bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-17 signaling inducibly degrades ABIN1, a constitutive inhibitor of inflammatory signaling
Authors: Cruz Juan Agustin , Childs Erin , Amatya Nilesh , Simpson-Abelson Michelle , Garg Abhishek , Beyaert Rudi , Aneskievich Brian , Ma Averil , Gaffen Sarah L
Published: January 2023

IL-17 is a pro-inflammatory cytokine that drives autoimmune diseases and promotes immunity to fungi. IL-17 binding to the IL-17 receptor initiates recruitment of adaptor proteins that activate NF-κB. IL-17 also induces expression of A20, a feedback inhibitor that restricts NF-κB activation and limits IL-17-dependent inflammation. ABIN1 is an A20 binding protein that has been implicated in autoimmunity in human genetic studies, so we hypothesized that ABIN1 might also play a role in restricting IL-17-dependent signaling. Indeed, ABIN1 inhibited the IL-17 pathway based on RNA silencing analyses. Specifically, the expression of the IL-17-dependent gene lipocalin 2 (Lcn2) increased at both basal levels and after stimulation upon ABIN1 knockdown. Consistently, reconstitution of ABIN1-deficient fibroblasts with ABIN1 also decreased both Lcn2 expression and promoter activation. However, while ABIN1 mRNA increased after IL-17A stimulation, ABIN1 protein progressively decreased following its inducible phosphorylation. Although feedback loops are commonly seen in signaling pathways, differential regulation of mRNA and protein of the same gene are not as common. Thus, we now propose that ABIN1 serves as a constitutive inhibitor of NF-κB signaling. While ABIN1 mRNA is induced by NF-κB at the mRNA level, removal of the ABIN1 protein is required to initiate signal transduction. Unexpectedly, we discovered that A20 is dispensable for both IL-17-mediated degradation of ABIN1 and for ABIN1-dependent inhibition of IL-17 signaling. Altogether, these findings suggest that ABIN1 is a tonic NF-κB inhibitor, and that IL-17 signaling induces its degradation independently of A20 to enhance expression of NF-κB-dependent genes such as Lcn2.
Th17 cells modulate the LN microenvironment through IL-17 dependent regulation of fibroblastic reticular cells
Authors: Majumder Saikat , Revu Shankar , Du Fang , McGeachy Mandy J
Published: January 2023

Integrins play a critical role in the function and migration of Th17 cells in CNS, and blockade of inflammatory T cell migration is used therapeutically in multiple sclerosis. Our previously published data demonstrated a critical role for αvβ3 in driving encephalitogenic Th17 cell migration in experimental autoimmune encephalomyelitis (EAE), in an IL-23R-dependent manner. Herein, we show that fibronectin, a ligand for integrin αvβ3, is significantly upregulated in the dLNs of EAE mice. Strikingly, the expression of fibronectin in dLN was significantly reduced in IL23R−/− mice following immunization. Fibronectin expression increased in dLNs during the progression of EAE with similar kinetics but delayed peak compared to IL-17. Since IL23R regulates IL-17 production, we analyzed LN from IL-17−/− and IL17Ra−/− mice immunized for EAE, and observed a similar defect in fibronectin expression in the absence of IL-17 signaling. CD45−gp38+CD31− fibroblastic reticular cells (FRCs) in the T cell zone are the major producers of fibronectin in the dLNs, and these stromal cells expand during EAE. Although IL-17RA−/− LN appeared similarly enlarged following immunization, indicating an inflammatory response, the number of FRCs was decreased in immunized IL17Ra−/− LN, corresponding to decreased fibronectin. Taken together, our data suggests that during priming of the immune response, and before migrating to their peripheral target tissue, Th17 cells modulate the local LN environment in which they are activated through production of IL-17 to promote FRCs expansion and induction of ECM. This could have long-term consequences in the host upon future infectious challenge, as well as influencing chronicity of Th17-mediated autoimmune disease.
Visualizing antigen-specific CD8 T cell dynamics in the small intestine
Authors: Thompson Emily A , Mitchell Jason , Beura Lalit K , Nelson Christine , Fife Brian T , Vezys Vaiva
Published: January 2023

The small intestine (SI) represents the largest mucosal barrier between the environment and the host. As such, the SI limits both normal and pathogenic microbe invasion and must balance immune tolerance and activation. CD8ab T cells constitute a dominant lymphocyte population in the SI and contribute to both of these processes but detailed investigation into their in situ behavior in response to various stimuli is lacking. We visualized Ag-specific CD8 T cell migration in the SI using two-photon microscopy to understand CD8 T cell immunosurveillance. We found that pathogen-specific CD8 T cells are initially slowed early after infection but regain motility directly after Ag clearance. At a memory time point, CD8 T cells exhibited slower migration concurrent with acquisition of a resident memory phenotype. Current studies are focused on determining what is required for CD8 T cell motility throughout the course of infection. Additionally, as tolerance reversal and expansion of self-specific CD8 T cells has recently become a focus of cancer therapy, we characterized in situ behavior of self-specific CD8 T cells. Using a mouse expressing a model Ag in the SI as a self-protein, we observed self-specific CD8 T cells before and after tolerance reversal. Tolerant CD8 T cells migrate significantly faster than pathogen-specific cells at acute time points after infection. Contrastingly, ex-tolerant CD8 T cells were comparable to pathogen-specific CD8 T cells indicating that indeed their in situ scanning behavior had been altered upon tolerance reversal. These observations have provided valuable insight into CD8 T cell motility within the SI and can inform targeted treatment options and vaccines to ensure effective immunosurveillance of this tissue.
HDAC3 controls homeostasis of retina-resident microglia derived from embryonic precursors
Authors: Mi Xiaofan , Yao Yi , Liu Queping , Kumar Ashok , Gao Hua , Zhou Li , Mi Qing-Sheng
Published: January 2023

