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Anthrax is a life-threatening disease caused by infection with Bacillus anthracis, which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2–Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2–Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4–JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.

Hypoxia is considered to be a contributor to the pathology associated with administration of anthrax lethal toxin (LT). However, we report here that serum lactate levels in LT-treated mice are reduced, a finding inconsistent with the anaerobic metabolism expected to occur during hypoxia. Reduced lactate levels are also observed in the culture supernatants of LT-treated cells. LT inhibits the accumulation of hypoxia-inducible factor (HIF)-1α, a subunit of HIF-1, the master regulator directing cellular responses to hypoxia. The toxin has no effect on the transcription or protein turnover of HIF-1α, but instead it acts to inhibit HIF-1α translation. LT treatment diminishes phosphorylation of eIF4B, eIF4E, and rpS6, critical components of the intracellular machinery required for HIF-1α translation. Moreover, blockade of MKK1/2-ERK1/2, but not p38 or JNK signaling, lowers HIF-1α protein levels in both normoxic and hypoxic conditions, consistent with a role for MKK1 and MKK2 as the major targets of LT responsible for the inhibition of HIF-1α translation. The physiological importance of the LT-induced translation blockade is demonstrated by the finding that LT treatment decreases the survival of hepatocyte cell lines grown in hypoxic conditions, an effect that is overcome by preinduction of HIF-1α. Taken together, these data support a role for LT in dysregulating HIF-1α and thereby disrupting homeostatic responses to hypoxia, an environmental characteristic of certain tissues at baseline and/or during disseminated infection with Bacillus anthracis.

The convergence of innate and adaptive immunity is critical for host defense, allowing for early protection and the generation of specific responses. STAT-4 is at that point of convergence, unifying the IFNalpha and IL-12 pathways. Activation of STAT-4 is crucial to T cell polarization, B cell and NK cell activation, and the control of intracellular pathogens. However, techniques to detect phosphorylated STAT-4 are cumbersome and require many cells. We have developed a flow cytometric detection technique to investigate IL-12 signaling in human peripheral blood mononuclear cells. Using different polyclonal antibodies that recognize either total STAT-4 protein or tyrosine-phosphorylated STAT-4, we can easily detect IL-12 and IFNalpha signaling in PHA/IL-2 blasts derived from peripheral blood lymphocytes. This technique not only allows us to evaluate IL-12 signaling, but it is also less time consuming and labor intensive than alternative methods. Using this flow cytometry-based method, we should be able to detect patients with defects in IL-12 receptor signal transduction, who typically present with disseminated nontuberculous mycobacterial infections.

We have described previously a family with an apparent genetic susceptibility to disseminated Mycobacterium avium complex infection and an underlying defect in IL-12 regulation leading to abnormally low interferon-gamma production. Their T cells appear to act normally when in the presence of normal accessory cells. Cell-to-cell contact was necessary for normal monocytes to complement the familial patient monocyte defect, suggesting the familial defect in interferon-gamma costimulation involves pathways requiring cell surface molecule interactions. In an effort to better characterize the abnormality in these patients, we examined the role of known costimulatory molecules in residual costimulation by patient PBMC compared to normals. Whereas normals utilized CD40/CD40L interactions and IL-12 production for optimal interferon-gamma costimulation in PHA-stimulated cocultures, familial patient interferon-gamma production was low and unaffected by their blockade. CD86 blockade caused a greater than 50% reduction in both normal and familial patient interferon-gamma production, implying that a majority of residual familial patient costimulation required this pathway. Furthermore, selected myelomonocytic cell lines (K562 and THP1) acted as potent accessory cells for interferon-gamma production by familial patient and normal T cells, largely independent of IL-12 production. However, CD86 blockade of K562 cell/familial cell cocultures resulted in less than a 20% reduction in interferon-gamma production, indicating that familial patient cells respond to IL-12- and CD86-independent costimulatory signals for interferon-gamma as well. Thus, we demonstrate that the familial defect also involves interferon-gamma costimulation pathways requiring both CD40/CD40L interaction and IL-12 production, while residual pathways remain that allow low-level interferon-gamma production. Familial Mycobacterium avium patient monocytes and certain myelomonocytic cell lines can be exploited to investigate IL-12-independent costimulation for interferon-gamma production.

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.