bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT6 and T-bet double deficient mouse serving as allograft recipient is a new viable animal model to study lung transplantation
Authors: Chen Rongjuan , Chen Qirui , Li Hui , Ochando Jordi , Li Liwu , Ding Yaozhong , Xu Jiangnan
Published: January 2023

The long-term success of lung transplantation is mainly limited by the development of obliterative bronchiolitis (OB) in which IL-17 plays a critical role. However, murine orthotopic allogeneic lung transplant model in wild type recipients develops IFNγ-dominant acute allograft rejection. Our previous research demonstrated that additional STAT6 deficiency to T-bet deficiency resulted in Th17-dominant immune responses, and also restored CD4 T cells to produce IFNγ which is essential to immunosuppression and immunological tolerance. In this study, murine orthotopic lung transplants were performed in C57BL/6 wild type or T-bet/STAT6 double knout-out (DKO) mice using BALB/c donors. Vigorous allograft rejection was observed in T-bet/STAT6 DKO recipients with much higher levels of IL-17 but comparable levels of IFNγ expression to that in wild type recipients. Analysis of the graft-infiltrating cells displayed a significantly decreased CD4:CD8 ratio in T-bet/STAT6 DKO recipients compared to wild type recipients, but not as extremely as that observed in T-bet KO recipients, demonstrating more balanced CD4 and CD8 responses in DKO recipients. Our results indicate T-bet and STAT6 double deficient recipients of lung allografts develop obliterative airway inflammation that is mediated by both CD4 and CD8 responses, more closely resembling OB pathogenesis, and provide a new viable model to study lung transplant rejection.
Silencing of lncRNA NEAT1 induces tolerogenic dendritic cells and immune tolerance in heart transplantation
Authors: Wu Jian , Li Shuang , Zheng Yang , Zhang Maomao , Zhang Hanlu , Sun Yong , Yu Bo
Published: January 2023

Objective

Increasing research have focused on the potential therapeutic value of tolerogenic DC (tol-DC) in clinical organ transplantation. However, the role of long non-coding RNA (lncRNA) in tol-DC remains to be elucidated. Our report aims to illuminate the relationship between lncRNA NEAT1 and DC function in inmune tolerance of heart transplantation.

Methods and Results

We identified lncRNA NEAT1 was upregulated in cardiac allografts of mice by microarray. Applying Flow Cytometry, we found that LPS-dependent maturation of DC was inhibited after siNEAT1 characterizing the low expression of costimulatory molecular CD80, CD86 and MHCII. A mixed lymphocytere action (MLR) was performed to dectect the function of DC in simulating T cells. CD4+CD25+FoxP3+T cells were significantly increased in siNEAT1-DC, however, the proliferation of T cells was suppressed. Bioinformatic databases and dual-luciferase reporter gene assay confirmed that Neat1 can regulate mircoRNA let-7i. In addition, BALB/c recipients transfused with siNEAT1-DC had significantly prolonged allograft survival. Histological examination of cardiac allografts showd slight cell infiltration in recipients transfused with siNEAT1-DC.

Conclusion

LncRNA NEAT1 can regulate DC maturation and function, and transfusion of LPS-induced DC transfected with siNEAT1 prolonged allograft survival, besides, we identified that NEAT1 can regulate mircoRNA let-7i. This study enriched the network of interaction between lncRNA and mircoRNA and may provide a new therapeutic target in heart transplantation.
KEAP1 gene editing using CRISPR/Cas9 for therapeutic NRF2 activation in human T cells
Authors: Noel Sanjeev , Lee Sul A , Sadasivam Mohanraj , Hamad Abdel Rahim A , Rabb Hamid
Published: January 2023

Background

T lymphocytes mediate ischemia reperfusion injury (IRI) during organ transplantation. We recently demonstrated that augmentation of NRF2 dependent antioxidant response in T lymphocytes by deleting KEAP1 significantly protects from IR induced kidney injury. In order to develop clinically translatable T cell specific antioxidant therapy, we used CRISPR technology to delete KEAP1, the inhibitor of NRF2, in human Jurkat T cells and primary T cells.

Methods

We targeted KEAP1 exon 2 using site specific guide RNA. Briefly, 5×105 cells were electroporated 1.5μM of Cas9 protein and 1.8μM crRNA:tracrRNA complex. Control cells were electroporated without cas9:guide RNA complex. Cells were harvested 72h after electroporation and assessed for cell number and viability, KEAP1 editing and qPCR based analysis of NRF2 target genes.

Results

Electroporation of cas9:guide RNA complex had no adverse effect on cell expansion and viability (≥ 95%) in either Jurkat or primary T cells. Furthermore, genomic cleavage analysis showed editing of KEAP1 in upto 75% Jurkat cells. qPCR based studies in Jurkat cells showed significant (p≤0.05) increases in NRF2 targets NQO1 (~10 fold), HO-1 (~13 fold) and GCLM (~2.6 fold) mRNA following KEAP1 deletion, compared to control cells. Similarly, CRISPR mediated KEAP1 editing in primary T cells resulted in significant increase (p≤0.05) in NQO1 (~24 fold), HO-1 (~48 fold), GCLM (~6 fold) and GCLC (~3.5 fold) compared to control cells.

