VSIG4(+) peritoneal macrophages induce apoptosis of double-positive thymocyte via the secretion of TNF-α in a CLP-induced sepsis model resulting in thymic atrophy.

Thymic atrophy in sepsis is a critical disadvantage because it induces immunosuppression and increases the mortality rate as the disease progresses. However, the exact mechanism of thymic atrophy has not been fully elucidated. In this study, we discovered a novel role for VSIG4-positive peritoneal macrophages (V4(+) cells) as the principal cells that induce thymic atrophy and thymocyte apoptosis. In CLP-induced mice, V4(+) cells were activated after ingestion of invading microbes, and the majority of these cells migrated into the thymus. Furthermore, these cells underwent a phenotypic shift from V4(+) to V4(-) and from MHC II(low) to MHC II(+). In coculture with thymocytes, V4(+) cells mainly induced apoptosis in DP thymocytes via the secretion of TNF-α. However, there was little effect on CD4 or CD8 SP and DN thymocytes. V4(-) cells showed low levels of activity compared to V4(+) cells. Thymic atrophy in CLP-induced V4(KO) mice was much less severe than that in CLP-induced wild-type mice. In addition, V4(KO) peritoneal macrophages also showed similar activity to V4(-) cells. Taken together, the current study demonstrates that V4(+) cells play important roles in inducing immunosuppression via thymic atrophy in the context of severe infection. These data also suggest that controlling the function of V4(+) cells may play a crucial role in the development of new therapies to prevent thymocyte apoptosis in sepsis.

Dictyostelium discoideum Ax2 as an Assay System for Screening of Pharmacological Chaperones for Phenylketonuria Mutations.

In this study, we developed an assay system for missense mutations in human phenylalanine hydroxylases (hPAHs). To demonstrate the reliability of the system, eight mutant proteins (F39L, K42I, L48S, I65T, R252Q, L255V, S349L, and R408W) were expressed in a mutant strain (pah(-)) of Dictyostelium discoideum Ax2 disrupted in the indigenous gene encoding PAH. The transformed pah- cells grown in FM minimal medium were measured for growth rate and PAH activity to reveal a positive correlation between them. The protein level of hPAH was also determined by western blotting to show the impact of each mutation on protein stability and catalytic activity. The result was highly compatible with the previous ones obtained from other expression systems, suggesting that Dictyostelium is a dependable alternative to other expression systems. Furthermore, we found that both the protein level and activity of S349L and R408W, which were impaired severely in protein stability, were rescued in HL5 nutrient medium. Although the responsible component(s) remains unidentified, this unexpected finding showed an important advantage of our expression system for studying unstable proteins. As an economic and stable cell-based expression system, our development will contribute to mass-screening of pharmacological chaperones for missense PAH mutations as well as to the in-depth characterization of individual mutations.

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.