Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Library size-normalized counts detected by RNA sequencing in mouse RPM and in M0-differentiated BMDM (3 mice each; only GPCRs with average count >15 displayed). B Gprc5b expression in RPM and lymph node-derived lymphocytes was determined by NanoString RNA analysis (cells pooled from two mice per data point). C Knockout efficiency in RPM from control mice (white) and M-G5b-KOs (gray) was analyzed by qRT-PCR (C, n = 4, data normalized to Gapdh and controls set to 1). D Knockout efficiency in RPM was analyzed by immunoblotting (unspecific band around 38 kDa; the higher of the two specific bands probably represents glycosylated GPRC5B ; GAPDH as loading control). E, F Expression of Nos2 (E) and Tnf (F) was determined in RPM by qRT-PCR under basal conditions and after 6 h of stimulation with 1 µg/ml LPS ( n = 11/12/12/12 in E, 12/10/11/12 in F), data normalized to Gapdh and control set to 1). G Basal and C5a (20 ng/ml)-induced transwell migration in RPM (all cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration) ( n = 3). H The distance travelled by individual RPM in response to different chemotactic factors (C5a, 20 nM; CCL5, 10 ng/ml; fMLP, 10 nM) was determined by live cell imaging ( n = 512 cells from 2 mice per group; cells pretreated with LPS 1 µg/ml for 3 h to facilitate migration, arb. units: arbitrary units). Phagocytic activity of LPS (1 µg/ml, 6 h)-stimulated RPM was determined by uptake of pHrodo E. coli bioparticles ( I , J , n = 5) or pHrodo-labeled apoptotic thymocytes ( K , L n = 10); I + K show original traces, J + L statistical evaluation of areas under the curve (AUC). Body weight change ( M ) and bacterial colony-forming units in peritoneal lavage fluid harvested 24 h after injection of fecal bacteria ( N ) ( n = 10). O, P Numbers of CD11b + , F4/80 + , MHCII - , Tim4 + RPM and CD11b + , F4/80 lo , MHCII + , CCR2 + BMDM before and 3, 24, and 54 h after i.p. injection of fecal bacteria ( n = 3/3/3/3/11/12/5/5). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t -test (C, J, L, N), two-way ANOVA (E-H) or two-way RM-ANOVA (M) with Sidak’s multiple comparison test, unpaired two-sided t-test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (O, P). * P < 0.05; *** P < 0.001; **** P < 0.0001; n , number of individual mice. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: RNA Sequencing, Expressing, Derivative Assay, Knock-Out, Control, Quantitative RT-PCR, Western Blot, Migration, Live Cell Imaging, Activity Assay, Labeling, Injection, Bacteria, Comparison

Journal: Nature Communications

Article Title: Orphan G protein-coupled receptor GPRC5B controls macrophage function by facilitating prostaglandin E receptor 2 signaling

doi: 10.1038/s41467-025-56713-0

Figure Lengend Snippet: A Knockout efficiency was determined by qRT-PCR in RPM and M0 BMDM (data normalized to Gapdh and RPM controls set to 1) ( n = 12). Analyses in resting and LPS (1 μg/ml, 6 h)-stimulated M0 BMDM: Expression of inflammatory genes ( B , C ; n = 15/14/15/15 in B, 15/14/15/15 in C), production of NOx ( D ; n = 3) or release of cytokines ( E , n = 3). F Transwell migration of M1 BMDM in response to different chemotactic factors ( n = 6) (CCL5: 75 ng/ml, CCL2: 10 ng/ml, SDF-1β: 100 ng/ml, C5a: 20 ng/ml, fMLP: 10 nM). Uptake of pHrodo E.coli fragments by M0 BMDM: G , exemplary curves; H , statistical analysis of AUC ( n = 6). I Flow cytometric analysis of CD11b-positive cells in the combined infarct and border zones of hearts harvested 4 days after infarction ( n = 5). J Echocardiographic analysis of ejection fraction (EF%) before and after infarction (8 controls, 4 KOs). K Histological analysis of scar size in hearts harvested 21 days after infarction ( n = 7 controls, 4 KOs), left ventricle (LV). L, M DSS colitis: Disease activity index integrating body weight change, stool consistency, intestinal bleeding (L) and colon length on day 6 (M) ( n = 7(L), 7/7/10/11 in M). Data are means ± SEM; comparisons between genotypes were performed using unpaired two-sided Student’s t- test (A, H, K), two-way ANOVA with Sidak’s multiple comparisons test (B-F, J, M), unpaired two-sided t -test corrected for multiple testing by two-stage step-up method Benjamini, Krieger and Yekutieli (I), two-way repeated measures ANOVA with Sidak’s multiple comparisons test (L). n, number of mice per group; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Complement component C5a (2150-C5-025) was from R&D systems.