Conclusions

These data show that KEAP1 editing is feasible using CRISPR technology to augment NRF2 regulated antioxidant response in primary human T cells. This approach could improve immune cell based therapy for IRI during transplantation and help treat allograft rejection.
Hyperlipidemia alters Treg function through effects on Akt
Authors: Hyde Michael , Bagley Jessamyn , Iacomini John
Published: January 2023

Hyperlipidemia is a common health condition in the transplant patient population. This condition causes end-stage heart disease in approximately 40% of all patients requiring a heart transplant, developing in 50% of heart transplant recipients after the first year and 95% of patients within 5 years of transplant. We have recently shown that hyperlipidemia promotes an aggressive rejection response that results in accelerated rejection of allogeneic heart transplants and resistance to tolerance induction. These results appear to be related to effects of hyperlipidemia on regulatory T cells (Tregs), including activation of Akt. Here we examined how activation of Akt might alter Treg function. Our data indicates that hyperlipidemia down-regulated regulators of Akt, allowing for increased Akt phosphorylation. Interestingly, hyperlipidemia preferentially affects the activation through phosphorylation of specific Akt isoforms and isoform specific targets. Akt isoforms activated because of hyperlipidemia in Tregs are not activated in CD4 effectors, suggesting a specific role in Tregs. Our data lead us to conclude that hyperlipidemia alters Treg function by altering specific Akt isoforms. These data may lead to new approaches that can be used to down or up-regulate Treg function.
Biomaterial scaffolds for localized IL-33 release to modulate the local immune environment and enhance extrahepatic allogeneic islet transplant
Authors: Liu Jeffrey M , Luo Xunrong , Zhang Xiaomin , Shea Lonnie D
Published: January 2023

Allogeneic islet transplantation represents a promising therapeutic option to restore endogenous insulin production in Type-1 diabetes patients. Clinically, new methods of regulating the immune response against the islet allograft without using general immunosuppression are required. We have previously demonstrated the viability of transplanting syngeneic islets on porous poly-lactide-co-glycolide (PLG) scaffolds within the epididymal fat pad to restore long-term euglycemia. The use of a biomaterial scaffold also provides an opportunity to incorporate novel immunomodulatory factors that can modify the local immune response to transplanted cells. Due to the prevalence of ST2+ regulatory T cells and M2 macrophages linked to promotion of an anti-inflammatory phenotype within adipose tissue, we hypothesized scaffold-mediated IL-33 release could promote a tolerogenic environment to extend islet allograft survival. We found incubation of an IL-33 loaded scaffold in vitro with naïve T cells undergoing anti-CD3 TCR stimulation stimulated release of IL-13, demonstrating maintenance of IL-33 bioactivity. We found that IL-33 loaded scaffolds implanted into the epididymal fat pad expanded the CD4+ Foxp3+ population within the graft site, most strikingly amongst CD4+ Foxp3+ ST2+ Tregs. Using an allogeneic murine islet transplant model, preliminary data showed diabetic C57BL/6 recipients receiving 250 Balb/c islets on IL-33 scaffolds had extended graft survival time compared to BSA-loaded control scaffolds. Collectively, these results indicate the potential for IL-33 to modulate the local immune response to support graft function, and support ongoing studies to investigate mechanisms to extend graft survival.
A Comparison of the Antigenic Properties of Different Strains of Bacillus Typhosus
Authors: Hooker Sanford B.
Published: January 2023

Summary and Conclusions

By means of the alexin-fixation reaction it has been found possible to demonstrate consistent antigenic differences among some strains of B. typhosus. In accordance with these differences a number of strains have been tentatively allocated in three fairly well-defined groups. Group I is made up of strains of rather recent isolation; no strain in this group has been under artificial cultivation for more than two years. Older strains, isolated from three to fifteen years ago, compose Group II. The third group is made up of those strains that fall neither in Groups I nor II, and that cross-fix irregularly with each other and with the members of the other groups. Group III strains are all more than three years old.

By means of agglutinin-absorption experiments, findings have been obtained that, in general, harmonize strikingly with the results of cross-fixation. This confirmatory evidence, secured by a widely different serologic method, tends strongly to prove that these antigenic differences among typhoid strains, as demonstrated by alexin-fixation, represent an actual condition and are not fortuitous.

It is considered that the evidence of antigenic differences thus far discovered among different strains of B. typhosus is sufficiently valid to warrant the presentation of these data, and sufficiently encouraging to justify extension of this work, especially with regard to the comparative protection afforded by sera obtained by immunization with strains of different character.

The facts that a serum immune to any recently isolated strain cross-fixes with all other strains, old or young, while sera immune to older strains do not so cross-fix, would lead to the seemingly justified assumption that only do the young strains contain all of the antigenic complexes of typhoid bacillary protein. The logical conclusion, then, would be that a young strain ought to afford the most efficient protection when used for prophylactic immunization against typhoid fever. However, the results of the work on agglutinin-absorption, if correctly interpreted, do not warrant the assumption that a univalent vaccine is sufficient. Therefore, although the grouping of strains here given is but tentative, it seems advisable, for the present, to recommend the use of a balanced polyvalent typhoid vaccine, for immunizing and therapeutic purposes, compounded in accordance with these groups. The experimental work here set forth distinctly does not uphold the prevailing practice of employing a single old strain for prophylactic immunization against typhoid fever.

