Muc5b is required for airway defence.

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

The DNA Damage Response and HIV-Associated Pulmonary Arterial Hypertension.

The HIV-infected population is at a dramatically increased risk of developing pulmonary arterial hypertension (PAH), a devastating and fatal cardiopulmonary disease that is rare amongst the general population. It is increasingly apparent that PAH is a disease with complex and heterogeneous cellular and molecular pathologies, and options for therapeutic intervention are limited, resulting in poor clinical outcomes for affected patients. A number of soluble HIV factors have been implicated in driving the cellular pathologies associated with PAH through perturbations of various signaling and regulatory networks of uninfected bystander cells in the pulmonary vasculature. While these mechanisms are likely numerous and multifaceted, the overlapping features of PAH cellular pathologies and the effects of viral factors on related cell types provide clues as to the potential mechanisms driving HIV-PAH etiology and progression. In this review, we discuss the link between the DNA damage response (DDR) signaling network, chronic HIV infection, and potential contributions to the development of pulmonary arterial hypertension in chronically HIV-infected individuals.

Widespread colonization of the lung by Tropheryma whipplei in HIV infection.

RATIONALE: Lung infections caused by opportunistic or virulent pathogens are a principal cause of morbidity and mortality in HIV infection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV. OBJECTIVES: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota. METHODS: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects. MEASUREMENTS AND MAIN RESULTS: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple's disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (>50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P = 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung-derived T. whipplei had similar gene content to two isolates obtained from subjects with Whipple's disease. CONCLUSIONS: Asymptomatic subjects with HIV infection have unexpected colonization of the lung by T. whipplei, which is reduced by effective antiretroviral therapy and merits further study for a potential pathogenic role in chronic pulmonary complications of HIV infection.

Adding Another Piece to the Puzzle of Why NTM Infections Are Relatively Uncommon despite Their Ubiquitous Nature.

Since nontuberculous mycobacteria (NTM) are pervasive in the environment and NTM infections are relatively uncommon, underlying hereditary or acquired host susceptibility factors should be sought for in most NTM-infected patients. To facilitate identification of underlying risk factors, it is useful to classify NTM disease into skin-soft tissue infections, isolated NTM lung disease, and extrapulmonary visceral/disseminated disease because the latter two categories have unique sets of underlying host risk factors. Nakajima and coworkers (M. Nakajima, M. Matsuyama, M. Kawaguchi, T. Kiwamoto, et al., mBio 12:e01947-20, 2021, https://doi.org/10.1128/mBio.01947-20) in a recent issue of mBio found that Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that is induced by oxidative stress but induces antioxidant molecules, provides protection against an NTM infection in a murine model. While they showed that Nrf2 induction of Nramp-1 enhanced phagosome-lysosome fusion, we discuss other potential mechanisms by which oxidative stress predisposes to and Nrf2 protects against NTM infections.

Nrf2 activator PB125® as a carnosic acid-based therapeutic agent against respiratory viral diseases, including COVID-19.

PB125® is a phytochemical composition providing potent Nrf2 activation as well as a number of direct actions that do not involve Nrf2. Nrf2 is a transcription actor that helps maintain metabolic balance by providing redox-sensitive expression of numerous genes controlling normal day-to-day metabolic pathways. When ordinary metabolism is upset by extraordinary events such as injury, pathogenic infection, air or water pollution, ingestion of toxins, or simply by the slow but incessant changes brought about by aging and genetic variations, Nrf2 may also be called into action by the redox changes resulting from these events, whether acute or chronic. A complicating factor in all of this is that Nrf2 levels decline with aging, leaving the elderly less able to maintain proper redox balance. The dysregulated gene expression that results can cause or exacerbate a wide variety of pathological conditions, including susceptibility to viral infections. This review examines the characteristics desirable in Nrf2 activators that have therapeutic potential, as well as some of the patterns of dysregulated gene expression commonly observed during pulmonary infections and the normalizing effects possible by judicious use of phytochemicals to increase the activation level of available Nrf2.

