bluuebunny/crossref_metadata_2025 huggingface dataset:
Dual roles of the bacterial siderophore enterobactin by inducing apoptosis in macrophages and promoting survival advantages to Salmonella typhimurium
Authors: Saha Piu , Xiao Xia , Yeoh Beng San , Chen Qiuyan , Katkere Bhuvana , Kirimanjeswara Girish Soorappa , Vijay-Kumar Matam
Published: January 2023

Iron, an essential transition metal ion, is required to facilitate the antimicrobial and redox activity of heme proteins expressed in macrophages (Mφs). In this study, we investigated whether the chelation of iron by enterobactin (Ent; a prototypical bacterial siderophore) can affect the immune responses of macrophages. Herein, we demonstrated that exogenous Ent confers protection to the intracellular pathogen Salmonella enterica serovar Typhimurium from Mφs by promoting M1 to M2 polarization, reducing the expression of pro-inflammatory cytokines and impairing their intracellular killing. Accordingly, WT Salmonella survived better compared to Ent-deficient strain (ΔentB) in Mφs. The addition of apo-Ent (iron-free), but not ferric-Ent, rescued both strains, thus suggesting that the immuno-modulatory effect of Ent could be likely mediated via chelation of Mφs cellular iron. To determine whether the effects of Ent on Mφs could be due to altered cell viability, we next assessed the Mφs for markers of apoptosis via flow cytometry. Interestingly, we observed that apo-Ent, but not ferric-Ent, promoted a time-dependent apoptosis in Mφs, which can be detected as early as 12h after Ent treatment. These findings corroborated with the time-dependent release of lactate dehydrogenase (cytotoxicity marker) from Ent-treated Mφs. Collectively, our findings demonstrate that Ent not only facilitates bacterial iron uptake but also serves to safeguard bacteria against Mφ innate immune responses, which could be mediated, in part, by inducing delayed cellular apoptosis.
TLR9 regulates host defense via modulation of peritoneal B1 cell recruitment and functions during intra-abdominal sepsis.
Authors: Deng Meihong , Xu Li , Hoffmann Rosemary , Billiar Timothy R.
Published: January 2023

Background

Toll like receptor 9 (TLR9) is identified as anintracellular DNA receptor playing important roles in immunity. However, the mechanisms of how TLR9 regulates host defense are unclear. B1 cells are aninnate-like B cell population predominately residing in peritoneum and highly express TLR9. Therefore, we hypothesize that TLR9 may regulate host defense via modulation of peritoneal B1 pathobiology during sepsis.

Method

C57BL/6 (WT) mice and TLR9−/− mice were subjected to cecal ligation and puncture (CLP) with for 18 hours.

Result

Deletion of TLR9 increased survival as well as decreased bacterial load and systemic inflammation after CLP. Interestingly, peritoneal B1 cell numbers were significantly higher in TLR9−/− than WT mice, associated with significant increases of peritoneal CXCL13 level in TLR9−/− miceat baseline and after CLP. Strikingly, the expression level of CXCL13, a B cell-attracting chemokine, in the mesenteric adipo tissue was 50 fold higher inTLR9−/− than WT mice after CLP. Treatment with CXCL13 neutralizing antibody impaired bacterial clearance and decreased peritoneal B1 cell numbersin TLR9−/− mice compared to the IgG control after CLP, suggesting that TLR9 regulated B1 cell recruitment via CXCL13. Furthermore, deletion of TLR9 significantly increased peritoneal natural antibody IgM levels compared to WT at baseline and after CLP. Importantly, adoptive transfer of TLR9−/−B1 cells to WT mice improved bacterial clearance and increased peritoneal IgM levels compared to adoptive transfer of WT B1 cells after CLP.

Conclusions

These findings revealed an unrecognized role of TLR9 in modulating host defense via regulating B1 cell recruitment and IgM production during intra-abdominal sepsis.
Targeting the inflammasome as host directed therapy for Mycobacterium tuberculosis and HIV co-infection
Authors: Naqvi Kubra Fatima , Huante Matthew B. , Saito Tais , Chauhan Sadhana , Walker Aida , Lisinicchia Joshua , Gelman Benjamin , Endsley Janice J.
Published: January 2023

Mycobacterium tuberculosis (Mtb) infection continues to pose a significant public health threat with 1.3 million deaths occurring annually. People with HIV are among the most susceptible to Mtb infection due to the microbial synergism occurring during co-infection which promotes inappropriate inflammation as a potential driver of lung pathology. However, the immunomodulatory mechanisms driving pulmonary inflammation and pathogenesis are not fully understood. Our preliminary studies suggest that synergistic inflammasome activation within macrophages, the cellular host of both Mtb and HIV, leads to a caspase-1 dependent increase in pro-inflammatory cytokines. Using a humanized mouse model of co-infection developed in our laboratory, we have evaluated the in vivo inhibition of caspase-1 using the pro-drug VX-765 to limit mature IL-1β secretion and therefore reduce pulmonary inflammation. Treatment with VX-765 in humanized and WT (BALB/C) mice resulted in a decrease of lung inflammatory cytokine and chemokine production. Mycobacterial burden in the lung and sites of dissemination were unaffected by VX-765 treatment, suggesting inhibition of caspase-1 activity did not suppress protective immune responses. By using monocyte-derived macrophages (MDM) for an in vitro co-infection assay, we observed reduced inflammatory responses to co-infection following caspase-1 inhibition. These preliminary studies suggest a potential role of VX-765 as a host directed therapeutic targeting inflammasome activation in co-infected patients. With host directed therapy emerging as a possible new line of “anti-AIDS” therapy, the impact of these drugs on Mtb infection and the course of treatment is of great clinical importance.
Collagen, a major extracellular matrix component, promotes NLRP3 inflammasome-dependent and -independent IL-1β production.
Authors: Poudel Barun , Gurung Prajwal
Published: January 2023

