CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages
Authors: Ming Yao
Nan Li
Chunmei Wang
Xingguang Liu
Xuetao Cao
Yuanyuan Zheng
Journal: Blood (2008)
Discipline: CLINICAL MEDICINE
Keywords: Calcium; CaMKII; TLR signaling; macrophages; cytokines
Affiliations: Institute of Immunology and National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai, China.

Abstract:  Calcium and its major downstream effector, calcium/calmodulin-dependent protein kinase II (CaMKII), are found to be important for the functions of immune cells. Lipopolysaccharide (LPS) has been shown to induce intracellular calcium release in macrophages; however, whether and how CaMKII is required for Toll-like receptor (TLR) signaling remain unknown. Here we demonstrate that TLR 4, 9, and 3 ligands markedly induce intracellular calcium fluxes and activate CaMKII-alpha in macrophages. Selective inhibition or RNA interference of CaMKII significantly suppresses TLR4, 9, 3-triggered production of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferon-alpha/beta (IFN-alpha/beta) in macrophages. Coincidently, overexpression of constitutively active CaMKII-alpha significantly enhances production of the above cytokines. In addition to the activation of mitogen-activated protein kinase and nuclear factor kappaB pathways, CaMKII-alpha can directly bind and phosphorylate transforming growth factor beta-activated kinase 1 (TAK1) and IFN regulatory factor 3 (IRF3; serine on 386) via the N-terminal part of its regulatory domain. Therefore, CaMKII can be activated by TLR ligands, and in turn promotes both myeloid differentiating factor 88 and Toll/IL-1 receptor domain-containing adaptor protein-inducing IFN-beta-dependent inflammatory responses by directly activating TAK1 and IRF3. The cross-talk with the calcium/CaMKII pathway is needed for full activation of TLR signaling in macrophages. 

Retracted: No

PubPeer comments:
 Figure 1A. The  unstimulated and  LPS panels in the BAPTA-AM row appear to look unexpectedly similar. Shown with red boxes. Could the authors please check? ![file] , 
 #1  Figure 1A. The  unstimulated and  LPS panels in the BAPTA-AM row appear to look unexpectedly similar. If I understand the caption correctly, the cells are the same in each row of Fig 1A, and their fluorescence is recorded without and then with the presence of LPS. And the cell field in the 3rd row are also exposed to 6 u03bcM BAPTA-AM, shielding them from the stimulatory effect of LPS. Is it possible that in this case, the level of similarity between the two panels is within the range of what could be expected? , 
 Thanks for the explanation, Hoya! , 
 Dear Dr. Bik, Iu2019m writing to you in response to recent inquiries you posted with regard to mentioned studies conducted at the National Key Laboratory of Medical Immunology and Institute of Immunology at the Second Military Medical University, with me being the corresponding author. I appreciate your interest in our work and your commitment to protecting the accuracy of scientific records and the integrity of research pursuit. They are of utmost importance to me all along as well. Upon being notified of your inquiries, I have made them our highest priority and immediately took steps to look into the concerns you raised with the team and collaborators and carefully re-examined our manuscripts and raw data and lab records. Weu2019ll work with the relevant journal editorial office(s) immediately if our investigation indicates any risk to the highest degree of accuracy of the published records. Based on our analyses up to this point (still ongoing) and additional feedback we received from colleagues and peers, I would like to add that I remain confident about the validity and strength of the scientific conclusions made in those publications and our worku2019s reproducibility. Nevertheless, there is no excuse for any lapse in supervision or laboratory leadership and the concerns you raised serve as a fresh reminder to me just how important my role and responsibility are as mentor, supervisor, and lab leader; and how I might have fallen short. I feel therefore very heavy-hearted and tremendously sorry, to my current and former students, my staff and colleagues, my peers, and the larger community. I most sincerely apologize for any oversight on my part and any inconvenience it might have caused. Iu2019ll use this as an invaluable learning opportunity to do better not only in advancing science, but also in safeguarding the accuracy and integrity of science. Yours sincerely, Xuetao Cao , 
 The cells from each row are the same cells under different exposure. They should be the same. Please be more careful when you are questioning. , 
 #5 That is probably what they are, but that is not what they are supposed to be, according to the figure legend: (A) RAW264.7 cells were loaded with Fluo 3 AM and stimulated with 0.1 u03bcg mL LPS in the absence or presence of 1.5 mM ethyleneglycoltetraacetic acid or 6 u03bcM BAPTA-AM, then imaged by Leica TCS SP2 confocal microscopy under a 10u00d7 0.40 CS objective lens at 10-second intervals. The basal (left panel) and peak (middle panel) fluorescence is displayed. Thus the right panels represent peak fluorescence for that field, and successive images were used to calculate fluorescence intensity. Again, without internal controls this is meaningless, even if the exact same microscope settings are used each time. , 
 @ #6 Hypanartia Dione I don t understand what do you mean by  is not what they are supposed to be, according to the figure legend . The figure legend seems quite clear to me: each row is same field of cells at two different times. I cant speak for #5 and can only guess what they mean by  exposure . If they mean photographic exposure then no, that is wrong. If they mean exposure to LPS - then yes, that s what  Unstimulated and  LPS labels refer to. This looks like a perfectly good figure to me - it seems Elisabeth Bik got a tad carried away in her hunt for image duplications in this instance. , 
 Dear Dr. Bik, Thanks for your concern. However, your question about u201cthe unexpected similarityu201d between these two images in BAPTA-AM treatment group is inappropriate. Obviously, the cells in left and right panels of each row in Figure 1A are the same from the same culture dish. For each group (each row), the calcium fluorescence of cells in the same field of culture dish was imaged at 10-second intervals after LPS stimulation, and the basal (left panel) and peak (right panel) fluorescence was displayed in Figure 1A. Because of the intracellular calcium chelator BAPTA-AM treatment, LPS-induced calcium fluorescence increasement was blocked (also see serial images below). Thus, the two images in BAPTA-AM treatment group display the same cells with the insignificant difference of fluorescence intensity at different time points after LPS stimulation, and they should look expectedly similar. Yours sincerely, Xingguang Liu ![file] **** , 
 Thank you, Dr. Liu. Hoya had already explained the experimental set up 3 months ago. See their comment in #2. I appreciate that you have now joined the discussion with additional figures. I respectfully disagree, however, that my question was  inappropriate . The labeling was different than most other time series (usually these are labeled with the amount of time passed, similar to how they are labeled in #8), so I simply did not understand the experimental set-up. Given the high amount of image concerns in papers from this lab, it did not seem inappropriate to ask the authors to clarify how these 2 images could look the same. , 
 Dear Dr. Bik, Thank you! ,