bluuebunny/crossref_metadata_2025 huggingface dataset:
Compartmentalized antigen presentation and inflammasome activation between epithelial cells and macrophages orchestrates gut immunity
Authors: Malik Ankit , Sharma Deepika , McGrath Shaina , Aguirre-Gamboa Raúl , Jabri Bana
Published: August 2023

All nucleated cells express MHCI and IFN-gR. It is suggested that structural cells express MHCI to prevent NK-cell mediated cytotoxicity and allow deletion by CD8+ T cells upon intracellular perturbations, but a cell-specific function of antigen presentation via MHCI has not been explored. We show here that upon sensing IFN-g, colonic epithelial cells productively present pathogen and self-derived antigens to the cognate intra-epithelial T cells, which are critically located at the epithelial barrier. Antigen presentation by the epithelial cells confers ATPase expression in the cognate intra-epithelial T cells which limits the accumulation of extracellular ATP and consequent activation of the NLRP3 inflammasome in tissue macrophages. In contrast, antigen presentation by the tissue macrophages alongside inflammasome-associated IL-1a and IL-1b production promotes a pathogenic transformation of CD4+ T cells into GM-CSF-producing T cells in vivo, which underlies colitis and colorectal cancer. Supporting the relevance of these findings in humans, we show that the IBD-associated IFN-gR SNP leads to defective IFN-g signaling. Furthermore, decreased expression of IFN-gR or ATPase and increased expression of GM-CSF are associated with IBD that is resistant to the standard therapies, and reduced survival with colorectal cancer. Taken together, our study has unraveled a critical checkpoint requiring IFN-g sensing and antigen presentation by epithelial cells that control the development of pathogenic CD4+ T cell responses in vivo.

Supported by the US National Institutes of Health (DK067180) to B.J, the University of Chicago’s Center for Interdisciplinary Study of Inflammatory Intestinal Disorders (C-IID) Pilot & Feasibility Award (NIDDK P30 DK042086) to A.M. and D.S, Crohn’s and Colitis Foundation Career Development Award #964209 to A.M. and G.I. Research Foundation Associates Board Award to A.M. and D.S.
Epigenetic mechanisms of epithelial – immune interactions that shape immune surveillance in the intestinal epithelium
Authors: Garipcan Aybuke , Eskiocak Onur , Subhash Santhilal , Ozler Kadir , Yueh Brian , Chung Charlie , Ergin Ilgin , Gautier Nelson , Habel Jill , Rubino Rachel , Van der Velden Adrianus W M , Beyaz Semir
Published: August 2023

Immune surveillance of the intestinal epithelium is regulated by the reciprocal interactions between intestinal epithelial cells, immune system and microbiome. This highly specialized compartment maintains the tolerance against dietary products and commensal bacteria while protecting the epithelium against pathogens, cancer risk and tissue damage. While recent studies identified epigenetic mechanisms that orchestrate the self-renewal and differentiation of intestinal epithelial cells (IECs), little is known about the mechanisms that govern epithelial – immune interactions and how they influence immune surveillance and tissue regeneration. Here we dissect epithelial-intrinsic epigenetic features including transcription factors and co-activators that govern immune surveillance of intestinal epithelium using genetically engineered mouse models and autologous human patient-derived organoid – immune co-cultures. Ablation of epigenetic regulators that associate with immune interaction gene modules specifically in IECs dampens intraepithelial lymphocyte-mediated immune surveillance. We identified precise epigenetic mechanisms of epithelial – immune interactions including promoter and enhancer regulation by transcription factors and co-activators both in mice and humans. Finally, disruption of the epigenetic regulators that maintain epithelial – immune interactions lead to impaired responses in clinically-relevant mouse models of infection, tissue regeneration and cancer. These results establish a strong foundation towards understanding the epigenetic mechanisms that govern multi-cellular interactions within the barrier tissues in physiology or disease states.

Supported by grants from Mathers Foundation, STARR Cancer Consortium (I13-0052), The Mark Foundation for Cancer Research (20-028-EDV), NIH (P30CA045508-33), CZI Ancestry Network for the Human Cell Atlas
Lipid sensing by gut CD1d calibrates peripheral serotonin release
Authors: Wu Chuan , Luo Jialie , Chen Zuojia , Castellano David , Han Wenyan , Lu Wei , Li Jian , Kim Girak
Published: August 2023

The crosstalk between the immune and the neuroendocrine system is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones release in response to environmental cues, such as self and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promotes peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. CD1d ligation on EC cells transduces a reverse signal and restrains potassium conductance, leading to calcium influx and 5-HT secretion. Together, we show that by coupling with iNKT cells, gut chemosensory cells perceive lipid antigens via CD1d to calibrate 5-HT release, modulating intestinal and systemic physiology.
Leaky gut mechanisms in giardiasis
Authors: Angelova Vanessa V , Li Erqiu , Fiedler Brooke , Wexelblatt Haley , Kosile Rita Taye , Darmadi Matthew , Miskovsky Eleanor , Singer Steven M
Published: August 2023

