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Bifidobacterium breve UCC2003 induces a distinct global transcriptomic programme in neonatal murine intestinal epithelial cells

Bifidobacterium is an important gut microbiota member during early life that is associated with improved gut health. However, the underlying health-driving mechanisms are not well understood, particularly how Bifidobacterium may modulate the intestinal barrier via programming of intestinal epithelial cells (IECs). In this study, we sought to investigate the global impact of model strain Bifidobacterium breve UCC2003 on the neonatal IEC transcriptome, including gene regulation and pathway modulation. Small IECs from two-week-old neonatal mice administered B. breve UCC2003 for three consecutive days or PBS (control group) were subjected to global RNASeq, with various bioinformatic approaches used to determine differentially expressed genes, pathways and affected cell types between control and experimental groups. Whilst colonisation with B. breve had minimal impacts on the neonatal microbiota, we observed extensive regulation of the IEC transcriptome; ~4,000 genes significantly up-regulated, including key genes associated with epithelial barrier function. Enrichment of cell differentiation and cell proliferation pathways were observed, along with an overrepresentation of stem cell marker genes, indicating an increase in the regenerative potential of the epithelial layer. Expression of distinct immune-associated pathway members (e.g. Toll-like Receptors) were also affected after neonatal B. breve colonisation. In conclusion, B. breve UCC2003 plays a central role in driving universal transcriptomic changes in neonatal IECs that enhances cell replication, differentiation and growth, predominantly in the stem cell compartment. This study enhances our overall understanding of the benefits of B. breve in driving intestinal epithelium homeostatic development during early life, with potential avenues to develop novel live biotherapeutic products.


Summary 25
Bifidobacterium is an important gut microbiota member during early life that is associated 26 with improved gut health. However, the underlying health-driving mechanisms are not well 27 understood, particularly how Bifidobacterium may modulate the intestinal barrier via 28 programming of intestinal epithelial cells (IECs). In this study, we sought to investigate the 29 global impact of model strain Bifidobacterium breve UCC2003 on the neonatal IEC 30 transcriptome, including gene regulation and pathway modulation. Small IECs from two-31 week-old neonatal mice administered B. breve UCC2003 for three consecutive days or PBS 32 (control group) were subjected to global RNASeq, with various bioinformatic approaches 33 used to determine differentially expressed genes, pathways and affected cell types between 34 control and experimental groups. Whilst colonisation with B. breve had minimal impacts on 35 the neonatal microbiota, we observed extensive regulation of the IEC transcriptome; ~4,000 36 genes significantly up-regulated, including key genes associated with epithelial barrier 37 function. Enrichment of cell differentiation and cell proliferation pathways were observed, 38 along with an overrepresentation of stem cell marker genes, indicating an increase in the 39 regenerative potential of the epithelial layer. Expression of distinct immune-associated 40 pathway members (e.g. Toll-like Receptors) were also affected after neonatal B. breve 41 colonisation. In conclusion, B. breve UCC2003 plays a central role in driving universal 42 transcriptomic changes in neonatal IECs that enhances cell replication, differentiation and 43 growth, predominantly in the stem cell compartment. This study enhances our overall 44 understanding of the benefits of B. breve in driving intestinal epithelium homeostatic 45 development during early life, with potential avenues to develop novel live biotherapeutic 46 products. 47

B. breve UCC2003 modulates cell maturation processes 173
