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Summary paragraphLung diseases due to infection and dysbiosis affect hundreds of millions of people world-wide1-4. Microbial communities at the airway mucosal barrier are conserved and highly ordered5, reflecting symbiosis and co-evolution with human host factors6. Freed of selection to digest nutrients for the host, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the results of the first systematic culture and whole-genome sequencing of the principal airway bacterial species, identifying abundant novel organisms within the genera Streptococcus, Pauljensenia, Neisseria and Gemella. Bacterial genomes were enriched for genes encoding antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. RNA-targeting CRISPR elements in some taxa suggest the potential to prevent or treat specific viral infections. Homologues of human RO60 present in Neisseria spp. provide a possible respiratory primer for autoimmunity in systemic lupus erythematosus (SLE) and Sjögren syndrome. We interpret the structure and biogeography of airway microbial communities from clinical surveys in the context of whole-genome content, identifying features of airway dysbiosis that may presage breakdown of homeostasis during acute attacks of asthma and chronic obstructive pulmonary disease (COPD). We match the gene content of isolates to human transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways that can sustain host interactions with the microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosal immunity in lung diseases of global significance.

Background: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A ( PDE3A ); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. Methods: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. Results: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. Conclusions: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.

Chronic kidney disease (CKD) is characterized by a sustained pro-inflammatory response. The underlying mechanisms are incompletely understood, but may be linked to gut dysbiosis. Dysbiosis has been described in adults with CKD; however, comorbidities limit CKD-specific conclusions. We analyzed the fecal microbiome, metabolites and immune phenotypes in children at three different CKD stages (G3-G4, G5 (hemodialysis), after kidney transplantation) and healthy controls. Serum TNF-α and sCD14 were stage-dependently elevated, indicating inflammation and gut barrier dysfunction. We observed microbiome alterations in CKD, including a diminished production of short-chain fatty acids. Bacterial tryptophan metabolites were increased in CKD. CKD serum activated the aryl hydrocarbon receptor and stimulated TNF-α production by monocytes, corresponding to a shift towards intermediate/non-classical monocytes. Unsupervised T cell analysis revealed pro-inflammatory shifts in MAIT and Treg cells. Thus, gut barrier dysfunction and microbial metabolites exacerbate inflammation and may therefore contribute to the increased cardiovascular burden in CKD.

AIMS: Hypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN. METHODS AND RESULTS: Four-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44 mg/kg/d) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory fecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice. CONCLUSIONS: Microbial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN. TRANSLATION PERSPECTIVE: To assess the potential of microbiota-targeted interventions to prevent organ damage in hypertension, an accurate quantification of microbial influence is necessary. We provide evidence that the development of hypertensive organ damage is dependent on colonization status and suggest that a healthy microbiota provides anti-hypertensive immune and metabolic signals to the host. In the absence of normal symbiotic host-microbiome interactions, hypertensive damage to the kidney in particular is exacerbated. We suggest that hypertensive patients experiencing perturbations to the microbiota, which are common in CVD, may be at a greater risk for target-organ damage than those with a healthy microbiome.

AimsHypertension (HTN) can lead to heart and kidney damage. The gut microbiota has been linked to HTN, although it is difficult to estimate its significance due to the variety of other features known to influence HTN. In the present study, we used germ-free (GF) and colonized (COL) littermate mice to quantify the impact of microbial colonization on organ damage in HTN.Methods and resultsFour-week-old male GF C57BL/6J littermates were randomized to remain GF or receive microbial colonization. HTN was induced by subcutaneous infusion with angiotensin (Ang) II (1.44mg/kg/d) and 1% NaCl in the drinking water; sham-treated mice served as control. Renal damage was exacerbated in GF mice, whereas cardiac damage was more comparable between COL and GF, suggesting that the kidney is more sensitive to microbial influence. Multivariate analysis revealed a larger effect of HTN in GF mice. Serum metabolomics demonstrated that the colonization status influences circulating metabolites relevant to HTN. Importantly, GF mice were deficient in anti-inflammatory fecal short-chain fatty acids (SCFA). Flow cytometry showed that the microbiome has an impact on the induction of anti-hypertensive myeloid-derived suppressor cells and pro-inflammatory Th17 cells in HTN. In vitro inducibility of Th17 cells was significantly higher for cells isolated from GF than conventionally raised mice.ConclusionsMicrobial colonization status of mice had potent effects on their phenotypic response to a hypertensive stimulus, and the kidney is a highly microbiota-susceptible target organ in HTN. The magnitude of the pathogenic response in GF mice underscores the role of the microbiome in mediating inflammation in HTN.Translation PerspectiveTo assess the potential of microbiota-targeted interventions to prevent organ damage in hypertension, an accurate quantification of microbial influence is necessary. We provide evidence that the development of hypertensive organ damage is dependent on colonization status and suggest that a healthy microbiota provides anti-hypertensive immune and metabolic signals to the host. In the absence of normal symbiotic host-microbiome interactions, hypertensive damage to the kidney in particular is exacerbated. We suggest that hypertensive patients experiencing perturbations to the microbiota, which are common in CVD, may be at a greater risk for target-organ damage than those with a healthy microbiome.

