Chitosan Protects Immunosuppressed Mice Against Cryptosporidium parvum Infection Through TLR4/STAT1 Signaling Pathways and Gut Microbiota Modulation

Cryptosporidium parvum infection is very common in infants, immunocompromised patients, or in young ruminants, and chitosan supplementation exhibits beneficial effects against the infection caused by C. parvum. This study investigated whether chitosan supplementation modulates the gut microbiota and mediates the TLR4/STAT1 signaling pathways and related cytokines to attenuate C. parvum infection in immunosuppressed mice. Immunosuppressed C57BL/6 mice were divided into five treatment groups. The unchallenged mice received a basal diet (control), and three groups of mice challenged with 1 × 106 C. parvum received a basal diet, a diet supplemented with 50 mg/kg/day paromomycin, and 1 mg/kg/day chitosan, and unchallenged mice treated with 1 mg/kg/day chitosan. Chitosan supplementation regulated serum biochemical indices and significantly (p < 0.01) reduced C. parvum oocyst excretion in infected mice treated with chitosan compared with the infected mice that received no treatment. Chitosan-fed infected mice showed significantly (p < 0.01) decreased mRNA expression levels of interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) compared to infected mice that received no treatment. Chitosan significantly inhibited TLR4 and upregulated STAT1 protein expression (p < 0.01) in C. parvum-infected mice. 16S rRNA sequencing analysis revealed that chitosan supplementation increased the relative abundance of Bacteroidetes/Bacteroides, while that of Proteobacteria, Tenericutes, Defferribacteres, and Firmicutes decreased (p < 0.05). Overall, the findings revealed that chitosan supplementation can ameliorate C. parvum infection by remodeling the composition of the gut microbiota of mice, leading to mediated STAT1/TLR4 up- and downregulation and decreased production of IFN-γ and TNF-α, and these changes resulted in better resolution and control of C. parvum infection.

Immune-Enhancing Effect of Submerged Culture of Ceriporia lacerata Mycelia on Cyclophosphamide-Induced Immunosuppressed Mice and the Underlying Mechanisms in Macrophages

The white-rot fungi Ceriporia lacerata is used in bioremediation, such as lignocellulose degradation, in nature. Submerged cultures and extracts of C. lacerata mycelia (CLM) have been reported to contain various active ingredients, including β-glucan and extracellular polysaccharides, and to exert anti-diabetogenic properties in mice and cell lines. However, the immunostimulatory effects have not yet been reported. This study aimed to identify the immunomodulatory effects, and underlying mechanisms thereof, of submerged cultures of CLM using RAW264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression in mice. Compared to CTX-induced immunosuppressed mice, the spleen and thymus indexes in mice orally administered CLM were significantly increased; body weight loss was alleviated; and natural killer (NK) cytotoxicity, lymphocyte proliferation, and cytokine (tumor necrosis factor [TNF]-α, interferon [IFN]-γ, and interleukin [IL]-2) production were elevated in the serum. In RAW264.7 macrophages, treatment with CLM induced phagocytic activity, increased the production of nitric oxide (NO), and promoted mRNA expression of the immunomodulatory cytokines TNF-α, IFN-γ, IL-1β, IL-6, IL-10, and IL-12. In addition, CLM increased the inducible NO synthase (iNOS) concentration in macrophages, similar to lipopolysaccharide (LPS) stimulation. Mechanistic studies showed that CLM induced the activation of the NF-κB, PI3k/Akt, ERK1/2, and JNK1/2 pathways. Moreover, the phosphorylation of NF-κB and IκB induced by CLM in RAW264.7 cells was suppressed by specific MAPKs and PI3K inhibitors. Further experiments with a TLR4 inhibitor demonstrated that the production of TNF-α, IL-1β, and IL-6 induced by CLM was decreased after TLR4 was blocked. Overall, CLM protected against CTX-induced adverse reactions by enhancing humoral and cellular immune functions, and has potential as an immunomodulatory agent.

Metabolomic Profiling and Immunomodulatory Activity of a Polyherbal Combination in Cyclophosphamide-Induced Immunosuppressed Mice

The study was aimed to develop a characterized polyherbal combination as an immunomodulator containing Phyllanthus emblica L., Piper nigrum L., Withania somnifera (L.) Dunal, and Tinospora cordifolia (Willd.) Miers. Through response surface methodology (RSM), the ratio of aqueous extracts of four plant materials was optimized and comprised 49.76% of P. emblica, 1.35% of P. nigrum, 5.41% of W. somnifera, and 43.43% of T. cordifolia for optimum immunomodulatory activity. The optimized combination showed antioxidant potential and contains more than 180 metabolites, out of which gallic acid, quercetin, ellagic acid, caffeic acid, kaempferitrin, and p-coumaric acid are some common and significant metabolites found in plant extracts and in polyherbal combination. Treatment with the polyherbal combination of different doses in cyclophosphamide-induced immunosuppressed mice significantly (p < 0.01) enhanced the subsets of immune cells such as natural killer (NK) cells (60%), B cells (18%), CD4 cells (14%), and CD8 cells (7%). The characterized polyherbal combination exhibited potent immunomodulatory activity, which can be further explored clinically for its therapeutic applicability.