Nrf2 Activator PB125® as a Potential Therapeutic Agent Against COVID-19.

Nrf2 is a transcription factor that regulates cellular redox balance and the expression of a wide array of genes involved in immunity and inflammation, including antiviral actions. Nrf2 activity declines with age, making the elderly more susceptible to oxidative stress-mediated diseases, which include type 2 diabetes, chronic inflammation, and viral infections. Published evidence suggests that Nrf2 activity may regulate important mechanisms affecting viral susceptibility and replication. We examined gene expression levels by GeneChip microarray and by RNA-seq assays. We found that the potent Nrf2 activating composition PB125® downregulates ACE2 and TMPRSS2 mRNA expression in human liver-derived HepG2 cells. ACE2 is a surface receptor and TMPRSS2 activates the spike protein for SARS-Cov-2 entry into host cells. Furthermore, in endotoxin-stimulated primary human pulmonary artery endothelial cells we report the marked downregulation by PB125 of 36 genes encoding cytokines. These include IL1-beta, IL6, TNF-α the cell adhesion molecules ICAM1, VCAM1, and E-selectin, and a group of IFN-γ-induced genes. Many of these cytokines have been specifically identified in the "cytokine storm" observed in fatal cases of COVID-19, suggesting that Nrf2 activation may significantly decrease the intensity of the storm.

Nrf2 Activator PB125® as a Potential Therapeutic Agent against COVID-19.

Nrf2 is a transcription factor that regulates cellular redox balance and the expression of a wide array of genes involved in immunity and inflammation, including antiviral actions. Nrf2 activity declines with age, making the elderly more susceptible to oxidative stress-mediated diseases, which include type 2 diabetes, chronic inflammation, and viral infections. Published evidence suggests that Nrf2 activity may regulate important mechanisms affecting viral susceptibility and replication. We examined gene expression levels by GeneChip microarray and by RNA-seq assays. We found that the potent Nrf2-activating composition PB125® downregulates ACE2 and TMPRSS2 mRNA expression in human liver-derived HepG2 cells. ACE2 is a surface receptor and TMPRSS2 activates the spike protein for SARS-CoV-2 entry into host cells. Furthermore, in endotoxin-stimulated primary human pulmonary artery endothelial cells, we report the marked downregulation by PB125 of 36 genes encoding cytokines. These include IL-1-beta, IL-6, TNF-α, the cell adhesion molecules ICAM-1, VCAM-1, and E-selectin, and a group of IFN-γ-induced genes. Many of these cytokines have been specifically identified in the "cytokine storm" observed in fatal cases of COVID-19, suggesting that Nrf2 activation may significantly decrease the intensity of the storm.

The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells.

Redox imbalance results in damage to cellular macromolecules and interferes with signaling pathways, leading to an inflammatory cellular and tissue environment. As such, the cellular oxidative environment is tightly regulated by several redox-modulating pathways. Many viruses have evolved intricate mechanisms to manipulate these pathways for their benefit, including HIV-1, which requires a pro-oxidant cellular environment for optimal replication. One such virulence factor responsible for modulating the redox environment is the HIV Transactivator of transcription (Tat). Tat is of particular interest as it is actively secreted by infected cells and internalized by uninfected bystander cells where it can elicit pro-oxidant effects resulting in inflammation and damage. Previously, we demonstrated that Tat regulates basal expression of Superoxide Dismutase 2 (sod2) by altering the binding of the Sp-transcription factors at regions relatively near (approx. -210 nucleotides) upstream of the transcriptional start site. Now, using in silico analysis and a series of sod2 promoter reporter constructs, we have identified putative clusters of Sp-binding sites located further upstream of the proximal sod2 promoter, between nucleotides -3400 to -210, and tested their effect on basal transcription and for their sensitivity to HIV-1 Tat. In this report, we demonstrate that under basal conditions, maximal transcription requires a cluster of Sp-binding sites in the -584 nucleotide region, which is extremely sensitive to Tat. Using chromatin immunoprecipitation (ChIP) we demonstrate that Tat results in altered occupancy of Sp1 and Sp3 at this distal Tat-sensitive regulatory element and strongly stimulated endogenous expression of SOD2 in human pulmonary artery endothelial cells (HPAEC). We also report altered expression of Sp1 and Sp3 in Tat-expressing HPAEC as well as in the lungs of HIV-1 infected humanized mice. Lastly, Tat co-immunoprecipitated with endogenous Sp3 but not Sp1 and did not alter the acetylation state of Sp3. Thus, here, we have defined a novel and important cis-acting factor in HIV-1 Tat-mediated regulation of SOD2, demonstrated that modulation of Sp1 and Sp3 activity by Tat promotes SOD2 expression in primary human pulmonary artery endothelial cells and determined that pulmonary levels of Sp3 as well as SOD2 are increased in the lungs of a mouse model of HIV infection.

