bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT3 in T cells Regulates Protective Capacity of Hybrid Type 1/Follicular helper T cells in Persistent Mouse Malaria Infection
Authors: Stephens Robin , Dent Alexander L. , Carpio Victor H.
Published: January 2023

In infection with Plasmodium spp., IFN-γ+ T helper (Th)1 cells control peak parasitemia, while antibody and IL-21+ T follicular helper (Tfh) cells effect final clearance. However, we show that hybrid Th1/Tfh cells (IFN-γ+IL-21+CXCR5+), reported to be sub-optimal Th cells and to inhibit Germinal Centers (GC), dominate the immune response to P. chabaudi infection. Th1/Tfh expand but plateau at low levels while GC form, with GC-Tfh numbers stable throughout. To understand molecular mechanisms of differentiation of Th1/Tfh, mice deficient in several transcription factors targeted in T cells were infected. We found that T cell-intrinsic Bcl6 and Blimp-1 regulated CXCR5 and IL-10 expression in Th1/Tfh cells. Bcl6, Blimp-1 and STAT3 all dampen T-bet expression. However, neither of the Signal Transducer and Activator of Transcription (STAT) proteins known to regulate T-bet expression, STAT4 or STAT1, were required for IFN-γ production by Th1/Tfh. On the other hand, T cell deficiency of the Tfh-promoting transcription factor, STAT3, reduced antibody while non-hybrid Th1 cells increased and mice were fully protected from re-infection. Conversely, deficiency in T-bet, reduced Th1 differentiation, including dramatically reducing production of IFN-γ and specific antibody of the IgG2b isotype, and showed reduced control of re-infection. We have previously shown that few committed Th1 memory cells are generated in Plasmodium infection, importantly here the increase in Th1 features in STAT3-deficient T cells can improve control of reinfection. In summary, a flexible T cell population regulates each effector function with multiple transcription factors, providing plasticity to allow continuous responsiveness to prolonged infection.
Ex vivo analysis of Plasmodium falciparum-specific B cell responses to natural malaria infection in children and adults
Authors: Hopp Christine , Andrews Sarah , Chambers Michael , Leggat David J , Traore Boubacar , Pierce Susan K , McDermott Adrian B , Crompton Peter D
Published: January 2023

In naturally acquired immunity to malaria, antibodies that reliably protect are only acquired after years of repeated Plasmodium falciparum (Pf) infections. We have shown in Mali that this inefficiency in humoral immunity to malaria is associated with a large expansion of CD21loCD27− ‘atypical’ MBCs. The function of atypical MBCs remains elusive, as they exhibit altered B cell receptor (BCR) signaling and when activated fail to secrete cytokines and antibodies. We set out to study whether atypical or classical B cells are recruited into the immune response to a febrile malaria infection: using Pf antigen probes, we characterized PfAMA1/PfMSP1-specific B cells in Malian children and adults (ages 2 to 36 years), at well-defined time points before, during and after a febrile malaria episode. Our preliminary analysis shows that Pf-specific atypical MBCs display signs of activation after a febrile malaria infection. Using antigen probes to track influenza hemagglutinin-specific B cells in the same individuals, we have gained insight into the relative role of Pf in driving atypical MBC expansion and have also compared the magnitude and kinetics of Pf-and influenza-specific atypical and classical MBC responses. We further show that classical Pf-specific IgG+MBCs are activated and expanded, but interestingly, that a large proportion of activated Pf-specific MBCs are unswitched, expressing surface IgM. Additionally, we have single cell sorted Pf- and influenza-specific MBCs to compare their BCR features (VH gene diversity and somatic hypermutation rates). This analysis provides important new insights into the mechanisms underlying the inefficient acquisition of naturally acquired immunity to malaria in children.
The Role of Neutrophils in Pseudomonas aeruginosa Chronic Suppurative Otitis Media
Authors: Khomtchouk Kelly M , Kouhi Ali , Maria Peter Santa
Published: January 2023

Chronic suppurative otitis media is largely due to biofilm-forming bacteria, of which a common pathogen is Pseudomonas aeruginosa. Neutrophils are well-known to have various contributions in both promoting and restricting bacterial growth in other biofilm-mediated diseases, as they are one of the first cells recruited to sites of infection. Herein we propose that 1) fluorescent P. aeruginosa develop into biofilms and form chronic infections in the middle ear of a mouse model of Eustachian tube obstruction and acute tympanic membrane wounds; 2) this biofilm reduces functional capacity of professional phagocytes; 3) biofilm initiates apoptosis of responding neutrophils; and 4) neutrophils contribute to disease burden. We also determine neutrophil subsets with a leading role in the establishment of bacterial biofilm-mediated disease, and test the capacity of polymorphonuclear leukocytes to suppress planktonic infection.

Neutrophil function is quantified as a measure of phagocytosis and the production of extracellular traps; we also measure cell survival and clearance via efferocytosis to determine host-mediated barriers to this recalcitrant infection. Using flow cytometry coupled with fluorescent-tagged P. aeruginosa, we are able to assess middle ear effusion post-inoculation and develop a timeline of response to chronic infection in our novel CSOM model.

Importantly, this study establishes a model system for further immunological and translational studies, including testing of topical therapeutics. The results of this work will also inform potential efforts of neutrophils as predictors for disease exacerbation and guide additional therapeutic development for an illness affecting millions of children globally.
TcdB-specific plasma cell and memory B cell responses in recurring Clostridioides difficile infection-associated disease
Authors: Amani Souwelimatou Amadou , Shadid Tyler , Ballard Jimmy D , Lang Mark
Published: January 2023

Recurrence of C. difficile infection with progressively worsening disease is a serious problem. Serum IgG specific to the secreted toxin B (TcdB) is the best-known correlate of protection against disease symptoms and pathology. However, little is known as to whether and how infection stimulates protective humoral immunity. We hypothesized that recurring disease is due to an inadequate development of a TcdB-specific memory B cell response following an initial infection. We measured TcdB-specific antibody and memory B-cell responses following two sequential infections in microbiome-disrupted B6 mice. A mild infection caused by a low dose spore challenge did not induce an isotype-switched antibody response against the toxin and failed to protect against recurring disease. However, a higher-dose infection resulted in a TcdB-specific primary serum IgG1 and IgG2b response but did not boost titers. Also, in mice with TcdB-specific serum IgG1 there was a strong correlation with fecal TcdB-specific IgG1 and an attenuation of subsequent disease. Following infection of TcdB-immunized mice, IgG1 titers did not increase and ELISPOT analyses detected TcdB-specific IgG1-secreting plasma cells primarily in the bone marrow. Infection of pre-immunized mice also resulted in increased amounts of TcdB-specific IgG1 reaching the gut lumen. These findings indicate that a more severe initial encounter with the pathogen may be required to elicit a protective systemic TcdB-specific IgG1 response. Re-infection or infection of pre-immunized mice does not stimulate a B cell memory response and protection appears to be mediated by transport of plasma cell-derived systemic IgG1 to the gut lumen.
CD160-HVEM signaling in intestinal epithelial cells modulates gut microbial homeostasis
Authors: Seo Goo-Young , Shui Jr-Wen , Mikulski Zbigniew , Wang Qingyang , Takahashi Daisuke , Giles Daniel A , Iwaya Hitoshi , Sethi Ashu , Kim Pyeung-Hyeun , Cheroutre Hilde , Kronenberg Mitchell
Published: January 2023

Intestinal epithelial cells (IEC) are a first barrier that segregates host and commensal bacteria to maintain intestinal homeostasis. Intestinal intraepithelial lymphocytes (IEL) are located beneath or between adjacent IEC and directly contact IEC. The herpes virus entry mediator (HVEM), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is highly expressed by IEC. Epithelial HVEM expression was previously reported as a regulator of innate immune defense during acute infections in the intestine (Shui et al., Nature, 2012). Here, we identify that HVEM signaling in IEC is important for the regulation of the gut microbiota at steady state. Mice with an epithelial-specific deletion of the gene encoding HVEM (HvemΔIEC) had significantly increased segmented filamentous bacteria (SFB) which caused an increase in Th17 cells in the ileum. Treatment with the antibiotic vancomycin eliminated SFB and decreased Th17 cells in HvemΔIEC mice. Additionally, mice with a deletion of the gene encoding CD160, which is a ligand for HVEM and is highly expressed by IEL, including intraepithelial innate lymphoid cells (ILC) and intraepithelial T cells, had increased SFB in the ileum. Our findings suggest that the interaction of CD160 expressed by IEL with HVEM expressed by IEC is important at steady state for shaping the microbiota in the intestine.
Zinc supplementation modulates T helper 17 cells via the gut microbiome
Authors: Gordon Sarah R , Vaishnava Shipra
Published: January 2023

