Fibroblasts exist within most loose and dense fibrous connective tissue. After endothelial and blood cells, fibroblasts are the most widespread cell types in the human body. They reside in the visceral organ capsules, skin, gastrointestinal and urogenital tracts, airway linings, central nervous system meninges, muscle and adipose tissues, as well as the musculoskeletal system. Fibroblasts are irregular-shaped cells (elongated, polygonal, or stellate), and their cytoplasm tapers off into several processes. The nucleus is usually centrally located, occupying almost 50% of the cell volume in resting conditions. In its proliferating stage, the nucleus occupies a larger portion of the cell, and there can be more than one nucleus (two to three). Mesenchymal cells found in the developing embryo and fetus are the progenitor cells of different types of fibroblasts, which are usually surrounded by collagen fibers, being produced by them (1). Along with collagen (of which collagen I is most abundant), fibroblasts produce a range of proteins that become the base of the extracellular matrix (ECM). Vimentin intermediate filaments, fibronectin, and interstitial collagens are predominant in the ECM. During tissue injury, fibroblasts transdifferentiate into myofibroblast phenotype with contractile properties to resolve the wound; in case the wound is not resolved, it leads to fibrosis (2).

At different stages of life, starting from the fetal to adult stage, fibroblasts undergo programmed phenotypic transitions to perform a diverse range of functions—from embryonic development to the aging process. This phenotypic differentiation is tightly regulated in a tissue- and developmental stage-specific manner. The relative distribution of fibroblasts at diverse locations throughout the human body suggests its role in coordinating with the adjacent resident cells. The heterogeneity in the phenotypic variations is reflected in the cellular morphology in both external and internal milieu, expression of surface receptors, and biosynthesis and secretion of growth factors and chemoattractant molecules (3–6). Fibroblast differentiation is governed by the degree of clonal expansion, clonal deletion/diminution, or inhibition of a particular subtype. This event is regulated by the release of humoral factors by the fibroblast itself as well as neighboring epithelial and infiltrating immune cells that instruct the migration and accumulation of the different subtypes that maintain the dynamic equilibrium of the heterogeneity and diversity of the fibroblasts (7). The markers that distinguish fibroblasts from other cell types are vimentin (VIM), fibroblast-specific protein 1 (FSP1), platelet-derived growth factor receptor-alpha (PDGFRα), fibroblast activation protein-alpha (FAP), CD90 (Thy 1), podoplanin (gp38), alpha smooth muscle actin (α-SMA), and CD34 (8). There is no single specific marker to discriminate fibroblasts from other cell types in different organs due to the profound heterogeneity of fibroblasts. Muhl et al have recently interrogated, via single-cell analysis, signatures that identify and distinguish between fibroblast, vascular smooth muscle cells and pericytes of heart, colon, bladder and skeletal muscle (9). Ninety gene signatures were identified with inter- and intra-organ fibroblast heterogeneity along with cross organ similarity, mostly related to genes of matrisome and ECM. Under various pathophysiological conditions, fibroblasts undergo differentiation into effector phenotypes, some specialized for ECM production and remodeling of tissues while others for carrying out immunological functions; these include the control of ECM synthesis via secretion of varied chemokines, cytokines, and growth factors.

Secondary lymphoid organs (SLOs) play an indispensable role in compartmentalizing immune cells for an effective environment for the maintenance of host immunity and simultaneously minimizing pathological damage. Here, resident fibroblasts coordinate the immune functioning with its vast reticular conduit network. This review highlights how fibroblasts function as sentinel cells, a source of scaffold for other immune cells during normal and inflammatory conditions, being a key regulator of germinal center (GC) formation, and in the maintenance of an overall core structural organization of the SLOs, such as lymph nodes, spleen, and Peyer’s patches.

