bluuebunny/crossref_metadata_2025 huggingface dataset:
Low NK Cell ADCC as a Risk Factor in Chronic Fatigue Syndrome (CFS): Familial Risk for CFS or Differences between Human Populations?
Authors: Hudig Dorothy , Tang Jennifer , Sung Alexander P. , Guglielmo Michael J. , Smith-Gagen Julie , Bateman Lucinda , Redelman Douglas D.
Published: January 2023

ADCC (antibody-dependent cell-mediated cytotoxicity) requires the cytotoxic subset of NK cells that can also recognize antibody. In vivo function may depend on the number of these ADCC NK cells. We report on ADCC activity and the number of IgG FcR-positive NK cells/ul blood as risk factors for chronic fatigue syndrome (CFS). ADCC capacity was measured as CX1:1 (PMID 29113954), the % Daudi B tumor cells killed at a 1:1 ratio of CD16A FcR-pos CD56pos NK effector cells to Daudi targets with obinutuzumab anti-CD20. CFS patients met the 1994 Fukuda diagnostic guidelines. We examined 5 families with 2 or more patients per family, comparing patients with their non-CFS family members and with unrelated healthy controls. ADCC activity and effector cell counts were similar between CFS patients and their family members. Surprisingly, the CX1:1 of both the CFS patients and their non-CFS family members was low, 77% of the controls (P=0.03). The ADCC effector cell counts/ul blood were also lower for both (two-tailed P=0.09, insignificant). The potential for synergistic interaction of low CX1:1 and low effector cell counts was 24-fold greater for CFS family members than for unrelated healthy controls (P<0.05). The ADCC similarity between patients and family members indicates that lower ADCC is unlikely to influence who is affected with CFS within families. The combined lower ADCC and lower effector cell counts of CFS family members could indicate a familial risk for CFS. However, caution is warranted by evidence that the CFS families are from a Utah population that is genetically skewed, while the controls have diversity representative of the USA. The ADCC differences could be between 2 human populations or between people more or less likely to suffer from CFS.
Different bactericidal and inflammatory activities of human and mouse blood
Authors: Lin Tian , Moorlag Simone J.C.F.M. , Liu Jialin , Ahmed Mohamed Yehia Hassan , Thundivalappil Sujatha R. , Riley Frank E. , Warren H. Shaw
Published: January 2023

Mouse models often serve as a selective gate for drugs developed to treat patients with inflammation and sepsis. However, mice are much more resistant than humans to intravenous challenge with LPS, a cell wall component of Gram-negative bacteria. It is common to detect up to 106 CFU/ml of live bacteria in the blood in many mouse models of bacterial challenge but less than 100 CFU/ml of bacteria in the blood of human adults with Gram-negative sepsis. These substantial differences between mice and humans during infections are unexplained, unstudied and usually ignored.

We studied and compared the growth of four encapsulated, clinically relevant strains of Escherichia coli (O4:K54:H5, O16:K1:H6, O18:K1:H7, O25:K5:H1) and Pseudomonas aeruginosa 12.4.4 in fresh human and mouse blood at 37°C for 30, 90 and 180 minutes. We also measured TNF and IL-6 levels in the supernatants, and performed a phagocytosis assay by using fluorescein isothiocyanate-labeled heat-killed bacteria incubated with human and mouse blood followed by flow cytometry. We found that E. coli and P. aeruginosa multiplied in mouse blood and plasma, but that the colony counts of each were decreased in human blood and plasma. More E. coli and P. aeruginosa were phagocytosed by leukocytes in human than those in mouse blood, but this difference reflected larger numbers of neutrophils in human blood than in mouse blood. Large amounts of pro-inflammatory cytokines TNF and IL-6 were detected in human but not in mouse blood after live bacterial inoculation. In conclusion, our results suggest that bacterial killing, phagocytosis, and cytokine induction in the blood compartment of patients with Gram-negative bacteremia may not be well mimicked in mouse models of bacterial challenge.
Histone h4 as a trigger for innate immune cells.
Authors: Hsieh I-Ni , White Mitchell , De Luna Xavier , Hartshorn Kevan L.
Published: January 2023

Extracellular histones have been considered to be responsible for several systemic inflammatory injuries, including lung, kidney and liver. We now show that histone H4 can directly activate neutrophil respiratory burst response, depolarization, and myeloperoxidase (MPO) release. In addition, histone H4 increased neutrophil adhesion activity and the expression of adhesion molecule. More interestingly, we found that histone H4 also induced a sustained rise in the intracellular calcium level in neutrophils, and that H4 increased caspase 3 activity in neutrophils over time, which can be inhibited by anti-H4 antibody treatment. Histone H4 induced IL-8 release from neutrophils and is also a strong inducer of IL-1 beta and TNF-alpha productions in monocytes and monocyte-derived macrophages. To counteract the effects of histone H4, we found that C reactive protein (CRP) and/or surfactant protein D (SP-D) could reduce histone H4-induced respiratory burst response, calcium flux, MPO release and caspase 3 activity in neutrophils. These findings suggest that extracellular histone H4 may play important roles in the innate immunity, and some endogenous peptides may have the ability to reduce H4-induced effects. This study will hopefully provide new treating targets for treating inflammatory diseases and novel strategies based on modification of CRP or SP-D with optimal histone binding site for treatment of severe infections.
Real-time visualisation and quantification of Neutrophil Extracellular Traps
Authors: Lovell Gillian F. , Bevan Nicola , Dale Tim , Trezise D. J.
Published: January 2023

The ability of neutrophils to release extracellular traps (NETs) is one of several mechanisms by which the body defends against infection. When neutrophils encounter invading pathogens, the cells release a mixture of antimicrobial proteins and chromatin to trap and degrade microbes. NETs have been implicated in a number of disorders including atherosclerosis, systemic lupus erythematosus (SLE) and thrombosis.

NETosis can be stimulated in vitro using a number of methods including chemical compounds, microbes and microcrystals. In order to better understand the signaling pathways involved, we developed a fully kinetic live-cell imaging assay for NETosis (96-well format, IncuCyte S3). Using a fluorescent cell impermeant DNA-binding reagent (IncuCyte CytoTox Green), NET release was visualised and quantified in real time. In both primary human neutrophils and differentiated ‘neutrophil-like’ dHL60 cells, PMA (100nM) induced rapid (2h onset, peak 4–6h), time-dependent increase in fluorescence and nuclear degradation. A concomitant increase in myeloperoxidase, elastase (immunofluorescence) and cell-free DNA (Picogreen) was observed, validating the NETosis signal. PMA-induced NETosis was ROS-dependent (CellRox Red) but did not cause externalisation of phosphatidylserine (PS, Annexin V). In contrast, ionomycin (5mM) induced NETosis more rapidly (onset time <15′), did not induce ROS but did externalise PS. Neutrophils and dHL60 cells were both able to phagocytose bacterial bioparticles (IncuCyte pHrodo-E-coli). Taken together, these data illustrate how NETs and neutrophil signaling pathways can be robustly, meaningfully and efficiently analysed using automated live-cell imaging and compatible detection reagents.
Cardiac fibroblast pretreated by ANG II induced M1 phenotype macrophage selective apoptosis through TNF-TNFR1 and survival M1 conversion into M2 depended on Leptin-PI3K-Akt pathway
Authors: Su Zhaoliang , Lu Hongxiang , Shao Xiaoyi , Chen Rong , Tian Yu , Zong Gangjun , Xu Huaxi
Published: January 2023

Inflammatory monocytes and tissue-resident macrophage are key regulators of tissue injury, would healing, organ remodeling and regeneration. After tissue injury, most monocytes and macrophages infiltrate into injury sites, undergo phenotypic and functional changes: differentiation into proinflammatory M1 phenotype and anti-inflammatory M2 in milieu. The reprogramming M1 and M2 macrophages involve in inflammatory initiation, development, resolution, and organ function remodeling, respectively. Disturbances in macrophage reprogramming can lead to aberrant repair or uncontrolled inflammatory response. Timely modulation of the reprogramming of macrophages is essential for the treatment of inflammatory diseases. Therefore, it becomes fatal to clarify the M1 fate or the function shift of M1 and M2 during tissue injury development. In the present work, we demonstrated that cardiac fibroblasts (MCF) pretreated by angiotensin II (ANG II) (myofibroblast, MFB) could not only promote macrophage reprogramming toward M1 but also induced M1 macrophage selective apoptosis through TNF/TNFR1 axis independent on Fas/FasL; furthermore, in co-culturing system, leptin produced by MFB or M1/M2 cells could promote the survival M1 conversion into M2 macrophage via PI3K/Akt pathway. In vivo, TNF-alpha blockade significantly ameliorated experimental autoimmune myocarditis (EAM) development. Our data highlighted a novel mechanism about the fates of M1 macrophage in inflammatory diseases and our data also suggested a potential check point for inflammatory disease therapy target on macrophage.
Efficient Enrichment of Functional ILC Subsets from Human PBMC by Immunomagnetic Selection
Authors: Valdez Yanet , Kyei Stephen K. , Poon Grace F.T. , Kokaji Andy , Woodside Steven M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

Innate lymphoid cells (ILCs) are exceedingly rare but important regulators of homeostatic and disease-associated immune processes. The frequency of ILCs in peripheral blood of healthy humans is ~0.07% of CD45+ leukocytes. ILCs lack specific cell surface markers but can be divided into distinct subsets (ILC1, 2 and 3) based on their differential expression of effector cytokines and master transcription factors.

Currently, cell sorting is the most widely used method to isolate ILCs, but it is time consuming, expensive and often results in low purities and recoveries. Pre-enrichment of ILCs would allow for reduced sorting times and improved purities. Accordingly, we have developed a fast immunomagnetic negative selection method to pre-enrich all ILCs subsets from human leukapheresis samples. Briefly, unwanted cells are labelled with antibodies and magnetic particles and placed into an EasySep™ magnet. Unwanted cells are retained in the magnet and the enriched ILC fraction is simply poured off into a new tube. We find that total ILCs (defined as Lineage− CD45+ CD127+) are enriched from a frequency of 0.01 – 0.23% (n=28) to a final frequency of 17 – 86%, an enrichment of 200 – 1500 fold with virtually no loss of ILCs. ILC1 were enriched from 0.01 – 0.2% to 4.5 – 14%. ILC2 were enriched from 0.01 – 0.1% to 5.8 – 51% and ILC3 were enriched from 0.01 – 0.1% to 6 – 16%. This pre-enrichment drastically decreases sort time, allowing sorting over 3.7 × 105 ILCs from 2 × 109 PBMCs in only 12 minutes. Sorted cells maintained their functionality; when stimulated, ILC1s produced IFNγ, ILC2s secreted IL-13 and ILC3s produced IL-22. Our newly developed method of ILC pre-enrichment should aid human ILC research by enabling their rapid isolation when combined with cell sorting
Complement controls antiviral CD8+T cell memory development and function
Authors: Jhun Kevin S. , van der Heide Verena , Heeger Peter S. , Homann Dirk
Published: January 2023