Repression of Nrf2/ARE regulated antioxidant genes and dysregulation of the cellular redox environment by the HIV Transactivator of Transcription.

Chronic HIV infection in the era of anti-retroviral therapy is associated with dramatically increased risk of developing severe cardio pulmonary disease. Common to these diseases is increased oxidative burden and chronic inflammation despite low viremia and restoration of CD4+ T-cell levels. Soluble viral factors are heavily implicated in these disease processes, including the HIV Transactivator of Transcription (Tat). Tat is produced in high levels during infection and secreted from infected cells into circulation where it is internalized by bystander cells and is known to regulate inflammatory pathways and elicit a pro-oxidant environment. We have examined the effects of Tat on the anti-oxidant regulatory network driven by the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in primary human pulmonary arterial endothelial cells, which are heavily involved in pathogenesis of HIV associated lung diseases including pulmonary arterial hypertension and COPD. Co-expression of Tat and a luciferase reporter construct driven by the Nrf2 activated anti-oxidant response element (ARE) demonstrated markedly reduced Nrf2/ARE activity, even when stimulated by the potent Nrf2 activating compound PB125. Additionally, Heme-oxygenase-1 (HO-1) transcription was potently repressed by Tat in a cell line as well as primary endothelial cells, and treatment with PB125 failed to restore transcriptional activity. Other anti-oxidant Nrf2 genes examined included NADPH Dehydrogenase Quinone 1 (NQO1) and Sulfiredoxin-1 (SRXN1). NQO1 was repressed basally by Tat, while SRXN1 transcription was refractory to activation by PB125 in the presence of Tat. Lastly, we demonstrated that Tat expressing cells have increased indicators of oxidative stress including elevated production of reactive oxygen species, measured by electron paramagnetic resonance spectroscopy, and increased levels of nitrotyrosine content. These observations suggest a novel mechanism by which HIV Tat increases oxidative burden by dysregulation of the Nrf2/ARE pathway.

Alterations in redox homeostasis and prostaglandins impair endothelial-dependent vasodilation in euglycemic autoimmune nonobese diabetic mice.

We report herein the novel observation that alterations in oxidant/antioxidant balance are evident and cause vascular dysfunction in aortae of prediabetic nonobese-diabetic mice (NOD). We found that nitrotyrosine, a biochemical marker of oxidant stress, was higher in the NOD aortae when compared to age-matched non-autoimmune BALB/c controls or the diabetes-resistant NOD congenic strain, NOD.Lc7. The oxidant stress was localized to the intimal and medial layers, and endothelium-dependent relaxation to acetylcholine was decreased in isolated aortic rings from NOD mice. Inhibition of nitric oxide synthesis caused an endothelium-dependent contraction, and treatment with either a selective thromboxane A2/prostaglandin H2 receptor antagonist or a non-isozyme-specific cyclooxygenase inhibitor reversed this effect. Aortic rings from NOD.Lc7 did not display the paradoxical vasoconstriction. Furthermore, the vascular dysfunction was caused by oxidative stress, as treatment with a superoxide dismutase mimetic in vivo or with native antioxidant enzymes ex vivo inhibited the tissue oxidant stress and restored endothelium-dependent relaxation. Endothelial function was also restored by the inhibitors of NAD(P)H oxidase, diphenylene iodonium or apocynin. Our studies indicate that an oxidant stress that occurs prior to the onset of diabetes in this mouse model contributes to endothelial dysfunction independently of overt diabetes.