Zinc is a dietary micronutrient that regulates various biological processes. Zinc is important for replication and survival of microbes and, as a result, a common strategy of the immune system is to steal zinc away from disease-causing microbes to prevent their growth. Recent studies show that changes in levels of dietary zinc can alter the composition of the gut microbiota, however how this impacts mucosal immune responses in the gut is not known. Here, we show that supplementing mice with zinc sulphate, a commonly used therapy for treating diarrheal episodes, in drinking water for a week resulted in significant changes in gut microbial composition as well drastic reduction in microbial diversity. More importantly. we saw that zinc supplementation attenuated the T helper17 cell number and activity in murine small intestine. To determine if zinc-dependent changes in gut microbiota are sufficient to limit Th17 response, we transplanted the microbiome from zinc treated mice into germ-free mice. We saw that germ-free mice that received cecal contents from zinc treated mice have reduced Th17 response, thus establishing that dietary zinc changes immune potential of the gut microbiome. Th17 cells mediate antimicrobial response in the gut and are involved in the pathogenesis of many autoimmune diseases. Our work thus provides a novel nutritional approach for remodeling microbiota to dampen inflammatory immune responses in the gut.
The adjuvant effects of MF59 on antigen-specific regulatory and effector T cells
Authors: Lin Pin-Hung , Yang Hung-Chih
Published: January 2023

Peptide vaccines are an ideal strategy to induce cross-protective T cell immunity against a broad range of influenza virus strains, but they are subimmunogenic and tend to induce regulatory T (Treg) cells. Our previous study has shown that adjuvanted peptide vaccines can suppress the development of antigen-specific Treg cells. MF59 is the first oil-in-water adjuvant approved for protein-based influenza vaccine, which enhances humoral and Th2 cellular immune responses. However, the role of MF59 in peptide-based vaccines regarding the induction of T cell immunity is unknown. We thus investigated the effects of MF59-adjuvanted peptide vaccines on antigen-specific T cell immunity against influenza virus infection. Using OT-I and OT-II TCR transgenic T cells, we found that the MF59-adjuvanted cognate OVA peptide vaccines induced a significant portion of OT-II Treg cells in primary immunization. However, MF59-adjuvanted peptide vaccine could prevent the expansion of pre-existing antigen-specific Treg cells. Of note, primary influenza virus infection induced a negligible portion of antigen-specific Treg cells, but secondary influenza virus infection drove the expansion of pre-existing Treg cells. Interestingly, swapping of the sequential immunizations with MF59-peptide vaccination and influenza virus infection resulted in very different levels of antigen-specific Treg cells, suggesting that the first immunization experience affects the development of antigen-specific Treg cells. Finally, MF59-peptide vaccines stimulated the production of IFN-γ and TNF-α of CD4 and CD8 T cells. In conclusion, MF59-peptide vaccine exhibits a unique feature in stimulation of antigen-specific T cell immunity.
Targeting antigen to lymph node lymphatic endothelial cells under steady-state to promote archiving and memory
Authors: Maulloo Chitavi D , Wilson David Scott , Hubbell Jeffrey A , Swartz Melody A
Published: January 2023

Lymphatic endothelial cells (LECs) have been shown to archive antigen (Ag), i.e. retain Ag in its native form, for weeks after exposure. We have developed a polymeric platform that promotes targeting of Ag to lymph node (LN)-resident LECs by exploiting the sugar-binding scavenger receptors expressed by LECs, leading to longer Ag retention over 2 weeks. This targeting results in enhanced Ag archiving in LECs, even in the absence of an adjuvant or other inflammatory stimuli previously shown to be necessary for Ag archiving in LECs. 2 weeks after subcutaneous (s.c.) delivery of unmodified OVA, we observed substantial proliferation and activation of adoptively transferred OVA-specific CD8+ T cells. Immunization and subsequent Ag archiving with our targeting polymer conjugated to OVA (without adjuvant) led to reduced CD8+ T cell proliferation but higher differentiation into a central memory phenotype, both by surface receptors as well as the ratio of EOMES vs. T-bet on activated T cells. We measured MHCI-mediated presentation of OVA (SIINFEKL presented on the H-2kb molecule) on LN-LECs vs. DCs over two weeks following s.c. injection and found that Ag presentation by LECs was comparable to DCs. This demonstrates that LECs are able to cross-present archived Ag directly to CD8+ T cells, with potential implications in the prolonged maintenance of memory CD8+ T cells and design of a more effective vaccine platform.
Proteogenomic characterization of the specific antibody repertoire induced by gold nanoparticles of differing shape and biomolecular surface: towards the development of nanoparticle-based vaccines
Authors: Pérez-Potti André , Giannone Valeria , Lara Sandra , Bigini Paolo , Dawson Kenneth A. , Yan Yan
Published: January 2023

Nanoparticles (NPs) in the size range of 1–100 nanometres (nm) present unique physical-chemical properties compared to bulk material making them suitable for a range of biomedical applications. Due to their size and shape, NPs engage in particular ways with cell receptors and by co-delivering antigens, adjuvants and immunomodulators grafter or encapsulated they can serve as new vaccine formulations for the treatment of several diseases such as cancer or HIV.

