Essential growth factor receptors for fibroblast homeostasis and activation: Fibroblast Growth Factor Receptor (FGFR), Platelet Derived Growth Factor Receptor (PDGFR), and Transforming Growth Factor ß Receptor (TGFßR).

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing studies on the influence of fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and transforming growth factor ß receptor (TGFßR) on fibroblast cell states.

Life of Pi: Exploring functions of Pi16+ fibroblasts.

Fibroblasts are mesenchymal cells that are responsible for creating and maintaining tissue architecture through the production of extracellular matrix. These cells also play critical roles in processes such as wound repair and immune modulation in normal tissues and various disease states including fibrosis, autoimmunity, and cancer. Fibroblasts have a complex repertoire of functions that vary by organ, inflammatory state, and the developmental stage of an organism. How fibroblasts manage so many functions in such a context-dependent manner represents a gap in our understanding of these cells. One possibility is that a tissue-resident precursor cell state exists that provides the fibroblast lineage with flexibility during growth, inflammation, or other contexts that require dynamic tissue changes. Recent work has suggested that a precursor fibroblast cell state is marked by expression of Peptidase inhibitor 16 ( Pi16). This review aims to concatenate and compare studies on fibroblasts that express Pi16 to clarify the roles of this cell state in fibroblast lineage development and other functions.

What's in a name? An emerging framework for cancer-associated fibroblasts, myofibroblasts, and fibroblasts.

In this issue of Cancer Cell, Foster and colleagues explore the heterogeneity in cancer-associated fibroblasts (CAFs) across tissue types and species, and they identify mechanoresponsive (MR), immunomodulatory (IM), and steady-state-like (SSL) CAFs. They show that altering the relative abundance of these CAF subtypes influences tumor progression and response to anti-tumor therapy.

LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Fibroblast-macrophage reciprocal interactions in health, fibrosis, and cancer.

Fibroblasts and macrophages are present in all tissues, and mounting evidence supports that these cells engage in direct communication to influence the overall tissue microenvironment and affect disease outcomes. Here, we review the current understanding of the molecular mechanisms that underlie fibroblast-macrophage interactions in health, fibrosis, and cancer. We present an integrated view of fibroblast-macrophage interactions that is centered on the CSF1-CSF1R axis and discuss how additional molecular programs linking these cell types can underpin disease onset, progression, and resolution. These programs may be tissue and context dependent, affected also by macrophage and fibroblast origin and state, as seen most clearly in cancer. Continued efforts to understand these cells and the means by which they interact may provide therapeutic approaches for the treatment of fibrosis and cancer.

Cross-tissue organization of the fibroblast lineage.

Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.

A Stromal Niche Defined by Expression of the Transcription Factor WT1 Mediates Programming and Homeostasis of Cavity-Resident Macrophages.

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6+ macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1+ stromal cells reduced the frequency of GATA6+ macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1+ mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages.

Adaptive adipose tissue stromal plasticity in response to cold stress and antibody-based metabolic therapy.

In response to environmental and nutrient stress, adipose tissues must establish a new homeostatic state. Here we show that cold exposure of obese mice triggers an adaptive tissue remodeling in visceral adipose tissue (VAT) that involves extracellular matrix deposition, angiogenesis, sympathetic innervation, and adipose tissue browning. Obese VAT is predominated by pro-inflammatory M1 macrophages; cold exposure induces an M1-to-M2 shift in macrophage composition and dramatic changes in macrophage gene expression in both M1 and M2 macrophages. Antibody-mediated CSF1R blocking prevented the cold-induced recruitment of adipose tissue M2 macrophages, suggesting the role of CSF1R signaling in the process. These cold-induced effects in obese VAT are phenocopied by an administration of the FGF21-mimetic antibody, consistent with its action to stimulate sympathetic nerves. Collectively, these studies illuminate adaptive visceral adipose tissue plasticity in obese mice in response to cold stress and antibody-based metabolic therapy.

Fibroblastic reticular cells initiate immune responses in visceral adipose tissues and secure peritoneal immunity.

