Macrophages marc(o) the difference in liver inflammation?

Miyamoto et al. report that Marco expression demarcates a population of IL-10-expressing immunosuppressive Kupffer cells (KCs) that are preferentially peri-portally located in the mouse liver, and which limit bacterial dissemination and liver inflammation.

A prognostic molecular signature of hepatic steatosis is spatially heterogeneous and dynamic in human liver.

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing in parallel with an obesity pandemic, calling for novel strategies for prevention and treatment. We defined a circulating proteome of human MASLD across ≈7000 proteins in ≈5000 individuals from diverse, at-risk populations across the metabolic health spectrum, demonstrating reproducible diagnostic performance and specifying both known and novel metabolic pathways relevant to MASLD (central carbon and amino acid metabolism, hepatocyte regeneration, inflammation, fibrosis, insulin sensitivity). A parsimonious proteomic signature of MASLD was associated with a protection from MASLD and its related multi-system metabolic consequences in >26000 free-living individuals, with an additive effect to polygenic risk. The MASLD proteome was encoded by genes that demonstrated transcriptional enrichment in liver, with spatial transcriptional activity in areas of steatosis in human liver biopsy and dynamicity for select targets in human liver across stages of steatosis. We replicated several top relations from proteomics and spatial tissue transcriptomics in a humanized "liver-on-a-chip" model of MASLD, highlighting the power of a full translational approach to discovery in MASLD. Collectively, these results underscore utility of blood-based proteomics as a dynamic "liquid biopsy" of human liver relevant to clinical biomarker and mechanistic applications.

Osteopontin characterizes bile duct-associated macrophages and correlates with liver fibrosis severity in primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated cholestatic liver disease for which pharmacological treatment options are currently unavailable. PSC is strongly associated with colitis and a disruption of the gut-liver axis, and macrophages are involved in the pathogenesis of PSC. However, how gut-liver interactions and specific macrophage populations contribute to PSC is incompletely understood. APPROACH AND RESULTS: We investigated the impact of cholestasis and colitis on the hepatic and colonic microenvironment, and performed an in-depth characterization of hepatic macrophage dynamics and function in models of concomitant cholangitis and colitis. Cholestasis-induced fibrosis was characterized by depletion of resident KCs, and enrichment of monocytes and monocyte-derived macrophages (MoMFs) in the liver. These MoMFs highly express triggering-receptor-expressed-on-myeloid-cells-2 ( Trem2 ) and osteopontin ( Spp1 ), markers assigned to hepatic bile duct-associated macrophages, and were enriched around the portal triad, which was confirmed in human PSC. Colitis induced monocyte/macrophage infiltration in the gut and liver, and enhanced cholestasis-induced MoMF- Trem2 and Spp1 upregulation, yet did not exacerbate liver fibrosis. Bone marrow chimeras showed that knockout of Spp1 in infiltrated MoMFs exacerbates inflammation in vivo and in vitro , while monoclonal antibody-mediated neutralization of SPP1 conferred protection in experimental PSC. In human PSC patients, serum osteopontin levels are elevated compared to control, and significantly increased in advanced stage PSC and might serve as a prognostic biomarker for liver transplant-free survival. CONCLUSIONS: Our data shed light on gut-liver axis perturbations and macrophage dynamics and function in PSC and highlight SPP1/OPN as a prognostic marker and future therapeutic target in PSC.

Studying Macrophages in the Murine Steatotic Liver Using Flow Cytometry and Confocal Microscopy.

The study of macrophage functions in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction associated steatohepatitis (MASH) has been hampered by the fact that until recently all macrophages in the liver were thought to be Kupffer cells, the resident macrophages of the liver. With the advent of single-cell technologies, it is now clear that the steatotic liver harbors many distinct populations of macrophages, likely each with their own unique functions as well as subsets of monocytes and dendritic cells which can be difficult to discriminate from one another. Here, we detail the protocols we utilize to (i) induce MASLD/MASH in mice, (ii) isolate cells from the steatotic liver, and (iii) describe reliable gating strategies, which can be used to identify the different subsets of myeloid cells. Finally, we also discuss the issue of increased autofluorescence in the steatotic liver and the techniques we use to minimize this both for flow cytometry and confocal microscopy analyses.

Transient Kupffer cell depletion and subsequent replacement by infiltrating monocyte-derived cells does not alter the induction or progression of hepatocellular carcinoma.

