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DOI: 10.1038/s41467-020-17179-4
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Citation for published version (APA): Bibby, J., Purvis, H., Hayday, T., Cope, A., & Perucha, E. (2020). Cholesterol metabolism drives regulatory B cell IL-10 through provision of geranylgeranyl pyrophosphate. Nature Communications, 11, [3412 ]. https://doi.org/10.1038/s41467-020-17179-4
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Download date: 25. Sep. 2021 1 Cholesterol metabolism drives regulatory B cell IL-10 through
2 provision of geranylgeranyl pyrophosphate
3 Jack A. Bibby1,5*, Harriet A. Purvis1, Thomas Hayday1, Anita Chandra2, Klaus Okkenhaug2, Sofia
4 Rosenzweig3, Ivona Aksentijevich3, Michael Wood4, Helen J. Lachmann4, Claudia Kemper1,5,6,
5 Andrew P. Cope1,7†*, Esperanza Perucha1,7†*
6
7 Affiliations:
8 1Centre for Inflammation Biology and Cancer Immunology, School of Immunology and Microbial
9 Sciences, King’s College London, London SE1 1UL, UK.
10 2Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK.
11 3Inflammatory Disease Section, National Human Genome Research Institute, National Institutes of Health,
12 Bethesda, MD, 20892
13 4National Amyloidosis Centre, Division of Medicine, University College London and Royal Free Hospital
14 London NHS Foundation Trust, London, NW3 2PF, UK.
15 5Complement and Inflammation Research Section (CIRS), National Heart, Lung, and Blood Institute,
16 National Institutes of Health, Bethesda, MD 20892
17 6Institute for Systemic Inflammation Research, University of Lübeck, Lübeck, Germany.
18 7Centre for Rheumatic Diseases, King’s College London, London SE1 1UL, UK
19 *Correspondence: [email protected]; [email protected]; [email protected]
20 †jointly supervised
1 21 22 Abstract
23 Regulatory B cells restrict immune and inflammatory responses across a number of contexts. This
24 capacity is mediated primarily through the production of IL-10. Here we demonstrate that the
25 induction of a regulatory program in human B cells is dependent on a metabolic priming event driven
26 by cholesterol metabolism. Synthesis of the metabolic intermediate geranylgeranyl pyrophosphate
27 (GGPP) is required to specifically drive IL-10 production, and to attenuate Th1 responses.
28 Furthermore, GGPP-dependent protein modifications control signalling through PI3Kδ-AKT-GSK3,
29 which in turn promote BLIMP1-dependent IL-10 production. Inherited gene mutations in cholesterol
30 metabolism result in a severe autoinflammatory syndrome termed mevalonate kinase deficiency
31 (MKD). Consistent with our findings, B cells from MKD patients induce poor IL-10 responses and
32 are functionally impaired. Moreover, metabolic supplementation with GGPP is able to reverse this
33 defect. Collectively, our data define cholesterol metabolism as an integral metabolic pathway for the
34 optimal functioning of human IL-10 producing regulatory B cells.
2 35 Introduction
36 Immunosuppressive B cells form a critical component of the immune regulatory compartment 1,2. It is
37 thought that their suppressive capacity derives mainly from their ability to produce IL-10, and in the
38 absence of any lineage marker, this is considered a hallmark of regulatory B cells 3–6. Their functional
39 importance has been well described in mouse models of disease, demonstrating a potent regulatory
40 capacity across a number of contexts including infection, cancer, and autoimmune disease 3,7–10.
41 Comparable suppressive activity has been demonstrated in human regulatory B cells in vitro,
42 suggesting that these cells also contribute toward regulating inflammatory responses in humans 5,6. In
43 support of this, IL-10 producing B cells have been demonstrated to be numerically depleted or
44 functional impaired, ex vivo, in patients with inflammatory disease 5,11,12.
45
46 Despite the importance of IL-10 production by B cells, relatively little is known about the molecular
47 mechanisms that govern its expression. Typically, induction of a regulatory phenotype in both human
48 and mouse B cells has been achieved through ligation of TLR9 or CD40 5,6,13. Downstream, PI3K and
49 ERK signals appear to be important for IL-10 expression 14,15. This is in broad agreement with in vivo
50 data from mouse models suggesting that both TLR and CD40 signals are required for the induction of
51 IL-10 in response to inflammatory stimuli 3,13. However, precise details of the signaling cascades or
52 cellular profiles underpinning the induction of a regulatory program in B cells are poorly understood.
53
54 Control of immune cell metabolism is critical in regulating fundamental immunological processes
55 16,17. However, in comparison to innate cell and T cell lineages, there has been relatively little work
56 aimed at understanding the metabolic regulation of B cell biology 18–20. Furthermore, there is currently
57 no understanding toward the metabolic requirements of regulatory B cells. An emerging concept from
58 studies of regulatory T cells and M2 macrophages is their heightened reliance on lipid metabolism in
59 comparison to the metabolic requirements of inflammatory immune cell lineages. Much of this work
60 has focused on fatty acid oxidation, defining this as a central pathway that underpins polarization and
61 effector functions in regulatory cells 21. In contrast, the contribution of cholesterol metabolism (the
62 multi-step conversion of HMG-CoA to cholesterol) to immune function is less well characterized. Our
3 63 recent studies, as well as those of others, suggest that cholesterol metabolism plays a role in restricting
64 inflammatory responses 22–24. These data are consistent with patients carrying mutations in the
65 pathway, who develop severe and recurring autoinflammatory fevers, associated with dysregulated B
66 cell responses, manifest by elevated serum immunoglobulin levels 25.
67
68 Given the immunoregulatory associations with cholesterol metabolism, we set out to address its role
69 in IL-10 producing B cells. In doing so, we reveal that cholesterol metabolism is a central metabolic
70 pathway required for the induction of a regulatory phenotype in human B cells, and define the specific
71 metabolic intermediate of the pathway that mediates critical signaling events for the induction of a
72 regulatory B cell program. In vivo evidence for this specific metabolic requirement is provided by
73 virtue of defective regulatory responses of B cells derived from patients with inherited defects in
74 cholesterol metabolism.
75
76
4 77 Results
78 Cholesterol metabolism drives regulatory B cell function
79 Given that patients with dysregulated cholesterol metabolism generate severe systemic inflammation
80 and hyperactive B cell responses, we investigated the role of cholesterol metabolism in regulatory B
81 cell function (the metabolic pathway is depicted in Supplementary Figure 1). Consistent with previous
82 observations, TLR9 ligation in human B cells induces a potent IL-10 response (Figure 1a-b,
83 Supplementary Figure 2a-b), which mediates their regulatory phenotype 5,11. In agreement, TLR9
84 activated B cells were able to suppress Th1 induction in CD4+ T cells in vitro (protocol outlined in
85 Figure 1c). Neutralization of IL-10 resulted in a complete loss of suppression, confirming the
86 dependence on IL-10 in mediating this function (Figure 1d). In line with this, concentrations of IL-10
87 produced by B cells upon TLR9 stimulation (>1ngml-1, Figure 1b) were sufficient to directly suppress
88 CD4+ T cell IFNγ expression (Supplementary Figure 2c). In contrast, pre-treatment of B cells with the
89 HMG-CoA reductase inhibitor atorvastatin, which inhibits an early step of the cholesterol metabolic
90 pathway, abrogated this suppressive capacity. Furthermore, supplementation of exogenous
91 mevalonate reversed this defect, suggesting the requirement for a specific metabolite downstream of
92 HMG-CoA (Figure 1d).
93
94 The loss of suppressive capacity by IL-10 neutralization and HMG-CoA reductase inhibition
95 indicated that cholesterol metabolism could be directly regulating IL-10 production. Indeed, inhibition
96 of HMG-CoA reductase impaired the ability of B cells to produce IL-10, both at the mRNA and
97 protein level (Figure 1e-f, Supplementary Figure 3a-b). Once again, and consistent with metabolic
98 depletion downstream of HMG-CoA, exogenous mevalonate was able to reverse this defect (Figure
99 1e-f). In contrast to IL-10, we observed a concurrent preservation in the inflammatory response, as
100 determined by TNF and IFNγ protein, and IL6, and LTA gene expression (Figure 1g-h, Supplementary
101 Figure 3c-d). Together, these data indicated that cholesterol metabolism was critical in mediating IL-
102 10 expression, and therefore the anti-inflammatory function of human B cells.
