Stromal cells in lymphoid tissues regulate lymphocyte recruitment and survival through the expression of specific chemokines and cytokines. During inflammation, the same signals recruit lymphocytes to the site of injury; however, the “lymphoid” stromal (LS) cells producing these signals remain poorly characterized. We find that mouse inflammatory lesions and tumors develop gp38+ LS cells, in recapitulation of the development of LS cells early during the ontogeny of lymphoid organs and the intestine, and express a set of genes that promotes the development of lymphocyte-permissive tissues. These gp38+ LS cells are induced by a robust pathway that requires myeloid cells but not known Toll- or NOD-like receptors, the inflammasome, or adaptive immunity. Parabiosis and inducible genetic cell fate mapping experiments indicate that local precursors, presumably resident fibroblasts rather that circulating precursors, massively proliferate and give rise to LS cells during inflammation. Our results show that LS cells are both programmed during ontogeny and reinduced during inflammation.
Stromal cells in lymphoid tissues, also termed lymphoid stromal (LS) cells3 (1), express chemokines and cytokines that regulate lymphocyte migration and promote their survival and differentiation. In B cell follicles of the spleen, lymph nodes (LNs), and Peyer’s patches (PPs), follicular dendritic cells (FDCs) express the B lymphocyte chemoattractant chemokine BLC (CXCL13) (2). In T cell zones, fibroblastic reticular cells (FRCs) express the chemokines CCL19 and CCL21, which recruit T cells and dendritic cells (DCs) (3, 4). FRCs also produce IL-7, which is essential for the survival of naive T cells and plays an important role in the homeostasis of naive T cells (5). During ontogeny, stromal cells in LN and PP anlagen have been characterized by their coexpression of the adhesion molecules ICAM-1 and VCAM-1. These fetal LS cells produce CXCL13, required for the development of lymphoid tissues (6), as well as CCL19 (7).
Similar signals recruit lymphocytes to inflammatory lesions. Numerous reports, both in mouse and man, document that “inflamed” stromal cells produce CXCL13, CCL19, and CCL21 (8, 9, 10, 11, 12) and express ICAM-1 and VCAM-1 (13). Additionally, fibroblasts from the synovia of rheumatoid arthritis patients express markers and display functional properties reminiscent of FDCs, suggesting that local fibroblasts differentiate into FDC-like cells during inflammation (14). Interestingly, FDCs and FRCs express desmin and α-smooth muscle actin (αSMA) (2, 5, 15), a characteristic trait of myofibroblasts that develop during wound healing and fibrosis (16, 17). Furthermore, inflammatory mediators such as membrane lymphotoxin (LT)α1β2 and TNF-α are required both for the differentiation and activation of FDCs and FRCs (2, 3, 18, 19), as well as for the development of lymphoid tissues (20). Thus, in accordance with the hypothesis that inflammation and developing lymphoid tissues engage similar pathways (21, 22), chronic inflammatory lesions develop ectopic (tertiary) lymphoid tissues (tLTs) (9, 23).
We demonstrate herein that inflammation induced by autoimmunity, adjuvants, infection, and tumors recapitulates the ontogeny of LS cells. These cells, expressing the mucin-type transmembrane glycoprotein gp38 (5, 24), develop early during the ontogeny of lymph nodes, thymus, and intestine and are reinduced during inflammation. Reinduction is dependent on local tissue-resident cells and does not involve the recruitment of circulating progenitors or epithelial-to-mesenchymal transition (EMT). A robust pathway induces gp38+ LS cells that is independent of individual innate receptors such as Toll- or NOD-like receptors and the inflammasome, but requires myeloid cells. Both fetal and adult gp38+ LS cells express genes essential in the recruitment and survival of leukocytes, in lymphoid tissue genesis and lymphangiogenesis, as well as in the growth and differentiation of fibroblasts and epithelial cells. Taken together, our data suggest that gp38+ LS cells are programmed during ontogeny, as well as reinduced during inflammation, to create a platform that supports lymphocyte recruitment through the production of specific cytokines, chemokines, and growth factors.
Materials and Methods
Bacterial artificial chromosome (BAC)-transgenic Rorc(γt)-GfpTG mice were described previously (25). The coding sequence for enhanced GFP (EGFP), including the stop codon, was inserted into exon 1 of Rorc(γt) in place of the endogenous ATG translation start codon, on a 200-kb BAC (Invitrogen) carrying at least 70 kb of sequence upstream of the Rorc(γt) translation start site. MyD88-, TRIF-, TLR2-, or TLR4-deficient mice were obtained from Dr. S. Akira, TLR3-deficient mice were from Dr. R. A. Flavell, NOD1 (card4−/−)-deficient mice were generated by Millennium Pharmaceuticals, NOD2- (card15−/−) deficient mice were provided by Dr. J.-P. Hugot, NALP3-deficient mice were from J. Tschopp, Asc-deficient mice were from Dr. V. Dixit, and LTβ-deficient mice were from K. Pfeffer. FoxP3sf, Apcmin/+, UbiquitinGFP, K14-CreERT2, and Rosa26Fl-STOP-Fl-Yfp reporter mice, as well as IFN-γ-, IL-12Rβ1-, or IL-6-deficient mice, were obtained from The Jackson Laboratory, and Nude mice were from Charles River Laboratories. Caspase-1- (26), IFN-αR- (27), IgM- (28), or RAG-2- (29) deficient mice have been described previously. Rip-TagTG mice were obtained from the NCI (National Cancer Institute) Mouse Models of Human Cancers Consortium. Parabiosis between C57BL/6 and UbiquitinGFP mice was performed as previously described (30). All mice were kept in specific pathogen-free conditions, and all animal experiments were approved by the committee on animal experimentation of the Institut Pasteur and by the French Ministry of Agriculture.
