bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-17 dependent fibroblastic reticular cell training boosts host-protective immunity to mucosal infection
Authors: Wu Dongwen , Majumder Saikat , McGeachy Mandy J
Published: February 2023

Prior experience of inflammatory stimuli is thought to reduce morbidity and mortality to newly encountered infections through innate immune training that can be enhanced by childhood vaccination. Here we demonstrate that IL-17 signaling in lymph node fibroblastic reticular cells (FRC) is required for efficient clearance and protective antibody-mediated immunity to the gut bacterial pathogen Citrobacter rodentium. Furthermore, prior experience of gut inflammation led to an enhanced antibody response to C. rodentium without altering the kinetics of bacterial infection. However, colon inflammation was paradoxically reduced by previous inflammatory exposure, due to IL-10 production by B cells. Mechanistically, FRC-intrinsic IL-17 dependent activation mediated the trained response, which includes enhanced metabolic response of FRC to infection. These data identify a new pathway for immune training through FRC that modulates the adaptive response, enhancing protective antibody while reducing gut inflammation, to more efficiently preserve host health.
Stochastic pooling of IKK/NEMO complexes describes information transmission in the NF-KB pathway in response to IL-1 and TNF stimulation.
Authors: Cruz J. Agustin , Mokashi Chaitanya , Lee Robin E.C.
Published: February 2023

A myriad of inflammatory cytokines regulates signaling pathways for normal cellular physiology. The IKK complex is an integration hub for extracellular cytokines that govern NF-κB signaling. In response to inflammation, IKK is activated through recruitment to receptor-associated protein assemblies. How and what information IKK complexes transmit about the milieu are open questions. Here we track dynamics of IKK complexes and nuclear NF-κB to identify upstream signaling features that determine same-cell responses. Quantitative imaging experiments and computational modeling of single complexes reveals that their size, number, and timing relays cytokine-specific information with feedback control that is independent of transcription. Our results provide evidence for variable-gain stochastic pooling, a noise-reducing motif that enables parsimonious and cytokine-specific signaling. We argue that emergent properties of stochastic pooling motif are general principles of receptor signaling, and we present new modules of regulation of NF-kB signaling in response to cytokine stimulation.
FMS-like tyrosine kinase 3 ligand (FLT3L) administration alters rat classical and plasmacytoid dendritic cell phenotype in a tissue-specific manner
Authors: Carlson Kristin N , Verhagen Joshua , Verhoven Bret , Pavan-Guimaraes Juliana , Al-Adra David
Published: February 2023

Background:

Characterization of tissue-resident dendritic cell (DC) subsets, including classical DCs (cDCs) and plasmacytoid DCs (pDCs), is complicated experimentally by their rare nature. Many investigators have artificially increased DC yields through administration of the cytokine FMS-like tyrosine kinase 3 ligand (FLT3L). Despite its previous use experimentally, no study has yet described the effects of in vivo FLT3L administration on rat tissue-resident DCs. This study provides an advanced characterization of rat tissue-resident cDCs and pDCs following FLT3L injection.

Methods:

Lewis rats were injected once daily for 10 days with PBS vehicle or with 10, 50, or 100μg of FLT3L. Animals were sacrificed on the 11th day and dissected to obtain liver, spleen, and mesenteric lymph nodes. Cells were stained for 13-color flow cytometry and data was acquired on the Cytek Aurora spectral cytometer.

Results:

cDC and pDC enumeration increased in a dose-dependent manner following FLT3L administration. Expression levels of MHC I, MHC II, CD40, CD80, CD86, and PD-L1 were evaluated on liver, spleen, and mesenteric lymph node-resident cells. MHC II, CD80, and CD86 levels were significantly decreased on liver-resident cDCs but remained unchanged or increased on spleen and lymph node cDCs. Changes in MHC I/II and costimulatory molecule expression levels on pDCs varied by tissue and marker.

Conclusions:

FLT3L administration in vivo increased cDC and pDC enumeration, generated immature cDCs in the liver, and increased cDC maturation in the spleen and mesenteric lymph nodes. This comprehensive characterization of rat liver, spleen, and lymph node-resident DCs is key to understanding the effects of FLT3L on cDC and pDC phenotype.
Lineage tracing and transplantation studies indicate endothelial origin of B-1a cells in the mouse embryo
Authors: Yoshimoto Momoko , Nishida Chika , Cornelius Samuel , Valiente Noemi , Kobayashi Michihiro
Published: February 2023

Innate-immune like B-1a cells are unique in its functions and developmental origin compared to conventional adaptive immune B-cells (B-2 cells). Especially, their origin and developmental pathway have been argued over four decades between two proposed models: the lineage model and the selection model. Although many transplantation studies support the lineage model in which B-1a cells are derived from fetal progenitors, contradictory results of fetal liver transplantation studies raise a question as to whether fetal liver hematopoietic stem cells (HSCs) are the primary source of the peritoneal B-1a cells. In order to address this question, we took two approaches using transplantation assays and lineage tracing mouse models that enable us to trace Tomato+ progeny of endothelial cells (ECs, Cdh5CreERT mice) and HSCs (Fgd5CreERT2 mice) at a time of Tamoxifen (Tam) injection, respectively.

Results:

First, we transplanted HSC-precursors (pre-HSCs) from the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic (E) 11.5 into irradiated NSG neonates. Interestingly, we found more B-1a repopulating capacity than multi-lineage in the pre-HSC population. The lineage tracing of hemogenic ECs revealed that B-1a progenitors and HSCs were simultaneously produced from ECs during E9.5 to 10.5, but B-1a production is diminished after E11.5 while HSC were still labeled at E11.5. The HSC-tracing model demonstrated the minimal contribution of FL HSCs to the postnatal B-1a cell pool.

Conclusion:

Our data addressed the long-lasting controversy and showed HSC-independent B-1a cell development in the mouse embryo.
The Inhibitor of MyoD Family A Promotes Terminal Myeloid Differentiation
Authors: Houser Jeremy S , Patel Maulin , Onopiuk Marta , Tsiokas Leonidas , Humphrey Mary Beth
Published: February 2023

The differentiation of hematopoietic stem and progenitor cells is tightly regulated to maintain immune and blood homeostasis while also responding to injury and immunological insult. Myeloid lineage-committed progenitor cells are highly proliferative, but do not self-renew and must be continually replenished by the differentiation of hematopoietic stem cells. The process of terminal differentiation is controlled by various cell-intrinsic and - extrinsic factors, and the interactions between these factors remain not well understood. We hypothesize that the Inhibitor of MyoD Family A (I-mfa) protein is a novel regulator of myeloid lineage differentiation through its interaction with other cell-intrinsic factors.

To examine this, mice which lack I-mfa expression (I-mfanull) were compared to wild type (WT) 129Sv age- and sex-matched controls. I-mfanull animals had reduced spleen size (both in weight and total cellularity), but had preserved relative proportions of B, T, and myeloid cells as analyzed by flow cytometry when compared to WT. Analysis of the bone marrow (BM) myeloid lineage-committed progenitors showed an accumulation of common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP) in I-mfanull animals when compared to WT. In serial colony forming unit assays I-mfanull BM produced fewer mature colonies and continued to produce colonies after 5 passages, suggesting aberrant self-renewal.

Using myeloid cell lines lacking or overexpressing I-mfa, the molecular mechanism by which I-mfa acts is being elucidated. These results taken together suggest I-mfa acts to promote differentiation of myeloid cells, an effect which may be important in the treatment of myeloid neoplasms or bone marrow failure.
Microbial exposure in early life alters the ontogeny and function of CD8+ T cells
Authors: Tabilas Cybelle , Reynaldi Arnold , Daly Ciarán W.P. , Mon Kristel Yee , Wesnak Samantha , Smith Norah L. , Davenport Miles P. , Grimson Andrew W. , Rudd Brian D.
Published: February 2023

Evidence has shown that microbial exposure in early life can have long-lasting effects on the health status of an individual. However, the underlying basis of how microbes shape the immune system is not well understood. In this study, we utilized a novel pet shop mouse model and compared the immune development and function of laboratory mice raised in either a clean (SPF) or dirty (pet-shop) environment. When we challenged the two groups with an intracellular pathogen in adulthood, we found that mice raised in the dirty environment mounted a more robust CD8+ T cell response and were highly resistant to infection. Adoptive transfer experiments with CD8+ T cells from clean and dirty mice into T cell deficient recipients indicated that differences in immune susceptibility were linked to changes in the CD8+ T cell compartment. Moreover, the dirty mice had an increased accumulation of antigen-specific CD8+ T cells and produced more effector molecules. To understand how the ontogeny of the CD8+ T cell compartment was altered in the dirty mice, we used our fate-mapping timestamp mice and found a higher proportion of fast-acting fetal-derived CD8+ T cells and fewer slow-acting adult-derived CD8+ T cells present in the dirty mice. We found that immune susceptibility could be normalized between clean and dirty mice by depleting the fetal layer of CD8+ T cells. Collectively, these findings suggest that microbial exposure leads to permanent changes in the immune system by altering the developmental layering of the peripheral T cell compartment. We are currently using a combination of RNA- and ATAC-seq to gain a deeper understanding of how the microbial environment mediates changes to CD8+ T cells at the gene regulatory level.
The distinct co-stimulatory requirements for thymic MAIT and NKT cell development
Authors: Celli Sofia I , Watanabe Masashi , Alkhaleel Farrah , Hodes Richard
Published: February 2023

Mucosal-associated invariant T (MAIT) cells are a unique lineage of T cells that develop in the thymus. MAIT cells are positively selected by TCR recognition of microbial metabolite / MHC Class I-like molecule MR1 complexes presented on CD4/CD8 double-positive thymocytes. The developmental program of MAIT cells has been characterized as similar to that of invariant Natural Killer T (NKT) cells. Both MAIT and NKT cells acquire innate-like effector phenotypes during thymic development, and in each case differentiate into multiple subsets, with mature NKT cells categorized as either NKT1, NKT2 or NKT17, and mature MAIT cells as MAIT1 or MAIT17. We previously found distinct B7/CD28 and CD40L/CD40 co-stimulatory requirements for the development of thymic NKT subsets in BALB/c mice. In the absence of B7/CD28 co-stimulation, all NKT subsets were decreased, while only the NKT2 subset was specifically abrogated when lacking CD40L/CD40 co-stimulation. In the present study, we investigated the co-stimulatory requirements for MAIT cell development. Interestingly, MAIT17 cells were significantly increased when lacking either B7 or CD40 co-stimulation, while MAIT1 development was largely unaffected. These results revealed distinct co-stimulatory requirements for the thymic development of MAIT and NKT subsets. We are currently investigating the underlying mechanisms leading to the increase of MAIT17 cells in the absence of co-stimulatory signals.
Chemically-induced colitis is more severe in mice expressing TLR4 SNPs: a novel model for studying human Inflammatory Bowel Disease
Authors: Vlk Alexandra , Vogel Stefanie N
Published: February 2023

Innate immune cells sense “danger” encountered in the environment using pattern recognition receptors (PRRs). The Toll-Like Receptors (TLRs) are a large family of PRRs, among which TLR4 is unique in the complexity of its downstream signaling. Single nucleotide polymorphisms (SNPs) expressed in the ectodomain of human TLR4 (D299G/T399I), have been shown to affect the functionality of this receptor. Population genetics studies have shown that these SNP frequencies vary geographically and among different ethnic backgrounds, with both SNPs co-segregating in ~10% of Caucasians. Humans expressing these TLR4 SNPs are LPS-hyporesponsive and exhibit increased susceptibility to certain bacterial infections and severe colitis. Our laboratory engineered a knock-in mouse strain that homozygously expresses homologous TLR4 SNPs (D298G/N397I). TLR4-SNP mice and their macrophages are LPS-hyporesponsive and exhibit altered susceptibility to bacterial and viral infections. To evaluate the mechanisms by which TLR4 SNPs contribute to Inflammatory Bowel Disease (IBD), we utilized a chemically-induced colonic inflammation and repair model to compare the responses of wild-type (WT) vs. TLR4-SNP mice. After 7 days of 3% dextran sodium sulfate (DSS) in the drinking water, followed by an additional 7 days of recovery, TLR4-SNP mice were found to be significantly more sensitive than WT mice to DSS-induced colitis (e.g., weight loss, fecal blood, colon length, histopathology, and a significant delay in recovery after DSS removal). Ongoing studies to determine if differences in the microbiota of TLR4-SNP mice vs. WT mice contribute to this phenotype are expected to provide insights into mechanisms underlying human IBD. Supported by NIH T32 AI095190.
Shared B cell but not T cell intratumoral immune repertoire among patients with low grade appendiceal mucinous neoplasms
Authors: Murthy Pranav , Weber Daniel , Sharma Sagar N. , Saini Pragosh , Pan Wenjing , Byrne-Steele Miranda , Han Jian , Pai Reetesh K. , Zureikat Amer H. , Choudry Haroon A. , Lotze Michael T.
Published: February 2023

Introduction: Low grade mucinous appendiceal neoplasms (LAMNs) are indolent tumors that present with widespread peritoneal metastases. Despite standard of care cytoreductive surgery and heated intraperitoneal chemotherapy (CRS-HIPEC), patients with LAMNs often recur within 1–3 years and respond poorly to chemotherapy.

Methods & Results: Histopathologic review identified clusters of lymphocytes associated with epithelial hypercellularity among larger pools of mucin. Utilizing dimer avoidance multiplex-PCR and next generation sequencing, we explored the adaptive immune response in patients with recurrent LAMNs (n=10). Bulk sequencing of tumor tissue following initial CRS-HIPEC revealed a predominant expression of B cell over T cell receptors (mean expression: 0.5% Vα, 1.4% Vβ, 0.01% Vδ, 12.6% IgH, 47.6% Igκ, 37.8% Igλ). Very interestingly, we observed a heightened IgE fraction (8.4%±1.1) in patients with LAMNs, greater than both healthy donor blood (0.57%±0.36, p<0.0001, n=239) and pretreated high-grade tumors (n=66, 3.3%±2.4, p<0.0001). Although the TCR repertoire was extremely private in patients, the BCR repertoire included several shared public and private clones: 11 IgH private CDR3s shared between 2 patients, 64 Igκ public CDR3s shared among >7 patients, 261 private Igκ CDR3s shared among 3 patients, 20 public Igλ CDR3s shared among >7 patients, and 137 private Igλ CDR3s shared between 2 patients, an intriguing and unexpected finding.

Conclusions:

This, we believe, is the first report of adaptive immunity in low grade mucinous neoplasms that identifies a skewed but diverse infiltration of T and B cells, that warrants study in additional patients.
Specific expression of a BTB-ZF transcription factor defines innate-like regulatory T cells that are essential for intestinal homeostasis
Authors: Krzyzanowska Agata K , Haynes Rashade A , Kovalovsky Damian , Edelblum Karen L , Corcoran Lynn M , Rabson Arnold B , Denzin Lisa K , Sant'Angelo Derek B
Published: February 2023

Regulatory T cells (Tregs) play a pivotal role in regulating immune responses and are a significant source of the anti-inflammatory cytokine IL-10 in the intestine. Defects in Tregs function lead to the development of systemic and intestinal inflammation. Using a single-cell BTB-ZF transcription factor expression analysis, we identified a distinct subset of Tregs. These cells have an activated phenotype (CD62Llo, CD44hi) and constitutively express the Il10. Moreover, these Tregs accumulate in the intestine and expand in number following DSS induced colitis. The targeted deletion of the defining transcription factor impaired these cells' ability to secrete IL-10, leading to significant disruptions in mucosal architecture and the development of intestinal inflammation following treatment with dextran sodium sulfate (DSS). Thus, the conditional knockout (cKO) mice developed much more severe symptoms leading to the death of 60% of them. In contrast, all wild type mice fully recovered. Notably, adoptive transfer of this Treg subset was sufficient to prevent the death of cKO mice. In comparison, the transfer of cKO Tregs was not able to rescue the mice. Collectively, our data show that we have identified a distinct subset of Tregs and determined a specific member of the BTB-ZF family that controls their function. This rare population of T cells has potent immunosuppressive abilities and, we propose, rather than acutely producing IL-10 in response to activation, these cells maintain intestinal homeostasis through continuous secretion of IL-10. Our findings imply that analogous to NKT cells, some thymic-derived Tregs have innate-like effector functions that are not dependent upon differentiation in the periphery.
Fibroblast-like synoviocytes shape inflammation and wound healing in Lyme arthritis
Authors: Wahhab Amanda , Danner Rebecca , Rouse Joseph , Timmler Sarah , Lochhead Robert B
Published: February 2023

Lyme arthritis (LA) is caused by infection with Borrelia burgdorferi (Bb) and, in many patients, can trigger autoimmune and autoinflammatory pathways. Despite proper treatment, some individuals can develop proliferative synovitis that persists for months or years, a condition known as post-infectious LA. Fibroblast-like synoviocytes (FLS) are the most abundant cell type found in the synovial lesion and are critical in wound healing. The aim of this study is to determine the role of FLS in dysregulated pro-inflammatory and tissue repair pathways in LA. FLS were cultured from joints of C57BL/6 (B6) mice and B6 Il10−/− mice and stimulated with interferongamma (IFNγ) and/or Bb. Cell culture supernatants were used for multiplex cytokine analysis, and RNA was isolated from cells for gene expression analysis. FLS were also used in flow cytometry and color flow imaging to identify immune markers, and in cell migration and proliferation assays as a measure of wound healing. When treated with IFNγ, FLS developed an immune-like phenotype. These immune fibroblasts were hyper-responsive to Bb and produced high levels of inflammatory mediators. FLS were also capable of internalizing Bb and expressing MHC-II molecules, suggesting the ability to present Bb antigen. Moreover, B6 FLS treated with IFNγ and/or Bb displayed an impaired wound healing phenotype, whereas this phenotype was seen intrinsically in both treated and untreated Il10−/− FLS. Under pro-inflammatory conditions seen in joints of patients, FLS likely play a key role in numerous pathogenic pathways, most notably in driving inflammation and blocking appropriate repair of damaged tissue. Further studies will examine the epigenetic pathways in both murine and human synovial tissue.
L-plastin supports macrophage NLRP3 inflammasome activation and pulmonary fibrosis
Authors: Joshi Hemant , Almgren-Bell Alison , Anaya Edgar , Todd Elizabeth M. , Morley Sharon Celeste
Published: February 2023

NLRP3 inflammasome induces IL-1β production and pyroptotic cell death in macrophages to fight against invading pathogens. However, excessive inflammasome activation promotes tissue fibrosis and inflammatory diseases such as acute lung injury, liver injury, and acute respiratory distress syndrome (ARDS). NLRP3 inflammasome activation has been extensively studied, but exact regulatory molecular mechanisms remain unclear.

We have found that the actin-bundling protein L-plastin (LPL) is required for efficient activation of the NLRP3 inflammasome pathway. In ex-vivo stimulation, lung resident alveolar macrophages (AMs) lacking LPL produced less IL-1β and IL-18 upon NLRP3 stimulation. LPL−/− AMs from ASC-citrine reporter mice exhibited reduced ASC speck formation after NLRP3 activation. Similarly, bone-marrow derived macrophages (BMDMs) displayed reduced ASC oligomerization. Downstream of ASC oligomerization, caspase-1 activity and downstream IL-1β and pyroptotic gasdermin-D processing were also reduced in LPL-deficient macrophages. Further, LPL−/− mice displayed reduced peritonitis after monosodium urate (MSU) treatment, an experimental NLRP3-induced inflammatory disease model. Since AMs are crucial for NLRP3-mediated lung fibrosis, we next examined NLRP3-dependent bleomycin-induced lung injury. We found that LPL−/− mice responded mildly to bleomycin and exhibited significantly reduced fibrosis development. Thus, we identify a previously unknown significant function of LPL in regulating NLRP3 inflammasome signaling in macrophages. This finding presents LPL as a potential target for diagnosis and treatment for NLRP3-associated inflammatory disorders such as ARDS, COPD and lung fibrosis.
Distinct lymph node entry efficiencies for CD8+ and CD4+ T cells are eliminated during malnourishment.
Authors: Bowman Lauren A , Goulmamine Syreen , Gibson David , Little Amie , Bupp Melanie Gubbels
Published: February 2023

Previous work suggests that glucocorticoids upregulate expression of CD127, an IL-7 receptor component, on peripheral naïve T cells (NT), even though the total number of peripheral NT declines dramatically during malnourishment. We proposed that CD127 up-regulation contributes to peripheral NT reduction by increasing the scavenge rate of IL-7, providing a mechanism to rapidly adjust the total number of NTs during malnutrition. Each malnourished NT would then receive a larger dose of IL-7 than control NTs. We next wondered if this larger dose might confer additional energy-saving behaviors. NT migration across HEV’s may be a high energy-consuming activity. Thus, we compared lymph node entry rates of adoptively-transferred malnourished and control NT in malnourished and control recipients. Control CD4+ NTs migrated more efficiently than control CD8+ NT, but malnourished CD4+ and CD8+ NT migrated at equivalent rates, regardless of recipient diet. We next analyzed expression of proteins known to be involved in NT migration to uncover the currently unknown mechanisms responsible for these various migration patterns. Flow cytometry analysis revealed malnutrition significantly reduces expression of both components of LFA-1 (CD18 and CD11a), CD49d (a VLA-4 component), and S1PR1 in CD4+ and CD8+ NT. We also compared expression levels of ICAM-1 (CD54), a protein expressed in HEV’s which binds to LFA-1 on migrating NT, in malnourished and control lymph node tissue via confocal microscopy. Improved understanding of altered migration molecule expression during malnourishment should enhance our knowledge of the energy-conserving behavior of NT, as well as uncover strategies to improve vaccination responses in malnourished children.
Podosome dissolution and focal adhesion assembly drive the enhanced migratory capacity of dendritic cells infected by ectromelia virus
Authors: Toka Felix N , Biernacka Zuzanna , Struzik Justyna , Gierynska Malgorzata , Szulc-Dabrowska Lidia
Published: February 2023

