Serine-threonine kinase, ROCK2, regulates germinal center B cell positioning and cholesterol biosynthesis

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Abstract

Germinal center (GC) responses require B cells to respond to a dynamic set of intercellular and microenvironmental signals that instruct B cell positioning, differentiation, and metabolic reprogramming. ROCK2, a serine-threonine kinase that can be therapeutically targeted by ROCK inhibitors or statins, is a key downstream effector of RHOA-GTPases. While RHOA-mediated pathways are emerging as critical regulators of GC responses, the role of ROCK2 in B cells is unknown. Here, we find that ROCK2 was activated in response to key T cell signals like CD40 and IL21 and that it regulated GC formation and maintenance. RNA-seq analyses revealed that ROCK2 controlled a unique transcriptional program in GC B cells that promoted optimal GC polarization and cholesterol biosynthesis. ROCK2 regulated this program by restraining AKT activation and subsequently enhancing FOXO1 activity. ATAC-seq and biochemical analyses revealed that the effects of ROCK2 on cholesterol biosynthesis were instead mediated via a novel mechanism. ROCK2 directly phosphorylated IRF8, a crucial mediator of GC responses, and promoted its interaction with SREBP2 at key regulatory regions controlling the expression of cholesterol biosynthetic enzymes, resulting in optimal recruitment of SREBP2 at these sites. These findings thus uncover ROCK2 as a multifaceted and therapeutically targetable regulator of GC responses.

Authors

Edd Ricker, Yurii Chinenov, Tania Pannellini, Danny M. Flores Castro, Chao Ye, Sanjay Gupta, Michela Manni, James K. Liao, Alessandra Pernis

Microenvironmental Th9– and Th17– lymphocytes induce metastatic spreading in lung cancer

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Abstract

Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the impact of tumor-infiltrating-lymphocyte subpopulations on lung cancer biology by studying in vitro co-cultures, in vivo mouse models and human lung cancer tissue. Lymphocyte conditioned media-(CM) induced epithelial-mesenchymal-transition (EMT), and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue, and correlated with poor survival. Co-culturing lung cancer cells with Th9/Th17 cells or exposing them to the respective-CM induced-EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL–9 and IL–17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Co-injection of Th9 and/or Th17 cells with tumor cells in wildtype, Rag1-/-, Il9r-/- and Il17ra-/- mice altered tumor growth and metastasis. Accordingly, inhibition of IL–9 or IL–17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration, and metastatic spreading and offering for novel therapeutic strategies.

Authors

Ylia Salazar, Xiang Zheng, David Brunn, Hartmann Raifer, Felix S.R. Picard, Yajuan Zhang, Hauke Winter, Stefan Günther, Andreas Weigert, Benno Weigmann, Laure Dumoutier, Jean-Christophe Renauld, Ari Waisman, Anja Schmall, Amanda Tufman, Ludger Fink, Bernhard Brüne, Tobias Bopp, Friedrich Grimminger, Werner Seeger, Soni Savai Pullamsetti, Magdalena Huber, Rajkumar Savai

Brain-specific repression of AMPKα1 alleviates pathophysiology in Alzheimer’s model mice

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Abstract

AMP-activated protein kinase (AMPK) is a key regulator at the molecular level to maintain energy metabolism homeostasis. Mammalian AMPK is a heterotrimeric complex and its catalytic α subunit exists in two isoforms: AMPKα1 and AMPKα2. Recent studies suggest a role of AMPKα over-activation in AD-associated synaptic failure. However, whether AD-associated dementia can be improved by targeting AMPK remains unclear, and roles of AMPKα isoforms in AD pathophysiology are not understood. Here we showed distinct disruption of hippocampal AMPKα isoform expression patterns in post mortem human AD patients and AD model mice. We further investigated the effects of brain- and isoform-specific AMPKα repression on AD pathophysiology. We found that repression of AMPKα1 alleviated cognitive deficits and synaptic failure displayed in two separate lines of AD model mice. In contrast, AMPKα2 suppression did not alter AD pathophysiology. Using unbiased mass spectrometry-based proteomics analysis, we identified distinct patterns of protein expression associated with specific AMPKα isoform suppression in AD model mice. Further, AD-associated hyper-phosphorylation of eukaryotic elongation factor 2 (eEF2) was blunted with selective AMPKα1 inhibition. Our findings reveal isoform-specific roles of AMPKα in AD pathophysiology, thus providing insights into potential therapeutic strategy for AD and related dementia syndromes.

