bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage inflammatory response increases in adult female exposed to neonate stress
Authors: Bouchard Karine , Patoine Dany , Fournier Stephanie , Lerdu Ophélie , Marsolais David , Kinkead Richard , Bissonnette Elyse , Lauzon-Joset Jean-Francois
Published: January 2023

Impacts of stress on the immune system vary depending on the nature, intensity, and time during which stress is experienced (eg, acute stress is pro-inflammatory vs chronic stress is anti-inflammatory). However, there is limited knowledge on the long-term impact of early life stress on immune responses. Given that immune programming occurs early in life and that immunity and stress both involve sexual dimorphisms, our hypothesis is that neonatal maternal separation (NMS) induces sex-specific immune alterations.

Objective

Our objective is to evaluate the influence of early life stress on immune response in adult and its dimorphism related to biological sex.

Methods

We used a well-established rat model of early life stress, NMS, in which we observed sex-specific modulations of the myeloid compartment. Thus, we evaluated the impact of NMS on macrophage activation using LPS stimulation of bone-marrow derived macrophages (BMDM) in vitro. Expression of activation (MHCII, CD40, CD86 and CD80) and maturation markers (SIRPα, CD11b, CD200 and CD200R) were analyzed by flow cytometry. TNF levels were measured by ELISA.

Results

LPS stimulation of female NMS BMDM had higher expression of activation markers compared to controls. Inflammatory cytokine levels, TNF, increased after LPS stimulation and further increased in female NMS, whereas male NMS level was lower than control male. Futhermore, LPS stimulation lowered CD200 receptor (CD200R) to a greater extent in male NMS than in female NMS.

Conclusion

These results suggest that NMS accentuates the pro-inflammatory response in adult female (but not in male) and will provide a strong base for improving sex-specific interventions in the context of diseases involving stress and the immune system.

Supported by grants from CIHR, QRHN and CRIUCPQ
Costimulation of TLR8 responses by CXCL4 in Human Monocytes Mediated by TBK1-IRF5 Signaling and Epigenomic Remodeling
Authors: Yang Chao , Bachu Mahesh , Brauner Caroline , Yuan Ruoxi , Du Yong , Ah Kioon Marie Dominique , Chesi Giancarlo , Barrat Franck J , Ivashkiv Lionel B
Published: January 2023

CXCL4 regulates responses of immune cells to endosomal TLRs and has been implicated in the pathogenesis of inflammatory and fibrotic diseases. However, mechanisms by which CXCL4 modulates TLR responses, and its functions in monocytes/macrophages, are still unclear. Here we report that CXCL4 changes the profile of the TLR8 response in human monocytes by selectively and dramatically amplifying inflammatory gene transcription and IL-1b production while partially attenuating the IFN response. Mechanistically, costimulation by CXCL4 and TLR8 synergistically activated TBK1/IKKe and repurposed these kinases towards an inflammatory response via coupling with IRF5, and by activating the NLRP3 inflammasome without the need for a second signal. CXCL4 strongly induced chromatin remodeling in a cooperative and synergistic manner with TLR8 signaling, inducing de novo enhancers associated with inflammatory genes. These findings identify signaling and epigenomic mechanisms that underly synergistic activation of inflammatory genes by CXCL4 and TLR8, provide a new paradigm for modulation of TLR responses that is relevant for cytokine storm, and suggest targeting the TBK1/IKKe-IRF5 axis may be beneficial in inflammatory diseases.
MicroRNAs modulate Chlamydia trachomatis infection- and vaccine-induced immunity in macrophages
Authors: Eko Francis , Bell Cortnee , Jones Leandra , Medhavi Fnu , Tanner Tayhlor , Omosun Yusuf , Igietseme Joseph U
Published: January 2023

Recent studies indicate that microRNAs (miRNAs), including immune cell type-specific miRNAs modulate inflammation and immune response to Chlamydia trachomatis (CT) genital infection. However, the role of these miRNAs in modulation of immune cell function following CT infection or vaccine stimulation has not been investigated. We hypothesized that pretreatment of CT-infected and vaccine-stimulated macrophages with miRNA mimics or inhibitors will modulate secretion of inflammatory cytokines and expression of IFN-γ pathway-related genes. Thus, THP-1-differentiated macrophages (TDM) transfected with miRNA mimics or inhibitors were infected with CT or stimulated with a Vibrio cholerae ghost (rVCG)-based vaccine (rVCG-MECA) and the level of inflammatory cytokines secreted in culture supernatants was assessed by cytokine ELISA. Also, TDM expression of IFN-γ pathway-related genes was assessed by RT-qPCR. The results show that CT infection of TDM resulted in the downregulation of miR-15a, miR-29a, miR-146a and miR-222 while stimulation with rVCG-MECA resulted in their upregulation, as we previously reported. The level of TNF-α, IL-8, IL-1β and IFN-γ secreted by miRNA mimic- or inhibitor-treated TDM following infection or vaccine stimulation was dependent on the miRNA type. Furthermore, CT infection of untreated macrophages increased mRNA expression of INFGR1, JAK2, STAT1 and SOCS1. However, pretreatment of CT-infected cells with miR-146a mimic or inhibitor showed significantly decreased expression of these genes. The study demonstrated that miRNAs differentially modulate macrophage secretion of inflammatory cytokines and expression of IFN-γ pathway-related genes following CT infection or vaccine stimulation.

