bluuebunny/crossref_metadata_2025 huggingface dataset:
PD-1 regulates response to bystander activation of memory CD8 T cells
Authors: Le Catherine , Dunai Cordelia , Khuat Lam T. , Murphy William J
Published: January 2023

Inflammatory cytokines, released in virally infected tissues, activate bystander memory T cells, expressing CD132 and CD122, in an antigen non-specific manner through signal 3 alone to provide early anti-viral protection in an NKG2D-dependent manner. We have previously observed that IL-2 stimulation expands bystander memory CD8 T cells that are NKG2D+/Programmed cell death protein 1(PD-1)-/CD25-. However, it is unclear if the presence of PD-1 regulates the ability of memory T cells to respond to signal 3 alone. In our study, IL-2 in vitro culture for 3 days results in proliferation of memory T cells in human PBMCs and preferential expansion of memory CD8 over CD4 T cells. PD-1- CD8 T cells had greater cycling of proliferation compared to PD-1+ CD8 T cells, as observed by dilution of CPeFluor670 staining. Similarly, mouse splenocytes proliferate in response to IL-2 in vitro. Ki67+ CD8 T cells were found to be predominantly PD-1−, while ConA-stimulated Ki67+ CD8 T cells were predominantly PD-1+. Adoptively transferred memory OTI CD8 T cells but not naïve OTI CD8 T cells expanded in vivo in the liver at 3–4 days post-infection with 2×10^3 PFU murine cytomegalovirus (MCMV), and a greater percentage of PD-1− OTI CD8 T cells were Ki67 positive compared to PD-1+ OTI CD8 T cells. We conclude that the expression of PD-1 on memory CD8 T cells can regulate response to signal 3 alone. Future studies will examine the impact of PD-1 blockade on the bystander response in anti-viral protection.
Hematopoietic stem progenitor cells prevent chronic stress-induced Tregs expansion in an IL12-dependent manner
Authors: Yin Deling , Caudle Yi , Zhang Haiju
Published: January 2023

Physical or psychological chronic stress induces the expansion of regulatory T cells (Tregs) to contribute to immunosuppression. However, the mechanisms remain to be defined. Recent studies from us and others have shown that hematopoietic stem progenitor cells (HSPCs) significantly modulate the immune system. Since allogeneic hematopoietic stem progenitor cell transplantation contributes to chronic graft-versus-host disease (cGVHD), but attenuation of cGvHD is associated with increased frequency of Treg. We hypothesized that HSPCs prevented the over-expansion of Tregs induced by chronic restraint stress to alleviate immunosuppression. Here we demonstrate that administration of HSPCs prevent Tregs expansion and alleviate the suppressive activity of Tregs following chronic stress. Moreover, we found this protective effect is dependent on IL-12. IL-12/STAT4 signal provides a critical role against the expansion of Tregs in chronic stress. When neutralizing antibody against IL-12 or treatment with selective STAT4 inhibitor was co-administered with HSPCs in chronic stressed mice, the protective effect of HSPCs was abrogated. Additionally, HSPCs treatment promotes IL-12 production by splenocytes in chronic stressed mice. These results suggest that HSPCs can prevent chronic stress-induced Tregs expansion in an IL-12-dependent manner, which might offer a novel strategy against immunosuppression.
c-Maf-dependent Treg cell control of intestinal TH17 cells and IgA establishes host-microbiota homeostasis
Authors: Rutz Sascha , Neumann Christian , Blume Jonas , Kalies Axel , Scheffold Alexander
Published: January 2023

Foxp3+T regulatory (Treg) cells are crucial to maintain immune homeostasis both in lymphoid and non-lymphoid tissues. Here we demonstrate that the ability of intestinal Treg cells to constrain microbiota-dependent interleukin 17–producing T helper cell (TH17) and immunoglobulin A (IgA) responses critically required the expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal Treg cell populations, including RORgt+Tregcells and T follicular regulatory cells, was c-Maf-dependent. c-Maf controlled Treg cell-derived interleukin 10 (IL-10) production and prevented excessive phosphatidylinositol-3-OH kinase (PI(3)K)–kinase Akt–mechanistic target of rapamycin (mTORC1) signaling and inflammatory cytokine expression in intestinal Treg cells. c-Maf-deficiency in Treg cells led to a profound dysbiosis of the intestinal microbiota, which when transferred to germ-free mice, was sufficient to induce exacerbated intestinal TH17 responses, even in a c-Maf-competent environment. Thus, c-Maf acts to preserve the identity and function of intestinal Treg cells, which is essential for the establishment of host-microbial symbiosis.
Deubiquitinase CYLD controls the plasticity of Treg cells by regulation of Scinderin expression
Authors: LEE JEEHO , Wan Qingqing , Han Jihye , el Baghdady Sarah , Geels Shannon , Elly Chris , Liu Yun-Cai
Published: January 2023

Regulatory T (Treg) cells are indispensable for the maintenance of immune tolerance and the prevention of autoimmunity; however, how Treg cells retain their homeostasis with immune suppressive function remains largely unclear. Here we report that deubiquitinase CYLD plays a critical role in the maintenance of Treg cells. Foxp3− specific CYLD knockout mice showed severe pulmonary inflammation due to preferential migration of Treg cells into the lung and increased interleukin-4 (IL-4) production compared to control mice, which was reversed by the deletion of IL-4. Genome-wide microarray analysis unveiled that Scinderin, a member of the actin-binding gelsolin family, was highly upregulated in CYLD-deficient Treg cells, which contributed to IL-4 production through complex formation with MEK/ERK. Increased IL-4 production by CYLD-deficient Treg cells was significantly rectified by Scinderin ablation. Our findings indicate that CYLD is essential to maintain Treg cell function through regulating Scinderin expression.
An agonist TIGIT mab suppresses regulatory T cell activity via an IL-2-mediated mechanism
Authors: Jones Brian H. , Liu Changbao , Swiecki Melissa , Strake Brandy , Chi Ellen , Bansal-Pakala Pratima
Published: January 2023

