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• W3183749649 abstract "•CMT rescued antibiotic-induced T1D enhancement in NOD mice•CMT restored potential T1D-protective bacterial taxa•CMT restored global patterns of gene expression and modifications in the ileum•The study reveals a regulatory network of the innate immunity-sensing intestinal signals Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury. Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury. IntroductionInfancy is a critical period for establishing a healthy gut microbiota and facilitating individual immune development (Gensollen et al., 2016Gensollen T. Iyer S.S. Kasper D.L. Blumberg R.S. How colonization by microbiota in early life shapes the immune system.Science. 2016; 352: 539-544Crossref PubMed Google Scholar; Gollwitzer and Marsland, 2015Gollwitzer E.S. Marsland B.J. Impact of early-life exposures on immune maturation and susceptibility to disease.Trends Immunol. 2015; 36: 684-696Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar; Pennisi, 2016Pennisi E. Microbiome. The right gut microbes help infants grow.Science. 2016; 351: 802Crossref PubMed Scopus (2) Google Scholar). Alteration of gut microbiota composition in infancy may change age-associated immunity and organ-specific inflammation, increasing the risk of immune-mediated diseases (Amenyogbe et al., 2017Amenyogbe N. Kollmann T.R. Ben-Othman R. Early-life host-microbiome interphase: the key frontier for immune development.Front. Pediatr. 2017; 5: 111Crossref PubMed Google Scholar; Aversa et al., 2021Aversa Z. Atkinson E.J. Schafer M.J. Theiler R.N. Rocca W.A. Blaser M.J. LeBrasseur N.K. Association of infant antibiotic exposure With childhood health outcomes.Mayo Clin. Proc. 2021; 96: 66-77Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar; Kemppainen et al., 2017Kemppainen K.M. Vehik K. Lynch K.F. Larsson H.E. Canepa R.J. Simell V. Koletzko S. Liu E. Simell O.G. Toppari J. et al.Association between early-life antibiotic use and the risk of islet or celiac disease autoimmunity.JAMA Pediatr. 2017; 171: 1217-1225Crossref PubMed Scopus (49) Google Scholar; Mullaney et al., 2019Mullaney J.A. Stephens J.E. Geeling B.E. Hamilton-Williams E.E. Early-life exposure to gut microbiota from disease-protected mice does not impact disease outcome in type 1 diabetes susceptible NOD mice.Immunol. Cell Biol. 2019; 97: 97-103Crossref PubMed Scopus (5) Google Scholar).Type 1 diabetes (T1D) is the most common autoimmune disease in childhood where the pancreatic insulin-producing β cells are destroyed by autoreactive T cells and other effectors (Atkinson and Eisenbarth, 2001Atkinson M.A. Eisenbarth G.S. Type 1 diabetes: new perspectives on disease pathogenesis and treatment.Lancet. 2001; 358: 221-229Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar; Wilson et al., 1998Wilson S.B. Kent S.C. Patton K.T. Orban T. Jackson R.A. Exley M. Porcelli S. Schatz D.A. Atkinson M.A. Balk S.P. et al.Extreme Th1 bias of invariant Vα24JαQ T cells in type 1 diabetes.Nature. 1998; 391: 177-181Crossref PubMed Scopus (0) Google Scholar). The triggers and intermediary molecular mechanisms of T1D remain unclear, but innate immunity may play a key role in the initial pathogenetic steps (Atkinson and Chervonsky, 2012Atkinson M.A. Chervonsky A. Does the gut microbiota have a role in type 1 diabetes? Early evidence from humans and animal models of the disease.Diabetologia. 2012; 55: 2868-2877Crossref PubMed Scopus (65) Google Scholar; Pino et al., 2010Pino S.C. Kruger A.J. Bortell R. The role of innate immune pathways in type 1 diabetes pathogenesis.Curr. Opin. Endocrinol. Diabetes Obes. 2010; 17: 126-130Crossref PubMed Google Scholar; Wen et al., 2008Wen L. Ley R.E. Volchkov P.Y. Stranges P.B. Avanesyan L. Stonebraker A.C. Hu C. Wong F.S. Szot G.L. Bluestone J.A. et al.Innate immunity and intestinal microbiota in the development of type 1 diabetes.Nature. 2008; 455: 1109-1113Crossref PubMed Scopus (1375) Google Scholar). In infants, both the gut microbiota (Dominguez-Bello et al., 2011Dominguez-Bello M.G. Blaser M.J. Ley R.E. Knight R. Development of the human gastrointestinal microbiota and insights from high-throughput sequencing.Gastroenterology. 