SLOs are intricately placed throughout the human body to perform various functions, such as receiving immunological signals from different locations, antigen sampling, and disseminating them via antigen-presenting cells (APCs). The signals received during the interaction between pattern recognition receptors (PRRs) with pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) present in pathogens, tumors, commensals, and inflamed tissues are sampled and presented to immune cells that are distributed in special niches inside the SLOs. The immune cells present in the different compartments of SLOs undergo effector functions in coordination with the lymphoid organ fibroblasts, which form the scaffold to provide the appropriate microenvironment and determine the strength of protective innate and adaptive immunity. SLOs maintain a similar organized architecture, segregated B- and T-cell compartments where lymphocyte priming and differentiation take place, and the antigen sampling zone, which harbors specialized myeloid cells. These compartments offer the microenvironments for immune cell-to-cell interaction (10).

GCs are specialized microstructures that are formed in SLOs during infection, or when challenged with T cell-dependent antigens (48). GC provides a dynamic microenvironment favoring the proliferation and differentiation of antigen-activated B cells, leading to the generation of high-affinity plasma cells and memory B cells. Mitotically active B cells undergo affinity maturation at the GCs, which are known as centroblasts/plasmablasts. The immunoglobulin modification at the variable genes of B cells takes place through somatic hypermutation to increase its affinity toward the antigen (49, 50). The emergence of plasmablasts at the interface of the germinal follicle is governed by IL-21 produced by T follicular helper (T_FH) cells and TNFSF13 produced by gp38⁺CD157^high FRC. Here, the plasmablasts express TNF receptor superfamily member 13B (TNFRSF13B), which can bind TNFSF13, enhancing its proliferation (51).

GCs produce memory B cells in preparation for reinfection. Once the cognate antigen is recognized by the B-cell receptor, the antigenic peptides are presented to CD4⁺ T helper cells, and this takes place at the border of the T-cell and B-cell follicle zones. B cells migrate to the center of the follicle and undergo clonal expansion (10, 48). This phenomenon demarcates the GC into two distinct compartments: dark zone (DZ) and light zone (LZ). A dense network of reticular cells expressing CXCL12 (CRCs) can be identified in the DZ (49), which promotes the homing of B cells during GC response and is rich in proliferating cells. Once the somatic hypermutation is complete in the DZ, CXCR4 (a ligand of CXCL12) is downregulated. B cells that express CXCR5 and migrate to LZ are known as centrocytes. The LZ is dominated by FDCs, which are specialized in internalizing and presenting the antigens and undergoing marked remodeling during the development of GCs. The FDCs secrete CXCL13 to attract and present antigens to B cells (51–53). B cells present the antigens retrieved from FDCs to the T cells, CD40/CD40L, and B- and T-cell interactions lead to the positive selection of LZ B cells (52, 53). Studies have suggested that these LZ B cells are Myc⁺ plasma cell precursors (54–58). CXCL13 helps the accumulation of B cells inside follicles and aids in the formation of GCs. The location of CXCL13 is more central than peripheral in the GC (59). In a study where diphtheria toxin (DTx) was injected in mice to induce short-term FDC, the follicular integrity was disorganized in both lymph node and spleen. T lymphocytes and DCs were found localized toward the boundary of the B-cell zone along with the CCL21, which functionally should be restricted in the T-zone area. Thus, in the absence of FDCs, the B-cell zone overlaps with the intermixing region of the CCL21-expressing T lymphocytes and DCs, causing severe loss of GC (60) ( Figure 3 ). Apoptosis of B cells in the GCs happens to eliminate self-reactive B cells, which express high levels of ELL (eleven-nineteen lysine-rich leukemia)-associated factor 2 (Eaf2). EAF2 is the apoptosis inducer specific for GC B cells (61).

