bluuebunny/crossref_metadata_2025 huggingface dataset:
AhR activation plays a detrimental role in airway epithelial injury after transplantation
Authors: Zhang Ruochan , Ochando Jordi , Li Liwu , Ding Yaozhong , Xu Jiangnan
Published: January 2023

Patients with tracheal tumors, trauma, infection, congenital malformations or injury after prolonged intubation may require partial or complete tracheal resection, and tracheal transplantation will be necessary when the length of resected trachea is longer than 30% in children or 50% in adults. Currently, obliterative bronchiolitis (OB) is a major obstacle that affects allograft function and survival after tracheal transplantation. Th17 polarization and inflammatory cytokine expression is crucially involved in airway transplant rejection, and activation of aryl hydrocarbon receptor (AhR) could modulate Th17 differentiation and inflammatory cytokine production. However, the role of AhR signal pathway in airway epithelial injury, a leading factor of OB development, after transplantation remains unclear. In this study, we performed murine orthotopic tracheal transplants in C57BL/6 wild type mice using BALB/c donors. The levels of IFNγ, IL-6 and IL-17 expression were significantly elevated in allografts, and the airway pseudostratified epithelia changed into flat epithelia with inflammation and fibroblast proliferation in submucosa tissue. Allografts of recipients administered with AhR antagonist (CH-223191) displayed inhibition of IL-17 and IFNγ, and some airway epithelial protection. Significant lamina fibrosis and higher levels of inflammatory cytokine production was observed in allografts of recipients administered with AhR agonist (FICZ). Together, our results demonstrate that AhR activation plays a detrimental role in airway epithelial injury after transplantation, pointing to a new therapeutic avenue for benefitting airway allograft survival through manipulating AhR pathway.
PD-L1/CD80 interaction differentially regulates CD8+ T cell metabolism, survival and GVHD capacity in the presence or absence of PD-1
Authors: Cassady Kaniel , Song Qingxiao , Zhang Yuankun , Martin Paul J. , Ann David , Riggs Arthur D. , Zeng Defu
Published: January 2023

Purified alloreactive CD8+ T cells induce no acute GVHD, but the mechanisms remain unknown. PD-L1 interacts with PD-1 and CD80. Here we show that, sorted C57BL/6 donor CD8+ T cells (1.25 ×106) expanded in wild-type MHC-mismatched BALB/c recipients but induced no acute GVHD. In PD-L1−/− recipients, donor CD8+ T cells expanded and induced lethal acute GVHD. PD-1−/−CD8+ T cells induced lethal acute GVHD in wild-type recipients. Blockade of PD-L1/CD80 interactions on days 0 and 2 prevented expansion of wild-type and PD-1−/− CD8+ T cells in wild type recipients and prevented acute GVHD caused by PD-1−/− CD8+ T cells. On the other hand, blockade of PD-L1/CD80 interaction after CD8+ T activation on day 5 caused lethal acute GVHD in wild-type recipients that usually have no acute GVHD. Naïve CD8+ T cells express much higher levels of CD80 than PD-1, whereas activated CD8+ T cells express higher levels of PD-1 than CD80. PD-L1/CD80 interaction augmented glycolysis in naïve WT and PD-1−/− CD8+T cells. This interaction was associated with increased phosphorylation of PLC-γ and activation of the AKT/mTOR pathway in a CD28-dependent manner. This interaction increased mitochondrial numbers and volume and augmented ROS production and apoptosis at the peak of CD8+ T cell activation, such that CD8+T cells did not over-expand to cause GVHD. Conversely, blockade of PD-L1/CD80 after CD8+ T activation in the presence of high PD-1 expression led to increased mitophagy, reduced apoptosis and overexpansion of CD8+ T cells, resulting in lethal GVHD. These results indicate that PD-L1/ CD80 interaction is dependent on PD-1 expression to differentially regulate glycolysis and mitophagy as well as expansion, survival and acute GVHD capacity of alloreactive CD8+ T cells.
Studies in Anaphylaxis
Authors: Weil Richard
Published: January 2023

The investigation of the effects of simultaneous intravenous injections of antigen and antibody occupies one of the most interesting and important chapters in anaphylaxis. About ten years ago Otto (1) first showed that passive sensitization is invariably accompanied by a latent period of several hours duration. He interpreted the latent interval simply as the period of time required by the cells to appropriate the introduced antibody, and drew the fundamental inference that the anaphylactic response could not take place except through the mediation of cellular antibody. This theory, which is quite in harmony with Besredka's views, was at first very generally accepted. The discovery of anaphylatoxin, however, gave rise to a rival and altogether irreconcilable theory of anaphylaxis. When it was found that the incubation of antigen with immune serum in the test tube gave rise to violent poisons, it seemed natural to infer that the same poisons would be produced when antigen was injected into the sensitized animals.
Studies in Protein Intoxication
Authors: Harris Boughton T.
Published: January 2023

The literature contains but few references to vascular lesions in acute anaphylaxis, and, so far as I can find, only one reference (7) to vascular lesions produced by repeated injections of foreign proteins. Gay and Southard (1) studying the lesions produced in guinea pigs in acute anaphylaxis, report finding fatty changes in capillary endothelium which they interpret as being the cause of the hemorrhages which are so constantly found in acute anaphylaxis. Rosenau and Anderson (2) were unable to find these lesions in acute anaphylaxis, but Longcope (3) and others mention finding them frequently. Longcope (3), (4), (5) in studying the histologic results of repeated injections of foreign proteins in various animals, found marked, and in some cases, even severe lesions in the parenchymatous cells of heart, liver, and kidney, with varying degrees of fibrosis, but, aside from perivascular infiltration, found no arterial lesions.
Fate of the Foreign Protein in the Acute Anaphylactic Reaction
Authors: Manwaring W. H. , Kusama Yoshio , Crowe Harold E.
Published: January 2023

