Specific binding sites on Rhesus rotavirus capsid protein dictate the method of endocytosis inducing the murine model of biliary atresia.

Biliary atresia (BA) is the leading indication for pediatric liver transplantation. Rhesus rotavirus (RRV)-induced murine BA develops an obstructive cholangiopathy that mirrors the human disease. We have previously demonstrated the "SRL" motif on RRV's VP4 protein binds to heat shock cognate 70 protein (Hsc70) facilitating entry into cholangiocytes. In this study, we analyzed how binding to Hsc70 affects viral endocytosis, intracellular trafficking, and uniquely activates the signaling pathway that induces murine BA. Inhibition of clathrin- and dynamin-mediated endocytosis in cholangiocytes following infection demonstrated that blocking dynamin decreased the infectivity of RRV, whereas clathrin inhibition had no effect. Blocking early endosome trafficking resulted in decreased viral titers of RRV, whereas late endosome inhibition had no effect. After infection, TLR3 expression and p-NF-κB levels increased in cholangiocytes, leading to increased release of CXCL9 and CXCL10. Infected mice knocked out for TLR3 had decreased levels of CXCL9 and CXCL10, resulting in reduced NK cell numbers. Human patients with BA experienced an increase in CXCL10 levels, suggesting this as a possible pathway leading to biliary obstruction. Viruses that use Hsc70 for cell entry exploit a clathrin-independent pathway and traffic to the early recycling endosome uniquely activating NF-κB through TLR3, leading to the release of CXCL9 and CXCL10 and inducing NK cell recruitment. These results define how the "SRL" peptide found on RRV's VP4 protein modulates viral trafficking, inducing the host response leading to bile duct obstruction.NEW & NOTEWORTHY In this study, we have determined that the presence of the "SRL" peptide on RRV alters its method of endocytosis and intracellular trafficking through viral binding to heat shock cognate 70 protein. This initiates an inflammatory pathway that stimulates the release of cytokines associated with biliary damage and obstruction.

Deletion of Interferon Lambda Receptor Elucidates Susceptibility to the Murine Model of Biliary Atresia.

Biliary atresia (BA) is a life-threatening cholangiopathy occurring in infancy, the most common indication for pediatric liver transplantation. The etiology of BA remains unknown; however, a viral etiology has been proposed as multiple viruses have been detected in explants of infants afflicted with BA. In the murine model of BA, Rhesus rotavirus (RRV) infection of newborn BALB/c pups results in a cholangiopathy that mirrors human BA. Infected BALB/c pups experience 100% symptomatology and mortality, while C57BL/6 mice are asymptomatic. Interferon-λ (IFN-λ) is an epithelial cytokine that provides protection against viral infection. We demonstrated that IFN-λ is highly expressed in C57BL/6, leading to reduced RRV replication. RRV-infection of C57BL/6 IFN-λ receptor knockout (C57BL/6 IFN-λR KO) pups resulted in 90% developing obstructive symptoms and 45% mortality with a higher viral titer in bile ducts and profound periportal inflammation compared to C57BL/6. Histology revealed complete biliary obstruction in symptomatic C57BL/6 IFN-λR KO pups, while C57BL/6 ducts were patent. These findings suggest that IFN-λ is critical in preventing RRV replication. Deficiency in IFN-λ permits RRV infection, which triggers the inflammatory cascade causing biliary obstruction. Further IFN-λ study is warranted as it may play an important role in infant susceptibility to BA.

The R2 non-neuroinvasive HSV-1 vaccine affords protection from genital HSV-2 infections in a guinea pig model.

Herpes simplex virus (HSV) infections are common and can cause severe illness but no vaccine is currently available. The recent failure of subunit HSV vaccines has highlighted the need for vaccines that present a diverse array of antigens, including the development of next-generation live-attenuated vaccines. However, most attenuated HSV strains propagate poorly, limiting their ability to elicit protective immune responses. A live-attenuated vaccine that replicates in non-neural tissue but is ablated for transmission into the nervous system may elicit protective immune responses without evoking neurologic complications or establishing life-long infections. Initial studies of R2, a live-attenuated vaccine that is engineered to be unable to invade the nervous system, used the guinea pig genital HSV model to evaluate the ability of R2 to replicate at the site of inoculation, cause disease and infect neural tissues. R2 was then evaluated as a vaccine using three routes of inoculation: intramuscular (IM), intradermal (ID) and intravaginal (IVag) and compared to IM administered gD2+MPL/Alum vaccine in the same model. R2 replicated in the genital tract but did not produce acute or recurrent disease and did not infect the neural tissue. The R2 vaccine-induced neutralizing antibody and decreased the severity of acute and recurrent HSV-2 disease as well as recurrent shedding. The ID route was the most effective. ID administered R2 was more effective than gD2+MPL/Alum at inducing neutralizing antibody, suppressing acute disease, and acute vaginal virus replication. R2 was especially more effective at reducing recurrent virus shedding, the most common source of HSV transmission. The live-attenuated prophylactic HSV vaccine, R2, was effective in the guinea pig model of genital HSV-2 especially when administered by the ID route. The use of live-attenuated HSV vaccines that robustly replicate in mucosal tissues but are ablated for neuroinvasion offers a promising approach for HSV vaccines.

