Intestinal epithelial cells (IECs) act as a physical barrier separating the commensal-containing intestinal tract from the sterile interior. These cells have found a complex balance allowing them to be prepared for pathogen attacks while still tolerating the presence of bacterial or viral stimuli present in the lumen of the gut. Using primary human IECs, we probed the mechanisms that allow for such a tolerance. We discovered that viral infections emanating from the basolateral side of IECs elicit a stronger intrinsic immune response in comparison to lumenal apical infections. We determined that this asymmetric immune response is driven by the clathrin-sorting adaptor AP-1B, which mediates the polarized sorting of Toll-like receptor 3 (TLR3) towards the basolateral side of IECs. Mice and human IECs lacking AP-1B showed an exacerbated immune response following apical stimulation. Together, these results suggest a model where the cellular polarity program plays an integral role in the ability of IECs to partially tolerate apical commensals while remaining fully responsive to invasive basolateral pathogens.
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This work was supported by a research grant from the Chica and Heinz Schaller Foundation and Deutsche Forschungsgemeinschaft (Project nos 240245660 (Project 14 of SFB 1129) and 278001972 (Project A09 of TRR186) to S.B.). This project received funding from the European Union’s Seventh Framework Programme under grant agreement no. 334336 (FP7-PEOPLE-2012-CIG). M.S. was supported by an Olympia Morata Fellowship from the Heidelberg University Hospital, the Brigitte-Schlieben Lange Program from the state of Baden Württemberg, Germany and the Dual Career Support from CellNetworks. M.M. is supported by the TRR186. K.P. and S.M. were supported by the SFB 1129. J.C.K. was supported by NIH grant no. 5R01AI093589.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
T84 cells were seeded onto transwell inserts. (a). The rate of establishment of barrier function was measured by the trans- epithelial electrical resistance (TEER) using a chop stick electrode. TEER greater than 1000 Ohm/cm2 indicates a tight barrier and is marked with a dashed line. Each point represents the mean +/- sd. n=6. (b). Barrier integrity was assessed by dextran diffusion assay. T84 cells were allowed to grow on transwells and at the indicated time points 4kDa fluorescent FITC-Dextran was added to the apical chamber. Three hours post-incubation, basolateral media was collected and analysed by fluorometry to assess the amount of dextran which has diffused to the basolateral compartment of the transwells. Results represent the mean +/- sd. n=3. n.d.= not determined. (c) pBMCs were isolated and treated with 10-fold serial dilutions of the indicated TLR agonists (starting concentrations: TLR1/2 (PAM3CSK4, 1 μg/mL); TLR3 (1:1 HMW and LMW poly(I:C), 10 μg/mL); TLR4 (LPS-EK, 1 μg/mL); TLR5 (Flagellin-ST, 1 μg/mL); TLR7/8 (R848, 1 μg/mL), TLR9 (ODN 2395 5 μM)). Supernatants were harvested 24 h post-stimulation and were tested for IL-6 by ELISA. Results represent the mean +/- sd. n=3. (d) Polarized T84 cells were fed FITC-labelled poly(I:C) in a side specific manner. Cells were harvested at indicated time points, were washed with a quick acid wash to remove any non-internalized poly(I:C) bound to the cell surface. Following acid wash, cells were lysed and the relative amount of internalized FITC-labelled poly(I:C) was measured by spectrofluorometry. Results represent the mean +/- sd. n=3. P=values were calculated from a two-tailed unpaired t test using GraphPad Prizm.
Extended Data Fig. 2 Apical infection of T84 cells by MRV leads to more de novo virus production compared to basolateral infection.
Polarized T84 cells were infected apically or basolaterally with MRV. (a) Fluorescently labelled MRV was added to polarized T84 cells in a side specific manner. Cells were collected at indicated times points and were washed with a quick acid wash to remove any non-internalized virus particles bound to the cell surface. Following acid wash, cells were lysed, and the relative amount of internalized virus particles was measured by spectrofluorometry. Results show the mean +/- sd. n=3. (b) Infected cells were collected at the indicated time points and viral replication was assayed by q-RT-PCR against the MRV μ2 genome segment. Results are normalized to non-infected controls and show the mean +/- sd. n=3. (c) Same as b except virus replication was addressed by Western blot against the MRV non-structural protein μNS. Actin was used as a loading control (left panel). Representative figure is shown. The relative expression of μNS is normalized to actin is shown (right panel). Results shown the mean +/- sd. n=3. (d) Infected cells were fixed at indicated time points and immunofluorescence was performed against the non-structural protein μNS. The number of infected cells per field of view was counted. 10 fields of view were counted for each time point for each replicate. Results shown the mean +/- sd. n=3. (e) Polarized T84 cells were infected apically or basolaterally with MRV. Infected cells were collected at the indicated time points and viral replication was assayed by q- RT-PCR against the MRV μ2 genome segment. Results are normalized to non-infected controls and show the mean +/- sd. n=3. (a-e) P-values were calculated from a two-tailed unpaired t test using GraphPad Prizm. Source Data
Extended Data Fig. 3 Side specific induction of IFN production upon viral infection in the human intestinal epithelial lines.