2011; 140: 1713-1719Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and the immune system are developing (Olin et al., 2018Olin A. Henckel E. Chen Y. Lakshmikanth T. Pou C. Mikes J. Gustafsson A. Bernhardsson A.K. Zhang C. Bohlin K. et al.Stereotypic immune system development in newborn children.Cell. 2018; 174: 1277-1292.e14Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Antibiotic exposure could potentially change the microbial interplay with immune system development, and experimental models in the non-obese diabetic (NOD) mouse indicate altered susceptibility to T1D development (Brown et al., 2016Brown K. Godovannyi A. Ma C. Zhang Y. Ahmadi-Vand Z. Dai C. Gorzelak M.A. Chan Y. Chan J.M. Lochner A. et al.Prolonged antibiotic treatment induces a diabetogenic intestinal microbiome that accelerates diabetes in NOD mice.ISME J. 2016; 10: 321-332Crossref PubMed Google Scholar; Candon et al., 2015Candon S. Perez-Arroyo A. Marquet C. Valette F. Foray A.P. Pelletier B. Milani C. Ventura M. Bach J.F. Chatenoud L. Antibiotics in early life alter the gut microbiome and increase disease incidence in a spontaneous mouse model of autoimmune insulin-dependent diabetes.PLoS One. 2015; 10: e0125448Crossref PubMed Scopus (118) Google Scholar; Hu et al., 2017Hu Y. Wong F.S. Wen L. Antibiotics, gut microbiota, environment in early life and type 1 diabetes.Pharmacol. Res. 2017; 119: 219-226Crossref PubMed Scopus (22) Google Scholar; Livanos et al., 2016Livanos A.E. Greiner T.U. Vangay P. Pathmasiri W. Stewart D. McRitchie S. Li H. Chung J. Sohn J. Kim S. et al.Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.Nat. Microbiol. 2016; 1: 16140Crossref PubMed Scopus (170) Google Scholar; Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar). Our recent studies showed that early-life antibiotic exposure induced gut microbiota perturbation, interfering with adaptive immune effectors, altering ileal gene maturational patterns and the incidence of T1D onset, especially in male NOD mice (Livanos et al., 2016Livanos A.E. Greiner T.U. Vangay P. Pathmasiri W. Stewart D. McRitchie S. Li H. Chung J. Sohn J. Kim S. et al.Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.Nat. Microbiol. 2016; 1: 16140Crossref PubMed Scopus (170) Google Scholar; Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar).Faced with massive disruption of the intestinal microbiota due to antibiotic exposures and other perturbations, clinicians have developed fecal microbiota transplantation (FMT), a restorative procedure to return to eubiosis and control the illness (Kelly et al., 2015Kelly C.R. Kahn S. Kashyap P. Laine L. Rubin D. Atreja A. Moore T. Wu G. Update on fecal microbiota Transplantation 2015: indications, methodologies, mechanisms, and outlook.Gastroenterology. 2015; 149: 223-237Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar; Malikowski et al., 2017Malikowski T. Khanna S. Pardi D.S. Fecal microbiota transplantation for gastrointestinal disorders.Curr. Opin. Gastroenterol. 2017; 33: 8-13Crossref PubMed Scopus (16) Google Scholar). This technique has been successful in permitting the recovery of patients with Clostridioides difficile colitis (van Nood et al., 2013van Nood E. Vrieze A. Nieuwdorp M. Fuentes S. Zoetendal E.G. de Vos W.M. Visser C.E. Kuijper E.J. Bartelsman J.F. Tijssen J.G. et al.Duodenal infusion of donor feces for recurrent Clostridium difficile.N. Engl. J. Med. 2013; 368: 407-415Crossref PubMed Scopus (2168) Google Scholar) and is being investigated for efficacy in numerous clinical conditions (Bowman et al., 2015Bowman K.A. Broussard E.K. Surawicz C.M. Fecal microbiota transplantation: current clinical efficacy and future prospects.Clin. Exp. Gastroenterol. 2015; 8: 285-291Crossref PubMed Scopus (1) Google Scholar; Kellermayer, 2019Kellermayer R. Fecal microbiota transplantation: great potential with many challenges.Transl. Gastroenterol. Hepatol. 2019; 4: 40Crossref PubMed Scopus (13) Google Scholar; Pathak et al., 2013Pathak R. Enuh H.A. Patel A. Wickremesinghe P. Treatment of relapsing Clostridium difficile infection using fecal microbiota transplantation.Clin. Exp. Gastroenterol. 2013; 7: 1-6PubMed Google Scholar).Since the antibiotic-perturbed microbiota T1D model that we have developed in NOD mice also involves substantial dysbiosis (Livanos et al., 2016Livanos A.E. Greiner T.U. Vangay P. Pathmasiri W. Stewart D. McRitchie S. Li H. Chung J. Sohn J. Kim S. et al.Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.Nat. Microbiol. 