Tissue fibroblasts are versatile immune regulators: An evaluation of their impact on the aging process

Fibroblasts are abundant stromal cells which not only control the integrity of extracellular matrix (ECM) but also act as immune regulators. It is known that the structural cells within tissues can establish an organ-specific immunity expressing many immune-related genes and closely interact with immune cells. In fact, fibroblasts can modify their immune properties to display both pro-inflammatory and immunosuppressive activities in a context-dependent manner. After acute insults, fibroblasts promote tissue inflammation although they concurrently recruit immunosuppressive cells to enhance the resolution of inflammation. In chronic pathological states, tissue fibroblasts, especially senescent fibroblasts, can display many pro-inflammatory and immunosuppressive properties and stimulate the activities of different immunosuppressive cells. In return, immunosuppressive cells, such as M2 macrophages and myeloid-derived suppressor cells (MDSC), evoke an excessive conversion of fibroblasts into myofibroblasts, thus aggravating the severity of tissue fibrosis. Single-cell transcriptome studies on fibroblasts isolated from aged tissues have confirmed that tissue fibroblasts express many genes coding for cytokines, chemokines, and complement factors, whereas they lose some fibrogenic properties. The versatile immune properties of fibroblasts and their close cooperation with immune cells indicate that tissue fibroblasts have a crucial role in the aging process and age-related diseases.


Introduction
Tissue fibroblasts are mesenchymal cells which have plasticity properties, i.e., they can display very different phenotypes to ensure the regulation of tissue homeostasis (LeBleu and Neilson, 2020;Plikus et al., 2021;Salminen, 2023a) (Fig. 1).Fibroblasts are the main organizers of the extracellular matrix (ECM) since they secrete its major components, such as collagens, fibronectin, elastins, proteoglycans, and many glycosaminoglycans.Furthermore, fibroblasts secrete hydrolytic enzymes, which can modify the structures of the ECM and thus control the integrity of tissues.Fibroblasts are stromal cells which regulate the integrity of connective tissues; in pathological conditions, they have a crucial role in the repair of tissue injuries since they trigger fibrosis and thus contribute to healing of damaged tissues (Plikus et al., 2021;Schuster et al., 2023).In addition to their fibrogenic properties, tissue fibroblasts act as immune regulators in a close cooperation with the cells of the immune system to maintain microenvironmental homeostasis.
Interestingly, fibroblasts can display both pro-inflammatory and immunosuppressive properties and in this way they strive to restore tissue homeostasis (Davidson et al., 2021;Schuster et al., 2021;Salminen, 2023b).Given their versatile immune properties, tissue fibroblasts are able to modulate inflammatory responses in cooperation with pro-inflammatory and immunosuppressive cells (Section 5.).There are also many tissue-specific fibroblast populations which are involved in the regulation of systemic immune responses, e.g., the fibroblastic reticulum cells in the lymphoid organs, or in disease-specific processes, such as cancer-associated fibroblasts and synoviocytes in inflammatory joints (Section 3.4.).Single-cell transcriptome studies on fibroblasts in different physiological and pathological conditions have confirmed the impressive heterogeneity of fibroblast populations which reflects their ability to adapt to diverse changes in the microenvironment.
The aging process is a progressive decline in the physiological abilities of both the tissues and organisms involving the deposition of pathological changes which promote the aging process.There are three major hallmarks of the aging process, i.e., the accumulation of senescent cells into tissues, the disruption of the ECM, and a chronic low-grade inflammation (Franceschi et al., 2018;Yousefzadeh et al., 2020;Selman and Pardo, 2021).There is abundant evidence that senescent cells display a pro-inflammatory phenotype, called the senescence-associated secretory phenotype (SASP).Given that inflammation is a stromal process within tissues, it is not surprising that the fibroblasts, the major cell type present in the stroma, have a dynamic role in the inflammatory processes occurring in the tissues.Moreover, fibroblast population can exhibit the markers of cellular senescence not only in aged tissues but especially in the age-related diseases.Although fibroblasts are master drivers of fibrogenic processes, it should be recalled that they are the blast-type cells, a kind of immature progenitor cells, which can adopt diverse phenotypes (Plikus et al., 2021).They can display not only the myofibroblast phenotype (Section 3.2.)but also exist in different inflammatory phenotypes (Section 4.).Here, we will examine in detail the immune properties of tissue fibroblasts and subsequently explore whether a chronic low-grade inflammation could remodel the properties of tissue fibroblasts to accelerate the age-related degenerative processes of the ECM and the host cells.

Structural cells as immune regulators
The main components of the immune system involve the myeloid and lymphoid cells which both originate from the haematopoietic progenitor cells.However, it is known that the structural cells present in tissues can also express many immune genes and closely interact with myeloid and lymphoid cells in times of tissue defence.This phenomenon has been called organ-specific immunity.Krausgruber et al. (2020) utilized single-cell multiomics in the profiling of fibroblasts as well as endothelial and epithelial cells across 12 tissues in normal mice.They detected not only the wide-spread expression of immune-related genes but also that this expression occurred in a cell-type-and organ-specific manner.Fibroblast populations expressed many genes coding for interleukins, immune checkpoint proteins, and complement C3 components.Moreover, they revealed that the structural cells studied appeared to be epigenetically primed to permit an activation of immune genes which might improve their responses to immune challenges.It seems that non-immune cells can also display a property called trained immunity, i.e., an innate immune memory which provides a rapid and enhanced response to future insults (Hamada et al., 2019).For example, Krausgruber et al. (2020) reported that the expression of many immune genes was robustly increased in structural cells after either a viral infection or an exposure to a cytokine although the immune responses were still cell-type-and tissue-specific outcomes.Several review articles have highlighted the role of structural cells in immune responses and their interactions with immune cells, e.g., hepatocytes (Zhou et al., 2016), skeletal muscle cells (Waldemer-Streyer et al., 2022), and smooth muscle cells (Salinthone et al., 2004).Next, we will examine the properties of tissue fibroblasts which enable these cells to act as immune regulators and we will emphasize many of the close interactions observed between fibroblasts and immune cells not only in the aging process but also in many age-related diseases.

Heterogeneity of fibroblasts
Fibroblasts are mesenchymal cells which originate from their progenitor cells via organ-specific lineages during development (LeBleu and Neilson, 2020;Plikus et al., 2021) (Fig. 1).Fibroblasts share their developmental lineages with certain other cells with a mesodermal origin, such as adipocytes, endothelial cells, pericytes, and perivascular smooth muscle cells.The transdifferentiation of these mesenchymal cells into fibroblasts and myofibroblasts in pathological states is thought to be attributable to their shared mesenchymal origin (Sun et al., 2016;Aujla and Kassiri, 2021).Fibroblasts are exceptionally plastic cells which are able to adapt to alterations within the tissue microenvironment in an attempt to maintain tissue homeostasis.Single-cell transcriptome techniques have revealed the extreme heterogeneity of tissue fibroblasts both in the steady-state and in several fibrotic and non-fibrotic diseases (Muhl et al., 2020;Buechler et al., 2021b;Deng et al., 2021;Lendahl et al., 2022).However, it is difficult to identify distinct fibroblast subpopulations by this method since the expression profiles are very variable not only between different tissues but also within diverse tissue locations.It seems that the heterogeneity of tissue fibroblasts reflects differences in the production and content of the ECM in diverse tissues (Muhl et al., 2020).However, certain functional categories of fibroblasts have been characterized, e.g., in many cancers ( Öhlund et al., 2017;Costa et al., 2018;Bryce et al., 2022).For instance, the inflammatory cancer-associated fibroblasts (iCAF) and the myofibroblastic myCAFs with immunosuppressive properties have been identified in human breast cancer (Costa et al., 2018).Several other studies on the tumor microenvironment have revealed not only the

Fibroblast-like synoviocytes
Fig. 1.Different phenotypes of fibroblasts.Tissue fibroblasts originate from mesenchymal progenitors.Specialized fibroblasts, i.e., lymph node fibroblastic reticular cells, hepatic stellate cells, and fibroblast-like synoviocytes, also originate from mesenchymal progenitors.Fibroblasts can become differentiated into myofibroblasts and subsequently myofibroblasts can be dedifferentiated into quiescent fibroblasts.Moreover, many cell types can be transdifferentiated into myofibroblasts which can display many phenotypes, such as cells with fibrogenic, pro-inflammatory, and immunosuppressive phenotypes.Both tissue fibroblasts and differentiated myofibroblasts can be converted into pro-inflammatory senescent fibroblasts.

Quiescent fibroblasts
Under steady-state conditions, most fibroblasts exist in the quiescent state, i.e., they are non-proliferating although they are metabolically highly active cells containing a wide network of rough endoplasmic reticulum and a rapid glycolytic flux, with especially intense activity along the pentose phosphate pathway (Lemons et al., 2010).Given that fibroblasts possess an impressive plasticity, they are important sensors of tissue homeostasis.For instance, in tissue injuries, such as myocardial infarction, fibroblasts switch to displaying a pro-inflammatory phenotype and secrete cytokines and chemokines with the aim to recruit immune cells into the damaged tissue (Section 4.1.).In addition, fibroblasts induce the differentiation of quiescent fibroblasts into myofibroblasts to enhance the fibrosis required for mechanical support (Section 3.2.)(Fig. 1).Intriguingly, there is convincing evidence that tissue fibroblasts not only possess the positional memory of their location in the tissue but also memories from their past experiences, i.e., a mechanical memory, a metabolic memory, and an inflammatory memory (Chang et al., 2002;Kirk et al., 2021).The ability of fibroblasts to recall memories from their past is probably attributable to epigenetics and metabolic reprogramming as has been observed in the trained immunity of macrophages and NK cells (Fanucchi et al., 2021;Dominguez-Andres et al., 2023).It seems that the chromatin of tissue fibroblasts is accessible for programming processes since several investigators have revealed that quiescent fibroblasts can be reprogrammed, e.g., into cardiomyocytes and neurons (Wada et al., 2013;Xu et al., 2020).For this reason, tissue fibroblasts are currently a promising therapeutic target in many diseases, such as cancers and rheumatoid arthritis.

