Carrot Pomace Polysaccharide (CPP) Improves Influenza Vaccine Efficacy in Immunosuppressed Mice via Dendritic Cell Activation

Despite the advancements in vaccination research and practices, influenza viruses remain a global health concern. Inducing a robust immune response by vaccination is especially challenging in the elderly, the immunocompromised, and persons with chronic illnesses. Polysaccharides derived from food may act as a safe and readily accessible means to boost the immune system during vaccination. In this study, we investigated whether crude polysaccharides derived from carrot pomace (CPP) could stimulate innate immune cell function and promote influenza vaccine immunogenicity. In bone marrow-derived dendritic cells (BMDCs), CPP increased the fraction of CD11c+MHCII+ cells and the expression of co-stimulatory molecules CD40 and CD80, indicative of enhanced maturation and activation. Functionally, CPP-treated BMDCs promoted inflammatory cytokine production in splenic lymphocytes. In a mouse model of immunosuppression induced by cyclophosphamide, animals given CPP before and after an influenza vaccine challenge showed increased frequencies of dendritic cells and natural killer cells in the spleen, in addition to the recovery of vaccine-specific antibody titers. Moreover, innate myeloid cells in CPP-fed mice showed evidence of phenotypic modification via markedly enhanced interleukin(IL)-12 and interferon(IFN)-γ production in response to lipopolysaccharide(LPS) stimulation ex vivo. Our findings suggest that the administration of carrot pomace polysaccharides can significantly enhance the efficacy of influenza vaccination.


Statistical Analysis
Unless otherwise specified, data are shown as the mean ± SD and each experiment was repeated two or three times. Data were analyzed by the two-tailed unpaired t-test or one-way ANOVA with Tukey's post hoc analysis using GraphPad Prism (v7.02, GraphPad, La Jolla, CA, USA).

Bone Marrow-Derived Dendritic Cell (BMDC) Maturation and Activation In Vitro Was Promoted by Food-Derived Polysaccharides
Given that dendritic cells (DCs) are critical for the initiation of innate and adaptive immune responses, we investigated the effects of food-derived polysaccharides on GM-CSF-driven DC maturation. In brief, bone marrow cultures were differentiated with GM-CSF, treated with food-derived polysaccharides, lipopolysaccharide (LPS), or vehicle, and measured for cell surface marker expression by flow cytometry. It has been reported that LPS treatment increases the expression of the costimulatory molecules and induces the maturation of DCs [17]. As such, LPS was used as a positive control that can induce DC maturation in vitro. In addition, green tangerine polysaccharide (GTP), known to enhance the production of IL-6, TNF-α, and nitric oxide (NO) in macrophage cell lines, was used as a positive control of immune-boosting food-derived polysaccharides [18]. CPP treatment up to 1000 µg/mL caused no significant cytotoxic effect in the BMDCs, whereas 100-200 µg/mL of GTP reduced cell viability (Figure 1a). The expression of CD11c (α integrin), a classic marker of myeloid dendritic cells, was unchanged in BMDCs treated with either polysaccharide. However, the fraction of mature DCs, as indicated by CD11c+ cells co-expressing MHCII+, was increased by both CPP and GTP (Figure 1a). In addition, 400 µg/mL CPP increased the expression of co-stimulatory molecules such as CD40 and CD80 in a dose-dependent manner, suggestive of enhanced DC activation (Figure 1b). A lower dose of GTP (100 µg/mL) also showed a strong effect on CD40 and CD80.
To assess whether these phenotypic changes translated into enhanced functionality, namely the initiation of the adaptive immune response, we performed a mixed (allogeneic) lymphocyte reaction (MLR). The MLR is a standard assay widely used to measure antigen presenting-cell activity. By mixing stimulator antigen-presenting cells from one strain with responder cells from another, the responder cells (usually T cells) can recognize the allogeneic stimulator cells as foreign and undergo potent activation. Subsequent cytokine levels can then be detected as a measure of lymphocyte activation and thereby provide indication of the antigen-presenting capacity of stimulator cells. Briefly, GM-CSF-derived BMDCs from C57BL/6 mice were used as stimulator cells and splenocytes from BALB/c mice as responder cells. BMDCs were treated with CPP, GTP, or vehicle for one day, washed, and co-cultured with the BALB/c splenocytes for 3 days. It is of note that basal cytokine production in the splenocytes + BMDC mixed reaction (lane 3, IL-17; 0.039, IL-6; 0.69 ng/mL) is a few times higher than those of BMDCs only (lane 1; IL-17; 0.003, IL-6; 0.11 ng/mL), indicating that the primary source of these cytokines are likely splenocytes (Figure 1c). The resulting medium was analyzed by capture bead assay for cytokine secretion. Remarkably, CPP-treated BMDCs induced a significant increase in the production of IL-6, TNFα, IL-17 and IL-10 when compared to the vehicle-treated group (Figure 1c). These results suggest that CPP treatment promotes BMDC maturation and lymphocyte-activation capacity.  Figure 3d). Altogether, these data suggest that CPP may enhance cytokine production in innate immune cells to promote a sustained, anti-viral response.

CPP Increases Dendritic Cell and Natural Killer(NK) Cell Population in Mouse Splenocytes
Based on the above in vitro observations, suggesting that CPP can enhance the maturation and activity of DCs, we sought to determine whether CPP could boost an immunosuppressed animal's immune response to an inactivated influenza vaccine. To induce immunosuppression, mice were treated with cyclophosphamide (CTX), an alkylating agent well known to rapidly deplete neutrophils and proliferating lymphocytes [19]. Mice were given IP injections of CTX for five days, then orally administered CPP, GTP, or control solutions for 10 days before (total 10 days) or both before and after (total 20 days) receiving an influenza vaccine (Figure 2a). The flow cytometry analysis of isolated mouse splenocytes revealed that 20 days of CPP treatment increased the percentage of CD11c+MHCII+ DCs to a level comparable to that of GTP treatment (Figure 2b,c). The population of CD11b+NK1.1+ natural killer cells in the spleen was also approximately 2.5 times higher in CPP-than in vehicle-treated mice ( Figure 2d). Interestingly, CPP given 10 days prior to vaccination did not affect the number of immune cells, suggesting that oral polysaccharides may be the most effective when given simultaneously to or following vaccination. These data suggest that innate immune cell expansion, such as that of DCs and NK cells, are sensitive to food-derived polysaccharides.

CPP Enhances the IL-12 Production Ability of Innate Immune Cells
An important modulator of the antiviral immune response is cytokine release by innate immune cells. IL-12 is particularly important for facilitating a coordinated immune response, as it is known to enhance the cytotoxic activity of NK cells and CD8+ T cells, as well as stimulate naïve T cells to differentiate into Th1 cells [20][21][22][23]. To examine whether CPP impacts IL-12 production in innate immune cells, we used flow cytometry to measure the fraction of IL-12+ cells in isolated CD11b+ and CD11c+ populations stimulated by LPS (Figure 3). Both populations showed significantly more IL-12+ cells after 20 days of CPP treatment at fractions comparable to that of GTP ( Figure S1). To investigate which innate immune cell types were responsible for this enhanced IL-12 production, we performed the same analysis in cell populations enriched in DC (CD11c+MHCII+), NK cells (CD11b+NK1.1+), or macrophages (CD11b+F4/80+) and stimulated with LPS. Remarkably, all three populations showed a striking increase in IL-12+ cells after 20 days of CPP treatment (Figure 3c). These cell types are also important producers of IFN-γ, a vital cytokine for protection against viral infection through mechanisms including the inhibition of viral entry, the disruption of viral replication, and the blockade of viral protein translation [24]. CPP treatment indeed impacted IFN-γ production, markedly enhancing the proportion of IFN-γ-producing cells in the DC-and macrophage-enriched (MP) populations in a dose-dependent manner (Figure 3d). Altogether, these data suggest that CPP may enhance cytokine production in innate immune cells to promote a sustained, anti-viral response.

