bluuebunny/crossref_metadata_2025 huggingface dataset:
A cancer-associated gene signature bifurcates healthy subjects into cancer-associated and cancer-distinct phenotypes
Authors: Scheid Adam , Van Keulen Virginia , Felts Sara , Zhang Yuji , Neier Steven C , Block Matthew , Markovic Svetomir , Mansfield Aaron , Pease Larry R
Published: January 2023

In the context of immunity, genomic sequence, epigenetic, and environmental factor diversity combine to propagate variable gene expression and immune function among individuals. We hypothesize that we can harness gene expression variability across people to study molecular immune phenotypes and predict individual responses to immune stimuli. To test this we used RNA-Seq to derive a set of genes whose expression is stable over time and differential among healthy people in peripheral blood CD4+ cells. This gene set, which we term the molecular immune response signature (MIRS), identifies transcriptomic “fingerprints” in each healthy person. The same gene set also identifies unique signatures in independent stage IV melanoma patients, and, intriguingly, groups the cancer patients with a particular subset of healthy people. We further clarified the subset by selecting MIRS genes that differentiate it from the other healthy subset. To test if a larger population of melanoma patients grouped with the same healthy subset we clustered additional independent patients with the healthy subsets using these genes. Each of the patient samples grouped with the cancer-associated healthy subset (p=0.00195). We also tested if the gene set would segregate an additional independent cohort of healthy donors into two subsets and found that it did. This, and the fact that no known demographics differentiate the healthy subsets, suggests the observed healthy phenotype may relate to a cancer-like phenotype. Pathway analysis implicates Th1-skewing in the cancer-associated healthy subset. Future directions include further understanding the biological impacts of the phenotype and using the MIRS to predict individual responses to immunotherapeutics.
The Role of Cbl-b in CD4+ T Cell Resistance to Regulatory T Cell Suppression
Authors: Han SeongJun , Chapatte Laurence , Ohashi Pamela S
Published: January 2023

Cancer immunotherapy has demonstrated enormous potential as a novel therapeutic modality in the management of multiple malignancies, including melanoma and leukemia. While cancer immunotherapy is designed to enhance effector T cell function and promote tumor destruction, one of its limitations is the negative influence of tumour-resident CD4+ FoxP3+ regulatory T (Treg) cells on T cell anti-tumour activity. Previous attempts to overcome this challenge have largely focused on the depletion of Treg cells, but this has proven to be a challenging and largely ineffectual process. An alternative approach to overcoming this problem involves generating effector T cell resistance against Treg cell-mediated suppression. Work in this area to date has shown that deficiency in the E3 ubiquitin ligase, Cbl-b, in mice results in the development of hyper-proliferative and pro-inflammatory effector T cells, even in the presence of Treg cells. This study investigates the cellular mechanisms behind this Treg resistance using Cbl-b deficient CD4+ FoxP3− and CD8+ T cells. We demonstrate that Cbl-b −/− CD4+ FoxP3− T cells exhibit both hyper-secretion of and hyper-sensitivity to IL-2, and that this serves as an important mechanism to escape suppression by Treg cells. Accordingly, we show that the blockade of IL-2Rα is sufficient to reverse the Treg cell-mediated suppression and excess IL-2 alone is sufficient to override suppressive signals by Treg cells. Overall, this study provides further insight into the potential development of Treg resistant effector T cells able to generate a more robust anti-tumor immune response.
Microbiota upregulate distinct APC functions in each intestinal dendritic cell subset, in a Myd88-independent fashion
Authors: Hammer Gianna , Liang Jie , Huang Hsin-I , Benzatti Fernanda , Karlsson Amelia , Hale Laura P.
Published: January 2023

While dendritic cells (DCs) are known to control T cell responses to pathogens, how dynamics between DCs and microbiota impact mucosal T cells is poorly defined. To understand how microbiota impact APC functions of intestinal DCs, we use DCs lacking A20, a signaling attenuator, to amplify DC responses to microbiota. We find each intestinal DC subset possesses unique APC functions that enforces strict subset-specific rules to DC-instruction of either IFNg+ or IL-17+ T cells. While CD103+ CD11b− DCs have potent abilities to instruct IFNg+ T cells, these DCs do not induce IL-17+ T cells. In mirror image, CD103+ CD11b+ DCs induce robust numbers of IL-17+ T cells, but induce no IFNg+ T cells. By contrast, CD103− CD11b+ DCs possess dual abilities to instruct IFNg+ and IL-17+ T cells. Interestingly, distinct mechanisms govern IFNg- and IL-17-inducing APC functions of intestinal DCs: While CD80/86 is required for DC-instruction of IFNg+ T cells, DC-instruction of IL-17+ T cells requires IL-6. We further show that MyD88, although notorious in ability to upregulate APC functions, are not required to upregulate APC functions of intestinal CD103+ DCs. In support of this, we find that mice with DC specific loss of both A20 and MyD88 spontaneously develop inflammation of small intestine. Finally, we ablate CD103− CD11b+ DCs and CD103+ CD11b+ DCs and find that these DCs are required for expansion of pathological Th17 cells. These results suggest that microbiota trigger distinct APC functions in different DC subsets and that in the context of intestinal inflammation and disease, there are defined teacher-student relationships between specific DC subsets and specific mucosal T cells.
TLR10 is a Negative Regulator of Human Monocyte Innate Immune Responses
Authors: Hess Nicholas J , Felicelli Christopher , Grage Jennifer , Mercado Reinaldo , Tapping Richard I.
Published: January 2023

