TNFR2 Deficiency Acts in Concert with Gut Microbiota To Precipitate Spontaneous Sex-Biased Central Nervous System Demyelinating Autoimmune Disease This information is current as of September 25, 2021. Patrick G. Miller, Michael B. Bonn, Craig L. Franklin, Aaron C. Ericsson and Susan C. McKarns J Immunol published online 16 October 2015 http://www.jimmunol.org/content/early/2015/10/16/jimmun ol.1501664 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 16, 2015, doi:10.4049/jimmunol.1501664 The Journal of Immunology

TNFR2 Deficiency Acts in Concert with Gut Microbiota To Precipitate Spontaneous Sex-Biased Central Nervous System Demyelinating Autoimmune Disease

Patrick G. Miller,* Michael B. Bonn,* Craig L. Franklin,† Aaron C. Ericsson,† and Susan C. McKarns*,‡

TNF-a antagonists provide benefit to patients with inflammatory autoimmune disorders such as Crohn’s disease, rheumatoid arthritis, and ankylosing spondylitis. However, TNF antagonism unexplainably exacerbates CNS autoimmunity, including mul- tiple sclerosis and neuromyelitis optica. The underlying mechanisms remain enigmatic. We demonstrate that TNFR2 deficiency results in female-biased spontaneous autoimmune CNS demyelination in myelin oligodendrocyte glycoprotein–specific 2D2 TCR 2/2 transgenic mice. Disease in TNFR2 2D2 mice was associated with CNS infiltration of T and B cells as well as increased Downloaded from production of myelin oligodendrocyte glycoprotein–specific IL-17, IFN-g, and IgG2b. Attenuated disease in TNF2/2 2D2 mice relative to TNFR22/2 2D2 mice identified distinctive roles for TNFR1 and TNFR2. Oral antibiotic treatment eliminated spon- taneous autoimmunity in TNFR22/2 2D2 mice to suggest role for gut microbiota. Illumina sequencing of fecal 16S rRNA identified a distinct microbiota profile in male TNFR22/2 2D2 that was associated with disease protection. Akkermansia muciniphila, Sutterella sp., Oscillospira sp., Bacteroides acidifaciens, and Anaeroplasma sp. were selectively more abundant in male TNFR22/2

2D2 mice. In contrast, Bacteroides sp., Bacteroides uniformis,andParabacteroides sp. were more abundant in affected female http://www.jimmunol.org/ TNFR22/2 2D2 mice, suggesting a role in disease causation. Overall, TNFR2 blockade appears to disrupt commensal bacteria–host immune symbiosis to reveal autoimmune demyelination in genetically susceptible mice. Under this paradigm, microbes likely contribute to an individual’s response to anti-TNF therapy. This model provides a foundation for host immune–microbiota- directed measures for the prevention and treatment of CNS-demyelinating autoimmune disorders. The Journal of Immu- nology, 2015, 195: 000–000.

