Small GTPase Rab7 shows altered expression during murine and human colitis correlative of disease severity

(A) Rab7 expression analyzed in whole colon tissue of healthy and DSS-treated mice. Graph represents densitometric analysis showing fold intensity of Rab7 expression calculated by normalizing to loading control (β actin). (B) Dynamics of Rab7 expression in intestinal epithelial cells isolated from the intestines of healthy and DSS-treated mice for different time durations. Corresponding graph shows densitometry analysis of Rab7 expression normalized to loading control (β actin). (C) RT-PCR analysis of relative fold expression of Rab7 gene in human UC patient colonic biopsies (n=22) relative to average control values (n=22). HPRT was used for normalization. (D) Immunoblotting of Rab7 protein in human UC (n=5) and control (n=3) biopsy samples. GAPDH was used as loading control. Graph shows densitometry analysis of Rab7 expression relative to control. (E) RT-PCR analysis of Rab7 gene expression variation with disease severity. (F) Immunoblot showing Rab7 protein expression dynamics of mild (n=3) and moderate (n=3) UC patient samples relative to controls (n=3). Graph represents densitometric analysis showing fold intensity of Rab7 expression calculated by normalizing to loading control (GAPDH). (G) Immunohistochemistry images of colon biopsies from control (n=3), UC mild (n=3) and UC moderate (n=3) patients stained for Rab7 (brown color) (Scale bar=100µm). Inset shows zoomed areas of the image (Scale bar=50µm). Each dot represents (A, B) one mouse or (C, D and F) one human. Error bars represent mean+SEM. Statistical analysis by Student’s t test. ns=non-significant. See also Fig S1 and Table S1.

Goblet cells of intestine display dominant expression of Rab7

(A-B) Confocal imaging of healthy and DSS-treated mice (7 days) colon sections showing Rab7 (red) expression within goblet cells (UEA1-FITC) marked with asterisk. Inset shows zoomed areas of the image (scale bar=20µm). Graph shows Rab7 fluorescence intensity within UEA1 positive goblet cells measured within region of interest (ROI). 20 goblet cells were selected randomly from 3 different fields of each mouse (n=3). (C-D) Representative co-immunofluorescence images of human UC and control colon biopsy sections stained for Rab7 (red) and Muc2 (green) for goblet cells (marked with asterisk). (Scale bar=50µm). Inset shows zoomed areas of the image (Scale bar=20µm). Graph shows Rab7 fluorescence intensity within Muc2 positive goblet cells measured within region of interest (ROI). 20 goblet cells were selected randomly from 5 different fields of each sample (n=3). (E) Co-immunofluorescence of Rab7 (green) and Muc2 (red) in undifferentiated and differentiated HT-29 cells (Scale bar=50µm). Graphs show Rab7 and Muc2 fluorescence intensity measured in 10 different fields of three independent experiments each. (F) Rab7 protein visualized in HT29-MTX-E12 cells by immune-EM. Arrows indicate presence of Rab7 on vacuoles of cells (Scale bar=1µm). Inset showed zoomed area of image (Scale bar=200nm). Error bars represent mean+SEM. Statistical analysis by Student’s t test. See also Fig S2.

Downregulation of Rab7 aggravates inflammation upon external trigger

(A) Schematic representation of the experimental plan for knock-down of Rab7 in C57Bl/6 mice showing different treatments (n=3 mice per group). (B) Graph showing body weight percent. (C) Representative photographs demonstrating rectal bleeding in mice. (D) Gross morphology of colon and caeca. Graph shows colon length quantification. (E) Representative spleens of different treatment mice groups. Graph showing spleen to body weight ratio. (F) Rab7 protein expression in isolated intestinal epithelial cells from mice colon. Graph represents densitometric analysis showing fold intensity of Rab7 expression calculated by normalizing to loading control (β actin). Significance value of each group is relative to untreated (C) group. (G-H) Hematoxylin and eosin staining of distal colon sections with histopathology scores showing increased characteristics of inflammation. (I) ELISA of TNFα from mucosal extracts of mice colon. Each dot represents one mouse. Error bars represent mean+SEM. Statistical analysis by (B) Two-way Anova or Student’s t test. ns=non-significant. See also Fig S3.

