Journal: Frontiers in Immunology

Article Title: Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer

doi: 10.3389/fimmu.2019.02019

Figure Lengend Snippet: CSF1 and IL34 regulate macrophage homeostasis in adult mice. (A) Effect of aCSF1 and/or IL34 antagonist antibodies on tissue-resident macrophages. C57/BL6 mice were treated with aCSF1 and/or aIL34, or anti-ragweed (aRW) IgG2a intraperitoneally for 1 month. Formalin-fixed tissues (intestine, liver, kidney, skin, and bone marrow) were stained with F4/80 and stained area was plotted and percentage of total tissue area to quantitate tissue-resident macrophages. (B) Quantitation of tissue-resident macrophages in spleen and liver after 7, 14, and 28 days of aCSF1 and/or aIL34 treatment stained with F4/80. (C) Staining of tissue-resident brain microglia (stained with anti-Iba-1) or skin epidermal Langerhans cells (anti-Langerin). Immuno-reactive cells were quantitated by digital image analysis as described in Materials and Methods. Results are displayed as group means ± SEM (standard error of the mean), and are representative of at least two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For measuring drug levels of aCSF1 in serum from dosed murine disease models, ELISA plates were coated with 0.5 mg/ml murine CSF1 (R&D 416-ML/CF) in PBS.

Techniques: Staining, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer

doi: 10.3389/fimmu.2019.02019

Figure Lengend Snippet: Reduced induced or spontaneous mouse arthritis treated with aCSF1 and/or IL34. (A) Reduced longitudinal clinical score in CIA. DBA/1J mice were randomized at day 24 and then treated with aIL34 and/or aCSF1 for 7 weeks. Murine TNFRII-Fc decoy was used as a standard of care control. Isotype control includes anti-ragweed (aRW). Mice were examined weekly for joint inflammation in each paw. (B) Joint cortical bone volume (JCBV) determined by micro-CT (μCT) was used to quantify bone remodeling at the base of metatarsal bones. One hundred percent bone volume was set for naive mice. Anti-RW control group had lowest JCBV (average 79%) due to the bone erosion. TNFRII-Fc improved it to 87% and aIL34 to 84%. aCSF1 or dual aCSF1/aIL34 maintained the bone volume at a healthy level (106 and 104%). (C) Cartilage injury histology score ( n = 38–40/group). (D) Analysis of cartilage damage by contrast-enhanced μCT imaging. Representative micro-CT images shows cartilage as dark rim at joint surface (left set of images). The cartilage scoring system employed a score range from 0 to 4 with the following scale: 0—thick continuous, 1—thick discontinuous, 2—thin continuous, 3—thin discontinuous, 4—no cartilage. All the five MTP joints were individually scored and averaged to get the total score per paw. The total cartilage score per mouse was obtained by averaging the total scores of both the hind limb paws ( n = 6 per group). Anti-CSF1 or anti-IL34 monotherapies were not tested in this study. (E) Joint inflammatory CD11b + F4/80 + macrophages are reduced with aCSF1/aIL34 treatment. Arthritic mice, with a disease score of 4, were randomized and received anti-IL34/anti-CSF1, aRW, or TNFRII-Fc for 7 days. Ankle joints were harvested and processed for FACS. Each data point represents samples pooled from three mice. (F) Reduced CD4 T central memory (TCM) and in draining lymph nodes. (G) TNFΔ ARE spontaneous arthritis, anti-CSF1 significantly reduces clinical arthritis score. (H) TNFΔ ARE arthritis paw lesion histology score is improved by aCSF1. (I) TNFΔ ARE arthritis JCBV is improved with aCSF1. Data are displayed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For measuring drug levels of aCSF1 in serum from dosed murine disease models, ELISA plates were coated with 0.5 mg/ml murine CSF1 (R&D 416-ML/CF) in PBS.

