Key Points: Human and mouse macrophages share partially conserved gene and protein expression programs in the resting or M2 activated state. TGM2 is a novel M2 marker consistently induced in human and mouse M2 macrophages.

Inflammation involves a coordinated, sequential, and self limiting sequence of events controlled by positive and negative regulatory mechanisms. Recent studies have shown that microRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide noncoding RNAs, contribute to the regulation of inflammation by repressing gene expression at the posttranscriptional level. In this study, we characterize the profile of miRNAs induced by LPS in human polymorphonuclear neutrophils (PMN) and monocytes. In particular, we identify miR-9 as the only miRNA (among 365 analyzed) up-regulated in both cell types after TLR4 activation. miR-9 is also induced by TLR2 and TLR7/8 agonists and by the proinflammatory cytokines TNF-α and IL-1β, but not by IFNγ. Among the 3 different genes encoding miR-9 precursors in humans, we show that LPS selectively induces the transcription of miR-9–1 located in the CROC4 locus, in a MyD88- and NF-κB-dependent manner. In PMN and monocytes, LPS regulates NFKB1 at both the transcriptional and posttranscriptional levels, and a conserved miR-9 seed sustained a miR-9-dependent inhibition of the NFKB1 transcript. Overall, these data suggest that TLR4-activated NF-κB rapidly increases the expression of miR-9 that operates a feedback control of the NF-κB-dependent responses by fine tuning the expression of a key member of the NF-κB family.

Tumor-associated macrophages (TAMs) are key orchestrators of the tumor microenvironment directly affecting neoplastic cell growth, neoangiogenesis, and extracellular matrix remodeling. In turn, the tumor milieu strongly influences maturation of TAMs and shapes several of their features. To address the early macrophage (Mϕ) differentiation phase in a malignant context, we mimicked a tumor microenvironment by in vitro coculturing human blood monocytes with conditioned media from different cancer cell lines. Only 2 out of 16 tumor cell lines induced Mϕ differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned Mϕ was performed. Comparison with other datasets (polarized M1-Mϕ, M2-Mϕ, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized Mϕ. The most expressed genes were selenoprotein 1, osteoactivin, osteopontin, and, interestingly, migration-stimulating factor (MSF), a poorly studied oncofoetal isoform of fibronectin. MSF (present in fetal/cancer epithelial and stromal cells but not in healthy tissues) was never identified in Mϕ. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGFβ but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of Mϕ. Tumor-conditioned Mϕ-derived MSFs strongly stimulated tumor cell migration, thus contributing to the motile phenotype of neoplastic cells. In conclusion, MSF is a new molecule associated with the M2 polarization of Mϕ and expressed by TAMs. Its biological function may contribute to Mϕ-mediated promotion of cancer cell invasion and metastasis.

Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.

Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed EROfor ER oxidoreductins, plays a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000)J. Biol. Chem. 275, 4827–4833). Here, we report the cloning and characterization of a novel human member of this family,ERO1-Lβ. Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lβ gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 °C and to alleviate the “unfolded protein response” when expressed inero1-1 cells indicates that ERO1-Lβ is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-Lβ display different tissue distributions. Furthermore, only ERO1-Lβ transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.