bluuebunny/crossref_metadata_2025 huggingface dataset:
MerTK mediates T cell tolerance in the pancreatic islets during type 1 diabetes
Authors: Friedman Rachel Sharon , Tracy Dayna , Lindsay Robin S
Published: January 2023

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease that results from destruction of the beta cells in the pancreatic islets. Curiously, infiltrated islets can continue to contain beta cells for years following disease onset, suggesting a tolerance checkpoint following islet infiltration. While T cells are required for T1D progression, other immune cell populations also infiltrate the islets, including CD11c+ antigen presenting cells (APCs). We depleted CD11c+ cells to determine their role in maintaining T cell tolerance in the islets. CD11c depletion resulted in increased CD8 T cell motility arrest and effector function. MerTK+ APCs have been associated with T cell tolerance induction following apoptotic cell uptake. We identified MerTK expression on a subset of islet-infiltrating CD11c+ APCs. To test if MerTK mediates T cell tolerance in the islets, we treated pre-diabetic non-obese diabetic (NOD) and non-autoimmune C57BL/6 mice with a MerTK/Flt3 inhibitor. Blood glucose remained normal in C57BL/6 mice. However, in 50% of NOD mice with pre-existing islet infiltration, MerTK/Flt3 inhibition resulted in extremely rapid diabetes onset. The rapid disease onset occurred prior to Flt3-dependent population shifts in the islets, suggesting a MerTK rather than Flt3 mediated effect. Within 24 hours of MerTK inhibition, islet-antigen specific CD4 and CD8 T cells arrested motility in the islets, and CD8 T cell activation increased in the islets. Thus, inhibition of MerTK signaling resulted in a break in T cell tolerance and rapid islet destruction. These results suggest that CD11c+ APCs are involved in a MerTK-dependent tolerance checkpoint during T1D, which maintains T cell tolerance in the islets following islet infiltration.
A20-deficient microglia and CD8 T cells in the brain
Authors: Mohebiany Alma N , Waisman Ari
Published: January 2023

Tumor necrosis factor alpha induced protein 3 (TNFAIP3), also known as A20, is a key negative regulator of the canonical NFkB pathway, and acts at various levels of the signaling cascade downstream of receptor stimulation, thereby regulating the pro-inflammatory response. As such, its absence leads to the development of spontaneous inflammation; indeed, a number of SNPs in the A20 gene have been associated with various autoimmune diseases. Cell-specific deletion of A20 in mice can also lead to the development of spontaneous disease. Mice lacking A20 in their astrocytes have stronger disease in a model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Furthermore, A20ko mice demonstrate spontaneous neuroinflammation, attributed to A20 deletion in either endothelial cells or microglia, the resident immune cells of the brain and spinal cord. We chose to examine the role of microglial A20 expression in the context of neuroinflammation by crossing A20fl/fl to CX3CR1-CreERT2 mice. The resulting A20fl/flCX3CR1-CreERT2 (A20Δmg) mice were injected with tamoxifen to induce A20 deletion. Without A20, microglia spontaneously upregulate a number of NFkB target genes and have an activated morphology accompanied by an increase in GFAP+ astrocytes in the cortex. Surprisingly, we found significantly more T cells (mainly CD8) infiltrate the brain and spinal cords of naïve A20Δmg mice relative to controls, after A20 deletion. Furthermore, upon EAE induction, A20Δmgmice develop symptoms sooner than controls, and have stronger disease. Overall, the deletion of A20 in microglia leads to a spontaneous activation, which may enhance the susceptibility of these animals to developing neuroinflammation under specific condtions.
Aberrant expression of CD11b and CD11a underlies impaired neutrophil adhesion in glucose-6-phosphate transporter-deficient mice
Authors: Jun Hyun Sik , Kim Goo-Young , Kwon Joon Hyun , Chou Janice
Published: January 2023

Neutrophils play an essential role in defense against intruding microorganisms. They are produced in great number in the bone marrow and circulate in blood where they are found in a quiescent state. In response to inflammatory stimuli, neutrophils take different steps-rolling, adhesion, and transmigration-to migrate towards inflammation sites. In this study, we investigated the underlying mechanisms of impaired neutrophil adhesion in glycogen storage disease type Ib (GSD-Ib) which is caused by a deficiency of the glucose-6-phosphate transporter (G6PT). GSD-Ib is characterized not only by disrupted glucose homeostasis but also by neutropenia and neutrophil dysfunction. GSD-Ib mice were infused with a recombinant adeno-associated virus (rAAV) vector expressing human G6PT only in liver to increase their survival. Unlike control mice, GSD-Ib-rAAV mice manifested severe neutropenia in both blood and bone marrow and neutrophils of GSD-Ib-rAAV mice were defective in adhesion to tumor necrosis factor-a-treated epithelial cells and intercellular adhesion molecule 1 and fibrinogen. The β2-integrins including CD11a/CD18 and CD11b/CD18 are recognized as vital players in neutrophil recruitment. Consistent with impaired neutrophil adhesion, the expression of CD11a and CD11b were found to be decreased in neutrophils of GSD-Ib-rAAV mice, compared with that of control mice. Particularly neutrophils of GSD-Ib-rAAV mice showed increased proteolytic degradation of CD11b. Given the central role of β2-integrins in neutrophil adhesion, this alternation of CD11a and CD11b molecules and their effect on neutrophil adhesion probably define a molecular mechanism to neutrophil dysfunction manifested in GSD-Ib.
Lung IL-33 overexpression does not cause inflammation, but increases allergen sensitivity and disrupts lung morphogenesis
Authors: Drake Li Y , Squillace Diane , Iijima Koji , Kobayashi Takao , Kephart Gail , O’Brien Daniel , Kita Hirohito
Published: January 2023