Myofibroblasts
The fibrogenic activation of tissue fibroblasts generates myofibroblasts, i.e., these are the fusiform large fibroblasts which contain contractile fibrillar structures, such as α-smooth muscle actin (α-SMA) proteins combined with an abundant stress fiber network, as well as vimentin intermediate filaments, and fibroblast activation proteins (FAP) at the cell surface (Hinz et al., 2003;Plikus et al., 2021).These proteins are common markers of myofibroblasts in histological samples.Myofibroblasts also contain an extensive amount of rough endoplasmic reticulum and a wide Golgi network.This is because myofibroblasts are highly secretory cells producing not only the components of the ECM but also growth factors and inflammatory mediators (Powell et al., 1999).However, there exist clear differences in the properties of myofibroblasts when they are present in fibrotic, inflammatory, and cancer microenvironments.The common structural and functional characteristics of myofibroblasts have been reviewed in detail elsewhere (Hinz et al., 2003;Darby et al., 2016;Schuster et al., 2023).The main function of myofibroblasts is to provide mechanical support for tissues both in acute and chronic tissue injuries.For example, α-SMA proteins and stress fibers are involved in the formation of mature focal adhesion (FA) sites which (i) facilitate myofibroblast adhesion, (ii) transfer the contractile force of myofibroblasts to ECM structures, and finally (iii) mediate the mechanosensing signaling from the ECM to myofibroblasts (Hinz et al., 2003;D'Urso and Kurniawan, 2020).For instance, the stiffness of the ECM, e.g., an increased level of the crosslinks in structural proteins, is a major enhancer of the conversion of quiescent fibroblasts into contractile myofibroblasts (Avery et al., 2018).Angelini et al. (2020) have reviewed the age-related dysregulation of fibroblast mechanosensing in mouse heart.Moreover, it is known that a modification of ECM proteins via either sialylation or glycation promoted the transition of fibroblasts into myofibroblasts (Retamal et al., 2017;Sasaki et al., 2017).However, there exist several other inducers of myofibroblast differentiation, e.g., oxidative stress via reactive oxygen species (ROS) (Toullec et al., 2010;Huang et al., 2020), endoplasmic reticulum stress (Baek et al., 2012), inflammatory mediators, e.g., those produced by senescent cells (Section 3.3.),telomere attrition (Razdan et al., 2018), and many alarmins, such as high-mobility group box 1 (HMGB1) proteins (Lee et al., 2015) (Fig. 2).There are several signaling pathways which are involved in the differentiation process of myofibroblasts, such as the TGF-β-driven pathways, JAK/STAT3 signaling, and the NF-κB/inflammasomes axis (Artlett et al., 2011;Lodyga and Hinz, 2020).The TGF-β signaling pathways are the major inducers of myofibroblast differentiation and they stimulate the expression of α-SMA proteins and many other ECM components as well as the expression of NADPH oxidases, especially NOX4 which activates the generation of ROS in tissues (Carmona--Cuenca et al., 2008;Lodyga and Hinz, 2020).
It is not only tissue fibroblasts which can be converted into myofibroblasts but several other cell types, mostly mesenchymal and myeloid cells, can be transdifferentiated into myofibroblasts (Fig. 1).For instance, adipocytes, pericytes, smooth muscle cells, and endo-and epithelial cells can be converted into myofibroblasts (Sava et al., 2017;LeBleu and Neilson, 2020;Plikus et al., 2021).In addition, in chronic pathological conditions, some myeloid cells, such as inflammatory macrophages and monocytes as well as fibrocytes, can be transdifferentiated into myofibroblasts to enhance fibrosis (Reilkoff et al., 2011;Meng et al., 2016;Vierhout et al., 2021).There is substantial evidence that the macrophage-to-myofibroblast transition (MMT) is mainly driven by the TGF-β/Smad3 signaling, e.g., in renal and subretinal fibrosis (Wang et al., 2016;Little et al., 2020).Myofibroblast deposition occurs within tissues in many fibrotic diseases, e.g., cardiac fibrosis, idiopathic pulmonary fibrosis, and systemic sclerosis (van Caam et al., 2018;Schuster et al., 2023), and these cells also accumulate in the microenvironment surrounding tumors (Otranto et al., 2012), wound healing sites (Talbott et al., 2022), and within aged tissues (Selman and Pardo, 2021).In addition to their fibrous functions, myofibroblasts are also versatile immune regulators cooperating with immune cells to coordinate the repair processes in chronic pathological states (Section 5.).

Senescent fibroblasts
Over sixty years ago, Hayflick and Moorhead (1961) discovered that human diploid fibroblasts isolated from fetuses were able to replicate about fifty times in cell culture before the cell cycle was arrested and the cells became exhausted.After this seminal observation, a plethora of articles has been published describing cellular senescence not only in fibroblasts but also in many other proliferating cells (Munoz-Espin and Serrano, 2014;Yousefzadeh et al., 2020;Roger et al., 2021).Most of the studies on cellular senescence have been focused on the senescence state in cultured cells although currently, it is not known whether cells in aged tissues undergo similar alterations as encountered in vitro experiments.For instance, fibroblasts cultured on plastic plates or on collagen-coated dishes display a myofibroblast-type phenotype with a robust expression of α-SMA protein (Baranyi et al., 2019).Briefly, senescent fibroblasts in vitro have been characterized by their flattened morphology with an irreversible cell growth arrest (Munoz-Espin and Serrano, 2014;Tigges et al., 2014).Senescent fibroblasts express many distinct markers of senescence, such as senescence-associated β-galactosidase (SA-β-gal), cyclin-dependent kinase inhibitor 4 A (p16INK4A), and senescence-associated heterochromatin foci (SAHF), which have also been used as markers of cellular senescence in tissues.Senescent fibroblasts suffer from several functional impairments, e.g., an increased resistance to apoptosis, an accumulation of dysfunctional mitochondria, and deficiencies in proteostasis.Interestingly, the senescence phenotype A. Salminen et al. of diverse cell types displays a pro-inflammatory SASP state which is associated with the expression of many cytokines, chemokines, and colony-stimulating factors (Freund et al., 2010).The SASP state of fibroblasts will be described more thoroughly in Section 4.1.Originally, it was believed that cellular senescence represented an endpoint of limited replication capacity and was the primary cause of tissue aging.However, currently it is known that many stress-related insults, e.g., those attacking DNA integrity, can trigger cellular senescence.
Cellular senescence is not only associated with aging but it is present, e.g., during mammalian embryonal development (Munoz-Espin et al., 2013), cancers (Yang et al., 2021) and many fibrotic states (Schafer et al., 2017;Shi et al., 2023a).During embryogenesis, programmed senescence of cells blocks their proliferation and subsequently promotes their clearance thus remodelling tissue growth (Munoz-Espin et al., 2013).In fibrosis, it seems that senescent fibroblasts act as pro-inflammatory cells to aggravate tissue fibrosis.For instance, Schafer et al. (2017) demonstrated in a mouse model that the senolytic clearance of senescent cells from bleomycin-induced fibrosis improved the pulmonary and physical health of animals.

Specialized fibroblasts
It is evident that all tissue fibroblasts possess many tissue-specific properties and heterogeneous subtypes, as discussed earlier (Buechler et al., 2021b;Plikus et al., 2021;Lendahl et al., 2022).Although all fibroblasts can display many immune properties, there are specialized fibroblasts or fibroblast-like cells in certain tissues which have distinctive functions with respect to both innate and adaptive immunity and thus they have an important role in the pathogenesis of many diseases (Fig. 1).

Fibroblastic reticulum cells (FRC) in secondary lymphoid organs
The fibroblastic reticulum cells (FRC) in secondary lymphoid organs, especially in lymph nodes, have many important immune functions, i.e., FRCs not only organize the microarchitecture of the lymph nodes but they also regulate several functions of the immune system (Brown and Turley, 2015;Fletcher et al., 2015;Perez-Shibayama et al., 2019).It is known that FRCs become located in the T cell-rich zones in lymphoid organs where they (i) control the activation and proliferation of T lymphocytes, (ii) facilitate the crosstalk between T cells and dendritic cells, and (iii) act to promote the induction of regulatory T cells (Treg).Moreover, FRCs provide a structural and co-operative niche for the resident macrophages in lymph nodes (D'Rozario et al., 2023).There are also studies indicating that FRCs regulate homeostasis of B cells by enhancing B cell homing and survival (Cremasco et al., 2014).The FRC population secretes a wide range of cytokines and chemokines targeted at the immune cells and in this way they support immune defence.Moreover, disturbances in the functions of FRCs can trigger some pathological conditions, such as chronic infections and immune deficiencies (Fletcher et al., 2015).Interestingly, Turner and Mabbott (2017) reported that the aging process induced significant changes in the microarchitecture and function of mouse lymph nodes.For instance, the size of the FRC network clearly declined, the number of T cells was reduced, and the retention of immune complexes decreased, whereas there was a robust increase in the numbers of macrophages.These changes might account for the enhancement of immune senescence observed in the inflammaging state.