Giardia duodenalis is a common intestinal pathogen transmitted via consumption of contaminated water. In addition to causing intestinal distress symptoms, Giardia infection is one of the top 5 infectious causes of growth stunting in children under two. Growth faltering is a complex pathology facilitated by changes in the intestinal barrier and enzymatic deficiencies. While infection is widely associated with barrier damage in vitro, in animal models, and in patient studies, the mechanisms behind this process are not well understood. Since PAR2 has a prominent role in increased intestinal permeability in Celiac disease, we are focusing on the involvement of the protease activated receptor 2 (PAR2) in giardiasis-facilitated intestinal defects. We are using human intestinal cell lines, murine intestinal organoids, and a mouse infection model to address the role of PAR2. Co-culture of Giardia and Caco2 cells results in altered tight junction morphology as revealed by staining for ZO-1; these changes are reduced by treatment with a PAR2 antagonist. Effects of PAR2 signaling on barrier function are being assessed by measuring transepithelial electrical resistance (TEER) in Caco2 cells and PAR2+/− and PAR2−/− organoids cultured on transwell filters. Additionally, while C57BL/6 mice fed a low-protein diet exhibit reduced weight gain when infected with Giardia, mice lacking PAR2 gain weight normally when infected. These data indicate that PAR2 signaling contributes to intestinal permeability defects and growth faltering. Identifying parasite factors that activate PAR2 will provide novel targets for development of pharmaceuticals and vaccines to improve child growth.

AI166467
Reduced γδ intraepithelial lymphocyte number and motility precede the onset of Crohn’s disease-like ileitis
Authors: Golovchenko Natasha B , Xu Weili , Fong Andrew , Zhang Lanjing , Edelblum Karen
Published: August 2023

Crohn’s disease (CD) is a relapsing, remitting disease resulting in chronic intestinal inflammation. Recent studies of resected ileal tissue from CD patients reported a loss of intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs). γδ IELs continuously survey the epithelial barrier and protect against experimental colitis; however, the role of γδ IELs in the initiation of ileitis is unknown. To this end, we profiled the IEL compartment in TNFΔARE/+ mice, which develop chronic CD-like ileitis at 8 wks old, prior to and during ileitis onset (4–10 wks). We observed a 2.5-fold reduction in the frequency of ileal γδ IELs in 5-wk-old TNFΔARE/+ mice compared to TNF+/+ (WT) littermates (p<0.001). Using intravital microscopy, we observed reduced γδ IEL surveillance behavior (10.6 vs 6.4 mm/min track speed mean, p<0.0001) in TNFΔARE/+ mice compared to WT at 6 wks. Analysis of cleared ileal tissue from 6-wk-old WT and TNFΔARE/+ mice revealed a reduction in the number of γδ IELs in villus tips in TNFΔARE/+ mice relative to WT, thereby increasing the proportion of γδ IELs found in the crypt region. Based on several reported links between γδ IELs and Paneth cells (PC), we quantified PCs by lysozyme immunostaining in ileal tissue. We found an increased proportion of morphologically abnormal PCs in 6-wk-old TNFΔARE/+ mice, reflecting PC dysfunction consistent with CD pathology. These findings are supported by a reduction in ileal Lyz1 expression in TNFΔARE/+ mice compared to WT prior to ileitis onset (p=0.02). Together, these studies demonstrate that γδ IELs exhibit impaired surveillance behavior and altered distribution along the crypt/villus axis prior to ileitis onset, which coincides with PC dysfunction in CD-like ileitis.

COCR23PRF017 NJCCR Predoctoral Fellowship

Golovchenko (PI)

June 1, 2022 – May 31, 2024

γδ IEL surveillance behavior in the onset of chronic ileitis
Maternal immune regulation of lactogenesis
Authors: Corral Dan , Chi Liang , Gine Eduard , Yong Daniel , Nie Jia , Sun Lilian , Bhandoola Avinash , Bosselut Remy , Jabri Bana , Belkaid Yasmine
Published: August 2023

Breastfeeding has long been associated with health benefits for the infant. Breast milk is composed of essential nutrients and bioactive molecules shaping the growth, and the immune development of the offspring. An optimal development of the mammary gland (MG) during pregnancy is critical to maintaining the beneficial effects of lactation. Nevertheless, how the lactation process is regulated by the immune system remains largely unexplored. Our data reveal that the commitment of mammary epithelial cells into milk-secreting cells is associated with the accumulation of T-bet+ lymphoid cells, preferentially localized at the proximity and inside the mammary epithelium. Among mammary T-bet+ cells, we characterized an unconventional T cell population exhibiting common features with gut CD8αα+ intra-epithelial lymphocytes and deriving from the same thymic progenitor. Such process is associated with thymus involution during pregnancy and specific enrichment in thymic precursor that preferentially migrates into the MG during pregnancy. These cells promote milk production and expression of β-casein, one of the main milk protein, resulting in an increase in newborn weight. This work uncovers a previously unappreciated contribution for the adaptive immune system in mammary gland development and lactation. Decoding the immune regulation of the MG will allow to understand the profound transformation of this compartment as a primary nutritional source for the offspring and may allow to understand how dysregulation of this process could affect the long-term development of newborns.