CFA, IFA, zymosan, BrdU, and 4-OH tamoxifen were purchased from Sigma-Aldrich. Mice were injected i.p. with 300 μl of BrdU dissolved in PBS at 10 mg/ml. The ears of K14-CreERT2 × Rosa26Fl-STOP-Fl-Yfp mice were covered with 1 mg of 4-OH tamoxifen dissolved in ethanol once a day the 5 days before CFA injection.
Ear inflammation and Leishmania infections
To induce ear inflammation, ears were injected intradermally with 25 μl of CFA, IFA, or zymosan, cut at different time points after injection, and processed for FACS or histology. Ear inflammation was also induced by the rubbing of the two skin surfaces by opposite motion for a few seconds. For Leishmania infection, parasite virulence was assessed following inoculation of Leishmania major Friedlin strain V1 (MHOM/IL/80/Friedlin) in the footpads of susceptible BALB/c mice. Metacyclic parasites (105) were injected s.c. into footpads and mice were sacrificed 4–6 wk later. Infections were monitored by comparing the thickness of the injected and uninjected footpads with a Vernier caliper. Before quantitative tests, parasites were passed twice through the mouse at high inoculating doses (5 × 107) and were thereafter maintained for less than three passages in vitro. Lesion parasites were enumerated by limiting dilution assay.
Abs and fusion protein
The following mAbs were purchased from BD Biosciences: PE-conjugated anti-CD157 (BP-3) and anti-CD4 (RM4-5), biotin-conjugated anti-VCAM-1 (429), and purified anti-CD11c (HL3). Purchased from eBioscience were: biotin-conjugated anti-CD45R/B220 (RA3-6B2), anti-CD45.2 (104), anti-CD31 (390), and anti-Gr-1 (RB6–8C5), and purified anti-Gr-1 (RB6-8C5), anti-IL-1α (ALF-161), anti-IL-1β (B122), anti-CD11b (M1/70), and rat IgG2b (B149/10H5). Purchased from Sigma- Aldrich were: Cy3-conjugated anti-αSMA (1A4) and streptavidin. Purchased from R&D Systems were: biotin-conjugated polyclonal anti-Lyve-1 (BAF2125) and anti-CXCL13 (BAF470). Purchased form Invitrogen were: purified anti-GFP (A-11122) and FITC-conjugated anti-rabbit polyclonal, biotin-conjugated anti-BrdU (PRB-1), Alexa 555-conjugated anti-rat, anti-goat, and anti-guinea pig, and Alexa 647- or Alexa 488-conjugated streptavidin. FITC- or Cy3-anti-armenian or syrian hamster were purchased from Jackson ImmunoResearch Laboratories, biotin-conjugated anti-neutrophils (MCA771B) were from Serotec, purified polyclonal anti-insulin (A0564) was from Dako, and anti-gp38 culture supernatant was a gift from A. Farr (Seattle, WA). Lymphotoxin-β receptor (LTβR)-Ig fusion protein was described previously (31) and has been provided by J. Browning (Biogen Idec).
Fragments of small intestine or colon were first incubated in PBS (Ca/Mg free) containing 15 ml of 1 mM DTT and 3 mM EDTA, and then 30 min at 37°C in DMEM medium containing 1 mg/ml collagenase (Roche), Blendzyme III (Roche), and 1 U/ml DNase (Invitrogen). Tissue suspensions were then pressed through a 100-μm mesh, pelleted, resuspended in a 40% isotonic Percoll solution (Pharmacia), and underlaid with an 80% isotonic Percoll solution. Centrifugation for 20 min at 2000 rpm yielded the mononuclear cells at the 40–80% interface. Ear skin was directly incubated 30 min at 37°C in DMEM medium containing collagenase, Blendzyme, and DNase and pressed through a 100-μm mesh. Cells were finally washed twice with PBS-F (PBS containing 2% FCS). Single-cell suspensions were prepared from thymus, spleen, and lymph nodes by pressing the tissues through a 100-μm mesh. All cells were first preincubated with mAb 2.4G2 to block Fcγ receptors, then washed and incubated with the indicated mAb conjugates for 40 min in a total volume of 100 μl of PBS-F, then with the appropriate secondary Ab or streptavidin when necessary, washed in 2 ml of PBS-F, and resuspended in 200 μl of PBS-F for FACS analysis. FACS analysis was performed with an Canto I analyzer (BD Biosciences) and cell sorting was performed with a MoFlo (Dako).
Tissues were washed and fixed overnight at 4°C in a fresh solution of 4% paraformaldehyde (Sigma-Aldrich) in PBS. The samples were then washed for 24 h in PBS, incubated for 2–4 h in a solution of 30% sucrose (Sigma-Aldrich) in PBS until the samples sank, embedded in OCT compound 4583 (Sakura Finetek), frozen in a bath of isopentane cooled with liquid nitrogen, and stored at −80°C. Frozen blocs were cut at 8-μm thickness and sections collected onto Superfrost Plus slides (VWR International). Slides were dried 1 h and processed for staining or stocked at −80°C. For staining, slides were first hydrated in PBS-XG (PBS containing 0.1% Triton X-100 and 1% normal bovine serum; VWR International) for 5 min and blocked with 10% bovine serum in PBS-XG for 1 h at room temperature. Endogenous biotin was blocked with a biotin blocking kit (Vector Laboratories). Slides were then incubated with primary polyclonal Ab or conjugated mAb in PBS-XG overnight at 4°C, washed three times for 5 min with PBS-XG, incubated with secondary conjugated polyclonal Ab or streptavidin for 1 h at room temperature, washed once, incubated with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min at room temperature, washed three times for 5 min, and mounted with Fluoromount-G (SouthernBiotech). For BrdU staining, slides were first incubated with 4 N HCl for 30 min, washed with PBS, incubated with 0.1 M Na2B4O7 (ph 8.5) for 10 min, washed, and stained with Abs as above. Slides were examined under an AxioImager M1 fluorescence microscope (Zeiss) equipped with a CCD camera, and images were processed with AxioVision software (Zeiss). Color pixel quantification was performed using Adobe Photoshop 7.0.