Microglia are tissue resident macrophages of the central nervous system (CNS) that function in CNS maintenance and innate immune defense. Macrophages are mediators of immune threshold and thus implicated in disease pathogenesis. Compared to research efforts on CNS microglia, relatively little is known about microglial development or homeostasis in the retina. Histone deacetylases (HDACs) are enzymes known to regulate gene expression by modifying chromatin structure. Here, we use a murine model with conditional deletion of HDAC3 induced by Colony Stimulating Factor 1 Receptor (Csf1r) Cre to determine the consequence of HDAC3 depletion on microglial development and homeostasis in the retina. Macrophages were identified using CD11b and F4/80 markers. Retinal microglial populations were assessed in mice at embryonic day 14.5 (E14.5) as well as at birth and 4-weeks old. A population of CD11b+F4/80bright retinal microglia were identified as early as E14.5, indicating that retinal microglia are derived from seeding of embryonic progenitors from the yolk sac or fetal liver. Although the frequency of retinal microglia was comparable at embryonic stage and at birth, it was significantly reduced (P = 0.008) in HDAC3 KO mice at 4 weeks old. Moreover, retinal microglia were partially radio-resistant, and could be repopulated by fetal liver cells transferred from WT but not HDAC3 KO embryos. This is the first report to show that retina-resident microglia are derived from embryonic precursors and that HDAC3 controls microglial homeostasis in adult retina. HDAC3 may serve as a promising therapeutic target for microglial immune-reactive pathologies such as age-related macular degeneration.
Utilizing self-specific CD8 T cells for the treatment of cancer
Authors: Nelson Christine , Quarnstrom Clare , Thompson Emily , Vezys Vaiva
Published: January 2023

CD8 T cells are potent effectors that monitor the body for infections as well as malignancies. CD8 T cell infiltration into tumors is correlated with better prognoses in many cancers. However, the tumor microenvironment is often toleragenic, which treatments like checkpoint inhibitors seek to overcome. Such treatments have been met with some success, but their benefits remain elusive for a subset of patients. Using a strict model of tolerance to intestinal self-antigen, we show that newly activated CD8 T cells respond best to checkpoint blockade therapy, while CD8 T cells in a toleragenic environment, even for a short time, do not respond. We have developed a therapeutic vaccination strategy that generates a targeted CD8 T cell response with renewed susceptibility to checkpoint blockade. Using heterologous prime boost vaccination model, we have refined tolerance reversal to a subset of self-antigen specific CD8 T cells, and eliminated the need for broadly immunostimulatory treatments. This model has been used to reverse tolerance in CD8 T cells specific for engineered antigens in the small intestine as well as naturally occurring self-antigens in the skin. The expanded populations of skin-specific CD8 T cells can be utilized for the treatment of melanoma. These melanocyte-specific CD8 T cells are functional and generate tissue resident memory (Trm) populations throughout the body, including the skin. Additionally, melanocyte-specific CD8 T cells can cooperate with neo-antigen specific CD8 T cells to aid in tumor clearance. By investigating tolerance reversal in self-specific CD8 T cells we have uncovered ways to improve upon current anti-cancer treatments and developed potentially translatable anti-cancer vaccine strategies.
Intestinal homeostasis governed by mesenchymal stromal cells
Authors: Nagashima Kazuki , Takayanagi Hiroshi
Published: January 2023

The mammalian intestine harbors numerous bacteria and contains a large number of immune cells. A continuous dialogue between immune cells and luminal microbes is critical for the maintenance of the intestinal homeostasis. The intestinal epithelium is a single-layered cell sheet that separates numerous microbes and the immune system. The appropriate development and maintenance of the epithelium are essential for the clearance of pathogens and the prevention of excess immune responses against commensal microbes. Microfold (M) cells are epithelial cells specialized for bacterial sampling and initiate germinal center formation and IgA production in the gut associated lymphoid tissues (GALTs). Here, we show that previously unrecognized mesenchymal stromal cells regulate an epithelial function and induce IgA production in the GALTs. The impairment of these mesenchymal stromal cells caused a reduction in IgA production against commensal bacteria, resulting in the altered composition of the gut microbiota. These results suggest that these mesenchymal stromal cells contribute to the maintenance of the intestinal homeostasis.
IL-13Rα2 modulates inflammatory and metabolic effects of IL-33 by regulation of tissue IL-13 responses
Authors: Duffen Jennifer Parker , Zhang Melvin , Hammerman Katherine , Morin Jeffrey , Nocka Karl , Kasaian Marion
Published: January 2023

In experimental models of obesity, IL-13 can overcome insulin resistance by promoting eosinophilic inflammation and M2 macrophage differentiation in adipose tissue. Endogenous IL-13 levels can be modulated by the IL-13 decoy receptor, IL-13Rα2, which inactivates and depletes the cytokine, and by IL-33, which triggers release of IL-13. We propose that elevation of tissue IL-13 levels resulting from IL-13Rα2 deficiency, with or without IL-33 administration, counteracts the metabolic stress of obesity produced by a high fat diet. IL-13Rα2 expression was found to be elevated in adipose tissue of obese mice. Mice deficient in IL-13Rα2 had high expression of M2 markers in adipose tissue, consistent with increased IL-13 activity. IL-33 administration raised serum and tissue IL-13 concentrations, produced splenomegaly, elevated tissue eosinophils, macrophages, and ILC2s, and induced an inflammatory gene expression signature in adipose tissue. IL-33 reduced body weight, adipocyte size and fasting blood glucose to a greater extent in IL-13Rα2-deficient mice compared to wild-type littermates, and these changes were attenuated in mice lacking IL-13. In contrast, IL-33-induced splenomegaly and eosinophilia were exacerbated in the absence of IL-13, associated with impaired induction of the soluble decoy receptor, ST2, and unrestrained IL-33 activity. In the context of high fat diet, IL-33 administration resulted in damaging systemic inflammation, which was enhanced in IL-13Rα2-deficient mice. These findings suggest that IL-13Rα2 regulation of tissue IL-13 levels can result in a skewed M2 bias under conditions of metabolic stress, and implicate IL-13Rα2 as a critical checkpoint to limit adverse effects of the IL-33/IL-13 axis.
Deciphering the role of Interleukin-33, a Janus-Faced cytokine, in mast cell-mediated responses in allergic asthma using systems immunological methodologies
Authors: Pushparaj Peter Natesan , Rasool Mahmood , Naseer Muhammad Imran , Abuzenadah Adel , Chaudhary Adeel , Al-Qahtani Mohammed , Gauthaman Kalamegam
Published: January 2023

