1 1 2 Stimulus-Selective Crosstalk Via the NF-Κb Signaling System

3 Stimulus-selective crosstalk via the NF-κB signaling system reinforces

4 innate immune responses to alleviate gut infection

6 Balaji Banoth1, Budhaditya Chatterjee1, Bharath Vijayaragavan1, MVR. Prasad1, Payel Roy1,

7 Soumen Basak1, *

9 1Systems Immunology Laboratory

10 National Institute of Immunology

11 Aruna Asaf Ali Marg

12 New Delhi-110067

13 India

15 *Correspondence should be addressed to S.B. (e-mail: [email protected])

16 tel: (91) (11) 26703853

17 fax: (91) (11) 26742626

18 1The authors declare no competing financial interest

20 Short title: Signal integration via the NF-κB system

22 Keywords: inflammation, innate immunity, lymphotoxin, Citrobacter rodentium, positive

23 feedback, RelA:p52/NF-κB

1 26 Abstract

27 Tissue microenvironment functions as an important determinant of the inflammatory

28 response elicited by the resident cells. Yet, the underlying molecular mechanisms remain

29 obscure. Our systems-level analyses identified a duration code that instructs stimulus specific

30 crosstalk between TLR4 activated canonical NF-κB pathway and lymphotoxin-β receptor

31 (LTβR) induced non-canonical NF-κB signaling. Indeed, LTβR costimulation synergistically

32 enhanced the late RelA NF-κB response to TLR4 prolonging NF-κB target gene-expressions.

33 Concomitant LTβR signal targeted TLR4-induced newly synthesized p100, encoded by

34 Nfkb2, for processing into p52 that not only neutralized p100 mediated inhibitions, but

35 potently generated RelA:p52/NF-κB activity in a positive feedback loop. Finally, Nfkb2

36 connected lymphotoxin signal within the intestinal niche in reinforcing epithelial innate

37 inflammatory RelA NF-κB response to Citrobacter rodentium infection, while Nfkb2-/- mice

38 succumbed to gut infections owing to stromal defects. In sum, our results suggest that signal

39 integration via the pleiotropic NF-κB system enables tissue microenvironment derived cues

40 in calibrating physiological responses.

2 51 Introduction

52 Tight regulation of inflammatory responses is important; uncontrolled inflammation

53 underlies various human ailments, while insufficient responses limit host defense to

54 pathogens. Tissue resident cells those participate in inflammatory immune activation also

55 exhibit functional differences by adapting to the repertoire of cell-differentiating cues present

56 in distinct microenvironments. Indeed, macrophages and dendritic cells present in different

57 anatomic niche display heterogeneity in inflammatory signatures1,2. Likewise, a requirement

58 for CD40, primarily involved in B-cell maturation, in inflammatory gene-expressions in

59 endothelial cells was documented3. Similarly, lymph node inducing LTβR was shown to be

60 critical for innate immune responses4,5. Yet, the cellular circuitry that function at the

61 intersection of tissue microenvironment derived signals and those impinged upon by pro-

62 inflammatory cytokines or pathogen-derived substances remains obscure.

63 The NF-κB family of transcription factors plays an essential role in activating

64 pathogen-responsive gene-expression program in tissue resident cells. In the canonical NF-

65 κB pathway, inflammatory cues engage NEMO-IKK2 (NEMO-IKKβ) kinase complex to

66 phosphorylate inhibitory IκB proteins, the major isoform being IκBα, bound to the

67 cytoplasmic RelA:p50 NF-κB dimers. Signal induced phosphorylation leads to proteasomal

68 degradation of IκBs and release of RelA:p50 dimers into the nucleus. The nuclear RelA

69 dimers activate the expressions of pro-inflammatory chemokine and cytokine genes as well

70 as its own inhibitor IκBα, which ensures proper attenuation of inflammatory responses in a

71 negative feedback loop. In contrast to canonical signaling, the non-canonical pathway

72 transduces signals from cell-differentiating cues those engage BAFFR, CD40 or LTβR. Non-

73 canonical signaling involves NIK and IKK1 (NIK-IKKα) mediated phosphorylation of Nfkb2

74 encoded precursor p100 bound to RelB6. Subsequent proteasomal processing removes the C-

3 75 terminal inhibitory domain of p100 from RelB:p100 complex to generate RelB:p52 NF-κB

76 dimer, which mediates the expressions of organogenic chemokine genes in the nucleus7.

77 Molecular interaction between the non-canonical signal transducer p100 and RelA has

78 also been charted. In its homo-oligomeric form, termed IκBδ, p100 was shown to utilize its

79 inhibitory domain to sequester a subpopulation of the RelA:p50 dimer8,9. LTβR through non-

80 canonical NIK-IKK1 signal inactivates IκBδ to induce a weak yet sustained RelA:p50

81 activity. Conversely, RelA induced synthesis of p100 and consequent accumulation of

82 inhibitory IκBδ was shown to exert negative feedback limiting canonical RelA activity10-12.

83 In addition, an alternate RelA:p52 dimer has been reported which is thought to constitute a

84 minor kappaB DNA binding activity13. Crosstalk between apparently distinct cell signaling

85 pathways are known to offer diversity in cellular responses. Despite these connectivities, a

86 plausible role of signal integration via the NF-κB system in regulating inflammatory RelA

87 NF-κB responses has not been investigated.

88 In a multidisciplinary study combining biochemistry, genetics and mathematical

89 modeling, here, we characterized a duration code that determines stimulus specific crosstalk

90 between canonical and non-canonical signaling in fine-tuning inflammatory RelA NF-κB

91 activity. Through such crosstalk, LTβR sustained TLR4 triggered RelA NF-κB responses by

92 supplementing RelA:p52 NF-κB dimer in a positive feedback loop. Finally, we established

93 the physiological significance of crosstalk control of RelA in intestinal epithelial cells (IECs),

94 where, the NF-κB system integrates gut microenvironment derived lymphotoxin signals

95 through Nfkb2 to calibrate innate immune responses to Citrobacter rodentium.

99 Results

4 100 A duration code controlling crosstalk between canonical and non-canonical NF-κB

101 signaling

102 Given the interconnectedness of the canonical and non-canonical arms (see

103 Introduction and Figure 1A), we asked if signal integration via the NF-κB system would

104 allow cell-differentiating cues to modulate inflammatory RelA NF-κB responses.

105 Mathematical reconstruction of dynamic networks illuminate emergent properties, such as

106 crosstalk14. To explore crosstalk control, we developed a mathematical model, which we

107 termed the NF-κB Systems Model v1.0 (Appendix-1), basing on previously published single

108 NF-κB dimer model versions9,15. In our mathematical model, however, we depicted nuclear

109 activation of both the major RelA:p50 dimer and RelA:p52 dimer, which is thought to

110 constitute a minor RelA NF-κB activity. As described in the preceding single dimer

111 models9,15, signal responsive degradation and resynthesis of IκBα, IκBβ, IκBε and inhibitory

112 p100/IκBδ dynamically controlled RelA activity. The model was parameterized based on

113 literature, our own measurements (Appendix-1, Appendix figure 1-5, Supplementary file 1-3)

114 and fitting procedures. Simulating individual TNFR or LTβR regime, we could recapitulate

115 experimentally observed strong, but temporally controlled, activation of RelA NF-κB

116 complexes during canonical IKK2 signaling or the weak induction of RelA dimers during

117 non-canonical NIK-IKK1 signaling, respectively (Figure 1B, Figure 1 -figure supplement 1).

118 Next, we examined potential crosstalk between IKK2 and NIK-IKK1 inputs in

119 augmenting RelA NF-κB response in silico. To this end, we generated a theoretical library11

120 of 356 kinase activity profiles, where each member posses distinct peak onset time, peak

121 amplitude and duration (Figure 1C, Figure 1 - figure supplement 2A). To screen for

122 permissive crosstalk conditions, we fed this library into the model through IKK2 or NIK-

123 IKK1 or both the arms and iteratively simulated respective RelA activities. Then we

124 computed RelA responses in the co-treatment regime relative to individual cell stimulations

5 125 to assign crosstalk indexes to different IKK2 and NIK-IKK1 combinations (Figure 1D).

126 Plotting the dynamic features of the crosstalk proficient kinase inputs, we could reveal a

127 critical duration threshold; where IKK2 activities sustained for more than 2h were more

128 likely to engage into crosstalk for varied peak amplitudes and inputs with shorter duration

129 were crosstalk inefficient (Figure 1E and Figure 1 - figure supplement 2B). Illustrating a

130 similar but more elaborate duration control, NIK-IKK1 activities longer than 8h selectively

131 participated into crosstalk with the canonical pathway.

