In situ expansion and reprogramming of Kupffer cells elicit potent tumoricidal immunity against liver metastasis.

Liver metastasis represents one of the most frequent malignant diseases with no effective treatment. Functional reprogramming of Kupffer cells (KCs), the largest population of hepatic macrophages, holds promise for treating liver cancer, but remains seldom exploited. Taking advantage of the superior capacity of KCs to capture circulating bacteria, we report that a single administration of attenuated Escherichia coli producing clustered regularly interspersed short palindromic repeats CasΦ (CRISPR/CasΦ) machinery enables efficient editing of genes of interest in KCs. Using intravital microscopy, we observed a failure of tumor control by KCs at the late stage of liver metastasis due to KC loss preferentially in the tumor core and periphery, resulting in inaccessibility of these highly phagocytic macrophages to cancer cells. Simultaneous disruption of MafB and c-Maf expression using the aforementioned engineered bacteria could overcome KC dysfunction and elicit remarkable curative effects against several types of metastatic liver cancer in mice. Mechanistically, bacterial treatment induced massive proliferation and functional reprogramming of KCs. These cells infiltrated into the tumor, dismantled macrometastases by nibbling cancer cells, and skewed toward proinflammatory macrophages to unleash antitumor T cell responses. These findings provide an immunotherapy strategy that could be applicable for treating liver metastasis and highlight the therapeutic potential of targeting tissue-resident macrophages in cancer.

Functional vulnerability of liver macrophages to capsules defines virulence of blood-borne bacteria.

Many encapsulated bacteria use capsules to cause invasive diseases. However, it remains largely unknown how the capsules enhance bacterial virulence under in vivo infection conditions. Here we show that the capsules primarily target the liver to enhance bacterial survival at the onset of blood-borne infections. In a mouse sepsis model, the capsules enabled human pathogens Streptococcus pneumoniae and Escherichia coli to circumvent the recognition of liver-resident macrophage Kupffer cells (KCs) in a capsular serotype-dependent manner. In contrast to effective capture of acapsular bacteria by KCs, the encapsulated bacteria are partially (low-virulence types) or completely (high-virulence types) "untouchable" for KCs. We finally identified the asialoglycoprotein receptor (ASGR) as the first known capsule receptor on KCs to recognize the low-virulence serotype-7F and -14 pneumococcal capsules. Our data identify the molecular interplay between the capsules and KCs as a master controller of the fate and virulence of encapsulated bacteria, and suggest that the interplay is targetable for therapeutic control of septic infections.

Single-cell profiling of the human decidual immune microenvironment in patients with recurrent pregnancy loss.

Maintaining homeostasis of the decidual immune microenvironment at the maternal-fetal interface is essential for placentation and reproductive success. Although distinct decidual immune cell subpopulations have been identified under normal conditions, systematic understanding of the spectrum and heterogeneity of leukocytes under recurrent miscarriage in human deciduas remains unclear. To address this, we profiled the respective transcriptomes of 18,646 primary human decidual immune cells isolated from patients with recurrent pregnancy loss (RPL) and healthy controls at single-cell resolution. We discovered dramatic differential distributions of immune cell subsets in RPL patients compared with the normal decidual immune microenvironment. Furthermore, we found a subset of decidual natural killer (NK) cells that support embryo growth were diminished in proportion due to abnormal NK cell development in RPL patients. We also elucidated the altered cellular interactions between the decidual immune cell subsets in the microenvironment and those of the immune cells with stromal cells and extravillous trophoblast under disease state. These results provided deeper insights into the RPL decidual immune microenvironment disorder that are potentially applicable to improve the diagnosis and therapeutics of this disease.

Voluntary-Opsonization-Enabled Precision Nanomedicines for Inflammation Treatment.

Nanomedicines that target specific blood cells represent an emerging strategy to improve drug biodistribution. However, the protein corona usually disrupts nanomedicine targeting to cells and tissues. Herein, instead of exploring synthetic methods to mitigate the impact of the protein corona, its natural interactions with blood cells are leveraged and turn the protein corona into an active ingredient in treating lung inflammation. It is discovered that molecularly engineered liposomes with inverse phosphocholine lipids rapidly enrich complement fragment iC3b by "voluntary opsonization," which triggers neutrophil hijacking through complement receptor 3 phagocytosis. This neutrophil targeting is cell-state dependent as only those activated by acute inflammation display efficient neutrophil reconstruction. The liposome-loaded neutrophils migrate across the alveolar-capillary barrier, accumulate in the inflamed lung parenchyma within hours, and release their payloads to kill the bacteria. This work shows that, in addition to biological cells, the protein corona can be a new platform for active and precision nanomedicine.

