NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Background: Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood. Methodology/Principal Findings: We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox-/- mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-kB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-kB activation. Conclusions/Significance: These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD

Upregulation of complement proteins in lung cancer cells mediates tumor progression

IntroductionIn vivo, cancer cells respond to signals from the tumor microenvironment resulting in changes in expression of proteins that promote tumor progression and suppress anti-tumor immunity. This study employed an orthotopic immunocompetent model of lung cancer to define pathways that are altered in cancer cells recovered from tumors compared to cells grown in culture.MethodsStudies used four murine cell lines implanted into the lungs of syngeneic mice. Cancer cells were recovered using FACS, and transcriptional changes compared to cells grown in culture were determined by RNA-seq.ResultsChanges in interferon response, antigen presentation and cytokine signaling were observed in all tumors. In addition, we observed induction of the complement pathway. We previously demonstrated that activation of complement is critical for tumor progression in this model. Complement can play both a pro-tumorigenic role through production of anaphylatoxins, and an anti-tumorigenic role by promoting complement-mediated cell killing of cancer cells. While complement proteins are produced by the liver, expression of complement proteins by cancer cells has been described. Silencing cancer cell-specific C3 inhibited tumor growth In vivo. We hypothesized that induction of complement regulatory proteins was critical for blocking the anti-tumor effects of complement activation. Silencing complement regulatory proteins also inhibited tumor growth, with different regulatory proteins acting in a cell-specific manner.DiscussionBased on these data we propose that localized induction of complement in cancer cells is a common feature of lung tumors that promotes tumor progression, with induction of complement regulatory proteins protecting cells from complement mediated-cell killing

Modeling the Combination of Amphotericin B, Micafungin, and Nikkomycin Z against Aspergillus fumigatus In Vitro Using a Novel Response Surface Paradigm

Response surface methods for the study of multiple-agent interaction allow one to model all of the information present in full concentration-effect data sets and to visualize and quantify local regions of synergy, additivity, and antagonism. In randomized wells of 96-well plates, Aspergillus fumigatus was exposed to various combinations of amphotericin B, micafungin, and nikkomycin Z. The experimental design was comprised of 91 different fixed-ratio mixtures, all performed in quintuplicate. After 24 h of drug exposure, drug effect on fungal viability was assessed using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) assay. First, we modeled each fixed-ratio combination alone using the four-parameter Hill concentration-effect model. Then, we modeled each parameter, including the 50% inhibitory concentration (IC(50)) effect, versus the proportion of each agent using constrained polynomials. Finally, we modeled the three-agent response surface overall. The overall four-dimensional response surface was complex, but it can be explained in detail both analytically and graphically. The grand model that fit the best included complex polynomial equations for the slope parameter m and the combination index (equivalent to the IC(50) for a fixed-ratio concentration, but with concentrations normalized by the respective IC(50)s of the drugs alone). There was a large region of synergy, mostly at the nikkomycin Z/micafungin edge of the ternary plots for equal normalized proportions of each drug and extending into the center of the plots. Applying this response surface method to a huge data set for a three-antifungal-agent combination is novel. This new paradigm has the potential to significantly advance the field of combination antifungal pharmacology

NADPH oxidase limits innate immune responses in the lungs in mice.

BackgroundChronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.Methodology/principal findingsWe found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47(phox-/-) mice and gp91(phox)-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-kappaB activation, and elevated downstream pro-inflammatory cytokines (TNF-alpha, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-kappaB activation.Conclusions/significanceThese studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD

Effectiveness of COVID-19 Vaccines in Preventing Hospitalization Among Adults Aged ≥65 Years - COVID-NET, 13 States, February-April 2021.

Clinical trials of COVID-19 vaccines currently authorized for emergency use in the United States (Pfizer-BioNTech, Moderna, and Janssen [Johnson & Johnson]) indicate that these vaccines have high efficacy against symptomatic disease, including moderate to severe illness (1-3). In addition to clinical trials, real-world assessments of COVID-19 vaccine effectiveness are critical in guiding vaccine policy and building vaccine confidence, particularly among populations at higher risk for more severe illness from COVID-19, including older adults. To determine the real-world effectiveness of the three currently authorized COVID-19 vaccines among persons aged ≥65 years during February 1-April 30, 2021, data on 7,280 patients from the COVID-19-Associated Hospitalization Surveillance Network (COVID-NET) were analyzed with vaccination coverage data from state immunization information systems (IISs) for the COVID-NET catchment area (approximately 4.8 million persons). Among adults aged 65-74 years, effectiveness of full vaccination in preventing COVID-19-associated hospitalization was 96% (95% confidence interval [CI] = 94%-98%) for Pfizer-BioNTech, 96% (95% CI = 95%-98%) for Moderna, and 84% (95% CI = 64%-93%) for Janssen vaccine products. Effectiveness of full vaccination in preventing COVID-19-associated hospitalization among adults aged ≥75 years was 91% (95% CI = 87%-94%) for Pfizer-BioNTech, 96% (95% CI = 93%-98%) for Moderna, and 85% (95% CI = 72%-92%) for Janssen vaccine products. COVID-19 vaccines currently authorized in the United States are highly effective in preventing COVID-19-associated hospitalizations in older adults. In light of real-world data demonstrating high effectiveness of COVID-19 vaccines among older adults, efforts to increase vaccination coverage in this age group are critical to reducing the risk for COVID-19-related hospitalization

