The effect of mindfulness-based stress reduction on the urinary microbiome in interstitial cystitis.

INTRODUCTION AND HYPOTHESIS: The objective was to investigate the impact of mindfulness-based stress reduction therapy on the urinary microbiome of patients with interstitial cystitis/bladder pain syndrome. METHODS: In this Institutional Review Board-approved prospective cohort study, patients with interstitial cystitis/bladder pain syndrome were recruited to attend an 8-week mindfulness-based stress reduction course involving yoga and meditation. Eligible participants were English-speaking women aged 18 or older with interstitial cystitis/bladder pain syndrome. All participants had a negative urinalysis within 2 months of enrollment and were currently undergoing first- or second-line treatment at the time of recruitment. The mindfulness-based stress reduction course met weekly for 1 h. A straight-catheter urine sample was obtained prior to and following the mindfulness-based stress reduction series. DNA from urine samples underwent bacterial 16S ribosomal gene sequencing at Johns Hopkins University Laboratories followed by taxonomic abundance and diversity analysis by Resphera Biosciences Laboratory. Participants completed validated symptom questionnaires pre- and post-intervention. RESULTS: A total of 12 participants completed the 8-week course and were included in the analysis. The average age was 59 and the majority identified as white. Patient symptoms, measured by the Urogenital Distress Inventory Short Form and Interstitial Cystitis Symptom and Pain Indices, improved significantly (all p < 0.05). Overall composition of the urinary microbiome changed significantly (p < 0.01) and demonstrated an increase in diversity following the intervention. CONCLUSIONS: Mindfulness-based stress reduction therapy improves patient symptoms and was associated with significant changes in the urinary microbiome in patients with interstitial cystitis/bladder pain syndrome.

Transcriptomics Underlying Pulmonary Ozone Pathogenesis Regulated by Inflammatory Mediators in Mice.

Ozone (O3) is the predominant oxidant air pollutant associated with airway inflammation, lung dysfunction, and the worsening of preexisting respiratory diseases. We previously demonstrated the injurious roles of pulmonary immune receptors, tumor necrosis factor receptor (TNFR), and toll-like receptor 4, as well as a transcription factor NF-κB, in response to O3 in mice. In the current study, we profiled time-dependent and TNFR- and NF-κB-regulated lung transcriptome changes by subacute O3 to illuminate the underlying molecular events and downstream targets. Mice lacking Tnfr1/Tnfr2 (Tnfr-/-) or Nfkb1 (Nfkb1-/-) were exposed to air or O3. Lung RNAs were prepared for cDNA microarray analyses, and downstream and upstream mechanisms were predicted by pathway analyses of the enriched genes. O3 significantly altered the genes involved in inflammation and redox (24 h), cholesterol biosynthesis and vaso-occlusion (48 h), and cell cycle and DNA repair (48-72 h). Transforming growth factor-ß1 was a predicted upstream regulator. Lack of Tnfr suppressed the immune cell proliferation and lipid-related processes and heightened epithelial cell integrity, and Nfkb1 deficiency markedly suppressed lung cell cycle progress during O3 exposure. Common differentially regulated genes by TNFR and NF-κB1 (e.g., Casp8, Il6, and Edn1) were predicted to protect the lungs from cell death, connective tissue injury, and inflammation. Il6-deficient mice were susceptible to O3-induced protein hyperpermeability, indicating its defensive role, while Tnf-deficient mice were resistant to overall lung injury caused by O3. The results elucidated transcriptome dynamics and provided new insights into the molecular mechanisms regulated by TNFR and NF-κB1 in pulmonary subacute O3 pathogenesis.

Sex Differences in Lung Imaging and SARS-CoV-2 Antibody Responses in a COVID-19 Golden Syrian Hamster Model.

In the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males than in females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8 to 10 weeks of age) were inoculated intranasally with 105 50% tissue culture infective dose (TCID50) of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developed more extensive pneumonia as noted on chest computed tomography, and recovered more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including interferon-ß (IFN-ß) and tumor necrosis factor-α (TNF-α), were comparable between the sexes. However, during the recovery phase of infection, females mounted 2-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole-inactivated SARS-CoV-2 and mutant S-RBDs as well as virus-neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2-associated sex differences seen in the human population. IMPORTANCE Men experience more severe outcomes from coronavirus disease 2019 (COVID-19) than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human isolate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in female than in male hamsters. Greater lung pathology during the acute phase combined with lower antiviral antibody responses during the recovery phase of infection in males than in females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection.

