Gut dysbiosis breaks immunological tolerance toward the central nervous system during young adulthood.

Multiple sclerosis (MS) is an autoimmune disease targeting the central nervous system (CNS) mainly in young adults, and a breakage of immune tolerance to CNS self-antigens has been suggested to initiate CNS autoimmunity. Age and microbial infection are well-known factors involved in the development of autoimmune diseases, including MS. Recent studies have suggested that alterations in the gut microbiota, referred to as dysbiosis, are associated with MS. However, it is still largely unknown how gut dysbiosis affects the onset and progression of CNS autoimmunity. In this study, we investigated the effects of age and gut dysbiosis on the development of CNS autoimmunity in humanized transgenic mice expressing the MS-associated MHC class II (MHC-II) gene, HLA-DR2a, and T-cell receptor (TCR) genes specific for MBP87-99/DR2a that were derived from an MS patient. We show here that the induction of gut dysbiosis triggers the development of spontaneous experimental autoimmune encephalomyelitis (EAE) during adolescence and early young adulthood, while an increase in immunological tolerance with aging suppresses disease onset after late young adulthood in mice. Furthermore, gut dysbiosis induces the expression of complement C3 and production of the anaphylatoxin C3a, and down-regulates the expression of the Foxp3 gene and anergy-related E3 ubiquitin ligase genes. Consequently, gut dysbiosis was able to trigger the development of encephalitogenic T cells and promote the induction of EAE during the age window of young adulthood.

dl-trans-3,4-Dihydroxy-1-selenolane (DHSred) heals indomethacin-mediated gastric ulcer in mice by modulating arginine metabolism.

BACKGROUND: The importance of the arginine metabolism in gastric ulcer-healing is given relatively less attention. Hence the role of controlling this pathway by dl-trans-3,4-dihydroxy-1-selenolane (DHSred) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated. METHODS: Swiss albino mice were ulcerated with indomethacin followed by treatment with the test samples, and the activities of myeloperoxidase (MPO), total nitric oxide synthase (NOS) and arginase, the expressions of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS), and the pro-/anti-inflammatory cytokine levels were assayed. NOS-inhibitors were also used to establish the biochemical mechanism. RESULTS: Indomethacin induced maximum ulceration in mice on the 3rd day, associated with reduced arginase activity, eNOS expression, along with increased MPO and total NOS activities, nitric oxide (NO) generation, iNOS expression, and pro-/anti-inflammatory (Th1/Th2) cytokine ratio. Treatment with DHSred (2.5mgkg(-1)×3days) restored the cytokine balance to shift the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity and eNOS expression, and reduced iNOS expression, total NOS activity and NO level. CONCLUSIONS: The ulcer-healing property of DHSred, but not of omeprazole was due to a favorable pro-/anti-inflammatory cytokine ratio that shifted the arginine metabolism to the polyamine pathway and increased the eNOS/iNOS ratio. The healing action of omeprazole was not significantly associated with these parameters. GENERAL SIGNIFICANCE: The shift in the ariginine-metabolism from the iNOS/NO axis to the arginase/polyamine axis is guided by Th1/Th2 cytokines ratio and plays an important role in gastric ulcer-healing. The favourable effects of the non-toxic and water-soluble compound, DHSred on these pathways and other COX-dependent and antioxidative parameters suggested it to be a promising anti-ulcer formulation for further studies.

Advances in the immunopathogenesis of multiple sclerosis.

PURPOSE OF REVIEW: Recent studies indicate a role for immune dysregulation in the pathogenesis of multiple sclerosis, an inflammatory demyelinating and degenerative disease of the central nervous system. This review addresses the current mechanisms of immune dysregulation in the development of multiple sclerosis, including the impact of environmental risk factors on immunity in both multiple sclerosis and its animal models. RECENT FINDINGS: CD4 T-helper (Th) cells have long been implicated as the main drivers of pathogenesis of multiple sclerosis. However, current studies indicate that multiple sclerosis is largely a heterogeneous disease process, which involves both innate and adaptive immune-mediated inflammatory mechanisms that ultimately contribute to demyelination and neurodegeneration. Therefore, B cells, CD8 T cells, and microglia/macrophages can also play an important role in the immunopathogenesis of multiple sclerosis apart from proinflammatory CD4 Th1/Th17 cell subsets. Furthermore, increasing evidence indicates that environmental risk factors, such as Vitamin D deficiency, Epstein-Barr virus, smoking, Western diet, and the commensal microbiota, influence the development of multiple sclerosis through interactions with genetic variants of multiple sclerosis, thus leading to the dysregulation of immune responses. SUMMARY: A better understanding of immune-mediated mechanisms in the pathogenesis of multiple sclerosis and the contribution of environmental risk factors toward the development of multiple sclerosis will help further improve therapeutic approaches to prevent disease progression.

