Impaired granulocytopoiesis in patients with chronic idiopathic neutropenia is associated with increased apoptosis of bone marrow myeloid progenitor cells

To probe the pathophysiologic mechanisms underlying neutropenia in patients with chronic idiopathic neutropenia (CIN) with hypoplastic and left-shifted granulocytic series in the bone marrow (BM), we have studied granulocytopoiesis in 32 adults with CIN by evaluating the number and survival characteristics of cells in several stages of granulocyte differentiation using flow cytometry and BM culture assays. We found that patients with CIN displayed a low percentage of CD34(+)/CD33(+) cells, defective granulocyte colony-forming unit (CFU-G) growth potential of BM mononuclear or purified CD34(+) cells, and low CFU-G recovery in long-term BM cultures (LTBMCs), compared with controls (n = 46). A low percentage of CD34(+)/CD33(+) cells in patients was associated with accelerated apoptosis and Fas overexpression within this cell compartment compared with controls. No significant difference was documented in the percentage of apoptotic cells or the Fas(+) cells within the fractionated CD34(+)/CD33(-), CD34(-)/CD33(+), and CD34(-)/CD33(-)/CD15(+) BM subpopulations or the peripheral blood neutrophils, suggesting that the underlying cellular defect in CIN probably concerns the committed granulocyte progenitors. LTBMC stromal layers from patients produced abnormally high amounts of tumor necrosis factor alpha and cytokine levels in culture supernatants inversely correlated with the number of myeloid progenitor cells and positively with the proportion of apoptotic CD34(+) cells. Patient LTBMC stromal layers displayed pathologic interferon gamma and Fas-ligand mRNA expression and failed to support normal myelopoiesis. These data suggest that impaired granulocytopoiesis in CIN is probably due to overproduction of inflammatory cytokines by immune cells within the BM microenvironment that may exert an inhibitory effect on myelopoiesis by inducing Fas-mediated apoptosis in the granulocyte progenitors.     

Induction of regulatory T cells by Opisthorchis viverrini

Opisthorchis viverrini causes public health problems in South‐East Asia. Recently, TGF‐β and IL‐10 have been reported to increase in O. viverrini‐infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4⁺ T cells in infected hamsters were investigated. It was demonstrated that IL‐4⁺CD4⁺ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL‐10⁺CD4⁺ T cells were also discovered at a significant level while Treg (T regulatory)‐like TGF‐ β⁺CD4⁺ T cells were in MLNs of infected hamsters. Moreover, the CD4⁺CD25⁺Foxp3⁺ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T‐cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF‐β⁺CD4⁺ while TCC35, TCC41 and TCC108 were IL‐4⁺CD4⁺ and TCC23 was IFN‐γ⁺CD4⁺. This TGF‐β⁺CD4⁺ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF‐β‐mediated mechanisms. This study indicated that O. viverrini‐infected hamsters could induce TGF‐β⁺ CD4⁺ Treg‐like cells. The CSAg‐specific Tregs secreted high TGF‐β, and limited immune cell proliferation.     

Increased levels of soluble flt-3 ligand in serum and long-term bone marrow culture supernatants in patients with chronic idiopathic neutropenia

The levels of serum and long-term bone marrow culture supernatant soluble flt-3 ligand (sFL) were determined in 54 patients with chronic idiopathic neutropenia (CIN) and 16 normal controls. Both serum and supernatant sFL levels were significantly increased in the patients compared with controls. Individual sFL values inversely correlated with the number of circulating neutrophils and the proportion of bone marrow CD34+ cells. Supernatant sFL values positively correlated with the levels of supernatant G-CSF. These findings suggest that the impaired myelopoiesis in CIN patients is accompanied by a compensatory mechanism attempting to increase the neutrophil production at the myeloid progenitor cell level.     

Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T-lymphocytes containing T-cell receptor excision circles

Objectives: Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T‐lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T‐cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T‐cell lymphopenia in CIN. Methods: We investigated parameters of T‐cell homeostasis namely the proliferation/apoptotic rate of naïve and memory T cells, the T‐cell senescence by telomere measurement, the recent thymic T‐cell production through quantification of T‐cell receptor rearrangement excision circles (TRECs), and the production of interleukin (IL)‐7. Results: Patients with CIN (n = 44) displayed lower proportion of naïve CD45RA⁺ cells within the CD4⁺ and CD8⁺ cells compared with controls (n = 15). The proportion of apoptotic cells within the CD8⁺ fraction was higher in patients compared with controls and was correlated with the percentage of Ki‐67⁺ cells, indicating an activation‐induced accelerated CD8⁺ cell death. The TREC content of CD4⁺ and CD8⁺ cells was lower in patients compared with controls and was correlated with the proportion of CD45RA⁺ CD4⁺ and CD8⁺ cells and with the levels of serum and BM IL‐7, which were significantly decreased in the patients. The mean relative telomere length of CD4⁺ and CD8⁺ cells was significantly lower in patients with CIN compared with age‐matched controls. Conclusions: The aberrant T‐cell expansions associated with the pathogenesis of CIN result in increased proliferation/apoptosis and possibly exhaustion of peripheral blood T cells which, in association with the inadequate compensatory thymic export of new TREC expressing T cells partially because of IL‐7 deficiency, may contribute to lymphopenia in CIN.     

Ascaris suum infection modulates inflammation: Implication of CD4+ CD25high Foxp3+ T cells and IL‐10

Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS‐induced inflammation. We found that infection with A. suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐6) and induced high levels of IL‐10 and TGF‐β. Augmented frequency of CD4⁺ CD25^high Foxp3⁺ T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4⁺ CD25⁺ T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS‐induced inflammation in air pouch model. In addition, adoptive transfer of CD4⁺ CD25⁺ T cells derived from IL‐10 knockout mice suggests that this suppressive effect of A. suum infection involves IL‐10 cytokine. In conclusion, our results demonstrated that A. suum experimental infection was capable of suppressing LPS‐induced inflammation by mechanisms, which seem to be dependent on responses of CD4⁺ CD25⁺ T cells and secretion of IL‐10 cytokine.     

Minor populations of paroxysmal nocturnal hemoglobinuria‐type cells in patients with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) is an acquired disorder of granulopoiesis characterized by increased apoptosis of the bone marrow (BM) granulocytic progenitor cells under the influence of pro‐inflammatory mediators and oligoclonal/monoclonal T‐lymphocytes. Because patients with immune‐mediated BM failure display frequently paroxysmal nocturnal hemoglobinuria (PNH)‐type cells in the peripheral blood (PB), we investigated the possible existence of PNH‐type cells in 91 patients with CIN using flow cytometry. The patients displayed increased proportions of PNH‐type glycophorin A⁺/CD59^dim and glycophorin A⁺/CD59^? red blood cells (RBCs), FLAER^?/CD24^? granulocytes, and FLAER^?/CD14^? monocytes, compared to controls (n = 55). A positive correlation was found between the proportions of PNH‐type RBCs, granulocytes, and monocytes and an inverse correlation between the number of PB neutrophils and the proportions of PNH‐type cell populations. The number of patients, displaying percentages of PNH‐type cells above the highest percentage observed in the control group, was significantly increased among patients with skewed compared to those with normal T‐cell receptor repertoire suggesting that T‐cell‐mediated immune processes underlie the emergence of PNH‐type cells in CIN. Our findings suggest that patients with CIN display PNH‐type cells in the PB at a high frequency corroborating the hypothesis that CIN belongs to the immune‐mediated BM failure syndromes.     

The immunosuppressive effects of CD4+CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B

The characteristics and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4⁺CD25⁺ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4⁺CD25⁺ Tregs on the maturation and function of monocyte‐derived DCs were examined. The results showed that CD4⁺CD25⁺ render the DCs inefficient as antigen‐presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL‐10 and TGF‐β. There were an increased IL‐10 and TGF‐β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4⁺T cells, CD4⁺CD25⁺ may modulate the immune response through DCs in CHB patients.     

Bone marrow progenitor cell reserve and function and stromal cell function are defective in rheumatoid arthritis: evidence for a tumor necrosis factor alpha-mediated effect