Identification of microbial agents in tissue specimens of ocular and periocular sarcoidosis using a metagenomics approach.

Background: Metagenomic sequencing has the potential to identify a wide range of pathogens in human tissue samples. Sarcoidosis is a complex disorder whose etiology remains unknown and for which a variety of infectious causes have been hypothesized. We sought to conduct metagenomic sequencing on cases of ocular and periocular sarcoidosis, none of them with previously identified infectious causes. Methods: Archival tissue specimens of 16 subjects with biopsies of ocular and periocular tissues that were positive for non-caseating granulomas were used as cases. Four archival tissue specimens that did not demonstrate non-caseating granulomas were also included as controls. Genomic DNA was extracted from tissue sections. DNA libraries were generated from the extracted genomic DNA and the libraries underwent next-generation sequencing. Results: We generated between 4.8 and 20.7 million reads for each of the 16 cases plus four control samples. For eight of the cases, we identified microbial pathogens that were present well above the background, with one potential pathogen identified for seven of the cases and two possible pathogens for one of the cases. Five of the eight cases were associated with bacteria ( Campylobacter concisus, Neisseria elongata, Streptococcus salivarius, Pseudopropionibacterium propionicum, and Paracoccus yeei), two cases with fungi ( Exophiala oligosperma, Lomentospora prolificans and Aspergillus versicolor) and one case with a virus (Mupapillomavirus 1). Interestingly, four of the five bacterial species are also part of the human oral microbiome. Conclusions: Using a metagenomic sequencing we identified possible infectious causes in half of the ocular and periocular sarcoidosis cases analyzed. Our findings support the proposition that sarcoidosis could be an etiologically heterogenous disease. Because these are previously banked samples, direct follow-up in the respective patients is impossible, but these results suggest that sequencing may be a valuable tool in better understanding the etiopathogenesis of sarcoidosis and in diagnosing and treating this disease.

Conservation of Intracellular Pathogenic Strategy among Distantly Related Cryptococcal Species.

The genus Cryptococcus includes several species pathogenic for humans. Until recently, the two major pathogenic species were recognized to be Cryptococcus neoformans and Cryptococcus gattii We compared the interaction of murine macrophages with three C. gattii species complex strains (WM179, R265, and WM161, representing molecular types VGI, VGIIa, and VGIII, respectively) and one C. neoformans species complex strain (H99, molecular type VNI) to ascertain similarities and differences in the yeast intracellular pathogenic strategy. The parameters analyzed included nonlytic exocytosis frequency, phagolysosomal pH, intracellular capsular growth, phagolysosomal membrane permeabilization, and macrophage transcriptional response, assessed using time-lapse microscopy, fluorescence microscopy, flow cytometry, and gene expression microarray analysis. The most striking result was that the intracellular pathogenic strategies of C. neoformans and C. gattii species complex strains were qualitatively similar, despite the species having separated an estimated 100 million years ago. Macrophages exhibited a leaky phagolysosomal membrane phenotype and nonlytic exocytosis when infected with either C. gattii or C. neoformans Conservation of the intracellular strategy among species that separated long ago suggests that it is ancient and possibly maintained by similar selection pressures through eons.

The Membrane Phospholipid Binding Protein Annexin A2 Promotes Phagocytosis and Nonlytic Exocytosis of Cryptococcus neoformans and Impacts Survival in Fungal Infection.

Cryptococcus neoformans is a fungal pathogen with a unique intracellular pathogenic strategy that includes nonlytic exocytosis, a phenomenon whereby fungal cells are expunged from macrophages without lysing the host cell. The exact mechanism and specific proteins involved in this process have yet to be completely defined. Using murine macrophages deficient in the membrane phospholipid binding protein, annexin A2 (ANXA2), we observed a significant decrease in both phagocytosis of yeast cells and the frequency of nonlytic exocytosis. Cryptococcal cells isolated from Anxa2-deficient (Anxa2(-/-)) bone marrow-derived macrophages and lung parenchyma displayed significantly larger capsules than those isolated from wild-type macrophages and tissues. Concomitantly, we observed significant differences in the amount of reactive oxygen species produced between Anxa2(-/-) and Anxa2(+/+) macrophages. Despite comparable fungal burden, Anxa2(-/-) mice died more rapidly than wild-type mice when infected with C. neoformans, and Anxa2(-/-) mice exhibited enhanced inflammatory responses, suggesting that the reduced survival reflected greater immune-mediated damage. Together, these findings suggest a role for ANXA2 in the control of cryptococcal infection, macrophage function, and fungal morphology.

