Regulatory T Cells Restrict Permeability to Bacterial Antigen Translocation and Preserve Short-Chain Fatty Acids in Experimental Cirrhosis

Mice

Male C57Bl/6J wild-type (WT) and Rag1^-/- mice on C57Bl/6J background (Jackson Laboratories, Bar Harbor, MN) were included in 16-week cirrhosis-induced protocols. Immunodeficient Rag1^-/- mice have a defective VDJ (variable, diversity, and joining) recombination, so the immune adaptive response is altered by the lack of production of mature T cells and B cells. All of the animals were subjected to a week of quarantine before the cirrhosis protocol. We selected Rag1^-/- mice, which are a T cell–free mouse model, to induce liver toxicity with a classical CCl₄ protocol. The cirrhotic Rag^-/- mice let us evaluate the activation of sorted naive T cells and Tregs from WT animals. Mice were fed a standard rodent chow and kept at a constant room temperature of 21°C in a 12:12 light/dark cycle. The cirrhosis protocol began with the treatment of animals with 0.25 mmol/L phenobarbital in tap water that was maintained along the study protocol. After 4 weeks, the animals received 2 weekly weight-controlled doses of CCl₄ by oral gavage for 12 weeks. The first CCl₄ dose was 100 μL/kg (2 μL per mouse) in mineral oil, and subsequent doses were adjusted based on changes in weight 48 hours after the previous dose, up to 100 μL per mouse. A group of naive WT mice not subjected to the CCl₄ protocol was used as a negative control for evaluating fibrosis.

Sample size was calculated according to preliminary TJ protein gene expression levels obtained by our group and published in abstract format (Juanola et al. Hepatology 2016;64:83A). Assuming an α error of 0.017, a β error of 0.2, an equal distribution of subjects between groups (0.5 each), and an observed mean difference (E) of colonic occludin gene expression between groups of 1 with a SD of 0.3, 4 animals per group per protocol were required. Typically, mortality rates in our work are about 20%. We increased the number of Rag1^-/- mice compared with control mice to prevent possible cell-transfer failures or an increased mortality rate. We included 40 to 45 mice in each protocol (30-35 Rag1^-/- and 10 WT) to make sure we were able to work with at least 5 to 6 mice per group.

Rag1^-/- cirrhotic mice were subjected to adoptive transfer experiments 48 hours before laparotomies, as described.29 A group of 24 male C57Bl/6 WT mice not subjected to cirrhosis induction protocol were used for isolating spleen-derived sorted naive T (CD4⁺CD25^-CD45RB^high) cells and Treg (CD4⁺CD25⁺CD45RB^low) cells for these experiments. Postsort purity was typically more than 98%. Rag1^-/- cirrhotic animals were classified into three groups according to transferred cells: (1) mice not receiving sorted cells; (2) mice transferred with naive T cells (2 × 10⁵ cells per mouse); and (3) mice receiving naive T and Treg cells (1 × 10⁵ each per mouse). Twenty-four hours prior to laparotomies, a subgroup of cirrhotic Rag1^-/- animals in each condition received oral Escherichia coli (E. coli), as described subsequently.

Laparotomies were performed under anesthesia with isoflurane. Whole blood was obtained from the cava vein for gut permeability experiments before liver perfusion. Livers were then perfused in situ with 6 mL of Hank’s balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺ (Life Technologies Corp., Grand Island, NY) at 37°C at a rate of 1.5 mL per minute. Detectable MLNs were removed, and the liver, spleen, complete small intestine, colon, and content from cecum were collected.

Animals received care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals. The study was approved by the Animal Research Committee of Universidad Miguel Hernández (Alicante, Spain).

Fluorescein Isothiocyanate–LPS Intestinal Permeability Assay

A subgroup of animals in each condition was intragastrically administered 100 µg of fluorescein isothiocyanate (FITC) conjugated LPS from E. coli 0111:B4 or dextran (Sigma-Aldrich, St. Louis, MO). Gut permeability was evaluated 2 hours after FITC-LPS or FITC-dextran administration as the percentage of recovery measured by fluorometry at 488 nm in 100 µL of serum samples collected from the cava vein. Dilutions of FITC-LPS and FITC-dextran in phosphate-buffered saline (PBS) were used for standard curves.