Different roles of Sp family members in HIV-1 Tat-mediated manganese superoxide dismutase suppression in hepatocellular carcinoma cells.

The expression of manganese superoxide dismutase (MnSOD) is regulated by agents associated with cancer development. It has been shown that infection with the human immunodeficiency virus type 1 (HIV-1) is associated with the development of liver cancer and that the transactivating transcriptional factor (Tat) of human HIV-1 reduces the expression of MnSOD in several cell types. However, the role of Tat in the expression of MnSOD in hepatocellular carcinoma is unknown. Furthermore, the precise mechanisms whereby Tat suppresses MnSOD expression in hepatocellular carcinoma cells remain unclear. In this report, we build on our original observations that Tat changes the distribution of Sp family members on the MnSOD promoter, which accounts for Tat-dependent changes in basal expression. In hepatic cells, Tat expression upregulates Sp1/Sp3, which play different roles in regulating MnSOD transcription. While overexpression of Sp1 stimulates, overexpression of Sp3 represses transcriptional activity. The transcription repression effect of Sp3 is not due to Sp3 competing for the binding site with Sp1 because only the full-length Sp3 but not the truncated Sp3 suppresses MnSOD promoter activity. These findings suggest a novel mechanism by which Tat modulates the repression of the MnSOD gene and establish a link between HIV infection and liver cancer.

HIV-1 Tat regulates the SOD2 basal promoter by altering Sp1/Sp3 binding activity.

Regulation of the basal manganese superoxide dismutase (SOD2) promoter depends on the transcriptional activity of the Sp family of transcription factors. Here we report that reduced expression in the presence of Tat is independent of induction with Tumor necrosis factor alpha and that Tat affects the interaction of Sp1 and Sp3 with the basal promoter. Footprinting and electrophoretic mobility shift assay (EMSA) analyses with extracts from HeLa cells showed that Sp1/Sp3 complexes populate the proximal SOD2 promoter, and that Tat leads to an increase in the binding activity of Sp3. In Drosophila S2 cells, both Sp1 and Sp3 activated the basal SOD2 promoter (88.1 +/- 39.4 fold vs. 10.3 +/- 3.5 fold, respectively), demonstrating a positive, yet lower transcriptional regulatory function for Sp3. Additionally, the inability of Sp3 to synergistically affect promoter activity indicates an efficient competition of Sp3 with Sp1 for the multiple Sp binding sites in the SOD2 basal promoter. Tat potentiated both Sp1 and Sp3 activation of the promoter in S2 cells, though the activity of Sp3 was still lower than that of Sp1. Thus, the consequence of a shift by Tat to increased Sp3-containing complexes on the basal SOD2 promoter is decreased SOD2 expression. Together, our studies demonstrate the functional importance of the interaction of Sp1, Sp3, and Tat, revealing a possible mechanism for the attenuation of basal manganese superoxide dismutase expression.

HIV-1 Tat increases oxidant burden in the lungs of transgenic mice.

Chronic human immunodeficiency virus infection is associated with higher incidence of pulmonary complications including hypertension, vasculopathy, lymphocytic alveolitis, and interstitial pneumonitis not attributed to either opportunistic infections or presence of the virus. The Tat (transactivator of transcription) protein, a required transactivator for expression of full-length viral genes, is pleiotropic and influences expression of cellular inflammatory genes. Tat-dependent transactivation of cellular genes requires specific mediators, including NF-κB, widely recognized as sensitive to changes in cellular oxidant burden. We hypothesized that overproduction of Tat leads to increased oxidant burden and to alterations in basal inflammatory status as measured by NF-κB activation. We engineered transgenic mouse lines that express Tat (86-amino-acid isoform) in the lung under the control of the surfactant protein C promoter. Tat-transgenic mice exhibit increased pulmonary cellular infiltration, increased nitrotyrosine and carbonyl protein modifications, and increased levels of NF-κB, MnSOD, and thioredoxin-interacting protein. These data indicate that Tat increases oxidant burden and resets the threshold for inflammation, which may increase susceptibility to secondary injuries.

