Background & Aims
Methods
Results
Conclusions
Graphical abstract

Keywords
Abbreviations used in this paper:
CD (Crohn’s disease), CXCL1 (chemokine (C-X-C motif) ligand 1), GEE (generalized estimating equation), GF (germ-free), IBD (inflammatory bowel disease), ITS (internal transcribed spacer), KO (knockout), NOD2 (nucleotide-binding oligomerization domain-containing protein 2), OTU (operational taxonomic unit), PCoA (principle coordinate analysis), qPCR (quantitative real-time polymerase chain reaction), rRNA (ribosomal RNA), SPF (specific pathogen free), WT (wild-type)- Ellinghaus D.
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- Spain S.L.
- Cortes A.
- Bethune J.
- Han B.
- Park Y.R.
- Raychaudhuri S.
- Pouget J.G.
- Hubenthal M.
- Folseraas T.
- Wang Y.
- Esko T.
- Metspalu A.
- Westra H.J.
- Franke L.
- Pers T.H.
- Weersma R.K.
- Collij V.
- D'Amato M.
- Halfvarson J.
- Jensen A.B.
- Lieb W.
- Degenhardt F.
- Forstner A.J.
- Hofmann A.
- Schreiber S.
- Mrowietz U.
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- Lazaridis K.N.
- Brunak S.
- Dale A.M.
- Trembath R.C.
- Weidinger S.
- Weichenthal M.
- Ellinghaus E.
- Elder J.T.
- Barker J.N.
- Andreassen O.A.
- McGovern D.P.
- Karlsen T.H.
- Barrett J.C.
- Parkes M.
- Brown M.A.
- Franke A.
- Chu H.
- Khosravi A.
- Kusumawardhani I.P.
- Kwon A.H.
- Vasconcelos A.C.
- Cunha L.D.
- Mayer A.E.
- Shen Y.
- Wu W.L.
- Kambal A.
- Targan S.R.
- Xavier R.J.
- Ernst P.B.
- Green D.R.
- McGovern D.P.
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- Weersma R.K.
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- McGovern D.P.
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- Anderson C.A.
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- Mitrovic M.
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- Cleynen I.
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- Ellinghaus D.
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- Franchimont D.
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- Haritunians T.
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- Targan S.R.
- Taylor K.D.
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- Verspaget H.W.
- De Vos M.
- Wijmenga C.
- Wilson D.C.
- Winkelmann J.
- Xavier R.J.
- Zeissig S.
- Zhang B.
- Zhang C.K.
- Zhao H.
- International I.B.D.G.C.
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- Hakonarson H.
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- Radford-Smith G.
- Mathew C.G.
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- Barrett J.C.
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- Chu H.
- Khosravi A.
- Kusumawardhani I.P.
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- Vasconcelos A.C.
- Cunha L.D.
- Mayer A.E.
- Shen Y.
- Wu W.L.
- Kambal A.
- Targan S.R.
- Xavier R.J.
- Ernst P.B.
- Green D.R.
- McGovern D.P.
- Virgin H.W.
- Mazmanian S.K.
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- Cuthbert A.
- Croucher P.J.
- Mirza M.M.
- Mascheretti S.
- Fisher S.
- Frenzel H.
- King K.
- Hasselmeyer A.
- MacPherson A.J.
- Bridger S.
- van Deventer S.
- Forbes A.
- Nikolaus S.
- Lennard-Jones J.E.
- Foelsch U.R.
- Krawczak M.
- Lewis C.
- Schreiber S.
- Mathew C.G.
- Ng K.M.
- Aranda-Diaz A.
- Tropini C.
- Frankel M.R.
- Van Treuren W.
- O'Laughlin C.T.
- Merrill B.D.
- Yu F.B.
- Pruss K.M.
- Oliveira R.A.
- Higginbottom S.K.
- Neff N.F.
- Fischbach M.A.
- Xavier K.B.
- Sonnenburg J.L.
