This work was supported by the National Natural Science Foundation of China (81973675).

The ethics committee of Guanganmen Hospital approved this experiment (animal experiment ethical approval number: IACUC-GAMH-2023-003).
1. Preparing mice for the experimental procedure
<ol>
	<li>Raise 22 SPF-grade male Balb/c mice (6-7 weeks old, body weight 20-25 g) in the animal facility of the hospital/research center. Divide the animals into control (n = 8) and model (n = 14) groups using the random number table method.</li>
	<li>After 1 week of adaptive rearing in the laboratory cage, feed the model group (IgAN group) with 0.1% bovine gamma globulin (BGG) solution in acidified water containing 6 mmol/L HCl for 9 weeks according to the modeling protocol of Zou et al.18.</li>
	<li>Inject 0.1 mL of 0.1% BGG solution in saline into the tail vein for 3 consecutive days while continuing to drink BGG solution to prepare an IgAN experimental mouse model18.</li>
	<li>Let the control group freely drink 6 mmol/L HCl acidified water without BGG for 9 weeks. Inject the corresponding volume of saline into the tail vein for consecutive 3 days. 
	NOTE: The control and model groups were fed the same quality as normal feed.</li>
	<li>After the tail vein injection, select 2 mice in the control group and 2 mice in the model group randomly and examine them by proteinuria, light microscopy, and immunofluorescence to determine whether the modeling was successful. 
	NOTE: No food is provided to the animals, but they are not prohibited from water; record urine output.</li>
	<li>Collect urine for 24 h by metabolic cages and centrifuge it at 400 x g for 5 min; discard urine sediment. After a 10-fold dilution of the supernatant, measure proteinuria concentration using a urine protein assay kit and then multiply by the dilution factor and urine volume to obtain 24 h of total urine protein. 
	NOTE: Microscopy and immunofluorescence methods are shown in sections 3 and 4, respectively.</li>
	<li>After successfully preparing the model, divide 12 mice in the model group into 6 mice, each in the model group (IgAN group) and DIO gavage group (DIO group), according to the random number table method.</li>
	<li>Let the control group continue to drink 6 mmol/L HCl acidified water without BGG, and the model group 0.1% BGG solution composed of acidified water containing 6 mmol/L HCl. Calculate the dose of DIO group gavage administration according to the dose conversion formula of pharmacological experimental methodology (converted according to the human body mass of 70 kg)19. Gavage DIO tablets 0.06 g/kg once a day for 8 weeks.</li>
	<li>After 8 weeks of gavage, anesthetize the mice intraperitoneally with 0.4% pentobarbital sodium (60 mg/kg), and after confirming proper anesthetization by toe pinch, isolate kidneys and Peyer&#39;s patch for subsequent light microscopy and immunohistochemistry analyses. 
	NOTE: The scheme for the in vivo model is in Supplementary Figure 1.</li>
</ol>
2. Histological analysis
<ol>
	<li>Paraffin sections for kidneys and Peyer&#39;s patch
	<ol>
		<li>Fix 3 mm thick kidney tissues or 1 Peyer&#39;s patch with 4% paraformaldehyde for 24 h, dehydrate with gradient ethanol and xylene. Dip in wax for 2 h, seal, and freeze.</li>
		<li>Cut 2 &#181;m thick kidney sections and 4 &#181;m thick Peyer&#39;s patch sections and spread them in warm water. Scoop up the unfolded slices with a clean glass slide and bake them in a constant temperature oven at 40 &#176;C for 1 h. Start staining after the pre-processing of the specimen. 
		NOTE: Take the coronal surface of the hilar part of the kidney, with a thickness of 3 mm tissue block.</li>
	</ol>
	</li>
	<li>Dewax and stain the paraffin sections at room temperature (RT) for 10 min with periodic acid solution, avoiding light. Rinse with distilled water and wipe dry-stained with Schiff&#39;s staining solution for 20-30 min, avoiding light. Rinse with distilled water until the sections were red under the microscope.</li>
	<li>Place the sections in hematoxylin staining solution, stain nuclei for 3 min (nuclei stained too profoundly can be divided by ethanol hydrochloride), and rinse with running water until the slides are colorless.</li>
	<li>Perform routine dehydration with gradient concentrations of ethanol and xylene, seal the sections with neutral gum, and observe under a microscope. PAS-positive is red, and the nucleus is blue.</li>
</ol>
3. Immunohistochemical analysis of the Peyer&#39;s patch
<ol>
	<li>Prepare paraffin sections of the Peyer&#39;s patch as described in step 2.1. 
	NOTE: The thickness of the sections for Immunohistochemical and subsequent immunofluorescence is 4 &#181;m.</li>
	<li>Dewax the paraffin sections:
	<ol>
		<li>Put the sections into xylene I for 5 min, xylene II for 5 min, and xylene III for 5 min.</li>
		<li>Further, rinse the slides in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min. Then rinse the slides in distilled water.</li>
	</ol>
	</li>
	<li>Antigen retrieval
	<ol>
		<li>Prepare 50x stock solution of sodium citrate and dilute with distilled water to 1x for use. Heat it in an autoclave for 2 min, then place the slices in the autoclave, ensuring that the liquid level exceeds the level of the slices.</li>
		<li>Heat at high temperature for 5 min, then allow the slides to cool naturally. Wash the slices three times with PBS solution for 5 min each.</li>
	</ol>
	</li>
	<li>Blocking of endogenous peroxidase: Mark the borders of the tissue in a circle with an immunohistochemical pen. Incubate the sections in 3% hydrogen peroxide solution for 15 min at RT, protected from light, and wash the sections three times with PBS solution for 5 min each time.</li>
	<li>Serum blocking: Block the sections by dropping 10% goat serum on the tissue sections marked for 30 min at RT. Ensure that the sections are evenly covered with the stain.</li>
	<li>Primary antibody incubation: Gently shake off the blocking solution and add a proportion of the prepared primary antibody (CD20 [1:800]; CXCR5 [1:800]) to the section. Place the section flat in a wet box and incubate overnight at 4 &#176;C. 
	NOTE: Add a small amount of water to the wet box to prevent evaporation of the antibody.</li>
	<li>Secondary antibody incubation: Wash the sections thrice with PBS solution for 5 min each time. Remove PBS by shaking the sections dry, cover the tissue with a drop of secondary antibody (HRP-label) of the related species of the primary antibody, and incubate at RT for 50 min.</li>
	<li>3,3&#39;-diaminobenzidine (DAB) homogenization: Wash the sections with PBS solution thrice for 5 min each. After shaking the sections dry, drop the freshly prepared DAB chromogenic solution on the sections. Observe the color development time under the microscope; the positive is brownish-yellow. Rinse with tap water to terminate the color development.</li>
	<li>Staining nuclei: Re-stain with hematoxylin for about 1 min, wash with tap water, and then rinse for 10 min with tap water to return to blue.</li>
	<li>Dehydration and sealing:
	<ol>
		<li>Place the sections in 75% alcohol for 5 min and 85% alcohol for 5 min. Place the sections in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and anhydrous ethanol III for 5 min.</li>
		<li>Wash the sections in xylene I for 5 min, take them out to dry slightly and seal the sections with neutral gum.</li>
	</ol>
	</li>
	<li>Image acquisition: Collect Images by microscopic examination and analyze by halo software for panoramic image analysis of the tissue. 
	NOTE: Hematoxylin-stained nuclei are blue, and DAB positive expression is observed as brownish-yellow.</li>
</ol>
4. Renal IgA immunofluorescence
<ol>
	<li>Prepare paraffin sections for kidneys as described in step 2.1.</li>
	<li>Dewax paraffin sections:
	<ol>
		<li>Put the sections into xylene I for 5 min, xylene II for 5 min, and xylene III for 5 min. Treat the sections in anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and 50% ethanol, each for 5 min, and wash with distilled water.</li>
	</ol>
	</li>
	<li>Proteinase K retrieval: Shake the sections dry and draw a circle around the tissue section with a histochemical pen. Add the proteinase K working solution (1:9 ratio of stock solution and PBS) dropwise to cover the tissue and incubate at 37 &#176;C for 30 min. Wash the sections three times with PBS for 5 min each.</li>
	<li>Penetrating the cell membrane: Shake the sections dry slightly and then cover them with 0.1% Triton. Incubate for 20 min at RT, and wash the sections three times with PBS for 5 min each.</li>
	<li>Blocking: Add 10% goat serum dropwise to cover the tissue evenly for blocking at RT for 30 min.</li>
	<li>Primary antibody incubation: Add an appropriate amount of goat anti-mouse AF488-conjugate IgA antibody (1:500) dropwise to cover the tissue uniformly and incubate overnight at 4 &#176;C.</li>
	<li>Staining nuclei: Wash the slices thrice with PBS for 5 min each. After removing the PBS, add the 4&#8242;,6-diamidino-2-phenylindole (DAPI) stain dropwise on the sections and incubate for 15 min at RT, protected from light.</li>
	<li>Wash and seal the sections: Wash the sections thrice with PBS for 5 min, then seal them with an antifade mounting medium.</li>
	<li>Microscopy and photography: Observe the sections under a fluorescent microscope and take images. 
	NOTE: For DAPI, ultraviolet excitation is wavelength 330-380 nm, and the emission wavelength is 420 nm, blue light. Fluorescein isothiocyanate (FITC) excitation wavelength is 465-495 nm, and the emission wavelength is 515-555 nm, green light.</li>
</ol>
5. Cell culture
<ol>
	<li>Obtain human B lymphocyte line DAKIKI from ATCC, USA. Culture DAKIKI cells in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin.</li>
	<li>Culture the cells in a 37 &#176;C, 5% CO2 incubator and sub-culture them every 2-3 days. Use cells at the logarithmic growth phase for all experiments.</li>
	<li>At 70%-80% confluency, collect the cells with a sterile pipette and centrifuge the cells at 140 x g for 5 min. Discard the supernatant, resuspend with the serum-free medium, and after 24 h, leave all cells in a quiescent period for subsequent treatment.</li>
</ol>
6. LDH cytotoxicity assays for screening safe concentrations of DIO on normal DAKIKI cells
<ol>
	<li>Seed DAKIKI cells in 96-well plates at a density of 4x105 cells/well and set up a Low control group, a High control group, and different concentrations of DIO (0.25, 0.5, 1.0, 2.0, 4.0, 8.0 &#181;g/mL). Incubate in 5% CO2, 37 &#176;C incubator for 24 h after corresponding treatment according to the grouping method.</li>
	<li>According to the instructions of the cytotoxicity detection kit, add 5 &#181;L of lysate per well in the high control group, and then place the plate in a 5% CO2, 37 &#176;C incubator for 15 min.</li>
