bluuebunny/crossref_metadata_2025 huggingface dataset:
Using wild mice to interrogate immunological mechanisms of asymptomatic malaria
Authors: Cornwall Douglas , Srey Mellina , Maloba Franklin , Evavold Birk , Olatunde Adesola C , Jensen Anne , Lamb Tracey , Evavold Brian
Published: January 2023

Malaria is a significant world health problem, with billions of people at risk and over 200 million clinical cases per year that result in over 200,000 deaths. However, the majority of the population in malaria endemic areas (>60%) is asymptomatic (without overt symptoms), even in high transmission areas. Although identified by Plasmodium-infected red blood cells in the circulation, the term asymptomatic malaria is a misnomer with individuals experiencing mild anemia, vascular activation and increased susceptibility to co-morbidities such as non-typhoidal Salmonella infections. The immunological mechanisms governing the development and maintenance of asymptomatic malaria are poorly understood and the influence of host genetics in endemic populations is unknown. Currently, no immunologically intact animal model is available to understand how host genetics influence the asymptomatic nature of Plasmodium infection. Here, we use wild-derived Mus musculus to show that the genetic diversity seen in the wild host population leads to increased variation in anemia upon infection with Plasmodium yoelii XNL with a subpopulation of mice experiencing asymptomatic malaria with mild anemia. Plasma TNF-a was positively correlated with the level of anemia, as might be expected from studies showing a positive correlation of TNF-a with anemia in children. Also, there was a negative correlation between monocyte populations and anemia experienced by the mice, which has also been observed in asymptomatic children in eastern Uganda. These results show that the wild derived mice can model asymptomatic malaria and allow us to interrogate the different immune profiles that lead to the range of phenotypes to malarial disease.
A novel variant in ubiquinone biosynthesis highly prevalent in Papua New Guinea children increases mortality following bacterial pneumonia
Authors: Walker Emma C , Ramani Rashmi , Javati Sarah , Todd Elizabeth , Chandra Pallavi , Matlam John-Paul , Anaya Edgar , Pomat William , Morley Sharon Celeste
Published: January 2023

To identify immune variants predisposing to severe pneumonia, we performed whole exome sequencing in a pediatric population highly susceptible to acute lower respiratory infections, identifying a candidate novel variant in the ubiquinone (CoQ10) biosynthetic pathway. To evaluate the effect of this variant on immune function during bacterial pneumonia, we generated a mouse line using CRISPR-Cas9 that expresses the homologous aspartate to tyrosine variant in the enzyme COQ6. Intra-tracheal S. pneumoniae infection leads to increased bacteremia and mortality in mice homozygous for the variant despite similar numbers of immune cells in the lung. Mechanistic studies show that macrophages expressing the variant have decreased mitochondrial activity at the ubiquinone-dependent reduction of cytochrome c by complex III, as well as decreased maximum respiratory capacity in response to acute stimulation. Variant-expressing macrophages also exhibit impaired generation of mitochondrial reactive oxygen species (mROS) causing a direct, intrinsic defect in intracellular killing of internalized bacteria. Thus, the novel variant in CoQ10 biosynthesis leads to changes in macrophage mitochondria and an intrinsic inability to kill internalized bacteria. As alveolar macrophages are the first responders in the lung to bacterial challenge, the inability of these macrophages to mount a sufficient immune response can explain the observed increase in mortality following bacterial pneumonia. Because variants in CoQ10 biosynthesis can be supplemented with CoQ10, a readily available therapy may be able to correct this defect and improve survival in children with this variant
Association of gut microbiota with spontaneous autoimmune uveitis studied by human flora reconstitution of germ-free mice
Authors: Zhang Amy , Horai Reiko , Salvador Ryan S. , O’hUigin Colm , Badger Jonathan , Yuan Wuxing , Thovarai Vishal , Sudo Katsuko , Atarashi Koji , Honda Kenya , Caspi Rachel R.
Published: January 2023

Autoimmune uveitis is a T cell driven, intraocular inflammation that affects the neuroretina and can lead to blindness. In a mouse model of spontaneous autoimmune uveitis (R161H), retina-specific T cells are primed in the gut through their TCR and trigger disease (PMID: 26287682). To support the relevance to human disease, we examined development of uveitis and its association with bacterial taxa in gnotobiotic R161H mice harboring human flora. Specifically, fecal samples from three healthy human donors were inoculated into germ-free R161H mice and their wild type (WT) littermates in separate isolators. Fecal pellets from reconstituted mice and their offspring were collected periodically and subjected to 16S amplicon sequencing. Uveitis scores were determined in R161H offspring by histology at termination of the experiment. Our results show that healthy human gut commensals can support uveitis in R161H mice, but to a lesser extent than native mouse flora from the NIH facility. Diversity analyses indicate that human flora-reconstituted mice retained a distinct but simplified gut microbial community compared to the original sample, and R161H mice tended to have a more complex microbiome profile than WT littermates. Among R161H offspring, mice with high disease scores appeared to harbor more diverse gut flora than those with low scores. Differentially abundant bacterial taxa were identified between mice with different disease scores, which may associate with intensity of autoreactive T cell activation and development of autoimmunity. R161H associated with human gut flora could provide a more translatable platform than mouse flora to explore microbial candidates and products that may modulate autoreactive T cells in uveitis.
IL–17 receptor signaling regulates Citrobacter rodentium growth
Authors: Matsunaga Yasuka , Wanek Alanna , Flemington Cathy , Song Kejing , Iwanaga Naoki , Clark Trevon , Bitoun Jacob P. , Good Misty , Kolls Jay K.
Published: January 2023

