The 50 most-cited articles in The Journal of Immunology, 2017

Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I molecules. Peptide binding to MHC molecules is the single most selective step in the Ag-presentation pathway. Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing number of MHC-presented peptides identified by mass spectrometry have been reported containing information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging the information from both data types. Large-scale benchmarking of the method demonstrates an increase in predictive performance compared with state-of-the-art methods when it comes to identification…

Macrophages become activated initiating innate immune responses. Depending on the signals, macrophages obtain an array of activation phenotypes, described by the broad terms of M1 or M2 phenotype. The PI3K/Akt/mTOR pathway mediates signals from multiple receptors including insulin receptors, pathogen-associated molecular pattern receptors, cytokine receptors, adipokine receptors, and hormones. As a result, the Akt pathway converges inflammatory and metabolic signals to regulate macrophage responses modulating their activation phenotype. Akt is a family of three serine-threonine kinases, Akt1, Akt2, and Akt3. Generation of mice lacking individual Akt, PI3K, or mTOR isoforms and utilization of RNA interference technology have revealed that Akt signaling pathway components have distinct and isoform-specific roles in macrophage biology and inflammatory disease regulation, by controlling inflammatory cytokines, miRNAs, and functions including phagocytosis, autophagy, and cell metabolism. Herein, we review the current knowledge on the role of the Akt signaling pathway in macrophages,…

Shohei Hori, Takashi Nomura, Shimon Sakaguchi; Pillars Article: Control of Regulatory T Cell Development by the Transcription Factor Foxp3. Science 2003. 2

Pathogenic human coronaviruses (CoVs), such as the severe acute respiratory syndrome (SARS)-CoV and the Middle East respiratory syndrome-CoV, cause acute respiratory illness. Epidemiological data from the 2002-2003 SARS epidemic and recent Middle East respiratory syndrome outbreak indicate that there may be sex-dependent differences in disease outcomes. To investigate these differences, we infected male and female mice of different age groups with SARS-CoV and analyzed their susceptibility to the infection. Our results showed that male mice were more susceptible to SARS-CoV infection compared with age-matched females. The degree of sex bias to SARS-CoV infection increased with advancing age, such that middle-aged mice showed much more pronounced differences compared with young mice. Enhanced susceptibility of male mice to SARS-CoV was associated with elevated virus titers, enhanced vascular leakage, and alveolar edema. These changes were accompanied by increased accumulation of inflammatory monocyte macrophages and neutrophils in the lungs of male mice, and depletion of inflammatory…

Commensal interactions between the enteric microbiota and distal intestine play important roles in regulating human health. Short-chain fatty acids (SCFAs), such as butyrate, produced through anaerobic microbial metabolism represent a major energy source for the host colonic epithelium and enhance epithelial barrier function through unclear mechanisms. Separate studies revealed that the epithelial anti-inflammatory IL-10 receptor α subunit (IL-10RA) is also important for barrier formation. Based on these findings, we examined if SCFAs promote epithelial barrier through IL-10RA-dependent mechanisms. Using human intestinal epithelial cells (IECs), we discovered that SCFAs, particularly butyrate, enhanced IEC barrier formation, induced IL-10RA mRNA, IL-10RA protein, and transactivation through activated Stat3 and HDAC inhibition. Loss and gain of IL-10RA expression directly correlates with IEC barrier formation and butyrate represses permeability-promoting claudin-2 tight-junction protein expression through an IL-10RA-dependent mechanism. Our findings provide a novel mechanism by which…

The healing of cutaneous wounds is dependent on the progression through distinct, yet overlapping phases of wound healing, including hemostasis, inflammation, proliferation, and resolution/remodeling. The failure of these phases to occur in a timely, progressive fashion promotes pathologic wound healing. The macrophage (MΦ) has been demonstrated to play a critical role in the inflammatory phase of tissue repair, where its dynamic plasticity allows this cell to mediate both tissue-destructive and -reparative functions. The ability to understand and control both the initiation and the resolution of inflammation is critical for treating pathologic wound healing. There are now a host of studies demonstrating that metabolic and epigenetic regulation of gene transcription can influence MΦ plasticity in wounds. In this review, we highlight the molecular and epigenetic factors that influence MΦ polarization in both physiologic and pathologic wound healing, with particular attention to diabetic wounds.