Programmed Death Ligand 1 Is Overexpressed in Liver Macrophages in Chronic Liver Diseases, and Its Blockade Improves the Antibacterial Activity Against Infections.

BACKGROUND AND AIMS: Bacterial infections are common and severe in cirrhosis, but their pathogenesis is poorly understood. Dysfunction of liver macrophages may play a role, but information about their function in cirrhosis is limited. Our aims were to investigate the specific profile and function of liver macrophages in cirrhosis and their contribution to infections. Macrophages from human cirrhotic livers were characterized phenotypically by transcriptome analysis and flow cytometry; function was assessed in vivo by single photon emission computerized tomography in patients with cirrhosis. Serum levels of specific proteins and expression in peripheral monocytes were determined by ELISA and flow cytometry. In vivo phagocytic activity of liver macrophages was measured by spinning disk intravital microscopy in a mouse model of chronic liver injury. APPROACH AND RESULTS: Liver macrophages from patients with cirrhosis overexpressed proteins related to immune exhaustion, such as programmed death ligand 1 (PD-L1), macrophage receptor with collagenous structure (MARCO), and CD163. In vivo phagocytic activity of liver macrophages in patients with cirrhosis was markedly impaired. Monocytes from patients with cirrhosis showed overexpression of PD-L1 that paralleled disease severity, correlated with its serum levels, and was associated with increased risk of infections. Blockade of PD-L1 with anti-PD-L1 antibody caused a shift in macrophage phenotype toward a less immunosuppressive profile, restored liver macrophage in vivo phagocytic activity, and reduced bacterial dissemination. CONCLUSION: Liver cirrhosis is characterized by a remarkable impairment of phagocytic function of macrophages associated with an immunosuppressive transcriptome profile. The programmed cell death receptor 1/PD-L1 axis plays a major role in the impaired activity of liver macrophages. PD-L1 blockade reverses the immune suppressive profile and increases antimicrobial activity of liver macrophages in cirrhosis.

Live Imaging of Innate and Adaptive Immune Responses in the Liver.

Immune response in the liver is determined by the spatial organization and cellular dynamics of hepatic immune cells. The liver vasculature accommodates abundant tissue-resident innate immune cells, such as Kupffer cells, natural killer cells, and natural killer T cells, to ensure efficient intravascular immunosurveillance. The fenestrated sinusoids also allow direct contact between circulating T cells and non-canonical antigen-presenting cells, such as hepatocytes, to instruct adaptive immune responses. Distinct cellular behaviors are exploited by liver immune cells to exert proper functions. Intravital imaging enables real-time visualization of individual immune cell in living animals, representing a powerful tool in dissecting the spatiotemporal features of intrahepatic immune cells during steady state and liver diseases. This review summarizes current advances in liver immunology prompted by in vivo imaging, with a particular focus on liver-resident innate immune cells and hepatic T cells.

Neutrophil Extracellular Traps Confine Pseudomonas aeruginosa Ocular Biofilms and Restrict Brain Invasion.

Bacterial biofilm infections are difficult to eradicate because of antibiotic insusceptibility and high recurrence rates. Biofilm formation by Pseudomonas aeruginosa, a leading cause of bacterial keratitis, is facilitated by the bacterial Psl exopolysaccharide and associated with heightened virulence. Using intravital microscopy, we observed that neutrophilic recruitment to corneal infections limits P. aeruginosa biofilms to the outer eye surface, preventing bacterial dissemination. Neutrophils moved to the base of forming biofilms, where they underwent neutrophil extracellular trap formation (NETosis) in response to high expression of the bacterial type-3 secretion system (T3SS). NETs formed a barrier "dead zone," confining bacteria to the external corneal environment and inhibiting bacterial dissemination into the brain. Once formed, ocular biofilms were resistant to antibiotics and neutrophil killing, advancing eye pathology. However, blocking both Psl and T3SS together with antibiotic treatment broke down the biofilm and reversed keratitis, suggesting future therapeutic strategies for this intractable infection.

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension.

BACKGROUND & AIMS: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes. METHODS: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or peptidyl arginine deiminase type IV (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice. RESULTS: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE-/- and Pad4-/- mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel. CONCLUSIONS: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.

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.

Visualizing Oncolytic Virus-Host Interactions in Live Mice Using Intravital Microscopy.

