Generation of macrophages with altered viral sensitivity from genome-edited rhesus macaque iPSCs to model human disease.

Because of their close biological similarity to humans, non-human primate (NHP) models are very useful for the development of induced pluripotent stem cell (iPSC)-based cell and regenerative organ transplantation therapies. However, knowledge on the establishment, differentiation, and genetic modification of NHP-iPSCs, especially rhesus macaque iPSCs, is limited. We succeeded in establishing iPSCs from the peripheral blood of rhesus macaques (Rh-iPSCs) by combining the Yamanaka reprograming factors and two inhibitors (GSK-3 inhibitor [CHIR 99021] and MEK1/2 inhibitor [PD0325901]) and differentiated the cells into functional macrophages through hematopoietic progenitor cells. To confirm feasibility of the Rh-iPSC-derived macrophages as a platform for bioassays to model diseases, we knocked out TRIM5 gene in Rh-iPSCs by CRISPR-Cas9, which is a species-specific HIV resistance factor. TRIM5 knockout (KO) iPSCs had the same differentiation potential to macrophages as did Rh-iPSCs, but the differentiated macrophages showed a gain of sensitivity to HIV infection in vitro. Our reprogramming, gene editing, and differentiation protocols used to obtain Rh-iPSC-derived macrophages can be applied to other gene mutations, expanding the number of NHP gene therapy models.

Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

Trehalose inhibits inflammatory cytokine production by protecting IkappaB-alpha reduction in mouse peritoneal macrophages.

OBJECTIVE: The aim of this study was to examine whether trehalose, a disaccharide, could inhibit Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS)-enhanced production of inflammatory cytokines in mouse peritoneal macrophages. DESIGN: Mouse peritoneal macrophages were treated with trehalose and stimulated with P. gingivalis LPS. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) levels in the culture supernatant were measured by ELISA. The mRNA levels of the cytokines in macrophages were analysed by semi-quantitative RT-PCR. DNA and protein synthesis were measured by incorporation of [(3)H] thymidine or [(14)C] praline into mouse peritoneal macrophages. IkappaB-alpha reductions were assessed by western blot. RESULTS: Treatment with trehalose suppressed LPS-induced IL-1beta and TNF-alpha production and downregulated transcription of these cytokines. Furthermore, trehalose inhibited LPS-induced reduction of IkappaB-alpha. In addition, we also observed expression of the trehalose receptor (T1R3) in mouse peritoneal macrophages. CONCLUSION: These results may suggest that trehalose inhibits LPS-induced production of IL-1beta and TNF-alpha in mouse peritoneal macrophages by inhibiting degradation of IkappaB-alphavia the trehalose receptor T1R3.