bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing the movement strategy of CD8+ T cells in finding malaria parasites in the liver
Authors: Ganusov Vitaly V , Rajakaruna Harshana , O’Connor James , Zenkov Viktor S , Cockburn Ian A
Published: January 2023

Movement strategies by which antigen-specific CD8+ T cells search for sites of infection in peripheral tissues are not fully understood. Measurable quantities, such as the distance traveled per unit of time, that follow Lévy statistics are common in nature, for example, in bacterial and higher animal foraging; in mussels, marine predators, monkeys. A recent study has shown that moving according to Lévy flights enables CD8+ T cells in the brain to find “rare targets” (Toxoplasma gondii-infected cells) in an order of magnitude more efficiently than using Brownian and other walking strategies. It has been, thus, suggested that such efficient strategy of T cell searching for infection may have been evolutionarily selected. However, T cells in the brain (or other tissues) are often constrained by the structure of the physical environment in which cells move. Whether the emerged statistical pattern of T cell movement is due to a cell-intrinsic program or a consequence of environmental constrains is, thus, unknown. Here, we address this question by using intravital imaging of movement of Plasmodium-specific CD8+ T cells in murine livers and mathematical modeling and analyses of the generated data. We investigate whether the physical structure of the movement paths in the liver plays a role in shaping the walking strategy of T cells in finding malaria parasites. Here, we map out liver sinusoids in 3D using intravital microscopy and use simulations to parameterize the alternative walking models by fitting them to empirical data having no references to the physical structure. Our preliminary data suggest that constraints imposed by the structure of liver sinusoids significantly impact the moving pattern of the T cells.
Upregulation of intestinal Tumor Growth Factor-β1 is associated with increased intestinal epithelial cell apoptosis in pathogenic simian immunodeficiency virus infection
Authors: Pahar Bapi , Pan Diganta , Srivastav Sudesh , Das Arpita
Published: January 2023

Mucosal Transforming Growth Factor-β1 (TGF-β1), a pleiotropic, potent immunoregulatory cytokine, demonstrated to manage phosphorylated AKT and IFNγ expressions, are associated with intestinal epithelial cells (iECs) survival in macaque colon explants and suggest a potential role of mucosal TGF-β1 in regulating intestinal homeostasis and iEC integrity. It is important to monitor and further explore the role of mucosal TGF-β1 in HIV/SIV pathogenesis and HIV/SIV enteropathy, which may lead to the development of improved therapeutic strategies to prevent iEC damage and systemic immune activation during acute and chronic infection. Our data showed an increased production of intestinal TGF-β1 in T-, B- and non-T/B cell populations during acute and chronic SIV infection in rhesus macaques, without a change in the expression of TGF-βRII. The increased levels of immunosuppressing TGF-β1 were also associated with increased production of IFNγ, suggest the lack of TGF-β1 mediated anti-inflammatory responses in SIV infection. An appropriate balance between inflammatory and anti-inflammatory cytokine responses are crucial for maintenance of a successful immune responses in HIV infection. TGF-β1 induced immune defects contribute to intestinal inflammation, loss of tight junction protein, and apoptosis by overexpression of SMAD3, and downregulation of inhibitory SMAD7 transcription factors. Together, these results indicate that SMAD mediated pathway play a crucial role in regulating TGF-β1 expression and that was thought to be a key contributor to the dysfunction of CD4+ and CD8+ T-cells, iEC apoptosis and disease progression.
Effect of malaria pathology on CD4 and immune cells
Authors: Amadi Emmanuel Chike , Eze Emmanuel , Chigor Vincent
Published: January 2023

Knowledge of immunity in malariology can help in understanding the pathology, treatment and vaccine production. The effect of malaria disorder on CD4 and immune cells counts was carried out at a Teaching Hospital in Enugu, Eastern Nigeria between October-December 2018. Patients on doctor’s provisional diagnosis of malaria were examined for Plasmodium infections and the degree of parasitaemia (0, +, ++, and +++). Positive samples and negative ones (0) were thereafter examined for their CD4 (Flow cytometry) and immune cells (Automated blood cell counter) counts. 45 patients were studied. All the Plasmodium-negative specimens were within reference ranges of CD4 cell count (464–1308); with mean value of 835. The (+) parasitaemia showed lower ranges of CD4 count (502–1282); mean = 678; with immune falls in one (427). The 12 (++) parasitaemia showed crash in 9 CD4 cells counts; range: 301–415; mean = 399. All the 7 (+++) parasitaemia showed crashes in CD4 cell counts, range: 160–357; mean = 225. The CD4 cells falls and crashes were detected only in Plasmodium falciparum parasitaemia infections. Depending on the CD4 cells count, also a reflex of parasitaemia, variances occur in the various immune cells’ percent and numeric. TWBC count fell in only 3 patients (1.99, 3.42 & 3.64) × 109 cells/L, corresponding to (+++), (++) & (+) parasitaemia, respectively. Low CD4 counts does not always produce low lymphocyte numeric, probably because other CD or killer cells compensates it. In conclusion, Plasmodium falciparum infection causes immuno-suppression in patients. Corollary, it means that malaria infection in the immunodeficients and AIDS patients will accelerate the complications as well as death, unlike prevailing reports.
CD28 Regulates Autophagy to Enhance Plasma Cells Survival
Authors: Peng Peng , Chavel Colin , Utley Adam , Carlson Louise , Lee Kelvin P
Published: January 2023