Charles D Mills, Kristi Kincaid, Jennifer M Alt, Michelle J Heilman, Annette M Hill; Pillars Article: M-1/M-2 Macrophages and the Th1/Th2 Paradigm. J. Immu

Since the pioneering work of Elie Metchnikoff and the discovery of cellular immunity, the phagocytic clearance of cellular debris has been considered an integral component of resolving inflammation and restoring function of damaged and infected tissues. We now know that the phagocytic clearance of dying cells (efferocytosis), particularly by macrophages and other immune phagocytes, has profound consequences on innate and adaptive immune responses in inflamed tissues. These immunomodulatory effects result from an array of molecular signaling events between macrophages, dying cells, and other tissue-resident cells. In recent years, many of these molecular pathways have been identified and studied in the context of tissue inflammation, helping us better understand the relationship between efferocytosis and inflammation. We review specific types of efferocytosis-related signals that can impact macrophage immune responses and discuss their relevance to inflammation-related diseases.

Stimulator of interferon genes (STING) was initially described as a sensor of intracellular bacterial and viral DNA and a promising adjuvant target in innate immune cells; more recently STING has also been shown to detect endogenous DNA and play a role in tumor immunity and autoimmune disease development. Thus far STING has been studied in macrophages and dendritic cells. In this study, to our knowledge we provide the first evidence of STING activation in T cells, in which STING agonists not only provoke type I IFN production and IFN-stimulated gene expression, mirroring the response of innate cells, but are also capable of activating cell stress and death pathways. Our results suggest a re-evaluation of STING agonist-based therapies may be necessary to identify the possible effects on the T cell compartment. Conversely, the effects of STING on T cells could potentially be harnessed for therapeutic applications.

The mammalian gastrointestinal tract and associated mucosal immune system harbor a large repertoire of metabolites of prokaryotic and eukaryotic origin that play important roles in eukaryotic development and physiology. These often bioactive small molecules originate from nutrition- and environmental-related sources, or are endogenously produced and modulated by the host and its microbiota. A complex network of interactions exists between the intestinal mucosal immune system and the microbiota. This intimate cross-talk may be driven by metabolite secretion and signaling, and features profound influences on host immunity and physiology, including the endocrine, metabolic, and nervous system function in health and disease. Alterations in microbiome-associated metabolite levels and activity are implicated in the pathogenesis of a growing number of illnesses. In this review we discuss the origin and influence of microbiome-modulated metabolites, with an emphasis on immune cell development and function. We further highlight the emerging data potentially implicating metabolite misbalance…

Abstract Substance P (SP) is an undecapeptide present in the CNS and the peripheral nervous system. SP released from the peripheral nerves exerts its biological and immunological activity via high-affinity neurokinin 1 receptor (NK1R). SP is also produced by immune cells and acts as an autocrine or paracrine fashion to regulate the function of immune cells. In addition to its proinflammatory role, SP and its metabolites in combination with insulin-like growth factor-1 are shown to promote the corneal epithelial wound healing. Recently, we showed an altered ocular surface homeostasis in unmanipulated NK1R−/− mice, suggesting the role of SP-NK1R signaling in ocular surface homeostasis under steady-state. This review summarizes the immunobiology of SP and its effect on immune cells and immunity to microbial infection. In addition, the effect of SP in inflammation, wound healing, and corneal epithelial homeostasis in the eye is discussed.

Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of…

Abstract GPBAR1 (TGR5 or M-BAR) is a G protein–coupled receptor for secondary bile acids that is highly expressed in monocytes/macrophages. In this study, we aimed to determine the role of GPBAR1 in mediating leukocyte trafficking in chemically induced models of colitis and investigate the therapeutic potential of BAR501, a small molecule agonist for GPBAR1. These studies demonstrated that GPBAR1 gene ablation enhanced the recruitment of classically activated macrophages in the colonic lamina propria and worsened the severity of inflammation. In contrast, GPBAR1 activation by BAR501 reversed intestinal inflammation in the trinitrobenzenesulfonic acid and oxazolone models by reducing the trafficking of Ly6C+ monocytes from blood to intestinal mucosa. Exposure to BAR501 shifted intestinal macrophages from a classically activated (CD11b+, CCR7+, F4/80−) to an alternatively activated (CD11b+, CCR7−, F4/80+) phenotype, reduced the expression of inflammatory genes (TNF-α, IFN-γ, IL-1β, IL-6, and CCL2 mRNAs), and attenuated the wasting syndrome and severity of colitis (≈70% reduction in…

