bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted over-expression of S100B in the retina of transgenic mice induced exacerbated experimental autoimmune uveitis
Authors: Yu Cheng-Rong , Ogbonna Oluchi , Lee Hyunsu , Choi Jin Kyeong , Jittayasothorn Yingyos , Li Lichao , Egwuagu Charles E
Published: January 2023

S100B, originally found in glial cells (astrocyte and microglial cells), acts as an intracellular Ca++-sensor and an extracellular regulator protein. Its neuronal trophic or toxic effect on cells might be dependent on its cellular concentrations. Human uveitis is an intraocular inflammatory disease of infectious or autoimmune etiology and neuroinflammatory responses orchestrated by retinal microglia cells are implicated in exacerbation of uveitis. The S100B protein is predominantly secreted by glial cells in central nerve system (CNS). In this study we examined whether S100B protein plays a role in regulating inflammatory responses in the neuroretina. To this end we generated retinal S100B transgenic mice and investigated whether experimental autoimmune uveitis (EAU) would be exacerbated in these transgenic mice. We monitored EAU progression and severity by fundoscopy and ERG (Electroretinogram)and found that over-expression of S100B+ promoted retinal inflammatory responses and vision loss during EAU. Results of FACS and ELISA analysis did not indicate significant difference in the numbers of uveitogenic Th1 and Th17 cells in the draining lymph-nodes. However, significant amounts of pathogenic Th1 and Th17 cells were detected in the retina of S100B transgenic mice. Conversely, the numbers of IL-10-producing CD4+Foxp3+Treg cells in the retina was significantly decreased in the S100B transgenic mice. These results suggest that S100B can serve as a biomarker for ocular inflammatory diseases.
Antigen dose affects disease kinetics in a murine model of demyelinating disease
Authors: Gavile Catherine Ann M , Alexander Amanda , Adler Fred , Evavold Brian D
Published: January 2023

Multiple sclerosis is an autoimmune disorder caused by immune cell damage to the myelin sheath lining nerve cells, leading to neuronal damage and dysfunction. The murine model of the disease - experimental autoimmune encephalomyelitis (EAE) - is induced by inoculation with MOG (myelin oligodendrocyte glycoprotein) peptide and an adjuvant, leading to recapitulation of disease that is mediated primarily by CD4 T cells. In this study, we look at the effect of MOG dosage on demyelinating disease kinetics in WT and MOG-deficient animals, as MOG-deficient mice have different numbers of MOG specific CD4 Teff and Tregs. The T cells from MOG deficient animals have a higher affinity for MOG, expand faster, and cause exacerbated disease in an adoptive transfer model compared to CD4 T cells from wildtype mice. Our results indicate that the dose of MOG peptide used to induce disease can also modulate disease course. Using tenfold lower doses changes T cell and disease kinetics resulting in disease that resolves compared to controls. Taken together, we can use varying doses of MOG as a tool to probe CD8 and CD4 T cell expansion and monitor migration of immune cells to the CNS. Further understanding of the cellular tides during disease course enables us to gain insight on how cell ratios in different sites contribute to disease pathogenesis. Using this information, we are developing mathematical modeling tools that could be used to analyze and predict disease progression.
Matrix metalloproteinase -28 alters cellular energetics in macrophages, influencing polarization and activation
Authors: Tokmina-Roszyk Dorota , Onwuha-Ekpete Lillian C. , Refai Mohammed , Tokmina-Lukaszewska Monika , Bothner Brian , Fields Gregg B.
Published: January 2023

Matrix Metalloproteinase -28 (MMP-28) is the newest and least characterized member of the matrixins family of proteolytic enzymes. These enzymes are associated with the degradation of the extracellular matrix affiliated with both physiological and pathological processes, including immune responses. In similar fashion to the rest of MMP family, a vast amount of literature on MMP-28 addresses its’ extracellular role. This study highlights the intracellular role of MMP-28 in macrophages, specifically its ability to modulate cellular energetics and consequently influence M0, M1, and M2 phenotype polarization.

In our preliminary studies, we utilized shotgun proteomics profiling of WT and MMP-28 null mice under both healthy conditions and following experimental autoimmune encephalomyelitis (EAE) induction. We identified two mitochondrial protein complexes dysregulated in MMP-28 null mice. We then isolated bone marrow derived macrophages to further elucidate differences using the Seahorse mitochondrial and glycolytic stress assays. We found that MMP-28 alters mitochondrial respiration and glycolysis across all tested phenotypes (M0, M1, and M2). Moreover, our preliminary data emphasis a significant role for MMP-28 in the efficient utilization of various fuel sources during mitochondrial respiration. Our findings show, for the first time, that MMP-28 has a profound effect on cellular bioenergetics affecting both oxidative phosphorylation, glycolytic pathways, and efficient macrophage activation. This suggests the potential of MMP-28 as both an immunomodulatory agent to further study macrophage bioenergetics and a therapeutic agent for pathologies associated with macrophages.
Blood Monocyte Subsets are Dysregulated and Respond with High IL-6 Secretion in Patients with Systemic Sclerosis
Authors: Friedrich Kathleen , Bearwald Christoph , Pierer Matthias , Rossol Manuela
Published: January 2023

Background

Most of the blood monocytes are classical monocytes (CM), minor subpopulations are intermediate monocytes (IM) and non-classical monocytes (NCM). CD56+ monocytes are a subpopulation of CM. Aim of the study was to analyze monocyte subpopulations in Systemic Sclerosis (SSc) patients and to evaluate their cytokine production potential.

Methods

25 SSc patients and 16 age-matched healthy controls were included in the study. Monocyte subpopulations were analyzed by flow cytometry (antibody staining of CD14, CD16, CD56). Magnetic bead separation was used to isolate monocytes. The cells were stimulated with TLR4 ligand LPS and TLR8 ligand ssRNA40 and cytokines were measured by ELISA.

