bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiologic stress molecules activate an acute phase response and enhance macrophage antimicrobial activity to Mycobacterium leprae (INM3P.411)
Authors: Scumpia Philip , Botten Giovanni , Kelly-Scumpia Kindra , Modlin Robert , Smale Stephen
Published: January 2023

Leprosy is a disease caused by the pathogen Mycobacterium leprae (mLEP). Patients can present with the progressive, lepromatous form of the disease or the self-limited, tuberculoid form of the disease. Physiologic stress is thought to inhibit host defense mediated by adrenergic catecholamines and neuroendocrine glucocorticoids. We hypothesize that acute treatment with adrenergic agents or glucocorticoids would inhibit in vitro macrophage mediated antimicrobial activity. Human monocyte-derived macrophages or mouse bone marrow-derived macrophages were treated with the adrenergic agonist metaproterenol hemisulfate (MPH, 1μM) or the glucocorticoid dexamethasone (Dex, 100nM) for two hours followed by LPS (100ng/ml) or mLEP (MOI 5). RNASeq was performed at various time points or an mLEP viability measurement was performed at 24 hours following infection. Unexpectedly, when compared to vehicle (DMSO), pretreatment with MPH resulted in greatly enhanced antimicrobial activity against mLEP, similar to interferon-γ pretreatment, whereas Dex resulted in a more modest induction of antimicrobial activity. Both Dex and MPH did not reduce the expression of the antimicrobial peptide cathelicidin, but did induce a dramatic increase in expression of genes involved in the acute phase response. Our findings suggest that molecules involved in the physiological stress response may be acutely beneficial during initial encounter with mLEP.
Dendritic cell homologous antigenic loading initiates AIMp1-mediated TH1 immunity (IRC7P.432)
Authors: Halpert Matthew , Liang Dan , Konduri Vanaja , Pautler Robia , Klumpp Sherry , Bhardwaj Ratan , Levitt Jonathan , Decker William
Published: January 2023

Attempts to treat cancer by vaccine immunotherapy have produced unsatisfactory results in part due to an inability to generate effective TH1 immunity. Here we demonstrate the existence of an elegant TH1 regulatory mechanism based upon release of the AIMp1 proinflammatory cytokine and elicited by the loading of dendritic cells (DC) with MHC class I and II antigenic epitopes of overlapping (homologous) amino acid sequence. Using a variety of distinct model systems, we demonstrate that DC possess a physical mechanism by which to compare amino acid sequence homology of bound class I and II epitopes, releasing AIMp1 when significant homology among such epitopes exists. AIMp1 release was shown to be upstream of microsomal CTLA-4 secretion, events that were inversely correlated and directly dependent upon the presence of homologous class I and II antigenic epitopes. Antigenic homology could mediate DC TH1 polarization and subsequent enhancement of CD8+ responses only when MHC molecules were present and able to be loaded, in an AIMp1-dependent manner, and when bound class I and II peptides shared uninterrupted sequence homology of at least five amino acid residues. Physiologic relevance and translational applicability to cancer were also demonstrated. The existence of this surprising and novel regulatory mechanism alters the paradigm by which regulation of cellular immunity is generally understood.
The tetraspanin CD151 is a T cell activation marker demarcating hyper-responsive CD8+ CD28- T cells in HIV patients (IRC7P.433)
Authors: Seu Lillian , Sabbaj Steffanie , Duverger Alexandra , Wagner Frederic , Goepfert Paul , kutsch olaf
Published: January 2023

HIV-infected patients, despite suppressive ART, exhibit signs of immune hyperactivation and premature senescence, compromising the success of long-term ART. Elucidation of the molecular mechanisms underlying immune dysfunction will be critical to develop future immuno-modulatory therapies to improve the long-term health status of HIV patients. To achieve this goal, biomarkers that resolve dysfunctional T cell populations are needed. To this end, we here demonstrate that the tetraspanin CD151 in HIV patients on ART is highly expressed on CD28- T cells (p<0.05 compared to healthy donors), a population that is massively increased in HIV patients and considered immunologically senescent. Functionally, we define CD151 as a T cell activation marker that is not affected by the presence of cytokines (e.g. TNF-a, IL-2), but is upregulated following TCR engagement. Surprisingly, the majority of CD8+ CD28- T cells responsive to CMV pp65 peptides were CD151+ as measured by Granzyme B+ and IFN-γ+. Blocking experiments with CD151 mAb led to a dose-dependent reduction of IFN-γ secretion from CMV pp65- stimulated PBMCs. In summary, CD151 is elevated on senescent CD8+ CD28- T cells that demonstrate enhanced antigen-responsiveness. As prior data shows that CD151 on T cells stabilizes the immune synapse and primes T cell activation, CD151 expression may confer responsiveness of otherwise senescent T cells and present one underlying cause of immunological hyperresponsiveness in HIV patients.
Rho kinase activity dampens the activation of CD4+ T cells (IRC7P.435)
Authors: Thauland Tim , Butte Manish
Published: January 2023

