bluuebunny/crossref_metadata_2025 huggingface dataset:
Using Tat-Cre to Generate Ex-Vivo Gene Knockouts in T-cells
Authors: Brown Rebecca Audrey , Byersdorfer Craig A
Published: January 2023

We have found unanticipated effects using CD4-driven Cre expression to generate gene knockouts, due to changes in T cells that occur between gene deletion and harvest. To bypass this, we are developing a method to delete genes in T cells just prior to experimentation using brief ex vivo exposure to a Tat-Cre fusion protein, where the TAT peptide drives translocation of Cre to excise loxP-flanked genes. We first defined an optimal concentration and length of treatment by measuring production of a knockout band in DNA extracted from treated T cells. Maximal knockout occurred with 30 minutes of Tat-Cre incubation, while longer treatment times led to no further increase in gene deletion. In contrast, increasing concentrations of Tat-Cre correlated with increasing gene deletion in a dose-dependent manner. 4.6 uM was determined to be the optimal concentration balancing deletion efficacy and resource allocation.

We next used T cells from td-Tomato (tdT)/green fluorescent protein (GFP) reporter mice, which express tdT at baseline, but delete tdT and turn on GFP when Cre is present, to determine if DNA deletions were measurable at the protein level. Immediately after Tat-Cre treatment there was no observable increase in GFP expression. However, by 2 hours post-treatment, 34% of treated cells expressed GFP, reaching 65% by 20 hours. We then sorted GFP+ T cells into a mixed leukocyte reaction, where they proliferated appropriately and where approximately 80% of treated T cells remained GFP+ while simultaneously losing tdT expression. Ultimately, this ex vivo method represents a promising approach for generating gene knockouts in T cells just prior to experimental manipulation avoiding changes that occur between deletion and harvest from donor animals.
Intravital Co-Imaging of Oxygen Tension and Immune Response in Leukemia using 2-Photon PLIM
Authors: Zal Tomasz , Rytelewski Mateusz , Harutyunyan Karine , Vinogradov Sergei , Ohtake Kazuki , Konopleva Marina
Published: January 2023

Tumor infiltrating lymphocytes (TILs) must compete with cancer cells for metabolic resources that become depleted in result of cancerous metabolism and compromised blood supply. In bone marrow, highly proliferative acute leukemias utilize oxidative phosphorylation and deplete local oxygen supply resulting in bone marrow hypoxia which contributes to therapeutic resistance. We aim to decipher the role that metabolic reprogramming of leukemic blasts plays in modulating the bone marrow microenvironment, particularly the impact on local oxygen tension, and how this affects the anti-leukemia immune response. To characterize the spatiotemporal dynamics of leukemia infiltrating T cells during disease progression, and determine the impact of oxygen tension, OxPhos, and OxPhos inhibition on T cell function inside the leukemic bone marrow, we have utilized a novel oxygen probe, PtP-C343, and intravital 2-photon phosphorescence lifetime microscopy (2p-PLIM). Our initial results demonstrate sufficient spatial and temporal resolution of the combination 2p-PLIM to simultaneously reveal intravital oxygen gradients and track lymphocytes in single cell resolution. The combination 2p-PLIM and in vivo cell tracking will enable to study the dynamic regulation of the migratory behavior of cancer-infiltrating T lymphocytes by the intratumoral oxygen tension gradients.
Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy
Authors: Yang Lili , Smith Drake J. , Lin Levina J. , Moon Heesung , Pham Alexander T. , Wang Xi , Liu Siyuan , Ji Sunjong , Rezek Valerie , Shimizu Saki , Ruiz Marlene , Lam Jennifer , Janzen Deanna M. , Memarzadeh Sanaz , Kohn Donald B. , Zack Jerome A. , Kitchen Scott G. , An Dong Sung
Published: January 2023

The humanized BLT (bone marrow-liver-thymus) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain HLA (human leukocyte antigen) haplotypes or monogene deficiencies. In order to circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ−/−) recipient mice, we were able to expand one primary BLT mouse into a colony of 4–5 proBLT (propagated BLT) mice in 6–8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to that of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2 restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable pre-clinical research tool.
IFN-λ4 induces a faster but more transient antiviral response compared to other type III interferons
Authors: Onabajo Olusegun O , Rao Nina , Obajemu Adeola A , Dilley Kari A , Sheikh Faruk , Donnelly Raymond , Shabman Reed S , Prokunina-Olsson Ludmila
Published: January 2023

IFN-λ4, a recently discovered type-III interferon (IFN), is genetically regulated, and has clinical significance because of its association with impaired clearance of hepatitis C virus (HCV) infection. Type-III IFNs play a critical role in the innate immune response to viral infections. Although IFN-λ4 induces a similar antiviral profile as other type-III IFNs, its specific contribution to the innate immune response is unclear. Delineating the functional properties of IFN-λ4 that distinguish it from other type-III IFNs may give insights into its unique role during HCV, and other infections. Here we compared antiviral responses induced by all four type-III IFNs in HepG2 cells. We show that IFN-λ4 attained 50% peak antiviral activity by 4 hours while other type-III IFNs were only at 20% of peak activity at this time. The rapid antiviral response induced by IFN-λ4 was followed by a sharp decline to less than 25% of peak activity within 24 hours, while activity of other type-III IFNs were still over 60% from peak.

