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Pseudomonas aeruginosa (P.a.) is a gram-negative, opportunistic bacterium abundantly present in the environment. Often P.a. infections cause severe pneumonia, if left untreated. Surprisingly, up to 30% of patients admitted to the hospital for community- acquired pneumonia develop adverse cardiovascular complications such as myocardial infarction, arrhythmia, left ventricular dysfunction, and heart failure. However, the underlying mechanism of infection-mediated cardiac dysfunction is not yet known. Recently, we demonstrated that P.a. infection of the lungs led to severe cardiac electrical abnormalities and left ventricular dysfunction with limited P.a. dissemination to the heart tissue. To understand the mechanism of cardiac dysfunction during P.a. infection, we utilized both in vitro and in vivo models. Our results revealed that inflammatory cytokines contribute but are not solely responsible for severe contractile dysfunction in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Instead, exposure of hiPSC-CMs with conditioned media from P.a. infected human monocyte-derived macrophages (hMDMs) was sufficient to cause severe contractile dysfunction and arrhythmia in hiPSC-CMs. Specifically, exosomes released from infected hMDMs and bacterial outer membrane vesicles (OMVs) are the major drivers of cardiomyocyte contractile dysfunction. By using LC-MS/MS, we identified bacterial proteins, including toxins that are packaged in the exosomes and OMVs, which are responsible for contractile dysfunction. Furthermore, we demonstrated that systemic delivery of bacterial OMVs to mice caused severe cardiac dysfunction, mimicking the natural bacterial infection. In summary, we conclude that OMVs released during infection enter circulation and drive cardiac dysfunction.

Lipopolysaccharide (LPS) in blood circulation causes endotoxemia and is linked to various disease conditions. Current treatments focus on preventing LPS from interacting with its receptor Toll-like receptor 4 (TLR4) and reducing inflammation. However, our body has a natural defense mechanism: reticuloendothelial cells in the liver rapidly degrade and inactivate much of the circulating LPS within minutes. But this LPS clearance mechanism is not perfect. Excessive LPS that escape this clearance mechanism cause systemic inflammatory damage through TLR4. Despite its importance, the role of reticuloendothelial cells in LPS elimination is not well-studied, especially regarding the specific cells, receptors, and mechanisms involved. This gap hampers the development of effective therapies for endotoxemia and related diseases. This review consolidates the current understanding of LPS clearance, narrates known and explores potential mechanisms, and discusses the relationship between LPS clearance and LPS signaling. It also aims to highlight key insights that can guide the development of strategies to reduce circulating LPS by way of bolstering host defense mechanisms. Ultimately, we seek to provide a foundation for future research that could lead to innovative approaches for enhancing the body's natural ability to clear LPS and thereby lower the risk of endotoxin-related inflammatory diseases, including sepsis.

Immune-checkpoint inhibitor (ICI) therapy has dramatically changed the clinical landscape for several cancers, and ICI use continues to expand across many cancer types. Low baseline clearance (CL) and/or a large reduction of CL during treatment correlates with better clinical response and longer survival. Similar phenomena have also been reported with other monoclonal antibodies (mAb) in cancer and other diseases, highlighting a characteristic of mAb clinical pharmacology that is potentially shared among various mAbs and diseases. Though tempting to attribute poor outcomes to low drug exposure and arguably low target engagement due to high CL, such speculation is not supported by the relatively flat exposure-response relationship of most ICIs, where a higher dose or exposure is not likely to provide additional benefit. Instead, an elevated and/or increasing CL could be a surrogate marker of the inherent resistant phenotype that cannot be reversed by maximizing drug exposure. The mechanisms connecting ICI clearance, therapeutic efficacy, and resistance are unclear and likely to be multifactorial. Therefore, to explore the potential of ICI CL as an early marker for efficacy, this review highlights the similarities and differences of CL characteristics and CL-response relationships for all FDA-approved ICIs, and we compare and contrast these to selected non-ICI mAbs. We also discuss underlying mechanisms that potentially link mAb CL with efficacy and highlight existing knowledge gaps and future directions where more clinical and preclinical investigations are warranted to clearly understand the value of baseline and/or time-varying CL in predicting response to ICI-based therapeutics., (©2023 American Association for Cancer Research.)

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: DHO reports research funding to institution from BMS, Merck, Genentech, Palobiofarma, Onc.AI, which is outside the scope of the current work. All other authors declare they have no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Rheumatoid arthritis is a chronic systemic autoimmune disease that involves transformation of the lining of synovial joints into an invasive and destructive tissue. Synovial fibroblasts become transformed, invading and destroying the bone and cartilage of the affected joint(s). Due to the significant role these cells play in the progression of the disease process, developing a therapeutic strategy to target and inhibit their invasive destructive nature could help patients who are afflicted with this debilitating disease. Gingival-derived mesenchymal stem cells are known to possess immunomodulatory properties and have been studied extensively as potential cell-based therapeutics for several autoimmune disorders., Methods: A chimeric human/mouse model of synovitis was created by surgically implanting SCID mice with a piece of human articular cartilage surrounded by RASF. Mice were injected once with either GMSC or GMSCExo at 5-7 days post-implantation. Histology and IHC were used to assess RASF invasion of the cartilage. Flow cytometry was used to understand the homing ability of GMSC in vivo and the incidence of apoptosis of RASF in vitro., Results: We demonstrate that both GMSC and GMSCExo are potent inhibitors of the deleterious effects of RASF. Both treatments were effective in inhibiting the invasive destructive properties of RASF as well as the potential for these cells to migrate to secondary locations and attack the cartilage. GMSC home to the site of the implant and induce programmed cell death of the RASF., Conclusions: Our results indicate that both GMSC and GMSCExo can block the pathological effects of RASF in this chimeric model of RA. A single dose of either GMSC or GMSCExo can inhibit the deleterious effects of RASF. These treatments can also block the invasive migration of the RASF, suggesting that they can inhibit the spread of RA to other joints. Because the gingival tissue is harvested with little difficulty, relatively small amounts of tissue are required to expand the cells, the simple in vitro expansion process, and the increasing technological advances in the production of therapeutic exosomes, we believe that GMSCExo are excellent candidates as a potential therapeutic for RA., (© 2023. The Author(s).)