	<li>Take out the plate, add 100 &#181;L of the reaction mixture to each well, incubate in the dark for 10 min at RT, and then add 50 &#181;L of the stop reaction solution. Measure the OD value at 490 nm on the microplate reader as soon as possible.</li>
	<li>Calculate the LDH release rate of different concentrations of DIO and &#8804;10% as the maximum dose administered according to the formula: LDH release rate = (experimental well LDH - low control LDH) / (high control LDH - low control LDH) x 100%.</li>
</ol>
7. CCK-8 assay for detecting the effect of DIO on DAKIKI cell proliferation
<ol>
	<li>Based on the results of our previous experiments20, establish an IgAN model using LPS 40 &#181;g/mL to induce DAKIKI cells.</li>
	<li>Then, seed 4x105 cells/well in 96-well plates and divide into the control, model, and DIO low, medium, and high concentration groups (0.25 &#181;g/mL, 0.5 &#181;g/mL, and 1.0 &#181;g/mL). Incubate the plates in a 5% CO2, 37 &#176;C incubator for 24 h.</li>
	<li>Then, add 20 &#181;L of CCK-8 reagent to each well and place the plates back in the incubator (5% CO2, 37 &#176;C) for 2 h. After the incubation, detect the OD at a wavelength of 450 nm on the microplate reader as soon as possible.</li>
</ol>
8. ELISA for detecting the effect of DIO on the secretion of IgA and Gd-IgA1 by DAKIKI cells
<ol>
	<li>Seed DAKIKI cells in 6-well plates at a density of 6x106 cells/well, and group and treat the cells according to step 7.2. Culture the cells for 24 h, then centrifuge at 850 x g for 10 min at 4 &#176;C to obtain the supernatant.</li>
	<li>Detect IgA and Gd-IgA1 concentrations according to the kit instructions.</li>
</ol>
9.qRT-PCR for detecting the effect of DIO on C1GALT1 and Cosmc mRNA levels in DAKIKI cells
<ol>
	<li>Seed the DAKIKI cells at a density of 6x106 cells/well in a 6-well plate, group and treat the cells as CCK8 mentioned in step 7.2, and incubate for 24 h. Extract total RNA from DAKIKI cells according to the instructions of the total RNA extraction kit.</li>
	<li>After taking 1 &#181;L of extracted RNA from each group of samples and measuring its concentration, transcribe reversely 1 &#181;g of total RNA from each sample into cDNA according to the kit instructions.</li>
	<li>Then, perform RT-PCR amplification to detect the expression of each gene (95 &#176;C for 15 min, 95 &#176;C for 10 s, and 60 &#176;C for 30 s). Calculate the expression level of each gene using the 2-&#916;&#916;CT method with &#946;-actin as the internal reference. 
	NOTE: The primer sequences were as follows: 
	C1GALT1: 5&#39;-AAGGTTGACACCCAGCCTAA-3&#39;, 5&#39;-CTTTGACGTGTTTGGCCTTT-3&#39;; 
	Cosmc: 5&#39;-GCTCCTTTTTGAAGGGTGTG-3&#39;, 5&#39;-TACTGCAGCCCAAAGACTCA-3&#39;; 
	&#946;-actin: 5&#39;-TCACCCACACTGTGCCCATCTACGA-3&#39;, 5&#39;-CAGCGGAACCGCTCATTGCCAATGG-3&#39;.</li>
</ol>
10. Western blotting for examining the effect of DIO on the expression of C1GALT1 and Cosmc proteins in DAKIKI cells
<ol>
	<li>Seed the DAKIKI cells at a density of 6x106 cells/well in a 6-well plate. Group and treat them as mentioned in step 7.2. After 24 h of incubation, collect each group of cells.</li>
	<li>Add an appropriate amount of cell lysis solution (PMSF: phosphatase inhibitor: RIPA lysis solution = 1:1:100) and incubate on ice for 30 min. Then centrifuge at 13,500 x g for 10 min at 4 &#176;C and collect the supernatant.</li>
	<li>Determine the protein concentration using the BCA protein concentration assay kit.</li>
	<li>Mix the protein samples with 5x SDS-PAGE loading buffer at 4:1 by vortexing, and heat the mixed samples at 100 &#176;C for 5 min to denature the protein.</li>
	<li>To detect proteins with different molecular weights, add the protein marker (5 &#181;L/well) and samples (20 &#181;g/well) into different lanes of a 12% SDS-PAGE gel, run SDS-PAGE electrophoresis, and transfer the gel to PVDF membranes.</li>
	<li>Block the membranes with 5% nonfat milk for 2 h at RT and incubate with the corresponding primary antibodies (C1GALT1 [1:1000], Cosmc [1:2000]) for 24 h. Use &#946;-actin antibody (1:100000) as an internal control.</li>
	<li>Wash the polyvinylidene fluoride (PVDF) membrane with 1x Tris-Buffered Saline, 0.1% Tween 20 detergent (TBST) three times (10 min/time), then incubate with the corresponding secondary goat anti-rabbit IgG antibody (1: 10000) at RT for 2 h.</li>
	<li>Wash the membrane with TBST again (three times for 10 min each), and treat it with an appropriate amount of enhanced chemiluminescence (ECL) working solution (per the manufacturer&#39;s instructions) for protein band detection.</li>
	<li>Capture the images using the chemiluminescence imaging system and perform a semi-quantitative analysis of the gray values of proteins using the Image J image analysis system.</li>
</ol>
11.&#160;Statistical analysis
<ol>
	<li>Use an appropriate software application for analyzing the data. Express all data as the means &#177; SD (standard deviation) and evaluate multiple samples by a one-way ANOVA test for comparison between groups. 
	NOTE: SPSS statistical software 26.0 was used for statistical analysis. The LSD method was used for two-way comparisons between groups when the variances were equal, and the Dunnett T3 method was used for two-way comparisons between groups when the variances were not equal. P&#60;0.05 was considered to indicate a statistically significant difference.</li>
</ol>

