Microbiota-Regulated IL-25 Increases Eosinophil Number to Provide Protection during Clostridium difficile Infection

Clostridium difficile infection (CDI) is the most common cause of hospital-acquired infection in the United States. Host susceptibility and the severity of infection are influenced by disruption of the microbiota and the immune response. However, how the microbiota regulate immune responses to mediate CDI outcome remains unclear. Here, we have investigated the role of the microbiota-linked cytokine IL-25 during infection. Intestinal IL-25 was suppressed during CDI in humans and mice. Restoration of IL-25 reduced CDI-associated mortality and tissue pathology even though equivalent levels of C. difficile bacteria and toxin remained in the gut. IL-25 protection was mediated by gut eosinophils, as demonstrated by an increase in intestinal eosinophils and a loss of IL-25 protection upon eosinophil depletion. These findings support a mechanism whereby the induction of IL-25-mediated eosinophilia can reduce host mortality during active CDI. This work may provide targets for future development of microbial or immune-based therapies

Inflammasome activation contributes to interleukin-23 production in response to Clostridium difficile

ABSTRACT   Clostridium difficile is the most common hospital-acquired pathogen, causing antibiotic-associated diarrhea in over 250,000 patients annually in the United States. Disease is primarily mediated by toxins A and B, which induce potent proinflammatory signaling in host cells and can activate an ASC-containing inflammasome. Recent findings suggest that the intensity of the host response to infection correlates with disease severity. Our lab has identified the proinflammatory cytokine interleukin-23 (IL-23) as a pathogenic mediator during C. difficile infection (CDI). The mechanisms by which C. difficile induces IL-23, however, are not well understood, and the role of toxins A and B in this process is unclear. Here, we show that toxins A and B alone are not sufficient for IL-23 production but synergistically increase the amount of IL-23 produced in response to MyD88-dependent danger signals, including pathogen-associated molecular patterns (PAMPs) and host-derived damage associated molecular patterns (DAMPs). Danger signals also enhanced the secretion of IL-1β in response to toxins A and B, and subsequent IL-1 receptor signaling accounted for the majority of the increase in IL-23 that occurred in the presence of the toxins. Inhibition of inflammasome activation in the presence of extracellular K+ likewise decreased IL-23 production. Finally, we found that IL-1β was increased in the serum of patients with CDI, suggesting that this systemic response could influence downstream production of pathogenic IL-23. Identification of the synergy of danger signals with toxins A and B via inflammasome signaling represents a novel finding in the mechanistic understanding of C. difficile-induced inflammation. IMPORTANCE Clostridium difficile is among the leading causes of death due to health care-associated infection, and factors determining disease severity are not well understood. C. difficile secretes toxins A and B, which cause inflammation and tissue damage, and recent findings suggest that some of this tissue damage may be due to an inappropriate host immune response. We have found that toxins A and B, in combination with both bacterium- and host-derived danger signals, can induce expression of the proinflammatory cytokines IL-1β and IL-23. Our results demonstrate that IL-1β signaling enhances IL-23 production and could lead to increased pathogenic inflammation during CDI

Role of Eosinophils and Tumor Necrosis Factor Alpha in Interleukin-25-Mediated Protection from Amebic Colitis

The parasite Entamoeba histolytica is a cause of diarrhea in infants in low-income countries. Previously, it was shown that tumor necrosis factor alpha (TNF-α) production was associated with increased risk of E. histolytica diarrhea in children. Interleukin-25 (IL-25) is a cytokine that is produced by intestinal epithelial cells that has a role in maintenance of gut barrier function and inhibition of TNF-α production. IL-25 expression was decreased in humans and in the mouse model of amebic colitis. Repletion of IL-25 blocked E. histolytica infection and barrier disruption in mice, increased gut eosinophils, and suppressed colonic TNF-α. Depletion of eosinophils with anti-Siglec-F antibody prevented IL-25-mediated protection. In contrast, depletion of TNF-α resulted in resistance to amebic infection. We concluded that IL-25 provides protection from amebiasis, which is dependent upon intestinal eosinophils and suppression of TNF-α

Tolerogenic Properties of Lymphatic Endothelial Cells Are Controlled by the Lymph Node Microenvironment

Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1). Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon) lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.</p

The binary toxin CDT enhances Clostridium difficile virulence by suppressing protective colonic eosinophilia