Echinacea purpurea Alleviates Cyclophosphamide-Induced Immunosuppression in Mice

Echinacea purpurea (EP) has been widely used to treat upper respiratory infections, influenza, and the common cold. It can also exert various pharmacological activities, such as anti-inflammatory and anti-allergic effects. However, the potential of EP to modulate immune reactions remains unclear. Therefore, we evaluated the immunostimulatory effects of EP in cyclophosphamide (CP)-induced immunosuppressed mice. In this study, EP extract (12.5, 25, or 50 mg/kg) was orally administered to cyclophosphamide-induced immunosuppressed BALB/c mice. Then, indexes of immune organs, including the spleen and thymus, were recorded. Splenocyte proliferation and natural killer (NK) cell activities were measured by lactate dehydrogenase assay. Subsets of T cells, such as CD4+ and CD8+, were measured by flow cytometry, and immuno-cytokines, such as interleukin (IL)-2, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, were measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. The immunosuppressed mice showed decreased thymus and spleen indexes and immune cell activities. Treatment of EP elevated the indexes of immune organs, splenocyte proliferation, and NK cell activities in CP-induced immunosuppressed mice. Simultaneously, administration of EP reversed the CP-induced decrease in T-lymphocyte subsets (CD4+ and CD8+) and immunocytokines (IL-2, TNF-α, and IFN-γ). Taken together, these findings suggest that EP could be used to enhance health and immunity in immunosuppressed conditions.

Histological Changes in the Kidneys and Heart in Experimental Acanthamoebiasis in Immunocompetent and Immunosuppressed Hosts

The course of Acanthamoeba spp. infection depends on the age and immune status of the host, and the virulence of the Acanthamoeba spp. strain. Some strains of free-living amoebae exhibit organ specificity, during the course of infection, while others may cause changes in many organs or completely lose pathogenicity. The aim of the current study was to investigate the pathological properties of Acanthamoeba spp. isolated from a patient with acute myeloid leukemia and atypical pneumonia (AM22). Moreover, the objective was to investigate the histopathological changes in the kidneys and heart of immunocompetent and immunosuppressed mice infected with Acanthamoeba spp. Amoebae were re-isolated from both the kidneys and hearts of the inoculated mice, although no cysts or trophozoites of the amoebae were detected in microscopic slides of the fragments of these organs. Acanthamoeba spp. induced changes in the kidney and heart weight of infected mice. In immunocompetent and immunosuppressed Acanthamoeba spp. infected mice, we found some histopathological changes, including areas with less acidic cytoplasm and a relaxation of muscle fibers. In further studies, it is important to analyze changes in gene and protein expressions in the heart and kidneys of hosts with disseminated acanthamoebiasis to better understand the course of infection in these organs, because the results of histological analysis varied depending on the immune status and duration of infection.

Effects of Platycodon grandiflorum on Gut Microbiome and Immune System of Immunosuppressed Mouse

Platycodon grandiflorum (PG) is a perennial plant that has been used as a traditional remedy to control immune-related diseases. PG was steamed and dried to improve its taste (PGS). The aim of the study was to investigate the effects of PG and PGS (PG-diets) on the gut microbiome and immune system. We treated PG-diets to immunosuppressed mice via cyclophosphamide (CPA) injection. After two weeks of the supplement, we evaluated specific genera related to body weight and serum immunoglobulin levels and analyzed 16S rRNA sequencing and metagenomics statistical analysis. PG-diets groups showed an increased abundance of microorganisms in immunodeficient mice compared to the control group (NC). Moreover, Akkermansia significantly decreased in response to the CPA in the NC group at the genus level, whereas its abundance increased in the PG-diets groups. We also found that the modulation of the gut microbiome by PG-diets was correlated with body weight, IgA, and IgM levels. The results demonstrate that PG-diets may improve the health benefits of immunosuppressed mice by altering the gut microbiome, though not much difference was found between PG and PGS treatments. Finally, this is the first study showing the effects of PGS-diets on the gut microbiome and immune system as a potential nourishing immunity supplement.

The Immunomodulatory Effects of Phellodendri Cortex Polysaccharides on Cyclophosphamide-Induced Immunosuppression in Mice

Cyclophosphamide is a commonly used anticancer drug, and immunosuppression is one of the most common side effects. How to recover the immunological function is important for cyclophosphamide-treated patients. In the present study, Phellodendri Cortex polysaccharides (CPP) could enhance the proliferation of mouse spleen lymphocytes in vitro. The immunoregulatory function of CPP was then investigated in cyclophosphamide-induced immunosuppressed mice. In CPP-treated groups, mice were orally treated with CPP at doses of 1, 0.5, and 0.25 g/kg bodyweight from 1 to 11 d, respectively. The cyclophosphamide was administrated in CPP and cyclophosphamide groups from 12 to 14 d. In the cyclophosphamide and normal control groups, the mice received equal volume of saline from 1 to 14 d. The results showed that CPP (1 g/kg) could significantly increase the bodyweight of mice, even during cyclophosphamide treatment. The organ coefficients of the spleen and thymus were recovered by CPP treatment. CPP upregulated the contents of cytokines (IL-2, IL-6, IFN-γ, and TNF-α) in serum, which were downregulated by cyclophosphamide. The mRNA levels of these cytokines were also elevated by CPP treatment in the spleen. Cyclophosphamide upregulated the expressions of NF-κB p65, TLR4, and MyD88, suggesting that the NF-κB signaling pathway was activated by cyclophosphamide. After CPP treatment, it was recovered to normal level. These results indicated that CPP alleviated the cyclophosphamide-induced immunosuppression.