The type 17 cytokine receptors are thought to play critical roles in mucosal immunity. We have shown that intestinal epithelial expression of IL–17 receptor control the development of microbiota in the terminal ileum and conditional deletion of these receptors results in overgrowth of segmented filamentous bacteria. To study the roles of these receptors in response to an attaching and effacing pathogen we challenged intestinal specific Il17ra mice and Il17rc mice with Citrobacter rodentium (C. rodentium). Conditional deletion of Il17ra and l17rc Iresulted in increased bacterial growth in the colon as well as enhanced dissemination to the liver suggesting that intestinal epithelial IL–17 receptors expression is required for control of this infection. This was supported by unbiased RNAseq analysis of the colon that showed diminished expression of Pigr, Tnfsf13 and Nox1 in the absence of Il17ra. IgA has crucial role in mucosal immunity to protect from pathogenic bacteria. PIGR binds dimeric IgA and translocation into lumen, while TNFSF13 activates induce differentiation to plasma cells following–crass switch. C. rodentium specific IgA in stool and C. rodentium specific IgA producing cells in lamina propria were reduced in Il17ra mice. These results suggested IL-17 receptor signaling regulates transcytosis of C. rodentium specific IgA as well as secretion of bacteria specific IgA. On the other hands Nox1 can produce apical hydrogen peroxide. Nox1 null mice were more sensitive for C. rodentium infection. Furthermore Nox1 was expressed by IL–17A stimulation in colonic organoid. Taken together, IL–17 receptor signaling control to produce C. rodentium specific IgA through PIGR and TNFSF13 as well as hydrogen peroxide through NOX1.
2B4 controls gut immune homeostasis by promoting IL-1β expression in Ly6Chigh monocytes that trigger IL-22/IL-18-mediated epithelial regeneration
Authors: Kim Tae-Jin , Kim Jeongmin , Park Bonggoo , Park Gayoung , Lim Seon Ah , Lee Kyung-mi
Published: January 2023

Impaired signaling, downstream of 2B4, is associated with colitis in human patients with XLP ; however, the precise mechanism by which 2B4 maintains gut integrity remains unknown. In dextran sodium sulfate (DSS)-fed reciprocal chimera mice, massive tissue destruction was observed due to lack of 2B4 in hematopoietic compartments, in particular Ly6ChighCD11b+ monocyte subsets within the lamina propria leukocytes. Not only did the number of Ly6ChighCD11b+ monocytes get significantly reduced, their intrinsic capacity to produce IL-1β during DSS stimulation was also drastically impaired in 2B4−/− mice. These defects led to an impaired IL-22 secretion in ILCs and IL-18 production in gut epithelial cells in the 2B4−/− mice. Furthermore, severe inflammatory phenotypes seen in 2B4−/− mice could only be rescued by adoptive transfer of Ly6ChighCD11b+ monocytes, but not with BMDC or NK cells. Therefore, 2B4 functions as an important receptor in inflammatory monocytes in response to commensal microbiota during acute colonic inflammation.
Ingestion of subtoxic doses of cadmium induces persistent dysbiosis and enhances susceptibility to DSS-induced colitis long after exposure
Authors: Kim Eunsoo , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Health officials often focus on the effect of high doses of heavy metal pollutants. However, most people will more likely be exposed to subtoxic doses via contaminated water or food. We addressed the effects exposure to subtoxic doses of heavy metal pollutants could have on the gut immune homeostasis. We report that chronic ingestion of environmentally relevant subtoxic doses of cadmium does not impair the barrier function of the intestine in mice. On the other hand, chronic exposure to subtoxic doses of this heavy metal stimulates the growth of intestinal epithelial cells including Paneth cells, and increased production of antimicrobial peptides by Paneth cells. We also found that subtoxic doses of cadmium induce a dysbiosis of the gut microbiota that persist months after the end of the exposure. Importantly, 12–14 months after exposure to cadmium, the cadmium-exposed mice weighted more and had more fat content than age-matched controls suggesting that these mice have developed a state of chronic inflammation. Cadmium is also known to accumulates in soft tissues with a half-life of 10–20 years. Thus, to investigate the long-term effect of exposure to subtoxic doses of cadmium, control and post-menopausal mice (> 15 months old) previously exposed to cadmium were treated with dextran sodium sulfate (DSS) in drinking water. Post-menopausal mice previously exposed to cadmium developed more severe signs of DSS-induced colitis including weight loss, shortened large intestine. Furthermore, they exhibited enhanced Th17 responses in mucosal tissues. Our results provide new insights the impact of chronic exposure to subtoxic doses of cadmium, and possibly other heavy metals on gut homeostasis and colitis or colon cancer.
ECM1 is an essential factor for the determination of M1 macrophage polarization in inflammatory bowel disease in response to LPS stimulation
Authors: Sun Bing , Zhang Yaguang
Published: January 2023

Inflammatory bowel disease (IBD) comprises chronic relapsing disorders of the gastrointestinal tract characterized pathologically by intestinal inflammation and epithelial injury. Here, we uncover a new function of extracellular matrix protein 1 (ECM1) in promoting the pathogenesis of human and mouse IBD. ECM1 was highly expressed in macrophages, particularly tissue-infiltrated macrophages under inflammatory conditions, and ECM1 expression was significantly induced during IBD progression. The macrophage-specific knockout of ECM1 resulted in increased arginase 1 (ARG1) expression and impaired polarization into the M1 macrophage phenotype following LPS treatment. A mechanistic study showed that ECM1 can regulate M1 macrophage polarization through the GM-CSF/STAT5 signalling pathway. Pathological changes in mice with dextran sodium sulphate-induced IBD were alleviated by the specific knockout of the ECM1 gene in macrophages. Taken together, our findings show that ECM1 has an important function in promoting M1 macrophage polarization, which is critical for controlling inflammation and tissue repair in the intestine.
Role of NFKB signaling in Respiratory Epithelial Cells in Vaccine-Induced T Cell Immunity to Influenza
Authors: Luu Tracy NK , Kingstad-Bakke Brock , Marinaik Chandramaik , Larsen Autumn Rose , Suresh Marulasiddappa R
Published: January 2023

