Publications by authors named "Maria G Tsokos"

31 Publications

IL-23 reshapes kidney resident cell metabolism and promotes local kidney inflammation.

J Clin Invest 2021 Jun;131(12)

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Interstitial kidney inflammation is present in various nephritides in which serum interleukin 23 (IL-23) is elevated. Here we showed that murine and human renal tubular epithelial cells (TECs) expressing the IL-23 receptor (IL-23R) responded to IL-23 by inducing intracellular calcium flux, enhancing glycolysis, and upregulating calcium/calmodulin kinase IV (CaMK4), which resulted in suppression of the expression of the arginine-degrading enzyme arginase 1 (ARG1), thus increasing in situ levels of free L-arginine. Limited availability of arginine suppressed the ability of infiltrating T cells to proliferate and produce inflammatory cytokines. TECs from humans and mice with nephritis expressed increased levels of IL-23R and CaMK4 but reduced levels of ARG1. TEC-specific deletion of Il23r or Camk4 suppressed inflammation, whereas deletion of Arg1 exacerbated inflammation in different murine disease models. Finally, TEC-specific delivery of a CaMK4 inhibitor specifically curbed renal inflammation in lupus-prone mice without affecting systemic inflammation. Our data offer the first evidence to our knowledge of the immunosuppressive capacity of TECs through a mechanism that involves competitive uptake of arginine and signify the importance of modulation of an inflammatory cytokine in the function of nonlymphoid cells, which leads to the establishment of an inflammatory microenvironment. New approaches to treat kidney inflammation should consider restoring the immunosuppressive capacity of TECs.
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http://dx.doi.org/10.1172/JCI142428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203450PMC
June 2021

MYOD1 as a prognostic indicator in rhabdomyosarcoma.

Pediatr Blood Cancer 2021 Apr 29:e29085. Epub 2021 Apr 29.

Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.

Background/objectives: Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis.

Design/methods: To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (<18 years of age) and adult (age range ≥18 to 35 years) ERMS and SRMS cases for presence or absence of MYOD1 immunoreactivity in correlation with clinical outcome and MYOD1 L122R mutations.

Results: Lack of MYOD1 immunoreactivity, identified in 23.8% of nonalveolar RMS (non-ARMS) cases, was more prevalent in SRMS (44%) than ERMS (17.2%) and was significantly associated with low overall survival and unfavorable tumor sites (p < .05). Lack of MYOD1 immunoreactivity was not associated with MYOD1 L122R mutations, which were identified in 3/37 (8%) cases including only two of 31 (6.5%) pediatric cases, one of 11 or 9% pediatric SRMS, and one case of infant ERMS.

Conclusion: These studies highlight the prognostic role of MYOD1 in non-ARMS. Lack of MYOD1 immunoreactivity is associated with poor prognosis in ERMS and SRMS. MYOD1 gene mutations are generally infrequent in pediatric RMS. Although mutations are predominant in SRMS, they may exceptionally occur in infantile ERMS.
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http://dx.doi.org/10.1002/pbc.29085DOI Listing
April 2021

Aberrantly glycosylated IgG elicits pathogenic signaling in podocytes and signifies lupus nephritis.

JCI Insight 2021 May 10;6(9). Epub 2021 May 10.

Department of Medicine and.

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of "liquid kidney biopsy" approaches to diagnose LN.
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http://dx.doi.org/10.1172/jci.insight.147789DOI Listing
May 2021

Activation of classical and alternative complement pathways in the pathogenesis of lung injury in COVID-19.

Clin Immunol 2021 05 24;226:108716. Epub 2021 Mar 24.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.

Lung inflammation and damage is prominent in people infected with SARS-Cov-2 and a major determinant of morbidity and mortality. We report the deposition of complement components in the lungs of people who succumbed to COVID-19 consistent with the activation of the classical and the alternative pathways. Our study provides strong rationale for the expansion of trials involving the use of complement inhibitors to treat patients with COVID-19.
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http://dx.doi.org/10.1016/j.clim.2021.108716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989127PMC
May 2021

The Regulatory Subunit PPP2R2A of PP2A Enhances Th1 and Th17 Differentiation through Activation of the GEF-H1/RhoA/ROCK Signaling Pathway.

