Ben Meijer

The Impact of Gut Microbiota on Gender-Specific Differences in Immunity

Males and females are known to have gender-specific differences in their immune system and gut microbiota composition. Whether these differences in gut microbiota composition are a cause or consequence of differences in the immune system is not known. To investigate this issue, gut microbiota from conventional males or females was transferred to germ-free (GF) animals of the same or opposing gender. We demonstrate that microbiota-independent gender differences in immunity are already present in GF mice. In particular, type I interferon signaling was enhanced in the intestine of GF females. Presumably, due to these immune differences bacterial groups, such as Alistipes, Rikenella, and Porphyromonadaceae, known to expand in the absence of innate immune defense mechanism were overrepresented in the male microbiota. The presence of these bacterial groups was associated with induction of weight loss, inflammation, and DNA damage upon transfer of the male microbiota to female GF recipients. In summary, our data suggest that microbiota-independent gender differences in the immune system select a gender-specific gut microbiota composition, which in turn further contributes to gender differences in the immune system.

Supplementation with Lactobacillus plantarum WCFS1 Prevents Decline of Mucus Barrier in Colon of Accelerated Aging Ercc1(-/Δ7) Mice

Although it is clear that probiotics improve intestinal barrier function, little is known about the effects of probiotics on the aging intestine. We investigated effects of 10-week bacterial supplementation of Lactobacillus plantarum WCFS1, Lactobacillus casei BL23, or Bifidobacterium breve DSM20213 on gut barrier and immunity in 16-week-old accelerated aging Ercc1−/Δ7 mice, which have a median lifespan of ~20 weeks, and their wild-type littermates. The colonic barrier in Ercc1−/Δ7 mice was characterized by a thin (< 10 μm) mucus layer. L. plantarum prevented this decline in mucus integrity in Ercc1−/Δ7 mice, whereas B. breve exacerbated it. Bacterial supplementations affected the expression of immune-related genes, including Toll-like receptor 4. Regulatory T cell frequencies were increased in the mesenteric lymph nodes of L. plantarum- and L. casei-treated Ercc1−/Δ7 mice. L. plantarum- and L. casei-treated Ercc1−/Δ7 mice showed increased specific antibody production in a T cell-dependent immune response in vivo. By contrast, the effects of bacterial supplementation on wild-type control mice were negligible. Thus, supplementation with L. plantarum – but not with L. casei and B. breve – prevented the decline in the mucus barrier in Ercc1−/Δ7 mice. Our data indicate that age is an important factor influencing beneficial or detrimental effects of candidate probiotics. These findings also highlight the need for caution in translating beneficial effects of probiotics observed in young animals or humans to the elderly.

Tryptophan restriction arrests B cell development and enhances microbial diversity in WT and prematurely aging Ercc1−/Δ7 mice

With aging, tryptophan metabolism is affected. Tryptophan has a crucial role in the induction of immune tolerance and the maintenance of gut microbiota. We, therefore, studied the effect of dietary tryptophan restriction in young wild‐type (WT) mice (118‐wk life span) and in DNA‐repair deficient, premature‐aged (Ercc1−/Δ7) mice (20‐wk life span). First, we found that the effect of aging on the distribution of B and T cells in bone marrow (BM) and in the periphery of 16‐wk‐old Ercc1−/Δ7 mice was comparable to that in 18‐mo‐old WT mice. Dietary tryptophan restriction caused an arrest of B cell development in the BM, accompanied by diminished B cell frequencies in the periphery. In general, old Ercc1−/Δ7 mice showed similar responses to tryptophan restriction compared with young WT mice, indicative of age‐independent effects. Dietary tryptophan restriction increased microbial diversity and made the gut microbiota composition of old Ercc1−/Δ7 mice more similar to that of young WT mice. The decreased abundances of Alistipes and Akkermansia spp. after dietary tryptophan restriction correlated significantly with decreased B cell precursor numbers. In conclusion, we report that dietary tryptophan restriction arrests B cell development and concomitantly changes gut microbiota composition. Our study suggests a beneficial interplay between dietary tryptophan, B cell development, and gut microbial composition on several aspects of age‐induced changes.

Warm ischemia time-dependent variation in liver damage, inflammation, and function in hepatic ischemia/reperfusion injury

BACKGROUND Hepatic ischemia/reperfusion (I/R) injury is characterized by hepatocellular damage, sterile inflammation, and compromised postoperative liver function. Generally used mouse I/R models are too severe and poorly reflect the clinical injury profile. The aim was to establish a mouse I/R model with better translatability using hepatocellular injury, liver function, and innate immune parameters as endpoints. METHODS Mice (C57Bl/6J) were subjected to sham surgery, 30min, or 60min of partial hepatic ischemia. Liver function was measured after 24h using intravital microscopy and spectroscopy. Innate immune activity was assessed at 6 and 24h of reperfusion using mRNA and cytokine arrays. Liver inflammation and function were profiled in two patient cohorts subjected to I/R during liver resection to validate the preclinical results. RESULTS In mice, plasma ALT levels and the degree of hepatic necrosis were strongly correlated. Liver function was bound by a narrow damage threshold and was severely impaired following 60min of ischemia. Severe ischemia (60min) evoked a neutrophil-dominant immune response, whereas mild ischemia (30min) triggered a monocyte-driven response. Clinical liver I/R did not compromise liver function and displayed a cytokine profile similar to the mild I/R injury model. CONCLUSIONS Mouse models using ≤30min of ischemia best reflect the clinical liver I/R injury profile in terms of liver function dynamics and type of immune response. GENERAL SIGNIFICANCE This short duration of ischemia therefore has most translational value and should be used to increase the prospects of developing effective interventions for hepatic I/R.