132 Inflammatory mediators activate canonical IKK2 with disparate temporal controls.

133 Consistent to the prior report16, our kinase assay (Materials and Methods) revealed that IL-

134 1β, an important pro-inflammatory cytokine, only transiently activates IKK2 in mouse

135 embryonic fibroblasts (MEFs) (left panel, Figure 1F). In contrast, bacterial LPS through

136 TLR4 induced IKK2 activity that persisted above the basal level even at 24h post-stimulation

137 (right panel, Figure 1F, Figure 1 - figure supplement 3B)17. Mimicking prolonged signaling

138 during cell-differentiation processes, LTβR engagement using agonistic αLTβR antibody led

139 to sustained activation of the non-canonical NIK-IKK1 (Figure 1G and Figure 1 - figure

140 supplement 3A and B). Using these experimental kinase activities as inputs, our

141 computational simulations revealed insulation of IL-1R signaling from LTβR mediated

142 crosstalk (left panel, Figure 1H), but robust crosstalk between TLR4 and LTβR that amplified

143 late RelA response upon costimulation (right panel, Figure 1H). Therefore, our mathematical

144 analyses predicted that a duration code selectively engages long lasting canonical kinase

145 activities into crosstalk with LTβR induced NIK signal to impart stimulus specificity.

146

147

148

6 149 Stimulus specific crosstalk allows LTβR signal to prolong TLR4 induced RelA NF-κB

150 response

151 To experimentally verify stimulus specificity of crosstalk control, we measured

152 nuclear RelA activities induced in MEFs by canonical or non-canonical inducers or co-

153 treatment regime that concomitantly activated both the pathways. IL-1R signal, in parallel to

154 transient IKK2 activation, elicited strong RelA activity at 30min in EMSA that was largely

155 attenuated within 1h, whereas, non-canonical LTβR signal only weakly induced RelA and

156 RelB dimers those persisted even at 24h (Figure 2A). Indeed, we were unable to detect any

157 significant enhancement of RelA activity, relative to IL-1 induced peak, upon costimulation

158 (Figure 2A). In comparison, canonical TLR4 induced a temporally distinct RelA NF-κB

159 activity with an early peak at 1h, subsequent descend and a progressively weakened late

160 phase between 8-24h (Figure 2B). Corroborating our mathematical prediction, concomitant

161 LTβR signal sustained NF-κB response triggered by TLR4 (Figure 2B). Signal integration

162 via the NF-κB system synergistically enhanced TLR4-induced late RelA activity at 24h in the

163 costimulation regime with mostly unaltered RelB response relative to solitary LTβR

164 engagement (Figure 2B, quantification and Figure 2C). Sequentially engaging MyD88 and

165 Trif, TLR4 was shown to produce extended IKK2 activity17. To determine if the observed

166 stimulus specificity of crosstalk is indeed due to the duration of IKK2, we utilized Trif-

167 deficient MEFs that only transiently activated IKK2 upon LPS treatment (Figure 2D).

168 Despite a functional non-canonical pathway (Figure 2 - figure supplement 1), LTβR was

169 restricted from crosstalk with TLR4 in Trif-deficient cells (Figure 2E), thereby, suggesting a

170 Trif dependent mechanism that relies on the duration of canonical IKK2 in imparting

171 stimulus specificity of crosstalk control.

172

7 173 Signal integration via the NF-κB system amplifies the late expressions of TLR4 induced

174 pro-inflammatory genes

175 Long-lasting kinase activities are expected to elicit sustained RelA responses. Then

176 what might be the significance of signal integration via the NF-κB system? Interestingly,

177 computational simulations demonstrated only a muted increment in the RelA activity with

178 increasing duration of IKK2 (Figure 3A). LTβR induced NIK-IKK1 signal relieved this

179 saturation to fully unravel the NF-κB activation potential of long duration IKK2 signals upon

180 crosstalk. We postulated that the difference in the RelA responses induced by long-lasting

181 IKK2 in the presence or absence of non-canonical signal would decode into differential gene

182 activities.

183 To evaluate the potential gene-effect of crosstalk, we measured the expressions of

184 several known RelA target chemokine and cytokine genes using quantitative RT-PCR. Our

185 analyses revealed that IL-1 treatment rapidly induces the expressions of TNF, IP-10 and

186 MIP-1α mRNAs within 1h with residual expressions at 24h post-stimulation (Figure 3B).

187 The levels of IL-1β and RANTES mRNAs were insensitive to IL-1 treatment. Also, weak

188 LTβR signal alone did not significantly induce the expressions of these pro-inflammatory

189 genes in MEFs. Indeed, LTβR costimulation was ineffective in augmenting IL-1 induced

190 early or late expressions of the chemokine and cytokine genes (Figure 3B). Solitary LPS

191 treatment not only robustly induced the expressions of TNF, IP-10 and MIP-1α mRNAs, but

192 also led to late accumulation of IL-1β and RANTES mRNAs at 24h (Figure 3C). Consistent

193 to our hypothesis, LTβR costimulation prolonged TLR4-induced gene-expressions with

194 further augmented late, but not early, expressions of IL-1β, IP-10, MIP-1α, and RANTES

195 mRNAs. TNF mRNA levels were insensitive to crosstalk regulation (Figure 3C).

8 196 Furthermore, we used microarray analyses to compare global gene expressions

197 activated by TLR4 or LTβR or both at 24h post-stimulation. Estimating normalized crosstalk

198 scores (bottom panel, Figure 3D, Figure 3 – source data 1, Materials and Methods), we could

199 reveal a synergistic effect of LTβR on TLR4 stimulated late gene-expressions in WT MEFs.

200 Out of 943 LPS induced genes, however, a select set of 114 genes was further upregulated

201 upon costimulation. Strikingly, gene set enrichment analysis (GSEA)18 (Figure 3 – source

202 data 2, Materials and Methods) demonstrated an enrichment of NF-κB targets among genes

203 positively controlled through crosstalk (middle and top, Figure 3D). We have also noted

204 downregulation of several LPS-induced genes in the costimulation regime those appeared

205 less likely to be NF-κB targets in GSEA. Taken together, these analyses substantiated an

206 important function of prolonged RelA activity in crosstalk-amplification of TLR4-induced

207 late expressions of NF-κB target genes, particularly those encode pro-inflammatory

208 chemokines and cytokines.

209

210 Non-canonical signal transducer Nfkb2 supplements RelA:p52 dimer to sustain

211 canonical RelA NF-κB responses

212 To determine the mechanism underlying signal integration via the NF-κB system, we

213 individually perturbed 105 model parameters and quantified relative changes in the crosstalk

214 index (Materials and Methods). Our parameter sensitivity analysis identified the rate constant

215 associated with the NF-κB induced transcription of Nfkb2 as the most critical parameter

216 underlying crosstalk control of RelA NF-κB activity (Figure 4A). As such, Nfkb2 encodes for

217 both NF-κB inhibitor and NF-κB precursor functions. Computationally simulating individual

218 RelA:p50 and RelA:p52 nuclear activities in the LPS, αLTβR or costimulation regimes, we

219 could further suggest that the Nfkb2 precursor function in generating RelA:p52 dimer is

220 important for augmenting RelA NF-κB activity during crosstalk in WT system (Figure 4B).

9 221 Consistently, our modeling analyses predicted complete abrogation of crosstalk in Nfkb2-/-

222 cells (Figure 4B).

223 Our biochemical studies revealed that LPS induces RelA NF-κB driven transcription

224 of Nfkb2 to produce p100 (top panel, Figure 4C and Figure 4 - figure supplement 1A), which

225 was shown to oligomerize as NF-κB inhibitory IκBδ 8,11,19. Concomitant LTβR signal instead

226 utilized TLR4-induced, newly synthesized p100 to potently generate p52, thereby,

227 neutralizing the inhibitory p100 function (Figure 4C). p50 levels were not discernibly altered

228 in these stimulation regimes in our experiments. Our immunoprecipitation based analysis

229 demonstrated that NIK-IKK1 signal relieves RelA from p100/IκBδ mediated inhibition

230 during crosstalk (Figure 4D). Intriguingly, LTβR costimulation of MEFs for 24h also

231 produced ~4 fold more RelA:p52 NF-κB dimer as compared to solitary LPS treatments

232 (Figure 4D). Limited transcriptional up-regulation of Nfkb2 by weak LTβR signal was

233 correlated with only modest p52 and RelA:p52 generation (Figure 4C and 4D). In supershift

234 assay, we could ascertain that RelA:p52 dimer generated upon LTβR costimulation appears

235 as a strong nuclear DNA binding activity at 24h (Figure 4E) to supplement to the TLR4-

236 induced RelA NF-κB responses. An important role of dimerization in stabilizing NF-κB

237 monomers from degradation has been reported earlier 20. Interestingly, RelA protein rapidly

238 accumulated in cells upon proteasome inhibition (Figure 4F) suggesting a robust constitutive

239 degradation mechanism that offsets basal synthesis of RelA monomers in maintaining the

240 steady-state level. Our study indicated that this enduring flux ensures copious supply of RelA

241 to bind to de novo generated p52, produced from the newly synthesized p100 during

242 crosstalk.