Bacterial DNA Translocation Assessment in MLNs

MLNs were harvested with sterile instruments in sterile PBS at laparotomies. All tissues were disrupted by using the Tissue Lyser LT (QIAgen, Hilden, Germany), and DNA was immediately isolated using the QIAamp DNA Mini Kit. Bacterial DNA was detected by performing a broad-range polymerase chain reaction (PCR) and partial sequencing analysis of the 16S ribosomal RNA gene, as described.14

Gene Expression Analysis

Total cellular RNA was isolated using the RNeasy Mini Kit (QIAgen). The qScript One-Step SYBR Green quantitative real-time PCR Kit (Quanta BioScience, Gaithesburg, MD) was used to perform the gene expression of claudin-1, claudin-2, ZO-1, occludin, collagen type I alpha-1 chain (Col1a1), tumor growth factor beta (TGF-β), matrix metalloproteinase 2 (MMP2), tissue inhibitor of metalloproteinase 1 (TIMP1), FFAR2 and FFAR3, and IL-10 in an IQ5 RT-PCR (Bio-Rad, Hercules, CA). β2-microglobulin was used as a housekeeping gene in all gene expression analyses. Primer pairs used in the study can be followed in Supporting Table S1.

Immunohistochemistry Analysis

Immunohistochemical (IHC) assays were carried out in 4-µm sections of paraffin-embedded colon tissue, and processed following standard procedures. The slides were incubated with primary antibodies claudin-1, claudin-2, occludin, and ZO-1 (Abcam PLC, Cambridge, United Kingdom). As secondary antibodies, we incubated the sections with the correspondent α-goat or α-rabbit biotinylated antibodies (Palex Medical SA, Sant Cugat del Vallés, Spain). Slides were incubated with avidin-biotin complex (Vector Laboratories Inc., Burlingame, CA) and revealed with peroxidase substrate 3,3′-diaminobenzidine (Vector Laboratories Inc.). Nuclei were stained by incubating the sections in Harris hematoxylin (Leica Biosystems Richmond Inc., Richmond, IL). As a negative control, staining was carried out in the absence of a primary antibody. Images were obtained in a camera-assisted optic Leica DM2000 LED microscope (Leica Biosystems, Richmond Inc.). All represented panels are shown at original magnification ×20. A semiquantitative analysis of protein expression was performed using the ImageJ software (https://rsbweb.nih.gov).

Western Blot Analysis

Colon homogenates were lysed with radioimmunoprecipitation assay buffer and protein concentration determined by Bradford protein assay (EMD Millipore Corp., Billerica, MA). Twenty micrograms of protein extracts were resolved under reducing conditions on 6% to 15% sodium dodecyl sulfate–polyacrylamide gels and transferred to Immobilon-P membranes (EMD Millipore Corp.). Primary antibodies used are β-actin (Sigma-Aldrich), ZO-1 (Thermo Fisher Scientific, Waltham, MA), and claudin-1, claudin-2, and occludin (Abcam PLC). Finally, membranes were incubated with the appropriate horseradish peroxidase (HRP)–conjugated secondary antibody (Cell Signaling Technology, Leiden, Netherlands). Immobilon Western Chemilum HRP Substrate (EMD Millipore Corp.) was used to detect the activity of the membrane-attached peroxidase, and images were obtained in ChemiDOC XRS+ operated by Image Lab software (Bio-Rad). Protein bands were quantified by densitometry using Scion Image software (Scion Corp., Frederick, MD). Band densities were expressed relative to total β-Actin protein.

E. Coli Culture Conditions and Administration to Mice

E. coli (CECT45) was grown in 9 mL of thioglycolate broth with resazurin (Biomérieux, Marcy l’Etoile, France) and incubated overnight at 37°C. Serial dilutions of incubated bacteria were performed before plating them in McConkey agar (Biomérieux) overnight at 37°C. Colony-forming units (CFUs) were then counted, and cells were resuspended in PBS. Each animal was administered 1 × 10⁶ CFUs orally.