Supported by None.
Anti-inflammatory Response Induced by Exopolysaccharide of the Probiotic Bacillus subtilis
Authors: Zamora-Pineda Jesus Bernabe , Knight Katherine L
Published: January 2023

Probiotics can lessen the severity of intestinal inflammatory diseases, although the mechanisms by which this occur are poorly understood. We showed that exopolysaccharide (EPS) from the probiotic Bacillus subtilis induces anti-inflammatory macrophages and protects mice from enteric infection and sepsis. Here, we examined the molecular mechanisms by which EPS induces an anti-inflammatory response. In co-cultures of OVA-loaded bone marrow-derived dendritic cells (BMDC) and OT-II T cells, EPS inhibits T cell activation in a TLR4-dependent manner. We hypothesize that EPS engages TLR4 and activates the noncanonical NF-kB signaling pathway, which up-regulates the expression of indoleamine 2,3-dioxygenase (IDO). IDO degrades the essential amino acid tryptophan into kynurenine, which leads to suppression of T-cell activation and induction of tolerance. Western blot analysis of BMDCs treated with EPS showed nuclear translocation of the transcription factor RelB, which is central to the noncanonical pathway and upregulation of IDO. Inhibition of IDO using the inhibitor 1-methyl tryptophan, and ido1−/− BMDCs reverse the effect of EPS. We conclude that EPS inhibition of T cell activation and proliferation is mediated by IDO. We have identified a novel mechanism for immune protection by a probiotic molecule.
Epigenetic regulation of inflammatory genes involving histone methylation is associated with hyperimmune response in COVID-19 patients
Authors: Yang Xiaoming , Yang Xiaoming C , Julian Julian , Schnell Timothy , Albrecht Helmut , Owens William , Yang Xiaoming S
Published: January 2023

One of the leading causes of the severe COVID-19 disease symptoms is the production of massive amounts of pro-inflammatory cytokines. Although the precise mechanisms that trigger such hyperimmune response is unclear, epigenetic mechanisms are known to play important roles in the regulation of cytokine production and inflammation. In this study we examined global gene expression and histone methylation profile in the PBMCs of hospitalized COVID-19 patients. Despite individual variations, the expressions of many inflammation-related genes such as Arginase 1 (ARG1) and IL1 receptor 2 (IL1R2) were significantly upregulated in COVID-19 patients. We also found the expressions of coagulation-related genes, VWF and Protein S, were altered in COVID-19 patients. The expression patterns of some genes such as IL1R2 correlated with their histone methylation marks. Pathway analysis revealed that most of these dysregulated genes were in the TGFb, IL1b, IL6 and IL17 pathway. Interestingly, we found that the majority of those altered genes were in the dexamethasone targeting pathway. We also found that the expression of BMX, a member of TEC family kinases, was significantly increased in the PBMCs of COVID-19 patient at both RNA level and protein level. Interestingly, some TEC kinase inhibitors have been used to treat COVID-19 patients in clinical trials. Overall, this study has identified genes in PBMCs of COVID-19 patients with altered histone mark and gene expression profile, providing important information towards identifying potential biomarkers and therapeutic targets for COVID-19 disease. (Supported by NIH grants P01AT003961, P20GM103641, R01AT006888, R01ES030144, R01AI129788 and R01AI123947 to P.S.N and M.N.)

Supported by NIH grants P01AT003961, P20GM103641, R01AT006888, R01ES030144, R01AI129788 and R01AI123947 to P.S.N and M.N.
12/15-lipoxygenase is required for inflammation resolution in a murine model of Lyme arthritis
Authors: Jackson Christa D , Brown Charles R
Published: January 2023

Lyme arthritis is a major sequela of disseminated Lyme disease that can persist despite antibiotic clearance of the etiological agent Borrelia burgdorferi. In the murine model of Lyme arthritis (mLA), infected mice develop an inflammatory arthritis characterized by robust innate cell infiltrate and synovial hyperplasia in the tibiotarsal joint, followed by spontaneous resolution around 4–5 weeks post-infection. Eicosanoids are bioactive lipid mediators that play a critical role in both the development and resolution of inflammation. As such, temporal regulation of eicosanoid class-switching from proinflammatory to proresolving is vital. In other inflammation models, late-phase cyclooxygenase (COX)-2 metabolite prostaglandin (PG) E2 skews the 5-lipoxygenase (LO) eicosanoid biosynthesis pathway away from leukotriene B4 production and towards proresolving lipoxin (LX) A4 production via 12/15-LO activity. Indeed, we have previously described a defect in mLA resolution in COX-2−/− and 5-LO−/− mice. Therefore, we hypothesize that 12/15-LO production of LXA4 is required to facilitate mLA resolution. To investigate this, we characterized mLA in 12/15-LO−/− mice and observed a pronounced resolution defect, as evidenced by significant ankle swelling and persistent innate immune infiltrate compared to wild-type mice after 4–5 weeks, despite efficient control of Borrelia burdens. We are currently investigating the mLA eicosanoid profile in 12/15LO−/− mice as well as whether exogenous treatment with LXA4 promotes mLA resolution. These experiments will determine how eicosanoids regulate inflammation resolution and may identify therapeutic targets for inflammatory diseases, including human Lyme arthritis.

