Isolation, Ex Vivo Expansion, and Lentiviral Transduction of Alveolar Macrophages.

Alveolar macrophages (AM) are resident macrophages of the lung and play important roles in the maintenance of tissue homeostasis as well as host defense. Here, we describe how they can be harvested from murine lungs, expanded in vitro, and transduced with lentiviral vectors.

M-CSF directs myeloid and NK cell differentiation to protect from CMV after hematopoietic cell transplantation.

Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony-stimulating factor (M-CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M-CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon-γ. This depended upon M-CSF-induced myelopoiesis leading to IL15Rα-mediated presentation of IL-15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M-CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G-CSF-mobilized hematopoietic stem and progenitor cells. Together, M-CSF-induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M-CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host-directed therapies.

CD150-dependent hematopoietic stem cell sensing of Brucella instructs myeloid commitment.

So far, hematopoietic stem cells (HSC) are considered the source of mature immune cells, the latter being the only ones capable of mounting an immune response. Recent evidence shows HSC can also directly sense cytokines released upon infection/inflammation and pathogen-associated molecular pattern interaction while keeping a long-term memory of previously encountered signals. Direct sensing of danger signals by HSC induces early myeloid commitment, increases myeloid effector cell numbers, and contributes to an efficient immune response. Here, by using specific genetic tools on both the host and pathogen sides, we show that HSC can directly sense B. abortus pathogenic bacteria within the bone marrow via the interaction of the cell surface protein CD150 with the bacterial outer membrane protein Omp25, inducing efficient functional commitment of HSC to the myeloid lineage. This is the first demonstration of direct recognition of a live pathogen by HSC via CD150, which attests to a very early contribution of HSC to immune response.

Trained immunity and epigenetic memory in long-term self-renewing hematopoietic cells.

Immunologic memory is a feature typically ascribed to the adaptive arm of the immune system. However, recent studies have demonstrated that hematopoietic stem cells (HSCs) and innate immune cells such as monocytes and macrophages can gain epigenetic signatures to enhance their response in the context of reinfection. This suggests the presence of long-term memory, a phenomenon referred to as trained immunity. Trained immunity in HSCs can occur via changes in the epigenetic landscape and enhanced chromatin accessibility in lineage-specific genes, as well as through metabolic alterations. These changes can lead to a skewing in lineage bias, particularly enhanced myelopoiesis and the generation of epigenetically modified innate immune cells that provide better protection against pathogens on secondary infection. Here, we summarize recent advancements in trained immunity and epigenetic memory formation in HSCs and self-renewing alveolar macrophages, which was the focus of the Spring 2022 International Society for Experimental Hematology (ISEH) webinar.

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.

Autofluorescence identifies highly phagocytic tissue-resident macrophages in mouse and human skin and cutaneous squamous cell carcinoma.

Macrophages from human and mouse skin share phenotypic and functional features, but remain to be characterized in pathological skin conditions. Skin-resident macrophages are known to derive from embryonic precursors or from adult hematopoiesis. In this report, we investigated the origins, phenotypes and functions of macrophage subsets in mouse and human skin and in cutaneous squamous cell carcinoma (cSCC) using the spectral flow cytometry technology that enables cell autofluorescence to be considered as a full-fledged parameter. Autofluorescence identifies macrophage subsets expressing the CD206 mannose receptor in human peri-tumoral skin and cSCC. In mouse, all AF+ macrophages express the CD206 marker, a subset of which also displaying the TIM-4 marker. While TIM-4-CD206+ AF+ macrophages can differentiate from bone-marrow monocytes and infiltrate skin and tumor, TIM-4 identifies exclusively a skin-resident AF+ macrophage subset that can derive from prenatal hematopoiesis which is absent in tumor core. In mouse and human, AF+ macrophages from perilesional skin and cSCC are highly phagocytic cells contrary to their AF- counterpart, thus identifying autofluorescence as a bona fide marker for phagocytosis. Our data bring to light autofluorescence as a functional marker characterizing subsets of phagocytic macrophages in skin and cSCC. Autofluorescence can thus be considered as an attractive marker of function of macrophage subsets in pathological context.

Long-term culture-expanded alveolar macrophages restore their full epigenetic identity after transfer in vivo.

Alveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs.