Intestinal Lymphocyte Isolation and T_h Differentiation

The whole small intestine from E. coli–challenged cirrhotic Rag1^-/- mice was obtained. Peyer’s patches, fatty tissue, and mesentery were removed from all intestines. The gut was cleaned using cold PBS without Ca²⁺ and Mg²⁺ (Euroclone, Milano, Italy), opened longitudinally, and cut into 1-cm pieces. Intraepithelial lymphocytes (IELs) were obtained by incubating the small intestine pieces twice with HBSS Ca²⁺ and Mg²⁺ free (Life Technologies Corp.) supplemented with 5 mM ethylene diamine tetraacetic acid (Bio-Rad), 1 mM dithiothreitol (Sigma-Aldrich), and 1% penicillin-streptomycin (Life Technologies Corp.) in Incubating Orbital Shake (VWR, Llinars del Vallés, Spain) for 30 minutes at 37°C. Thereafter, the tissue pieces were washed with PBS and then incubated three times in HBSS with 0.5 mg/mL collagenase D (Roche Diagnostics GmbH, Mannheim, Germany), 3 mg/mL dispase II (Sigma-Aldrich), 1 mg/mL DNase I (Roche Diagnostics GmbH), and 1% penicillin-streptomycin with orbital agitation for 30 minutes at 37°C to collect the lamina propria lymphocyte (LPL) cells. All supernatant fractions were filtered with 70-µm nylon cell strainers (Corning Incorporated Life Sciences, Oneonta, NY), washed with PBS 3% fetal bovine serum (FBS) (Life Technologies Corp.), and centrifugated to harvest cell suspensions. IELs and LPLs were pooled and constituted the intestinal lymphocyte content. Live lymphocytes were obtained by preparing a 40%/80% Percoll (Sigma-Aldrich) gradient and then cultured in advanced Roswell Park Memorial Institute (RPMI)-1640 (Life Technologies Corp.) supplemented with 10% FBS, 1% L-glutamine (Life Technologies), and 1% penicillin-streptomycin. Cells were treated and marked according to manufacturer’s instructions in the Mouse T_h1/T_h2/T_h17 Phenotyping Kit (BD Biosciences, San Diego, CA). To induce a complete T-cell activation, E. coli was administered according to the conditions described here. T_h differentiation was evaluated as intracellular production of interferon (IFN)-γ, IL-4, and IL-17. Data acquisition and analysis of marked intestinal lymphocytes were performed using a FACSCanto II flow cytometer operated by FACSDiva software (BD Biosciences).

Gas Chromatography–Mass Spectrometry Analysis of SCFAs in Intestinal Content

Intestinal content samples with PBS were homogenized, and 300 µL were mixed with 300 µL of 0.5% phosphoric acid. Intestinal content suspensions were sonicated in an ultrasonic bath for 5 minutes and centrifuged for 10 minutes at 17,949g. The aqueous supernatant was extracted with the same volume of methyl tert-buthyl ether for 5 minutes and centrifuged in the same conditions. The upper organic phase was transferred into a tube and 4-methyl valeric acid added as internal standard at a final concentration of 500 µM. Gas chromatography–mass spectrometry (GC-MS) system and chromatographic and mass parameter conditions were followed, as reported.31

Statistical Analysis

Continuous variables are reported as mean ± SD and categorical variables as frequency or percentages. Quantitative data were analyzed using the Mann-Whitney U test for simple comparisons with the post hoc Bonferroni correction for multiple comparisons. Differences in qualitative variables were analyzed using the χ2 test. All reported P values are 2-sided, and P values less than 0.05 indicate significance. All calculations were performed using SPSS Statistics 19 (IBM, Chicago, IL).

Cirrhosis Induction Protocols and Mice Groups

Five independent protocols of CCl₄-induced cirrhosis were run to complete the different experiments performed. Each protocol included a group of WT mice and three groups of Rag1^-/- mice (nontransferred Rag1^-/- mice, naive T cell–transferred Rag1^-/- mice, and naive T plus Treg cell–cotransferred Rag1^-/- mice). Table 1 lists the number of mice included in each experiment distributed by groups and protocol.

Gene expression levels of profibrogenic markers were evaluated in all CCl₄ protocols and combined in Supporting Fig. S1. Results in Rag1^-/- mice were similar to those present in WT mice and significantly increased compared with WT mice without liver damage.

