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Genetic and Inflammatory Influences on the Microbiota: Insights from Colitic Mice

Christy Harrison1,2, Daniel Laubitz1, Rajalakshmy Ramalingam1, Monica Kiela1, Fayez Ghishan1, Pawel Kiela1,2 1Department of Pediatrics, Steele Children’s Research Center, 2Department of Immunology,

University of Arizona Health Sciences Center

Abstract

Introduction

NHE3 is an electroneutral sodium/hydrogen exchanger that exists on the apical membrane of the intestinal and renal epithelia. It is responsible for the majority of intestinal water absorption, and helps to mediate homeostasis by balancing intestinal pH.

NHE3 is inhibited by several inflammatory and microbial agents, including TNFα, IFNγ, and Clostridium diffiicile toxin B.1 When this happens, sodium is no longer drawn into the cell, which also osmotically excludes water. Hydrogen export is also inhibited, contributing to offset pH and mild systemic acidosis. Barrier function of the mucosa is also disturbed.2 Loss of functioning NHE3 is thought to be central to inflammation and IBD associated diarrhea.

Our group has shown that the loss of NHE3 in murine models is sufficient itself to cause a distal, bacterially mediated colitis that is responsive to antibiotics.3 Moreover, NHE3 knockout mice experience a profound IBD-like dysbiosis in which the Firmicutes phylum is significantly decreased.4 Clostridia clusters IV and XIVa, butyrate-producing subsets within Firmicutes, are associated with regulatory T-cell induction. We have also previously shown that lower abundances of these clusters correlate with greater disease severity in NHE3 knockout mice.4

References

Results Con’t

Future Directions

• Completion of all time points within the study to assess complete dynamics over time.

• Antibiotic treatment of T-cell/PBS recipient mice• Next-generation sequencing of DNA samples to get full

microbial profile

Sodium Hydrogen Exchanger 3 (NHE3) is one of the primary mediators of intestinal water absorption, and a target of inhibition for pro-inflammatory cytokines and bacterial toxins associated with colitis and inflammatory bowel disease (IBD). However, not only is NHE3 repressed by inflammation, but the lack of NHE3 on its own is sufficient to cause spontaneous T-cell mediated distal colitis in mice. Mice lacking NHE3 do not develop colitis when treated with antibiotics, which indicates an important bacterial role in the development of NHE3-associated disease. Indeed, NHE3 knockout mice develop a profound IBD-like dysbiosis characterized by significant changes in Firmicutes and clusters of Clostridia (IV and XIVa), indicating that NHE3 modulates the microflora in turn. Whether NHE3 primarily influences the microflora to drive colitis or whether dysbiosis precipitates NHE3 inhibition in human IBD is not thoroughly understood. In this experiment, we show that adoptive transfer of naïve T-cells into NHE3xRag double knockout mice causes an accelerated colitis that reaches critical severity within two weeks. By this time, pro-inflammatory cytokines are significantly upregulated and the morphology of the intestinal mucosa altered into an inflamed IBD-like state. Through real-time PCR analysis of Firmicutes, Clostridia cluster IV, and Clostridia cluster XIVa, we indicate that the NHE3-null genotype and the inflammation state both influence dysbiosis in a cluster dependent and at times additive manner.

Introduction Con’t

Methods

Results Con’t

Results

Rag2 -/- NHE3xRag2 -/- Distal Colon

Real-Time PCR FirmicutesBoth genotype and inflammation appear to correlate with

the relative abundance of the Firmicutes phylum, as day 0 indicates a pre-existing genetic difference between the relative abundance of double and single knockouts. However, by the end of the study, the T-cell injected animals demonstrated the lowest relative abundance, indicating the influence of inflammation as well.

Clostridia IVNHE3xRag double-knockout mice showed a large initial

genetic difference in relative abundance, but these differences were reduced over the course of the study.

Clostridia XIVaLike Clostridia IV, there was a reduction by day 3 that

either stabilized or reversed by the end of the study. Unlike cluster IV, the difference in relative abudance of NHE3xRag double-knockouts remained consistent and even increased over time. d0 d3 d12

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Firmicutes Change Over TimeNormalized to Rag -/- d0

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Clostridia IV Change Over TimeNormalized to Rag -/- d0

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Clostridia XIVa Change Over TimeNormalized to Rag -/- d0

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Using stool samples gathered at different time points through the T-cell transfer demonstrates the inherent differences between Firmicutes, and Clostridia clusters IV and XIVa due to genotype (day 0), and how these populations change over time in correlation with the colonic inflammation. Together these data indicate whether genetics or inflammation state are affecting the composition of the microbial groups studied. Although mice were only kept for two weeks before critical severity of disease was achieved, preliminary results seem to indicate that changes in these clusters are dynamic over time, correlating most consistently with the genotype of the mouse. It appears that influences on the microbiota - either genetic or inflammation induced - are unique to each bacterial subset. Firmicutes may be influenced by both inflammation and genotype because different species within this group react differently. Changes in both Clostridia clusters appeared to correlate more strongly with genotype than with inflammation, although this difference was most pronounced in cluster XIVa. Greater numbers, more diversity of groups studied, and a larger variety of time points will be needed to address the question more fully.

Discussion & Conclusions

However, it is unclear whether NHE3 is downregulated in response to these and other changes in the microflora, or whether the microflora respond to changes in NHE3. Our studies so far indicate that NHE3 may act as a modifying gene for the severity of colitis.

Because colitis in NHE3 knockout mice is largely CD4 T-cell driven, we chose an adoptive transfer model to accelerate colitis in these mice. Here we adoptively transferred naïve T-cells into Rag and NHE3xRag knockout mice to induce accelerated colitis and assess changes Firmicutes and Clostridia clusters IV and XIVa. Our results indicate that both genotype and inflammation correlate with the dynamic changes seen in the groups studied.

Four groups of mice, either Rag2 -/- or NHE3xRag2 -/- were injected with either naïve T-cells or phosphate buffered saline (PBS). Stool was collected at day of injection and every couple days until sacrifice. Mice were sacrificed when several in the experimental group had reached critical 20% weight loss, which occurred within two weeks of injection.

Stool pellets were collected every couple days and frozen at -80°C until the time of DNA extraction. This poster focuses on time points at day 0, day 3, and sacrifice (circled). DNA from stool was extracted using a phenol/chloroform extraction protocol, quantified, and diluted to 10ng/ul for qPCR amplification using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a CFX96 Real-Time PCR Detection System (BioRad).

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Firmicutes Relative Abundance at Day 0Normalized to Rag -/-

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Clostridia IV Relative Abundance at Day 0

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Clostridia XIVa Relative Abundance at Day 0

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1. Amin, M. R. et al (2006). Cell Physiology, 291(5), C887–96. doi:10.1152/ajpcell.00630.2005

2. Kiela, P. R. et al. (2009). Gastroenterology, 137(3), 965–75– 975.e1–10. doi:10.1053/j.gastro.2009.05.043

3. Laubitz, D. et al. (2008) American Journal of Physiology. Gastrointestinal and Liver Physiology, 295(1), G63–G77. doi:10.1152/ajpgi.90207.2008

4. Larmonier, C. B. et al. (2013). American Journal of Physiology. Gastrointestinal and Liver Physiology, 305(10), G667–77. doi:10.1152/ajpgi.00189.2013

This work was funded by the National Institutes of Health, Grant # 2R01 DK041274 (to FKG and PRK)