Lack of Histamine H4-Receptor Expression Aggravates TNBS-Induced Acute Colitis Symptoms in Mice

Abstract

Inflammatory bowel diseases (IBD) are a growing health problem worldwide, severely affecting patients' life qualities and life expectancies. Therapeutic options, which are rare and focus on symptoms associated with the disease, suffer from increasing numbers of patients refractory to the established strategies. Thus, in order to generate new therapeutic regimens, the detailed understanding of the pathogenic mechanisms causing IBD is necessary. Histamine is an inflammatory mediator associated with IBD. Four histamine receptors are currently known of which the histamine H₄-receptor (H₄R) has been shown to possess a pro-inflammatory function in several experimental models of inflammatory diseases, including dextran sodium sulfate (DSS)-induced colitis in mice. No single model reflects the complexity of human IBD, but each model provides valuable information on specific aspects of IBD pathogenesis. While DSS-induced colitis mostly relies on innate immune mechanisms, trinitrobenzene sulfonic acid (TNBS)-induced colitis rather reflects T-cell mechanisms. Consequently, an observation made in a single model has to be verified in at least one other model. Therefore, in the present study we investigated the effect of genetic blockade of H₄R-signaling in mice subjected to the model of TNBS-induced acute colitis. We analyzed severity and progression of clinical signs of colitis, as well as histopathologic alterations in the colon and local cytokine production. Genetic ablation of H₄R expression worsened clinical signs of acute colitis and histological appearance of colon inflammation after TNBS application. Moreover, TNBS instillation enhanced local synthesis of inflammatory mediators associated with a neutrophilic response, i.e., CXCL1, CXCL2, and interleukin-6. Lastly, also myeloperoxidase concentration, indicative for the presence of neutrophils, was elevated in cola of TNBS-treated mice due to the absence of H₄R expression. Our results indicate an anti-inflammatory role of histamine via H₄R in TNBS-induced acute neutrophilic colitis in mice, thus questioning the strategy of pharmacological H₄R blocked as new therapeutic option for patients suffering from IBD.

Keywords: GPCR; TNBS-induced colitis; histamine; inflammation; mouse models; receptor.

FIGURE 1

Lack of H₄R expression worsens TNBS-induced colitis. Wild type (WT) or H₄R-deficient (H₄R^-/-) BALB/cJ mice were treated intra-rectally with 2 mg/100 μl^∗mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH) at day 0. Mice were inspected for their health status every 6 h for a total of 3 days. (A) Every 24 h the disease activity of each single mouse was evaluated according to a scoring system which takes into account the body weight, the stool consistency, and the degree of anal bleeding and assigns a numeric value to the respective status (Alex et al., 2009). The sum of the single values of a mouse refers to as its disease activity index (DAI)-score, being the higher the worse is the health condition of a mouse. ^∗pWT; TNBS vs. H₄R^-/-; TNBS < 0.05 (Two-Way ANOVA), n = 7/group. (B) Mice which had to be euthanized due to severely impacted health status throughout the 3 days observation period were recorded. On day 3 all remaining mice were killed for analysis. The relative numbers of surviving mice were plotted against the time. ^∗pWT; TNBS vs. H₄R^-/-; TNBS < 0.05 (Log-rank (Mantle-Cox) test), n = 7/group. (C) The cola of the mice were prepared and their lengths recorded. ^∗∗∗<0.005 (One-Way ANOVA), TNBS: n = 7/group; EtOH: n = 4/group.

FIGURE 2

Lack of H₄R expression enhances histopathological findings of colonic inflammation. Wild type (WT) or H₄R-deficient (H₄R^-/-) BALB/cJ mice were treated with 2 mg/100 μl^∗mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH). After dissection, the cola were cut in three sections, proximal, medial, and distal. Parts of the sections were fixed in formaldehyde, embedded in paraffin, cut into slices, and stained with hematoxylin/eosin. (A) Representative micro-photographs of tissue slices of medial colon sections of each experimental group are demonstrated. (B) The tissue slices as demonstrated in (A) were analyzed in a blinded fashion for pathological derangements as indicated above the single graphs applying a scoring matrix, i.e., the higher the score, the worse the histological appearance. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005 (One-Way ANOVA), TNBS: n = 7/group; EtOH: n = 4/group. (C) Representative micro-photographs in enhanced magnification of sections obtained from cola of TNBS-treated mice.

FIGURE 3

Lack of H₄R expression enhances mRNA expression of inflammation-associated cytokines and chemokines. Wild type (WT) or H₄R-deficient (H₄R^-/-) BALB/cJ mice were treated with 2 mg/100 μl^∗mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH). After dissection, the cola were cut in three sections, proximal, medial, and distal. From parts of the medial sections mRNAs were extracted and analyzed by RT-qPCR array. Relative abundancies of individual mRNAs as indicated on the abscissa were calculated relative to the abundancy of the GAPDH mRNA. Reported are the values comparative to those observed in solvent-treated WT mice, which were set to 1. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005 (Student’s t-test), n = 4/group.

FIGURE 4

Lack of H₄R expression enhances protein expression of inflammation-associated cytokines and chemokines. Wild type (WT) or H₄R-deficient (H₄R^-/-) BALB/cJ mice were treated with 2 mg/100 μl^∗mouse TNBS (TNBS) or with an equivalent volume of the solvent mixture EtOH/PBS (EtOH). After dissection, the cola were cut in three sections, proximal, medial, and distal. From parts of the medial sections proteins were extracted and analyzed by Luminex Array (cytokines, chemokines) and ELISA (MPO). Analyte concentrations are reported relative to the total protein concentrations of the colon lysates. ^∗∗∗p < 0.005 (One-Way ANOVA), TNBS: n = 7/group; EtOH: n = 4/group.