103
5 104 Cholesterol metabolism drives IL-10 independent of phenotype
105 We next aimed to understand how cholesterol metabolism was able to mediate IL-10 production.
106 Certain populations of human B cells have been proposed as primary producers of IL-10. The most
107 well characterized of these are CD24hiCD27+ (B10) and CD24hiCD38hi B cells 5,6. In agreement with
108 previous observations, we observed that all populations measured (B10, CD24hiCD38hi, naïve,
109 memory, and plasmablast) contribute to the pool of IL-10 expressing cells to varying degrees after
110 stimulation with CpG (IL-10+ cells ranging from 1-12% of B cell populations, Supplementary Figure
111 4a-b). Furthermore, B10 and CD24hiCD38hi B cells produced higher levels (2-3-fold) of IL-10 in
112 response to TLR9 stimulation (Supplementary Figure 4b). Acquiring the capacity to produce IL-10
113 showed no dependence on proliferation, as IL-10 production was seen irrespective of proliferation
114 state (Supplementary Figure 4c). Following inhibition of HMG-CoA reductase we observed no
115 change in frequencies of B cell populations, viability, or cell surface markers (HLA-DR, CD86, or
116 CD40), excluding the possibility that perturbation of cholesterol metabolism was depleting specific B
117 cell subsets that possess a greater propensity to express IL-10 (Supplementary Figure 4d-f).
118 Furthermore, HMG-CoA reductase inhibition resulted in a 2-3-fold reduction in IL-10 expression
119 irrespective of B cell population (either naïve, memory, B10, or CD24hiCD38hi, Supplementary Figure
120 4g). Therefore, these data indicated a role for cholesterol metabolism in regulating IL-10 production
121 that is shared across B cell populations, rather than an effect on specific populations.
122
123 Cholesterol metabolism drives IL-10 via GGPP
124 To more precisely understand the mechanistic control by cholesterol metabolism, we next sought to
125 investigate if a specific pathway metabolite downstream of HMG-CoA was regulating IL-10.
126 Cholesterol metabolism encompasses a number of metabolic pathways implicated in immune function
127 including mevalonate, isoprenyl and sterol metabolism (Supplementary Figure 1), all of which are
128 attenuated by HMG-CoA reductase inhibition to varying degrees. Given that defects in the isoprenyl
129 branch have been demonstrated to result in hyperinflammatory responses in vivo 23,26, we investigated
130 if isoprenylation was regulating IL-10. To this end, we targeted geranylgeranyltransferase (GGTase)
131 and farnesyltransferase (FTase), enzymes known to post-translationally modify proteins with
6 132 geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP) groups respectively
133 (enzymes and metabolites outlined in Figure 2a). Inhibition of GGTase, but not FTase, substantially
134 reduced TLR9-induced IL-10 production, indicating that geranylgeranyl dependent modifications
135 regulate IL-10 expression (Figure 2b, Supplementary Figure 5a-b). In keeping with the effects of
136 HMG-CoA reductase inhibition, inflammatory cytokine production was preserved (Figure 2c).
137 Furthermore, we observed no or little effect on the proliferation, differentiation, and antibody
138 production by B cells after TLR9 ligation in the presence of either atorvastatin or GGTi during longer
139 cultures (5-7 days, Supplementary Figure 5c). In experiments to test GGTase specificity, we also
140 observed that IL-10 was dependent on GGTase-I, but not GGTase-II, as inhibition of GGTase-II upon
141 TLR9 ligation did not affect IL-10 expression (Supplementary Figure 5d).
142
143 To support the notion of GGPP dependency, we depleted metabolites within the pathway with
144 atorvastatin, and found that specifically supplementing the geranyl branch with exogenous GGPP
145 prevented the blockade of IL-10 production (Figure 2d, Supplementary Figure 5e). In agreement with
146 a specific effect of GGPP, supplementation with squalene – a metabolite downstream of the
147 isoprenylation branch – was unable to rescue IL-10 production (Supplementary Figure 5f). Finally, to
148 understand if cellular utilization of GGPP was dependent on the enzymatic activity of GGTase, we
149 supplied cells with exogenous GGPP together with inhibition of GGTase. We found that GGPP was
150 unable to rescue levels of IL-10 (Figure 2e), consistent with the idea that both GGTase activity and
151 GGPP sufficiency are required for TLR9 induced expression of IL-10.
152
153 GGPP regulates signaling through TLR9
154 The most well characterized targets of isoprenylation are Ras superfamily proteins27,28. Ras-dependent
155 pathways downstream of TLR9 include Raf-MEK-ERK and PI3K-AKT cascades (Figure 3a).
156 Signaling through both pathways is critically required for IL-10 production, as inhibition of either
157 pathway is sufficient to block TLR9-dependent IL-10 induction (Figure 3b-c). Therefore, we sought
158 to address if activation of these pathways is dependent on GGPP. Following GGTase inhibition, AKT
159 phosphorylation on Ser473 was severely impaired, whereas ERK phosphorylation was modestly
7 160 reduced at early timepoints (Figure 3d, quantification shown in Supplementary Figure 6a-b).
161 Downstream, AKT restricts GSK3 activity through an inhibitory phosphorylation event targeting Ser9
162 29. In keeping with the idea that GSK3 suppression is required for IL-10 production, chemical
163 inhibition of GSK3 enhanced TLR9-induced IL-10 expression (Figure 3e). Blocking GGTase activity
164 resulted in reduced Ser9 phosphorylation on GSK3. This indicated preserved activation of GSK3, and
165 that GGTase activity negatively regulates GSK3, which in turn is necessary for IL-10 production
166 (Figure 3f). We then examined if inhibition of GSK3 was sufficient to rescue an upstream
167 perturbation of geranylgeranylation. Indeed, bypassing GGTase through GSK3 inhibition was able to
168 fully rescue IL-10 expression, without affecting TNF (Figure 3g, Supplementary Figure 6c). Together
169 these data suggest that following TLR9 engagement, IL-10 induction through AKT-GSK3, and
170 possibly ERK, is dependent on GGTase activity.
171
172 PI3Kδ regulates IL-10 expression in human and mouse B cells
173 We next aimed to determine how geranylgeranyl-driven phosphorylation cascades regulate AKT-
174 GSK3 signaling. PI3K mediates Ras-dependent AKT signaling, which suggests that isoprenylation-
175 driven phosphorylation cascades through AKT are dependent on PI3K activity. Accordingly, pan
176 inhibition of PI3K blocked expression of IL-10 upon TLR9 stimulation (Figure 4a). We found, by
177 selective inhibition of either δ, α, or γ isoforms of PI3K, that IL-10 is primarily regulated through
178 PI3Kδ downstream of TLR9 (Figure 4b). To further support the importance of PI3Kδ, we examined
179 IL-10 production in splenic B cells derived from mice expressing either a hyperactive (E1020K) or
180 catalytically inactive (D910A) PI3Kδ subunit. Following TLR9 activation, presence of the
181 hyperactive PI3Kδ mutant resulted in 3-fold increased expression of IL-10, whereas the catalytically
182 inactive PI3Kδ resulted in almost a complete loss of IL-10 production (Figure 4c). Furthermore, and
183 in agreement with our previous observations, TNF production was preserved (Figure 4c). These data
184 suggest that isoprenylation-dependent interactions between Ras and PI3Kδ are required for IL-10
185 production, and likely underpin the regulatory function of B cells.
