bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium tuberculosis modulates DC-T cell cross-talk and Th17 polarization by dampening Notch signaling
Authors: Enriquez Ana B , Sia Jonathan K , Dhkar Hedwin K , Goh Shu Ling , Quezada Melanie J , Rengarajan Jyothi
Published: February 2023

Tuberculosis (TB) is among the leading causes of death worldwide. While IFN-γ and CD4+ Th1 T cell responses are necessary to mount an immune response to Mycobacterium tuberculosis (Mtb), the causative agent of TB, they are not sufficient to provide protection. Studies from several groups, including our own, have highlighted an important role for IL-17 and Th17 responses in immunity to Mtb infection. Previous research from the laboratory has shown that Mtb restricts Th17 responses by dampening dendritic cell (DC) responses and has identified CD40-mediated co-stimulation as critical for generating Th17 responses in response to Mtb. We showed that exogenous CD40 engagement on Mtb-infected DCs enhances Th17 polarization and reduces Mtb burden in the lungs in a vaccination model. However, the DC mechanisms that mediate CD40-dependent Th17 polarization and protection have not been defined. Here we show that Notch signaling in DCs modulates DC-T cell crosstalk and influences T cell polarization during infection. Engaging the CD40 pathway on Mtb-infected DCs increases the mRNA and protein expression of Notch ligands DLL4 and Jagged1 over the course of infection. Blockade of Jagged1 during a DC-T cell co-culture lowers Th1 responses while blockade of both DLL4 and Jagged1 significantly limits Th17 polarization. These results reveal that during infection, Mtb restricts expression of Notch ligands, which impedes Th17 responses during TB. Insights from this study could be applied to designing more efficacious TB vaccines and adjuvants.
The infection-chronic disease link: chronic kidney disease abate immunity in Porphyromonas gingivalis infection leading to pathogen overgrowth and kidney degeneration
Authors: Adamowicz Karina , Ribeiro Andrea , Wadowska Marta , Potempa Jan , Koziel Joanna , Lech Maciej
Published: February 2023

Objectives:

Various studies provide evidence confirming that chronic diseases can derive from infections. Vice versa, chronic disorders impair immune responses and affect the incidence and severity of infections.

Methods:

We investigated the progress of Porphyromonas gingivalis (P. g) infection during chronic kidney disease (CKD) and the effects of P.g-overgrowth on CKD. Two infectious models: the alveolar bone-loss or chamber model were applied in CKD mice. We stimulated primary macrophages, B-lymphocytes as well as osteoclasts with P. g in the presence of uremic toxins to characterize immune responses.

Results:

We showed that CKD significantly increased survival and organ-distribution of P.g. We observed a decreased IgGs in sera of CKD mice infected with P.g, indicating an immune depression caused by uremia. Levels of pro-inflammatory factors indicated changes in inflammation and homeostasis. The bone-loss was significantly increased in the CKD group, indicating that uremia affects bone formation. In vitro results revealed that microbiome/pathogen-derived indoxyl sulfate significantly inhibited the formation of TRAP-positive multinucleated osteoclasts. These effects of indoxyl sulfate might be associated with a higher rate and severity of the gingival infections observed in the CKD group. Moreover, infected mice showed a significantly higher progression of CKD.

Conclusion:

This study represents a link between infection and chronic disease. It shows that controlling pathogens’ overgrowth might reduce the morbidity of preexisting chronic kidney disease. Moreover, the efficient removal of indoxyl sulfate by dialysis could be a therapeutic approach to restore local homeostasis and immune responses.
The origin and fate of malaria-associated atypical memory B cells
Authors: Bunnik Evelien M , Batugedara Gayani , Braddom Ashley , Sullivan Richard , Gonzales Jake , Reyes Raphael , Ssewanyana Isaac , Greenhouse Bryan , Bol Sebastiaan
Published: February 2023

Chronic and frequently recurring infectious diseases, such as malaria, are commonly associated with expanded populations of atypical memory B cells (atMBCs, here defined as CD19+CD20+CD21−CD27− B cells). While the phenotype and conditions leading to the generation of atMBCs in malaria-experienced individuals have extensively been studied, the origin and fate of these cells remain largely elusive. Using B cell receptor (BCR) sequencing to track clonally related cells present before and after a malaria episode in 5-year-old Ugandan children, we observed that IgM+ atMBCs were predominantly derived from naïve B cells, while IgG+ atMBCs present after a malaria episode mainly originated from FcRL5+IgG+ classical MBCs (cMBCs, CD19+CD20+CD21+CD27+ B cells). IgM+ atMBCs showed fewer connections with other B cell subsets and higher turnover than IgG+ atMBCs. Single-cell transcriptomics analysis revealed that IgG+ atMBCs could be divided into two distinct clusters, one of which was maintained up to 6 months after infection. These results underscore the heterogeneity among atMBCs and highlight differences in their longevity. Finally, we observed that atMBCs from malaria-naïve and malaria-experienced donors showed many similarities in BCR characteristics, such as levels of somatic hypermutation, suggesting that atMBCs undergo similar differentiation pathways in response to different types of pathogens. In terms of BCR characteristics, IgM+ atMBCs of all donors closely resembled NBCs, while IgG+ atMBCs were more similar to IgG+ cMBCs. Collectively, our results highlight differences between unswitched and switched atMBC populations and shed light on the origin and fate of atMBCs in malaria-experienced individuals.
Dysregulated neutrophils failed to control bacteria in an intradermal murine model of Orientia tsutsugamushi infection
Authors: Liang Yuejin , Card Galen , Fisher James Robert , Sun Jiaren , Walker David Hughes , Soong Lynn
Published: February 2023

Scrub typhus is a vector-borne febrile illness caused by the obligately intracellular bacterium, Orientia tsutsugamushi that are transmitted by the larvae of trombiculid mites. Approximately 1 million people are infected every year in an Asia-Pacific region. Patients without appropriate treatment may develop severe damage in various organs and lead to death. However, the host protective immunity against this infection is still not understood. Using an intradermal infection mouse model and CFSE-labeled bacteria, we found that neutrophils and monocytes were the majority of infected cells in the skin infection site at 4 and 8 hours post-infection (hpi). However, very few of them can migrate to local lymph node at 24 hpi. Instead, the infected langerhans cells were observed in local lymph nodes, but not in other organs including lung, bone marrow and spleen, as well as blood. To investigate the role of neutrophils in Orientia infection, we treated the infected mice with Anti-Ly6G antibody daily from −1 to 3 dpi. However, the bodyweight loss was comparable between control and neutrophil-depleted mice until 14 dpi. No significant difference of bacterial loads were found in organs including lung and brain. Our in vitro study showed that Orientia infection induced an immunosuppressive neutrophil phenotype and increased neutrophil death. Collectively, our data suggest that infected-neutrophil may undergo the functional dysregulation and not be able to control bacteria efficiently, while antigen-presenting cells may contribute to bacterial dissemination from skin to systemic infection in scrub typhus.
The corruption of innate immunity by Streptococcus anginosus group
Authors: Budziaszek Joanna , Pilarczyk-Zurek Magdalena , Sitkiewicz Izabela , Koziel Joanna
Published: February 2023

Streptococcus anginosus group (SAG), formerly known as Streptococcus milleri, consists of three distinct streptococcal species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. SAG was considered as commensal bacteria of colon, oral cavity and vagina. Recent observation reported those bacteria as potent pathogens found in brain or liver abscess. Mechanism of SAG pathogenesis is still unknown, despite the strong set of clinical data. In our study, we determined the virulence of different SAG clinical isolates by examining the activity of hemolysins, DNAses and proteases. By using Dictyostelium discoideum as model for screening SAG virulence we selected a few high and low virulence strains out of 41 SAG isolates. Finally, we established and characterized the infection process of selected SAG strains by wax worm Galleria mellonella. Therefore, we aimed to characterize how those bacteria can interact with human innate immunity. In our experiments, using both human serum and whole blood we demonstrated, that these bacteria are resistant to killing by non-specific mechanisms of the immune response. Our studies have demonstrated that SAG have numerous virulence factors which differ among examined strains, and which allow bacteria to effectively colonize the human body and avoid elimination by the immune system.

Supported by National Science Centre, Poland 2018/29/B/NZ6/00624
Ineffective CD4 T cell polarization reveals a novel immune evasion target of Borrelia burgdorferi
Authors: Hammond Elizabeth M , Olsen Kimberly J. , Baumgarth Nicole
Published: February 2023

Appropriate polarization of effector CD4 T cells is essential for eliminating invading pathogens. Borrelia burgdorferi (Bb), the spirochetal agent of Lyme disease, evades immune clearance in infected mice and humans, establishing bacterial persistence. Here we aimed to explore the functionality of CD4 T cell as a possible target of Borrelia-induced immune evasion. In response to Bb infection, CD4 T cell polarization is considered to be Th1 dominant. However, when we stimulated CD4 T cells from Bb infected mice ex vivo with Bb surface Ag Arthritis Related Protein (Arp), we were unable to detect IFNγ, prompting us to re-examine the polarization state of lymph node and blood effector CD4 T cells throughout Bb infection by quantitative RT-PCR and flow cytometry. In contrast to prevailing views, we found only modest numbers of Th1-polarized activated CD4 T cells, as measured by expression of Tbet (Tbx21), and little to no induction of Ifng, including in Bb-specific CD4 T cells, identified by a novel I-Ab tetramer to Arp. This could not be explained by enhanced polarization to Th2 or Th17, as GATA-3 and RORγt expression were also largely absent. Nor could it be explained by polarization at a T cell effector site, the skin, as skin-resident T cells also showed no polarization even 4 months after infection. Furthermore, Th1 polarization was not necessary for controlling bacterial dissemination, as Bb infection of Tbet−/− mice showed significant reductions in Borrelia numbers in the tibiotarsal joints, which correlated with enhanced Th17 polarization as measured by RORγt expression. Together, our data demonstrate a failure of CD4 T cell polarization after Bb infection, indicating effector CD4 T cell polarization as a novel target of Bb immune evasion.
Dual transcriptomics to decipher the interferon-gamma independent responses to intestinal Cryptosporidium parvum infection.
Authors: GALLEGO-LOPEZ GINA MARCELA , Mendoza-Cavazos Carolina , Tibabuzo-Perdomo Andres Mauricio , OConnor Roberta , Knoll Laura Jean
Published: February 2023

Background:

This study characterizes the IFNγ independent host immune response against C. parvum to discover new targets to halt the disease. We used transcriptomic analysis of ileum and cecum intestinal sections of IFNγ-deleted mice that were treated with Soluble Toxoplasma gondii antigen (STAg) or PBS upon C. parvum infection.

Results:

STAg treatment reduced the oocyst shedding in C. parvum infected IFNγ deleted mice. To understand the IFNγ independent mechanism of STAg treatment against C. parvum, we performed 12 RNAseq comparisons between tissues, treatments, and infection status for Mus musculus and C. parvum. In the ileum, regardless of STAg or PBS treatment, more than 100 genes had a significant differential expression. Within STAg treated mice, a total of 129 genes for ileum and 78 for the cecum were classified as differentially expressed. We found in high abundance at least 28 genes related to IFN type I response in infected mice treated with STAg, among them, the oligoadenylate synthetase and Schlafen family gene members. For C. parvum transcriptome analysis, we observed in the ileum high abundance of the oocyst wall protein 1 and some mucins; while in the cecum, the RRM domain containing-protein and Tryptophan synthase beta chain transcripts were the more abundant transcripts.

Conclusions:

STAg treatment and C. parvum infection induced a type I interferon response that would be the cause of the reduction of oocyst shedding in STAG treated mice in an IFNy independent manner. While the depth of sequence did not allow us to observe in detail differential expression of genes in C. parvum, we found differences in parasite gene expression between the ileum and the cecum.
Effector function prior to establishment of the phagosomal pathogen niche is required for protective CD4+ T cell-mediated immunity against Leishmania
Authors: Hohman Leah S , Carneiro Matheus B.H. , Kratofil Rachel M. , Peters Nathan C.
Published: February 2023

Leishmania is an appealing model organism to study CD4+ T cell-mediated protective immunity against phagosomal pathogens which features localized primary and secondary infection sites with defined innate and adaptive responses. Upon secondary challenge of chronic L. major-infected C57Bl/6 mice, rapid CD4+T effector (TEFF) function via IFN-g-mediated activation of infected monocytes is associated with optimal immunity. However, the requirement for immediate effector function has yet to be demonstrated. Thus, we isolated time as a variable in the delivery of CD4+ TEFF effector function. We adoptively transferred (AT) chronic mouse-derived Ly6C+CD4+TEFFs into naïve recipients immediately (D0) or 4 days (D4) post-L. major challenge. In this time window Leishmania establishes an intracellular niche but does not proliferate. At day 21 post-challenge, D4 AT resulted in a total loss of the parasite control mediated by D0 transfer. To address whether parasite niche establishment modulated Th1 TEFF cell recruitment, we employed intravital imaging. Ag-sp T cells were present in the infected dermis at significantly lower numbers following D4 vs D0 transfer at 4 days post-T cell transfer, indicating a recruitment deficit. Rapid CD4+ Th1 effector function was required for circulating Th1 TEFF cells to capitalize on an early recruitment window associated with cxcl9 and cxcl10 expression and to prevent parasite niche establishment through altered cell recruitment, gene expression, and functional capacity of both innate and adaptive immune cells. Thus, acute TEFF cell availability is required to prevent host immunomodulation by Leishmania and is a key consideration for vaccination against phagosomal pathogens reliant on Th1 immunity.
Murine iNOS expression is essential for antifungal defenses in kidneys during disseminated cryptococcosis
Authors: Goughenour Kristie D , Zhao Jessica , Xu Jintao , Zhao Peter , Freeman Christine , Ganguly Anutosh , Olszewski Michal A
Published: February 2023

C. neoformans, is one of the top 3 fungal pathogens, causing disseminated disease with high mortality in the absence of immunoprotecion. The protective Th1 immune response leads to M1 macrophage polarization that upregulates inducible nitric oxide synthase (iNOS) generating microbicidal nitric oxide. The role of iNOS has been studied in pulmonary defenses to C. neoformans, while its role in disseminated infection remains unknown. Here we study the effect of iNOS deletion in mouse model of disseminated cryptococcosis. The iNOS−/− and the WT, C57BL/6 mice were infected with 106 CFU of C. neoformans 52D via retro-orbital intravenous inoculation. Infected iNOS−/− mice showed significantly reduced survival times, however, they lacked significant increases in fungal burdens in the common infection sites (lung, spleen and CNS). Interestingly, iNOS deletion resulted in nearly 100-fold increase of the kidney fungal burden. Kidney histology revealed extensive lesions resulting in almost complete destruction of the kidney cortical area in iNOS−/− mice and a corresponding loss of kidney function was observed. The lack of fungal control was not due to a failure to recruit immune cells as iNOS−/− mice had increased kidney leukocyte recruitment as well as increases in monocytes and neutrophils. BMDMs from iNOS−/− mice were able to kill Cryptococcus despite a slight M2 bias seen in vivo. iNOS−/− mice had no defect in T-cell polarization, but a slight decrease in surface markers of activation. We conclude that iNOS expression is particularly important for local fungal control in kidneys, although its deletion does not induce a shift in a systemic immunophenotype in the iNOS−/− mice; rather it serves as an effector molecule in a local kidney environment.
Heterogeneity of human intestinal intraepithelial T cells and their abnormal distribution in Crohn’s disease revealed at high resolution
Authors: Jaeger Natalia , Gamini Ramya , Cella Marina , Schettini Jorge L. , Bugatti Mattia , Zhao Shanrong , Rosadini Charles V. , Esaulova Ekaterina , Di Luccia Blanda , Kinnett Baylee , Vermi Willam , Artyomov Maxim N. , Wynn Thomas A , Xavier Ramnik J , Jelinsky Scott A. , Colonna Marco
Published: February 2023

Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Recurrent/relapsing CD and resistance to medical treatments result in complications requiring surgery. High-dimensional single-cell profiling approaches, such as scRNA-seq and mass cytometry, have been recently performed on intestinal specimens from patients with IBD and controls. However, most of these studies have analyzed whole mucosal biopsies or the lamina propria (LP) compartment, while few have addressed the intraepithelial lymphocytes (IEL) compartment. Here, we profiled T cells purified from the IEL and LP from terminal ileum resections of adult severe CD cases by single cell technologies. Our study defined a vast heterogeneity of T cell lineages in the IEL compartment. IEL included, among others, unique γδT cell subsets: NKp30+γδ T cells expressing RORγt, which produced IL-26 upon NKp30 engagement and a subset expressing PDGFD and CSF1, which may act on epithelial cells, IEL ILC1s, and macrophages, respectively. We have also observed long-lived memory TCF7+CD8+ T cells expressing DC chemoattractants and TFH subsets that may respond to distinct glutathione-conjugated lipids. CD IEL showed a significant increase of activated TH17, coupled with decreased CD8+ T cells, γδT cells, TFH, and Treg. Conversely, the LP showed increased CD8+ T cells and reduced CD4+ T cells with a relative increase of TH17 over Treg/TFH. Results provide an unbiased view of diversity of cell lineages and their functional states in the intestinal mucosa of controls and CD and identify an altered spatial distribution of T cell subsets between the IEL and the LP compartments.
Th17 immunity in the colon is controlled by two novel subsets of colon-specific mononuclear phagocytes
Authors: Huang Hsin-I , Jewell Mark , Hammer Gianna
Published: February 2023

Intestinal immunity is coordinated by specialized populations of mononuclear phagocytes. Although the cell subsets that constitute these populations are thought uniform throughout the intestine, small and large intestine have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity is unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14+CD24+ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14+CD24+ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.
Role of membrane mucin MUC17 in enterocytic barrier dysfunction associated with Crohn’s disease
Authors: Hernandez Elena Layunta , Jäverfelt Sofia , Pelaseyed Thaher
Published: February 2023

The aim of this study is to determine the role of membrane mucin MUC17 in Crohn’s disease (CD). The incidence of CD is increasing in Western countries. However, the epithelial defects that cause disease are unclear. CD is characterized by the disruption of intestinal barriers leading to detrimental bacterial challenges that result in chronic intestinal inflammation. Our recent studies show that enterocytes are coated by the membrane mucin MUC17 that creates a glycocalyx barrier that protects enterocytes against luminal bacteria (Layunta et al. 2021 Accepted). Despite recent advances in defining intestinal barriers, the role of MUC17 and glycocalyx in CD remains unknown.

In order to determine the role of MUC17 and glycocalyx in CD, we studied MUC17 and glycocalyx in ileum biopsies from CD (n=10) and non-CD (n=29) patients collected at Sahlgrenska University Hospital (Gothenburg). Our preliminary data show that MUC17 is downregulated in CD. In mouse ileum, MUC17 forms the glycocalyx barrier. Then, we asked if decrement of MUC17 levels could affect glycocalyx barrier function in CD. To address this question we designed a glycocalyx permeability assay in which we can measure the barrier function of MUC17 in glycocalyx by incubating patient biopsies with live GFP-expression E.coli (GFP+ E.Coli) and exogenous molecules such as fluorescent dextran. Our results showed that the glycocalyx in ileum of CD patients was more penetrable to GFP+ E.Coli and dextran compared to non-CD patients.

In summary, we propose that changes in MUC17 levels in the glycocalyx contributes to disruption of epithelial barrier function during CD. In addition, our novel glycocalyx permeability assay can be used to diagnose CD, and to evaluate treatment response in patients.
The contributions of NAIP/NLRC4 inflammasome activation in Tuft cells to host barrier defense and immunity.
Authors: Churchill Madeline J , Rauch Isabella
Published: February 2023

The small intestine is a key interface between host cells and potentially pathogenic agents, and as such has developed an array of host defense mechanisms to mitigate disease. The role of the epithelium in preventing systemic disease is becoming more appreciated, however the function of a subset of epithelial cells, the tuft cells, outside of parasitic sensing remains unclear.

Tuft cells express all of the NAIP/NLRC4 inflammasome components, suggesting that tuft cells have the ability to recognize bacterial pathogens. The NAIP/NLRC4 inflammasome is a multi-protein complex that forms when bacterial flagellin or type III secretion systems are detected in the cytosol of the host cell, leading to induction of pyroptosis. The exact contributions of NAIP/NLRC4 inflammasome activation in tuft cells towards host barrier defense and immunity are not known.

Data generated by my lab demonstrates that activation of the NAIP/NLRC4 inflammasome in tuft cells facilitates robust release of prostaglandin D2. The specific function of prostaglandin D2 release after NAIP/NLRC4 inflammasome activation in not currently known. However, it has been demonstrated that a subset of innate lymphoid cells (ILCs), ILC3s express the receptor for prostaglandin D2 and after activation, secrete IL-17 and IL-22, key cytokines that promote host defense against bacterial infections. We find that after specific activation of NAIP/NLRC4 inflammasome in tuft cells, IL-22 protein is increased in the small intestine. These findings support our hypothesis that tuft cells are not simply parasitic sensors but contribute to host barrier defense and immunity against bacterial pathogens.
Genetically Engineering ocular probiotics to manipulate ocular immunity and disease
Authors: Rigas Yannis Elias , Treat Benjamin R , Shane Jackie , Shanks Robert QM , Leger Anthony J St.
Published: February 2023

Recently, our lab discovered that the eye harbors a microbiome that includes Corynebacterium mastitidis, which induces the recruitment of gdT cells and the production of interleukin 17 (IL-17). This local immune response to C. mast can protect the eye from more serious infections, such as Candida albicans and Pseudomonas aeruginosa. Because of C. mast’s ability to stably colonize the ocular surface while inducing a beneficial immune response, we believe that this bacterium has the potential to act as a natural vehicle to deliver therapeutics locally at the ocular surface to alleviate ocular surface disease. Before this can become a reality, a reliable method to genetically modify C. mast is required. Therefore, we took the initial steps toward achieving this goal by genetically modifying C. mast to express genes of interest using transposon mutagenesis. This allowed us to create a mutant library with 1,000+ distinct candidates that expressed the fluorescent protein, mCherry, and kanamycin resistance. Using FACS, we were able to select the brightest C. mast mutants. The brightest four mutants were used to inoculate the eyes of C57BL/6 mice, so that we could: 1) track C. mast in vivo and 2) ensure that the promoters upstream of the genes of interest remain active when C. mast has colonized the ocular mucosa. We were able to image recombinant bacteria directly ex vivo and also confirmed that there was no decrease in bacterial fitness or the protective immune response elicited by C. mast. We concluded from these studies that C. mast can be engineered to express proteins of interest without a reduction in bacterial fitness or protective immunity.
Myeloid cells direct recruitment of colon intraepithelial lymphocytes to the epithelium, where they undergo functional maturation through MHC class II interactions
Authors: Lefferts Adam R , Mann Sarah , Van Der Kraak Lauren A , Canna Scott , Kuhn Kristine A
Published: February 2023

Intraepithelial lymphocytes (IELs) are T cells defined by direct contact with epithelial surfaces. While important for barrier integrity, the mechanisms of recruitment and functional maturation remain unknown. We identified sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) as highly enriched in the IEL population compared to lamina propria T cells by microarray, suggesting importance in IEL recruitment. Blockade of S1P signaling using FTY720 significantly reduced recruitment of TCRab+ IELs, but not TCRgd+ IELs, following bacterial recolonization of antibiotic-treated mice, which is known to recruit IELs. Since mice lacking either Myd88 or an intact microbiome lack IELs, we hypothesized that either the epithelium or mononuclear phagocytes responded to microbial TLR ligands and produced S1P. Myeloid lineage cells were deemed the cells responsible for Myd88/microbe dependent IEL recruitment. In vitro, cecal contents stimulated LysM-Cre+ Myd88fl/fl bone-marrow derived macrophages do not upregulate sphk1, the enzyme responsible for most S1P production, and fail to induce S1P dependent T cell migration in a transwell assay. To understand how the IEL-IEC synapse affects IEL function, we utilized Villin-Cre+ MHCIIfl/fl mice. We observed altered maturation in both CD4+ and CD4− CD8− IEL compartments, associated with increased CD44− CD62L+ naive and CD44+ CD62L+ central memory IELs, and decreased CD44+ CD62L− effector, and CD44− CD62L− “revertant” IELs, suggesting a block in T cell maturation. Together these data show a coordinated, multi-system process by which IELs are first recruited to, and then functionally matured by the IECs. Further work will investigate how inflammation of the mucosal barrier alters these processes.
Type 2 Diabetes Increased the Risk of Hyposmia in COVID-19 Mild Pneumonia Patients by Suppressing Nasal Immunity
Authors: Zhang Jun , Tao Ye , Xu Zhaohui , Liu Yehai
Published: February 2023

Type 2 diabetes mellitus (T2DM) can impair the function of nasal-associated lymphoid tissue (NALT) and result in olfactory dysfunction in patients with COVID-19. Exploring the causative alterations of T2DM within the nasal mucosa and NALT could provide insight into the pathogenic mechanisms and fill the knowledge gap between innate immunity and adaptive immunity for virus clearance. Here, a case-control study was used to compare the olfactory function (OF) among the groups of normal control (NC), COVID-19 mild pneumonia (MP), and MP patients with T2DM (MPT) after a 6–8 months’ recovery. Our clinical data suggested that MPT had a higher risk of hyposmia than MP and NC. No significant difference was found between the MP and NC. This elevated risk of hyposmia indicated that T2DM increased COVID-19 susceptibility in the nasal cavity. Furthermore, T2DM animal model (db/db mice) was used to evaluate how T2DM increased COVID-19 associated susceptibilities in the nasal mucosa and lymphoid tissues. Our animal data demonstrated upregulated microvasculature ACE2 expression and significant alterations in lymphocytes component of NALT. Specifically, db/db mice NALT had increased immune-suppressive TCRγδ+CD4-CD8- T and decreased immune-effective CD4+/CD8+TCRβ+ T cells and decreased mucosa-protective CD19+ B cells. These results indicated that T2DM increased the risk of hyposmia and COVID-19 susceptibility in the nasal cavity by suppressing the first-line defense of nasal immunity.
Functional characterization of a self-replicating and genetically tractable murine alveolar macrophage model
Authors: Thomas Sean M , Wierenga Kathryn , Pestka James , Olive Andrew J
Published: February 2023

Alveolar macrophages (AMs) are a critical element of the innate immune response to inhaled agents, yet functional and genetic studies of this unique macrophage population are lacking. Current strategies to obtain large quantities of AMs are cumbersome and inefficient. This is due largely to both the high cost of time and resources involved in the extraction of AMs and the inability to effectively culture AMs ex vivo for extended periods of time. While bone marrow derived macrophages (BMDMs) are modeled in numerous immortalized cell lines, AMs currently lack an acceptable model that can be used in vitro. Recently, self-replicating cells derived from the fetal mouse liver, termed “MPI” cells, have been shown to possess AM-like characteristics. Here, we show that early after isolation, these cells are SiglecFhi, Cd11chi, and Cd14low, while also expressing high levels of Pparg, Marco, Itgax, and Car4, akin to AMs. Additionally, like AMs, MPI cells effectively efferocytose dead cell debris and phagocytose silica particles. While these cells lose their “AM-likeness” over time, addition of the cytokine TGF-β dramatically delays this shift away from the AM-like phenotype. Gene expression analysis shows that in contrast to cells treated with TGF-β, untreated MPI cells cease expressing Tgfbr1, the receptor for TGF-β, concurrent with the shift away from the AM-phenotype. Further, these cells are amenable to viral transduction, and we have successfully employed CRISPR/Cas9 targeted genetic editing in MPI cells. These findings further our understanding of MPI cells as an accessible and genetically tractable model for AMs that allow for long-term and large-scale studies that are not possible with AMs isolated ex vivo.
ATG16L1 deficiency exacerbates acute small intestinal epithelial damage.
Authors: Foerster Elisabeth G , Streutker Catherine J , Philpott Dana J
Published: February 2023

Polymorphisms of the autophagy-related gene ATG16L1 are known to contribute to the genetic risk for developing Crohn’s Disease (CD), an inflammatory bowel disease that can affect the entire gastrointestinal tract. Previous studies have shown a role for autophagy in the intestinal epithelium both in Paneth cell function and in protection from intestinal damage, but no spontaneous disease at baseline. Here, we challenged mice specifically deleted for Atg16l1 in intestinal epithelial cells (Atg16l1ΔIEC) with the anti-CD3 mediated model of acute small intestinal injury. Rapid T cell activation by anti-CD3 injection results in small intestinal damage within 24 hours of injection, this typically self-resolves in wild-type mice 5 days post treatment. We found that autophagy sufficiency was protective in anti-CD3 induced injury. Atg16l1ΔIEC mice had higher mortality and worsened pathology in comparison to their wild-type littermates. We found indications of increased intestinal barrier permeability and crypt cell apoptosis in Atg16l1ΔIEC mice. In addition, expression of interferon gamma (IFNγ) and tumour necrosis factor (TNF) was significantly increased in Atg16l1ΔIEC mice, cytokines that have been implicated in driving intestinal injury in autophagy-deficient mice. Antibody-mediated blockade of either TNF or IFNγ rescued survival of Atg16l1ΔIEC mice. Interestingly, anti-IFNγ abolished both the crypt cell apoptosis and Paneth cell death at 24 hours, whereas anti-TNF did not decrease the crypt cell apoptosis or Paneth cell death, suggesting IFNγ is the primary contributor to pathology in this model. Overall, this work implicates intestinal epithelial autophagy in the protective response to acute intestinal injury.
Blocking IL1, IL33 and IL36 signaling by an anti-IL1RAP antibody is an efficient anti-inflammatory treatment that improves heart function in a model of autoimmune myocarditis
Authors: Rattik Sara , Jakobsson Gabriel , Grönberg Caitriona , Birkedal Gabriel Svensson , Millrud Camilla Rydberg , Thuring Camilla , Pettersson Nina Fransén , Falk Hanna Håkansson , Schiopu Alexandru , Liberg David
Published: February 2023

The IL1 receptor accessory protein (IL1RAP) is a coreceptor for the IL1, IL33, and IL36 receptors. We have developed a fully humanized antibody (CAN10) that binds IL1RAP with high affinity and disrupts IL1α, IL1β, IL33, IL36α, IL36β and IL36γ signaling without inducing ADCC. CAN10 is currently undergoing preclinical development in preparation for clinical studies.

The efficacy of IL1RAP-blockade as an anti-inflammatory treatment was investigated in a mouse model of acute peritonitis (i.p injection of MSU crystals). A CAN10 surrogate (mCAN10) decreased the recruitment of neutrophils and monocytes to the peritoneum as well as the production of e.g IL6 and KC. Blocking only IL1α/β had similar effects but notably, mCAN10 also induced additional anti-inflammatory responses including reductions in e.g IL5 and eotaxin. In addition, mCAN10, but not anti-IL1β, reduced skin inflammation in a mouse model of psoriasis. Taken together, these results indicate that blocking IL1RAP is a potent anti-inflammatory strategy that is qualitatively different from blocking IL1α/β alone.

IL1, IL33 and IL36 may have disease promoting roles in myocarditis, an inflammatory heart disease with no available treatment. Therefore, we investigated if IL1RAP blockade can counteract inflammation and decline in heart function in a model of autoimmune myocarditis (EAM). EAM was induced by immunizing Balb/C mice with αMHC peptide on day 0 and 7. Treatment with mCAN10 or isotype control started day 7 and heart function was analyzed by echocardiography at days 0, 28 and 42. Interestingly, anti-inflammatory treatment with mCAN10 potently counteracted the decline in heart function. These studies highlight the potential of blocking IL1RAP to treat inflammatory diseases.
Use of an Orally Delivered Oxysterol, a Lipid Membrane-Order (Lo) Disruptor, to Relieve IBD Symptoms in IL-10 Deficient Mouse Model
Authors: Lawrence Audrey Pearl , Bamezai Anil K
Published: February 2023

Inflammatory bowel disease (IBD) is a life-long condition that currently has no cure. Existing treatments fail in a significant proportion of IBD patients who are either nonresponsive to treatment or lose their initial responsiveness to these therapies over time. Genetic susceptibility of an individual in combination with environmental triggers can contribute to inflammation in the gastrointestinal (GI) tract and disease onset. Increasingly, microbiome composition is considered critical for chronic disease expression; IBD patients show altered microbiome composition as compared to healthy individuals. While IBD is of unknown explicit cause, disease etiology is perpetrated by the heightened response of immune cells (T, Innate Lymphoid, Natural Killer, B) and their secreted mediators interacting with the GI tract’s epithelial cells. Given the complexity of IBD and involvement of multiple cell types in triggering and fueling inflammation, we have tested the effectiveness of 7-Ketocholesterol (7-KC), an oxysterol, to treat IBD symptoms in IL-10 gene-deficient mice. 7-KC is known to disrupt membrane order in cells and inhibit cellular responses. IL-10 knockout mice gavaged with 7-KC show significant reduction in inflammatory gut pathology. Examination of the microbiome composition using metagenomic shotgun sequencing showed restoration of microbiome constitution in treated mice that was comparable to the normal non-disease wildtype mouse. These preliminary findings shed light on the potential for 7-KC in treating IBD symptoms. Mechanism of 7-KC action, either its effects on the microbiota and/or gut immune/non-immune cell membrane for its immune suppressing effect is currently under investigation.
Deficiency in B Cell Maturation Antigen reveals sex differences in Experimental Autoimmune Encephalomyelitis
Authors: Kumar Gaurav , Ko Rose M. , Bhatt Neal , Axtell Robert C.
Published: February 2023

Multiple sclerosis (MS) is immune mediated demyelinating disease of the central nervous system (CNS) leading to neuronal damage. The disease is heterogeneous with respect to onset and clinical course, being more prevalent in women then in men with a ratio of 3:1. Further sex differences have been observed with males having a more severe and debilitating disease course compared to females. The complex interplay of immune cells, sex hormones or genetic differences could be contributing to these clinical phenomena, which remains to be elucidated. In experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, several reports have demonstrated no sex differences in disease onset or severity in C57BL/6 mice. Herein we report that deficiency in B Cell Maturation Antigen (BCMA) in C57BL/6 mice reveals a sex difference in EAE. BCMA plays a prominent role in B cell function, however the role BCMA plays in MS and EAE is unknown. Our initial goal of this project was to determine the impact BCMA deficiency has on EAE. In doing so we confirmed that EAE disease was not different in males and females from BCMA+/+mice. Strikingly in the BCMA−/− mice, there was a significant increase in EAE disease severity in males compared to females. The increased EAE in the BCMA−/− male mice was associated with increased demyelination and infiltration of inflammatory T cells and macrophages into CNS. Also, we observed decreased splenic transitional B cells in male BCMA−/− mice compared to females. Castration of mice significantly reduces EAE in BCMA−/− mice as compared to sham controls. Altogether, our data demonstrate that male hormones exacerbates EAE in BCMA−/− mice by augmenting inflammatory responses compared to females leading to increased neuro-inflammation.
CD40 on B cells and on dendritic cells mediates experimental autoimmune encephalomyelitis pathogenesis through distinct and complementary mechanisms
Authors: Lu Ying , Xu Max , Zhang Ray , Wagner David H , Hodes Richard
Published: February 2023

Costimulatory CD40 plays an essential role in autoimmune disease models including experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). However, the mechanism underlying CD40 function are not well defined in these processes. Conditional knockout of CD40 on either dendritic cells (DCs) or B cells led to profoundly reduced severity of EAE induced by recombinant human MOG (rhMOG). CD40 expression on DCs, but not on B cells, was required for priming of pathogenic T helper (Th) cells in peripheral draining lymph nodes and for appearance of these pathogenic T cells in the CNS. In marked contrast, CD40 on B cells, but not on DCs, was essential for class-switched MOG-specific antibody production. The distinct function of CD40 on B cells and DC was confirmed by the ability of transferred MOG-immune serum to restore sensitivity to EAE in mice lacking CD40 on B cells but not in mice lacking CD40 on DC. Thus, CD40 expressed on B cells and on DC provides distinct and complementary pathways essential for EAE pathogenesis, providing multiple targets for intervention in EAE, and potentially for MS and other autoimmune diseases.
Evaluating the Effects of Intestinal Bacteria’s Production of GABA Neurotransmitter on an Animal Model of Multiple Sclerosis
Authors: Kohl Hannah Marie , Hoffman Kristina , Staben Kendall , Shi Xutong , Long Tyrel , Castillo Andrea , Gibson K. Michael , Roullet Jean-Baptiste , Ochoa-Repáraz Javier
Published: February 2023

Past studies show that the production of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, by gut bacteria is decreased in patients with multiple sclerosis (MS). Furthermore, the administration of GABAergic compounds ameliorates the progression of experimental autoimmune encephalomyelitis (EAE), a murine model of MS. We investigated whether modifying the microbiota of mice with GABA-producing bacteria reduced the severity of EAE. We genetically modified a Lactococcus lactis with increased copies of the genes encoding the enzyme glutamic acid decarboxylase (GAD), which synthesizes GABA, and the GABA/glutamate antiporter (GadC). This modified L. lactis strain (GAD-L. lactis) produced significantly increased GABA, measured by ELISA, compared to control L. lactis strain, with an unmodified plasmid (P-L. lactis). These strains were compared with sham treatment as probiotic treatments against EAE. EAE-induced C57BL/6 mice were divided into three groups: sham group (EAE induced, treated with autoclaved media; n = 10); EAE mice treated with P-L. lactis (n = 10); and EAE mice treated with GAD-L. lactis (n = 10). The treatments occurred five times a week via oral gavage (5 x 108 CFU/mouse). The oral administration of GAD-L. lactis significantly reduced the severity of EAE and body weight loss during disease, compared to both the P-L. lactis and sham groups. Our results indicate that oral treatment with a probiotic strain that produces enhanced GABA levels protects against the progression of CNS demyelination. Immunophenotyping studies will help us understand the mechanism of action of GAD-L. lactis and its potential as a novel approach to treat autoimmune disorders.
ST8Sia6 expression in beta cells mitigates onset of autoimmune diabetes in the murine NOD model
Authors: Choe Justin , Belmonte Paul , Rajcula Matthew , McCue Shaylene , Shapiro Michael J , Shapiro Virginia
Published: February 2023

Cell surface hypersialylation is used by malignant cells as a survival mechanism, leveraging interactions with inhibitory Siglec receptors to dampen immune rejection. In this study, we harnessed this mechanism to prevent autoimmune rejection of beta cells in the context of Type 1 Diabetes. Mice were engineered to express the sialyltransferase ST8Sia6 in a beta cell specific manner (βST mice) and crossed to the non-obese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Both female and male littermates on the NOD background developed diabetes by 300 days (56% and 80.8%, respectively). βST mice, however, demonstrated minimal occurrence of disease (5.3% and 16.6%). Additionally, histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration at 4 and 8 weeks of age, but the severity of infiltration was drastically reduced only in the βST mice at 20 weeks of age. IHC for various leukocyte markers suggested no difference in composition of immune infiltrates at the 8 week time point. To determine whether protection was due to central or peripheral mechanisms, euglycemic βST and littermate mice at 16 weeks of age were challenged with anti-PD-L1 monoclonal antibodies; both groups resulted in similar induction of disease, suggesting that ST8Sia6 dependent protection occurs peripherally. Physiologically, an insulin tolerance test demonstrated no difference between βST mice and littermates, suggesting no systemic effects of beta cell ST8Sia6 expression. The induced expression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD mouse and presents a novel path forward for improving the success of curative islet transplant into diabetic patients.
BT-11 (omilancor), a first-in-class, topical therapeutic for psoriasis, ameliorates disease severity and inflammation through activation of LANCL2 pathway
Authors: Tubau-Juni Nuria , Leber Andrew , Hontecillas Raquel , Chauhan Jyoti , Bassaganya-Riera Josep
Published: February 2023

BT-11 (omilancor) is a small molecule therapeutic that activates the LANCL2 pathway in immune and epithelial cells, resulting in regulatory effects at the intersection of immunity and metabolism. LANCL2 modulates the plasticity between Th17 and regulatory T cells (Tregs), favoring the stability and suppressive capacity of Tregs. Given the implication of the LANCL2 pathway in autoimmune disease and the demonstrated clinical efficacy and safety of BT-11 in a Phase 2 clinical trial of UC, we evaluated the therapeutic efficacy of BT-11 in psoriasis. In an imiquimod-induced mouse model of psoriasis, pharmacological activation of LANCL2 through topical BT-11 treatment ameliorates disease severity, resulting in over 60% reduction of PASI score. Indeed, BT-11 treatment decreased acanthosis and immune cell infiltration, with no signs of parakeratosis. The loss of LANCL2 in mice resulted in increased hyperkeratosis and parakeratosis. Local LANCL2 activation by BT-11 resulted in decreased TNF, IL17, IL6 and IL21-producing cells in the spleen. Additionally, LANCL2 activation regulated keratinocyte metabolism and function. Keratinocytes from BT-11-treated mice presented lower metabolic activation, reported as decreased ATP production, mitochondrial respiration and glucose metabolism. In translational studies with human PBMCs, BT-11 modulated the metabolic profile of CD4+ T cells at late state glycolysis, favoring oxidative pathways, and decreased expression of proinflammatory cytokines (IFNγ, TNFα), while upregulating IL-10. These results merit further exploration into the implication of LANCL2 signaling in human psoriasis and leverages the development of BT-11, as a first-in-class, topical therapeutic for psoriasis.