Immune cell migration requires extracellular matrix (ECM) components that include collagen, fibronectin and laminin. As such ECM components regulate cellular processes including cell signaling, trafficking and differentiation. Excessive collagen production during disorders such as atherosclerosis, cancer, and renal and hepatic fibrosis has been known to have adverse immunological effects. However, the role of collagen in regulating inflammatory processes remain largely unexplored. Our data demonstrate that Nigericin-induced activation of the NLRP3 inflammasome in collagen-I-primed bone-marrow derived macrophages (BMDM) was not inhibited at chronic time points. In contrast, chronic TLR stimulation dampens ATP/Nigericin-induced NLRP3 inflammasome activation in BMDM. Collagen-I induced similar levels of pro-inflammatory cytokines TNF and IL-6 when compared to LPS-stimulated BMDM suggesting similar activation of signaling pathways. Moreover, our data demonstrate that chronic collagen-I stimulation was sufficient to induce IL-1β production in the absence of activating signals. This IL-1β production was dependent on the kinase activity of RIPK1 and RIPK3 but not the NLRP3 inflammasome. Current studies are focused on identifying, 1) mechanisms for chronic activation of the NLRP3 inflammasome and, 2) proteases that induce maturation and production of IL-1β in collagen-I treated BMDM. Altogether, our studies show that collagen-I can promote IL-1β production independently of NLRP3 inflammasome; however, in presence of Nigericin, collagen-I can chronically activate the NLRP3 inflammasome. Our study has the potential to identify novel targets to treat collagen-associated inflammatory disorders.
Id3 expression defines anatomically and functionally distinct regulatory T cells
Authors: Sullivan Jenna M. , Hoellbacher Barbara , Campbell Daniel J.
Published: January 2023

The Id (or inhibitor of DNA binding) proteins are small proteins that contain a helix-loop-helix domain and can heterodimerize with E-protein transcription factors. However, the Id proteins lack a DNA binding domain and act as natural dominant negatives of E-protein function. As a powerful transcriptional regulator, Id3 helps control the functional differentiation of CD8+ effector T cells and its expression is essential for regulatory T cell (TR) maintenance and function. Here we show that expression of Id3 is dynamically regulated in TR in a subset- and tissue-specific fashion. Within the TR compartment cTR (CD62LhiCD44lo) uniformly express Id3, however the eTR (CD62LloCD44hi) compartment can be further subdivided into Id3+ and Id3− eTR. Id3− eTR are found in low abundance in lymphoid tissues but are the majority of TR found in non-lymphoid tissue such as the skin and fat. These Id3− eTR express higher levels of inhibitory surface markers and have similar molecular profiles to the recently described ST2+ tissue TR. We propose that Id3 maintains a lymphoid-tissue TR phenotype and loss of Id3 promotes localization and retention of TR in non-lymphoid tissue.
SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-β signaling
Authors: Cortez Victor S. , Ulland Tyler K. , Cervantes-Barragan Luisa , Bando Jennifer K. , Robinette Michelle L. , Wang Qianli , White Andrew J. , Gilfillan Susan , Cella Marina , Colonna Marco
Published: January 2023

Among the characteristics that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of ‘imprinting’ by cytokines of the TGF-β family. We studied mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-β family. While SMAD4 deficiency did not impact ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-β signaling mediated by the cytokine receptor TGFβR1 in NK cells. NK cells from a SMAD4-deficient person were also hyper-responsive to TGF-β. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-β signaling in NK cells.
Kinetics of the liver resident NK cell compartment
Authors: Dodard Garvin , Erick Timothy , Tata Angela , Brossay Laurent
Published: January 2023

The presence of tissue-resident NK (trNK) cells in the liver has only recently been appreciated. While the role of conventional NK cells (cNK) has been well-characterized, the trNK cell function is only beginning to be elucidated. Using the MCMV model of infection, we found that in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction phase is independent of the virus itself as a similar phenotype is observed following Poly I:C/CpG or α-GalCer injection. Interestingly, this initial contraction phase is independent of other liver lymphocyte subsets as it occurs even in infected lymphopenic animals, suggesting it is provoked by the inflammatory milieu. We also found that the contraction phase is partially due to cell apoptosis while the recovery phase occurs via proliferation in situ. These findings underscore the unique homeostatic properties and kinetics of liver resident NK cell compartment.
Transcription Factor TX Is Required for Liver-resident NK cells development
Authors: Song Jiaxi , Peng Hui , Zheng Xiaodong , Chen Yongyan , Wei Haiming , Sun Rui , Tian Zhigang
Published: January 2023

Hepatic NK cells are heterogeneous populations, consisting of DX5+CD49a− NK cells and CD49a+DX5− NK cells. CD49a+DX5− NK cells, also termed “tissue-resident NK (trNK) cells”, are distinct from DX5+CD49a− conventional NK (cNK) cells and possess unique hallmarks. Recent study shows that trNK cell differentiation is programmed by transcription factor T-bet, instead of Eomes, which is critical for cNK cell development. T-bet is highly expressed by both trNK and cNK cells, its deficiency leads to dramatic reduction of trNK cell and terminal mature cNK cells, suggesting a non-specific role of T-bet in the development of NK cell subsets.