Inhibition of TNF-α, and NF-κB and JNK pathways accounts for the prophylactic action of the natural phenolic, allylpyrocatechol against indomethacin gastropathy.

BACKGROUND: The gastro-intestinal disorders, induced by the NSAIDs including indomethacin (IND) remain unresolved medical problems. Herein, we disclose allylpyrocatechol (APC) as a potential agent against IND-gastropathy and rationalize its action mechanistically. METHODS: Mice were pre-treated with APC for 1h followed by IND (18mgkg(-1)) administration, and the ulcer-prevention capacity of APC was evaluated on the 3rd day by histology. Its effect on the inflammatory (MPO, cytokines, adhesion molecules), ulcer-healing (COX, prostaglandins, growth factors and their receptors) and signaling parameters (NF-κB and MAPKs) were assessed by immunoblots/mRNA, and ELISA at the time points of their maximal changes due to IND administration. RESULTS: IND induced oxidative stress, triggering mucosal TNF-α that activated NF-κB and JNK MAPK signaling in mice. These increased the pro-inflammatory biochemical parameters, but reduced the healing factors. APC reversed all the adverse effects to prevent gastric ulceration. APC (5mgkg(-1)), trolox (50mgkg(-1)) and NAC (250mgkg(-1)) showed similar protection that was better than that by misoprostol (5µgkg(-1)) and omeprazole (3mgkg(-1)). CONCLUSIONS: The anti-ulcer effect of APC can be primarily attributed to its antioxidant action that helped in controlling various inflammatory parameters and augmenting angiogenesis. GENERAL SIGNIFICANCE: Given that APC is an effective, non-toxic antioxidant with appreciable natural abundance, further evaluation of its pharmacokinetics and dynamics would help in promoting it as a new anti-inflammatory agent.

Fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis and intestinal inflammation in multiple sclerosis.

Multiple Sclerosis (MS) has been reported to be associated with intestinal inflammation and gut dysbiosis. To elucidate the underlying biology of MS-linked gut inflammation, we investigated gut infiltration of immune cells during the development of spontaneous experimental autoimmune encephalomyelitis (EAE) in humanized transgenic (Tg) mice expressing HLA-DR2a and human T cell receptor (TCR) specific for myelin basic protein peptide (MBP87-99)/HLA-DR2a complexes. Strikingly, we noted the simultaneous development of EAE and colitis, suggesting a link between autoimmune diseases of the central nervous system (CNS) and intestinal inflammation. Examination of the colon in these mice revealed the infiltration of MBP-specific Th17 cells as well as recruitment of neutrophils. Furthermore, we observed that fecal Lipocalin-2 (Lcn-2), a biomarker of intestinal inflammation, was significantly elevated and predominantly produced by the gut-infiltrating neutrophils. We then extended our findings to MS patients and demonstrate that their fecal Lcn-2 levels are significantly elevated compared to healthy donors (HDs). The elevation of fecal Lcn-2 levels correlated with reduced bacterial diversity and increased levels of other intestinal inflammation markers including neutrophil elastase and calprotectin. Of interest, bacteria thought to be beneficial for inflammatory bowel disease (IBD) such as Anaerobutyricum, Blautia, and Roseburia, were reduced in fecal Lcn-2-high MS patients. We also observed a decreasing trend in serum acetate (a short-chain fatty acid) levels in MS Lcn-2-high patients compared to HDs. Furthermore, a decrease in the relative abundance of Blautia massiliensis was significantly associated with a reduction of acetate in the serum of MS patients. This study suggests that gut infiltration of Th17 cells and recruitment of neutrophils are associated with the development of gut dysbiosis and intestinal inflammation, and that fecal Lcn-2 level is a sensitive biological indicator for gut dysbiosis in multiple sclerosis.

Effect of switching glatiramer acetate formulation from 20 mg daily to 40 mg three times weekly on immune function in multiple sclerosis.