Supported by a grant from the NIH (R21 AR175160) and the G. Ellsworth Huggins Graduate Fellowship.
Determining chicken immunoglobulin-like receptors (CHIRs) expression and their effect on immune response in a macrophage disease model
Authors: Sparling Brandi Ann Terrance Elize , Drechsler Yvonne
Published: January 2023

Immunoglobulin-like receptors are large transmembrane glycoproteins who direct the balance of the immune response. However, the full repertoire and effect of putatively activating, inhibiting, or dually working CHicken Immunoglobulin-like Receptors (CHIR-A, -B, and -AB, respectively) on immune responses in chickens, are not well understood. The objective was to characterize CHIRs expression and role in immune responses in the B-type homozygous chicken. B2B2 and B19B19 chicken lines were selected as model for their remarkable differences in disease susceptibility to coronavirus and other viral infections associated with the selectively bred major-histocompatibility complex class I (MHC-I), B locus. We first identified CHIRs from the new GRCg7b genome assembly by amino acids charge and protein motifs of CHIR -A, -B, or -AB. Subsequently, the transcriptomes of IFN-stimulated monocyte-derived macrophages from B2B2 or B19B19 were analyzed for CHIR abundance and ratio of CHIR-A:CHIR-B expression. Also, pooled siRNAs targeting CHIR-AB and or CHIR-B were generated for application in a monocyte-derived macrophage and T cell co-culture model. A higher median expression of CHIR-A in comparison to CHIR-B transcripts was observed in the B2B2 obtained macrophages; and inversely so for the B19B19 genotype 48-hours post-stimulation. Too, preliminary work show that CHIR-Bi enhances macrophage-mediated anti-viral response, endorsing CHIR-Bs suppressive role. These characterizations align with disease susceptibility traits of the B19B19 chicken line such that they link MHC and CHIRs, and highlight CHIRs in active infection and immunity.
Relationships between inflammation, coagulation, and oxidative stress in malaria-induced pregnancy compromise
Authors: Andrew Alicer Keendalyn , Moore Julie M
Published: January 2023

Plasmodium falciparum malaria in pregnancy contributes to poor birth outcomes in association with placental malaria (PM), a syndrome initiated by parasitized erythrocyte binding, precipitating placental oxidative stress, dysregulated inflammation, and coagulation. We have tested the hypothesis that these pathogenic processes are synergistic in PM using C57BL/6J (B6) mice infected with P. chabaudi. Ablation of tumor necrosis factor (Tnf), anticoagulant treatment, and infection in Tnf−/− and tissue factor-deficient (F3flox/flox Tie-2Cre) mice improves pregnancy outcomes. Relative to infected B6 controls, infected Tnf−/− mouse embryos show two to four-fold reduced transcript expression of a key molecular link between coagulation and inflammation, protease activated receptor 2 (F2rl1). Tissue factor (F3) expression is 50% reduced, while endothelial protein C receptor (Procr) transcripts are increased three to five-fold, supporting the bidirectional nature of coagulation and inflammation in PM. Moreover, embryonic transcripts for markers of inflammation (Ifng, Tnf, Il-10, Il-1β, and Ccl2), antioxidants (Sod1, Sod2, and Nfe2l2) and F3 are significantly downregulated in anticoagulant-relative to sham-treated, infected B6 mice, whereas Procr transcripts are elevated, suggesting functionally linkages of the three pathways. Ongoing antioxidant therapeutic studies are expected to confirm perturbations of inflammation and coagulation responses. In sum, this work is contributing to understanding of molecular interactions between the major pathogenic responses to PM, how synergy thereto galvanizes pregnancy compromise, and promises to reveal potential therapeutic options to improve pregnancy outcomes.
Sodium butyrate prevented mortality in Acute Respiratory Distress Syndrome through induction of anti-inflammatory response by regulation of epigenetic pathway
Authors: Sultan Muthanna Ali , Nagarkatti Prakash
Published: January 2023

Acute Respiratory Distress Syndrome (ARDS) is a life-threatening disease that has been identified following pneumonia, inflammation, as well as accompained with COVID19 cases. In this study, we investigated the effect of Sodium Butyrate (BUT) in ARDS induced by Staphylococcus Enterotoxin B (SEB). To that end, C3H/HeJ mice were administered BUT through oral gavage with a dose of 200mg/kg and 30mins later followed by a dual dose of SEB, starting with an intranasal dose of 7ug (i.n.) and 2hrs later with another dose of 2ug intraperitoneally (i.p.). The adminstration of BUT continued as same daily and the sacrifice was 72 hrs after SEB exposure. Our data demonstrated that the mortality rate was 80% in SEB+VEH mice compared to 0% mortality in SEB+BUT mice. Plethysmography data indicated a significant improvement in the clinical pulomonary functions. ELISA results indicated that the cytokine storm parameters including IFNγ were significantly decreased after BUT treatment compared to SEB+VEH. Furthermore, flow cytometry data indicated that CD4+ T cells, Vβ8+ T cells and NK T cells were significantly decreased in SEB+BUT compared to SEB+VEH. Analysis of miRNA from MNCs of lungs indicated that anti-inflammatory genes related to T-regulatory cells (Tregs) and Myeloid Derived Suppressor Cells (MDSCs) were targeted by many downregulated miRNAs which were then validated by qRT-PCR. Furthermore, flowcytometry data indicated a significant increase in T regs and MDSCs. Together, our data indicated that BUT prevented mortality and improved Lungs clinical functions in ARDS through modulation of the miRNAs in MNCs in lungs leading to induction of anti-inflammatory genes and suppression of inflammatory T cell subsets as well as increase in Tregs and MDSCs.

Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01ES030144, R01AI160896 and R01AI123947
Increased mitochondrial metabolism profile is required for immunologic function in age associated B cells
Authors: Kim Sun Jung , De Oleo Ivan Ramirez , Diamond Betty
Published: January 2023

Specific metabolic programs are activated by immune cells to fulfill their functional roles. Age-associated B cells (ABCs) are a subset of B cells that are identified by expression of non-canonical B lineage marker, CD11c. An expansion of ABCs has been identified in various autoimmune diseases, including systemic lupus erythematosus (SLE). They have many distinct properties, such as capacity to stimulate T cells and the ability to secrete autoreactive antibodies when they are stimulated in vitro. The metabolic pathways that support these functions are unknown. Using mouse models of human SLE, we confirmed ABCs induce T cell activation effectively and these activated T cells acquire follicular helper T (Tfh) cell phenotypes, including BCL6 expression, up-regulation of surface expression of CXCR5 and PD-1 and IL-21 production. Metabolically, ABCs are bioenergetically more active than follicular (FO) B cells, with higher rates of glycolysis and oxidative phosphorylation. They have increased mitochondrial activation with an increased production of reactive oxygen species (mtROS). Treatment of ABCs with mitochondrial complex I inhibitor or mtROS scavenger leads to a selective loss of their capacity to induce Tfh differentiation but not T cell activation or proliferation. The aberrant activation of mitochondrial metabolism and mtROS production was also consistently observed in ABCs from SLE patients as well. Therefore, ABCs have evolved a distinct metabolism adapted to their specific functional properties.

Supported by NIH NIAMS R01 AR075565 NIH NIAMS U19 AI144306
Rag GTPase critically contributes to humoral immunity independent of canonical mTORC1 signaling
Authors: Zhu Xingxing , Zhou Xian , Li Chaofan , Li Yanfeng , Sun Jie , Raybuck Ariel , Boothby Mark Robin , Zeng Hu
Published: January 2023

The humoral immune response requires that B cells undergo a rapid metabolic shift and high demand of nutrients, which are vital to sustain the formation of germinal center. Rag GTPase senses amino acid availability to activate mechanistic target of rapamycin complex 1(mTORC1) pathway and modulate the function of transcription factor EB (TFEB), a member of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, little is known about how Rag GTPase coordinates amino acid sensing, mTORC1 activation and TFEB activity in humoral immune response. Here, we show that B cell intrinsic Rag GTPase is critical to the development and activation of B cells. Disruption of Rag GTPase complex, but not mTORC1 complex, abrogates germinal center formation, antibody production as well as plasma cell generation upon respiratory influenza infection. Mechanistically, the Rag GTPase complex senses specific amino acids to suppress TFEB activity, independent of canonical mTORC1 activation. Collectively, our data support the idea that Rag GTPase critically contributes to humoral immunity partly through suppressing TFEB and it is largely not necessary for canonical mTORC1 signaling.

Supported by Mayo Foundation for Medical Education and Research
Mechanisms of TRAF3 regulation of TLR signaling in B lymphocytes
Authors: Ybarra Tiffany K , Bishop Gail A
Published: January 2023

TRAF3 is an adaptor protein that binds to a variety of receptor types on immune cells and exerts multiple regulatory functions in signaling pathways. TRAF3 functions are cell type and context dependent. B cell-TRAF3 inhibits homeostatic NFκB2 activation and other pro-survival signaling pathways; restraint of survival is a B cell-unique TRAF3 function. Loss of B cell-TRAF3 in vivo contributes to early autoimmunity and later lymphomagenesis. TRAF3 inhibits B cell TLR functions, including cytokine production (IL-6, IL-10, IL-12p40 and TNFα), Ig isotype switching, and antibody production in response to ligands for TLRs 3, 4, 7/8 and 9. A key knowledge gap is identifying the molecular mechanisms by which TRAF3 mediates this regulation. Our working hypothesis is that B cell-TRAF3 regulates the activity, recruitment, and/or post-translational modification of specific TLR signaling proteins. Our recent data show that B cell-TRAF3 forms a heterocomplex with TRAF6, associating with the tyrosine kinase Syk and inhibiting early TLR signals, such as pIRAK1 degradation. In the absence of TRAF3, TRAF6 shows a greater association with several TLR signaling proteins. These results suggest that TRAF3 may inhibit TRAF6 access to the TLR signaling complex, and thus early TLR signaling. Further studies aimed at providing new insights into the molecular mechanisms contributing to the regulation of B cell-TRAF3 in TLR signaling can inform selection and development of more targeted therapies for B cell malignancies and autoimmunity.

Supported by VA Merit Review IO1 BX001702 NIH RO1 AI62656 NIH P50 CA97274 NIH T32 Training Grant AI07485
Follicular B cells from old mice are hyper-responsive and produce non-specific antibodies.
Authors: Ticas Rodas Carlos J , Greer Braxton D , Maul Robert W , Gearhart Patricia J
Published: January 2023

Immunosenescence is the increase in immune defects that occurs during aging. To identify any intrinsic defects in old B lymphocytes, we devised a transfer system to introduce follicular B cells from old mice (FOO, >22 months) or young mice (FOY, 8–12 weeks) into young mMT mice which have no B cells. Using this system, we observed that, compared to FOY cells, FOO cells do not produce antigen-specific antibodies to nitrophenyl (NP)-chicken gamma globulin after immunization. Examination of the germinal center (GC) response shows that FOO cells produced increased numbers of GC B cells which have divided faster than their FOY counterparts, but they have significantly decreased NP+ λ+ antigen-specificity. To understand the differences between FOO and FOY cells, single-cell RNAseq was performed on naïve and day 14 GC B cells. Comparing naïve populations revealed very few differences with only 387 differentially expressed genes between FOO and FOY cells. Interestingly, Ccr6 was upregulated in the FOO cells, suggesting that the cells were pre-activated before transfer into mMT mice. Antibody repertoire analysis also confirmed the lack of antigen-specificity in FOO cells, as the canonical NP-specific IgH V-gene, 1–72, was utilized in fewer cells and had zero instances of the CDR2 W33L mutation known to increase affinity. Taken together, old mice produce B cells which are pre-activated and hyper-responsive upon immunization, causing a lack of proper selection and affinity maturation in the GC.