2016; 1: 16140Crossref PubMed Scopus (170) Google Scholar; Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar), we now ask whether we can restore the microbiota and return the enhanced disease risk to baseline. The underlying questions are to determine which microbiota might be optimal for restoration and to assess the intermediate steps affected by the perturbation and restored by the transplantation. This experimental approach establishes the feasibility of the process and identifies key microbiota members, metagenomic pathways, metabolites, host genes, and signaling mechanisms that can be harnessed for future focused interventions. These discoveries provide insights into analogous human pathophysiology.ResultsCecal microbiota transfer normalizes post-antibiotic gut microbiome compositionsWe first evaluated whether specific microbiota transfer can restore changes in the intestinal microbiota and intestinal innate immunity induced by an early-life antibiotic course in NOD mice (Figure 1A). To reflect microbial populations relevant to the maturation of the recipient pups, we prepared cecal microbiota pools from dams or pups from four different donor types: (1) pups on their fifth day of life (Pup-P5), (2) dams on pup day of life 5 (Mom-P5), (3) dams at parturition (Mom-P0), and (4) dams at parturition, who received an antibiotic (tylosin) course during pregnancy to disrupt their microbiome (Mom-T-P0). These inocula contained ∼107 copies of 16S rRNA genes and varied in their α-diversity, with the Mom-P0 inoculum showing the highest values (Table S1). Community structure (β-diversity) using unweighted UniFrac showed the dam pool inocula clustering together, whereas the pup pool inoculum was distinct (data not shown). We then performed cecal microbiota transfer (CMT) via gavage to recipient mice at P15 that had received the standard antibiotic treatment (1PAT, with tylosin) from P5–P10 and evaluated the gut microbiota and the intestinal innate responses at two early time points after the CMT or control transfers (Figure 1). Pups exposed to 1P followed by the control gavage showed markedly diminished α-diversity, persisting to P42 (Figures 1B and 1C). Donor pool i CMT(Pup−P5) did not significantly change 1PAT cecal microbiota α-diversities for either time point (Figure 1B). Donor pool ii CMT(Mom−P5) significantly increased 1P cecal microbiota α-diversities at P42 but not at P28 (Figure 1B). Donor pool iii CMT(Mom−P0) significantly increased recipient cecal microbiota α-diversities at both P28 and P42 (Figure 1B). Compared with donor pool 3 CMT(Mom−P0), donor pool iv CMT(Mom−T−P0) did not significantly increase cecal microbiota α-diversities at P28 (Figure 1B).The 1PAT mice that received the Mom-P0 CMT or control broth had significantly different microbial compositions from each other (Figure 1C). The other maternal inocula (Mom-P5 and Mom-T-P0, but not the Pup-P5) yielded similar results (data not shown). Compared with control mice without antibiotic exposure, 1PAT depleted particular Firmicutes (including genera Oscillospira, Ruminococcus) and enriched genus Akkermansia in the cecal microbiota from early time points (Figure S1A), consistent with our prior observation (Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar). Based on these short-term restoration (STR) analyses, we concluded that Mom-P0 CMT best restored early-life α-diversity, β-diversity, and specific taxa of interest, making it the optimal transfer material among those studied for long-term restoration (LTR) experiments.Maternal CMT normalized ileal gene expression and the metabolic pathways regulating intestinal immune markers in antibiotic-treated miceNext, we asked whether the partially restored microbiota affected gene expression in the ileum. 