Human Alveolar and Monocyte-Derived Human Macrophage Responses to Mycobacterium tuberculosis.

Alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) mediate early lung immune responses to Mycobacterium tuberculosis. Differences in the response of these distinct cell types are poorly understood and may provide insight into mechanisms of tuberculosis pathogenesis. The objective of this study was to determine whether M. tuberculosis induces unique and essential antimicrobial pathways in human AMs compared with MDMs. Using paired human AMs and 5-d MCSF-derived MDMs from six healthy volunteers, we infected cells with M. tuberculosis H37Rv for 6 h, isolated RNA, and analyzed transcriptomic profiles with RNA sequencing. We found 681 genes that were M. tuberculosis dependent in AMs compared with MDMs and 4538 that were M. tuberculosis dependent in MDMs, but not AMs (false discovery rate [FDR] < 0.05). Using hypergeometric enrichment of DEGs in Broad Hallmark gene sets, we found that type I and II IFN Response were the only gene sets selectively induced in M. tuberculosis-infected AM (FDR < 0.05). In contrast, MYC targets, unfolded protein response and MTORC1 signaling, were selectively enriched in MDMs (FDR < 0.05). IFNA1, IFNA8, IFNE, and IFNL1 were specifically and highly upregulated in AMs compared with MDMs at baseline and/or after M. tuberculosis infection. IFNA8 modulated M. tuberculosis-induced proinflammatory cytokines and, compared with other IFNs, stimulated unique transcriptomes. Several DNA sensors and IFN regulatory factors had higher expression at baseline and/or after M. tuberculosis infection in AMs compared with MDMs. These findings demonstrate that M. tuberculosis infection induced unique transcriptional responses in human AMs compared with MDMs, including upregulation of the IFN response pathway and specific DNA sensors.

A pediatric randomized, controlled trial of German cockroach subcutaneous immunotherapy.

BACKGROUND: Cockroach allergy contributes to morbidity among urban children with asthma. Few trials address the effect of subcutaneous immunotherapy (SCIT) with cockroach allergen among these at-risk children. OBJECTIVES: We sought to determine whether nasal allergen challenge (NAC) responses to cockroach allergen would improve following 1 year of SCIT. METHODS: Urban children with asthma, who were cockroach-sensitized and reactive on NAC, participated in a year-long randomized double-blind placebo-controlled SCIT trial using German cockroach extract. The primary endpoint was the change in mean Total Nasal Symptom Score (TNSS) during NAC after 12 months of SCIT. Changes in nasal transcriptomic responses during NAC, skin prick test wheal size, serum allergen-specific antibody production, and T-cell responses to cockroach allergen were assessed. RESULTS: Changes in mean NAC TNSS did not differ between SCIT-assigned (n = 28) versus placebo-assigned (n = 29) participants (P = .63). Nasal transcriptomic responses correlated with TNSS, but a treatment effect was not observed. Cockroach serum-specific IgE decreased to a similar extent in both groups, while decreased cockroach skin prick test wheal size was greater among SCIT participants (P = .04). A 200-fold increase in cockroach serum-specific IgG4 was observed among subjects receiving SCIT (P < .001) but was unchanged in the placebo group. T-cell IL-4 responses following cockroach allergen stimulation decreased to a greater extent among SCIT versus placebo (P = .002), while no effect was observed for IL-10 or IFN-γ. CONCLUSIONS: A year of SCIT failed to alter NAC TNSS and nasal transcriptome responses to cockroach allergen challenge despite systemic effects on allergen-specific skin tests, induction of serum-specific IgG4 serum production and down-modulation of allergen-stimulated T-cell responses.

Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.

Mycobacterium tuberculosis -induced monocyte transcriptional responses associated with resistance to tuberculin skin test/interferon-γ release assay conversion in people with HIV.

