Resistance of Human Alveolar Macrophages toBacillus anthracisLethal Toxin

Abstract

The etiologic agent of inhalational anthrax, Bacillus anthracis, produces virulence toxins that are important in the disease pathogenesis. Current studies suggest that mouse and human macrophages are susceptible to immunosuppressive effects of one of the virulence toxins, lethal toxin (LT). Thus a paradigm has emerged that holds that the alveolar macrophage (AM) does not play a significant role in the innate immune response to B. anthracis or defend against the pathogen as it is disabled by LT. This is inconsistent with animal models and autopsy studies that show minimal disease at the alveolar surface. We examined whether AM are immunosuppressed by LT. We found that human AM were relatively resistant to LT-mediated innate immune cytokine suppression, MEK cleavage, and induction of apoptosis as compared with mouse RAW 264.7 macrophages. Mouse AM and murine bone marrow-derived macrophages were also relatively resistant to LT-mediated apoptosis despite intermediate sensitivity to MEK cleavage. The binding component of LT, protective Ag, does not attach to human AM, although it did bind to mouse AM, murine bone marrow-derived macrophages, and RAW 264.7 macrophages. Human AM do not produce significant amounts of the protective Ag receptor anthrax toxin receptor 1 (TEM8/ANTXR1) and anthrax toxin receptor 2 (CMG2/ANTXR2). Thus, mature and differentiated AM are relatively resistant to the effects of LT as compared with mouse RAW 264.7 macrophages. AM resistance to LT may enhance clearance of the pathogen from the alveolar surface and explain why this surface is relatively free of B. anthracis in animal models and autopsy studies.

Targeted Silencing of Anthrax Toxin Receptors Protects against Anthrax Toxins

“…Poor or undetectable levels of Tem8 transcripts in Raw 264.7 cells have been previously reported (16,24). Furthermore, primary mouse and human alveolar macrophages also express marginal levels of TEM8 protein (24). However, although TEM8 may not be important as an ANTXR in mouse macrophages, it may be more noteworthy in the anthrax toxin entry of other cell types, as is the case in differentiated THP-1 cells.…”

“…It was not possible for us to evaluate silencing of Tem8 in Raw 264.7 cells because Tem8 transcript levels were undetectable in these cells. Poor or undetectable levels of Tem8 transcripts in Raw 264.7 cells have been previously reported (16,24). Furthermore, primary mouse and human alveolar macrophages also express marginal levels of TEM8 protein (24).…”

Opening moves: early events in anthrax pathogenesis and their consequences for dissemination

“…Further, a SNP that causes a P357A substitution in the human CMG2 region involved with PA internalization reduces the amount of PA uptake in transgenic RAW264.7 cells [132]. It is likely, however, that AMs are uniquely resistant to LT-mediated death as the AMs in Wu's studies were likely collected from several individuals [204]. AMs from Cynomolgus macaques, however, do have reductions in cytokine production when intoxicated with purified LT in vitro, likely due to the effects of MEK1 cleavage [205].…”

“…However, human AMs are more resistant to MEK1 cleavage and LT-mediated cytokine inhibition than peritoneal macrophages or the murine RAW264.7 macrophage-like cell line [204]. Further, PA does not bind AMs well, despite similar transcription levels of CMG2 compared to RAW264.7 cells.…”

“…Therefore, in addition to differential responses between human and mouse AMs, important species difference in the intoxication of immune cells exist even in very closely related non-human primates and humans. Despite the difference in cytokine production, both studies demonstrate stark differences in the viability of AMs when compared to circulating monocytes/macrophages and macrophage-like cell lines [175,204,206]. ET intoxication of Guinea pig AMs with purified ET reduces their secretion of Type-IIA phospholipase A2 [207].…”