J Immunol 2021 Apr 24;206(8):1719-1728. Epub 2021 Mar 24.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215

Protein phosphatase 2A (PP2A) composed of a scaffold subunit, a catalytic subunit, and multiple regulatory subunits is a ubiquitously expressed serine/threonine phosphatase. We have previously shown that the PP2A catalytic subunit is increased in T cells from patients with systemic lupus erythematosus and promotes IL-17 production by enhancing the activity of Rho-associated kinase (ROCK) in T cells. However, the molecular mechanism whereby PP2A regulates ROCK activity is unknown. In this study, we show that the PP2A regulatory subunit PPP2R2A is increased in T cells from people with systemic lupus erythematosus and binds to, dephosphorylates, and activates the guanine nucleotide exchange factor GEF-H1 at Ser, which in turn increases the levels of RhoA-GTP and the activity of ROCK in T cells. Genetic PPP2R2A deficiency in murine T cells reduced Th1 and Th17, but not regulatory T cell differentiation and mice with T cell-specific PPP2R2A deficiency displayed less autoimmunity when immunized with myelin oligodendrocyte glycoprotein peptide. Our studies indicate that PPP2R2A is the regulatory subunit that dictates the PP2A-directed enhanced Th1 and Th17 differentiation, and therefore, it represents a therapeutic target for pathologies linked to Th1 and Th17 cell expansion.
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http://dx.doi.org/10.4049/jimmunol.2001266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026731PMC
April 2021

Cell derived extracellular matrix-rich biomimetic substrate supports podocyte proliferation, differentiation and maintenance of native phenotype.

Adv Funct Mater 2020 Oct 19;30(44). Epub 2020 Feb 19.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02215, United States.

Current technologies and available scaffold materials do not support long-term cell viability, differentiation and maintenance of podocytes, the ultra-specialized kidney resident cells that are responsible for the filtration of the blood. We developed a new platform which imitates the native kidney microenvironment by decellularizing fibroblasts grown on surfaces with macromolecular crowding. Human immortalized podocytes cultured on this platform displayed superior viability and metabolic activity up to 28 days compared to podocytes cultured on tissue culture plastic surfaces. The new platform displayed a softer surface and an abundance of growth factors and associated molecules. More importantly it enabled podocytes to display molecules responsible for their structure and function and a superior development of intercellular connections/interdigitations, consistent with maturation. The new platform can be used to study podocyte biology, test drug toxicity and determine whether sera from patients with podocytopathies are involved in the expression of glomerular pathology.
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http://dx.doi.org/10.1002/adfm.201908752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939063PMC
October 2020

PPP2R2D suppresses IL-2 production and Treg function.

JCI Insight 2020 10 2;5(19). Epub 2020 Oct 2.

Department of Medicine, Beth Israel Deaconess Medical Center (BIDMC) and Harvard Medical School, Boston, Masschusetts, USA.

Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that comprises a scaffold, a catalytic, and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of IL-2 in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2-enhancing transcription factors remain open, while the levels of the enhancer phosphorylated CREB are high. Mice with T cell-specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to Treg function do not change in the absence of PPP2R2D, Tregs exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs.
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http://dx.doi.org/10.1172/jci.insight.138215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566706PMC
October 2020

N-glycosylated IgG in patients with kidney transplants increases calcium/calmodulin kinase IV in podocytes and causes injury.

Am J Transplant 2021 01 6;21(1):148-160. Epub 2020 Jul 6.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3β (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.
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http://dx.doi.org/10.1111/ajt.16140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8188503PMC
January 2021

Systemic lupus erythematosus favors the generation of IL-17 producing double negative T cells.

Nat Commun 2020 06 5;11(1):2859. Epub 2020 Jun 5.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Mature double negative (DN) T cells are a population of αβ T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8 T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE.
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http://dx.doi.org/10.1038/s41467-020-16636-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275084PMC
June 2020

Serine/threonine phosphatase PP2A is essential for optimal B cell function.

JCI Insight 2020 03 12;5(5). Epub 2020 Mar 12.

