Lymphoid tissues.

The specialization of fibroblasts is intrinsically linked to their positioning in space. Fibroblasts are sessile compared to leucocytes, and this places them in an ideal position to control the positioning and localization of cells within tissues. Secondary lymphoid tissues such as lymph nodes and spleen are prototypical examples of how fibroblasts control boundaries and help provide barriers within tissues. Within a naïve lymph node, B cells and T cells are highly segregated into well-defined areas, producing a baseline immunosuppressive environment by reducing communication between B cells and T cells that work together to trigger immunity. These spatial environments are produced by a series of distinct fibroblast populations that generate different chemokine gradients. For instance, FDCs localize B cells to the follicles through the expression of CXCL13, whereas T-zone reticular cells (TRCs) attract T cells and DCs to the T-zone by expressing CCL19 and CCL21. TRCs not only generate migratory cues, but they also produce growth factors to sustain immune cell niches and construct and maintain the ECM-rich conduit network so that small molecules can rapidly diffuse throughout the lymph node [25,26] (Fig 3).

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Fig 3. Fibroblasts in lymphoid and peripheral tissues.

Fibroblasts have different roles in the lymphoid and peripheral tissues. They mainly define and maintain cellular niches within the tissue microenvironment to ensure smooth functioning of the tissues, but when they go rogue, they can drive tissue damage and orchestrate pathological inflammation. Figure is created with BioRender.com.

https://doi.org/10.1371/journal.pbio.3001954.g003

In addition to lymph nodes, other specialized lymphoid tissues are found throughout the body, such as mucosal-associated lymphoid tissues in healthy gut and lung. In pathological conditions, tertiary lymphoid structures (TLSs) (Glossary) can form within peripheral tissues. Patterns of immune cell niches and chemokine networks observed in secondary lymphoid structures are broadly conserved in TLSs. During pathology, such as occurs in chronic inflammation, infection, and cancer, tissue-resident fibroblasts shift their phenotype to support leucocyte migration and TLS organization [27]. Given that TLSs are associated with worse outcomes in autoimmune diseases but with better survival in cancer, it is critical that the location and spectrum of fibroblast functions are tightly regulated. An open question that remains is: To what extent do the rules of intratissue patterning of immune cells by lymphoid tissue immune fibroblasts apply to peripheral tissues?

Peripheral tissues.

As in lymphoid tissues, it is now clear that fibroblasts are imprinted with positional identity within peripheral tissues. For instance, gut fibroblasts located at the top of crypts promote epithelial differentiation, whereas fibroblasts at the bottom of crypts secrete factors that regulate maintenance, self-renewal, and differentiation of the stem cell niche (Fig 3) [21,28]. Similarly, in the synovium, synovial lining and sublining fibroblasts are two distinct populations, expressing different markers and holding different roles. For example, in rheumatoid arthritis (RA) (Glossary), lining fibroblasts drive cartilage destruction and bone damage, whereas the sublining fibroblasts are responsible for driving inflammation. New research findings suggest that lining and sublining fibroblasts might not be stable long-term clusters. Instead, they exist along a transcriptional gradient corresponding to their proximity to the vasculature and their anatomical location within the synovium [29]. Classically, lymphocytes are only found in the synovial sublining region in RA, where perivascular cuffing of lymphocytes is a well-known phenomenon observed at many sites of chronic inflammation. What confines lymphocytes to the sublining around perivascular microdomains and excludes them from the synovial lining remains unknown. As humans have evolved the ability to orchestrate precise localization and compartmentalization of immune cells in lymphoid organs, it is tempting to speculate that the peripheral organs also harness the same mechanisms to triage immune cells into different compartments.

