bluuebunny/crossref_metadata_2025 huggingface dataset:
Androgen deprivation therapy allows for effective anti-TIGIT immunotherapy in murine model of castration resistant prostate cancer.
Authors: Polesso Fanny , Guan Xiangnan , Xia Zheng , Moran Amy E
Published: January 2023

Prostate cancer is the second leading cause of cancer associated mortality in men, and for reasons that remain unclear, immunotherapy treatments have largely failed in patients with advanced prostate cancer. Standard of care therapy for these patients relies on androgen deprivation therapies (ADT) to block testosterone production combined with small molecules that inhibit androgen synthesis and/or the function of the androgen receptor (AR). Importantly, androgens are reported to be immunosuppressive; therefore, we postulated that androgen axis blockade might promote response to immunotherapy. In the recent years, the co-inhibitory receptor TIGIT has emerged as a promising immunotherapy target, and we observed that TIGIT is highly expressed on tumor infiltrating T cells and NK cells in metastatic castration resistant prostate cancer (CRPC) patients treated with the AR antagonist enzalutamide. Likewise, in a mouse model of CRPC, we show that tumor infiltrating T cells express TIGIT and that treatment with enzalutamide increases its expression. In this model, neither treatment with anti-TIGIT alone nor with chemical castration and enzalutamide affects tumor growth. Remarkably, the combination of anti-TIGIT with chemical castration and enzalutamide leads to delayed tumor growth and long-term cures in about 60% of the animals. Taken together, our data suggest that androgen receptor blockade sensitizes immune cells in the tumor to respond to anti-TIGIT immunotherapy.

This work was largely supported by an OHSU Foundation grant and a sponsored research agreement with AstraZeneca to A.E.M. Additional funding was provided by Collins Medical Trust and Prostate Cancer Foundation.
Opposite effects of CXCR1 and CXCR2 overexpression in prostate tumorigenesis
Authors: Richardson Micheler , Adekoya Timothy , Smith Nikia , Kothari Parag
Published: January 2023

Chemokines and their receptors play important role in tumor progression and metastasis. In prostate cancer (PCa), some members of the CXC chemokine receptor have been shown to enhance angiogenesis, proliferation and metastasis. In this study we assess the roles of the interleukin-8 (IL-8/CXCL8) receptors CXCR1 and CXCR2 in prostate tumorigenesis, using MDA PCa 2b and LNCaP cell lines. Our results show that overexpression of CXCR2 enhanced in-vitro cell proliferation, soft agar growth and in-vivo tumor xenograft in nude mice, whereas overexpression of CXCR1 inhibited cell proliferation and tumor growth, relative to control cells. CXCR1 cells showed decrease prostate specific antigen (PSA) expression whereas CXCR2 exhibited decrease androgen receptor (AR) expression. Interestingly, tumor lysates from CXCR1 cells displayed significant increase in ERK and AKT activation as well as chemokines production relative to control and CXCR2 tumors. CXCR1 tumors also exhibited a more mesenchymal phenotype, characterized by reduced E-Cadherin but increased N-Cadherin and Vimentin expression relative to CXCR2 or control tumors. Altogether, these results indicate that CXCR1 and CXCR2 may couple to distinct pathways to modulate prostate tumorigenesis.

Supported by CA156735, MD012392
Single-cell myeloid diversity in human fallopian tube and its implications for early high grade serous ovarian cancer
Authors: Brand Joshua , Haro Marcela , Abbasi Forough , Lin Xianzhi , Siedhoff Matthew , Li Andrew , Rimel Bobbie , Medeiros Fabiola , Lawrenson Kate , Dinh Huy
Published: January 2023

The 5-year survival rate of high grade serous ovarian cancer (HGSOC) has remained below 40% for decades. This rises significantly to 90% when detected early. Increasing evidence suggests that HGSOC tumors originate in the fallopian tube (FT). However, little is known about FT’s immune cell heterogeneity and how it contributes to HGSOC development. Here we show a single-cell transcriptome analysis of non-malignant FT samples, revealing high monocyte diversity that is lost in HGSOC. FT monocyte subsets display distinct inflammatory gene signatures such as IL1A, CCL3/4 and others. Trajectory inference showed potential monocyte maturation steps that started with early cytokine/chemokine secreting monocytes that gain MHCII expression and develop into macrophages or dendritic cells. We found the macrophage-to-monocyte ratio is remarkably elevated in tumors compared to adjacent normal ovary and non-cancerous FT tissues in ours and publicly available scRNA-Seq. Intriguingly, analysis of FT of patients with BRCA1/2 germline mutations revealed half of these high-risk individuals are more like those found in HGSOC tumors than in normal FT. They also showed distinct T cell phenotypes in their FTs. Our data suggested that myeloid diversity, and their interactions with T cells in the FT, might contribute to early HGSOC development. Understanding tissue-of-origins immune microenvironment has a potential contribution to early cancer detection. We anticipate this work provides evidence of how myeloid cell plasticity induces tumorigenesis and can shape different tumor microenvironments.

Supported by startup grants from UW-Madison School of Medicine and Public Health and UW-Madison Cellular and Molecular Pathology Graduate Training Program travel awards
Complement Factor H Immunoproteins Increase Complement-mediated Opsonophagocytic Killing of Methicillin-resistant Staphylococcus aureus
Authors: Sharp Julia A , Cranmer Katelyn A , Sage Megan G , Wycoff Keith L
Published: January 2023

Background

As a major cause of a myriad of superficial and invasive infections worldwide, Staphylococcus aureus is a master of immune evasion, with the complement system being a primary target. Previously, we have shown that S. aureus surface protein SdrE binds the regulator Factor H (FH) to inhibit complement. Taking advantage of this interaction, we tested the effect of a fusion protein comprised of FH(18–20) fused to an IgG Fc on complement-mediated opsonophagocytosis and killing of methicillin resistant S. aureus.

