The 50 most-cited articles in The Journal of Immunology, 2021

Lymph node stromal cells coordinate the adaptive immune response in secondary lymphoid organs, providing both a structural matrix and soluble factors that regulate survival and migration of immune cells, ultimately promoting Ag encounter. In several inflamed tissues, resident fibroblasts can acquire lymphoid-stroma properties and drive the formation of ectopic aggregates of immune cells, named tertiary lymphoid structures (TLSs). Mature TLSs are functional sites for the development of adaptive responses and, consequently, when present, can have an impact in both autoimmunity and cancer conditions. In this review, we go over recent findings concerning both lymph node stromal cells and TLSs function and formation and further describe what is currently known about their role in disease, particularly their potential in tolerance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

African swine fever virus (ASFV) is a devastating infectious disease in pigs, severely threatening the global pig industry. To efficiently infect animals, ASFV must evade or inhibit fundamental elements of the innate immune system, namely the type I IFN response. In this study, we identified that ASFV MGF-505-7R protein exerts a negative regulatory effect on STING-dependent antiviral responses. MGF-505-7R interacted with STING and inhibited the cGAS-STING signaling pathway at STING level. MGF-505-7R overexpression either degraded STING or STING expression was reduced in ASFV-infected cells via autophagy, whereas STING expression was elevated in MGF-505-7R-deficient ASFV-infected cells. We further found that MGF-505-7R promoted the expression of the autophagy-related protein ULK1 to degrade STING, whereas ULK1 was elevated in MGF-505-7R-deficient ASFV-infected cells. Moreover, MGF-505-7R-deficient ASFV induced more IFN-β production than wild-type ASFV and was attenuated in replication compared with wild-type ASFV. The replicative ability of MGF-505-7R-deficient ASFV was also…

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with…

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune…

Abstract Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16–25.91 μM for human ASAH2 and 18.6–30.2 μM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione…

Abstract Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from Pseudomonas aeruginosa infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow–derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen…

Abstract Gut microbiota is increasingly linked to the development of various pulmonary diseases through a gut–lung axis. However, the mechanisms by which gut commensal microbes impact trafficking and functional transition of immune cells remain largely unknown. Using integrated microbiota dysbiosis approaches, we uncover that the gut microbiota directs the migration of group 2 innate lymphoid cells (ILC2s) from the gut to the lung through a gut–lung axis. We identify Proteobacteria as a critical species in the gut microbiome to facilitate natural ILC2 migration, and increased Proteobacteria induces IL-33 production. Mechanistically, IL-33–CXCL16 signaling promotes the natural ILC2 accumulation in the lung, whereas IL-25–CCL25 signals augment inflammatory ILC2 accumulation in the intestines upon abdominal infection, parabiosis, and cecum ligation and puncture in mice. We reveal that these two types of ILC2s play critical but distinct roles in regulating inflammation, leading to balanced host defense against infection. Overall results delineate that Proteobacteria in gut microbiota…

Abstract It has been well established that the presence of diabetes is accompanied by a chronic inflammatory state promoting various diabetes-associated complications. One potential driver of this enhanced inflammatory state in patients with diabetes is hyperglycemia. Even after blood glucose control is achieved, diabetes-associated complications persist, suggesting the presence of a “hyperglycemic memory.” Innate immune cells, critically involved in various complications associated with diabetes, can build nonspecific, immunological memory (trained immunity) via epigenetic regulation. We examine the potential involvement of hyperglycemia-induced trained immunity in promoting inflammation. Our results show that hyperglycemia induces a trained phenotype in vivo in mice and in vitro in human monocytes, representative by an increased TNF-α secretion after ex vivo stimulation with LPS. These effects were largely mediated by epigenetic changes controlled by the mixed lineage leukemia (MLL) family because treatment with the MLL inhibitor menin-MLL during the process of trained immunity…

ABSTRACT Over the last two decades, there have been three deadly human outbreaks of Coronaviruses (CoVs) caused by emerging zoonotic CoVs: SARS-CoV, MERS-CoV, and the latest highly transmissible and deadly SARS-CoV-2, which has caused the current COVID-19 global pandemic. All three deadly CoVs originated from bats, the natural hosts, and transmitted to humans via various intermediate animal reservoirs. Because there is currently no universal pan-Coronavirus vaccine available, two worst-case scenarios remain highly possible: (1) SARS-CoV-2 mutates and transforms into a seasonal “flu-like” global pandemic; and/or (2) Other global COVID-like pandemics will emerge in the coming years, caused by yet another spillover of an unknown zoonotic bat-derived SARS-like Coronavirus (SL-CoV) into an unvaccinated human population. Determining the antigen and epitope landscapes that are conserved among human and animal Coronaviruses as well as the repertoire, phenotype and function of B cells and CD4 + and CD8 + T cells that correlate with resistance seen in asymptomatic COVID-19 patients should…

