IL-17 metabolically reprograms activated fibroblastic reticular cells for proliferation and survival

T_H17 cells drive increased ECM in inflamed LNs

Increased production of ECM components such as fibronectin and collagen are features of T_H17-mediated inflammation, including the central nervous system (CNS) during multiple sclerosis (MS) or its animal model experimental autoimmune encephalomyelitis (EAE)^{26, 27}. Following immunization with the myelin oligodendrocyte glycoprotein peptide MOG(aa35–55) in complete Freund’s adjuvant (CFA) to induce EAE, we observed that expression of Fn1 (encoding fibronectin) increased along with Il17a in draining LNs (Supplementary Fig. 1a). Immunization-induced Fn1 required IL-23R (Fig. 1a), implicating ‘type-17’ IL-23R⁺ cells that could be T_H17 cells, type 3 innate lymphoid cells (ILC3) or γδ T cells. We therefore dissected the role of de novo generated antigen-specific T_H17 cells in driving Fn1 expression following immunization. Wild-type or Il23r^−/− CD4⁺ TCR-transgenic OT-II cells were transferred into wild-type or Il23r^−/− hosts, which were then immunized with the OT-II-activating ovalbumin peptide OVA(aa323–339) in CFA. As expected²⁸, IL-17 was only strongly induced when OT-II cells expressed IL-23R (i.e. wild-type OT-II), and did not require host IL-23R (Fig. 1b). Similarly, Fn1 expression was only increased when OT-II cells were able to successfully produce IL-17 (Fig. 1c).

To determine whether IL-17 itself was required for Fn1 increases in inflamed LNs, we immunized Il17a^−/− mice with MOG(35–55) in CFA. Indeed, Fn1 expression was impaired in dLNs of Il17a^−/− mice compared to wild-type following immunization (Fig. 1d). Immunization-elicited Fn1 was dependent on IL-17RA and the signaling adaptor Act1, further confirming the requirement for the IL-17 signaling pathway (Fig. 1e, f). To confirm these gene expression changes resulted in protein changes, we performed immunofluorescence (IF) staining. Fibronectin expression again increased in wild-type but not Il17ra^−/− dLNs following immunization (Fig. 1g). Expression of Col1a1, encoding collagen type 1a was also increased in dLNs in an IL-17RA-dependent manner following immunization (Fig. 1h), but Col3a1 did not change indicating some specificity to the ECM components that were regulated by IL-17 (Supplementary Fig. 1b). Correspondingly, collagen deposition was increased in wild-type but not Il17ra^−/− mouse dLNs (Fig 1I). Despite these defects in ECM, the gross appearance, mass and cell numbers were similar between dLNs from immunized Il17ra^−/− , Il17^−/− and wild-type controls (Fig 1J-L, Supplementary Fig. 1c). These data thus suggested that T_H17 cells promote expression of ECM in inflamed dLNs through IL-17 signaling, independently of LN size or hypercellularity.

IL-17 signaling is required for FRC population expansion

FRCs are the major cell type that expresses fibronectin and type I collagen in LN⁷, and IF staining confirmed co-localized expression of the FRC marker ERTR7 with fibronectin in dLN (Supplementary Fig. 2a). We therefore analyzed the stromal cell composition of naïve LNs and inflamed dLNs from immunized wild-type and Il17ra^−/− mice. CD45^–CD31^–PDPN⁺ FRC were significantly increased in number in wild-type inflamed LN and spleen from immunized animals compared to naïve LN, and this increase required IL-17 signaling as dLN from immunized Il17ra^−/− or Il17^−/− had significantly reduced FRC numbers compared to wild-type (Fig. 2a, b and Supplementary Fig. 2b, c). As further confirmation of the importance of IL-17 signaling in inflamed LN, FRC numbers were reduced in absence of the IL-17R signaling adaptor Act1 (Fig 2c). Regnase-1 is a potent negative regulator of IL-17 signaling²⁹, and accordingly mice with reduced Regnase-1 expression had increased numbers of FRCs following immunization (Fig 2d). In contrast, numbers of CD45^–PDPN⁺CD31⁺ lymphoid endothelial cells (LECs) and CD45^–PDPN^–CD31⁺ blood endothelial cells (BECs) were not different in dLN from immunized l17ra^−/− compared to controls (Fig. 2e, f, Supplementary Fig. 2b). PDPN⁺CD21⁺CD35⁺ follicular dendritic cells (FDC) and PDPN⁺MadCAM1⁺ marginal zone reticular cells (MRC), were unchanged by IL-17 deficiency (Fig 2g,h). While not an exhaustive characterization of stromal cell subsets, these data suggested that the CD21^–MadCAM1^–CCL19⁺ TRC population, which is also the largest and best positioned to interact with T_H17 cells, may be a functional target of IL-17 signaling in lymphoid tissues.

