Aggravation of experimental autoimmune neuritis in TNF-alpha receptor 1 deficient mice

The role of tumor necrosis factor (TNF)-alpha and its receptors in the pathogenesis of experimental autoimmune neuritis (EAN) induced by P0 peptide 180-199 in TNFR1 (p55) deficient (TNFR1-/-) mice was investigated. Compared to wild type EAN mice, TNFR1-/- EAN mice developed significantly more severe clinical signs, in parallel with enhanced numbers of inflammatory infiltrating cells in peripheral nerves and splenic P0-reactive T cell proliferation, as well as increased obviously MHC class II and CCR3 expression on the macrophages in the cauda equina. Our data indicated that TNF-alpha might have anti-inflammatory effect preventing the development of EAN in this mouse model.     

趋化因子mRNA在实验性变态反应性神经炎中表达的研究

目的:实验性变态反应性神经炎(EAN)是一类T细胞介导的周围神经系统的自身免疫病,可用牛坐骨神经加完全氟氏佐剂诱导而成。本文研究趋化因子mRNA在实验性变态反应性神经炎(EAN)中的表达并探索其可能的作用。方法:用兔坐骨神经匀浆免疫Wistar大鼠,诱导格林巴利综合症(GBS)的动物模型EAN;采用地高辛标记的寡核苷酸探针检测EAN病变神经组织浸润细胞上趋化因子单核细胞趋化蛋白-1(MCP-1)及巨噬细胞炎性蛋白-1β(MIP-1β)mRNA表达情况。结果:MCP-1mRNA在临床症状出现前1-2天(14天)水平最高,随后逐渐下降;MIP-1 βmRA在临床症状出现前1-2天水平开始升高,在临床症状达到高峰时(21天)最高,进入恢复期后降至基础水平。结论:趋化因子在EAN的炎性细胞迁移及浸润进入神经细胞过程中起到重要作用。     

Suppression of autoimmune neuritis in IFN-gamma receptor-deficient mice

Experimental autoimmune neuritis (EAN) is an animal model of the human disease Guillain-Barré syndrome. In this autoimmune inflammatory disease, CD4(+) T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-gamma are intimately involved in causing pathogenic effects. To investigate the role of IFN-gamma in cell-mediated EAN, IFN-gamma receptor-deficient mutant (IFN-gammaR(-/-)) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. IFN-gammaR(-/-) mice exhibited later onset of clinical disease. The disease was also less severe than in wild-type mice. Fewer IL-12-producing but more IL-4-producing cells were found in sciatic nerve sections from IFN-gammaR(-/-) mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-gammaR(-/-) mice had less infiltration of inflammatory cells, including macrophages, CD4(+) T cells, and monocytes, into sciatic nerve tissue and less demyelination. However, numbers of IFN-gamma-secreting cells from the spleen were significantly augmented in the IFN-gammaR(-/-) mice, reflecting a failure of negative feedback circuits. The IFN-gammaR deficiency did not affect the production of anti-P0 peptide 180-199-specific antibodies. These results indicate that IFN-gamma contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.     

Dynamics of production of MIP-1alpha, MCP-1 and MIP-2 and potential role of neutralization of these chemokines in the regulation of immune responses during experimental autoimmune neuritis in Lewis rats.

Experimental autoimmune neuritis (EAN) is an inflammatory autoimmune demyelinating disease of the peripheral nervous system (PNS) and represents an animal model of Guillain-Barré syndrome (GBS), which is a major inflammatory demyelinating disease of the PNS in humans. In the present study, the dynamics of the expression of the chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-2 and monocyte chemotactic protein-1 (MCP-1) were determined in the sciatic nerves of EAN rats. Additionally, the effect of neutralizing antibodies against MIP-1alpha, MIP-2 and MCP-1 on the clinical course of EAN and the chemokine expression was investigated. The maximum of MIP-1alpha positive cells in the sciatic nerves was seen on day 14 post immunization (p.i.) correlating with the development of severe clinical signs. Administration of an anti-MIP-1alpha antibody suppressed the clinical signs of EAN and inhibited inflammation and demyelination in the sciatic nerve. Peak numbers of MCP-1 positive cells in the sciatic nerves were detected on day 7 p.i. Administration of an anti-MCP-1 antibody caused a delay of onset of EAN. However, 4 of the 6 EAN rats receiving the anti-MCP-antibody showed the same degree of inflammatory cell infiltration and demyelination in the sciatic nerves as sham-treated EAN rats, whereas only 2 EAN rats had less inflammation and demyelination. The numbers of MIP-2 positive cells reached a maximum on day 21 p.i. Anti-MIP-2 antibody failed to suppress the clinical signs of EAN and the inflammation and demyelination in the sciatic nerves. Only administration of the anti-MIP-1alpha antibody resulted in a significant reduction in the number of chemokine (MIP-1alpha)-positive cells and ED1-positive macrophages in the sciatic nerves. The present results demonstrate that MIP-1alpha and MCP-1 may play a role in the immunopathogenesis of EAN, and that MIP-1alpha induced trafficking of inflammatory cells can be inhibited by immunoneutralization. Further elucidation of the regulation and coordination of MIP-1alpha and MCP-1 production may lead to new therapeutic approaches to GBS in humans.     