We next sought to understand the biological functions of up-regulated DEGs by employing 174 PANTHER GO-Slim functional assignment, and process/pathway enrichment analysis (Fig. 175 S1; Table S5-S9). DEGs were predominantly involved in general biological processes 176 including cellular process (901 genes) and metabolic process (597 genes; Table S5). At the 177 molecular function level, DEGs were primarily assigned to binding (868 genes) and catalytic 178 activity (671 genes; Table S6), with Olfactory Signalling Pathway and Cell Cycle (biological) 179 pathways also found to be enriched (Table S7). 180 To delve further into the data, we constructed a signaling network based on up-regulated 181 DEGs (n=3,996) with the aim of identifying specific gene networks involved in important 182 signalling pathways (Fig. 5a). Overall, 1,491 DEGs were successfully mapped (37.3%) to a 183 signalling network that comprised 8,180 genes. Four individual clusters of genes were 184 detected, with functional assignment and pathway analysis implemented on these clusters 185 ( Fig. 5a). All gene clusters were associated with cell differentiation and maturation, with 186 cluster 1 (68 genes) linked specifically with DNA replication and transcription, cluster 2 (26 187 genes) with cell growth and immunity, cluster 3 (11 genes) with cell replication, and cluster 4 188 (72 genes) related to cell cycle and cell division (Table S10). 189 Intestinal cell type analysis on DEGs identifies significant enrichment of epithelial stem 190 cells 191 IECs include several absorptive and secretory cell types, namely enterocytes, Paneth cells,192 goblet cells, enteroendocrine cells, tuft cells and stem cells. As these cells perform different 193 functions in the gut, it was important to understand whether B. breve UCC2003 had a cell 194 type specific effect on the intestinal epithelium. Using known cell type specific gene markers 195 [24], we identified cell type gene signatures modulated within the UCC2003 group . Importantly, all cell type markers were well represented in the expressed genes of the 197 whole IEC transcriptomics data from both groups (control + UCC2003), thus validating the 198 presence of all IEC types in our study data (Fig. 5b). Cell type analysis of genes differentially 199 expressed after B. breve UCC2003 supplementation, revealed that stem cell marker genes 200 were significantly enriched (30%; P < 0.05) among the six IEC types (Table S11). Signatures 201 of other cell types were also present (linking to marker genes in the DEG list) but not 202 significantly overrepresented: Tuft cells (22%), enteroendocrine cells (18%), goblet cells 203 (15%), Paneth cells (15%) and enterocytes (13%; Fig. 5c). These data indicated that intestinal 204 epithelial stem cells, cells primarily involved in cell differentiation, were the primary cell 205 type whose numbers and transcriptomic programme were regulated by B. breve UCC2003. 206 Further investigation of this stem cell signature revealed that of the 37 differentially 207 expressed marker genes, 35 are up-regulated in the presence of B. breve UCC2003. This 208 indicates an increase in the quantity of stem cells or semi-differentiated cells in the 209 epithelium, consistent with the overrepresentation of cell cycle and DNA replication 210 associated genes observed in the whole differential expression dataset. Functional analysis of 211 the 37 stem cell signature genes revealed only one overrepresented process -Regulation of 212 Frizzled by ubiquitination (P < 0.05), which is a subprocess of WNT signalling. WNT 213 signalling is important in maintaining the undifferentiated state of stem cells [25]. 214 Finally, we employed a network approach to predict key transcription factor (TF) regulators 215 of the differentially expressed stem cell marker genes, through which B. breve UCC2003 may 216 be acting (Fig. 5d). Using the TF-target gene database, DoRothEA, we identified expressed 217 TFs known to regulate these genes [26,27]. Five genes had no known and expressed 218 regulator, thus were excluded. Hypergeometric significance testing was used to identify 219 which of these TFs are the most influential (see Methods for details). This analysis identified 220 32 TF regulators (Fig. 5d). Of these regulators, 12 were differentially expressed in the IEC 221 dataset (all up-regulated): Fos, Gabpa,Rcor1,Arid2,Tead1,Mybl2,Mef2a,Ahr,Pgr,Kmt2a,222 Ncoa2 and Tcf12. Functional analysis of all the TF regulators and their targeted genes 223 together, revealed overrepresented functions relating to WNT signalling, histone methylation 224 for self-renewal and proliferation of hematopoietic stem cells and nuclear receptor (incl. 225 estrogen) signalling (Table S12). These data provide evidence that B. breve UCC2003 226 directly affects key transcriptomic programmes regulating drives specific signalling 227 processes, particularly within stem cells. 228