This work was supported in part by intramural funds of NIAID, NIH.
Butyrate silences MUC5AC expression in airway epithelial cells by mediating histone deacetylation at its transcription start site
Authors: Jo Sang Hee , Bae Ikhyeon , Sim Myeong Seong , Kim Hye Jeong , Kim Chun , Chung Il Yup
Published: August 2023

Secreted airway mucins form a mucous gel consisting of MUC5AC and MUC5B and maintain airway homeostasis for appropriate mucociliary clearance and airway defense. There is conceivably a global mechanism to counteract excessive mucin production in response to incessant exposure to external and internal stimuli, which otherwise leads to goblet metaplasia and mucus hyperproduction. Here, we report that butyrate, a short-chain fatty acid (SCFA), strongly suppresses MUC5AC production at as low as sub-to micromolar concentrations in NCI-H292, airway epithelial cells. Butyrate transcriptionally inhibits MUC5AC mRNA and protein that are induced by a variety of stimuli including EGF and LPS. In line, butyrate upregulates FOXA2, which is known to block goblet cell metaplasia. The mode of the inhibition by butyrate appears to be independent of the known receptors of butyrate. In addition, butyrate does not alter the EGFR signaling pathway. Chromatin immunoprecipitation analysis demonstrates that histone H3K27 acetylation level is increased upon stimulation with EGF and returns to baseline in the presence of butyrate. We propose that butyrate, a metabolic product of gut microbiota, plays a central role in maintaining homeostasis of airway mucous gel by counteracting mucin production resulting from repeated provocation of airway epithelium.

National Research Foundation of Korea (NRF) funded by the Korean government (MSIT), NRF-2022R1A2B5B01002127.
TL1A control of eczema and psoriasis
Authors: Cheng Jason Liang , Steele Hope , Breckenridge Joey , Dell Garrison , Ghastine Andrew , Willicut Ashley , Herro Rana
Published: August 2023

Psoriasis is a Th17-induced dermatitis, characterized by epidermal thickening. Psoriasis increases the risk of developing arthritis and cardiovascular-related morbidities, hence the need to stop its progression early. Why the disease is patchy, and what activates the keratinocytes in the psoriatic plaques driving a Th17-type inflammation, remain unknown. Chronic exposure to imiquimod (IMQ) drives psoriasis in mice reminiscent of human disease. We have preliminary data implicating the TNFSF (tumor necrosis factor superfamily) member TL1A (tumor necrosis factor-like cytokine 1A) in psoriasis. Our loss of function studies show that the genetic deletion of TL1A receptor, protects from IMQ-induced psoriasis. Further, TL1A is known to be enriched in the psoriatic lesions and serum of psoriatic patients. These findings suggest that TL1A may play a central role in the development and maintenance of psoriasis. Therefore, targeting TL1A could be beneficial to treat Psoriasis.
Dedicator of cytokinesis 8 (DOCK8) is a negative regulator of skin mast cell function
Authors: Molina Irina Miralda , Samanas Nyssa B , Seo Albert J , Foronda Jake , Deutsch Gail H , Baris Safa , Unal Ekrem , Eken Ahmet , James Richard G , Piliponsky Adrian M
Published: August 2023

Dysregulated expansion and/or activation of mast cells have detrimental consequences in allergic disease. In humans, homozygous or compound heterozygous deletions of DOCK8 cause a combined immunodeficiency characterized by allergic disorders. Based on this evidence, we hypothesized that DOCK8 may be a negative regulator of mast cell function. To address this hypothesis, we used mice with a loss-of-function mutation in Dock8 (primuris, Dock8pri/primice) and conditional mice in which DOCK8 is ablated in connective tissue mast cells (Mcpt5-Cre+; Dock8fl/fl mice). Dock8pri/primice exhibited increased plasma levels of mast cell protease-1 (MCPT1) in homeostatic conditions. Furthermore, Dock8pri/primice and Mcpt5-Cre+; Dock8fl/fl mice experienced a more severe IgE-dependent passive cutaneous anaphylactic reaction (PCA) than control mice. This data suggests that DOCK8 deficiency is linked to increased mast cell activation in homeostatic conditions and upon stimulation by IgE-dependent mechanisms. Fetal skin derived cultured mast cells (FSMCs) generated from Dock8pri/primice released increased amounts of β-hexosaminidase upon IgE-dependent activation. Consistent with this finding, naïve DOCK8-deficient FSMCs exhibited alterations in cytoskeletal dynamics that may facilitate mast cell degranulation.

Finally, we observed an increased number of tryptase positive cells in skin biopsies obtained from three pediatric patients diagnosed with hyper IgE syndrome (HIES) due to DOCK8 mutations, suggesting that DOCK8 immunodeficiency may contribute to skin mast cell expansion in humans.

Our findings provide strong evidence that DOCK8 can contribute to negative regulation of skin mast cell activation and expansion.

R01 AI140626-05
Cell-Intrinsic Effects of TGF-b Signaling in Mast Cell Effector Function that Modulate Allergic Inflammation
Authors: Haque Tamara , Leak Leshawna , Laky Karen , Guererrio Pamela
Published: August 2023

IgE mediated mast cell activation is a key feature of allergic disease, although the mechanisms that govern mast cell homeostasis are not fully understood. The TGFb signaling pathway has been shown to regulate mast cell effector function. Furthermore, variants in the TGFb signaling pathway are associated with allergic diseases; TGFb mediated mast cell regulation is likely involved in the allergy diathesis. Patients with Loeys Dietz Syndrome (LDS), a disorder caused by loss of function variants in TGFBR1 and TGFBR2, are predisposed to develop allergic diseases, thus provide an opportunity to study the role of mast cell TGFb signaling in allergic diseases. Mast cells are activated through the high affinity IgE receptor FcERI causing the release of granules containing mediators that are directly involved in anaphylaxis and other allergic symptoms. IgE mediated activation can be modulated by other co-stimulatory signals such as the type 2 alarmin IL-33. We examined murine mast cells carrying an LDS mutation, or with conditional deletion of Tgfbr1, and found that they degranulated less in response to IgE/antigen, in vivo and in vitro. This phenotype was not tied to changes in the IgE and SCF receptor expression or mast cell tissue distribution in mice and was recapitulated in human LDS mast cells, in vitro. Additionally, LDS mice responded more to IL-33 stimulation. Mechanistically, the LDS anaphylaxis phenotype was linked to IL-33, as the reduction of anaphylaxis in LDS mice was partially restored in IL-33RKO LDS mice. Thus, the TGFb-IL-33R axis likely plays a major role in controlling IgE mediated mast cell functions. Taken together, TGFb signaling upregulates mast cell effector function by disrupting an IL-33/ST2 mediated regulatory pathway.