Respiratory epithelial cells (RECs) express Toll-like receptors (TLRs), produce both pro-inflammatory and anti-inflammatory molecules and function as immunological rheostats in the respiratory tract. The role of RECs in T cell responses to mucosal vaccination remains unknown. We have identified a combination adjuvant consisting of acrylic acid polymers in a nanoemulsion (Adjuplex; ADJ) and TLR4 agonist (glucopyranosyl lipid adjuvant; GLA) that elicits potent CD8 and CD4 T cell responses in the respiratory tract. Using mice (NFKB-REC) that conditionally lack NF-KB signaling in the RECs, we addressed the role of NFKB-dependent REC responses in regulating primary T cell responses to influenza virus nucleoprotein (NP), following intranasal vaccination with ADJ, GLA or ADJ+GLA. Surprisingly, we found that loss of REC NFKB signaling did not affect the magnitude of primary or recall antigen-specific T cell responses in the lungs and airways. Further, mucosal imprinting of CD8 T cells, as evidenced by expression of CD103 and CD49a, was minimally affected in NFKB-REC mice. Greater percentages of effector CD8 T cells in NFKB-REC mice expressed KLRG-1, regardless of the adjuvant but effector CD8 T cells in GLA mice showed elevated levels of CX3CR1, CD49a and granzyme B following challenge with influenza A virus. Preliminary evidence suggests that vaccine-induced T cell-dependent protection against influenza virus might be impaired in NFKB-REC mice.
Baby’s First Macrophage: characterizing Hofbauer Cell phenotype through gestation using unsupervised dimensionality reduction
Authors: Swieboda Dominika , Beaver Jacob , Johnson Erica LaShai , Skountzou Ioanna , Chakraborty Rana
Published: January 2023

Fetal placental macrophages (Hofbauer cells (HCs)) appear at 18 days post-conception and are maintained throughout pregnancy. Despite their identification more than a century ago, there are few studies characterizing the evolution of human HC phenotype through gestation. Using high-dimensional flow cytometry, we measured expression of CD68, CD80, CD86, HLA-DR, CD163, CD206, and CD209 on human HCs ex vivo. This panel includes markers previously validated on term human HCs for immune activation (CD80, CD86) and modulation (DC-SIGN, HLA-DR). To best capture the spectrum of HC diversity across gestation, we employed an unbiased approach to analyze processed datasets from HCs isolated from placentae 12–17 weeks gestational age (n=5), 17–24 weeks gestational age (n=7) and at term (n=5) using t-distributed stochastic neighbor embedding analysis (tSNE). HC populations were more diverse early in pregnancy and at term, compared to the second trimester; this is anticipated given the changing microenvironment following initial placental anchoring to eventual parturition. Marker expression heatmaps were generated to assess gestation-dependent changes in HC phenotype; HCs expressing activation markers were most frequent in early gestation, and reduced by term. HCs bearing markers of immune modulation were most frequent at mid-gestation, and least in early gestation. These results demonstrate the power of utilizing computational methods to analyze high-dimensional flow cytometry data collected from an understudied cell type and demonstrate how HCs phenotypically evolve during human pregnancy.
Targeting interferon regulatory factor 5 (IRF5) activation with a peptide inhibitor ameliorates clinical lupus in MRL/lpr mice
Authors: Song Su M , LaPan Margaret , Chopra Samin , Kampta Kyle , Guo Qin , Barnes Betsy J
Published: January 2023

Systemic lupus erythematosus (SLE) is associated with high levels of inflammatory cytokines as well as increased autoantibodies. Accumulating evidence show that loss and/or inhibition of the transcription factor interferon regulatory factor 5 (Irf5) ameliorates disease severity. Previously, we characterized a peptide inhibitor targeting Irf5 activation in the NZB/W F1 murine model of lupus and showed therapeutic protection when treated before disease onset. Here, we expanded our study to include a clinical treatment arm in the MRL/lpr lupus model after disease onset. 8 week-old MRL/lpr female mice were IP-injected with PBS or inhibitor at a dose of 100ug/mouse/injection on D0, D1, D4, D7 and D14. Proteinuria was monitored weekly; serum auto-antibodies were measured by ANA-HEp-2; anti-dsDNA IgG by ELISA, and kidney pathology was assessed. Blood was collected weekly to examine Irf5 expression and activation in immune cell subsets by imaging flow cytometry. At 8 weeks-old and before treatment, MRL/lpr mice were positive for anti-nuclear antibodies (ANA). At week 17, IgG and IgM staining on HEp-2 cells showed stronger fluorescence intensity in the control group (p=0.0053 for IgG; p<0.0001 for IgM). Serum anti-dsDNA IgG levels were also higher in the control group (782179 ± 337490 U/mL, n=7) as compared to treated (385947 ± 156487 U/mL, n=9). By week 17, 2 out of 9 mice in the control group had died, while all mice in the treated group survived. Proteinuria and body weights remained similar between groups. Data indicate that direct inhibition of murine Irf5 in multiple lupus models (NZB/W F1 and MRL/lpr) ameliorates disease severity. The inhibitor was also well-tolerated supporting the therapeutic utility of IRF5 inhibitors in the clinic.
Immune-phenotyping IRF5 genetic risk and in vivo targeting of Irf5 hyper-activation in mouse models of lupus
Authors: Barnes Betsy J , Song Su , Li Dan , Matta Bharati , De Saurav
Published: January 2023

