The FGF/FGFR system in the microglial neuroinflammation with Borrelia burgdorferi: intersectionality with other neurological conditions

Background Lyme neuroborreliosis, caused by the bacterium Borrelia burgdorferi affects both the central and peripheral nervous systems (CNS, PNS). The CNS manifestations, especially at later stages, can mimic/cause many other neurological conditions including psychiatric disorders, dementia, and others, with a likely neuroinflammatory basis. The pathogenic mechanisms associated with Lyme neuroborreliosis, however, are not fully understood. Methods In this study, using cultures of primary rhesus microglia, we explored the roles of several fibroblast growth factor receptors (FGFRs) and fibroblast growth factors (FGFs) in neuroinflammation associated with live B. burgdorferi exposure. FGFR specific siRNA and inhibitors, custom antibody arrays, ELISAs, immunofluorescence and microscopy were used to comprehensively analyze the roles of these molecules in microglial neuroinflammation due to B. burgdorferi. Results FGFR1- 3 expressions were upregulated in microglia in response to B. burgdorferi. Inhibition of FGFR 1, 2 and 3 signaling using siRNA and three different inhibitors showed that FGFR signaling is proinflammatory in response to the Lyme disease bacterium. FGFR1 activation also contributed to non-viable B. burgdorferi mediated neuroinflammation. Analysis of the B. burgdorferi conditioned microglial medium by a custom antibody array showed that several FGFs are induced by the live bacterium including FGF6, FGF10 and FGF12, which in turn induce IL-6 and/or IL-8 in a dose dependent manner, indicating a proinflammatory nature. To our knowledge, this is also the first-ever described role for FGF6 and FGF12 in CNS neuroinflammation. FGF23 upregulation, in addition, was observed in response to the Lyme disease bacterium. B. burgdorferi exposure also downregulated many FGFs including FGF 5,7, 9, 11,13, 16, 20 and 21. Some of the upregulated FGFs have been implicated in major depressive disorder or dementia development, while the downregulated ones have been demonstrated to have protective roles in epilepsy, Parkinson’s disease, Alzheimer’s disease, spinal cord injury, blood-brain barrier stability, and others. Conclusions In this study we show that FGFRs and FGFs are novel mediators of inflammatory pathogenesis in Lyme neuroborreliosis. It is likely that an unresolved, long-term (neuro)-Lyme infection can contribute to the development of other neurologic conditions in susceptible individuals either by augmenting pathogenic FGFs or by suppressing ameliorative FGFs or both.


INTRODUCTION
the same medium supplemented with 0.5 ng/mL granulocyte macrophage colony stimulating 126 factor (GM-CSF, Millipore Sigma), to the required multiplicity of infection (MOI).

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Isolation and culture of primary microglia 129 Primary microglia were isolated from frontal cortex tissues of rhesus macaques (Macaca mulatta) 130 as described previously [22]. Briefly, brain tissues were obtained from un-inoculated young 131 animals from the breeding colony that were euthanized due to injury or persistent idiopathic 132 diarrhea. Euthanasia protocols, all performed by veterinarians, were approved by the Tulane 133 Institutional Animal Care and Use Committee (Tulane IACUC). The leptomeningeal blood vessels 134 and the leptomeninges were removed first with fine tweezers, followed by mincing of the tissue 135 with scalpels. The finely minced tissue was then subjected to enzymatic digestion with 0.25% for 20 minutes. Following digestion, the tissue was centrifuged at 335 x g, for 10 minutes, upper 138 layer of cells removed and filtered through a 20µm Nitex filter. The filtrate was resuspended in 139 DMEM: F12 supplemented with 10% FBS, 1% penicillin-streptomycin and 0.5 ng/mL  The aggregate cultures were seeded in T-75 flasks and incubated at 37C, 5% CO2. Medium was 141 changed every four days for about 4 weeks, prior to harvesting of microglia. Microglia were 142 isolated by vigorous tapping of the sides of the T-75 flasks, counted and seeded at the desired 143 density. Typical yield of microglia was between 90-95%, unless otherwise stated. Microglial 144 identity was verified by microglial marker Iba1 (1:10 to 1:25-mouse monoclonal #sc-32725, Santa 145 Cruz Biotechnology; 1:100-rabbit polyclonal, #019-19741, FujiFilm Wako Pure Chemical Corp., 146 Richmond, VA), as well as relative cellular size. All cell assays were conducted 2-3 days after 147 seeding. Microglia were isolated from 9 frontal cortex tissues, obtained from animals ranging in 148 age from 1.21 to 6.26, through the multi-year course of this study. Silencing of the FGFR transcripts by siRNA was carried out as follows. Microglia were seeded on 152 24-well plates at a density of ~2 x 10 4 / well. Cells were allowed to adhere for 48h (37C, 5% CO2), 153 after which medium was removed and replaced with 100 µL antibiotic-free medium. siRNA-154 transfection reagent complexes were generated using 2 µL HiPerfect transfection reagent (Qiagen,155 Germantown, MD) and 25-50 nM siRNA (non-specific control siRNA (sc-37007) or FGFR 156 specific (FGFR1/Flg-sc-29316, FGFR2/Bek-sc-29218, FGFR3-sc-29314); Santa Cruz 157 Biotechnology) in antibiotic and serum-free medium. The complexes were allowed to incubate at 158 room temperature for 30 minutes, and 100 µL of the complex was added to each well. Cells 159 containing the transfection complexes were incubated at 37C, 5% CO2 for 6h, followed by 160 8 addition of 400 µL of antibiotic-free medium. After a further 18h incubation, B. burgdorferi (MOI 161 10:1) or medium alone was added. Cells were incubated for an additional 24h, prior to collection 162 of supernatants (3000 rpm, 10 minutes at 4C).

