Objective-To genetically and functionally characterize mutations of c-Mpl, that lead to thrombocytopenia in a child with congenital amegakaryocytic thrombocytopenia (CAMT).Methods-We identified two c-Mpl mutations in a child with clinical features of CAMT, one a previously described mutation in the extracellular domain (R102P) and the other a novel mutation leading to truncation of the receptor after the box 1 homology domain (541Stop). Cell line models were created to examine the ability of the mutant receptors to signal in response to thrombopoietin and thrombopoietin-like agonists.Results-Data from cell line models indicate that c-Mpl R102P does not support significant signaling in response to thrombopoietin due to impaired trafficking of the mutant receptor to the cell surface. Alternative thrombopoietic agents do not circumvent this block to signaling, likely due to the inaccessibility of the receptor. In addition, previous data indicate that c-Mpl 541Stop does not support intracellular signaling due to the loss of critical intracellular domains.Conclusions-This case demonstrates two different mechanisms by which c-Mpl mutations can impair thrombopoietin signaling, and suggests that mutations in the extracellular domain will not be rescued by c-Mpl agonists if they interfere with normal receptor expression.

Objective-To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists.Methods-We created cell line models of type II c-Mpl mutations identified in CAMT. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing WT or F104S c-Mpl in response to thrombopoetin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD 4665) domains of the receptor by MTT assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species. Results-Cells expressing F104S c-Mpl proliferated in response toLGD 4665 but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD 4665 stimulates signaling with delayed kinetics in both WT and F104S c-Mpl expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen bonding interactions that are critical for binding to thrombopoietin.Conclusions-These findings suggest that a small subset of patients with CAMT might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin.

Thrombopoietin (TPO) is essential for normal megakaryopoiesis, and mice and humans lacking the TPO receptor c-Mpl have significantly impaired platelet production. However, in the c-Mpl-null mouse model platelet counts, while reduced to ~10% of normal, are not zero, suggesting that another cytokine is able to support some degree of residual thrombopoiesis. We and others have reported that elimination or severe reduction of stem cell factor, G-CSF, IL-3, IL-6 or IL-11 does not eliminate residual thrombopoiesis. Because megakaryocytes (MKs) and erythrocytes are derived from a common progenitor, we asked if erythropoietin (EPO) can stimulate thrombopoiesis in c-Mpl-null mice. We administered 90 u recombinant EPO or vehicle by subcutaneous injection every 3 days to c-Mpl-null or WT control mice and measured baseline and weekly platelet counts. In three independent experiments, at 2 weeks platelet counts in c-Mpl-null mice receiving EPO were significantly higher that at baseline (5–7 mice per group, average of mean platelet counts 425,000/mm3 vs. 285,000/mm3, p=0.0015). There was a trend towards higher platelet counts in WT mice receiving EPO but this did not reach statistical significance. No difference in platelet counts was observed in mice injected with vehicle. In one experiment c-Mpl-null or WT mice were injected with EPO for 4 weeks and the platelet response in the c-Mpl-null animals was sustained for the duration of the experiment. Western blotting showed that murine MKs express the EPO receptor. To determine if EPO stimulates MK production directly we stimulated WT murine MKs in vitro with either 6 u/ml EPO, 100 ng/ml rhTPO or both and monitored activation of ERK and STAT5 signaling by immunoblotting. Stimulation of MKs with EPO resulted in phosphorylation of ERK and STAT5 (15- and 14-fold above baseline, respectively), compared to TPO (97- and 75-fold above baseline). Stimulation with EPO and TPO together had an additive effect (phospho-ERK increased 121-fold and phospho-STAT5 increased 100-fold). To determine if EPO acts primarily on early or late MKs, we harvested bone marrow from c-Mpl-null mice after 2 weeks of treatment with EPO or vehicle and measured CFU-MK frequency and MK ploidy. Although there was a small increase in the frequency of CFU-MK in mice treated with EPO compared to vehicle, these differences were not significant (n=3, p=0.7), possibly due to the difficulty in assaying CFU-MK in vitro without TPO. In addition, EPO did not significantly enhance MK ploidy in c-Mpl-null mice, although MKs in the 32N and greater peaks were slightly more numerous. Therefore, we conclude that EPO can augment platelet production in the absence of c-Mpl signaling, although it is not yet clear if EPO primarily acts on early or late cells. Additional experiments are underway to determine if ablating EPO receptors in a TPO-null mouse model will eliminate residual thrombopoiesis. These findings may have clinical relevance for treating patients with congenital amegakaryocytic thrombocytopenia and other causes of thrombocytopenia in which c-Mpl signaling is impaired.

In the renal collecting duct, sodium reabsorption through the epithelial sodium channel (ENaC) creates an osmotic driving force for water reabsorption resulting in increased blood volume and pressure. Investigators have previously shown that prolactin (PRL) stimulated ENaC activity in bullfrog skin cells. However, studies in mammalian systems show no effect of PRL on ENaC function. PRL is present in two forms: a 23kDa full length form (PRL) and a 14‐16kDa cleaved form (PRL‐C). We hypothesized that these two PRL molecules may exert different effects on ENaC activity. To investigate this hypothesis, we used a cell culture model of principal cells from the cortical collecting duct (mpkCCD) which express ENaC subunits and exhibit amiloride‐sensitive current. We measured transepithelial voltage and resistance across cell monolayers before and after basolateral treatment with vehicle (water) or 1‐2 ug/mL of PRL or PRL‐C and calculated transepithelial current using Ohm’s law. We found that PRL produced only a small, variable effect on ENaC activity in mpkCCD cells. However, PRL‐C approximately doubled the amiloride‐sensitive current in these cells within 24 hrs. Compared to kidney lysates, mpkCCD cells exhibit a low abundance of the PRL receptor which may explain the need for large PRL concentrations in these cells. Our results indicate that cleaved PRL stimulates ENaC activity in mpkCCD cells. In future studies, we will examine the effect of PRL and PRL‐C on single channel properties of ENaC in isolated collecting ducts and the effect of these hormones on blood pressure in vivo. Grant Funding Source: Supported by NIH K12GM000680 and T32HL076118

The role of IL-17/Th17 response in host resistance against Chlamydia trachomatis (Ct) infection is investigated in C57BL/6 and C3H/HeN mice. We showed that C3H/HeN mice developed a skewed Th17/Th1 immune profile which led to a massive accumulation of neutrophils in the lung, impaired bacterial clearance, severe body weight loss and high mortality rate. In comparison, a relatively moderate IL-17/Th17 response was developed in C57BL/6 mice. By using recombinant adenovirus vector-mediated IL-17 transgene delivery approach, we have found that IL-17/Th17 response has both protective and detrimental roles in host resistance against Ct. On one hand, IL-17 protects host from Ct infection via stimulating pulmonary DC function for establishing a protective immunity. On the other hand, IL-17 stimulates neutrophils which counteract the protective effect of IL-17 via promoting intracellular growth of Ct. Of importance, the final outcome of two opposing effects manifests in a time-dependent and dose-dependent manner. Over expressing IL-17 at later stages of Ct infection in the airway could convert C57BL/6 mice into Ct-susceptible phenotype, indicating that an exacerbated Th17 response is responsible for an enhanced Ct susceptibility. This is the first report that a multi-facet role of IL-17 is documented in modifying host immune responses via divergent immune mechanisms in a single disease model.