Podosomes and focal adhesions (FAs) are both adhesive cytoskeletal structures engaged in cell-matrix adhesion of many different cell types, including dendritic cells (DCs). Despite sharing almost, the same proteins, e.g. vinculin, paxillin, talin and Src family proteins, podosomes and FAs show differences in their architecture, dynamics and function. While highly dynamic podosomes are organized in an actin-rich core surrounded by a ring of adhesive molecules with a perpendicular orientation to the extracellular matrix (ECM), more stable FAs have elongated structure and exhibit tangential orientation to the substrate surface. A switch from podosomes to FAs is often observed during TLR4-mediated DC maturation and is associated with an increased motility of these cells. Ectromelia virus (ECTV) is an orthopoxvirus that can control several different aspects of the cytoskeleton organization and dynamics in DCs and, thus, provides a useful model to study virus-cytoskeleton interactions and cell motility in these specialized immune cells, in which ECTV can productively replicate. Our results show that DCs infected with ECTV completely lose podosomes during early stages of infection, within the viral factory formation phase in the cytoplasm (4 hpi). Instead of podosomes, ECTV-infected DCs develop FAs, which are larger but less numerous than in uninfected control cells. FAs are predominantly observed within long-branched cellular projections formed extensively during later stages of infection (18–24 hpi). Loss of podosomes is associated with enhanced migration of infected DCs in vitro, suggesting that ECTV may potentially exploit DC migration to facilitate dissemination of the virus in vivo.
Inhibition of Epigenetic Reader BET Proteins by Apabetalone Counters Inflammation in Activated Innate Immune Cells from Fabry Disease Patients Receiving ERT
Authors: Fu Li , Wasiak Sylwia , Tsujikawa Laura , Rakai Brooke D. , Stotz Stephanie C. , Wong Norman C.W. , Johansson Jan O. , Sweeney Michael , Mohan Connie M. , Khan Aneal , Kulikowski Ewelina
Published: February 2023

Fabry disease (FD) is a rare genetic disorder with a deficit in the degradation pathway of a glycolipid, globotriaocylceramide (Gb3). Gb3 deposits in various tissues evoke immune-mediated systemic inflammation, driving the progression of FD complications. In particular, cardiac and renal problems, leading causes of mortality in FD patients, are insufficiently managed by enzyme replacement therapy (ERT) in the long term. Here, we examined immune profiles in unstimulated peripheral blood mononuclear cells (PBMCs) from FD patients on ERT relative to untreated patients. The results showed an upregulation of the CCR2/MCP-1 axis, particularly in the presence of renal dysfunction. We also observed an increase of IL12B transcripts in unstimulated PBMCs when tracking the immune status over two years of ERT in patients with early-stage chronic kidney disease (CKD). Interestingly, pro-inflammatory responses in LPS-stimulated PBMCs were countered by apabetalone, a clinical-stage candidate that inhibits Bromodomain and Extra Terminal (BET) proteins, epigenetic readers. Apabetalone treatment dose dependently inhibited MCP-1 and IL-12 production by up to 90%. Apabetalone downregulated the transcription of other pro-inflammatory cytokines including TNF-α, IL-6 and IL-8 by 70%, 48% and 54%, respectively. Reactive oxygen species (ROS) are an indicator of oxidative damage caused by intracellular Gb3 deposits in FD patients. Apabetalone suppressed ROS levels by up to ~70% in LPS-stimulated neutrophils. Hence, apabetalone treatment may reduce pathological inflammation and oxidative stress in FD patients and thus complement ERT to optimize patient outcomes, warranting further investigation of apabetalone as a therapeutic for FD.
A single-cell atlas of the lung mononuclear cell response in Staphylococcus Enterotoxin B-induced Acute Respiratory Distress Syndrome
Authors: Wilson Kiesha Maria , Cannon Alkeiver S , Sultan Muthanna , Nagarkatti Prakash , Nagarkatti Mitzi
Published: February 2023

The emergence of SARS-CoV-2 variants will change the landscape of what we already know about COVID-19, making it important to understand the immunological and molecular mechanism of this illness. The virulent nature of the virus, disease outcomes and lack of suitable facilities for studying virus-infected samples has made the need for small animal models urgent. Staphylococcus enterotoxin B (SEB)-induced Acute Respiratory Distress Syndrome (ARDS) mimics the cytokine storm presented in patients with severe COVID-19 and is a safer alternative than working with infected samples. To identify pathways in pulmonary cells that potentially cause ARDS, we utilized single-cell RNA sequencing (scRNASeq) to profile the whole lung cells of C3H/HeJ mice treated with SEB and healthy controls. While it is well established that SEB induces robust proliferation of CD4+ T cells with the receptor Vβ8.2 by binding of these cells with antigen presenting cells, effects in other cell types is not well described. We found that intranasal administration of SEB induced the proliferation of three subsets of macrophages, neutrophils, and cytotoxic T cells in the lungs. Our data also illustrated a loss of multiple cell types including resident macrophages, multiple subsets of B cells, and club cells. Macrophage subsets displayed increased expression of genes related to interferon signaling and lipid metabolism. This data emphasizes the importance of understanding the role of macrophages in ARDS, and can further provide insights into elucidating the mechanisms not only in SEB-induced ARDS but also in severe COVID-19. This may serve as a model for severe case of the disease. (Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01 ES030144 and R01AI123947)
The chromatin reader SP140 regulates intestinal innate immune responses to microbes
Authors: Fraschilla Isabella Rose , Jeffrey Kate L
Published: February 2023

Genome-wide association studies (GWAS) identified single nucleotide polymorphisms (SNPs) within SP140 that associate with Crohn’s disease (CD) in addition to multiple sclerosis (MS) and chronic lymphocytic leukemia (CLL). SP140 is a transcriptional regulator expressed exclusively in immune cells. It is part of the Speckled Protein family that also contains SP100, SP110, and SP140L and shares high homology with autoimmune regulator (Aire). We have previously shown that SP140 is a critical orchestrator of macrophage identity by occupying and repressing silenced lineage-inappropriate genes. However, the role for SP140 functional domains in regulating macrophages identity and transcriptional responses to microbial sensing remained unknown. Here we show how SP140 influences innate immune responses to microbes. By engineering domain mutants, we investigate how the chromatin-binding domains (SAND, plant homeodomain, and bromodomain) and the caspase activation and recruitment domain (CARD) within SP140 dictate macrophage cytokine production. Furthermore, we determine how SP140 localization to promyelocytic leukemia (PML)-nuclear bodies affects function. Global deficiency of SP140 results in exacerbated colitis in mice, and a loss of SP140 specifically in the hematopoietic compartment results in microbial dysbiosis. Collectively, our work provides functional characterization of the novel chromatin regulator SP140 and how disease variants may contribute to immunological disease.
Regulating the unregulated: what host factors are required for GSDMD pore formation?
Authors: Evavold Charles Lee , Hafner-Bratkovic Iva , Devant Pascal , Kagan Jonathan C.
Published: February 2023

Cleavage of gasdermin D (GSDMD) by inflammatory caspases results in pore formation at the plasma membrane. GSDMD pores are recognized to mediate pyroptotic lysis of the cell or direct translocation of IL-1 family cytokines from the cytosol into the extracellular space. While several regulators of GSDMD cleavage have been identified, positive regulators of pore formation that act after cleavage have occurred are unknown. To identify proteins that promote GSDMD pore formation at the plasma membrane after GSDMD cleavage, we utilized a genome-wide CRISPR/Cas9 screening platform in macrophages. This screen uncovered a novel link between host metabolism and cell death at the level of GSDMD pore oligomerization. Several hits in this screen belong to the Ragulator-Rag complex that serves to activate mTORC1. Our data suggests that this Ragulator-Rag-mTORC1 axis promotes pyroptosis through enforcing functional GSDMD pore formation.
NLRP3 and AIM2 inflammasome-triggered Th17 cell response promotes severe immunopathology in schistosomiasis
Authors: Morales Yoelkys , Miller Emily A , Shecter Ilana , Fitzgerald Katherine A , Golenbock Douglas T , Stadecker Miguel J , Kalantari Parisa
Published: February 2023

Infection with the helminth Schistosoma mansoni can cause exacerbated morbidity and mortality via a pathogenic host CD4 T cell-mediated immune response directed against parasite egg antigens, with T helper (Th) 17 cells playing a major role in the development of increased granulomatous hepatic immunopathology. A role of inflammasomes in intensifying disease has been reported, however, neither the types of inflammasomes involved, nor their impact on the Th17 response are known. Here we show that the observed enhanced egg-induced IL-1β secretion and pyroptotic cell death required both NLRP3 and AIM2 inflammasome activation. Schistosome genomic DNA activated AIM2, whereas reactive oxygen species, as well as potassium efflux, were the major activators of NLRP3. In addition, dual activation of caspase-1 and caspase-8 was required for egg-induced IL17 production. NLRP3 and AIM2 deficiency led to a significant reduction in Th17 responses, suggesting that both play a crucial role in triggering inflammation. We also determined that inflammasome-induced IL-1β promotes the anti-inflammatory Th2 cytokine IL-4. Our findings establish NLRP3 and AIM2 inflammasomes as central inducers of pathogenic Th17 responses in schistosomiasis.
The SARS-CoV-2 proteins NSP1 and NSP13 inhibit interferon activation through distinct mechanisms
Authors: Vazquez Christine , Swanson Sydnie , Negatu Seble , Dittmar Mark , Ramage Holly , Cherry Sara , Jurado Kellie
Published: February 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, infecting over 100 million people and claiming over 2 million lives globally. This drastically highlights the need for understanding the host-virus protein-protein interactions and immune evasion strategies governing SARS-CoV-2 pathogenesis. Our overall aim was to identify antiviral immune targeting during SARS-CoV-2 infection and protein overexpression. To understand if SARS-CoV-2 can block immune signaling, we first examined nuclear translocation of the transcription factors, IRF3 and NF-κB, as nuclear translocation is one step in interferon induction, during SARS-CoV-2 infection of the human airway epithelial cells. We found that in infected cells, there was limited IRF3 and NF-κB nuclear translocation, suggesting SARS-CoV-2 can limit immune activation. Additionally, through promoter luciferase signaling assays, the viral proteins NSP1, the host translation shutoff factor, and NSP13, the RNA helicase, can block signaling to the IFN-β and NF-κB promoters. To further investigate this reduction in promoter signaling, we examined nuclear translocation of NF-κB in the context of NSP1 and NSP13 plasmid overexpression. Surprisingly, we found that while NSP13 reduced NF-κB nuclear translocation, NSP1 was unable to despite its ability to limit promoter signaling. Instead, we found, utilizing Click-it chemistry, that NSP1 reduces host protein synthesis. Last, we found NSP13 targets TBK1 activation by limiting its phosphorylation in a dose-dependent manner. Collectively, our data illustrate that SARS-CoV-2 can block multiple innate immune signaling pathways through distinct mechanisms.
Blockade of IFN-I improves adaptive immune responses and immune coverage after vaccination
Authors: Palacio Nicole M , Dangi Tanushree , Wang Yidan , Chung Young Rock , Sanchez Sarah , Penaloza-MacMaster Pablo
Published: February 2023

IFN-I is a main innate antiviral defense critical for the activation of adaptive immunity. However, we hypothesized that acute viral clearance by IFN-I could limit antigen availability following initial antigen encounter, which could in turn, reduce the priming of the adaptive immune system. To test this hypothesis, we immunized mice with different vaccines or acute viruses together with a low dose of an IFN-I receptor-blocking antibody to induce a short-term abrogation of IFN-I signaling. This resulted in transient increase in antigen levels followed by antigen clearance. Importantly, short-term IFN-I blockade during coronavirus, flavivirus, rhabdovirus, or arenavirus immunization resulted in a potent improvement of adaptive immune responses, which conferred improved protection following future re-infections. Short-term IFN-I modulation also improved immune coverage elicited by viral vaccines or acute viral infections. In particular, administration of an HIV-1 vaccine together with the IFN-I blocker generated highly cross-reactive antibody responses against other HIV-1 clades; and similarly, immunization with a mouse coronavirus (MHV) together with the IFN-I blocker generated more cross-reactive antibody responses against other related coronaviruses, including SARS CoV-2. These data demonstrate a novel mechanism by which innate immunity modulates the magnitude and breadth of adaptive immune responses, providing insights for rational vaccine design.
Titers, prevalence and duration of SARS-CoV-2 antibodies in two waves of a local COVID-19 outbreak
Authors: Hedges Jodi F , Thomspon Macy A. , Snyder Deann T. , Robison Amanda , Taylor Matthew P. , Jutila Mark A
Published: February 2023

Information concerning the development of neutralizing antibodies and their duration will be critical to establishing herd immunity for COVID-19. We sought to evaluate SARS-CoV-2 spike protein receptor binding domain (RBD)-specific antibodies, their duration and capacity for SARS-CoV-2 neutralization in volunteers while the pandemic spread within our community starting in March 2020. Those participants with the highest starting titers had the longest lasting response, up to 9 months post-diagnosis. SARS-CoV-2 neutralization capacity was correlated with anti-RBD antibody levels. The majority of our participants with confirmed COVID-19 diagnosis had very mild or asymptomatic infections. We also detected low and largely non-neutralizing anti-RBD IgG titers in a few participants with no known COVID-19 diagnosis. Finally, we found that antibody responses induced by vaccination were significantly higher than those induced by natural infection. Thus, our study suggests that vaccination is still critical even for those naturally infected or diagnosed with COVID-19.
Heterogenicity of Tissue Distribution Recognized by A panel of Monoclonal Antibodies
Authors: Zhang Hong , Lin Mongshang , Wen Chiaokai , Kim Daniel , Frey Avery , Chronopoulou Efthalia , Yang Xifeng , Ni Jessie
Published: February 2023

ACE2 (Angiotensin Converting Enzyme 2) is the main viral entry point for SARS-CoV and SARS-CoV-2. Human ACE2 is widely expressed on different organs but if virus attached all the organs that highly expressed ACE2 is not fully studied. Most recent finding showed that a novel short isoform of ACE2 expressed in the airway epithelium is substantially upregulated in response to interferon stimulation, but not SARS-CoV-2 infection due to this short isoform lacking SARS-CoV-2 spike high-affinity binding sites. This arouses our interest in whether ACE2 has more isoforms expressed in different tissues that directly affect different organs response to virus infection. To support understanding ACE2 expression in response to viral infection, we generated human ACE2 monoclonal antibody by immunizing rat with recombinant human ACE2 protein covering extracellular domain (Gln18-Ser740). We found that there is discrepancy in different clones on different tissues. After three rounds subcloning, four clones have been selected based on chromosomally stable IgG producing hybridoma cells. Our data showed that all four clones showed equivalent signals on ACE2 transfectants but staining signals varied on tubules of kidney and seminiferous tubules and leyding cells on testis, two clones showed medium level staining signals on intestine and colon, but only one clone showed positive staining signals on spleen vascular and red pulp sinus endothelium. In addition, different neutralization potency and Western blot detection performance were observed among the collections of clones. Our data indicated that different clones might recognize different epitopes of ACE2 and indirectly suggested that ACE2 might have different isoforms in different tissues.
Chronic air pollution exposure exaggerates obesity-induced tissue inflammation and insulin resistance by activating inflammasome
Authors: Rao Xiaoquan , Zhong Jixin
Published: February 2023

Background:

Air pollution has been associated with cardiometabolic diseases such as obesity and insulin resistance. However, the underlying mechanisms were not fully understood. In this study, we examined the role of NLRP3 inflammasome in chronic air pollution-induced tissue inflammation and insulin resistance.

Methods:

8 weeks old male WT and NLRP3−/− mice were exposed to filtered air (FA) or concentrated ambient particles (CAPS) in a small animal whole-body exposure system with or without high fat diet for 6 hours/day, 5 days/week, for a total of 12 weeks (n= 5–8 per group).

Results:

CAPS exposure enhanced insulin resistance in the WT mice but not in the NLRP3−/− mice. Cytokine array and ELISA assays indicated that IL-1β secretion was increased in the culture supernatant of macrophages isolated from CAPS-exposed WT mice, compared to that of FA-exposed mice. Electronic microscopy showed particulate deposites in the peritoneal macrophages isolated from the peritoneal cavity of CAPS-exposed animals. The gene expression of Nlrp3 and Il-1β, the rate-limiting factor of NLRP3 inflammasome activation, were also increased in CAPS-exposed WT mice. Deficiency of NLRP3 blocked CAPS exposure-induced lung inflammation, inflammatory gene expression, and IL-1β secretion in mice. Subsequent ex vivo experiments by culturing macrophages with particulate matters indicate that air pollution particles not only initiate the transcription of Nlrp3 and Il-1β, the first signal for inflammasome activation, but also induce the activation of caspase-1, the second signal for inflammasome activation.

Conclusions:

Our data suggest that NLRP3 inflammasome activation mediates air pollution-induced tissue inflammation and insulin resistance.
Controlling for Confounding Factors when Diagnosing Cancer from T Cell Receptor Repertoires
Authors: Ostmeyer Jared L , Christley Scott , Reuben Alexandre , Zhang Jianjun , Cowell Lindsay
Published: February 2023

Our publications have shown that T cell receptor (TCR) repertoires sequenced from tissue biopsies can be used to distinguish tumor from control tissue with > 90% classification accuracies, suggesting that TCR repertoires can be used to diagnose cancer.

TCR repertoires sequenced from blood samples of cancer patients are now publicly available, and it is tempting to use these existing samples to develop blood-based assays for diagnosing cancer. Already, research groups including us obtained promising results. However, these results are obtained by combining cancer and control samples from mismatching studies. Without controlling for age, sex, race, and even the reduced amount of blood volume collected from cancer patients relative to controls, these exciting results could merely be an artifact of confounding factors.

In this study, we control for possible confounding factors and observe that our ability to diagnose cancer from TCR repertoires sequenced from blood does not vanish. On patient-holdout cross-validations, we achieve diagnostic accuracies of 90% for colorectal cancer (controlling for depth coverage), 84% for breast cancer (controlling for depth coverage and age), and 83% for lung cancer (controlling for age, sex, race, and depth coverage).

While it is impossible to control for all potential confounding factors like variations in geographic locations, these results are encouraging. We propose future studies that (i) collect matching controls and cases and (ii) confirm TCR repertoire sequencing provides a distinct signal for diagnosing cancer independent of other blood-based assays and therefore can improve the diagnostic performance of these other blood-based assays.
Near infrared light reduces neutrophil chemokine production during ischemia-reperfusion injury
Authors: Compton Tyler Compton , Weihrauch Dorothee , Ninomiya James T , Lochhead Robert
Published: February 2023

Background:

Ischemia reperfusion injury and tissue necrosis commonly follows administration of a tourniquet. Previously, we have shown that exposure to non-thermal infrared (NIR) light decreases neutrophil and macrophage mediated inflammation and tissue necrosis in a mouse hindlimb model. This study tests whether exposure to NIR light during a period of ischemia limits tissue damage by reducing secretion of chemoattractant proteins and phenotypic switching of tissue macrophages from a pro-inflammatory (M1) to a wound healing (M2) phenotype.

Methods and Results: Mouse hindlimbs received 3 hours of tourniquet-induced ischemia followed by a period of reperfusion with or without 15 or 30 minutes of NIR exposure. Hindlimbs receiving injury and NIR treatment showed a significant decrease in the neutrophil chemoattractants CXCL1 and CXCL5 by ELISA, compared with mice receiving ischemia reperfusion injury alone and control mice receiving no injury or NIR. Protein isolated from mouse hindlimbs receiving NIR treatment and injury had a significant decrease in M1 macrophage marker CD68 and significant increase in M2 macrophage marker CD206, normalized to CD14, compared with mice receiving injury alone and uninjured mice.

Conclusion:

We have shown that NIR light affects neutrophil chemokine secretion and tissue macrophage polarization in a murine hindlimb ischemia and reperfusion model. These data indicate that NIR light treatment during tourniquet administration limits inflammatory responses, reduces tissue necrosis, and promotes wound healing following removal of the tourniquet. Thus, NIR treatment may improve limb preservation following vascular injury and inform timing of tourniquet application during surgical procedures.
Itolizumab-induced antigenic modulation of CD6 inhibits T cell activity
Authors: Chu Dalena Ngoc , Ampudia Jeanette , Connelly Stephen , Ng Cherie
Published: February 2023

CD6 is an immunomodulatory receptor on T cells that promotes immune synapse formation, T-cell activation, and cell migration into tissues by binding activated leukocyte cell adhesion molecule (ALCAM). Excessive activation through the CD6-ALCAM pathway has been implicated in the pathogenesis of multiple autoimmune and inflammatory diseases. Hence, the ability to modulate the level of activation is beneficial to disease resolution. Itolizumab is a humanized anti-CD6 monoclonal antibody that is specific for the membrane distal domain 1 of CD6. Previously, itolizumab was believed to sterically hinder the CD6-ALCAM interaction, thereby blocking T-cell co-stimulation. Here we describe antigenic modulation as an additional mechanism whereby binding of itolizumab to CD6 induces proteolytic cleavage of the extracellular portion of CD6. Upon treatment with itolizumab, surface levels of CD6 decreased in a dose- and time-dependent manner, as monitored by flow cytometry using a noncompetitive anti-CD6 monoclonal antibody. This loss was inhibited in the presence of protease inhibitors and the decrease in surface levels of CD6 was accompanied by an increase in levels of soluble CD6 in the supernatant. Furthermore, we assessed the effect of surface levels of CD6 on the response of cells to T cell stimulation in the presence of ALCAM. CD6low cells showed reduced T-cell activity compared to CD6highcells as measured by cell surface activation makers, including CD25, CD69 and PD-1, and cytokine production. These findings demonstrate that CD6 is an important regulator of T-cell activity and that modulating surface levels of CD6 is an effective method for fine-tuning the activity of T-cells.