IL-33 is an IL-1 cytokine family member that promotes inflammatory responses via activation of many immune cell types. IL-33 is expressed in epithelial barrier tissues such as lung epithelium under both resting and inflammatory conditions. Secreted IL-33 plays an important role in type 2 innate immunity and adaptive immune responses. To characterize the roles of IL-33 in tissue and immune homeostasis in lung epithelium, we generated a novel transgenic mouse in which IL-33 was overexpressed only in lung epithelial cells driven by the tetracycline-sensitive Club cell secretory protein (CCSP) promoter. When IL-33 expression was induced in adult mice, overexpression of IL-33 protein approximately 3-fold above normal levels produced no pathological or immunological changes in the lungs. However, these animals developed significant increases in innate type 2 immune responses when exposed to natural or model allergens. When IL-33 expression was induced in pre- and post-natal mice, these mice had increased mortality, which was accompanied by lung hemorrhage and lung lobe malformation without detectable signs of inflammation and immune responses. Furthermore, IL-33 transgenic mice that survived to adulthood exhibited simplified and enlarged alveoli resembling bronchopulmonary dysplasia. Correspondingly, the epithelial cells from IL-33 transgenic mice displayed altered expression of genes involved in tissue and hematological system development. These data indicate dual effects of IL-33 overexpression depending on the developmental stage of the lungs, namely impairment of lung morphogenesis in the neonatal period and heightened sensitivity of innate type 2 immunity in adult mice.
High sensitivity multiplex cytokine panel used to identify unique signatures in serum associated with gallbladder cancer.
Authors: Corbel Amanda Faith , Kemp Troy J , Pinto Ligia A , Koshiol Jill
Published: January 2023

Gallstones (GS) and chronic inflammation are two of the strongest risk factors associated with gallbladder cancer (GBC). Since these conditions are frequently asymptomatic, GBC detection often occurs incidentally and when the cancer has reached a progressive stage. Gaining a better understanding of the mechanisms involved in the development and progression of GBC could help identify the subpopulation of gallstone patients that will go on to develop GBC. Therefore, we sought to measure 16 key inflammatory markers in serum samples with a high sensitivity bead-based multiplex cytokine assay of population-based controls (PBC), patients with gallstone(s) (GS), and patients with gallbladder cancer (GBC), from Shanghai, China. Wilcoxon’s rank sum test was used to compare median concentrations. We found 11 markers (IL-1β, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, IL-21, and IL-23) that were significantly (p value <0.01) elevated in GS compared to PBC; fold changes ranged from 1.3 to 1.7. We found 6 markers (MIP-1α, MIP-1β, IL-6, IL-7, IL-8, and IL-10) that were significantly elevated in GBC compared to GS; fold changes ranged from 1.2 to 3.6. Moreover, only 3 markers (IL-6, IL-7, and IL-10) were elevated in both comparison groups. The unique set of markers highlighted between the two comparison groups suggests that there is a transitional stage in the progression of disease leading up to GBC. Future studies are needed to further evaluate the common pathway or mechanism associated with these cytokine signatures. Futhermore, such studies should be replicated in bile to assess the local inflammatory response that develops in the gallbladder.
IL-22RA1 signaling regulates intestinal secretory cell development and protects against infectious colitis
Authors: Good Misty , Kumar Pawan , Parks Olivia , Ma Congrong , Horne William , Kolls Jay K
Published: January 2023

The gastrointestinal tract harbors a complex immune system to contend with both normal flora and invasive pathogens. Recently, interleukin (IL)-22 signaling has proven to play a critical role in maintaining the gut barrier, promoting epithelial healing and controlling inflammation from attaching and effacing pathogens. Specifically, IL-22 produced by Th17 cells and group 3 innate lymphoid cells (ILCs) is critical for host resistance to the attaching and effacing gut pathogen Citrobacter rodentium. However, the importance of intestinal epithelial IL-22Ra1 signaling in regulating secretory cell development and protecting against infectious colitis remains unknown. We hypothesize that intestinal IL-22 signaling plays an important role in mucosal host defense against infectious colitis by regulating the intestinal cell types that maintain the gut barrier. To test this hypothesis, we generated mice that selectively lack the receptor for IL-22, IL-22RA1, and discovered these mice have a striking phenotype of intestinal cell differentiation. We demonstrate that abrogation of intestinal IL-22 signaling led to a decreased number of goblet and paneth cells and decreased expression of the antimicrobial peptide Reg3g. Further, the IL-22RA1 intestinal specific knock-out mice were more susceptible to C. rodentium colitis than WT mice as demonstrated by increased weight loss that correlated with increased spleen bacterial burden, decreased epithelial cell proliferation and disrupted mucosal architecture. These findings offer insights into the protective role that IL-22 plays on mediating intestinal mucosal host defense and how critical IL-22 signaling is to intestinal cell differentiation and attenuating infectious colitis.
FVIII tolerance induction in hemophilia A mice utilizing low dose IL2 treatment
Authors: Lyle Meghan J , Fu Richard Y. , Miao Carol H.
Published: January 2023

Hemophilia A patients lack or possess low levels of functional FVIII protein, which results in an inability for the blood to clot when injury occurs. The current treatment for hemophilia A patients is FVIII protein replacement, but this treatment is expensive and often results in anti-FVIII immune responses, neutralizing the clotting effect.