186
8 187 GGPP regulates IL-10 induction via BLIMP1
188 Currently there is no defined transcription factor that regulates IL-10 in human B cells. Therefore, we
189 sought to understand how IL-10 is transcriptionally regulated, and to clarify the role for GGPP in this
190 process. Stimulation of B cells in the presence of actinomycin D indicated that a transcriptional event
191 within the first 24 hours was necessary for IL-10 production (Supplementary Figure 7a). Therefore,
192 we suspected that under conditions of GGTase inhibition, expression of a transcription factor
193 necessary for IL-10 may be compromised. To test this, we performed RNA-sequencing on TLR9-
194 stimulated human B cells in the presence or absence of GGTase inhibition. Principal component
195 analysis demonstrated that the main variation in the transcriptional profile of the cells was dependent
196 on GGTase activity, as expected (Figure 5a, Supplementary Figure 7b). We then interrogated the list
197 of differentially expressed genes (±1.5 fold, FDR<0.05) for those known or likely to encode human
198 transcription factors (as defined by 30). Among the 73 differentially expressed transcription factors, 9
199 have been shown to regulate IL-10 expression in other cell types (Figure 5b-d, Supplementary Figure
200 7c), either through activation or repression (defined in 31). We decided to focus on BLIMP1 for two
201 reasons. First, its transcript is enriched in IL-10+ human B cells 32. Secondly, interrogation of a mouse
202 B cell ChIP- and RNA-seq data set 33 demonstrated that binding of BLIMP1 to the Il10 locus
203 correlates with increased IL-10 expression (Supplementary Figure 7d). We first confirmed reduced
204 expression of BLIMP1 protein upon GGTase inhibition (Supplementary Figure 7e), and consistent
205 with previous data, we observed increased expression of BLIMP1 protein within the IL-10+ B cell
206 population (Figure 5e). To more thoroughly address its role, we performed gene targeting experiments
207 on BLIMP1 in primary human B cells. TLR9 stimulation strongly upregulated BLIMP1 expression in
208 multiple B cell populations, including, unexpectedly, recently activated naïve and CD24hiCD38hi B
209 cells (Figure 5f, Supplementary Figure 7f-g), whilst siRNA knockdown was able to reduce TLR9-
210 dependent protein level expression by ~60% (Figure 5g-h). In agreement with a central role in
211 regulating IL-10, siRNA-mediated knockdown of BLIMP1 reduced IL-10 expression by 50-90%,
212 whereas TNF production was preserved (Figure 5i). Together, these data demonstrate that BLIMP1
213 regulates IL-10 in human B cells, and its expression is dependent on cholesterol metabolism for its
214 provision of GGPP.
9 215
216 MKD patients generate poor regulatory B cell responses
217 Our data demonstrated that cholesterol metabolism is essential for B cell derived IL-10 production.
218 We therefore sought to validate our findings in the context of human inflammatory disease. To
219 address this, we studied MKD patients, who carry a partial loss-of-function mutation in the
220 mevalonate kinase gene, whose cognate substrate lies directly downstream of HMG-CoA in the
221 metabolic pathway (highlighted in Supplementary Figure 1a). These patients present with a complex
222 and severe autoinflammatory syndrome 25. We first compared peripheral regulatory B cells from
223 MKD patients to age and sex matched healthy controls (Patient information in supplementary Figure
224 8a). We observed similar frequencies of B10 cells, whereas CD24hiCD38hi B cells were reduced
225 (Figure 6a-b). This is in line with a general shift from a naïve to memory like B cell phenotype in
226 MKD patients (Supplementary Figure 8b). In longer term in vitro cultures, we observed that B cells
227 from MKD patients possess a normal capacity to proliferate, differentiate, and to produce antibodies
228 (Supplementary Figure 8c).
229
230 We next examined if dysregulated cholesterol metabolism in patients with MKD leads to an inability
231 to mount an IL-10 response, which could contribute to disease persistence or exacerbation. In line
232 with this hypothesis, MKD patients generated poor IL-10 responses after stimulation through TLR9
233 (30-70% reduction versus controls, Figure 6c), whilst TNF expression was enhanced in 2 of 4 donors
234 (Supplementary Figure 8d). Interestingly, the defect in IL-10 production could be reversed by the
235 addition of GGPP, indicating that at least in part, this defect was due to a relative deficiency of the
236 isoprenyl metabolite (Figure 6c). As with our previous observations, IL-10 expression was reduced
237 across all B cell phenotypes, including a ~3-fold reduction within B10 and CD24hiCD38hi B cells
238 (Figure 6d, gating in Supplementary Figure 4a and d).
239
240 We previously identified GSK3 as a key node in IL-10 production (Figure 3e-g). Therefore, we
241 addressed if reduced IL-10 levels in MKD patients could be rescued through inhibition of GSK3,
242 which would provide a rescue to signaling pathways further downstream of GGPP supplementation.
10 243 Indeed, GSK3 restriction in MKD patients was able to fully rescue IL-10 production (Fig. 6e). As
244 with our previous data, supplementation with squalene was unable to restore IL-10 expression in
245 MKD patients, supporting evidence that the IL-10 defect is specifically due to poor GGPP production
246 (Supplementary Figure 8e). To examine the dependency of IL-10 expression on BLIMP1 in MKD
247 patients, given our previous findings in healthy donor B cells, we analysed the capacity of MKD
248 patients to upregulate BLIMP1 in response to TLR9 stimulation. In spite of the increased proportions
249 of memory B cells present in MKD patients, we demonstrated a reduced capacity to induce BLIMP1
250 compared to healthy B cells in 2 of 2 donors (Figure 6f), providing additional mechanistic data to
251 explain why these patients show reduced B cell derived IL-10.
252
253 We then addressed if the defect in IL-10 production from MKD patients was associated with a
254 functional impairment. Indeed, we observed that MKD B cells were unable to suppress IFNγ
255 production by autologous CD4+ T cells, when compared to B cells from healthy controls (Figure 6g).
256 Moreover, pre-treatment of MKD B cells with exogenous GGPP prior to co-culture, was able to
257 restore regulatory capacity in 2 of 2 donors tested (Supplementary Figure 8f), suggesting that a GGPP
258 deficiency in MKD patients impairs both IL-10 expression and results in defective suppressive
259 function. Given our B cell phenotype, we examined whether MKD imposed more a systemic defect
260 on IL-10 producing immune cells we investigated whether T cell responses from MKD patients were
261 also defective in IL-10 expression. In contrast to B cells, T cells were able to induce effective IL-10
262 responses, alongside comparable levels of other cytokines including TNF, IFNγ, IL-2 and IL-17
263 (Supplementary Figure 8g).
264
265 Finally, to corroborate our results, we interrogated an independent dataset (GSE43553 taken from 34)
266 that performed global gene expression analysis in ex vivo PBMCs from healthy controls (n=20) and
267 MKD patients (n=8). Gene set enrichment analysis identified defective Ras (KRAS) and PI3K-Akt-
268 mTOR signaling pathways in MKD patients when compared to healthy donors, in line with our own
269 observations (Figure 6h). Collectively, and consistent with our in vitro findings, these data suggest
11 270 that dysregulated cholesterol metabolism in vivo, as seen in MKD patients, results in impaired
271 regulatory B cell responses.
272
273
274
12 275 Discussion
276 This study provides the first description of the metabolic requirements for IL-10 producing B cells,
277 arguing for a central reliance on cholesterol metabolism. Our data point to a model in which synthesis
278 of GGPP prior to stimulation permits transduction of receptor signaling cascades necessary for IL-10
279 expression. As a consequence, the transcription factor BLIMP1 is induced, which then promotes IL-
280 10 gene expression. We propose that this has direct relevance in vivo, as IL-10 producing B cells from
281 patients who carry mutations in the cholesterol metabolic pathway phenocopy our in vitro findings
282 (Outlined in Figure 7).
283
284 Investigations into B cell metabolism have focused primarily on either antibody production or
285 activation induced metabolic reprogramming 18–20,35. As alluded to above, there was no understanding
286 of the metabolic requirements for regulatory B cells. Here we demonstrate that cholesterol
287 metabolism is critical in mediating the regulatory capacity of human B cells through its control of IL-
288 10. We have also previously shown that T cells require an intact cholesterol biosynthesis pathway to
289 switch from inflammatory Th1 cells (IFNγ+IL-10-) to IFNγ+IL-10+ cells22, although this appears to be
290 regulated by an alternative mechanism, as inhibition of isoprenylation did not affect this switch.
291 Interestingly, cholesterol metabolism has been implicated in regulatory T cell function through a
292 mechanism dependent on ICOS and CTLA-4, whilst having no effect on IL-10 expression 36.
