Opposing roles of resident and infiltrating immune cells in the defense against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here, we investigated the specific roles of tissue-resident alveolar macrophages (AMs) and infiltrating phagocytes during infection with this pathogen. AMs were the predominant cell type that internalized bacteria 1 day after infection. A total of 3 and 5 days after infection, AM numbers were greatly reduced, whereas there was an influx of neutrophils and, later, monocyte-derived cells (MCs) into lung tissue. AMs carried greater numbers of viable L. longbeachae than neutrophils and MCs, which correlated with a higher capacity of L. longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection, whereas neutrophils and MC were required for efficient bacterial clearance. Interleukin (IL)-18 was important for interferon-γ production by IL-18R+ natural killer cells and T cells, which, in turn, stimulated reactive oxygen species-mediated bactericidal activity in neutrophils, resulting in the restriction of L. longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L. longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L. longbeachae infection that may be manipulated to improve protective responses.

Dual fluorescence reporter mice for Ccl3 transcription, translation, and intercellular communication.

Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.

Rapid protection against viral infections by chemokine-accelerated post-exposure vaccination.

Introduction: Prophylactic vaccines generate strong and durable immunity to avoid future infections, whereas post-exposure vaccinations are intended to establish rapid protection against already ongoing infections. Antiviral cytotoxic CD8+ T cells (CTL) are activated by dendritic cells (DCs), which themselves must be activated by adjuvants to express costimulatory molecules and so-called signal 0-chemokines that attract naive CTL to the DCs. Hypothesis: Here we asked whether a vaccination protocol that combines two adjuvants, a toll-like receptor ligand (TLR) and a natural killer T cell activator, to induce two signal 0 chemokines, synergistically accelerates CTL activation. Methods: We used a well-characterized vaccination model based on the model antigen ovalbumin, the TLR9 ligand CpG and the NKT cell ligand α-galactosylceramide to induce signal 0-chemokines. Exploiting this vaccination model, we studied detailed T cell kinetics and T cell profiling in different in vivo mouse models of viral infection. Results: We found that CTL induced by both adjuvants obtained a head-start that allowed them to functionally differentiate further and generate higher numbers of protective CTL 1-2 days earlier. Such signal 0-optimized post-exposure vaccination hastened clearance of experimental adenovirus and cytomegalovirus infections. Conclusion: Our findings show that signal 0 chemokine-inducing adjuvant combinations gain time in the race against rapidly replicating microbes, which may be especially useful in post-exposure vaccination settings during viral epi/pandemics.

Conditional NKT Cell Depletion in Mice Reveals a Negative Feedback Loop That Regulates CTL Cross-Priming.

NKT cells are unconventional T cells whose biological role is incompletely understood. Similar to TH cells, activated NKT cells can cause dendritic cell (DC) maturation, which is required for effective CTL responses. However, it is unclear whether and how NKT cells affect CTLs downstream of the DC maturation phase. This is partially due to the lack of techniques to conditionally deplete NKT cells in vivo. To overcome this problem, we have developed two approaches for this purpose in mice: the first is based on mixed bone marrow chimeras where Jα18 knockout and depletable CD90 congenic bone marrow is combined, and the second used PLZFCre × iDTR bone marrow chimeras, which target innate-like T cells. Using these tools, we found that NKT cell depletion at 20 h, that is, after initial DC activation, did not render CTLs helpless, as CD40L signaling by non-NKT cells sufficed. Instead, NKT cell depletion even augmented CD8 T cell expansion and cytotoxicity by mechanisms distinct from reduced STAT6 signaling. These findings revealed a negative feedback loop by which NKT cells control CTL cross-priming downstream of DC maturation. The techniques described in this study expand the toolbox to study NKT cells and other unconventional T cell subsets in vivo and uncovered a hidden immunoregulatory mechanism.

Interferon-induced IL-10 drives systemic T-cell dysfunction during chronic liver injury.