To find a transcription factor that potentially impacts trNK cell development in a specific manner, we analyzed differentially expressed transcription factor genes between liver trNK and cNK cells by microarrays. We found that the transcription factor TX is expressed on liver trNK cells at much higher levels than on cNK cells. In order to figure out whether TX has an effect on liver trNK cells, we overexpressed TX on HSCs, and transferred the cells into lethally irradiated mice. Surprisingly, the frequency and number of liver trNK cells increased in TX-overexpressed mice. Then, we knocked down TX expression on HSCs, and transferred the cells to lethally irradiated mice. Although the frequency of liver trNK cells was invariant, the number decreased significantly. Therefore, these results suggest that the transcription factor TX affects liver trNK cells development, and the underlying mechanisms await further investigation.
Protection from lethal Staphylococcus aureus infection by probiotic exopolysaccharide
Authors: Paik Wonbeom , Alonzo Francis , Knight Katherine L.
Published: January 2023

Staphylococcus aureus is a bacterium that causes severe systemic infection with high mortality. Antibiotic resistance poses major problems for treatment of S. aureus infections, and novel therapeutics are needed. We previously showed that the probiotic Bacillus subtilis, produces an exopolysaccharide (EPS) that induces anti-inflammatory M2 macrophages. Transfer of EPS-induced M2 macrophages to naïve mice protects them from colitis due to an enteric pathogen. We tested if EPS could also protect from lethal S. aureus bloodstream infection. We treated mice with EPS before infection, and found that they survived and had reduced bacterial burden in the liver, and reduced levels of systemic inflammatory cytokines. We sought to determine the mechanism by which EPS protected the mice, and found that the EPS-induced M2 macrophages not only take-up S. aureus, but also inhibit the growth of S. aureus. We used an inhibitor of reactive oxygen species (ROS), and found that the growth inhibition of S. aureus by M2 macrophages was abrogated. In addition, we found that these M2 macrophages retain the capacity to inhibit activation of T cells by S. aureus supernatants which contain superantigens and numerous pro-inflammatory molecules. We suggest that the EPS-induced M2 macrophages represent “hybrid” macrophages capable of both M1- and M2-like functions, and could be used to protect hosts from lethal S. aureus infection by bolstering anti-bacterial immunity, while simultaneously limiting inflammation.
Type 3 interferons expressed in tuberculous granulomas may influence signaling in epithelioid macrophages
Authors: Mattila Joshua T. , Talukdar Priyanka , Junecko Beth A.
Published: January 2023

Type 3 interferons, also known as interferon lambda (IFNλ), play important roles in mucosal immunity and protection against viral infection, but their roles in tuberculosis (TB) remain to be investigated. IFNλ have properties that partially overlap with type 1 interferons (IFNα/β), a group of cytokines whose expression positively correlates with TB severity. Conversely, IFNλ can downregulate neutrophilic inflammation, and because neutrophils can be pathologic in TB, IFNλ may have currently-unappreciated protective effects. To better understand IFNλ expression in tuberculous lung granulomas, we performed RT-PCR analysis and immunohistochemistry on cynomolgus macaques with TB, a nonhuman primate with human-like pathology, to determine whether IFNλ is expressed in granulomas and to identify IFNλ-expressing cells at the site of disease. We found that mRNAs for the IFNλ isoforms IFNλ1 (IL-29), IFNλ3 (IL-28B), and IFNλ4 were expressed in normal lung tissue and granulomas, and there was a significant amount of heterogeneity in granuloma IFNλ expression. In normal lung tissue, IFNλ protein expression was most pronounced in ciliated epithelial cells, but there were differences in subcellular localization between isoforms. We identified differences in the subcellular localization of IFNλ4 in different granuloma macrophage subsets, with IFNλ4 in lymphocyte cuff-region macrophages present in the macrophage cytoplasm while in epithelioid macrophages, IFNλ4 was most abundant inside the macrophage nuclei. These data suggest that IFNλ4 may be differentially-expressed by granulomas macrophage subsets and have a previously-unappreciated signaling role in epithelioid macrophages.
Immune-Medicated Angiopoietin/Tie2 Expression and Renal Injury in Murine Orientia tsutsugamushi Infection Models
Authors: liu jinjun , Trent Brandon J. , Liang Yuejin , Brehm Tyler Y. , Xing Yan , Cai Jiyang , Sun Jiaren , Soong Lynn
Published: January 2023

Scrub typhus caused by O. tsutsugamushi infection is a life-threatening disease endemic in the Asia-Pacific region; however, its molecular pathogenesis remains unclear. Using new mouse models, we have reported that endothelial cell (EC)-targeted O. tsutsugamushi infection, acute tissue injury, and kidney bacterial persistency are pathological hallmarks, which mimic human scrub typhus. In this study, we focused on the regulation of vascular growth factor angiopoietin-1 (Ang1), its antagonist Ang2, and their specific Tie2 receptor in the kidneys of C57BL/6 mice. At D6 (disease onset) and D10 (prior to host death), infected kidneys had high Ang2/Ang1 ratio, but low Tie2 levels, and a progressive loss of glomerulus integrity. These renal injury signs were associated with increased influx of MPO+ neutrophils and CD3+T cells and expression of Th1 cytokines (TNF-α, IFN-γ, CXCL9) in the kidneys. Notable, we found a significant, time-dependent increase in alarmin expression (IL-33 and IL-36γ) preferentially in the kidneys, rather than in other organs, even though kidneys contained much lower bacterial loads than other organs. To test if host alarmin molecules contribute to disease severity, we compared pathology in lethally-infected WT and IL-33−/− mice, as well as rIL-33-injected WT mice. IL-33 deficiency promoted BCL2, Ang1, and eNOS expression and reduced cellular apoptosis in inflamed kidneys. In contrast, rIL-33 injunctions markedly exacerbated vascular stress and renal injury and increased host susceptibility to sublethal infection. Together, this study reveals that an immune-regulated expression of the Ang1/Tie2 axis can influence renal injury during infection. It helps understand the pathogenesis of severe scrub typhus.
Dysregulated Th1 Immune and Endothelial Responses during Severe Scrub Typhus
Authors: Soong Lynn , Trent Brandon J. , Xing Yan , Wei Yang , Liang Yuejin , Brehm Tyler Y. , Cho Nam-Hyuk , Cai Jiyang , Bouyer Donald H. , Sahni Sanjeev , Walker David H. , Sun Jiaren
Published: January 2023