Hepatic stellate cells and portal fibroblasts
The role of fibroblasts in the pathogenesis of liver fibrosis and hepatocellular carcinoma has been under intensive research (Iwaisako et al., 2014;Baglieri et al., 2019).Currently, it is known that in pathological conditions hepatic stellate cells (HSC) and portal fibroblasts (PF)

Senescent fibroblasts
Fig. 2. Modification of the properties of tissue fibroblasts towards either the pro-inflammatory or immunosuppressive phenotypes.Microenvironmental insults can alter the properties of quiescent fibroblasts so that they display phenotypes with pro-inflammatory or immunosuppressive features.Inflammatory cytokines, chemokines, and complements promote the shift of fibroblasts into the pro-inflammatory phenotype, whereas anti-inflammatory cytokines, ROS, adenosine/lactate, and the PD-L1 checkpoint protein can direct fibroblasts to adopt an immunosuppressive phenotype.Several age-related signaling factors can promote the switch of tissue fibroblasts into modified phenotypes.Abbreviations: AGE/RAGE, advanced glycation endproduct/receptor for AGE; CCL/CXCL, chemokines; CSF, colony-stimulating factor; ECM, extracellular matrix; ER, endoplasmic reticulum; HMGB1, high mobility group box 1; IL, interleukin; PD-L1, programmed death-ligand 1; ROS, reactive oxygen species; SASP, senescence-associated secretory protein; TGF-β, transforming growth factor-β.
are the major sources of myofibroblast formation in liver fibrosis.There are quiescent HSCs located in the perisinusoidal space of the liver, but different kinds of injuries activate HSCs and transdifferentiate them into myofibroblasts which induce the deposition of fibrotic lesions within the liver.While normally PFs surround the portal veins and maintain the integrity of bile ducts, in pathological states they can trigger biliary fibrosis (Karin et al., 2016).There are indications that the activation of HSCs can stimulate the development of CAFs which subsequently have been revealed to enhance the pathogenesis of hepatocellular carcinoma (Baglieri et al., 2019).Zhao et al. (2014) demonstrated that the experimental activation of HSCs promoted tumor angiogenesis and lymphangiogenesis in mouse liver and robustly expanded the populations of immunosuppressive Tregs and MDSCs both in the tumor microenvironment as well as in the spleen and the bone marrow.Chou et al. (2011) revealed that activated HSCs/myofibroblasts stimulated the generation of MDSCs via the secretion of inflammatory mediators, such as INFγ cytokines.Sun et al. (2022) reported that HSCs secreted programmed death-ligand 1 (PD-L1), an immune checkpoint protein, which enhanced the TGF-β-induced differentiation of HSCs into myofibroblasts and thus promoted hepatic carcinogenesis.These studies indicated that the inflammation-associated activation of HSCs has an important role not only in liver diseases but it is also able to stimulate immunosuppressive responses in the spleen and the bone marrow.It is known that a systemic immunodeficiency is a trait associated with liver cirrhosis (Tuchendler et al., 2018).

Fibroblast-like synoviocytes
The fibroblasts in the joints, i.e., the fibroblast-like synoviocytes (FLS), have a crucial role in the pathogenesis of rheumatoid arthritis (RA) (Kemble and Croft, 2021;Smith et al., 2023).RA is a chronic inflammatory disorder with an autoimmune origin although the specific mechanisms leading to arthritis still need to be clarified.Single-cell transcriptome investigations have revealed that functionally different subsets of FLSs are localized in the RA synovium (Mizoguchi et al., 2018;Croft et al., 2019).Mizoguchi et al. (2018) demonstrated that the synovium of human RA and osteoarthritis (OA) patients contained three major FLS populations but their proportions were differently affected between RA and OA patients.The synovium of RA patients contained more fibroblasts that were positive for thymus cell antigen 1 (THY1, also called CD90) than the synovium of OA patients, whereas the THY1-negative subpopulation was markedly abundant in OA patients sufferring from bone and cartilage damages.They also reported that the CD34-positive fibroblasts, the third subpopulation, displayed a high expression and secretion of chemokines, such as IL-6, CXCL12, and CCL2, indicating that these fibroblasts recruited monocytes into the inflamed synovium.Subsequently, Croft et al. (2019) reported that the different FLS subsets promoted either inflammatory responses or tissue damage in mouse RA model.They revealed that the FAPα + FLS cells were present both in the synovial lining and the sub-lining layers.However, the FAPα + population contained two subpopulations which were either positive or negative for the THY1 phenotype, i.e., the immune active THY1 + fibroblasts were located in the synovial sub-lining layer, whereas the THY1-negative cells were restricted to the synovial lining layer.Next, Croft et al. (2019) utilized a cell transfer technique and demonstrated that the injection of the FAPα + THY1 + fibroblasts into the joints of healthy mice induced intense inflammatory response in the joints but exerted only a slight effect on bone and cartilage, whereas the transfer of the FAPα + THY1 -fibroblasts inflicted damages in bone and cartilage but triggered only minimal inflammation in the joints.These studies indicated that due to the heterogeneity of fibroblasts, there might be a need for different therapies in RA and OA diseases.
There are close interactions between synovial fibroblasts and immune cells in the progress of RA pathology (Kemble and Croft, 2021;Marsh et al., 2021;Smith et al., 2023).The inflammation in human RA is associated with an expansion of synovial fibroblasts which express many pro-inflammatory cytokines and chemokines, such as IL-6 and IL-8.Smith et al. (2023) demonstrated that the exposure of human synovial fibroblasts to the major myeloid and T cell-derived cytokines, i.e., IL-1β, IFN-γ, and TNFα, stimulated signaling via the IRF, STAT, and NF-κB pathways inducing four distinct inflammatory states in the synovial fibroblast population.This heterogeneity in the fibroblast population was spatially constrained in human synovium.In the resting state, it is thought that tissue resident fibroblasts and specialized macrophages maintain homeostasis in the joints (Kemble and Croft, 2021).In the RA environment, synovial fibroblasts act as immune effector cells modifying the activities of resident macrophages within the joint.For instance, activated synovial fibroblasts express several pattern recognition receptors, such as Toll-like receptors (TLR) and alarmins, as well as secrete several chemokines thus recruiting T and B lymphocytes and monocytes into the inflamed synovium (Marsh et al., 2021;Smith et al., 2023).The interactions between fibroblasts and immune cells will be examined in detail in Section 5. Currently, it is known that there are clear changes in the epigenetic profiles of synovial fibroblasts during the progress of RA and it seems that epigenetics are driving the changes in the phenotype of synovial fibroblasts and thus promoting the pathogenesis of RA (Karami et al., 2020).

Immune properties of tissue fibroblasts
There is convincing evidence that tissue fibroblasts are versatile immune regulators which can display many different immune properties as they attempt to maintain tissue homeostasis in pathological conditions (Davidson et al., 2021;Cavagnero and Gallo, 2022).Single-cell studies investigating the phenotypes of fibroblasts in the microenvironments of cancers and inflammatory diseases have revealed that there are simultaneously both pro-inflammatory and immunosuppressive fibroblast subpopulations (Costa et al., 2018;Monteran and Erez, 2019;Mhaidly and Mechta-Grigoriou, 2020;Korsunsky et al., 2022) (Fig. 2).Accordingly, there exists an abundant fibrogenic myofibroblast population in fibrotic states, such as cardiac fibrosis and idiopathic pulmonary fibrosis.Considering that fibroblasts are sensors of tissue injuries and major drivers of the repair processes, it seems likely that tissue fibroblasts are able to change their phenotypes in cooperation with immune cells during the repair process, such as after myocardial infarction (Section 5.2.).