© 2016 Macmillan Publishers Limited. All rights reserved. Clostridium difficile is the most common hospital acquired pathogen in the USA, and infection is, in many cases, fatal. Toxins A and B are its major virulence factors, but expression of a third toxin, known as C. difficile transferase (CDT), is increasingly common. An adenosine diphosphate (ADP)-ribosyltransferase that causes actin cytoskeletal disruption, CDT is typically produced by the major, hypervirulent strains and has been associated with more severe disease. Here, we show that CDT enhances the virulence of two PCR-ribotype 027 strains in mice. The toxin induces pathogenic host inflammation via a Toll-like receptor 2 (TLR2)-dependent pathway, resulting in the suppression of a protective host eosinophilic response. Finally, we show that restoration of TLR2-deficient eosinophils is sufficient for protection from a strain producing CDT. These findings offer an explanation for the enhanced virulence of CDT-expressing C. difficile and demonstrate a mechanism by which this binary toxin subverts the host immune response

Tolerogenic Properties of Lymphatic Endothelial Cells Are Controlled by the Lymph Node Microenvironment

<div><p>Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1). Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon) lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.</p></div

LtβR-Ig signaling and B cells do not control tyrosinase expression but influence the kinetics of FH T cell deletion.

<p><b><i>a.</i></b> LEC were purified from the LN of mice treated with PBS or LtβR-Ig for 4 weeks, or from WT (C57BL/6) or µMT^−/− mice. RNA was purified and 40-cycle qPCR was performed for tyrosinase. Graphs represent results from 2 independent experiments. Data are represented as mean +/− SEM. <b><i>b.</i></b> LNSC were purified by enzymatic digestion of pooled LN from the indicated mice, obtained via magnetic bead separation, and were analyzed by flow cytometry. Plots are gated on CD45^neg gp38⁺ CD31⁺ cells. <b><i>c.</i></b> 1 × 10⁶ CD8-enriched FH T cells were labeled with cell trace violet (CTV)-labeled and adoptively transferred into WT, µMT^−/−, or Tyr^neg (albino) recipients. At day 3 post-transfer, pooled peripheral LN were harvested and stained for CD8 and Tyr₃₆₉-tetramer, and assessed for CTV dilution. <i>Left panel</i>, representative histogram plot gated on CD8⁺ Tet⁺ cells. <i>Right panel,</i> summary graph of percent FH T cells divided per division representative of 4 independent experiments. Data are represented as mean +/− SEM. *p<0.05, **p<0.01, ^#p = 0.06 ns = not significant. <b><i>d.</i></b> Experiment performed as in (c), except FH T cells were identified using CD45.1 and LN were harvested at 7 days post transfer.</p

PD-L1 in combination with ICAM-1, MAdCAM-1, and LtβR define distinct LEC subsets that vary in location in the LN.

<p><b><i>a.</i></b> Single cell suspensions of CD45^neg LN cells were stained with antibodies against PD-L1, ICAM-1, MAdCAM-1, and LtβR. Number in the gp38 vs. CD31 2-D plot indicates percentage of total CD45^neg cells. Number in the 2-D PD-L1 vs. ICAM-1 plot indicates percentage of total LEC. Number in the 2-D MAdCAM-1 vs. LtβR plots indicates percentage of total PD-L1^hi ICAM-1^hi LEC (top) or PD-L1^int ICAM-1^int LEC (bottom). <b><i>b.</i></b> Summary graph of 7 independent experiments of the indicated LEC subpopulation percentage of total LEC. Data are represented as mean +/− SEM. <b><i>c, d.</i></b> Frozen axillary LN sections were stained with antibodies specific for SIGN-R1, PD-L1, Lyve-1, CD31, and B220 (c) or SIGN-R1, MAdCAM-1, and Lyve-1 (d). Dashed line represents regions of SIGN-R1 staining. S = subcapsule, C = cortex. Scale bar = 200 µm. Staining is representative of multiple fields from 3 experiments consisting of 2 separate LN from 3 mice. Graphs represent PD-L1 (c) or MAdCAM-1 (d) MFI gated on Lyve-1⁺ pixels in the indicated LN compartments. Data are represented as mean +/− SEM. *p<0.05, ***p<0.001, ns = not significant.</p