Distinct Host–Mycobacterial Pathogen Interactions between Resistant Adult and Tolerant Tadpole Life Stages of Xenopus laevis

Mycobacterium marinum is a promiscuous pathogen infecting many vertebrates, including humans, whose persistent infections are problematic for aquaculture and public health. Among unsettled aspects of host–pathogen interactions, the respective roles of conventional and innate-like T (iT) cells in host defenses against M. marinum remain unclear. In this study, we developed an infection model system in the amphibian Xenopus laevis to study host responses to M. marinum at two distinct life stages, tadpole and adult. Adult frogs possess efficient conventional T cell–mediated immunity, whereas tadpoles predominantly rely on iT cells. We hypothesized that tadpoles are more susceptible and elicit weaker immune responses to M. marinum than adults. However, our results show that, although anti–M. marinum immune responses between tadpoles and adults are different, tadpoles are as resistant to M. marinum inoculation as adult frogs. M. marinum inoculation triggered a robust proinflammatory CD8+ T cell response in adults, whereas tadpoles elicited only a noninflammatory CD8 negative- and iT cell–mediated response. Furthermore, adult anti–M. marinum responses induced active granuloma formation with abundant T cell infiltration and were associated with significantly reduced M. marinum loads. This is reminiscent of local CD8+ T cell response in lung granulomas of human tuberculosis patients. In contrast, tadpoles rarely exhibited granulomas and tolerated persistent M. marinum accumulation. Gene expression profiling confirmed poor tadpole CD8+ T cell response, contrasting with the marked increase in transcript levels of the anti–M. marinum invariant TCR rearrangement (iVα45-Jα1.14) and of CD4. These data provide novel insights into the critical roles of iT cells in vertebrate antimycobacterial immune response and tolerance to pathogens

Pharmacologic inhibition of MLK3 kinase activity blocks the in vitro migratory capacity of breast cancer cells but has no effect on breast cancer brain metastasis in a mouse xenograft model.

Brain metastasis of breast cancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3) in controlling the in vitro migratory capacity of breast cancer cells, as well as the metastasis of MDA-MB-231 breast cancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasis of breast cancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration of breast cancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model of breast cancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size of breast cancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce the in vitro migratory capacity of breast cancer cells, but that it has no effect on either the frequency or size of breast cancer brain metastases, in a mouse xenograft model

MLK3 inhibition decreases wound healing recovery in a dose dependent manner.

<p>Relative change of wound recovery was measured for MDA MB-231, eGFP8.4, HS578T and MCF10A cells. Each value is the mean of 4 wells, error bars denote the standard error of the mean; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108487#s2" target="_blank">Methods</a> for details.</p

MLK3 inhibition decreases transwell migration of breast cancer cells.

<p>(<b>A</b>) MDA MB-231 or (<b>B</b>) eGFP8.4 migrated toward 10% FBS in presence of 200 nM URMC099 or vehicle during 24 hours. (<b>C</b>) eGFP8.4 cells migration toward 10% FBS reduced in dose-dependent manner; migration allowed for 6 hours. Each value is the mean of 3 wells, error bars denote the standard error of the mean. *, ** and # denote p<0.05 and p<0.005 and p = 0.058, two-tailed unpaired <i>t</i>-test.</p

Total number of brain metastases is not reduced in mice treated with URMC099.

<p>The total number of metastases was counted in 8 serial coronal sections of each brain. Each dot represents an individual mouse. The bars represent mean values. Asterisk denotes statistically significant difference (p<0.05, two-tailed t-test).</p