Histamine receptor H1 is required for TCR-mediated p38 MAPK activation and optimal IFN-γ production in mice (original) (raw)
H1R expression is required for IFN-γ production by CD4+ T cells. MOG35–55 peptide–immunized H1RKO splenocytes produce less IFN-γ and more IL-4 than do splenocytes from immunized WT mice (17). However, it is not clear whether this immune deviation is caused by lack of H1R signaling in CD4+ T cells or in APCs. To investigate the role of H1R in regulating IFN-γ production and Th1 differentiation, CD4+ T cells were purified from WT and H1RKO mice and activated with anti-CD3 and anti-CD28 mAbs in the presence of recombinant IL-12 and neutralizing anti–IL-4 mAb. After 4 days, Th1 effector cells were extensively washed and counted, and equal numbers of cells were restimulated with anti-CD3 mAb for 24 h. Th1 effector cells from H1RKO mice produced considerably less IFN-γ than did WT Th1 cells (Figure 1A). We also examined the production of IL-4 upon restimulation of Th2 effector cells generated in the presence of IL-4 and anti–IFN-γ mAb. A marginal increase in IL-4 production was observed in cells from H1RKO mice compared with cells from WT mice (Figure 1B). Recent studies have established IL-17 as an important cytokine in EAE (20). Consequently, we examined IL-17 production by Th17 cells generated in the presence of IL-6 and TGF-β and anti–IFN-γ and anti–IL-4 mAbs. There was no difference in IL-17 production between H1RKO and WT Th17 cells (Figure 1C). Moreover, we examined the role of H1R in nonpolarized effector cells, generated by stimulating cells in the absence of exogenous cytokines for 4 days. Effector cells were then restimulated with anti-CD3 mAb for 24 h. CD4+ T effector cells from H1RKO mice produced significantly less IFN-γ than did cells from WT mice (Figure 1D). Thus, under these conditions, IFN-γ production in H1RKO effector CD4+ T cells was impaired.
H1R is required for IFN-γ production by CD4+ T cells. Purified CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs either (A) in the presence of IL-12 (4 ng/ml) and anti–IL-4 mAb (10 μg/ml), (B) in the presence of IL-4 (30 ng/ml) and anti–IFN-γ mAb (10 μg/ml), or (C) in the presence of TGF-β (1 ng/ml), IL-6 (30 ng/ml), and anti–IFN-γ (10 μg/ml) and anti-IL4 mAbs (10 μg/ml). After 4 days, the cells were restimulated with anti-CD3 mAb (5 μg/ml) for 24 h. Production of (A) IFN-γ, (B) IL-4, and (C) IL-17 was determined by ELISA in triplicate. *P < 0.05, Student’s t test. (D) CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) mAbs. After 4 days, cells were restimulated with anti-CD3 mAb (5 μg/ml) for 24 h, and IFN-γ production was determined by ELISA. **P = 0.002, Student’s t test. (E and F) WT and H1RKO CD4+ T cells were stimulated as in D for the indicated periods of time. Supernatants were analyzed for (E) IFN-γ (F = 168.8, P < 0.0001, 2-way ANOVA; **P < 0.01, ***P < 0.001, Bonferroni corrected post-hoc comparison) and (F) IL-2 by ELISA. (G) CD4+ T cells from WT and H1RKO mice were stimulated as in E, and 18 h [3H]-thymidine incorporation was measured in total 72 h culture. Data are representative of at least 2 independent experiments.
IFN-γ production by CD4+ T cells contributes to their differentiation into Th1 effector cells (25). To examine the role of H1R signaling in this process, purified CD4+ T cells from H1RKO and WT mice were stimulated with anti-CD3 and anti-CD28 mAbs for different periods of time, and IFN-γ production was quantified. CD4+ T cells from H1RKO mice produced significantly lower IFN-γ than did those from WT mice at all time points examined (Figure 1E). In contrast, no difference in IL-2 production between WT and H1RKO CD4+ T cells was observed (Figure 1F). Furthermore, proliferation was comparable between WT and H1RKO CD4+ T cells (Figure 1G). Taken together, these results demonstrated that H1R expression in CD4+ T cells plays a critical role in regulating IFN-γ production during the activation and differentiation of these cells.
Hrh1 expression is downregulated early upon TCR activation. In order to demonstrate that the reduced secretion of IFN-γ by CD4+ T cells is caused by the absence of a functional H1R in these cells, we carried out H1R complementation in H1RKO CD4+ T cells by retroviral transduction. We generated a retroviral construct using the pEGZ-HA vector where H1R was subcloned downstream of a HA tag and upstream of IRES-EGFP. To confirm that the HA-H1R was properly expressed, we transiently transfected HEK293T cells with the pEGZ-HA-H1R construct and examined its expression by Western blot analysis using anti-HA mAb. A band corresponding to the HA-H1R size (~55 kDa) was present only in HA-H1R–transfected cells (Figure 2A). To demonstrate that the HA-H1R was expressed on the cytoplasmic membrane, the HA-H1R–transfected HEK293T cells were stained using anti-HA mAb and examined by confocal microscopy. HA-H1R was indeed found to be expressed on the cytoplasmic membrane only in HA-H1R–transfected cells (Figure 2B).
Expression and function of HA-H1R in HEK293T cells. (A) HEK293T cells were transfected with empty pEGZ (control) and pEGZ-HA-H1R plasmids, and the expression of HA-H1R was determined by Western blot using an anti-HA mAb. Data are representative of at least 3 independent experiments. (B) HEK293T cells were transfected as in A, fixed, permeabilized, and stained with an anti-HA mAb (red) and Topro-nuclear dye (blue). EGFP expression (green) represents transfected cells. Cells were visualized by confocal microscopy. Data are representative of at least 3 independent experiments. (C) HEK293 cells were transfected with pHA-H1R-Gα11 fusion construct, and membrane fractions were isolated and used in [35S]GTPγS binding assays in the absence (basal) or presence of 10–4 M histamine. Samples were then used in immunoprecipitation using Gα11 antiserum, and the bound [35S]GTPγS was measured by liquid-scintillation spectrometry. ***P < 0.001, Student’s t test.
H1R coupling to second messenger pathways is primarily via Gαq/11 (1). The ability of the transfected HA-H1R to activate Gα11 was tested in a [35S]GTPγS binding assay. When membrane fractions from transfected HEK293 cells were used in the [35S]GTPγS binding assay, HA-H1R activated Gα11 in response to histamine (Figure 2C). Taken together, these results show that HA-H1R is properly expressed and is functional.
We next performed retroviral transduction: CD4+ T cells were isolated from H1RKO and WT mice, activated with anti-CD3 and anti-CD28 mAbs for 16 h, and transduced with either pEGZ or pEGZ-HA-H1R retroviruses. Expression of HA-H1R in transduced CD4+ T cells was confirmed by confocal microscopy and flow cytometry (data not shown). After 2 days, transduced CD4+ T cells were isolated by cell sorting based on EGFP expression, and equal numbers of cells were activated with anti-CD3 mAb for an additional 24 h. Both pEGZ- and pEGZ-HA-H1R–transduced CD4+ T cells from H1RKO mice produced significantly lower levels of IFN-γ than did those from WT mice (Figure 3A). These results indicate that the expression of H1R in activated CD4+ T cells does not restore IFN-γ production by H1R-deficient cells.
Hrh1 is downregulated upon activation in CD4+ T cells. (A) CD4+ T cells from WT and H1RKO mice were stimulated in the presence of anti-CD3 and anti-CD28 mAbs for 16 h and then retrovirally transduced with pEGZ-HA-H1R or with empty pEGZ control plasmids. Transduced, sorted EGFP+ cells were then restimulated with anti-CD3 mAb, and 24 h later, the supernatants were harvested for determination of IFN-γ by ELISA in triplicate. Data are representative of 2 independent experiments. ***P < 0.001, Student’s t test. (B) Freshly isolated CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 and anti-CD28 mAbs. After 24 h, IFN-γ production was determined by ELISA. Data are representative of at least 3 independent experiments. ***P < 0.001, Student’s t test. (C and D) CD4+ T cells were isolated from WT mice and stimulated with anti-CD3 and anti-CD28 mAbs. Cells were harvested at the indicated time points; total RNA was isolated and used to examine Hrh1 expression by conventional RT-PCR with Hprt1 as the endogenous control (C) and by quantitative real-time RT-PCR relative to B2m as the endogenous control (D). Data (representative of at least 3 independent experiments) are presented as expression relative to the unstimulated CD4+ T cells.