Induction of human tolerogenic dendritic cells by 3′-sialyllactose via TLR4 is explained by LPS contamination

Abstract The human milk oligosaccharide 3′-sialyllactose (3′SL) has previously been shown to activate murine dendritic cells (DC) in a Toll-like receptor (TLR) 4-mediated manner ex vivo. In this study we aimed to investigate whether 3′SL has similar immunomodulatory properties on human DC. 3′SL was shown to induce NF-κB activation via human TLR4. However, LPS was detected in the commercially obtained 3′SL from different suppliers. After the removal of LPS from 3′SL, we studied its ability to modify DC differentiation in vitro. In contrast to LPS and 3′SL, LPS-free 3′SL did not induce functional and phenotypical changes on immature DC (iDC). iDC that were differentiated in the presence of LPS or 3′SL showed a semi-mature phenotype (i.e., fewer CD83+CD86+ DC), produced IL-10 and abrogated IL-12p70 and tumor necrosis factor-alpha levels upon stimulation with several TLR ligands. Differentiation into these tolerogenic DC was completely abrogated by LPS removal from 3′SL. In contrast to previous reports in mice, we found that LPS-free 3′SL does not activate NF-κB via human TLR4. In conclusion, removing LPS from (oligo)saccharide preparations is necessary to study their potential immunomodulatory function.

Hypothermic perfusion with retrograde outflow during right hepatectomy is safe and feasible

Background. In situ hypothermic perfusion during liver resection performed under vascular inflow occlusion decreases hepatic ischemia‐reperfusion injury, but technical limitations have restricted its widespread use. In situ hypothermic perfusion with retrograde outflow circumvents these impediments and thus could extend the applicability of in situ hypothermic perfusion. The safety and feasibility of in situ hypothermic perfusion with retrograde outflow were analyzed in selected patients undergoing right (extended) hepatectomy and compared to intermittent vascular inflow occlusion, the gold standard method, in this randomized pilot study. Methods. Patients were first screened for parenchymal liver disease (exclusion criteria: steatosis ≥30%, cirrhosis, or cholestasis). Study participants were randomized intraoperatively to undergo in situ hypothermic perfusion with retrograde outflow (n = 9) or intermittent vascular inflow occlusion (n = 9). The target liver core temperature during in situ hypothermic perfusion with retrograde outflow was 28°C. The primary end point was ischemia‐reperfusion injury (expressed by peak postoperative transaminase levels). Secondary outcomes included functional liver regeneration (assessed by hepatobiliary scintigraphy) and clinical outcomes. Results. Peak transaminase levels, total bilirubin, and the international normalized ratio were similar between both groups, although a trend toward more rapid normalization of bilirubin levels was noted for the in situ hypothermic perfusion with retrograde outflow group. Functional liver regeneration as evaluated by hepatobiliary scintigraphy was improved on postoperative day 3 fafter in situ hypothermic perfusion with retrograde outflow but not after intermittent vascular inflow occlusion. Furthermore, in situ hypothermic perfusion with retrograde outflow (requiring continuous ischemia) was comparable to intermittent vascular inflow occlusion for all clinical outcomes, including postoperative complications and hospital stay. Conclusion. The use of in situ hypothermic perfusion with retrograde outflow appears to be safe and feasible in selected patients with healthy liver parenchyma and may benefit early functional liver regeneration. Future applications of in situ hypothermic perfusion with retrograde outflow include patients with damaged liver parenchyma who would require major hepatic resection with a prolonged vascular inflow occlusion duration.

BAFF augments IgA2 and IL‐10 production by TLR7/8 stimulated total peripheral blood B cells

Class‐switching of B cells to IgA can be induced via both T‐cell‐dependent and T‐cell‐independent mechanisms. IgA is most predominantly produced mucosally and is important for combating infections and allergies. In contrast to mice, humans have two forms of IgA; IgA1 and IgA2 with diverse tissue distribution. In early life, IgA levels might be sub‐optimal especially during the fall season when bacterial and viral infections are more common. Therefore, we investigated using human B cells whether T‐cell‐independent factors ‐promoting cell survival, class switching and immunoglobulin secretion‐ BAFF, APRIL, IL‐10 and retinoic acid can boost IgA production in the context of viral or bacterial infection. To this end total and naive peripheral blood B cells were stimulated with these factors for 6 days in the presence or absence of TLR7/8 agonist R848 (mimicking viral infection) or TLR9 agonist CpG‐ODN (mimicking bacterial infection). We show that BAFF significantly augments IgA2 production in TLR7/8 stimulated mature, but not naïve B cells. In addition, BAFF augments IL‐10 production and viability in TLR7/8 and TLR9 stimulated mature B cells. These data warrant further investigation of its role in immune regulation both in the periphery and mucosal tissues in early life or during disease.