bluuebunny/crossref_metadata_2025 huggingface dataset:
Composition of the gut microbiota influences infection severity and pregnancy outcome in Plasmodium chabaudi-infected pregnant mice
Authors: Smith Catherine Diane , Cooper Caitlin A. , Moore Julie M.
Published: January 2023

Placental malaria, a severe clinical manifestation of Plasmodium falciparum infection, is a major cause of pregnancy loss, neonatal mortality, and severe maternal illness. We have developed a novel mouse model for pregnancy maintenance during maternal malaria infection utilizing outbred Swiss Webster mice. When infected with Plasmodium chabaudi in early gestation, several inbred mouse strains will abort their pregnancies at midgestation. However, outbred Swiss Webster mice infected with P. chabaudi in early gestation carry their pregnancies to term, allowing us to explore the immunological balance between parasite clearance and pregnancy success. The composition of the gut microbiota may alter this balance. As described by Villarino et al., C57BL/6 mice sourced from different vendors display microbiota-dependent differences in Plasmodium infection severity resulting in a susceptible or resistant phenotype. Similarly, pregnant Swiss Webster mice with susceptible gut microbes develop higher parasite burdens than mice with resistant gut microbes, although both cohorts produce live pups at term. Despite the lower parasite burdens observed in resistant mice, these dams tend to produce fewer viable pups per litter, suggesting a trade-off between accelerated parasite clearance and pregnancy success. We hypothesize that the difference in litter size between susceptible and resistant dams is a result of the elevated levels of pro-inflammatory cytokines observed in resistant dams. The levels of systemic TNF and IFN-γ, which have previously been associated with the clearance of blood-stage malaria infection and pregnancy loss in P. chabaudi-infected pregnant C57BL/6 mice, are expected to be higher in resistant mice.
Vitamin A deficiency in mice results in a hypermetabolic state, dysbiosis and alterations in bacterial metabolism
Authors: Tian Yuan , Nichols Robert G , Snyer Lindsay , Weaver Veronika , Cai Jingwei , Allen Joselyn Natasha , Vijay Anitha , Patterson Andrew D , Cantorna Margherita T
Published: January 2023

Vitamin A deficiency (A−) is the leading cause of preventable childhood blindness in developing countries and is associated with decreased ability to fight infection. A− mice are susceptible to Citrobacter rodentium infection and surviving mice fail to clear the infection. To better understand how vitamin A status influences the metabolome and gut microbiota, we systematically analyzed biological samples from vitamin A sufficient (A+, n = 6) and vitamin A deficient (A−, n = 6) mice using 1H NMR-based metabolomics and 16S rRNA gene sequencing. 1H NMR analyses showed that A− mice had disturbances in multiple metabolic pathways including alterations in energy metabolism (hyperglycemia, glycogenesis, TCA cycle, and lipoprotein biosynthesis) and the A− host showed overall signs of a hypermetabolic state. 16S rRNA gene sequencing indicated that the microbiota in A− mice had phenotypic as well as functional shifts (based on PICRUSt). Evaluation of bacterial metabolism showed enhanced amino acid metabolism, carbohydrate metabolism, DNA replication and repair, and signal transduction in A− mice. In addition, the A− microbiota had lower amino acid biosynthesis, vitamin metabolism, and cell motility. These findings suggest that vitamin A deficiency is associated with hyperglycemia, liver disease, and dysbiosis of the gut microbial communities. Bacteria do not express receptors for vitamin A and therefore it seems likely that the changes in bacterial communities and bacterial metabolism are indirectly regulated by vitamin A. How these changes might compromise the ability of the A− mice to clear a gastrointestinal infection with C. rodentium is an area of continuing investigation.
Role of Acinetobacter baumannii thioredoxin in bacterial dissemination by modulation of mucosal oxidative homeostasis
Authors: May Holly , Yu Jieh-Juen , Gupta Rishein , Guentzel M. Neal , Arulanandam Bernard
Published: January 2023

Acinetobacter baumannii is an increasing cause of hospital-acquired infections and a prominent cause of combat-related infections in the Middle East. Infections with this bacterium lead to a TLR4 response that initiates a potent innate immune response. Bacterial coinfection may occur in hospitalized patients, and endotoxin (LPS) released from one Gram-negative bacterium may have profound effects on pathogenesis of other co-infecting bacteria. Specifically, LPS induced oxidative stress has a significant impact on mucosal barrier function, leading to enhanced permeability and bacterial translocation. To delineate the importance of LPS in E. coli and Ab coinfection, we utilized a murine model of pulmonary Ab infection with oxidative stress induced via LPS injection. While the bacterial factors involved in translocation under oxidative stress are largely unknown, one plausible factor, based on previous research in our lab, is thioredoxin. Thioredoxin-1 (Trx1/TrxA) is a member of the thioredoxin protein superfamily that can be reversibly oxidized and reduced to facilitate reduction of disulfide bonds, as well as for protecting against free-radical damage. Our lab has created a mutant of Ab Clinical isolate 79 (Ci79) that lacks thioredoxin-1 (ΔTrxA). Using this mutant, the effects of thioredoxin on bacterial translocation during oxidative stress can be determined. Mice given E. coli LPS showed significantly increased translocation of Ci79 via organ burden. Mice infected with ΔTrxA showed significantly less translocation than the WT strains. Additionally, when Ci79 was treated with a TrxA blocker before infection, survival was increased and organ burden decreased.
Δ9-tetrahydrocannabinol (THC) activation of cannabinoid receptors induces unique changes in the murine gut microbiome and associated induction of myeloid-derived suppressor cells and Th17 cells.
Authors: Becker William J , Nagarkatti Mitzi , Nagarkatti Prakash S
Published: January 2023