Perfusions of isolated organs and tissues with dilute foreign proteins furnish no evidence of a measurable destruction of the foreign protein by the blood serum, nor of an appreciable destruction or binding of the foreign protein by the fixed tissues during the acute anaphylactic reaction.
Anaphylaxis
Authors: Sherwood Noble P. , Stoland O. O.
Published: January 2023

Summary and Conclusion

Our results indicate that in heart-lung perfusion experiments on normal and sensitized dogs, the phenomena consisting of a 50 to 75 per cent reduction in rate of flow of perfusion fluid, the development of a rubbery consistency of the lungs and the appearance of a tracheal exudate, all in four to seven minutes are not peculiar to the sensitized dog as judged by the blood pressure and coagulation time phenomena. While these do not correlate with other and at present accepted criteria of sensitization, yet a quantitative study of our data suggests that a probable change in permeability in heart-lung tissue, perhaps independent of anaphylaxis, does frequently result from the injection of horse serum in a dog. Experiments in passive sensitization show that marked variation exists in either ability to take up antibody or refractoriness to the shocking dose of antigen. This observation indicates that negative results on a single recipient are not an accurate criterion of deficiency or absence of antibodies in the donor's blood.
Isohemagglutination: The Work of Jan Janský with a Critical Analysis
Authors: Kennedy James A.
Published: January 2023

Summary and Conclusions

Janský's classification is based upon 3160 “cross-agglutinations” in which he utilized sera from 30 (32) individuals and corpuscles from 99. These “cross-agglutinations” are reproduced from his work, appearing herein as table 1. A more orderly arrangement of his “cross-agglutinations” has been made by me and appears as table 2. Janský's classification is based upon the 30 (32) individuals whose sera he used in the “cross-agglutinations.” This is reproduced as table 3 and shows: Group I, 12 individuals; Group II, 10; Group III, 6; and Group IV, 2. In table 4, I have given the result of my analysis of his series of “cross-agglutinations.” It is evident that Janský failed to realize that, in addition to grouping the sera that he was using in his work, he was also grouping the other individuals whose corpuscles only he employed. This is clearly shown in table 2. Consequently his results should have been Group I, 40 individuals; Group II, 38; Group III, 20; and Group IV, 2; with a racial index of 1.8. It seems more logical to me to assume that the irregularity shown by the blood of individual 7524 F. B. is due to a low isoagglutinin “b” titer which has been made ineffective by dilution, very short incubation period and possibly temperature of incubation. This is contrary to the interpretation of Guthrie and Huck (15). The blood of individual 7627 Š. J. is exceptional. However, due to the fact that Janský did not include the serum of this individual's blood in his series, one is hardly justified in drawing any definite conclusions regarding it.
Production of Potent Antitetanic Serum
Authors: Predtechensky S. N.
Published: January 2023

A method for producing a potent antitetanus serum in horses is described. A ground-immunity was obtained by a few injections of toxin and antitoxin; then the horses rested during three or four months and, after that, they were immunized with doses of toxin doubled each time. The content of antitoxin in the serums reached 1500 units per cubic centimeter and even more.
Diphtheria Antibodies in the Urine of a Child After Intramuscular Injection of Antitoxic Horse Globulin
Authors: Neill James M. , Gaspari Emidio L. , Sugg John Y.
Published: January 2023

Tests for diphtheria antibodies were made upon the urine of a child with a mistaken diagnosis of diphtheria who had received intramuscular injections of a large amount of the usual therapeutic antitoxic horse globulin. The presence of the antitoxin antibody was proved both by the capacity of the urine to neutralize the toxin in vitro, and also by in vivo neutralization of toxin injected subcutaneously in guinea pigs several hours subsequent to the intravenous injection of the urine.

Therapeutic antitoxin solutions contain not only the antitoxin antibody, but also diphtheria antibodies of the antibacterial sort, recognizable by agglutination of a degraded strain of diphtheria bacilli; a certain amount of these antibacterial antibodies were also contained in the urine of the child who had received the therapeutic solution.

No serum protein was detected in the urine either by the usual chemical tests or by anaphylaxis tests. The failure to detect the globulin in the urine was not considered evidence of a separation of the antitoxin from the globulin with which it was originally injected, because both the chemical and anaphylaxis tests proved unable to detect the globulin in dilutions of therapeutic antitoxic globulin equivalent in antitoxin concentration to that found in the child's urine.
The Influence of Hypersensitiveness to Horse Serum on the Duration of Local Passive Antitoxic Immunity in Man
Authors: Hooker Sanford B. , Follensby Edna M.
Published: January 2023

Previous experimental work on this subject has been here reviewed.

The duration of a local passive immunity produced by injecting scarlatinal antitoxin into the skin was compared in human subjects who were separable into 3 groups according to their endermal reactivity to horse serum. Those who responded negatively or with a reaction of the delayed type, usually retained an effective portion of the test dose of antitoxin for more than twenty-four hours; the immediate reactors disposed of antitoxin so much more rapidly that in less than four hours an equally effective portion was no longer demonstrable.

That this accelerated disposal of antitoxin is specifically dependent upon the allergic state seems highly probable; certain evidences that were not directly conformable to this hypothesis have been discussed.
Extirpation of the Antigenic Depot and Antibody Production
Authors: Roberts Edward F.
Published: January 2023

Within the past decade, the whole subject of the antigen-antibody complex has been approached in an entirely new way and the astounding results obtained have threatened to overthrow many of the earlier concepts. Excision of the area of antigen deposition has raised the question whether or not antigen needs to come in contact with the antibody-producing tissues and if not, what the nature of the stimulus might be.

The pioneer in this field of investigation was Oshikawa (11). He not only confirmed the observation of Friedberger and Girgoloff (8) that transplants of organs of immune animals stimulate antibody production in animals of the same species, but he also added his own observation to the effect that antibody production proceeded perfectly well in the rabbit whose area of antigen deposition (ear) was excised. A small antigenic dose and short intervals between injection and extirpation were found to be instrumental in assuring a good antibody response.
The Speed of Flocculation of Diphtheria Toxin
Authors: Bunney William E. , Kiamil Mumtaz
Published: January 2023

A toxin which on acidification yields a slight precipitate flocculating slowly in the Ramon test can be made to yield a rapidly flocculating precipitate by the addition to the toxin of a protein foreign to the diphtheria bacillus but precipitating at the same pH as the toxin itself.
STIM1 contributes to mast cell functional response via MrgprX2
Authors: Occhiuto Christopher J. , Kammala Ananth Kumar , Subramanian Hariharan
Published: January 2023

Mast cells are inflammatory immune cells whose activation is critically regulated by intracellular Ca2+ concentrations. MrgprX2 is a G-protein coupled receptor (GPCR) expressed on mast cells that has been implicated in pseudo-allergic reactions in humans. Though it is known that MrgprX2 activation leads to an increase in intracellular Ca2+ levels, the Ca2+ mobilizing mechanisms utilized by this receptor are largely unknown. Previous reports have demonstrated that the stromal interaction molecule 1 (STIM1) calcium sensor regulates GPCR-mediated Ca2+ responses in mast cells. Therefore, the objective of this study was to determine whether STIM1 contributed to MrgprX2 activation in mast cells. We treated human mast cells with the STIM1 inhibitor SKF 96365 (SKF) prior to MrgprX2 stimulation. As a complementary approach, we also silenced the expression of STIM1. Inhibition or deletion of STIM1 expression resulted in blunted Ca2+mobilization to two different MrgprX2 agonists; Cortistatin-14 and Cathelicidin LL-37. Accordingly, degranulation and inflammatory cytokine production were also attenuated. An in vivo mouse-paw edema model utilizing SKF for inflammation prevention further supplemented the in vitro data suggesting that STIM1 also regulated Ca2+ responses in the mouse ortholog of the human receptor (MrgprB2). Thus, MrgprX2 activation is dependent upon STIM1, and employing STIM1 inhibitors could be a potential strategy to alleviate pseudo-allergic reactions.
IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from human skin mast cells by a STAT3-dependent mechanism.
Authors: McHale Cody C. , Gomez Gregorio
Published: January 2023

Mast cells are tissue resident immune cells that cause allergies. The agents responsible for allergic reactions are pre-formed mediators like histamine and inflammatory lipids such as Prostaglandin D2 (PGD2) that are rapidly biosynthesized following crosslinking of FcɛRI with allergen. In humans, increased IL-6 levels are consistently correlated with allergic diseases like asthma and urticaria in which mast cells play a vital role. However, the exact role of IL-6 in allergic disease is not known. In this study, we show that human in situ-matured skin mast cells (huSMCs) express functional membrane-bound receptors for IL-6, and demonstrate our novel finding that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis. Treatment of huSMCs with IL-6 alone induced the phosphorylation of STAT3 at the activating tyrosine 705, and the expression of SOCS3 and VEGF mRNA. IL-6 alone did not induce PGD2 secretion, but significantly and dose-dependently enhanced PGD2 secretion from FcɛRI-activated huSMCs. Accordingly, IL-6 enhanced the FcɛRI-induced expression of COX-2, which is directly involved in PGD2 biosynthesis. In addition, phosphorylation of STAT3 at both tyrosine 705 and serine 727, which is required for maximal transcriptional activity, was increased in huSMCs treated with IL-6 + FcɛRI crosslinking. Moreover, the IL-6-mediated increase in FcɛRI-induced COX-2 expression was inhibited with C188-9, the small molecule inhibitor of STAT3 tyrosine phosphorylation. Together, these data demonstrate that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from huSMCs by a STAT3-dependent mechanism. Thus, we hypothesize that IL-6 contributes to allergic disease by enhancing the production of inflammatory PGD2 from mature mast cells.
Mast cell corticotropin releasing factor receptor CRF2 regulates mast cell function in response to psychological and immunological stress
Authors: Moeser Adam James , D’Costa Susan , Ayyadurai Saru , Mackey Emily , Wilson Neco
Published: January 2023