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Infection assays with FGFR inhibitors 165 Microglia were seeded on 24-well plates or 4-well chamber slides at a density of ~2 x10 4 cells/well. 166 After 48h, cells were pretreated with specific FGFR inhibitors or solvent control (dimethyl 167 sulfoxide (DMSO)) for about 2 h. The medium was discarded and fresh medium without 168 antibiotics containing B. burgdorferi at an MOI of 10:1 was added, followed by addition of 169 inhibitors or DMSO. Medium only group served as controls. After 24h at 37C, 5% CO2, 170 supernatants were collected as before and stored at -20C until analysis. The following inhibitors 171 were used-FGFR1 inhibitor PD166866 (#341608-Millipore Sigma); FGFR1-3 (and likely

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To determine whether secreted factors trigger FGFR activation, supernatants after infection 175 assays were collected as before. They were thawed, re-centrifuged, and filtered through a 0.22 µm 176 filter and applied to freshly cultured microglia from the same tissue grown on chamber slides.

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Cells were fixed after 24 h for immunofluorescence. Microglial-conditioned medium without the 178 bacteria was similarly collected and used as a negative control.

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To determine the effect of FGFs on inflammatory mediator production, various doses of 180 specific FGFs were added to fresh microglia for 24h and supernatants, and cells analyzed as before.     Primary rhesus microglia were exposed to live B. burgdorferi for 24h and analyzed for FGFR1, 2, 250 and 3 expressions by immunofluorescence. The results are shown in Fig. 1. Fig.1a shows the 251 percent of microglia derived from three different animal tissues, respectively, that express specific 252 FGFRs. Expression levels varied among tissues, but were significantly higher than medium alone 253 controls across all tissues. Immunofluorescence photographs of FGFR1, FGFR2 and FGFR3 254 expression in microglia in response to B. burgdorferi is shown in Fig.1b, and the relative increase 255 in expression over medium controls for FGFR2 and FGFR3 is shown in Fig.1c. Fig.1c also shows 256 that FGFR2 and 3 expressions (green) is confined to microglia. This is shown through Iba1 staining 257 (red). Other than Iba1 as a marker for microglial specificity, confirmation was also through the     We next looked at whether B. burgdorferi-conditioned medium can induce expression of FGFR1 332 or pFGFR1. As seen in Fig. 4a, supernatants obtained from B. burgdorferi exposed cells were able 333 to activate FGFR1 and pFGFR1, while the supernatants obtained from medium alone controls did values were in single digits (not shown), and was not considered to be a real upregulation, but an 341 artifact of fold-change. FGF17, 18 and 19 did not show any distinct pattern, while FGFs 342 4, 5,7,8,9,11,13-1B, 16,20,21   Specific FGFs are expressed in primary rhesus microglia in response to B. burgdorferi 354 We next sought to verify some of the antibody array data with additional lines of evidence. As we 355 were mostly interested in factors that likely induced FGFR expression, we focused on the 356 upregulated FGFs. These were FGF6, 10, 12, and FGF23. Fig. 5a shows upregulated expression 357 of FGF6, FGF10 FGF12 and FGF23 in microglial cells in response to B. burgdorferi exposure, as 358 verified through immunofluorescence, using antibodies from a different company. Fig.5b shows 359 confocal microscopy of FGF staining in microglial cells, stained for Iba1. Staining was seen along 360 the surface indicating possible engagement with