Immune Responses in the Liver.

The liver is a key, frontline immune tissue. Ideally positioned to detect pathogens entering the body via the gut, the liver appears designed to detect, capture, and clear bacteria, viruses, and macromolecules. Containing the largest collection of phagocytic cells in the body, this organ is an important barrier between us and the outside world. Importantly, as portal blood also transports a large number of foreign but harmless molecules (e.g., food antigens), the liver's default immune status is anti-inflammatory or immunotolerant; however, under appropriate conditions, the liver is able to mount a rapid and robust immune response. This balance between immunity and tolerance is essential to liver function. Excessive inflammation in the absence of infection leads to sterile liver injury, tissue damage, and remodeling; insufficient immunity allows for chronic infection and cancer. Dynamic interactions between the numerous populations of immune cells in the liver are key to maintaining this balance and overall tissue health.

Patrolling Alveolar Macrophages Conceal Bacteria from the Immune System to Maintain Homeostasis.

During respiration, humans breathe in more than 10,000 liters of non-sterile air daily, allowing some pathogens access to alveoli. Interestingly, alveoli outnumber alveolar macrophages (AMs), which favors alveoli devoid of AMs. If AMs, like most tissue macrophages, are sessile, then this numerical advantage would be exploited by pathogens unless neutrophils from the blood stream intervened. However, this would translate to omnipresent persistent inflammation. Developing in vivo real-time intravital imaging of alveoli revealed AMs crawling in and between alveoli using the pores of Kohn. Importantly, these macrophages sensed, chemotaxed, and, with high efficiency, phagocytosed inhaled bacterial pathogens such as P. aeruginosa and S. aureus, cloaking the bacteria from neutrophils. Impairing AM chemotaxis toward bacteria induced superfluous neutrophil recruitment, leading to inappropriate inflammation and injury. In a disease context, influenza A virus infection impaired AM crawling via the type II interferon signaling pathway, and this greatly increased secondary bacterial co-infection.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

A Reservoir of Mature Cavity Macrophages that Can Rapidly Invade Visceral Organs to Affect Tissue Repair.

A key feature of inflammation is the timely recruitment of leukocytes, including monocytes, from blood into tissues, the latter maturing into macrophages over a period of 2-3 days. Using multi-channel spinning disk microscopy, we identified a rapid pathway of macrophage recruitment into an injured organ via a non-vascular route requiring no maturation from monocytes. In response to a sterile injury in liver, a reservoir of fully mature F4/80(hi)GATA6(+) peritoneal cavity macrophages rapidly invaded into afflicted tissue via direct recruitment across the mesothelium. The invasion was dependent on CD44 and DAMP molecule ATP and resulted in rapid replication and switching of macrophage toward an alternatively activated phenotype. These macrophages dismantled the nuclei of necrotic cells releasing DNA and forming a cover across the injury site. Rapid invasion of mature macrophages from body cavity with capacity for induction of reparative phenotype may impact altered tissues ranging from trauma to infections to cancer. VIDEO ABSTRACT.

Sex-hormone-driven innate antibodies protect females and infants against EPEC infection.

Females have an overall advantage over males in resisting Gram-negative bacteremias, thus hinting at sexual dimorphism of immunity during infections. Here, through intravital microscopy, we observed a sex-biased difference in the capture of blood-borne bacteria by liver macrophages, a process that is critical for the clearance of systemic infections. Complement opsonization was indispensable for the capture of enteropathogenic Escherichia coli (EPEC) in male mice; however, a faster complement component 3-independent process involving abundant preexisting antibodies to EPEC was detected in female mice. These antibodies were elicited predominantly in female mice at puberty in response to estrogen regardless of microbiota-colonization conditions. Estrogen-driven antibodies were maternally transferrable to offspring and conferred protection during infancy. These antibodies were conserved in humans and recognized specialized oligosaccharides integrated into the bacterial lipopolysaccharide and capsule. Thus, an estrogen-driven, innate antibody-mediated immunological strategy conferred protection to females and their offspring.

Gata6+ Pericardial Cavity Macrophages Relocate to the Injured Heart and Prevent Cardiac Fibrosis.

Macrophages play an important role in structural cardiac remodeling and the transition to heart failure following myocardial infarction (MI). Previous research has focused on the impact of blood-derived monocytes on cardiac repair. Here we examined the contribution of resident cavity macrophages located in the pericardial space adjacent to the site of injury. We found that disruption of the pericardial cavity accelerated maladaptive post-MI cardiac remodeling. Gata6+ macrophages in mouse pericardial fluid contributed to the reparative immune response. Following experimental MI, these macrophages invaded the epicardium and lost Gata6 expression but continued to perform anti-fibrotic functions. Loss of this specialized macrophage population enhanced interstitial fibrosis after ischemic injury. Gata6+ macrophages were present in human pericardial fluid, supporting the notion that this reparative function is relevant in human disease. Our findings uncover an immune cardioprotective role for the pericardial tissue compartment and argue for the reevaluation of surgical procedures that remove the pericardium.