Genetic variations within interferon regulatory factor 5 (IRF5) associate with risk of systemic lupus erythematosus (SLE) and mice lacking Irf5 are protected from lupus onset and severity. Exactly how IRF5 functions in the context of SLE disease progression remains unclear. We sought to determine how IRF5 genetic risk contributes to SLE disease onset and severity, and whether targeting IRF5 with select inhibitors would alleviate disease severity and mortality. Studies were performed in blood from genotyped healthy donors, SLE patients and mouse models of murine lupus. In NZB/W F1 lupus mice, we found that Irf5 is already hyper-activated before clinical onset in a cell type-specific manner. In healthy donors carrying IRF5 genetic risk, we detected IRF5 hyper-activation in the myeloid compartment that drives a pre-symptomatic SLE immune-phenotype. In SLE patients, IRF5 hyper-activation correlates with SLEDAI and dsDNA titers. To test whether IRF5 hyper-activation is a targetable function, we developed novel inhibitors that are cell permeable, non-toxic and selectively bind to the inactive IRF5 monomer. Treatment of NZB/W F1 and MRL/lpr mice with inhibitor attenuated lupus pathology by reducing serum ANA and dsDNA titers. In NZB/W F1 mice, we detected significant reductions in the number of circulating plasma cells and age- or autoimmune-associated B cells (ABCs), which alleviated kidney pathology and improved overall survival. In ex vivo human studies, the inhibitor blocked SLE serum-induced IRF5 activation in healthy immune cells and reversed basal IRF5 hyper-activation in SLE immune cells. This study provides the first in vivo pre-clinical support for treating SLE patients with an IRF5 inhibitor.
Cell therapy targeting follicular T cells as an innovative approach for treating autoimmune disease
Authors: Rangel Kelly M , Reighard Seth D , Ochayon David E , Krishnamurthy Durga , Shao Wen-Hai , Waggoner Stephen N
Published: January 2023

Systemic lupus erythematosus (SLE) is a debilitating and incurable autoimmune disease characterized by the uncontrolled production of autoantibodies and inflammatory damage to multiple organ systems. Dysregulated follicular helper T cell (TFH) responses are a cardinal feature and putative driver of disease in both mice and humans, where these cells contribute to unrestrained inflammation and autoantibody production by B cells. There is currently no safe and effective way to selectively eliminate TFH as a means to therapeutically alleviate diseases caused by these cells. Although cell surface biomarkers that are unique (lineage-specific targets) to TFH remain undefined, these cells express markedly more programmed cell death protein 1 (PD-1) than other immune cells. We recently engineered a novel programmed death-ligand 1 (PD-L1)-based chimeric antigen receptor (CAR) that when expressed on human natural killer (NK) cells, facilitated discriminate targeting of PD-1high follicular T cells while sparing other PD-1-expressing lymphocytes. The inherent specificity of this approach lies in the lower affinity interactions between PD-L1 and PD-1 compared to that likely achieved with a PD-1-specific single-chain variable fragment-based CAR. Here, we develop a mouse T cell version of the PD-L1 CAR platform to show that selective depletion of PD-1high T cells is safe and effective at undermining harmful autoreactive T and B cell responses in mouse models of SLE-like disease. We believe this affinity-based CAR approach for PD-1high target cell elimination represents a novel clinical tool for treatment of TFH-driven diseases like SLE.
Prevention of Type 1 Diabetes via a lipid Nanoparticle-Based Oral Immunotherapy that Targets Selective Lymphoid Tissues
Authors: ZHANG YICHUAN , Wang Jialu , Calderon-Colon Xiomara , Tiburzi Olivia , Lozano Marcos Iglesias , Patrone Julia B. , Raimondi Giorgio
Published: January 2023

Type 1 diabetes (T1D) is an untreatable autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Antigen presenting cells (APCs) malfunctions and aberrant accumulation of type 1 interferons contribute to T1D, and the inhibition of inflammatory cytokines signaling via JAK inhibitors like Tofacitinib (Tofa) has shown promise in the prevention of the disease. However, maintenance of a therapeutic concentration of these drugs is challenging and their prolonged use leads to significant side effects. Our objective was to design a controlled and localized delivery of Tofa via biocompatible lipid nanoparticles, Nanostructured Lipid Carrier (NLC), and assess if, via this strategy, a short-term administration would impact T1D development.

We identified a formulation of NLC that had negligible toxicity, could be readily taken up by multiple immune cells, and had a favorable Tofa encapsulation efficiency. Live animal imaging confirmed these particles have the unique property of accumulating in lymphoid tissues, particularly in spleen, pancreatic and mesenteric lymph nodes when administrated via oral gavage. Ex-vivo, Tofa-NLC rapidly delivered Tofa to mouse APCs preventing their maturation and the release of inflammatory cytokines. Importantly, short-term administration of Tofa-NLC via oral gavage at early (3-week-old) or late (10-week-old) stage in disease prone NOD mice promoted a significant delay of T1D onset. Cell profiling and in vivo challenges indicated that this therapeutic effect is likely due to localized inhibition of APCs maturation and promotion of anergy in diabetogenic T cells. These results highlight the versatility of this lipid nanoparticle-based drug delivery strategy.
Saliva plebeia ameliorates articular carttilage degration of osteoarthritis induced rats
Authors: Lee Sung Hyen , Lee Eun-Byeol , Choi Ji-Hye , Ku Sae-Kwang , Choi Beom-Rak , Kim Jung-Bong , Jang Hwan-Hee , Hwang Kyung-Ah , Park Shin-Young
Published: January 2023