It is a pleasure here to acknowledge my gratitude to Dr. F. P. Gay for the helpful interest he has taken in this work.
The Hecht (Gradwohl Modification) Complement Fixation Reaction in Syphilis with Special Reference to Cholesterinized Antigens
Authors: Kolmer John A.
Published: January 2023

Various modifications of the Wassermann reaction utilizing the complement and natural antisheep hemolysin of human serum, have been advocated from time to time for the purpose of conducting the test in a more economical manner or, more particularly, to increase its delicacy and obviate certain sources of error ascribed to the inactivation or heating of the serum.

Of these methods, that of Hecht (1, 2 and 3) received considerable attention. As originally proposed and with later modification, this test employs an alcoholic extract of guinea-pig heart, human fetal heart and other extracts as antigen using 1 cc. of 1:50, 1:100 and 1:200 dilutions with a constant dose of 0.1 to 0.2 cc. of fresh active serum. The fourth tube of the series serves as the control. After a primary incubation of a half to one hour, 1 cc. of a 2 per cent suspension of sheep cells is added to all tubes followed by re-incubation for one to two hours.
Methods of Determining the Optimal Amount of “Antigen” for the Wassermann Reaction
Authors: Ottenberg Reuben
Published: January 2023

In the practical performance of the Wassermann reaction the doses of all the ingredients have to be adjusted to each other. The original Wassermann reaction and many of its modifications follow the principle of employing a fixed dose of complement and as large a dose of antigen as possible without danger of non-specific fixation. Usually those who follow this method also determine the least amount of antigen capable of giving a positive reaction with positive serum (the antigenic unit). They require that the dose actually used shall be a considerable multiple of this unit and state that the more multiples of the smallest antigenic unit contained in the dose used, the better the antigen.

On the other hand there are many authors, such as Browning and McKenzie, Thomas and Ivy, Thomsen, Boas, Stern, who use an arbitrary fixed dose of antigen and adjust the complement to the dose of antigen in such a way that the complement is capable of giving complete hemolysis in the presence of the dose of antigen used.
A Study of Von Dungern's Indigo Test for Syphilis
Authors: Flood Edward P.
Published: January 2023

Von Dungern described an alkaline indigo solution that when added to an inactivated serum would inhibit its normal coagulation by heat. He believed that the indigo present was the active inhibiting substance. We have shown that neither the indigo nor the salt content of the reagent takes part in the inhibition of the coagulation. The alkali is the sole inhibiting agent. Von Dungern further stated that with similar amounts of the reagent this inhibition was less marked in syphilitic than in normal sera. In our experience such a difference was present in but very slight degree and with no constancy. The method is of no value in the serum diagnosis of syphilis for in our entire series of 140 sera, 40 of which were evidently syphilitic, coagulation occurred n but 3, with the prescribed amount of reagent.
The Influence of Typhoid Vaccine on Tuberculous Guinea-Pigs
Authors: Baldwin Helen , L'Esperance Elise S.
Published: January 2023

In the protection of large groups of men by typhoid vaccination, it has been customary to avoid giving the vaccine to those having active tuberculous lesions and often, also, to those with latent forms of tuberculosis. The reason for this has presumably been the dread of the effect of any febrile reaction or of any temporary lowering of resistance in the tuberculous patients and also the reported exacerbations of tuberculous lesions following attacks of typhoid fever. On account of the few cases of tuberculosis which have been injected with typhoid vaccine and because of the confusing complexity of conditions often found in clinical work, it has seemed of value to observe the effect of typhoid vaccination upon susceptible animals which had been inoculated with active tubercle bacilli.

The preliminary experiment of this series was made on two guinea-pigs of approximately the same age and weight.
Cutaneous Antibodies in the Guinea-Pig
Authors: Cook M. W. , Smith G. H.
Published: January 2023

In explaining the mechanism of cutaneous reactions it is frequently assumed that the response called forth in the cutaneous tissue by the introduction of an antigen, to which the individual is sensitized, is due to the presence in the skin of antibodies for that antigen. As guinea-pigs, whether immunized or sensitized, do not give pronounced cutaneous reactions, and as, at the same time, these animals are particularly susceptible to anaphylactic shock when an intravenous injection is made into sensitized individuals, it was considered of interest to discover whether or not antibodies can be demonstrated in their skin.

A possible method of establishing the presence of antibodies is found in the precipitin procedure. In employing this test in work with cutaneous tissue it is essential that circulating antibody be excluded. If the blood serum is eliminated from the skin and a positive reaction is then obtained by using skin emulsions as precipitin with an homologous antigen, we have fairly complete evidence that the skin does possess antibodies.
An Antigenic Classification of the Group IV Pneumococci
Authors: Olmstead Miriam
Published: January 2023

A biologic classification of pneumococci into four groups has been established by the work of Dochez, Gillespie, Cole and Avery. A study of type IV, which has been considered a heterogeneous series of independent strains, was undertaken at The Presbyterian Hospital in connection with an investigation of post-operative pneumonia, in which the type IV pneumococci have been found to be an important etiological factor. Many of the strains examined have been obtained, by mouse passage from sputum or saliva of surgical cases before operation and may be considered normal mouth inhabitants. The others have been recovered from the sputum of post-operative pneumonia cases, of pneumonia cases in the medical wards of the hospital, of bronchitis cases, from lung cultures at autopsies, from blood cultures, from spinal fluid, from chest fluid and from abscess cultures. All the strains when isolated failed to react with serum of types I and II.
Intracutaneous Reaction in Infectious Diarrhea
Authors: Baker Horace Mitchell
Published: January 2023

In the above series of cases the organism of the infectious diarrhea group was isolated in 55 per cent of the cases. Agglutinins for the dysentery group were found in 12 per cent of the cases which were found to be negative from the bacteriological examination and a characteristic smear, as stained by Gram's method, with a typical history in 25 per cent of the other cases.