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.
Memory B cells are a heterogenous population regulated by epigenetic programming
Authors: Wiggins Keenan J , Scharer Christopher , Boss Jeremy M
Published: January 2023

Memory B cells (MBC) are a heterogenous population that consist of immunoglobin class switched and non-class switched MBC, and these populations can arise via germinal center dependent or independent mechanisms. The timing of MBC development influences the fate outcome of MBC populations. These different populations of MBC are regulated by cell signaling, but it is not clearly defined what epigenetic factors influence MBC differentiation. EZH2 is an important histone methyltransferase that catalyzes H3K27me3 resulting in gene repression. EZH2 has been shown to regulate B cell differentiation into germinal centers and plasma cells; however, it is unknown if EZH2 regulates MBC development. To address this, a knockout model has been established where EZH2 is conditionally deleted using the CD19 and AICDA driven CRE expression. Here we used the influenza PR8 model to ascertain the kinetics of MBC differentiation and formation in the spleen, dLN, and lungs following a live infection in wild-type and EZH2-KO cohorts. Using B cell tetramers, antigen-specific MBC will be analyzed to define the kinetics of MBC development and determine the similarities and differences between the different populations of MBC. Overall, these data define the early kinetics of MBC establishment and the developmental timing that EZH2-dependent H3K27me3 remodeling is required for MBC formation.

Supported by NIH/NIAID to CDS (R01 AI148471)
Decreased CD40L through disrupted transcriptional stability affects the autoimmune response in a sex-dependent manner in a GVHD model of SLE
Authors: De Maio Diego Javier Prado , Ganapathi Usha , Covey Lori
Published: January 2023

Increased levels of CD40L have been observed on CD4 T cells of patients with systemic lupus erythematosus (SLE) and in several murine models of lupus-like disease. Notably, elevated/prolonged expression of CD40L corresponds to the expansion and survival of autoreactive B cells. However, targeting CD40L as a potential therapeutic has been challenging due to the development of off-target events. We have developed a unique mouse model termed CD40LΔ5 (Δ5) which lacks a stability element in the 3’ UTR of the CD40L message and upon immunization, shows decreased CD40L expression on Tfh cells leading to both reduced GC B cell survival and a critically disproportional loss of pre-memory B cells. Using our Δ5 model, we sought to determine whether reduced CD40L could substantially alter the development of disease in an adoptive transfer model of lupus based upon MHC II incompatibility. B6 WT mice were engrafted with CD4 T cells from bm12-WT or bm12-Δ5 mice and analyzed for splenic lymphocyte subsets. In mice that received Δ5 T cells, total CD4 and CD8 subsets were unchanged compared to mice receiving WT cells, however, there was an increase and decrease of Tfh cells in male and female mice, respectively. This was confirmed by a corresponding change in Tfh proliferation as measured by staining for Ki67. Total CD19 B cells were increased in male mice receiving D5 T cells. Finally, intracellular staining of T cell cytokines was different in male and female mice receiving the Δ5 T cells as was the distribution of GC B cell subsets, including pre-memory B cells. Together our results suggest that limiting CD40L in T cells at an early stage of bm12-induced lupus has a significant effect on the maturation of GC subsets that is closely aligned with the sex of the WT recipient mice.

Supported by AAI Careers in Immunology Fellowship 2019-2020
Tet2-mediated programing balances T follicular helper cell and T helper 1 cell differentiation.
Authors: Hale J Scott , Baessler Andrew , Novis Camille L , Shen Zuolian , Perovanovic Jelena , Wadsworth Mark , Sircy Linda M , Harrison-Chau Malia , Varley Katherine E , Tantin Dean R
Published: January 2023

The differentiation of CD4+ T cell subsets in response to infection has been studied extensively, however the epigenetic programs that regulate these processes remain poorly understood. Active demethylation by tet methylcytosine dioxygenases of CpG dinucleotides within DNA is a key component of epigenetic programing that promotes lineage specific gene expression and contributes to cellular differentiation and function. Here we report that Tet2 acts to restrict the differentiation of T follicular helper (Tfh) cells in CD4+ T cells responding to viral infection. Using an adoptive transfer model of virus-specific CD4+ cells we found that Tet2-deficient CD4+ T cells skew away from the Th1 lineage and instead preferentially differentiate into highly functional germinal center (GC) Tfh cells that provide enhanced help for B cell responses. We found that the impact of Tet2-mediated programing on CD4+ T cell differentiation is cell intrinsic and the shift in lineage differentiation occurs as early as 2 days post infection. Using genome-wide expression, DNA methylation and transcription factor binding analyses, we found that Tet2 coordinates with multiple transcription factors to mediate the demethylation and expression of their target genes following activation. Our findings establish Tet2 as an important regulator of Tfh cell differentiation and reveal pathways that could be targeted to enhance GC responses against infectious disease.

