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Recognition of Influenza A virus (IAV) by the innate immune system triggers pathways that restrict viral replication, activates innate immune cells, and regulates adaptive immunity. However, excessive innate immune activation can exaggerate disease. The pathways promoting excessive activation are incompletely understood, with limited experimental models to investigate mechanisms driving influenza-induced inflammation in humans. Interferon regulatory factor (IRF5) is a transcription factor that plays important roles in induction of cytokines after viral sensing. In an in vivo model of IAV infection, IRF5 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine production, specifically reducing cytokine-producing myeloid cell populations in Irf5 -/- mice, but not impacting type 1 IFN production or virus replication. Using cytometry by time-of-flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses by myeloid cells to IAV, we employed human induced pluripotent stem cells (hIPSCs) with biallelic mutations in IRF5, demonstrating for the first time iPS-derived dendritic cells (iPS-DCs) with biallelic mutations can be used to investigate regulation of human virus-induced immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or macrophages, corresponded with reduced virus-induced inflammatory cytokine production, with IRF5 acting downstream of TLR7 and, possibly, RIG-I after viral sensing. Thus, IRF5 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in mice and humans, and drives immune-mediated viral pathogenesis independently of type 1 IFN and virus replication.ImportanceThe inflammatory response to Influenza A virus (IAV) participates in infection control but contributes to disease severity. After viral detection intracellular pathways are activated, initiating cytokine production, but these pathways are incompletely understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN and virus replication, implying that IRF5 could be specifically targeted to treat influenza-induced inflammation. We show for the first time that human iPSC technology can be exploited in genetic studies of virus-induced immune responses. Using this technology, we deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired influenza-induced cytokine production and revealed that IRF5 acts downstream of Toll-like receptor 7 and possibly retinoic acid-inducible gene-I. Our data demonstrate the importance of IRF5 in influenza-induced inflammation, suggesting genetic variation in the IRF5 gene may influence host susceptibility to viral diseases.

Invasive candidiasis, mainly caused by Candida albicans, is a serious healthcare problem with high mortality rates, particularly in immunocompromised patients. Innate immune cells express pathogen recognition receptors (PRRs) including C-type lectin-like receptors (CLRs) that bind C. albicans to initiate an immune response. Multiple CLRs including Dectin-1, Dectin-2 and Mincle have been proposed individually to contribute to the immune response to C. albicans. However how these receptors collaborate to clear a fungal infection is unknown. Herein, we used novel multi-CLR knockout (KO) mice to decipher the individual, collaborative and collective roles of Dectin-1, Dectin-2 and Mincle during systemic C. albicans infection. These studies revealed an unappreciated and profound role for CLR co-operation in anti-fungal immunity. The protective effect of multiple CLRs was markedly greater than any single receptor, and was mediated through inflammatory monocytes via recognition and phagocytosis of C. albicans, and production of C. albicans-induced cytokines and chemokines. These CLRs were dispensable for mediating similar responses from neutrophils, likely due to lower expression of these CLRs on neutrophils compared to inflammatory monocytes. Concurrent deletion of Dectin-1 and Dectin-2, or all three CLRs, resulted in dramatically increased susceptibility to systemic C. albicans infection compared to mice lacking a single CLR. Multi-CLR KO mice were unable to control fungal growth due to an inadequate early inflammatory monocyte-mediated response. In response to excessive fungal growth, the multi-CLR KO mice mounted a hyper-inflammatory response, likely leading to multiple organ failure. Thus, these data reveal a critical role for CLR co-operation in the effective control of C. albicans and maintenance of organ function during infection., Author summary Fungal infections including invasive candidiasis are a serious healthcare problem particularly for immunocompromised patients. Mortality rates for invasive candidiasis are very high and complex anti-fungal immune responses are poorly understood, hindering the development of novel immunotherapies. Dectin-1, Dectin-2 and Mincle are three cell surface receptors that are proposed to be involved in the immune response to fungal pathogens. However, if or how these receptors work together during infection is currently unknown. Here we demonstrate that these receptors, in particular Dectin-1 and Dectin-2, work together to promote fungal clearance by a group of innate immune cells called inflammatory monocytes. Furthermore, we found that mice lacking these three receptors are dramatically susceptible to systemic Candida albicans infection due to defective early innate immune responses. These mice develop hyper-inflammation to try to control excessive fungal growth likely resulting in multi-organ failure. Our work helps explain how these receptors work together to clear/control invasive candidiasis. Our improved knowledge of the interactions between these receptors could be used to help design novel anti-fungal immunotherapies.

