bluuebunny/crossref_metadata_2025 huggingface dataset:
Disease-specific antigen presentation on MHC class II is inhibited by a small molecule in Sjögren’s Syndrome
Authors: Gupta Shivai , Nguyen Cuong Q
Published: February 2023

Sjogren’s Syndrome (SjS) is one of the more common autoimmune diseases that has a strong genetic linkage. To date, no vaccine or therapeutic exists to cure SjS, and patients must rely on lifelong therapies to treat symptoms. Class II MHC are the primary susceptibility loci that form the genetic basis for many auto-immune disorders including SjS. I-Ag7 is the MHC in NOD mice that is used for the presentation of autoantigens and plays a critical role in the adaptive immune response. HLA-DQ8 the MHC in humans, is analogous to I-Ag7, in the NOD mouse model and is an important model for the onset of SjS. Using an in-silico molecular docking program a chemical library was screened that defined small molecules that were capable of occupying specific structural pockets along the I-Ag7 binding groove, with the objective of influencing auto-antigen peptide presentation to T cells. In this study we show, that the small molecule TATD can inhibit TCR signaling based on the structural pocket targeting mechanism. TATD previously proven to inhibit the onset of diabetes by blocking the auto-antigen presentation on I-Ag7, was analyzed to observe effects of prevention of SjS by, preventing the accumulation of B and T cells within the salivary gland of NOD mice. Treated mice also showed reduction in the incidence of inflammatory cell infiltration into lacrimal and salivary glands as compared to control mice with notable reduction in of the hallmark SjS anti-nuclear antibodies of Ro52, Ro60 and La. As a result this study presents a novel method for identifying small molecules capable of inhibiting T cell responses, with potentially therapeutic applications.
A cell-free antigen processing system reveals antigenic factors critical for HIV-1 epitope dominance and informs vaccine design
Authors: Sengupta Srona , Reed Madison , Yu Jeanna , Boronina Tatiana , Yang Weiming , Timmons Andrew , Hoh Rebecca , Deeks Steve , Cole Robert N. , Siliciano Janet , Siliciano Robert , Sadegh-Nasseri Scheherazade
Published: February 2023

Certain CD4+T cell epitopes have been associated with immune control of HIV-1 replication and the presence of anti-Env neutralizing antibodies. However, it remains unclear what antigen-dependent factors influence the dominance of certain epitopes in HIV-1 proteins. To study HIV-1 epitope dominance, we used a cell-free antigen processing system previously developed by our group that utilizes soluble HLA-DR1*01:01 (DR1), HLA-DM (DM), protein antigen and three cathepsins in a reducing and low pH environment to allow for antigen binding and cleavage. We subjected HIV-1 antigens from Gag, Pol, Env and accessory proteins Vif, Nef, Tat, and Rev to this system. Immunoprecipitation of pMHC-II complexes followed by peptide elution and identification via LC-MS/MS revealed a map of DR1-bound epitopes across the near-full length HIV-1 proteome. Most identified epitopes were DM-resistant, and in some cases, DM narrowed the epitope diversity to one species. Importantly, we identified 28 novel epitopes, including several glycopeptides eluted from DR1. We noted epitope “hot spots” in HIV-1 antigens and striking similarities in epitopes from HIV polyproteins (Gag precursor) versus individual subunits (Gag capsid). Analysis of the location of several epitopes from smaller HIV-1 proteins based on their known crystal structures mapped to regions of low protein stability and higher solvent accessibility. Finally, we found that several epitopes induced memory CD4+T cell responses in persons living with HIV-1. Thus, by using a minimalist in vitro processing system, we can interrogate novel HIV-1 epitopes and understand how DM-resistance and antigen structure influence epitope dominance, which has important implications for vaccine design.
Akkermansia muciniphila induces Th17 cells by mediating tryptophan metabolism and exacerbates experimental autoimmune encephalomyelitis (EAE)
Authors: Lin Xun , Tawch Suzanne , Singh Ankita , Morgun Andriy , Shulzhenko Natalia , Kumar Pawan
Published: February 2023

Multiple sclerosis patients have been shown to have increased Akkermansia muciniphila (Akk) colonization. However, the role of Akk to regulate MS remains poorly understood.

Here, we utilized fecal microbiota transplantation (FMT) model and generated Akk+ and Akk− SPF C57BL/6 mice. When subjected to EAE, Akk+ mice displayed worse clinical scores. An increase of spinal cord infiltrating IL-17A-producing and GM-CSF-producing CD4+ T cells was observed in Akk+ mice at 15 days post EAE induction. Moreover, ileum explant culture showed an elevated IL-17A production in Akk+ mice. Furthermore, we found dysregulated tryptophan in the cecal contents of naïve Akk+ mice. Additionally, using Kovac’s reagent, Akk was confirmed to utilize tryptophan and produce indole, an aryl hydrocarbon receptor (AhR) ligand. AhR activation has previously been shown to promote Th17 differentiation. Therefore, we performed Th17 differentiation in vitro. The treatment of Akk-derived tryptophan metabolites significantly induced IL-17A response. CH-223191, an AhR antagonist, abrogated the induction of Akk-mediated Th17 differentiation, suggesting that Akk-mediated Th17 differentiation was dependent on AhR. In addition, increased AhR agonists were observed in the feces of MOG immunized Akk+ mice as compared to Akk− mice.