FRC respond directly to IL-17 during inflammation

We next sought to determine the factors that regulate IL-17-dependent increases in FRC numbers. Lymphotoxin (LT)α and LTβ are critical for development, expansion and maintenance of FRC populations in LN¹¹. Lta and Ltb expression increased in response to immunization, but were not different between wild-type and Il17ra^−/− mice, suggesting that IL-17 does not regulate lymphotoxin availability (Fig. 3a,b). FRC numbers in LN from naïve Il17ra^−/− mice were similar to wild-type mice (Supplementary Fig. 3a), further supporting an IL-17-independent function of lymphotoxins in FRC development. To rule out any contribution of developmental defects to the impaired FRC response in Il17^−/− mice, we immunized wild-type mice with MOG(35–55) in CFA and administered IL-17 neutralizing antibodies (Supplementary Fig. 3b). FRC numbers were again significantly reduced in LN from anti-IL-17 treated mice compared to isotype controls (Fig. 3c), despite similar LN size between these groups (Supplementary Fig. 3c). Moreover, Fn1 expression was significantly reduced when IL-17 was neutralized (Supplementary Fig. 3d).

These data confirmed a role for IL-17 in promoting FRC proliferation in mature SLO following immunization. However, they did not rule out an indirect effect of IL-17 within the LN. For example, IL-17 signaling leads to neutrophil recruitment³⁰, and accordingly neutrophils were decreased in immunized Il17ra^−/− dLN compared to wild-type (Fig. 3d). To determine whether IL-17 signaling is required in FRC, FRC^ΔIl17ra mice were generated by crossing Il17ra^fl/fl mice with the FRC-specific CCL19^{Cre11, 22, 31} (Supplementary Fig. 3e). Ccl19^CreIl17ra^fl/+ littermates were analyzed as controls (FRC^ctrl). FRC^ΔIl17ra showed no defect in neutrophil recruitment following immunization (Fig. 3e), indicating that FRC are not a necessary source of neutrophil-attracting chemokines in response to IL-17 signaling in LN. As observed in Il17ra^−/− dLN, LEC and BEC numbers were unchanged in FRC^ΔIl17ra (Supplementary Fig. 3f, g). Expression of Fn1 and Col1a1, but not Col3a1, were also significantly decreased in FRC^ΔIl17ra mice compared to FRC^ctrl (Supplementary Fig. 3h-j). Furthermore, numbers of FRC in LN of FRC^ΔIl17ra mice were significantly decreased on day 12 and 15 post-immunization (Fig. 3f,g). Thus we concluded that FRC-intrinsic IL-17 signaling is required for their successful proliferation during LN inflammation.