Control of infectious disease is predicated on the combined efforts of both innate and adaptive immune defenses including the generation of pathogen-specific CD8+effector T cells (CD8+TE). Upon resolution of inflammation and pathogen clearance, the CD8+TE population contracts and the preferential survivors mature into CD8+TM, which provide further protection against recurrent infection. In this context, the precise properties of CD8+TM populations emerge as a consequence of the unique conditions during initial T cell priming and thus make the generation of T cell memory contingent upon a variety of co-stimulatory and -inhibitory interactions. Here, we demonstrate a non-redundant role for complement, an innate immune defense pathway, in the development of CD8+T cell memory generated in response to an acute lymphocytic choriomeningitis virus (LCMV) infection. Mice deficient for the complement component 3 (C3) mount a relatively normal LCMV-specific CD8+T cell response, yet the subsequent development of CD8+T cell memory is compromised by impaired phenotypic, metabolic and functional CD8+TM maturation. Interestingly, the contraction phase is also impaired which results in an increased abundance of CD8+TM. Despite this observation, C3-deficient CD8+TM exhibit a decreased capacity to undergo in vivo secondary expansion when adoptively transferred into naïve hosts for rechallenge. We propose that C3, but no other individual complement component evaluated (C3aR, C5aR, and DAF/CD55), exerts a unique co-stimulatory function that generates a balanced formation of CD8+T cell memory and recall responses.
Atrophied thymus produces altered repertoire of tTregs with potential to break the balance of peripheral tolerance
Authors: Thomas Rachel Ruth , Oh Jiyoung , Wang Weikan , Su Dongming
Published: January 2023

We have previously shown that in the age-related atrophied thymus, there is an increased ratio of thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells, suggesting that the aged thymus has an enhanced tTreg generation. It is also known that in the periphery of aged mice and humans, there is an accumulation of peripheral Tregs. We raise the following question: why is the increased Treg population unable to suppress self-reactivity in the elderly? We utilized a sub-lethally irradiated chimeric mouse model in which OT-II TCR transgenic bone-marrow cells were transplanted into RIP-mOVA host mice. Therefore, OVA serves as a mock self-antigen, and because this transgene is driven by the rat insulin promotor, the pancreas of these mice expresses OVA. Further, the mOVA mice also carry a FoxN1-floxed gene, which can be conditionally knocked-out via CreERT transgene activation by injection(s) with tamoxifen to induce thymic atrophy. We observed that chimera mice with induced thymic atrophy show total Tregs (termed pan-Tregs) were increased but OT-II specific Tregs were slightly decreased in the spleen. Additionally, there is a dramatic decrease in the OTII-specific Tregs found in the pancreas, and pancreatic atrophy was observed in these mice compared to those with normal thymus. Our initial results suggest that thymic atrophy leads to a biased tTreg generation that manifests in decreased selftissue specific-Tregs. Therefore, although pan-Tregs may accumulate and be enhanced with age, tissue-specific peripheral tolerance may be poised to fail.
Mechanisms of maintenance of protection from persistent malaria infection by CD4 effector and effector memory T cells
Authors: Stephens Robin , Ibitokou Samad , Opata Michael M.
Published: January 2023

Generation of CD4 effector memory T cells (Tem) is associated with persistent infections. However, two counterintuitive features we identified obscure any mechanism for the predominance of Tem in chronic infection: that Tem differentiation begins very early in infection, and that Tem do not require persistent infection to survive. Within the first five days of Plasmodium chabaudi infection, the relative proportions of MPEC to SLEC (CD127− CD62Llo) are determined, and predict the ratio of Tcm to Tem. Furthermore, Tem survive as well as Tcm, and better than SLEC, in uninfected recipients. In addition, we determined that Tem are derived from the same memory precursor effector T cells (MPEC, CD127− CD62Lhi) as central memory T cells (Tcm). We have now identified three mechanisms promoting Tem numbers in chronic infection: 1) Tcm can be programmed by chronic infection to produce Tem, even after infection is over low-level, 2) persistent infection promotes turnover of Tem, and 3) some SLEC can also survive to generate Tem. These findings support the feasibility of generating protective Tem by vaccination. Another caveat to generating protection to chronic infection by vaccination, is that protection from disease can be improved by ongoing infection, suggesting a role for SLEC. Therefore, we tested both SLEC and Tmem subsets for contributions to protection, and found that SLEC protect best. Also, decay of SLEC coincides with declining protection as chronic infection is cleared. Nevertheless, one subset of Tem (CD127hiCD62LloCD27−) reduces parasitemia and pathology, and maintains Ifng expression in chronic infection. These findings provide important insight into mechanisms of development and maintenance of immunity to chronic infection.
Infiltrating CCR2+ Monocytes Drive Lethal Immunopathology during Cryptococcal Meningoencephalitis
Authors: Xu Jintao , Neal Lori , Ivey Mike , Lopez Rafael , Segal Benjamin , Osterholzer John J. , Williamson Peter R. , Olszewski Michal A.
Published: January 2023

Cryptococcal meningoencephalitis (CM) is a major cause of CNS-related mortality worldwide. While immunosuppression can lead to cryptococcal infection, immune responses are recently also reported to promote CNS injury during CM. The role of monocytes in both anti-cryptococcal protection and the CNS pathology during CM remains unknown, but CCL2 level in CNS are linked to mortality risk in HIV-associated CM.

