Microglial Function Is Distinct in Different Anatomical Locations during Retinal Homeostasis and Degeneration.

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity.

A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFα-Mediated Recruitment of Neutrophils.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor α (VEGF-α) by a minor subset of activated EpCAM+CD59+Ly-6D+ cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guérin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Hemogenic Endothelial Fate Mapping Reveals Dual Developmental Origin of Mast Cells.

Hematopoiesis occurs in distinct waves. "Definitive" hematopoietic stem cells (HSCs) with the potential for all blood lineages emerge in the aorta-gonado-mesonephros, while "primitive" progenitors, whose potential is thought to be limited to erythrocytes, megakaryocytes, and macrophages, arise earlier in the yolk sac (YS). Here, we questioned whether other YS lineages exist that have not been identified, partially owing to limitations of current lineage tracing models. We established the use of Cdh5-CreERT2 for hematopoietic fate mapping, which revealed the YS origin of mast cells (MCs). YS-derived MCs were replaced by definitive MCs, which maintained themselves independently from the bone marrow in the adult. Replacement occurred with tissue-specific kinetics. MCs in the embryonic skin, but not other organs, remained largely YS derived prenatally and were phenotypically and transcriptomically distinct from definite adult MCs. We conclude that within myeloid lineages, dual hematopoietic origin is shared between macrophages and MCs.

TSPO expression in a Zika virus murine infection model as an imaging target for acute infection-induced neuroinflammation.

INTRODUCTION: Zika virus (ZIKV) is a neurotropic human pathogen that causes neuroinflammation, whose hallmark is elevated translocator protein (TSPO) expression in the brain. This study investigates ZIKV-associated changes in adult brain TSPO expression, evaluates the effectiveness of TSPO radioligands in detecting TSPO expression, and identifies cells that drive brain TSPO expression in a mouse infection model. METHODS: The interferon-deficient AG129 mouse infected with ZIKV was used as neuroinflammation model. TSPO expression was evaluated by tissue immunostaining. TSPO radioligands, [3H]PK11195 and [18F]FEPPA, were used for in vitro and ex vivo detection of TSPO in infected brains. [18F]FEPPA-PET was used for in vivo detection of TSPO expression. Cell subsets that contribute to TSPO expression were identified by flow cytometry. RESULTS: Brain TSPO expression increased with ZIKV disease severity. This increase was contributed by TSPO-positive microglia and infiltrating monocytes; and by influx of TSPO-expressing immune cells into the brain. [3H]PK11195 and [18F]FEPPA distinguish ZIKV-infected brains from normal controls in vitro and ex vivo. [18F]FEPPA brain uptake by PET imaging correlated with disease severity and neuroinflammation. However, TSPO expression by immune cells contributed to significant blood pool [18F]FEPPA activity which could confound [18F]FEPPA-PET imaging results. CONCLUSIONS: TSPO is a biologically relevant imaging target for ZIKV neuroinflammation. Brain [18F]FEPPA uptake can be a surrogate marker for ZIKV disease and may be a potential PET imaging marker for ZIKV-induced neuroinflammation. Future TSPO-PET/SPECT studies on viral neuroinflammation and related encephalitis should assess the contribution of immune cells on TSPO expression and employ appropriate image correction methods to subtract blood pool activity.

Mast cells-fetal mast cells crosstalk with maternal interfaces during pregnancy: Friend or foe?

Mast cells (MC) are hematopoietic immune cells that play a major role during allergic reactions in adults by releasing a myriad of vasoactive and inflammatory mediators. MC seed all vascularized tissues and are most prominent in organs with a barrier function such as skin, lungs, and intestines. These secreted molecules cause mild symptoms such as localized itchiness and sneezing to life-threatening symptoms (i.e., anaphylactic shock). Presently, despite the extensive research on Th2-mediated immune responses in allergic diseases in adults, we are still unable to determine the mechanisms of the role of MC in developing pediatric allergic (PA) disorders. In this review, we will summarize the most recent findings on the origin of MC and discuss the underappreciated contribution of MC in the sensitization phase to maternal antibodies during pregnancy in allergic reactions and other diseases such as infectious diseases. Then, we will lay out potential MC-dependent therapeutic strategies to be considered in future investigations to understand the remaining gaps in MC research for a better quality of life for these young patients.