Lifelong humoral immunity depends on the longevity of long-lived plasma cells (LLPC). We have shown that CD28 signaling in LLPC is critical to their survival but the molecular mechanisms are unclear. Here, we report that CD28 mediates a pro-survival response through autophagy in multiple myeloma (MM). As the transformed LLPC, MM is similar to LLPC in CD28 signaling and survival regulation. CD28 in MM is activated by antibody crosslinking or co-culture with dendritic cells that express CD80/CD86. With chemotherapy (melphalan) treatment or serum starvation, CD28 activation increases MM viability from 30% to 70%. Pharmacologic inhibition of autophagy (Baf-A1 or 3-MA) or ATG5 knockdown, a critical autophagy protein, abrogates the CD28 pro-survival effects. CD28 activation up-regulates the autophagy marker LC3II as well as increases autophagosome numbers assessed by Cyto-ID. Conversely, knockdown of CD28 decreases these effects. CD28 signals via GRB2-VAV-PLC in LLPC, and we found that blocking PKC activity in MM by inhibitor Gö6983 diminishes CD28 pro-survival effects and CD28-mediated LC3II up-regulation. Upon activation of CD28, ATG5 protein levels are elevated not through transcriptional up-regulation but enhanced by ATG5 protein modification. Taken together, these data suggest CD28 signaling via GRB2-VAV-PLC-PKC induces autophagy by ATG5 induction. CD28 regulates metabolic fitness in T cells, and we also found inhibition of autophagy by 3-MA minimizes CD28-mediated increase of oxidative phosphorylation. This indicates that CD28-mediated autophagy maintains metabolic homeostasis to improve survival. Our findings suggest a novel mechanism by which CD28 signaling regulates metabolism, survival and function in LLPC.
O-GlcNAcylation of c-Myc dynamically regulates pre-B cell proliferation
Authors: PARK SUNG-KYUN
Published: January 2023

O-GlcNAc modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes such as gene expression, signaling and cell growth. However, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation is largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affect the growth of large pre-B cells transiently expressing pre-B cell receptors on their surface membrane, which are known to rapidly proliferate for the expansion of functional clones that express successfully rearranged heavy chains during early B cell development. Our study first show that overall O-GlcNAc levels in these proliferative pre-B cells are highly induced compared to pro-B cells, depending on pre-B cell receptor expression. Mechanistically, inhibition of O-GlcNAcylation in pre-B cells markedly decreases c-Myc expression, which results in downregulation of cell proliferation through the blocking of E- and A-type cyclin expressions. In addition, restriction of glucose uptake in pre-B cells also reduces cellular O-GlcNAc levels as well as c-Myc expression and subsequently decreases cell proliferation. Furthermore, the population of large pre-B cells in the bone marrow is significantly reduced by the administration of O-GlcNAc inhibitor to mice. Thus, our study strongly suggests that O-GlcNAcylation dependent expression of c-Myc is a new component in regulation of pre-B cell proliferation and also a potential therapeutic target for the treatment of pre-B cell acute lymphoblastic leukemia.
Differential gene expression of the B Cell Receptor (BCR) signaling pathway during pregnancy in mice
Authors: Valeff Natalin Jimena , Muzzio Damian , Abba Martin , Zygmunt Marek , Jensen Federico
Published: January 2023

The aim of this work was to characterize BCR signaling pathway gene expression in B cells during pregnancy. B-cell receptor (BCR) signaling regulates critical cell-fate decisions in B cells like activation, proliferation, expression of costimulatory molecules and cytokine receptors. Changes on the dynamic of BCR signaling pathway during pregnancy might have an impact on the way how the immune system fight against infections or regulate autoimmunity during this critical period of time.

We performed a genome-wide transcriptome profile (GeneChip™ Mouse Gene 1.0 ST Array, Affymetrix) on pure splenic B cells isolated from pregnant and non-pregnant mice. Results were analyzed and significant gene expression regulation was confirmed by qPCR.

In silico analysis showed that genes associated with B cell receptor signaling pathway were significantly down-regulated in B cells from pregnant mice compared to non-pregnant animals (≈1.5–2 fold changes, Limma eBayes test, p<0.0001, n=4). Further qPCR analysis showed that genes of the BCR pathway involved in B cell activation, proliferation and antibodies production were down-regulated (Unpaired t test, one-tailed, *p<0.05, n=8) in B cell from pregnant mice as compared to non-pregnant females. In contrast, genes of the BCR pathway that inhibits B cells activation presented no significant differences.

We showed here that genes associated with BCR signaling leading to B cell activation and proliferation were significantly down-regulated during pregnancy. These results might help to understand why pregnant women are more susceptible to infections as well as some B cell associated autoimmune diseases experience a significant improvement in the symptoms during pregnancy.
Human plasma cells

switch on

and

turn off

antibody secretion
Authors: Nguyen Doan C , Saney Celia , Ley Ariel , Sanz Iñaki , Lee F. Eun-Hyung
Published: January 2023