243 Next, our genetic analyses revealed that canonical RelA:p50 response to TLR4 signal,

244 primarily controlled through classical IκBs, is largely intact in Nfkb2-/- with early induction

245 and diminished late activities comparable to WT MEFs (Figure 4G and Figure 4 - figure

10 246 supplement 1B). Consistent to the prediction based on computational modeling studies, a lack

247 of RelA:p52 dimer generation in Nfkb2-/- cells, however, ablated LTβR mediated

248 enhancement of TLR4-induced late RelA DNA binding activity (Figure 4G) as well as

249 crosstalk amplification of RelA target pro-inflammatory gene-expressions (bottom panel,

250 Figure 4H). LTβR costimulation not only enhanced TLR4 induced RelA DNA binding, but

251 also activated RelB dimers through the non-canonical pathway. Prior reports have indicated

252 cell-type specific inhibitory as well as activating role of RelB in chemokine gene-

253 expressions21,22. Importantly, costimulation of Relb-/- MEFs led to similar hyperactivation of

254 LPS-induced late expressions of IL-1β, IP-10, and RANTES mRNAs as in WT cells

255 (compare top panel, Figure 4H with Figure 3C). Although, the crosstalk effect on MIP-1α

256 expressions was somewhat muted owing to prolonged expression of this gene in Relb-/- MEFs

257 in response to solitary LPS treatment. Therefore, our analyses confirmed that the precursor

258 function encoded by Nfkb2 in generating RelA:p52 NF-κB dimer is critical for integrating

259 lymphotoxin derived signals to the pro-inflammatory RelA NF-κB pathway. Our analyses

260 also suggested that LTβR costimulation led to the hyperactivation of LPS-induced

261 expressions of chemokine and cytokine genes in an Nfkb2 dependent manner with only a

262 minor, if any, role for Relb.

263

264 Inducible synthesis of Nfkb2 by canonical signal triggers a positive feedback loop during

265 crosstalk

266 Given the computational prediction for an important role of NF-κB induced

267 transcription of Nfkb2, we compared the inducible expression of the crosstalk-mediator Nfkb2

268 in response to LPS or IL-1 to understand the molecular basis of stimulus specific control. As

269 opposed to rapid expression of Nfkbia mRNA, which encodes IκBα, LPS induced Nfkb2

270 mRNA with a delay (top panel, Figure 5A). Similarly, chronic TNF treatment induced Nfkb2

11 271 mRNA in WT MEFs with an explicit 1h delay (Figure 5B) that was also observed earlier and

272 incorporated in both the previous9 as well as the current mathematical model versions.

273 Analogous time lags were observed in the expression of several inflammatory genes those

274 require additional chromatin modifications for the initiation of RelA induced transcription23.

275 When Nfkb2 transgene was stably expressed in Nfkb2-/- MEFs from an exogenous NF-κB

276 responsive promoter, Nfkb2 mRNA was readily induced by TNF without a delay (Figure 5B).

277 Remarkably, IL-1 treatment was ineffective in activating the expression of Nfkb2 mRNA in

278 WT MEFs, despite the early induction of Nfkbi (bottom, Figure 5A). Only upon eliminating

279 the transcriptional delay, our mathematical model could simulate Nfkb2 mRNA induction by

280 IL-1 treatment (Figure 5C). Indeed, we could also experimentally rescue the defect in Nfkb2

281 mRNA induction by IL-1 treatment in the engineered Nfkb2-/- cell-line, which expresses

282 Nfkb2 transgene from the NF-κB responsive promoter without the delay (Figure 5D).

283 Consistent to our computational identification that NF-κB inducible transcription of Nfkb2 is

284 important, disruption of NF-κB inducible synthesis by expressing p100 from a constitutive

285 promoter in Nfkb2-/- MEFs abrogated crosstalk-amplification of TLR4 induced late NF-κB

286 activity by concomitant LTβR signal.(Figure 5E and Figure 5 - figure supplement 1A). While

287 the NF-κB responsive expression of Nfkb2 transgene in Nfkb2-/- cells restored the crosstalk

288 effect at the level of RelA NF-κB activation (Figure 5E) by potentiating RelA:p52 induction

289 in LPS+αLTβR costimulation regime (Figure 5F, Figure 5- figure supplement 1B).

290 Our results confirmed that signal induction of Nfkb2 is important for crosstalk and

291 suggested that a promoter intrinsic delay necessitates persistent canonical signal for RelA

292 mediated induction of pro-synergistic Nfkb2. Such delay encoding insulated IL-1R signaling,

293 which transiently activates IKK2 and RelA by restricting Nfkb2 mRNA expressions, and

294 accounted for the abrogated crosstalk in Trif-deficient MEFs that transiently activated the

295 NF-κB pathway. Our studies also explained the requirement for the long-duration NIK-IKK1

12 296 signals in targeting this late acting p100 Nfkb2 feedback for RelA:p52 dimer generation. In

297 contrast, IL-1 signal led to early induction of Nfkb2 mRNA expressions in the engineered

298 cells, which inducibly express Nfkb2 transgene without the delay (Figure 5D). Pretreatment

299 of these engineered cells with αLTβR for 8h and subsequent IL-1 stimulation that effectively

300 converged the non-canonical signal to IL-1 induced Nfkb2 feedback, potentiated p52

301 production (Figure 5 - figure supplement 1C) and prolonged IL-1 induced RelA response

302 (Figure 5G and Figure 5 - figure supplement 1D). Correlating with the early onset of Nfkb2

303 mRNA induction in response to IL-1 treatment, observed crosstalk effects were indeed

304 obvious within 1h of IL-1 treatment in these engineered cells.

305 In sum, we elucidate a crosstalk mechanism that discriminates between TLR4

306 engagement and concomitant cell activation through TLR4 and LTβR (Figure 5H). Negative

307 feedbacks by IκBα and p100/IκBδ coordinately terminate canonical TLR4 response. But,

308 Nfkb2 functions pro-synergistically upon costimulation; in a positive feedback loop, non-

309 canonical LTβR signal targets the newly synthesized p100, abundantly produced by TLR4, to

310 potently generate p52 and RelA:p52 dimers in sustaining inflammatory RelA NF-κB

311 responses. Importantly, RelA:p50 and RelA:p52 heterodimers were shown to share DNA

312 binding and gene-expression specificities24,25. Our experimental data also indicated that

313 RelA:p52 dimer has comparable efficiency in inducing the expression of Nfkb2 mRNA as the

314 RelA:p50 dimer (Appendix-1, Appendix figure 4C). Although, emergent crosstalk is

315 expected to be controlled by several biochemical constrains, the transcriptional delay intrinsic

316 to the Nfkb2 promoter appears to be critical for the duration code and thereby the stimulus

317 specificity.

318

319 Nfkb2 integrates lymphotoxin signal within intestinal niche to reinforce epithelial NF-

320 κB responses to Citrobacter rodentium

13 321 In addition to its role in lymph node development during embryogenesis, recent

322 studies have illustrated a requirement for LTβR in innate immune responses in adult mice.

323 Disruption of LTβR signal using LTβR-Ig fusion protein was shown to compromise innate

324 immune responses upon subsequent infection with Citrobacter rodentium, a natural mouse

325 enteric pathogen that led to mortality4,5. IEC specific deletion of LTβR similarly obliterated

326 bacterial clearance5. The engagement of ligand-expressing RORγt+ innate lymphoid cell is

327 thought to provide the critical lymphotoxin signal in colon during the course of bacterial

328 infection26. A requirement of epithelial RelA activity in the chemokine gene-expressions has

329 been documented earlier27. Given our identification of a costimulatory function of LTβR in

330 inflammatory RelA activation, we asked if signal integration via the NF-κB system could

331 explain the epithelial requirement of LTβR in innate immune responses in vivo.

332 First, a functional non-canonical pathway downstream of LTβR (Figure 6 - figure

333 supplement 1A) augmented the RelA activity induced by pathogen sensing TLR4 in

334 otherwise hypo-responsive MSIE colon epithelial cell-line (Figure 6A). Next, we

335 biochemically analyzed NF-κB activation in IECs derived from WT mice intraperitoneally

336 injected with antagonistic LTβR-Ig or a control-Ig one day prior to oral infection with C.

337 rodentium. Upon colonization, Citrobacter initially triggered epithelial accumulation of p100

338 that was fully processed into p52 by day5 (Figure 6B) generating RelA:p52 dimer (Figure

339 6C) in control-Ig, but not LTβR-Ig, treated mice. Bacterial infection elicited RelA DNA

340 binding activity in IECs that gradually accumulated in the nucleus (Figure 6D and 6E) with

341 substantial contribution from RelA:p52 dimer along with RelA:p50 dimer at day5 post-

342 infection (Figure 6 – figure supplement 1B). Our supershift analyses further confirmed

343 complete absence of RelB containing NF-κB DNA binding activity in IECs derived from

344 infected mice (Figure 6E). Perturbing LTβR signal attenuated RelA NF-κB activation with

14 345 more obvious defects at day5 (Figure 6D). Likewise, pathogen-responsive RelA activation in

346 IECs derived from Nfkb2-/- mice was severely weakened at day5 (Figure 6F) that led to

347 significantly reduced expressions of the RelA target chemokines encoding KC and MIP-2α

348 as compared to WT mice (Figure 6G). Indeed, infected Nfkb2-/- mice exhibited diminished

349 neutrophil recruitment in the lamina propria, as revealed by anti-myeloperoxidase

350 immunostaining of the colon sections (Figure 6H). Sustained epithelial RelA activity that

351 relies on LTβR mediated processing of pathogen-induced p100 into p52, therefore, mirrored

352 our MEF based analyses depicting crosstalk between canonical and non-canonical signaling.

