Nancy B. Ray

Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia

Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue–enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.

Induction of an invasive phenotype by human parvovirus B19 in normal human synovial fibroblasts

OBJECTIVE To investigate the possible role of human parvovirus B19 as an etiologic agent in rheumatoid arthritis (RA), with particular emphasis on its ability to induce invasiveness in human synovial fibroblasts. METHODS We established an experimental in vitro system in which normal primary human synovial fibroblasts were treated with or without parvovirus B19-containing human sera for 7 days. The fibroblasts were then tested for their ability to degrade reconstituted cartilage matrix using a well-characterized cartilage invasion assay system. RESULTS Incubation with parvovirus B19-containing serum induced an invasive phenotype in normal human synovial fibroblasts. B19 serum-treated synovial fibroblasts exhibited an increase in invasion of up to 248% compared with the activity of fibroblasts in media alone, in contrast to B19-negative sera-treated synovial fibroblasts, which exhibited no significant change compared with that in media alone. In addition, preincubation of viremic serum with a neutralizing antibody to B19 abrogated the observed effect. CONCLUSION These results provide direct evidence regarding the ability of parvovirus B19 to induce invasive properties in normal human synovial fibroblasts. Parvovirus B19 has been proposed as an etiologic agent of RA, and our data provide the first biologic link between exposure to B19 and phenotypic changes in normal human synovial fibroblasts.

Chronic ethanol induces inhibition of antigen-specific CD8+ but not CD4+ immunodominant T cell responses following Listeria monocytogenes inoculation.

Chronic ethanol consumption results in immunodeficiency. Previous work with chronic ethanol‐fed mice has shown reduced splenic weight and cellularity, including reduced numbers of CD8+ T cells. However, antigen‐specific CD8+ and CD4+ T cell responses in chronic ethanol‐fed mice have been studied relatively little. We have used an attenuated Listeria monocytogenes strain DPL 1942 (LM ΔactA) to inoculate mice and subsequently used CD4+ and CD8+ immunodominant peptides of LM to measure the CD4+ and CD8+ T cell responses after chronic ethanol exposure. We found no major differences between control and ethanol‐fed mice in the kinetics and persistence of antigen‐specific CD4+ T cells in response to an immunodominant LM peptide, as measured by intracellular IFN‐γ staining. In contrast to CD4+ responses, three methods of in vitro antigen presentation indicated that the primary response of CD8+ T cells to several different epitopes was reduced significantly in mice chronically fed ethanol. Antigen‐specific CD8+ T cells were also reduced in chronic ethanol‐fed mice during the contraction phase of the primary response, and memory cells evaluated at 29 and 60 days after inoculation were reduced significantly. BrdU proliferation assays showed that in vivo proliferation of CD8+ T cells was reduced in ethanol‐fed mice, and IL‐2‐dependent in vitro proliferation of naive CD8+ T cells was also reduced. In conclusion, these results suggest that antigen‐specific CD4+ T cell responses to LM are affected little by chronic ethanol consumption; however, antigen‐specific CD8+ T cell responses are reduced significantly, as are in vivo and in vitro proliferation. The reduction of antigen‐specific CD8+ T cells may contribute strongly to the immunodeficiency caused by ethanol abuse.

Oral pretreatment of mice with CpG DNA reduces susceptibility to oral or intraperitoneal challenge with virulent Listeria monocytogenes.

ABSTRACT Listeria monocytogenes is an enteroinvasive intracellular bacterial pathogen that infects humans and other animals, including mice, sometimes resulting in severe systemic infections. Previous studies showed that intraperitoneal (i.p.) pretreatment of susceptible BALB/c mice with immune-stimulatory CpG DNA 48 to 96 h prior to i.p. challenge with virulent L. monocytogenes reduces bacterial numbers in livers by greater than 100-fold, correlating with recovery from infection. Here we show that oral pretreatment of BALB/c mice with CpG DNA results in decreased susceptibility to either oral or i.p. challenge with L. monocytogenes. A single dose of 200 μg of CpG DNA administered to BALB/c mice orally by gavage 48 h or 7 days before oral challenge with virulent L. monocytogenes reduces bacterial numbers approximately 10- to 100-fold in livers and spleens. Lymphotoxin alpha knockout mice lacking Peyers patches (PPs) and pretreated orally with CpG DNA 48 h prior to oral challenge with L. monocytogenes also have reduced susceptibility to infection, suggesting that PPs are required neither for oral infection nor for CpG-induced resistance against oral infection with L. monocytogenes. Surprisingly, 48-h oral pretreatment of BALB/c mice with 100 to 200 μg of CpG DNA results in approximately 100-fold-decreased bacterial numbers in livers following i.p. challenge with L. monocytogenes, suggesting, along with other data in this report, that orally delivered CpG DNA induces systemic resistance to infection. These results indicate that oral administration of CpG DNA induces systemic innate immune defenses against either oral or systemic infection with virulent L. monocytogenes.

Reply to "The flip side of cardiolipin import"

To the Editor: We read with interest the paper by Ray and colleagues1 reporting that elevated cardiolipin levels during pulmonary infection impair lung and lung surfactant function. They suggest that Atp8B1 is a cardiolipin importer that helps clear this lipid from lung surfactant and that it contains a 40-amino-acid cardiolipin-binding domain that reduces lung injury when introduced into infected lungs. We do appreciate the inference that elevated cardiolipin contributes to the etiology of pneumonia and the observation that Atp8B1 overexpression improves lung function after experimental pneumonia. However, the mechanism of pulmonary cardiolipin accumulation and the role of Atp8B1 therein is not illuminated by the experiments of Ray and colleagues1. Atp8B1 deficiency not only causes progressive familial intrahepatic cholestasis type 1 but also extrahepatic symptoms arising from effects on the apical membranes of epithelial cells2. For instance, Atp8B1deficient hepatocytes are prone to apical membrane damage resulting from extraction of membrane constituents by hydrophobic bile salts3–5, and stereocilia and cochlear hair cells suffer from progressive degeneration probably as a result of impaired Ca2+-ATPase (PMCA2) activity6. In vitro, Atp8B1-deficient Caco-2 cells show loss of microvilli and reduced membrane protein expression7. Collectively, these data support a role for Atp8B1 in preserving a stable apical membrane environment and suggest that enhanced pulmonary cardiolipin in Atp8B1 deficiency may result from damaged apical membranes in lung epithelia. Ray and colleagues suggest that Atp8B1 is a cardiolipin transporter, on the basis of measurements of cardiolipin internalization1. To be transported, cardiolipin must traverse the aqueous space to reach the membrane. The authors used fluorescently labeled nitrobenzoxadiazole (NBD)-cardiolipins with four long fatty acid side chains. Such molecules are essentially insoluble in water, and thus are unable to partition into membranes. If the solubility of NBD-cardiolipin were unexpectedly high enough to permit exchange between membranes, the probe transported to the cytosolic leaflet would exchange into interior membranes. In Figure 4a of Ray et al.1, there is no sign of such redistribution, suggesting that the cardiolipin remains trapped at the external surface. Because cardiolipin is a nonbilayer lipid, which prefers the so-called invertedhexagonal phase, it has fusogenic properties8, so that cardiolipin at the cell surface may result from fusion and not transport. Together, the experiments provide no clear evidence for cardiolipin transport into cells, and it therefore remains unclear that Atp8B1 can be described as a cardiolipin importer. The isolation of the cardiolipin-binding domain peptide (CBD) from Atp8B1 might seem to confirm the idea that Atp8B1 is a cardiolipin importer. Atp8B1 is a P-type ATPase, and structural studies have shown that these transporters combine transmembrane domains, which bind the transported substrate, with three cytoplasmic domains that hydrolyze ATP to drive transport9,10. The CBD is a hydrophilic amino acid sequence in the ATP-hydrolyzing domain in the aqueous cytoplasm, and it cannot be reached by cardiolipin molecules that must be imported from the external membrane leaflet, where they are anchored by their four fatty acid side chains. The data presented by Ray and colleagues1 do not justify the conclusion that Atp8B1 is a cardiolipin transporter. However, the discovery of excess cardiolipin in lung surfactant is noteworthy as cardiolipin is normally confined to the mitochondrial inner membrane, and it thus will be of interest to discover how the cardiolipin reaches the surface. The mechanisms behind the beneficial effects of CBD administration in pulmonary infection will await further investigation.