Landon S. King

Aquaporin water channels – from atomic structure to clinical medicine

The water permeability of biological membranes has been a longstanding problem in physiology, but the proteins responsible for this remained unknown until discovery of the aquaporin 1 (AQP1) water channel protein. AQP1 is selectively permeated by water driven by osmotic gradients. The atomic structure of human AQP1 has recently been defined. Each subunit of the tetramer contains an individual aqueous pore that permits single‐file passage of water molecules but interrupts the hydrogen bonding needed for passage of protons. At least 10 mammalian aquaporins have been identified, and these are selectively permeated by water (aquaporins) or water plus glycerol (aquaglyceroporins). The sites of expression coincide closely with the clinical phenotypes ‐ ranging from congenital cataracts to nephrogenic diabetes insipidus. More than 200 members of the aquaporin family have been found in plants, microbials, invertebrates and vertebrates, and their importance to the physiology of these organisms is being uncovered.

Functional requirement of aquaporin-5 in plasma membranes of sweat glands

The distribution and function of aquaporins (AQPs) have not previously been defined in sweat glands. In this study, AQP1, AQP3, and AQP5 mRNA were demonstrated in rat paw by reverse transcription (RT)–PCR, but AQP2 and AQP4 were not. AQP1, AQP3, and AQP5 protein were confirmed in these tissues by immunoblotting. AQP1 was identified in capillary endothelial cells by immunohistochemical labeling, but not in sweat glands or epidermis. Abundant AQP3 expression was seen in basal levels of epidermis, but not in sweat glands. AQP2 and AQP4 were not observed in either skin or sweat glands. Immunohistochemical labeling revealed abundant AQP5 in secretory parts of rat and mouse sweat glands, where immunoelectron microscopy demonstrated abundant AQP5 labeling in the apical plasma membrane. AQP5 immunolabeling of human sweat glands yielded a similar pattern. To establish the role of AQP5 in sweat secretion, we tested the response of adult mice to s.c. injection of pilocarpine, as visualized by reaction of secreted amylase with iodine/starch. The number of active sweat glands was dramatically reduced in AQP5-null (−/−) mice compared with heterozygous (+/−) and wild-type (+/+) mice. We conclude that the presence of AQP5 in plasma membranes of sweat glands is essential for secretion, providing potential insight into mechanisms underlying mammalian thermoregulation, tactile sensitivity, and the pathophysiology of hyperhidrosis.

Aquaporins in health and disease

The molecular basis of membrane water-permeability remained elusive until the recent discovery of the aquaporin water-channel proteins. The fundamental importance of these proteins is suggested by their conservation from bacteria through plants to mammals. Ten mammalian aquaporins have thus far been identified, each with a distinct distribution. In the kidney, lung, eye and brain, multiple water-channel homologs are expressed, providing a network for water transport in those locations. It is increasingly clear that alterations in aquaporin expression or function can be rate-limiting for water transport across certain membranes. Aquaporins are likely to prove central to the pathophysiology of a variety of clinical conditions from diabetes insipidus to various forms of edema and, ultimately, they could be a target for therapy in diseases of altered water homeostasis.

Regulatory T cells as immunotherapy

Regulatory T cells (Tregs) suppress exuberant immune system activation and promote immunologic tolerance. Because Tregs modulate both innate and adaptive immunity, the biomedical community has developed an intense interest in using Tregs for immunotherapy. Conditions that require clinical tolerance to improve outcomes – autoimmune disease, solid organ transplantation, and hematopoietic stem cell transplantation – may benefit from Treg immunotherapy. Investigators have designed ex vivo strategies to isolate, preserve, expand, and infuse Tregs. Protocols to manipulate Treg populations in vivo have also been considered. Barriers to clinically feasible Treg immunotherapy include Treg stability, off-cell effects, and demonstration of cell preparation purity and potency. Clinical trials involving Treg adoptive transfer to treat graft versus host disease preliminarily demonstrated the safety and efficacy of Treg immunotherapy in humans. Future work will need to confirm the safety of Treg immunotherapy and establish the efficacy of specific Treg subsets for the treatment of immune-mediated disease.

Regulatory T Cells Reduce Acute Lung Injury Fibroproliferation by Decreasing Fibrocyte Recruitment

Acute lung injury (ALI) causes significant morbidity and mortality. Fibroproliferation in ALI results in worse outcomes, but the mechanisms governing fibroproliferation remain poorly understood. Regulatory T cells (Tregs) are important in lung injury resolution. Their role in fibroproliferation is unknown. We sought to identify the role of Tregs in ALI fibroproliferation, using a murine model of lung injury. Wild-type (WT) and lymphocyte-deficient Rag-1(-/-) mice received intratracheal LPS. Fibroproliferation was characterized by histology and the measurement of lung collagen. Lung fibrocytes were measured by flow cytometry. To dissect the role of Tregs in fibroproliferation, Rag-1(-/-) mice received CD4(+)CD25(+) (Tregs) or CD4(+)CD25(-) Tcells (non-Tregs) at the time of LPS injury. To define the role of the chemokine (C-X-C motif) ligand 12 (CXCL12)-CXCR4 pathway in ALI fibroproliferation, Rag-1(-/-) mice were treated with the CXCR4 antagonist AMD3100 to block fibrocyte recruitment. WT and Rag-1(-/-) mice demonstrated significant collagen deposition on Day 3 after LPS. WT mice exhibited the clearance of collagen, but Rag-1(-/-) mice developed persistent fibrosis. This fibrosis was mediated by the sustained epithelial expression of CXCL12 (or stromal cell-derived factor 1 [SDF-1]) that led to increased fibrocyte recruitment. The adoptive transfer of Tregs resolved fibroproliferation by decreasing CXCL12 expression and subsequent fibrocyte recruitment. Blockade of the CXCL12-CXCR4 axis with AMD3100 also decreased lung fibrocytes and fibroproliferation. These results indicate a central role for Tregs in the resolution of ALI fibroproliferation by reducing fibrocyte recruitment along the CXCL12-CXCR4 axis. A dissection of the role of Tregs in ALI fibroproliferation may inform the design of new therapeutic tools for patients with ALI.

