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Li, X., Singh, K., Luo, Z., Mejia Cordova, M., Jamalpour, M., Lindahl, B., . . . Welsh, M. (2018). Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas. OncoTarget, 9(27), 18720-18733
Open this publication in new window or tab >>Pro-tumoral immune cell alterations in wild type and Shb-deficient mice in response to 4T1 breast carcinomas
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2018 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 9, no 27, p. 18720-18733Article in journal (Refereed) Published
Abstract [en]

To assess mechanisms responsible for breast carcinoma metastasis, 4T1 breast carcinomas were grown orthotopically in wild type or Shb knockout mice. Tumor growth, metastasis, vascular characteristics and immune cell properties were analyzed. Absence of Shb did not affect tumor growth although it increased lung metastasis. Shb knockout mouse tumors showed decreased redness and less developed vascular plexa located at the periphery of the tumors. No difference in overall tumor vascular density, leakage or pericyte coverage was noted between the genotypes although the average vessel size was smaller in the knockout. Tumors induced an increase of CD11b+ cells in spleen, lymph node, thymus, bone marrow and blood. Numbers of Shb knockout CD11b/CD8+ cells were decreased in lymph nodes and bone marrow of tumor bearing mice. Mice with tumors had reduced numbers of CD4+ lymphocytes in blood/lymphoid organs, whereas in most of these locations the proportion of CD4+ cells co-expressing FoxP3 was increased, suggesting a relative increase in Treg cells. This finding was reinforced by increased blood interleukin-35 (IL-35) in wild type tumor bearing mice. Shb knockout blood showed in addition an increased proportion of IL-35 expressing Treg cells, supporting the notion that absence of Shb further promotes tumor evasion from immune cell recognition. This could explain the increased number of lung metastases observed under these conditions. In conclusion, 4T1 tumors alter immune cell responses that promote tumor expansion, metastasis and escape from T cell recognition in an Shb dependent manner. 

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-347019 (URN)10.18632/oncotarget.24643 (DOI)
Available from: 2018-03-23 Created: 2018-03-23 Last updated: 2018-04-12Bibliographically approved
Espes, D., Singh, K., Sandler, S. & Carlsson, P.-O. (2017). Increased Interleukin-35 Levels in Patients With Type 1 Diabetes With Remaining C-Peptide. Diabetes Care, 40(8), 1090-1095
Open this publication in new window or tab >>Increased Interleukin-35 Levels in Patients With Type 1 Diabetes With Remaining C-Peptide
2017 (English)In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 40, no 8, p. 1090-1095Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE Many patients with long-standing type 1 diabetes have remaining functional β-cells. This study investigated immunological differences between patients with or without measurable remaining endogenous insulin production after ≥10 years duration of disease.

RESEARCH DESIGN AND METHODS Patients (n = 113; ≥18 years of age) with type 1 diabetes and with disease duration of ≥10 years were recruited at Uppsala University Hospital. Residual β-cell function was determined with an ultrasensitive C-peptide ELISA. Circulating cytokines, including interleukin-35 (IL-35), were determined in plasma. Additional blood samples were collected from 14 of the identified C-peptide–positive patients and 12 of the C-peptide–negative patients, as well as from 15 healthy control subjects, and were used for immediate investigation of peripheral blood mononuclear cells.

RESULTS The blood concentration of the cytokine IL-35 was markedly lower in C-peptide–negative patients, and this was associated with a simultaneous decrease in the proportion of IL-35+ regulatory T cells (Tregs), IL-35+ regulatory B cells, and IL-35–producing CD8+Foxp3+ cells. IL-35 has previously been shown to maintain the phenotype of Tregs, block the differentiation of T-helper 17 cells, and thereby dampen immune assaults to β-cells. We found that the proportions of IL-17a+ cells among the Tregs, CD4+ T cells, and CD8+ T cells were lower in the C-peptide–positive patients.

CONCLUSIONS Patients with remaining endogenous β-cell function after >10 years duration of type 1 diabetes differ immunologically from other patients with long-standing type 1 diabetes. In particular, they have a much higher IL-35 production.