Department of Medicine, Beth Israel Deaconess Medical Center (BIDMC), Boston, Massachusetts, USA.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, has been shown to control T cell function. We found that in vitro-activated B cells and B cells from various lupus-prone mice and patients with systemic lupus erythematosus display increased PP2A activity. To understand the contribution of PP2A to B cell function, we generated a Cd19CrePpp2r1afl/fl (flox/flox) mouse which lacks functional PP2A only in B cells. Flox/flox mice displayed reduced spontaneous germinal center formation and decreased responses to T cell-dependent and T-independent antigens, while their B cells responded poorly in vitro to stimulation with an anti-CD40 antibody or CpG in the presence of IL-4. Transcriptome and metabolome studies revealed altered nicotinamide adenine dinucleotide (NAD) and purine/pyrimidine metabolism and increased expression of purine nucleoside phosphorylase in PP2A-deficient B cells. Our results demonstrate that PP2A is required for optimal B cell function and may contribute to increased B cell activity in systemic autoimmunity.
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http://dx.doi.org/10.1172/jci.insight.130655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141385PMC
March 2020

Complement and coagulation cascades in trauma.

Acute Med Surg 2019 Oct 25;6(4):329-335. Epub 2019 Apr 25.

Department of Medicine Beth Israel Deaconess Medical Center Harvard Medical School Boston Massachusetts.

Trauma remains a major cause of death throughout the world, especially for patients younger than 45 years. Due to rapid advances in clinical management, both in the acute and prehospital settings, trauma patients survive devastating injuries at unprecedented rates. However, these patients can often face life threatening complications that stem from the robust innate immune response induced by severe hemorrhage, leading to further tissue injury rather than repair. The complement and coagulation cascades are key mediators in this disordered reaction, which includes the development of trauma-induced coagulopathy. There is increasing evidence that cross-talk between these two pathways allows rapid amplification of their otherwise targeted responses and contributes to overwhelming and prolonged systemic inflammation. In this article, we summarize the initial steps of innate immune response to trauma and review the complex complement and coagulation cascades, as well as how they interact with each other. Despite progress in understanding these cascades, effective therapeutic targets have yet to be found and further research is needed both to improve survival rates as well as decrease associated morbidity.
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http://dx.doi.org/10.1002/ams2.426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773636PMC
October 2019

Hyaluronic Acid Synthesis Contributes to Tissue Damage in Systemic Lupus Erythematosus.

Front Immunol 2019 13;10:2172. Epub 2019 Sep 13.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.

Hyaluronic acid (HA), a component of the extracellular matrix, is the ligand for CD44 and has been implicated in the pathogenesis of kidney inflammation in patients with systemic lupus erythematosus (SLE), but its direct role and mechanism of action have not been studied. Here we show that administration of hymecromone (4-Methylumbelliferone, 4-MU), an HA synthesis inhibitor, to lupus-prone mice suppressed dramatically lupus-related pathology. Interestingly, 4-MU stopped the appearance of disease when administered prior to its onset and inhibited the progression of disease when administered after its appearance. Inhibition of HA synthesis reduced tissue damage and the number of intrarenal lymphoid cell infiltrates including double negative CD3+CD4-CD8- T cells which are known to be involved in the pathogenesis of SLE. Exposure of human peripheral blood mononuclear cells to HA increased the generation of CD3+CD4-CD8- T cells through a mechanism involving Rho-associated kinase. Our results signify the importance of the HA-rich tissue microenvironment in the activation of lymphocytes to cause tissue damage in SLE and suggest the consideration of inhibition of HA synthesis to treat patients.
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http://dx.doi.org/10.3389/fimmu.2019.02172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753633PMC
October 2020

TARGETING TARGETED TREATMENT FOR IMMUNE AND NON-IMMUNE KIDNEY DISEASES.

Trans Am Clin Climatol Assoc 2019 ;130:88-99

BOSTON, MASSACHUSETTS.

We have found that calcium calmodulin kinase IV is increased in T cells, podocytes, and mesangial cells from patients with systemic lupus erythematosus, as well as in lupus-prone mice, podocytes of patients with focal segmental glomerulosclerosis, and in mice injected with doxorubicin. We showed that this accounts for aberrant T cell function and glomerular damage. Using nanoparticles (nlg) loaded with a small drug inhibitor of calcium calmodulin kinase IV and tagged with antibodies directed to CD4 we have been able to show inhibition of autoimmunity and lupus nephritis. Also, using nlg tagged with antibodies to nephrin, we showed suppression of nephritis in lupus-prone mice and of glomerular damage in mice exposed to doxorubicin. We propose the development of approaches to deliver drugs to cells in a targeted and precise manner.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735968PMC
February 2020

Pyruvate kinase M2 is requisite for Th1 and Th17 differentiation.

JCI Insight 2019 06 20;4(12). Epub 2019 Jun 20.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston Massachusetts, USA.

