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Cigarette smokers have an increased risk of infectious diseases involving the respiratory tract. Some effects of smoking on specific respiratory tract bacteria have been described, but the consequences for global airway microbial community composition have not been determined. Here, we used culture-independent high-density sequencing to analyze the microbiota from the right and left nasopharynx and oropharynx of 29 smoking and 33 nonsmoking healthy asymptomatic adults to assess microbial composition and effects of cigarette smoking. Bacterial communities were profiled using 454 pyrosequencing of 16S sequence tags (803,391 total reads), aligned to 16S rRNA databases, and communities compared using the UniFrac distance metric. A Random Forest machine-learning algorithm was used to predict smoking status and identify taxa that best distinguished between smokers and nonsmokers. Community composition was primarily determined by airway site, with individuals exhibiting minimal side-of-body or temporal variation. Within airway habitats, microbiota from smokers were significantly more diverse than nonsmokers and clustered separately. The distributions of several genera were systematically altered by smoking in both the oro- and nasopharynx, and there was an enrichment of anaerobic lineages associated with periodontal disease in the oropharynx. These results indicate that distinct regions of the human upper respiratory tract contain characteristic microbial communities that exhibit disordered patterns in cigarette smokers, both in individual components and global structure, which may contribute to the prevalence of respiratory tract complications in this population.

Defining the biogeography of bacterial populations in human body habitats is a high priority for understanding microbial-host relationships in health and disease. The healthy lung was traditionally considered sterile, but this notion has been challenged by emerging molecular approaches that enable comprehensive examination of microbial communities. However, studies of the lung are challenging due to difficulties in working with low biomass samples. Our goal was to use molecular methods to define the bacterial microbiota present in the lungs of healthy individuals and assess its relationship to upper airway populations. We sampled respiratory flora intensively at multiple sites in six healthy individuals. The upper tract was sampled by oral wash and oro-/nasopharyngeal swabs. Two bronchoscopes were used to collect samples up to the glottis, followed by serial bronchoalveolar lavage and lower airway protected brush. Bacterial abundance and composition were analyzed by 16S rDNA Q-PCR and deep sequencing. Bacterial communities from the lung displayed composition indistinguishable from the upper airways, but were 2 to 4 logs lower in biomass. Lung-specific sequences were rare and not shared among individuals. There was no unique lung microbiome. In contrast to other organ systems, the respiratory tract harbors a homogenous microbiota that decreases in biomass from upper to lower tract. The healthy lung does not contain a consistent distinct microbiome, but instead contains low levels of bacterial sequences largely indistinguishable from upper respiratory flora. These findings establish baseline data for healthy subjects and sampling approaches for sequence-based analysis of diseases.

SARS-CoV-2 infection causes COVID-19 disease, which can result in consequences ranging from undetectable to fatal, focusing attention on the modulators of outcomes. The respiratory tract microbiome is thought to modulate the outcomes of infections such as influenza as well as acute lung injury, raising the question to what degree does the airway microbiome influence COVID-19? Here, we review the results of 56 studies examining COVID-19 and the respiratory tract microbiome, summarize the main generalizations, and point to useful avenues for further research. Although the results vary among studies, a few consistent findings stand out. The diversity of bacterial communities in the oropharynx typically declined with increasing disease severity. The relative abundance of Haemophilus and Neisseria also declined with severity. Multiple microbiome measures tracked with measures of systemic immune responses and COVID outcomes. For many of the conclusions drawn in these studies, the direction of causality is unknown—did an alteration in the microbiome result in increased COVID severity, did COVID severity alter the microbiome, or was some third factor the primary driver, such as medication use. Follow-up mechanistic studies can help answer these questions. -hPNxGBEUu4dZv1p37KLx1 Video Abstract

Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg −1 of mRNA into mice results in ∼170 μg ml −1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg −1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml −1 . Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. Monoclonal antibodies are highly effective therapeutics that can be delivered as proteins or encoded DNA or mRNA. Here the authors develop lipid nanoparticle-formulated nucleoside-modified mRNA encoding an HIV-1 neutralizing antibody and see sustained and protective antibody levels in treated mice.

Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.

Significance The innate immune system provides a first line of defense against invading pathogens. The inflammasome is an innate immune complex that activates inflammatory caspases upon infection, causing cell death and IL-1 cytokine release, which initiate defense against gram-negative bacterial pathogens but also mediate septic shock. Many inflammasome studies have been performed using cells from mice, but mice and humans differ in their complement of inflammatory caspases. Instead of caspase-11, humans encode the putative orthologs caspase-4 and caspase-5. Here, we show that caspase-4 plays a conserved role in inflammasome activation in response to virulent gram-negative pathogens in primary human macrophages. Our findings provide important insight into how inflammasomes are regulated in human cells. Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.

Background Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. Results Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. Conclusions LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

Long-term survival after lung transplantation is limited by infectious complications and by bronchiolitis obliterans syndrome (BOS), a form of chronic rejection linked in part to microbial triggers. To define microbial populations in the respiratory tract of transplant patients comprehensively using unbiased high-density sequencing. Lung was sampled by bronchoalveolar lavage (BAL) and upper respiratory tract by oropharyngeal wash (OW). Bacterial 16S rDNA and fungal internal transcribed spacer sequencing was used to profile organisms present. Outlier analysis plots defining taxa enriched in lung relative to OW were used to identify bacteria enriched in lung against a background of oropharyngeal carryover. Lung transplant recipients had higher bacterial burden in BAL than control subjects, frequent appearance of dominant organisms, greater distance between communities in BAL and OW indicating more distinct populations, and decreased respiratory tract microbial richness and diversity. Fungal populations were typically dominated by Candida in both sites or by Aspergillus in BAL but not OW. 16S outlier analysis identified lung-enriched taxa indicating bacteria replicating in the lower respiratory tract. In some cases this confirmed respiratory cultures but in others revealed enrichment by anaerobic organisms or mixed outgrowth of upper respiratory flora and provided quantitative data on relative abundances of bacteria found by culture. Respiratory tract microbial communities in lung transplant recipients differ in structure and composition from healthy subjects. Outlier analysis can identify specific bacteria replicating in lung. These findings provide novel approaches to address the relationship between microbial communities and transplant outcome and aid in assessing lung infections.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Here, we investigated the respiratory tract microbiome in coronavirus disease 2019 (COVID-19) and its relationship to disease severity, systemic immunologic features, and outcomes. We examined 507 oropharyngeal, nasopharyngeal, and endotracheal samples from 83 hospitalized COVID-19 patients as well as non-COVID patients and healthy controls. Bacterial communities were interrogated using 16S rRNA gene sequencing, and the commensal DNA viruses Anelloviridae and Redondoviridae were quantified by qPCR. We found that COVID-19 patients had upper respiratory microbiome dysbiosis and greater change over time than critically ill patients without COVID-19. Oropharyngeal microbiome diversity at the first time point correlated inversely with disease severity during hospitalization. Microbiome composition was also associated with systemic immune parameters in blood, as measured by lymphocyte/neutrophil ratios and immune profiling of peripheral blood mononuclear cells. Intubated patients showed patient-specific lung microbiome communities that were frequently highly dynamic, with prominence of Staphylococcus . Anelloviridae and Redondoviridae showed more frequent colonization and higher titers in severe disease. Machine learning analysis demonstrated that integrated features of the microbiome at early sampling points had high power to discriminate ultimate level of COVID-19 severity. Thus, the respiratory tract microbiome and commensal viruses are disturbed in COVID-19 and correlate with systemic immune parameters, and early microbiome features discriminate disease severity. Future studies should address clinical consequences of airway dysbiosis in COVID-19, its possible use as biomarkers, and the role of bacterial and viral taxa identified here in COVID-19 pathogenesis. IMPORTANCE COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized COVID-19 patients and found that each was markedly abnormal compared to that in healthy people and differed from that in critically ill patients without COVID-19. Early airway samples tracked with the level of COVID-19 illness reached during hospitalization, and the airway microbiome also correlated with immune parameters in blood. These findings raise questions about the mechanisms linking SARS-CoV-2 infection and other microbial inhabitants of the airway, including whether the microbiome might regulate severity of COVID-19 disease and/or whether early microbiome features might serve as biomarkers to discriminate disease severity.

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.