293 Although the authors did not explore this, we anticipate that regulatory T cells may also possess
294 significant GGPP dependency, as regulatory T cell function is especially reliant on PI3Kδ activity 37.
295 Therefore, whilst cholesterol metabolism appears to regulate different effector molecules between the
296 cell types, it may be that regulatory T and B cells rely more heavily on cholesterol metabolism due to
297 the necessity for potent GGPP-dependent PI3Kδ activity.
298
299 Much of the experimental data regarding immunity and metabolism have suggested a paradigm in
300 which the integration of metabolic pathways controlling cell fate arises as a direct consequence of
301 immune cell activation. Based on the ability of cholesterol metabolism to control induction of a
13 302 regulatory program in human B cells by modulating TLR9 signaling, we propose that the
303 programming of immune responses through cholesterol metabolism may differ in this regard. We
304 propose that isoprenyl modifications constitute a metabolic pre-programming event. This pre-
305 programming requires the generation of GGPP prior to cellular stimulation (rather than upon or as a
306 direct consequence of stimulation), that has the effect of fine-tuning signaling cascades. This notion of
307 pre-programming is consistent with data showing the GGPP-dependent constitutive localization of
308 Ras family proteins at the cell membrane and endosomes, and that blockade of cholesterol
309 metabolism retains Ras in the cytoplasm 38,39. In other words, the state of signaling intermediates is
310 preset by the state of cholesterol metabolism of the quiescent cell at any given point.
311
312 Our finding that either GGPP deficiency or inhibition of its cognate enzyme GGTase was sufficient to
313 block IL-10 production suggested that both the metabolite and its enzyme are absolutely required for
314 the induction of a regulatory phenotype. Notably, inhibition of FTase was unable to inhibit IL-10
315 production, likely due to the differing targets for geranylgeranylation and farnesylation 27. This
316 suggests that cholesterol metabolism drives the anti-inflammatory function of B cells primarily
317 through the synthesis of the isoprenyl group GGPP. These observations contrast with previous reports
318 suggesting that isoprenylation largely mediates the restriction of pro-inflammatory cytokines,
319 including TNF, IL-6, and IL-1β. It should be noted however, that these data were derived from
320 studies of mouse macrophages and intestinal epithelial cells 23,40,41. Nonetheless, whilst human B cells
321 are known to co-express inflammatory cytokines TNF and IL-6 alongside IL-10 42, we saw no
322 alteration in their capability to produce these upon blockade of GGTase. This suggests that pro-
323 inflammatory cytokine production relies less heavily on cholesterol metabolism. Our finding that
324 cholesterol metabolism regulates PI3K signaling may somewhat explain this, given that PI3K can
325 differentially regulate pro- versus anti-inflammatory cytokine production upon TLR ligation 43–45.
326
327 We found that the mechanistic control of cholesterol metabolism revolves around its ability to
328 regulate PI3Kδ-AKT signaling downstream of TLR9, although this is likely not the only target as we
14 329 also saw an effect on pERK activation. Although we documented expression of all isoforms of PI3K,
330 we observed that PI3Kδ, to a greater extent than PI3Kα or PI3Kγ, contributed to IL-10 expression in
331 human B cells upon TLR9 ligation. This finding is consistent with a recent observation identifying IL-
332 10 producing B cells as the primary pathological cell type in a model of activated PI3Kδ syndrome,
333 driving Streptococcus pneumoniae persistence 14. We further suggest that GSK3 inactivation
334 downstream of GGPP-dependent PI3Kδ-AKT signaling is also required for optimal IL-10 expression.
335 Consistent with this, AKT-driven phosphorylation of GSK3 on Ser9 is known to differentially
336 regulate cytokine production in monocytes 29. Moreover, we observed that reduced IL-10 expression
337 induced in the context of defective isoprenylation could be rescued through inhibition of GSK3. In
338 line with our findings, GSK3 inhibition in both innate cells 29 and Th1/2/17 cells 46 potentiates IL-10
339 expression. This provides a common framework in all immune cells, whereby restriction of GSK3
340 activity upon receptor ligation is necessary for IL-10 expression.
341
342 Downstream of TLR9 ligation, BLIMP1 was shown to positively regulate IL-10 expression,
343 suggesting its role as a transcriptional regulator of IL-10 in human B cells. Indeed, BLIMP1 has been
344 previously demonstrated to control IL-10 induction in T cells47, and in a recent study in regulatory B
345 cells48. Analysis of publicly available ChIP-seq datasets in both mouse and human B cells are in line
346 with this finding, suggesting that IL-10 is a significantly enriched target of BLIMP1 33,49. This
347 observation is reminiscent of recent work highlighting the role for BLIMP1-expressing regulatory
348 plasmablasts and plasma cells in vivo 3,4,50,51, but also in humans ex vivo 51. Whilst this previous work
349 provided a correlative link between BLIMP1 and IL-10, we suggest that BLIMP1 is a direct regulator
350 of IL-10 in B cells. Therefore, it will be interesting in future work to further delineate a direct link and
351 understand if there is a common mechanism regulating B cell derived IL-10 in both mouse and human
352 B cells. Surprisingly, we observed expression of BLIMP1 in recently activated IgD+CD27- (naïve) B
353 cells upon TLR9 stimulation (Supplementary Figure 7F). This has also been observed previously,
354 where expression of BLIMP1 alongside a conformationally open chromatin arrangement in naïve B
355 cells has been demonstrated52. This raises the question of whether BLIMP1 plays an earlier role in B
15 356 cell function at much earlier stages of activation, distinct from its more classical function linked to the
357 development of terminally differentiated B cells. In addition to BLIMP1, several interesting
358 candidates such as AHR and BATF were also identified in our screen, which warrant further
359 investigation.
360
361 To validate our findings in the context of human disease, we investigated patients with dysregulated
362 cholesterol metabolism. MKD patients carry a mutation in the mevalonate kinase gene. As a
363 consequence, their ability to convert mevalonate to mevalonate-5-phosphate is severely impaired. In
364 keeping with our findings following perturbation of cholesterol metabolism in healthy B cells in vitro,
365 we observed poor regulatory responses in B cell from these patients. This anti-inflammatory defect
366 uncovered an unappreciated dimension to the spectrum of MKD. This included a reduced ability to
367 produce IL-10, associated with a functional impairment in restricting T cell responses. In both cases,
368 supplementation of GGPP was able to reverse this defect, suggesting that a lack of metabolic flux
369 through the pathway might be contributing. Interestingly, GSK3 inhibition was also able to reverse
370 the reduced IL-10 production observed in MKD patients, providing a specific signaling node
371 downstream of GGPP deficiency that is dysregulated in patients. In agreement with GGPP deficiency,
372 MKD patients have been demonstrated to accumulate unprenylated Ras proteins 53. Further
373 characterization of MKD B cells demonstrated that, despite their increased frequencies of memory B
374 cells, these cells do not effectively induce BLIMP1 expression in response to TLR9. This data is in
375 line with our observations demonstrating that a deficiency in geranylgeranylation results in an
376 inability to properly induce BLIMP1 expression.
377
378 To date, the causative factor driving disease pathology has been defined as excessive inflammatory
379 cytokine production, driven through increased macrophage driven IL-1β production 23,54, but also
380 through the induction of a trained immunity phenotype driven by accumulated mevalonate 55. Here we
381 also provide evidence that CD4+ T cells in MKD patients induce cytokine responses equivalent to
382 those in healthy donors, suggesting this effect is intrinsic to B cells. Given the role of regulatory B
383 cells in restricting cellular responses, we propose this B cell defect could contribute to the relapsing
16 384 and remitting nature of the disease. Moreover, it has been shown that B cell derived IL-10 is
385 important in T follicular helper organization, and restricting excessive antibody responses via
386 interaction with Tfh cells 56,57. It is therefore tempting to speculate that these patients generate
387 inappropriately inflammatory B cell responses in lymphoid organs. This would go some way to
388 explain the clinical observations in MKD patients who are typically diagnosed in early childhood with
389 high circulating IgD and IgA levels 25.
390
391 In summary, these results argue that cholesterol metabolism acts as a central metabolic pathway
392 promoting an intrinsic anti-inflammatory program in B cells, driving IL-10 production and subsequent
393 suppression and restriction of immune responses.