Psychological stress is a significant risk factor in a number of inflammatory and allergic diseases. We have previously demonstrated that CRF1 receptors expressed on mast cells (MCs) can potentiate psychological stress-induced MC degranulation intestinal permeability, and IgE-mediated anaphylaxis. In this study, we investigated the role of the CRF2 receptors in regulating MC activation in response to psychological and immunological stress. CRF2-deficient (CRF2−/−) bone marrow derived MCs exhibited enhanced IgE-and c48/80-mediated mast cell degranulation, determined by β-hex and histamine release. Heightened degranulation in CRF2−/− MCs were mediated by enhanced intracellular Ca2+ mobilization from endoplasmic reticulum stores. Similar results were obtained with human LAD2 mast cells treated with the CRF2 antagonist Astressin 2B. In vitro findings translated to in vivo studies as MC-deficient KitW-sh/W-sh mice systemically engrafted with CRF2−/− BMMCs exhibited a 3-fold increase in serum histamine levels and intestinal permeability following acute restraint stress or passive systemic anaphylaxis. Contrasting effects of CRF2 were observed with de novo synthesized mediator release as IL-4, IL-6, MCP-1 and prostaglandin D2 release were reduced (by ~40–50%) in CRF2−/− BMMCs following stimulation with IgE-antigen, LPS and IL-33. In summary, these data show that CRF2 signaling inhibits MC degranulation and associated pathophysiology induced by acute psychological stress and IgE-mediated anaphylaxis, while enhancing the release of de novo synthesized mediators. Therefore, targeting CRF2 may represent a novel therapeutic approach for preventing stress-related allergic and inflammatory disease flares.
Toll-like receptor 9 signaling antagonizes B cell antigen capture, processing and presentation resulting in a failure to activate helper T cells
Authors: Akkaya Munir , Akkaya Billur , Kim Ann S. , Miozzo Pietro , Sohn Haewon , Pena Mirna , Roesler Alexander S. , Theall Brandon P. , Lu Jinghua , Pierce Susan K.
Published: January 2023

Key events in T cell-dependent antibody responses, including the formation of germinal centers and affinity selection, are dependent on the ability of B cells to capture antigen and present it to follicular helper T cells. Despite its importance, little is known about the regulation of this critical B cell function. Here we show that although TLR9 signaling induced B cell proliferation, secretion of cytokines and IgM, it blocked the ability of antigen-specific B cells to capture, process and present antigen. In the presence of the TLR9 agonist, CpG, B cells were less able to form synapses with antigen-containing membranes and to pull antigen from them. CpG also diminished the ability of the BCR to deliver internalized antigen to antigen processing compartments resulting in fewer MHC-peptide complexes on the B cell surface. Additionally CpG blocked the BCR-induced increases in the expression of both MHC class II and important co-receptors, including CD86, resulting in the inability of B cells to activate antigen-specific CD4+ T cells. A principle component analysis of RNA seq data showed little overlap between B cells stimulated through the BCR or TLR9 alone or together indicating that TLR9 antagonism of BCR-dependent antigen presentation is through the activation of a novel transcriptional program. In a chimeric mouse model and in a human clinical trial the TLR9 agonist, CpG, enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling may enhance the magnitude of an antibody response at the expense of the ability of B cells to engage in germinal center events that are highly dependent on antigen capture and presentation.
Deconstructing T-B interactions by manipulation of IRF4 to understand regulation of T follicular helper cells.
Authors: Sievert Evelyn P. , Cook Sarah L. , Sciammas Roger
Published: January 2023

Germinal center (GC) derived high affinity antibody is important for protecting against infectious disease. Interactions between T follicular helper (Tfh) cells and GC B cells are requisite for affinity maturation. While it is clear that bi-directional signals are important for the generation of Tfh and GC B cells, a full understanding of the signaling dynamics that lead to efficient T and B cell differentiation is not known. Studying these B cell-derived signals and their role in Tfh cell commitment, maintenance, and function during GC reactions will lead to an improved understanding of this tightly regulated system. The transcription factor IRF4 is necessary and sufficient for generating GC B and plasma cell fates through graded expression. We have shown previously through gene ontology analysis that IRF4 targets include genes involved in T and B cell interaction. This raises the possibility that beyond cell-specific control of B cell fates, IRF4-regulated genes in B cells may facilitate interactions between B and T cells. We hypothesize that IRF4 controls a gene program in B cells necessary to generate efficient T cell activation and Tfh cell differentiation. Using in-vitro co-culture, we assessed changes in T and B cell activation and functionality by flow cytometry. We found that Irf4−/− B cells processed antigen poorly and led to quantitatively and qualitatively reduced T cell activation compared to Irf4+/+ B cells. This indicates a major role for IRF4 in controlling a B cell gene program necessary for efficient T cell activation. We are currently investigating the nature of these signals through in-vitro and in-vivo approaches in order to deconstruct the molecular nature of T-B interactions during the GC program.
Elucidating synergistic and antagonistic effects of B cell receptor and toll-like receptors 3, 4, and 9 in B cell activation
Authors: Theall Brandon P. , Henke Travis A. , Roesler Alexander S. , Akkaya Billur , Pena Mirna , Cimperman Clare K. , Tadesse Yeabsera , Pierce Susan K. , Akkaya Munir
Published: January 2023