- Huang K.C.
Results
Loss of NOD2 Alters Antibiotic-Induced Body Weight Loss and Recovery From Bacterial Dysbiosis


Matrix (comparison) | Psuedo F | P value |
---|---|---|
20-week-old mice | ||
WTday0–WTday50 | 7.704 | .01000 |
KOday 0–KOday 50 | 10.607 | .00500 |
WTday0–KOday0 | 1.273 | .23300 |
WTday50–KOday50 | 9.156 | .03500 |
1-year-old mice | ||
WTday0–WTday86 | 3.723 | .000999 |
KOday 0–KOday 86 | 3.608 | .000999 |
WTday0–KOday0 | 0.606 | .9411 |
WTday86–KOday86 | 1.955 | .01199 |

Bacteroidetes | Proteobacteria | Verrucomicrobia | Firmicutes | Tenericutes | |
---|---|---|---|---|---|
Genotype (WT vs Nod2-KO) | 6.26E-01 | 1.38E-01 | 0.576497 | 8.23E-01 | 5.10E-01 |
Day 2 vs day 0 | 1.29E-05 | 5.72E-11 | 0.302459 | 2.94E-08 | 3.31E-01 |
Day 9 vs day 0 | 1.40E-11 | 0.00E+00 | 0.001512 | 1.11E-11 | 8.29E-03 |
Day 12 vs day 0 | 0.00E+00 | 0.00E+00 | 0.000808 | 3.87E-11 | 9.03E-03 |
Day 14 vs day 0 | 1.74E-14 | 0.00E+00 | 0.016084 | 2.96E-03 | 4.29E-03 |
Day 21 vs day 0 | 1.30E-01 | 8.27E-01 | 0.190371 | 3.10E-01 | 3.00E-01 |
Day 28 vs day 0 | 5.46E-02 | 5.50E-01 | 0.025025 | 8.18E-01 | 2.35E-02 |
Day 49 vs day 0 | 5.33E-03 | 5.38E-01 | 0.932214 | 2.24E-02 | 1.40E-02 |
Day 70 vs day 0 | 9.37E-01 | 1.80E-01 | 0.290782 | 5.25E-01 | 7.10E-01 |
Day 86 vs day 0 | 3.58E-01 | 1.04E-01 | 0.023226 | 1.71E-01 | 2.35E-01 |
Barnesiella | Parabacteroides | Uncl. porphyromonadaceae | Prevotella | Alistipes | Bacteroides | Uncl. Bacteroidales | Parasutterella | Escherichia Shigella | Anaeroplasma | Akkermansia | Lactobacillus | Clostridium XlVa | Uncl. Lachnospiraceae | Uncl. Ruminococcaceae | Uncl. Clostridiales | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Genotype (WT vs Nod2-KO) | 0,045312 | 0,4866 | 2,34E-01 | 2,08E-01 | 2,41E-01 | 2,16E-01 | 0,40942 | 0,69085 | 0,70228 | 0,74512 | 4,95E-01 | 7,01E-01 | 2,27E-01 | 2,23E-01 | 7,72E-01 | 3,62E-01 |
day 2 vs day 0 | 0 | 0,182 | 1,20E-08 | 8,29E-06 | 2,69E-01 | 1,89E-01 | 0,00702 | 0,55719 | 0 | 0,125309 | 3,87E-02 | 6,14E-01 | 1,83E-05 | 1,94E-13 | 1,95E-08 | 6,03E-01 |
day 9 vs day 0 | 0 | 0,0396 | 2,22E-16 | 6,63E-10 | 7,44E-15 | 1,67E-06 | 0,21807 | 0,72818 | 0 | 0,000455 | 1,31E-07 | 3,44E-04 | 1,12E-02 | 1,94E-10 | 1,78E-05 | 1,11E-03 |