Based on previous reports for impaired hematopoiesis in rheumatoid arthritis (RA), and in view of the current interest in exploring the role of autologous stem cell transplantation (ASCT) as an alternative treatment in patients with resistant disease, we have evaluated bone marrow (BM) progenitor cell reserve and function and stromal cell function in 26 patients with active RA. BM progenitor cells were assessed using flow cytometry and clonogenic assays in short-term and long-term BM cultures (LTBMCs). BM stroma function was assessed by evaluating the capacity of preformed irradiated LTBMC stromal layers to support the growth of normal CD34(+) cells. We found that RA patients exhibited low number and increased apoptosis of CD34(+) cells, defective clonogenic potential of BM mononuclear and purified CD34(+) cells, and low progenitor cell recovery in LTBMCs, compared with healthy controls (n = 37). Patient LTBMC stromal layers failed to support normal hematopoiesis and produced abnormally high amounts of tumor necrosis factor alpha (TNF alpha). TNF alpha levels in LTBMC supernatants inversely correlated with the proportion of CD34(+) cells and the number of colony-forming cells, and positively with the percentage of apoptotic CD34(+) cells. Significant restoration of the disturbed hematopoiesis was obtained following anti-TNF alpha treatment in 12 patients studied. We concluded that BM progenitor cell reserve and function and BM stromal cell function are defective in RA probably due, at least in part, to a TNF alpha-mediated effect. The role of these abnormalities on stem cell harvesting and engraftment in RA patients undergoing ASCT remains to be clarified.     

The −509C/T polymorphism of transforming growth factor‐β1 is associated with increased risk for development of chronic idiopathic neutropenia

Objective: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro‐inflammatory and pro‐apoptotic mediators, such as tumor necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β1, and Fas‐Ligand (Fas‐L). In this study, we evaluated the frequency of TNF‐α, TGF‐β1 and Fas‐L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. Methods: The TNF‐α?308G/A, TGF‐β1 ?509C/T, +869T/C, +915G/C, and Fas‐L ?844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well‐defined area with genetically homogeneous population, using a polymerase chain reaction‐based restriction fragment length polymorphism assay. Results: The mutant genotype C/T or T/T of TGF‐β1 ?509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild‐type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18–27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF‐β1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF‐α?308G/A, TGF‐β1 +869T/C and +915G/C and Fas‐L ‐844T/C polymorphisms. Conclusions: The TGF‐β1 ?509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF‐β1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.     

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self‐limiting hepatitis E

Hepatitis E virus (HEV) is a major cause of self‐limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4⁺CD25⁺Foxp3⁺, CD4⁺CD25⁻Foxp3⁺ and rise in IL‐10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real‐time PCR and by flow cytometry, (ii) the levels of antigen‐specific IL‐10 and TGF‐β by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg‐associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3⁺ cells in both patient groups were significantly elevated. The levels of IL‐10 and TGF‐β in the acute patients and TGF‐β in the recovered individuals were elevated. Significantly higher expression of CTLA‐4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg‐mediated suppression.     

Assessment of bone marrow stem cell reserve and function and stromal cell function in patients with severe congenital neutropenia

OBJECTIVE: To investigate further the cellular defect responsible for impaired granulopoiesis in severe congenital neutropenia (SCN), we have evaluated bone marrow (BM) stem cell reserve and function and BM stromal cell myelopoiesis supporting capacity in two patients with SCN. METHODS: BM primitive stem cells and myeloid progenitor cells were assessed using flow cytometry, limiting dilution assay, clonogenic assays, and long-term BM cultures (LTBMC). BM stroma function was assessed by evaluating the ability of irradiated stromal layers from the patients to induce granulocyte-macrophage colony formation (CFU-GM) by normal CD34+ cells. RESULTS: Compared to the normal controls (n = 37), SCN patients displayed a low percentage of CD34+/CD38+ cells (P < 0.05), low CFU-GM colony formation by highly purified CD34+ cells (P < 0.05), low CFU-GM recovery in LTBMC (P < 0.05), and normal primitive stem cells as indicated by the frequency of CD34+/CD38- cells and the number of long-term culture initiating cells. Patient BM stromal layers exhibited normal myelopoiesis supporting capacity as shown by the CFU-GM content of irradiated LTBMC recharged with normal CD34+ cells. In addition, patient LTBMC supernatants displayed 20-fold normal granulocyte colony stimulating factor and 2-fold normal granulocyte-macrophage colony stimulating factor levels. CONCLUSION: These data show that primitive BM stem cells and stromal cells are not affected in SCN patients, while they support further the concept of a primary defect at the myeloid progenitor cell level. To know the differentiation stage at which the underlying defect causes the malfunction will be relevant for further elucidation of its nature at the molecular level.     

