bluuebunny/crossref_metadata_2025 huggingface dataset:
TREX1-induced chromosome fragmentation at the interface of innate immunity and genomic instability
Authors: Mohr Lisa , Chu Kevan , Maciejowski John
Published: January 2023

The cGAS-STING pathway, a crucial component of innate immunity, recognizes viral DNA in the cytosol. cGAS binds dsDNA and catalyzes the production of the second messenger cGAMP, resulting in the expression of Type I interferons and other pro-inflammatory genes. To prevent autoimmunity, activation of cGAS by cytosolic self-DNA is normally counteracted by the cytosolic nuclease TREX1. In many chromosomally instable cancers, cytosolic self-DNA accumulates as DNA bridges or micronuclei (MN), whose nuclear envelopes are prone to rupture, exposing contained DNA to the cytosol. cGAS activation at ruptured MN is implicated in anti-tumor immunity. However, mechanisms regulating cGAS activation at ruptured MN are yet unknown. Here, we show that TREX1 partially degrades chromosomal DNA in the cytosol to suppress cGAS activation. We observed localization of cGAS and TREX1 to ruptured MN and DNA bridges, strongly suggesting an unknown role for TREX1 in regulating cGAS activation in cancer. After induction of MN or DNA bridge formation, TREX1 KO causes dramatic increases in cGAMP production and cGAS-STING pathway signaling. Furthermore, ruptured MN exhibit TREX1-dependent DNA damage and ssDNA foci. TREX1 is bound to the ER, which is known to invade ruptured MN. We found that an ER binding mutant of TREX1 does not localize to MN anymore and was not able to rescue the TREX1 KO phenotype, suggesting a role for the ER to recruit TREX1 to MN.

In summary, we propose that TREX1 enters MN as a consequence of nuclear envelope rupture to partially degrade DNA, thereby suppressing cGAS activation. By eliminating the immunogenic potential of these DNA species, TREX1 may dampen anti-tumor immunity and thereby help cancer cells escape the immune system.
Enhanced anti-tumor response upon deficiency of Msn kinase MINK1 in mice
Authors: Zhu Kaixiang , Lu Linrong
Published: January 2023

Misshapen (Msn)/NIK-related kinase 1 (MINK1) is a serine/threonine kinase belongs to the germinal center kinase (GCK) family. Previous studies indicated the correlation of MINK1 with tumor cell senescence and Th17 differentiation. Its dual role in modulating both tumor growth and T cell function indicated MINK1 a possible intervention target in tumor immunotherapy. We have utilized the MINK1 deficient mice to explore the possible role of MINK1 in the anti-tumor immune response. By using the subcutaneous tumor-xenograft model, we have found that MINK1 deficiency in mice resulted in slower tumor growth. We have also accumulated evidence in adoptive transfer and CD8+ T cells depletion experiments to show that the genetic ablation of MINK1 suppressed tumor growth primarily via CD8+ T cells without affecting other immune cells. Along with which, MINK1 deficient CD8+ T cells are superior in both tumor infiltration and the expression of effector markers such as TNF-α, IFN-γ, and Granzyme-B when compared to WT CD8+ T cells. Lower expression of exhaustion markers such as PD-1, TIM-3, and TIGIT were also observed in MINK1 KO CD8+ T cells. We are currently exploring the possible mechanisms of how MINK1 regulated CD8+ T cell function, and examine the possibility to target MINK1 in experimental tumor immunotherapy.
Protein Phosphatase 2A (PP2A) is essential for CD8+ T cell mediated anti-tumor immunity
Authors: Rohila Deepak , Lu Linrong
Published: January 2023

PP2A controls a wide variety of cellular pathways responsible for proliferation, growth, differentiation, and apoptosis. However, its role in immune cells, particularly in CD8+ T cells, still remains a mystery. We therefore sought to characterize the influence of PP2A on CD8+ T cell function in anti-tumor response. T cell specific genetic ablation of PP2A in mouse resulted in aberrant anti-tumor activity evidenced by higher tumor volume in both skin melanoma B16F10 and lymphoma E.G7 tumor graft model. Similarly, inhibition of PP2A using small molecule inhibitor LB100 showed similar effect. In addition, lower expression of effector cytokines including IFN-g, TNF-a and Granzyme B was also observed in PP2A-deleted CD8+ T Cells isolated from tumor infiltrate. Importantly, the defect in anti-tumor response is due to deficiency in CD8+ T cell function, which was proven in adoptive transfer experiment, in which CD8+ T cells from both WT and PP2A KO mice into tumor bearing RAG1 KO mice. PP2A KO CD8+ T cells-transferred mice show markedly impaired anti-tumor response. Further mechanism study suggested a possible role of PP2A in regulating CD28 signaling. PP2A KO lead to lower AKT phosphorylation upon CD28 stimulation, along with lower proliferative capability of CD8+ T cells upon stimulation with anti-CD28 and suboptimal anti-CD3 concentration. Thus this study highlights the potentially important role of PP2A in CD8+ T cells and its therapeutic consideration for designing combinatorial immunotherapy.
Identification of a core miRNA signature unique to exhausted cytotoxic CD8+ T cells
Authors: Kiernan Caoimhe H. , Hope Jennifer L , Leon Leticia G. , Zhao Manzhi , van Meurs Marjan , Brouwers-Haspels Inge , van Strien Paulette , Bindels Erik , Hoogenboezem Remco M. , Li Yunlei , Stubbs Andrew P. , Zehn Dietmar , Mueller Yvonne M , Erkeland Stefan J. , Katsikis Peter D
Published: January 2023

Cytotoxic CD8+ T lymphocytes (CTLs) play a major role in protection against chronic viral infections and tumors. Chronic antigen stimulation in both conditions leads to CTL exhaustion and failure of CTLs to efficiently eradicate tumors and virally infected cells. miRNAs are small, non-coding RNA molecules that participate in post-transcriptional gene regulation. This study aimed to determine the core miRNA signature of CTL exhaustion in mouse models of both tumors and chronic infection. Exhausted anti-tumor CTLs were isolated from tumors of mice that received 10^6 mouse mesothelioma cells (AE17sOVA) followed by an adoptive transfer of 10^4 CD45.1+ OT-I cells. Effector anti-viral CTLs were isolated from mice adoptively transferred with 10^4 CD45.1+ OT-I cells or P14 cells and infected with WSN-OVA or LCMV Armstrong respectively. For exhausted anti-viral CTLs, mice were adoptively transferred with 10^4 CD45.1+ P14 cells and infected with LCMV-Cl13. Single cell suspensions were prepared from the different tissues, live CTLs were FACS sorted, RNA isolated and small RNA-seq was performed. Principle component analysis (PCA) of miRNA expression revealed that exhausted anti-tumor CTLs and anti-LCMV Cl13 CTLs cluster apart from effector anti-viral CTLs (WSN-OVA and LCMV Armstrong). Differential expression analysis identified 73 miRNAs commonly shared between the exhausted day anti-tumor CTL and anti-viral CTL. Using RNA sequencing of two different models of exhaustion, distinct RNA profiles common in exhausted CTLs were found. This core miRNA signature can now be used to manipulate the expression of specific miRNA to potentially prevent or revert CTL exhaustion and improve the CTL response against tumors and/or chronic infections.
Differential recruitment and activation of mucosal and systemic ILC for improved vaccine design
Authors: Khanna Mayank , Sheehan Jared , Nichols Olga , Ranasinghe Charani , Ramsay Alistair
Published: January 2023

Innate lymphoid cells (ILC) have emerging roles in immune modulation, tissue homeostasis, and inflammation. ILC may function as innate counterparts of T cells, influencing early host responses against infection or vaccination, potentially modulating development of adaptive immune responses. Little is known, however, of their recruitment and activation following immunization. Here, we investigated the activity of ILC populations after immunization with poxvirus-based vaccine vectors. Our data suggest that fowlpox virus (FPV) and modified vaccinia Ankara strain (MVA) vectors can differentially polarize ILC responses at mucosae. Intranasal (IN) delivery of FPV vectors increased numbers of pulmonary ILC1 and ILC3 subsets expressing IL-17a and IL-22 through 72 hours post-immunization. In contrast, MVA vectors given IN enhanced pulmonary ILC2 numbers and their expression of IL-13. Our previous work indicated that mucosal immunization with these vectors induced high avidity T cell responses associated with transient inhibition of IL-4/13 expression by local ILC2. Since IL-25 has been shown to down regulate IL-12 and promote ILC2/IL-13 expression, we delivered FPV encoding an IL-25 binding protein (IL-25BP) in order to sequester host IL-25 at the site of immunization. While mucosal delivery of FPV-IL-25BP appeared to induce ILC2/IL-13 expression in the lungs, IM delivery reduced ILC2/IL-13 expression, increased IL-17a expression by local ILC1 and ILC3, and enhanced Th1-type adaptive immune responses against co-expressed vaccine antigens. Together, our findings suggest that manipulation of ILC responses by vaccine vectors, co-expressed immune modulators, and delivery route may be exploited for improved vaccine design.
Development of a reverse genetics system to generate vaccine strain of Hepatitis A virus
Authors: Seo Dongwon , Kang Hyeon Jeong , Choi Jung Ah , Seo Sang Hwan , Song Man Ki
Published: January 2023

Objective

Reverse genetic system have been used to generate mutant viruses to investigate the genetic features as well as to produce vaccine strain of various viruses. However, the virus producing system has not been applied to produce vaccine strain of Hepatitis A virus (HAV). Here, we established reverse genetic system to generate vaccine strain of HAV and assessed the generated HAV in terms of immunogenicity.

Methods

To produce HAV, MRC-5 cells were transfected with HAV RNA generated via in vitro transcription. To confirm the virus generation, we performed RT-PCR, Western blot, Rapid amplification of cDNA ends (RACE) and transmission electron microscopy (TEM) analysis using the supernatant of the HAV RNA transfected cells. To assess the immunogenicity of the generated HAV, mice were immunized with the generated and inactivated HAV and performed enzyme-linked immunosorbent assay (ELISA) to assess anti-HAV IgG using sera from the immunized mice.

Results

From the supernatant of HAV RNA transfected cells, HAV-specific RNA and protein were detected. TEM analysis showed that there are two types of HAV particles as shown in a previous report: naked and enveloped HAV, which is one of HAV features. Although seven nucleotide sequences are different in the coding sequence for structural protein compared to those of wildtype vaccine strain of HAV, genomic RNA analysis using RACE showed that the generated HAV have HAV-specific RNA as genome. In the immunogenicity study using mouse, the generated HAV induced HAV-specific IgG and the titer was comparable to wild-type vaccine strain of HAV.

Conclusions

We established the reverse genetic system to produce vaccine strain of HAV and confirmed that the generated HAV can be used as vaccine strain.
Chlamydia recombinant MOMP encapsulated in PLGA 85/15 nanoparticles admixed with Escherichia coli dmLT mucosal adjuvant enhanced protection against a genital challenge in subcutaneously immunized BALB/c mice
Authors: Sahu Rajnish , Joshi Sameer , Norton Elizabeth B. , Singh Shree R. , Dennis Vida A.
Published: January 2023

There is an urgent need to develop a vaccine against Chlamydia trachomatis, the most reported bacterial-sexually transmitted infection globally. We developed a Chlamydia nanovaccine by employing PLGA [poly (D, L-lactic-co-glycolic acid) 85/15 nanoparticles encapsulated with recombinant MOMP (major outer membrane protein) of Chlamydia](named PLGA-rMOMP) that partially protected mice against a genital challenge. Here we hypothesized that admixing PLGA-rMOMP with the Escherichia coli dmLT (double-mutant heat-labile enterotoxin (LT)-R192G/L211A) mucosal adjuvant will improve the protective efficacy. Six groups of BALB/c mice were subcutaneously immunized three-times at two-week intervals with various immunogens and then challenged intravaginally with 105 inclusions forming units (IFUs) of C. muridarum. Vaginal swabs were collected every three days up to three weeks from all mice and analyzed by indirect immunofluorescence to compare the IFUs between groups of mice. Our results showed maximal protection provided in mice immunized with PLGA-rMOMP admixed with dmLT evidently by their lower IFUs as compared with the other mice groups. Moreover, the PLGA-rMOMP adjuvanted mice produced elevated MOMP-specific serum IgG2b (Th1) as compared to IgG1 (Th2) antibodies, which is indicative of an effective Th1 immune response. Interestingly splenic T-cells from these mice also produced enhanced IL-17 and IL-2 MOMP-specific cytokines after the genital challenge in comparison to the other groups of mice. Our results indicate that the E. coli dmLT mucosal adjuvant improves the immunogenicity and the protective efficacy of the PLGA-rMOMP nanovaccine against a genital challenge in mice.
The effective immunization of plasmid DNA vaccine targeted for smallpox virus
Authors: Son Wonrak , Kim Nayoung , Lee Minhoon , Sim Euni , Song Donghyun , Yu Chiho , Song YoungJo , Hur GyeungHaeng , Jeong SeongTae , Hong Sungyoul , Shin YoungKee , Shin Sungho , Choi JunYoung
Published: January 2023

Smallpox, caused by variola virus, could be a bio-terrorism agent although eradicated from the WHO program in the 1980s. DNA vaccine is a third generation vaccine system which contains DNA sequence with specific antigen codes that activates the immune system. DNA vaccines show great potential in inducing immune response with strong triggered-humoral, and cellular immunity. DNA vaccines have emerged as an attractive approach for infectious disease because of rapid manufacture, fast adaptation to newly emerging pathogens and high stability at ambient temperatures. However, since the potency of naked DNA vaccines is limited to immune efficacy, adjuvant incorporation could be a good option to increase the immune response. Several different compound-composed adjuvants are developed to help DNA vaccine efficacy. In other aspects, for inducing a more effective immune response, global vaccine manufacturer focused on targeting dendritic cell (DC) through the DC-specific surface protein, DEC-205. In this study, with plasmid construction modification, we developed a DNA vaccine vector that have effective antigen expression and confirmed immunogenicity against smallpox. For enhancing in vivo immune responses, we screened various adjuvant strategies and observed effective DNA vaccination response with various adjuvant studies and time-pointed reaction. Also, we tested the enhancement of immune response using DNA vaccine containing the smallpox antigen fused with DEC-205 and revealed a th1–th2 cellular immune response. Our results propose how to regulate modified-DNA immunization efficacy within DC targeting stimulation and adjuvant strategy against DNA vaccine development.
Understanding how immune maturity shapes responses to cancer therapy
Authors: Serrano Daniel , Tzelepis Fanny , Chen Andrew , Diallo Jean-Simon
Published: January 2023

Clinical translation of cancer therapies from mice to human remains a constant challenge. Cancer immunotherapy aims develop a sustained long-term adaptive immune response to tumors. This raises the question of whether the typical pathogen-free animal models reflect the human immunological context and cancer pathology. Recent research suggests that exposure of pathogen-free mice to pet shop mice leads to their immunological maturation. We hypothesize that such a model may be more reflective of a human adult context. Specific-pathogen-free (SPF) Balb/c mice were co-housed with pet shop mice for 40 days to generate the “dirty mouse” model. Dirty mice were challenged with Listeria m. infection to test immune maturity. In parallel, CT26LacZ, CT26WT, EMT6 and K7M2 tumor cell lines were used to measure tumor growth in dirty mice. In particular, CT26LacZ tumor model was treated with oncolytic virus to assess the impact of immune maturity over efficacy of therapy. Complementary comparative experiments were performed in SPF mice. Dirty mouse immune response to Listeria challenge was more robust than in clean mice. Immune phenotyping revealed a different distribution of immune populations. A matured immune system impacted the growth of tumors to varying extents, with some models experiencing complete tumor rejection. Finally, oncolytic virus in dirty mice implanted with CT26-LacZ showed an improved efficacy on survival and tumor burden. Immune response to infections and tumor models between SPF mice and dirty mice reveals the potential impact of a mature immune system on immune-therapeutic responses. Therefore, establishment of model that better recreates the state of immune maturity may result in higher chances of translational impact.
Development of a prophylactic vaccine for lung squamous cell carcinoma
Authors: Riolobos Laura , Gad Ekram , Treuting Piper M. , Timms Andrew , Disis Mary Lenora
Published: January 2023

High-grade bronchial dysplasia is a marker for high risk of lung squamous cell carcinoma (SCC). Cancer vaccines targeting dysplasia could prevent the progression to SCC and decrease lung cancer incidence in population at risk. In order to develop a vaccine to prevent lung SCC we need to identify antigens and epitopes within them able to elicit a potent Type I anti-tumor immune response. One caveat to develop a prophylactic vaccine targeting dysplasia is that driver mutations (neo-antigens) are not known. Many mutations appear late in lung cancer and are not shared between patients. However, upregulated non-mutated proteins are needed from the early stages of cancer to support the new proliferation requirements of the cells, are common between patients and are good candidates for a preventive vaccine. Type I CD4+ T cells (Th1) secreting interferon-gamma (IFN-g) are necessary for enhanced function of antigen presenting cells, epitope spreading and activation of cytotoxic CD8+ T cells. However, non-mutated tumor self-antigens frequently induce Type II CD4+ T cells (Th2), which secrete cytokines that inhibit the function of Th1 and CD8+ T cells. To develop a prophylactic lung cancer vaccine we have: (1) searched in public datasets genes that are upregulated in bronchial dysplasia and maintain upregulation in SCC; (2) used web based algorithms for class II epitope prediction followed by functional ELISPOT screening to identified epitopes eliciting IFN-g (Th1) and/or IL-10 (Th2) responses; (3) selected only the Th1 epitopes (excluding the Th2 epitopes) to evaluated efficacy of the vaccines in a chemically induced rodent model of SCC. Preliminary data shows that we can reduce dysplasia frequency in vaccinated mice treated with the carcinogen.
Loss of the Class II Accessory Molecule HLA-DO leads to presentation of lower abundance of DM-resistant epitopes
Authors: Welsh Robin A , Song Nelson , Foss Catherine , Sadegh-Nasseri Scheherazade
Published: January 2023

HLA-DO (DO in human; H2-O in mice) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. Although DO forms a stable complex with HLA-DM, its biological function remains poorly understood. Previously, our lab provided biochemical evidence in support of a model where DO enhances the binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 (DR1) molecules in vitro. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both the thymic deletion, and peripheral activation of CD4 T cells. Here, using in vivo antigen presentation in an autoimmune as well as infectious model, we found that DO-KO mice present a lower abundance of MOG(35-55)/I-Ab and H5N1-HA(259-274)/DR1 complexes by B cells. Using 2D2 Tg T cells specific for MOG(35-55)/I-Ab as a readout system, we observed a less effective T cell activation upon co-culture of brain recovered APCs from diseased DO-KO mice. When we vaccinated DR1+DO-KO and DR1+DO-WT mice with flu vaccine, we found that HA(259-274)/DR1 specific CD4 T cells had a less avid interaction with B cells presenting HA(259-274)/DR1 from DR1+DO-KO as compared to B cells from vaccinated DR1+DO-WT mice as measured by a novel avidity measurement tool, z-MoviTM.
Identification and characterization of adipose tissue-derived human antibodies with “anti-self” specificity
Authors: Frasca Daniela , Diaz Alain , Romero Maria , Garcia Denisse , Jayram Diya , Thaller Seth , Blomberg Bonnie
Published: January 2023

We have previously shown that the human obese adipose tissue (AT) contributes to increased secretion of adipocyte-specific IgG antibodies in individuals with obesity. We have also shown that this occurs without any exogenous stimulation, because the ongoing process of cell death in the obese AT leads to the release of “self” antigens able to induce chronic stimulation of B cells. Here, we confirm and extend our initial observation on a different cohort of individuals, and we show that also the plasma of obese individuals is enriched in IgG antibodies with specificities for adipocyte-derived antigens. Adipocyte-specific IgG secreted in the obese AT are significantly correlated with those present in plasma. Using immunoprecipitation and mass spectrometry, we have identified these antigenic specificities. The antigens are almost exclusively intracellular or cell-associated, usually not “self” antigens, but they are released by cells dying in the AT. These antigens will be used in protein arrays to screen plasma from individuals with obesity and also with autoimmune diseases. We have indeed preliminary evidence that the plasma of Rheumatoid Arthritis patients is enriched in adipocyte-specific IgG antibodies, suggesting that obesity may contribute to pathogenicity in autoimmune patients. Finally, we show for the first time that not only macrophages but also adipocytes in the obese AT are efficient antigen-presenting cells and stimulate the secretion of IgG autoimmune antibodies. They do so because they express the antigen-presenting molecules CD1d and, to a lesser extent, MHC class II, as well as the co-stimulatory molecule CD86. These results may lead to the development of novel therapeutic strategies to controlautoimmunity.
Insulin-specific regulatory T cell receptor alpha clonotypes restricted to HLA-DQ6 (DQB1*06:02) are shared between blood and islets
Authors: Mitchell Angela M , Alkanani Aimon K , McDaniel Kristen A , Landry Laurie G , Nakayama Maki , Michels Aaron W
Published: January 2023

The major genetic determinant in susceptibility or protection from many autoimmune diseases resides in the human leukocyte antigen (HLA) region. Specific class II alleles (e.g., HLA-DQ8) increase the risk for developing type 1 diabetes (T1D), a prototypical organ specific autoimmune disease, whereas HLA-DQ6 leads to dominant protection. We hypothesize that DQ6 is protective via its ability to present insulin peptides to regulatory CD4+ T cells (Tregs), resulting in anti-inflammatory responses.

We expanded insulin-specific Tregs from peripheral blood mononuclear cells (PBMCs) of DQ6+ non-diabetic individuals using an insulin peptide. Insulin-expanded Tregs were flow sorted, and paired single-cell TCR/RNA-seq (10X Genomics) was performed.

Of 2,096 insulin-expanded Tregs from three DQ6+ individuals, 883 αβTCRs (42%) were present ≥2 times, and we confirmed the antigen specificity of dominant clonotypes. The single-cell RNA-seq data showed 4 clusters of Treg cells based on the expression levels of FOXP3 and IL-2RA. Additionally, we flow-sorted CD4+ T cells from the pancreatic islets of a non-diabetic DQ6+ organ donor and performed single-cell TCR sequencing. Of the 67 CD4+ cells, TCR clonotypes from three cells possessed identical TCRα chains (including CDR3 sequences) with Tregs sequenced from the PBMCs of the separate DQ6+ non-diabetic individuals.

These data indicate the potential presence of insulin-specific Tregs within the pancreatic islets of non-diabetic organ donors. Our findings provide a mechanistic basis for understanding HLA-linked protection from autoimmune diabetes development and have implications for manipulating self-antigen-specific T cell responses to modify the disease course in T1D.
High-salt modulates cellular metabolism of human regulatory T cells
Authors: Kleinewietfeld Markus , Côrte-Real Beatriz F , Hamad Ibrahim
Published: January 2023

High-salt intake has been linked with shifts in the immune cell balance, mainly by promoting proliferation and activity of pro-inflammatory cells, such as T helper 17 (Th17) and M1 macrophages, and by impairing the functions of anti-inflammatory cells such as regulatory T cells (Tregs) and M2 macrophages. However, the precise molecular mechanisms that contribute to this phenomenon are still unknown. The role of metabolic regulation in shaping immune responses has gained increasing attention in recent years and can be greatly influenced by environmental factors such as diet. High-salt intake was previously shown to promote metabolic changes in M2 macrophages by decreasing their mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis necessary for their activation. So we hypothesize that this phenomenon could likely be extrapolated to other cell types such as Tregs. We analyzed changes on the metabolic pathways of human Tregs after high-salt exposure. Our results show significant salt-induced changes in the cellular metabolism of Tregs and since these changes are known to alter suppressive function both in vitro and in vivo, we further hypothesize that the observed metabolic alterations might be linked to the loss of suppressive function seen in human Tregs upon high-salt challenge. Thus, the interference with these pathways may have the potential for targeting Tregs in salt-sensitive diseases.
Dietary tryptophan alters CD4+ T cell function in the lupus-prone B6. Sle1.Sle2.Sle3 (TC) mouse model
Authors: Brown Josephine Michelle , Choi Seung-Chul , Morel Laurence
Published: January 2023

Tryptophan (Trp) metabolism is altered in lupus patients with higher production of kynurenine (Kyn). We show that TC mice have the same alterations. To ascertain whether Trp plays a role in autoimmune pathogenesis, TC and B6 control mice were fed with either high or low Trp. High Trp exacerbated disease in TC mice while low Trp was protective. Expression of IDO1, the enzyme producing Kyn from Trp, was similar between TC and B6 mice, and its inhibition by 1MT did not change disease outcomes with and without dietary Trp variations. To identify the mechanism by which Trp plays a role in this process, we analyzed CD4 T cell phenotypes. Regulatory T cell (Treg) functional markers were altered by dietary Trp. Further, Tregs from TC mice consuming high Trp lost their suppressive capacity in vitro, whereas Tregs from TC mice on low Trp were as suppressive as that of B6 controls. Additionally, increasing Trp concentrations affected B6 and TC T cell polarization in vitro differently. A high Trp concentration increased the percentage of IFNg+ and FoxP3+ CD4+ T cells from B6 and TC mice under Th1 and Treg polarizing conditions, respectively. On the other hand, B6 Treg polarization increased with a higher Kyn concentration while TC Treg polarization decreased. These findings suggest that Trp and some of its metabolites modulate lupus pathogenesis in TC mice potentially by altering T cell phenotypes. Thus, these data emphasize a novel pro-inflammatory role for Trp metabolism in lupus.

Supported by a grant from the NIH (RO1 AI143313) to LM.
NLRP3 inflammasome activation profile in CD4+ T and CD19+ B lymphocytes from HIV-infected patients
Authors: Leal Vinícius Nunes Cordeiro , Reis Edione Cristina , Fernandes Fernanda Pereira , Pontillo Alessandra
Published: January 2023

Background

HIV-1 triggers inflammasome activation in myeloid cells, through NLRP3, which contributes to the first line defense against infection. However, it was postulated that NLRP3 activation could be involved in chronic inflammation and immune exhaustion in HIV-infected patients. But whether its activation occurs only in myeloid cells or also in lymphocytes is not yet understood. Considering that NLRP3 inflammasome was recently described in CD4+ T and CD19+ B lymphocytes, we hypothesize that NLRP3 inflammasome could be activated also in lymphoid cells during chronic HIV infection.

Aim

To evaluate NLRP3 activation in peripheral blood lymphocytes from HIV-infected patients.

Methods

NLRP3 inflammasome activation was evaluated in Antirretroviral-treated HIV-infected patients and healthy donors (HD) peripheral blood lymphocytes by the meaning of caspase-1 cleavage, IL-1β and IL-18 production and NLRP3+ASC+ “specks” formation.

Results

CD19+ B lymphocytes present a constitutive activation of NLRP3 inflammasome. But, differently from myeloid cells, LPS and β-glucan induced a higher NLRP3+ASC+ “specks” formation, caspase-1 activation and IL-1β production in B lymphocytes from HIV-infected patients compared to HD, as well as CpG induced a higher IgM production in a NLRP3-dependent way. Differently from CD19+ B lymphocytes, CD4+ T lymphocytes from HIV-infected patients present a NLRP3-independent activation of caspase-1 and IL-1β production, which was amplified by αCD3-αCD28 stimulation.

Conclusions

Our findings suggest that inflammasome is more activated in lymphocytes from HIV-infected patients in a cell and receptor specific manner, which could contribute to disease outcome during chronic infection.
Autocrine prostaglandin feedback determines the kinetics of bacterial type I interferon induction by restricting endosomal TLR4/TRIF complex assembly
Authors: Perkins Darren , Richard Katharina , Hansen anne-marie , Koller Beverly , Vogel Stefanie
Published: January 2023

TLR4 is unique in its capacity to initiate two qualitatively distinct transcriptional programs from two cellular locations. Cell surface TLR4/MyD88 complexes drive pro-inflammatory cytokine transcription through NF-κB, while endosomal TLR4/TRIF complexes activate IRF3 and a type I interferon response. Feedback circuits to limit signals from both locations are necessary to balance the inflammatory response to Gram negative bacteria, yet how endosomal TLR4/TRIF signals are terminated has remained an open question. We find that activation of TLR4 triggers rapid prostaglandin PGE2 production, necessary to limit the kinetics of endosomal TRIF, but not MyD88, signaling. Mechanistically, autocrine PGE2 activates the specific EP4 receptor to inhibit small GTPase mediated TLR4 trafficking to endosomes and to accelerate autophagic degradation of active TLR4/TRIF complexes. Inhibition of EP4 receptor function does not impair cell surface MyD88 signals, but leads to hyper TRIF dependent type I interferon production in response to bacterial LPS or to macrophage infection by live Gram negative pathogens.
NOD2 suppresses chronic intestinal inflammation through dampening TLR4 signaling
Authors: Khan Shahanshah , Zaki Hasan
Published: January 2023

The cytosolic NOD-like receptor (NLR) family protein NOD2 activates NF-kB and MAPK pathways in response to MDP. 15–20% of inflammatory bowel disease (IBD) patients carry mutations in NOD2. However, the mechanism of NOD2-mediated suppression of colitis is poorly understood. We recently showed that Nod2−/− mice are highly susceptible to chemical-induced acute colitis. Further, we observed that NOD2 suppresses NF-kB and MAPK pathways in LPS stimulated macrophages. However, it is not known whether such as a crosstalk between NOD2 and TLR4, sensor for LPS, occurs during chronic inflammation. We, therefore, primed wild-type and Nod2−/− macrophages with LPS or other TLR ligands overnight and then re-stimulated with LPS. Interestingly, we observed increased activation of NF-kB and MAPK pathways as well as higher expression of inflammatory molecules in primed Nod2−/− macrophages compared to wild-type. Increased inflammatory response in Nod2−/− macrophages was accompanied by reduced expression of IRF4, a negative regulator of NF-kB and MAPK pathways. To observe in vivo relevance of NOD2-mediated suppression of inflammatory responses, we crossed Nod2−/− mice with Il10−/− mice and induce colitis in them with piroxicam fortified diet. Mice double deficient in Nod2 and Il10 showed increased body weight loss and histopathological changes compared to Il10−/− mice. Higher colitis susceptibility of Nod2−/−Il10−/− mice was associated with increased activation of NF-kB and MAPK pathways and reduced expression of IRF4. These data, therefore, suggest that NOD2-mediated protection against colitis involves suppression of TLR4 downstream inflammatory signaling pathways via induction of IRF4.
Mitochondrial Calcium Signaling Facilitates Bacterial Survival by Restraining LC3-associated Phagocytosis
Authors: Wen Haitao , Li Tianliang , Li Xinghui , Lei Yu , Green Douglas R
Published: January 2023

Mitochondria in eukaryotic cells are believed to have originated from proteobacteria through endosymbiosis 2.5 billion years ago. Accumulating evidence suggests essential roles of mitochondria in host defense response against invading pathogens. Whether intracellular bacteria can hijack mitochondria to promote their survival remains elusive. Here, we demonstrate a previously unappreciated pro-survival strategy employed by intracellular bacteria Listeria monocytogenesthat involves the suppression of LC3-associated phagocytosis (LAP) by mitochondrial Ca2+ signaling. Invasion of macrophages by L. monocytogenes caused a robust mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU), leading to elevated acetyl-coenzyme A (acetyl-CoA) production via the pyruvate dehydrogenase (PDH). Acetylation of LAP effector Rubicon with the use of acetyl-CoA antagonized LAP formation. Genetic ablation of Mcuimproved bacterial killing due to elevated LAP formation. Our study indicates that modulation of mitochondrial Ca2+ signaling is a beneficial strategy for bacterial survival and highlights the importance of mitochondrial metabolism in host-microbial interaction.
Mechanistic investigations of Granzyme A-mediated TB protective effects
Authors: Rasi Valerio , Xia Mei , Wood David , Eickhoff Christopher , Hoft Daniel F
Published: January 2023

One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA when released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen. To elucidate the mechanism(s) involved in this inhibition, the lab has purified human GzmA. We have proven that Granzyme A’s intact active site is necessary to inhibit the intracellular replication of mycobacteria. The substitution of serine to alanine in the active site rendered the protein unable to control the mycobacterial infection as measured by mycobacterial growth inhibition assays, and the measurement of the pro-inflammatory marker TNF. Conversely, the effects of highly pure and active GzmA on mycobacterial replication inside human macrophages have been investigated using systems biology. A 2D-DIGE proteomic approach using adherent human monocytes has been performed to identify the pathways stimulated by GzmA involved in control of mycobacterial replication. Our analysis indicates that ER-stress responses (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Modulation of key proteins in these pathways are currently being targeted by pharmaceutical intervention and gene alteration to test the effects on GzmA-mediated inhibitory effects. Confirmation that these pathways are involved in pathogen clearance will lead to the development of novel host-directed therapies for control of Mtb infection.
A naturally-occurring mutation in IL-17F reveals a protective role for the IL-17AF heterodimer in oropharyngeal candidiasis (OPC)
Authors: Zhou Chunsheng , Aggor Felix E.Y. , Monin Leticia A , Gordon Rachael A , Edwards Tara N , Kaplan Daniel H , Shlomchik Mark J , Gingras Sebastien , Gaffen Sarah L
Published: January 2023

IL-17A is the original member of the IL-17 family of cytokines. Among the IL-17 family, IL-17F shares the most homology with IL-17A at the amino acid level. IL-17A and IL-17F exist as homodimers and also form a heterodimer (IL-17AF). All of these cytokine dimers signal through the same IL-17RA:IL-17RC receptor complex, but the ligands exhibit different signaling strengths (IL-17A > IL-17AF > IL-17F). We previously showed that IL-17 signaling is critical for immunity against oropharyngeal candidiasis (OPC), an opportunistic infection of the oral mucosa caused by the commensal fungus C. albicans. Mice lacking IL-17RA, IL-17RC, or the adaptor ACT1 all have higher oral fungal burdens than wild type (WT) following oral infection with C. albicans. IL-17A deficient mice are also mildly susceptible to C. albicans oral infection, but blockade of IL-17F does not cause disease susceptibility. Furthermore, double blockade of IL-17A and IL-17F during OPC reveals a cooperative antifungal activity of IL-17A and IL-17F. However, the role of the IL-17AF heterodimer still remains poorly understood. Here, we took advantage of a dominant-negative mutation (IL-17F.S65L) that was previously identified in chronic mucocutaneous candidiasis disease (CMCD) patients. This mutation blocks the signals of IL-17F and IL-17AF but not IL-17A. Using CRISPR/Cas9 technology, we created mice with the analogous IL-17F S65L mutation. These IL-17FS65L/S65L mice showed a similar degree of susceptibility as IL-17A−/− mice though less than IL-17RA−/− mice upon C. albicans oral infection. This result suggests that IL-17AF contributes to protection against OPC.
Serine phosphorylation of CREB-binding protein (CBP) is critical to intestinal homeostasis but not CD4 T cell differentiation
Authors: Miaw Shi-Chuen , Lin Yi-Ting , Hsu Yu-Hua , Liu Chi , Shun Chia-Tung , Chen Ching-Chow
Published: January 2023

CREB-binding protein (CBP) is a transcriptional coactivator involved in the regulation of DNA accessibility to transcription factors. Phosphorylation of human CBP by IKKα at Ser1382/1386 plays a critical role in the regulation of cell fate by suppressing p53-mediated gene expression. Therefore, we evaluated whether impaired serine phosphorylation of CBP is involved in the pathogenesis of disease. We generated mutated serine 1383/1387 to alanine knock-in mice (AA mice) to study whether they exhibit any phenotype alteration. AA mice showed an IBD-like phenotype with softer, looser and hemoccult stools and shorter colon length compared to the wild type mice. Increased p53 accompanied with apoptosis in colonic tissues of AA mice was demonstrated by IHC and TUNEL staining. In addition, male AA mice were more vulnerable to DSS-induced colitis with severe crypt loss and epithelial disruption. Elevated immune cells in mesenteric lymph nodes are more prominent in AA mice with infiltration of neutrophil and macrophage. However, the differentiation of CD4+ T cells was not affected with Cbp mutation (AA) ex vivo. Taken together, deficiency of NF-kB signaling in AA mice did not affect immune cells but modulated apoptosis of IECs. Our findings suggested that serine phosphorylation of CBP by IKKα might be a critical regulator to maintain intestinal homeostasis.
Transcriptomic approaches elucidate novel aspects of the host immune response to candidemia
Authors: Steinbrink Julie Marie , Myers Rachel , Johnson Melissa D. , Alexander Barbara D. , McClain Micah T.
Published: January 2023

Objective

Examination of the human transcriptome in circulating leukocytes during invasive candidiasis can provide an innovative approach to defining immunologic mechanisms driving the host response to this deadly infection.

Methods

RNA sequencing was performed on peripheral blood samples from 50 hospitalized patients with blood culture positive invasive candidiasis, along with 30 control subjects. Generalized linear hypothesis testing and weighted gene co-expression network analysis (WGCNA) were used to define biological networks contributing to the host response to Candida infection.

Results

1,748 genes were differentially upregulated and 2,149 genes downregulated in the presence of candidemia compared to matched controls. These genes reflected many canonical antifungal immune response pathways including phagocytosis, neutrophil activation, type I interferon responses, and T cell signaling. However, the data also demonstrated activation of some less well described antifungal immune mechanisms, including B cell activation, upregulation of humoral immune response pathways, and a profound alteration in the host regulation of heme synthesis and iron metabolism. While iron is known to be critical for fungal pathogens such as Candida in vitro, this analysis offers evidence that the human host manipulates this system as a part of the overall response to fungal infection.

Conclusion

Analysis of the transcriptome in circulating white blood cells of infected human subjects offers unique and novel insight into the complex immunologic response to invasive candidiasis.
Vaccine elicited ASGM1+ CD8+ T cells are essential for protective immunity against Toxoplasma gondii infection
Authors: Gigley Jason P. , Ivanova Daria L , Denton Stephen
Published: January 2023

The importance of enriched asialylated sphingolipid GM1 (ASGM1) in CD8+ T cell dependent protective immunity against T. gondii infection is unknown. Using a vaccine challenge model of T. gondii infection, we observed treatment of animals with anti-ASGM1 resulted in 100% death and greater parasite burden at the site of infection. Upon further dissection of this phenotype, we found that anti-ASGM1 treatment depleted not only NK cells but also T cells in vaccinated mice. While both CD8+ and CD4+ T cells were affected, CD8+ T cells were more susceptible to the anti-ASGM1 treatment. ASGM1 on the surface of the T cells was expressed on a higher percentage of CD8+ T cells than CD4+ T cells. ASGM1 was highly enriched on the surface of memory CD8+ T cells (Tem and Tcm). However, Tem were more susceptible to anti-ASGM1 treatment. Anti-ASGM1 treatment during rechallenge resulted in greater depletion of activated CD8+ IFNγ+, Granzyme B+, Tem and Tef than Tcm cells. In addition, anti-ASGM1 depleted IFNγ+ CD4+ T cells. Recombinant IFNγ supplementation prolonged the survival of anti-ASGM1 treated mice, demonstrating that this antibody eliminated the protective T. gondii specific IFNγ-producing T cells important for control of the parasite. This study demonstrates that ASGM1 is enriched on effector T cells during T. gondii infection and the potential importance of changes in sialylation in lipid rafts for T cell activation during T. gondii infection.
Immunity to Invasive Infection by Intestinal Commensal Microbes
Authors: Shao Tzu-Yu , Ang W.X. Gladys , Huang Felicia Scaggs , Haslam David , Way Sing Sing
Published: January 2023

Commensal microbes that colonize the intestine are an important cause of invasive infection. However, how colonization of individuals impacts their susceptibility to invasive infection remains undefined. This knowledge gap reflects technical roadblocks in models of long-term commensal colonization by microbes that can also cause invasive infection, and conceptual appreciation for systemic antigen-specific immune modulation primed by commensals. To address this knowledge gap, a model of intestinal colonization with the human commensal and pathogenic fungi, Candida albicans was developed. We show C. albicans intestinal colonization protects against subsequent invasive C. albicans infection. These benefits require tonic stimulation since protection is eliminated in mice eradicated of commensal fungi. Protection against invasive infection parallels the systemic expansion of C. albicans-specific RORγt+ Th17 cells and enhanced IL-17 responsiveness by circulating neutrophils. Reciprocally, the protective benefits of intestinal colonization are overturned by depletion of CD4+ T cells or Ly6G+ neutrophils, or IL-17 neutralization. These results highlight a new facet in the symbiotic relationship between host and microbe in mucosal tissues
Streptococcus pneumoniae coinfection dictates susceptibility to oral Listeria monocytogenes through suppressive Natural Killer (NK) cell activity
Authors: Bortell Nikki , Lenz Laurel L
Published: January 2023

Coinfection with viral and bacterial pathogens is a major health concern and results in significant morbidity and mortality. Recent research has aimed to understand the complex interplay between multiple pathogens and the immune response during viral:bacterial coinfections. However, studies using bacterial-bacterial coinfections are lacking. Streptococcus pneumoniae (Spn) and Listeria monocytogenes (Lm) are both bacterial pathogens that are capable of causing severe infections in young, elderly, and immunocompromised populations. Here we show that coinfection with intratracheal (IT) Spn increases susceptibility of typically resistant mice to oral Lm infection. On day 3 post infection (p.i.), the presence of Spn increased oral Lm burdens in the spleen, liver, and lungs of coinfected C57/BL6 mice. In addition, while the anti-inflammatory cytokine Interleukin-10 (IL-10) is limited during oral Lm infection, Spn infection induced the production of IL-10 from Natural Killer (NK) cells, and that this correlated with an increase in Lm burdens during coinfection. To assess the importance of NK cell IL-10 in the absence of an ongoing infection, mice were inoculated with a recombinant Lm polypeptide, designated L1S, which stimulates NK cell IL-10 production. In the presence of L1S, the induction of NK cell IL-10 similarly increased systemic burdens of oral Lm. Lastly, BMMs stimulated with rIL-10 and infected with Lm have increased bacterial burdens when compared to untreated but infected controls. Overall, our data show that coinfection impairs host protection through the induction of NK cell production of IL-10 and dictates susceptibility to oral Lm infection.
Male microbiota-associated metabolites restore macrophage apoptotic cell clearance function in female lupus-prone mice
Authors: Harder James W , Ma Jing , Alard Pascale , Zhang Xiang , Yuan Fang , Kosiewicz Michele M
Published: January 2023

Systemic lupus erythematosus (SLE) has been linked to microbiota dysbiosis. We have previously shown that lupus-prone female and non-lupus-prone male BWF1 mice have significantly different gut microbiota compositions and metabolomic profiles. Transferring microbiota from male into female BWF1 mice suppresses disease. This protective phenotype is androgen dependent, as microbiota from castrated male BWF1 mice (i.e., lupus-prone) fails to protect against disease. This protection could be mediated via differential metabolite production. Metabolomic analyses of female, and castrated and intact male BWF1 feces identified >100 differentially produced metabolites including phytol and phytanic acid, byproducts of chlorophyll degradation that can act as RXR agonists. These metabolites were produced at higher levels in non-lupus-prone male mice than in lupus-prone female and castrated male mice. Their presence in male BWF1 mice correlated with higher levels of a bacterial family, Lachnospiraceae, which contains species that express chlorophyll-degrading enzymes. Feeding phytanic acid to female mice delayed kidney disease onset. This male microbiota metabolite may confer protection, in part, by improving splenic macrophage phagocytosis of apoptotic cells. Macrophage clearance of apoptotic debris (efferocytosis) is defective in SLE patients, and this is thought to drive disease progression. We found lupus-prone female and castrated male BWF1 mice also have defective macrophage efferocytosis and this defect could be corrected in vitro by phytanic acid treatment. Taken together, our data indicate that BWF1 male microbiota produce metabolites that enhance macrophage efferocytosis and protect against disease.