394
395
396 Methods
397 Cell isolation and culture
398 Blood samples were obtained locally from healthy donors, and mevalonate kinase deficient patients
399 were recruited from Royal Free Hospital NHS Foundation Trust. Human: Peripheral blood
400 mononuclear cells were isolated by density gradient centrifugation using Lymphoprep (Alere
401 Technologies). Fresh CD19+ B cells and CD4+ T cells were subsequently isolated by magnetic cell
402 sorting, based on CD19 and CD4 positive or CD4 negative selection (MACS, Miltenyi Biotech);
403 purity was consistently >97%. Cells were cultured in RPMI-1640 (Sigma), 10% Fetal Bovine Serum
404 (Sigma) with 2mM L-glutamine (Sigma), 0.1gL-1 sodium bicarbonate (Sigma), supplemented with
405 100UmL-1 penicillin and 0.1mgmL-1 Streptomycin (PAA), and 10mM Hepes (PAA), in round bottom
406 96-well Nunclon plates (Thermo Scientific) at 0.3 x 106 cells/well. B cells were activated with TLR9
407 ligand CpG ODN 2006 (1μM, Invivogen), alongside recombinant human IL-2 (25Uml-1, Proleukin,
408 Novartis) typically for 40 hours, or as indicated. Where indicated, CellTrace Violet (Thermofisher
409 Scientific) labelling of cells was conducted as per manufacturer’s instructions, used at a final
410 concentration of 2μM prior to stimulation. All inhibitors and metabolites were extensively titrated
17 411 prior to use, and reagent details are as follows: MK-2206 (Akt, 100nM), FTi-277 (Farnesyl
412 transferase, 5μM), GGTi-298 or GGTi-2133 (Geranylgeranyl transferase-II, 5μM), Psoromic acid
413 (Geranylgeranyl transferase-I, 0.1-10μM) CHIR-99021 (GSK3α/β, 5μM), atorvastatin (HMG-CoA
414 reductase, 5μM), SCH772984 (ERK, 1μM), HS-173 (p110α, 1pM-1μM), IP-549 (p110α, 1pM-1μM),
415 nemiralisib (p110δ, 1pM-1μM), (R)-mevalonic acid (250μM), Squalene (1-10μM), and
416 geranylgeranyl pyrophosphate (2μM). Mouse: Total splenocytes were isolated from 8-15-week-old
417 mice, red blood cells were lysed (Biolegend), and resulting single cell suspensions were cultured in
418 the above media in flat bottom 48-well Nunclon plates (ThermoFisher), at 1 x 106 cells/well. Wild
419 type, p110δ E1020K and p110δ D910A mice used in the study have been described previously58. B
420 cells were stimulated with CpG ODN 1826 (1μM, Invivogen) in the presence of IL-2 (25Uml-1,
421 Proleukin, Novartis) for 48 hours. Other ligands used for stimulation include: CD40L (6245-CL,
422 1μgml-1, R&D Systems), LPS (10-500ngml-1, Invivogen), and IFNα (200-1000Uml-1).
423
424 T and B cell co-culture
425 Autologous CD4+ T and CD19+ B cells were isolated from healthy donors or MKD patients as
426 described above. B cells were incubated overnight with CpG (1μM), IL-2 (25Uml-1), +/- atorvastatin,
427 +/- mevalonic acid. In parallel, CD4+ T cells were stimulated with plate bound αCD3 and αCD28
428 (both 1μgml-1, Biolegend). After 12 hours, B cells were washed twice, added to the T cells at a 1:1
429 ratio, and cultured for 4 days +/- αIL10 antibody +/- isotype control (5μgml-1, both Biolegend).
430 Assessment of the direct effect of IL-10 on human CD4 T cells was performed by plate bound
431 αCD3/28 (1μgml-1) stimulated, cell trace violet labelled T cells, alongside incubation of the indicated
432 concentration of IL-10 for 4 days.
433
434 Flow Cytometry
435 Antibodies used were as follows: IL-10-PE (dilution 1:800, JES3-19F1), TNF-BV421 (dilution 1:400,
436 Mab11), IFNγ-PE/Cy7 (dilution 1:400, 4S.B3), CD20-AF647 (dilution 1:400, 2H7), CD24-BV605
437 (dilution 1:400, ML5), CD27-FITC (dilution 1:200, M-T271), CD38-PE/Cy7 (dilution 1:400, HB-7),
18 438 IgD-BV421 (dilution 1:400, IA6-2), CD4-FITC (dilution 1:200, A161A1), HLA-DR-PerCP/Cy5.5
439 (dilution 1:200, L243), CD86-BV421 (dilution 1:400, IT2.2), CD40-PE (dilution 1:400, 5C3): all
440 Biolegend, pGSK3ser9-PE (dilution 1:10, REA436, Miltenyi), and BLIMP1-APC (dilution 1:10,
441 646702, R&D Systems). For intracellular cytokine detection, cells were restimulated in the final 3
442 (human) or 5 (mice) hours of culture with phorbol 12-myristate 13-acetate (50ngmL-1, Sigma) and
443 ionomycin (500ngmL-1, Sigma) in the presence of Brefeldin A, and GolgiStop (both 1μlml-1, both BD
444 Biosciences). For surface staining, cells were harvested and incubated with Fixable Viability Dye
445 eFluor780 (dilution 1:4000, eBiosciences) for 15 minutes in phosphate buffered saline (PBS),
446 followed by the appropriate volume of antibody diluted in 0.5% bovine serum albumin (BSA) in PBS
447 for 20 minutes, all at 4°C. Cells were then washed and fixed in 3% paraformaldehyde (Electron
448 Microscopy Sciences) for 15 minutes at room temperature. For intracellular cytokine staining, cells
449 were incubated with the appropriate volume of antibody, diluted in 0.1% saponin in 0.5% BSA in
450 PBS for 45 minutes at room temperature or 4°C overnight. For staining of transcription factors, or
451 phospho-flow, FoxP3/Transcription factor buffer set was used, as per manufacturer’s instructions
452 (eBiosciences). Staining using a secondary antibody against the primary was undertaken either at
453 room temperature for 1 hour, or 4°C overnight. Cells were acquired using a BD LSRFortessa or
454 FACSCanto II (BD Biosciences), and analysis conducted using FlowJo V.10.1 software (Tree Star
455 Inc.).
456
457 RNA extraction and qRT-PCR analysis
458 Harvested cells were lysed in TRIzol (Ambion, Life Technologies), and stored at -20°C until RNA
459 extraction by phenol-chloroform phase separation. Briefly, RNA was eluted by chloroform, and
460 subsequently precipitated using ice cold isopropanol for 1 hour at -20°C. If necessary, RNA clean-up
461 was conducted by reprecipitation of RNA in 0.5 volumes of 7.5M ammonium acetate, 2.5 volumes
462 100% ice cold ethanol, and 1μl GlycoBlue overnight at -20°C. 40ng of RNA was added for reverse
463 transcription, using qPCRBIO cDNA Synthesis Kit (PCR Biosystems) as per manufacturer’s
464 instructions. qPCR was conducted using 7900HT Fast Real-Time PCR System (ThermoFisher
19 465 Scientific). Primers used were: IL10 (Hs00961622_m1), IL6 (Hs00174131_m1), or LTA
466 (Hs04188773_g1), multiplexed with a VIC- labelled 18S probe (all Thermofisher Scientific) in PCR
467 Biosystems mastermix.
468
469 ELISA
470 Sandwich ELISA was used to detect supernatant analytes. All cell free supernatants harvested at
471 indicated time points were stored at -20°C until analysis. ELISAs for IL-10, IFNγ (both R&D
472 systems, cat nos. DY217B and DY285B, respectively), TNF (Biolegend, cat no. 430201), or IgM
473 (ThermoFisher Scientific, cat no. BMS2098) were conducted according to manufacturer’s protocols
474 and detected on a Victor 1420 multilabel counter (Perkin Elmer) quantifying concentrations drawn
475 from a standard curve on each plate.
476
477 Western Blotting
478 Antibodies used were as follows: BLIMP1 (dilution 1:1000, 646702, R&D Systems), pAktser473
479 (dilution 1:1000, D9E), pERKThr202/Tyr204 (dilution 1:1000, D12.14.4E), and GAPDH (dilution
480 1:5000, 14C10, all Cell Signaling Technologies). After culture, cells were harvested, washed in ice
481 cold PBS, and lysates immediately extracted, or pellets were snap frozen on dry ice for 1 minute and
482 kept at -80°C until lysis. Whole cell lysates were extracted using RIPA Buffer (Cell Signaling
483 Technology) with 1X Protease inhibitor cocktail. Lysates were added to 4X Laemmli Sample Buffer
484 (Bio Rad) with 10% 2-mercaptoethanol, and resolved on SDS-PAGE gels, transferred to PVDF
485 membranes, blocked (Tris, 5% BSA, 0.05% Tween20) and probed with the desired antibody.
486 Immunoblots were developed with anti-rabbit-HRP (Dako) secondary antibody, and proteins
487 visualized by SuperSignal chemiluminescent reaction (Pierce) in a ChemiDoc station (BioRad).
488
489 siRNA knockdown
490 siRNA knockdown was conducted by Amaxa nucleofection using the nucleofector 2b machine, as per
491 manufacturers protocol. siRNA knockdown was optimized, resulting in the used of 2-3 million cells
20 492 per condition, and 500nM of either BLIMP1 targeted siRNA (Silencer Select assay ID s1992) or
493 scrambled control (Silencer Select Negative control, both from ThermoFisher Sceintific). Cells were
494 electroporated using program U-015, resuspended in warm fully supplemented culture medium, and
495 left to recover for 15 minutes before stimulation. Knockdown efficiency was determined by western
496 blot at 40 hours post stimulation.
497
498 RNA sequencing
499 CD19+ B cells were stimulated with CpG (1μM) and IL-2 (25Uml-1) +/- GGTi-298 for 12 hours. RNA
500 was isolated by column centrifugation using RNeasy Plus mini kit (with gDNA removal step) as per
501 manufacturer’s instructions (Qiagen). Library preparation was completed using NEBNext Ultra
502 Directional RNA Library Prep Kit for Illumina. Depletion of ribosomal RNA was performed using
503 Next rRNA Depletion kit (New England BioLabs). RNA quality was confirmed by bioanalyser
504 (Agilent 2100 Bioanalyzer G2938B), resulting in a mean RIN score of 8.1, ranging from 7.3-8.7.
505 Paired-end sequencing was then conducted using the HiSeq 2500 platform (Illumina). Raw data was
506 checked for quality using FASTQC. Processing of the raw data involving alignment (STAR) and
507 annotation (hg19) were done using Partek. After annotation, TMM-normalised data were used for
508 downstream statistical analysis using the exact test in the edgeR package. Inclusion criteria for
509 significantly differentially expressed genes was a false discovery rate of <0.05 and a fold change of
510 greater than 1.5x. Subsequent processing and visualization of the data was completed in RStudio or
511 Morpheus (Broad Institute, Boston, MA).
512
513 Gene set enrichment analysis
514 Gene set enrichment analysis used on dataset GSE4355334 was conducted with GSEA v4.0.2 (Broad
515 Institute). Gene expression data was used from 20 healthy controls (GSM1065214 to GSM1065233)
516 and 8 MKD patients (GSM1065234 to GSM1065241). Gene sets were compared to the gene set
517 database h.all.v7.0.symbols.gmt [Hallmarks], using 1000 permutations, the ‘weighted’ enrichment
518 statistic, and ‘Signal2Noise’ for gene ranking.
519
21 520 Statistics
521 All statistical analysis was conducted using GraphPad Prism v7.0 (GraphPad, San Diego, CA, USA).
522 Paired data was analysed through either two-way ANOVA with Dunnett’s test for multiple
523 comparisons, or Freidman’s test with Dunn’s test for multiple comparisons. Unpaired data was
524 assessed through ANOVA with Tukey’s test for multiple comparisons. Finally, two-group
525 comparison was performed either through unpaired or paired T test. A P value of < 0.05 was
526 considered statistically significant, and all statistically significant values are shown.
527
528 Study approval
529 Blood samples were obtained locally from healthy donors, and mevalonate kinase deficient patients
530 were recruited from Royal Free Hospital NHS Foundation Trust, London, UK or National Human
531 Genome Research Institute/Inflammatory Disease Section, Bethesda, USA. Written informed consent
532 was received from all healthy donors and patients used in this study, approved by the Bromley
533 Research Ethics Committee (REC06/Q0705/20) and by the NIDDK/NIAMS Institutional Review
534 Board. Animal experiments were performed according to the Animals (Scientific Procedures) Act
535 1986, license PPL 70/7661 and approved by the Babraham Institute Animal Welfare and Ethics
536 Review Body.
537
538 Data Availability
539 Sequence data generated in this study have been deposited in GEO under the accession code
540 GSE150245 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150245]. Publicly available
541 datasets used for analysis are available at GEO under the accession codes GSE43553
542 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43553] (used in GSEA analysis), and
543 GSE71698 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE71698] (used for ChIP
544 and RNA-seq analysis).
545
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681
682
27 683 Acknowledgements
684 We thank all the healthy donors and patients for their support. This research was funded/supported by
685 the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St
686 Thomas’ NHS Foundation Trust and King’s College London and/or the NIHR Clinical Research
687 Facility. The views expressed are those of the author(s) and not necessarily those of the NHS, the
688 NIHR or the Department of Health and Social Care This work was also supported by: the IMI-funded
689 project BeTheCure (115142-2); EU/EFPIA Innovative Medicines Initiative 2 Joint Undertaking
690 RTCure (777357) (both E.P. and A.P.C.); the National Institute for Health Research Biomedical
691 Research Centre, at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London
692 (J.A.B.); Intramural Research Programs of the National Human Genome Research Institute (I.A. and
693 S.R.); MRC (MR/M012328/2, K.O.); Wellcome Trust (206618/Z/17/Z, A.C.)
694
695 Author contributions
696 JAB designed, performed, and interpreted experiments, and wrote the manuscript, HAP contributed to
697 experimental design, performed experiments, and contributed to manuscript preparation, TH
698 performed experiments, AC and KO contributed to experimental design and provided mice, MW, SR,
699 HJL, and IA, recruited patients and extracted patient samples, HJL contributed to experimental design
700 and provided patient samples, CK supervised research, and contributed to experimental design, APC
701 and EP conceived the project, designed and performed experiments, supervised research, and
702 contributed to manuscript preparation.
703
704 Competing interests
705 The authors declare no competing interests
706
707 Figure. 1. Cholesterol metabolism drives regulatory B cell function. a-b. Kinetics of IL10 mRNA transcript
708 (a) and IL-10 secreted protein (b) expression at various time points in human B cells after TLR9 stimulation (n=4
709 for mRNA or n=4-5 for protein). IL10 mRNA was measured by qRT-PCR, and calculated relative to 18S. c.