FRC-intrinsic IL-17 signaling promotes antibody production

It might be expected that reduced numbers of FRCs would impact the adaptive immune system. However, numbers of CD4⁺ T cells and B cells were not different between immunized FRC^ΔIl17ra and FRC^ctrl mice (Fig. 4a, b). The disruption of FRC stromal architecture in immunized FRC^ΔIl17ra dLN did not disrupt B and T cell zones, and frequencies of naïve and effector T cell populations, as well as IL-17-producing T_H17 cells, were unaffected (Fig. 4c, Supplementary Fig. 4a-c). Since already-differentiated T_H17 cells produce IL-17 to then potentiate FRC proliferation, this is perhaps unsurprising in this acute model. However, there was a significant decrease in the frequency of germinal center B cells from immunized dLN when FRCs could not respond to IL-17 (Fig. 4d, Supplementary Fig. 4c), and a corresponding defect in induction of total immunoglobulin G (IgG) as well as MOG-specific IgG production (Fig. 4e, f). B cell activating factor (BAFF) is produced by FRCs to support B cell survival under homeostatic conditions¹⁰. IL-17 did not stimulate increased BAFF production in FRC in vitro (Fig. 4g). However, corresponding to reduced numbers of FRC, BAFF levels were significantly decreased in LN from immunized FRC^ΔIl17ra mice (Fig. 4h). Hence, lack of IL-17 signaling in FRC does not lead to global immune defects, but does cause impaired B cell responses, most likely through reduced availability of BAFF at a critical juncture for germinal center formation and maintenance.

Colitis increases local IL-17-dependent FRC numbers

CFA is a potent long-acting adjuvant, and we wanted to test whether the IL-17-dependent increase in FRC numbers was more broadly applicable to inflamed LN. We therefore analyzed FRC in gut-draining mesenteric LN (MLN) during DSS colitis, a colon injury model in which IL-17 plays a protective role in restoring epithelial barrier integrity^{3, 4}. As expected, Il17ra^–/– mice developed more severe colitis, as determined by weight loss and colon length, while FRC^ΔIL17RA mice developed colitis of similar severity to controls since the gut epithelium response to IL-17 was intact (Fig. 5a, b, c, Supplementary Fig. 5a). Naïve mice with FRC-specific ablation of IL-17RA showed no difference in MLN FRC numbers compared to littermate controls, again confirming that IL-17 is not required for FRC development (Supplementary Fig. 5b). Numbers of FRC increased in the inflamed MLN of wild-type mice after administration of DSS compared to water controls (Fig. 5d). Strikingly, the colitis-driven MLN FRC population expansion was critically dependent on IL-17RA expression by FRC, rather than differing gut epithelial cell IL-17RA expression and disease severity in Il17ra^−/− and FRC^ΔIL17RA mice (Fig. 5d). Therefore, these data support the conclusion that FRC-intrinsic IL-17RA expression is required for FRC remodeling in the dLN during inflammation that elicits an IL-17 response, regardless of the type of peripheral tissue insult.

IL-17 potentiates FRC proliferation and survival

To further understand how IL-17 promotes FRC activation, we performed transcriptomic analysis of dLN FRC isolated at day 12 post-immunization (Supplementary Fig. 6a). Known IL-17 targets were increased in wild-type compared to Il17ra^−/− FRC (Fig. 6a), including genes encoding CXCL1, CXCL2, s100a8, s100a9, C/EBPβ and IκBζ³². Fitting with decreased FRC numbers, gene set enrichment analysis (GSEA) showed significant dysregulation of the cell cycle pathway in Il17ra^−/− FRC (Fig. 6b). There was a trend towards reduced expression of the cell cycle-associated protein ki67 in Il17ra^−/− FRC in LN and a significant decrease in Ki67 in spleen (Fig. 6c and Supplementary Fig. 6b, c). Ki67 marks all cells that have initiated cell cycle. In fact, a number of the genes identified by GSEA to be increased in Il17ra^−/− FRC are negative regulators of cell cycle, including Trp53, Nek2, Tfdp1, Chek2 and Rad17 (Fig. 6d). We therefore analyzed cell cycle stages and found that similar proportions of cells were in G2/M phase, but significantly fewer FRC^ΔIl17ra cells were in S phase compared to FRC^ctrl, with an inverse trend towards increased G0/G1, which includes resting cells (Fig. 6e, Supplementary Fig. 6d). Fibroblasts have been shown to actively express genes that maintain cellular quiescence and suppress proliferation following in vitro triggers of quiescence such as growth factor withdrawal or cell-contact inhibition³³. Il17ra^−/− FRC had high expression of these fibroblast quiescence-associated genes when compared to wild-type FRC (Supplementary Fig. 6e), further supporting that cell cycle was stalled.

