The Annexin A1/FPR2 pathway controls the inflammatory response and bacterial dissemination in experimental pneumococcal pneumonia.

Streptococcus pneumoniae is a major cause of community-acquired pneumonia leading to high mortality rates. Inflammation triggered by pneumococcal infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Annexin A1 (AnxA1) mainly acts through Formyl Peptide Receptor 2 (FPR2) inducing the resolution of inflammation. Here, we have evaluated the role of AnxA1 and FPR2 during pneumococcal pneumonia in mice. For that, AnxA1, Fpr2/3 knockout (KO) mice and wild-type (WT) controls were infected intranasally with S pneumoniae. AnxA1 and Fpr2/3 KO mice were highly susceptible to infection, displaying uncontrolled inflammation, increased bacterial dissemination, and pulmonary dysfunction compared to WT animals. Mechanistically, the absence of AnxA1 resulted in the loss of lung barrier integrity and increased neutrophil activation upon S pneumoniae stimulation. Importantly, treatment of WT or AnxA1 KO-infected mice with Ac2-26 decreased inflammation, lung damage, and bacterial burden in the airways by increasing macrophage phagocytosis. Conversely, Ac2-26 peptide was ineffective to afford protection in Fpr2/3 KO mice during infection. Altogether, these findings show that AnxA1, via FPR2, controls inflammation and bacterial dissemination during pneumococcal pneumonia by promoting host defenses, suggesting AnxA1-based peptides as a novel therapeutic strategy to control pneumococcal pneumonia.

Immune and metabolic shifts during neonatal development reprogram liver identity and function.

BACKGROUND & AIMS: The liver is the main hematopoietic site in embryos, becoming a crucial organ in both immunity and metabolism in adults. However, how the liver adapts both the immune system and enzymatic profile to challenges in the postnatal period remains elusive. We aimed to identify the mechanisms underlying this adaptation. METHODS: We analyzed liver samples from mice on day 0 after birth until adulthood. Human biopsies from newborns and adults were also examined. Liver immune cells were phenotyped using mass cytometry (CyTOF) and expression of several genes belonging to immune and metabolic pathways were measured. Mortality rate, bacteremia and hepatic bacterial retention after E. coli challenge were analyzed using intravital and in vitro approaches. In a set of experiments, mice were prematurely weaned and the impact on gene expression of metabolic pathways was evaluated. RESULTS: Human and mouse newborns have a sharply different hepatic cellular composition and arrangement compared to adults. We also found that myeloid cells and immature B cells primarily compose the neonatal hepatic immune system. Although neonatal mice were more susceptible to infections, a rapid evolution to an efficient immune response was observed. Concomitantly, newborns displayed a reduction of several macronutrient metabolic functions and the normal expression level of enzymes belonging to lipid and carbohydrate metabolism was reached around the weaning period. Interestingly, early weaning profoundly disturbed the expression of several hepatic metabolic pathways, providing novel insights into how dietary schemes affect the metabolic maturation of the liver. CONCLUSION: In newborns, the immune and metabolic profiles of the liver are dramatically different to those of the adult liver, which can be explained by the differences in the liver cell repertoire and phenotype. Also, dietary and antigen cues may be crucial to guide liver development during the postnatal phase. LAY SUMMARY: Newborns face major challenges in the extra-uterine life. In fact, organs need to modify their cellular composition and gene expression profile in order to adapt to changes in both microbiota and diet throughout life. The liver is interposed between the gastrointestinal system and the systemic circulation, being the destination of all macronutrients and microbial products from the gut. Therefore, it is expected that delicately balanced mechanisms govern the transformation of a neonatal liver to a key organ in adults.

Susceptibility to Infections During Acute Liver Injury Depends on Transient Disruption of Liver Macrophage Niche.