To study the role of CCR2-axis recruited monocytes and monocyte derived cells (MDC) we evaluated outcomes of CM in WT versus CCR2-deficient mice. Fungal burdens, behavioral scores, pathology, immune parameters and mouse survival were compared. WT mice developed symptomatic disease and neurological deterioration from 3 weeks with 100% mortality by wk 5, despite a reduction in fungal burdens from wk 3 to wk 5. CCL2 production and MDC accumulation peaked at week 3, corresponding to onsets of symptoms and mouse mortality. Infiltrating MDC showed classical activation phenotypes (high level of iNOS/CD80 and low level of Arg1/CD206). In contrast, CCR2-deficient mice displayed a marked reduction in MDC recruitment relative to WT mice after infection. T cell inflammatory responses were diminished and switched from a Th1 response to mixed Th1/Th2/Th17 responses in CCR2 deficient mice. Furthermore, MDC activation shifted from M1 towards M2-type. These results show that CCR2-axis recruited MDC contributed to exuberant and detrimental Th1 inflammation. Survival of CCR2-deficient mice significantly improved despite impaired fugal clearance relative to WT mice. Taken together, we conclude that CCR2+ monocyte/MDC, while important for fungal clearance in CNS, are also critical mediators of inflammatory damage in CM.
Novel epitope derived from Plasmodium berghei liver-stage antigen induces a subset of protective CD8 T cells against sporozoite challenge
Authors: Pichugin Alexander V. , Zarling Stasya , Perazzo Leah , Duffy Patrick E. , Ploegh Hidde L. , Krzych Urszula
Published: January 2023

CD8 T cells specific for Plasmodium liver stage antigens (LS Ags) are considered key effectors in protective immunity induced by immunization with radiation-attenuated sporozoites (RAS). Sporozoite (spz)-stage epitopes that induce protective CD8 T cells have previously been identified, however, fine specificities of LS Ags-induced protective CD8 T cells remain unknown. Using transcriptome data based on LS forms of Plasmodium falciparum parasites, we identified several orthologue Plasmodium berghei (Pb) LS Ags that, as plasmid DNA vaccines delivered by Gene Gun immunization, significantly reduce liver parasite burden (LPB). Using algorithms that predict binding of peptides to H-2b alleles, we designed ~600 8–9 mer peptides derived from protective Pb LS Ags and screened them utilizing caged MHC class I-tetramer technology. Spleen cells from C57Bl/6 mice immunized thrice with Pb RAS and challenged with infectious spz were tested for binding to the conditional tetramers. We identified an H-2Kb-restricted 9-mer peptide, Kb-17, derived from the novel protective Pb LS Ag, MIF4G-like protein, that formed MHC class I-tetramer specifically binding CD8 T cells; the Kb-17 peptide also recalled IFN-γ responses in splenocytes and liver mononuclear cells at different time points after RAS immunization/challenge. The Kb-17 epitope, delivered as a minigene expressed in adenovirus 5, expanded Kb-17 specific CD8 T cells expressing effector memory phenotype and significantly reduced Pb LPB after infectious spz challenge. Identification of protective MHC class I-restricted epitopes on LS Ags as well as establishing a role for LS Ags-specific CD8 T cells in protection are crucial elements for vaccine development against malaria.
Requirement of CXCL11 chemokine production for induction of protection against pulmonary cryptococcosis
Authors: Castro-Lopez Natalia , Campuzano Althea , Hole Camaron , Leopold Wager Chrissy M. , Wozniak Karen , Wormley Floyd L.
Published: January 2023

Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal pneumonia in immunocompromised and immunocompetent individuals. We previously have shown that mice given an experimental pulmonary infection with a genetically modified strain of H99 that produces murine IFN-γ, H99γ, are able to completely resolve the pulmonary infection via the upregulation of the IFN-γ/STAT1 signaling pathway. Activation of STAT1 signaling leads to the production of CXCL9, CXCL10 and CXCL11 chemokines. These chemokines each serve as ligands for the CXCR3 receptor that is present on host immune cells including T cells, NK cells, dendritic cells (DCs) and plasmacytoid DCs and facilitate their chemotaxis to the site of infection. C57BL/6 (B6) mice produce a non-functional CXCL11 protein. Our studies show that B6 mice infected with C. neoformans strain H99γ have a 40% survival, compared to 100% survival in BALB/c mice and an 80% survival of F1 generation mice (first generation of BALB/c × B6) at day 60-post inoculation. After 60 days post-inoculation F1 mice infected with H99γ had a significant decrease in fungal burden compared to B6 mice. We also demonstrated that B6 mice do not produce CXCL11, as expected, at day 7 and 14 post-inoculation whereas F1 mice show a significant increase in the production of CXCL11 compared to B6 mice. Lastly, treatment of B6 mice given a pulmonary C. neoformans infection with recombinant CXCL11 resulted in a significant decrease in pulmonary fungal burden compared to untreated B6 mice infected with C. neoformans. These data suggest that activation of the STAT1-mediated signaling pathway and CXCL11 chemokine production is necessary for optimal protection against pulmonary C. neoformans infection.
Pathologic inflammation and neuronal damage during murine neurocysticercosis associates with increased expression of IL-1β
Authors: Cardona Astrid E. , Vanegas Difernando , Church Kaira , Mishra Pramod , Mares Chris , Cardona Sandra M.
Published: January 2023