The co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) is a 26 kDa immunoglobulin domain-containing transmembrane protein that contains both intracellular ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif) and ITT (Immunoreceptor Tail Tyrosine) like domains and is found on the surface of Natural Killer and T cells. CD155 (or the poliovirus receptor) and CD112 (or Nectin-2) are natural ligands for both TIGIT and the activating receptor CD226 (or DNAM-1). In addition to the complexity of the TIGIT/DNAM-1 interplay, TIGIT has been demonstrated to drive both cell intrinsic and extrinsic functions. TIGIT binding to CD155 on Dendritic cells can drive reduction of pro-inflammatory cytokines while inducing tolerogenic cytokine secretion (Yu, 2009). More recently regulatory T cells have been demonstrated to express high levels of TIGIT and upon ligation have been demonstrated to induce effector molecules like FGL2 and reported to promote suppressive function (Joller, 2014).

We’ve identified an agonist anti-TIGIT monoclonal antibody, TGTB227, that inhibits T cell responses in vitro in both cellular reporter assays and primary memory T cell assays. TGTB227 is a human IgG1k mab that reduced the expression of the transcription factor, Foxp3, on Tregs. Moreover, TGTB227, but not the related TGTB277, reduced the functional suppressive ability of both natural and induced Tregs. Finally, these data support a TIGIT driven intrinsic CD25/IL-2-dependent mechanism for the modulation of Treg activity.
Fatty acid transporter 2 regulates the suppressive functions of PMN-MDSC in cancer
Authors: Veglia Filippo , Tyurin Vladimir , Blasi Maria , De Leo Alessandra , Donthireddy Laxminarasimha , DiRusso Concetta , Black Paul , Kagan Valerian , Gabrilovich Dmitry I
Published: January 2023

Polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) are critically important for the regulation of immune responses in cancer, promotion of tumor progression, and metastases. PMN-MDSC contribute to the failure of cancer therapies and their presence in cancer patients correlates with a poor prognosis. However, despite the recent advances in understanding of the PMN-MDSC biology, the mechanisms responsible for their suppressive functions are not well defined. The understanding of these mechanisms is critical important for the development of therapeutic strategies for the specific targeting of PMN-MDSC in cancer. Here, we report a massive accumulation of lipids in PMN-MDSC from tumor bearing mice and from cancer patients, compared to normal neutrophils from tumor free mice and healthy donors, respectively. The content of lipids was much higher in PMN-MDSC infiltrating the tumor. The accumulation of lipids was mediated by the exclusive up-regulation of the fatty acid transporter 2 (FATP2) in PMN-MDSC. The deletion of FATP2 abrogated the suppressive activity of PMN-MDSC in spleens and tumors. The selective pharmacological inhibition of FATP2 altered the activity of PMN-MSC and delayed tumor progression in mice. Importantly, FATP2 inhibition in PMN-MDSC favored tumor regression in mice when combined with checkpoint inhibitors. In conclusion, we show that FATP2 regulates the immune suppressive activity of PMN-MDSC and may represent a new target to selectively inhibit the functions of PMN-MDSC and to improve the effect of immunotherapy.
Rapid Isolation of untouched mouse MDSCs
Authors: Antignano Frann , Messing Melina , Hughes Michael R , McNagny Kelly , Kokaji Andy I , Woodside Steven M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that regulate immune responses in cancer, chronic infections and inflammatory conditions. In an individual with a tumor burden, MDSCs accumulate in peripheral lymphoid organs and within the tumor microenvironment. In several human cancers, and in mouse tumor models, the presence of high numbers of MDSCs correlates with tumor growth, metastasis and an overall poor prognosis. Approaches that impair the activity or deplete MDSCs have shown promise in animal models. Thus, MDSCs are an attractive target for therapeutic intervention.

We have developed an immunomagnetic method for the isolation of untouched mouse MDSCs. Using CyTOF analysis of cell surface antigens on spleen cells from tumor bearing mice, we developed a cocktail of antibodies that targets non-MDSCs for removal using a column-free, negative selection protocol. Our method isolates CD45+CD11b+Gr1+ cells with the following purity and recovery from spleen, BM and blood:

MDSCs (CD11b+Gr1+) from tumor-bearing miceTissuePurity (±SD)Recovery (±SD)Spleen94 ± 2.1%50 ± 12%Blood99 ± 0.6%55 ± 7.0%Bone marrow96 ± 1.0%68 ± 5.6% (naïve)

As a functional test, naïve mouse splenocytes were labeled with a proliferation dye and the T cells were activated with anti-CD3 and anti-CD28 antibodies. MDSCs isolated from tumor-bearing mice were added at different ratios and after 3 days of culture proliferation of CD4+ and CD8+ T cells was assessed by flow cytometry. This assay showed a dose-dependent suppression of both CD4+ and CD8+ T cells by the isolated MDSCs.

