Combinatorial roles for histamine H1-H2 and H3-H4 receptors in autoimmune inflammatory disease of the central nervous system

Abstract

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts its effects through four different G protein-coupled receptors designated H(1)-H(4). We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H(1) R and H(2) R are propathogenic, while H(3) R and H(4) R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease-modifying therapy (DMT) in MS. To test this hypothesis, we generated H(1) H(2) RKO and H(3) H(4) RKO mice and studied them for susceptibility to EAE. Compared with wild-type (WT) mice, H(1) H(2) RKO mice developed a less severe clinical disease course, whereas the disease course of H(3) H(4) RKO mice was more severe. H(1) H(2) RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H(3) H(4) RKO mice. Additionally, splenocytes from immunized H(1) H(2) RKO mice produced less interferon(IFN)-γ and interleukin(IL)-17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.

Figure 1

H₁H₂RKO and H₃H₄RKO mice exhibit differential susceptibility to EAE. B6 (n = 12), H₁H₂RKO (n = 23), and H₃H₄RKO (n = 15) were immunized twice with MOG_35–55-CFA. (A) The clinical EAE scores following immunization were recorded and the significance of the differences between the clinical courses of disease was calculated by regression analysis. The best fit curve is shown. (B and C) The severity of CNS pathology in (B) brain and (C) spinal cord was determined and shown as mean + standard deviation. The significance of differences observed was determined using the Kruskal–Walis test followed by Dunns post-hoc multiple comparison test (*p < 0.05; **p < 0.01; ***p < 0.001). (D) BBB permeability was compared and expressed as mean + standard error of the mean (SEM) of n = 10 mice per strain. The significance of differences in BBB permeability indexes of B6, H₁H₂RKO, and H₃H₄RKO mice at d10 post immunization was calculated by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc multiple comparison test (***p < 0.001). (I_CSF/I_Blood - Permeability index, expressed as the ratio of the fluorescence intensity of the CSF divided by the fluorescence intensity of the plasma). Data are representative of two independent experiments.

Figure 2

Naïve H₁H₂RKO and H₃H₄RKO mice exhibit normal immune systems. Single-cell suspensions of thymus, lymph node, and spleen were generated from B6, H₁H₂RKO and H₃H₄RKO mice. (A) The total number of thymic, lymph node, and spleen cells was enumerated and shown as mean + SEM of n = 10 per strain. Flow cytometric analysis of immune cell subpopulations in (B) thymus, (C) lymph node and (D) spleen of B6, H₁H₂RKO, and H₃H₄RKO mice was also performed and shown as mean + SEM of n = 10 per strain. (E) The frequency of Foxp3⁺ cells in thymus, spleen, and lymph node was also determined and shown as mean + SEM of n = 10 per strain. Significance of differences was determined by ANOVA followed by Bonferroni post-hoc multiple comparison (**p < 0.01; ***p < 0.001). Data are representative of two independent experiments.

Figure 3

Ex vivo MOG_35–55-specific T-cell proliferation and cytokine profiles of immunized B6, H₁H₂RKO, and H₃H₄RKO mice. (A) MOG_35–55-specific T-cell proliferative responses were evaluated by ³H-thymidine incorporation. Mean counts per minute + standard deviation were calculated from triplicate wells of n = 10 per strain. The significance of differences was calculated by ANOVA followed by Bonferroni post-hoc multiple comparisons. (B) IFN-γ and (C) IL-17 production by MOG_35–55-stimulated DLNs and splenocytes from B6, H₁H₂RKO, and H₃H₄RKO mice were evaluated by ELISA and shown as mean + SEM of n = 10 per strain. Significance of differences observed in cytokine production was determined by one-way ANOVA followed by Bonferroni’s post-hoc multiple comparison test (**p < 0.01; ***p < 0.001). Data are representative of two independent experiments.

Figure 4

HRs influence the polarization of in vitro activated CD4⁺ T cells. CD4⁺T cells from B6, H₁H₂RKO, and H₃H₄RKO mice were activated with anti-CD3 and anti-CD28 mAbs. (A) IFN-γ, (B) IL-4, and (C) IL-2 production at 24, 48, and 72 h poststimulation was determined by ELISA and shown as mean + SEM of n = 4 per strain. In (A) and (C) significance of differences in cytokine production was determined by ANOVA followed by Bonferroni post-hoc multiple comparison. In (B) the significance of differences in IL-4 production was determined using a rank transformed nonparametric two-way ANOVA followed by Bonferroni post-hoc multiple comparison (**p < 0.01; ***p < 0.001). Data are representative of two independent experiments.

Figure 5

Compensatory upregulation of HRs in H₁H₂RKO and H₃H₄RKO CD4⁺T cells. Hrh1 Hrh2 Hrh3 Hrh4 and Hdc mRNA expression levels were determined by qRT-PCR and analyzed using comparative threshold cycle method with β2-microglobulin as an endogenous control. Receptor expression levels are normalized to that of WT levels. (A) HR expression was evaluated in individual HRKO CD4⁺T cells. (B) Hrh4 expression was evaluated in H₁RKO, H₂RKO, and H₁H₂RKO CD4⁺ T cells. (C) Hrh1 and (D) Hrh2 expression was evaluated in H₃RKO, H₄RKO, and H₃H₄RKO CD4⁺ T cells. (E and F) CD4⁺T cells from B6, H₁H₂RKO, and H₃H₄RKO mice were activated with anti-CD3 and anti-CD28 mAbs. (E) Hdc expression was analyzed by qRT-PCR and (F) HA production was determined by EIA at 24, 48, and 72 h poststimulation. Data are representative of two independent experiments. In (B), (C), and (D), significance of differences in HR expression levels was determined by one-way ANOVA followed by Bonferroni post-hoc multiple comparison. In (E and F) significance of differences in HA production was determined by two-way ANOVA followed by Bonferroni post-hoc multiple comparison (*p = 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). All data are shown as mean + standard error of the mean of n = 3 per strain and are representative of two independent experiments.