Biology subjects

Olive, A.

Publications and source records attributed to Olive, A..

6 recordsLinked to original sources

bioRxiv2026

Inflammasome Activation and IL-1β Release in Alveolar Macrophages Infected with Pseudomonas aeruginosa is Reduced in Hypoxia

Pseudomonas aeruginosa is an important agent of acute or chronic airway infections. A complex set of genetic and environmental factors determine the outcome of a P. aeruginosa airway infection. P. aeruginosa can undergo genetic adaptation to fine tune the expression of a type III secretion system (T3SS) and other virulence factors in response to selective pressures encountered during infection. Genetic and environmental factors predispose many patient groups for P. aeruginosa infections, including people with cystic fibrosis (pwCF). CF is a genetic disorder resulting in areas of hypoxia and thick mucus that fosters P. aeruginosa airway infection. Resident alveolar macrophages (AMs) help coordinate immune responses to P. aeruginosa in airways by producing proinflammatory cytokines such as IL-1{beta}, which can be important for infection resistance. AMs infected with laboratory strains of P. aeruginosa detect the T3SS and activate the NLRC4 inflammasome, resulting in IL-1{beta} release. Studies of inflammasome responses in AMs to a clinical CF isolate of P. aeruginosa have not been reported. Here, we characterized the CF clinical isolate DH1137 and found that it has a downregulated but functional T3SS, is adapted to grow in hypoxia, and induces significant production of the inflammasome cytokines IL-1{beta} and IL-1 during lung infection in a CF mouse model. Inflammasome gene expression was primed in AMs by LPS stimulation, and upon DH1137 infection these cells released IL-1[beta]. Intriguingly, we found that infection of AMs with DH1137 in hypoxia resulted in significantly dampened inflammasome activation compared to normoxia. These data describe a new mechanism that P. aeruginosa may exploit to evade immune detection by tissue-resident lung macrophages in the context of CF.

immunology↗

bioRxiv2026

Myeloid-derived alveolar-like macrophages are a tractable model to understand the role of ontogeny in alveolar macrophage function ex vivo and in the lungs.

Alveolar macrophages (AMs) are tissue-resident and the primary immune cells in the airspace. Following perturbations in the lungs, these AMs that are derived from the fetal liver, become depleted and are transiently replaced by myeloid cells that use lung-specific cues to differentiate into myeloid-derived AMs. While these myeloid-derived AMs are critically important in a range of pulmonary diseases, including post-influenza bacterial pneumonia, it remains challenging to fully understand their function due to a lack of ex vivo models that recapitulate key differences observed in vivo between AMs and myeloid-derived AMs. Here, we overcome this limitation by expanding our recently developed model of fetal liver-derived alveolar macrophages (FLAMs) to differentiate myeloid progenitors in the presence of GM-CSF and TGF{beta}, key cytokines that drive tissue resident AM functions. These myeloid-derived alveolar-like macrophages (MAMs) express AM surface markers and look similar morphologically to FLAMs, however, they remain more inflammatory than FLAMs. Mechanistic studies found that differential CpG methylation at inflammatory loci, basal transcriptional expression, and metabolic flux all contribute to the hyperinflammatory state of MAMs. Importantly, we find that while FLAMs are highly dependent of lipid metabolism, MAMs are more glycolytic and this hardwired metabolism is not easily overcome to mute their inflammatory state. Finally, we found that MAMs and FLAMs both function within the lung environment following transfer into mice lacking AMs. While both MAMs and FLAMs stably seed the lungs and reverse pulmonary proteinosis, MAMs remain highly inflammatory in the lungs following an LPS model of acute lung injury. Taken together our results find that MAMs are a reproducible model of myeloid-derived AMs and lays the groundwork to better understand how these important immune cells contribute to pulmonary homeostasis and responses to lung perturbations. These future studies will help to identify new targets that can be modulated to prevent severe pulmonary disease outcomes.

immunology↗

bioRxiv2026

A genome-wide CRISPR screen defines host determinants of early Brucella infection in human macrophage-like cells

Brucella spp. are widespread intracellular animal pathogens that cause brucellosis, a significant zoonosis. Despite the global impact of brucellosis on animal and human health, the host genes that support Brucella infection remain incompletely defined. To address this knowledge gap, we developed a flow cytometry-based infection assay with fluorescent Brucella and performed a genome-wide CRISPR-Cas9 loss-of-function screen in human macrophage-like cells. Disruption of >150 host genes significantly reduced intracellular B. abortus burden at 3 h post-infection. In addition to recovering known host factors, the screen revealed previously unappreciated genes linked to endosomal trafficking, cytoskeletal remodeling, and lipid homeostasis. The screen was robust, as validation within these functional categories confirmed that the small GTPase RAB14, the Src-family kinase regulator CSK, and the phospholipid flippase subunit TMEM30A support early infection by B. abortus and B. ovis without impairing general phagocytosis. Gene set enrichment analysis further revealed positive regulators of mTORC1 signaling as key host factors; this result was validated through targeted disruption of LAMTOR2 and AKT1, and pharmacologic inhibition of AKT1. Thus, the AKT-Ragulator-mTORC1 signaling axis contributes to the establishment of a permissive intracellular niche during early Brucella infection. Finally, to assess whether these host requirements extend beyond Brucella, we examined infection by the unrelated intracellular pathogen Mycobacterium abscessus. CSK, AKT1, and LAMTOR2 were required for efficient M. abscessus infection, whereas RAB14 was dispensable. Together, these results define host genes that support early Brucella infection and distinguish shared versus pathogen-specific host dependencies exploited by intracellular bacteria.

microbiology↗

bioRxiv2025

Ku limits aberrant mRNA splicing promoted by intronic antisense Alu elements

Alu elements are short repeats that occupy approximately 10% of the human genome 1,2. Saturation of primate genomes with Alu sequences occurred at the prosimian/new-world monkey evolutionary juncture. Alu elements have clearly driven unique aspects of higher primate evolution, but their presence can be detrimental to genomic stability 3. The expansion of Alu sequences in the genomes of higher primates precisely coincides with a substantial increase in the ubiquitous expression of the three polypeptides of the DNA-dependent protein kinase (DNA-PK), the Ku70/80 heterodimer and DNA-PKcs 4. Previous work suggests that the elevated levels of Ku70/80 are required to prevent the activation of innate immune signaling pathways triggered by RNA molecules derived from Alu elements 5. Here we demonstrate that Ku ablation dramatically alters mRNA splicing, by allowing the use of alternative splice sites contained in intronic antisense Alu elements, which are known to directly associate with Ku70/80 5. Dysregulation of mRNA splicing precedes cell death and preferentially impacts genes involved in essential RNA metabolism processes, including splicing and ribosome biogenesis, likely impacting cell viability. In addition, we demonstrate that cell death after Ku70 depletion cannot be rescued by expression of its prosimian homologue, which suggests that primate Ku70 has evolved specific molecular features to suppress deleterious effects of an Alu element rich genome. We propose a model in which Ku binding of antisense Alu elements in introns of nascent RNAs modulates the use of alternative splice sites to balance beneficial and detrimental contributions of Alu repeats within primate genomes.

molecular biology↗

bioRxiv2021

FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies

Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGF{beta}. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

immunology↗

bioRxiv2020

A genome-wide screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation

AbstractCytokine-mediated activation of host immunity is central to the control of pathogens. A key cytokine in protective immunity is interferon-gamma (IFN{gamma}), which is a potent activator of antimicrobial and immunomodulatory effectors within the host. A major role of IFN{gamma} is to induce major histocompatibility complex class II molecules (MHCII) on the surface of cells, which is required for CD4+ T cell activation. Despite its central role in host immunity, the complex and dynamic regulation of IFN{gamma}-induced MHCII is not well understood. Here, we integrated functional genomics and transcriptomics to comprehensively define the genetic control of IFN{gamma}-mediated MHCII surface expression in macrophages. Using a genome-wide CRISPR-Cas9 library we identified genes that control MHCII surface expression, many of which have yet to be associated with MHCII. Mechanistic studies uncovered two parallel pathways of IFN{gamma}-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3{beta}) or the mediator complex subunit MED16. Both pathways are necessary for IFN{gamma}-mediated induction of the MHCII transactivator CIITA, MHCII expression, and CD4+ T cell activation. Using transcriptomic analysis, we defined the regulons controlled by GSK3{beta} and MED16 in the presence and absence of IFN{gamma} and identified unique networks of the IFN{gamma}-mediated transcriptional landscape that are controlled by each gene. Our analysis suggests GSK3{beta} and MED16 control distinct aspects of the IFN{gamma}-response and are critical for macrophages to respond appropriately to IFN{gamma}. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+ T cell responses by macrophages. These discoveries will aid in our basic understanding of macrophage-mediated immunity and will shed light on mechanisms of failed adaptive responses pervasive in infectious disease, autoimmunity, and cancer.

immunology↗