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.

CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity. Using quantitative imaging, scRNAseq, and flow cytometry, we demonstrate that Mtb infection in the absence of prior immunity elicits dysregulated neutrophil recruitment and necrotic granulomas. In contrast, prior immunity induces rapid recruitment and activation of T cells, local macrophage activation, and diminished late neutrophil responses. Depletion studies at distinct infection stages demonstrated that neutrophils are required for early necrosis initiation and necrosis propagation at chronic stages, whereas early CD4 T cell responses prevent neutrophil feedforward circuits and necrosis. Together, these studies reveal fundamental determinants of tuberculosis lesion structure and pathogenesis, which have important implications for new strategies to prevent or treat tuberculosis.

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.

Genetic Modification of Primary Human Myeloid Cells to Study Cell Migration, Activation, and Organelle Dynamics.

Myeloid dendritic cells (DCs) and macrophages are mononuclear phagocytes with key roles in the immune system. As antigen-presenting cells, they link innate detection of microbes with programming adaptive immune responses. Myeloid DCs and macrophages also play critical roles in development, promote tissue homeostasis, and direct repair in response to injury and inflammation. As cellular migration and organelle dynamics are intimately connected with these processes, it is necessary to develop tools to track myeloid cell behavior and function. Here, we build on previously established protocols to isolate primary human myeloid cells from peripheral blood and report an optimized method for their genetic modification with lentiviral vectors to study processes related to cell migration, activation, and organelle dynamics. Specifically, we provide a protocol for delivering genetically encoded fluorescent markers into primary monocyte-derived DCs (MDDCs) and monocyte-derived macrophages (MDMs) to label mitochondria, peroxisomes, and whole cells. We describe the isolation of primary CD14+ monocytes from peripheral blood using positive selection with magnetic beads and, alternatively, isolation based on plastic adherence. Isolated CD14+ cells can be transduced with lentiviral vectors and subsequently cultured in the presence of cytokines to derive MDDCs or MDMs. This protocol is highly adaptable for cotransduction with vectors to knock down or overexpress genes of interest. These tools enable mechanistic studies of genetically modified myeloid cells through flow cytometry, fluorescence microscopy, and other downstream assays. © 2022 Wiley Periodicals LLC. Basic Protocol: Transduction of MDDCs and MDMs with lentiviral vectors encoding fluorescent markers Alternate Protocol 1: Isolation of monocytes by plastic adhesion Alternate Protocol 2: Transduction of MDDCs and MDMs with lentiviral vectors to knock down or overexpress genes of interest Support Protocol 1: Production and purification of lentiviral vectors for transduction into primary human myeloid cells Support Protocol 2: Flow cytometry of MDDCs and MDMs Support Protocol 3: Fixed and live-cell imaging of fluorescent markers in MDMs and MDDCs.

A genome-wide CRISPR-Cas9 screen identifies CENPJ as a host regulator of altered microtubule organization during Plasmodium liver infection.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources, likely delivered by vesicles. Here, we demonstrate that host microtubules (MTs) dynamically reorganize around the developing liver stage (LS) parasite to facilitate vesicular transport to the parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, and ER/Golgi stress that regulate LS development. Foci of γ-tubulin localized to the parasite periphery; depletion of centromere protein J (CENPJ), a novel regulator identified in the screen, exacerbated this re-localization and increased infection. We demonstrate that the Golgi acts as a non-centrosomal MT organizing center (ncMTOC) by positioning γ-tubulin and stimulating MT nucleation at parasite periphery. Together, these data support a model where the Plasmodium LS recruits host Golgi to form MT-mediated conduits along which host organelles are recruited to PVM and support parasite development.

Use of a Contained Mycobacterium tuberculosis Mouse Infection Model to Predict Active Disease and Containment in Humans.

Previous studies have identified whole-blood transcriptional risk and disease signatures for tuberculosis; however, several lines of evidence suggest that these signatures primarily reflect bacterial burden, which increases before symptomatic disease. We found that the peripheral blood transcriptome of mice with contained Mycobacterium tuberculosis infection (CMTI) has striking similarities to that of humans with active tuberculosis and that a signature derived from these mice predicts human disease with accuracy comparable to that of signatures derived directly from humans. A set of genes associated with immune defense are up-regulated in mice with CMTI but not in humans with active tuberculosis, suggesting that their up-regulation is associated with bacterial containment. A signature comprising these genes predicts both protection from tuberculosis disease and successful treatment at early time points where current signatures are not predictive. These results suggest that detailed study of the CMTI model may enable identification of biomarkers for human tuberculosis.

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.

ILF3 Is a Negative Transcriptional Regulator of Innate Immune Responses and Myeloid Dendritic Cell Maturation.

APCs such as myeloid dendritic cells (DCs) are key sentinels of the innate immune system. In response to pathogen recognition and innate immune stimulation, DCs transition from an immature to a mature state that is characterized by widespread changes in host gene expression, which include the upregulation of cytokines, chemokines, and costimulatory factors to protect against infection. Several transcription factors are known to drive these gene expression changes, but the mechanisms that negatively regulate DC maturation are less well understood. In this study, we identify the transcription factor IL enhancer binding factor 3 (ILF3) as a negative regulator of innate immune responses and DC maturation. Depletion of ILF3 in primary human monocyte-derived DCs led to increased expression of maturation markers and potentiated innate responses during stimulation with viral mimetics or classic innate agonists. Conversely, overexpression of short or long ILF3 isoforms (NF90 and NF110) suppressed DC maturation and innate immune responses. Through mutagenesis experiments, we found that a nuclear localization sequence in ILF3, and not its dual dsRNA-binding domains, was required for this function. Mutation of the domain associated with zinc finger motif of ILF3's NF110 isoform blocked its ability to suppress DC maturation. Moreover, RNA-sequencing analysis indicated that ILF3 regulates genes associated with cholesterol homeostasis in addition to genes associated with DC maturation. Together, our data establish ILF3 as a transcriptional regulator that restrains DC maturation and limits innate immune responses through a mechanism that may intersect with lipid metabolism.

Multi-Omics Resolves a Sharp Disease-State Shift between Mild and Moderate COVID-19.

We present an integrated analysis of the clinical measurements, immune cells, and plasma multi-omics of 139 COVID-19 patients representing all levels of disease severity, from serial blood draws collected during the first week of infection following diagnosis. We identify a major shift between mild and moderate disease, at which point elevated inflammatory signaling is accompanied by the loss of specific classes of metabolites and metabolic processes. Within this stressed plasma environment at moderate disease, multiple unusual immune cell phenotypes emerge and amplify with increasing disease severity. We condensed over 120,000 immune features into a single axis to capture how different immune cell classes coordinate in response to SARS-CoV-2. This immune-response axis independently aligns with the major plasma composition changes, with clinical metrics of blood clotting, and with the sharp transition between mild and moderate disease. This study suggests that moderate disease may provide the most effective setting for therapeutic intervention.

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.

Multiomic Immunophenotyping of COVID-19 Patients Reveals Early Infection Trajectories.

Host immune responses play central roles in controlling SARS-CoV2 infection, yet remain incompletely characterized and understood. Here, we present a comprehensive immune response map spanning 454 proteins and 847 metabolites in plasma integrated with single-cell multi-omic assays of PBMCs in which whole transcriptome, 192 surface proteins, and T and B cell receptor sequence were co-analyzed within the context of clinical measures from 50 COVID19 patient samples. Our study reveals novel cellular subpopulations, such as proliferative exhausted CD8 + and CD4 + T cells, and cytotoxic CD4 + T cells, that may be features of severe COVID-19 infection. We condensed over 1 million immune features into a single immune response axis that independently aligns with many clinical features and is also strongly associated with disease severity. Our study represents an important resource towards understanding the heterogeneous immune responses of COVID-19 patients and may provide key information for informing therapeutic development.

Crippling life support for SARS-CoV-2 and other viruses through synthetic lethality.

With the rapid global spread of SARS-CoV-2, we have become acutely aware of the inadequacies of our ability to respond to viral epidemics. Although disrupting the viral life cycle is critical for limiting viral spread and disease, it has proven challenging to develop targeted and selective therapeutics. Synthetic lethality offers a promising but largely unexploited strategy against infectious viral disease; as viruses infect cells, they abnormally alter the cell state, unwittingly exposing new vulnerabilities in the infected cell. Therefore, we propose that effective therapies can be developed to selectively target the virally reconfigured host cell networks that accompany altered cellular states to cripple the host cell that has been converted into a virus factory, thus disrupting the viral life cycle.

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.

PPARα exacerbates necroptosis, leading to increased mortality in postinfluenza bacterial superinfection.

Patients infected with influenza are at high risk of secondary bacterial infection, which is a major proximate cause of morbidity and mortality. We have shown that in mice, prior infection with influenza results in increased inflammation and mortality upon Staphylococcus aureus infection, recapitulating the human disease. Lipidomic profiling of the lungs of superinfected mice revealed an increase in CYP450 metabolites during lethal superinfection. These lipids are endogenous ligands for the nuclear receptor PPARα, and we demonstrate that Ppara-/- mice are less susceptible to superinfection than wild-type mice. PPARα is an inhibitor of NFκB activation, and transcriptional profiling of cells isolated by bronchoalveolar lavage confirmed that influenza infection inhibits NFκB, thereby dampening proinflammatory and prosurvival signals. Furthermore, network analysis indicated an increase in necrotic cell death in the lungs of superinfected mice compared to mice infected with S. aureus alone. Consistent with this, we observed reduced NFκB-mediated inflammation and cell survival signaling in cells isolated from the lungs of superinfected mice. The kinase RIPK3 is required to induce necrotic cell death and is strongly induced in cells isolated from the lungs of superinfected mice compared to mice infected with S. aureus alone. Genetic and pharmacological perturbations demonstrated that PPARα mediates RIPK3-dependent necroptosis and that this pathway plays a central role in mortality following superinfection. Thus, we have identified a molecular circuit in which infection with influenza induces CYP450 metabolites that activate PPARα, leading to increased necrotic cell death in the lung which correlates with the excess mortality observed in superinfection.

The Ratiometric Transcript Signature MX2/GPR183 Is Consistently Associated With RTS,S-Mediated Protection Against Controlled Human Malaria Infection.

The RTS,S/AS01 vaccine provides partial protection against Plasmodium falciparum infection but determinants of protection and/or disease are unclear. Previously, anti-circumsporozoite protein (CSP) antibody titers and blood RNA signatures were associated with RTS,S/AS01 efficacy against controlled human malaria infection (CHMI). By analyzing host blood transcriptomes from five RTS,S vaccination CHMI studies, we demonstrate that the transcript ratio MX2/GPR183, measured 1 day after third immunization, discriminates protected from non-protected individuals. This ratiometric signature provides information that is complementary to anti-CSP titer levels for identifying RTS,S/AS01 immunized people who developed protective immunity and suggests a role for interferon and oxysterol signaling in the RTS,S mode of action.

A Comprehensive Map of the Monocyte-Derived Dendritic Cell Transcriptional Network Engaged upon Innate Sensing of HIV.

Transcriptional programming of the innate immune response is pivotal for host protection. However, the transcriptional mechanisms that link pathogen sensing with innate activation remain poorly understood. During HIV-1 infection, human dendritic cells (DCs) can detect the virus through an innate sensing pathway, leading to antiviral interferon and DC maturation. Here, we develop an iterative experimental and computational approach to map the HIV-1 innate response circuitry in monocyte-derived DCs (MDDCs). By integrating genome-wide chromatin accessibility with expression kinetics, we infer a gene regulatory network that links 542 transcription factors with 21,862 target genes. We observe that an interferon response is required, yet insufficient, to drive MDDC maturation and identify PRDM1 and RARA as essential regulators of the interferon response and MDDC maturation, respectively. Our work provides a resource for interrogation of regulators of HIV replication and innate immunity, highlighting complexity and cooperativity in the regulatory circuit controlling the response to infection.

Liver stage malaria infection is controlled by host regulators of lipid peroxidation.

The facets of host control during Plasmodium liver infection remain largely unknown. We find that the SLC7a11-GPX4 pathway, which has been associated with the production of reactive oxygen species, lipid peroxidation, and a form of cell death called ferroptosis, plays a critical role in control of Plasmodium liver stage infection. Specifically, blocking GPX4 or SLC7a11 dramatically reduces Plasmodium liver stage parasite infection. In contrast, blocking negative regulators of this pathway, NOX1 and TFR1, leads to an increase in liver stage infection. We have shown previously that increased levels of P53 reduces Plasmodium LS burden in an apoptosis-independent manner. Here, we demonstrate that increased P53 is unable to control parasite burden during NOX1 or TFR1 knockdown, or in the presence of ROS scavenging or when lipid peroxidation is blocked. Additionally, SLC7a11 inhibitors Erastin and Sorafenib reduce infection. Thus, blocking the host SLC7a11-GPX4 pathway serves to selectively elevate lipid peroxides in infected cells, which localize within the parasite and lead to the elimination of liver stage parasites.

Denisovan, modern human and mouse TNFAIP3 alleles tune A20 phosphorylation and immunity.

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

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.

CD8+ T cells induce cachexia during chronic viral infection.

Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). In the present paper we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8+ T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8+ T cells caused morphologic and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8+ T cell response and required T cell-intrinsic type I interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8+ T cells in IAC.