bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutralizing CXCL13 attenuates neuropsychiatric manifestations in lupus-prone mice
Authors: Huang Michelle , Stock Ariel D , Putterman Chaim
Published: January 2023

The pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE) remains unclear, despite the large number of lupus patients affected by neuropsychiatric manifestations. We recently showed that MRL/lpr mice, a classic SLE mouse model with neuropsychiatric symptoms, develop tertiary lymphoid structures (TLS) (a.k.a. ectopic germinal centers). TLS structurally and functionally resemble lymph nodes and form at the site of chronic inflammation to create more localized and specialized immune responses, which may promote a local immune response against self-antigen and thus contribute to the pathogenesis. Since CXCL13 is a key chemokine that drives lymphoid formation, we injected female MRL/lpr mice intraperitoneally with an anti-CXCL13 antibody, an IgG isotype control antibody, or PBS three times weekly for 12 weeks starting at 6–8 weeks of age. Cognitive dysfunction and affective deficits were assessed at the end of treatment. Anti-CXCL13 mAb treated MRL/lpr mice showed significant improvement in spatial memory, recognition memory, and depression-like behavior as compared to the two control groups (p=0.034, p=0.013, and p=0.0087, respectively). Furthermore, mice receiving the antibody by intracerebroventricular delivery showed similar results (p=0.055, p=0.20, and p=0.018, respectively). There were, however, no apparent differences in indicators of systemic disease, including autoantibody titers. Our results suggest that TLS play an important role in the pathophysiology of affective deficits and cognitive dysfunction, and that CXCL13 neutralization may be a potential therapeutic strategy to target NPSLE.
Transferable Immune Reactive Microbiota Determine Clinical and Immunologic Outcome of FMT in UC
Authors: Lima Svetlana Ferreira , Gogokhia Lasha , Viladomiu Monica , Woo Viola , Marderstein Andrew , Putzel Gregory , Scherl Ellen J , Brown Su-Ellen , Hambor John , Rosenthal Melissa , Jacob Vinita , Crawford Carl , Chou Lance , Longman Randy
Published: January 2023

Background

Over two million people worldwide suffer from ulcerative colitis (UC). Biologic therapy has significantly improved treatment, but nearly two-thirds of patients attenuate response. Fecal microbiota transplant (FMT) is an emerging therapy for the treatment of UC, but the microbial mechanism responsible for clinical response is poorly understood. Using samples from our pilot FMT study (Jacob V, et al 2017), we aim to define the core transferable microbiota (CTM) in UC patients responsive to FMT therapy and its therapeutic mechanism.

Methods

IBD disease activity scores were used to define clinical response. Metagenomic sequencing of donor, recipient, and 4 week post-FMT fecal samples was performed to define the CTM and strain level transferability. To define the transferable immune-reactive microbiota (TIM), IgA-seq was also performed on donor and recipient samples. Patient TIM strains were isolated and tested in gnotobiotic mouse models to evaluate their impact on mucosal immunity and colitis.

Results

We defined a CTM associated with clinical response to FMT. CTM strain tracking confirmed that clinical response correlated with strain transferability. We defined a core TIM by IgA-seq that correlated with clinical response. In humanized mouse models, TIM induced IgA in a T cell independent manner. Colonization of germ-free mice with a core TIM strain, Odoribacter splanchnicus, robustly induced mucosal Th17 and RORgt+/Foxp3+ iTreg cells and reduced the severity of transfer T cell colitis.

Our data highlights a core TIM in UC responders to FMT and the mechanistic impact of it in shaping mucosal immunity and guiding the response to UC. This provides a framework for rational selection of TIM for microbial-therapy in IBD.
Myelin-specific CD8 T cells require IFNγ responsiveness to optimally suppress CNS autoimmunity
Authors: Boyden Alexander W. , Brate Ashley A. , Karandikar Nitin J.
Published: January 2023

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) and for developing successful immunotherapies. Our group has demonstrated that central nervous system (CNS) myelin-specific CD8 T cells unexpectedly harbor striking regulatory capacity in both mouse and human. In particular, PLP178–191-specific CD8 T cells (PLP-CD8) robustly suppress the MS mouse model experimental autoimmune encephalomyelitis (EAE). We have recently shown this depends on PLP-CD8 elaborating IFNγ and perforin in a coordinated suppression program over time. However, the cellular target and downstream effects of CD8 T cell-elaborated IFNγ remains poorly understood. Here, we show that while WT PLP-CD8 were robustly suppressive in IFNγR-deficient mice, IFNγR-deficient PLP-CD8 exhibited defective suppression in WT mice. Further, compared to WT counterparts, IFNγR-deficient PLP-CD8 were defective in suppressing disease in IFNγ-deficient recipients, a scenario in which the only IFNγ WT PLP-CD8 “see” is their own. These data indicate that myelin-specific CD8 T cells must sense their own IFNγ in an autocrine/paracrine manner to optimally perform autoregulatory function in vivo. Further mechanistic dissection showed that IFNγR-deficient PLP-CD8 exhibited altered degranulation/granzyme profiles, altered migration in recipients, and potential deficits in killing capacity. Collectively, this work suggests that myelin-specific CD8 T cells require IFNγ-responsiveness to optimally regulate CNS autoimmunity and may help elucidate future adoptive immunotherapeutic approaches in MS.
Metformin reduces PD-L1 expression in the tumor and enhances the efficacy of vaccine generated CD8 T cells in a murine model of triple negative breast cancer
Authors: Munoz Luis E , Huang Haley Lei , Guin Rohini Neha , Bommireddy Ramireddy , Selvaraj Periasamy
Published: January 2023

