Human adipose-derived mesenchymal stem cells (hASCs) are mesenchymal stem cells (MSCs) with reduced immunogenicity and capability to modulate immune responses. Whereas the immunosuppressive activity of bone marrow-MSCs has received considerable attention during the last few years, the specific mechanisms underlying hASC-mediated immunosuppression have been poorly studied. Recent studies comparing both cell types have reported differences at transcriptional and proteomic levels, suggesting that hASCs and bone marrow-MSCs, while having similarities, are quite different. This suggests that different mechanisms of immunosuppression may apply. Here, we report that hASCs inhibit peripheral blood mononuclear cells (PBMCs), and CD4(+) and CD8(+) T cell proliferation in both cell-cell contact and transwell conditions, which is accompanied by a reduction of proinflammatory cytokines. We demonstrate that hASCs do not constitutively express immunomodulatory factors. Conditioned supernatants from hASCs stimulated by IFN-gamma, PBMCs, or activated PBMCs highly inhibited PBMC proliferation, indicating that inhibitory factors are released upon hASC activation. Many factors have been involved in MSC-mediated immunosuppression, including IFN-gamma, IL-10, hepatocyte growth factor, prostaglandin E2, transforming growth factor-beta1, indoleamine 2,3-dioxygenase (IDO), nitric oxide, and IL-10. Using pharmacological inhibitors, neutralizing antibodies, and genetically modified hASCs that constitutively express or silence IDO enzyme, we demonstrate that, in the case of hASCs, the IFN-gamma/IDO axis is essential. Taken together, our data support the key role of IDO in the therapeutic use of hASC on immunomediated diseases.

Mesenchymal stem cells (MSCs) have differentiation and immunomodulatory properties that make them interesting tools for the treatment of degenerative disorders, allograft rejection, or inflammatory and autoimmune diseases. Biological properties of MSCs can be modulated by the inflammatory microenvironment they face at the sites of injury or inflammation. Indeed, MSCs do not constitutively exert their immunomodulating properties but have to be primed by inflammatory mediators released from immune cells and inflamed tissue. A polarization process, mediated by Toll-like receptors (TLRs), toward either an anti-inflammatory or a pro-inflammatory phenotype has been described for MSCs. TLRs have been linked to allograft rejection and the perpetuation of chronic inflammatory diseases (e.g., Crohn’s disease, rheumatoid arthritis) through the recognition of conserved pathogen-derived components or endogenous ligands (danger signals) produced upon injury. Interest in understanding the effects of TLR activation on MSCs has greatly increased in the last few years since MSCs will likely encounter TLR ligands at sites of injury, and it has been proven that the activation of TLRs in MSCs can modulate their function and therapeutic effect.

This report describes the distribution of conventional nuclear localization sequences (NLS) and of a beta-stranded so-called nuclear localization motif (NLM) in the two proteins (VP1, 82 kDa; VP2, 63 kDa) forming the T‫1؍‬ icosahedral capsid of the parvovirus minute virus of mice (MVM) and their functions in viral biogenesis and the onset of infection. The approximately 10 VP1 molecules assembled in the MVM particle harbor in its 142-amino-acid (aa) N-terminal-specific region four clusters of basic amino acids, here called BC1 (aa 6 to 10), BC2 (aa 87 to 90), BC3 (aa 109 to 115), and BC4 (aa 126 to 130), that fit consensus NLS and an NLM placed toward the opposite end of the polypeptide (aa 670 to 680) found to be necessary for VP2 nuclear uptake. Deletions and site-directed mutations constructed in an infectious MVM plasmid showed that BC1, BC2, and NLM are cooperative nuclear transport sequences in singly expressed VP1 subunits and that they conferred nuclear targeting competence on the VP1/VP2 oligomers arising in normal infection, while BC3 and BC4 did not display nuclear transport activity. Notably, VP1 proteins mutated at BC1 and -2, and particularly with BC1 to -4 sequences deleted, induced nuclear and cytoplasmic foci of colocalizing conjugated ubiquitin that could be rescued from the ubiquitin-proteasome degradation pathway by the coexpression of VP2 and NS2 isoforms. These results suggest a role for VP2 in viral morphogenesis by assisting cytoplasmic folding of VP1/VP2 subviral complexes, which is further supported by the capacity of NLM-bearing transport-competent VP2 subunits to recruit VP1 into the nuclear capsid assembly pathway regardless of the BC composition. Instead, all four BC sequences, which are located in the interior of the capsid, were absolutely required by the incoming infectious MVM particle for the onset of infection, suggesting either an important conformational change or a disassembly of the coat for nuclear entry of a VP1-associated viral genome. Therefore, the evolutionarily conserved BC sequences and NLM domains provide complementary nuclear transport functions to distinct supramolecular complexes of capsid proteins during the autonomous parvovirus life cycle.The nuclear membrane offers a second barrier to those viruses that, upon specific cell surface recognition and internalization, need components of the replication and transcription machinery of the host cells for their multiplication. Indeed the structural components of karyophilic viruses reach the nucleus at two stages of the life cycle, first when the incoming particle delivers the genome and late in the infection during the nuclear accumulation of viral components leading to the biogenesis of the virions. As for the cellular components, the nuclear import of viral macromolecules must proceed across the central aqueous channel of the nuclear pore complex (NPC) (18, 58), a large structure with an eightfold rotational symmetry built from proteins called nucleoporins. Cytoplasmic-nuclear transport is directed by the interac...