Pro-inflammatory fibroblasts
Given that several alarmins and damage-associated molecular patterns (DAMPs) as well as many inflammatory mediators are activators of tissue fibroblasts, it is not surprising that the cells secrete several cytokines, chemokines, and inflammatory mediators to enhance immune responses to retain tissue homeostasis (Pang et al., 1994;Powell et al., 1999;Bräuninger et al., 2021).For instance, Pang et al. (1994) reported that a treatment of human duodenal fibroblasts with either lipopolysaccharide (LPS) or IL-1α robustly increased the expression of GM-CSF, IL-6, IL-8, IL-1α, IL-1β, and ICAM-1 in a time and dose-dependent manner.It is known that myofibroblasts can also secrete many inflammatory mediators, such as NO, H 2 O 2 , PGE 2 , prostacyclin, and HETE (Powell et al., 1999).Thus, it seems that fibroblasts can regulate their inflammatory status both in an autocrine and paracrine manner since they also express the receptor proteins for several inflammatory factors, such as IL-1, IL-6, IL-8, TNF-α, and prostaglandins (Powell et al., 1999).Moreover, pro-inflammatory fibroblasts secrete several chemokines which facilitate immune cell generation in the BM and subsequently direct their recruitment into inflamed tissues (Section 5.2.).It is known that fibroblasts secrete a wide array of chemokines, e.g., CCL2,3,5,8 and CXCL2,8,10,12 (Smith et al., 1997;Powell et al., 1999;Bräuninger et al., 2021;Caetano et al., 2023) (Fig. 2).There is substantial evidence that in inflammatory states fibroblasts cooperate with tissue macrophages and other immune cells to enhance inflammatory responses (Van Linthout et al., 2014;Correa-Gallegos et al., 2021;Schuster et al., 2021) (Fig. 2) (Section 5.2.).Bräuninger et al. (2021) reported that the treatment of human fibroblasts with TNF-α, a pro-inflammatory factor, significantly increased the expression of several inflammatory cytokines and chemokines, whereas the exposure to TGF-β, an anti-inflammatory/immunosuppressive factor, significantly augmented the secretion of collagen components and the inhibitors of matrix proteinases.These observations indicate that tissue fibroblasts respond to different stimuli inducing either pro-inflammatory or pro-fibrotic responses.
Not only acute tissue injuries induce a pro-inflammatory phenotype of fibroblasts but also cellular senescence observed with aging and many chronic inflammatory conditions can trigger the inflammatory SASP state in tissue fibroblasts (Fig. 1).Several other cell types, in addition to fibroblasts, can also express the molecular markers of senescent state during the aging process and diverse age-related diseases.The secretory phenotype of senescent fibroblasts not only involves pro-inflammatory factors, but it also includes anti-inflammatory markers, such as TGF-β, IL-6, and IL-10, growth factors, matrix metalloproteinases, and components of the ECM (Freund et al., 2010;Maciel-Baron et al., 2016;Zorina et al., 2022).At the single-cell level, cellular senescence displays a wide heterogeneity which seems to be dependent on insulting stimuli and differences in the microenvironment.For instance, inflammatory cytokines stimulate NF-κB signaling which is known to induce cellular senescence in human fibroblasts (Chien et al., 2011;Salminen et al., 2012).Moreover, immunosuppressive cells recruited into inflamed tissues secrete TGF-β factor which not only triggers myofibroblast differentiation (Section 3.2.)but it is also a potent inducer of cellular senescence, especially in cancer microenvironment (Acosta et al., 2013;Tominaga and Suzuki, 2019;Matsuda et al., 2023).Acosta et al. (2013) demonstrated that an exposure to the conditioned medium from the senescent fibroblasts induced the senescence of normal human IMR90 fibroblasts.They revealed that this paracrine senescence of human IMR90 cells was induced by secreted TGF-β and certain agonists of TGF-β signaling via the activation of NLRP3 inflammasomes.It is known that a bystander effect induced by senescent fibroblasts could also be mediated via extracellular vesicles (Wijesinghe et al., 2022).It seems that senescent pro-inflammatory fibroblasts are able to switch their phenotypes from an acute resolving inflammation to the chronic inflammatory state by propagating cellular senescence and recruiting immunosuppressive cells into inflamed tissues (Section 5.2.).

Immunosuppressive fibroblasts
There is convincing evidence that CAFs have a crucial role in the generation of the immunosuppressive microenvironment in tumors and subsequently they provide for tumor cells an evasion from immune surveillance (Monteran and Erez, 2019;Mhaidly and Mechta-Grigoriou, 2020).Single-cell transcriptome investigations have demonstrated that there exists an extensive heterogeneity in the CAF populations although most studies have identified both the pro-inflammatory (iCAF) and the myofibroblastic (myCAF) subpopulations.Costa et al. (2018) observed in human breast cancer four different CAF subsets of which CAF-S1 involved the fibroblasts possessing significant immunosuppressive activity and resistance to immunotherapy.For instance, the immunosuppressive CAF-S1 fibroblasts displayed an abundant expression of FAP and CD73 proteins.The CD73 protein is an ecto-5'-nucleotidase which can convert AMP and ADP into adenosine.It is known that extracellular adenosine stimulates the immunosuppressive activity of MDSCs and Tregs (Ryzhov et al., 2011;Ohta and Sitkovsky, 2014).The FAP protein is a membrane-bound prolyl endopeptidase which is highly expressed in CAF-S1 cells as well as in fibroblasts in many pathological conditions, e. g., rheumatoid arthritis and liver fibrosis.FAP signaling activated the production of CCL2 chemokine via STAT3 signaling and subsequently CCL2 promoted the recruitment of MDSCs into inflamed tissue (Yang et al., 2016) (Fig. 2).
Interestingly, in pathological conditions, myofibroblasts utilize aerobic glycolysis for their energy production (Guido et al., 2012;Xie et al., 2015).Myofibroblasts benefit from the secretion of lactic acid since it enhances the pH-dependent activation of latent TGF-β and subsequently TGF-β promotes the activity of myofibroblasts.However, it is known that lactate is a potent inducer of the immunosuppressive microenvironment (Husain et al., 2013;Nolt et al., 2018) (Fig. 2).For instance, Husain et al. (2013) reported that lactate exposure increased the proliferation and immunosuppressive activity of mouse MDSCs and accordingly it suppressed the cytotoxicity of NK cells.Moreover, lactate exposure induced the polarization of mouse pro-inflammatory M1 macrophages into the immunosuppressive M2 phenotype in mouse model of intestinal inflammation (Zhou et al., 2022).There is also clear evidence that fibroblasts express on their cell surface the programmed death ligand 1 (PD-L1) protein which targets the PD-1 receptor present on the surface of many immune cells (Pinchuk et al., 2008;Ghosh et al., 2021).The PD-1 receptor is an immune checkpoint protein which inhibits the activities of many immune cells (Section 5.3.).For instance, the immunosuppression induced by the PD-1/PD-L1 axis has an important role in the prevention of autoimmune diseases but on the other hand, it allows cancer cells to avade immune attacks.Overall, these examples indicate that immunosuppressive fibroblasts have close interactions with the cells of the immunosuppressive network as they attempt to counteract excessive immune responses.

Antigen-presenting fibroblasts
Due to their immune properties, many researchers have also categorized the antigen-presenting fibroblasts and complement-secreting fibroblasts into their own subgroups (Bryce et al., 2022;Lavie et al., 2022).The expression of major histocompatibility complex class II (MHCII) molecules is a hallmark of professional antigen-presenting cells, e.g., dendritic cells and macrophages.There are a number of reports indicating that tissue fibroblasts express MHCII proteins in several pathological states, such as in human tumors (Huang et al., 2022) and tissue inflammation (Wassenaar et al., 1997;Ngwenyama et al., 2022).Moreover, it is known that the antigen presentation by fibroblasts has important functions in many specialized tissues, such as mouse lymphoid organs (Kündig et al., 1995), human colonic mucosa (Saada et al., 2006), and human rheumatoid synovium (Boots et al., 1994).In all these studies, it was confirmed that the antigen presentation was functionally active, i.e., foreign antigens were processed and presented for T cells leading to their activation.These observations indicate that tissue fibroblasts can initiate the antigen-specific form of immunity in pathological conditions.For instance, Barrera et al. (2004) reported that human intestinal myofibroblasts were able to induce the MHCII-mediated antigen presentation after an exposure to Staphylococcal enterotoxin.Ngwenyama et al. (2022) demonstrated that IFNγ exposure also stimulated the expression of MHCII proteins in isolated normal rat cardiac fibroblasts and subsequently it induced a robust proliferation of CD4+ T cells.IFNγ is a common inducer of the expression of MHCII proteins and an enhancer of antigen presentation.However, Huang et al. (2022) revealed that the apCAF present in human pancreatic cancer specimens induced the formation of immunosuppressive Tregs from the CD4 + T cells which was attributed to a lack of certain co-stimulatory molecules in the apCAFs.Currently, it is not known whether the presence of an immunosuppressive microenvironment can affect antigen presentation by fibroblasts since it is known that MDSCs and Tregs reduce the antigen uptake and lower the presenting capacity of dendritic cells.It seems that tissue fibroblasts possess the potential to be involved in antigen-specific immunity but that property seems to be a context-dependent process.

Complement-secreting fibroblasts
The complement system is an important branch of the innate immune system, e.g., it promotes inflammation and enhances the clearance of immune complexes, microbes, and damaged cells but it can also aggravate chronic inflammatory diseases.Interestingly, it is not only liver and certain immune cells but also the fibroblasts present in several tissues can secrete complement components including those involved in the classical, alternative, or lectin pathways (Kulics et al., 1994;Ezure et al., 2019;Le Fournis et al., 2020;Friscic et al., 2021) (Fig. 2).For instance, Le Fournis et al. (2020) demonstrated that after an injury or bacterial attack in the dental pulp tissue, fibroblasts stimulated the synthesis of complement components and activated the complement system which produced several active molecules, e.g., C3b, C5a, and the membrane attack complex.This complement activation not only eliminated the invading bacteria but also initiated a local inflammatory reaction which was controlled by dental pulp fibroblasts via the complement cascades.Moreover, Friscic et al. (2021) reported that synovial fibroblasts acted as the checkpoints between the remission and relapse phases in mouse arthritis by triggering an activation of the complement system.Repeated inflammatory insults induced a fibroblast-mediated priming, i.e., sensitization, in inflammatory synovium.They revealed that the priming enhanced the accumulation of inflammatory THY + fibroblasts (Section 3.4.3.)into mouse synovium as well as increasing the expression of complement components, especially those of C3, C3a, and C3a receptor in the primed fibroblasts.The priming of synovial fibroblasts increased the complement-mediated metabolic activity and the activation of the NLRP inflammasomes that intensified the invasiveness of fibroblasts and promoted osteoclastogenesis.This indicated that the priming of fibroblasts into an inflammatory state aggravated the severity arthritis in mouse model.A similar priming phenomenon was observed in the synovial fibroblasts isolated from patients with arthritis.Chen et al. ( 2021) utilized single-cell transcriptomics and identified a csCAF population in human pancreatic ductal adenocarcinoma.For instance, a robust expression of C3, C7, CFB, and CFD complements was associated with increased inflammatory responses in the tumor microenvironment.Tissue fibroblasts can also deliver complement components via their packaging into exosomal vesicles.Kumar et al. (2021) demonstrated that the perivascular fibroblasts from calf pulmonary hypertension secreted extracellular vesicles which were enriched in complement components, especially the C3 component.They revealed that these exosomes significantly augmented the expression of proinflammatory cytokines and chemokines in the BM-derived macrophages.Fibroblast-derived exosomes were critical mediators in the complement-induced perivascular inflammation associated with pulmonary hypertension in calves.Currently, it is not known whether the so-called intracellular complosomes are involved in the immune functions of fibroblasts although the complement C3 and C3a receptors as well as NLRP3 inflammasomes are required for the activation of the complosomes (West and Kemper, 2023).