Pseudomonas aeruginosa (P.a.) infection accounts for nearly 20% of all cases of hospital acquired pneumonia with mortality rates >30%. P.a. infection induces a robust inflammatory response, which ideally enhances bacterial clearance. Unfortunately, excessive inflammation can also have negative effects, and often leads to cardiac dysfunction with associated morbidity and mortality. However, it remains unclear how P.a. lung infection causes cardiac dysfunction. Using a murine pneumonia model, we found that P.a. infection of the lungs led to severe cardiac left ventricular dysfunction and electrical abnormalities. More specifically, we found that neutrophil recruitment and release of S100A8/A9 in the lungs activates the TLR4/RAGE signaling pathways, which in turn enhance systemic inflammation and subsequent cardiac dysfunction. Paradoxically, global deletion of S100A8/A9 did not improve but aggravated cardiac dysfunction and mortality likely due to uncontrolled bacterial burden in the lungs and heart. Our results indicate that P.a. infection induced release of S100A8/9 is double-edged, providing increased risk for cardiac dysfunction yet limiting P.a. growth., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Rheumatoid arthritis is a chronic systemic autoimmune disease that involves transformation of the lining of synovial joints into an invasive and destructive tissue. Synovial fibroblasts become transformed, invading and destroying bone and cartilage of the affected joint(s). Due to the significant role these cells play in the progression of the disease process, developing a therapeutic strategy to target and inhibit their invasive destructive nature could help patients who are affiicted with this debilitating disease. Gingival-derived mesenchymal stem cells are known to possess immunomodulatory properties and have been studied extensively as potential cell-based therapeutics for several autoimmune disorders., Methods: A chimeric human/mouse model of synovitis was created by surgically implanting SCID mice with a piece of human articular cartilage surrounded by RASF. Mice were injected once with either GMSC or GMSCExo at 5-7 days post-implantation. Histology and IHC were used to assess RASF invasion of the cartilage. Flow cytometry was used to understand the homing ability of GMSC in vivo and the incidence of apoptosis of RASF in vitro., Results: We demonstrate that both GMSC and GMSCExo are potent inhibitors of the deleterious effects of RASF. Both treatments were effective in inhibiting the invasive destructive properties of RASF as well as the potential of these cells to migrate to secondary locations and attack the cartilage. GMSC home to the site of the implant and induce programmed cell death of the RASF., Conclusions: Our results indicate that both GMSC and GMSCExo can block the pathological effects of RASF in this chimeric model of RA. A single dose of either GMSC or GMSCExo can inhibit the deleterious effects of RASF. These treatments can also block the invasive migration of the RASF, suggesting that they can inhibit the spread of RA to other joints. Because the gingival tissue is harvested with little difficulty, relatively small amounts of tissue are required to expand the cells, the simple in vitro expansion process, and the increasing technological advances in the production of therapeutic exosomes, we believe that GMSCExo are excellent candidates as a potential therapeutic for RA., Competing Interests: Declarations Competing interests: The authors declare that they have no competing interests.

Overactivation of immune responses is a hallmark of autoimmune disease pathogenesis. This includes the heightened production of inflammatory cytokines such as Tumor Necrosis Factor α (TNFα), and the secretion of autoantibodies such as isotypes of rheumatoid factor (RF) and anticitrullinated protein antibody (ACPA). Fcγ receptors (FcγR) expressed on the surface of myeloid cells bind Immunoglobulin G (IgG) immune complexes. Recognition of autoantigen-antibody complexes by FcγR induces an inflammatory phenotype that results in tissue damage and further escalation of the inflammatory response. Bromodomain and extra-terminal protein (BET) inhibition is associated with reduced immune responses, making the BET family a potential therapeutic target for autoimmune diseases such as rheumatoid arthritis (RA). In this paper, we examined the BET inhibitor PLX51107 and its effect on regulating FcγR expression and function in RA. PLX51107 significantly downregulated expression of FcγRIIa, FcγRIIb, FcγRIIIa, and the common γ-chain, FcϵR1-γ, in both healthy donor and RA patient monocytes. Consistent with this, PLX51107 treatment attenuated signaling events downstream of FcγR activation. This was accompanied by a significant decrease in phagocytosis and TNFα production. Finally, in a collagen-induced arthritis model, PLX51107-treatment reduced FcγR expression in vivo accompanied by a significant reduction in footpad swelling. These results suggest that BET inhibition is a novel therapeutic approach that requires further exploration as a treatment for patients with RA.