The prevalence of Allergy and Asthma has been increasing over the past decade, and about 300 million people suffer from these diseases worldwide. Interleukin-33 (IL-33), an Interleukin-1 family of cytokine, binds with the IL-33 receptor (IL-33R) to elicit an array of molecular and cellular processes in immune cells. Here, we have studied the role of IL-33 in mast cell-mediated responses and its impact on allergic asthma using systems immunological methodologies. We have obtained all the microarray datasets from the Gene Expression Omnibus (GEO) related to the IL-33 treated mast cells and allergic asthma. The datasets were analyzed using Genespring GX12.6 (Agilent Technologies, USA) to classify the differentially expressed genes (P<0.05) compared to untreated controls. The ingenuity pathway analysis (Qiagen, USA) of the IL-33 triggered differentially expressed genes in mast cells have revealed that the canonical pathways such as T-helper cell differentiation, the role of macrophages, fibroblasts, endothelial cells in autoimmunity, and chemokine signaling were significantly (P<0.05) upregulated. Besides, in-depth analyses of the IL-33 induced differentially expressed genes in mast cells and allergic asthma, using the comparison analysis module in IPA, showed a significant attenuation in HCK and other related signaling networks. However, the stimulation of both Th1 and Th2 associated cytokines by IL-33 could differentially regulate allergic asthma. Thus, IL-33 is a Janus-Faced cytokine in mast cell mediated allergic responses and, hence, requires further in vitro and in vivo studies to evaluate the therapeutic importance.
The role of eosinophils in adipose tissue inflammation and metabolic function
Authors: Bolus William Reid , Gutierrez Dario , Anderson-Baucum Emily , Kennedy Arion , Hasty Alyssa
Published: January 2023

Adipose tissue (AT) inflammation in obesity is mediated by immune cells and correlates with metabolic disorders (e.g. insulin resistance & type 2 diabetes). After the seminal 2011 paper by Wu et al., implicating eosinophils improve AT health, a series of studies sought to define mechanisms of this initial discovery. In lean AT, eosinophils are abundant & promote M2-like macrophages (MΦ). In obesity the proportion of AT eosinophils declines, concomitant with increased inflammation & M1-like MΦ. Using eosinophil deficient mice (ΔdblGATA) fed high fat diet, Wu et al. showed a lack of AT eosinophils correlated with increased weight & impaired glucose tolerance. In contrast, IL-5 transgenic (IL5tg) mice with excess AT eosinophils weigh less & have improved glucose tolerance. We recently published that CCR2−/− mice have an unexpected surge in AT eosinophils during obesity, associated with M2MΦs, but without the metabolic benefits of IL-5tg mice. This was the first study to link CCR2 function & AT eosinophil accumulation. In new studies, we elevated AT eosinophils via injections of IL-5 protein. The elevated AT eosinophils were not able to reduce weight or improve glucose tolerance. Thus, the mere presence of AT eosinophils is insufficient to alter metabolic heath. Eosinophils are not traditionally viewed as very plastic cells (like MΦs), however eosinophils of metabolic tissues such as AT & the GI tract may indeed respond differently than classically understood eosinophils of parasitic infections & allergy/asthma. Our data suggest the phenotype of AT eosinophils, and not just their quantity, is responsible for their impact on AT health, providing impetus for future studies to determine whether eosinophils also demonstrate plasticity.
Diffusional analysis of cytokines in lymph node tissue on a microfluidic chip
Authors: Ross Ashley E , Pompano Rebecca R
Published: January 2023

Cytokines must diffuse between immune synapses, to neighboring cells, and across the lymph node in order to propagate specific immune responses. However, cytokine diffusion in the tissue environment has never been measured, making it difficult to model cell-cell signaling or the localization of cytokine therapeutics. Here, we analyzed the diffusion of TNF-α, IFN-γ, and IL-2, selected because they have central roles in immunity, they represent two different structural classes, and they vary in molecular weight and multimerization. We utilized a microfluidic platform to deliver picogram quantities of fluorescently labelled cytokines to specific regions of 300-μm thick slices of live murine lymph node tissue ex vivo and analyzed spread by fluorescent microscopy. The analysis method was compared to FRAP and microinjection techniques by analyzing diffusion of 10 kDa and 40 kDa Dextrans in both agarose and tissue slices. With the device, we were able to analyze diffusion of cytokines within the B-cell and T-cell zones of the tissue separately. On average, both TNF-α and IFN-γ diffused at the same rate in both the T-cell and B-cell zone (TNF-α: 2.5 ± 0.8 and 2.5 ± 1.6 × 10−7 cm2/s and IFN-γ: 6.0 ± 1.8 and 4.9 ± 1.5 × 10−7 cm2/s respectively, n = 7–9, p > 0.05); however, IL-2 diffused more rapidly in the T-cell zone (8.1 ±1.1 × 10−7 cm2/s, n = 7–8) than in the B-cell zone (4.5 ± 0.3 × 10−7 cm2/s, n = 7–8) yet the difference was not significant (p>0.05). Variation in diffusion between different zones suggests that cytokines interact differently within specific environments. This data provides the first diffusional analysis of cytokines in intact lymph node tissue and validates a new method to enhance the understanding of cell signaling kinetics.
Immunoparalysis is mediated by a dysfunctional IL-10/IL-12 axis in severe burn injury
Authors: Stepp Wesley H , Malfitano Madison , Long Clayton , Maile Robert , Cairns Bruce A
Published: January 2023