Δ9-tetrahydrocannabinol (THC) is a partial agonist for cannabinoid receptors (CB1 & CB2), is the main psychoactive ingredient found in the Cannabis plant, and can modulate the nervous and immune system. Our intestine harbors a diverse array of bacteria, referred to as the microbiome, which is known to impact many bodily functions, including the immune system. In the current study, we investigated the effect of THC on the gut microbiome and the naïve immune response. We tested the effect of acute or chronic exposure of C57BL/6 mice to THC on the immune system, and correlated said changes with a flux in intestinal bacteria. Intraperitoneal exposure to THC resulted in migration of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) from the bone marrow to the peritoneal cavity. These MDSCs remained in the peritoneum throughout chronic THC administration, where they proliferated and produced IL-6. The peritoneal IL-6 led to an increase in T helper 17 (Th17) cells in the mesenteric lymph node (mLN), as well as increases of IL-17A in the serum and mLN supernatant of THC-treated mice. Indeed, an adoptive transfer of THC MDSCs increased the number of Th17 cells compared to bone marrow (BM) MDSCS, and in-vitro polarization experiments revealed THC MDSCs to be more potent inducers of Th17 cells than BM MDSCs. The alterations in the gut immune cells occurred in tandem with an increase in the number of Alphaproteobacteria in the cecum and feces of mice treated with THC compared to vehicle and naïve mice, and this effect was replicated after an adoptive transfer of THC MDSCS, but not BM MDSCs. Together, these data demonstrated that marijuana use can alter the gut microbiota as well as cause significant changes in the naïve immune system.
Neutrophil maturation and responses to infectious pathogens are regulated by microbiota
Authors: Kugadas Abirami , Geddes-McAlister Jennifer , Gadjeva Mihaela
Published: January 2023

It has long been considered that neutrophils are cells with prepacked anti-microbial granules whose responses to various infectious challenges are innately pre-determined. Here, we provide data that clearly demonstrates definition of neutrophil transcriptomes and proteomes modulated by the microbiota. Using RNAseq and quantitative proteomic approaches we defined mature neutrophil transcriptome and proteome signatures at steady state and during ocular infection with Pseudomonas aeruginosa. Following in-solution digestion and LC-MS/MS analysis, we quantified 3585 proteins from mature neutrophils derived from either non–infected or infected germ free (GF) and specific pathogen free (SPF) mice. We found that the proteomic signatures of mature neutrophils derived from the GF and SPF were significantly different at steady state. Similarly, RNAseq analysis revealed significant differential expression of 327 genes at steady state. During infection with P. aeruginosa, GF-derived neutrophils responded with significant alterations in transcriptional regulation, ncRNA processing, rRNA metabolic processes, mRNA processing, and splicing, while the SPF-derived neutrophils responded with alterations in acetylation. Consistently, GF mice were more susceptible to ocular keratitis compared to SPF controls. Cumulatively, the data support the concept that microbiota drives neutrophil maturation by defining not only the quantity, but also the quality of mature neutrophils. Based on our data, we predict that neutrophil responses can be diverse and specifically tailored to pathogens. In conclusion, neutrophil responses, although innately determined, are constantly adapted and molded by the commensal presence.
The microbiome of NLR knockout animals reveals insights into susceptibility to experimental colitis.
Authors: Ringel-Scaia Veronica Marie , Thomas Cassidy A. , Qin Yufeng , Wade Paul A. , Allen Irving C.
Published: January 2023

Upon sensing particular pathogen associated molecular patterns (PAMPs), inflammasome-forming NOD-like receptors (NLRs) initiate pyroptosis and the cleavage of pro IL-18 and pro IL-1β to their active forms. Consequently, the absence of an NLR or its malfunction in the gut has been shown to result in dysbiosis. There is significant evidence linking dysbiosis with chronic inflammation in the gut and colon cancer. Prior work in our laboratory has revealed that mice lacking NLR inflammasomes, specifically Nlrp1−/− mice, are highly susceptible to experimental colitis and colitis-associated cancer. Here, we extend these findings by evaluating the microbiome of mice lacking either Asc or Nlrp1. 16s rRNA sequencing revealed 6 unique genera prevalent in Nlrp1−/− mice compared to WT and 9 genera in Asc−/− mice compared to WT. Remarkably, the microbiome composition in both knockout animals was more pathogenic, including such genera as Helicobacter, Mucispirillum, and Clostridium. The abundance of volatile free fatty acids was assessed to investigate functional metabolic differences due to the uniqueness of the microbiome in these animals, and interestingly the abundance of acetate, butyrate, and proprionate were distinctly different in Nlrp1−/− and Asc−/− mice compared to WT. Crucially, our analyses were conducted prior to any experimental colitis model. The differences in the microbiomes of these mice have the potential to resolve conflicting data in the field regarding inflammasome deficient mice and subsequent predisposition to or protection from colitis and cancer. Future work will establish which NLR is responsible for sensing the specific bacteria present in the microbiome of the inflammasome-deficient mice at rest.
Identification of CNS-specific biomarkers for monitoring the progression of multiple sclerosis
Authors: Chase Carol Ann , Raphael Itay , Forsthuber Thomas
Published: January 2023