day 12 vs day 0 | 0 | 0,1362 | 0,00E+00 | 5,55E-16 | 0,00E+00 | 5,60E-09 | 0,09343 | 0,00278 | 0 | 0,000339 | 1,54E-07 | 8,98E-06 | 6,55E-04 | 1,84E-13 | 8,18E-11 | 2,44E-06 |
day 14 vs day 0 | 0 | 0,6734 | 0,00E+00 | 0,00E+00 | 0,00E+00 | 1,43E-02 | 0,00563 | 0,46939 | 0 | 0,633845 | 2,43E-05 | 1,22E-01 | 2,26E-06 | 4,35E-14 | 4,24E-12 | 5,31E-07 |
day 21 vs day 0 | 0,000144 | 0,0241 | 5,32E-07 | 4,81E-02 | 3,98E-01 | 2,56E-01 | 0,13552 | 0,31411 | 0,00301 | 0,142477 | 2,44E-02 | 6,33E-01 | 1,40E-02 | 6,48E-01 | 4,23E-02 | 4,28E-01 |
day 28 vs day 0 | 0,031842 | 0,0887 | 2,72E-02 | 3,98E-04 | 4,26E-01 | 5,28E-01 | 0,02707 | 0,2149 | 0,05153 | 0,018377 | 1,01E-01 | 7,80E-02 | 9,71E-03 | 9,53E-01 | 3,11E-01 | 6,40E-01 |
day 49 vs day 0 | 0,535091 | 0,3141 | 5,96E-01 | 3,07E-10 | 9,81E-01 | 1,07E-01 | 0,1357 | 0,86042 | 0,03436 | 0,03386 | 3,19E-01 | 3,92E-02 | 3,62E-03 | 2,77E-01 | 2,70E-01 | 2,02E-02 |
day 70 vs day 0 | 0,085452 | 0,1169 | 3,41E-01 | 4,62E-07 | 8,92E-04 | 8,38E-03 | 0,09318 | 0,96517 | 0,26435 | 0,765982 | 5,02E-02 | 2,39E-01 | 1,91E-02 | 8,26E-01 | 4,44E-01 | 1,89E-01 |
day 86 vs day 0 | 0,415301 | 0,1269 | 7,12E-01 | 7,83E-11 | 3,53E-01 | 1,57E-03 | 0,14705 | 0,22099 | 0,68781 | 0,07713 | 1,54E-01 | 1,40E-01 | 2,14E-03 | 1,73E-01 | 1,85E-01 | 8,62E-02 |
OTU | WT | Nod2-/- | ||||
---|---|---|---|---|---|---|
Day 0 | Day 9 | Day 86 | Day 0 | Day 9 | Day 86 | |
Clostridium IV #125 | 3 | 0 | 2 | 2 | 0 | 1 |
Barnesiella #012 | 7 | 0 | 13 | 0 | 0 | 2 |
Ruminococcus #029 | 7 | 0 | 4 | 5 | 0 | 1 |
Unclassified Ruminococcaceae #166 | 1 | 0 | 1 | 1 | 0 | 0 |
Escherichia #001 | 0 | 1171 | 0 | 0 | 1154 | 1 |
Streptococcus #067 | 0 | 1 | 0 | 0 | 0 | 0 |
Unclassified Porphyromonadaceae #017 | 23 | 0 | 13 | 32 | 0 | 20 |
Barnesiella #009 | 6 | 0 | 73 | 4 | 0 | 24 |
Unclassified Ruminococcaceae #032 | 6 | 0 | 15 | 5 | 0 | 7 |
Barnesiella #073 | 4 | 0 | 3 | 3 | 0 | 3 |
Oscillibacter #023 | 16 | 0 | 7 | 6 | 0 | 10 |
Emergence of Antibiotic Resistance Occurs Independently of NOD2 Signaling
- Hasler R.
- Kautz C.
- Rehman A.
- Podschun R.
- Gassling V.
- Brzoska P.
- Sherlock J.
- Grasner J.T.