Intramural NIAID Support
The induction of histamine synthesis from rat bone marrow derived mast cells with rat stem cell factor
Authors: Cho Sung-Weon , Sur Bong Jung , Shin Kyung Sook
Published: August 2023

Mast cells are the main cells to produce histamine regarded as the main factor to induce allergy. To comprehend the allergy by mast cell activation, The mast cells of bone marrow derived mast cells (BMMC) are usually prepared by culturing bone marrow cells including rodent hematopoietic stem cells, in the presence of both IL-3 and stem cell factor (SCF). However, BMMC was reported not to produce the high levels histamine comparable to mature mast cells in host tissue. In this study, rat BMMC (rBMMC) were cultured in 96 well plate to induce diverse conditions in cell numbers per unit culture media, rat IL-3 and rat SCF to find the conditions to synthesize high level histamine. Cells of rat bone marrow cells were cultured in the cell density of 1.1×105/ml (low density, lowD) and 5.5 × 105/ml (high D), rat IL-3 was added to the levels of 10 ng/ml (low 3) or 50 ng/ml (high 3), Rat SCF was adjusted to the levels of 12.5 (lows), 25 (midS) and 50 (highS) ng/ml. Histamine included in 5×104 BMMC were extracted by perchloric acid, n-butanol, and heptane. The levels of histamine were measured by ophthalialdehyde solution and read for fluorometric intensity. The levels at culture day 4 (D 4) were in the range of 12~45 ng. The significant elevation was observed at D 11 in the conditions of lowD high3 (153 ng), however, D 8 in highD high3 (253 ng). The average histamine level was 74 ng in the condition of low Dlow 3, but 112 ng in low Dhigh 3 at D 11. That was 106 ng in high Dlow 3 but 183 ng in high Dhigh 3 at culture D 8. These results indicated that rat IL-3 increased the level of histamine synthesis and shorten the histamine production from undifferentiated hematopoietic stem cells.

The induction of histamine synthesis from rat bone marrow derived mast cells with rat stem cell factor
Anti-inflammatory and anti-allergic activity of Jacareubin derivatives
Authors: Castillo-Arellano Jorge Ivan , Hernandez Rosario Tavera , Camacho Antonio Nieto , Campos Omar Noel Medina , Lagunes Rodolfo Perez , Oyola Juan Francisco Leon , Chaverri José Pedraza , Gonzalez-Espinosa Claudia , Chilpa Ricardo Reyes , Estrada Manuel Jimenez
Published: August 2023

Xanthone Jacareubin (JAC) isolated from heartwood of Calophyllum brasiliense has shown good anti-inflammatory against spike protein of SARS-CoV2 and anti-allergic activity. Methoxylated (Met) and acetylated (Ac) derivatives of JAC and its precursor xanthone V (X-V) were analyzed in this work to identify the structural functional groups that confer their biological activity.

Met- and Ac-derivatives of JAC and X-V were synthesized and identified by 1H and 13C-NMR and MS. All compounds were evaluated for β-hexosaminidase degranulation (β-hex) of Bone Marrow Mast Cells. Both xanthones and Met-JAC derivative (3metJac) were evaluated on their inhibitory activity of xanthine oxidase (XO) and inhibition of passive cutaneous anaphylaxis (PCA). Also, both xanthones, 3metJac, and 4AcX-V were evaluated for inhibitory activity of Myeloperoxidase (MPO) and TPA-induced edema in mice.

JAC showed better inhibition of β-hex than X-V, while Met-derivatives showed greater inhibitory activity on β-hex than JAC and X-V, but Ac-derivatives showed reduced inhibitory activity. Inhibition of XO and MPO of both xanthones showed the highest activity at 1 μmol/ear compared to Met- and Ac-derivatives evaluated. Also, both xanthones showed higher anti-inflammatory activity in the TPA-induced edema compared to the Met- and Ac-derivatives. While in the PCA, JAC and 3metJac showed similar inhibition and twice the inhibition of X-V and their derivatives.

These results suggest that the fourth ring in the JAC is an important structural group for anti-allergic activity because X-V does not possess the fourth cyclized ring and has a fourth hydroxyl substituent group, showing poor inhibition of the allergic response. Supported by Conacyt Grant CF-2019-51488.