There are currently no reliable methods for assessing the progression of multiple sclerosis (MS) from the relapsing-remitting to the secondary progressive form. This gap in knowledge hinders the ability for therapeutic intervention and results in continued relapses and physiological deterioration. To begin to address the urgent need for biomarkers of progressive MS we investigated proteome changes over the disease course of progressive experimental autoimmune encephalomyelitis (EAE) in NOD mice as a preclinical model of the disease. Our lab has pioneered a high-throughput quantitative proteomic technique, which we have previously used to quantify expression levels of central nervous system (CNS) proteins over the course of monophasic EAE in C57BL/6 mice, producing a predictive protein biomarker fingerprint for clinical relapses. In this study, we utilized this established proteomic technique as well as bioinformatics tools to prioritize key proteins whose expression level in the CNS correlated specifically with the progressive phase of disease in the NOD EAE model. Bioinformatics analysis identified 11 proteins with differential expression in the CNS of mice across the time course of NOD EAE, three of which are CNS specific. We are seeking to detect corollary changes in these three CNS-specific proteins in the serum, pointing to a minimally invasive means of monitoring disease progress and measuring drug efficacy. Our studies will provide a proof-of-concept for identifying homologous human biomarkers to guide treatment in individual patients. Furthermore, our results may provide insights into mechanisms that contribute to disease pathology and offer additional therapeutic targets for slowing the progression of MS.
RGC-32 enhances experimental autoimmune encephalomyelitis by promoting Il-17 and GM-CSF production
Authors: Rus Violeta , Nguyen Vinh , Tatomir Alexandru , Mekala Armugam Prasad , Boodhoo Dallas , Rus Horea G
Published: January 2023

Response Gene to Complement (RGC)-32 is an intracytoplasmatic protein expressed in a variety of cells in response to sublytic complement activation, TGF-β and several growth factors. Upon activation it translocates into the nucleus and plays an important role in cell proliferation, migration, angiogenesis and TGF-β induced epithelial mesenchymal transition and fibrosis. We have recently reported that RGC-32 promotes Th17 but not Treg, Th1 or Th2 differentiation in murine CD4 T cells in vitro. To evaluate the relevance of RGC-32 in an IL-17 dependent inflammatory condition we induced EAE in WT and RGC-32−/− mice by immunization with MOG35–55 peptide emulsified in CFA. The timing of disease onset was similar in both groups of mice. However, at peak disease, RGC-32−/− mice had significantly lower clinical scores. The attenuated EAE phenotype was confirmed by histopathologic examination of the spinal cords that showed fewer inflammatory infiltrates and demyelination foci in RGC-32−/− than in WT EAE mice. The percentage and absolute number of IL-17+ and GM-CSF+CD4+ T cells were significantly lower in the CNS of RGC-32−/− EAE mice. The percentage and number of IFNγ+ and FoxP3+ CD4+ T cells did not differ between WT and RGC-32−/− EAE mice. RGC-32 deficiency did not alter antigen specific expansion of RGC-32−/− spleen cells in response to MOG35-55 peptide. Transfer of WT CD4+ T cells into WT or RGC-32−/− mice before MOG immunization resulted in EAE of similar severity suggesting that the attenuated EAE phenotype in RGC-32−/− mice is T-cell-mediated. These data suggest that RGC-32 exerts a proinflammatory role in EAE by promoting Th17 immune responses and the generation of GM-CSF and that RGC-32 blockade might be a novel therapeutic strategy in MS.
Signaling via TNFR2 mediates CNS remyelination in EAE through regulation of oligodendrocyte progenitor cells
Authors: Gomez-Rivera Francisco , Raphael Itay , Forsthuber Thomas
Published: January 2023

Tumor necrosis factor-alpha (TNF) is a pleiotropic inflammatory cytokine that has been associated with the pathogenesis of several autoimmune diseases, including multiple sclerosis (MS). Consequently, TNF-blocking drugs have been widely used to treat many inflammatory conditions and have proven highly effective. However, treatment of MS patients with anti-TNF drugs leads to disease exacerbation and severe demyelination. This effect has been specifically associated with lack of TNF signaling through its receptor, TNFR2. However, the underlying mechanisms are not fully understood. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model used to study MS. Our lab has recently generated TNFR2−/− DR2b+/+ mice to study the role of TNFR2 signaling in EAE in the context of the HLA-DR2b (DRB1*1501), a haplotype strongly associated with MS. We found that these mice developed progressive EAE characterized by increased demyelinating lesions. Strikingly, this phenotype was not due to lack of TNFR2 expression in T cells, but rather was associated with a decreased numbers of oligodendrocyte progenitor cells (OPCs) in the CNS. Moreover, we demonstrated that TNFR2 signaling is critical for expression of chemokines in the CNS, suggesting its involvement in OPC function and recruitment. Our studies provide key insights into CNS repair and regulatory mechanisms controlled by TNF during inflammation, and this information may help develop novel therapeutic strategies.
The neurokinin 1 receptor and its agonists recruited at the DC-T cell synapse are necessary to promote the survival of activated T cells in vivo
Authors: Larregina Adriana T , Sumpter Tina L , Rojas-Canales Darling M , Tkacheva Olga , Shufesky William J , Falo Louis D , Morelli Adrian E
Published: January 2023

Initiation of cellular immunity relies on T cell activation by Ag-presenting dendritic cells (DCs). By signaling via the neurokinin-1 receptor (NK1R), the neuropeptides hemokinin-1 (HK-1) and substance P (SP) potentiate cellular immunity by mechanisms not fully elucidated. Although SP is mainly secreted by sensory nerves, it is also synthesized and released by T cells. Here, we investigated the role of NK1R and autocrine SP and HK-1 on T cell activation. Using Imagestream flow cytometry (Amnis) to visualize couplets of OVA loaded WT or SP/HK1double KO DCs and responder OT-II CD4 T cells, we observed that the NK1R, SP and HK-1 colocalize with phalloidin at the site of DC-T cell contact. Following CD3/CD28 activation in vitro, we demonstrate that T cells express the full-length NK1R, and that NK1R signaling induces Ca2 flux, activation of calcineurin, NFAT1/2 and NFκB, which results in IL-2 secretion and survival of CD4 and CD8 T cells. These effects were abrogated in NK1RKO orSP/HK1double KO T cells, or by soluble NK1R antagonists. Addition of exogenous SP or HK-1 to CD3/CD28 activated WT T cells did not increase IL-2 secretion indicating that autocrine SP and HK1 released at the DC-T cell synapse suffice to promote T cell survival. In a model of skin DTH in NK1RKO or HK-1/SPdouble KOT cell chimeras, 73 ± 5% of activated (CD44high) CD4 and CD8 T cells die in the draining lymph nodes after priming, and 79%± 7% of the remaining T cells recruited to the skin die in situ following elicitation. In summary, NK1R signaling by autocrine SP and HK1 at the DC-T cell synapse is necessary for the survival of activated T cells and the development of potent cellular immunity.
Leukocyte immunoglobulin-like receptor A3 (LILRA3) displays anti-inflammatory and neuro-regenerative functions through interaction with multiple ligands
Authors: Tedla Nicodemus , An Hongyan , Fath Thomas , Lord Megan , Bryant Katherine
Published: January 2023

LILRA3 belongs to a family of highly homologous surface activating and inhibitory receptors that are emerging as critical regulators of immune cell activation by transducing opposing signals. Uniquely, LILRA3 is soluble and so cannot transduce intracellular signals, hence its functions are unknown, despite its ubiquitous presence in sera and its strong clinical association with various inflammatory diseases. This is primarily due lack of knowledge of its ligands, absence of a LILRA3 homolog in rodents and difficulties in producing functional protein. We produced full-length rLILRA3 protein typical of the native protein and used it in a proteomic approach to concurrently identify two novel ligands; LAMR1 and Nogo 66, and one known MHC I ligand, HLA-B2705 that were validated by expression cloning. Importantly, LAMR1 and Nogo 66 independently interacted with LILRA3 with high affinities of 9.1 pmol and 22 pmol respectively. LAMR1 is a multifunctional protein involved in LPS-induced inflammation and HLA-B2705 is strongly associated with seronegative arthritis (SpA). Nogo 66 is potent inhibitors of neurite outgrowth in the central nervous system. We discovered that LILRA3 promoted neurite outgrowth and synapse formation in central nervous system by blocking Nogo 66; strongly suppressed LPS-mediated inflammation in vivo via interaction with LAMR1, and inhibited activation of peripheral blood T cell by competitively blocking interaction of HLA-B2705 with a closely related activating receptor (LILRA1) which is highly expressed on a subset of T cells in HLA-B27 +ve patients with SpA. These results led to a new concept that LILRA3 may bind multiple ligands with varying affinities and display clinically relevant tissue-specific functions.
Suppression of foreign body giant cell formation by phospholipase Cγ1 via RUNX1/PU.1/ DC-STAMP axis in macrophages
Authors: Kim Ye Seon , Ok Chang Youp , Park Joon Seong , Lee Ha Young , Bae Yoe-Sik
Published: January 2023

Foreign body giant cell (FBGC), a fused macrophage, formation is associated with the inflammatory response following material implantation. However, the intracellular signaling events that regulate the process remain unclear. Here, we investigated the potential role of phospholipase C (PLC)γ1, a crucial enzyme required for growth factor-induced signaling, on FBGC formation. Depletion of PLCγ1 using shRNA induced FBGC formation accompanied by increased expression of fusogenic genes such as cathepsin K, DC-STAMP and CD36. Re-addition of PLCγ1 decreased FBGC formation. PLCγ1-deficiency caused a reduce in RUNX1 and subsequent increased expression of PU.1 while subsequent rescue of RUNX1 in sh-PLCγ1-transfected cells strongly inhibited FBGC formation. FBGC generated by knock-down of PLCγ1 using shRNA resulted in strongly increased TNF-α production, with augmented activation of ERK, p38 MAPK and JNK, and subsequently NF-kB. Collectively, we suggest that an important cell signaling molecule PLCγ1 plays a role in the foreign body response by regulating the RUNX1/PU.1/ DC-STAMP axis in macrophages.
Dynamic programming and memory of monocytes by varying signal strengths of innate stimulants
Authors: Geng Shuo , Yuan Ruoxi , Li Liwu
Published: January 2023