Gold NPs (AuNPs) are interesting due to their ease of synthesis and control of size and shape, being the later considered a relevant parameter driving interactions with cell receptors. Here is shown the work to study the capabilities of AuNPs with different shape to induce antibody responses in vivo. Spherical and branched AuNPs were repeatedly inoculated subcutaneously to CD rats. The levels of Immunoglobulins G and M (IgG and IgM) were analyzed by Enzyme-Linked Immunosorbent assay (ELISA) and the B cell receptor (BCR) V(D)J rearrangements were assessed by Ig-Sequencing of the BCR transcripts from different lymphoid tissues. Mass spectrometry (MS) analysis of enriched IgG from treated rats plasma revealed the functional antibody response.

In the case of branched AuNPs, the levels of total IgG and IgM appeared higher and increased autoantibody levels were found. V(D)J gene and allele usages were more diverse, showing considerable shifts in the repertoire, and B cell clonality was higher in comparison to spherical AuNPs and controls. Immune tolerance seems to be disrupted by branched NPs with particular biomolecular surfaces. These findings represent a step forwards in the development of NP-based vaccines towards motifs on the surface (e.g. tumor antigens) of such NPs.
Evaluation of the safety profile of ssRNA adjuvant derived from the IGR IRES region of CrPV
Authors: Park Hyeongjun , Kim Hye-Jung , Ko Hae Li , Kwak Hye Won , Won Dong-Hoon , Hwang Da-Bin , Shin Yoo-Sub , Yun Jun-Won , Nam Jae-Hwan
Published: January 2023

Adjuvants enhance the efficacy of vaccines by stimulating immune response relate gene expression and pathways. However, development of an appropriate adjuvant for a specific vaccine type is difficult, because of their unwanted systemic and local adverse effects. Despite intensive efforts for more than 80 years, there are only a few approved adjuvants. Poly I:C, a double stranded RNA was experimentally used as an adjuvant since 1970s. Although poly I:C dramatically stimulates the innate immune response, which is a prerequisite of a good adjuvant, inducing effective immune response while avoiding adverse events is a major challenge. In this study, we evaluated the safety and toxicological profile of a high-dose (200ug/mouse) single stranded RNA (ssRNA) derived from the intergenic region (IGR) internal ribosome entry site (IRES) of cricket paralysis virus (CrPV) as a novel adjuvant in a mouse model. The results indicate that there were no significant differences in body weight and food intake pattern in mice immunized with ssRNA adjuvant and the control group. Hematological parameters like ALT and AST levels appeared to be normal in both treated and control group. Histology of organs such as liver, spleen, lung, and kidney appeared to be normal. There was no evidence of tissue damage in mice treated with ssRNA adjuvant and control group. IgE levels, which is marker of autoimmune disease, is also not different compared with treated group and control group. In addition, the adjuvant effect is similar in both high-dose and low-dose (20ug/mouse) treated groups. In conclusion, novel ssRNA adjuvant exhibited a safe toxicological profile in mouse model. The results obtained may be utilized for conducting further clinical trials in humans.
Designing pathogen-like particles to augment vaccine potency through the induction of broadened and synergistic innate immune responses
Authors: Toy Randall , Pradhan Pallab , Keenum M. Cole , Jhita Navdeep , Blanchard Emmeline , Atalis Alexandra , Narang Neha , Chukwu Chinwendu , Cortes Gabriela , Suthar Mehul S , Santangelo Philip , Shayakhmetov Dmitry , Roy Krishnendu
Published: January 2023

