Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal bone marrow stem cell disorders characterized by ineffective erythropoiesis with dysplastic bone marrow leading to peripheral blood cytopenias a variable propensity for progression to acute myelogenous leukemia (AML). Neutropenia occurs in 50 % of newly diagnosed patients, more frequently in those with higher-risk (70–80 %) than lower-risk (15–20 %) MDS. Pneumonia was identified as the leading cause of death in a recent investigation of untreated MDS patients with low or intermediate risk. The pathogen was mainly bacterial, although sometimes fungal or more rarely viral. Using hypomethylating agents and lenalidomide in MDS patients will transiently worsen neutropenia and has stimulated investigation into infection causes and strategies to reduce risk.

The infection has been attributed mainly to neutrophil dysfunction rather than neutropenia since early studies showed that neither infections nor mortality was related to the low neutrophil count. A recent study in Asian MDS patients using the French–American–British (FAB) classification and International Prognostic Scoring System (IPSS) confirmed that neutrophil count did not predict prognosis in persistent severe neutropenia (neutrophil count < 500 μ/L) and that even a neutrophil count <200 μ/L showed only borderline prognostic significance.

Early studies reported that granulocyte function (mainly neutrophils in circulation) was reduced across FAB classifications. Subsequent investigations revealed variable impairment in different neutrophil functions independent of neutrophil number, including reduced chemotaxis, decreased adhesion molecule expression, and loss of microbicidal activity, especially in patients with recurrent infection. Scientists identified mechanisms of neutrophil dysfunction in MDS as disturbed chemokine receptor-induced response and impaired migration of circulating neutrophils toward the chemoattractant interleukin-8 (IL-8). Impaired migration in blood could be traced to defects in early hematopoietic progenitors in the bone marrow.

Intracellular generation of highly reactive oxygen species (ROS) in the respiratory burst pathway is produced by the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase complex. ROS activation is triggered by receptor-mediated binding of soluble chemotactic agents, such as the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) and receptor-independent phorbol ester phorbol-myristate-acetate (PMA). Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and cytokines prime the ROS response. Early studies showed that ROS activity varied in MDS patients from normal to abnormal independent of neutrophil number. Priming with G-CSF and GM-CSF could partially enhance fMLP-stimulated ROS activity in MDS patients’ neutrophils compared with healthy control responses. Subcutaneous administration of GM-CSF to MDS patients enhanced neutrophil function, assessed ex vivo as microbicidal activity. G-CSF treatment of high-risk MDS patients showed a borderline protective effect against infections. However, the use of GM-CSF and G-CSF treatment in MDS has been limited due to concerns about effects on progression, safety, and side effects.

We recently reported that oral administration of Maitake beta-glucan, a dietary supplement, stimulated hematopoiesis in bone marrow, recovery of circulating monocyte/neutrophil numbers, and normalized ROS production compared with no treatment or G CSF treatment in a mouse model of acute hemotoxic bone marrow injury. Since Maitake beta-glucan induced G-CSF in human umbilical cord blood monocytes stimulating colony-forming unit-granulocyte/macrophage (CFU-GM) differentiation in circulating hematopoietic progenitors, we hypothesized that Maitake would be effective in MDS.

Maitake extract, derived from the fruit body of the edible mushroom Grifola frondosa, contains beta-glucans with a 1,6-glucan main chain and 1,3 branches. Maitake beta-glucan does not show direct cytotoxic or cytocidal activity but inhibits lung metastasis when given an intraperitoneal injection, enhancing IL-12 production and activating natural killer cells. Maitake increased messenger ribonucleic acid expression of GM-CSF, G-CSF, M-CSF, interferon, and IL-12 p40 and attenuated the decrease in CFU-GM colonies of cisplatin-treated mice. Maitake had dose-dependent hematopoietic effects on mouse bone marrow cells in vitro, protecting CFU-GM progenitor cells from doxorubicin toxicity. Maitake enhanced granulopoiesis and mobilized granulocytes and progenitors by stimulating G-CSF production in cyclophosphamide-induced granulocytic mice. Oral Maitake stimulated homing and engraftment of transplanted donor cord blood cells into recipient mice. At the same time, Maitake administered i.p. caused downregulation of chemokine receptor CXCR4 and the ligand stromal-derived factor-1 in the bone marrow of granulocytic mice, causing granulocyte mobilization. Maitake enhances the differentiation and migration of hematopoietic cells, including progenitors, enhancing peripheral myeloid cell ROS function.