Human fetal dendritic cells promote prenatal T-cell immune suppression through arginase-2.

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Preclinical evaluation of [18F]FDG-PET as a biomarker of lymphoid tissue disease and inflammation in Zika virus infection.

PURPOSE: Zika (ZIKV) is a viral inflammatory disease affecting adults, children, and developing fetuses. It is endemic to tropical and sub-tropical countries, resulting in half the global population at risk of infection. Despite this, there are no approved therapies or vaccines against ZIKV disease. Non-invasive imaging biomarkers are potentially valuable tools for studying viral pathogenesis, prognosticating host response to disease, and evaluating in vivo efficacy of experimental therapeutic interventions. In this study, we evaluated [18F]fluorodeoxyglucose ([18F]FDG)-positron emission tomography (PET) as an imaging biomarker of ZIKV disease in a mouse model and correlated metabolic tracer tissue uptake with real-time biochemical, virological, and inflammatory features of tissue infection. METHODS: [18F]FDG-PET/CT imaging was performed in an acute, lethal ZIKV mouse infection model, at increasing stages of disease severity. [18F]FDG-PET findings were corroborated with ex vivo wholemount-tissue autoradiography and tracer biodistribution studies. Tracer uptake was also correlated with in situ tissue disease status, including viral burden and inflammatory response. Immune profiling of the spleen by flow cytometry was performed to identify the immune cell subsets driving tissue pathology and enhancing tracer uptake in ZIKV disease. RESULTS: Foci of increased [18F]FDG uptake were consistently detected in lymphoid tissues-particularly the spleen-of ZIKV-infected animals. Splenic uptake increased with disease severity, and corroborated findings in tissue pathology. Increased splenic uptake also correlated with increased viral replication and elevated expression of pro-inflammatory cytokines within these tissues. ZIKV-infected spleens were characterized by increased infiltration of myeloid cells, as well as increased proliferation of both myeloid and lymphoid cells. The increased cell proliferation correlated with increased tracer uptake in the spleen. Our findings support the use of [18F]FDG as an imaging biomarker to detect and track ZIKV disease in real time and highlight the dependency of affected tissue on the nature of the viral infection. CONCLUSION: [18F]FDG uptake in the spleen is a useful surrogate for interrogating in situ tissue viral burden and inflammation status in this ZIKV murine model.

Granzyme B PET Imaging Stratifies Immune Checkpoint Inhibitor Response in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a notoriously difficult cancer to treat. The recent development of immune checkpoint inhibitors has revolutionised HCC therapy; however, successful response is only observed in a small percentage of patients. Biomarkers typically used to predict treatment response in other tumour types are ineffective in HCC, which arises in an immune-suppressive environment. However, imaging markers that measure changes in tumour infiltrating immune cells may supply information that can be used to determine which patients are responding to therapy posttreatment. We have evaluated [18F]AlF-mNOTA-GZP, a radiolabeled peptide targeting granzyme B, to stratify response to ICIs in a HEPA 1-tumours, a syngeneic model of HCC. Posttherapy, in vivo tumour retention of [18F]AlF-mNOTA-GZP was correlated to changes in tumour volume and tumour-infiltrating immune cells. [18F]AlF-mNOTA-GZP successfully stratified response to immune checkpoint inhibition in the syngeneic HEPA 1-6 model. FACS indicated significant changes in the immune environment including a decrease in immune suppressive CD4+ T regulatory cells and increases in tumour-associated GZB+ NK+ cells, which correlated well with tumour radiopharmaceutical uptake. While the immune response to ICI therapies differs in HCC compared to many other cancers, [18F]AlF-mNOTA-GZP retention is able to stratify response to ICI therapy associated with tumour infiltrating GZB+ NK+ cells in this complex tumour microenvironment.

Translocator protein (TSPO) is a biomarker of Zika virus (ZIKV) infection-associated neuroinflammation.