African swine fever is a severe animal infectious disease caused by African swine fever virus (ASFV), and the morbidity and mortality associated with virulent ASFV isolates are as high as 100%. Previous studies showed that the ability of ASFV to antagonize IFN production is closely related to its pathogenicity. Here, we report that ASFV HLJ/18 infection induced low levels of type I IFN and inhibited cGMP-AMP-induced type I IFN production in porcine alveolar macrophages that were isolated from specific pathogen-free Landrace piglets. Subsequently, an unbiased screen was performed to screen the ASFV genes with inhibitory effects on the type I IFN production. ASFV pI215L, a viral E2 ubiquitin-conjugating enzyme, was identified as one of the strongest inhibitory effectors on the production of type I IFN. Knockdown of pI215L expression inhibited ASFV replication and enhanced IFN-β production. However, inhibition of type I IFN production by pI215L was independent of its E2 enzyme activity. Furthermore, we found that pI215L inhibited type I IFN production and K63-linked polyubiquitination…

-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG-Fab regions, allowing neighboring IgG-Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1-Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1-Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1-Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the…

Abstract Short-chain fatty acids, such as butyrate, are major gut microbial metabolites that are beneficial for gastrointestinal health. Clostridium butyricum MIYAIRI588 (CBM588) is a bacterium that produces a robust amount of butyrate and therefore has been used as a live biotherapeutic probiotic in clinical settings. Clostridioides difficile causes life-threatening diarrhea and colitis. The gut resident microbiota plays a critical role in the prevention of C. difficile infection (CDI), as the disruption of the healthy microbiota by antibiotics greatly increases the risk for CDI. We report that CBM588 treatment in mice significantly improved clinical symptoms associated with CDI and increased the number of neutrophils and Th1 and Th17 cells in the colonic lamina propria in the early phase of CDI. The protective effect of CBM588 was abolished when neutrophils, IFN-γ, or IL-17A were depleted, suggesting that induction of the immune reactants is required to elicit the protective effect of the probiotic. The administration of tributyrin, which elevates the concentration of butyrate in…

Abstract Human plasmacytoid dendritic cells (pDCs) play a vital role in modulating immune responses. They can produce massive amounts of type I IFNs in response to nucleic acids via TLRs, but they are also known to possess weak Ag-presenting properties inducing CD4+ T cell activation. Previous studies showed a cross-regulation between TNF-α and IFN-α, but many questions remain about the effect of TNF-α in regulating human pDCs. In this study, we showed that TNF-α significantly inhibited the secretion of IFN-α and TNF-α of TLR-stimulated pDCs. Instead, exogenous TNF-α promoted pDC maturation by upregulating costimulatory molecules and chemokine receptors such as CD80, CD86, HLA-DR, and CCR7. Additionally, RNA sequencing analysis showed that TNF-α inhibited IFN-α and TNF-α production by downregulating IRF7 and NF-κB pathways, while it promoted Ag processing and presentation pathways as well as T cell activation and differentiation. Indeed, TNF-α–treated pDCs induced in vitro higher CD4+ T cell proliferation and activation, enhancing the production of Th1 and Th17 cytokines. In…

Chimeric Ag receptor (CAR) T cell therapy has shown astonishing potency in treating a variety of hematological malignancies in recent years. Along with this lifesaving potential comes the life-threatening toxicities of cytokine release syndrome (CRS) and neurotoxicity. This work seeks to consolidate biomarker candidates with the potential to predict the severity of CRS and neurotoxicity in patients receiving CD19-targeted CAR T cell therapy. In this systematic review, 33 clinical trials were evaluated for biomarkers that can predict the severity of posttreatment CRS and neurotoxicity. CRS and neurotoxicity occurred in 73.4 and 37% of the reviewed patients, respectively. Identified biomarker candidates included tumor burden, platelet count, C-reactive protein, ferritin, IFN-γ, IL-2, IL-6, IL-8, IL-10, IL-15, and TGF-β. Combinatorial algorithms based on cytokine levels and clinical parameters show excellent promise in predicting CAR-T-cell-therapy-associated toxicities, with improved accuracy over the component biomarkers.