Abstract Danger-associated molecular patterns derived from damaged or dying cells elicit inflammation and potentiate antitumor immune responses. In this article, we show that treatment of breast cancer cells with the antitumor agent topotecan (TPT), an inhibitor of topoisomerase I, induces danger-associated molecular pattern secretion that triggers dendritic cell (DC) activation and cytokine production. TPT administration inhibits tumor growth in tumor-bearing mice, which is accompanied by infiltration of activated DCs and CD8+ T cells. These effects are abrogated in mice lacking STING, an essential molecule in cytosolic DNA–mediated innate immune responses. Furthermore, TPT-treated cancer cells release exosomes that contain DNA that activate DCs via STING signaling. These findings suggest that a STING-dependent pathway drives antitumor immunity by responding to tumor cell–derived DNA.

Mouse models have enabled breakthroughs in our understanding of the immune system, but it has become increasingly popular to emphasize their shortcomings when translating observations to humans. This review provides a brief summary of mouse natural history, husbandry, and the pros and cons of pursuing basic research in mice versus humans. Opportunities are discussed for extending the predictive translational value of mouse research, with an emphasis on exploitation of a "dirty" mouse model that better mimics the diverse infectious history that is typical of most humans.

Immune tolerance is necessary to prevent the immune system from reacting against self, and thus to avoid the development of autoimmune diseases. In this review, we discuss key findings that position dendritic cells (DCs) as critical modulators of both thymic and peripheral immune tolerance. Although DCs are important for inducing both immunity and tolerance, increased autoimmunity associated with decreased DCs suggests their nonredundant role in tolerance induction. DC-mediated T cell immune tolerance is an active process that is influenced by genetic variants, environmental signals, as well as the nature of the specific DC subset presenting Ag to T cells. Answering the many open questions with regard to the role of DCs in immune tolerance could lead to the development of novel therapies for the prevention of autoimmune diseases.

Macrophages are abundant in almost all tissues of all multicellular organisms, estimated to total 1010 cells in adult humans. Macrophage function is essential for health, as strikingly demonstrated by the severe pathologies of macrophage-deficient animal models. Therefore, understanding the

Mucosal IgA or secretory IgA (SIgA) are structurally equipped to resist chemical degradation in the harsh environment of mucosal surfaces and enzymes of host or microbial origin. Production of SIgA is finely regulated, and distinct T-independent and T-dependent mechanisms orchestrate Ig α class switching and SIgA responses against commensal and pathogenic microbes. Most infectious pathogens enter the host via mucosal surfaces. To provide a first line of protection at these entry ports, vaccines are being developed to induce pathogen-specific SIgA in addition to systemic immunity achieved by injected vaccines. Mucosal or epicutaneous delivery of vaccines helps target the inductive sites for SIgA responses. The efficacy of such vaccines relies on the identification and/or engineering of vaccine adjuvants capable of supporting the development of SIgA alongside systemic immunity and delivery systems that improve vaccine delivery to the targeted anatomic sites and immune cells.

Influenza viral infections often lead to increased mortality in older people. However, the mechanisms by which aging impacts immunity to influenza lung infection remain unclear. We employed a murine model of influenza infection to identify these mechanisms. With aging, we found reduced numbers of alveolar macrophages, cells essential for lung homeostasis. We also determined that these macrophages are critical for influenza-induced mortality with aging. Furthermore, aging vastly alters the transcriptional profile and specifically downregulates cell cycling pathways in alveolar macrophages. Aging impairs the ability of alveolar macrophages to limit lung damage during influenza infection. Moreover, aging decreases alveolar macrophage phagocytosis of apoptotic neutrophils, downregulates the scavenging receptor CD204, and induces retention of neutrophils during influenza infection. Thus, aging induces defective phagocytosis by alveolar macrophages and increases lung damage. These findings indicate that therapies that enhance the function of alveolar macrophages may improve outcomes in…

Celiac disease is a human T cell-mediated autoimmune-like disorder caused by exposure to dietary gluten in genetically predisposed individuals. This review will discuss how CD4 T cell responses directed against an exogenous Ag can cause an autoreactive B cell response and participate in the licensing of intraepithelial lymphocytes to kill intestinal epithelial cells. Furthermore, this review will examine the mechanisms by which intraepithelial cytotoxic T cells mediate tissue destruction in celiac disease.