Aquaporins and disease: lessons from mice to humans

Recent discovery of a family of water-specific membrane channel proteins, the aquaporins, has provided new insights into the molecular basis of membrane water permeability. Eleven mammalian aquaporins have been identified to date, with homolog present across the spectrum of life, including bacteria, yeast and plants. The distribution of the mammalian aquaporins predicts their participation in a range of pathophysiological events. Empirical evidence of a physiological role for aquaporins is emerging from studies in both mice and humans, and suggests that aquaporins are likely to play significant roles in human pathophysiology.

A critical role for muscle ring finger-1 in acute lung injury-associated skeletal muscle wasting

RATIONALEnAcute lung injury (ALI) is a debilitating condition associated with severe skeletal muscle weakness that persists in humans long after lung injury has resolved. The molecular mechanisms underlying this condition are unknown.nnnOBJECTIVESnTo identify the muscle-specific molecular mechanisms responsible for muscle wasting in a mouse model of ALI.nnnMETHODSnChanges in skeletal muscle weight, fiber size, in vivo contractile performance, and expression of mRNAs and proteins encoding muscle atrophy-associated genes for muscle ring finger-1 (MuRF1) and atrogin1 were measured. Genetic inactivation of MuRF1 or electroporation-mediated transduction of miRNA-based short hairpin RNAs targeting either MuRF1 or atrogin1 were used to identify their role in ALI-associated skeletal muscle wasting.nnnMEASUREMENTS AND MAIN RESULTSnMice with ALI developed profound muscle atrophy and preferential loss of muscle contractile proteins associated with reduced muscle function in vivo. Although mRNA expression of the muscle-specific ubiquitin ligases, MuRF1 and atrogin1, was increased in ALI mice, only MuRF1 protein levels were up-regulated. Consistent with these changes, suppression of MuRF1 by genetic or biochemical approaches prevented muscle fiber atrophy, whereas suppression of atrogin1 expression was without effect. Despite resolution of lung injury and down-regulation of MuRF1 and atrogin1, force generation in ALI mice remained suppressed.nnnCONCLUSIONSnThese data show that MuRF1 is responsible for mediating muscle atrophy that occurs during the period of active lung injury in ALI mice and that, as in humans, skeletal muscle dysfunction persists despite resolution of lung injury.

Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation

Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4(+)CD25(hi)Foxp3(+) Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1-null mice and Treg-depleted (diphtheria toxin-treated Foxp3(DTR)) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 10(5) DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS.

Resolution of Experimental Lung Injury by Monocyte-Derived Inducible Nitric Oxide Synthase

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the lung at day 4 after LPS. When iNOS−/− mice were exposed to intratracheal LPS, early lung injury was attenuated; however, recovery was markedly impaired compared with WT mice. iNOS−/− mice had increased mortality and sustained increases in markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS−/− mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule CD86 in iNOS−/− mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS−/− mice improved survival and enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung repair.

Foxp3 + regulatory T cells promote lung epithelial proliferation

Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality each year. There is a paucity of information regarding the mechanisms necessary for ARDS resolution. Foxp3+ regulatory T cells (Foxp3+ Treg cells) have been shown to be an important determinant of resolution in an experimental model of lung injury. We demonstrate that intratracheal delivery of endotoxin (lipopolysaccharide) elicits alveolar epithelial damage from which the epithelium undergoes proliferation and repair. Epithelial proliferation coincided with an increase in Foxp3+ Treg cells in the lung during the course of resolution. To dissect the role that Foxp3+ Treg cells exert on epithelial proliferation, we depleted Foxp3+ Treg cells, which led to decreased alveolar epithelial proliferation and delayed lung injury recovery. Furthermore, antibody-mediated blockade of CD103, an integrin, which binds to epithelial expressed E-cadherin decreased Foxp3+ Treg numbers and decreased rates of epithelial proliferation after injury. In a non-inflammatory model of regenerative alveologenesis, left lung pneumonectomy, we found that Foxp3+ Treg cells enhanced epithelial proliferation. Moreover, Foxp3+ Treg cells co-cultured with primary type II alveolar cells (AT2) directly increased AT2 cell proliferation in a CD103-dependent manner. These studies provide evidence of a new and integral role for Foxp3+ Treg cells in repair of the lung epithelium.