umor necrosis factor a antagonists provide significant transverse myelitis (4, 5). Continuation of anti-TNF therapy after benefit in the treatment of autoimmune inflammatory the development of neurologic symptoms is associated with per- T disorders such as Crohn’s disease, rheumatoid arthritis, manent disease, whereas discontinuation of the therapy usually by guest on September 25, 2021 and ankylosing spondylitis. In contrast, despite a correlation be- results in complete or partial recovery from neurologic deficits. tween the level of TNF and disease severity (1–3), anti-TNF The etiology of CNS demyelinating inflammatory disorders re- agents paradoxically exacerbate CNS demyelinating autoim- mains poorly understood. However, in accordance with clinical mune disorders, including optic neuritis, multiple sclerosis (MS), observations, anti-TNF therapy is currently contraindicated in neuromyelitis optica (NMO), Guillain–Barre´ syndrome, and patients with a pre-existing or family history of CNS demyelin- ating inflammation. Optic neuritis, an acute demyelinating disease of the optic nerve, *Laboratory of TGF-b Biology, Epigenetics, and Cytokine Regulation, Center for Cellular and Molecular Immunology, Department of Surgery, University of Missouri can rapidly lead to blindness within days and is often one of the first School of Medicine, Columbia, MO 65212; †Department of Veterinary Pathobiology, symptoms of chronic incurable CNS demyelinating autoimmune ‡ University of Missouri, Columbia, MO 65201; and Department of Microbiology and diseases, including MS and NMO. MS and NMO each manifests as Immunology, University of Missouri, Columbia, MO 65212 a heterogeneous relapsing–remitting neurodegenerative disorder in ORCID: 0000-0001-6746-4908 (S.C.M.). which periods of demyelination, in response to migration of Received for publication July 27, 2015. Accepted for publication September 20, activated lymphocytes across the blood–brain barrier and attack 2015. on oligodendrocytes and neuronal processes, are followed by pe- This work was supported by National Institutes of Health Grant R01 ES022966 (to S.C.M.) and a University of Missouri Richard Wallace Faculty Incentive Grant Award riods of recovery and repair of the damaged myelin (6, 7). MS (to S.C.M.). affects the brain, optic nerves, and spinal cord. In contrast, NMO The sequences presented in this article have been submitted to the National Center characteristically targets optic nerves and the spinal cord. Both for Biotechnology Information Sequence Read Achive database (http://www.ncbi. diseases typically present with varying degrees of sensory motor nlm.nih.gov/Traces/sra) under accession number PRJNA289572. disturbances, bladder–bowel dysfunction, and visual loss. The Address correspondence and reprint requests to Dr. Susan C. McKarns, Department of Surgery, University of Missouri School of Medicine, M616 Medical Sciences development of MS and experimental autoimmune encephalo- Building, One Hospital Drive, Columbia, MO 65212. E-mail address: mckarnss@health. myelitis (EAE), an animal model of MS, has been mostly attrib- missouri.edu uted to the activation of autoreactive CD4+ T cells secreting IFN-g Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; AQP4, aquaporin (Th1 cells) and IL-17 (Th17 cells) (8, 9). Although their role is 4; EAE, experimental autoimmune encephalomyelitis; GA, glatirmer acetate; IBD, inflammatory bowel disease; LN, lymph node; MOG, myelin oligodendrocyte glycopro- poorly understood, B cells have also been implicated. Current tein; MS, multiple sclerosis; NMO, neuromyelitis optica; OTU, operational taxonomic evidence suggests that B cells are not required for myelin oligo- unit; PC, principal component; Teff, T effector cell; TMS, trimethoprim/sulfamethoxazole; dendrocyte glycoprotein (MOG) –induced EAE (10), but they Treg, T regulatory cell. 35–55 are required for EAE that is induced by human, but not mouse Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 or rat, MOG protein (11, 12). In contrast to EAE and MS,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501664 2 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY complement-activating autoantibodies against aquaporin 4 sp., Bacteroides acidifaciens, Anaeroplasma sp., and Sutterella sp. in (AQP4), the predominant CNS water channel, have been implicated in male TNFR22/2 2D2 mice suggest possible microbial influences on the induction of inflammatory demyelination in humans with NMO disease causation and protection, respectively. Increased IL-17, IFN-g, (13). More recently, AQP4-specific T cells exhibiting a Th17 bias and and IgG2b are consistent with autoreactive cell and humoral immune displaying cross-reactivity to the gut commensal bacteria Clostridium responses in the female TNFR22/2 2D2 mice. Overall, these data perfringens have also been implicated (14, 15). suggest that TNFR2 potentiates autoimmune reactions that are driven The underlying mechanisms for the unpredictable exacerbation by cross-reactivity between commensal microbiota and autoantigens of disease in response to TNF blockade remain enigmatic. How- in female mice. Understanding whether selective TNF blockade in- ever, it is now appreciated that TNF exists in two bioactive forms fluences the composition of the microbiota in the human gut may and signals through two distinct receptors (16, 17). TNFR1 is produce a paradigm shift for anti-TNF therapy in autoimmune dis- ubiquitously expressed, activated by soluble TNF, and promotes eases. This model provides a foundation for host immune–gut apoptosis and inflammation. In contrast, TNFR2 expression is microbiota-directed measures for the prevention and treatment of CNS mostly restricted to endothelial, hematopoietic, microglial, and demyelinating autoimmune disorders. some neuronal cells, is selectively activated in response to trans- membrane TNF, and promotes cell survival and proliferation, in- Materials and Methods cluding expansion of CD4+Foxp3+ T regulatory cells (Tregs) (5, 18– Mice 20). The gene encoding TNFR1 has recently been identified as an MS risk allele (21), thus suggesting that the onset of autoimmune TNF and closely linked lymphotoxin-a and lymphotoxin-b genes are lo- disease following nonselective TNF blockade may be a consequence cated within the MHC, and thus all mice used in these studies were ob- Downloaded from tained on the C57BL/6J (H-2b) background and crossbred to avoid genetic TNFR signaling diversity. Consistent with this hypothesis, TNF heterogeneity introduced through backcrossing (31). Homozygous plays dual roles during autoimmune demyelination, namely exac- TNFR22/2 C57BL/6J mice were purchased from The Jackson Laboratory erbation of inflammation at the onset of disease and facilitation of (Bar Harbor, ME) and bred with Foxp3gfp 2D2 TCR transgenic reporter gfp the resolution of inflammation and myelin repair during ongoing mice to generate MOG35–55-specific TNFR2-deficient Foxp3 reporter 2/2 disease (18, 22). In agreement, selective pharmacological inhibition mice (henceforth referred to as 2D2 TNFR2 mice). The 2D2 TCR mice were generated from a clone (2D2) that expresses a TCR combination of of soluble TNF prevents transcriptional changes associated with the http://www.jimmunol.org/ Va3.2 and Vb11 with specificity for the MOG35–55 peptide in the context onset of CNS inflammatory demyelination and protects against of MHC class II (I-Ab) (32, 33). The GFP expressed in these mice is not MOG-induced EAE (23). Therefore, it is conceivable that selective fused with Foxp3 but is concomitantly expressed from a bicistronic 2/2 blockade of TNFR1 may minimize undesirable side effects from mRNA, thus leaving Foxp3 intact and functional. TNF C57BL/6J mice were purchased from The Jackson Laboratory and bred with Foxp3gfp 2D2 anti-TNF therapy. mice to generate homozygous Foxp3gfp 2D2 TNF2/2 reporter (2D2 TNF2/2) EAE rarely develops spontaneously in mice, but it can be induced mice. Timed-pregnancy surrogate CD-1 female mice were purchased from by injection of myelin Ags or by adoptive transfer of CD4+ T cells Charles River Laboratories (Portage, MI). These mice were housed in the isolated from mice primed with myelin Ags. Disrupted homeostasis Area P10 barrier at Charles River Laboratories prior to purchase. After between encephalitogenic lymphocytes and Tregs plays a key role in arrival at the University of Missouri, they were housed in a separate limited-access quarantine room. All animals were bred and housed under by guest on September 25, 2021 the disease pathogenesis of both MS and EAE (24–26). Given that specific pathogen-free conditions in University of Missouri facilities that Tregs regulate MOG35–55-specific EAE and TNFR2 modulates Treg are accredited by the Association for Assessment and Accreditation of function, a genetic loss-of-function approach was used to study the Laboratory Animal Care International. All experimental procedures using influence of selective TNFR2 blockade on the control of autoreactive animals were approved by the University of Missouri Institutional Animal 2/2 Care and Use Committee and were performed in accordance with the T cells. Accordingly, C57BL/6J TNFR2 mice were crossbred Guide for the Use and Care of Laboratory Animals. with 2D2 Foxp3gfp reporter mice to generate 2D2 TNFR22/2 Foxp3gfp reporter mice (TNFR22/2 2D2) that recognize the CD4+ Clinical scoring of CNS demyelinating autoimmune disease b T cell epitope, MOG35–55, presented by MHC class II (I-A ). Un- Clinical paralysis was scored using the following scale: 0, clinically normal; 2 2 expectedly, TNFR2 / 2D2 mice demonstrated a breakdown in 1, decreased tail tone or weak tail only; 2, hindlimb weakness (paraparesis); functional tolerance of autoreactive T and B cells with development 3, hindlimb paralysis (paraplegia) and/or urinary incontinence; 4, weakness of highly penetrant fulminant spontaneous CNS demyelinating au- of forelimbs with paraparesis or paraplegia (quadriparesis) and/or atonic bladder; and 5, paralysis of all limbs (quadriplegia) toimmunity with a pronounced sex bias. Although highly implicated in the pathogenesis of NMO, B cells have only recently received Oral antibiotic administration attention as a major contributor to EAE, and the involvement of Trimethoprim/sulfamethoxazole (TMS) (MWI Animal Health, Chicago, B cells in the induction of spontaneous EAE is rare. Inflammation, IL) was added to drinking bottles at a targeted dosage of 80 mg/kg/d. demyelination, and neuronal loss in the optic nerves and spinal Water bottles were changed weekly. TMS has bactericidal activity cord of the diseased female TNFR22/2 2D2 mice are also against a broad spectrum of Gram-positive and Gram-negative bacteria, including Streptococcus pyogenes, Streptococcus agalactiae, Streptococ- consistent with NMO pathogenesis. We further correlate spon- 2/2 cus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, taneous demyelinating autoimmunity in female TNFR2 2D2 Listeria monocytogenes, Nocardia asteroides, Mycobacterium fortuitum, mice with increased IL-17, IFN-g,andIgG2bproductionand Escherichia coli, Shigella dysenteriae, Salmonella enterica, Klebsiella sex- and genotype-dependent changes in the composition of the pneumoniae, Enterobacter cloacae, Serratia marcescens, Proteus mira- gut microbiota. bilis, Stenotrophomonas maltophilia, Haemophilus influenzae, Pasteurella multocida, Bordetella pertussis, Brucella melitensis, Neisseria gonorrheae, Emerging data strongly suggest an interaction between the gastro- and Neisseria meningitidis. Sulfamethoxazole inhibits dihydrofolic intestinal system and CNS disorders. The composition of the gut acid synthesis, trimethoprim inhibits thymidine and DNA synthesis, microbiota varies between monozygotic twins (27) and influences the and together these two agents act synergistically to inhibit folic acid development of the immune system (28). The composition of the gut synthesis. commensal microbiota has recently been identified as an environ- Cross-foster rederivation mental factor that can trigger CNS demyelinating autoimmune disease Before, during, and after rederivation, barrier mice were housed in auto- (14, 29, 30). Increased abundance of Bacteroides sp., Bacteroides 2/2 claved microisolator cages with autoclaved compressed pelleted paper uniformis,andParabacteroides sp. in female TNFR2 2D2 mice bedding and nestlets with ad libitum access to irradiated diet (breeder diet, and increased abundance of Akkermansia muciniphila, Oscillospira LabDiet 5058; stock diet, LabDiet5053) and acidified, autoclaved water, The Journal of Immunology 3 under a 12:12-h light/dark cycle. Cross-fostering was carried out in a plus adjuvant for 10 d. Rat anti-mouse IgM (II/41), IgG1 (A85-3), IgG2a separate limited-access quarantine room. After rederivation, mice were (R11-89), and IgG2b (R9-91) capture Abs and rat anti-mouse IgM (R6-60.2), housed on ventilated racks (Thoren Industries, Hazleton, PA). All cages IgG1 (A85-1), IgG2a (R19-15), and IgG2b (R12-3) detection Abs were were changed in class II type A biosafety cabinets. Dedicated staff were purchased from BD Pharmingen. The absorbance (450 nm) minus back- assigned to barrier and quarantine rooms. Four TNFR2+/2 2D2 C57BL/6 3 ground (570 nm) of the colorimetric reactions was quantified using a BioTek TNFR2+/2 2D2 C57BL/6 breeding pairs were moved from barrier housing Synergy 4 spectrophotometry microplate reader. and mated in quarantine. Within 12 h after delivery, pups were removed from donor cages and placed with a CD-1 surrogate recipient litter that was Necropsy and sample collection born within 2 d of the transfer. Within 1 wk of cross-fostering, the re- cipient’s pups were removed so that the total number of pups in her cage Mice were euthanized by an inhaled overdose of carbon dioxide. Imme- was ,10. At weaning, cross-fostered pups were tested for Pasteurella diately after death, the thoracic cavity was opened and the heart located. A pneumotropica and Helicobacter sp., and each surrogate dam was sub- butterfly catheter was placed in the heart, and mice were perfused with mitted for a comprehensive necropsy profile. Two weeks after weaning, a saline until blood draining from incised jugular veins cleared. Mice were second Helicobacter PCR test was performed on all cross-fostered pups then perfused with 4% paraformaldehyde. The brain, spinal column before they were transferred to restricted-access barrier housing. In the (containing the spinal cord), spleen, LNs (cervical, inguinal, axillary, and barriers, sentinel testing was routinely performed to confirm that colonies mesenteric), pancreas, liver, and kidneys were removed and immersion remained free of opportunistic pathogens, including Bordetella bronchiseptica, fixed in paraformaldehyde for 48–72 h. The spinal column was decalcified cilia-associated respiratory bacillus, Citrobacter rodentium, Clostridium in formic acid in a sodium citrate buffer for 48–72 h. Sections of spleen, piliforme, Corynebacterium bovis, Corynebacterium kutscheri, Helicobacter LNs, pancreas, liver, and kidneys were paraffin embedded, and 7-mm- sp., Mycoplasma sp., P. pneumotropica, Pneumocystis carinii, Salmonella sp., thick, H&E-stained sections were prepared for histologic examination. Streptobacillus moniliformis,andS. pneumoniae; adventitious viruses, Brains were sectioned into four coronal sections and the decalcified spinal including H1, Hantaan, KRV, LCMV, MAD1, MNV, PVM, RCV/SDAV, cord was sectioned into 14 sections. These sections were paraffin em-

REO3, RMV, RPV, RTV, and Sendai viruses; intestinal protozoa, in- bedded and slides were prepared and stained with H&E. To further char- Downloaded from cluding Spironucleus muris, Giardia muris, Entamoeba muris, tricho- acterize lesions, selected sections were also stained with Luxol fast blue (myelin) and Bielschowsky (axon) stains and probed, by immunohisto- monads, and other large intestinal flagellates and amoebae; intestinal + + + parasites, including pinworms and tapeworms; and external parasites, chemistry, for CD3 (T cells), CD45R (B220) (B cells), and CD68 cells including all species of lice and mites. All testing, including sentinels, (macrophages). was performed by Charles River Laboratories and IDEXX BioResearch (Columbia, MO). Immunohistochemistry