229
The early life developmental window represents a crucial time for microbe-host interactions 230 that impacts health both in the short-and longer-term. Understanding how specific 231 microbiota members modulate host responses during these life stages is crucial if we are to 232 develop next-stage targeted microbiota therapies. Here we investigated how B. breve 233 UCC2003 induces genome-wide transcriptomic changes in small intestine IECs of neonatal 234 mice. We observed that B. breve had a global impact on the IEC transcriptome, evidenced by 235 the large number of significantly up-regulated genes and pathways related to cell 236 differentiation and cell proliferation, including genes associated with epithelial barrier 237 function. We propose that B. breve is a key early life microbiota member driving fundamental 238 cellular responses in IECs, particularly within the stem cell compartment, and thus drives 239 epithelial barrier development and maintenance during neonatal life stages. 240 B. breve UCC2003 is a model strain that was previously isolated from the stool of a breast-241 fed infant [28,29]. Although human-associated, numerous previous studies have shown this 242 strain can efficiently colonise (long-term) the mouse gastrointestinal tract, which we also 243 observed in this study [30,31]. Importantly, although at lower levels (~10 5 CFU/g), we 244 observed B. breve UCC2003 within the small intestine, linking to our subsequent 245 observations of significant impacts on the IEC transcriptome from this intestinal region. 246 Furthermore, our microbiota profiling suggests minimal impacts on the wider microbiota (at 247 genus level) after supplementation, suggesting that B. breve UCC2003 is principally driving 248 specific transcriptomic outcomes. However, we cannot discount that B. breve is driving more 249 nuanced microbiota changes, which may also be contributing to downstream IEC responses. 250 B. breve is known to confer beneficial effect on gut health, however our knowledge related to 251 the mechanisms underlying these responses are limited. Most studies have focused on 252 targeted immune cells or pathways (during disease and/or inflammation), and to our 253 knowledge no studies have probed global transcriptomic changes within IECs -the frontline 254 physical barrier between bacteria and host [32,33]. Our presented findings: ~4,000 up-255 regulated DEGs and ~450 down-regulated DEGs within the B. breve group indicate that this 256 Bifidobacterium strain modulates whole-scale changes within this critical single cell layer. 257 Notably, we also examined how B. breve modulates adult IEC responses, however, we did 258 not observe any significantly differentially regulated genes when compared to control 259 animals. The striking differences in DEGs between these two life points indicates that B. with increased risk and/or symptoms of ulcerative colitis and Crohn's disease [41][42][43][44][45]. A 282 wide range of TJ-related genes were up-regulated after UCC2003 supplementation, 283 particularly Tjp1 (that encodes ZO-1), Jam2 and Claudin34c1, with a previous study 284 indicating other Bifidobacterium species (i.e. B. bifidum) also modulate TJ expression via 285 . These data indicated that specific strains of Bifidobacterium may modulate key 286 barrier integrity systems during the neonatal period, and therefore absence of this key initial 287 bacterial-host crosstalk may correlate with increased risk of chronic intestinal disorders in 288 later-life [44]. Intestinal mucus, encoded by Muc genes (up-regulated due to B. breve 289 UCC2003 in this study), plays a crucial role in colonic protection via formation of a physical 290 barrier between the gut lumen and IECs, and deficiencies in MUC-2 has been linked with 291 experimental colitis and increased inflammation in IBD patients [47,48]. We have also 292 observed that B. breve UCC2003 significantly increases goblet cell numbers and mucus 293 production (in gnotobiotic and SPF mice; data not shown Further network and functional analysis indicated clusters of up-regulated DEGs were 300 associated with cell maturation and cell differentiation (as confirmed by cell type specific 301 analysis), suggesting neonatal B. breve exposure positively modulates