Abstract Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that focal sites of inflammation around dying adipocytes, so-called crown-like structures, exhibit a unique microenvironment for macrophage proliferation. Interestingly, locally proliferating macrophages were not classically activated (M1), but they exhibited a rather alternatively activated (M2) immune phenotype. In this study, we established organotypic cell cultures of AT explants to study the impact of cytokine treatment on local ATM proliferation, without the bias of early monocyte recruitment. We show that exposure of AT to Th2 cytokines, such as IL-4, IL-13, and GM-CSF, stimulates ATM proliferation, whereas Th1 cytokines, such as TNF-α, inhibit local ATM proliferation. Furthermore, AT from obese mice exhibits an increased sensitivity to IL-4 stimulation, indicated by an increased phosphorylation of STAT6. In line with this, gene expression of the IL-4 receptor (Il4ra) and its…

New attention to sexual dimorphism in normal mammalian physiology and disease has uncovered a previously unappreciated breadth of mechanisms by which females and males differentially exhibit quantitative phenotypes. Thus, in addition to the established modifying effects of hormones, which prenatally and postpubertally pattern cells and tissues in a sexually dimorphic fashion, sex differences are caused by extragonadal and dosage effects of genes encoded on sex chromosomes. Sex differences in immune responses, especially during autoimmunity, have been studied predominantly within the context of sex hormone effects. More recently, immune response genes have been localized to sex chromosomes themselves or found to be regulated by sex chromosome genes. Thus, understanding how sex impacts immunity requires the elucidation of complex interactions among sex hormones, sex chromosomes, and immune response genes. In this Brief Review, we discuss current knowledge and new insights into these intricate relationships in the context of viral infections.

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independently of gasdermin-D.

Cytosolic dsDNA activates the cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway to produce cytokines, including type I IFNs. The roles of many critical proteins, including NEMO, IKKβ, and TBK1, in this pathway are unclear because of the lack of an appropriate system to study. In this article, we report that lower FBS concentrations in culture medium conferred high sensitivities to dsDNA in otherwise unresponsive cells, whereas higher FBS levels abrogated this sensitivity. Based on this finding, we demonstrated genetically that NEMO was critically involved in the cGAS-STING pathway. Cytosolic DNA activated TRIM32 and TRIM56 to synthesize ubiquitin chains that bound NEMO and subsequently activated IKKβ. Activated IKKβ, but not IKKα, was required for TBK1 and NF-κB activation. In contrast, TBK1 was reciprocally required for NF-κB activation, probably by directly phosphorylating IKKβ. Thus, our findings identified a unique innate immune activation cascade in which TBK1-IKKβ formed a positive feedback loop to assure robust cytokine production during cGAS-STING…

Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex…

The existence of a neonatal window was first highlighted by epidemiological studies that revealed the particular importance of this early time in life for the susceptibility to immune-mediated diseases in humans. Recently, the first animal studies emerged that present examples of early-life exposure-triggered persisting immune events, allowing a detailed analysis of the factors that define this particular time period. The enteric microbiota and the innate and adaptive immune system represent prime candidates that impact on the pathogenesis of immune-mediated diseases and are known to reach a lasting homeostatic equilibrium following a dynamic priming period after birth. In this review, we outline the postnatal establishment of the microbiota and maturation of the innate and adaptive immune system and discuss examples of early-life exposure-triggered immune-mediated diseases that start to shed light on the critical importance of the early postnatal period for life-long immune homeostasis.

RNA-based vaccines have recently emerged as a promising alternative to the use of DNA-based and viral vector vaccines, in part because of the potential to simplify how vaccines are made and facilitate a rapid response to newly emerging infections. SAM vaccines are based on engineered self-amplifying mRNA (SAM) replicons encoding an Ag, and formulated with a synthetic delivery system, and they induce broad-based immune responses in preclinical animal models. In our study, in vivo imaging shows that after the immunization, SAM Ag expression has an initial gradual increase. Gene expression profiling in injection-site tissues from mice immunized with SAM-based vaccine revealed an early and robust induction of type I IFN and IFN-stimulated responses at the site of injection, concurrent with the preliminary reduced SAM Ag expression. This SAM vaccine-induced type I IFN response has the potential to provide an adjuvant effect on vaccine potency, or, conversely, it might establish a temporary state that limits the initial SAM-encoded Ag expression. To determine the role of the early type I…

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system.