Due to a combination of rapid disease progression and the lack of curative treatment options, hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide. Infiltrated, monocyte-derived, tumor-associated macrophages are known to play a role in HCC pathogenesis, but the involvement of Kupffer cells (KCs) remains elusive. Here, we used the Clec4F-diphteria toxin receptor transgenic mouse model to specifically investigate the effect of KC depletion on HCC initiation, progression and neoplastic growth following liver resection. For this purpose, several HCC mouse models with varying underlying etiologies were used and partial hepatectomy was performed. Our results show that in HCC, developed on a fibrotic or non-alcoholic steatohepatitis background, depletion of embryonic KCs at the onset of HCC induction and the subsequent replacement by monocyte-derived KCs does not affect the tumor burden, tumor microenvironment or the phenotype of isolated KCs at end-stage disease. In non-chronic liver disease-associated diethylnitrosamine-induced HCC, ablation of Clec4F+ KCs did not alter tumor progression or neoplastic growth following liver resection. Our results show that temporal ablation of resident KCs does not impact HCC pathogenesis, neither in the induction phase nor in advanced disease, and indicate that bone marrow-derived KCs are able to swiftly repopulate the available KC niche and adopt their phenotype.

A20 is a master switch of IL-33 signaling in macrophages and determines IL-33-induced lung immunity.

BACKGROUND: IL-33 plays a major role in the pathogenesis of allergic diseases such as asthma and atopic dermatitis. On its release from lung epithelial cells, IL-33 primarily drives type 2 immune responses, accompanied by eosinophilia and robust production of IL-4, IL-5, and IL-13. However, several studies show that IL-33 can also drive a type 1 immune response. OBJECTIVE: We sought to determine the role of A20 in the regulation of IL-33 signaling in macrophages and IL-33-induced lung immunity. METHODS: We studied the immunologic response in lungs of IL-33-treated mice that specifically lack A20 in myeloid cells. We also analyzed IL-33 signaling in A20-deficient bone marrow-derived macrophages. RESULTS: IL-33-induced lung innate lymphoid cell type 2 expansion, type 2 cytokine production, and eosinophilia were drastically reduced in the absence of macrophage A20 expression, whereas neutrophils and interstitial macrophages in lungs were increased. In vitro, IL-33-mediated nuclear factor kappa B activation was only weakly affected in A20-deficient macrophages. However, in the absence of A20, IL-33 gained the ability to activate signal transducer and activator of transcription 1 (STAT1) signaling and STAT1-dependent gene expression. Surprisingly, A20-deficient macrophages produced IFN-γ in response to IL-33, which was fully STAT1-dependent. Furthermore, STAT1 deficiency partially restored the ability of IL-33 to induce ILC2 expansion and eosinophilia in myeloid cell-specific A20 knockout mice. CONCLUSIONS: We reveal a novel role for A20 as a negative regulator of IL-33-induced STAT1 signaling and IFN-γ production in macrophages, which determines lung immune responses.

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes.

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages.

Liver macrophages in health and disease.

Single-cell and spatial transcriptomic technologies have revealed an underappreciated heterogeneity of liver macrophages. This has led us to rethink the involvement of macrophages in liver homeostasis and disease. Identification of conserved gene signatures within these cells across species and diseases is enabling the correct identification of specific macrophage subsets and the generation of more specific tools to track and study the functions of these cells. Here, we discuss what is currently known about the definitions of these different macrophage populations, the markers that can be used to identify them, how they are wired within the liver, and their functional specializations in health and disease.

Modulating hepatic macrophages with annexin A1 in non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) and associated end-stage liver disease is a growing cause of concern throughout the Western world. It constitutes a significant clinical burden for which therapeutic approaches are very limited. Over the last years, considerable attention has therefore been paid to identifying potential therapeutic strategies to reduce this burden. Annexin A1 (AnxA1), a calcium-phospholipid binding protein, has been proposed to be a negative regulator of inflammation in the context of NASH. In a recent publication, Gadipudi, Ramavath, Provera et al. investigated the therapeutic potential of Annexin A1 treatment in preventing the progression of NASH. They demonstrate that treatment of mice with NASH with recombinant human AnxA1 can reduce inflammation and fibrosis without affecting steatosis or metabolic syndrome. This was proposed to be achieved through the modulation of the macrophage populations present in the liver. Here, we discuss the main findings of this work and raise some outstanding questions regarding the possible mechanisms involved and the functions of distinct macrophage populations in NASH.

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Welcoming c-MAF to the macrophage transcription factor VAM-ily.

The transcription factor c-MAF regulates perivascular macrophage phenotypes across tissues, including white adipose tissue, where its loss from murine vasculature­associated macrophages leads to increased vascularization and protection from metabolic syndrome (see the related Research Article by Moura Silva et al.).

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

In matters of the heart, (cellular) communication is key.

To maintain cardiac output, the failing heart undergoes significant remodeling. However, the mechanisms regulating this remain unclear. In this issue of Immunity, Zaman et al. and Wong, Mohan, and Kopecky et al. uncover an interaction between resident cardiac macrophages and cardiomyocytes governing this process.

Hepatic Macrophage Responses in Inflammation, a Function of Plasticity, Heterogeneity or Both?