Toll-like receptors (TLRs) are important pattern recognition receptors involved in the activation of innate immune responses against foreign pathogens. Humans possess ten TLRs, of which TLR10 is the only remaining TLR without a known ligand, signaling pathway or cellular function. Recent data by our lab has shown for the first time a suppression of cellular innate immune responses in primary human monocytes that is mediated by a TLR10 monoclonal antibody. This study sought to expand on our initial observations by investigating the signaling pathways, gene expression and long-term consequences of TLR10 engagement on primary human monocytes. Our data shows that TLR10 engagement is sufficient to suppress both IL-6 and TNF-α induction in response to TLR4, TLR7/8 or CD40 stimulation. Cellular lysates show that the primary target of TLR10 signaling may be to inhibit phosphorylation of Akt and its downstream targets. To further this hypothesis, we have performed RNA sequencing on cellular extracts to determine which pathways are targeted by TLR10 signaling. Our data also suggests that these events also affect long-term differentiation of monocytes into macrophages and dendritic cells. Macrophages and dendritic cells differentiated in the presence of a TLR10 antibody exhibit suppressed differentiation markers such as CD80/CD86 and HLA-DR/CD16 respectively after four days of differentiation. These data show that TLR10 is a novel regulator of innate immune responses on primary human monocytes and may have eventual therapeutics benefits in chronic inflammatory and auto-immune diseases.
Comparative proteomic analysis of chicken macrophages stimulated with Salmonella Lipopolysaccharide (LPS) and Monosodium Urate (MSU) Crystals
Authors: Makkar Sarbjeet K , Liyanage Rohana , lay jackson o , RATH NARAYAN C
Published: January 2023

Immune system cells are armed with recognition receptors by means of which they can sense pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). Interaction of pathogen as well as damage associated ligands with their cognate receptors drives transcriptional and translational changes. To study the proteomic differences between signal transduction pathways involved in microbial induced and sterile injury, we used chicken macrophages HTC as a model system. Cells were labeled and treated with LPS or MSU by using SILAC proteomic approach. After 20 hrs of stimulation, the protein was extracted from secretome and cell lysate, quantified and subjected to in-solution digestion followed by liquid chromatography/tandem mass spectrometry. We quantified 150 proteins using Skyline software, 30 were upregulated and 18 down regulated in LPS treated cells and 40 upregulated and 11 down regulated in MSU treated cells. Interleukin 8, a secretory protein is differentially upregulated in LPS group. Cathepsin K, vimentin, kinases are some of the significantly upregulated cell lysate proteins in LPS group. Lactate dehydrogenase, heat shock, microtubule, annexin are significantly upregulated in MSU stimulated cell lysate. Functional annotation of proteins in both the groups by gene ontology software shows most of the proteins are involved in cytoskeleton remodeling, cell migration and cell signaling, which may be important in immune activation except nucleic acid binding transcription activity factor proteins which are uniquely expressed in LPS group. In conclusion our study gives an insight about the functional differences between pathogen and sterile associated inflammation at cellular level.
Different characteristics of Myeloid-derived suppressor cells from septic and tumor-bearing mice
Authors: Shu Qiang , Qin Chaojin
Published: January 2023

Background

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells and immature myeloid cells. These cells have immunosuppressive properties and express myeloid differentiation markers Gr-1 and CD11b in mice. The characteristics of MDSCs in different pathological conditions remain unclear.

Methods

Polymicrobial sepsis was induced by cecal ligation and puncture. For tumor model, mice were injected s.c. in the flank with 5×105 3LL or B16 tumor cells. Splenic Gr-1+ CD11b+ MDSCs were harvested from 7-day septic mice and three-week tumor-bearing mice, which purified using magnetically assisted cell sorting.

Results

MDSCs from tumor (3LL and B16)-bearing and septic mice shared a common phenotype and morphology. In contrast to tumor-bearing MDSCs, septic MDSCs were mainly characterized as neutrophil like-MDSCs, which expressed lower levels of CD115. Tumor bearing MDSCs have a greater inhibitory effect on Ag-induced CD4+ T cell proliferation than those from septic mice, and expressed higher levels of ARG-1. In addition, septic MDSCs significantly induced macrophage to M1 (F4/80+ CD206−) and expressed proinflamatory cytokine IL-6, whereas tumor-bearing MDSCs induced macrophage to M2 (F4/80+ CD206+) and expressed anti-inflammatory cytokine TGF-beta.

Conclusion

Sepsis derived MDSCs are functionally different from tumor-bearing MDSCs, which suggests there different functions in the disease’s progress.
Notch-1 regulation of microRNAs alter the expression of proinflammatory cytokines and Th1 response
Authors: Chandiran Karthik , Minter Lisa M
Published: January 2023

Notch signaling is required for activation and differentiation of T cells. Upon its S3 cleavage by gamma secretase, Notch intracellular domain triggers the expression of various molecules depending on its binding partners. The data published from our lab states that Notch1 can induce Th1 immune response and regulates the expression of IFNγ by modulating the expression of the transcription factor T-bet. However the detailed mechanism behind Notch1 mediated Th1 differentiation is not clear. Here we report that Notch1 can regulate Th1 differentiation by targeting microRNAs. miR-29 is a family of micro RNAs (miR29a,b,c) that targets ifng and both its transcription factors tbx21 and eomes directly by binding to their respective 3′ UTRs. By using novel gamma secretase inhibitors (GSIs) we show that Notch can modulate the expression of miR-29. Overexpression of Notch1 in T cell hybridoma cell lines further validates Notch1- mediated mirR-29 regulation. These results indicate a novel regulatory mechanism of T cell differentiation through Notch1 mediated microRNA expression.
Short-chain fatty acid HDAC inhibitor-mediated downregulation of AID expression and class switch DNA recombination is relieved by estrogen through modulation of selected microRNAs
Authors: shen Tian , Sanchez Helia Nasrollahi , Zan Hong , Casali Paolo
Published: January 2023

