Journal: Transplantation and cellular therapy

Article Title: A Low Level of Plasmacytoid Dendritic Cells at Engraftment Is a Valuable Prognostic Indicator in Children Receiving Allogeneic Hematopoietic Stem Cell Transplantation.

doi: 10.1016/j.jtct.2021.04.012

Figure Lengend Snippet: Figure 4. High biomarker levels indicates the occurrence of II-IV aGVHD in the ST2 and Reg3a cohort. Plasma was isolated before the isolation of PBMCs. Biomarkers were detected using ELISA at engraftment and 2 weeks after engraftment. (A) ST2 levels at engraftment and 2 weeks after engraftment. (B) Reg3a levels at engraft- ment and 2 weeks after engraftment. (C) ROC curves for ST2 levels in plasma for the prediction of II-IV aGVHD at engraftment and 2 weeks after engraftment. (D) ROC curves for Reg3a levels in plasma for the prediction of II-IV aGVHD at engraftment and 2 weeks after engraftment. *P < .05; **P < .01; ***P < .001.

Article Snippet: Reg3a was analyzed using the Human Reg3a DuoSet ELISA Kit (R&D Systems, Minneapolis, MN).

Techniques: Biomarker Discovery, Clinical Proteomics, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: The levels of regenerating islet-derived protein 3-alpha (REG3A) was increased and miR-146a was decreased in polymyositis and dermatomyositis (PM/DM) patients. (A) Peripheral blood mononuclear cells (PBMCs) were isolated from patients ( n = 25) and the healthy controls ( n = 20) and the messenger RNA (mRNA) levels of interferon gamma (IFN-γ), interleukin (IL)-17A, REG3A, and miRNA-146a were determined by real-time PCR. The relative mRNA expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/U6. (B) The levels of IFN-γ and IL-17A in serum from patients and the healthy controls were determined by ELISA assay. (C) The REG3A levels from patients and the healthy controls were determined by immunohistochemistry. Data are shown as means ± SEM. * p < 0.05, ** p < 0.01 in comparison with the healthy controls. PBMCs were obtained from 20 healthy controls and 25 patients with PM/DM.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Isolation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Comparison