Metabolic programming is integrally linked to immune cell function. Nowhere is this clearer than in the differentiation of macrophages. Proinflammatory M1 macrophages primarily use glycolysis as a rapid energy source but also to generate antimicrobial compounds, whereas alternatively activated M2 macrophages primarily rely on oxidative phosphorylation for the longevity required for proper wound healing. mTOR signaling has been demonstrated to be a key regulator of immune cell metabolism and function. mTORC2 signaling is required for the generation of M2 macrophages, whereas the role of mTORC1 signaling, a key regulator of glycolysis, has been controversial. By using genetic deletion of mTORC1 signaling in C57BL/6 mouse macrophages, we observed enhanced M1 macrophage function in vitro and in vivo. Surprisingly, this enhancement occurred despite a significant defect in M1 macrophage glycolytic metabolism. Mechanistically, enhanced M1 function occurred because of inhibition of the class III histone deacetylases the sirtuins, resulting in enhanced histone acetylation. Our findings…

Abstract Targeting interactions between α4β7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEβ7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. β1 and β7 integrin expression on circulating lymphocytes was similar across groups. TGF-β1 treatment induced expression of αE on both β7+ and β7− T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4β7 can become…

Abstract Upon Ag encounter, T cells can rapidly divide and form an effector population, which plays an important role in fighting acute infections. In humans, little is known about the molecular markers that distinguish such effector cells from other T cell populations. To address this, we investigated the molecular profile of T cells present in individuals with active tuberculosis (ATB), where we expect Ag encounter and expansion of effector cells to occur at higher frequency in contrast to Mycobacterium tuberculosis–sensitized healthy IGRA+ individuals. We found that the frequency of HLA-DR+ cells was increased in circulating CD4 T cells of ATB patients, and was dominantly expressed in M. tuberculosis Ag–specific CD4 T cells. We tested and confirmed that HLA-DR is a marker of recently divided CD4 T cells upon M. tuberculosis Ag exposure using an in vitro model examining the response of resting memory T cells from healthy IGRA+ to Ags. Thus, HLA-DR marks a CD4 T cell population that can be directly detected ex vivo in human peripheral blood, whose frequency is increased during ATB…

The discovery of IL-10 more than 30 years ago marked the beginning of our understanding of how cytokines regulate immune responses, based on cross-regulation between Th1 and Th2 cytokines. Although multiple cell types were shown to produce IL-10, its identity as a Th2 cytokine remained strong because it was rigidly associated with Th2 clones in mice, whereas both Th1 and Th2 clones could secrete IL-10 in humans. However, as new Th1/Th2 cell functionalities emerged, anti-inflammatory action of IL-10 gained more attention than its inhibitory effect on Th1 cells, which may occur as an indirect consequence of suppression of APCs. This notion is also supported by the discovery of regulatory T cells, whose suppressor functions involve the mediation of IL-10, among other molecules. From this perspective, we discuss the functionalities of IL-10 by highlighting important differences between mice and humans with an emphasis on the Th1 and Th2 paradigm.

Abstract The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare. This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria.…

Abstract Classic T cell subsets are defined by a small set of cell surface markers, while single cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq Clustered-Populations (scCPops) and cell surface marker-defined classic T cell subsets remain unclear. Here, we interrogated 6 bead-enriched T cell subsets with 62,235 single cell transcriptomes and re-grouped them into 9 scCPops. Bead-enriched CD4 Naïve and CD8 Naïve were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops including mucosal-associated invariant T cells and natural killer T cells. The multiple T cell subsets that form a single scCPop exhibited similar expression pattern, but not vice versa, indicating scCPops are much homogeneous cell populations with similar cell states. Interestingly, we discovered and named IFN hi T, a new T cell subpopulation that highly expressed Interferon Signaling Associated Genes (ISAGs). We further enriched IFN hi T by FACS sorting of BST2 for…

Abstract The IL-1 receptor antagonist, anakinra, may represent a therapeutic option for acute respiratory distress syndrome (ARDS) associated with coronavirus disease 2019 (COVID-19). In this study, COVID-19 ARDS patients admitted to the Azienda Socio Sanitaria Territoriale of Lecco, Italy, between March 5th to April 15th, 2020, and who had received anakinra off-label were retrospectively evaluated and compared with a cohort of matched controls who did not receive immunomodulatory treatment. The primary end point was survival at day 28. The population consisted of 112 patients (56 treated with anakinra and 56 controls). Survival at day 28 was obtained in 69 patients (61.6%) and was significantly higher in anakinra-treated patients than in the controls (75.0 versus 48.2%, p = 0.007). When stratified by continuous positive airway pressure support at baseline, anakinra-treated patients’ survival was also significant compared with the controls (p = 0.008). Univariate analysis identified anakinra usage (odds ratio, 3.2; 95% confidence interval, 1.47–7.17) as a significant survival…

Over the past decade, T cell immunotherapy has changed the face of cancer treatment, providing robust treatment options for several previously intractable cancers. Unfortunately, many epithelial tumors with high mortality rates respond poorly to immunotherapy, and an understanding of the key impediments is urgently required. Cancer-associated fibroblasts (CAFs) comprise the most frequent nonneoplastic cellular component in most solid tumors. Far from an inert scaffold, CAFs significantly influence tumor neogenesis, persistence, and metastasis and are emerging as a key player in immunotherapy resistance. In this review, we discuss the physical and chemical barriers that CAFs place between effector T cells and their tumor cell targets, and the therapies poised to target them.