Osteoarthritis (OA) is the most prevalent articular disease in the elderly. The process is characterized by changes in the structure and function of the articulation, mainly due to a degeneration that takes place in the articular cartilage (AC). OA is characterized by destruction of AC and is the most common clinical syndrome of joint pain accompanied by varying degrees of functional limitation and reduced quality of life. This study was conducted to evaluate the effects of Saliva plebeia (SP) on AC degration of osteoarthritis induced rats. Salvia plebeia R.Br is an annual or biennial plant that grows in numerous countries, including Korea, China, and India. It is used as a traditional medicine to treat inflammatory diseases. The active components of SP comprise flavonoids, diterpenoids, and lignin. It is considered that appropriate treatment of SP inhibits surgical-induced OA through anti-inflammatory and AC preserve effects. In the histopathological observation, OA control rats showed marked increases of surface cartilage damages and clone formations, and decreases of chondrocytes and Safranin O stain intensities on the both femur and tibia AC. Significant increases of the Mankin scores were detected in surgical-induced OA control rats as compared with those of normal control rats. However, SP significantly improved surface cartilage damages, Mankin scores, mean femur and tibia AC thicknesses, and inflammatory cell numbers. It is expected that SP may be used as a novel potent therapeutic regime for various OA though detail mechanism studies should be needed in near future.
ST8Sia6 overexpression in beta cells prevents onset of autoimmune diabetes in the murine NOD model
Authors: Choe Justin , Belmonte Paul , Rajcula Matthew , McCue Shaylene , Shapiro Michael J , Shapiro Virginia
Published: January 2023

Cell surface hypersialylation is used by malignant cells as a survival mechanism, leveraging interactions with Siglec receptors to dampen the immune response. In this study, we aimed to harness this mechanism to prevent autoimmune rejection of beta cells in the context of type 1 diabetes. Mice engineered to overexpress the sialyltransferase ST8Sia6 in beta cell (BST) were generated and crossed to the non-obese diabetic (NOD) mouse for 10 generations. The blood glucose of these mice compared to non-overexpressing littermates was assessed twice weekly to determine diabetes incidence, defined as 3 consecutive blood glucose readings above 250 mg/dL. Analysis of female BST mice aged 15 weeks or greater showed no incidence of overt diabetes, while littermates developed diabetes. BST males aged 15 weeks or greater also had no disease incidence, while littermate controls also became diabetic. An insulin tolerance test demonstrated no significant difference between BST mice and littermates, though the BST had significantly higher blood glucose during a glucose challenge, possibly representing insulin hypersecretion by littermate islets in a stressed pre-diabetic state. Histologic analysis of young BST and littermate pancreata showed no differences, with similar levels of insulitis at 8 weeks. Thus, the overexpression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD model and presents a novel path forward for prevention of islet transplant rejection.
Blockade of the neurokinin-1 receptor in keratinocytes prevents neuroinflammation and decreases innate and adaptive immune responses in the skin
Authors: Bandyopadhyay Mohna , Morelli Adrian E , Erdos Geza , Sumpter Tina L , Tkacheva Olga , Shufesky William , Falo Louis D , Larregina Adriana T
Published: January 2023

Substance P (SP) is a proinflammatory neuropeptide that following Ag entrance in peripheral tissues signals via the neurokinin 1 receptor (NK1R) to initiate innate and support adaptive cellular immune responses. These mechanisms underlie chronic skin inflammatory disorders like contact dermatitis (CD). Here we propose to develop an immunosuppressive method to prevent and treat CD by blocking the effects of SP during skin sensitization with haptens. We utilized microneedle arrays to efficiently deliver the hapten 2,4-dinitrocholorobencene (DNCB) and NK1R antagonists simultaneously to the skin of C57/BL6 mice. This approach, restrained neuroinflammation, increased T regulatory cells and decreased the viability of Th1 and Tc1 biased cells in the draining lymph nodes. Together these effects inhibited local and systemic CD and prevented its relapses. Using the Cre-Lox P system, we demonstrate that specific deletion of the NK1R in keratinocytes but not in leukocytes inhibited the sensitization phase of CD by blocking the release of IL-1β and IL-6. Whereas deletion of the receptor in keratinocytes or in dendritic cells was necessary to abrogate the adaptive cellular immunity. Our data demonstrate the possibility of preventing the development of cellular immunity by engineering the skin microenvironment to restrain the effects of neuroinflammatory peptides accounting for the onset of chronic skin inflammatory diseases.
The Endocannabinoid System: a new anti-inflammatory target in rheumatoid arthritis
Authors: TAN KYLE BOON CHIN , Murray Elaine K , Watterson Steven , Alexander Denis , Linden James , Gibson David S
Published: January 2023