National Category
Medical and Health Sciences
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-329523 (URN)10.2337/dc16-2121 (DOI)000406014200026 ()28620093 (PubMedID)
Funder
Swedish Research Council, 55X-15043Swedish Research Council, 921-2014-7054EXODIAB - Excellence of Diabetes Research in SwedenSwedish Diabetes AssociationTorsten Söderbergs stiftelseNovo NordiskSwedish Child Diabetes Foundation
Available from: 2017-09-18 Created: 2017-09-18 Last updated: 2017-11-02Bibliographically approved
Luo, Z., Varli, S., Enström, E., Thorvaldson, L., Blixt, M., Hansell, P., . . . Singh, K. (2017). Kinetics of innate immune and regulatory T cells responses in experimental diabetic nephropathy. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 304-304
Open this publication in new window or tab >>Kinetics of innate immune and regulatory T cells responses in experimental diabetic nephropathy
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 304-304Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-346962 (URN)000411865200135 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Available from: 2018-03-23 Created: 2018-03-23 Last updated: 2018-03-23Bibliographically approved
Mejia Cordova, M., Soläng, C., Luo, Z., Blixt, M., Thorvaldson, L., Sandler, S. & Singh, K. (2017). Kinetics of the innate immune cell responses in experimental Type 1 Diabetes. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 303-304
Open this publication in new window or tab >>Kinetics of the innate immune cell responses in experimental Type 1 Diabetes
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 303-304Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-346961 (URN)000411865200134 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Available from: 2018-03-23 Created: 2018-03-23 Last updated: 2018-03-23Bibliographically approved
Digre, A., Singh, K., Åbrink, M., Reijmers, R. M., Sandler, S., Vlodavsky, I. & Li, J.-P. (2017). Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis. Scientific Reports, 7, Article ID 46229.
Open this publication in new window or tab >>Overexpression of heparanase enhances T lymphocyte activities and intensifies the inflammatory response in a model of murine rheumatoid arthritis
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 46229Article in journal (Refereed) Published
Abstract [en]

Heparanase is an endo-glucuronidase that degrades heparan sulfate chains. The enzyme is expressed at a low level in normal organs; however, elevated expression of heparanase has been detected in several inflammatory conditions, e.g. in the synovial joints of rheumatoid arthritis (RA) patients. Herein, we have applied the model of collagen-induced arthritis (CIA) to transgenic mice overexpressing human heparanase (Hpa-tg) along with wildtype (WT) mice. About 50 % of the induced animals developed clinical symptoms, i.e. swelling of joints, and there were no differences between the Hpa-tg and WT mice in the incidence of disease. However, Hpa-tg mice displayed an earlier response and developed more severe symptoms. Examination of cells from thymus, spleen and lymph nodes revealed increased innate and adaptive immune responses of the Hpa-tg mice, reflected by increased proportions of macrophages, antigen presenting cells and plasmacytoid dendritic cells as well as Helios-positive CD4+ and CD8+ T cells. Furthermore, splenic lymphocytes from Hpa-tg mice showed higher proliferation activity. Our results suggest that elevated expression of heparanase augmented both the innate and adaptive immune system and propagated inflammatory reactions in the murine RA model.

Keywords
Collagen-induced arthritis, Heparanase, Heparan Sulfate, Flow cytometry, T cells
National Category
Immunology in the medical area
Research subject
Immunology; Medical Science
Identifiers
urn:nbn:se:uu:diva-315694 (URN)10.1038/srep46229 (DOI)000398988100001 ()28401953 (PubMedID)
Funder
Swedish Research Council, 2015-02595; K2012-56X-15046-09-4Swedish Research Council, 521-2011-3533Swedish Heart Lung Foundation, 20140131Swedish Cancer Society, 150815Swedish Diabetes AssociationSwedish Child Diabetes Foundation
Available from: 2017-02-20 Created: 2017-02-20 Last updated: 2018-01-13Bibliographically approved
Singh, K. (2017). Regulatory T cells in type 1 diabetes: the role of IL-35 in counteracting the disease. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Regulatory T cells in type 1 diabetes: the role of IL-35 in counteracting the disease
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1 diabetes (T1D) is etiologically considered as an autoimmune disease, where insulin-producing β-cells are damaged by autoimmune attacks. Regulatory T (Treg) cells are immune homeostasis cells. In the present thesis I aimed to investigate the role of Treg cells and other immune cells in the early development of T1D. In order to do that, we first determined which immune cells that are altered at an early stage of the T1D development. We found that dendritic cells and plasmacytoid dendritic cells induce the initial immune response.