- Hoppenstedt G.
- Schubert S.
- Ferlinz A.
- Lieb W.
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- Heinsen F.A.
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Family/genus | Target | Target gene | Assay ID | Context sequence |
---|---|---|---|---|
Enterobacteriaceae Phylum: Proteobacteria | ESBL | CTX-M group9 | Pa04646127_s1 | GCTTAATCAGCCTGTCGAGATCAAG |
SHV | Pa04646134_s1 | CGCTTTCCCATGATGAGCACCTTTA | ||
TEM | Pa04646128_s1 | GCGGCCAACTTACTTCTGACAACGA | ||
OXA-1 | Pa04646133_s1 | TCATACACCAAAGACGTGGATGCAA | ||
AmpC resistance | BIL/LAT/CMY | Pa04646135_s1 | TAAGACGTTTAACGGCGTGTTGGGC | |
ACT/MIR | Pa04646124_s1 | ACCGTTACGCCGCTGATGAAAGCGC | ||
Carbapenem resistance | VIM | Pa04646155_s1 | GATGGTGATGAGTTGCTTTTGATTG | |
KPC | Pa04646152_s1 | CCTCGTCGCGGAACCATTCGCTAAA | ||
Enterococcus Phylum: Firmicutes | Vancomycin resistance | vanA2 | Pa04646147_s1 | AGCTACTCCCGCCTTTTGGGTTATT |
vanB | Pa04646150_s1 | AACTTAACGCTGCGATAGAAGCGGC | ||
vanC2-C3-2 | Pa04646122_s1 | TTGAGATCGGTTGCGGTATTTTGGG |
Target gene | Target | Associated phylum | Individual mouse | Day resistance gene was present |
---|---|---|---|---|
ACT/MIR | AmpC resistance | Proteobacteria | #46 WT | 14 |
ACT/MIR | AmpC resistance | Proteobacteria | #55 WT | 71 |
vanC2-C3-2 | Vancomycin resistance | Firmicutes | #24 WT | 21 and 71 |
vanA2 | Vancomycin resistance | Firmicutes | #32 KO | 0 |
vanC2-C3-2 | Vancomycin resistance | Firmicutes | #35 KO | 14 |
vanC2-C3-2 | Vancomycin resistance | Firmicutes | #11 KO | 21 |
BIL/LAT/CMY | AmpC resistance | Proteobacteria | #7 KO | 21 |
The Fungal Gut Communities Change Long Term Irrespective of NOD2 Deficiency





The Postantibiotic Nod2-KO Microbiota Elicits a Proinflammatory Response Upon Transfer to GF WT Mice


Discussion
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- Pons N.
- David O.
- Schaeffer B.
- Lepage P.
- Martin P.
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- Seksik P.
- Beaugerie L.
- Ehrlich S.D.
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- Van Dorsselaer A.
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- Ito G.
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- Lipinski S.
- Kuiper J.W.
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- Saveljeva S.
- Bhattacharyya J.
- Hasler R.
- Bartsch K.
- Luzius A.
- Jentzsch M.
- Falk-Paulsen M.
- Stengel S.T.
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- Schwarzer R.
- Rabe B.
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- Krautwald S.
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- Blumberg R.S.
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- Kaser A.
- Rosenstiel P.
- Couturier-Maillard A.
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- Rehman A.
- Normand S.
- De Arcangelis A.
- Haesler R.
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- Grandjean T.
- Bressenot A.
- Delanoye-Crespin A.
- Gaillot O.
- Schreiber S.
- Lemoine Y.
- Ryffel B.
- Hot D.
- Nunez G.
- Chen G.
- Rosenstiel P.
- Chamaillard M.
- Ng K.M.
- Aranda-Diaz A.
- Tropini C.
- Frankel M.R.
- Van Treuren W.
- O'Laughlin C.T.
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- Yu F.B.
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- Neff N.F.
- Fischbach M.A.
- Xavier K.B.
