Mycobacterium abscessus-Induced Granuloma Formation Is Strictly Dependent on TNF Signaling and Neutrophil Trafficking

Mycobacterium abscessus is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with M. abscessus are increasingly found in patients with chronic lung diseases, especially cystic fibrosis, and are often refractory to antibiotic therapy. M. abscessus has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring infected transparent zebrafish embryos. Loss of TNFR1 function resulted in increased mortality with both variants, and was associated with unrestricted intramacrophage bacterial growth and decreased bactericidal activity. The use of transgenic zebrafish lines harboring fluorescent macrophages and neutrophils revealed that neutrophils, like macrophages, interact with M. abscessus at the initial infection sites. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization, and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted bacterial growth and subsequent larval death. Our findings emphasize the central role of neutrophils for the establishment and maintenance of the protective M. abscessus granulomas. These results also suggest that the TNF/IL8 inflammatory axis is necessary for protective immunity against M. abscessus and may be of clinical relevance to explain why immunosuppressive TNF therapy leads to the exacerbation of M. abscessus infections

Capturing sequence diversity in metagenomes with comprehensive and scalable probe design.

Metagenomic sequencing has the potential to transform microbial detection and characterization, but new tools are needed to improve its sensitivity. Here we present CATCH, a computational method to enhance nucleic acid capture for enrichment of diverse microbial taxa. CATCH designs optimal probe sets, with a specified number of oligonucleotides, that achieve full coverage of, and scale well with, known sequence diversity. We focus on applying CATCH to capture viral genomes in complex metagenomic samples. We design, synthesize, and validate multiple probe sets, including one that targets the whole genomes of the 356 viral species known to infect humans. Capture with these probe sets enriches unique viral content on average 18-fold, allowing us to assemble genomes that could not be recovered without enrichment, and accurately preserves within-sample diversity. We also use these probe sets to recover genomes from the 2018 Lassa fever outbreak in Nigeria and to improve detection of uncharacterized viral infections in human and mosquito samples. The results demonstrate that CATCH enables more sensitive and cost-effective metagenomic sequencing

Early type I Interferon response induces upregulation of human β-defensin 1 during acute HIV-1 infection

HIV-1 is able to evade innate antiviral responses during acute infection to establish a chronic systemic infection which, in the absence of antiretroviral therapy (ART), typically progresses to severe immunodeficiency. Understanding these early innate immune responses against HIV-1 and their mechanisms of failure is relevant to the development of interventions to better prevent HIV-1 transmission. Human beta defensins (HBDs) are antibacterial peptides but have recently also been associated with control of viral replication. HBD1 and 2 are expressed in PBMCs as well as intestinal tissue, but their expression in vivo during HIV-1 infection has not been characterized. We demonstrate that during acute HIV-1 infection, HBD1 but not HBD2 is highly upregulated in circulating monocytes but returns to baseline levels during chronic infection. HBD1 expression in monocytes can be induced by HIV-1 in vitro, although direct infection may not entirely account for the increase in HBD1 during acute infection. We provide evidence that HIV-1 triggers antiviral IFN-α responses, which act as a potent inducer of HBD1. Our results show the first characterization of induction of an HBD during acute and chronic viral infection in humans. HBD1 has been reported to have low activity against HIV-1 compared to other defensins, suggesting that in vivo induced defensins may not significantly contribute to the robust early antiviral response against HIV-1. These data provide important insight into the in vivo kinetics of HBD expression, the mechanism of HBD1 induction by HIV-1, and the role of HBDs in the early innate response to HIV-1 during acute infection

HIV-1 induces HBD1 <i>in vitro</i> but is unlikely to be sufficient <i>in vivo</i>.