Isolation of CNS mononuclear cells Five-micrometer-thick sections were mounted onto ProbeOn Plus micro- http://www.jimmunol.org/ scope slides (Fisher Scientific, Pittsburgh, PA). Sections were dewaxed in Mice were euthanized by inhaled carbon dioxide and perfused with cold xylene, rehydrated through graded concentrations of ethanol, and rinsed in PBS. Brains and spinal cords were minced and incubated with 0.2 U distilled water. Ag retrieval was performed by immersing sections in heated Liberase R1 (Roche, Basel, Switzerland) and 50 mg/ml DNase I 10 mM citrate buffer (pH 6.0) for 30 min and then allowed to cool to room (Invitrogen/Life Technologies, Grand Island, NY). Digested pieces were temperature. Slides were treated with 3% hydrogen peroxide (to inactivate filtered through 40-mm Sefar Nitex (Small Parts, Logansport, IN) to yield endogenous peroxidase activity) and rinsed prior to incubation in blocking single-cell suspensions. Mononuclear cells were isolated by a discontin- buffer with 5% BSA for 20 min to minimize nonspecific Ab binding. uous Percoll (GE Healthcare Life Sciences, Pittsburgh, PA) gradient (34). Sections were then incubated for 60 min at room temperature with each of the following Abs: anti-CD3 (rabbit polyclonal anti-CD3 at a 1:200 dilution Flow cytometry [clone G7; A0452], Dako, Carpenteria, CA), anti-CD45R (rat monoclonal anti-CD45R/B220 at a 1:500 dilution [RA3-6B2; RM6200], Invitrogen/Life by guest on September 25, 2021 Cells were incubated with anti-CD16/32 prior to staining to prevent non- Technologies), and anti-CD68 (rat monoclonal anti-CD68 at a 1:200 di- specific Ab capture by FcRs. Cells were then stained directly with con- lution [FA-11; MCA1957], AbD Serotec, Oxford, U.K.). Sections of mouse jugated Abs in FACS buffer (PBS containing 1% FCS, 0.05% EDTA, and spleen were similarly probed with all three Abs for positive control samples 0.02% sodium azide). 7-Aminoactinomycin D (7-AAD) was used to ex- whereas sections of experimental tissue were incubated with normal rabbit clude dead cells for data analyses. FACSCalibur (BD Biosciences) and and rat Abs as negative controls. Sections were rinsed and then incubated for FlowJo v8.8.7 (FlowJo Ashland, OR) software were used. 30 min with one of two detection protocols. Sections probed with anti-CD3 Cytokine and cytokine receptor determinations were incubated with a HRP-labeled polymer conjugated to anti-rabbit Abs. Sections probed with anti-CD45R and anti-CD68 were incubated with a To determine IFN-g and IL-17 secretion, cells isolated from CNS draining biotinylated rabbit anti-rat Ab (catalog no. E0464 at 1:200 dilution; Dako), lymph nodes (LNs) were adjusted to 5 3 106 cells/ml in RPMI 1640 rinsed, and incubated with HRP-labeled streptavidin (Dako) for 30 min. medium, containing 8.6% FCS, 4 mM glutamine, 1 mM sodium pyruvate, Bound Abs were visualized following incubation with 3,39-diaminobenzidine 0.1 mM MEM nonessential amino acids, 200 mg/ml penicillin, 200 mg/ml solution (0.05% with 0.015% H2O2 in PBS; Dako) for 3–5 min. Sections streptomycin, 25 mg/ml gentamicin, and 50 mM 2-ME and stimulated with were counterstained with Meyer’s hematoxylin, dehydrated, and cover- MOG35–55 (MEVGWYRSPFSRVVHLYRNGK) (AnaSpec, Fremont, CA) slipped for microscopic examination. in Costar 96-well round-bottom culture plates (Thermo Fisher Scientific, Waltham, MA) for 96 h. Cell-free supernatants were collected and cyto- DNA extraction kines were quantified by ELISA using Ready-SET-Go! ELISA kits (eBioscience, San Diego, CA). To determine TNF, TNFR1, and TNFR2 Two fresh fecal pellets were collected from each mouse directly into a 2-ml round-bottom tube containing lysis buffer adapted from Yu and Morrison concentrations, serum samples were collected and quantified using eBio- 2 science Ready-SET-Go! ELISA kits (TNF and TNFR2) or the mouse (35) and a 0.5-cm-diameter stainless steel bead and stored at 80˚C. TNFR1 DuoSet ELISA kit (R&D Systems, Minneapolis, MN). All cell- Following mechanical disruption using a TissueLyser II (Qiagen, Venlo, 2 the Netherlands), tubes were incubated at 70˚C for 15 min and then free culture supernatants and serum samples were stored at 80˚C until 3 use. The absorbance (450 nm) minus background (570 nm) of the color- centrifuged at 16,000 g for 5 min at room temperature. The supernatant imetric reactions was quantified using a Synergy 4 spectrophotometry was transferred to a clean 1.5-ml Eppendorf tube and mixed with am- microplate reader (BioTek, Winooski, VT). The lower limit of sensitivity monium acetate (10 M, 200 ml), incubated on ice for 5 min, and then centrifuged as above. Supernatant was mixed with one volume of chilled of the TNF assay was 8 pg/ml. The recognition of TNF by the Ready-SET- 2 Go! assay is not affected by 1000-fold excess soluble TNFR1 or TNFR2. isopropanol and incubated at 20˚C, while mixing, for 30 min, and then centrifuged at 16,000 3 g at 4˚C for 15 min. The supernatant was aspirated Detection of MOG-specific Abs by ELISA and the DNA pellet was washed three times with 70% ethanol and resuspended in Tris-EDTA (150 ml) with proteinase K (20 mg/ml, 15 ml) MOG35–55 (7 mg/ml) was coated onto 96-well plates and incubated overnight and buffer AL (200 ml, DNeasy blood and tissue kit, Qiagen). Following at 4˚C. The plates were washed and saturated with PBS/3% BSA. Sera incubation at 70˚C for 10 min, 100% ethanol (200 ml) was added and the (1:100 dilution) was added and incubated overnight at 4˚C. After extensive contents were transferred to a spin column (DNeasy kit) for DNA isolation washes, bound Ig was detected by ELISA, consisting of biotinylated isotype- according to the manufacturer’s instructions. DNA was eluted in Tris-HCl specific anti-mouse Ig and a HRP complex (eBioscience) with 3,39,5,59- (10 mM, 100 ml). The Quant-iT BR dsDNA reagent kit (Qubit, Life tetramethylbenzidine (eBioscience) as a color substrate (measured at 450 Technologies, Carlsbad, CA) and NanoDrop (Thermo Fisher Scientific) nm). Control serum was from a C57BL/6 mouse immunized with MOG35–55 were used to obtain the quantity and purity, respectively. 4 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY

16S rRNA library preparation and sequencing induced EAE. Because TNF blockade can unexplainably augment Fecal DNA was processed at the University of Missouri DNA Core Facility. MS, we selected the MOG35–55-specific 2D2 TCR transgenic mouse Bacterial 16S rDNA amplicons were constructed via amplification of the V4 model to test our hypothesis. We first validated that TNFR2 ex- + hypervariable region of the 16S rDNA gene using universal primers pression was indeed upregulated by MOG35–55-specific CD4 (U515F/806R) previously designed against the V4 region, flanked by T cells in response to cognate Ag. For these experiments, T cells Illumina (San Diego, CA) standard adapter sequences (36, 37); sequences from 2D2 Foxp3gfp reporter mice were stimulated with increasing are available at probeBase (38). A single forward primer and reverse primers with a unique 12-base index were used in all reactions. PCR re- concentrations of MOG35–55 for 66 h and cell surface TNFR2 ex- actions (50 ml) contained 100 ng genomic DNA, forward and reverse pression was quantified by flow cytometry (Fig. 1A). As expected, primers (0.2 mM each), dNTPs (200 mM each), and Phusion high-fidelity prior to activation, TNFR2 was constitutively expressed on the DNA polymerase (1 U). Thermal cycling parameters were 98˚C for 3 min; surface of Foxp3 gfp Tregs but not CD4+ T effector cells (Teffs). 25 cycles of 98˚C for 15 s, 50˚C for 30 s, and 72˚C for 30 s; followed by 72˚C for 7 min. Amplified product (5 ml) from each reaction was combined Following MOG35–55 activation, TNFR2 expression was upregu- and thoroughly mixed; pooled amplicons were purified by addition of lated by both Tregs and Teffs in an Ag concentration– (Fig. 1A) Axygen AxyPrep Mag PCR clean-up beads to an equal volume of 50 ml and time-dependent (Fig. 1B) manner. The level of TNFR2 ex- amplicons and incubated for 15 min at room temperature. Products were pression was elevated on Tregs obtained from LNs in comparison washed with 80% ethanol, air dried, resuspended in 32.5 ml EB buffer with splenic Tregs (Fig. 1A, 1B). TNFR2 is readily cleaved from (Qiagen) for 2 min at room temperature, and placed on a magnetic stand for an additional 5 min. The final amplicon pool was evaluated using the the cell surface by matrix metalloproteases, including TNF Fragment Analyzer automated electrophoresis system (Advanced Analyt- cleavage enzyme (also called ADAM17), and therefore we ex- ical, Ames, IA), quantified with a Qubit fluorometer using the Quant-iT HS panded our studies to ask whether decreased soluble TNFR2 as- dsDNA reagent kit (Invitrogen), and diluted according to MiSeq specifi- sociated with increased cell surface TNFR2 expression by Tregs in Downloaded from cations (Illumina). comparison with Teffs. For these experiments, LN and splenic Informatics analysis Teffs and Foxp3gfp Tregs were FACS purified (.95% purity) and Assembly, binning, and annotation of DNA sequences were performed at stimulated separately with plate-bound anti-TCRb plus soluble the University of Missouri Informatics Research Core Facility. Briefly, anti-CD28. At times thereafter, cells were harvested and processed contiguous sequences of DNAwere assembled using FLASH software (39), for quantification of membrane-bound TNFR2 by flow cytometry.