We recently established murine Orientia tsutsugamushi infection models, which mimic immunological features of human scrub typhus. A hallmark of lethal Orientia infection is Th1-skewed, but Th2-impaired, immune responses, accompanied by endothelial dysfunction and multi-organ failure. Notably, while tissue bacterial loads peak at D6 (disease onset) in mice, lung neutrophil and CD8 T cell influx/activation reach peaks at D10 (prior to host death). We proposed a two-stage mechanism for disease pathogenesis: 1) Orientia replication in phagocytes and endothelial cells (EC) triggers inflammatory responses and host defense machinery; 2) infection-mediated release of DAMP molecules further exacerbates vascular damage, even after the control of bacterial replication. This hypothesis was supported by WB, qRT-PCR, and immunofluorescent co-staining of bacteria with leukocyte/EC/platelet-specific markers. During infection, progressive loss of vasculature and tight junctions (occludin, VE-cad, lectin positivity), vascular malfunction (reduced angiopoietin-1, Tie2, pTie2), and reduced CD41+ platelets in the lungs were positively linked to influx/activation of neutrophils and CD8 T cells, rather than bacterial loads. Immune dysregulation mechanisms were further examined by using infected human EC cultures, patient sera, IL-33−/− mice, and mouse macrophages/neutrophils. While Orientia infection can directly alter the angiopoietin/Tie2 axis in EC, endogenous IL-33/IL-36-like DAMPs contribute to vascular and tissue damage at the late stages. Our studies provide new insights into infection- vs. immune-mediated dysregulation and prognostic biomarkers, which will help the control of this neglected tropical disease.
Aryl hydrocarbon receptor activation differentially impacts Cholera toxin-specific IgA levels in feces and serum of male and female mice
Authors: Dekrey Gregory K. , King Aspen E. , Gawronska Eleanour , Metten Maddie R.
Published: January 2023

Secretory IgA plays a critical role in neutralizing enteric toxins and protecting against some intestinal pathogens. In mice, activation of the aryl hydrocarbon receptor (AhR) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to suppress antigen-specific systemic IgM and IgG responses, whereas the impact of AhR activation on mucosal IgA responses is less clear. Previous studies in our laboratory have demonstrated that Cholera toxin (CT)-specific fecal IgA levels in female mice are initially suppressed by TCDD exposure, but they recover to control levels within three weeks, unlike serum IgA levels. The goal of this study was to determine if TCDD exposure similarly alters CT-specific IgA levels in male mice. Male and female C57Bl/6 mice were administered peanut oil (vehicle) or TCDD on day 0, and each animal was immunized with CT (10 μg/Kg) on days 1, 8 and 15. Feces were collected on days 14 and 21, and antibody levels were measured by ELISA. Relative to controls at week 2, CT-specific fecal IgA levels were significantly decreased (P ≤ 0.05) by TCDD exposure at 40 μg/Kg (but not lower doses) in both male and female mice. Suppressed antibody levels persisted at 3 weeks in male mice only. In serum, significant suppression of CT-specific IgA was observed at week 3 in male mice (40 μg/Kg TCDD) and at week 4 in female mice (10 and 40 μg/Kg TCDD). These results suggest that antigen-specific fecal IgA responses in female mice recover from AhR-mediated suppression more rapidly than systemic responses and more rapidly than in male mice.
Fluid absorption modulates Peyer’s patch homeostasis and mucosal antibody responses
Authors: Chang Jonathan , Gressier Elise , Turley Shannon J. , Carroll Michael C.
Published: January 2023

Peyer’s patches (PPs) are B cell-rich lymphoid tissues situated throughout the small intestine which play an important role in mucosal antibody responses. PP architecture and stromal cell composition closely resemble that of peripheral lymph nodes despite geographical and functional differences. Notably, fibroblastic reticular cells (FRCs) located in small intestinal PPs form a network of collagen-rich reticular fibers similar to conduits found in lymph nodes. In the PP, these conduits extend from the basement membrane of the epithelial cell lining into the PP follicle, and terminate along blood and efferent lymphatic vessels.

Unlike lymph nodes, PPs lack a conventional source of afferent lymph that would normally contribute fluid flow through the conduit network. Instead fluid flow through PP conduits depends largely on water absorbed across the intestinal epithelium. We find that by disrupting water absorption, we can limit or prevent the contribution of absorbed lumenal fluids to the flow of PP conduits. Disruption of fluid absorption subsequently has profound effects on the structural integrity of the high endothelial venule (HEV) and surrounding perivascular FRCs. These alterations correlate with a striking defect in the recruitment of naïve lymphocytes to the PP.