Vaccines have protected against many deadly diseases, but they are less effective at defending against pathogens with weakly immunogenic or rapidly mutating antigen epitopes. Adjuvants that specifically trigger Toll-like and cytosolic receptors have been developed to enhance vaccine potency against antigens with poor immunogenicity. We hypothesized that adjuvant combinations delivered on pathogen-like particles (PLPs) could further improve the breadth and potency of the innate immune response. PLPs were synthesized by encapsulating hydrophobic adjuvants inside PLGA microparticles and nanoparticles, which were functionalized with polyethylenimine to facilitate surface loading of charged RNA or ssDNA adjuvants. The immune response of a virus PLP (TLR7/RIG-I adjuvants) and a Gram-negative bacteria PLP (TLR4/TLR9 adjuvants) was evaluated in bone marrow-derived dendritic cells (BMDCs). The viral PLP induced a broadened immune response by simultaneously inducing cytokine profiles representative of the individual adjuvants. The Gram-negative bacteria PLP induced synergistic upregulation of IFN-b and IL12-p70 at both the gene and protein level. Studies in BMDCs from knockout mice revealed that TRIF signaling cooperates with MyD88 signaling and IRF5, not IRF3 or IRF7, is the primary interferon regulatory factor driving synergistic cytokine responses. Moreover, the Gram-negative bacteria PLP could induce upregulation of pro-inflammatory genes and synergistic DC activation in vivoafter IM injection. Ongoing work will evaluate the molecular mechanisms governing adjuvant synergy with PLPs and assess if PLPs can enhance flu vaccine potency in challenge studies
New role of Epstein-Barr virus in pathogenesis of acute and chronic lymphocytic leukemia
Authors: Laurynenka Viktoryia , Carter Martha , Parameswaran Sreeja , Chen Xiaoting , Kottyan Leah C. , Weirauch Matthew T. , Harley John B
Published: January 2023

Acute lymphoblastic leukemia (ALL) is the most common mortal cancer in children. Chronic lymphocytic leukemia (CLL) is the most prevalent form of adult leukemia in western countries. B lymphocytes dominate the origin of both diseases. We have found a possible role for Epstein-Barr virus (EBV) in the origins of both ALL and CLL.

We applied our strategy (Nat Genet 50:699, 2018) to determine whether the binding of EBV transcription factors (TFs) was concentrate at the 84 known risk loci for CLL and 16 loci for ALL. We evaluated 52 virally encoded TF ChIP-seq (chromatin immunoprecipitation with DNA sequencing) datasets and complemented this analysis with the results from 1535 human TF ChIP-seq datasets.

We found that Epstein-Barr nuclear antigen leader protein (EBNALP), EBNA3C and EBNA2 were concentrated in the CLL loci by 3.7, 3.7 and 3.5-fold with p=4.89*10−19, p=2.74*10−11 and p=1.07*10−8, respectively. The viral (n=3) and human TFs (n=40) cluster together in an optimal subset of ~15 of the 84 known loci in CLL at p<10−6. Eighty percent of the most highly associated viral and human TF ChIP-seq datasets were collected from EBV transformed B cell lines in the Latency III program of viral expression, for which EBNALP, EBNA3C and EBNA2 are viral gene products.

In ALL 3 of 16 loci were occupied by EBNA3ABC by a 17.7-fold enrichment with p=2.0*10−10. Among human TFs only c-MYC reached statistical significance binding 2 non-overlapping risk loci with 28.4-fold enrichment with p=2.9*10−10.

These results nominate EBV for a role in the pathogenesis of CLL and ALL by a mechanism operating in transformed B cells through the EBV Latency III program of viral expression.
Axl promotes ZIKV entry and modulates antiviral state of human Sertoli cells
Authors: Strange Daniel P , Jiyarom Boonyanudh , Zarandi Nima Pourhabibi , Xie Xuping , Sadri-Ardekani Hooman , Shi Pei-Yong , Verma Saguna
Published: January 2023

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in its ability to be sexually transmitted. The ability of ZIKV to establish persistent infection in the testes clearly suggests involvement of unique immune suppressive pathways, however the underlying mechanisms are not defined. We demonstrated that human Sertoli cells (SC), the major cell type of seminiferous tubules, an immune privileged compartment of the testes, support prolonged ZIKV replication without compromising cell survival. Our recent study also provides evidence that human testicular organoids (HTO) can be used as a tool to study ZIKV immunopathogenesis. Recent reports have identified the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase Axl, a critical regulator of innate immunity and apoptosis, as an entry receptor and/or immune modulator for ZIKV infection depending on cell type. Interestingly Axl receptor is highly expressed in the testes, however, the role of Axl in ZIKV infection of SC has not been determined. In this study, we show that Axl was highly expressed in SC as compared to Leydig cells that correlated with robust infection of ZIKV only in SC. Further, neutralizing Axl receptor and its ligand Gas6 strongly attenuated virus entry and replication in SC. Blocking of Axl kinase and downstream signaling was associated with decrease in the negative regulators of antiviral immunity including pSTAT1 and SOCS1 that led to increased expression of IFIT1 and MxA, and reduced virus replication in the SC and HTO. Together, our data demonstrates that Axl promotes ZIKV entry and negatively modulates antiviral state of SC to augment long-term infection of the testes and provides new insights into testis immunology in the context of ZIKV persistence.
Quantifying Virus Escape from T Cells in the Latent HIV Reservoir
Authors: Warren Joanna A , Zhou Shuntai , Xu Yinyan , Moeser Matthew , Kirchherr Jenn , Sung Julia , Roan Nadia , Adimora Adaora , Kuruc JoAnn , Gay Cindy , Margolis David , Archin Nancie , Swanstrom Ronald , Goonetilleke Nilu
Published: January 2023