1PAT significantly affected P42 ileal expression of 817 of the ∼30,000 detected genes, downregulating 483 and upregulating 334 (Figure 1D, top); Mom-P0 CMT affected expression of 403 genes compared with 1PAT, downregulating 211, upregulating 192; Figure 1D, bottom); KEGG analysis showed genes related to retinol, cysteine, methionine, pyruvate, and sulfur metabolism as well as cytochromes and steroid hormone biosynthesis (Figure S1C). The genes most repressed by CMT compared with 1PAT included a group of Antp homeobox family transcription factor genes (Hoxb5, Hoxb6, Hoxb7, and Hoxb8) and Ifi202b (autoimmune-development-associated interferon-activated gene), and those most induced included Gata4 (β cell transcription factor), Trim 38 (inhibitor of TLR3/4-mediated inflammation), and Ighv1-47 (immunoglobulin heavy-chain gene) (Figure 1D; Table S2). In total, these findings indicate that Mom-P0 CMT changed host ileal gene expression, including genes important in early-life immunological development.Next, we evaluated the effects of Mom-P0 CMT on specific ileal innate and adaptive immune early marker signature genes that we previously identified as 1PAT-perturbed (Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar) (Figure 1E). Ileal expression of NOS2 (innate immune-inducible nitric oxide synthase), downregulated by 1PAT, was partially restored by Mom-P0 CMT. Similar trends were seen for MUC2 and MUC4 (genes central for mucin synthesis), TNF-α, RUNX1 (early-life transcription factor) and two of its downstream genes (FOXP3, CD3G) critically involved in adaptive immunity (Figure 1E). Serum amyloid A1 (SAA1) was significantly downregulated by 1P at P28 in the ileum, but Mom-P0 CMT had little effect (Figure 1E). CMT with antibiotic-perturbed maternal microbiota (Mom-T-P0) had effects similar to Mom-P0 CMT, but generally to a lesser extent. Collectively, findings from the STR experiment provided evidence that Mom-P0 CMT significantly restored both microbiota population structure and host responses, suggesting the utility of an experiment to assess whether the 1PAT-enhanced T1D could be blocked.Maternal CMT largely rescued the antibiotic-induced T1D enhancementTo assess whether the Mom-P0 CMT protected NOD mice against antibiotic-enhanced T1D, we used male NOD mice, which showed stronger antibiotic effects than females (Livanos et al., 2016Livanos A.E. Greiner T.U. Vangay P. Pathmasiri W. Stewart D. McRitchie S. Li H. Chung J. Sohn J. Kim S. et al.Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.Nat. Microbiol. 2016; 1: 16140Crossref PubMed Scopus (170) Google Scholar; Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar). In the LTR experiment, male mice received 1PAT (or not as C, Control) at P5-10 and then were gavaged with Mom-P0 CMT or with blank medium (PBS) on P13-17 and then were monitored for 30 weeks for T1D development (Figure 2A). By the end of the study, control (C) mice were significantly more often TID-free (50%) than were the 1P mice (15%) (Figure 2B), consistent with our prior studies (Livanos et al., 2016Livanos A.E. Greiner T.U. Vangay P. Pathmasiri W. Stewart D. McRitchie S. Li H. Chung J. Sohn J. Kim S. et al.Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.Nat. Microbiol. 2016; 1: 16140Crossref PubMed Scopus (170) Google Scholar; Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar). Mom-P0 CMT restored week 30 survival (45%) similar to that observed in C mice (50%) (Figure 2B). Moreover, according to the Kaplan-Meier analyses and weighted log-rank tests, there were significant differences in disease-free rates across the three experimental groups (p = 0.005), especially during the middle and late time points. Disease-free rates for the three groups were similar at the beginning phase (week 0 to ∼20), but the rate for group 1P dropped during the middle phase compared with those of C (p = 0.009) and CMT (p = 0.045) (Figure 2B). Thus, we now confirmed our model of T1D enhancement with a single early-life antibiotic course and showed the ability to significantly reverse the phenotype. Since the interval between the early-life antibiotic exposure (ending on P10) and gavages (ending on P17), and T1D onset (earliest at P85, median at P180) represents a long latency period, we focused on early time points to better understand the initial mechanisms in T1D pathogenesis.Figure 2Effect of CMT on T1D development and early-life fecal microbiome in an LTR experimentShow full caption(A) Design of long-term restoration (LTR) experiment. Pregnant NOD/ShiLtJ male mice were randomized into three groups: control mice gavaged with PBS at P13-17 (group C); antibiotic-treated (1P) mice receiving either PBS (group 1P) or gavages of Mom-P0 cecal contents (group CMT). Mice (n = 20/group) were tested weekly for diabetes by blood glucose measurement from weeks 11–30. In addition, to provide specimens for the intermediate analyses of mechanism, 72 mice were sacrificed earlier (12 each in the C, 1P, and CMT groups at P23, and six in each group at P42 [n = 18] and at P70 [n = 18]). Symbols indicate times at which the fecal microbiome was analyzed by ∗16S rRNA sequencing, and +shotgun metagenomic sequencing, or a subset was #sacrificed for examining ileal and cecal tissues and contents, and metabolomics.