Supported by grants from NIH (R01 AI137238 to J.S.H., T32 AI055434 to L.M.S., and T32 AI138945 to A.B.
Microbiota regulation of development of thymic microbiota-specific T cells
Authors: Ruiz Daniel Fernando Zegarra , Kim Dasom V , Saldana-Morales Fatima B , Chen Amanda , Wu Wan-Jung , Longman Randy S , Bettini Matthew L , Diehl Gretchen E
Published: January 2023

While exposure to microbial antigens in peripheral sites causes expansion of antigen-specific T cells, a role for antigen-specific expansion had not been identified during T cell development in the thymus. We recently found that intestinal colonization induced intestinal dendritic cells migration to the thymus, driving thymic expansion of microbiota-specific T cells early in life. In contrast, colonization in adulthood leads to expansion of peripheral but not thymic microbiota-specific T cells. Thymic microbiota-specific T cells developed early in life were undifferentiated and caused intestinal inflammation in immunocompromised hosts upon antigen reencounter. We sought to understand regulation of this developmental window and define if thymic expansion of microbiota-specific T cells could be restarted in adult mice. We found that microbiota manipulation reestablished expansion of microbiota-specific T cells in the thymus of adult mice. We are in the process of characterizing these T cells in adulthood to assess their impact on intestinal inflammation. Our data suggests that intestinal colonization controls the generation of thymic microbiota-specific T cells which has the potential to modulate inflammatory processes.
B cells drive tertiary lymphoid organ formation in ileal inflammation
Authors: Erlich Emma , Czepielewski Rafael , Kumar Shashi , Field Rachael , Zhang Xinya , Saleh Leila , Guilak Farshid , Brestoff Jonathan , Ellebedy Ali , Randolph Gwendalyn J
Published: January 2023

Crohn’s disease [CD] is one of the two most common forms of inflammatory bowel disease, affecting over half a million Americans. Like many other diseases with chronic inflammation, some patients with CD develop tertiary lymphoid organs [TLO] in areas of the gastrointestinal tract with active disease. TLOs are organized clusters of lymphocytes, similar in structure to secondary lymphoid organs, though they develop after birth and their contribution to pathogenesis in CD, or other diseases, is unclear. We, and others, have also found B cell rich lymphoid aggregates in the mesenteric fat of CD patients along dramatically remodeled lymphatic vessels. TNFΔARE/+ is a murine model of ileal inflammation that recapitulates several key features of ileal CD, including development of mesenteric tertiary lymphoid organs. Use of this model revealed that mesenteric TLOs block cellular and molecular export from the gut, leading us to wonder if mechanisms that interfere with their development might reduce ileitis. TNFΔARE/+ mice that lack B cells revealed that B cells are required for tertiary lymphoid organ formation in this model. Without these TLOs, lymphatic outflow from the intestine was restored. Nonetheless, histological and flow cytometric approaches reveal no difference in local inflammation in the ileum. However, systemic inflammation, as assessed by metabolic cages and changes in body weight over time, increased. This suggests that TLOs may act to trap inflammatory signals locally, preventing systemic dissemination of inflammatory cells or mediators.

Work supported by NIH grants DP1-DK109668-04 and T32-DK077653-27 and the Kenneth Rainin Foundation
Expression of a BTB-ZF transcription factor controls the function of an innate-like Tregs essential for intestinal homeostasis
Authors: Krzyzanowska Agata K , Haynes Rashade A , Kovalovsky Damian , Lin Hsinching , Edelblum Karen L , Corcoran Lynn M , Rabson Arnold B , Denzin Lisa K , Sant’Angelo Derek B
Published: January 2023

The BTB-ZF genes are a family of 49 transcription factors that control the lineage specification and development of key effector functions of different subsets of lymphocytes. Using a single-cell BTB-ZF transcription factor expression analysis, we identified a unique subset of T regulatory cells (Tregs). These Tregs are genetically and phenotypically distinct from the larger conventional Treg population. For instance, analogous to natural killer (NKT) cells they have an activated phenotype (CD62Llo, CD44hi) and constitutively express IL-10. Interestingly, these characteristics are acquired early during T cell development. The defining transcription factor is induced during thymic development, binds the IL-10 promoter, and enables T cells to rapidly produce the cytokine. The subset of Tregs is enriched in the intestine and expands during acute colitis. The deletion of the defining transcription factor in T cells results in intestinal epithelial layer damage causing loss of the barrier function making mice highly vulnerable to severe disease and death from induced colitis. Moreover, in the absence of the transcription factor in T cells, normal intestinal macrophage responses are disrupted most likely due to decreased production of IL-10 by the Tregs. Therefore, by profiling BTB-ZF transcription factor expression, we have identified a distinct subset of T cells with potent immunosuppressive abilities that are essential for the health of the intestine. Numerous aspects of these Tregs are parallel to NKT cells, indicating an existence of “naïve” Tregs that have innate-like effector functions acquired independently from the differentiation in the periphery.

Supported by NIH/NIAID R01 AI122757-01 Careers in Immunology Fellowships – 2021
Regulatory T cell coordination of the mucosal NK cell response during viral infection.
Authors: Vick Sarah C , Lund Jennifer M
Published: January 2023