Long-lived plasma cells (LLPC) are terminally-differentiated, non-dividing cells that reside in a protected and supportive bone marrow (BM) microenvironment. After terminal differentiation, antibody (Ab)-secreting cells (ASC) or plasma cells (PC) are believed to constitutively secrete Ab for a life-time. Recently, by integrated bioinformatics and using a novel in vitro cell-free BM microniche system, we demonstrated that protein factors such as fibronectin, YWHAZ, GAPDH, HSPD1, and hnRNPA1 are required for human ASC/PC survival. Specific blockade or inhibition of GAPDH, HSPD1, or hnRNPA1 resulted in loss of human ASC/PC IgG ELISpot frequencies. To understand whether ASC/PC secretion or survival was affected, we sequentially removed the inhibitors and replated the previously-treated ASC/PC in fresh conditions. Upon removal of the inhibitors, the inhibitory effects of Ab secretion were reversed, suggesting a functional recovery of Ab secretion of the inhibited ASC/PC. No evidence of additional ASC/PC proliferation was observed by BrdU uptake. Given the physiological functions of GAPDH, HSPD1, and hnRNPA1, it appeared that mechanisms involved in glucose or mitochondrial metabolism, or protein synthesis, can modulate Ab secretion of human ASC/PC. Together, these data suggest that human ASC/PC do not produce Ab constitutively, but can actually modulate Ab secretion based on their local microenvironment. Moreover, Ab secretion is not linked to survival. In essence, human ASC/PC switch on or turn off Ab secretion, but the exact mechanisms require further validation.
The bona fide structure of IgM pentamer and its binding mode with AIM/CD5L molecule
Authors: Miyazaki Toru , Hiramoto Emiri , Arai Satoko
Published: January 2023

Soluble IgM forms a predominantly pentameric complex that also contains a small polypeptide J chain. While the IgM pentamer has various immune functions to defend against foreign pathogens, it also behaves as a carrier of the circulating apoptosis inhibitor of macrophage (AIM, also called CD5L). The binding to IgM pentamer protects AIM from renal excretion, leading to high serum AIM levels. When AIM dissociates from IgM, it becomes functionally active and facilitates the repair of a variety of diseases including acute kidney injury and hepatocellular carcinoma. Since the precise manner of AIM-IgM pentamer binding remains unknown, we examined the structure using a latest single-particle negative-stain electron microscopy. Surprisingly, the IgM pentamer shapes “one-piece missing” hexagon containing one large gap, which is markedly different from the textbook prediction of a symmetric pentagon model. A single AIM molecule stably fits into the gap, cross-bridging two IgM-Fc through a disulfide bond and a charge-based interaction. We also found that the association of AIM with IgM pentamer not only inactivates AIM but also progresses inflammatory immune-complex formation in certain autoimmune diseases such as IgA nephropathy. Thus, our current findings of the bona fide structure of the AIM/IgM pentamer complex provides an important insight into the releasing mechanism of AIM from IgM, which could be the basis for the development of new therapeutic strategies against multiple diseases, via the increase of active AIM as well as the disruption of immune-complex formation.
Effects of antiorthostatic suspension, tetanus toxoid, and CpG on the bone marrow antibody repertoire in C57BL/6 mice
Authors: Rettig Trisha A , Nishiyama Nina C , Pecaut Michael J , Chapes Stephen K
Published: January 2023

Each B cell produces an antibody composed of two heavy chains paired with two light chains. During development, B cells rearrange V-, D- (heavy chain only), and J-gene segments. These rearrangements along with the imprecisions in recombination and mutations create diversity in the antibody repertoire.

The physical and psychological stressors of spaceflight are known to affect both the innate and adaptive immune systems. We used a ground-based model, antiorthostatic suspension (AOS), to analyze the impact of spaceflight environmental stressors on the antibody repertoire. Eight-week-old female C57BL/6J mice were suspended for two weeks and immunized via ip injection with saline, tetanus toxoid (TT) and/or CpG, and resuspended. Two weeks after immunization, animals were sacrificed, and bone marrow was collected. RNA was extracted and sequenced using MiSeq 2×300bp. Resulting sequences were analyzed using ImMunoGeneTic’s High-V quest program to identify antibody characteristics.

We identified significant variations in VH, Vκ, D, JH, and Jκ-gene segment usage and CDR3 length due to AOS, TT, and CpG treatment. Changes were also detected in the class-switched repertoire for VH-gene segment usage. We also focused our analysis on the loaded saline, loaded TT+CpG, and AOS+TT+CpG treatment groups to isolate the TT-specific antibody response. Statistically significant changes were detected in VH-gene segment usage and gene segment combinations among treatment groups. These results demonstrate that treatment with a physiological model of spaceflight leads to alterations in the development of the TT-specific antibody repertoire in the bone marrow.
Epigenetic priming underpins enhanced memory B cell differentiation
Authors: Price Madeline J , Scharer Christopher D. , Kania Anna K , Mi Tian , Hicks Sakeenah L , Randall Troy D , Boss Jeremy
Published: January 2023