(a-b) SKCO15 cells were seeded on transwells. Following establishment of barrier function, cells were infected apically or basolaterally with MRV. (a) 16 hpi, the production of type I (IFNβ) and type III (IFNλ2/3) IFNs was assessed by q-RT-PCR. Results are normalized to mock infected samples and show the mean +/- sd. n=3. (b) Efficiency of the primary MRV infection in SKCO15 cells was controlled by immunofluorescence against the non-structural protein μNS (red). Cell nuclei were stained using DAPI (blue). Three biological replicates were performed. Representative images are shown. Scale bar =100 μm. (c) T84 cells were infected apically or basolaterally with MRV. RNA samples were collected at the indicated times post-infection and production of type I (IFNβ) and type III (IFNλ2/3) interferons was measured by q-RT-PCR. Results are normalized to mock infected samples and show the mean +/- sd. n=3. (d) T84 cells were infected apically or basolaterally with MRV. Supernatants were collected at the indicated times post- infection and analysed by ELISA for the secretion of type III (IFNλ2/3) interferon. Results show the mean +/- sd. n=3. Black line indicates the limit of detection. (a-d) A=apical infection. B =basolateral infection. (a,d) P values were calculated using a two-tailed unpaired t test in GraphPad Prizm.
Extended Data Fig. 4 Stronger immune response observed during basolateral infection of hIECs is not due to differences in transcript stability.
(a) Polarized T84 cells were infected apically or basolaterally with MRV. 16 hpi cells were treated with actinomycin D to block transcription. RNA samples were collected at the indicated times post-actinomycin treatment. (b) Relative amounts of type I (upper panel) and type III IFN (lower panel) transcripts (normalized to the time of actinomycin treatment) were evaluated by q-RT-PCR (left panel). The half-life of each transcripts was determined (right panel). Results indicate the mean +/- sd. n=3. P values were determined from a two-tailed unpaired t test using GraphPad Prizm.
Extended Data Fig. 5 Basolateral infection of T84 cells by MRV leads to the upregulation of many immune related genes.
Polarized T84 cells were infected apically or basolaterally with MRV. 16 hpi, RNA was collected and subjected to microarray analysis. Expression values were quantile-normalized after background correction and log2-transformed. Unspecific filtering was performed by discarding genes with non- significant p-values in any sample. (a) Differentially expressed genes between apical infection and mock infected cells as well as between basolateral infection and mock infected cells were identified using the empirical Bayes approach and are shown in a Venn diagram. (b) Heatmap of expression values of significantly upregulated genes in apically and basolaterally infected cells were scaled across samples and hierarchical clustering was performed using euclidean distance and complete linkage. Gene values and their reference numbers can be found in the Supplementary Dataset. (c) KEGG analysis of significantly upregulated genes upon basolateral infection of T84 cells.
Extended Data Fig. 6 Basolateral infection of human organoids by MRV generates a stronger intrinsic immune response compared to apical infection.
(a) Schematic of the microinjection approach used to infect organoids specifically from either their apical or basolateral sides. (b-c) Human intestinal organoids were infected by MRV apically or basolaterally through microinjection. Virus infection was assessed 16 hpi for (b) the production of the non-structural viral protein μNS using Western blot analysis. Representative western blot is shown. and (c) the production of the MRV μ2 genome segment by q-RT-PCR. Results shown the mean +/- sd. n=3. (d) Generation of intrinsic immune response was assessed by quantifying the production of type I (IFNβ) and type III (IFNλ2/3) IFNs using q-RT-PCR. Results shown the mean +/- sd. n=3. (c-d) P values were calculated with a two-tailed unpaired t test using GraphPad Prizm. (e) The relative expression of TLR2-9 was evaluated in human intestinal organoids. Results are expressed as a ratio to the house keeping gene HPRT1. Results shown the mean +/- sd. n=3. Source Data
Extended Data Fig. 7 Knockdown of the clathrin adaptor AP-1B impacts the TLR3 mediated polarized immune response.