Supported by Conacyt Grant CF-2019-51488
Evaluation of salt-soluble protein extract from an ancient wheat progenitor (Aegilops tauschii) for intrinsic allergenicity in an adjuvant-free mouse model of wheat allergy
Authors: Gangur Venugopal , Gao Haoran , Jorgensen Rick Dean , Raghunath Rajsri , Chandra Shivam , Othman Aqilah , Olson Eric , Ng Perry K W
Published: August 2023

Wheat allergies are a growing significant health concern around the globe. The ancient diploid wheat progenitor, Aegilops tauschii (genome DD), contributed to the genetic diversity of the common soft white wheat (Triticum aestivum, genomes AABBDD). The allergenic potential of Ae. tauschii is largely unknown. Here we tested the hypothesis that the the salt-soluble protein extract (SSPE) from Ae. tauschii will be intrinsically allergenic in an adjuvant-free mouse model of wheat allergy. The ancient wheat progenitor was grown at the Michigan State University greenhouse using the seeds stored at our wheat breeding program. The SSPE was prepared, characterized for quality, and then tested in the mouse model that uses skin sensitization followed by oral allergen challenge. Balb/c mice were produced and maintained on a plant protein-free diet. Groups of adult mice (n=10/group) were repeatedly exposed to SSPE or vehicle via the skin. Allergic sensitization was assessed by specific IgE (sIgE) response. Oral anaphylaxis was quantified by hypothermic shock response (HSR). The mucosal mast cell response (MMCR) was quantified by measuring MMCP-1 in the blood. Repeated skin exposure to SSPE but not the vehicle elicited robust sIgE response. Oral SSPE challenge but not vehicle challenge elicited significant, but a modest HSR as well as MMCR. In summary, we report the characterization of intrinsic allergenicity potential of the SSPE obtained from the Ae. tauschii wheat progenitor for the first time. Funding: USDA/NIFA.

The United States Department of Agriculture (USDA)/National Institute of Food and Agriculture (NIFA); Hatch project MICL02486 (Accession Number: 1012322); Hatch project MICL01699; Agricultural and Food Research Initiative Competitive Program, grant number: 2018-67017-27876
Age and sex influence cultured rodent mast cell and basophil responses to antigen
Authors: Kulka Marianna , Wagner Ashley
Published: August 2023

Mast cells initiate allergic inflammatory immune responses and play a role in disease by releasing various inflammatory and immunomodulatory mediators. Several mast cell-lines and primary cultured cells have been used as mast cell models with inconsistent results among research groups. Bone marrow-derived mast cells (BMMC) cultured from mouse bone marrow progenitor cells are often used as a representative model of mucosal mast cell behaviour, however their reported phenotype is variable due to inconsistent culture protocols. RBL-2H3 is a rat basophilic histamine-releasing cell line that has some characteristics of both mast cells and basophils but is not a true representation of either cell type. The murine mast cell line MC/9 is an IL-3-dependent mucosal mast cell model but has limited mast cell characteristics. In this study, we have compared the response of BMMC (derived from C57BL/6 male or female mice), two sources of RBL-2H3 (purchased directly from ATCC and a lab curated culture), and MC/9 (ATCC) at several critical stages to some common stimuli (IgE/Ag, A23187) and analyzed mast cell morphology, expression level of common mast cell surface markers (CD117 and FcɛRI), protease expression, and function (growth kinetics, viability, ROS production, degranulation, cytokine release and FcɛRI signaling). The objective of this study was to provide insight into the effects of culture conditions, biological sex, and age of the cells on variability among reported phenotypes and, to determine optimal conditions for activation of these cells. Factors that are often overlooked such as source, age and biological sex of mast cells play an integral role in phenotypic outcomes and may account for the reported variability in their function.

National Research Council Canada
The role of Monocyte Chemoattractant Protein-Induced Protein-1 in the regulation of the immune responses to mycobacterial infection
Authors: Ramos Octavio , Wang Qinghong , Wei Jiawei , Eickhoff Christopher , Ning Huan , Hoft Daniel , Liu Jianguo
Published: August 2023

Mycobacterium tuberculosis (Mtb) infection is mainly controlled by protective cell-mediated immunity. TNFα and other mediators, such as nitric oxide (NO), are crucial to the macrophage’s antimicrobial activity. The RNA-binding protein MCPIP1 is a zinc-finger protein with RNase activity that targets the 3’UTR region of cytokine’s mRNA for degradation like IL-6, IL-1β, and IL-12. Here, we aim to investigate the role of MCPIP1 during infection and its contribution to Mtb’s immune evasion. Our observations show that MCPIP1 increases after Mtb infection, correlating with disease progression. Similarly, global MCPIP1 KO (MCPIP1−/−) mice showed lower lung bacterial burden than WT mice after 30 days. To address wheater the reduced number of bacteria was due to the macrophage’s absent MCPIP1 expression, we generated myeloid-specific MCPIP1 KO mice (M-MCPIP1−/−). M-MCPIP1−/−mice showed a similar reduction of bacterial growth as the MCPIP1−/−mice compared with their WT littermates. Infection of bone marrow-derived macrophages confirmed that myeloid-MCPIP1 deficient cells had improved control of bacterial growth with increased IL-1β, IL-6, IL-12, iNOS, and TNFα. To evidence the mechanism by which M-MCPIP1−/− macrophages contribute to Mtb growth control, we blocked TNFα and iNOS activity. Results showed that protection in M-MCPIP1−/− is partly due to these molecule’s high expression, as the blockade of these mediators increased the bacterial burden. Our preliminary data suggest that the induction of MCPIP1 contributes to the bacteria’s immune evasion by targeting critical anti-TB molecules. Further experiments are needed to understand the mechanisms involved and consider MCPIP1 as a target for host-directed therapy for TB.