Methods

Fusions were produced via the ΔXT/FT Nicotiana benthamiana plant expression system. Binding dynamics were examined via immunoblotting, fluorescence and ELISA. The effect of fusions on complement activation was determined by examining C3-fragment opsonization of S. aureus (total C3, C3b, iC3b) and subsequent C5a generation. The effect of fusions on S. aureus survival was assessed using an opsonphagocytosis assay with human polymorphonuclear cells.

Results

S. aureus bound significantly more FH-Fc compared to Fc-controls, and competed with serum FH for S. aureus binding. FH-Fc outperformed a complement inactive fusion variant in C3-fragment deposition and C5a generation, signifying an active role for the Fc region of FH-Fc. For 75% (3/4) of clinical isolates tested, FH-Fc significantly reduced S. aureus survival. Clinical isolates were all sdrE positive, sequence type 8, SCCmec IVa and Panton-Valentine Leukocidin (pvl) positive).

Conclusion

FH-Fc competitively inhibited serum FH from S. aureus binding and increased complement-mediated opsonophagocytosis and killing of CA-MRSA. Future studies will focus on enhancing the efficacy of FH-Fc fusion molecules as potential anti-staphylococcal therapeutics.

Supported by a grant from the US Department of Defense, USAMRDC (W81XWH-19-1-0119).
Evaluation of the protective efficacy of a novel rVCG-based Chlamydia abortus vaccine in a pregnant mouse model
Authors: Richardson Shakyra S , Bell Courtnee , Jones Leandra , Medhavi F N U , Tanner Tayhlor , Lundy Stephanie , Omosun Yusuf , Eko Francis O
Published: January 2023

Chlamydia abortus (Cab) is the cause of ovine enzootic abortion (OEA) in small ruminants and poses a zoonotic risk to pregnant women, which may result in spontaneous abortion. Although antibiotics are effective against Cab infection, most infections are asymptomatic, suggesting alternative strategies may be required for controlling these infections. We tested the ability of a recombinant Vibrio cholerae ghost (rVCG)-based Cab subunit vaccine expressing the truncated Cab polymorphic membrane protein 18D (rVCG-Pmp18.3) to protect against Cab-induced abortion in a pregnant mouse model. Thus, groups of female C57bl/6J mice were immunized intranasally (IN) with rVCG-Pmp18.3 and mated with males 3 weeks post immunization. Immunized and control mice were infected IN or transcervically (TC) at day 10 of pregnancy and the number of pups per mouse was enumerated 7 days postpartum. Mice were sacrificed on day 8 and Cab burden in the lungs and spleens was determined. The results showed IN immunization protected mice against abortion and neonatal death while IN infected control mice lost 20% of their litter and 60% of TC infected controls lost their entire litter 7 days post-parturition. Immunized mice completely cleared the infection in the lungs and showed a significant reduction (p ≤0.0001) in bacterial burden in the spleen compared to non-immunized, infected controls. Furthermore, immunized mice were protected from uterine dilation compared to 60% of non-immunized controls that developed tubal pathology. These results demonstrate the ability of rVCG-Pmp18.3 to prevent respiratory/systemic infection and protect against Cab-induced abortion and tubal pathologies in mice, suggesting its potential development as a vaccine for ruminants.

Supported by PHS grant R01AI41231 and NIH R01AI26897
Assessing how biological sex effects tissue-resident memory T cell responses to influenza infection
Authors: Bachnak Louay , Godwin Matthew , McLachlan James B
Published: January 2023

The severity and outcome of influenza virus infection are dependent on various host factors including sex. During the 1918 influenza pandemic, males succumbed to viral infections more than females. Following seasonal influenza vaccination, haemagglutination inhibition titers are significantly higher in females than males. Additionally, the Tlr7 gene that recognizes influenza viruses is located on the X chromosome, resulting in higher expression in females. It is not known whether sex influences non-lymphoid tissue residency of lymphocytes during influenza infection. To explore this, we infected male and female mice intratracheally with 20 PFU influenza virus (A/PR8/H1N1). No significant change was noted in weight loss; however, females displayed a higher survival rate and milder clinical disease nine days post-infection. Lungs of female mice contained greater numbers of influenza-specific tissue-resident CD4+ T-cells compared to males. To assess the role of extra-lymphoid tissues in tissue residency in both sexes, we infected male and female lymphotoxin-a knockout (LTa KO) splenectomized mice that resulted in a total lack of all lymphoid tissues. Physiological differences trended the same for both males and females, with no significant weight difference; however, LTa KO females generated more tissue-resident CD4+ T-cells, while males displayed greater numbers of tissue-resident CD8+ T-cells. Overall, females generated a stronger immune response in the absence of lymphoid tissues in response to the virus. Taken together, our data reveal a novel sex-based effect of viral infection on tissue-resident lymphocytes and non-lymphoid tissue immunity.

