The immunoregulatory role of CD4⁺ FoxP3⁺ CD25⁻ regulatory T cells in lungs of mice infected with Bordetella pertussis

The identification of regulatory T (Treg) cells was originally based on CD25 expression; however, CD25 is also expressed by activated effector T cells. FoxP3 is a more definitive marker of Treg cells, and CD4(+) FoxP3(+) CD25(+) T cells are considered the dominant natural Treg (nTreg) population. It has been suggested that certain CD4(+) FoxP3(+) Treg cells do not express CD25. In this study, we used a murine model of respiratory infection with Bordetella pertussis to examine the role of Treg cells in protective immunity in the lung. We first demonstrated that CD4(+) FoxP3(+) CD25(-) cells are the dominant Treg population in the lung, gut and liver. Pre-activated lung CD4(+) FoxP3(+) CD25(-) cells suppressed CD4(+) effector T cells in vitro, which was partly mediated by IL-10 and not dependent on cell contact. Furthermore, CD4(+) FoxP3(+) CD25(-) IL-10(+) T cells were found in the lungs of mice at the peak of infection with B. pertussis. The rate of bacterial clearance was not affected by depletion of CD25(+) cells or in IL-10-deficient (IL-10(-/-) ) mice, but was compromised in CD25-depleted IL-10(-/-) mice. Our findings suggest that IL-10-producing CD4(+) FoxP3(+) CD25(-) T cells represent an important regulatory cell in the lung.     

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei

The epithelial Na? channels (ENaCs) are present in kidney and contribute to Na? and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.     

NK cell development, homeostasis and function: parallels with CD8⁺ T cells

Natural killer (NK) cells survey host tissues for signs of infection, transformation or stress and, true to their name, kill target cells that have become useless or are detrimental to the host. For decades, NK cells have been classified as a component of the innate immune system. However, accumulating evidence in mice and humans suggests that, like the B and T cells of the adaptive immune system, NK cells are educated during development, possess antigen-specific receptors, undergo clonal expansion during infection and generate long-lived memory cells. In this Review, we highlight the many stages that an NK cell progresses through during its remarkable lifetime, discussing similarities and differences with its close relative, the cytotoxic CD8(+) T cell.     

Gut sensing of dietary K⁺ intake increases renal K⁺excretion

Dietary K(+) intake may increase renal K(+) excretion via increasing plasma [K(+)] and/or activating a mechanism independent of plasma [K(+)]. We evaluated these mechanisms during normal dietary K(+) intake. After an overnight fast, [K(+)] and renal K(+) excretion were measured in rats fed either 0% K(+) or the normal 1% K(+) diet. In a third group, rats were fed with the 0% K(+) diet, and KCl was infused to match plasma [K(+)] profile to that of the 1% K(+) diet group. The 1% K(+) feeding significantly increased renal K(+) excretion, associated with slight increases in plasma [K(+)], whereas the 0% K(+) diet decreased K(+) excretion, associated with decreases in plasma [K(+)]. In the KCl-infused 0% K(+) diet group, renal K(+) excretion was significantly less than that of the 1% K(+) group, despite matched plasma [K(+)] profiles. We also examined whether dietary K(+) alters plasma profiles of gut peptides, such as guanylin, uroguanylin, glucagon-like peptide 1, and glucose-dependent insulinotropic polypeptide, pituitary peptides, such as AVP, α-MSH, and γ-MSH, or aldosterone. Our data do not support a role for these hormones in the stimulation of renal K(+) excretion during normal K(+) intake. In conclusion, postprandial increases in renal K(+) excretion cannot be fully accounted for by changes in plasma [K(+)] and that gut sensing of dietary K(+) is an important component of the regulation of renal K(+) excretion. Our studies on gut and pituitary peptide hormones suggest that there may be previously unknown humoral factors that stimulate renal K(+) excretion during dietary K(+) intake.     

The interaction between megalin and ClC-5 is scaffolded by the Na⁺-H⁺ exchanger regulatory factor 2 (NHERF2) in proximal tubule cells

