Journal: Nutrients

Article Title: Ghee Butter from Bovine Colostrum Reduces Inflammation in the Mouse Model of Acute Pancreatitis with Potential Involvement of Free Fatty Acid Receptors

doi: 10.3390/nu13093271

Figure Lengend Snippet: MPO activity level in pancreas ( A ) and lungs ( B ) of healthy mice, mice with evoked acute pancreatitis (AP), and mice with AP after administration of 100% GBBC and FFAR1 or FFAR4 antagonist. Data presented as raw data (circles) and means (horizontal bolded lines) with SEM (boxes). Significance estimated with the use of one-way ANOVA followed by Dunnett’s post hoc test. # (hash) denotes comparison to control. * (asterisk) indicates comparison to AP. n = 4–8; * denotes p < 0.05; ** denotes p < 0.01; ###/*** denotes p < 0.001.

Article Snippet: The following antibodies were used: primary rabbit anti-mouse FFAR1 polyclonal antibody (AFR-001; dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR2 polyclonal antibody (AFR-032, dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR3 polyclonal antibody (PA5-97747; dil. 1:1000; Thermo Scientific, Rockford, IL, USA), FFAR4 polyclonal antibody (E-AB-31,576; dil. 1:1000; Elabscience, Wuhan, China), and mouse beta actin (β-actin; sc-47778; dil. 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activity Assay, Comparison, Control

Journal: Nutrients

Article Title: Ghee Butter from Bovine Colostrum Reduces Inflammation in the Mouse Model of Acute Pancreatitis with Potential Involvement of Free Fatty Acid Receptors

doi: 10.3390/nu13093271

Figure Lengend Snippet: Microphotographs of the pancreas. H&E staining: control group—normal architecture of the exocrine pancreas with functional acinar cells rich in zymogen granules ( A ); acute pancreatitis (AP) group—diffuse inflammatory infiltrates in periacinar area with the predominance of neutrophils ( B ); AP + 100% ghee butter from bovine colostrum (GBBC) group—acinar cell vacuolization, minimal atrophy of acinar cells with no evidence of inflammatory cell infiltrates ( C ); AP + FFAR1 antagonist + 100% GBBC group—dissociation and moderate atrophy of acinar cells ( D ); AP + FFAR4 antagonist + 100% GBBC group—dissociation of acinar cells, strong remodeling of the stroma, and marked atrophy of acinar cells ( E ). Masson’s trichrome staining: control group—minimal collagen deposition as a delicate framework for acinar cells ( F ); AP group—minimal collagen deposition in inflamed and edematous stroma ( G ); AP + 100% GBBC group—minimal collagen deposition between well-compacted acinar cells ( H ); AP + FFAR1 antagonist + 100% GBBC group—moderate collagen deposition in periacinar area and between individual acinar cells ( I ); AP + FFAR4 antagonist + 100% GBBC group—marked deposition of collagen between atrophied acinar cells ( J ). Immunoreactivity of α-SMA protein: control group—negative reaction ( K ); AP group—positive reaction in individual myofibroblast-like cells ( L ); AP + 100% GBBC group—moderate positive cytoplasmic reaction in migrating myofibroblast-like spindle-shaped cells penetrating periacinar spaces ( M ); AP + FFAR1 antagonist + 100% GBBC group—strong immunohistochemical reaction in myofibroblast-like cells ( N ); AP + FFAR4 antagonist + 100% GBBC group—strong immunohistochemical reaction in numerous myofibroblast-like cells ( O ). Nikon’s Eclipse E600 light microscope (Nikon Instruments Inc., Tokyo, Japan) was used to examine tissue samples. Microphotographs were taken with a digital imaging system that included a microscopy digital camera (Nikon DS-Fil, Nikon Instruments Inc., Tokyo, Japan) and image analysis software (NIS-Elements BR-2.20, Laboratory Imaging, Praha, Czech Republic); 400× magnification.