FDCs were earlier considered phagocytic cells, but later, it appeared that the antigens were not internalized but rather trapped in the filiform dendritic processes of the cell, forming a web-like pattern (62–64). Hence, FDCs are non-phagocytic reticular cells that do not express MHC class-II (HLA-DR) molecules, and they have the unique capacity to retain the native antigens in an intact form in the B-cell follicles for a long period (50, 65–67). FDCs endocytose immune complexes containing the antigens acquired from non-cognate B cells and recycle them to their surface in their native form for long-term display (68). This provides an opportunity for the B cells to encounter the antigen for an extended period, FcγRIIB, which binds with IgG expressed on FDC is crucial for trapping the immune complexes (50) ( Figure 3 ). FDCs also use complement receptors for trapping antigens; this is explained. Electron microscopy revealed that in some FDCs, the dendritic processes had a chain of interconnected immune complex bodies, termed iccosomes (0.3- to 0.7-μm diameter). Iccosomes derived from FDCs act as a novel mechanism for the delivery of antigens to B cells (69). Iccosomes form a lamellar labyrinth-like structure varying between filiform and glomerulated. In the GCs, these iccosomal antigens are endocytosed by B cells (B220⁺), processed, and presented to T cells, leading to their activation and proliferation (70). Cognate B cells can also acquire FDC-bound immune complexes by capturing CD157 present in FDC or through surface Igs (71).

The complement system is a crucial part of the innate immune system as a first line of defense against infections. The activation of the complement system occurs through three different pathways: classical, lectin, and alternative pathways. These pathways converge at the formation of C3 convertase that culminates in inflammation, opsonization, or cell lysis, through the release of C3a and C5a, the generation of C3b, and the formation of membrane attack complex (MAC), respectively. Excessive activation and tissue damage may be prevented by strict regulation of complement activation by complement receptors that facilitate immune cell interactions and pathogen clearance. Fibroblasts have a significant role in complement regulation and expression of complement receptors (CRs). CRs like CR1 (CD35), CR2 (CD21), CR3, and CR4 (CD11b/CD18) mediate complement-driven cellular responses and contribute to the diverse functions of fibroblasts in immune regulation and tissue homeostasis (72, 73). Excessive complement activation on fibroblasts is implicated in fibrotic diseases (74).

Complement proteins play an indispensable role in the antigen presentation by FDCs. In the GCs of the spleen, aggregates of human IgG are found to be C3-dependent (50, 75). Adult thymectomized mice, when treated with cobra snake venom to deplete C3 level in serum following immunization with dinitrophenylated keyhole limpet hemocyanin (DNP-KLH), exhibited C3 depletion, which led to the impairment of B-cell memory, affecting the precursor proliferation in the splenic lymphoid follicles. The localization of the antigen (DNP-KLH) was also found to be C3 and antibody-dependent (76). Thus, antigen–antibody–C3 complexes formed on FDCs are necessary for the development of B-cell memory; however, the depletion of C3 had minimal effect on the functioning of the already primed B cells. Complement receptors CR1 (CD35), which can bind C3d, CR2 (CD21) with C4b, and Fc receptor FcγR, which binds IgG, are expressed on FDCs and are crucial for trapping the immune complexes (ICs) (50, 77–81) ( Figure 3 ). In addition, the expression of CR1 and CR2 on FDC is one of the prerequisites for a strong IgG response (82). FDCs express FcγRIIB at high levels in the GCs but not in the primary follicles. In the lymph node and spleen of FcγRII^−/− mice, FDCs exhibit reduced antigen trapping. In a cell culture model when immune complexes were added to antigen-specific T and B cells, antibody response was elevated only when co-cultured with FDCs. Similarly, lymph node lymphocytes from ovalbumin (OVA)-immunized mice were injected into FcγRIIB^−/− and irradiated wild-type mice and then after 24 hours injected with anti-OVA immune complexes (preformed ICs). The serum collected after 2 weeks showed robust anti-OVA IgG in the wild type, confirming that the binding of ICs to FcγRIIB makes them highly immunogenic to stimulate B cells. Therefore, the expression of FcγRIIB on FDCs in the GCs acts as a positive regulator of B-cell activation (83).

Efficient capture of ICs by FDCs likely limits the dissemination of pathogens and provides an opportunity for the non-cognate B cells to leave the lymph node without the IC cargo (50, 84). The MZ of the white pulp provides the first line of defense in the spleen. The IgM-bound ICs first localize themselves in the MZ and then associate with MZ B cells with the help of CR1/CR2 receptors. These marginal B cells at the marginal zone between white and red pulp present these IgM-ICs to FDCs in the follicle (85). FDCs release chemoattractant CXCL13 to recruit B lymphocytes and engage them with its CXCR5 receptors; this shuttling is regulated by the balance between CXCR5 and sphingosine-1-phosphate (S1P) receptor, S1P₁ (86). The localization of IgM-IC is affected in Cr2 ^−/− and C3-depleted mice, suggesting that IgM-IC binding to MZ B cells and antigen deposition on FDCs are complement-dependent (87). To understand the molecular signals specific to FDCs, transgenic mice expressing Cre recombinase controlled by CD21 locus were used. This CD21-cre transgenic mice, where the cre gene was inserted at the CD21/35 locus on a bacterial artificial chromosome, showed Cre expression independent of an inducer in mature B cells (88). Thus, the development of the FDC network and B-cell follicle formation are dependent on the expression of p55TNFR (TNF receptor p55) on FDCs that restore VCAM-1, ICAM-1, and CXCL-13 expression in FDCs. However, the effective antibody-mediated immune response is critically dependent on the FDC-specific IKK2 (IκB kinase 2) expression (89). DCs present within the B-cell follicle are capable of sampling FDC-retained antigens before they migrate to the T-cell zone (90, 91). Dysfunction in the FDCs to retain ICs may also limit the peripheral deletion of autoreactive T cells. An increase in the number of autoreactive T cells and their subsequent activation can lead to the loss of B-cell tolerance (92).

Infection or antigenic challenge leads to a huge influx of immune cells into the lymph node parenchyma. The lymphatic vessel increases in size to accommodate more lymphatic fluid, which further activates FRCs to overexpress CCL21 to attract migratory DCs (CCR7⁺) and naïve T lymphocytes, leading to an inflammatory response. To make room for the infiltrating cells, the lymph node undergoes remodeling, i.e., loosening of its reticular network. DCs loaded with antigenic peptides get activated and increase the expression of CLEC-2 (C-type lectin-like receptor-2), which interacts with podoplanin on FRCs (93). This induces the FRCs to lose their contractibility and become elongated, causing the reticular network to relax and the conduit network to expand, enhancing the DC and T-cell mobility and proliferation. IL-1β expression by monocytes and DCs can stimulate the early proliferation of FRCs during antigenic challenge. The inhibition of CCR2 and CCR7 can attenuate the expansion of LNSCs (8). When C57BL/6 mice were challenged with Escherichia coli LPS (lipopolysaccharides), and OVA intravenously after 18 hours of T-cell transfer, the expression of several genes was upregulated in FRCs within 12 hours. Among them were the pro-inflammatory chemokines, CCL5 and CXCL9, α-2-macroglobulin, serum amyloid A3 and serine (or cysteine) peptidase inhibitor, clade A, and member 1B (Serpina1d). The upregulation of several interferons (IFNs) and TLR-4 inducible/regulatory genes (e.g., LY6 and LCN2) was also observed. In FRCs as well as BECs, genes related to MHC class II antigen presentation were upregulated (11).

Type I IFNs are secreted by a range of cell types to mediate antiviral response. IFN signaling is mediated through the JAK/STAT signaling pathway, which finally upregulates the expression of IFN-stimulated genes (ISGs) that are crucial for effective antiviral immune response. Approximately 104 ISGs were analyzed in 11 FC clusters in the spleen. Among all the FCs in the spleen, four Ly6c1 ⁺ FC clusters (Ly6c1⁺/Hmox1⁺C1q⁺ , Ly6c1⁺/Hmox1⁺C1q⁻ , Ly6c1⁺/Hmox1⁻Ccl5lo, and Ly6c1⁺/Hmox1⁻Ccl5 ^hi) showed maximum elevated ISGs (29). Protein kinase R or eukaryotic translation initiation factor 2 alpha kinase 2, Eif2ak2 (which binds to viral nucleic acid and inhibits protein synthesis at the initiation stage of translation); mixed lineage kinase domain-like pseudokinase, Mlkl (a terminal mediator of necroptosis that activates inflammasome synthesis); interferon-induced protein 44, Ifi44 (upregulated in a wide range of viral infections to restrict viral proliferation); and members of the Oas, Gbp, Ifit, and Ifitm families [which are antiretroviral host restriction factors (RFs)] as well as various mediators of viral RNA and DNA sensors such as Dhx58, Ddx60 and Ifih1, Irf7, and Zbp1 were overexpressed when Ly6c1 ⁺ FC was infected with murine cytomegalovirus (29, 94–97). Pezoldt et al. also validated that the upregulation of antiviral signatures in Ly6c1 ⁺ FC present in red pulp stroma was Stat1-dependent, reaffirming the crucial antiviral role played by splenic red pulp stroma (29).

During chronic tissue inflammation in viral or bacterial infections, autoimmunity (rheumatoid arthritis, Sjogren’s syndrome, multiple sclerosis, Hashimoto’s thyroiditis, and autoimmune encephalitis), cancer, age-related disorders chronic allograft rejection, and tertiary lymphoid structures (TLS), ectopic lymphoid tissues are formed in non-lymphoid organs as an immunoprotective response (98). These lymphoid aggregate structures comprising B and T lymphocytes and DCs are supported by a complex network of resident fibroblasts aiding in lymphocyte infiltration through high endothelial venules (99). Various mouse and human experiments so far have identified two subsets of stromal fibroblasts, CXCL13⁺ FDC and CCL21⁺ TRC (100). In the lungs, both bacteria (Staphylococcus aureus or Mycobacterium tuberculosis) and viruses such as IAV can induce TLS development (98, 101). TLS can also be formed in the liver due to the hepatitis C virus and in the stomach due to Helicobacter pylori chronic infection (102). These act as sites for antigen presentation and pathogen clearance. IL-22 is considered a regulatory cytokine for TLS organization in the salivary glands of mice that stimulates the production of CXCL12 and CXCL13 in a virus-induced autoantibody production model (103). Lung fibroblasts during IAV infection express CXCL13 and can drive CXCR5-dependent B-cell recruitment to provide a cross-reactive antibody response (101). In the case of kidney injury during aging, fibroblasts in TLSs get activated and differentiate into a distinct phenotype expressing p75NTR, a neural crest marker, and mature into CXCL13-producing FDCs (104). In human tumors, cancer-associated fibroblasts (CAFs) can organize TLS in a CXCL13-dependent manner, activating lymphotoxin-α₁β₂-expressing B lymphocytes as well as tumor-infiltrating lymphocytes (105).

Fibroblast-driven innate immune responses can be immunostimulatory or immunosuppressive, depending on the nature and the location of the insult in different tissues, resulting in the upregulation of trans-differentiated effector fibroblast phenotypes that modulate the expression and functioning of various innate immune components (PRRs, cytokines, chemokines, and complements) to either resolve or facilitate disease persistence. There is recent surge in studies examining fibroblasts as a therapeutic target in infectious and non-infectious diseases. However, it is vital to fully understand the interacting markers and mediators between fibroblast and immune cells in the heterogeneous milieu of various diseases. The fibroblast heterogeneity needs to be corroborated in different physiological and pathological conditions. Different fibroblast populations program and regulate themselves in order to recreate a lymphocyte-permissive tissue condition and facilitate the spatial orientation and distribution of the cellular infiltrates.