Next, we investigated the role of Treg cells in multiple low dose streptozotocin (MLDSTZ) induced T1D and in NOD mice. We found that the numbers of Treg cells were increased in both MLDSTZ and NOD mice when the MLDSTZ mice were hyperglycemic. However, the increased Treg cells showed a decreased production of anti-inflammatory cytokines (IL-10, IL-35 and TGF-β) and an increased expression of pro-inflammatory cytokines (IFN-γ and IL-17a). These results revealed that Treg cells switch their phenotype under T1D conditions.

IL-35 administration effectively prevented the development of, and reversed established MLDSTZ induced T1D. Treg cells from IL-35 treated mice showed an increased expression of the Eos transcription factor, accompanied by an increased expression of IL-35 and a decreased expression of IFN-γ and IL-17a. These data indicate that IL-35 administration counteracted the early development of T1D by maintaining the phenotype of the Treg cells. Furthermore, IL-35 administration reversed established T1D in the NOD mouse model by maintaining the phenotype of Treg cells, seemingly by inducing the expression of Eos. Moreover, the circulating level of IL-35 was significantly lowered in both new onset and long-standing T1D patients compared to healthy controls. In addition, patients with T1D with remaining C-peptide had significantly higher levels of IL-35 than patients lacking C-peptide, suggesting that IL-35 might prevent the loss of β-cell mass. In line with this hypothesis, we found that LADA patients had a higher proportion of IL-35+ tolerogenic antigen presenting cells than T1D patients.

Subsequently, we determined the proportions of IL-35+ Treg cells and IL-17a+ Treg cells in T1D patients with diabetic nephropathy (DN), which were age, sex and BMI matched with healthy controls and T1D patients. The proportion of IL-35+ Treg cells was decreased in DN and T1D patients, but IL-17a+ Treg cells were more abundant than in healthy controls. Furthermore, we found that the number of Foxp3+ Treg cells was increased in the kidneys of MLDSTZ mice. However, infiltration of mononuclear cells was seen in kidneys of these mice. In addition, kidney tissues of IL-35 treated MLDSTZ mice did not show any mononuclear cell infiltration. These results demonstrate that IL-35 may be used to prevent mononuclear cell infiltration in kidney diseases.