Chronic inflammatory reactions have a predilection for certain anatomical sites. For example, RA occurs mainly in synovial joints and not the gut, whereas inflammatory bowel diseases (IBDs) (Glossary) occur in the gut and not the skin. Although these diseases are classified under the same umbrella of immune-mediated inflammatory diseases, we are only now starting to appreciate that fibroblasts in gut and joints can be very different and that this fibroblast heterogeneity may contribute to the site specificity. A good example is vitiligo, which is an autoimmune disease characterized by bilateral symmetric skin depigmentation. It has been a puzzle why vitiligo commonly presents on the face, neck, hands, and in skin creases. The answer has now been revealed by a recent piece of research that showed that anatomically distinct dermal fibroblasts have different abilities to recruit and activate cytotoxic T cells depending on their response towards cytokines (in this case, interferon gamma (IFNγ)), which in turn determines the patterns of vitiligo lesions on the skin [30].

When fibroblasts behave.

In peripheral tissues, fibroblasts help maintain homeostasis, but they can also contribute towards pathology in disease states. Traditionally, fibroblast function has been thought to be limited to providing structural and biochemical support to surrounding cells by producing and remodeling ECM. Fibroblasts were thought to be invariant across tissues, despite the fact that, for some time, it has been clear that fibroblasts in different tissues produce distinct types of ECM with optimized mechanical properties best suited for the respective tissue function, for instance, respiration in the lung and gut motility [31,32].

Fibroblasts also have a central role in environmental sensing. Like macrophages, they act as sentinel cells and possess specialized sensory functions that detect changes in environmental factors such as mechanical forces, oxygen and nutrient levels [19]. In response to bacterial infection, intestinal fibroblasts produce chemokines that recruit pro-inflammatory monocytes and neutrophils, which in turn activates an adaptive immune response to eradicate the bacteria [33]. On the other hand, specialized kidney fibroblasts in the peritubular interstitium help maintain healthy systemic oxygen level by sensing hypoxia and producing erythropoietin as needed, which in turn stimulates red blood cell production in the bone marrow [34]. Fibroblasts also have a role in maintaining the correct cell numbers and ratio in each tissue compartment. As tissue density changes, fibroblasts sense the difference in mechanical force through actin-dependent mechanisms and then direct macrophage proliferation by regulating growth factor expression. In real time, cell loss due to injury or tissue damage results in increased space availability, and this is detected by the fibroblasts, which then increase macrophage numbers by increasing growth factor production [19,20]. While macrophages clear up the cell debris [35], fibroblasts work together with neutrophils to repair and regenerate the ECM, facilitating wound closure [36]. Given fibroblasts have important roles in the healthy behavior of tissues, it follows that their dysfunction should contribute to disease.

When fibroblasts go rogue.

In disease, fibroblasts often acquire pathological features that facilitate disease progression, persistence, and recurrence at specific anatomical sites. One commonly pathological form of fibroblasts comes in the form of myofibroblasts (Glossary), a differentiated, contractile form of fibroblast that is found in tissue repair and fibrosis [37]. During wound healing, the myofibroblast phenotype is transient [38], but it becomes persistent in many fibrotic diseases, including idiopathic pulmonary fibrosis and Dupuytren’s disease [39,40]. The contractile and ECM-producing phenotype of myofibroblasts, as well as their pathogenic persistence, results in increased tissue stiffness in fibrotic tissues.

In addition to fibrosis, fibroblasts also contribute to chronic inflammation. Studies across multiple inflammatory diseases show that disease-related fibroblasts share common properties and communicate with both immune and vascular compartments, which then contribute to persistent inflammation [41]. In RA, synovial fibroblasts undergo metabolic reprogramming and mediate tissue priming by enhancing inflammasome activity, which means that sensitized synovial tissues are more prone to reinflammation, a concept that has been termed “stromal memory” [42]. More recently, in the case of Coronavirus Disease 2019 (COVID-19), a single-cell RNA lung atlas revealed higher fractions of pathological fibroblasts in the lungs of patients with COVID-19 as compared with healthy controls, with COVID-19 lung fibroblasts expressing collagen-producing and fibrosis-related signatures [43]. This insight would not have been possible without the latest advances in single-cell sequencing technology.