Results

The absolute number of circulating monocytes was significantly increased in SSc patients compared to healthy controls (684/μl ± 61 vs. 512/μl ± 38, p=0.033). Calculated absolute number of CM and CD56+ monocytes was not different between SSc patients and controls. In contrast, the calculated absolute numbers of IM (72/μl ± 13 vs. 32/μl ± 5, p=0.0003) and NCM was increased in SSc patients compared to controls (97/μl ± 12 vs. 45/μl ± 8, p=0.0011). The cytokine response of SSc monocytes to TLR4 ligand LPS was equal to control monocytes but SSc monocytes produced significantly more IL-6 in response to TLR8 ligand ssRNA40 than control monocytes. In addition, SSc monocytes already secrete IL-6 without further stimulation. IL-6 was also increased in the serum of SSc patients compared to healthy controls.

Conclusion

Total monocytes are expanded in the blood of SSc patients, and this increase is caused by an expansion of IM and NCM. The monocytes might contribute to the presence of IL-6 in SSc patients due to their increased potential to secrete this cytokine.
Defining the role of interferon signaling in mouse models of Down syndrome
Authors: Tuttle Kathryn , Waugh Katherine A. , Minter Ross , Sullivan Kelly D , Espinosa Joaquin M
Published: January 2023

Down syndrome (DS) is caused by triplication of chromosome 21 (chr21) and is the most common chromosomal abnormality (1/700 live births). Our lab recently identified interferons (IFNs) as a core signaling pathway hyperactivated among diverse lineages of primary immune cells from individuals with DS. Notably, four of the six IFN receptor (IFNR) subunits for Types I, II, and III IFNs are encoded on chr21. Heightened IFN signaling in the disomic population is associated with developmental defects, cognitive impairments, and autoimmunity, a clinical profile highly similar to individuals with DS. However, the relationship between IFN signaling and such phenotypes has not been investigated in DS. We stimulated three different mouse models of DS with toll-like receptor (TLR) agonists, which potently induce interferon production. Both chronic and acute administration of TLR agonists caused lethal hyperresponsiveness only in the DS strain that harbors three copies of the IFNR locus. The resulting inflammation impacts multiple organ systems. This observation has profound implications for understanding how people with DS may differentially experience inflammation in response to infection during their lifetime. These studies represent the first steps in establishing a causative relationship between IFN signaling and DS-associated immune dysfunction to identify potential therapeutic targets for improving health outcomes in DS.
Human bone marrow plasma cell survival is independent of APRIL
Authors: Nguyen Doan C , Saney Celia , Sanz Iñaki , Lee F. Eun-Hyung
Published: January 2023

A fraction of the circulating antibody (Ab)-secreting cells (ASC) matures into long-lived plasma cells (LLPC) in the bone marrow (BM) microniches. Previous studies showed that ASC survival and longevity require APRIL, which upon binding its receptors, BCMA or TACI, activates PI3K/Akt (and its downstream mTOR) pathways and upregulates the expression of antiapoptotic proteins Mcl-1 and Bcl-2. Later work revealed that APRIL binding to SDC-1 (CD138), a cell-surface heparin sulfate proteoglycan (HSPG), also enables delivery of survival signals. Recently, we showed that APRIL is crucial in the ex vivo survival of early-minted human blood ASC. Here, using an in vitro cell-free BM microniche system, we demonstrate the differential roles of APRIL in the survival of human blood ASC and BM plasma cells (PC) and LLPC. APRIL substantially enhanced the survival of both CD138+ and CD138− ASC populations in the blood, which highly expressed BCMA. In contrast, it had no survival advantage on BM PC or LLPC, despite their high BCMA expression. Additionally, anti-CD138 Ab inhibited the survival of both blood ASC and BM PC and LLPC, including CD138− cell subsets, suggesting inhibiting the later CD138 upregulation also affects blood ASC survival. These data suggest that APRIL is important for the survival of early-minted blood ASC but not BM PC or LLPC. In conclusion, APRIL is an important survival and imprinting factor of blood ASC but is not required for LLPC maintenance.
TNF-α induces a pro-inflammatory phenotypic shift in monocytes through ACSL1: Relevance to metabolic inflammation
Authors: Alrashed Fatema , Ahmad Zunair , Iskandar Mina Amin , Tuomilehto Jaakko , Ahmad Rasheed
Published: January 2023

Background

TNF-α mediated proinflammatory phenotypic change in monocytes is known to be implicated in the pathogenesis of metabolic inflammation and insulin resistance. However, the mechanism by which TNF-α induces inflammatory phenotypic shift in monocytes is poorly understood. Since long-chain acyl-CoA synthetase 1 (ACSL1) is associated with inflammatory monocytes/macrophages, we investigated the role of ACSL1 in the TNF- α driven inflammatory phenotypic shift in the monocytes.

Methods

Monocytes (Human monocytic THP-1 cells) were stimulated with TNF-α. Inflammatory phenotypic markers (CD16, CD11b, CD11c and HLA-DR) expression was determined with real time RT-PCR and flow cytometry. IL-1b and MCP-1 were determined by ELISA. Signaling pathways were identified by using ACSL1 inhibitor, ACSL1 siRNA and NF-kB reporter monocytic cells. Phosphorylation of NF-kB was analyzed by western blotting and flow cytometry.

Results

Our data show that TNF-α induced significant increase in the expression of CD16, CD11b, CD11c and HLA-DR. Inhibition of ACSL1 activity in the cells with triacsin C significantly suppressed the expression of these inflammatory markers. Using ACSL-1 siRNA, we further demonstrate that TNF-α induced inflammatory markers expression in monocytic cells requires ACSL1. In addition, IL-1b and MCP-1 production by TNF-α activated monocytic cells was significantly blocked by the inhibition of ACSL-1 activity. Interestingly, elevated NF-κB activity resulting from TNF-α stimulation was attenuated in ACSL1 deficient cells.