CPP Treatment Enhanced Antibody Production to Vaccine Challenge
Given that CPP may stimulate the anti-viral activity of the innate immune system, we were interested in the humoral immune response to an influenza vaccine challenge. Consistent with previous literature [25,26], vaccine-specific antibody titers were almost abolished in cyclophosphamide-injected animals given a quadrivalent inactivated influenza vaccine (Figure 4). Remarkably, treatment with CPP significantly recovered the total IgG response to a level comparable to that of GTP. Given that CTX-induced immunosuppression was not fully recovered by CPP treatment, however, further investigation is warranted on CPP's role in influenza viral vaccine-mediated adaptive immunity (Figure 4a). Subclass-specific antibody concentrations for IgG1 and IgG2 also demonstrated modest recovery, although without reaching statistical significance. These findings suggest that CPP can enhance protection against influenza viral infection.
Remarkably, treatment with CPP significantly recovered the total IgG response to a level comparable to that of GTP. Given that CTX-induced immunosuppression was not fully recovered by CPP treatment, however, further investigation is warranted on CPP's role in influenza viral vaccinemediated adaptive immunity (Figure 4a). Subclass-specific antibody concentrations for IgG1 and IgG2 also demonstrated modest recovery, although without reaching statistical significance. These findings suggest that CPP can enhance protection against influenza viral infection.

Discussion
In this study, we sought to explore a novel influenza vaccination strategy in a mouse model of immunosuppression. Importantly, we showed that oral treatment with carrot pomace-derived polysaccharides (CPP) can partially restore the vaccine-specific total IgG response in immunosuppressed mice. This immune-boosting effect may be attributable to not only a marked expansion of total macrophage, DC, and NK cells in the spleen of CPP-treated mice, but also a dramatic increase in those positive for IL-12 and IFN-γ. In addition, BMDC cultures treated with CPP showed an increased expression of maturation markers and stimulated co-cultured lymphocytes to release elevated levels of cytokines. Altogether, CPP may improve influenza vaccine immunogenicity in immunosuppressed mice by enhancing antigen presentation and cytokine production in innate immune cells.
Dendritic cells are traditionally considered to be the bridge between the two immune responses, presenting antigens to adaptive immune cells and generating copious amounts of cytokines to orchestrate both innate cell activity and adaptive cell differentiation [27,28]. Our data showed that CPP can induce the upregulation of MHC II, CD11c, CD80, and CD40 in BMDC cultures, suggesting greater antigen presentation and co-stimulation functionality [29,30]. It is noteworthy that performing these experiments with cDCs or other dendritic cell types may provide additional information to that of BMDCs [31]. The polysaccharide adjuvant derived from delta inulin, Advax, has been reported to induce similar phenotypic changes in APCs, enhancing antigen presentation to promote cellular and humoral responses specific to a trivalent influenza vaccine [32]. Various other