Supported by a grant from the W.M. Keck Foundation
CD4+ CD151+ T cells may serve as preferential reservoir cells of latent HIV infection events.
Authors: Tidwell Christopher M , Perez Mildred , Zhang Mingce , Duverger Alexandra , Wagner Frederic , Cron Randy Q , Sabbaj Steffanie , Kutsch Olaf
Published: January 2023

We have demonstrated that the tetraspanin CD151 marks a hyperactivated, pro-inflammatory human CD4+ T cell population. Expansion of this population in HIV patients on ART may explain the associated risk of inflammation-related co-morbidities. However, CD151+ CD4 T cells can also proliferate independent of cognate antigen recognition, driven by just IL-2, a feature that is reminiscent of latently infected T cells which have been reported to maintain the latent reservoir size by homeostatic proliferation mechanisms. The objective of this study was to test our hypothesis that the CD4+CD151+ T cell population is more likely to host latent HIV infection events. First, we noted that all latently HIV-1 infected T cell clones we investigated had upregulated CD151 expression. Using samples from PLWH on ART (n=5) with undetectable viral loads and CD4 count >500 cells/mm3 we enriched CD4+CD151+ and CD4+CD151− T cell populations. Using a nested ALU/GAG qPCR assay to determine the level of proviral HIV-1 integration events, we found a 3.6-fold increase in integration events in the enriched CD4+CD151+ T cell population. Given the demonstrated correlation of proviral DNA frequencies and latent reservoir size, this would suggest that CD151+ T cells disproportionately harbor latent HIV infection, and that CD151+ T cells could be used as a surrogate population to study pertinent features of latently HIV-1 infected T cells. We here begin to address the conundrum of how T cells with a hyperactivated phenotype can host latent HIV-1 infection events and dissect the influence of CD151 expression on latency establishment and reactivation. Characterization of this T cell compartment may reveal new molecular targets to efficiently trigger HIV-1 reactivation.

Supported by grants from NIH: NIH R01-AI122842 NIH R33-AI116188 NIH R33-AI133679
The characterization of MCMV specific Qa-1-restricted CD8+ T cells
Authors: Reilly Shanelle , Anderson Courtney , Brossay Laurent
Published: January 2023

While MHC class Ia-restricted classical CD8+ T cells have been widely shown to participate in the immune response to viral infection, little is known about the MHC class Ib-restricted non-classical CD8+ T cell response. Our laboratory recently demonstrated that during murine cytomegalovirus (MCMV), a well-established model system for human CMV, adoptively transferred non-classical CD8+ T cells are sufficient to protect Kb Db RAG1−/− mice from lethality. We sought to further characterize this protective population, which is largely Qa-1-restricted. Using Kb Db−/− and Kb Db−/−. SJL mice, we are able to track the same original population of T cells through iterative adoptive transfers in multiple hosts. Following MCMV infection of new hosts, these T cells continue to play an active role in viral protection and exhibit both effector and memory phenotypes. Strikingly, following multiple challenges, an enriched clonal CD8+ T cell response is observed. While certain strains of human CMV encode a mimic of the canonical peptide binding partner for Qa-1, MCMV does not have a similar mechanism. Rather, our data show that these T cells are stimulated by an MCMV-derived antigen that is recognized by CD8+ T cells with limited αβ TCR diversity. The population we describe may act as a compensatory immune mechanism when MHC class Ia is targeted by CMV immunoevasins. Taken together, these data demonstrate that an MCMV-derived peptide is presented by Qa-1, necessary for an MCMV-specific activation of non-classical CD8+ T cells, and may be of importance for viral protection when classical CD8+ T cell immunity is compromised.

This work is supported by NIH Research Grants AI046709 and AI12221, and was supported by a T32 Training Grant in Molecular and Cell Biology and Biochemistry in years 2019-2020. Shanelle Reilly is also supported by research supplement R01 3AI046709-18S1 to promote diversity.
GC Tfh Cell Metabolism and Survival of HIV-1 Infection
Authors: Syed Fahim , Li Wei , Yu Qigui
Published: January 2023

CD4 T cells are the primary targets for HIV-1 infection and their loss is a hallmark of HIV-1 disease. However, T follicular helper (Tfh) cells, a distinct subset of CD4 T cells that are specialized in helping B cells form germinal centers (GCs) for the generation of high-affinity class-switched antibodies, undergo expansion in people living with HIV-1 (PLHIV). Indeed, Tfh cells with latent HIV-1 are enriched in PLHIV, whether the PLHIV are on antiretroviral therapy (ART) or not. This shows that Tfh cells are preferentially infected by HIV-1 and are able to survive infection. Through total RNAseq of tonsillar subsets of T cells, including Tfh cells, our lab was able to find a sharp upregulation of an inhibitor of apoptosis family member gene (BIRC5) and a distinct metabolic gene profile in Tfhs compared to the other subsets. We further were able to find and confirm that (1) BIRC5 was highly expressed in human tonsil Tfh cells and the expression was upregulated upon HIV-1 infection, (2) human tonsil Tfh cells had high levels of Fatty Acid Oxidation related genes, but low levels of glycolytic genes, when compared with non-Tfh CD4 T cells, and (3) inhibition of FaO and BIRC5 led to changes in the survival of Tfh cells following HIV-1 infection. Our results show that Tfh survival of HIV-1 is associated with both the metabolic profile and expression of BIRC5 due to their ability to inhibit virus-induced direct and bystander apoptosis.

Supported by grants from the NIH T32: Immunology and Infectious Disease Training Program
Severe acute respiratory syndrome coronavirus 2 vaccination induces short-term polyclonal antibody response against seasonal endemic coronaviruses
Authors: Adhikari Bindu , Oltz Eugene M , Gumina Richard J , Saif Linda J , Vlasova Anastasia N
Published: January 2023

Antibody (Ab) responses induced by endemic human coronaviruses (HCoVs) may affect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific Ab production due to cross-reactivity among these HCoVs. To explore this hypothesis, we collected serum samples of healthy healthcare workers (HCW) at four time-points: pre-vaccination, post-vaccination (1st and 2nd dose) and 6 months post-vaccination. We used N protein peptide-based enzyme-linked immunosorbent assays (ELISA) specific for alphaCoVs (NL63 and 229E) and betaCoVs (OC43 and HKU1) as well as N and S peptide-based ELISA for SARS-CoV-2. Low levels of SARS-CoV-2 N protein-specific Abs were present in some individuals prior to vaccination. The level remained constant following one or two doses of vaccine and declined after 6 months. SARS-CoV-2 S protein-specific Abs level were low in most individuals prior to vaccination, increased sharply following 1st and 2nd vaccinations and declined by 6 months.