Zika is a systemic inflammatory disease caused by infection with Zika virus (ZIKV). ZIKV infection in adults is associated with encephalitis marked by elevated expression of pro-inflammatory cytokines and chemokines, as well as increased brain infiltration of immune cells. In this study, we demonstrate that ZIKV encephalitis in a mouse infection model exhibits increased brain TSPO expression. TSPO expression on brain-resident and infiltrating immune cells in ZIKV infection correlates with disease and inflammation status in the brain. Brain TSPO expression can also be sensitively detected ex vivo and in vitro using radioactive small molecule probes that specifically bind to TSPO, such as [3H]PK11195. TSPO expression on brain-resident and infiltrating immune cells is a biomarker of ZIKV neuroinflammation, which can also be a general biomarker of acute viral neuroinflammatory disease.

Mast Cell Interaction with Foxp3+ Regulatory T Cells Occur in the Dermis after Initiation of IgE-Mediated Cutaneous Anaphylaxis.

Mast cells (MCs) are well-known for their role in IgE-mediated cutaneous anaphylactic responses, but their regulatory functions in the skin are still under intense scrutiny. Using a Red MC and Basophil reporter (RMB) mouse allowing red fluorescent detection and diphtheria toxin mediated depletion of MCs, we investigated the interaction of MCs, Foxp3+ regulatory T lymphocytes (Tregs) and Langerhans cells (LCs) during passive cutaneous anaphylaxis (PCA) responses. Using intravital imaging we show that MCs are sessile at homeostasis and during PCA. Breeding RMB mice with Langerin-eGFP mice revealed that dermal MCs do not interact with epidermal-localized LCs, the latter showing constant sprouting of their dendrites at homeostasis and during PCA. When bred with Foxp3-eGFP mice, we found that, although a few Foxp3+ Tregs are present at homeostasis, many Tregs transiently infiltrated the skin during PCA. While their velocity during PCA was not altered, Tregs increased the duration of their contact time with MCs compared to PCA-control mice. Antibody-mediated depletion of Tregs had no effect on the intensity of PCA. Hence, the observed increase in Treg numbers and contact time with MCs, regardless of an effect on the intensity of PCA responses, suggests an anti-inflammatory role dedicated to prevent further MC activation.

Tissues and Tumor Microenvironment (TME) in 3D: Models to Shed Light on Immunosuppression in Cancer.

Immunosuppression in cancer has emerged as a major hurdle to immunotherapy efforts. Immunosuppression can arise from oncogene-induced signaling within the tumor as well as from tumor-associated immune cells. Understanding various mechanisms by which the tumor can undermine and evade therapy is critical in improving current cancer immunotherapies. While mouse models have allowed for the characterization of key immune cell types and their role in tumor development, extrapolating these mechanisms to patients has been challenging. There is need for better models to unravel the effects of genetic alterations inherent in tumor cells and immune cells isolated from tumors on tumor growth and to investigate the feasibility of immunotherapy. Three-dimensional (3D) organoid model systems have developed rapidly over the past few years and allow for incorporation of components of the tumor microenvironment such as immune cells and the stroma. This bears great promise for derivation of patient-specific models in a dish for understanding and determining the impact on personalized immunotherapy. In this review, we will highlight the significance of current experimental models employed in the study of tumor immunosuppression and evaluate current tumor organoid-immune cell co-culture systems and their potential impact in shedding light on cancer immunosuppression.

Granzyme B PET Imaging of Combined Chemotherapy and Immune Checkpoint Inhibitor Therapy in Colon Cancer.

PURPOSE: Chemotherapeutic adjuvants, such as oxaliplatin (OXA) and 5-fluorouracil (5-FU), that enhance the immune system, are being assessed as strategies to improve durable response rates when used in combination with immune checkpoint inhibitor (ICI) monotherapy in cancer patients. In this study, we explored granzyme B (GZB), released by tumor-associated immune cells, as a PET imaging-based stratification marker for successful combination therapy using a fluorine-18 (18F)-labelled GZB peptide ([18F]AlF-mNOTA-GZP). METHODS: Using the immunocompetent CT26 syngeneic mouse model of colon cancer, we assessed the potential for [18F]AlF-mNOTA-GZP to stratify OXA/5-FU and ICI combination therapy response via GZB PET. In vivo tumor uptake of [18F]AlF-mNOTA-GZP in different treatment arms was quantified by PET, and linked to differences in tumor-associated immune cell populations defined by using multicolour flow cytometry. RESULTS: [18F]AlF-mNOTA-GZP tumor uptake was able to clearly differentiate treatment responders from non-responders when stratified based on changes in tumor volume. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumor-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells and GZB+ NK+ cells. CONCLUSIONS: [18F]AlF-mNOTA-GZP tumor uptake, driven by changes in immune cell populations expressing GZB, is able to stratify tumor response to chemotherapeutics combined with ICIs. Our results show that, while the immunomodulatory mode of action of the chemotherapies may be different, the ultimate mechanism of tumor lysis through release of Granzyme B is an accurate biomarker for treatment response.