Passive transfer of interferon-γ over-expressing macrophages enhances resistance of SCID mice to Mycobacterium tuberculosis infection.

Infection with Mycobacterium tuberculosis (M.tb) is associated with increased deaths worldwide. Alveolar macrophages (AMs) play a critical role in host defense against infection with this pathogen. In this work we tested the hypothesis that passive transfer of normal AMs, IFN-γ activated AMs, or macrophages transduced to over-express IFN-γ into the lungs of immunosuppressed SCID mice, where resident macrophages are present but not functional, would enhance alveolar immunity and increase clearance of pulmonary M.tb infection. Accordingly, SCID mice were infected with M.tb intratracheally (I.T.), following which they received either control macrophages or macrophages overexpressing IFN-γ (J774A.1). The extent of M.tb infection was assessed at 30days post-M.tb infection. SCID mice administered macrophages over-expressing IFN-γ showed a significant decrease in M.tb burden and increased survival compared to J774A.1 control macrophages or untreated mice. This was further associated with a significant increase in IFN-γ and TNF-α mRNA and protein expression, as well as NF-κB (p65) mRNA, in the lungs. The increase in IFN-γ and TNF-α lung levels was inversely proportional to the number of M.tb organisms recovered. These results provide evidence that administration of macrophages overexpressing IFN-γ inhibit M.tb growth in vivo and may enhance host defense against M.tb infection.

Airway delivery of interferon-γ overexpressing macrophages confers resistance to Mycobacterium avium infection in SCID mice.

Mycobacterium avium (M. avium) causes significant pulmonary infection, especially in immunocompromised hosts. Alveolar macrophages (AMs) represent the first line of host defense against infection in the lung. Interferon gamma (IFN-γ) activation of AMs enhances in vitro killing of pathogens such as M. avium We hypothesized that airway delivery of AMs into the lungs of immunodeficient mice infected with M. avium will inhibit M. avium growth in the lung and that this macrophage function is in part IFN-γ dependent. In this study, normal BALB/c and BALB/c SCID mice received M. avium intratracheally while on mechanical ventilation. After 30 days, M. avium numbers increased in a concentration-dependent manner in SCID mice compared with normal BALB/c mice. Airway delivery of IFN-γ-activated BALB/c AMs or J774A.1 macrophages overexpressing IFN-γ into the lungs of SCID mice resulted in a significant decrease in M. avium growth (P < 0.01, both comparisons) and limited dissemination to other organs. In addition, airway delivery of IFN-γ activated AMs and macrophages overexpressing IFN-γ increased the levels of IFN-γ and TNF-α in SCID mice. A similar protective effect against M. avium infection using J774A.1 macrophages overexpressing IFN-γ was observed in IFN-γ knockout mice. These data suggest that administration of IFN-γ activated AMs or macrophages overexpressing IFN-γ may partially restore local alveolar host defense against infections like M. avium, even in the presence of ongoing systemic immunosuppression.

HcZrt2, a zinc responsive gene, is indispensable for the survival of Histoplasma capsulatum in vivo.

Histoplasma capsulatum (Hc) exists in the soil and is capable of adapting to the shift in environment during infection to ensure survival. Yeast encounter a restrictive host environment low in nutrients such as zinc. In this study we functionally analyzed a putative zinc regulated transporter, HcZrt2, in zinc limiting conditions by complementation of HcZrt2 and gene knockdown through RNA interference (RNAi). Complementation analysis demonstrated HcZrt2's ability to functionally replace the characterized Saccharomyces cerevisiae zinc plasma membrane transporters Zrt1 and Zrt2 in zinc deficient medium. Gene silencing revealed that HcZrt2 is essential for growth in zinc deficient medium and plays a role in zinc accumulation. Fungal burden was reduced in mice infected with HcZrt2 silenced strains compared to a control strain. Sixty-seven percent of mice infected with a lethal dose of HcZrt2-RNAi#1 survived, and 100% of mice infected with HcZrt2-RNAi#2 withstood lethal infection. Our data suggest that HcZrt2 is a vital part of zinc homeostasis and essential for the pathogenesis of histoplasmosis.