In response to changing external signal strength, innate monocytes may be dynamically programmed into distinct functional states, which may bear significance relevance in host pathophysiology. However, molecular mechanisms that control innate memory dynamics are not well understood nor extensively studied. Using lipopolysaccharide (LPS), the model stimulant of Toll-Like-Receptor 4 (TLR4), we observed that the expressions of pro-inflammatory mediators are preferentially sustained in monocytes adapted by subclinical super-lower dose LPS, and suppressed/tolerized in monocytes programmed by higher dosages of LPS. These phenomena are well conserved in both mice and human monocytes. At the molecular level, we observed that monocytes programmed by super-low dose LPS maintain higher levels of transcription factor IRF5. We also observed that the inflammatory monocyte programming is dependent upon TRAM/TRIF but not MyD88. Similar to LPS, we also observed biphasic inflammatory adaptation and tolerance in monocytes challenged with varying dosages of TLR7 agonist. In sharp contrast, rising doses of TLR3 agonist preferentially caused inflammatory adaptation without inducing tolerance. At the functional level, we observed that in vivo programming of monocytes by subclinical dose LPS exacerbates the progression of atherosclerosis. Taken together, our study provides novel mechanisms for the dynamic programming/memory of innate monocytes and their pathological implications in atherosclerosis.
The role of neutrophils in organ tissue damage in SLE
Authors: Deng Guo-Min , Guo Xuanxuan , Fang Xiang , Qiao Wei , Fei Xibing
Published: January 2023

Neutrophils are the most abundant leukocytes in Human blood, but it is not clear whether neutrophils exert an important role in the pathogenesis of organ tissue damage in systemic lupus erythematosus (SLE). We used lupus-prone mouse model and model of lupus serum-induced tissue inflammation in mouse to investigate the role of neutrophils in the organ damage of SLE. We found that there was a little neutrophil infiltration in the inflammatory sites of skin, liver, brain and joint in lupus-prone mice. We also found that there was also little neutrophil infiltration in the site of skin inflammation induced by lupus serum in normal mouse. The severity of skin inflammation induced by lupus serum was not significantly decreased in mice with neutrophil depletion compared to ones without neutrophil depletion. But we found that neutrophils were actually involved in tissue injury induced by lupus IgG. Further studies showed that lupus IgG stimulated and activated neutrophils, and cause the death of neutrophils. Studies also confirmed that Fas plays an important role in neutrophils apoptosis. Our study indicates that neutrophils participate in the early stage of lupus organ damage, and then they died through activation-mediated apoptosis. These findings promote the understanding of the role of neutrophils in the tissue injury with SLE.
The human lung mucosa determines Mycobacterium tuberculosis infection outcome in the alveolar epithelium
Authors: Torrelles Jordi B , Scordo Julia M , Sidiki Sabeen , Moliva Juan I. , Arcos Jesus
Published: January 2023

Mycobacterium tuberculosis (M.tb) deposited into the alveolar space encounters the human lung mucosa, i.e. alveolar lining fluid, or ALF. We have shown that M.tb exposure to ALF alters the M.tb cell wall surface and subsequently its interactions with human phagocytes, allowing these cells to control better the infection. Understudied host cells in M.tb pathogenesis are alveolar epithelial cells (ATs), nonprofessional phagocytes which are thought to participate in the initial phase and subsequent outcome of M.tb infection. Based on our previous results with phagocytes, we hypothesized that exposure of M.tb to human ALF will allow ATs to control better the infection. However, our results show that exposure of M.tb to ALF drives highly variable rates of bacterial uptake by ATs. Furthermore, independently of this uptake variability, M.tb exposure to ALF resulted in significant differences in M.tb intracellular growth in ATs. Based on these results, we defined two different types of human ALFs: Low-ALF (L-ALF, upon exposure allows better control of the M.tb infection) and High-ALF (H-ALF, upon exposure exacerbates M.tb infection). We further show that H-ALF exposed M.tb reduces the ATs release of pro-inflammatory cytokines and chemoattractants, as well as reduces ATs cytotoxicity. Moreover, H-ALF exposed M.tb affects the crosstalk between infected ATs and phagocytes by downregulating their inflammatory response. These results suggest a novel role for human ALF, where exposure of M.tb to ALF during infection defines ATs as potential M.tb stable reservoirs promoting intracellular bacterial replication and growth, while limiting local inflammation, and phagocyte infiltration and activation.
Autoantibody cross-reactivity with a microbial protein from a prevalent human gut commensal in antiphospholipid syndrome
Authors: ruff william , Roth Alexander S , Dehner Carina Anja , Vieira Silvio Manfredo , Goodman Andrew , Kriegel Martin A
Published: January 2023

Given the vast antigenic source of the gut microbiota, we hypothesized that human gut commensal bacteria induce and sustain autoreactivity via cross-reactivity. As a paradigm, we studied the antiphospholipid syndrome (APS), which is a serious autoimmune clotting disorder of unknown etiology but with a well-defined major autoantigen, b2-glycoprotein I (b2GPI). Infectious triggers are implicated in transient autoantibody production, but the persistent stimuli for anti-b2GPI antibodies remain unknown. To this end, we characterized b2GPI-specific monoclonal autoantibody reactivity to an in silico identified candidate commensal, Roseburia intestinalis (R.int), that is abundant in human stool from APS patients as detected by a species-specific PCR. Using an APS-derived monoclonal antibody, P1-117, that binds to the major B cell epitope in domain I of b2GPI (RGGMR), we tested for cross-reactivity to anaerobically cultured R.int and its candidate cross-reactive R.int protein (WP_006857340.1). P1-117 bound significantly to R.int lysates whereas a control antibody specific for another epitope within b2GPI did not. Further, P1-117 displayed significant binding to the recombinantly expressed R.int cross-reactive candidate protein. BALB/cJ mice immunized with whole heat-killed R.int displayed significant cross-reactive splenocytes and low titers of cross-reactive IgG autoantibodies. In conclusion, we present here in vitro and in vivo studies supporting cross-reactivity of pathogenic b2GPI-specific autoantibody with a common gut commensal. This study provides a concept for persistently colonizing gut commensals as chronic cross-reactive triggers in genetically susceptible autoimmune hosts such as APS patients.
Mapping the chromatin and RNA expression landscapes of human macrophages to interrogate the function of disease-associated genetic polymorphisms
Authors: Martins Andrew J , Narayanan Manikandan , Fixsen Bethany , Tsang John S
Published: January 2023