Immunological memory is a hallmark of the adaptive immune response. Memory B cells (MBC) have increased affinity, expression of costimulatory molecules, and capacity to proliferate and differentiate than their naïve counterparts; however, the epigenetic mechanisms that control enhanced MBC functions are currently unknown. Using a murine model of influenza infection, nucleoprotein (NP)-specific MBC and follicular naïve B cells (nB) were isolated and the chromatin accessibility and transcriptional landscape determined by ATAC and RNA-seq, respectively. MBC displayed distinct gene expression profiles from nB, including an upregulation of plasma cell (PC) signature genes and significant overall increases in total mRNA content. Furthermore, MBC display an open and primed chromatin conformation in gene regulatory regions that map to transcription factors controlling either PC (Prdm1 and Irf4), germinal center (Aicda), or MBC (Zbtb32) fates. These data reveal additional novel MBC-specific transcription factor networks and describe an epigenetic “antigen experience” signature that correlates with enhanced MBC function. To confirm the importance of these molecular changes, naïve and memory mice were challenged with a heterosubtypic influenza infection or isolated and cultured with CD40L, IL-4, and IL-5. In vivo, MBC form germinal centers earlier and to a higher frequency than nB. MBC also form significantly more NP+IgG+ PCs by flow cytometry and ELISPOT, corroborating the open chromatin signature. Ex vivo, MBCs upregulated primed transcription factors earlier than nB, validating the enhanced formation of CD138+ PCs. These data describe an epigenetic basis for enhanced the differentiation capacity and function of MBC.
Transcriptional regulation of IL-10 in T helper cells
Authors: Zhang Huiyuan , Madi Asaf , Yosef Nir , Chihara Norio , Awasthi Amit , Pot Caroline , Apetoh Lionel , Srivastava Amitabh , Wang Chao , Regev Aviv , Kuchroo Vijay
Published: January 2023

IL-10 produced by CD4 T cells plays an essential role in limiting inflammation and autoimmunity. All T help cell subsets can co-produce IL-10 to mitigate unwanted immunopathology but naïve CD4 T cells can also be directly polarized to IL-10 producing Type 1 regulatory (Tr1) cells by the immunoregulatory cytokine, IL-27. Although several transcription factors (TFs) have been shown to regulate IL-10 expression in different contexts, a comprehensive transcriptional network that induce and maintain IL-10 in various CD4 T cells is lacking.

Here we utilized two complementary approaches to systemically study the transcriptional mechanisms driving IL-10 production in T helper cells: 1) We combined novel computational tools with transcriptomic profiling of in-vitro Tr1 cell differentiation at high temporal resolution to build a comprehensive and dynamic transcriptional network that regulates IL-10 production in CD4 T cells. We identified four transcription waves that govern Tr1 differentiation: dynamic early phase, stable early phase, induction phase and maintenance phase. Based on the network analysis, we computationally predicted 78 TFs that regulate different waves and experimentally validated 24 of them using knock-out mice. 2) To identify TFs associated with IL-10 production across T helper cell subsets, we compared RNA-seq data of IL-10+ versus IL-10− compartments of in-vitro differentiated Th1, Th2 and Th17 cells. Using a comprehensive ranking scheme, we identified two TFs that regulates IL-10 production in all helper T cell subsets. Conditional deletion of both the TFs but not either alone with CD4-Cre abolishes IL-10 production from Tr1 cells in vitro and leads to spontaneous colitis in vivo.
Lymph node-fibroblastic reticular cells regulate differentiation of CD4+ T cells through CD25
Authors: Kim Tae Woo , Kim Dong-Eon
Published: January 2023

Lymph node-fibroblastic reticular cells (LN-FRCs) provide functional structure to LNs and play important roles in interactions between T cells and antigen-presenting cells. However, the direct impacts of LN-FRCs on naïve CD4+ T cell differentiation have not been explored. Here, we show that FRCs in T cell zones of LNs express CD25 (IL-2 receptor α chain (IL-2Rα) of the IL-2R hetero-trimer). LN-FRCs trans-presented IL-2 to naïve CD4+ T cells through CD25, thereby facilitating early IL-2-mediated signaling. CD25-deficient LN-FRCs attenuated STAT5 phosphorylation of naïve CD4+ T cells during CD4+ T cell lineage differentiation, promoted T helper 17 (Th17) cell differentiation, and induced Th17 immune response-related gene expression. In an experimental autoimmune encephalomyelitis (EAE) disease model, Th17 cell-mediated EAE severity was elevated in mice lacking CD25 in LN-FRCs. Therefore, our results suggest that CD25 expression in LN-FRCs regulates CD4+ T cell differentiation by modulating early IL-2 signaling of neighboring naive CD4+ T cells, influencing immune responses.
Helios predisposes human fetal CD4+ naive T cells towards regulatory T cell differentiation
Authors: Ng Melissa , Roth Theodore , Mendoza Ventura , Marson Alexander , Burt Trevor
Published: January 2023

Activation of naïve CD4+ T cells by T cell receptor (TCR) stimulation and cytokine cues lead to differentiation into effector T cell populations with distinct pro-inflammatory or regulatory functions. Unlike adult naïve T cells, human fetal naïve CD4+ T cells uniquely differentiate into FOXP3+ regulatory T (Treg) cells upon TCR activation independent of exogenous cytokine signalling. This facility for Treg differentiation is crucial for generating tolerance in utero; however, the mechanisms underlying this ability in fetal naïve cells are largely unknown. Here, we reveal FOXP3-independent transcriptional and epigenetic programs shared between fetal naive T cells and committed adult Treg cells that are inactive in adult naive T cells. We show that a subset of adult Treg-specific super-enhancers is active within fetal naive T cells, including two super-enhancers at Helios, a thymic Treg gene. Helios is expressed in fetal naive T cells, but not in adult naïve T cells, and only fetal-derived induced Treg (iTreg) cells continue to express Helios. Fetal, but not adult iTreg cells, have suppressed IL-2 production, which is regulated by Helios in committed Treg cells. CRISPR-Cas9 ablation of Helios in fetal naive T cells then resulted in increased IL-2 production in fetal iTreg cells. Crucially, the loss of Helios expression in fetal naïve T cells impaired their differentiation into Treg cells upon TCR stimulation, indicating Helios as a critical contributor to the cell-intrinsic predisposition of fetal naive T cells for Treg cell differentiation. Treg-biased transcriptional and epigenetic programs within fetal naive T cells identified here could be utilized to engineer enhanced adult iTreg populations for adoptive cellular therapies.
DNA Methylation impairs monocyte function in tuberculosis leading to disease progression
Authors: Frantz Fabiani G , Castro Ricardo Cardoso , Fontanari Caroline , Bollela Valdes Roberto , Zambuzi Fabiana Albani
Published: January 2023