Rab7 downregulation in intestinal epithelium modulates mucus layer thickness and permeability

(A-B) Alcian blue staining in mice distal colon sections displaying IML (blue) marked with dotted lines and nuclei (red). Thickness of IML was measured using Image J software (15 measurements per section of each mice). Scale bar=100µm and 20µm (inset). (C-D) Representative images of FISH staining for bacteria detection in mucus layer using general bacterial probe EUB338-Alexa Fluor 647 (red). Arrow heads demarcate presence of bacteria in IML (dotted lines). Graph shows bacteria count detected in IML (6 regions per section of each mouse). Scale bar=100µm and 20µm (inset). Error bars represent mean+SEM. Statistical analysis by Student’s t test.

Gut microbiota in Rab7 knockdown mice is altered alike DSS-colitis mice

Microbial composition of CScr (Scr siRNA), Rab7KD (Rab7 siRNA) and DSS+CScr (DSS+Scr siRNA) was analyzed by 16S metagenomic profiling. (A) Alpha diversity quantified as Shannon index. Significance was calculated using the Kruskal-Wallis test followed by the improved Benjamini-Hochberg procedure from false discovery rate (FDR) correction. (B) Principle Coordinates (PCoA) plot calculated from distance matrices obtained from Bray-Curtis. (C) Venn diagram representation of shared and unique OTUs between groups. (D-F) Mean relative abundance of each taxon. Relative abundance of top 10 Phylum (D) and Class (F) of each group is depicted in the figure. Each bar represents the mean of the merged OTUs from three mice. Taxonomic lineages not included in top 10 were collapsed as “Others”, while the ones which have not been classified has been placed under the category of “unclassified”. Relative abundance (in %) of some important taxa showing significant differences between experimental groups (E). (G) Plot showing relative abundance of 8 different taxa, identified with Kruskal-wallis test. Each dot represents one mouse. See also Fig S4.

Rab7 perturbation alters mucus composition of colon

(A) Schematic representing steps for mucus isolation from mice colon followed by sample preparation for total mucin measurement through mucin gels and mucus layer composition analysis using mass spectrometry (Created with BioRender.com). (B) Mucin gel showing the amount of total mucins (blue) stained with alcian blue dye.(C-E) Coomassie staining of mucus samples isolated from different experimental groups. Table listing the total number of proteins identified in the mucus samples through mass spectrometry and venn diagram displaying the number of individual proteins common and unique in different samples. (F-G) Heat maps of the proteins differentially expressed without FDR (F) and with FDR (G). (H-I) Volcano plots of proteome in mucus samples of different mice groups showing differential expression of proteins in Rab7KD mice verses CScr (H) and DSS+ Rab7KD mice verses DSS control group (I). See also Fig S5.

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Rab7 mediates CLCA1 degradation via lysosomal pathway in goblet cells

