Inhibition of Nox2 oxidase activity ameliorates influenza A virus-induced lung inflammation

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2(-/y) mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8(+) T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2(-/y) mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ～3-fold higher in Nox2(-/y) mice. The numbers of influenza-specific CD8+D(b)NP(366)+ and D(b)PA(224)+ T cells in the BALF and spleen were comparable in WT and Nox2(-/y) mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner.     

The Initial Draining Lymph Node Primes the Bulk of the CD8 T Cell Response and Influences Memory T Cell Trafficking after a Systemic Viral Infection

Lymphocytic choriomeningitis virus (LCMV) causes a systemic infection in mice with virus replication occurring in both peripheral tissues and secondary lymphoid organs. Because of the rapid systemic dissemination of the virus, the secondary lymphoid organs responsible for the induction of the LCMV-specific CD8 T cell response are poorly defined. We show that the mediastinal lymph node (MedLN) serves as the primary draining lymph node following LCMV infection. In addition, we demonstrate that the MedLN is responsible for priming the majority of the virus-specific CD8 T cell response. Following resolution of the acute infection, the draining MedLN exhibits characteristics of a reactive lymph node including an increased presence of germinal center B cells and increased cellularity for up to 60 days post-infection. Furthermore, the reactive MedLN harbors an increased frequency of CD62L⁻ effector memory CD8 T cells as compared to the non-draining lymph nodes. The accumulation of LCMV-specific CD62L⁻ memory CD8 T cells in the MedLN is independent of residual antigen and is not a unique feature of the MedLN as footpad infection with LCMV leads to a similar increase of virus-specific CD62L⁻ effector memory CD8 T cells in the draining popliteal lymph node. Our results indicate that CD62L⁻ effector memory CD8 T cells are granted preferential access into the draining lymph nodes for an extended time following resolution of an infection.     

Induction and Role of Indoleamine 2,3 Dioxygenase in Mouse Models of Influenza A Virus Infection

Influenza infection stimulates protective host immune responses but paradoxically enhances lung indoleamine 2,3 dioxygenase (IDO) activity, an enzyme that suppresses helper/effector T cells and activates Foxp3-lineage regulatory CD4 T cells (Tregs). Influenza A/PR/8/34 (PR8) infection stimulated rapid elevation of IDO activity in lungs and lung-draining mediastinal lymph nodes (msLNs). Mice lacking intact IDO1 genes (IDO1-KO mice) exhibited significantly lower morbidity after sub-lethal PR8 infection, and genetic or pharmacologic IDO ablation led to much faster recovery after virus clearance. More robust influenza-specific effector CD8 T cell responses manifested in lungs of PR8-infected IDO1-KO mice, though virus clearance rates were unaffected by IDO ablation. Similar outcomes manifested in mice infected with a less virulent influenza A strain (X31). IDO induction in X31-infected lungs was dependent on IFN type II (IFNγ) signaling and was restricted to non-hematopoietic cells, while redundant IFN type 1 or type II signaling induced IDO exclusively in hematopoietic cells from msLNs. Memory T cells generated in X31-primed IDO1-KO mice protected mice from subsequent challenge with lethal doses of PR8 (100×LD₅₀). However recall T cell responses were less robust in lung interstitial tissues, and classic dominance of TCR Vβ8.3 chain usage amongst memory CD8⁺ T cells specific for influenza nucleoprotein (NP₃₆₆) did not manifest in IDO1-KO mice. Thus, influenza induced IDO activity in lungs enhanced morbidity, slowed recovery, restrained effector T cell responses in lungs and shaped memory T cell repertoire generation, but did not attenuate virus clearance during primary influenza A infection.     

Infection with a Mouse-Adapted Strain of the 2009 Pandemic Virus Causes a Highly Severe Disease Associated with an Impaired T Cell Response

Despite a relatively low fatality rate, the 2009 H1N1 pandemic virus differed from other seasonal viruses in that it caused mortality and severe pneumonia in the young and middle-aged population (18–59 years old). The mechanisms underlying this increased disease severity are still poorly understood. In this study, a human isolate of the 2009 H1N1 pandemic virus was adapted to the mouse (MAp2009). The pathogenicity of the MAp2009 virus and the host immune responses were evaluated in the mouse model and compared to the laboratory H1N1 strain A/Puerto Rico/8/1934 (PR8). The MAp2009 virus reached consistently higher titers in the lungs over 14 days compared to the PR8 virus, and caused severe disease associated with high morbidity and 85% mortality rate, contrasting with the 0% death rate in the PR8 group. During the early phase of infection, both viruses induced similar pathology in the lungs. However, MAp2009-induced lung inflammation was sustained until the end of the study (day 14), while there was no sign of inflammation in the PR8-infected group by day 10. Furthermore, at day 3 post-infection, MAp2009 induced up to 10- to 40-fold more cytokine and chemokine gene expression, respectively. More importantly, the numbers of CD4⁺ T cells and virus-specific CD8⁺ T cells were significantly lower in the lungs of MAp2009-infected mice compared to PR8-infected mice. Interestingly, there was no difference in the number of dendritic cells in the lung and in the draining lymph node. Moreover, mice infected with PR8 or MAp2009 had similar numbers of CCR5 and CXCR3-expressing T cells, suggesting that the impaired T cell response was not due to a lack of chemokine responsiveness or priming of T cells. This study demonstrates that a mouse-adapted virus from an isolate of the 2009 pandemic virus interferes with the adaptive immune response leading to a more severe disease.     

