Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

The gut microbiome and intestinal immune system are engaged in a dynamic interplay that provides myriad benefits to host health. However, the microbiome can also elicit damaging inflammatory responses, and thus establishing harmonious immune-microbiome interactions is essential to maintain homeostasis. Gut microbes actively coordinate the induction of anti-inflammatory responses that establish these mutualistic interactions. Despite this, the microbial pathways that govern this dialogue remain poorly understood. We investigated the mechanisms through which the gut symbiont Bacteroides thetaiotaomicron exerts its immunomodulatory functions. Our data reveal that B. thetaiotaomicron stimulates production of the cytokine IL-10 via secreted factors that are packaged into outer membrane vesicles, in a TLR2 and MyD88 dependent manner. Using a transposon mutagenesis based screen, we identified a key role for the B. thetaiotaomicron encoded NQR complex, which regenerates NAD+ during respiration, in this process. Finally, we found that disruption of NQR reduces the capacity of B. thetaiotaomicron to induce IL-10 by impairing biogenesis of outer membrane vesicles. These data identify a microbial pathway with a previously unappreciated role in gut microbe mediated immunomodulation that may be targeted to manipulate the capacity of the microbiome to shape host immunity. Key points The B. theta NQR complex coordinates OMV-driven TLR2-dependent IL-10 expression.

The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg ) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg -driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.

Cardiac sarcoidosis is a component of an often multiorgan granulomatous disease of still uncertain cause. It is being recognized with increasing frequency, mainly as the result of heightened awareness and new diagnostic tests, specifically cardiac magnetic resonance imaging and 18F-fluorodeoxyglucose positron emission tomography scans. The purpose of this case-based review is to highlight the potentially life-saving importance of making the early diagnosis of cardiac sarcoidosis using these new tools and to provide a framework for the optimal care of patients with this disease. We will review disease mechanisms as currently understood, associated arrhythmias including conduction abnormalities, and atrial and ventricular tachyarrhythmias, guideline-directed diagnostic criteria, screening of patients with extracardiac sarcoidosis, and the use of pacemakers and defibrillators in this setting. Treatment options, including those related to heart failure, and those which may help clarify disease mechanisms are included.

Over the last few years data from our group have indicated that α-synuclein is important in development of immune cells as well as potentially erythrocytes and platelets. The latter is important since this protein may work as negative regulator of granule release. Thus, we sought to begin to understand the structure of this protein in platelets. Flow cytometric analysis of this protein using region-specific (N-terminus, central region and C-terminus) monoclonal antibodies was performed. Antibody to the central region gave the strongest shift among all three antibodies, with the C-terminus having intermediate shift and N-terminus minimal shift. Western blotting using the same antibodies showed similar binding of all antibodies to α-synuclein. These results suggest a similar arrangement of this protein in platelets as seen in neurons. Future studies ought to look at the role that each protein region plays in platelets.

A pandemic of historic impact, coronavirus disease 2019 (COVID-19) has potential consequences on the cardiovascular health of millions of people who survive infection worldwide. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, can infect the heart, vascular tissues, and circulating cells through ACE2 (angiotensin-converting enzyme 2), the host cell receptor for the viral spike protein. Acute cardiac injury is a common extrapulmonary manifestation of COVID-19 with potential chronic consequences. This update provides a review of the clinical manifestations of cardiovascular involvement, potential direct SARS-CoV-2 and indirect immune response mechanisms impacting the cardiovascular system, and implications for the management of patients after recovery from acute COVID-19 infection.

A pandemic of historic impact, coronavirus disease 2019 (COVID-19) has potential consequences on the cardiovascular health of millions of people who survive infection worldwide. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, can infect the heart, vascular tissues, and circulating cells through ACE2 (angiotensin-converting enzyme 2), the host cell receptor for the viral spike protein. Acute cardiac injury is a common extrapulmonary manifestation of COVID-19 with potential chronic consequences. This update provides a review of the clinical manifestations of cardiovascular involvement, potential direct SARS-CoV-2 and indirect immune response mechanisms impacting the cardiovascular system, and implications for the management of patients after recovery from acute COVID-19 infection.