and contigs were culled when found to be short after trimming for a base Analyses of cell-free culture supernatants revealed greater pro- http://www.jimmunol.org/ , quality of 31. QIIME software v1.7 (40) was used to perform de novo duction of soluble TNFR2 by Foxp3gfp Tregs in comparison with and reference-based chimera detection and removal, and remaining contigs , were assigned to operational taxonomic units (OTUs) using a criterion of Teffs (Fig. 1C) (p 0.001). In contrast to unfractionated splenic 97% nucleotide identity. Taxonomy was assigned to selected OTUs using and LN cells (Fig. 1A, 1B), TNFR2 expression did not differ be- BLAST (41) against the Greengenes database (42) of 16S rRNA sequences tween purified LN and splenic Foxp3gfp Tregs (Fig. 1C, 1D). It and taxonomy. Samples yielding ,10,000 sequences were removed from remains to be determined whether enhanced TNF cleavage enzyme all analyses based on QIIME-generated rarefaction curves. Principal component analyses and loading plots were generated using a nonlinear activity augments TNFR2 shedding in spleens compared with LNs. iterative partial least square algorithm, implemented in a macro-enabled Collectively, these results show MOG35–55-specific induction of Excel worksheet provided by Hiroshi Tsugawa of the Riken Institute TNFR2 expression by Foxp3gfp Tregs and CD4+ Teffs. Further- (Wako, Japan). Mean Chao1 indices were calculated using QIIME- more, impaired shedding of membrane-bound TNFR2 does not by guest on September 25, 2021 generated rarefaction data, that is, iterative subsampling of sequence contribute to elevated cell surface TNFR2 expression on Foxp3gfp data at the maximum coverage attained by all samples. All microbiome + sequence data were uploaded to the National Center for Biotechnology Tregs compared with CD4 Teffs. Information Sequence Read Achive database (http://www.ncbi.nlm.nih. 2/2 gov/Traces/sra) under accession number PRJNA289572. For statistical Female, but not male, MOG35–55-specific TNFR2 2D2 mice analyses, a two-way ANOVA was used to test for differences between develop fulminant spontaneous inflammatory CNS groups in the relative abundance of taxa, overall richness, and the a-di- demyelination versity (Chao1) index, with sex and TCR (2D2 TCR+ versus 2D2 TCR2) as independent variables. Previous studies have demonstrated attenuated or exacerbated MOG-induced EAE with TNFR1 or TNFR2 deficiency, respec- Statistical analyses tively (44–46). In the present study, we used a genetic loss-of- All error bars represent mean 6 SEM. For comparisons in the relative function approach to determine whether selective ablation of abundance of individual taxa, richness, and a-diversity and identification TNFR2 signaling is sufficient to augment MOG35–55-specific of interactions between variables, two-way ANOVA was performed using CD4+ T cell autoreactivity. TNFR22/2 C57BL/6J mice were SigmaPlot 12.3 (Systat Software, San Jose, CA) with sex and TCR as gfp independent variables. Elsewhere, means were compared using either the crossbred with MOG35–55-specific Foxp3 2D2 reporter mice to 2/2 gfp two-tailed unpaired Student t test for single comparisons or ANOVA fol- generate TNFR2 2D2 Foxp3 mice (henceforth referred to as lowed by Student–Newman–Keuls, or in some cases Bonferroni, posttest TNFR22/2 2D2) with the expectation of exacerbated disease upon correction for multiple group comparisons using GraphPad InStat v.3 active induction of EAE. (GraphPad Software, La Jolla, CA). For all comparisons, p , 0.05 was considered to be significant. Unexpectedly, we observed spontaneous disease development in most female, but few male, TNFR22/2 2D2 mice. Following this initial observation, mice were monitored, beginning at 14 d of age, Results for clinical signs of spontaneous disease development. A striking Concentration- and time-dependent induction of TNFR2 by 2/2 gfp + difference between female and male TNFR2 2D2 mice was Foxp3 Tregs and CD4 T effector cells in response to observed. At birth, female and male TNFR22/2 2D2 mice were MOG35–55 indistinguishable from TNFR2+/+ 2D2 and TNFR22/2 2D2 Previously, we determined that CD4+ T cell–intrinsic TNFR2 littermates (Fig. 2A). However, 92% (59 of 64) of the female promotes Il2 expression (43). Because IL-2 is required for the TNFR22/2 2D2 mice eventually developed severe, nonremitting expansion and function of Tregs and impairs Th17 differentiation, paralyses between 29 and 79 d of age (Fig. 2B, 2C, Table I). It is we hypothesized that a loss of TNFR2 signaling may be sufficient not clear why disease developed earlier in some female mice than to break functional tolerance of autoreactive T cells. MOG35–55 is in others, but differences in the frequency of MOG35–55-specific a potent encephalitogenic peptide in C57BL/6 mice. Tregs have CD4+ T cells or 2D2 TCR expression by individual CD4+ T cells been implicated in the pathogenesis of MS and suppress MOG35–55- are possible contributing factors. Regardless of the age of the The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

+ + + FIGURE 1. Concentration- and time-dependent induction of TNFR2 by CD4 Foxp3 Tregs versus CD4 Teffs in response to MOG35–55 stimulation. (A) Flow cytometric analysis of concentration-dependent induction of cell surface TNFR2 expression on 7-AAD2CD4+ Foxp3gfp+ Tregs and 7-AAD2CD4+ Foxp3gfp2 Teffs from 2D2 Foxp3gfp reporter mice. Total LNs or splenic cells isolated from three adult (6–12 wk of age) female mice were pooled and 6 2 + stimulated (2 3 10 c/ml) with 0, 5, 10, or 20 mM MOG35–55 peptide for 66 h. Cells were then stained for CD4, 7-AAD, and TNFR2; then 7-AAD CD4 Foxp3gfp+ and 7-AAD2CD4+ Foxp3gfp2 cells were gated to quantify surface TNFR2 expression. Representative histograms (left) and mean fluorescence intensity (MFI) 6 SEM (n = 3 independent experiments) (right) of TNFR2 expression are shown. (B) Flow cytometric analysis of time-dependent induction of cell surface TNFR2 expression on 7-AAD2CD4+ Foxp3gfp+ Tregs and 7-AAD2CD4+ Foxp3gfp2 Teffs. Total LN or splenic cells isolated from three mice 6 were pooled and stimulated (2 3 10 cells/ml) with 10 mM MOG35–55 for 0, 8, 16, 32, 40, 48, and 66 h. Cells were then stained for CD4, 7-AAD, and TNFR2; then 7-AAD2CD4+ Foxp3gfp+ and 7-AAD2CD4+ Foxp3gfp2 cells were gated to quantify surface TNFR2 expression. A representative histogram of each time point for each population of gated cells is shown (left). The TNFR2 MFI (mean 6 SEM) of three independent experiments is shown (right). (C) Flow cytometric analysis of time-dependent induction of cell surface TNFR2 expression on FACS-purified CD4+ Foxp3gfp+ Tregs and CD4+ Foxp3gfp2 Teffs. Total LN or splenic cells were isolated from three mice, FACS purified (purity . 95%), and then stimulated (2 3 106 cells/ml) with 10 mg/ml plate- bound anti-TCRb (H57) and soluble anti-CD28 for 0, 12, 24, 40, and 56 h. Cells were collected, stained for CD4 and TNFR2, and 7-AAD2CD4+ Foxp3gfp+ and 7-AAD2CD4+ Foxp3gfp2 cells were gated for analyses. The TNFR2 MFI (mean 6 SEM) from three independent experiments is shown. (D) Time- dependent secretion of soluble TNFR2 by FACS-purified CD4+ Foxp3gfp+ Tregs and CD4+ Foxp3gfp2 Teffs. Soluble TNFR2 was quantified by ELISA. Secreted TNFR2 (ng/ml; mean 6 SEM) from three independent experiments is shown. *p , 0.05. mouse at disease onset, the severity of the disease in affected onset and decreased disease severity, as determined by the mean female mice rapidly progressed to hind- and forelimb paralyses, peak score, in the few male TNFR22/2 2D2 mice that did exhibit without remission, and mice were euthanized once they reached clinical signs (Table I), the disease was similar to female this stage. In contrast, only a few male TNFR22/2 2D2 littermates TNFR22/2 2D2 littermates. The incidence of spontaneous disease developed clinical signs (Fig. 2C). Except for a delay in disease in female TNFR2+/2 2D2 littermates (7.5%; n = 40) did not differ 6 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Female 2D2 TNFR22/2 mice develop fulminant spontaneous inflammatory demyelination. (A) Female C57BL6/J 2D2 TNFR22/2 mice appear normal at birth and at early age; shown are representative 14-d-old female TNFR22/2 and TNFR2+/+ mice. (B) Ninety-two percent of female 2D2 TNFR22/2 mice spontaneously developed disease between 28 and 79 d of age. Representative affected 65-d-old female 2D2 TNFR22/2 and unaffected age- and gender-matched 2D2 TNFR2+/+ mice are shown. (C) The cumulative incidence of 2D2 TNFR22/2 mice with clinical manifestations of disease with a score $2 (female 2D2 TNFR22/2, 59 of 64; male 2D2 TNFR22/2, 5 of 60; female 2D2 TNFR2+/+, 5 of 73; male 2D2 TNFR2+/+, 4 of 72). Remission of disease severity was not observed in any of the mice studied. (D) Soluble TNF (pg/ml) was determined in sera collected from 4- to 5-wk-old unaffected male and female 2D2 TNFR2+/+ and 2D2 TNFR22/2 mice by ELISA. The data are reported as means 6 SEM; n = 3 per group. (E) The cumulative incidence of 2D2 TNF2/2 mice with a score $2 (female, 5 of 63; male, 4 of 55). Remission of disease severity was not observed in any of the mice studied. (F) Soluble TNFR1 (pg/ml) was determined by ELISA from sera collected from the unaffected mice described in (D). The data are reported as means 6 SEM; n =3.(G) Soluble TNFR2 (pg/ml) was determined by ELISA from sera collected from the unaffected 2D2 TNFR2+/+ mice described in (D). The data are reported as means 6 SEM; n =3.(H) Representative paraffin-embedded spinal cord sections from unaffected female 2D2 TNFR22/2 mice 2 2 (top) or age- and gender-matched affected 2D2 TNFR2 / mice (bottom) stained with H&E, Luxol fast blue, or Bielschowsky (Figure legend continues) The Journal of Immunology 7 from TNFR2+/+ 2D2 controls (6.8%; n = 73), showing a lack of a ation. Eosinophils were scattered throughout the parenchyma of gene dosage effect. Collectively, these results demonstrate that the optic nerve, but they were rarely found in the spinal cord and genetic ablation of TNFR2 predisposes female, but not male, 2D2 were undetected in the brain (data not shown). CD3+ T cells and mice to develop a greater incidence of spontaneous disease. B220+ B cells were identified as the dominant inflammatory in- Because circulating levels of TNF are elevated in asymptomatic filtrates in the spinal cord and optic nerves of the symptomatic TNFR22/2 mice showing no signs of disease (Fig. 2D), and in- 2D2 TNFR22/2 mice (Fig. 2J, 2K, respectively). B220+ B cell creased TNF has been linked to autoimmunity, we next bred infiltrates were mostly confined to the perivascular space, with C57BL/6J TNF2/2 mice with 2D2 Foxp3gfp mice to generate fewer of these cells infiltrating into the parenchyma. CD68+ MOG35–55-specific mice that completely lacked TNF signaling. macrophages/microglia were also found scattered throughout the These mice were monitored for disease development for up to 6 parenchyma (Fig. 2J, 2K). Overall, these results suggest that mo after birth. As shown in Fig. 2E and Table I, the cumulative TNFR2 deficiency does not appear to alter the pattern of inflam- incidence of spontaneous disease in TNF2/2 2D2 mice did not matory demyelination observed in the 2D2 transgenic mice (7, increase above the background incidence observed in TNF+/+ 2D2 50), but rather it potentiates a remarkable sex bias increase in mice. A low incidence (4%) of spontaneous EAE has previously disease incidence (23-fold) and severity. Given the similar phe- been reported for 2D2 mice (32), but this is remarkably different notypes of TNFR22/2 2D2 and double-transgenic mice that ex- from the sex-biased 92% incidence observed in female TNFR22/2 press MOG-specific receptors on T and B cells (33, 47), genetic 2D2 mice. Spontaneous NMO-like disease has also been seen in deficiency of TNFR2 in 2D2 mice appears to be sufficient to 2D2 mice crossed with MOG35–55-specific Ig H chain knock-in overcome the requirement for the MOG-specific BCR transgene mice (IgHMOG), in which 20% of the peripheral B cells recognize for spontaneous autoimmune disease development (33, 47). De- Downloaded from the same autoantigen as 95% of the CD4+ T cells (33, 47). spite similarities between TNFR22/2 2D2 mice and the 2D2 3 However, TNFR22/2 2D2 mice differ strikingly from these MOGBCR mice, these two experimental models differ markedly models with respect to sex bias, cumulative incidence, and se- with regard to sexual dimorphic disease incidence and severity. verity of disease. The mortality rate of the female TNFR22/2 2D2 Overall, these results suggest that TNFR2 deletion is sufficient to mice that develop disease is 100%. break functional peripheral tolerance of autoreactive MOG-