Oncolytic virus (OV) therapy is an emerging cancer treatment that uses replicating viruses to infect and kill tumor cells and incite anticancer immunity. While the approach shows promise, it currently fails most patients, indicating strategies to improve OV activity are needed. Developing these will require greater understanding of OV biology, particularly in the context of OV delivery and clearance, the infection process within a complex tumor microenvironment, and the modulation of anticancer immunity. To help achieve this, we have established a technique for high-resolution 4D imaging of OV-host interactions within intact tissues of live mice using intravital microscopy (IVM). We show that oncolytic vesicular stomatitis virus (VSV) directly labeled with Alexa Fluor dyes is easily visualized by single- or multiphoton microscopy while retaining bioactivity in vivo. The addition of fluorophore-tagged antibodies and genetically encoded reporter proteins to image target cells and the virus infection enables real-time imaging of dynamic interactions between VSV and host cells in blood, tumor, and visceral organs of live mice. The method has sufficient in vivo resolution to observe leukocytes in blood binding to and transporting VSV particles, foci of VSV infection spreading through a tumor, and antigen-presenting cells in the spleen interacting with and being infected by VSV. Visualizing OV-host interactions by IVM represents a powerful new tool for studying OV therapy.

α-Toxin Induces Platelet Aggregation and Liver Injury during Staphylococcus aureus Sepsis.

During sepsis, small blood vessels can become occluded by large platelet aggregates of poorly understood etiology. During Staphylococcal aureus infection, sepsis severity is linked to the bacterial α-toxin (α-hemolysin, AT) through unclear mechanisms. In this study, we visualized intravascular events in the microcirculation and found that intravenous AT injection induces rapid platelet aggregation, forming dynamic micro-thrombi in the microcirculation. These aggregates are retained in the liver sinusoids and kidney glomeruli, causing multi-organ dysfunction. Acute staphylococcal infection results in sequestration of most bacteria by liver macrophages. Platelets are initially recruited to these macrophages and help eradicate S. aureus. However, at later time points, AT causes aberrant and damaging thrombosis throughout the liver. Treatment with an AT neutralizing antibody (MEDI4893∗) prevents platelet aggregation and subsequent liver damage, without affecting the initial and beneficial platelet recruitment. Thus, AT neutralization may represent a promising approach to combat staphylococcal-induced intravascular coagulation and organ dysfunction.

Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody.

Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.

CRIg Functions as a Macrophage Pattern Recognition Receptor to Directly Bind and Capture Blood-Borne Gram-Positive Bacteria.

Kupffer cells (KCs), the vast pool of intravascular macrophages in the liver, help to clear blood-borne pathogens. The mechanisms by which KCs capture circulating pathogens remain unknown. Here we use intra-vital imaging of mice infected with Staphylococcus aureus to directly visualize the dynamic process of bacterial capture in the liver. Circulating S. aureus were captured by KCs in a manner dependent on the macrophage complement receptor CRIg, but the process was independent of complement. CRIg bound Staphylococcus aureus specifically through recognition of lipoteichoic acid (LTA), but not cell-wall-anchored surface proteins or peptidoglycan. Blocking the recognition between CRIg and LTA in vivo diminished the bacterial capture in liver and led to systemic bacterial dissemination. All tested Gram-positive, but not Gram-negative, bacteria bound CRIg in a complement-independent manner. These findings reveal a pattern recognition role for CRIg in the direct capture of circulating Gram-positive bacteria from the bloodstream.

Interferon-γ facilitates hepatic antiviral T cell retention for the maintenance of liver-induced systemic tolerance.

Persistent exposure to liver pathogens leads to systemic antigen-specific tolerance, a major cause of chronicity during hepatotropic infection. The mechanism regarding how this systemic tolerance is maintained remains poorly elucidated. In a well established mouse model of hepatitis B virus (HBV) persistence-induced systemic tolerance, we observed that interferon-γ (IFN-γ) deficiency led to complete loss of tolerance, resulting in robust anti-HBV responses upon peripheral vaccination. The recovery of vaccine-induced anti-HBV responses was mainly caused by the retained antigen-specific CD4(+) T cells rather than decreased functional inhibitory cells in the periphery. Mechanistically, HBV persistence induced sustained hepatic CD4(+) T cell-derived IFN-γ production. IFN-γ was found to promote CXCL9 secretion from liver-resident macrophages. This T cell chemokine facilitated the retention of antiviral CD4(+) T cells in the liver in a CXCR3-dependent manner. Hepatic sequestrated antiviral CD4(+) T cells subsequently underwent local apoptotic elimination partially via cytotoxic T lymphocyte-associated protein 4 ligation. These findings reveal an unexpected tolerogenic role for IFN-γ during viral persistence in the liver, providing new mechanistic insights regarding the maintenance of systemic antigen-specific tolerance during HBV persistence.