Close interactions between tissue fibroblasts and immune cells
As described above, tissue fibroblasts display remarkably different immune properties in diverse pathological conditions in their attempts to prevent excessive tissue injuries and subsequently to initiate efficient repair processes.Tissue fibroblasts possess an impressive capability to cooperate with professional immune cells to restore tissue homeostasis.Currently, it is recognized that there are close interactions between fibroblasts and immune cells in the pathogenesis of both tissue injuries and many fibrotic and inflammatory diseases as well as during wound healing.Given that these pathological states are very common disorders, the role of fibroblasts in these pathological states has been rather well characterized and frequently reviewed in detail elsewhere (van Caam et al., 2018;Monteran and Erez, 2019;Davidson et al., 2021;Huaux, 2021;Schuster et al., 2023).Here, we will focus on some important interactions between tissue fibroblasts and immune cells which might be involved in the age-related fibrotic lesions and tissue degeneration.

Interactions via the modulation of the ECM
A robust remodelling of the ECM is commonly observed during developmental processes, in many pathological states, and in the aging process (Lu et al., 2011;Gordon-Weeks and Yuzhalin, 2020;Selman and Pardo, 2021).Interestingly, the ECM provides an important niche through which fibroblasts and immune cells can reciprocally communicate with each other.Tissue fibroblasts regulates the composition of the ECM by secreting not only the components of the ECM but also the enzymes which modify the structural integrity of the ECM.Accordingly, the properties of the ECM control many activities of tissue fibroblasts, e. g., the stiffness of the ECM as well as protein modifications promote the differentiation of fibroblasts into myofibroblasts (Section 3.2.).Intriguingly, the immune cells present in tissues, e.g., macrophages, are not only able to respond to the molecular changes in the ECM but they secrete molecules which modify the structures of the ECM, such as ROS, many proteases, and lysosomal enzymes (Werb and Gordon, 1975;Hakala et al., 2003;Canli et al., 2017) and thus regulate the functions of fibroblasts.This mutual interaction between fibroblasts and immune cells via the ECM niche has an important role in tumour growth and many inflammatory diseases (Sorokin, 2010;Gordon-Weeks and Yuzhalin, 2020;Marangio et al., 2022).The ECM-related mechanisms in the regulation of the functions of immune cells have been explored in many review articles (Sorokin, 2010;Boyd and Thomas, 2017;Gordon-Weeks and Yuzhalin, 2020).For instance, an increase in the stiffness of the ECM promoted the polarization of pro-inflammatory M1 macrophages into immunosuppressive M2 macrophages in human and mouse macrophages (Wang et al., 2022b;Taufalele et al., 2023).The M2 polarization of macrophages also has a crucial role in the myofibroblast-driven fibrosis, e.g., in human systemic sclerosis (Hu et al., 2023).
The structural changes occurring in the ECM also regulate the activities of T cells, NK cells and Tregs (Salmon et al., 2012;Bunting et al., 2022;Shi et al., 2023b).For instance, Salmon et al. (2012) demonstrated that the matrix architecture regulated the recruitment and migration of T cells in the stroma of human lung tumors.Warren et al. (2014) reported that laminins, especially laminin α4, controlled the trafficking of T cells and their differentiation into Tregs in mouse lymph nodes.Accordingly, Shi et al. (2023b) revealed that an increase in the stiffness of the matrix promoted the induction and metabolism of human Tregs.Recently, Bunting et al. (2022) demonstrated in mouse skin graft rejection model that the deposition of collagen and elastin inhibited the cytotoxicity of NK cells, whereas it promoted the production of chemokines and cytokines.Moreover, in mouse melanoma, the cytotoxicity of NK cells was robustly increased by a blockage of collagen deposition (Bunting et al., 2022).In addition to the NK cells, also other tissue-resident innate lymphoid cells (ILC) are able to display bidirectional immunomodulation via the ECM (An et al., 2020).As stated in Section 3.2., the M2 macrophages and several other immunosuppressive cells secreted TGF-β which not only promoted the differentiation of myofibroblasts but was also a major inducer of the cross-linking of ECM proteins and thus it enhanced the stiffness and rigidity of the ECM (Cho et al., 2018;Semkova and Hsuan, 2021).Furthermore, it is known that fibroblasts and immune cells secrete proteolytic enzymes which can cleave the DAMP peptides from the proteins of the ECM, e.g., aggrecan, biglycan, and decorin, which locally control innate immunity (Frevert et al., 2018).Moreover, the elastin-derived peptides, which are matrikines, are able to modulate several immune responses in tissues (Fulop et al., 2012;Le Page et al., 2019).Considering inflammaging, it is interesting to remember that structural changes in the ECM with the accompanying increased stiffness of tissues are hallmarks of the aging process.

Interactions between fibroblasts and immune cells in acute inflammation
As described earlier (Section 4.1), in the conditions of acute A. Salminen et al. inflammation, there is a close crosstalk between pro-inflammatory fibroblasts and immune cells, either with tissue-resident macrophages and ILCs or with the immune cells recruited into tissues (Shinde and Frangogiannis, 2014;Elemam et al., 2017;Cooper et al., 2021;Correa-Gallegos et al., 2021;Davidson et al., 2021).It seems that tissue macrophages and fibroblasts can mutually stimulate their inflammatory activities by secreting an arsenal of inflammatory mediators (Buechler et al., 2021a).For instance, fibroblasts secrete many important chemokines, e.g., CCL2, CXCL8, CXCL10, CXCL12, which are known to be chemotactic factors for monocytes and lymphocytes (Smith et al., 1997;Powell et al., 1999;Caetano et al., 2023).The chemokines released from inflamed tissues induce hematopoiesis in the bone marrow generating both myeloid and lymphoid cells which are released into the circulation and subsequently can be recruited into inflamed tissues.
Myocardial infarction has been an extensively studied model on the versatile functions of fibroblasts in different phases during the recovery process (Prabhu and Frangogiannis, 2016;Mouton et al., 2019;Venugopal et al., 2022).After myocardial infarction, fibroblasts are activated and they adopt a pro-inflammatory phenotype secreting diverse cytokines and chemokines as well as proteolytic enzymes which remodel the post-infarct ECM of the myocardium (Prabhu and Frangogiannis, 2016;Daseke et al., 2020;Venugopal et al., 2022).During acute inflammatory phase there are mutual interactions between fibroblasts and macrophages which potentiate inflammatory activities of both cell types.After the acute inflammatory phase, tissue fibroblasts become the dominant cells, proliferating and differentiating into myofibroblasts which not only secrete the components of the ECM but also release many anti-inflammatory cytokines.In addition, these fibroblasts secrete diverse pro-angiogenic factors in an attempt to prevent the expansion of myocardial injuries.During this proliferation/maturation phase, there is a close collaboration between tissue-resident cells and recruited immunosuppressive cells, such as MDSCs and Tregs (Saxena et al., 2014;Mouton et al., 2019;Zhang et al., 2023).For instance, (i) M1 macrophages will become polarized into immunosuppressive M2 macrophages, (ii) MDSCs and Tregs enhance the conversion of fibroblasts as well as macrophages into fibrogenic myofibroblasts by secreting TGF-β and IL-10, and (iii) dead cells will be phagocytized by macrophages and myofibroblasts thus augmenting the resolution of the inflammatory condition (Saxena et al., 2014;Nakaya et al., 2017;Lu et al., 2020;Zhang et al., 2023).There is mounting evidence that it is the cooperation between myofibroblasts, M2 macrophages, as well as between recruited MDSCs and Tregs which has a crucial role in determining whether the resolution and repair processes will be successful or whether there will be an excessive accumulation of fibrotic lesions into the post-infarcted myocardium (Saxena et al., 2014;Kurose and Mangmool, 2016;Prabhu and Frangogiannis, 2016).