Neurocysticercosis (NCC) is the leading cause of acquired epilepsy worldwide caused by Taenia solium larvae. Disease progression involves an initial asymptomatic phase followed by a symptomatic period, in which dying organisms induce inflammation and symptoms such as seizures, intracranial hypertension, or cognitive decline. A mixed Th1/Th2 response develops in the brain, contrasting the canonical Th2 pathway associated with peripheral helminthic infections. We found that disease progression was more severe in BALB/c mice, correlating with increased parasite burdens and enhanced eosinophil and macrophage infiltration, although comparable numbers of T cells were detected. Moreover, elevated IL-1β levels were significantly elevated in BALB/c mice. Notably, Mrc1-deficient mice which exhibited a more resistant phenotype with decreased mortality and morbidity displayed low IL-1β expression. Therefore, we hypothesize that early up regulation of IL-1β in response to antigen recognition by macrophages plays a key role in damage to the nervous tissue. Comparison of glial and neuronal distribution in naïve and infected wild type C57BL6 and Balb/c mice, and also in strains with defective trafficking of eosinophils to the CNS, revealed that expression of IL-1β was detected early during infection and dramatically increased by 2 wks, colocalizing to macrophages and astrocytes. Notably, calbindin+ neurons were also significantly reduced in cerebellar regions where severe pockets of inflammatory cells are detected. Thus, our results suggest that IL-1β is a central proinflammatory mediator with neurotoxic potential during NCC infection.
IL-4 signaling is required for optimal Th1 responses during Leishmania major infection
Authors: Gurung Prajwal , Poudel Barun
Published: January 2023

Leishmania spp. is a global health problem that affects more than 2 million people every year with 300 million people at risk worldwide. It is well established that a dominant Th1 response (IFN-gamma, a hallmark Th1 cytokine) provides resistance, while a dominant Th2 response (IL-4, a hallmark Th2 cytokine) confers susceptibility during Leishmania spp. infections. Given the important role of IL-4 during Leishmania major infections, we used IL-4 neutralizing antibody to study and investigate the cellular and molecular events regulated by IL-4 signaling. As previously published, neutralization of IL-4 in Leishmania major-infected BALB/c mice (a Leishmania susceptible strain) provided protection when compared to control Leishmania major-infected BALB/c mice. Interestingly, despite the protection, IFN-gamma production by T cells was dramatically reduced. This demonstrates two important and previously unknown points-1) the protection against Leishmania major does not depend on the amount of IFN-gamma, but rather on the amount of IL-4, and 2) IL-4 is required for optimal production of IFN-gamma by T cells. In conclusion, our studies break the pre-existing dogma of resistance/susceptibility to Leishmania spp. infections and open new research grounds to investigate the unsuspecting role of IL-4 in controlling Th1 responses. A complete understanding of these cellular and molecular crosstalk will hopefully uncover a much-needed novel therapeutic target to treat Leishmania major infections.
Clustering of CD8 T cells around malaria-infected hepatocytes is rapid and is driven by antigen-specific T cells
Authors: Ganusov Vitaly V. , Kelemen Reka , Rajakaruna Harshana , Cockburn Ian
Published: January 2023

Malaria, a disease caused by parasites of the Plasmodium genus, begins when Plasmodium-infected mosquitoes inject malaria sporozoites while searching for blood. Sporozoites migrate from the skin via blood to the liver, infect hepatocytes, and form liver stages. In mice, vaccine-induced CD8 T cells are capable of locating and eliminating all liver stages in 48 hours, thus preventing the blood-stage disease. We recently reported formation of clusters consisting of variable numbers of activated CD8 T cells around Plasmodium yoelii (Py)-infected hepatocytes. Using a combination of experimental data and mathematical models we now provide additional insights into mechanisms of formation of these clusters. First, we show that a model in which cluster formation is driven exclusively by T-cell-extrinsic factors, such as variability in “attractiveness” of different Py-infected cells, cannot explain distribution of cluster sizes in different experimental conditions. In contrast, the model in which cluster formation is driven by the positive feedback loop (i.e., larger clusters attract more T cells) can accurately explain the available data. Second, while both Py-specific CD8 T cells and T cells of irrelevant specificity (non-specific T cells) are attracted to the clusters, we found no evidence that non-specific T cells play a role in cluster formation. Third and finally, mathematical modeling suggested that formation of clusters occurs rapidly, within few hours after adoptive transfer of T cells, thus illustrating high efficiency of T cells in locating their targets in complex peripheral organs such as the liver. Our analysis thus clarifies details of the formation of clusters of antigen-specific CD8 T cells in the liver.
Microbiota metabolites short-chain fatty acid butyrate conditions intestinal epithelial cells to promote development of Treg cells and T cell IL-10 production
Authors: Xu Leiqi , Ma Chunyan , Huang Xiangsheng , Yang Wenjing , Chen Liang , Bilotta Anthony James , Yao Suxia , Cong Yingzi
Published: January 2023

Gut microbiota greatly impacts host immune response and intestinal homeostasis. However, the mechanisms are still largely unclear. Short chain fatty acids (SCFAs), the most abundant metabolites in intestines derived from gut bacterial fermentation of dietary fibers, either existing free in gut lumen or being absorbed into circulation, perform multiple functions in maintenance of intestinal epithelial integrity and immune homeostasis, and have been shown to directly induce Treg cells and ameliorate the colitis. However, the low concentration of SCFAs in bloodstream makes them hard to directly act on T cells in physiological condition. In this study, we investigated whether SCFAs regulate T cell development through interaction with intestinal epithelial cells (IECs), which are in constantly contact with high concentration of luminal SCFAs. We report here that treatment with SCFA butyrate induced IEC production of TGF-β in both primary IECs and cell line MSIE. We then cultured MSIE cells with or without butyrate for 24 and 48 hrs to generate condition media (CM). When added to T cell cultures under different polarization conditions, butyrate treated CM significantly enhanced the expression of Foxp3 under Treg conditions, and IL-10 production in Th1, Th17, and Treg cells. Furthermore, by using GPR43−/− IECs and the HDAC inhibitor, we found butyrate promotion of TGF-β production was partially mediated by its activity of HDAC inhibition but not through GPR43. Our study thus demonstrated that SCFA butyrate regulates host T cell responses through interaction with IECs. The trialogues of microbiota metabolites SCFAs, IECs, and T cells could play a crucial role in intestinal homeostasis.
Exposure to microbial antigens during early life is required for the establishment of tolerance to commensal bacteria
Authors: Knoop Kathryn A. , Gustafsson Jenny K. , McDonald Keely G. , Hsieh Chyi-Song , Hogan Simon P. , Elson Charles O. , Tarr Phillip I. , Newberry Rodney D.
Published: January 2023