OBJECTIVE: To determine whether Mycobacterium tuberculosis (Mtb)-induced monocyte transcriptional responses differ in people with HIV (PWH) who do (RSTR) or do not (LTBI) resist tuberculin skin test/interferon-γ (IFN-γ) release assay (TST/IGRA) conversion after exposure. DESIGN: We compared ex-vivo Mtb-induced monocyte transcriptional responses in a Ugandan tuberculosis (TB) household contact study of RSTR and LTBI individuals among PWH. METHODS: Monocytes were isolated from peripheral blood mononuclear cells from 19 household contacts of pulmonary TB patients, and their transcriptional profiles were measured with RNA-Seq after a 6 h infection with Mtb (H37Rv) or media. Differentially expressed genes (DEGs) were identified by a linear mixed effects model and pathways by gene set enrichment analysis that compared RSTR and LTBI phenotypes with and without Mtb stimulation. RESULTS: Among PWH, we identified 8341 DEGs that were dependent on Mtb stimulation [false discovery rate (FDR) <0.01]. Of these, 350 were not significant (FDR >0.2) in individuals without HIV. Additionally, we found 26 genes that were differentially expressed between RSTR and LTBI monocytes in PWH, including 20 which were Mtb-dependent (FDR <0.2). In unstimulated monocytes, several gene sets [TGF-ß signaling, TNF-α signaling via NF-κB, NOTCH signaling, coagulation, and epithelial mesenchymal transition (EMT)] were enriched in RSTR relative to LTBI monocytes (FDR <0.1). These patterns were not observed in individuals without HIV. CONCLUSION: RSTR monocytes in PWH show different gene expressions in response to Mtb infection when compared with those with LTBI and RSTR without HIV. These differential expression patterns are enriched in inflammatory pathways.

Kimma: flexible linear mixed effects modeling with kinship covariance for RNA-seq data.

MOTIVATION: The identification of differentially expressed genes (DEGs) from transcriptomic datasets is a major avenue of research across diverse disciplines. However, current bioinformatic tools do not support covariance matrices in DEG modeling. Here, we introduce kimma (Kinship In Mixed Model Analysis), an open-source R package for flexible linear mixed effects modeling including covariates, weights, random effects, covariance matrices, and fit metrics. RESULTS: In simulated datasets, kimma detects DEGs with similar specificity, sensitivity, and computational time as limma unpaired and dream paired models. Unlike other software, kimma supports covariance matrices as well as fit metrics like Akaike information criterion (AIC). Utilizing genetic kinship covariance, kimma revealed that kinship impacts model fit and DEG detection in a related cohort. Thus, kimma equals or outcompetes current DEG pipelines in sensitivity, computational time, and model complexity. AVAILABILITY AND IMPLEMENTATION: Kimma is freely available on GitHub https://github.com/BIGslu/kimma with an instructional vignette at https://bigslu.github.io/kimma_vignette/kimma_vignette.html.

Type 2 inflammation reduces SARS-CoV-2 replication in the airway epithelium in allergic asthma through functional alteration of ciliated epithelial cells.

BACKGROUND: Despite well-known susceptibilities to other respiratory viral infections, individuals with allergic asthma have shown reduced susceptibility to severe coronavirus disease 2019 (COVID-19). OBJECTIVE: We sought to identify mechanisms whereby type 2 inflammation in the airway protects against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by using bronchial airway epithelial cells (AECs) from aeroallergen-sensitized children with asthma and healthy nonsensitized children. METHODS: We measured SARS-CoV-2 replication and ACE2 protein and performed bulk and single-cell RNA sequencing of ex vivo infected AEC samples with SARS-CoV-2 infection and with or without IL-13 treatment. RESULTS: We observed that viral replication was lower in AECs from children with allergic asthma than those from in healthy nonsensitized children and that IL-13 treatment reduced viral replication only in children with allergic asthma and not in healthy children. Lower viral transcript levels were associated with a downregulation of functional pathways of the ciliated epithelium related to differentiation as well as cilia and axoneme production and function, rather than lower ACE2 expression or increases in goblet cells or mucus secretion pathways. Moreover, single-cell RNA sequencing identified specific subsets of relatively undifferentiated ciliated epithelium (which are common in allergic asthma and highly responsive to IL-13) that directly accounted for impaired viral replication. CONCLUSION: Our results identify a novel mechanism of innate protection against SARS-CoV-2 in allergic asthma that provides important molecular and clinical insights during the ongoing COVID-19 pandemic.