Interactions between fibroblasts and the immunosuppressive network in chronic inflammation
Over twenty years ago, it was claimed that tissue fibroblasts regulated the switch from the acute inflammatory state to the chronic inflammatory process (Buckley et al., 2001).Currently, it is known that tissue fibroblasts play a major role in the development of tumors, diverse chronic inflammatory and fibrotic diseases, and wound healing.However, fibroblast populations are very heterogeneous in different diseases since they reflect the tissue type and features of the pathological state.The presence of senescent fibroblasts as well as both pro-inflammatory and immunosuppressive subsets of fibroblasts augment the heterogeneity of fibroblast populations.Interestingly, in chronic diseases, either inflammatory or fibrotic disorders, there is an increase in the numbers of immunosuppressive cells, such as M2 macrophages, MDSCs, and Tregs, in affected tissues.For example, Fernandez et al. (2016) observed the number of MDSCs was augmented in the circulation of patients with idiopathic pulmonary fibrosis.The level of MDSCs in the blood correlated with the severity of the disease.Liu et al. (2022) also reported that the presence of MDSCs and Tregs was significantly increased in the circulation of the patients with idiopathic pulmonary fibrosis.Moreover, they reported that the recruitment of MDSCs into the lungs of mice suffering from the bleomycin-induced fibrosis was robustly increased and the level of fibrogenic myofibroblasts was upregulated.Birjandi et al. (2016) demonstrated that an injection of Tregs into mice exacerbated the bleomycin-induced pulmonary fibrosis, whereas Chakraborty et al. (2018) revealed that a depletion of Tregs ameliorated the progression of the bleomycin-induced fibrotic pathology.Moreover, it is known that the M2-polarized macrophages are associated with the enhanced development of fibrotic lesions in several experimental models of tissue fibrosis (Braga et al., 2015;Lis-Lopez et al., 2021).Currently, there is mounting evidence that the recruitment and activation of immunosuppressive cells, especially MDSCs, Tregs, and M2 macrophages, evoke an excessive conversion of fibroblasts into myofibroblasts and thus aggravate the severity of tissue fibrosis (Braga et al., 2015;Huaux, 2021;van Geffen et al., 2021).
Currently, it is known that the tissue fibroblasts in tumor sites, i.e., the CAFs, have a crucial role in the generation of the immunosuppressive microenvironment which impairs the immune surveillance of cancer cells (Monteran and Erez, 2019;Yoshida et al., 2019;Mhaidly and Mechta-Grigoriou, 2020;Mao et al., 2021).Single-cell transcriptome studies have revealed that the CAF populations consist of distinctive subsets which possess the properties of several phenotypes, such as iCAFs, myCAFs, immunosuppressive CAFs, and pro-metastatic CAFs (Mhaidly and Mechta-Grigoriou, 2020).The CAFs are specialized myofibroblasts which can be activated through diverse mechanisms from either tissue fibroblasts or via transdifferentiation from many mesenchymal cell types (Section 3.2.).In general, the inflammatory signals, such as cytokines and especially chemokines, are essential initial triggers in cancers since they can recruit MDSCs and Tregs into tumor sites and stimulate the differentiation of macrophages into immunosuppressive TAMs as well as inducing the transition of fibroblasts into myofibroblasts.However, certain non-inflammatory signals can also induce the differentiation of myofibroblasts, such as DNA damage, ROS, metabolic disorders, and ECM stiffness (Section 3.2.).Interestingly, it is known that there is immune cooperation between activated myofibroblasts and recruited immunosuppressive cells since both of these cell types secrete immunosuppressive cytokines, such as TGF-β, IL-6, and IL-10, which potentiate their immunosuppressive properties (Monteran and Erez, 2019;Mhaidly and Mechta-Grigoriou, 2020;Salminen, 2023b).Moreover, ROS, lactate, and PGE2 are immunosuppressive mediators secreted by cancer cells, myofibroblasts, and immunosuppressive cells and they enhance the efficiency of the immunosuppressive network in the tumor microenvironment (Yoshida et al., 2019) (Fig. 2).
The interactions between tissue fibroblasts and immune cells have been studied in many chronic inflammatory diseases, e.g., in rheumatoid arthritis (Section 3.4.3),chronic kidney and pulmonary diseases, and vascular inflammatory states (Enzerink and Vaheri, 2011;Sato and Yanagita, 2017;Korsunsky et al., 2022;Ghonim et al., 2023).For instance, the single-cell transcriptome study conducted by Korsunsky et al. (2022) on four different chronic inflammatory diseases revealed the presence of two shared fibroblast phenotypes across the diseases and in many affected tissues, i.e., the immune-interacting (CXCL10 + /CCL19 + ) and vascular-interacting (SPARC + /COL3A1 + ) phenotypes.In addition to those common subgroups, they also observed some disease-specific inflammatory fibroblast subsets.Many other studies, e.g., on rheumatoid arthritis (Section 3.4.3),have also revealed that chronic inflammatory diseases contain inflammatory fibroblast populations which express and secrete many cytokines and chemokines.These fibroblast subsets recruit immune cells into inflamed tissues and thus maintain a chronic inflammatory state (Van Linthout et al., 2014).On the other hand, invading immune cells can promote the fibroblast transition into myofibroblasts and it is known that fibrotic lesions are associated with many chronic inflammatory diseases (Hartupee and Mann, 2016).
A. Salminen et al.There are several immune checkpoint proteins, either inhibitory or stimulatory receptors, which regulate the activity of the immune system.The PD-1/PD-L1 axis is one of the inhibitory checkpoints and it suppresses many important functions of T, B, natural killer (NK), and dendritic cells (Ghosh et al., 2021;Zhao et al., 2023).The activation of the PD-1 receptor after binding of the PD-L1 protein triggers an inhibitory signaling cascade which inhibits many immune-related functions of the PD-1-positive cells and consequently promotes immunosuppression in the microenvironment (Ghosh et al., 2021;Laba et al., 2022).For instance, an exposure to PD-L1 proteins inhibits the proliferation and functions of the antigen-specific T cells, reduces the secretion of cytokines, such as IL-2, and evokes their exhaustion or even apoptosis.In CD8 + T cells, PD-L1 exposure decreases their cytotoxicity and thus impairs the immune surveillance of cancer cells.Interestingly, the PD-L1 protein is expressed not only in immune cells, such as MDSCs and TAMs, but also in tissue fibroblasts, CAFs, and many cancer cells (Pinchuk et al., 2008;Kang et al., 2020;Wang et al., 2022a;Kawasaki et al., 2023).However, the expression level of PD-L1 protein is highly regulated and it has a crucial role in the immune efficiency of the PD-L1-positive cells (Shen et al., 2019).Moreover, it is known that the PD-L1 protein can be secreted from the PD-L1-positive cells or packed into exosomal vesicles (Kang et al., 2020).The secretion of PD-L1 proteins, especially those in exosomes, enhances the immunosuppressive potential of the PD-L1-positive cells.Kang et al. (2020) demonstrated that TGF-β exposure induced the expression of the immunoinhibitory PD-L1 protein in human and mouse fibroblasts.They also reported that the knockdown of PD-L1 in fibroblasts decreased the expression of α-SMA and other components of the ECM.Moreover, Kang et al. (2020) revealed that the treatment of human lung fibroblasts with TGF-β increased the production of the PD-L1-containing exosomes which inhibited the proliferation of T cells and activated the migration of human fibroblasts.These results accounting for the mechanism causing a stimulation of the PD-L1-mediated immunosuppression by tissue fibroblasts are interesting observations because immunosuppressive cells secrete TGF-β and thus, via an increased PD-L1 expression, they enhance the role of fibroblasts in the immunosuppressive network.Wang et al. (2022a) reported that the presence of the PD-L1-positive fibroblasts promoted the healing of excisional wounds in mouse skin by inhibiting inflammation.For instance, the PD-L1-positive fibroblasts induced the polarization of M1 macrophages into M2 macrophages and thus they enhanced the resolution of inflammation in wound locations.Li et al. (2019) reported that CAFs secreted the CXCL5 chemokine which promoted the expression of PD-L1 in diverse cancer cells, thus enhancing their immune escape.On the other hand, Kawasaki et al. (2023) revealed that in cocultures, human cancer cells stimulated the expression of the PD-L1 protein in human primary fibroblasts.It seems that fibroblasts are capable of augmenting immunosuppression via the PD-1/PD-L1 axis in different tissue microenvironments.

Fibroblasts are drivers of tertiary lymphoid structures in chronic inflammation
Recent studies have revealed that tissue fibroblasts have an important role in the formation and maintenance of the tertiary lymphoid structures (TLS), also called tertiary lymphoid organs, in tissues suffering from chronic inflammation (Nayar et al., 2019;Asam et al., 2021;Sato et al., 2023).The TLS foci are ectopic immune organs which locate in the peripheral, non-lymphoid tissues during a persistent inflammatory state, e.g., autoimmune diseases, atherosclerosis, chronic infections, chronic kidney disease, and tumors (Bery et al., 2022;Sato et al., 2023).These TLS foci contain a heterogeneous cell population with compartments for T and B lymphocytes although the clusters can also harbor ILC3 cells, dendritic cells, monocytes, macrophages, fibroblastic reticular cells, and fibroblasts.The immune function of the TLS hub is very flexible depending on the microenvironment, e.g., it can range from antibody production against local antigens, T cell priming, up to cytokine secretion.In this respect, TLS foci can be viewed as powerhouses of adaptive immunity in non-lymphoid tissues (Bery et al., 2022;Sato et al., 2023).While the TLS hubs can have certain beneficial context-dependent responses, mostly their effects are detrimental causing an aggravation of the tissue pathology.Accordingly, Tregs are potent inhibitors for the development and function of TLS by suppressing the activities of T and B lymphocytes (Jones et al., 2016).Tissue fibroblasts have a crucial role not only in the recruitment of immune cells into the TLS hubs but they also organize the clusters of TLS within inflamed tissues.In brief, it has been reported that several inflammatory cytokines, e.g., IL-13, IL-17, and IL-22, induced the priming of fibroblasts into the immunofibroblasts (Nayar et al., 2019;Asam et al., 2021).IL-22 cytokine was the major driver of the expansion of immunofibroblasts in the TLS foci.Immunofibroblasts represented the podoplanin (PDPN) and FAP-positive fibroblast population in the salivary glands of the patients with primary Sjögren syndrome (Nayar et al., 2019).In a model of inflammation in mouse salivary glands, the priming and proliferation of immunofibroblasts were noted to be dependent on IL-4 and IL-22 signaling (Nayar et al., 2019).Interestingly, Nayar et al. (2019) demonstrated that depletion of the pdpn + /Fap + fibroblasts abrogated the formation of mouse TLS clusters and also prevented the development of local inflammation.These studies clearly indicated that tissue fibroblasts aggravated an existing chronic inflammatory state by establishing TLS foci.Currently, it is not known whether tissue fibroblasts can promote the formation of TLS clusters into aged tissues although there are reports indicating that in age-related chronic inflammation there is an increased infiltration of B and T lymphocytes into mouse liver and lacrimal glands and also evidence for the formation of ectopic immune cell clusters (Singh et al., 2008;Galletti et al., 2023).