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20^AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20^AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20^AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20^AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20^AEC-KO mice during later stages of infection. When A20^AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20^AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.     

Sequential activation of CD8+ T cells in the draining lymph nodes in response to pulmonary virus infection

We have used a TCR-transgenic CD8+ T cell adoptive transfer model to examine the tempo of T cell activation and proliferation in the draining lymph nodes (DLN) in response to respiratory virus infection. The T cell response in the DLN differed for mice infected with different type A influenza strains with the onset of T cell activation/proliferation to the A/JAPAN virus infection preceding the A/PR8 response by 12-24 h. This difference in T cell activation/proliferation correlated with the tempo of accelerated respiratory DC (RDC) migration from the infected lungs to the DLN in response to influenza virus infection, with the migrant RDC responding to the A/JAPAN infection exhibiting a more rapid accumulation in the lymph nodes (i.e., peak migration for A/JAPAN at 18 h, A/PR8 at 24-36 h). Furthermore, in vivo administration of blocking anti-CD62L Ab at various time points before/after infection revealed that the virus-specific CD8+ T cells entered the DLN and activated in a sequential "conveyor belt"-like fashion. These results indicate that the tempo of CD8+ T cell activation/proliferation after viral infection is dependent on the tempo of RDC migration to the DLN and that T cell activation occurs in an ordered sequential fashion.     

Antibody-forming cell response to virus challenge in mice immunized with DNA encoding the influenza virus hemagglutinin.

Immunization of mice with DNA encoding the influenza virus hemagglutinin (HA) affords complete protection against lethal influenza virus infection and the means to investigate the mechanisms of B-cell responsiveness to virus challenge. Using a single-cell enzyme-linked immunospot assay, we sought to determine the localization of HA-specific antibody-forming cells (AFCs) during the development of humoral immunity in mice given HA DNA vaccine by gene gun. At 33 days postvaccination, populations of AFCs were maintained in the spleen and bone marrow. In response to lethal challenge with influenza virus, the AFCs became localized at the site of antigenic challenge, i.e., within the draining lymph nodes of the lung compartment. Immunoglobulin G (IgG)- and IgA-producing AFCs were detected in lymph nodes of the upper and lower respiratory tracts, underscoring their importance in clearing virus from the lungs. Response to challenge required competent CD4+ T cells, without which no AFCs were generated, even those producing IgM. By contrast, in mice vaccinated with an HA-containing subunit vaccine, fewer AFCs were generated in response to challenge, and these animals were less capable of resisting infection. Our findings demonstrate the comparable localization of AFCs in response to challenge in mice vaccinated with either HA DNA or live virus. Moreover, the former strategy generates both IgG- and IgA-producing plasma cells.     

Cellular immune responses of mice to influenza virus infection

Mice infected with influenza virus develop cytotoxic T lymphocytes (CTL) specific for viral antigens prior to the appearance of virus-specific antibody-forming cells (AFCs). Effector T cells were detected at a time coincident with a precipitous decline in pulmonary virus titer. CTLs of draining lymph nodes and spleen were found to be cross-reactive among H-2 compatible cells infected with influenza type A virus subtypes. AFCs were observed to be primarily hemagglutinin specific. Virus-specific IgA-secreting AFCs were detected in mediastinal lymph nodes of infected mice.     

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.     

Protection against influenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.     

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

Modeling of influenza-specific CD8+ T cells during the primary response indicates that the spleen is a major source of effectors

The biological parameters that determine the distribution of virus-specific CD8(+) T cells during influenza infection are not all directly measurable by experimental techniques but can be inferred through mathematical modeling. Mechanistic and semimechanistic ordinary differential equations were developed to describe the expansion, trafficking, and disappearance of activated virus-specific CD8(+) T cells in lymph nodes, spleens, and lungs of mice during primary influenza A infection. An intensive sampling of virus-specific CD8(+) T cells from these three compartments was used to inform the models. Rigorous statistical fitting of the models to the experimental data allowed estimation of important biological parameters. Although the draining lymph node is the first tissue in which Ag-specific CD8(+) T cells are detected, it was found that the spleen contributes the greatest number of effector CD8(+) T cells to the lung, with rates of expansion and migration that exceeded those of the draining lymph node. In addition, models that were based on the number and kinetics of professional APCs fit the data better than those based on viral load, suggesting that the immune response is limited by Ag presentation rather than the amount of virus. Modeling also suggests that loss of effector T cells from the lung is significant and time dependent, increasing toward the end of the acute response. Together, these efforts provide a better understanding of the primary CD8(+) T cell response to influenza infection, changing the view that the spleen plays a minor role in the primary immune response.     