Long-term bone marrow culture in persons with Fanconi anemia and bone marrow failure

Fanconi anemia is an autosomal recessive disease characterized by a high risk of developing bone marrow (BM) failure and acute myelogenous leukemia. We studied growth of hematopoietic progenitor cells in long- term BM culture (LTBMC) in 8 persons with Fanconi anemia and BM failure. Although LTBMC were initiated with very few BM cells, an adherent layer formed in cultures from 7 persons. In these cultures, the number of nonadherent cells increased for 10 to 15 days. Cell growth continued until cultures were terminated at day 35 to 40. During the first 2 weeks of culture, most nonadherent cells were differentiated myeloid cells. By days 35 to 40, the adherent layer contained cells able to initiate secondary LTBMCs. These data indicate that hematopoietic precursors cells able to proliferate and differentiate in vitro are present in the BM of persons with Fanconi anemia and BM failure. They suggest that mechanisms other than absent precursor cells are responsible for BM failure in Fanconi anemia.     

CD133 allows elaborated discrimination and quantification of haematopoietic progenitor subsets in human haematopoietic stem cell transplants

The success of haematopoietic stem cell (HSC) transplantation largely depends on numbers of transplanted HSCs, which reside in the CD34⁺ populations of bone marrow (BM), peripheral blood stem cells (PBSC) and umbilical cord blood (UCB). More specifically HSCs reside in the CD38^low/? subpopulation, which cannot be objectively discriminated from mature CD34⁺ CD38⁺ progenitors. Thus, better marker combinations for the quantification of more primitive haematopoietic stem and progenitor cells in transplants are required. Recently, by combining CD34 and CD133 we could clearly distinguish CD133⁺ CD34⁺ multipotent and lympho‐myeloid from CD133^low CD34⁺ erythro‐myeloid progenitors in UCB samples. To qualify the assessment of CD133 for routine quality control of adult HSC sources, we analysed the developmental potentials of CD133⁺ and CD133^low subpopulations in BM and PBSC. Similar to UCB, CD133 expression objectively discriminated functionally distinct subpopulations in adult HSC sources. By implementing anti‐CD45RA staining, which separates multipotent (CD133⁺ CD34⁺ CD45RA^?) from lympho‐myeloid (CD133⁺ CD34⁺ CD45RA⁺) progenitor fractions, UCB was found to contain 2–3 times higher multipotent progenitor frequencies than BM and PBSC. To test for the consistency of CD133 expression, we compared CD133⁺ CD34⁺ contents of 128 UCB samples with maternal and obstetrical factors and obtained similar correlations to related studies focusing on CD34⁺ cell contents. In conclusion, implementation of anti‐CD133 staining into existing routine panels will improve the quality control analyses for HSC transplants.     

CD8α− DC is the major DC subset which mediates inhibition of allergic responses by Schistosoma infection

Our and others' previous studies have shown that Schistosoma japonicum (SJ) infection can inhibit allergic reactions. We recently reported that DCs played an important role in SJ infection‐mediated inhibition of allergy, which was associated with enhanced IL‐10 and T regulatory cell responses. Here, we further compared the role of CD8α⁺ DC and CD8α^? DC subsets for the inhibitory effect. We sorted CD8α⁺ DC (SJCD8α⁺ DC) and CD8α^? DC (SJCD8α^? DC) from SJ‐infected mice and tested their ability to modulate allergic responses in vivo. The data showed that the adoptive transfer of SJCD8α^? DC was much more efficient than SJCD8α⁺ DC for the suppression of allergic airway eosinophilia, mucus overproduction, antigen‐specific IgE responses, and Th2 cytokines (IL‐4 and IL‐5). More importantly, we found that the transfer of SJCD8α^? DC, but not SJCD8α⁺ DC, significantly increased IL‐10 and TGF‐β production following OVA exposure. As control, the transfer of DC subsets from naïve mice had no significant effect on allergic inflammation. In addition, SJCD8α‐DC expressed significantly higher IL‐10 but lower IL‐12, CD80 and CD86 than SJCD8α⁺ DC, fitting a tolerogenic phenotype. The results suggest that CD8α^? DC is the predominant DC subset which is involved in the parasitic infection‐mediated inhibition of allergic inflammation and possibly through enhancing immunomodulatory cytokine (IL‐10 and TGF‐β) production.     