receptors, or at least localized there. Since ELISA 361 assays require a substantial volume of sample materials, we verified only specific FGFs using 362 supernatants from B. burgdorferi or medium exposed microglia. FGF6 secretion was additionally 363 verified by ELISA (supplemental material 6 (SM6). FGF12 was only seen by immunofluorescence 364 and not by ELISA. But overall, there was consensus in terms of specificity of induced FGFs with 365 the antibody array. On a technical note, FGF6 was only detected by ELISA when fresh media with 366 fresh serum was used in experiments and detected quickly. Dilution agents also affected detection 367 by ELISA, with PBS being better than standard diluents. The latter reduced detection by 368 approximately 50%. Surprisingly, addition of protease inhibitor phenylmethylsulphonyl fluoride 369 (PMSF) (1mM) lowered the detection levels as well.  Increasing doses of FGF6 either increased inflammatory mediator output or remained almost the 379 same. Lower doses of FGF10 (10 ng/ml or 30 ng/ml) did not have any specific effect on 380 cytokine/chemokine levels, but at higher doses (≥50 ng/ml) significantly upregulated IL-8 but not 381 IL-6. FGF12 at lower doses (5 ng/ml) significantly induced IL-6 and IL-8, and also at higher doses 382 (50 ng/ml) but the levels induced were much lower than those induced at 5 ng/ml. This showed 383 that FGFs have proinflammatory effects but in a dose dependent manner. This proinflammatory 384 effect was also seen when FGFs were added in combination. FGF6, 10, and 12 at 5 ng/ml, 20 ng/ml 385 18 and 5 ng/ml respectively or FGF6, 10, and 12 at 25 ng/ml,60 ng/ml,25 ng/ml respectively, elicited 386 significantly elevated IL-6 and IL-8 compared to controls with no FGFs (not shown). 387 We next confirmed that the effect of FGFs, particularly for FGF6 was through FGFR1. 388 Fig.6b shows upregulation of pFGFR1 in Iba1 stained microglia in response to FGF6, and that 389 inhibition of FGFR1 by PD166866 downregulated the FGF6 mediated upregulation of IL-6 and 390 IL8 (Fig. 6c). We show in this study using primary rhesus microglia, a rarely available resource, that members 398 of the FGF/FGFR system are novel mediators of pathogenesis in Lyme neuroborreliosis. We show 399 here that FGFRs 1,2 and 3 are activated in response to B. burgdorferi (Fig. 1) 416 We also show that secreted factors in the supernatants activate FGFRs, particularly FGFR1 417 (Fig. 4). And that several FGFs especially FGF6, secreted into the supernatant can activate the 418 FGFR1 pathway (Fig. 6b and 6c). However, the effect with B. burgdorferi supernatant alone was 419 much more profound than that with FGF6 alone (Fig. 5 and Fig.6b) or in combination with other 420 FGFs (FGF10, 12, not shown). It is possible that FGF23, which we did not test, might contribute 421 in activating FGFR1. But it is also likely that non-FGFs activate this pathway. One type of such and others, which in-turn contribute to IL6 and IL-8 levels. An overview of the data obtained from 451 this study is shown in Fig.7a Lower doses did not have any significant effect, while higher doses (≥ 50 ng/ml) significantly 467 induced IL-8, but had no significant effect on IL-6. Since 100 ng/ml was used in BV2 cells, it is 468 not clear if its protective at very high doses, or if its due to species difference, or cell line effects.

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It should be noted that deletion of an FGF gene may result in a completely different phenotype 470 than when its levels are modulated. Therefore, functions attributed to FGFs due to gene deletions 471 may or may not reflect disease pathogenesis where its levels can vary.