Influenza A virus (IAV) and SARS-CoV-2 virus are both acute respiratory viruses currently circulating in the human population. This study aims to determine the impact of IAV infection on SARS-CoV-2 pathogenesis and cardiomyocyte function. Infection of human bronchial epithelial cells (HBEC), A549 cells, lung fibroblasts (HLF), monocyte derived macrophages (MDMs), cardiac fibroblasts (HCF) and hiPSC-derived cardiomyocytes with IAV enhanced the expression of ACE2, the SARS-CoV-2 receptor. Similarly, IAV infection increased levels of ACE2 in the lungs of mice and humans. Of interest, we detected heavily glycosylated form of ACE2 in hiPSC-CMs and poorly glycosylated ACE2 in other cell types. Also, prior IAV infection enhances SARS-CoV-2 spike protein binding and viral entry in all cell types. However, efficient SARS-CoV-2 replication was uniquely inhibited in cardiomyocytes. Glycosylation of ACE2 correlated with enzymatic conversion of its substrate Ang II, induction of eNOS and nitric oxide production, may provide a potential mechanism for the restricted SARS-CoV-2 replication in cardiomyocytes., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The immune pathways that define treatment response and non-response in lupus nephritis (LN) are unknown. To characterize these intra-kidney pathways, transcriptomic analysis was done on protocol kidney biopsies obtained at flare (initial biopsy (Bx1)) and after treatment (second biopsy (Bx2)) in 58 patients with LN. Glomeruli and tubulointerstitial compartments were isolated using laser microdissection. RNA was extracted and analyzed by nanostring technology with transcript expression from clinically complete responders, partial responders and non-responders compared at Bx1 and Bx2 and to the healthy controls. Top transcripts that differentiate clinically complete responders from non-responders were validated at the protein level by confocal microscopy and urine ELISA. At Bx1, cluster analysis determined that glomerular integrin, neutrophil, chemokines/cytokines and tubulointerstitial chemokines, T cell and leukocyte adhesion genes were able to differentiate non-responders from clinically complete responders. At Bx2, glomerular monocyte, extracellular matrix, and interferon, and tubulointerstitial interferon, complement, and T cell transcripts differentiated non-responders from clinically complete responders. Protein analysis identified several protein products of overexpressed glomerular and tubulointerstitial transcripts at LN flare, recapitulating top transcript findings. Urine complement component 5a and fibronectin-1 protein levels reflected complement and fibronectin expression at flare and after treatment. Thus, transcript analysis of serial LN kidney biopsies demonstrated how gene expression in the kidney changes with clinically successful and unsuccessful therapy. Hence, these insights into the molecular landscape of response and non-response may help align LN management with the pathogenesis of kidney injury., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The neonatal Fc receptor (FcRn) is responsible for recycling of IgG antibodies and albumin throughout the body. This mechanism has been exploited for pharmaceutic delivery across an array of diseases to either enhance or diminish this function. Monoclonal antibodies and albumin-bound nanoparticles are examples of FcRn-dependent anti-cancer therapeutics. Despite its importance in drug delivery, little is known about FcRn expression in circulating immune cells. Through time-of-flight mass cytometry (CyTOF) we were able to characterize FcRn expression in peripheral blood mononuclear cell (PBMC) populations of pancreatic ductal adenocarcinoma (PDAC) patients and non-cancer donors. Furthermore, we were able to replicate these findings in an orthotopic murine model of PDAC. Altogether, we found that in both patients and mice with PDAC, FcRn was elevated in migratory and resident classical dendritic cell type 2 (cDC2) as well as monocytic and granulocytic myeloid-derived suppressor cell (MDSC) populations compared to tumor-free controls. Furthermore, PBMCs from PDAC patients had elevated monocyte, dendritic cells and MDSCs relative to non-cancer donor PBMCs. Future investigations into FcRn activity may further elucidate possible mechanisms of poor efficacy of antibody immunotherapies in patients with PDAC.

Background: Cancer cachexia exhibits decreased albumin and associates with short overall survival (OS) in patients with non-small cell lung cancer (NSCLC), but whether on-treatment albumin changes associate with OS in NSCLC patients treated with immune checkpoint inhibitors (ICIs) and combination chemoimmunotherapy has not been thoroughly evaluated., Patients and Methods: We conducted a single-center retrospective study of patients with advanced NSCLC who received first-line ICI with or without chemotherapy between 2013 and 2020. The association of pretreatment albumin and early albumin changes with OS was evaluated using Kaplan-Meier method and Cox regression models., Results: A total of 210 patients were included: 109 in ICI cohort and 101 in ICI + Chemo cohort. Within a median of 21 days from treatment initiation, patients with ≥ 10% of albumin decrease had significantly shorter OS compared to patients without albumin decrease in ICI cohort. Pretreatment albumin and albumin decrease within the first or second cycle of treatment were significantly and independently associated with OS in ICI cohort, but not in ICI + Chemo cohort. The lack of association between albumin and OS with the addition of chemotherapy was more pronounced among patients with ≥ 1% PD-L1 expression in subgroup analysis., Conclusion: Pretreatment serum albumin and early albumin decrease in ICI monotherapy was significantly associated with OS in advanced NSCLC. Early albumin change, as a routine lab value tested in clinic, may be combined with established biomarkers to improve outcome predictions of ICI monotherapy. The underlying mechanism of the observed association between decreased albumin and ICI resistance warrants further investigation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Lipopolysaccharides (LPSs) cause lethal endotoxemia if not rapidly cleared from blood circulation. Liver sinusoidal endothelial cells (LSEC) systemically clear LPS by unknown mechanisms. We discovered that LPS clearance through LSEC involves endocytosis and lysosomal inactivation via Stabilin-1 and 2 (Stab1 and Stab2) but does not involve TLR4. Cytokine production was inversely related to clearance/endocytosis of LPS by LSEC. When exposed to LPS, Stabilin double knockout mice (Stab DK) and Stab1 KO, but not Stab2 KO, showed significantly enhanced systemic inflammatory cytokine production and early death compared with WT mice. Stab1 KO is not significantly different from Stab DK in circulatory LPS clearance, LPS uptake and endocytosis by LSEC, and cytokine production. These data indicate that (1) Stab1 receptor primarily facilitates the proactive clearance of LPS and limits TLR4-mediated inflammation and (2) TLR4 and Stab1 are functionally opposing LPS receptors. These findings suggest that endotoxemia can be controlled by optimizing LPS clearance by Stab1., Competing Interests: The authors declare no competing interests.

