Interleukin-10 enhances recruitment of immune cells in the neonatal mouse model of obstructive nephropathy.

Urinary tract obstruction during renal development leads to inflammation, leukocyte infiltration, tubular cell death, and interstitial fibrosis. Interleukin-10 (IL-10) is an anti-inflammatory cytokine, produced mainly by monocytes/macrophages and regulatory T-cells. IL-10 inhibits innate and adaptive immune responses. IL-10 has a protective role in the adult model of obstructive uropathy. However, its role in neonatal obstructive uropathy is still unclear which led us to study the role of IL-10 in neonatal mice with unilateral ureteral obstruction (UUO). UUO serves as a model for congenital obstructive nephropathies, a leading cause of kidney failure in children. Newborn Il-10-/- and C57BL/6 wildtype-mice (WT) were subjected to complete UUO or sham-operation on the 2nd day of life. Neonatal kidneys were harvested at day 3, 7, and 14 of life and analyzed for different leukocyte subpopulations by FACS, for cytokines and chemokines by Luminex assay and ELISA, and for inflammation, programmed cell death, and fibrosis by immunohistochemistry and western blot. Compared to WT mice, Il-10-/- mice showed reduced infiltration of neutrophils, CD11bhi cells, conventional type 1 dendritic cells, and T-cells following UUO. Il-10-/- mice with UUO also showed a reduction in pro-inflammatory cytokine and chemokine release compared to WT with UUO, mainly of IP-10, IL-1α, MIP-2α and IL-17A. In addition, Il-10-/- mice showed less necroptosis after UUO while the rate of apoptosis was not different. Finally, α-SMA and collagen abundance as readout for fibrosis were similar in Il-10-/- and WT with UUO. Surprisingly and in contrast to adult Il-10-/- mice undergoing UUO, neonatal Il-10-/- mice with UUO showed a reduced inflammatory response compared to respective WT control mice with UUO. Notably, long term changes such as renal fibrosis were not different between neonatal Il-10-/- and neonatal WT mice with UUO suggesting that IL-10 signaling is different in neonates and adults with UUO.

Impaired microtubule dynamics contribute to microthrombocytopenia in RhoB-deficient mice.

Megakaryocytes are large cells in the bone marrow that give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids, and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics, were not affected in the absence of RhoB. However, in vitro-generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, nonredundant functions in the megakaryocyte lineage.

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1.

Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.