Bennett Davenport

The Channel Kinases TRPM6 and TRPM7 Are Functionally Nonredundant

TRPM7 and its closest homologue, TRPM6, are the only known fusions of an ion channel pore with a kinase domain. Deletion of TRPM7 in DT40 B-lymphocytes causes growth arrest, Mg2+ deficiency, and cell death within 24–48 h. Amazingly, in analogy to TRPM6-deficient patients who can live a normal life if provided with a Mg2+-rich diet, TRPM7-deficient DT40 B-lymphocytes show wild type cell growth if supplied with 5–10 mm Mg2+ concentrations in their extracellular medium. Here we have investigated the functional relationship between TRPM6 and TRPM7. We show that TRPM7 deficiency in DT40 cells cannot be complemented by heterologously expressed TRPM6. Nevertheless, both channels can influence each others biological activity. Our data demonstrate that TRPM6 requires TRPM7 for surface expression in HEK-293 cells and also that TRPM6 is capable of cross-phosphorylating TRPM7 as assessed using a phosphothreonine-specific antibody but not vice versa. TRPM6 and TRPM7 coexpression studies in DT40 B-cells indicate that TRPM6 can modulate TRPM7 function. In conclusion, although TRPM6 and TRPM7 are closely related and deficiency in either one of these molecules severely affects Mg2+ homeostasis regulation, TRPM6 and TRPM7 do not appear to be functionally redundant but rather two unique and essential components of vertebrate ion homeostasis regulation.

Altered functional properties of a TRPM2 variant in Guamanian ALS and PD

Two related neurodegenerative disorders, Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism–dementia (PD), originally occurred at a high incidence on Guam, in the Kii peninsula of Japan, and in southern West New Guinea more than 50 years ago. These three foci shared a unique mineral environment characterized by the presence of severely low levels of Ca2+ and Mg2+, coupled with high levels of bioavailable transition metals in the soil and drinking water. Epidemiological studies suggest that genetic factors also contribute to the etiology of these disorders. Here, we report that a variant of the transient receptor potential melastatin 2 (TRPM2) gene may confer susceptibility to these diseases. TRPM2 encodes a calcium-permeable cation channel highly expressed in the brain that has been implicated in mediating cell death induced by oxidants. We found a heterozygous variant of TRPM2 in a subset of Guamanian ALS (ALS-G) and PD (PD-G) cases. This variant, TRPM2P1018L, produces a missense change in the channel protein whereby proline 1018 (Pro1018) is replaced by leucine (Leu1018). Functional studies revealed that, unlike WT TRPM2, P1018L channels inactivate. Our results suggest that the ability of TRPM2 to maintain sustained ion influx is a physiologically important function and that its disruption may, under certain conditions, contribute to disease states.

Analysis of T cell receptor beta chains that combine with dominant conserved TRAV5D-4*04 anti-insulin B:9-23 alpha chains.

OBJECTIVE The objective of this study was to define the spectrum of TCR beta chains permissive for T cells with alpha chains containing the conserved TRAV5D-4*04 sequence to target the insulin B:9-23 peptide, a major epitope for initiation of diabetes in the NOD mouse. MATERIALS AND METHODS We produced T cell hybridomas from mice with single T cell receptors (BDC12-4.1 TCR alpha(+)beta(+) double transgenic mice and BDC12-4.4 TCR alpha(+)beta(+) double retrogenic mice) or from mice with only the corresponding alpha chains transgene or retrogene and multiple endogenous TCR beta chains. RESULTS Hybridomas with the complete BDC12-4.1 and BDC12-4.4 T cell receptors, despite having markedly different TCR beta chains, responded to similar B:9-23 peptides. Approximately 1% of the hybridomas from mice with the fixed TRAV5D-4*04 alpha chains and multiple endogenous beta chains responded to B:9-23 peptides while the majority of hybridomas with different beta chains did not respond. There was no apparent conservation of TCR beta chain sequences in the responding hybridomas. CONCLUSIONS Approximately 1% of hybridomas utilizing different TCR beta chains paired with the conserved TRAV5D-4*04 containing alpha chains respond to insulin peptide B:9-23. Therefore, TCR beta chain sequences make an important contribution to insulin B:9-23 peptide recognition but multiple beta chain sequences are permissive for recognition.

Multiple layers of CD80/86-dependent costimulatory activity regulate primary, memory, and secondary lymphocytic choriomeningitis virus-specific T cell immunity.

