Anna Vossenkämper

Association of innate immune activation with latent Epstein-Barr virus in active MS lesions

Objective: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. Methods: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. Results: We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFNα in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFNα was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFNα production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFNα production in vitro. Conclusion: These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFNα production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.

Inhibition of NF-κB Signaling in Human Dendritic Cells by the Enteropathogenic Escherichia coli Effector Protein NleE

Intestinal dendritic cells (DCs) send processes between epithelial cells into the gut lumen to sample pathogens. Noninvasive enteropathogenic Escherichia coli (EPEC) colonize the gut using a type three secretion system (T3SS) to inject effector proteins into epithelial cells. We hypothesized that EPEC might also inject proteins into DC processes to dampen immune recognition. Using a T3SS-linked fluorescence resonance energy transfer-based system we show that EPEC injects effectors into in vitro grown human myeloid DCs. Injected cells emit a blue signal due to cleavage of the green fluorescence resonance energy transfer-based substrate CCF2/AM by β-lactamase. When cultured with a mutant EPEC unable to translocate effector proteins, myeloid DCs show rapid activation of NF-κB, secrete large amounts of proinflammatory cytokines and increase expression of CD80, CD83, and CD86, whereas wild-type EPEC barely elicits cytokine production and shuts off nuclear translocation of NF-κB p65. By deleting effector protein genes, we identified NleE as being critical for this effect. Expression of NleE in HeLa cells completely prevented nuclear p65 accumulation in response to IL1-β, and luciferase production in an NF-κB reporter cell line. DCs cocultured with wild-type EPEC or NleE-complemented strains were less potent at inducing MLR. EPEC was also able to inject effectors into DCs sending processes through model gut epithelium in a transwell system and into Peyer’s patch myeloid DCs. Thus, EPEC translocate effectors into human DCs to dampen the inflammatory response elicited by its own pathogen-associated molecular patterns.

Reciprocal Regulation of Aquaporin-2 Abundance and Degradation by Protein Kinase A and p38-MAP Kinase

Arginine-vasopressin (AVP) modulates the water channel aquaporin-2 (AQP2) in the renal collecting duct to maintain homeostasis of body water. AVP binds to vasopressin V2 receptors (V2R), increasing cAMP, which promotes the redistribution of AQP2 from intracellular vesicles into the plasma membrane. cAMP also increases AQP2 transcription, but whether altered degradation also modulates AQP2 protein levels is not well understood. Here, elevation of cAMP increased AQP2 protein levels within 30 minutes in primary inner medullary collecting duct (IMCD) cells, in human embryonic kidney (HEK) 293 cells ectopically expressing AQP2, and in mouse kidneys. Accelerated transcription or translation did not explain this increase in AQP2 abundance. In IMCD cells, cAMP inhibited p38-mitogen-activated protein kinase (p38-MAPK) via activation of protein kinase A (PKA). Inhibition of p38-MAPK associated with decreased phosphorylation (serine 261) and polyubiquitination of AQP2, preventing proteasomal degradation. Our results demonstrate that AVP enhances AQP2 protein abundance by altering its proteasomal degradation through a PKA- and p38-MAPK-dependent pathway.

Both IL-12 and IL-18 contribute to small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii, but IL-12 is dominant over IL-18 in parasite control

Oral infection of C57BL/6 mice with Toxoplasma gondii results in small intestinal Th1‐type immunopathology mediated by local production of IFN‐γ, TNF‐α, and NO. To analyze whether the proinflammatory cytokines IL‐12 and IL‐18 play a role in the induction of immunopathology, IL‐12p35/p40–/– and IL‐18–/– mice were orally infected with T. gondii. Wild‐type mice developed massive necrosis in their small intestines and died 7–10 days post infection. Even though IL‐12p35/40–/– mice did not develop the necrosis they all died between day 9 and 11 after infection. In contrast, 50% of IL‐18–/– mice died during the acute phase of infection. Compared to wild‐type mice, IL‐12p35/p40–/– but not IL‐18–/– mice showed significantly higher parasite numbers in their small intestines and significantly higher numbers of parasite‐associated inflammatory foci in their livers. IFN‐γ production was similar in infected wild‐type and IL‐18–/– mice but significantly decreased in IL‐12p35/p40–/– mice. Treatment of mice with anti‐IL‐12‐ or anti‐IL‐18 antibodies after infection prevented the development of intestinal necrosis. These results reveal that both IL‐12 and IL‐18 play an important role in the development of intestinal immunopathology following oral infection with T. gondii. However, IL‐12 is dominant over IL‐18 in the host defense against parasite replication. Therefore, neutralization of IL‐18 (rather than TNF‐α, IL‐12, and IFN‐γ) may be a safe strategy for the treatment of Th1‐associated diseases.

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease

Background and aim Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohns disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohns blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohns lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. Methods To define the optimum population for Treg cell therapy in CD, CD4+CD25+CD127loCD45RA+ and CD4+CD25+CD127loCD45RA− Treg subsets were isolated from patients’ blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. Results Tregs can be expanded from the blood of patients with CD to potential target dose within 22–24 days. Expanded CD45RA+ Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA− Tregs. CD45RA+ Tregs highly express α4β7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA+ Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA+ Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohns mucosa. Conclusions CD4+CD25+CD127loCD45RA+ Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.

IgA-Producing Plasma Cells Originate From Germinal Centers That Are Induced by B-Cell Receptor Engagement in Humans

BACKGROUND & AIMS IgA contributes to homeostatic balance between host and intestinal microbiota. Mechanisms that initiate the IgA response are unclear and likely to differ between humans and animal models. We used multiple experimental approaches to investigate the origin of human intestinal plasma cells that produce IgA in the gastrointestinal tract. METHODS Complexity of IgA-producing plasma cell populations in human gastrointestinal mucosa and bone marrow and the specific response to oral cholera vaccine were compared by analysis of immunoglobulin genes. Flow cytometry, gene expression analysis, and immunohistochemistry were used to analyze signaling pathways induced by B-cell receptor engagement in human gut-associated lymphoid tissue (GALT) and involvement of innate immunity in B-cell activation in GALT compared with nonintestinal sites. RESULTS Human intestinal IgA-producing plasma cells appeared to be of germinal center origin; there was no evidence for the population complexity that accompanies multiple pathways of derivation observed in bone marrow. In germinal center B cells of human GALT, Btk and Erk are phosphorylated, CD22 is down-regulated, Lyn is translocated to the cell membrane, and Fos and Jun are up-regulated; these features indicate B-cell receptor ligation during germinal center evolution. No differences in innate activation of B cells were observed in GALT, compared with peripheral immune compartments. CONCLUSIONS IgA-producing plasma cells appear to be derived from GALT germinal centers in humans. B-cell receptor engagement promotes formation of germinal centers of GALT, with no more evidence for innate immune receptor activation in the mucosa than nonintestinal immune compartments. Germinal centers in GALT should be targets of mucosal vaccinations because they are the source of human intestinal IgA response.

A role for gut-associated lymphoid tissue in shaping the human B cell repertoire

Transitional 2 B cells home to gut-associated lymphoid tissue and present an activated phenotype in healthy subjects, but gut immune compartments are depleted in SLE.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

Translational Mini-Review Series on B cell subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies

OTHER ARTICLES PUBLISHED IN THIS MINI‐REVIEW SERIES ON B CELL SUBSETS IN DISEASE

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation

Background & Aims Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results IL17A- and IL22-producing, natural cytotoxicity receptor–negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor–positive cells, in a dose-dependent manner. Conclusions IL6 contributes to activation of colonic natural cytotoxicity receptor–negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor–positive cells.