Local injection of dsRNA targeting calcitonin receptor-like receptor (CLR) ameliorates Clostridium difficile toxin A-induced ileitis

Abstract

Enteritis caused by Clostridium difficile toxin (Tx) is a nosocomial disease of increasing clinical concern, but the local mediators of C. difficile TxA inflammation are unknown. The potent vasodilator calcitonin gene-related peptide mediates neurogenic inflammation via the calcitonin receptor-like receptor (CLR). Here we examined the ileum-specific effects of reducing CLR on TxA ileitis by local preinjection of double-stranded RNAs. Treatment with CLR dsRNA for 7 d decreased CLR immunoreactivity, whereas treatment with non-CLR dsRNA did not. Subsequent injection of TxA in the same location increased CLR in rats treated with non-CLR dsRNA but not in rats treated with CLR dsRNA, documenting that local injection of dsRNA is effective in preventing the increase in CLR immunoreactivity in response to local TxA. After non-CLR dsRNA pretreatment, TxA induced robust intestinal secretion, myeloperoxidase activity, and histopathologic indications of inflammation including epithelial damage, congestion, neutrophil infiltration, loss of mucin from goblet cells, and increase in mast cell numbers. After CLR dsRNA pretreatment, TxA-induced changes in intestinal secretion and histopathologic inflammation were improved, including normal mucin staining and fewer resident mast cells. Loss of CLR prevented TxA-mediated activation of NF-κB and concomitant increases in pERK1/2 and TNF-α mRNA. Locally produced CLR plays a proinflammatory role in TxA ileitis via MAPK signaling and TNF-α. The results reported here strongly suggest that a local injection of dsRNA targeting CLR could be an effective local therapeutic approach at the inflammation site in the treatment of a growing, clinically relevant hospital-acquired disease, C. difficile infection.

Fig. 1.

CLR dsRNA decreases basal CLR-IR and prevents TxA-induced increase in CLR. Seven days after pretreatment with vehicle, non-CLR dsRNA, or CLR dsRNA, the rats received an injection of vehicle or TxA into ileal loops and were killed after 4 h. Loop tissue from rats treated with non-CLR dsRNA and vehicle had basal CLR-IR as determined by confocal microscopy; pretreatment with non-CLR dsRNA had no effect. TxA increased staining for CLR in the ileum of rats treated with non-CLR dsRNA. Loop tissue from rats injected with CLR dsRNA had minimal CLR-IR. TxA did not increase CLR-IR in the ileum of rats pretreated with CLR dsRNA. Arrowheads point to CLR-IR. L, lumen; myn, myenteric neurons. (Scale bar, 50 µm.)

Fig. 2.

Local injection of CLR dsRNA decreases ileitis mediated by TxA. After 7 d of pretreatment with non-CLR dsRNA or CLR dsRNA, an ileal loop spanning the dsRNA injection site was infused with vehicle or TxA (5 µg). (A) After 4 h of treatment, intestinal secretion was determined as the weight/length (mg/cm) of the excised loop. Secretion was significantly decreased (P < 0.05) with CLR dsRNA pretreatment. (B) MPO activity was also decreased in loop fluid (U/L) after CLR dsRNA pretreatment. Values are mean ± SEM per loop, n = 4–12 loops per group. *P < 0.05 vs. vehicle, ^#P < 0.05 vs. rats pretreated with non-CLR dsRNA and injected with TxA.

Fig. 3.

Local injection of CLR dsRNA decreases histopathologic changes induced by TxA. A portion of loop tissue was sectioned and stained with hematoxylin and eosin. (A) Graphical representation of the histologic score is shown. Duplicate sections were scored for epithelial damage, edema and congestion, and neutrophil infiltration (0–5 per end point) (17). *P < 0.05 vs. vehicle, ^#P < 0.05 vs. rats treated with non-CLR dsRNA, followed by TxA treatment 7 d later. Values are mean ± SEM per loop, n = 8–12 loops per group. PMNs, polymorphonuclear leukocytes. (B) Pretreatment with CLR dsRNA minimized histopathologic indications of inflammation induced by TxA. In the non-CLR dsRNA controls, TxA induced severe damage (note dilated blood vessels; arrows). Pretreatment with CLR dsRNA preserved epithelial integrity even after TxA treatment (arrowheads). lp, lamina propria (n = 6–8 per group). (Scale bar, 100 µm.)

