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. 2010 Apr 6;18(6):1143–1154. doi: 10.1038/mt.2010.53

Detrimental Effect of the Proteasome Inhibitor, Bortezomib in Bacterial Superantigen- and Lipopolysaccharide-induced Systemic Inflammation

Ashenafi Y Tilahun 1, Jayne E Theuer 1, Robin Patel 2, Chella S David 1, Govindarajan Rajagopalan 1
PMCID: PMC2889741  PMID: 20372109

Abstract

Bacterial superantigen (BSAg)–induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)–induced shock are characterized by severe systemic inflammation. As nuclear factor κB (NFκB) plays an important role in inflammation and bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NFκB activation, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using murine models. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, at 3 hours, serum level of TNF-a, an important cytokine implicated in TSS, was significantly reduced but not abolished. At 6 hours, there was no difference in the serum TNF-a levels between bortezomib treated and untreated mice challenged with staphylococcal enterotoxin B (SEB). Paradoxically, all mice treated with bortezomib either before or after BSAg challenge succumbed to TSS. Neither bortezomib nor BSAg was lethal if given alone. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB-challenged mice treated with bortezomib showed a significant reduction in NFκB activation. Because NFκB-dependent antiapoptotic pathways protect hepatocytes from TNF-α-induced cell death, inhibition of NFκB brought forth by bortezomib in the face of elevated TNF-α levels caused by BSAg or LPS is detrimental.

Introduction

Bacterial superantigens (BSAgs) are a family of exotoxins produced chiefly by the Gram-positive cocci, Staphylococcus aureus and Streptococcus pyogenes. BSAgs are unique in that they are probably the most potent biological activators of T lymphocytes.1 BSAg, in their native conformation, bind directly to cell surface major histocompatibility complex (MHC) class II molecules outside of the peptide-binding groove. Subsequently, they activate T cells by interacting with the variable region of the β chain (and in rare cases, α chain) of the T-cell receptor (TCR). Their ability to activate a large pool of T cells (30–70% of the total T cells) in an MHC class II–dependent, MHC-unrestricted, CD4, CD8 co-receptor-independent, TCR Vβ-specific, but antigen nonspecific manners, differentiate them from mitogens and conventional antigens.1

BSAgs can cause a spectrum of human diseases, ranging from self-limiting food poisoning to severe acute toxic shock syndrome (TSS)1 and could be used as biological weapons.2 TSS (either menstrual or nonmenstrual) has a rapid onset, often associated with high morbidity/mortality and is characterized by systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS).3 In spite of their clinical significance and their potential use as biological weapons, there are no specific therapies available for treating the acute systemic diseases caused by BSAg and they are treated only symptomatically. Because a robust superantigen-induced T-cell activation and the concomitant cytokine production are believed to be the underlying causes for TSS, it is theoretically possible to inhibit SIRS/MODS using inhibitors of T-cell activation and the cytokine cascade. In this context, the transcription factor, nuclear factor κB (NFκB), would be an ideal target for such inhibition because several proinflammatory pathways utilize NFκB.

The intracellular levels of transcriptionally activated NFκB is tightly maintained by the multicatalytic protease complexes called proteasomes, through controlling the proteolysis of the NFκB inhibitory protein, IκB. Therefore, proteasomes can strongly influence the production of proinflammatory cytokines through regulation of NFκB pathway,4,5 and several studies have shown that administration of proteasome inhibitors can suppress systemic cytokine storm in sepsis and related inflammatory conditions.6,7 However, the therapeutic role of proteasome inhibitors in BSAg-induced TSS has not been investigated. In this context, bortezomib is a novel proteasome inhibitor approved for clinical use (reviewed extensively by Terpos et al.8). It is a dipeptidyl boronic acid analogue that potently and selectively inhibits the chymotryptic activity of the proteasome. This is the first molecule in its class to be approved for clinical trials in cancer chemotherapy, particularly for the treatment of multiple myeloma. Bortezomib has been shown to effectively block TNF-α-induced activation of NFκB. As a result, bortezomib sensitizes cells to apoptotic death.8 By virtue of its ability to block activation of NFκB, bortezomib has been shown to effectively dampen systemic cytokine storm in certain animal models.9

Bortezomib is a reversible inhibitor of proteasome and is distributed very rapidly following parenteral delivery.8 It is metabolized primarily in the liver followed by the kidneys. Therefore, bortezomib has to be used with caution in patients with hepatic and renal complications. In addition, bortezomib can also cause other reversible side effects such as gastrointestinal toxicity, neuropathy, and reduction in blood cell counts.8 In the current study, we evaluated the role of bortezomib in BSAg-induced TSS with two main objectives. First, because bortezomib is known to block NFκB activation and possesses excellent pharmacokinetic properties,8 it could be used as an anti-inflammatory agent in TSS. Second, the toxicities associated with it might negate the anti-inflammatory effects of bortezomib and therefore could be contraindicated in TSS.

We have established that endogenous MHC class II–null mice transgenically expressing human MHC [human leukocyte antigen (HLA)] class II molecules (either HLA-DR3 or HLA-DQ8) mount a robust immune response to BSAgs when compared to conventional mice strains, and unlike conventional mice, these are readily susceptible to TSS without sensitizing agents.10,11 Therefore, we used HLA class II transgenic mice to screen bortezomib for therapy of BSAg-induced TSS. In this context, our recent study on the effect of bortezomib on staphylococcal enterotoxin B (SEB)-induced gene expression changes using microarrays showed that bortezomib was able to suppress the expression of several proinflammatory cytokine genes induced by SEB.10 Encouraged by these findings, we undertook the current study to further evaluate the use of bortezomib in superantigen-induced TSS. Paradoxically, our studies revealed that bortezomib sensitizes HLA class II transgenic mice to BSAg-induced TSS as well as lipopolysaccharide (LPS)-induced shock rather than conferring protection.

Results

Immune response elicited by SEB in HLA-DR3 transgenic mice and its suppression by bortezomib

BSAgs first bind to MHC class II molecules and subsequently activate T cells.1 It is known that BSAg interact more efficiently with human MHC class II molecules than to mouse class II molecules. Therefore, we and others have shown that HLA class II transgenic mice mount a more robust immune response to BSAg including SEB, when compared to the conventional laboratory mice expressing endogenous MHC class II molecules.10,11,12 As seen in Figure 1a, the extent of 5-(6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution was far greater in CD4+ and CD8+ T cells from HLA-DR3 transgenic mice at 48 and 72 hours when compared to B6 mice. T cells expressing TCR Vβ8, which are specifically stimulated by SEB, underwent 3–6 divisions in HLA-DR3 transgenic mice at 48 and 72 hours, respectively. However, T cells from B6 mice showed no signs of proliferation, and there was a complete overlap of CFSE histogram profiles of cells cultured with medium or SEB. Even though SEB primarily activates TCR Vβ8+ cells in HLA-DR3 transgenic mice, at 72 hours, minimal CFSE dilution could be noticed in TCR Vβ6+ T cells, which are not stimulated by SEB, probably due to bystander activation.