Cell cycle analysis also revealed increased apoptotic cells in IL-17RA-deficient FRC (Fig. 6e). There was no difference in total cell viability of digested wild-type and Il17ra^−/− LN, in accord with similar total LN cell numbers (Supplementary Fig. 6f). However, GSEA pathway analysis supported significant upregulation of apoptotic pathways in IL-17RA-deficient FRC (Fig. 6f). Immunofluorescence staining confirmed significantly increased frequencies of cells that were positive for the apoptosis markers TUNEL and active caspase 3 in dLN of immunized Il17ra^–/– mice compared to wild-type (Fig. 6g, h, Supplementary Fig. 6g, h). As further confirmation of the FRC-intrinsic role of IL-17 in maintaining FRC viability, there were significantly increased frequencies of apoptotic FRC in dLN from immunized FRC^ΔIl17ra compared to controls (Fig. 6i, Supplementary Fig. 6i). Taken together, these data indicate a critical role for IL-17 in promoting survival and continued proliferation of activated FRC.

IL-17 drives metabolic reprogramming in proliferating FRC

Proliferation requires increased energy, and insufficient nutrient availability can be a cause of halted cell cycle and increased apoptosis in proliferating cells. Nutrient stress, including low glucose, leads to induction of autophagy through activation of AMPK³⁴. Il17ra^−/− FRC had increased expression of genes regulating autophagy (Fig. 7a), corresponding with increased activation of AMPK (Fig. 7b). GSK3β phosphorylation can also be an indicator of glucose uptake and glycolysis³⁵, and IL-17 signaling has previously been associated with regulation of GSK3β activity³². Indeed, Il17ra^−/− FRC had decreased phosphorylation of GSK3β (Fig. 7c), further supporting a possible defect in energy metabolism in absence of IL-17 signaling. We therefore directly assessed the ability of FRC to take up glucose in vivo by injecting the fluorescent glucose analog 2-NBDG on day 12 post-immunization. FRC from immunized LN had significantly increased 2-NBDG uptake compared to naïve controls (Fig. 7d, e Supplementary Fig. 7a), while FRC from immunized Il17ra^–/–, as well as FRC^ΔIl17ra LN and spleen, showed a striking defect in 2-NBDG uptake (Fig. 7d-g, Supplementary Fig. 7b). These data show that IL-17 signaling is required to increase the capacity for glucose uptake in proliferating FRC.

To determine the functional metabolic capacity of FRC activated in presence or absence of IL-17, we performed Seahorse extracellular flux analysis on pooled LN and spleen FRC from immunized wild-type and Il17ra^–/– mice and compared to naïve wild-type controls. Corresponding to increased glucose uptake, aerobic glycolysis (as assessed by extracellular acidification rate (ECAR)), was increased following immunization in wild-type but not Il17ra^−/− FRC (Fig. 7h). Similarly, wild-type FRC from immunized mice showed a strong increase in basal oxygen consumption rate (OCR), indicating enhanced oxidative phosphorylation (OXPHOS) (Fig. 7i), and correspondingly had a high spare respiratory capacity (SRC) compared to naïve FRC (Fig. 7i, j). In contrast, FRC from immunized Il17ra^−/− LN had relatively lower basal OXPHOS rates with minimal SRC, more similar to naïve FRC (Fig. 7i ,j). Gene expression data supported the IL-17RA-dependent increases in metabolic activity: compared to wild-type, FRC from Il17ra^−/− immunized LN had low expression of the inflammation-induced glycolytic enzyme Hk2, and the rate limiting enzyme of mitochondrial fatty acid oxidation Cpt1a, along with several subunits of mitochondrial complex I NADH:ubiquitinone oxidoreductase (NDUF) (Fig. 7k). However, mitochondrial mass per FRC was not dependent on IL-17RA (not shown). We further confirmed that Cpt1a protein expression was reduced when IL-17RA was specifically deleted in FRC (Fig. 7l and Supplementary Fig. 7c). These data thus demonstrate that IL-17 signaling is required for activated FRC to increase their glucose uptake, glycolysis and particularly mitochondrial bioenergetic activity, corresponding with the requirement for IL-17 signaling in promoting successful proliferation of FRC.