We observed low levels of pre-existing IgG Abs against endemic HCoVs prior to COVID-19 vaccination. However, like SARS-CoV-2-specific IgG Ab titers, they increased (1.6–1.9-fold) following SARS-CoV-2 vaccination and waned 6 months later. IgG Abs surge targeting HCoV N protein suggest polyclonal activation of memory B cell responses induced by the S gene based COVID-19 vaccines. The levels of IgG Abs against endemic HCoVs were 1.1–1.5 times higher in SARS-CoV-2 seropositive vs. seronegative individuals, suggesting serological cross-reactivity between SARS-CoV-2 and other HCoVs. In summary, short-term immunity (six months) against common cold causing HCoVs can be reactivated following COVID-19 vaccination however it requires booster dose for long term protection.
Developing a model of autoimmune diseases with human tonsil organoids
Authors: Chen Xin , Ghanizada Mustafa , Sola Elsa , Davis Mark M
Published: January 2023

Mouse models have contributed significantly to our knowledge of the pathogenesis of autoimmune diseases. However, differences in mouse and human immune systems limit the translation of the knowledge in mechanism and therapeutic outcomes from mouse to human. Therefore, a human model of autoimmune diseases is urgently needed. Recently, our lab has developed a functional tonsil organotypic system that can recapitulate key T cells responses and germinal center response to influenza vaccine in vitro. We now want to utilize this tonsil system to study autoimmunity.

Regulatory T cells (Tregs) have been suggested to play a central role in maintaining self-tolerance. Reduced Treg cell frequencies and impaired suppressive function have been reported in a wide range of autoimmune diseases. In this study, we show that we can efficiently knock out FOXP3 in T cells from human tonsil organoids by using CRISPR technology. We also show that tonsil organoids with FOXP3 knocked-out T cells are able to induce humoral immune responses towards autoantigens stimulation and influenza vaccination. This system could be useful for studying underlying mechanisms in the functions of Tregs in the autoimmune condition in humans and provides insights into the therapeutic strategy for autoimmune diseases.
Class II Phosphatidylinositol-3-kinases Promotes CCR7 Recycling to the Cell Membrane in Human T-Cells
Authors: Brissette Michael Abraham , Cardona Cesar I , Bill Colin A , Vines Charlotte M
Published: January 2023

C-C Chemokine Receptor 7 (CCR7) and its ligands (CCL19 and CCL21) are important for immune cell migration/co-localization during the primary and subsequent immune responses. We have previously shown both in humans and in mice that signaling through CCR7/CCL19 promotes rapid receptor internalization via arrestin-3. Therefore, we questioned whether internalized CCR7/CCL19 recycles to the membrane in T-cells, and if so, what are the drivers for recycling. G protein-coupled receptors can activate phosphatidylinositol-3-kinase (PI3K) family members to regulate membrane remodeling events, which include receptor recycling to the cell surface. To better understand the role of PI3K in the recycling of CCR7, we used wortmannin at 5μM to block class I and class II PI3K’s and at 0.5μM to block class II PI3K during CCR7 recycling. We found that under both conditions, receptor recycling was reduced by 20% suggesting a role for PI3K family members in recycling of CCR7 to the cell membrane. Treatment of the cells with the class I δ specific inhibitor, GS-1101, however, did not affect recycling, suggesting that CCR7/CCL19 recycling to the cell membrane is dependent on class II PI3K. To determine the site within the cells where CCR7 was retained following inhibition of only class II PI3K, we used HEK-293 cells transfected with CCR7-eGFP and the trans-Golgi network (TGN) marker, TPST2-mCherry, ± 1μM wortmannin, which is required to inhibit class II PI3K in HEK-293 cells. We found that after receptor internalization with CCL19 and 30 minutes of recycling, CCR7-eGFP remained within the TGN in the presence of wortmannin, in contrast CCR7-eGFP, which recycled to the cell membrane in the absence of wortmannin, demonstrating a role for class II PI3K in CCR7 recycling.

Supported by NIH-SC1GM111172 NIH-2U54MD007592 NSF-HRD-182675 NIH-5UL1GM118970
Interleukin-2 signaling engages de novo cholesterol biosynthesis in regulatory T cells
Authors: Crouch Acacia , Malek Thomas R
Published: January 2023

In the absence of IL-2R signaling, Treg homeostasis is severely disrupted. This is accompanied by a decrease in the expression of enzymes related to cholesterol biosynthesis, but the function of this pathway in Tregs is unknown. To investigate this, we selectively ablated Sterol Regulatory Binding Element Protein 2 by crossing Srebp2flox/flox mice to Foxp3cre mice (Srebp2cKO). Consistent with published studies, homozygous Srebp2cKO mice are outwardly normal. However, when Srebp2cKO Tregs are placed in competition with wild-type Tregs, they are found at a reduced frequency in the spleen, lymph nodes and gut mucosa. In this setting, Srebp2cKO Tregs have reduced Foxp3 expression, suggesting that their function and/or stability may be impacted by altered cholesterol biosynthesis. These defects persist in lymphoid and non-lymphoid tissues when Tregs in competition are treated with an IL-2R agonist. In addition, a marked reduction in effector Treg differentiation is associated with Srebp2cKO Tregs, as expression of CD39, CD73, CD103, Klrg1 and ICOS is reduced while Bcl-2 and CD62L are increased. In response to IL-2R signaling, Srebp2cKO Tregs do not optimally upregulate CD25, which suggests a function for cholesterol biosynthesis in supporting the high affinity IL-2R. These results are consistent with a model whereby IL-2R signaling coordinates Treg cholesterol biosynthesis, which contributes to effector Treg development and raises the possibility that cholesterol biosynthesis could be a target in the therapeutic manipulation of Tregs.