Aging-mediated immune dysregulation affects the normal cardiac immune cell phenotypes and functions, resulting in cardiac distress. During cardiac inflammation, immune activation is critical for mounting the regenerative responses to maintain normal heart function. We investigated the impact of aging on myeloid cell phenotype and function during cardiac inflammation induced by a sub-lethal dose of LPS. Our data show that hearts of old mice contain more myeloid cells than the hearts of young mice. However, while the number of monocytic-derived suppressor cells did not differ between young and old mice, monocytic-derived suppressor cells from old mice were less able to suppress T-cell proliferation. Since cardiac resident macrophages (CRMs) are important for immune surveillance, clearance of dead cells, and tissue repair, we focused our studies on CRMs phenotype and function during steady state and LPS treatment. In the steady state, we observed significantly more MHC-II low and MHC-II high CRMs in the hearts of old mice; however, these populations were decreased in both young and aged mice upon LPS treatment and the decrease in CRM populations correlated with defects in cardiac electrical activity. Notably, mice treated with a liver X receptor (LXR) agonist showed an increase in MerTK expression in CRMs of both young and old mice, which resulted in the reversal of cardiac electrical dysfunction caused by lipopolysaccharide (LPS). We conclude that aging alters the phenotype of CRMs, which contributes to the dysregulation of cardiac electrical dysfunction during infection in aged mice., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Neutralizing Abs suppress HIV infection by accelerating viral clearance from blood circulation in addition to neutralization. The elimination mechanism is largely unknown. We determined that human liver sinusoidal endothelial cells (LSEC) express FcγRIIb as the lone Fcγ receptor, and using humanized FcγRIIb mouse, we found that Ab-opsonized HIV pseudoviruses were cleared considerably faster from circulation than HIV by LSEC FcγRIIb. Compared with humanized FcγRIIb-expressing mice, HIV clearance was significantly slower in FcγRIIb knockout mice. Interestingly, a pentamix of neutralizing Abs cleared HIV faster compared with hyperimmune anti-HIV Ig (HIVIG), although the HIV Ab/Ag ratio was higher in immune complexes made of HIVIG and HIV than pentamix and HIV. The effector mechanism of LSEC FcγRIIb was identified to be endocytosis. Once endocytosed, both Ab-opsonized HIV pseudoviruses and HIV localized to lysosomes. This suggests that clearance of HIV, endocytosis, and lysosomal trafficking within LSEC occur sequentially and that the clearance rate may influence downstream events. Most importantly, we have identified LSEC FcγRIIb-mediated endocytosis to be the Fc effector mechanism to eliminate cell-free HIV by Abs, which could inform development of HIV vaccine and Ab therapy., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The mechanisms that promote local inflammatory injury during lupus nephritis (LN) flare are largely unknown. Understanding the key immune cells that drive intrarenal inflammation will advance our knowledge of disease pathogenesis and inform the development of new therapeutics for LN management. In this study, we analyzed kidney biopsies from patients with proliferative LN and identified a novel inflammatory dendritic cell (infDC) population that is highly expressed in the LN kidney, but minimally present in healthy human kidneys. During an agnostic evaluation of immune transcript expression in the kidneys of patients with proliferative LN, the most abundantly overexpressed transcript from isolated glomeruli was FCER1G , which encodes the Fc receptor gamma chain (FcRγ). To identify the cell types expressing FcRγ that infiltrate the kidney in LN, studies were done on kidney biopsies from patients with active LN using confocal immunofluorescence (IF) microscopy. This showed that FcRγ is abundantly present in the periglomerular (PG) region of the kidney and to a lesser extent in the tubulointerstitium (TI). Further investigation of the surface markers of these cells showed that they were FcRγ + , MHC II + , CD11c + , CD163 + , CD5 - , DC-SIGN + , CD64 + , CD14 + , CD16 + , SIRPα + , CD206 - , CD68 - , CD123 - , CD3 - , and CD11b - , suggesting the cells were infDCs. Quantification of the infDCs showed an average 10-fold higher level of infDCs in the LN kidney compared to the healthy kidneys. Importantly, IF identified CD3 + T cells to be adjacent to these infDCs in the PG space of the LN kidney, whereas both cell types are minimally present in the healthy kidney. Thus, we have identified a previously undescribed DC in lupus kidneys that may interact with intrarenal T cells and play a role in the pathogenesis of kidney injury during LN flare., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Parikh, Malvar, Shapiro, Turman, Song, Alberton, Lococo, Mejia-Vilet, Madhavan, Zhang, Yu, Satoskar, Birmingham, Jarjour, Rovin and Ganesan.)

Background: Tuberculosis is caused by Mycobacterium tuberculosis. Recent emergence of multidrug-resistant (MDR) tuberculosis strains seriously threatens tuberculosis control and prevention. However, the role of macrophage multidrug resistance gene MDR1 on intracellular M. tuberculosis survival during antituberculosis drug treatment is not known., Methods: We used the human monocyte-derived macrophages to study the role of M. tuberculosis in regulation of MDR1 and drug resistance., Results: We discovered that M. tuberculosis infection increases the expression of macrophage MDR1 to extrude various chemical substances, including tuberculosis drugs, resulting in enhanced survival of intracellular M. tuberculosis. The pathway of regulation involves M. tuberculosis infection of macrophages and suppression of heat shock factor 1, a transcriptional regulator of MDR1 through the up-regulation of miR-431. Notably, nonpathogenic Mycobacterium smegmatis did not increase MDR1 expression, indicating active secretion of virulence factors in pathogenic M. tuberculosis contributing to this phenotype. Finally, inhibition of MDR1 improves antibiotic-mediated killing of M. tuberculosis., Conclusion: We report a novel finding that M. tuberculosis up-regulates MDR1 during infection, which limits the exposure of M. tuberculosis to sublethal concentrations of antimicrobials. This condition promotes M. tuberculosis survival and potentially enhances the emergence of resistant variants., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objective: The aim of this study was to investigate the differences in adherence to the Mediterranean diet, assessed by the alternative Mediterranean Diet (aMED) score, and diet quality, assessed by Healthy Eating Index 2015 to 2020 (HEI-2015), between presence and type of arthritis (rheumatoid arthritis and osteoarthritis). Additionally, the study investigated the association between aMED scores and HEI-2015 scores and the presence of arthritis., Methods: Cross-sectional data from four cycles (2007-2014) of the National Health and Nutrition Examination Survey were used and weighted to produce a nationally representative sample. Arthritis information was extracted from the Medical Conditions file and recoded into relevant variables. Food group and nutrient data from the 24-h recall was transformed to provide aMED and HEI-2015 scores., Results: Individuals with arthritis had significantly worse adherence to the Mediterranean diet and diet quality. aMED scores were 3.43 ± 0.04 for individuals with arthritis and 3.54 ± 0.03 for individuals without arthritis (P = 0.014). HEI-2015 scores were also lower in individuals with arthritis (51.41 ± 0.37) than in those without (53.50 ± 0.28; P < 0.001). There were no significant differences in aMED or HEI-2015 scores between individuals with rheumatoid arthritis and those with osteoarthritis. There were also no associations between aMED scores or HEI-2015 scores and the presence of arthritis., Conclusions: Individuals diagnosed with arthritis can take steps to improve their diet quality as a possible route to reduce arthritis symptoms and maintain a healthy body weight. Further research on dietary patterns and their potential to treat and manage arthritis is warranted., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Biological aging dynamically alters normal immune and cardiac function, favoring the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased instances of cardiac distress. Cardiac failure is the primary reason for hospitalization of the elderly (65+ years). The elderly are also increasingly susceptible to developing chronic bacterial infections due to aging associated immune abnormalities. Since bacterial infections compound the rates of cardiac failure in the elderly, and this phenomenon is not entirely understood, the interplay between the immune system and cardiovascular function in the elderly is of great interest. Using Mycobacterium avium, an opportunistic pathogen, we investigated the effect of mycobacteria on cardiac function in aged mice. Young (2-3 months) and old (18-20 months) C57BL/6 mice were intranasally infected with M. avium strain 104, and we compared the bacterial burden, immune status, cardiac electrical activity, pathology, and function of infected mice against uninfected age-matched controls. Herein, we show that biological aging may predispose old mice infected with M. avium to mycobacterial dissemination into the heart tissue and this leads to cardiac dysfunction. M. avium infected old mice had significant dysrhythmia, cardiac hypertrophy, increased recruitment of CD45 + leukocytes, cardiac fibrosis, and increased expression of inflammatory genes in isolated heart tissue. This is the first study to report the effect of mycobacteria on cardiac function in an aged model. Our findings are critical to understanding how nontuberculous mycobacterium (NTM) and other mycobacterial infections contribute to cardiac dysfunction in the elderly population., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