Figure 1.

Figure 1

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Superior SEB-induced immune activation in HLA-DR3 transgenic mice and its inhibition by bortezomib in vitro. (a) Splenic mononuclear cells from C57Bl/6 and HLA-DR3 transgenic mice were labeled with CFSE and stimulated with SEB. At the indicated time points, the intensity of CFSE in TCR Vβ6- (SEB-nonresponsive) and TCR Vβ8-gated (SEB-responsive) CD4+ and CD8+ T-cell subsets was analyzed by flow cytometry. Representative histograms are shown. (b) Splenic mononuclear cells from HLA-DR3 transgenic mice were cultured in 96-well tissue culture plates with indicated concentrations of SEB and bortezomib. Cell proliferation was measured by standard tritiated thymidine incorporation assay. Each bar represents mean ± SE from triplicate wells. (c) Supernatants from splenic mononuclear cells cultured as in b were analyzed by sandwich ELISA for indicated cytokines. Representative results of four similar experiments were shown. CFSE, 5-(6-) carboxyfluorescein diacetate succinimidyl ester; SEB, staphylococcal enterotoxin B; TCR, T-cell receptor.

We next studied whether SEB-induced T-cell proliferative response in HLA-DR3 transgenic mice could be abrogated by bortezomib in vitro. For this, splenic mononuclear cells from HLA-DR3 transgenic mice were cultured with varying concentrations of SEB in the absence or presence of decreasing concentrations of bortezomib. First of all, SEB induced a robust, dose-dependent proliferation in HLA-DR3 transgenic mice (Figure 1b), confirming the results in Figure 1a. Second, at concentrations between 10,000 and 10 ng/ml, bortezomib completely abrogated SEB-induced T-cell proliferation (Figure 1b). This inhibitory effect was lost at bortezomib concentrations ≤1 ng/ml. SEB-induced cytokine production was also significantly inhibited by bortezomib (Figure 1c). Whereas culture supernatants from SEB-stimulated HLA-DR3 splenocytes had very high levels of IL-2 and IL-6, inclusion of bortezomib at 1 µg/ml but not 1 ng/ml, significantly inhibited SEB-induced cytokine production. These results indicated that proteasome inhibition could block the immunostimulating effects of SEB, at least in the in vitro settings.

Suppression of SEB-induced systemic cytokine storm by bortezomib

We have shown previously that SEB can elicit a SIRS-like syndrome in HLA-DR3 transgenic mice similar to that seen in humans and that these mice succumb to TSS induced by SEB. We have also shown that systemic cytokine levels remain elevated for at least 6 hours and generally becoming undetectable by 24 hours.10 Because the majority of the proinflammatory cytokines that are implicated in TSS are under the transcriptional control of NFκB, we next investigated whether prior treatment of HLA class II transgenic mice with bortezomib on days 1 and 0 would dampen the SEB-induced systemic cytokine storm and confer protection from TSS.

As depicted in Figure 2, compared to naive HLA-DR3 transgenic mice, HLA-DR3 transgenic mice treated with SEB had significantly elevated serum levels of several cytokines and chemokines. As expected, serum cytokine levels were negligible in HLA-DR3 transgenic mice treated with bortezomib alone and in SEB-challenged mice that lack HLA-DR3 expression (i.e., transgene negative littermates). Prior conditioning with bortezomib significantly reduced the levels of most of the cytokines/chemokines tested in SEB-challenged HLA-DR3 transgenic mice. Nonetheless, it should be noted that SEB-challenged mice treated with bortezomib still had significant levels of cytokines in their sera when compared to naive HLA-DR3 transgenic mice. Cytokines such as IL-12p40, IL-12p70, IL-6, IFN-γ, MCP-1, IL-1α, GM-CSF, RANTES, eotaxin, MCP-1, IL-3, IL-4, and IL-5 were significantly reduced. At 3 hours, serum levels of TNF-α, an important cytokine implicated in TSS, were reduced but not abolished, and at 6 hours, there was no difference in the TNF-α levels between bortezomib-treated and untreated mice challenged with SEB. Even though statistically insignificant, the systemic levels of the proinflammatory cytokines IL-17 and IL-1β were appreciably decreased. Interestingly, CXCL1 was the only chemokine whose level was significantly increased in SEB-challenged mice treated with bortezomib at both 3 and 6 hours. For example, at 6 hours, HLA-DR3 mice challenged with SEB had about 750 pg/ml of CXCL1 in their sera, whereas HLA-DR3 mice pretreated with bortezomib and challenged with SEB had nearly 4,000 pg/ml of CXCL1 in their sera.

Figure 2.

Figure 2

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Systemic cytokine/chemokine storm following in vivo SEB challenge and its inhibition by bortezomib. Age-matched HLA-DR3 transgenic mice and the HLA-DR3 transgene negative littermates were treated with bortezomib (1 mg/kg) on days 1 and 0. Immediately following bortezomib treatment on day 0, mice were challenged with a single intraperitoneal injection of 10 µg of SEB or phosphate-buffered saline. Mice were bled at 3 and 6 hours later, and serum cytokine levels were determined using a multiplex suspension array system (Bio-Plex, Bio-Rad). Each bar represents the mean ± SE of five mice. SEB, staphylococcal enterotoxin B.

Increased mortality in SEB-challenged HLA-DR3 transgenic mice treated with bortezomib

Paradoxically, even though HLA-DR3 transgenic mice treated with bortezomib had significantly reduced systemic levels of several cytokines and chemokines following challenge with SEB, all mice in that treatment group were found dead (mortality: 6/6; Supplementary Table S1). When we carefully monitored the mortality pattern, about 90% of the mortality in the SEB- and bortezomib-treated group occurred within 18 hours, with the remaining occurring mostly within 48 hours and seldom extending beyond 72 hours. None of the mice that received either SEB alone or bortezomib alone died (mortality: 0/6 in each group; Supplementary Table S1). We next determined whether just one dose of bortezomib administered immediately following SEB challenge also rendered HLA-DR3 transgenic mice susceptible to SEB-induced TSS. As indicated in Supplementary Table S1, bortezomib rendered HLA-DR3 transgenic fully susceptible to TSS and mortality even if given immediately after SEB challenge (mortality: 6/6).