Laser capture microdissection
Embryos or tumors were embeded in OCT compound 4583 (Sakura Finetek), frozen in a bath of isopentane cooled with liquid nitrogen, and stocked at −80°C. Frozen blocs were cut at 10-μm thickness and serial sections collected onto Superfrost Plus slides (VWR International). Sections were immediately fixed 5 min in acetone at −20°C, dried, and stored at −80°C. Some sections of a series were stained and mounted as described in the preceding paragraph, except that sections were hydrated, stained, and washed in PBS containing only 1% normal bovine serum, and all stainings were performed at room temperature for 1 h. Histological structures of interest or groups of cells were identified by immunofluorescence histology, and their position in the tissue was marked and stored. Serial sections were then thawed and immediately stained for 5 s with Histogen (Molecular Devices), washed briefly in RNase-free water supplemented with ProtectRNA (Sigma-Aldrich), dehydrated successively in one bath of 70% ethanol for 30 s, two baths of 95% ethanol for 1 min, two baths of water-free ethanol (VWR International) for 2 min, and two baths of xylene for 5 min, and air-dried. Slides were transferred immediately into a Veritas laser capture microdissection system (Molecular Devices), and the regions previously identified by immunofluorescence were marked, microdissected, and captured with CapSure Macro LCM Caps (Molecular Devices). RNA was isolated using the PicoPure RNA isolation kit (Molecular Devices), and its quality assessed using the 2100 Bioanalyzer system (Agilent Technologies).
Gene expression analysis
To obtain RNA for gene expression analysis by real-time RT-PCR, 500–5000 cells were directly FACS-sorted into vials containing RLT buffer (Qiagen) supplemented with 2-ME, and total RNA was extracted using an RNeasy Micro Kit (Qiagen). The quality of total RNA was assessed using the 2100 Bioanalyzer system (Agilent Technologies). Two hundred fifty to 500 pg of high-quality total RNA was subjected to one linear mRNA amplification cycle using the MessageBooster kit for qRT-PCR (Epicentre Biotechnologies). Fifty to 100 ng of amplified mRNA was then converted to cDNA using SuperScript III (Invitrogen). All procedures were performed according to the manufacturers’ protocols. The expression of 84 different genes was measured using custom-made RT2 Profiler PCR Array (SABiosciences) and confirmed for individual genes using specific primers pairs (SABiosciences). Real-time PCR was performed on a PTC-200 thermocycler equipped with a Chromo4 detector (Bio-Rad Laboratories). Data were analyzed using Opticon Monitor software (Bio-Rad Laboratories).
Gp38+ LS cells are constitutive in lymphoid organs and intestine
In adult organs, gp38/podoplanin is expressed on a number of epithelial, endothelial, and stromal cell types (32), including lymphatic endothelial cells (LECs), medullary thymic epithelial cells (33), and cells of the intestinal lamina propria (33). Nonepithelial and nonendothelial gp38+ stromal cells have been best described in lymphoid tissues as FRCs in the T cell zones of LNs, PPs, and spleen (24). Recently, Link and colleagues showed that FRCs also express αSMA (5), a characteristic trait of myofibroblasts (16, 17). We found that, in addition to the T cell zone of secondary lymphoid organs, gp38+ stromal cells expressing αSMA were also found in the thymic medulla (supplemental Fig. S1),4 as well as in most of the intestinal lamina propria (Fig. 1). Thus, in addition to T cell zones in secondary lymphoid organs, large populations of gp38+ myofibroblasts are present in the thymus and in a nonlymphoid organ such as the intestine. As the intestinal lamina propria harbors a major proportion of the total lymphocyte population (34), it might be suggested that intestinal gp38+ stromal cells function like FRCs in the recruitment, survival, and differentiation of lymphocytes (5).
Gp38+ LS cells develop early during the ontogeny of lymphoid organs and intestine
As gp38+ FRC-like cells are constitutive in adult lymphoid organs and intestine, we examined when and where these cells developed during ontogeny. LN anlagen were visualized in transgenic mice expressing EGFP under control of the Rorc(γt) gene (Rorc(γt)-GfpTG mice) on a BAC (25). RORγt is a nuclear hormone receptor expressed in the fetus by lymphoid tissue inducer (LTi) cells, and it is required for the generation of LNs and PPs. LTi cells are recruited to LN anlagen starting on embryonic day 12.5, and they form clusters and activate stromal cells through membrane LTα1β2. Its receptor on stromal cells, LTβR, induces the expression of the “structural” chemokines CXCL13, CCL19, and CCL21 and of the adhesion molecules ICAM-1 and VCAM-1 (20, 35).