710 Schematic protocol for the co-culture for T and B cells. Briefly, human B cells were stimulated through TLR9 ±
711 atorvastatin (AT) ± mevalonate (MA) overnight, before washing and addition to anti-CD3/28 stimulated human
28 + + 712 CD4 T cells for 4 days ± αIL-10 antibody. d. IFNγ production in human CD4 T cells after co-culture with
713 autologous TLR9-activated B cells (n=6, pval = 0.03 and 0.02). IFNγ suppression was calculated by: ((x-y)/x)*100
714 where x = IFNγ production by T cells alone, y = IFNγ production in co-cultured T cells. e. IL-10 expression in
715 human B cells after stimulation through TLR9 ± AT ± MA (n=7, pval = 0.003). f. IL-10 mRNA expression over
716 time in human B cells after stimulation through TLR9 ± AT ± MA (n=4). g. TNF expression in human B cells after
717 stimulation through TLR9 ± AT ± MA (n=7). h. IL6 or LTA mRNA expression, relative to 18S, in human B cells
718 after stimulation through TLR9 ± AT (n=4). Each data point represents individual donors. All data presented are
719 mean ± SD. Statistical testing in all figures was done by a Friedman’s test with Dunns’s multiple comparisons
720 test, or for (f) a mixed-effects model with Dunnett’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001,
721 and all significant values are shown
722
723 Figure. 2. Cholesterol metabolism drives IL-10 via GGPP. a. Schematic diagram showing key metabolites and
724 enzymes of the isoprenylation route in cholesterol metabolism. b-c. IL-10 (b) and TNF (c) expression in human B
725 cells after stimulation through TLR9 ± geranylgeranyl transferase inhibition (GGTi, GGTi-298) ± farnesyl
726 transferase inhibition (FTi, FTi-277) (n=5 for IL-10, pval = 0.001; n=6 for TNF). d. IL-10 expression in human B
727 cells after stimulation through TLR9 ± atorvastatin (AT) ± geranylgeranyl pyrophosphate (GGPP) (n=4, pval =
728 0.009). e. IL-10 expression in human B cells after stimulation through TLR9 ± GGTi ± GGPP (n=4, pval both
729 0.03). Each data point represents individual donors. All data presented are mean ± SD. Statistical testing in all
730 figures was done by a Friedman’s test with Dunns’s multiple comparisons test. *P<0.05, **P<0.01, and all
731 significant values are shown.
732
733 Figure. 3. GGPP regulates signalling through TLR9. a. Schematic of Ras-dependent signalling cascades
734 downstream of TLR9. b-c. IL-10 expression in human B cells stimulated through TLR9 ± inhibition of (b) AKT
735 (AKTi, MK-2206) or (c) ERK (ERKi, SCH772984) (n=4, pval = 0.005, and 0.001). d. Phosphorylation of AKT
736 (ser473) or ERK1/2 (Thr202/Tyr204) over time in human B cells upon stimulation through TLR9 ± inhibition of
737 geranylgeranyl transferase (GGTi) (representative of n=3). e. IL-10 expression in human B cells stimulated
738 through TLR9 ± inhibition of GSK3 (GSK3i, CHIR-99021) (n=9, pval<0.001). f. Phosphorylation of GSK3 (ser9)
739 over time in human B cells upon stimulation through TLR9 ± inhibition of geranylgeranyl transferase (GGTi-298),
740 relative to unstimulated controls (n=4, pval = 0.02). g. IL-10 and TNF expression in human B cells stimulation
741 through TLR9 ± GGTi ± GSK3i (n=7, pval = 0.0001). Each data point represents individual donors. All data
742 presented are mean ± SD. Statistical testing in figures in (b), (c), and (e) was done by a paired t test, (f) using a
743 twoway ANOVA with Sidak’s multiple comparisons test, and (g) by Friedman’s test with Dunns’s multiple
744 comparisons test **P<0.01, ***P<0.001, ****P<0.0001 and all significant values are shown.
29 745
746 Figure. 4. PI3Kδ regulates IL-10 expression in human and mouse B cells. a. IL-10 expression in human B
747 cells stimulated through TLR9 ± pan inhibition of PI3K (PI3Ki, ZSTK474) (n=4, pval = 0.008). b. IL-10 expression
748 in human B cells stimulated through TLR9 ± inhibitors for specific family members of PI3K: these being δ
749 (Nemiralisib), γ (IP-549), or α (HS-173), over a range of concentrations (n=4). c. IL-10 and TNF expression in
750 mouse B cells after stimulation through TLR9. Mice used were wild type (WT), hyperactive PI3Kδ (E1020K), or
751 catalytically inactive PI3Kδ (D910A) (n=3, pval = 0.002 and <0.0001). Each data point represents individual
752 donors or mice. Data presented are mean ± SD. Statistical testing in figures in (a) was done by a paired t test, or
753 in (c) by a one-way ANOVA with Dunnet’s multiple comparisons test. **P<0.01, ****P<0.0001 and all significant
754 values are shown.
755
756 Figure. 5. GGPP regulates IL-10 induction via BLIMP1. a. Principal component analysis of human B cells after
757 stimulation through TLR9 ± geranylgeranyl transferase inhibition (GGTi), with analysis performed on total
758 normalised counts generated from RNA-sequencing data, before statistical analysis (n=7). b. Workflow for the
759 identification of putative IL-10 transcription factors. First, differentially expressed genes were cross referenced
760 with known or likely human transcription factors, and the subsequently identified differentially expressed
761 transcription factors were cross referenced with previously validated IL-10 transcription factors in either
762 macrophages or T cells. Differentially regulated IL-10 transcription factors are shown in (c) and all differentially
763 regulated transcription factors are shown in (d) (n=7). e. Expression of BLIMP1 in human B cells within either IL-
+ - 764 10 or IL-10 B cells after stimulation through TLR9 (n=6, pval = 0.003). f. Western blot showing BLIMP1
765 expression in human B cells either in unstimulated or stimulated through TLR9 (CpG) after 40 hours
766 (representative of three independent experiments). g-h. Western blot showing expression of BLIMP1 in human B
767 cells after stimulation of TLR9 and nucleofection with either a scrambled control (Scr) or BLIMP1 siRNA (n=5,
768 pval = 0.001). i. IL-10 and TNF expression in human B cells stimulated through TLR9 after nucleofection with
769 either Scr or BLIMP1 siRNA (n=6 for flow cytometry, pval = 0.002; and 5 for ELISA, pval = 0.009). Each data
770 point represents individual donors. All data presented are mean ± SD where average values are shown.
771 Statistical analysis in all figures was done using a paired t test. **P<0.01, ***P<0.001 and all significant values
772 are shown.
773
hi + 774 Figure. 6. MKD patients generate weak regulatory B cell responses. a-b. Frequencies of B10 (CD24 CD27 )
hi hi 775 and CD24 CD38 regulatory B cells in mevalonate kinase deficient (MKD) patients, compared to age and sex
776 matched healthy donors (HD). c. IL-10 expression in MKD patients and HD after stimulation through TLR9 ±
hi hi 777 geranylgeranyl pyrophosphate (GGPP). d. IL-10 expression within B10, CD24 CD38 , naïve, and memory
30 778 populations, showing the differential contribution of B cell phenotypes to IL-10 production (n=3, pval = <0.0001,
779 0.002, and 0.01). e. IL-10 expression in MKD patients and HD after stimulation through TLR9 ± GSK3i (CHIR-
780 99021). f. BLIMP1 expression in naïve and memory HD or MKD patient B cells after stimulation through TLR9
+ 781 (n=2). g. IFNγ production in human CD4 T cells of healthy controls and MKD patients after co-culture with
782 autologous TLR9-activated B cells. Protocol is outlined in Fig. 1C. h. Gene set enrichment analysis conducted on
783 gene expression profiles from an independent dataset (GSE43553) on PBMCs ex vivo in MKD patients (n=8) or
784 HD (n=20). Heatmap shows all genes involved in ‘Hallmark’ PI3K-Akt-mTOR gene set collection from MSigDB,
785 and their relative expression in MKD vs HD. Enrichment plots show PI3K-Akt-mTOR and KRAS DN (genes
786 downregulated by KRAS activation) in MKD vs HD. Each data point represents individual donors. All data
787 presented are mean +/- SD where average values are shown. Statistical analysis in (d) was conducted using a
788 paired t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 and all significant values are shown.