IEC cell 302 differentiation, growth and maturation. Somewhat surprisingly, we did not observe the same 303 type of striking responses in immune pathways, which may be masked by the sheer number 304 of DEGs involved in cellular differentiation and processes. However, pathways such as Toll-305 like Receptor TLR1 or TLR2 pathways do appear to be enriched (cluster 2 of signalling 306 network analysis). This may link to previous work indicating that the UCC2003 EPS directly 307 signals via TLR2 to induce MyD88 signalling cascades to protect IECs during intestinal 308 inflammation [17]. B. breve M-16V was also shown to interact with TLR2 to up-regulate 309 ubiquitin-editing enzyme A20 expression that correlated with increased tolerance to a TLR4 For preparation of gavage inoculums, 5 ml of inoculated broth was incubated overnight 360 followed by sub-culturing into 5 ml De Man, Rogosa and Sharpe (MRS) medium (Oxoid). 361 After an additional overnight incubation, another sub-culturing into 40 ml RCM was 362 performed. Inoculums were prepared from cultures by 3 rounds of centrifugation at 3220 g 363 for 10 min followed by three PBS washes before dilution in 4 ml (adult mice) or 2 ml 364 (neonatal mice) sterile PBS. Bacterial concentration of inoculum was enumerated by plating 365 serial dilutions in sterile PBS on RCA plates and enumerating colonies following two-day 366 incubation to calculate CFU/ml. 367

Bacterial treatment and gut colonisation 368
Neonatal mice were colonised with B. breve UCC2003 by oral gavage with bacterial 369 inoculations of 10 8 CFU/ml in 50 μl every 24 h for 3 consecutive days. Control mice 370 received oral gavages of sterile PBS. B. breve UCC2003 colonisation was confirmed by 371 collection of fresh faeces or intestinal content homogenised with 1 ml sterile PBS followed 372 by serial-dilution plating in sterile PBS on RCA supplemented with 50 mg/L mupirocin and 373 counting of colonies following 2-day incubation to calculate CFU/mg. 374

Gut microbiota profiling by 16S rRNA amplicon sequencing and analysis 375
Genomic DNA extraction of mouse caecal samples on day 4 was performed with FastDNA 376 Spin Kit for Soil following manufacturer's instructions and extending the bead-beating step 377 to 3 min as described previously [56]. Extracted DNA was quantified, normalised and 378 sequenced on Illumina MiSeq platform using a read length of 2 × 300 bp, sequencing reads 379 were analysed using OTU clustering methods (QIIME v1.9.1) to assign bacterial taxonomy 380 and visualised as described previously [57,58] to QIAshredder spin columns (QIAGEN) followed by centrifugation at 9,300 g for 1 min. 403 Follow-through was mixed with 600 μl RLT lysis buffer and used for subsequent RNA 404 isolation. Homogenised sample in RLT buffer from both tissue and IEC isolations were 405 processed by adding 700 μl of 70% ethanol and mixing by pipetting. Sample was then added 406 into RNeasy spin column and spun at 8,000 g for 15 sec. Flow through was discarded and 407 process repeated until all of sample was filtered through column. Then 700 μl of buffer RW1 408 was added to column and centrifuge at 8,000 g for 30 s. Again, flow through was discarded 409 and filter placed in a new collection tube. To the filter, 500 μl RPE was added and spun at 410 8,000 g for 30 s followed by discarding of flow through. An additional 500 μl RPE was 411 pipetted into column and centrifuged at 8,000 g for 2 min. Spin column was then placed in a 412 new collection tube and centrifuged at 8,000 g for 2 min. Columns were transferred to a RNA 413 low-bind Eppendorf tube and 30 μl of RNase free water added to directly to the filter. After 414 an incubation of 1 min at RT, sample was centrifuged at 8,000 g for 1 min and flow through 415 containing RNA stored at -80˚C. 416 Purified RNA was quantified, and quality controlled using RNA 6000 Nano kit on a 2100 417 obtain clusters in the network: degree cutoff = 3, haircut = true, fluff = false, node score 458 cutoff = 0.5, k-core = 3 and max depth = 100. 459 The nodes of each individual module were tested for functional enrichment based on both 460 Reactome and PANTHER annotations using PANTHER Classification System as described 461 in previous sub-section [70,77,78]. 462   Hall LJ, Clarke P: Incidence of necrotising enterocolitis before and after 553 introducing routine prophylactic Lactobacillus and Bifidobacterium probiotics.