(a) Knock-down efficiency AP-1B was evaluated by q-RT-PCR. Results show the mean +/- sd. n=3. (b-d) The integrity of the barrier function of the AP-1B knock down T84 cells was evaluated by (b) indirect immunofluorescence of the tight junction protein Z-O1 (red). Representative image for immunofluorescence is shown. Scale bar =100 μm. (c) Dextran diffusion assay. Results show the mean +/- sd. n=3. and (d) TEER measurement. Results show the mean +/- sd. n=6. (e) Polarized WT and AP-1B knock-down T84 cells were infected with MRV in a side specific manner. Supernatants were collected at indicated time points and subjected to ELISA to monitor the amount of secreted type III IFNs. Results show the mean +/- sd. n=3. (f) AP-1B knock-down T84 cells were seeded on transwells and treated with indicated TLR agonists: TLR1/2 (PAM3CSK4, 1 μg/mL); TLR3 (1:1 HMW and LMW poly(I:C), 10 μg/mL); TLR4 (LPS-EK, 0.01 μg/mL); TLR5 (Flagellin-ST, 1 μg/mL); TLR7/8 (R848, 1 μg/mL), TLR9 (ODN 2395 5 μM). 6 h post-treatment RNA was harvested and evaluated for the upregulation of IL-6 by q-RT-PCR. Results are normalized to mock-treated samples and show the mean +/- s.d. n=3. (g) Same as f except type III IFN (IFNλ2/3) was evaluated. Results are normalized to mock-treated samples and show the mean +/- s.d. n=3. (e-g) P values were calculated from a two-tailed unpaired t test using GraphPad Prizm.
Extended Data Fig. 8 Inhibition of TRIF interferes with the polarized immune response generated by T84 cells.
(a-b) WT and AP-1B knock-down T84 cells were seeded on transwell inserts. (a) WT cells were treated with a TRIF inhibitor or a control peptide. T84 cells were infected with MRV in a side specific manner. RNA samples were harvested at indicated time points post-MRV infection and the intrinsic innate immune induction was analysed by q-RT-PCR for type III IFN (IFNλ2/3). Results show the mean +/- sd. n=4. (b) Same as a, except AP-1B knock-down cells were evaluated. Results show the mean +/- sd. n=4. (a-b) P values were calculated from a two-tailed unpaired t test using GraphPad Prizm. ns=not significant, * P < 0.05. ** P < 0.01. Statistics in panel b are between AP-1B and AP-1B+TRIFi. Exact p-values can be found in Supplementary Table 2.
(a) TLR3 antibody specificity was controlled by transfecting HEK 293T (naturally non-TLR3 expressing) cells with a plasmid expressing TLR3. 48 h post-transfection cells were fixed and stained for TLR3 expression (red). Nuclei were stained with DAPI (blue). Representative images are shown. (b) T84 cells were seeded as spheroids. Five days post-seeding spheroids were fixed and stained for Laminin (red), and either TLR3 (green) or AP-1 (green). Nuclei were stained with DAPI (blue). Representative image is shown. Scale bar represents 20μm. (c) Same as b, except TLR3 and AP-1 were co-stained. Laminin (red), TLR3 (green), AP-1 (magenta), and nuclei were stained with DAPI (blue). Two representative images are shown. Scale bar represents 20μm in full size image and 5μm in zoom in.
Extended Data Fig. 10 The clathrin sorting adaptor protein AP-1 localizes TLR3 to the basolateral side of hIECs leading to an asymmetric IFN- mediated immune response.
Schematic showing TLR3 localization in polarized intestinal epithelial cells and its implication for side specific interferon induction upon viral infection.
Microarray genes and values. Table shows expression values and gene reference numbers for microarray data shown in Extended Data 5.
Tables 1 and 2 include the P values from Fig. 6 and Extended Data Fig. 8. Table 3 includes the primer sequences and accession numbers for the primers used in qPCR.
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Stanifer, M.L., Mukenhirn, M., Muenchau, S. et al. Asymmetric distribution of TLR3 leads to a polarized immune response in human intestinal epithelial cells. Nat Microbiol (2019) doi:10.1038/s41564-019-0594-3