We next tested whether mortality in HLA-DR3 transgenic mice could be prevented by reducing the quantity of SEB administered. Administration of even 2 µg of SEB was lethal in HLA-DR3 transgenic mice when they were treated with bortezomib immediately following SEB challenge (mortality: 6/6; Supplementary Table S1). Sensitization to SEB-induced mortality by bortezomib appeared to be dependent on the expression of HLA-DR3 as neither the B6 mice nor any of the HLA-DR3 transgene negative littermates challenged even with a very high quantity (50 µg) of SEB died of TSS (mortality: 0/4 in each group; Supplementary Table S1). Thus, the heightened immune response to SEB, facilitated by expression of HLA-DR3 molecules, appeared to be necessary for sensitization to SEB-induced lethality by bortezomib.

We next studied whether reducing the dose of bortezomib would still render mice susceptible to SEB-induced mortality. For this, HLA-DR3 transgenic mice were challenged with 2 µg of SEB (for each mouse) and immediately treated with 0.5 mg/kg or 0.1 mg/kg of bortezomib. All HLA-DR3 transgenic mice (mortality: 5/5), challenged with SEB and treated with 0.5 mg/kg of bortezomib died, whereas none (mortality: 0/5) of the SEB-challenged HLA-DR3 transgenic mice treated with 0.1 mg/kg of bortezomib succumbed (additional data not shown). It should be noted that the routinely used pharmacologic dose of bortezomib is 0.8–1 mg/kg. Therefore, absence of mortality at 0.1 mg/kg might be due to lack of its pharmacological activity, i.e., lack of proteasome inhibition.

In the next set of experiments, we determined whether delaying the administration of bortezomib would reverse this detrimental effect and protect from TSS. Because, even in the clinical settings if bortezomib is to be used as a therapeutic agent in TSS, it would be administered at various time points after exposure to superantigens. Therefore, HLA-DR3 transgenic mice were challenged with 2 µg of SEB (for each mouse). Mice were treated with bortezomib at 0, 3, 6, or 24 hours after SEB challenge. As indicated in Supplementary Table S2, although none of the HLA-DR3 transgene negative littermates succumbed (mortality: 0/4 at each time point tested), all transgenic mice expressing HLA-DR3 died even when bortezomib was administered 24 hours after SEB challenge (mortality: 4/4 at each time point tested). Thus, sensitization to SEB-induced mortality by bortezomib occurred even up to 24 hours after exposure to SEB.

Bortezomib is a reversible inhibitor of proteasomes. Therefore, to further specify whether the mortality is actually due to proteasome inhibition, we administered bortezomib (1 mg/kg) to HLA-DR3 transgenic mice. These mice were challenged with SEB immediately afterward, or after a 24- or 48-hour delay. Only mice challenged immediately with SEB died (mortality: 4/4), whereas those challenged at 24 and 48 hours later survived (mortality: 0/4 mice in each group) (additional data not shown). However, these surviving SEB-challenged mice succumbed to TSS when they were administered with another dose of bortezomib the next day (after 18 hours of SEB challenge) (additional data not shown). These experiments confirmed that bortezomib-mediated proteasome inhibition sensitized mice to TSS.

Effect of lactacystin on SEB-induced TSS

Given the paradoxical observations made with bortezomib, we next studied whether the other well-known proteasome inhibitor lactacystin has the similar property. We tested two different concentrations of lactacystin, i.e., 10 and 40 µg/mouse. Immediately following SEB challenge (2 µg/mouse), HLA-DR3 transgenic mice received lactacystin and were closely monitored. During the follow-up, none of the SEB-challenged HLA-DR3 transgenic mice treated with lactacystin succumbed (mortality: 0/4 mice for each dose of lactacystin) (additional data not shown).

NBD inhibitor peptide does not sensitize HLA class II transgenic mice to BSAg-induced TSS

We next addressed whether other known inhibitors of NFκB pathway also sensitize mice to TSS. We used the IκB kinase-γ NEMO-binding domain (NBD) inhibitor peptide for this study. This peptide has been shown to effectively inhibit NFκB activation in vivo in mice in several inflammatory disease models, at concentrations much lower than that used in this study (reviewed in ref. 13).

Briefly, HLA-DR3 transgenic mice were challenged with 2 µg of SEB followed by 500 µg of wild-type (WT)-NBD or mutant-NBD peptide. None of the animals treated with WT-NBD (or mutant-NBD) peptide and SEB died indicating that blockade of NFκB per se does not sensitize to SEB-induced TSS (data not shown). Furthermore, a recent study has suggested that bortezomib could activate NFκB pathway at least in certain cell types.14 Therefore, we next addressed the question whether sensitization to TSS by bortezomib is due to induction of NFκB by bortezomib and whether this can be reversed by NFκB blockade. HLA-DR3 transgenic mice were challenged with 2 µg of SEB and subsequently treated with 1 mg/kg of bortezomib along with 500 µg WT-NBD or mutant-NBD peptide. None of the animals survived (data not shown). In another set of experiments, we increased the frequency of NBD peptide administration. WT-NBD or mutant-NBD peptides (250 µg) were administered 30 minutes prior to and 3 hours after SEB and bortezomib challenge. Animals were monitored daily. None of the animals survived this regimen as well (data not shown).

Neutralization of CXCL1 does not protect HLA-DR3 mice from bortezomib-sensitized SEB-induced mortality

We wished to understand the mechanisms by which bortezomib uniformly sensitized mice to SEB-induced mortality. Because elevated systemic CXCL1 level was the only factor that could possibly be associated with this effect, we tested whether in vivo neutralization of CXCL1 could reverse this effect. As a first step, we established that the commercially available polyclonal goat anti-mouse CXCL1 antibody was capable of significantly neutralizing SEB-induced CXCL1 levels (Figure 3). We next challenged HLA-DR3 mice with SEB and treated them with bortezomib followed by goat anti-CXCL1 or goat IgG. All SEB-challenged mice treated with bortezomib died irrespective of whether they received goat anti-CXCL1 (mortality: 4/4 mice) or goat-IgG control antibodies (mortality: 4/4 mice), implying that sensitization to SEB-induced mortality by bortezomib is not mediated through elevated CXCL1 levels.

Figure 3.