Glial cell line‐derived neurotrophic factor is expressed by inflammatory cells in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

Neurotrophic factors, including glial cell line‐derived neurotrophic factor (GDNF), have been known to play a role in neuroprotection in the injured peripheral nervous system (PNS). To evaluate the involvement of GDNF in experimental autoimmune neuritis (EAN) pathogenesis, the expression of GDNF in rat sciatic nerves with EAN was studied. Western blot analysis showed that the level of GDNF protein significantly increased 1.8‐fold at the paralytic stage of EAN at day 12 post‐immunization (PI) (p < 0.01), and its level further increased approximately 2.5‐fold at day 21 PI (p < 0.001) in the sciatic nerves of EAN‐affected rats compared with those of control rats, and then declined thereafter at day 28 PI. Immunohistochemical analysis showed that axons and Schwann cells constitutively contained GDNF in normal controls. In sciatic nerves with EAN at day 12 PI, GDNF was immunostained in infiltrating inflammatory cells including macrophages and T cells. Collectively, we postulate that GDNF plays a regulatory role in EAN paralysis. A paradoxical role of inflammatory cells to ameliorate PNS inflammation remains to be further studied in EAN, an animal model of human demyelinating disease.     

Neutralizing antibodies to IL-18 ameliorate experimental autoimmune neuritis by counter-regulation of autoreactive Th1 responses to peripheral myelin antigen

Experimental autoimmune neuritis (EAN) is a demyelinating disease of the peripheral nervous system (PNS). This acute inflammatory disease is mediated by CD4+ T cells and bears significant similarities to the Guillain-Barré syndrome of humans. In the present study, we investigated the function of IL-18 in T cell-mediated autoimmunity of EAN in mice induced by P0 peptide 180-199 and Freund's complete adjuvant. Our data indicate that in 2 different therapeutic regimens, anti-IL-18 monoclonal antibody (mAb) effectively ameliorates the clinical and pathological signs of EAN. The suppression is associated with reduced inflammatory cell infiltration into the PNS and an insufficiency of autoreactive Th1 cells, as reflected by a reduced mononuclear cell proliferation and IFN-gamma-secretion in the spleen. Increased numbers of IL-4 expressing cells and decreased numbers of IFN-gamma and TNF-alpha expressing cells were found in the PNS. Our results suggest that shifting the Th1/Th2 balance towards Th2 cells may be one mechanism underlying EAN suppression by anti-IL-18 mAb. In addition, anti-IL-18 mAb treatment reduced anti-P0 peptide 180-199 autoantibody responses, which may also contribute to EAN suppression. We conclude that endogenous IL-18 plays a critical role in the pathogenesis of autoimmune demyelinating disease of the PNS and that IL-18 antagonists may provide a new therapy for these diseases.     

CD28-B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28-/-) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180-199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28-/- mice manifested clinical signs of disease. Additionally, CD28-/- mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-gamma secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28-/- mice had milder infiltration of such inflammatory cells as macrophages, CD4+ T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180-199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.     

Suppression of Autoimmune Neuritis in IFN-γ Receptor-Deficient Mice

Experimental autoimmune neuritis (EAN) is an animal model of the human disease Guillain–Barré syndrome. In this autoimmune inflammatory disease, CD4⁺ T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-γ are intimately involved in causing pathogenic effects. To investigate the role of IFN-γ in cell-mediated EAN, IFN-γ receptor-deficient mutant (IFN-γR^−/−) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180–199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. IFN-γR^−/− mice exhibited later onset of clinical disease. The disease was also less severe than in wild-type mice. Fewer IL-12-producing but more IL-4-producing cells were found in sciatic nerve sections from IFN-γR^−/− mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-γR^−/− mice had less infiltration of inflammatory cells, including macrophages, CD4⁺ T cells, and monocytes, into sciatic nerve tissue and less demyelination. However, numbers of IFN-γ-secreting cells from the spleen were significantly augmented in the IFN-γR^−/− mice, reflecting a failure of negative feedback circuits. The IFN-γR deficiency did not affect the production of anti-P0 peptide 180–199-specific antibodies. These results indicate that IFN-γ contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.     

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade enhances incidence and severity of experimental autoimmune neuritis in resistant mice

Experimental autoimmune neuritis (EAN), an autoimmune inflammatory demyelinating disease of the peripheral nervous system, represents an animal model of the human Guillain-Barré syndrome. EAN can be induced by active immunization in several animals, including Lewis rats. In contrast, most strains of mice including the widely used C57BL/6 (B6) strain are reputedly resistant to the induction of EAN. In the present study, we demonstrate that in B6 mice, anti-CTLA-4 monoclonal antibody administration in conjunction with immunization with the P0 protein derived peptide 180-199 can induce clinical and pathological definite EAN. Upregulating effects of CTLA-4 blockade on initial and ongoing EAN are demonstrated. CTLA-4 blockade augmented cellular infiltration and enhanced demyelination in the target organ sciatic nerves as well as increased T cell proliferation in lymph node cells. Moreover, serum levels of IFN-gamma and IL-4 were increased. Thus, manipulation of CTLA-4/B7 costimulatory pathway by CTLA-4 blockade can promote autoreactivity and break the relative tolerance to peripheral autoantigen P0 in resistant B6 mice.     

P0 glycoprotein peptides 56-71 and 180-199 dose-dependently induce acute and chronic experimental autoimmune neuritis in Lewis rats associated with epitope spreading

Two synthetic peripheral nerve myelin P0 protein peptides, an immunodominant (amino acids 180-199) and a cryptic (amino acids 56-71) one, induced an acute or chronic course of experimental autoimmune neuritis (EAN) in Lewis rats, when given at low dose (50-100 microg/rat) or high dose (250 microg/rat), respectively. Corresponding to the different clinical course, pathological changes and immune responses were found: (1) Onset of clinical signs of P0 peptide 56-71 (P0 56-71) induced EAN was 1-3 days later than in P0 peptide 180-199 (P0 180-199) induced EAN at all immunizing doses, whereas the peak of the disease occurred at a similar time point post immunization (p.i.), i.e. at days 14-16 p.i. in P0 56-71 induced EAN and at day 16 p.i. in P0 180-199 induced EAN. (2) Intramolecular epitope spreading as assessed by delayed type hypersensitivity response occurred in P0 56-71 induced EAN at both low and high antigen doses and in P0 180-199 induced EAN at high antigen dose (250 microg/rat) only. (3) P0 180-199 stimulated higher levels of interferon-gamma production in P0 180-199 induced EAN than in P0 56-71 induced EAN and vice versa. (4) Histopathologic evaluation revealed a similar grade of mononuclear cell infiltration in the sciatic nerves of both types of EAN, but more severe demyelination was found in P0 180-199 induced EAN compared to P0 56-71 induced EAN. The results support the hypothesis that high dose autoantigen immunization induces extensive determinant spreading and chronic course of autoimmune diseases.     

IL-18 deficiency inhibits both Th1 and Th2 cytokine production but not the clinical symptoms in experimental autoimmune neuritis

IL-18 deficient (IL-18-/-) mice were used to investigate the role of IL-18 in the pathogenesis of experimental autoimmune neuritis (EAN) which was induced by immunization of the mice with P0 protein peptide 180-199. The clinical course was not different between IL-18-/- and wild-type mice. The splenic mononuclear cell (MNC) proliferation was also similar in both animal groups. However, the percentages of IFN-gamma, IL-10 and IL-12 positive cells were decreased among infiltrating MNC of cauda equine in IL-18-/- mice. This indicates that IL-18 deficiency inhibits the production of both Th1 and Th2 cytokines in the target organ of mice with EAN.     

Activation of p38 mitogen-activated protein kinase in the early and peak phases of autoimmune neuritis in rat sciatic nerves

To examine the involvement of p38 mitogen-activated protein kinase (MAPK) in autoimmune disorders of the peripheral nerve system, we analyzed the phosphorylation of p38 MAPK protein in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that phosphorylated p38 (p-p38) MAPK protein was significantly increased in the sciatic nerves of rats in the early and peak phases of EAN, and declined gradually thereafter. Immunohistochemistry showed that p-p38 MAPK levels were increased in the infiltrating inflammatory cells, including T cells and macrophages, as well as in blood vessels and some Schwann cells in EAN-affected sciatic nerves, as compared to the sciatic nerves of controls. Some inflammatory cells and a few Schwann cells were also positive for TUNEL reaction at the peak and recovery phases of EAN. In conclusion, we postulate that the phosphorylation of p38 MAPK is involved in the elimination of infiltrating inflammatory cells during the course of EAN and may possibly modulate recovery in autoimmune disorders of the peripheral nervous system.     