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.
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http://dx.doi.org/10.1172/jci.insight.127395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629104PMC
June 2019

Complement Deposition on the Surface of RBC After Trauma Serves a Biomarker of Moderate Trauma Severity: A Prospective Study.

Shock 2020 01;53(1):16-23

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.

Background: Activation of the complement system and complement deposition on red blood cells (RBCs) contribute to organ damage in trauma. We conducted a prospective study in subjects with traumatic injuries to determine the pattern of complement deposition on RBC and whether they are associated with clinical outcomes.

Method: A total of 124 trauma patients and 42 healthy controls were enrolled in this prospective study. RBC and sera were collected at 0, 6, 24, and 72 h from trauma patients and healthy controls during a single draw. Presence of C4d, C3d, C5b-9, phosphorylation of band 3 and production of nitric oxide were analyzed by flow cytometry.

Results: RBC from trauma patients at all time points up to 24 h displayed significantly higher deposition of C4d on their RBC membrane as compared with healthy donors. Incubation of normal RBC with sera from trauma patients resulted in significant increase of C4d deposition (at 0, 6, 24, and 72 h), C5b-9 deposition (at 0 and 6 h), phosphorylation of band 3 (at 0 and 24 h), and nitric oxide production up to 24 h compared with sera from healthy subjects. Deposition of C4d and C5b-9 in patients with an Injury Severity Score (ISS) of 9 and above remained elevated up to 72 h.

Conclusions: Our study demonstrates that the presence of C4d, C3d, and C5b-9 on the surface of RBC is linked to increased phosphorylation of band 3 and increased production of nitric oxide. Deposition of C4d and C5b-9 decreased faster over course of 3-day study in subjects with ISS less than 9.
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http://dx.doi.org/10.1097/SHK.0000000000001348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6790152PMC
January 2020

PP2A enables IL-2 signaling by preserving IL-2Rβ chain expression during Treg development.

JCI Insight 2019 03 26;5. Epub 2019 Mar 26.

Division of Rheumatology and Clinical Immunology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Tregs require IL-2 signaling for signal transducer and activator of transcription 5 (STAT5)-mediated induction of Foxp3. While phosphatase 2A (PP2A) is a negative regulator of IL-2 production in effector T cells and Tregs do not produce IL-2, it is not known whether PP2A controls IL-2 signaling in Tregs. To address the role of PP2A in IL-2 signaling in Tregs we studied mice engineered to lack PP2A in all Foxp3-expressing cells. We report that PP2A is required to enable Foxp3 expression and to maintain sufficient numbers of Tregs in the thymus. We show for the first time that PP2A prevents the selective loss of surface IL-2Rβ and preserves IL-2R signaling potency in Tregs. The loss of IL-2Rβ in thymus- and spleen-derived Tregs that lack PP2A is due to increased sheddase activity. Pan-sheddase or selective A disintegrin and metalloproteinase 10 (ADAM10) inhibition, like forced expression of IL-2Rβ in PP2A-deficient Tregs restored IL-2Rβ expression and signaling. Thus, PP2A restrains the sheddase activity of ADAM10 in Treg cells to prevent the cleavage of IL-2Rβ from the cell surface to enable competent IL-2R signaling which is essential for Tregs development and homeostasis.
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http://dx.doi.org/10.1172/jci.insight.126294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538314PMC
March 2019

FGF21 underlies a hormetic response to metabolic stress in methylmalonic acidemia.

JCI Insight 2018 12 6;3(23). Epub 2018 Dec 6.

Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland, USA.

Methylmalonic acidemia (MMA), an organic acidemia characterized by metabolic instability and multiorgan complications, is most frequently caused by mutations in methylmalonyl-CoA mutase (MUT). To define the metabolic adaptations in MMA in acute and chronic settings, we studied a mouse model generated by transgenic expression of Mut in the muscle. Mut-/-;TgINS-MCK-Mut mice accurately replicate the hepatorenal mitochondriopathy and growth failure seen in severely affected patients and were used to characterize the response to fasting. The hepatic transcriptome in MMA mice was characterized by the chronic activation of stress-related pathways and an aberrant fasting response when compared with controls. A key metabolic regulator, Fgf21, emerged as a significantly dysregulated transcript in mice and was subsequently studied in a large patient cohort. The concentration of plasma FGF21 in MMA patients correlated with disease subtype, growth indices, and markers of mitochondrial dysfunction but was not affected by renal disease. Restoration of liver Mut activity, by transgenesis and liver-directed gene therapy in mice or liver transplantation in patients, drastically reduced plasma FGF21 and was associated with improved outcomes. Our studies identify mitocellular hormesis as a hepatic adaptation to metabolic stress in MMA and define FGF21 as a highly predictive disease biomarker.
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http://dx.doi.org/10.1172/jci.insight.124351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328030PMC
December 2018

Calcium/Calmodulin Kinase IV Controls the Function of Both T Cells and Kidney Resident Cells.