- Sonnenburg J.L.
- Huang K.C.
Materials and Methods
Specific Pathogen–Free Mice
Antibiotic Treatment
Colonization of GF Mice
- Pan W.H.
- Sommer F.
- Falk-Paulsen M.
- Ulas T.
- Best P.
- Fazio A.
- Kachroo P.
- Luzius A.
- Jentzsch M.
- Rehman A.
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- Walter J.
- Kunzel S.
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- Rosenstiel P.
16S Amplicon Sequencing
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- Pontarollo G.
- Grill A.
- Kuijpers M.J.E.
- Wilms E.
- Weber C.
- Sommer F.
- Nagy M.
- Neideck C.
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- Ascher S.
- Formes H.
- Karwot C.
- Bayer F.
- Kollar B.
- Subramaniam S.
- Molitor M.
- Wenzel P.
- Rosenstiel P.
- Todorov H.
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- Walter U.
- Jurk K.
- Heemskerk J.W.M.
- van der Vorst E.P.C.
- Doring Y.
- Reinhardt C.
- Schloss P.D.
- Westcott S.L.
- Ryabin T.
- Hall J.R.
- Hartmann M.
- Hollister E.B.
- Lesniewski R.A.
- Oakley B.B.
- Parks D.H.
- Robinson C.J.
- Sahl J.W.
- Stres B.
- Thallinger G.G.
- Van Horn D.J.
- Weber C.F.
- Schloss P.D.
- Westcott S.L.
- Ryabin T.
- Hall J.R.
- Hartmann M.
- Hollister E.B.
- Lesniewski R.A.
- Oakley B.B.
- Parks D.H.
- Robinson C.J.
- Sahl J.W.
- Stres B.
- Thallinger G.G.
- Van Horn D.J.
- Weber C.F.
Fungal ITS1 Amplicon Sequencing
- Bengtsson-Palme J.
- Ryberg M.
- Hartmann M.
- Branco S.
- Wang Z.
- Godhe A.
- De Wit P.
- Sánchez-García M.
- Ebersberger I.
- de Sousa F.
- Amend A.
- Jumpponen A.
- Unterseher M.
- Kristiansson E.
- Abarenkov K.
- Bertrand Y.J.K.
- Sanli K.
- Eriksson K.M.
- Vik U.
- Veldre V.
- Nilsson R.H.
- Nilsson R.H.
- Tedersoo L.
- Ryberg M.
- Kristiansson E.
- Hartmann M.
- Unterseher M.
- Porter T.M.
- Bengtsson-Palme J.
- Walker D.M.
- de Sousa F.
- Gamper H.A.
- Larsson E.
- Larsson K.H.
- Koljalg U.
- Edgar R.C.
- Abarenkov K.
Detection of Antibiotic Resistance Genes Using Real-Time qPCR
Histopathology
- Adolph T.E.
- Tomczak M.F.
- Niederreiter L.
- Ko H.J.
- Bock J.
- Martinez-Naves E.
- Glickman J.N.
- Tschurtschenthaler M.
- Hartwig J.
- Hosomi S.
- Flak M.B.
- Cusick J.L.
- Kohno K.
- Iwawaki T.
- Billmann-Born S.
- Raine T.
- Bharti R.
- Lucius R.
- Kweon M.N.
- Marciniak S.J.
- Choi A.
- Hagen S.J.
- Schreiber S.
- Rosenstiel P.
- Kaser A.
- Blumberg R.S.