Abstract Adoptive T cell therapies have achieved significant clinical responses, especially in hematopoietic cancers. Two types of receptor systems have been used to redirect the activity of T cells, normal heterodimeric TCRs or synthetic chimeric Ag receptors (CARs). TCRs recognize peptide-HLA complexes whereas CARs typically use an Ab-derived single-chain fragments variable that recognizes cancer-associated cell-surface Ags. Although both receptors mediate diverse effector functions, a quantitative comparison of the sensitivity and signaling capacity of TCRs and CARs has been limited due to their differences in affinities and ligands. In this study we describe their direct comparison by using TCRs that could be formatted either as conventional αβ heterodimers, or as single-chain fragments variable constructs linked to CD3ζ and CD28 signaling domains or to CD3ζ alone. Two high-affinity TCRs (KD values of ∼50 and 250 nM) against MART1/HLA-A2 or WT1/HLA-A2 were used, allowing MART1 or WT1 peptide titrations to easily assess the impact of Ag density. Although CARs were expressed at…

Regulatory signals provide negative input to immunological networks promoting resolution of acute and chronic inflammation. Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, displays broad anti-inflammatory and proresolving activities by targeting multiple immune cell types. Within the innate immune compartment, Gal-1 acts as a resolution-associated molecular pattern by counteracting the synthesis of proinflammatory cytokines, inhibiting neutrophil trafficking, targeting eosinophil migration and survival, and suppressing mast cell degranulation. Likewise, this lectin controls T cell and B cell compartments by modulating receptor clustering and signaling, thus serving as a negative-regulatory checkpoint that reprograms cellular activation, differentiation, and survival. In this review, we discuss the central role of Gal-1 in regulatory programs operating during acute inflammation, autoimmune diseases, allergic inflammation, pregnancy, cancer, and infection. Therapeutic strategies aimed at targeting Gal-1-glycan interactions will contribute…

Over the last decade, danger-associated molecular pattern molecules, or alarmins, have been recognized as signaling mediators of sterile inflammatory responses after trauma and injury. In contrast with the accepted passive release models suggested by the "danger hypothesis," it was recently shown that alarmins can also directly sense and report damage by signaling to the environment when released from live cells undergoing physiological stress, even without loss of subcellular compartmentalization. In this article, we review the involvement of alarmins such as IL-1α, IL-33, IL-16, and high-mobility group box 1 in cellular and physiological stress, and suggest a novel activity of these molecules as central initiators of sterile inflammation in response to nonlethal stress, a function we denote "stressorins." We highlight the role of posttranslational modifications of stressorins as key regulators of their activity and propose that targeted inhibition of stressorins or their modifiers could serve as attractive new anti-inflammatory treatments for a broad range of diseases.

Abstract Dimethyl fumarate (DMF; trade name Tecfidera) is an oral formulation of the fumaric acid ester that is Food and Drug Administration approved for treatment of relapsing-remitting multiple sclerosis. To better understand the therapeutic effects of Tecfidera and its rare side effect of progressive multifocal leukoencephalopathy, we conducted cross-sectional and longitudinal studies by immunophenotyping cells from peripheral blood (particularly T lymphocytes) derived from untreated and 4–6 and 18–26 mo Tecfidera-treated stable relapsing-remitting multiple sclerosis patients using multiparametric flow cytometry. The absolute numbers of CD4 and CD8 T cells were significantly decreased and the CD4/CD8 ratio was increased with DMF treatment. The proportions of both effector memory T cells and central memory T cells were reduced, whereas naive T cells increased in treated patients. T cell activation was reduced with DMF treatment, especially among effector memory T cells and effector memory RA T cells. Th subsets Th1 (CXCR3+), Th17 (CCR6+), and particularly those expressing both…

NLRP3 inflammasome activiation requires two sequential signals. The priming signal 1 from TLRs or cytokine receptors induces the transcription of NLRP3 and IL-1β, and concomitantly promotes transcription-independent activation of caspase-1. The activating signal 2 can be provided by microbial products or danger signals. In this study we found that TRAF6 is necessary for the nontranscriptional priming of NLRP3 inflammasome by TLR/IL-1R derived signals. Deficiency of TRAF6 specifically inhibited TLR/IL-1R priming-initiated caspase-1 cleavage, pyroptosis, and secretion of presynthesized IL-18. Mechanistically, TRAF6 promoted NLRP3 oligomerization as well as the interaction between NLRP3 and apoptosis-associated speck-like protein containing a CARD. Of note, the nontranscriptional priming via TRAF6 did not involve mitochondrial reactive oxygen species or the phosphorylation of Jnk, Erk, and Syk, whereas the ubiquitin E3 ligase activity of TRAF6 was required. Our findings thus extended cognition on the mechanism of NLRP3 inflammasome activation, and provided a novel target for…