Our new EasySep™ Mouse MDSC (CD11b+Gr1+) Isolation Kit offers a fast and easy method that could facilitate MDSC discoveries that impact a wide range of diseases.
TRPV4 Mediates the Macrophage Phagocytic and Cytokine Response to Leptin
Authors: Scheraga Rachel Greenberg , Perelas Apostolos , Abraham Susamma , Grove Lisa , Southern Brian D , Crish James , Olman Mitchell
Published: January 2023

Obesity is characterized by chronic activation of the innate immune system. Leptin is an adipokine upregulated in obesity that has been shown to stimulate macrophage function through an unidentified signal. Our lab recently showed that the mechanosensitive calcium channel, transient receptor potential vanilloid 4 (TRPV4) mediates macrophage phagocytosis in response to infection. This study seeks to determine if TRPV4 also mediates the macrophage response to leptin. Bone marrow derived macrophages were isolated from 7–10 week old WT and TRPV4 KO mice and incubated in the presence of pathophysiological range leptin concentrations (0.1–1μg/mL; 24 hr) ± LPS (E coli 0111:B4 as a positive control) ± TRPV4 inhibitor. Macrophage phagocytosis was measured as uptake of fluorescently-labeled E coli particles. Intracellular calcium was measured using the FLIPR Calcium 5 Assay in the presence of a selective TRPV4 agonist (GSK). MIP-2 and IL-8 secretion was measured by ELISA. Leptin increases phagocytosis of E coli particles by 17% (59% of the LPS response) and TRPV4 activity by 24% (51% of the LPS response). The phagocytic response and calcium influx are abrogated by either pharmacologic inhibition or deletion of TRPV4. Secretion of MIP-2 and IL-8 in response to leptin increases by 8-fold after pharmacologic inhibition of TRPV4. Our data show that TRPV4 mediates macrophage phagocytosis of E coli particles, and suppresses proinflammatory cytokine release, in response to leptin. In addition, we show that TRPV4 function is increased by leptin. In vitro results predict that TRPV4 enhances host defense and suppresses inflammation in the setting of obesity-induced hyperleptinemia, thus potentially modulating pneumonia severity in obese patients.
Dietary Soluble Fiber Induces HCC in Dysbiotic Mice Through a Spectrum of Immunosuppressive Mediators
Authors: Vijay-Kumar Matam , Yeoh Beng San , Singh Vishal , Golonka Rachel M , Saha Piu
Published: January 2023

Dietary fermentable, soluble fibers (e.g. inulin) are known for promoting health; yet, ~40% of Toll-like receptor 5 deficient (T5KO) mice fed inulin-containing diet (ICD) develop icteric hepatocellular carcinoma (HCC) (Cell, 2018). Specifically, the disease progresses starting with hepatic cholestasis and neutrophilic inflammation at one month, followed by HCC at 6 months of ICD feeding. This diet-driven HCC was dependent on the gut microbiota and was mediated, largely in part, via surfeit production of immunosuppressive short-chain fatty acids (SCFA) and secondary bile acids, presumably as compensatory mechanisms against inflammation in the 40% pre-HCC T5KO mice. What was most striking was the upregulation of systemic bilirubin (a host immunosuppressive metabolite) within one week of ICD feeding. The hyperbilirubinemia was accompanied with an increase in IgA by ~50 and ~10-fold in the serum and liver, respectively, compared to the 60% non-HCC T5KO mice. Markers for anti-tumor T cell exhaustion, i.e. hepatic PD-L1 and layilin, were also upregulated, where the former was elevated within a week of feeding and persisted, while the latter was observed at 6 months. Hepatic immune cell phenotyping after the onset of HCC revealed substantial increase in CD4+FOXP3+PD-L1+T cells and IgA+IL10+PD-L1+Bcells, with the latter denoting a recently identified subset of highly immunosuppressive B cells. Our unprecedented findings collectively demonstrate that dietary fibers and SCFA —which are widely-held to be anti-inflammatory— could potentiate immunosuppression and impede anti-tumor immune surveillance in the tumor microenvironment in mice with gut microbiota dysbiosis.
BHLHE40 programs mitochondrial regulation of resident CD8+ T cell fitness and functionality
Authors: Li Chaofan , ZHU BIBO , Son YoungMin , Wang Zheng , Edelson Brian T , Hitosugi Taro , Rathmell Jeffrey C , Dong Haidong , Sun Jie
Published: January 2023

Tissue-resident memory CD8+ T cells (TRM) share characteristics and core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic and epigenetic regulation of TRM and TIL differentiation, maintenance and function are largely undefined. Furthermore, it is unknown whether metabolic and epigenetic manipulations can be employed to promote TRM/TIL functional programs for cancer immunotherapy. Here we report a critical mechanism programming the mitochondrial and epigenetic regulation of TRM/TIL functionality in situ. We find that mouse and human TRM/TIL express the transcription factor BHLHE40 and the BHLHE40-associated gene signature. Bhlhe40 is specifically required for TRM and TIL development and poly-functionality. Local PD-1 signaling inhibits TIL Bhlhe40 expression, and Bhlhe40 is critical for TIL reinvigoration following anti-PD therapy. Mechanistically, Bhlhe40 sustains TRM/TIL mitochondrial fitness for the promotion of a functional epigenetic state. Building on these findings, we also identify an epigenetic and metabolic regimen that promotes TRM/TIL gene signatures associated with tissue residency and poly-functionality through in vitro T cell screening. Strikingly, this regimen empowers the anti-tumor activity of CD8+ T cells and possesses therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights on the local regulation of TRM and TIL function, and offer a viable strategy for developing novel immunotherapeutic means for cancer.
Novel expression of Bone Morphogenic Protein 10 (BMP10) in lymphocytes
Authors: Tsiagbe Vincent K , Avina Samantha , Mills Steven , Patel Sehrish , Li Yu
Published: January 2023