HIV-1 Nef is associated with complex pulmonary vascular lesions in SHIV-nef-infected macaques.

RATIONALE: HIV-infected patients with pulmonary arterial hypertension have histologic manifestations that are indistinguishable from those found in patients with idiopathic pulmonary arterial hypertension. In addition, the role of pleiotropic viral proteins in the development of plexiform lesions in HIV-related pulmonary hypertension (HRPH) has not been explored. Simian immunodeficiency virus (SIV) infection of macaques has been found to closely recapitulate many of the characteristic features of HIV infection, and thus hallmarks of pulmonary arterial hypertension should also be found in this nonhuman primate model of HIV. OBJECTIVES: To determine whether pulmonary arterial lesions were present in archived SIV-infected macaque lung tissues from Johns Hopkins University and two National Primate Research Centers. METHODS: Archived macaque and human lung sections were examined via immunohistochemistry for evidence of complex vascular lesions. RESULTS: Complex plexiform-like lesions characterized by lumenal obliteration, intimal disruption, medial hypertrophy, thrombosis, and recanalized lumena were found exclusively in animals infected with SHIV-nef (a chimeric viral construct containing the HIV nef gene in an SIV backbone), but not in animals infected with SIV. The mass of cells in the lesions were factor VIII positive, and contained cells positive for muscle-specific and smooth muscle actins. Lung mononuclear cells were positive for HIV Nef, suggesting viral replication. Endothelial cells in both the SHIV-nef macaques and patients with HRPH, but not in patients with idiopathic pulmonary arterial hypertension, were also Nef positive. CONCLUSIONS: The discovery of complex vascular lesions in SHIV-nef- but not SIV-infected animals, and the presence of Nef in the vascular cells of patients with HRPH, suggest that Nef plays a key role in the development of severe pulmonary arterial disease.

The human immunodeficiency virus-1 Tat protein activates human umbilical vein endothelial cell E-selectin expression via an NF-kappa B-dependent mechanism.

Human immunodeficiency virus infection is associated with inflammation and endothelial cell activation that cannot be ascribed to direct infection by the virus or to the presence of opportunistic infections. Factors related to the virus itself, to the host and/or to environmental exposures probably account for these observations. The HIV protein Tat, a viral regulator required for efficient transcription of the viral genome in host cells is secreted from infected cells and taken up by uninfected by-stander cells. Tat can also act as a general transcriptional activator of key inflammatory molecules. We have examined whether Tat contributes to this endothelial cell activation by activating NF-kappaB. Human endothelial cells exposed to Tat in the culture medium activated E-selectin expression with delayed kinetics compared with tumor necrosis factor (TNF). Tat-mediated E-selectin up-regulation required the basic domain of Tat and was inhibited by a Tat antibody. Transfection of human E-selectin promoter-luciferase reporter constructs into Tat-bearing cells or into endothelial cells co-transfected with a Tat expression vector resulted in induction of luciferase expression. Either Tat or TNF activated p65 translocation and binding to an oligonucleotide containing the E-selectin kappaB site 3 sequence. Tat-mediated p65 translocation was also delayed compared with TNF. Neither agent induced new synthesis of p65. A super-repressor adenovirus (AdIkappaBalphaSR) that constitutively sequesters IkappaB in the cytoplasm as well as cycloheximide or actinomycin D inhibited Tat- or TNF-mediated kappaB translocation and E-selectin up-regulation.

A novel class of microbial phosphocholine-specific phospholipases C.

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.