Treating Immunoglobulin A Nephropathy (IgAN) with Dioscin in a Murine Model

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R., You, Y., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+pharmacology,+molecular+docking,+and+in+vitro+experiments+to+explore+the+molecular+mechanism+of+Dioscorea+Nipponica+Makion+in+the+treatment+of+IgA+nephropathy.">Network pharmacology, molecular docking, and in vitro experiments to explore the molecular mechanism of Dioscorea Nipponica Makion in the treatment of IgA nephropathy.</a> World Journal of Integrated Traditional and Western Medicine. 16 (12), 2246-2254 (2021).</li><li>Mestecky, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA+nephropathy:+molecular+mechanisms+of+the+disease.">IgA nephropathy: molecular mechanisms of the disease.</a> Annual Review of Pathology. 8, 217-240 (2013).</li><li>Novak, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgA1-containing+immune+complexes+in+IgA+nephropathy+differentially+affect+proliferation+of+mesangial+cells.">IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells.</a> Kidney International. 67 (2), 504-513 (2005).</li><li>Nihei, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+IgA+autoantibodies+targeting+mesangial+cells+redefines+the+pathogenesis+of+IgA+nephropathy.">Identification of IgA autoantibodies targeting mesangial cells redefines the pathogenesis of IgA nephropathy.</a> Science Advances. 9 (12), (2023).</li><li>Raska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+CMP-NeuAc:+GalNAc-IgA1+alpha2,6-sialyltransferase+in+IgA1-producing+cells.">Identification and characterization of CMP-NeuAc: GalNAc-IgA1 alpha2,6-sialyltransferase in IgA1-producing cells.</a> Journal of Molecular Biology. 369 (1), 69-78 (2007).</li></ol>

The authors declare that they have no competing financial interests.

The characteristic pathological feature of IgAN is the deposition of IgA1 and GD-IgA1-containing immune complexes in the mesangial region of the glomerulus21,22. Reducing the formation of immune complexes can reduce renal injury and alleviate the clinical symptoms of IgAN. In an in vivo experiment, we studied the therapeutic effects of DIO on IgAN, and we found that DIO can reduce IgA deposition in the kidney of IgAN model mice. It is demonstrated that the accumulation of IgA-secreting cells in the kidney is related to the pathogenesis of IgAN23. As an important site of proliferation and activation of B lymphocytes, Peyer's patch is an important source of IgA-secreting cells, so we examined the expression of B lymphocyte markers (CD20, CXCR5) in Peyer's patch and found that DIO could inhibit the expression of B lymphocytes in the Peyer's patch of IgAN mice model. These experimental results could provide a basis for applying DIO in treating IgAN.
We performed the following experiments In vitro to further investigate the action mechanism of DIO on IgAN. Firstly it has been demonstrated earlier that DAKIKI, an EBV-immortalized B cell line that secretes IgA1, part of which is GD-IgA124, is ideal for in vitro research of the drug's mechanism of action on IgAN. We chose DAKIKI cells to investigate the molecular mechanism of DIO in the treatment of IgAN. In addition, the mucosal inflammatory immune response plays an integral role in the pathogenesis of IgAN. As mentioned above, we use LPS to stimulate DAKIKI cells, which can release pro-inflammatory factors and mediate inflammatory responses, which can better mimic the mechanism of mucosal immune responses in IgAN. The in vitro cellular model may help investigate the possibility and mechanism of other drugs for treating IgAN. The results showed that DIO inhibited the proliferation of DAKIKI cells stimulated by LPS in a concentration-dependent manner. DIO could inhibit the secretion of IgA and Gd-IgA1 in DAKIKI cells caused by LPS stimulation and up-regulate the expression of mRNA and protein of C1GalT1 and its chaperone Cosmc in DAKIKI cells, suggesting that DIO could reduce the secretion of Gd-IgA1 by up-regulating C1GALT1/Cosmc expression and thus inhibit the excessive activation of DAKIKI cells. 
Key steps should be noted during the experimental procedures. The concentration of Gd-IgA1 in the DAKIKI cell supernatant is not within the detection range of the ELISA kit (1.56~100 ng/mL), and the collected supernatant must be centrifuged by an ultrafiltration tube to obtain the concentrated Gd-IgA1. Also, ensure the volume of supernatant starting from each group is the same and the final volume of concentrate obtained after ultrafiltration is the same.
In this study, we used both in vitro and in vivo methods simultaneously, which can mutually support each other in pharmacological effects and provide an example for studying the effects and their mechanisms of herbal medicine. Some things could be improved in this protocol. Firstly, We did not detect blood concentrations in the mice's DIO gavage group; therefore, the concentration of DIO equivalent to blood concentrations is not used in vitro experiments. Secondly, only the DIO monomer, the active component of DNR, was investigated; the effects of other components of DNR on IgAN still need further study.
In conclusion, this study provides an experimental basis for treating IgAN with DIO, the active ingredient of DNR. This study established a cellular pathological model of IgAN by mimicking the mucosal immune response of IgAN both in vitro and in vivo. It gives a new idea for studying traditional Chinese medicine to prevent and treat IgAN.