Rheumatoid arthritis (RA) is characterised by the loss of regulatory T cell (Treg) mediated suppression of inflammatory T helper cells. RA patients with loss of immune tolerance display elevated levels of circulating pro-inflammatory cytokines. The endocannabinoid system (ES) present on immune cells may represent a novel target for development of anti-inflammatory treatments. We screened the therapeutic potential of a phytocannabinoid compound, GL1b, in an ex vivo, peripheral blood mononuclear cell (PBMC) inflammatory model. PBMCs were cultured with CD3/CD28 and IL-2 to activate and expand T cells. Inflammation was induced by phorbol 12-myristate-13-acetate (PMA)/ionomycin activation, over 4 hours. Inflamed PBMCs were treated with 0, 10, 20 and 50 μM GL1b for 24 hours, then harvested for flow cytometry analysis of Tregs. Cytokine mRNA expression and conditioned media concentration were analysed with qRT-PCR and ELISA, respectively. Results will be fed into in silico model. 50 μM GL1b significantly suppressed the total Treg population (p<0.05), and relative numbers of CD69+, FOXP3+ and CTLA4+ Treg subpopulations decreased in a dose-responsive manner. A trend toward increased mean fluorescent intensity (MFI) of FOXP3 and CTLA4 was observed at 10 μM, relative to vehicle. A significant (p<0.01) decrease was observed in IL-6 mRNA relative to vehicle with 50 μM GL1b, whereas at 10 – 20 μM concentrations, a trend toward lower IL-6, TNFα, IL-10 expression was evident. Conversely, secreted cytokine concentrations were increased at 10 – 20 μM GL1b, relative to vehicle. Although the phytocannabinoid GL1b reduces the overall Treg population, activated and suppressive Treg subpopulations are elevated and cytokine gene expression reduced.
In Vivo Generation of Autoantigen-specific Treg Cells in Treating Experimental Autoimmune Uveitis Without Compromising Anti-Tumor Immunity
Authors: Chen Jun , Chen Zilin , Chong Wai Po , Zhang Tian , Zhou Hongyan , Chen Wanjun
Published: January 2023

Generation of autoantigen-specific regulatory T (Treg) cells that suppress tissue-specific autoimmunity without compromising beneficial immune responses is the holy-grail for immunotherapy to autoimmune diseases. In a model of experimental autoimmune uveitis (EAU), we show here that we successfully generated retinal autoantigen-specific Treg cells in vivo by inducing CD4+ T cell apoptosis with antibody followed by administration of retinal autoantigen peptides (e.g., interphotoreceptor retinoid-binding protein) in mice with established EAU. We demonstrated that the therapeutic approach resulted in long-term remission of ocular inflammation and rescue of retinal function in the eyes of the mice with EAU. Mechanistically, this therapeutic effect was mediated by the generation of retinal antigen-specific Treg cells that inhibited uveitogenic Th17 response in TGF-β and IL-10 dependent manner. Importantly, these tolerized mice did not compromise their anti-tumor immunity in a model of melanoma. Thus, we have discovered a specific immunotherapy of EAU by in vivo generation of retinal antigen-specific Treg cells, which should have implication for patients with autoimmune uveitis.
Inhibition of Aurora-A recruited CD8+ T cells infiltration via mediating IL-10 production of cancer cells
Authors: Yang Jing , Han Jing , Jiang Zhen , Wang Chennan , Chen Xin , Li Rongqing , Sun Na , Liu Xiangye , Zheng Kuiyang , Ikezoe Takayuki
Published: January 2023

It has been shown that approximately 10% of infiltrated CD8+ T cells could be the tumor-specific intratumoral CD8+ T cells in colorectal cancer. While, inhibition of Aurora-A, a member of serine/threonine Aurora kinase family, by alisertib promoted the percentage of infiltrated intratumoral CD8+ T cells. We therefore assumed that targeting Aurora-A could be a therapeutic strategy for recruitment of intratumoral CD8+ T cells.

In CT-26 tumor model, blockade of Aurora-A with alisertib slowed the tumor growth in association with an increased infiltration, activation and expansion of intratumoral CD8+ T cells. We further analyzed the role of Aurora-A in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model, and found that AURKA deficiency significantly decreased tumor formation, in parallel with an increase in infiltration of intratumoral CD8+ T cells. Gene expression profiling indicated that Il10ra expression was obviously increased in intratumoral CD8+ T cells sorted from alisertib-treated tumors. Antibody-mediated blockade of IL-10Ra dramatically attenuated the anti-tumor ability of alisertib in association with reducing the percentage of intratumoral CD8+ T cells. IL-10Ra was the key mediator of IL-10. We herein examined the levels of IL-10 and found that the levels of IL-10 in the serum of AURKA deficiency mice treated with AOM/DSS were elevated. Additionally, IL-10 obviously promoted migration of CD8+ T cells in vitro. Blockade of Aurora-A increased the transcriptional levels of IL-10.

In conclusion, inhibition of Aurora-A could be useful for recruiting infiltration of intratumoral CD8+ T cell by mediating IL-10 production.
CD4 Cytotoxic T cells play a critical role in anti-tumor protection
Authors: Thiault Nicolas , Husain Mushtaq , Chen Angeline , Larange Alexandre , Cheroutre Hilde
Published: January 2023

The CD4 T cell lineage is established in the thymus by the transcription factor, ThPOK. In the periphery, ThPOK maintains the CD4 T helper commitment and allows the differentiation to different helper fates in response to stimulation with cognate antigens. We demonstrated that in the intestine, chronic stimulation of CD4 T cells results in the loss of ThPOK expression leading to the reprogramming of the T helper cells to cytotoxic T lymphocytes (CTL), expressing notably CD8 T cells attributes. Continuous activation in a non-immunogenic environment is also a hallmark of tumor progression.