353 Collectively, our results connected the previously reported epithelial requirement of LTβR5

354 and NIK28 in innate immune response to the NF-κB system in reinforcing RelA activity

355 through Nfkb2 mediated crosstalk control. Subdued epithelial NF-κB activation, and not

356 hyper-induction, in IECs from infected Nfkb2-/- mice also suggested that a dominant

357 precursor function of p100 supplying RelA:p52 dimer prolongs RelA response within the

358 intestinal niche.

359

360 Stromal expression of Nfkb2 is required for limiting C. rodentium infection

361 While WT mice efficiently eliminated infections, increased fecal excretion of bacteria

362 at day10 post-infection in Nfkb2-/- mice (Figure 7A) indicated an inadequacy in limiting local

363 infection, thereby, correlating with the observed defects in the early innate inflammatory

364 response in this knockout (Figure. 6). Histological analysis of the shrunken colons (Figure 7 -

365 figure supplement 1A and B) derived from the infected Nfkb2-/- mice further revealed

366 exacerbated damage with signatures of submucosal leukocyte infiltration (Figure 7B). Breach

367 in the intestinal barrier was accompanied by systemic bacterial dissemination with increased

368 count in blood (left panel, Figure 7C) and liver (right, Figure 7C). Finally, bacterial colitis

369 induced in Nfkb2-/- mice resulted in significant body weight loss (Figure 7D) and onset of

15 370 mortality as early as day10 post-infection (Figure 7E). Next, we performed reciprocal bone

371 marrow transfer experiments between WT and Nfkb2-/- mice to ensure that the observed

372 sensitivity was not due to previously reported hematopoietic defects in Nfkb2-/- mice29. WT

373 bone marrow cells in Nfkb2-/- recipients (Figure 7 - figure supplement 1C) were unable to

374 prevent the infection-related colon pathology (Figure 7F), reductions in the body weight

375 (Figure 7G) and morality (Figure 7H). In contrast, WT recipients receiving either WT or

376 Nfkb2-/- bone marrow resolved infections with comparable efficiencies (Figure 7F-7H).

377 In addition to modulating RelA activity through crosstalk, Nfkb2 also mediates

378 RelB:p52 activation in response to singular non-canonical stimuli. Heightened RelB:p52

379 activation in B-cells was shown to strengthen host defense in Otu7b-/- mice30. Our inability to

380 rescue the infection-related mortality in Nfkb2-/- mice using WT hematopoietic cells indicated

381 that the protective function of Nfkb2 lies within stromal cells. Despite the presence of

382 mesenteric lymph nodes31, additional stromal defects in Peyer’s patches in Nfkb2-/- mice32

383 could impair mucosal IgA responses. Although IgM levels were comparable, very low IgA

384 levels at day7, prior to the onset of mortality in Nfkb2-/- recipients, precluded bona fide

385 comparisons in the radiation chimeras. Interestingly, adult mice, similar to those utilized in

386 our study, lacking IgA or its transporter pIgR efficiently resolved Citrobacter infections33.

387 μMT mice depleted of peripheral B-cells presented few mucosal changes in the first two

388 weeks of infection and were completely free of mortality34. Finally, Rag1-/- mice succumbed

389 to Citrobacter only late during infections in the fourth week5. Though, our study does not

390 rule out crosstalk independent engagement of the Nfkb2 pathway in activating RelB:p52

391 dimer in the hematopoietic compartment at a later stage of infection, epithelial RelA NF-κB

392 activation defects coupled to aggravated early colon pathology and early onset of mortality in

393 Nfkb2-/- mice suggested that the stromal requirement of Nfkb2, at least in part, lies within the

16 394 intestinal epithelial cells in the initial events controlling early innate immunity and involves

395 crosstalk regulation of RelA NF-κB activity (Figure 7I, also discussed later).

396

17 397 Discussion

398 The NF-κB system transduces signals from a variety of cell-activating stimuli. Our

399 study suggested that such pleiotropic system enables tuning of cellular response to an

400 instructive signal by microenvironment-derived additional costimulatory signals. However, a

401 duration code selectively integrates costimulatory LTβR signal with the TLR4 pathway,

402 insulating transient cytokine signaling, secondary to microbial infections, from crosstalk

403 amplifications. Recent studies have identified important positive feedback regulations

404 underlying dose threshold control of NF-κB response in B-cells35 or in myeloid cells36. Our

405 crosstalk study illuminated a role of a positive feedback loop in sustaining NF-κB response;

406 where non-canonical LTβR signal prolonged canonical TLR4 response by targeting RelA

407 induced p100 for the generation of RelA:p52 NF-κB dimer. Indeed, a dominant precursor

408 function of p100 in producing RelA:p52 led to ablated crosstalk in Nfkb2-/- cells. As such,

409 non-canonical signal generates RelB:p52 by removing the C-terminal domain of p100 present

410 in the preexisting RelB:p100 dimeric complex6 or liberates RelA:p50 dimer from the

411 p100/IκBδ inhibited complexes9. Signal generation of the RelA:p52 dimer requires both

412 canonical induction of p100/Nfkb2 expressions and concomitant processing of the newly

413 synthesized p100 into p52 by non-canonical signal. Previous report demonstrating that p100

414 could be efficiently processed cotranslationally37 explained the requirement of canonical

415 signals in amply synthesizing nascent p100 as a substrate for the abundant production of p52

416 subunit during crosstalk. More so, mature p52 could readily dimerise with RelA, despite a

417 preference of full-length p100 for RelB binding20. These observations along with our current

418 study, thereby, elaborated a requirement of convergence of canonical and non-canonical

419 signals in synergistically generating and activating RelA:p52 dimer. Nevertheless, consistent

420 to the observed gene-effect of crosstalk in our study, a significant overlap between RelA:p50

421 and RelA:p52 dimers in DNA binding25 and pro-inflammatory gene expressions13 was

18 422 reported earlier. Of note, our results rely on bulk measurements of signaling intermediates

423 and deterministic modeling approaches. Given studies documenting cell-to-cell variations in

424 signal induced NF-κB responses38, it would be interesting to examine the potential

425 implication of signal integration at the single cell level in determining cellular heterogeneity.

426 TLR4 activation of epithelial RelA was implicated in the chemokine gene expressions

427 and neutrophil recruitment upon bacterial infections39. Yet, epithelial LTβR5 was also

428 important for effective innate immune responses to Citrobacter. In our proposed model

429 (Figure 7I), we could clarify that LTβR provides a critical costimulatory signal through

430 Nfkb2 to sustain RelA NF-κB response to pathogens in otherwise hyporesponsive colonic

431 epithelial cells. Such signal integration ameliorated innate immune functions by enhancing

432 pro-inflammatory gene-expressions. Interestingly, p100-/- mice, which lacked the expression

433 of p100, but aberrantly produced p52, revealed hyperplasia of gastric epithelial cells and

434 elevated expressions of RelA target genes40. In Nlrp12-/- mice, robust p52 generation in

435 stromal cells through the non-canonical pathway led to colon cancer associated

436 inflammation41, a hallmark for aberrant RelA activity. These studies indeed support a

437 possible role of Nfkb2 in mucosal epithelial cells in strengthening RelA activity. LTβR

438 engagement in the dendritic cells within colonic patches was shown to trigger IL-22

439 production by innate lymphoid cells involving cell-cell communications to potentiate gut

440 immunity42. Although, a defect in the colonic patches in Nfkb2-/- mice could impair IL-22

441 mediated protective responses, our cell-intrinsic crosstalk model explained that the reported

442 epithelial requirement of LTβR5 is in sustaining RelA NF-κB response during bacterial

443 infection. In future, tissue-specific knockouts may help to further distinguish between innate

444 immune functions of Nfkb2 in different cell-types.

445 The underlying mechanism and biological functions of RelB:p52 dimer activated by

446 non-canonical signal is well-established. From the perspective of signaling crosstalk, our

19 447 study offers a significant revision of our understanding of non-canonical signaling in

448 amplifying canonical RelA responses. It extends the intriguing possibility that cell-

449 differentiating cues present in the tissue microenvironment may play a more direct role,

450 separate from merely determining the differentiation states of the resident cells, in calibrating

451 innate immune responses by engaging into cell-autonomous signaling crosstalks. Future

452 studies ought to further examine the signal integration via Nfkb2 in potentiating immune

453 responses against other microbial pathogens. More so, the potential involvement of the

454 deregulated crosstalk control in the pathophysiology of inflammatory disorders, particularly

455 those involving gut, remains to be addressed.

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20 472 Materials and Methods

473 Mice, cells and recombinant DNA. Wild-type or gene-deficient C57BL/6 mice were

474 housed at NII small animal facility and used in accordance with the IAEC guidelines.

475 Primary MEFs were generated from E12.5–14.5 embryos. Late passage Trif-deficient and

476 NIK-deficient MEFs have been described 9. MSIE cell-line was a gift from R. Whitehead,

477 Ludwig Cancer Research. Mouse Nfkb2 was stably expressed from a promoter containing

478 five tandem kappaB sites from HRS.puro or from a constitutive promoter from pBabe.puro

479 retroviral constructs.

480

481 Biochemical analyses. Cells were stimulated using 0.3μg/ml αLTβR (a gift from J.

482 Browning and A. Papandile, Biogen, Cambridge, MA, USA), 100ng/ml recombinant LTα1β2

483 (Sigma, St. Louis, MO, USA), 1ng/ml TNF (Roche, BASEL, Switzerland), 1ng/ml IL-1β

484 (Biosource, Carlsbad, CA, USA) or 1μg/ml LPS (Enzo, NY, USA), either individually or in

485 combination. EMSA, Immunoblot analyses and IKK assay have been described earlier 9. NIK

486 was immunoprecipitated (Cell Signaling Technology, Danvers, MA, USA) from cytoplasmic

487 extracts and immunopellets were examined for kinase activity using GST-IκBδ as substrate

488 (GST-p100406-899, BioBharati Life Sciences, Kolkata, India). The gel images were acquired

489 using PhosphorImager (GE, Amersham, UK) and quantified in ImageQuant. Immunobloting

490 of Immunoprecipitates was done using TrueBlot (eBioscience, San Diego, CA, USA).

491

492 Gene expression studies. Total RNA was isolated using RNeasy Kit (Qiagen, Venlo,

493 Netherlands). For microarray analysis, labeling, hybridization of RNA samples to the

494 Illumina MouseRef-8 v2.0 Expression BeadChip, data processing and quantile normalization

495 was performed by Sandor Pvt. Ltd (Hyderabad, India). We have considered genes that are

496 induced at least 1.3 fold by LPS at 24h in representative data sets and has a detection p-

21 497 value<0.05 for LPS, αLTβR and co-treatment regimes. Next, LPS response genes in WT 498

MEFs were ranked based on the merit of their normalized crosstalk scores, which is defined 499 below and also described earlier43.

500 Crosstalk score = [(Δco-treatment – (ΔLPS+ΔαLTβR))/0h_int], where 0h_int indicates the

501 signal intensity of a given gene in untreated cells and Δtreatment signifies the 502 differences in signal intensities between treated and untreated cells,

503 Normalized crosstalk score = Crosstalk Score * [{(ΔL+ ΔB))/0h_int/|(ΔL+ ΔB))/0h_int|],

504 As implied, positive crosstalk scores signify hyperactivation, whereas negative

505 crosstalk scores imply diminished gene-expressions in the co-treatment regime as compared

506 to cell treatment with the individual stimuli. The ordered gene set was examined in GSEA

507 v2.0.12 (Broad Institute at MIT)18. The MIAME version of the microarray data set discussed

508 in this publication are available on NCBI Gene Expression Omnibus (accession number

509 GSE62301). For quantitative RT-PCR, total RNA was reversed transcribed with Transcriptor

510 cDNA kit and amplified using Sybr Green PCR mix (Roche) in ABI7500 cycler. The

511 Relative gene expressions were quantified using ΔΔCT method upon normalizing to β-actin

512 mRNA level. Absolute quantification was done using plasmid DNA constructs encoding

513 respective genes as standards and normalized to express as gene/actin mRNA level.

514

515 Murine infection model. Sex matched, 8-10 week old mice, fasting for 8h, were orally

516 gavaged with 1.2x1010 cfu of C. rodentium strain DBS100 (ATCC 51459). In certain

517 instances, mice were intraperitoneally injected with 200μg of murine LTβR-IgG1 fusion

518 protein or MOPC21 isotype control (Biogen Idec) one day prior to infection, as described 5.

519 IECs, isolated following published procedure 44, were utilized for biochemical analyses. For

520 histology, dissected colons were fixed in 10% neutral buffered formalin. Paraffin-embedded

521 tissue sections were stained with anti-myeloperoxidase antibody (Pierce, Waltham,

22 522 Massachusetts, USA) for neutrophil recruitment or with Hematoxylin and Eosin (H&E) for

523 tissue pathology evaluation. Fecal samples were weighted, homogenized and serially diluted

524 homogenates were plated on MacConkey agar (HiMedia, Mumbai, India) to score for C.

525 rodentium. Similarly, spleens and livers were aseptically removed and assessed for bacterial

526 load. For bone marrow chimera experiment, recipient WT or Nfkb2-/- mice were lethally

527 irradiated and marrow cells from the indicated donor mice were transferred. After 6-8 weeks,

528 mice were infected.

529

530 Computational modeling. The NF-κB Systems Model v1.0 was simulated in Matlab (v.

531 2012b, Mathworks, Natick, MA, USA) using the ode15s 9. A detailed description of the

532 model has been provided in the Appendix-1. To estimate crosstalk sensitivity, each parameter

533 values were individually increased and decreased by 10%, euclidean distances were used to

534 determine the resultant changes in the crosstalk indexes as compared to the unperturbed

535 system, averaged for a given parameter and normalized to nominal crosstalk index.

536

537 Statistical analysis. Data are expressed as mean of 3-5 quantified biological replicates ±

538 SEM. Statistical significance was calculated by two-tailed Student’s t-test. For survival

539 curves log rank (Mantel-Cox) test were conducted.

540

23 541 Author contributions

542 B.B., B. V. and P. R. did the wet lab experiments, B. C., and M. P., carried out the in

543 silico work, S.B. supervised the research and wrote the manuscript with B. B. The authors

544 declare no competing interest.

545

546 Additional files

547 This article additionally contains i) ten figure supplements associated with the main

548 text, ii) three Supplementary tables (Supplementary file 1-3) and five Appendix figures

549 associated with the an Appendix file (Appendix-1), which provides a detailed description of

550 the mathematical model, as well as a file describing Matlab source codes.

551

552 Acknowledgements

553 We thank Alexander Hoffmann, UCLA for insightful discussions, Gouri Ghosh,

554 UCSD and Satyajit Rath, NII for critical reading of the manuscript. We thank P. Nagarajan,

555 SAF and V. Kumar, SIL for technical help. This study was supported by an intermediate

556 fellowship to SB from the Wellcome Trust DBT India Alliance and funding from NII-Core.

557 BB and PR thank CSIR for research fellowships.

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565

24 566 References

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27 703 Appendix-1

704 Appendix-1 includes, a description of the mathematical model, supporting references, and

705 associated Appendix figures 1-5, as well as Supplementary files 1-3.

706

707 i) Description of the Mathematical Model

708 The NF-κB Systems Model Version 1.0 builds upon a previously constructed computational

709 model9. In the preceding version, canonical IKK2 signals degraded classical IκBs, while

710 non-canonical NIK-IKK1 targeted IκBδ to liberate the RelA:p50 NF-κB dimers, represented

711 as a singular species, into the nucleus. Although, biochemical analyses defined IκBδ as an

712 oligomer of p100, the previous versions represented IκBδ as a composite species that is

713 produced directly from an NF-κB responsive gene. In the current version, we have

714 elaborated biochemical reactions associated with p100 in the model. We have explicitly

715 described p100 as a species that is generated from the transcripts encoded by NF-κB

716 responsive Nfkb2. Monomer p100 was subjected to i) constitutive degradation or ii)

717 oligomerization to form IκBδ or iii) NIK-IKK1 dependent processing that generated

718 RelA:p52 dimer as a composite species. In contrast, RelA:p50 dimer was constitutively

719 produced as a singular species. As described in the preceding single dimer model, the

720 activation dynamics of these two RelA NF-κB dimers was coordinately regulated by signal

721 responsive degradation and resynthesis of IκBα, IκBβ, IκBε and IκBδ during inflammatory

722 signaling. The newly built model consists of 34 species, 138 reactions with 105 parameters ;

723 those describe the synthesis and degradation of NF-κB and IκBs, association and dissociation

724 of IκB:NF-κB complexes, nuclear import and export of NF-κB and IκB species as well as

725 processing of p100 (see a detailed wiring in Appendix figure 1). Constrains generated based

726 on our quantitative biochemical analyses (Appendix figure 2-4) was used along with

727 published literature and fitting procedures for parameterizing the model (see the description

28 728 of the parameters in Supplementary files 1-2). The following assumptions were implicit in

729 the wiring.

730 1. RelA forms two distinct dimers RelA:p50 and RelA:p52;

731 2. RelA:p50 dimers are regulated at the level of nuclear translocation;

732 3. NIK-IKK1 induced processing of p100 generates p52 that forms RelA:p52, which is then

733 regulated by IκBs akin to RelA:p50 dimer.

734 Based on the existing literature and our own analyses (Appendix figure 2-4), we have

735 assumed that RelA:p50 and RelA:p52 dimers exhibit mostly similar biochemical properties,

736 except for IκBα binding (note Appendix figure 4D). By generating RelA:p52 dimer from

737 p100 upon processing, we also captured the mutual stabilization of NF-κB monomers, where,

738 de novo synthesized p52 rescued RelA monomer from degradation to generate RelA:p52

739 dimer in response to NIK-IKK1 signal. We have summarized the new reactions incorporated

740 into the model version published in Basak et. al., 2007 to arrive onto the Systems Model v1.0

741 and provided justification for the parameter values in Supplementary file 3.

742 Simulations were performed in MatLab version 2012b (Mathworks) using the built-in ode15s

743 solver at default settings. We have introduced a time delay of 65 and 37 mins in the NF-κB

744 induced transcription of Nfkb2 and those encoding IκBε, respectively, to recapitulate

745 experimental observations. The transcriptional delay function has been executed in the

746 Systems Model v1.0 as follows: for an explicit delay of t1 min, a given NF-κB dependent

747 transcription reaction assumes a NF-κB concentration at basal level up to t1 min.

748 Subsequently, nuclear concentration of NF-κB has been used as such from (t1+Δt1) min and

749 onwards for computing transcription rates. As previously described, an equilibrium phase

750 was introduced prior to the onset of stimulation phase9. Matlab model codes have been

751 provided as an additional source code file with this manuscript. As such, the NF-κB Systems

752 Model version 1.0 was able to reproduce temporal profiles of nuclear RelA:p50 and

29 753 RelA:p52 dimer activities (Appendix figure 5), as measured experimentally in response to

754 TNFR as well as LTβR stimulation (Figure 1 - figure supplement 1).

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777 Legends to Figures in the Main Text

30 778 Figure 1. Computational simulations predicting a duration code underlying crosstalk

779 control. (A) A current model for RelA NF-κB activation via the canonical (IKK2) or the

780 non-canonical (NIK-IKK1) pathways, respectively. RelA dimers represent both RelA:p50

781 and RelA:p52. Regulation of RelA NF-κB activities through crosstalk between these two

782 pathways has not been addressed. (B) Computational simulations of nuclear RelA NF-κB

783 induction by TNFR induced IKK2 (top, magenta) or LTβR induced NIK-IKK1 signals

784 (bottom, green). (C) A theoretical library of 356 distinct kinase activity profiles. (D) A

785 schematic describing in silico crosstalk studies. The kinase inputs were fed into the model

786 through the IKK2 or NIK-IKK1 or both the arms. The RelA NF-κB responses, quantified as

787 baseline corrected total area under the respective activity curves, were used for computing

788 crosstalk indexes. (E) Based on their respective crosstalk indexes, top 10% combinations of

789 theoretical IKK2 and NIK1 activity profiles were identified and duration as well as amplitude

790 of the associated crosstalk proficient IKK2 (top) or NIK-IKK1 (bottom) profiles were plotted.

791 (F) and (G) The IKK2 (F) or NIK-IKK1 (G) activities were monitored by incubating GST-

792 IκBα or GST-IκBδ with NEMO or NIK co-immunoprecipitates derived from MEFs treated

793 with IL-1β, LPS (F, left and right panels) or αLTβR (G), respectively. For IKK2 assay, co

794 immunoprecipitated IKK1 and for NIK-IKK1 assay, actin present in the cell extracts was

795 used as loading controls. (H) Computational simulations predicting augmented RelA activity

796 in LPS+αLTβR (right panel) and a lack of crosstalk in IL-1+αLTβR (left) co-treatment

797 regimes.

798

799 Figure 2. LTβR signal sustains TLR4, but not IL-1R, induced RelA NF-κB response.

800 (A) Nuclear NF-κB activities induced in MEFs by IL-1β or αLTβR or co-treatment were

801 resolved in EMSA using a κB site containing DNA probe. The faster migrating complex,

802 indicated with an arrowhead, consists of RelB and the slower migrating complex activated by

31 803 both canonical or non-canonical signaling, denoted with an arrow, consists of RelA dimers.

804 The compositions of the DNA binding complexes were ascertained in Figure 2C. Right,

805 signal corresponding to RelA NF-κB activities were quantified and graphed relative to the

806 respective IL-1 induced peak value. Data were expressed as mean of 3 quantified biological

807 replicates ± SEM. (B) EMSA result, representative of three independent biological

808 replicates, revealing augmented late NF-κB activities in the co-treatment regime as compared

809 to cell treatment with LPS or αLTβR alone. Right, signal corresponding to RelA NF-κB

810 activities were similarly quantified and graphed relative to LPS induced peak value. Note,

811 late RelA activities in the LPS+αLTβR co-treatment regime were significantly augmented

812 from of the LPS induced activities. (C) Supershift analysis distinguishing RelA and RelB

813 dimers induced in MEFs treated with LPS (L) or αLTβR (B) or both (LB) for 24h. (D)

814 Kinase assay revealing transient IKK2 activities in response to LPS in Trif-deficient MEFs.

815 (E) EMSA data, representative of three independent experiments, revealing a lack of NF-κB

816 crosstalk between TLR4 and LTβR in Trif-deficient MEFs.

817

818 Figure 3. LTβR signal augments the late expressions of TLR4 induced NF-κB target

819 genes. (A) Computational simulation revealing total RelA activities induced by IKK2 inputs

820 of various durations, in the absence or presence of LTβR induced NIK-IKK1. (B)

821 Quantitative RT-PCR measuring early (1h) and late (24h) expressions of chemokine and

822 cytokine genes in WT MEFs by IL-1R or LTβR or costimulation. (C) Gene-expression

823 analyses similarly revealing early (3h) and late (24h) expressions of chemokine and cytokine

824 genes in WT MEFs by TLR4 or LTβR or costimulation. In (B) and (C), data are expressed as

825 mean of 3 quantified biological replicates ± SEM. The statistical significance was determined

826 using two-tailed Student’s t-test. (D) LPS induced genes, identified in representative

827 microarray experiments at 24h post-stimulation, were ranked based on their normalized

32 828 crosstalk score (bottom panels), which reflects synergistic gene activation in the co-treatment

829 regime as compared to individual cell treatments for a positive value. GSEA demonstrated

830 statistically significant enrichment of NF-κB targets (top), with enrichment score of 0.44 for

831 WT MEFs, among genes positively controlled through crosstalk. Hits (middle) indicate NF-

832 κB response genes.

833

834 Figure 4. Signal generation of RelA:p52 NF-κB dimer underlies a pro-synergistic

835 function of Nfkb2. (A) Local sensitivity analysis revealing the effect of perturbation of the

836 individual biochemical parameters on crosstalk between TLR4 and LTβR. (B) Computational

837 simulations of total RelA:p50 and RelA:p52 activities between 8-24h in response to LPS,

838 αLTβR or both in WT or Nfkb2 deficient systems. (C) Immunoblot charting cellular

839 abundance of NF-κB/IκB proteins during signaling. Right, signal corresponding to p100 and

840 p52 levels at 24h post-stimulation were quantified and graphed. (D) Immunoblot of RelA co-

841 immunoprecipitates, normalized for the RelA content, obtained using whole cells extracts

842 derived from MEFs treated with LPS (L) or αLTβR (B) or both (LB) for 24h. The quantified

843 data demonstrates the level of RelA-p100 or RelA-p52 complexes at 24h post-stimulation.

844 (E) Supershift analysis revealing the composition of the RelA dimers induced upon indicated

845 cell treatments for 24h. Right, signal corresponding to RelA:p50 or RelA:p52 NF-κB

846 activities were quantified and graphed. (F) Representative immunoblot demonstrating an

847 increase in the RelA protein level in MEFs, in parallel to phospho-IκBα accumulation, upon

848 proteasome inhibition using MG-132. (G) EMSA revealing RelA activities induced in WT or

849 Nfkb2-/- MEFs upon indicated cell-stimulations by supershifting RelB. Below, quantified late

850 (24h) RelA activities were plotted for different genotypes subsequent to normalizing against

851 the respective LPS induced early 1h peak. (H) mRNA analyses comparing late (24h)

852 expressions of chemokines/cytokines in Relb-/- (top) and Nfkb2-/- (bottom) MEFs upon

33 853 indicated cell stimulations. Quantified data for both biochemical and gene-expression

854 analyses presented in this figure are expressed as mean of 3 biological replicates ± SEM.