Regulatory T (Treg) cells help balance T effector (Teff) cells through their suppressive function during an immune response and keep autoimmunity in check. Treg cells possess a high affinity epitope of the IL2 receptor. With a low dose of IL2, the Treg population outcompetes the Teff cells, leading to an increase in activation and number. The increased suppressive activity may induce tolerance in hemophilia A mice treated with FVIII. Hemophilia A mice were hydrodynamically injected with IL2 and FVIII plasmids. Cell staining data showed a marked increase in Treg cell population and activation. In addition, the Treg/Teff ratio was significantly increased in the first 3 weeks and maintained at increased levels over several weeks afterwards. On Day 28-post FVIII injection, control mice had started to develop inhibitors associated with significant decrease of FVIII expression, while mice treated with IL2 showed no inhibitors with persistent FVIII expression. The treated mice are monitored to determine the potential long term tolerance induced by low dose IL2. Combined gene transfer of FVIII and IL2 plasmids can produce therapeutic FVIII and simultaneously prevent inhibitory antibody formation, thus providing a potentially effective treatment of hemophilia A.
PSMP, a novel chemotactic cytokine, triggers inflammatory Ly6ChiCCR2+ monocyte migration and promotes colitis in a CCR2-dependent manner
Authors: She Shaoping , Pei Xiaolei , Zheng Danfeng , Ma Jing , Guo Changyuan , Zhang Yan , Peng Xinjian , Chen Dixin , Wang Pingzhang , Mo Xiaoning , Zhang Yingmei , Song Quansheng , Ma Dalong , Wang Ying
Published: January 2023

Chemokines plays a crucial role in the migration of leukocytes, which are involved in immune and inflammatory reactions with their receptors. To find novel chemotactic cytokines, we carried out genome-wide bioinformatics analysis and PBMC chemoattractant platform screen. Seven novel potential chemotactic cytokines were picked up, one of them was PC3-secreted microprotein (PSMP). The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, lymphocytes and THP-1 cells. CCR2 was identified as a high-affinity receptor for PSMP by in vitro chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays. Furthermore, we found that PSMP was extensively expressed at low levels in normal human tissues, but was significantly up-regulated in intestinal biopsies from patients with inflammatory bowel disease and mouse DSS-induced colitic tissue in the early stage prior to IL-6, TNF-α and CCL2 up-regulation. Moreover, the results showed that PSMP triggered inflammatory Ly6ChiCCR2+ monocyte migration from the circulation into the lamina propria in a CCR2-dependent manner and promoted the progression of inflammation. A PSMP neutralizing monoclonal antibody significantly mollified inflammation in a mouse DSS colitis model. Taken together, our study provides the first evidence that PSMP is a novel chemotactic cytokines whose receptor is CCR2 and PSMP plays a vital and irreplaceable role in triggering and promoting DSS colitis. Our finding not only reveals the function and regulating mechanism of PSMP, but also provides a promising therapeutic strategy for treating inflammatory bowel disease.
IL-33-Driven Innate Tissue-Protective Function of ST2+ Treg Cells
Authors: Liu Quan , Lott Jeremy M. , Mathews Lisa R. , Li Huihua , Zhu Qiang , Matta Benjamin M. , Delgoffe Greg M. , Fan Ming-Hui , Turnquist Heth R.
Published: January 2023

Non-lymphoid tissue-resident CD4+ Foxp3+ regulatory T (Treg) cells with the capacity to modulate non-immunological processes including organismal metabolism and tissue repair have been recently described. Notably, a large fraction of non-lymphoid tissue-resident Treg cells express ST2, the receptor for the tissue-derived cytokine and alarmin, IL-33. However, the relationship between IL-33 and ST2+ Treg cells in quiescent and pathogenic states is only starting to be understood. Using FACS and Foxp3 reporter mice, we demonstrate that ST2+ Treg cells from naïve animals are phenotypically and functionally unique relative to ST2− Treg isolated from the same location. Yet, our studies establish that peripheral and lymphoid tissue ST2+ Treg cells are highly comparable. Interestingly, ST2+ Treg exhibit a high level of aerobic glycolysis, which supports their augmented potential for cytokine secretion, especially that of IL-5, IL-10, and IL-13. IL-5 and IL-13 secretion in response to IL-33 is an innate function of ST2+ Treg cells as it does not require TCR-stimulation. Conversely, ST2+ Treg release of other cytokines, including IL-10, is antigen-restricted and amplified by IL-33. Among Treg subsets, ST2+ Treg cells are the exclusive source of Type 2 cytokines, especially IL-13, which we reveal supports Treg reparative responses. Using mice deficient in IL-33 or Treg cells, we establish that Treg cells and IL-33 are both required to protect mice from lethal lung injury. In line with these data, delivery of IL-13, but not IL-33, at the time of lung injury rescues Treg-depleted mice. This study establishes that ST2+ Treg cells possess an innate capacity for cytokine production that is crucial to tissue repair and inflammation control.
Down-regulation of E protein activity augments an ILC2 differentiation program in the thymus
Authors: Sun Xiao-Hong , Wang Hong-Cheng , Qian Miranda Liangyue , Zhao Ying , Zhuang Yuan , Urban Joseph F. , Fung Kar-Ming
Published: January 2023