Human cytomegalovirus (HCMV) is a β-herpesvirus with high sero-prevalence within the human population. Primary HCMV infection and life-long carriage are typically asymptomatic. However, HCMV is implicated in exacerbation of chronic conditions and associated damage in individuals with intact immune systems. Furthermore, HCMV is a significant cause of morbidity and mortality in the immunologically immature and immune-compromised where disease is associated with tissue damage. Infection-induced inflammation, including robust cytokine responses, is a key component of pathologies associated with many viruses. Despite encoding a large number of immune-evasion genes, HCMV also triggers the induction of inflammatory cytokine responses during infection. Thus, understanding how cytokines contribute to CMV-induced pathologies and the mechanisms through which they are regulated may inform clinical management of disease. Herein, we discuss our current understanding based on clinical observation and in vivo modeling of disease of the role that cytokines play in CMV pathogenesis. Specifically, in the context of the different tissues and organs in which CMV replicates, we give a broad overview of the beneficial and adverse effects that cytokines have during infection and describe how cytokine-mediated tissue damage is regulated. We discuss the implications of findings derived from mice and humans for therapeutic intervention strategies and our understanding of how host genetics may influence the outcome of CMV infections.

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen-presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte-derived macrophages we noted differential responses to the immune-modulatory cytokine IL-10. In contrast to the suppressive actions of IL-10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL-10 may thus have limited impact on development of potentially tissue-damaging adaptive immune responses, whilst restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune-mediated membrane dysfunction, treatment failure and increased morbidity.

Interferon lambda (IFNλ) is a group of cytokines that belong to the IL-10 family. They exhibit antiviral activities against certain viruses during infection of the liver and mucosal tissues. Here we report that IFNλ restricts in vitro replication of the β-herpesvirus murine cytomegalovirus (mCMV). However, IFNλR1-deficient (Ifnλr1-/-) mice were not preferentially susceptible to mCMV infection in vivo during acute infection after systemic or mucosal challenge, or during virus persistence in the mucosa. Instead, our studies revealed that IFNλ influences NK cell responses during mCMV infection. Ifnλr1-/- mice exhibited defective development of conventional interferon-gamma (IFNγ)-expressing NK cells in the spleen during mCMV infection whereas accumulation of granzyme B-expressing NK cells was unaltered. In vitro, development of splenic IFNγ+ NK cells following stimulation with IL-12 or, to a lesser extent, IL-18 was abrogated by IFNλR1-deficiency. Thus, IFNλ regulates NK cell responses during mCMV infection and restricts virus replication in vitro but is redundant in the control of acute and persistent mCMV replication within mucosal and non-mucosal tissues.

Since the development of Vaccinia virus as a vaccine vector in 1984, the utility of numerous viruses in vaccination strategies has been explored. In recent years, key improvements to existing vectors such as those based on adenovirus have led to significant improvements in immunogenicity and efficacy. Furthermore, exciting new vectors that exploit viruses such as cytomegalovirus (CMV) and vesicular stomatitis virus (VSV) have emerged. Herein, we summarize these recent developments in viral vector technologies, focusing on novel vectors based on CMV, VSV, measles and modified adenovirus. We discuss the potential utility of these exciting approaches in eliciting protection against infectious diseases. This article is protected by copyright. All rights reserved.

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.