A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

Neutrophils during SARS-CoV-2 infection: Friend or foe?

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has resulted in more than 6 million deaths worldwide. COVID-19 is a respiratory disease characterized by pulmonary dysfunction leading to acute respiratory distress syndrome (ARDs), as well as disseminated coagulation, and multi-organ dysfunction. Neutrophils and neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of COVID-19. In this review, we highlight key gaps in knowledge, discuss the heterogeneity of neutrophils during the evolution of the disease, how they can contribute to COVID-19 pathogenesis, and potential therapeutic strategies that target neutrophil-mediated inflammatory responses.

The Neutrophil's Role During Health and Disease.

Neutrophils have always been considered as uncomplicated front-line troopers of the innate immune system equipped with limited proinflammatory duties. Yet recently, the role of the neutrophil has been undergoing a rejuvenation of sorts. Neutrophils are now considered complex cells capable of a significant array of specialized functions, and as an effector of the innate immune response, they are able to regulate many processes such as acute injury and repair, cancer, autoimmunity, and chronic inflammatory processes. Furthermore, evidence exists to indicate that neutrophils also contribute to adaptive immunity by aiding the development of specific adaptive immune responses or guiding the subsequent adaptive immune response. With this revived interest in neutrophils and their many novel functions, it is prudent to review what is currently known about neutrophils and, even more importantly, understand what information is lacking. We discuss the essential features of the neutrophil, from its origins, lifespan, subsets, margination and sequestration of the neutrophil to the death of the neutrophil. We highlight neutrophil recruitment to both infected and injured tissues and outline differences in recruitment of neutrophils between different tissues. Finally, we examine how neutrophils use different mechanisms to either bolster protective immune responses or negatively cause pathological outcomes at different locations.

Gata6+ large peritoneal macrophages: an evolutionarily conserved sentinel and effector system for infection and injury.

There are striking similarities between the sea urchin cavity macrophage-like phagocytes (coelomocytes) and mammalian cavity macrophages in not only their location, but also their behaviors. These cells are crucial for maintaining homeostasis within the cavity following a breach, filling the gap and functioning as a barrier between vital organs and the environment. In this review, we summarize the evolving literature regarding these Gata6+ large peritoneal macrophages (GLPMs), focusing on ontogeny, their responses to perturbations, including their rapid aggregation via coagulation, as well as scavenger receptor cysteine-rich domains and their potential roles in diseases, such as cancer. We challenge the 50-year old phenomenon of the 'macrophage disappearance reaction' (MDR) and propose the new term 'macrophage disturbance of homeostasis reaction' (MDHR), which may better describe this complex phenomenon.

iNKT Cells Orchestrate a Switch from Inflammation to Resolution of Sterile Liver Injury.

After traumatic injury, some cells function as detectors to sense injury and to modulate the local immune response toward a restitution phase by affecting the local cytokine milieu. Using intravital microscopy, we observed that patrolling invariant natural killer T (iNKT) cells were initially excluded from a site of hepatic injury but subsequently were strategically arrested first via self-antigens and then by cytokines, circumscribing the injured site at exactly the location where monocytes co-localized and hepatocytes proliferated. Activation of iNKT cells by self-antigens resulted in the production of interleukin-4 (IL-4) but not interferon-γ (IFN-γ). This promoted increased hepatocyte proliferation, monocyte transition (from Ly6Chi to Ly6Clo), and improved healing where IL-4 from iNKT cells was critical for these processes. Disruption of any of these mechanisms led to delayed wound healing. We have shown that self-antigen-driven iNKT cells function as sensors and orchestrators of the transformation from inflammation to tissue restitution for essential timely wound repair.

A Functional Assessment of Fetal Liver and Monocyte-Derived Macrophages in the Lung Alveolar Environment.

It is becoming clear that every organ is seeded by a population of fetal liver-derived macrophages that are replaced at different rates by monocyte-derived macrophages. Using the Ms4a3tdTomato reporter mouse that reports on monocyte-derived alveolar macrophages (Mo-AMs) and our ability to examine AM function using our multichannel intravital microscopy, we examined the fetal-liver derived alveolar macrophage (FL-AM) and Mo-AM populations within the same mouse under various environmental conditions. The experiments unveiled that AMs migrated from alveolus to alveolus and phagocytosed bacteria identically regardless of ontogenic origin. Using 50 PFU of influenza A virus (IAV) determined using the Madin-Darby canine kidney (MDCK) cell line, we noted that both populations were susceptible to IAV-induced immunoparalysis, which also led to impaired phagocytosis of secondary bacterial infections. Both FL-AMs and Mo-AMs were trained by ß-glucan to resist IAV-induced paralysis. Over time (40 wk), Mo-AMs began to outperform FL-AMs, although both populations were still sensitive to IAV. Our data also show that clodronate depletion of AMs leads to replenishment, but by FL-AMs, and these macrophages do show some functional impairment for a limited time. Overall, the system is designed such that new macrophages rapidly assume the function of tissue-resident macrophages when both populations are examined in an identical environment. These data do differ from artificial depletion methods that compare Mo-AMs and FL-AMs.