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: The levels of regenerating islet-derived protein 3-alpha (REG3A) and miR-146a in an experimental autoimmune myositis (EAM) model. (A) The messenger RNA (mRNA) expression of interferon gamma (IFN-γ), interleukin (IL)-17A, REG3A, and miRNA-146a in muscle tissues were determined by real-time PCR. The relative mRNA expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/U6. ( n = 9) (B) . The levels of IFN-γFand IL-17A in serum from the EAM mice were determined by ELISA assay ( n = 9). (C) The protein levels of REG3A in muscle tissues were determined by Western blot ( n = 3). GAPDH was used as the internal controls for Western blot analysis. Bands were quantified using ImageJ. Data are shown as means ± SEM. * p < 0.05, ** p < 0.01 in comparison with the control group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: The effects of miR-146a and regenerating islet-derived protein 3-alpha (REG3A) on macrophage migration. (A) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were transfected with the negative control (NC) microRNA (miRNA) and miR-146a mimics, miR-146a inhibitors for 24 h. (B) Monocyte-derived macrophages from the peripheral blood mononuclear cells (PBMCs) of the healthy donors ( n = 3) were transfected with the NC siRNA, REG3A siRNA, pcDNA3.1-NC, and pcDNA3.1-REG3A plasmids for 24 h. All treated cells (2 × 10 5 ) were suspended and added to the upper chamber of transwell. The medium containing 10% human serum was used as a chemoattractant in the lower chamber. After incubation for 24 h, the invaded cells into the lower chamber were stained with crystal violet. The migrated cells were counted, and photomicrographs were taken under an Olympus inverted microscope (IX71, Olympus, Japan). Data are shown as means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 in comparison with the control group. ## p < 0.05 in comparison with pcDNA3.1-NC group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Migration, Transfection, Negative Control, Incubation, Staining, Inverted Microscopy, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: Interleukin (IL)-17A induced regenerating islet-derived protein 3-alpha (REG3A) expression in macrophage. (A) Monocyte-derived macrophages from the peripheral blood mononuclear cells (PBMCs) of the healthy donors ( n = 3) were treated with the different concentrations of IL-17A for 24 h. The messenger RNA (mRNA) expression of REG3A was determined by real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal controls. (B) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were treated with the different concentrations of IL-17A for 24 h. The mRNA expression of miR-146a was determined by real-time PCR. U6 was used as the internal controls. (C) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were treated with the different concentrations of IL-17A for 24 h. The protein levels of REG3A were determined by Western blot. GAPDH was used as the internal controls. (D) Monocyte-derived macrophages were transfected with the NC small-interfering RNA (siRNA) and IL-17RA siRNA in the absence or presence of IL-17A for 24 h. The protein levels of REG3A were determined by Western blot. GAPDH was used as the internal controls. Western blot bands were quantified using ImageJ and normalized to GAPDH. Data are shown as means ± SEM of three independent experiment. * p < 0.05, ** p < 0.01 in comparison with the control group. # p < 0.05 in comparison with IL-17A + NC siRNA group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Small Interfering RNA, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: miR-146a regulates regenerating islet-derived protein 3-alpha (REG3A) expression in macrophage. (A,B) Monocyte-derived macrophages from the peripheral blood mononuclear cells (PBMCs) of the healthy donors ( n = 3) were transfected with negative control (NC) microRNA (miRNA), miR-146a mimics, or miR-146a inhibitor for 24 h. The messenger RNA (mRNA) and protein levels of REG3A were determined by real-time PCR and Western blot. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal controls. (C) The cells were transfected with NC small-interfering RNA (siRNA), REG3A siRNA, pcDNA3.1-NC, and pcDNA3.1-REG3A plasmids for 24 h. The mRNA levels of miR-146a were determined by real-time PCR. U6 was used as the internal controls. (D,E) The cells were transfected with NC miRNA or miR-146a mimics in the absence or presence of IL-17A for 24 h. The mRNA and protein levels of REG3A were determined by real-time PCR and Western blot. GAPDH was used as the internal controls. Data are shown as means ± SEM of three independent experiments. * p < 0.05, ** p < 0.01 in comparison with the control group. # p < 0.05, ## p < 0.01 in comparison with IL-17A treatment group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Derivative Assay, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Small Interfering RNA, Comparison, Control

Journal: Frontiers in Immunology

Article Title: Reduced miR-146a Promotes REG3A Expression and Macrophage Migration in Polymyositis and Dermatomyositis

doi: 10.3389/fimmu.2020.00037

Figure Lengend Snippet: miR-146a inhibited macrophage migration through suppression of regenerating islet-derived protein 3-alpha (REG3A) expression. (A) Monocyte-derived macrophages from the PBMCs of the healthy donors ( n = 3) were transfected with the NC siRNA or REG3A siRNA in the absence or presence of miR-146a inhibitor for 24 h. (B) Monocyte-derived macrophages were transfected with the pcDNA3.1-NC or pcDNA3.1-REG3A plasmids in the absence or presence of miR-146a mimics for 24 h. All treated cells (2 × 10 5 ) were suspended and added to the upper chamber of transwell well. The medium containing 10% human serum was used as a chemoattractant in the lower chamber. After incubation for 24 h, the invaded cells into the lower chamber were stained with crystal violet. The migrated cells were counted, and photomicrographs were taken under an Olympus inverted microscope (IX71, Olympus, Japan). Data are shown as means ± SEM of three independent experiment. * p < 0.05, ** p < 0.01 in comparison with NC + NC siRNA or NC + pcDNA3.1 group. # p < 0.05, ## p < 0.01 in comparison with inhibitor + NC siRNA or NC + pcDNA3.1-REG3A group.

Article Snippet: The sections were immunostained with primary REG3A antibody (Novus biologicals, #NBP2-24763) at room temperature for 1 h and secondary antibody for 30 min in a humidified chamber.