B cells are key components of the humoral immune response. Two forms of B cell activation come from engagement of the B cell receptor (BCR) with foreign antigens and from pathogen-associated molecular patters (PAMPs) binding to toll-like receptors (TLRs). For TLRs, MyD88 and TRIF are the major adaptors that lead to NFkB and IRF3 activation respectively. Among various TLRs that are present in B cells, TLR9 is only able to use MyD88 and TLR3 only signals through TRIF, while TLR4 uses both pathways. Therefore, these TLRs provide excellent tools to investigate the roles of TRIF and MyD88 pathways in B cell function and the interplay between them. Here, using stimulation through these three different TLRs either individually or in combination with each other and with/or without anti-IgM, we observed that there is synergy and antagonism between these pathways in terms of activation induced metabolic boost, cytokine production, IgM secretion, expression of various cell surface markers, and antigen presentation. We identified the TRIF pathway as an inducer of Type-I interferon secretion by B cells, which eventually favors the expression of costimulatory molecules such as CD86. On the other hand, MyD88 stimulation antagonized the expression of costimulatory molecules and dramatically induced rapid proliferation and production of IgM as well as proinflammatory cytokines such as IL-6 and TNF. Using B cells obtained from MyD88 or TRIF KO mice, we showed that the interplay between pathways is of limited scale; however, for TLR4 signaling MyD88 has a largely permissive effect while TRIF determines the magnitude of response. These results further our understanding of the complex TLR signaling machinery in B lymphocytes.
Hypoxia-induced reactive oxygen species contribute to immune checkpoint molecule expression in T cells undergoing rapid clonal proliferation
Authors: OConnor Roddy , Guo Lili , Ghassemi Saba , Snyder Nathaniel , Worth Andrew , Weng Liwei , OBrien Shaun , Kam Yoonseok , Philipson Benjamin , Nunez-Cruz Selene , Lee Joyce , Wellen Kathryn , Busch Theresa , Moon Edmund , Blair Ian , June Carl H. , Milone Michael
Published: January 2023

In response to antigen and costimulation, T cells undergo a series of metabolic transitions supporting clonal expansion and differentiation. We recently showed how CD28 and 4-1BB intracellular co-stimulatory domains induce glycolytic vs oxidative metabolic programs, respectively, in the context of a chimeric antigen receptor. Understanding how endogenous co-stimulatory pathways, in particular CD28, supports differentiation and memory cell development despite encountering hostile environments characterized by varying oxygen tension and metabolic checkpoints is of clear importance to cellular immunotherapy.

Immune checkpoint molecule (ICM) expression is associated with T cell exhaustion characterized by reduced proliferative capacity and diminished effector function. While studying the role of LCFAO in primary human T cell differentiation, we uncovered that the CPT1a inhibitor, etomoxir (ETO), induces expression of the PD-1, Tim-3, and Lag-3 ICMs. This is mediated by non-specific effects on oxidative metabolism, culminating in reactive oxygen species (ROS) production. The induction of ICM expression by ETO is calcineurin-dependent and reversed by the antioxidant N-acetylcysteine (NAC). We show that Tim-3 is upregulated by T cells in highly hypoxic xenograft tumors independent of TCR signaling. Transition of highly activated T cell cultures from normoxia (21% O2) to hypoxia (1% O2) induces Tim-3, which is reversed by NAC. These results suggest that ICMs, especially Tim-3, are part of a natural regulatory system that responds to oxidative stress and tempers T cell activation and associated metabolism to minimize oxidative damage to T cell clones expanding following antigen receptor activation.
Adipose T cell microRNAs influence the T cell expansion, microbiome and macrophage function during obesity
Authors: Singh Udai P. , Singh Narendra Prasad , Murphy E. Angela , Singh Santosh Kumar , Price Robert L. , Nagarkatti Mitzi , Nagarkatti Prakash S.
Published: January 2023

Obesity is a global health problem that has reached epidemic proportions and the World Health Organization (WHO) estimates 500 million adults are obese. Obesity facilitates the development and progression of metabolic disorder diseases. Extensive evidence suggests that composition of the intestinal microbiota can influence susceptibility to obesity. T helper cells and macrophage in adipose tissue (AT) are strongly linked to the initiation of obesity-associated chronic inflammation. In this study, we investigated how high fat diet (HFD) impacts homeostatic expansion of T cells, adipose T cell microRNAs (miRs), macrophage and the intestinal microbiota during obesity. HFD consumption alters the body weight, fat mass and lean mass of mice as compared with a normal diet (ND). HFD also increases the homeostatic expansion of T cells in the epididymal AT as compared with a ND. Epididymal AT in mice fed a HFD exhibited significant downregulation of miR-10a, miR-125b, and miR-1247 when compared to mice fed a ND. These obesity-induced changes in miRs were validated by RT-PCR analysis. Pathways analysis also validated that downregulated miRs and gene target mRNAs are associated with T cells homeostatic expansion and macrophage function. We also noticed striking changes in microbial composition of firmicutes, bacteriodates, and ruminococeae during consumption of HFD as compared to ND. Taken together, these results indicate that HFD modulates AT miRs and intestinal microbiome to induce T cells homeostatic expansion and changes in macrophage function to sustain inflammation. This study supports a key role of AT miRs in mediating chronic inflammation during HFD induced obesity.
Self-reactivity controls the basal metabolism and in vivo function of CD4 T cells
Authors: Milam Ashley Viehmann , Bartleson Juliet , Donermeyer David , Horvath Stephen , Chang Chih-Hao , Buck Michael , Lam Wing , Durai Vivek , Raju Saravanan , Yu Haiyang , Zinselmeyer Bernd , Murphy Kenneth M. , Pearce Erika L. , Allen Paul M.
Published: January 2023