Soluble TNF, but not TNFR1, concentrations were elevated in specific lymphocytes to yield spontaneous disease in female TCR http://www.jimmunol.org/ TNFR22/2 mice (Fig. 2D, 2F), but elevated soluble TNF levels 2D2 transgenic mice. 2/2 did not differ between male and female TNFR2 mice. In 2/2 agreement with our previous studies (48), the levels of soluble Ex vivo analysis of encephalitogenic T and B cells in TNFR2 TNFR2 were consistently greater than those of soluble TNFR1 2D2 mice (p , 0.001). Collectively, these data suggest that increased Infiltration of T and B cells into the CNS of TNFR22/2 2D2 mice was TNFR1 signaling alone does not account for the sex bias sponta- confirmed ex vivo by flow cytometry. Cells were isolated from the neous disease in female 2D22/2 mice. Taken together, our studies spinal cord, LNs, and spleen of unaffected (healthy) TNFR22/2 2D2 reveal a profound and previously unrecognized sex bias regulatory mice and compared with affected TNFR22/2 2D2 mice. Consistent role for TNFR2 in the development of autoimmune disease. with immunohistochemistry results, T and B cells were undetected in by guest on September 25, 2021 We next examined the optic nerves, brain, and spinal cord of the brain (data not shown). Optic nerves were not studied ex vivo. affected female 2D2 TNFR22/2 mice for pathological lesions CD4+ T and B cells were present in the spinal cords of unaffected as consistent with EAE. No gross lesions were observed upon visual well as affected TNFR22/2 2D2 mice (Fig. 3A, 3F), but they increased examination of any tissues, with the exception of the optic nerves. in the affected mice (p , 0.05). The ratio of spinal cord B cells/T cells In all cases, either one or both nerves were smaller in size com- was also increased in affected mice (unaffected, 0.8:1; affected, 1.4:1) pared with asymptomatic 2D2 TNFR22/2 and 2D2 TNFR2+/+ (Fig. 3A). The low number of spinal cord infiltrates in unaffected mice but similar to symptomatic 2D2 TNFR2+/+ mice (32). His- TNFR22/2 2D2 mice likely contributed to their lack of detection by tological examination identified extensive inflammation, demye- immunohistochemistry. In contrast to TNFR22/2 2D2 mice, B220+ lination, and axonal loss in the spinal cord (Fig. 2H) and optic B cells were not detected in the spinal cords of unaffected 2D2 mice nerves (Fig. 2I). The disease presented unilaterally in the optic (data not shown). The frequency of spinal cord CD4+ T cells nerves of some 2D2 TNFR22/2 mice but was bilateral in others. expressing the Vb11 TCR (p , 0.05) declined from 97% in unaf- Consistent with a predominant localization of MOG-mediated fected mice to 69% in affected mice (Fig. 3C, 3F). This reduction in + immune responses to the optic nerve and spinal cord (32, 49), Vb11 TCR frequency, and presumably MOG35–55 specificity, was we did not observe lesions in the brain. The pathological lesions in evident in Teffs as well as Foxp3 gfp Tregs. The frequency of Tregs, the spinal cord were multifocal and segmental, and they varied regardless of Vb11 specificity, increased in affected TNFR22/2 2D2 from mild perivascular lymphocyte cuffing to severe demyelin- mice compared with unaffected healthy cohorts.

staining to visualize inflammatory cell infiltration, demyelination, and axonal damage, respectively. H&E-stained infiltrates are evident in perivascular cuffs (arrowhead) and the parenchyma (arrow). Luxol fast blue shows limited loss of myelin in the central white matter (arrowhead), but extensive demyelination of peripheral white matter (arrow). Bielschowsky staining shows mild (arrowhead) to severe (arrow) axonal loss. Scale bars, 200 mm. (I) Representative paraffin-embedded optic nerve sections from unaffected female 2D2 TNFR22/2 mice (top) or age- and gender-matched affected 2D2 TNFR22/2 mice (bottom) stained with H&E, Luxol fast blue, or Bielschowsky staining demonstrate profound inflammation (left) and demyelination (middle) and moderate axonal loss (right), respectively. A representative monocyte (arrow) and eosinophil infiltrate (arrowhead) are indicated. Scale bars, 50 mm. (J) T cell (anti- CD3), B cell (anti-B220), and macrophage/microglia (anti-CD68) infiltration into the spinal cord of unaffected (top) or age- and gender-match affected (bottom) female 2D2 TNFR22/2 mice. Representative paraffin-embedded spinal cord sections are shown. Localization of cells in the perivascular cuffs of the spinal cord (arrowheads) and localization to the parenchyma (arrows) are shown. Scale bars, 200 mm. (K) T cell (anti-CD3), B cell (anti-B220), and macrophage/microglia (anti-CD68) infiltration into the optic nerves of unaffected (top) or age- and gender-match affected (bottom) female 2D2 TNFR22/2 mice. Representative paraffin-embedded optic nerve sections are shown with representative T cells and macrophages/microglia indicated (arrowheads). Scale bars, 50 mm. *p , 0.05. 8 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY

Table I. Summary of spontaneous clinical paralyses in C57BL/6 2D2 TNFR22/2 and TNF2/2 mice

Genotype Gender % Incidencea Mean Day of Onsetb Mean Peak Scorec 2D2 TNFR2/2 ♀ 92 (59/64) 48.6 6 1.5 3.88 6 0.04 2D2 TNFR2/2 ♂ 8.3 (5/60) 75.2 6 2.6 3.00 6 0.09 2D2 TNF2/2 ♀ 7.9 (5/63) 86.6 6 2.4 2.80 6 0.11 2D2 TNF2/2 ♂ 7.2 (4/55) 85.8 6 2.9 2.50 6 0.08 2D2 TNFR2/2 ♀ 7.5 (3/40) 87.7 6 1.1 2.33 6 0.09 2D2 TNFR2/2 ♂ 4.1 (1/24) 79.0 2 2D2 TNFR2/2 ♀ 6.8 (5/73) 87.0 6 1.7 2.40 6 0.06 2D2 TNFR2/2 ♂ 5.5 (4/72) ND ND aThe percentage incidence was calculated by dividing the number of mice that developed clinical paralyses within 6 mo of age by the total number of mice scored in the group. bThe mean day of onset (mean 6 SEM) was calculated by adding the first day of clinical signs of individual mice and dividing by the number of mice in the group, not including mice that did not develop EAE. cThe mean peak score (mean 6 SEM) was calculated by adding the peak scores of individual mice and dividing by the number of mice in each group. Mice that did not develop clinical symptoms of paralyses were not included in the analyses.

