Graft-versus-host disease of the CNS is mediated by TNF upregulation in microglia

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Abstract

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes, MHC-II- and CD80-upregulation occurred upon GVHD induction. RNA-sequencing-based analysis of purified microglial obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1creER:Tnffl/-mice reduced MHC-II-expression, decreased CNS T-cell infiltrates and VCAM-1+ endothelial cells. GVHD increased microglia TGF-β-activated kinase-1 (TAK1) activation and NF-κB/p38-MAPK-signaling. Selective Tak1-deletion in microglia using Cx3cr1creER:Tak1fl/fl-mice resulted in reduced TNF-production, microglial MHC-II, and improved neurocognitive-activity. Pharmacological TAK1-inhibition reduced TNF-production and MHC-II-expression by microglia, Th1 and Th17 T-cell infiltrates, VCAM-1+ endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF-production of microglia in the CNS of GVHD-patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as mediator of CNS-GVHD and provide a novel TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.

Authors

Nimitha R. Mathew, Janaki M. Vinnakota, Petya Apostolova, Daniel Erny, Shaima’a Hamarsheh, Geoffroy Andrieux, Jung-Seok Kim, Kathrin Hanke, Tobias Goldmann, Louise Chappell-Maor, Nadia El-Khawanky, Gabriele Ihorst, Dominik Schmidt, Justus Duyster, Jürgen Finke, Thomas Blank, Melanie Boerries, Bruce R. Blazar, Steffen Jung, Marco Prinz, Robert Zeiser

Myalgic encephalomyelitis/chronic fatigue syndrome patients exhibit altered T cell metabolism and cytokine associations

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Abstract

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex disease with no known cause or mechanism. There is an increasing appreciation for the role of immune and metabolic dysfunction in the disease. ME/CFS has historically presented in outbreaks, often has a flu-like onset, and results in inflammatory symptoms. Patients suffer from severe fatigue and post-exertional malaise. There is little known about the metabolism of specific immune cells in ME/CFS patients. To investigate immune metabolism in ME/CFS, we isolated CD4+ and CD8+ T cells from 53 ME/CFS patients and 45 healthy controls. We analyzed glycolysis and mitochondrial respiration in resting and activated T cells, along with markers related to cellular metabolism, and plasma cytokines. We found that ME/CFS CD8+ T cells have reduced mitochondrial membrane potential compared to healthy controls. Both CD4+ and CD8+ T cells from ME/CFS patients had reduced glycolysis at rest, while CD8+ T cells also had reduced glycolysis following activation. ME/CFS patients had significant correlations between measures of T cell metabolism and plasma cytokine abundance that differed from healthy control subjects. Our data indicate that patients have impaired T cell metabolism consistent with ongoing immune alterations in ME/CFS that may illuminate the mechanism behind this disease.

Authors

Alexandra H. Mandarano, Jessica Maya, Ludovic Giloteaux, Daniel L. Peterson, Marco Maynard, C. Gunnar Gottschalk, Maureen R. Hanson

Glibenclamide reverses cardiovascular abnormalities of Cantu Syndrome driven by K_ATP channel overactivity

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Abstract

Cantu Syndrome (CS) is a complex disorder caused by gain-of-function (GoF) mutations in ABCC9 and KCNJ8, which encode the SUR2 and Kir6.1 subunits, respectively, of vascular smooth muscle (VSM) KATP channels. CS includes dilated vasculature, marked cardiac hypertrophy, and other cardiovascular abnormalities. There is currently no targeted therapy, and it is unknown whether cardiovascular features can be reversed once manifest. Using combined transgenic and pharmacological approaches in a knock-in mouse model of CS, we have shown that reversal of vascular and cardiac phenotypes can be achieved (1) by genetic downregulation of KATP channel activity specifically in VSM, and (2) by chronic administration of the clinically-used KATP channel inhibitor, glibenclamide. These findings demonstrate (i) that VSM KATP channel GoF underlies CS cardiac enlargement, (ii) reversibility of CS-associated abnormalities and (iii) evidence of in vivo efficacy of glibenclamide as a therapeutic agent in CS.

Authors

Conor McClenaghan, Yan Huang, Zihan Yan, Theresa Harter, Carmen M. Halabi, Rod Chalk, Attila Kovacs, Gijs van Haaften, Maria S. Remedi, Colin G. Nichols

CD8⁺ T cells target cerebrovasculature in children with cerebral malaria

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Abstract

BACKGROUND. Cerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics. METHODS. Using multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or –), iRBC sequestration (Seqhi, lo, or 0) and HIV status (HIV+ or –). RESULTS. We identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV– children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ vs –, P = 0.004) and sequestration level (Seqhi > lo, P = 0.010). HIV co-infection significantly increased T cell numbers and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution. CONCLUSION. Within the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease. FUNDING. This research was supported by the Intramural Research Program of the National Institutes of Health.