BACKGROUND:Accumulation of iron is a consistent feature of Alzheimer's disease (AD) brains. The underlying cause, however, remains debatable. OBJECTIVE:To explore whether local hepcidin synthesized by brain cells contributes to iron accumulation in AD brains. METHODS:Brain tissue from the cingulate cortex of 33 cases of AD pre-assigned to Braak stage I-VI, 6 cases of non-dementia, and 15 cases of non-AD dementia were analyzed for transcriptional upregulation of hepcidin by RT-qPCR and RT-PCR. Change in the expression of ferritin, ferroportin (Fpn), microglial activation marker Iba1, IL-6, and TGFβ2 was determined by western blotting. Total tissue iron was determined by colorimetry. RESULTS:Significant transcriptional upregulation of hepcidin was observed in Braak stage III-VI relative to Braak stage I and II, non-AD dementia, and non-dementia samples. Ferritin was increased in Braak stage V, and a significant increase in tissue iron was evident in Braak stage III-VI. The expression of Iba1 and IL-6 was also increased in Braak stage III-VI relative to Braak stage I and II and non-AD dementia samples. Amyloid-β plaques were absent in most Braak stage I and II samples, and present in Braak stage III-VI samples with few exceptions. CONCLUSION:These observations suggest that upregulation of brain hepcidin is mediated by IL-6, a known transcriptional activator of hepcidin. The consequent downregulation of Fpn on neuronal and other cells results in accumulation of iron in AD brains. The increase in hepcidin is disease-specific, and increases with disease progression, implicating AD-specific pathology in the accumulation of iron.

Mycobacterium tuberculosis (Mtb) utilizes a number of immune evasion mechanisms in order to persist inside of host antigen-presenting cells. Dendritic cells (DCs) are important in restricting Mtb growth by migrating to draining lymph nodes and activating antigen-specific T cell responses, but the roles of DCs in Mtb infection require further study. This study investigated DC activation and functional outcomes following Mtb H37Ra-driven tumor progression locus 2 (Tpl2) signaling. The role of Tpl2 was interrogated genetically, utilizing bone marrow-derived DCs from Tpl2−/− mice. We assessed cytokine production via ELISA, mRNA levels via qRT-PCR, and expression of cell surface molecules via flow cytometry. In Mtb-treated DCs, genetic depletion of Tpl2 increased production of pro-inflammatory cytokines such as IL-12p40 and IL-6. Loss of Tpl2 in Mtb-treated DCs also led to decreased E-cadherin expression, and increased expression of Icam-1 (Cd54) and Mmp2, which are molecules involved in DC transmigration. Expression of Ccr7 and Ccr4 was also enhanced in Mtb-treated Tpl2−/− DCs, which correlated with improved migration of Tpl2−/− DCs towards CCL19 and CCL21 in vitro (assessed using a trans-well assay). When antigen-specific CD4+ T cells were co-cultured with Mtb-infected DCs, deletion of Tpl2−/− in the DCs resulted in increased T cell production of IFNγ and IL-2, as well as increased Tbet expression, indicative of enhanced Th1 polarization. Together, these data indicate that Mtb-induced Tpl2 signaling suppresses certain aspects of DC activation, leading to blunted Th1 responses against the pathogen.

This study investigated responses to Toll-like receptor 2 (TLR2)-driven extracellular signal-related kinase (ERK) signaling in dendritic cells (DCs) versus macrophages. TLR2 signaling was induced with Pam(3)Cys-Ser-Lys(4), and the role of ERK signaling was interrogated pharmacologically with MEK1/2 inhibitor U0126 or genetically with bone marrow-derived macrophages or DCs from Tpl2(-/-) mice. We assessed cytokine production via enzyme-linked immunosorbent assay (ELISA) or V-Plex, and mRNA levels were assessed via reverse transcriptase quantitative PCR (qRT-PCR). In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited interleukin 10 (IL-10) expression and increased expression of the p40 subunit shared by IL-12 and IL-23 (IL-12/23p40). In DCs, blockade of ERK signaling similarly inhibited IL-10 expression but decreased IL-12/23p40 expression, which is opposite to the effect of ERK signaling blockade on IL-12/23p40 in macrophages. This difference in IL-12/23p40 regulation correlated with the differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12 family members, including IL-12 and IL-23. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and DCs, potentially regulating their distinctive functions in the immune system. ERK-mediated suppression of IL-12/23p40 in macrophages may prevent excessive inflammation and associated tissue damage following TLR2-stimulation, while ERK-mediated induction of IL-12/23p40 in DCs may promote priming of T helper 1 (Th1) responses. A greater understanding of the role that ERK signaling plays in different immune cell types may inform the development of host-directed therapy and optimal adjuvanticity for a number of infectious pathogens.

This study investigated responses to TLR2-driven ERK signaling in dendritic cells versus macrophages. TLR2 signaling was induced with Pam3Cys, and the role of ERK signaling was interrogated pharmacologically with a MEK1/2 inhibitor (U0126) or genetically using bone-marrow-derived macrophages or dendritic cells from Tpl2−/− mice. We assessed cytokine production via ELISA and mRNA levels by qRT-PCR. In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited IL-10 production and increased IL-12p40 production significantly. In dendritic cells, blockade of ERK signaling similarly inhibited IL-10 production but decreased IL-12p40 production, opposite to the effect of ERK signaling blockade in macrophages. This difference in IL-12p40 regulation correlated with differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and dendritic cells, potentially regulating their distinctive functions in the immune system. ERK-mediated suppression of IL-12p40 in macrophages may prevent excess inflammation and associated tissue damage following TLR2-stimuation, while ERK-mediated induction of IL-12p40 in dendritic cells may promote priming of Th1 responses. Greater understanding of the role that ERK signaling plays in different immune cell types may inform the development of host-directed therapy for a number of infectious pathogens.