Immune protection of the body cavities depends on the swift activation of innate and adaptive immune responses in nonclassical secondary lymphoid organs known as fat-associated lymphoid clusters (FALCs). Compared with classical secondary lymphoid organs such as lymph nodes and Peyer's patches, FALCs develop along distinct differentiation trajectories and display a reduced structural complexity. Although it is well established that fibroblastic reticular cells (FRCs) are an integral component of the immune-stimulating infrastructure of classical secondary lymphoid organs, the role of FRCs in FALC-dependent peritoneal immunity remains unclear. Using FRC-specific gene targeting, we found that FRCs play an essential role in FALC-driven immune responses. Specifically, we report that initiation of peritoneal immunity was governed through FRC activation in a myeloid differentiation primary response 88 (MYD88)-dependent manner. FRC-specific ablation of MYD88 blocked recruitment of inflammatory monocytes into FALCs and subsequent CD4+ T cell-dependent B-cell activation and IgG class switching. Moreover, containment of Salmonella infection was compromised in mice lacking MYD88 expression in FRCs, indicating that FRCs in FALCs function as an initial checkpoint in the orchestration of protective immune responses in the peritoneal cavity.

Testosterone is an endogenous regulator of BAFF and splenic B cell number.

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.

BCAP inhibits proliferation and differentiation of myeloid progenitors in the steady state and during demand situations.

B-cell adaptor for phosphatidylinositol 3-kinase (BCAP) is a signaling adaptor expressed in mature hematopoietic cells, including monocytes and neutrophils. Here we investigated the role of BCAP in the homeostasis and development of these myeloid lineages. BCAP-/- mice had more bone marrow (BM) monocytes than wild-type (WT) mice, and in mixed WT:BCAP-/- BM chimeras, monocytes and neutrophils skewed toward BCAP-/- origin, showing a competitive advantage for BCAP-/- myeloid cells. BCAP was expressed in BM hematopoietic progenitors, including lineage-Sca-1+c-kit+ (LSK), common myeloid progenitor, and granulocyte/macrophage progenitor (GMP) cells. At the steady state, BCAP-/- GMP cells expressed more IRF8 and less C/EBPα than did WT GMP cells, which correlated with an increase in monocyte progenitors and a decrease in granulocyte progenitors among GMP cells. Strikingly, BCAP-/- progenitors proliferated and produced more myeloid cells of both neutrophil and monocyte/macrophage lineages than did WT progenitors in myeloid colony-forming unit assays, supporting a cell-intrinsic role of BCAP in inhibiting myeloid proliferation and differentiation. Consistent with these findings, during cyclophosphamide-induced myeloablation or specific monocyte depletion, BCAP-/- mice replenished circulating monocytes and neutrophils earlier than WT mice. During myeloid replenishment after cyclophosphamide-induced myeloablation, BCAP-/- mice had increased LSK proliferation and increased numbers of LSK and GMP cells compared with WT mice. Furthermore, BCAP-/- mice accumulated more monocytes and neutrophils in the spleen than did WT mice during Listeria monocytogenes infection. Together, these data identify BCAP as a novel inhibitor of myelopoiesis in the steady state and of emergency myelopoiesis during demand conditions.

Cutting Edge: Direct Sensing of TLR7 Ligands and Type I IFN by the Common Myeloid Progenitor Promotes mTOR/PI3K-Dependent Emergency Myelopoiesis.

During infection, recognition of pathogens and inflammatory cytokines skews hematopoiesis toward myeloid development, although the precise mechanisms responsible for this are unclear. In this study, we show that accelerated myeloid differentiation, known as emergency myelopoiesis, involves recognition of pathogen-associated molecular patterns by the common myeloid progenitor (CMP) and is dependent on type I IFN for monocyte/macrophage differentiation. Direct sensing of TLR agonists by CMP induced rapid proliferation and induction of myeloid-differentiation genes. Lack of type I IFN signaling in CMP abrogated macrophage differentiation in response to TLR stimuli, whereas exogenous type I IFN amplified this process. Mechanistically, TLR7 induced PI3K/mammalian target of rapamycin signaling in CMP, which was enhanced by type I IFN, and this pathway was essential for emergency myelopoiesis. This work identifies a novel mechanism by which TLR and type I IFN synergize to promote monocyte/macrophage development from hematopoietic progenitors, a process critical in triggering rapid immune responses during infection.