Current pediatric guidelines recommend breastfeeding and avoidance of oral antibiotics in the first years of life to reduce the risk of development of allergies. Additionally, animal models have shown exposure to the microbiota prior to weaning is necessary for the prevention of asthma or inflammatory diseases later in life. These studies suggest exposure to commensal microbes prior to weaning supports the development of tolerogenic responses in the intestinal tract. This interval of life coincides with the development of regulatory T cells in the colon which can suppress inflammatory responses. We have recently described a critical window for the development of tolerance in mice and found exposure to microbial antigens between 10 and 21 days of life occurred via the colon and induced long lived antigen specific FoxP3+ regulatory T cell responses. This window of tolerance is defined and regulated by ligands found in breastmilk and in the microbial milieu, and exposure to the microbial antigens required goblet cell associated antigen passages. Regulatory T cells developing during early life were required to restrain inflammatory responses against commensals later in life in a model of colitis. Additionally, exposure to commensal antigens via the colon after day of life 21 resulted in robust antigen-specific T effector responses and exacerbated inflammation in a model of colitis, exhibiting the critical nature of regulating this window of tolerance. Thus, exposure to microbial antigens early in life is an imperative element to the development of tolerance, and is highly regulated to prevent inflammation against the commensal microbiota.
IL-4 signaling regulates the IL-9 producing mast cells (MMC9) gene expression and function in food-induced anaphylaxis in mice
Authors: Tomar Sunil , Ganesan Varsha , Zeng Chang , Waggoner Lisa , Smith Andrew , Shik Dana , Wang Yui-Hsi , Hogan Simon
Published: January 2023

We have recently described an IL-9 producing mucosal mast cell population (MMC9s) that drives intestinal mastocytosis and food-induced anaphylaxis. The cytokine signaling pathways involved in the regulation of MMC9 function in food allergic reactions are currently not well understood. Employing a murine model of food-induced anaphylaxis and WT, gain-of-function Il4RaF709mice, and Il4Rα−/− mice we revealed an important role for IL-4 signaling in the enhancement of MMC9 frequency and this positively correlated with elevated serum levels of MCPT-1, CD4+TH2 cells, intestinal mastocytosis and increased susceptibility to food-induced anaphylaxis. Employing an adoptive transfer model of food allergy, we show direct IL-4 signaling is required for increased levels of MMC9 and development of food-induced anaphylaxis. RNA-seq analysis of purified WT, Il4RaF709and Il4Rα−/−MMC9s identified that IL-4-signaling pathway differentially regulates 238 genes that were identified to be associated with inflammatory responses and cytokine signaling processes by GO network analyses. Functional analysis revealed that IL-4 upregulates IL-9 and not IL-13 production in MMC9s. These results suggest that IL-4 signaling directly regulates MMC9 gene expression and function and onset of food-induced anaphylaxis. These studies suggest that targeting MMC9s through IL-4 signaling blockade maybe therapeutically beneficial for prevention and treatment of food allergy.
Dietary Early Glycation Products Protect Type 1 Diabetic Mice against Hyperglycemia through Altering Immune Homeostasis
Authors: Chen Yingjia , Guo Tai L.
Published: January 2023

Early glycation products (EGPs) are proteins modified with reducing sugar moieties. They are generated in the first step of Maillard reaction/glycation, and are precursors for the advanced glycation end-products (AGEs). This modification enhances the physiochemical properties of proteins (e.g. solubility), and has been proposed as a strategy to improve food protein qualities. However, the health effects of EGPs are rarely known. Our previous studies suggested that EGPs are anti-inflammatory. In contrast, AGEs are pro-inflammatory, and tightly associated with aging and hyperglycemia. It was hypothesized that EGPs were protective for type 1 diabetes (T1D) through modulating the immune homeostasis.

Food-borne EGPs were produced from a whey protein isolate-glucose model system. Six-week-old nondiabetic non-obese diabetic (NOD) mice were dosed with water, non-reacted samples (NR) and EGPs up for 8 wks. Body weight and blood glucose levels (BGLs) were measured weekly; diabetic incidence (BGLs > 200 mg/dL) was recorded; glucose tolerance test and insulin tolerance test were conducted monthly; sera were separated; and spleen and thymus were harvested for flow cytometry analysis of the immune cell populations. The results showed that EGP-treated NOD mice exhibited significantly lower BGLs, delayed diabetes onset, less responsive to glucose challenge, and more sensitive to insulin challenge than water and NR-treated mice. Moreover, the diabetic NOD females displayed lower or even normal BGLs after EGP treatment. The flow cytometric data showed that EGP-treated mice had a significantly higher CD4/CD8 ratio than NR-treated mice. In conclusion, EGPs protected T1D mice against hyperglycemia through altering immune balance.
Delineating tissue-specific alloimmunity during acute GVHD
Authors: Tkachev Victor , Furlan Scott Nicholas , Potter E. Lake , Zheng Betty H. , Hunt Daniel J. , Colonna Lucrezia , Taraseviciute Agne , Carlson Judith , Betz Kayla , Yu Alison , Hoffman Michelle , Herrin Sarah , Olvera Joe , Littlewood Chad , Blazar Bruce R. , Roederer Mario , Kean Leslie S.
Published: January 2023