Figure 3

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Induction of CXCL1 by SEB and its neutralization by anti-CXCL1 antibodies. Age-matched HLA-DR3 transgenic mice were challenged with a single intraperitoneal injection of 10 µg of SEB, immediately treated with affinity-purified goat anti-mouse CXCL1 antibodies or the control antibody (100 µg/mouse). Mice were bled 3 hours later, and serum CXCL1 levels were measured using a multiplex suspension array system (Bio-Plex, Bio-Rad). Each bar represents the mean ± SE of four mice. PBS, phosphate-buffered saline; SEB, staphylococcal enterotoxin B.

Sensitization to SEB-induced mortality by bortezomib—dependence on HLA class II, T-cell subsets, cytokines, and co-stimulation

We next determined whether bortezomib could sensitize mice to SEB-induced TSS only in the context of HLA-DR3 or whether it is applicable to other HLA class II molecules as well. For this, we challenged HLA-DQ8 transgenic mice with either 10 or 2 µg of SEB alone, bortezomib alone (1 mg/kg), or SEB followed by bortezomib. It is known that SEB binds to HLA-DQ with lower affinity than to HLA-DR,15 therefore, higher amounts of SEB is required to induce mortality in HLA-DQ8 transgenic mice.16 Nonetheless, all HLA-DQ8 mice that received either 10 or 2 µg of SEB followed by bortezomib died within 48 hours, whereas none of the mice challenged with SEB alone or bortezomib alone died (Supplementary Table S3). As discussed earlier, none of the B6 mice treated with even 50 µg of SEB followed by bortezomib died, suggesting the specificity toward HLA class II molecules.

We have developed HLA-DQ8 transgenic mice deficient in CD4+ or CD8+ T-cell subsets to delineate their roles in the immunopathogenesis of TSS. We determined whether sensitization to SEB-induced TSS by bortezomib is dependent on either of these T-cell subsets. As indicated in Supplementary Table S3, deficiency of either CD4+ or CD8+ T-cell subsets did not protect HLA-DQ8 transgenic mice from death suggesting independent roles for CD4+ and CD8+ T cells. We also used the STAT4 and STAT6-deficient HLA-DQ8 transgenic mice to determine whether alterations in the Th1 and Th2 type cytokines could modulate bortezomib-enhanced sensitivity to TSS. STAT4 is required for signaling through IL-12 and IL-23 receptors, whereas STAT6 is required for signaling through IL-4 receptors.17 As depicted in Supplementary Table S3, deficiency in STAT4 or STAT6 did not make any difference in the sensitivity of HLA-DQ8 transgenic mice to SEB-induced mortality potentiated by bortezomib, suggesting that this effect is not due to cytokine imbalance. Similarly, all HLA-DR3.IFN-γ−/− transgenic mice challenged with SEB and treated with bortezomib died, suggesting that the effect is independent of IFN-γ.

We have shown that HLA-DQ8 transgenic mice lacking the co-stimulatory molecule CD28 are resistant to lethality induced by SEB at doses that are uniformly lethal to WT HLA-DQ8 transgenic mice due to reduced systemic cytokine production.16 Nonetheless, it should be noted that SEB-induced immune responses are more robust in HLA-DQ8.CD28−/− mice than in B6 mice. Therefore, we next studied whether bortezomib could render HLA-DQ8.CD28−/− mice susceptible to SEB-induced lethality. As indicated in Supplementary Table S3, every one of HLA-DQ8.CD28−/− mice succumbed to TSS even at 10 or 2 µg of SEB/mouse when given along with bortezomib, whereas none of the mice receiving either SEB or bortezomib alone died. These consistent observations imply that bortezomib sensitizes mice to SEB-induced TSS in HLA class II transgenic mice because they mount a strong immune response to BSAg.

Induction of lymphocyte apoptosis by bortezomib and the effect of immunostimulation with SEB

Proteasome inhibitors such as bortezomib have been shown to induce apoptosis in lymphocytes.18 As could be inferred from Figure 1b, not only SEB-induced proliferation, but the thymidine counts in lymphocyte cultures incubated with medium alone were also dramatically reduced at bortezomib concentrations between 10,000 and 10 ng/ml (Supplementary Figure S1). This indicated that bortezomib is toxic to splenic mononuclear cells. Therefore, we investigated whether sensitization to SEB-induced lethality by bortezomib is because of its ability to induce apoptosis in vitro and in vivo. For this, splenic mononuclear cells from HLA-DR3 transgenic mice were incubated with 0, 1, or 0.001 µg/ml of bortezomib in the presence or absence of SEB (1 µg/ml). After 18 hours of culture, the percentage of apoptotic cells was determined by flow cytometry by annexin V/7AAD staining. As indicated in Supplementary Figure S1b, at 1 µg/ml, bortezomib induced apoptosis in about 85% of the mononuclear cells, whereas at 0.001 µg/ml, the extent of apoptosis was comparable to that seen with media alone. This indicated that bortezomib induces apoptosis at higher concentrations. We next studied the ability of bortezomib to induce apoptosis in SEB-stimulated cells.

In the absence of bortezomib, inclusion of SEB in cultures significantly reduced the percentage of annexin V+ cells (Supplementary Figure S1b). However, in the presence of bortezomib (1 µg/ml), the protective effect of SEB was lost and about 85% of the cells were still annexin V+. This implies that the extent of apoptosis induced by bortezomib is not modulated by the activation status of the cells. At the lower concentration of bortezomib (0.001 µg/ml, which does not significantly induce apoptosis), SEB was able to rescue a significant percentage of cells from apoptosis. Similar results were obtained when the data were analyzed by multiple methods such as analysis of forward scatter/side scatter profiles (Supplementary Figure S1b–e), including manual counting of live and dead cells by Trypan blue dye exclusion test (data not shown). These results indicated that bortezomib at higher concentration was capable of inducing apoptosis in vitro.

We next studied whether bortezomib-induced apoptosis in splenic/thymic mononuclear cells in vivo at the systemic concentrations achieved following intraperitoneal administration. For this, mice were challenged with a single dose of bortezomib at 1 mg/kg and killed 18 hours later. Annexin V staining of mononuclear cells from spleens or thymus did not reveal any significant increase in the percentage of apoptotic cells either in the ungated or CD3-gated populations indicating at therapeutic concentrations, bortezomib does not cause apoptosis in vivo (Supplementary Figure S2).

