During blood-stage Plasmodium infection, effective immune control hinges on the IL12β-IFN-γ axis, yet how this pathway is transcriptionally tuned in vivo remains incompletely defined. Innate sensing of parasite-derived ligands by pattern-recognition receptors, including Toll like receptors, in dendritic cells and macrophages induces IL-12β production that drives IFN-γ mediated control of infection. Emerging evidence implicates cellular nucleic acid-binding protein (CNBP), a zinc-finger transcriptional regulator, in control of IL12β gene expression in myeloid cells exposed to bacterial and viral infections. Here, we defined the contribution of CNBP in cytokine-driven immunity to Plasmodium infection including both P. falciparum (the major cause of malaria), as well as P. chabaudi and P. berghei ANKA, two rodent species that model human disease. Upon exposure to Plasmodium-infected erythrocytes, CNBP rapidly translocated to the nucleus in mouse and human dendritic cells, bound IL12β promoter, and was required for optimal IL12β induction. Genetic ablation of CNBP in mice and siRNA knockdown of CNBP in human monocyte-derived dendritic cells markedly reduced IL12β production and downstream IFN-γ responses, while TNF-α and several other innate cytokines were largely unaffected. In vivo, hematopoietic-specific deletion of CNBP (using vav-iCre; Cnbp fl/fl) resulted in elevated peak parasitemia, impaired parasite clearance, and relapse after initial resolution. Consistent with these outcomes, spleens from mice lacking CNBP in hematopoietic cells exhibited reduced inflammatory remodeling, altered T-cell composition, and transcriptional reprogramming characterized by selective regulation of IL12β-IFN-γ transcripts alongside upregulation of distinct cytotoxic genes. Paradoxically, mice lacking CNBP in hematopoietic cells showed delayed mortality in the lethal infection model, underscoring its context-dependent contributions to host protection and inflammatory pathology. Collectively, these findings position CNBP as a pivotal modulator of the IL12β-IFN-γ axis during malaria, extending its functional repertoire beyond microbial contexts, with potential as a therapeutic target to fine-tune immune responses for enhanced protection with limited immunopathology.

During infection, foreign DNA is sensed by cyclic GMP-AMP synthase (cGAS) leading to the production of cGAMP, STING-dependent type I interferon and proinflammatory cytokine expression, and autophagy. To prevent a response to self-DNA, cGAS activity is tightly regulated. Dysregulation of cGAS underpins interferonopathies, such as Aicardi–Goutières syndrome, as well as Lupus and neurodegenerative diseases like Parkinson’s disease. Thus, cGAS and its product cGAMP are therapeutic targets. However, if cGAS functions independently of cGAMP signaling is undefined. Here, we identified an alternative signaling pathway that cGAS engages independent of cGAMP synthesis. We demonstrate that alternative cGAS signaling promotes hyperexpression of CXCL1 and enhanced neutrophil recruitment that facilitates viral dissemination during herpes simplex encephalitis. Our study reports of an alternative cGAS response independent of cGAMP, highlighting a previously uncharacterized scaffold function for cGAS.

Subclinical parasitemia constitutes the predominant proportion of Plasmodium spp. infections in hyperendemic regions of the world. Elevated levels of serum interleukin-10 (IL-10) are observed in both acute symptomatic and chronic subclinical Plasmodium spp. infections. The role of IL-10 in acute infection has been extensively studied; however, the role of sustained elevated levels of IL-10 in chronic subclinical plasmodial infections remains to be determined. We investigated the role of IL-10 in a long-term subclinical and patent Plasmodium chabaudi chabaudi-AS (Pc) infection using mice lacking humoral immunity (µMT-/- mice). Pc-infected µMT-/- mice exhibit a long-term (99 days) chronic infection, with microscopic levels of parasitemia and without any outward signs of disease. We found that chronically infected mice have slightly elevated levels of tumor necrosis factor α (TNFα) and interferon-γ (IFNγ), and high levels of IL-10 in the circulation. The source of IL-10 was CD4+ T cells. We found that elevated IL-10 levels were mechanistically linked to subclinical Plasmodium infection by blocking IL-10 signaling. Anti-IL-10R resulted in a marked, albeit transient, reduction of the parasitemia that was accompanied by a robust pro-inflammatory response and death of chronically infected µMT-/- mice. A similar outcome was observed in infected µMT-/- mice after CD4+ T cell depletion with anti-CD4 antibody. CD4-depleted infected µMT-/- mice exhibited reduced IL-10 and rapid weight loss, succumbing to infection by day 6 after CD4 neutralization. Our results showed that IL-10 from CD4+ T cells limits immune-mediated pathology in chronic subclinical Pc infection in µMT-/- mice by protecting against excessive inflammatory responses to blood-stage parasites.

The female genital mucosa expresses the hormone-dependent type I interferon (IFN), IFN-epsilon (IFN-ε), which protects against chlamydia and herpes infection. Surprisingly, we found that IFN-ε knockout (Ifnε-/-) mice and type I IFN receptor knockout (Ifnar1-/-) mice exhibited enhanced clearance of Neisseria gonorrhoeae (Ng). This result was phenocopied using blocking anti-IFNAR monoclonal antibody (mAb). Ng colonization of the Ifnε-/- urogenital tract was restored by exogenous recombinant IFN-ε or IFN-β. Clearance of Ng in anti-IFNAR-treated mice required the expression of the cathelicidin mCRAMP. Ng deploys a unique mechanism to evade cathelicidins and other innate defenses by sialylating its lipooligosaccharide (LOS) using host-derived cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac or CMP-sialic acid). Ifnε-/- mice expressed reduced levels of CMP-sialic acid synthetase mRNA in genital tissues. Accordingly, Ng recovered from IFN-deficient mice were hyposialylated. In conclusion, Ng exploits type I IFNs to obtain CMP-sialic acid for LOS sialylation, resulting in innate immune evasion and enhanced colonization.

Alcohol use disorder (AUD) has been associated with Alzheimer’s disease (AD) and dementia, yet the underlying mechanisms and specific role of ethanol in AD progression remain poorly understood. Neuroinflammation has emerged as a key contributor to both AD pathogenesis and ethanol-induced brain damage. Activation of innate immune cells and signaling pathways, in particular NLRP3 inflammasome, plays a pivotal role in both AD and ethanol-induced inflammation. Thus, we postulated that excessive ethanol consumption could contribute to AD progression via amplified neuroinflammation. The 12–15-month-old WT and APP/PS1 mice received water or ethanol (3.5 g/kg) binge every alternate day for a period of one month. The effects of ethanol on amyloid pathology, microglia and astrocyte activation, and NLRP3 inflammasome activation were evaluated in the mouse brains. The effect of ethanol and amyloid β on NLRP3 inflammasome signaling was further studied in primary glial cells. In this study, we show that repeated ethanol binges aggravate the amyloid pathology and plaque burden in the hippocampus of APP/PS1 mice. Furthermore, we demonstrate the additive effect of ethanol administration on NLRP3 inflammasome activation, IL-1β release, and ASC aggregation in the brains of APP/PS1 mice and primary glia cultures. Our study also reveals a strong astrocyte activation by ethanol in the hippocampus of APP/PS1 mice as demonstrated by significantly increased GFAP and ALDH1L1 protein levels. Further in vitro analysis revealed that ethanol potentiates the effect of amyloid β to increase the NLRP3 inflammasome activation in both primary astrocytes and microglia. Lastly, we demonstrate that glia-produced ASC specks induce IL-1β in microglia and astrocytes and induce ROS in SH-SY5Y neurons, contributing to sustained neuroinflammation in AD. Collectively, our results demonstrate that ethanol consumption exacerbates features of AD pathology associated with amplified neuroinflammation and NLRP3/ASC inflammasome activation, which may play an important role in the disease progression and severity.

Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.

Multiphoton intravital microscopy (MP-IVM) is an imaging technique used for the observation of living organisms at a microscopic resolution. The tissue of interest is exposed through a window allowing imaging of cells in real time. Using MP-IVM, the temporospatial kinetics of leukocyte transendothelial migration can be visualized and quantitated using reporter mice and cell-specific fluorophore-conjugated monoclonal antibodies to track the leukocytes within and outside of vascular beds. Here we describe a method used to study neutrophil transendothelial migration and blood-brain barrier permeability in a mouse model of herpes simplex virus I (HSV) encephalitis.

Subclinical (asymptomatic) parasitemia is very common amongst Plasmodium -infected individuals. The immunological mechanisms underlying subclinical parasitemia remain elusive. We investigated the immune regulatory mechanisms behind chronic asymptomatic Plasmodium infection using mice lacking humoral immunity (µMT −/− mice). µMT −/− mice became chronically infected, despite lacking outward signs of disease, and exhibited increased macrophage numbers, decreased dendritic and CD4 cells, massive hemozoin accumulation in the spleen and bone marrow, and inadequate hematopoiesis. These changes were accompanied by high circulating levels of interleukin-10 (IL-10), enhanced chromatin accessibility of the STAT3 promoter, and enhanced STAT3 binding to the IL-10 promoter in macrophages. Inhibition of IL-10 signaling, despite promoting parasite clearance, resulted in a proinflammatory response, weight loss, and mortality. These results suggest that epigenetic changes induced by chronic P. chabaudi infection lead to high levels of circulating IL-10, protecting chronically infected mice against an excessive inflammatory response to high levels of blood-stage parasites. Author summary Malaria is a life-threatening disease with a range of symptoms, and it is induced in humans by infections with different species of Plasmodium . Highly prevalent in endemic regions, asymptomatic Plasmodium infections are related to long-term exposure to the parasite due to multiple infections and have been demonstrated in human and mouse studies to be associated with elevated levels of IL-10. However, how IL-10 levels remain elevated in the circulation in individuals over the long term has not been determined. We used a mouse model of chronic asymptomatic Plasmodium infection to investigate the mechanisms by which IL-10 levels are elevated during chronic asymptomatic infection. Our results show that epigenetic changes in immune genes of myeloid origin could be responsible for the elevated levels of IL-10, and that IL-10 signaling protected chronically infected mice from a severe inflammatory response induced by the infection.

Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum–infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell–depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum–trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum–trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.

Interleukin-1 receptor-associated kinases (IRAKs) -4, -2, and -1 are involved in transducing signals from Toll-like receptors (TLRs) via the adaptor myeloid differentiation primary-response protein 88 (MYD88). How MYD88/IRAK4/2/1 complexes are formed, their redundancies, and potential non-enzymatic roles are subjects of debate. Here, we examine the hierarchical requirements for IRAK proteins in the context of TLR4 activation and confirmed that the kinase activity of IRAK4 is essential for MYD88 signaling. Surprisingly, the IRAK4 scaffold is required for activation of the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) by both MYD88 and TIR domain-containing adaptor protein inducing IFN-β (TRIF), a unique adaptation in the TLR4 response. IRAK4 scaffold is, therefore, essential in integrating MYD88 and TRIF in TLR4 signaling.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to emerge, causing surges, breakthrough infections, and devastating losses-underscoring the importance of identifying SARS-CoV-2 antivirals. A simple, accessible human cell culture model permissive to SARS-CoV-2 variants is critical for identifying and assessing antivirals in a high-throughput manner. Although human alveolar A549 cells are a valuable model for studying respiratory virus infections, they lack two essential host factors for SARS-CoV-2 infection: angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). SARS-CoV-2 uses the ACE2 receptor for viral entry and TMPRSS2 to prime the SARS-CoV-2 spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a suitable human cell line for SARS-CoV-2 studies by transducing human ACE2 and TMPRSS2 into A549 cells. We show that subclones highly expressing ACE2 and TMPRSS2 ("ACE2plus" and the subclone "ACE2plusC3") are susceptible to infection with SARS-CoV-2, including the delta and omicron variants. These subclones express more ACE2 and TMPRSS2 transcripts than existing commercial A549 cells engineered to express ACE2 and TMPRSS2. Additionally, the antiviral drugs EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir strongly inhibit SARS-CoV-2 variants in our infection model. Our data show that ACE2plusC3 cells are highly permissive to SARS-CoV-2 infection and can be used to identify anti-SARS-CoV-2 drugs.

Abstract Background Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection. Methods We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection. Results Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14−/− HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production. Conclusions We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication.