Seoul virus suppresses NF-kappaB-mediated inflammatory responses of antigen presenting cells from Norway rats.

Hantavirus infection reduces antiviral defenses, increases regulatory responses, and causes persistent infection in rodent hosts. To address whether hantaviruses alter the maturation and functional activity of antigen presenting cells (APCs), rat bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were generated and infected with Seoul virus (SEOV) or stimulated with TLR ligands. SEOV infected both DCs and macrophages, but copies of viral RNA, viral antigen, and infectious virus titers were higher in macrophages. The expression of MHCII and CD80, production of IL-6, IL-10, and TNF-alpha, and expression of Ifnbeta were attenuated in SEOV-infected APCs. Stimulation of APCs with poly I:C prior to SEOV infection increased the expression of activation markers and production of inflammatory cytokines and suppressed SEOV replication. Infection of APCs with SEOV suppressed LPS-induced activation and innate immune responses. Hantaviruses reduce the innate immune response potential of APCs derived from a natural host, which may influence persistence of these zoonotic viruses in the environment.

Common and divergent immune response signaling pathways discovered in peripheral blood mononuclear cell gene expression patterns in presymptomatic and clinically apparent malaria.

Using genome-wide expression profiles from persons either experimentally challenged with malaria-infected mosquitoes or naturally infected with Plasmodium falciparum malaria, we present details of the transcriptional changes that occur with infection and that either are commonly shared between subjects with presymptomatic and clinically apparent malaria or distinguish these two groups. Toll-like receptor signaling through NF-kappaB pathways was significantly upregulated in both groups, as were downstream genes that function in phagocytosis and inflammation, including the cytokines tumor necrosis factor alpha, gamma interferon (IFN-gamma), and interleukin-1beta (IL-1beta). The molecular program derived from these signatures illuminates the closely orchestrated interactions that regulate gene expression by transcription factors such as IRF-1 in the IFN-gamma signal transduction pathway. Modulation of transcripts in heat shock and glycolytic enzyme genes paralleled the intensity of infection. Major histocompatibility complex class I molecules and genes involved in class II antigen presentation are significantly induced in 90% of malaria-infected persons regardless of group. Differences between early presymptomatic infection and natural infection involved genes that regulate the induction of apoptosis through mitogen-activated protein (MAP) kinases and signaling pathways through the endogenous pyrogen IL-1beta, a major inducer of fever. The induction of apoptosis in peripheral blood mononuclear cells from patients with naturally acquired infection impacted the mitochondrial control of apoptosis and the activation of MAP kinase pathways centered around MAPK14 (p38alpha and p38beta). Our findings confirm and extend findings regarding aspects of the earliest responses to malaria infection at the molecular level, which may be informative in elucidating how innate and adaptive immune responses may be modulated in different stages of infection.

Mycobacterium-induced potentiation of type 1 immune responses and protection against malaria are host specific.

Malaria and tuberculosis are endemic in many regions of the world, and coinfection with the two pathogens is common. In this study, we examined the effects of long- and short-term infection with Mycobacterium tuberculosis on the course of a lethal form of murine malaria in resistant (C57BL/6) and susceptible (BALB/c) mice. C57BL/6 mice coinfected with M. tuberculosis CDC1551 and Plasmodium yoelii 17XL had a lower peak parasitemia and increased survival compared to mice infected with P. yoelii 17XL alone. Splenic microarray analysis demonstrated potentiation of type 1 immune responses in coinfected C57BL/6 mice, which was especially prominent 5 days after infection with P. yoelii 17XL. Splenocytes from coinfected C57BL/6 mice produced higher levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha than splenocytes from mice infected with either pathogen alone. Interestingly, mycobacterium-induced protection against lethal P. yoelii is mouse strain specific. BALB/c mice were significantly more susceptible than C57BL/6 mice to infection with P. yoelii 17XL and were not protected against lethal malaria by coinfection with M. tuberculosis. In addition, M. tuberculosis did not augment IFN-gamma responses in BALB/c mice subsequently infected with P. yoelii 17XL. These data indicate that M. tuberculosis-induced potentiation of type 1 immune responses is associated with protection against lethal murine malaria.

Role of Toll-like receptor-4 in genetic susceptibility to lung injury induced by residual oil fly ash.

