Ultrastructural features of lymphocyte suppression induced by anthrax lethal toxin and treated with chloroquine

Abstract

Antibacterial therapy does not fully protect against anthrax because of severe systemic intoxication. Lysosomal processing of anthrax lethal toxin (LTX) is a key event in the disease pathogenesis, and agents interfering with this process, like chloroquine (CQ), may have practical applications. Although LTX is known to induce T-cell suppression, precise mechanisms of this phenomenon are not completely characterized. In the present study, we investigated alterations of lymphocyte ultrastructure caused by LTX and associated with favorable effect of CQ on the LTX-related dysfunction. Peripheral blood lymphocytes were activated via CD3 crosslinking in the presence or absence of LTX and CQ, and examined by transmission electron microscopy, flow cytometry and immunoblotting. Crosslinking of CD3 induced ultrastructural signs of lymphocyte activation, mostly disappeared after LTX treatment. The cell ultrastructure was well preserved in LTX-treated cells, despite dose-and time-dependent inhibition of T-cell function associated with impaired activation of mitogen-activated protein kinase. Regardless of intracellular signaling abnormalities, LTX did not decrease T-cell viability. CQ restored expression of CD69 (Po0.001) and improved phosphorylation of p38 (P ¼ 0.022) in LTXexposed T lymphocytes. The exposure of cells to CQ, with or without LTX, led to appearance of many phagolysosomes with heterogeneous content, possibly representing unprocessed internalized material. In conclusion, LTX suppressed Tcell functions, but did not affect the viability and caused no ultrastructural damage. Ultrastructural observations indicated that CQ reduced harmful effects of LTX, possibly by interfering with lysosomal activity.

Debridement Increases Survival in a Mouse Model of Subcutaneous Anthrax

“…Accordingly, spores from B. anthracis Sterne or the TKO mutant were injected subcutaneously into the left ear of mice at a concentration of 1×10 6 spores and at 24 hours cLN were removed, homogenized, and stained for flow cytometric analysis. Expression of the activation markers CD69, CD80 and CD86 were chosen as measures of activation since previous publications have demonstrated that these cell markers are down regulated by B. anthracis exotoxins [36], [37], [38]. Specifically, CD80 and CD86 are down regulated in dendritic cells [37], [39], and CD69 is down regulated in response to LT in T cells at 24 hours [36].…”

“…Expression of the activation markers CD69, CD80 and CD86 were chosen as measures of activation since previous publications have demonstrated that these cell markers are down regulated by B. anthracis exotoxins [36], [37], [38]. Specifically, CD80 and CD86 are down regulated in dendritic cells [37], [39], and CD69 is down regulated in response to LT in T cells at 24 hours [36]. Flow cytometry revealed that expression of CD69 and CD86, but not CD80, were significantly up-regulated in mice that had been infected with Sterne or TKO spores as compared to internal control lymph nodes (Figure 3A to C).…”

Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung

“…Indeed, CD69 transcripts were elevated in LeTx-treated lungs of A/J mice at this early time point. In contrast, several groups have demonstrated that LeTx instead impairs lymphocyte proliferation and activation [11,12,66,67]. However, all of these studies either examined responses of isolated lymphocytes in vitro or examined restimulation of lymphocytes previously exposed to LeTx.…”

Current and Future Medical Approaches To Combat the Anthrax Threat

“…(d) Chloroquine is a commonly used antimalarial compound. Chemically it is a weak base that reduces the suppressive effect of LT on lymphocytes, possibly by interfering with intracellular acidification steps (neutralizes the acidic heptameric PA 63 compartments). ,, It may augment current treatment and prophylaxis options for this otherwise lethal infection.…”