In the cancer field, researchers are starting to appreciate the importance of cancer-associated fibroblasts (CAFs) (Glossary), which are generally described as the fibroblastic population that accompanies solid tumors. They are complex, heterogeneous, and surprisingly plastic in their functional states. There are many potential sources of CAFs, including tissue-resident fibroblasts [44], bone marrow–derived fibroblasts [45], adipocytes [46], endothelial cells [47], and pericytes [48]. Not only do they arise from different sources, they also exist in different functional states and are described as inflammatory CAFs (iCAFs) [49], myfibroblastic CAFs (myCAFs) [49], antigen-presenting CAFs (apCAFs) [50], and vascular CAFs (vCAFs) [48]. MyCAFs tend to be found adjacent to the tumor and show a strong bias towards an ECM remodeling phenotype, whereas iCAFs tends to be more distal from tumor edge and express pro-inflammatory cytokines and chemokines that can activate and traffic immune cells [49]. On the other hand, apCAFs have the capacity to present antigens to T cells and promote immunity [50,51], and vCAFs are involved in vascular development and regulating angiogenesis [48]. Being the main cellular component of the tumor microenvironment for most solid tumors, CAFs have an important role in tumor initiation, progression, metastasis, local tissue metabolic reprogramming, and therapeutic resistance [11,12]. One of the open questions remains: Can we skew CAFs from an immunosuppressive to an inflammatory and immunomodulatory phenotype, which in turn could improve cancer immunotherapies?

Fibroblasts can swing between functional states.

Cells can have both positional identity within tissues and a functional state (for instance, inflamed, replicating, or quiescent). As fibroblasts were thought to be mainly structural and inert, their functional states have until now not been well studied.

Fibroblasts in steady-state lymph nodes are fundamentally tolerance-promoting cells through a variety of mechanisms. In addition to segregating lymphocytes into distinct T cell and B cell niches to prevent unintended cell–cell interactions, fibroblasts also promote tolerance by presenting peripheral self-antigens. Lymph node fibroblasts also aid in the induction of regulatory T cells, both directly through TGF-β and indirectly by skewing DCs towards a tolerogenic phenotype [52,53]. This tolerogenic phenotype of lymph nodes varies across tissues, with mesenteric lymph nodes having a higher propensity towards tolerance promotion than skin-draining lymph nodes, most likely due to constant exposure to a high volume of gut microbiota.

Critically, lymph node fibroblasts have the capacity to transform the lymph node from a quiescent environment into one that supports highly efficient conditions for generating adaptive immune responses. Upon infection or immunization, lymph nodes can expand rapidly to up to 10 times their initial volume while maintaining their structural integrity [54]. The FRCs stretch and relax, which loosens the collagen-based reticular network, and this enhances the motility and interactions between immune cells [55]. Interactions between B cells and T cells at the border between the follicles and the T-cell zone are critical for the development of adaptive immunity. Cells are guided to this zone by chemokine gradients produced by fibroblasts that are laid down in the steady state [56]. Infection skews lymph node fibroblasts from tolerogenic towards an inflammatory phenotype with increases in antigen presentation, and chemokine and cytokine production [13].

Cytokine networks that drive pathogenesis in diseases such as RA and IBD are now well established [57,58]. However, the cellular origins of these cytokine culprits remain unclear. We need a better understanding of the cells producing the pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin 6 (IL-6), so that we can target inflammatory disease at their cellular root. There is a consensus in the stromal field that fibroblasts in their activated states express fibroblast activation protein (FAP) and PDPN [59,60]. What regulates the switch between these distinct functional states is less clear, but pathogenic amplificatory loops involving fibroblast-driven regulatory networks, such as IL-6–JAK–STAT3, have been strongly implicated [17,41,57]. Could it be due to cytokines and growth factors produced by other tissue-resident cells such as macrophages, endothelial and epithelial cells in the microenvironment? As cellular metabolism is the cornerstone of all biological activities, could fibroblasts change their phenotype or functional state by undergoing metabolic reprogramming? With the latest tools and algorithms, such as COMPASS, we can not only sequence genes at a single-cell level, but are also now able to characterize their metabolism at the resolution of single cells [61], which should help in answering some of the questions posed above.