T cells respond to increasing doses of antigen with enhanced proliferation and cytokine production, both in vitro and in vivo. Furthermore, effector T cells respond more robustly to limiting amounts of peptide than naïve cells. Although much is known about the signaling pathways downstream of the T cell receptor, the molecular mechanism responsible for maintaining the threshold for activation in T cells remains obscure. We have previously shown that the T cell cytoskeleton softens upon T cell activation. Here, we show that effector T cells form larger interfaces with antigen presenting cells (APCs) than naïve cells after correcting for cell volume. We also demonstrate that the RhoA effector Rho-associated protein kinase (ROCK) is more active in naïve than activated T cells. Inhibition of ROCK activity with a small molecule inhibitor, glycyl-H-1152, softened the actin cytoskeleton, independent of stimulation through the T cell receptor. The size of T-APC contacts were increased upon ROCK inhibition, and calcium flux was enhanced. These data demonstrate that ROCK has a central part in tuning the response of T cells to antigen. In the future, we will identify and examine the downstream targets of ROCK that are involved in modulating T cell activation.
Genome-wide profiling of H3K4me3 and H3K27me3 dynamics reveals fundamental epigenetic influences upon CD4 T cell activation (IRC7P.423)
Authors: LaMere Sarah
Published: January 2023

H3K4me3 and H3K27me3 are frequently studied in the context of their association with gene expression, but their cause and effect relationship during CD4 T cell activation has not been determined. Using ChIPseq for H3K4me3 and H3K27me3 alongside RNAseq in naïve and memory human CD4 T cells at rest and after activation at 24 hours, 5 days, and 2 weeks, we have defined the roles these modifications are playing throughout the process of activation. Our results demonstrate that promoter H3K4me3 is acting in a feed-forward fashion, where lower expression genes containing H3K4me3 at rest increase expression more rapidly than genes without H3K4me3, and H3K4me3 deposition acts to reinforce increased expression. Conversely, promoters containing H3K27me3 at rest generally show a decrease in expression after activation, particularly in higher expressing genes. However, changes to promoter H3K27me3 after activation act to reinforce basal expression. H3K27me3 demethylation is a prominent finding in both naïve and memory cells during activation, and inhibiting this demethylation leads to decreased proliferation, blunted upregulation of IL2RA, and changes to the cell cycle, suggesting H3K27me3 demethylation is integral to achieving full T cell activation. Our results enhance our understanding of the role these histone modifications play during CD4 T cell activation and underscore key differences between naïve and memory cells in their activation dynamics.
CTLA4-Ig treatment improves RBP4-induced adipose tissue inflammation and insulin resistance triggered by MyD88, JNK, ERK and p38 pathways (IRC8P.443)
Authors: Vieira Pedro , Castoldi Angela , Aryal Pratik , Wellenstein Kerry , Yore Mark , Peroni Odile , Kahn Barbara
Published: January 2023

Adipose tissue (AT) inflammation and impaired insulin action is a major cause of type 2 diabetes. RBP4 is an adipocyte- and liver-derived protein that has an important role in insulin resistance, metabolic syndrome and AT inflammation. RBP4 elevation causes AT inflammation by activating innate immunity that elicits an adaptive immune response. Our aims are to determine the signaling pathways involved in RBP4-induced macrophage activation and the resulting antigen presentation and Th1 polarization and whether the blockade of antigen presentation improves AT inflammation and insulin resistance. RBP4-overexpressing mice (RBP4-Ox) are insulin resistant and glucose intolerant and have increased AT macrophage and Th1 cell infiltration. In RBP4-Ox, AT macrophages display enhanced JNK, ERK and p38 phosphorylation, and in vitro inhibition of these pathways reduces macrophage activation and macrophage-induced CD4 T cell proliferation and Th1 polarization. Moreover, macrophages obtained from MyD88 knockout mice and activated with RBP4 do not secrete TNF, IL12 and IL-1b and fail to induce CD4 T cell proliferation and Th1 polarization. Treatment of RBP4-Ox mice with CLTA4-Ig reduces AT inflammation and improves insulin resistance. Thus, RBP4 causes insulin resistance, at least partly, through MyD88 pathway and downstream by activating JNK, ERK and p38 pathways. These pathways induce macrophage activation and Th1 polarization, which can be blocked by inhibiting antigen presentation.
miR-146a as a negative regulator in controlling humoral immunity (IRM10P.612)
Authors: Lee Hyang-Mi , Choi Youn , Cho Sunglim , Lin Ling-Li , Kang Seung , Aly Khan , Xiao Changchun , Crotty Shane , Lu Li-Fan
Published: January 2023

The germinal center (GC) reaction is the hallmark of B cell-mediated immune responses to T cell-dependent antigens Recently, a specialized immune population of follicular helper T (Tfh) cells has emerged as a central player in driving GC B cell response and promoting humoral immunity. It has been shown that many genes that are critically involved in the generation of GC B cells also play an indispensable role in the differentiation of Tfh cell population from naive T cells. Here, we show miR-146a is highly upregulated in both Tfh and GC B cells. Our analysis of mixed bone marrow chimeras as well as mice with cell-type-specific deletion of miR-146a demonstrated that elevated levels of miR-146a in both Tfh and B cells are pivotal to control corresponding responses during GC reactions in a cell intrinsic manner. Moreover, deletion of both miR-146a and its paralog, miR-146b resulted in further enhance phenotypes, suggesting that both miR-146 family members contribute to the negative regulation of humoral immunity. Finally, combining transcriptional profiling and biochemical approaches including HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation), several miR-146a targets with potential roles in controlling Tfh and GC B cell responses have been identified. In sum, our study identifies a miRNA family highly enriched in GC cell subsets with the capacity to control GC reaction by limiting differentiation of Tfh and GC B cells.
Constitutive IL-10 production by normal and malignant B-1 cells is dependent on B cell receptor signaling (IRM10P.620)
Authors: Alhakeem Sara , McKenna Mary , Gachuki Beth , Wang Chi , Liu Jinpeng , Byrd John , Muthusamy Natarajan , Bondada Subbarao
Published: January 2023