Prolonged blockade of water absorption additionally impacts mucosal antibody responses. Antigen-specific fecal IgA titers are reduced, and germinal center responses are decreased in scale. These findings reveal a critical role for fluid absorption and conduit-mediated fluid flow in the maintenance of PP function and may have important implications for mucosal health under conditions of fluid malabsorption, long-term laxative use, and diarrhoeal diseases.
Rise in invariant Natural Killer T-17 cells during Bordetella pertussis infection in mice
Authors: Segal-Abramson Tzvia , Santos Daniel
Published: January 2023

Bordetella pertussis is a Gram-negative bacterium known for causing the respiratory disease whooping cough. Whooping cough has recently seen resurgence, especially among children, with the disease’s resolution being delayed. Here, we observed the contribution of invariant Natural Killer T (iNKT) cells in the response to B. pertussis infections. Invariant NKT cells are so called for their invariant chain (Vα14Jα18 in mice) within the T cell receptor, which allows these cells to respond to lipid antigens presented by CD1d molecules. iNKT cells are known to express a variety of transcription factors and release different cytokines, as well as interact with numerous cells of the immune system. Here we hypothesize that IL-17 producing iNKT (iNKT17) cells rise in number during B. pertussis infections to aid in the resolution via an IL-17 driven inflammation. However, due to a pertussis toxin-dependent inhibition, there is a delay in the resolution of the infection. The population of iNKT17 cells, as well as dendritic cells and their expression of CD1d, was assessed via comparison of uninfected to B. parapertussis- and B. pertussis -infected C57BL/6 mice, using a multicolor flow cytometry analysis. The mice infected with pertussis displayed a significant rise in RORγt-positive iNKT cells when compared to uninfected mice, while having reduced IL-17 secretion. These findings indicate a role for iNKT17 cells in the pathogenesis and potential resolution of B. pertussis infection, with the potential for future analysis into iNKT cell knockout studies to determine further understanding of the cell’s function during the infection.
Novel role of intestinal Goblet cells and Goblet Cell Associated Antigen Passages in induction of tolerance to dietary antigens and promoting intestinal homeostasis
Authors: Kulkarni Devesha Hrishikesh , Gustafsson Jenny K. , McDonald Keely G. , Knoop Kathryn A. , Davis Jazmyne , Miller Mark J. , Newberry Rodney D.
Published: January 2023

Mucosal tolerance towards luminal antigens is a process fundamental to intestinal homeostasis, the breakdown of which results in onset of inflammatory bowel disease. How luminal antigens cross the intestinal epithelium to interact with the immune system in a manner capable of induction and maintenance of oral tolerance is unknown. We have identified a new pathway of luminal antigen delivery to dendritic cells (DC) in the lamina propria (LP), which was mediated by goblet cells (GC) associated antigen passages (GAPs). The role of GCs and GAPs in promoting mucosal tolerance is unexplored. We studied tolerogenic responses to dietary antigen, ovalbumin (Ova) in mice where GCs were deleted, or when GAPs are inhibited but GCs remain and compared to corresponding control mice. In the absence of GCs or functional GAPs, LP-DCs could not acquire luminal antigens in a manner capable of inducing antigen specific T cell responses in the draining mesenteric lymph nodes (MLN). Moreover, deletion of GCs/GAPs resulted in loss of induction of regulatory T cells in the MLN and abrogated tolerance to dietary antigens. In addition, loss of GCs/GAPs resulted in rapid reduction of pre-existing induced Treg population in the SI-LP. Notably, the in absence of GCs/GAPs resulted in rapid expansion of IL-17 producing T cells in the small intestine, indicating that GCs and GAPs play a crucial role in balancing the Treg/Th-17 axis in the intestine. Hence, suggesting that GCs and GAPs play a crucial role in the induction and maintenance of oral tolerance. These findings identify a previously unappreciated role of GCs in maintaining the Treg/Th17 balance and that processes resulting in inadequate formation of GAPs may underlie the loss of tolerance to luminal antigens.
Engineering of Targeted Mucoadhesive Chitosan Based Salmonella Nanovaccine for Oral Delivery in Poultry
Authors: Renu Sankar , Markazi Ashley D. , Dhakal Santosh , Lakshmanappa Yashavanth Shaan , Shanmugasundaram Revathi , Selvaraj Ramesh K. , Renukaradhya Gourapura J.
Published: January 2023

Salmonellosis in poultry is a serious economic burden. The major concern is the public health hazard by consumption of salmonella contaminated poultry meat and egg. Currently used salmonella vaccines are ineffective in combating the Salmonellosis of chicken thus warranting the need of a potent vaccine, especially an oral vaccine that can elicit robust local intestinal immunity. Biodegradable and biocompatible natural polymers are FDA approved vehicles for vaccine delivery. We prepared a candidate subunit chitosan nanoparticle vaccine containing the immunogenic outer membrane proteins (OMPs) and flagellar (F) protein of salmonella, and surface decorated with F-protein (OMPs-F-CS NPs). Physicochemical and biocompatibility properties of OMPs-F-CS NPs were studied in detail including its stability in stomach pH conditions. Salmonella targets the microfold (M) cells in the ileum Peyer’s patches (PPs) of chicken. We designed the oral OMPs-F-CS NPs vaccine to target ileal PPs to induce specific local intestinal immunity, and demonstrated that by ex vivo and in vivo studies. Layer chickens vaccinated orally with OMPs-F-CS NPs had significantly higher OMPs- specific intestinal mucosal antibody and OMPs-specific lymphocytes proliferation responses. Further, OMPs-F-CS NPs vaccination upregulated the expression of toll-like receptor (TLR) -2, TLR-4, IFN-g, IL-4 and GATA-3 mRNA levels in cecal tonsils of birds. In conclusion, we engineered chitosan based oral salmonella nanovaccine targeting intestinal PPs immune cells of birds, and demonstrated its ability to induce antigen specific mucosal antibody and T cell responses. Thus, our candidate oral salmonella vaccine has the potential to mitigate Salmonellosis in poultry.
Self-tolerance to an ileal antigen requires both IL-17A and IFNγ in mice predisposed to spontaneous ileocolitis
Authors: Jeschke Jonathan C. , Mayne Christopher G. , DeCiantis Christopher L. , Kumar Suresh N. , Drobyski William R. , Salzman Nita H. , Williams Calvin B.
Published: January 2023

