NF-kappaB activation suppresses host cell apoptosis during Rickettsia rickettsii infection via regulatory effects on intracellular localization or levels of apoptogenic and anti-apoptotic proteins (original) (raw)
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Infection and immunity, 2003
Apoptotic host cell death is a critical determinant in the progression of microbial infections and outcome of resultant diseases. The potentially fatal human infection caused by Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, involves the vascular endothelium of various organ systems of the host. Earlier studies have shown that survival of endothelial cells (EC) during this infection depends on their ability to activate the transcription factor nuclear factor kappa B (NF-kappa B). Here, we investigated the involvement of caspase cascades and associated signaling pathways in regulation of host cell apoptosis by NF-kappa B. Infection of cultured human EC with R. rickettsii with simultaneous inhibition of NF-kappa B induced the activation of apical caspases 8 and 9 and also the executioner enzyme, caspase 3, whereas infection alone had no significant effect. Inhibition of either caspase-8 or caspase-9 with specific cell-permeating peptide inhibitors caused a...
Infection and …, 2005
Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-κB (NF-κB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-κB subunits, p65 and p50, and IκB proteins, IκBα and IκBβ. The transcript and protein levels of p50, p65, and IκBβ remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IκBα at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IκBα mRNA. The level of IκBα mRNA gradually returned toward baseline, whereas that of total IκBα protein remained lower than the corresponding controls. The activities of IKKα and IKKβ, the catalytic subunits of IκB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-κB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKα and IKKβ, leading to attenuation of nuclear translocation of NF-κB. Also, increased activity of IKKα was evident later during the infection, coinciding with the late phase of NF-κB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-κB activation. Since NF-κB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.
Infection and Immunity, 2005
Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-B (NF-B) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-B subunits, p65 and p50, and IB proteins, IB␣ and IB. The transcript and protein levels of p50, p65, and IB remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IB␣ at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IB␣ mRNA. The level of IB␣ mRNA gradually returned toward baseline, whereas that of total IB␣ protein remained lower than the corresponding controls. The activities of IKK␣ and IKK, the catalytic subunits of IB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-B response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKK␣ and IKK, leading to attenuation of nuclear translocation of NF-B. Also, increased activity of IKK␣ was evident later during the infection, coinciding with the late phase of NF-B activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-B activation. Since NF-B is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.
Infection and immunity, 1998
Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor kappaB (NF-kappaB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-kappaB activation in cultured human umbilical vein endothelial cells consequent to infection with Rickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786-2791, 1997). In the present study, we describe activation of NF-kappaB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of R. rickettsii organisms added. Antibody supershift ...
International Journal of Medical Microbiology, 2005
Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5-28-fold increase in IL-8 and 4-16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein IkBa and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF-kB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of IkBa (to render NF-kB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1a or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF-kB activation and IL-8/ MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF-kB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF-kB, subtle autocrine effects of newly synthesized IL-1a may contribute, in part, to the control of NF-kB activation and chemokine production at later times. These findings implicate a prominent role for host EC in recruiting immune cells to the site of inflammation during Rickettsia
Infection and Immunity
Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor B (NF-B) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-B activation in cultured human umbilical vein endothelial cells consequent to infection with Rickettsia rickettsii, an obligate intracellular gramnegative bacterium and the etiologic agent of Rocky Mountain spotted fever (Infect. Immun. 65:2786-2791, 1997). In the present study, we describe activation of NF-B in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of R. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-B subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-B pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATP␥S, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IB␣. This lack of IB␣ involvement was supported by the finding that R. rickettsii can induce NF-B activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IB␣, rendering NF-B inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-B activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.
Journal of Medical Microbiology, 2007
The Gram-negative intracellular bacteria Rickettsia conorii and Rickettsia typhi are the aetiological agents of Mediterranean spotted fever and endemic typhus, respectively, in humans. Infection of endothelial cells (ECs) lining vessel walls, and the resultant vascular inflammation and haemostatic alterations are salient pathogenetic features of both of these rickettsial diseases. An important consideration, however, is that dramatic differences in the intracellular motility and accumulation patterns for spotted fever versus typhus group rickettsiae have been documented, suggesting the possibility of unique and potentially different interactions with host cells. This study characterized and compared R. conorii- and R. typhi-mediated effects on cultured human ECs. The DNA-binding activity of nuclear transcription factor-κB (NF-κB) and the phosphorylation status of stress-activated p38 kinase were determined as indicators of NF-κB and p38 activation. R. conorii infection resulted in a...