Estrogen contributes to the elevated antibody response to vaccines in women and the female bias of autoimmune responses, likely by increasing class-switched and hypermutated antibodies and pathogenic autoantibodies. As we have shown, estrogen potentiates the induction of AID, which is critical for class switch DNA recombination (CSR) and somatic hypermutation (SHM), through upregulation of HoxC4. Estrogen may also alter AID gene expression through epigenetic modifications. As we have also shown, short-chain fatty acid (SCFA) histone deacetylase inhibitors (HDIs), including valproic acid and butyrate, upregulated miRNAs that silence AID expression, resulted in reduction of T-dependent and T-independent antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone mice. Here we found that the HDI-mediated downregulation of antibody and autoantibody responses was reversed by estrogen but significantly enhanced by deletion or inhibition of estrogen receptor α (ERα). In B cells, estrogen downregulated the expression of miR-26a, which we found to target the 3′UTR Aicda mRNA. miR-26a was highly expressed in activated B cells and significantly upregulated by SCFA HDIs. Estrogen enhanced cytoplasm to nuclear translocation of HDAC4/HDAC5, and decreased histone acetylation of the host gene of miR-26, and possibly, other selective miRNAs. Finally, The estrogen-mediated enhancement of AID was abrogated by selective inhibition of HDAC4/HDAC5 by LMK258, which decreased CSR. Collectively, our findings provide mechanistic insights into the immunomodulating activity of estrogen and its potential use as therapeutic target in systemic autoimmunity.

Supported by the NIH grants AI 105813, AI 079705.
IL-10-induced miR-155 targets SOCS1 to enhance IgE-mediated mast cell function
Authors: Qayum Amina Abdul , Paranjape Anuya R , Abebayehu Daniel , Kolawole Elizabeth Motunrayo , Haque Tamara , McLeod Jamie J , Spence Andrew J , Caslin Heather , Chumanevich Alena P , Baker Bianca , Taruselli Marcela T , Oskeritzian Carole A , Ryan John J
Published: January 2023

Interleukin 10 (IL-10) is an important regulatory cytokine that modulates a wide range of immune cells. While it is best known for its ability to suppress inflammation, IL-10 has been found to be pathogenic in several human and animal studies of inflammatory diseases. There is a considerable gap in our understanding of the molecular mechanisms behind the pro-inflammatory effects of IL-10. Long term (3–4 day) IL-10 treatment has been shown to suppress mast cell function. In this study we report the effect of short-term (24 hour) IL-10 treatment, which enhanced IgE-mediated histamine and cytokine production both in vitro and in vivo. This pro-inflammatory effect was consistent in mouse and human skin mast cells. IL-10 enhanced activation of the key FcɛRI signaling proteins Stat5, JNK, and ERK. We demonstrate that IL-10 effects are dependent on Stat3 activation, eliciting miR-155 expression, with a resulting loss of SOCS-1. The importance of miR-155 was demonstrated by the inability of IL-10 to enhance anaphylaxis in miR-155-deficient mice. Taken together, our results reveal an IL-10-induced, Stat3-miR-155 signaling pathway that can promote mast cell inflammatory responses.
Short-chain fatty acid (SCFA) histone deacetylase inhibitors produced by gut microbiota regulate selected microRNAs to modulate local and systemic antibody responses
Authors: Sanchez Helia N. , shen Tian , Taylor Julia Renee , Munoz Kristina , Zan Hong , Casali Paolo
Published: January 2023

Epigenetic marks and factors, such as histone modifications and miRNAs, interact with genetic programs to regulate intrinsic B cell functions and inform antibody responses. As shown by our transcriptome and miRNome analyses, histone deacetylase inhibitors (HDIs) selectively downregulate AID, which is critical for class switch DNA recombination (CSR) and somatic hypermutation (SHM), and Blimp-1, the master transcription factor for B cells differentiation to plasma cells, by selectively upregulating miRNAs that target Aicda and Prdm1 mRNAs. As we have also shown, HDI-mediated impairment of CSR, SHM and plasma cell differentiation blunted T-dependent and T-independent antibody responses in wild type mice and autoantibody responses in lupus-prone mice. Potent HDI activity is displayed by butyrate and propionate, two abundant SCFA molecules generated by gut microbiota through fermentation of dietary fibers. We found that these SCFAs synergistically downregulate AID and Blimp-1 expression through selective miRNA upregulation. Feeding a no-fiber diet, or decreasing the load of SCFA-producing microbiota by treating mice with vancomycin reduced gut and serum SCFAs and resulted in increased IgA and IgG1 titers in serum and feces, and IgA+, IgG1+ B cells and plasma cells in Peyer’s patches, mesenteric lymph nodes and spleen. This outcome was reversed by administration of butyrate and propionate, which also synergistically inhibited CSR to IgG and IgA in purified human and mouse B cells in a dose-dependent fashion. Thus, endogenous HDIs integrate cues from metabolites by gut microbiota and relay them to selective downstream effectors, e.g. miRNAs, thereby modulating B cell differentiation and antibody/autoantibody responses.
Elucidating the contribution of IRF3 to effector and memory cytotoxic T cell responses after influenza virus infection
Authors: Vogel Alexander J , Petro Thomas , Brown Deborah
Published: January 2023