The intracutaneous reaction, on the other hand, showed 100 per cent negative results in the control cases. In cases with history of infectious diarrhea positive reactions were obtained in 85 per cent of the cases.

I wish to thank Dr. Henry I. Bowditch, chief of the visiting staff of the Boston Floating Hospital for the free use of the clinic and wards, as well as for the many helpful suggestions which he has given me and also Dr. Cleveland Floyd for his valuable aid and advice.
A New Diluent for Diphtheria Toxin in the Schick Test
Authors: Bunney William Edward
Published: January 2023

A 2 per cent solution of peptone (Difco-“Proteose”), containing 0.85 per cent sodium chloride and 0.5 per cent phenol, has been found to maintain the toxicity of certain diphtheria toxins diluted to ¼ m.l.d. per cubic centimeter under ordinary conditions for periods long enough to permit warranting no loss of toxicity in two months. Schick tests done with toxin so diluted gave satisfactory results in children whether or not they had received toxin-antitoxin six months previously, and in adults who had not received toxin-antitoxin.
Studies on the Antigenic Properties of the Ultraviruses
Authors: Schultz E. W. , Quigley J. S. , Woolsey E. L.
Published: January 2023

Summary and Conclusions

Judged by plaque counts, the neutralization of 'phage by antiphagic serum was found to follow quite closely the curve of the velocity of an adsorption reaction. No evidence was obtained to indicate that the reaction follows a logarithmic curve. Results were obtained which indicate that in the neutralization of 'phage by antiphagic serum the reaction under set conditions takes place in definite proportions, provided the reaction is allowed to go to completion. The Danysz-Bordet effect was elicited, when 'phage was added in small increments to serum and, similarly, when serum was added to 'phage in small increments. Neutral serum-'phage mixtures could not be dissociated by dilution with physiological saline.
Local Skin Reaction Obtained by Intravenous Injection of Agar Following Intracutaneous Inoculation of Meningococcus Toxin
Authors: Sickles Grace M.
Published: January 2023

Intravenous injections of agar may be followed by reactions in skin areas prepared by injections of meningococcus toxic filtrate.

Local reactions did not occur at the site of intracutaneous injections of agar when followed by intravenous injections of agar or of toxin.

No reactions were obtained after the injection of other nonbacterial substances such as galactose, gelatin, serum and India ink, intravenously, following intracutaneous injection of meningococcus toxic filtrate.
Tissue Specificity in Anthrax Infection
Authors: Burke Victor , Barnes Laverne A.
Published: January 2023

The ability of Bacillus anthracis to start an infection in various tissues of the body has been questioned. According to Besredka this organism can start an infection only in the skin. He contends that, “outside the skin the anthrax bacillus acts as a saprophyte” and further that, “in order to produce a fatal anthrax infection it is necessary to injure the skin.” It follows that if the injured skin is protected infection should not occur following introduction of the organism through it into the body. Of the various attempts made to detect pathogenicity of Bacillus anthracis for tissues other than the skin none have, so far as we know, included the protection of punctured skin by the use of antiseptics.

In the following experiments gentian violet was used to protect the injured skin at the site of needle puncture. Gentian violet proved to be a very satisfactory antiseptic for this purpose.
The Chemical Composition of the Active Principle of Tuberculin
Authors: Lewis Julian H. , Seibert Florence B.
Published: January 2023

The proteins of acid-fast bacteria isolated with the newer methods described by Long and Seibert (1, 2) are excellently adapted to the study of the general problem of the relation of biological specificity to chemical constitution. First, because they are derived from a group of organisms that are biologically related, and, secondly, because they can be isolated in comparatively large amounts from filtrates of cultures of the bacteria grown on antigenically indifferent synthetic media by procedures which avoid chemical and physical manipulations that are likely to alter or denature proteins.

The methods devised by the above authors for isolating the proteins of tuberculin consist of growing the tubercle bacilli in a synthetic fluid medium for six to eight weeks, removing the bacteria by filtration through Berkefeld filters and obtaining the proteins either by ultrafiltration and washing or by precipitation with ammonium sulphate, either of which processes yields a concentrated final product that is apparently in the same chemical state as it was in the original culture, since it exhibits the entire antigenic properties, as far as the tuberculin reaction goes, of the entire tubercle bacillus filtrate.
Transmission to the Third Generation of Antitoxin Derived by Active Immunization of the First Generation
Authors: Sugg John Y. , Richardson Lurline V. , Neill James M.
Published: January 2023

A guinea pig of the second generation transmitted to its own offspring (third generation) small amounts of antitoxin originally derived by active immunization of the first generation. Previous reports of failure of transmission were probably due to quantitative inadequacy of the immunity originally transmitted to the females of the second generation to persist to the time of birth of the third generation.
Studies in Contact Dermatitis
Authors: Brown Aaron , Milford E. L. , Coca A. F.
Published: January 2023

It has been thought that the dermatitis caused by pollen and experimentally reproducible by cutaneous testing with pollen oil, is due to the presence in the oil of the atopic excitant. We have been able to show that the dermatitis is not due to the atopic excitant but to the oil itself. Hypersensitiveness to the pollen oil occurs in 15 per cent of hay fever subjects, in 15 per cent of atopic persons not subject to hay fever and in 15 per cent of nonatopic persons. It is thus to be classified as a contact dermatitis similar in its nature to dermatitis due to poison ivy, sumac and primrose.