IL-17 promotes metabolic changes through IκBζ

The data so far clearly implicate FRC-intrinsic IL-17 signaling in driving metabolic changes. However, FRC isolated from naive LN and stimulated in vitro with IL-17 did not increase their glucose uptake, as assessed by 2-NBDG (Fig. 8a). In contrast, dLN FRC from mice immunized 6 days previously did increase 2-NBDG uptake in response to IL-17 stimulation (Fig. 8b). These data therefore suggest that IL-17 modulation of glucose absorption requires that cells have first received ‘priming’ signals in an inflamed LN.

We then tested the converse question: does prior IL-17 signaling in vivo alter the threshold for glucose uptake in response to distinct stimuli? Lipopolysaccharide (LPS), a prototypical TLR agonist known to drive glycolysis in myeloid cells, strongly increased glucose uptake in wild-type FRC from immunized LN, but not from naïve LN (Fig. 8c). Interestingly, FRC from immunized Il17ra^−/− dLN did respond to LPS, but with significantly lower rates of glucose uptake compared to wild-type FRC (Fig. 8c), supporting the idea that FRC receiving activating signals that render them responsive to further stimuli, but require IL-17 to reach full bioenergetic potential.

One intriguing link between the IL-17 and LPS signaling pathways is that they both induce IκBζ, a co-activator of the NF-κB signal transduction pathway, to drive a subset of target inflammatory genes³²,³⁶. Nfkbiz, which encodes IκBζ, was accordingly decreased in FRC from immunized Il17ra^−/− mice compared to wild-type controls (Fig. 6a). IL-17 stimulation induced Nfkbiz expression in FRC from day 6 primed LN, as expected, and Nfkbiz expression was effectively depleted by siRNA (Fig. 8d). IL-17-induced expression of Fn1 and Col1a1 were unaffected by Nfkbiz deletion, confirming that IL-17 signaling was intact and that IκBζ does not regulate all IL-17 target genes (Fig. 8e, f). However, IL-17-stimulated glucose uptake was significantly decreased in Nfkbiz-deleted FRC (Fig. 8g). Furthermore, IL-17 induced Cpt1a expression in an Nfkbiz-dependent manner (Fig. 8h). Therefore we conclude that IL-17 directs metabolic shifts in activated FRC through induction of the transcriptional co-activator IκBζ, leading to increased glucose uptake and expression of rate-limiting enzymes for mitochondrial oxidative phosphorylation.

Mice

The following mice were used: C57BL/6, Il17a^−/−, OT-II, Il17ra^{fl/fl 51}mice (Jackson Laboratory), Act1^−/− (Traf3ip2^−/−)mice⁵², CCL19Cre mice (EMMA repository, Infrafrontier), Il23r^−/− mice⁵³, Il17ra^−/− (Amgen, Inc), Regnase1^+/– (Zc3h12a^+/–) mice⁵⁴. All experiments included age- and sex-matched controls. Mice were housed under SPF conditions in an AAALAC-approved facility. Protocols were approved by the University of Pittsburgh IACUC and adhered to guidelines in the Guide for the Care and Use of Laboratory Animals of the NIH.

Experimental Autoimmune Encephalomyelitis

Mice were immunized subcutaneously in 2 sites on the back with 100 μg myelin oligodendrocyte glycoprotein (MOG) peptide (35–55) (Biosynthesis) emulsified with Complete Freund’s Adjuvant (CFA) with M. tuberculosis strain H37Ra (DIFCO Laboratories). Mice also received 100 ng pertussis toxin (List Biological Laboratories) i.p. on day 0 and day 2. Mice were assessed daily and scored as follows: 1, flaccid tail; 2, impaired righting reflex and hindlimb weakness; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, hindlimb paralysis with partial forelimb paralysis; 6, moribund. For IL-17 neutralization studies, mice were treated i.p. with 100 μg of anti-IL-17A (clone 17F3, BioXCell) or mouse IgG1 isotype control anitbody (clone MOPC-21, BioXCell) every 3 days starting at day of EAE immunization till day 12 of immunization.