Immunotherapy has emerged as a promising treatment of breast cancer, however, response rates to therapy remain far from ideal. We have developed a new platform for personalized vaccine immunotherapy that utilizes tumor tissue to generate tumor membrane vesicles (TMVs). These TMVs can be incorporated with immunostimulatory molecules such as IL-12 and B7-1 and used for immunization. In this study, we investigated the combinatorial effect of TMV-based vaccine immunotherapy, to induce an anti-tumor immune response, with the type 2 diabetes drug metformin, which has been reported to exert anti-tumor effects by promoting CD8 T cell activity in the tumor microenvironment. Our results show that TMV vaccination inhibits primary tumor growth in preclinical tumor models, while the combination with metformin resulted in greater inhibition of primary growth. TMV vaccination reduced the metastatic burden in the lungs while the combination with metformin further abrogated metastatic spread. Interestingly, TMV vaccination alone induced a significant increase in T cell infiltration to the tumor, and combination with metformin caused a decrease in the accumulation of T cells, despite having better control of tumor growth. Further, we show that metformin can reduce PD-L1 expression within the tumor microenvironment and result in more IFN-γ producing CD8 T cells within the tumor, providing a mechanistic insight for the improvement of TMV-based vaccine immunotherapy.
Combining a novel dual RAF/MEK inhibitor with immunomodulation to promote an anti-tumor response
Authors: Dong Lauren , Choi Hyejin , Budhu Sadna , Mehanna Nezar , Falik Nouraiz , Rosen Neal , Merghoub Taha , Wolchok Jedd D
Published: January 2023

The therapeutic scope of MEK inhibitors (MEKis) is currently limited to use in BRAF mutant melanoma. Therefore, we aim to develop new strategies to extend their usage to MEKi resistant RAS mutant cancers, which represent an unmet clinical need. A strategy we investigated is to balance the positive and negative immunomodulatory effects of MEKis for optimal combination with immunotherapy. In Ras mutant murine lung cancers, CH5126766 (CKI27) is novel due to its ability to inhibit both RAF and MEK, preventing the rebound of ERK that normally results from the relief of negative feedback in the MAPK pathway. We observed that CKI27 increased MHC expression on tumor cells and T cell mediated killing. Yet, CKI27 also decreased T cell proliferation, activation, and cytolytic activity. Implementing a break for T cells to recover with intermittent dosing of CKI27 partially relieved these inhibitory effects. Further combination with co-stimulatory agonist antibodies targeting OX40 and GITR completely alleviated these T cell toxicities and increased combination efficacy with checkpoint blockade antibody anti-CTLA-4. Understanding the immunomodulatory effects of combining CKI27 with immunotherapy will elucidate the mechanism behind their increased efficacy. This will allow us to make more informed decisions in dosing regimens, overcoming resistance, and generating long-term immune responses in current and future clinical trials treating patients with RAS mutant cancers.
Androgen receptor targeted therapy sensitizes tumor bearing mice to effective immunotherapy
Authors: Polesso Fanny , Hipfinger Christina , Sehrawat Archana , Moran Amy
Published: January 2023

Sex hormones impact both the innate and adaptive immune response, contributing to differences in autoimmune diseases, response to infection, and cancer incidence between males and females. Androgens are known to exert a general immunosuppressive effect. Historically, immune checkpoint blockade (ICB) has not proven to be efficacious in patients with prostate cancer despite standard of care consisting of androgen deprivation therapy (ADT). Upon disease progression, men will be treated with second line ADT therapy; some of which can prevent androgen receptor transcriptional activity. One such drug is enzalutamide. Given T cells express AR, we hypothesized that enzalutamide treatment would relieve the immunosuppressive effects of androgens on T cells, rendering them more responsive to immunotherapy. In the studies presented, we sensitize castration resistant prostate cancer tumor bearing mice to effective ICB with a combination of castration and enzalutamide. This combination therapy leads to ~40% tumor rejection and long-term survival; an effect that is dependent on CD8 T cells. These tumor infiltrating CD8 T cells made 6-fold more IFNγ within the tumor in the triple therapy group compared to control group. Surprisingly, this combination therapy showed efficacy in the androgen independent sarcoma tumor model MCA-205. Using in vitro and in vivo assays, we show that enzalutamide has a direct effect on T cell function. Taken together, our data suggests that ADT, and specifically enzalutamide treatment, can sensitize tumor bearing animals to effective immune checkpoint blockade and allow for tumor control by T cells.
DNMTi plus anti-4-1BB therapy reverses suppressive tumor microenvironment and reduces tumor burden in ovarian cancer
Authors: Gomez Stephanie , Palmer Erica , Kobyra Julie , Hadley Melissa , Chiappinelli Katherine B.
Published: January 2023

Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Better OC prognosis is associated with high tumor-infiltrating CD8 T cells, but OC is generally characterized by an immunosuppressive tumor microenvironment (TME), with less than 10% of patients responding to immune checkpoint blockade therapy in clinical trials. DNA methyltransferase inhibitors (DNMTi) activate transcription of repetitive elements to induce a type I interferon response in OC, which recruits and activates host immune cells to fight the tumor. Additionally, changes in DNA methylation in T cells regulate T cell memory. Thus, DNMTi increase anti-tumor immunity through 1) tumor IFN response and 2) reversal of T cell exhaustion.

