (TRAF6), IκB-α, β, p105 degradation (responsible for biosynthesis of p50 subunits), and MAPK because of changes in proteasomal activities, proteasome subunits (priming, LMP mode), ubiquitination/deubiquitination and expression of proinflammatory cytokines (5, 11), This also leads to a net increase in ubiquitinated proteins and increased activation of transcription factors, such as NF-κB (33); however, HIF-1 (34), Keap/NRF2 (35), and PPAR-γ are inhibited following proteasome's activation. In contrast, during tolerance, very low proteasome subunit expression and activity leads to inhibition in NF-κB activation, but activation of HIF-1, PPAR-γ, and NrF2 transcription activities. Thus, the proteasome regulates the degradation of regulatory proteins and activities of transcription factors.

The proteasome has been shown to directly regulate the structure and function of chromatin and chromatin regulatory proteins (non-canonical pathway) (36). DNA and nuclear proteins histones (H2A, H2B, H3, and H4) form the nucleosomes that are present in chromatin. Post-translational modifications of histones such as acetylation, phosphorylation, methylation, ubiquitylation, and SUMOylation can affect the properties of chromatin to enhance or suppress gene transcription (36). The ubiquitin-proteasome pathway has been suggested to be involved in elongation phase of transcription to chromatin remodeling/modification (37); and also in protein degradation of unbound RNA polymerase II associated factor (38). Therefore, downregulation of proteasome's proteases can have a profound effect on gene expression via histone modification, chromatin remodeling, and RNA polymerase II associated factor.

Proteasome inhibitors such as lactacystin bind to the active site of proteasome subunit X and prevent the activation of CT-like activity and thus inhibit the transcription of both inflammatory and anti-inflammatory LPS-induced genes and >90% of the LPS-induced genes (total 800 genes) are proteasome-dependent (12). Although 5 μM lactacystin (specific for X, high doses 20 μM affects other subunits) and LMP7 knockdown was unable to completely inhibit the upregulation of TNF-α in response to LPS (14), it now appears that cells containing either X, Y, Z, or LMP type proteasomes can induce TNF-α expression; therefore, blocking one subunit may not be sufficient to inhibit TNF-α. Thus, proteasome inhibitors may be useful in alleviating the effects of agonist-induced early inflammation. We have shown that proteasome inhibitors and antibiotics can prevent LPS-induced septic shock in a cecal-ligation and puncture model. A selective inhibitor of LMP7 has been used to reduce arthritis in mice (39). Our present data indicate that proteasomes play a vital role in both inflammation and tolerance. These findings support the role of proteasome's protease-sites as potentially valuable immunotherapy targets in treatment of initial stages of sepsis, and other diseases in which excessive inflammation may play a key role, such as cancer, asthma, cardiac problems, irritable bowel syndrome, and autoimmune diseases (40, 41); whereas proteasome activators such as IFN-γ may be needed when the cells become tolerant during septic shock, or in immunocompromised individuals. These studies are vital for understanding function of proteasome's subunits during inflammation and tolerance in mouse and human cells, and for design of therapeutic strategies for all diseases based on inflammation.

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The authors thank Dr David C. Morrison for helpful suggestions and Dr Stefanie Vogel for helpful discussions and critical review of the manuscript. The authors thank Xiu Qin Guan for excellent technical assistance with Western blots.

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Cytokines; differentiation; endotoxin shock; inflammation; macrophages; proteasomes

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