<p>(A-C) CD14+ monocytes isolated using Miltenyi MACS technology were incubated with either HIV-1 R5, poly I:C (TLR3 ligand), 5’ppp-dsRNA (RIG-I ligand), R837 (TLR7 ligand), LPS (TLR4 ligand) or FLA-ST (TLR5 ligand), or left untreated. After 24 h cells were harvested and RNA extracted. Relative expression of HBD1 (A, C) or HBD2 (B) was assessed using quantitative PCR. HBD1 expression was found to be significantly increased in monocytes when incubated with HIV-1 R5, poly I:C or 5’pppdsRNA (A), whereas LPS and FLA-ST induced HBD2 (B). (C) Significant upregulation of HBD1 by HIV-1 was only achieved at 1x10^6 infection HIV-1 particles (i.p.) (Kruskal-Wallis and Dunn’s multiple post comparison test). Data points are presented with median and interquartile range which each dot representing an independent experiment from a different healthy control subject. (D) HBD1 transcription in PBMCs of subjects with acute HIV-1 infection (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.g001" target="_blank">Fig 1A</a>) was plotted against the corresponding viral load which each dot representing one subject. No significant correlation was found between viral load and HBD1 transcription (Spearman r correlation). (E, F) CD4+ T cells or CD14+ monocytes were isolated from PBMCs and incubated with either β-lactamase packaged HIV-1 X4 (CXCR4 tropic)/ HIV-1 R5 (CCR5 tropic) or left untreated. Where indicated cells were pre-treated with the CCR5 antagonist maraviroc (Mav) for 30min before HIV-1 treatment. All virus contained Vpr-BlaM which allowed assessment of HIV-1 entry into cells 12h after exposure. The highest entry was observed in CD4+ T cells by HIV-1 X4, whereas HIV-1 R5 entered CD4+ T cells as well CD14+ monocytes at low frequency. The graph shows the median and interquartile range with each dot representing one independent experiment from a different healthy control subject (F).</p

HBD1 is expressed by circulating monocytes and gut epithelial cells.

<p>(A, B) Cells were sorted from 5 different bulk colon excess tissue (n = 5) or PBMCs (n = 5), RNA extracted and HBD1 transcription quantified by qPCR. For colon, epithelial cells were identified using the surface marker CD326 and CD45 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). For PBMCs, monocytes were identified as CD14+, DCs as CD11c+ (mDCs) or CD123+ (pDCs) and lymphocytes as CD3+CD19+ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). Epithelial cells were the main producers of HBD1 in colon whereas CD14+ cells were the main producers in PBMCs. (C, D) The frequency of HBD1 producing cells is not altered during acute infection. Frequency of monocytes (C) and DC (D) populations in PMBCs from HIV-1- (n = 9) or acutely (Acute; n = 8) infected individuals was determined by flow cytometry. Conventional monocytes were identified as CD14+ CD16-, inflammatory monocytes as CD14+ CD16+ and mDCs as well as pDCs as described above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173161#pone.0173161.s002" target="_blank">S2 Fig</a>). No significant difference in cell frequencies was found between HIV-1- and sample from subjects with acute HIV-1 infection for any of the analyzed cell populations (Kruskal-Wallis and Dunn’s multiple post comparison test). (A-D) Data points are presented with median and interquartile range.</p

HBD1 is upregulated in PBMCs during acute HIV-1 infection.

<p>Samples from HIV-1 uninfected (HIV-), HIV-1 untreated chronic progressors (PG), HIV-1 chronic ART treated (CT) and acutely HIV-1 infected (Acute) individuals were analyzed for HBD1 expression. HBD1 transcription in PBMCs (A, B) or gut pinch biopsies (C, D) was determined by qPCR. Grey color indicates subjects with matched gut biopsy samples. (A) HBD1 expression in blood of acutely infected subjects (n = 32) was found to be significantly higher compared to HIV-1 individuals from PG (n = 8), CT (n = 9) or HIV- (n = 17) subjects (Kruskal-Wallis and Dunn’s multiple comparison test with error bars indicate min and max of boxplots). (B) Longitudinal sampling of individuals (n = 10) showed significant downregulation of HBD1 in chronic HIV-1 infection compared to acute infection (Wilcoxon matched-pairs signed rank test). (C, D) No significant differences in HBD1 expression in ileum (C) or colon (D) was found between HIV- (n = 5), PG (n = 4), CT (n = 9) and HIV acute infected subjects (n = 4), (Kruskal-Wallis and Dunn’s multiple comparison test and median with interquartile range).</p