Article Snippet: The following antibodies were used: primary rabbit anti-mouse FFAR1 polyclonal antibody (AFR-001; dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR2 polyclonal antibody (AFR-032, dil. 1:1000; Alomone Labs, Jerusalem, Israel), FFAR3 polyclonal antibody (PA5-97747; dil. 1:1000; Thermo Scientific, Rockford, IL, USA), FFAR4 polyclonal antibody (E-AB-31,576; dil. 1:1000; Elabscience, Wuhan, China), and mouse beta actin (β-actin; sc-47778; dil. 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Staining, Control, Functional Assay, Immunohistochemical staining, Light Microscopy, Imaging, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Neutrophils in the Spotlight—An Analysis of Neutrophil Function and Phenotype in ARDS

doi: 10.3390/ijms252312547

Figure Lengend Snippet: Overview of fluorescently labeled antibodies. Abbreviations: phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin (APC).

Article Snippet: fMLP-Receptor , APC , APC Anti-human, fMLP receptor Antibody Clone: REA169 Miltenyl Biotec, Bergisch Gladbach, Deutschland.

Techniques: Labeling

Journal: Scientific Reports

Article Title: Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

doi: 10.1038/s41598-024-71180-1

Figure Lengend Snippet: Fpr1 and 2 expression in the lungs is associated with TB disease. Wild type (Wt) and Il1r1 −/− mice were infected via aerosol with Mtb HN878 smyc’:: mCherry (Mtb), delivering approximately 100 CFU to the lungs. At 26 dpi, Fpr1 ( a ) and Fpr2 ( b ) expressions were quantified in the lungs using qPCR. Expression levels in Il1r1 −/− mice were calculated relative to those in wild type C57BL/6 (Wt) mice. Error bars represent Mean ± SD. Statistical analysis was conducted using an unpaired t-test, with * p < 0.05 and “ns” denotes non-significant results. ( c , d ) Representative immunofluorescence images of formalin-fixed paraffin-embedded (FFPE) lung sections from Mtb-infected wild-type (Wt) and Il1r1 −/− mice are shown. The top panels ( c ) display DAPI (blue) staining the nuclei and Fpr1 (red), while the bottom panels ( d ) show DAPI (blue) and Fpr2 (yellow). The expression of Fpr1 and Fpr2 in the lung sections is quantified as corrected total fluorescence intensity (CTCF) from three fields of view per group, representing one of two experiments. Zoomed-in areas for individual lung samples are provided in thumbnails. The data represent n = 3 samples per time point, with error bars showing the Mean ± SEM. Statistical analysis was conducted using an unpaired t-test, with * p < 0.05 indicating significance. ( e ) qPCR analysis of FPR1 and FPR2 expressions in rabbit lungs post Mtb infection with strains HN878 and CDC1551 at 3 h and 4 weeks post infection. Data represent n = 3 samples per time point. Error bars show Mean ± SEM. Statistical analysis was performed using two-way ANOVA. * p < 0.05. ( f ) FPR1 and 2 expression in human TB before and after anti-TB therapy: RNA-seq data was extracted from publicly available dataset previously published (GSE19435). Data is comprised of whole blood transcriptional signatures obtained from two different cohorts. Data sets were downloaded from NCBI as the Longitudinal TB Treatment in a UK cohort (GSE19435) and ( g ) whole blood transcriptional signatures in latent TB (LTBI) and active TB in a South African Cohort (GSE19442). Genes were identified based on their Ilumina IDs; FPR1(ILMN_2092118), FPR2 (ILMN_2392569, ILMN_1740875), extracted, plotted and analysed by unpaired student t-test versus the indicated groups. ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Primary antibodies used were: anti-FPR1 antibody (Cat: FPR1-101AP, Fabgennix), and anti-FPRL1/FPR2 antibody (Cat: NLS1878SS, Novus Biologicals).

Techniques: Expressing, Infection, Aerosol, Immunofluorescence, Formalin-fixed Paraffin-Embedded, Staining, Fluorescence, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

