Cathelicidin-mediated lipopolysaccharide signaling via intracellular TLR4 in colonic epithelial cells evokes CXCL8 production

Abstract

We hypothesized that the antimicrobial peptide cathelicidin has a physiological role in regulating gut inflammatory homeostasis. We determined that cathelicidin synergizes with LPS to facilitate its internalization and signaling via endosomic TLR4 in colonic epithelium, evoking synthesis of the human neutrophil chemoattractant, CXCL8 (or murine homolog, CXCL1). Interaction of cathelicidin with LPS in the control of CXCL8/CXCL1 synthesis was assessed in human colon epithelial cells, murine colonoids and cathelicidin-null mice (Camp −/-). Mechanistically, human cathelicidin (LL-37), as an extracellular complex with LPS, interacted with lipid raft-associated GM1 gangliosides to internalize and activate intracellular TLR4. Two signaling pathways converged on CXCL8/CXCL1 production: (1) a p38MAPK-dependent pathway regulated by Src-EGFR kinases; and, (2) a p38MAPKindependent, NF-κB-dependent pathway, regulated by MEK1/2-MAPK. Increased cathelicidindependent CXCL8 secretion in the colonic mucosa activated human blood-derived neutrophils. These cathelicidin effects occurred in vitro at concentrations well below those needed for microbicidal function. The important immunomodulatory role of cathelicidins was evident in cathelicidinnull/Camp −/mice, which had diminished colonic CXCL1 secretion, decreased neutrophil recruitment-activation and reduced bacterial clearance when challenged with the colitis-inducing murine pathogen, Citrobacter rodentium. We conclude that in addition to its known microbicidal action, cathelicidin has a unique pathogen-sensing role, facilitating LPS-mediated intestinal responses, including the production of CXCL8/CXCL1 that would contribute to an integrated tissue response to recruit neutrophils during colitis.

Early immune innate hallmarks and microbiome changes across the gut during Escherichia coli O157: H7 infection in cattle

“…Since BMAP28 is mostly derived from myeloid leukocytes, this cathelicidin could appear later when leukocytes arrive and massively infiltrate the gut. In that case, cathelicidins could promote further neutrophil influx to injury sites; as cathelicidin was chemoattractant in the skin 12 and induced the expression of CXCL8 in keratinocytes 59 and colonocytes 60 .…”

Interleukin-4 Programmed Macrophages Suppress Colitis and Do Not Enhance Infectious-Colitis, Inflammation-Associated Colon Cancer or Airway Hypersensitivity

“…Calgary) were injected with M(IL4)s (1×10 6 in 500 mL PBS, ip.). Two days later, mice were orally gavaged with C. rodentium (strain DBS 100; 5×10 8 CFU in 200 µL PBS) (26,27). Feces were collected at 3-, 5-, and 7-days post-infection (dpi), homogenized at 0.1g/1 mL of sterile PBS, then serial dilutions were added to MacConkey agar plates.…”

“…Feces were collected at 3-, 5-, and 7-days post-infection (dpi), homogenized at 0.1g/1 mL of sterile PBS, then serial dilutions were added to MacConkey agar plates. After 24h of aerobic culture at 37°C, colonies were counted to obtain bacterial CFU/g (26). Mice were euthanized at 7 dpi, colon length measured, and segments of mid-colon processed for H&E staining.…”

“…Mice were euthanized at 7 dpi, colon length measured, and segments of mid-colon processed for H&E staining. Fecal lipocalin-2 was measured as a general marker of inflammation by ELISA (R&D Systems) and following the provided manufactures instructions (26,27).…”

“…After each incubation step, tubes were shaken for 10 seconds and medium containing epithelial cells was discarded. The lamina propria cells were centrifuged at 400xg for 10 min at 4°C, and the pellet re-suspended in PBS (26). The cells were stained with antibodies against CD45 (563891; BD Biosciences), CD3e (557984; BD Biosciences), CD4 (561104; BD Biosciences), Ly6G (561104; BD Biosciences) and CD17a (506915; BioLegend) for 30 min at 4°C at the concentrations shown in Supplementary Table 2.…”