Innate lymphoid cells (ILCs) have gained recognition as important regulators in various immune responses. How these cells are generated is just beginning to be understood. Current paradigm states that all three subsets of ILCs originate from common lymphoid progenitors (CLP) in the bone marrow through an array of downstream ILC progenitors. Id2, a member of the Id family that inhibits the function E protein transcription factors, is expressed in these ILC progenitors and is indispensable for ILC differentiation. Unexpectedly, we found that specifically down-regulating E protein function in the thymus by either Id1 expression or conditional deletion of two E protein genes resulted in massive expansion of ILC2s in the thymus as well as other organs where ILC2 normally reside. Consequently, the mutant mice exhibit augmented spontaneous infiltration of eosinophils and heightened responses to allergen, papain, in the lung. Furthermore, these mice also display increased ability in the expulsion of helminth parasites, N. Brasiliensis. These results raise an interesting question as to whether the thymus is a natural site for ILC2 production and the phenotypes of our mutant mice simply highlight such capacity. Indeed, lineage tracing experiments using lck-Cre/ROSA26-stop-tdTomato mice demonstrate that tdTomoto specifically marks a fraction of ILC2s in the thymus as well as in the lung, suggesting that thymus-derived ILC2s preferentially home to the lung. We also found that thymic progenitors were able to differentiate into ILC2 cells in vitro. Together, our data reveal a new ILC2 differentiation program present in the thymus, which enriches our knowledge about ILC2 ontogeny and has potential biological significance.
Differential role of ID2 in the group 3 innate lymphoid cells for the development of different gut associated lymphoid tissues.
Authors: Guo Xiaohuan , Wang Wenyang , Fu Yang-Xin
Published: January 2023

Gut associated lymphoid tissues (GALTs), including the mesentery lymph nodes (mLNs), Peyer’s patches (PPs), and isolated lymphoid follicles (ILFs), are important locations for the initiation of adaptive immune responses in the intestine. Group 3 Innate lymphoid cells (ILC3s) are defined by their expression of RORγt and include lymphoid tissue inducer (LTi) cells, which play a critical role in the development of GALT. However, the function of ID2 in these well-differentiated ILCs is still unclear. In our study, RorccreId2fl/fl mice showed normal LNs, but completely lacked PPs and ILFs compared with WT mice. And ILC3s were significantly reduced in the gut of RorccreId2fl/fl mice compared with WT mice, but were actually increased in mLNs, indicating that the development or maintenance of ILC3s in the gut, but not LNs, requires ID2 regulation. Moreover, ID2 deficient ILC3s exhibited significantly reduction of the lymphotoxin expression in the gut, which is important for the formation of GALT. We also found IL-7 stimulation could induce lymphotoxin expression in WT ILC3s, while the IL-7Rα expression was dramatically reduced in ID2 deficient ILC3s, suggesting that ID2 may regulate the lymphotoxin expression through IL-7 signaling. On the other hand, we found that the expression of Rank was upregulated in ID2 deficient ILC3s, which indicates that the elevated Rank may rescue ILC3s’ survival and lymphotoxin expression through NF-κB pathway. Taken together, our results suggest that ID2 could regulate the function and maintenance of ILC3s through IL-7 and Rank signaling to further determine the differential development of GALTs.
The synthesis of VEGF in allergen-stimulated mast cells is through a leukotriene B4 receptor-2-dependent signaling pathway
Authors: Lee A-Jin , Ro MyungJa , Park JaeIn , Jang Jae-Hyun , Kim Jae-Hong
Published: January 2023

Mast cells are among the principal effector cells in the pathogenesis of allergic asthma. In allergic reactions, allergen(Ag)-induced cross-linking of IgE bound to FcɛRI on mast cells results in the production of vascular endothelial growth factor(VEGF), which is essential for the initiation and development of the allergic response. Despite the central role of VEGF in allergic asthma, the signaling events responsible for the production of VEGF remain unclear, particularly in Ag-stimulated mast cells. In the present study, we observed that blocking leukotriene B4 receptor-2(BLT2) completely abrogated the production of VEGF in Ag-stimulated bone marrow-derived mast cells(BMMCs). The synthesis of BLT2 ligands (leukotriene B4 [LTB4] and 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]) was also required for VEGF production, suggesting a mediating role of an autocrine BLT2 ligands-BLT2 axis in the production of VEGF in mast cells. The Nox1-reactive oxygen species(ROS)-NF-kB cascade is downstream of BLT2 during Ag signaling to VEGF synthesis in mast cells. Furthermore, the level of VEGF synthesis in genetically mast cell-deficient Kit(W/Wv) mice was significantly lower than that in wild-type(WT) mice in the ovalbumin(OVA)-induced asthma model, suggesting that mast cells play a critical role in the synthesis of VEGF in OVA-induced allergic asthma. Importantly, VEGF production was restored to the levels observed in WT mice after adoptive transfer of normal BMMCs into Kit(W/Wv) mice but was not restored in BLT2−/− BMMC-reconstituted Kit(W/Wv) mice in the OVA-induced asthma model. Taken together, our results suggest that BLT2 expression in mast cells is essential for the production of VEGF in OVA-induced allergic asthma.
Epitope spreading in experimental autoimmune encephalomyelitis by the infection of periodontitis pathogen porphyromonas gingivalis
Authors: Guan Hongbing , Zheng Zhichao , Li Zhengmao
Published: January 2023

Deep cervical lymph nodes (dcLNs) were reported to be the first location for the initiation of encephalitogenic CD4+ T cells in experimental autoimmune encephalomyelitis (EAE). However, the precise mechanisms are not crystal clear. Here, we explored the epitope spreading in the two anatomically adjacent diseases, periodontitis and EAE. C57bl/6 mice were immunized with porphyromonas gingivalis (p.gingivalis), a virulent peridontopathogen, in the buccal submucosal tissue. Activation of T cells and MOG-specific CD4+ T cells in dcLNs and superficial cervical lymph nodes were detected with respect to the expression of CD69, CD44, and MOG-tetramer. A clinical symptom was observed in p.gingivalis-immunzed mice similar to that in MOG35-55 peptide-immunized mice. Dendritic cells in the buccal submucosal tissue and dcLNs were analyzed for the spreading of MOG epitope. Our results demonstrated the existence of the epitope spreading to the endogenous myelin in the inflammatory reponse of periodontitis caused by infection of p.gingivalis.
Visualization of IFN-β mRNA in Chlamydia-infected and adjacent uninfected cells and the contribution of CX43 in intercellular transfer of cGAMP
Authors: Tripathy Manoj Kumar , Zhang Yugen , Rodriguez-Nieves Jennifer , Bodnar Wanda M , Nagarajan Uma
Published: January 2023

Background

IFNβ has been implicated a key effector of oviduct pathology after genital chlamydial infection in mice. We reported that the host DNA sensor cGAS (cGAMP synthase) is essential for IFNβ expression during Chlamydia trachomatis infection. Sensing during infection leads to generation of the second messenger, 2′3-cGAMP, which can migrate to uninfected adjacent cells via gap-junction mediated transfer. cGAMP binding to central adaptor molecule, STING drives IFNβ induction. The goals of this study were to visualize the mechanism of c-GAMP transfer and to identify the DNA source triggering this response.

Methods

cGAMP production was detected by mass spectrometry. The contribution of gap junction proteins, connexins CX43 and CX45 in intercellular transfer of cGAMP was assayed by siRNA depletion and subsequent co-culture experiments. view RNATM methodology was used to detect IFNβ mRNA transcripts in Hela cells. Mitochondrial DNA as IFNβ inducer was assessed in mtDNA depleted cell lines.

Results

Mass spectroscopy confirmed cGAMP in infected host cytosol. Connexin depletion studies revealed that CX43 but not CX45 was essential for optimal IFNβ expression during infection. IFNβ mRNA transcripts were visualized in infected and adjacent, uninfected cells. In contrast, distal uninfected cells lacking cell-cell contact were negative. IFNβ induction after chlamydia infection was unchanged in mtDNA depleted cells.

Conclusion

Overall, our results confirm the production of cGAMP in chlamydia-infected cells and its transfer via CX43 to adjacent cells resulting in IFNβ production. Our studies exclude mtDNA as a potential source initiating this response. We are currently investigating the role of Chlamydial-DNA as an initiator.
Protein kinase C dependent development of dendritic cells.
Authors: Chavel Colin A , Lee Kelvin P
Published: January 2023

Dendritic cells (DC) are professional antigen presenting cells that are important activators of adaptive immune responses and play a role in the activation of CD8+ T cells that target tumor cells. Our lab has also previously shown that protein kinase C (PKC) βII is essential for DC differentiation and that tumor derived factors, particularly IL-6, inhibit DC differentiation by repressing the expression of PKCβII through a Stat3 dependent mechanism. However, the pathways activated downstream of PKCβII during DC differentiation have yet to be fully characterized. To study this, we used the K562 cell line, which differentiates into DCs with addition of PMA, a known chemical activator of conventional PKC isoenzymes. We have also shown previously that PMA specifically activates PKCβII in K562 cells. Addition of PMA to K562 cells showed the activation of the ERK1/2 and NFκB pathways, including both the canonical and non-canonical NFκB pathways. Chemical inhibition of either the ERK1/2 or NFκB pathway yielded immature DCs with a reduced capacity to stimulate T-cell proliferation when incubated with PMA. Inhibition of ERK1/2 also showed a change in levels of RelB, a NFκB transcription factor, with PMA treatment and during PMA induced DC differentiation. Our lab has also shown that matured DCs reduce Foxo3a protein levels with the stimulation of CD80/CD86 through an Akt dependent mechanism. Inhibition of either the ERK1/2 or NFκB pathways showed an increase in Foxo3a levels, indicating a role for the ERK1/2 and NFκB pathways in Foxo3a regulation during differentiation into mature DCs outside of Akt. Taken together, these results show an integral role for both the ERK and NFκB pathways downstream of PKCβII activation during DC differentiation.
ItpkB regulates CCR7 expression on dendritic cells prior to lymph node trafficking
Authors: Vella Jennifer L , Wang Xinxin , Huang Yina H
Published: January 2023

Dendritic cells (DC) are fundamental in the initiation of the adaptive immune response. Following antigen uptake, tissue-resident DCs undergo maturation in preparation for their migration to lymph nodes where they present processed antigen to T cells. Down-regulation of tissue-homing receptors and upregulation of lymphoid-homing receptors, including CCR7 is required for dendritic cell entry into afferent lymphatic vessels. CCR7 binding to its ligands, CCL19 and CCL21, provides the appropriate directional cues for DCs to traffic to lymph nodes. Although both maturation and migration of DCs have been extensively studied, it still remains unclear what signaling pathways are responsible for inducing CCR7 expression. We found that DCs deficient in inositol-trisphosphate 3-kinase B (ItpkB) express higher levels of CCR7. ItpkB-deficient BMDCs migrate to draining lymph nodes more efficiently in both competitive and noncompetitive environments compared to their wild-type counterparts. We have previously shown that ItpkB deficiency leads to enhanced Akt activation in NK cells. Western blot analysis showed that ItpkB-deficiency in DCs also leads to increased basal and LPS-induced Akt1 activation. To determine whether hyper-Akt activity is sufficient to trigger enhanced lymph node homing, we expressed two forms of constitutively active Akt1, Myr-Akt1 and Akt1 T308E, S473D, in wildtype DCs. Akt1 hyperactivity was sufficient to promote CCR7 expression but did not affect DC maturation. Our findings suggest that ItpkB regulates CCR7 expression on dendritic cells as they prepare to traffic to draining lymph nodes for the purpose of primary T cell responses.
Transcriptional regulation of basophil development and function in Th2 immunity
Authors: Zhang Sai , Henry Everett , Siracusa Mark C , Sant’Angelo Derek B
Published: January 2023