Despite treatment and cure, Tuberculosis (TB) is still considered a world health public problem. It is known that interaction between bacteria and host immune mechanisms results in changes in gene expression, which could be a result of epigenetic changes, as DNA methylation. Recent studies suggest that alterations in host epigenome could be promoted by Mtb infection and influence immune cells function. In this context, we aimed to determine the methylation profile of monocytes from tuberculosis patients and correlate with immune function of these cells. Monocytes were isolated from active TB patients (APTB) with treatment-time less than one month (n=10) and non-disease controls (CTRL) (n=16) - Ethics Committee approval FMRP-USP (Protocol #6481/2013). The monocytes were used to evaluate the global DNA methylation content and immune function, through cytokine and ROS production after heat killed Mtb challenge. When we evaluated the group of tuberculosis patients according to their lung injury degree, we found an increased methylation in those with more severe disease. Also, it was possible to observe that monocytes from patients with increased methylation profile, presented a reduction in secretion of anti-inflammatory cytokine, IL-10 and increased pro-inflammatory IL-12, indicating impairment in monocytes ability to regulate the excess of inflammation, which mediates the lung injury often observed in these patients. Hence, we suggested that global DNA methylation content might act as a clinic prognostic toll for active tuberculosis disease.
Macropinocytosis of amino acids regulates T cell growth by promoting the sustained activation of mTORC1
Authors: Charpentier John , Lapinski Philip E. , Turner Jackson , Grigorova Irina , Swanson Joel A. , King Philip D
Published: January 2023

Macropinocytosis, a non-selective form of bulk endocytosis, has been associated with diverse processes in mammalian cells, including antigen presentation and regulation of receptor density. In Ras-transformed tumor cells, macropinocytosis of protein regulates growth when amino acids are limiting in the extracellular space. A requirement of macropinocytosis for the growth of non-transformed cells has not been described previously. Here we show that primary mouse and human T cells engage in constitutive macropinocytosis that is enhanced approximately three-fold in response to CD3/CD28 antibody stimulation. We further show that macropinocytosis is essential for the growth of CD3/CD28 antibody-stimulated T cells during the G1 phase of the cell cycle, even under amino acid-replete conditions. Macropinocytosis in activated T cells provides a means of access of extracellular amino acids to an endolysosomal compartment necessary for sustained activation of the mechanistic target of rapamycin complex 1 (mTORC1), which in part explains its function in G1 growth. In addition, macropinocytosis promotes activated T cell growth by means other than activation of mTORC1, possibly by furnishing bulk substrates for anabolism and anaplerosis. Regulation of mammalian cell growth by macropinocytosis, therefore, is not limited to Ras-transformed tumor cells but may instead represent a more general means of promoting sustained mTORC1 activation in highly proliferative cells. These findings provide important new information upon the mechanisms by which T cells acquire nutrients for growth and suggest means by which T cell growth might be manipulated for therapeutic benefit.
A ‘sweet spot’ for complement receptor CD46 activity via direct regulation of glycolytic vs. moonlighting functions of GAPDH
Authors: Kunz Natalia , Le Friec Gaelle , Singh Parul , Lavender Paul , Kazemian Majid , Hess Christoph , Kemper Claudia
Published: January 2023

Autocrine engagement of the complement receptor CD46, driven by T cell receptor (TCR) activation on human CD4+ T cells, is critical for human Th1 induction: Processing of the intracellular CD46 domains by γ-secretase and their nuclear translocation mediates the expression of nutrient transporters GLUT1 and LAT1, increases glycolysis and oxidative phosphorylation, and enables metabolic reprogramming required for cell growth, expansion and effector function. How, at the molecular level, CYT-1 enables this fundamental cellular adaptation remains unknown. To probe for CYT-1 interacting proteins we generated antibodies against cleaved CYT-1 and performed co-immunoprecipitation experiments. Unexpectedly, mass spectrometry revealed association of CYT-1 with several glycolytic enzymes, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Although ELISA and MST-based experiments confirmed a strong, dose-dependent CYT-1/GAPDH interaction, glycolytic activity was hardly affected by CYT-1. Rather, CYT-1 modulated the activity of a key ‘moonlighting function’ of GAPDH, namely the posttranscriptional regulation of mRNA stability/translation: CYT-1 binding fostered desoligomerization of glycolytically active GAPDH tetramers to monomers with altered mRNA binding abilities – particularly for mRNAs encoding metabolic enzymes. These data identify CD46 as a ‘shape-shifter’ of GAPDH activity, critically balancing its canonical (glycolysis) versus non-canonical (posttranscriptional regulation) activity – further substantiating the notion that autocrine complement is a critical regulator of normal cell physiology.
Regulation of IL-10-producing T cells by polyunsaturated fatty acids
Authors: Escalante Jaileene Hernandez , Jones Albert , Sedletsky Victoria Volfson , Dooms Hans
Published: January 2023