Gp38+ stromal cells could be detected as early as embryonic day12.5 at the site where LTi cells were recruited, but they remained diffusely distributed (Fig. 2,A). Between embryonic days 14.5 and 15.5, clusters of gp38+ stromal cells formed together with LTi cells on one side of large veins showing polarized expression of the lymphatic marker Lyve-1. By embryonic day 15.5, gp38+ stromal cells formed a compact tissue colonized by large numbers of LTi cells. In agreement with previous work on stromal cells of LN and PP anlagen (7, 36), gp38+ stromal cells expressed VCAM-1 and CXCL13, and like FRCs (5), expressed αSMA and BP3 (Fig. 2,B). By embryonic day 16.5, LN anlagen consisted of large clusters of gp38+ stromal cells and LTi cells, clearly delimited by LECs (Fig. 2 A). These data show that gp38+ stromal cells are involved early in the development of LN anlagen, days before lymphocytes are recruited (20), and they constitute most of the LTi-recruiting stroma.
In the absence of LTβR-mediated signaling in stromal cells, LTi cells are recruited to LN anlagen until embryonic day 15.5 but do not activate the stromal cells to express ICAM-1 and VCAM-1 (36, 37). As a consequence, LTβ-deficient mice or embryos born from mothers treated with LTβR-Ig fusion protein fail to develop LNs (31). We therefore assessed whether the development of gp38+ stromal cells early in LN anlagen was also dependent on LTβR-mediated signaling. Pregnant mothers were treated with LTβR-Ig fusion protein from day 13.5 postcoitus, and embryos were examined at embryonic day 15.5. Even though VCAM-1 was not detected within LN anlagen (Fig. 2,C), as consequence of LTβR blockage, the development of gp38+ stromal cells and their expression of CXCL13 were not affected, in accordance with the suggested role of CXCL13 early in the recruitment of LTi cells (20). However, gp38+ stromal cells in LTβR-Ig-treated mice did not mature into FRC-like cells, as they failed to express detectable levels of αSMA or BP3 (Fig. 2 C). Taken together, these data suggest that gp38+ stromal cells develop early in LN anlagen, independently of LTα1β2 delivered by LTi cells, and they recruit LTi cells through chemotactic factors such as CXCL13 but require LTi cells to develop into fully mature FRC-like LN organizers (38).
Gp38+ stromal cells were also detected during the development of the intestinal lamina propria, starting at day embryonic day 15.5, and as small clusters in the thymic medulla (supplemental Fig. S2). In this latter case, gp38+ cells likely included both stromal and epithelial cells (33).
Gp38+ LS cells are induced in inflammation and tumors
A large body of literature shows that stromal cells in inflammatory lesions produce CXCL13, CCL19, and CCL21 (8, 9, 10, 11, 12) and express ICAM-1 and VCAM-1 (13). We therefore examined different types of inflammatory lesions, as well as tumors, for the presence of gp38+ FRC-like cells. Of note, gp38/podoplanin has been used previously in inflammation and tumors as a marker of LECs together with Lyve-1 and Prox-1, thus as evidence for lymphangiogenesis (32, 39). In the following experiments, gp38+ stromal cells were thus distinguished from LECs by concomitant Lyve-1 staining.
We first examined mice bearing the Scurfy mutation in the FoxP3 gene (FoxP3sf mice). As a consequence, these mice lack regulatory T cells and develop autoimmune inflammatory lesions in all tissues (40). Whereas no gp38+ stromal cells were found in control nonlymphoid tissues such as the liver and the pancreas, a substantial gp38+ stroma developed in the proximity of LECs in inflamed regions infiltrated with CD45+ cells (Fig. 3,A). Second, we assessed the development of gp38+ stromal cells in a model of ear inflammation that allowed for convenient longitudinal analyses. Mouse ears were injected with CFA and collected after different periods of time. Massive thickening of the ear developed 1 day after CFA injection and peaked after 1 wk (Fig. 3,B), concomitant with a heavy neutrophil infiltration starting as early as 3–6 h after CFA injection, whereas DCs and lymphocytes were recruited in significant numbers only after 1 wk (supplemental Fig. S3A–C). Before inflammation, gp38 expression was confined to LECs and a monolayer of cells lining the central ear cartilage. Gp38+ stromal cells were detected starting 12 h after CFA injection, and they expanded massively within 2–3 days. After 5–7 days, gp38+ stromal cells started to express αSMA, as well as BP-3 (CD157) (Fig. 3,C), reminiscent of the expression of αSMA and BP-3 by FRCs (5). Finally, a prominent gp38+ stroma expressing αSMA (data not shown) also developed in the inflamed footpad of mice infected for 1 mo with Leishmania major (Fig. 3 D).
Gp38 is expressed by tumor cells at the invasive front of human tumors (41). We thus assessed whether gp38+ stromal cells also developed during tumor progression in mouse models for pancreatic islet (Rip-TagTG) (42) or intestinal (Apcmin/+) (43) tumors. Rip-TagTG mice, which express the SV40 T Ag under control of the rat insulin promoter, develop progressive islet dysplasia starting at 4–5 wk of age and large and highly vascularized tumors by the age of 12–14 wk (42). Whereas gp38 expression was limited to LECs in normal pancreas and dysplastic islets, gp38+ stromal cells progressively developed around large (SV40+) tumors after 10–12 wk, concomitant with the induction of local lymphangiogenesis, and gradually expressed αSMA at the latest stages (Fig. 3 E). Similarly, gp38+ stromal cells expanded within intestinal polyps of 6-mo-old Apcmin/+ mice, and they also supported expansion of a local lymphatic network (supplemental Fig. S3D). These observations are in accordance with recent reports showing gp38 expression in the stroma of human tumors (44, 45).
Collectively, our data show that inflammation and tumors induce the development of a large population of gp38+ stromal cells displaying a phenotype similar to that of FRCs in secondary lymphoid tissues. As during ontogeny (Fig. 2), the accumulation of gp38+ stromal cells is accompanied by the development of LECs (Fig. 3).
Gp38+ LS cells promote the development of lymphocyte-permissive tissues
FRCs express Il7 and Ccl19, which play important roles in the recruitment, survival, and homeostasis of naive T cells (5). We therefore assessed whether gp38+ stromal cells developing during ontogeny or inflammation also express the Il-7 and Ccl19 genes. Gp38+ CD31− cells (thus excluding endothelial cells such as LECs) were isolated by flow cytometry (supplemental Fig. S4) from adult small intestine, LNs, and inflamed ears, and by laser capture microdissection from tumors and LN anlagen (Fig. 4,B). We found that Il7 and Ccl19 transcripts were up-regulated, although to varying degrees, in all subsets of gp38+ stromal cells, except in stromal cells isolated from Apcmin/+ tumors (Fig. 4 C).
To further establish the FRC-like nature of gp38+ stromal cells, we generated a gene expression pattern of FRCs for 84 cytokines and chemokines by using quantitative RT-PCR arrays. Genes overexpressed by FRCs (Fig. 4,A) were then analyzed in the different populations of gp38+ stromal cells. In addition to Il7 and Ccl19, most gp38+ stromal cells expressed elevated levels of transcripts coding for CCL21 (5), as well as for CXCL13, TRANCE, and CXCL12 (Fig. 4 C and supplemental Fig. S5A), all involved in the development of lymphoid tissues (6, 46, 47). Notably, CXCL13 is a marker of FDCs rather than FRCs (2). However, a subset of CD35+ FDCs in B cell follicles has recently been found to coexpress gp38 (5), indicating that gp38+ stromal cells are not a homogeneous population of cells. Other overexpressed transcripts coded for the proinflammatory cytokines thymic stromal lymphopoietin (TSLP) (48) and IL-17d (49), the neutrophil or eosinophil chemoattractants CXCL1 and CCL11 (eotaxin), and the immunomodulator TWEAK (50) (Fig. S5A). Thus, based on their expression pattern of cytokines and chemokines, which appears to be similar to that of FRCs, gp38+ stromal cells might be rightly termed gp38+ LS cells.
FRCs and most gp38+ stromal cell populations express the myofibroblast marker αSMA (Figs. 1–3). Myofibroblasts have been extensively described in wound healing and fibrosis, where they provide mechanical tension and generate extracellular matrix (17). Accordingly, gp38+ stromal cells overexpressed transcripts coding for HAS-1 (supplemental Fig. S5B), which synthesize hyaluronan, an important component of the extracellular matrix (51). Furthermore, using quantitative RT-PCR arrays, we found that most subsets of gp38+ stromal cells expressed, although to varying degrees, transcripts coding for connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 (also basic FGF), both fibroblast and endothelial cell growth factors promoting angiogenesis (52, 53, 54) (Fig. 4,D and supplemental Fig. S5B). Also expressed were transcripts coding for FGF-7 (keratinocyte growth factor, or KGF), hepatocyte growth factor (HGF/scatter factor), and platelet-derived growth factor (PDGF)-D, which play important roles in epithelial homeostasis and tissue regeneration, as well as for insulin-like growth factor (IGF)-1, a pleiotrophic stromal growth factor (55). It is noteworthy that FGF-2, HGF, and IGF-1 are also involved in lymphangiogenesis (56, 57, 58). Additionally, gp38+ stromal cells overexpressed transcripts coding for the potent lymphangiogenic factors vascular endothelial growth factor (VEGF)-C and VEGF-D (55) (Fig. 4,D and supplemental Fig. S5B), reminiscent of recent findings by Chyou and colleagues (59). Interestingly, Vegfd was expressed only early (at embryonic day 14.5) in the development of LN anlagen, when the first LECs developed (Fig. 2). Thus, gp38+ LS cells also express genes that promote connective tissue genesis through extracellular matrix build-up, fibroblast and epithelial cell growth and differentiation, and angio- and lymphangiogenesis. Together with their chemokine and cytokine gene expression profile, we conclude that the genetic program unfolding in gp38+ LS cells in ontogeny and in inflammation contributes to create organized vascularized tissues that actively recruit immune cells.
The generation of gp38+ LS cells during inflammation requires myeloid cells but not TLRs, Nod-like receptors (NLRs), the inflammasome, or adaptive immunity
In the sterile environment of the fetus, a genetic program leads to the development of gp38+ LS cells in LN anlagen, the thymus, and the intestine. In contrast, during inflammation in the adult mouse, gp38+ LS cells are induced by a trigger that remains to be identified. CFA, which we used to induce gp38+ LS cells in ears, contains Mycobacteria-derived ligands for the innate TLR2 and TLR4 (60). Gp38+ stromal cells were also induced by zymosan, prepared from the cell wall of the yeast Saccharomyces cerevisiae and a TLR2 ligand (61) (data not shown). We therefore assessed whether mice deficient for TLR2 or TLR4, or the TLR-associated signaling molecules MyD88 or TRIF (62), developed gp38+ stromal cells upon CFA injection. In all cases, the induction of gp38+ stromal cells was unaffected (Fig. 5, A and B). Gp38+ LS cells were also induced in mice deficient for the pattern recognition molecules of the NLR family NOD-1 and NOD-2 (63), for the type I IFN receptor IFN-αR, which is activated downstream of the TLR pathway (62), for essential components of the inflammasome, such as ASC, NALP3, or caspase-1, for cytokines or cytokine receptors involved in inflammation, such as IFN-γ, IL-1, IL-6, and IL-12Rβ1, and in the absence of B and T lymphocytes (Fig. 5, A and B). Similar to the development of gp38+ stromal cells in LN anlagen, the development of these cells during inflammation was also independent of LTβR-mediated signaling in LTβ-deficient mice (Fig. 5,B) or mice treated with LTβR-Ig fusion protein (data not shown). Additionally, gp38+ stromal cells isolated from the inflamed ears of different mutant mice expressed normal levels of LS cell-specific genes, such as Ccl19 and Il7 (5) (data not shown). IFA, which lacks the Mycobacteria Ags contained in CFA, also induced the development of gp38+ stromal cells (Fig. 5,B), in the presence or absence of the molecules listed above (data not shown). Furthermore, internal injury generated in ears by friction of the two skin surfaces in opposite motions induced gp38+ stromal cells (Fig. 5, A and B), demonstrating that exogenous molecular signals are not necessary for their induction. Thus, during inflammation, gp38+ LS cells are induced by a robust pathway that does not depend on known TLRs or NODs, the inflammasome, or adaptive immunity. However, these pathways might be redundant in the generation of gp38+ LS cells, as multiple pathways have been shown to induce myofibroblast differentiation (16, 17, 64).
Large numbers of neutrophils, as well as CD11b+ myeloid cells but few macrophages, were recruited hours after CFA injection into the ear (supplemental Fig. S3, A–C). We therefore assessed whether myeloid cells were required for the generation of gp38+ stromal cells during CFA-mediated inflammation. Myeloid cells were ablated by the injection before and during inflammation of anti-CD11b or anti-Gr-1 mAb (Fig. 5 C). Both treatments significantly reduced the induction of gp38+ stromal cells, which was correlated with the number of recruited neutrophils and was most reduced upon anti-Gr-1 treatment. Of note, cells other than neutrophils express Gr-1, such as a subset of monocytes (65), and their specific contribution to the induction of gp38+ stromal cells remains to be assessed, even though the vast majority of Gr-1+ cells in the inflamed ear were neutrophils (supplemental Fig. S3, A–C). The mechanisms by which myeloid cells induce gp38+ LS cells are presently under investigation.
Inflammation induces the generation of gp38+ LS cells from local precursors
In the fetus, it is expected that the progressive differentiation of mesenchymal cells gives rise to gp38+ LS cells (7). During inflammation, gp38+ LS cells might develop from a variety of circulating or resident precursors. Fibrocytes, or circulating bone marrow-derived mesenchymal precursors, have been shown to differentiate into fibroblasts and myofibroblasts during wound healing (66, 67) and to give rise to FDCs (68, 69). On the other hand, resident fibroblasts were found to proliferate and differentiate into myofibroblasts during wound healing or fibrosis (17, 70), and in vitro in the presence of TGFβ and growth factors such as CTGF and IGF-2 (52). In a parabiosis experiment, we therefore determined whether the massive number of gp38+ stromal cells induced by CFA was derived from recruited or local precursors. A C57BL/6 mouse and a UbiquitinGFP mouse were sutured together and the efficiency of parabiosis was confirmed 4 days later by the presence of 50% GFP+ cells in the blood of both mice. CFA was injected, and after 4 days, we observed that none of the gp38+ stromal cells of the C57BL/6 partner expressed GFP, whereas all gp38+ stromal cells of the UbiquitinGFP partner expressed GFP (Fig. 6 A), demonstrating that gp38+ stromal cells were not derived from circulating progenitors during inflammation, but were most likely from local precursors.
The vast increase in the numbers of gp38+ stromal cells, and the consequent ear thickening, indicated that local precursors had to undergo proliferation. To demonstrate proliferation, BrdU was injected at different time points during CFA-induced inflammation (Fig. 6,B), and BrdU+gp38+ stromal cells were quantified (Fig. 6,C). The highest proportion of BrdU+ cells among gp38+ stromal cells, reaching 30%, was observed during the first day after CFA injection. This proportion decreased thereafter, even though the highest absolute number of BrdU+gp38+ stromal cells was found during the second day after CFA injection (data not shown). After a pulse of BrdU during the second day after CFA injection, resulting in the staining of 15–20% of gp38+ stromal cells, the accumulated proportion of BrdU+ cells among gp38+ stromal cells reached 50% (Fig. 6 C) after 4 days, demonstrating that several rounds of proliferation accounted for the massive increase in the number of gp38+ LS cells.
Notably, an important fraction of BrdU+ cells (Fig. 6,B) or cells expressing the proliferation marker Ki67 (Fig. 6,D) resided within the basal layer of epithelial cells in the skin. Basal epithelial cells, expressing keratin 14 (K14), generate the upper layers of epithelial cells through proliferation and differentiation (71). It has been suggested that epithelial cells give rise to myofibroblasts during wound healing and tumorigenesis in a process termed epithelial-to-mesenchymal transition (EMT) (72). This process is paramount for the generation of mesenchymal cells at different stages of embryogenesis and may channel the progression of carcinoma toward malignancy (73). We therefore assessed whether gp38+ stromal cells generated during CFA-induced inflammation were derived from K14+ epithelial cells. Transgenic mice expressing the tamoxifen-inducible recombinase Cre-ERT2 under control of the K14 promoter (74) crossed to Rosa26Fl-STOP-Fl-Yfp reporter mice were first treated with tamoxifen and then injected with CFA (Fig. 6 D). Whereas all epithelial cells from the skin and hair follicle expressed the YFP reporter and thus were progeny of K14+ epithelial cells, no gp38+ stromal cells expressing YFP could be detected. Taken together, these data indicate that inflammation induces the generation of gp38+ LS cells from local nonepithelial precursors, most likely resident fibroblasts, or from local mesenchymal precursors that remain to be identified.
In this study, we have shown that inflammation induces the generation of gp38+ LS cells, in an apparent recapitulation of the ontogeny of gp38+ LS cells during the development of secondary lymphoid tissues and intestinal stroma. However, whereas ontogeny programs the generation of gp38+ LS cells, inflammation induces massive generation of gp38+ LS cells from resident fibroblasts or mesenchymal precursors through a robust pathway involving myeloid cells. Gp38+ LS cells were first characterized in T cell zones of lymph nodes and spleen, and they express cytokines and chemokines for the recruitment and survival of lymphocytes. Additionally, we find that gp38+ LS cells express a number of genes involved in the growth and differentiation of stromal cells, as well as in lymphangiogenesis. We therefore suggest that gp38+ LS cells are generated during ontogeny and inflammation to create an optimal platform for the recruitment and survival of lymphocytes.
The generation of LS-like stromal cells during inflammation is well documented (8, 9, 10, 11, 12). However, characterization of these LS-like cells remains circumstantial, and no direct comparison with stromal cells from lymphoid tissue has been performed. We isolated programmed or inflammation-induced gp38+ stromal cells by FACS or laser capture microdissection from fetal LN anlagen, LNs, intestine, inflamed ears, and tumors and demonstrated that all subsets of gp38+ stromal cells shared an FRC-like phenotype (5) by virtue of their expression of gp38, CD157, αSMA, IL-7, and CCL19. We therefore suggest naming gp38+ stromal cells more generally gp38+ lymphoid stromal (LS) cells. However, expression of these markers and of an additional panel of genes coding for cytokines and chemokines varied significantly between gp38+ stromal cells of different origins, suggesting that gp38+ LS cells include distinct subsets of stromal cells, or cells at different maturation stages. For example, gp38+ stromal cells in LNs also include CD35+ FDCs of the B cell follicle (5), a cell subset that may account for the expression of the B cell chemoattractant Cxcl13 we report in gp38+ stromal cells (Fig. 4,C), but that is distinct from gp38+ FRC-like cells expressing Il7 and Ccl19. Furthermore, the varying levels of αSMA expression observed in gp38+ stromal cells (Figs. 1–3) indicated that different populations of gp38+ cells were at different stages of maturation from fibroblasts to myofibroblasts.
Most types of gp38+ LS cells expressed elevated levels of transcripts coding for “structural” chemokines and cytokines involved in the development of lymphoid tissues, such as CCL19, CCL21, CXCL13, CXCL12, IL-7, and TRANCE (20). This is in accordance with the hypothesis that inflammation and developing lymphoid tissues engage similar pathways (21, 22), and with the observation that chronic inflammatory lesions develop ectopic (tertiary) lymphoid tissues (tLTs) (9, 23). Furthermore, ears that have endured CFA-induced inflammation for 1 wk or more develop a massive gp38+ lymphoid stroma, as well as numerous DCs and T cells clustered around small vessels (supplemental Fig. S3A and data not shown), reminiscent of a rudimentary lymphoid organization that may prefigure the formation of neo- (or tertiary) lymphoid tissues during chronic inflammation (9, 13). Interestingly, the early development of gp38+ stromal cells both during ontogeny and inflammation was independent of LTβR-mediated signals, required for the development of secondary (31) and tertiary lymphoid tissues (75), as such signals induce the expression of the structural chemokines (35). Nevertheless, gp38+ stromal cells that formed in the absence of LTβR-mediated signals expressed CXCL13 but failed to express αSMA, VCAM-1, and BP3 (Fig. 2 C), suggesting that these cells are programmed to develop first and exert chemotactic activity through CXCL13, but require LTβR signaling for maturation into mature LS cells.
Gp38+ LS cells expressed αSMA, a characteristic trait of myofibroblasts. Previously, FRCs have been shown to express αSMA and desmin and to develop tensile force capable of creating wrinkles in the surface of a deformable collagen-coated silicon substrate, definitely earning them the designation of myofibroblasts (5). The role of myofibroblasts has been extensively described in wound healing and fibrosis to provide mechanical tension and produce extracellular matrix (17). This latter function is also suggested to nurture tumor progression (76). Accordingly, we found that gp38+ stromal cells overexpressed transcripts coding for HAS-1 (supplemental Fig. S5B), which synthesize hyaluronan, an important component of extracellular matrix (51). Furthermore, the function of gp38+ LS cells as inferred from their gene expression profile was not restricted to metabolism of the extracellular matrix, but also included the production of an array of growth factors that favor the proliferation and differentiation of fibroblasts and endothelial and epithelial cells. One particular factor, CTGF, is required for the TGF-β-induced proliferation and differentiation of myofibroblasts (52). Notably, FDCs have been shown to express transcripts for CTGF (77). Most important in establishing structures that enable the recruitment of leukocytes, gp38+ LS cells also express an array of lymphangiogenic factors, including VEGF-C, VEGF-D, FGF-2, HGF, IGF-1, and CTGF (55, 56, 57, 58). Accordingly, we observed that the development of LECs accompanied the generation of gp38+ LS cells both during ontogeny and in inflammation.
In our model of CFA- or IFA-induced inflammation in the ear, development of gp38+ LS cells did not depend on the activation of innate immune pathways engaged by TLRs, NLRs, or the inflammasome. First, this suggest that induction of gp38+ LS cells, as well as the “storm” of cytokines, chemokines, and growth factors that they produce, is very robust and is unlikely to fail if one of these pathway is defective or blocked by pathogen-derived virulence factors (78). Therefore, gp38+ LS cells are likely to form part of an early system to detect insult and drive the subsequent inflammatory reaction and recruitment of leukocytes. Second, it is possible that a yet unknown pathway induces the generation of gp38+ LS cells upon tissue injury. However, a plethora of signals have been shown to induce the generation of myofibroblasts, including TLR4-mediated signals (79) and mechanical stress (16, 17), suggesting that the pathways leading to the generation of myofibroblasts, and of gp38+ LS cells, are highly redundant. Third, we find that Gr-1+ and CD11b+ cells, presumably neutrophils, as they are recruited in large numbers that correlate with gp38+ LS cell induction, are required for the induction of gp38+ LS cells. Other Gr-1+ and/or CD11b+ cells might be involved, such as so-called “inflammatory monocytes” (65), a possibility that remains to be assessed. The mechanism through which myeloid cells might induce gp38+ LS cells is presently under investigation. Interestingly, colon-derived myofibroblasts induce the recruitment of neutrophils through proteolytic activation of the chemokine CXCL7 (80). Thus, together, myofibroblasts and neutrophils might establish a positive feedback loop for the generation of the former and the recruitment of the latter.
The massive de novo generation of gp38+ LS cells during inflammation triggers the question of their origin. Myofibroblasts have been suggested to derive locally from resident fibroblasts, “protomyofibroblasts”, pericytes, or smooth muscle cells, or from circulating fibrocytes during wound healing and fibrosis (16, 17, 64, 66, 67). In liver, fat-storing cells (also known as Ito cells or hepatic stellate cells) have been reported to differentiate into myofibroblasts upon LPS challenge and cause hepatic fibrosis (79). Furthermore, epithelial cells have the capacity to trans-differentiate by EMT into fibroblasts and myofibroblasts during wound healing and tumorigenesis, a process essential during ontogeny (72). Our data clearly show that during CFA-induced inflammation, gp38+ LS cells are derived from local rather than from circulating progenitors. However, we cannot rule out that in other settings, gp38+ LS cells might be generated from recruited precursors, as suggested for the generation of FDCs (68, 69). In our case, local progenitors did not include cells of the epithelial lineage, but they might include cells of the endothelial or fibroblastic lineages. Given the rapid incorporation of BrdU by gp38+ stromal cells during the first day of inflammation and the massive generation of gp38+ LS cells within days, we favor the hypothesis of resident fibroblasts being activated to differentiate into myofibroblasts, a process that can be recapitulated in vitro in the presence of TGFβ and CTGF (52). Such a differentiation pathway, generating gp38+ LS cells from resident fibroblasts, would ensure the rapid mobilization of large numbers of stromal cells to drive and support an ongoing immune response.
Collectively, our data show that inflammation recapitulates the generation of gp38+ LS cells that is programmed during ontogeny. Given their gene expression profile, gp38+ LS cells most likely contribute to create an environment that promotes the expansion and differentiation of myofibroblasts, epithelial cells, and endothelial cells, the generation of lymphatic vessels, the recruitment of leukocytes, and, during chronic inflammation, the organization of lymphocytes into tertiary lymphoid tissues. As gp38+ LS cells are induced early during inflammation, they might represent an important pharmacological and immunological target for the modulation of inflammatory diseases or the inhibition of tumor progression, or, conversely, they might be exploited to reinforce immunity to local infection or tumors.
We thank A. Farr for anti-gp38 Ab; A. Freitas for FoxP3sf and μ−/− mice; S. Akira, M. Chignard, and V. Balloy for Myd88−/−, Trif−/−, Tlr2−/−, Tlr3−/−, and Tlr4−/− mice; J. P. Hugot for card15−/− mice; Millennium Pharmaceuticals for card4−/− mice; B. Ryffel and J. Tschopp for Nalp3−/− and Caspase1−/− mice; V. Dixit for Asc−/− mice; A. Zychlinsky for Casp1−/− mice; M. Albert for Il6−/−, Ifng−/−, and Ifnar−/− mice; K. Pfeffer and R. Golub for Ltb−/− mice; and J. Di Santo for Il12rb1−/− mice. We also thank L. Polomack for technical assistance.
The authors have no financial conflicts of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Institut Pasteur, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Agence Nationale de la Recherche, Fondation de la Recherche Médicale, Mairie de Paris and a Marie Curie Excellence grant.
L.P. designed and performed all experiments and wrote the paper; S.D. performed qRT-PCR; M.L. performed FACS sorting; G.F.S. and M.A.M. provided Leishmania-infected mice; A.C. performed FACS sorting; G.E. designed and directed the study and wrote the paper; and all authors critically reviewed the manuscript.
Abbreviations used in this paper: LS cell, lymphoid stromal cell; LN, lymph node; PP, Peyer’s patch; FDC, follicular dendritic cell; FRC, fibroblastic reticular cell; DC, dendritic cell; αSMA, α smooth muscle actin; LTα1β2, lymphotoxin α1β2; tLT, tertiary lymphoid tissue; EMT, epithelial to mesenchymal transition; BAC, bacterial artificial chromosome; EGFP, enhanced GFP; LTβR, lymphotoxin-β receptor; LEC, lymphatic endothelial cell; LTi cell, lymphoid tissue inducer cell; NLR, Nod-like receptor.
The online version of this article contains supplemental material.