By comparing antiviral activity of IFN-λ4 and IFN-λ3 against Sendai virus infection in primary human hepatocytes, we show that IFN-λ4 had stronger antiviral activity than IFN-λ3 early in the course of infection, but comparable activity at later time points. We also show that despite its poor secretion, IFN-λ4 was significantly more potent than IFN-λ3 in inducible cell lines engineered to express either protein. In summary, we show that IFN-λ4 is more active during the early stages of viral infection compared to other type-III IFNs, which maybe a clinically important window for restricting viral infection in the host, and affecting disease outcome.
Interleukin-36 cytokines as countermeasures to viral immune evasion
Authors: Jensen Liselotte E
Published: January 2023

IL-36α, IL-36β and IL-36γ engage the same receptor, IL-36R, and are related to the pro-inflammatory IL-1α, IL-1β, IL-18 and IL-33. While there is increasing evidence that the IL-36s play important roles in host immunity, the redundancy remains an enigma. Here we explore the possibility that the IL-36s have evolved to counteract viral immune evasion mechanisms. Many skin tropic viruses inhibit IL-1β and IL-18 as immune evasion prevent protein synthesis, accelerate mRNA and protein turnover, block inflammasome activation, inhibit caspase-1, trap cytokines and/or impede signaling. IL-36γ production is induced by poly(I:C); however, IL-36γ does not appear to play a significant role in the flank skin HSV-1 infection mouse model. This outcome may be due to the release of IL-36γ being dependent upon the inflammasome pathway, which is blocked by HSV-1. Similar observations were made using IL-36α KO mice. Additional data reveal that IL-1α is released in an inflammasome independent manner from HSV-1 infected keratinocytes; an event important for early recruitment of inflammatory cells to sites of HSV-1 infection in vivo. IL-36β appears to play an additional role in the skin, as IL-36β KO mice develop larger skin lesions than wildtype mice. Furthermore, the survival rate was significantly reduced. Complementary studies involving the TLR7 ligand imiquimod were performed. Imiquimod induced skin inflammation was dependent upon IL-36α, but independent of IL-36β and IL-36γ. This could suggest an important role for IL-36α in immunity against viruses that activate TLR7 yet evade immune responses targeting IL-1/IL-18 signaling, e.g., the measles virus. Our data may suggest that the IL-36 cytokines have evolved to counteract viral immune evasion.
CXCL11 production in cerebrospinal fluid distinguishes herpes simplex meningitis from herpes simplex encephalitis
Authors: Lind Liza , Studahl Marie , Berg Linn Persson , Eriksson Kristina
Published: January 2023

The closely related herpes simplex viruses 1 and 2 can cause inflammations of the central nervous system (CNS), where type 1 most often manifest as encephalitis (HSE), and type 2 as meningitis (HSM). HSE is associated with severe neurological complications, while HSM is benign in adults. We proposed that the chemokine and cytokine production in cerebrospinal fluid (CSF) and serum could indicate why two closely related viruses exhibit different severity of their accompanied CNS inflammation. Secretion patterns of 30 chemokines and 10 cytokines in CSF of adult patients with acute HSE and HSM in the initial stage of disease were analyzed and compared to control subjects without viral central nervous system infections and to levels in serum. Most measured chemokines and cytokines increased in CSF of HSE and HSM patients. Overall, the CSF chemokines levels were higher in CSF of HSM patients compared to HSE patients. However, only five chemokines reached levels in the CSF that exceeded those in serum facilitating a positive CSF-serum chemokine gradient. Of these, CXCL8, CXCL9 and CXCL10 were present at high levels both in HSE and HSM whereas CXCL11 and CCL8 were present in HSM alone. Several chemokines were also elevated in serum of HSE patients but only one in HSM patients. No chemokine in- or efflux between CSF and serum was indicated as the levels of chemokines in CSF and serum did not correlate. We show that HSM is associated with a stronger and more diverse inflammatory response in the CNS compared to HSE in the initial stage of disease. The chemokine patterns were distinguished by the exclusive local CNS production of CXCL11 and CCL8 in HSM. Inflammation in HSM appear to be restricted to the CNS whereas HSE also was associated with systemic inflammation.
Investigation of Macrophages Serving as a Viral Reservoir in Pediatric SAIDS
Authors: Merino Kristen Michelle , Sugimoto Chie , Cai Yanhui , Allers Carolina , Didier Elizabeth , Kuroda Marcelo J
Published: January 2023

In SIV-infected adult Macaca mulatta, increases in monocyte turnover indicates death of short-lived tissue macrophages and predicts SAIDS. Increased turnover also correlates with infection, not death, of long-lived tissue macrophages. Higher baseline monocyte turnover is measured in neonates, and faster disease progression. Our data shows increase in monocyte turnover and tissue macrophage infection is associated with rapid SAIDS progression in infants. We hypothesize early and high infection of macrophages results in establishment of viral reservoir in long-lived subsets, in infants not controlling viral load despite antiretroviral therapy (ART). In rhesus infants infected with SIVmac251, cell turnover is measured using BrdU uptake. Flow cytometry and confocal imaging allow phenotypic characterization of cells in blood and tissues. PCR and in situ hybridization are used to quantify SIV. Plasma cytokine analysis is performed using Milliplex bead panel. Infant macaques euthanized post-infection (no ART) are used to quantify macrophage infection. Animals euthanized after various lengths of ART provide data on reservoir seeding, viral persistence and immune activation status. ART-treated infants with discordant viral loads are evaluated to identify the source of viral replication. In treated animals, regardless of viral load, no differences are observed in activation status, absolute number, or kinetics of CD4 and CD8 T cells. Confocal imaging and viral RNA and DNA analysis of tissues and sorted cells should confirm that viral replication during ART is due to infected macrophages. Ongoing works suggest magnitude of long-lived macrophage infection may dictate effectiveness of ART in pediatric SIV infection.
The host response to murine norovirus infection induces significant engagement of IFN and TNF-a immunological programs
Authors: Levenson Eric Andrew , Martens Craig , Kanakabandi Kishore , Turner Charles , Sosnovtsev Stanislav V. , Ricklefs Stacey , Porcella Stephen , Green Kim Y.
Published: January 2023