Retroviral transduction requires prior activation of CD4+ T cells for at least 16 h to induce cell cycling. Thus, if H1R is normally required during the early phase of activation concomitant with TCR engagement, the retroviral transduction would not rescue the H1R deficiency. The results presented above (Figure 1C) indicated that IFN-γ production was already reduced at 36 h in H1RKO CD4+ T cells compared with WT cells. We therefore examined IFN-γ production by H1RKO CD4+ T cells earlier during activation with anti-CD3 and anti-CD28 mAbs. Although lower levels of IFN-γ were present in WT CD4+ T cells at 24 h of activation, H1RKO CD4+ T cells still produced significantly less IFN-γ (Figure 3B), indicating that H1R plays a role early during the activation of CD4+ T cells.
To our knowledge, H1R expression during mouse T cell activation has not previously been investigated. We therefore analyzed Hrh1 expression in WT CD4+ T cells stimulated for different periods of time with anti-CD3 and anti-CD28 mAbs. Relative levels of Hrh1 were examined by conventional and quantitative real-time RT-PCR. CD4+ T cells markedly downregulated Hrh1 expression by 24 h after activation (Figure 3, C and D), further indicating that H1R plays a role early (i.e., less than 24 h) after TCR engagement and that it is not required for IFN-γ production by CD4+ T cells once the cells are activated.
Selective H1R expression in T cells in Tg mice restores IFN-γ production. To examine the role of H1R during the initial activation of CD4+ T cells, we generated transgenic mice expressing H1R under the control of distal lymphocyte protein tyrosine kinase (dlck) promoter, which drives expression in T cells (26). Tg mice were generated directly on the C57BL/6J background. Two Tg founders were identified and crossed to H1RKO mice to obtain H1RKO mice expressing H1R selectively in T cells (H1RKO-Tg mice). The expression of the transgene in CD4+ T cells from 2 lines (H1RKO-Tg1 and H1RKO-Tg3) was confirmed by RT-PCR using transgene-specific primers (Figure 4A). We examined the surface expression of the transgene in CD4+ T cells by immunostaining using anti-HA mAb and confocal microscopy (Figure 4B). The transgene was expressed in CD4+ T cells from both transgenic lines. No differences in the total numbers or distribution of T cell subpopulations in the thymus and peripheral lymphoid tissues were observed among the WT, H1RKO, H1RKO-Tg1, and H1RKO-Tg3 lines (data not shown).
Transgenic expression of H1R in H1RKO CD4+ T cells complements IFN-γ production. (A) Hrh1 transgene expression was analyzed by RT-PCR in CD4+ T cells from WT and H1RKO mice and the 2 independent lines of H1R transgenic mice crossed with H1RKO mice, HIRKO-Tg1 and HIRKO-Tg3. (B) CD4+ T cells were stained with anti-HA mAb (red) and visualized by confocal microscopy. Nuclear stain Topro (blue) is shown. (C) CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs for 72 h, and IFN-γ was determined by ELISA. Data are expressed as IFN-γ production relative to that by WT cells (set as 100%). (D) CD4+ T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated as in C for the indicated periods of time, and IFN-γ was determined by ELISA. F = 55.1, P < 0.0001, 2-way ANOVA. **P < 0.01, ***P < 0.001, Bonferroni corrected post-hoc comparison. (E) CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of IL-12 (4 ng/ml) and anti-IL-4 mAb (10 μg/ml). After 4 days, cells were restimulated and IFN-γ production was determined. F = 25.4, P < 0.001; 1-way ANOVA. **P < 0.01, Bonferroni corrected post-hoc comparison. (F) CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs. After 4 days, cells were restimulated with anti-CD3 mAb for 24 h, and IFN-γ production was determined by ELISA. F = 288.0, P < 0.0001; 1-way ANOVA. ***P < 0.001, Bonferroni corrected post-hoc comparison. Data are representative of at least 3 independent experiments.
We then examined whether the expression of H1R in H1RKO CD4+ T cells restored IFN-γ production. CD4+ T cells from WT, H1RKO, and H1RKO-Tg mice were stimulated with anti-CD3 and anti-CD28 mAbs, and IFN-γ levels were quantified. The levels of IFN-γ in CD4+ T cells from HIRKO-Tg3 mice were comparable to those of WT CD4+ T cells, whereas IFN-γ production by HIRKO-Tg1 CD4+ T cells remained slightly lower than that in WT CD4+ T cells but nevertheless significantly higher than that in H1RKO CD4+ T cells (Figure 4C). Analyses at different periods of time after activation confirmed that the transgenic expression of H1R in H1RKO-Tg3 CD4+ T cells fully restored IFN-γ production (Figure 4D).