In the past year alone, burn injuries accounted for greater than 500,000 medical encounters in the United States. Few molecular markers exist that can detail the extent of burn severity or predict outcomes in burn patients. The identification of biomarkers that can accurately predict patients who are at a higher risk for in-hospital complications and would improve patient outcomes. In this study, we identified means to objectively quantify the degree of a patient’s burn injury and developed a gene-expression profile that identifies the degree of immune compromise in patients following significant burn trauma.

In a prospectively collected cohort of 54 burn patients, we identified that the amount of cell-free DNA in a patient’s plasma significantly correlates with the degree of burn severity (as determined by % total body surface area affected, %TBSA) (R2=0.4256; p<0.0001) and length of hospital stay (R2=0.1895; p=0.0018). Using these data, we stratified patients into either low-moderate injury (<15% TBSA) or severe injury (>15% TBSA) for gene expression analysis of key immunoregulatory molecules. When compared to uninjured, healthy control subjects (N=13), patients with low-moderate injuries (N=33) had 10-fold upregulation in IL-10 expression (p=0.0004), while patients with severe burn injuries (N=18) had a more pronounced upregulation in IL-10 gene expression (25.85-fold increase, p=0.0029). Conversely, IL-12 gene expression remained constant in low-moderate burn patients but was significantly decreased in patients with severe burn injuries. These data suggest that severe burn injury generates a profoundly dysfunctional immune response that may prove a significant challenge to recovery.
Elevated expression of TLR10 in the adipose tissue in obesity: Implication in metabolic inflammation
Authors: THOMAS REEBY SARA , Kochumon Shihab , Sindhu Sardar , Ahmad Rasheed
Published: January 2023

Obesity is marked with larger adipocytes and increasing number of infiltrating immune cells within adipose tissue. These infiltrated immune cells are considered to be involved in the production of inflammatory mediators which cause chronic low grade inflammation called metabolic inflammation (MI). MI plays a key role in the development of insulin resistance (IR) and type-2 diabetes (T2D). Research has shown that the innate immune toll-like receptors (TLRs) specifically TLR2/TLR4 are involved in MI/IR. TLR10 is identified as a pattern recognition receptor without known ligand specificity or biological function and is highly expressed in tissue rich in immune cells. Recent studies have shown that TLR10 has been involved in innate immune responses against infections. However, its role in obesity/T2D remains to be elucidated. Therefore, we investigated the changes in the TLR10 expression and analyzed their association with inflammatory markers in the subcutaneous adipose tissues (AT) obtained from adults with/without T2D classified as lean, overweight, and obese based on BMI. Our data show upregulation of TLR10 expression (P<0.05) in obese as compared with lean/overweight individuals and these changes correlated with BMI (P=0.0008) and body fat percentage (P=0.002). Increased TLR10 expression also correlated positively with various inflammatory signatures including TNF-α (r=0.71), CXCL10 (r=0.79), IL-23 (r=0.6), CCL5 (r=0.69), CCR5 (r=0.79); CCR2 (r= 0.95), IL-12A (r=0.73) and TLR2 (r=0.74). In T2D, TLR10 correlated only with BMI (P=0.001) and CCL5 (P=0.01). Collectively, our data show that increased AT-TLR10 expression is associated with signature inflammatory markers which may have significance in MI.
Molecular determinants of noncanonical inflammasome activation by LPS
Authors: Behl Bharat , Vanaja Sivapriya Kailasan , Rathinam Vijay
Published: January 2023

Septic shock is the leading cause of morbidity and mortality in intensive care units worldwide. Gram-negative bacteria constitute one of the most common causes of sepsis that results in high fatality by initiating an excessive and uncontrolled host inflammatory response. At the epicenter of this response is the innate immune detection of lipopolysaccharides (LPS). In addition to TLR4 recognition of LPS, recent studies revealed a new LPS sensing mechanism in the cytosol. Inflammatory caspases, such as caspase-11, detect LPS to execute pyroptosis, an inflammatory form of cell death, and caspase-1 activation. A prerequisite for the activation of noncanonical inflammasome is the transcriptional induction of caspase-11, which is mediated by TLR4-TRIF, type I interferon, and complement signaling. Additionally, guanylate binding proteins (GBPs), interferon regulatory factor 1, and IRGB10 orchestrate the release of LPS from the vacuolar bacterial pathogens into the cytosol. While the host factors involved in LPS activation of caspase-11 are fairly characterized, the role of bacterial factors in this process is not clear. Our new findings from this study delineate the bacterial components necessary for the optimal engagement of the cytosolic LPS sensing pathway and eliciting noncanonical inflammasome responses.
Lamina propria lymphocytes in sanroque mice produce proinflammatory cytokines that drive chronic small intestinal inflammation
Authors: Klein John R , Williams Alexander , Vigneswaran Nadarajah , Montufar-Solis Dina
Published: January 2023

