Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: BCR editing and Nod1 increase in mature B cells. (A) B220 + B cell analysis of V H 3609t knockout mice crossed with V K 21-5 Tg mice (V H 3609 +/− V k 21-5 +/− ) on either Thy WT or Thy KO background. Gated unedited ATA B in the spleen and PerC (red diagonal, with percentage) for phenotype analysis, and in vitro 2 d Thy M antigen stimulation outcome analysis with spleen ATA B. Thy M , thymocyte plasma membrane . PerC IgM + IgD lo/− ATA B cells in Thy WT were CD5 + as B1a. (B) Relative ATA serum titer determined by a thymocyte staining assay. Nontransgenic CB17 mouse sera (at 1:100 dilution) was set to 1.0 as the staining background. (C) V H 3609t × V K 21-5 Tg.Thy KO mouse spleen B cell analysis comparing transitional and mature B cells. Percentage of endogenous IgM (IgM b+ ), VDJ engineered IgM (IgM a+ ), VDJ-expressing B cells (V H 3609id + ), and unedited ATA B cells (ATAid hi ), out of total IgM (IgM a + IgM b ). ATAid hi B cell decrease with V H 3609id retention (center; n = 3; *, P < 0.01), generating L chain–edited ATAid lo FO B and MZ B cells (right). (D) Auto + AGcA B cell presence in L chain–edited B cells, including in FO B cells under Thy KO (Table S1). (E) In FO B cells under Thy KO , ATA B (1) and edited B cell fractions (2 and 3) for Nod1 quantitative PCR analysis, together with WT FO B cells ( n = 5; mean ± SE; *, P < 0.05; **, P < 0.005; ***, P < 0.001), and 6-h stimulation with CiE-DAP or anti-IgM. Unstimulated B cells shown as a dotted line. (F) Nonedited ATA B and edited B cell frequencies in total PBL B cells during aging in V H 3609t × V K 21-5 Tg.Thy KO mice ( n = 10–15 of each age group; mean ± SE).

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: Cell Analysis, Knock-Out, In Vitro, Clinical Proteomics, Membrane, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: Nod1 ligand signal–sensitive mature B cells increase with age in comparison to Xid mice . (A) Nod1 quantitative PCR in WT neonatal and adult B-lineage cells relative to self-antigen–unexposed adult ATA FO B cells from ATAμκTg.Thy KO mice (ATA FOB.Thy KO ), set to 1 ( n = 3–5, each group; mean ± SE; *, P < 0.05; **, P < 0.01; ***, P < 0.001). mat B, mature B; pB1a, PerC B1a; sB1a, spleen B1a. Right: Nod1 RT-PCR. (B) Anti-Nod1 cytoplasmic staining level comparison between ATA FO B.Thy KO cells and WT FO B cells ( n = 5; mean ± SE; *, P = 0.01). (C) CiE-DAP reactivity comparison between ATA FO B.Thy KO and WT mouse B cell subsets (representative of n = 4 each). (D) Top: Western blot of Nod1. Bottom: CiE-DAP–stimulated spleen cells in culture for 6 h. (E) Spleen cells stimulated with CiE-DAP, LPS, CpG, or medium alone (gray). Gating was based on IgM and IgD levels, together with AA4/CD93 and CD23. CiE-DAP–induced CD69 + cell frequency is shown. C.B17 mouse data were similar to B6 data (two mice each). (F) CiE-DAP–stimulated spleen cells from different-aged mice were stained with IgM, IgD, and CD69, and the frequency of CD69 + -activated cells among IgM med/lo IgD hi FO B cells (square region) is shown. Purified 5-wk and 2-mo FO B cells stimulated with CiE-DAP (right). C.B17, BALB, and C57BL/6 mice (three mice or more each) were analyzed, confirming similar results; data from C.B17 mice are shown. (G) Spleen and purified FO B cells in 2-mo-old Xid mice stimulated with CiE-DAP (left and middle). Nod1 quantitative PCR of Xid compared with WT mouse FO B cells (right; n = 3 each; mean ± SE; *, P < 0.003; **, P < 0.008).