After allogeneic hematopoietic cell transplantation (allo-HCT), donor T cells undergo priming in secondary lymphoid tissues followed by migration to different target organs, where they mediate inflammation and cause acute Graft-versus-Host Disease (aGVHD). Here, we characterized T cells infiltrating GVHD-target organs using a model of aGVHD in non-human primates in order to delineate tissue-specific patterns of the alloimmune response. We found that aGVHD profoundly shifted the T cell phenotype from the naïve state toward an effector-memory state in both the blood and secondary lymphoid organs, with an attenuated shift in non-lymphoid organs. However, in all compartments, tissue-infiltrating T cells demonstrated an aGVHD-specific activated phenotype characterized by a high rate of proliferation and elevated effector functions. In addition, transcriptomic profiles of aGVHD- and tissue-residency developed in T cells residing the colon, liver and lungs as well as in the blood. To further delineate the patterns of T cell trafficking and activation during aGVHD, we directly labeled leukocytes in vivo using fluorescent anti-CD45 antibodies. Using this approach, we detected notable changes in T cell trafficking patterns which included increased T cell trafficking to lymph nodes, lungs and kidneys after allo-HCT, with rates of T cell migration to the GI tract unchanged even during aGVHD. These data provides novel insights into the spatial organization of systemic alloimmunity during aGVHD and the organ-specific impact of this disease on T cell trafficking, activation and functional maturation.
Bendamustine Conditioning Induces Phenotypic and Functional Changes in Host Antigen-Presenting Cells Which May Confer Protection Against GvHD
Authors: Molina Megan Stanley , Stokes Jessica , Hoffman Emely , Eremija Jelena , Zeng Yi , Katsanis Emmanuel
Published: January 2023

Background

In an MHC-mismatched mouse bone marrow transplantation (BMT) model, we have found that substituting the pre-conditioning chemotherapeutic cyclophosphamide (CY) with an alternative alkylating agent, bendamustine (Ben), can significantly reduce graft-versus-host disease (GvHD) while maintaining engraftment. The mechanisms by which Ben achieves this effect are not understood, but evidence suggests that the early cellular interactions that occur in the first week after BMT can be responsible for GvHD severity later on.

Objective

We hypothesized that Ben may be affecting the function and/or phenotype of host antigen presenting cells (APCs) in the early post-BMT period (before complete myeloablation is achieved).

Design/Method

We applied a murine myeloablative MHC-mismatched model (C57BL/6 > BALB/c) to study the effect of Ben versus CY pre-conditioning in conjunction with total body irradiation (TBI) on host APCs. Specifically, we evaluated the phenotype and activation state of splenic host CD11c+ dendritic cells (DCs) on days 1, 3, and 5 following BMT.

Results

We found a significant increase in number and proportion of CD8a+ DCs in Ben versus CY. We also found that the Ben CD8a+ DC subset express differing levels of activation markers versus CY CD8a+ DCs. Furthermore, functional assays revealed that Ben CD11c+ DCs induce less T cell proliferation than either CY or naïve CD11c+ DCs, and that Ben CD8a+ DCs are more suppressive than either CY or naïve CD8a+ DCs.

Conclusion

These results suggest that pre-transplant Ben engenders phenotypic and functional changes in host CD11c+CD8a+ splenic DCs, inducing a suppressive phenotype, signifying a possible mechanism by which Ben induces protection against GvHD.
NOD2 acts as a master regulator of regeneration pathways in the thymus
Authors: Kinsella Sinéad , Cooper Kirsten , deRoos Paul , Iovino Lorenzo , Jain Reema , Dudakov Jarrod A.
Published: January 2023

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following stress, infection and cytotoxic cancer treatments. Although there is continuous thymic involution and regeneration in response to stress and infection in the healthy state, prolonged T cell deficiency is common after profound thymic damage, such as that caused by the conditioning required for hematopoietic stem cell transplant. Our previous studies have revealed two pathways important for thymic repair, centered around the production of IL-22 and BMP4 by innate lymphoid cells and endothelial cells (ECs), respectively. However, the specific mechanisms that trigger these pathways are poorly understood. Here we reveal an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in governing multiple pathways of thymic regeneration. Mice deficient in NOD2 showed increased intrathymic levels of L-22, IL-23 and BMP4 and increased thymus cellularity after damage caused by total body irradiation (TBI). Although NOD2 is classically defined as a bacterial sensor, members of the Rho GTPase family have been implicated to promote NOD signaling, even in the absence of pattern recognition. Consistent with this, inhibition of RhoA or Rac1 can induce the production of BMP4 by ECs in vitro, and after TBI there is a significant reduction in these two Rho GTPases. These studies not only enhance our understanding of endogenous tissue regeneration, but in identifying a master regulator of multiple pathways, offers a superior therapeutic strategy to boost thymic function in patients whose immune system has been decimated due to age, infection or cancer therapies.
Immunosuppression reverse engineered from cancer: Reducing nicotinamide adenine dinucleotide (NAD) impairs effector T cell function through glycolysis
Authors: Beier Ulf H. , Quinn William J. , Jiao Jing , TeSlaa Tara , Dahiya Satinder , Wang Zhonglin , Levine Matthew H. , Hancock Wayne W. , Rabinowitz Joshua D. , Baur Joseph A.
Published: January 2023