Roquin is an E3 ligase that regulates mRNA stability. Mice with a mutation in the Rc3h1 gene and Roquin protein, referred to as Roquinsan/san or sanroque mice, develop extensive chronic inflammation and autoimmune pathologies. Our laboratory recently reported that beginning at 8 weeks of age sanroque mice also develop inflammation that is localized to the small intestine but is rare in the colon. Here, we demonstrate that small intestinal intraepithelial lymphocytes (IELs) are present in the epithelium of sanroque mice, but that cell recoverability is low using standard extraction techniques even though lamina propria lymphocytes (LPLs) can be recovered in normal numbers. In characterizing T cell costimulatory markers and activation molecules on LPLs in sanroque mice, we identified Ly6C and 4-1BB (CD137) as being expressed at elevated levels on small intestinal LPLs, and we show that both subsets, in conjunction with cells expressing the KLRG1 T cell activation molecule, are sources of the IL-17A, IFN-γ, and TNFα proinflammatory cytokines. TNFα was primarily produced by 4-1BB+, KLRG1− cells, but was also made by some 4-1BB−, KLRG1− cells, and 4-1BB−, KLRG1+ cells. These findings collectively suggest that the small intestinal inflammatory response in sanroque mice is driven, at least in part, by LPL activation through Ly6C, 4-1BB, and KLRG1 signaling, and they provide further evidence demonstrating the utility of using the sanroque mouse as an animal model of chronic inflammation in the small intestine.
Gut microbiota fermentation and inflammasome responses determine the effects of dietary fiber on intestinal inflammation
Authors: Singh Vishal , Saha Piu , Yeoh Beng San , Walker Rachel , Cai Jingwei , Xiao Xia , Shearer Gregory C. , Patterson Andrew D , Vijay-Kumar Matam
Published: January 2023

Short-chain fatty acids (SCFAs), major microbial fermentation products of dietary fiber in the gut, are shown to improve gut health. We hypothesized that soluble fiber (inulin and pectin) which are more accessible for microbiota fermentation may offer more pronounced mucoprotection than insoluble fiber (cellulose) in a murine model of chronic colitis. WT (BL6) mice receiving dietary cellulose exhibited robust colitis upon IL-10R neutralization as examined by serological, biochemical, histological and immunological parameters. Similar results were observed in colitis-prone toll-like receptor 5 knockout (Tlr5KO) mice. Surprisingly, prebiotic fiber inulin failed to protect against colitis in both WT and Tlr5KO mice. Remarkably, pectin ameliorates colitis development. GC-MS analysis of cecal content revealed that cecal butyrate, an inflammasome (NLRP3) response modulator in the gut, was augmented in inulin-fed colitic mice. Serum cytokine analysis showed that dietary inulin elevated IL-18, but diminished the IL-1 receptor antagonist (sIL-1Ra). On the flip side, dietary pectin reduced IL-18 and augmented sIL-1Ra. More importantly, suppressing butyrate production by metronidazole, which specifically depletes the butyrate producers in the gut, protected the mice from chronic colitis, suggesting that SCFAs-inflammasome axis shapes the beneficial effects of dietary fiber on the gut health. Collectively, mitigation of chronic colitis by dietary pectin can be partly explained by reduced cecal butyrate and elevated IL-1β inhibitor, sIL-1Ra. A detailed mechanistic study on how pectin-derived metabolites modulate inflammasome signaling may yield development of novel therapeutics to treat intestinal inflammation.
CCR2 promotes intestinal tumorigenesis through enhancing inflammation via IL-17 and IL-23 pathway
Authors: Jala Venkatakrishna R , Bodduluri Sobha Rani , Chilton Paula , Chheda Zinal S , Sharma Rajesh K , Singh Rajbir , Fleming Christopher Jay , Yan Jun , Bodduluri Haribabu
Published: January 2023

Chemokines and their receptors have been implicated in several cancers and metastasis including colon cancer. CCL2, a classic chemokine mediates biological functions via CCR2 receptor. In this study, we investigated the role of CCR2 in progression of intestinal tumors using spontaneous intestinal tumorigenesis mouse model, ApcMin/+ mice. Ablation of CCR2 in ApcMin/+ mice significantly increased the survival, reduced tumor burden and corrected immune abnormalities caused by ApcMin/+ mutation. Immune cell analysis in spleen and mesenteric lymph nodes (mLNs) suggested that CCR2−/−ApcMin/+ mice exhibited significant reduction in myeloid cell population and increase in IFNγ producing T-cell populations compared to ApcMin/+ mice. CCR2−/−ApcMin/+ mice also displayed decreased tumor infiltrating F4/80+ and increased CD8+ cells. Polyps in CCR2−/−ApcMin/+ displayed significant reduction in IL-17 and IL-23 cytokines compared to polyps of ApcMin/+. Adoptive transfer of splenic CD4+ T cells from ApcMin/+ mice into Rag2−/− mice develops severe colitis. However, transfer of CD4+ T cells from CCR2−/− ApcMin/+ failed to develop colitis in Rag2−/− mice. The levels of IL-17 in these mice significantly reduced suggesting importance of CCR2 expression on CD4+cells to promote IL 17 mediated colonic inflammation. Microbiota analysis revealed that ablation of CCR2 in ApcMin/+ lead to decrease in tumor promoting Bacteroides genus compared to ApcMin/+. In conclusion, CCR2 plays a critical role in enhancing tumor-promoting inflammation via IL-17 and IL-23 leading to increased intestinal tumorigenesis in ApcMin/+ mice. Blocking CCR2-CCL2 axis potentially offers an alternative therapeutic option to control intestinal tumorigenesis.
Mucosal immune responses to b-lactoglobuin (βLG)-immunized mice are altered following colitis challenge
Authors: Zlotkowska Dagmara Anna , Wasilewska Ewa , Wroblewska Barbara
Published: January 2023

Food allergens induce mucosal immune responses, and their continual exposure to sensitive hosts leads to health problems, e.g. intestinal inflammation. We tested dextran sodium sulfate (DSS) to disrupt intestinal homeostasis in mice immunized with βLG.