IgA Nephropathy (IgAN) is the most common type of primary glomerulonephritis, for which there is no specific treatment, and it remains a significant cause of end-stage renal disease1. Although the pathogenesis of IgAN is still not fully understood, the &#34;multi-hit hypothesis&#34; is generally accepted and supported by a large body of clinical and experimental research evidence2. The pathogenesis of IgAN involves activating B cells and producing Galactose-deficient IgA1 (Gd-IgA1)3. The increase in circulating Gd-IgA1 due to the excessive proliferation and activation of IgA-secreting cells during the mucosal immune response is a critical link in the pathogenesis of IgAN4,5,6. As the central place for the proliferation and activation of B lymphocyte phenotype conversion to IgA-secreting cells, Peyer&#39;s patch is the primary source of IgA secretion, closely related to the occurrence and development of IgAN7,8. In addition, the proliferation of IgA1-secreting cells, as well as expression of Core 1&#946;-1,3-galactosyltransferase(C1GalT1) and C1GalT1-specific molecular chaperone (Cosmc), were associated with abnormal glycosylation of IgA1, which causes GD-IgA1 production in IgAN patients6,9.
Clinical study on IgAN treatment with herbal medicine has progressed in recent years. Yiqi Qingjie Formula is an essential formula for treating IgAN by the Department of Nephrology of Guang&#39;anmen Hospital. The previous study of our group found that Gd-IgA1 decreased in the serum of IgAN patients after treatment with Yiqi Qingjie Formula. As one of the most used herbs in the Yiqi Qingjie Formula, Dioscoreae Nipponicae Rhizoma (DNR) is the dried rhizome of Dioscorea Nipponica Makino, which has various functions such as regulating immunity, suppressing inflammation, relieving cough and asthma10,11. Several scholars treated IgAN with DNR and achieved good results12,13,14. As the main active ingredient in DNR15, Dioscin (DIO) lowers uric acid, inhibits fibrosis, inhibits inflammatory response, and anti-oxidative stress16,17. Therefore, DIO may have a novel action mechanism to inhibit the cellular secretion of excessive Gd-IgA1 and exert specific kidney protection effects. Still, no study has been reported on DIO&#39;s action mechanism for treating IgAN.
To explore the potential therapeutic mechanism of DIO on IgAN and provide a new method for the treatment of IgAN, we carried out experiments for the therapeutic effects of DIO on IgAN in vivo and in vitro.

<table><tbody><tr><td>Anti-CD20/MS4A1 Antibody</td><td>Boster Biotechnology Company</td><td>A03780-3</td><td></td></tr><tr><td>Antifade mounting medium</td><td>Beyotime, Shanghai, China</td><td>P0128S</td><td></td></tr><tr><td>Balb/c mice</td><td>Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.</td><td>110322220101424000</td><td></td></tr><tr><td>blocking serum</td><td>Solarbio, Beijing, China</td><td>SL038</td><td></td></tr><tr><td>Bovine gamma globulin</td><td>ShangHai YuanYe Biotechnology Company</td><td>S12031</td><td></td></tr><tr><td>C1GALT1 polyclonal antibody</td><td>Proteintech Group, Inc,USA</td><td>27569-1-AP</td><td></td></tr><tr><td>Citrate antigen retrieval solution(50&amp;times;)</td><td>Phygene Biotechnology Company</td><td>PH0422</td><td></td></tr><tr><td>COSMC polyclonal antibody</td><td>Proteintech Group, Inc,USA</td><td>19254-1-AP</td><td></td></tr><tr><td>Cytotoxiciy detection kit</td><td>Roche Company</td><td>4744926001</td><td></td></tr><tr><td>Dako REAL EnVision detection system, Peroxidase/DAB+</td><td>Dako</td><td>K5007</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Dioscin</td><td>National Institute For Food and Drug Control</td><td>111707-201703</td><td></td></tr><tr><td>DIO tablets</td><td>Chengdu No 1 Pharmaceutical Co. Ltd.</td><td>H51023866</td><td></td></tr><tr><td>ECL working solution</td><td>Merck Biotechnology, Inc</td><td>WBKLS0100&amp;nbsp;</td><td></td></tr><tr><td>Enhanced cell counting kit-8</td><td>Beyotime, Shanghai, China</td><td>C0043</td><td></td></tr><tr><td>Fasking one-step removal of gene cDNA first-strand synthesis premix</td><td>TIANGEN,Beijing, China</td><td>KR118-02</td><td></td></tr><tr><td>Glycogen Periodic acid Schiff (PAS) stain kit&amp;nbsp;</td><td>BaSO Biotechnology Company</td><td>BA4080A</td><td></td></tr><tr><td>Goat anti-mouse IgA-AF488</td><td>SouthernBiotech</td><td>1040-30</td><td></td></tr><tr><td>Goat anti-rabbit IgG antibody (H+L), HRP conjugated</td><td>BeiJing Bioss Biotechnology Company</td><td>BS-0295G-HRP</td><td></td></tr><tr><td>Human Gd-IgA1 ELISA kit</td><td>IBL</td><td>27600</td><td></td></tr><tr><td>Human IgA ELISA kit</td><td>MultiSciences (LiankeBio)</td><td>70-EK174-96</td><td></td></tr><tr><td>Pierce BCA protein assay kit</td><td>Thermo Scientific</td><td>23227</td><td></td></tr><tr><td>PMSF solution</td><td>Beyotime, Shanghai, China</td><td>ST507</td><td></td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>Phygene Biotechnology Company</td><td>PH1521</td><td></td></tr><tr><td>Rabbit anti-CXCR5 polyclonal antibody&amp;nbsp;</td><td>BeiJing Bioss Biotechnology Company</td><td>bs-23570R</td><td></td></tr><tr><td>RIPA lysis buffer</td><td>Beyotime, Shanghai, China</td><td>P0013B</td><td></td></tr><tr><td>RNAsimple total RNA extraction kit</td><td>TIANGEN,Beijing, China</td><td>DP419</td><td></td></tr><tr><td>RPMI Medium 1640</td><td>Solarbio, Beijing, China</td><td>31800</td><td></td></tr><tr><td>Super-Bradford protein assay kit</td><td>CWBIO, Beijing, China</td><td>CW0013</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Beyotime, Shanghai, China</td><td>ST795</td><td></td></tr><tr><td>&amp;beta;-Actin Rabbit mAb</td><td>Abclonal, Wuhan, China</td><td>AC026</td><td></td></tr></tbody></table>