In conclusion, our data showed that Akk exacerbated EAE possibly by tryptophan-dependent induction of IL-17A-producing CD4+ T cells.
Interleukin-17A deficient mice microbiota induced regulatory T cells and suppressed experimental autoimmune encephalomyelitis (EAE) in WT HLA-DR3 transgenic mice
Authors: Shahi Shailesh K , Jensen Samantha N. , Ghimire Sudeep , Carter CheyAnne Q. , Guseva Natalya V. , Gibson-Corley Katherine , Bossler Aaron D. , Ganesan Sukirth M. , Karandikar Nitin J. , Mangalam Ashutosh K.
Published: February 2023

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system, and among all the genes associated with MS, HLA class II genes have the highest relative risk. Although IL-17A is thought to be a major cytokine in the pathogenesis of MS, the exact role of IL-17A in the MS is still poorly understood. Specifically, it is unclear whether IL-17A is required for disease initiation or disease severity or in both. Therefore, in the present study, utilizing HLA-DR3 transgenic mice lacking IL-17A (HLA-DR3.IL-17A−/−), we investigated the importance of IL-17A in disease development and progression in experimental autoimmune encephalomnyelitis (EAE) an animal model of MS. We observed that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ regulatory T cells (Treg)s and developed milder EAE compared to IL-17 sufficient HLA-DR3 mice. Further characterization of mechanism identified a novel role of IL-17A in regulating the disease progression through modulation of immunoregulatory responses. We also identified an important role of gut microbiota in IL-17A dependent disease regulation specifically depletion of gut bacteria with ability to promote Treg population in IL-17A sufficient HLA-DR3 mice. Fecal transplantation of HLA.DR3 mice with HLA-DR3. IL17A−/− showed milder EAE and an increase in Treg cells in HLA-DR3 mice confirming a role of gut microbiota in shaping Treg repertoire and function. Additionally, we observed higher frequency of metabolic pathway involved with Treg promoting short chain fatty acid (SCFA) metabolism in HLA-DR3.IL17A−/− mice. Thus our study shows a novel role of IL-17A in immune homeostasis and inflammation by regulating levels of Tregs through modulation of gut microbiota.
Gut commensal Lactobacillus reuteri exacerbates CNS autoimmunity in a dietary tryptophan-dependent manner.
Authors: Montgomery Theresa L , Eckstrom Korin , Künstner Axel , Kennedy Josephine J. , Busch Hauke , Krementsov Dimitry
Published: February 2023

Gut commensal bacteria exert profound effects on the immune system of the mammalian host. In particular, Lactobacillus species have a long-standing history as putative probiotics, generally being characterized as safe and/or beneficial for the mammalian host. Despite this fact, current literature points to both pro- and anti-inflammatory roles for this genus, with little consensus as to the mechanisms governing these divergent outcomes. Using mouse models, we and others have previously shown that, unexpectedly, stable commensal colonization with Lactobacillus reuteri (L. reuteri) is correlated with, and sufficient to enhance host susceptibility to experimental autoimmune encephalomyelitis (EAE), in association with alterations in tryptophan derived metabolites in host circulation. To identify the underlying mechanisms, we characterized the genomes of commensal L. reuteri isolates, along with two other representative Lactobacillus species, L. murinus and L. johnsonii. Whole genome sequencing revealed an enrichment in the enzymatic machinery necessary to catabolize dietary tryptophan into immunomodulatory indole derivatives in L. reuteri. Mechanistically, supplementation with high dietary tryptophan sustained an L. reuteri-dependent exacerbation of EAE and augmented inflammatory T cell populations, while depletion of dietary tryptophan profoundly suppressed EAE in a microbiome-specific manner. Taken together, our data reveal considerable variation in the genetic landscape of commensal Lactobacilli, and demonstrate mechanisms responsible for differential modulation of autoimmunity by gut microbiota, including an unexpected disease-promoting role for catabolism of host dietary tryptophan.
Germinal center checkpoint restrains anti-insulin B lymphocytes in type 1 diabetes-prone mice
Authors: McNitt Dudley H , Joose Bryan A , Thomas James W , Bonami Rachel H
Published: February 2023

Anti-insulin B lymphocytes (AIBCs) are key drivers of the autoimmune attack in type 1 diabetes (T1D) but their exact functions in T1D pathogenesis, and the point(s) during their development where tolerance is lost, are unknown. In the class-switch-competent anti-insulin B cell receptor transgenic non-obese diabetic (VH125SD.NOD) mice, 1–2% of B lymphocytes are insulin-specific, which accelerates diabetes development despite limited insulin autoantibody (IAA) production. Here we used VH125SD.NOD mice to determine where immune tolerance is lost throughout AIBC class-switch, germinal center entry, and antibody-secreting cell differentiation. We find an increased percentage of AIBCs enter germinal centers relative to non-insulin binding B lymphocytes in the same mice. In addition, an increased percentage of AIBCs are found within the pancreas, class-switch to IgG1, and proliferate relative to non-insulin binding B lymphocytes. We also find that AIBCs are activated in the gut to a greater extent than non-insulin binding B lymphocytes. These data suggest that AIBCs escape anergy and are able to class-switch, enter GCs, and proliferate, but maintain functional silencing preventing spontaneous IAA-production. Moreover, the escape from anergy may occur within the pancreas and/or the gastrointestinal tract. Our data has implications in immunotherapy treatments for T1D, as these GC checkpoints may provide an opportunity for T1D intervention.
Simultaneous Measurement of 67 Non-Human Primate Immunological, Metabolic and Hormonal Biomarkers using Luminex® xMAP® Technology
Authors: Keith Robert H , Luo Hong , Raeber Tina , Kornmeier Christine , Gilliam Brooke , Xiao Qiang
Published: February 2023

Immunology, metabolic, and pituitary biomarker research in non-human primates (NHP) is essential for the study of several diseases and biological regulation. To meet this need, we developed three NHP MILLIPLEX® multiplex panels: 12-plex NHP Metabolic Hormone, 7-plex NHP Pituitary Panel 1, and our new 48-plex NHP Cytokine/Chemokine Panel A for the simultaneous measurement of picogram levels of 67 immune, metabolic and hormone factors, in small volumes of serum, plasma or cell culture samples using Luminex® xMAP® technology. Serum from in vivo LPS-challenged animals (n=2 cynomolgus, n=2 rhesus) demonstrated increased levels of 19 cytokines/chemokines with peak elevated levels at 1 hr for 4 analytes (e.g. IL-10, TNFα), at 3 hr for 11 analytes (e.g. IL-6, IFNγ) and at 8 hr (e.g. Fractalkine, I-TAC). Metabolic markers, GIP and GLP-1 (active) in rhesus, also increased significantly at 8 hr and 3 hr, respectively (p<0.02). Significant differences between fasted and fed serum sample values (n=5 cynomolgus, n=5 rhesus) were found for several metabolic markers, including C-peptide, GIP, Insulin and PP, which increased with feeding (P<0.05). In cynomolgus, I-TAC and IP-10 significantly increased after feeding (P<0.003). Testing healthy serum samples from three additional NHP species (African green monkey (n=5), baboon (n=5), chimpanzee (n=5)) exhibited overall sample detectability of 89%, 77%, and 64% for the MILLIPLEX®Cytokine/Chemokine Panel A, Pituitary, and Metabolic multiplex panels, respectively. Thus, the MILLIPLEX®NHP Metabolic Hormone, NHP Pituitary Panel 1 and our latest and largest NHP panel, the 48-plex NHP Cytokine/Chemokine Panel A, are valuable tools for profiling 67 biomarkers in 5 NHP species for disease research.
Type I interferon-induced β-catenin promotes hematopoietic stem cell activation in persistent viral infection in mice
Authors: NGUYEN TRIEU HAI , KWARTENG EDWARD , LAULHE XAVIER , CHARTRAND KARINE , LAMARRE ALAIN , HEINONEN KRISTA
Published: February 2023

Quiescent hematopoietic stem cells (HSCs) become activated and exit their bone marrow niches in response to infections or sterile inflammation to initiate extramedullary hematopoiesis at peripheral sites. While these processes have been described in acute infections, the impact of persistent viral infections on HSC activation and mobilization remains less clear. Our results demonstrate that an active persistent lymphocytic choriomeningitis virus (LCMV) clone 13 infection resulted in type I interferon-dependent stabilization of β-catenin in HSCs, promoting their activation, mobilization and functional exhaustion that was sustained in time and could be seen even after transfer to uninfected hosts. To further examine the functional role of β-catenin in HSC activation, we generated bone marrow chimeras to follow β-catenin-deficient (Ctnnb1−/−) and wild type HSC response to LCMV Cl13 in the same environment. Ctnnb1−/− HSCs and multipotent progenitor cells failed to expand in response to the infection, while still retaining their ability to reconstitute secondary hosts. Moreover, through the course of the infection, Ctnnb1−/− cells produced myeloid progeny at a similar level to the wild type. These findings lead us to propose that β-catenin is required for HSC activation by interferons in the early phase of the infection; however, it appears dispensable for LCMV-induced emergency myelopoiesis.
Fetal liver hematopoietic stem cells possess the mast cell repopulation ability within a limited time window
Authors: Kobayashi Michihiro , Kosters Astrid , Ghosn Eliver Eid Bou , Yoshimoto Momoko
Published: February 2023

Recent progress in developmental immunology has revealed the hematopoietic stem cell (HSC)-independent ontogeny for the various hematopoietic lineages, including mast cells (MC). Adult bone marrow (BM) HSCs fail to generate MC in both lineage tracing and transplantation assays. However, it is unclear whether fetal liver (FL) HSCs can generate MC in vivo. We investigated the MC potential of HSCs from FL and the aorta-gonad-mesonephros (AGM) in lineage tracing and transplantation assays. We employed different genetic mouse models that allowed us to identify the Tomato+ progeny of hematopoietic precursors (Runx1mer-cre-mer), endothelial cells (EC, Cdh5ERT2Cre) and HSCs (Fgd5ERT2Cre) by injecting tamoxifen (TAM) at different embryonic days starting at E7.5. TdTomato (Tom)+ HSCs and MCs were quantified after birth. In addition, we transplanted HSCs from AGM (11.5), FL (E12.5 and E15.5), and adult BM (>6 wks) into irradiated mice.

Results:

The EC-labeling at E7.5 efficiently generated Tomato+ MC, but not HSCs (similar results were obtained for the Runx1mer-cre-mer model). EC-labeling at E9.5 made equivalent Tom+ labeling efficiency in MC and HSCs (~85%). However, E11.5 EC mostly failed to mark MC, but still generated Tom+ HSCs (~25%). HSC-labeling at E14 or postnatal day 2 efficiently marked HSCs, but not MC. Collectively, our results indicate that active MC production terminates before E12 and that HSCs after E14 lack MC potential. Our transplantation study also shows lack of MC repopulation by HSCs after E15. However, E11.5 AGM and E12.5 FL HSCs repopulated MC, revealing a transient MC production potential of HSCs.

Conclusion:

MC production by hemogenic ECs terminates before E12 and early HSCs produce MC within a limited time window.
Genomic Tools and Models for Investigating the Role of Germline Diversity in Mouse Antibody Repertoire Development
Authors: Kos Justin T , Watson Corey T
Published: February 2023

In mouse, the immunoglobulin (IG) loci span several Mb of the genome and contain hundreds of repeated, highly homologous sets of variable (V), diversity (D), and joining (J) genes that recombine in B cells to produce an individual’s expressed Ab repertoire. Similar to what has been reported in humans, recent data has shown significant levels of haplotype variation in the IG loci between commonly used inbred mouse strains, challenging the assumption that the IG loci are conserved across all strains of mice. We are using this intra-strain diversity to develop new models for studying the role of IG germline variation on Ab repertoire dynamics and function, questions that remain difficult to address in outbred human populations and pre-existing animal models.

Given the diversity and complexity within the IG loci, the development of effective mouse models first requires the characterization of intra-strain differences and the construction of high-quality reference assemblies for the IG loci in several representative strains. To address this problem, we are using the Pacific Biosciences SMRT sequencing to sequence BAC clones spanning the IGH, IGK, and IGL loci in NOD/ShiLtJ and BALB/cByJ strains. We are also gaining additional insight on intra-strain diversity by profiling the expressed IGM, IGK, and IGL repertoires of 18 commonly used laboratory mouse strains. We have used this data to guide the construction of congenic lines on the C57BL/6 background, carrying divergent IGH or IGK loci from BALB/cByJ or NOD/ShiLtJ, respectively. Together, our data show significant germline diversity in our new IG assemblies, as well as divergent Ab repertoires in common lab mouse strains.
Splenic B-1 Cells Shows Diversified IgM Repertoire in Aged Atherosclerotic Mice.
Authors: Srikakulapu Prasad , Upadhye Aditi , Marshall Melissa A , Holodick Nichol , Rothstein Thomas L , McNamara Coleen A
Published: February 2023

B-1 cell derived IgM attenuates chronic inflammatory diseases like atherosclerosis. The spleen is a major lymphoid tissue where B-1 cells (both B-1a and B-1b) produce natural IgM. B-1 derived natural IgM has long been thought to contain restricted predominantly germline encoded V-D-J gene segments with low number of non-template encoded nucleotide (N)-additions, which are added at the junctions between V-D and D-J segments. However, differences in VDJ gene usage and N-addition frequency between peritoneal cavity (PerC) and spleen B-1 cells in aged atherosclerotic mice are unknown. B-1a and B-1b cells were single-cell sorted from PerC and spleen of 100-week-old, chow-fed ApoE−/− mice. Single cell sequencing of the immunoglobulin heavy chain variable region was performed. Splenic B-1 cells displayed increased diversity at the V-D and D-J junctions, as evidenced by increased average number of N-additions. Sequences containing >1 N-additions at both V-D and D-J junctions were 2% and 27% for PerC B-1a and B-1b sequences as compared to 44% and 44% of sequences in splenic B-1a and B-1b respectively. B-1 cells from PerC and spleen showed marked differences in VH, D and JH gene segment usage. Importantly, the most commonly expressed heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence in spleen B-1a (AREVTTMYYFDY) and B-1b (AREDYYGSSYYFDY) cells was different from PerC B-1a (AGDYDGYWYFDV) and B-1b (AGDRDGYWYFDV) cells. Results provide clear evidence of a diversified IgM repertoire expressed by spleen B-1 cells compared to PerC B-1 cells during advanced atherosclerosis. The CDR-H3 sequences of splenic B-1 cells suggest underlying differences in antigen specificity which could further regulate disease progression.
mCherry-expressing Mycobacterium tuberculosis gives insight into host cell tropism and immune pressure at the single bacterium level in macaques with tuberculosis
Authors: Mattila Joshua T , Pisu Davide , Junecko Beth A. , Flynn JoAnne L , Russell David G.
Published: February 2023

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb) and is associated with granuloma formation in the lungs and other tissues. These lesions can limit bacterial replication but also contain host cells for Mtb and support bacterial persistence and dissemination in poorly controlled disease. Macrophages are often viewed as primary host cells for Mtb but work in mice demonstrates that other cell types host Mtb in acute infection. Less is known about host cell tropism in human TB, especially in the early stages of infection but these early interactions may have implications for later stages of disease. To identify which cells are infected in early and established TB, we infected two cynomolgus macaques with an mCherry-expressing Mtb strain and used flow cytometry and microscopy to identify and phenotype the infected cells at four- and ten-weeks post infection. This approach also allowed us to compare bacterial mCherry expression and size to determine if granulomas exert different levels of pressure on Mtb that vary by timepoint or lesion type. We found that granulomas contained numerous Mtb-infected neutrophils and macrophages at four weeks post infection but fewer infected neutrophils at ten weeks post infection. When the fluorescence and size of individual bacteria were compared across granulomas and timepoints, we found that bacteria in high Mtb burden granulomas were more fluorescent than bacteria in granulomas that contained fewer bacteria, and the bacteria in the four-week granulomas were longer, suggesting that early granulomas are better at supporting Mtb growth than granulomas in established disease. These results provide new insights into Mtb-host cell dynamics in a nonhuman primate model that replicates human TB.
Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogram the host immune response during persistent infection
Authors: Heim Cortney , Bosch Megan E , Yamada Kelsey J , Aldrich Amy L , Chaudhari Sujata S , Klinkebiel David , Gries Casey M , Alqarzaee Abdulelah A , Li Yixuan , Thomas Vinai C , Seto Edward , Kielian Tammy
Published: February 2023

Staphylococcus aureus (S. aureus) is a leading cause of prosthetic joint infection (PJI) typified by biofilm formation. Our laboratory has identified preferential myeloid-derived suppressor cell (MDSC) recruitment as a critical mechanism for biofilm persistence, as MDSCs are a main source of IL-10. We screened the Nebraska Transposon Mutant Library to identify S. aureus mutants impaired in their ability to trigger MDSC IL-10 production. Significant hits in lactate biosynthesis were identified. A S. aureus Δddh/ldh1/ldh2 mutant, defective in both D- and L-lactate production, decreased leukocyte IL-10 expression and biofilm-associated monocytes were reprogrammed to a pro-inflammatory state, resulting in reduced biofilm burden. Histone acetylation plays a central role in regulating gene expression and histone deacetylase (HDAC) activity was significantly increased in leukocytes recovered from mice infected with Δddh/ldh1/ldh2 compared to WT, resulting in decreased global histone 3 (H3) acetylation at the IL-10 promoter in MDSCs from Δddh/ldh1/ldh2-infected mice. Lactate inhibited HDAC11, resulting in unchecked activity of HDAC6, a positive regulator of Il-10 transcription, and increased IL-10 production. MDSCs and macrophages produced significantly less IL-10 when treated with HDAC6 inhibitor tubastatin A during co-culture with WT but not Δddh/ldh1/ldh2 biofilm. D-lactate was elevated in synovial fluid of patients with PJI compared to aseptic controls and lactate promoted IL-10 production by human monocyte-derived macrophages. Collectively, S. aureus lactate-mediated inhibition of HDAC11 plays a critical role during biofilm infection, leading to epigenetic changes that reprogram the host immune response.
Uncovering the role of CBLB for innate immune responses regulating systemic dissemination during a Non-Tuberculous Mycobacterial infection
Authors: Sharma Jaishree , Mudalagiriyappa Srinivasu , Abdelaal Hazem F.M. , Kelly Thomas C. , Choi Woosuk , Vieson Miranda D. , Talaat Adel M. , Nanjappa Som Gowda
Published: February 2023

Non-Tuberculous Mycobacteria (NTM) are ubiquitous and cause chronic lung infections to fatal disseminated diseases in immunocompromised individuals. The incidence and prevalence of NTM infections are increasing globally, mainly due to the surging numbers of immunocompromised patients. The diagnosis is often misleading, and the emergence of multi-drug resistant NTM has deprived the treatment options. Innate immune responses, dictated by immune regulators, shape the clinical outcomes of NTM infections. Here, we describe the role of an E3 ubiquitin ligase, CBLB, for innate immunity against an NTM infection. We found that the absence of CBLB, under T-cell deficiency, enhanced the bacterial burden and dissemination following both intravenous and intratracheal infections. Strikingly, CBLB fostered an early induction of granulomatous inflammation, which thwarts the dissemination. CBLB-deficiency caused a significant reduction of NK cell numbers, but not their intrinsic functions. Further, we found that CBLB was required to augment the numbers of inflammatory monocytes and conventional dendritic cells, the latter, additionally, for their activation. Thus, our findings suggest that CBLB plays an essential role in eliciting protective innate immune responses to control the growth and dissemination of NTM during T-cell deficiency.
Diminished monocyte response in severe COVID-19 patients.
Authors: Davis-Gardner Meredith E , Edara Venkata , Lai Lilin , Jin Fengzhi , Floyd Katharine , Nyhoff Lindsay , Mantus Grace , Adelman Max , Pellegrini Kathryn , Chirkova Tatiana , Kissick Haydn , Bosinger Steven , Anderson Larry , Rouphael Nadine , Ahmed Rafi , Wrammert Jens , Grakoui Arash , Suthar Mehul S
Published: February 2023

The COVID-19 pandemic has caused a global public health crisis of unprecedented proportions. SARS-CoV-2, the etiological agent of COVID-19, primarily infects the respiratory tract, leading to a range of symptoms including dry cough, fever, and shortness of breath. While many cases remain mild or asymptomatic, a subset progress to acute respiratory distress syndrome (ARDS) requiring ICU admission and ventilation support. Rapid progress has been made to understand the pathogenesis and immune response to SARS-CoV-2 infection; however, there remain a number of unanswered questions.

Recent evidence has indicated that mild cases of COVID-19 are controlled by an effective innate immune response. However, in moderate and severe cases, the immune response to infection appears to be dysfunctional leading to low levels of interferon and high inflammatory responses.

Here, we examined a cohort of patients admitted to the Emory University Hospital with severe COVID-19. Patients had variable neutralization titers measured by FRNT assay, which did not correlate age or disease severity. Flow cytometry immune subset analysis, cytokine and chemokine quantification, and single cell RNA sequencing (scRNAseq) revealed an inflammatory phenotype including a significant shift in the myeloid compartment compared to healthy donors. Numbers of pDCs and non-classical (CD14−CD16+) monocytes in the blood were decreased, while intermediate monocytes (CD14+CD16+) cells were increased. Additionally, stimulated CD14+ cells from healthy donors were able to limit SARS-CoV-2 infection in a human airway epithelial culture system. This suggests a role for monocytes to combat infection by SARS-CoV-2 that may be dysregulated in severe patients.
Constitutive immune surveillance of the nasal mucosa by neutrophils
Authors: Gonzalez Rodrigo J. , Alvarez David , Mazo Irina B , Matysyak Colette , Garg Rohit , Helble Jennifer , Starnbach Michael N , von Andrian Ulrich H
Published: February 2023

The nasal mucosa is a versatile tissue where functions such as olfaction and conditioning of air take place. A particularly relevant and less explored function of this tissue relates to its capacity to respond to insult. Air-borne pathogens can use the nasal mucosa as a portal of entry to further disseminate into deeper tissues and little is known about the immune cell populations these pathogens encounter upon infection. We asked what immune cell populations are present in the murine nasal mucosa and unexpectedly found extravascular neutrophils to be a constitutive and prominent population in the steady state. These neutrophils were found in comparable numbers in conventional vs. germ-free mice and can be divided into three well-defined populations based on the expression of specific cell surface markers and mRNA transcript profiles. One of the populations was traced to nasal mucosa-associated pockets of bone marrow and determined to contribute to the nasal mucosa neutrophil extravascular pool. A second population was found to preferentially phagocytose bacteria during infection. In the absence of infection, this population differentiates into a third population, which interacts with other cells of the nasal mucosa in a manner that resembles trogocytosis. Lastly, we found extravascular granulocyte hematopoietic precursors in the nasal mucosa that likely contribute to the extravascular neutrophil pool. Thus, the nasal mucosa encompasses a constitutive population of extravascular neutrophils that show diverse origins and specialization of function. These findings contribute to our understanding of the composition and development of the microenvironment that air-borne pathogens encounter upon infection in the nasal mucosa.
An Atlas of the Complement System in Developing and Adult Mouse Brains
Authors: Zhang Yingying , Moffitt Jeffrey , Carroll Michael
Published: February 2023

The complement system is traditionally known as a key pathway of peripheral innate immunity. Recent studies reveal distinct functions of the complement system in normal development of mammalian central nervous system (CNS). Over activation of the complement system has been associated with increased risks for Schizophrenia in humans by causing excessive synapse pruning. However, the source of complement and its regulation in the CNS remains largely unknown. To this end, we applied MERFISH (Multiplexed Error Robust Fluorescent in situ Hybridization), a novel spatial transcriptomic approach, to establish a brain atlas of the complement system during development. We found that most components of the complement pathway are locally expressed in the brain. They are expressed by particular types of cells in the brain with distinct spatial and temporal distribution. Notably, the lectin pathway showed striking age-dependent differences in expression level and spatial heterogeneity in the brain. Moreover, suggesting an active role of complement in brain development, we found the spatial distribution of a subset of complement regulatory genes coincides with that of neural development-related genes in the cerebral cortex. To further investigate this hypothesis, we are currently studying the effect of complement inactivation on CNS development by comparing neuronal populations from wild type mice to those with C3 or C4 deficiency. Ultimately, we aim to use our approach to understand molecular changes and abnormalities in CNS development caused by overactivation of the complement cascade, and how these changes increase risks for developing Schizophrenia. This work is supported by the Conte Center Grant NIH 1P50MH112491.
Single-cell transcriptome analysis reveals CNS innate immune landscape plasticity in diet-induced obesity and type 2 diabetes
Authors: Yu I-Chen Ivorine , Paraiso Hallel C. , Kuo Ping-Chang , Scofield Barbara A. , Chang Fen-Lei , Yen Jui-Hung
Published: February 2023

Midlife obesity and type 2 diabetes are strong risk factors for late-life cognitive impairment and dementia. Previous studies show that obesity is associated with a heightened state of neuroinflammation, indicating that systemic factors profoundly influence brain innate immunity. Here, we reported that the diversity and plasticity of brain myeloid cells are reshaped in middle-aged mice fed with a high-fat diet (HFD). Using single-cell RNA sequencing, we revealed the heterogeneity of brain-resident myeloid cells, including microglia (MG, Tmem119/P2ry12/Hexb) and border-associated macrophages (BAMs, Mrc1/Ms4a7/Pf4). MG from HFD-fed mice exhibited changes of genes involved in oxidative phosphorylation and lipoprotein metabolism, suggesting a modulated cellular metabolism in MG responding to HFD. A subset of MG displayed induced disease-associated MG (DAM) gene signature during HFD feeding. We found that Apoe was predominantly expressed in BAM clusters and up-regulated in HFD-fed MG clusters. Several distinct subsets of BAMs enriched in MHC-II (H2-Eb1, H2-Aa, and Cd74), or leukocyte adhesion and migration (Clec4n, Ccl7, Vcam1) genes, displayed the increased cell proportion in HFD-fed mice. In addition, HFD feeding led to the IL-10 downregulation in MHC-II-expressing BAMs. These data suggested that pro-inflammatory BAMs might incorporate T cell immunity to potentiate cerebrovascular inflammation during chronic HFD consumption. Overall, our results highlighted the heterogeneity and plasticity of the brain myeloid cells in middle-aged obesity. The identified BAM subsets provide insights into crosstalk between CNS innate immune cells and T cells to feature neuroinflammation in obesity-associated cognitive decline.
The transcription factor Bach1 plays an important role in regulating macrophage ferroptosis upon iron supplementation
Authors: Aberman Kate , Fisher Logan , Oland Sandra , Sher Alan , Amaral Eduardo P.
Published: February 2023

Ferroptosis is a novel iron-mediated necrotic cell death pathway associated with accumulation of lipid peroxidation, reduced levels of glutathione (GSH) and glutathione peroxidase-4 (Gpx4). This form of cell death has been implicated in different diseases, including tuberculosis. Our group previously described that Mtb-induced macrophage necrosis is associated with hallmarks of ferroptosis, facilitating bacterial spread extracellularly. Nevertheless, the mechanisms underlying the effect of iron in modulating cell death remain poorly delineated. In this study, we used an iron overload model of ferroptosis on bone-marrow derived macrophages (BMDMs) to investigate molecular mediators of antioxidant generation and lipid peroxidation, and their role in modulating ferroptotic cell death. Our findings showed that BMDMs deficient in Nrf2, a transcription factor that regulates many antioxidant genes, were highly susceptible to iron-mediated ferroptosis. In the opposite direction, BMDMs deficient in lipoxygenase-5 (5-LO) as well as in Bach1, a Nrf2 suppressor, displayed enhanced resistance to the cytotoxic effects of iron overload. Interestingly, ~50% of the ferroptotic death triggered by iron supplementation was blocked by 5-LO deficiency while in Bach1-/- macrophage cultures this suppression reached ~94%. Together, these findings indicate that 5-LO plays a partial role in lipid peroxidation driven by iron overload and reveal Bach1 as a master regulator of ferroptosis in macrophages. This study additionally provides a conceptual basis for further investigation of the role of ferroptosis-induced cellular and tissue damage in Mtb infection.
SHIP-1 Inhibitors improve human microglial-like cell function
Authors: Ramakrishnan Gautham S , Humphrey Mary Beth
Published: February 2023

Alzheimer’s disease (AD) is associated with progressive formation of Aβ plaques and neuronal cell death in the brain. Microglia maintain brain homeostasis and their dysfunction is implicated in AD pathology. Triggering receptor expressed on myeloid cells #2 (TREM2) promotes phagocytosis, migration, and metabolic fitness of microglia. TREM2 signaling is inhibited by src-homology 2(SH2) domain containing inositol phosphatase 1 (SHIP-1), encoded by INPP5D, in macrophages and osteoclasts, and the rs669 SNP is associated with increased brain SHIP-1 expression and confers AD risk. We hypothesize that overexpression of SHIP-1 leads to reduced TREM2 signaling and subsequent microglial dysfunction. To test this hypothesis, we treated human microglial like (HMC3) cells, expressing endogenous TREM2, SHIP-1, Iba-1, and CD68, with small molecule inhibitors of SHIP-1 (SHIPi) (3AC) and its paralog SHIP-2 (K161). Treatment with SHIPi significantly increases the surface expression of TREM2 and CD68 as detected by flow cytometry, while not changing Iba-1 expression. Additionally, we demonstrate that treatment of HMC3 cells with SHIPi significantly improves their ability to phagocytose fluorescently tagged Aβ. At concentrations that promote phagocytosis, SHIPi were non-toxic at 72 hours. Together, these data have given us early evidence that SHIPi can promote human microglia-like cell phagocytosis. Further studies are underway to determine the effect of SHIPi on metabolic fitness. SHIPi are safe and effective in mouse models of metabolic dysfunction and obesity and could be an ideal treatment for AD alone or in combination with stimulating TREM2 antibodies.
Interferon-Lambda: A Key Cytokine for Dendritic Cell Development?
Authors: Baffit Mariam , Pang Ee Shan , Wright Mark , O’Keeffe Meredith , Hertzog Paul
Published: February 2023

In light of the current pandemic, COVID-19, our understanding of how the immune system combats viral infection has become more important than ever. Interferons (IFNs) are an integral component of the immune system’s anti-viral response. While the functions of Type I Interferons have been extensively studied, the newest member of the Interferon family, Interferon-Lambda (IFN-λ), is yet to be fully investigated. Currently, it is believed that the canonical signalling pathway of the IFN-λ receptor (R, defined as IFN-λR1 and IL-10Rb) is shared with Type I IFN-αR signalling and leads to the transcription of Interferon Stimulated Genes (ISGs), key genes involved in antiviral immunity. However, there is a growing body of evidence that indicates that IFN-λR1 is involved in signalling pathways that differ to IFN-αR. Notably amongst this evidence is IFN-λR1’s restricted expression to epithelial and some haematopoietic cells as well as non-redundant roles being discovered at mucosal barriers. Building on novel preliminary data, this project seeks to differentiate IFN-λR1 signalling from this canonical pathway and determine whether other receptor chains are involved. Dendritic Cells (DCs), key mediators linking innate and adaptive immunity, are the focus of this project as they are major producers of IFN-λ and are also one of the few haematopoietic cells which express IFN-λR1. Using DCs, this project will investigate the signalling pathways regulated by IFN-λR1 and other potential pathways instigated by the interaction between IFN-λR1 and other receptors. Additionally, this work will focus on how IFN-λ signalling contributes to cellular function in the context of DC differentiation and development.
Innate immune profiling discriminates between liver regeneration and liver failure following partial hepatectomy
Authors: Navarro-Alvarez Nalu , Huang Christene A. , Kovacs Elizabeth J , Yang Yong-Guang
Published: February 2023

Post-hepatectomy liver failure is a major complication following liver resection and while the basic mechanisms are not well understood, limited regeneration of the remnant tissue contributes to its development. Using an established murine model of 70% partial hepatectomy, we studied the innate immunological profile associated with liver regeneration and liver failure. Following partial hepatectomy, there is a significant increase in circulating CD11b+ myeloid derived cells from (52±10 to 70±12 p>0.01). Further characterization of the CD11b+ cells revealed three different subpopulations: Gr-1hiF4/80, Gr-1hiF4/80+, and Gr-1loF4/80+. Liver failure was accompanying with markedly prolonged and increased mobilization and liver infiltration of activated CD11b+ cells (6±2% on day 0 to 55±12% on day 1 p>0.001). Specifically, the CD11b+Gr-1hiF4/80− cells identified as neutrophils were predominantly increased in these mice from (25±5% to 80±10% p>0.001) on day 1. And persistently remained high until day 7. We also found high IL-6 serum levels in these mice (300±150pg/mL) on day 1. The other CD11b+ cell populations, including Gr-1hiF4/80+ and Gr-1loF4/80+ cells, were markedly reduced in peripheral blood of mice with impaired liver regeneration. In contrast, all CD11b+ populations rapidly recovered to normal levels within 2 days in mice showing normal liver regeneration. In conclusion, following partial hepatectomy, significantly prolonged infiltration of CD11b+ neutrophils and marked reduction of other CD11b+ populations are a hallmark of liver failure. Thus, controlling neutrophil infiltration/activation may help ease recovery from the surgical procedure and improve liver regeneration after partial liver resection.
Activation of stimulator of interferon genes in dendritic cells induces interferon-lambda and subset- and species-specific dendritic cell death
Authors: Pang Ee Shan , Daraj Ghazal , Macri Christophe , Balka Katherine , de Nardo Dominic , Hochrein Hubertus , Masterman Kelly-Anne , Zhan Yifan , Kile Benjamin , Lawlor Kate , Radford Kristen , Wright Mark , O’Keeffe Meredith
Published: February 2023

Stimulator of Interferon (IFN) Genes (STING), a cytosolic DNA sensor that recognises cyclic dinucleotides (CDNs), has been an adjuvant target in many cancer immunotherapies. Activation of STING results in the production of Type I IFN through the phosphorylation of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) and it is thought this enhances cross-presentation of tumour antigens by dendritic cells (DCs). However, DCs interrogated in many of these studies use in vitro-generated DCs instead of putative ex vivo DCs, and the direct effects of STING activation on different DC subsets are not completely understood. Here, we report that mouse and humanised mouse splenic DC subsets as well as human blood DCs are activated by CDN stimulation and produce Type III IFN (IFN-lambda) but only type 2 conventional DCs (cDC2s) and plasmacytoid DCs (pDCs) produce Type I IFN in response to CDN stimulation. However, only mouse pDCs aberrantly express extremely high levels of CD86 and CD80 and are ablated rapidly after STING activation. Some DC death was also observed in mouse and human cDC2s, but not in human pDCs. This ablation is STING-dependent and occurs via a cell-intrinsic mechanism involving intrinsic apoptosis. These observations demonstrate the differential effects STING activation has on DC subsets as well as highlight a discordance amongst mouse and human DCs during activation, which serve as an important consideration in the translation of animal cancer models and the use of STING ligands as adjuvants in cancer immunotherapy.
Till cell death do us part: The protective role of inflammasome-mediated pyroptosis and genomic DNA recognition during brucellosis
Authors: Tupik Juselyn D , Coutermarsh-Ott Sheryl L , Benton Angela H , King Kellie A , Kiryluk Hanna D , Caswell Clayton C , Allen Irving C
Published: February 2023

Brucellosis, a zoonotic disease instigated by the bacteria Brucella, results in chronic inflammatory symptoms. Despite mechanisms utilized by Brucella for immune evasion, some pattern recognition receptors, specifically NOD-like receptors (NLRs), sense Brucella pathogen-associated molecular patterns (PAMPS) and form a multi-protein complex termed the inflammasome. The inflammasome attenuates Brucella pathogenesis through caspase-1 (canonical) or caspase-11 (non-canonical) mediated cleavage of pro-inflammatory cytokines IL-18 & IL-1β. It can also activate inflammatory cell death known as pyroptosis, a mechanism rarely implicated with canonical inflammasome activation in brucellosis. Here, we evaluated in vivo canonical (Asc−/−) and non-canonical (Caspase-11−/−) inflammasome mouse models infected with Brucella abortus and determined that wildtype mice displayed increased survival and recruitment of WBCs with decreased bacteria load in comparison with Asc−/− mice. This indicates that the canonical inflammasome plays a necessary, defensive role against Brucella. Conducting in vitro B. abortus infections in Asc−/− macrophages, we further determined this protective mechanism of canonical inflammasome activation results from the activation of pyroptosis. We also found that the Brucella PAMP genomic (g)DNA instigated a high inflammatory response in wildtype macrophages that was suppressed in Asc−/− and amplified in anti-inflammatory NLR knockouts. This suggests that NLR activation occurs through sensing gDNA and may involve mechanisms of interaction among NLRs. Ultimately, these results suggest a greater importance and continued exploration of pyroptosis and the recognition of gDNA during brucellosis.
MicroRNA and periodontal pathogen interaction in shaping innate immune response
Authors: Valverde Araceli Maria , Schaller Samantha , Naqvi Afsar Raza
Published: February 2023

MicroRNA (miR) play a critical role in the pathogenesis of immune-mediated diseases, however, their role in periodontal disease (PD) remain poorly studied. We hypothesize that periopathogens (P. gingivalis [Pg] and A. actinomycemtemcomitans [Aa]) impair host immune response by perturbing miR expression. Naïve CD14+ monocytes were isolated from human PBMC and differentiated to M1 MΦ or M2 MΦ in presence of GM-CSF or M-CSF for 7 days. Cells were challenged with Pg or Aa (both 100 MOI) for 4 h and 24 h. Total RNA was isolated to quantitate expression of pro- and anti-inflammatory miRNAs, cell lysate was prepared for protein expression of NFκB, PU.1 and CEBP/β by western blot and supernatants were collected to analyze cytokine profiles. We observed that expression of pro-inflammatory miRNAs viz., miR-125a and miR-155a are increased in M1-MΦ and M2-MΦ challenged with Pg and Aa. Surprisingly, the expression of anti-inflammatory miR-146a was differentially expressed in both MΦ, but miR-511 expression was reduced. miR-142-3p expression in response to Pg and Aa challenge was decreased suggesting its possible anti-inflammatory function. To examine the impact of miR-142-3p on Pg and Aa-mediated immune responses, we examined PU.1 levels, a known TF regulating miR-142 expression, and noticed a significant decrease in PU.1 and it correlates with mature miR-142-3p levels. miR-142-3p transfected MΦ challenged with Pg or Aa exhibit altered secretion of inflammatory cytokines. In conclusion, periopathogen-mediated activation of NFκB and miR-155 augments inflammatory signaling by suppressing expression of anti-inflammatory miR-142-3p by decreasing PU.1 levels. Perturbation of miR by periodontal bacteria is central to the pathogenesis of PD.