Our findings indicate that the numbers of Foxp3+ Treg cells are increased in T1D, but that these Treg cells fail to counteract the ongoing immune assault in islets and kidneys of hyperglycemic mice. This could be explained by a phenotypic shift of the Treg cells under hyperglycemic conditions. IL-35 administration reversed established T1D in two different animal models of T1D and prevented mononuclear cell infiltration in the kidneys by maintaining the phenotype of Treg cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1373
Keywords
IL-35, regulatory T cells, type 1 diabetes, LADA, kidney and diabetic nephropathy
National Category
Medical and Health Sciences
Research subject
Medical Science; Immunology; Endocrinology and Diabetology
Identifiers
urn:nbn:se:uu:diva-329524 (URN)978-91-513-0084-9 (ISBN)
Public defence
2017-11-14, hall B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Available from: 2017-10-20 Created: 2017-09-18 Last updated: 2017-10-30
Lundin, S., Espes, D., Luo, Z., Blixt, M., Mejia Cordova, M., Carlsson, P.-O., . . . Singh, K. (2017). Role of regulatory B cells in clinical and experimental type 1 diabetes. Paper presented at 44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN. Scandinavian Journal of Immunology, 86(4), 349-349
Open this publication in new window or tab >>Role of regulatory B cells in clinical and experimental type 1 diabetes
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2017 (English)In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 86, no 4, p. 349-349Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-346972 (URN)000411865200233 ()
Conference
44th Annual Meeting of the Scandinavian-Society-for-Immunology (SSI), OCT 17-20, 2017, Stockholm, SWEDEN
Available from: 2018-03-28 Created: 2018-03-28 Last updated: 2018-03-28Bibliographically approved
Varli, S., Thorvaldson, L., Sandler, S. & Singh, K. (2016). Kinetics of the Foxp3(+) cell response in kidney tissue of both autoimmune and non-autoimmune induced hyperglycemia in mice. Paper presented at International Congress of Immunology (ICI), AUG 21-26, 2016, Melbourne, AUSTRALIA. European Journal of Immunology, 46, 1065-1065
Open this publication in new window or tab >>Kinetics of the Foxp3(+) cell response in kidney tissue of both autoimmune and non-autoimmune induced hyperglycemia in mice
2016 (English)In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 46, p. 1065-1065Article in journal, Meeting abstract (Other academic) Published
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-308497 (URN)000383610402537 ()
Conference
International Congress of Immunology (ICI), AUG 21-26, 2016, Melbourne, AUSTRALIA
Available from: 2016-11-28 Created: 2016-11-28 Last updated: 2018-01-13Bibliographically approved
Singh, K., Hjort, M., Thorvaldson, L. & Sandler, S. (2015). Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naive mice. Scientific Reports, 5, 7767
Open this publication in new window or tab >>Concomitant analysis of Helios and Neuropilin-1 as a marker to detect thymic derived regulatory T cells in naive mice
2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, p. 7767-Article in journal (Refereed) Published
Abstract [en]

Regulatory T (Treg) cells are characterized by the expression of CD4, CD25 and the intracellular Foxp3. However, these markers do not indicate whether Treg cells are thymic derived Treg (tTreg) cells or peripherally induced Treg (pTreg) cells. Recently, Helios and Neuropilin-1 (Nrp1) has been reported as potential markers for tTreg cells. Herein, we used flow cytometry to examine the proportion of CD4(+)CD8(-)CD25(+) Treg cells expressing Helios, Nrp1 and Foxp3 in thymus, pancreatic draining lymph nodes (PDLNs) and spleen of CD-1 mice, and thymus of NOD and C57BL/6 mice. The frequency of Helios(+) cells was higher than that of Nrp1(+) cells in CD4(+)CD8(-)CD25(+) and CD4(+)CD8(-)CD25(+)Foxp3(+) Treg cells in thymus. Interestingly, the proportion of IL-10(+), Ebi3(+) and CTLA-4(+) cells was higher in Helios(+) than Nrp1(+) tTreg cells. The anti-apoptotic activity of Helios(+) tTreg cells was higher in thymus compared to Nrp1(+) tTreg cells. Nrp1 seems to be expressed at a later developmental stage compared to Helios and Foxp3. Furthermore, the expression of Nrp1 in CD4(+)CD25(+) T cells of younger mice did not increase after stimulating them in vitro with anti-CD3 and -CD28. Thus, under these conditions, Helios could be considered a more reliable marker for distinguishing tTreg cells from pTreg cells than Nrp1.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-244481 (URN)10.1038/srep07767 (DOI)000347838500002 ()
Available from: 2015-02-25 Created: 2015-02-17 Last updated: 2017-12-04Bibliographically approved
Oskarsson, M. E., Singh, K., Wang, J., Vlodavsky, I., Li, J.-p. & Westermark, G. T. (2015). Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis. Journal of Biological Chemistry, 290(24), 15121-15132
Open this publication in new window or tab >>Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 24, p. 15121-15132Article in journal (Refereed) Published
Abstract [en]

Deposition of beta cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mM glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that beta cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of beta cells.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-257645 (URN)10.1074/jbc.M114.631697 (DOI)000356177300027 ()25922077 (PubMedID)
Funder
Swedish Cancer Society, 120762
Available from: 2015-07-09 Created: 2015-07-06 Last updated: 2017-12-04Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6771-7757

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