Role of fibroblasts as immune regulators in the aging process
According to the great plasticity of fibroblasts, the populations of tissue fibroblasts are extremely heterogeneous and they possess diverse fibrogenic and immune properties.For practical purposes, it is more appropriate to use the categories based on the disease state, such as CAFs, FAFs, and WAFs, although sometimes it is better to define the location of fibroblasts, e.g., adventitial fibroblasts and synovial fibroblasts (LeBleu and Neilson, 2020).While the aging-associated fibroblasts (AAF) could be categorized as the basis of the age-related pathology, however, at the single-cell level the characterization has turned out to be difficult, e.g., due to significant differences between fibroblasts in a distinct tissue and moreover, due to alterations in the identity of fibroblasts with aging.

Aging-associated fibroblasts
Recently, single-cell transcriptome techniques have provided a novel possibility to reveal age-related changes in the properties of the fibroblast population in diverse tissues.For instance, these techniques have been applied in several extensive screening trials on the alterations in the expression profiles of fibroblasts in human and mouse skin (Salzer et al., 2018;Mahmoudi et al., 2019;Sole-Boldo et al., 2020;Zou et al., 2021), and mouse heart (Vidal et al., 2019) as well as in human and mouse arteries (van Kuijk et al., 2023).The major observations have been that the expression of fibrous components of the ECM significantly declined in fibroblasts with aging, whereas the expression of inflammatory mediators clearly increased in fibroblasts of aged skin.In the hearts of aged mice, fibroblasts also displayed a significant increase in the expression of genes related to immune and inflammatory functions, while there were some indications that cardiac fibroblasts exhibited several antiangiogenic traits and they adopted many osteogenic properties (Vidal et al., 2019).Moreover, Salzer et al. (2018) reported that there was clear evidence that the fibroblasts in the skin of old mice lost certain characteristics of their identity, e.g., fibrogenic properties, but simultaneously there was an increase in the expression of immune markers and fibroblasts gained some adipogenic properties.It is known that the presence of lipofibroblasts has been associated with the process of myofibroblast differentiation and later with dedifferentiation during the resolution of lung fibrosis in mice (El Agha et al., 2017).Interestingly, Salzer et al. (2018) demonstrated that a long-term caloric restriction, a well-known anti-aging treatment, partially prevented these age-related alterations in the phenotype of skin fibroblasts.It seems that the aging process in vivo is accompanied by alterations resembling the senescence of cultured fibroblasts.The induction of cellular senescence of human myofibroblasts in vitro revealed a robust decrease in the expression of α-SMA and collagen proteins, whereas the expression levels of pro-inflammatory markers, IL-6 and IL-8, were clearly upregulated (Lopez-Antona et al., 2022).Most probably the age-related decrease in the expression of fibrous proteins was linked with the significant decline in the expression and secretion of TGF-β, a potent enhancer of tissue fibrosis.Brun et al. (2016) revealed that when human dermal fibroblasts isolated from old donors were cultured, they exhibited an increase in the expression of several senescence markers, such as SA-β-gal and p16INK4A, while the expression of α-SMA and collagens were robustly down-regulated.They reported that the activation of the TGF-β/SMAD3 signaling pathway was impaired in fibroblasts of aged skin, probably attenuating the expression of fibrous components.Thus, fibroblasts in aged tissue seem to undergo a switch from fibrogenic properties towards cells with an inflammatory phenotype.
Results from the studies with single-cell transcriptomics have indicated that the fibroblasts in aged tissues express many of the common features observed in the senescence models of fibroblasts in cell culture conditions (Section 3.3.).However, it seems that the changes observed in aged tissues represent only a trend toward their senescence phenotype.Moreover, single-cell studies on aged tissues, especially in the skin, have revealed that the heterogeneity of fibroblasts has robustly increased with aging.Nonetheless, these properties of fibroblasts in aged tissue impact directly on the integrity of the ECM, thus augmenting the accumulation of fibrotic lesions into tissues with aging.These agerelated fibrotic lesions can be attributed to an increase in the SASP activity including higher levels of matrix proteinases, ROS, and many inflammatory mediators (Section 4.1.).Single-cell transcriptome studies have also revealed that the fibroblasts in aged tissues possess a robust expression and secretion of chemokines (Section 6.2.) and complement components (Section 6.3.).This indicates that chemokines and complements not only recruit but also activate immunosuppressive cells which subsequently suppress the expansion of inflammation but they also promote degenerative processes in aged tissues (Section 6.4.).Another process which enhances the deposition of fibrotic lesions is the significant loss of the ability to resolve an undesirable fibrosis, as observed in human fibroblasts with aging (Horowitz and Thannickal, 2019;Kato et al., 2020).For example, Kato et al. (2020) demonstrated that the human senescent fibroblasts in culture and the mouse fibroblasts isolated from the idiopathic pulmonary fibrosis displayed an impaired capacity of myofibroblasts to dedifferentiate as well as an increased resistance of fibroblasts to apoptosis.Apoptosis and dedifferentiation of myofibroblasts are common mechanisms to induce a resolution of fibrotic deposits (Horowitz and Thannickal, 2019).It is known that an increased resistance to apoptosis with aging enhances the accumulation of senescent cells which promote inflammatory responses and subsequently disturb the integrity of the ECM (Salminen et al., 2011b).

Role of chemokine secretion
In pathologies involving acute inflammation, e.g., myocardial infarction, pro-inflammatory fibroblasts play a crucial role in the initiation of the repair process by secreting chemokines which are known to recruit immunosuppressive cells into inflamed tissue (Section 5.2.).Interestingly, several studies on fibroblast senescence have revealed that the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) and many chemokines, e.g., CCL2,3,5,8 and CXCL1,2,5,8,10,12 are the most highly upregulated genes in senescent fibroblasts in different cell culture models (Begley et al., 2005;Eyman et al., 2009;Rodier et al., 2009;Freund et al., 2010;Acosta et al., 2013).Moreover, there is a robust increase in the expression and secretion of chemokines in the fibroblasts isolated from aged tissues, especially from mouse and human skin (Salzer et al., 2018;Mahmoudi et al., 2019;Sole-Boldo et al., 2020).For example, the expression levels of CCL2, CCL11, CXCL1, and CXCL12 were significantly increased in the population of fibroblasts obtained from old donors.GM-CSF is a potent mediator of tissue inflammation since it can stimulate the survival, growth, and differentiation of myeloid cells in chronic inflammation as well as control myelopoiesis and trained immunity (Ingelfinger et al., 2021).For instance, GM-CSF treatment of mice robustly increased the level of immunosuppressive Tregs in the blood and was able to prevent the development of an experimental myastenia gravis (Sheng et al., 2011).Chemokines, also those secreted by senescent fibroblasts, activate hematopoiesis, especially myelopoiesis, in the BM.For example, the development of MDSCs via the myelopoiesis is coordinated by GM-CSF and certain chemokines, such as CCL2, CCL5, CXCL5, and CXCL12 (Li et al., 2022), i.e., the chemokines produced abundantly by senescent fibroblasts.The chemokine system is also able to control the activation and polarization of macrophages in inflammatory conditions (Mantovani et al., 2004).It seems that the secretion of chemokines by senescent fibroblasts stimulate myelopoiesis and subsequently myeloid cells are recruited into the aging/inflamed tissues.In tumor sites, CAFs have a crucial role in the recruitment and activation of immunosuppressive cells, thus promoting local immunosuppression (Section 4.2.).For instance, Yang et al. (2016) demonstrated that in mouse tumors, the FAP-positive CAFs induced immunosuppression by secreting the CCL2 chemokine which recruited MDSCs into tumors.These examples indicate that the chemokines secreted by senescent fibroblasts have an important role in the recruitment of immune cells into aged tissues in an attempt to counteract excessive inflammation and enhance resolution.