To test the hypothesis that combination epigenetic and immune therapy could increase the anti-tumor response in OC, we treated an immunocompetent mouse model of OC with DNMTi +/− anti-CD137. The ID8 MOSE P53−/− model grows intraperitoneally and produces ascites, mimicking human OC which is 90% P53 mutant. The TME of this model is immunosuppressive and it is not responsive to immune checkpoint blockade therapy. At 9 weeks post tumor injection, we observed the following survival percentages: 50% for the combination group, 30% for the DNMTi group, and 0% survival for the both the anti-CD137 and mock groups. We also observed statistically significant increases in %CD3+IFNg+ T cells in the spleen for the DNMTi + anti-CD137 group compared to the mock group. We hypothesize that anti-CD137 treatment is activating immune cells recruited by DNMTi. Future experiments will manipulate cytokine expression to delineate mechanism. We thus describe a novel immunotherapeutic combination for ovarian cancer.
Characterizing the mechanism of action of a HER2xCD3 bispecific T-cell engager (BiTE) antibody in 2D and 3D immune cell killing assays using a combined live-cell imaging and flow cytometry workflow
Authors: Bevan Nicola , McBain Kirsty , Kelsey Lauren , Szybut Clare , Trezise Del , Dale Tim
Published: January 2023

Solid cancers have proved to be a challenge for immunotherapies and targeted approaches are needed. Evaluation of these therapies using relevant models plays a large part in their development. A workflow that combines live-cell imaging and flow cytometry was used to characterize immune cell killing induced by a BiTE antibody.

Immune cell co-culture assays were set up in 2D or 3D formats with green nuclear labeled AU565 HER2 positive breast cancer cells and various effector cell types. Immune cell activation was induced using HER2xCD3 BiTE or CD3/CD28 Dynabeads. Images were captured every 4 h with IncuCyte and quantified for numerous cellular parameters. Cytokine analysis of supernatant samples was performed using a multiplex QBead panel alongside assessment of immune cell activation markers using the iQue3.

Quantification of PBMC mediated cytotoxicity showed a comparable 70 ± 2% and 62 ± 1% reduction in spheroid growth with BiTE (10 ng mL−1) and Dynabead (1:1 bead-to-cell ratio) activation. In contrast to Dynabeads, BiTE activation did not induce immune cell shape change or upregulation of T cell activation markers; 1 ± 0.2% CD69 expression was observed on CD3+CD8+ cells, compared to 53 ± 0.6%. Further evaluation found BiTE activated isolated CD56+ cells induced less target cell death compared to PBMCs and granzyme B secretion was not detected, suggesting a lack of involvement of NK cells. Release of IL-6 and CCL2 cytokines indicate immune cell mediated cytotoxicity via an inflammatory pathway.

These data suggest HER2xCD3 BiTE activity may not be mediated through T cell or NK cell subtypes. At present the mechanism of target cell killing is unclear, however further studies combining imaging and flow cytometry may provide the required insight.
Heterosubtypic human antibody response to influenza H7N9 in survivors of natural infection
Authors: Gilchuk Iuliia M , Dong Jinhui , Li Sheng , Turner Hannah L , Irving Ryan P , Ward Andrew B , Crowe James E
Published: January 2023

H7N9 avian influenza virus causes severe infections in humans after cross-species transmission from bird reservoirs. Previous studies revealed that infection with H7N9 elicits a heterosubtypic polyclonal antibody (Ab) response between H7 and H3 or H1 hemagglutinins (HAs) in humans. However, relatively little is known about the functional activity, targeted epitopes, and breadth of reactivity of human monoclonal Abs (mAbs) against H7N9. In this study, we isolated 18 H7-reactive mAbs from survivors of natural H7N9 infection. The majority (14 of 18) of H7-reactive mAbs within the panel possessed neutralizing activity, mediated by inhibition of attachment or egress of the virus. Binding analysis of the isolated mAbs against fourteen influenza virus HAs representing group 1 and 2 revealed cross-reactivity for diverse strains within the H7 subtype and an extended response to H15 HA. Seven identified cross-reactive mAbs targeted the HA head domain, including neutralizing and non-neutralizing mAbs. Two neutralizing mAbs that are specific to the HA stem region revealed heterosubtypic reactivity against H7, H15 and H3 HAs. Further analysis identified the epitopes for heterosubtypic mAbs. This study provides insights into the basis for heterosubtypic antibody responses against H7N9 in human infection survivors and may inform efforts to incorporate novel epitope targets in broadly protective influenza vaccines.
Codelivery of antigens with TLR7/8 agonists leads to an improved immune response to influenza vaccination
Authors: Lathrop Stephanie K , Massena Casey J , Davison Clara J , Schoener Roman , Burkhart David J , Evans Jay T
Published: January 2023