855

856 Figure 5. Induction of Nfkb2 expressions by canonical signal is required for crosstalk.

857 (A) Relative levels of Nfkb2 mRNA and Nfkbia mRNA, which encodes IκBα, in WT MEFs

858 during LPS or IL-1 signaling. (B) TNF induced delayed expression of Nfkb2 mRNA in WT

859 MEFs and rapid production in an engineered Nfkb2-/- cell-line from an exogenous NF-κB

860 dependent promoter. Data presented in (A) and (B) are expressed as mean of 3 quantified

861 biological replicates ± SEM. (C) Simulations comparing IL-1 induced Nkfb2 mRNA

862 expressions in the presence or absence of transcriptional delay. (D) Quantitative RT-PCR

863 revealing IL-1 induced expression of Nfkb2 and Nfkbia mRNAs in the engineered Nfkb2-/-

864 cell-line with transgenic expressions of Nfkb2 from the NF-κB inducible promoter. Data are

865 expressed as mean of 3 quantified biological replicates ± SEM. (E) EMSA comparing NF-κB

866 activities induced in Nfkb2-/- MEFs expressing Nfkb2 from either a constitutive (lane1-7) or

867 an NF-κB responsive (lane2-14) transgenic (Tg) promoter. (F) Supershift analyses comparing

868 nuclear abundance of different NF-κB dimers activated upon costimulation with LPS+αLTβR

869 for 24h in these two engineered Nfkb2-/- cell-lines. (G) The engineered Nfkb2-/- cell-line,

870 which expresses Nfkb2 from the NF-κB inducible promoter, was pretreated for 8h with

871 αLTβR and subsequently treated with IL-1. Supershifting RelB, representative RelA

872 activities were captured in EMSA. (H) A graphical depiction of the proposed crosstalk

873 control; two negative feedback loops coordinately attenuate TLR4 responses. However, one

874 of these negative feedback loops is converted into a positive feedback loop by non-canonical

875 signals to generate crosstalk at the level of RelA NF-κB activation. Magenta and green

876 arrows indicate canonical IKK2 and non-canonical NIK-IKK1 inputs, respectively, and line

877 thickness signifies relative strength of feedbacks.

34 878

879 Figure 6. Nfkb2 dependent LTβR crosstalk prolongs RelA NF-κB response in the colon

880 of Citrobacter rodentium infected mice. (A) EMSA data, representing two independent

881 experiments, revealing LPS-induced total RelA NF-κB activities induced in MSIE colon

882 epithelial cell-line at 12h in the absence or presence of 1mgm/ml of αLTβR. (B), (C) and (D)

883 WT mice were injected with control-Ig or LTβR-Ig (n=2) one day prior to infection with C.

884 rodentium. IECs were isolated at day3 and day5 post-infection and analyzed for p52 and

885 p100 levels by immunoblotting (B), RelA:p52 complex formation by immunoprecipitation

886 based assay (C) or NF-κB DNA binding activities in EMSA (D). OCT1 DNA binding

887 activity served as loading control. (E) Supershift analyses revealing that exclusively

888 RelA/NF-κB dimer are activated in IECs derived from mice infected with C. rodentium. (F)

889 NF-κB activities induced in IECs derived from infected WT and Nfkb2-/- mice (n=2) were

890 similarly measured. (G) Epithelial expressions of KC and MIP-2a mRNA derived from WT

891 and Nfkb2-/- mice (n=5) at day5 post-infection. Data are expressed as mean of 3 quantified

892 biological replicates ± SEM. The statistical significance was determined using two-tailed

893 Student’s t-test. (H) Representative data showing antimyeloperoxidase staining of neutrophils

894 in colons of WT and Nfkb2-/- mice at day4 post-infection. Colon sections from three animals

895 per set and five fields/section were used for quantification and presented as mean ± SEM.

896 The panels with 40X magnification have been presented using scale bars that represent

897 200μm.

898

899 Figure 7. A protective role of pro-synergistic Nfkb2 in the non-hematopoietic

900 compartment to Citrobacter infection. (A) and (C) Bacterial titers in the fecal homogenate

901 (A), blood (C, left panel) or spleen and liver homogenate (C, right) derived from WT and

902 Nfkb2-/- mice (n=4) at day10 post-infection. Data are expressed as mean ± SEM. The

35 903 statistical significance was determined using two-tailed Student’s t-test. (B) and (F) H&E

904 staining of the representative colon sections derived from WT and Nfkb2-/- mice at day10

905 (n=4, five fields/section) (B) or indicated bone marrow chimeras at day7 (n=2, five

906 fields/section) (F) after inoculation. The panel shows 20X magnification with scale bars

907 representing 200μm. (D) and (G) Average change in the body weight of WT and Nfkb2-/-

908 mice (n=7) (D) or indicated bone marrow chimeras (n=4) (G) upon infection. Data are

909 expressed as mean± SEM. The statistical significance was determined using two-tailed

910 Student’s t-test. (E) and (H) Survival rates of WT and Nfkb2-/- mice (n=7) (E) or indicated

911 bone marrow chimeras (n=4) (H) infected with C. rodentium. The statistical significance was

912 determined using log rank (Mantel-Cox) test. (I) A model depicting the proposed regulatory

913 role of crosstalk in sustaining colonic inflammatory immune responses.

914

915

916 Legends to Figure Supplements

917 Figure 1 - figure supplement 1. Analyzing the composition of signal induced NF-κB

918 dimers. The dynamic profiles, and the composition of the nuclear RelA NF-κB DNA binding

919 activities induced upon TNFR (top panel) or LTβR (bottom) engagement were analyzed in

920 EMSA and supershift assay using the indicated antibodies. A longer exposure of EMSA gel

921 was used in the bottom panel to reveal otherwise weak LTβR induced DNA binding

922 activities.

923

924 Figure 1 - figure supplement 2. Mathematical modeling revealing a duration code

925 underlying signaling crosstalk. (A) A schematic of a typical kinase activity profile. For the

926 indicated values of ‘a’, ‘b’ and ‘h’, a theoretical library of activity profiles, as presented in

927 Figure 1C, was generated. (B) Representative long or short duration as well as low or high

36 928 amplitude kinase profiles were selected from the library to simulate RelA activity in response

929 to singular IKK2 or NIK-IKK1 inputs or co-treatment regimes. All sixteen NF-κB activity

930 plots were enclosed together in a blue rectangle. Crosstalk index for different IKK2 and

931 NIK-IKK1 combinations have been indicated in the respective NF-κB plots which indicates a

932 dominant role of duration of the activating kinases in crosstalk.

933

934 Figure 1 - figure supplement 3. Kinase assays probing cellular activation of NEMO-

935 IKK2 and NIK-IKK1. (A) NIK associates with and stimulate an NEMO independent pool

936 of IKK1, which participates in non-canonical signaling. By immunoprecipitating NIK, we

937 could reveal LTβR induced kinase activity in the immunopellet at 8h post-stimulation (lane 2

938 and 3) that specifically phosphorylated GST-IκBδ (lane 3 and 4). The specificity of the NIK-

939 IKK1 kinase assay was further confirmed using αLTβR treated Nik-/- (lane 1) or Ikk1-/- (lane

940 5, 6) MEFs. (B) Quantification of the data presented in Figure 1F revealing IKK2 activation

941 in response to IL-1 or LPS (top) and Figure 1G revealing NIK-IKK1 activation in response to

942 αLTβR (bottom) in WT MEFs.

943

944 Figure 2 - figure supplement 1. Analyzing crosstalk control of in Trif-deficient MEFs.

945 Immunoblot revealing p100 processing into p52 upon LTβR engagement in Trif-deficient

946 MEFs to confirm a functional non-canonical pathway in these cells.

947

948 Figure 4 - figure supplement 1. An important role of the non-canonical signal

949 transducer Nfkb2 in crosstalk. (A) The level of Nfkb2 mRNA (top) and Nfkbia mRNA,

950 which encodes IκBα (bottom) were monitored in a time course during TLR4 or LTβR

951 signaling or in the co-stimulation regime. Strong RelA activity, observed in EMSA, led to

952 potent induction of both the NF-κB targets in response to LPS treatment or in the co-

37 953 treatment regime. LTβR that only weakly activated RelA, only subtly altered the levels of

954 these two mRNAs. Data are expressed as mean of 3 quantified biological replicates ± SEM.

955 (B) EMSA revealing NF-κB DNA binding activities in Nfkb2-/- MEFs upon cell-treatment

956 with indicated stimuli. Note constitutive RelB DNA binding activity that largely substituted

957 LTβR induction of RelB dimers.

958

959 Figure 5 - figure supplement 1. Induction of Nfkb2 expression by canonical signal is

960 important for crosstalk. (A) Comparing LPS induced changes in Nfkb2 mRNA levels in

961 two engineered Nfkb2-/- cell-lines expressing Nfkb2-Tg from either a constitutive or an NF-

962 κB responsive promoter. Our analyses confirmed a lack of LPS inducible expression of

963 Nfkb2 from the constitutive promoter, but 3.5 fold induced expression from the inducible

964 promoter. (B) Immunoblot comparing p52 generation upon LPS+LTβR costimulation in

965 these two engineered Nfkb2-/- cell-lines. (C) Left panel, immunoblot comparing p52

966 production in WT cells upon 8h of αLTβR treatment with or without subsequent 3h IL-1

967 stimulation. Similarly, the engineered Nfkb2-/- cell-line, which expresses Nfkb2 from the NF-

968 κB responsive promoter, was subjected to IL-1 treatment subsequent to 8h of LTβR pre-

969 treatment (right panel). (D) Quantification of the data presented in Figure 5G and two more

970 experimental replicates. Data are expressed as mean of 3 quantified biological replicates ±

971 SEM.

972

973 Figure 6 - figure supplement 1. Control of epithelial RelA NF-κB activation through

974 signaling crosstalk. (A) Immunoblot showing functional non-canonical LTβR pathway

975 generating p52 in MSIE colon epithelial cell line. ‘*’ indicates a non-specific protein band.