Homeostasis in the ileum involves ongoing immune responses. To study how homeostatic processes of the ileum impact CD4+T cell responses, we used TCR transgenic tools to breed “Bigenic” mice that spontaneously produced CD4+T cells reactive to a model antigen expressed in the ileum. Bigenic mice were grossly similar to control littermates at weaning. By DOL 50, the Bigenic cohort showed reduced weight, increased circulating CD4+TEM frequencies and TH17-associated ileal hyperplasia. An extended natural history study to DOL 150 revealed that only half of Bigenic mice exhibit chronic wasting disease marked by mucosal infiltration by TH17 and TH1 cells expressing non-transgenic clonotypes, colitis and loss of ileal crypt hyperplasia. By contrast, adult Bigenic mice with normal growth maintained the TH17-associated ileal crypt hyperplasia phenotype and additionally accumulated ileal-reactive Treg cells. Adoptive transfer of nTreg cells prevented both ileal crypt hyperplasia and ileal-reactive Treg cell accumulation. Both IL-17A and IFNγ were required for ileal-reactive Treg accumulation and regulation of ileal-reactive CD4+Tconv cells. Ifng−/− Bigenic mice also exhibited an IL-23R-dependent, acutely progressive wasting disease that was >95% penetrant by DOL 50. Thus, our studies identify an IL-17A and IFNγ-dependent homeostatic inflammatory process that mobilizes ileal-reactive Treg cells and is disrupted by IL-23.
Single-cell RNA-seq identifies the neuropeptide NMU as a novel regulator of ILC2 function
Authors: Wallrapp Antonia , Riesenfeld Samantha J. , Burkett Patrick R. , Abdulnour Raja-Elie E. , Nyman Jackson , Dionne Danielle , Hofree Matan , Cuoco Michael S. , Rodman Christopher , Farouq Daneyal , Haas Brian J. , Tickle Timothy L. , Trombetta John J. , Baral Pankaj , Klose Christoph S. N. , Mahlakõiv Tanel , Artis David , Rozenblatt-Rosen Orit , Chiu Isaac M. , Levy Bruce D. , Kowalczyk Monika S. , Regev Aviv , Kuchroo Vijay K.
Published: January 2023

Type 2 innate lymphoid cells (ILC2s) are important for both mucosal homeostasis as well as for initiation and progression of allergy and asthma. Alarmin cytokines produced by damaged or stressed epithelial cells activate ILC2s, however, the role of other pathways regulating the homeostatic versus inflammatory functions of ILC2s remain poorly defined. We generated transcriptional profiles of thousands of lung-resident ILCs isolated from mice challenged with either PBS or the alarmin cytokines IL-25 or IL-33 using massively parallel droplet-based single-cell RNA sequencing (scRNA-seq). We found significant transcriptional heterogeneity within activated ILCs and identified differentially expressed genes as potential novel regulators of ILC function. One of the genes, neuromedin U receptor 1 (NMUR1), is a receptor for the neuropeptide neuromedin U (NMU) and is specifically expressed by ILC2s and not by other immune cells. Nmur1 is particularly expressed by ILC2s at steady-state and after IL-25 treatment. In vivo NMU synergized with IL-25 to induce more severe airway inflammation than was seen with either IL-25 or NMU alone and this effect was dependent on ILCs. Mechanistically, IL-25 + NMU expanded highly proliferative and inflammatory ILCs with distinct transcriptional profiles and increased expression of IL-5 and IL-13. We observed reduced ILC2 frequency and function in NMU- or NMUR1-deficient mice after allergen challenge and found that ILCs from these mice had a lower score for an inflammatory ILC2 gene signature. Our data therefore establish a novel neuro-immune pathway that amplifies ILC2-driven airway inflammation.
Bioengineered organoid models of human germinal centers
Authors: Quizon Nicolas , Kwak Kihyuck , Shah Shivem , Singh Ankur , Pierce Susan
Published: January 2023

B cells undergo affinity maturation to T cell-dependent antigens within specialized microenvironments in secondary lymphoid organs termed germinal centers (GCs) resulting in the selective proliferation and differentiation of high-affinity B cells. Selection is driven by the ability of antigen-specific GC B cells to extract antigen presented on follicular dendritic cells and subsequently recruit stimuli from T follicular helper cells. Successful GC B cells ultimately differentiate into plasma cells that secrete large amounts of antigen-specific antibody, or memory B cells that contribute to long-term immunity. However, the factors that influence selection and differentiation of GC B cells in humans are not yet well understood. One challenge arises from the rapid loss of viability of human primary GC B cells ex vivo under conventional cell culture conditions. To overcome this, we bioengineered organoids using a customizable matrix along with modified feeder cell lines. When encapsulated in these organoids, primary human GC B cells exhibit enhanced viability similar to that of B cells in GCs in vivo. The low auto-fluorescent properties of these organoids allow for imaging of cellular interactions and migration. Furthermore, the customizable chemistry of the organoid matrix allows us to screen for factors that influence the differentiation and fate of human GC B cells. Improving our understanding of these processes may provide important insights in areas such as therapeutics and vaccine development.
The potential application of ctDNA technology as a cancer diagnostic tool within the Socialist Republic of Vietnam
Authors: Sika-Paotonu Dianne , Guilford Parry
Published: January 2023