When designing an effective and safe vaccine, inducing a protective immune response to a pathogen without also inducing unacceptable levels of immunopathology is a major challenge. One promising strategy is the use of purified or recombinant antigens along with adjuvants that specifically engage pathogen-associated molecular pattern (PAMP) receptors. This targeted approach aims to minimize immune-mediated reactogenicity. Our group has developed libraries of novel toll-like receptor (TLR)7 and/or TLR8 ligands which show a range in receptor activity. Previous studies have shown that the high potency of TLR7/8 adjuvants leads to unacceptable toxicity in human clinical trials. One strategy to overcome this toxicity is direct adjuvant-antigen conjugation, which serves to limit systemic distribution of the adjuvant through the codelivery of the antigen and adjuvant to APCs. We have demonstrated that covalent conjugation of a TLR7/8 agonist to an antigen results in improved humoral and cell-mediated responses, with decreased systemic and local inflammation. This approach allows for the use of a reduced adjuvant dose by preventing the engagement of PAMPs in the absence of antigen. We hypothesize that antigen/adjuvant codelivery boosts the engagement of CD4+ T cells and elicits the necessary cytokines to promote a Th1-related switch to antigen-specific IgG2a antibodies. In addition, engagement of TLR7/8 appears to improve cross-presentation, thereby boosting the CD8+ T cell response, which is desirable for improved immunity to influenza and other viruses. We find this approach has great potential to enhance vaccine efficacy, while reducing the immunopathology often associated with highly active TLR7/8 adjuvants.
Efficacy of agonist αOX40 and antagonist αCTLA-4 cancer immunotherapy is increased by ibrutinib treatment through enhanced Eomes expression and T-cell response
Authors: Emerson Dana A , Redmond William L
Published: January 2023

Antagonistic monoclonal antibodies targeting individual checkpoint receptors on T cells, such as CTLA-4 and PD-1, are used to treat patients with metastatic disease; however, only a subset of patients respond. Combination therapies, such as CTLA-4 blockade with agonist αOX40 mAb, are synergistic in preclinical models. αOX40/αCTLA-4 therapy synergistically enhances expression of Eomesodermin (Eomes), a transcription factor that drives CD8 T cell differentiation and memory function. We hypothesize that Eomes expression plays a critical role in the synergistic efficacy of αOX40/αCTLA-4 therapy. Compared to Eomeslo CD8 T cells, Eomeshi CD8 T cells induced by αOX40/αCTLA-4 therapy express lower levels of checkpoint receptors (PD-1, TIM-3, LAG-3) and higher levels of tumor trafficking and activation markers (CXCR3, CD44), and intratumoral cytokines (IFN-γ, TNF-α). CD8 T cell-specific Eomes−/− tumor-bearing mice did not respond to αOX40/αCTLA-4 therapy, demonstrating the necessity of Eomes expressing CD8 T cells. Interleukin-2 inducible T cell kinase (ITK) negatively regulates T cell Eomes expression following T cell receptor ligation. Thus, we asked whether enhancing T cell Eomes expression by combining ibrutinib, an FDA-approved ITK inhibitor, with αOX40/αCTLA4 therapy would further improve anti-tumor responses by increasing Eomeshi CD8 T cells. This triple combination increased CD8 T cell-specific Eomes expression, tumor-specific CD8 T cells, Ki-67 (proliferation), and Th1 cytokines (IFN-γ, TNF-α) leading to reduced tumor growth and improved survival over αOX40/αCTLA-4 alone, making ibrutinib/αOX40/αCTLA-4 a potential novel triple combination therapy.
CD4+ T cells are dispensable for induction of heterologous tier 2 HIV neutralizing antibodies in rhesus macaques
Authors: Kobie James , Barnette Philip , Pandey Shilpi , Spencer David , Basu Madhubanti , Piepenbrink Michael , Keefer Michael , Hessell Ann J , Haigwood Nancy L , Sarkar Sanghita
Published: January 2023

Induction of a sustained and broad neutralizing antibody (Ab) response is a major goal in developing a protective HIV vaccine. Many HIV Envelope (Env)-specific broadly neutralizing antibodies isolated to date exhibit substantial somatic hypermutation, and their development in HIV infected individuals has been correlated with T follicular helper responses. This has bolstered the rational that a robust CD4+ T helper cell response will likely be required for the induction of such HIV broadly neutralizing antibody responses by vaccination. Using the VC10014 DNA-protein co-immunization vaccine platform consisting of gp160 envelope plasmids and gp140 trimeric envelope proteins derived from a clade B HIV-1 infected subject who developed broadly neutralizing serum Abs, and which has been previously shown to induce Tier 2 heterologous neutralizing Abs in rabbits and rhesus macaques, we determined the influence of CD4+ T cell depletion during the vaccination regimen on the characteristics of the Env-specific humoral response in rhesus macaques. Both CD4+ depleted and non-depleted animals developed Tier 2 heterologous neutralizing plasma antibodies, with no inferiority in titers among the CD4+ T cell depleted animals. Similarly, there was no diminishment of titers of HIV Env-specific cross-clade binding antibodies, antibody dependent cellular phagocytosis or antibody dependent complement deposition in the CD4+ depleted animals. These results suggest that HIV neutralizing antibodies can be induced in the absence of robust CD4+ T cell help, which may have implications for developing effective HIV vaccine strategies.
Unique Features of Immunity within the Immunoglobulin Heavy Chain Locus of Egyptian Rousette Bats
Authors: Bartlett Maggie , Larson Peter A. , Palacios Gustavo , Sanchez-Lockhart Mariano
Published: January 2023