Front Immunol 2018 1;9:2113. Epub 2018 Oct 1.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.

Calcium calmodulin kinase IV (CaMK4) regulates multiple processes that significantly contribute to the lupus-related pathology by controlling the production of IL-2 and IL-17 by T cells, the proliferation of mesangial cells, and the function and structure of podocytes. CaMK4 is also upregulated in podocytes from patients with focal segmental glomerulosclerosis (FSGS). In both immune and non-immune podocytopathies, CaMK4 disrupts the structure and function of podocytes. In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to CD4 T cells suppresses both autoimmunity and the development of nephritis. Targeted delivery though to podocytes averts the deposition of immune complexes without affecting autoimmunity in lupus-prone mice and averts pathology induced by adriamycin in normal mice. Therefore, targeted delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies.
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http://dx.doi.org/10.3389/fimmu.2018.02113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176098PMC
October 2019

Regulatory T cells in the treatment of disease.

Nat Rev Drug Discov 2018 11 12;17(11):823-844. Epub 2018 Oct 12.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

Regulatory T (T) cells suppress inflammation and regulate immune system activity. In patients with systemic or organ-specific autoimmune diseases or those receiving transplanted organs, T cells are compromised. Approaches to strengthen T cell function, either by expanding them ex vivo and reinfusing them or by increasing the number or capacity of existing T cells, have entered clinical trials. Unlike the situation in autoimmunity, in patients with cancer, T cells limit the antitumour immune response and promote angiogenesis and tumour growth. Their immunosuppressive function may, in part, explain the failure of many immunotherapies in cancer. Strategies to reduce the function and/or number of T cells specifically in tumour sites are being investigated to promote antitumour immunity and regression. Here, we describe the current progress in modulating T cells in autoimmune disorders, transplantation and cancer.
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http://dx.doi.org/10.1038/nrd.2018.148DOI Listing
November 2018

Pyruvate dehydrogenase phosphatase catalytic subunit 2 limits Th17 differentiation.

Proc Natl Acad Sci U S A 2018 09 27;115(37):9288-9293. Epub 2018 Aug 27.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215

Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4 T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4 cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4 T cells from lupus-prone MRL/ mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.
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http://dx.doi.org/10.1073/pnas.1805717115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140513PMC
September 2018

Precision DNA demethylation ameliorates disease in lupus-prone mice.

JCI Insight 2018 08 23;3(16). Epub 2018 Aug 23.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Defective DNA methylation in T cells leads to a series of T cell abnormalities in lupus; however, the full effect of T cell lineage-specific DNA methylation on disease expression has not been explored. Here, we show that 5-azacytidine, a DNA methyltransferase inhibitor, targeted to either CD4 or CD8 T cells in mice with established disease using a nanolipogel delivery system dramatically ameliorates lupus-related pathology through distinct mechanisms. In vivo targeted delivery of 5-azacytidine into CD4 T cells favors the expansion and function of Foxp3+ Tregs, whereas targeted delivery to CD8 T cells enhances the cytotoxicity and restrains the expansion of pathogenic TCR-αβ+CD4-CD8- double-negative T cells. Our results signify the importance of cell-specific inhibition of DNA methylation in the treatment of established lupus.
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http://dx.doi.org/10.1172/jci.insight.120880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141184PMC
August 2018

CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease.

J Clin Invest 2018 08 9;128(8):3445-3459. Epub 2018 Jul 9.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin-dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3β, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.
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http://dx.doi.org/10.1172/JCI99507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063476PMC
August 2018

Potential Value of YAP Staining in Rhabdomyosarcoma.

J Histochem Cytochem 2018 08 29;66(8):577-584. Epub 2018 Mar 29.

Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts.