BACKGROUND & AIMS: Patients with chronic liver disease (CLD), including cirrhosis, are at increased risk of intractable viral infections and are hyporesponsive to vaccination. Hallmarks of CLD and cirrhosis include microbial translocation and elevated levels of type I interferon (IFN-I). We aimed to investigate the relevance of microbiota-induced IFN-I in the impaired adaptive immune responses observed in CLD. METHODS: We combined bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver injury with vaccination or lymphocytic choriomeningitis virus infection in transgenic mice lacking IFN-I in myeloid cells (LysM-Cre IFNARflox/flox), IFNAR-induced IL-10 (MX1-Cre IL10flox/flox) or IL-10R in T cells (CD4-DN IL-10R). Key pathways were blocked in vivo with specific antibodies (anti-IFNAR and anti-IL10R). We assessed T-cell responses and antibody titers after HBV and SARS-CoV-2 vaccinations in patients with CLD and healthy individuals in a proof-of-concept clinical study. RESULTS: We demonstrate that BDL- and CCL4-induced prolonged liver injury leads to impaired T-cell responses to vaccination and viral infection in mice, subsequently leading to persistent infection. We observed a similarly defective T-cell response to vaccination in patients with cirrhosis. Innate sensing of translocated gut microbiota induced IFN-I signaling in hepatic myeloid cells that triggered excessive IL-10 production upon viral infection. IL-10R signaling in antigen-specific T cells rendered them dysfunctional. Antibiotic treatment and inhibition of IFNAR or IL-10Ra restored antiviral immunity without detectable immune pathology in mice. Notably, IL-10Ra blockade restored the functional phenotype of T cells from vaccinated patients with cirrhosis. CONCLUSION: Innate sensing of translocated microbiota induces IFN-/IL-10 expression, which drives the loss of systemic T-cell immunity during prolonged liver injury. IMPACT AND IMPLICATIONS: Chronic liver injury and cirrhosis are associated with enhanced susceptibility to viral infections and vaccine hyporesponsiveness. Using different preclinical animal models and patient samples, we identified that impaired T-cell immunity in BDL- and CCL4-induced prolonged liver injury is driven by sequential events involving microbial translocation, IFN signaling leading to myeloid cell-induced IL-10 expression, and IL-10 signaling in antigen-specific T cells. Given the absence of immune pathology after interference with IL-10R, our study highlights a potential novel target to reconstitute T-cell immunity in patients with CLD that can be explored in future clinical studies.

Cigarette smoke depletes alveolar macrophages and delays clearance of Legionella pneumophila.

Legionella pneumophila is the main etiological agent of Legionnaires' disease, a severe bacterial pneumonia. L. pneumophila is initially engulfed by alveolar macrophages (AMs) and subvert normal cellular functions to establish a replicative vacuole. Cigarette smokers are particularly susceptible to developing Legionnaires' disease and other pulmonary infections; however, little is known about the cellular mechanisms underlying this susceptibility. To investigate this, we used a mouse model of acute cigarette smoke exposure to examine the immune response to cigarette smoke and subsequent L. pneumophila infection. Contrary to previous reports, we show that cigarette smoke exposure alone causes a significant depletion of AMs using enzymatic digestion to extract cells, or via imaging intact lung lobes by light-sheet microscopy. Furthermore, treatment of mice deficient in specific types of cell death with smoke suggests that NLRP3-driven pyroptosis is a contributor to smoke-induced death of AMs. After infection, smoke-exposed mice displayed increased pulmonary L. pneumophila loads and developed more severe disease compared with air-exposed controls. We tested if depletion of AMs was related to this phenotype by directly depleting them with clodronate liposomes and found that this also resulted in increased L. pneumophila loads. In summary, our results showed that cigarette smoke depleted AMs from the lung and that this likely contributed to more severe Legionnaires' disease. Furthermore, the role of AMs in L. pneumophila infection is more nuanced than simply providing a replicative niche, and our studies suggest they play a major role in bacterial clearance.

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing.

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased levels of cGAMP and enhanced IFN-I signature. To identify disease mechanisms and potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-ß, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-ß-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I, or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcomes.

Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19.

Anorexia and fasting are host adaptations to acute infection, and induce a metabolic switch towards ketogenesis and the production of ketone bodies, including ß-hydroxybutyrate (BHB)1-6. However, whether ketogenesis metabolically influences the immune response in pulmonary infections remains unclear. Here we show that the production of BHB is impaired in individuals with SARS-CoV-2-induced acute respiratory distress syndrome (ARDS) but not in those with  influenza-induced ARDS. We found that BHB promotes both the survival of and the production of interferon-γ by CD4+ T cells. Applying a metabolic-tracing analysis, we established that BHB provides an alternative carbon source to fuel oxidative phosphorylation (OXPHOS) and the production of bioenergetic amino acids and glutathione, which is important for maintaining the redox balance. T cells from patients with SARS-CoV-2-induced ARDS were exhausted and skewed towards glycolysis, but could be metabolically reprogrammed by BHB to perform OXPHOS, thereby increasing their functionality. Finally, we show in mice that a ketogenic diet and the delivery of BHB as a ketone ester drink restores CD4+ T cell metabolism and function in severe respiratory infections, ultimately reducing the mortality of mice infected with SARS-CoV-2. Altogether, our data reveal that BHB is an alternative source of carbon that promotes T cell responses in pulmonary viral infections, and highlight impaired ketogenesis as a potential confounding factor in severe COVID-19.

Type 1 conventional dendritic cells maintain and guide the differentiation of precursors of exhausted T cells in distinct cellular niches.

Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology.

ICAMs are dispensable for influenza clearance and anti-viral humoral and cellular immunity.

