The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research.

BACKGROUND: Macrophages are the primary innate immune cells encountered by the invading coronaviruses, and their abilities to initiate inflammatory reactions, to maintain the immunity homeostasis by differential polarization, to train the innate immune system by epigenic modification have been reported in laboratory animal research. METHODS: In the current in vitro research, murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus, a coronavirus existed in mouse. At 3-, 6-, 12-, 24-, and 48-h post infection (hpi.), the attached cells were washed with PBS and harvested in Trizol reagent. Then The harvest is subjected to transcriptome sequencing. RESULTS: The transcriptome analysis showed the immediate (3 hpi.) up regulation of DEGs related to inflammation, like Il1b and Il6. DEGs related to M2 differential polarization, like Irf4 showed up regulation at 24 hpi., the late term after viral infection. In addition, DEGs related to metabolism and histone modification, like Ezh2 were detected, which might correlate with the trained immunity of macrophages. CONCLUSIONS: The current in vitro viral infection study showed the key innated immunity character of macrophages, which suggested the replacement value of viral infection cells model, to reduce the animal usage in preclinical research.

[Effects of human alpha-mannosidase Man2c1 transgene on growth and metastasis of transplanted tumor in mice].

OBJECTIVE: To study the effect of human alpha-mannosidase Man2c1 transgene on tumor growth and metastasis in mice. METHODS: Hepatoma cell H22 or squamous epithelial carcinoma cell S180 was subcutaneously inoculated into the right armpit of mice (wild type mice and 28#, 35#, and 54# transgenic mice). Tumor size was measured every week. Mice were sacrificed on day 9 or 10 and then the tumors were exercised and weighted. Tumors and lungs were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The red blood cells in spleen were destroyed by Tris-NH4Cl. Natural killer (NK) cell activity was detected with Yac-1 cell as target. RESULTS: H22 and S180 tumors grew faster in all the three transgenic mice (28#, 35#, and 54#) than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P<0.05). Most tumors in the transgenic mice invaded the surrounding tissues. In contrast, nearly all the tumors in wild type mice were capsulized. Three of 10 28# transgenic mice, 5 of 10 35# transgenic mice, 3 of 10 54# transgenic mice, and 1 of 10 wild type mice showed lung metastasis of H22 tumor. Two of 6 28# transgenic mice, 3 of 6 35# transgenic mice, 1 of 6 54# transgenic mice, and 0 of 6 wild type mice showed lung metastasis of S180 tumor. No difference of NK activity in spleen cells was observed between the transgenic mice and wild type mice. CONCLUSIONS: hMan2c1 transgene promotes growth, invasion, and metastasis of transplanted H22 and S180 tumors in mice. hMan2cl transgene does not affect NK activity in splenocytes.

Inhibition of the alpha-mannosidase Man2c1 gene expression enhances adhesion of Jurkat cells.

Protein N-glycosylation plays very important roles in immunity and alpha-mannosidase is one of the key enzymes in N-glycosylation. This paper reports that inhibition of alpha-mannosidase Man2c1 gene expression enhances adhesion of Jurkat T cells. In comparison to the controls with normal expression of the enzyme, Jurkat cells with the inhibition of Man2c1 gene expression (AS cell) formed larger aggregates in culture, indicating an enhancement of adhesion between the cells. mRNA differential display analysis discovered up-regulation of several adhesion molecule genes in the AS cell. Because of the pivotal role played by CD54-LFA-1 interaction in immune cell interaction, this study focused on the contribution of enhanced expression of CD54 and LFA-1 to the enhanced adhesion of AS Jurkat cells. These facts, including increased binding of AS cells to ICAM-1-Fc, Mg(2+) activation of the binding of AS cells to ICAM-1-Fc and enhanced aggregation of AS cells, together with the inhibiting effect of a blocking CD11a mAb on the binding to ICAM-1-Fc and aggregation of the cells demonstrate an important contribution of enhanced CD54-LFA-1 interaction to increased adhesion between AS cells. The enhanced CD54-LFA-1 interaction also resulted in increased adhesion between AS Jurkat T cells and Raji B cells. In addition, AS cells showed cytoskeletal rearrangement. The data imply a biological significance of MAN2C1 in T-cell functioning.