Albumin endocytosis in the proximal tubule is mediated by a number of proteins, including the scavenger receptor megalin/cubilin and the PSD-95/Dlg/ZO-1 (PDZ) scaffolds NHERF1 and NHERF2. In addition, in a number of in vitro and in vivo models, the loss of ClC-5 results in a decreased cell surface expression and whole cell level of megalin, suggesting an interaction between these two proteins in vivo. We investigated if ClC-5 and megalin interact directly, and as ClC-5 binds to NHERF2, we investigated if this PDZ scaffold was required for a megalin/ClC-5 complex. GST-pulldown and immunoprecipitation experiments using rat kidney lysate demonstrated an interaction between ClC-5 and megalin, which was mediated by their C-termini. As this interaction may be controlled by a scaffold protein, we characterised any interaction between megalin and NHERF2. Immunoprecipitation experiments indicated that megalin interacts with NHERF2 in vivo, and that this interaction was via an internal NHERF binding domain in the C-terminus of megalin and PDZ2 and the C-terminus of NHERF2. Silencing NHERF2 had no effect on megalin protein levels in the whole cell or plasma membrane. Using siRNA against NHERF2, we demonstrated that NHERF2 was required to facilitate the interaction between megalin and ClC-5. Using fusion proteins, we characterised a protein complex containing ClC-5 and megalin, which is scaffolded by NHERF2, in the absence of any other proteins. Importantly, these observations are the first to describe an interaction between megalin and ClC-5, which is scaffolded by NHERF2 in proximal tubule cells.     

Partial depletion of natural CD4⁺CD25⁺ regulatory T cells with anti-CD25 antibody does not alter the course of acute influenza A virus infection

Foxp3⁺ CD4⁺ regulatory T cells represent a T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, and there is emerging evidence to suggest these cells temper pulmonary inflammation in response to acute viral infection. Recent studies have demonstrated treatment with PC61 CD25-depleting antibody potentiates inflammation in a murine model of RSV infection, while paradoxically delaying recruitment of CD8⁺ T cells to the site of inflammation. The present study therefore sought to examine the role of these cells in a murine model of acute influenza A virus infection through the administration of PC61 CD25-depleting antibody. PC61 antibody is able to partially deplete CD25⁺Foxp3⁺ regulatory T cells to a comparable degree as seen within previous work examining RSV, however this does not alter influenza A-virus induced mortality, weight loss, viral clearance and cellularity within the lung. Collectively, these data demonstrate that partial depletion of CD4⁺CD25⁺ regulatory T cells with PC61 antibody does not alter the course of influenza A virus infection.     

Identification and characterization of a novel multipotent sub-population of Sca-1⁺ cardiac progenitor cells for myocardial regeneration

Methods and Results

The cardiac stem/progenitor cells from adult mice were seeded at low density in serum-free medium. The colonies thus obtained were expanded separately and assessed for expression of stem cell antigen-1 (Sca-1). Two colonies each with high Sca-1 (CSH1; 95.9%; CSH2; 90.6%) and low Sca-1 (CSL1; 37.1%; CSL2; 17.4%) expressing cells were selected for further studies. Sca-1⁺ cells (98.4%) isolated using Magnetic Cell Sorting System (MACS) from the hearts were used as a control. Although the selected populations were similar in surface marker expression (low in c-kit, CD45, CD34, CD31 and high in CD29), these cells exhibited diverse differentiation potential. Unlike CSH1, CSH2 expressed Nanog, TERT, Bcrp1, Nestin, Musashi1 and Isl-1, and also showed differentiation into osteogenic, chondrogenic, smooth muscle, endothelial and cardiac lineages. MACS sorted cells exhibited similar tendency albeit with relatively weaker differentiation potential. Transplantation of CSH2 cells into infarcted heart showed attenuated infarction size, significantly preserved left ventricular function and anterior wall thickness, and increased capillary density. We also observed direct differentiation of transplanted cells into endothelium and cardiomyocytes.

Conclusions

The cardiac stem/progenitor cells isolated by a combined clonal selection and surface marker approach possessed multiple stem cell features important for cardiac regeneration.     

Early activated Th-1 type and dominantly diverse natural killer T (CD3⁺CD161⁺Vα24⁻) cells in bone marrow among visceral leishmaniasis patients

Lipid antigens of Leishmania donovani-like lipophosphoglycans (LPG) are demonstrated to be a potent ligand for natural killer T (NKT) cell activation. Little is known about the phenotype or function of these cells and their trafficking pattern to the bone marrow (BM) of visceral leishmaniasis (VL) patients. Their precise role in humans still requires pathological validation. The study included 42 parasitologically confirmed patients (mean age 24.80 ± 16.26 years; range 3-70 years; 25 males and 17 females), 33 healthy contact subjects (family/non-family members) and normal BM specimens (NBM; n = 9). Enumeration of NKT cells and quantification of parasites (before and after therapy) were performed for the recruited patients. Results established that non-CD1d restricted, diverse cells are the dominant population among resident but not enriched NKT (CD3⁺CD161⁺) cells at the disease site (BM). Expression profiles for various markers are indicative of their early activated (CD69⁺, CD62L^low, CD11a^high) CCR5⁺ phenotype at the BM. Functionally, BM-derived NKT cells were dominantly producing IFN-γ in response to L. donovani antigen in vitro. Given these observations, these data indicate that CD3⁺CD161⁺ diverse NKT cells are heterogeneous in function and of the dominant Th-1 phenotype at the disease site.     