OT-II transfer

Wild-type or Il23r^−/− recipient mice received 1 × 10⁵ Il23r^−/− or Il23r^+/+ OT-II CD4⁺ T cells i.p. 1 day before being immunized subcutaneously in the flank with 100 μg OVA (323–339) in CFA.

DSS colitis

Mice received 2.5% DSS (36,000–50,000 M.W.; MP Biomedicals) in their drinking water for 7 days, followed by 3 days of distilled water without DSS. Control animals received distilled water for the entire period. Mice were monitored daily for body weight and sacrificed on day 10 for MLN isolation.

qPCR

RNA isolated with RNeasy Mini Kits (QIAGEN), cDNA generated with Superscript III First Strand kits (Invitrogen), followed by real-time RT-PCR (qPCR) using SYBR Green Master mix with ROX (Invitrogen) and RT² qPCR Primers (QIAGEN) on a 7300 Real Time instrument (Applied Biosystems). Gene expression normalized to GAPDH.

FRC preparation for flow cytometry

LNs, and spleens where indicated, were incubated in digestion medium (RPMI, 0.1 mg/ml DNase I (Invitrogen), 0.1–0.2 mg/ml liberase (Roche) and 0.8 mg/ml dispase (Roche)¹⁹. Collected single-cell suspensions were filtered. Spleen samples underwent red blood cell lysis. For flow cytometry, cells were stained with Ghostdye510 viability dye (Tonbo Biosciences) in PBS, followed by labeling with CD45 (30-F11, Invitrogen), CD45.2 (104, BD Biosciences), PDPN (8.1.1, Biolegend), CD31 (MEC 13.3, BD Biosciences) and Ki67 (SolA15, eBioscience), Madcam-1 (MECA-89, BD), CD21/CD35 (Clone: 7G6, BD), Cpt1a (8F6AE9, Abcam), CD4 (GK1.5, Invitrogen), B220 (RA3–6B2, BD), IL-17RA (PAJ-17R, Invitrogen) and CountBright™ Absolute Counting Beads (Molecular Probes). For detection of apoptosis, FRC were stained with FITC Active Caspase-3 Apoptosis Kit (BD Biosciences), as per manufacturer’s protocol. For in vivo cell cycle analysis, mice were injected i.p. with 1 mg of bromodeoxyuridine (BrdU flow kit; BD Biosciences) 24 h before sacrifice. Data acquired with a FACS Fortessa (BD Biosciences), analyzed using FlowJo (Tree Star).

Histology and Immunocytochemistry

Sections from frozen LNs were fixed in 4% paraformaldehyde, stained with Masson Trichrome (Sigma) to detect collagen deposition according to manufacturer’s instructions; Primary antibodies used for immunofluorescence staining: ERTR7 (Santacruz; sc73355), Fibronectin (Thermoscientific; FBN11), CD4 (BioLegend; GK1.5), B220 (BioLegend; RA3–6B2) and cleaved caspase-3 (Cell Signaling; D175), TUNEL staining performed using apoptosis detection kit (Millipore). Images acquired with EVOS FL Auto microscope (Life Technologies). TUNEL⁺ cells enumerated in 6–10 randomly selected high-powered fields (400X) per slide.

RNA sequencing and GSEA

Live CD45^–CD31^–PDPN⁺ FRC from wild-type and Il17ra^−/− mice (4/group, day 12 post-immunization) were sorted on a FACSAria directly into SmartSeq low-input RNA kit lysis buffer. DNA libraries were prepared (Nextera XT kit) and RNA-Sequencing was performed on Illumina NextSeq500 by Health Sciences Sequencing Core at University of Pittsburgh. Raw sequence reads were cleaned for adapter sequences using Trimmomatic with default parameters ⁵⁵. Trimmed reads were mapped onto mouse genome build mm9 using TopHat2.1.1 and gene expression values (FPKM; fragments per kilobase exon per million mapped reads) calculated using Cufflinks2.2.1 ⁵⁶. Unbiased hierarchical clustering of differentially expressed genes with P < 0.05 calculated using Partek software. Relative expression shown in heatmaps was calculated as fpkm for each sample divided by mean expression of that gene in all samples. Gene set enrichment analysis (GSEA) from the Broad Institute (http://www.broad.mit.edu/gsea) was used to calculate enrichment of genes in each set. Cell cycle and apoptosis pathway gene lists were assembled using Qiagen’s SABiosciences pathway PCR arrays.