P0 protein peptide 180-199 together with pertussis toxin induces experimental autoimmune neuritis in resistant C57BL/6 mice

The C57BL/6 mice strain is known to be reputedly resistant to induction of experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in human by bovine peripheral myelin (BPM), and P2 protein or the P2 protein peptide 57-81. The P0 peptide 180-199 is a stronger neuritogenic antigen than the P2 peptide 57-81. We found that this synthetic peptide induced both clinical and pathological characteristics of an acute monophasic EAN in C57BL/6 mice. Only male mice were more sensitive to EAN induction with the P0 peptide 180-199. Intravenously administrated pertussis toxin (PT) had an adjuvant effect that increased the incidence of P0 peptide 180-199-induced EAN as well as the inflammation and demyelination in the peripheral nerves. Spontaneous and P0 peptide 180-199 stimulated proliferation of peripheral T-cells were enhanced by PT-treatment as well. The enhancing effect was lower before onset of the disease (Day 6 post immunization) (p.i.) as compared to the early phase of the disease (Day 22 p.i.). Thus, P0 peptides together with PT are able to break tolerance to myelin in C57BL/6 mice.     

Antigen-specific immunosuppression: nasal tolerance to P0 protein peptides for the prevention and treatment of experimental autoimmune neuritis in Lewis rats.

Experimental autoimmune neuritis (EAN) is an autoimmune inflammatory demyelinating disease of the peripheral nervous system (PNS), and represents an animal model of the human Guillain-Barré syndrome (GBS). In this study, we report that nasal administration of the neuritogenic peptide 180-199 and of the cryptic peptide 56-71 of the rat neuritogenic P0 protein of peripheral nerve myelin prevents EAN and attenuates ongoing EAN. Both peptides effectively decreased the severity and shortened clinical EAN. Both a prophylactic and a therapeutic approach proved to be beneficial. These effects were associated with T and B cells hyporesponsiveness to the peptide antigens, reflected by downregulated Th1 cell responses (interferon-gamma secretion) and macrophage function, whereas Th2 cell responses (IL-4 secretion) and transforming growth factor-beta mRNA expression were upregulated.     

CD4 and CD8 T cells,but not B cells,are critical to the control of murine experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune disease of the peripheral nervous system that duplicates the clinical, pathological, and electrophysiological features of Guillain-Barré syndrome in humans. However, the molecular pathogenesis of EAN remains controversial. Therefore, for this study, we induced EAN with P0 protein peptide 180-199 in CD4(-/-), CD8(-/-), CD4(-)8(-), and B cell knockout (microMT) mice to further investigate the roles of these cells in EAN. Our results showed that the severity of clinical signs and histopathological manifestations of EAN and the T cell response to P0 peptide 180-199 in CD4(-/-) mice were significantly lower than those in their wild-type counterparts. CD8(-/-) mice also had a milder clinical course, less histopathological change, and a diminished T cell response to P0 peptide 180-199. However, more severe clinical and histopathological manifestations, a stronger T cell response to P0 peptide 180-199, and enhanced IFN-gamma production in the spleen were observed in the EAN of CD4(-)8(-) and microMT mice, but these were not obviously different from those of wild-type mice. Levels of IgG production were similar in sera from CD4(-/-), CD8(-/-), and CD4(-)8(-), and wild-type mice. These findings suggest that the induction and control of murine EAN are dependent on both CD4(+) and CD8(+) T cells and that B cells apparently do not perpetuate the related inflammatory demyelination.     

A study of associated cell-mediated immune mechanisms in experimental autoimmune neuritis rats

The aims of this study are to investigate the optimal antigens used to induce acute or chronic EAN and the associated cell-mediated immune mechanisms. Lewis rats were grouped into EAN rats and control rats. EAN rats were immunized by injection into both hind footpads of inoculums containing 100 microg/200 microg of P2 peptide 57-81 and FCA, or 200 mug of P0 peptide 180-199 and FCA. Control rats were immunized by FCA. Clinical scores were compared at the maximum of disease. On the 14th day after immunization, we examined lymphocyte proliferation, fractions of CD4+ T cells within lymph node mononuclear cells (MNC), frequencies of CD4+CD25+ T cells within CD4+ T cells, supernatant productions of IFN-gamma, IL-4, IL-10 and TGF-beta1 secreted by lymphocytes. Histopathology of sciatic nerves was assessed. Our findings indicated: (1) 100 microg of P2 peptide 57-81 and 200 microg of P0 peptide 180-199 may induce an acute EAN and 200 microg of P2 peptide 57-81 may induce a chronic EAN; (2) Lewis rats were more sensitive to P2 peptide 57-81 than P0 peptide 180-199; (3) clinical disease had nothing to do with a change of relative CD4+ T cells number in lymph node MNC; (4) frequencies of CD4+CD25+ T cells and levels of TGF-beta1 secreted by lymphocytes negatively paralleled clinical EAN, while levels of IFN-gamma secreted by lymphocytes roughly paralleled clinical EAN at the acute phase; (5) sciatic nerve sections from the chronic EAN rats didn't show any inflammatory cells, but showed remaining segmental demyelination and axonal collapse at the chronic phase; (6) self-limitation of acute EAN may owe to the rising levels of IL-4 and IL-10, while a longer duration of chronic EAN may owe to the decreasing levels of IL-4 and IL-10.     

