bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement activation by C-reactive protein is essential for protection against pneumococcal infection
Authors: Singh Sanjay Kumar , Ngwa Donald N , Agrawal Alok
Published: January 2023

C-reactive protein (CRP), a component of the innate immune system, is an antimicrobial plasma protein. The primary ligand-binding specificity of CRP is for phosphocholine-containing substances and ligand-complexed human CRP activates the complement system in both human and mouse sera. It has been shown that human CRP protects mice against infection with lethal doses of Streptococcus pneumoniae; the protection is due to CRP-dependent decrease in bacteremia and increase in survival time. In this study, we tested a decades old hypothesis that the complement-activating function of ligand-complexed CRP contributes to protection of mice against pneumococcal infection. We employed site-directed mutagenesis of CRP, guided by its three-dimensional structure, and identified a mutant H38R which, unlike wild-type CRP, did not activate C3 in both human and mouse sera. The calcium-dependent binding to phosphocholine on pneumococci, the half-life in mouse circulation, and the overall pentameric structure of H38R mutant CRP were similar to that of wild-type CRP. Employing a murine model of pneumococcal infection, we found that passively administered H38R mutant CRP did not protect mice from infection. Infected mice injected with H38R mutant CRP showed no reduction in bacteremia and did not survive longer, as opposed to infected mice receiving wild-type CRP. Thus, the hypothesis that complement activation by ligand-complexed CRP is an antimicrobial effector function is true. We conclude that complement activation by ligand-complexed CRP is essential for CRP-mediated protection against pneumococcal infection. (NIH AI117730)
Staphylococcus aureus biofilm-derived lactate inhibits HDAC11 to augment leukocyte IL-10 production and promote infection persistence
Authors: Heim Cortney , Bosch Megan E , Yamada Kelsey J , Aldrich Amy L , Chaudhari Sujata S , Klinkebiel David , Gries Casey M , Alqarzaee Abdulelah A , Li Yixuan , Seto Edward , Karpf Adam R , Kielian Tammy
Published: January 2023

Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infections (PJIs), where leukocyte IL-10 production is critical for biofilm persistence. We screened a S. aureus mutant library that identified the importance of lactate biosynthetic pathways to elicit IL-10 production. A S. aureus Δddh/ldh1/ldh2 mutant, defective in both D- and L-lactate production, led to significantly decreased leukocyte IL-10 expression and re-programmed biofilm-associated monocytes to a pro-inflammatory state, resulting in reduced biofilm burden. Histone acetylation plays a central role in regulating gene expression, and we found that histone deacetylase (HDAC) activity was increased in Δddh/ldh1/ldh2-infected mice compared to WT concomitant with significant decreases in global histone 3 (H3) acetylation and H3 acetylation at the IL-10 promoter, demonstrating that S. aureus-derived lactate is a HDAC inhibitor. Lactate was found to inhibit HDAC11, which resulted in unregulated HDAC6 activity and increased IL-10 production. MDSCs and macrophages produced significantly less IL-10 in the presence of the HDAC6 inhibitor tubastatin A during co-culture with WT but not Δddh/ldh1/ldh2 biofilms, while HDAC11 KO MDSCs and macrophages co-cultured with Δddh/ldh1/ldh2 biofilms produced significantly more IL-10 compared to WT cells due to unchecked HDAC6 action. D-lactate was elevated in synovial fluid of patients with PJI compared to aseptic controls and lactate also promoted IL-10 production by human monocyte-derived macrophages. Collectively, S. aureus lactate-mediated inhibition of HDAC11 plays a critical role during biofilm infection, leading to epigenetic changes that reprogram the host immune response.
Suppression of caspase-11-mediated cytosolic LPS sensing pathway by Shiga toxin
Authors: Mendonça Morena , Ta Atri , Russo Ashley , Behl Bharat , Rathinam Vijay , Vanaja Sivapriya Kailasan
Published: January 2023

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS). Shiga toxin (Stx), the primary virulence factor of EHEC, mediates much of the morbidity and mortality associated with EHEC infection. Although Stx is well characterized in terms of its toxic activity, it is not clear if Stx has a specific role in immune cells during EHEC infection. In this study, we identified an inflammasome inhibitory role for Stx during EHEC infection. Stx specifically suppressed the activation of caspase-11-mediated cytosolic LPS sensing pathway in macrophages and mice. Stx did not inhibit the production of inflammasome-independent cytokines, TNF and IL-6. Similarly, Stx did not impair the activation of AIM2, NLRC4, or the canonical NLRP3 inflammasomes. Multiples subtypes of stx were able to suppress the caspase-11-mediated inflammasome responses. Stx was also capable of suppressing inflammasome responses induced by intraperitoneal injection of LPS as well as by Citrobacter rodentium infection in mice. Together, these data reveal that Stx plays an important role in overcoming inflammasome responses during EHEC infection.
Regulatory T cells integrate combinatorial cytokine signaling to promote immune homeostasis
Authors: Zhou Julie Y , Alvarez Carlos A , Cobb Brian A
Published: January 2023