Gut Permeability to LPS Is Reduced in Cirrhotic Rag1^-/- Mice Cotransferred With Treg Cells

We aimed to study in vivo gut permeability to orally administered FITC-LPS. The results of this functional test are described in Fig. 1A. Rag1^-/- mice showed a significantly increased permeability to FITC-LPS compared with WT mice. The recovery rate of LPS in Rag1^-/- mice was not reduced after injection with spleen-derived naive T cells. On the contrary, only the coinjection of naive T and Treg cells significantly reduced gut permeability to FITC-LPS. In agreement with this, the translocation of bacterial DNA to MLNs of Rag1^-/- mice cotransferred with naive T and Treg cells was significantly down-regulated to levels present in WT cirrhotic mice (Fig. 1B).

Treg Cells Are Associated With a Restoration of TJ Protein Expression in Cirrhotic Rag1^-/- Mice

The observed reduction in gut permeability led us to study the status of TJ proteins in cirrhotic mice. The expression of TJ proteins ZO-1, occludin, claudin 1, and claudin 2 was measured by gene expression, IHC, and western blot (WB) in all study groups (Fig. 2). Messenger RNA (mRNA) levels of all studied genes were significantly decreased in cirrhotic Rag1^-/- mice either nontransferred or transferred with naive T cells compared with cirrhotic WT mice. The cotransfer with naive T and Treg cells restored transcript expression of all genes to levels shown in cirrhotic WT mice (Fig. 2A).

Cirrhotic Rag1^-/- mice without any transferred cells showed a significant reduction in the protein expression of the different TJ proteins compared with the cirrhotic WT, as shown by IHC and WB analyses (Fig. 2B,C, respectively). None of the TJ protein levels significantly increased when these mice were transferred with naive T cells. However, the cotransfer with naive T and Treg cells restored all TJ protein expression to the levels shown in WT. Visually observed differences between groups in TJ protein expression were shown to be statistically significant after a semiquantitative analysis of IHC and WB images.

Transferred Treg Cells Modulate IL Proinflammatory T_h1 and T_h17 Responses to Oral E. Coli Administration in Cirrhotic Rag1^-/- Mice

Because Treg cells are functionally required to maintain gut barrier integrity and reduced permeability in CCl₄-cirrhotic mice, we were interested in evaluating whether transferred Treg cells in cirrhotic Rag1^-/- mice restrained the induced proinflammatory T_h differentiation as a possible explanation for the function of Treg cells in gut barrier improvement. Figure 3A shows the flow cytometry gating strategy for determining T_h subpopulations in ILs of Rag1^-/- mice in response to oral E. coli administration. Rag1^-/- mice showed a low T_h differentiation from residual intestinal CD4⁺ cells, as expected (Fig. 3B). The injection of T naive cells induced exacerbated proinflammatory T_h1 and T_h17 responses that were significantly down-regulated by the cotransfer with Treg cells. The T_h2 profile was not altered in any experimental condition.

Because Treg cells exert their action through IL-10 production, we set out to determine transcript expression levels of IL-10 in intestinal lymphocytes. IL-10 levels in cirrhotic Rag1^-/- mice cotransferred with naive T and Treg cells were significantly increased in response to E. coli compared with the rest of the groups, providing a counterbalance for the observed T_h proinflammatory polarization (Fig. 3C).

Treg Cells Contribute to Sustain SCFA Levels in the Intestinal Content of Cirrhotic Rag1^-/- Mice After Exposure to E. Coli

Microbiota products such as SCFAs also play a role in maintaining gut homeostasis. We evaluated whether Treg cells are also relevant in SCFA modification in response to E. coli, as a model of induced BT, in the intestinal content of cirrhotic Rag1^-/- mice. The concentration of all measured SCFAs resulted in a significant reduction after E. coli administration, but for acetic acid in nontransferred Rag1^-/- mice. The concentration of all SCFAs remained unaltered in the intestinal content of cirrhotic Rag1^-/- mice cotransferred with naive T and Treg cells except for valeric and isovaleric acids (Fig. 4A). The gene expression levels of receptors FFAR2 and FFAR3 for these SCFAs were measured in colonic tissue homogenates (Fig. 4B). Both receptors were significantly reduced in response to E. coli in cirrhotic Rag1^-/- mice either nontransferred or transferred with naive T cells. The cotransfer with naive T and Treg cells was able to reestablish FFAR2 and FFAR3 levels shown in cirrhotic Rag1^-/- mice not challenged with E. coli.