Techniques: Migration, Derivative Assay, Expressing, Transfection, Incubation, Staining, Inverted Microscopy, Comparison

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Scheme of caerulein injection to induce acute pancreatitis. Acute pancreatitis was induced by intraperitoneal (i.p.) injection with caerulein dissolved in PBS at a dose of 50 μg/kg at hourly intervals eight times daily, for two consecutive days. PBS injection alone served as control. b The size of pancreas of PBS-treated mice appear smaller than those of caerulein-treated mice ( n = 6, student’s t -test). c Percentage of pancreas/body weight of caerulein-treated mice versus PBS-treated mice ( n = 6, student’s t -test). d Abundant ADM formation in caerulein-treated mice (H&E, 40×, Scale bar: 500 μm). e Difference in the extent of ADM between PBS-treated mice and caerulein-treated mice, as measured by percentage of ADM area in total pancreatic area on H&E stained tissue sections ( n = 6, student’s t -test). f Western blot analysis with quantifications showed higher REG3B protein level and lower AMYLASE protein level in the pancreatic tissue of caerulein-treated mice than that of PBS-treated mice ( n = 6, student’s t -test). GAPDH served as a loading control. g RT-qPCR analysis showed higher Ck19 and lower Mist1 mRNA levels in the pancreatic tissue of caerulein-treated mice than that of PBS-treated mice ( n = 5, student’s t -test). h H&E staining of caerulein-induced ADM in mice, with corresponding REG3B IHC staining. ADM areas were highlighted by dashed lines, with arrows indicating naïve ADM and asterisks indicating relatively mature ADM. Scale bar: 200 μm. Data are represented as means ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001; Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Injection, Control, Staining, Western Blot, Quantitative RT-PCR, Immunohistochemistry, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Scheme of intraperitoneal injection of different reagents in the five experimental groups. b At day 10 (D10), no ADM phenotype was observed in WT mice injected with caerulein. WT mice injected with both recombinant mouse REG3B protein and caerulein, and the REG3B TG mice injected with caerulein showed persistent ADM as indicated by the ductal morphology (black arrow heads) in H&E staining. Focal PanIN (marked by black asterisk) was observed in two out five REG3B TG mice injected with caerulein. Scale bar: 200 μm. c WT mice injected with recombinant REG3B and caerulein and the REG3B TG mice injected with caerulein demonstrate higher levels of Ck19 and lower levels of Mist1 mRNA. ( n = 5, one-way ANOVA). d Immunofluorescence staining shows gain of CK19 expression (red) and loss of AMYLASE (green) in the ADM area of WT mice injected with recombinant REG3B and caerulein and in the Reg3b TG mice injected with caerulein. Scale bars: 20 μm. White arrows indicate ADM. e Persistence of ADM in WT mice injected with recombinant REG3B 2, 4, and 10 weeks after caerulein injection. ( n = 5, magnification ×200, scale bar: 200 μm). Values in graphs are means ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001. Non-significant (n.s.) if P > 0.05. n = 5 per group, WT wild type, TG REG3B transgenic mice, Cae caerulein.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Injection, Recombinant, Staining, Immunofluorescence, Expressing, IF-P, Transgenic Assay