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: Comparison, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining, Western Blot, Purification

Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: Nod1 up-regulation in BCR engaged B cells in vivo. (A) Rag1 and Rag2 quantitative PCR of ATA B cells under Thy KO and Thy WT in ATAμκTg mice ( n = 3; mean ± SE; *, P = 0.03; **, P = 0.003). (B) Rag1 and Rag2 quantitative PCR of ATA B cells and edited ATAid lo B cells in V H 3609t × V k 21-5Tg.Thy KO mice ( n = 3; mean ± SE; *, P = 0.1; **, P = 0.03). (C) Quantitative PCR of WT (C.B17) FO B cells stimulated with anti-IgM, LPS, or CpG during 24-h culture. Similar data were obtained by B6 FO B cell simulation (two samples each). (D) Ptpn22 quantitative PCR in B-lineage cells ( n = 3–5, each group; mean ± SE; *, P < 0.01; **, P < 0.002) and Western blot. (E) Nod1 and Ptpn22 quantitative PCR of ATAid + FO B cells sorted from ATAμκTg.Thy KO mice exposed to lck -Thy-1 Tg cells, Thy 10, or Thy 60 by breeding ( n = 3 each, 2–3 mo old; mean ± SE; *, P < 0.05), comparing ATAid + FO B cells without lck -Thy-1 Tg, as (−) 1.0, and Thy-1 Tg + in littermates. CiE-DAP reactivity comparison by ATAid + FO B cells without (−) and with lck -Thy 60 Tg littermates (middle). Ptpn22 up-regulation resulting from a BCR ligand signal, followed by Nod1 up-regulation, occurred in vivo (right). (F) Mature (AA4 − CD21 med CD23 + ) B cell Nod1 and Ptpn22 mRNA comparison ( n = 3 each; mean ± SE). WT mice used for comparison with LTα −/− .B6 mice were B6. CiE-DAP reactivity by mature B cells in CD40 −/− and Ltα −/− mice (right). (G) Comparison of Nod1 and Ptpn22 mRNA levels in immature stages of ATA B cells from V H 3609t × V K 21-5 Tg mice on Thy WT (black square) and Thy KO (open circle) backgrounds, and mature edited FO B cells (gray; n = 4; mean ± SE; *, P < 0.005; **, P < 0.01 for T1 comparison). (H) Edited B cells with Nod1 up-regulation as the outcome of BCR ligand engagement.

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Western Blot, Comparison

Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: Nod1 signaling promotes survival of BCR ligand–experienced B cells. (A) Viability of FO B cells, 3 and 7 d in culture, stimulated with CiE-DAP, BAFF, or both. Total cell numbers were similar ( n = 3, mean ± SE). Data obtained in C57BL/6 mouse FO B cells were similar (two samples). (B) Analysis at 6 h and 3 d after stimulation of purified B cell subsets from WT mouse. After 6 h, comparison of CD69 level by PE–anti-CD69 staining (PE mean level). For 3-d analysis, CSFE labeling of purified B cells was used for analysis of cell division and viability (%). Representative data (C.B17 mice) from seven experiments with C.B17 and BALB/c, and five experiments with C57BL/6 are shown. All showed increased viability with CiE-DAP ( n = 2, mean ± SE). (C) Nod1 and Ptpn22 mRNA comparison between (1) ATA FO B from ATAμκTg.Thy KO mice (auto − ), (2) FO B cells from WT mice, and (3 and 4) AGcA FO B and MZ B cells from AGcAμκTg mice (auto + ), respectively (all on a C.B17 background; n = 3 each; mean ± SE; *, P < 0.05). (D) B cells, listed in C, were stimulated for 6 h to 3 d by CiE-DAP (thin line), anti-IgM (thick line), and CiE-DAP plus anti-IgM (dotted line). On day 3, cell size (flow cytometry scatter) and percent viability were determined. Four experiments showed similar results. (E) Increased viability and cell size by CiE-DAP stimulation through the Nod1–Ripk2 signal in WT (B6) FO B cells.