dioscin mediated iga nephropathy alleviation by inhibiting b cell activation in vivo and decreasing galactose-deficient iga1 production in vitro

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal immune responses, which is a critical link in the pathogenesis of IgA nephropathy (IgAN). Peyer&#39;s patch, the prominent place where B lymphocytes are transformed into IgA-secreting plasma cells, is the primary source of IgA. In addition, the lower expression of core 1&#946;-1,3-galactosyltransferase (C1GalT1) and its molecular chaperone, C1GalT1-specific molecular chaperone (Cosmc), is related to abnormal glycosylation of IgA1 in IgAN patients. Our clinical experience shows that Dioscoreae Nipponicae Rhizoma&#39;s (DNR) herbal medicine can relieve proteinuria and hematuria and improve renal function in IgAN patients. Dioscin (DIO) is one of the main active ingredients of DNR, which has various pharmacological activities. This study explores DIO&#39;s possible mechanism in treating IgAN.The IgAN model mouse was established by mucosal immune induction. The mice were divided into the control, model, and DIO gavage groups. The glomerular IgA deposition in mice, renal pathological changes, and B cell markers CD20 and CXCR5 expression in Peyer&#39;s patch were detected by immunofluorescence and immunohistochemistry. After lipopolysaccharide (LPS) stimulation, DIO&#39;s effects on DAKIKI cells proliferation, IgA and Gd-IgA1 secretion, C1GalT1, and Cosmc expression were studied by cell counting kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA) test, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting (WB). In in vivo studies, IgA deposition accompanied by glomerular mesangial hyperplasia and increased expression of CD20 and CXCR5 in Peyer&#39;s patch in the IgAN model mouse was alleviated by DIO. In vitro studies showed 0.25 &#181;g/mL to 1.0 &#181;g/mL DIO inhibited LPS-induced DAKIKI cell proliferation, IgA and Gd-IgA1 secretion, and up-regulated the mRNA and protein expression of C1GalT1 and Cosmc. This study demonstrates that DIO may reduce Gd-IgA1 production by inhibiting excessive activation of IgA-secreting cells and up-regulating C1GALT1/Cosmc expression.