Abstract Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation and cytokine storm. Exploring the immune-inflammatory characteristics of COVID-19 patients is essential to reveal pathogenesis and predict progression. In this study, COVID-19 patients showed decreased CD3+, CD4+, and CD8+ T cells but increased neutrophils in circulation, exhibiting upregulated neutrophil-to-lymphocyte and neutrophil-to-CD8+ T cell ratio. IL-6, TNF-α, IL-1β, IL-18, IL-12/IL-23p40, IL-10, Tim-3, IL-8, neutrophil extracellular trap–related proteinase 3, and S100A8/A9 were elevated, whereas IFN-γ and C-type lectin domain family 9 member A (clec9A) were decreased in COVID-19 patients compared with healthy controls. When compared with influenza patients, the expressions of TNF-α, IL-18, IL-12/IL-23p40, IL-8, S100A8/A9 and Tim-3 were significantly increased in critical COVID-19 patients, and carcinoembryonic Ag, IL-8, and S100A8/A9 could serve as clinically available hematologic indexes for identifying COVID-19 from influenza. Moreover, IL-6, IL-8, IL-1β, TNF-α, proteinase 3, and…

The thymus is an intricate primary lymphoid organ, wherein bone marrow-derived lymphoid progenitor cells are induced to develop into functionally competent T cells that express a diverse TCR repertoire, which is selected to allow for the recognition of foreign Ags while avoiding self-reactivity or autoimmunity. Thymus stromal cells, which can include all non-T lineage cells, such as thymic epithelial cells, endothelial cells, mesenchymal/fibroblast cells, dendritic cells, and B cells, provide signals that are essential for thymocyte development as well as for the homeostasis of the thymic stroma itself. In this brief review, we focus on the key roles played by thymic stromal cells during early stages of T cell development, such as promoting the homing of thymic-seeding progenitors, inducing T lineage differentiation, and supporting thymocyte survival and proliferation. We also discuss recent advances on the transcriptional regulation that govern thymic epithelial cell function as well as the cellular and molecular changes that are associated with thymic involution and regeneration.

Abstract Autosomal recessive (AR) STAT1 deficiency is a severe inborn error of immunity disrupting cellular responses to type I, II, and III IFNs, and IL-27, and conferring a predisposition to both viral and mycobacterial infections. We report the genetic, immunological, and clinical features of an international cohort of 32 patients from 20 kindreds: 24 patients with complete deficiency, and 8 patients with partial deficiency. Twenty-four patients suffered from mycobacterial disease (bacillus Calmette–Guérin = 13, environmental mycobacteria = 10, or both in 1 patient). Fifty-four severe viral episodes occurred in sixteen patients, mainly caused by Herpesviridae viruses. Attenuated live measles, mumps, and rubella and/or varicella zoster virus vaccines triggered severe reactions in the five patients with complete deficiency who were vaccinated. Seven patients developed features of hemophagocytic syndrome. Twenty-one patients died, and death was almost twice as likely in patients with complete STAT1 deficiency than in those with partial STAT1 deficiency. All but one of the eight…

Siglec-8 is an inhibitory receptor expressed on eosinophils and mast cells. In this study, we took advantage of a novel Siglec-8 transgenic mouse model to assess the impact of modulating IgE-dependent mast cell degranulation and anaphylaxis using a liposomal platform to display an allergen with or without a synthetic glycan ligand for Siglec-8 (Sig8L). The hypothesis is that recruitment of Siglec-8 to the IgE-FcεRI receptor complex will inhibit allergen-induced mast cell degranulation. Codisplay of both allergen and Sig8L on liposomes profoundly suppresses IgE-mediated degranulation of mouse bone marrow-derived mast cells or rat basophilic leukemia cells expressing Siglec-8. In contrast, liposomes displaying only Sig8L have no significant suppression of antigenic liposome-induced degranulation, demonstrating that the inhibitory activity by Siglec-8 occurs only when Ag and Sig8L are on the same particle. In mouse models of anaphylaxis, display of Sig8L on antigenic liposomes completely suppresses IgE-mediated anaphylaxis in transgenic mice with mast cells expressing Siglec-8 but has…

Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a serine protease that degrades damaged or misfolded mitochondrial proteins. CLPP-null mice exhibit growth retardation, deafness, and sterility, resembling human Perrault syndrome, but also display immune system alterations. However, the molecular mechanisms and signaling pathways underlying immunological changes in CLPP-null mice remain unclear. In this study, we report the steady-state activation of type I IFN signaling and antiviral gene expression in CLPP-deficient cells and tissues, resulting in marked resistance to RNA and DNA virus infection. Depletion of the cyclic GMP-AMP (cGAS)-stimulator of IFN genes (STING) DNA sensing pathway reduces steady-state IFN-I signaling and abrogates the broad antiviral phenotype of CLPP-null cells. Moreover, we report that CLPP deficiency leads to mitochondrial DNA (mtDNA) instability and packaging alterations. Pharmacological and genetic approaches to deplete mtDNA or inhibit cytosolic release markedly reduce antiviral gene expression, implicating mtDNA stress as the…

Abstract Understanding the function of SARS-CoV-2 Ag-specific T cells is crucial for the monitoring of antiviral immunity and vaccine design. Currently, both impaired and robust T cell immunity is described in COVID-19 patients. In this study, we explored and compared the effector functions of SARS-CoV-2–reactive T cells expressing coinhibitory receptors and examine the immunogenicity of SARS-CoV-2 S, M, and N peptide pools in regard to specific effector T cell responses, Th1/Th2/Th17, in COVID-19 patients. Analyzing a cohort of 108 COVID-19 patients with mild, moderate, and severe disease, we observed that coinhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, VISTA, CD39, CD160, 2B4, TIGIT, Gal-9, and NKG2A) were upregulated on both CD4+ and CD8+ T cells. Importantly, the expression of coinhibitory receptors on T cells recognizing SARS-CoV-2 peptide pools (M/N/S) was associated with increased frequencies of cytokine-producing T cells. Thus, our data refute the concept of pathological T cell exhaustion in COVID-19 patients. Despite interindividual variations in the T cell response to…

Abstract Memory T cells (Tmem) rapidly mount Ag-specific responses during pathogen reencounter. However, Tmem also respond to inflammatory cues in the absence of an activating TCR signal, a phenomenon termed bystander activation. Although bystander activation was first described over 20 years ago, the physiological relevance and the consequences of T cell bystander activation have only become more evident in recent years. In this review, we discuss the scenarios that trigger CD8 Tmem bystander activation including acute and chronic infections that are either systemic or localized, as well as evidence for bystander CD8 Tmem within tumors and following vaccination. We summarize the possible consequences of bystander activation for the T cell itself, the subsequent immune response, and the host. We highlight when T cell bystander activation appears to benefit or harm the host and briefly discuss our current knowledge gaps regarding regulatory signals that can control bystander activation.

Sepsis is a life-threatening organ dysfunction caused by a dysfunctional host response to infection. Neutrophils play a protective role by releasing antibacterial proteins or by phagocytizing bacteria. However, excess neutrophils can induce tissue damage. Recently, a novel intercellular communication pathway involving extracellular vesicles (EVs) has garnered considerable attention. However, whether EVs secreted by macrophages mediate neutrophil recruitment to infected sites has yet to be studied. In this study, we assessed the chemotactic effect of EVs isolated from mouse Raw264.7 macrophages on mouse neutrophils and found that CXCL2 was highly expressed in these EVs. By regulating CXCL2 in Raw264.7 macrophages, we found that CXCL2 on macrophage EVs recruited neutrophils in vitro and in vivo. The CXCL2 EVs activated the CXCR2/PKC/NOX4 pathway and induced tissue damage. This study provides information regarding the mechanisms underlying neutrophil recruitment to tissues and proposes innovative strategies and targets for the treatment of sepsis.

ABSTRACT COVID-19, caused by SARS-CoV-2, is an acute self-resolving disease in most of the patients, but some patients can develop a severe illness or even death. To characterize the host responses and identify potential biomarkers during disease progression, we performed a longitudinal transcriptome analysis for peripheral blood mononuclear cells (PBMCs) collected from 4 COVID-19 patients at 4 different time points from symptom onset to recovery. We found that PBMCs at different COVID-19 disease stages exhibited unique transcriptome characteristics. SARS-CoV-2 infection dysregulated innate immunity especially type I interferon response as well as the disturbed release of inflammatory cytokines and lipid mediators, and an aberrant increase of low-density neutrophils may cause tissue damage. Activation of cell death, exhaustion and migratory pathways may lead to the reduction of lymphocytes and dysfunction of adaptive immunity. COVID-19 induced hypoxia may exacerbate disorders in blood coagulation. Based on our analysis, we proposed a set of potential biomarkers for monitoring…

Abstract Autoimmune diseases develop when autoantigens activate previously quiescent self-reactive lymphocytes. Gene–gene interaction between certain HLA class I risk alleles and variants of the endoplasmic reticulum aminopeptidase ERAP1 controls the risk for common immune-mediated diseases, including psoriasis, ankylosing spondylitis, and Behçet disease. The functional mechanisms underlying this statistical association are unknown. In psoriasis, HLA-C*06:02 mediates an autoimmune response against melanocytes by autoantigen presentation. Using various genetically modified cell lines together with an autoreactive psoriatic TCR in a TCR activation assay, we demonstrate in this study that in psoriasis, ERAP1 generates the causative melanocyte autoantigen through trimming N-terminal elongated peptide precursors to the appropriate length for presentation by HLA-C*06:02. An ERAP1 risk haplotype for psoriasis produced the autoantigen much more efficiently and increased HLA-C expression and stimulation of the psoriatic TCR by melanocytes significantly more than a protective haplotype.…