Authors

Helena R. Zimmermann, Wenzhong Yang, Nicole P. Kasica, Xueyan Zhou, Xin Wang, Brenna C. Beckelman, Jingyun Lee, Cristina M. Furdui, C. Dirk Keene, Tao Ma

Sensory nerves regulate mesenchymal stromal cell lineage commitment by tuning sympathetic tones

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Abstract

Sensory nerve was recently identified as being involved in regulation of bone mass accrual. We previously discovered that PGE2 secreted by osteoblastic cells could activate sensory nerve EP4 receptor to promote bone formation by inhibiting sympathetic activity. However, the fundamental units of bone formation are active osteoblasts, which originate from skeletal stem cells. Here, we found that after sensory denervation, knockout of the EP4 receptor in sensory nerves, or knockout of cyclooxygenase-2 (COX2) in osteoblasts could significantly promote adipogenesis and inhibit osteogenesis in adult mice. Furthermore, injection of SW033291 (a small molecule that locally increases PGE2 level) or propranolol (a beta-blocker) significantly promoted osteogenesis and inhibited adipogenesis. This effect of SW033291, but not propranolol, was abolished in conditional EP4 knockout mice under normal conditions or in the bone repair process. We conclude that the PGE2-EP4 sensory nerve axis could regulate skeletal stem cell differentiation in bone marrow of adult mice.

Authors

Bo Hu, Xiao Lv, Hao Chen, Peng Xue, Bo Gao, Xiao Wang, Gehua Zhen, Janet L. Crane, Dayu Pan, Shen Liu, Shuangfei Ni, Panfeng Wu, Weiping Su, Xiaonan Liu, Zemin Ling, Mi Yang, Ruoxian Deng, Yusheng Li, Lei Wang, Ying Zhang, Mei Wan, Zengwu Shao, Huajiang Chen, Wen Yuan, Xu Cao

Complement activated interferon-γ-primed human endothelium transpresents interleukin-15 to CD8+ T cells

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Abstract

Alloantibodies in pre-sensitized transplant candidates deposit complement membrane attack complexes (MAC) on graft endothelial cells (ECs), increasing risk of CD8+ T cell-mediated acute rejection. We recently showed (a) human ECs endocytose MAC into Rab5+ endosomes, creating a signaling platform that stabilizes NF-κB–inducing kinase (NIK) protein; (b) endosomal NIK activates both non-canonical NF-κB signaling to synthesize pro-IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β; and (c) IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4+ T (TEM) cells. Here, we report IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB-dependent process, where IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8+ TEM. Blocking NLRP3 inflammasome assembly, IL-1 receptor or IL-15 on ECs inhibited the augmented CD8+ TEM responses, indicating this pathway was not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice and enhanced intimal CD8+ T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Authors

Catherine B. Xie, Bo Jiang, Lingfeng Qin, George Tellides, Nancy C. Kirkiles-Smith, Dan Jane-wit, Jordan S. Pober

B cell-intrinsic TLR9 expression is protective in murine lupus

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Abstract

Toll-like receptor 9 (TLR9) is a regulator of disease pathogenesis in systemic lupus erythematosus (SLE). Why TLR9 represses disease while TLR7 and MyD88 have the opposite effect remains undefined. To begin to address this question, we created two novel alleles to manipulate TLR9 expression, allowing for either selective deletion or overexpression. We used these to test cell type-specific effects of Tlr9 expression on the regulation of SLE pathogenesis. Notably, Tlr9 deficiency in B cells was sufficient to exacerbate nephritis while extinguishing anti-nucleosome antibodies, whereas Tlr9 deficiency in dendritic cells (DCs), plasmacytoid DCs, and neutrophils had no discernable effect on disease. Thus, B cell-specific Tlr9 deficiency unlinked disease from autoantibody production. Critically, B cell-specific Tlr9 overexpression resulted in ameliorated nephritis, opposite of the effect of deleting Tlr9. Our findings highlight the non-redundant role of B cell-expressed TLR9 in regulating lupus and suggests therapeutic potential in modulating and perhaps even enhancing TLR9 signals in B cells.

Authors

Jeremy S. Tilstra, Shinu John, Rachael A. Gordon, Claire Leibler, Michael Kashgarian, Sheldon Bastacky, Kevin M. Nickerson, Mark J. Shlomchik

Semaphorin 3F signaling actively retains neutrophils at sites of inflammation

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Abstract

Neutrophilic inflammation is central to disease pathogenesis, e.g. in chronic obstructive pulmonary disease, yet the mechanisms retaining neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 Semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to pro-inflammatory mediators and following recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whilst neutrophil specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous- (F-) actin subsequently showed SEMA3F mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.