Modulation of bortezomib-induced apoptosis by pan-caspase inhibitor Z-VAD-FMK

Although apoptosis was not significantly high in bortezomib-treated mice and appeared not to be the cause for increased mortality, we nevertheless wished to completely rule out this possibility. Apoptosis induced by bortezomib has been shown to be abolished by the pan-caspase inhibitor Z-VAD-FMK.18 Therefore, we challenged HLA-DR3 mice with SEB, treated with bortezomib and administered Z-VAD-FMK or its control peptide Z-FA-FMK. All of HLA-DR3 transgenic mice challenged with SEB and bortezomib died irrespective of whether they received Z-VAD-FMK (mortality: 5/5) or Z-FA-FMK (mortality: 5/5). None of the mice receiving these compounds individually died (data not shown). This further confirmed that sensitization to SEB-induced TSS by bortezomib was not due to enhanced caspase-dependent apoptosis.

Potentiation of LPS-induced mortality by bortezomib

We next studied whether bortezomib could sensitize mice only to BSAg-induced mortality or to other acute systemic inflammatory conditions. Unlike to BSAg, B6 mice respond robustly to LPS. HLA class II transgenic mice also respond efficiently to LPS as they express endogenous murine TLR4 as in B6 mice. Therefore, we challenged them with LPS and administered bortezomib. As shown in Supplementary Table S4, bortezomib sensitized B6 as well as HLA-DQ8 transgenic mice to LPS-induced shock at concentrations of LPS that are otherwise nonlethal. Thus, bortezomib potentiated lethal effects of BSAg as well as LPS.

Liver dysfunction in bortezomib-treated mice following SEB challenge

To gain insights into the possible mechanisms behind sensitization to mortality by bortezomib, HLA-DR3 transgenic mice were challenged with SEB and treated with bortezomib at time 0. Body temperature, general activity, and body weights were recorded once every 3 hours. Groups of mice were killed every 3 hours and sera collected for biochemical analyses; tissues and organs were collected in buffered formalin for histopathology. In this experimental cohort, majority of the mice challenged with SEB and treated with bortezomib died at 6-hour time point or were killed humanely as they were moribund. Thus, we had only two time points, i.e., 3 and 6 hours.

As shown in Figure 4, the body temperature of the mice that received SEB and bortezomib dropped drastically. The physical activity also significantly reduced with time, thus correlating with high mortality in this group. Biochemical analyses of sera indicated a dramatic elevation in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in sera of SEB+bortezomib group, especially at 6-h time point (Figure 4), indicating significant hepatocyte death. There were no differences in serum creatinine levels between different groups at these time points (data not shown). As liver enzymes were significantly elevated, we performed TUNEL staining to detect apoptosis and also evaluated the hematoxylin and eosin–stained liver sections from various groups of mice.

Figure 4.

Figure 4

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Clinical parameters in SEB-challenged mice treated with bortezomib. Age-matched HLA-DR3 transgenic mice were challenged with SEB (2 µg/mouse) or PBS. Mice were further divided into two groups, received either bortezomib (1 mg/kg) or PBS immediately after SEB. Physical activity and rectal temperature were recorded at 3-hour intervals. Groups of mice were killed at the indicated time points, sera collected for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) determination. Temperature and activity data—mean ± SE from 6 to 8 mice per group. AST and ALT data—sera pooled from 3 to 4 mice per group. The entire experiment was repeated at least three times with similar results. PBS, phosphate-buffered saline; SEB, staphylococcal enterotoxin B.

At 3 hours, although phosphate-buffered saline- and SEB-treated mice showed no apoptotic cells, liver sections from mice treated with bortezomib alone showed some cells that were stained positive. Interestingly, mice receiving both SEB and bortezomib showed similar but not increased distribution of apoptotic cells as compared to mice that received bortezomib alone (Figure 5). This correlated well with similar serum AST and ALT levels seen in these two groups at 3 hours (Figure 4). We did not notice any significant apoptosis at 3 hours in kidneys or lungs (Figure 5). Based on the serum AST and ALT levels, we expected a more widespread hepatocyte apoptosis at 6 hours than at 3 hours. On the contrary, we did not see increased apoptosis in SEB+bortezomib group or in bortezomib group at 6 hours by TUNEL staining. However, liver sections from SEB+bortezomib-treated group showed significant necrotic changes by light microscopy. Under lower magnification (Figure 6, left panels), almost all of the vessels were filled with fibrinoid material and red blood cells. Under higher magnification (Figure 6, right panels), extensive hepatocyte damage was noticed suggesting necrotic cell death. These histological parameters correlated well with elevated serum AST and ALT levels suggestive of hepatic dysfunction as a possible cause of death in SEB-challenged mice treated with bortezomib. Liver sections from phosphate-buffered saline- and bortezomib-treated mice had normal liver histology. Although sections from SEB-challenged mice showed mild hyperemia, hepatocytes were largely intact. We did not observe any appreciable histopathological changes in other tissues/organs, including lung, kidneys, heart, and the gut at 6 hours (data not shown).

Figure 5.

Figure 5

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Hepatocyte apoptosis in bortezomib-treated mice. Lung, liver, and kidneys were collected 3 hours following SEB challenge, and bortezomib treatment was fixed in paraffin, sectioned and stained by TUNEL technique. Positive and negative controls for TUNEL were set up as per the manufacturer's protocol. SEB, staphylococcal enterotoxin B.

Figure 6.

Figure 6

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Liver pathology in SEB-challenged mice treated with bortezomib. Formalin-fixed, paraffin-embedded liver sections from the indicated groups of mice treated as in Figure 5 were collected at 6 hours and stained with H&E for (a) light microscopy or TUNEL stained for (b) fluorescent microscopy. Positive and negative controls for TUNEL were set up as per the manufacturer's protocol. PBS, phosphate-buffered saline; SEB, staphylococcal enterotoxin B. PC, positive control; NC, negative control.

Decreased hepatic NFκB activation in bortezomib-treated mice following SEB challenge

Given the known effect of bortezomib on NFκB pathway, we next studied whether NFκB activation occurs in liver following SEB challenge and whether bortezomib has any influence on this process. Liver tissue from different groups of mice were harvested 1 hour after respective treatment, and the levels of NFκB subunits p65 and p50 in nuclear extracts were quantified by ELISA as described in Materials and Methods. As shown in Figure 7, liver tissue from naive mice and mice treated with bortezomib alone had very low levels of p65 and p50 in their nuclear extracts. Nuclear extracts from mice challenged with SEB had significantly elevated levels of these two subunits suggesting robust NFκB activation following SEB challenge. However, bortezomib treatment significantly blunted this activation, suggesting that inhibition of NFκB activation in liver is probably responsible for the detrimental effect of bortezomib seen in SEB-challenged mice.

Figure 7.