The bone morphogenic protein family (~20 BMPs in humans), are members of the transforming growth factor-β (TGF-β) superfamily. BMPs have been shown to exert important roles during development and organogenesis by delivering positional information in both vertebrates and invertebrates. We had previously observed early induction of mRNA for BMP10 in rat B cells soon after Aggregatibacter actinomycetemcomitans (Aa) bacterial infection, in studies conducted in a rat model for human periodontal disease. However, prior to the onset of bone resorption, BMP10 mRNA precipitously declined. Being the first demonstration that mRNA for BMP10 is expressed by lymphocytes, we examined the expression of BMP10, at the protein level. Peripheral blood lymphocytes from humans, rats and mice were purified, by micro-immunomagnetic bead separation, into B cell, CD4, and CD8 T cell fractions. Flow cytometry analysis conducted with anti-BMP10 antibody revealed cellular expression of BMP10 in all the fractions examined. Interestingly, activation of CD4 T cells by Con A, rodent B cells by LPS, or human B cells by S. Aureus, resulted in drastic shut-down of BMP10 expression at mRNA and protein levels. In conclusion, our studies demonstrate, for the first time, that lymphocytes express BMP10 protein. Furthermore, while activation of B cells by bacterial antigen induced early BMP10 expression, further chronic stimulation resulted in decline in BMP10 expression. Intriguingly, mitogen stimulation of B cells and CD4 T cells, led to a drastic reduction in BMP10 expression at mRNA and protein levels, suggesting that BMP10 might be a novel marker for naïve lymphocytes.
High Spare Respiratory Capacity is a Canonical Feature of Virtual Memory, but not Conventional Memory CD8 T Cells, and is Driven by IL-15 Signalling
Authors: La Gruta Nicole L , Quinn Kylie M , Kraus Felix , Formosa Luke , Dagley Michael , Saunders Eleanor , Hussain Tabinda , Assmus Lisa , Costin Adam , Clark Joan , Ramm Georg , Good-Jacobson Kim , McConville Malcolm , Ryan Michael
Published: January 2023

Virtual memory T (TVM) cells are antigen-naïve CD8 T cell cells that exist in a semi-differentiated state. They exhibit some characteristics that are classically associated with conventional memory T cells (TMEM), including elevated expression of CD44, accelerated TCR-driven proliferation, and enhanced cytokine responsiveness. Despite rapid and robust proliferation of young TVM cells after TCR-mediated stimulation, aged TVM cells exhibit marked proliferative dysfunction. Recently, high spare respiratory capacity (SRC) was proposed as a defining metabolic characteristic of antigen-experienced memory T cells (TMEM) cells, thought to promote both their longevity and rapid functionality. Given the semi-differentated state of TVM cells, we sought to determine whether the SRC of TVM cells also corresponded to their longevity and functionality. Surprisingly, we found that elevated SRC was a feature of TVM cells, but not TMEM cells, and that SRC increased substantially with age in both subsets, repudiating a link between SRC and functionality. We further showed that SRC was closely linked with responsiveness to IL-15, since TVM cells alone were dependent on IL-15 for survival/accumulation, and showed increased receptor (CD122) expression, and IL-15 mediated STAT5 phosphorylation. Moreover, recent infection caused an increase in SRC in TVM cells but inhibition of IL-15 during infection was sufficient to prevent this increase. Collectively, these findings indicate that elevated SRC is a feature of TVM and not TMEM cells and is a consequence of their heightened IL-15 sensitivity. Moreover, elevated SRC is not associated with T cell functionality in young or aged cells, but may be associated with survival of TVM cells.
TIM-3-dependent regulation of restimulation-induced cell death sensitivity flips changes in early versus late-stage human effector T cells
Authors: Lake Camille , Jiang Timothy , Bauman Bradley , Voss Kelsey , Snow Andrew L
Published: January 2023

In healthy individuals, the balance between immune-mediated pathogen clearance and tissue damage is tightly regulated through multiple processes. One such mechanism is restimulation-induced cell death (RICD), a propriocidal apoptotic mechanism that constrains T cell responses. Activated T cells must surpass a high threshold of TCR signal strength to induce pro-apoptotic genes responsible for executing RICD. Because co-receptors such as TIM-3 are thought to modulate TCR signal strength, we hypothesized that TIM-3 can shape the T cell response by tuning RICD sensitivity. We utilized siRNA knockdowns and transfection of wild-type and mutant TIM-3 expression constructs to assay for changes in RICD sensitivity in both immortalized and primary human T cells in vitro. Strikingly, we observed that the effect of altering TIM-3 expression varied temporally during the effector T cell response. Whereas TIM-3 promoted RICD resistance in newly activated T cells, TIM-3 enhanced RICD sensitivity in late-stage effector T cells by boosting TCR-induced FASL and BIM expression in a ligand-independent fashion. Preliminary evidence suggests that this dichotomy may relate to substantial temporal changes in the expression, cellular localization, and glycosylation of TIM-3 in early versus late-stage effector T cells. Overall, these results indicate that TIM-3 influences apoptosis sensitivity differently at distinct phases of the human T cell response, with important implications for the use of checkpoint blockade immunotherapies targeting TIM-3. Our findings may also help to clarify discrepancies in the literature regarding TIM-3 signaling and function in T cells.
Maximizing engagement and critical thinking with stories – case studies of all shapes and sizes
Authors: Lukin Kara R , Aviszus Katja
Published: January 2023

When done purposely, case studies blend desires of modern students for relevance and social engagement with educators needs to convey detailed content and develop critical thinking skills. By selecting or creating cases that include both local and international settings as well as characters from broad socio-economic and cultural backgrounds, we elevate discussions of diversity and students’ feelings of inclusivity. In addition, even terse mini-scenarios can require students to analyze situations and apply their knowledge instead of memorize facts. For many, finding or writing exciting narratives and integrating case study strategies can seem daunting. However, barriers to adopting this pedagogical tool are reduced when educators recognize that cases are 1) highly variable in depth (from a few sentences to several pages), and 2) can be used very flexibly. Here, we explain examples of how we have integrated cases of various lengths throughout an undergraduate immunology course. Two examples of how a single, well selected case can address topics through an entire term will be detailed. We will explain a simple strategy for converting standard exam or practice questions into mini-cases, leveraging summaries of primary literature and the course text.

All classroom ready resources will be available for immediate access through AAI’s preferred method.
Role modelling Immunology to support STEM education outreach efforts for Pacific College students
Authors: Sika-Paotonu Dianne
Published: January 2023

Educators are important role models for their students. Specialists and professionals working within STEM related areas can support outreach related education activities as role models for children and young people. To support improved education and health outcomes for Māori and Pacific young people in New Zealand (NZ), various strategies and interventions have been proposed. The importance of role modelling in this context is recognised. The purpose of this work was to support a STEM-related outreach education event organised by an external organisation, with a teaching contribution delivered by a Pacific Biomedical Scientist that was Immunologically based. The female Pacific Biomedical Scientist was invited to contribute to this work as a role model specifically to support improved interest and participation with STEM related learning in a workshop classroom environment. Given that context relevant science related learning was a primary focus, clinical research work was presented to the Pacific College students aged 14–16 years. The research work in part related to the autoimmune condition known as Acute Rheumatic Fever (ARF) that can trigger permanent heart damage known as Rheumatic Heart Disease (RHD) if left untreated. It is recognised that Māori and Pacific Peoples in NZ and in the Pacific Region are affected disproportionately by ARF and RHD. Verbal and written feedback from the College students highlighted the Immunology teaching session was well-received and enjoyed by the students. This work also demonstrated the importance of role modelling in the classroom blended with scientific knowledge communication in supporting positive interactions in the classroom for Pacific College students.
Crosstalk between lung epithelial cells and myeloid cells in innate immune defense
Authors: Liu Xin , Boyer Mark A , Shin Sunny
Published: January 2023

Pathogens have evolved a wide range of strategies to allow survival and subsequently cause disease in humans. However, it is still poorly understood how the immune system successfully overcomes pathogen-induced disturbance and enable robust immune responses against infection. The intracellular bacterium Legionella pneumophila has the ability to block global protein synthesis in target macrophages, but the host can still strongly evoke an IL-1-dependent inflammatory response that clears infection. Here, we show that IL-1 signals directly to alveolar epithelial cells, which potently drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by these cells. Importantly, GM-CSF-mediated JAK2/STAT5 signaling promotes inflammatory gene expression in myeloid cells. We found that STAT5 was enriched at the 5′ upstream regions of inflammatory cytokine genes. In addition, GM-CSF metabolically reprogrammed myeloid cells to undergo aerobic glycolysis, which was required for maximal inflammatory cytokine production. Our findings reveal that lung epithelial cells act as a key intermediary to facilitate communication between infected and bystander myeloid cells and orchestrate antimicrobial defense.
Insulin-like growth factor 1 regulates a pulmonary niche promoting type 3 innate lymphoid cell development necessary for protection against pneumonia in the newborn
Authors: Gray Jerilyn Kaye , Oehrle Katherine , Acker Elizabeth Grace , Lang Ian , Cloud Natalee , Deshmukh Hitesh
Published: January 2023

Bacterial pneumonia kills more than a million infants worldwide each year. Type 3 innate lymphoid cells (ILC3) are critical for lung mucosal defense against bacterial pneumonia in the neonatal period when T-cell mediated immunity is weak. The signals that guide the development of pulmonary ILC3 remain incompletely understood. Bone marrow-derived ILC3 are proposed to populate the lungs. We demonstrate that neonatal lung is the principal site of pulmonary ILC3 biogenesis. Mature pulmonary ILC3 are derived from ILC precursors that populate a spatially distinct niche, defined by fibroblasts in developing alveoli. Insulin-like growth factor (IGF) 1, produced by alveolar fibroblasts, instructed expansion and maturation of pulmonary ILC precursors throughout infancy. Conditional ablation of IGF1 in alveolar fibroblasts during postnatal development or deletion of the IGF-1 receptor from ILC precursors interrupted ILC3 biogenesis and rendered newborn mice susceptible to pneumonia. Finally, we show that developmental signals active in alveolar fibroblasts link the postnatal lung development with the expansion and maturation of pulmonary ILC3s, thus coordinating the simultaneous development of lungs and pulmonary mucosal defenses. Accordingly, premature infants with bronchopulmonary dysplasia, a disorder characterized by interrupted postnatal alveolar development and increased morbidity to respiratory infections, had reduced levels of IGF1 and pulmonary ILC3. These data highlight the importance of newborn period as ‘critical window’ in the development of pulmonary mucosal immunity and explain why disrupted lung development in prematurely born infants has an enduring effect on host resistance to respiratory infections.
Type 1 interferon expression and signaling occur in spatially-distinct regions in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques
Authors: Mattila Joshua T
Published: January 2023