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Bright field images showing an increase in ADM events (depicted by black arrows) in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (Magnification, ×200). TGFα-treated mouse primary acinar cells served as a positive control. b Bar graph showing increase in ADM quantity in 3D culture of mouse and human primary acinar cells during the 5-day REG3B or REG3A or TGFα treatment ( n = 3, 15 fields each group, one-way ANOVA and student’s t -test). c Bright field images in the upper row showing ADM events in cultured mouse primary acinar cells after 5 days of REG3B treatment and in cultured human primary acinar cells after 5 days of REG3A treatment (magnification, ×630). Lower four rows are corresponding confocal immunofluorescence images showing a decrease in AMYLASE protein expression (green) and an increase in CK19 (red) protein in REG3B-induced, REG3A-induced, or TGFα-induced ADM (magnification, ×630. Scale bars: 20 μm). d RT-qPCR analysis showed a decrease in acinar-specific mRNA (Ptf1a, Cpa, and Mist1) and an increase in duct-specific mRNA (Ck19 and Nestin) in mouse and human primary acinar cells after 48 h of REG3B or REG3A treatment. ( n = 3 per group, student’s t -test). Values are represented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Cell Culture, Positive Control, Immunofluorescence, Expressing, Quantitative RT-PCR, Standard Deviation, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Western blot showing increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in the pancreatic tissue of REG3B-treated WT mice with caerulein-induced pancreatitis (WT cae+REG3B) and a moderate increase in TG mice with caerulein-induced pancreatitis (TG cae) ( n = 3, one-way ANOVA). b Western blots demonstrating increased expression of p-ERK, p-MEK, p-BRAF, KRAS, and active RAS in cultured human primary acinar cells and mouse acinar cell line 266-6 after REG3A or REG3B treatment respectively, for 48 h ( n = 3, student’s t -test). c Western blot showing the reduced expression of phosphorylated ERK, MEK, BRAF, and total KRAS in the 266-6 cell line after Reg3b gene knockdown by siRNA ( n = 3, student t -test). d In the upper panel, LY3009120 inhibited ERK, MEK, BRAF phosphorylation in a dose-dependent manner in 266-6 cell line. In the lower panel, LY3009120 (5 μM) blocked REG3B-induced MEK and ERK phosphorylation ( n = 3, one-way ANOVA). e Upper panel, Trametinib efficiently attenuated ERK phosphorylation in a dose-dependent manner in the AR42J cell line ( n = 3, one-way ANOVA). Lower panel, Trametinib (100 nM) blocked REG3B-induced ERK phosphorylation. f , g Bright field images ( g ) with quantification ( f ) show that Trametinib (100 nM) and LY3009120 (5 μM) treatment hindered REG3B-induced ADM in 3D cultures of mouse primary acinar cells (200×, n = 3, one-way ANOVA). h Confocal microscopy shows that Trametinib (100 nM) and LY3009120 (5 μM) treatment reduced CK19 protein expression (red) and increased AMYLASE protein expression (green) in 3D culture of mouse primary acinar cells (Scale bar: 20 μm). Data are represented as means ± SD, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Non-significant (n.s.) if P > 0.05.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Western Blot, Expressing, Cell Culture, Knockdown, Phospho-proteomics, Confocal Microscopy, IF-P

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Co-localization of EXTL3 (green) with human REG3A (red) or rodent REG3B (red) in ADM zones derived from human primary acinar cells in 3D culture, and AR42J and 266-6 cell lines in 2D culture by immunofluorescence microscopy. (magnification: ×630, Scale bars: 10 μm). b Co-immunoprecipitation of REG3B and EXTL3 in mouse primary acinar cells, rat AR42J and mouse 266-6 acinar cell lines. Lysate-bead/antibody conjugate mixture was eluted with sample buffer without DTT for 10 min at 50 °C (elution 1). Sample buffer with DTT (100 mM) was added to the pelleted beads from elution 1 and boiled for 5 min (elution 2). c Confocal immunofluorescence microscopy showed that EXTL3 monoclonal antibody treatment effectively blocks REG3B-induced ADM as indicated by increased expression of the acinar marker AMYLASE and decreased expression of the ductal marker CK19. (magnification: ×630, Scale bar: 20 μm). d – f Extl3 siRNA or neutralizing antibody treatment reduced the protein expression of KRAS and phosphorylated ERK, MEK, and BRAF in the presence or absence of REG3B for 48 h. d Western blotting analysis of the knockdown of Extl3 by siRNA (20 nM) in 266-6 cell line, e knockdown of Extl3 by siRNA (20 nM) in the context of the 266-6 cell line treated with REG3B for 48 h, f Western blotting analysis of EXTL3 neutralizing antibody treatment (2 μg/ml) in 266-6 and AR42J cell lines and mouse primary acinar cells treated with or without REG3B for 48 h. Human primary acinar cells were treated for 30 min. TGFα treatment serves as a positive control for ADM induction.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Derivative Assay, Immunofluorescence, Microscopy, Immunoprecipitation, Expressing, Marker, Western Blot, Knockdown, Positive Control