Macrophages are known to show highly dynamic responses to diverse stimuli, and have been implicated in metabolic syndrome and related diseases. Genome-wide association studies have identified many single-nucleotide polymorphisms (SNPs) associated with these common conditions, largely in non-coding regions; how these SNPs influence disease processes remains largely unknown. We hypothesized that examining RNA expression and chromatin state in macrophages across diverse activation conditions would elucidate the roles of macrophages in mediating the effects of disease-linked SNPs on the development of disease. We measured gene expression via RNAseq and histone 3 lysine 27 acetylation via ChIPseq as an indicator of promoter/enhancer activity in human blood monocyte-derived macrophages stimulated in vitro with a range of inflammatory and disease-relevant stimuli. Over 11,000 genes and 43,000 regulatory elements show condition-dependent activity in this dataset. Functional enrichment in modules of co-expressed transcripts confirmed expected stimuli-specific biology and reveal potentially novel relationships between distinct stimulation states. Correlation of regulatory element activity with module gene expression shows broad inflammatory/anti-inflammatory separation, and metabolic-trait associated SNPs tend to be embedded within regulatory elements whose activity is associated with both non-inflammatory and, surprisingly, some anti-viral macrophage gene modules. Ongoing work includes using these data to infer the transcriptional networks underlying these states. This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.
A heterozygous mutation in the coiled-coil domain of STAT5B is associated with immune deficiency and dysfunction
Authors: Fritz Jill Marie , Majri Sonia , Villarino Alejandro , Zheng Lixin , Lenardo Michael J
Published: January 2023

The signal transducer and activator of transcription (STAT) family of transcription factors play a critical role in regulating gene expression required for immune development and function. In a patient diagnosed with primary immunodeficiency characterized by chronic lymphadenopathy, splenomegaly, immune thrombocyotopenia and necrotizing granulomas, we discovered a heterozygous missense mutation in STAT5B (c.617A>G, p.Q206R). Cotransfection of Q206R and wild-type STAT5B in HEK293 cells reduced STAT5B-mediated transcriptional activity compared to wild-type STAT5B alone, suggesting that Q206R STATB dominantly-interfered with wild-type STAT5B function. Patient T cell blasts exhibited reduced phosphorylation of STAT5B in response to IL-2, IL-7 and IL-15 compared to healthy controls. Further, IL-2/STAT5-dependent gene transcription was impaired in patient T cell blasts. The patient exhibited increased lymphocytes (as well as monocytes and neutrophils) in the peripheral blood associated with a higher frequency and number of CD4+ T effector memory cells (TEMs). Further, CD4+ TEMs isolated from the patient were resistant to restimulation-induced cell death compared to healthy control TEMs in vitro. Collectively, these data suggest that the Q206R STAT5B variant reduced IL-2/STAT5B-mediated gene expression in human T cells, and defects in this signaling pathway are associated with increased survival of TEMs, disrupted immune homeostasis and disease.
ST8Sia6 deficiency is insufficient to induce complement-mediated attack on recent thymic emigrants
Authors: Belmonte Paul , Hsu Fan-Chi , Dash Barsha , Chung Ji Young , Arocha Sinibaldo Romero , Shapiro Virginia
Published: January 2023

T cells require additional developmental steps after positive selection before they are functionally competent and can enter the long-lived peripheral T cell pool. During maturation, T cells gain the ability to produce cytokines upon stimulation, and the ability to protect themselves from TNF and complement-mediated cell death. In three different mouse models with defects in T cell maturation (CD4-cre NKAP conditional knockout mice, CD4-cre HDAC3 conditional knockout mice and CD4-cre RUNX1 conditional knockout mice), recent thymic emigrant (RTE) numbers are greatly reduced due to complement-mediated attack initiated by the binding of IgM and activation of the classical pathway. These models have deficiencies in the addition of sialic acid modifications, which is normally increased during the later stages of T cell development. As stripping cell surface sialic acids by neuraminidase on peripheral T cells leads to binding of natural IgM and complement activation, it was hypothesized complement-mediated attack is due to a defect in sialylation. Transcriptionally, the sialic acid transferase ST8Sia6 is upregulated concurrently with T cell maturation after positive selection. To understand the role of ST8Sia6 in T cell maturation, we generated ST8Sia6 knockout mice. These mice are healthy and fertile. RTEs and mature T cells are present in the periphery in normal numbers, and do not show evidence of complement deposition. Therefore, loss of ST8Sia6 alone is insufficient to induce complement-mediated attack on RTEs and other co-expressed sialyltransferases may also be required to protect from complement deposition.
The Specific and Non-Specific Action of Rabbit Blood Serum in the Complement Fixation Test
Authors: Huddleson I. F.
Published: January 2023

There is not sufficient evidence to condemn naturally infected milk as dangerous to rabbits by ingestion since it has shown no pathogenic action and no antigenic action.