TLR7 Signaling Drives the Development of Sjögren's Syndrome.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease that affects predominately salivary and lacrimal glands. SS can occur alone or in combination with another autoimmune disease like systemic lupus erythematosus (SLE). Here we report that TLR7 signaling drives the development of SS since TLR8-deficient (TLR8ko) mice that develop lupus due to increased TLR7 signaling by dendritic cells, also develop an age-dependent secondary pathology similar to associated SS. The SS phenotype in TLR8ko mice is manifested by sialadenitis, increased anti-SSA and anti-SSB autoantibody production, immune complex deposition and increased cytokine production in salivary glands, as well as lung inflammation. Moreover, ectopic lymphoid structures characterized by B/T aggregates, formation of high endothelial venules and the presence of dendritic cells are formed in the salivary glands of TLR8ko mice. Interestingly, all these phenotypes are abrogated in double TLR7/8-deficient mice, suggesting that the SS phenotype in TLR8-deficient mice is TLR7-dependent. In addition, evaluation of TLR7 and inflammatory markers in the salivary glands of primary SS patients revealed significantly increased TLR7 expression levels compared to healthy individuals, that were positively correlated to TNF, LT-α, CXCL13 and CXCR5 expression. These findings establish an important role of TLR7 signaling for local and systemic SS disease manifestations, and inhibition of such will likely have therapeutic value.

C/EBPß-Dependent Epigenetic Memory Induces Trained Immunity in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) maintain life-long production of immune cells and can directly respond to infection, but sustained effects on the immune response remain unclear. We show that acute immune stimulation with lipopolysaccharide (LPS) induced only transient changes in HSC abundance, composition, progeny, and gene expression, but persistent alterations in accessibility of specific myeloid lineage enhancers occurred, which increased responsiveness of associated immune genes to secondary stimulation. Functionally, this was associated with increased myelopoiesis of pre-exposed HSCs and improved innate immunity against the gram-negative bacterium P. aeruginosa. The accessible myeloid enhancers were enriched for C/EBPß targets, and C/EBPß deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPß-dependent chromatin accessibility, resulting in HSC-trained immunity, during secondary infection. This establishes a mechanism for how infection history can be epigenetically inscribed in HSCs as an integral memory function of innate immunity.

Tissue-resident macrophages in omentum promote metastatic spread of ovarian cancer.

Experimental and clinical evidence suggests that tumor-associated macrophages (TAMs) play important roles in cancer progression. Here, we have characterized the ontogeny and function of TAM subsets in a mouse model of metastatic ovarian cancer that is representative for visceral peritoneal metastasis. We show that the omentum is a critical premetastatic niche for development of invasive disease in this model and define a unique subset of CD163+ Tim4+ resident omental macrophages responsible for metastatic spread of ovarian cancer cells. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and pharmacological tools prevented tumor progression and metastatic spread of disease. These studies describe a specific role for tissue-resident macrophages in the invasive progression of metastatic ovarian cancer. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence.

Bhlhe40 and Bhlhe41 transcription factors regulate alveolar macrophage self-renewal and identity.

Tissues in multicellular organisms are populated by resident macrophages, which perform both generic and tissue-specific functions. The latter are induced by signals from the microenvironment and rely on unique tissue-specific molecular programs requiring the combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs)-a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41 deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity.

Characterization of Mouse Adult Testicular Macrophage Populations by Immunofluorescence Imaging and Flow Cytometry.

Testicular macrophages (tMΦ) are the most abundant immune cells residing in the testis, an immune-privileged organ. TMΦ are known to exhibit different functions, such as protecting spermatozoa from auto-immune attack by producing immunosuppressive cytokines and trophic roles in supporting spermatogenesis and male sex hormone production. They also contribute to fetal testicular development. Recently, we characterized two distinct tMΦ populations based on their morphology, localization, cell surface markers, and gene expression profiling. Here, we focus and describe in detail the phenotypical distinction of these two tMΦ populations by fluorescence-activated cell sorting (FACS) using multicolor panel antibodies combining with high-resolution immunofluorescence (IF) imaging. These two techniques enable to classify two tMΦ populations: interstitial tMΦ and peritubular tMΦ.

Publisher Correction: c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

In the version of this article initially published, the Supplementary Data file was an incorrect version. The correct version is now provided. The error has been corrected in the HTML and PDF version of the article.

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages.

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for in vitro culture and expansion of AM. In addition, we describe specific culture conditions, under which AM proliferate long-term in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor. This method can be used to obtain large numbers of AM for in vivo transplantation or for in vitro experiments with primary mouse macrophages.