αLß2 (LFA-1) mediated interactions with ICAM-1 and ICAM-2 predominate leukocyte-vascular interactions, but their functions in extravascular cell-cell communications is still debated. The roles of these two ligands in leukocyte trafficking, lymphocyte differentiation, and immunity to influenza infections were dissected in the present study. Surprisingly, double ICAM-1 and ICAM-2 knock out mice (herein ICAM-1/2-/- mice) infected with a lab adapted H1N1 influenza A virus fully recovered from infection, elicited potent humoral immunity, and generated normal long lasting anti-viral CD8+ T cell memory. Furthermore, lung capillary ICAMs were dispensable for both NK and neutrophil entry to virus infected lungs. Mediastinal lymph nodes (MedLNs) of ICAM-1/2-/- mice poorly recruited naïve T cells and B lymphocytes but elicited normal humoral immunity critical for viral clearance and effective CD8+ differentiation into IFN-γ producing T cells. Furthermore, whereas reduced numbers of virus specific effector CD8+ T cells accumulated inside infected ICAM-1/2-/- lungs, normal virus-specific TRM CD8+ cells were generated inside these lungs and fully protected ICAM-1/2-/- mice from secondary heterosubtypic infections. B lymphocyte entry to the MedLNs and differentiation into extrafollicular plasmablasts, producing high affinity anti-influenza IgG2a antibodies, were also ICAM-1 and ICAM-2 independent. A potent antiviral humoral response was associated with accumulation of hyper-stimulated cDC2s in ICAM null MedLNs and higher numbers of virus-specific T follicular helper (Tfh) cells generated following lung infection. Mice selectively depleted of cDC ICAM-1 expression supported, however, normal CTL and Tfh differentiation following influenza infection, ruling out essential co-stimulatory functions of DC ICAM-1 in CD8+ and CD4+ T cell differentiation. Collectively our findings suggest that lung ICAMs are dispensable for innate leukocyte trafficking to influenza infected lungs, for the generation of peri-epithelial TRM CD8+ cells, and long term anti-viral cellular immunity. In lung draining LNs, although ICAMs promote lymphocyte homing, these key integrin ligands are not required for influenza-specific humoral immunity or generation of IFN-γ effector CD8+ T cells. In conclusion, our findings suggest unexpected compensatory mechanisms that orchestrate protective anti-influenza immunity in the absence of vascular and extravascular ICAMs.

Leukocyte trafficking to the lungs and beyond: lessons from influenza for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Understanding of the fundamental processes underlying the versatile clinical manifestations of COVID-19 is incomplete without comprehension of how different immune cells are recruited to various compartments of virus-infected lungs, and how this recruitment differs among individuals with different levels of disease severity. As in other respiratory infections, leukocyte recruitment to the respiratory system in people with COVID-19 is orchestrated by specific leukocyte trafficking molecules, and when uncontrolled and excessive it results in various pathological complications, both in the lungs and in other organs. In the absence of experimental data from physiologically relevant animal models, our knowledge of the trafficking signals displayed by distinct vascular beds and epithelial cell layers in response to infection by SARS-CoV-2 is still incomplete. However, SARS-CoV-2 and influenza virus elicit partially conserved inflammatory responses in the different respiratory epithelial cells encountered early in infection and may trigger partially overlapping combinations of trafficking signals in nearby blood vessels. Here, we review the molecular signals orchestrating leukocyte trafficking to airway and lung compartments during primary pneumotropic influenza virus infections and discuss potential similarities to distinct courses of primary SARS-CoV-2 infections. We also discuss how an imbalance in vascular activation by leukocytes outside the airways and lungs may contribute to extrapulmonary inflammatory complications in subsets of patients with COVID-19. These multiple molecular pathways are potential targets for therapeutic interventions in patients with severe COVID-19.

BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

A high-salt diet compromises antibacterial neutrophil responses through hormonal perturbation.

The Western diet is rich in salt, which poses various health risks. A high-salt diet (HSD) can stimulate immunity through the nuclear factor of activated T cells 5 (Nfat5)-signaling pathway, especially in the skin, where sodium is stored. The kidney medulla also accumulates sodium to build an osmotic gradient for water conservation. Here, we studied the effect of an HSD on the immune defense against uropathogenic E. coli-induced pyelonephritis, the most common kidney infection. Unexpectedly, pyelonephritis was aggravated in mice on an HSD by two mechanisms. First, on an HSD, sodium must be excreted; therefore, the kidney used urea instead to build the osmotic gradient. However, in contrast to sodium, urea suppressed the antibacterial functionality of neutrophils, the principal immune effectors against pyelonephritis. Second, the body excretes sodium by lowering mineralocorticoid production via suppressing aldosterone synthase. This caused an accumulation of aldosterone precursors with glucocorticoid functionality, which abolished the diurnal adrenocorticotropic hormone-driven glucocorticoid rhythm and compromised neutrophil development and antibacterial functionality systemically. Consistently, under an HSD, systemic Listeria monocytogenes infection was also aggravated in a glucocorticoid-dependent manner. Glucocorticoids directly induced Nfat5 expression, but pharmacological normalization of renal Nfat5 expression failed to restore the antibacterial defense. Last, healthy humans consuming an HSD for 1 week showed hyperglucocorticoidism and impaired antibacterial neutrophil function. In summary, an HSD suppresses intrarenal neutrophils Nfat5-independently by altering the local microenvironment and systemically by glucocorticoid-mediated immunosuppression. These findings argue against high-salt consumption during bacterial infections.