Small fragments of tumor DNA can be found in the blood circulation and can be monitored and analysed as part of a novel cancer detection method known as circulating tumor DNA (ctDNA). These tumor DNA fragments can be isolated and quantified to produce detailed information related to cancer progression and treatment responses in patients. The possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth and behavior is highly desirable given that gaining access to and obtaining biopsy samples from solid cancers in people is not always possible or as straightforward as needed. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer care for patients, and will eventuate into a particularly useful tool within resource constrained environments. The purpose of this work was to identify the diagnostic methods, treatment and pharmacological options currently available for cancer patients within the Socialist Republic of Vietnam and to explore the potential utility and applicability of ctDNA as a mechanism to support the management, treatment and diagnosis of cancer patients. Favorable interest was expressed by medical, health, community and lay persons regarding the development of ctDNA as a potential diagnostic tool within the health setting in the Socialist Republic of Vietnam and remains an attractive tool with potential to support cancer patients in Vietnam.
The application of ctDNA technology for early Cancer diagnostics in Samoa
Authors: Sika-Paotonu Dianne , Viali Satupa’itea , Naseri Leausa Toleafoa Take K. , So’o Fui Le’apai Tu’ua Ilaoa Asofou , Lameko Toleafoa Viali , Punivalu Monalisa , Burich Shelley , Maiava Fuatai , Ah-Ching Salausa John , Guilford Parry
Published: January 2023

Fragmented pieces of tumor DNA can be found in the human blood circulation. These tumor DNA fragments can be isolated and quantified to produce detailed information related to cancer progression and treatment responses in patients. This monitoring and analysis comprises a novel cancer detection method known as circulating tumor DNA (ctDNA). Given that gaining access to and obtaining biopsy samples from solid cancers in people is not always possible or as straightforward as needed, the utilization of a simple blood sample to allow detection and monitoring of cancer growth and behavior is highly desirable. This simple detection and monitoring technology is likely enhance the precision of cancer care for patients, and support early cancer detection. The purpose of this work was to explore in detail the potential for ctDNA to be utilized as an early cancer detection tool within the healthcare setting within the Pacific in Samoa. Consultation was sought with senior Government officials, Medical, Nursing, Health and Community research staff concerning the development and implementation of ctDNA as a diagnostic tool within the clinical health care setting throughout Samoa. The application of the ctDNA technology as an early cancer detection tool within the clinical healthcare setting was explored in depth and received with approval, given issues that currently exist concerning resource constraints and late cancer presentations. It is anticipated that the utility of ctDNA as an early cancer detection tool will support improved cancer management within the health care setting in Samoa.
A simple and rapid method for measuring human natural killer cell function in whole blood by anti-NKp30-conjugated nanoparticles
Authors: Kato Kazunori , Moriya Masaaki , Kato Eri
Published: January 2023

Natural killer (NK) cells play a central role as innate immune effector cells to virus infected cells and tumor cells. The standard assay to evaluate NK activity is 51Cr release assay using radio-labeled K562 cells, however, new methods using fluorescence-labeled targets or flow cytometry to examine degranulation markers have been developed recently. In addition to cytotoxicity, NK cells enable to produce various cytokines to regulate the adaptive immune responses in infection, inflammation and cancer. Here we generated nanoparticles conjugated with various antibody for NK receptors and examined to stimulate human whole blood with these nanoparticles instead of K562 target cells. We found that various cytokines such as IFN-γ, IL-6, IL-1β and CCL2 were produced by NK cells in whole blood by anti-NKp30-conjugated nanoparticles in dose-dependent manner more than by anti-NKp46 or anti-NKG2D nanoparticles. Production of IFN-γ was detected within 4 hours and reached maximum level at 24 hours after stimulation. IFN-γ production was relatively high at incubation temperature of 37°C, but that was significantly decreased over 39°C. Importantly, we could evaluate the reproducible cytokine production in same donors at another blood collection date because of the stable stimulus of nanoparticles and less variation data by human manipulation for lymphocyte separation. Additionally, cytokine secretion did not correlate with NK cell-mediated lysis of K562 target cells and NK cell numbers in whole blood. These data indicated that this method using NKp30 nanoparticles might be useful for measurement of individual differences of NK function especially correlated in adaptive immune response and inflammation.
Gene Therapy induced Tregs: A treatment for relapsing-remitting MS in mice
Authors: Keeler Geoffrey D. , Hoffman Brad E. , Cote Isabelle , Gaddie Cristina D.
Published: January 2023

Autoimmune disease is caused by a breakdown of tolerance against endogenous protein(s). New therapies are being developed that utilize the natural abilities of regulatory T (Treg) cells to suppress autoimmune effector cells in an antigen-specific manner. Recently, we demonstrated a clinically relevant in vivo gene immunotherapy approach for restoring Ag-specific tolerance using hepatic adeno-associated virus (AAV) gene transfer. This approach was shown to be remarkably successful at preventing and reversing a monophasic, MOG-induced EAE disease. Additionally, we have shown the dynamic ability of this treatment to prevent and/or reverse disease induced with various MOG epitopes, in genetically diverse mouse strains. However, it is unclear if this approach would be effective in the more complicated, PLP induced relapsing-remitting disease model. Thus, we re-engineered our therapeutic vector to contain the full-coding sequence of PLP. This is critically important as the established RR-EAE (SJL) model is characterized by epitope spreading; a phenomenon that may more closely resemble multiple sclerosis. SJL mice received a single peripheral injection of AAV. PLP vector, or control, 2 weeks prior to PLP 139–151 induced EAE. By day 14, control mice developed significant neurological deficits followed by a relapsing-remitting disease course. In contrast, the treated mice failed to exhibit any neurological symptoms of disease throughout the duration of the experiment, ~75 days. The long-term prevention of PLP induced disease, provides the first evidence of an effective Treg based therapy for combating epitope drift in RR-EAE, and potentially a new paradigm for treating the autoimmune disease multiple sclerosis.
B Cell Maturation Antigen impacts the efficacy of B-cell targeting therapies in neuro-inflammation.
Authors: Kumar Gaurav , Ko Rose M. , Axtell Robert C.
Published: January 2023