Kupffer cells are the primary liver resident immune cell responsible for the liver firewall function, including clearance of bacterial infection from the circulation, as they are strategically positioned inside the liver sinusoid with intimate contact with the blood. Disruption in the tissue-resident macrophage niche, such as in Kupffer cells, can lead to a window of susceptibility to systemic infections, which represents a significant cause of mortality in patients with acetaminophen (APAP) overdose-induced acute liver injury (ALI). However, how Kupffer cell niche disruption increases susceptibility to systemic infections in ALI is not fully understood. Using a mouse model of ALI induced by APAP overdose, we found that Kupffer cells upregulated the apoptotic cell death program and were markedly reduced in the necrotic areas during the early stages of ALI, opening the niche for the infiltration of neutrophils and monocyte subsets. In addition, during the resolution phase of ALI, the remaining tissue macrophages with a Kupffer cell morphology were observed forming replicating cell clusters closer to necrotic areas devoid of Kupffer cells. Interestingly, mice with APAP-induced liver injury were still susceptible to infections despite the dual cellular input of circulating monocytes and proliferation of remaining Kupffer cells in the damaged liver. Therapy with bone marrow-derived macrophages (BMDM) was shown to be effective in occupying the niche devoid of Kupffer cells following APAP-induced ALI. The rapid BMDM migration to the liver and their positioning within necrotic areas enhanced the healing of the tissue and restored the liver firewall function after BMDM therapy. Therefore, we showed that disruption in the Kupffer cell niche and its impaired function during acute liver injury are key factors for the susceptibility to systemic bacterial infections. In addition, modulation of the liver macrophage niche was shown to be a promising therapeutic strategy for liver injuries that reduce the Kupffer cell number and compromise the organ function.

Corrigendum: Susceptibility to infections during acute liver injury depends on transient disruption of liver macrophage niche.

[This corrects the article DOI: 10.3389/fimmu.2022.892114.].

High-dimensional intravital microscopy reveals major changes in splenic immune system during postnatal development.

Spleen is a key organ for immunologic surveillance, acting as a firewall for antigens and parasites that spread through the blood. However, how spleen leukocytes evolve across the developmental phase, and how they spatially organize and interact in vivo is still poorly understood. Using a novel combination of selected antibodies and fluorophores to image in vivo the spleen immune environment, we described for the first time the dynamics of immune development across postnatal period. We found that spleens from adults and infants had similar numbers and arrangement of lymphoid cells. In contrast, splenic immune environment in newborns is sharply different from adults in almost all parameters analysed. Using this in vivo approach, B cells were the most frequent subtype throughout the development. Also, we revealed how infections - using a model of malaria - can change the spleen immune profile in adults and infants, which could become the key to understanding different severity grades of infection. Our new imaging solutions can be extremely useful for different groups in all areas of biological investigation, paving a way for new intravital approaches and advances.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

Chronic ingestion of Primex-Z, compared with other common fat sources, drives worse liver injury and enhanced susceptibility to bacterial infections.

OBJECTIVES: The aim of this study was to investigate putative different outcomes on the development of non-alcoholic fatty liver disease in mice using fat options regularly used in human nutrition. METHODS: Male C57BL/6 mice were fed a control diet, and four different high-fat diets (HFD: 40% calories from fat; Research Diet, Inc., New Brunswick, New Jersey, USA) for 16 and 30 wk. HFDs had different common fat sources, including trans-fat, non-trans-fat palm oil (Primex-Z), palm oil alone, and corn oil alone. Mice were sacrificed and samples were collected for analysis. RESULTS: Using an unprecedented combination of in vivo imaging with immunometabolic phenotyping, we revealed that a HFD induced a major increase in hepatic lipid droplet deposition compared with control mice, being significantly higher in Primex-Z-fed mice. All HFD mice had similar or less weight gain as control mice; however, Primex-Z ingestion led to a higher increase in adiposity index (~90% increase) compared with other fat sources. Gene expression of isolated liver immune cells revealed large changes in expression of several inflammatory pathways, which were also more elevated in Primex-Z-fed mice, including Tnf (~20-fold), Il1b (~60-fold), and Tgfb (2.5-fold). Immunophenotyping and in vivo analysis showed that the frequency of hepatic immune cells was also disturbed during different HFD contents, rendering not only Kupffer cell depletion, but also reduced bacterial arresting ability. CONCLUSION: Different fat dietary sources imprint different immune and metabolic effects in the liver during consumption of an HFD. The present data highlighted that Primex-Z-a novel non-trans-fat-is not only able to damage hepatocytes, but also to impair liver ability to clear blood-borne infections.