R21AI151936 R21AI171734
Airway goblet cells resulting from conditional YAP/TAZ deletion promote distinct pulmonary inflammation
Authors: Cheng Nan , Hicks-Berthet Julia , Varelas Xaralabos
Published: August 2023

The aberrant presence of goblet cells, which are epithelial cells of the conducting airways that have specialized roles in secreting mucus, is frequently associated with distinct innate and adaptive immune changes that together are hallmarks of various pulmonary diseases. The transcriptional regulators YAP and TAZ, key effectors of Hippo pathway signaling, play a central role in suppressing the differentiation of airway goblet cells. Here we investigated the consequences of goblet cell production, resulting from the YAP/TAZ conditional depletion in Scgb1a1+ secretory cells, on lung immunity. We observed that loss of YAP/TAZ resulted in the production of goblet cells promoting local CD45+ immune cell expansion. To gain insight into immune changes associated with the production of YAP/TAZ-deleted goblet cells, we performed a single-cell RNA sequencing experiment on CD45+ cells isolated from control and Yap/Taz-cNull mouse lungs. Data analyses, along with follow-up validation experiments, demonstrated significant immune changes in Yap/Taz-cNull mice including an expansion of type 2 innate lymphoid cells (ILC2s), CD4+ T cells Th2 polarization, and alternative activation of alveolar macrophages, accompanied with elevated type 2 cytokine production. Gene expression analysis of YAP/TAZ-deleted airway cells revealed altered expression of notable immune-modulating factors, many of which are implicated in lung diseases. Collectively our study demonstrates that key immune-modulating signals arise from the secretory lung epithelium through reduced YAP/TAZ activity, raising interesting questions about the functions of YAP/TAZ in lung homeostasis and about the contributions of goblet cells to shaping lung immune responses.
System biology or pattern recognition of the immune response for offering immunotherapeutic cures for human diseases
Authors: Manjili Masoud H , Koelsch Nicholas
Published: August 2023

Our brain interprets observations by computations in the form of imaginations and creating patterns based on distinct networks of interactions. Nevertheless, collective patterns of the immune response as a system are hidden from our brain’s spontaneous computations, and should be discovered through research. Nevertheless, highly reductionist approaches hinder the discovery of immunological patterns because of getting lost in the details and seeking for a cause-effect direction. Such reductionist approaches have not been fruitful in offering a cure beyond alleviating the symptoms for immune-related diseases. Recent advances in system biology using big data, deep sequencing at single cell levels, multi-omics and artificial intelligence offer unique opportunities for pattern discovery research in immunology. Here, we provide a new interpretation for the existing data by suggesting that distinct internetwork interactions of the immune cells without any cause-effect direction create distinct immunological patterns manifesting collective functions independent from their cellular constituents. Such immunological patterns emerge from distinct proportions of the immune cell types transforming one another for producing collective functions. Based on empirical data, we propose that discovery of immunological patterns in immune-related diseases could offer better understanding of the immune response as a system as well as novel immune modulatory therapeutics for human diseases.

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense.
The therapeutic role of fibronectin in the re-epithelialization response following wounding in a human skin/mouse xenografting model
Authors: Zhang Wenwu , Akhtar Nahid , Zhao Jennifer , Spandau Dan , Kaplan Mark H
Published: August 2023

The atopic environment diminishes wound re-epithelialization and the defect in the wound healing process is IL-4 dependent. IL-4 represses fibronectin secretion from keratinocytes and impairs the ability of keratinocytes in culture to migrate into a scratch and to close a wound in vivo. Addition of fibronectin to culture increased closure of the scratch assay in vitro and re-epithelialization of wound tissue in vivo when fibronectin was applied topically. In this study, a human skin/mouse xenografting model was used to test the ability of IL-4 to impair re-epithelialization in human skin and to test whether fibronectin can mitigate the effects of IL-4. Human skin was grafted onto immunodeficient SCID/NOD mice. Approximately 10–12 weeks post-transplantation, human skin was fully integrated onto the mouse. A shallow punch biopsy was performed on the human skin to generate a wound that did not penetrate into mouse tissue. Human IL-4 was intravenously injected twice a week before the punch and recombinant fibronectin was spread over the wound twice daily after the wounding. Anatomic wound healing was monitored, and EDC gene expression and skin histology were assessed. Our results showed that mice injected with human IL-4 had delayed wound healing, decreased human EDC gene expression and increased MMP19 and CCL26 expression in human skin tissue. Treatment of skin with fibronectin attenuated the effect of IL-4 on human gene expression and promoted wound healing and re-epithelialization. These results suggest that fibronectin has a potential therapeutic role in the treatment of atopic lesions.

Supported by grants from NIH (R01AI095282)
ADSC-derived exosomes inhibit cbl-cathepsin K-dependent osteoclast activation in inflammatory arthritis
Authors: Liao Hsiu-Jung , Shen Yi-Shan , Lu Ssu-Jung , Jiang Ni-En , Shen Chia-Ning , Chang Chih-Hung
Published: August 2023

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes joint inflammation and bone destruction due to increased osteoclast differentiation and activity. Adipose-derived stem cells (ADSCs) are used to treat tissue injury because they are easy to harvest and exhibit immunomodulatory and regenerative capacity. Previous studies demonstrated that ADSC-derived exosomes (ADSC-Exo) protect articular cartilage from damage and exert anti-inflammation effects. Thus, we investigated the underlying mechanisms of ADSC-Exo in regulating osteoclast activation in inflammatory arthritis. RANKL is a TNF-family cytokine that binds to the RANK required for mature osteoclasts. However, whether ADSC-Exo is functionally involved in the inhibition of osteoclast formation via regulating RANK-mediated signaling in these pathological conditions is unclear.