Figure 7

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SEB-induced NFκB activation in liver and its modulation by bortezomib. Nuclear extracts from the indicated groups of mice were collected 1 hour after respective treatment. Levels of p65 and p50 were quantified using TransAM ELISA as per the manufacturer's protocol. Values indicate mean ± SE from two different experiments. SEB, staphylococcal enterotoxin B.

Discussion

Superantigen exotoxins produced by S. aureus and S. pyogenes bind directly to MHC class II molecules without undergoing any antigen processing. MHC class II–bound BSAg can vigorously activate large number of CD4+ as well as CD8+ T cells expressing certain TCR Vβ families irrespective of their antigen specificities. Exposure to BSAg that are either preformed or produced in vivo following bacterial infection can cause a robust immune activation, systemic cytokine storm, and multiple organ dysfunction.1 TSS is an acute systemic illness caused by BSAgs and could cause high mortality, if not treated promptly. Unfortunately, specific therapies for TSS are still lacking. Given the role of immune activation and proinflammatory cytokines in the pathogenesis of TSS, drugs that can block or dampen the inflammatory pathways would be of choice for treating TSS.

Bortezomib, a highly selective, reversible inhibitor of 26S proteasomes, has been approved for clinical use to treat certain malignancies and displays a wide array of biological functions.8,19 However, inhibition of NFκB pathway is a well-established outcome of bortezomib-mediated proteasome inhibition. Because NFκB pathway plays an important role in several inflammatory pathways, bortezomib has been shown to attenuate chronic inflammatory diseases like arthritis, experimental models of multiple sclerosis,20 and other systemic inflammatory responses.21,22 Given these established anti-inflammatory activities of bortezomib, we evaluated its role in the prevention/treatment of BSAg-induced TSS, using HLA class II transgenic mice. Contrary to our expectations, all mice challenged with SEB and treated with bortezomib died due to liver failure possibly by the following mechanism.

The superantigen SEB induced a robust systemic cytokine storm in vivo and bortezomib significantly dampened this process, as has been reported in other models of inflammation.9 Paradoxically, all of the HLA class II transgenic mice challenged with SEB and treated with bortezomib succumbed to TSS. Bortezomib sensitized only HLA class II transgenic mice to SEB-induced TSS at doses of SEB that are otherwise seldom lethal. This effect was HLA class II dependent as HLA class II transgene negative littermates and B6 mice were resistant to SEB-induced mortality. Thus, a heightened immune response to SEB imposed by HLA class II molecules is a mandatory event. Because HLA class II transgenic mice mount a robust immune response to SEB, bortezomib was not able to attenuate all of this response. Even though some proinflammatory cytokines were reduced, these mice still had significant levels of cytokines when compared to naive mice. More importantly, serum levels of important cytokines, such as TNF-α, were reduced but not completely abolished by bortezomib in mice challenged with SEB (Figure 2a).

TNF-α plays a very important role in BSAg-induced TSS as well as LPS-induced shock.23,24 TNF-α can bring forth two completely opposite outcomes in hepatocytes.25 Binding of TNF-α to its predominant receptor TNF-R1 can elicit a pro- or an antiapoptotic response depending upon the signaling complex recruited to the receptor.26 Signalosome complex 1 (SC1) consisting of TNF-R-associated death domain, TNF-R associated factor-2, and receptor interacting protein (RIP)1 results in activation of IκB kinase, phosphorylation, ubiquitination, proteasome-dependent cleavage of IκB, and activation of NFκB (reviewed in ref. 27). Activation of NFκB results in upregulation of a variety of antiapoptotic molecules, which provide the survival signal. On the contrary, in the absence of SC1 and NFκB activation, a distinct signalosome complex 2 (SC2), consisting of Fas-associated death domain and pro-caspase-8 without TNF-R1 is formed in the cytosol that leads to activation of caspases culminating in cell death.28 Challenging mice with SEB alone induces TNF-α, which would bind to TNF-R1. This would elicit signaling through the SC1 and upregulation of antiapoptotic molecules through the NFκB pathway and very little cell death. In mice challenged with SEB and treated with bortezomib, which still have high levels of TNF-α particularly at 6 hours (Figure 2a), the TNF-R1-dependent, SC1-mediated upregulation of antiapoptotic molecules through the NFκB pathway will not occur as bortezomib would inhibit NFκB activation. Therefore, TNF-R1-mediated signaling would occur through SC2, resulting in massive hepatocyte cell death culminating in mortality of animals.

It is well known that hepatocytes are extremely sensitive to NFκB depletion29 and inhibition of NFκB in the hepatocytes can render them extremely sensitive to the cytotoxic effects of proinflammatory cytokines (such as TNF-α) in different models of liver injury.30,31,32 For example, Beraza et al. have shown that mice in which the nonenzymatic regulatory subunit NEMO (NFκB essential modulator, a necessary component for activation of NFκB) is selectively deleted in liver, 50% of the animals die within 6 hours of inducing ischemia/reperfusion injury, and the remaining 50% succumb within the next 18 hours. Hepatocyte apoptosis, necrosis, and elevated AST/ALT levels strikingly similar to our current study were seen in these mice hepatocyte-specific NEMO-deleted mice. However, they observed no mortality in mice that can normally upregulate NFκB in their hepatocytes.33 Other studies have also shown that hepatocyte apoptosis can rapidly progress to necrosis.25,33,34 This explains the presence of very few apoptotic cells at 6 hours, and histological sections showed evidence of massive liver necrosis. Put together, in our model, SEB alone induces a heightened systemic inflammatory response characterized by elevated cytokines including TNF-α. However, without further pharmacological manipulation, TNF-α activates NFκB pathway (Figure 7) and transduces a survival signal in hepatocytes. However, in SEB-challenged mice treated with bortezomib, as the NFκB pathway is inhibited by bortezomib (Figure 7) as shown in other mouse models,35,36,37 the prosurvival pathway fails to initiate, the death pathway is activated, and massive hepatocyte cell death ensues.

It is also well known that TNF-R1-mediated activation of RIP1 in the signalosome 1 in the presence of caspase inhibitors results in a different form of cell death called necroapoptosis (reviewed in ref. 38). In mice challenged with SEB and treated with bortezomib, signaling through TNF-R would activate RIP1. However, the hepatocytes cannot produce RIP1-induced NFκB due to bortezomib-mediated proteasome inhibition as discussed above. Normally, these cells would die by caspase-dependent apoptosis. However, in the presence of caspase inhibitors, the hepatocytes cannot proceed to die by apoptosis by caspase-dependent pathway and the RIP1-RIP3-dependent cell death process would ensue.39 This explains the inability of Z-VAD-FMK, a caspase inhibitor, to protect SEB-challenged HLA-DR3 transgenic mice from death caused by bortezomib. Because HLA-DR3 transgene negative littermates as well as B6 mice do not respond to superantigens and do not have very high levels of proinflammatory cytokines, they are not sensitized to death by bortezomib. As C57Bl/6 as well as HLA class II transgenic mice can respond to LPS, bortezomib sensitized them to mortality. Another study has also reported that combination of LPS and bortezomib can result in very high mortality in mice.40 It should be noted that at equimolar concentrations, SEB is far more efficient in activating the immune response than LPS. This explains why even extremely smaller dose of SEB is sufficient to sensitize HLA class II transgenic mice to mortality in the presence of bortezomib.

Bortezomib is predominantly metabolized in the liver by a cytochrome P450-dependent process.8 This might result in preferential accumulation of the drug in the hepatocytes and sensitize them to death. In addition, bortezomib has been shown to sensitize hepatocellular carcinoma cells to TRAIL-induced apoptosis.41 Although bortezomib does not sensitize normal human hepatocytes to TRAIL-induced apoptosis,41 we cannot rule out this possibility in the inflammatory settings of TSS. In similar lines, bortezomib could also sensitize hepatocytes to apoptosis through inhibition of phosphatidylinositol 3-kinase/Akt pathway.42 Thus, there are several mechanisms by which bortezomib can cause hepatocyte cell death. Bortezomib is known to cause certain conditions such as ischemic colitis in cancer patients.8 However, in our study, we did not see any histopathological evidence for death due to gastrointestinal involvement. Based on the rapidity at which the deaths occurred, serum biochemical parameters, histopathology, and NFκB levels, we believe that massive hepatocyte death might be the primary reason for mortality.

Elevated CXCL1 level is unlikely to be solely responsible for the detrimental effect of bortezomib as neutralization of CXCL1 did not revert this effect. It is also possible that bortezomib may be directly toxic to mice. Nevertheless, several mouse studies have used the dose of 1 mg/kg and have administered bortezomib repeatedly to mice without adverse effects.20,43,44 Also, none of the mice treated with bortezomib alone nor any of the SEB-treated B6 mice or HLA-DR3 transgene negative littermates receiving the same dosing of bortezomib died in our study. These observations argued against the toxicity of bortezomib. The pharmacokinetics and pharmacodynamics of lactacystin, the other proteasome inhibitor used in this study, is very different from bortezomib.45,46 Thus, bortezomib but not lactacystin has been approved for clinical use. Therefore, we cannot directly compare the two agents and warrants a separate investigation. Similar arguments would apply for the NBD inhibitor used in this study.

Traditionally, proteasome inhibitors are believed to have beneficial effects in a variety of inflammatory conditions.21 However, in the current study, we show the paradoxical toxic effects of a proteasome inhibitor in acute systemic inflammatory conditions such as TSS and sepsis. Whether our observation has any implication regarding the outcome of bacterial infections/sepsis in patients who are already on bortezomib therapy remains to be addressed. Because patients receiving bortezomib have several other underlying complications and receive other therapies, the effect of bortezomib could be underappreciated. Incidentally, case reports of severe hepatitis with elevated ALT and AST levels in patients with bacterial sepsis receiving bortezomib underscore the clinical significance of our findings.47,48 In conclusion, we have shown that the bortezomib therapy rendered mice susceptible to the lethal effects of BSAg and LPS. Although proteasomes regulate variety of intricately linked cellular/molecular processes,49 the detrimental effect of bortezomib appears to be mediated through inhibition of NFκB.49 Our results suggest a careful evaluation of therapeutic use of proteasomal inhibitors in systemic inflammatory conditions.21

Materials and Methods

Mice. Previously described AE.HLA-DR3 transgenic mice expressing functional HLA-DRA1*0101 and HLA-DRB1*0301 transgenes on the complete mouse MHC class II–deficient background were used in this study. HLA-DQ8 transgenic mice and HLA-DQ8.CD4−/−, HLA-DQ8.CD8−/−, HLA-DQ8.STAT4−/−, HLA-DQ8.STAT6−/−, and HLA-DQ8.CD28−/− have been described elsewhere.10,12 HLA-DR3.IFN-γ−/− transgenic mice were generated by crossing HLA-DR3 transgenic mice and B6.IFN-γ−/− mice (Jackson Laboratory, Bar Harbor, ME). C57Bl/6 mice, originally from Jackson Laboratory, were bred and maintained in our animal facility. Mice were bred within the barrier facility of Mayo Clinic Immunogenetics Mouse Colony (Rochester, MN) and moved to a conventional facility after weaning. All experiments were approved by the Institutional Animal Care and Use Committee.

Reagents and antibodies. Bortezomib (Velcade; Millennium Pharmaceuticals, Cambridge, MA) was obtained from Mayo Clinic Pharmacy (Mayo Clinic, Rochester, MN). It was used either within 2 days of reconstitution or stored frozen in aliquots at −80 °C. Frozen bortezomib was used within 15 days. Lactacystin was obtained from Sigma (St Louis, MO). Pan-caspase inhibitor Z-VAD-FMK and Z-FA-FMK (an inhibitor of cathepsins B and L used routinely as a control for Z-VAD-FMK) were purchased from MP Biomedicals (Solon, OH), reconstituted in dimethyl sulfoxide and stored frozen in aliquots. Endotoxin-reduced highly purified SEB (Toxin Laboratories, Sarasota, FL) was dissolved in phosphate-buffered saline at 1 mg/ml and stored frozen at −80 °C in aliquots. Goat anti-CXCL1 neutralizing antibody and its isotype controls were from R&D Systems (Minneapolis, MN). Escherichia coli (055:B5) lipopolysaccharide (LPS) was obtained from Sigma. The following antibodies were used for flow cytometry. The IκB kinase-γ NBD WT inhibitor peptide and the mutant-NBD peptide that lacks the inhibitory potential were purchased from Imgenex, San Diego, CA. Anti-TCR Vβ8 (F23.1), anti-TCR Vβ6 (RR4-7), anti-CD4 (RM 4.5), and anti-CD8 (53.67) (BD Biosciences, San Diego, CA).

Determination of cell proliferation by CFSE dilution. Splenic mononuclear cells from C57Bl/6 or HLA-DR3 transgenic mice were labeled with 5 µmol/l of CFSE (Molecular Probes, Invitrogen, Carlsbad, CA) as per manufacturer's directions. Subsequently, cells were washed twice with RPMI-1640, plated in 24-well plates and cultured with medium alone or SEB (1 µg/ml). Cells were collected at 48 and 72 hours, and the extent of CFSE fluorescence in CD4+ and CD8+ T-cell subsets expressing TCR Vβ6 and Vβ8 was determined by flow cytometry.

T-cell proliferation and cytokine assay. Single-cell suspensions of splenocytes from HLA-DR3 transgenic mice were depleted of red blood cells by buffered ammonium chloride lysis. Cells were cultured in HEPES-buffered RPMI-1640 containing 5% horse serum, serum supplement, streptomycin, and penicillin, at a concentration of 10 × 105 cells/well in 100 µl volumes in 96-well flat-bottomed tissue culture plates. SEB and bortezomib were diluted in complete medium and added to cells. Cell proliferation was determined by standard [3H]thymidine incorporation. Concentrations of cytokines in the culture supernatant were estimated by sandwich ELISA using BD Biosciences capture antibodies, biotinylated detection antibodies and standards, per standard procedures.

In vivo administration of bortezomib, lactacystin, SEB, caspase inhibitor, and chemokine neutralizing antibodies. Bortezomib was used at a dose of 1 mg/kg as has been used in several murine studies, without any apparent toxicity. This dose even given repeatedly is well tolerated by mice.50 SEB was injected as indicated in a final volume of 200 µl. In some experiments, mice received either 10 or 40 µg of lactacystin intraperitoneally immediately following SEB challenge. Z-VAD-FMK and Z-FA-FMK were injected at 500 µg/mouse at times 0 and 12 hours. In some experiments, immediately following SEB challenge, mice were injected with 100 µg of goat anti-mouse CXCL1 antibody or its isotype control, along with bortezomib or phosphate-buffered saline. Body temperature (rectal), body weight, and physical activity/alertness of the mice were recorded where indicated. Physical activity was scored on a scale of 1–4, with normal activity being 4.

Serum biochemical studies, cytokine quantification, and histopathology. At the time of killing at the indicated time points, blood from experimental mice was collected in serum separation tubes (BD Biosciences), sera separated and stored frozen at −80 °C in aliquots. Lung, liver, and kidneys were collected in buffered formalin for histopathological analysis. Serum levels of AST, ALT, and creatinine were determined at the Mayo Clinic clinical chemistry laboratory. Sera from 3 to 4 mice from each group were pooled for biochemical studies. The cytokine concentrations in individual serum samples were determined using a multiplex bead assay, per the manufacturer's protocol and using their software and hardware (Bio-Plex; Bio-Rad, Hercules, CA).

Detection of apoptosis by annexin V staining and TUNEL. Apoptosis of cells in suspension was determined by annexin V and 7-AAD staining as per the vendor's instructions (BD Biosciences) and analyzed by flow cytometry immediately following staining. Apoptosis in tissue preparations were ascertained on thin sections of the paraffin-embedded blocks on microscopic glass slides using in situ cell death detection kits following manufacturer's directions (Roche, Indianapolis, IN). As a positive control for the TUNEL reaction, sections were incubated with DNAse I to introduce DNA strand breaks, prior to adding the reaction mix. Negative control sections were handled the same samples except that the terminal deoxyribonucleotidyl transferase was omitted from the reaction mix. After staining sections were evaluated using an Olympus AX70 research microscope (Olympus America, Center Valley, PA). Images were acquired using an Olympus DP70 camera (Olympus America).

Detection of NFκB activation in liver by transcription factor ELISA. Liver tissue from naive, SEB, bortezomib, or SEB+bortezomib treated HLA-DR3 transgenic mice were collected 60 minutes later, finely chopped using a sharp blade and collected in a prechilled 7 ml Wheaton glass homogenizer. Nuclear extracts from these tissues were obtained using the nuclear extract kit following the manufacturer's protocol (Active Motif, Carlsbad, CA). NFκB activation in the liver extracts was quantified using a commercial ELISA kit (TransAM; Active Motif) as per the manufacturer's protocol.

SUPPLEMENTARY MATERIALFigure S1. Induction of apoptosis in splenic mononuclear cells in vitro by bortezomib.Figure S2. Induction of apoptosis in splenic mononuclear cells in vivo by bortezomib.Table S1. Potentiation of SEB-induced mortality by bortezomib.Table S2. Potentiation of SEB-induced mortality by bortezomib: time kinetics.Table S3. Potentiation of SEB-induced mortality by bortezomib in HLA-DQ8 transgenic mice.Table S4. Potentiation of LPS-induced mortality by bortezomib.

Supplementary Material

Figure S1.

Induction of apoptosis in splenic mononuclear cells in vitro by bortezomib.

Click here for supplemental data (6.4MB, tiff)
Figure S2.

Induction of apoptosis in splenic mononuclear cells in vivo by bortezomib.

Click here for supplemental data (490.1KB, tiff)
Table S1.

Potentiation of SEB-induced mortality by bortezomib.

Click here for supplemental data (33KB, doc)
Table S2.

Potentiation of SEB-induced mortality by bortezomib: time kinetics.

Click here for supplemental data (29.5KB, doc)
Table S3.

Potentiation of SEB-induced mortality by bortezomib in HLA-DQ8 transgenic mice.

Click here for supplemental data (37KB, doc)
Table S4.

Potentiation of LPS-induced mortality by bortezomib.

Click here for supplemental data (27.5KB, doc)

Acknowledgments

We thank Julie Hanson and Michele Smart for mouse husbandry and mouse genotyping. We also thank Gregory Gores, Professor of Medicine and Physiology, Mayo Clinic, Rochester, MN for his valuable suggestions and histopathological evaluations. This study was funded by NIH grant 1R01AI068741. The authors declared no competing financial interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Induction of apoptosis in splenic mononuclear cells in vitro by bortezomib.

Click here for supplemental data (6.4MB, tiff)
Figure S2.

Induction of apoptosis in splenic mononuclear cells in vivo by bortezomib.

Click here for supplemental data (490.1KB, tiff)
Table S1.

Potentiation of SEB-induced mortality by bortezomib.

Click here for supplemental data (33KB, doc)
Table S2.

Potentiation of SEB-induced mortality by bortezomib: time kinetics.

Click here for supplemental data (29.5KB, doc)
Table S3.

Potentiation of SEB-induced mortality by bortezomib in HLA-DQ8 transgenic mice.

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Table S4.

Potentiation of LPS-induced mortality by bortezomib.

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