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: Purification, Comparison, Staining, Labeling, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: Nod1-mediated NF-κB, PI3K, and MAPK signals in B cells. (A) Western blot of WT (B6) mouse (left) and WT, Ripk2 −/− , and Nod1 −/− FO B cells (right) stimulated with CiE-DAP, showing Nod1- and Ripk2-dependent IκBα phosphorylation. (B) Activation of MAPK by CiE-DAP stimulation. (C) Nod1-dependent Akt Ser473/Thr308 phosphorylation in CiE-DAP–stimulated FO B cells. (D) The PI3K inhibitor LY294002 inhibits Akt phosphorylation induced by CiE-DAP stimulation. (E) FO B cell stimulation with CiE-DAP from 5 min to 24 h, and kinetic analysis of phosphorylated Akt, FoxO1, and Gsk3β. Initial phosphorylated Akt was transient, declining after 1 h and then recovering. (F) Comparison of CiE-DAP–mediated Nod1 signal–induced IκBα degradation in different B cell subsets. Samples 1–4 were the same as in (1–4). Sample 5 was autoreactive B1a from V H 11 knock-in mice (auto + ; ) and the lowest (or negative) by auto − FO B cells. (G) Summary of Western blot results of Nod1, BCR, and Nod1-BCR signaling of FO B, MZ B, and B1a B cells. β-Actin levels were all similar within each Western blot. Nod1-BCR co-stimulation consistently increased the NF-κB and JNK signal for WT FO B cells and overrode autoreactive B cell tolerance. Shown are representative data from two to three experiments for each B cell stimulation.

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: Western Blot, Phospho-proteomics, Activation Assay, Cell Stimulation, Comparison, Knock-In

Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: Continued B cell survival in aged mice by transduced Nod1 expression. (A) Transfer of nonedited auto − ATAμκTg.Thy KO mouse BM with Nod1-GFP or pMIG (without Nod1)-GFP as a control. Comparison of GFP + cell frequency in ATA B cells between the FO B cell and T1 stage (as a ratio) in PBLs at 3 wk ( n = 6 each; mean ± SE) and spleen at 6 wk. In the 6-wk GFP histogram, immature B cells in BM (1) are shown in gray, along with GFP + frequency. Data are representative of n = 6 each. (B) Increase in the number of Nod1-GFP + cells in the ATA FO B cell pool under Thy KO . (C) Transfer of edited V H 3609t × V k 21-5 Tg.Thy KO mouse BM with Nod1-GFP or control-GFP into Rag KO Thy KO recipients. Spleen B cell analysis 6 wk after transfer, comparing GFP + and GFP − B cell subsets, with percentage of edited ATAid lo cells (middle). Nod1-GFP transfected B cell data (right; n = 3 each; mean ± SE). (D) PBL B cell analysis during aging (and spleen in aged mice) after transfer with Nod1-GFP V H 3609t × V K 21-5 Tg.Thy KO mouse BM. After transfer, the WT/knockout mouse B cell ratio levels in spleen were based on the WT/knockout mouse B cell ratio level data of T1 (CD19 + IgM hi AA4 + CD21 − CD23 − as 1.0. IgM/IgD phenotype of GFP + B cells (right). Unchanged IgM med level by Nod1-GFP + hi auto − ATA B cells 12 mo after transfer, in comparison with edited GFP + med B cells with IgM lo B cells. Data are representative of three recipients. (E) Increased survival outcome by FO B cells in aged mice by Nod1 up-regulation. IgM lo IgD + FO B cells are predominantly BCR signal–experienced Nod1 + B cells.