Supported by grants from NIH (F30 AI157211-01A1, RO1 AI148675-01)
Sustained NPSLE development in the absence of systemic lupus-like disease in TLR7- deficient B6.Nba2
Authors: Kwun Audrey , Sullivan James , Mey Gabrielle , DeSilva Tara M , Jorgensen Trine N
Published: January 2023

Neuropsychiatric Lupus (NPSLE) is a common manifestation of systemic lupus erythematosus (SLE) which includes presentations of cognitive learning and memory deficits and anxiety disorders. The B6.Nba2 mouse model of SLE presents with anxiety and depression. Toll-like receptor (TLR7), a known driver of type I interferons and SLE, drives systemic autoimmunity in the B6.Nba2 model, but whether TLR7-deficient B6.Nba2 mice develop NPSLE remains unknown.

Age-matched female B6, B6.Nba2, and B6.Nba2.TLR7 knockout mice (KO) were tested in open field (OFT), elevated plus maze (EPM), Y maze (YM) and novel object recognition (NOR) tests between the ages of 8–32 weeks. To determine levels of systemic autoimmunity and inflammation serum autoantibody and tumor necrosis factor-related of apoptosis (TWEAK) levels were determined by ELISA.

Both B6.Nba2 and B6.Nba2.TLR7 KO mice presented with anxiety (OFT, EPM) and dysfunctional spatial memory (YM). B6.Nba2 mice displayed a general memory defect (NOR), but this was not seen in TLR7-KO mice. Only B6.Nba2 mice presented with elevated levels of anti-dsDNA autoantibodies and corresponding DWEYS-reactive antibodies. In contrast, serum levels of TWEAK were elevated in both B6.Nba2 and B6.Nba2.TLR7 KO mice, suggesting that signals other than TLR7 may drive inflammation in this model.

In conclusion, both B6.Nba2 and B6.Nba2.TLR7KO presented with elevated serum TWEAK levels, anxiety and memory deficits, despite only B6.Nba2 mice displaying anti-dsDNA and DWEYS-reactive autoantibodies. Thus in this model, NPSLE appear to develop in an autoantibody-independent fashion. Studies are ongoing to determine the nature of TLR7/autoantibody-independent NPSLE.

Supported by a grant from the Department of Defense (LR 180040)
Validation of Ferret Luminex Cytokine Assays with serum from ferrets infected with flu, stimulated whole blood and PBMCs
Authors: Race Tori C , Fletcher Ashley , Damour Marc , Brown Deborah M , Rutgers Robyn S , Stephen Laurie L
Published: January 2023

Ferrets represent excellent models of infectious disease due to their natural susceptibility to many human respiratory viruses including influenza A virus (IAV), respiratory syncytial virus and severe acute respiratory syndrome (SARS). Ferrets can reproduce a similar course of disease and display similar symptoms of respiratory infection as humans, while mice do not exhibit key features of disease such as sneezing, nasal discharge and fever. There is pressing need to develop reagents to better understand and characterize the immune response to infection and support biomarker and countermeasure development through immunologic analyses of respiratory infections in the ferret. IL-1β, IL-2, IL-5, IL-6, IL-12 p40, IL-12p35, IL-17, IFNγ, TNFα, & IP-10 were expressed in HEK293 cells and purified. Monoclonal antibodies were generated and screened by Luminex for matched pairs to the recombinant protein and presumed positive samples. Capture antibodies were coupled to Luminex beads and detection antibodies were biotinylated. Serum from ferrets infected with flu had 10-fold higher levels of IP-10 and 3-fold higher levels of IL-6 than control serum. Stimulation of ferret whole blood with LPS showed increases in IL-2 and IL-6 at 2, 4 and 24 hrs. IL-12p40 and TNFα increased at 24hrs after stimulation with LPS. A validated cytokine panel in the study of immune related diseases in the ferret model.

Supported by SBIR contract 75N93020C00022: Development of Ferret Reagents for use in the Characterization of Immune Responses to Respiratory Infections in the Ferret Model
Candida albicans Induces Foaming and Inflammation in Macrophages through FABP4: Its Implication for Atherosclerosis
Authors: Alrashed Fatema , Haider Mohammed , Albaqsumi Zahraa , Alobaid Khalid , Alqabandi Rawan , Al-Mulla Fahd , Ahmad Rasheed
Published: January 2023

Atherosclerosis is a chronic degenerative disorder characterized by lipid-dense plaques and low-grade inflammation affecting arterial walls. Foamy macrophages are important in the formation of atherosclerotic plaques and the induction of low-grade inflammation. Candida albicans is the major cause of candidiasis in immunocompromised patients, including those with diabetes mellitus. However, the role played by C. albicans in macrophage foaming and the associated inflammation is poorly understood. We investigated whether C. albicans induces foaming along with inflammation in macrophages and, if so, by which mechanism(s). We incubated THP-1 macrophages with heat-killed C. albicans (HKCA). HKCA-induced lipid accumulation in macrophages along with increased expression of inflammatory markers, including CD11b and CD11c or expression and secretion of IL-1β. HKCA also increased the expression of PPAR, CD36, and FABP4 in macrophages. Mechanistically, we found that the foamy and inflammatory macrophage phenotype induced by HKCA requires FABP4 because disruption of FABP4 in macrophages either by chemical inhibitor BMS309404 or small interfering RNA (siRNA) abrogated foam cell formation and expression of inflammatory markers CD11b, CD11c, and IL-1β. Furthermore, HKCA-treated macrophages displayed high expression and secretion of MMP-9. Inhibition of FABP4 resulted in suppression of HCKA-induced MMP-9 production. Overall, our results demonstrate that C. albicans induces foam cell formation, inflammation, and MMP-9 expression in macrophages via the upregulation of FABP4, which may constitute a novel therapeutic target for treating C. albicans-induced atherosclerosis.