Fetal mast cells mediate postnatal allergic responses dependent on maternal IgE.

Mast cells (MCs) are central effector cells in allergic reactions that are often mediated by immunoglobulin E (IgE). Allergies commonly start at an early age, and both MCs and IgE are detectable in fetuses. However, the origin of fetal IgE and whether fetal MCs can degranulate in response to IgE-dependent activation are presently unknown. Here, we show that human and mouse fetal MCs phenotypically mature through pregnancy and can be sensitized by maternal IgE. IgE crossed the placenta, dependent on the fetal neonatal Fc receptor (FcRN), and sensitized fetal MCs for allergen-specific degranulation. Both passive and active prenatal sensitization conferred allergen sensitivity, resulting in postnatal skin and airway inflammation after the first allergen encounter. We report a role for MCs within the developing fetus and demonstrate that fetal MCs may contribute to antigen-specific vertical transmission of allergic disease.

Liver fibrosis and CD206+ macrophage accumulation are suppressed by anti-GM-CSF therapy.

BACKGROUND & AIMS: Chronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206+ macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease. METHODS: Liver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206+ monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations in vitro and in vivo in humanised mice. RESULTS: Using multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206+ macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206+ macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV+ patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated in vitro that monocytes converted to TNFα-producing CD206+ macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the in vivo normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206+ macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice. CONCLUSIONS: While the direct involvement of CD206+ macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease. LAY SUMMARY: Liver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes.

Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult.

The murine epidermis harbors two immune cell lineages, Langerhans cells (LCs) and γδ T cells known as dendritic epidermal T cells (DETCs). LCs develop from both early yolk sac (YS) progenitors and fetal liver monocytes before locally self-renewing in the adult. For DETCs, the mechanisms of homeostatic maintenance and their hematopoietic origin are largely unknown. Here, we exploited multicolor fate mapping systems to reveal that DETCs slowly turn over at steady state. Like for LCs, homeostatic maintenance of DETCs is achieved by clonal expansion of tissue-resident cells assembled in proliferative units. The same mechanism, albeit accelerated, facilitates DETC replenishment upon injury. Hematopoietic lineage tracing uncovered that DETCs are established independently of definitive hematopoietic stem cells and instead originate from YS hematopoiesis, again reminiscent of LCs. DETCs thus resemble LCs concerning their maintenance, replenishment mechanisms, and hematopoietic development, suggesting that the epidermal microenvironment exerts a lineage-independent influence on the initial seeding and homeostatic maintenance of its resident immune cells.

Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment.

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection.

Mast cells aggravate sepsis by inhibiting peritoneal macrophage phagocytosis.

Controlling the overwhelming inflammatory reaction associated with polymicrobial sepsis remains a prevalent clinical challenge with few treatment options. In septic peritonitis, blood neutrophils and monocytes are rapidly recruited into the peritoneal cavity to control infection, but the role of resident sentinel cells during the early phase of infection is less clear. In particular, the influence of mast cells on other tissue-resident cells remains poorly understood. Here, we developed a mouse model that allows both visualization and conditional ablation of mast cells and basophils to investigate the role of mast cells in severe septic peritonitis. Specific depletion of mast cells led to increased survival rates in mice with acute sepsis. Furthermore, we determined that mast cells impair the phagocytic action of resident macrophages, thereby allowing local and systemic bacterial proliferation. Mast cells did not influence local recruitment of neutrophils and monocytes or the release of inflammatory cytokines. Phagocytosis inhibition by mast cells involved their ability to release prestored IL-4 within 15 minutes after bacterial encounter, and treatment with an IL-4-neutralizing antibody prevented this inhibitory effect and improved survival of septic mice. Our study uncovers a local crosstalk between mast cells and macrophages during the early phase of sepsis development that aggravates the outcome of severe bacterial infection.