The role of interferon regulatory factor (IRF) 3 in initiating the type I interferon response in innate immune cells has been well studied in the context of viral infection. However, it is less clear what impact IRF3 has in shaping and maintaining adaptive immune responses. Previous reports have demonstrated that when restimulated, both CD4 and CD8 T cells from IRF3 knockout (KO) mice have significantly reduced expression of Granzyme B 30 days after infection or following secondary influenza A virus (IAV) challenge. To determine whether deficiencies in Granzyme B correlate with CTL function, we are examining the cytotoxic potential and specific antiviral lytic activity after primary and secondary challenge with IAV in CD4 and CD8 cells. Additionally, as early signals derived from antigen presenting cells (APCs) influence effector and memory T cell development, the innate immune response following IAV infection was examined. Our results demonstrate the innate response is not compromised in IRF3 KO mice at day 1 or 3 post IAV infection in the draining lymph node. Interestingly, macrophages and dendritic cells had decreased expression of MHCII and CD86 at both time points in the lung. These results suggest that while initial priming is sufficient in the draining lymph nodes, secondary factors in the lung could provide T cells with the signals needed for effective memory development. Together, this work provides insight into the molecular regulation of T cell responses to viral infections and highlights the role IRFs play in the development of high quality effector and memory T cells.
The molecular basis of CD4+ T cell priming for CD8+ T cell memory formation
Authors: Cullen Jolie G , Olshansky Moshe , Turner Stephen
Published: January 2023

A long-lived pool of potent memory cells is the defining feature of adaptive immunity. Memory CD8 T cells offer protection for the life of the host due to their unique capabilities to survive in the absence of antigen and respond rapidly to secondary challenge. Therefore, effective CD8 T cell memory is the goal of cell-mediated vaccination strategies. While it is well established that CD4 help is required for CD8 T cell memory formation, it is unclear when during CD8 differentiation this help is required. Further, the affect that CD4 help has on the transcriptional profiles of CD8 T cells and the molecular pathways they use during the generation and maintenance of memory CD8 T cells remains elusive. Using a mouse model of Influenza A virus infection, where priming occurs in the presence or absence of CD4 T cell help, we have pinpointed that help is required during the initial priming of CD8 T cells, and not during memory maintenance or recall. Genome wide RNA-sequencing analysis of the transcriptional signatures between resting “helped” and “ unhelped” memory CD8 T cells reveals surprisingly few differentially expressed genes. However, upon reactivation, “helped” memory CD8 T cells exhibited greater transcriptional up regulation than their “unhelped” counterparts, and utilization of alternate molecular pathways. Our analysis revealed that CD4 help during initial priming is essential for establishing a memory cell pool with enhanced transcriptional potential. Intriguing metabolism differences are currently being further dissected based on this RNA-sequencing data. Thus, CD4 T cell dependent programming likely underpins rapid responsiveness, a key characteristic of memory CD8 T cells.
ent-kaur-16-en-19-oic acid protects mice from neutrophilic lung inflammation and sepsis
Authors: Joo Myungsoo , Kim Kyun Ha , Lee Ji-Yeon , Kwun Min Jung , Han Chang Woo , Choi Jun-Yong
Published: January 2023

The roots of Aralia continentalis Kitagawa (Araliaceae), a remedy to treat patients with inflammatory diseases in traditional Asian medicine, contain kaurenoic acid (ent-kaur-16-en-19-oic acid: KA) that activates Nrf2, a key transcription factor that suppresses inflammation. Here, we explored a possible therapeutic usage of KA against acute lung injury (ALI), a neutrophilic inflammatory lung disease. When administered intratracheally (i.t.) to mouse lungs 2 h after intraperitoneal lipopolysaccharide injection that induces lung inflammation, differential amounts of KA (0.3, 3, or 30 μg/kg) significantly suppressed cellular and neutrophil infiltrations and the expression of pro-inflammatory cytokines in mouse lungs. In a mouse model of sepsis, a major cause of ALI, single i.t. KA (3 μg/kg) administered 2 h after the onset of sepsis significantly decreased the mortality of the mice. KA treatment increased Nrf2-dependent gene expression in the lung. However, while KA could not significantly suppress cellular infiltration to the lung of Nrf2 knockout (KO) mice, KA suppressed neutrophil infiltration to the lung of Nrf2 KO mice, suggesting that Nrf2 is not prerequisite for KA to suppress neutrophil infiltration. Together, these results suggest that KA, likely via multiple pathways, exerts a suppressive effect against neutrophilic lung inflammation and sepsis in mice.
Phospholipase C mediates therapeutic effects of WKYMVm against sepsis
Authors: Kim Hyung Sik , Lee Sung Kyun , Park Min Young , Kim Ji Cheol , Bae Yoe-Sik
Published: January 2023

Sepsis is a serious life-threatening complicated clinical syndrome that results from a detrimental or damaging host reaction to infection. Previously we reported that an immune stimulating peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), strongly induced therapeutic effects against polymicrobial sepsis via formyl peptide receptor 2. In this study, we investigated the mechanism involved in WKYMVm-induced anti-septic activity focusing on phospholipase C (PLC). WKYMVm strongly stimulated mouse neutrophils, resulting in calcium increase which was completely blocked by a PLC-selective inhibitor (U-73122). We found that PLC-specific inhibitor U-73122 markedly blocked therapeutic effects of WKYMVm against sepsis. WKYMVm-induced inhibition of lung inflammation and lymphocyte apoptosis in cecal ligation and puncture sepsis model were markedly blocked by U-73122. We also found that WKYMVm-stimulated bactericidal activity and neutrophil migration to event area were also strongly inhibited by the PLC-selective inhibitor (U-73122). Taken together, we suggest that PLC enzymatic activity is essentially required for the therapeutic activity of WKYMVm against sepsis.
TNF deficiency leads to splenomegaly during Cutaneous Leishmaniasis
Authors: Hu Shanshan , Marshall Cameron , Darby Jocelyn , Korner Heinrich , Lyons Bruce
Published: January 2023