Supported by Kuwait Foundation for the Advancement of Sciences (KFAS) (RA CB-2019-002)
A rare case of Liraglutide-induced iatrogenic amyloidosis
Authors: Aujla Sumeet K , Jasti Pallavi
Published: January 2023

Iatrogenic amyloidosis has been linked to injectable drugs such as Enfuvirtide and insulin, which can cause cutaneous deposition of insoluble proteins. We present a rare case in which Liraglutide injections resulted in an iatrogenic amyloidosis.

Our patient is a 63 year-old female with a 12-year history of type 2 diabetes mellitus complicated by peripheral neuropathy, on Liraglutide for 10 years, who presented with a 4-month history of developing abdominal subcutaneous masses. She underwent abdominal soft tissue mass excision and biopsy, which revealed Congo red stain positive for amyloid and apple green birefringence on polarization. Lab work and skeletal survey showed no abnormalities. Serum protein electrophoresis revealed no monoclonal protein. Mass spectrometry of amyloid tissue at Mayo clinic revealed Liraglutide detection by immunohistochemistry using glucagon antibodies, dense amyloid deposits typical of amyloid caused by injectable drugs, and a peptide profile inconclusive for an amyloid type-all confirming a diagnosis of iatrogenic amyloidosis caused by Liraglutide injection.

This case highlights a very rare side effect of Liraglutide injections. If biopsy confirms amyloid deposits, further workup is absolutely necessary to differentiate the forms of amyloidosis, as the treatment varies based on the kind of amyloidosis. Diabetic patients who are on Liraglutide can have other signs and symptoms which overlap with symptoms of AL amyloidosis. Hence further workup including mass spectroscopy to type the amyloid are vital.
Loss of Mitochondrial Protease CLPP Activates Type I IFN Responses through the Mitochondrial DNA–cGAS–STING Signaling Axis
Authors: Torres-Odio Sylvia , Lei Yuanjiu , Gispert Suzana , Maletzko Antonia , Key Jana , Menissy Saeed , Wittig Ilka , Auburger Georg , West A Phillip
Published: January 2023

Caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) is a serine protease that degrades damaged or misfolded mitochondrial proteins. CLPP-null mice exhibit growth retardation, deafness, and sterility, resembling human Perrault syndrome (PS). Absence of CLPP also leads to immune system alterations but the molecular mechanisms and underlying signaling pathways remain unclear. In this study, we report the steady-state activation of type I IFN signaling and antiviral gene expression in CLPP-deficient cells and tissues, resulting in marked resistance to RNA and DNA virus infection. Depletion of the cyclic GMP-AMP (cGAS)-stimulator of IFN genes (STING) DNA sensing pathway reduces steady-state IFN-I signaling and abrogates the broad antiviral phenotype of CLPP-null cells. We report that CLPP deficiency leads to mitochondrial DNA (mtDNA) instability and packaging alterations, a phenotype also observed in CLPP mutant fibroblasts from PS patient. Our work places the cGAS-STING-IFN-I innate immune pathway downstream of CLPP and may have implications for understanding PS and other human diseases involving CLPP dysregulation and proteostasis.

Office of the Assistant Secretary of Defense for Health Affairs, U.S. Department of Defense (W81XWH-17-1-0052; W81XWH-20-1-0150); National Heart, Lung, and Blood Institute, National Institutes of Health (R01HL148153)
Leukocyte associated immunoglobulin-like receptor 1 (LAIR1) is immunoprotective in S. aureus skin infection
Authors: Dorando Hannah K , Tomaszewski Kelly , Tian Ling , Wurtz Mellisa , Quinn Chaz , Mutic Evan , Wardenburg Juliane Bubeck , Payton Jacqueline
Published: January 2023

Background

Inhibitory immune receptors are important for maintaining immune homeostasis. We recently identified a member of this group, LAIR1, in advanced cutaneous T-cell lymphoma, a cancer of skin-invasive T cells associated with recurrent and often fatal S. aureus skin and soft tissue infections. Based on these data, we predicted that LAIR1 plays a role in immune response to S. aureus infection.

Objective

Determine the impact of LAIR1 loss on S. aureus skin infection.

Methods

Wild type (WT) and Lair1 knock-out (KO) mice were subjected to subcutaneous S. aureus skin infection. In skin lesions, we measured size, bacterial clearance, immune cell recruitment (flow cytometry), and cytokine production (ELISA). We compared changes in gene expression (qRT-PCR), cytokine production (ELISA), and complement-mediated phagocytosis (flow cytometry) in WT and Lair1 KO bone marrow-derived macrophages (BMDMs) infected with S. aureus. Finally, we conducted RNAseq from S. aureus skin lesions and bone marrow neutrophils and monocytes 2 and 5 days post-infection (dpi), and from in vitro-infected BMDMs.

Results

Lair1 KO mice had significantly larger areas of abscess and dermonecrosis by 1 dpi and decreased bacterial clearance at 2 dpi. BMDMs showed defects in proinflammatory cytokine production and complement-mediated phagocytosis.