BACKGROUND: Many RRMS patients who had been treated for over 20 years with GA 20 mg/ml daily (GA20) switched to 40 mg/ml three times-a-week (GA40) to reduce injection-related adverse events. Although GA40 is as effective as GA20 in reducing annualized relapse rate and MRI activity, it remains unknown how switching to GA40 from GA20 affects the development of pathogenic and regulatory immune cells. OBJECTIVE: To investigate the difference in immunological parameters in response to GA20 and GA40 treatments. METHODS: We analyzed five pro-inflammatory cytokines (IL-1ß, IL-23, IL-12, IL-18, TNF-α), and three anti-inflammatory/regulatory cytokines (IL-10, IL-13, and IL-27) in serum. In addition, we analyzed six cytokines (IFN-γ, IL-17A, GM-CSF, IL-10, IL-6, and IL-27) in cultured PBMC supernatants. The development of Th1, Th17, Foxp3 Tregs, M1-like, and M2-like macrophages were examined by flow cytometry. Samples were analyzed before and 12 months post switching to GA40 or GA20. RESULTS: Pro- and anti-inflammatory cytokines were comparable between the GA40 and GA20 groups. Development of Th1, Th17, M1-like macrophages, M2-like macrophages, and Foxp3 Tregs was also comparable between the two groups. CONCLUSIONS: The immunological parameters measured in RRMS patients treated with GA40 three times weekly are largely comparable to those given daily GA20 treatment.

Dimethyl Fumarate Suppresses Demyelination and Axonal Loss through Reduction in Pro-Inflammatory Macrophage-Induced Reactive Astrocytes and Complement C3 Deposition.

Dimethyl fumarate (DMF) is an oral agent for relapsing-remitting multiple sclerosis (RRMS). In this study, we investigated the therapeutic mechanism of DMF using experimental autoimmune encephalomyelitis (EAE). DMF treatment decreased the proliferation of T cells and the production of IL-17A and GM-CSF. DMF treatment also decreased the development and/or infiltration of macrophages in the central nervous system (CNS), and reduced the ratio of iNOS+ pro-inflammatory macrophage versus Ym1+ immunomodulatory macrophages. Furthermore, DMF treatment suppressed the deposition of complement C3 (C3) and development of reactive C3+ astrocytes. The decrease in iNOS+ macrophages, C3+astrocytes, and C3 deposition in the CNS resulted in the reduction in demyelination and axonal loss. This study suggests that the beneficial effects of DMF involve the suppression of iNOS+ pro-inflammatory macrophages, C3+ astrocytes, and deposition of C3 in the CNS.

Surface Layer Protein A Expressed in Clostridioides difficile DJNS06-36 Possesses an Encephalitogenic Mimotope of Myelin Basic Protein.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Recent studies suggest that migration of Th1 and Th17 cells specific for enteric bacteria from the gut to the CNS may lead to the initiation and/or exacerbation of autoimmune diseases including MS. Human leukocyte antigen (HLA)-DR15 is an MHC class II (MHCII) haplotype highly associated with the development of MS that contains the two HLA-DRB* genes, DRB1*1501 (DR2b) and DRB5*0101 (DR2a). To identify enteric bacteria which harbor antigenic epitopes that activate myelin-specific T cells and drive CNS inflammation, we screened for enteric bacteria which express cross-reactive epitopes ('mimotopes') of an immunodominant myelin basic protein 89-98 (MBP89-98) epitope. Based on known MHCII HLA-DR2a amino acid binding motifs and cultivation with splenic T cells isolated from MBP-T cell receptor (TCR)/DR2a transgenic (Tg) mice, we discovered that a certain variant of surface layer protein A (SLPA), which is expressed by a subtype of Clostridioides difficile, contains an amino acid sequence that activates MBP89-98-reactive T cells. Furthermore, activation of MBP-specific T cells by SLPA upon active immunization induced experimental autoimmune encephalomyelitis (EAE) in MBP-TCR/DR2a Tg mice. This study suggests that a unique strain of C. difficile possesses an encephalitogenic mimotope of MBP that activates autoreactive, myelin-specific T cells.

A bis-resorcinol resveratrol congener prevents indomethacin-induced gastric ulceration by inhibiting TNF-α as well as NF-κB and JNK pathways.

The cytoprotective action of the synthetic resveratrol (Resv) congener, E-3,3',5,5'-tetrahydroxystilbene (designated as HST-1) against indomethacin (IND)-induced stomach ulceration has been established using a mice model. HST-1 reversed the adverse effects of IND on several inflammatory (myeloperoxidase, cytokines, adhesion molecules etc.) and ulcer-healing (cyclooxygenases, prostaglandin, growth factors and their receptors etc.) parameters in mice. More importantly, HST-1 down-regulated TNF-α and the TNF-α-mediated activation of NF-κB and JNK/MAPK pathways that are the key determinants in the IND-gastropathy. The effect of HST-1 on all these factors was significantly better than that of Resv, misoprostol, and omeprazole. HST-1 also did not induce small intestinal mucosal injury, unlike some of the proton pump inhibitors. On the other hand, Resv reduced activation of the prosurvival ERK1/2 pathway that may explain its contraindicative property in the gastrointestinal tract.