Imaging reveals novel innate immune responses in lung, liver, and beyond.

Highly dynamic immune responses are generated toward pathogens or injuries, in vivo. Multiple immune cell types participate in various facets of the response which leads to a concerted effort in the removal and clearance of pathogens or injured tissue and a return to homeostasis. Intravital microscopy (IVM) has been extensively utilized to unravel the dynamics of immune responses, visualizing immune cell behavior in intact living tissues, within a living organism. For instance, the phenomenon of leukocyte recruitment cascade. Importantly, IVM has led to a deep appreciation that immune cell behavior and responses in individual organs are distinct, but also can influence one another. In this review, we discuss how IVM as a tool has been used to study the innate immune responses in various tissues during homeostasis, injury, and infection.

Immune surveillance by the liver.

Receiving both portal vein blood and arterial blood, the liver is an important and critical component in the defense against blood-borne infection. To accomplish this role, the liver contains numerous innate and adaptive immune cells that specialize in detection and capture of pathogens from the blood. Further, these immune cells participate in coordinated immune responses leading to pathogen clearance, leukocyte recruitment and antigen presentation to lymphocytes within the vasculature. Finally, this role in host defense must be tightly regulated to ensure that inappropriate immune responses are not raised against nonpathogenic exogenous blood-borne molecules, such as those derived from food. It is this balance between activation and tolerance that characterizes the liver as a frontline immunological organ.

Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.

Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection.

Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: 'intrahepatic myeloid-cell aggregates for T cell population expansion') without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.

Acute skin exposure to ultraviolet light triggers neutrophil-mediated kidney inflammation.

Photosensitivity to ultraviolet (UV) light affects up to ∼80% of lupus patients. Sunlight exposure can exacerbate local as well as systemic manifestations of lupus, including nephritis, by mechanisms that are poorly understood. Here, we report that acute skin exposure to UV light triggers a neutrophil-dependent injury response in the kidney characterized by upregulated expression of endothelial adhesion molecules as well as inflammatory and injury markers associated with transient proteinuria. We showed that UV light stimulates neutrophil migration not only to the skin but also to the kidney in an IL-17A-dependent manner. Using a photoactivatable lineage tracing approach, we observed that a subset of neutrophils found in the kidney had transited through UV light-exposed skin, suggesting reverse transmigration. Besides being required for the renal induction of genes encoding mediators of inflammation (vcam-1, s100A9, and Il-1b) and injury (lipocalin-2 and kim-1), neutrophils significantly contributed to the kidney type I interferon signature triggered by UV light. Together, these findings demonstrate that neutrophils mediate subclinical renal inflammation and injury following skin exposure to UV light. Of interest, patients with lupus have subpopulations of blood neutrophils and low-density granulocytes with similar phenotypes to reverse transmigrating neutrophils observed in the mice post-UV exposure, suggesting that these cells could have transmigrated from inflamed tissue, such as the skin.

Neutrophils in host defense, healing, and hypersensitivity: Dynamic cells within a dynamic host.

Neutrophils are cells of the innate immune system that are extremely abundant in vivo and respond quickly to infection, injury, and inflammation. Their constant circulation throughout the body makes them some of the first responders to infection, and indeed they play a critical role in host defense against bacterial and fungal pathogens. It is now appreciated that neutrophils also play an important role in tissue healing after injury. Their short life cycle, rapid response kinetics, and vast numbers make neutrophils a highly dynamic and potentially extremely influential cell population. It has become clear that they are highly integrated with other cells of the immune system and can thus exert critical effects on the course of an inflammatory response; they can further impact tissue homeostasis and recovery after challenge. In this review, we discuss the fundamentals of neutrophils in host defense and healing; we explore the relationship between neutrophils and the dynamic host environment, including circadian cycles and the microbiome; we survey the field of neutrophils in asthma and allergy; and we consider the question of neutrophil heterogeneity-namely, whether there could be specific subsets of neutrophils that perform different functions in vivo.

Complex regulation of alarmins S100A8/A9 and secretion via gasdermin D pores exacerbates autoinflammation in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.

Ductular reaction-associated neutrophils promote biliary epithelium proliferation in chronic liver disease.

BACKGROUND & AIMS: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. METHODS: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. RESULTS: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. CONCLUSIONS: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. IMPACT AND IMPLICATIONS: Our results indicate that neutrophils are highly plastic and can have an extended lifespan. Moreover, we identify a new role of neutrophils as triggers of expansion of the biliary epithelium. Overall, the results of this study indicate that ductular reaction-associated neutrophils (or DRANs) are new players in the maladaptive tissue-healing response in chronic liver injury and may be a potential target for therapeutic interventions to reduce ductular reaction expansion and promote tissue repair in advanced liver disease.