Census tract socioeconomic indicators and COVID-19-associated hospitalization rates-COVID-NET surveillance areas in 14 states, March 1-April 30, 2020.

ObjectivesSome studies suggested more COVID-19-associated hospitalizations among racial and ethnic minorities. To inform public health practice, the COVID-19-associated Hospitalization Surveillance Network (COVID-NET) quantified associations between race/ethnicity, census tract socioeconomic indicators, and COVID-19-associated hospitalization rates.MethodsUsing data from COVID-NET population-based surveillance reported during March 1-April 30, 2020 along with socioeconomic and denominator data from the US Census Bureau, we calculated COVID-19-associated hospitalization rates by racial/ethnic and census tract-level socioeconomic strata.ResultsAmong 16,000 COVID-19-associated hospitalizations, 34.8% occurred among non-Hispanic White (White) persons, 36.3% among non-Hispanic Black (Black) persons, and 18.2% among Hispanic or Latino (Hispanic) persons. Age-adjusted COVID-19-associated hospitalization rate were 151.6 (95% Confidence Interval (CI): 147.1-156.1) in census tracts with >15.2%-83.2% of persons living below the federal poverty level (high-poverty census tracts) and 75.5 (95% CI: 72.9-78.1) in census tracts with 0%-4.9% of persons living below the federal poverty level (low-poverty census tracts). Among White, Black, and Hispanic persons living in high-poverty census tracts, age-adjusted hospitalization rates were 120.3 (95% CI: 112.3-128.2), 252.2 (95% CI: 241.4-263.0), and 341.1 (95% CI: 317.3-365.0), respectively, compared with 58.2 (95% CI: 55.4-61.1), 304.0 (95%: 282.4-325.6), and 540.3 (95% CI: 477.0-603.6), respectively, in low-poverty census tracts.ConclusionsOverall, COVID-19-associated hospitalization rates were highest in high-poverty census tracts, but rates among Black and Hispanic persons were high regardless of poverty level. Public health practitioners must ensure mitigation measures and vaccination campaigns address needs of racial/ethnic minority groups and people living in high-poverty census tracts

Intratracheal zymosan caused increased lung inflammation and NF-κB activation, in gp91<i>^phox-</i>deficient (X-linked CGD) versus wildtype mice.

<p>A) Representative lung section of gp91<i>^phox-</i>deficient mouse 6 days after i.t. zymosan showing extensive inflammation (H&E, 100x). Similarly treated wildtype (WT) mice had no lung inflammation (not shown). B) On day 6 after i.t. zymosan, both NADPH oxidase deficient genotypes (p47<i>^phox−/−</i> and gp91<i>^phox−/</i>) had similar BALF neutrophilic leukocytosis, whereas monocytes predominated in WT BALF. C) gp91<i>^phox-</i>deficient/HLL mice had increased whole lung NF-κB activation compared to WT/HLL mice.</p

Intratracheal zymosan treatment results in higher levels of pro-inflammatory cytokines and NF-κB activation in lungs of p47<i>^phox−/−</i> mice.

<p>A) BALF levels of TNF-α, IL-17, and G-CSF in wild type (WT) and p47<i>^phox−/−</i> (CGD) mice administered i.t. zymosan. Note that the Y-axes in the TNF-α and IL-17 graphs are in log-scale. The interaction of genotype (p47<i>^phox−/−</i> vs. WT) and time was assessed by 2-way ANOVA and was significant for each of the 3 cytokines (p<0.001). Bonferroni post-test was used to test for significance at each time point (*, p<0.05). B) Whole lung NF-κB activation measured by bioluminescence imaging over the chest after i.v. luciferin in NF-κB reporter mice (p47<i>^phox−/−</i>/HLL and WT/HLL). C) NF-κB dependent luciferase activity in bone marrow-derived macrophages (BMDMs) from p47<i>^phox−/−</i>/HLL and WT/HLL mice after <i>in vitro</i> stimulation with zymosan (20 µg/ml). For (B) and (C), 2-way ANOVA indicated p<0.0001 between genotypes with significant differences at the indicated time points by Bonferroni post-test. *, p<0.05; **, p<0.01; ***, p<0.001).</p