Abstract Macrophages are polarized into functional classically activated and alternatively activated (M2) phenotypes depending on their microenvironment, and these cells play an important role in the immune system. M2-like polarization of tumor-associated macrophages (TAMs) is activated by various secretions from cancer cells; however, the interaction between cancer cells and TAMs is not well understood. Recent studies showed that cancer cell–derived extracellular vesicles (EVs) contribute to tumor development and modulation of the tumor microenvironment. In the current study, we investigated colorectal cancer–derived EVs containing miR-145 with respect to the polarization of TAMs. Colorectal cancer cells positively secreted miR-145 via EVs, which were taken up by macrophage-like cells. Interestingly, colorectal cancer–derived EVs polarized macrophage-like cells into the M2-like phenotype through the downregulation of histone deacetylase 11. An in vivo study showed that EV-treated macrophages caused significant enlargement of the tumor volumes. These findings suggest that colorectal…

Abstract Eosinophils are multifunctional cells of the innate immune system linked to allergic inflammation. Asthmatics were more likely to be hospitalized but less likely to suffer severe morbidity and mortality during the 2009 influenza pandemic. These epidemiologic findings were recapitulated in a mouse model of fungal asthma wherein infection during heightened allergic inflammation was protective against influenza A virus (IAV) infection and disease. Our goal was to delineate a mechanism(s) by which allergic asthma may alleviate influenza disease outcome, focused on the hypothesis that pulmonary eosinophilia linked with allergic respiratory disease is able to promote antiviral host defenses against the influenza virus. The transfer of eosinophils from the lungs of allergen-sensitized and challenged mice into influenza virus–infected mice resulted in reduced morbidity and viral burden, improved lung compliance, and increased CD8+ T cell numbers in the airways. In vitro assays with primary or bone marrow–derived eosinophils were used to determine eosinophil responses to the virus…

Allergic asthma is a chronic Th2 inflammation in the lungs that constricts the airways and presents as coughing and wheezing. Asthma mostly affects boys in childhood and women in adulthood, suggesting that shifts in sex hormones alter the course of the disease. Alveolar macrophages have emerged as major mediators of allergic lung inflammation in animal models as well as humans. Whether sex differences exist in macrophage polarization and the molecular mechanism(s) that drive differential responses are not well understood. We found that IL-4-stimulated bone marrow-derived and alveolar macrophages from female mice exhibited greater expression of M2 genes in vitro and after allergen challenge in vivo. Alveolar macrophages from female mice exhibited greater expression of the IL-4Rα and estrogen receptor (ER) α compared with macrophages from male mice following allergen challenge. An ERα-specific agonist enhanced IL-4-induced M2 gene expression in macrophages from both sexes, but more so in macrophages from female mice. Furthermore, IL-4-stimulated macrophages from female mice exhibited…

Activation of cells in the adaptive immune system is a highly orchestrated process dictated by multiples cues from the innate immune system. Although the fundamental principles of innate control of adaptive immunity are well established, it is not fully understood how innate cells integrate qualitative pathogenic information to generate tailored protective adaptive immune responses. In this review, we discuss complexities involved in the innate control of adaptive immunity that extend beyond TCR engagement, costimulation, and priming cytokine production but are critical for the generation of protective T cell immunity.

Abstract T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.

Abstract Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1β, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell-fate decisions in various inflammatory scenarios. In this study, we identify the G-protein–coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macrophages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis–induced bioactive IL-1β production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven…

Abstract Age-related alterations in immunity have been linked to increased incidence of infections and decreased responses to vaccines in the aging population. Human peripheral blood monocytes are known to promote Ag presentation and antiviral activities; however, the impact of aging on monocyte functions remains an open question. We present an in-depth global analysis examining the impact of aging on classical (CD14+CD16−), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16+) monocytes. Monocytes sorted from nonfrail healthy adults (21–40 y) and old (≥65 y) individuals were analyzed after stimulation with TLR4, TLR7/8, and retinoic acid–inducible gene I agonists. Our data showed that under nonstimulated conditions, monocyte subsets did not reveal significant age-related alternations; however, agonist stimulated-monocytes from adults and old subjects did show differences at the transcriptional and functional levels. These alternations in many immune-related transcripts and biological processes resulted in reduced production of IFN-α, IFN-γ, IL-1β, CCL20, and CCL8, and higher…