ABSTRACT The lymphocytic choriomeningitis virus (LCMV) system constitutes one of the most widely used models for the study of infectious disease and the regulation of virus-specific T cell immunity. However, with respect to the activity of costimulatory and associated regulatory pathways, LCMV-specific T cell responses have long been regarded as relatively independent and thus distinct from the regulation of T cell immunity directed against many other viral pathogens. Here, we have reevaluated the contribution of CD28-CD80/86 costimulation in the LCMV system by use of CD80/86-deficient mice, and our results demonstrate that a disruption of CD28-CD80/86 signaling compromises the magnitude, phenotype, and/or functionality of LCMV-specific CD8+ and/or CD4+ T cell populations in all stages of the T cell response. Notably, a profound inhibition of secondary T cell immunity in LCMV-immune CD80/86-deficient mice emerged as a composite of both defective memory T cell development and a specific requirement for CD80 but not CD86 in the recall response, while a related experimental scenario of CD28-dependent yet CD80/86-independent secondary CD8+ T cell immunity suggests the existence of a CD28 ligand other than CD80/86. Furthermore, we provide evidence that regulatory T cells (TREGs), the homeostasis of which is altered in CD80/86−/− mice, contribute to restrained LCMV-specific CD8+ T cell responses in the presence of CD80/86. Our observations can therefore provide a more coherent perspective on CD28-CD80/86 costimulation in antiviral T cell immunity that positions the LCMV system within a shared context of multiple defects that virus-specific T cells acquire in the absence of CD28-CD80/86 costimulation.

High Efficiency of Antiviral CD4+ Killer T Cells

The destruction of infected cells by cytotxic T lymphocytes (CTL) is integral to the effective control of viral and bacterial diseases, and CTL function at large has long been regarded as a distinctive property of the CD8+T cell subset. In contrast, and despite their first description more than three decades ago, the precise contribution of cytotoxic CD4+T cells to the resolution of infectious diseases has remained a matter of debate. In particular, the CTL activity of pathogen-specific CD4+ “helper” T cells constitutes a single trait among a diverse array of other T cell functionalities, and overall appears considerably weaker than the cytolytic capacity of CD8+ effector T cells. Here, using an in vivo CTL assay, we report that cytotoxic CD4+T cells are readily generated against both viral and bacterial pathogens, and that the efficiency of MHC-II-restricted CD4+T cell killing adjusted for effector:target cell ratios, precise specificities and functional avidities is comparable in magnitude to that of CD8+T cells. In fact, the only difference between specific CD4+ and CD8+T cells pertains to the slightly delayed killing kinetics of the former demonstrating that potent CTL function is a cardinal property of both antiviral CD8+ and CD4+T cells.

Neutrophil transfer of miR-223 to lung epithelial cells dampens acute lung injury in mice

Intercellular transfer of miR-223 from neutrophils to alveolar epithelial cells reduces lung inflammation in a mouse model of ventilator-induced lung injury or pulmonary bacterial infection. Starting an intercellular conversation In a new study, Neudecker et al. show that transfer of microRNA-223 (miR-223) from neutrophils to lung alveoli helps to dampen lung inflammation and promotes the resolution of ventilator-induced lung injury in mice. The authors suggest that neutrophils secrete microRNAs in microvesicles that are then taken up by alveolar epithelial cells. They show that miR-223–deficient mice are prone to lung injury, whereas overexpression of miR-223 is protective. Intercellular transfer of microRNAs can mediate communication between critical effector cells. We hypothesized that transfer of neutrophil-derived microRNAs to pulmonary epithelial cells could alter mucosal gene expression during acute lung injury. Pulmonary-epithelial microRNA profiling during coculture of alveolar epithelial cells with polymorphonuclear neutrophils (PMNs) revealed a selective increase in lung epithelial cell expression of microRNA-223 (miR-223). Analysis of PMN-derived supernatants showed activation-dependent release of miR-223 and subsequent transfer to alveolar epithelial cells during coculture in vitro or after ventilator-induced acute lung injury in mice. Genetic studies indicated that miR-223 deficiency was associated with severe lung inflammation, whereas pulmonary overexpression of miR-223 in mice resulted in protection during acute lung injury induced by mechanical ventilation or by infection with Staphylococcus aureus. Studies of putative miR-223 gene targets implicated repression of poly(adenosine diphosphate–ribose) polymerase–1 (PARP-1) in the miR-223–dependent attenuation of lung inflammation. Together, these findings suggest that intercellular transfer of miR-223 from neutrophils to pulmonary epithelial cells may dampen acute lung injury through repression of PARP-1.

