Histamine drives severity of innate inflammation via histamine 4 receptor in murine experimental colitis

Abstract

Ulcerative colitis (UC) patients exhibit elevated histamine, but how histamine exacerbates disease is unclear as targeting histamine 1 receptor (H1R) or H2R is clinically ineffective. We hypothesized that histamine functioned instead through the other colon-expressed histamine receptor, H4R. In humans, UC patient biopsies exhibited increased H4R RNA and protein expression over control tissue, and immunohistochemistry showed that H4R was in proximity to immunopathogenic myeloperoxidase-positive neutrophils. To characterize this association further, we employed both the oxazolone (Ox)- and dextran sulfate sodium (DSS)-induced experimental colitis mouse models and also found upregulated H4R expression. Mast cell (MC)-derived histamine and H4R drove experimental colitis, as H4R^-/- mice had lower symptom scores, neutrophil-recruitment mediators (colonic interleukin-6 (IL-6), CXCL1, CXCL2), and mucosal neutrophil infiltration than wild-type (WT) mice, as did MC-deficient Kit^W-sh/W-sh mice reconstituted with histidine decarboxylase-deficient (HDC^-/-) bone marrow-derived MCs compared with WT-reconstituted mice; adaptive responses remained intact. Furthermore, Rag2^-/- × H4R^-/- mice had reduced survival, exacerbated colitis, and increased bacterial translocation than Rag2^-/- mice, revealing an innate protective antibacterial role for H4R. Taken together, colonic MC-derived histamine initiates granulocyte infiltration into the colonic mucosa through H4R, suggesting alternative therapeutic targets beyond adaptive immunity for UC.

Figure 1

H4R is increased in active UC and regionally co-localizes with MPO⁺ cells. (A) Quantitative real-time PCR of RNA isolated from biopsy lysates from non-UC individuals (Control) and UC patients displayed as fold induction vs control for H1R, H2R, and H4R. (B) Relative intensity quantification of H4R IHC from non-UC individuals and UC patients displayed as relative units/mm². (C) Relative intensity quantification of MPO IHC for non-UC individuals and UC patients in relative units/mm². (D) Example of H4R/MPO staining on serial sections of colon tissue (circle indicates area of regional co-localization). *P < 0.05.

Figure 2

H4R drives clinical and histologic severity in Ox- and DSS-induced experimental colitis. (A–D, I) Ox-induced and (E–H, J) DSS-induced colitis experiments. (A, E) Percentage weight change in BALB/c vs H4R^−/− mice (n = 16–26 mice/group). (B, F) Disease activity index (DAI) score for WT and H4R^−/− mice. (C, G) Composite histology score for WT and H4R^−/− mice. (D, H) Component histology score for cellular infiltrate and mucosal damage in WT and H4R^−/− mice. (I, J) Representative histology in WT and H4R^−/− mice. *P < 0.05, **P < 0.01, ***P < 0.001. Data represented as mean ± SEM from 3–4 independent experiments.

Figure 3

H4R signaling allows for neutrophil recruitment into the mucosa during experimental colitis. (A) Percentage neutrophil abscesses/hpf present within colon sections (3–5 sections/animal). (B) Representative MPO IHC in WT and H4R^−/− Ox-treated mice. (C) Mucosal MPO⁺ cells/mm² quantification in WT vs H4R^−/− Ox-treated mice. (D) Colonic IL-6, CXCL1, and CXCL2 protein quantification by ELISA. *P < 0.05, ***P < 0.001. Data represented as mean ± SEM from 3 independent experiments.

Figure 4

MC-derived histamine drives clinical and histologic severity of experimental colitis. (A) Percentage weight change in Ox- or vehicle-treated WT and HDC^−/− mice (n = 11–24 mice/group). (B) DAI score in Ox- or vehicle-treated WT and HDC^−/− mice (day 2). (C) Histology score in Ox- or vehicle-treated WT and HDC^−/− mice (day 3). (D) Percentage weight change in Ox- or vehicle-treated WT or HDC^−/− BMMC–reconstituted Sash mice (n = 7–10 mice/group). (E) DAI score in Ox- or vehicle-treated WT or HDC^−/− BMMC–reconstituted Sash mice (day 2). (F) Histology score in Ox- or vehicle-treated WT or HDC^−/− BMMC–reconstituted Sash mice (day 3). *P < 0.05, **P < 0.01, ***P < 0.001. Data represented as mean ± SEM from 3 independent experiments.

Figure 5

MC-derived histamine aids in neutrophil recruitment into the mucosa during experimental colitis. (A) Percentage neutrophil abscesses/hpf present within colon sections (n = 3–5 sections/animal) from Ox- or vehicle-treated WT and HDC^−/− mice. (B) Quantitative real-time PCR of colon lysates for IL-6, CXCL1, and CXCL2 from Ox- or vehicle-treated WT and HDC^−/− mice; fold induction shown relative to vehicle. (C) Percentage neutrophil abscesses/hpf present within colonic sections (n = 3–5 sections/animal) from Ox- or vehicle-treated WT and HDC^−/− BMMC–reconstituted Sash mice. (D) Quantitative real-time PCR of colon lysates for IL-6, CXCL1, and CXCL2 from Ox- or vehicle-treated WT and HDC^−/− BMMC–reconstituted Sash mice. **P < 0.05, **P < 0.01, ***P < 0.001. Data represented as mean ± SEM from 3 independent experiments.

Figure 6

T cell responses during experimental colitis are unaffected by MC-derived histamine or H4R. (A) Quantitative real-time PCR for T cell–derived cytokines in colonic lysates from Ox-or vehicle-treated WT and H4R^−/− mice (day 3). (B) Frequency of CD4⁺CD45⁺ LPMCs isolated from colon from Ox- or vehicle-treated WT and H4R^−/− mice (day 3). (C) Quantitative real-time PCR for IL-17A in colonic lysates from DSS- or vehicle-treated WT and H4R^−/− mice (day 9). (D) Quantitative real-time PCR from colonic lysates from Ox- or vehicle-treated WT vs HDC^−/− mice (day 3). (E) Quantitative real-time PCR from colonic lysates from Ox- or vehicle-treated WT or HDC^−/− BMMC–reconstituted Sash mice (day 3). *P < 0.05, **P < 0.01. Data represented as mean ± SEM from 3 independent experiments, except B (2 independent experiments).

Figure 7

In the absence of functional adaptive immunity, H4R protects against weight loss, death, and increased bacterial translocation to the MLN after DSS-induced colitis. (A) Percentage weight change for DSS- or vehicle-treated Rag2^−/− and Rag2^−/−×H4R^−/− mice (n = 5–17 mice/group). (B) Survival curve to day 7 in DSS- or vehicle-treated Rag2^−/− and Rag2^−/−×H4R^−/− mice. (C) CFU counts from sterile MLN on blood agar plates on day 7 from DSS- or vehicle-treated Rag2^−/− and Rag2^−/−×H4R^−/− mice. *P < 0.05. Data represented as mean ± SEM from 3 independent experiments.