As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ Mϕ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ Mϕ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ Mϕ and…

The signal regulatory protein α (SIRPα)/CD47 axis has emerged as an important innate immune checkpoint that enables cancer cell escape from macrophage phagocytosis. SIRPα expression is limited to macrophages, dendritic cells, and neutrophils-cells enriched in the tumor microenvironment. In this study, we present novel anti-SIRP Abs, SIRP-1 and SIRP-2, as an approach to targeting the SIRPα/CD47 axis. Both SIRP-1 and SIRP-2 bind human macrophage SIRPα variants 1 and 2, the most common variants in the human population. SIRP-1 and SIRP-2 are differentiated among reported anti-SIRP Abs in that they induce phagocytosis of solid and hematologic tumor cell lines by human monocyte-derived macrophages as single agents. We demonstrate that SIRP-1 and SIRP-2 disrupt SIRPα/CD47 interaction by two distinct mechanisms: SIRP-1 directly blocks SIRPα/CD47 and induces internalization of SIRPα/Ab complexes that reduce macrophage SIRPα surface levels and SIRP-2 acts via disruption of higher-order SIRPα structures on macrophages. Both SIRP-1 and SIRP-2 engage FcγRII, which is required for single-agent…

Abstract Seneca Valley virus (SVV), a newly emerging virus belonging to the Picornaviridae family, has caused vesicular disease in the swine industry. However, the molecular mechanism of viral pathogenesis remains poorly understood. This study revealed that SVV infection could induce pyroptosis in SK6 cells in a caspase-dependent and -independent manner. SVV may inhibit caspase-1 activation at late infection because of 3Cpro cleavage of NLRP3, which counteracted pyroptosis activation. Further study showed that 3Cpro targeted porcine gasdermin D (pGSDMD) for cleavage through its protease activity. 3Cpro cleaved porcine GSDMD (pGSDMD) at two sites, glutamine 193 (Q193) and glutamine 277 (Q277), and Q277 was close to the caspase-1–induced pGSDMD cleavage site. pGSDMD1–277 triggered cell death, which was similar to N-terminal fragment produced by caspase-1 cleavage of pGSDMD, and other fragments exhibited no significant inhibitory effects on cellular activity. Ectopic expression of pGSDMD converted 3Cpro-induced apoptosis to pyroptosis in 293T cells. Interestingly, 3Cpro did not cleave…

Abstract Ag-specific immunotherapy is a long-term goal for the treatment of autoimmune diseases; however developing a means of therapeutically targeting autoimmune T cells in an Ag-specific manner has been difficult. Through the engineering of an HLA-DR1 chimeric Ag receptor (CAR), we have produced CD8+ CAR T cells that target CD4+ T cells in an Ag-specific manner and tested their ability to inhibit the development of autoimmune arthritis in a mouse model. The DR1 CAR molecule was engineered to contain CD3ζ activation and CD28 signaling domains and a covalently linked autoantigenic peptide from type II collagen (CII; DR1-CII) to provide specificity for targeting the autoimmune T cells. Stimulation of the DR1-CII CAR T cells by an anti-DR Ab induced cytokine production, indicating that the DR1-CAR functions as a chimeric molecule. In vitro CTL assays using cloned CD4+ T cells as target cells demonstrated that the DR1-CII CAR T cells efficiently recognize and kill CD4+ T cells that are specific for the CII autoantigen. The CTL function was highly specific, as no killing was observed…

Most of the variation in outcome following severe traumatic brain injury (TBI) remains unexplained by currently recognized prognostic factors. Neuroinflammation may account for some of this difference. We hypothesized that TBI generated variable autoantibody responses between individuals that would contribute to outcome. We developed a custom protein microarray to detect autoantibodies to both CNS and systemic Ags in serum from the acute-phase (the first 7 d), late (6-12 mo), and long-term (6-13 y) intervals after TBI in human patients. We identified two distinct patterns of immune response to TBI. The first was a broad response to the majority of Ags tested, predominantly IgM mediated in the acute phase, then IgG dominant at late and long-term time points. The second was responses to specific Ags, most frequently myelin-associated glycopeptide (MAG), which persisted for several months post-TBI but then subsequently resolved. Exploratory analyses suggested that patients with a greater acute IgM response experienced worse outcomes than predicted from current known risk factors,…