Effect of DIO on kidney tissue in IgAN mice model
Compared with the control group, the mucosal immune-induced IgAN mice model (model group) had a significant increase in proteinuria (Supplementary Figure 2), IgA deposition was visible in the mesangial region, fluorescence was uniformly distributed in clusters throughout the entire mesangial region (Figure 1A), PAS staining of the renal tissue showed mesangial cells proliferation and stromal hyperplasia (Figure 1B), which was reduced in the DIO gavage group (DIO group).
Effect of DIO on B lymphocytes in Peyer&#39;s patch
Peyer&#39;s patch is the leading site of the conversion of B lymphocytes into IgA-secreting cells. We took Peyer&#39;s patch as the research object to observe the effect of DIO on B lymphocytes by detecting the expression of B cell markers CD20 and CXCR5. Immunohistochemical results showed that the expression of CD20 and CXCR5 was significantly higher in the model group compared with the control group. DIO could inhibit the expression of the above molecular markers (Figure 2A,B).
The safe concentration range of DIO on DAKIKI cells
LDH is a marker of plasma membrane integrity and an indicator of cell death, with higher LDH release rates indicating more severe cell damage. The LDH release assay was used to determine the safe concentration range of DIO. The maximum safe concentration of DIO was determined by an LDH release rate below 10%. The results (Figure 3) showed no significant cytotoxicity induced by DIO at concentrations of 0.25 to 1.0 &#181;g/mL. Therefore, the following study used 0.25, 0.5, and 1.0 &#181;g/mL DIO as the dosing level.
Effects of DIO on DAKIKI cell proliferation
The experimental results (Figure 4) showed that compared with the model group (LPS-stimulated group), DIO inhibited LPS-induced DAKIKI cell proliferation in a concentration-dependent manner. DIO at 0.5 and 1.0 &#181;g/mL concentrations significantly inhibited LPS-induced DAKIKI cell proliferation (P &#60; 0.01).
Effects of DIO on the secretory function of DAKIKI cells
Gd-IgA1 levels are closely related to the pathological process of IgAN, and total IgA is tested together as an indicator of cellular secretory function. An ELISA assay was used to detect IgA and Gd-IgA1 content in the supernatant of the DAKIKI cell culture. The results showed (Figure 5A,B) that DAKIKI cells stimulated by LPS secreted more IgA compared with the control group (P &#60; 0.01). In comparison, DIO significantly inhibited DAKIKI cells from secreting IgA (P &#60; 0.01) in a concentration-dependent manner. Compared with the control group, DAKIKI cells stimulated by LPS secreted more Gd-IgA1 with a statistical tendency (P &#60; 0.10), and DIO inhibited Gd-IgA1 secretion from LPS-stimulated DAKIKI cells in a concentration-dependent manner (P &#60; 0.05 and P &#60; 0.01), among which DIO at 1.0 &#181;g/mL significantly inhibited the secretion of Gd-IgA1 with the inhibited rate of 25%.
The mechanism of DIO inhibits Gd-IgA1 secretion by DAKIKI cells
To further investigate the possible mechanism of DIO inhibiting excessive Gd-IgA1 secretion by DAKIKI cells, the levels of glycosylated transferase C1GALT1 and chaperone protein Cosmc mRNA in DAKIKI cells were detected by qRT-PCR, and the results showed (Figure 6A,B) that the relative mRNA expression of C1GALT1 and Cosmc was down-regulated in DAKIKI cells in the model group compared with the control group (P &#60; 0.01). DIO up-regulated the relative mRNA expression of C1GALT1 and Cosmc to different degrees compared with the model group, with DIO 1.0 &#181;g /mL significantly up-regulated the relative mRNA expression of C1GALT1 and Cosmc (P &#60; 0.05).
At the same time, the WB method was used to detect the effect of DIO on the protein expression of C1GALT1 and Cosmc in DAKIKI cells. Compared with the control group, the protein expression of C1GALT1 and Cosmc in DAKIKI cells in the model group decreased obviously (P &#60; 0.05). Compared with the model group, the protein expression of C1GALT1 and Cosmc after DIO intervention was up-regulated. The protein expression of C1GALT1 and Cosmc was significantly up-regulated by DIO at a concentration of 1.0 &#181;g/mL (P &#60; 0.05) (Figure 7A-C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65719/65719fig1.jpg" /> 
Figure 1: Histopathology of the kidneys. (A) Immunofluorescence microscope. Kidney sections of mice in each group were stained with anti-IgA (green) and DAPI (blue). The above picture scale bar = 200 &#956;m. The below picture scale bar = 50 &#956;m. n = 6 per group. (B) Representative pictures of PAS staining of kidney tissue from mice in the Control, Model, and DIO groups. Scale bar = 30 &#956;m. The downward arrow shows the mesangial cells and the upward arrow shows the stroma. Scale bar = 30 &#956;m. n = 6 per group. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65719/65719fig2.jpg" /> 
Figure 2: Effect of DIO on B lymphocyte markers. (A) The expression of CD20 in the Peyer&#39;s patch. Scale bar = 200 &#956;m. n = 6 per group. (B) The expression of CXCR5 in the Peyer&#39;s patch. The scale bars are in the lower right corner of the image. Scale bar = 200 &#956;m. n = 6 per group. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65719/65719fig3.jpg" /> 
Figure 3. Screening of the safe concentration of DIO on DAKIKI cells. Statistical values are expressed as the mean &#177; SD from three independent experiments. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65719/65719fig4.jpg" /> 
Figure 4. Different concentrations of DIO affect the proliferation of DAKIKI cells. The data were expressed as mean &#177;SD. Compared with the control group, **P &#60; 0.01; compared with the model group, #P &#60; 0.05&#8218;&#160;##P &#60; 0.01; The results of all experiments were repeated three times. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65719/65719fig5.jpg" /> 
Figure 5. DIO inhibits IgA and Gd-IgA1 secretion by DAKIKI cells. (A) ELISA method detected the expression of IgA in each group. (B) ELISA method detected the expression of Gd-IgA1 in each group. The data were expressed as mean &#177; SD. Compared with the control group, **P &#60; 0.01; compared with the model group, #P &#60; 0.05, ##P &#60; 0.01; all experimental results were repeated three times. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65719/65719fig6.jpg" /> 
Figure 6. The mechanism of DIO inhibits excessive Gd-IgA1 secretion by DAKIKI cells. (A) QRT-PCR detected the mRNA expression of C1GALT1. (B) QRT-PCR detected the mRNA expression of Cosmc. The data were expressed as mean &#177; SD. Compared with the control group, **P&#60;0.01; compared with the model group, #P &#60; 0.05, ##P &#60; 0.01; all experimental results were repeated three times. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65719/65719fig7.jpg" /> 
Figure 7. DIO affects protein expression of C1GALT1 and Cosmc in DAKIKI cells. (A) WB verified the up-regulation of protein expression of C1GALT1 and Cosmc by DIO. (B) Semi-quantitative analysis of C1GALT1 expression was performed using Image J. (C) Semi-quantitative analysis of Cosmc expression using Image J. The data were expressed as mean &#177;SD. Compared with the control group,*P &#60; 0.05; compared with the model group, #P &#60; 0.05,&#160;##P &#60; 0.01, all experimental results were repeated three times. <a href="https://www.jove.com/files/ftp_upload/65719/65719fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. The schema for the in vivo model. <a href="https://www.jove.com/files/ftp_upload/65719/Supplementary_Figure_1.pdf" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2. Changes in proteinuria. The data were expressed as mean &#177; SD; n = 6 per group. <a href="https://www.jove.com/files/ftp_upload/65719/Supplementary_Figure_2.jpg" target="_blank">Please click here to download this figure.</a>