Type 1 interferons (IFN1s) are implicated in the pathogenesis of tuberculosis (TB), a disease caused by infection with Mycobacterium tuberculosis (Mtb). Most of the human data on IFN1 in TB come from transcriptional profiling on peripheral blood or human airway cells, but little is known about IFN1 expression or signaling in granulomas, the multicellular lesions associated with TB. Identifying which cells express, or are regulated by, IFN1 in granulomas may enable IFN1-targeting host directed therapies in TB to improve outcomes in TB. To expand our understanding of IFN1 in TB, we examined granulomas from macaques, nonhuman primates that replicate the pathology and course of human TB, to identify how IFN1 is regulated and expressed at the site of disease. We found that granulomas expressed more interferon-stimulated genes (ISGs) and contained more IFN1 protein than uninfected lung but there was substantial heterogeneity between granulomas, even within the same tissue. Epithelioid macrophages in regions commonly infected with Mtb, and a subset of neutrophils in necrotic granuloma regions, had phospho-IRF3+ nuclei, a transcription factor upstream of IFN1 expression and expressed greater quantities of IFN1 protein than cells in other granuloma regions. In contrast, a subset of cells in the lymphocyte cuff strongly expressed the IFN1 receptor IFNAR1 and expressed MX1, an ISG downstream of IFN1. Alveolar macrophages adjacent to granulomas had diverse IFN1-expressing and IFN1-activated phenotypes. These results indicate that IFN1’s biology differs by granuloma microenvironment and that IFN1 expression may be driven by Mtb antigens in granulomas.
Caspase 11 Negatively Regulates Carbapenem-resistant Klebsiella Pneumoniae (CRKP)-induced Pneumonia
Authors: Ghimire Laxman , Paudel Sagar , Jin Liliang , Jeyaseelan Samithamby
Published: January 2023

Background

CRKP remains one of the major causes of health-care associated infections worldwide. Due to its multi-drug resistance property, host-targeted immunotherapy is a promising alternative to treat CRKP-infections. Caspase11, a non-canonical inflammasome, has been shown to protect against cytosolic bacterial pathogens. However, its role in CRKP-induced pneumonia remains elusive.

Methods

We used C57BL/6 (WT) and Caspase 11 knock out (KO) mice to induce pneumonia by intratracheal inoculation of 108 colony forming units (CFUs) of CRKP per mouse. In E coli-induced pneumonia model, 106 CFUs per mouse was used.

Results

Using a mouse model of CRKP-induced pneumonia, we found that Caspase 11 KO mice were protected from intratracheal CRKP infection. The KO mice had significantly less bacterial burden in lungs and BALF compared to their wild-type counterparts. Total leukocytes and neutrophil recruitment into the air spaces were significantly higher in KO mice. To determine whether the phenotype is specific to CRKP infection, we induced pneumonia in WT and KO mice using E. coli through intratracheal route. Consistent with CRKP model, the KO mice displayed protective phenotype during pulmonary E coli challenge suggesting that the phenotype observed in the KO mice is not specific to single bacterium. Further, the KO mice had less IL-1β, CXCL1, and CXCL2 but higher amount of IL-17A secreted in the lungs suggesting that IL-17A could mediate neutrophil recruitment in the KO mice during pulmonary CRKP infection.

Conclusions

Our findings together demonstrate that Caspase 11 negatively regulates CRKP-induced pneumonia and blocking Caspase 11 could be a potential therapeutic intervention for treating CRKP-infected pneumonic patients.
IL-25 Regulates Alveolar Macrophage Exosome Release in Response to LPS
Authors: Fan Jie , Li Yuehua , Li Zhigang
Published: January 2023

Background

Acute lung injury (ALI) is a major component of multiple organ dysfunction syndrome (MODS) following pulmonary and systemic infection. Alveolar macrophages (AMf) are at the center of the pathogenesis of ALI. Emerging evidence has shown that cell-cell interactions in the lungs play an important regulatory role in the development of acute lung inflammation. However, the exact mechanisms remain poorly addressed. In this study, we explore a novel function of lung epithelial cells (LEPC)-derived IL-25 in the regulation of AMf release of exosomes following LPS stimulation.

Methods

For in vivo experiments, C57BL/6 (WT) and TLR4 knockout (TLR4−/−) mice were treated with LPS (2 mg/kg B.W.) in 0.2 ml saline via intratracheal aerosol administration. Bronchoalveolar lavage fluid was then collected at 0 – 24 h after LPS treatment, and the AMf-derived exosomes were measured. For in vitro studies, LEPC and bone marrow-derived Mf (BMDM) were isolated from WT or TLR4−/− mice, and then cocultured in the Transwell™ system. At 0 – 24 h after the coculture, the BMDM and supernatant were harvested for the measurements of exosomes and cytokines.

Results

We demonstrate that LPS induces AMf release of exosomes, which are then internalized by neighboring Mf to promote TNF-a expression. Secretion of IL-25 from LEPC downregulates Rab27a and Rab27b expression in Mf, resulting in suppressed exosome release, thereby attenuating exosome-induced TNF-a expression and secretion.