Norovirus, a positive stranded RNA virus in the family Caliciviridae, is a major cause of acute gastroenteritis. Outbreaks occur primarily in locations such as schools, nursing homes, and cruise ships where individuals are in close proximity. An acute norovirus infection can become chronic in immunocompromised individuals, and an effective antiviral drug is needed. The 7.5 kb genome encodes a polyprotein in ORF1 that is co-translationally cleaved by the viral protease into six nonstructural proteins. The two structural proteins, VP1 and VP2, are encoded in ORF2 and ORF3, respectively. The viral proteins mediate replication and packaging of the genome into icosahedral capsids. The host cell provides additional proteins and building blocks for replication, but only a few essential host factors have been elucidated. To gain insight into the host response to norovirus infection, we performed next generation sequencing-based RNA sequencing on murine macrophages infected with murine norovirus. We obtained RNA from a time course of infection at 0, 8, 14, and 20 hours post infection with mock infections at 0 and 20 hours. Analysis of the host transcriptome reveals full activation of cellular immune response pathways, with NF-kβ, STAT1, and STAT3-based signaling evident. In addition, we observed transcriptional evidence of IRF3 activation with a transition to IRF3/IRF7 signaling. TNF-a-based activation is also clear with most downstream effectors up-regulated. These observations correlate well with the known cytokine response from patient serum samples, disease progression, and symptomatology of human norovirus. We are currently applying these findings toward drug discovery efforts.
B cell Intrinsic and Extrinsic Function of STING in the Antibody Response
Authors: Walker Melissa Marie , Crute Bergren W , Getahun Andrew , Cambier John C
Published: January 2023

Generation of protective immune responses requires coordinate stimulation of innate and adaptive immune systems. An important mediator of innate immunity is STimulator of INterferon Genes (STING), a ubiquitously expressed adaptor molecule that functions in the relay of signals initiated by sensing of cytosolic DNA and bacterial ci-di-nucleotides. Allelic variants of STING, HAQ-STING and R232H-STING, found in 15–20% of humans appear, based on in vitro studies, to be defective in signal relay, suggesting that carriers may be predisposed to certain infections. Our laboratory is engaged in studying the functions of STING, HAQ-STING and R232H-STING.

Here we show that STING promotion of in vivo IgG1 and IgA antibody responses requires both systemic and B cell expression of STING. Analyses of the possible direct effects ci-di-GMP on B cells revealed that CDNs stimulate upregulation of activation markers (CD69 and CD86) as well as apoptotic death, and these responses are dependent on STING expression. Coordinate BCR signals synergize in induction of B cell activation but partially spare cells from apoptosis.

Consistent with defective function of HAQ-STING, B cells from a newly-produced human HAQ and R231H knockin mouse were defective in mounting responses to ci-di-GMP.

Taken together, these data indicate that STING and BCR function coordinately in B cells to promote cell activation and antibody responses. Human carriers of HAQ and R231H STING may be defective in responses to certain immunogens.
Spontaneous Translocation of a Gut Pathobiont Drives Autoimmunity that is Preventable by Intramuscular Vaccination
Authors: Vieira Silvio Manfredo , Hiltensperger Michael , Kumar Varun , Dehner Carina , Zegarra-Ruiz Daniel , Ruff William , Khan Nafeesa , Goodman Andrew , Kriegel Martin A
Published: January 2023

The gut microbiota contributes to the pathogenesis of autoimmunity. The mechanisms and sites of host-microbiota interactions are, however, largely unknown. Dietary antigens reach the mesenteric lymph node (MLN) via the lymphatics and the liver via the portal vein. We hypothesized that commensals reach lymphocytes beyond the gut barrier in autoimmune-prone hosts. We first altered the gut microbiota with single antibiotics in the (NZWxBXSB)F1 model of systemic autoimmunity that is characterized by a leaky barrier based on oral gavage with FITC-dextran. To test commensal translocation, we cultured anaerobically MLN and livers. Compared to controls, vancomycin alone lowered autoantibodies, Th17 and Tfh cells, and protected mice from autoimmune deaths. Cultures of MLN and liver of (NZWxBXSB)F1 mice revealed significant anaerobic growth, which was suppressed by vancomycin. Sequencing of colonies identified E. gallinarum, a human gut commensal, that grew uniquely in tissues of autoimmune but not control mice. Species-specific PCR of liver tissues from autoimmune hepatitis patients also revealed E. gallinarum. 16S rDNA sequencing confirmed the presence of Enterococcus genus in the gut of (NZWxBXSB)F1 mice. Germ-free C57BL/6 mice monocolonized with E. gallinarum also led to translocation and induction of autoantibodies as well as Th1/Th17 responses. Remarkably, an intramuscular vaccine against E. gallinarum prevented autoimmune deaths in (NZWxBXSB)F1 mice. These data support that an innocuous gut commensal translocates spontaneously to initiate autoimmunity that is preventable by a vaccine targeted at the translocating pathobiont. This approach represents a novel therapeutic avenue for immune-mediated diseases.
Engineered immunomodulatory activity in therapeutic nucleic acids
Authors: Johnson Morgan B , Khisamutdinov Emil F , Afonin Kirill A. , Marriott Ian
Published: January 2023