Authors

Tracie Plant, Suttida Eamsamarng, Manuel A. Sanchez-Garcia, Leila Reyes, Stephen A. Renshaw, Patricia Coelho, Ananda S. Mirchandani, Jessie-May Morgan, Felix E. Ellett, Tyler Morrison, Duncan Humphries, Emily R. Watts, Fiona Murphy, Ximena L. Raffo-Iraolagoitia, Ailiang Zhang, Jenna L. Cash, Catherine Loynes, Philip M. Elks, Freek Van Eeden, Leo M. Carlin, Andrew J. W. Furley, Moira K. B. Whyte, Sarah R. Walmsley

c-Rel gain in B cells drives germinal center reactions and autoantibody production

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Abstract

Single nucleotide polymorphisms and locus amplification link the NF-κB transcription factor c-Rel to human autoimmune diseases and B cell lymphomas, respectively. However, the functional consequences of enhanced c-Rel levels remain enigmatic. Here, we overexpressed c-Rel specifically in mouse B cells from BAC-transgenic gene loci and demonstrate that c-Rel protein levels linearly dictated expansion of germinal center (GC) B cells and isotype-switched plasma cells. c-Rel expression in B cells of otherwise c-Rel-deficient mice fully rescued terminal B cell differentiation, underscoring its critical B cell-intrinsic roles. Unexpectedly, in GCB cells transcription-independent regulation produced the highest c-Rel protein levels amongst B cell subsets. In c-Rel overexpressing GCB cells this caused enhanced nuclear translocation, a profoundly altered transcriptional program and increased proliferation. Finally, we provide a link between c-Rel gain and autoimmunity by showing that c-Rel overexpression in B cells caused autoantibody production and renal immune complex deposition.

Authors

Maike Kober-Hasslacher, Hyunju Oh-Strauß, Dilip Kumar, Valeria Soberón, Carina Diehl, Maciej Lech, Thomas Engleitner, Eslam Katab, Vanesa Fernandez Saiz, Guido Piontek, Hongwei Li, Björn Menze, Christoph Ziegenhain, Wolfgang Enard, Roland Rad, Jan P. Böttcher, Hans-Joachim Anders, Martina Rudelius, Marc Schmidt-Supprian

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL

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Abstract

Children and adults with Philadelphia chromosome-like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of tyrosine kinase inhibitor (TKI) addition to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor-like factor 2 (CRLF2)-rearranged Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe a novel adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK and PI3K signaling consistent with activated B-cell receptor (BCR) signaling, although they do not express cell surface mu heavy chain (uHC). Combinatorial targeting of JAK/STAT, PI3K, and ‘BCR-like’ signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.

Authors

Christian Hurtz, Gerald B. Wertheim, Joseph P. Loftus, Daniel Blumenthal, Anne Lehman, Yong Li, Asen Bagashev, Bryan Manning, Katherine D. Cummins, Janis K. Burkhardt, Alexander E. Perl, Martin Carroll, Sarah K. Tasian

E4BP4-mediated inhibition of T follicular helper cells is compromised in autoimmune disease

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Abstract

T follicular helper (Tfh) cells are indispensable for the formation of germinal center (GC) reactions, while T follicular regulatory (Tfr) cells inhibit Tfh-mediated GC responses. Aberrant activation of Tfh cells contributes significantly to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). Nonetheless, the molecular mechanisms mitigating excessive Tfh cell differentiation, which in turn trigger autoimmunity, are not fully understood. Herein we demonstrate that the adenovirus E4 promoter-binding protein (E4BP4) mediates a feedback loop and acts as a transcriptional brake to inhibit Tfh cell differentiation. Furthermore, we show that such an immunological mechanism is compromised in patients with SLE. Establishing mice with either conditional knock-out (cKO) or knock-in (cKI) of the E4bp4 gene in T cells reveals that E4BP4 strongly inhibits Tfh cell differentiation. Mechanistically, E4BP4 deregulates Bcl6 transcription by recruiting the repressive epigenetic modifiers HDAC1 and EZH2. E4BP4 phosphorylation site mutants had limited capability with regard to inhibiting Tfh cell differentiation. In SLE, we detected impaired phosphorylation of E4BP4, finding that this compromised transcription factor is positively correlated with disease activity. These findings unveiled molecular mechanisms by which E4BP4 restrains Tfh cell differentiation, whose compromised function is associated with uncontrolled autoimmune reactions in SLE.

Authors

Zijun Wang, Ming Zhao, Jinghua Yin, Limin Liu, Longyuan Hu, Yi Huang, Aiyun Liu, Jiajun Ouyang, Xiaoli Min, Shijia Rao, Wenhui Zhou, Haijing Wu, Akihiko Yoshimura, Qianjin Lu