Rhabdomyosarcoma (RMS) is a common malignancy of soft tissue, subclassified as alveolar (ARMS), pleomorphic (PRMS), spindle cell/sclerosing (SRMS), and embryonal (ERMS) types. The Yes-associated protein (YAP) is a member of the Hippo pathway and a transcriptional regulator that controls cell proliferation. We have studied the immunohistochemical expression of YAP in different RMSs, arranged in tissue microarray (TMA) and whole slide formats. Pertinent clinical data including patient age, gender, tumor location, and clinical stage were collected. Out of 96 TMA cases, 30 cases (31%) were pleomorphic, 27 (28%) were embryonal, 24 (25%) alveolar, and 15 (16%) spindle cell. Positive nuclear YAP staining was seen in the PRMS (17/30, 56.7%), SRMS (7/15, 46.7%), ERMS (19/27 or 70%), and less in ARMS (37.5%). YAP nuclear staining was significantly more prevalent in ERMS than ARMS ( p=0.02). Of the 41 whole slide cases, nuclear staining was detected in all ARMS but was restricted in distribution to <30% of the cells, in contrast to ERMS and SRMS, which had diffuse or >30% staining. These results highlight the role of YAP in RMS tumorigenesis, a fact that can be useful in engineering targeted therapy. Restricted nuclear YAP staining (<30% of cells) may be of value in the diagnosis of ARMS.
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http://dx.doi.org/10.1369/0022155418766515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071181PMC
August 2018

C3a Enhances the Formation of Intestinal Organoids through C3aR1.

Front Immunol 2017 4;8:1046. Epub 2017 Sep 4.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States.

C3a is important in the regulation of the immune response as well as in the development of organ inflammation and injury. Furthermore, C3a contributes to liver regeneration but its role in intestinal stem cell function has not been studied. We hypothesized that C3a is important for intestinal repair and regeneration. Intestinal organoid formation, a measure of stem cell capacity, was significantly limited in C3-deficient and C3a receptor (C3aR) 1-deficient mice while C3a promoted the growth of organoids from normal mice by supporting Wnt-signaling but not from C3aR1-deficient mice. Similarly, the presence of C3a in media enhanced the expression of the intestinal stem cell marker leucine-rich repeat G-protein-coupled receptor 5 (Lgr5) and of the cell proliferation marker Ki67 in organoids formed from C3-deficient but not from C3aR1-deficient mice. Using mice we showed significant expression of C3 in Lgr5 intestinal stem cells whereas C3aR1 was expressed on the surface of various intestinal cells. C3 and C3aR1 expression was induced in intestinal crypts in response to ischemia/reperfusion injury. Finally, C3aR1-deficient mice displayed ischemia/reperfusion injury comparable to control mice. These data suggest that C3a through interaction with C3aR1 enhances stem cell expansion and organoid formation and as such may have a role in intestinal regeneration.
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http://dx.doi.org/10.3389/fimmu.2017.01046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591398PMC
September 2017

IL-17A Produced by Innate Lymphoid Cells Is Essential for Intestinal Ischemia-Reperfusion Injury.

J Immunol 2017 10 6;199(8):2921-2929. Epub 2017 Sep 6.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115;

Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response that is characterized by villous damage and neutrophil infiltration. IL-17A, a cytokine produced by a variety of cells in response to inflammatory cytokines released following tissue injury, has been implicated in IR injury. Using , , and mice and administration of anti-IL-17A and anti-IL-23 neutralizing Abs to wild-type mice, we demonstrate that intestinal IR injury depends on IL-17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using bone marrow chimeras, we demonstrate that the IL-17A required for intestinal IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into and mice, we demonstrate that innate lymphoid cells are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by innate lymphoid cells is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation.
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http://dx.doi.org/10.4049/jimmunol.1700655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807067PMC
October 2017

Intracellular Activation of Complement 3 Is Responsible for Intestinal Tissue Damage during Mesenteric Ischemia.

J Immunol 2017 01 2;198(2):788-797. Epub 2016 Dec 2.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B-deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.
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http://dx.doi.org/10.4049/jimmunol.1502287DOI Listing
January 2017

Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas.

Cancer Immunol Res 2016 10 22;4(10):869-880. Epub 2016 Aug 22.

Pediatric Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, Maryland. Department of Pediatrics, Stanford University School of Medicine, Stanford, California.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2 sarcoma and neuroblastoma cell lines in vitro However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2 sarcoma, despite effectively controlling GD2 neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869-80. ©2016 AACR.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5050151PMC
http://dx.doi.org/10.1158/2326-6066.CIR-15-0230DOI Listing
October 2016

Phosphatase PP2A is requisite for the function of regulatory T cells.