Fig. 4.

Local injection of CLR dsRNA blocks goblet and mast cell involvement in TxA-induced enteritis. Triplicate sections were stained with Alcian Blue and Fast Red to identify goblet cell mucin, or toluidine blue to identify mast cells. (A) Pretreatment with CLR dsRNA decreased mucin loss due to TxA treatment. In areas adjacent to epithelial cell loss, TxA, but not vehicle, induced marked loss of mucin in goblet cells of rats pretreated with non-CLR dsRNA (arrows). (Scale bar, 100 µm.) (B) Pretreatment with CLR dsRNA prevented a TxA-induced change in mast cell numbers and appearance. In rats pretreated with non-CLR dsRNA, TxA induced mast cell accumulation in the mesenteric fat adjacent to the ileal loop (arrows). Often these cells were associated with dispersed metachromatic granules suggestive of degranulation. (Scale bar, 54 µm.) (C) Quantitation of mast cells showed basal levels occurred after CLR dsRNA and TxA treatment. *P < 0.05 vs. vehicle, ^#P < 0.05 vs. rats pretreated with non-CLR dsRNA and treated with TxA. Values are mean ± SEM, n = 8–12 per group.

Fig. 5.

Local injection of CLR dsRNA modifies TxA-induced signaling pathways. (A–C) Western blot analysis of the cytosolic fraction showed decreases in NF-κB (50%, *P = 0.003) and IκBα (63%, ^#P = 0.029) protein levels (actin, a housekeeping gene, was used as a normalization control) after TxA treatment in non-CLR dsRNA controls (A and C). Pretreatment with CLR dsRNA appears to prevent phosphorylation of IκBα, and thus protein levels of both IκBα and NF-κB in the cytosol remain unchanged even after TxA treatment (B). Confocal images of ileal villi showed diffuse cytoplasmic staining of NF-κB (p65) in buffer-treated rats that were pretreated with non-CLR dsRNA. TxA treatment results in nuclear translocation of NF-κB where it colocalizes with DAPI, a nuclear stain, and loss of CLR prevents activation of NF-κB and translocation to the nucleus. (B) Dashed box area of the tissue is shown in adjacent box at 3× magnification. Arrows show nuclear localization of NF-κB in nonCLRdsRNA tissue and arrowheads show cytoplasmic retention of NF-κB after pretreatment with CLR dsRNA. (D) Agarose gel showed TNF-α mRNA products amplified by RT-PCR of ileal tissue; cyclophilin, a housekeeping gene, was used as a normalization control. TxA treatment increased TNF-α mRNA levels in non-CLR dsRNA controls, whereas pretreatment with CLR dsRNA decreased this effect. Quantitation of RT-PCR data showed that TxA increased TNF-α mRNA levels over that seen with vehicle treatment in controls. Pretreatment with CLR dsRNA decreased this effect. (E) Western blot analysis of the cytosolic fraction from ileal tissue showed increases in pERK1/2 but not total ERK levels after treatment with TxA. TxA treatment increased pERK1/2 levels in controls, whereas pretreatment with CLR dsRNA reduced activation of total ERK to pERK1/2. Quantitation of pERK1/2 normalized to total ERK showed that TxA increased pERK1/2 over vehicle in controls, and pretreatment with CLR dsRNA decreased this effect. *P < 0.05 vs. vehicle, ^#P < 0.05 vs. rats pretreated with non-CLR dsRNA and injected with TxA. Values are mean ± SEM, n = 8–12 per group, two to four experiments.