We crafted human immunodeficiency virus (HIV)-like particles of diameter about 140 nm, which expressed two major HIV-1 proteins, namely, env and gag gene products, and used this reagent to simulate the rate of decay of HIV from the blood stream of BALB/c male mice. We found that most (~90%) of the particles were eliminated (cleared) from the blood by the liver sinusoidal endothelial cells (LSECs), the remainder from Kupffer cells; suggesting that LSECs are the major liver scavengers for HIV clearance from blood. Decay was rapid with kinetics suggesting second order with respect to particles, which infers dimerization of a putative receptor on LSEC. The number of HIV-like particles required for saturating the clearance mechanism was approximated. The capacity for elimination of blood-borne HIV-like particles by the sinusoid was 112 million particles per minute. Assuming that the sinusoid endothelial cells were about the size of glass-adherent macrophages, then elimination capacity was more than 540 particles per hour per endothelial cell.

During Gram-negative bacterial infections, excessive LPS induces inflammation and sepsis via action on immune cells. However, the bulk of LPS can be cleared from circulation by the liver. Liver clearance is thought to be a slow process mediated exclusively by phagocytic resident macrophages, Kupffer cells (KC). However, we discovered that LPS disappears rapidly from the circulation, with a half-life of 2-4 min in mice, and liver eliminates about three quarters of LPS from blood circulation. Using microscopic techniques, we found that ∼75% of fluor-tagged LPS in liver became associated with liver sinusoidal endothelial cells (LSEC) and only ∼25% with KC. Notably, the ratio of LSEC-KC-associated LPS remained unchanged 45 min after infusion, indicating that LSEC independently processes the LPS. Most interestingly, results of kinetic analysis of LPS bioactivity, using modified limulus amebocyte lysate assay, suggest that recombinant factor C, an LPS binding protein, competitively inhibits high-density lipoprotein (HDL)-mediated LPS association with LSEC early in the process. Supporting the previous notion, 3 min postinfusion, 75% of infused fluorescently tagged LPS-HDL complex associates with LSEC, suggesting that HDL facilitates LPS clearance. These results lead us to propose a new paradigm of LSEC and HDL in clearing LPS with a potential to avoid inflammation during sepsis., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background: Sepsis patients with cardiac dysfunction have significantly higher mortality. Although several pathways are associated with myocardial damage in sepsis, the precise cause(s) remains unclear and treatment options are limited. This study was designed to develop a new model to investigate the early events of cardiac damage during sepsis progression., Methods and Results: Francisella tularensis subspecies novicida (Ft.n) is a Gram-negative intracellular pathogen causing severe sepsis syndrome in mice. BALB/c mice (N=12) were sham treated or infected with Ft.n through the intranasal route. Serial electrocardiograms were recorded at multiple time points until 96 hours. Hearts were then harvested for histology and gene expression studies. Similar to septic patients, we illustrate both cardiac electrical and structural phenotypes in our murine Ft.n infection model, including prominent R' wave formation, prolonged QRS intervals, and significant left ventricular dysfunction. Notably, in infected animals, we detected numerous microlesions in the myocardium, previously observed following nosocomial Streptococcus infection and in sepsis patients. We show that Ft.n-mediated microlesions are attributed to cardiomyocyte apoptosis, increased immune cell infiltration, and expression of inflammatory mediators (tumor necrosis factor, interleukin [IL]-1β, IL-8, and superoxide dismutase 2). Finally, we identify increased expression of microRNA-155 and rapid degradation of heat shock factor 1 following cardiac Ft.n infection as a primary cause of myocardial inflammation and apoptosis., Conclusions: We have developed and characterized an Ft.n infection model to understand the pathogenesis of cardiac dysregulation in sepsis. Our findings illustrate novel in vivo phenotypes underlying cardiac dysfunction during Ft.n infection with significant translational impact on our understanding of sepsis pathophysiology., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with β-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.

FcRn, a non-classical MHCI molecule, transports IgG from mother to young and regulates the rate of IgG degradation throughout life. Brambell proposed a mechanism that unified these two functions, saying that IgG was pinocytosed nonspecifically by the cell into an FcRn-expressing endosome, where, at low pH, it bound to FcRn and was exocytosed. This theory was immediately challenged by claims that FcRn specificity for ligand could be conferred at the cell surface in neonatal jejunum. Assessing Brambell's hypothesis we found abundant nonspecifically endocytosed IgG present in the cytoplasm of FcRn(-/-) enterocytes. Further, IgG was present in the intercellular clefts and the cores of FcRn(+/+) but not FcRn(-/-) jejunum. FcRn specificity for ligand could be determined within the cell.