Identification and spontaneous immune targeting of an endogenous retrovirus K envelope protein in the Indian rhesus macaque model of human disease.

BACKGROUND: Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections that have invaded the germ line of both humans and non-human primates. Most ERVs are functionally crippled by deletions, mutations, and hypermethylation, leading to the view that they are inert genomic fossils. However, some ERVs can produce mRNA transcripts, functional viral proteins, and even non-infectious virus particles during certain developmental and pathological processes. While there have been reports of ERV-specific immunity associated with ERV activity in humans, adaptive immune responses to ERV-encoded gene products remain poorly defined and have not been investigated in the physiologically relevant non-human primate model of human disease. FINDINGS: Here, we identified the rhesus macaque equivalent of the biologically active human ERV-K (HML-2), simian ERV-K (SERV-K1), which retains intact open reading frames for both Gag and Env on chromosome 12 in the macaque genome. From macaque cells we isolated a spliced mRNA product encoding SERV-K1 Env, which possesses all the structural features of a canonical, functional retroviral Envelope protein. Furthermore, we identified rare, but robust T cell responses as well as frequent antibody responses targeting SERV-K1 Env in rhesus macaques. CONCLUSIONS: These data demonstrate that SERV-K1 retains biological activity sufficient to induce cellular and humoral immune responses in rhesus macaques. As ERV-K is the youngest and most active ERV family in the human genome, the identification and characterization of the simian orthologue in rhesus macaques provides a highly relevant animal model in which to study the role of ERV-K in developmental and disease states.

Hematopoietic and nonhematopoietic cells promote Type I interferon- and TLR7-dependent monocytosis during low-dose LCMV infection.

Release of inflammatory monocytes from the bone marrow (BM) into the blood is an important physiological response to infection, but the mechanisms regulating this phenomenon during viral infection are not completely defined. Here, we show that low-dose infection with lymphocytic choriomeningitis virus (LCMV) caused rapid, transient inflammatory monocytosis that required type I interferon (IFN) and Toll-like receptor (TLR) 7 signaling. Type I IFN and TLR7 signals were critical for induction of IFN-stimulated gene expression and CCR2 ligand upregulation in the BM microenvironment in response to LCMV infection. Experiments utilizing BM chimeric mice demonstrated that type I IFN and TLR7 signaling on either hematopoietic or nonhematopoietic cells was sufficient to initiate monocytosis in response to LCMV infection. BM plasmacytoid dendritic cells (pDCs) generated type I IFN directly ex vivo, suggesting that pDCs are a hematopoietic contributor of type I IFN in the BM early during LCMV infection. Overall, we describe novel roles for type I IFN and TLR7 signaling in nonhematopoietic cells and BM pDCs in directing IFN-stimulated gene and CCR2 ligand expression in the BM to initiate an increase in blood inflammatory monocytes during viral infection.

Vaccination against endogenous retrotransposable element consensus sequences does not protect rhesus macaques from SIVsmE660 infection and replication.