Therapeutic nucleic acids (TNAs) can be engineered to deliver functional moieties such as siRNAs to target cells. An additional benefit of TNAs is that they can be complexed with amphiphilic molecules for delivery into sites such as the central nervous system (CNS) that are difficult to access due to the blood-brain barrier. However, previous TNA constructs have failed clinical trials due to the induction of severe systemic inflammatory responses. We have developed a new generation of TNAs in which immunomodulatory activity, in addition to chemical and thermodynamic stability, can be engineered. In this study, we have assessed the immunomodulatory activity of various TNAs on human CNS cell types. We have determined the ability of 16 TNAs of differing nucleic acid composition and polygon type to elicit cytokine and chemokine production by transfected human microglia and astrocyte-like cells. Our results indicate that, in general, our TNA formulations do not elicit immune responses in human astrocyte-like cells, but do induce significant release of the type I interferon, IFN-β, by cultured microglia with minimal release of the inflammatory cytokine IL-6 and the chemokine IL-8. Interestingly, we observed that the TNA nucleic acid composition and polygon type affect the immune mediator amount and profile released by human microglia-like cells. Specifically, IFN-β responses to TNAs composed of RNA are robust while those composed of DNA are minimal. Furthermore, triangle and pentagon TNA structures stimulate more IFN-β release than square and hexagon compositions. Together these results suggest these novel TNAs may be specifically engineered to minimize detrimental inflammatory responses while promoting beneficial host immunity.
Swine as a Surrogate Model for Human Brucellosis Following Oropharyngeal Vaccination with a Live Attenuated Brucella melitensis Mutant
Authors: Clapp Beata , Yang Guan , Artiaga Bianca L. , Hoffman Carol , Yang Xinghong , Driver John P , Pascual David W
Published: January 2023

Human brucellosis is a global health problem, and no human vaccines exist. Although Brucella infections primarily occur via the oropharynx and the upper respiratory tract mucosa, mucosal aspects of Brucella’s pathogenesis are often ignored. Mucosal vaccination with our live attenuated mutant was found to confer exceptional protection against Brucella challenge. While mice have been instrumental to study immunity to Brucella, they may be less suitable to evaluate mucosal infections. Domestic pigs’ close resemblance to human immune system and anatomical features of the oropharyngeal (OPG) mucosa enables studying mucosal brucellosis in a natural host. To assess our mutant’s immunogenicity, 8 wk-old pigs were vaccinated by directly applying 109 CFUs onto the tonsils and sublingual mucosa plus a buccal injection with 108 CFUs on days 0, 2, and 4, and study was terminated on day 60. Isolated peripheral blood mononuclear cells (PBMCs) on days 0, 15, 23, 35, and 60 as well as terminal head and neck lymphoid tissue lymphocytes were evaluated for IFN-g responses by flow cytometry. The greatest IFN-g came from CD8+ and CD4+ CD8+ T cells, as well as NK cells. Changes in peripheral blood T cells over the course of the response showed a significant reduction in the percentage of total CD4+ T cells, and a significant increase in the percentage of total CD8+ and CD4+ CD8+ T cells. Among the lymphoid tissues examined, with the exception of the tonsils where the IFN-g was mostly derived from CD8+ T cells, IFN-g came from CD4+CD8+ T cells, particularly in the mandibular lymph nodes. Thus, our Brucella mutant is immunogenic in swine and capable of eliciting elevated IFN-g responses following OPG vaccination. Work supported by R03 AI128123 & USDA-NIFA2013-01165.
Establishment of a Regulatory Microenvironment by Lactococcus Expressing Colonization Factor Antigen I (CFA/I) Fimbriae Ameliorates Type 1 Diabetes (T1D) in Non-Obese Diabetic (NOD) Mice
Authors: Nelson Andrew Scott , Maddaloni Massimo , Hoffman Carol , Abbott Jeffrey , Pascual David W
Published: January 2023

Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae protect in murine models of multiple sclerosis and rheumatoid arthritis. Although CFA/I fimbriae initial mode of action is in a bystander or in an antigen (Ag)-independent fashion, protection was found to be ultimately dependent upon the induction and/or activation of auto-Ag-specific regulatory T cells (Tregs). Our recent findings show that oral dosing with a L. lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45% with a concomitant 8-fold increase in their splenic Foxp3+CD25+ Tregs. However, little is known about how protection transitions from bystander suppression to Ag-specific Tregs. We hypothesized that LL-CFA/I stimulates dendritic cells (DCs) to establish a regulatory microenvironment since DCs play an integral role in fate decisions for T cells becoming inflammatory or tolerogenic. In this study, 4 wk-old NOD mice were orally dosed with LL-CFA/I and treated every 2 wks; control groups were given L. lactis vector or PBS. At 11 wks of age, frequency of insulitis was reduced by more than half, and insulin-specific T cells were reduced in the pancreatic lymph nodes (PaLNs; P < 0.05). To discern how this was mediated, groups of mice were examined at 3, 7, 14, and 21 days post-treatment for changes in DC and T cell phenotypes. As early as 3 days post-treatment, DCs exhibited significantly less expression of costimulatory molecules, CD40 and CD86, and stably maintained this phenotype for at least 7 wks. Splenic DCs showed reduced IL-6 production. These data show that orally dosing with LL-CFA/I ameliorates T1D in NOD mice by establishing a regulatory microenvironment via DCs. Work is supported by AI121745.
IL-6 producing intraepithelial lymphocytes in the colon modulate epithelial barrier integrity to protect against C. rodentium infection
Authors: Kuhn Kristi , Schulz Hanna M. , Whitney Alyssa K. , Campbell Eric L , Colgan Sean P.
Published: January 2023