Discussion
In this study, we sought to explore a novel influenza vaccination strategy in a mouse model of immunosuppression. Importantly, we showed that oral treatment with carrot pomace-derived polysaccharides (CPP) can partially restore the vaccine-specific total IgG response in immunosuppressed mice. This immune-boosting effect may be attributable to not only a marked expansion of total macrophage, DC, and NK cells in the spleen of CPP-treated mice, but also a dramatic increase in those positive for IL-12 and IFN-γ. In addition, BMDC cultures treated with CPP showed an increased expression of maturation markers and stimulated co-cultured lymphocytes to release elevated levels of cytokines. Altogether, CPP may improve influenza vaccine immunogenicity in immunosuppressed mice by enhancing antigen presentation and cytokine production in innate immune cells.
Dendritic cells are traditionally considered to be the bridge between the two immune responses, presenting antigens to adaptive immune cells and generating copious amounts of cytokines to orchestrate both innate cell activity and adaptive cell differentiation [27,28]. Our data showed that CPP can induce the upregulation of MHC II, CD11c, CD80, and CD40 in BMDC cultures, suggesting greater antigen presentation and co-stimulation functionality [29,30]. It is noteworthy that performing these experiments with cDCs or other dendritic cell types may provide additional information to that of BMDCs [31]. The polysaccharide adjuvant derived from delta inulin, Advax, has been reported to induce similar phenotypic changes in APCs, enhancing antigen presentation to promote cellular and humoral responses specific to a trivalent influenza vaccine [32]. Various other plant-derived polysaccharides have been shown to upregulate DC function, including the active polysaccharide in aloe vera, acemannan [33], and polysaccharides from Astragalus mongholicus [34]. Polysaccharides derived from mushroom and barley were also shown to increase HLA-DR, CD40, CD80, and CD86 expression in human DCs [35].
We also observed phenotypic changes in innate immune cells in vivo, where oral CPP significantly increased the percentage of IL-12-and IFN-γ-producing DCs and macrophages in immunosuppressed mice. Given that IL-12 and IFN-γ are critical for the early polarization and sustained effector activity of CD4+ Th1 cells [36], CPP may induce a Th1-skewed response. DCs are also important regulators of NK cells, the representative innate lymphoid cells and first line of defense against virus-infected host cells [37,38]. IL-12 released from DCs is a potent inducer of IFN-γ production in NK cells [39,40]. Although the proportion of IFN-γ-expressing NK cells was unaffected by CPP treatment, the absolute number of such cells was likely increased due to the expansion of the total NK cell population. NK cells also display features of antigen-specific memory, undergoing robust secondary expansion and degranulation in response to exposure to a familiar virus [41,42].
Interestingly, the influenza vaccine alone did not induce NK cell expansion in either control or immunosuppressed mice although it has been previously reported in infections with cytomegalovirus, influenza A, and vaccinia virus [41,43,44]. Only mice treated with CPP before and after vaccination demonstrated heightened innate immune cell activation. Although the underlying mechanism requires further study, our results indicate that unlike alum, CPP does not need to be associated with the antigen to boost immunogenicity. Like Advax, however, simply priming local APCs prior to antigen presentation is insufficient [32]. The effects of CPP may be related to a relatively new concept termed "trained immunity", which describes the innate immune cells that produce a heightened, secondary response to encounters with the same or novel pathogen up to several months after the initial exposure [45]. In our study, the innate immune cells from mice that received CPP treatment before and after vaccination mounted a significant cytokine response to an LPS challenge, suggestive of trained immunity. Furthermore, the significant expansion and activation of myeloid and NK cells in CPP-treated mice was observed at four weeks post-vaccine challenge, even though pro-inflammatory transcriptional programs in innate immune cells are thought to only last in the order of days. A recent study in non-human primates observed similarly late phenotypic modifications, such as increased an expression of maturation and activation markers, in innate immune cells between 2 weeks and 2 months post vaccination with modified Vaccinia Ankara [46]. In another study, circulating monocytes in healthy volunteers given the bacille Calmette-Guérin (BCG) vaccine showed an increased and sustained production of pro-inflammatory cytokines and activation markers in response to various bacterial and fungal pathogens [47]. The polysaccharide β-glucan has also been shown to induce trained immunity in a process that may be dependent on the accumulation of mevalonate [48,49].
In summary, we demonstrated that food-derived polysaccharides such as CPP can enhance the antigen presentation capacity of innate immune cells. Given that influenza vaccine efficacy is dependent on the host's ability to rapidly recruit competent immune cells and generate long-lasting immunological memory, food-based polysaccharides may be a safe but effective means of boosting vaccine-mediated protection in immunocompromised individuals.