In our previous dose-escalation trial, breast cancer patients receiving Maitake extract orally at 5–7 mg/kg daily over 3 weeks showed significant dose-related changes in immune function with no serious adverse events or dose-limiting toxicity. Based on these dose effects, the present study was launched using Maitake extract at 6 mg/kg (i.e., 3 mg/kg twice daily) to assess neutrophil and monocyte function in MDS patients.

Infections are the most common cause of death in patients with lower-risk MDS. Although antibacterial and antifungal prophylaxis may be employed, the efficacies are limited. We have previously shown that Maitake beta-glucan promotes the maturation of hematopoietic progenitor cells (HPC) in vitro and enhances the recovery of peripheral blood leukocyte numbers and ROS function after chemotoxic bone marrow injury in vivo. The present study examined the effects of oral Maitake extract treatment on peripheral blood neutrophil and monocyte function in untreated, lower-risk MDS patients.

Impaired host defense in MDS is associated with functional defects in the myeloid lineage, including HPC, and presents as aberrant functioning and has selective effects on host defense mechanisms. Neutrophil and monocyte production of ROS during the respiratory burst is essential for bactericidal activity. Scientists using a similar whole blood flow cytometric assay reported that monocyte and neutrophil ROS production defects were characteristic of MDS patients, independent of neutrophil numbers, and worsened with more advanced disease. Isolated neutrophils displayed reduced microbicidal activity, explaining the high frequency of recurrent infections in MDS patients who were not neutropenic. They demonstrated progressive loss of microbicidal activity against gram-negative E. coli but not gram-positive lactobacilli or fungus (Candida albicans) throughout monthly studies in patients compared with corresponding functions at diagnosis, suggesting that immune dysfunction in MDS may be pathogen-specific and involve selective host defense pathways.

Our results demonstrate for the first time that monocyte ROS response to physiological E. coli is reduced in lower-risk MDS patients but could be restored after 12 weeks of Maitake treatment. Furthermore, monocyte ROS response to fMLP, the bacterial peptide analog, was significantly increased. Both neutrophils and monocytes showed increased basal production of ROS after Maitake treatment. Recent studies show that LPS from E. coli activates neutrophils to prime monocyte ROS production, releasing proinflammatory cytokines and further priming neutrophils. Our data suggest that monocyte ROS dysfunction may be an early marker of impaired microbicidal activity due to defective monocyte–neutrophil interaction. Maitake has been shown to stimulate human neutrophil phagocytosis in vitro.

Endogenous ROS production is necessary to maintain basal activity and is regulated by cytokines and the growth factors GM-CSF and G-CSF. Our observation that MDS patients showed increased neutrophil ROS response after Maitake treatment suggests that G-CSF induction in bone marrow leads to HPC maturation and release of more functionally competent cells.

We did plan to measure serum G-CSF (and GM-CSF for comparison) on an exploratory basis. Still, we could not experiment due to concerns about specimen integrity after the freezer malfunction. In our breast cancer trial with oral Maitake beta-glucan, we assessed circulating levels of G-CSF in a subset of 6 patients, comparing double baseline levels with three follow-up visits over 12 weeks. All patients showed positive or elevated G-CSF at baseline visits (11–80 pg/mL). While marked changes occurred in some, baseline and post-treatment differences were insignificant.

Others have reported that ROS is higher in RBCs but not in neutrophils in MDS patients, compared with normal cells. Moreover, increased ROS in RBCs from MDS patients correlated with increased serum ferritin due to iron accumulation. Although serum ferritin levels did not change during our study, it is possible that Maitake does not increase RBC ROS or that a more extended treatment period is needed to produce an increase in ferritin levels.

Maitake extract appeared to be safe in this MDS population. We found no evidence of altered disease activity or progression. Mild eosinophilia was noted in four patients, including one patient with associated diarrhea. In this context, the etiology and clinical significance of peripheral eosinophilia are unclear and could have been an allergic reaction to the mushroom. Eosinophilia and basophilia predicted reduced survival without affecting leukemia-free survival in intermediate-2-risk MDS patients. No patient in our study showed increased basophils.

Our study has some limitations, including single-arm design and the potential for patients to have similar improvements without the intervention. A future study with a non-treatment control arm is needed to rule this out. In addition, due to the number of tests performed, it is possible that some changes between baseline and 12 weeks were due solely to chance. However, this effect would also have affected the double baseline values and should have reduced the detection of treatment differences.

Maitake beta-glucan consumption improves neutrophil and monocyte function in lower-risk MDS patients. The enhanced ROS response to E. coli ex vivo in response to Maitake extract treatment suggests that Maitake may enhance immune responses against bacterial infection in MDS patients.