Coronavirus disease 2019 (COVID-19) has presented substantial challenges to patient care and impacted healthcare delivery, including cardiac electrophysiology practice throughout the globe. Based upon the undetermined course and regional variability of the pandemic, there is uncertainty as to how and when to resume and deliver electrophysiology services for patients with arrhythmia. This joint document from representatives of the Heart Rhythm Society, American Heart Association, and American College of Cardiology seeks to provide guidance for clinicians and institutions reestablishing safe electrophysiological care. To achieve this aim, we address regional and local COVID-19 disease status, the role of viral screening and serological testing, return-to-work considerations for exposed or infected health care workers, risk stratification and management strategies based on COVID-19 disease burden, institutional preparedness for resumption of elective procedures, patient preparation and communication, prioritization of procedures, and development of outpatient and periprocedural care pathways.

Objectives: To report methods and findings of 2 autopsies with molecular evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive individuals. Methods: Postmortem examination was completed following Centers for Disease Control and Prevention public guidelines. Numerous formalin-fixed paraffin-embedded (FFPE) tissue types from each case were surveyed for SARS-CoV-2 RNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR). SARS-CoV-2 viral genome was sequenced by next-generation sequencing (NGS) from FFPE lung tissue blocks. Results: Postmortem examinations revealed diffuse alveolar damage, while no viral-associated hepatic, cardiac, or renal damage was observed. Viral RNA was detected in lungs, bronchi, lymph nodes, and spleen in both cases using qRT-PCR method. RNA sequencing using NGS in case 1 revealed mutations most consistent with Western European Clade A2a with ORF1a L3606F mutation. Conclusions: SARS-CoV-2 testing and viral sequencing can be performed from FFPE tissue. Detection and sequencing of SARS-CoV-2 in combination with morphological findings from postmortem tissue examination can aid in gaining a better understanding of the virus's pathophysiologic effects on human health.

Background: SARS-CoV-2 enters cells by binding of its spike protein to angiotensin-converting enzyme 2 (ACE2). Angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) have been reported to increase ACE2 expression in animal models, and worse outcomes are reported in patients with co-morbidities commonly treated with these agents, leading to controversy during the COVID-19 pandemic over whether these drugs might be helpful or harmful. Methods: : Animal, in vitro and clinical data relevant to the biology of the renin-angiotensin system (RAS), its interaction with the kallikrein-kinin system (KKS) and SARS-CoV-2, and clinical studies were reviewed. Findings and Interpretation: SARS-CoV-2 hijacks ACE2to invade and damage cells, downregulating ACE2, reducing its protective effects and exacerbating injurious Ang II effects. However, retrospective observational studies do not show higher risk of infection with ACEI or ARB use. Nevertheless, study of the RAS and KKS in the setting of coronaviral infection may yield therapeutic targets.

Stimulation of dendritic cells and macrophages by pathogen-related products activates a variety of immune responses. Pathogen-induced cell signaling and associated outcome research is abundant in macrophages, but minimal in dendritic cells. We investigated the role of ERK signaling in the induction of cytokines such as IL-12p40 and IL-10 following activation by Pam3Cys or Mycobacterium tuberculosis (Mtb) H37Ra. ERK ablation was accomplished pharmacologically with MERK1/2 inhibitor U0126 or genetically by using cells from Tpl2−/− mice (TPL2 connects TLR to ERK). We utilized western blotting to examine ERK activation, ELISA to assess cytokine production, and qRT-PCR to investigate mRNA levels. In macrophages, blockade of ERK signaling inhibited IL-10 production and increased IL-12p40 production (6–10 fold increase in IL-12p40 mRNA and 3–4 fold increase in protein). In dendritic cells, ERK blockade similarly inhibited IL-10 production, but produced a very different change in IL-12p40 expression: ERK blockade decreased IL-12p40 production (2-fold decrease in IL-12p40 mRNA and protein). This result suggests that the impact of ERK signaling in response to these stimuli differs between macrophages and dendritic cells for a subset of ERK-regulated genes. ERK-mediated suppression of IL-12p40 in macrophages may prevent excess inflammation and associated tissue damage, while ERK-mediated induction of IL-12p40 in dendritic cells may promote priming of Th1 responses. Future studies will examine other genes and proteins involved in infection responses. Greater understanding of the role that ERK signaling plays in different cell types may inform the development of host-directed therapy for infections such as tuberculosis.

