Growth phase of orally administered Lactobacillus strains differentially affects IgG1/IgG2a ratio for soluble antigens: Implications for vaccine development

Lactobacillus strains with probiotic activity are major constituents of numerous common food products. Due to their 'generally regarded as safe'-status (GRAS-status), Lactobacillus strains can also be genetically engineered for use in oral immunotherapeutic applications, such as vaccination and T lymphocyte tolerance induction in autoimmune disease. In the current study, we demonstrate that the growth phase of orally administered individual Lactobacillus strains can differentially affect antigen-specific antibody subclasses IgG1 and IgG2a, which might reflect skewing of systemic activity of T helper cell type 2 (Th2) and T helper cell type 1 (Th1) pathways, respectively. Mice were orally fed different wild type Lactobacillus strains in log phase or stationary phase and immunized intraperitoneally with a T-cell dependent protein antigen. Sera were evaluated for the ratio of antigen-specific IgG1 and IgG2a antibodies. Stationary Lactobacillus murines and Lactobacillus casei cultures, but not two other Lactobacillus strains, evoked significantly higher IgG1/IgG2a ratios than log phase cultures, possibly relating to increased activity of the Th2-pathway. Despite normal variation in antibody responses against TNP-CGG among individual mice, a high correlation was found between the IgG1 and IgG2a responses of mice within experimental groups. This differential antibody response is likely due to growth phase-dependent differences in bacterial cell composition. Since Lactobacillus growth phase dependent skewing of antibody responses possibly reflecting T-cell pathways can inadvertently affect allergic and (auto)-immune responses, the current findings strongly caution against unidimensional views on the oral administration of individual Lactobacillus strains for probiotic or immunotherapeutic purposes, but also suggest additional possibilities for immune modulation

Reduced experimental autoimmune encephalomyelitis after intranasal and oral administration of recombinant lactobacilli expressing myelin antigen

Oral administration of autoantigens is a safe and convenient way to induce peripheral T-cell tolerance in autoimmune diseases like multiple sclerosis (MS). To increase the efficacy of oral tolerance induction and obviate the need for large-scale purification of human myelin proteins, we use genetically modified lactobacilli expressing myelin antigens. A panel of recombinant lactobacilli was constructed producing myelin proteins and peptides, including human and guinea pig myelin basic protein (MBP) and proteolipid protein peptide 139-151 (PLP139-151). In this study we examined whether these Lactobacillus recombinants are able to induce oral and intranasal tolerance in an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Lewis rats received soluble cell extracts of Lactobacillus transformants intranasally three times prior to induction of EAE. For the induction of oral tolerance, rats were fed live transformed lactobacilli for 20 days. Ten days after the first oral administration EAE was induced. Intranasal administration of extracts containing guinea pig MBP (gpMBP) or MBP72-85 significantly inhibited EAE in Lewis rats. Extracts of control transformants did not reduce EAE. Live lactobacilli expressing guinea pig MBP72-85 fused to the marker enzyme β-glucuronidase (β-gluc) were also able to significantly reduce disease when administered orally. In conclusion, these experiments provide proof of principle that lactobacilli expressing myelin antigens reduce EAE after mucosal (intranasal and oral) administration. This novel method of mucosal tolerance induction by mucosal administration of recombinant lactobacilli expressing relevant autoantigens could find applications in autoimmune disease in general, such as multiple sclerosis, rheumatoid arthritis and uveitis

The JAK1/JAK2- inhibitor ruxolitinib inhibits mast cell degranulation and cytokine release

Background: Mastocytosis is characterized by the accumulation of aberrant mast cells (MC). Patients suffering from mastocytosis suffer from a wide range of symptoms due to increased levels of MC mediators. It would therefore be of great benefit to inhibit MC mediator release. However, to date there are few drugs available that are known to effectively lower MC mediator levels. The evidence for the involvement of the janus kinase 2 (JAK2)—signal transducer and activation of transcription 5 (STAT5) signalling pathway in MC activation is slowly accumulating. Interference with the JAK2-STAT5 pathway might inhibit MC mediator release. Ruxolitinib, a JAK1/JAK2 inhibitor, indeed decreases symptoms like pruritus and fatigue in patients with myeloproliferative neoplasms. Yet, detailed studies on how ruxolitinib affects human mast cell activity are lacking. Objective: To investigate the effect of JAK1/2-inhibition with ruxolitinib in the human mast cell lines LAD2 and HMC1. Methods: LAD2 and HMC1 were stimulated with substance P, codeine or the calcium ionophore A23817. The effect of ruxolitinib on mast cell degranulation (via measurement of β-hexosaminidase, histamine release and CD63 membrane expression) and IL-6, IL-13, MCP-1 and TNF-α production was investigated. The involvement of STAT5 activation was explored using the selective STAT5 inhibitor pimozide. Results: Ruxolitinib effectively inhibited codeine- and substance P-induced degranulation in a concentration-dependent manner. Ruxolitinib also significantly inhibited the production of IL-6, TNF-α and MCP-1 as induced by A23817 and substance P. Selective STAT5 inhibition with pimozide resulted in diminished degranulation and inhibition of cytokine production as induced by A23817 and substance P. Conclusions & clinical relevance: This study demonstrates that the JAK1/JAK2 inhibitor ruxolitinib can inhibit MCactivity, possibly through prevention of STAT5 activation. This renders the JAK-STAT pathway as an interesting target for therapy to release symptom burden in mastocytosis and many other MC mediator-related diseases