The modern day western diet has led to an increase of n-6 polyunsaturated fatty acid (n-6 PUFA) intake and is considered an environmental factor resulting in negative health outcomes in different disease settings. In relation to the immune system, it is not well understood how changes in dietary factors like n-6 PUFAs can influence immune responses, for example during autoimmune disease. PUFAs have a variety of roles within cells such as being used as an energy source, incorporation in cell membranes, and conversion into lipid mediators that influence cell signaling and gene expression. We initially sought to understand how n-6 PUFAs influence the phenotype and function of T cell subsets from the Type 1 Diabetes mouse model (NOD) and non-diabetic control mouse model (B6.NOD). By treating splenocytes from these mouse models in vitro with n-6 PUFA linoleic acid we discovered by flow cytometry a dose response inhibition of interleukin 10 (IL-10), an anti-inflammatory cytokine, in both CD4+ and CD8+ T cell subsets. To the contrary, interferon γ production was not diminished after linoleic acid treatment. While IL-10 inhibition was not strain-specific, further studies by gene expression analysis displayed a decrease of IL-10 mRNA expression and of transcription factors involved in regulation of IL-10 (c-maf, IRF4, and Bhlhe40) after linoleic acid treatment. These results demonstrate that dietary n-6 PUFAs regulate T cell cytokine production and can influence the balance of pro- and anti-inflammatory cytokines. Further studies will provide more insight into the regulation of T cell subsets by fatty acids present in the organismal environment, with potential implications for the development of new therapeutics to modulate autoimmune responses.
Serum Amyloid P Component Bound to Fungal Functional Amyloid Is Anti-Phagocytic and Anti-inflammatory
Authors: Bradley Nicole Elizabeth , Lipke Peter N , Pilling Darrell , Gomer Richard H , Klotz Stephen A
Published: January 2023

Invasive fungal disease is often characterized by a sparse cellular inflammatory response, and we have proposed that deposition of serum amyloid P component (SAP, also called PTX2) on fungal surfaces dampens the innate immune response. Binding of SAP to fungi was dependent upon fungal expression of cell surface functional amyloid, with 92% of Candida albicans yeast cells binding SAP, whereas only 18% of yeasts of an amyloid-depleted strain of Saccharomyces cerevisiae bound SAP. Furthermore, SAP bound to 40% of S. cerevisiae cells expressing the amyloidogenic C. albicans adhesin Als5pWT, but only 19% of cells expressing a non-amyloidogenic single site mutant, Als5pV326N. Macrophages phagocytosed fungi in proportion to expression of surface amyloid: C. albicans > S. cerevisiae expressing Als5pWT >>>S. cerevisiae expressing Als5pV326N or S. cerevisiae. Macrophages incubated with C. albicans yeasts secreted significantly increased amounts of IFNγ, IL-6, TNFα and IL-10. Pre-treatment of macrophages with SAP or yeasts with SAP reduced C. albicans phagocytosis by more than 80% within 30 minutes, and led to reductions of IFNγ, IL-6, TNFα, but to an increase in the secretion of the anti-inflammatory cytokine IL-10. The structurally similar pentraxin, C-reactive protein, demonstrated negligible binding to Candida (7% on flow cytometry) and slightly increased macrophage phagocytosis of yeasts. Thus SAP, whether added exogenously to macrophages or coating fungi, rapidly dampened the host cellular immune response. These findings support the hypothesis that amyloid-dependent SAP binding diminishes cellular innate immunity in invasive candidiasis in humans.
Complement C3 increases cardiomyocyte resistance to apoptosis in response to hypoxia
Authors: Fang Zhou , Li Xiang , Xu Fou , Elvington Michelle , Kulkarni Hrishikesh , Atkinson John P. , Zhang Ming
Published: January 2023

Hypoxia takes places in pathogenic conditions, such as heart ischemia. Complements factors have been reported to play an important role in ischemia-reperfusion injury in various animal models. We studied the cellular response to complement under hypoxia condition using a human cardiomyocyte cell line AC16. Under hypoxia condition without serum (thus no exogenous complements provided), these cardiomyocytes underwent apoptosis. However, when pure human C3 was supplemented after hypoxia, these cells had less apoptosis and maintained a reasonable survival rate. The results indicate that complement C3 can increase the cardiomyocytes resistance to apoptosis under the hypoxia condition.
The Functional Role of CD169+ Macrophages in Septic Shock
Authors: Yeung Stephen T , Khanna Kamal Mohan
Published: January 2023

Sepsis affects more than 30 million people and accounts for approximately 6 million deaths worldwide each year. Sepsis is often associated with gram-positive, gram-negative, or fungal infections. Currently, there are no known cures for sepsis; however, current therapeutic strategies consist of supportive care to keep vital organs functional and antibiotics to combat infections, with survival prognosis contingent on early detection. Our current knowledge of the cellular and molecular mechanisms that regulate the inflammatory pathways resulting in sepsis is limited. Notably, we know little about the precise subpopulations of immune cells that play a central role during sepsis. Here we examined the specific role of CD169+ macrophages and its immunological receptors in septic shock. We utilized the CD169-DTR mouse to selectively and temporally deplete CD169+ macrophages. Using this model, we examined survival, cytokine/chemokine milieu, and cellularity in response to gram-negative LPS. We report the critical role of CD169+ macrophages and the CD169 receptor in protection against LPS induced septic shock. We show that in the absence of these macrophages, mice stimulated with sublethal LPS fail to survive and exhibit increased inflammatory cytokines and impaired IL-10 production. Protection against LPS induced lethality is rescued by supplemental treatment with recombinant IL-10. Lastly, we show that following LPS treatment, CD169+ macrophages are the initial targeted cell. These findings not only reveal a pivotal role for CD169+ macrophages but also the role for the CD169 receptor as a critical mediator of protection from sepsis induced lethality, and provide new therapeutic targets against sepsis.
Neutrophils are more effective than monocytes at containment and killing of Listeria monocytogenes
Authors: Okunnu Busola , Berg Rance E
Published: January 2023