A non-specific absorption of complement takes place in the presence of inactivated rabbit blood serum and a bacterial antigen of Bact. abortus.
Identification of immunological pathways altered by a pro-autoimmune genetic risk variant, PTPN22 1858C>T, that enhances the prevalence of Type 1 diabetes
Authors: Schmiel Shirdi E. , Marquardt Kristi , Lin Xiaotian , Sherman Linda A.
Published: January 2023

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) encodes lymphoid tyrosine phosphatase (Lyp) adaptor molecule that is involved in numerous biochemical pathways in hematopoietic cells. Studies have shown an association with the minor allelic variant of PTPN22, R620W, and increased incidence of autoimmune disorders such as type 1 diabetes (T1D). Approximately 10% of the North American population carry the R620W minor allele, which increases the risk of T1D by as much as 2–4-fold. The exact mechanisms by which this pro-autoimmune risk allele enhances the incidence of T1D is unknown. To address this gap of knowledge our lab introduced the murine ortholog of the human R620W allele, R619W, into the endogenous locus of Non-obese diabetic (NOD) mice that develop spontaneous T1D. NOD mice harboring this mutation (NOD 619W/W) demonstrated a similar phenotype seen in affected humans with accelerated onset of T1D and enhanced anti-islet autoantibodies (IAA). Clinical manifestation of T1D in humans is preceded by IAA in the serum. IAA affinity can often identify individuals at risk of developing diabetes. Germinal centers (GC) are lymphoid structures where affinity maturation occurs with the help of follicular helper T cells (Tfh) cells. An increase in circulating Tfh cells has been reported in newly diagnosed and pre-diabetic individuals, thus suggesting a role for GC reactivity in T1D. Similar to human observations, NOD 619W/W mice also show enhanced IAA and increased Tfh and GC B cell numbers before onset of T1D therefore, our lab is currently examining the immunological pathways altered by this pro-autoimmune risk alleles and their contributions to T1D.
Aire-dependent thymic FoxP3+ regulatory T cells in Type 1 Diabetes
Authors: Bridge Jennifer Anne , Yuen Benjamin , Dong Shen , Ye Jimmy , Kappler John W. , Bluestone Jeffrey A. , Anderson Mark S.
Published: January 2023

Aire expression within the specialized mTECs has been found to be important for enforcing negative selection of T cells, however, mounting evidence has demonstrated the importance of Aire in Treg differentiation and development. Autoimmune diseases like T1D arise from the failure to restrain immune responses against self. Utilizing a novel mouse model, where the Aire locus drives the selected expression of the high affinity DT receptor, revealed a significant reduction in the number of FoxP3+ Tregs within the thymus following treatment. Thus, we hypothesize that alterations in the thymic self-antigen expression and presentation via ablation of Aire-expressing mTECs, will affect the repertoire and function of Aire-dependent FoxP3+ Tregs. New high-throughput platforms and single-cell DNA barcoding technology will allow us to assess several thousand individual Tregs that have undergone Aire-dependent selection in the thymus. The identification of these unique clones and gene signatures will be further interrogated retrogenically, allowing for qualitative assessment of their function within the model of T1D. The antigen specificities of the Aire-dependent TCRs will be assessed via screening against peptide libraries using MHCII molecule strongly associated with NOD mouse T1D. We anticipate identification of novel sequences that are recognized by Tregs, which may lead to discovery of new self-peptides that contribute to T1D. Our proposed study aims to expand our knowledge on TCR repertoire of Aire-dependent FoxP3+ Tregs and the antigen-specificity in T1D, improving our understanding of the respective function of thymic-derived antigen-specific Treg suppression in peripheral tissues associated with autoimmune T1D.
IL-27 Protects from Autoimmunity by Suppressing IL-17 Production and Promoting Treg Suppressor Functions
Authors: Bockerstett Kevin A. , Wong Chun Fung , Ford Eric L. , DiPaolo Richard J.
Published: January 2023

IL-27 has been implicated in the regulation of several autoimmune diseases including multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. However, a better mechanistic understanding of how IL-27 regulates individual autoimmune diseases is needed. Autoimmune gastritis involves chronic inflammation and tissue damage caused by self-reactive T cells and autoantibodies. Serious consequences of autoimmune gastritis include pernicious anemia and a significant increase in the risk of developing gastric cancer. We used a TCR transgenic mouse model (TxA23) in which mice develop autoimmune gastritis due to CD4+ T cells that express a transgenic TCR specific for a peptide expressed by parietal cells in the gastric mucosa. Disease in this model mimics many aspects of human disease including the development of autoantibodies and precancerous lesions. We recently discovered a critical role for IL-27 in regulating autoimmune gastritis. IL-27 deficient mice have more severe inflammation and tissue damage, and develop precancerous lesions much faster compared to control mice. Further analyses revealed that IL-27 expression suppressed IL-17A secretion by immune cells in vivo. In vitro we observed that IL-27 acts directly on autoreactive CD4+ T cells and reduced IL-17A secretion. Finally, IL-27 expression increased inhibitory receptors (LAG3, CD39, CD73, GITR, CD25) on CD4+FoxP3+ regulatory T cells (Tregs) and supported their ability to suppress autoreactive CD4+ T cells in vitro. These data elucidate an important role for IL-27 in protecting from autoimmune gastritis by suppressing IL-17A secretion and promoting suppressor functions of Tregs.
Antigen discovery for regulatory T cells (Tregs) in type-1 diabetes (T1D)
Authors: Dong Shen , Xu Daqi , Bridge Jennifer Anne , Purtha Whitney , Spence Allyson , Wells Danny , Wang Xiao hua , Tang Qizhi , Anderson Mark S. , Bluestone Jeffrey A.
Published: January 2023