BALB/c mice were ip. sensitised with βLG/PBS, thereafter orally challenged with βLG. DSS was delivered in drinking water et libitum. Humoral response and T cell profile were monitored for differences in immune response between groups. The highest level of Ag-specific serum IgG was observed in the i.p. immunized group without dependence on DSS presence. The level of Ag-specific IgA in sera and fecal extracts was low in each group. We found the greatest amount of CD4+ and FoxP3+CD25+CD4+ T cells in the spleens, mesenteric and peripheral lymph nodes obtained from mice orally immunized with βLG and receiving DSS treatment. DSS also induced a subset of CD4+ T cells producing a high amount of IFN-γ in all experimental groups. An elevated number IFN-γ+ CD4+ T cells was found in the lymphoid tissues of βLG-immunized mice (ip and orally) and treated with DSS. The profile of peripheral blood lymphocytes showed a two-fold increase in FoxP3+CD25+CD4+ T cells after DSS stimulation.

The proposed experimental model of the inflamed intestine provoked with allergens showed differences in humoral responses and lymphocyte profiles, demonstrating that this regimen can be used to monitor immunoreactivity to food compounds.
MERTK inhibition in the leukemia microenvironment promotes a therapeutic immune response to acute lymphoblastic leukemia
Authors: Jacobsen Kristen M , Lee-Sherick Alisa B. , Huey Madeline G. , Page Lauren S. , Zhang Weihe , Wang Xiaodong , Fyre Stephen V. , Earp H. Shelton , Jordan Craig T. , DeRyckere Deborah , Graham Douglas K.
Published: January 2023

T cell immunotherapy is effective in some, but not all cancers, and targeting the immunosuppressive, innate immune tumor microenvironment is one key to further advancement. During efferocytosis, activation of MERTK tyrosine kinase receptor in innate immune cells suppresses proinflammatory cytokines, stimulates immunosuppressive cytokines and upregulates co-inhibitory ligands. MERTK has also been validated as a therapeutic target in the tumor microenvironment in solid tumor models. To investigate immunoregulatory roles for MERTK in leukemia, we utilized pharmacologic and genetic inhibition of MERTK in a MERTK-negative syngeneic murine model of acute lymphoblastic leukemia (ALL). Treatment with MRX-2843, a potent, orally bioavailable, small molecule MERTK inhibitor, significantly reduced leukemic burden and prolonged median survival in wildtype mice (38 days post-treatment vs. 24 days in vehicle-treated mice, p>0.01). Similarly, overall survival was increased more than four-fold in Mertk−/− mice transplanted with leukemia (>80 days vs. 21 days in wild-type mice, p>0.0001), suggesting that the observed therapeutic activity is immune-mediated. Indeed, treatment with MRX-2843 abrogated expression of the immune co-inhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the ALL microenvironment. Although T cells do not express MERTK, expression of the cognate PD-1 receptor was also reduced on CD8+ T cells and the frequency of FOXP3+CD4+ regulatory T cells was decreased in the leukemia microenvironment following MRX-2843 treatment. These data demonstrate several mechanisms by which MERTK can suppress anti-leukemia immunity and validate MERTK as a novel immunotherapeutic target in ALL.
Topical chemo-immunotherapy converts a pro-tumorigenic tumor microenvironment to a pro-inflammatory phenotype in human cutaneous squamous cell carcinoma
Authors: Friedman Blake , Carey Cara , Erdos Geza , Falo Louis D.
Published: January 2023

We have previously demonstrated that microneedle array (MNA) delivery of anti-tumor agents improves survival in mouse models of melanoma and non-melanoma skin cancers and induces cell death in freshly excised human cutaneous squamous cell carcinomas (cSCCs). As tumors employ strong immunosuppressive and tumor-promoting mechanisms that prevent their elimination by immune surveillance mechanisms and support their proliferation, we sought to determine if application of MNAs containing anti-tumor agents could abrogate their protumorigenic phenotype and promote an anti-tumor phenotype in a human model of cSCC, the second most common human malignancy worldwide. To accomplish this, we compared Nanostring gene expression data from human cSCCs treated with MNAs containing doxorubicin (MNA-Dox) to matched, untreated cSCC controls and unmatched normal skin. As compared to matched, untreated cSCC controls, MNA-Dox-treated cSCCs demonstrated significantly decreased expression of genes associated with a pro-tumorigenic environment, including IDO, ARG2, S100A7 and VEGF-A. Further, MNA-Dox-treated cSCCs demonstrated significantly increased expression of genes associated with an anti-tumor phenotype, including CXCL14, HMGB1, P53, and Caspase-8. Comparison of MNA-Dox-treated cSCCs and unmatched, untreated normal skin suggested a reversion of a pro-tumorigenic phenotype to a normal skin phenotype, with similarly low expression of pro-tumorigenic genes seen in both groups. Together, these results suggest the potential of MNA-administered doxorubicin to effectively transform a pro-tumor microenvironment to one favoring the induction of tumor immunity in a human cSCC.
IL-22 Promotes Pancreatic Cancer Tumorigenesis through Induction of Stemness and Epithelial to Mesenchymal Transition
Authors: Lanfranca Mirna Perusina , Kryczek Ilona , Rhim Andrew , Girgis Alexander , Lazarus Jenny , Di Magliano Marina Pasca , Zou Weiping , Frankel Timothy
Published: January 2023

Elevated levels of Interleukin 22 (IL-22) and its receptor (IL-22R) are associated with poor prognosis in pancreatic ductal adenocarcinoma (PDAC) and the underlying mechanism is currently unknown. Our aim is to investigate the role of the IL-22 axis in PDAC initiation and progression.