We also studied IFN-γ production by Th1 polarized and nonpolarized effector cells from H1RKO-Tg mice. CD4+ T cells from WT, H1RKO, and H1RKO-Tg mice were differentiated in the absence of exogenous cytokines (nonpolarized) or in the presence of recombinant IL-12 and anti–IL-4 mAb (Th1). After 4 days, effector cells were restimulated with anti-CD3 mAb for 24 h, and IFN-γ production was measured. Compared with H1RKO effectors, both Th1 polarized (Figure 4E) and nonpolarized CD4+ effector T cells (Figure 4F) from H1RKO-Tg3 mice produced significantly more IFN-γ. Furthermore, the levels of IFN-γ in H1RKO-Tg cells were comparable to those in WT CD4+ T cells. Together, these data demonstrate that the presence of H1R at the time of activation of CD4+ T cells under both polarizing and nonpolarizing conditions regulates IFN-γ production and Th1 differentiation.
Impaired activation of p38 MAPK by TCR ligation in H1RKO CD4+ T cells. In order to dissect the molecular mechanism of H1R signaling in regulating IFN-γ production by CD4+ T cells, we examined the signaling pathways that have been previously associated with H1R in other cell types. NF-κB has been shown to be activated through H1R in green monkey kidney cells (5) and has been associated with regulation of IFN-γ expression in CD4+ T cells (27). Therefore, we performed EMSA to examine NF-κB DNA binding. CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for different periods of time. A significant difference in NF-κB activation between WT and H1RKO CD4+ T cells was not detected (Figure 5A). STAT1 is also known to regulate IFN-γ expression in CD4+ T cells (28), and it has recently been shown that H1R signaling regulates STAT1 phosphorylation in splenocytes (6). Therefore we examined activation of STAT1 by Western blot analysis in stimulated CD4+ T cells. Prior to 3 h of activation, STAT1 phosphorylation was not detected in WT or H1RKO cells (data not shown); however, phospho-STAT1 was detected after 3 h of activation, but there was no difference in STAT-1 phosphorylation between WT and H1RKO CD4+ T cells (Figure 5B). Although H1R signaling has also been reported to regulate STAT4 phosphorylation in splenocytes (7), phospho-STAT4 was not detected in WT and H1RKO CD4+ T cells after activation with anti-CD3 and anti-CD28 mAbs (data not shown).
Activation of p38 MAPK by TCR ligation requires H1R signals. (A) Purified CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and nuclear extracts were prepared and analyzed for NF-κB DNA binding by EMSA. (B) CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-STAT1 (P-STAT1) and total STAT1 by Western blot analysis. Actin was used as loading control. (C) CD4+ T cells from WT and H1RKO mice were treated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-p38 MAPK and total p38 by Western blot analysis. (D) CD4+ T cells from WT and H1RKO mice were activated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were prepared and analyzed for phospho-ERK and total ERK by Western blot analysis. (E) CD4+ T cells from WT, H1RKO, and HIRKO-Tg3 mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for phospho-p38, total p38, and actin by Western blotting. Data are representative of at least 2 independent experiments.
H1R ligation has recently been shown to lead to the phosphorylation of p38 MAPK in DDT1MF-2 cells (10) and in human aortic endothelial cells (29). Activation of the p38 MAPK pathway is required for IFN-γ production and Th1 differentiation (30). We therefore examined the activation of p38 MAPK by Western blot analysis. CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for different periods of time. p38 MAPK was activated in WT CD4+ T cells but was markedly impaired in H1RKO CD4+ T cells (Figure 5C). In contrast, no difference in ERK MAPK activation was observed between WT and H1RKO CD4+ T cells (Figure 5D). As we reported previously (31), activation of JNK MAPK was not detected at earlier time points in both WT and H1RKO CD4+ T cells stimulated with anti-CD3 and anti-CD28 mAbs (data not shown). We further examined the activation of p38 MAPK by TCR ligation in H1RKO-Tg CD4+ T cells. Unlike H1RKO CD4+ T cells, the levels of phospho-p38 MAPK in H1RKO-Tg CD4+ T cells were equivalent to those in the WT CD4+ T cells (Figure 5E). Thus, TCR-mediated activation of p38 MAPK required the presence of H1R in CD4+ T cells.
Activation of p38 MAPK by TCR is mediated by histamine/H1R binding. To understand the mechanism by which H1R may regulate TCR-mediated p38 MAPK activation, we examined whether histamine itself could activate the p38 MAPK in CD4+ T cells. Histamine was already present at low concentrations (about 10–7 M) in the serum used for the culture medium. Therefore, we assessed p38 MAPK phosphorylation in response to histamine using medium prepared with histamine-depleted serum (14). CD4+ T cells from WT and H1RKO mice were resuspended in the histamine-free medium and subsequently treated with histamine. p38 MAPK was activated by histamine in WT CD4+ T cells but not in H1RKO CD4+ T cells (Figure 6A), indicating that histamine activates this pathway in CD4+ T cells through H1R.
Activation of p38 MAPK by TCR ligation is mediated by histamine/H1R binding. (A) CD4+ T cells from WT and H1RKO mice were treated with histamine (10–7 M) for the indicated periods of time in the histamine-free medium. Whole-cell extracts were used to analyze phospho-p38, total p38, and actin by Western blotting. (B) CD4+ T cells were isolated from WT and H1RKO mice and stimulated with anti-CD3 and anti-CD28 mAbs in the histamine free-medium for the indicated periods of time. CD4+ T cells stimulated in medium containing 10–7 M histamine (Hist) are shown as positive control for p38 MAPK activation. Phospho-p38, total p38, and actin are shown. (C) CD4+ T cells from WT and H1RKO mice were incubated with anti-CD3 and anti-CD28 mAbs, 10–7 M histamine, or both in the histamine-free medium for 30 minutes, and whole-cell lysates were analyzed for phospho-p38, total p-38, and actin by Western blotting. (D) CD4+ T cells from WT and H1RKO mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and whole-cell lysates were analyzed for T-bet expression by Western blot. Actin is shown as loading control. (E) Purified CD4+ T cells from WT, H1RKO, and H1RKO-MKK6Glu Tg mice were stimulated with anti-CD3 and anti-CD28 mAbs for the indicated periods of time, and supernatants were analyzed for IFN-γ production by ELISA in triplicate. F = 21.7, P < 0.0001, 2-way ANOVA. ***P < 0.001, Bonferroni corrected post-hoc comparison. Data are representative of at least 2 independent experiments.
Because histamine was present in the normal medium used to activate CD4+ T cells with anti-CD3 and anti-CD28 mAbs (Figure 5, C and E), it was possible that the activation of p38 MAPK by TCR ligation was codependent upon histamine signaling through the H1R. To test this possibility, we examined p38 MAPK activation upon anti-CD3 and anti-CD28 mAb stimulation in histamine-free media. TCR ligation failed to activate p38 MAPK in both WT and H1RKO CD4+ T cells in histamine-free media (Figure 6B). In contrast, the absence of histamine did not affect TCR-mediated ERK activation (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI32792DS1) or intracellular calcium mobilization (data not shown) in WT CD4+ T cells. To further demonstrate the selective requirement for histamine in TCR-mediated p38 MAPK activation, WT and H1RKO CD4+ T cells were stimulated in histamine-free media with anti-CD3 and anti-CD28 mAbs in the presence of histamine. TCR-mediated p38 MAPK activation was restored by histamine in WT CD4+ T cells but not in H1RKO CD4+ T cells (Figure 6C), indicating that binding of histamine to H1R was required for activation of p38 MAPK upon TCR ligation. Interestingly, the levels of phospho-p38 MAPK in WT CD4+ T cells treated with anti-CD3 and anti-CD28 mAbs and histamine were similar to the levels obtained when the cells were treated with histamine alone (Figure 6C). The inability of TCR stimulation to activate p38 MAPK in H1R-deficient cells in normal histamine-containing media (Figure 5C), activate p38 MAPK in the histamine-free media (Figure 6B), or further increase p38 MAPK activation when histamine was added back to the histamine-free media strongly suggest that the activation of p38 MAPK observed upon TCR ligation is dependent upon concomitant H1R signaling.
Although the precise mechanism by which p38 MAPK regulates IFN-γ production in CD4+ T cells remains unclear, recent studies have suggested that the activation of the this MAPK pathway is required for T-bet expression (32, 33), and T-bet regulates IFN-γ production (34). We therefore examined T-bet expression by Western blot analysis during activation of WT and H1RKO CD4+ T cells. T-bet levels were lower in activated H1RKO CD4+ T cells than in WT CD4+ T cells (Figure 6D). Thus, the impairment in p38 MAPK activation in the absence of H1R reduces the T-bet expression and thereby IFN-γ production by CD4+ T cells during TCR activation.