doi: 10.1038/s41598-024-71180-1

Figure Lengend Snippet: Protective effects of Fpr1 deletion on tuberculosis outcomes in mice. Wt and Fpr1 −/− mice on a C57BL/6 background were aerosol-infected with Mtb HN878 reporter bacteria as per the protocol in Fig. . Necropsies and tissue analyses were conducted at 32 dpi, with 4–5 mice per group. ( a ) Percentage of weight change upto 32 dpi in Mtb-infected Wt and Fpr1 −/− mice. ( b ) Bacterial load in the lungs and spleens measured in CFU per ml at 32 dpi in both Wt and Fpr1 −/− mice. ( c ) Flow cytometry assessment of myeloid cells, including neutrophils, macrophages, and monocytes at 32 dpi in both Wt and Fpr1 −/− mice. ( d ) Flow cytometry assessment of Mtb infected live and dead/dying neutrophils and ( e ) macrophages at 32 dpi in both Wt and Fpr1 −/− mice. Infected live neutrophils were identified as viability dye -ve CD11b + Ly6G + smyc’::mCherry+, infected dead/dying neutrophils were identified viability dye +ve CD11b + Ly6G + smyc’::mCherry+, while infected macrophages were detected as viability dye -ve CD11b + Ly6G-CD11c + MHCII + SiglecF-smyc’::mCherry+. ( f ) Flow cytometry assessment of T-lymphocytes (CD4+, CD8+) and B lymphocytes (CD19+) at 32 dpi in Wt and Fpr1 −/− mice. ( g ) Quantification of cytokines (IL-1α, IL-1β, IL-6, TNF-α, IFNγ) in lung homogenates from both Wt and Fpr1 −/− mice at 32 dpi. n = 3 per group. ( h ) Lung Histopathology: Representative H&E images from 3 lung sections from Wt and Fpr1 −/− mice at 32 dpi. n = 3 mice per group. ( i ) Quantification of lesion area was shown as percentage of the entire lung lobe (n = 6, pooled from two independent experiments). Error bars represent Mean ± SD for panel ( a , i ) and Mean ± SEM for panel ( b – g ). Statistical analysis involved a two-way ANOVA for panel a , with significance determined by Tukey’s multiple comparison test (**** p < 0.0001). Unpaired t-tests were conducted for panels b - g . * p < 0.05, *** p < 0.001, and “ns” denotes non-significant results.

Article Snippet: Primary antibodies used were: anti-FPR1 antibody (Cat: FPR1-101AP, Fabgennix), and anti-FPRL1/FPR2 antibody (Cat: NLS1878SS, Novus Biologicals).

Techniques: Aerosol, Infection, Bacteria, Flow Cytometry, Histopathology, Comparison

Journal: Scientific Reports

Article Title: Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

doi: 10.1038/s41598-024-71180-1

Figure Lengend Snippet: Blocking Fpr1 in susceptible mice increased bacterial growth in the lungs. ( a ) Experimental setup: Il1r1 −/− mice were exposed to an aerosol containing approximately 100 CFU of Mtb HN878 reporter bacteria. Starting one day before infection (day -1), the mice were given either a vehicle or Fpr1 inhibitors every other day. The Fpr1 inhibitors used were a combination of Cyclosporin H (4 mg/kg) and HCH6-1 (4 mg/kg), administered orally. Measurements were taken on days 14, 21, and 25 post-infection. ( b ) Bacterial load in the lungs was determined by CFU counts. ( c ) Flow cytometry was used to assess live and dead/dying neutrophils among total lung cells at different time points post-Mtb infection. dpi: days post infection. ( d ) Flow cytometry was used to assess Mtb-infected neutrophils (live), ( e ) dead/dying neutrophils and ( f ) macrophages in the lung. Data are from n = 3–7 mice per group. Results from day 25 are combined from two separate experiments. Error bars represent Mean ± SEM. Statistical significance was assessed using an unpaired t-test compared to respective controls. * p < 0.05; **** p < 0.0001; ‘ns’ denotes non-significant results. ( g ) Lung Histopathology: Representative images of H&E-stained FFPE lung sections are shown for the specified infection times. ( h ) Quantification of necrotic lesion areas illustrates the progression of disease over time in both vehicle-treated and inhibitor-treated mouse lungs. Error bars show Mean ± SD.

Article Snippet: Primary antibodies used were: anti-FPR1 antibody (Cat: FPR1-101AP, Fabgennix), and anti-FPRL1/FPR2 antibody (Cat: NLS1878SS, Novus Biologicals).