Conclusion

These findings reveal a previously unidentified pathway of LEPC-Mf cross-talk, which negatively regulates Mf inflammatory responses to LPS. Modulating IL-25 signaling and targeting exosome release may present a new therapeutic strategy for the treatment of ALI.
Dysregulated CARD9 Signaling in Neutrophils Drives Inflammation in a Mouse Model of Neutrophilic Dermatoses
Authors: Tartey Sarang , Kanneganti Thirumala-Devi
Published: January 2023

Mice homozygous for the Tyr208Asn amino acid substitution in the carboxy terminus of SHP-1 (referred to as Ptpn6spin mice) spontaneously develop a severe inflammatory disease resembling neutrophilic dermatoses in humans. Disease in Ptpn6spin mice is characterized by persistent footpad swelling and suppurative inflammation. This IL-1α dependent inflammation is mediated through SYK-RIPK1-TAK1-ASK1 kinases in Ptpn6spin mice independent of inflammasome activation. In the current study we have identified previously unknown role for CARD9 signaling as a critical regulator for Ptpn6spin-mediated footpad inflammation. Genetic deletion of CARD9 significantly rescued thePtpn6spin-mediated footpad inflammation. Mechanistically, enhanced IL-1α mediated signaling in Ptpn6spin mice neutrophils was dampened in Ptpn6spinCard9−/−mice. Collectively, this study identifies SHP-1 and CARD9 crosstalk as a novel regulator of IL-1α driven inflammation and opens future avenues for finding novel drug targets to treat neutrophilic dermatosis in humans.
Modeling the stimulatory network of CD16, 2B4 and NKG2D in natural killer cell activation
Authors: Makaryan Sahak Z , Finley Stacey
Published: January 2023

Natural killer (NK) cells are innate immune effector cells that play an immediate role in host defense by releasing cytotoxic chemicals upon activation. A better understanding of the mechanisms regulating NK cell activation can improve NK cell-based therapies. However, due to the diversity of stimulatory pathways, each with a complex network of signaling species, a systems biology approach is required to understand NK cell activation. We constructed a mathematical model that mechanistically describes the signaling network of CD16, 2B4 and NKG2D. The model was fit to published experimental data and validated with a separate dataset. Baseline model predictions demonstrate that CD16 and NKG2D are qualitatively similar in that they activate the downstream species to a greater degree and at a faster rate when compared to 2B4. Contrastingly, 2B4 activates the signaling species over a longer time interval. In silico pairwise co-stimulation of the receptors, using various ligand concentrations, preferentially impacts the kinetics of species activation: the species are activated faster but the time interval of species activation becomes shorter as more ligands are introduced. Perturbing the stimulatory pathways by altering kinase (activator) and phosphatase (inhibitor) activity, highlights the phosphatases’ predominant control of the network and shows that decreasing their level of activity greatly improves the rate of species activation. Thus, the model predicts (1) qualitative differences between the pathways, (2) increasing receptor stimulation impacts the kinetics of species activation and (3) inhibiting the inhibitor significantly augments signal transduction. This insight can help design NK cell immunotherapies.
The suppressor of cytokine signaling 1 (SOCS1) impairs skin host defense in healthy and hyperglycemic mice
Authors: Klopfenstein Nathan , Serezani Henrique
Published: January 2023

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of skin and soft tissue infections in both healthy individuals and those with preexisting conditions such as diabetes. MRSA skin infections are characterized by the formation of a neutrophilic abscess that prevents bacterial spread to deeper tissues. Although we and others have shown that skin resident macrophages initiate this immune response, the molecular events that lead to optimal neutrophil recruitment and abscess formation remain to be fully elucidated. Here we examined the role of the protein SOCS1 and its impact on host defense during MRSA skin infection in both hyperglycemic and euglycemic mice. SOCS1 is a known STAT1 inhibitor and negatively influences TLR activation, possibly influencing early skin host defense. We have previously demonstrated that hyperglycemic mice are unable to control MRSA skin infection, which was accompanied by poor abscess formation. Here, we demonstrate that SOCS1 expression in macrophages isolated from infected hyperglycemic mice is greatly enhanced in a manner dependent on histone deacetyltransferase (HDAC) activity. Delivery of SOCS1 inhibitor peptide (iKIR) as well as myeloid cell-specific SOCS1 deletion decreased both lesion size and bacterial burden in the skin. SOCS1 inhibition enhanced the production of pro-inflammatory cytokines TNF-a and IL-1b. Importantly, SOCS1 inhibition enhanced bacterial ingestion and killing in macrophages from both euglycemic and hyperglycemic mice. Overall, these data indicate that the threshold of SOCS1 expression may negatively impact skin host defense in both hyperglycemic and other susceptible patient populations.
Lactic acid suppresses LPS-induced mast cell cytokine production by limiting glycolysis and ATP availability
Authors: Ryan John J , Hoeferlin L. Alexis , Chalfant Charles E. , Caslin Heather
Published: January 2023

Sepsis, a life threatening response to infection, has a well-studied initial cytokine storm, with a less understood secondary immunosuppressive phase. Elevated blood lactate and slow lactate clearance are associated with mortality, however, any regulatory role is unknown. We hypothesized that lactic acid contributes to the late phase of sepsis and is not solely a consequence of bacterial infection. Thus far, no studies have examined the effect of lactic acid on sepsis models in vivo or a mechanism by which they suppress LPS activation in vitro. Because mast cells can be activated systemically and contribute to sepsis, we examined lactic acid effects on the mast cell response to the bacterial product LPS. Lactic acid significantly suppressed LPS-induced cytokine production and NFκB-mediated transcription in mouse bone marrow derived mast cells and cytokine production in peritoneal mast cells. Suppressive effects were MCT-1-dependent and observed with sodium lactate or formic acid. Further, lactic acid significantly suppressed cytokine induction following LPS-induced endotoxemia in mice. Because glycolysis is linked to inflammation and lactic acid is a by-product of this process, we examined changes in glucose metabolism. Lactic acid treatment reduced glucose uptake and lactate export during LPS stimulation. Lactic acid effects were mimicked by glycolytic inhibitors and reversed by increasing ATP availability. These results indicate that glycolytic suppression and ATP production are necessary and sufficient for lactic acid effects. Our work suggests that enhancing glycolysis and ATP production could improve immune function, counteracting lactic acid suppressive effects in the immunosuppressive phase of sepsis.
Single-cell analysis of γδ T cells reveals limited TCR delta chain diversity in mouse lung Vγ4 γδ T cells
Authors: Mistri Somen K , Hampel Kenneth J , Dienz Oliver , Boyson Jonathan E
Published: January 2023