Within the CNS draining and nondraining LNs, the numbers of munity within the gut is established early in life, we hypothesized Downloaded from CD4+ Teffs (Vb112 and Vb11+) and B220+ B cells as well as the a role for gut microbiota in the precipitation of sex-biased auto- B/T cell ratio increased in affected versus unaffected TNFR22/2 immunity in TNFR22/2 2D2 mice. To determine whether disease 2D2 mice (Fig. 3A, 3F). Although the frequencies of Foxp3gfp susceptibility in female TNFR22/2 2D2 mice associated with a Tregs were comparable, the total numbers of lymph nodal Tregs change in gut microbiota composition, fecal samples were col- were elevated in affected versus unaffected TNFR22/2 2D2 mice lected from 4- to 8-wk-old male and female TNFR22/2 and (Fig. 3D–F). The frequency of splenic Foxp3gfp Tregs did not TNFR22/2 2D2 mice for Illumina-based sequencing of the http://www.jimmunol.org/ differ between affected and unaffected TNFR22/2 2D2 mice hypervariable V4 region of bacterial 16S rRNA genes. Male and (Fig. 3D, 3F). In the thymus, the frequency and number of CD4+ female mice were housed separately, but 2D2 TCR+ and 2D2 CD82 Foxp3gfp2, CD4+CD82 Foxp3gfp+, and CD4+CD8+ double- TCR2 littermates were cohoused. At fecal collection, none of the positive T cells were comparable between affected and unaffected female TNFR22/2 2D2 mice had clinical signs of disease, but all TNFR22/2 2D2 mice (p . 0.05) (Fig. 3B, 3F). of them eventually developed paralyses. No other mice in the study developed clinical signs of disease. After filtering, a total of Elevated ex vivo MOG -specific cytokine production and 35–55 4,846,601 quality reads comprising 18 different bacterial classes circulating Abs from nine phyla (Actinobacteria, Bacteroidetes, Cyanobacteria, by guest on September 25, 2021 To gain further insight into the mechanisms underlying the sex- Deferribacteres, Firmicutes, Proteobacteria, TM7, Tenericutes, biased spontaneous CNS-demyelinating autoimmunity in female and Verrucomicrobia) were detected in samples from 46 mice (13 2/2 + TNFR2 2D2 mice, we next examined ex vivo CD4 T cell re- female TNFR22/2 2D2, 14 female TNFR22/2, 9 male TNFR22/2 2 2 / 2/2 sponsiveness to MOG35–55 and circulating Abs. Female TNFR2 2D2, and 10 male TNFR2 mice) with an average coverage of 2/2 2D2 mice were compared with healthy male TNFR2 2D2 mice, 105,360 reads per sample. Microbial richness, as determined by +/+ +/+ female TNFR2 2D2 mice, and male TNFR2 2D2 mice. Cells the total number of OTUs detected, differed in a sex-independent 2/2 isolated from female TNFR2 2D2 mice produced significantly manner between TNFR22/2 (42.00 6 0.65, mean 6 SEM) and 2/2 more MOG35–55-dependent IFN-g and IL-17 (Fig. 4A). We next TNFR2 2D2 (45.74 6 0.73, mean 6 SEM) mice. Conversely, investigated the differentiation state of B cells from the affected a-diversity, estimated using the Chao1 index, revealed a restricted 2 2 / 2/2 TNFR2 2D2 mice. For these studies, MOG35–55-specific Abs microbial diversity in female TNFR2 mice compared with were quantified from sera collected from female and male TNFR22/2 2D2 mice (Fig. 5B). TNFR22/2 2D2 or 2D2 mice. Without immunization, female TNFR22/2 2D2 mice, regardless of whether they were affected or Sex- and TCR-dependent differences in the relative abundance of microbial taxa unaffected, had elevated titers of MOG35–55 IgG2b, MOG35–55 IgM, and total IgG2b (p , 0.05) (Fig. 4B, 4C). In contrast, We next characterized the taxonomic variation across groups at 2/2 MOG35–55 IgG1 titers were unaltered in female TNFR2 2D2 the phylum level. For all four experimental groups, Bacteroidetes mice. MOG35–55 IgG2a was not detected in any of the mice. Anti- and Firmicutes were the predominant phyla, and they ranged in IgG2b sera titers were comparable between 4-wk-old and 12-wk- abundance from 65 to 84% and from 13 to 29%, respectively old TNFR22/2 2D2 mice (data not shown). Taken together, ele- (Fig. 5C). Neither the abundance of Bacteroidetes or Firmicutes vated IFN-g, IL-17, and MOG35–55-specific IgG2b titers associate nor the ratio of Firmicutes/Bacteroidetes differed between any with spontaneous CNS demyelination in female TNFR22/2 2D2 of the groups (p . 0.05) (Fig. 5D). Sex-dependent differences mice. were detected in three of the remaining seven phyla (Proteo- bacteria, Tenericutes, and Verrucomicrobia), with male mice Antibiotic treatment reduces the incidence and severity of 2 2 harboring significantly greater proportions of microbes than did spontaneous CNS demyelination in TNFR2 / 2D2 mice females in all three phyla (p , 0.05), regardless of TCR ge- In response to an unexplainable production decline and onset of notype (Fig. 5E). early mortality in our barrier-housed breeding colonies, we initi- ated a 6-wk course of therapeutic TMS for opportunistic pathogen Relative abundance at taxonomic level of family, genus, and prophylaxis and observed a loss of disease in offspring born during species a 10-mo span (Fig. 5A), which was reversed by cross-fostering to When resolved to the OTU level, in which all sequences met a surrogate Harlan CD-1 recipients. Given that the microbial com- threshold of 97% nucleotide identity, several taxa differed between The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Ex vivo analysis of encephalitogenic T and B cells in TNFR22/2 2D2 mice. (A) Freshly isolated mononuclear cells from the spinal cord, CNS draining LNs (DLNs), or CNS non-DLNs (pooled axillary and brachial) of unaffected (top) or age- and gender-matched affected (bottom) 2D2 TNFR22/2 mice are compared. FACS plots of CD4+ T cells and B220+ B cells from 7-AAD2 lymphocyte-gated cells are shown. (B) Freshly isolated cells from the thymus of unaffected 2D2 TNFR22/2 mice (left) are compared with age-matched affected 2D2 TNFR22/2 mice (right). FACS plots of CD4+ T cells versus CD8+ T cells (top) and CD4+ versus Foxp3gfp+ Tregs (bottom) are shown. The cells shown in top panels are gated on 7-AAD2 lymphocytes. 2 2 The cells shown in the bottom panels are gated on CD4+ CD8 7-AAD lymphocytes. (C) Freshly isolated mononuclear cells (Figure legend continues) 10 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Elevated ex vivo MOG35–55-specific cytokine production and circulating Abs. (A) MOG35–35-specific Th1 and Th17 responses. Splenic or cervical LN cells (1 3 106) from affected 2D2 TNFR22/2 mice (n = 3) compared with unaffected age-matched 2D2 TNFR2+/+ mice (n = 4) were cultured with increasing concentrations (0, 0.1, 0.3, 1, 3, or 10 mg/ml) of MOG35–55 for 96 h. Cell-free supernatants were collected and IFN-g and IL-17 production were determined by ELISA. Shown are the means 6 SEM. (B) MOG35–35-specific B cell responses were determined by ELISA. Relative concentrations are 2/2 shown of MOG35–55-specific sera Ig Abs (1:100 diluted serum) from female affected 2D2 TNFR2 mice (indicated as female KO; n = 5) and age- matched unaffected female TNFR2+/+ mice (indicated as female 2D2; n = 4 or 5) and unaffected male 2D2 TNFR22/2 (indicated as male KO; n =5) control mice as determined by isotype specific sandwich ELISA. Individual values and mean absorbance at OD of 450 nm are shown. Sera from MOG35–55- immunized C57BL/6 mice was used as a control: IgM, 0.27 6 0.05; IgG1, 0.88 6 0.11; IgG2a, 0.82 6 0. 02; and IgG2b, 1.97 6 0.04 (mean 6 SEM). (C) Total serum IgG2b was determined by ELISA. Sera collected from affected 2D2 TNFR22/2 mice was compared with unaffected female TNFR2+/+ mice and unaffected female and male 2D2 TNFR22/2 mice. Purified IgG2b was used in the ELISA to generate a curve for quantification. Mean values 6 SEM are indicated for each group. *p , 0.05. KO, knockout. groups (Table II). There were also several interactions detected compared with female TNFR22/2 2D2 mice (Fig. 6B). Anaero- suggesting that the influence of one variable was dependent on the plasma sp. and A. muciniphila were also more abundance in male status of the other variable. With regard to sex-dependent differ- TNFR22/2 2D2 mice compared with male TNFR22/2 mice (p , ences, microbes in the genera Oscillospira and Sutterella were 0.001), but no TCR-dependent difference was detected in female present at greater relative abundance in males (p , 0.05) (Fig. 6A). mice (Fig. 6B). B. acidifaciens and B. ovatus were present at greater Similarly, B. acidifaciens, B. ovatus, Anaeroplasma sp., and levels in both male and female TNFR22/2 2D2 mice relative to A. muciniphila were more abundant in male TNFR22/2 2D2 mice TNFR22/2 mice. Bacteroides sp., B. uniformis,andParabacteroides

from the spinal cords of unaffected (top) or age- and gender-matched affected (bottom) 2D2 TNFR22/2 mice are indicated. FACS plots of Vb11 TCR and GFP expression from 7-AAD2 lymphocytes are shown. (D) Comparison of CD4 Foxp3gfp+ Tregs isolated form the DLNs (top) or spleen (bottom)of unaffected (left) and age-matched affected (right) 2D2 TNFR22/2 mice. FACS plots of CD4 and Foxp3 expression from 7-AAD2 lymphocytes are shown. (E) CD4+7-AAD2 gated T cells from (D) were further gated for Vb11 TCR expression. Shown are the frequencies of Vb11+ and Vb112 CD4+ Foxp3gfp2 Teffs and CD4+ Foxp3gfp+ Tregs representative of DLNs (top) or spleens (bottom) from unaffected (left) and age-matched affected (right) 2D2 TNFR22/2 mice. Plots shown are representative of three mice per genotype. (F) The graphs depict the numbers and frequencies of cells shown in (A)–(E). Mean values 6 SEM are indicated for each group. DLN, draining LN. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 5. Sex-specific microbiota profiles define disease-resistant male TNFR22/2 2D2 mice and disease-susceptible female TNFR22/2 2D2 mice. (A) The spontaneous development of paralyses in TNFR22/2 2D2 and 2D2 mice is lost following antibiotic (oral TMS) treatment. Both males and females were affected. The bar graph indicates the percentage of disease incidence. Shown are the numbers of mice that succumb to disease relative to the total numbers of mice studied. All mice were monitored for disease onset for a minimum of 90 d of age. (B) Bacterial 16S rRNA amplicon sequencing of fecal samples was used to define the microbiome profiles of female TNFR22/2 2D2 (red, n = 13), female TNFR22/2 (green, n = 14), male TNFR22/2 2D2 (blue, n = 9), and male TNFR22/2 2D2 (purple, n = 10) mice. Chao1 indices in fecal microbiota are shown. Statistical analysis was performed using a Mann– Whitney rank sum tests. (C) Relative abundance at the taxonomic level of phylum. Bar charts compare the fecal bacterial composition between unaffected male and female TNFR22/2 and TNFR22/2 2D2 mice. (D) Firmicutes/Bacteroidetes ratios were determined for each individual mouse by dividing percentage abundance of Firmicutes by percentage abundance of Bacteroidetes. The means 6 SEM of these determinations are shown. (E) Annotation at the level of phylum for each individual mouse is shown to compare the difference of bacterial abundance between groups. The solid line depicts the mean percentage abundance. *p , 0.05, **p , 0.01. 12 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY

Table II. Sex-biased taxa dominating the bacterial microbiota of TNFR22/2 2D2 mice

Phylum Class Order Family Genus Species Fold Increase Increased in Males Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides acidifaciens 1.81 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides ovatus 1.52 Firmicutes Clostridia Clostridiales Ruminococcaceae Oscillospira 1.53 Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobiaceae Akkermansia muciniphila 5.87 Tenericutes Mollicutes Anaeroplasmatales Anaeroplasmataceae Anaeroplasma 3.31 Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Sutterella 3.45 Increased in Females Bacteroidetes Bacteroidia Bacteroidales Porphyromonadaceae Parabacteroides 3.69 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides uniformis 7.65 Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides 1.38

sp.weremoreabundantinfemalemicecomparedwithmales the microbiota in the male TNFR22/2 2D2 that relates to disease (p , 0.05) (Fig. 6C). Turicibacter sp. and the family Clos- protection. tridiaceae were more abundant in female TNFR22/2 mice 2/2 relative to male TNFR2 mice (p , 0.01), but no difference Discussion Downloaded from 2 2 was detected between female and male TNFR2 / 2D2 mice TNF blockade has become a mainstay of therapy for inflammatory (Fig. 6D). bowel disease (IBD) and rheumatoid arthritis. However, TNF antagonists, including infliximab (Remicade), etanercept (Enbrel), Relative abundance of key bacterial players in intestinal and adalimumab (Humira), sometimes precipitate CNS autoim- immunity and host health mune demyelinating diseases such as NMO and MS. Why? Various

During the past decade, selected gut commensal bacteria, includ- theories, including failed antagonist entry into the CNS (52), http://www.jimmunol.org/ ing Candidatus arthromitus, Lactobacillus, Clostridium, B. fragilis, downregulated TNFR2-mediated immune suppression (53), and and Bifidobacterium, have been identified as key regulators of abrogated TNFR2-mediated remyelination (54) have been pro- immunity and host health (51). None of these bacteria differed posed. Although these postulates provide plausible explanations (p . 0.05) with respect to sex or genotype between male or female for the failure to treat demyelinating autoimmunity, they do not 2 2 2 2 TNFR2 / and TNFR2 / 2D2 mice. It is well accepted that in- explain the onset of new or worsened CNS demyelination that teractions between commensal bacteria are important for overall occurs in IBD or rheumatoid arthritis patients. In the present study, robustness. Therefore, it is possible that nonsignificant (p . 0.05) we provide one possible explanation. We present evidence to trends of these individual bacteria may contribute to a larger col- suggest that TNFR2 is key to maintain immunological tolerance lective effect. C. arthromitus, commonly called segmented fila- against commensal bacteria in mice that are at high genetic risk by guest on September 25, 2021 mentous bacteria, was detected in all groups of mice, but its for autoimmune disease. Taking into account the interactions be- abundance was not sex- or TCR-dependent (Fig. 7A). B. fragilis tween the immune system, the gut microbiota, and the nervous 2 2 2 2 was detected in most TNFR2 / 2D2 mice but in few TNFR2 / system, we hypothesize that TNFR2 blockade disrupts this sym- mice, and it was more abundant in female compared with male biotic relationship to unmask autoimmune demyelination in ge- 2 2 TNFR2 / 2D2 mice (Fig. 7B). Clostridium sp. were detected in netically susceptible individuals. Under this paradigm, individuals 2 2 all groups, with greater abundance in TNFR2 / 2D2 mice without respond differently to anti-TNF therapy, not because of their sex dependence (Fig. 7C). C. perfringens was detected in only one genes, but because of the microbes they carry. There are multiple of the 46 mice studied (Fig. 7C). Bifidobacterium was not detected direct and indirect pathways through which TNF might modulate 2 2 in any of the male TNFR2 / mice, but it was expressed in all immune–microbial–neuroendocrine communication to facilitate 2 2 male TNFR2 / 2D2 and most female mice (Fig. 7D). Lactoba- sex-biased CNS autoimmune demyelination. These include im- 2 2 cillus was detected in all mice with greater abundance in TNFR2 / mune activation, microbiota, and endocrine and neurotransmitter male mice (Fig. 7E). Coprococcus, previously associated with signaling. MS, was detected in all mice without either TCR or sex depen- Sexual dimorphism is characteristic of many autoimmune dis- dence (Fig. 7F). Dorea, another member of the Lachnospiraceae orders with disease incidence more often higher in females compared 2 2 family, was found in greater abundance in female TNFR2 / 2D2 with males. In humans, there is a significant female predominance in mice (Fig. 7F). both NMO and MS, with the female-to-male ratio being even greater

2/2 2/2 in NMO, ranging from 5:1 to 11:1 (55). The reasons for differences Gut microbiota in TNFR2 and TNFR2 2D2 mice between females and males in autoimmune disease susceptibility clusters according to sex and genotype remain an enigma. Sex hormones, the presence or absence of a To assess b-diversity among the gut microbiota and characterize second X chromosome, microbiota, and disruption at the blood– the differences between experimental groups, taking into account spinal cord and blood–brain barriers have been implicated as con- all taxa detected, principal component (PC) analysis was per- tributing factors (56–58). Although progress has been made in formed using a nonlinear iterative partial least squares algorithm. identifying the genetic determinants of autoimmunity (59), ad- Comparison of PC1 and PC2, explaining 21.40 and 18.52% var- vances in identifying how sex hormones, microbiota, and their in- iation, respectively, demonstrated a distinct separation of male teractions with the immune system influence gender-bias autoim- TNFR22/2 2D2 and TNFR22/2 mice (Fig. 8A) but not female munity remain limited. Realization that the environment is an even TNFR22/2 2D2 and TNFR22/2 mice. In contrast, when PC1 was greater factor than genes in an immune response (60) exemplifies visualized against PC3, explaining 13.07% variation, female the importance of unraveling these complex interactions. Our pres- TNFR22/2 2D2 and TNFR22/2 mice did separate along PC3 ent work does not explain fully the mechanisms for the sex dif- (Fig. 8B). Overall, these results identify a distinct composition of ferences in TNFR22/2 mice. However, our studies do provide a The Journal of Immunology 13 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Relative abundance at taxonomic level of family, genus, and species. 16S bacterial rRNA sequencing of fecal samples was used to define the microbiome profiles of female TNFR22/2 2D2 (red, n = 13), female TNFR22/2 (green, n = 14), male TNFR22/2 2D2 (blue, n =9),andmaleTNFR22/2 2D2 (purple, n =10)mice.(A) The microbes that were significantly more abundant in male mice relative to female mice are shown. (B) The microbes that were sig- nificantly more abundant in unaffected disease-resistant male TNFR22/2 2D2 mice relative to unaffected disease-susceptible female TNFR22/2 2D2 mice are shown. (C) The microbes that were significantly more abundant in female mice relative to male mice are shown. (D) The microbes that were significantly less abundant in unaffected disease-resistant male TNFR22/2 2D2 mice relative to unaffected disease-susceptible female TNFR22/2 2D2 mice are shown. Each datum point represents an individual mouse. The horizontal line indicates the mean of each group. The vertical line represents the SEM of each group. *p , 0.05, ***p , 0.001. foundation to explore sexual dimorphism in microbiota-targeted have a low incidence of spontaneous disease. Rather, TNFR2 approaches to treat CNS autoimmunity. In particular, we show deficiency in combination with autoreactive MOG35–55-specific that TNFR2 deficiency alone is not responsible for the observed T cells precipitates a nearly complete penetrance of disease in sex-related differences, as TNFR22/2 malesandfemalesboth female mice. The high disease incidence in sexually dimorphic 14 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY

FIGURE 7. Relative abundance of key bacterial players in intestinal immunity and host health. 16S bacterial rRNA sequencing of fecal samples was used to define the microbiome profiles of female TNFR22/2 2D2 (red, n = 13), female TNFR22/2 (green, n = 14), male TNFR22/2 2D2 (blue, n = 9), and male TNFR22/2 2D2 (purple, n = 10) mice. The relative abun- dance of (A) C. arthromitus,(B) B. fragilis, (C) Clostridium sp. and C. perfringens,(D) Bifidobacterium sp., (E) Lactobacillus sp., and (F) Coprococcus sp. and Dorea sp. are shown. Each datum point represents an in- dividual mouse. The horizontal line indi- cates the mean of each group. The vertical line represents the SEM of each group. *p , 0.05, **p , 0.01. Downloaded from