The increasing prevalence of inflammatory and autoimmune diseases worldwide fuels a growing need to develop new therapeutics. Here we identify and adopt a strategy utilized by a common commensal microbe, Bacteroides fragilis, to induce immune homeostasis, by which its dominant antigen Polysaccharide A (PSA) drives classical MHCII:αβTCR engagement and elicits a strong IL-10 response via effector memory T cell (Tem) to regulatory T cell (Treg) communication. We recently discovered that IL-2 and IL-4 are critical factors driving PSA-initiated Tem->Treg communication. By tracking Tregs isolated from novel FoxP3RFP/IL-10GFP mice over time, we reveal here that these cytokines in combination elicit a profoundly divergent response from that of single cytokine stimulation. The Tregs resulting from combinatorial cytokine stimulation were more IL-10 expressing and proliferative, with IL-10+ Tregs expanding preferentially. We found that these Tregs were more effective in suppressing the proinflammatory behavior of conventional T cells, with changes in STAT5 and STAT6 signaling appearing to underlie this response. The robust effect of the combinatorial cytokines is further illustrated by their ability to suppress both HDM-induced asthma and experimental autoimmune encephalomyelitis (EAE), including clinical score, demyelination, MOG-specific conventional T cells, and production of pathogenic IL-17A in preventative, concomitant, and therapeutic delivery schedules. These data indicate that Tregs integrate multiple cytokine signaling to produce a biological outcome divergent from the two individual cytokines, and that this phenomenon holds therapeutic promise for multiple inflammatory and autoimmune diseases.
Sub-lethal Dose of Alcohol Modulates Innate Immune-Mediated Regulatory T-Cell Differentiation
Authors: Sharma Apoorv R , Venkatadri Rajkumar , Sabapathy Vikram , Mohammad Saleh , Sharma Rahul
Published: January 2023

The use of alcohol for consumption and other purposes is widely prevalent in our society. High concentration of alcohol (ethanol) in the household and clinical cleaners is employed for disinfecting. Deleterious effects of alcohol abuse in terms of death and dysfunction of cells, including immune-cells, has been extensively documented. However, moderate consumption of alcoholic beverages has been indicated to be beneficial. In this study, we sought to investigate the effects of sub-lethal doses of alcohol on the interaction of innate and adaptive immunity. We used murine bone marrow-derived macrophages (BMM) as antigen presenting cells and naïve T-cells isolated from spleens of C57BL/6 mice for in vitro T-helper differentiation assays. We found that doses of alcohol at 3% (v/v) or higher induces severe cell death, whereas lower concentrations of alcohol (<2% v/v) modulates the BMM activity for increased differentiation of T-regulatory cells (Tregs), which play a major role in preventing inflammation. Further, alcohol at lower doses also reversed the inhibitory effect of the endotoxin lipopolysaccharide (LPS) on Treg differentiation. Interestingly, BMM treated with alcohol also increased differentiation of TH17 cells. We also investigated the effects of alcohol-treated BMM on immune-modulation of T-helper differentiation in vivo. The data provide insights for immunomodulatory effects of low doses of alcohol and have implications for the regulation of Tregs.
Examining immunological tolerance through the lens of reproduction
Authors: Kinder Jeremy Michael , Way Sing Sing
Published: January 2023

Pregnancy is generally considered an “immunological conundrum” whereby a mother must circumvent immunological rules dictating binary “self” vs “non-self” antigen distinction to tolerate her genetically-foreign baby. However, considering reproductive success is the primary biological imperative for all species, it has likely served as an important driver of immune development across mammalian evolution. In other words, reproductive tolerance is not an exception to these rules but serves as a selective pressure continually refining our immune development. To this end, we have begun investigating the long-term impacts of the bi-directional exchange of intact maternal and fetal cells during pregnancy that persist systemically at low, microchimeric levels in mother and child decades after delivery. We believe these genetically-foreign microchimeric cells are not accidental souvenirs of pregnancy, but are instead intentionally preserved to promote tolerogenic memory to both fetal-paternal and non-inherited maternal alloantigens encountered during pregnancy. Thus, we have developed animal pregnancy models and cutting-edge tools that allow selective manipulation of maternal and fetal microchimeric cells together with dynamic tracking of antigen-specific immune responsiveness to these genetically-foreign cells during and after pregnancy. Viewing reproductive success as a strong driver of our immunological evolution, uncovering tolerance mechanisms employed during and after pregnancy will not only inform strategies for amelioration of pregnancy complications but applies in other clinical settings where the ability to regulate immune tolerance is desired (e.g. transplantation, cancer, persistent infection).
A Neural Circuit for the Suppression of Peripheral Inflammation by Hunger
Authors: Klima Michelle , Alhadeff Amber , Kruger Kayla , Pulido Santiago , McKnight Aaron , Betley J. Nicholas
Published: January 2023

Hunger is a primal biological drive that can initiate behavioral changes. Intuitively, hunger can inhibit behaviors such as sleep while simultaneously promoting foraging. Because pain can prevent an animal from seeking food we questioned if hunger could suppress pain. We found that food deprived mice reduce their response to inflammatory nociceptive stimuli. This reduction in nocifensive behavior during hunger is specific to inflammatory pain as the response to acute thermal and mechanical nociceptive stimuli remains intact. During hunger, agouti-related protein expressing (AgRP) neurons in the hypothalamus become active. AgRP neuron activation is both necessary and sufficient for feeding behavior and is thus a way to model hunger without the peripheral complications. Importantly, activating AgRP neurons recapitulates the analgesic effects of hunger.

