Biology subjects

Murphy, P. M.

Publications and source records attributed to Murphy, P. M..

7 recordsLinked to original sources

bioRxiv2024

Cxcl10 is required for survival during SARS-CoV-2 infection in mice

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, remains endemic worldwide [~]5 years since the first documented case. Severe COVID-19 is widely considered to be caused by a dysregulated immune response to SARS-CoV-2 within the respiratory tract. Circulating levels of the chemokine CXCL10 are strongly positively associated with poor outcome; however, its precise role in pathogenesis and its suitability as a therapeutic target have remained undefined. Here, we challenged 4-6 month old C57BL/6 mice genetically deficient in Cxcl10 with a mouse-adapted strain of SARS-CoV-2. Infected male, but not female, Cxcl10-/- mice displayed increased mortality compared to wild type controls. Histopathological damage, inflammatory gene induction and virus load in the lungs of male mice 4 days post infection and before death were not broadly influenced by Cxcl10 deficiency. However, accumulation of B cells and both CD4+ and CD8+ T cells in the lung parenchyma of infected mice was reduced in the absence of Cxcl10. Thus, during acute SARS-CoV-2 infection, Cxcl10 regulates lymphocyte infiltration in the lung and confers protection against mortality. Our preclinical model results do not support targeting CXCL10 therapeutically in severe COVID-19.

immunology↗

bioRxiv2024

Chemokines Kill Bacteria by Binding Anionic Phospholipids without Triggering Antimicrobial Resistance

Classically, chemokines coordinate leukocyte trafficking during immune responses; however, many chemokines have also been reported to possess direct antibacterial activity in vitro. Yet, the bacterial killing mechanism of chemokines and the biochemical properties that define which members of the chemokine superfamily are antimicrobial remain poorly understood. Here we report that the antimicrobial activity of chemokines is defined by their ability to bind phosphatidylglycerol and cardiolipin, two anionic phospholipids commonly found in the bacterial plasma membrane. We show that only chemokines able to bind these two phospholipids kill Escherichia coli and Staphylococcus aureus and that they exert rapid bacteriostatic and bactericidal effects against E. coli with a higher potency than the antimicrobial peptide beta-defensin 3. Furthermore, our data support that bacterial membrane cardiolipin facilitates the antimicrobial action of chemokines. Both biochemical and genetic interference with the chemokine-cardiolipin interaction impaired microbial growth arrest, bacterial killing, and membrane disruption by chemokines. Moreover, unlike conventional antibiotics, E. coli failed to develop resistance when placed under increasing antimicrobial chemokine pressure in vitro. Thus, we have identified cardiolipin and phosphatidylglycerol as novel binding partners for chemokines responsible for chemokine antimicrobial action. Our results provide proof of principle for developing chemokines as novel antibiotics resistant to bacterial antimicrobial resistance mechanisms.

microbiology↗

bioRxiv2023

Atypical Chemokine Receptor 1 (Ackr1)-deficient Mice Resist Lethal SARS-CoV-2 Challenge

High pro-inflammatory chemokine levels have been reported in blood and lung in patients with COVID-19. To investigate specific roles in pathogenesis, we studied the regulation of chemokine ligands and receptors in the lungs of 4-6-month-old wild type C57BL/6 mice infected with the MA10 mouse-adapted strain of SARS-CoV-2. We found that atypical chemokine receptor 1 (Ackr1, also known as Duffy antigen receptor for chemokines/DARC) was the most highly upregulated chemokine receptor in infected lung, where it localized to endothelial cells of veins and arterioles. In a screen of 7 leukocyte chemoattractant or chemoattractant receptor knockout mouse lines, Ackr1-/- mice were unique in having lower mortality after SARS-CoV-2 infection, particularly in males. ACKR1 is a non-signaling chemokine receptor that in addition to endothelium is also expressed on erythrocytes and Purkinje cells of the cerebellum. It binds promiscuously to both inflammatory CC and CXC chemokines and has been reported to control chemokine availability which may influence the shape of chemotactic gradients and the ability of leukocytes to extravasate and produce immunopathology. Of note, erythrocyte ACKR1 deficiency is fixed in sub-Saharan African populations where COVID-19 has been reported to result in low mortality compared to worldwide data. Our data suggest the possibility of a causal contribution of ACKR1 deficiency to low sub-Saharan COVID-19 mortality and identify ACKR1 as a possible drug target in the disease.

immunology↗

bioRxiv2023

CXCR4 signaling strength regulates hematopoietic multipotent progenitor fate through extrinsic and intrinsic mechanisms

How cell-extrinsic niche-related and cell-intrinsic cues drive lineage specification of hematopoietic multipotent progenitors (MPPs) in the bone marrow (BM) is partly understood. We show that CXCR4 signaling strength regulates localization and fate of MPPs. In mice phenocopying the BM myeloid skewing of patients with WHIM Syndrome (WS), a rare immunodeficiency caused by gain-of-function CXCR4 mutations, enhanced mTOR signaling and overactive Oxphos metabolism were associated with myeloid rewiring of lymphoid-primed MPPs (or MPP4). Fate decision of MPP4 was also affected by molecular changes established at the MPP1 level. Mutant MPP4 displayed altered BM localization relative to peri-arteriolar structures, suggesting that extrinsic cues contribute to their myeloid skewing. Chronic treatment with CXCR4 antagonist AMD3100 or mTOR inhibitor Rapamycin rescued lymphoid capacities of mutant MPP4, demonstrating a pivotal role for the CXCR4-mTOR axis in regulating MPP4 fate. Our study thus provides mechanistic insights into how CXCR4 signaling regulates the lymphoid potential of MPPs.

cell biology↗

bioRxiv2022

NF kappa B Regulator Bcl3 Controls Development and Function of Classical Dendritic Cells in Toxoplasma gondii

The atypical I{kappa}B family member Bcl3 associates with p50/NF-{kappa}B1 or p52/NF-{kappa}B2 homodimers in the nucleus, and positively or negatively modulates transcription in a context-dependent manner. In mice lacking Bcl3 globally or specifically in CD11c+ cells, Toxoplasma gondii infection is uniformly fatal and is associated with an impaired Th1 immune response. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, we investigated the role of Bcl3 specifically in cDCs in T. gondii infection in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. The conditional cDC Bcl3 KO was as susceptible to lethal T. gondii infection as the total Bcl3 KO and generated poor Th1 responses. Splenocyte single cell RNA seq in the model revealed defective Bcl3-dependent expression of genes involved in antigen processing. Consistent with this, soluble toxoplasma antigen presentation was impaired in Bcl3-deficient cDCs, and tetramer staining demonstrated defective T. gondii antigen-specific splenic CD4+ and CD8+ T cell responses in infected cDC Bcl3-/- mice. In vitro differentiation of bone marrow progenitors from wildtype and cDC Bcl3-/- mice using Flt3L, NOTCH and IFN-{gamma} stimulation recapitulated the defective Bcl3-dependent cDC antigen-presentation activity observed in vivo. Splenocyte single cell RNA seq also revealed the existence of a unique subpopulation of Zbtb46+LysM+ DC which exhibited Bcl3-dependent expansion after infection. We also detected cDCs coexpressing the monocytic markers CD64 and Ly6C (designated icDC1 and icDC2) mainly in infected spleen, which were less abundant in Bcl3flx/flx Zbtb46 cre mice. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infected mice, and shapes DC ontogeny. Author SummaryDendritic cells initiate immune responses against invading pathogens. As professional antigen presenting cells they process and present antigen via the major histocompatibility complex to T cells and thus activate them. Bcl3, an atypical member of the I{kappa}B family regulates the APC function of dendritic cells. In this study we show that expression of Bcl3 specifically in classical DCs is critical for host protection against a protozoan parasite, Toxoplasma gondii. Host protective proinflammatory mechanisms are compromised in mice deficient in Bcl3 in classical DCs leading to an elevated organ parasite load and eventually death of the infected animals. We also found the emergence of Bcl3-dependent hybrid DCs upon T. gondii infection, which have mixed phenotypic markers from DCs and monocytes. Antigen processing genes are significantly downregulated in Bcl3-deficient cDCs, which may account for defective cross presentation of T. gondii antigens. In an in vitro differentiation model, we showed that development of XCR1+cross presenting cDC1s is critically regulated by Bcl3. Overall, this study reveals the complexity of dendritic cell ontogeny and the role of Bcl3 in classical DC function in the context of Toxoplasma infection.

immunology↗

bioRxiv2020

Chemokines as phosphatidylserine-bound 'find-me' signals in apoptotic cell clearance

Chemokines are positively charged cytokines that attract leukocytes by binding to anionic glycosaminoglycans (GAGs) on endothelial cells for efficient presentation to leukocyte G protein-coupled receptors (GPCRs). The atypical chemokine CXCL16 has been reported to also bind the anionic phospholipid phosphatidylserine (PS), but the biological relevance of this interaction remains poorly understood. Here we demonstrate that PS binding is in fact a widely shared property of chemokine superfamily members that, like GAG binding, induces chemokine oligomerization. PS is an essential phospholipid of the inner leaflet of the healthy cell plasma membrane but it is exposed in apoptotic cells to act as an eat-me signal that promotes engulfment of dying cells by phagocytes. We found that chemokines can bind PS in pure form as well as in the context of liposomes and on the surface of apoptotic cells and extracellular vesicles released by apoptotic cells, which are known to act as find-me signals that chemoattract phagocytes during apoptotic cell clearance. Importantly, we show that GAGs are severely depleted from the surface of apoptotic cells and that extracellular vesicles extracted from apoptotic mouse thymus bind endogenous thymic chemokines and activate cognate chemokine receptors. Together these results indicate that chemokines tethered to surface-exposed PS may be responsible for the chemotactic and find-me signal activity previously attributed to extracellular vesicles, and that PS may substitute for GAGs as the anionic scaffold that regulates chemokine oligomerization and presentation to GPCRs on the GAG-deficient membranes of apoptotic cells and extracellular vesicles. Here, we present a new mechanism by which extracellular vesicles, currently recognized as essential agents for intercellular communication in homeostasis and disease, can transport signaling cytokines.

immunology↗

bioRxiv2019

Discovery of several thousand highly diverse circular DNA viruses

Although it is suspected that there are millions of distinct viral species, fewer than 9,000 are catalogued in GenBanks RefSeq database. We selectively enriched for and amplified the genomes of circular DNA viruses in over 70 animal samples, ranging from cultured soil nematodes to human tissue specimens. A bioinformatics pipeline, Cenote-Taker, was developed to automatically annotate over 2,500 circular genomes in a GenBank-compliant format. The new genomes belong to dozens of established and emerging viral families. Some appear to be the result of previously undescribed recombination events between ssDNA viruses and ssRNA viruses. In addition, hundreds of circular DNA elements that do not encode any discernable similarities to previously characterized sequences were identified. To characterize these "dark matter" sequences, we used an artificial neural network to identify candidate viral capsid proteins, several of which formed virus-like particles when expressed in culture. These data further the understanding of viral sequence diversity and allow for high throughput documentation of the virosphere.

microbiology↗