IL-27: a potential biomarker for responders to glatiramer acetate therapy.

Glatiramer acetate (GA) is an FDA-approved efficacious drug for the treatment of relapsing-remitting multiple sclerosis (RRMS). However, this treatment is not effective for all RRMS patients. Therefore, it is important to identify reliable biomarkers that can predict a beneficial clinical response to GA therapy. Since an increase in IL-27 has been demonstrated to suppress autoimmune and allergic diseases of inflammatory origin, we examined the effect of GA on the production of IL-27. We observed that IL-27 production in PBMCs cultured with GA was heterogeneous amongst MS patients and healthy donors (HD), and thus, defined these MS patients as either efficient, weak, or non-IL-27 producers. Interestingly, GA could induce the expression of the IL-27p28 subunit more efficiently than the IL-27 EBI3 subunit, and the production of IL-27 depended on MHC class II binding by GA. In addition, we found that GA could augment Toll-like receptor (TLR)-mediated IL-27 production. Importantly, serum production of IL-27 and IL-10 was significantly increased at 6months during GA therapy in clinical responders to GA, but not in GA non-responders. Altogether, our data suggest that GA-induced IL-27 may represent a therapeutic mechanism of GA-mediated immunomodulation and that GA-mediated IL-27 production in PBMCs is worth exploring as a biomarker to screen for GA responders prior to the initiation of GA treatment.

Molecular mechanism of indomethacin-induced gastropathy.

The probable cross talk among large numbers of inflammatory and angiogenic parameters in indomethacin (IND)-induced gastropathy and the associated signaling mechanism were studied in a mouse model. A single dose of IND (18 mg/kg, po) produced robust gastric ulceration in mice without any mortality, which peaked on the third day, but started healing from the fifth day onward. The ulceration was associated with increased myeloperoxidase activity and expression of proinflammatory (TNF-α, adhesion molecules, COX-2) and antiangiogenic (endostatin) parameters. The levels of proangiogenic factors such as COX-1, prostaglandin E, VEGF, and von Willebrand factor VIII were downregulated by IND. Our results revealed that although the maximal and minimal levels of these parameters were attained sequentially at different time points, TNF-α upregulation was the primary event to initiate and induce gastric ulceration. IND also activated NF-κB and all the MAP kinases, but only the inhibitors of TNF-α, NF-κB, and JNK MAP kinase could abrogate the IND-induced damages. Further TNF-α inhibition also reduced the IND-mediated activation of NF-κB and JNK MAP kinase. All this evidence strongly suggests that mitigation of TNF-α may offer a potential solution to IND-mediated gastropathy.

DL-trans-3,4-dihydroxy-1-selenolane (DHS(red)) accelerates healing of indomethacin-induced stomach ulceration in mice.

Management of the gastro-toxicity of non-steroidal anti-inflammatory drugs (NSAIDs) remains a crucial problem, because the commercially available anti-ulcer drugs have side effects and are often expensive. Hence, the potential of a new water-soluble GPx mimic, DL-trans-3,4-dihydroxy-1-selenolane (DHS(red)) in healing the indomethacin-induced stomach ulceration in mice was examined. Administration of indomethacin (18 mg/kg, p. o.) induced ulceration in the glandular portion of the gastric mucosa, accompanied by increased lipid peroxidation (1.3-fold, p <0.001) and protein oxidation (1.5-fold, p < 0.001), depletion of thiol-defense (42.5%, p < 0.01), plasma total antioxidant status (53.4%, p < 0.001) and mucin (47.5%, p < 0.01), as well as reduced expressions of cyclooxygenases and prostaglandin synthesis (54.7%, p < 0.001) in the gastric tissues of mice. Daily oral administration of DHS(red) (2.5 mg/kg) or omeprazole (Omez) (3 mg/kg) for 3 days respectively produced ˜74% and 69% (p < 0.001) healing of the acute gastric ulceration. The test samples also significantly reversed all the adverse effects of indomethacin on the biochemical parameters. Apparently, the gastric ulcer healing action of DHS(red) and Omez was due to their antioxidant action and their ability to protect mucin and augment PG synthesis by upregulation of the COX isozymes. The results suggested that the non-toxic and inexpensive compound, DHS(red), may be a good candidate for further evaluation as a potent anti-ulcer drug.

Black tea and theaflavins suppress various inflammatory modulators and i-NOS mediated nitric oxide synthesis during gastric ulcer healing.