With the increasing availability and accessibility of single cell technologies, much attention has been given to delineating the specific populations of cells present in any given tissue. In recent years, hepatic macrophage heterogeneity has also begun to be examined using these strategies. While previously any macrophage in the liver was considered to be a Kupffer cell (KC), several studies have recently revealed the presence of distinct subsets of hepatic macrophages, including those distinct from KCs both under homeostatic and non-homeostatic conditions. This heterogeneity has brought the concept of macrophage plasticity into question. Are KCs really as plastic as once thought, being capable of responding efficiently and specifically to any given stimuli? Or are the differential responses observed from hepatic macrophages in distinct settings due to the presence of multiple subsets of these cells? With these questions in mind, here we examine what is currently understood regarding hepatic macrophage heterogeneity in mouse and human and examine the role of heterogeneity vs plasticity in regards to hepatic macrophage responses in settings of both pathogen-induced and sterile inflammation.

ILC3s control splenic cDC homeostasis via lymphotoxin signaling.

The spleen contains a myriad of conventional dendritic cell (cDC) subsets that protect against systemic pathogen dissemination by bridging antigen detection to the induction of adaptive immunity. How cDC subsets differentiate in the splenic environment is poorly understood. Here, we report that LTα1ß2-expressing Rorgt+ ILC3s, together with B cells, control the splenic cDC niche size and the terminal differentiation of Sirpα+CD4+Esam+ cDC2s, independently of the microbiota and of bone marrow pre-cDC output. Whereas the size of the splenic cDC niche depended on lymphotoxin signaling only during a restricted time frame, the homeostasis of Sirpα+CD4+Esam+ cDC2s required continuous lymphotoxin input. This latter property made Sirpα+CD4+Esam+ cDC2s uniquely susceptible to pharmacological interventions with LTßR agonists and antagonists and to ILC reconstitution strategies. Together, our findings demonstrate that LTα1ß2-expressing Rorgt+ ILC3s drive splenic cDC differentiation and highlight the critical role of ILC3s as perpetual regulators of lymphoid tissue homeostasis.

Osteopontin Expression Identifies a Subset of Recruited Macrophages Distinct from Kupffer Cells in the Fatty Liver.

Metabolic-associated fatty liver disease (MAFLD) represents a spectrum of disease states ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs), are suggested to play important roles in the pathogenesis of MAFLD through their activation, although the exact roles played by these cells remain unclear. Here, we demonstrated that KCs were reduced in MAFLD being replaced by macrophages originating from the bone marrow. Recruited macrophages existed in two subsets with distinct activation states, either closely resembling homeostatic KCs or lipid-associated macrophages (LAMs) from obese adipose tissue. Hepatic LAMs expressed Osteopontin, a biomarker for patients with NASH, linked with the development of fibrosis. Fitting with this, LAMs were found in regions of the liver with reduced numbers of KCs, characterized by increased Desmin expression. Together, our data highlight considerable heterogeneity within the macrophage pool and suggest a need for more specific macrophage targeting strategies in MAFLD.

Macrophage Subsets in Obesity, Aligning the Liver and Adipose Tissue.

The increasing prevalence of obesity is accompanied by a rising incidence in metabolic syndrome and related pathologies such as non-alcoholic fatty liver disease. Macrophages are hypothesized to play central roles in these diseases, through their role as inflammatory mediators and as such are thought to be potential targets for future therapies. Recently, single cell technologies have revealed significant heterogeneity within the macrophage pool in both liver and adipose tissue in obesity. Thus current efforts are focused on dissecting this heterogeneity and understanding the distinct functions of the individual subsets. In this review, we discuss the current knowledge regarding macrophage heterogeneity, ontogeny and functions in the context of obese adipose tissue and fatty liver disease and attempt to align the distinct populations described to date.

Inflammatory Type 2 cDCs Acquire Features of cDC1s and Macrophages to Orchestrate Immunity to Respiratory Virus Infection.

The phenotypic and functional dichotomy between IRF8+ type 1 and IRF4+ type 2 conventional dendritic cells (cDC1s and cDC2s, respectively) is well accepted; it is unknown how robust this dichotomy is under inflammatory conditions, when additionally monocyte-derived cells (MCs) become competent antigen-presenting cells (APCs). Using single-cell technologies in models of respiratory viral infection, we found that lung cDC2s acquired expression of the Fc receptor CD64 shared with MCs and of IRF8 shared with cDC1s. These inflammatory cDC2s (inf-cDC2s) were superior in inducing CD4+ T helper (Th) cell polarization while simultaneously presenting antigen to CD8+ T cells. When carefully separated from inf-cDC2s, MCs lacked APC function. Inf-cDC2s matured in response to cell-intrinsic Toll-like receptor and type 1 interferon receptor signaling, upregulated an IRF8-dependent maturation module, and acquired antigens via convalescent serum and Fc receptors. Because hybrid inf-cDC2s are easily confused with monocyte-derived cells, their existence could explain why APC functions have been attributed to MCs.

Profiling peripheral nerve macrophages reveals two macrophage subsets with distinct localization, transcriptome and response to injury.

While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relmα+Mgl1+ snMacs in the epineurium and Relmα-Mgl1- snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.