Cutaneous leishmaniasis in resistant C57BL/6 (B6.WT) mice is resolved spontaneously by Th-1 mediated production of TNF-α and IFN-γ, with consequent induction of inducible nitric oxide synthase. Intriguingly, C57BL/6 mice with a TNF gene knockout (B6.TNF−/−) show uncontrolled fatal infection involving the liver and spleen, which are the two main targets in visceral leishmaniasis. The exact role of TNF or specific mechanism, which is responsible for the fatal outcome of Leishmania infection in this gene-deficient mouse strain, is not yet understood.

B6.TNF−/− mice spleen significantly enlarged from Day 28 post-infection, and they were more than 2-fold larger than B6.WT mice on Day 35 and Day 42. To confirm Leishmania infection in the spleen, limiting dilution assay was recruited and showed Leishmania existed since Day 21 in B6.TNF−/− mice, but there was none evident in B6.WT mice. Diff Quik stain results confirmed that architecture of spleen in B6.TNF−/− was disorganized and white and red pulps were no longer clearly separated. Cells infected with Leishmania were dispersed throughout the spleen and lymph cells infiltrated. A gradually increasing population CD45+CD11b+CD11chighCCR2+Ly6C+F4/80high was emerged in B6.TNF−/− mice, which had both dendritic cell and macrophages like phenotype. In vitro, at steady state, macrophages from B6.TNF−/− mice showed no difference comparing to B6.WT. After exposure with Leishmania promastigotes, they showed potential M2-like phenotype and more easily succumbed to infection

We are currently sorting this population from B6.TNF−/− mice and examining the characteristics and potential mechanisms that may affect the spleen immunity against Leishmania major infection in the absence of TNF.
Constitutive and inducible expression of the neurokinin-1 receptor for the inflammatory neuropeptide substance P in human microglia and astrocytes
Authors: Burmeister Amanda Rae , Johnson Morgan Brittany , Ramesh Geeta , Martinez Alejandra , Philipp Mario , Marriott Ian
Published: January 2023

We have previously demonstrated that murine microglia and astrocytes express the neurokinin-1 high affinity receptor (NK-1R) for the neuropeptide substance P. Importantly, we have also shown that substance P/NK-1R interactions augment the inflammatory responses of isolated murine glia to bacterial challenge and exacerbate in vivo neuroinflammatory damage within the central nervous system in mouse models of meningitis. Here, we show the constitutive expression of NK-1R in acutely isolated non-human primate (NHP) brain tissue and ex vivo brain slices. Interestingly, NK-1R protein levels were increased in brain tissue as rapidly as 4 hours following ex vivo infection with Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis. Furthermore, we observed higher NK-1R protein levels in NHP brain tissue at 2 and 4 weeks following in vivo infection, increases that correlated with decreases in the expression of glial fibrillary acidic protein. These in vivo changes were prevented by treatment with a specific NK-1R antagonist suggesting a key role for substance P/NK-1R interactions in these effects. We then investigated the expression of NK-1R in human glial cells. While human microglia showed robust NK-1R expression at rest, astrocytes constitutively expressed only low levels. However, challenge with bacteria or their products elicited rapid increases in whole cell and cell surface NK-1R protein expression by primary human astrocytes and cell lines. Together, these data indicate that human microglia and astrocytes show constitutive and/or inducible expression of the substance P receptor and may therefore be susceptible to this pro-inflammatory neuropeptide.
Impact of Klebsiella pneumoniae on efferocytosis of polymorphonuclear cells
Authors: Jondle Christopher Ned , Mishra Bibhuti , Sharma Jyotika
Published: January 2023

Efficient clearance of invading pathogen and the infiltrating cells by phagocytic cells are two key events that are required for successful elimination of a pathological insult. Clearance of the pathogen is usually achieved by phagocytosis and dead cells are cleared by a process called efferocytosis. While there is substantial information on various strategies employed by a number of pathogens to subvert phagocytosis, our knowledge about inhibition of efferocytosis by microbes is extremely limited. In that regard, we observed that, polymorphonuclear cells (PMNs) infected with Klebsiella pneumoniae (KPn), an opportunistic pathogen and a frequent cause of nosocomial infections leading to sepsis, were efferocytosed less than their uninfected counterparts. This inhibition did not correlate with the infection induced effect on phosphatidylserine (PS) exposure, a well-studied “eat me” signal recognized by efferocytic cells or p53 expression, as a marker of apoptosis in PMNs. We further observed that bacterial replication is required for the full inhibitory effect on efferocytosis since heat-killed KPn were not as effective in inhibiting efferocytosis of PMNs. Next we examined the possible role of LPS in diminishing effecrocytosis, given that KPn is a Gram-negative bacterium. By utilizing cells deficient in TLR-4 (a receptor for LPS) and direct stimulation with LPS we determined that LPS is able to directly inhibit efferocytosis of PMNs. To our knowledge, this study is the first to show a modulatory effect of KPn infection and LPS on PMNs leading to inhibition of their efferocytic uptake by macrophages. This could have implications in acute and chronic airway inflammatory diseases such as pneumonia, sepsis, cystic fibrosis, and COPD.
Social stress-induced priming of innate immunity is associated with translocation of commensal lactobacilli to the spleen
Authors: Lafuse William P , Gearinger Rachel , Fisher Sydney E. , Bailey Michael T.
Published: January 2023