(B) Kaplan-Meier analysis of T1D incidence in mice (n = 20/group). The first T1D case occurred at P84; after P154, the 3 groups were clearly differentiated in T1D outcomes. Statistical significance was determined by the Gρ,γ weighted log-rank test (Fleming and Harrington, 1991Fleming T. Harrington D. Counting Processes and Survival Analysis. John Wiley & Sons, 1991Google Scholar). Post-hoc pairwise comparisons were adjusted by a Benjamini-Hochberg procedure.(C) α-diversity over time measuring Faith’s PD and Pielou evenness. Top: ∗∗∗p < 0.001, one-way ANOVA; bottom: ∗p < 0.01, ∗∗∗p < 0.001, ns p > 0.05, one-way ANOVA with Tukey’s HSD post-test.(D) β-diversity, as determined by unweighted UniFrac analysis of the fecal microbiota of the three groups. Inter-group UniFrac distances were all significant (p < 0.0001), one-way-ANOVA with Tukey’s HSD post-test.(E) Early-life taxa significantly under-(red) or over-(blue) represented in either the STR or LTR experiments in CMT compared with 1P mice, according to MaAsLin2 analysis. The abundance of taxa marked with ° were significantly different in 1P compared with C and also were restored by CMT (also see Figure S1A). Analysis identified a set of genera (from family S24-7, orders Clostridiales and RF32 and genera Dorea and Oscillospira) significantly decreased by 1P but increased by CMT, and also identified a set of genera (from family Enterobacteriaceae, and genera Blautia and Proteus) significantly increased by 1P but decreased by CMT. “g” indicates unclassified genus below the family or Order levels.Related to Figures S1 and S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CMT significantly restored fecal microbiome community characteristics toward baselineNext, we assessed the effects of maternal CMT on the pup microbiome. Community richness, measured by Faith’s PD, was markedly diminished by 1PAT compared with C mice, but was intermediate in the CMT mice and community richness increased faster for C and CMT from P23 to P49 than for 1P mice (Figure 2C, top). Community evenness (Pielou score) was also markedly reduced in the 1P mice, and P23 to P49 trends differed for each group (Figure 2C, bottom); however, CMT restored community evenness to the control level. Community structure (β-diversity) significantly differed between C and 1P mice, and CMT led to an intermediate group, significantly distinct from 1P mice and C mice (Figure 2D). Estimating the total number of bacterial cells in P23 fecal samples using universal bacterial qPCR showed that 1PAT significantly decreased total bacterial abundance, but CMT was restorative (Figure S1B, left). All these metrics indicate that CMT restored antibiotic-perturbed global bacterial populations toward normal. We also noted substantial 16S count heterogeneity in C mice, with low numbers associated with early (<week 22) T1D development (Figure S1B, right).Effect of post-antibiotic CMT on specific taxaNext, we assessed the specific taxa restored by CMT. Across the 267 fecal and cecal samples obtained at early timepoints in the Mom-P0 CMT STR and LTR experiments, we identified 194 individual taxa at the genus level. Applying the MaAsLin2 algorithm to the pre-T1D STR cecal samples and LTR fecal samples, we observed 1PAT differentially selected taxa (Figure S1A) with significantly differential taxa after CMT (Figure 2E). The Bacteroidales family S24-7, dominant in the NOD mouse gut, was depleted in 1P mice (Figure S1A) and was restored by CMT (Figures 2E and 3B ). Firmicutes unclassified species from genera Oscillospira and Dorea, and unclassified species from orders Clostridiales and RF32, were under-represented in 1P mice, respectively, and restored by CMT (Figures 2E, S1A, and S2D). Among them, Oscillospira relative abundances in C mice that developed T1D early (<week 22) were significantly lower at all three early time points than those developing T1D late (>week 28) or never (Figure 3A, right), and unclassified species within Order Clostridiales at P35 and P49 was also negatively associated with T1D-development (Figure S2A), independently in all three treatment groups (Figure S2B). In contrast, Blautia producta and two unclassified species from Enterobacteriaceae (a Proteus species and an unclassified genus) were enriched in 1P compared with C, consistent with prior findings (Zhang et al., 2018Zhang X.S. Li J. Krautkramer K.A. Badri M. Battaglia T. Borbet T.C. Koh H. Ng S. Sibley R.A. Li Y. et al.Antibiotic-induced acceleration of type 1 diabetes alters maturation of innate intestinal immunity.eLife. 