The β-human chorionic gonadotropin-related peptide LQGV exerts anti-inflammatory effects through activation of the adrenal gland and glucocorticoid receptor in C57BL/6 mice

The systemic inflammatory response syndrome is a complex host response to a variety of clinical insults, generally leading to severe pathology. The human chorionic gonadotropin β-chain-related tetrapeptide leucine-glutamine- glycine-valine (LQGV) reduces hemorrhagic and LPS-induced systemic inflammatory response syndrome, but its mechanisms of action are not yet fully understood. Through the combination of in vivo, in vitro, and ex vivo approaches, we demonstrate that LQGV actively stimulates corticosterone production in mice and thereby suppresses in vivo TLR4-directed inflammation upon LPS administration. Blocking in vivo glucocorticosteroid receptor signaling reduced the prosurvival effect of LQGV. Also, upon multiple TLR activation by heat-killed Listeria monocytogenes, splenocytes from LQGV-treated mice produced significantly less TNF-α and IL-6, which was absent after in vitro blockage of the glucocorticosteroid receptor. Using adrenal gland and adrenal cell line cultures, we show that LQGV stimulates corticosterone production. Moreover, by using specific pharmacological inhibitors of the adrenocorticotropic hormone (ACTH) and luteinizing hormone receptors as well as of cAMP signaling, we demonstrate that LQGV stimulates the ACTH receptor. These data show that the b-human chorionic gonadotropin-related tetrapeptide LQGV stimulates adren

Growth phase of orally administered Lactobacillus strains differentially affects IgG1/IgG2a ratio for soluble antigens:implications for vaccine development

Lactobacillus strains with probiotic activity are major constituents of numerous common food products. Due to their 'generally regarded as safe'-status (GRAS-status), Lactobacillus strains can also be genetically engineered for use in oral immunotherapeutic applications, such as vaccination and T lymphocyte tolerance induction in autoimmune disease.In the current study, we demonstrate that the growth phase of orally administered individual Lactobacillus strains can differentially affect antigen-specific antibody subclasses IgG1 and IgG2a, which might reflect skewing of systemic activity of T helper cell type 2 (Th2) and T helper cell type 1 (Th1) pathways, respectively. Mice were orally fed different wild type Lactobacillus strains in log phase or stationary phase and immunized intraperitoneally with a T-cell dependent protein antigen. Sera were evaluated for the ratio of antigen-specific IgG1 and IgG2a antibodies. Stationary Lactobacillus murines and Lactobacillus casei cultures, but not two other Lactobacillus strains, evoked significantly higher IgG1/IgG2a ratios than log phase cultures, possibly relating to increased activity of the Th2-pathway. Despite normal variation in antibody responses against TNP-CGG among individual mice, a high correlation was found between the IgG1 and IgG2a responses of mice within experimental groups. This differential antibody response is likely due to growth phase-dependent differences in bacterial cell composition.Since Lactobacillus growth phase dependent skewing of antibody responses possibly reflecting T-cell pathways can inadvertently affect allergic and (auto)-immune responses, the current findings strongly caution against unidimensional views on the oral administration of individual Lactobacillus strains for probiotic or immunotherapeutic purposes, but also suggest additional possibilities for immune modulation

Histamine induces NF-κB controlled cytokine secretion by orbital fibroblasts via histamine receptor type-1

Mast cells and their products are likely to be involved in regulating orbital fibroblast activity in Graves' Ophthalmopathy (GO). Histamine is abundantly present in granules of mast cells and is released upon mast cell activation. However, the effect of histamine on orbital fibroblasts has not been examined so far. Orbital tissues from GO patients and controls were analyzed for the presence of mast cells using toluidine blue staining and immunohistochemical detection of CD117 (stem cell factor receptor). Orbital fibroblasts were cultured from GO patients and healthy controls, stimulated with histamine and cytokines (IL-6, IL-8, CCL2, CCL5, CCL7, CXCL10 and CXCL11) were measured in culture supernatants. Also hyaluronan levels were measured in culture supernatants and hyaluronan synthase (HAS) and hyaluronidase (HYAL) gene expression levels were determined. In addition, histamine receptor subtype gene expression levels were examined as well as the effect of the histamine receptor-1 (HRH1) antagonist loratadine and NF-κB inhibitor SC-514 on histamine-induced cytokine production. Mast cell numbers were increased in GO orbital tissues. Histamine stimulated the production of IL-6, IL-8 and CCL2 by orbital fibroblasts, while it had no effect on the production of CCL5, CCL7, CXCL10, CXCL11 and hyaluronan. Orbital fibroblasts expressed HRH1 and loratadine and SC-514 both blocked histamine-induced IL-6, IL-8 and CCL2 production by orbital fibroblasts. In conclusion, this study demonstrates that histamine can induce the production of NF-κB controlled-cytokines by orbital fibroblasts, which supports a role for mast cells in GO