signature channels of excitability no more: L-type channels in immune cells

Although the concept of Ca2+ as a universal messenger is well established, it was assumed that the regulatory mechanisms of Ca2+-signaling were divided along the line of electric excitability. Recent advances in molecular biology and genomics have, however, provided evidence that non-excitable cells such as immunocytes also express a wide and diverse pool of ion channels that does not differ as significantly from that of excitable cells as originally assumed. Ion channels and transporters are involved in virtually all aspects of immune response regulation, from cell differentiation and development to activation, and effector functions such as migration, antibody-secretion, phagosomal maturation, or vesicular delivery of bactericidal agents. This comprises TRP channel family members, voltage- and Ca2+-gated K+- and Na+-channels, as well as unexpectedly, components of the CaV1-subfamily of voltage-gated L-type Ca2+-channels, originally thought to be signature molecules of excitability. This article provides an overview of recent observations made in the field of CaV1 L-type channel function in the immune context, as well as presents results we obtained studying these channels in B-lymphocytes.

Molecules in medicine mini review: the αβ T cell receptor

As an integral part of the mammalian immune system, a distributed network of tissues, cells, and extracellular factors, T lymphocytes perform and control a multitude of activities that collectively contribute to the effective establishment, maintenance, and restoration of tissue and organismal integrity. Development and function of T cells is controlled by the T cell receptor (TCR), a heterodimeric cell surface protein uniquely expressed on T cells. During T cell development, the TCR undergoes extensive somatic diversification that generates a diverse T cell repertoire capable of recognizing an extraordinary range of protein and nonprotein antigens presented in the context of major histocompatibility complex molecules (MHC). In this review, we provide an introduction to the TCR, describing underlying principles that position this molecule as a central regulator of the adaptive immune system involved in responses ranging from tissue protection and preservation to pathology and autoimmunity.

Inflammatory monocytes mediate control of acute alphavirus infection in mice

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause debilitating acute and chronic musculoskeletal disease. Monocytes are implicated in the pathogenesis of these infections; however, their specific roles are not well defined. To investigate the role of inflammatory Ly6ChiCCR2+ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administration of diptheria toxin (DT). DT-treated CCR2-DTR mice displayed more severe disease following CHIKV and RRV infection and had fewer Ly6Chi monocytes and NK cells in circulation and muscle tissue compared with DT-treated WT mice. Furthermore, depletion of CCR2+ or Gr1+ cells, but not NK cells or neutrophils alone, restored virulence and increased viral loads in mice infected with an RRV strain encoding attenuating mutations in nsP1 to levels detected in monocyte-depleted mice infected with fully virulent RRV. Disease severity and viral loads also were increased in DT-treated CCR2-DTR+;Rag1-/- mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. Monocytes and macrophages sorted from muscle tissue of RRV-infected mice were viral RNA positive and had elevated expression of Irf7, and co-culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was Irf3;Irf7 and Mavs-dependent. Consistent with these data, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in Mavs-/- mice. Finally, reconstitution of Irf3-/-;Irf7-/- mice with CCR2-DTR bone marrow rescued mice from severe infection, and this effect was reversed by depletion of CCR2+ cells, indicating that CCR2+ hematopoietic cells are capable of inducing an antiviral response. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1, the viral RNA capping protein, counteract this response.

Aging Boosts Antiviral CD8+T Cell Memory Through Improved Engagement Of Diversified Recall Response Determinants

The determinants of protective CD8+ memory T cell (CD8+TM) immunity remain incompletely defined and may in fact constitute an evolving agency as aging CD8+TM progressively acquire enhanced rather than impaired recall capacities. Here, we show that old as compared to young antiviral CD8+TM more effectively harness disparate molecular processes (cytokine signaling, trafficking, effector functions, and co-stimulation/inhibition) that in concert confer greater secondary reactivity. The relative reliance on these pathways is contingent on the nature of the secondary challenge (greater for chronic than acute viral infections) and over time, aging CD8+TM re-establish a dependence on the same accessory signals required for effective priming of naïve CD8+T cells in the first place. Thus, our findings are consistent with the recently proposed “rebound model” that stipulates a gradual alignment of naïve and CD8+TM properties, and identify a diversified collection of potential targets that may be exploited for the therapeutic modulation of CD8+TM immunity.