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The…

IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ…

Stromal cells have for a long time been viewed as structural cells that support distinct compartments within lymphoid tissues and little more. Instead, an active cross-talk between endothelial and fibroblastic stromal cells drives the maturation of lymphoid niches, a relationship that is recapitulated during lymph node organogenesis, steady-state conditions, and following inflammation. In this review, we go over recent advances in genetic models and high-resolution transcriptomic analyses that have propelled the finer resolution of the stromal cell infrastructure of lymph nodes, revealing that the distinct subsets are strategically positioned to deliver a catered mixture of niche factors to interacting immune cell populations. Moreover, we discuss how changes in the activation state of poised stromal cell-underpinned niches rather than on-demand differentiation of new stromal cell subsets govern the efficient interaction of Ag, APC, and cognate B and T lymphocytes during adaptive immune responses.

Abstract Expression and activity of serum- and glucocorticoid-inducible kinase 1 (SGK1) are associated with many metabolic and inflammatory diseases. In this study, we report that SGK1 promotes alternative macrophage polarization and restrains inflammation in the infectious milieu of the gingiva. Inhibition of SGK1 expression or activity enhances characteristics of classically activated (M1) macrophages by directly activating the transcription of genes encoding iNOS, IL-12P40, TNF-α, and IL-6 and repressing IL-10 at message and protein levels. Moreover, SGK1 inhibition robustly reduces the expression of alternatively activated (M2) macrophage molecular markers, including arginase-1, Ym-1, Fizz1, and Mgl-1. These results were confirmed by multiple gain- and loss-of-function approaches, including small interfering RNA, a plasmid encoding SGK1, and LysM-Cre–mediated sgk1 gene knockout. Further mechanistic analysis showed that SGK1 deficiency decreases STAT3 but increases FoxO1 expression in macrophages under M2 or M1 macrophage–priming conditions, respectively. Combined with decreased…

Abstract The major role of chemokines is to act as a chemoattractant to guide the migration of immune cells to the infectious sites. In the current study, we found that CiCXCL20a, a teleost-specific chemokine from grass carp (Ctenopharyngodon idella), demonstrates broad-spectrum, potent, direct bactericidal activity and immunomodulatory functions to bacterial infections, apart from the chemotaxis. CiCXCL20a kills bacteria by binding, mainly targeting acid lipids, perforating bacterial membrane, resulting in bacterial cytoplasm leakage and death. CiCXCL20a aggregates and neutralizes LPS, agglutinates Gram-negative bacteria, and binds to peptidoglycan and Gram-positive bacteria, but not agglutinate them. All the complexes may be phagocytized and cleared away. CiCXCL20a chemoattracts leukocytes, facilitates phagocytosis of myeloid leukocytes, not lymphoid leukocytes, and enhances the bacteria-killing ability in leukocytes. We further identified its receptor CiCXCR3.1b1. Furthermore, we investigated the physiological roles of CiCXCL20a against Aeromonas hydrophila infection in vivo. The…

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic porcine enteropathogenic coronavirus causing severe enteritis and lethal watery diarrhea in piglets. PEDV infection suppresses the synthesis of type I IFN, and multiple viral proteins of PEDV have been shown to target the adaptors of innate immune pathways to inhibit type I IFN production. In this study, we identified PEDV membrane (M) protein as a new antagonist of type I IFN production in both human embryonic kidney HEK293T cells and porcine kidney PK-15 cells and determined the antagonistic mechanism used by M protein to target IFN regulatory factor 7 (IRF7), an important regulator of type I IFN production. IRF7 is phosphorylated and activated by TBK1 and IKKε in response to viral infection. We found that PEDV M protein interacted with the inhibitory domain of IRF7 and significantly suppressed TBK1/IKKε-induced IRF7 phosphorylation and dimerization of IRF7, leading to the decreased expression of type I IFN, although it did not affect the interaction between TBK1/IKKε and IRF7. As expected, overexpression of M protein…

Abstract Radiation is associated with tissue damage and increased risk of atherosclerosis but there are currently no treatments and a very limited mechanistic understanding of how radiation impacts tissue repair mechanisms. We uncovered that radiation significantly delayed temporal resolution programs that was associated with decreased efferocytosis in vivo . Resolvin D1 (RvD1), a known pro-resolving ligand, promoted swift resolution and restored efferocytosis in sub-lethally irradiated mice. Irradiated macrophages exhibited several features of senescence, including increased expression of p16 INK4A and p21, heightened levels of SA-β-gal, COX-2, and oxidative stress (OS) in vitro , and when transferred to mice exacerbated inflammation in vivo . Mechanistically, heightened OS in senescent macrophages led to impairment in their ability to carry out efficient efferocytosis and treatment with RvD1 reduced OS and improved efferocytosis. Sub-lethally irradiated Ldlr -/- mice exhibited increased plaque necrosis and p16 INK4A cells compared with non-irradiated controls and treatment with…