In order to demonstrate that reduced p38 MAPK activation in H1RKO CD4+ T cells is responsible for decreased IFN-γ production by these cells, we crossed H1RKO mice with the previously described _dlck_-MKK6Glu Tg mice (30). These mice express a constitutively active form of MKK6, a specific upstream activator of p38 MAPK, under the control of dlck promoter. Thus p38 MAPK is constitutively and selectively active in T cells in these mice. Anti-CD3 and anti-CD28 mAb–stimulated CD4+ T cells from H1RKO-MKK6Glu Tg mice produced significantly more IFN-γ than did CD4+ T cells from littermate H1RKO mice (Figure 6E), indicating that the diminished activation of p38 MAPK in H1RKO CD4+ T cells is responsible for the reduced IFN-γ production by these cells.
H1R signaling directly in CD4+ T cells regulates encephalitogenic Th1 effector responses. As a shared autoimmune disease susceptibility gene, Hrh1 has been shown to control numerous disease associated subphenotypes, including blood-brain barrier permeability, antigen presentation, and delayed-type hypersensitivity responses (13, 35). To assess whether H1R signaling in CD4+ T cells influences EAE by regulating encephalitogenic Th1 responses, we examined the susceptibility of H1RKO and H1RKO-Tg mice to EAE using the classical MOG35–55-CFA plus pertussis toxin (PTX) model and the 2× MOG35–55-CFA model (36), which does not use PTX as an ancillary adjuvant. Regression analysis (36) revealed that the clinical disease courses elicited with the MOG35–55-CFA plus PTX protocol (Figure 7A) was significantly more severe in WT and H1RKO-Tg mice compared with H1RKO mice (WT, F = 132.1, P < 0.0001; HIRKO-Tg1, F = 127.5, P < 0.0001; HIRKO-Tg3, F = 83.8, P < 0.0001). Identical results were obtained with the 2× MOG35–55 protocol (Figure 7B; WT, F = 226.9, P < 0.0001; HIRKO-Tg1, F = 134.0, P < 0.0001; HIRKO-Tg3, F = 215.8, P < 0.0001).
H1R signaling directly in CD4+ T cells regulates encephalitogenic Th1 effector responses. Clinical EAE course (A and B), severity of CNS pathology (C and D), and ex vivo cytokine responses (E and F) of WT, H1RKO, and H1RKO-Tg mice were compared following immunization with MOG35–55-CFA plus PTX (A, C, and E) or 2× MOG35–55 and CFA (B, D, and F). Cytokine production was assessed by stimulating splenocytes with MOG35–55 on day 10 after injection, and supernatants were collected and quantified by ELISA in triplicate. The significance of differences in the course of clinical disease, CNS pathology indices, and cytokine responses were assessed by regression analysis (63), χ2 test, or ANOVA followed by Bonferroni corrected post-hoc comparisons. With the exception of TNF-α and IL-17 production, significant differences among the strains were detected for all parameters at P < 0.0001 — WT, H1RKO-Tg1, and H1RKO-Tg3 groups were equivalent and all significantly different from the H1RKO group.
Analysis of EAE-associated clinical quantitative trait variables (37) revealed that the mean day of onset, cumulative disease score, number of days affected, overall severity index, and peak score were significantly different among the strains immunized with either MOG35–55-CFA plus PTX or 2× MOG35–55-CFA (Table 1). Bonferroni corrected post-hoc multiple comparisons for each trait variable revealed that the values were not significantly different among WT, HIRKO-Tg1, and HIRKO-Tg3 mice, all of which were significantly greater than those in H1RKO mice. Additionally, compared with H1RKO mice, both HIRKO-Tg1 and HIRKO-Tg3 mice immunized with MOG35–55-CFA plus PTX (Figure 7C) and 2× MOG35–55-CFA (Figure 7D) exhibited significantly more severe overall CNS pathology (38), which was equivalent in severity to that seen in WT mice. Therefore, H1R expression in CD4+ T cells alone was capable of complementing EAE susceptibility in H1R-deficient animals.
Clinical disease traits following immunization with MOG35–55-CFA plus PTX or with 2× MOG35–55-CFA
We also examined cytokine production following ex vivo stimulation of splenocytes from mice immunized with MOG35–55-CFA plus PTX and 2× MOG35–55-CFA. The H1R transgene fully complemented IFN-γ production by H1RKO CD4+ T cells and restored IL-4 production to WT levels (Figure 7, E and F). In contrast, no significant differences in TNF-α or IL-17 production were detected among WT, H1RKO, and H1RKO-Tg mice. Together, these data indicate that H1R signaling in CD4+ T cells complements EAE severity independently of TNF-α and IL-17 production.