The Egyptian fruit bat (Rousettus aegyptiacus, ERB) is reported to be the reservoir of Marburg virus (MARV). Indeed, MARV infection is almost completely asymptomatic in ERBs while eliciting a severe hemorrhagic fever in humans and non-humans primates (NHPs). To understand the biological mechanisms that explain these differential outcomes to MARV, we aim to investigate the differences between both animals’ immune systems. One of the antiviral mechanisms to control viruses is the humoral response. The B cell receptor (BCR) is composed of two heavy and two light chains heterodimers. Both immunoglobulin (Ig) chains undergo DNA rearrangement to generate a diverse repertoire, crucial for diverse antigenic recognition. The Ig heavy chain locus is comprised of the variable (V), diversity (D), joining (J), and constant (C) genes. Utilizing high throughput sequencing to resolve the V(D)J rearrangements we can characterize the BCR repertoire and define the clonotypes that are generated in response to vaccination or infection. To define the BCR repertoire with DNA sequencing, a thorough representation of the Ig heavy chain (IGH) locus is necessary. We were able to resolve on contiguous IGH locus containing all BCR genes that were previously on 21 different scaffolds. In addition, we identified a unexpected expansion of IgE genes not described in another species to date. We defined the tissue expression of these novel IgE genes as well as IgGs, IgA, and IgM. Finally, we predicted differential functions between subclasses in silico. These data support unique immune response capabilities of ERBs that may aid in their control of pathogenic human viruses.
Interleukin-6 induces immunometabolic reprogramming in human rheumatoid arthritis synovial fibroblasts to promote osteoclastogenesis
Authors: Ahmed Salah-Uddin , Singh Anil K , Haque Mahamudul , Basit Abdul , Shi Yuanyuan , Wildman Benjamin J , Prasad Bhagwat , Hassan Quamarul
Published: January 2023

Interleukin-6 (IL-6) plays an important role in progressive bone loss in rheumatoid arthritis (RA). However, the molecular mechanisms through which IL-6 propels RA synovial fibroblasts (RASFs) to contribute to bone loss are not fully understood. Treatment of human RASFs with IL-6 and IL-6 receptor (IL-6/IL-6R, 100 ng/ml each) alone or in combination with M-CSF (2 ng/ml) and RANKL (5 ng/ml) for 9 days resulted in the phenotypic changes to osteoclast-like features as determined by increase in TRAP staining and Dentin pit resorption area, which were comparable to M-CSF/RANKL group. IL-6/IL-6R induced a dose-dependent increase in the expression of transcription factor Ets2 and enhanced its nuclear translocation alone or combinatorially in M-CSF/RANKL-differentiated human RASFs. Chromatin immunoprecipitation (ChIP) analysis of Cathepsin K and B promoters showed Ets2 binding and transcriptional activation upon IL-6/IL-6R stimulation, which was further increased with M-CSF/RANKL. Interestingly, the untargeted proteomics analysis of the conditioned media from activated RASFs using Mass-Spectrometry (MS) technique and Trans-Proteomic Pipeline tool showed the production of 113 analytes unique to IL-6/IL-6R stimulation. Further analysis of the proteomics data using STRING database for pathway analysis showed the impact of IL-6/IL-6R on immunometabolic reprogramming of human RASFs towards osteoclast-like phenotype, which was partly mediated through Ets2. These preliminary studies identified a novel role of IL-6/IL-6R-mediated transsignaling in the metabolic reprogramming in RASFs that could potentially be targeted by inhibiting Ets2 to limit their role in bone resorption in RA.
Identifying innate immune receptors causing production of inflammatory cytokines by infection of Francisella tularensis
Authors: Gutierrez Stephanie K , Setzu Nicole R , Spencer Charles T
Published: January 2023

Francisella tularensis is a pathogenic species and a causative agent of tularemia. This bacterium is incredibly infectious and often lethal in that F. tularensis causes the overproduction of pro-inflammatory cytokines. The objective of this study is to identify the innate immune receptors responsible for the production of inflammatory cytokines in response to F. tularensis infection. The study uses an immortalized C57BL/6 Bone Marrow Macrophage (BMMac) cell line as the control and knockout BMMac that are deficient in various innate immune receptors. The cells are passaged and seeded before infecting them with F. tularensis for 2 hours. The cells are treated with a high dose of gentamicin for 2 hours followed by a low dose of gentamicin for an incubation period of 24 hours. The supernatant from these cells are then collected and analysis processed by ELISA, an enzyme-linked immunosorbent assay, to measure production of inflammatory cytokines. These data were compared with the positive control of wild type BMMac and the negative control of uninfected BMMac cells. Analysis of these mutants reveal the involvement of innate immune receptors in triggering the pro-inflammatory cytokine storm in response to F. tularensis infection that leads to death of the host.
C3 complement mediates neutrophil control of cryptococcal lung infection and dissemination
Authors: Ogbomo Henry , Li ShuShun , Stack Danuta , Chojnacki Andrew K , Wojcik Katarzyna , Polyak Maria J , Colarusso Pina , Kubes Paul , Mody Christopher H
Published: January 2023

The encapsulated budding yeast Cryptococcus neoformans causes meningitis and pneumonia particularly in immunocompromised individuals including HIV-infected patients. The organism is acquired by the pulmonary route where it disseminates via the bloodstream from the lung to the brain and causes meningitis. Since the lung vasculature is replete with several innate immune cells, it is conceivable that the host would mount an immune response in the pulmonary vasculature to prevent or at least limit the dissemination of C. neoformans to other sites of the body. Hence, we used intravital microscopy to study the host cell immune interactions that occur within the lung vasculature in a living mouse. We found that neutrophils, but not monocytes, interacted with and phagocytosed C. neoformans. Interestingly, additional neutrophils underwent accretion around the phagocytosed C. neoformans, forming a cast of the vasculature. Phagocytosis of C. neoformans was impaired by the presence of cryptococcal capsule, since acapsular strain of C. neoformans were rapidly phagocytosed by neutrophils compared with encapsulated strain. Neutrophil phagocytosis of C. neoformans and subsequent accretion was mediated by complement C3 in both acapsular and encapsulated strains. Mice deficient in C3 complement showed reduced clearance of C. neoformans from the lungs and brain, compared to C3 sufficient mice. These findings highlight the importance of neutrophils in the control of C. neoformans in the pulmonary vasculature and subsequent dissemination to the distal organs including the brain.
Age-related chromatin landscapes in mouse macrophages
Authors: Oh Kyu-Seon , Babagana Mahamat , Pacholewska Alicja , Chakraborty Sayantan , Ha Jisu , Baek Songjoon , Sung Myong-Hee
Published: January 2023

