The Gut-pancreas axis in autoimmune diabetes

The gut microbiota has been suggested to be an important factor in the development of autoimmune diseases such as type 1 diabetes (T1D). Priming of islet specific T cells in the pancreatic lymph nodes (PaLN) and their migration to the pancreatic islets are critical steps in the destruction of insulin producing β-cells that leads the development of autoimmune diabetes. Perturbation of gut homeostasis by pathogenic microbes or imbalance of the gut microbiota during dysbiosis may provoke the autoimmune reactions that are associated with T1D. However, the mechanisms by which altered gut microbiota and its interaction with the immune system affect autoimmune diabetes development are unclear. In this thesis, we investigated how gut microbiota composition influences the progression of autoimmune diabetes in nonobese (NOD) mice and whether dysbiosis aggravates the immunological events associated with T1D pathogenesis. This thesis will show how compromised intestinal barrier integrity and dysbiosis promote islet-specific T cell activation and their attraction to pancreatic islets. First, we show that healthy microbiota is associated with enhanced intestinal integrity and has a beneficial effect on the progression of autoimmune diabetes. We show that induction of dysbiosis with pathogenic microbes impairs intestinal barrier function, where among other factors plasmacytoid dendritic cells (pDCs) have an important regulatory role, and promote the autoimmune responses associated with the β-cell destruction. We describe a novel shared lymphatic drainage between the gut and pancreas that allows dendritic cell and bacterial migration to PaLNs during dysbiosis. Dysbiosis enhances the activation of islet-specific T cells and their priming with chemokine receptor CXCR3 in PaLNs. Additionally, we show that dysbiosis, followed by elevated endotoxin levels, enhances CXCL10 production within the pancreatic islets, which leads to enhanced attraction of CXCR3+ lymphocytes into pancreatic islets. Our results indicate that dysbiosis may be the initial factor that promotes lymphocyte infiltration into healthy pancreatic islets. This thesis will give new insight into the effects of gut microbiota on the development of autoimmune diabetes and the role of dysbiosis in actuating the autoimmune responses associated with T1D pathogenesis.-- Suolistomikrobiston uskotaan olevan tärkeä tekijä autoimmuunisairauksien, kuten tyypin 1 diabeteksen (T1D) kehittymisessä. Tyypillisiä vaiheita autoimmuunidiabeteksen kehittymisessä ovat saareke-spesifisten T-solujen aktivoituminen haiman imusolmukkeissa ja niiden siirtyminen haiman saarekkeisiin, johtaen insuliinia tuottavien β-solujen tuhoutumiseen. Patogeenisten mikrobien tai mikrobiston epätasapainon eli dysbioosin aiheuttama suoliston homeostaasin häiriintyminen voi edesauttaa näitä T1D:ssä havaittuja autoimmuunivasteita. On kuitenkin vielä epäselvää mitkä ovat ne mekanismit, joilla epätasapainoinen suolistomikrobisto ja sen vuorovaikutus immuunijärjestelmän kanssa vaikuttavat autoimmuunidiabeteksen kehittymiseen. Tämän tutkimuksen tarkoituksena oli selvittää, miten suolistomikrobiston koostumus vaikuttaa autoimmuunidiabeteksen kehittymiseen ja kuinka dysbioosi voi pahentaa T1D-patogeneesiin liittyviä immunologisia vasteita. Tämä väitöskirja osoittaa, kuinka heikentynyt suolen seinämän eheys ja dysbioosi edistävät saareke-spesifisten T-solujen aktivaatiota ja niiden houkuttelua haiman saarekkeisiin. Osoitamme, että terveellä mikrobistolla ja hyvällä suolen seinämän eheydellä on hidastava vaikutus autoimmuunidiabeteksen kehittymisessä. Vastaavasti osoitamme, että dysbioosi edistää β-solujen tuhoutumiseen johtavia autoimmuunivasteita heikentämällä suoliston seinämän eheyttä, jossa muiden tekijöiden ohella plasmasytoidi dendriittisoluilla (pDC) on tärkeä säätelevä tehtävä. Osoitamme ennen kuvaamattoman imutieyhteyden suoliston ja haiman välillä, joka mahdollistaa dendriittisolujen ja bakteerien siirtymisen haiman imusolmukkeisiin dysbioosin aikana. Dysbioosi tehostaa saareke-spesifisten T-solujen aktivaatiota ja niiden kemokiinireseptori CXCR3 ilmentymistä. Lisäksi näytämme, kuinka dysbioosi ja siitä johtuva kohonnut endotoksiinitaso indusoi CXCL10 tuotantoa haiman saarekkeissa, mikä lisää CXCR3+ lymfosyyttien houkuttelua haiman saarekkeisiin. Tuloksemme osoittavat, että dysbioosi voi olla yksi ensimmäisistä tekijöistä lymfosyyttien tunkeutumisessa terveisiin haiman saarekkeisiin. Tämä väitöskirja tuottaa uutta tietoa suolistomikrobiston vaikutuksista autoimmuunidiabeteksen kehittymisessä ja siitä, kuinka dysbioosi voi olla merkittävä laukaiseva tekijä T1D patogeneesille tyypillisissä autoimmuunivasteissa