Depletion of CD25⁺ cells during acute toxoplasmosis does not significantly increase mortality in Swiss OF1 mice

The interleukin (IL)-2R alpha chain (CD25) is expressed on regulatory T cells (Treg), which constitute more than 85% of the CD25+ T cell population in a na?ve mouse. CD25 is also expressed on effector T cells in mice suffering from an acute infection by the obligate intracellular protozoan parasite, Toxoplasma gondii. Lethal toxoplasmosis is accompanied by a significant loss of Treg in mice naturally susceptible to toxoplasmosis. The present study was done to explore the role of Treg cells using an anti-CD25 antibody-mediated depletion in mice naturally resistant to toxoplasmosis. Although a significant decrease in the percentage of Treg cells was observed following anti-CD25 monoclonal antibody injections, the depletion of CD25+ cells during acute toxoplasmosis did not significantly increase the mortality of Swiss OF1 mice and no significant difference was observed in the brain parasitic load between the mice in the depleted-infected and isotype-infected groups. We found no significant difference between the titres of total IgG in the sera of the mice from the two groups in the chronic phase. However, CD25+ cells depletion was followed by significantly higher levels of IL-12 in the serum of depleted mice than in that of mice injected with the isotype control antibody.     

The a3 isoform vacuolar type H⁺-ATPase promotes distant metastasis in the mouse B16 melanoma cells

Accumulating evidence indicates that the acidic microenvironments critically influence malignant behaviors of cancer including invasiveness, metastasis, and chemoresistance. Because the vacuolar-type H(+)-ATPase (V-ATPase) has been shown to cause extracellular acidification by pumping protons, we studied the role of V-ATPase in distant metastasis. Real-time PCR analysis revealed that the high-metastatic B16-F10 melanoma cells strongly expressed the a3 isoform V-ATPase compared to the low-metastatic B16 parental cells. Consistent with this, B16-F10 cells created acidic environments in lung metastases by acridine orange staining and strong a3 V-ATPase expression in bone metastases by immunohistochemistry. Immunocytochemical analysis showed B16-F10 cells expressed a3 V-ATPase not only in cytoplasm but also plasma membrane, whereas B16 parental cells exhibited its expression only in cytoplasm. Of note, knockdown of a3 V-ATPase suppressed invasiveness and migration with reduced MMP-2 and MMP-9 expression in B16-F10 cells and significantly decreased lung and bone metastases, despite that tumor growth was not altered. Importantly, administration of a specific V-ATPase a3 inhibitor FR167356 reduced bone metastasis of B16-F10 cells. These results suggest that a3 V-ATPase promotes distant metastasis of B16-F10 cells by creating acidic environments via proton secretion. Our results also suggest that inhibition of the development of cancer-associated acidic environments by suppressing a3 V-ATPase could be a novel therapeutic approach for the treatment of cancer metastasis.     

Human CD8⁺ and CD4⁺ T cell memory to lymphocytic choriomeningitis virus infection

Although cellular immunity to acute lymphocytic choriomeningitis virus (LCMV) infection has been well characterized in experimental studies in mice, the T cell response to this virus in humans is incompletely understood. Thus, we analyzed the breadths, magnitudes, and differentiation phenotypes of memory LCMV-specific CD8(+) and CD4(+) T cells in three human donors displaying a variety of disease outcomes after accidental needle stick injury or exposure to LCMV. Although only a small cohort of donors was analyzed at a single time point postinfection, several interesting observations were made. First, we were able to detect LCMV-specific CD8(+) and CD4(+) T cell responses directly ex vivo at 4 to 8 years after exposure, demonstrating the longevity of T cell memory in humans. Second, unlike in murine models of LCMV infection, we found that the breadths of memory CD8(+) and CD4(+) T cell responses were not significantly different from one another. Third, it seemed that the overall CD8(+) T cell response was augmented with increasing severity of disease, while the LCMV-specific CD4(+) T cell response magnitude was highly variable between the three different donors. Next, we found that LCMV-specific CD8(+) T cells in the three donors analyzed seemed to undergo an effector memory differentiation program distinct from that of CD4(+) T cells. Finally, the levels of expression of memory, costimulatory, and inhibitory receptors on CD8(+) and CD4(+) T cell subsets, in some instances, correlated with disease outcome. These data demonstrate for the first time LCMV-specific CD8(+) and CD4(+) T cells in infected humans and begin to provide new insights into memory T cell responses following an acute virus infection.     

Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F⁺ cells

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO(3)(-)) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3'-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3'-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK(1)-F(+) cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.