Oxysterol binding protein regulates the resolution of TLR-induced cytokine production in macrophages.

Toll-like receptors (TLRs) on macrophages sense microbial components and trigger the production of numerous cytokines and chemokines that mediate the inflammatory response to infection. Although many of the components required for the activation of the TLR pathway have been identified, the mechanisms that appropriately regulate the magnitude and duration of the response and ultimately restore homeostasis are less well understood. Furthermore, a growing body of work indicates that TLR signaling reciprocally interacts with other fundamental cellular processes, including lipid metabolism but only a few specific molecular links between immune signaling and the macrophage lipidome have been studied in detail. Oxysterol-binding protein (Osbp) is the founding member of a family of lipid-binding proteins with diverse functions in lipid sensing, lipid transport, and cell signaling but its role in TLR responses is not well defined. Here, we demonstrate that altering the state of Osbp with its natural ligand, 25-hydroxycholesterol (25HC), or pharmacologically, sustains and thereby amplifies Tlr4-induced cytokine production in vitro and in vivo. CRISPR-induced knockdown of Osbp abrogates the ability of these ligands to sustain TLR responses. Lipidomic analysis suggested that the effect of Osbp on TLR signaling may be mediated by alterations in triglyceride production and treating cells with a Dgat1 inhibitor, which blocks triglyceride production and completely abrogates the effect of Osbp on TLR signaling. Thus, Osbp is a sterol sensor that transduces perturbations of the lipidome to modulate the resolution of macrophage inflammatory responses.

Exposure to Mycobacterium remodels alveolar macrophages and the early innate response to Mycobacterium tuberculosis infection.

Alveolar macrophages (AMs) play a critical role during Mycobacterium tuberculosis (Mtb) infection as the first cells in the lung to encounter bacteria. We previously showed that AMs initially respond to Mtb in vivo by mounting a cell-protective, rather than pro-inflammatory response. However, the plasticity of the initial AM response was unknown. Here, we characterize how previous exposure to Mycobacterium, either through subcutaneous vaccination with Mycobacterium bovis (scBCG) or through a contained Mtb infection (coMtb) that mimics aspects of concomitant immunity, impacts the initial response by AMs. We find that both scBCG and coMtb accelerate early innate cell activation and recruitment and generate a stronger pro-inflammatory response to Mtb in vivo by AMs. Within the lung environment, AMs from scBCG vaccinated mice mount a robust interferon-associated response, while AMs from coMtb mice produce a broader inflammatory response that is not dominated by Interferon Stimulated Genes. Using scRNAseq, we identify changes to the frequency and phenotype of airway-resident macrophages following Mycobacterium exposure, with enrichment for both interferon-associated and pro-inflammatory populations of AMs. In contrast, minimal changes were found for airway-resident T cells and dendritic cells after exposures. Ex vivo stimulation of AMs with Pam3Cys, LPS and Mtb reveal that scBCG and coMtb exposures generate stronger interferon-associated responses to LPS and Mtb that are cell-intrinsic changes. However, AM profiles that were unique to each exposure modality following Mtb infection in vivo are dependent on the lung environment and do not emerge following ex vivo stimulation. Overall, our studies reveal significant and durable remodeling of AMs following exposure to Mycobacterium, with evidence for both AM-intrinsic changes and contributions from the altered lung microenvironments. Comparisons between the scBCG and coMtb models highlight the plasticity of AMs in the airway and opportunities to target their function through vaccination or host-directed therapies.

Neo-sex chromosome evolution shapes sex-dependent asymmetrical introgression barrier.