976 (B Supershift analyses to confirm the induction of both p50 as well as p52 containing

38 977 RelA/NF-κB dimers in IECs derived from mice infected with C. rodentium. Right, quantified

978 data representing the relative abundance of individual RelA/NF-κB complexes.

979

980 Figure 7 - figure supplement 1. Nfkb2 controls gut immune response to Citrobacter

981 infection. (A) and (B) Comparison of colons from WT and Nfkb2-/- mice infected (B) with

982 C. rodentium revealing shrinkage with signatures of hyperplasia in the absence of Nfkb2.

983 Colon from uninfected mice had no discernible differences (A). (C) Representative FACS

984 plots confirming repopulation of CD45.1 hematopoietic cells (~90%) from WT donor into

985 Nfkb2-/- mice with CD45.2 congenic background and vice versa after 5 weeks of bone

986 marrow transplant.

987

988

989 Legends to Appendix figures and Supplementary files

990 Appendix figure 1. A detailed wiring diagram (A) A wiring diagram depicting the

991 biochemical connectivities between the molecular species included in the NF-κB Systems

992 Model v1.0 to capture signal responsive activation of RelA:p50 (Ap50) and RelA:p52 (Ap52)

993 dimers. The number adjacent to each reaction arrow is the corresponding parameter number,

994 as described in the associated supplementary table-1 and supplementary table-2. For the ease

995 of presentation, three classical IκBs, IκBα, -β and -ε, were presented as IκBs on certain

996 occasions. Similarly, RelA:p50 and RelA:p52 dimers were collectively depicted as A:NFκB.

997

998 Appendix figure 2. Nfkb2 mRNA analyses in unstimulated cells and during signaling

999 (A) The NF-kB independent constitutive transcription of Nfkbia mRNA, which encodes

1000 IκBα, and Nfkb2 mRNA in the late passage MEFs was measured using quantitative RT-PCR.

1001 The mRNA levels of Nfkbia and Nfkb2 were reduced to 10% and 25% in NF-κB deficient

39 1002 (Rela-/-Crel-/-Relb-/-) cells as compared to WT cells. Transcript level of b-actin was used for

1003 normalization. (C) Quantitative RT-PCR to measure half-life of Nfkb2 mRNA. WT cells

1004 were treated with 5 mg/ml of transcription inhibitor actinomycin-D in a time-course,

1005 harvested, absolute mRNA levels of Nfkb2 and b-actin were determined and presented as

1006 Nfkb2 mRNA copy numbers per thousand copies of b-actin mRNA. The curve fitting was

1007 done assuming first-order decay of Nfkb2 mRNA to arrive onto a half-life of ~10.5h. (D)

1008 LTβR induced expression of Nfkbia and Nfkb2 mRNAs was analyzed in a time course. Due

1009 to weak NF-κB activation, LTβR activation using 0.3mg/ml agonist antibody only subtly

1010 altered the levels of these two NF-κB target genes in MEFs.

1011

1012 Appendix figure 3. Analyzing p52 and p100 protein encoded by Nfkb2 in unstimulated

1013 cells and during signaling (A) Constitutive degradation of NF-κB bound (left panel) or free

1014 (right) IκBα (top panel) and p100/IκBδ (middle) were evaluated by immunobloting

1015 respective extracts derived from WT (left) or NF-κB deficient (right) cells treated with

1016 10mg/ml of protein synthesis inhibitor cycloheximide. As compared to the stable NF-κB

1017 bound form, free IκBα degraded rapidly with a half-life ~ 5 min. In contrast, both NF-κB

1018 bound p100/IκBδ present in WT cells (top) and free p100/IκBδ in NF-κB deficient cells

1019 (bottom) were stable with a half-life>12h. Actin (bottom panel) was used as loading control.

1020 The data represents two biological replicates. (B) Immunoblot revealing TNFR induced

1021 IKK2 mediated degradation of NF-κB bound IκBα in WT (left panel, top) or free IκBα in

1022 NF-κB deficient cells (right, top). Based on these data, IKK2 mediated degradation rates of

1023 the free and NF-κB bound classical IκBs were assigned comparable values in the model.

1024 Both in its bound (left, middle) or free (right, middle) form, p100/IκBδ were insensitive to

1025 IKK2 signals. (C) Immunoblot showing LTβR induced NIK-IKK1 mediated degradation of

40 1026 NF-κB bound (top panel) and free (bottom) p100/IκBδ in WT and NF-κB deficient cells,

1027 respectively. Importantly, signal induced p52 accumulation was evident at 24h in WT, but

1028 not in NF-κB deficient, cells suggesting that newly generated p52 requires other NF-κB

1029 monomers for mutual stabilization. (D) Immunoblot of RelA co-immunoprecipitates

1030 revealing liberation of RelA from inhibitory p100/IκBδ (top panel) and generation of

1031 RelA:p52 complexes (middle) during LTβR signaling. Nfkb2-/- cell extracts were used as

1032 control (bottom).

1033

1034 Appendix figure 4. Comparing RelA:p50 and RelA:p52 NF-κB dimers (A) EMSA

1035 revealing TNF activation of RelA:p50 (left panel) or RelA:p52 (right) dimers in Relb-/-Nfkb2-

1036 /- or Relb-/-Nfkb1-/- cells, respectively. Quantification of the band intensities (bottom panel)

1037 revealed that basal and TNF induced RelA:p52 activities in Relb-/-Nfkb1-/- are ~ 10% of the

1038 corresponding RelA:p50 activities in Relb-/-Nfkb2-/-. These values were used as constrains to

1039 describe constitutive RelA:p52 dimer generation in WT cells in the model. Both RelA:p50

1040 and RelA:p52 dimers were induced within 30min of TNF treatment indicating that NEMO-

1041 IKK mediated degradation rates of IκBs bound these RelA heterodimers are comparable. Of

1042 note, TNF induced fold changes in the activity of the respective RelA dimers were

1043 comparable in Relb-/-Nfkb2-/- and Relb-/-Nfkb1-/- cells. The result represents three biological

1044 replicates. (B) NF-κB responsive expression of Nfkbia mRNA, encoding IκBα, induced by

1045 either RelA:p50 or RelA:p52 dimers in Relb-/-Nfkb2-/- or Relb-/-Nfkb1-/- cells upon TNF

1046 treatment, respectively. Comparable mRNA fold induction along with our EMSA data

1047 suggested similar DNA binding and transcriptional proficiency of RelA:p50 and RelA:p52.

1048 Data are expressed as mean of 3 quantified biological replicates ± SEM. (C) Gene-expression

1049 analyses revealing comparable induction of Nfkb2 mRNA upon LPS treatment in WT MEFs,

1050 those mostly activate RelA:p50 dimer and Relb-/-Nfkb1-/- MEFs, which exclusively activates

41 1051 RelA:p52 dimer. Our results confirm that RelA:p52 dimer is capable of inducing the

1052 expressions of Nfkb2 mRNA as effectively as RelA:p50 dimers. (D) Biochemical analyses

1053 indicating weaker IκBα binding to the RelA:p52 dimer. Briefly, nuclear extracts derived

1054 from Relb-/-Nfkb2-/- or Relb-/-Nfkb1-/- cells treated with TNF was used as a source of free

1055 RelA:p50 or RelA:p52, respectively, and normalized to obtain comparable DNA binding

1056 activities. Nuclear RelA dimers were incubated with a gradient of recombinant IκBα for

1057 30min to facilitate the formation of IκB:NF-κB complex, which does not bind DNA in

1058 EMSA. The abundance of unbound NF-κB dimers was then scored using EMSA. Incubation

1059 with 25nM IκBα was sufficient to completely inhibit RelA:p50 DNA binding. While, more

1060 than 200nM of IκBα was required to inhibit RelA:p52 and RelA:RelA DNA binding, owing

1061 to weaker IκBα binding by these dimers. The data represents three experimental replicates.

1062

1063 Appendix figure 5. Simulating RelA:p50 and RelA:p52 activation in response to

1064 canonical and non-canonical signals. Computational simulations of nuclear RelA:p50

1065 (grey) and RelA:p52 (black) induction by TNFR induced IKK2 (top) or LTβR induced NIK-

1066 IKK1 signals (bottom). Our computational data effectively recapitulates relative abundance

1067 of these two RelA dimers during canonical and non-canonical signaling, as experimentally

1068 observed and demonstrated in Figure 1 - figure supplement 1.

1069

1070 Supplementary file 1. List of biochemical species and their initial concentrations in the

1071 model.

1072

1073 Supplementary file 2. List of biochemical reactions and rate constants in the model

1074

42 1075 Supplementary file 3. List of newly described biochemical parameters in the Systems

1076 Model v1.0

1077

1078 Source Data files

1079 Figure 3 – source data 1. List of LPS target genes positively regulated through

1080 crosstalk.

1081

1082 Figure 3 – source data 2. A pre-determined list of 290 NF-B response genes used in

1083 GSEA.

1084