It has long been known that the ITIM-bearing IgG Fc receptor (FcγRIIb, RIIb) is expressed on liver sinusoidal endothelial cells (LSEC) and that the liver is the major site of small immune complex (SIC) clearance. Thus, we proposed that RIIb of LSEC eliminates blood-borne SIC, thereby controlling immune complex-mediated autoimmune disease. Testing this hypothesis, we found most RIIb of the mouse, fully three-quarters, to be expressed in liver. Moreover, most (90%) liver RIIb was expressed in LSEC, the remainder in Kupffer cells. An absent FcRγ in LSEC implied that RIIb is the sole FcγR expressed. Testing the capacity of liver RIIb to clear blood-borne SIC, we infused mice intravenously with radio-iodinated SIC made of OVA and rabbit IgG anti-OVA. Tracking decay of SIC from the blood, we found the RIIb knockout strain to be severely deficient in eliminating SIC compared with the wild-type strain, terminal half-lives being 6 and 1.5 h, respectively. RIIb on LSEC, a major scavenger, keeps SIC blood concentrations low and minimizes pathologic deposition of inflammatory immune complex.

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.

While generally accepted that FcRn of the human syncytiotrophoblast and the mouse yolk sac endoderm is the major IgG transporter, the finding of a different Fc receptor FcgammaRIIb (RIIb) in the human placental endothelium has suggested the existence of an additional IgG transporter. Testing our hypothesis in mouse, we found that while RIIb is expressed in the yolk sac vasculature, IgG concentrations in fetuses of wild-type mice (RIIb(+/+)) and mice with a null mutation in the gene encoding RIIb (RIIb(-/-) mice) are not different, and we thus reject our hypothesis that yolk sac RIIb transports IgG in utero in the mouse. However, the capillary bed in the mouse yolk sac is structurally more complex than in human placenta, consisting of three types of cells: an RIIb-negative endothelium, a unique RIIb-bearing cell that also expresses 2 out of 4 macrophage markers but not endothelial cell or pericyte markers, and pericytes. As in the human placenta the b2 isoform of RIIb predominates in the mouse yolk sac. Remarkably only a single capillary channel rather than 2 channels with a loop is found in each yolk sac villus, which, along with intracapillary erythrocytes, suggests that blood flow is peristaltic, mediated by pericytes. It is not clear whether RIIb in the human placental villus might mediate an IgG transport function in light of the mouse yolk sac equivalent failing to do so., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

In adults, the nonclassical MHC class I molecule, FcRn, binds both IgG and albumin and rescues both from a degradative fate, endowing both proteins with high plasma concentrations. FcRn also transports IgG from mother to young during gestation. Anticipating that a detailed understanding of gestational IgG transport in the mouse may give us a useful model to understand FcRn function in the human placenta, we have studied FcRn in the mouse yolk sac placenta in detail. Analyzing day 19-20 fetuses of the three FcRn genotypes resulting from matings of FcRn(+/-) parents, we found that FcRn(-/-) fetuses showed negligible IgG concentrations (1.5 microg/ml), whereas IgG concentrations in FcRn(+/-) fetuses were about a half (176 microg/ml) that of FcRn(+/+) fetuses (336 microg/ml), indicating that FcRn is responsible for virtually all IgG transport from mother to fetus. Immunofluorescence and immunoblotting studies indicated that FcRn is expressed in the endoderm of the yolk sac placenta but not in other cells of the yolk sac placenta or in the chorioallantoic placenta. IgG was found in the endoderm of both FcRn(+/+) and FcRn(-/-) yolk sac placentas and in the mesenchyme of FcRn(+/+) but was missing from the mesenchyme of FcRn(-/-) yolk sac placentas, indicating that IgG enters the endoderm constitutively but is moved out of the endoderm by FcRn. The similarities of these results to human placental FcRn expression and function are striking.

Macrophages are important effectors in the clearance of antibody-coated tumor cells. However, the signaling pathways that regulate macrophage-induced ADCC are poorly defined. To understand the regulation of macrophage-mediated ADCC, we used human B cell lymphoma coated with Rituximab as the tumor target and murine macrophages primed with IFNgamma as the effectors. Our data demonstrate that the PtdIns 3-kinase/Akt pathway is activated during macrophage-induced ADCC and that the inhibition of PtdIns 3-kinase results in the inhibition of macrophage-mediated cytotoxicity. Interestingly, downstream of PtdIns 3-kinase, expression of constitutively active Akt (Myr-Akt) in macrophages significantly enhanced their ability to mediate ADCC. Further analysis revealed that in this model, macrophage-mediated ADCC is dependent upon the release of nitric oxide (NO). However, the PtdIns 3-kinase/Akt pathway does not appear to regulate NO production. An examination of the role of the PtdIns 3-kinase/Akt pathway in regulating conjugate formation indicated that macrophages treated with an inhibitor of PtdIns 3-kinase fail to polarize the cytoskeleton at the synapse and show a significant reduction in the number of conjugates formed with tumor targets. Further, inhibition of PtdIns 3-kinase also reduced macrophage spreading on Rituximab-coated surfaces. On the other hand, Myr-Akt expressing macrophages displayed a significantly greater ability to form conjugates with tumor cells. Taken together, these findings illustrate that the PtdIns 3-kinase/Akt pathway plays a critical role in macrophage ADCC through its influence on conjugate formation between macrophages and antibody-coated tumor cells.

Francisella tularensis, the causative agent of tularemia, is phagocytosed by immune cells such as monocytes and macrophages. Instead of being destroyed in the phagolysosome, the bacterium escapes the phagosome and replicates within the host cytosol. Recent studies indicate that phagosomal escape may have a major impact on the nature of the inflammatory cytokine response to infection. To better understand the host cell response to Francisella infection, we exposed human peripheral blood monocytes to Francisella novicida and analyzed transcriptional changes using high-density oligonucleotide microarrays. Results showed a nearly 300-fold up-regulation of transcripts for the p19 subunit of IL-23, and a nearly 18-fold up-regulation for the p40 subunit of IL-12. IL-23 is formed by the heterodimerization of p19 and p40, and is an important cytokine of the innate immune response. Up-regulation of p19 and p40 was confirmed at the protein level by Western blotting and ELISA analyses, and was found to be largely dependent on PI3K and NF-kappaB activity. Studies using medium from infected monocytes with or without a p19 blocking Ab showed that the secreted IL-23 induced IFN-gamma production from NK cells, suggesting a potential biologically important role for IL-23 in host defense. Finally, infection of human monocytes by the highly virulent Francisella SCHU S4 strain likewise led to IL-23 production, suggesting that the IL-23 response may be relevant during tularemia.