Because food deprivation reduces inflammatory pain responses, we next explored the influence of hunger and AgRP neuron activity on peripheral inflammation. To address this question, we measured paws after an injection of Complete Freund’s Adjuvant (CFA) in food deprived mice. We found that food deprivation was able to reduce CFA-induced paw inflammation compared to ad libitum fed controls. To assess the role of the AgRP circuit on inflammation, we measured CFA injected paws during optogenetic stimulation of AgRP neurons. Optogenetic activation of AgRP neurons rapidly reduces paw inflammation. This rapid and robust reduction in peripheral inflammation following activity in a CNS circuit suggests that AgRP neuron activity interacts with the immune system. Current experiments are aimed to determine the CNS à immune pathway that quickly reduces peripheral inflammation.
Divergent roles of retinoic acid signaling in T regulatory cell function
Authors: Thangavelu Govindarajan , Andrejeva Gabriela , Loschi Michael , Aguilar Ethan G , Lee Yu-Chi , Furlan Scott N , McDonald-Hyman Cameron , Zaiken Michael C , Feser Colby J , Panoskaltsis-Mortari Angela , Kean Leslie S , Rathmell Jeffrey C , Chi Hongbo , Noelle Randolph J , Blazar Bruce R
Published: January 2023

Regulatory T cells (Tregs) modulate immune responses to maintain immune homeostasis. Retinoic acid (RA) signaling promotes Treg generation and stability. Paradoxically, we observed that dominant negative RA receptor α expressed only in donor T cells (DNRARαfl/flCD4Cre) dampened inflammation and graft-versus-host disease (GVHD), while increasing Tregs. We found that diminishing RA signaling significantly enhanced their in vitro suppressive capacity. In vivo the adoptive transfer of DNRARαfl/flCD4Cre Tregs at the time of transplant was significantly (p<0.01) more effective than wild type (WT) controls in ameliorating GVHD. Mechanistically, enhanced suppression was associated with elevated expression of suppressive (CTLA-4, GITR, CD39) and fitness (CD25, pSTAT5) markers. To evaluate the intrinsic requirement of RA signaling in Tregs, we generated transgenic mice with conditional DNRARα expression in Tregs (DNRARαfl/fl x Foxp3YFPCre). Surprisingly, the extent of ablation of RA signaling resulted in divergent phenotypes. Heterozygous ablation (DNRARαfl/wt) was permissive of enhanced Treg suppression. In striking contrast and unexpectedly, homozygous ablation (DNRARαfl/fl) impaired Treg suppressive function and caused multi-organ (lung, liver) autoimmunity in >90% of females and males by 3 months of age. Poor suppression is not cell intrinsic as DNRARαfl/fl Tregs isolated from mixed bone marrow chimeras of congenic WT and DNRARαfl/fl x Foxp3YFP Cre had enhanced function. Our studies indicate that RA signaling in Tregs can be targeted to prevent autoimmunity by enhancing Treg suppression or alternatively, promote antitumor responses by reducing Treg function.
Increased regulatory T cells suppress traumatic injury induced inflammation and control adaptive immune cell expansion in an experimental model of burn trauma
Authors: Cahill Laura , Guo Fei , Zhang Fan , Ng Julie , Griffith Alec , Howard Kaitlyn , Keegan Joshua , Lederer James
Published: January 2023

Trauma-induced inflammatory dysfunction is a lesser appreciated field of conventional immunology. In fact, traumatic injury is the leading cause of death in the US for people under 44 years old and resultantly disrupts immune homeostasis that puts injured patients at risk for developing opportunistic infections and succumbing to post-injury sepsis. Regulatory T cells (Tregs) are unique cells that contribute to immune balance but play a key role in the undesirable post-trauma immune suppression by repressing important Th-1 type immunity. Interestingly however, several reports demonstrate that Tregs confer a survival advantage in trauma. Therefore, it raises the question, do injured patients with quantitively enhanced Tregs fare better than those with less Tregs? Accordingly, we examined the role of increased Tregs on immune cell subsets in a murine burn-trauma model.

C57BL/6 mice were treated with anti-DR3 to increase Tregs before burn trauma. Mice treated with anti-DR3 prior to burn and secondary pseudomonas aeruginosa infection showed improved survival compared with untreated mice. CyTOF analysis revealed burn-injured mice given anti-DR3 showed increased counts of Ki-67+ splenic PMN-MDSCs compared to sham and burn-injured control mice. This corresponded with higher levels of PMN chemoattractants by stimulated splenocytes from burn-injured mice given DR3. Sham and burn-injured mice with higher Tregs also had increased numbers of activated γδ T cells compared to untreated mice, suggesting that these cells are regulated by an increase in Tregs. This study indicates that in trauma, it may be necessary to maintain high Treg levels to alleviate trauma-induced immune dysregulation and restore immune homeostasis.
Super-induction of immunosuppressive glioblastoma extracellular vesicles by IFN-g through PD-L1 and IDO1
Authors: Jung Mi-Yeon , Brown Desmond A. , Himes Benjamin , Garcia Luz Milbeth Cumba , Parney Ian F.
Published: January 2023