(A) CLCA1 expression in mucus samples of Rab7KD-DSS mice. Graph represents densitometric analysis showing fold intensity of CLCA1 expression. (B) RT-PCR analysis of relative fold expression of CLCA1 gene in human UC (n=22) patient colonic biopsies relative to average control (n=22) values. HPRT was used for normalization. (C) Immunoblotting of CLCA1 protein in human UC (n=5) and control (n=3) mucus and biopsy samples. Graphs represent densitometric analysis showing fold intensity of CLCA1 expression in mucus and tissue samples calculated by normalizing to loading control (GAPDH shown in Fig1F). (D) Correlation graph showing expression of Rab7 and CLCA1 in mucus and tissue samples of human UC patients relative of controls plotted using fold change from immunoblots. (E) Representative immunohistochemistry images of Rab7 and CLCA1 staining (brown color) in human UC patient (n=3) and control biopsy (n=3) sections and cell nuclei (blue color) (Scale bar=100µm). Inset shows zoomed areas of the image (Scale bar=50µm). (F) CLCA1 protein expression in HT29-MTX-E12 cells transfected with either scrambled siRNA (control) or Rab7 siRNA (Rab7Knockdown). Graph represents densitometric analysis showing fold intensity of CLCA1 expression calculated by normalizing to loading control (β actin). (G) Immunoblot showing CLCA1 protein expression change in HT29-MTX-E12 cells overexpressed with EGFP empty vector (control), Rab7-GFP (Rab7WT), Rab7-GFP GTP locked form (Rab7GTP) and Rab7-GFP GDP locked form (Rab7GDP). Graph represents densitometric analysis showing fold intensity of CLCA1 expression calculated by normalizing to loading control (β actin). (H) Immunoblot showing CLCA1 protein expression after treatment of bafilomycin in HT29-MTX-E12 cells. Graph represents densitometric analysis showing fold intensity of CLCA1 expression calculated by normalizing to loading control (β actin). (I) Representative image of structured illumination microscopy showing images of HT29-MTX-E12 cells transfected with Rab7GTP (green) and stained with CLCA1 (Red) using anti-CLCA1 antibody and lysosomes with LysoTracker Red DND-99 (Magenta) (Scale bar=5µm). Inset shows zoomed areas of colocalization marked with arrows. (J-K) Representative confocal images of HT29-MTX-E12 cells transfected with either scrambled siRNA (control) or Rab7 siRNA (Rab7Knockdown). Cells are stained with CLCA1 (green) using anti-CLCA1 antibody and lysosomes with LysoTracker Red DND-99 (red). Graph shows quantitation of colocalization between CLCA1 and lysosomes from images (n=120) using Mander’s overlap coefficient. (Scale bar=100µm). Inset shows zoomed areas of the image with colocalization puncta (yellow) marked with arrows (Scale bar=50µm). Each dot represents (A) one mouse or (B, C and E) one human or (F, G and H) one independent experiment. Error bars represent mean+SEM. Statistical analysis by (C) Welch’s t test or (A, B, F, G, H and K) Student’s t test. ns=non-significant. See also Fig S6.

Rab7 maintains mucus layer dynamics in intestine by regulating degradation of CLCA1 protein via lysosomal fusion

Healthy intestine inhabits lumen microbes well separated by mucus layer secreted by goblet cells along with CLCA1 protein in balanced levels (1). During colitis, Rab7 downregulates along with increased expression of CLCA1 resulting in diffused mucus layer penetrable to microbes (2). In a goblet cell, CLCA1 filled vacuoles destined for secretion are rerouted for degradation pathway by Rab7 and fuse with lysosomes (3) leading to a balanced release outside the cell (4). However, during inflammation the loss of Rab7 consequently impedes CLCA1 degradation (5) fostering its increased secretion from the cell (6) (Created with BioRender.com).

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Rab7 expression in various states of murine and human colitis (related to Fig 1)

(A) Immunohistochemistry images of Rab7 staining (brown color) in distal colon sections of healthy and DSS-treated mice (Scale bar=100µm). Inset shows zoomed areas of the image (Scale bar=50µm).

(B) Schematic representation of DSS treatment to mice for different time durations.

(C) Graph represents body weight percent of mice in different groups.

(D) Gross morphology of colon and caeca. Graph shows colon length quantification.

(E) Spleen to body weight ratio indicating increased splenomegaly and inflammation.

(F) Dynamics of Rab7 expression in whole tissue and crypts isolated from the intestines of healthy and DSS-treated mice for different time durations. Corresponding graph shows densitometry analysis of Rab7 expression normalized to loading control (β actin).

(G) A pilot study of Rab7 expression in CD patients. RT-PCR analysis of relative fold expression of Rab7 gene in human CD patient colonic biopsies (n=9) relative to average control values (n=10). HPRT was used for normalization.

(H) Immunoblotting of Rab7 protein in CD (n=3) and control (n=3) biopsy samples. GAPDH was used as loading control.

(I) Immunoblotting of Rab7 protein in human UC remission (n=3) and control (n=3) biopsy samples. GAPDH was used as loading control.

Each dot represents (D, E) one mouse or (F, G and H) one human. Error bars represent mean+SEM. Statistical analysis by Student’s t test. ns=non-significant.