Metabolites produced by the microbiome such as short chain fatty acids (SCFAs) and tryptophan metabolites have been shown to impact the pathogenesis of hypertension. The microenvironment of the host gastrointestinal (GI) tract acts as the site-of-action for many host-microbiome interactions, although this space is not readily accessible. Fecal or serum samples are commonly used as a proxy, relying on the assumption that the obtained information should in some respect represent what is seen at the host epithelial interface. We surmised that it would be feasible to overcome the limitations of such indirect analysis by isolating interstitial fluid (IF) from the gut mucosa. We have established two methodologies to isolate IF from small segments of along GI tract, a centrifugation-based and elution-based approach in rats and mice. For rats and mice, 51 Cr-EDTA tracer experiments were used to demonstrate that fluid obtained was from an extracellular origin and can be reliably considered IF. Using GC-MS, we could identify several SCFAs (acetate, butyrate, propionate, valerate) within eluted IF samples. We were able to empirically measure the enrichment of these metabolites in eluted IF from the colon of rats compared to the duodenum (for propionate; mean difference=59.57 μM, p-value < 0.0001) or the serum (for propionate; mean difference= 60.25 μM, p-value < 0.0001). LC-MS based shotgun proteomics revealed that proteins annotated to the extracellular phase were site-specifically identifiable in IF and were differentially expressed when compared to matched serum samples. Furthermore, we could demonstrate that the use of tissue for IF isolation does not impede the use of other methods such as immunophenotyping or histology. Absolute CD45+ cells from the lamina propria measured using flow cytometry were not influenced by IF methods (p-value = 0.8344) but were unsurprisingly influenced by the gut segment which was analyzed (p-value = 0.0010). The ability to collect IF and directly measure metabolites at the site-of-action overcomes the limitations of indirect analysis of fecal samples or serum from the systemic circulation, and thus offers direct insight into this hitherto underexplored compartment.

Introduction: The gut microbiota is suspected to play a role in hypertension and hypertensive end organ damage. In the present study, we used germ-free mice to demonstrate that microbial colonization modulates the response to a hypertensive stimulus. Methods: Four-week-old male germ-free C57BL6/J littermates were randomized to remain germ-free (GF) or to receive microbiota transfer from SPF donor mice to achieve full colonization status (COL). At 12 weeks, Angiotensin (Ang) II was infused s.c. for 14 days (1.44mg/kg/d, osmotic minipumps) and 1% NaCl added to the drinking water; sham-treated mice served as control. After 14 days of AngII we assessed inflammation and organ damage. Results: Fecal bacterial load in COL mice was similar to SPF donor mice (qPCR). Shotgun metagenomic sequencing of fecal samples revealed hypertension-induced alterations in microbiome composition confirming previous reports. Serum metabolome analysis ( Biocrates MxP Quant 500) confirmed the absence of microbiota-dependent metabolites in GF. Interestingly, microbiota-dependent metabolites relevant for cardiovascular risk (TMAO, indoxyl sulfate) were elevated in hypertensive COL mice compared to sham-treated. Hypertensive kidney damage was aggravated in GF mice. However, marker genes for tubular damage ( Lcn2 ), inflammation ( Ccl2 ), and fibrosis ( Col1a3 ) showed a stronger increase in GF mice (fold changes [fc] COL vs. GF: 7.5 vs 11.0, 1.2 vs 3.3, 1.3 vs 2.2, respectively). Albuminuria (fc 2 vs 25) and histology for kidney fibrosis (fc 1.1 vs 1.4) confirmed the aggravated kidney damage in GF mice. Similarly, we observed an aggravated cardiac damage in GF mice. Flow cytometry of splenic lymphocytes showed that the adaptive immune response to AngII + 1% NaCl, as evidenced by Th17 (fc 1.4 vs 2) and CD8+ central memory cells, was intensified in GF mice. In vitro , naïve T cells isolated from GF mice more readily polarized into Th17 (26 ± 5%) compared to T cells from SPF mice (19 ± 1%). Conclusion: The bacterial colonization status has potent effects on the phenotypic response to a hypertensive stimulus, evident to varying degrees in hearts and kidneys. The inflammatory response and the end organ damage in GF compared to COL mice demonstrates the importance of the gut microbiota in hypertension.

Aims Recent technical developments have allowed the study of the human microbiome to accelerate at an unprecedented pace. Methodological differences may have considerable impact on the results obtained. Thus, we investigated how different storage, isolation, and DNA extraction methods can influence the characterization of the intestinal microbiome, compared to the impact of true biological signals such as intraindividual variability, nutrition, health, and demographics. Methods and results An observative cohort study in 27 healthy subjects was performed. Participants were instructed to collect stool samples twice spaced by a week, using six different methods (naive and Zymo DNA/RNA Shield on dry ice, OMNIgene GUT, RNALater, 95% ethanol, Zymo DNA/RNA Shield at room temperature). DNA extraction from all samples was performed comparatively using QIAamp Power Fecal and ZymoBIOMICS DNA Kits. 16S rRNA sequencing of the gut microbiota as well as qPCRs were performed on the isolated DNA. Metrics included alpha diversity as well as multivariate and univariate comparisons of samples, controlling for covariate patterns computationally. Interindividual differences explained 7.4% of overall microbiome variability, whereas the choice of DNA extraction method explained a further 5.7%. At phylum level, the tested kits differed in their recovery of Gram-positive bacteria, which is reflected in a significantly skewed enterotype distribution. Conclusion DNA extraction methods had the highest impact on observed microbiome variability, and were comparable to interindividual differences, thus may spuriously mimic the microbiome signatures of various health and nutrition factors. Conversely, collection methods had a relatively small influence on microbiome composition. The present study provides necessary insight into the technical variables which can lead to divergent results from seemingly similar study designs. We anticipate that these results will contribute to future efforts towards standardization of microbiome quantification procedures in clinical research.