Sex chromosomes play a special role in the evolution of reproductive barriers between species. Here we describe conflicting roles of nascent sex chromosomes on patterns of introgression in an experimental hybrid swarm. Drosophila nasuta and Drosophila albomicans are recently diverged, fully fertile sister species that have different sex chromosome systems. The fusion between an autosome (Muller CD) with the ancestral X and Y gave rise to neo-sex chromosomes in D. albomicans, while Muller CD remains unfused in D. nasuta. We found that a large block containing overlapping inversions on the neo-sex chromosome stood out as the strongest barrier to introgression. Intriguingly, the neo-sex chromosome introgression barrier is asymmetrical and sex-dependent. Female hybrids showed significant D. albomicans­biased introgression on Muller CD (neo-X excess), while males showed heterosis with excessive (neo-X, D. nasuta Muller CD) genotypes. We used a population genetic model to dissect the interplay of sex chromosome drive, heterospecific pairing incompatibility between the neo-sex chromosomes and unfused Muller CD, neo-Y disadvantage, and neo-X advantage in generating the observed sex chromosome genotypes in females and males. We show that moderate neo-Y disadvantage and D. albomicans specific meiotic drive are required to observe female-specific D. albomicans­biased introgression in this system, together with pairing incompatibility and neo-X advantage. In conclusion, this hybrid swarm between a young species pair sheds light onto the multifaceted roles of neo-sex chromosomes in a sex-dependent asymmetrical introgression barrier at a species boundary.

Dynamics and Impacts of Transposable Element Proliferation in the Drosophila nasuta Species Group Radiation.

Transposable element (TE) mobilization is a constant threat to genome integrity. Eukaryotic organisms have evolved robust defensive mechanisms to suppress their activity, yet TEs can escape suppression and proliferate, creating strong selective pressure for host defense to adapt. This genomic conflict fuels a never-ending arms race that drives the rapid evolution of TEs and recurrent positive selection of genes involved in host defense; the latter has been shown to contribute to postzygotic hybrid incompatibility. However, how TE proliferation impacts genome and regulatory divergence remains poorly understood. Here, we report the highly complete and contiguous (N50 = 33.8-38.0 Mb) genome assemblies of seven closely related Drosophila species that belong to the nasuta species group-a poorly studied group of flies that radiated in the last 2 My. We constructed a high-quality de novo TE library and gathered germline RNA-seq data, which allowed us to comprehensively annotate and compare TE insertion patterns between the species, and infer the evolutionary forces controlling their spread. We find a strong negative association between TE insertion frequency and expression of genes nearby; this likely reflects survivor bias from reduced fitness impact of TEs inserting near lowly expressed, nonessential genes, with limited TE-induced epigenetic silencing. Phylogenetic analyses of insertions of 147 TE families reveal that 53% of them show recent amplification in at least one species. The most highly amplified TE is a nonautonomous DNA element (Drosophila INterspersed Element; DINE) which has gone through multiple bouts of expansions with thousands of full-length copies littered throughout each genome. Across all TEs, we find that TEs expansions are significantly associated with high expression in the expanded species consistent with suppression escape. Thus, whereas horizontal transfer followed by the invasion of a naïve genome has been highlighted to explain the long-term survival of TEs, our analysis suggests that evasion of host suppression of resident TEs is a major strategy to persist over evolutionary times. Altogether, our results shed light on the heterogenous and context-dependent nature in which TEs affect gene regulation and the dynamics of rampant TE proliferation amidst a recently radiated species group.

Contained Mycobacterium tuberculosis infection induces concomitant and heterologous protection.

Progress in tuberculosis vaccine development is hampered by an incomplete understanding of the immune mechanisms that protect against infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Although the M72/ASOE1 trial yielded encouraging results (54% efficacy in subjects with prior exposure to Mtb), a highly effective vaccine against adult tuberculosis remains elusive. We show that in a mouse model, establishment of a contained and persistent yet non-pathogenic infection with Mtb ("contained Mtb infection", CMTB) rapidly and durably reduces tuberculosis disease burden after re-exposure through aerosol challenge. Protection is associated with elevated activation of alveolar macrophages, the first cells that respond to inhaled Mtb, and accelerated recruitment of Mtb-specific T cells to the lung parenchyma. Systems approaches, as well as ex vivo functional assays and in vivo infection experiments, demonstrate that CMTB reconfigures tissue resident alveolar macrophages via low grade interferon-γ exposure. These studies demonstrate that under certain circumstances, the continuous interaction of the immune system with Mtb is beneficial to the host by maintaining elevated innate immune responses.

Patterns of Genomic Differentiation in the Drosophila nasuta Species Complex.

The Drosophila nasuta species complex contains over a dozen recently diverged species that are distributed widely across South-East Asia, and which shows varying degrees of pre- and postzygotic isolation. Here, we assemble a high-quality genome for D. albomicans using single-molecule sequencing and chromatin conformation capture, and draft genomes for 11 additional species and 67 individuals across the clade, to infer the species phylogeny and patterns of genetic diversity in this group. Our assembly recovers entire chromosomes, and we date the origin of this radiation ∼2 Ma. Despite low levels of overall differentiation, most species or subspecies show clear clustering into their designated taxonomic groups using population genetics and phylogenetic methods. Local evolutionary history is heterogeneous across the genome, and differs between the autosomes and the X chromosome for species in the sulfurigaster subgroup, likely due to autosomal introgression. Our study establishes the nasuta species complex as a promising model system to further characterize the evolution of pre- and postzygotic isolation in this clade.

Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation.

The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA Using Path-seq and regulatory network analyses, we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/desA2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection.

MiR-155-regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis.

The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.

Absence of c-Maf and IL-10 enables Type I IFN enhancement of innate responses to low-dose LPS in alveolar macrophages.

Alveolar macrophages (AMs) are lower-airway resident myeloid cells and are among the first to respond to inhaled pathogens. Here, we interrogate AM innate sensing to Pathogen Associated Molecular Patterns (PAMPs) and determine AMs have decreased responses to low-dose LPS compared to other macrophages, as measured by TNF, IL-6, Ifnb, and Ifit3. We find the reduced response to low-dose LPS correlates with minimal TLR4 and CD14 surface expression, despite sufficient internal expression of TLR4. Additionally, we find that AMs do not produce IL-10 in response to a variety of PAMPs due to low expression of transcription factor c-Maf and that lack of IL-10 production contributes to an enhancement of pro-inflammatory responses by Type I IFN. Our findings demonstrate that AMs have cell-intrinsic dampened responses to LPS, which is enhanced by type I IFN exposure. These data implicate conditions where AMs may have reduced or enhanced sentinel responses to bacterial infections.

Host and pathogen genetic diversity shape vaccine-mediated protection to Mycobacterium tuberculosis.

To investigate how host and pathogen diversity govern immunity against Mycobacterium tuberculosis (Mtb), we performed a large-scale screen of vaccine-mediated protection against aerosol Mtb infection using three inbred mouse strains [C57BL/6 (B6), C3HeB/FeJ (C3H), Balb/c x 129/SvJ (C129F1)] and three Mtb strains (H37Rv, CDC1551, SA161) representing two lineages and distinct virulence properties. We compared three protective modalities, all of which involve inoculation with live mycobacteria: Bacillus Calmette-Guérin (BCG), the only approved TB vaccine, delivered either subcutaneously or intravenously, and concomitant Mtb infection (CoMtb), a model of pre-existing immunity in which a low-level Mtb infection is established in the cervical lymph node following intradermal inoculation. We examined lung bacterial burdens at early (Day 28) and late (Day 98) time points after aerosol Mtb challenge and histopathology at Day 98. We observed substantial heterogeneity in the reduction of bacterial load afforded by these modalities at Day 28 across the combinations and noted a strong positive correlation between bacterial burden in unvaccinated mice and the degree of protection afforded by vaccination. Although we observed variation in the degree of reduction in bacterial burdens across the nine mouse/bacterium strain combinations, virtually all protective modalities performed similarly for a given strain-strain combination. We also noted dramatic variation in histopathology changes driven by both host and bacterial genetic backgrounds. Vaccination improved pathology scores for all infections except CDC1551. However, the most dramatic impact of vaccination on lesion development occurred for the C3H-SA161 combination, where vaccination entirely abrogated the development of the large necrotic lesions that arise in unvaccinated mice. In conclusion, we find that substantial TB heterogeneity can be recapitulated by introducing variability in both host and bacterial genetics, resulting in changes in vaccine-mediated protection as measured both by bacterial burden as well as histopathology. These differences can be harnessed in future studies to identify immune correlates of vaccine efficacy.

Type I interferon decreases macrophage energy metabolism during mycobacterial infection.

Metabolic reprogramming powers and polarizes macrophage functions, but the nature and regulation of this response during infection with pathogens remain controversial. In this study, we characterize the metabolic and transcriptional responses of murine macrophages to Mycobacterium tuberculosis (Mtb) in order to disentangle the underlying mechanisms. We find that type I interferon (IFN) signaling correlates with the decreased glycolysis and mitochondrial damage that is induced by live, but not killed, Mtb. Macrophages lacking the type I IFN receptor (IFNAR) maintain glycolytic flux and mitochondrial function during Mtb infection in vitro and in vivo. IFNß itself restrains the glycolytic shift of inflammatory macrophages and initiates mitochondrial stress. We confirm that type I IFN acts upstream of mitochondrial damage using macrophages lacking the protein STING. We suggest that a type I IFN-mitochondrial feedback loop controls macrophage responses to mycobacteria and that this could contribute to pathogenesis across a range of diseases.

Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice.

Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for understanding how innate immune cells respond to infection but is technically challenging due to the small number of bacteria that initially infect the lung. Previous studies either evaluated Mtb-infected cells at later stages of infection when the number of bacteria in the lung is much higher or used in vitro model systems to assess the response of myeloid cells to Mtb. Here, we describe a method that uses fluorescent bacteria, a high-dose aerosol infection model, and flow cytometry to track Mtb-infected cells in the lung immediately following aerosol infection and fluorescence-activated cell sorting (FACS) to isolate naïve, bystander, and Mtb-infected cells for downstream applications, including RNA-sequencing. This protocol provides the ability to monitor Mtb-infection and cell-specific responses within the context of the lung environment, which is known to modulate the function of both resident and recruited populations. Using this protocol, we discovered that alveolar macrophages respond to Mtb infection in vivo by up-regulating a cell protective transcriptional response that is regulated by the transcription factor Nrf2 and is detrimental to early control of the bacteria.

Alveolar macrophages generate a noncanonical NRF2-driven transcriptional response to Mycobacterium tuberculosis in vivo.

Alveolar macrophages (AMs) are the first cells to be infected during Mycobacterium tuberculosis (M.tb.) infection. Thus, the AM response to infection is the first of many steps leading to initiation of the adaptive immune response required for efficient control of infection. A hallmark of M.tb. infection is the slow initiation of the adaptive response, yet the mechanisms responsible for this are largely unknown. To study the initial AM response to infection, we developed a system to identify, sort, and analyze M.tb.-infected AMs from the lung within the first 10 days of infection. In contrast to what has been previously described using in vitro systems, M.tb.-infected AMs up-regulate a cell-protective antioxidant transcriptional signature that is dependent on the lung environment but not bacterial virulence. Computational approaches including pathway analysis and transcription factor motif enrichment analysis identify NRF2 as a master regulator of the response. Using knockout mouse models, we demonstrate that NRF2 drives expression of the cell-protective signature in AMs and impairs the control of early bacterial growth. AMs up-regulate a substantial pro-inflammatory response to M.tb. infection only 10 days after infection, yet comparisons with bystander AMs from the same infected animals demonstrate that M.tb.-infected AMs generate a less robust inflammatory response than the uninfected cells around them. Our findings demonstrate that the initial macrophage response to M.tb. in the lung is far less inflammatory than has previously been described by in vitro systems and may impede the overall host response to infection.