CD4+ T cells support host defence against herpesviruses and other viral pathogens. We identified that CD4+ T cells from systemic and mucosal tissues of hosts infected with the β-herpesviridae human cytomegalovirus (HCMV) or murine cytomegalovirus (MCMV) express the regulatory cytokine interleukin (IL)-10. IL-10+CD4+ T cells co-expressed TH1-associated transcription factors and chemokine receptors. Mice lacking T cell-derived IL-10 elicited enhanced antiviral T cell responses and restricted MCMV persistence in salivary glands and secretion in saliva. Thus, IL-10+CD4+ T cells suppress antiviral immune responses against CMV. Expansion of this T-cell population in the periphery was promoted by IL-27 whereas mucosal IL-10+ T cell responses were ICOS-dependent. Infected Il27rα-deficient mice with reduced peripheral IL-10+CD4+ T cell accumulation displayed robust T cell responses and restricted MCMV persistence and shedding. Temporal inhibition experiments revealed that IL-27R signaling during initial infection was required for the suppression of T cell immunity and control of virus shedding during MCMV persistence. IL-27 production was promoted by type-I IFN, suggesting that β-herpesviridae exploit the immune-regulatory properties of this antiviral pathway to establish chronicity. Further, our data reveal that cytokine signaling events during initial infection profoundly influence virus chronicity., Author Summary Viruses including the pathogenic β-herpesvirus human cytomegalovirus (HCMV) can replicate within and disseminate from mucosal tissues. Understanding how to improve antiviral immune responses to restrict virus replication in the mucosa could help counter virus transmission. Studies in the murine cytomegalovirus (MCMV) model have demonstrated the importance of the CD4+ T cells in control of mucosal MCMV replication. However, this process is inefficient, allowing virus persistence. Herein, we reveal that production by CD4+ T cells of the immune-suppressive soluble protein, or cytokine, interleukin (IL)-10 facilitates virus persistence in mucosal tissue. Mice deficient in T cell-derived IL-10 mounted heightened T cell responses and reduced virus replication in the salivary glands and shedding in the saliva. The cytokine IL-27 induced IL-10-producing CD4+ T cells in the periphery whereas a cell surface-expressed protein, ICOS, promoted mucosal IL-10+ T cell responses. IL-27 acted in the initial stages of infection to impinge on T cell responses and antiviral control. In turn, IL-27 production in response to viral infection was triggered by type-I interferon, a prototypic antiviral cytokine. Thus, our data suggest that herpesviruses may exploit immune-suppressive properties of this early antiviral cytokine response to facilitate persistence within and shedding from mucosal tissue.

Summary Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation., Highlights • Repeated acute resolving inflammation leads to excessive tissue damage • IL-6 regulates profibrotic IFN-γ-secreting T cells • IFN-γ increases detrimental STAT1 signaling in stromal tissue • STAT1 activity alters homeostatic control of extracellular matrix to promote fibrosis

Decline of cell-mediated immunity is often attributed to decaying T-cell numbers and their distribution in peripheral organs. This study examined the hypothesis that qualitative as well as quantitative changes contribute to the declining efficacy of CD8(+) T-cell memory. Using a model of influenza virus infection, where loss of protective CD8(+) T-cell immunity was observed 6 months postinfection, we found no decline in antigen-specific T-cell numbers or migration to the site of secondary infection. There was, however, a large reduction in antigen-specific CD8(+) T-cell degranulation, cytokine secretion, and polyfunctionality. A profound loss of high-avidity T cells over time indicated that failure to confer protective immunity resulted from the inferior functional capacity of remaining low avidity cells. These data imply that high-avidity central memory T cells wane with declining antigen levels, leaving lower avidity T cells with reduced functional capabilities.

The beta-herpesvirus cytomegalovirus induces a substantial and progressive expansion of virus-specific memory CD8 T cells, which protect the host against viral reactivation from latency. Here, we report that this expansion, or “inflation”, of memory T cells is amplified dramatically during mouse cytomegalovirus (MCMV) infection of interleukin-10 (IL-10) knockout (IL-10−/−) mice. T cells from IL-10−/− mice were oligoclonal, exhibited a highly activated phenotype, expressed antiviral cytokines and degranulated in response to cognate antigen encounter ex vivo. Moreover, latent viral load was reduced in IL-10−/− mice. Importantly, these results were recapitulated by IL-10 receptor blockade during chronic/latent infection of wild-type mice. These data demonstrate that regulatory immune mechanisms can influence cytomegalovirus-specific T cell memory and suggest a possible rationale for the acquisition of functional IL-10 orthologs by herpesviruses.

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.