Aging is associated with changes in gene expression. Age-related changes in chromatin structure may contribute to alterations in gene expression and to aging phenotypes in human. Macrophages play essential roles in tissue homeostasis and repair. Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor (TF) families. We previously showed that chromatin accessibility of macrophages is massively increased after LPS treatment. Similar programs may also contribute to the chronically elevated inflammatory state in aging. Therefore, it is important to understand how macrophages are epigenetically and functionally altered during aging. To address the age-associated differences in the transcriptome and chromatin landscape of macrophages, we have performed RNA-seq and ATAC-seq of tissue-resident macrophages in the presence or absence of LPS. Our results revealed that age effect in macrophages seems cell type-specific with the strongest effect in microglia. The majority of age-dependent TF footprints in macrophages showed reduced TF occupancy, but with either reduced or increased chromatin accessibility. We are investigating the functional relevance of the candidate TFs identified in our analysis. Our data highlight the tissue-specificity of aging features that occur in resident macrophages in vivo.
Macrophage-associated lipin-1 regulates lipid catabolism to promote effective efferocytosis
Authors: Schilke Robert Michael , Blackburn Cassidy M.R. , Rao Shashanka , Krzywanski David M. , Woolard Matthew D.
Published: January 2023

Failure to resolve inflammation leads to numerous chronic diseases. Disease resolution requires the effective removal of dead cells by macrophage-mediated efferocytosis. Excess lipid accumulation within macrophages can lead to dysfunction that promotes disease pathogenesis. Efferocytosis results in a significant accumulation of lipid inside the macrophage, yet macrophage continue to function. This suggest that during efferocytosis, macrophages have pathways to ameliorate the high lipid load. We have identified that lipin-1, a regulator of lipid metabolism, is critical to proper macrophage responses during efferocytosis. Lipin-1 is a phosphatidic acid phosphatase that also functions as a transcriptional coregulator. We used mice that lack either lipin-1 enzymatic activity or both functions in myeloid cells to define how lipin-1 regulates excess lipids during efferocytosis. We have demonstrated that mice lacking myeloid-associated lipin-1 have diminished apoptotic cell (AC) clearance in a zymozan model of efferocytosis. Clearance of lipids during efferocytosis is accomplished through beta-oxidation. Bone marrow derived macrophages lacking lipin-1 have reduced oxidative respiration in response to both AC and purified palmitate (lipid), indicating defective lipid catabolism. These data suggest that lipin-1 regulates mitochondrial lipid catabolism to reduce lipid burden during efferocytosis. These studies highlight regulation of lipid metabolic pathways in macrophages during efferocytosis that allow them to handle excess lipid burden and promote disease resolution.
Lipin-1 coordinates macrophage lipid metabolism to allow macrophage wound healing function
Authors: Woolard Matthew D , Blackburn Cassidy M.R. , Schilke Robert Michael , Chandran Sunitha , Vozenilek Aimee E
Published: January 2023

Excess lipid burden within macrophages disrupts cellular metabolism and alters their cellular function allowing these macrophages to contribute to both inappropriate and/or unresolving inflammation. The molecular mechanisms that align lipid metabolism to macrophage function are not well understood. Lipin-1 is a phosphatidic acid phosphatase that regulates the penultimate step of glycerolipid metabolism. Lipin-1 is also a transcriptional coregulator that regulates the expression of lipid catabolism genes including beta-oxidation. We and others have demonstrated that lipin-1 promotes macrophage pro-inflammatory responses. The objective of this study was to determine the contribution of lipin-1 transcriptional coregulator function within macrophages activity. We used mice that lack either lipin-1 enzymatic activity or both lipin-1 functions in myeloid cells to define how lipin-1 transcriptional coregulator function contributes to macrophage function. We demonstrated that lipin-1 transcriptional coregulator function is required for IL-4 mediated macrophage polarization and effective efferocytosis through the promotion of beta-oxidation/oxidative phosphorylation via activation of PPARγ. Loss of both lipin-1 activities from myeloid cells leads to increased atherosclerotic burden and reduction in wound closure. Furthermore, we observed enhanced pro-inflammatory responses both in the plaques and the serum of atherosclerotic mice lacking myeloid associated lipin-1. Our work provides evidence that lipin-1 aligns cellular lipid metabolism to promote effective macrophage responses.
Single-cell transcriptome profiling reveals heterogeneity of brain myeloid cells and unique subsets that regulate T cell immunity and cerebrovascular inflammation in diet-induced obesity
Authors: Yu I-Chen Ivorine , Paraiso Hallel C. , Kuo Ping-Chang , Furnas Destin J , Scofield Barbara A. , Weng Wen-Tsan , Sweazey Robert D , Chang Fen-Lei , Yen Jui-Hung
Published: January 2023