Authors

Brittany A. Riggle, Monica Manglani, Dragan Maric, Kory R. Johnson, Myoung-Hwa Lee, Osorio Lopes Abath Neto, Terrie E. Taylor, Karl B. Seydel, Avindra Nath, Louis H. Miller, Dorian B. McGavern, Susan K. Pierce

DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis

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Abstract

Background: DICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist. Methods: Whole exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome wide analyses were performed on 13 tissue samples from familial and non-familial DGCR8-E518K positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTC) and Wilms Tumors. MiRNA profiles of four tissue types were compared, and sequencing of miRNA, pre-miRNA and mRNA was performed in a subset of 9 schwannomas, four of which harbor DGCR8-E518K. Results: We identified c.1552G>A;p.E518K in DGCR8, a microprocessor located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms Tumors and has been identified in two PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTC, MNG, schwannomas and Wilms Tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons. Conclusions: We identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis. Funded by CIHR, Compute Canada, Alex’s Lemonade Stand Foundation, and the Mia Neri Foundation for Childhood Cancer.

Authors

Barbara Rivera, Javad Nadaf, Somayyeh Fahiminiya, Maria Apellaniz-Ruiz, Avi Saskin, Anne-Sophie Chong, Sahil Sharma, Rabea Wagener, Timothée Revil, Vincenzo Condello, Zineb Harra, Nancy Hamel, Nelly Sabbaghian, Karl Muchantef, Christian Thomas, Leanne de Kock, Marie-Noëlle Hébert-Blouin, Angelia V. Bassenden, Hannah Rabenstein, Ozgur Mete, Ralf Paschke, Marc P. Pusztaszeri, Werner Paulus, Albert Berghuis, Jiannis Ragoussis, Yuri E. Nikiforov, Reiner Siebert, Steffen Albrecht, Robert Turcotte, Martin Hasselblatt, Marc R. Fabian, William D. Foulkes

Insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease

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Abstract

Background. An increase in intrahepatic triglyceride (IHTG) is the hallmark feature of nonalcoholic fatty liver disease (NAFLD) and is decreased by weight loss. Hepatic de novo lipogenesis (DNL) contributes to steatosis in people with NAFLD. The physiological factors that stimulate hepatic DNL and the effect of weight loss on hepatic DNL are not clear.Methods. Hepatic DNL, 24-h integrated plasma insulin and glucose concentrations, and both liver and whole-body insulin sensitivity were determined in people who were lean (n = 14), obese with normal IHTG content (Obese, n = 26) and obese with NAFLD (Obese-NAFLD, n = 27). Hepatic DNL was assessed by using the deuterated water method corrected for the potential confounding contribution of adipose tissue DNL. Liver and whole-body insulin sensitivity were assessed by using the hyperinsulinemic-euglycemic clamp procedure in conjunction with glucose tracer infusion. Six subjects in the Obese-NAFLD group were also evaluated before and after 10% diet-induced weight loss.Results. The contribution of hepatic DNL to IHTG-palmitate was 11%, 19% and 38% in the Lean, Obese and Obese-NAFLD groups, respectively. Hepatic DNL was inversely correlated with hepatic and whole-body insulin sensitivity, but directly correlated with 24-h plasma glucose and insulin concentrations. Weight loss decreased IHTG content, in conjunction with a decrease in hepatic DNL and 24-h plasma glucose and insulin concentrations. Conclusions. These data suggest hepatic DNL is an important regulator of IHTG content, and that increases in circulating glucose and insulin stimulate hepatic DNL in people with NAFLD. Weight loss decreases IHTG content, at least in part, by decreasing hepatic DNL.

Authors

Gordon I. Smith, Mahalakshmi Shankaran, Mihoko Yoshino, George G. Schweitzer, Maria Chondronikola, Joseph W. Beals, Adewole L. Okunade, Bruce W. Patterson, Edna Nyangau, Tyler Field, Claude B. Sirlin, Saswata Talukdar, Marc K. Hellerstein, Samuel Klein

IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

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Abstract

Epidermal growth factor receptor (EGFR) and MEK inhibitors (EGFR/MEKi) are beneficial for the treatment of solid cancers but are frequently associated with severe therapy-limiting acneiform skin toxicities. The underlying molecular mechanisms are poorly understood. Using gene expression profiling we identified IL-36γ and IL-8 as candidate drivers of EGFR/MEKi skin toxicity. We provide molecular and translational evidence that EGFR/MEKi in concert with the skin commensal bacterium Cutibacterium acnes act synergistically to induce IL-36γ in keratinocytes and subsequently IL-8, leading to cutaneous neutrophilia. IL-36γ expression was the combined result of C. acnes-induced NF-κB activation and EGFR/MEKi-mediated expression of the transcription factor Krüppel-like factor 4 (KLF4), due to the presence of both NF-κB- and KLF4-binding sites in the human IL-36γ gene promoter. EGFR/MEKi increased KLF4 expression by blockade of the EGFR-MEK-ERK pathway. These results provide an insight into understanding the pathological mechanism of the acneiform skin toxicities induced by EGFR/MEKi and identify IL-36γ and the transcription factor KLF4 as potential therapeutic targets.