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Migration, Activity Assay

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 1. Ca2+ influx after stimulation of PMN cells with C5a is required for efficient chemo- taxis. (A, B) PMN cells from C57Bl/6 mice were loaded with Fura2/AM and stimulated or not (w/o C5a) with either C5a (50 ng/ml) in CaCl2 (1 mM) containing buffer (C5a) or in buffer sup- plemented with EGTA (0.5 mM) (EGTA + C5a). (A) One representative measurement of n = 4 spectrofluorometric measurements are pre- sented. (B) In the same setting, maximal ([Ca2+]i ± SEM) values were calculated of n = 4 spectrofluorometric measurements per group; *P < 0.05, determined by 2-tailed Stu- dent´s t test. (C) PMN cells from C57Bl/6 mice were incubated with a Ca2+-specific chelator, EGTA and assayed for efficient C5a (50 ng/ml)- elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each per- formed in duplicate; **P < 0.01, determined by 2-tailed Student´s t test.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Incubation, Chemotaxis Assay, Migration

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 2. Differential involvement of Orai1 and STIM1 in C5a-induced Ca2+ influx and SOCE in PMN. (A, C) Orai1−/−or Stim1−/−PMN cells loaded with Fura2/AM and stimulated with C5a (50 ng/ml) in CaCl2 (1 mM) containing buffer. One representative spec- trofluorometric measurement of n = 4 measure- ments and maximal ([Ca2+]i ± SEM) values are presented (*P < 0.05, determined by 2-tailed Stu- dent´s t test). (B, D) Orai1−/−or Stim1−/−PMN cells loaded with Fura2/AM and treated with Tg (2 μM) in EGTA-containing buffer (w/o [Ca2+]e) followed by the addition of CaCl2 (1 mM [Ca2+]e) and monitoring of [Ca2+]i. Representative spectrofluorometric measure- ments and maximal ([Ca2+]i ± SEM) values (n = 4 per group, in the presence of 1 mM CaCl2 in measuring buffer are shown) (**P < 0.01, determined by 2-tailed Student´s t test).

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques:

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 3. Orai1 is required for efficient C5a- elicited chemotaxis of PMN in vitro and peri- toneal recruitment in vivo. (A) PMN cells from Stim1−/−or Orai1−/−and matched wild- type (Stim1+/+ and Orai1+/+) BM chimeras were assayed for efficient C5a (50 ng/ml)-elicited chemotaxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (**P < 0.01, determined by 2-tailed Student´s t test). (B) Orai1−/−or Orai1+/+ BM chimeras were injected with 200μl of C5a (1 μg/ml) or 200 μl of PBS. Two hours later PMN cells infiltrating the peritoneal cavity were counted. Results are expressed as mean ± SEM of n = 4-5 mice per group (*P < 0.05, deter- mined by 2-tailed Student´s t test). Groups of 2– 3 Orai1−/−BM chimeras and two matched wild- type mice were assayed together. (C) Orai1−/−or Orai1+/+ Gr1-positive PMN were characterized for C5aR expression by flow cytometry. Rep- resentative histogram (left) and mean fluores- cence intensity (MFI) ± SEM of n = 3 measure- ments (right) are depicted. P: ns = non signifi- cant, determined by 2-tailed Student´s t test.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Chemotaxis Assay, In Vitro, In Vivo, Migration, Injection, Expressing, Cytometry

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 4. The requirement of extracellular Ca2+ and Orai1 for efficient PMN chemotaxis activated by LPS and IgG IC primed biological fluids is not related to CXCL2. (A) C57Bl/6 mice were injected with (LPS 10 mg/ kg) i.p. Two hours later mice were sacrificed and PLF sam- ples were evaluated. Measurement of CXCL2 content of PLF by ELISA (left) and PLF bioactive C5a (right). Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (***P < 0.001, deter- mined by 2-tailed Student´s t test). (B) C57Bl/6 mice were exposed to IgG IC hypersensitivity alveolitis. Four hours later mice were sacri- ficed and BALF samples were evaluated. Mea- surement of CXCL2 content of BALF by ELISA (left) and BALF bioactive C5a (right). Results are expressed as mean ± SEM of n = 4 experi- ments (***P < 0.001, determined by 2-tailed Stu- dent´s t test) (C) PMN cells from C57Bl/6 mice were incubated with EGTA and assayed for effi- cient PLF- (left) or BALF-elicited (right) chemo- taxis in Transwell migration assays. Results are expressed as mean ± SEM of n = 4 indepen- dent experiments, each performed in dupli- cate (*P < 0.05; **P < 0.01, determined by 2- tailed Student´s t test). (D) PMN cells from C57Bl/6 mice were incubated with a Ca2+- specific chelator, EGTA and assayed for efficient CXCL2 (25 ng/ml)-elicited chemotaxis in Tran- swell migration assays. Results are expressed as mean ± SEM of n = 4 independent exper- iments, each performed in duplicate. (E) PMN cells from Orai1−/−and matched Orai1+/+ BM chimeras were assayed for efficient CXCL2 (25 ng/ml)-elicited chemotaxis in Transwell migra- tion assays. Results are expressed as mean ± SEM of n = 4 independent experiments, each performed in duplicate (ns = nonsignificant, determined by 2-tailed Student´s t test). (F) Orai1−/−or Orai1+/+ BM chimeras were injected with 200μl of CXCL2 (1 μg/ml) or 200 μl of PBS. 2 hours later PMN cells infiltrating the peritoneal cavity were counted. Results are expressed as mean ± SEM of n = 4 mice per group (ns = non- significant, determined by 2-tailed Student´s t test).