Conclusion

Our findings provide an evidence that TNF-α associated inflammatory polarization in monocytes is an ACSL1 dependent, which indicates its central role in metabolic inflammation.
Defining the Phenotype and Function of Granzyme A Expressing T-Helper Cells
Authors: Hayes Tristan A , Kaplan Mark H
Published: January 2023

Granzymes have been implicated in numerous diseases including arthritis, viral and bacterial infections, hypersensitivity, autoimmune diabetes, and some cancers. In a STAT6 transgenic mouse model of atopic dermatitis, we identified a population of Granzyme A expressing CD4-T- cells. The role that these cells play in disease is unclear. To define their phenotype, we generated granzyme A (GrA)-expressing T helper cells in vitro using a combination of IL-4/IL-6 and IL-21. GrA-expressing TH cells did not express other TH subsetassociated cytokines, and other TH subsets did not appreciably express GrA. To further examine the development and function of GrA+ TH cells, we developed a Granzyme A-GFP reporter allele termed Grama (GrA Monitoring Allele) mouse. Grama+ cells were identified in the spleen and intestine. Using RNA-seq analysis of in vitro derived cells, we additionally identified several genes, including Arg1, Zpbp2, and Rpl39, that were enriched in GrA-expressing T cells. Thus, GrA-expressing CD4 T cells may represent a novel TH subset and it will be interesting to define the function of this subset in inflammatory diseases.
Complement activation causes oncogene expression in Gaucher disease
Authors: Pandey Manoj K , Magnusen Albert F , McKay Mary A , Nyamajenjere Tsitsi C , DiPasquale Betsy Ann , Magnusen Daniel N , Rani Reena , Witte David , Grabowski Gregory A , Köhl Jörg
Published: January 2023

GBA1 mutations lead to defective lysosomal glucocerebrosidase resulting in accumulation of glucosylceramide (GC) in Gaucher disease (GD). Patients with GD have an increased risk to develop B cell lymphomas. The exact mechanistic bases for this propensity remain elusive. Recently, we uncovered formation of GC-specific IgG autoantibodies in Gba1 D409V/knockout (Gba19V/−) mice, which recapitulate features of human GD, and in humans with untreated GD. In vivo formation of IgG-GC immune complexes induced massive complement activation and C5a generation. Importantly, C5a-mediated activation of its cognate C5a receptor 1 (C5aR1) on immune cells enhanced GC synthesis, thereby fueling GC accumulation and excess tissue recruitment and activation of inflammatory myeloid and lymphoid immune cells, leading to visceral tissue damage in GD. Here, the expression of Runt-related transcription factor 1 (RUNX-1) was determined in Gba19V/− mice, to evaluate if C5a/C5aR1 axis activation may control the development of lymphomas in GD. RUNX-1 is a member of the Runt oncogene family linked to hematologic malignancies. We determined RUNX-1 expression in tissue from C5aR1 sufficient (+/+) and deficient (−/−) Gba19V/− mice as well as strain-matched control WT and C5aR1−/− mice. Compared to WT, Gba19V/− mice had increased RUNX-1 expression. Strikingly, RUNX-1 expression was markedly downregulated in C5aR−/−Gba19V/− vs. C5aR1+/+Gba19V/− mice. Our findings suggest that the C5a-C5aR1 axis activation in GD drives RUNX1 expression as a novel mechanism to control the development of hematologic malignancies in GD that may be diminished by targeting the C5aR1 axis in GD.
Functional characterization of an activating anti-TLR4 monoclonal antibody (UT18) with a demonstrated role in reversal of new-onset type I diabetes in NOD mice
Authors: LOCKER KATHRYN CS , Hoebe Kasper , Ridgway William M , Herr Andrew B
Published: January 2023

Type I diabetes (T1D) is an autoimmune disorder characterized by inflammation and cellular infiltration of the pancreatic islets, resulting in insulitis and damage to islet beta-cells. T1D pathogenesis involves soluble and cellular mediators of both the innate and adaptive arms of the immune system; thus, finding therapeutic targets for reversal of acute disease has proved challenging. We have shown that an activating monoclonal antibody (UT18) targeting mouse TLR4/MD2 reverses acute T1D in non-obese diabetic (NOD) mice. UT18 preserves insulin-producing islet beta-cells and decreases insulitis; however, the molecular mechanisms underlying disease reversal remain unclear. Using AMNIS imaging flow cytometry, we observed an atypical internalization event in which UT18-bound TLR4 persists in EEA1-enriched endosomes rather than recycling back to the cell surface as seen with the TLR4 ligand LPS. Because the subcellular localization of TLR4 alters its proximity to adaptor proteins, we performed immunoblots of downstream signaling mediators. UT18 activates both MyD88 and TRIF pathways; however, the magnitude of MAPK activation is dampened and the kinetics of signaling differ from the rapid, robust activation characteristic of pro-inflammatory LPS. Our data suggest that UT18 functions as a mild TLR4 agonist, eliciting an intermediate signaling profile hypothesized to contribute to tolerance of antigen presenting cells. Knowledge of how UT18 manipulates the TLR4 signaling axis may be applicable for the development of novel immunotherapies for T1D and other autoimmune or inflammatory diseases in which innate immune receptors contribute to pathogenesis.
Age associated insulin resistance is controlled by microbiota through a monocyte-B1a cell pathway
Authors: Bodogai Monica , O'Connell Jennifer , Kim Yoo , Egan Josephine , Biragyn Arya
Published: January 2023

Aging is associated with increased hyperglycemia, insulin resistance and dysregulation of the immune system, but the causative factors underlying their association remain unknown. We show that IR is induced by activated innate 4-1BBL+ B1a (4BL) cells that accumulate as a response to age induced changes in gut microbial composition and subsequent decrease in beneficial microbial metabolites. The loss of the commensal bacterium Akkermansia muciniphila impairs intestinal integrity, causing leakage of bacterial products such as endotoxin, which activate CCR2+ monocytes in an environment with reduced butyrate concentration. These CCR2+ monocytes infiltrate in the omentum where they induce 4-1BBL expression on B1a cells. The cascade continues with 4BL cells inducing IR, presumably by triggering 4-1BB receptor signaling as in obesity-induced metabolic disorders. This pathway and IR are reversible, as manipulation of gut microbiota can restore normal insulin response in aged mice and macaques. In addition, treatments that target monocytes or 4BL cells had the same effect on IR. These results underscore the pathological function of B1a cells and suggest that the microbiome-monocyte- B cell axis could potentially be targeted to reverse age-associated IR.
Peripheral Blood Th40 cells Reflect Disease Progression and Are Controlled by Rituximab in Human Multiple Sclerosis
Authors: Wagner David H , Vaitaitis Gisela , Waid Dan
Published: January 2023

Multiple Sclerosis (MS) is a neuro-degenerative autoimmune disease, progressive in nature, that lacks biomarkers for diagnosis or progression. We described a population of CD4+ T cells that express the CD40 receptor, Th40 cells, which proved causal in murine models of autoimmunity.

Hypothesis

Peripheral blood Th40 numbers reflect MS progression and their control correlates with improvement. Peripheral blood and cerebral spinal fluid from MS patients and disease controls were examined for HLA haplotype and PBMC were characterized by flow cytometry. Random sampling of CIS, RRMS, SPMS and PPMS patients demonstrated diverse HLA haplotypes; DR2 / DQ6 were highly represented but surprisingly DR4, DR3 and DQ2 were prominent. In antigen recall, each HLA haplotype preferred unique myelin-based antigens when presenting to Th40 cells. Percentages of Th40 cells in peripheral blood of CIS and RRMS had a wide range while the range in PPMS and SPMS was tight yet significantly elevated. CSF from RRMS patients had Th40 cell percentages mirroring peripheral blood. RRMS subjects had elevated B cells that surprisingly were the major source of IL-17. Rituximab treated subjects demonstrated a significant but temporal reduction in Th40 cell numbers; FoxP3+ cells were elevated while Th40 inflammatory-cytokine production was reduced.

Conclusions

The wide range of Th40 cell numbers in CIS and RRMS but universally high numbers in SPMS suggest that Th40 cell number expansions in MS reflect progression. Th40 cells in CSF demonstrate access to the CNS and an important correlation between peripheral blood and CNS. Antigen recall to myelin demonstrates Th40 pathogenicity. Rituximab treatment ablates B cells that then affect pathogenic effector Th40 cells.
Long-range cis-regulatory architecture of the human IL2 gene
Authors: Sharma Prabhat K , Thomas Rajan M , Johnson Matthew , Wells Andrew
Published: January 2023

Interleukin-2 is a potent T cell growth factor with crucial roles in both immunity and tolerance. The IL2 gene is regulated by multiple signaling pathways, transcription factors, and epigenetic processes operating at the 500 bp upstream regulatory region (URR). Genome-wide association studies (GWAS) in humans have shown that genetic variation in the IL2 region is associated with susceptibility to autoimmune diseases. This region harbors several conserved noncoding sequences. We are performing a comprehensive epigenomic and functional interrogation of the human IL2-IL21 multi-locus region, using a combination of open chromatin mapping (ATAC-seq), recombinant promoter-reporter assays, 3-dimensional chromosome conformation capture (Capture-C), and CRISPR/CAS9 genome editing. ATAC-seq analysis identified TCR/CD28 activation-dependent chromatin remodeling at −46, −51, −80, −83, −-85, −121, and −128 kb upstream of IL2 in human T cells. Chromatin conformation capture indicates that several of these distal regions physically interact with IL2, and are capable of enhancing transcription from the IL2-URR in recombinant reporter assays. The −128 kb element showed autoimmune disease-associated allelic variation in enhancer activity, and CRISPR/CAS9-mediated deletion of this element in human T cells resulted in a 2–3 fold reduction in activation-induced IL-2 production. These results suggest that the IL2 locus has evolved an extensive regulatory architecture that allows for variable production of IL-2, and provide insight into why most autoimmune IL2 polymorphisms are located far away from the gene.
PAK6- A novel kinase in the maintenance of T cell anergy
Authors: Torres Annabel , Tang Alice , Vyas Radha , Su Chen , Johnson Matthew , Wells Andrew
Published: January 2023

Costimulation of T cells through the antigen receptor and the CD28 receptor engages an ‘effector’ gene expression program and a burst of proliferation. Conversely, antigen receptor engagement in the absence of costimulation induces a hypo-responsive state, referred to as anergy, that is associated with active silencing of effector genes. Our ATAC-seq analysis of CD28-dependent chromatin remodeling in anergic and effector CD4+ T cells has identified p21-activated kinase 6 (PAK6) as a gene that becomes accessible in anergic but not effector T cells. PAK6 is a member of a family of kinases comprised of two groups, group I (PAK1-3) and group II (PAK4-6). PAK6 and other group II members lack a regulatory domain that is present in group I, causing it to function differently. Previous studies by others have shown that PAK2, a group I PAK, was a positive regulator of CD3/CD28 T cell activation. Here, we demonstrate that PAK6 has the opposite role in T cell activation as PAK2, where it inhibits IL2 secretion, a hallmark characteristic of T cell anergy. Knockdown of PAK6 in anergic and effector T cells, using a CRISPR/CAS9 approach, resulted in a 50% increase in IL2 secretion, and 5-fold more cell expansion in anergic cultures upon restimulation with CD3/CD28. This suggests that PAK6 may be involved in a signaling pathway which inhibits cellular proliferation or promotes apoptosis. The highest level of PAK6 expression was observed upon re-stimulation of anergic cells, suggesting that PAK6 may be involved in the maintenance of T cell anergy. This study identifies a novel role for PAK6 in T cell anergy and a potential new target for regulating T cell function in autoimmune diseases.
JNK inhibition by Transforming Growth Factor-β Type I Receptor (TGF-β RI) in T Cells
Authors: Jacks Ramiah , Iwashima Makio
Published: January 2023

T cells are a critical component of the adaptive immune system and their responses in pathogenic and steady state conditions require strict regulation. One mechanism involved in this regulation is Transforming Growth Factor-β (TGF-β) signaling. TGF-β acts on activated T cells to facilitate the differentiation of some T helper subsets or the suppression of activation and proliferation. Our recent data demonstrate that TGF-β plays a role in the survival of Tregs against continuous antigen exposure. TGF-β signaling is propagated by initially binding to the TGF-β type II receptor, which activates TGF-β type I receptor that phosphorylates the transcription factors SMAD2/SMAD3. This induces nuclear localization of SMAD complexes and transcription of target genes. To understand how TGF-β signaling is coupled to T cell specific events, we generated TGF-βRI deficient Jurkat T cells using the CRISPR/cas9 system. Unexpectedly, TGF-βRI deficient Jurkat demonstrate a hyperactivation phenotype featuring a highly elevated expression of surface antigens (CD69, CD25), and secreted significantly increased levels of IL-2 and TNF. Analysis of signaling events in TGF-βRI deficient Jurkat demonstrate constitutive activity of activator protein-1 (AP-1), constitutive activation of jun-amino-terminal kinase (JNK) and increased expression of JUN. Similarly, in primary T cells transient expression of a dominant negative mutant of TGF-βRI elevated AP-1 activity while a constitutively active mutant reduced this activity. Together, these data show that TGF-βRI signaling plays a novel role in controlling the activity of JNK and AP-1 in T cells. This signaling pathway may play a critical role in TGF-β mediated differentiation and/or survival of T cells.
Genome-wide analysis identifies pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity
Authors: Zhang Qiong , Chao Ti-chun , Patil Veena S. , Qin Yue , Tiwari Shashi Kant , Chiou Joshua , Dobin Alexander , Tsai Chih-Ming , Li Zhonghan , Dang Jason , Gupta Shagun , Urdahl Kevin B , Nizet Victor , Gingeras Thomas R. , Gaulton Kyle J , Rana Tariq M.
Published: January 2023

Long noncoding RNAs (lncRNAs) can regulate target gene expression by acting in cis (locally) or in trans (non-locally). Here, we performed genome-wide expression analysis of Toll-like receptor (TLR)-stimulated human macrophages to identify pairs of cis-acting lncRNAs and protein-coding genes involved in innate immunity. A total of 229 gene pairs were identified, many of which were commonly regulated by signaling through multiple TLRs and were involved in the cytokine responses to infection by group B Streptococcus. We focused on elucidating the function of one lncRNA, named lnc-MARCKS or ROCKI (Regulator of Cytokines and Inflammation), which was induced by multiple TLR stimuli and acted as a master regulator of inflammatory responses. ROCKI interacted with APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1) to form a ribonucleoprotein complex at the MARCKS promoter. In turn, ROCKI–APEX1 recruited the histone deacetylase HDAC1, which removed the H3K27ac modification from the promoter, thus reducing MARCKS transcription and subsequent Ca2+ signaling and inflammatory gene expression. Finally, genetic variants affecting ROCKI expression were linked to a reduced risk of certain inflammatory and infectious disease in humans, including inflammatory bowel disease and tuberculosis. Collectively, these data highlight the importance of cis-acting lncRNAs in TLR signaling, innate immunity, and pathophysiological inflammation.
Identification and characterization of fish-specific IL-23 isoforms and their roles in fish immunity
Authors: Qin Lei , Zhang Shengnan , Zhou Hong
Published: January 2023

IL-23 is well known as a regulator in inflammation response and host defense by promoting the development of Th17 cells. In fish, three genes coding p40 (p40a, p40b and p40c) are originated from fish-specific genome duplication, thereby generating three IL-23 isoforms (IL-23a, IL-23b and IL-23c). Considering the functional diversity of fish IL-23, it is possible that the fish-specific immunoregulatory mechanism of IL-23 may have existed. In this study, we isolated and identified the subunits of IL-23 from grass carp. In response to bacterial infection in vivo, the mRNA levels of gcp19 were upregulated in headkidney, liver and gill, while those of p40a were significantly induced in the selected tissues except liver. Surprisingly, the levels of gcp40b and gcp40c did not show marked change in tissues. What’s more, LPS, PHA stimulation in vitro, the mRNA levels of these subunits also showed a significant increase in periphery blood lymphocytes. Meanwhile, the result of Co-IP showed p19 could couple with each of the three p40 to form IL-23 and be secreted into the supernatants, verifying the existence of three IL-23 isoforms in grass carp. In order to assess their functions in fish immunity, we constructed plasmids containing gcp19 CDS combined with gcp40a/b/c CDS by a linker sequence and over-expressed them in CHO cells. The positive stable transfectants were selected, and the recombinant proteins were produced and purified from the culture medium. At present, we have gained these grass carp IL-23 proteins. Their bioactivities were investigated in grass carp headkidney cells, showing same or different functions in the immune regulation of grass carp. Finally, our work has provided a basis for elucidating the roles of IL-23 isoforms in fish immunity.
Cannabinoid receptor 1 blockade attenuates obesity and adipose tissue type 1 inflammation through miR-30e-5p regulation of Delta-like-4
Authors: Miranda Kathryn , Mehrpouya-Bahrami Pegah , Nagarkatti Prakash S , Nagarkatti Mitzi
Published: January 2023