We investigated if the tumor environment supports the generation of CD4 CTL and moreover if they play essential roles in anti-tumor protection. Using the well-defined melanoma B16 model, we observed that CD4 T cells responding to the tumor uniquely lose ThPOK expression. To address the importance of the CD4 CTL in the tumor immune surveillance, we engineered two unique mouse models, in which either all the CD4 T cells are forced to reprogram to CTL or else, prevented from reprogramming to CTL. Using these models, we observed that the forced CD4 CTL eradicate melanoma cells, whereas the CD4 cells which cannot lose ThPOK and reprogram to CTL are ineffective against the tumor growth. These findings underscore the importance of CD4 CTL reprogramming in protective immunity. Finally, we developed a culture system to induce the reprograming of CD4 Th to CTL in vitro. We injected in vitro-generated CD4 CTL alone or with a co-transfer of CD8 T cells into Rag deficient hosts bearing tumor. As compared to the transfer of the CD4 CTL or CD8 CTL alone, the cooperation of the cytotoxic effector cells displayed a strong synergized effect on long term protection against tumor growth.
Response to FOLFIRINOX neoadjuvant chemotherapy is associated with a high prevalence of Tbet expressing T cells in pancreatic cancer patients
Authors: Peng Hui , James Alston , Cullinan Darren R , Mudd Jacqueline , Takchi Rony , DeNardo David G , Fields Ryan C , Goedegebuure Simon Peter , Hawkins William G
Published: January 2023

FOLFIRINOX is the first line treatment option for patients with pancreatic ductal adenocarcinoma (PDAC). Neoadjuvant chemotherapy has a beneficial effect on overall survival in PDAC in comparison with upfront surgery and adjuvant therapy. However, chemotherapy has not yet been successfully combined with immune therapy in PDAC. Herein, we characterize the immunologic effect of FOLFIRINOX and thus provide a rationale for future combination with immune therapy in PDAC. Peripheral Blood Mononuclear Cells were obtained from treatment-naïve (n=20) and FOLFIRINOX-treated patients (n=15) with primary PDAC tumors. Immune cell subset composition was assessed by using mass cytometry (CyTOF). Response to FOLFIRINOX was defined as a reduction in tumor size by more than 30%. 8 of 15 patients were considered responders with a range from 32 to 60% reduction in tumor volume. Responders to FOLFIRINOX showed a significantly higher proportion of CD8+ T cells and lower proportion of monocytes in their peripheral blood compared to treatment naïve patients (both p<0.05). Responders to FOLFIRINOX treatment also showed a significantly lower percentage of CD39+ Treg cells and a significantly higher percentage of CXCR3+CCR6+ Th1/Th17 cells compared to treatment naïve patients (both p<0.05). Lastly, FOLFIRINOX responders showed significantly higher proportions of Tbet expressing CD4+ and CD8+ T cells compared to non-responder patients (both p<0.05). Taken together, our study reveals that neoadjuvant chemotherapy with FOLFIRINOX could enhance functional T cells and down-regulate suppressor cells in circulation. Thus, combination neoadjuvant FOLFIRINOX chemotherapy with immunotherapy may improve clinical outcome.
CD40 signaling induces type I interferon and immune control in mouse pancreatic cancer lacking the CXCL12-coat
Authors: Li Jiayun , Wang Zhikai , Fearon Douglas T
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) has a low 5 year survival rate of 8.5% because of its late diagnosis and resistance to the existing therapies. Patients with PDA who are microsatellite stable do not respond to check point blockade immunotherapy due to impaired T cell infiltration, which is mediated by the CXCL12-coat on cancer cells (Wang Z, et al. bioRχiv). In mouse hepatic metastases established with PDA cells from the KPC model of PDA, Krt19-CRISPR/Cas9-edited tumors lacking the CXCL12-coat showed a type I interferon (IFN) response as well as T cell infiltration and activation compared to the control scramble tumors. By RNA FISH, we found that IFNb1 is dominantly produced by CD40+ cells, and not by SiglecH+ plasmacytoid dendritic cells. RNA sequencing data showed up-regulation of Cd40lg in the Krt19-edited tumors compared to the scramble tumors. Administration of antagonistic anti-CD40 antibody prior cancer cells inoculation abolished IFNb1 production, T cell accumulation and activation. To confirm the role of CD40, administration of agonistic anti-CD40 antibody to mice bearing wild type PDA tumors elicited intra-tumoral IFNb1 production in CD11b+ CD11c+ myeloid cells. The T cell infiltration and activation induced by agonistic anti-CD40 antibody treatment sensitized both subcutaneous pancreatic tumors and hepatic metastases to anti-PD1 treatment. These findings indicate that the expression of CD40L elicits an intra-tumoral type I IFN response that is required for immune control of PDA tumors in which CXCR4 signaling is impaired.
Promoting antitumor immunity and increased T-cell function by depletion and blockade of the immunosuppressive cell compartment in murine cancer
Authors: Smith Timothy M , Camarena Carmen , Bear Harry , Martin Rebecca
Published: January 2023

Tumor growth occurs due to a failure of the immune system to recognize and destroy malignant cells. The cause of breakdown in immune surveillance is at the systemic level and at in the tumor microenvironment. These changes can even occur in the tumor cells themselves to make them less detectable by anticancer immune cells such as downregulation of tumor-associated antigens or decreased expression of antigen presenting molecules such as MHCI. In addition, many cancer types will upregulate immune checkpoint molecules, such as PD-L1 which binds to PD-1 on cytotoxic T-cells to suppress antitumor immune cell function. Immune surveillance can also be curtailed by the presence of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and T-regulatory cells (T-regs) which both serve to dampen antitumor immunity. Our lab utilizes a metronomic low-dose therapeutic approach to examine the effects of anticancer drugs against murine breast cancer and the effects on the immunosuppressive cell compartments. Recent data has shown a reduction in T-regs following treatment with cyclophosphamide, a common chemotherapy. We have also shown that guadecitabine, a second-generation DNA methyl-transferase inhibitor, depletes MDSCs. Here we show that combining low-dose metronomic treatments of guadecitabine and cyclophosphamide leads to a depletion in splenic and tumor MDSCs and T-regs, increased CD8+ T-cell function in the tumor, as well as a reduction in tumor size in FoxP3 reporter mice bearing triple-negative 4T1 tumors. In addition, preliminary studies suggest this antitumor effect is amplified by combining guadecitabine and cyclophosphamide with checkpoint inhibitors such as anti-PD-1, in vivo.
A new Natural Killer cell therapy to target relapse Acute Lymphoblastic Leukemia
Authors: Vicioso Yorleny M , Beck Rose , Gram Herman , Zhang Keman , Asthana Abhishek , Wong Derek , Letterio John , Parameswaran Reshmi
Published: January 2023

Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer. Drug resistance and relapse are two major problems in ALL. Pre-B ALL cells express B-cell activating factor receptor (BAFF-R) on their surface, which is not present on normal pre-B cells. This makes it an attractive therapeutic target for ALL. We used a BAFF-R antibody optimized for antibody dependent cell cytotoxicity (ADCC) to enhance NK cell mediated killing of drug resistant and relapsed ALL cells, which are difficult to treat by conventional chemotherapy. We found that anti-BAFF-R antibody enhanced NK cell mediated killing of drug resistant ALL cells in vitro. Early treatment with anti-BAFF-R antibody and NK cells significantly reduced disease burden in xenograft ALL models and increased overall mice survival. However, in advanced disease, the efficacy of NK cells to mediate ADCC is reduced. ALL cells are known to produce TGF beta, a cytokine that causes NK cell dysfunction. We found that ALL cells produce TGF-β and co-culturing ALL cells with NK cells lead to a decrease in CD16 expression on NK cells, which is reversible by addition of EW-7197, a potent TGF-beta receptor I inhibitor. We found that EW-7197 treatment improved anti-BAFF-R antibody mediated ADCC of drug resistant ALL cells, in vivo, even if treatment is started late. Our data validate this novel BAFF-R antibody as a very promising tool for pre-B ALL therapy.
Combination of TEW-7197 and adoptively transferred NK cells leads to Improved Antitumor Responses in vivo
Authors: Kim Jungwon , Lim Seon Ah , Kim Tae-Jin , Lim Siyoung , Kim Jiyoung , Kim Jeongsoo , Kim Junghee , Lee Eunju , Park Sunghee , Kim Seong-Jin , Lee Kyung-mi
Published: January 2023

Transforming growth factor beta (TGF-β) supports tumor cell proliferation, metastasis, and invasion in later stages of cancer while suppresses the proliferation and survival of lymphocytes including Natural Killer (NK) cells. The current therapeutic approaches of targeting TGF-β include inhibiting its transcription through anti-sense oligonucleotides, blocking TGF-β receptor binding through the use of antibodies, or suppressing the TGF-beta signaling pathways using small molecules, such as TEW-7197. Here, we report the anti-tumorigenic role of TEW-7197, an orally administered TGF-b receptor kinase ALK5 inhibitor, by synergizing with adoptively transferred NK cells in the human xenograft A375 melanoma model. Addition of TEW-7197, at its therapeutic doses, in cultures of A375 melanoma cells did affect neither the growth nor the apoptotic killing of tumor cells in vitro, however, it caused reduction of tumor cell growth in vivo. This was possibly due to the upregulation of TGF-b mRNA upon tumor implantation in the xenograft tissues, which became a target of TEW-7197. Adoptive transfer of ex-vivo expanded human NK cells into these mice also inhibited tumor growth but co-administration of TEW-7197 resulted in significantly higher degree of tumor regression, by both inhibiting TGFb signaling as well as increasing the NK cell’s availability within tumor tissues. These data highlight that the anti-tumor response exhibited by TEW-7197 and NK cells are distinct, and can be synergized by the combination of the two. Therefore, our data provide the scientific rationale for the combination therapies of TGF-b small molecule kinase inhibitors with adoptively transferred NK cells in advanced cancer patients.
Immune microenvironments of early onset vs late onset colorectal cancers
Authors: Anand Sudarshan , Gardner Ivy , Siddarthan Raga , Watson Kate , Ruhl Rebecca , Tsikitis Liana V
Published: January 2023

Over the past 40 years, the incidence of rectal cancer in patients 20–39 years old has quadrupled. The immune microenvironment in colorectal cancer is a key player in disease progression, therapy response, and overall survival. However, the role of the immune microenvironment in early onset rectal cancer (less than 50 years old) has not been elucidated. We profiled the immune microenvironments of a cohort of early onset vs late onset (over 65 years) colorectal cancers from FFPE surgical specimens and biopsies from patients. We compared our dataset to a publicly available database (GSE8721) of gene expression profiles for 203 rectal cancer and 160 matched normal mucosa samples. We identified genes that were differentially expressed between early and late onset rectal cancer groups and validated our results with qPCR. Interestingly, we noted differences in immune signatures depending on the anatomical site (colon vs rectum). Two genes, FLT3 and SAA1, were found to have increased expression in both our patient samples and the GSE8721 rectal cancer database. FLT3 codes for fms-like tyrosine kinase receptor and activating mutations of this gene are present in 30% of acute myeloid leukemia cases and is associated with a worse prognosis. SAA1 codes serum amyloid A1 which is an acute phase protein that is highly expressed in response to inflammation. While FLT3 inhibitors did not directly affect tumor cell proliferation and cell death in vitro, we observed a decrease in tumor burden in vivo. Our data indicates that there are distinct changes in the immune microenvironment between early and late onset rectal cancer. We propose that understanding these changes will enable the development of age dependent immune modulation for this disease.