Th1-Th2 Cross-Regulation Controls Early Leishmania Infection in the Skin by Modulating the Size of the Permissive Monocytic Host Cell Reservoir.

The impact of T helper (Th) 1 versus Th2 immunity on intracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility. However, observations in multiple infectious settings demonstrate deficiencies in mediators of Th1-Th2 immunity, which have paradoxical or no impact. We report that prior to influencing activation, Th1/Th2 immunity first controls the size of the permissive host cell reservoir. During early Leishmania infection of the skin, IFN-γ- or STAT6-mediated changes in phagocyte activation were counteracted by changes in IFN-γ-mediated recruitment of permissive CCR2+ monocytes. Monocytes were required for early parasite expansion and acquired an alternatively activated phenotype despite the Th1 dermal environment required for their recruitment. Surprisingly, STAT6 did not enhance intracellular parasite proliferation, but rather modulated the size and permissiveness of the monocytic host cell reservoir via regulation of IFN-γ and IL-10. These observations expand our understanding of the Th1-Th2 paradigm during infection.

Prolonged neutrophil survival at necrotic sites is a fundamental feature for tissue recovery and resolution of hepatic inflammation.

Neutrophils were classically described as powerful effectors of acute inflammation, and their main purpose was assumed to be restricted to pathogen killing through production of oxidants. As consequence, neutrophils also may lead to significant collateral damage to the healthy tissues, and after performing these tasks, these leukocytes are supposed to die within tissues. However, there is a growing body of evidence showing that neutrophils also play a pivotal role in the resolution phases of inflammation, because they can modulate tissue environment due to secretion of different kind of cytokines. Drug-induced liver injury (DILI) is a worldwide concern being one of the most prevalent causes of liver transplantation, and is well established that there is an intense neutrophil recruitment into necrotic liver during DILI. However, information if such abundant granulocyte infiltration is also linked to the tissue repairing phase of hepatic injury is still largely elusive. Here, we investigated the dynamics of neutrophil trafficking within blood, bone marrow, and liver during hepatic inflammation, and how changes in their gene expression profile could drive the resolution events during acetaminophen (APAP)-induced liver injury. We found that neutrophils remained viable during longer periods following liver damage, because they avidly patrolled necrotic areas and up-regulated pro-resolutive genes, including Tgfb, Il1r2, and Fpr2. Adoptive transference of "resolutive neutrophils" harvested from livers at 72 h after injury to mice at the initial phases of injury (6 h after APAP) significantly rescued organ injury. Thus, we provide novel insights on the role of neutrophils not only in the injury amplification, but also in the resolution phases of inflammation.

Imaging and immunometabolic phenotyping uncover changes in the hepatic immune response in the early phases of NAFLD.

BACKGROUND & AIMS: The precise determination of non-alcoholic fatty liver disease (NAFLD) onset is challenging. Thus, the initial hepatic responses to fat accumulation, which may be fundamental to our understanding of NAFLD evolution and clinical outcomes, are largely unknown. Herein, we chronologically mapped the immunologic and metabolic changes in the liver during the early stages of fatty liver disease in mice and compared this with human NAFLD samples. METHODS: Liver biopsies from patients with NAFLD (NAFLD activity score [NAS] 2-3) were collected for gene expression profiling. Mice received a high-fat diet for short periods to mimic initial steatosis and the hepatic immune response was investigated using a combination of confocal intravital imaging, gene expression, cell isolation, flow cytometry and bone marrow transplantation assays. RESULTS: We observed major immunologic changes in patients with NAS 2-3 and in mice in the initial stages of NAFLD. In mice, these changes significantly increased mortality rates upon drug-induced liver injury, as well as predisposing mice to bacterial infections. Moreover, deletion of Toll-like receptor 4 in liver cells dampened tolerogenesis, particularly in Kupffer cells, in the initial stages of dietary insult. CONCLUSION: The hepatic immune system acts as a sentinel for early and minor changes in hepatic lipid content, mounting a biphasic response upon dietary insult. Priming of liver immune cells by gut-derived Toll-like receptor 4 ligands plays an important role in liver tolerance in initial phases, but continuous exposure to insults may lead to damage and reduced ability to control infections. LAY SUMMARY: Fatty liver is a very common form of hepatic disease, leading to millions of cases of cirrhosis every year. Patients are often asymptomatic until becoming very sick. Therefore, it is important that we expand our knowledge of the early stages of disease pathogenesis, to enable early diagnosis. Herein, we show that even in the early stages of fatty liver disease, there are significant alterations in genes involved in the inflammatory response, suggesting that the hepatic immune system is disturbed even following minor and undetectable changes in liver fat content. This could have implications for the diagnosis and clinical management of fatty liver disease.