2 2 TNFR2 / 2D2 mice exceeds that of double-transgenic 2D2 3 been found in low abundance in IBD patients and has been shown MOG IgH mice that produce MOG35–55-specific B and T cells (33, to ameliorate experimental colitis, at least in part, by promoting 2/2 61). Resistance to disease development in male TNFR2 2D2, Treg differentiation and enhancing gut barrier integrity (74, 75). http://www.jimmunol.org/ both sexes of TNFR22/2,andTNF2/2 2D2 mice implicates sex, Bacteroidaceae, Ruminococcus, Lactobacillaceae, Clostridium, CNS Ag-reactive CD4+ T cells, and TNFR2 specificity as im- Coprococcus,andA. muciniphilia all share a similar capacity to portant factors in disease development. A role for microbiota is promote immune tolerance by targeting T cell differentiation and implicated by the loss of disease with antibiotic treatment and gut barrier integrity. An association between commensal bacteria segregation of microbiota profiles that interact with sex and and host immunity is less well established in NMO patients, but a genotype. Disease protection in male TNFR22/2 2D2 mice re- link is evident. For example, AQP4 is considered a primary au- lates to increased abundance of A. muciniphila, B. acidifaciens, toimmune target in NMO (76), and AQP4-specific T cells show B. ovatus, Anaeroplasma sp., Sutterella sp., and Oscillospira sp. cross-reactivity to a protein product of C. perfringens (14). Disease susceptibility in female TNFR22/2 2D2 mice correlates In our studies, A. muciniphilia was the most abundant bacteria that by guest on September 25, 2021 with increased Parabacteroides sp., B. uniformis,andBacteroides was selectively increased in protected male TNFR22/2 2D2 mice. sp. A distinct gut microbiota profile in TNFR22/2 2D2 mice seems A. muciniphilia, a Gram-negative bacteria, constitutes 3–5% of the to parallel male microbiome-mediated protection similar to that microbiome in healthy humans, is characterized as a mucin degrader, previously reported in the NOD type 1 diabetes model (62, 63). and also enhances gut barrier integrity (77). It remains to be deter- Elevated production of MOG35–55-specific IFN-g, IL-17, and IgG2b mined whether A. muciniphilia provides similar protection at blood– suggests the loss of functional T as well as B cell tolerance. On- brain or blood–spinal cord barriers. A. muciniphilia has also been going studies aim to explain the unexpectedly increased frequency implicated in the regulation of genes involved in host lipid metab- of CD4+ Foxp3+ Tregs with loss of functional immune tolerance. olism and inflammation, including histone deacetylases and perox- Infectious pathogenic viruses and bacteria have long been isome proliferator-activated receptor g (78, 79). Furthermore, speculated to contribute to human CNS autoimmune pathogenesis A. muciniphilia abundance has been inversely correlated with MS (64, 65). However, no etiologic or causal effect has been firmly (73), type 1 diabetes (80), type 2 diabetes (81), and metabolic established. In contrast, our results are consistent with numerous disorders associated with obesity (82), but positively correlated other more recent studies indicating that commensal, rather than with the neurologic disorder, autism (83). It is not known how pathogenic, bacteria facilitate CNS autoimmune demyelination TNFR2 deficiency impacts A. muciniphilia abundance, but intra- (29, 30, 66–68). Attenuated MOG-induced EAE development in cellular cross-talk between short-chain fatty acid and sex-hormone antibiotic-treated and germ-free mice has linked gut microbiota to signaling and gene regulation is one possibility. Sutterella, also CNS demyelination (29, 30). Segmented filamentous bacteria have selectively more abundant in male TNFR22/2 2D2 mice, has been been positively correlated with EAE (29). In contrast, B. fragilis, positively correlated with autism and Down syndrome (84), as Bifidobacterium sp., and some strains of Lactobacillus negatively well as degradation of secretory IgA (85). In addition to providing correlate with EAE (69–72). Evidence is now also emerging for a a first line of defense against enteric toxins and pathogens, in- role by commensal microbiota in the etiology of human MS. testinal secretory IgA, promotes Ag clearance, quenches bacterial Faecalibacterium (phylum Firmicutes) has been found in low virulence factors, and changes the composition of the intestinal abundance in untreated MS patients, and this was reversed by microbiota (86). Therefore, Sutterella-mediated protection in male glatimer acetate (GA) treatment (73). GA treatment was also shown TNFR22/2 2D2 mice is plausible. Less in known about how other to increase Bacteroidaceae, Ruminococcus, Lactobacillaceae, and bacteria found in high abundance in the male TNFR22/2 2D2 Clostridium in these MS patients. In contrast, vitamin D sup- mice may benefit health to provide protection against autoim- plementation increased Faecalibacterium, A. muciniphilia,and munity. These bacteria include, in order of increasing abundance, Coprococcus in untreated, but not GA-treated, MS patients. When B. acidifaciens, Oscillospira sp., Anaeroplasma sp., and B. ovatus. considering these microbes in disease etiology, Faecalibacterium With that being said, carbohydrate fermentation by Bacteroides prausnitzii, a highly abundant butyrate-producing commensal, has and other bacteria provide a significant proportion of the host’s The Journal of Immunology 15

Toward defining a mechanism, the intestine is an important site for peripheral lymphocyte development. Exposure to different gut microbiota products, including short-chain fatty acid and poly- saccharide A, may differentiate MOG-reactive CD4+ T cells into an autoreactive phenotype through molecular mimicry (89). It is also plausible that the induction of autoimmunity in the TNFR22/2 2D2 mice relies on a direct effect of gut microbiota on B cell function. B cell development in the intestinal mucosa is influ- enced by commensal bacteria, including Sutterella, and alterations to the gut microbiome may modulate B cell function (30, 70). B cell activation might be also be explained by the recruitment of MOG-specific B cells from the naive repertoire by autoreactive CD4+ T cells (61). MOG expression in the periphery, perhaps as debris from trafficking of activated CD4+ T cells, might also promote MOG-specific B cell expansion (90). In considering a mechanism for the unexpected Treg expansion, increased abundance of B. fragilis and Lactobacillus sp. in female TNFR22/2 2D2 mice is consistent microbial-mediated CD4+ Foxp3 + Treg differentiation (70,

91, 92). It remains to be determined whether Foxp3 + Treg function Downloaded from differs between male and female TNFR22/2 2D2 mice. Changes to the gut microbiome may be a consequence, and not a cause, of autoimmunity (93). For instance, it is plausible that fe- male TNFR22/2 2D2 mice have increased disease susceptibility as a direct consequence of estrogen-induced IFN-g expression and

Th1-mediated IgG2b class switching (94–96). Estrogen has been http://www.jimmunol.org/ also reported to suppress TNF production (97). However, we ob- served comparable levels of TNF in male and female mice to suggest that elevated concentrations of TNF alone are insufficient to link gut microbiota and spontaneous sexual dimorphic auto- immunity in 2D2 TNFR22/2 mice. Interestingly, male rheumatoid arthritis patients respond better than females to anti-TNF therapy (98–101), and female mice respond better than male mice to TNFR2-dependent cardioprotection (102). Therefore, a similar sex-bias role of TNFR2-mediated immune modulation may be by guest on September 25, 2021 applicable to other diseases such as myocarditis, colitis, and ar- thritis (103–105). Regardless of whether males are protected or females are sus- ceptible, our data demonstrate that genetic loss of TNFR2 in 2D2 FIGURE 8. Sex-specific microbiota profiles define disease-resistant male TNFR22/2 2D2 mice. PC analysis of gut microbial communities TCR mice is sufficient to break functional tolerance of both T cells detected in the fecal samples are shown from female TNFR22/2 (open red and B cells. Taken together, we propose disruption of immune circles), female TNFR22/2 2D2 (closed red circles), male TNFR22/2 modulation of the CNS–gut–microbe axis as a plausible expla- (open blue triangles), and male TNFR22/2 2D2 (closed blue triangles) nation for some of the dichotomous effects of nonselective TNF mice. (A) Depiction of PC1 versus PC2 separates male TNFR22/2 and blockade in patients. Our results shed new light on a growing body 2 2 TNFR2 / 2D2 mice. (B) Depiction of PC1 versus PC3 separates fe- of evidence suggesting that TNFR1-selective antagonism may 2/2 2/2 2/2 male TNFR2 and TNFR2 2D2 mice as well as male TNFR2 and enhance treatment of CNS demyelinating autoimmune disorders. 2/2 TNFR2 2D2 mice. Although the mechanistic basis remains unclear, the TNFR22/2 2D2 model provides a unique experimental paradigm to investi- daily energy requirements. Moreover, different colonization likely gate the involvement of microbiotia on T and B cell tolerance in reflects survival and functional interactions between commensal sexual dimorphic CNS autoimmune disorders. Lastly, gender bacteria within the intestine. For example, interactions among should be considered as a variable in the design and imple- acetate or butyrate producers, poly- or oligosaccharide de- mentation of microbiota-directed therapeutic measures to treat graders, or even sulfate-reducing bacteria and other microbes CNS autoimmunity. can be affected by the carbon sources available. In contrast to the males, the bacteria that were selectively more abundant in fe- Acknowledgments 2 2 male TNFR2 / 2D2 mice were all Bacteroidetes of the genera We sincerely thank Dr. Nancy H. Ruddle (Yale University) for the many en- Parabacteroid and Bacteroides, and studies to define their con- lightening discussions and numerous manuscript critiques; Drs. Lon Dixon, tribution to disease are ongoing. Previous studies have positively Erin O’Connor, and Dana Weir, from the University of Missouri Office of correlated F. prausnitzii, a potent butyrate producer and histone Animal Resources, for assistance with cross-fostering and animal care; Dr. deacetylase inhibitor, with Crohn’s disease (87) and type 2 di- Habib Zaghouani for sharing of mice and reagents; Nathan Bivens, from the University of Missouri DNA Core Facility, for Illumina sequencing; Scott abetes (81). Although Faecalibacterium sp. was undetected in Givan, Christopher Bottoms, and Bill Spollen, from the University of Mis- our study, Dorea, another Lachnospiraceae family member, was souri Bioinformatics Core Facility, for assistance with bioinformatics; Jacob 2/2 foundinhighabundanceinfemaleTNFR2 2D2 mice. Dorea Lucas, Azeem Khan, Wesley Reznicek, and Giedre Turner for excellent has also been linked to autoimmune and metabolic disorders technical assistance; and the staff of the University of Missouri Cell and (81, 88). Immunobiology Core Facility for flow cytometry and cell sorting. 16 TNFR2 AND MICROBES REGULATE CNS AUTOIMMUNITY

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