SARS coronavirus 1 (SARS-CoV-1) causes a respiratory infection that can lead to acute respiratory distress characterized by inflammation and high levels of cytokines in the lung tissue. In this study we constructed a herpes simplex virus 1 replication-defective mutant vector expressing SARS-CoV-1 spike protein as a potential vaccine vector and to probe the effects of spike protein on host cells. The spike protein expressed from this vector is functional in that it localizes to the surface of infected cells and induces fusion of ACE2-expressing cells. In immunized mice, the recombinant vector induced antibodies that bind to spike protein in an ELISA assay and that show neutralizing activity. The spike protein expressed from this vector can induce the expression of cytokines in an ACE2-independent, MyD88-dependent process. These results argue that the SARS-CoV-1 spike protein intrinsically activates signaling pathways that induce cytokines and contribute directly to the inflammatory process of SARS.

Herpes simplex virus (HSV)-1 encephalitis has significant morbidity partly because of an over-exuberant immune response characterized by leukocyte infiltration into the brain and increased blood-brain barrier (BBB) permeability. Determining the role of specific leukocyte subsets and the factors that mediate their recruitment into the brain is critical to developing targeted immune therapies. In a murine model, we find that the chemokines CXCL1 and CCL2 are induced in the brain following HSV-1 infection. Ccr2 (CCL2 receptor)-deficient mice have reduced monocyte recruitment, uncontrolled viral replication, and increased morbidity. Contrastingly, Cxcr2 (CXCL1 receptor)-deficient mice exhibit markedly reduced neutrophil recruitment, BBB permeability, and morbidity, without influencing viral load. CXCL1 is produced by astrocytes in response to HSV-1 and by astrocytes and neurons in response to IL-1α, and it is the critical ligand required for neutrophil transendothelial migration, which correlates with BBB breakdown. Thus, the CXCL1-CXCR2 axis represents an attractive therapeutic target to limit neutrophil-mediated morbidity in HSV-1 encephalitis.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunomodulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 18 patients with acute influenza infection. Microscopy reveals activated platelets, many containing viral particles and extracellular-DNA associated with platelets. To understand the mechanism, we isolate human platelets and treat them with influenza A virus. Viral-engulfment leads to C3 release from platelets as a function of TLR7 and C3 leads to neutrophil-DNA release and aggregation. TLR7 specificity is confirmed in murine models lacking the receptor, and platelet depletion models support platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but can also lead to thrombotic vascular occlusion.

Influenza infection increases the incidence of myocardial infarction but the reason is unknown. Platelets mediate vascular occlusion through thrombotic functions but are also recognized to have immunoregulatory activity. To determine if platelet processes are activated during influenza infection, we collected blood from 22 patients with acute influenza infection. Microscopy revealed activated platelets, some of them containing viral particles. Influenza infected patients had elevated complement C3 (C3) levels and, in some patients, we observed unattached neutrophil-DNA associated with platelets. To understand the mechanism, we isolated human platelets and treated them with infectious influenza A virus. Transmission electron micrographs (TEM) showed that platelets engulf the virus as early as 5 min post incubation and one platelet can engulf up to 10 viral particles in 30 min. TEMs of influenza incubated platelets, as a function of time, also showed that viral engulfment is achieved mostly by phagocytosis. Biochemically, viral engulfment led to C3 release from platelets in a Toll-like receptor 7 (TLR7)-specific manner. Released C3 led to neutrophil-DNA release and the formation of DNA aggregates that was abrogated by either the TLR7-specific antagonist, IRS661, or the C3-specific activation inhibitor, Compstatin. TLR7 specificity was confirmed in murine models lacking the receptor, and murine platelet depletion models supported platelet-mediated C3 and neutrophil-DNA release post-influenza infection. These findings demonstrate that the initial intrinsic defense against influenza is mediated by platelet-neutrophil cross-communication that tightly regulates host immune and complement responses but, in cases of dysregulation, can lead to thrombotic vascular occlusion.

Viral myocarditis is a leading cause of death in the United States, contributing to numerous unexplained deaths in people ≤35 years old. Enteroviruses contribute to many cases of human myocarditis. Encephalomyocarditis virus (EMCV) infection causes viral myocarditis in rodent models, but its receptor requirements have not been fully identified. CRISPR-Cas9 screens can identify host dependency factors essential for EMCV infection and enhance our understanding of key events that follow viral infection, potentially leading to new strategies for preventing viral myocarditis. Using a CRISPR-Cas9 screen, we identified a d isintegrin a nd m etalloproteinase 9 domain (ADAM9) as a major factor required for the early stages of EMCV infection in both human and murine infection.