Our data revealed that ADSC-Exo ameliorated the RA severity and bone loss in vivo and inhibited osteoclast differentiation, RANK downstream signaling, NFATc1, cathepsin K, and cbl-b. Moreover, ADSC-Exo could significantly suppress the population of RANKL-expressing TH17 cells. In brief, ADSC-Exo protects articular cartilage and bone destruction from damage and ameliorates gait abnormality in CIA rats, which may be related to the inhibition of the RANK-signaling pathway in osteoclasts. As it is relatively feasible to obtain human adipose tissue, ADSC-Exo may be a potential therapy for RA.

This study could comprehensively understand the immunomodulatory effects of ADSC-Exo on suppressing osteoclastogenesis. This finding is critical for understanding the bone homeostasis modulated by MSC-Exo and provides biological evidence of ADSC-Exo in inflammatory arthritis.

Supported by grants from the Ministry of Science and Technology (111-2321-B-418-002, 111-2314-B-418-004), FEMH (2022-C-042 and 2022-C-123), NYCU-FEMH Cooperation grants (111DN12, 111DN22), NTUH-FEMH Cooperation (111-FTN0012).
Investigating HIV-1 TAT protein role in Atherogenesis
Authors: Kirk Cassandra Lee , Moriarty Alina , Stahr Natalie Ann , Keeter W Coles , Ma Shelby , Kim Woong-Ki , Guo Ming-Lei , Galkina Elena
Published: August 2023

With the recent development of antiretroviral therapy, HIV patients can live a long and normal lifespan. Recent evidence demonstrates an increased incidence of developing cardiovascular diseases (CVD) specifically atherosclerosis in aged HIV-infected patients. The adaptive and innate immune responses play a complex specific role in atherosclerotic plaque burden. However, specific mechanisms of the implication of the immune system in HIV-accelerated atherosclerosis are unclear. The protein Trans-Activator of Transcription–TAT is strongly associated with HIV infection as it enhances the efficiency of viral transcription. To understand the contributions of TAT protein in HIV-associated atherogenesis, a doxycycline-inducible astrocyte-specific HIV-1 TAT transgenic mouse was used. An injection of recombinant adeno-associated virus vector (AAV) encoding a gain-of-function mutant PCSK9 into mice and feeding with high fat with added cholesterol (DDC) and doxycycline (Dox) was used to promote atherosclerosis and TAT expression. After 16 wks of feeding, various tissues were collected and used for flow cytometry analysis (FACS) for immune profiling. Our preliminary data suggest that TAT is involved in the regulation of peripheral blood neutrophil numbers and therefore can serve as a regulator of inflammation in HIV-associated atherosclerosis. Future analysis will provide insight into potential mechanisms of HIV TAT-dependent involvement in atherosclerosis development and progression.

This work was supported by project –initiation funds from the Center for Integrative Neuroscience and Inflammatory Diseases at EVMS and NIH NHLBI R01HL142129-04S1 grant.
RORγt-Raftlin1 complex regulates the pathogenicity of Th17 cells and intestinal inflammation
Authors: Singh Amir Kumar , Kumar Ritesh , Brooks John F , Conlon Kevin P , Basrur Venkatesha , Chen Zhe , Han Xialin , Hooper Lora , Burstein Ezra , K Venuprasad
Published: August 2023

Th17 cells that produce IL-17 are pathogenic in many human diseases, including inflammatory bowel disease (IBD), but are, paradoxically, essential for maintaining the integrity of the intestinal barrier in a non-inflammatory state. However, the intracellular mechanisms that regulate distinct transcriptional profiles and functional diversity of Th17 cells remain unclear. We have found that Raftlin1, a lipid raft protein, is specifically upregulated in and forms a complex with RORγt via its conserved LLNSL motif in pathogenic Th17 cells. Disruption of the RORγt-Raftlin1 complex in Raftlin1ΔLLNSL knock-in mice resulted in attenuated pathogenic Th17 cells elicited by Citrobacter rodentium; however, there was no effect on nonpathogenic Th17 cells induced by commensal segmented filamentous bacteria (SFB). Mechanistically, we found that Raftlin1 recruits distinct phospholipids to RORγt and promotes the pathogenicity of Th17 cells. Thus, we have identified a fundamental mechanism that drives the pathogenic function of Th17 cells, which could provide a platform for new therapeutic strategies to dampen Th17-mediated inflammatory diseases.

This work was supported by funds from the National Institutes of Health (R01-DK115668, R01-AI155786) and Cancer Prevention Research Institute of Texas (RP190527) and a stimulus grant from the Harold C. Simmons Comprehensive Cancer Center, UT Southwestern Medical Center.
Repercussions of high extracellular K+ on T-cell antitumor responses
Authors: Wong Brandon Han Siang , Ong Seow Theng , Goay Stephanie Shee Min , Ho Ying Swan , Chen Shuwen , Bi Xuezhi , Webster Richard D , Shelat Vishalkumar G , Verma Navin Kumar
Published: August 2023

Tumours create an immunosuppressive microenvironment that limits the antitumor function of infiltrating T-cells. Dying/necrotic tumor cells extrude intracellular potassium (K+) into the local extracellular milieu, raising the extracellular K+ ([K+]e) by 8–15 fold, which then accumulates in tumor-infiltrating T-cells. Here, we determined the functional impact of high-[K+]e (50 mM) on human T-cells. Using molecular assays, we demonstrated that prolonged exposure to high-[K+]e reduced CD3ɛ and CD25 (intermediate activation marker) surface expression, suggesting weakened T-cell activation. High-[K+]e perturbed T-cell receptor signalling and the downstream glycolysis pathway. Using metabolomics analysis, we identified dysregulation in pathways involving fatty acid oxidation, TCA cycle and choline metabolism under high-[K+]e. Exposure to high-[K+]e impeded T-cell motility and skewed T-cell polarization towards T-helper 2 (Th2) and regulatory T-cell (Treg) subsets. A similar Th2 subset trend, although not significant, was seen towards the core of patient-derived tumor biopsies (n=7) along with the amassment of Treg cells intratumorally. Collectively, our findings substantiate the immunosuppressive effects of high-[K+]e on T-cell functions, which underlines the potential use of K+ efflux channel activators in reversing dampened T-cell antitumor responses.