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Chemotaxis Assay, Injection, Enzyme-linked Immunosorbent Assay, Incubation, Migration

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 6. Normal C5a and CXCL2 production, but reduced versus enhanced LPS-induced peritoneal neutrophil accumulation in Orai1−/−and Stim1−/−

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques:

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 7. Orai1 and STIM1 act at distinct levels of the inflammatory cascade in IgG IC-induced hypersensi- tivity pneumonitis. Orai1−/−(A-F) or Stim1−/−(G-L) and matched wild-type chimeras were either exposed to IgG immune complex lung injury. 4 hours later, mice were sacrificed and lung tissue injury and BAL fluid samples were evaluated. (A, G) Functional detection of bioactive C5a in BALF from IC-challenged BM chimeras. Chemotactic activity was determined with Transwell migration assays of neutrophils (PMNs isolated from BM of C57BL/6 and C5aR–/– mice). (B, H) Measurement of CXCL2 content of BALF by ELISA. (C, I) Measurement of TNF-α content of BALF by ELISA. (D, J) Lung H&E section (original magnification 40x). (E, K) Evaluation of lung interstitial PMN infiltration by measurement of MPO activity, normalized to the weight of the lavaged lung. (F, L) Evaluation of PMN accumulation in BALF. Results are expressed as mean ± SEM of n = 5 mice per group (*P < 0.05; **P < 0.01;***P < 0.001, determined by 2-tailed Student´s t test). Groups of 2–3 Orai1−/−or Stim1−/−BM chimeras and 2–3 matched wild-type mice were assayed together.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Functional Assay, Activity Assay, Migration, Isolation, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Orai1 controls C5a-induced neutrophil recruitment in inflammation.

doi: 10.1002/eji.201445337

Figure Lengend Snippet: Figure 8. A summarized model of the distinct roles of STIM1 and Orai1 in IC inflammation and of SOCE vs. nonSOCE in the activation of neutrophils. (A), 1: Initial contact between IC and resident effector cells induces C5a and CXCL2 production that is STIM1-dependent [21]; 2: PMN migration induced by C5a and CXCL2 involves the activation of distinct calcium channels, Orai1 and TRPC6 [30]; 3: TNF-α-mediated regulation of endothelial ICAM-1 promotes PMN adhesion through LFA-1 and Orai1 calcium dynamics [22]; all these steps contribute to the recruitment of PMN to sites of inflammation, but CXCL2 appears not sufficient to overcome C5a dysfunction in the absence of Orai1 (indicated by the dashed arrow). (B) Binding of C5a to the Gαi-coupled C5aR activates Orai1 at the plasma membrane independent from STIM1-mediated SOCE, implicating the existence of a nonSOCE pathway for increased Ca2+ in the regulation of C5aR-mediated PMN migration. The minor role of (IP3-IP3R) Ca2+ store depletion and SOCE pathway for C5a-induced neutrophil chemotaxis is indicated by the dashed arrows.

Article Snippet: After labeling, cells were washed twice and treated with 2 μM Thapsigargin (Tg; Invitrogen, Darmstadt, Germany) or stimulated with recombinant mouse C5a (50 ng/ml) (R&D Systems, Wiesbaden, Germany) in buffer supplemented or not with 0.5 mM EGTA as indicated.

Techniques: Activation Assay, Migration, Binding Assay, Clinical Proteomics, Membrane, Chemotaxis Assay

Journal: Medicina

Article Title: Complement Properdin Regulates the Metabolo-Inflammatory Response to a High Fat Diet

doi: 10.3390/medicina56090484

Figure Lengend Snippet: Biochemical and inflammatory markers in properdin-deficient (KO) and wild type (WT) mice fed a high-fat diet (HFD).

Article Snippet: C5a was measured by sandwich ELISA using purified rat anti-mouse C5a (BD Pharmingen), serum samples were diluted 1:20 and purified recombinant mouse C5a (BD Pharmingen) was used to generate a standard curve.

Techniques: