bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping the timeline of dendritic cell migration and crosstalk in the CNS-lymphatic axis
Authors: Laaker Collin , Hsu Martin , Herbath Melinda , Sandor Matyas , Fabry Zsuzsanna
Published: February 2023

Recent studies show that meningeal lymphatic vessels surrounding the brain are quite reactive during neuroinflammatory disease states and can undergo changes in morphology to regulate the changing CNS microenvironment. It has been shown by our lab that cribriform plate lymphatic endothelial cells (cpLECs) functionally expand, proliferate, and change expressional profile in response to neuroinflammation. This shift in cpLEC phenotype is also associated with an increased affinity for leukocyte interactions, specifically dendritic cells (DCs). We hypothesize that these DC-cpLEC interactions are an important source of these lymphatic cell modifications and thus CNS homeostasis. However, the timing, origin, and full functional impact of peripheral DCs communication with cpLECs is unknown. In this study we attempted to track the arrival of peripherally stained DCs during neuroinflammation as they migrate to a cellular synapse with cpLECs. Utilizing a method of intravascular staining we found that peripherally stained DCs from the blood do not have immediate access to the cribriform lymphatics but can be observed bound to these lymphatic cells as early as 6hrs post injection, with steadily increases populations thereafter. Additionally, we mapped the migration of peripherally stained DCs across CNS parenchyma along key points of migration and infiltration like the choroid plexus, olfactory bulbs, and periventricular areas. Finally using novel methods of transgenic mice with photoconvertible cells, we validate that leukocytes bound to cpLECs have passed through the CNS parenchyma. Together these data attempt to characterize the complete circuit of migratory DCs pathway from the blood and through the CNS-lymphatic network.
STAT2 Deficiency Protects from Acute Colitis by Enhancing IL-22 Signals
Authors: Gamero Ana M , Cremers Tess , Pryimych Nataliya
Published: February 2023

Ulcerative colitis (UC) is one of two major forms of inflammatory bowel disease (IBD). This debilitating chronic condition causes inflammation in the colon and heightens the risk of colorectal cancer. What causes IBD remains largely unclear. Type I and Type III interferons are cytokines that protect from colitis via activation of the ISGF3 transcriptional complex composed of STAT1/STAT2/IRF9. Yet, our previous studies have shown that STAT2 promotes colitis-associated carcinogenesis suggesting that STAT2 may also be implicated in IBD. Therefore, we investigated the role of STAT2 in intestinal inflammation by employing the dextran sodium sulfate (DSS) model of human UC-like acute colitis. DSS-induced Stat2KO mice experienced less body weight loss compared to wild type mice. Pathology scoring of Stat2KO colons revealed reduced damage to the epithelial gut barrier, mild inflammation, and less immune cell infiltration. Conversely, DSS-induced Stat2KO bone marrow chimaeras harboring wild type immune cells developed severe intestinal inflammation indicating a STAT2 mediated immune response. Furthermore, DSS-induced Stat2KO colons showed reduced induction of inflammatory IL6 and Lcn2. In contrast, early induction of the antimicrobial cytokine IL-22 and associated IL-22 target genes was observed together with Stat3 activation in colonic epithelial cells. In addition, IL-22 treatment of Stat2KO colonic epithelial cells led to enhanced STAT3 activation. Therefore, STAT2 is damaging- in part- by attenuating the protective effects of IL-22. Collectively, this bring to the forefront an unrecognized but detrimental role of STAT2 in the pathogenesis of UC, that when deregulated may cause progression to colorectal cancer.
Innate Lymphocytes in Young Splenocytes Promote Protection Against Plasmodium chabaudi Infection
Authors: Opata Michael M. , Smith Margaret Rose , Richard Lyndsay Blake
Published: February 2023

Immunity to malaria requires an elongated time to develop and understanding of the immune response to malaria, especially in children under five, is limited due to the lack of a reliable animal model to study the pathogenesis of the disease. By utilizing a newly developed young rodent model in our lab, we have observed that both splenocytes and purified CD4+ T cells from 8-week adult mice proliferate faster than day 15 old young mice (pups) when stimulated in vitro. Based on the proliferation pattern, we concluded that as the mice age, their cells have the potential to proliferate faster. When transferred to immunocompromised RAG1 knockout (RAG1KO) mice, both pup and adult cells protected from death. This data suggests that there is both a proliferative and responsive cell population in the splenocytes of pups that may promote protection against malaria infection in mice. To better understand this responsive cell population, we adoptively transferred splenocytes from both pups and adult mice into RAG1KO mice and infected the cell recipients with P. chabaudi. We observed higher proportions and numbers of innate lymphocytes including gamma delta (γδ) T cells and natural killer (NK) cells in the pup splenocyte recipients when compared to the adult counterparts on day 9 post-infection. However, there were significantly higher proportions of macrophages in the adult spleen cell recipients. Taken together, our findings suggest that pup cells are enriched with innate lymphocytes that may promote protection against P. chabaudi infection.
The role of intracellular Pattern Recognition Receptors (PRRs) in recognition and immunomodulation of Dendritic cells (DC) in response to Daptomycin (Dap) resistant MRSA
Authors: Jahan Nazneen , Jiang Jhih-Hang , Peleg Anton Y. , O’Keeffe Meredith
Published: February 2023

Methicillin resistant Staphylococcus aureus (MRSA) infection is one of the leading causes of hospital-acquired infections and is more difficult to treat than other bacterial infections. In recent years, resistance to last line anti-staphylococcal antibiotic daptomycin has been observed during treatment of MRSA bacteremia. Protection against MRSA infection is challenging and requires both innate and adaptive immune effector mechanisms. Being at the interface between innate and adaptive immune responses, DCs are thus central to the immune protection against S. aureus. We have previously shown that distinct and paired clinical isolates of MRSA have differential capacity to induce DC activation, with MRSA strains resistant to the last_line antibiotic daptomycin inducing a compromised response to MRSA. However, this study explores the molecular mechanism by which MRSA strains impede DC activation. We explored the role of intracellular PRRs in the recognition and immunomodulation of these clinical isolates. We found that the cytosolic sensors cGAS and STING are implicated in the sensing of clinical isolates of MRSA, but their use is more prominent in response to DapS strains. We also determined that the TLR3 ligand poly(I:C) induced a highly synergistic increase in the activation of DC in response to both Daptomycin sensitive (DapS) and Daptomycin resistant (DapR) MRSA. Our work, thus provides important insights for the mechanism of differential recognition of clinical isolates of MRSA and immunomodulation through engagement of PRR pathways which are critical to achieve effective and comprehensive immune defense against this pathogen.
Bcl3 dependent regulation of Dendritic Cell function in Toxoplasma gondii infection
Authors: Guha June , Kang Byunghyun , Vazquez Estefania , Siebenlist Ulrich , Murphy Philip M
Published: February 2023

The atypical IκB family member Bcl-3 associates with p50/NF-κB1 or p52/NF-κB2 homodimers in nuclei, thereby either positively or negatively modulating transcription in a context-dependent manner. Bcl-3 is critical for host resistance to Toxoplasma gondii and Bcl-3-deficient mice succumbed within 3–5 weeks after infection, correlating with an apparently impaired Th1-type adaptive immune response rather than a defect in the innate immune response. Surprisingly, mice lacking Bcl-3 in CD11c+ dendritic cells (DC) were as susceptible as mice globally deficient for Bcl-3, demonstrating that Bcl-3 is required in these cells to prime protective T-cell-mediated immunity to T. gondii. It remains to be determined however whether conventional or monocyte derived DCs are critical mediators of a successful T cell response against the invading pathogen.

We generated mice specifically lacking Bcl-3 in conventional DCs (Zbtb46 cre knockout [KO]) or in monocyte derived DCs (LysM cre KO). Surprisingly, both conditional KO strains were as susceptible to T. gondii infection as total KO mice. Interestingly, we detected the presence of a unique subpopulation of conventional DCs that expressed the monocytic markers CD64 and Ly6C in infected spleen, which was reduced in the absence of Bcl3. Single cell RNA sequencing was done with infected splenic DCs to understand the developmental regulation of DC across genotypes. Functionally Bcl3 depleted DC are poor presenter of antigen and are defective in cross presenting toxoplasma specific antigens. Consequently, the T cells are poorly primed in these KO mouse leading to inadequate generation of inflammatory cytokines such as IFNg, which is indispensable for host protection.
Microenvironment controls inflammatory response in macrophages
Authors: Zhou Xu , Pope Scott D , Medzhitov Ruslan
Published: February 2023

Inflammatory response often changes tissue environment, such as pH, oxygen level or composition of cells. Sustained deviation in these variables from homeostasis leads to tissue dysfunction and pathology, as collateral damage of inflammation. Meanwhile, we begin to realized that immune cells also constantly monitor tissue environment and maintain tissue homeostasis. This raises an intriguing question: do changes in tissue environment feedback to modulate immune functions during inflammation?

pH is normally maintained within a narrow homeostatic range. Decrease in pH, or known as acidosis, is associated with diverse inflammatory conditions. Using macrophages, we found that acidic pH modulates their inflammatory response to lipopolysaccharide (LPS, TLR4 agonist) in a gene-specific manner, independent of known pH-sensing receptors. Employing a computational deconvolution algorithm, we systemically characterized how change in pH and activation of TLR4 regulate inflammatory gene expression. This revealed three distinct gene classes: a group insensitive to environment, and two groups representing alternative inflammatory programs, whose control by LPS is either antagonistic or synergistic with acidic pH. Surprisingly, this influence by pH correlates with differential control by NF-kB and IRF3, but is not due to difference in pathway activity. Through profiling chromatin landscape, we discovered that pH regulates gene expression via the activity of specific enhancers and chromatin remodeling. Our findings highlight a critical role of tissue microenvironment in controlling inflammatory gene expression, and reveal qualitatively different responses encoded by transcription circuits and epigenetic mechanisms.
IL-33 sensitizes mast cells to respond to a second alarmin, ATP, but not PAMPs.
Authors: Straus David , Pryor Destiny T. , Kee Sydney Ann , Haque Tamara T , Ryan John J
Published: February 2023

Barrier defenses must respond rapidly to pathogen infection but avoid responses to innocuous antigens or microbes. Aberrant recognition can lead to hypersensitivity and/or chronic inflammatory conditions. Barrier tissues actively release alarmins such as IL-33 in response to cell stress helping to identify pathogenic stimuli. Additional alarmin molecules such as ATP, uric acid crystals, and HMBG protein have also been identified but it remains unclear how the innate immune system integrates these multiple signals. We have used mast cells to address how IL-33 and ATP signals interact. While best known for their role in IgE-mediated allergic responses, mast cells readily produce inflammatory cytokines in response to either IL-33 or ATP. Measuring secreted IL-6, TNF alpha and IL-13 we found that bone marrow-derived and peritoneal mast cells had a 3–6-fold increased response to ATP when sensitized by pretreatment with IL-33. This synergistic effect was observed at relatively low ATP concentrations (<300μM) and required <1hr exposure to IL-33. Stimulating with PAMPs (LPS, LTA, poly I:C, ß-glucan) failed to show any enhancement with IL-33 pretreatment. Inhibitor studies indicated that the P2X3 receptor and the NFAT transcription factor mediated the ATP response. To test IL-33 and ATP synergy in vivo, we assessed eosinophil mobilization to the peritoneal cavity in mice. Consistent with the in vitro studies, co-administration of IL-33 and ATP increased eosinophil numbers compared to either alarmin alone. These results support the hypothesis that alarmins collaborate to surpass a threshold level of barrier stress necessary to initiate an inflammatory immune response.
Transcriptional regulation of type 2 innate lymphoid cells and precursors by interleukin-7 receptor signalling
Authors: Lu Julia , Sheikh Abdalla , Abraham Ninan
Published: February 2023

Innate lymphoid cells (ILCs) are a rare population of innate immune cells that act as the first line of defense against pathogens. These cells arise from the lymphoid lineage that B and T cells also belong to. Type 2 ILCs (ILC2s) are a subset of ILCs that reside in the lungs, mucosal layers, and skin, and have roles in clearing helminth infections, triggering allergic responses, and promoting lung tissue repair after influenza infections. The functions of ILC2s mirror those of TH2 cells but lack antigen recognition. However, their full development background is still unknown. It is believed that ILC2s develop in the fetal liver and adult bone marrow from common lymphoid progenitors and differentiate into several intermediates including helper ILC precursors. From this, ILC2 progenitors arise and express interleukin-7 (IL-7) receptor with the aid of the transcription factor GATA3. Our lab has found that IL-7 is critical for ILC2 development as mutations to the IL-7 receptor show a reduction in the ILC2 numbers and GATA3 expression. Conversely, overexpression of IL-7 results in the expansion of ILC2s and elevated GATA3 expression. We hypothesize that IL-7 signalling dictates ILC2 development through the transcriptional regulation of lineage determining factors. We aim to identify the transcriptional and epigenetic landscape regulated by IL-7 in ILC2s and their progenitors using single cell RNA sequencing and chromatin immunoprecipitation sequencing. We will also investigate how IL-7 influences airway immune responses generated by ILC2s using flow cytometry and RNA sequencing. This study will increase our understanding of a vital cell population and contribute to the development of therapeutics for allergic asthma and viral infections.
Postnatal ozone exposure disrupts alveolar development, exaggerates mucoinflammatory responses, and suppresses bacterial clearance in developing Scnn1b-Tg+ mice lungs.
Authors: Choudhary Ishita , Vo Thao , Paudel Kshitiz , Yadav Radha , Mao Yun , Patial Sonika , Saini Yogesh
Published: February 2023

Increased levels of ambient ozone result in the respiratory tract injury and worsening of ongoing lung diseases. However, the effect of ozone exposure on the respiratory tract undergoing active lung development and simultaneously experiencing mucoinflammatory lung diseases such as cystic fibrosis (CF) remains unclear. To address these questions, we exposed sodium channel non-voltage gated 1, beta subunit overexpressing transgenic mice (Scnn1b-Tg+), a mouse model of CF-like lung disease, and littermate wild type (WT) mice to ozone from postnatal day (PND) 3–20 and examined the lung phenotypes at PND21. As compared to filtered air (FA)-exposed WT mice, the ozone-exposed WT mice exhibited marked alveolar space enlargement, significant eosinophilic infiltration, type-2 inflammation, and mucous cell metaplasia. Ozone-exposed Scnn1b-Tg+ mice also exhibited significantly increased alveolar space enlargement, exaggerated granulocytic infiltration, type-2 inflammation, and greater degree of mucoobstruction. The alveolar space enlargement in ozone-exposed WT, and ozone-exposed Scnn1b-Tg+ mice was accompanied by elevated levels of MMP12 protein in macrophages and Mmp12 mRNA in the lung homogenates. Finally, while bacterial burden was largely resolved by PND21 in FA-exposed Scnn1b-Tg+ mice, ozone-exposed Scnn1b-Tg+ mice exhibited compromised bacterial clearance which was also associated with increased levels of IL-10, an immunosuppressive cytokine, and marked mucoobstruction. Taken together, our data show that ozone exposure results in alveolar space remodeling during active phases of lung development and markedly exaggerates the developmental history of an ongoing mucoinflammatory pediatric onset lung disease.
Neutrophils contribute to Doxorubicin-Induced Cardiotoxicity
Authors: Bhagat Anchit , Kleinerman Eugenie S
Published: February 2023

Doxorubicin (Dox) is a widely used chemotherapy agent for treating childhood cancer. However, Dox causes damage to the heart and as a result childhood cancer survivors are at higher risk of developing a cardiovascular disease at an earlier age. Elucidating the mechanisms by which Dox induces cardiotoxicity is crucial to identify therapeutic interventions. The innate immune system, in particular: neutrophils have been linked to several cardiac diseases. However, their role in Dox-induced cardiotoxicity has not been examined. Here, we hypothesize that neutrophils contribute to early damage caused by Dox treatment. We first evaluated whether Dox treatment induced neutrophil infiltration in heart. Mice were treated with Dox for 2 weeks and then neutrophil infiltration was quantified 24 hours after therapy by flow cytometry. There was an increase in neutrophils in heart tissue of Dox-treated mice as compared to controls. Additionally, when heart function was quantified by echocardiography, we found a decrease in ejection fraction [EF] and fractional shortening [FS]), and therefore decreased heart function. Next, we depleted neutrophils using an anti-Ly6G antibody and confirmed depletion in the blood and hearts of Dox-treated mice by flow cytometry. We found that neutrophil depletion prevented Dox-induced decrease in both EF and FS both short (24 hours after therapy) and long term (12 weeks after therapy) in the mice. Together these findings demonstrate that targeting neutrophils is a potential strategy to prevent Dox-induced cardiotoxicity.
Polymorphisms in LILRB-1 affects its ligand interactions
Authors: Azaizeh Bara’ah
Published: February 2023

Leukocyte immunoglobulin-like receptor B-1 (LILRB-1) is an inhibitory receptor of the immunoglobulin super family expressed on a subset of natural killer (NK) cells. LILRB-1 binds a wide range of classical and non-classical MHC class I molecules, and a glycoprotein product of the human cytomegalovirus (HCMV), UL18. UL18 is a structural homolog of MHC class I expressed on the surface of infected cells. The two most distal domains of LILRB-1, denoted as D1 and D2, are the main contributors at the interaction interface of LILRB-1 with its ligands. Four non-synonymous polymorphisms within D1 and D2 domains produce LILRB-1’s natural variants. Interestingly, these variants have shown different expression and binding trends on NK cells and were suggested to affect susceptibility to HCMV infection in a cohort of kidney transplant recipients. Differences in the interaction of different LILRB-1 variants with their ligands such as, classical (HLA-Cw15) and non-classical MHC class I ligands (HLA-G) as well as UL18 have are assessed using a flowcytometry based binding assay and confirmed with functional assays. Our experiments show a trend of tight binding range of most LILRB-1 variants with HLA-G. However, the strength of binding is more variant with other classical MHC class I ligands, showing a constant trend based on the protein introduced by each of the four non-synonymous polymorphisms in different genotypes. Such findings may highlight an evolutionary pressure to maintain a relatively constant interaction of LILRB-1 with HLA-G, which is expressed on fetal trophoblasts to maintain maternal tolerance.
Transcription factors NHR-49/PPARα and HLH-30/TFEB promote host defense in C. elegans via a flavin-containing monooxygenase
Authors: Wani Khursheed A. , Goswamy Debanjan , Taubert Stefan , Ratnappan Ramesh , Ghazi Arjumand , Irazoqui Javier E.
Published: February 2023

During bacterial infection, the host is confronted with multiple overlapping signals that are integrated at the organismal level to produce defensive host responses. How multiple infection signals are sensed by the host and how they elicit the transcription of host defense genes is much less understood at the whole-animal level than at the cellular level. The model organism Caenorhabditis elegans is known to mount transcriptional defense responses against intestinal bacterial infections that elicit overlapping starvation and infection responses, but the regulation of such responses is not well understood. Direct comparison of C. elegans that were starved or infected with Staphylococcus aureus revealed a large infection-specific transcriptional signature. This signature was almost completely abrogated by the deletion of transcription factor hlh-30/TFEB, except for six genes including a flavin-containing monooxygenase (FMO) gene, fmo-2/FMO5. Deletion of fmo-2/FMO5 severely compromised infection survival, thus identifying the first FMO with innate immunity functions in animals. Further, fmo-2/FMO5 was specifically induced by Gram-positive pathogens and Gram-positive natural microbiota of C. elegans. Moreover, the mechanism of fmo-2/FMO5 induction required the nuclear receptor, NHR-49/PPARα, which induced fmo-2/FMO5 and host defense cell non-autonomously. These findings for the first time reveal an infection-specific host response to S. aureus, identify HLH-30/TFEB as its main regulator, reveal that FMOs are important innate immunity effectors in animals, and identify the mechanism of FMO regulation through NHR-49/PPARα in C. elegans, with important implications for innate host defense in higher organisms.
Sepsis-induced immune dysregulation and dysbiosis impairs cognitive abilities
Authors: Giridharan Vijayasree Vayalanellore , Generoso Jaqueline S , Lee Juneyoung , Petronilho Fabricia , Dal-Pizzol Felipe , Morales Rodrigo , Barichello Tatiana
Published: February 2023

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to microbial infection. More than 50% of sepsis survivors suffer from long-term cognitive deficits. Recently the disruption of gut microbiota at both compositional and functional levels was reported in sepsis patients. In this context, we aimed to explore if experimental sepsis-induced immune dysregulation and dysbiosis accelerates Alzheimer’s disease (AD) pathology. Male, 50-day-old APP/PS1 mice were subjected to cecal ligation and perforation (CLP) surgery for sepsis induction. Longitudinally, at 3, 30, and 120 days after sepsis or sham surgery, feces were collected for 16S rRNA sequencing and short-chain fatty acid (SCFA) evaluation. The brain was removed to measure Aβ expression and inflammatory cytokines levels. Cognition was tested using novel object recognition (NOR) task at 30 and 120 days after sepsis. Alpha and beta diversity analyses revealed higher disruption of intestinal communities at 30 and 120 days after sepsis. The levels of SCFA, such as acetic acid, butyric acid, isovaleric acid, and isobutyric acid, decreased in sepsis mice. At 30 days, IFN-γ, IL1-α, IL-12 (p40), eotaxin, and TNF-α were increased; at 120 days, anti-inflammatory cytokines IL-6, IL-10, and IL-13 were decreased in the hippocampus of sepsis mice as compared to control. At 120 days after sepsis, the AD pathological marker, Aβ expression, was elevated in the sepsis group. Further, at 30 and 120 days, the sepsis group demonstrated a significant cognitive decline. Sepsis-induced dysbiosis precedes overt cognitive impairment in APP/PS1 mice. Therefore gut microbiota can be targeted to impact outcomes from sepsis-related long-term cognitive decline positively.
Elucidating the mechanism of Interferon Beta (IFNβ) mediated neuroprotection in HIV associated neurocognitive disorder (HAND)
Authors: Koury Jeffrey , Singh Hina , Kaul Marcus
Published: February 2023

37 million people worldwide are diagnosed with Human Immunodeficiency Virus (HIV). An estimated 15–55% of these individuals develop HIV-associated neurocognitive disorder (HAND). Previous studies, including work in our lab identifies a transient increase of interferon beta (IFNβ), an anti-inflammatory and anti-viral type 1 interferon, preceding any behavioral or neuropathological signs in the HIVgp120 transgenic mouse and Simian Immunodeficiency Virus (SIV) models, suggesting a neuroprotective role for IFNβ in early HIV infection. We have previously shown that exogenous intranasal IFNβ treatment is sufficient to confer neuronal protection in HIVgp120 transgenic mice. However, the mechanism by which IFNβ mediates neuroprotection remains unclear. Here we focus primarily on the microglial contribution to IFNβ mediated neuroprotection, as HIV productively infects macrophage/microglia in the CNS.

In this study, we show that abrogating IFNβ down regulates expression of viral sensor, RIG-I, as well as interferon regulatory factors, IRF9, and most robustly IRF7 in the cortex of IFNβ KO HIVgp120tg mice. In addition, using the HMC3 human microglial cell line we show a robust dose dependent increase in innate immune anti-viral genes including IRF7, IFIT1, GBP1 and GBP4. Beyond its anti-viral effects, IFNβ treated microglia are potent inducers of the neuroprotective beta chemokines MIP-1a (CCL3), MIP-1b (CCL4) and RANTES (CCL5). Taken together, this data suggests that IFNβ may be conferring neuroprotection through microglial specific induction of anti-viral genes and neuroprotective beta chemokines CCL3, -4 and -5.
The Parkinson’s disease-associated variant Lrrk2 G2019S confers hyper-susceptibility to inflammasome-mediated cell death and Mycobacterium tuberculosis pathogenesis
Authors: Weindel Chi Gidley , Vail Krystal , Bell Samantha , Martinez Eduardo , Patrick Kristin , Watson Robert Owen
Published: February 2023

Leucine-rich repeat kinase 2 (LRRK2), a multifunctional kinase linked to Parkinson’s disease (PD), has been repeatedly implicated in mycobacterial infection, although it’s function outside the nervous system is poorly understood. To create a replicative niche within the harsh environment of a macrophage, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), needs to manipulate two major innate immune pathways, the type I IFN response and regulated cell death (RCD). Previously, we found that loss of LRRK2 (Lrrk2−/−) in macrophages results in type I IFN defects during Mtb infection in vitro and in vivo. We mechanistically linked this type I IFN reprogramming to LRRK2-dependent mitochondrial dysfunction and leakage of mtDNA into the macrophage cytosol. To further investigate the role of LRRK2 in Mtb pathogenesis, we shifted focus to the G2019S variant of LRRK2, which is the greatest genetic risk factor for PD. While the type I IFN response is normal in Lrrk2G2019S macrophages, their mitochondrial network is fragmented and depolarized. These macrophages also are more prone to a pyroptotic form of regulated cell death (RCD) that is dependent on mtDNA. Importantly, during Mtb infection in vivo, Lrrk2G2019S mice experience hyperinflammation and elevated signatures of pyroptotic cell death. Collectively, these data suggest that Lrrk2G2019S impairs mitochondrial health and enhances inflammatory cell death (pyroptosis) with severe in vivo consequences during Mtb infection. The potent enhanced inflammatory response present in the periphery of G2019S mice in vivo might begin to suggest a model where increased inflammation in the periphery could trigger or exacerbate neurodegeneration in carriers of this allele.
Clostridia metabolites promote intestinal barrier integrity to protect from food allergy.
Authors: Kemter Andrea Maria , Patry Robert T. , Hesser Lauren A. , Arnold Jack , Littmann Eric , Campbell Evelyn , Nagler Cathryn
Published: February 2023

We previously reported that a consortium of bacteria from the Clostridia class modulates intestinal barrier function through induction of IL-22 and prevents allergic sensitization to food in mice. We further investigated the properties of these bacteria and the mechanisms that underly their protective impact. Shotgun metagenomic sequencing of the anaerobically cultured Clostridia consortium identified genes which suggest the presence of flagellated bacteria. Motility was then confirmed using agar-based assays. Lysates generated from the consortium stimulated ileal tissue to produce IL-23 and IL-22 in vitro in a MyD88 and TLR5-dependent manner, specifically MyD88 signaling in CD11c+ cells. Together, these experiments support the presence of flagellin in our consortium. Flagellin is a TLR5 ligand that induces IL-23 production by CD11c+ dendritic cells, which stimulates IL-22 production in RORγt+ innate lymphoid cells (ILC3). In addition, we found that the Clostridia consortium produced indoles, known ligands for the aryl hydrocarbon receptor (AhR). In vitro stimulation of ileal tissue with indoles induced IL-22 production, and oral administration to antibiotic treated mice improved intestinal barrier function. Lastly, we observed impaired intestinal IL-22 production and barrier function, and exacerbated food allergy in mice that lack AhR signaling in RORγt+ cells such as ILC3, confirming the relevance of AhR signaling in our food allergy model. Production of flagellin and indoles are two potentially beneficial aspects of allergy-protective commensal bacteria and our knowledge of these pathways and metabolites will allow us to develop targeted therapeutics for the prevention or treatment of food allergy.
Gastrointestinal dysbiosis caused by a lack of Paneth cells promotes diet-induced obesity and trafficking of inflammatory immune cells into adipose tissues.
Authors: Joldrichsen Marisa R , Kim Eunsoo , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: February 2023

Paneth cells regulate many aspects of gastrointestinal health through the antimicrobial products and cytokines they produce. Loss or defective Paneth cell function leads to dysbiosis and is one of the causes of Inflammatory Bowel Disease (IBD). The prevalence of obesity is growing in the general population and among recently diagnosed IBD patients, leading to the speculation that obesity increases IBD incidence, but the underlying mechanisms remain to be elucidated. We address whether gastrointestinal dysbiosis caused by a lack of Paneth cells influences obesity. Analysis of their fecal bacterial composition showed that Sox9ΔIEC mice, which lack Paneth cells due to a Sox9 gene deletion within the intestinal epithelium, have more Firmicutes bacteria (p<0.001) in their feces. These mice were then fed a high fat diet for 13 weeks. When Sox9ΔIEC mice were fed a high-fat diet, they exhibited increased bacterial dysbiosis. Furthermore, intestinal permeability measured by FITC-dextran absorption in vivo was increased (p<0.05) in Sox9ΔIEC mice. Finally, Sox9ΔIEC mice gained weight faster and ultimately became more obese than the wild-type mice and displayed impaired glucose tolerance (p<0.0001) indicating the presence of a metabolic disorder. The Sox9ΔIEC mice developed larger abdominal fats, which included increased numbers of inflammatory immune cells such as macrophages (p<0.05), neutrophils (p<0.05), and T cells (p<0.05). Interestingly, B cells were the most increased (p<0.0001) immune cell population in abdominal fat tissues. These results suggest a new role for Paneth cells as regulators of diet-induced obesity via their effect on gastrointestinal dysbiosis and immune cell infiltration of omental fat tissues.
High-sugar diet predisposes colitis via promoting mucus degrading bacteria in the gut
Authors: Zaki Hasan , Khan Shahanshah
Published: February 2023

The Western diet characterized by high fat and high sugar is considered a risk factor for many inflammatory disorders including colitis. However, the impact of dietary sugars, such as glucose, fructose, and sucrose, on colitis remains poorly understood. We, therefore, investigated the role of dietary simple sugars in colitis using dextran sulfate sodium (DSS) and Il10−/− mice models. We fed WT mice with high sugar in drinking water or diet, followed by administration of 2.5% DSS plus high-sugar diet. Sugar-fed wild-type mice showed extreme sensitivity to DSS-induced colitis. Consistently, Il10−/− mice spontaneously developed severe colitis with the consumption of high sugar. To understand the underlying mechanism of detrimental effect of dietary sugar on colitis, we measured pathophysiological changes in the healthy gut. Interestingly, while there was no induction of cell death, inflammatory mediators, and activation of inflammatory pathways, gut microbiota composition was significantly altered in sugar-fed healthy mice. Notably, the abundance of mucus degrading bacteria Akkermansia muciniphila and Bacteroides fragilis was increased. Consistently, bacteria-derived mucolytic enzymes were enriched and the colonic mucus layers were eroded in sugar-fed mice. Furthermore, germ-free mice colonized with microbiota from sugar-treated mice showed higher abundance of mucus degrading bacteria and increased colitis susceptibility. By demonstrating the complex interplay between high-sugar diet, gut microbiota composition, and intestinal mucus barrier, this study, therefore, elucidates a novel mechanism of colitis pathogenesis.
The role of B cell-derived acetylcholine in the regulation of antiviral immunity
Authors: Prieto Antonio Cembellin , Murray Kaitlin , Reardon Colin , Baumgarth Nicole
Published: February 2023

Acetylcholine (ACh) is a neurotransmitter of signals from pre to post-synaptic neurons and from neural to muscle and gland cells, requiring the enzyme choline acetyltransferase (ChAT) for production. ChAT-GFP reporter mice recently showed that ACh is produced also by B and T cells. The physiological role of B cell-derived ACh remains largely unknown. Using ChAT-GFP mice, we show that naïve, conventional (B-2) as well as B-1 cells are major contributors to ChAT expression in spleen, lymph nodes, lung and pleural cavities, with B-1 cells expressing ChAT at the highest frequencies. In contrast, naive CD4 and CD8 T cells are ChATneg. To study the function of B cell-derived ACh we first infected ChAT reporter mice with influenza A/Puerto Rico/8/34. Infection led to increased ChAT+ B-1, but not B-2 cell numbers in the lungs at 3 days post-infection and of ChAT+ CD4 T cells at day 7. ChAT+ CD4 T cells were CD44hi, while the resting CD44lo naïve CD4 and CD8 T cells remained ChATneg. Mice with a B cell-specific deletion in Chat showed changes in lung cell composition, had decreased weight loss, and significant increases in influenza-specific serum IgG1 at day 14 but not later, compared to their wild-type controls. The data suggest that B cell-derived ACh significantly affects lung inflammation during influenza A infection and regulates the size of early extrafollicular B cell responses.
Diverse Functional Autoantibodies that Underlie Immune Perturbations in COVID-19
Authors: Mao Tianyang , Wang Eric Y. , Klein Jon , Dai Yile , Huck John D. , Liu Feimei , Zheng Neil S. , Zhou Ting , Goldman-Israelow Benjamin , Wong Patrick , Lucas Carolina , Silva Julio , Oh Ji Eun , Song Eric , Perotti Emily S. , Fischer Suzanne , Campbell Melissa , Fournier John B. , Wyllie Anne L. , Vogels Chantal B. F. , Ott Isabel M. , Kalinich Chaney C. , Petrone Mary E. , Watkins Anne E. , Cruz Charles Dela , Farhadian Shelli F. , Schulz Wade L. , Grubaugh Nathan D. , Ko Albert I. , Iwasaki Akiko , Ring Aaron M.
Published: February 2023

Infection with SARS-CoV-2 results in a broad spectrum of immunological and clinical outcomes for patients with COVID-19. While dysregulated innate immunity has been extensively explored in severe disease, autoantibody responses and their impact on disease trajectories remain uncharted. Here, we enrolled a cohort of 194 SARS-CoV-2 infected patients and uninfected individuals, and employed a high-throughput autoantibody discovery platform, Rapid Extracellular Antigen Profiling (REAP), to assess the prevalence of autoantibodies against 2,770 extracellular and secreted proteins in COVID-19 disease. We found that COVID-19 patients harbor dramatically expanded autoantibody repertoires compared to uninfected controls. We further established that immunomodulatory proteins including cytokines, chemokines, complements, and cell surface receptors, are frequent targets of autoantibodies. Longitudinal analysis revealed the existence of both pre-existing autoantibodies, as well as a broad subset of autoantibodies induced upon SARS-CoV-2 infection. We uncovered distinct mechanisms by which immune-targeting autoantibodies perturb immunological functions, including interference of immunoreceptor signaling, depletion of circulating leukocytes, and modulation of antiviral antibody responses. Concordantly, murine surrogates of these autoantibodies hinder immune activation and exacerbate disease severity in a mouse model of SARS-CoV-2 infection. In summary, through the lens of an unbiased proteome-scale survey for autoantibody targets, these findings implicate humoral immunopathology as an integral aspect of COVID-19 pathogenesis with diverse impacts on immune functionality and clinical outcomes.
Adoptive B cell therapy improves viral control during a chronic viral infection.
Authors: Chung Young Rock , Dangi Tanushree , Sanchez Sarah , Palacio Nicole M , Penaloza-MacMaster Pablo
Published: February 2023

T cell-based therapies have been explored for the treatment of cancer and chronic infection. However, B cell-based therapies have remained largely understudied. To evaluate the antiviral effect of adoptive B cell therapy, we adoptively transferred virus-specific B cells into mice that were chronically infected with lymphocytic choriomeningitis virus (LCMV). This resulted in a significant increase in antibody titers and reduction of viral loads. Interestingly, blockade of the IFN-I pathway using IFNAR1 blocking antibodies abrogated the antiviral effect of adoptive B cell therapy, suggesting that IFN-I signaling is necessary to control infection after adoptive B cell therapy. These findings provide a proof-of-concept that adoptive B cell therapy can be effective for the treatment of chronic infections.
Intrinsic antibody mediated enhancement of ZIKV infection in placental macrophages
Authors: Moore Kathryn , Zimmerman Matthew G , Kauffman Robert C , Wrammert Jens , Suthar Mehul S
Published: February 2023

Zika virus (ZIKV) vertical transmission from mother to child in utero is associated with a number of neonatal abnormalities, including congenital microcephaly. Yet, the mechanism by which ZIKV infects the placenta and subsequently the fetus remains poorly understood. ZIKV and Dengue virus (DENV) endemic regions are commonly overlapping, and DENV specific antibodies can cross-react with ZIKV. We and others have previously demonstrated that cross-reactive DENV IgG can mediate ZIKV infection of placental cells though increased viral entry via Fc receptors. Specifically, we found that infection with ZIKV immune complexes (ZIKV-IC) generated from cross-reactive anti-DENV monoclonal IgG was enhanced in human placental macrophages called Hofbauer cells, which are found within the chorionic villus stroma near the fetal vasculature. In this study, we aimed to characterize the intrinsic cellular changes that occur in Hofbauer cells as a result of ZIKV-IC infection. We performed RNA sequencing on Hofbauer cells infected with ZIKV complexed with different concentrations of anti-DENV monoclonal IgG. Principal component analysis revealed that while ZIKV incubated with anti-influenza IgG had a similar gene expression profile to ZIKV alone, ZIKV-IC infection with increasing anti-DENV IgG skewed gene expression towards a unique phenotype. We identified a number of transcriptional profiles related to anti-DENV IgG dose. Notably, we observed a pattern of decreased interferon stimulated genes with increasing anti-DENV IgG dose. Yet, increasing anti-DENV IgG also increased ZIKV viral RNA. These results may indicate that ZIKV-IC infection makes Hofbauer cells more permissive to ZIKV replication by modulating the innate antiviral response.
Differential use of complementarity-determining regions by synthetic nanobodies identifies multiple epitopes on receptor binding domain of SARS-CoV2
Authors: Dhobi Javeed Ahmad , Jiang Jiansheng , Boyd Lisa F , Natarajan Kannan , Margulies David H
Published: February 2023

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, enters host cells through the receptor angiotensin-converting enzyme 2 (ACE2). Identification of numerous variant strains of SARS-CoV-2 have demanded unprecedented attention in developing rapid diagnostics, effective therapies, and safe vaccines to bring the current pandemic under control. Rapid progress is underway in the characterization of fundamental aspects of the structure and mechanisms of viral adsorption to cells, entry, and replication as well as in studies of the immune response to the virus. Here we report binding studies and crystal structures of the spike protein receptor binding domain (RBD) in complex with two individual synthetic nanobodies (sybodies) (Sb16 and Sb45). These bind the RBD at the previously identified ACE2 interface, positioning their complementarity determining region (CDR)2 and CDR3 in diametrically opposite orientations with large buried surface area. We also solved a structure of the RBD simultaneously bound by two sybodies, Sb45 and Sb68. In this structure, Sb45 binds at the ACE2 interface and Sb68 interacts at a second site. Comparison of structures of Sb16 both free and bound to RBD reveals a large movement of its CDR2 loop. Structural insights gained from these synthetic nanobody complexes will be helpful in designing new vaccines and expand the possibilities of using high affinity antibodies or linked bifunctional antibodies for therapy.
T-bet expression marks a transcriptionally and functionally distinct population of memory B cells
Authors: Risley Christopher A , Scharer Christopher D. , Boss Jeremy M. , Lund Frances E
Published: February 2023

Memory B cells (Bmem) rapidly differentiate and mount antibody (Ab) responses to previously encountered antigen (Ag). Mouse Bmem can be subdivided using BCR isotype or expression of CD73, CD80, and PD-L2. Many of these Bmem populations were discovered following vaccination with alum-adjuvanted Ag, which drives a distinct immune response from those generated after infection with IFN-inducing viruses. We previously demonstrated that the IFNγ-inducible transcription factor T-bet is required for Bmem recall responses to influenza but not nematode infections, suggesting that T-bet might regulate anti-viral Bmem. Therefore, we hypothesized that the anti-viral Bmem compartment likely contained cells that were distinct from the established subpopulations. Here, we show that T-bet expression within the flu-specific Bmem is heterogeneous and not restricted to any of the previously described Bmem subsets. Furthermore, B cell-intrinsic T-bet ablation caused the loss of one of these Bmem subpopulations, and inducible deletion of T-bet in Bmem caused the loss of the same subpopulation. To determine how T-bet regulates the Bmem compartment, we performed single-cell RNAseq and CITEseq on flu infection-induced Bmem. We identified numerous transcriptionally distinct clusters of Bmem, several of which express T-bet as well as genes associated with plasma cell function. Consistent with this, in vitro assays show that T-bet+ Bmem differentiate more robustly than T-betneg Bmem. These data show that the anti-viral Bmem compartment is heterogeneous and includes a T-bet expressing subpopulation that appears poised for rapid Ab responses. Thus, T-bet appears to be important for the maintenance and function of some, but not all, Bmem.
Natural infection with SARS-CoV-2 generates durable Spike S1 Receptor-Binding-Domain-specific memory B cell and neutralizing antibody responses.
Authors: Mishra Pankaj Kumar , Bruiners Natalie , Ukey Rahul , Datta Pratik , Onyuka Alberta , Handler Deborah , Singh Sukhwinder , Honnen William , Chang Theresa Li-Yun , Pinter Abraham , Gennaro Maria L
Published: February 2023

Since human infection with coronavirus SARS-CoV-2 was first detected in December 2019, we are still developing an understanding of the nature and duration of protection against this infection. Most neutralizing antibodies, which are a key component of the protective response against SARS-CoV-2 infection, target the Receptor Binding Domain (RBD) of the Spike glycoprotein and critically prevent binding of the virus to the host cell receptor, and viral entry. Thus, it is of vital importance to monitor the presence of neutralizing antibodies and RBD-specific B cells that are key for rapid production of protective antibodies upon reinfection with SARS-COV2 infection. In this study, we developed a multicolor flow cytometric assay to enumerate the RBD-specific memory B cell and memory B cell subsets. We collected peripheral blood cells and plasma from 22 subjects 1–2 months since COVID-19 diagnosis (early time point - ET) and then again after 5–7 months (late time point – LT). Comparing the data collected from these two time points, we observed a significant decrease in plasma blasts and double-negative memory B cell and an increase in the IgG+ switched-memory B cells and decrease in the IgM+ switched-memory B cells at LT relative to ET. We concurrently observed a trend toward decreased anti-RBD IgG titers over time. When we tested plasma neutralizing activity employing ACE2-expressing HeLa cell lines infected with mNeonGreen(mNG)SARS-CoV-2, we also observed reduced neutralizing antibody titers over time. Thus, a correlation exists between titers of RBD-specific IgG antibody and neutralizing titers. In addition, the presence of RBD-specific B-cell memory in circulating blood is a strong indication of a durable protective response.
Anti-pseudomonas antibodies contribute to lung disease in cystic fibrosis.
Authors: Lu Shiping , Bomberger Jennifer , Kolls Jay K
Published: February 2023

Cystic fibrosis (CF) is an inherited lethal disease caused by mutations in CFTR. Approximately 60% of patients have homozygous F508del mutation, but their lung function is quite variable. Gene modifier studies identified I MHC II alleles are associated with the age of first Pseudomonas aeruginosa (PA) infection and lung function. Thus, we assessed serological responses to PA among CF subjects with poor lung function (FEV1<70%) versus good lung function (FEV1>90%). Immunoprecipitation-proteomics using outer membrane proteins (OMPs) from PA and human serum identified OprI as a potential important B cell antigen. We found increased anti-mucoid PA antibodies in CF and higher titers against OMPs and recombinant OprI in patients with poor lung function. Mice that were immunized with either recombinant OprI or inactivated whole PA subcutaneously developed high IgG titers in serum. Immunized mice that were challenged with mucoid PA had more weight loss and more severe lung pathology compared to unimmunized controls. There was minimal weight loss in PA immunized Rag2KO mice (impaired T and B cells), suggesting a role of adaptive immunity in the pathology. We observed more sensitive responses in FABP-hCFTR mice (CFTR knockout except GI tract) that OprI-immunized group exhibited the highest anti-OprI IgG titer and the most significant weight loss after challenge. These data suggest that one mechanism by which class II MHC is a modifier gene in CF is through antigen presentation and determining the types of anti-PA antibodies are generated in CF. Anti-PA antibodies may contribute to CF lung disease that we are conducting systems serology studies to understand which antibody functions are exacerbating disease.