Antigen specific Tregs are more efficient than polyclonal Tregs in their suppressive capacity, which makes them attractive tools for further targeted tolerogenic therapy. Autoreactive T cells specificity has been well characterized in type 1-diabetes but little is known about autoreactive Treg specificity. Whether Tregs recognize the same tissue-specific self-antigens as autoreactive T cell, or they recognize different self-antigens to regulate local immune responses via bystander suppression is unclear. Our goal is to identify antigen specificity from enriched islet infiltrated Treg by using previously validated peptide-major histocompatibility complex (pMHC) library displayed on yeast. We first performed single cell TCR sequencing on Tregs from the pancreatic islet of 12 weeks old NOD mice. Data show an abundance of the TCR allele TRAV5D4 usage in islet infiltrated Tregs that has been characterized to target a primary insulin peptide. Moreover, we also performed single cell RNAseq together with TCR sequencing on islet infiltrated CD4+ T cells from a T1D patient from the Network for Pancreatic Organ Donors with Diabetes. We were able to identify enriched TCRs with Treg gene expression profile that will be used to screen for antigen recognition on a DR4 pMHC yeast library. New antigen identification for pancreatic islet-infiltrated Tregs will allow us to improve our understanding of Treg deficiencies in T1D and may lead us to design new immunosuppressive therapies.
Dissecting the genetic networks that control regulatory T cell stability using pooled CRISPR screens
Authors: Cortez Jessica T. , Shifrut Eric , Lee Youjin , Mumbach Maxwell R. , Satpathy Ansuman T. , Granja Jeffrey , Subramaniam Meena , Roth Theodore , Simeonov Dimitre , Ye Chun J. , Chang Howard Y. , Van Gool Frederic , Marson Alexander
Published: January 2023

Regulatory T cells (Tregs) are a specialized subset of CD4+ T cells that suppress inflammation to maintain homeostasis and prevent autoimmunity. Treg development and function depend on expression of the master transcription factor Foxp3. While Tregs have been thought to be irreversibly committed to suppressive functions, lineage tracing studies have challenged this by revealing that Tregs can exhibit plasticity. Tregs that lose Foxp3 expression, termed ‘exTregs’, have been shown to acquire cytokine production capabilities of pro-inflammatory effector T cells and exacerbate autoimmunity. However, the gene regulatory programs that promote or disrupt Foxp3 stability in Tregs under various physiological conditions are not well understood. Here we have leveraged improved functional genetic tools, including pooled CRISPR screens, to identify nuclear factors that regulate Treg plasticity. A Foxp3 lineage-tracing reporter mouse model was used to confidently distinguish Foxp3− exTregs from contaminating Foxp3−T effectors. Using HiChIP, we identified Treg and exTreg-specific chromatin loops and systematically assessed their contribution to Foxp3 stability. Furthermore, we investigated transcriptional signatures of distinct exTreg populations residing in the peripheral organs using single-cell RNA sequencing. Collectively, this work helps us better understand the genetic networks that control stable expression of Foxp3 in Tregs as they encounter changing inflammatory environments.
CCR2 mediates transendothelial migration of human pathogenic Th17 cells
Authors: Parween Farhat , Kathuria Noshin , Zhang Hongwei H. , Farber Joshua M.
Published: January 2023

Leukocyte extravasation is a multistep process characterized by rolling and arrest on the endothelium followed by transendothelial migration (TEM). Arrest and TEM by T cells typically requires the activities of chemokines and their receptors. Previously, we showed that CCR2 identifies highly differentiated, readily activated CD4+ memory T cells in human blood with diverse effector capabilities, and we showed that CCR6 is the signature chemokine receptor for Th17 cells. Here we describe studies of the migratory capabilities and effector profiles of CD4+ CCR6+ CCR2+ as compared with CD4+ CCR6+ CCR2− and CD4+ CCR6− CCR2− T cells from human blood. Using flow chamber assays with TNFa-activated human umbilical vein endothelial cells, we found that among the CCR6+ cells, only the CCR2+ subset was able to cross the endothelial monolayer efficiently. Using antibodies against the CCR6 ligand, CCL20, and a specific inhibitor of CCR2, we found that CCR6 was important for arrest and that CCR2 was critical for subsequent TEM. Using intracellular staining of cells activated ex vivo, we found that although IL-17-expressing cells were found within all CCR6+ subsets, the CCR6+CCR2+ cells were distinguished by their production of IFNg, GM-CSF, and TNFa, thereby demonstrating the cytokine profile of pathogenic Th17 cells as identified in mouse models of autoimmune disease. Taken together, our studies suggest that chemokine receptors on memory Th cells play non-redundant roles in the egress from blood, that providing T cells with CCR6 and CCR2 could enhance their trafficking into tissue, and that, on the other hand, blocking CCR2 alone could prevent the extravasation of pathogenic Th17 cells and ameliorate tissue damage in Th17-cell driven disease.