Obesity is characterized by chronic low-grade inflammation that contributes to development of cardiometabolic disorders. Cannabinoid receptor 1 (CB1) antagonists attenuate diet-induced obesity (DIO) and related inflammation, although the precise anti-inflammatory mechanisms involved have not been fully explored. In the current study we used a mouse model of DIO intervention to determine the microRNA (miR)-mediated anti-obesity and anti-inflammatory effects of the CB1 antagonist AM251. DIO mice that were fed high-fat diet (HFD) for 12 weeks were treated with AM251 (10mg/kg) for an additional four weeks. HFD+AM251 mice experienced rapid and prolonged weight loss and reduced inflammatory M1 adipose tissue macrophage (ATM) infiltration. To investigate miRNA-mediated regulation of ATMs, F4/80+ cells from stromal vascular fractions of epididymal fat were subjected to miR microarray analysis. Several miRs were differentially expressed in AM251-treated mice that were independent of calorie restriction. Prominently, miR-30e-5p was upregulated in ATMs from HFD+AM251 mice while the miR-30e-5p targeting DLL4 was downregulated. Consistent with a decrease in DLL4-Notch signaling, fat storage and pro-inflammatory cytokine/chemokine expression was reduced following AM251 treatment. Furthermore, we demonstrate that AM251-treated macrophages can suppress DLL4-mediated Th1 polarization in CD4+ T cells. Together these data indicate that weight-loss due to AM251 treatment mediates anti-inflammatory effects and amelioration of obesity through miR-30e-5p regulation of DLL4-Notch signaling-induced type 1 inflammation. These data support therapeutic potential of miR-30 in the treatment of cardiometabolic disorders.
A host-associated antimicrobial agent exhibits both immunoprotective and immunotherapeutic effects
Authors: Dawes Maisie Ernestene , Xie Chen , Chitko-McKown Carol G.
Published: January 2023

Inflammation is a critical component of both the defense against injury and the pathophysiology of disease. Despite advances in anti-inflammatory therapy and critical care, Gram-negative infections remain a significant cause of mortality in multiple species, and are a primary cause of diarrhea, endotoxemia, sepsis and ultimately death in neonatal calves. Given the impact of host response to disease on the health of humans and food animals alike, we examined the effects of supplemental bovine lactoferrin (bLF) and its functional peptide lactoferricin B (LFcin B), on the dysregulated immune response to lipopolysaccharide (LPS). We hypothesized that these antibiotic agents have therapeutic value as low-risk anti-inflammatory agents, a characteristic that is attributable to their regulatory role on intracellular signaling in innate cells.

Although present in colostrum, saliva and mucosal secretions, bLF is notably at its highest concentration in the secondary granules of neutrophils. In the host, the 25 AA peptide LFcin B is released following the activity of proteolytic enzymes in the stomach as well as at sites of infection.

Viable peripheral monocytes and neutrophils were isolated from calves and stimulated in vitro with LPS, in the presence or absence of bLF/LFcin B. Samples were analyzed using qRT-PCR, Meso Scale Discovery Electrochemiluminescence, immunoprecipitation and western immunoblot detection. The effects of bLF and LFcin B on pro-inflammatory cytokine and inflammatory mediator production will be expressed as the mean ± SD and will be considered significant at P<0.05. Although each exerted differential effects, the p38 MAP kinase pathway appears to be a likely target.
Bisphenol-A leads to overexpression of estrogen receptors and diminishes protective chemokine responses in a zebrafish model
Authors: Walser-Kuntz Debby , Tieu Tam , Scott Tamara , Khan Pavana
Published: January 2023

Bisphenol-A (BPA), widely used in thermal receipts and food containers, binds to both estrogen receptor (ER) α and β, disrupting estrogen activity and presenting an environmental challenge to the immune system of humans and wildlife. We are exploring the impact of in vivo BPA exposure on zebrafish innate immunity. Zebrafish exposed to 100 ng/ml BPA immediately following fertilization (day 0) show a two to fourfold reduction in gene expression of CXCL8, the chemokine that recruits neutrophils to the site of infection on days 3 through 6 post fertilization (dpf). To determine if reduced chemokine expression has a functional outcome, we studied MPO::GFP zebrafish expressing GFP under a neutrophil-specific myeloperoxidase promoter, which allows tracking of neutrophil movement to a tail cut injury. Compared to controls, 4 dpf BPA-exposed zebrafish show significantly reduced neutrophil recruitment to the site of injury (p=0.004). Control zebrafish receiving a tail cut injury at 4 dpf show a fourfold upregulation in CXCL8 expression one hour post-injury, whereas the BPA-exposed embryos display a blunted CXCL8 response. Because BPA-exposure at 0 dpf leads to a more than twofold increase in CXCR2 expression at 3 and 4 dpf compared to controls, neutrophil migration is likely reduced due to diminished CXCL8. Estradiol is known to reduce CXCL8 expression via the ER, and we found that BPA upregulates ERα and β gene expression by 3 dpf. During the first week of development, control zebrafish show an overall pattern of expression in all four tested genes that differs from that of BPA-exposed embryos. Our findings suggest that BPA leads to the overexpression of ERs, thus reducing CXCL8 expression and diminishing key protective innate immune responses.
Developmental and Environmental Regulation of Lung Macrophage Immune Response
Authors: Yamamura Asami , Lund Sean J , Lakhdari Omar , Sakai Mashito , Glass Christopher K , Prince Lawrence S
Published: January 2023