The mechanisms of susceptibility to particle-induced lung injury are not clearly understood. To evaluate the contribution of genetic background to pulmonary pathogenesis, we compared the lung injury responses to residual oil fly ash (ROFA) in inbred mouse strains and calculated heritability estimates. Significant interstrain (genetic) variation was observed in ROFA-induced lung inflammation and hyperpermeability phenotypes; broad-sense heritability ranged from approximately 0.43 to 0.62, and the coefficient of genetic determination ranged from 0.28 to 0.45. C3H/HeJ (HeJ) mice were most resistant to the ROFA-induced injury responses. This was particularly important, as HeJ mice contain a dominant negative mutation in Toll-like receptor-4 (Tlr4). We then characterized ROFA-induced injury and TLR4 signaling in HeJ mice and its coisogenic strain C3H/HeOuJ (OuJ; Tlr4 normal) to understand the potential role of Tlr4 in this model. ROFA-induced lung injury was significantly greater in OuJ mice compared with HeJ mice. ROFA also significantly enhanced transcript and protein levels of lung TLR4 in OuJ but not in HeJ mice. Greater activation of downstream signal molecules (i.e., MYD88, TRAF6, IRAK-1, NF-kappaB, MAPK, AP-1) was observed in OuJ mice than in HeJ mice before the development of ROFA-induced pulmonary injury. Putative TLR4-dependent inflammatory genes that were differentially induced by ROFA in the two strains include interleukin-1beta and tumor necrosis factor-alpha. Results support an important contribution of genetic background to particle-mediated lung injury, and Tlr4 is a candidate susceptibility gene.

Role of NRF2 in protection against hyperoxic lung injury in mice.

NRF2 is a transcription factor important in the protection against carcinogenesis and oxidative stress through antioxidant response element (ARE)-mediated transcriptional activation of several phase 2 detoxifying and antioxidant enzymes. This study was designed to determine the role of NRF2 in the pathogenesis of hyperoxic lung injury by comparing pulmonary responses to 95-98% oxygen between mice with site-directed mutation of the gene for NRF2 (Nrf2-/-) and wild-type mice (Nrf2+/+). Pulmonary hyperpermeability, macrophage inflammation, and epithelial injury in Nrf2-/- mice were 7.6-fold, 47%, and 43% greater, respectively, compared with Nrf2+/+ mice after 72 h hyperoxia exposure. Hyperoxia markedly elevated the expression of NRF2 mRNA and DNA-binding activity of NRF2 in the lungs of Nrf2+/+ mice. mRNA expression for ARE- responsive lung antioxidant and phase 2 enzymes was evaluated in both genotypes of mice to identify potential downstream molecular mechanisms of NRF2 in hyperoxic lung responses. Hyperoxia-induced mRNA levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST)-Ya and -Yc subunits, UDP glycosyl transferase (UGT), glutathione peroxidase-2 (GPx2), and heme oxygenase-1 (HO-1) were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice. Consistent with differential mRNA expression, NQO1 and total GST activities were significantly lower in Nrf2-/- mice compared with Nrf2+/+ mice after hyperoxia. Results demonstrated that NRF2 has a significant protective role against pulmonary hyperoxic injury in mice, possibly through transcriptional activation of lung antioxidant defense enzymes.

Linkage analysis of susceptibility to hyperoxia. Nrf2 is a candidate gene.

A strong role for reactive oxygen species (ROS) has been proposed in the pathogenesis of a number of lung diseases. Hyperoxia (> 95% oxygen) generates ROS and extensive lung damage, and has been used as a model of oxidant injury. However, the precise mechanisms of hyperoxia-induced toxicity have not been completely clarified. This study was designed to identify hyperoxia susceptibility genes in C57BL/6J (susceptible) and C3H/HeJ (resistant) mice. The quantitative phenotypes used for this analysis were pulmonary inflammatory cell influx, epithelial cell sloughing, and hyperpermeability. Genome-wide linkage analyses of intercross (F2) and recombinant inbred cohorts identified significant and suggestive quantitative trait loci on chromosomes 2 (hyperoxia susceptibility locus 1 [Hsl1]) and 3 (Hsl2), respectively. Comparative mapping of Hsl1 identified a strong candidate gene, Nfe2l2 (nuclear factor, erythroid derived 2, like 2 or Nrf2) that encodes a transcription factor NRF2 which regulates antioxidant and phase 2 gene expression. Strain-specific variation in lung Nrf2 messenger RNA expression and a T --> C substitution in the B6 Nrf2 promoter that cosegregated with susceptibility phenotypes in F2 animals supported Nrf2 as a candidate gene. Results from this study have important implications for understanding the mechanisms through which oxidants mediate the pathogenesis of lung disease.