Paradoxical Role of Matrix Metalloproteinases in Liver Injury and Regeneration after Sterile Acute Hepatic Failure.

Acetaminophen (APAP) poisoning is one of the leading causes of acute hepatic failure and liver transplantation is often the only lifesaving alternative. During the course of hepatocyte necrosis, an intense accumulation of neutrophils is often observed within the liver microenvironment. Despite the classic idea that neutrophil accumulation in tissues causes collateral tissue damage, there is a growing body of evidence showing that neutrophils can also orchestrate the resolution of inflammation. In this work, drug-induced liver injury was induced by oral administration of APAP and pharmacological intervention was made 12 h after this challenge. Liver injury and repair kinetics were evaluated by a novel combination of enzyme quantifications, ELISA, specific antagonists of neutrophil enzymes and confocal intravital microscopy. We have demonstrated that neutrophil infiltration is not only involved in injury amplification, but also in liver tissue repair after APAP-induced liver injury. In fact, while neutrophil depletion led to reduced hepatic necrosis during APAP poisoning, injury recovery was also delayed in neutropenic mice. The mechanisms underlying the neutrophil reparative role involved rapid degranulation and matrix metalloproteinases (MMPs) activity. Our data highlights the crucial role of neutrophils, in particular for MMPs, in the resolution phase of APAP-induced inflammatory response.

The multifaceted role of commensal microbiota in homeostasis and gastrointestinal diseases.

The gastrointestinal tract houses a complex and diverse community of microbes. In recent years, an increased understanding of the importance of intestinal microbiota for human physiology has been gained. In the steady state, commensal microorganisms have a symbiotic relationship with the host and possess critical and distinct functions, including directly influencing immunity. This means that recognition of commensal antigens is necessary for the development of complete immune responses. Therefore, the immune system must face the challenge of maintaining mucosal homeostasis while dealing with undue passage of commensal or pathogenic microbes, as well as the host nutritional status or drug use. Disruption of this fine balance has been associated with the development of several intestinal inflammatory diseases. In this review, we discuss the mechanisms involved in the modulation of host-microbe interactions and how the breakdown of this homeostatic association can lead to intestinal inflammation and pathology.

IFN-γ-Dependent Recruitment of CD4(+) T Cells and Macrophages Contributes to Pathogenesis During Leishmania amazonensis Infection.

Interferon gamma (IFN-γ) is a key factor in the protection of hosts against intracellular parasites. This cytokine induces parasite killing through nitric oxide and reactive oxygen species production by phagocytes. Surprisingly, during Leishmania amazonensis infection, IFN-γ plays controversial roles. During in vitro infections, IFN-γ induces the proliferation of the amastigote forms of L. amazonensis. However, this cytokine is not essential at the beginning of an in vivo infection. It is not clear why IFN-γ does not mediate protection during the early stages of infection. Thus, the aim of our study was to investigate the role of IFN-γ during L. amazonensis infection. We infected IFN-γ(-/-) mice in the footpad and followed the development of leishmaniasis in these mice compared with that in WT mice. CD4(+) T lymphocytes and macrophages migrated earlier to the site of infection in the WT mice, and the earlier migration of these 2 cell types was associated with lesion development and parasite growth, respectively. These differences in the infiltrate populations were explained by the increased expression of chemokines in the lesions of the WT mice. Thus, we propose that IFN-γ plays a dual role during L. amazonensis infection; it is an important inducer of effector mechanisms, particularly through inducible nitric oxide synthase expression, and conversely, it is a mediator of inflammation and pathogenesis through the induction of the expression of chemokines. Our data provided evidence for a pathogenic effect of IFN-γ production during leishmaniasis that was previously unknown.