Proliferative expansion and acquisition of effector activity by memory CD4+ T cells in the lungs following pulmonary virus infection

The memory CD4+ T cell response to the respiratory syncytial virus (RSV) attachment (G) protein in the lungs of primed BALB/c mice undergoing challenge pulmonary RSV infection is dominated by effector T cells expressing a single Vbeta-chain, Vbeta14. We have used Vbeta14 expression to examine the kinetics of the activation, accumulation, and acquisition of the effector activity of memory CD4+ T cells responding to pulmonary infection. This analysis revealed that proliferative expansion and effector CD4+ T cell differentiation preferentially occur in the respiratory tract following rapid activation within and egress from the lymph nodes draining the respiratory tract. These findings suggest that, in response to natural infection at a peripheral mucosal site such as the lungs, memory CD4+ T cell expansion and differentiation into activated effector T cells may occur predominantly in the peripheral site of infection rather than exclusively in the lymph nodes draining the site of infection.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

PC61 (Anti-CD25) Treatment Inhibits Influenza A Virus-Expanded Regulatory T Cells and Severe Lung Pathology during a Subsequent Heterologous Lymphocytic Choriomeningitis Virus Infection

Prior immunity to influenza A virus (IAV) in mice changes the outcome to a subsequent lymphocytic choriomeningitis virus (LCMV) infection and can result in severe lung pathology, similar to that observed in patients that died of the 1918 H1N1 pandemic. This pathology is induced by IAV-specific memory CD8⁺ T cells cross-reactive with LCMV. Here, we discovered that IAV-immune mice have enhanced CD4⁺ Foxp3⁺ T-regulatory (Treg) cells in their lungs, leading us to question whether a modulation in the normal balance of Treg and effector T-cell responses also contributes to enhancing lung pathology upon LCMV infection of IAV-immune mice. Treg cell and interleukin-10 (IL-10) levels remained elevated in the lungs and mediastinal lymph nodes (mLNs) throughout the acute LCMV response of IAV-immune mice. PC61 treatment, used to decrease Treg cell levels, did not change LCMV titers but resulted in a surprising decrease in lung pathology upon LCMV infection in IAV-immune but not in naive mice. Associated with this decrease in pathology was a retention of Treg in the mLN and an unexpected partial clonal exhaustion of LCMV-specific CD8⁺ T-cell responses only in IAV-immune mice. PC61 treatment did not affect cross-reactive memory CD8⁺ T-cell proliferation. These results suggest that in the absence of IAV-expanded Treg cells and in the presence of cross-reactive memory, the LCMV-specific response was overstimulated and became partially exhausted, resulting in a decreased effector response. These studies suggest that Treg cells generated during past infections can influence the characteristics of effector T-cell responses and immunopathology during subsequent heterologous infections. Thus, in humans with complex infection histories, PC61 treatment may lead to unexpected results.     

Infection with Human Metapneumovirus Predisposes Mice to Severe Pneumococcal Pneumonia

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes respiratory tract infections. Prior clinical studies have highlighted the importance of respiratory viruses, such as influenza virus, in facilitating secondary bacterial infections and increasing host immunopathology. The objective of the present work was to evaluate the effects of initial viral infection with hMPV or influenza A virus followed by Streptococcus pneumoniae superinfection 5 days later in a murine model. Both groups of superinfected mice demonstrated significant weight loss (mean of 15%) and higher levels of airway obstruction (mean enhanced pause value of 2.7) compared to those of mice infected with hMPV, influenza virus, or pneumococcus alone. Bacterial counts increased from 5 × 10² CFU/lung in mice infected with pneumococcus only to 10⁷ and 10⁹ CFU/lung in mice with prior infections with hMPV and influenza A virus, respectively. A more pronounced interstitial and alveolar inflammation correlated with higher levels of inflammatory cytokines and chemokines such as interleukin-1α (IL-1α), IL-1β, IL-6, IL-12, monocyte chemotactic protein 1, macrophage inflammatory protein 1α, KC, and granulocyte colony-stimulating factor, as well as greater expression of Toll-like receptor 2 (TLR2), TLR6, TLR7, and TLR13 in the lungs of superinfected animals compared to results for single infections, with similar immunological effects seen in both coinfection models. Prior infection with either hMPV or influenza A virus predisposes mice to severe pneumococcus infection.     

Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea

Background

In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction.

Methods and Findings

The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox ^_/Y) and wild-type littermates. On a standard place training task, gp91phox ^_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA) controls, while no changes emerged in gp91phox ^_/Y mice. Additionally, wild-type mice, but not gp91phox ^_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures.

Conclusions

The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Murine model of BCG lung infection: Dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes

C57B1/6 female mice were infected with an intrapulmonary dose of 2.5 × 10⁴ BCG(Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1,2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4–6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3⁺ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1⁺) per lung increased by about 50%, 5–6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNγ producing non-T cells outnumbered T cells producing IFNγ. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNγ producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNγ response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNγ, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine