Biology subjects

Parks, A.

Publications and source records attributed to Parks, A..

5 recordsLinked to original sources

bioRxiv2023

Expansion of Disease Specific Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis

BackgroundImmune checkpoint inhibitors (ICIs), antibodies targeting PD-1/PD-L1 or CTLA4 have revolutionized cancer management but are associated with devastating immune-related adverse events (irAEs) including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI-myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. While much has been learned regarding the role of T-cells in ICI-myocarditis, little is understood regarding the identity, transcriptional diversity, and functions of infiltrating macrophages. MethodsWe employed an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization and molecular imaging and antibody neutralization studies. ResultsWe observed marked increases in CCR2+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9, Cxcl10, Gbp2b, and Fcgr4 that originated from CCR2+ monocytes. Importantly, a similar macrophage population expressing CXCL9, CXCL10, and CD16 (human homologue of mouse FcgR4) was found selectively expanded in patients with ICI myocarditis compared to other forms of heart failure and myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-{gamma} and CXCR3 signaling pathways. Depleting CD8+T-cells, macrophages, and blockade of IFN-{gamma} signaling blunted the expansion of Cxcl9+Cxcl10+macrophages in the heart and attenuated myocarditis suggesting that this interaction was necessary for disease pathogenesis. ConclusionThese data demonstrate that ICI-myocarditis is associated with the expansion of a specific population of IFN-{gamma} induced inflammatory macrophages and suggest the possibility that IFN-{gamma} blockade may be considered as a treatment option for this devastating condition.

immunology↗

bioRxiv2022

Reprogramming Alveolar Macrophage Responses to TGF-β Reveals CCR2+ Monocyte Activity that Promotes Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-{beta} bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes are engulfed primarily by alveolar macrophages (AM), which become reprogrammed to become less responsive to TGF-{beta} and reduce TGF-{beta} bioavailability through secretion of the TGF-{beta} antagonist Decorin. In untreated recipients, high airway TGF-{beta} activity stimulates AM to express CCL2 leading to CCR2+ monocyte-driven BOS development. Moreover, we find TGF-{beta} receptor 2-dependent differentiation of CCR2+ monocytes is required for the generation of monocyte-derived AM, which in turn promote BOS by expanding tissue-resident memory CD8+ T cells that inflict airway injury through Blimp-1-mediated Granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-{beta}-dependent network, which couples CCR2+ monocyte recruitment and differentiation to alloimmunity and BOS. Alveolar macrophage plasticity can be harnessed to prevent Bronchiolitis Obliterans Syndrome.

immunology↗

bioRxiv2020

Bacteriophage SP01 Gene Product 56 (gp56) Inhibits Bacillus subtilis Cell Division by Interacting with DivIC/FtsL to Prevent Pbp2B/FtsW Recruitment

Previous work identified gp56, encoded by the lytic bacteriophage SP01, as responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here we show that expression of the predicted 9.3-kDa gene product 56 (gp56) of SP01 inhibits latter stages of B. subtilis cell division without altering FtsZ ring assembly. GFP-tagged gp56 localizes to the membrane at the site of division. While its localization permits recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analysis suggest that gp56 localization and activity depends on its interaction with mid-recruited proteins DivIC and/or FtsL. Together these data support a model where gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis. IMPORTANCEResearch over the past decades has uncovered bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. Phage factors that cause cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanism of several identified phage factors that inhibit cytokinesis remain unexplored, including gp56 of bacteriophage SP01 of Bacillus subtilis. Here, we show that unlike related published examples of phage inhibition of cyotkinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and block recruitment of proteins needed for the septal cell wall synthesis.

microbiology↗

bioRxiv2019

Large-scale transgenic Drosophila resource collections for loss- and gain-of-function studies

The Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

genetics↗

bioRxiv2019

Tumor cell-organized fibronectin is required to maintain a dormant breast cancer population

Tumors can undergo long periods of dormancy, with cancer cells entering a largely quiescent, non-proliferative state before reactivation and outgrowth. For a patient, these post-remission tumors are often drug resistant and highly aggressive, resulting in poor prognosis. To understand the role of the extracellular matrix (ECM) in regulating tumor dormancy, we created an in vitro cell culture system that combines carefully controlled ECM substrates with nutrient deprivation to observe entrance into and exit from dormancy with live imaging. We saw that cell populations capable of surviving entrance into long-term dormancy were heterogeneous, containing quiescent, cell cycle arrested, and actively proliferating cells. Cell populations that endured extended periods of serum-deprivation-induced dormancy formed an organized, fibrillar fibronectin matrix via v{beta}3 and 5{beta}1 integrin adhesion, ROCK-generated tension, and TGF{beta}2 stimulation. We surmised that the fibronectin matrix was primarily a mediator of cell survival, not proliferation, during the serum-deprivation stress, bacause cancer cell outgrowth after dormancy required MMP-2-mediated fibronectin degradation. Given the difficulty of animal models in observing entrance and exit from dormancy in real-time, we propose this approach as a new, in vitro method to study factors important in regulating dormancy, and we used it here to elucidate a role for fibronectin deposition and MMP activation.

cancer biology↗