The most common human leukemia is B-cell chronic lymphocytic leukemia (B-CLL), which can have immunosuppressive effects. CLL cells share multiple phenotypic markers with B-1 B cells such as their surface expression of CD5. This study identifies some functional similarities between B-1 and CLL cells. B-1 B cells have been shown to produce a high level of interleukin-10 (IL-10) constitutively and upon toll-like receptor stimulation, a characteristic property of regulatory B cells. Eμ-Tcl1 mice, which express the Tcl1 oncogene in a B cell specific manner, spontaneously develop B-CLL. We show that Eμ-Tcl1 mouse B-CLL cells also produce IL-10 constitutively. The constitutively produced IL-10 was not required for CLL survival in vitro. This study describes a novel role of B cell receptor (BCR) signaling pathway in constitutive IL-10 secretion by normal and malignant B-1 cells. Thus inhibition of Src family kinases, spleen tyrosine kinase (Syk) or Bruton’s tyrosine kinase (Btk), all of which are downstream of BCR, reduces constitutive IL-10 production by both normal and malignant B-1 cells. Analysis of a published microarray study showed that IL-10 levels were higher in peripheral blood of CLL patients than normal human blood samples, which correlated positively with STAT3 levels. Interestingly, we found that BCR signaling induced IL-10 production is independent of STAT3. Current studies are directed at understanding the mechanisms by which BCR signaling affects IL-10 production.
Regulation of the HSH2 adaptor protein turnover via an indirect NF-κB-dependent mechanism is involved in determining the nature of the class switched antibody response. (IRM10P.609)
Authors: VanValkenburg Arthur , King R. , Justement Louis
Published: January 2023

The Hematopoietic SH2 containing adaptor protein (HSH2) is differentially expressed in mature, peripheral B cell subpopulations, exhibiting low level expression in germinal center (GC) B cells versus high-level expression in B1 and marginal zone B cells. Studies have demonstrated an inverse relationship between its expression and the production of class-switched antibodies due to effects on terminal differentiation. Therefore, delineation of molecular mechanism that controls its expression is critical for understanding the class-switched antibody response. Studies demonstrated that upregulation of HSH2 following TNF family receptor or TLR-mediated signals, as well as its maintenance, requires NF-κB activity. Using both pharmacological and genetic approaches, it was shown that the canonical pathway is critical for maintenance of HSH2 expression. Inhibition of NF-kB selectively results in the rapid loss of HSH2, whereas actinomycin D and cycloheximide treatment do not. Analysis of the canonical pathway revealed that maintenance of HSH2 requires translocation of NF-κB to the nucleus. Because HSH2 mRNA levels are not altered under any conditions, it is hypothesized that NF-κB indirectly regulates the function of a short-lived protease that regulates HSH2 expression at the protein. These results reveal a potentially novel mechanism, in which NF-κB regulates HSH2 expression via one or more proteases and may represent a novel paradigm for controlling GC B cell function.
The role of Fli1 in lupus T cell function and nephritis in MRL/lpr lupus prone mice (IRM11P.634)
Authors: Nowling Tamara , Sundararaj Kamala , Thiyagarajan Thirumagal , Molano Ivan
Published: January 2023

The Ets factor Fli1 is implicated as a key modulator of lupus disease expression. Over-expressing Fli1 in healthy mice, results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of Fli1 in two lupus mouse models significantly improved kidney disease and prolonged survival. We recently demonstrated that lowering the global levels of Fli1 in MRL/lpr lupus mice (Fli1+/-) reduced T cell activation and IL-4 production. This was likely a result of decreased glycosphingolipid (GSL) metabolism, specifically decreased Neuraminidase1 (Neu1) expression and activity and decreased levels of the GSL lactosylceramide (LacCer) in the Fli1+/- T cells. GSLs are a heterogeneous class of lipids in the sphingolipid family that play a role in the regulation of cellular processes. Neu1 is an enzyme that mediates the GSL catabolic pathway. Here we demonstrate that MRL/lpr Fli1+/- T cells have significantly decreased: 1) levels of CXCR3 message, 2) percentage that are CXCR3+, 3) migration to the kidney and 4) renal Neu1 and LacCer levels, which we showed are significantly elevated in nephritic mice and humans with lupus. IHC staining shows that increased LacCer levels co-localize with T cells in the glomeruli. Together, our data demonstrate that reducing Fli1 levels in lupus T cells partially normalizes GSL metabolism in the kidney that is likely due in part to a reduction in T cell activation and migration to the kidney.
Stat3 promotes kidney disease in Stat1-KO mice with lupus-like cGVHD (IRM11P.635)
Authors: Shao Wenhai , Gamero Ana , Zhen Yuxuan , Lobue Monica , Priest Stephen , Albandar Hazem , Cohen Philip
Published: January 2023

Lupus nephritis (LN) is one of the most serious manifestations of SLE with high incidence of morbidity and mortality. Opposing effects of activated Stat1 and Stat3 have been described for different diseases. Stat1 activation was reported upregulated in the kidney of lupus-prone mice with nephritis, while inhibition of Stat3 in the kidney attenuated kidney inflammation. The present study was undertaken to investigate the role of Stat1 in an experimental animal model of lupus. We induced bm12-B6 chronic graft-versus-host disease (cGVHD) in Stat1-KO and WT mice. WT mice developed anti-dsDNA autoantibodies starting at week 2 as expected, with a decline noted after week 4. In contrast, Stat1-KO mice exhibited a prolonged and significant increase of anti-dsDNA and anti-chromatin autoantibody responses compared to WT mice (week 4 to week 8). Enhanced IgG1 and IgG2b responses but absent IgG3 response were demonstrated in the sera of Stat1-KO mice as compared to WT mice given the same treatment. To our surprise, Stat1-KO mice developed severe nephritis starting at week 8. Three months after cGVHD-induction, 40% Stat1-KO mice were dead. Immunofluorescent staining of kidney sections from the cGVHD Stat1-KO mice revealed an enhanced glomerular Stat3 expression, which correlated with increased IL-6 expression. The observation that altered expression of Stat3 influences IL-6 expression in the glomeruli in this lupus model suggests possible molecular targets for treatment of LN.
Signal transducer and activator of transcription 3 (STAT-3) is a critical transcription factor in regulating germinal center IgE+ B-cell class switching
Authors: Dascani Paul Lorenzo , Ding Chuanlin , Yan Jun
Published: January 2023

A mutation in the Signal Transducer and Activator of Transcription 3 (STAT-3) has been linked to incidence of Autosomal Dominant Hyper Immunoglobulin E Syndrome (AD-HIES), a disease characterized by elevated serum IgE antibody and susceptibility to respiratory and skin infection. However, how this genetic mutation leads to the phenotype has not been fully understood. In this study we investigated the specific role of STAT-3 in the germinal center (GC), the site of B-cell proliferation and isotype switching. Through the use of CD2-Cre or CD19-Cre driven STAT-3 conditional knockout (cKO) mice in a Th2-type immunization model, we aimed to determine the specific cell subset(s) and signaling mechanism by which STAT-3 mediated isotype switching is regulated. We demonstrated in vivo and in vitro that CD2-Cre driven STAT-3 cKO mice showed elevated IgE and decreased IgG1 in the serum, and a reduction in GC formation. Within the GC, IgG1+ GC B cells were decreased while IgE+ GC B cells were more prevalent. Additionally, these mice exhibited reduced IgG1 and elevated IgE populations of antibody-producing plasma cells. Subsequent experiments using a CD19-Cre B-cell specific cKO mouse established this effect to be B-cell dependent. Our data also revealed other transcription factors, including Bcl-6 and Aicda, function as downstream signals of STAT-3 regulation. This model suggests a role for STAT-3 in regulating class switching of the GC B cells from the IgG1 to the IgE producing state, which may serve as a potential therapeutic target for treatment of AD-HIES and other immune disorders.
IL-1 uses distinct mechanisms to enhance CD8+ T cell expansion and differentiation.
Authors: Lee Ping-Hsien , Germain Ronald N , Paul William E
Published: January 2023

IL-1 is a potent proinflammatory cytokine with pleiotropic functions within and outside the immune system. We previously showed that IL-1 promotes antigen-specific CD8+ T cell expansion, effector function, and memory differentiation but the underlying mechanisms remain unclear. Here we report that IL-1 likely utilizes distinct pathways to drive CD8 T cell expansion and effector differentiation. Maximal expansion in the peripheral tissues (spleen, lung, and liver) was only achieved when IL-1 acted on both CD8 T cells and non-T cells, which involved cells of hematopoietic and non-hematopoietic origins. Although IL-1did not affect the abundance of CD8 T cells in the draining lymph nodes (dLNs), these IL-1-treated, dLN-derived CD8 T cells expanded more robustly than their control treated counterparts after getting into the tissues. IL-1 treatment also enhanced cytotoxic function, as evidenced by increased granzyme B expression, of CD8 T cells in both dLNs and tissues. This effect was mediated by IL-1R1 signaling in non-hematopoietic cells, especially vascular endothelial cells, but not in CD8 T cells or other hematopoietic cells. Furthermore, CD8 T cells were endowed with a T-bet(hi)Eomes(low) phenotype by IL-1 and did not succumb to apoptosis but rather generated better memory cells both quantitatively and qualitatively than did their control treated counterparts. Collectively, this study demonstrates novel and complex mechanisms used by IL-1 to enhance antigen-specific CD8 T cell responses, which may shed light into developing novel strategies for optimizing CTL responses in treating cancer and infectious diseases.
Expression of IL-15 is differentially regulated during lymphopenia depending on the method of induction
Authors: Schluns Kimberly S , Rivas Sarai , Stonier Spencer W , Colpitts Sara L , Anthony Scott M
Published: January 2023

Induction of lymphopenia has been exploited therapeutically to improve immune responses to cancer therapies and vaccinations. IL-15 has a well-established role in mediating lymphocyte responses after lymphodepletion; however, the mechanisms regulating these IL-15 responses are unclear. We report that cell surface IL-15 expression is upregulated during lymphopenia induced by total body irradiation (TBI), cyclophosphamide, or Thy1 antibody (Ab)-mediated T cell depletion, as well as in RAG−/− mice; interestingly, the cellular profile of surface IL-15 expression is distinct in each model. In contrast, soluble (s)IL-15 complexes were upregulated only after TBI and αThy1Ab. Analysis of cell specific IL-15Rα conditional knock out mice revealed that macrophages and DCs were important sources of sIL-15 complexes after TBI but provided a minimal contribution after αThy1Ab treatment. Unlike with TBI, induction of sIL-15 complexes after αThy1 Ab was sustained and only partially dependent on type I Interferons. The Stimulator of IFN Genes (STING) pathway was discovered to be a potent inducer sIL-15 complexes and was required for optimal induction of sIL-15 complexes in response to Ab-mediated T cell depletion and TBI suggesting products of cell death drive production of sIL-15 complexes after lymphocyte depletion. Lastly, we provide evidence that IL-15 induced by inflammatory signals in response to lymphodepletion drive lymphocyte responses as proliferation of memory CD8 T cells was strongly dependent on IL-15. Overall, these studies demonstrate that the form in which IL-15 is expressed, it’s kinetics and cellular source, and the inflammatory signals involved are differentially dictated by the manner in which lymphopenia is induced.
High fat diet influences T cell homeostasis and macrophage phenotype to maintain chronic inflammation
Authors: Singh Udai P , Mehrpooya Pegah , Marpe Bam , Singh Narendra P , Murphy E. Angela , Mishra Manoj K , Price Bob L , Nagarkatti Mitzi , Nagarkatti Prakash S
Published: January 2023

Over the past 20 years obesity has become a global health problem affecting the life expectancy of people at epidemic proportions. Obesity is characterized as a state of low-grade chronic inflammation that influences the development and progression of many chronic diseases. A unique role of T cells in adipose tissue has been shown in the initiation and regulation of the inflammatory cascade. However, the mechanisms responsible for the obesity-associated inflammation are not known. We investigated how high fat diet may influence homeostatic expansion of T cells, macrophage behavior and inflammation. High fat diet consumption alters the body weight, fat mass, and lean mass of mice when compared with those on a normal diet. The high fat diet increases the frequency of CD44+ and TCR αβ+T cells in the epididymal adipose tissues as compared with a normal diet. In mice consuming a high fat diet, we also found a significant increase in the frequency of CXCR3+ activated CD8T cells, CD8+KLRG1 cells and pro-inflammatory cytokines in mucosal and epididymal adipose tissues. High fat diet consumption resulted in greater than 2 fold changes in 85 gene and 142 miRs in epididymal adipose immune cells. Among these, ten inflammatory, obesity miRNAs and genes were validated by RT-PCR analysis. Pathway analysis also validated that differentially regulated miRNAs and gene target mRNAs are associated with T cell homeostatic expansion and macrophage function. Taken together, these results indicate that high fat diet modulates T cell homeostatic proliferation, macrophage phenotype, inflammatory miRNAs and genes to sustain inflammation. This study supports a key role of T cell homeostasis and macrophages to induce inflammation during high fat diet-induced obesity.
Natural Killer cell-mediated antibody-dependent cellular cytotoxicity towards red blood cells infected by Plasmodium falciparum
Authors: Arora Gunjan , Hart Geoffrey T. , Desai Sanjay A. , Long Eric O
Published: January 2023

In areas of high Plasmodium transmission, humans develop clinical immunity only after years of recurrent exposure. This naturally acquired immunity depends primarily on antibodies specific for parasite antigens. The underlying basis of this protective response remains unclear. In particular, the contribution of antibody-dependent cellular cytotoxicity (ADCC) to malaria immunity remains largely unexplored. Peripheral blood natural killer (NK) cells exhibit potent ADCC through FcγRIII (CD16) binding to antibody-coated target cells. We have developed sensitive and quantitative in vitro assays to evaluate outcome of infected red blood cells (RBC) incubation with NK cells. Without specific antibodies, NK cell-mediated natural cytotoxicity was undetectable towards both uninfected and infected RBCs. Addition of serum antibodies raised against human RBCs in immunized rabbits, however, produced NK cell-dependent lysis of both uninfected and infected RBCs. Rabbit serum antibodies specific for PfEMP1, an immunodominant P. falciparum variant antigen at the surface of infected RBCs, yielded selective lysis of infected RBCs. Plasma from malaria-immune individuals also triggered selective NK-mediated ADCC of infected RBCs. Finally, incubation of infected RBCs with NK cells in the presence of plasma from malaria-immune individuals inhibited P. falciparum growth, as determined by reinvasion of fresh, uninfected RBCs. Therefore, primary human NK cells can limit growth of bloodstream P. falciparum through specific ADCC-mediated lysis of infected RBCs.
Regulation of plasma cell differentiation, migration and class-switch by c-Myb and T-bet
Authors: Good-Jacobson Kim , Di Pietro Andrea , Tempany Jessica , Tarlinton David
Published: January 2023

Humoral responses are tailored to respond most effectively to the invading pathogen. To achieve this, long lasting, protective antibodies of an affinity and isotype appropriate for the immunising antigen or infecting pathogen are produced mainly within germinal centres (GC). Here, we detail the critical, interdependent roles of the transcription factors c-Myb and T-bet in regulating GC egress and plasma cell differentiation. Deletion of c-Myb in mature B cells led to aberrant up-regulation of plasma cell transcription factors within the GC. Indeed, a subset of cycling cells within the GC secreted antibody and were CD138+ Blimp-1-GFP+. T-bet-regulated genes were also modulated, resulting in significantly elevated serum IgG2c and CXCR3 expression on plasma cells despite a Th2-biased immunization with NP-KLH in alum. These aberrant plasma cells within the GC were no longer evident upon deletion of T-bet in c-Myb-deficient mice. Surprisingly, this interplay between transcription factor networks also occurred during a response to influenza infection, in which T-bet expression is a normal component of B cell responses in wild-type mice. Chromatin accessibility studies of the T-bet promoter revealed that wild-type GC B cells from flu-infected mice had an increase in accessibility in comparison to naïve B cells, as expected. In line with the cellular data, however, there was an even further increase in chromatin accessibility in c-Myb-deficient GC B cells. Therefore, c-Myb establishes a transcription factor network during an immune response that can modulate the transcriptional network of T-bet and subsequent coordination of GC egress with plasma cell differentiation.
Ubiquilin1 is uniquely required for cell cycle entry and proliferation downstream of B-cell receptor signaling.
Authors: Greer Alexandra , Abbas Alexander , Haley Benjamin , Peng Ivan , Roose-Girma Merone , Brown Eric J
Published: January 2023

Ubiquilins are a family of ubiquitin-like proteins thought to be chaperones for proteasome-bound ubiquitinated substrates. Recently, ubiquilins have been implicated in neurodegenerative disease, as UBQLN2 mutations cause ALS in some humans and variants in the ubiquitously expressed UBQLN1 have been associated with Alzheimers disease. The roles for ubiquilins in immune function, however, are unknown. In this study, we studied a mouse deleted for UBQLN1 to investigate the role of the gene in B-cell immunity. While adult knockout (ko) mice appeared generally healthy, we found a specific defect in peritoneal B1a cells and a dysregulation of the marginal zone/follicular B-cell proportions in the spleen. When activated in vitro, ko B-cells were profoundly hyporesponsive to BCR stimulation, but normally responsive to TLR4 stimulation. Despite a lack of proliferation downstream of BCR aggregation, ko B-cells had normal signal transduction and no significant changes in gene transcription at 4 hours post-stimulation. Instead, ko B-cells failed to accumulate cyclin D2 protein, a necessary event for cell proliferation, without any effect on BCR-stimulated upregulation of cyclin D2 mRNA. Moreover, knockdown of UBQLN1 via a doxycycline-inducible shRNA profoundly inhibited proliferation of BJAB, a Burkitt’s lymphoma line, in a rapid and reversible manner. These results demonstrate that UBQLN1 is required downstream of BCR stimulation for cell cycle entry by allowing accumulation of cyclin D2. We hypothesize that UBQLN1 may be involved in degradation of specific cell cycle checkpoint control proteins, or more generally the translation or stabilization of cell cycle control proteins upregulated upon certain mitogenic stimulation.
Identification of IFN-γ-producing innate B cells
Authors: Bao Yan , Cao Xuetao
Published: January 2023

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be investigated further. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus, and TLR ligands, we identified an inducible CD11ahiFcγRIIIhi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of pathogen infection. This subpopulation of B cells can promote macrophage activation via IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B-cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of innate B cells from follicular B cells via CD40L-CD40 ligation, and increased Btk activation was found to be responsible for the increased activation of noncanonical NF-κB in innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.
A novel molecule purified from Staphylococcus aureus mutant produces γδ T cell-mediated IL-17A in mice peritoneum and protects MRSA-derived skin abscess expansion
Authors: Lee Bok Luel , Khan Adnan , Lee Min-Ja , Sung Min-Young , Lee Bo-Ram , Yun Hye-Soo
Published: January 2023

Staphylococcus aureus, a commensal bacterium in human, causes a wide range of human diseases from soft tissue infections to lethal systemic infections. But, immune-competent people are highly resistant to these diseases, indicating that unknown host defense response(s) can protect host. An effective immune response to S. aureus requires a rapid activation of innate immunity in the shape of γδ-T cells mediated IL-17 production for bacterial clearance. We hypothesized that an unidentified staphylococcal-cell wall derived molecule(s) that can activate host innate γδ-T cells is generated after S. aureus infection in healthy people. To purify this molecule(s) in mice, we generated 11 different S. aureus mutant strains, in which genes involved in staphylococcal cell wall components bio-syntheses were mutated. The mutant cells were acetone-treated to inactivate and extract toxic molecules, and then injected intra-peritoneal cavity of mice. When peritoneal lavage fluid collected 5 hr after injection was examined which mutant bacteria can produce IL-17A, three mutants produced IL-17A. To purify active fractions capable of γδ T cell-mediated IL-17A production, acetone-treated mutant bacterium was solubilized with two different peptidoglycan hydrolyzing enzymes. Then, active molecules were purified to homogeneity on ion-exchange columns. By pre-injection of purified molecule or acetone-treated mutant bacteria, the expansion of USA 300 MRSA-mediated skin abscess was inhibited, leading to a lower bacterial numbers and decrease of abscess lesion size at the site of infection. These results suggest that a novel molecule producing γδ T cell-derived IL-17A in peritoneum protected MRSA-derived skin abscess expansion in mice.
A novel role of the Type I CRISPR-Cas system in impairing host immunity by targeting endogenous genes
Authors: Wu Min , Li Rongpeng
Published: January 2023

The CRISPR-Cas9 of bacterial adaptive immune systems has garnered intense interests due to its potent and flexible ability to edit genes. However, the role and mechanism of CRISPR-Cas function in bacterial invasion of hosts have remained elusive. We demonstrate that the type I CRISPR-Cas system mediates evasion from host defense via targeting and regulation of bacterial endogenous genes. Our work shows that the CRISPR-cas type I has such a function in Pseudomonas aeruginosa (PA14 strain). The type I CRISPR-Cas system of PA14 modulates quorum-sensing mast regulator LasR to dampen host immunity and inflammatory responses by evading TLR4 recognition. These findings provide insight into the role of Type I CRISPR-Cas system of pathogenic bacteria in the evasion of host immune response, reducing inflammatory response and less resistance to infection. Because we observed a similar evasion mechanism of host defense in other bacteria such as Klebsiella pneumoniae and Escherchia coli, we reasoned that this phenomenon of “self-priming (endogenous target regulation)” is likely applicable other in pathogenic bacteria. Since the type I CRISPR-Cas system and quorum sensing system ubiquitously exist in a variety of bacteria, our findings indicate a potentially common strategy employed by bacteria to evade host immune function, which may have potential therapeutic interest.
Bait and Switch: A strategy to avoid host defense by the ticks
Authors: Bakshi Mariam , Kim Tae , Radulovic Zeljko , Porter Lindsay , Mwangi Waithaka , Mulenga Albert
Published: January 2023

Ticks are obligate haematophagous ectoparasites of animals and humans. In the livestock industry, economic losses due to ticks and tick borne diseases (TBD) amount to million of dollars. In public health, ticks are second to mosquitoes in terms of impact of human diseases. CDC has listed 14 TBD causative agents of which, 9 are transmitted by Amblyomma americanum and Ixodes scapularis. With few exceptions, the transmission of TBD agents requires successful tick feeding. Thus understanding tick-feeding mechanism is a critical step in developing novel methods to stop TBD agent transmission. We know that ticks secrete numerous tick saliva proteins that suppress host immunity to facilitate feeding and disease agent colonization. However, mechanisms of how the tick suppresses host immunity remain unanswered. We investigated the effect of 15 A. americanum and I. scapularis recombinant tick saliva proteins (rTSPs) on murine macrophage function as revealed by expression of pro-inflammatory co-stimulatory markers (CD40, C80, and CD86). Additionally, the effect of rTSP on macrophage secretion of pro- and anti-inflammatory cytokines (TNFa, IL1, IL6, IL-10, TGF beta) was also investigated. Our analysis identified five pro-inflammatory and two anti-inflammatory tick saliva proteins. The two anti-inflammatory proteins appear to reverse the expression of co-stimulatory markers activated by LPS and rTSPs. These data collectively demonstrate the presence of a “bait and switch” mechanism through which the tick may evade host immunity. We propose that ticks secrete two types of immune-modulating proteins: those that stimulate host immune cells and those that dampen the activated cells.
Wnt5a signaling antagonizes Leishmania donovani infection.
Authors: Chakraborty Arijit , Roy Syamal , Sundar Shyam , Sen Malini
Published: January 2023

Leishmania donovani, the causative agent for visceral leishmaniasis creates a niche within host immune cells to sustain itself. In light of the fact that Wnt5a utilizes a Rac1–NFκB circuitry for sustaining immune homeostasis in macrophages and alters actin assembly (Naskar.et.al 2014, Witze et.al 2008) we deciphered whether there is any influence of Wnt5a signaling on L. donovani infection. Upon infection of macrophages with L.donovani, we noted significant decrease in Wnt5a protein levels. Infection with both SAG sensitive and SAG resistant parasite strains yielded similar results. Interestingly, upon treatment of macrophages with recombinant Wnt5a prior to infection separately with SAG senstive and SAG resistant L.donovani, there was significant decrease in the intracellular load of both strains of parasite, as judged by microscopic analysis and expression of Leishmania specific genes (Amastin and G6PDH). Depleting Wnt5a from the macrophages by si-RNA in-vitro or by use of Inhibitor of Wnt Production (IWP2) in-vivo, on the other hand, led to an increase in parasite load. Using specific inhibitors of the Wnt5a signaling intermediates Rac1 and NFκB, we demonstrated that Wnt5a induced reduction in parasite load is due to Wnt5a dependent Rac1 activity but not NFκB signalling. In light of the known involvement of Rac1 in actin organization, the altered actin assembly of Wnt5a pre-treated macrophages, as evident from flow cytometric analysis corroborated the observed Rac1 mediated inhibition of L.donovani infection. Taken together, our results indicate that (i) Wnt5a signaling is detrimental to L. donovani infection and (ii) the parasite evades host immune response at least partly through obliteration of Wnt5a signaling.
Gene expression profile changes in NB4 cells induced by Retinoic Acid and pathogenic bacteria
Authors: Lin Lin , Wang Amy , Bai Ray
Published: January 2023

For several years, we have been developing a cellular immunotherapy based on differentiation of NB4 to neutrophils to treat neutropenia fungal and bacterial infection in neutropenia patients. Here, we report the study of gene expression profiles changed in activated NB4 cells and to elucidate the molecular level associated with retinoic acid (RA) and lethal bacteria. The total RNA was isolated from following samples for cDNA microarray: NB4 cells, induced mature neutrophils from NB4 cells (NB4-PMN), NB4-PMN+Acinetobacter baumannii (AB), NB4-PMN+Staphylococcus aureus (LAC), NB4+AB and NB4+LAC. The analysis of gene expression profiles indicated that after the treatment of RA, RA+AB and RA+LAC, 2 genes were dramatically up-regulated and the other 2 gene were significantly down-regulated; these genes may be related to the differentiation of NB4 cells induced by RA plus pathogenic bacteria and help us improve the NB4 cells differentiation. In addition, this can help us understand the molecular mechanism of activated NB4 cells on how it protects against lethal bacterial infection. Finally, we found that the RA+AB treatment’s result showed similar gene expression when compared to RA’s induction. In contrast, RA+LAC treatment’s result showed different gene expression when compared to the RA’s induction. However, AB and LAC stimulation by themselves didn’t change inactivated NB gene expression profiles.
Nox2-derived oxidative stress results in inefficacy of antibiotics against post-influenza S. aureus pneumonia.
Authors: Sun Keer , Yajjala Vijaya Kumar , Bauer Christopher , Talmon Geoffrey A , Fischer Karl K , Kielian Tammy L , Metzger Dennis W
Published: January 2023

Clinical post-influenza S. aureus pneumonia is characterized by extensive lung inflammation, associated with severe morbidity and mortality even after appropriate antibiotic treatment. In this study, we found that antibiotics rescue NADPH oxidase 2 (Nox2)-deficient mice but fail to fully protect WT animals from influenza and S. aureus coinfection. Further studies indicate that the inefficacy of antibiotics against coinfection is attributable to oxidative stress-associated inflammatory lung injury. However, Nox2-induced lung damage during coinfection was not associated with aggravated inflammatory cytokine response or cell infiltration but rather due to reduced survival of myeloid cells. Specifically, oxidative stress increased necrotic death of inflammatory cells, thereby resulting in lethal damage to surrounding tissue. Collectively, our results demonstrate that influenza infection disrupts the delicate balance between Nox2-dependent antibacterial immunity and inflammation. This disruption not only leads to increased susceptibility to S. aureus infection but also extensive lung damage. Importantly, we show that combination treatment of antibiotic and NADPH oxidase inhibitor significantly improved animal survival from coinfection. These findings suggest that treatment strategies that target both bacteria and oxidative stress will significantly benefit patients with influenza-complicated S. aureus pneumonia.
Innate Immune Response to Respiratory Syncytial Virus-like Particles: Comparison with the Parental Virus
Authors: Walpita Pramila
Published: January 2023

Respiratory Syncytia Virus (RSV) virus-like particles (VLPs) composed of of the two surface glycoproteins G, and F, and M protein self-assemble into VLPs. Both morphologically and immunologically, and are seen by the host cell as the real virus. RSV VLPs with adjuvant showed protection of the the lung and nose while RSV without adjuvant no protection in either site, while RSV without adjuvant provided to no protection in either site. To unravel the mystery behind the poor protection, we evaluated RSV VLPs-induced innate immunity and characterized RSV VLP-mediated cytokine response using human monocytic THP-1 cell line. Using PCR array we tested innate immune gene expression. We show that both the virus and the VLPs induced IFN-1β, pSTAT2 and interferon-stimulated genes (ISGs), namely ISG-15, ISG-56, IP-10 OAS-1 and MxA, suggesting the antiviral state in both cases. RSV-mediated induction of IFN-1β and ISG-15 seemed partially dependent on TLR-4, whereas RSV VLP-mediated induction of IFN-1β and the same ISGs was entirely dependent on TLR-4. Array data demonstrated that RSV signaled through MYD88 while RSV VLPs signaled through IRAK2, inducing NFKB activation. Furthermore, both RSV VLPs and RSV induced a Th1-biased cytokine response; however the RSV-induced responses were much weaker. Taken together, these findings suggest that RSV VLPs have several intrinsic properties that would make an effective and safe vaccine candidate when formulated with adjuvants.
Functional characterization of T cells that recognize modified epitopes in patients with type 1 diabetes
Authors: Nguyen Hai T , Arribas-Layton David , Chow I-Ting , Greenbaum Carla , Marre Meghan L. , Piganelli Jon D , Kwok William , James Eddie A
Published: January 2023

Selective recognition of self-proteins that have undergone enzymatic post-translational modification (PTM) by CD4+ T cells is increasingly implicated as a means of potentiating the loss of tolerance in autoimmune diseases such as type 1 diabetes (T1D). In particular, enzymatic deamidation and citrullination of beta cell derived peptides at HLA anchor residues enhances their presentation by high risk alleles (DR4 and DQ8). Modification of residues outside of binding pockets alter T cell recognition, resulting in enhanced proliferation and increased cytokine production. Our work demonstrates that CD4+ T cells specific for PTM epitopes are present in the peripheral blood of patients with T1D at significantly higher ex vivo frequencies than in HLA matched controls. Upon re-activation, PTM specific T cells from T1D patients produce elevated levels of effector cytokines, most notably interferon gamma. Among T1D patients, we observed heterogeneous frequencies of PTM specific T cells with a range of functional profiles. This led us to hypothesize that activation of a PTM specific T cell response is of particular significance in a subset of T1D patients. To test this hypothesis we compared the ex vivo frequency and functional profiles of PTM specific T cells in a cross sectional cohort of patients selected to include diverse levels of residual c-peptide. Our findings implicate a mechanistic role for PTMs in exacerbating autoreactive T cell responses in T1D.