Neutrophils and monocytes have previously been shown to be important for host protection during infection with Listeria monocytogenes (Lm). Previous studies have shown that simultaneous depletion of neutrophils and monocytes with the Gr-1 antibody leads to susceptibility to Lm infection. The purpose of these studies is to delineate differences in function between neutrophils and monocytes during Lm infection. Neutrophils obtained from the bone marrow, liver and spleen of C57BL/6 mice were more effective at phagocytosis of Lm in comparison to monocytes as they had a higher total bacteria MFI than the monocytes. To determine differences in the ability of the cells to keep bacteria contained in the phagosome, the cells were infected with a strain of Lm that only expresses GFP when the bacteria escapes out of the phagosome into the cytosol. Comparison of the MFI of total bacteria present vs escaped bacteria showed that monocytes allowed for more bacteria to escape in comparison to neutrophils. Therefore, monocytes are less effective at bacterial containment in comparison to neutrophils. To ascertain differences in killing ability, bone marrow neutrophils and monocytes were sorted for a killing assay and neutrophils were also more effective than monocytes at bacterial killing. In conclusion, although both cell types are important for protection during Lm infection, neutrophils appear to be essential for protection as they are more effective at phagocytosis, phagosomal containment and bacteria killing. Future studies, such as measurement of ROS/RNS as well as cytokine production, will aid in further defining specific functional differences between neutrophils and monocytes during intracellular bacterial infection.
Anaphylatoxin receptor C3aR on platelets participates in platelet function and links innate immunity with thrombosis
Authors: Sauter Reinhard Joerg , Sauter Manuela , Reis Edimara S. , Emschermann Frederic , Nording Henry , Kraft Peter , Mauler Maximilian , Muenzer Patrick , Rheinlaender Johannes , Edlich Frank , Schäffer Tilman E , Meuth Sven G , Duerschmied Daniel , Borst Oliver , Kleinschnitz Christoph , Lambris John D , Langer Harald
Published: January 2023

Background

Beyond the superficial function of blood coagulation, platelets play an important role in the genesis of vascular inflammatory processes. Interestingly, platelets express the complement receptor C3aR. Here, we examine the role of the complement receptor C3aR on platelets for platelet function and in diseases.

Methods

We detected the expression of C3aR on murine and human platelets using western blot and flow cytometry. We examined proteins, that are in close connection to the C3aR receptor on platelets by mass spectrometry to elucidate the intracellular signaling. We examined the role of the platelet C3aR for platelet function using different platelet function tests. To investigate the relevance of our findings in vivo, we made use of mice that are deficient for C3aR. After performing bleeding time and intravital microscopy experiments, we induced myocardial infarction and stroke in knockout animals.

Results

We found that C3aR on platelets plays an important role in platelet function and thrombus formation. We discovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis using C3aR−/− mice or C3−/− mice with re-injection of C3a. After induction of a myocardial infarction or stroke in mice, C3aR−/− mice showed a better outcome in comparison to WT mice. When analyzing the intracellular signaling, we discovered that those effects were PI3 kinase and Rap-1 dependent.

Conclusions

Taken together, we identified the C3a receptor on platelets as an important player in platelet function and thrombus formation. Linking the systems of innate immunology and hemostasis, the C3a receptor on platelets could be a possible therapeutic target in diseases featuring thromboinflammation.
Monocytes subsets and cytokine levels in chronic Chagas disease
Authors: González John M , Gómez-Olarte Sergio , Bolaños Natalia , Echeverry Mariana , Rodríguez Ayda , Cuellar Adriana , Puerta Concepción J. , Mariño Alejandro
Published: January 2023

Monocytes are classified according to CD14 and CD16 expression into classical, intermediate and non classical. Variations in monocytes subsets are involved in the pathogenesis of protozoan and helminthic infections. This study assessed the relationship of monocyte subsets percentages and the level of cytokines in blood from Colombian chagasic patients. Donors included asymptomatic or indeterminate (IND), symptomatic or chronic cardiac chagasic (CCC) and heart transplant (HTCC) patients; also, non-chagasic cardiopathy patients (NCC) and healthy individuals (HI) as controls. Peripheral blood mononuclear cells (PBMCs) were isolated. Fc receptors were blocked using anti-CD16/CD32 and human AB serum. Cells were labeled with antibodies for CD14, CD16 and HLA-DR, and acquired in a flow cytometer. Cytokines were measured in plasma with a Human M1/M2 Macrophage kit. Comparisons were done with Kruskal-Wallis followed by Dunn’s post hoc test and correlations were evaluated by Spearman’s coefficient. Percentages of CD14+ CD16+ and CD14+ HLA-DR+ monocytes were higher in patients with heart involvement (CCC, HTCCC, and NCC). Percentage of intermediate monocytes (inflammatory) increased in CCC patients and was positively correlated with IL-6 concentration and negatively correlated with IL-12p40 levels. IND donors had higher percentages of classical monocytes and an increased production of CCL17, and they positively correlate. The results suggest that variations in the monocyte subsets and cytokines levels might be associated with protection (classical monocytes and CCL17) or the development (intermediate monocytes and IL-6) of chagasic myocardiopathy
Vitamin D promotes anti-microbial activity of macrophages via Complement Receptor Immunoglobulin
Authors: Small Annabelle , Harvey Sarah , Kaur Jaspreet , Putty Trishni , Quach Alex , Munawara Usma , Hii Charles , Ferrante Antonio
Published: January 2023