Infection with the enteric pathogen C. rodentium promotes islet-specific autoimmunity by activating a lymphatic route from the gut to pancreatic lymph node

In nonobese diabetic (NOD) mice, C. rodentium promotes priming of islet-specific T-cells in pancreatic lymph nodes (PaLN), which is a critical step in initiation and perpetuation of islet-autoimmunity. To investigate mechanisms by which C. rodentium promotes T-cell priming in PaLN, we used fluorescent imaging of lymphatic vasculature emanating from colon, followed dendritic cell (DC) migration from colon using photoconvertible-reporter mice, and evaluated the translocation of bacteria to lymph nodes with GFP-C. rodentium and in situ hybridization of bacterial DNA. Fluorescent dextran injected in the colon wall accumulated under subcapsular sinus of PaLN indicating the existence of a lymphatic route from colon to PaLN. Infection with C. rodentium induced DC migration from colon to PaLN and bacterial DNA was detected in medullary sinus and inner cortex of PaLN. Following infection with GFP-C. rodentium, fluorescence appeared in macrophages and gut-derived (CD103+) and resident (CD103-/XCR1+) DC, indicating transportation of bacteria from colon to PaLN both by DC and by lymph itself. This induced proinflammatory cytokine transcripts, activation of DC and islet-specific T-cells in PaLN of NOD mice. Our findings demonstrate the existence of a direct, enteric pathogen-activated route for lymph, cells, and bacteria from colon, which promotes activation of islet-specific T-cells in PaLN.</p

Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling

Abnormally high gamma delta T cell numbers among individuals with atypical SCID have been reported but detailed immunopheno typing and functional characterization of these expanded gamma delta T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large gamma delta T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRv gamma sequencing, and target cell killing. In contrast to his alpha beta T cells, the patient's gamma delta T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. V delta 2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's gamma delta T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRv gamma repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to gamma delta T cells. Over time this variant became predominant in gamma delta T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of gamma delta T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.Peer reviewe

Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients

Peer reviewe

Novel Hemizygous IL2RG p.(Pro58Ser) Mutation Impairs IL-2 Receptor Complex Expression on Lymphocytes Causing X-Linked Combined Immunodeficiency

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient's clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.Peer reviewe

Persistent T cell-mediated immune responses against Omicron variants after the third COVID-19 mRNA vaccine dose

IntroductionThe prime-boost COVID-19 mRNA vaccination strategy has proven to be effective against severe COVID-19 disease and death. However, concerns have been raised due to decreasing neutralizing antibody levels after COVID-19 vaccination and due to the emergence of new immuno-evasive SARS-CoV-2 variants that may require additional booster vaccinations.MethodsIn this study, we analyzed the humoral and cell-mediated immune responses against the Omicron BA.1 and BA.2 subvariants in Finnish healthcare workers (HCWs) vaccinated with three doses of COVID-19 mRNA vaccines. We used enzyme immunoassay and microneutralization test to analyze the levels of SARS-CoV-2 specific IgG antibodies in the sera of the vaccinees and the in vitro neutralization capacity of the sera. Activation induced marker assay together with flow cytometry and extracellular cytokine analysis was used to determine responses in SARS-CoV-2 spike protein stimulated PBMCs.ResultsHere we show that within the HCWs, the third mRNA vaccine dose recalls both humoral and T cell-mediated immune responses and induces high levels of neutralizing antibodies against Omicron BA.1 and BA.2 variants. Three weeks after the third vaccine dose, SARS-CoV-2 wild type spike protein-specific CD4+ and CD8+ T cells are observed in 82% and 71% of HCWs, respectively, and the T cells cross-recognize both Omicron BA.1 and BA.2 spike peptides. Although the levels of neutralizing antibodies against Omicron BA.1 and BA.2 decline 2.5 to 3.8-fold three months after the third dose, memory CD4+ T cell responses are maintained for at least eight months post the second dose and three months post the third vaccine dose.DiscussionWe show that after the administration of the third mRNA vaccine dose the levels of both humoral and cell-mediated immune responses are effectively activated, and the levels of the spike-specific antibodies are further elevated compared to the levels after the second vaccine dose. Even though at three months after the third vaccine dose antibody levels in sera decrease at a similar rate as after the second vaccine dose, the levels of spike-specific CD4+ and CD8+ T cells remain relatively stable. Additionally, the T cells retain efficiency in cross-recognizing spike protein peptide pools derived from Omicron BA.1 and BA.2 subvariants. Altogether our results suggest durable cellmediated immunity and protection against SARS-CoV-2

Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients

The emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it more difficult to prevent the virus from spreading despite available vaccines. Reports of breakthrough infections and decreased capacity of antibodies to neutralize variants raise the question whether current vaccines can still protect against COVID-19 disease. We studied the dynamics and persistence of T cell responses using activation induced marker (AIM) assay and Th1 type cytokine production in peripheral blood mononuclear cells obtained from BNT162b2 COVID-19 mRNA vaccinated health care workers and COVID-19 patients. We demonstrate that equally high T cell responses following vaccination and infection persist at least for 6 months against Alpha, Beta, Gamma, and Delta variants despite the decline in antibody levels.</p

Plasmacytoid dendritic cells regulate host immune response to Citrobacter rodentium induced colitis in colon-draining lymph nodes

Dendritic cells (DCs) are first in line to sense invading microbes and to deliver signals to other immune cells. Plasmacytoid DCs (pDC) produce high amounts of type I interferons (IFNs) but also regulate immune responses. Using the Clec4C (BDCA2)-diphtheria toxin receptor mouse model allowing conditional pDC depletion, we identified an essential role for pDCs in regulating intestinal inflammation locally in the gut. In pDC-depleted mice, Citrobacter rodentium infection led to enhanced activation of conventional DCs and induction of IFN-γ-producing Th1-cells in colon-draining lymph nodes, while induction of Foxp3+/CD25+ Treg and IL-17-producing Th17 cells was impaired. Concomitantly, F4/80+ macrophages accumulated into the colon lamina propria in excess, and levels of Il-1β and Tnf transcripts increased and Foxp3+ Treg were fewer. Our results indicate that pDCs control inflammation in the gut during C. rodentium infection and that they have an important immune regulatory role in colon-draining lymph nodes

Akkermansia muciniphila induces gut microbiota remodelling and controls islet autoimmunity in NOD mice

Objective Intestinal microbiota is implicated in the pathogenesis of autoimmune type 1 diabetes in humans and in non-obese diabetic (NOD) mice, but evidence on its causality and on the role of individual microbiota members is limited. We investigated if different diabetes incidence in two NOD colonies was due to microbiota differences and aimed to identify individual microbiota members with potential significance. Design We profiled intestinal microbiota between two NOD mouse colonies showing high or low diabetes incidence by 16S ribosomal RNA gene sequencing and colonised the high-incidence colony with the microbiota of the low-incidence colony. Based on unaltered incidence, we identified a few taxa which were not effectively transferred and thereafter, transferred experimentally one of these to test its potential significance. Results Although the high-incidence colony adopted most microbial taxa present in the low-incidence colony, diabetes incidence remained unaltered. Among the few taxa which were not transferred, Akkermansia muciniphila was identified. As A. muciniphila abundancy is inversely correlated to the risk of developing type 1 diabetes-related autoantibodies, we transferred A. muciniphila experimentally to the high-incidence colony. A. muciniphila transfer promoted mucus production and increased expression of antimicrobial peptide Reg3., outcompeted Ruminococcus torques from the microbiota, lowered serum endotoxin levels and islet toll-like receptor expression, promoted regulatory immunity and delayed diabetes development. Conclusion Transfer of the whole microbiota may not reduce diabetes incidence despite a major change in gut microbiota, but single symbionts such as A. muciniphila with beneficial metabolic and immune signalling effects may reduce diabetes incidence when administered as a probiotic.Peer reviewe