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Author Spotlight: Investigating the Potential of Chinese Herbal Medicinal Active Dioscin in Treating IgA Nephropathy

This study provides experimental data for treating immunoglobulin A nephropathy (IgAN) with Dioscin (DIO), the active ingredient of Dioscoreae Nipponicae Rhizoma (DNR), and a paradigm for studying herbal medicine's effects and underlying mechanisms in vivo and in vitro.

Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

The increase of circulating galactose-deficient IgA1 (Gd-IgA1) is caused by excessive activation of IgA-positive secretory cells in the process of mucosal ...

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1.7K Views.  Guanganmen Hospital. Ons belangrijkste onderzoeksgebied is hoe Chinese kruidengeneeskunde werkt om chronische nierziekten zoals IGA-nefropathie te behandelen. In deze studie, gebaseerd op onze klinische ervaring en behandeling van IGA-nefropathie, met behulp van Dioscorea Nipponica Rhizoma, afkorting DNR, en ons onderzoeksresultaat van de belangrijkste werkzame stof DNR, proberen we de potentiële waarde van dioscine als een van de belangrijkste werkzame stoffen van DNR te bevest...

Video: Auteur in de schijnwerpers: onderzoek naar het potentieel van Chinese kruidenmedicinale actieve dioscine bij de behandeling van IgA-nefropathie

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Immunology and Infection

Effects of Taste Signaling Protein Abolishment on Gut Inflammation in an Inflammatory Bowel Disease Mouse Model

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects the life quality of millions of people worldwide. IBD symptoms include abdominal pain, diarrhea, and rectal bleeding, which may result from the interactions among gut microbiota, food components, intestinal epithelial cells, and immune cells. It is of particular importance to assess how each key gene expressed in intestinal epithelial and immune cells affects inflammation in the colon. G protein-coupled taste receptors, including G protein subunit &#945;-gustducin and other signaling proteins, have been found in the intestines. Here, we use &#945;-gustducin as a representative and describe a dextran sulfate sodium (DSS)-induced IBD model to evaluate the effect of gustatory gene mutations on gut mucosal immunity and inflammation. This method combines gene knockout technology with the chemically induced IBD model, and thus can be applied to assess the outcome of gustatory gene nullification as well as other genes that may exuberate or dampen the DSS-induced immune response in the colon. Mutant mice are administered with DSS for a certain period during which their body weight, stool, and rectal bleeding are monitored and recorded. At different timepoints during administration, some mice are euthanized, then the sizes and weights of their spleens and colons are measured and gut tissues are collected and processed for histological and gene expression analyses. The data show that the &#945;-gustducin knockout results in excessive weight loss, diarrhea, intestinal bleeding, tissue damage, and inflammation vs. wild-type mice. Since the severity of induced inflammation is affected by mouse strains, housing environment, and diet, optimization of DSS concentration and administration duration in a pilot experiment is particularly important. By adjusting these factors, this method can be applied to assess both anti- and pro-inflammatory effects.

Here we present a protocol to investigate the effect of the nullification of gustation-related genes on immune responses in a dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) mouse model.

Inflammatory bowel disease (IBD) is one of the immune-related gastrointestinal disorders, including ulcerative colitis and Crohn's disease, that affects ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn's disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

A Cell-Based Therapy for Autoimmune Arthritis in a Murine Model

Source: Haque, M. et al., <a href="https://app.jove.com/v/54720">Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity.</a>&#160;J. Vis. Exp. (2016)
This video showcases a cell-based therapy for autoimmune arthritis in mice. iPSC-derived T regulatory cells expressing ovalbumin-specific receptors are injected with methylated bovine serum albumin (mBSA) emulsified in an adjuvant. Murine antigen-presenting cells (APCs) process and present the mBSA to T cells, prompting T cell activation and migration to the knee joint&#39;s synovial membrane. Subsequent injections involve mBSA alone in the left knee and a combination of mBSA and ovalbumin in the right knee. In the left knee, the immune response to mBSA triggers arthritis, while in the right knee, T-regulatory cells interacting with ovalbumin mitigate inflammation, improving arthritis symptoms.

Dioscin Mediated IgA Nephropathy Alleviation by Inhibiting B Cell Activation In Vivo and Decreasing Galactose-Deficient IgA1 Production In Vitro

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