Antibody ELISA

Total IgG concentrations in serum detected by mouse IgG ELISA kit (Invitrogen). For detection of MOG-specific antibody in serum, 96-well-plates were coated overnight with recombinant MOG protein (AnaSpec) (10 μg/ml), blocked for 2 h, then incubated with sera (serial dilutions starting from 1:100) for 3 h. After washing, antibody bound to rMOG were detected by HRP-labeled anti-IgG antibody, and developed using mouse IgG ELISA kit (Invitrogen), O.D. values of 1:333 dilution were normalized relative to standard curve for anti-IgG for each experiment, since standards for anti-MOG IgG are not commercially available.

FRC isolation

LNs and spleens digested as described above. Resulting cell suspension was incubated with anti-CD31-APC (MEC13.3; BioLegend) and anti-CD45-APC (30-F11; BioLegend), followed by anti-APC selection cocktail and magnetic particles (EasySep; StemCell Technologies). After negative selection (MACS column, Miltenyi Biotech), the flow-through containing FRC was further incubated with anti-PDPN-PE (8.1.1; BioLegend) and anti-PE selection cocktail and magnetic particles (EasySep; StemCell Technologies) followed by positive selection. This typically resulted in 80–95% CD45^–PDPN⁺CD31^– FRCs.

Immunoblotting

CD45^–CD31^– cells isolated from LNs as described above were immediately lysed on ice in lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 2 mM NaF and 0.01% NP40) containing 1 mM sodium orthovanadate (phosphatase inhibitor), 50 mM PMSF, and protease inhibitor cocktail (Calbiochem). Samples were boiled in 4× sample buffer (Bio-Rad) and immunoblotted using anti-Phospho-AMPKα and anti-AMPKα (Cell Signaling Technology).

Seahorse metabolic assay

FRC were plated on Cell-Tak coated Seahorse culture plates (50,000 cells/well) in DMEM with 25 mM glucose, 1% BSA, 1 mM pyruvate, 2 mM glutamine, and analyzed using a Seahorse XFe96 (Agilent). EACR (Basal extracellular acidification) and OCR (oxygen consumption rates) for 160 min, as cells were stimulated with oligomycin (2 mM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP); 0.5 mM, 2-deoxyglucose (100 mM) and rotenone/antimycin A (100 mM) to obtain maximal and control OCR values. SPR (Spare respiratory capacity) measured as the difference between basal and maximal OCR values before and after addition of FCCP.

siRNA transfection

Isolated FRC were rested overnight then transfected with 50nM ON-TARGETplus SMARTpool siRNAs targeting Nfkbiz or scrambled control (Dharmacon) in DharmaFECT Reagent 1 (Dharmacon) for 24 h before stimulation with 200ng/ml IL-17 (Peprotech) for 12hr.

Glucose uptake

In vivo, 500 nM of 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (Cayman Chemicals) was injected via tail vein 30 min prior to sacrifice. In vitro, 2-NBDG was added for final 30 min. of culture. Uptake of 2-NBDG (a fluorescent glucose analog) was then assessed by flow cytometry. Glucose uptake following LPS stimulation ex vivo measured by Glucose Uptake-Glo™ Assay kit (Promega) according to the manufacturer’s instructions.

Statistics

Experimental results were analyzed for significance using ONE-WAY ANOVA (for multiple groups) or Student’s t-test, except EAE clinical data analyzed by Mann-Whitney test on each day of scoring. Statistical analyses were performed using GraphPad Prism. P values are shown as *, P <0.05, **, P < 0.01, and ***, P < 0.001, where statistical significance was found, and all data are represented as mean ± standard deviation.