Vitamin D deficiency remains of major global concern, affecting up to half of the population at some stage of life. Although the use of vitamin D to treat infections has been studied over several decades, the mechanisms involved still remain to be elucidated. There has been a major focus on the effects of vitamin D on innate immunity and macrophages, one of the few cell types that can synthesize active vitamin D. Here, we examined whether vitamin D exerts its anti-microbial effects through Complement Receptor Immunoglobulin (CRIg), a phagocytosis-promoting receptor unique to macrophages. Using a series of in vitro culture methods of human macrophages, we show that CRIg expression by macrophages is increased at the mRNA and protein level by the active derivative 1,25-dihydroxyvitamin D (1,25D), while having no effect on the expression of the classical complement receptors, CR3 (CD11b) and CR4 (CD11c). This upregulation of CRIg was associated with an enhanced anti-microbial capability by the cell against the pathogens Staphylococcus aureus and Candida albicans. Furthermore, using the synthetic triacylated lipopeptide Pam3CSK4, we show that macrophages require activation to convert vitamin D to the active 1,25D, which then in turn acts in an autocrine manner to upregulate expression of CRIg. These findings suggest that a major innate defense mechanism against both bacterial and fungal pathogens controlled by vitamin D is through the regulation of CRIg expression in macrophages.
Mitochondrial alterations in NK lymphocytes from ME/CFS patients
Authors: Silvestre Isabel Barao , Dagda Raul Y , Dagda Ruben K , Darley-Usmar Victor
Published: January 2023

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease characterized by profound fatigue, flu-like symptoms, trouble concentrating, and autonomic problems, all of which worsen after exertion. ME/CFS patients have impaired natural killer (NK) cell activity. NK lymphocytes are a critical first defense against viruses and cancer. ME/CFS patients have difficulties controlling viral infections and many develop non-Hodgkin’s lymphoma. Mitochondrial metabolism is crucial for immune cell function. Mitochondria dysfunction has been previously reported in ME/CFS, but it is not known whether the NK cells of these patients have altered mitochondrial metabolism that affect their activity and contribute to ME/CFS pathogenesis. More importantly, there is currently no efficient method to diagnose ME/CFS or assess efficacy of therapeutic interventions. The Bioenergetic Health Index (BHI) has been developed as promising and reliable surrogate readout of human health by measuring the bioenergetic status of immune cells. Variations in bioenergetic function in patient’s immune cells can reflect both metabolic stress and the mutable role of these cells in ME/CFS immunity and pathogenesis. In our study, we observed that the two main energy-generating mitochondrial pathways, oxidative phosphorylation and glycolysis (bioenergetics parameters), are deregulated in ME/CFS NK cells and in PBMCs. Moreover, we observed alterations in the morphology and membrane potential of the mitochondria of NK cells. These mitochondrial features can affect NK cell function and contribute to the severity of disease. To date, this is the first metabolism assessment of NK cells in ME/CFS and as potential new diagnostic tool for the disease.
Unique Innate Functions of Fetal Macrophage Populations
Authors: Prince Lawrence Stephen , Lakhdari Omar , Anderson Kathryn
Published: January 2023

In fetal and newborn tissues, macrophages play key roles in generating the innate immune response to pathogens. Distinct macrophage populations arise from hematopoietic precursors in the embryonic yolk sac and fetal liver. Here we tested the hypothesis that fetal macrophage populations possess unique innate immune function based on their cellular origin. Innate immune activation following TLR agonist exposure was tested using FACS sorted fetal macrophage populations or total single cell suspension from fetal lung or fetal liver. TLR agonists increased inflammatory cytokine expression in CD11bHI fetal liver macrophages. However F4/80HI yolk sac derived macrophages failed to respond to TLR activation and expressed very low levels of both Il1b and Il6. Similar data were obtained with ELISA measurement of secreted IL-1beta and IL-6 protein. When total liver cells were treated with TLR agonists, again IL-1beta expression was restricted to CD11bHI macrophages. F4/80HI macrophages from the yolk sac were also less likely to phagocytose labeled E. coli. Expression of multiple TLRs was low or undetectable in F4/80HI macrophages, suggesting a unique overall functional program for this distinct macrophage population. Expression profiling showed similar levels of multiple macrophage signature genes in CD11bHI and F4/80HI cells. However expression of C1q family members and Igf1 were much higher in F4/80HI macrophages. Expression of these genes was more consistent with an anti-inflammatory phenotype and removal of damaged cells during developmental morphogenesis. These important mechanistic differences suggest important cellular and molecular strategies for treating neonatal inflammatory disease.