Natural Killer Cells-Produced IFN-γ Improves Bone Marrow-Derived Hepatocytes Regeneration in Murine Liver Failure Model.

Bone-marrow transplantation (BMT) can repopulate the liver through BM-derived hepatocyte (BMDH) generation, although the underlying mechanism remains unclear. Using fumarylacetoacetate hydrolase-deficient (Fah(-/-)) mice as a liver-failure model, we confirmed that BMDHs were generated by fusion of BM-derived CD11b(+)F4/80(+)myelomonocytes with resident Fah(-/-) hepatocytes. Hepatic NK cells became activated during BMDH generation and were the major IFN-γ producers. Indeed, both NK cells and IFN-γ were required for BMDH generation since WT, but not NK-, IFN-γ-, or IFN-γR1-deficient BM transplantation successfully generated BMDHs and rescued survival in Fah(-/-) hosts. BM-derived myelomonocytes were determined to be the IFN-γ-responding cells. The IFN-γ-IFN-γR interaction contributed to the myelomonocyte-hepatocyte fusion process, as most of the CD11b(+) BMDHs in mixed BM chimeric Fah(-/-) hosts transplanted with a 1:1 ratio of CD45.1(+) WT and CD45.2(+) Ifngr1(-/-) BM cells were of CD45.1(+) WT origin. Confirming these findings in vitro, IFN-γ dose-dependently promoted the fusion of GFP(+) myelomonocytes with Fah(-/-) hepatocytes due to a direct effect on myelomonocytes; similar results were observed using activated NK cells. In conclusion, BMDH generation requires NK cells to facilitate myelomonocyte-hepatocyte fusion in an IFN-γ-dependent manner, providing new insights for treating severe liver failure.

A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury.

Monocytes are recruited from the blood to sites of inflammation, where they contribute to wound healing and tissue repair. There are at least two subsets of monocytes: classical or proinflammatory (CCR2(hi)CX3CR1(low)) and nonclassical, patrolling, or alternative (CCR2(low)CX3CR1(hi)) monocytes. Using spinning-disk confocal intravital microscopy and mice with fluorescent reporters for each of these subsets, we were able to track the dynamic spectrum of monocytes that enter a site of sterile hepatic injury in vivo. We observed that the CCR2(hi)CX3CR1(low) monocytes were recruited early and persisted for at least 48 h, forming a ringlike structure around the injured area. These monocytes transitioned, in situ, from CCR2(hi)Cx3CR1(low) to CX3CR1(hi)CCR2(low) within the ringlike structure and then entered the injury site. This phenotypic conversion was essential for optimal repair. These results demonstrate a local, cytokine driven reprogramming of classic, proinflammatory monocytes into nonclassical or alternative monocytes to facilitate proper wound-healing.

IL-12-based vaccination therapy reverses liver-induced systemic tolerance in a mouse model of hepatitis B virus carrier.

Liver-induced systemic immune tolerance that occurs during chronic hepadnavirus infection is the biggest obstacle for effective viral clearance. Immunotherapeutic reversal of this tolerance is a promising strategy in the clinic but remains to be explored. In this study, using a hepatitis B virus (HBV)-carrier mouse model, we report that IL-12-based vaccination therapy can efficiently reverse systemic tolerance toward HBV. HBV-carrier mice lost responsiveness to hepatitis B surface Ag (HBsAg) vaccination, and IL-12 alone could not reverse this liver-induced immune tolerance. However, after IL-12-based vaccination therapy, the majority of treated mice became HBsAg(-) in serum; hepatitis B core Ag was also undetectable in hepatocytes. HBV clearance was dependent on HBsAg vaccine-induced anti-HBV immunity. Further results showed that IL-12-based vaccination therapy strongly enhanced hepatic HBV-specific CD8(+) T cell responses, including proliferation and IFN-γ secretion. Systemic HBV-specific CD4(+) T cell responses were also restored in HBV-carrier mice, leading to the arousal of HBsAg-specific follicular Th-germinal center B cell responses and anti-hepatitis B surface Ag Ab production. Recovery of HBsAg-specific responses also correlated with both reduced CD4(+)Foxp3(+) regulatory T cell frequency and an enhanced capacity of effector T cells to overcome inhibition by regulatory T cells. In conclusion, IL-12-based vaccination therapy may reverse liver-induced immune tolerance toward HBV by restoring systemic HBV-specific CD4(+) T cell responses, eliciting robust hepatic HBV-specific CD8(+) T cell responses, and facilitating the generation of HBsAg-specific humoral immunity; thus, this therapy may become a viable approach to treating patients with chronic hepatitis B.