Basophils are the most rare granulocyte population, accounting for less than 1% of circulating leukocytes in mice and human. Recent studies have shown that basophils are associated with multiple immunological processes, such as allergic diseases, autoimmunity, and protective immunity against parasites. It has been show that the rapid secretion of Th2 cytokines by basophils in response to various stimuli plays key roles in immunity against parasitic infections. Basophils also release histamine and, therefore, are key contributors to allergy. In addition, basophil responses are associated with multiple human diseases such as asthma, atopic dermatitis and cancer. Given their important roles in inflammatory signaling and human diseases, remarkably little is known about the molecular regulation of basophil effector functions. Therefore, we have been exploring transcription factors that may play a direct role in controlling basophil development and effector functions. In this study, we have developed multiple in vitro and in vivo systems to study the influence of the loss of a potentially key transcription factor on the basophil response to allergen-induced Th2 immunity. We are in the process of performing gene expression arrays to understand the molecular mechanisms underlying the transcriptional regulation. Collectively, our studies provide new insight into the transcriptional network controlling basophil effector functions in Th2 immune response and lead to potential therapies that modulate basophil activities during the treatment of allergic diseases.
The advancing age affects the frequency, functions and antibody repertoire of human B-1 cells
Authors: Hernandez Ana Maria , Rodríguez-Zhurbenko Nely , Quach Tam , Hopkins Thomas J , Rothstein Thomas
Published: January 2023

Aging decreases the efficiency of several physiological processes, including immune function, which has been associated with an increased incidence of infections, autoimmune diseases and cancer. Human B cell populations, like naïve and memory B cells, suffer significant quantitative and qualitative changes in the elderly. The frequency and function of human B-1 cells, which play critical housekeeping and anti-microbial defensive roles, have not been studied. In the present work we analyzed how the frequency and function of human B-1 cells (CD20+CD27+CD43+CD38low) change with age. Our results show that not only the percentage of B-1 cells but also their capacity to spontaneously secrete IgM decreased with age. In fact, expression levels of Xbp1 and Blimp1, associated with a secreting B cell phenotype, were significantly lower, while those of Pax5, characteristic of non-secreting B cells, were significantly higher in elderly donors. Since aging affects the humoral immune response both quantitatively and qualitatively, next we studied the antibody repertoire of B-1 cells of healthy young and elderly donors. Elderly donors showed a higher number of repeated clones and therefore, a reduction of the antibody repertoire variability in comparison with young individuals. Furthermore it was interesting to observe in aged individuals an increased frequency of VH3-23 and VH4-34 usage, genes which have been linked to autoimmune diseases and hematopoietic malignancies. Moreover, there is a significant reduction in older individuals of clones expressing the sequence VH3-33, frequently found on antibodies against PC, oxidized LDL, apoptotic cells and induced after vaccination with Pneumavax23.
Neutrophil extracellular traps (NETs) promote disseminated intravascular coagulation in sepsis.
Authors: McDonald Braedon , Davis Rachelle , Jenne Craig N
Published: January 2023

Neutrophil extracellular traps (NETs; webs of DNA coated in anti-microbial proteins) are released into the vasculature during sepsis and contribute to organ damage. Various components of NETs have been shown to modulate the coagulation cascade in vitro. We hypothesized that NETs released into the vasculature during sepsis activate intravascular coagulation, leading to microvascular hypoperfusion and end-organ damage. Multi-color confocal intravital microscopy was used in mouse models of sepsis (endotoxemia and E. coli peritonitis) to visualize and quantify neutrophil and platelet trafficking, NETs production, real-time perfusion analysis, and intravascular thrombin activity (using novel in vivo zymography). In vivo imaging of the liver microcirculation in septic mice revealed spatial co-localization between NETs and intravascular coagulation (thrombin activity, fibrin deposition, and platelet aggregation). Inhibition of NETs using i.v. DNase infusion (to digest NETs), or PAD4-deficient mice (that have impaired NETs production) resulted in significantly lower quantities of intravascular thrombin activity, and improved microvascular perfusion. In a model of Gram-negative sepsis (E. coli peritonitis), PAD4-deficient mice had reduced markers of disseminated intravascular coagulation (intravascular thrombin, PAI-1), and end-organ damage (serum lactate, ALT) compared to wild-type controls. Together, these data demonstrate that NETs activate coagulation in vivo, and promote disseminated intravascular coagulation in sepsis. Inhibition of NETs in septic animals reduces intravascular coagulation, improves microvascular perfusion, and attenuates end-organ damage.
The effects of agonistic α-4-1BB antibody on natural killer cell function
Authors: Lee Seung-Hwan , Hahm Dahn
Published: January 2023