Supported by Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 1 Grant (2020-T1-001-062) and Tier 2 (MOE2017-T2-2-004) grants. #LINK# #ENDLINK#
Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3
Authors: Bauman Bradly M , Frank Andrew , Sukumar Gauthaman , Saucier Nermina , Dalgard Clifton L , Chan Alice Y , Cooper Megan A , Snow Andrew L
Published: August 2023

Although prior research implicated c-Jun N-terminal kinase (JNK) in helper CD4+ T cell differentiation, mechanisms governing JNK signaling and function in human lymphocytes remain nebulous. CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor (AgR) engagement to transcriptional programs necessary for effector cell survival, proliferation, and differentiation. In addition to NF-κB, CARD11 is also required for AgR-induced mTORC1 and JNK2 signaling. We sought to define the impact of dominant interfering (DI) CARD11 variants on JNK signaling and elucidate transcriptomic changes governed by CARD11-dependent JNK activation in T cells. We transfected WT and CARD11 knockout (KO) Jurkat T cells with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling and performed RNA sequencing on mitogen-activated WT and CARD11 KO Jurkat T cells ± JNK inhibition. Our results revealed that AgR-dependent JNK1 and JNK2 activation are CARD11-dependent. Additionally, heterozygous DI CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, mirroring the extent of impaired NF-κB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4+ T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. Our novel findings suggest that defects in CARD11-dependent JNK signaling in CD4+ T cells may contribute to severe allergic disease noted in CADINS patients, unveiling a new potential therapeutic target.

Supported by funding from the Jeffrey Modell Specific Defect Research Program (ALS) and the Henry M. Jackson Foundation for the Advancement of Military Medicine (BMB).
Kinetic regulation of TCR activation as a potential mechanism for CD28 co-stimulation
Authors: Raychaudhuri Kumarkrishna , Rangu Rohita , Ma Alison , Samelson Lawrence E
Published: August 2023

T cell receptor (TCR) engagement initiates a chain of proximal TCR signaling events including phosphorylation of TCRζ, recruitment of upstream kinases, followed by recruitment of signaling and adapter proteins. These events occur within seconds of TCR engagement and are important determinants of the T cell response. Yet, we lack a precise understanding of the spatiotemporal relationships between these molecular events due to the limitations of conventional light microscopy. We used stimulatory antibodies and ligands against the TCR to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Formation of these signaling subunits was then studied using TIRF and TIRF-SIM to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events and subsequent effects on the immunoresponse.

Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in the MCs, as revealed by a decreased kinetic lag between detection of TCRζ and the recruitment of ZAP70. We also observed that CD28 co-stimulation markedly changed Lck dynamics in T cells, resulting in Lck enrichment within MCs and a concomitant increase in co-localization of Lck with ZAP70 and TCRζ. CD28 co-stimulation led to spatial segregation between activated and inhibited species of Lck, raising the possibility of formation of localized Lck activation sites within MCs. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs, thereby decreasing the energy barrier for a rate-limiting kinetic step in TCR proximal signaling.
Establishing novel and unique technology to uncover how Activation-Induced Deaminase specifically targets immunoglobulin regions
Authors: Ruiz Stormy Elizabeth , Heltzel Justin M H , Maul Robert W , Gearhart Patricia J
Published: August 2023

Activation-induced deaminase (AID) is the critical enzyme required for somatic hypermutation (SHM) and class switch recombination (CSR). Upon exposure to antigen, B cells become activated and express AID, initiating the process of affinity maturation. While we understand how AID is targeted to the switch region to initiate CSR, relatively little is known about why AID is targeted to the variable region to initiate SHM. The difficulty lies in the requirement to study SHM in vivo in a small population of germinal center B cells. To address this, we developed a knock-in mouse model called JH1, which utilizes a VDJ containing endogenous gene segments in the Variable (V) gene locus where upstream and downstream sequence isintact and intervening DNA is deleted. This unique model’s V region represents what is observed in endogenous B cells generated from the bone marrow. I have used and validated CUT&RUN on our low cell inputs, expanding possibilities for probing critical targets in precise populations of interest. Our lab and others have shown that SPT5 (a subunit of the RNA Polymerase II modulator, DSIF) is required for AID targeting and thus SHM. Unlike a typical gene, promoter-proximal pausing occurs up to 2kb downstream of the V promoter and is necessary for AID activity. We hypothesize distinctive regulation of RNA Polymerase II activity is what allows AID targeting and SHM. I am currently performing analysis of my generated CUT&RUN datasets for well-known transcriptional regulatory proteins to determine if there is a unique signature for SHM. Understanding how AID is targeted to the V region will help us to understand the elusive, yet indispensable process of SHM, which may be useful to fine-tune humoral responses via vaccines and other treatments.

This work was supported entirely through the Intramural Research Program at the National Institutes of Health (NIH), National Institute on Aging (NIA).