Glioblastoma is the most common and aggressive primary brain tumor. Median survival is 15 months despite surgery, radiation, and chemotherapy. Immunotherapy is promising but glioblastoma-mediated immunosuppression remains a barrier. Recently, extracellular vesicles (EVs) have been implicated in glioblastoma-mediated immunosuppression through expression of the immune checkpoint molecules PD-L1 and IDO1 which have been implicated in T cell function suppression and decreased proliferation in some cancers. Our data demonstrate that PD-L1 and IDO1 expression are increased in most nucleated cells following IFN-γ exposure. IFN-γ exposure did not alter EV production or expression of common EV markers but increased PD-L1 and IDO1 expression in EVs from differentiated but not stem-like GBM cells. Differentiated glioblastoma cell derived EVs induced myeloid-derived suppressor cell and non-classical monocyte differentiation from normal monocytes. This was increased following IFN-γ exposure and was dependent upon both PD-L1 and IDO1 expression but not stem-like GBM cell. Monocytes exposed to differentiated but not stem-like GBM cell EVs inhibited T cell proliferation in a similar manner (increased with IFN-γ exposure, decreased with PD-L1 or IDO1 knockdown). Thus, IFN-γ exposure results in super-induction of immunosuppressive EVs from differentiated but not stem-like GBM cells that increase MDSC and NCM differentiation in normal monocytes and increase their ability to inhibit T cell proliferation. These effects are dependent upon PD-L1 and IDO1 up-regulation induced by IFN-γ. This may be an important mechanism GBMs utilize to suppress anti-tumor T cell responses that are typically accompanied by increased IFN-γ expression.
Immunological function of Foxo6 in hypersensitive immune reaction
Authors: Kim Mi Eun , Lee Jun Sik
Published: January 2023

Forkhead box O(FoxO) is a transcription factor which is involved in cell cycle progression, cell survival and DNA-repair. Dendritic cells(DCs) are professional antigen-presenting cells which present the antigens to T lymphocytes and regulate the adaptive immunity. Previous studies indicated that FoxO1 and 3 are plays a critical role in DC maturation and DC-T cell interaction. FoxO6, a member of FoxO family, is less attended than other FoxO families, because limited expression region of FoxO6. Also, the effect of FoxO6 is still unknown. Here, we investigated the effect of FoxO6 in dendritic cells. Phosphorylated FoxO6 is increased and acetylated FoxO6 is decreased during dendritic cell differentiation. Moreover, deficiency of Foxo6 altered antigen capacity of dendiritc cells. Therefore, we expect that FoxO6 has important role in DC differentiation and maturation, and will further investigate the functions and the mechanisms of FoxO6 on DC. And alteration of FoxO6 expression level had effect on DCs maturation.
Lymphotoxin β receptor signaling and CD11c-positive dendritic cells form high endothelial venule-like vessels in the skin in murine atopic dermatitis model
Authors: Kanameishi Shuto , Ono Sachiko , Honda Tetsuya , Kabashima Kenji
Published: January 2023

High endothelial venules (HEVs), which are characterized by the expression of peripheral node addressin (PNAd), are essential routes for the trafficking of naïve T cells and central memory T cells (Tcms) into secondary lymphoid organs. In lymph nodes (LNs), lymphotoxin (LT) α1β2-LTβ receptor (LTβR) signaling from dendritic cells (DCs) is essential for the formation of HEVs. Although it has been reported that PNAd+ HEV-like vessels appear in the skin under some inflammatory condition, such as atopic dermatitis (AD), the formation mechanism of HEV-like vessels and their functions in the skin have remained unclear.

To clarify these issues, we established a system to induce PNAd+ HEV-like vessels in the skin, using a mouse AD model induced by calcipotriol. Flow cytometric (FCM) analysis revealed that around 20–30% of vessels were PNAd+ in the AD-like skin lesions. Electron microscopic analysis showed prominent formation of rough endoplasmic reticulum, Golgi apparatus and mitochondria in the HEV-like vessels similar to HEVs in LNs. These vessels were mainly detected in post-capillary venules, around which dermal DCs formed clusters. Administration of LTβR-Fc fusion protein or inhibition of the DC clusters by either pertussis toxin or depletion of DCs using CD11c diphtheria toxin receptor transgenic mice abolished the formation of HEV-like vessels in the skin. Naïve T cells and Tcms infiltration were detected in both AD patients and murine AD model skin by immunohistochemistry and FCM.

Collectively, our results suggest that LTβR signaling and CD11c+ DCs mediate the formation of HEV-like vessels in the skin, which may possess functions of HEV and regulate the transmigration of naïve T cells and Tcms.
MicroRNA-9 fine tunes dendritic cell function by suppressing negative regulators
Authors: Cordeiro Brendan , Jeon Peter , Boukhaled Giselle , Corrado Mario , Lapohos Orsolya , Roy Dominic G , Williams Kelsey , Jones Russell G , Gruenheid Samantha , Sagan Selena M. , Krawczyk Connie M.
Published: January 2023

MicroRNAs (miRNA) are emerging as important regulators of immune function due to their fast action and ability to regulate programs of gene expression. Dendritic cell (DC) responses to stimuli involves a rapid transition from steady-state an activated state, leading to numerous phenotypic changes underpinned by changes in gene expression. We have found that microRNA-9 (miR-9) expression is rapidly increased upon LPS stimulation. Pathway analysis on predicted miR-9 targets show a significant enrichment of negative regulators of gene expression. We investigated whether miR-9 promotes DC activation through targeting negative regulators. We found that DCs overexpressing miR-9 showed an increased activation phenotype whereas DCs sequestering miR-9 showed blunted activation. Co-culture of miR-9 sequestering DCs with T cells led to decreased T-cell activation, whereas miR-9 overexpressing DCs promoted T cell activation. Mice immunized with miR-9 overexpressing DCs following injection with B16-OVA melanoma cells displayed decreased tumour volume compared to controls. This work demonstrates that miR-9 promotes the activation and function of DCs, adding to the growing evidence that miRNAs are involved in the regulation of immune responses.
Identification of splice isoforms in T helper cells using Nanopore-based cDNA sequencing
Authors: Mir Quoseena , Srivastava Rajneesh , Ulrich Benjamin J , Kaplan Mark H , Janga Sarath Chandra
Published: January 2023

