Effect of Cytoskeletal Disruption on Mechanotransduction of Hydrostatic Pressure by C3H10T1/2 Murine Fibroblasts

Cyclic hydrostatic pressure of physiological magnitude (< 10 MPa) stimulates chondrogenic differentiation of mesenchymal stem cells, but mechanotransduction mechanisms are not well understood. It was hypothesized that an intact cytoskeleton would be required for uninhibited mechanotransduction of hydrostatic pressure. Therefore we examined the effects of drugs which selectively interfere with actin and tubulin polymerization on pressure-induced upregulation of aggrecan and col2a1 (type II collagen) mRNA expression. C3H10T1/2 cells were cultured as pellets in either 4µM cytochalasin D or 4µM nocodazole and subjected to 3 days of cyclic hydrostatic compression (1 Hz, 5 MPa, 2 h per day). Phalloidin staining and indirect immunostaining with anti α-tubulin antibody confirmed disruption of microfilament and microtubule assemblies, respectively. Real time RT-PCR revealed that both drugs substantially lowered the basal level of aggrecan and col2a1 mRNA, but that neither drug prevented a pressure-stimulated increase in gene expression relative to the altered basal state. Thus upregulation of macromolecular gene expression by cyclic hydrostatic pressure did not require a completely intact cytoskeleton

Imbalance of placental regulatory T cell and Th17 cell population dynamics in the FIV-infected pregnant cat

Abstract Background An appropriate balance in placental regulatory T cells (Tregs), an immunosuppressive cell population, and Th17 cells, a pro-inflammatory cell population, is essential in allowing tolerance of the semi-allogeneic fetus. TGF-β and IL-6 are cytokines that promote differentiation of Tregs and Th17 cells from a common progenitor; aberrant expression of the cytokines may perturb the balance in the two cell populations. We previously reported a pro-inflammatory placental environment with decreased levels of FoxP3, a Treg marker, and increased levels of IL-6 in the placentas of FIV-infected cats at early pregnancy. Thus, we hypothesized that FIV infection in the pregnant cat causes altered placental Treg and Th17 cell populations, possibly resulting in placental inflammation. Methods We examined the effect of FIV infection on Treg and Th17 populations in placentas at early pregnancy using quantitative confocal microscopy to measure FoxP3 or RORγ, a Th17 marker, and qPCR to quantify expression of the key cytokines TGF-β and IL-6. Results FoxP3 and RORγ were positively correlated in FIV-infected placentas at early pregnancy, but not placentas from normal cats, indicating virus-induced alteration in the balance of these cell populations. In control cats the expression of IL-6 and RORγ was positively correlated as predicted, but this relationship was disrupted in infected animals. TGF-β was reduced in infected queens, an occurrence that could dysregulate both Treg and Th17 cell populations. Co-expression analyses revealed a highly significant positive correlation between IL-6 and TGF-β expression in control animals that did not occur in infected animals. Conclusion Collectively, these data point toward potential disruption in the balance of Treg and Th17 cell populations that may contribute to FIV-induced inflammation in the feline placenta.</p