B cells have emerged as a therapeutic target for multiple sclerosis (MS). Depleting B cells with anti-CD20 antibodies are effective in treating MS. Yet, blockade of BAFF and APRIL, two cytokines important for B cell development and function, paradoxically increased disease activity in MS patients. The stark differences in clinical outcomes with these therapies demonstrate that B cells have both inflammatory and regulatory effects in MS. B cell Maturation Antigen (BCMA), a receptor that binds both BAFF and APRIL, plays a prominent role in B cell function. The role BCMA plays in MS remains enigmatic and therefore we designed this study to explore the impact BCMA deficiency has on the development and progression of experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. We found that EAE induced with MOG35–55 was more severe in BCMA−/− mice compared to BCMA+/+ mice. The increased disease was associated with increased CNS inflammation and a skewing of B-cells toward a mature/inflammatory phenotype. Using bone marrow chimera strategies, we found that BCMA deficiency in B-cells was responsible for the increase in disease severity. Next, we found that anti-CD20 treatment significantly reduced EAE in BCMA−/− mice but had no effects on BCMA+/+ mice. Conversely, we found that TACI-Ig treatment had no effect on BCMA−/− mice but did reduce EAE in BCMA+/+ mice. Our data demonstrate that BAFF and APRIL constrains the development and function of inflammatory B-cells through BCMA. Furthermore, our data provide clues into the paradoxical results of clinical trials with anti-CD20 and TACI-Ig in MS.
Immune modulation by combination type I interferon and checkpoint blockade therapy in murine urothelial carcinoma
Authors: Plote Devin E , Choi Woonyoung , Mokkapati Sharada , McConkey David , Schluns Kimberly S , Dinney Colin
Published: January 2023

Type I interferon (IFN) has potent antitumor effects in bladder cancer and provides an essential alternative treatment option for patients who do not respond to Bacillus Calmette-Guerin (BCG). However, the mechanism of the IFN-stimulated immune response in bladder cancer is not well understood. We hypothesized that type I IFN enhances recruitment and activation of immune cells in murine bladder cancer tumors, and makes them more responsive to anti-PD-1 mAb therapy. To incite an IFN-driven inflammatory response, poly(I:C) was given to murine MB49 bladder cancer tumors peritumorally alone and in combination with anti-PD-1 mAb to determine effects on tumor growth and animal survival. IFN induction in MB49 tumors significantly inhibited tumor growth. This response was reliant on both innate and adaptive immune subsets, shown in the increased influx of intratumoral Ly6G+ neutrophils and the increase in IFNy+CD8 T cells. In addition, depleting specific immune cell subsets indicated that no one type of immune cell was critical for the IFN-mediated anti-tumor response. When used in combination with anti-PD-1 mAb, poly(I:C) prolonged mouse survival and enriched gene pathways involved in metabolism, extracellular matrix formation and organization, and PI3K and AKT signaling. To examine effects of IFN-I in BCG-unresponsive patients, tumor samples were analyzed for RNA expression and immunohistochemical (IHC) staining both before and after treatment with adenoviral interferon-α. Among these samples, 25% showed increased expression of T cell and checkpoint markers, similarly reflected in IHC analysis. Our findings suggest type I IFN grooms the tumor-immune landscape in bladder cancer by activating both innate and adaptive immune cells.
Combination therapy of adoptive T cell therapy and immune checkpoint blockades engages distinct mechanisms in CD4+ and CD8+ T cells
Authors: Shi Lewis Zhichang , Gao Jianjun , Allison James P. , Sharma Padmanee
Published: January 2023

Adoptive transfer of tumor-reactive T cells (ACT) has led to significant clinical responses in hematological malignancies. However, its efficacy in solid tumors has been dismal, likely due to the immunosuppression imposed by the tumor microenvironment (TME) of solid tumors. A proven effective strategy to revert the immunosuppressive TME is immune checkpoint blockades (ICBs) including anti-CTLA-4 and anti-PD-1/PD-L1 that have been recently approved by FDA for cancer therapy. Here, using the orthotopic B16-F10 melanoma model, we combine ACT of PMEL-1 Tg+ CTLs that recognize the endogenous melanoma antigen gp100 with dual blockade of CTLA-4 and PD-1, the highly-pursued effective combinatorial ICB, to treat established tumors. Although ACT alone transiently suppresses tumor growth, complete tumor regression requires additional dual anti-CTLA-4 and anti-PD-1 blockade. Mechanistically, we demonstrate that the ICOS-Eomes-IFN-g axis in the transferred CTLs plays a predominant role in mediating the therapeutic effects of ACT and ICBs, distinct from the dominant ICOS-T-bet-IFN-g axis in CD4+ T cells that we previously identified. Our data provide direct evidence that combining ACT of CTLs with ICBs confers long-term protection to solid tumors, which largely depends on CTL-intrinsic expression of ICOS.