bluuebunny/crossref_metadata_2025 huggingface dataset:
Priming of microvascular endothelium by microbiota-derived metabolites regulates neutrophil trafficking to the liver during sepsis
Authors: Zucoloto Amanda , Yu Katrina , McCoy Kathy , McDonald Braedon
Published: February 2023

During systemic bacterial infections and sepsis, neutrophils are recruited en mass into the microcirculation of the liver and lungs as part of a coordinated intravascular response to defend the bloodstream against bacterial pathogens. The mechanisms that program this response have not been elucidated. The gut microbiota regulates immune cell development and function. In extra-intestinal organs, the microbiota conducts long-distance communication with immune cells through the release of metabolites into the bloodstream. While gut microbes augment granulopoiesis in the bone marrow, the role the microbiota and commensal-derived metabolites on neutrophil trafficking is poorly understood. Using confocal intravital microscopy, we observed that germ-free (GF) mice had impaired neutrophil recruitment to the liver, but not the lungs, during sepsis. Analysis of microbial metabolites in the gut, portal circulation (between gut and liver), and systemic circulation revealed that commensal-derived D-lactate levels are high in the gut and portal circulation, but nearly absent in the systemic circulation. Reconstitution of gut-to-liver D-lactate signalling by enteral administration restored neutrophil trafficking to the liver. In contrast, D-lactate did not impact neutrophil infiltration to the lungs, systemic levels of pro-inflammatory mediators or granulopoiesis. Instead, commensal D-lactate primed liver endothelium to express adhesion molecules required for neutrophil adhesion and crawling. Taken together, we show that compartmentalized molecular communication between gut microbes and the liver primes the microvascular endothelium for neutrophil recruitment in response to systemic bacterial infection and sepsis.
hRSV nonstructural protein 1 (NS1) binds chromatin at regulatory regions of immune response genes.
Authors: Beri Nina , Pei Jingjing , Amarasinghe Gaya , Leung Daisy , Payton Jacqueline
Published: February 2023

Human respiratory syncytial virus (hRSV) causes severe acute respiratory tract infections in infants, elderly and immunocompromised people worldwide and few effective treatments are available. The hRSV nonstructural protein NS1 is known to inhibit host interferon responses in the cytoplasm. We and others have used in vitro interactome studies to identify host proteins that interact with NS1. Among these is the Mediator complex, a transcriptional coactivator localized in the nucleus, suggesting that NS1 may have a more direct role in modulating IFN-stimulated gene expression. We performed chromatin immunoprecipitation of NS1 or Mediator followed by sequencing (ChIP-seq) to map the genomic localization of hRSV NS1 or Mediator in A549 cells infected with hRSV or exogenously expressing hRSV-NS1. We identified 1756 NS1 binding sites, of which 82% (1442) of these are located within 10 KB of at least one gene. 42% (739 / 1756) of NS1 binding sites coincide with Mediator binding sites identified in the same cells, and 73% (1277 / 1756) overlap a known enhancer element (Enhancer Atlas). Moreover, NS1 binding sites often coincide with transcription factor binding sites in A549 cells (ENCODE). Comparison of RNA-seq from hRSV infected cells showed that nearly one-fourth of NS1 binding sites are located near genes significantly up- or down-regulated by hRSV infection. Moreover, these genes are enriched for IFN response genes. Finally, expression of NS1 reduced reporter gene expression when regulated by NS1-bound genetic elements. Together, our findings suggest that NS1 partially suppresses the expression of IFN immune response genes via directly interfering with transcriptional mechanisms, thereby contributing to immune evasion by hRSV.
Interferon-gamma receptor (IFN-γR) signaling is critical for bacterial containment during Staphylococcus aureus craniotomy infection
Authors: Kak Gunjan , Horn Christopher M. , Heim Cortney E. , Kielian Tammy
Published: February 2023

Neurosurgery for brain tumor or epilepsy resection requires a craniotomy to access the brain. Infectious complications occur at a frequency of 1–3%, with approximately half caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap. Our recent scRNA-seq study revealed the transcriptional heterogeneity of infiltrating leukocyte populations during S. aureus craniotomy infection, several of which were enriched for IFN-γ-regulated genes. In the current report, we examined the functional importance of IFN-γ signaling during S. aureus craniotomy infection in IFN-γR knockout (KO) mice. Bacterial burden was significantly increased in the subcutaneous galea of IFN-γR KO compared to WT mice throughout the 28 day time course, whereas titers in the brain and bone flap were similar, highlighting a compartmentalized immune response. Leukocyte infiltrates in the galea were equivalent in IFN-γR KO and WT animals, which led us to utilize scRNA-seq to interrogate alterations in leukocyte activation following IFN-γR loss. Bioinformatics revealed the predominance of granulocyte-myeloid-derived suppressor cell (G-MDSC)-like subsets in the galea of both IFN-γR KO and WT mice. Hallmark pathway analysis identified a significant reduction in not only the IFN-γ pathway in G-MDSC-like subsets, but also a decrease in IFN-α and inflammatory (IL-1β, IL-18, and PLAUR) pathways in IFN-γR KO mice. Collectively, these results demonstrate the importance of IFN-γR signaling in preventing S. aureus outgrowth in the galea and a likely synergism between Type I and Type II IFN pathways in controlling craniotomy infection.
Capsule-dependent metabolic immunosuppression of host cells by Francisella tularensis
Authors: Stromberg Kaitlin A , Jessop Forrest , Schwarz Benjamin , Wehrly Tara , Bosio Catharine M
Published: February 2023

Francisella tularensis subsp. tularensis (Ftt) is a highly pathogenic, intracellular bacterium capable of replicating while avoiding detection by the host immune system. An O-antigen polysaccharide capsule has been identified as an important virulence factor involved in suppressing the immune response. Previous studies have shown that Ftt capsule limits the metabolic shift from oxidative phosphorylation to glycolysis needed for activation of host defense mechanisms. The specific mechanism by which Ftt capsule alters host metabolism is not fully understood but involves improvement of mitochondrial function. In this study, central metabolism in primary cell culture was mapped using targeted metabolomics following TLR stimulation and capsule treatment. Consistent with previous findings, capsule stimulated TCA cycle activity to increase mitochondrial respiration. Ftt capsule treatment did not inhibit increased glycolytic flux associated with TLR-induced metabolic activation. However, capsule limited accumulation of lactate and depletion of acetyl-CoA pools associated with TLR-driven uncoupling of glycolysis and the TCA cycle. These data point to capsule-dependent manipulation of pyruvate dehydrogenase, the enzyme responsible for converting pyruvate made during glycolysis into acetyl-CoA for utilization within the TCA-cycle. Together, these results demonstrate a novel mechanism by which a bacterial capsule can limit inflammation by maintaining coupling of central metabolic pathways.
Functional CFTR channel is essential for effective antimicrobial activities of neutrophils
Authors: Avila Frank Hanz Robledo , Kopp Benjamin T , Partida-Sanchez Santiago
Published: February 2023

Patients with Cystic Fibrosis (CF) are highly susceptible to infections caused by opportunistic pathogens including Burkholderia cenocepacia. In response to infection, CF neutrophils are deficient in clearing bacteria and release inflammatory molecules that contributes to aggravated lung-tissue damage in people with CF (PWCF). Although CF has no cure, novel therapeutic drugs have emerged targeting defective CFTR channel (CFTR modulators). However, it is unknown whether CFTR modulators may restore the basic antimicrobial functions of phagocytes in PWCF.

Here, we first evaluated the expression and subcellular localization of CFTR. Neutrophils from PWCF and non-CF donors were treated in vitro with CF modulators, before infection with B. cenocepacia, and then, CFTR cellular localization was evaluated by confocal microscopy followed by the analysis of antimicrobial killing, and the production of Neutrophil Extracellular Traps (NETs). Furthermore, CFTR-mediated currents were measured by patch-clamp in non-CF and CF neutrophils after treatment with the CFTR modulators.

We found that CFTR modulators increased the expression of functional CFTR channel at the plasma membrane, and also potentiated CFTR-mediated channel activity in CF and non-CF neutrophils, as recorded by whole-cell patch clamp. The production of NETs was also increased in both CF and non-CF neutrophils treated with CFTR modulators, those changes were comparable to cells stimulated with B. cenocepacia or PMA. Finally, CF neutrophils treated with the CFTR modulators restored their antimicrobial killing against B. cenocepacia. Together, these data indicate that functional CFTR channel is essential for regulating the antimicrobial mechanisms of neutrophils.
Maternal high fat diet interacts with the maternal microbiome to expand IL-17 producing type 3 innate lymphoid cells in offspring.
Authors: Mirpuri Julie , Lu Dongmei , Niu Xinying , Mahendroo Mala
Published: February 2023

Introduction: Previously, we demonstrated that maternal high fat diet (mHFD) exposure results in a unique pattern of microbial colonization in offspring and subsequent expansion of IL-17 producing type 3 innate lymphoid cells (ILC3). We hypothesized that in utero mechanisms involving the maternal microbiome were responsible for the phenotype in offspring.

Methods:

To assess if the maternal microbiome was responsible for altering the mHFD offspring phenotype, we delivered mHFD and regular diet (RD)-exposed pups at birth by C-section (CS) and exposed mHFD mothers to broad-spectrum antibiotics (ABX) throughout pregnancy. We cross-fostered the offspring to RD mothers at birth. Microbial colonization in 2-week-old offspring was determined by 16s-based rRNA analysis, intestinal cytokines were quantified by qRT-PCR and lamina propria cells (LP) were profiled by flow cytometry. To determine if innate immunity is altered prenatally by maternal diet, we delivered mHFD and RD offspring by CS and examined the small intestine (SI) at birth for cytokine expression, TLR signaling and LP profiling. Global MyD88 KO mice on mHFD or RD were also examined for the same endpoints.

Results:

mHFD offspring at 2-weeks showed an expansion of Firmicutes and IL-17 producing ILC3, independent of the mode of delivery. ABX in mHFD mothers ameliorated the mHFD offspring phenotype, with microbial colonization and ILC3 similar to controls. mHFD offspring had altered TLR signaling at birth in SI. ILC3 were expanded at birth in mHFD offspring (14.2% vs 9.5%). MyD88−/− offspring exposed to mHFD did not have an expansion of ILC3.

Conclusion:

mHFD prenatally alters the offspring microbiome and ILC3 and this effect is dependent on the maternal microbiome and TLR signaling.
B cells in the mesenteric fat contribute to Crohn’s disease
Authors: Erlich Emma , Czepielewski Rafael Sanguinetti , Kumar Shashi , Andhey Prabhakar Sairam , Artyomov Maxim N. , Ellebedy Ali H. , Randolph Gwendalyn J
Published: February 2023

Crohn’s disease [CD] is one of the two common forms of inflammatory bowel disease and it affects over half a million Americans. Like many other diseases marked by chronic inflammation, some patients with CD develop tertiary lymphoid organs [TLO] in regions of the gut with active disease. TLOs are organized clusters of adaptive immune cells, similar in function and structure to secondary lymphoid organs. Recently, we found B cell rich lymphoid aggregates in the mesenteric fat of CD patients positioned along dramatically remodeled lymphatic vessels, but their function, specifically of the B cells within these aggregates, is completely unknown. Using complementary imaging, flow cytometry, and single cell RNA sequencing approaches, we analyzed B cells present in the mesenteric fat from CD patients. Whereas B cells were essentially not present in the mesentery under homeostatic conditions, they were significantly increased in the inflamed mesenteric fat of CD patients. Single cell RNA sequencing data revealed differences in B cell populations in the mesenteric fat that potentially implicate B cells as contributing to lymphangiogenesis associated with TLOs. Simultaneously, we used a murine model of ileal inflammation to probe mechanism. Imaging and flow cytometry showed that the B cells in TLOs from TNFΔARE/+mice were comparable to the B cells from CD patients. Additionally, TNFΔARE/+/μMT mice, which lack B cells, further revealed that B cells may be required for the lymphatic remodeling seen in the mesentery.
Cross-neutralizing antibody recognition of sarbecoviruses
Authors: Liu Hejun , Yuan Meng , Lee Chang-Chun D. , Wu Nicholas C. , Wilson Ian A.
Published: February 2023

The ongoing COVID-19 pandemic has been devasting to public health and global economy, with more than 82 million confirmed cases and 1.8 million deaths in 2020. Vaccines and antibody therapies are currently under emergency use authorization and provide hope to slow down and eventually stop the spread of SARS-CoV-2 infection. However, the continued identification of SARS-CoV-2 mutants as well as reciprocal transmission between human and farmed mink raise concerns on antigenic drift of SARS-CoV-2 and potential escape from current vaccines. Despite potent and versatile antibody responses to the receptor binding site of the SARS-CoV-2 spike protein, cross-neutralizing antibodies isolated from COVID-19 patients are relatively rare. We have been characterizing these rare cross-neutralizing antibodies using high-resolution X-ray crystallography, as well as other biophysical methods. These antibodies neutralize SARS-CoV-2 and SARS-CoV pseudotyped viruses and are cross-reactive with the receptor binding domain of several sarbecoviruses tested. Our structures have revealed the location of the conserved epitopes that lead to cross-neutralization and the different neutralization mechanisms used by these antibodies. Our findings provide insights for developing more universal SARS-like coronavirus vaccines and therapies to combat antigenic drift in SARS-CoV-2 as well as potential epidemics of other zoonotic coronaviruses.
Glycan-reactive natural antibodies mediate blockade of endogenous retroviruses emergence
Authors: Yang Yexin , Treger Rebecca S. , Hernandez-Bird Juan , Iwasaki Akiko
Published: February 2023

Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential are able to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. B-1 cells specificity is biased toward conserved epitopes shared by bacterial- and self-antigens due to distinct developmental pathways in fetal haematopoiesis. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies targeting glycan structures exhibited by bacterial and viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence.
Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses
Authors: Altin John , Ladner Jason T , Kainov Denis , Shiryaev Sergey , Henson Sierra , Boyle Annalee , Rahee Fatima , Engelbrektson Anna
Published: February 2023

The SARS-CoV-2 proteome shares regions of conservation with endemic human Coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly-multiplexed peptide assay (‘PepSeq’) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have potential to raise broadly-neutralizing responses.
IgG Antibodies against SARS-CoV-2 Correlate with Days from Symptom Onset, Viral Load and IL-10
Authors: Young Mary K , Kornmeier Christine , Carpenter Rebecca , Natale Nick , Sasson Jennifer , Solga Michael , Mathers Amy , Poulter Mendy , Qiang Xiao , Petri William A.
Published: February 2023

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a pandemic of coronavirus disease 2019 (COVID-19). Antibody testing is essential to identify persons exposed to the virus and potentially predicting immunity. 183 COVID-19 patients (68 mechanically ventilated) and 41 controls were tested for plasma IgG, IgA and IgM against the SARS-CoV-2 S1, S2, receptor binding domain (RBD) and N proteins using the MILLIPLEX® SARS-CoV-2 Antigen Panels. COVID-19 positive patients had high levels of IgG, IgA and IgM anti-SARS-CoV-2 antibodies against viral proteins. Sensitivity of anti-S1 IgG increased from 60% to 93% one week after symptom onset. S1-IgG and S1-IgA had specificities of 98%. Ventilated COVID-19 patients had higher antibody levels than the COVID-19 patients who were not ventilated. IgG antibody levels against S1 protein had the strongest correlation to days from symptom onset. We found that patients with the highest IgG levels had the lowest viral load. Finally, there was a correlation of high plasma IL-10 with low anti-SARS-CoV-2 IgG.
ScRNA-seq of Rashes Shows Recurrent Downregulation of the RNA-binding protein ZFP36L2, Revealing Its Role In Mediating Human T cell Cytokine Repression
Authors: Cheng Jeffrey , Liu Yale , Cook Christopher , Cho Raymond
Published: February 2023

The mechanisms driving inflammatory disease are incompletely understood. We performed single-cell RNA-seq on immune cells isolated from 24 human inflammatory skin disease samples of atopic dermatitis, psoriasis, and lichen planus and identified the RNA-binding protein ZFP36L2 as a recurrently repressed transcript in T cells (log FC = −0.85; adjPval < 0.001). CRISPR/Cas9-mediated disruption of ZFP36L2 in unstimulated primary human T cells resulted in a greater than 5-fold induction of inflammatory cytokines (e.g. TNF-α (p = 0.008), GM-CSF, (p = 0.002), IFN-γ (p = 0.030), and IL-13 (p = 0.044)), with minimal effect in stimulated T cells. This greater effect prior to stimulation suggests a role for ZFP36L2 in repressing the onset of inflammation. Consistent with this, temporal analysis of ZFP36L2 and cytokine T cell response to inflammatory stimuli displays ZFP36L2 transcript downregulation prior to induction of cytokine transcripts. These results reveal a previously unknown early, central regulatory role for ZFP36L2 in human inflammation and cutaneous disease, suggesting a potential target for therapeutic exploitation.
Respiratory exposure to putative Gulf War toxins induces phenotypic and transcriptional alterations with effector dysfunction in alveolar macrophages
Authors: Powers Amy A , Jones Katherine E , Eisenberg Seth H , Rigatti Lora H , Luketich James D , Lotze Michael T. , LaRue Amanda C , Dhupar Rajeev , Soloff Adam C
Published: February 2023

Respiratory disorders including chronic obstructive pulmonary disease, asthma, and increased susceptibility to lung infections are a prominent component of Gulf War Illness (GWI). Although the mechanisms of GWI are unknown, chronic immune dysfunction is a consistent factor of this multi-symptomatic disorder. Alveolar macrophages (AMs) represent the predominant mononuclear phagocytes of the pulmonary mucosa, orchestrating the host response to pathogens and environmental stimuli via balanced induction of inflammatory, anti-inflammatory, and pro-resolution mediators. Utilizing commercially available instrumentation, we evaluated the effect of aerosolized exposure to two putative GW toxins, the pesticide malathion and diesel exhaust particulate (DEP), on the immune composition and inflammatory response of the lung in FVB/N mice. Toxin exposure resulted in gross lung pathology with variable increases in protein, lactose dehydrogenase, and cytokines in bronchoalveolar lavage (BAL). Aerosolized toxin exposure reduced AMs in the lung and BAL, associated with a shift to wound-healing phenotypes and concomitant influx of monocyte-derived progenitors from the circulation. Exposure to DEP and malathion, either alone or in combination, induced varying levels of dysfunction in flow-sorted AMs ex vivo as determined by inhibited migration, cytokine production, and phagocytosis. Toxin-induced alterations were associated with unique transcriptional profiles following RNA sequencing analysis. Collectively, these findings suggest that respiratory toxin-induced macrophage dysfunction may establish a state of frustrated immune resolution underlying the chronic immunopathology of respiratory GWI.
The Impact of Aging on Antiviral Pathways and Consequences for Immune Function
Authors: Connors Jennifer R , Kutzler Michele A. , Haddad Elias
Published: February 2023

It is known that aging leads to increased morbidity and mortality, however, this has become more of a concern as the world population shifts to an increased percentage of older adults (>65) especially for COVID-19. These deaths may be from immunosenescence, a condition of chronic increased level of basal inflammation which can also result in diminished vaccine responses and immune system dysregulation, leading to COVID-19 complications. Current research shows a decrease in pro-inflammatory cytokines but the downstream signaling pathways in the context of aging have yet to be evaluated. Two critical pathway points in the production of type I IFN are the phosphorylation of pIRF7 in the TLR 7/8 pathway and of pTBK-1 in the RIG-I and cGAS-STING pathways. We show a decrease in pIRF7 and pTBK-1 in cDC1s, cDC2s, and CD14dimCD16+vascular patrolling monocytes from older adults (n=12) following stimulation with pathway specific agonists in comparison with healthy controls (n=12). This decrease correlates with decreased phagocytosis in the same cell subsets. These data support our previous reports of a delayed and decreased pro-inflammatory output following stimulation and demonstrate potential targets of antiviral pathways that can resolve dysfunctions of the aging immune system for an effective vaccination response. Therefore, we further hypothesize that cDC1, cDC2, and CD14dimCD16+ from older adults will have reduced antigen presentation and ability to cross-present viral peptides. We will utilize COVID-19 patient samples (n=110) to assess antigen-specificity of DCs and T cells in older COVID-19 pos. patients vs healthy controls (n=12).
CD40 intracellular TRAF binding domains modulate B cell activation and experimental autoimmune encephalomyelitis development
Authors: Xu Max , Lu Ying , Zhang Ray , Chiang Jeffrey , Hodes Richard J.
Published: February 2023

CD40 is a costimulatory surface protein expressed on multiple immune cells, including B cells, where it is required for their full activation. Upon interacting with CD40 ligand, CD40 initiates downstream signaling that culminates in activation of naïve B cells. Previous work from our lab revealed that cell autonomous expression of CD40 on B cells regulates their activity, including germinal center development and antibody class switching. Intracellular (IC) domains of the CD40 protein are crucial for signaling. Several of these domains are known to be associated with TNF receptor associated factor proteins (TRAFs) which facilitate further downstream signaling. These include a TRAF6 binding domain and a TRAF2/3 binding domain. We analyzed the CD40 IC domain requirements for CD40 signaling in B cells and for induction of experimental autoimmune encephalomyelitis (EAE). To do so, multiple mouse strains were generated with elimination of the putative TRAF6 or TRAF2/3 domains by CRISPR technology. Whereas eliminating the putative TRAF6 binding site (Domain 1) had little to no effect on B cell viability and in vitro activation by anti-CD40 antibody, eliminating the putative TRAF2/3 binding site (Domain 2) impaired both these functions. Similarly, generation of high affinity IgG in vivo was also dependent on Domain 2 but not Domain 1. In contrast, both Domain 1 and Domain 2 were required for full development of EAE. Interestingly, mixed BM chimeras revealed that the ameliorating effect of CD40 mutations on EAE was mediated by hematopoietic derived non-B cells. Our results indicate that the CD40 TRAF2/3 binding Domain 2 is critical for CD40 dependent B cell function, while both Domain 1 and Domain 2 expression by other APCs function in EAE induction.
Complement Factor H regulates kidney function by modulating macrophages
Authors: Alexander Jessy John , Okamura Kazuki , Jacob Alexander , Chaves Lee Daniel
Published: February 2023

The complement system is a powerful arm of the innate immune system. Complement factor H (CFH) is a critical regulator of the complement system and CFH abnormalities have been implicated in diseases such as dense deposit disease, atypical hemolytic syndrome and age related macular degeneration. Earlier studies from our lab showed that absence of CFH rendered the, normally resistant, C57BL6 mouse susceptible to immune complex mediated glomerulonephritis. CFH knockout mice developed proliferative glomerulonephritis with endocapillary F4/80+ macrophage (Mϕs) infiltration. Nanostring assessment of wild type (WT) and CFHKO kidneys using mouse myeloid panel and stringent analysis revealed that of the 754 genes on the array, 18 macrophage related genes were decreased with at least 1.5 fold change and p<0.05. We therefore studied the role of Mϕs in this setting. Depletion of Mϕs with liposomal clodronate was protective from disease pathology as assessed by BUN, histology, and reduced expression of laminin. For the first time, our studies show that Mϕs isolated from WT mice and grown in culture express intrinsic CFH. Therefore to understand whether the changes are induced by intrinsic Mϕ CFH or CFH in circulation, we have grown bone marrow derived Mϕs from CFH KO and WT mice. Metabolic bioenergetics was markedly impaired in Mϕs deficient in CFH. Since oxidative phosphorylation enables repolarization, our results suggest that modulation of mitochondrial function by complement and thereby reprogramming of inflammatory Mϕs could be a therapeutic avenue in this setting.
Role of CCR2+ Myeloid Cells in Pulmonary Inflammation Responses Driven by Expression of a Surfactant Protein-C Mutant in the Alveolar Epithelium
Authors: Venosa Alessandro , Cowman Sophie , Tomer Yaniv , Katzen Jeremy , Armstrong Brittnie , Beers Michael
Published: February 2023

Acute inflammatory exacerbations (AIE) represent precipitous deteriorations of a number of chronic lung conditions, including pulmonary fibrosis (PF), COPD, and asthma. AIEs are marked by diffuse and persistent polycellular alveolitis that profoundly accelerate lung function decline and mortality. Excess monocyte mobilization during AIE and their persistence in the lung are linked to poor disease outcome. Guided by clinical evidence, we have developed a mutant model of pulmonary fibrosis leveraging the PF-linked missense isoleucine to threonine substitution at position 73 [I73T] in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene [SFTPC]. With this toolbox at hand, the present work investigates the role of resident alveolar macrophages and peripheral monocytes during the initiation and progression of AIE-PF. Genetic ablation of CCR2+ monocytes (SP-C I73TCCR2KO) resulted in improved lung histology, mouse survival, and reduced inflammation compared to SP-C I73TCCR2WT cohorts FACS analysis of SigF+CD11b− alveolar macrophages and Ly6Chi monocytes isolated 3 d and 14 d after SP-CI73T induced injury reveals dynamic transcriptional changes associated with “Innate Immunity’ and ‘ECM Remodeling” signaling. In situ hybridization analysis revealed comparable levels of tgfb1 mRNA expression localized primarily in parenchymal cells in parenchymal cells found nearby foci of injury. We also observed partial tgfb1 mRNA colocalization in iNOS+ and Arg1+ activated cells in SP-CI73TCCR2WT mice, but not in SP-CI73TCCR2KO lungs. Together, these results provide a detailed picture on the role of resident macrophages and recruited monocytes in the context of AIE-PF driven by alveolar epithelial dysfunction.
RNF5-deficiency inhibits CD8+ T cell exhaustion in chronic antigen stimulation
Authors: Bae Eun-ah , Hope Jennifer L , Otero Dennis C , Faso Hannah A , Palete Ashley B , Ronai Ze’ev A , Bradley Linda M
Published: February 2023

Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.
DNA-PK senses DNA virus infection in human cells
Authors: Ferguson Brian J , Hristova Dayana , Oliveira Marisa , Wagner Emma
Published: February 2023

The type I interferon (IFN-I) response to virus infection is initiated by the detection of nucleic acids by intracellular pattern recognition receptors (PRRs). The sensing of DNA viruses by PRRs is carried out by a number of DNA-binding PRRs that signal via the adaptor protein stimulator of interferon genes (STING) to drive IFN-I transcription. We previously described the role of DNA-dependent protein kinase (DNA-PK) as a viral DNA sensor in murine fibroblasts. In this study we show that DNA-PK is essential for the host response to DNA and DNA viruses in human fibroblasts. In the absensce of the catalytic subunit of the DNA-PK heterotrimer, DNA-PKcs, fibroblasts are deficient in their ability to activate a IFN-I response to DNA. DNA-PKcs is activated rapidly following exogenous DNA transfection or DNA virus infection and is required for signalling via STING and the kinase TBK-1 to the transcription factor interferon regulator factor 3 (IRF-3). Most wild-type DNA viruses combat intracellular DNA PRRs using immunomodulatory proteins encoded in their genomes and are effective at blocking IFN-I responses in infected cells. Here we make use of attenutated vaccina and herpes simplex 1 viruses that are lacking the immunomodulators that target DNA sesining mechanisms. We show that DNA-PKcs can sense these viruses and is required for triggering the IFN-I response in infected cells. These data cement the role of DNA-PK in the sensing of DNA virus infections in human cells.
Contribution of the NADase function of bacterial TIR domain proteins to the inhibition of innate immune responses
Authors: Palm Cheyenne , Scudder Alexis , Schmidt Katrina , Harberts Erin M , Snyder Greg A , Snyder Michelle
Published: February 2023

Pathogenic microbes have evolved various mechanisms to evade recognition by host immune responses, which can lead to the prevalence of disease and infection. Many strains of pathogenic bacteria contain genes encoding Toll/Interleukin-1 receptor (TIR) domain-containing proteins that function through the inhibition of TLR and myeloid differentiation factor 88 (MyD88) signaling. Previous research has also revealed that prokaryotic TIR domains may regulate bacterial and host metabolic pathways by acting as NAD+ glycohydrolases (NADases). We hypothesize that this NADase activity may contribute to the role of bacterial TIRs as virulence factors. In order to test this hypothesis, we observed the effects of TIR NADase activity on the binding interactions between bacterial TIRs and host MyD88, and on the ability of bacterial TIRs to block NF-kB signaling. For this, we have identified amino acids predicted to play a role in TIR NADase activity and have expressed and purified recombinant bacterial TIR proteins that have incorporated these mutations. We then tested the NADase activity of these mutants in comparison with that of wild-type TIR proteins and TIRs with mutations already known to inhibit NADase activity. We are now using mutant TIRs with deficiencies in NADase activity in pull-down assays to test their binding to MyD88 and utilizing reporter assays to analyze the effects of these mutant TIRs on NF-kB signaling. This information will allow us to gain a deeper understanding into the relationship between NADase activity in bacterial TIRs and their virulence activity, and can potentially uncover new mechanisms for pathogenic evasion of host immune defenses.
Phosphorylation and Chromatin Tethering Prevent cGAS activation During Mitosis
Authors: Li Tuo , Huang Tuozhi , Chen Zhijian J
Published: February 2023

The cyclic GMP-AMP synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs. cGAS also associates with chromatin especially after nuclear envelop breakdown when cells enter mitosis. How cGAS is regulated during cell cycle transition is not clear. In this presentation, we will provide direct biochemical data showing that cGAS activity is selectively suppressed during mitosis, and we report two parallel mechanisms underlying this suppression. First, we discovered that cGAS is hyperphosphorylated at the N-terminus by mitotic kinases, including Aurora kinase B. We show that the N-terminus of cGAS is critical for sensing nuclear chromatin, but not mitochondrial DNA and that chromatin sensing is blocked by hyperphosphorylation. Second, we demonstrate that chromatin-bound cGAS is prevented from oligomerization, which is required for its activation. Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis to prevent autoimmune reaction.
B-Cell Responses in the Lung during Acute Pneumocystis Infection
Authors: Noell Kristin M , Kolls Jay K
Published: February 2023

Pneumocystis jiroveci is an opportunistic fungal organism that causes Pneumocystis pneumonia in humans with HIV-AIDS or in other immunocompromised individuals. A unique aspect of Pneumocystis is although it is associated with T cell immunodeficiency, recent evidence shows that B cells play a key role as antigen presenting cells in the lung to prime fungal T cell responses (Lund et.al, 2006; Lund et.al, 2003; and Opata et.al, 2015). Our lab has used the murine model of Pneumocystis as many mutations that predispose to Pneumocystis infections in humans replicate in this model (Elsegeiny et al. 2018). In contrast to other infections where dendritic cells are critical antigen presentation cells (APCs), it has been shown that in the context of Pneumocystis, B cells are the key APCs. Our lab has previously shown that IgM−/− mice have delayed CD4+ T cell priming as well as delayed fungal clearance. Therefore we hypothesize that germline encoded B cells react to specific Pneumocystis antigens, and that these B cells are critical to prime Pneumocystis specific CD4+ T cells. We have found that both C57BI/6 and Aicda−/− mice have anti-Pneumocystis IgM prior to infection. Moreover, we found anti-Pneumocystis antibodies in human cord blood and using proteomics we have identified that cord blood pulled down several antigens including PNEG_01454. In the murine model, we found that IgM reacts with a fungal protein involved in ascospore assembly, Meu10. Additionally, using recombinant Meu10 we found Meu10 specific IgM B cells in spleen by Elispot. Current studies are assessing if Meu10 is a T cell epitope and whether Meu10 specific B cells facilitate T cell mediated clearance of the fungus in vivo.
IL-12p40 and Type 1 immunity are induced by Toxoplasma gondii in the absence of the TLR-MyD88 signaling cascade
Authors: Snyder Lindsay , Doherty Claire M. , Mercer Heather L. , Denkers Eric Y.
Published: February 2023

Toxoplasma gondii is an orally acquired pathogen that induces strong, protective IFN-γ based immunity. The response in mice is driven in part by well-characterized MyD88-dependent signaling pathways. The objective of this study was to determine the induction of immune responses that do not involve this Toll-like receptor (TLR)/IL-1 family receptor adaptor molecule, with particular focus on events in the intestinal mucosa. Using eYFP-IL-12p40 reporter mice on an MyD88−/− background, we identified dendritic cells, macrophages, and neutrophils as cellular sources of MyD88-independent IL-12 after peroral T. gondii infection. While infection-induced IL-12 was lower in the absence of MyD88, the cytokine was nonetheless required to induce type I immunity. Type 1 and type 3 innate lymphoid cells (ILC), CD4+ T cells, and CD8+ T cells each contributed to the IFN-γ pool. Furthermore, bacterial flagellin and Toxoplasma specific CD4+T cell populations in the lamina propria expanded to similar frequencies observed in the wildtype small intestine. ILC3 were expanded in both naïve and infected MyD88−/− mice relative to their MyD88+/+ counterparts, suggesting a compensatory response triggered by loss of MyD88. This study identifies MyD88-independent intestinal immune pathways induced by T. gondii including myeloid cell derived IL-12 production, downstream type I immunity, and IFN-γ production by ILC1, ILC3, and T lymphocytes. Collectively, these data reveal an underlying network of immune responses that do not involve MyD88 but that contribute to pathways associated with resistance to T. gondii.
MALNUTRITION IMPACTS PROTECTIVE IMMUNE RESPONSES AND MUCOSAL IMMUNITY DURING VISCERAL LEISHMANIASIS
Authors: Sacramento Laís Amorim , Lombana Cláudia , Scott Phillip
Published: February 2023

Protein malnutrition is a risk factor for developing visceral leishmaniasis (VL) due to defective immune responses that culminate in early visceralization. The intestine is a target of both malnutrition and infection, but the immunological events that occur there during infection are unknown. We postulated that malnutrition exacerbates VL due to defective immunological mechanisms to control parasite replication in the spleen and liver, and by modifying intestinal mucosal immunity. To study the effect of malnutrition on immune response in chronic VL we used a polynutrient deficiency (deficient protein, energy, zinc, and iron) diet which mimics moderate human malnutrition followed by Leishmania infantum infection. The polynutrient deficient diet leads to growth stunting, reduced mass, and cellularity of the spleen, liver, and mesenteric lymph node (MLN). Malnourished-infected mice were more susceptible to infection, harboring more parasites in the spleen and liver. Flow cytometry analysis revealed a reduced number of T lymphocytes, and reduced production of IFN-γ by T cells in malnourished-infected mice. In the MLN, L. infantum infection induced the production of IFN-γ and TNF, while malnourished-infected mice exhibited a significant decrease in the IFN-γ production by T cells. Also, malnourished-infected mice presented a reduction in the IL-17 and IL-22 production by CD4+ cells, which are critical components for intestinal integrity and homeostasis. Together, malnutrition causes a defective IFN-γ-mediated response and promotes dysfunctional mucosal immunity which increases susceptibility in VL.
Nod2-deficient CD4+ T cells protect against Candida albicans infection
Authors: Lashley Sydney J , Lee Ellen J , Vance Emily E , Dugan Jae W , Philpott Dana J , Davey Michael P , Rosenzweig Holly L , Napier Ruth J
Published: February 2023

Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) is a pattern recognition receptor known to promote anti-bacterial immunity yet its function in fungal disease is less understood. Here we sought to investigate the role of Nod2 in regulating fungal infection. Our results demonstrate that Nod2−/− mice infected with 105 (LD50) Candida albicans had increased survival and reduced kidney pathology compared to WT mice, indicating an important role for Nod2-deficiency in protection against infection. In corroboration, Nod2−/− mice had increased fungal clearance in the kidney within 24–72 h post-infection. T cell-deficient Rag−/− or Nod2−/− Rag−/− mice infected with C. albicans had similar fungal burdens, indicating a cellular role for Nod2 outside of innate and non-hematopoietic cells. In support, Nod2−/− mouse kidneys had increased frequencies of CD4+ T cells 48h post-infection, indicating a potential protective function for Nod2−/− T cells. To evaluate a putative T cell-intrinsic function of Nod2 we infected Rag1−/− mice reconstituted with CD4+ T cells from conditional knockout mice deficient in Nod2 in CD4+ T cells (Nod2fl/fl/CD4-cre) or control mice (CD4-cre). Mice harboring CD4+ T cells from Nod2fl/fl/CD4-cre mice had decreased fungal burden within 48 h and increased Th17 cells in the kidneys akin to the global Nod2−/− mice. Protection conferred by Nod2-deficient CD4+ T cells was abrogated by neutralization of IL-17, indicating the functional importance of the Nod2/IL-17 response in CD4+ T cells in C. albicans pathogenesis. Cumulatively, our data suggest that in opposition to the known protective role of Nod2 in bacterial infections, T cell-intrinsic Nod2 promotes fungal infection.
Airway bacteria prime innate immune protection against Streptococcus pneumoniae
Authors: Clark Sarah E , Schopper Melissa , Horn Kadi
Published: February 2023

Commensal (non-pathogenic) bacteria play an important role in protecting against disease. In both humans and animal models, the depletion of commensal bacteria by antibiotics increases susceptibility to pathogen infection of the lung. However, the specific commensal bacteria which contribute to this protective effect remain undefined. In the respiratory tract, Prevotella species are frequently associated with health rather than disease, indicating a potentially beneficial role. Specifically, some Prevotella species negatively correlate with infection with the bacterial pathogen Streptococcus pneumoniae, the most common cause of bacterial pneumonia. We recapitulated this relationship in mice, where we find that exposure to an airway commensal Prevotella species improves rapid clearance of S. pneumoniae from the lung. The use of heat-killed Prevotella was sufficient for inducing protection against S. pneumoniae, which was lost in mice depleted of the pro-inflammatory cytokine TNF-α. These results suggest that Prevotella exposure primes an immune response that increases protection against S. pneumoniae infection in the lung. We further find that mouse neutrophils both secrete TNF-α and are improved in their ability to kill S. pneumoniae following Prevotella exposure. Together, these findings indicate that airway Prevotella species can enhance protection against S. pneumoniae lung infection by activating an innate immune response that increases bacterial clearance.
Colitis alters the antigen-specific response to skin commensal bacteria and predisposes to neutrophilic skin inflammation
Authors: Merana Geil Ramvielle , Dhariwala Miqdad , Dwyer Laura , Scharschmidt Tiffany
Published: February 2023

Both gut and skin house microbial communities capable of influencing host immunity. During homeostasis, resident microbes are thought to have a dominant impact on local immune cell function. However, the prevalence of skin disorders among IBD patients suggests that this compartmentalized control may not hold under disease conditions. We hypothesize that an altered immune response to gut-resident microbes during colitis may facilitate excessive inflammation directed at skin commensals. Our lab has previously shown that colonization of neonatal mice with Staphylococcus epidermidis expressing the model antigen 2W (S. epi-2W) results in establishment of tolerance, as denoted by a higher percentage of 2W-specific regulatory T cells (Tregs) in the skin and skin-draining lymph nodes (SDLNs) and fewer skin neutrophils upon later-life S. epi-2W re-exposure. To test whether colitis perturbs tolerance to commensal skin bacteria, we colonized mice with S. epi-2W as neonates and subjected them to colitis as adults in conjunction with re-exposure. Colitic mice exhibited increased skin neutrophils and reduced percentages of S. epi-specific Tregs in skin and SDLNs compared to controls. Notably, no difference in S. epi-specific Tregs was noted in mice subjected to LPS-induced sepsis. Increased intra-intestinal presence of S. epi-2W during colitis indicates initiation of this response in the gut. Consistent with this, adoptive transfer experiments revealed a colitis-induced increase in CD4+ T cell trafficking from gut-draining LN to SDLNs. Recovery of S. epi-specific Tregs in colitic Cd4CreIl1r1flox/flox mice indicates an additional role for circulating IL-1 cytokines in shaping the skin commensal response during colitis.