Neonates are uniquely susceptible to lung infection and injury. Alveolar (AM) and interstitial (IM) lung macrophages are the major cell populations protecting the lung against inhaled microbes and facilitating wound repair. AM and IM express unique cell surface markers and transcriptional profiles. We therefore hypothesized that neonatal and adult lung environments lead to differences in macrophage function. When lung macrophages were isolated by flow cytometry and stimulated with LPS in vitro for 2 h, neonatal AM expressed higher levels of Il1b and Il6 compared to adult AM. We next tested our hypothesis in vivo, challenging neonatal and adult mice with intranasal LPS. Contrary to our in vitro experiments, LPS induced higher cytokine expression in AM compared to IM. In addition, the adult AM response was higher than neonatal AM. To specifically test how the lung microenvironment regulated macrophage response, we stimulated neonatal and adult AM and IM with LPS either immediately after isolation or after 10–20 h of tissue culture. Consistent with our hypothesis, expression of key AM transcription factors fell quickly during cell culture. While neonatal AM and IM had similar LPS responses immediately after isolation, adult IM had higher cytokine expression compared to adult AM. Intriguingly, for both adults and neonates, longer culture time enhanced cytokine expression in activated AM while dampening the expression in IM. Our data suggest that the developmentally regulated lung microenvironment regulates the macrophage innate immune response.
Regulatory roles of suppressors of cytokine signaling 1 during type I vs type II macrophage polarization and phenotype determination
Authors: Lee Choong-eun , Jeong Hana , Kim Ga-young
Published: January 2023

During infection, activated macrophages display type 1 pro-inflammatory (M1) and type II anti-inflammatory (M2) features for pathogen clearence and the subsequent tissue repair, respectively. While suppressors of cytokine signaling (SOCS) play a role in the regulation of inflammation in diverse systems, the regulatory role of SOCS in M1 vs M2 activity has not been clearly established. We have studied SOCS action in macrophage phenotype determination in vitro employing SOCS gene transfer and knock-down using THP1 cells differentiated with PMA. For the LPS-induced M1 polarization, ROS signaling was crucial as the early ROS generation, MAPK activation, and the production of pro-inflammatory cytokines were all subject to inhibition by NAC, an anti-oxidant. Meanwhile, the dexamethasone (Dex)-induced M2 polarization involved ROS generation and MAPK/MPK1 activation in delayed kinetics, leading to the production of anti-inflammatory cytokines, which was also sensitive to NAC. Our previous studies demonstrated anti-oxidant functions of SOCS1 through the induction of thioredoxin and Nrf-2. In line with this, SOCS1 over-expression significantly inhibited M1 induction with ROS down-regulation, while SOCS1 ablation promoted ROS and pro-inflammatory/M1 cytokine production. In contrast, Dex-induced anti-inflammatory/M2 cytokines were severely reduced by SOCS1 knock-down with the impaired p38 and GILZ induction. The data support the anti-inflammatory action of SOCS1, while suggesting that SOCS1 exerts differential effects on M1 vs M2 polarization. The identification of distinct target molecules of SOCS1 action in M1vs M2 macrophage phenotype determination is under investigation.
Chronic Viral Infection Induces Metabolic Reprograming of Plasmacytoid Dendritic Cells
Authors: Greene Trever T , Jo Yeara , Zuniga Elina
Published: January 2023

Viruses that cause chronic infections, including Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV), infect more than 500 million people worldwide and represent a major health burden. Chronic viral infections suppress immune responses to promote their own persistence. Plasmacytoid Dendritic Cells (pDCs) are an innate cell type which are specialized to produce exceptionally high levels of type I interferon (IFN-I) in response to viral infection, however in the context of chronic viral infection they lose the capacity to produce IFN-I. The metabolic program of pDCs has previously been implicated to be crucial for their function, but it is unclear how chronic infection modifies pDC metabolism. We demonstrate that chronic infection stimulates metabolic reprogramming in pDCs. Specifically, chronic infection is associated with sustained suppression of respiratory metabolism in pDCs. Furthermore, we demonstrate that respiratory metabolism in pDCs is highly correlated to their ability to produce cytokine, and that byproducts of respiratory metabolism (mitochondrial derived reactive oxygen species) can promote cytokine production in pDCs. These data suggest that chronic infection drives changes in pDC metabolism that could be associated with infection driven pDC exhaustion.
Clinical manifestations following intramuscular exposure of mice to a lethal dose of ricin
Authors: Sapoznikov Anita , Falach Reut , Gal Yoav , Evgy Yentl , Kronman Chanoch , Sabo Tamar
Published: January 2023

Ricin a plant toxin derived from seeds of Ricinus communis, irreversibly inactivates ribosomes by site-specific depurination, thereby arresting cell protein synthesis, while its clinical manifestations following intramuscular exposure have not yet been investigated in depth. Here we report an extensive pathological study of intramuscular intoxication with a lethal dose of ricin in a murine model.

Ricin injection caused neutrophilia, thrombocytopenia and coagulopathy. Although histopathological alternations including hemorrhages, vessel congestion and focal necrosis were observed in various organs, major insults were limited to the spleen, bone marrow (BM) and cardiovascular system. Intensive atrophy, evident in the BM and splenic white pulps, were accompanied be severe depletion of megakaryocytes in both organs. Moreover, splenic T and B lymphocytes, NK cells, macrophages and dendritic cells decreased significantly. The most striking damage was in the heart, where we observed diffuse hemorrhages, myocyte disconnection, loss of striation, interstitial edema, collagen deposition and elevated levels of serum cardiac troponin. Finally, echocardiography revealed marked thickening of the walls, along with sequential decrease in left ventricular volume. These were accompanied by a significant reduction in stroke volume and cardiac output, while the ejection fraction remained unaltered. Measurements of the toxin direct catalytic performance in the spleen, BM and heart of the ricin-intoxicated mice revealed no substantial 28S rRNA depurination. The lack of measurable ricin catalytic activity in conjunction with the striking clinical disorders observed, suggests that ricin affects these organs in an indirect manner.
NLRP12 regulates intestinal inflammation and tumorigenesis independent of gut microbiota
Authors: Khan Shahanshah , Zaki Hasan
Published: January 2023

Cytosolic NOD-like receptor NLRP12 plays a key role in suppressing intestinal inflammation and tumorigenesis via downregulation of NF-kB and MAPK pathways. Recent studies showed that altered gut microbiota in Nlrp12−/− mice is responsible for higher activation of NF-kB and MAPK pathways. Therefore, the precise function of NLRP12 in the intestinal remains poorly defined. Here we reinvestigated the contribution of gut microbiota in NLRP12-dependent regulation of colitis and colorectal cancer (CRC). We analyzed the microbiota composition of wild-type and Nlrp12−/− mice by 16S rRNA sequencing. Although there was a considerable difference in different microbial taxa between WT and Nlrp12−/− mice, no major difference was noted in Lachnospiraceae, which was previously been linked to hyperinflammatory responses in Nlrp12−/− mice. We next assessed the contribution of altered microbiota in colitis and CRC susceptibility of Nlrp12−/− mice. WT and Nlrp12−/− mice were co-housed before induction of experimental colitis and colorectal tumorigenesis. Separate or co-housed Nlrp12−/− mice developed significantly heightened colitis and increased burden of colorectal tumors. Higher activation of NF-kB, ERK, and STAT3 pathways was observed in both separate and co-housed Nlrp12−/− mouse colons during acute colitis. Further, macrophages from littermate or co-housed Nlrp12−/− mice exhibited hyperinflammatory responses along with higher activation of NF-kB and ERK pathways upon stimulation with LPS. Taken together, NLRP12 is an intrinsic regulator of NF-kB and MAPK pathways downstream of TLRs and suppresses inflammatory and tumorigenic responses in the intestine independent of gut microbiota.
MyD88 mediates the pathogenesis of dermatitis in SHARPIN-deficient mice by regulating TNF production on the skin
Authors: Sharma Bhesh Raj , Karki Rajendra , Kanneganti Thirumala-Devi
Published: January 2023

Mice deficient in SHANK-associated RH domain–interacting protein (SHARPIN), a component of the linear ubiquitin chain assembly complex (LUBAC), develop a spontaneous inflammatory disorder with pathologic hallmarks similar to atopic dermatitis and psoriasis in humans. Previous studies identified the crucial role of components of the TNF and IL-1 signaling pathways in the progression of disease in SHARPIN-deficient mice. However, an innate adaptor or sensor that relates to the disease progression has remained unknown. In this study, we found that the genetic ablation of myeloid differentiation primary response 88 (MyD88) completely rescued skin inflammation in SHARPIN-deficient (Sharpincpdm) mice. Systemic inflammation and immune cell dysregulation were also rescued. Fibroblasts derived from SharpincpdmMyd88−⁄− micefailed to provide protection against TNF-induced cell death. SharpincpdmMyd88−⁄− mice had reduced TNF production in their skin. Furthermore, depletion of the microbiota through the oral administration of antibiotics partially rescued both the skin inflammation and systemic inflammation, demonstrating a role for the gut microbiota in SHARPIN-deficient mice. Our findings suggest a detrimental role for the innate immune adaptor MyD88 in instigating skin inflammation in Sharpincpdm mice.
The class II peptide editor, H2-M, affects the development and function of B-1 cells
Authors: Ghosh Debopam , He Xiao , O’Mara Mary E , Kantor Aaron B , Sengupta Deepanwita , Yang Yang , Eisenlohr Laurence C , Jensen Peter E , Herzenberg Leonore A , Mellins Elizabeth D
Published: January 2023

B-1 cells are known for their innate, polyreactive BCR repertoire, and proficiency at polarizing T cells towards inflammatory effector T cells, a critical component in host defense and autoimmunity. B-1 cells are antigen presenting cells (APCs) and express MHCII proteins to bind and display self and foreign peptides to CD4 T cells. H2-M edits peptide-MHCII complexes to ensure display of stably bound peptides by APCs. Absence of H2-M has most widely been studied for its impact on T cell activation, but knowledge of how this protein affects the development and function of APCs is lacking. We found that absence of H2-M down-regulated the surface expression and altered the distribution of MHCII molecules in B cells across lymphoid organs. Importantly, in H2-M KO mice, compared to the wild-type C57BL/6 (B6) mice, the frequency and abundance of B-1 cells, but not conventional B-2 cells was affected. Interestingly, the H2-M mediated effect on B-1 cell population was only evident in I-Ab expressing B6, not in Balb/c (I-Ad/I-Ed), indicating an MHCII haplotype dependent effect. Decrease in B-1 cell number was evident in both immature and mature B-1 cells, further indicating an H2-M mediated developmental defect of B-1 cells. In H2-M KO mice, B-1 cells display a significantly lower self-renewal capacity and higher rate of apoptosis compared to WT B6. Despite a lower total B-1 cell number, the frequency of B-1 cells specific for predominant self-antigens (like – phosphatidylcholine) is increased in H2-M KO mice, indicating skewing of B-1 BCR repertoire in absence of H2-M. Collectively, these data identify a novel impact of H2-M/MHCII interaction that regulates the development of B-1 cells and influences the selection of mature B-1 cell clones in the periphery.