Midlife obesity and type 2 diabetes mellitus (T2DM) are linked to decreased cognition and strong risk for Alzheimer’s disease in late life. Previous studies show that obesity/T2DM is associated with a heightened state of neuroinflammation in aging mice. However, it remains unclear how diverse immune cell populations in the brain contribute to the cognitive decline. To address this, we fed middle-aged mice with high-fat diet (HFD) to induce visceral obesity and hyperglycemia. Chronic HFD consumption resulted in cognitive impairment and activation of brain resident microglia (MG). The CD45hiCD11b+ peripheral myeloid cells and CD3+ T cells were significantly increased in the brain of obese mice. Single-cell RNA sequencing was performed to characterize CD11b+ cells isolated from the brain. Unsupervised clustering singled out distinct myeloid cell clusters. Using Tmem119 and P2ry12 as markers to distinguish MG from infiltrating cells, we observed a 2-fold increase of peripheral myeloid cells, comprising distinct clusters in HFD-fed mice. Multiple subsets of MG were identified in HFD-fed mice, showing Ms4a, Apoe and gene signatures of perivascular macrophages. Notably, these subsets expressed high levels of MHC class-II-related molecules, H2-Eb1, H2-Ab1, and Cd74, and with special functions in lipid metabolism. These data suggested possible interactions between MG and T cells to potentiate neuroinflammation in HFD-fed mice. Overall, our results highlighted heterogeneity of brain myeloid cells in obesity during aging. The identified subsets of MG provide insights into brain MG controlling T cell immunity and cerebrovascular inflammation, which might lead to cognitive impairment associated with obesity and T2DM during aging.
A novel role for indoleamine 2,3-dioxygenase (IDO) in supporting the survival of long lived plasma cells (LLPC)
Authors: Lightman Shivana M , Carlson Louise M , Holling G. Aaron , Lee Kelvin P
Published: January 2023

Protective antibodies against diseases such as measles and polio primarily come from bone marrow (BM) resident long lived plasma cells (LLPC) and are a key component of durable humoral immunity. As LLPC are not innately long lived a major question remains about how these cells maintain survival. Our lab has previously demonstrated that the T cell receptor, CD28, is essential for the survival and function of LLPC. Here we further demonstrate that when CD28 on LLPC ligates with CD80/CD86 on DC, it induces upregulation and activation of indoleamine 2,3-dioxygenase (IDO). IDO’s primary action is catabolizing tryptophan into kynurenine, a well-known ligand of the aryl hydrocarbon receptor (AhR). A nuclear transcription factor, AhR is known to modulate immune responses and is expressed in PC. Unexpectedly we observed that IDO KO mice have significantly less long lived antigen specific antibody titers and a decrease in LLPC specifically in the BM post immunization. Specific deletion of IDO+DC also significantly decreases LLPC in vivo. Furthermore, treatment with kynurenine in LLPC causes upregulation of CYP1A1, the downstream target of AhR. Moreover, we see an increase in CD28 expression with kynurenine treatment in murine and human PC. We propose a model where the interaction of CD28 on LLPC with CD80/CD86 on DC activates IDO, producing kynurenine, which activates AhR to further regulate CD28 expression on LLPC. These findings challenge the traditional paradigm of IDO as immunosuppressive and uncover a novel role in promoting immunity. Filling this gap in knowledge furthers our understanding of diseases that involve LLPC, but also has direct implications in future vaccine and therapeutic drug development.

Support: T32 CA085183, R01CA121044
Solid micro particle mediated sterile type2 inflammation is mediated through cell death and A2b adenosine receptor signaling pathways
Authors: Mishra Pankaj Kumar , Davra Viralkumar , Palma Mark , Lothstein Katherine E , El-Naccache Darine W. , Benevenia Joseph , Beebe Kathleen , Hallab Nadim J , Hasko George , Birge Raymond B , Gause William
Published: January 2023

Sterile solid microparticles (MPs) trigger an innate type 2 inflammatory response dependent on a SYK-BTK-IL33 signaling axis (Nature Materials,18, 289,2019). Inoculation with MPs trigger innate inflammation with influx of neutrophils, eosinophils and alternatively activated macrophages (M2). MPs also trigger cell death in macrophages leading to release of IL33 and the development of a type 2 response with increased IL4,IL5 and IL13 and expression of M2 markers including Arg1, Relmα and Chil3. Blockade of cell death by inhibiting necroptosis (necrox-2) or apoptosis (ZVED-FMK) alone or in combination inhibited the MP mediated release of IL33 by macrophages ex vivo. We also examined the role of adenosine in MP-mediated IL-33 release by inoculating LysMCreA2bfl/flwith MP.MP induced inflammation was blocked in LysMCreA2bfl/fmice compared to LysMCre controls, with significant decreases in type 2 cytokines and IL33. Adoptive transfer of wild type macrophages to LysMCreA2bfl/flrestored the MP-mediated type 2 inflammation. These findings indicate that programmed cell death and A2b receptor signaling pathways play a major role in particulate induced sterile inflammation.
Degranulation-enhanced presynaptic membrane packing protects NK cells from perforin-mediated autolysis
Authors: Li Yu , Orange Jordan Scott
Published: January 2023

NK cells are resistant to autolysis when they kill a target cell using perforin secretion into the lytic immunological synapse. Perforin makes pores in target cell membranes allowing delivery of pro-apoptotic enzymes. Despite the fact that the perforin-containing organelles (lytic granules) are released in close range to both the NK and target cell, the NK cell membrane is protected. How NK cells avoid autolysis during degranulation is perplexing.

We demonstrate that NK cells are protected from perforin by their densely packed presynaptic membranes. When treated with 7-ketocholesterol lipid packing is reduced in NK cells, making them susceptible to perforin mediated autolysis after degranulation.