Authors

Takashi K. Satoh, Mark Mellett, Barbara Meier-Schiesser, Gabriele Fenini, Atsushi Otsuka, Hans-Dietmar Beer, Tamara Rordorf, Julia-Tatjana Maul, Jürg Hafner, Alexander A. Navarini, Emmanuel Contassot, Lars E. French

Oral N-acetylcysteine improves cone function in retinitis pigmentosa patients in phase 1 trial

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Abstract

Background: In retinitis pigmentosa (RP) rod photoreceptors degenerate from one of many mutations after which cones are compromised by oxidative stress. N-acetylcysteine (NAC) reduces oxidative damage and increases cone function/survival in RP models. We tested the safety, tolerability, and visual function effects of oral NAC in RP patients. Methods: Subjects (n = 10 per cohort) received 600 mg (cohort 1), 1200 mg (cohort 2), or 1800 mg (cohort 3) NAC BID for 12 weeks and then TID for 12 weeks. Best-corrected visual acuity (BCVA), macular sensitivity, ellipsoid zone (EZ) width, and aqueous NAC were measured. Linear mixed effects models were used to estimate the rates of changes during the treatment period. Results: There were 9 drug-related gastrointestinal adverse events which resolved spontaneously or with dose reduction (MTD 1800 mg bid). During the 24 week treatment period, mean BCVA significantly improved at 0.4 (95% CI 0.2–0.6, P < 0.001), 0.5 (95% CI 0.3–0.7, P < 0.001) and 0.2 (95% CI 0.02–0.4, P = 0.03) letters/month in cohorts 1, 2 and 3, respectively. There was no significant improvement in mean sensitivity (MS) over time in cohorts 1 and 2, but there was in cohort 3 (0.15 dB/month, 95%CI 0.04–0.26). There was no significant change in mean EZ width in any cohort. Conclusion: Oral NAC is safe and well-tolerated in patients with moderately advanced RP and may improve suboptimally functioning macular cones. A randomized, placebo-controlled trial is needed to determine if oral NAC can provide long term stabilization and/or improvement in visual function in patients with RP.

Authors

Peter A. Campochiaro, Mustafa Iftikhar, Gulnar Hafiz, Anam Akhlaq, Grace Tsai, Dagmar Wehling, Lili Lu, G. Michael Wall, Mandeep S. Singh, Xiangrong Kong

Neuromuscular junctions are stable in patients with cancer cachexia

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Abstract

Cancer cachexia is a major cause of patient morbidity and mortality, with no efficacious treatment or management strategy. Despite sharing pathophysiological features with a number of neuromuscular wasting conditions, including age-related sarcopenia, the mechanisms underlying cachexia remain poorly understood. Studies of related conditions suggest that pathological targeting of the neuromuscular junction (NMJ) may play a key role in cachexia, but this has yet to be investigated in human patients. Here, high-resolution morphological analyses were undertaken on NMJs of rectus abdominis obtained from patients undergoing upper GI cancer surgery compared with controls (N=30; n=1,165 NMJs). Cancer patients included those with cachexia and weight-stable disease. Despite the low skeletal muscle index and significant muscle fibre atrophy in patients with cachexia, NMJ morphology was fully conserved. No significant differences were observed in any of the pre- and post-synaptic variables measured. We conclude that NMJs remain structurally intact in rectus abdominis in both cancer and cachexia, suggesting that denervation of skeletal muscle is not a major driver of pathogenesis. The absence of NMJ pathology is in stark contrast to related conditions, such as age-related sarcopenia, and supports the hypothesis that intrinsic changes within skeletal muscle, independent of any changes in motor neurons, represent the primary locus of neuromuscular pathology in cancer cachexia.

Authors

Ines Boehm, Janice Miller, Thomas M. Wishart, Stephen J. Wigmore, Richard J.E. Skipworth, Ross A. Jones, Thomas H. Gillingwater

Chikungunya virus replication in skeletal muscle cells is required for disease development

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Abstract

Chikungunya virus (CHIKV) is an arbovirus capable of causing a severe and often debilitating rheumatic syndrome in humans. CHIKV replicates in a wide variety of cell types in mammals, which has made attributing pathologic outcomes to replication at specific sites difficult. To assess the contribution of CHIKV replication in skeletal muscle cells to pathogenesis, we engineered a CHIKV strain exhibiting restricted replication in these cells via incorporation of target sequences for skeletal muscle cell-specific miR-206. This virus, which we term SKE, displayed diminished replication in skeletal muscle cells in a mouse model of CHIKV disease. Mice infected with SKE developed less severe disease signs, including diminished swelling in the inoculated foot and less necrosis and inflammation in the interosseous muscles. SKE infection was associated with diminished infiltration of T cells into the interosseous muscle as well as decreased production of IL-1b, IL-6, IP-10, and TNFa. Importantly, blockade of the IL-6 receptor led to diminished swelling of a control CHIKV strain capable of replication in skeletal muscle, reducing swelling to levels observed in mice infected with SKE. These data implicate replication in skeletal muscle cells and release of IL-6 as important mediators of CHIKV disease.

Authors

Anthony J. Lentscher, Mary K. McCarthy, Nicholas A. May, Bennett J. Davenport, Stephanie A. Montgomery, Krishnan Raghunathan, Nicole McAllister, Laurie A. Silva, Thomas E. Morrison, Terence S. Dermody