The T helper (Th) cell compartment of the adaptive immune response provides a central mechanism of balance between inflammation and immune tolerance. The differentiation of naïve Th cells into both regulatory and effector Th subsets is regulated through complex gene regulatory networks that lead to distinct transcriptomes. Alternative RNA splicing is a major component of cellular differentiation that allows protein heterogeneity and mechanistic diversity across cell types. However, our understanding of alternative splicing control is still limited. Here we show differential expression of RNA binding Proteins (RBP) genes among Th cell subsets suggesting a regulatory role of RBPs in RNA splicing and Th differentiation. In order to interrogate the regulatory gene network, we utilized innovative Nanopore-based sequencing to generate a single molecule transcriptome map of naïve CD4 T cells, and differentiated Treg and Th1 cells to characterize full-length cDNA transcripts and alternatively spliced isoforms. Gene expression was compared across four major gene categories; RBPs, Transcription Factors (TFs), Other Protein Coding (OPC) and LncRNA. Our study revealed that RBPs exhibited high splicing potential while LncRNA exhibited relatively less splicing potential in comparison to other gene categories in all cell types. Overall, we identify RBPs that are both differentially expressed in Th subsets and differentially spliced themselves. This might represent an important control mechanism of functional protein diversity in regulating immune responses.
How do CD8+ T cells complete DNA replication in less than 4 hours?
Authors: Sugitani Norie , Moiseeva Tatiana , Wang Yiyan , Palmer Drake , Ford Rhodes , Schamus Sandra , Singh Ankita , Lemon Kathryn , Kammula Udai , Vendetti Frank P , Poholek Amanda , Osmanbeyoglu Hatice , Delgoffe Greg M. , Bakkenist Christopher J
Published: January 2023

We demonstrate that activated CD8+ T cells from Pmel mice replicate their genome within 4 hours which is significantly faster than most cancer cells. Many genotoxic chemotherapeutic agents target the rapid proliferation of cancer cells. However, activated T cells also expand rapidly and this can lead to lymphopenia in patients receiving these chemotherapeutic agents. Understanding how CD8+ T cells complete DNA replication in less than 4 hours could lead to the development of treatment schedules that spare CD8+ T cells.

Three possible mechanisms may underlie the rapid DNA replication in CD8+ T cells: 1) faster replication fork velocity, 2) higher density of origin firing, or 3) modified replication timing program, allowing for simultaneous replication of early- and late-replicating regions of the genome. Using DNA combing, we show that the replication fork velocity and inter-origin distance is similar in unperturbed CD8+ T cells and B16 cancer cells. Repli-seq analysis, a NGS genome-wide replication timing analysis, suggest that regions of the genome replicated late in S phase in fibroblasts are replicated earlier in CD8+ T cells, suggesting that the rapid DNA replication in CD8+ T cells is a consequence of a modified, truncated replication timing program. We also show for the first time that actively replicating CD8+ T cells have dormant origins suggesting that this mechanism of genome stability is maintained in activated CD8+ T cells.
Dissecting the mechanisms responsible for the generation of regulatory versus pathogenic human CD4+ T cells by TLR9-activated plasmacytoid dendritic cells
Authors: Caielli Simone , Banchereau Jacques , Pascual Virginia
Published: January 2023

Understanding the mechanisms underlying how regulatory and pathogenic CD4+ T cells are generated will accelerate therapeutic target identification in autoimmune diseases such as Systemic Lupus Erythematosus (SLE), where this balance is altered. We recently described that in vitro priming of naïve CD4+ T cells with plasmacytoid DCs (pDCs) activated with either CpGA or Oxidized mitochondrial DNA (Ox mtDNA) leads to the generation of Type 1 regulatory T cells (Tr1) or of a novel T helper subset (Th10 cells), respectively. Th10 cells are expanded in the blood of SLE patients and accumulate within the tubuloinsterstitial areas of proliferative nephritis (PLN) lesions. These cells produce IL10 and mitochondrial ROS (mtROS) as the result of reverse electron transport (RET) fueled by the tricarboxylic acid (TCA) cycle intermediate succinate. Functionally, Th10 cells are not suppressive, but they provide B cell help through the synergistic effect of IL10 and succinate. The mechanisms responsible for the acquisition of a regulatory versus a helper phenotype remain elusive. We now show that naïve CD4+ T cells require the activation of the Delta Like Canonical Notch Ligand 4 (Dll4)-Notch pathway in order to acquire a TH10 phenotype. Conversely, the TLR7-IRF7 pathway is necessary for the generation of Tr1 cells. These pathways might be exploited for therapeutic intervention in human autoimmune diseases.
High-affinity tonic TCR:self-pMHC interactions inhibit T follicular helper cell development
Authors: Bartleson Juliet M , Viehmann Milam Ashley A , Donermeyer David L , Horvath Stephen , Allen Paul M
Published: January 2023

T cells receive tonic signals before encountering their immunogenic antigen, but whether or not peripheral, tonic TCR:self-pMHC interactions can affect a T cell’s response to foreign antigen remains relatively unexplored. We interrogated potential T effector functions controlled by tonic signaling with the use of a transgenic TCR system composed of two distinct CD4+ T cells that recognize the same Listerolysin O epitope but lie at opposite ends of the TCR:self-pMHC affinity spectrum. Remarkably, studies revealed differences in the ability of these two cells to produce Tfh cells during a primary immune response. The cells with lower-affinity tonic TCR:self-pMHC interactions generated a significantly greater frequency and total number of Tfh cells when compared to the high-affinity cells, which directly impacted the humoral immune response. To test whether affinity for self-pMHC controls Tfh development through tonic signaling, we generated two mouse lines: Scn5a/CD4-creERT2 and H2-DMf/f/UBC-creERT2. These mouse lines allow us to temporally increase or decrease tonic signaling, respectively. Results showed decreasing tonic self-pMHC availability did not allow for the survival of cells accustomed to high-affinity tonic TCR:self-pMHC interactions; however, increasing tonic signaling in cells that normally experience low-affinity TCR:self-pMHC interactions did indeed inhibit Tfh development. Furthermore, the H2-DMf/f mouse line was additionally crossed to various cre-strains to reveal that the CD11c+ antigen presenting cell subset is responsible for tonic signaling maintenance through presentation of self-pMHC. These studies reveal critical roles tonic signaling plays in Tfh development and CD4+ T cell survival.
T cell receptor repertoire features display universal rules for selection and plasticity in the functional CD4 T cell subsets
Authors: Kasatskaya Sofya A , Ladell Kristin , Egorov Evgeny S. , Price David A. , Chudakov Dmitriy
Published: January 2023

Helper T cell choice of functional subset may depend on the mode and strength of T cell receptor interaction with peptide-MHC complex on antigen presenting cells. However, the extent, complexity, and homogeneity of this influence of TCR structure on the selection into a functional subset, as well as further stability of acquired clonal programs remain poorly studied. Here we present the first analysis of deep sequenced T-cell receptor repertoires of the eight effector CD4 T cell subsets (Th1, Th2, Th2a, Th17, Th1-17, Th22, Treg, and Tfh) from peripheral blood of 5 healthy donors, revealing unexpectedly prominent, multivariate subset-specific differences that are highly reproducible across unrelated donors. Similar analysis performed for the sorted naive CD4 T cell subsets showed that specific TCR features of Tregs (short and strongly interacting CDR3 region) are rooted in thymic selection, while differences between other subsets accumulate later upon T cell functional engagement. We next provide the first deep analysis of the plasticity of human effector CD4 subsets ex vivo. We demonstrate high plasticity within Th17 and Th22 functional subsets and prominent clonal exchange of both Th17 and Th22 with Th2. Th1-17 subset shares clones with Th1 but not with the Th17 subset. Finally, we investigate relative publicity of effector CD4 subset repertoires across donors. We reveal high publicity of Treg and Tfh, and high privacy of Th22 and Th2a repertoires, which corresponds to the number of added N-nucleotides for these subsets, suggesting their earlier fetal and evolutionary origin. Altogether, we provide the first detailed picture of distinct repertoire features, plasticity, and publicity of helper CD4 T cell subsets.
Gut microbial composition and expression of host immune-related genes are tightly linked in the model teleost system, Gasterosteus aculeatus (three-spined stickleback)
Authors: Fuess Lauren E , Ling Fei , Steinel Natalie C , Weber Jesse , Bolnick Daniel I
Published: January 2023

Associations between eukaryotic organisms and microbial symbionts contribute to a diversity of host functions, including growth and immune system development. Despite these broad effects of the microbiome on host function, the mechanisms linking host processes and microbiome composition are still being elucidated. Using a model teleost species, Gasterosteus aculeatus (three-spined stickleback), we investigated patterns of co-variation between gut microbiome composition and host gene expression, focusing specifically on immune-related processes. First, we tested hypotheses of co-variation between microbiome diversity and expression of immune-related genes, and found strong positive associations between microbial alpha diversity and immune gene expression. Next we examined correlations between abundance of specific microbial taxa and gene expression. We identified 15 microbial families that were highly correlated with approximately 1200 host genes. These 15 families fell into three categories: those positively correlated, negatively correlated, and neutrally related to immune processes. Positively immune-correlated families included unclassified Firmicutes, while negatively immune-correlated families included many families within the phylum Proteobacteria. Increased functional understanding of these specific families will help disentangle mechanisms of correlation between microbial families and host immune processes. This is, to our knowledge, the first comprehensive study of co-variation between gut microbiome composition and host gene expression. Our data supports predominate hypotheses of an intimate link between gut microbial composition and host immune function.
Memory-like innate immunity following infection with Pneumocystis murina
Authors: Samet Shelly , Bishop Lisa , Kovacs Joseph
Published: January 2023

Pneumocystis pneumonia (PCP) continues to be a life-threatening infection in HIV-infected patients, renal transplant patients, and other immunocompromised individuals. Dendritic cells are key antigen presenters to CD4+ T cells, which are critical to controlling Pneumocystis infection. β-1,3 glucans are found in the cell wall of the cyst form of Pneumocystis and are a known activator of innate immune cells via the dectin-1 receptor. β-glucans derived from other fungi have been shown to induce an enhanced “memory-like” immune response, termed trained immunity, in innate cells, including monocytes, macrophages, and dendritic cells. Therefore, we explored if Pneumocystis infection can induce an enhanced innate immune response in mice following exposure via a natural route of infection. We conducted mixing experiments using purified CD4+ T cells and dendritic cells from unexposed C57BL/6 mice and mice that spontaneously cleared infection. We found that CD4+ T cells from naïve mice exhibited a significantly greater proliferation after 5-day cultures when a crude Pneumocystis antigen was presented by CD11c+ dendritic cells from exposed mice compared to unexposed mice. In support of this, CD4+ T cells from C57BL/6 mice that were immunized with Freund’s adjuvant alone exhibited significantly greater proliferation when a crude Pneumocystis antigen was presented by either CD11c+ dendritic cells or CD11b+ macrophages from mice immunized with Pneumocystis antigen mixed with Freund’s adjuvant vs. adjuvant alone. These results suggest that Pneumocystis infection stimulates a memory-like innate immune response which may be important in controlling subsequent exposure to Pneumocystis, as well as exposure to other pulmonary pathogens.
Developmental immaturity of sialic acid recognition mediates neonatal susceptibility to GBS pneumonia
Authors: Lund Sean J , Patras Kathryn A , Kimmey Jacqueline M , Yamamura Asami , Butcher Lindsay D , Del Rosario Pamela G B , Hernandez Gilberto E , McCoy Alyssa M , Lakhdari Omar , Nizet Victor , Prince Lawrence S
Published: January 2023

Group B Streptococcus (GBS) is a major neonatal pathogen but rarely causes disease in adults. We previously showed in mice that GBS escapes killing in the neonatal lung via its heavily sialylated capsule. Immune cells detect sialic acid moieties via expression of a repertoire of Siglec receptors. Combinatory expression of proinflammatory and anti-inflammatory Siglec receptors allows differentiation between host and pathogenic microbial sialic acid modifications. We test here the hypothesis that neonatal alveolar macrophages (AMs) fail to detect and kill GBS due to developmental immaturity of Siglec receptor expression. Adult AMs express the proinflammatory sialic acid receptor Sialoadhesin (Sn, Siglec-1) and the anti-inflammatory receptors Siglec-E and Siglec-F. However, real time PCR, immunofluorescence, and FACS detected only Siglec-E in neonatal lung macrophage populations. Sn expression increased soon after birth and was restricted to AMs. The timing of increased Sn expression in newborn mice correlated with susceptibility to GBS. Mice infected with GBS on day 1 suffered early onset mortality, while mice infected on day 2 displayed late onset disease. Mice infected on day 3 survived GBS infection. Further implicating AM immaturity, Csf2−/− mice, which have defects in AM differentiation, lacked Sn expression and had reduced GBS clearance following infection. The presence of Siglec-E but absence of Sn in newborn AMs appeared to promote tolerance to GBS. Newborn SigE−/− mice had increased GBS phagocytosis and killing compared to WT controls. We therefore conclude that GBS exploits developmental immaturity of Siglec expression in AMs via its sialic acid capsule in causing neonatal disease.
Initial phospholipid recognition on Toxoplasma vacuoles elicits IFN-inducible GTPase-mediated host defense
Authors: Yamamoto Masahiro , Lee Youngae
Published: January 2023

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals by ingestion. The organism enters host cells and resides in the cytoplasm in a membrane-bound parasitophorous vacuole (PV). Inducing an interferon response enables IFN-γ-inducible immunity-related GTPase (IRG protein) to accumulate on the PV and to restrict parasite growth. However, little is known about the mechanisms by which IRG proteins recognize and destroy T. gondii PV. We characterized the role of IRG protein Irgb6 in the cell-autonomous response against T. gondii, which involves vacuole ubiquitination and breakdown. We show that Irgb6 is capable of binding a specific phospholipid on the PV membrane. Furthermore, the absence of Irgb6 causes reduced targeting of other effector IRG proteins to the PV. This suggests that Irgb6 has a role as a pioneer in the process by which multiple IRG proteins access the PV. Irgb6-deficient mice are highly susceptible to infection by a strain of T. gondii avirulent in wild-type mice.
Identification and characterization of Escherichia coli genes associated with grazing resistance to the social amoeba Dictyostelium discoideum
Authors: Perez Vazquez Marta G , Snyder Michelle , Johnson De’Sha , Rasko David
Published: January 2023

Pathogenic Escherichia coli strains remain a significant health challenge, particularly in developing countries. The social amoeba, Dictyostelium discoideum, which phagocytizes bacteria for nutritional purposes, serves as a host model for several pathogenic bacteria, including E. coli. Here we have characterized the interactions of D. discoideum with 35 representative E. coli strains and identified strains sensitive and resistant to D. discoideum grazing in each E. coli pathotype. Whole genome phylogenomic analysis revealed that grazing resistance is distributed among the phylotypes, although resistant strains are enriched in the B2 phylogenomic group. Comparative genomic analysis identified genes more prevalent among strains resistant to D. discoideum grazing, including genes associated with adhesion, carbohydrate modification, iron transport, signaling and metabolism. Interestingly, the gipA (growth in Peyer’s Patches) gene, which has been found associated with microbiota in patients with Crohn’s disease, was widely present in E. coli strains resistant for D. discoideum grazing. We are currently working to characterize the role of E. coli genes associated with resistance to phagocytosis and killing by D. discoideum, through creation and analysis of E. coli strains manipulated for expression of the identified genes. Such characterization is aimed at providing insight into potentially novel E. coli virulence factors.
Anti-Vibrio cholerae responses in the human periphery and intestine following a live-attenuated vaccine
Authors: Nyhoff Lindsay E , Adekunle Oluwaseyi , Kauffman Robert C , Keilin Steven , Rouphael Nadine , Wrammert Jens
Published: January 2023

Vibrio cholerae is a prototypical mucosal pathogen that causes cholera, a severe diarrheal disease. Cholera affects an estimated 3 to 5 million people annually, resulting in over 100,000 deaths. Infection leads to serotype-specific immunity that can last for up to ten years. Immunity is likely mediated by intestinal antibodies, which primarily target the immunodominant antigens lipopolysaccharide (LPS) and cholera toxin (CT). However, how immunity is generated and maintained is not well understood. Here we have used the live-attenuated cholera vaccine, Vaxchora, to characterize the primary immune response in both the periphery and the human duodenum. Previously, this vaccine showed 80% protective efficacy 90 days after vaccination of naïve volunteers, and induced early antibody responses in levels similar to infection. We developed new technology to identify and characterize V. cholerae-specific plasmablasts and plasma cells by flow cytometry, and found that antigen-specific plasmablasts in peripheral blood quickly expand and upregulate mucosal trafficking markers CCR9 and CCR10. These B cells either develop locally or home to the small intestine, where anti-LPS and anti-CT antibody-secreting cells are present by day 28. Surprisingly, our preliminary data shows that vaccinees may exhibit robust anti-CT responses in the duodenum even when peripheral anti-CT antibody secreting cells are not detected, which provides novel insight into the generation of sustained protection. These data will help us understand not only protective immunity to V. cholerae, but also how the human mucosal plasma cell compartment is developed and maintained.
Lymph Node Priming Licenses Intestinal CD103+ CD8 Tissue-Resident Memory T Cell Development
Authors: Qiu Zhijuan , Khairallah Camille , Imperato Jessica Nancy , Chu Timothy H , Xu Daqi , Romanov Galina , Puddington Lynn , Sheridan Brian S
Published: January 2023

CD8 tissue-resident memory T (TRM) cells provide front-line protective immunity at barrier tissues. Understanding TRM cell development will provide significant insights for vaccine design targeting infections and cancers at barrier tissues. Here, we demonstrate that pathogen-induced inflammation and pathogen-derived cognate antigen had minimal impact on intestinal CD103+ TRM cell differentiation. Instead, T cell priming in the mesenteric lymph nodes (MLN) was the principal determinant of CD103+ TRM cell differentiation in the intestine. In contrast, T cells primed in the spleen were incapable of differentiating into intestinal CD103+ TRM cells. Foodborne rechallenge of spleen-primed memory T cells was unable to induce intestinal CD103+ TRM cell differentiation, suggesting initial priming promoted a lasting fate. Moreover, both CD103− and CD103+ naïve T cells were highly efficient in differentiating into CD103+ TRM cells in the intestine after priming in the MLN, suggesting preconditioning of naïve T cells by TGF-b during homeostasis had little impact on intestinal CD103+ TRM cell differentiation. We further demonstrate that MLN priming initiated a CD103+ TRM cell program before effector T cell migration to the intestine and promoted CD103+ TRM cell differentiation in situ in part by promoting CCR9 expression and the ability to respond to TGF-b in a cell-extrinsic manner. Thus, mesenteric lymph node priming is specialized to license intestinal CD103+ CD8 TRM cell development.
The Effects of Quantum Dots Nanoparticles on Caenorhabditis elegans infected with Microsporidia
Authors: Elfert Amr , Meiner Kavan , Idris Sally , Cotlet Mircea , Delaney Tetyana
Published: January 2023

Caenorhabditis elegans are model organisms that have intestinal lining similar to that of humans. They are transparent and can be easily cultivated in the lab. Nematocida parisii is an intracellular parasitic species of microsporidia that infect the intestines of C. elegans. Similar species cause microsporidiosis in humans. Microsporidiosis can cause severe digestive system symptoms including life-threatening diarrhea in immunocompromised as well as in immunocompetent individuals. Currently available treatments are not always sufficient and can be toxic.

In this experiment, the effects of different nanoparticles (NPs) on the intestinal lining of C. elegans and N. parisii within are tested. It was reported that NPs can diffuse through cellular membranes and cause oxidative stress, DNA damage, and macrophage activation. Some NPs showed anti-bacterial and anti-fungal effects.

If the treated C. elegans have a longer lifespan and a larger population density than untreated ones, then NPs can preferentially diffuse through the epithelial cells of the intestines and affect the infection. The number of thrashes of the C. elegans was used as a measure of their response to stimuli. Irritating and disrupting stimuli cause an initial increase in the movements but it is then followed with a sharp drop and the organisms may eventually die. The imaging of C. elegans was done using confocal microscopy with DIC. The preliminary results showed that the quantum dots of 655 nm emission wavelength and at a concentration of 0.8μM showed no significant difference that indicated a harmful effect on the C. elegans. Thus, different concentrations and longer exposure times will be implemented to compare their effects on the microsporidia.