Enzyme-Linked Immunosorbent Assay
Real-Time PCR Quantification of the Microbiota
Primer | Sequence 5’ to 3’ | Probe 5’ to 3’ | Reference |
---|---|---|---|
All bacteria, Bac338_F | ACTCCTACGGGAGGCAG | TGCCAGCAGCCGCGGTAATAC | PMID: 9687486 |
All bacteria, Bac805_R | GACTACCAGGGTATCTAATCC | ||
Bacteroidetes, Bac 32F | AACGCTAGCTACAGGCTTAACA | CAATATTCCTCACTGCTGCCTCCCGTA | PMID: 11010920 |
Bacteroidetes, BactR | ACGCTACTTGGCTGGTTCA | ||
Firmicutes, 8F | AGAGTTTGATCCTGGCTCAG | CTGATGGAGCAACGCCGCGT | PMID: 10461381 |
Firmicutes, 534R | ATTACCGCGGCTGCTGG | PMID: 9687486 | |
Prevotalla, F_Bacter 11 | CCTWCGATGGATAGGGGTT | AAGGTCCCCCACATTG | PMID: 19302550 |
Prevotalla, R_Bacter 08 | CACGCTACTTGGCTGGTTCAG | ||
FungiQuant-F | GGRAAACTCACCAGGTCCAG | TGGTGCATGGCCGTT | PMID: 23136846 |
FungiQuant-R | GSWCTATCCCCAKCACGA |
Availability of Data and Materials
Statistical Analysis
Acknowledgments
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Article info
Publication history
Footnotes
Author contributions Jacqueline Moltzau Anderson, Simone Lipinski, Felix Sommer, and Philip Rosenstiel designed the research; Simone Lipinski, Olivier Boulard, and Maren Falk-Paulsen managed the specific pathogen free mouse breeding, monitored the mice, and collected fecal pellets; Sven Künzel and John F. Baines managed the germ-free mouse breeding and provided and maintained the germ-free mouse facility; Simone Lipinski and Felix Sommer performed the germ-free mouse experiment and collected samples; Jacqueline Moltzau Anderson, Simone Lipinski, Maren Falk-Paulsen, Stephanie T. Stengel, and Ateequr Rehman conducted the wet laboratory experiments; Jacqueline Moltzau Anderson, Simone Lipinski, Olivier Boulard, Ateequr Rehman, Maren Falk-Paulsen, Stephanie T. Stengel, Konrad Aden, Felix Sommer, Mathias Chamaillard, and Philip Rosenstiel analyzed and interpreted the data; Jacqueline Moltzau Anderson, Wei-Hung Pan, Ateequr Rehman, Felix Sommer, and Richa Bharti performed the bioinformatics analyses; Jacqueline Moltzau Anderson, Simone Lipinski, Wei-Hung Pan, Felix Sommer, and Robert Häsler prepared the figures; Mathias Chamaillard and Philip Rosenstiel obtained funding; and Jacqueline Moltzau Anderson, Simone Lipinski, Felix Sommer, Mathias Chamaillard, and Philip Rosenstiel co-wrote the manuscript.
Conflicts of interest The authors disclose no conflicts.
Funding This work was supported by the International Max Planck Research Schools of the Max-Planck-Institute for Evolutionary Biology and Christian-Albrechts-University Kiel, the Origin and Function of Metaorganisms Collaborative Research Centre 1182 (C2), the Precision Medicine in Chronic Inflammation Excellence Cluster 2167, the Inflammation at Interfaces Excellence Cluster 306 of the Deutsche Forschungsgemeinschaft and the Schleswig-Holstein Excellence Chair Program (P.R. and J.F.B.), and by Institut national de la santé et de la recherche médicale (M.C.) and by the Fondation pour la Recherche Médicale grant (grant number DEQ20130326475 to M.C.).
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- The Hidden Effect of Nod2 in the Host/Microbiota RelationshipCellular and Molecular Gastroenterology and HepatologyVol. 10Issue 2
- PreviewIn this issue of Cellular and Molecular Gastroenterology and Hepatology, Anderson et al1 present new insights on the role played by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor in shaping and regulating the intestinal microbiota. NOD2 is a pattern recognition receptor expressed by specialized cells of the epithelium, such as Paneth cells, and some immune cells, and which has been involved in inflammatory disorders (first susceptibility gene identified in Crohn’s disease).
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