Suppression of adaptive NK cell expansion by macrophage-mediated phagocytosis inhibited by 2B4-CD48.

Infection of mice by mouse cytomegalovirus (MCMV) triggers activation and expansion of Ly49H+ natural killer (NK) cells, which are virus specific and considered to be "adaptive" or "memory" NK cells. Here, we find that signaling lymphocytic activation molecule family receptors (SFRs), a group of hematopoietic cell-restricted receptors, are essential for the expansion of Ly49H+ NK cells after MCMV infection. This activity is largely mediated by CD48, an SFR broadly expressed on NK cells and displaying augmented expression after MCMV infection. It is also dependent on the CD48 counter-receptor, 2B4, expressed on host macrophages. The 2B4-CD48 axis promotes expansion of Ly49H+ NK cells by repressing their phagocytosis by virus-activated macrophages through inhibition of the pro-phagocytic integrin lymphocyte function-associated antigen-1 (LFA-1) on macrophages. These data identify key roles of macrophages and the 2B4-CD48 pathway in controlling the expansion of adaptive NK cells following MCMV infection. Stimulation of the 2B4-CD48 axis may be helpful in enhancing adaptive NK cell responses for therapeutic purposes.

Germicidal efficacy of continuous and pulsed ultraviolet-C radiation on pathogen models and SARS-CoV-2.

Ultraviolet radiation's germicidal efficacy depends on several parameters, including wavelength, radiant exposure, microbial physiology, biological matrices, and surfaces. In this work, several ultraviolet radiation sources (a low-pressure mercury lamp, a KrCl excimer, and four UV LEDs) emitting continuous or pulsed irradiation were compared. The greatest log reductions in E. coli cells and B. subtilis endospores were 4.1 ± 0.2 (18 mJ cm-2) and 4.5 ± 0.1 (42 mJ cm-2) with continuous 222 nm, respectively. The highest MS2 log reduction observed was 2.7 ± 0.1 (277 nm at 3809 mJ cm-2). Log reductions of SARS-CoV-2 with continuous 222 nm and 277 nm were ≥ 3.4 ± 0.7, with 13.3 mJ cm-2 and 60 mJ cm-2, respectively. There was no statistical difference between continuous and pulsed irradiation (0.83-16.7% [222 nm and 277 nm] or 0.83-20% [280 nm] duty rates) on E. coli inactivation. Pulsed 260 nm radiation (0.5% duty rate) at 260 nm yielded significantly greater log reduction for both bacteria than continuous 260 nm radiation. There was no statistical difference in SARS-CoV-2 inactivation between continuous and pulsed 222 nm UV-C radiation and pulsed 277 nm radiation demonstrated greater germicidal efficacy than continuous 277 nm radiation. Greater radiant exposure for all radiation sources was required to inactivate MS2 bacteriophage. Findings demonstrate that pulsed irradiation could be more useful than continuous UV radiation in human-occupied spaces, but threshold limit values should be respected. Pathogen-specific sensitivities, experimental setup, and quantification methods for determining germicidal efficacy remain important factors when optimizing ultraviolet radiation for surface decontamination or other applications.

Dysregulation of B lymphocyte development in the SKG mouse model of rheumatoid arthritis.

Rheumatoid arthritis is a chronic and systemic inflammatory disease that affects approximately 1% of the world's population and is characterised by joint inflammation, the destruction of articular cartilage and bone, and many potentially life-threatening extraarticular manifestations. B lymphocytes play a central role in the pathology of rheumatoid arthritis as the precursors of autoantibody secreting plasma cells, as highly potent antigen-presenting cells, and as a source of various inflammatory cytokines, however, the effects of rheumatoid arthritis on B lymphocyte development remain poorly understood. Here, we analyse B lymphocyte development in murine models of rheumatoid arthritis, quantifying all the subsets of B cell precursors in the bone marrow and splenic B cells using flow cytometry. We demonstrate a severe reduction in pre-B cells and immature B cells in the bone marrow of mice with active disease, despite no major effects on the mature naïve B cell numbers. The loss of B cell precursors in the bone marrow of the affected mice was associated with a highly significant reduction in the proportion of Ki67+ cells, indicating impaired cell proliferation, while the viability of the B cell precursors was not significantly affected. We also observed some mobilisation of the B cell precursor cells into the mouse spleen, demonstrated with flow cytometry and pre-B colony forming units assays. In summary, the current work demonstrates a severe dysregulation in B lymphocyte development in murine rheumatoid arthritis, with possible implications for B cell repertoire formation, tolerance induction, and disease mechanisms.

Nanoplasmonic amplification in microfluidics enables accelerated colorimetric quantification of nucleic acid biomarkers from pathogens.

Deployment of nucleic acid amplification assays for diagnosing pathogens in point-of-care settings is a challenge due to lengthy preparatory steps. We present a molecular diagnostic platform that integrates a fabless plasmonic nano-surface into an autonomous microfluidic cartridge. The plasmonic 'hot' electron injection in confined space yields a ninefold kinetic acceleration of RNA/DNA amplification at single nucleotide resolution by one-step isothermal loop-mediated and rolling circle amplification reactions. Sequential flow actuation with nanoplasmonic accelerated microfluidic colorimetry and in conjugation with machine learning-assisted analysis (using our 'QolorEX' device) offers an automated diagnostic platform for multiplexed amplification. The versatility of QolorEX is demonstrated by detecting respiratory viruses: SARS-CoV-2 and its variants at the single nucleotide polymorphism level, H1N1 influenza A, and bacteria. For COVID-19 saliva samples, with an accuracy of 95% on par with quantitative polymerase chain reaction and a sample-to-answer time of 13 minutes, QolorEX is expected to advance the monitoring and rapid diagnosis of pathogens.

Identifying Markers of Emerging SARS-CoV-2 Variants in Patients With Secondary Immunodeficiency.

Since the end of 2019, the world has been challenged by the coronavirus disease 2019 (COVID-19) pandemic. With COVID-19 cases rising globally, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, resulting in the emergence of variants of interest (VOI) and of concern (VOC). Of the hundreds of millions infected, immunodeficient patients are one of the vulnerable cohorts that are most susceptible to this virus. These individuals include those with preexisting health conditions and/or those undergoing immunosuppressive treatment (secondary immunodeficiency). In these cases, several researchers have reported chronic infections in the presence of anti-COVID-19 treatments that may potentially lead to the evolution of the virus within the host. Such variations occurred in a variety of viral proteins, including key structural ones involved in pathogenesis such as spike proteins. Tracking and comparing such mutations with those arisen in the general population may provide information about functional sites within the SARS-CoV-2 genome. In this study, we reviewed the current literature regarding the specific features of SARS-CoV-2 evolution in immunocompromised patients and identified recurrent de novo amino acid changes in virus isolates of these patients that can potentially play an important role in SARS-CoV-2 pathogenesis and evolution.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

The NF-κB Transcription Factor c-Rel Modulates Group 2 Innate Lymphoid Cell Effector Functions and Drives Allergic Airway Inflammation.

Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and orchestration of early type 2 immune responses. Upon tissue damage, ILC2s are activated by alarmins such as IL-33 and rapidly secrete large amounts of type 2 signature cytokines. ILC2 activation is governed by a network of transcriptional regulators including nuclear factor (NF)-κB family transcription factors. While it is known that activating IL-33 receptor signaling results in downstream NF-κB activation, the underlying molecular mechanisms remain elusive. Here, we found that the NF-κB subunit c-Rel is required to mount effective innate pulmonary type 2 immune responses. IL-33-mediated activation of ILC2s in vitro as well as in vivo was found to induce c-Rel mRNA and protein expression. In addition, we demonstrate that IL-33-mediated activation of ILC2s leads to nuclear translocation of c-Rel in pulmonary ILC2s. Although c-Rel was found to be a critical mediator of innate pulmonary type 2 immune responses, ILC2-intrinsic deficiency of c-Rel did not have an impact on the developmental capacity of ILC2s nor affected homeostatic numbers of lung-resident ILC2s at steady state. Moreover, we demonstrate that ILC2-intrinsic deficiency of c-Rel alters the capacity of ILC2s to upregulate the expression of ICOSL and OX40L, key stimulatory receptors, and the expression of type 2 signature cytokines IL-5, IL-9, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Collectively, our data using Rel-/- mice suggest that c-Rel promotes acute ILC2-driven allergic airway inflammation and suggest that c-Rel may contribute to the pathophysiology of ILC2-mediated allergic airway disease. It thereby represents a promising target for the treatment of allergic asthma, and evaluating the effect of established c-Rel inhibitors in this context would be of great clinical interest.

The c-Rel transcription factor limits early interferon and neuroinflammatory responses to prevent herpes simplex encephalitis onset in mice.

Herpes simplex virus type 1 (HSV-1) is the predominant cause of herpes simplex encephalitis (HSE), a condition characterized by acute inflammation and viral replication in the brain. Host genetics contribute to HSE onset, including monogenic defects in type I interferon signaling in cases of childhood HSE. Mouse models suggest a further contribution of immune cell-mediated inflammation to HSE pathogenesis. We have previously described a truncating mutation in the c-Rel transcription factor (RelC307X) that drives lethal HSE in 60% of HSV-1-infected RelC307X mice. In this study, we combined dual host-virus RNA sequencing with flow cytometry to explore cell populations and mechanisms involved in RelC307X-driven HSE. At day 5 postinfection, prior to HSE clinical symptom onset, elevated HSV-1 transcription was detected together with augmented host interferon-stimulated and inflammatory gene expression in the brainstems of high-responding RelC307X mice, predictive of HSE development. This early induction of host gene expression preceded pathological infiltration of myeloid and T cells in RelC307X mice at HSE onset by day 7. Thus, we establish c-Rel as an early regulator of viral and host responses during mouse HSE. These data further highlight the importance of achieving a balanced immune response and avoiding excess interferon-driven inflammation to promote HSE resistance.

Bisphosphoglycerate Mutase Deficiency Protects against Cerebral Malaria and Severe Malaria-Induced Anemia.

The replication cycle and pathogenesis of the Plasmodium malarial parasite involves rapid expansion in red blood cells (RBCs), and variants of certain RBC-specific proteins protect against malaria in humans. In RBCs, bisphosphoglycerate mutase (BPGM) acts as a key allosteric regulator of hemoglobin/oxyhemoglobin. We demonstrate here that a loss-of-function mutation in the murine Bpgm (BpgmL166P) gene confers protection against both Plasmodium-induced cerebral malaria and blood-stage malaria. The malaria protection seen in BpgmL166P mutant mice is associated with reduced blood parasitemia levels, milder clinical symptoms, and increased survival. The protective effect of BpgmL166P involves a dual mechanism that enhances the host's stress erythroid response to Plasmodium-driven RBC loss and simultaneously alters the intracellular milieu of the RBCs, including increased oxyhemoglobin and reduced energy metabolism, reducing Plasmodium maturation, and replication. Overall, our study highlights the importance of BPGM as a regulator of hemoglobin/oxyhemoglobin in malaria pathogenesis and suggests a new potential malaria therapeutic target.

Mechanisms of Natural Killer Cell Evasion Through Viral Adaptation.

The continuous interactions between host and pathogens during their coevolution have shaped both the immune system and the countermeasures used by pathogens. Natural killer (NK) cells are innate lymphocytes that are considered central players in the antiviral response. Not only do they express a variety of inhibitory and activating receptors to discriminate and eliminate target cells but they can also produce immunoregulatory cytokines to alert the immune system. Reciprocally, several unrelated viruses including cytomegalovirus, human immunodeficiency virus, influenza virus, and dengue virus have evolved a multitude of mechanisms to evade NK cell function, such as the targeting of pathways for NK cell receptors and their ligands, apoptosis, and cytokine-mediated signaling. The studies discussed in this article provide further insights into the antiviral function of NK cells and the pathways involved, their constituent proteins, and ways in which they could be manipulated for host benefit.

ZBTB7B (ThPOK) Is Required for Pathogenesis of Cerebral Malaria and Protection against Pulmonary Tuberculosis.

We used a genome-wide screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mice to identify genes in which recessive loss-of-function mutations protect against pathological neuroinflammation. We identified an R367Q mutation in the ZBTB7B (ThPOK) protein in which homozygosity causes protection against experimental cerebral malaria (ECM) caused by infection with Plasmodium berghei ANKA. Zbtb7bR367Q homozygous mice show a defect in the lymphoid compartment expressed as severe reduction in the number of single-positive CD4 T cells in the thymus and in the periphery, reduced brain infiltration of proinflammatory leukocytes in P. berghei ANKA-infected mice, and reduced production of proinflammatory cytokines by primary T cells ex vivo and in vivo Dampening of proinflammatory immune responses in Zbtb7bR367Q mice is concomitant to increased susceptibility to infection with avirulent (Mycobacterium bovis BCG) and virulent (Mycobacterium tuberculosis H37Rv) mycobacteria. The R367Q mutation maps to the first DNA-binding zinc finger domain of ThPOK and causes loss of base contact by R367 in the major groove of the DNA, which is predicted to impair DNA binding. Global immunoprecipitation of ThPOK-containing chromatin complexes coupled to DNA sequencing (ChIP-seq) identified transcriptional networks and candidate genes likely to play key roles in CD4+ CD8+ T cell development and in the expression of lineage-specific functions of these cells. This study highlights ThPOK as a global regulator of immune function in which alterations may affect normal responses to infectious and inflammatory stimuli.

CYRI/FAM49B negatively regulates RAC1-driven cytoskeletal remodelling and protects against bacterial infection.

Salmonella presents a global public health concern. Central to Salmonella pathogenicity is an ability to subvert host defences through strategically targeting host proteins implicated in restricting infection. Therefore, to gain insight into the host-pathogen interactions governing Salmonella infection, we performed an in vivo genome-wide mutagenesis screen to uncover key host defence proteins. This revealed an uncharacterized role of CYRI (FAM49B) in conferring host resistance to Salmonella infection. We show that CYRI binds to the small GTPase RAC1 through a conserved domain present in CYFIP proteins, which are known RAC1 effectors that stimulate actin polymerization. However, unlike CYFIP proteins, CYRI negatively regulates RAC1 signalling, thereby attenuating processes such as macropinocytosis, phagocytosis and cell migration. This enables CYRI to counteract Salmonella at various stages of infection, including bacterial entry into non-phagocytic and phagocytic cells as well as phagocyte-mediated bacterial dissemination. Intriguingly, to dampen its effects, the bacterial effector SopE, a RAC1 activator, selectively targets CYRI following infection. Together, this outlines an intricate host-pathogen signalling interplay that is crucial for determining bacterial fate. Notably, our study also outlines a role for CYRI in restricting infection mediated by Mycobacterium tuberculosis and Listeria monocytogenes. This provides evidence implicating CYRI cellular functions in host defence beyond Salmonella infection.

The complex of MCMV proteins and MHC class I evades NK cell control and drives the evolution of virus-specific activating Ly49 receptors.

CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and demonstrates how this forced the evolution of virus-specific activating MHC I-restricted Ly49 receptors.

Rel-Dependent Immune and Central Nervous System Mechanisms Control Viral Replication and Inflammation during Mouse Herpes Simplex Encephalitis.

Herpes simplex encephalitis (HSE), caused by HSV type 1 (HSV-1) infection, is an acute neuroinflammatory condition of the CNS and remains the most common type of sporadic viral encephalitis worldwide. Studies in humans have shown that susceptibility to HSE depends in part on the genetic make-up of the host, with deleterious mutations in the TLR3/type I IFN axis underlying some cases of childhood HSE. Using an in vivo chemical mutagenesis screen for HSV-1 susceptibility in mice, we identified a susceptible pedigree carrying a causal truncating mutation in the Rel gene (RelC307X ), encoding for the NF-κB transcription factor subunit c-Rel. Like Myd88-/- and Irf3-/- mice, RelC307X mice were susceptible to intranasal HSV-1 infection. Reciprocal bone marrow transfers into lethally irradiated hosts suggested that defects in both hematopoietic and CNS-resident cellular compartments contributed together to HSE susceptibility in RelC307X mice. Although the RelC307X mutation maintained cell-intrinsic antiviral control, it drove increased apoptotic cell death in infected fibroblasts. Moreover, reduced numbers of CD4+CD25+Foxp3+ T regulatory cells, and dysregulated NK cell and CD4+ effector T cell responses in infected RelC307X animals, indicated that protective immunity was also compromised in these mice. In the CNS, moribund RelC307X mice failed to control HSV-1 viral replication in the brainstem and cerebellum, triggering cell death and elevated expression of Ccl2, Il6, and Mmp8 characteristic of HSE neuroinflammation and pathology. In summary, our work implicates c-Rel in both CNS-resident cell survival and lymphocyte responses to HSV-1 infection and as a novel cause of HSE disease susceptibility in mice.