The modulation of the cyclooxygenase-independent pathway by black tea (BT) and its constituent theaflavins (TFs) during their healing action against indomethacin-induced stomach ulceration in mice was investigated. On the 3(rd) day of its administration, indomethacin (18 mg/kg) induced maximum stomach ulceration, which was associated with increased myeloperoxidase (MPO) activity (93.3%, p<0.001), and inducible nitric oxide synthase (iNOS) expression (1.6-fold, p<0.001), along with augmented levels of serum nitrite (1.5-fold, p<0.001), selectins and cell adhesion molecules (CAMs), as well as reduced endothelial nitric oxide synthase (eNOS) expression (60%, p<0.001). Treatment with BT (40 mg/kg) and TF (1 mg/kg) for 3 days reversed these parameters and provided excellent (78-81%) ulcer healing. However, alterations of NOS expressions and levels of selectins and CAMs were only partially responsible for the excellent healing capacity (∼80%) of omeprazole (3 mg/kg × 3 days).

Epigallocatechin gallate accelerates healing of indomethacin-induced stomach ulcers in mice.

Management of the gastric toxicity of non-steroidal anti-inflammatory drugs (NSAIDs) remains a crucial problem because the commercially available drugs have side effects and are often expensive. Therefore, we examined the potential of the green tea-derived polyphenol epigallocatechin gallate (EGCG) to treat indomethacin-induced stomach ulcers in mice. Administration of indomethacin (18 mg/kg, po) to mice induced ulceration in the glandular portion of the gastric mucosa, accompanied by increased lipid peroxidation (LPO) and protein oxidation and reductions in thiol defense, mucin, cyclooxygenase (COX) expression and prostaglandin (PG) synthesis in the gastric tissues. Daily oral administration of EGCG (2 mg/kg) or omeprazole (3 mg/kg) for 3 days produced similar (≈ 72-75%, p < 0.001) beneficial effects on the acute gastric ulceration. Treatment with the test samples partially reversed all the adverse oxidative effects of indomethacin. In addition, EGCG, but not omeprazole, enhanced expression of the COX isoforms and PG synthesis. The results suggest that the non-toxic and inexpensive tea polyphenol EGCG may be an excellent candidate for further evaluation as a potent anti-ulcer drug.

Role of the COX-independent pathways in the ulcer-healing action of epigallocatechin gallate.

The modulation of the cyclooxygenase-independent pathway by the green tea-derived polyphenol, epigallocatechin gallate (EGCG) during its healing action against indomethacin (IND)-induced stomach ulceration in mice was investigated. On the 3rd day of its administration, IND (18 mg kg(-1)) induced maximum stomach ulceration which was associated with increased myeloperoxidase (MPO) activity (2.1-fold, p < 0.001), and inducible nitric oxide synthase (iNOS) expression (2.5-fold, p < 0.001), along with augmented levels of serum nitrite (1.3-fold, p < 0.001), selectins and cell adhesion molecules (CAMs), as well as reduced endothelial nitric oxide synthase (eNOS) expression (53%, p < 0.001). Treatment with EGCG (2 mg kg(-1)) and omeprazole (3 mg kg(-1)) for 3 days reversed these parameters, and provided excellent (76-77%) ulcer healing.

The gastric ulcer-healing action of allylpyrocatechol is mediated by modulation of arginase metabolism and shift of cytokine balance.

The role of the ariginine-metabolism in the healing action of the Piper betle phenol, allylpyrocatechol (APC) and omeprazole against indomethacin-induced stomach ulceration in mouse was investigated. Indomethacin (18 mg/kg) was found to induce maximum stomach ulceration in Swiss albino mice on the 3rd day of its administration, which was associated with reduced arginase activity (21.6%, P<0.05), endothelial nitric oxide synthase (eNOS) expression (72%, P<0.001), and IL-4 and TGF-beta levels, along with increased inducible nitric oxide synthase (iNOS) (9.3 folds, P<0.001) expression, nitrite (2.29 folds, P<0.001), IL-1beta and IL-6 generation. Besides providing comparable healing as omeprazole (3 mg/kg x 3 days), APC (5 mg/kg x 3 days) shifted the iNOS/NO axis to the arginase/polyamine axis as revealed from the increased arginase activity (73.1%, P<0.001), eNOS expression (67.8%, P<0.001), and reduced iNOS expression (65.6%, P<0.001) and nitrite level (53.2%, P<0.001). These can be attributed to a favourable anti-/pro-inflammatory cytokines ratio, generated by APC. The healing by omeprazole was however, not significantly associated with those parameters.