Citrobacter rodentium infection of mice is frequently used as a model to study host responses during pathogenic gastrointestinal infection. IL-6 signaling in the colon is required for protection against C. rodentium infection; however the cellular source of IL-6 for this protection is unclear. Our previous work has identified IELs as one source of IL-6 in the colon. Therefore, we hypothesized that IL-6 produced by colonic IELs was sufficient to provide protection during C. rodentium infection. To test our hypothesis, we orally infected mice with C. rodentium following transfer of donor IL-6+/+ or IL-6−/− IELs into IL-6+/+ or IL-6−/− recipients. IL-6−/− mice that received IL-6−/− IELs had significantly more weight loss, increased intestinal histopathology, and increased bacterial translocation after 12 days of infection compared to transfer of IL-6+/+ IELs into recipient IL-6−/− mice and IL-6+/+ IELs into IL-6+/+ mice. IEL-derived IL-6 is functionally important in the maintenance of the epithelial barrier as IL-6−/− mice were noted to have increased paracellular permeability, decreased claudin-1 expression, and a thinner mucus-gel layer, all of which were reversed by transfer of IL-6+/+ IELs. In vitro studies utilizing model epithelia confirmed IL-6 was able to signal and increase claudin-1 and mucin-2 expression. Therefore, we conclude that IL-6 expression by IELs is sufficient to restore protection against C. rodentium infection through modulation of the epithelial barrier integrity.
Interleukin-33 promotes REG3γ expression in intestinal epithelial cells via activation of mTOR, STAT3 and ERK1/2
Authors: Xiao Yi , Chen Feidi , Zhao Ye , Sun Mingming , Huang Xiangsheng , Chen Liang , Yao Suxia , Liang Yuejin , Sun Jiaren , Yan Fang , Cong Yingzi
Published: January 2023

REG3γ, an antimicrobial peptides typically expressed by intestinal epithelial cells (IEC), plays crucial roles in maintaining intestinal mucosal barrier functions against microbiota and potentially pathogenic microorganisms in the intestines. However, the mechanisms by which regulate IEC expression of REG3γ are still not fully understood. IL-33, a member of the IL-1 cytokine family, has been implicated in maintenance of intestinal homeostasis. In this study, we investigated whether and how IL-33 regulates IEC expression of REG3γ, thus contributes to the maintenance of intestinal homeostasis. We report here that the expression of REG3γ in IEC of IL-33−/− mice is significantly lower than that in the wild type (WT) mice at both mRNA and protein levels. Treatment with IL-33 induced IEC expression of REG3γ in mouse MSIE intestinal epithelial cell line. Consistently, IL-33 also induced expression of REG3α, the human ortholog of mouse REG3γ, in human epithelial cells. Mechanistically, rIL-33 activated phosphorylation of STAT3, mTOR and ERK1/2 in MSIE cells. Knockdown of STAT3, mTOR and ERK-activating enzyme MEK by using siRNA or CRISPR abrogated IL-33 induction of REG3γ expression in IEC, indicating that IL-33 promoted IEC expression of REG3γ though activating STAT3, mTOR and ERK1/2 pathways. REG3γ has been shown to be able kill gram-positive gut bacteria. Interestingly, total gut bacteria were significantly lower in IL-33−/− mice compared to WT mice, indicating that IL-33 regulation of gut microbiota, possibly through induction of IEC REG3γ. Put all together, our study demonstrated a novel crosstalk between IL-33 and REG3γ in maintenance of intestinal homeostasis.
Antimicrobial Cytotoxic Immune Proteins in Human Milk
Authors: Filgueira Luis , Kueffer Gwendoline , Twigger Alecia-Jane , Kharoubi Solange , Walch Michael , Geddes Donna T
Published: January 2023

Human milk (HM) contains a large variety of immune components providing protection to the infant. The presence of the cytotoxic immune proteins perforin (Per), granulysin (Grly)and granzymes (Grzm)has not yet been shown in HM cells.

Per, Grnly and Grzm (A, B, H and M) were examined in cells isolated from samples of prepartum HM (PS) collected during pregnancy and HM collected longitudinally (2–5 sampling occasions) from mothers (n=31) during the first year postpartum, including 3 samples from breast infections. Gene expression was analyzed in RNA sequencing data and using qRT-PCR. Linear modeling and principle component analysis between the genes was conducted. Flow cytometry (n=5) was performed to investigate the presence of these immune proteins in HM cells.

Gene expression of all immune proteins was confirmed in PS and in HM during month 1 of lactation, where variation of expression was found between women and time lactation. Strong correlations were found between GrzmA – Per (r2=0.86), GrzmA – CD45 (r2=0.76), Per – CD45 (r2=0.68) and Per – GrzmB (r2=0.67). Comparison between healthy and mastitis samples showed a higher expression of CD45 and all immune peptides in mastitis. Moreover, flow cytometry analysis showed an increase in CD45 positive cells in mastitis in comparison with healthy participants (15.1% and 4.7% respectively of total cell amount).