Impact of IL-22 on tumor initiation was assayed by subcutaneous and intravenous inoculation of PDAC cells into wild-type and IL-22−/− mice. The PKCY (Pdx1-Cre; KrasG12D; p53fl/+; RosaYFP) model of pancreas cancer was used to study the in-vivo presence and significance of IL-22 in spontaneous tumors. Results were confirmed in human specimens of surgically resected pancreas cancer.

IL-22R was present in all tested PDAC lines and IL-22 treatment led to STAT3 phosphorylation and subsequent increased expression of EMT (Epithelial to Mesenchymal Transition) transcription factors. Cells transitioned to a mesenchymal phenotype and robust tumor sphere formation was observed. While tumors readily formed in wild-type mice, initiation, establishment and growth were impaired in IL-22−/− mice and PKCY-IL-22−/− mice. Increased levels of IL-22 were found in both spontaneous murine tumors and surgical specimens compared to control tissue. IHC of tumors showed diffuse IL-22R staining with increasing intensity of pSTAT3 and EMT markers as tumors progressed to invasive cancer. FACS analysis identified type 3 innate lymphoid cells and TH22 cells as the source of IL-22 in both human and murine PDAC.

Our data suggests that IL-22 is integral in the initiation, progression and establishment of pancreatic cancer, positioning it as an attractive target for cancer therapy.
Activation of cannabinoid receptors alters the expression of miRNAs in Granulocytic Myeloid Derived Suppressor cells (G-MDSCs) and Splenic Resident Granulocytes (SRG) thereby identifying shared and unique pathways of differentiation
Authors: Singh Narendra Prasad , Miskin Arianna , Singh Udai P , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

THC (Δ9-Tetrahydrocannabinol) is the main psychotropic component in marijuana and has been shown to have potent anti-inflammatory properties. Recent studies from our laboratory have shown that THC triggers massive induction of myeloid derived suppressor cells (MDSCs) through modulation of miRNA(miR). In this study, we investigated the effect of THC on the regulation of miRs in Granulocytic MDSCs (G-MDSCs) and splenic resident Granulocytes (SRG) in mice. To this end, mice (C57BL/6) were treated with vehicle or THC (20 mg/kg bw) and 24 hrs post injection, G-MDSCs from peritoneal wash and SRG from spleen were sorted (>95% purity) and microRNA arrays were performed using total RNAs including miRs. There was significant alterations in the expression of about 75 miRs in both G-MDSCs and SRG. Out of the 75 miRs, 31 miRs were upregulated and 9 miRs were downregulated in both G-MDSCs and SRG. There were 21 miRs that were downregulated in G-MDSCs but upregulated in SRG. The remaining 14 miRs were upregulated in G-MDSCs but downregulaed in SRG. Upon analysis of dysregulaed miRs using IPA, there were more than 20 diseases and pathways that were regulated by these miRs. We also observed that dysregulatd miRs played a common role in the development of several diseases including cancer and inflammatory diseases. This study demonstrate for the first time that THC regulates miRs that are both shared and unique to G-MDSCs and SRGs, thereby suggesting that while G-MDSCs are similar in some ways to SRGs, they may be at different stages of differentiation.

The present study was supported in part by NIH grants (P20GM103641, P01AT003961, R01AT006888, R01ES019313, and R01MH094755).
Mincle regulates autophagy to control neutrophil extracellular trap formation
Authors: Sharma Atul , Simonson Tanner J , Jondle Christopher N. , Mishra Bibhuti B. , Sharma Jyotika
Published: January 2023

“Neutrophil extracellular traps (NETs) constitute antimicrobial function of neutrophils but have also been linked to perpetuation of inflammation. Despite this evident physiological relevance, mechanistic understanding of NET formation is poor. In the current study we examined the mechanism by which Mincle, a C-type lectin receptor, regulates NET formation. We found that Mincle mediates NET formation in response to several activation stimuli in-vitro and in-vivo during pneumoseptic infection with Klebsiella pneumoniae, indicating its regulatory role in NET formation. Mechanistically, we show that attenuated NET formation in Mincle−/− neutrophils correlates with an impaired autophagy activation in-vitro and in-vivo, while reactive oxygen species (ROS) formation in these neutrophils remained intact. The requirement of autophagy in Mincle-mediated NET formation was further supported by exogenous treatment with autophagy inducer tamoxifen, which rescued the NET formation defect in Mincle−/− neutrophils. Our findings identify a previously unrecognized role of Mincle as a regulator of autophagy which mediates NET formation without affecting ROS generation. Our study addresses a major challenge in the field by positing this pathway to be targeted for modulation of NETs, while preserving ROS production, an important innate immune defense.”
Prediction, molecular modelling and dynamic simulations of Vascular endothelial growth factor receptor 1 as a potential molecular target for 2,3,7,8-tetrachlorodibenzo-pdioxin
Authors: Chitrala Kumarswamy naidu , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Dioxins are ubiquitous, persistent environmental pollutants known to accumulate in fat. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immunotoxicity and promotes the differentiation regulatory T cells and inhibits the Th17 cells. To date, studies on only few molecular targets for TCDD are available, thereby limiting the understanding of the exact mechanism of action on immune response. To that end, in the present study, we have predicted the molecular targets for TCDD using knowledge-based approach and reverse pharmacophore mapping approach, and analyzed the mechanism underlying receptor conformational changes following the binding of TCDD using molecular modelling and molecular dynamic simulation (MDS) studies. Results showed that 11 potential target candidates predicted in Mus musculus and 20 in Homo sapiens with Vascular endothelial growth factor receptor 1 (VEGFR1) being the high probability target for TCDD in both Mus musculus and Homo sapiens. Results from molecular docking studies showed that TCDD interacts with a binding energy of −7.37 kcal/mol with mouse VEGFR1 and −6.55 kcal/mol with human VEGFR1. Four independent MDS studies revealed key interactions and major secondary structural changes for both mouse and human VEGFR1-TCDD complexes, specifically at residues in α-helices and coils for human VEGFR1-TCDD complex. In conclusion, this study defines VEGFR1 as a potential molecular target candidate for TCDD and establishes the structural basis for the VEGFR1-TCDD interactions as well as reveals that conformational changes at the onset of ligand binding may play a role in immunomodulation.
TRIM29 promotes persistence of oncogenic DNA viruses by inhibiting innate immune response
Authors: Zhang Ao , Xing Junji , Zhang Zhiqiang
Published: January 2023