Yeast Transcriptome and In Vivo Hypoxia Detection Reveals Histoplasma capsulatum Response to Low Oxygen Tension.

Although there is growing understanding of the microenvironmental conditions fungal pathogens encounter as they colonize their host, nothing is known about Histoplasma capsulatum's response to hypoxia. Here we characterized hypoxia during murine histoplasmosis using an in vivo hypoxia detection agent, Hypoxyprobe-2 (HP-2); and analyzed H. capsulatum's transcriptional profile in response to in vitro hypoxia. Immunohistopathology and flow cytometry analyses revealed distinct regions of hypoxia during infection. Granuloma cells, enriched with macrophages and T-cells isolated from infected livers were 66-76% positive for HP-2, of which, 95% of macrophages and 55% of T-cells were hypoxic. Although inhibited, H. capsulatum was able to survive under in vitro hypoxic conditions (<1% O2), and restored growth when replaced in normoxia. Next-generation sequencing (RNA-seq) analysis after 24 hours of hypoxia demonstrated a significant increase in NIT50 (swirm domain DNA binding protein), a predicted ABC transporter (ABC), NADPH oxidoreductase (NADP/FAD), and guanine nucleotide exchange factor (RSP/GEF); and other genes with no known designated function. Computational transcription factor binding site analysis predicted human sterol regulatory element binding protein (SREBP) binding sites upstream of NIT50, ABC, NADP/FAD and RSP/GEF. Hypoxia resulted in a time-dependent increase in the H. capsulatum homolog of SREBP, here named Srb1. Srb1 peaked at 8 hours and returned to basal levels by 24 hours. Our findings demonstrate that H. capsulatum encounters and survives severe hypoxia during infection. Additionally, the hypoxic response may be regulated at the level of transcription, and these studies contribute to the understanding of hypoxic regulation and adaptation in H. capsulatum.

Keratinocyte growth factor administration attenuates murine pulmonary mycobacterium tuberculosis infection through granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent macrophage activation and phagolysosome fusion.

Augmentation of innate immune defenses is an appealing adjunctive strategy for treatment of pulmonary Mycobacterium tuberculosis infections, especially those caused by drug-resistant strains. The effect of intranasal administration of keratinocyte growth factor (KGF), an epithelial mitogen and differentiation factor, on M. tuberculosis infection in mice was tested in prophylaxis, treatment, and rescue scenarios. Infection of C57BL6 mice with M. tuberculosis resulted in inoculum size-dependent weight loss and mortality. A single dose of KGF given 1 day prior to infection with 10(5) M. tuberculosis bacilli prevented weight loss and enhanced pulmonary mycobacterial clearance (compared with saline-pretreated mice) for up to 28 days. Similar effects were seen when KGF was delivered intranasally every third day for 15 days, but weight loss and bacillary growth resumed when KGF was withdrawn. For mice with a well established M. tuberculosis infection, KGF given every 3 days beginning on day 15 postinoculation was associated with reversal of weight loss and an increase in M. tuberculosis clearance. In in vitro co-culture experiments, M. tuberculosis-infected macrophages exposed to conditioned medium from KGF-treated alveolar type II cell (MLE-15) monolayers exhibited enhanced GM-CSF-dependent killing through mechanisms that included promotion of phagolysosome fusion and induction of nitric oxide. Alveolar macrophages from KGF-treated mice also exhibited enhanced GM-CSF-dependent phagolysosomal fusion. These results provide evidence that administration of KGF promotes M. tuberculosis clearance through GM-CSF-dependent mechanisms and enhances host defense against M. tuberculosis infection.

Gallium nitrate is efficacious in murine models of tuberculosis and inhibits key bacterial Fe-dependent enzymes.

