mTORC1-mediated polarization of M1 macrophages and their accumulation in the liver correlate with immunopathology in fatal ehrlichiosis.

A polarized macrophage response into inflammatory (M1) or regenerative/anti-inflammatory (M2) phenotypes is critical in host response to multiple intracellular bacterial infections. Ehrlichia is an obligate Gram-negative intracellular bacterium that causes human monocytic ehrlichiosis (HME): a febrile illness that may progress to fatal sepsis with multi-organ failure. We have shown that liver injury and Ehrlichia-induced sepsis occur due to dysregulated inflammation. Here, we investigated the contribution of macrophages to Ehrlichia-induced sepsis using murine models of mild and fatal ehrlichiosis. Lethally-infected mice showed accumulation of M1 macrophages (iNOS-positive) in the liver. In contrast, non-lethally infected mice showed polarization of M2 macrophages and their accumulation in peritoneum, but not in the liver. Predominance of M1 macrophages in lethally-infected mice was associated with expansion of IL-17-producing T, NK, and NKT cells. Consistent with the in vivo data, infection of bone marrow-derived macrophages (BMM) with lethal Ehrlichia polarized M0 macrophages into M1 phenotype under an mTORC1-dependent manner, while infection with non-lethal Ehrlichia polarized these cells into M2 types. This work highlights that mTORC1-mediated polarization of macrophages towards M1 phenotype may contribute to induction of pathogenic immune responses during fatal ehrlichiosis. Targeting mTORC1 pathway may provide a novel aproach for treatment of HME.

MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.

The dormancy dilemma: quiescence versus balanced proliferation.

Metastatic dissemination with subsequent clinical outgrowth leads to the greatest part of morbidity and mortality from most solid tumors. Even more daunting is that many of these metastatic deposits silently lie undetected, recurring years to decades after primary tumor extirpation by surgery or radiation (termed metastatic dormancy). As primary tumors are frequently curable, a critical focus now turns to preventing the lethal emergence from metastatic dormancy. Current carcinoma treatments include adjuvant therapy intended to kill the cryptic metastatic tumor cells. Because such standard therapies mainly kill cycling cells, this approach carries an implicit assumption that metastatic cells are in the mitogenic cycle. Thus, the pivotal question arises as to whether clinically occult micrometastases survive in a state of balanced proliferation and death, or whether these cells undergo at least long periods of quiescence marked by cell-cycle arrest. The treatment implications are thus obvious--if the carcinoma cells are cycling then therapies should target cycling cells, whereas if cells are quiescent then therapies should either maintain dormancy or be toxic to dormant cells. Because this distinction is paramount to rational therapeutic development and administration, we investigated whether quiescence or balanced proliferation is the most likely etiology underlying metastatic dormancy. We recently published a computer simulation study that determined that balanced proliferation is not the likely driving force and that quiescence most likely participates in metastatic dormancy. As such, a greater emphasis on developing diagnostics and therapeutics for quiescent carcinomas is needed.

Modeling boundary conditions for balanced proliferation in metastatic latency.

PURPOSE: Nearly half of cancer metastases become clinically evident five or more years after primary tumor treatment; thus, metastatic cells survived without emerging for extended periods. This dormancy has been explained by at least two countervailing scenarios: cellular quiescence and balanced proliferation; these entail dichotomous mechanistic etiologies. To examine the boundary parameters for balanced proliferation, we conducted in silico modeling. EXPERIMENTAL DESIGN: To illuminate the balanced proliferation hypothesis, we explored the specific boundary probabilities under which proliferating micrometastases would remain dormant. A two-state Markov chain Monte Carlo model simulated micrometastatic proliferation and death according to stochastic survival probabilities. We varied these probabilities across 100 simulated patients each with 1,000 metastatic deposits and documented whether the micrometastases exceeded one million cells, died out, or remained dormant (survived 1,218 generations). RESULTS: The simulations revealed a narrow survival probability window (49.7-50.8%) that allowed for dormancy across a range of starting cell numbers, and even then for only a small fraction of micrometastases. The majority of micrometastases died out quickly even at survival probabilities that led to rapid emergence of a subset of micrometastases. Within dormant metastases, cell populations depended sensitively on small survival probability increments. CONCLUSIONS: Metastatic dormancy as explained solely by balanced proliferation is bounded by very tight survival probabilities. Considering the far larger survival variability thought to attend fluxing microenvironments, it is more probable that these micrometastatic nodules undergo at least periods of quiescence rather than exclusively being controlled by balanced proliferation.