In many infections the mucosal barrier is the site of pathogen exposure, yet the regulation of mucosal immunity is not well understood. Herpes simplex virus-2 (HSV-2) is one of the most prevalent mucosal infections, making this disease an ideal model to study mucosal anti-viral immune response. My goal is to define how regulatory T cells (Tregs) shape anti-viral immune responses through modulation of natural killer (NK) cells during mucosal virus infection. To accomplish my goal, I utilized a mouse model of intravaginal HSV-2 infection. NK cell phenotypes were examined using high parameter flow cytometry. To define how transient Treg depletion during HSV-2 infection impacts NK cells, I utilized Foxp3DTR mice. Treg depletion led to increased maturation of NK cells in the vaginal tract (VT). KLRG1 is associated with NK cell maturation and is a marker of terminally mature NK cell subsets. I found a significant increase in the frequency and total number of KLRG1+ NK cells in the VT two days after HSV-2 infection in the absence of Tregs. I further analyzed NK cell maturation using the expression of the two surface markers CD11b and CD27 and found a significant increase in CD11b+CD27+ NK cells after Treg depletion. This subset of NK cells is known to be significant producers of pro-inflammatory cytokines. The cytokine IL-15 is necessary for NK cell development and maturation. I found that in the VT, CD11b+Ly6C+ monocyte-derived DCs display increased IL-15 transpresentation after Treg depletion. My data suggests that Tregs have a modulatory effect on NK cell development in the VT as Treg depletion leads to altered NK cell maturation and development. This demonstrates the role of Tregs in mediating an effective immune response while limiting immune pathology.

Supported by grants from NIH (R01 AI141435-01, T32 AI 7509-21)
Imaging the lung microenvironment during Coccidioidomycosis
Authors: Miranda Nadia Esmeralda , Diep Anh , Tejeda-Garibay Susana , Hoyer Katrina K
Published: January 2023

Valley fever is a respiratory disease caused by the fungal pathogen Coccidioides. Very little is known about how the fungus interacts with the immune system and the lung microenvironment, hampering vaccine and therapy development. There is a critical need to identify which immune cells are interacting with the fungus in the lungs and how these cells control infection. Granulomas form within the lung to control infection, but the formation, maintenance, and molecular and immune players in these processes are largely unexplored. Granuloma cell mass can be identified in X-rays and MRI scans only to be misdiagnosed for tumors, until a lung biopsy is analyzed. To investigate immune cells present in Coccidioides granulomas, we utilized tissue immunohistochemistry via fixed-formalin paraffin-embedded (FFPE) and fresh frozen embedded samples to assess immune cell and Coccidioides interactions. Various labeling methods have been investigated using infected mouse lungs to optimize procedures before switching to patient samples. Hematoxylin and eosin (H&E) staining combined with periodic acid–Schiff (PAS) identifies Coccidioides spread within the lung following fungal inhalation in mice. Lung imaging challenges include the presence of endogenous peroxidases that cause non-specific antibody binding. Antigen retrieval following paraffin embedding, have now been optimized to fit the lung and specific to the antibody that is being used. Here we highlight various troubleshooting methods with successful immunofluorescence FFPE lung imaging.

Supported by University of California Office of the President grant VFR-19-633952 & R21
Lung-resident memory CD4 T cells recognizing antigen in the airway enhance the kinetics of antigen presentation and naive T cell activation in draining lymph nodes
Authors: Finn Caroline M , Prokop Emily , McKinstry Karl Kai
Published: January 2023

CD4+ tissue-resident memory (TRM) T cells act as sentinels at previous sites of infection and rapidly initiate protective inflammatory responses upon antigen re-encounter. Here, we tested if beyond local impacts in the lung, TRM responses lead to faster egress of antigen-loaded dendritic cells (DC) resulting in their more rapid accumulation in draining lymph nodes (DLN). To do so, we used a mouse model originally developed to examine CD4+ TRM-mediated protection against influenza virus infection. We established a TRM population in otherwise naïve mice by intranasal transfer of memory CD4 T cells specific for ovalbumin (OVA). The mice were then challenged with fluorescently-labelled OVA protein intranasally. Faster migration from the lung to the DLN by OVA+ DC was seen within 48 hours of protein administration in mice receiving TRM compared to controls without TRM transfer. We hypothesized that the enhanced kinetics of OVA presentation in DLN mediated by TRM antigen recognition would result in more rapid systemic activation of OVA-specific CD4 T cells. We thus transferred an indicator population of naïve OVA-specific CD4 T cells intravenously to mice receiving TRM transfer or not prior to OVA protein administration. Faster kinetics of naïve T cell activation were seen in recipients of TRM within 3 days as judged by expression of activation markers and proliferation. Most importantly, by day 4, responding naïve cells reached the lung in greater numbers and were more functional in mice receiving TRM. Our results reveal that CD4+ TRM activation can enhance the kinetics of antigen presentation in DLNs and suggest this mechanism can be harnessed by vaccines to fully integrate protective local and systemic CD4 T cell responses.

Supported by grants from NIH (R21 R21AI148882)
Mycobacterium avium via oral route provide enhanced protection against pulmonary tuberculosis: An essential role of B cells and germinal centers
Authors: Dutt Taru S , Karger Burton Richard , Fox Amy , Youssef Nathan , Dadhwal Rhythm , Obregon-Henao Andres , Henao-Tamayo Marcela
Published: January 2023

Human tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis (Mtb) and represents an enormous challenge to global health because of the inadequacy of currently available drugs and vaccines. The most common clinical manifestation is pulmonary TB, and the Bacille Calmette Guerin (BCG) is the only licensed vaccine for protection against TB; however, its efficacy is highly variable. Today at least 52 countries have reported MDR-TB and XDR-TB cases which cannot be cured or contained by current TB therapy. Thus, there is an urgent need to develop new therapies/vaccines that effectively prevent or cure TB. In the last decade, lymphoid follicles (LFs) having ectopic GCs (eGC) have been identified in the lungs of Mtb infected animals and humans; however, a mechanism of its formation and role has not been adequately characterized. Our studies found that mice vaccinated with BCG and exposed to Mycobacterium avium [a non-tuberculous mycobacterium (NTM)] via drinking water provide long-term protection from pulmonary TB than BCG alone as determined by reduced Mtb bacterial burden and inflammatory progression of infection. Interestingly, these mice also developed eGC in the lungs and have increased B cells, anti-Mtb cell lysate specific IgA, and IgG antibodies. These findings suggest that NTM and B cells play a critical role in generating protective immunity against pulmonary Mtb infection, and the formation of eGC in these mice is a crucial factor in this improved immunity. Investigating the mechanism of eGC formation and the role of NTM and B cells in its stimulation is an important question to understand TB pathogenesis and to develop effective vaccines and therapies.