The results of this investigation naturally suggest a similar differentiation of the excitant of atopy and of dermatitis in other materials, and it is obvious that this principle must be important to the diagnosis and also to the treatment of dermatitis caused by materials containing atopic excitants.
Studies in the Transmission of Sensitization Fromm Other to Child in Human Beings
Authors: Bell Samuel D. , Eriksson Zaida
Published: January 2023

Five cases of asthma and five of hay fever in pregnant women were studied by the Prausnitz-Küstner method as to the passage of antibodies through the placenta from mother to child. In each case the ability of the mother's serum, while she was carrying the child, to give a local passive transfer to a normal individual was established, in most cases with the serum diluted 1:40 and in 1 case as high as 1:320. In no case could similarproperties be shown in the undiluted cord blood or the undiluted child's blood.
Loss of Immune Substances from the Body
Authors: Sugg John Y. , Neill James M.
Published: January 2023

The paper dealt with diphtheria antitoxin in the salivas of “naturally” immune persons and of a toxoid-immunized person. The data owe much of their significance to the unusually high degree of antitoxic immunity of the people used as subjects with whom it was possible to establish ratios between the saliva and serum antitoxin more accurately than would be possible with subjects possessing the more common low degrees of immunity.

The concentration of antitoxin in the saliva was directly related tothe concentration in the blood; the average ratio of was 1.4 × 10-3; the ratio remained of the same order of magnitude in spite of the wide range in serum antitoxin concentrations (1.25 to 60 units) occurring in the group of people studied. Exception to the usual ratio appeared to be characteristic of certain individual persons.

The data indicate that during a year's time the antitoxin swallowed with the saliva would be equivalent to approximately 25 per cent of the total amount in the blood. Unless reabsorption occurs the gastro-intestinal tract must be considered an important channel of loss.
A novel role for the adapter molecule NHERF1 in regulating mast cell-mediated anaphylaxis
Authors: Kammala Ananth Kumar , Subramanian Hariharan
Published: January 2023

Anaphylaxis is a life threatening allergic reaction that is classically elicited by crosslinking of antigen-specific IgE bound to the high affinity IgE receptor FcɛRI on mast cells. Proteases released from FcɛRI-activated mast cells result in the accumulation of the anaphylatoxin, C3a that can further amplify the anaphylactic response via interaction with C3a receptor (C3aR), a G-protein coupled receptor (GPCR). This autocrine loop resulting in C3aR activation as a consequence of FcɛRI stimulation initiates anaphylaxis in humans. C3aR possess a class I PDZ binding motif on its cytoplasmic tail. Adapter proteins such as the Na+/H+ exchanger regulatory factor 1 (NHERF1) have been implicated in regulating GPCR functions by binding to the class I PDZ motifs in these receptors. In our previous study, we had demonstrated that NHERF1 promotes C3a-induced degranulation and chemokine production in human mast cells. The aim of this study was to determine if NHERF1 regulates mouse mast cell response following FcɛRI stimulation. Our data suggests that NHERF1 enhances intracellular Ca2+ mobilization, activation of the MAP kinase pathway (ERK and P38) and production of cytokines (IL-6 and IL-13) following IgE/antigen-mediated activation in bone marrow derived mouse mast cells. In agreement with our in vitro data, NHERF1 promotes mast cell-mediated passive systemic anaphylaxis and passive cutaneous anaphylaxis induced by IgE and antigen in clinically relevant murine models. These results thus indicate that the NHERF1 plays a vital role in regulating mast cell-mediated anaphylaxis and that NHERF1 could be a potential novel target for anaphylaxis and other allergic diseases.
Identification of an Il9 gene locus regulatory element in mast cells and basophils
Authors: Qayum Amina Abdul , Koh Byunghee , Kaplan Mark H.
Published: January 2023

Interleukin 9 (IL-9) is a pleiotropic cytokine that plays a central role in immunological disorders. Extensive work has been done to describe the Il9 gene locus and its transcriptional regulation in Th9 and other T cell subsets. Some innate immune cells, such as mast cells and basophils, can also produce a substantial amount of IL-9 in an inflammatory milieu. However, very little is known about how the Il9 gene is regulated in these cells. In this study, we show for the first time that IL-33, and not IgE cross linking, induces IL-9 production in mast cells and basophils and that this is an IL-3 dependent effect. Using the CRISPR-Cas9-mediated deletion approach, we show that IL-9 production in mast cells and basophils is dependent on a conserved non-coding sequence -25 kb (CNS-25) upstream of the IL-9 promoter. Our results show that the CNS-25 region is important for mast cell and basophil IL-9 production in vitro and in vivo. ChIP assays show that the CNS-25 can bind multiple transcription factors at a higher magnitude than the Il9 promoter. Additionally, this region has high chromatin remodeling and p300, an acetyl transferase, binding in mast cells. We show that p300 is essential for IL-33 induced IL-9 production in mast cells and basophils. Taken together, our data define a mechanism for Il9 gene regulation orchestrated by IL-33 and IL-3 signaling in mast cells and basophils.
DC subsets are functionally specialized to induce GC-dependent or -independent humoral immune responses
Authors: Bouteau Aurélie , Zurawski Sandra , Zurawski Gerard , Igyártó Botond
Published: January 2023

Our knowledge of humoral immunity mainly comes from experimental vaccine models that use high antigen doses injected into the periphery in combination with adjuvants. This allows extended exposure to antigen/adjuvant by all of the immune cells. Thus, the contribution of a specific immune cell to humoral immune responses cannot be easily determined. To better understand the roles of different dendritic cell (DC) subsets in regulating humoral immunity, we developed a system to limit antigen availability and presentation to certain skin DC subsets. We found that antigen delivery without adjuvants to Langerhans cells (LCs) through the c-type lectin Langerin led to higher antibody titers and affinity, involved GC formation and was dependent on antigen dose and CD40 and IL-10 signaling, unlike the response initiated by CD103+DCs that produced lower antibody titers in a GC-independent manner and was characterized by long-lived PC formation. Interestingly, co-delivery of IFNa or poly-IC enabled CD103+DCs to drive GC-dependent humoral immune responses. Surprisingly, when both DC subsets had the antigen, the humoral immune response was like the one induced by the CD103+DCs alone but was dominated by memory/PB formation. Consistent with the B cell responses, the LC-induced Tfh cells were CD69+ S1PR1low and efficiently migrated to the B cell area, while the ones induced by the CD103+DCs were CD69− S1PR1high and localized mainly to the T cell zone. CD69 expression on Th cells inversely correlated with the antigen presentation capacity of the DCs. LCs, unlike CD103+DCs, migrated into the B cell area including B cell follicles and GCs.

Thus, pre-programmed DC-intrinsic features allow DC subsets to induce distinct B cell responses.
Dampening the B Cell Response, a modified lipid story.
Authors: Waseem Tayab C. , Gjurich Breanne , Butcher Matthew J. , Moriarty Alina , Keeter William Coles , Galkina Elena
Published: January 2023

Atherosclerosis is a multifaceted chronic inflammatory disease characterized by the accumulation of modified low density lipoproteins (mLDL) and immune cells in the aortic wall leading to vascular dysfunction. Uptake of mLDL by macrophages (MF) and the formation of foam cells are key events in the induction of atherosclerosis. Our data shows that B cells can also uptake acetylated and oxidized LDL (acLDL and oxLDL, respectively), but the functional consequences of this uptake are currently not well-understood. The transcriptional profiles of sorted B cells that took up acLDL in vivo by RNAseq suggests that acLDL+ B cells assume a role distinct from MF, and reduces activation. Upon mLDL uptake, B cells dampen activating signaling molecule such as SYK, ERK, and BTK, but upregulate inhibitory signaling molecules such as phospho-Lyn (Tyr507). Importantly, the uptake of mLDL affects the ability of B cells to be activated both in vivo and in vitro. mLDL+ B cells mobilize less intracellular calcium in response to B cell receptor cross-linking. Additionally, after mLDL uptake, B cells are less efficient in response to TI-2 antigen DNP-Ficoll and B cells produce significantly lower levels of anti DNP-IgM Abs after the immunization. Upon several TLR stimulations B cells that took up acLDL also produce significantly less proinflammatory cytokines (IFN-g, IL-6). Together the results suggest that mLDL uptake by B cells results in an upregulation of threshold for B cell activation in pro-inflammatory atherosclerosis-prone conditions. Thus, the uptake of mLDL by B cells may play an unexpected protective role in atherosclerosis.
Secondary damage or viral infection following mild traumatic brain injury impedes tissue repair induced by distinct myeloid cell subsets
Authors: Russo Matthew V. , McGavern Dorian B.
Published: January 2023

Mild traumatic brain injury (mTBI) causes severe meningeal cell death and vascular injury, leading to a robust peripheral immune response featuring neutrophils and monocytes/macrophages. Complications such as repetitive head injuries or viral infections following mTBI have the potential to significantly alter the immune response and impact wound repair. Using a closed-skull compression model of mTBI, we found that meningeal vasculature can regenerate within 7 days of injury, coinciding with a dynamic, bifurcated myeloid cell response. Macrophages derived from inflammatory monocytes scavenged dead cells while non-classical monocyte-derived macrophages promoted angiogenesis. The CD206+ angiogenic macrophages were positioned around the lesion perimeter where they aided in fibrinogen clearance and matrix remodeling. A peripheral infection by lymphocytic choriomeningitis virus (LCMV) during peak vascular damage delayed wound repair and promoted a significant meningeal CD8+ T cell response. The infected environment also shifted the myeloid cells to a more inflammatory state, reducing the amount of CD206+ macrophages around the lesion. Interestingly, we found that a secondary injury also interrupted wound repair only when experienced during the acute “inflammatory” but not the “wound-healing” phase. In fact, the mTBI lesion and infiltrating macrophages were resistant to further damage when reinjured during the wound-healing phase. Our findings demonstrate that meningeal vasculature can undergo robust regeneration following mTBI orchestrated by distinct myeloid cell populations. However, this wound repair process can be significantly disrupted by both sterile and viral insults following the initial injury.
CD5 expression influences T cell metabolism and mice behavior
Authors: Freitas Claudia T. , Cox Tyler , Johnson Deborah , Weber K. Scott
Published: January 2023

T cells are key players in the adaptive immune response and undergo metabolic changes upon activation. CD5 is a co-receptor found on T cells and plays a significant role in regulating T cell thymic development, intracellular signaling and cytokine production. Previous studies have found that naïve T cells with high CD5 expression (CD5hi) have increased TCR signal strength and enhances immune response to foreign peptide in the periphery. Additionally, we have reported that CD5hi naïve T cells have higher calcium mobilization and improved T cell activation compared to CD5lo T cells. Calcium influx levels can modulate and influence metabolic changes in T cells. Thus, we hypothesized that CD5hi, CD5lo and CD5 deficient T cells have different bioenergetic demands that affect metabolic pathways and T cell activation. We evaluated the effects of CD5 levels on metabolism using CD5 deficient mice vs wild type controls and found CD5 deficient T cells had significant differences in metabolic function. Recently published work has described a connection between increased T cell metabolism and altered cognitive function in PD-1 deficient mice. We have also found significant differences between CD5 deficient and wild type mice in marble burying rates, elevated plus and water maze behavior and open field activity. These behavioral test results suggest CD5 deficient mice have altered cognitive function and higher levels of anxiety. Thus, CD5 deficiency alters T cell metabolic and cognitive function.
The Mechanosensitive Ion Channel, Transient Receptor Potential Vanilloid 4 (TRPV4) in Macrophages Regulates the Host Defense Response to Bacterial Pneumonia
Authors: Scheraga Rachel Greenberg , Abraham Susamma , Grove Lisa M. , Southern Brian D. , Crish James , Lumpkin Timothy Brian , Hamilton Thomas A. , McDonald Christine , Olman Mitchell A.
Published: January 2023

Macrophage phagocytosis and cytokine production are sensitive to the surrounding matrix and thereby mediate host defense and lung tissue injury. The consequences and mechanism of this matrix sensitivity are unknown. We have determined that the mechanosensitive channel, TRPV4, responds to extracellular matrix biophysical properties and thereby modulates the macrophage response to pathogens. We undertook this study to determine the in vivo consequences and the intracellular signaling pathway by which TRPV4 modulates macrophage responses. In order to evaluate the role of TRPV4 in chronic pneumonia/lung injury, WT and TRPV4 KO mice were administered agarose bead embedded-Pseudomonas aeruginosa. Loss of TRPV4 led to decreased phagocytosis by macrophages (6-fold), and increased lung injury as measured by inflammatory cell infiltration (≥ 80 ± 3%), vascular permeability (total protein ≥ 63 ± 6%), and cytokine secretion (IL-1β ≥ 71 ± 4%). In vivo, lung alveolar macrophages predominantly expressed TRPV4 and were the key phagocytic cell, as assessed by FACS and immunofluorescence. Known LPS signaling pathways were investigated in vitro. Loss of TRPV4 abrogates LPS-induced p38 activation and phagocytosis of E. coli particles. Additionally, loss of TRPV4 increased basal pro-/active IL-1β expression (2-fold), thereby enhancing IL-1β secretion independent of caspase 1 cleavage and blockade. These findings demonstrate that TRPV4 is important for bacterial clearance, lung injury and cytokine production in macrophages. TRPV4 mediates these effects through p38 and through upregulation of IL-1β protein expression in an inflammasome-independent manner. The results implicate macrophage TRPV4 as a key component of the host defense.
Heatr9 is upregulated during influenza virus infection in lung alveolar epithelial cells
Authors: Stairiker Christopher , Mueller Yvonne , van Meurs Marjan , Brouwers-Haspels Inge , Erkeland Stefan , Katsikis Peter D.
Published: January 2023

Influenza virus infection poses a serious threat to public health. Influenza virus infects lung alveolar epithelial cells in the respiratory tract and utilizes them to produce more virus, spreading the infection. Understanding genes that are dynamically regulated during influenza virus infection may help elucidate essential genes and pathways that affect influenza virus infection. We examined by RNAseq the in vivo gene expression profiles of alveolar epithelial cells sorted from A/Puerto Rico/8/1934-GFP (PR8-GFP) expressing influenza virus infected mice. We identified a novel influenza virus-induced gene, Heatr9 (also known as Gm11435), that was upregulated by >200-fold in vivo in mouse alveolar epithelial cells. The upregulation of Heatr9 by influenza virus was further confirmed in vitro in PR8-GFP influenza virus infected human lung A549 cells. In vivo Heatr9 upregulation was found to be an indirect effect of influenza virus infection as bystander alveolar epithelial cells in lungs exhibited similar levels of Heatr9 induction as directly infected cells. Furthermore, supernatants of influenza virus infected A549 cells were capable of potently inducing Heatr9 mRNA even in the absence of infection. To identify factors that upregulate Heatr9 we examined the effect of cytokines on Heatr9 expression in vitro. Heatr9 expression was not induced by type I IFN, TNFα, and IL-1β alone in A549 cell, however when used in combination IFNβ and TNFα or IFNβ and IL-1β potently induced Heatr9 mRNA. Currently, we are generating Heatr9 deficient cell lines to examine the function of this gene in infection. In summary, we have identified Heatr9 as a cytokine-induced gene during influenza virus infection, the function of which has a yet to be determined.
Effect of the blocking by monoclonal antibodies of TLR2 and CD14 receptors on the expression of intracellular signaling molecules and the production of cytokines in THP-1 macrophages infected with Mycobacterium tuberculosis
Authors: Mendoca Alma Yolanda Arce , Rosas-Taraco Adrian Geovanni , Rodriguez Victor Garcia , Zapata Edgar Ivan Montes , Flores Alberto Yairh Limon
Published: January 2023

INTRODUCTION

There are several reports that indicate the importance of TLR2 and CD14 in the response against Mycobacterium tuberculosis since they activate the cascade of intracellular signaling and the production of inflammatory cytokines necessary for the infiltration of neutrophils and macrophages in the focus of the infection, however it can remain the live bacillus inside the granuloma.

MATERIAL AND METHODS

In this study, monoclonal antibodies (mAbs) against TLR2 and CD14 were used to block these receptors in the macrophages of the THP-1 cell line infected with Mycobacterium tuberculosis. Molecules of the intracellular signaling cascade were measured: MYD88 and TRAF6 and the transcription factor NFKB by RT-PCR, preinflammatory and anti-inflammatory cytosines by ELISA, also CFU and nitric oxide were measured.

RESULTS

An increase in the expression of Myd88, TRAF6 and NFKB was found; a decrease in CFU, increased production of TNF-α and INF-γ and low production of nitric oxide.

CONCLUSION

The results suggest that the mechanisms of intracellular signaling were modified by helping macrophages kill the intracellular bacillus, so we suggest that the use of these mAbs as an immunotherapy that together with the antimicrobial can help to resolve the infection in the patients with tuberculosis. Blockade of TLR2 and CD14 favored the expression of MyD88, TRAF6 and NFkB. Cytosine was also produced, such as TNF-α, which helps the confinement of bacilli in the granuloma and INF-γ, which probably also helped the intracellular death of Mycobacterium tuberculosis.
FOXP3 protects human T cells from restimulation-induced cell death via CD48 upregulation and autophagy
Authors: Voss Kelsey , Dalgard Clifton , Arjunaraja Swadhinya , Dorjbal Batsukh , Snow Andrew L.
Published: January 2023

The adaptive immune response relies on specific apoptosis programs to regulate effector T cell expansion and maintain homeostasis. Restimulation-induced cell death (RICD) is an apoptotic pathway triggered by repeated stimulation through the T cell receptor (TCR), which ensures that effector T cell proliferation remains in check. Constitutive fork head box P3 (FOXP3) expression renders regulatory T cells (Tregs) resistant to RICD. Although FOXP3 is also expressed transiently in human conventional T cells (Tcons) during initial rounds of activation-induced proliferation, its function in this context remains unclear. Here we describe a novel role for FOXP3 in protecting both CD4+ and CD8+ human Tcons from premature RICD during the expansion phase. SiRNA-mediated silencing of FOXP3 sensitized early effector Tcons to RICD, and was dependent on de novo transcription. RNA-Seq analysis revealed FOXP3-dependent effects on genes involved in metabolic pathways, autophagy, and cell surface receptors. FOXP3 silencing increased glycolysis and reduced autophagy in Tcons, which has been linked to changes in apoptosis sensitivity. Indeed, blocking autophagy increased RICD in control Tcons without augmenting death of FOXP3-silenced cells, suggesting FOXP3 reduces RICD sensitivity through an autophagy-dependent mechanism. Additionally, FOXP3 knockdown markedly reduced expression of CD48, a SLAM-family receptor. We now show that CD48 protects expanding Tcons from RICD. Surprisingly, CD48 also promoted protective autophagy, illuminating a novel connection between SLAM receptor signaling, autophagy regulation, and RICD sensitivity that is governed by FOXP3 expression in early Tcons.
The cytoplasmic domain of CTLA-4 inhibits effector T cell responses and autoimmunity via increasing regulatory T cells in mice
Authors: Lim Sangho , Lee Hong-Gyun , Kim Gil-Ran , Choi Je-Min
Published: January 2023

Recently, a critical role of CTLA-4 in a regulatory T cell (Treg) function has been highlighted both in peripheral and follicular regulatory T cells. However, the importance of cytoplasmic domain CTLA-4 signaling in Treg function and differentiation is not clearly understood. In this study, we utilized a recombinant extracellular domain (CTLA-4-Ig) and cytoplasmic domain of CTLA-4 (dNP2-ctCTLA-4) to elucidate the mechanism of CTLA-4 in Tregs. We found dNP2-ctCTLA-4 increased follicular regulatory T (Tfr) cells in draining lymph node (dLN), accompanying by decreased level of follicular helper T (Tfh) cells and germinal center (GC) B cells, while CTLA-4-Ig only reduced Tfh cells and GC B cells without affects on Tfr cells. In addition, dNP2-ctCTLA-4 induces Foxp3 positive regulatory T cells in Th17 and Th1 conditions with TGF-β, while CTLA-4-Ig inhibited effector cytokine productions without affecting Foxp3 levels. One of the molecular mechanism of dNP2-ctCTLA-4 in Foxp3 expression would be inhibition of Jak2/STAT3 pathway via binding to Jak2 which inhibits Th17. In addition, TGF-β signaling was enhanced by it via inhibition of phosphorylation on Erk and Smad2 linker region to results increasing Foxp3 expression in T cells. These results suggest that the cytoplasmic domain CTLA-4 signaling would be critical to support immune homeostatic environments in dLN via enhancing Foxp3 expression in T cells.