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FGF12 gene expression has been shown to be induced in BV2 microglia in response to 473 LPS [51], however no role for it has been described until this study. The main focus of FGF12 474 studies has been with its genetic alterations and associated epileptic changes through its ability to 475 bind voltage-gated sodium ion channels [52,53]. Its expression has also been shown to be elevated interesting anomaly is that FGF12 (along with FGF11,13, and 14) is considered intracellular. A 478 pioneering study in human embryonic kidney cells (HEK 293, epithelial morphology) showed that 479 transfection of the cells with FGF12 gene caused accumulation of the protein in the nucleus with 480 no detectable secretion [55]. In our study, microglial supernatants were analyzed by the antibody 481 array and FGF12 was found to be elevated in the extracellular environment. Confocal microscopy 482 also showed likely surface location of FGF12 in microglia (Fig. 5b). However, we could not detect 483 FGF12 in the supernatants by ELISA. So we cannot confirm whether FGF12 is actually secreted 484 outside, like interleukins. It is possible it is secreted but surface located and not truly in the 485 extracellular environment. Our hypotheses for this anomaly between array and ELISA are 1) As 486 the array is much more sensitive than ELISA, intracellular FGF12 was detected by the array due 487 to possible breach of cellular contents, or presence of some cells in the supernatants. 2) FGF12 is 488 secreted, and as the array procedure biotinylates the proteins prior to detection, FGFs are stabilized 489 and better detected. In ELISA assays due to long incubations prior to detection, the natural 490 confirmation destabilizes quickly and hence is not detected. The latter hypothesis could be tested 491 by ELISA of the cell lysates, but await tissue availability. More studies in other primary cell types 492 are needed to clarify this issue, and not just immortalized cells. The intracellular class of FGFs or 493 FGF homologous factors (FHFs) as they are known, were also thought to not activate FGFRs [56]. 494 Recent studies shown that is not the case [57] and in our study cells did respond to exogenous 495 addition of FGF12 in inducing IL-6 and IL-8, and is possibly through FGFRs as well. Therefore, 496 the characterization of these factors is far from complete. This is also the first study (to our 497 knowledge) to show a role for FGF12 in the brain glial cells.

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FGF23, an endocrine hormone secreted by osteocytes is required for maintaining 499 phosphate homeostasis. Due to this function, it has long known for its role in chronic kidney  With respect to the other FGFs that were downregulated in response to B. burgdorferi 507 exposure, only some of the salient ones will be discussed here. In terms of modulating 508 neuroinflammatory mediators per se, not much data exist for much of the FGFs. FGF20 was shown 509 to be protective in blood brain barrier disruption by upregulating tight junction proteins, increasing  as AD or PD is unclear and remains to be tested using single factorial approach in appropriate 551 animal models. As microglia share similar functionality with macrophages, we expect similar FGF 552 modulations in the periphery also. Since this study utilized a single glial cell type to study 553 FGF/FGFR system, we hope to conduct follow-up studies in vivo in relevant animal models. We 554 also hope to assess the FGF system in human Lyme disease patients to correlate specific FGFs 555 with symptomology. As FGFR1 also contributed towards neuroinflammation mediated by non-556 viable B. burgdorferi, it poses an attractive target for anti-inflammatory treatments in antibiotic 557 refractive conditions. In conclusion, in this study we show a novel molecular basis for 558 neuroinflammation associated with Lyme neuroborreliosis and its sequelae.   To test the hypothesis that secreted factors activate the FGFR1 pathway, 24h supernatants from 896 microglia exposed to either B. burgdorferi or medium alone were collected, filtered and added to 897 35 fresh microglial cells from the same tissue for an additional 24 h. Cells were fixed and analyzed 898 for FGFR1 or pFGFR1 by immunofluorescence as before. a) shows the activation of FGFR1 or 899 pFGFR1 (both green) by B. burgdorferi-exposed microglial conditioned media, indicating 900 activation of this pathway through secreted factors. Bar represents 50 µm. As FGFs are the likely 901 ligands for FGFR1 activation, supernatants from microglia derived from 4 different brain tissues 902 were analyzed for FGF secretion by a custom antibody Array (b). Experiment (Exp.) 1: A-Bb  FGFR2 and FGFR1 [85]. As FGF6, 10 and 12 only activated IL-6 and/or IL-8, but the inhibition 946 of FGFR1 individually by siRNA downregulated IL-6, IL-8 as well as MCP-1, it is likely that 947 other than FGF23, non-FGF molecules present in the supernatant also likely activate this receptor.

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It should be noted that only autocrine effects of FGF binding FGFRs in microglia are shown. It is 949 possible that some paracrine effects on other glial cells also occur and will be tested in future