789
790 Figure 7. B cell IL-10 regulation by GGPP. IL-10 production by B cells is regulated by cholesterol metabolism
791 though geranylgeranyl pyrophosphate, controlling signalling cascades and BLIMP1 induction downstream of
792 TLR9 engagement. MKD = Mevalonate kinase deficiency
793
31 a b c
0.6 2.0
1.5 0.4
1.0 0.2
IL-10 (ng/mL) 0.5
0.0
IL10 mRNA relative to 18S 0.0 0 16 20 24 36 48 64 0 16 20 24 36 48 64 Time (hours) Time (hours) T:B - B cell pre-treatment d 60 T alone T:B T:B + αIL-10 AT AT + MA * 15.5 9.4 17.1 16.2 10.3 40 Cy7 - suppression
20 γ PE - γ 0 % IFN IFN AT - - + + CD4 MA - - - + αIL-10 - + - - CD4-FTIC e Control AT AT + MA ** f 7 0.06 0 AT *** 6 AT + MA 5 0.04 4.2 2.0 4.3 **
B cells 4 + PE - 3
10 0.02 * - 2 IL % IL-10 1 0.00
0 IL10 mRNA relative to 18S AT - + + 0 16 20 24 36 48 MA - - + Time (hours) FSC-A g h 60 0.04 IL6 LTA
0.03 41 45 43 40 B cells BV421 + - 0.02 α α 20 0.01 TNF % TNF mRNA relative to 18S 0 0.00 AT - + + CpG + + + + CD20-APC MA - - + AT - + - + a b d 5 Control GGTi FTi 4 ** ** 4 3 3 3.3 0.5 2.9 B cells B cells + +
PE 2 - 2 10
- 1 % IL-10 1 % IL-10 IL
0 0 GGTi - + - AT - + + FTi - - + GGPP - - + c 60 e 5 * 38 46 40 4
BV421 40 - 3 B cells B cells α + + α 2
TNF 20
% IL-10 1 % TNF
0 0 GGTi - + - GGTi - + + CD20-APC FTi - - + GGPP - - + a b c d 400 ** 500 ** 15’ 30’ 60’ 120’ - - GTi - GTi - GTi - GTi 400 0’ 15’ 30’30’ 60’60’ 120’ 300 pAkt - GGTi - -- GTi+ - GTi+ -- GTi+ -- GTi+ kDa 300 pERKpAkt - 56 200 pAkt 200 pERKpERKERK - 44 IL-10 (pg/mL) IL-10 (pg/mL) 100 100 GAPDHGAPDHERK - 37 GAPDH 0 0 - AKTi - ERKi e f 30’ Minutes 90’ Control GSK3i 8 **** 2.5 Unstim 2.0 2.8 5.8 Control 6 GGTi 1.5 * PE - B cells + 4
10 1.0 - IL 0.5 - GGTi 2 gMFI pGSK3 ser9 + GGTi % IL-10 0.0 0 0 30 60 CD20-APC - GSK3i pGSK3ser9-PE Time (minutes) g Control GGTi GGTi + GSK3i 3 60 *** 38 2 46 0 55 2 1 0 1 2 40 B cells B cells + + BV421 α -
α 1 20 % IL-10 % TNF TNF
0 0 GTi - + + GTi - + + GSK3i - - + GSK3i - - + IL-10-PE c a TNFα-BV421 IL-10 (pg/mL) 200 400 600 0 0 ** WT PI3Ki 36 b 1 4
IL-10 (pg/mL) 200 400 600 800 0 -6 -4 log (concentration)nM IL E1020K Nemiralisib ( - 10 IC -2 50 - =0.4pM PE 21 0 δ ) 13 1 2 4 -6 IC -4 log (concentration)nM 50 HS-173 ( D910A =36nM -2 α 44 ) 0 0 1 2
+ % IL-10 B cells 4 10 15 -6 0 5
WT
E1020K IC **** 50 -4 log (concentration)nM =264nM IP-549 ( ** D910A -2 γ ) 0 % TNFα+ B cells 20 40 60 0 2 WT E1020K 4
D910A i e TNFα-BV421 a PC2 = 11.21% IL BLIMP1 - 10 PC1 = 34.42% =PC1 Control - Scr - APC IL - 10+ 55 GGTi 4 1 IL - 10 -
PE Relative Blimp1 gMFI 0 1 2 3 4 IL-10 b BLIMP1 ** - IL-10 + 65 2 0 f g kDa kDa 35 35 95 95 - - - % IL-10+ B cells - Scr 0 1 2 3 4 5 - siRNA CpG Blimp1 ** + Scrambled GAPDH BLIMP1 GAPDH BLIMP1 % TNFα+ B cells 20 40 60 80 0 h c BLIMP1 siRNA BLIMP1 BLIMP1 relative to Scr 0.0 0.2 0.4 0.6 0.8 1.0 1.2 HMGA1 PRDM1 FOXO4 RXRA ETS1 BATF ATF3 AHR IL-10 (pg/mL) FOS 100 150 50 -6 Scr 0 -4 Fold change *** BLIMP1 -2 0 ** 2 4 6 d row min GGTi row min GGTi -
Control------+
- GGTi + + +
+ + row max + +
+ row max + + + id NR1D2 HBP1 KLF11 ZEB1 ARID5B KLF7 NR4A3 FOXO3 CREM PRDM2 POU6F1 ESR2 BHLHE40 GLMP BHLHE41 HES1 HNF1B NME2 ZNF485 PAX5 SP140 POU2F2 GTF2I ETS1 PHF19 RUNX1 BATF BCL6 ZNF296 ZNF692 PRDM1 HIC1 AHR ATF3 DNTTIP1 ZNF219 RXRA SREBF1 ZHX2 SETDB2 SKI LYL1 ZBTB16 MNT GTF2IRD1 KLF9 DDIT3 ZNF704 TCF7 ZBTB10 MBNL2 KLF3 TSC22D1 TGIF1 FOSL2 JUND THRA MXI1 FOXO4 FOS MAFF PRDM4 NR4A2 NR3C1 ZBTB1 + ZNF385C SOX5 ZNF318 TFDP1 HMGA1 YBX1 PA2G4 GTF3A + id ZBTB1 NR1D2 HBP1 KLF11 ZEB1 ARID5B KLF7 NR4A3 FOXO3 CREM PRDM2 POU6F1 ESR2 BHLHE40 GLMP BHLHE41 HES1 HNF1B NME2 ZNF485 PAX5 SP140 POU2F2 GTF2I ETS1 PHF19 RUNX1 BATF BCL6 ZNF296 ZNF692 PRDM1 HIC1 AHR ATF3 DNTTIP1 ZNF219 RXRA SREBF1 ZHX2 SETDB2 SKI LYL1 ZBTB16 MNT GTF2IRD1 KLF9 DDIT3 ZNF704 TCF7 ZBTB10 MBNL2 KLF3 TSC22D1 TGIF1 FOSL2 JUND THRA MXI1 FOXO4 FOS MAFF PRDM4 NR4A2 NR3C1 ZNF385C SOX5 ZNF318 TFDP1 HMGA1 YBX1 PA2G4 GTF3A IL Repressor Activator - 10: a hi + hi hi b CD24 CD27 (B10) CD24 CD38 20 HD 40 8 HD HD MKD HD MKD + 15 MKD hi hi MKD 30 6
7.5 3.1 CD27 - 10 CD38 CD24 hi + FITC
- 24.4 30.6 20 4
% IgD 5 CD24 10 2 CD27 % CD24 % CD27 0 0 0 MKD 1 MKD 2 MKD 3
CD24-BV605 CD38-PE-Cy7 MKD 1 MKD 2 MKD 3 MKD 1 MKD 2 MKD 3
c d MKD + GGPP HD MKD MKD + GGPP HD MKD 14 6 **** HD 5.5 1.8 3.4 12 MKD 10 * 4
PE 8 ** - B cells +
10 6 -
IL 2 4 % IL-10+ B cells
% IL-10 2 0 0 B10 Trans Naive CD27+ CD27- MKD 1 MKD 2 MKD 3 MKD 4 CD20-APC IgD- e f 20 BLIMP1-APC 1250 HD HD MKD MKD + GSK3i HD MKD MKD + GSK3i + 15 MKD 10 Unstim 1000 5.5 3.3 5.2 CD27 8 - 10 750 Naive HD
PE 6 B cells - % IgD + Memory 5 500 10
- 4 BLIMP1 gMFI IL Naive 0 250
% IL-10 2 MKD 0 0 Memory MKD 1 MKD 2 MKD 3 FSC-A MKD 2 MKD 3 MKD 5 Naive Naive Mem Mem
g h HD MKD HD MKD 60 HD 0.3 FDR=0.112 T T:B T T:B MKD mTOR -
23 14 27 23 40 Akt - Cy7 - suppression PE PI3K
- γ γ 20 FDR<0.001 mTOR genes mTOR - IFN % IFN Akt
- -0.5
0 DN KRAS CD4-FITC PI3K MKD MKD 1MKD 2MKD 3MKD 4 HD Row min Row max