Insights into the pathogenesis of herpes simplex encephalitis from mouse models.

A majority of the world population is infected with herpes simplex viruses (HSV; human herpesvirus types 1 and 2). These viruses, perhaps best known for their manifestation in the genital or oral mucosa, can also cause herpes simplex encephalitis, a severe and often fatal disease of the central nervous system. Antiviral therapies for HSV are only partially effective since the virus can establish latent infections in neurons, and severe pathological sequelae in the brain are common. A better understanding of disease pathogenesis is required to develop new strategies against herpes simplex encephalitis, including the precise viral and host genetic determinants that promote virus invasion into the central nervous system and its associated immunopathology. Here we review the current understanding of herpes simplex encephalitis from the host genome perspective, which has been illuminated by groundbreaking work on rare herpes simplex encephalitis patients together with mechanistic insight from single-gene mouse models of disease. A complex picture has emerged, whereby innate type I interferon-mediated antiviral signaling is a central pathway to control viral replication, and the regulation of immunopathology and the balance between apoptosis and autophagy are critical to disease severity in the central nervous system. The lessons learned from mouse studies inform us on fundamental defense mechanisms at the interface of host-pathogen interactions within the central nervous system, as well as possible rationales for intervention against infections from severe neuropathogenic viruses.

The mitochondrial protease HtrA2 restricts the NLRP3 and AIM2 inflammasomes.

Activation of the inflammasome pathway is crucial for effective intracellular host defense. The mitochondrial network plays an important role in inflammasome regulation but the mechanisms linking mitochondrial homeostasis to attenuation of inflammasome activation are not fully understood. Here, we report that the Parkinson's disease-associated mitochondrial serine protease HtrA2 restricts the activation of ASC-dependent NLRP3 and AIM2 inflammasomes, in a protease activity-dependent manner. Consistently, disruption of the protease activity of HtrA2 results in exacerbated NLRP3 and AIM2 inflammasome responses in macrophages ex vivo and systemically in vivo. Mechanistically, we show that the HtrA2 protease activity regulates autophagy and controls the magnitude and duration of inflammasome signaling by preventing prolonged accumulation of the inflammasome adaptor ASC. Our findings identify HtrA2 as a non-redundant mitochondrial quality control effector that keeps NLRP3 and AIM2 inflammasomes in check.

Discovery of Variants Underlying Host Susceptibility to Virus Infection Using Whole-Exome Sequencing.

The clinical course of any viral infection greatly differs in individuals. This variation results from various viral, host, and environmental factors. The identification of host genetic factors influencing inter-individual variation in susceptibility to several pathogenic viruses has tremendously increased our understanding of the mechanisms and pathways required for immunity. Next-generation sequencing of whole exomes represents a powerful tool in biomedical research. In this chapter, we briefly introduce whole-exome sequencing in the context of genetic approaches to identify host susceptibility genes to viral infections. We then describe general aspects of the workflow for whole-exome sequence analysis together with the tools and online resources that can be used to identify and annotate variant calls, and then prioritize them for their potential association to phenotypes of interest.

Mouse Chromosome 4 Is Associated with the Baseline and Allergic IgE Phenotypes.

Regulation of IgE concentration in the blood is a complex trait, with high concentrations associated with parasitic infections as well as allergic diseases. A/J strain mice have significantly higher plasma concentrations of IgE, both at baseline and after ovalbumin antigen exposure, when compared to C57BL/6J strain mice. Our objective was to determine the genomic regions associated with this difference in phenotype. To achieve this, we used a panel of recombinant congenic strains (RCS) derived from A/J and C57BL/6J strains. We measured IgE in the RCS panel at baseline and following allergen exposure. Using marker by marker analysis of the RCS genotype and phenotype data, we identified multiple regions associated with the IgE phenotype. A single region was identified to be associated with baseline IgE level, while multiple regions wereassociated with the phenotype after allergen exposure. The most significant region was found on Chromosome 4, from 81.46 to 86.17 Mbp. Chromosome 4 substitution strain mice had significantly higher concentration of IgE than their background parental strain mice, C57BL/6J. Our data presents multiple candidate regions associated with plasma IgE concentration at baseline and following allergen exposure, with the most significant one located on Chromosome 4.

USP15 regulates type I interferon response and is required for pathogenesis of neuroinflammation.

Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.