Exposure to the social disruption (SDR) stressor, which involves repeated social defeat in subordinate mice, increases serum cytokine levels, results in accumulation of CD11b+ myeloid cells in the spleen, and primes macrophages for increased cytokine and microbicidal activity. Our previous studies showed that the intestinal microbiota are necessary for SDR-enhancement of splenic macrophage microbicidal activity. The current study determined whether SDR-induced translocation of microbes to the spleen was associated with changes in spleen myeloid cell populations and cytokine mRNA. The SDR stressor increased the numbers of spleen neutrophils and inflammatory monocytes compared to control mice, while having no effect on numbers of non-classical monocytes and red pulp macrophages. Using an RNA flow cytometry assay, we found that neutrophils and inflammatory monocytes from SDR mice express higher levels of IL-1β mRNA compared to control mice. To detect translocated bacteria, qRT-PCR was used with bacterial group specific primers to 16S rRNA in addition to traditional bacterial cultures. SDR induced the translocation of Lactobacillus to the spleen, but translocation of other taxa, such as Bacteroides and Bifidobacterium, was not increased in stressor-exposed mice. Levels of Lactobacillus 16s rRNA in the spleen positively correlated with increased levels of IL-1β and IL-23 mRNA in stressor-exposed mice. Our results indicate that stressor exposure increases translocation of Lactobacillus from the gut to the spleen and primes the innate immune system for enhanced reactivity. These studies suggest that commensal microbes are key players in stressor-induced immunopotentiation. Funded by NIH grant R21AI107238.
Antigen Presenting Cells as Drivers of Specialized Immune Responses
Authors: Devarajan Priyadharshini , Vong Allen M , Bautista Bianca , Castonguay Catherine H , Swain Susan L
Published: January 2023

We have defined the “Memory Checkpoint” as the period when CD4 effector T cells (TEFF) require cognate antigen (Ag) recognition for further differentiation into CD4 memory T cells. Using an influenza A virus (IAV) infection model, we find that generation of CD4 cytotoxic T cells (ThCTL) from TEFF requires MHC Class-II restricted Ag at this checkpoint, 5–7 days after initial infection. Ag recognition at this checkpoint also determines CD4 follicular helper T cell (TFH) generation in our model. We have used transgenic mouse models in which Ag presentation is restricted to specific antigen presenting cells (APC), bone marrow chimeras and addition of various APC subsets at the memory checkpoint to study which APC drive TEFF to TFH vs. ThCTL. Our studies indicate that distinct APC, during this checkpoint, promote different fates resulting in generation of distinct effector subsets. While, as expected, B cell APC are necessary for TFH responses, the APC subset(s) that promote ThCTL generation are not yet clear. Our data indicate that a specialized hematopoietic subset of CD11c+ APC, that are preferentially localized in the infected tissue, is required at this checkpoint, to support ThCTL generation. Once we identify the APC subset(s) needed, we plan to target Ag to particular APC at the checkpoint, to uniquely promote distinct functional subsets and evaluate their function. We suggest this may allow us to tailor the quality of the effector response to better protect against distinct pathogens.
Plasticity along with differentiation of Tc17 cells is controlled by CTLA-4 (CD152)
Authors: Brunner-Weinzierl Monika C , Lingel Holger , Pick Jonas , Huber Magdalena , Pierau Mandy , Arra Aditya
Published: January 2023

Tc17 cells are known to express low IFN-γ and granzyme B, resulting in diminished cytotoxicity. Adoptive transfer into Ag-bearing hosts converts them partially towards IFN-γ producing phenotype retaining Tc17 characteristics like increased persistence of survival. Nevertheless molecular mechanisms involved in Tc17 lineage plasticity and stability are yet to be identified. CTLA-4, a homologue to co-stimulatory molecule CD28, diminishes cytotoxicity of Tc1 cells by selective downregulation of GranzymeB, Eomes, and IFN-γ production, but its impact on Tc17 differentiation and plasticity is not known. CTLA-4+/+ and −/− CD8+ T cells were stimulated under Tc17 conditions. Tc1 and Tc17 characteristics in these cells were analysed by flow cytometry, western blot and real-time-PCR. For plasticity studies, cells were re-stimulated and analyzed for expression of Tc17 and Tc1 characteristics. Listeria Monocytogenes(LM) infection model and B16 melanoma model were used to investigate the effects of CTLA-4 on Tc17 cell mediated clearance of infection and controlling tumor growth. CTLA-4 showcased a cell intrinsic ability to enhance Tc17 differentiation program, by sustaining the levels of IL-17 inducing factors like STAT3, RORγ T and IRF4. Adoptively transferred Tc17 cells lacking CTLA-4 signal were more effective in clearing LM infection and controlling tumor growth. Re-stimulated CTLA-4 incompetent Tc17 cells revealed that higher cytotoxicity of these cells is due to their enhanced plasticity nature to develop Tc1 characteristics. Inactivation of CTLA-4 could be a unique target for Tc17 cells, to enhance lineage plasticity and develop Tc1 characteristics with increased persistence of survival and immunity.
The fate of CD8+ T cells during infection is linked to their developmental origin
Authors: Smith Norah L , Wang Jocelyn , Nzingha Kito , Reynaldi Arnold , Davenport Miles P , Rudd Brian D
Published: January 2023