Techniques: Blocking Assay, Aerosol, Bacteria, Infection, Flow Cytometry, Histopathology, Staining

Journal: Scientific Reports

Article Title: Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

doi: 10.1038/s41598-024-71180-1

Figure Lengend Snippet: Fpr1 blockade impairs bacterial control in C3HeB mice. ( a ) Experimental design: C3HeB mice were infected with Mtb HN878 reporter bacteria via aerosol and treated with either a vehicle or Fpr1 inhibitors according to the schematic. Evaluations were conducted at 14- and 35 dpi. ( b ) Bacterial burden in the lungs and spleens of both vehicle-treated and inhibitor-treated mice was measured and expressed as colony-forming units (CFU). ( c ) Flow cytometry was used to assess live and dead/dying neutrophils among total lung cells at different time points post-Mtb infection. dpi: days post infection. ( d ) Flow cytometry was used to assess Mtb-infected live neutrophils; ( e ) dead/dying neutrophils and ( f ) macrophages at the specified time points post-infection. n = 4 per group. ( g ) Representative histopathology images of H&E-stained FFPE lung sections. ( h ) Quantification of necrotic lesion areas in the lungs at 14 and 35 dpi for both vehicle- and inhibitor-treated mice. ( i ) Effect of Fpr1 blockade on neutrophilic lesions in C3HeB mice 35 days post Mtb infection. Neutrophil clusters were indicated by arrows. Error bars represent Mean ± SEM ( b – f ) and Mean ± SD ( h ). Statistical significance was assessed using an unpaired t-test compared to respective controls. * p < 0.05; *** p < 0.001; 'ns' denotes a non-significant result.

Article Snippet: Primary antibodies used were: anti-FPR1 antibody (Cat: FPR1-101AP, Fabgennix), and anti-FPRL1/FPR2 antibody (Cat: NLS1878SS, Novus Biologicals).

Techniques: Control, Infection, Bacteria, Aerosol, Flow Cytometry, Histopathology, Staining

Journal: Scientific Reports

Article Title: Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection

doi: 10.1038/s41598-024-71180-1

Figure Lengend Snippet: Fpr1 plays a contrasting role in macrophages and neutrophils during Mtb infection. ( a ) Experimental setup: Bone marrow-derived neutrophils from Wt and Fpr1 −/− mice were isolated using magnetic cell sorting (MACS) and infected with Mtb HN878 at a multiplicity of infection (MOI) of 3.0 for 4 h. After removing extracellular bacteria through washing, cells were further incubated for 20 h. The intracellular bacterial load was then assessed using CFU analysis. ( b ) Intracellular bacterial load in neutrophils is presented as CFU counts. ( c ) Experimental setup: Bone marrow-derived neutrophils from Wt, Pad4 −/− , and Cybb −/− mice were isolated using MACS and infected with Mtb HN878 at an MOI of 3.0, as described in ( a ), with or without the addition of 100 nM fMLP. ( d – f ). Bacterial burden in these neutrophils was measured at 24 h post-infection and is shown as CFU. ( g ) Experimental setup: Bone marrow-derived macrophages (BMDMs) from Wt and Fpr1 −/− mice were infected with Mtb HN878 at a MOI of 3.0. ( h ) Bacterial load in BMDMs was determined at various time points post-infection and expressed as CFU counts. The experiments were conducted with n = 3 replicates per group and are representative of two independent experiments. Error bars represent Mean ± SEM. Statistical analysis was performed using unpaired t-tests. * p < 0.05; 'ns' denotes a non-significant result. ( i ) Model depicting the differential roles of Fpr1 in myeloid cell anti-mycobacterial functions. This model illustrates the contrasting effects of Fpr1 expression in neutrophils and macrophages during Mtb infection. Fpr1 expression in neutrophils is essential for effectively controlling Mtb growth within these cells. Conversely, in macrophages, Fpr1 expression impedes their ability to control intracellular Mtb growth, as evidenced by a reduced bacterial burden in macrophages lacking Fpr1. The graphics were generated using www.biorender.com .

Article Snippet: Primary antibodies used were: anti-FPR1 antibody (Cat: FPR1-101AP, Fabgennix), and anti-FPRL1/FPR2 antibody (Cat: NLS1878SS, Novus Biologicals).

Techniques: Infection, Derivative Assay, Isolation, FACS, Bacteria, Incubation, Expressing, Control, Generated