Conclusions

Taken together, these data support a previously unrecognized role for LAIR1 in myeloid response to bacterial infection.
Lactic acid bacteria derived-bioactive molecules condition an in vitro cell model into a tolerogenic dendritic-like cell phenotype
Authors: Saleem Lin , Jeffrey Michael P , Clarke Sandra T , Green-Johnson Julia M
Published: January 2023

Retinoic acid (RA) is a vitamin A metabolite; CD103+ dendritic cells (DCs) acquire a tolerogenic phenotype through an RA-dependent mechanism, driving the differentiation of gut-homing regulatory T cells in the gut mucosa. Growing evidence shows bioactive molecules derived from lactic acid bacteria exhibit immunomodulatory activity by interacting with antigen presenting cells (APCs), stimulating their differentiation and altering their phenotype to further shape adaptive immunity. We utilized the monocytic THP-1 cell line as an in vitro model to examine the impact of the Lacticaseibacillus rhamnosus secretome (LrS) and interactions with RA on APC differentiation into a tolerogenic dendritic-like cell (DLC) phenotype. THP-1 monocytes were treated with Phorbol 12-myristate 13-acetate to drive differentiation into macrophages, then conditioned with the LrS and/or RA for 24, 48 or 72 hours. Expression of key DC markers CD11b, CD11c, DC-SIGN, CD83, CD80 and CD103 increased, while expression of proinflammatory marker CD64, co-stimulatory marker CD86 and MHC class II molecule HLA-DR was reduced. Cytokine profiling revealed LrS + RA co-treatment increased the production of immunoregulatory cytokines IL-10 and IL-1RA. Expression of NFKB1 and NFKB2, key genes involved in the activation of the NF-κB canonical and non-canonical pathways was induced by 48 hours of LrS conditioning, accompanied by increased expression of CD40 and IDO1, indicating an ability to drive DLC maturation and induce co-stimulatory capacity. Deciphering the mechanisms behind these types of microbiota-host interactions gives insight into how microbial metabolites can influence immune maturation and maintain immune homeostasis.

Supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN-2017-05237
Innate Memory Following Intravenous BCG in Neonatal Pigs
Authors: Byrne Kristen A , Hill Dana C , Loving Crystal L
Published: January 2023

In adults and rodents, Bacillus Calmette-Guerin (BCG) inoculation alters innate immune cells via epigenetic and metabolic modifications resulting in heightened responses to subsequent microbial exposure (innate memory). Neonatal BCG vaccination can correlate to improved disease resistance in some human populations, but studies with human infants are extremely limited. Pigs are a relevant biomedical model for human disease and offer a path to directly asses neonatal innate memory to improve disease resilience. BCG administration in a weaned (3 week old) pig model elicited monocyte memory only with intravenous (IV) but not subcutaneous administration. Therefore, to evaluate induction of innate memory in neonates, 3–5 day old piglets received intravenous (IV) BCG or mock and ex vivo monocyte memory was assessed. At 3 and 6 weeks post-BCG, monocytes from the IV-BCG group had elevated TNF but not IL-1β production in response to LPS stimulation ex vivo, indicating neonatal BCG exposure induced innate memory. Interestingly, broncho-alveolar lavage cells from IV-BCG group had minimal differences compared to mock group in response to LPS, suggesting IV-BCG alters responses in peripheral monocytes differently than tissue resident myeloid-lineage cells. Overall, IV-BCG administration induced monocyte memory in neonatal pigs, highlighting piglets as a valuable model for continued study on the utility of BCG induced innate memory to improve disease resilience.

This work was funded by USDA-NIFA AFRI grant # 2019-07511 as part of the joint USDA-NIFA-NIH Dual Purpose with Dual Benefit: Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Animal Species Program and USDA-ARS CRIS project 5030-32000-225-000D
In vitro assessment of neutrophil response to proteins and antibodies of SARS-CoV-2
Authors: Almeida Nathalie Bonatti Franco , Fantone Kayla Marie , Sarr Demba , Grenfell Rafaella Fortini Queiroz , Rada Balazs
Published: January 2023

The SARS-CoV-2 virus caused a worldwide epidemic of respiratory disease (COVID-19). Acute respiratory distress syndrome (ARDS) has been associated with severe COVID-19. Activation of neutrophils have been linked to ARDS in COVID-19. While emerging results indicate that SARS-CoV-2 leads to neutrophil activation, it remains unknown which components of the virus are responsible for this. We assessed in vitro how the spike (S) protein and anti-spike antibodies affect neutrophils. Human neutrophils were isolated from healthy donors. Different concentrations of recombinant S protein, a monoclonal antibody (mAb) produced against the S protein and their immunocomplex were used to stimulate neutrophils. Unstimulated and PMA-treated neutrophils were used as negative and positive controls. Reactive oxygen species (ROS) production was measured by Diogenes chemiluminescence, and cell death was measured by fluorescence. While no significant increase of cell death was observed, neutrophils responded with a dose-dependent increase in the production of ROS when stimulated with either the S protein or the mAb. Neutrophils produced significantly (p<0.05) more ROS when activated by 1,000 ng of the mAb. Immune complex stimulation (1,000 ng each component) of human neutrophils also lead to significantly (p<0.005) enhanced ROS production compared to stimulations by the protein or the mAb alone. Neutrophil cell death was also significantly enhanced when stimulated by the immune complex (500 ng and 1,000 ng each component) (p<0.05, p<0.005, respectively). Our work indicates that the S protein and its immune complex formed with mAb could stimulate neutrophils in vitro and contribute to neutrophil-mediated tissue damage in COVID-19 patients.

Supported by grants from: Oswaldo Cruz Foundation, International Graduate Programme (PrInt FIOCRUZ-CAPES), University of Georgia and NIH.
Type III IFNs (IFNλs) expressed in tuberculosis (TB) granulomas enhance anti-mycobacterial activity of macrophages
Authors: Talukdar Priyanka , Junecko Beth Fallert , Mattila Joshua T
Published: January 2023

Granulomas are the pathologic hallmark of Mycobacterium tuberculosis (Mtb) infection. How a granuloma functions at limiting Mtb growth depends on the qualitative balance of pro- and anti-inflammatory cytokines in these lesions. Understanding which cytokines constitute protective and detrimental inflammation in TB is important for the improvement of current TB therapeutics. The type III interferon (IFN) family is composed of four isoforms and includes IL-29, IL-28A, IL-28B, and IFNλ4. These cytokines may be important regulators of inflammation in TB, yet little is known about their relation with TB or whether they have different properties in granulomas than type I IFN (IFNα/β), which is associated with severe TB. We identified that IFNλ is expressed at a significantly higher level in TB granulomas than non-disease lung, and negatively correlates with the CFU/granuloma. IHC characterization of IFNλ expression in granulomas revealed neutrophils and macrophages expressing IFNλ1 and IFNλ4, with neutrophils having a higher percentage of IFNλ expression. IFNλ1 and IFNλ4 differed in their intracellular localization, IFNλ4 having a significantly higher nuclear localization in macrophages than IFNλ1. IFNλ1 treatment of monocyte-derived macrophages induced the expression of different proinflammatory genes at the transcript level and costimulatory molecules like CD86 at the protein level. Further, pretreatment of macrophages with IFNλ1 inhibited the transcriptional activity in a reporter Mtb strain and follow-up experiments suggested this was accompanied by increased acidification of macrophage lysosomes. Our findings suggest that IFNλs may have unappreciated roles in regulating inflammation at the site of disease in TB.

Supported by Grant from NIH entitled Pitt HIV-TB Research and Training Program in India (grant number: D43 TW010039)
Leptospira spp. are a potent activator of Type I IFN responses via the cGAS-STING pathway
Authors: Gupta Suman , Matsunaga James , Cassel Suzanne L , Haake David A , Sutterwala Fayyaz S
Published: January 2023

Leptospirosis is a widespread zoonotic disease which can be potentially fatal in infected humans and dogs. The spirochetal bacteria Leptospira interrogans is the major causative agent of leptospirosis. Rodents serve as reservoir hosts for L. interrogans and once colonized they continue to shed bacteria in their urine. The host immune response to Leptospira spp. remains poorly defined. Leptospiral lipopolysaccharide and lipoproteins can activate TLR4 and TLR2. In addition, leptospires have been shown to activate the NLRP3 inflammasome in macrophages. We observed that when compared to other Gram-negative bacteria, L. interrogans induced a poor inflammasome response with little or no notable pyroptosis. In contrast, L. interrogans induced a robust type I IFN responses in both mouse macrophages and human monocytes. The L. interrogans-induced type I IFN response was dependent on both cGAS and STING and resulted in rapid phosphorylation of TBK1 and IRF3. Furthermore, supernatants from infected macrophages were capable of inducing IFNβ expression in uninfected macrophages, suggesting paracrine signaling from infected cells. These studies demonstrate that L. interrogans can drive a cGAS-STING dependent type I IFN response that may contribute to the host defense against this pathogen.
Role of foxn1 in Xenopus laevis thymopoiesis.
Authors: Dimitrakopoulou Dionysia dionysia , Bellin Eleanor , Horb Marko , Robert Jacques
Published: January 2023

The transcription factor forkhead box N1 (FOXN1) plays a pivotal role in differentiation of thymic epithelial cells (TECs) essential for intrathymic T cell education and keratinocyte development. Mutations in murine foxn1 are associated with T− B+ NK+ nude/severe combined immunodeficiency (SCID) phenotype. In humans FOXN1 mutations also lead to thymic aplasia or hypoplasia, hair loss and dysmorphic nails. In the amphibian Xenopus tropicalis, foxn1−/− line exhibited loss of thymi formation and absence of mature T-cells, similar with Foxn1−/− murine line. Due to expanded resources in immunobiology of X. laevis (a close relative of X. tropicalis), we generated a novel foxn1-deficient X. laevis model by using the CRISPR/Cas9. foxn1 crispants shows a high mutational efficiency (30–90%) and about 25% of tadpoles (stage 55–56), exhibit hypoplastic thymi. foxn1 disruption resulted in significant reduction of T-cell markers transcript levels, detected by RT-PCR. Furthermore, spleen of foxn1 crispants had markedly reduced CD3e staining compared to controls. Interestingly, Mycobacterium marinum (M. marinum) infection of tadpoles results in noninflammatory CD8 negative- and iT cell–mediated response, while foxn1 mutations in X. laevis appeared to affect tolerigenic innate immune response of infected tadpoles. This foxn1-deficient transgenic line will empower X. laevis as nonmammalian alternative experimental organism for studying host interaction with to non-tuberculosis mycobacteria. Currently, we are investigating mechanisms by which foxn1 mutation alter innate immunity of M. marinum inoculated tadpoles and evaluating tolerance/resistance by analysing the inflammatory/anti-inflammatory cytokine profile.
NLRC3 Localizes to the Endoplasmic Reticulum via Interactions with a Novel ER-Resident Protein
Authors: Davis Beckley K , Chapman Elisabeth , Gawon Kalida , Petrecca Izzy , Baker Taylor , Rechnitzer Alma
Published: January 2023

Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 (and others) are not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen recognition. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein in particular localizes to the endoplasmic reticulum (ER) and might serve as a localization-dependent anchor for NLRC3, thus facilitating its interaction with STING and TBK1. Here we show NLRC3 is distributed to the ER where it can interact with several important signaling molecules involved in type I interferon production.

Supported by a grant from the NIH (R15 GM134430)