Key features of the CD8+ T cell response to infection are the robust clonal expansion and phenotypic diversification of naïve precursors into effector and memory cells. However, mechanisms that regulate the fate of naïve CD8+ T cells after microbial challenge remain a subject of controversy. Previous studies have suggested heterogeneity in the effector pool is driven by differences in TCR avidity, asymmetric cell division, and environmental cues. However, one variable that has not been closely examined is the developmental origin of naïve CD8+ T cells. Notably, the pool of naïve CD8+ T cells in the adult is comprised of clonotypes that were produced during various stages of life. To determine whether the developmental origin of naïve CD8+ T cells in adults plays a deterministic role in their fate after infection, we developed a system to ‘timestamp’ CD8+ T cells from various stages of development (1 day, 1 week, and 4 weeks) and examined their phenotype and behavior in 8 week-old adult mice. Interestingly, we found naïve CD8+ T cells made early in life predominantly display a CD44hi CD122hi memory phenotype whereas cells made later in life are CD44lo CD122lo. In vitro stimulation of stamped populations showed that fetal- and neonatal-derived CD8+ T cells are hypersensitive to IL12/IL18 and proliferate more rapidly than cells made in older animals. In response to infection in adulthood, both polyclonal and monoclonal CD8+ T cells produced in early life skew towards a terminally differentiated short-lived effector phenotype (KLRG1hiCD127lo), indicating this phenomenon is TCR independent. These data indicate that the spectrum of CD8+ T cell differentiation observed after infection is influenced by when the responding cells were initially made.
Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist
Authors: DeVault Victoria L , Dienz Oliver , Lilley Graham WJ , Benoit Patrick J , Schwartzberg Pamela L , Boyson Jonathan E
Published: January 2023

Signaling lymphocyte activation marker family member 6 (Slamf6) is a cell surface signaling receptor that plays an important role in NKT cell development. Upon activation with the CD1d ligand α-galactosylceramide (αGalCer), NKT cell Slamf6 expression increases dramatically (~10-fold) and it maintains this high level of expression for at least 5 days after activation. The mechanisms through which Slamf6 regulates NKT cell function are largely unknown. To investigate the effect of Slamf6 on peripheral NKT cell populations, we challenged C57BL/6 (B6) or B6.Slamf6−/− mice with the NKT-specific agonist αGalCer. Examination of liver NKT cell numbers 3 days after challenge revealed a 50-fold increase in B6 mice over vehicle-treated controls. In contrast, we observed a 270-fold increase in NKT cells in B6.Slamf6−/− mice versus controls. An analysis of spleen NKT cells yielded similar results. A comparison of in vivo BrdU uptake by NKT cells between B6 and B6.Slamf6−/− mice revealed no significant differences in proliferation. In addition, NKT cell intracellular IFN-γ, IL-4, and TNF production between B6 and B6.Slamf6−/− mice after administration of αGalCer revealed no difference in the production of these cytokines. We next evaluated the Slamf6-specific effect on NKT cell expansion using an in vitro co-culture system. Cross-linking of Slamf6 on purified B6 NKT cells resulted in diminished NKT cell expansion and increased numbers of TUNEL+ NKT cells. Taken together, these data support a model where Slamf6 acts as a negative regulator of NKT cell expansion in the presence of a strong agonist by regulating the death threshold of NKT cells. These data suggest that Slamf6 blockade could be a useful tool to manipulate the expansion of NKT cells in vivo.
CD4+ T Cell Differentiation is Determined During an Initial Priming Phase and Occurs Independently of Effects due to Division or Survival.
Authors: van Panhuys Nicholas J , Palmer Douglas
Published: January 2023

The differentiation of naïve CD4+ T cells into discrete effector subsets, each able to mediate specific aspects of immunity is a central tenet of the adaptive immune response. Current dogma suggests that the generation of committed effector CD4+ T cells takes place over a 3–4 day period following initial engagement of the TCR. Activation of CD4+ T cells is thought to occur in three distinct functional phases, priming, proliferation and differentiation. Here, we present data indicating that CD4+ T cells acquire the disposition to commit to either Th1, Th2 or Treg lineages very soon after activation, prior to the induction of division. Further, we show that division is not required for the induction of a full program of differentiation to occur. We demonstrate that TCR signaling is responsible for two distinct activation programs whereby the strength of signal that a naïve T cell receives works in concert with cytokines at an early phase to induce differentiation, whereas the length of TCR signal can be construed as a secondary component of activation which controls the ability of CD4+ T cells to enter into division.
Absence of preferential high-affinity TCR expansion during CD4 T cell responses
Authors: Martinez Ryan J , Andargachew Rakieb , Martinez Hunter , Evavold Brian D
Published: January 2023

Current views assume higher-affinity T cells, as identified by pMHCII tetramers, possess enhanced capabilities while lower-affinity T cells are inconsequential to the immune response. We set out to formally address the contribution of low-affinity T cells to the expanded repertoire. To perform this study we employed a micropipette based assay and T cell receptor signaling functional reporter to quantify the total number of antigen-specific CD4 T cells from naïve precursors through their expansion to antigen. In the naïve state, an in vivo limiting dilution assay revealed hundreds more precursor T cells than defined by pMHCII tetramers, finding high-affinity T cells comprise only 5–30% of the total naïve repertoire. Upon immunization, low-affinity T cells expand similarly to higher-affinity counterparts, demonstrating no affinity maturation or preferential expansion of the highest-affinity T cells as the immune response progressed. These findings demonstrate affinity diversity of CD4 T cells is maintained without a selective shift favoring the highest-affinity cells.
Reciprocal regulation of IL-17 and IL-22 secretion by CD4 T cells through the Aryl Hydrocarbon Receptor
Authors: McAleer Jeremy , Lewis Alisha Milton , Nguyen John
Published: January 2023