Acquiring iron (Fe) is critical to the metabolism and growth of Mycobacterium tuberculosis. Disruption of Fe metabolism is a potential approach for novel antituberculous therapy. Gallium (Ga) has many similarities to Fe. Biological systems are often unable to distinguish Ga(3+) from Fe(3+). Unlike Fe(3+), Ga(3+) cannot be physiologically reduced to Ga(2+). Thus, substituting Ga for Fe in the active site of enzymes may render them nonfunctional. We previously showed that Ga inhibits growth of M. tuberculosis in broth and within cultured human macrophages. We now report that Ga(NO3)3 shows efficacy in murine tuberculosis models. BALB/c SCID mice were infected intratracheally with M. tuberculosis, following which they received daily intraperitoneal saline, Ga(NO3)3, or NaNO3. All mice receiving saline or NaNO3 died. All Ga(NO3)3-treated mice survived. M. tuberculosis CFU in the lungs, liver, and spleen of the NaNO3-treated or saline-treated mice were significantly higher than those in Ga-treated mice. When BALB/c mice were substituted for BALB/c SCID mice as a chronic (nonlethal) infection model, Ga(NO3)3 treatment significantly decreased lung CFU. To assess the mechanism(s) whereby Ga inhibits bacterial growth, the effect of Ga on M. tuberculosis ribonucleotide reductase (RR) (a key enzyme in DNA replication) and aconitase activities was assessed. Ga decreased M. tuberculosis RR activity by 50 to 60%, but no additional decrease in RR activity was seen at Ga concentrations that completely inhibited mycobacterial growth. Ga decreased aconitase activity by 90%. Ga(NO3)3 shows efficacy in murine M. tuberculosis infection and leads to a decrease in activity of Fe-dependent enzymes. Additional work is warranted to further define Ga's mechanism of action and to optimize delivery forms for possible therapeutic uses in humans.

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite.

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ∼7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.

Airway delivery of silica increases susceptibility to mycobacterial infection in mice: potential role of repopulating macrophages.

Silica exposure results in an increased lifelong risk of developing mycobacterial pulmonary infections. To date, there are no animal models that replicate this finding to permit assessment of the mechanisms underlying susceptibility to mycobacterial infection. To test the hypothesis that prior silica exposure increases risk of mycobacterial infection, we intratracheally (I.T.) administered silica, a control dust (Al(2)O(3)) or saline into mechanically ventilated C57BL/6 mice. Later, the mice received Mycobacterium avium or Mycobacterium tuberculosis I.T. Mice were sacrificed at defined time points and mycobacteria in lung homogenates were quantified. M. avium or M. tuberculosis infection was markedly increased in silica-exposed mice compared with mice exposed to either Al(2)O(3) or saline beginning 3 wk after silica exposure. Similarly, lung sections from silica-exposed mice had many more acid fast bacilli(+) (AFB(+)) organisms than from control mice. Alveolar macrophages (AMs) from bronchoalveolar lavage of silica-exposed mice also revealed a higher number of mycobacteria compared with mice treated with Al(2)O(3) or saline. In addition, passive transfer of AMs from silica-exposed mice to control mice increased M. tuberculosis susceptibility. These results indicate that silica exposure converts mycobacteria-resistant mice into mycobacteria-susceptible mice via a process that likely involves a new population of AMs that are more susceptible to mycobacterial infection.

Susceptibility of Hermansky-Pudlak mice to bleomycin-induced type II cell apoptosis and fibrosis.

Pulmonary inflammation, abnormalities in type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS), a recessive disorder associated with intracellular trafficking defects. We have previously reported that "Pearl" (HPS2) and "Pale Ear" (HPS1) mouse models have pulmonary inflammatory dysregulation and constitutive alveolar macrophage (AM) activation (Young LR et al., J Immunol 2006;176:4361-4368). In the current study, we used these HPS models to investigate mechanisms of lung fibrosis. Unchallenged HPS1 and HPS2 mice have subtle airspace enlargement and foamy AMs, but little or no histologic evidence of lung fibrosis. Seven days after intratracheal bleomycin (0.025 units), HPS1 and HPS2 mice exhibited increased mortality and diffuse pulmonary fibrosis compared to strain-matched C57BL/6J wild-type (WT) mice. HPS mice had significantly increased collagen deposition, and reduced quasi-static and static compliance consistent with a restrictive defect. The early airway and parenchymal cellular inflammatory responses to bleomycin were similar in HPS2 and WT mice. Greater elevations in levels of TGF-beta and IL-12p40 were produced in the lungs and AMs from bleomycin-challenged HPS mice than in WT mice. TUNEL staining revealed apoptosis of type II cells as early as 5 h after low-dose bleomycin challenge in HPS mice, suggesting that type II cell susceptibility to apoptosis may play a role in the fibrotic response. We conclude that the trafficking abnormalities in HPS promote alveolar apoptosis and pulmonary fibrosis in response to bleomycin challenge.

A novel approach to restore lung immunity during systemic immunosuppression.

Systemic immunosuppression accounts for significant morbidity and mortality worldwide. Pulmonary infections represent the most common cause of death in these patients. The resident inflammatory cell in the lung is the alveolar macrophage (AM) and its function is markedly diminished by immunosuppression. We hypothesized that AMs from normal mice with or without gene transfer of the gamma interferon gene inside the macrophages, can restore alveolar immunity in immunosuppressed mice with severe combined immunodeficiency syndrome (SCID). To test this hypothesis we intratracheally instilled normal and IFN-gamma activated macrophages to the lungs of SCID mice. We demonstrated that airway delivery of macrophages results in widespread alveolar distribution, improved phagocytic function and ability to clear opportunistic infections such as Pneumocystis carinii. Airway delivery of IFN-gamma expressing macrophages further increased the function of alveolar macrophages. Reconstitution of the lungs of immunosuppressed mice with normal or activated AMs can restore alveolar immunity despite ongoing systemic immunosuppression.

Alveolar macrophages from HIV-infected subjects are resistant to Mycobacterium tuberculosis in vitro.

HIV-infected individuals frequently develop Mycobacterium tuberculosis (MTB) infection. Alveolar macrophages (AM) are the initial host defense against this organism. We measured MTB growth in AM from normal and HIV-infected subjects after in vitro exposure. Intracellular growth of MTB was reduced in AM from HIV-infected subjects compared with normal macrophages. This was confined to subjects with CD4 counts greater than 200/microl. Growth of avirulent mycobacteria in HIV macrophages was significantly less than virulent MTB. Because avirulent MTB is more sensitive to tumor necrosis factor-alpha (TNF-alpha), we examined the relationship between cytokine secretion and mycobacterial growth. Higher AM spontaneous TNF-alpha secretion was associated with reduced MTB growth in normal AM. This relationship was not seen in HIV-infected subjects, suggesting that other factors contributed to mycobacteria resistance. Mycobacteria-induced TNF-alpha secretion was inversely associated with growth in normal AM but not in HIV-infected subjects. Finally, binding and internalization of MTB was augmented in HIV macrophages compared with normal, demonstrating that reduced intracellular MTB growth was not due to impaired phagocytosis. In conclusion, the increased incidence of MTB infection in HIV-infected subjects does not appear to be due to a defect in macrophage innate immunity.

Morphologic detection and functional assessment of reconstituted normal alveolar macrophages in the lungs of SCID mice.

Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for approximately 50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense.

Fibronectin facilitates Mycobacterium tuberculosis attachment to murine alveolar macrophages.

Mycobacterium tuberculosis remains a major cause of pulmonary infection worldwide. Attachment of M. tuberculosis organisms to alveolar macrophages (AMs) represents the earliest phase of primary infection in pulmonary tuberculosis. In this study fibronectin (Fn), an adhesive protein, is shown to bind M. tuberculosis organisms and facilitates attachment of M. tuberculosis to murine AMs. A monoclonal antibody (MAb) specific to the heparin binding domain (HBD) of Fn decreases (125)I-Fn binding to M. tuberculosis; whereas MAbs specific to either the cell binding domain (CBD) or the gelatin binding domain (GBD) have no effect on Fn binding to M. tuberculosis. In the presence of exogenous Fn (10 microg/ml) M. tuberculosis attachment to AMs increased significantly from control levels (means +/- standard errors of the means) of 11.5% +/- 1.1% to 44.2% +/- 4.2% (P < 0.05). Fn-enhanced attachment was significantly decreased from 44.2% +/- 4.2% to 10.8% +/- 1.2% (P < 0.05) in the presence of anti-Fn polyclonal antibodies. The attachment is also inhibited in the presence of MAbs specific for the HBD and CBD, whereas MAbs specific to GBD did not affect the attachment. Further, an Fn cell binding peptide, Arg-Gly-Asp-Ser (RGDS), decreased the attachment from 44.2% +/- 4.2% to 15.3% +/- 1.2% (P < 0.05), whereas addition of a control peptide, Arg-Gly-Glu-Ser (RGES) did not affect the attachment (40.5% +/- 1.8%). These results suggest that Fn-mediated attachment of M. tuberculosis can occur through the binding of Fn to the AM via the CBD and to M. tuberculosis organisms via the HBD.