Splenic Red Pulp Macrophages Cross-Prime Early Effector CTL That Provide Rapid Defense against Viral Infections.

Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow-derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1- CD127- CX3CR1- Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

Charcot-Leyden Crystals Activate the NLRP3 Inflammasome and Cause IL-1ß Inflammation in Human Macrophages.

Charcot-Leyden crystals (CLCs) are Galectin-10 protein crystals that can form after eosinophils degranulate. CLCs can appear and persist in tissues from patients with eosinophilic disorders, such as asthma, allergic reactions, and fungal and helminthic infections. Despite abundant reports of their occurrence in human disease, the inflammatory potential of CLCs has remained unknown. In this article, we show that CLCs induce the release of the proinflammatory cytokine IL-1ß upon their phagocytosis by primary human macrophages in vitro. Chemical inhibition and small interfering RNA knockdown of NLRP3 in primary human macrophages abrogated their IL-1ß response to CLCs. Using C57BL/6 ASC-mCitrine transgenic inflammasome reporter mice, we showed that the instillation of CLCs into the lungs promoted the assembly of ASC complexes in infiltrating immune cells (neutrophils and inflammatory monocytes) and resulted in IL-1ß accumulation into the bronchoalveolar lavage fluid. Our findings reveal that CLCs are recognized by the NLRP3 inflammasome, which may sustain inflammation that follows eosinophilic inflammatory processes.

Prolonged IKKß Inhibition Improves Ongoing CTL Antitumor Responses by Incapacitating Regulatory T Cells.

Regulatory T cells (Tregs) prevent autoimmunity but limit antitumor immunity. The canonical NF-κB signaling pathway both activates immunity and promotes thymic Treg development. Here, we report that mature Tregs continue to require NF-κB signaling through IκB-kinase ß (IKKß) after thymic egress. Mice lacking IKKß in mature Tregs developed scurfy-like immunopathology due to death of peripheral FoxP3+ Tregs. Also, pharmacological IKKß inhibition reduced Treg numbers in the circulation by ∼50% and downregulated FoxP3 and CD25 expression and STAT5 phosphorylation. In contrast, activated cytotoxic T lymphocytes (CTLs) were resistant to IKKß inhibition because other pathways, in particular nuclear factor of activated T cells (NFATc1) signaling, sustained their survival and expansion. In a melanoma mouse model, IKKß inhibition after CTL cross-priming improved the antitumor response and delayed tumor growth. In conclusion, prolonged IKKß inhibition decimates circulating Tregs and improves CTL responses when commenced after tumor vaccination, indicating that IKKß represents a druggable checkpoint.

Targeting myeloid derived suppressor cells with all-trans retinoic acid is highly time-dependent in therapeutic tumor vaccination.

Tumor immune escape is a critical problem which frequently accounts for the failure of therapeutic tumor vaccines. Among the most potent suppressors of tumor immunity are myeloid derived suppressor cells (MDSCs). MDSCs can be targeted by all-trans-retinoic-acid (atRA), which reduced their numbers and increased response rates in several vaccination studies. However, not much is known about the optimal administration interval between atRA and the vaccine as well as about its mode of action. Here we demonstrate in 2 different murine tumor models that mice unresponsive to a therapeutic vaccine harbored higher MDSC numbers than did responders. Application of atRA overcame MDSC-mediated immunosuppression and restored tumor control. Importantly, atRA was protective only when administered 3 d after vaccination (delayed treatment), whereas simultaneous administration even decreased the anti-tumor immune response and reduced survival. When analyzing the underlying mechanisms, we found that delayed, but not simultaneous atRA treatment with vaccination abrogated the suppressive capacity in monocytic MDSCs and instead caused them to upregulate MHC-class-II. Consistently, MDSCs from patients with colorectal carcinoma also failed to upregulate HLA-DR after ex vivo treatment with TLR-ligation. Overall, we demonstrate that atRA can convert non-responders to responders to vaccination by suppressing MDSCs function and not only by reducing their number. Moreover, we identify a novel, strictly time-dependent mode of action of atRA to be considered during immunotherapeutic protocols in the future.