4-1BB is a member of the Tumor Necrosis Factor Receptor (TNFR) family. The TNFR family regulates a variety of functions including immune response, inflammation, and cell life/death. 4-1BB is a co-stimulatory molecule known best for its ability to increase survival, proliferation, anti-viral/tumor effects on T cells, and thus is currently undergoing clinical trials. However, the effects of 4-1BB stimulation in other cell types, such as natural killer cells (NK), are not well studied. Using a murine cytomegalovirus (MCMV) infected mouse model, we investigated the effects of agonistic α-4-1BB antibody stimulation on NK cell function and its impact on viral resistance. We hypothesized that 4-1BB stimulation will induce robust activation and proliferation of NK cells, resulting in an increased resistance against MCMV. To the contrary, treatment of the α-4-1BB antibody increased viral burden on 4 days post infection (p.i.) with highly increased NK cell proportion in spleens and livers. Further studies showed higher viral burden on day 1.5 p.i. with a reduced NK cell frequency. This suggests that an uncontrolled viral replication due to low NK numbers on early days of MCMV infection allows rapid expansion of residual NK cells by day 4. In vitro, treatment with α-4-1BB along with α-FasL antibodies rescued NK cell reduction, indicating that the NK cell loss is induced by Fas-mediated death signaling. Taken together, our data demonstrates that 4-1BB stimulation induces cell death signaling in the NK cell population, and results in an impaired anti-viral response. Further studies will provide a more complete view of the effects of 4-1BB stimulation as a cancer therapeutic, and also into the mechanism of NK cell life/death through TNFR signaling.
An Nlrc4 mutation associated with systemic hyperinflammation drives spontaneous non-hematopoietic IL-18 hyperproduction
Authors: Tariq Zeshan , McGavern Dorian B , O’Shea John J , Canna Scott
Published: January 2023

Macrophage activation syndrome (MAS) is a life-threatening sepsis-like hyperinflammatory state arising in certain infectious and rheumatic diseases. Previous genetic associations have implicated genes critical for perforin-mediated cytotoxicity. However, we recently identified that activating mutations in inflammasome component NLRC4 lead to MAS and early-onset enterocolitis. Patients with NLRC4-MAS also have chronically and extraordinarily elevated serum IL-18. Notably, disease-causing mutations in other inflammasomes do not cause MAS or chronic IL-18 elevation.

In vitro, IL-1b production by THP1 cells expressing various NLRC4 patient mutations correlated with disease severity and identified pathogenic clusters in the molecular structure. To further study the function of NLRC4, we created a mouse bearing the T337S point mutation. Like NLRC4-MAS patients, NLRC4-T337S mice demonstrate elevated serum IL-18 in the absence of systemic inflammation. However, macrophages from NLRC4-T337S mice did not demonstrate enhanced IL-1β or IL-18 production. Correspondingly, bone marrow chimeras demonstrated that IL-18 hyperproduction was derived from non-hematopoietic cells. NLRC4-T337S mice did not demonstrate more severe disease in response to repeated systemic stimulation through TLR9 or TLR4, nor upon LCMV infection, but developed colitis with IL-10 receptor blockade. These results suggest intestinal epithelia, which express both NLRC4 and proIL-18, may be a source of pathogenic IL-18 in this model. Future work will clarify the source(s) of excessive IL-18 and identify how chronic IL-18 effects on gut homeostasis and T and NK cell responses may promote systemic hyperinflammation.
Autophagy and Inflammasome activation triggered by LPS-negative Ehrlichia is dependent on both mTOR activation and MyD88 signaling
Authors: Vorhauer Jennie , Alaoui-El-Azher Mounia , Wells Alan , Ismail Nahed
Published: January 2023

Lipopolysaccharide (LPS) positive pathogens trigger autophagy and inflammasome activation. Autophagy is an innate anti-microbial host defense that maintains tissue homeostasis and is regulated by the nutrient-sensing mTORC1 complex. However it is unknown how the LPS-negative, Gram negative Ehrlichia elicits the same response. Monocytic Ehrlichia triggers Type I IFN-dependent NLRP3 inflammasome activation, which detrimentally effects host response to Ehrlichia. In this study, we examined the regulatory mechanisms of autophagy and inflammasome activation. With toll like receptor (TLR)-adaptor molecule MYD88 deficient mice, we reveal a crucial role for MYD88 in mediation of Ehrlichia-dependent inflammasome activation and hepatic inflammation. This role is demonstrated by decreased Caspase 1 and IL-1β levels in infected liver tissues, and decreased inflammatory cytokines (TNF-α and IL-6) when compared to wild type. Ehrlichia-induced inflammasome activation in wild type mice correlated with inhibition of autophagy. Ehrlichia-induced inhibition of autophagy in infected macrophages was dependent on TLR9, TLR2, and their adaptor MYD88. Inhibition was enhanced by IFN-β stimulation. We also demonstrate that mTORC1 autophagy inhibition acts downstream of MYD88. These data suggest that LPS-negative Ehrlichia inhibit autophagy and activate the inflammasome through mTORC1 activation and MYD88 signaling, contributing to Ehrlichia-induced inflammatory disregulation and liver injury.
Guanylate-binding proteins and other interferon-inducible proteins are essential for the activation of the inflammasomes
Authors: Man Si Ming , Karki Rajendra , Kanneganti Thirumala-Devi
Published: January 2023

Recognition of pathogens by the cell is paramount for the initiation of an appropriate immune response. A subset of pattern-recognition receptor family, including NLRP3, NLRC4 and AIM2, are cytosolic sensors which induce formation of the inflammasome, a multi-protein complex which drives the release of the pro-inflammatory cytokines IL-1β and IL-18. Here, we found that guanylate-binding proteins and other novel interferon-inducible GTPases were required for activation of the DNA-sensing AIM2 inflammasome during infection with Francisella novicida. These proteins were recruited to the cell membrane of the bacteria to mediate bacteriolysis, and thereby, liberating DNA for sensing by AIM2. However, engagement of the AIM2 inflammasome by mouse cytomegalovirus or transfected double-stranded DNA did not require interferon-inducible GTPases. Our results reveal a specific mechanistic link between cell-autonomous immunity and innate immune sensing pathways in response to bacterial infection.
Differential epigenetic regulation of encephalitogenic T cells in relapse-remitting and chronic-progressive mouse models of multiple sclerosis
Authors: Gandy K. Alexa Orr , Nagarkatti Mitzi , Nagarkatti Prakash S
Published: January 2023

In this study, we used a mouse model of multiple sclerosis (MS), experimental autoimmune encephalitis (EAE), to evaluate the role of microRNA (miRNA) in modulating T helper cell function in chronic-progressive (CP) versus relapse-remitting (RR) forms of the disease. We hypothesized that encephalitogenic T cell function in RR-EAE and CP-EAE is differentially regulated my microRNA. Microarray analysis of brain-derived CD4+ T cells revealed significant up-regulation of 106 and down-regulation of 42 miRNAs in the chronic-progressive (relative to relapse-remitting) form of disease. In silico analysis of the top up-regulated miRNAs and their target genes revealed significant overlap with signaling pathways imperative for the generation and maintenance of immune tolerance. Specifically miRNA-34, -148, -326 and -6691 were found to be up-regulated in the chronic-progressive form of disease. These miRNAs were predicted to target genes required for TGF-β signaling (ITGB8, ITGAV, NOTCH1, NOTCH2, SMAD2 & SMAD3), and T regulatory (T reg) function (CREB1, RUNX1 & FOXP3). Given the role for T regs in suppression of inflammation and the inverse correlation between T regs and inflammatory resolution, like that which occurs in MS remission, it is fitting that miRNAs governing this process are up-regulated in a chronic versus relapse-remitting form of EAE, promoting perpetuation of T-cell-mediated neuroinflammation. The current study demonstrates a role for miRNA in differentially shaping the function of T cells in models of RRMS and CPMS. Information regarding tolerance–promoting or –inhibiting miRNAs can be harnessed for the potential development of miRNA-directed therapies for diseases possessing important inflammatory components.
Development of a Therapeutic to Preserve Vaccine Memory Following Ionizing Radiation Exposure
Authors: Aryankalayil Joseph B , McFarland Hugh I , Rosenberg Amy S
Published: January 2023

We have demonstrated that in a mouse model of Listeria monocytogenes (LM) infection, revaccination within 3 days after exposure to sub-lethal ionizing radiation prevents the death of memory effector T lymphocytes and preserves vaccine memory. Although revaccination immediately after radiation exposure circumvents an increased risk of infection, it is impractical to revaccinate victims of radiation exposure to the innumerable agents to which they have developed immunity. Thus, we are seeking to develop a single therapeutic or a combination that mimic the effects of T cell activation via revaccination. We have selected our array of potential therapeutics based on their differential targets within T cell survival pathways. Broad spectrum therapeutics, stimulating numerous cell types such as Nicotine, Cimetidine, and Valproic Acid activate AKT and inhibit GSK3-beta to prevent subsequent apoptotic signals. Common gamma chain cytokines such as IL-15 and IL-21 are also candidates because of their ability to potently and specifically promote lymphocyte activation and survival. In addition, we have included Lovastatin to increase levels of P21 by proteasome inhibition. P21 may improve survival through temporary cycle arrest and enhanced DNA repair in activated T cells. We also utilized supplements to our therapeutics such as the FOXP3 inhibiting peptide- P60 and the checkpoint inhibitors anti-CTLA4 and anti-CD25 since regulatory T cells are a radio-resistant population that hinder the immune response to infection. We show, through comparisons of Listeria burden in spleens, that these candidates, either alone or in combination, are able to preserve vaccine memory following radiation exposure.
Defining the contribution of T cell exhaustion to failed immunity during TB
Authors: Jacques Miye K , Jayaraman Pushpa , Kuchroo Vijay K , Behar Samuel M
Published: January 2023

Tuberculosis is a pulmonary disease, caused by Mycobacterium tuberculosis (Mtb) infection, which affects approximately one third of the global population and is a major public health concern. Although T cells are essential in mediating protection against Mtb infection, they are often unable to sterilize infected tissues. One possible reason for an ineffective immune response is the onset of T cell exhaustion or dysfunction. T cell exhaustion is characterized by the progressive loss of T cell effector function accompanied by an increase in expression of inhibitory receptors. Here we show that during Mtb infection, T cells express the inhibitory molecules Tim3 and PD1, and T cells that express both Tim3 and PD1 have decreased IFNγ and TNF production. In addition, antibody blockade of Tim3 improves T cell function and decreases bacterial burden. These results indicate that T cells are functionally exhausted during chronic Mtb infection and that blockade of inhibitory receptors can re-invigorate T cell function and improve the outcome of infection. Based on a possible role for CEACAM1 in the T cell inhibitory signal mediated by Tim3, we are currently performing experiments to determine how CEACAM1 affects host defense against Mtb. Understanding the roles of inhibitory receptors, such as Tim3 and PD1, during Mtb infection is providing insight into why T cell immunity is insufficient to eradicate Mtb infection.