Techniques: Micro-CT, Imaging

Journal: Frontiers in Immunology

Article Title: Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer

doi: 10.3389/fimmu.2019.02019

Figure Lengend Snippet: Reduced induced or spontaneous mouse colitis treated with aCSF1 and/or IL34. (A) Dual blockade of CSF1 and IL34 reduced DSS-induced colitis. Acute colitis was induced by administration of 3% DSS in drinking water at day 0 for total 6 days. Mice were treated with aCSF1 and/or IL34 1 day prior to DSS administration. Cyclosporine A (CSA) was used as a positive control treatment arm. Dual blockade of CSF1 and IL34 improved the disease (32%, * p < 0.05) compared to aRW disease control. (B) Colon rectum cellularity: Single or dual blockade of CSF1 and IL34 reduces macrophages. Anti-CSF1 or aIL34 reduces macrophages compared to aRW isotype control group. However, dual blockade showed superior to single blockade with aIL34 alone. (C) Serum cytokines: Reduction of TNFα and IL6 by aCSF1 or aCSF1/aIL34 blockade. (D) TNFΔ ARE ileitis: Improved body weight in TNFRII-Fc or aCSF1/IL34 combination treatment. (E) Reduced ileitis histology score with aIL34 and/or aCSF1 as well as TNFRII-Fc compared to control aRW group. Data are displayed as mean ± SEM. (F) Reduced colon histology score in IL10 null colitis model treated with aCSF1/IL34 compared to TNFRII-Fc. Positive control includes anti-p40. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For measuring drug levels of aCSF1 in serum from dosed murine disease models, ELISA plates were coated with 0.5 mg/ml murine CSF1 (R&D 416-ML/CF) in PBS.

Techniques: Positive Control

Journal: Frontiers in Immunology

Article Title: Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer

doi: 10.3389/fimmu.2019.02019

Figure Lengend Snippet: CSF1, but not IL34, is required for TAM and immune homeostasis within MC38 tumors. Female C57Bl/6 inoculated with MC38 tumors were treated with control, aCSF1, aIL34, or aCSF1/aIL34. MC38 tumors were harvested on day 12 post-initial treatment and the following parameters were evaluated: (A) total CD45 + cells; (B) TAMs per gram (g) tumor; (C) monocytic and granulocytic MDSC populations (mMDSC or gMDSC, respectively); (D) Ki67 + TAMs; (E) functional marker analysis within TAMs including proinflammatory NOS2, CD86, as well as the anti-inflammatory marker arginase 1 (Arg1); (F) MHCII and PD-L1 TAM expression; (G) Number of NK, CD8, and CD4 T cells within tumors; (H) CD4 + Teff and Foxp3 + Tregs within tumors; (I) ratio of CD8 T cells to Tregs within tumors; and (J) survival of mice following control antibody aRW or aCSF1/aIL34 (left), and individual tumor growth curves (right). Functional and phenotypic data were derived from 6 to 7 mice per treatment group (A – I) . Each symbol indicates data from a single tumor harvested from an individual mouse. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For measuring drug levels of aCSF1 in serum from dosed murine disease models, ELISA plates were coated with 0.5 mg/ml murine CSF1 (R&D 416-ML/CF) in PBS.

Techniques: Functional Assay, Marker, Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer

doi: 10.3389/fimmu.2019.02019

Figure Lengend Snippet: Assessment of infection and liver toxicity risks with aIL34 and/or aCSF1. (A) Infection risk: Pre-treatment with aCSF1, aIL34, or aCSF1/aIL34 antibodies prior to L. monocytogenes infection resulted in an increase in survival rate compared to TNFRII-Fc. C57BL6 female mice ( n = 10/group) were treated with aRW, aCSF1, aIL34, aCSF1/aIL34, or mTNFRII-Fc starting 2 days prior to infection. Mice were infected via intravenous route with two doses of L. monocytogenes [12.5K or 25K colony-forming units (cfu)/mouse] and animals were monitored for 14 days. (B) B6C3F1 mice were treated with aRW or a-CSF1/a-IL34 antibodies for 7, 14, or 28 days ( n = 5/group/time point). Following high-dose APAP (1,200 mg/kg APAP for 6 h), increased ALT, AST, SDH, GLDH, and miR-122 values were observed. Treatment with a-CSF1/a-IL34 antibodies resulted in mild to moderate increases in liver injury biomarkers ALT and AST compared to concurrent anti-RW control values with no significant changes in liver injury biomarkers miR122 or GLDH. Following dosing, animals were necropsied, serum or plasma was collected for liver biomarker analysis, and liver tissue was processed for microscopic evaluation or enumeration of Kupffer cells as described in Materials and Methods. (C) Analysis of liver Kupffer cell numbers using F4/80+ IHC. Data in C are displayed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For measuring drug levels of aCSF1 in serum from dosed murine disease models, ELISA plates were coated with 0.5 mg/ml murine CSF1 (R&D 416-ML/CF) in PBS.