Relatively little is known with regard to CD4 T cell memory formation and metabolism, in part because CD4 T cell numbers remain low during a recall infection. Further, the contribution of self-peptide-MHC signaling (during both thymic T cell development and peripheral maintenance) to metabolic programming, if any, remains unknown. To address these questions, we have employed LLO-Hi5 and LLO-Lo5, two TCRtg CD4 T cells that recognize the same Listeria epitope with the same affinity. LLO-Hi5 CD4 T cells are highly self-reactive and respond poorly in a primary infection but robustly in a secondary infection. Less self-reactive LLO-Lo5 cells respond well during primary infection but poorly during secondary infection. We performed metabolic profiling to determine whether differences in response during infection were linked to metabolic differences between LLO-Lo5 and LLO-Hi5. We found that LLO-Lo5 had dramatically higher respiration and glycolytic rates relative to LLO-Hi5, and hypothesized that the decreased metabolism resulting from a strong interaction with self was mediated through TCR signaling. To test this hypothesis, we generated a knockin mouse expressing the Scn5a voltage gated sodium channel. This channel, when normally expressed in DP thymocytes, enhances TCR-mediated signaling. We found that overexpression of Scn5a in peripheral T cells increased TCR-proximal signaling. Further, Scn5a-expressing LLO-Lo5 cells displayed an impaired response during a primary infection. In this way, we demonstrate that tuning of TCR sensitivity to self can be used to alter in vivo immune responses. These studies highlight the critical relationship between TCR:self-pMHC interaction, metabolism, and the immune response to infection.
Triaging the innate immune system: Responding to lung infections suppresses the dermal wound healing response
Authors: Jamieson Amanda Mercedes , Crane Meredith , Xu Yun , Henry William , Albina Jorge
Published: January 2023

The development of pneumonia is a risk for hospitalized patients. Retrospective analysis of patient data revealed a strong correlation with the development of pneumonia and poor wound healing. Responding to lung infections and the healing wound response involve many of the same innate immune cell types, in particular macrophages, monocytes, and neutrophils. This study addresses the impact of lung infections on the ability to heal a dermal wound at a distal site. Using two in vivo models of injury combined with either bacterial (Klebsiella or Streptococcus) or viral (IAV) lung infections, this study unequivocally demonstrates that lung infections suppress the ability to heal wounds. Cutaneous wound healing is delayed in mice that have an ongoing lung infection. There is a decrease in trafficking of innate immune cells, in particular neutrophils and monocytes, into wounds of mice that have an ongoing lung infection. There are also decreased chemokines and cytokines in wounds from infected mice. Systemic changes of the innate immune system occur after wounding and infection that impact immune cell trafficking into wounds, but allow the immune response in the lung to remain intact. There are also alterations in the wound environment when there are simultaneous wounds and lung infections. By redirecting the innate immune response to the wounds we are able to increase wound healing. This information will allow for the better monitoring and supportive care for patients with this type of complex disease sequelae.
Gut dysbiosis and immunological profile in endometriosis
Authors: Alghetaa Hasan F. , Mohammed Amira , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Ectopic growth of normal cells or debris originating from endometrium, the inner layer of female reproductive tube leads to endometriosis. The main symptoms are dyspareunia, dysmenorrhea, and sometimes chronic pelvic pain. Unfortunately, it has no cure or treatment, only symptoms relief medication or surgical removal. Endometriosis causes estrogen imbalance leading to intestinal bacterial estrobolome dysregulation and subsequent change in immune response. Our study aims to investigate the linkage between gut dysbiosis and immunological response in endometriotic mice. Balb/c mice were ovariectomized (OVX) 7 days before intraperitoneal transplantation of 2mm2 endometrial tissue from OVX donors to OVX- or intact ovary recipients. Colonic content was collected 2 weeks post transplantation for 16s rRNA pyrosequencing. Peritoneal exudate was collected and stained to determine the phenotype of infiltrating cells by flow cytometry. Results revealed a significant increase in the number of exudate cells, natural killer (NK) cells and myeloid derived suppressor cells (MDSCs) in endometriotic mice. Phylogenetic taxonomy showed an increase in the biomass of the bacteria in endometriotic OVX mice in comparison with endometriotic nonovariectomized mice. However, the abundance of Phylum Tenericutes, Order Clostridiales, Dehalobacterium sp. and Anaeroplasma sp. were decreased. Collectively, endometriosis changed estrogen metabolism and gut microbiota which resulted in the immune modulation. (Supported by NIH P01AT003961, R01AT006888, R01MH094755, P20GM103641, R01AI129788 and R01AI123947 to PN and MN, and MoHESR to AKM).
Regulation and In Vivo Function of Dendritic Cell CTLA-4
Authors: Halpert Matthew , Vasquez-Perez Jonathan , Konduri Vanaja , Chen Yunyu , Liang Dan , Levitt Jonathan M. , Decker William K.
Published: January 2023

CTLA-4, the target of the blockbuster cancer immunotherapeutic ipilimumab, is the best characterized of the T-cell immune checkpoints. In contrast to T-cell expressed CTLA-4, almost nothing is known about CTLA-4 expression or function in non-lymphoid cell types. Recently we reported expression and secretion of microvesicle-bound CTLA-4 from myeloid lineage dendritic cells. Previous data indicated that microvesicular CTLA-4 bound B7, leading to vesicle internalization and downregulation of B7 expression in vitro. Further, siRNA knockdown of DC-expressed CTLA-4 resulted in upregulation of CD8+ cell proliferation and enhanced antitumor and antiviral immunity in vitro and in vivo. Here we report that DC CTLA-4 can be regulated by interferons a, g, and especially b, whereas TLR ligands appeared to have little effect on regulation. Additional experiments suggested negative regulation of DC CTLA-4 by miR-223 and the transcription factor C/EBP-b. To better understand the role of CTLA-4 in dendritic cell function in vivo, Balb/c conditional knockout animals were generated and characterized under conditions of homeostasis and following nonlethal challenge with H3N2 A/HongKong/8/68 influenzavirus. T-cells derived from conditional knockout animals lacked expression of CD62L and exhibited downregulated CCR7 expression with upregulation of exhaustion markers PD-1 and GITR. Knockout animals challenged with influenza exhibited higher levels of NP tetramer-specific CD8+CD25+ cells and a lengthily extended period of morbidity. The sum total of the data reflect the broad hypothesis that dendritic cell CTLA-4 serves as a regulatory checkpoint to prevent non-specific, inappropriate, or excessively pathologic T-cell priming.
The NLRC4 inflammasome regulates extra- and intra-macrophage expression of its own pathogen-derived ligand flagellin to impact Salmonella survival.
Authors: Akhade Ajay Suresh , Atif Shaikh M. , Lakshmi Bhavna S. , Qadri Ayub , Subramanian Naeha
Published: January 2023

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but whether the process of inflammasome activation per se regulates expression of microbial effectors is unknown. Here we show that inflammasome activation regulates expression of the NLRC4 ligand, flagellin, on Salmonella. Lysophospholipids released upon macrophage pyroptosis increase expression of flagellin on Salmonella and its ability to induce further NLRC4 inflammasome-dependent pyroptosis, establishing a positive feedback loop that potentiates Salmonella detection and clearance. As infection progresses, this positive feedback is checked by inhibition of NLRC4 expression and lysophospholipid biosynthesis, thus switching Salmonella to a flagellin-negative phenotype inside macrophages. While inhibition of inflammasome activation could be a host strategy aimed at preventing overt cell death and enabling generation of a productive adaptive immune response post infection, our study demonstrates that Salmonella hijacks this host response to facilitate its intracellular survival. Our data reveal a host-dependent mechanism by which Salmonella downregulates flagellin within macrophages and identify the modulation of expression of a pathogen-derived inflammasome ligand by the very process of inflammasome activation as a novel mode of host-pathogen cross talk during infection with a bacterial pathogen.
STAT5b Isoform Controls IgE-mediated Mast Cell Function
Authors: Kiwanuka Kasalina N. , Ryan John J.
Published: January 2023

Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that control survival, proliferation and many other cellular functions. This protein family consists of seven members, including the closely related STAT5A and STAT5B, which show 96% amino acid sequence homology. We previously found that Stat5B is required for IgE-mediated cytokine production by mast cells in vitro, but have not tested in vivo correlates to this finding. In the current study, we show that Stat5B deficiency (KO) reduces IgE-induced passive systemic anaphylaxis (PSA) and IL-13 secretion in vivo, without altering mast cell tissue distribution. These results were corroborated by reduced PSA in response to anti-FceRI antibody injection. However, we were surprised to find that reduced PSA was not matched by lower plasma histamine levels. Further investigation revealed that Stat5B KO mice have a >5-fold increase in basal IgE levels and are hyperresponsive to histamine-induced anaphylaxis. These data support the conclusion that Stat5B is critical for IgE-induced mast cell activation in vivo, but also indicate that it contributes to basal IgE production and suppresses vascular responses to histamine. Thus Stat5B is involved in several overlapping aspects of the systemic mast cell response that warrant further study.
Chromatin accessibility modulates the induction of innate immunity in cells of myeloid lineage.
Authors: Song Ran , Dozmorov Igor , Wakeland Benjamin , Arana Carlos , Liang Chaoying , Zhang Bo , Zhou Jinchun , Pasare Chandrashekhar , Wakeland Edward K.
Published: January 2023

The Toll-like receptors (TLRs) of the innate immune system play a key role in the recognition of pathogens. TLRs signal through the recruitment of specific adaptor molecules, leading to activation of the transcription factors, such as AP-1, NF-κB and interferon regulatory factors (IRFs). Although TLRs are mainly expressed in immune cells, a systematic comparison of immune cells activation programs induced by specific individual TLRs is not yet available. Here, we used whole transcriptome sequencing to evaluate global changes in gene expression on monocytes, monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) of five human donors before and after 18 hours stimulation through the TLR2, TLR4, TLR7 and TLR7/8 agonists (Pam3CSK4, LPS, R837 and R848). Cluster analysis demonstrated eight distinct clusters of unresponsive or responsive genes by the four TLR agonists in monocytes, MDM and MDDC. TLR7/8 stimulation by R848 led to the most robust cytokine and chemokine transcription in all cell types, especially in monocytes. Interestingly, we found that interferon-stimulated genes were strongly induced only by TLR4 activation in MDM and only by TLR7 and TLR8 activation in monocytes. Unexpectedly, there was no difference of activation of MAPK, NF-κB and IRF by LPS or R848 in these cell types. Surprisingly, we revealed that chromatin accessibility is significantly correlated with genes that are induced by specific TLR agonists on specific cell type(s), using ATAC-seq. Therefore, our results suggest in spite of robust early signal transduction events, factor that regulate chromatin accessibility play a critical role in regulation of inflammatory gene expression by TLR agonists in cells of the innate immune system.
Separation of RORgammat function in Thymocyte development and Th17 differentiation
Authors: Sun Zuoming , He Zhiheng , Ma Jian
Published: January 2023

RORgt regulates Th17 differentiation, thymic T cell development, and lymph node genesis. Although elimination of RORgt prevents Th17-mediated experimental autoimmune encephalomyelitis (EAE), it also disrupts thymocyte development, eventually leading to lethal thymic lymphoma. To specifically targeting RORgt-dependent Th17 autoimmunity, it is thus essential to distinguish between RORgt-regulated mechanisms in Th17 differentiation and thymocyte development. We identified two amino acid mutations within the hinge region of RORgt (RORgtM) that preferentially disrupted Th17 differentiation but not thymocyte development. Mice expressing RORgtM were resistant to Th17-mediated autoimmune EAE that was associated with defective Th17 differentiation, but maintained normal thymic T cell development and lymph node genesis, except for Peyer’s patches. The RORgtM mouse is thus an effective model for specifically evaluating RORgt-dependent Th17 immunity. We further showed that RORgtM ubiquitination at lysine 69 (K69) was greatly reduced. Mutation of wild-type RORgt K69 to arginine to prevent ubiquitination preferentially impaired Th17 differentiation but not T cell development, similar to RORgtM. Therefore, our study distinguished between important RORgt-regulated functions via a critical ubiquitination event. This study will inform the development of RORgt-based treatments that selectively target Th17-mediated autoimmunity without interrupting the development of T cells and peripheral lymphoid organs.
Prenatal exposure to cadmium alters the transciptome of regulatory T cells in C57BL/6 mice and may contribute to immunological health of the offspring
Authors: McCall Jamie L. , Elliot Meenal , Nowak Evan , Damron F. Heath , Barnett John B.
Published: January 2023

Cadmium (Cd) is a carcinogen present at superfund sites, in battery manufacturing facilities, in and around zinc smelters, and in cigarettes (the most common exposure pathway). In humans, prenatal Cd exposure (preCd) is associated with differential DNA methylation patterns in blood leukocytes of mother-baby pairs. C57BL/6 mice chronically exposed to low doses of Cd in utero develop abnormal immune phenotypes including reduced regulatory T cell (Treg) numbers in the spleen, but not thymus, of adult animals. These data suggest that the probable immune consequences of preCd exposure are cause for alarm. To further define the mechanisms behind these phenotypic differences, we isolated Treg and CD4+ T conventional (Tconv) cells from the spleens of preCd offspring at 8 woa. Target cells were enriched using magnetic isolation prior to purification by flow cytometry. RNA-seq and Ingenuity Pathway analyses were conducted to define the Treg and Tconv transcriptomes and identify mechanisms dysregulated with preCd exposure. Treg cells exhibited more changes in gene expression than Tconv cells. In the splenic Tregs, there were 289 significantly changed genes (166 upregulated and 135 downregulated) in females and 135 (82 upregulated and 53 downregulated) in males. Of note, only 23 upregulated genes and 3 downregulated genes were shared between females and males. However, common pathways, including CREB1 signaling, were disrupted in both sexes. As CREB signaling plays an important role in TGFβ-mediated generation and maintenance of FoxP3 Tregs, inhibition of this pathway may promote Treg instability and contribute to the decrease in Treg numbers observed in the periphery of preCd-exposed mice.
AP-1 transcription factor remodels chromatin during T cell activation.
Authors: Barski Artem , Yukawa Masashi , Jagannathan Sajjeev , Kartashov Andrey , Chen Xiaoting , Weirauch Mathew
Published: January 2023

Upon encountering an antigen, naïve T helper cells activate, differentiate into several lineages and contribute to immune response. Epigenetic changes at cytokine and other effector genes during activation have been described, but the underlying mechanism is not understood. We profiled open chromatin during human T cell activation using ATAC-Seq and identified regions of epigenetic changes genome-wide. Open chromatin correlated with expression at nearby genes, which were enriched for functions related to T cell activation and immune response. As AP-1 and NFAT motifs were enriched in these newly open chromatin regions, we profiled their binding using ChIP-Seq. AP-1 was present at the majority of activation-specific open chromatin regions, which were often co-bound by both transcription factors. Notably, super enhancer formation was associated with AP-1 binding and chromatin opening during T cell activation. We also found that autoimmune disease-associated SNPs were significantly enriched in the newly open chromatin regions. Our data suggest that AP-1 activity contributes to the formation of the activated T cell epigenome and that mutations at the activation-specific open chromatin regions may contribute to autoimmune diseases.
GPR65, a critical regulator of Th17 cell pathogenicity, is regulated by the CRTC2/CREB pathway
Authors: Hernandez Jeniffer B.
Published: January 2023

The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of the pro-inflammatory T cell, Th17. Although Th17 cells protect us against specific pathogens, Th17 cells have been found to cause destruction of tissue in patients with autoimmune diseases. Using RNAseq analysis, we have identified several genes that may be regulated by CREB in Th17 cells. Of these potential CREB targets, the G protein coupled receptor 65 is uniquely expressed in Th17 cells and mice deficient in GPR65 are resistant to autoimmunity. In our study, we confirm our RNAseq analysis by quantitative QPCR and chromatin immunoprecipitation. In addition, mice deficient in CRTC2, a CREB co-activator, have decreased expression of GPR65 in Th17 cells, indicating a role for not only CREB in regulating GPR65 expression, but also for CRTC2. Using a GPR65 agonist, we see an increase in GPR65 expression and Th17 cell differentiation. Therefore, blockade of GPR65 may lead to inhibition of pathogenic Th17 cells while simultaneously leaving non-pathogenic Th17 cells untouched, protecting patients against opportunistic infection.