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: Expressing, Control, Comparison, Cell Analysis, Transfection, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome

doi: 10.1084/jem.20170497

Figure Lengend Snippet: Competitive survival by mature B cells promoted by Nod1–Ripk2 signaling. (A) FO B/T1 GFP + AGcA B cell ratio in spleen 5–6 wk after transfer of AGcATg mouse BM with shNod1-GFP or LMP (without shNod1)-GFP as a control ( n = 3 each; mean ± SE; *, P = 0.008). (B) Spleen cell analysis (IgM/IgD) and stimulation with CiE-DAP, muramyl dipeptide (Nod2 ligand), and LPS, comparing WT (B6) and Nod1/2 −/− mice. (C) Ripk2 −/− mouse FO B cell Nod1 and Ptpn22 levels in comparison with Ripk2 +/+ littermates (middle; quantitative PCR; n = 3 each; mean ± SE). Ripk2 −/− FO B stimulation analysis (right). (D) Chimeric transfer of WT (Ly5.1) and Nod1/2 −/− (Ly5.2) mouse BM at a 1:1 ratio. Initial (4 wk) chimeric transfer ratio was set to 1:1 at the IgM + IgD lo AA4 + transitional cell stage (“1”) in PBL. In spleen, the WT/Nod1/2 −/− ratio was based on the T1 in each mouse ( n = 3; mean ± SE). (E) Chimeric transfer of WT and Ripk2 −/− mouse BM, as done with Nod1/2 −/− mice in D ( n = 4; mean ± SE). (F) Splenic B cells in AGcAμκTg.B6 mice without or with Ripk2 deficiency. Similar AGcA B cell dominance per number and CD21 hi MZ B cell frequency. Representative of three mice each. (G) Chimeric transfer of WT (B6) and AGcAμκTg (Ripk2 +/+ or Ripk2 −/− ) BM at a 3:1 ratio, followed by splenic B cell analysis. Frequencies of AGcA B cells (as marked, also V k 19-17 recognizing 13H8k + ) in total B cells and CD21 hi cells among AGcA B cells are shown ( n = 3; mean ± SE). (H) 1:1 chimeric transfer of purified B cells from Ripk2 +/+ WT and Ripk2 −/− mouse spleen, followed by analysis of PBL B cells and IgM hi IgD lo AA4 − cells in PBL at 12 wk ( n = 3, mean ± SE). (I) The microenvironment, including BCR ligands, BAFF, and microbial products, plays a role in mature B cell survival. BCR ligand–mediated Nod1 up-regulation, but without maturation arrest, leads to preferential mature B cell survival as a competitive survival, increasing the Nod1 + B cell pool with age.

Article Snippet: Hairpin sequences specific for Nod1 were from the Broad Institute public TRC portal.

Techniques: Control, Cell Analysis, Comparison, Real-time Polymerase Chain Reaction, Purification

Journal: Molecular Vision

Article Title: Constitutive and UV-B modulated transcription of Nod-like receptors and their functional partners in human corneal epithelial cells

doi:

Figure Lengend Snippet: Expression of Nod subtypes in corneal epithelial cells. A : The relative expression of Nod1 , Nod2 , NLRC5 , and NLRX1 mRNA in HCE-T cells and in primary corneal epithelial cells derived from five individuals were measured and documented as described in the legend of . B : Detection of phosphorylated IκB using western blotting is illustrated. HCE-T cells were treated with 4 μg/ml ultrapure PGN-ECndss for the indicated time periods. The control THP-1 cells (c) were treated with 1 μg/ml LPS for 4 h. Equal amount of sample loading was verified by detecting β-actin protein expression.

Article Snippet: The following ABI-TaqMan assays were used in the studies: ASC-HS00203118, Caspase-1-Hs00354836, Caspase-5-Hs00362072, Cardinal-Hs00209095, Nalp1-Hs00248187, Nalp3-Hs00366461, Nalp2-Hs215284, Nalp7-Hs00373683, Nalp10-Hs738590, NLRC5-Hs00260008, NLRX1-Hs00226360, Nod1-Hs00196075, and Nod2-Hs00223394.