Supported by NIH/NIAID - 1R01AI127475-01AI, Colorado State University’s MIP research team grant
Low and transient salivary neutralizing antibodies after COVID vaccination
Authors: Nahass Georgie , Salomon-Shulman Rachel , Sheikh-Mohamed Salma , Blacker Grace , Haider Kazim , Blish Catherine A , Sanders Erin , Gommerman Jennifer L , Weissman Irving L , Tal Michal Caspi
Published: January 2023

Neutralizing activity found in the blood following intramuscular vaccination protects against systemic disease from SARS-CoV-2 infection. Mild breakthrough mucosal infections in vaccinated individuals can contribute to transmission chains of infection, and as such the contribution of intramuscular vaccination to mucosal immunity is important to understand. We assessed vesicular stomatitis virus (VSV) SARS-CoV-2 SPIKE protein neutralization activities as well as anti-viral spike and RBD IgG and IgA isotype antibody binding, in saliva from individuals who had recovered from SARS-CoV-2 infection and people who received immunization. The various vaccination strategies deployed globally showed key differences in the extent of neutralizing activity that could be measured. Even in subjects with significant peak activity 2–4 weeks following a second dose of mRNA vaccination, this neutralizing activity was transient and significantly reduced within 3–6 months. Among adenoviral vectors, ChAdOx1 elicited significantly more salivary neutralizing activity and antibody levels compared to Ad26.S. A large range of salivary neutralizing activity is induced following different intramuscular SARS-COV-2 vaccination strategies. All were orders of magnitude lower than neutralization levels observed in blood, and it remains to be determined if any of the observed neutralizing activity in the saliva can neutralize virus in the upper respiratory tract upon exposure. Based on studies where high levels of mucosal IgA antibodies were induced in response to stimulation with aerosol intranasal vaccines, such vaccines could be important in lowering transmission of SARS-COV-2.

Supported by The Fairbairn Foundation and SPARK
RAMP3 unexplored relevance for innate-T cell immunity
Authors: Ascui-Gac Gabriel Antonio , Mendis Alba , Phung Eleni , Chandra Shilpi , Han Jihye , Caron Kathleen , Kronenberg Mitchell
Published: January 2023

RAMP3 is a chaperon protein that aids in the transport, activity modulation and pharmacological switch of G-protein coupled receptors (GPCR). Chemokine receptors involved in immune cell recruitment and activation are GPCRs, but the role of RAMP3 in the function of the immune system has not been explored. Our lab found that Ramp3 gene is highly expressed in innate-like T cells in the lungs of mice, particularly for Mucosal-associated invariant T (MAIT) cells. Ramp3 KO mice have an increased absolute number of MAIT cells, while other innate T cells are not affected. These differences are even higher after vaccinations with a live attenuated Salmonella strain containing MAIT-cell antigens. Both at steady-state and after vaccination, Ramp3 KO mice MAIT cells have increased levels of CXCR6, a chemokine receptor that has been associated to lung homing and T cell maintenance in the periphery. Ramp3 KO mice also have a decreased proportion of IL17A-producing MAIT cells in the lung; IL-17-producing innate cells have been shown to be protective, for example following Streptococcus pneumoniae infection. Consistent with the MAIT17 decrease, there was increased bacterial burden after lung infections. While Ramp3 has been reported in several gene signatures related to T cell regulation, its role for T cell immunity has not been explored. This is the first evidence of the relevance of Ramp3 for immune cell function and host protection, influencing innate-like T cell mediated immune function and protection against lung bacterial infections.

Supported by grants from NIH 5R01AI137230-04
Leveraging Resident Memory T Cells to Fortify Oral Immunity
Authors: Stolley Michael , Masopust David
Published: January 2023

Resident memory T cells (TRM) durably survey barrier tissues for reinfection and orchestrate collaborative immune responses upon activation. Importantly, peptides derived from previously encountered viral infections can locally reactivate TRM. This biology may have convenient therapeutic ramifications for augmenting tissue-specific immunity. Yet, while TRM have been well-characterized in the gut, skin, and urogenital mucosa, the mouth remains a barrier tissue largely overlooked by T cell biologists. We asked whether TRM in the oral mucosa could be reactivated using virus-mimicking peptides, and if so, how that might impact oral immunity. This question was addressed using a novel prime-pull model for generating preternaturally abundant TRM in the mouths of SPF mice to manipulate and study. Oral TRM reactivation perpetuated a robust oral anti-pathogen state, including induction of key antiviral and interferon-stimulated genes, and recruitment of innate and adaptive cells into the oral mucosa. Oral peptide pre-exposure thwarted infection with an antigenically unrelated virus. Thus, mouth-resident T cells are amenable to local peptide reactivation, and their proinflammatory potential can be intentionally deployed to bolster oral immunity.

Supported by T90 DE 022732 K99DE031014 OMIC Pilot Grant