CD4 T cells are critical for host defense against microorganisms and direct immune responses by differentiating into specialized subsets. While Th17 cells secreting IL-17 and IL-22 contribute to vaccine immunity, they have also been implicated in autoimmune inflammation. Our goal is to understand how endogenous factors regulate Th17 cell cytokine secretion. Murine models have demonstrated that the aryl hydrocarbon receptor (AhR) can directly promote Th17 cell differentiation or provide systemic anti-inflammatory effects, suggesting that pleiotropic functions of AhR may be dependent the ligand and/or cell type studied. Tryptophan metabolites have been identified as endogenous AhR agonists that can modulate T cell immunity. Here, we examined a potential link between tryptophan metabolism and the AhR on cytokine secretion by primary human CD4 T cells cultured under Th17 conditions. L-tryptophan decreased IL-17 secretion and RORgt expression in T cells, but did not significantly affect IL-22 secretion. Similar results were observed with the tryptophan metabolite L-kynurenine. Conversely, AhR blockade decreased IL-22 with no effect on IL-17 secretion. These data suggest that IL-17 and IL-22 production are reciprocally regulated in human CD4 T cells through tryptophan availability and the AhR, and provide an avenue for therapeutic modulation of pro-inflammatory T cell responses.
‘Efferocytosis of infected apoptotic cells inhibits Th17 differentiation via PGE2-EP4 signaling’
Authors: Dejani Naiara , Fortino Felipe , Nino Victoria , Medeiros Alexandra Ivo
Published: January 2023

Clearance of infected apoptotic cells (IAC) by dendritic cells (DC) triggers the production of Th17-related cytokines. Moreover, we have found that prostaglandin E2 (PGE2) is also produced during efferocytosis containing microbial products, but the effect of this prostanoid on Th17-differentiation is still unknown. The purpose of this study was to evaluate the role of PGE2, produced during recognition of IAC by DC, on Th17 differentiation. As source of IAC, Raw 264-7 cells were cultured with Escherichia coli during 2h for phagocytosis and apoptosis was induced by UV irradiation. BMDC were co-culture with IAC during 18h, and the supernatant from this co-culture was used to differentiate naïve CD4+ T cells for 4d, plus anti-CD3/anti-CD28. Phagocytosis of IAC by DC promotes the production of high levels of PGE2 (15±0.7 ng/ml), IL-6 (26±0.4 ng/ml), TGF-β (3.5±0.09 ng/ml) and IL-1β (5.4±0.1 ng/ml), when compared to phagocytosis of uninfected-AC. The treatment with COX inhibitor significantly decreased PGE2 level and promotes Th17 cell differentiation at least 4 times higher (16%) than untreated co-cultures (4%). Moreover, adding back exogenous PGE2 decreased drastically the percentage of Th17 cells (2%). Moreover, the treatment of naive T cells with antagonists of PGE2 receptor (EP1/EP2/EP4) suggests that the suppressor effect of this prostanoid on Th17 cell differentiation is mainly mediated by EP4/cAMP/PKA activation pathway. Taken all together, our results show that PGE2 is able to suppress Th17 differentiation in vitro through EP4 signaling. Further studies are needed to clarify how PGE2 can modulate the Th17 differentiation using in vivo model.

Financial Support: FAPEP 14/17374-3, 12/23580-0, 11/17611-7.
Chronic antigen-specific T helper 17 inflammatory responses in the lungs require persistent adjuvant exposure to be sustained
Authors: Shalaby Karim H , Whitehead Gregory S. , Thomas Seddon Y. , Cook Donald N.
Published: January 2023

Allergic asthma is characterized by exaggerated lung T helper 2 (Th2) and Th17 responses which require signals that stimulate the innate immune system, provided by exposure to environmental adjuvants with an allergen. Once airway disease has developed, it is unclear as to whether the same mechanisms that initiated the disease remain important in sustaining it. We investigated the contribution of persistent innate danger signals in sustaining a chronic antigen-specific Th17 cell response. Animals were sensitized and challenged acutely by aspiration of ovalbumin (OVA) with different adjuvants, including house dust extract (HDE). They were then challenged for a further 5 weeks with OVA alone or OVA + adjuvant to test whether the adjuvant becomes redundant in sustaining airway inflammation once it has already been established. Removal of the adjuvant during the chronic challenges resulted in the animals displaying minimal airway inflammation, whereas continuous challenge with adjuvant and OVA caused significant inflammation. HDE was particularly potent at promoting airway neutrophilia, lymphocytosis and greater IL-17A production from lung explants restimulated ex vivo with OVA, suggesting that persistent exposure to this adjuvant amplified the antigen-specific Th17 response. Furthermore, CD4+ cells from OVA-receptor transgenic (OT-II) IL-17A-fate mapping mice were differentiated under Th17 polarizing conditions and adoptively transferred to recipients which were then challenged with either OVA alone, OVA + HDE or HDE alone. Persistent exposure to HDE and OVA, compared to either one alone, amplified the lung antigen-specific memory Th17 response, independently of the enzymatic activity of HDE but rather via TLR4 signaling.