Expression of Rab7 in goblet cells of different types and regions of gastrointestinal tract (related to Fig 2)

(A) Immunohistochemistry images of staining of Rab7 in different regions of intestine: small intestine, proximal colon and distal colon; showing Rab7 expression in vacuolated cells (marked with asterisk) (n=3). Scale bar=50µm.

(B) Confocal images of mice distal colon stained with Rab7 (red) and goblet cell specific marker UEA1 (green) showing presence of Rab7 in UEA1 positive cells (marked with asterisk) (n=3). Scale bar=100µm.

(C) RT-PCR analysis of relative fold expression of Lgr5, Muc2 and Rab7 genes in differentiated HT29 cells relative to undifferentiated cells. HPRT was used for normalization.

(D) Immunoblot showing Rab7 protein expression in differentiated HT29 cells relative to undifferentiated cells. Graph represents densitometric analysis of Rab7 expression calculated by normalizing to loading control (β actin).

(E) TEM analysis of HT29-MTX-E12 cells showing presence of vacuoles (V) in the cytoplasm (Cy) along with nucleus (Nu) nucleolus (Ns) and mitochondrion (M). Each dot represents one individual experiment. Error bars represent mean+SEM. Statistical analysis by Student’s t test. ns=non-significant.

Rab7 perturbation changes goblet cell number and Muc2 distribution (related to Fig 3 and Fig 4)

(A) Rab7 protein expression in lysates of spleen, MLN and liver collected from CScr and Rab7KD mice. Graph represents densitometric analysis showing fold intensity of Rab7 expression calculated by normalizing to loading control (β actin).

(B-C) Alcian blue staining in sections of distal colon show mucus filled goblet cells (blue) and nuclei (red). Number of goblet cells per crypt were counted manually (15-20 crypts per mouse).

(D) Representative immunofluorescence images of HEK WT and Rab7-/- cells stained for Muc2 (green), Phalloidin (red) and cell nuclei (blue) (Scale bar=10µm).

Each dot represents one mouse. Error bars represent mean+SEM. Statistical analysis by Student’s t test. ns=non-significant.

Gut microbiota composition analysis of Rab7KD mice using 16 S rRNA sequencing (related to Fig 5)

(A) Non-metric dimensional scaling (NMDS) calculated from distance matrices obtained from Bray-Curtis.

(B) Heat map showing beta diversity index obtained from Bray-Curtis.

(C) Mean relative abundance of top 10 Family, Order, Genus and species.

(D) Heat map representing mean relative abundance of all species across different groups (blue represents low abundance, red represents more abundance).

Mucus proteome analysis of Rab7KD mice (related to Fig 6)

(A) Principle component analysis (PCA) of different experimental groups.

(B-C) Fold change of proteins enriched in Rab7KD mice group compared to CScr mice group based on Gene ontology analysis of biological process (B) and molecular function (C).

(D-E) Table summarizing fold change of differentially expressed proteins in Rab7KD versus CScr mice group

(D) and DSS+ Rab7KD versus DSS+CScr treated mice group (E).

(F-H) Relative fold expression of CLCA1 (F), APOA1 (G) and TFRC (H) in Rab7KD, DSS+CScr and DSS+Rab7KD treated mice relative to CScr mice (n=3). HPRT was used for normalization.

Each dot represents one mouse. Error bars represent mean+SEM. Statistical analysis by Student’s t test. ns=non-significant.

CLCA1 protein degrades via lysosomal degradation pathway (related to Fig 7)

(A) Schematic illustrating mucus isolation steps from human colon biopsy samples. (Created with BioRender.com)

(B) Immunoblot showing CLCA1 protein expression after MG132 treatment. Graph represents densitometric analysis showing fold intensity of CLCA1 expression calculated by normalizing to loading control (β actin).

(C) Representative zoomed in SIM images of HT29-MTX-E12 cells showing Rab7, CLCA1 and Lysosomes in grey scale. The left panel show the merged image.

Each dot represents one individual experiment. Error bars represent mean+SEM. Statistical analysis by Student’s t test. ns=non-significant.

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List of patient clincial parameters

List of primers used in qRT-PCR