Early gene expression changes induced by the bacterial superantigen staphylococcal enterotoxin B and its modulation by a proteasome inhibitor

Abstract

Toxic shock syndrome (TSS) is an acute, serious systemic illness caused by bacterial superantigens. Nonavailability of a suitable animal model until recently has hampered an in-depth understanding of the pathogenesis of TSS. In the current study, we characterized the early molecular events underlying TSS using our HLA-DR3 transgenic mouse model. Gene expression profiling using DNA microarrays identified a rapid and significant upregulation of several pro- as well as anti-inflammatory mediators, many of which have never been previously described in TSS. In vivo administration of staphylococcal enterotoxin B (SEB) led to an increase in the expression of Th0- (IL-2, 240-fold); Th1- (IFN-gamma, 360-fold; IL-12, 8-fold); Th2- (IL-4, 53-fold; IL-5, 4-fold) as well as Th17-type cytokines (IL-21, 19-fold; IL-17, 5-fold). The immunoregulatory cytokines (IL-6, 700-fold; IL-10, 18-fold); CC chemokines (such as CCL 2, 11, 3, 24, 17, 12, 7), CXC chemokines (such as CXCL 1, 2, 5, 11, 10, 19); and several proteases (matrix metalloproteinases 13, 8, 3, and 9) were also upregulated. Serum levels of several of these cytokines/chemokines were also significantly elevated. Pathway analyses revealed significant modulation in a variety of biochemical and cellular functions, providing molecular insights into the pathogenesis of TSS. Administration of bortezomib, a clinically approved proteasome inhibitor capable of blocking NF-kappaB pathway, was able to significantly modulate the expression of a variety of genes induced by SEB. Thus, our study showed that TSS is a complex process and emphasized the potential of use of bortezomib in the therapy of superantigen-induced TSS.

Fig. 1.

Systemic cytokine storm following in vivo staphylococcal enterotoxin B (SEB) challenge. Age-matched HLA-DR3 transgenic mice were challenged with a single intraperitoneal injection of 10 μg of SEB or PBS. Prior to death at 3 h, animals were bled. Serum cytokine levels were determined using a multiplex suspension array system (Bio-Plex, Bio-Rad). Each bar represents the mean ± SE of 4 mice.

Fig. 2.

Molecular and cellular functions modulated by SEB. Microarray data containing genes that were significantly upregulated by SEB [false discovery rate (FDR) <0.005] were analyzed by Ingenuity Pathway Analysis (IPA) tool for IPA functional analysis. Depicted are molecular and cellular functions that are significantly modulated by SEB. The black solid line represents the cutoff −log(P value).

Fig. 3.

Modulation of physiological system development and function by SEB. Microarray data containing genes that were significantly upregulated by SEB (FDR <0.005) were analyzed by IPA tool for IPA functional analysis. Figure depicts physiological system development and functions that are significantly modulated by SEB. The black solid line represents the cutoff −log(P value).

Fig. 4.

Diseases and disorders functions modulated by SEB. Microarray data containing genes that were significantly upregulated by SEB (FDR <0.005) were analyzed by IPA tool for IPA functional analysis. Figure depicts diseases and disorder functions that are significantly modulated by SEB. The black solid line represents the cutoff −log(P value).

Fig. 5.

Toxicity profile of SEB-induced TSS. Microarray data containing genes that were significantly upregulated by SEB (FDR <0.005) were analyzed by IPA tool for IPA Tox analysis for hepato-, nephro-, and cardiotoxicities. Figure depicts toxicity functions that are significantly modulated by SEB. The black solid line represents the cutoff −log(P value).

Fig. 6.

Bortezomib-induced alteration in gene expression. Splenic mRNA from different experimental groups were processed and hybridized to Affymetrix 430A 2.0 arrays as described in materials and methods. A: changes in protein ubiquitination pathway. Statistical significance [−log(P values)] of each comparison group is depicted. The vertical line (“Threshold”) indicates the threshold value to attain statistical significance. Protein ubiquitination pathway is significantly affected in all groups except in the one that did not receive bortezomib (i.e., PBS+SEB vs. PBS+PBS). B: modulation of signaling pathways by bortezomib during toxic shock syndrome (TSS). Figure represents changes (increase or decrease) expression of genes involved in given signaling pathways in SEB-challenged mice treated or untreated with bortezomib. Statistical significance [−log(P values)] of each pathway is depicted.

Fig. 7.

Modulation of SEB-induced gene expression by bortezomib. Diagrammatic representation of transcriptional profiles of genes between different groups.