The presence of Per, Grly and Grzm has been confirmed in HM cells. An increase in gene expression of these immune proteins has been confirmed in 3 participants suffering from breast infection. Further investigations are required to elucidate the roles of these immune peptides for the infant and/or mother
IL-10 Can Enhance FcγR-mediated Mast Cell Activation
Authors: Haque Tamara , Ryan John J
Published: January 2023

Mast cells are involved in many inflammatory diseases, partly by their production of cytokines that promote inflammation. IgG signaling through FcγRIII/IIb in mast cells is linked to autoimmune disease such as rheumatoid arthritis, but factors controlling this signaling remain unclear. Our group, as well as others, has previously shown that IL-10 suppresses mast cell IgE signaling, consistent with known anti-inflammatory effects of IL-10. However, we recently found that in IL-10 has unexpected pro-inflammatory effects on IgE-mediated mast cell functions. Therefore, we designed a study to determine the effects of IL-10 on FcγR signaling. Our data show that IL-10 enhances IgG-mediated IL-6 and MCP-1 production in bone marrow derived mast cells. Interestingly, we also found that IgG-induced cytokine production is suppressed by IL-10 in bone marrow derived macrophages under the same conditions. Current work is examining this different response to IL-10 to have a better understanding of the overall affect of IL-10 in the inflamed microenvironment. These studies suggest that mast cell and macrophage responses are altered by cytokines in their microenvironment, with distinct and opposite effects.
Partial protection from lupus-like disease in B-cell specific IFNAR-deficient B6.Nba2 mice
Authors: Keller Emma J , Patt Madeline M , Nemunaitis Teddy , Reth Michael , Sen Ganes C , Jorgensen Trine N
Published: January 2023

A majority of patients presenting with systemic lupus erythematosus (SLE) have increased serum concentrations of interferon alpha (IFNα). IFNα has previously been shown to affect lymphocyte activities such as B cell differentiation and T cell maintenance, but whether IFNα signaling specifically in B cells is required for disease pathology remains unknown. In order to examine the role of IFNα stimulation on B cells, we backcrossed mice lacking the IFNα receptor on B cells, Mb1.cre IFNARflx/flx, with the B6.Nba2 lupus mouse model. B6.Nba2.BΔIFNAR mice were evaluated alongside littermate control mice for splenomegaly, abnormal spleen subset distribution, IgG immune-complex deposition in the kidney glomeruli. Serum total IgM, total IgG and anti-chromatin IgG levels were determined monthly. BΔIFNAR mice display normal IgM and IgG levels, but decreased serum antinuclear autoantibody levels (p<0.05), while deposition of IgG immune complexes in kidney glomeruli was similar to that of control mice. Both spleen weight and total splenocyte number were found to be significantly decreased in BΔIFNAR mice (p<0.05). Analyses of spleen cell subsets at 4 months of age revealed a trend towards decreased plasma cells and GC B cells in spleens of BΔIFNAR mice. Despite IFNAR being removed from B cells only, we observed a shift in the balance of effector/memory to naïve CD4 T cells in BΔIFNAR mice. In summary, we found that B6.Nba2.BΔIFNAR mice were partly protected from lupus pathology including splenomegaly, autoantibody production and B cell hyperactivation. Most surprisingly, T cell activation appeared to be diminished in B6.Nba2.BΔIFNAR mice, suggesting that B cells play a central role as both APCs and ASCs in the B6.Nba2 mouse model of lupus.
RELT binds HIC and induces apoptosis by a mechanism distinct from TNFR1.
Authors: Cusick John K , Moua Pachai , Talbott George , Sumida Cara , Jacobs Aaron
Published: January 2023

Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) that is expressed most prominently in cells and tissues of the hematopoietic system. RELT has two identified homologous binding partners RELL1 and RELL2. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. Human I-mfa domain-containing protein (HIC), also known as MDFIC, was identified in a yeast two-hybrid genetic screen using RELL1 as bait. HIC is a transcription factor that was initially identified to differentially regulate HTLV and HIV gene expression. HIC is now known to regulate many other cellular processes and the hic gene is located proximally to regions of chromosome 7 (7q31.1) frequently lost in Acute Myeloid Leukemia (AML) patients. HIC physically interacts with both RELT and RELL1, as determined by in vitro co-immunoprecipitations. HIC co-localizes with RELL1 at the plasma membrane and co-localizes with RELT in intracellular compartments. Deletion mutants of RELT were utilized to determine regions of RELT required for both the activation of p38 and the induction of apoptosis in HEK-293 cells, two previously described functions of RELT. Co-immunoprecipitation experiments utilizing the deletion mutants indicate that regions of RELT proximal to the plasma membrane are sufficient for physical interaction with HIC. Overexpression of RELT induces cleavage of PARP and Caspase-3 as determined by western blotting. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented if signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from death-domain containing TNFRs such as TNFR1.
Evolution of interleukin-1 receptor-like 1 and its role in rainbow trout (Oncorhynchus mykiss) resistance to Flavobacterium psychrophilum
Authors: Shaw Cassidy H. , Palti Yniv , Gao Guangtu , Wiens Gregory D
Published: January 2023

Rainbow trout exhibit extensive phenotypic variation in innate disease resistance and we have divergently selected lines with either increased or reduced survival following exposure to the gram-negative bacterium, Flavobacterium psychrophilum (Fp). Following five generations of selection, gene expression was analyzed between the resistant and susceptible lines by RNA-seq and RT-PCR in either whole body or spleen tissue samples. Lower base-line expression of interleukin-1 receptor-like 1 (il1rl1) was identified in susceptible line fish as well as decreased expression in response to infection. Genomic analyses identified three tandem, il1rl1 genes on chromosome 3 located within a previously identified, bacterial cold water disease QTL. Here, we report the genomic organization and evolution of the three il1rl1 genes, and develop gene specific assays to measure the expression of the individual il1rl1 genes in susceptible and resistant rainbow trout lines. Sequence homology between these three putative genes is approximately 95%. Each gene contains a signal peptide, three IG/IG-like domains, a transmembrane region and a TIR domain. Two il1rl1 genes and one pseudogene are present in Atlantic salmon (Salmo salar) genome with 90–94% sequence identity. Phylogenetic analyses suggest that the salmonid il1rl1 genes expanded by tandem duplication within the salmonid linage. Our working model is that il1rl1 gene copy number or differential expression may contribute to specific disease resistance.
Irgm1 suppresses NLRP3 Inflammasome-mediated IL-1β production
Authors: pei chunying , wang chun , xu hongwei
Published: January 2023

Immune-related GTPase M (Irgm1) plays a crucial role in resolving immune responses during infection. The molecular mechanism underlying the immunoregulatory function of Irgm1 is not fully understood. NLRP3 inflammasome triggered by variety of stimulus is actively involved in immune responses during both physiological and pathological conditions. In the current study, we investigate the potential role of Irgm1 in regulating NLRP3-Caspase-1-IL-1β signal cascades. To test that, bone marrow derived-macrophage and peritoneal macrophage from either Irgm1-deficient (Irgm1−/−) or wild-type (WT) mice were challenged with bacterial LPS and ATP. Secretion of IL-1β was measured by ELISA. NLRP3 Inflammasome activity was measured by ASC oligomerization, caspase-1 activity. While the expression of pro-IL-1β is not different between Irgm1-deficient (Irgm1−/−) or wild-type (WT), mature IL-1β secretion by Irgm−/− macrophage is strongly up-regulated upon stimulation as compared to macrophage from WT mouse. Furthermore, we found that the expression NLRP3, ASC and cleaved caspase-1 is also increased in the Irgm−/− macrophage. In keeping with that, ASC oligmerization as well as caspase-1 activity is significantly enhanced in the macrophage from Irgm1-deficient mice. We blocked NLRP3 inflammasome activity by using specific inhibitors against NLRP3 (MCC950) and caspase-1 (VX765). Interestingly, the addition of NLRP3 inflammasome inhibitors normalized the up-regulation of IL-1β observed in Irgm−/− macrophage. Taken together, these data suggested that Irgm1 negatively regulates NLRP3 inflammasome, which may be involved in the resolution of immune responses.
A novel method for the detection of IFN-lambda 3 binding to cells to quantify IFN-lambda receptor expression on epithelial and immune cell subsets
Authors: Santer Deanna , Minty Gillian , Mohamed Adil , Baldwin Lesley , Bhat Rakesh , Joyce Michael , Egli Adrian , Tyrrell D. Lorne , Houghton Michael
Published: January 2023

Type III interferons (IFN-lambdas) are important antiviral cytokines that also modulate immune responses by acting through a unique IFN-λR1/IL-10R2 heterodimeric receptor. Conflicting data has been reported for which human cells express the IFN-λR1 subunit and directly respond to IFN-λ. Since the commercially available anti-IFN-λR1 flow cytometry antibodies we tried were suboptimal, we developed a novel method to measure IFN-λ3 binding to IFN-λR1/IL-10R2 on the surface of cells via flow cytometry. We found that Huh7.5 hepatoma cells bound IFN-λ3 to the greatest extent with the lowest Kd(app) (83.2nM), and had the corresponding highest induction of IFN stimulated genes (ISGs). Raji and Jurkat cell lines, representing B and T cells, respectively, moderately bound IFN-λ3 and had lower ISG responses. U937 cells, representing monocytes, did not bind IFN-λ3 and therefore, did not have detectable ISG induction. We confirmed that IFN-λ3 was bound to the surface of cells through imaging flow cytometry. Importantly, lentivirus shRNA knockdown of IFNLR1 in Huh7.5 cells decreased our binding signal proportionally and reduced ISG induction by up to 93%. IFN-λ3 responsiveness increased over time with maximal ISG responses seen at 24 hrs for all but one gene. We next applied our assay to human peripheral blood mononuclear cells and saw that only specific immune cell subsets bound IFN-λ3 (eg. plasmacytoid dendritic cells and B cells). These data confirm our new IFN-λ3 binding assay can be used to quantify where the IFN-λ receptor is expressed and reflects IFN-λ3 responsiveness. Knowing which cells express the IFN-λ receptor will be crucial for determining how IFN-λ3 modulates the adaptive immune response.
Recombinant Fasciola hepatica fatty acid binding protein down regulates the expression of inflammatory cytokines from macrophages induced by LPS in vitro
Authors: Ramos-Benítez Marcos Javier , Espino Ana M.
Published: January 2023

Gram-negative sepsis develops as a consequence of an uncontrolled bacterial infection that constantly release LPS into the bloodstream triggering an unrestrained systemic inflammatory response that leads to multi-organ failure and death. Molecules that can neutralize inflammation induced by LPS are highly pursued by the scientific community. Our research group revealed that a 12kDa F. hepatica FABP (Fh12) exhibit potent anti-inflammatory properties and prevented the septic shock development in mice. Recently, we optimized the expression and purification of a recombinant variant of Fh12, termed Fh15 and demonstrated that both molecules possess immunological identity and similar capacity to inhibit the NF-κB activation induced by LPS in THP1-CD14 cells. In the current work we investigated whether Fh15 can suppress the expression of inflammatory cytokines from murine bone marrow-derived macrophages (BMDM) and also examined whether the integrity of the secondary structure is determinant for the anti-inflammatory role of this molecule. qRT-PCR results demonstrated that Fh15 significantly suppressed the expression of TNF-α and IL-1β cytokines (p<0.0041) from BMDM in response to LPS. Moreover, Fh15 significantly repressed the expression of CD14 co-receptor (p<0.0145), reinforcing our notion that one of mechanisms used by Fh15 to suppress the inflammatory response is by targeting this co-receptor. Additionally, anti-Fh12 antibodies does not inhibit the capacity of Fh15 to block the NF-κB activation in THP1-CD14 cells. After a thermal treatment, Fh15 capacity to suppress NF-κB activation was not reduced. Circular dichroism analysis confirmed that the secondary structure of Fh15 was not altered by thermal denaturation.
Metformin Suppresses LPS-induced CXCL8 Expression
Authors: Xiao Zhihui , Wu Wenjun , Poltoratsky Vladimir
Published: January 2023

Human chemokine CXCL8 plays an important role in cancer development by promoting tumor growth, angiogenesis, and tumor cell migration. It also recruits monocytes that, after differentiation into tumor associated macrophages, further increase tumor progression. Anti-diabetic therapy with Metformin reduces cancer incidence and mortality and has a potential application in anti-cancer therapies. In this study we tested the effects of metformin on LPS-induced CXCL8 expression, cell proliferation, and migration. We used the modified HEK293 cell line which express the Toll like receptor 4 signaling pathway receptor complex, but lack expression of CXCL8 receptors.

We demonstrated that Metformin significantly suppresses the LPS-induced CXCL8 expression in a dose-dependent manner. Metformin reduced LPS-induced CXCL8 activation on a transcriptional level through the suppression of NF-κB, but not AP-1 or C/EBP transcription factor activities. Immunofluorescent analysis revealed that Metformin suppresses the NF-κB translocation into the nucleus. In addition, Metformin suppresses LPS-independent HEK293/TLR4 cell migration, but not cell proliferation. In conclusion, our data suggested that metformin suppresses CXCL8 expression via inhibiting NF-κB activities, and inhibits HEK293/TLR4 cell migration without affecting cell proliferation.
Local host level of cytokines and gene expression are associated with successful healing of combat-related extremity wounds with persistent critical colonization
Authors: Lisboa Felipe A. , Bradley Matthew J. , Forsberg Jonathan A. , Potter Benjamin K , Muñoz Beau J. , Hueman Matthew T. , Elster Eric A.
Published: January 2023

Persistent critical colonization (CC) may lead to wound healing failure (WF) despite antibiotic treatment. We investigated the local host gene expression and cytokine response of critically colonized wounds to identify early signs of WF.

Seventy-three patients with combat-related extremity wounds requiring serial operative debridements were assessed to determine whether there was an association between CC and WF. A total of 116wounds were identified. A subset of 44% (51/116) of these wounds developed CC (considered as growth of 105 bacterial CFU/g of tissue or μL of wound effluent). Samples were collected at each debridement and tested for 32 cytokines and 192 genes associated with wound healing. A two-tailed alpha < 0.05 was considered statistically significant.

Wounds closed with CC had increased odds of failing to heal OR=3.3 (1.225 to 8.9). A. baumannii was the most commonly isolated species. A subset of 12 out of 51 (24%) wounds which developed CC, failed. Of those, 10 had CC present on day of wound closure. However, 10 additional wounds with CC present at time of closure healed successfully after a similar number of debridements. Comparing these wounds, WF was associated with a decrease in the level of EGF (p<0.05) and lower gene expression of IFNB1 (p=0.01), FGF-8 (p=0.01) and HMGB1 (p=0.01). Successful healing was associated with an increase in the level of VEGF (p<0.01).

Resolution of persistent CC before closure may prevent failure. Local host gene expression and cytokine response may provide additional insight for successful closure. Further studies investigating microbiology data and possible antibiotic resistant genes are needed.
IL-22-deficiency mediates resolution of colitis in Il10
−/− mice
Authors: Gunasekera Dilini C , Ramakrishnan Amritha , Ma Jinxia , Uprety Priyanka , Vacharathit Vimvara , Sheh Alexander , Whary Mark T. , Fox James G. , Bream Jay H.
Published: January 2023

The cytokines IL-10 and IL-22 play important, but presumably distinct roles in promoting intestinal homeostasis. Although the anti-inflammatory actions of IL-10 in the gut are well established, the role(s) of IL-22 remain unclear. Il10−/− mice develop chronic colitis characterized by rectal prolapse, colonic thickening and inflammatory infiltrates, including abundant Th17 cells. We have also observed a modest, but consistent elevation in IL-22 (mRNA and IL-22+CD4 T cells) in colons of Il10−/− mice. This is accompanied by an increased expression of IL-22-target bactericidal genes such as Reg3g, s100a8, and s100a9 and reduced intestinal microbial diversity in Il10−/− mice. To define a potential role for IL-22 in spontaneous colitis, we generated Il10−/− Il22−/− mice by backcrossing. Unexpectedly, concurrent IL-22 deficiency prevented colitis in Il10−/− mice, normalizing histopathology inflammatory scores and restoring microbial diversity to WT levels in Il10−/− Il22−/− mice, despite endemic Helicobacter spp. infection, an established risk factor for colitis in Il10−/− mice. Interestingly, prevention of disease was not accompanied by a reduction in Th17 cells. Importantly, introduction of recombinant IL-22 into the GI tracts of both Il10−/− Il22−/− and WT mice resulted in colonic shortening and thickening after only 2 weeks of treatment, suggesting that IL-22 is at least partially responsible for the immunopathology observed in Il10−/− mice. Thus, although beneficial during certain bacterial infections and in response to acute tissue damage, our data suggest that left unchecked, IL-22 expression can contribute to chronic colitis and appears to be negatively regulated by IL-10.