γδ T cells are innate-like T cells that are one of the first responders in the defense against pathogens through their rapid production of pro-inflammatory cytokines like IL-17 and IFN-γ. Currently, the mechanisms that direct γδ T cells to exert these distinct effector functions are unknown. The limited TCR diversity of tissue-specific γδ T cells is often associated with specific effector functions. We have recently found that IL-17- and IFN-γ-producing Vγ4 γδ T cells in the lung are marked by SLAM1 and SLAM6 cell surface receptors, respectively. Our long-term goal is to investigate the link between γδ T cell effector function, specific SLAM family receptor expression, and TCR clonotype usage. The objective of this study was to identify the major Vγ4 γδ TCR clonotype(s) in the mouse lung. Vγ4 γδ T cells were single cell-sorted and paired TCR clonotypes were identified using next-generation sequencing of TCR amplicon libraries. Out of 789 sorted lung C57BL/6 Vγ4 T cells, we obtained paired TCR γ and δ chain sequences from 396 cells. These data revealed that Vδ5 and Vδ2 chains accounted for 56.20% and 35.77% of the productive TCRδ chain rearrangements, respectively. A significant fraction of the Vδ5 sequences were characterized by invariant germline-encoded (28.57%) or highly restricted (9.10%) Vδ5Dδ2Jδ1 sequences. Likewise, 55.10% of the Vδ2 sequences were characterized by a highly restricted Vδ2Dδ2Jδ1 sequence. These data reveal that the lung Vγ4 γδ T cell population is characterized by extremely limited delta chain usage. Since lung Vγ4 T γδ cells are programmed to be SLAM1+IL-17+ or SLAM6+IFN-γ+, future studies will determine whether there is a link between this limited TCRδ chain usage, cytokine production, and SLAM receptor expression.
Redox regulation of autophagy in thymic stromal cell function
Authors: Semwal Manpreet Kaur , Hester Allison K , Xiao Yangming , Venables Thomas , Griffith Ann
Published: January 2023

T lymphocytes develop in the thymus, where mutually inductive signaling between lymphoid progenitors and thymic stromal cells directs the progenitors along a well-characterized program of differentiation. However, the biology of the stromal cells comprising the lymphopoietic thymic microenvironment remains relatively under-characterized because stromal cells are rare and difficult to isolate. Using a deconvolution technique to study gene expression essentially in situ, we previously identified a deficiency in the peroxide quenching enzyme catalase (CAT) in thymic stromal cells, and found that CAT deficiency results in high reactive oxygen (ROS) levels in this population, eventually leading to thymic atrophy. Our current studies address the possibility that high ROS levels serve physiological functions in stromal cells in the young, steady-state thymus. Autophagy is critical for self-antigen presentation and T cell selection in the thymus, and is induced by many stressors, including oxidative stress. Our preliminary data indicate that catalase overexpression targeted to mitochondria in transgenic mice (mCAT Tg) results in diminished autophagy in thymic stromal cells and causes diminished negative selection in the thymus. Our results suggest that, while accumulated oxidative damage ultimately undermines healthy thymus function, a high ROS environment in stromal cells promotes autophagy critical for stromal induction of T cell tolerance in the young, steady-state thymus.

Supported by N.I.H. grants R01AI121367 and R21AI103685
Catalase expression mediates redox regulation of promiscuous gene expression in thymic stromal cells
Authors: Hester Allison Katherine , Semwal Manpreet K , Yangming Xiao , Cepeda Sergio , Venables Thomas , Griffith Ann
Published: January 2023

The primary site for the development of T lymphocytes is the thymus, where cross-talk between thymic stromal cells (TSCs) and T cell progenitors mediates the development and maintenance of both populations. However, the thymus begins to atrophy relatively early in life, resulting in diminished T cell output, and corresponding immunodeficiencies in aged individuals. Our previous studies revealed that thymic stromal cells express conspicuously low levels of the peroxide quenching enzyme catalase (CAT), which results in high reactive oxygen species (ROS) levels and accumulated oxidative damage in thymic stromal cells, ultimately promoting thymic atrophy. In our current studies, we find that when catalase deficiency is complemented by overexpression targeted to mitochondria in transgenic mice (mCAT Tg), stromal function declines in young mice relative to non-transgenic littermates. TSC transcriptome analysis reveals decreased expression of tissue-restricted antigen (TRA) genes in young mCat Tg mice relative to controls. Stromal TRA expression is required for self-antigen presentation, and therefore promotes negative selection of potentially auto-reactive T cells. We propose that oxidative stress generated by low catalase levels in stromal cells promotes this key physiological function in the young, steady state thymus; in contrast however, the resulting accumulated oxidative damage ultimately impairs function in the aged thymus.