Nat Immunol 2016 May 14;17(5):556-64. Epub 2016 Mar 14.

Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Homeostasis of the immune system depends on the proper function of regulatory T cells (T(reg) cells). Compromised suppressive activity of T(reg) cells leads to autoimmune disease and graft rejection and promotes anti-tumor immunity. Here we report a previously unrecognized requirement for the serine-threonine phosphatase PP2A in the function of T(reg) cells. T(reg) cells exhibited high PP2A activity, and T(reg) cell-specific ablation of the PP2A complex resulted in a severe, multi-organ, lymphoproliferative autoimmune disorder. Mass spectrometry revealed that PP2A associated with components of the mTOR metabolic-checkpoint kinase pathway and suppressed the activity of the mTORC1 complex. In the absence of PP2A, T(reg) cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of T(reg) cells and the prevention of autoimmunity.
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http://dx.doi.org/10.1038/ni.3390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4837024PMC
May 2016

Recurrent Mutations in the Basic Domain of TWIST2 Cause Ablepharon Macrostomia and Barber-Say Syndromes.

Am J Hum Genet 2015 Jul 25;97(1):99-110. Epub 2015 Jun 25.

NIH Undiagnosed Diseases Program, Common Fund, Office of the Director, NIH and National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.

Ablepharon macrostomia syndrome (AMS) and Barber-Say syndrome (BSS) are rare congenital ectodermal dysplasias characterized by similar clinical features. To establish the genetic basis of AMS and BSS, we performed extensive clinical phenotyping, whole exome and candidate gene sequencing, and functional validations. We identified a recurrent de novo mutation in TWIST2 in seven independent AMS-affected families, as well as another recurrent de novo mutation affecting the same amino acid in ten independent BSS-affected families. Moreover, a genotype-phenotype correlation was observed, because the two syndromes differed based solely upon the nature of the substituting amino acid: a lysine at TWIST2 residue 75 resulted in AMS, whereas a glutamine or alanine yielded BSS. TWIST2 encodes a basic helix-loop-helix transcription factor that regulates the development of mesenchymal tissues. All identified mutations fell in the basic domain of TWIST2 and altered the DNA-binding pattern of Flag-TWIST2 in HeLa cells. Comparison of wild-type and mutant TWIST2 expressed in zebrafish identified abnormal developmental phenotypes and widespread transcriptome changes. Our results suggest that autosomal-dominant TWIST2 mutations cause AMS or BSS by inducing protean effects on the transcription factor's DNA binding.
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http://dx.doi.org/10.1016/j.ajhg.2015.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4572501PMC
July 2015

Mesothelioma patient derived tumor xenografts with defined BAP1 mutations that mimic the molecular characteristics of human malignant mesothelioma.

BMC Cancer 2015 May 8;15:376. Epub 2015 May 8.

Thoracic and GI Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Background: The development and evaluation of new therapeutic approaches for malignant mesothelioma has been sparse due, in part, to lack of suitable tumor models.

Methods: We established primary mesothelioma cultures from pleural and ascitic fluids of five patients with advanced mesothelioma. Electron microscopy and immunohistochemistry (IHC) confirmed their mesothelial origin. Patient derived xenografts were generated by injecting the cells in nude or SCID mice, and malignant potential of the cells was analyzed by soft agar colony assay. Molecular profiles of the primary patient tumors, early passage cell cultures, and patient derived xenografts were assessed using mutational analysis, fluorescence in situ hybridization (FISH) analysis and IHC.

Results: Primary cultures from all five tumors exhibited morphologic and IHC features consistent to those of mesothelioma cells. Mutations of BAP1 and CDKN2A were each detected in four tumors. BAP1 mutation was associated with the lack of expression of BAP1 protein. Three cell cultures, all of which were derived from BAP1 mutant primary tumors, exhibited anchorage independent growth and also formed tumors in mice, suggesting that BAP1 loss may enhance tumor growth in vivo. Both early passage cell cultures and mouse xenograft tumors harbored BAP1 mutations and CDKN2A deletions identical to those found in the corresponding primary patient tumors.

Conclusions: The mesothelioma patient derived tumor xenografts with mutational alterations that mimic those observed in patient tumors which we established can be used for preclinical development of novel drug regimens and for studying the functional aspects of BAP1 biology in mesothelioma.
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http://dx.doi.org/10.1186/s12885-015-1362-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4431029PMC
May 2015