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: a Western blot analysis of related protein expression in 266-6 and AR42J cell lines 30 min and 72 h after different stimulations, with quantification data. b , c Expression level of JAK2/STAT3 signaling components in the 266-6 cell line under different conditions for 30 min ( b ) and 72 h ( c ). d , e Expression level of JAK2/STAT3 signaling components in the AR42J cell line under different conditions for 30 min ( d ) and 72 h ( e ) ( n = 3, one-way ANOVA test). REG3B treatment alone did not increase p-JAK nor p-STAT3 expression at two timepoints in two cell lines but did increase their expression once the receptor EXTL3 was blocked.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Western Blot, Expressing

Journal: Communications Biology

Article Title: REG3A/REG3B promotes acinar to ductal metaplasia through binding to EXTL3 and activating the RAS-RAF-MEK-ERK signaling pathway

doi: 10.1038/s42003-021-02193-z

Figure Lengend Snippet: REG3B/REG3A binds to its receptor, EXTL3 receptor on the acinar cell membrane, and promotes ADM by activating the downstream RAS-RAF-MEK-ERK signaling pathway, in the absence of oncogenic Kras mutation. Targeting REG3B/REG3A, neutralizing its receptor EXTL3, or inhibiting downstream signaling molecules, such as B-RAF (LY3009120) or MEK1/2 (Trametinib), could interrupt the ADM process and potentially prevent early PDAC carcinogenesis.

Article Snippet: Recombinant human REG3A and mouse REG3B protein were purchased from R&D Systems (5940-RG and 5110-RG).

Techniques: Membrane, Mutagenesis

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A) Schematic of stool extract preparation and analysis. B) Western blots of REG1A, REG3A, REG3G, and REG4 in stool extracts from representative 3 non-IBD (NIBD) and 5 IBD patients. C) Proportion of stool specimens from individual NIBD (n = 23) and IBD (n = 26) patients in which REG1A, REG3A, REG3G, or REG4 were detectable by western blot. D) Quantification of REG3A in NIBD and IBD patients-derived stool extracts by ELISA. E) Fold changes in colony forming units (CFU) of Enterococcus faecalis ( Efl ) and Salmonella Typhimurium ( S Tm) following 24 hrs of culture in stool extracts from NIBD (n = 56) and IBD (n = 56) patients. Results obtained with Efl were confirmed with E. faecium ( Efm ) (n = 18 for NIBD and n = 19 for IBD). F) Correlation between REG3A concentration and CFU fold changes of Efl (upper), S Tm (middle), and Efm (lower) cultured in stool extract from NIBD and IBD patients. G) Fold change in Efm CFU at 24 hr-post culture in NIBD (left) and IBD (right) patient-derived stool extract or PBS in the presence of anti-REG1A, REG3A, and REG3G antibodies. N = 13 for NIBD and IBD. H) Correlation between REG3A concentration and Crohn’s disease activity index (CDAI) for CD patients (n = 31, left) or total Mayo score for ulcerative colitis (UC) patients (n = 24, right). Data points in D-H represent individual patients. Bars represent mean ± SEM and at least three independent experiments were performed. NIBD, non-IBD; Efl , E. faecalis ; S Tm, S. Typhimurium; Efm , E. faecium ; CDAI, Crohn’s disease activity index; UC, ulcerative colitis; r, Pearson correlation coefficient. Indicated p values by Fisher’s exact test in C, unpaired t test, two-tailed in D, E, and G, and simple linear regression analysis in F and H.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Activity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A) REG3A concentration in stool extracts from NIBD and IBD patients from separated by sex. B) REG3A concentration in stool extracts from Crohn’s disease (CD) or ulcerative colitis (UC) patients. C) Correlation between age of NIBD and IBD patients and REG3A concentration. D) REG3A concentration in stool extracts segregated into indicated age groups. E) Proportion of patients in which Enterococcus and E. faecium ( Efm ) were detected in stool. F) Proportion of Enterococcus that are Efm in NIBD and IBD stools. Data points in A-D and F represent individual patients. Bars represent mean ± SEM and at least two independent experiments were performed. r, Pearson correlation coefficient. Indicated p values by unpaired t test, two-tailed in A, D, and F, simple linear regression analysis in C, and Fisher’s exact test in E.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A and B) 16S rRNA sequencing of stool from NIBD and IBD patients from . Alpha diversity values calculated as Shannon (right), Faith’s phylogenetic diversity (PD) (middle), and Pielou-evenness (right) indices (A). Principle coordinate analyses of beta diversity determined by Bay-Curtis, Jaccard, and Unweighted and Weighted unifrac methods (B). C) Proportion of sequencing reads representing Enterococcus in NIBD and IBD patient stool. One NIBD sample contained >80% Enterococcus indicative of an overabundance, which is shown as a reference on the graph but excluded from statistical analysis and downstream assays. D) Total Enterococcus (left) and Efm (right) CFUs in stool from NIBD (n = 39) and IBD (n = 43) patients. E) Correlation between REG3A concentration and the burden of total Enterococcus and Efm in IBD stool. F) Correlation between the burden of total Enterococcus and CDAI of CD patients (n = 24, left) and total Mayo score of UC patients (n = 18, right). Data points represent individual patients. Bars represent mean ± SEM and at least three independent experiments were performed. r, Pearson correlation coefficient. Indicated p values by Kruskal-Wallis test in A, unpaired t test, two-tailed in C and D, and simple linear regression analysis in E and F.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Sequencing, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization

doi: 10.1101/2023.01.29.526128

Figure Lengend Snippet: A) NOD2 R702W allele frequency according to ethnic groups. The frequencies were retrieved from 1000 Genomes Project and Allele Frequency Aggregator. B) Schematic of genotyping PCR product (left) and representative genotyping gel image (right) for Nod2 Q675W knock-in mouse. C) Sequencing of the Nod2 locus from the above mice confirmed successful gene targeting. Figure shows exon 4 DNA and amino acid sequences from Nod2 Q675W knock-in mouse sequencing results aligned to the WT sequences. Red boxes indicate the mutated region. D) Western blot image of NOD2 and β-actin (ACTB) in colonic tissue lysates from Nod2 -/- , Nod2 Q675W/+ , and Nod2 Q675W/Q675W mice. E and F) DSS treatment of Nod2 Q675W/+ and Nod2 Q675W/Q675W mice from room 6 following 2-week administration of Efm in drinking water or control. The mice were examined for the burden of Efm (E) and LCN2 concentration (F) in the stool samples at the indicated time points. G) Endogenous Enterococcus burden among mice with different genotypes on day 0 (upper). The p -values relative to Nod2 Q675W/+ or Nod2 Q675W/Q675W mice were indicated in lower table. H) Schematic of the mechanism by which Efm activates NOD2 to suppress inflammation, and how this process is disrupted when either REG3A is overproduced or NOD2 is genetically inactivated. Lines in E and data points in F and G represent individual mice. Bars in F and G represent mean ± SEM and at least three independent experiments were performed. Het, heterozygotes; Homo, homozygotes. Indicated p values by unpaired t test, two-tailed in F and G.

Article Snippet: The following primary antibodies were used for western blotting studies: anti-REG1A (R&D systems, MAB4937), REG3A (R&D systems, MAB5965), REG3G (Abcam, ab233480), REG4 (Abcam, ab255820), NOD2 (Invitrogen, MA1-16611), and β-actin (Sigma-Aldrich, A5441).

Techniques: Knock-In, Sequencing, Western Blot, Control, Concentration Assay, Two Tailed Test