Role of the complement system
In many tissues, fibroblasts are an important local source of complement components (Human Protein Atlas) (Section 4.4.).Although the complement system has a crucial role in the defence against foreign invaders, such as viruses and bacteria, or damaged cells in tissues, it seems that the activation of the complement system with aging promotes degenerative changes in tissues.There is compelling evidence that the level of complement components, such as C3, C5, and C1q peptides, significantly increases with aging both in the circulation (Naito et al., 2012;Gaya da Costa et al., 2018;Hasegawa et al., 2019;Cao et al., 2020) and within several tissues (Naito et al., 2012;Stephan et al., 2013;Wu et al., 2020).Knockout studies on the component C3 have convincingly revealed that its deficiency, which evokes a blockade of all complement pathways, exerts many protective effects against the aging process.For instance, Wu et al. (2020) reported that in mice a lack of component C3 inhibited many age-related changes in the kidney, e.g., it reduced chronic inflammation, delayed cellular senescence, and significantly decreased the level of fibrosis.The deficiency of component C3 in mice also prevented the age-related hippocampal degeneration and improved the hippocampus-dependent cognitive performance and synaptic functions (Perez-Alcazar et al., 2014;Shi et al., 2015).Naito et al. (2012) demonstrated that the age-related increase in the expression of the C1q protein in several mouse tissues augmented the activity of Wnt signaling which is commonly upregulated with aging.In the satellite cells (myoblasts) and fibroblasts isolated from mouse skeletal muscle, C1q exposure attenuated the proliferation of satellite cells, whereas C1q treatment stimulated fibroblast proliferation and enhanced the secretion of collagen proteins.
Several studies on tissue fibroblasts in vivo have revealed that the complement system is activated during the aging process.For instance, Salzer et al. (2018) demonstrated that the activation of the complement and coagulation cascade (Gene Ontology analysis) was one of the hallmarks of dermal fibroblasts in aged mice.Increased expression included C2, C3, C5, C3aR, C5aR, and CFB components.Moreover, Ezure et al. (2019) revealed that the expression and secretion of the complement factor D (CFD), the rate-limiting enzyme in the alternative pathway (Barratt and Weitz, 2021), were significantly elevated in human dermal fibroblasts of aged humans.In the fibroblast cocultures, the CFD derived from senescent fibroblasts increased the expression of matrix metalloproteinases in young fibroblasts.Ezure et al. (2019) also reported that the expression of the CFD enzyme was significantly increased in UV-radiation-induced senescent human fibroblasts.There is abundant evidence that the activation of complement signaling occurring after UV-radiation induced immunosuppression in the skin (Hammerberg et al., 1998;Stapelberg et al., 2009).It seems that complement factors are involved in UVB-induced immunosuppression and dermal photoaging (Salminen et al., 2022).These studies clearly indicate that the activation of the complement system in senescent/old fibroblasts can promote degenerative processes in aging tissues.

Collaboration of fibroblasts with the immunosuppressive network during the aging process
As described above, there is a close collaboration between tissue fibroblasts and the cells of immunosuppressive network in many pathological states involving both inflammatory conditions and fibrotic disorders (Section 5.3.).Given that fibroblasts in aged tissues possess pro-inflammatory properties with increased expression of many cytokines, chemokines, and complement components, it seems likely that fibroblasts from aged tissues recruit or activate immunosuppressive cells in aged tissues while attempting to counteract the inflammatory challenges encountered with aging (Section 4.1.and 5.2.).The role of the fibroblast-secreted chemokines and complement factors in the aging process has been addressed above (Section 6.2. and 6.3.).There is convincing evidence that the aging process is associated with an increase in immunosuppressive activity which promotes a decline in the efficiency of the immune system, i.e., a state called immunosenescence (Frasca and Blomberg, 2016;Ladomersky et al., 2020;Salminen, 2020Salminen, , 2021a;;Mogilenko et al., 2022).It has been demonstrated that the aging process increases (i) myelopoiesis in the BM of both mice and humans (Pang et al., 2011;Elias et al., 2017), (ii) the expansion of MDSC population in the circulation of mice and humans (Enioutina et al., 2011;Verschoor et al., 2013), and (iii) the number of MDSCs in the BM, spleen, lymph nodes, and skin (Enioutina et al., 2011;Ruhland et al., 2016;Flores et al., 2017;Salminen et al., 2018).Moreover, the level of Tregs increases with aging in the circulation as well as in the spleen, lymph nodes, and skin in mice and humans (Gregg et al., 2005;Sharma et al., 2006;Lages et al., 2008;Ruhland et al., 2016).In addition, higher numbers of immunosuppressive M2 macrophages were detected with aging in many mouse tissues, such as BM, spleen, and lungs (Jackaman et al., 2013).In aged tissues, it seems that there is a close cooperation between myofibroblasts and immunosuppressive cells (Section 5.3.),e. g., via TGF-β signaling, which is a potent enhancer of immunosuppressive activity (Flavell et al., 2010) as well as being a major inducer of myofibroblast differentiation (Lodyga and Hinz, 2020).As stated above (Section 4.2.), it is not only CAFs which excessively secrete TGF-β and many immunosuppressive cytokines, also myofibroblasts release many immunosuppressive factors, such as TGF-β, IL-6, IL-10, and PGE2 (Powell et al., 1999), an indication of immunosuppressive activities.Thus, it seems that myofibroblasts and immunosuppressive cells can reciprocally enhance each other's functions by secreting immunosuppressive factors (Mhaidly and Mechta-Grigoriou, 2020;Salminen, 2023b) (Section 5.3.).For instance, it is known that MDSCs and M2 macrophages can stimulate the activity of myofibroblasts and this can lead to an increased accumulation of fibrotic lesions in many fibrotic disorders (Section 5.1.).The aging process is associated with a deposition of fibrotic lesions and many other detrimental alterations in the ECM (Selman and Pardo, 2021), most probably attributable to disturbances in the functions of myofibroblasts (Salminen, 2023b).In addition, there is substantial evidence that the accumulation of immunosuppressive cells into tissues impairs the homeostasis of the host tissues through the secretion of TGF-β, IL-4, IL-10, ROS, and PGE2 as well as by depleting certain amino acids, e.g., L-arginine and threonine, from the tissue microenvironment (Salminen, 2021b).
The close collaboration between fibroblasts and the immunosuppressive network has been highlighted by the observations that AMPactivated protein kinase (AMPK) inhibits the differentiation of fibroblasts into myofibroblasts (Salminen, 2024) as well as suppressing the function of MDSCs thus impairing the activity of the immunosuppressive network (Qin et al., 2018;Salminen et al., 2019).There is clear evidence that AMPK signaling can delay the aging process and thus it has been able to extend the lifespan of Caenorhabditis elegans (Curtis et al., 2006) and improve the health span and lifespan of mice (Martin-Montalvo et al., 2013).Currently, there is a debate whether the activation of AMPK signaling with metformin, a drug used to treat type 2 diabetes, might increase the health span and even extend lifespan in humans.It is known that AMPK signaling inhibits not only the TGF-β/SMAD3-driven signaling but also activity along the NF-κB and JAK/STAT3 pathways (Salminen et al., 2011a;Lin et al., 2015;Ji et al., 2023).The inhibition of NF-κB signaling via the activation of AMPK has a crucial role in acute inflammatory conditions since increased AMPK signaling can prevent the generation of pro-inflammatory fibroblasts which aggravate inflammation by secreting chemokines and complement components (Section 6.2. and 6.3.).For example, Kriete et al. (2008) reported that the DNA-binding activity of the NF-κB complex was significantly upregulated in fibroblasts isolated from the skin of old human donors when compared to that from their young counterparts.This type of constitutive activation of NF-κB signaling was associated with an elevated expression of many cytokines, chemokines, and complement components in the fibroblasts isolated from aged tissues (Section 6.1).It is known that the presence of pro-inflammatory fibroblasts enhanced the polarization of M1 macrophages into immunosuppressive M2 fibroblasts and moreover, they recruited and activated MDSCs and Tregs in inflamed tissues (Section 5.2.).The chronic inflammatory microenvironment was also associated with the generation of the myofibroblast-driven tissue fibrosis and the potentiation of immunosuppression in tumors by CAFs.It is known that AMPK signaling attenuates the severity of many fibrotic diseases, e.g., myocardial and pulmonary fibrosis (Jiang et al., 2017).This is attributed not only to the AMPK-mediated inhibition of the myofibroblastic transition but also to a decline in the presence of M2 macrophages and MDSCs in fibrotic tissues.Moreover, metformin, an activator of AMPK signaling, was reported to inhibit the growth of tumors by inhibiting the functions of CAFs (Xu et al., 2018).These examples imply that tissue fibroblasts in collaboration with immunosuppressive cells promote the age-related accumulation of fibrotic lesions and tissue degeneration, processes which can be alleviated by the activation of AMPK signaling.

Future perspectives
Fibroblasts have been utilized for decades in cell culture studies investigating cellular and molecular phenomena.For instance, cultured fibroblasts have been the most widely used model for elucidating the mechanisms underpinning cellular senescence.Nonetheless, it has been claimed that the senescence of fibroblasts in cell culture conditions does not correspond to the age-related changes occurring in tissues.However, recent single-cell transcriptome studies on the fibroblasts isolated from aged tissues have revealed many changes reminiscent of those previously observed in senescent fibroblasts in vitro, e.g., a pro-inflammatory phenotype, although it is difficult to model in vitro the conditions related to aged tissues.It must be recalled that most of the senescence studies have been performed using fibroblastic cell lines even though primary fibroblasts isolated from tissues would be a more physiological choice.Moreover, many investigators have applied stress-induced senescence models which might reveal different results than replicative senescence experiments.Currently, single-cell transcriptome experiments have tended to focus on fibrogenic and inflammatory parameters of the fibroblasts in aged tissues.Future single-cell studies should be expanded from the skin to other tissues and in addition, the focus should be expanded to conducting a more detailed characterization of the immune properties of fibroblasts.Given that tissue fibroblasts have close interactions with many immune cells, cocultures between primary fibroblasts and immune cells might reveal many novel mechanisms through which fibroblasts and immune cells communicate with each other, especially the crosstalk between fibroblasts and the cells of the immunosuppressive network.This approach might improve the drug discovery process for different chronic fibrotic and inflammatory diseases.