The trillions of bacteria that constitutively colonize the human gut collectively generate thousands of unique small molecules. These microbial metabolites can accumulate both locally and systemically and potentially influence nearly all aspects of mammalian biology, including immunity, metabolism, and even mood and behavior. In this review, we briefly summarize recent work identifying bioactive microbiota metabolites, the means through which they are synthesized, and their effects on host physiology. Rather than offering an exhaustive list of all known bioactive microbial small molecules, we select a few examples from each key class of metabolites to illustrate the diverse impacts of microbiota-derived compounds on the host. In addition, we attempt to address the microbial logic behind specific biotransformations. Finally, we outline current and emerging strategies for identifying previously undiscovered bioactive microbiota metabolites that may shape human health and disease.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes that hydrolyze nucleotides such as ATP, UTP, ADP, and UDP to monophosphates derivates such as AMP and UMP. The NTPDase family consists of eight enzymes, of which NTPDases 1, 2, 3, and 8 are expressed on cell membranes thereby hydrolyzing extracellular nucleotides. Cell membrane NTPDases are expressed in all tissues, in which they regulate essential physiological tissue functions such as development, blood flow, hormone secretion, and neurotransmitter release. They do so by modulating nucleotide-mediated purinergic signaling through P2 purinergic receptors. NTPDases 1, 2, 3, and 8 also play a key role during infection, inflammation, injury, and cancer. Under these conditions, NTPDases can contribute and control the pathophysiology of infectious, inflammatory diseases and cancer. In this review, we discuss the role of NTPDases, focusing on the less understood NTPDases 2-8, in regulating inflammation and immunity during infectious, inflammatory diseases, and cancer.

Abstract Excessive release of heme from red blood cells is a key pathophysiological feature of several disease states including bacterial sepsis, malaria, and sickle cell disease. This hemolysis results in an increased level of free heme that has been implicated in the inflammatory activation of monocytes, macrophages, and endothelium. Here, we show that extracellular heme engages the human inflammatory caspases, caspase-1, caspase-4, and caspase-5, resulting in the release of IL-1β. Heme-induced IL-1β release was further increased in macrophages from patients with sickle cell disease. In human primary macrophages, heme activated caspase-1 in an inflammasome-dependent manner, but heme-induced activation of caspase-4 and caspase-5 was independent of canonical inflammasomes. Furthermore, we show that both caspase-4, and caspase-5 are essential for heme-induced IL-1β release, while caspase-4 is the primary contributor to heme-induced cell death. Together, we have identified that extracellular heme acts as a damage associated molecular pattern (DAMP) that can engage canonical and…

Abstract The formation of neutrophil extracellular trap (NET) is a critical host defense when neutrophils migrate to infection sites. Pyroptosis is a newly identified programmed cell death, which is tightly regulated by inflammasome activation. However, the mechanism of pyroptotic signaling participating in NET production remains to be elucidated. In this study, the zebrafish larvae otic vesicle microinjection model was used to infect larvae with hemolysin-overexpressing Edwardsiella piscicida (EthA+), and a rapid migration of neutrophils to infection sites was observed. Intriguingly, EthA+ infection effectively induced significant neutrophil membrane rupture in vivo, which was dependent on caspase-B (caspy2) and gasdermin Eb (GSDMEb) but not caspase-A or gasdermin Ea. Specifically, the EthA+ E. piscicida infection induced pyroptosis along with NETosis in vitro, and depletion of either caspy2 or GSDMEb impaired NET formation in vivo. Consequently, inhibition of the caspy2–GSDMEb axis-gated NETosis impaired bacterial clearance in vivo. Altogether, these data provide evidence that…

Abstract The lungs harbor multiple resident microbial communities, otherwise known as the microbiota. There is an emerging interest in deciphering whether the pulmonary microbiota modulate local immunity, and whether this knowledge could shed light on mechanisms operating in the response to respiratory pathogens. In this study, we investigate the capacity of a pulmonary Lactobacillus strain to modulate the lung T cell compartment and assess its prophylactic potential upon infection with Mycobacterium tuberculosis, the etiological agent of tuberculosis. In naive mice, we report that a Lactobacillus murinus (Lagilactobacillus murinus) strain (CNCM I-5314) increases the presence of lung Th17 cells and of a regulatory T cell (Treg) subset known as RORγt+ Tregs. In particular, intranasal but not intragastric administration of CNCM I-5314 increases the expansion of these lung leukocytes, suggesting a local rather than systemic effect. Resident Th17 and RORγt+ Tregs display an immunosuppressive phenotype that is accentuated by CNCM I-5314. Despite the well-known ability of M. tuberculosis…