Using advanced imaging, we showed that lytic granules themselves have endogenously densely packed membranes. Importantly, during degranulation lytic granule-cell membrane fusion further reinforces local presynaptic membrane packing. This provides enhanced membrane protection at the specific sites of degranulation where NK cells face maximum local concentrations of secreted perforin.

Additionally, we found that an aggressive breast cancer cell line utilizes the same strategy to evade NK cellmediated killing. These cells are perforin-resistant owing to a densely packed postsynaptic membrane. By disrupting membrane packing, we effectively switched them to an NK-susceptible state.

Overall, we reveal an unexpected role for lipid membranes in NK cell functionality and that degranulation uses lytic granule membrane packing to create local “shields” against autolysis. Furthermore, lipid membrane modulation could alter the susceptibility of perforin-resistant cancer cells and should be considered in cytotoxic cell therapies.
Liver-derived mitochondria drive CD8 T-cell dysfunction, alter purinergic signaling and impair bacterial clearance in the lung in vivo
Authors: Tiwari-Heckler Silpa , Lee Ghee Rye , Harbison James , Csizmadia Eva , Ledderose Carola , Hauser Carl J , Otterbein Leo E , Longhi Maria Serena , Robson Simon C.
Published: January 2023

Trauma is the leading cause of death in young adults. The pathogenetic mechanisms resulting in posttraumatic immunosuppression with increased susceptibility to infection remain unclear. Mitochondria (MT) are released by cells after trauma and can generate danger molecules, such as extracellular ATP (eATP). The vascular and immune cell ectonucleotidase CD39 scavenges eATP to generate immunosuppressive derivatives such as adenosine. Here, we examined the role of eATP generated by exogenous mitochondria in modulating the immune response and provoking immunosuppression in the context of experimental pneumonia models. ATP probes and MitoSOX revealed that liver-derived MT generate ATP and ROS ex vivo. When injected into recipient mice, MT isolated from mouse liver induced upregulation of CD39 and CD73 expression on circulating CD8 T cells and promoted immune cell exhaustion. Next, wild-type mice were injected intraperitoneally with liver MT, followed by intratracheal instillation of pathogenic S. aureus. Mice receiving MT were unable to clear bacteria, as demonstrated by elevated CFU counts in the bronchoalveolar lavage, when compared to mice with bacterial instillation alone. Histological analysis confirmed more pronounced lung injury with infiltration of inflammatory neutrophils in mice administered both MT and bacteria, when compared to mice instilled only with bacteria. Lastly, the MT injection with concomitant bacteria administration resulted in loss of lung-derived CD8 T-cells.

Our data indicate that ATP generation by exogenous mitochondria boost CD39 expression in circulating CD8 T-cells and drive dysfunctional immune responses in mice that result in increased susceptibility to bacterial infection of the lungs.
Analysis of the role of helminth TGF-β mimic in tissue repair
Authors: Lothstein Katherine E , Smyth Danielle J , Maizels Rick , Gause William C
Published: January 2023

Intestinal parasites express excretory/secretory (ES) molecules, which modulate the type-2 immune response, including anti-inflammatory and tissue repair mechanisms. TGF-β mimic (TGM), an ES molecule secreted by H. polygyrus (Hp), binds TGF-β receptors yet lacks structural homology to TGF-β and exhibits distinct receptor interactions. Studies suggest TGM generates higher expression of immunoregulatory Foxp3+ Tregs and decreased fibrotic markers in vitro compared to TGF-β. Host TGF-β also influences wound healing. In cutaneous wounds, effective tissue repair requires rapid suppression of inflammation and local production of wound healing factors. We investigated whether TGM modulates wound healing using an in vivo wound biopsy model during which TGM is injected beneath a Tegaderm® layer to mimic cutaneous wound treatment. We showed that TGM accelerates wound healing when applied to the wound surface at multiple doses and modulates the associated inflammatory response. LC-MS/MS analysis of TGM-treated wound exudate revealed increases in proteins mediating leukocyte chemotaxis and proliferation, collagen deposition and fibrotic regulation. There was also enhanced immune cell infiltration of alternatively activated CD206+F4/80+ macrophages and epithelial cells concurrent with accelerated wound closure. These macrophages are CD301b+ and are essential for effective wound healing and potentially regulating fibrosis. Our findings indicate that TGM can enhance skin wound healing through enhanced production of proteins facilitating wound repair and through recruitment of specific macrophage subsets. This study will investigate TGM as a potential novel and effective therapeutic option for enhanced wound healing.
Developmental plasticity allows outside-in immune responses by resident memory T cells
Authors: Quarnstrom Clare F , Fonseca Raissa , Beura Lalit K , Ghoneim Hazem E. , Fan Yiping , Zebley Caitlin C , Scott Milcah C , Fares-Frederickson Nancy J , Wijeyesinghe Sathi P , Thompson Emily A , da Silva Henrique Borges , Vezys Vaiva , Youngblood Benjamin A , Masopust David
Published: January 2023

Central memory T (TCM) cells patrol lymph nodes and perform conventional memory responses upon re-stimulation: proliferation, migration, and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (TRM) cells are parked within single organs, share properties with terminal effectors, and contribute to rapid host protection. We observed that reactivated TRM cells rejoined the circulating pool. Epigenetic analyses revealed that TRM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated TRM cells isolated from small intestine epithelium exhibited the potential to differentiate into TCM, TEM, and TRM cells upon recall. Ex-TRM cells, former intestinal TRM that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin upon subsequent reactivation and a heightened capacity to re-differentiate into TRM cells. Thus, TRM cells can rejoin the circulation but are advantaged to re-form local TRM when called upon.