The Gram-negative bacterium Francisella novicida infects primarily monocytes/macrophages and is highly virulent in mice. Macrophages respond by producing inflammatory cytokines that confer immunity against the infection. However, the molecular details of host cell response to Francisella infection are poorly understood. In this study, we demonstrate that F. novicida infection of murine macrophages induces the activation of Akt. Inhibition of Akt significantly decreases proinflammatory cytokine production in infected macrophages, whereas production of the anti-inflammatory cytokine IL-10 is enhanced. Analysis of the mechanism of Akt influence on cytokine response demonstrated that Akt promotes NF-kappaB activation. We have extended these findings to show that Akt activation may be regulated by bacterial genes associated with phagosomal escape. Infection with mglA mutants of F. novicida elicited sustained activation of Akt in comparison to cells infected with wild-type F. novicida. Concomitantly, there was significantly higher proinflammatory cytokine production and lower IL-10 production in cells infected with the mglA mutant. Finally, transgenic animals expressing constitutively active Akt displayed a survival advantage over their wild-type littermates when challenged with lethal doses of F. novicida. Together, these observations indicate that Akt promotes proinflammatory cytokine production by F. novicida-infected macrophages through its influence on NF-kappaB, thereby contributing to immunity against F. novicida infection.

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate.

Phagocytosis of IgG-coated particles via FcgammaR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcgammaR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcgammaR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1beta and IL-6 production in response to FcgammaR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1beta production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcgammaR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1beta production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways.

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida-induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida-induced cytokine production through the inhibition of NFkappaB. Consistently, macrophages lacking SHIP displayed enhanced NFkappaB-driven gene transcription, whereas overexpression of SHIP led to decreased NFkappaB activation. Thus, we propose that SHIP negatively regulates F. novicida-induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFkappaB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia.

Trimetazidine (TMZ), an anti-ischemic metabolic drug, is used to treat chest pain (angina pectoris). We hypothesized that derivatives of TMZ with antioxidant functions may improve the cardiac dysfunction caused by ischemia-reperfusion (I/R) above that observed with TMZ alone. Isolated rat hearts perfused with Krebs-Henseleit buffer according to the Langendorff method were subjected to 30 min of global ischemia followed by 45 min of reperfusion. Trimetazidine, TMZ-NH (TMZ modified with a pyrroline moiety), or TMZ-PhiNH (TMZ-NH with a phenyl substitute) were infused (50 microM) for 1 min before the onset of ischemia. Untreated (control) hearts at the end of 45 min of reperfusion showed a significant decrease in the recovery of coronary flow (42%), left ventricular-developed pressure (22%), and rate-pressure product (25%) compared with preischemic baseline values. The I/R hearts also showed markedly increased lactate dehydrogenase and creatine kinase activities in the coronary effluent, significant myocardial infarction (46% of risk area), and activation of Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. Pretreatment of hearts with TMZ-NH or TMZ-PhiNH significantly enhanced the recovery of heart function and decreased infarct size. The I/R-induced activation of Akt was further enhanced by TMZ-PhiNH. The present study demonstrated that TMZ-NH and TMZ-PhiNH significantly protected hearts against I/R-mediated cardiac dysfunction and injury. The protective effect of the TMZ derivatives could be due to the combined effects of antioxidant and anti-ischemic activities as well as enhanced pro-survival Akt activity.

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.

The bacterial endotoxin lipopolysaccharide (LPS), is a potent inducer of the inflammatory response. Previous studies demonstrated that LPS-induced toxicity is reversed upon FcgammaR clustering by IgG immune complexes (IC) through upregulation of the anti-inflammatory cytokine IL-10. The PI3K-Akt pathway is also reported to reverse LPS-induced inflammation. In this study, we have examined the role of Akt in LPS-induced IL-10 production. First, we compared Akt activation in macrophages stimulated with either LPS alone, or with a combination of LPS and ICs. Our experiments revealed that while Akt was activated under both conditions, the level of activation was significantly higher in cells stimulated with LPS and ICs, suggesting that Akt may be involved in IC-induced upregulation of IL-10 production. Using several independent models we have then tested the notion that enhanced Akt activation may lead to enhanced LPS-induced IL-10 production. Over-expression of constitutively active Myr-Akt in the mouse macrophage cell line Raw 264.7 led to significant increase in IL-10 production in response to LPS. In addition, down-regulation of Akt by siRNA resulted in a decrease in LPS-induced IL-10 production. Peritoneal macrophages from transgenic mice with macrophage-specific expression of Myr-Akt produced significantly higher levels of IL-10 when stimulated with LPS, compared to their wild-type counterparts. Consistent with this observation, serum levels of IL-10, post-LPS challenge, was higher in the Myr-Akt transgenic mice compared to the wild-type mice. Taken together, these data demonstrate that Akt plays a critical role in LPS-induced production of IL-10.

The inhibitory receptor FcgammaRIIb becomes tyrosine phosphorylated and associates with the inositol phosphatase SHIP to downregulate phagocytosis. The two splice variants of FcgammaRIIb, b1 and b2, are differentially expressed in hematopoetic cells. Both isoforms of FcgammaRIIb are expressed in human myeloid cells although FcgammaRIIb2 predominates. In murine B cells FcgammaRIIb2 associates with clathrin-coated pits and undergoes endocytosis, whereas FcgammaRIIbl is excluded from the coated pits, indicating that the two isoforms serve partially differing functions. In humans, there are conflicting reports with regard to the ability of FcgammaRIIb2 to become tyrosine phosphorylated, and the functional capacities of the two isoforms are poorly understood. We, and others, have previously reported that the expression of FcgammaRIIb is upregulated in human monocytes by the anti-inflammatory cytokine IL-4. Here, we extend these findings to demonstrate that the IL-4-induced upregulation of FcgammaRIIb is synergistically enhanced by the addition of IL-10, both at the protein and the mRNA level. The upregulated receptors are functional as assessed by their ability to become tyrosine phosphorylated and to downregulate phagocytosis. Interestingly, both b1 and b2 isoforms are upregulated by anti-inflammatory cytokines. Transfection experiments expressing human FcgammaRIIbl or b2 in Raw 264.7 murine macrophage cells revealed that both isoforms are tyrosine phosphorylated and promote SHIP phosphorylation. Finally, both b1 and b2 isoforms of FcgammaRIIb downregulate phagocytosis to a similar extent. Thus we conclude that FcgammaRIIbl and b2 are both functional inhibitory receptors in the phagocytic process.