T-cell deficiency and hyperinflammatory monocyte responses associate with Mycobacterium avium complex lung disease.

Immunological mechanisms of susceptibility to nontuberculous mycobacterial (NTM) disease are poorly understood. To understand NTM pathogenesis, we evaluated innate and antigen-specific adaptive immune responses to Mycobacterium avium complex (MAC) in asymptomatic individuals with a previous history of MAC lung disease (MACDZ). We hypothesized that Mav-specific immune responses are associated with susceptibility to MAC lung disease. We measured MAC-, NTM-, or MAC/Mtb-specific T-cell responses by cytokine production, expression of surface markers, and analysis of global gene expression in 27 MACDZ individuals and 32 healthy controls. We also analyzed global gene expression in Mycobacterium avium-infected and uninfected peripheral blood monocytes from 17 MACDZ and 17 healthy controls. We were unable to detect increased T-cell responses against MAC-specific reagents in MACDZ compared to controls, while the responses to non-mycobacteria derived antigens were preserved. MACDZ individuals had a lower frequency of Th1 and Th1* T-cell populations. In addition, MACDZ subjects had lower transcriptional responses in PBMCs stimulated with a mycobacterial peptide pool (MTB300). By contrast, global gene expression analysis demonstrated upregulation of proinflammatory pathways in uninfected and M. avium-infected monocytes, i.e. a hyperinflammatory in vitro response, derived from MACDZ subjects compared to controls. Together, these data suggest a novel immunologic defect which underlies MAC pathogenesis and includes concurrent innate and adaptive dysregulation which persists years after completion of treatment.

Gut Metabolites Are More Predictive of Disease and Cohoused States than Gut Bacterial Features in a Polycystic Ovary Syndrome-Like Mouse Model.

Polycystic ovary syndrome (PCOS) impacts ∼10% of reproductive-aged women worldwide. In addition to infertility, women with PCOS suffer from metabolic dysregulation which increases their risk of developing type 2 diabetes, cardiovascular disease, and nonalcoholic fatty liver disease. Studies have shown differences in the gut microbiome of women with PCOS compared to controls, a pattern replicated in PCOS-like mouse models. Recently, using a letrozole (LET)-induced mouse model of PCOS, we demonstrated that cohousing was protective against development of metabolic and reproductive phenotypes and showed via 16S amplicon sequencing that this protection correlated with time-dependent shifts in gut bacteria. Here, we applied untargeted metabolomics and shotgun metagenomics approaches to further analyze the longitudinal samples from the cohousing experiment. Analysis of beta diversity found that untargeted metabolites had the strongest correlation to both disease and cohoused states and that shifts in metabolite diversity were detected prior to shifts in bacterial diversity. In addition, log2 fold analyses found numerous metabolite features, particularly bile acids (BAs), to be highly differentiated between placebo and LET, as well as LET cohoused with placebo versus LET. Our results indicate that changes in gut metabolites, particularly BAs, are associated with a PCOS-like phenotype as well as with the protective effect of cohousing. Our results also suggest that transfer of metabolites via coprophagy occurs rapidly and may precipitate changes in bacterial diversity. This study joins a growing body of research linking changes in primary and secondary BAs to host metabolism and gut microbes relevant to the pathology of PCOS. IMPORTANCE Using a combination of untargeted metabolomics and metagenomics, we performed a comparative longitudinal analysis of the feces collected in a cohousing study with a PCOS-like mouse model. Our results showed that gut metabolite composition experienced earlier and more pronounced differentiation in both the disease model and cohoused mice compared with the microbial composition. Notably, statistical and machine learning approaches identified shifts in the relative abundance of primary and secondary BAs, which have been implicated as modifiers of gut microbial growth and diversity. Network correlation analysis showed strong associations between particular BAs and bacterial species, particularly members of Lactobacillus, and that these correlations were time and treatment dependent. Our results provide novel insights into host-microbe relationships related to hyperandrogenism in females and indicate that focused research into small-molecule control of gut microbial diversity and host physiology may provide new therapeutic options for the treatment of PCOS.