Increased susceptibility to experimental autoimmune neuritis after upregulation of the autoreactive T cell response to peripheral myelin antigen in apolipoprotein E-deficient mice

Experimental autoimmune neuritis (EAN), an acute demyelinating inflammatory disease of the peripheral nervous system (PNS), is a good model for the human counterpart, Guillain-Barré syndrome. Apolipoprotein E (ApoE), a 34 kDa glycosylated protein with multiple biological properties, has been linked both with the innate immune response of mice and with neurological disease. The present study investigated the previously unexplored role of ApoE in autoimmune-mediated demyelination. ApoE-deficient (apoE -/-) mice exhibited a greater susceptibility to EAN induced by the PNS myelin P0 protein peptide 180-199, as compared to wild type (apoE +/+) mice. The augmented susceptibility seen in apoE -/- mice was associated with increased inflammatory cell infiltrates in the PNS during the effector phase. Although the 2 groups of mice exhibited no quantitative or proportional differences in splenic lymphocyte populations, the apoE -/- mice showed enhanced antigen-specific proliferation of T cells of spleen, which is related to modified macrophage function, upregulation of Th1 and downregulation of Th2-autoreactive responses to P0 peptide. These effects were shown as increased numbers of IFN-gamma expressing cells in the spleen and of IFN-gamma, IL-12 and TNF-alpha expressing cells in the PNS, as well as a decreased IL-10 production by splenic cells in apoE -/- mice. In addition, apoE -/- mice had enhanced antigen-specific antibody responses, which might have contributed to their aggravated EAN. These data provide strong evidence that apoE acts as an inhibitor of this inflammatory and demyelinating disease by upregulating IL-10, as well as by inhibiting Th1 responses and antigen-specific antibody formation. These data may aid the development of new and more effective therapeutic strategies for inflammatory and demyelinating diseases such as Guillain-Barré syndrome.     

Hyperpolarisation-activated cyclic nucleotide channel 4 (HCN4) involvement in Tourette's syndrome autoimmunity

Previous studies have shown that interferon-gamma (IFN-γ) is a proinflammatory cytokine that contributes to the pathogenesis of Guillain-Barré syndrome and its animal model, experimental autoimmune neuritis (EAN). Treatments with anti-IFN-γ antibodies improve clinical outcome in GBS patients and EAN animals and administration of IFN-γ markedly worsens EAN. Paradoxically, the mice deficient in IFN-γ remain susceptible to experimental autoimmune encephalomyelitis, an analogous disease in the central nervous system. These observations raise a question whether IFN-γ might be protective in autoimmune demyelinating diseases. To clarify the role of IFN-γ in the pathogenesis of autoimmune demyelinating diseases, we used P0 protein peptide 180-199 to induce EAN in IFN-γ knockout (KO) mice. After the acute phase of EAN, the clinical signs of IFN-γ KO mice were significantly more severe than those of wild type (WT) controls. After antigenic stimulation, the proliferation of splenic mononuclear cells was significantly higher in IFN-γ KO than in WT mice with EAN. At the peak of EAN, the proportion of interleukin (IL)-17A expressing cells in cauda equina (CE) infiltrating cells, and the levels of IL-17A in sera were elevated in IFN-γ KO mice when compared with their WT counterparts. The proportions of major histocompatibility complex (MHC) II, macrosialin, and IL-12/IL-23p40 expressing cells, relative to total CE infiltrating cells were correspondingly higher in IFN-γ KO than in WT mice with EAN. However, IFN-γ deficiency reduced the production of NO by cultured macrophages in response to proinflammatory stimuli and induced a systemic Th2-oriented immune response. In conclusion, IFN-γ deficiency exacerbates EAN via upregulating Th17 cells despite a mitigated systemic Th1 immune response.