Distinct host‐related dendritic cell responses during the early stage of Plasmodium yoelii infection in susceptible and resistant mice

The diverse outcomes of experimental murine infection with Plasmodium parasites, ranging from spontaneous cure to death, depend largely on the establishment of an effective Th1 immune response during the early stages of infection. However, the molecular and cellular factors responsible for the induction and regulation of this response are poorly understood. As immunity is initiated by dendritic cells (DCs), we compared their phenotype and function during the early stages of infection with Plasmodium yoelii 17XL (P.y 17XL) strain in susceptible (BALB/c) and resistant (DBA/2) mice. Resistant DBA/2 mice developed a greater number of myeloid (CD11c⁺CD11b⁺) and mature DCs, which were fully functional and capable of secreting IL‐12p40. In contrast, susceptible BALB/c mice produced more plasmacytoid (CD11c⁺CD45R/B220⁺) and less mature DCs, resulting in high levels of IL‐10 and TGF‐β1. In addition, an in vitro experiment confirmed that splenic DCs from the two strains of mice differ in their ability to prime CD4⁺T cells in response to P.y 17XL stimulation. These findings indicate that the subset, the phenotype and the type of inflammatory and anti‐inflammatory signals of splenic DCs are critical factors responsible for the discrepancy in the ability to induce or regulate Th1 immune responses in different hosts.     

Toll-like receptor 4 mediates alcohol-induced steatohepatitis through bone marrow-derived and endogenous liver cells in mice

Background: Excessive alcohol intake causes an increase in intestinal permeability that induces translocation of gut‐derived lipopolysaccharide (LPS) to the portal vein. Increased LPS in the portal vein stimulates Kupffer cells through Toll‐like receptor (TLR) 4 in the liver. Activated TLR4 signaling in Kupffer cells induces various inflammatory mediators including TNF‐α, IL‐1β, and reactive oxygen species, resulting in liver injury. Hepatic stellate cells (HSCs) also express TLR4. This study investigates whether TLR4 on bone marrow (BM)‐derived cells, including Kupffer cells, or non–BM‐derived endogenous liver cells, including HSCs, contributes to the progression of alcohol‐induced steatohepatitis and fibrogenesis in mice. Methods: TLR4 BM chimera (wild‐type [WT] mice with TLR4^?/? BM or TLR4^?/? mice with WT BM) were generated by the combination of liposomal clodronate injection with whole body irradiation and BM transplantation, followed by treatment with intragastric alcohol feeding. Results: WT mice transplanted with WT BM exhibited liver injury, steatosis, inflammation, and a fibrogenic response. Conversely, TLR4^?/? mice with TLR4^?/? BM displayed less steatosis, liver injury, and inflammation. Notably, steatosis, macrophage infiltration, and alanine aminotransferase levels in both TLR4‐chimeric mice showed intermediate levels between WT mice transplanted with WT BM and TLR4^?/? mice transplanted with TLR4^?/? BM. Hepatic mRNA expression of fibrogenic markers (collagen α1(I), TIMP1, TGF‐β1) and inflammatory cytokines (IL‐1β, IL‐6) were markedly increased in WT mice with WT BM, but there was less of an increase in both TLR4‐chimeric mice and in TLR4^?/? mice transplanted with TLR4^?/? BM. Conclusions: TLR4 signaling in both BM‐derived and non–BM‐derived liver cells is required for liver steatosis, inflammation, and a fibrogenic response after chronic alcohol treatment.     

The effect of Echinococcus granulosus on spleen cells and TGF‐β expression in the peripheral blood of BALB/c mice

Cystic echinococcosis (CE) caused by the cestode Echinococcus granulosus (E. granulosus) is a zoonotic parasitic disease. The effective immune evasion mechanisms of E. granulosus allow it to parasitize its hosts. However, the status of the innate and adaptive immune cells and their contributions to E. granulosus progression remain poorly understood. In this study, we aimed to determine the impact of E. granulosus infection on T cells, NK cell responses and TGF‐β expression during the early infection phase in BALB/c mice. In E. granulosus infections, there was an increasing tendency in the percentage of CD4⁺CD25⁺ T cells and CD4⁺Foxp3⁺ T cells and peripheral blood TGF‐β levels and relative expression of the Foxp3 gene. Moreover, there were a decreasing tendency in the percentage of NK cells and NK cell cytotoxicity and the expression of NKG2D on NK cells. The TGF‐β1/Smad pathway was activated by E. granulosus in mice. Above results can be reversed by the inhibitor SB‐525334 (potent activin receptor‐like kinase 5 inhibitor). These results suggest that the TGF‐β/Smad pathway plays an important role in changes of T‐cell or NK cell responses. These results may contribute to revealing the preliminary molecular mechanisms in establishing hydatid infection.