We appreciate the viewpoint of Sun and colleagues in support of the training of MD–PhD students in the humanities and social sciences. MD–PhD training in these fields is a relatively recent development in the context of the history of MD–PhD training and is offered by only a subset of MD–PhD programs. We agree that MD–PhDs trained in the humanities and social sciences provide an important, additional benefit to the physician–scientist workforce. Our report is largely agnostic with respect to the scholarly pursuits of medical scientist training program (MSTP) trainees. Our primary aim was to analyze the effect of MSTP training in comparison with PhD-only training for outcomes such as attaining medical school faculty positions and National Institutes of Health (NIH) grant funding. We analyzed these two outcomes as a function of decade of appointment to an MSTP training grant and demographics (race, ethnicity, gender). We recognize that these outcome measures do not capture the breadth of scholarly activities pursued by MSTP program graduates; however, graduates who received training in the humanities and social sciences were included in the analysis. Our analysis did not compare different areas of PhD study or scholarly field beyond reporting the NIH institute or center involved in funding of MSTP alumni and the general nature of research in terms of subjects as defined by NIH data (e.g., animal subjects, human subjects, clinical trials). The trainees in these datasets have pursued a wide range of research fields—basic, translational, and clinical—which in turn include many subsets and variations, potentially including the social sciences and humanities. We agree that diversity of the field of scholarly training is important for the development of the physician–scientist workforce; training could include not only the humanities and social sciences but also other areas of scholarly diversity: basic science, engineering, epidemiology, emerging fields (e.g., bioinformatics, computational genomics, systems biology), and more. Clifford V. Harding, MD, PhDJoseph R. Kahn Professor, chair of pathology, and director, Medical Scientist Training Program, Case Western Reserve University and University Hospitals Cleveland Medical Center, Cleveland, Ohio; e-mail: [email protected]; ORCID: http://orcid.org/0000-0002-6333-162X. Myles H. Akabas, MD, PhDProfessor of physiology and biophysics and director, Medical Scientist Training Program, Albert Einstein College of Medicine, Bronx, New York; ORCID: http://orcid.org/0000-0001-8781-7846. Olaf S. Andersen, MDProfessor of physiology and biophysics, Weill Cornell Medical College, and director, Weill Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD–PhD Program, New York, New York; ORCID: http://orcid.org/0000-0002-3026-6710.

ObjectivesIn platelets, -synuclein is important in calcium-dependent granule release. Notably, cells release -synuclein in setting of cell damage or death. Therefore, we investigated -synuclein levels in plasma of single donor platelet (SDP) units during storage. MethodsAliquots were obtained from same SDP units for 7days from day of donation. Additionally, randomly sampled SDP units at same storage time points were also assayed by enzyme-linked immunosorbent assay. Results-Synuclein in SDP plasma increased continuously over time at each assayed time point. Significant increases were measured on day 3 (11.79.6ng/mL, P=0.025), day 5 (15.3 +/- 5.9ng/mL, P=0.002), and highest on day 7 (23.7 +/- 5.6ng/mL, P<0.0001) compared to day 0 (1.1 +/- 0.8ng/mL). Similar significant results were obtained in randomly sampled SDP units at same corresponding time points. Flow cytometry showed that platelets had strong expression of -synuclein and lacked expression of other synucleins. ConclusionsIncreases of -synuclein during SDP storage is a steady and continuous process that increases with time. Our findings indicate that -synuclein may represent a biomarker of platelet biological state during storage. Further research will be needed to determine how -synuclein increases correlate with platelets' function.

Mycobacterium tuberculosiscauses persistent infection due to its ability to evade host immune responses.M. tuberculosisinduces Toll-like receptor 2 (TLR2) signaling, which influences immune responses toM. tuberculosis.

People living with HIV/AIDS on antiretroviral therapy have increased risk of non-AIDS-defining cancers (NADCs). However, the underlying mechanism for development and progression of certain NADCs remains obscure. Here we show that exosomes released from HIV-infected T cells and those purified from blood of HIV-positive patients stimulate proliferation, migration and invasion of oral/oropharyngeal and lung cancer cells. The HIV transactivation response (TAR) element RNA in HIV-infected T-cell exosomes is responsible for promoting cancer cell proliferation and inducing expression of proto-oncogenes and Toll-like receptor 3 (TLR3)-inducible genes. These effects depend on the loop/bulge region of the molecule. HIV-infected T-cell exosomes rapidly enter recipient cells through epidermal growth factor receptor (EGFR) and stimulate ERK1/2 phosphorylation via the EGFR/TLR3 axis. Thus, our findings indicate that TAR RNA-containing exosomes from HIV-infected T cells promote growth and progression of particular NADCs through activation of the ERK cascade in an EGFR/TLR3-dependent manner.