2018; 7e37816Crossref PubMed Scopus (31) Google Scholar), and reduced by CMT (Figures 2E, S1C, and S2D). We also identified unclassified species from families Erysipelotrichaceae and genera Lactobacillus depleted by 1PAT but significantly restored by CMT, whereas Akkermansia muciniphila was enriched by 1PAT but partially repressed by CMT (Figure S2D). Comparing taxa abundances across groups, 1PAT significantly decreased the Lachnospiraceae/Enterococcus ratio from that in C, whereas CMT was significantly restorative (Figure S2C). In total, these analytical methods identified taxa associated or not with protection against T1D development.Figure 3CMT effects on specific bacterial taxa and on bacterial metabolic pathwaysShow full caption(A) Oscillospira genus relative abundance with respect to the average microbe in groups (C, 1P, and CMT) at P23, P35, and P49 (left), changing with time (middle); and in the mice that developed T1D early (E) or late or never (L) within each treatment group (right). Mean ± SD shown; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; Mann-Whitney U test.(B) S24-7 family relative abundance in groups (C, 1P, and CMT), at P23, P35, and P49; Analysis was performed by QIIME2 based on serial fecal samples from 19 ∼ 20 mice/group. Mean ± SD shown; ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; Mann-Whitney U test.(C) Metagenomic (MetaCyc) pathway distribution (Caspi et al., 2018Caspi R. Billington R. Fulcher C.A. Keseler I.M. Kothari A. Krummenacker M. Latendresse M. Midford P.E. Ong Q. Ong W.K. et al.The MetaCyc database of metabolic pathways and enzymes.Nucleic Acids Res. 2018; 46: D633-D639Crossref PubMed Scopus (285) Google Scholar) at P23 and P35 in C (blue, n = 6), or 1P (red, n = 6), or CMT mice (green, n = 8) visualized by principal component analysis.(D) Unsupervised clustering of 48 fecal microbial pathways with significant differences at P23. A group of 29 pathways, which were restored to control levels by CMT, is highlighted within the red box (see Table S2). A subgroup of 5 pathways with the greatest consistency between CMT and C is highlighted within the blue box, including three fatty acid salvage, and two fatty acid β-oxidation pathways (also see Figures S1D and S1E).(E) Comparison of the relative abundance of the most abundant genes in the fatty acid metabolism-related pathways most restored by CMT. Of the 5 pathways, the eight genes with highest relative abundance (D4ILA3-B4EVV3) encode long-chain acyl-CoA synthetases and ligases (E.C.6.2.1.3; n = 5) or 3-ketoacyl-CoA thiolases (E.C.2.3.1.16; n = 3) and long-chain fatty acid CoA ligases (n = 2). Mean ± SD shown from determinations in 5.2 ± 1.7 individual mice/group (also see Figures S1D and S1E).Related to Figures S1, S2, and S3 and Tables S3 and S4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)CMT partially restores gut microbial metabolic pathwaysTo determine whether the altered microbiota following CMT differed in its metabolic functions, we used shotgun sequencing to examine post-gavage fecal metagenomes. Based on the Bray-Curtis analysis of 325 metabolic pathways identified at P23 and P35, 1PAT globally affected metagenomic composition, whereas CMT was at least partially restorative (Figure 3C). The P23 and P35 results clustered together within each treatment group, indicating the stability of the findings. CMT restored 29 (71%) of the 41 pathways that differentiated the 1P and C mice (Figure 3D; Table S3), as shown in the heatmap red box (from pathway 52 to pathway 73, and Table S3). The restoration of gut microbial me" @default.
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• W3183749649 date "2021-08-01" @default.
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• W3183749649 title "Maternal cecal microbiota transfer rescues early-life antibiotic-induced enhancement of type 1 diabetes in mice" @default.
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