Techniques: Expressing, Derivative Assay, Western Blot

Journal: Molecular Vision

Article Title: Constitutive and UV-B modulated transcription of Nod-like receptors and their functional partners in human corneal epithelial cells

doi:

Figure Lengend Snippet: The effect of UV-B treatment on the expression of Nod subtypes in HCE-T cells. Changes of Nod mRNA expression 6 h ( A ) or 24 h ( B ) after UV-B treatment are shown. The relative expression of Nod1 , Nod2 , Nod4/Nod27 , and Nod5/Nod9 was measured in 85%–90% confluent HCE-T cells irradiated with 30 mJ/cm 2 UV-B as described in the legend of and is shown as compared to cells incubated in the same way but not irradiated. Mean values and ±SD were calculated from six independent measurements. The asterisk indicates a p<0.005.

Article Snippet: The following ABI-TaqMan assays were used in the studies: ASC-HS00203118, Caspase-1-Hs00354836, Caspase-5-Hs00362072, Cardinal-Hs00209095, Nalp1-Hs00248187, Nalp3-Hs00366461, Nalp2-Hs215284, Nalp7-Hs00373683, Nalp10-Hs738590, NLRC5-Hs00260008, NLRX1-Hs00226360, Nod1-Hs00196075, and Nod2-Hs00223394.

Techniques: Expressing, Irradiation, Incubation

Journal: BMC Evolutionary Biology

Article Title: A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

doi: 10.1186/1471-2148-8-42

Figure Lengend Snippet: Human NLR sequences used for analyses

Article Snippet: Gallus gallus , Chr 2 , GG-NOD1 , NOD1 , ENSGALG00000011535 , CARD a.

Techniques:

Journal: BMC Evolutionary Biology

Article Title: A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

doi: 10.1186/1471-2148-8-42

Figure Lengend Snippet: NLR-SUBFAMILY A in zebrafish

Article Snippet: Gallus gallus , Chr 2 , GG-NOD1 , NOD1 , ENSGALG00000011535 , CARD a.

Techniques:

Journal: BMC Evolutionary Biology

Article Title: A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

doi: 10.1186/1471-2148-8-42

Figure Lengend Snippet: NLR subfamily genes identified in other non-mammalian genomes

Article Snippet: Gallus gallus , Chr 2 , GG-NOD1 , NOD1 , ENSGALG00000011535 , CARD a.

Techniques:

Journal: BMC Evolutionary Biology

Article Title: A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

doi: 10.1186/1471-2148-8-42

Figure Lengend Snippet: Schematic diagram depicting the deduced protein structures of zebrafish NLRs. Zebrafish NLR subfamily-A have structures similar to the NOD subfamily in mammals, with NOD1/NLR-A1 possessing one CARD motif while NOD2/NLR-A2 possesses two. While only the NACHT domain was identified for most members of the NLR-B subfamily, one member of this group (NLR-B2) was predicted with a putative N-terminal CARD effector domain. All NLR-C subfamily members were predicted to have an N-terminal effector domain, a central NACHT domain and a LRR domain. In addition, some of the NLR-C molecules were identified with a C-terminal B30.2 domain. The predicted effector domains of molecules within the NLR-C subfamily varied; some had a pyrin (P) effector domain, while others had a distinct uncharacterized effector domain (X). B30.2 domains are also found in other important immune related molecules such as certain TRIMs and the Pyrin molecule, whose structures are also shown. C = card domain, P = pyrin domain, X = other domain, N = NACHT domain, L = LRR region, B = B30.2/PRY-SPRY domain, R = ring finger domain, BB = B-box, CC = coiled coil.

Article Snippet: Gallus gallus , Chr 2 , GG-NOD1 , NOD1 , ENSGALG00000011535 , CARD a.

Techniques: