Biology subjects

Bou Ghanem, E. N.

Publications and source records attributed to Bou Ghanem, E. N..

12 recordsLinked to original sources

bioRxiv2026

CD73 controls neutrophil responsiveness to type I interferon impairing antibacterial responses during influenza A virus/ Streptococcus pneumoniae co-infection

Streptococcus pneumoniae (pneumococcus) are asymptomatic colonizers of the nasopharynx but can progress to pulmonary and systemic pathogens upon influenza A virus (IAV) infection. Polymorphonuclear cells (PMNs) are required to control bacterial numbers, but we previously found that IAV infection impairs their ability to kill S. pneumoniae. Here, using a model that allows transition of pneumococci from colonizers to disease-causing pathogens upon IAV co-infection, we examined the signaling pathways impairing PMN responses. When we investigated the effect of type I interferons (IFN) produced upon IAV infection on PMN antibacterial activity, we found that PMNs treated with IFN were unable to kill S. pneumoniae ex vivo, and that in vivo blocking of IFN receptor 1 (IFNAR1) in IAV infected mice rescued PMN antibacterial function. In exploring what controls PMN responsiveness to IFN, we examined CD73, an ectonucleotidase that is known to regulate PMN function in primary pneumococcal pneumonia. To test if there is an intersection between CD73 and IFN signaling, we examined receptor levels and IFN production in wildtype versus CD73KO mice and found no difference in IFNAR expression on PMNs or IFN[a] and IFN levels in the lungs and circulation. However, CD73KO PMNs expressed significantly lower levels of the interferon stimulated protein IFIT1. When we looked at ex vivo PMN responsiveness to IFNs, CD73KO PMNs were less responsive to IFN-mediated inhibition of antimicrobial activity. In exploring mechanisms, we found that CD73 expressing PMNs had elevated production of reactive oxygen species in response to IAV challenge, that paradoxically impaired their ability to kill S. pneumoniae. Importantly, despite similar pathogen loads in the respiratory tract, co-infected CD73KO mice cleared bacteremia and survived significantly better than wildtype controls. These findings suggest that CD73 impairs host defense against IAV/S. pneumoniae co-infection in part by sensitizing PMNs to type I IFN-mediated inhibition of antibacterial function. Author SummaryDespite available therapeutics and vaccines, secondary bacterial pneumonia following influenza A virus (IAV) infection remains a major cause of disease. A common cause of secondary bacterial pneumonia are Streptococcus pneumoniae (pneumococcus), bacteria that resides asymptomatically in the nasopharynx, but upon viral infection can transition to cause severe disease in susceptible hosts. In this study we examined how host responses change during single versus polymicrobial infections. We focused on neutrophils, which are innate immune cells that are required for effective clearance of S. pneumoniae, and proper control of IAV. We found that the immune response to IAV, mediated by type I interferons (IFN), impair the ability of neutrophils to kill bacteria. We identified an enzyme called CD73 to be required for the ability of neutrophils to respond to IFN. In exploring mechanisms, we found that IFN and CD73 result in dysregulated reactive oxygen species production by neutrophils. Importantly, this impairs the ability of the host to clear bacteria that spread from the lungs to the blood upon viral co-infection and results in overall worse host outcome. This study describes a novel interaction between CD73 and type I interferons and provides a new therapeutic target to treat secondary pneumococcal pneumonia.

immunology↗

bioRxiv2025

Neutrophils induce effective antibody responses to the pneumococcal conjugate vaccine by inhibiting regulatory T cells

Neutrophils are required for production of protective antibodies in response to the pneumococcal conjugate vaccine (PCV), however, the mechanisms behind that are not known. Here, in exploring mechanisms, we found that depletion of neutrophils at the time of vaccination in female mice led to increased regulatory T cells (Tregs) in the spleen that was accompanied by a dysregulated cytokine environment. Following vaccination, neutrophils homed into secondary lymphoid organs where they increased expression of T cell engaging/inhibiting markers. Depletion of Tregs in neutrophil deficient mice restored antibody function against pneumococcal infection. Importantly, PCV vaccination of human female participants altered neutrophil phenotype and in vitro coculture of donor neutrophils with PBMCs at one week following vaccination resulted in lower percentage and proliferation of FOXP3+ T cells, showing the clinical relevance of this phenotype. This work reveals a new mechanism by which neutrophils ensure protective antibody responses to vaccination by suppressing Tregs.

immunology↗

bioRxiv2025

Adenosine 2B receptor signaling impairs vaccine-mediated protection against pneumococcal infection in young hosts by blunting neutrophil killing of antibody opsonized bacteria

Background/ObjectiveNeutrophils are essential for vaccine-mediated protection against pneumococcal infection and impairment in their antibacterial function contributes to reduced vaccine efficacy during aging. However, the signaling pathways controlling neutrophil responses in vaccinated hosts are not fully understood. The extracellular adenosine pathway is a known regulator of neutrophils in naive hosts. The aim of this study was to test the role of this pathway in neutrophil function and protection against infection upon vaccination across host age. MethodsTo test the role of adenosine in the antimicrobial activity of neutrophils against antibody-opsonized pneumococci, we used bone marrow derived neutrophils isolated from wild type or specific adenosine receptors knock-out mice. To measure the effect of adenosine receptor signaling in vivo, we treated vaccinated mice with agonists or antagonists specific to the different adenosine receptors prior to pulmonary challenge with pneumococci and assessed bacterial burden and clinical score post infection. ResultsWe found that signaling via the adenosine 2B (A2BR) but not A2A or A1 receptor diminished intracellular pneumococcal killing following antibody-mediated uptake in young hosts. In vivo, agonism of A2BR significantly worsened pneumococcal infection outcome in young, vaccinated mice. In contrast, A2BR signaling had no effect on intracellular bacterial killing by neutrophils from aged mice. Further, in vivo A2BR inhibition had no effect on pneumococcal disease progression in aged, vaccinated mice. ConclusionsA2BR signaling reduced pneumococcal vaccine-mediated protection by impairing neutrophil antimicrobial activity against antibody-opsonized bacteria in young hosts. However, inhibiting this pathway was not sufficient to boost responses in aged hosts.

immunology↗

bioRxiv2024

Sex-based difference in immune responses and efficacy of the pneumococcal conjugate vaccine

Vaccine-mediated protection and susceptibility to Streptococcus pneumoniae (pneumococcus) infections are influenced by biological sex. The incidence of invasive pneumococcal disease remains higher in males compared to females even after the introduction of the pneumococcal conjugate vaccine (PCV). However, sex-based differences in the immune response to this conjugate vaccine remain unexplored. To investigate those differences, we vaccinated adult male and female mice with PCV and assessed cellular and humoral immune responses. Compared to females, male mice displayed lower levels of T follicular helper cells, germinal center B cells and plasmablasts, which are all required for antibody production following vaccination. This was linked to lower IgG and IgM levels against pneumococci and lower isotype switching to IgG3 in vaccinated males. Additionally, sera of vaccinated male mice had lower efficacy in several anti-pneumococcal functions including neutralization of bacterial binding to pulmonary epithelial cells as well as direct cytotoxicity against S. pneumoniae. Importantly, while the vaccine was highly protective in females, vaccinated males succumbed to infection more readily and were more susceptible to both lung-localized infection and systemic spread following S. pneumoniae challenge. These findings identify sex-based differences in immune responses to PCV that can inform future vaccine strategies.

immunology↗

bioRxiv2024

Activating A1 adenosine receptor signaling boosts early pulmonary neutrophil recruitment in aged mice in response to Streptococcus pneumoniae infection

BackgroundStreptococcus pneumoniae (pneumococcus) is a leading cause of pneumonia in older adults. Successful control of pneumococci requires robust pulmonary neutrophil influx early in infection. However, aging is associated with aberrant neutrophil recruitment and the mechanisms behind that are not understood. Here we explored how neutrophil recruitment following pneumococcal infection changes with age and the host pathways regulating this. ResultsFollowing pneumococcal infection there was a significant delay in early neutrophil recruitment to the lungs of aged mice. Neutrophils from aged mice showed defects in trans-endothelial migration in vitro compared to young controls. To understand the pathways involved, we examined immune modulatory extracellular adenosine (EAD) signaling, that is activated upon cellular damage. Signaling through the lower affinity A2A and A2B adenosine receptors had no effect on neutrophil recruitment to infected lungs. In contrast, inhibition of the high affinity A1 receptor in young mice blunted neutrophil recruitment to the lungs following infection. A1 receptor inhibition decreased expression of CXCR2 on circulating neutrophils, which is required for transendothelial migration. Indeed, A1 receptor signaling on neutrophils was required for their ability to migrate across endothelial cells in response to infection. Aging was not associated with defects in EAD production or receptor expression on neutrophils. However, agonism of A1 receptor in aged mice rescued the early defect in neutrophil migration to the lungs and improved control of bacterial burden. ConclusionsThis study suggests age-driven defects in EAD damage signaling can be targeted to rescue the delay in pulmonary neutrophil migration in response to bacterial pneumonia.

immunology↗

bioRxiv2021

Liposomal encapsulation of polysaccharides (LEPS) as an effective vaccine strategy to protect aged hosts against S. pneumoniae infection

Despite the availability of licensed vaccines, pneumococcal disease caused by the bacteria Streptococcus pneumoniae (pneumococcus), remains a serious infectious disease threat globally. Disease manifestations include pneumonia, bacteremia, and meningitis, resulting in over a million deaths annually. Pneumococcal disease disproportionally impacts elderly individuals [≥]65 years old. Interventions are complicated through a combination of complex disease progression and 100 different bacterial capsular polysaccharide serotypes. This has made it challenging to develop a broad vaccine against S. pneumoniae, with current options utilizing capsular polysaccharides as the primary antigenic content. However, current vaccines are substantially less effective in protecting the elderly. We previously developed a Liposomal Encapsulation of Polysaccharides (LEPS) vaccine platform, designed around limitations of current pneumococcal vaccines, that allowed the non-covalent coupling of polysaccharide and protein antigen content and protected young hosts against pneumococcal infection in murine models. In this study, we modified the formulation to make it more economical and tested the novel LEPS vaccine in aged hosts. We found that in young mice (2-3 months), LEPS elicited comparable responses to the pneumococcal conjugate vaccine Prevnar-13. Further, LEPS immunization of old mice (20-22 months) induced comparable antibody levels and improved antibody function compared to Prevnar-13. Importantly, LEPS protected old mice against both invasive and lung localized pneumococcal infections. In summary, LEPS is an alternative and effective vaccine strategy that protects aged hosts against different manifestations of pneumococcal disease.

microbiology↗

bioRxiv2021

Transcriptome profiling reveals CD73 and age-driven changes in neutrophil responses against Streptococcus pneumoniae

Neutrophils are required for host resistance against Streptococcus pneumoniae but their function declines with age. We previously found that CD73, an enzyme required for antimicrobial activity, is down-regulated in neutrophils from aged mice. This study explored transcriptional changes in neutrophils induced by S. pneumoniae to identify pathways controlled by CD73 and dysregulated with age. Ultrapure bone marrow-derived neutrophils isolated from wild type (WT) young, old, and CD73KO young mice were mock-challenged or infected with S. pneumoniae ex vivo. RNA sequencing was performed to identify differentially expressed genes (DEGs). We found that infection triggered distinct global transcriptional changes across hosts, that were strongest in CD73KO neutrophils. Surprisingly, there were more down-regulated than up-regulated genes in all groups upon infection. Down-regulated DEGs indicated a dampening of immune responses in old and CD73KO hosts. Further analysis revealed that CD73KO neutrophils expressed higher numbers of long non-coding RNAs (lncRNAs) compared to WT controls. Predicted network analysis indicated that CD73KO specific lncRNAs control several signaling pathways. We found that genes in the JNK-MAPK-pathway were up-regulated upon infection in CD73KO and WT old but not in young mice. This corresponded to functional differences, as phosphorylation of the downstream AP-1 transcription factor component c-Jun was significantly higher in infected CD73KO and old mice neutrophils. Importantly, inhibiting JNK/AP-1 rescued the ability of these neutrophils to kill S. pneumoniae. Altogether, our findings revealed that neutrophils modify their gene expression to better adapt to bacterial infection and that this capacity declines with age and is regulated by CD73.

immunology↗

bioRxiv2020

A Murine Model for the Transition of Streptococcus pneumoniae from Asymptomatic Colonizer to Systemic Pathogen

Streptococcus pneumoniae (pneumococcus) resides asymptomatically in the nasopharynx but can progress from benign colonizer to lethal pulmonary or systemic pathogen. Both viral infection and aging are risk factors for serious pneumococcal infections. Previous work established a murine model that featured the movement of pneumococcus from the nasopharynx to the lung upon nasopharyngeal inoculation with influenza A virus (IAV) but did not fully recapitulate the severe disease associated with human co-infection. We built upon this model by first establishing pneumococcal nasopharyngeal colonization, then inoculating both the nasopharynx and lungs with IAV. In young (2 months) mice, co-infection triggered bacterial dispersal from the nasopharynx into the lungs, pulmonary inflammation, disease and mortality in a fraction of mice. In old mice (20-22 months), co-infection resulted in earlier and more severe disease. Aging was not associated with greater bacterial burdens but rather with more rapid pulmonary inflammation and damage. Both aging and IAV infection led to inefficient bacterial killing by neutrophils ex vivo. Conversely, aging and pneumococcal colonization also blunted IFN- production and increased pulmonary IAV burden. Thus, in this multistep model, IAV promotes pneumococcal pathogenicity by modifying bacterial behavior in the nasopharynx, diminishing neutrophil function, and enhancing bacterial growth in the lung, while pneumococci increase IAV burden likely by compromising a key antiviral response. Thus, this model provides a means to elucidate factors, such as age and co-infection, that promote the evolution of S. pneumoniae from asymptomatic colonizer to invasive pathogen, as well as to investigate consequences of this transition on antiviral defense.

microbiology↗

bioRxiv2020

Extracellular adenosine signaling reverses the age-driven decline in the ability of neutrophils to kill S. pneumoniae

The elderly are susceptible to serious infections by Streptococcus pneumoniae (pneumococcus), which calls for a better understanding of the pathways driving the decline in host defense in aging. We previously found that extracellular adenosine (EAD) shaped polymorphonuclear cell (PMN) responses, which are crucial for controlling infection. EAD is produced by CD39 and CD73, and signals via A1, A2A, A2B and A3 receptors. The objective of this study was to explore the age-driven changes in the EAD pathway and its impact on PMN function. We found in comparison to young mice, PMNs from old mice expressed significantly less CD73, but similar levels of CD39 and adenosine receptors. PMNs from old mice failed to efficiently kill pneumococci ex vivo; however, supplementation with adenosine rescued this defect. Importantly, transfer of PMNs expressing CD73 from young mice reversed the susceptibility of old mice to pneumococcal infection. To identify which adenosine receptor(s) is involved, we used specific agonists and inhibitors. We found that A1 receptor signaling was crucial for PMN function as inhibition or genetic ablation of A1 impaired the ability of PMNs from young mice to kill pneumococci. Importantly, activation of A1 receptors rescued the age-associated defect in PMN function. In exploring mechanisms, we found that PMNs from old mice failed to efficiently kill engulfed pneumococci and that A1 receptor controlled intracellular killing. In summary, targeting the EAD pathway reverses the age-driven decline in PMN antimicrobial function, which has serious implications in combating infections.

immunology↗

bioRxiv2020

Neutrophils are required during immunization with the pneumococcal conjugate vaccine for protective antibody responses and host defense against infection

Neutrophils can shape adaptive immunity, however their role in vaccine-induced protection against infections in vivo remains unclear. Here, we tested their role in the clinically relevant polysaccharide conjugate vaccine against Streptococcus pneumoniae (pneumococcus). We antibody depleted neutrophils during vaccination, allowed them to recover, and four weeks later challenged mice with pneumococci. We found that while isotype-treated vaccinated controls were protected against an otherwise lethal infection in naive mice, full protection was lost upon neutrophil depletion. Compared to vaccinated controls, neutrophil-depleted mice had higher lung bacterial burdens, increased incidence of bacteremia and lower survival rates. Sera from neutrophil-depleted mice had less anti-pneumococcal IgG2c and IgG3, were less efficient at inducing opsonophagocytic killing of bacteria by neutrophils in vitro and worse at protecting naive mice against pneumococcal pneumonia. In summary, neutrophils are required during vaccination for optimal host protection, which has important implications for future vaccine design against pneumococci and other pathogens.

immunology↗

bioRxiv2019

Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production

PMNs are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the anti-microbial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its anti-microbial activity by inhibiting IL-10 production. PMNs from wild type mice did not increase IL-10 production in response to S. pneumoniae, however, CD73-/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of wild type PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73-/- PMNs as well as host resistance of CD73-/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake and intracellular killing or production of anti-microbial Neutrophil Elastase and Myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal ROS production by PMNs. ROS contributed to PMN anti-microbial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN anti-microbial phenotype during S. pneumoniae infection.

immunology↗

bioRxiv2018

PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause invasive diseases in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a bona fide CW protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.\n\nIMPORTANCENeutrophils are innate immune cells needed to effectively clear Streptococcus pneumoniae (Spn). Neutrophil serine proteases (NSPs) are important for killing phagocytosed Spn, however, the identity of the Spn proteins that are degraded by NSPs are unknown. This study identifies a Spn cell wall protein, aminopeptidase N (PepN) that is degraded by the NSP, neutrophil elastase (NE). We demonstrate that PepN is a bona fide cell wall protein and mutants lacking PepN are significantly more resistant than wild-type to killing by purified NE and human neutrophils. This study demonstrates that PepN is a NE substrate and its degradation contributes to effective Spn killing. By better understanding how neutrophils kill Spn, we aim to inform the development of improved therapeutic interventions.

microbiology↗