Mesenteric Lymphatic Alterations Observed During DSS Induced Intestinal Inflammation Are Driven in a TLR4-PAMP/DAMP Discriminative Manner

Abstract

Background: Inflammatory bowel disease (IBD) is characterized by both acute and chronic phase inflammation of the gastro-intestinal (GI) tract that affect a large and growing number of people worldwide with little to no effective treatments. This is in part due to the lack of understanding of the disease pathogenesis and also the currently poorly described involvement of other systems such as the lymphatics. During DSS induced colitis, mice also develop a severe inflammation of terminal ileum with many features similar to IBD. As well as inflammation within the ileum we have previously demonstrated lymphatic remodeling within the mesentery and mesenteric lymph nodes of DSS-treated mice. The lymphatic remodeling includes lymphangiogenesis, lymphatic vessel dilation and leakiness, as well as cellular infiltration into the surrounding tissue and peripheral draining lymph nodes. Methods: Intestinal inflammation was induced in C57BL/6 mice by administration of 2.5% DSS in drinking water for 7 days. Mice were treated with TLR4 blocker C34 or Polymyxin-B (PMXB) daily from days 3 to 7 of DSS treatment via I.P. injection, and their therapeutic effects on disease activity and lymphatic function were examined. TLR activity and subsequent effect on lymphangiogenesis, lymphadenopathy, and mesenteric lymph node cellular composition were assessed. Results: DSS Mice treated with TLR4 inhibitor, C34, had a significantly improved disease phenotype characterized by reduced ileal and colonic insult. The change correlated with significant reduction in colonic and mesenteric inflammation, resolved mesenteric lymphangiectasia, and CD103 + DC migration similar to that of healthy control. PMXB treatment however did not resolve inflammation within the colon or associated mesenteric lymphatic dysfunction but did however prevent lymphadenopathy within the MLN through alteration of CCL21 gradients and CD103 + DC migration. Conclusions: TLR4 appears to mediate several changes within the mesenteric lymphatics, more specifically it is shown to have different outcomes whether stimulation occurs through pathogen derived factors such as LPS or tissue derived DAMPs, a novel phenomenon.

Ultra-purification of Lipopolysaccharides reveals species-specific signalling bias of TLR4: importance in macrophage function

“…A shift in balance of LPS-TLR4 signalling from unbiased NF-κB/IRF3 toward the more anti-inflammatory IRF3 cascade is a known feature in regulating tolerance to LPS 32 . The role of this balance shift in chronic diseases such as: inflammatory bowel disease, amyotrophic Lateral Sclerosis, liver disease, or rheumatic diseases, is still poorly understood, but creates an avenue of research for future investigation especially in diseases where there is known involvement of bacterial products and/or NF-κB/IRF3 signalling 15 , 33 – 35 . In the expanding realm of the microbiota and its involvement in disease progression, we theorise that this discriminative capacity may be defunct in certain patient cohorts and infections.…”

“…Multiple analyses were performed using a one-way Anova with post-hoc Tukeys test where indicated. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 15 .…”

“…One nanogram of the converted cDNA samples was added to Bright-Green SYBR qPCR master-mix and qPCR analysis was performed in an ABI StepOne Plus PCR system. The PCR conditions were as follows: 95 °C for 1 min followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, and with a final round of extension at 72 °C for 10 min 15 . Sequences of primers used are detailed in Table 1 .…”

Cellular and molecular mediators of lymphangiogenesis in inflammatory bowel disease

“…The importance of the balancing role of TLR4 in intestinal injury and repair is also reported by Yun-Jie and colleagues, who found that moderate activation of TLR4 signaling both promotes inflammation and repairs the intestinal epithelium in DSS-induced colitis and radiation damage [ 124 ]. TLR4-mediated signaling is important for the recruitment of immune cells to the site of inflammation, promoting reparative mechanisms, but can be described as a double-edged sword, as aberrant stimulation can induce chronic inflammation and mesenteric lymphatic alterations in a TLR4-PAMP (pathogen-associated molecular patterns)/DAMP (-associated molecular patterns) discriminative manner [ 125 ]. The observation that reduced lymphangiogenesis improves DSS-induced phenotype implies that the restoration of lymphatic function to a “normal” phenotype, significantly aids the repair of DSS-induced disease activity, which is achieved through TLR4 blockade with C34 [ 125 ].…”

“…TLR4-mediated signaling is important for the recruitment of immune cells to the site of inflammation, promoting reparative mechanisms, but can be described as a double-edged sword, as aberrant stimulation can induce chronic inflammation and mesenteric lymphatic alterations in a TLR4-PAMP (pathogen-associated molecular patterns)/DAMP (-associated molecular patterns) discriminative manner [ 125 ]. The observation that reduced lymphangiogenesis improves DSS-induced phenotype implies that the restoration of lymphatic function to a “normal” phenotype, significantly aids the repair of DSS-induced disease activity, which is achieved through TLR4 blockade with C34 [ 125 ]. This apparent contradiction of the general observation that increased lymphangiogenesis improves IBD implies that a balance is required in the therapeutic induction of lymphangiogenesis to arrive at a maximum benefit.…”