Fc gamma receptor (Fc gamma R) clustering by immune complexes activates multiple signaling pathways leading to phagocytosis. We and others have previously reported that Akt is phosphorylated in response to Fc gamma R clustering. However, the functional consequence of Akt activation by Fc gamma R is not known. Using Raw 264.7 macrophage cells transfected to overexpress either constitutively active myristoylated (Myr)-Akt or a dominant-negative CAAX-Akt and bone marrow macrophages (BMMs) from wild-type and transgenic mice expressing macrophage-specific Myr-Akt, we analyzed the function of Akt in phagocytosis. We report that overexpression of Myr-Akt resulted in significant increase in phagocytic efficiency, whereas CAAX-Akt down-regulated phagocytosis in Raw 264.7 cells. Likewise BMMs expressing Myr-Akt displayed enhanced phagocytic ability. Analyzing the downstream effectors of Akt, we demonstrate that p70S6 kinase is constitutively phosphorylated in Myr-Akt-expressing BMMs. p70S6 kinase is reported to influence actin cytoskeleton and cell migration, suggesting that Akt may influence phagocytosis through the activation of p70S6 kinase. Consistent with this, overexpression of either wild-type or constitutively active but not a kinase-inactive p70S6 kinase in Raw 264.7 cells significantly enhanced phagocytosis. Likewise suppression of p70S6 kinase with rapamycin down-regulated phagocytic efficiency conferred by the expression of constitutively active Akt. These findings demonstrate a novel role for Akt in phagocytosis through the activation of p70S6 kinase.

LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators. Recent reports indicated that the inflammatory response to LPS is diminished by PI3K, through the activation of the serine/threonine kinase Akt. SHIP is an inositol phosphatase that can reverse the activation events initiated by PI3K, including the activation of Akt. However, it is not known whether SHIP is involved in TLR4 signaling. In this study, we demonstrate that LPS stimulation of Raw 264.7 mouse macrophage cells induces the association of SHIP with lipid rafts, along with IL-1R-associated kinase. In addition, SHIP is tyrosine phosphorylated upon LPS stimulation. Transient transfection experiments analyzing the function of SHIP indicated that overexpression of a wild-type SHIP, but not the SHIP Src homology 2 domain-lacking catalytic activity, up-regulates NF-kappaB-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP(+/+) and SHIP(-/-) mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP(+/+) cells, whereas Akt phosphorylation is enhanced in SHIP(-/-) cells compared with SHIP(+/+) cells. Finally, LPS-induced TNF-alpha and IL-6 production was significantly lower in SHIP(-/-) bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation.

Fc gamma RIIa is a low affinity IgG receptor uniquely expressed in human cells that promotes phagocytosis of immune complexes and induces inflammatory cytokine gene transcription. Recent studies have revealed that phagocytosis initiated by Fc gamma RIIa is tightly controlled by the inositol phosphatase SHIP-1, and the protein-tyrosine phosphatase SHP-1. Whereas the molecular nature of SHIP-1 involvement with Fc gamma RIIa has been well studied, it is not clear how SHP-1 is activated by Fc gamma RIIa to mediate its regulatory effect. Here we report that Fc gamma RIIa clustering induces SHP-1 phosphatase activity in THP-1 cells. Using synthetic phosphopeptides, and stable transfectants expressing immunoreceptor tyrosine-based activation motif (ITAM) tyrosine mutants of Fc gamma RIIa, we demonstrate that SHP-1 associates with the phosphorylated amino-terminal ITAM tyrosine of Fc gamma RIIa, whereas the tyrosine kinase Syk associates with the carboxyl-terminal ITAM tyrosine. Association of SHP-1 with Fc gamma RIIa ITAM appears to suppress total cellular tyrosine phosphorylation. Furthermore, Fc gamma RIIa clustering results in the association of SHP-1 with key signaling molecules such as Syk, p85 subunit of PtdIns 3-kinase, and p62dok, suggesting that these molecules may be substrates of SHP-1 in this system. Finally, overexpression of wild-type SHP-1 but not catalytically deficient SHP-1 led to a down-regulation of NF kappa B-dependent gene transcription in THP-1 cells activated by clustering Fc gamma RIIa.

SHIP-2, a recently identified inositol 5'-phosphatase, shares high level homology with SHIP-1. Although the role of SHIP-1 has been extensively studied, the role of SHIP-2 in myeloid cell functions is not known. Here, we have analyzed the expression patterns, molecular mechanism of activation, and function of SHIP-2 in human myeloid cell Fcgamma receptor (FcgammaR) signaling. We report that SHIP-2 is expressed in transformed myeloid cells and in primary macrophages, but not in peripheral blood monocytes. Treatment of peripheral blood monocytes with bacterial lipopolysaccharide induced expression of SHIP-2 in a dose-dependent manner. FcgammaRIIa clustering in THP-1 cells induced SHIP-2 tyrosine phosphorylation, suggesting a role for SHIP-2 in modulating FcgammaR-mediated function. Consistent with this notion, overexpression of wild-type SHIP-2 (but not catalytically deficient SHIP-2) in THP-1 cells almost completely abrogated NFkappaB-mediated gene transcription in response to FcgammaRIIa clustering. Furthermore, FcgammaRIIa-induced Akt activation was blocked by wild-type SHIP-2, but not by a catalytically deficient mutant of SHIP-2. Additional experiments analyzing the molecular mechanism of SHIP-2 induction by FcgammaRIIa revealed that SHIP-2 associated with the phosphorylated FcgammaRIIa immunoreceptor tyrosine-based activation motif via the SHIP-2 SH2 domain. Thus, an SH2 domain mutant of SHIP-2 failed to associate with FcgammaRIIa or to become tyrosine-phosphorylated upon FcgammaRIIa clustering. Finally, we also demonstrate that SHIP-2 phosphorylation was induced by FcgammaRI clustering in THP-1 cells. These findings unravel a novel level of regulation of FcgammaR-mediated activation of human myeloid cells by the expression and function of the inositol phosphatase SHIP-2.