Abstract Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure, with high morbidity and mortality. The literature implies that autophagy is a crucial regulator of inflammation in sepsis. In this article, we report that autophagy-related protein 7 (Atg7) is involved in inflammasome activation in Pseudomonas aeruginosa abdominal infection. Following i.p. challenge with P. aeruginosa, atg7fl/fl mice showed impaired pathogen clearance, decreased survival, and widespread dissemination of bacteria into the blood and lung tissue compared with wild-type mice. The septic atg7fl/fl mice also exhibited elevated neutrophil infiltration and severe lung injury. Loss of Atg7 resulted in increased production of IL-1β and pyroptosis, consistent with enhanced inflammasome activation. Furthermore, we demonstrated that P. aeruginosa flagellin is a chief trigger of inflammasome activation in the sepsis model. Collectively, our results provide insight into innate immunity and inflammasome activation in sepsis.

Abstract Infection or inflammation of the skin recruits effector CD8+ T cells that enter the epidermis and form populations of long-lived tissue-resident memory T (TRM) cells. These skin TRM cells migrate within the constrained epidermal environment by extending multiple dynamic dendritic projections and squeezing between keratinocytes to survey the tissue for pathogens. In this study, we examined the signals required for this distinctive mode of T cell migration by inhibiting key cytoskeletal components and performing intravital two-photon microscopy to visualize TRM cell behavior. We found that TRM cell motility and dendrite formation required an intact actomyosin cytoskeleton and the Rho-associated coiled-coil containing kinases. We also identified an essential role for microtubules for maintaining skin TRM cell shape and cellular integrity. We reveal a role for pertussis toxin–sensitive signaling for TRM cell dendritic morphology and migration that is independent of CXCR3 or CXCR6, or the skin-selective chemokine receptors CCR10 and CCR8. However, we found that CXCR6 and CCR10…

Th17 cells are an important therapeutic target in autoimmunity. However, it is known that Th17 cells exhibit considerable plasticity, particularly at sites of autoimmune inflammation. Th17 cells can switch to become ex-Th17 cells that no longer produce IL-17 but produce IFN-γ. These ex-Th17 cells are also called nonclassical Th1 cells because of their ability to produce IFN-γ, similar to Th1 cells; however, it is unclear whether they resemble Th1 or Th17 cells in terms of their function and regulation, and whether they have a pathogenic role in autoimmunity. We compared the phenotypic and functional features of human Th17, Th1, and ex-Th17 cell populations. Our data showed that despite their loss of IL-17 expression, ex-Th17 cells were more polyfunctional in terms of cytokine production than either Th1 or bona fide Th17 cells, and produced increased amounts of proinflammatory cytokines. The proliferative brake on Th17 cells appeared to be lifted because ex-Th17 cells proliferated more than Th17 cells after stimulation. In contrast with Th1 and Th17 cells, ex-Th17 cells were highly…

Abstract Th1 and Th17 cells have an established role in protective immunity to Bordetella pertussis, but this evidence is based largely on peripheral T cells. There is emerging evidence that local tissue-resident memory T (TRM) cells that accumulate in tissue following mucosal infection may be crucial for long-term immunity. In this study, we examined the role of respiratory CD4 TRM cells in immunity to B. pertussis. Natural immunity to B. pertussis induced by infection is considered long lasting and effective at preventing reinfection. Consistent with this, we found that convalescent mice rapidly cleared the bacteria after reinfection. Furthermore, CD4 T cells with a TRM cell phenotype (CD44+CD62L−CD69+ or CD44+CD62L−CD69+CD103+) accumulated in the lungs of mice during infection with B. pertussis and significantly expanded through local proliferation following reinfection. These CD4 TRM cells were B. pertussis specific and secreted IL-17 or IL-17 and IFN-γ. Treatment of mice with FTY720, which prevented migration of T and B cells from lymph nodes to the circulation, significantly…

Abstract Multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis, are neuroinflammatory diseases driven by autoreactive pathogenic TH cells that elicit demyelination and axonal damage. How TH cells acquire pathogenicity and communicate with myeloid cells and cells of the CNS remain unclear. IL-1β is recognized to play an important role in experimental autoimmune encephalomyelitis (EAE) and perhaps MS. Clinical EAE is significantly attenuated in IL-1R–deficient and IL-1β–deficient mice, and IL-1β is found in the blood, cerebrospinal fluid, and CNS lesions of MS patients. In this article, we focus on new reports that elucidate the cellular sources of IL-1β and its actions during EAE, in both lymphoid tissues and within the CNS. Several immune cell types serve as critical producers of IL-1β during EAE, with this cytokine inducing response in both hematopoietic and nonhematopoietic cells. These findings from the EAE model should inspire efforts toward investigating the therapeutic potential of IL-1 blockade in MS.

Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data. Although several hierarchical clustering-based methods have been proposed, they vary in distance and linkage methods and have not yet been rigorously compared. In this study, we use a combination of human experimental and simulated data to characterize the performance of hierarchical clustering-based methods for partitioning sequences into clones. We find that single linkage clustering has high performance, with specificity, sensitivity, and positive predictive value all >99%, whereas other linkages result in a significant loss of…

Mass cytometry has revolutionized the study of cellular and phenotypic diversity, significantly expanding the number of phenotypic and functional characteristics that can be measured at the single-cell level. This high-dimensional analysis platform has necessitated the development of new data analysis approaches. Many of these algorithms circumvent traditional approaches used in flow cytometric analysis, fundamentally changing the way these data are analyzed and interpreted. For the beginner, however, the large number of algorithms that have been developed, as well as the lack of consensus on best practices for analyzing these data, raise multiple questions: Which algorithm is the best for analyzing a dataset? How do different algorithms compare? How can one move beyond data visualization to gain new biological insights? In this article, we describe our experiences as recent adopters of mass cytometry. By analyzing a single dataset using five cytometry by time-of-flight analysis platforms (viSNE, SPADE, X-shift, PhenoGraph, and Citrus), we identify important considerations and…

Abstract The role of peroxisome proliferator–activated receptor α (PPAR-α) in innate host defense is largely unknown. In this study, we show that PPAR-α is essential for antimycobacterial responses via activation of transcription factor EB (TFEB) transcription and inhibition of lipid body formation. PPAR-α deficiency resulted in an increased bacterial load and exaggerated inflammatory responses during mycobacterial infection. PPAR-α agonists promoted autophagy, lysosomal biogenesis, phagosomal maturation, and antimicrobial defense against Mycobacterium tuberculosis or M. bovis bacillus Calmette–Guérin. PPAR-α agonists regulated multiple genes involved in autophagy and lysosomal biogenesis, including Lamp2, Rab7, and Tfeb in bone marrow–derived macrophages. Silencing of TFEB reduced phagosomal maturation and antimicrobial responses, but increased macrophage inflammatory responses during mycobacterial infection. Moreover, PPAR-α activation promoted lipid catabolism and fatty acid β-oxidation in macrophages during mycobacterial infection. Taken together, our data indicate that PPAR-α…

Early-life respiratory viral infection is a risk factor for asthma development. Rhinovirus (RV) infection of 6-d-old mice, but not mature mice, causes mucous metaplasia and airway hyperresponsiveness that are associated with the expansion of lung type 2 innate lymphoid cells (ILC2s) and are dependent on IL-13 and the innate cytokine IL-25. However, contributions of the other innate cytokines, IL-33 and thymic stromal lymphopoietin (TSLP), to the observed asthma-like phenotype have not been examined. We reasoned that IL-33 and TSLP expression are also induced by RV infection in immature mice and are required for maximum ILC2 expansion and mucous metaplasia. We inoculated 6-d-old BALB/c (wild-type) and TSLP receptor-knockout mice with sham HeLa cell lysate or RV. Selected mice were treated with neutralizing Abs to IL-33 or recombinant IL-33, IL-25, or TSLP. ILC2s were isolated from RV-infected immature mice and treated with innate cytokines ex vivo. RV infection of 6-d-old mice increased IL-33 and TSLP protein abundance. TSLP expression was localized to the airway epithelium, whereas…

NKT cells recognize lipid Ags presented by a class I MHC-like molecule CD1d, a member of the CD1 family. Although most initial studies on NKT cells focused on a subset with semi-invariant TCR termed invariant NKT cells, the majority of CD1d-restricted lipid-reactive human T cells express diverse TCRs and are termed type II NKT cells. These cells constitute a distinct population of circulating and tissue-resident effector T cells with immune-regulatory properties. They react to a growing list of self- as well as non-self-lipid ligands, and share some properties with both invariant NKT and conventional T cells. An emerging body of evidence points to their role in the regulation of immunity to pathogens/tumors and in autoimmune/metabolic disorders. An improved understanding of the biology of these cells and the ability to manipulate their function may be of therapeutic benefit in diverse disease conditions.