Understanding the mechanisms by which cancers evade immune responses may lead to new immunosuppressive therapies. The metabolic environment surrounding solid tumors is often enriched in lactic acid and glucose depleted, which impairs effector T cells (Teff). We observed that sodium L- and D-lactate inhibit T cell proliferation in vivo and increase Foxp3+ formation independent of acidity, while sodium pyruvate achieved the opposite. Inhibiting lactate dehydrogenase (GSK 2837808A) negated the effects of L-lactate on Teff and regulatory T cells (Treg). These findings suggested a role of NAD in controlling Teff function and Foxp3 stability. NAD is a co-factor for GAPDH (glycolysis), poly (ADP-ribose) polymerase-1 and sirtuin-1 (Foxp3 stability). We impaired NAD oxidation through oligomycin, rotenone, antimycin, or blocking NAD recycling (FK866). We used mitochondrial uncoupling, β-lapachone, and α-ketobutyrate to augment NAD oxidation, or added nicotinamide riboside to restore NAD after FK866 treatment. Reducing NAD led to decreased glycolytic flux and accumulation of pre-GAPDH glycolytic metabolites, which was reversed by increasing oxidized NAD. T cell IFN-γ production and Foxp3 turnover were increased by NAD oxidation, and impaired by NAD reduction. Blocking NAD recycling also impaired Teff proliferation and IFN-γ production, which was reversed by restoring NAD. Reducing NAD in vivo with sodium D-lactate (9 μmol/g/d x 5 d) or phenformin (70 mg/kg/d x 14 d) prolonged MHC-mismatched BALB/c to C57BL/6 cardiac allograft survival. We conclude that NAD controls T cell function through glycolysis and Foxp3 stability, which may be exploited for therapeutic immunosuppression.
Tannic acid-encapsulated islets delay immune rejection following transplantation
Authors: Barra Jessie M. , Kozlovskaya Veronika , Kharlampieva Eugenia , Tse Hubert Ming
Published: January 2023

Islet transplantation can restore β-cell function in patients with Type 1 diabetes (T1D), however, limited islet availability, toxicity of immunosuppressants, and poor graft survival are major hurdles for clinical application. We hypothesize that islet encapsulation with a nanothin multilayer coating consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), will provide an immunoprotective barrier and maintain β-cell function after transplantation. (PVPON/TA)-encapsulated syngeneic NOD.Rag islets restored euglycemia after transplantation under the kidney capsule of streptozotocin-treated diabetic NOD mice by more than 30 days in comparison to non-encapsulated islets. Allogeneic encapsulated B6 islets also restored euglycemia in diabetic NOD recipients and delayed rejection by 7.5 days in contrast to non-encapsulated islets. Gene expression of allogeneic B6 islets at 5 days post-transplant displayed an increase in Ins2 (6-fold), Gcg (6-fold), Pdx1 (5-fold), Ccl22 (3-fold), and Arg1 (2.5-fold) mRNA accumulation with (PVPON/TA)-encapsulated islet grafts, suggesting a potential shift toward an alternatively-activated M2 macrophage phenotype. To address limited islet availability, the use of (PVPON/TA)-encapsulated differentiated neonatal porcine islets restored euglycemia and maintained β-cell function for more than 200 days after transplantation into diabetic NOD. scid mice, displaying a potential intervention to delay xenotransplant rejection. Our results support the hypothesis that (PVPON/TA) coatings are immunoprotective and may promote islet graft acceptance following transplantation.
Obesity results in marked increases in gut permeability, inflammatory cytokines, and acute gut graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.
Authors: Khuat Lam T. , Pai Chien-Chun Steven , Le Catherine T. , Dunai Cordelia , Chen Mingyi , Raybould Helen E. , Abedi Mehrdad , Murphy William J.
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the most applicable curative option for treating hematologic diseases. In this study, we assessed the impact of obesity on recipient graft-versus-host disease (GVHD) outcome post allogeneic HSCT using a minor histocompatibility antigens (miHA)-mismatched mouse model. Control or diet-induced obese (DIO) BALB/c mice received HSCT from H2-identical B10.D2 mice which is known for resulting in chronic skin GVHD. Strikingly, DIO mice rapidly succumbed to gut GVHD while control mice survived and developed sclerodermatous GVHD much later post-HSCT. The DIO recipient mice had higher expression of pro-inflammatory cytokines (IL-6 and TNF-α) in the gut and serum, as well as higher serum ST2. There were increased numbers of donor CD4+ T cells in the mesenteric lymph nodes of DIO mice compared to control mice. Untreated DIO mice also displayed increased gut permeability and lipopolysaccharide translocation, as well as having less-diverse microbiota which potentially contributed to the severe GVHD post-HSCT. Importantly, administration of anti-IL-6R monoclonal antibody and Enbrel significantly protected DIO mice from lethal gut GVHD, correlating with lower pro-inflammatory cytokine concentrations (IL-6 and TNF-α) and ST2 in the serum. In an initial evaluation, treating DIO mice with broad-spectrum antibiotics 2 weeks before HSCT also ameliorated GVHD. Taken together, these results indicate that obesity exacerbates GVHD, manifested primarily as gut pathology, and therapeutic intervention by blockade of pro-inflammatory cytokine signaling ameliorates GVHD post-HSCT.