Techniques: Infection, Biomarker Assay

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of testicular sections from mice under four different treatments in the short term (W2, 2 weeks) and long term (W8, 8 weeks) experiments. Scale bar, 200 μm. b ELISA of CSF1 in mice serum of the four different groups in W2 and W8 experiments. c RT-qPCR detected Csf1 expression in mice testicular tissue under the four different treatments in W2 and W8 experiments. d CSF1 expression level detected by western blot analysis in the four groups in W2 and W8. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. (The results are shown as the mean ± S.E.M of at least three mice ( n = 3) and the statistical significance was expressed as follows: * p < 0.05; ** p < 0.01). C control group, M normal group treated with MLT, D diabetic group, DM MLT-treated diabetic group

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Melatonin attenuates detrimental effects of diabetes on the niche of mouse spermatogonial stem cells by maintaining Leydig cells

doi: 10.1038/s41419-018-0956-4

Figure Lengend Snippet: a CSF1 expression with Hoechst 33342 and their merges of MLTC-1 under three different ERS states (C control, Tm Tm treatment induced excessive ERS, Tm + 4PBA Tm treatment in combination with 4PBA for alleviated ERS). b CSF1 concentration in culture medium of MLTC-1 under the three different treatments was detected using ELISA. c RT-qPCR detected the mRNA expression level of Csf1 in MLTC-1 under the three treatments. d Expression of CSF1 in MLTC-1 under different treatments was measured by Western blot. Histograms are the statistical result of Image J (V1.48d) gradation analysis for the western blot experiments. e A graphical conclusion of this study is displayed: DM-induced high glucose activated ERS response factors (Grp78 and CHOP) in Leydig cells, resulting in apoptosis of Leydig cells and growth arrest of SSCs in testes. In response to these changes, melatonin treatment alleviated the apoptosis of Leydig cells via inhibition of ERS and recovered the CSF1 secretion in testes. CSF1 then resumed the self-renewal capacity of SSCs. (The results are expressed as the mean ± S.E.M of three separated wells of cells ( n = 3) in at least three different experiments and the statistical significance is expressed as follows: * p < 0.05; ** p < 0.01)

Article Snippet: Mouse CSF1 level of mouse plasma was quantified using an ELISA kit (Boster, Wuhan, China) according to the instructions.

Techniques: Expressing, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Inhibition

Journal: Frontiers in Immunology

Article Title: Identification of biomarkers related to immune and inflammation in membranous nephropathy: comprehensive bioinformatic analysis and validation

doi: 10.3389/fimmu.2023.1252347

Figure Lengend Snippet: Analysis of immune-associated hub genes. (A) Lollipop plot of CSF1R correlation with 28 immune cells. The horizontal coordinate indicates the correlation size, and the color indicates the P-value. (B) Immune-related GSEA analysis of CSF1R with different expression levels. (C) Distribution of 28 immune cells in CSF1R with different expression levels. (D-F) Analysis of CYBB. **P <0.01, ***P <0.001, ****P <0.0001.

Article Snippet: To measure the protein levels of CSF-1 and CYBB/NOX2, we used specific ELISA kits for human CSF-1 (KE00184; Proteintech, USA) and CYBB/NOX2 (EK13559; Signalway Antibody, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of biomarkers related to immune and inflammation in membranous nephropathy: comprehensive bioinformatic analysis and validation

doi: 10.3389/fimmu.2023.1252347

Figure Lengend Snippet: Validation of CSF-1 and CYBB. Each point represents a sample. (A, B) ELISA measurement of CSF-1 and CYBB. (C, D) mRNA expression of CSF1R and CYBB was analyzed by RT-qPCR. *** P < 0.001, vs Control.

Article Snippet: To measure the protein levels of CSF-1 and CYBB/NOX2, we used specific ELISA kits for human CSF-1 (KE00184; Proteintech, USA) and CYBB/NOX2 (EK13559; Signalway Antibody, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR