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

Replication in Cells of Hematopoietic Origin Is Necessary for Dengue Virus Dissemination

Dengue virus (DENV) is a mosquito-borne pathogen for which no vaccine or specific therapeutic is available. Although it is well established that dendritic cells and macrophages are primary sites of DENV replication, it remains unclear whether non-hematopoietic cellular compartments serve as virus reservoirs. Here, we exploited hematopoietic-specific microRNA-142 (miR-142) to control virus tropism by inserting tandem target sites into the virus to restrict replication exclusively in this cell population. In vivo use of this virus restricted infection of CD11b+, CD11c+, and CD45+ cells, resulting in a loss of virus spread, regardless of the route of administration. Furthermore, sequencing of the targeted virus population that persisted at low levels, demonstrated total excision of the inserted miR-142 target sites. The complete conversion of the virus population under these selective conditions suggests that these immune cells are the predominant sources of virus amplification. Taken together, this work highlights the importance of hematopoietic cells for DENV replication and showcases an invaluable tool for the study of virus pathogenesis

Noncanonical cytoplasmic processing of viral microRNAs

Cellular utilization of RNA interference (RNAi) as a mechanism to combat virus infection is thought to be restricted to plants and invertebrates. In vertebrates, antiviral defenses are largely dependent on interferons (IFNs), with the use of small RNAs restricted to microRNA (miRNA)–mediated targeting of host transcripts. Here we demonstrate that incorporation of a primary miRNA into a cytoplasmic virus results in the formation of a Dicer-dependent, DGCR8-independent, mature miRNA capable of conferring RNAi-like activity. Processing of the viral mirtron-like product (virtron) is indistinguishable from endogenous miRNA maturation and elicits post-transcriptional gene silencing, albeit at a reduced level. Furthermore, virtrons impose Dicer-dependent, microprocessor-independent, and IFN-independent interference on virus replication in a sequence-specific manner. Taken together, these results suggest the existence of a noncanonical, small-RNA-based activity capable of processing cytoplasmic hairpins and perhaps contributing to the cell's antiviral arsenal

DENV-2 does not block miRNA function.

<p>(A) Fluorescence microscopy of human fibroblasts cotransfected with a plasmid expressing GFP targeted by miR-142 (pEGFP-142t) and either a construct expressing miR-142 (p142) or an empty vector control (vector). Cells were mock treated or infected with DENV-2 at an MOI of 1 48 hrs prior to analysis. (B) Fluorescence-activated cell sorting of samples treated as in (A). (C) Quantitative RT-PCR on samples described in (A). Data depicted as NS5 over tubulin levels. (Statistical significance: ****, P<0.0001, ***, P<0.001).</p

<i>In vivo</i> knockdown of miR-142-targeted DENV-2.

<p>(A) Quantitative RT-PCR (qRT-PCR) on NS5 and tubulin from CD11b⁺ and CD11c⁺ cells from splenocytes of <i>Ifnar1^−/−/Il28r^−/−</i> mice inoculated with either ctrl or 142t strains via intravenous (IV) injection. (B) qRT-PCR for DENV capsid in CD45⁻ versus CD45⁺ splenocytes represented as a ratio for ctrl and 142t virus infections in <i>Ifnar1^−/−/Il28r^−/−</i> mice. (C) qRT-PCR on spleens from <i>Ifnar1^−/−/Il28r^−/−</i> mice infected intraveneously (IV), intraperitonealy (IP), and subcutaneously (SC) for 48 hrs. (D) Viral titers from spleens of <i>Ifnar1^−/−/Il28r^−/−</i> mice infected as in (A) for 24, 48, and 72 hpi. (Statistical significance: *, P<0.05, **, P<0.01) Each data point represents an individual animal.</p

Generation and characterization of miR-142-targeted DENV-2.

<p>(A) Cloning strategy for the insertion of miR-142 target sites into the 3′untranslated region (UTR) of a T7-driven DENV-2 cDNA clone. A 157 nucleotide (nt) insert containing four tandem target sites were cloned into the variable region of DENV-2 (142t virus). A control (ctrl) virus containing four reverse sites is also depicted. (B) Northern blot for miR-142 expression in <i>Aedes albopictus</i> mosquito (C6/36) cells, mammalian baby hamster kidney cells (BHKs), murine bone-marrow-derived macrophages (BMMs), and a human B cell line (Raji). (C) Quantitative RT-PCR and western blot analysis on C6/36 cells infected with wt, ctrl, and 142t viruses at the indicated time points. Wild type (wt) virus refers to a clone encompassing no modifications. (D) Same as described in (C) for BHK cells.</p

<i>In vitro</i> knockdown of miR-142-targeted DENV-2 during exogenous and endogenous miR-142 expression.

<p>(A) Northern blot confirming exogenous expression of miR-142 by plasmid transfection. BHKs and HEK293s were transfected with vector or p142 and analyzed 24 hrs post transfection (hpi). Bone marrow-derived macrophages (BMMs) were included as a positive control for miR-142 expression. (B) Western blot for NS5 and actin expression in BHKs transfected with vector (−) or p142 (+) and treated with wt, ctrl, or 142t <i>in vitro</i> transcription (IVT) products. (C) BHKs treated as in (B) were infected with wt, ctrl, or 142t viruses and analyzed as in (B). (D) Western blot for NS5 and actin expression in BHKs treated as in (B) with wt, ctrl, 142t, or 142tm IVT products. (E) Quantitative RT-PCR on DENV-2 transcript in BHKs treated as in (B) with ctrl, 142t, or 142tm IVT products. (F) Western blot for NS5 and actin expression in hematopoietic cells (Raji B cells and BMMs) and non-hematopoietic fibroblasts (HEK293s) infected with the indicated recombinant DENV-2 strains.</p

PKR Transduces MDA5-Dependent Signals for Type I IFN Induction.

Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVΔE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluΔNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2α, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVΔE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism