bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of NK cells in circadian regulation of lung inflammation
Authors: Sengupta Shaon , Naik Amruta , Issah Yasmine , Forrest Kaitlyn
Published: January 2023

Influenza is a leading cause of respiratory mortality and morbidity. While inflammation is necessary for fighting influenza, a fine balance of anti-viral defense and host tolerance is crucial to successful recovery from the same. Circadian rhythms form an anticipatory system wherein various aspects of cellular physiology oscillate across a 24hrs period; these rhythms have been known to modulate various facets of immunity.

We have recently demonstrated that circadian rhythms control lung inflammation and injury in influenza A virus (IAV) infection. A hallmark of circadian regulation is a difference in outcomes based on time of day of infection. Indeed, we found that C57bl6 mice infected with IAV (PR8) at the beginning of their active phase (ZT11) had 3-fold higher mortality than littermates infected at the beginning of the rest phase (ZT23). This difference disappeared when NK cells were depleted (with NK1.1 antibody) prior to the infection, confirming the role of NK cells in mediating the circadian regulation of IAV infection.

Next, we investigated the mechanism underlying the role of NK cells in circadian regulation of lung injury. We determined viral titers in the lungs from days 1, 2, 4 and 6 following IAV infection and found that there was no difference between the NK depleted and control animals. This suggests that NK directed circadian control of lung inflammation is not mediated through control of viral replication or burden. We then quantified lung pathology in NK depleted and control animals and found that histological injury was worse in the NK depleted group.

In summary, these data suggest that NK cells mediate circadian regulation of outcomes in IAV through control of host tolerance rather than through direct influence on IAV burden.
The spatiotemporal dynamics of type I IFN response determine viral infection outcome
Authors: Casella Valentina , Argilaguet Jordi , Domenjo Eva , Pedragosa Mireia , Encabo Hector Huerta , Esteve-Codina Anna , Vidal Enric , Bocharov Gennady , López-Rodríguez Cristina , Meyerhans Andreas
Published: January 2023

Type I interferons (IFN-I) play a critical role in shaping the antiviral immune response early after an infection. However, the dynamics by which different immune cell subsets regulate the IFN-I response during the early stages of acute and chronic infections is not completely understood. Here we used the Lymphocytic Choriomeningitis Virus (LCMV)-infection mouse model system to characterize the dynamics of the IFN-I response in acute and chronic infections. Time-resolved spleen-transcriptomes revealed that during an acute infection, IFN-I showed two peaks of expression at days 2 and 5 post-infection. In contrast, in chronically infected mice a single peak of IFN-I genes appeared at day 1. Further analysis revealed that IFN-I genes in acute infection were co-expressed with genes related to inflammatory macrophages, suggesting an important role of these cells determining infection fate. Indeed, we identified metallophilic macrophages as an important source of Ifnb only during acute infection and demonstrated that the subsequent IFN-I receptor (IFNAR) signaling is necessary to induce pro-inflammatory macrophages. In contrast, during chronic infection, early depletion of marginal zone macrophages results in a lack of IFN-I production and the inflammatory response is not induced. Importantly, blockage of the second peak of IFN-I response by IFNAR blockage during an acute infection also resulted in exhaustion of virus-specific CD8 T cells and prevention of lymphoid tissue fibrosis. Further studies are ongoing to decipher the regulatory mechanisms underlying the characterized events, thus revealing universal concepts related to infection fate decisions that are also relevant for persistent human infections such as HIV or HCV.
In vivo visualization of immune responses during rejection of allogeneic tissue in the anterior eye chamber
Authors: Chang Hsin-Fang , Krause Elmar , Paloma Saskia , Rettig Jens
Published: January 2023

The anterior chamber of the eye (ACE) was recently described as a novel imaging site in diabetes research by observing transplanted pancreatic islet activity in the eye of a living animal. The ACE technology is ideally suited for non-invasive and longitudinal in vivo imaging. We take advantage of the ACE window to observe immune responses, especially allorejection of islet cells by cytotoxic T lymphocytes. We transplant pancreatic islets from DBA/1J mice into C57BL/6 recipient knock-in mouse which endogenously express a fluorescent protein (GzmB-mTFP or Bonzo) in T cells. We follow the onset of the rejection after vascularization on islets, when immune cells start to infiltrate into ACE, until the end of the rejection process where the islets are cleaned up for roughly a month by repetitive two-photon microscopy. We find that T cells show less migration velocity on islets of foreign mice than on autologous tissue, indicating T cell activation and recognition on the islets. Interestingly, the temporal infiltration pattern of T cells during the rejection process is precisely regulated, showing enrichment of CD4+ T cells on the islets before arrival of CD8+ T cells in histological studies. We use the ACE platform as a more comprehensive environment to study immune response and focus on T cell killing. We aim to understand the mechanism and regulation of T cell-mediated killing in vivo and to further investigate the killing in gene-deficient mice (i.e. munc13-4 and perforin) that resemble to severe human immune disease.
GPR68 promotes CD4 T cell survival by increasing sensitivity to CD3 signaling
Authors: McAleer Jeremy P. , Griggs Nosakhere , Foster William , Zha Xiangming , Xu Yan
Published: January 2023

CD4 T cells contribute to host defense against pathogens, but are also implicated in autoimmunity and allergies when directed against innocuous antigens. Since Th17 cells are upregulated in several autoimmune diseases, identifying genes involved in their activation, proliferation or survival may lead to novel therapies for inflammatory diseases. We previously demonstrated that the aryl hydrocarbon receptor increases expression of the proton sensing receptor GPR68 in human CD4 T cells. Since T cell clonal expansion is associated with a metabolic shift towards anaerobic glycolysis resulting in extracellular acidification, we tested the hypothesis that GPR68 is required for optimal T cell proliferation and survival following activation. To delete GPR68 in T cells, GPR68-floxed mice were crossed with a strain expressing Cre-recombinase under the CD4 promoter. Purified CD4 T cells from CD4-Cre+ and Cre-negative littermate controls were activated in vitro with anti-CD3 and Th17-inducing cytokines (IL-1b, IL-6, IL-23, TGF-b) for 1–5 days. In the control (Cre-negative) group, T cell numbers increased over the first 2 days following activation and then stabilized from days 3–5. In the GPR68-deficient group (Cre+), T cell numbers significantly decreased after day 2, demonstrating a role for GPR68 in promoting CD4 T cell survival. Nevertheless, IL-17 production was GPR68-independent, demonstrating that GPR68 did not regulate Th17 differentiation. GPR68 was especially critical for survival when low doses of anti-CD3 were used (<2.5ug/mL), but not at a high dose (10ug/mL). Overall, our data demonstrate that GPR68 increases the sensitivity of CD4 T cells to survival signals induced by CD3 stimulation.
The Regulatory Role of PIRB in CD4+ Th17 Mediated Colitis
Authors: Uddin Jazib , Waggoner Lisa , Vanoni Simone , Hoebe Kasper , Divanovic Senad , Munitz Ariel , Hogan Simon
Published: January 2023

Rationale

CD4+T-cell differentiation into effector or memory populations is intrinsically regulated by the delicate balance between activating and inhibitory signals. Previously we identified a role of the inhibitory receptor, Paired Immunoglobulin-like Receptor (PIR) B in the negative regulation of innate immune responses in colitis. The aim of this study is to define the involvement of PIRB in CD4+ Th17 development and regulation of CD4+ Th17-dependent colitis.

Results

We demonstrate an intrinsic deficiency in PirB−/− Th17 in vitro polarization and cell survival. Specifically, polyclonal activation of PirB−/− Th17 cells lead to increased cell death and enhanced caspase activation (# of Dead cells, WT vs PirB−/−: 17.6 ± 2.3 × 103 vs 38.5 ± 5.8 × 103; # of Caspase 3/7+ cells: 9.8 ± 1.8 × 102 vs 19.7 ± 1.2 × 102; mean ± SEM, p < 0.05). In vivo, PirB−/− Il10−/− mice were protected from development of Il10−/− spontaneous colitis phenotype (Clinical Score, Il10−/− vs PirB−/− Il10−/−: 3.3 ± 0.6 vs 0.14 ± 0.08, p < 0.01; Histological Score: 2.3 ± 0.4 vs 1.2 ± 0.2; mean ± SEM, p < 0.01) and this was associated with reduced CD4+ Th17 cells in the mesenteric lymph nodes (% IL-17a+ cells, Il10−/− vs PirB−/− Il10−/−: 2.5 ± 0.3 vs 1.2 ± 0.1; mean ± SEM, p < 0.001). To test the intrinsic effects to the CD4+ T-cell compartment, we performed the CD4+ CD45RBhi T-cell transfer model of colitis. Rag−/− mice which received PirB−/− naïve CD4+ T-cells were protected from T-cell mediated colitis (% Change in Body Weight: −3.5 ± 4.9 % WT; 20.7 ± 4.6 % PirB−/−; p < 0.001; Histological Score: 3.8 ± 0.2 WT; 1.0 ± 0.4 PirB−/−; mean ± SEM, p < 0.001).

Conclusions

These data support the concept that PIRB regulates CD4+ Th17 differentiation and development of T-cell-dependent colitis phenotype.
Role of PRMT5 in cholesterol metabolism and Th17 pathogenicity
Authors: Sengupta Shouvonik , Webb Lindsay M , Laliotis Georgios , Tsichlis Philip N , Guerau-de-Arellano Mireia
Published: January 2023

Protein Arginine Methyltransferase (PRMT) 5 is an enzyme catalyzing symmetric dimethylation (SDM) of arginine. This post-translational modification is known to be involved in oncogenesis and embryonic development. Our previous work has shown that PRMT5 is induced during T cell activation. However, the role and mechanisms by which PRMT5 modulates T helper (Th) cell polarization and autoimmune disease have not yet been elucidated. To study the impact of PRMT5 on gene expression programs induced in activated T cells, we developed a conditional PRMT5 knockout (KO) mouse model in which PRMT5 deletion can be induced in CD4+ T cells after thymic development and performed RNA sequencing (RNAseq) analyses in 3-day activated naïve CD4 T cells. We found that PRMT5 promoted expression of cholesterol biosynthetic pathway enzymes that produce Retinoid-Related Orphan Receptor (ROR) agonists that activate ROR-gt and promote Th17 differentiation. Indeed, Th17 differentiation was blunted in PRMT5 KO T cells. Finally, we identify the cholesterol biosynthesis pathway regulator SREBP-1 as a target of PRMT5 SDM. This work shows that PRMT5 expression in activated T cells is needed for the cholesterol biosynthesis gene expression program, generating ROR-gt agonistic activity and promoting Th17 differentiation. These results point to T cell PRMT5 and the downstream cholesterol biosynthesis pathway as promising therapeutic targets in Th17 cell-based diseases.
Smad signaling determines the fate of activated CTLs via multiple intersecting signaling pathways
Authors: Chandiran Karthik , Suarez-Ramirez Jenny E , Hu Yinghong , Ugur Zeynep , Kaech Susan M , Cauley Linda S
Published: January 2023

Immune responses to an infection are controlled by various factors including antigens, co-stimulatory molecules and cytokines present in the tissue micro-environment. TGFβ is a pleiotropic cytokine that modulates immune response and cellular differentiation usually through SMAD (Smad 2,3 & 4)-mediated signaling cascade. Interestingly, TGFβ and Smad4 have independent and opposing roles in determining the fate of activated CTLs. While TGFβ-receptor mediated signaling is required for resident memory (TRM) cells, Smad4 is required for effector (TEff) and central memory subsets (TCM). However, the mechanism behind TGFβ as well as Smad4 mediated differentiation is yet to be unraveled. Using transgenic animal models and pharmacological inhibitors, we investigate the role of TGFβ and multiple SMAD proteins in determining the fate of activated CTLs. We identified that while canonical TGFβ signaling, through Smad2 and Smad3, induces CD103 expression (TRM), Smad4 inhibits CD103 expression thus preventing TRM differentiation. In addition, targeted-ablation of Smad4 alters the expression of various transcription factors that participate in fate determination, including Eomes and KLF2. Although Eomes has previously been implicated in negative regulation of CD103, over-expression of Eomes does not affect CD103 expression in the absence of Smad4. This suggests that Smad4 is required for negative regulation of CD103. These results reveal a novel central role for Smad4 in guiding the fate decisions of activated CTLs and tissue localization via multiple intersecting signaling pathways.
Bone marrow T cell sequestration as a novel mode of CNS immune privilege
Authors: Wilkinson Daniel S , Champion Cosette , Chongsathidkiet Pakawat , Wang Haichen , Laskowitz Daniel , Fecci Peter
Published: January 2023

The central nervous system (CNS), although a site of relative immune “privilege,” is continuously monitored by immune cells. The CNS must possess means for limiting excess inflammation that can otherwise cause irreparable damage or death. Recently, we characterized a unique mode of cancer-induced immunosuppression whereby T cells become sequestered in high numbers in the bone marrow (BM) of humans and mice with intracranial tumors. Sequestration of T cells is mediated in part by the loss of sphingosine-1-phosphate receptor 1 (S1P1) from the T cell surface. Interestingly, this phenomenon occurs only when tumors are located intracranially, but not peripherally. This suggests that sequestration is not mediated by brain tumors themselves, but instead represents tumors usurping of a novel mechanism for CNS immune privilege designed to limit T-cell access to the CNS. Consequently, we investigated whether BM T cell sequestration occurs in other CNS disorders, such as stroke. In a murine model of stroke, immune alterations recapitulated those observed in intracranial tumors. These changes include severe but transient lymphopenia, lymphoid organ shrinkage, and BM T cell sequestration. As with intracranial tumors, BM T cell sequestration was mediated by loss of surface S1P1 on T cells. A mechanistic understanding of CNS-driven BM T cell sequestration will enable the development of novel therapeutics to limit neuroinflammation and damage in the context of CNS pathologies.
Functional dissection of the Il2ra superenhancer reveals developmentally regulated and cell type specific elements
Authors: Spolski Rosanne , Li Peng , Oh Jangsuk , Liu Chengyu , Ren Min , Enomoto Yutaka , West Erin , Shin Boyoung , Rothenberg Ellen , Leonard Warren
Published: January 2023

The expression of the IL-2 receptor a chain is tightly regulated during early T cell development and in mature lymphoid populations by either cytokine stimulation or T cell receptor interactions. IL-2Ra expression can be either constitutive, as in some double negative thymocytes and T regulatory cells, or inducible, as in CD4+and CD8+mature T cells. Previously, our lab identified a super-enhancer in the mouse Il2ra locus that extensively bound STAT5 and controlled expression of this gene in mature T cells. To further dissect developmental and cell type specific expression of this super-enhancer, Crispr-Cas9 mediated deletion was used to generate mutant mice lacking either individual or groups of STAT5 binding sites in either the upstream region or the first intron. Deletion of the upstream region led to the absence of CD25 expression in DN2 and DN3 thymocytes as well as a significant reduction of CD25 expression on regulatory T cells, but minimal effect on inducible CD25 expression in mature T cells. Analysis of individual upstream elements in reporter assays revealed a specific element that controlled high activity in DN3 cells and a Treg cell line, with markedly diminished activity in the presence of Notch inhibitors. In contrast, deletion of an intronic region markedly diminished both IL-2 and TCR-induced expression of CD25 in vitro, while having little if any effect on CD25 expression in DN thymocytes or Treg cells. These data reveal that the Il2rasuper-enhancer is composed of multiple elements that preferentially mediate either constitutive or inducible Il2ragene expression. Further analysis of these elements will define mechanisms for regulation and potentially manipulation of CD25 expression in autoimmune and other disease states.
Blimp1 suppresses proliferation in human regulatory T cells as a potential homeostasis regulator
Authors: Ding Ying , Malek Thomas R
Published: January 2023

B lymphocyte-induced maturation protein 1 (Blimp1), encoded by the PRDM1 gene, is a multifunctional transcriptional regulator that has established roles in mouse T cells. However, the mechanisms underlying its function in the proliferation, suppressive function, and homeostasis of human regulatory T cells (Tregs) are not known. To directly evaluate the role of Blimp1 in human Tregs, Blimp1α, the active isoform of Blimp1 that has dominant expression, was knocked out by Crispr-Cas9 using dual-targeting sgRNAs. High knockout efficiency was verified at DNA, RNA, and protein level by T7E1, RT-PCR, and Western blot analyses, respectively. Knockout of Blimp1α in cultured human Tregs resulted in increased cell number, upregulated Ki67 expression, and enhanced [3H]thymidine incorporation, consistent with a normal inhibitory role of Blimp1α in the proliferation of human Tregs. Compared with control cells, Blimp1α-knockout Tregs showed high CD25 expression and enhanced tyrosine phosphorylation of STAT5 (pSTAT5), suggesting a potential role of Blimp1 in regulation of IL-2R signaling. In vitro suppression assay showed that knockout of Blimp1α slightly decreased the suppressive function of Tregs. Knockout of Blimp1α also increased the expressions of Tigit and CD62L, but decreased the expressions of Foxp3, CD73, and Bcl-2, suggesting that Blimp1α may contribute to maintaining Treg identity. Collectively, our results are consistent with a model whereby Blimp1α regulates homeostasis of human Tregs by constraining proliferation in part by limiting IL-2R signaling while promoting Treg function and identity.
A ‘sweet spot’ for complement receptor CD46 activity: direct regulation of glycolytic vs. moonlighting functions of glycolytic enzymes
Authors: Kunz Natalia , Le Friec Gaelle , Singh Parul , Lavender Paul , Kazemian Majid , Hess Christoph , Kemper Claudia
Published: January 2023

Autocrine engagement of the complement receptor CD46, driven by T cell receptor (TCR) activation on human CD4 T cells, is critical for human Th1 induction: Processing of the intracellular CD46 domains by gamma-secretase and their nuclear translocation mediates the expression of nutrient transporters and enables metabolic reprogramming required for cell growth, expansion and effector function. How, at the molecular level, CYT-1 enables this fundamental cellular adaptation remains unknown. Co-immunoprecipitation experiments with antibodies against cleaved CYT-1 revealed an association of CYT-1 with the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). A strong, dose-dependent interaction was confirmed via ELISA and Microscale Thermophoresis- (MST−) based experiments in vitro and via proximity ligation assays (PLAs) in CD4 T cells ex vivo. Unexpectedly, subsequent experiments demonstrated that CYT-1 did not affect metabolic activity of GAPDH but rather fostered its des-oligomerization. CYT-1-driven des-oligomerization of GAPDH ‘unleashed’ its non-canonical ‘moonlighting’ function: CYT-1-generated GAPDH monomers displayed augmented mRNA binding abilities and stabilized metabolic mRNAs in living CD4 T cells. Importantly, this CYT-1capacity is not confined to GAPDH: CYT-1 binds and des-oligomerizes several other glycolytic enzymes. These data identify CD46 as a ‘shape-shifter’ of glycolytic enzyme activity, critically balancing the canonical (glycolysis) versus non-canonical (e.g. posttranscriptional regulation) activity – further substantiating the notion that autocrine complement is a critical regulator of normal cell physiology.
Chronic stress-induced lymphocyte reduction in a nitric oxide dependent manner
Authors: Yin Deling , Yao Bifeng , Caudle Yi
Published: January 2023

Psychological and physical stress can suppress the immune system in both humans and animals. The mechanism by which stress affects immune responses, however, remains poorly defined. Recent evidence demonstrated that nitric oxide (NO) modulates cell-mediated immune responses. Inducible nitric oxide synthase (iNOS) plays a major role in immune regulation. To investigate the role of NO in the chronic stress-induced lymphocyte depletion, we utilized a selective inhibitor of iNOS activity, NG-monomethyl-L-arginine (L-NMMA), which has been widely used, including from our laboratory and others, to evaluate the role of NO in immune responses both in vitro and in vivo. Here we demonstrate that administration of L-NMMA prevents chronic stress-induced lymphocyte reduction. Moreover, inhibition of iNOS prevents the alterations of cleaved-caspase-1 and p-JNK following chronic stress. These results reveal that chronic stress-induced lymphocyte reduction in a NO dependent manner.
Sphingosine-1-phosphate lyase inhibition exacerbates DSS-induced colitis via promoting the disruption of epithelial barrier
Authors: Lundborg Luke R , Guzman Mauricio , Tyler Christopher J , Yeasmin Shaila , Rivera-Nieves Jesus
Published: January 2023

S1P is a bioactive sphingolipid that participates as a critical regulator of many physiological processes, mainly in lymphocyte egress from lymphoid tissue, mediated by S1P gradients through the activity of S1P lyase (SPL). Inhibition of both S1P-specific receptors and Lyase activity has been shown to improve inflammatory conditions during Crohn’s-like ileitis. In contrast to this, initial observations show acute DSS-induced colitis was aggravated by SPL inhibition. Here we investigate whether the inhibition of Lyase in the intestinal epithelium is the cause of the exacerbation of colitis.

To induce global or epithelial cell specific SPL deletion, Gt(rosa)-cre or Villin-cre Sgpl1 transgenic mice were administered with tamoxifen (80 mg/kg) by oral gavage for 5 consecutive days. Then, mice were treated with DSS (2.5%) in drinking water for 7 days. Clinical features and histological scores were recorded. Permeability assays were performed to evaluate integrity of the intestinal epithelium with fluorescein isothiocyanate (FITC)-dextran 4 kDa.

Significant differences in the histopathological features (inflammation and injury) in colon were observed between Gt(rosa)Cre+/−/SPL−/− or VillinCre+/−/SPL−/− mice compared to their respective controls (without tamoxifen treatment) (n>10, p<0.001). In addition, these animals presented higher levels of epithelial permeability since the concentration of serum (FITC)-dextran was higher than their respective controls (n>10, p<0.001).

In conclusion, in the acute model of DSS-induced colitis the presence of S1P in the intestinal epithelium exacerbates inflammatory conditions, demonstrating that there could be differences in the mechanisms with chronic models of IBD.
Evolutionary conserved sequence patterns that determine functional specialization of metazoan TIR domains
Authors: Toshchakov Vladimir Y , Neuwald Andrew F
Published: January 2023

Toll-Interleukin-1R Resistance (TIR) domains represent a type of protein fold ubiquitously present in all forms of cellular life. The TIR domains are present in functionally and architecturally diverse proteins. A large subgroup of metazoan TIRs is involved in signaling that initiate and regulate the immune response elicited by infection or damaged self. This study applies Bayesian partitioning with pattern selection (BPPS) to classify the TIR domains based on sequence patterns that best distinguish individual groups. The algorithm was applied to >26000 phylogenetically diverse TIR domain sequences selected for non-redundancy from publicly available protein sequence banks. The analysis has revealed 10 metazoan groups, nine of which comprise either orthologous proteins or a protein family. Available structural data suggest that the identified groups may be uniquely characterized by regional backbone fold variants, which can be associated with a corresponding group-specific sequence motif. In addition to these fold-defining motifs, the BPPS also identified groups of surface-exposed residues conserved in TIRAP and MyD88 orthologs, which correspond to predicted sites that mediate the signal-dependent TIR oligomerization. This analysis also has clarified the functional significance of locally conserved sequence motifs, named Box 2 and Box 3, as each of these motifs is a part of a larger block of conserved, intramolecularly interacting residues. Finally, this study has identified the sequence patterns that differentiate TIRs of different classes, such TIRs of TLRs, from TIRs of the IL-1R family, or their adapter proteins, and highlights the differences between metazoan, plant, and bacterial TIRs.
Deficiency of the methylcytosine dioxygenase Tet2 enhances CD4 T cell memory generation
Authors: Baessler Andrew Stephen , Novis Camille Leite , Sircy Linda , Thiede Kendall , Newman Jordan , Hale J Scott
Published: January 2023

In response to viral infection, antigen-specific CD4 T cells differentiate into Th1 and T follicular helper (Tfh) cell subsets that acquire distinct DNA demethylation changes which act to enforce their lineage commitment and functions. Following viral clearance, the populations of Th1 and Tfh cells contract and form distinct memory populations that maintain their specific methylation programming. Previously, we found that deficiency of Tet2, an oxygenase that mediates CpG demethylation, skews the balance of Th1 and Tfh cells at 7 days post infection leading to the formation of significantly more germinal center (GC) Tfh cells. Here we examined whether Tet2 impacts CD4 T cell memory formation and the recall of lineage specific functions upon re-challenge. At days 15, 30, and 45 post infection with lymphocytic choriomeningitis virus (LCMV) there was a greater frequency of Tet2 deficient cells positive for memory markers in the blood when compared to WT cells. To assess the recall ability of Tet2 deficient memory CD4 T cells, Tet2KO and WT memory cells were purified from spleens of recipient mice at 90 days post-infection and equivalent numbers of WT and Tet2KO cells were then re-transferred into naive C57BL6/J recipient mice followed by LCMV infection. At 7 days post re-challenge, we observed a greater secondary expansion of Tet2KO cells compared to WT. Tet2KO memory cells also retained the ability to generate higher frequencies of GC Tfh cells in response to re-challenge. Ongoing work will identify whether Tet2 deficient memory Tfh cells can provide enhanced help for B cells during secondary infection and improve the antibody response. Taken together, these data suggest that Tet2 can act to restrict memory differentiation in CD4 T cells.
FcγRIIb signaling by germinal B cells following Borrelia burgdorferi infection
Authors: Tran Vuvi G. , Olsen Kimberly , Baumgarth Nicole
Published: January 2023

Tick-borne infection with Borrelia burgdorferi (Bb) causes Lyme disease in humans and persistent infection of mice, its natural reservoir host. IgG responses critically control Bb tissue burden but do not clear the infection. We previously showed induction of germinal centers (GC) in mice after Bb infection followed by their rapid collapse within 30 days, despite ongoing infection. This was consistent with the lack of long-lived and high-affinity antibody responses in these mice. Our recent experiments showed that serum IgG from Bb-infected mice bound more strongly to the inhibitory FcγRIIb on B cells, as well as recombinant FcγRIIb than those from uninfected mice, as assessed by flow cytometry and ELISA, respectively. The data prompted us to investigate whether the interaction between Bb infection-induced IgG and FcγRIIb results in the suppression of GC. Indeed, Bb-infected mice lacking FcγRIIb signaling (FcγRIIb−/−) maintained GC B cells and CD4 TFH for at least 90 days after infection, and GC B cells showed reduced apoptosis, as assessed by staining for Annexin V. Furthermore, transfer of serum from Bb-infected wild type but not AID−/−sIgM−/− mice lacking IgG, IgM and from non-infected wild type mice, into 14-day Bb-infected wild type mice resulted in reduced GC at day 21. However, despite GC maintenance and the resulting increases in Bb-specific, T cell-dependent IgG responses, enhanced antibody affinity maturation was not observed and Bb tissue burdens of FcγRIIb−/− and control mice were comparable. The data show the importance for FcγRIIb in the regulation of GC during Bb infection, while additional mechanisms currently under investigation seem to underlie their functional deficits, enabling the establishment of Bb persistence.
Experimental evolution identifies an IL-1α dependent host response against Aspergillus fumigatus regulated by the fungal SakA-HOG1 pathway
Authors: Mitchell Marina E Kirkland , Caffrey-Carr Alayna , Stannard McKenzie , Kowalski Caitlin H , Mould Dallas L , Temple Rachel , Cramer Robert A , Obar Joshua J
Published: January 2023

Fungal infections are emerging global health threats, costing the United States $7.2 billion annually in health care costs, with Aspergillus fumigatus accounting for $1.2 billion of those costs, the highest for a single fungal species and tied with Candida spp. infections. A. fumigatus has an inexcusably high mortality rate of 40–90%, disproportionately affecting those with compromised immune systems or impaired pulmonary clearance disorders such as CF and COPD. It is critical that the mechanisms behind A. fumigatus pathogenesis is elucidated. In this study, we explore A. fumigatus divergent pathologies amongst multiple strains which induce distinctly different host immune responses based on germination rate. In vivo and in vitro germination of fungal conidia segregate into two distinct groups of “fast” germinators, which elicit an IL-1α dependent host response, and “slow” germinators, which invoke a type I and III interferon dominant response. Here we use experimental evolution to find that the SakA-HOG1 stress response pathway regulates fungal growth in the lung, and serves as a switch for the induction of IL-1α dominant defense. This study demonstrates a need for revision of clinical protocols for treatment of A. fumigatus based on the strain and type of inflammation on-going in patients with A. fumigatus infections as health care standards move toward individualized patient care and antifungal stewardship.
B-Cell Responses in the Lung during Acute Pneumocystis Infection
Authors: Noell Kristin M , Kolls Jay K
Published: January 2023

Pneumocystis jiroveci is an opportunistic fungal organism that causes Pneumocystis pneumonia (PCP) in humans with HIV-AIDS or other immunocompromised individuals. Pneumocystis can replicate in the alveoli where it disrupts gas exchange and promotes inflammation in the lung. Although Pneumocystis association is with T cell immunodeficiency, recent evidence shows that B cells play a key role as APCs in the lung to prime fungal T cell responses. We hypothesized that B cells are recruited and undergo tissue specific CSR during PCP infection and that these cells are critical to the priming of PCP specific T cells. To test this hypothesis we inoculated 4 to 6wk old male and female C57Bl/6 mice with Pneumocystis murina by oral pharyngeal aspiration. Flow cytometry analysis revealed that IgG+ and IgA+ B cells were at higher abundance in infected lung tissue at days 14 and 24. Single cell BCR-sequencing revealed an increase in class switched B cell clonotypes (both IgG and IgA) at day 14 compared to spleen. Our lab has previously shown that germ encoded IgM has specificity to PC antigens. To understand the role of germline encoded IgM and class-switched antibody responses, 4 to 6 wk old Aicda, Ighm −/− or C57Bl/6 mice were inoculated with P. murina for 14 days. We found at day 14 that fungal burden loads were slightly higher in the Aicda,Ighm −/− mice than C57Bl/6 mice, but H&E staining showed that Aicda,Ighm −/− mice possessed high levels of immunopathology. Bulk RNA sequencing revealed a lack of Type II immune responses, which have been shown to control Pneumocystis lung burden. Our data suggests that both germline encoded IgM and class switched antibody responses are critical in controlling pneumocystis infection.
Leishmania donovani modulates host macrophage mitochondrial metabolism, integrity, and function
Authors: Ospina Hamlet Acevedo , Descoteaux Albert
Published: January 2023

To colonize macrophages, Leishmania promastigotes employ virulence factors, including lipophosphoglycan (LPG), to impair host cell processes. Whereas previous studies revealed that Leishmania alters signaling axes that regulate mitochondrial function, scarce attention has been paid to the characterization of host cell mitochondrial metabolism during Leishmania infection and to the effectors involved therein. In this study, we addressed the hypothesis that L. donovani modulates host cell mitochondrial metabolism and function in an LPG-dependent manner. To this end, we infected bone-marrow-derived macrophages with metacyclic promastigotes and we assessed the expression kinetics of host cell nuclear and mitochondrial genes that control mitochondrial biogenesis, and we measured host mitochondrial metabolic fluxes. We found that host cell nuclear and mitochondrial genes that control mitochondrial biogenesis are upregulated in an LPG-dependent manner. We also observed that IRG-1, the enzyme that synthesizes itaconate, is highly induced during infection in an LPG-dependent manner and this response was independent of endosomal TLRs. We next found that L. donovani induces a doubling in the mtDNA/nDNA ratio in an endosomal TLRs and IFNAR-dependent manner, suggesting that L. donovani promotes host mitochondrial biogenesis in an inflammatory context. Metabolic flux analyzes showed that the OCR/ECAR ratio is modulated multiple times during infection, independently of β-oxidation, suggesing that L. donovani promastigotes induce the Warburg effect to promote energetic metabolic changes. Collectively, our data indicate that L. donovani alters host cell mitochondrial dynamics during the colonization process.
Defining the role of CD8 T cell subsets in tuberculosis in non-human primates
Authors: Winchell Caylin G , Maiello Pauline , Nyquist Sarah , Shalek Alex , Lin Philana , Flynn JoAnne L
Published: January 2023

Tuberculosis (TB) is the number one cause of death by a single infectious agent with no protective vaccine available. Challenges in vaccine development are partially attributed to a deficiency in knowledge regarding specific immune cells’ contribution to protection. CD8 T cells comprise ~30–40% of T cells in the lung granuloma, an organized collection of immune cells that is a hallmark of Mycobacterium tuberculosis (Mtb) infection. However, few studies have explored the diversity, function and contribution to protection of CD8 subsets during TB. We investigated the roles of conventional CD8αβ T cells and unconventional CD8αα T cells during early Mtb infection. Cynomolgus macaques were CD8-depleted using anti-CD8α or anti-CD8β antibodies prior to and during Mtb infection and necropsied at 6 weeks post-infection. CD8α depleted NHPs (CD8αα and CD8αβ depletion) had higher bacterial burdens, worse pathology and overall poorer disease outcome compared to IgG controls, while CD8β depleted (CD8αβ only depletion) displayed an intermediate phenotype. This provides the first evidence for an important early role for non-conventional CD8 T cells in TB. Using spectral flow cytometry we identified multiple CD8 subsets and assessed their functions in granulomas for the first time in NHPs. After CD8α depletion, the repertoire of CD8 subsets dramatically changed in granulomas. This was associated with functional alterations in other immune cell populations which we validated and expanded upon using single-cell RNA sequencing. These studies provide a functional immune landscape of early events in Mtb granulomas as well as the effects of CD8 depletion, shedding light on the protective potential of CD8 T cell subsets during primary Mtb infection.
Vancomycin intermediate strains of Staphylococcus aureus have dampened induction of type I interferon production
Authors: Peignier Adeline Carole Marine , Parker Dane
Published: January 2023

Studies by our group have shown that interferon (IFN) signaling contributes to pathogenesis of Staphylococcus aureus pneumonia and that different strains of S. aureus can activate type I IFN signaling via distinct receptors, such as TLR9 and NOD2, at various amplitudes. In this study we investigated the capacity of a wide range of S. aureus strains to induce both type I & III IFN. We screened 78 S. aureus strains from various clinical scenarios for their ability to induce IFN-β and IFN-λ in bone marrow-derived dendritic cells (BMDC) and found that both cytokines are differentially induced by various S. aureus strains independently of their isolation sites or methicillin resistance profiles. These induction patterns persisted over time and type I & III IFN differentially correlated with tumor necrosis factor α (TNF-α) production. Furthermore, vancomycin intermediate S. aureus (VISA) strains induced up to 45-fold less (P<0.001) IFN-β than the current epidemic methicillin-resistant S. aureus strain USA300 and displayed increased in vitro survival (up to 9-fold) after 4-hour exposure to BMDC. Further investigation of one VISA strain, HIP07930, showed decreased autolysis and increased resistance to lysostaphin compared to USA300, consistent with its reduction in IFN β induction. Interestingly, this strain also displayed decreased in vivo virulence compared to USA300 in a murine acute pneumonia model of infection (4.9-fold and 54.5-fold decrease in colony forming units in bronchoalveolar lavage fluid and lung homogenates, respectively, 24 hours post infection). Overall, this study demonstrates the heterogeneity of IFN induction by S. aureus and uncovered a unique property of VISA strains in their inability to induce type I IFN production.
Oral Probiotic Lactobacillus kefiri prevents Lipopolysaccharide-induced preterm birth, reduces inflammation and modulates the vaginal microbiota inpregnant mice
Authors: VENTIMIGLIA MARIA SILVIA , ALBAN MARLON IRWIN POZO , Valeff Natalin Jimena , Schander JULIETA , Quadrana Florencia , DE LOS ANGELES SERRADELL MARIA , Jensen Federico
Published: January 2023

Prevention and treatment of preterm birth (PTB) are an unresolved global challenge in obstetric care. Human PTB is defined as delivery prior to 37 weeks of gestation. It is known that inflammation is a contributing factor where an inflammatory cascade within the gestational tissues followed by a massive influx of leukocytes takes place. The efficacy of actual interventions are largely unsatisfactory. We have shown that treatment with probiotic Lactobacillus Kefiri (Lk) completely prevented PTB (100%) in a lipopolysaccharide (LPS)-induced PTB mice model. Given this, we sought to elucidate the mechanisms behind PB-Lk protective role in PTB induction.

C57BL/6 females were oral treated every 48h during a week with Lk or vehicle before being mated with BALB/c males and during pregnancy. Females were challenged with LPS on gd 16 (~3 days before expected term). Samples from gestational tissues were harvest 18 h after LPS. Leukocytes influx was evaluated by flow cytometry. Metalloproteinase-9 activity was measured by zymography. In addition, histological examination by light microscopy was performed. The impact on vaginal microbiota was evaluated by a qualitative analysis (PCR-DGGE).

Treatment with Lk, significantly lowered leucocytes influx induced by LPS as well as MMP-9 activity. Histological studies confirmed these findings. In placenta, Lk treatment ameliorated changes in micro and macrocirculation and DGGE profiles allowed differentiation of groups in two clusters, suggesting that Lk induced changes in the vaginal community composition. Our results demonstrate that Lk administration may have the potential to serve as a prophylactic therapy for inflammation-associated conditions during pregnancy, including PTB.
Gut microbial metabolites as therapeutics to treat of alcoholic liver disease
Authors: Jala Venkatakrishna R , Singh Rajbir , Chandrashekharappa Sandeep , Joshi-Barve Swathi , McClain Craig , Bodduluri Bodduluri , Vemula Praveen Kumar
Published: January 2023

Alcoholic liver disease (ALD) is resultant of excessive consumption of alcohol. ALD is the world’s third largest risk factor for diseases and disabilities and accounting for 5.9% of all deaths worldwide. ALD patients exhibit increased gut permeability, gut microbial dysbiosis, endotoxemia and hepatic steatosis. Recent studies, unequivocally demonstrated the importance of gut microbiota and their metabolites in regulating host pathophysiology. Urolithin A (UroA) is a gut microbial metabolite derived from dietary ellagic acid and ellagitannins containing foods. Previously, we showed that UroA act as an AhR agonist and activate AhR-Nrf2 pathways to induce tight junction proteins and enhance gut barrier function. In the current study, we tested the hypothesis that correcting gut barrier dysfunction, blocking inflammation and reducing hepatic steatosis simultaneously by UroA would provide better therapeutic options to treat ALD. Our studies suggested that UroA significantly induced expression of tight junction (TJ) proteins (Occludin, Cldn4 and ZO1) in AhR-Nrf2 dependent manner and protected from alcohol-induced downregulation of TJ proteins and reduced gut barrier leakage. Further, UroA attenuated ALD pathogenesis in both acute and chronic experimental mouse models by reducing alcohol induced barrier permeability, systemic endotoxin levels and inflammatory mediators. In these models, UroA treatment also significantly reduced alcohol induced liver ALT/AST levels, inflammatory mediators (TNF-α, IL-6, IL-1β) and triglyceride levels as well as prevented liver damage. In summary, these studies highlight that the mechanisms of microbial metabolite, UroA and its multi-pronged beneficial effects in attenuating ALD.
Discovery of a protective, transmissible, and type 2 inflammation-skewing intestinal microbiome
Authors: Grifka-Walk Heather M , Pinkham Nicholas V , Balasubramanian Narayanaganesh , Jenkins Brittany R , Looby Nicholas , Deuling Benjamin , Swain Steve D , Liss Hailey , Walk Seth T , Kominsky Douglas J
Published: January 2023

We recently discovered a transmissible, dominant, and remarkably protective intestinal microbiome (a “Magical Microbiome,” MM) in the setting of a murine model of colitis. The objective of this project is to fill current knowledge gaps regarding the molecular mechanisms underlying protection in this novel model of microbiome-mediated protection. Protection can be transmitted to unmanipulated conventional or germ-free C57Bl/6 mice via cohousing or oral gavage of MM+ stool. During DSS-induced colitis, MM+ mice develop less weight loss, intestinal pathology, and production of proinflammatory molecules relative to genetically identical C57Bl/6 MM-controls. Preliminary results suggest that the eukaryotic and bacterial microbiome under investigation re-programs the host inflammation to prevent disease or promote repair. MM-mediated protection is IL-13-dependent and associated with enhanced type 2 inflammation, as transcript levels of type 2-associated genes including IL-4, IL-13, and Fizz1 were significantly higher in colons of MM+ mice. 16s rRNA gene sequencing and eukaryote screening demonstrated that MM+ mice have a bacterial microbiome distinct from controls and host a novel Tritrichomonas species. Transfer of Tritrichomonas to wild-type mice correlated with protection, but vancomycin treatment ameliorates the protection while maintaining Tritrichomonas, suggesting a possible interaction between bacteria and protist. Stool metabolites are also distinct between MM+ and MM− hosts, further supporting a distinct microbiome function. Overall, we possess a unique microbiome that can be used to understand protective host-microbe interactions and identify novel therapeutic targets for inflammatory diseases.
Contribution of the Microbiome to Maintenance of the CD4:CD8 Ratio in Inbred and Congenic Mouse Strains
Authors: Sarafova Sophia D , Soden Kaitlin , Dankwa Sedem , Watt Aren , Thurtle-Schmidt Debbie
Published: January 2023

The gut microbiome is known to play an integral role in the regulation of the immune system. Previous studies indicate that species diversity and overall abundance of commensal bacteria in the gut influences immune system function by altering total T cell counts and the ratio of CD4+ to CD8+ T cells in peripheral lymphoid organs and blood. In normal individuals, the CD4:CD8 ratio is greater than one. However, the congenic B10.A mouse strain in our conventional facility consistently demonstrates an inverted CD4:CD8 ratio in a sex- and age-dependent manner, in contrast to both the parental C57Bl/10 strain and B10.A kept under SPF conditions. To determine whether this phenomenon is due to the composition and diversity of the gut microbiome affecting the homeostasis of the CD4:CD8 ratio, we altered the microbiome by supplementing the diet with the prebiotic inulin and looked for changes in the number of Peyer’s patches and changes in the CD4:CD8 ratio in gut lymphoid organs and blood in both B10.A and C57Bl/10 strains. In addition, we followed the changes in the microbiome upon treatment. We find that the mice in our facility have lower microbiome diversity and very different composition from mice kept at SPF conditions. The inulin treatment significantly increased the CD4:CD8 ratio in the blood of the C57Bl/10 mice, but not the B10.A mice. In addition, males of both strains had a significant increase in Peyer’s patch number, while the females had a significant increase in the CD4:CD8 ratio in the Peyer’s patches.
Novel decidual innate lymphoid cells display lineage plasticity
Authors: Vazquez Jessica , Fisher Rachel Catherine , Stanic Aleksandar K
Published: January 2023

Immune cells at the maternal-fetal interface play a complex role in regulation of vascular remodeling, fetal tolerance and protection from infection. We have previously reported the identification of two novel CD56high innate lymphoid cells (ILCs) based on Eomes (lo/hi) expression in the decidua. Preliminary experiments suggest that cytokines can modify the expression of canonical transcription factors (TF) by decidual ILCs.

Decidual specimens were dissected from term placentas and mononuclear cells (MCs) were isolated by mechanical/enzymatic disruption, then cultured in the presence of IL7/IL15, IL1β/IL23, or IL2/TGFβ for 7 days, with matched samples processed without culturing for ex vivo assessment. MCs were labeled with fluorochrome-conjugated antibodies against CD3, 14, 16, 19, 34, 45, 49a, 56, 94, 117, 127, GATA3, Eomes, RORγt, and Tbet. Data was acquired using the BD Fortessa flow cytometer and analyzed using FlowJo 10.2.

Our assessment of TF expression revealed significant differences between cytokine-stimulated cultured cells and direct ex vivo samples. GATA3 expression was highest in EomesLo (C2) and EomesHi (C10) cells cultured with IL7/IL15, compared to ex vivo cells. Interestingly, Eomes expression did not change across treatments, while Tbet expression was highest in C2/C10 cells cultured with IL7/IL15. Contrary to our expectations, culturing with IL1β/IL23 did not lead to higher RORγt expression, which was instead upregulated by IL7/IL15.

Preliminary results suggest that both C10 and C2 decidual ILCs demonstrate a level of lineage plasticity. Further analysis will reveal if these novel subsets have the capacity of transdifferentiating fully or if they maintain a limited phenotypic and functional capacity.