C57BL/6 female mice (6–8 weeks old) were purchased from Orient Bio (Daejeon, South Korea) and maintained at the Hanyang University mouse facilities under pathogen-free conditions with ad libitum feeding. All animal protocols were approved by the Animal Experimentation Ethics Committee of Hanyang University, and the experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Hanyang University (2016-0141).

NP-OVA (50 μg, Bioresearch Technologies, Novato, CA, USA), diluted in Complete Fruend's Adjuvant (Chondrex, Redmond, WA, USA) was subcutaneously injected into the dorsal region of mice. Seven days after immunization, the mice were sacrificed, and the inguinal lymph node cells were isolated and analyzed by flow cytometry. Curcumin was purchased from Abcam (141921; Cambridge, England) and dissolved in DMSO. Curcumin (200 μg) was injected intra-peritoneally (i.p.) daily following the indicated experimental scheme.

To analyze the T_FH cells and GC responses, the inguinal lymph node cells were isolated and stained with anti-mouse CXCR5-biotin for 30 min at 4°C, followed by anti-mouse CD44-Ag-presenting cell (APC)/Cy7 and CD4-Percp/Cy5.5 and streptavidin-APC staining. After fixation and permeabilization using a Foxp3 Staining Kit (eBioscience, San Diego, CA, USA), intracellular Bcl-6 and Foxp3 was stained using anti-mouse Bcl-6-PE (BD Bioscience, San Jose, CA, USA) and Foxp3-FITC (eBioscience) for 40 min at room temperature. To analyze GC B cells, the isolated single cells were stained with anti-mouse B220-Percp/Cy5.5, GL-7-FITC, and CD95-PE for 30 min at 4°C. Then, the cells were examined using a FACSCanto II system (BD Bioscience); the data were analyzed using FlowJo software (Treestar, Ashland, OR, USA).

The total Ab production in mouse serum was measured using the IgM ELISA kit (eBioscience) and IgG ELISA kit (Abcam). To measure low and high affinity Abs, NP₃₀-conjugated BSA or NP₇-conjugated BSA (Biosearch Technologies, Petaluma, CA, USA) were coated, respectively. For the isotype Ab analysis, HRP-conjugated goat anti-mouse IgG1, IgG2b, IgG2c, and IgG3 Abs (Southern Biotech, Birmingham, AL, USA) were used to detect the Abs.

Data were analyzed using Student's t-test by Prism5 software (GraphPad, San Diego, CA, USA). The p-values less than 0.05 were considered statistically significant.

To examine curcumin function in the T_FH and GC response to a specific Ag, 6-week-old female C57BL/6 mice were subcutaneously immunized with NP-OVA in complete adjuvant on day 0, followed by daily administration of 200 μg curcumin by i.p. injection until day 6 (Fig. 1). At day 7, inguinal lymph node cells were isolated and analyzed by flow cytometry. As shown in Fig. 2A, NP-OVA immunization, with DMSO vehicle, led to a significant increase in Bcl-6⁺CXCR5⁺ T_FH cells compared to non-immunized group. Curcumin treatment significantly increased Bcl-6⁺CXCR5⁺ T_FH cells, which was gated from CD4⁺CD44⁺ cells in comparison with the DMSO vehicle control group. The CD44 expression level of CD4 T cells (Fig. 2B) and percentage of Bcl-6⁺Foxp3⁺ T follicular regulatory cells (Fig. 2C) was comparable between the two groups. The expression level of T_FH cell marker molecules such as Bcl-6 or CXCR5 (Fig. 2D) was significantly increased, collectively suggesting that curcumin administration significantly induces T_FH cells in the draining lymph nodes during Ag immunization.

Due to the observed increased proportion of T_FH cells and the results of a previous study that showed enhanced B cell function in sheep RBC immunized mice (27), we hypothesized that there would be elevated levels of GC B cells. The inguinal lymph node cells were analyzed by flow cytometry. With no difference in the proportion of B cells (Fig. 3A), NP-OVA immunization of the DMSO-treated group led to an increase in the GL-7⁺CD95⁺ cells, whereas curcumin administration significantly increased the proportion of GC B cells in the draining lymph nodes (Fig. 3B). In addition, there was an increase in the high affinity total IgG and IgM Abs in the serum upon curcumin treatment (Fig. 3C), suggesting that increased T_FH cells by curcumin administration could result in elevated GC B cells and Ab production.

As T_FH cells are key players in affinity maturation and isotype class-switching by the stimulation of GC B cells, we examined various NP-specific isotypes of the Abs in the serum to confirm the consequences of increased T_FH and GC responses by curcumin administration. Fig. 4A and B shows the significant increase in IgG1 and IgG2b Ab production upon curcumin treatment, whereas no significant differences were observed for other isotypes or low affinity Abs. These results suggest that curcumin treatment stimulates high affinity Ab production with isotype class-switching.

Curcumin, one of the most widely studied natural compounds, has diverse biological functions in attenuating chronic inflammatory diseases, including cancer, asthma, inflammatory bowel disease, and rheumatoid arthritis (3031). These therapeutic functions of curcumin have been mainly associated with the suppression of the production of inflammatory cytokines TNF, IL-1, IL-6, and IL-8 (1832). However, recent studies have reported that curcumin can directly regulate T cells by inhibiting IL-2 signaling and NF-κB activation (3334). Moreover, curcumin inhibited Th1 differentiation by blocking JAK-STAT signal activation (3536). Here, we demonstrate for the first time, to our knowledge, the positive biological function of curcumin in T_FH cell differentiation and GC B cell formation. We found that curcumin administration in vivo increases Bcl-6⁺CXCR5⁺ T_FH cells and GL-7⁺CD95⁺ GC B cells, with high affinity Ab production. As curcumin has a direct effect on the biological functions of T cells, we further examined curcumin treatment in T cell differentiation and found that curcumin appears to specifically inhibit Th1 and Th2 differentiation rather than Th17 (Supplementary Fig. 1). Collectively, these results indicate that curcumin modulates multiple molecular targets and has potent anti-inflammatory activities regulating effector T cell functions (T_FH, Th1, and Th2 cells). Direct regulation of the NF-κB and JAK-STAT signaling pathway by curcumin might be the clue to elucidating the potential mechanisms involved in the generation of T_FH cells and GC responses. Furthermore, considering previous reports that curcumin can enhance B cell function (2137), dissecting the mechanism underlying the Ab production increasing effect of curcumin should be addressed in further studies.

In conclusion, this study is the first to report that the administration of curcumin increases humoral immunity by Ab production, which is presumably mediated by increased T_FH cells in the draining lymph nodes. Interestingly, curcumin also contributes in the production of high affinity Abs of the IgG1 and IgG2b isotypes during immunization. Although the molecular mechanisms of curcumin's action on the T_FH response should be further evaluated in detail, we believe that curcumin could be an advantageous supplement, to enhance protective immunity via increased Ab production, in the treatment of infectious diseases or cancer.