The enormous sequence diversity of HIV remains a major roadblock to the development of a prophylactic vaccine and new approaches to induce protective immunity are needed. Endogenous retrotransposable elements (ERE) such as endogenous retrovirus K (ERV)-K and long interspersed nuclear element-1 (LINE-1) are activated during HIV-1-infection and could represent stable, surrogate targets to eliminate HIV-1-infected cells. Here, we explored the hypothesis that vaccination against ERE would protect macaques from acquisition and replication of simian immunodeficiency virus (SIV). Following vaccination with antigens derived from LINE-1 and ERV-K consensus sequences, animals mounted immune responses that failed to delay acquisition of SIVsmE660. We observed no differences in acute or set point viral loads between ERE-vaccinated and control animals suggesting that ERE-specific responses were not protective. Indeed, ERE-specific T cells failed to expand anamnestically in vivo following infection with SIVsmE660 and did not recognize SIV-infected targets in vitro, in agreement with no significant induction of targeted ERE mRNA by SIV in macaque CD4+ T cells. Instead, lower infection rates and viral loads correlated significantly to protective TRIM5α alleles. Cumulatively, these data demonstrate that vaccination against the selected ERE consensus sequences in macaques did not lead to immune-mediated recognition and killing of SIV-infected cells, as has been shown for HIV-infected human cells using patient-derived HERV-K-specific T cells. Thus, further research is required to identify the specific nonhuman primate EREs and retroviruses that recapitulate the activity of HIV-1 in human cells. These results also highlight the complexity in translating observations of the interplay between HIV-1 and human EREs to animal models.

Overexpression of TLR7 promotes cell-intrinsic expansion and autoantibody production by transitional T1 B cells.

Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.

Cutting edge: Type I IFN drives emergency myelopoiesis and peripheral myeloid expansion during chronic TLR7 signaling.

Mice overexpressing TLR7 (TLR7.1 mice) are a model of systemic lupus erythematosus pathogenesis and exhibit peripheral myeloid expansion. We show that TLR7.1 mice have a dramatic expansion of splenic cells that derive from granulocyte/macrophage progenitors (GMP) compared with wild-type mice. In the bone marrow, TLR7.1 mice exhibited hallmarks of emergency myelopoiesis and contained a discrete population of Sca-1(+) GMP, termed emergency GMP, which are more proliferative and superior myeloid precursors than classical Sca-1(-) GMP. The emergency myelopoiesis and peripheral myeloid expansion in TLR7.1 mice was dependent on type I IFN signaling. TLR7 agonist administration to nontransgenic mice also drove type I IFN-dependent emergency myelopoiesis. TLR7.1 plasmacytoid dendritic cells were cell-intrinsically activated by TLR7 overexpression and constitutively produced type I IFN mRNA. This study shows that type I IFN can act upon myeloid progenitors to promote the development of emergency GMP, which leads to an expansion of their progeny in the periphery.

Simian immunodeficiency virus-specific CD8+ T cells recognize Vpr- and Rev-derived epitopes early after infection.

The kinetics of CD8(+) T cell epitope presentation contribute to the antiviral efficacy of these cells yet remain poorly defined. Here, we demonstrate presentation of virion-derived Vpr peptide epitopes early after viral penetration and prior to presentation of Vif-derived epitopes, which required de novo Vif synthesis. Two Rev epitopes exhibited differential presentation kinetics, with one Rev epitope presented within 1 h of infection. We also demonstrate that cytolytic activity mirrors the recognition kinetics of infected cells. These studies show for the first time that Vpr- and Rev-specific CD8(+) T cells recognize and kill simian immunodeficiency virus (SIV)-infected CD4(+) T cells early after SIV infection.

Gag- and Nef-specific CD4+ T cells recognize and inhibit SIV replication in infected macrophages early after infection.

The precise immunological role played by CD4(+) T cells in retroviral infections is poorly defined. Here, we describe a new function of these cells, the elimination of retrovirus-infected macrophages. After experimental CD8(+) cell depletion, elite controlling macaques with set-point viral loads < or = 500 viral RNA copies/mL mounted robust Gag- and Nef-specific CD4(+) T cell responses during reestablishment of control with > or = 54% of all virus-specific CD4(+) T cells targeting these 2 proteins. Ex vivo, these simian immunodeficiency virus (SIV)-specific CD4(+) T cells neither recognized nor suppressed viral replication in SIV-infected CD4(+) T cells. In contrast, they recognized SIV-infected macrophages as early as 2 h postinfection because of presentation of epitopes derived from virion-associated Gag and Nef proteins. Furthermore, virus-specific CD4(+) T cells displayed direct effector function and eliminated SIV-infected macrophages. These results suggest that retrovirus-specific CD4(+) T cells may contribute directly to elite control by inhibiting viral replication in macrophages.