Certain double-stranded DNA (dsDNA) virus such as Epstein-Barr virus (EBV) often infect nasopharyngeal and airway epithelial cells (AECs), which is etiologically linked to nasopharyngeal carcinoma in humans. But the underlying mechanisms remain poorly understood. Here we report that in human AECs, EBV employs TRIM29, a member of TRIM family of proteins, to evade immune attacks by inhibiting innate immune activation. We found that the human AECs selectively express TRIM29 and are highly sensitive to EBV infection in TRIM29 upregulation. Indeed, knockdown of TRIM29 in AECs markedly enhanced type I interferon (IFN-I) production, and in human nasopharyngeal cancer cells resulted in almost complete EBV clearance. We also found TRIM29 was highly induced by cytosolic dsDNA in myeloid dendritic cells (mDCs) to control immune response. In animal models, TRIM29−/− mice showed strong adenovirus clearance in the lung, and resistance to lethal HSV-1 viral infections due to enhanced production of IFN-I. Mechanistically, TRIM29 induced K48-linked ubiquitination of STING, a key adaptor in dsDNA sensing pathway, followed by its rapid degradation, thus effectively shutting down the anti-viral innate immunity in favor of viral persistence. These data demonstrate that EBV and possible other dsDNA viruses use TRIM29 to suppress local innate immunity, and key process in viral persistence and development of nasopharyngeal carcinoma.
Role of Notch signaling in basophils in Type 2 inflammation
Authors: Webb Lauren M , Cubitt Rebecca , Wojno Elia Dion Tait
Published: January 2023

Basophils are rare innate granulocytes, making up just 1% of circulating immune cells. Despite their rarity, basophils expand and contribute to the development of host-protective type 2 inflammation and worm expulsion following infection with Trichuris muris, a murine model of human whipworm. However, the pathways that regulate basophil expansion and function in this setting remain unclear. Previous work has shown that the Notch signaling pathway is important in lymphopoiesis and the function of T helper 2 cells, but little is known about its role in innate immune cells. Here, we have investigated the importance of the Notch signaling pathway in controlling the function of basophils during Type 2 inflammation. We show for the first time that basophils selectively upregulate Notch under Type 2 inflammatory conditions. Further, Notch is required for optimal cytokine production from basophils in response to such damage signals as IL-33. Using mice that selectively lack Notch signaling in basophils, we demonstrate that basophil expansion during T. muris infection depends on Notch signaling. Further, efficient clearance of T. muris also depends on effective Notch signaling in basophils. We are now actively investigating how other facets of basophil function are affected by inhibition of Notch signaling. These findings have wide reaching implications for our understanding of innate cell function in Type 2 immunity and for the potential development of therapeutics aimed at targeting Type 2 inflammation. Given that Notch inhibitors are on their way to the clinic, a clear understanding on their impact on Type 2 effector responses is essential. Our work suggests that these therapies could be re-purposed to control aberrant Type 2 immunity.
IL-33 is required for a protective myeloid cell response to T. gondii infection in the brain
Authors: Still Katherine M. , Thompson Jeremy A. , Batista Samantha J. , O’Brien Carleigh A. , Harris Tajie H.
Published: January 2023

Monocytes can traffic to the brain in response to a variety of CNS insults. During infection with the parasite Toxoplasma gondii, monocyte-derived myeloid cells cluster in areas of free parasite and are required to control infection. While the functions of these cells are being investigated, the mechanisms of their recruitment to the infected brain are largely unknown. In regions of focal inflammation, we observe a loss of brain-resident cells and hypothesize that damage to these cells is an initial step in monocyte recruitment. We are specifically exploring the alarmin IL-33 and its potential for immune amplification upon extracellular release. Although largely characterized in barrier tissues, IL-33 is expressed by oligodendrocytes in the CNS. We find that the absence of IL-33 signaling in infected mice results in decreased expression of ccl2, an important chemokine for monocyte recruitment. Using a CCL2-mcherry reporter mouse line we have visualized that CCL2 production greatly increases upon infection and occurs in areas of resident brain cell loss and inflammation. Aligning with a loss of ccl2 we see a decrease in the number of recruited macrophages in IL-33R knockout mice. Though IL-33 has been connected with Type 2 immune responses, IL-33R knockout mice exhibit functional defects in our model’s Th1-dominated environment of intracellular pathogen killing. Specifically, infiltrating myeloid cells in these mice have a decreased capacity to produce iNOS, which is critical for limiting parasite replication. Thus, infected mice lacking IL-33 signaling have an increase in brain tissue cyst numbers. Together, our results demonstrate the impact of one alarmin, IL-33, on monocyte recruitment and control of T. gondii CNS infection.