There is no cure for HIV, largely because HIV establishes a small but sustained pool of latently infected cells that are not cleared by antiretroviral therapy (ART). Current strategies include firstly reactivating the latent HIV reservoir and then using T cell immunotherapy to clear reactivated cells. However, pre-ART CD8 T cell escape variants have been reported in the HIV reservoir, which may limit CD8 T cell recognition and clearance of HIV-infected cells. The extent of virus escape from T cells in the latent reservoir is unclear.

HIV-specific T cell responses were comprehensively mapped across the Clade B HIV proteome by IFN-g ELISpot in 25 ART-suppressed participants. In parallel, replication competent viruses derived from supernatants of autologous resting CD4 T cells following mitogenic reactivation were sequenced. Peptides spanning virus variants within reactive T cell epitopes were synthesized and examined by ELISpot for evidence of escape, defined as ≥50% difference in T cell magnitude between peptide variants.

No correlations were observed between the size of the latent HIV reservoir and either HIV-specific T cell breadth (1–19 epitopes) or magnitude (156–2855 SFU/M PBMCs). T cell escape was assessed in 17 participants. 39% of reactive T cell epitopes (48/124) harbored ≥1 amino acid variants in the sequenced latent reservoir. Of those 48, 20 afforded T cell escape, providing an overall escape frequency in the reservoir of 16% (20/124).

These data show that the majority of replication competent latent HIV viruses do not harbor CD8 T cell escape mutants, suggesting that immunotherapy approaches that boost CD8 T cell responses can successfully target the latent reservoir in HIV curative and or remission strategies.
Stochastic HIV Gene Expression is a Novel Mechanism for HIV Persistence
Authors: Matus Nicodemos Rodrigo A , Douek Daniel , Koup Richard
Published: January 2023

In HIV-infected people, the virus persists in resting memory CD4 T cells as a latent provirus integrated into transcribed genes. HIV has 9 gene products that are generated from 7 alternatively spliced mRNAs whose expression is regulated by the cell’s transcriptional state. Furthermore, transcription of genes accessible by RNA pol II occurs in bursts, giving rise to the stochastic expression of mRNAs in cells.

The HIV protein Nef allows infected cells to evade CD8 T cell recognition through the downregulating of peptide-MHC complexes (pMHCs). The precise kinetics of pMHC downregulation by Nef upon reactivation of a latent provirus remains unclear. We explored this question by direct infection and longitudinal analysis of primary resting CD4 T cells with a CCR5-tropic intact replication-competent virus in which GFP reports the expression of Nef.

We detected GFP+ cells 3 to 4 days after infection. These GFP+ cells were resting memory CD4 T cells and had downregulated CD4 and pMHCs. We then performed single-cell RNA-seq on sorted GFP+ cells to examine both cellular and virus gene expression, as well as HIV integration sites. Our dataset identified various transcribed host genes that had an integrated provirus. Strikingly we found GFP+ cells either (1) had no HIV mRNAs or (2) had HIV mRNAs encoding Nef, Vpr, Vif or Vpu-Env, but never HIV mRNAs encoding Gag-Pol, Tat or Rev.

We conclude that in resting memory CD4 T cells, an integrated HIV genome is transcribed and alternatively spliced for mRNAs encoding Nef, Vif, Vpr or Vpu-Env. HIV expression is stochastic because infected cells didn’t always express HIV mRNAs. Therefore we propose stochastic HIV gene expression is a novel mechanism for HIV persistence by hiding the infected cells from the immune system.
Age-dependent Susceptibility To Virus Infection Is Governed by Host Genetics
Authors: Menachery Vineet D , McAnarney Eileen T , Ferris Martin T
Published: January 2023

While improved sanitation and medical care have reduced the burden of infectious disease, it remains the 4th leading cause of death in the elderly. Coupled with the expansion of the aged population over the next two decades, understanding the interplay between infection and the senescent host is critical. Both the microbiome and host genetics have been predicted to play critical roles in aging immunity. In this work, we explore the role of each in age-dependent susceptibility to virus infection. Severe acute respiratory syndrome coronavirus (SARS-CoV), the first outbreak virus of the 21st century, produces increased disease in aged patients as compared to the young. Importantly, age-dependent susceptibility is observed in mouse models of SARS-CoV infection. Our results indicate that despite microbiome variation between young and aged animals, limiting these differences had only a marginal effect on disease in the aged following infection. In contrast, age-dependent susceptibility was not fully conserved across genetically diverse Collaborative Cross (CC) mice. We found that while the majority of CC lines maintained increased disease as they aged, several lines showed no distinction between young and aged animals. In one CC line, we found that aged mice were more resistant to SARS-CoV infection than their younger equivalent. Notably, age-dependent susceptibility was not simply associated with higher viral loads driving augmented disease. Instead, the results suggest that host factors including tissue and inflammation responses govern disease in the senescent host. Together, the work highlights the importance of genetic elements in driving age-dependent disease following infection.
Respiratory syncytial virus strains differentially activate the inflammasome eliciting a pathogenic Th17 response
Authors: Waldstein Kody , Weiss Kayla A. , Hartwig Stacey , Quick Hannah , Poudel Barun , Gurung Prajwal , Varga Steven M
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide with no licensed vaccine available. RSV-induced disease ranges from mild to severe, with severe disease correlated with elevated levels of pro-inflammatory cytokines, including the Th17-associated cytokine IL-17. To better understand the virus factors that influence the induction of a Th17 response, we compared the inflammatory responses elicited by different RSV strains. In contrast to the commonly used RSV A2 strain, RSV 2–20 infection resulted in increased levels of IL-6 and IL-1β, which were required to induce a pathogenic Th17 response. Th17 induction was associated with increased mucus production and airway hyperreactivity following RSV 2–20 infection indicating a strain-dependent increase in disease severity. A recombinant RSV A2 virus expressing the 2–20 fusion (F) protein induced IL-1β to similar levels as wild-type RSV 2–20 suggesting that the 2–20 F protein is necessary for increased pro-inflammatory cytokine production. Sequencing of the RSV A2 and 2–20 F protein genes revealed three point mutations in a region likely to interact with host cells. Reversion of these three amino acids in the RSV A2/2–20 F recombinant virus to the A2 sequence significantly reduced RSV-induced IL-6 and IL-1β protein production. Together our results demonstrate that the RSV F protein plays an essential role in the induction of pro-inflammatory cytokines which are critical for CD4 Th17 cell differentiation. Genetic variations in the RSV F protein between RSV strains play a crucial role in modulating RSV-induced disease severity in the animal model and may contribute to the differential RSV pathogenesis observed in humans.
Evaluating the role of candidate gene, csf3r, for sex-linked susceptibility to lupus-like disease in mice
Authors: Gubbels Bupp Melanie R , Woodfin Anne E.
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disorder that primarily affects women and this trend is also observed in BWF1 lupus-prone mice. Although the factors that contribute to sex-specific lupus incidence are complex, evidence suggests that sex hormone regulation of genetic targets likely play a role. Several candidate genes have been identified by their disproportionate expression within splenocytes isolated from male and female BWF1 mice and because their expression is regulated by the sex hormone, testosterone. One candidate gene in particular, csf3-r, is predominantly expressed in CD11b+ cells and is the focus of our current studies. Previous research has indicated that Gr1low CD11b+ cells accumulate in male lupus-prone mice, and are associated with disease suppression. We hypothesize that sex-specific expression of csf3-r contributes to the different frequencies of specific neutrophil subpopulations in BWF1 lupus prone mice and may contribute to disease protection in male mice. To test this hypothesis, we are examining the expression levels of csf3r in neutrophils in lupus-prone and nonautoimmune-prone male and female mice and assessing potential correlations between sex-specific expression of csf3r and the frequencies of particular neutrophil subpopulations. The elucidation of testosterone-regulated pathways that contribute to lupus-like disease may point to new therapies that may be beneficial in autoimmune diseases with sex-specific incidences.
Educational Program Impacts Suicidal Thoughts of Individuals with Autoimmune Conditions
Authors: Ramirez Francisco E , Nedley Neil , Queva Timothy
Published: January 2023

Rationale

Some research has suggested a relationship between suicidality and autoimmune diseases. This study documents the effects of an 8-week community educational program on suicidal ideation severity in participants with autoimmune conditions.

Methods

Participants were educated in healthy lifestyle habits during a once a week for 8 weeks program, each program lasted 2 hours. Instruction included control of thoughts, benefits of a plant-based diet, proper exercise, adequate sunlight, and sleep hygiene. No patient-doctor relationship was established. Each individual took the depression and anxiety test before and after the program which measures depression, symptoms of depression, suicidal ideation, demographics and autoimmune conditions. Suicidal thoughts were reported as None or rare, About 1/4 of the days of week, 1/2 of the days of a week and almost every day.

Results

From n=431 participants that had an autoimmune condition and finished the 8 week program, at baseline 62% (n=267) participants reported no suicidal thoughts. Those with suicidal thoughts, 15% (n=65) reported suicidal thoughts 1/4 of the days, 13% (n=56) reported suicidal thoughts 1/2 of the days, and 10% (n=43) reported suicidal thoughts almost every day.

Upon completion of the 8 week program: 85% (n=368) had no suicidal thoughts, 8% (n=36) had suicidal thoughts 1/4 of the days, 4% (n=17) had suicidal thoughts 1/2 of days, and 2% (n=10) had suicidal thoughts almost every day.

Conclusion

The program was effective in decreasing quantity and quality of suicidal thoughts. Only a small number of participants did not improve (6%) and only 3% became worse.
Modulation of ILC2 trafficking and effector functions in ILC2-driven airway hyperreactivity
Authors: Hurrell Benjamin P , Galle-Treger Lauriane , Howard Emily , Jahani Pedram Shafiei , Santosh Swetha , Soroosh Pejman , Akbari Omid
Published: January 2023

Background

Asthma is a heterogeneous disease of the airways characterized by increased airway hyperreactivity (AHR) to specific and unspecific stimuli. Group 2 innate lymphoid cells (ILC2s) are type 2 secreting cells that are sufficient to induce eosinophilic lung inflammation and AHR independent of adaptive immunity in murine models. Identifying pathways that modulate ILC2 homeostasis and function are therefore an important step towards treating ILC2-dependent asthma.

Objective

We examined the expression of adhesion molecule lymphocyte-associated antigen 1 (LFA-1) and ligand intercellular adhesion molecule (ICAM)-1 on ILC2s and their effect on the development of lung inflammation and AHR in murine models.

Methods

Genetically modified mice constitutively lacking LFA-1 or ICAM-1 were assessed for the development of lung inflammation and AHR. We further generated mixed bone marrow chimeras and performed transcriptomic analysis to characterize their role on ILC2 homeostasis and function.

Results

LFA-1 is expressed on murine naïve and IL-33-activated ILC2s, while ICAM-1 is surprisingly expressed – and upregulated – upon activation. Compared to WT mice, both LFA-1−/− and ICAM-1−/− mice develop less lung inflammation and AHR in response to intranasal IL-33 challenge. Furthermore, experiments with chimeric mice revealed that LFA-1−/− ILC2s significantly traffic less to the lungs, unlike ICAM-1−/− ILC2s. Strikingly, ICAM-1−/− ILC2s however show a defect in proliferation and cytokine production, ultimately affecting lung inflammation.

Conclusions

The LFA-1-ICAM-1 interaction is involved in lung ILC2 trafficking, homeostasis and function. Such findings could lead to the development of new therapeutic targets.
Epithelial membrane protein 2 regulates transepithelial migration of neutrophils into the inflamed airspace
Authors: Lin Wan-Chi , Gowdy Kymberly , Madenspacher Jennifer , Zemans Rachel , Yamamoto Kazuko , Lyons-Cohen Miranda , Nakano Hideki , Janardhan Kyathanahalli , Williams Carmen , Cook Donald , Mizgerd Joseph , Fessler Michael
Published: January 2023

Neutrophil (PMN) migration from the circulation to the airway lumen is critical for pulmonary host defense but also causes bystander tissue injury that underlies a wide array of acute and chronic lung diseases. Whereas good evidence supports a role for intestinal epithelial cells in control of transmigration of extravasated PMNs into the intestinal lumen, little is known about whether and how respiratory epithelial cells regulate PMN influx into the airspace. Here, we identify epithelial membrane protein 2 (EMP2), a tetraspan membrane protein highly expressed in alveolar epithelial type 1 (AT1) cells but of unknown function in lung biology, as a master regulator of the terminal entry of PMNs into the airspace. EMP2−/− mice had reduced PMN influx into the airspace after multiple inhaled exposures and as a result exhibited attenuated lung injury and improved survival during bacterial pneumonia. Bone marrow chimeras, intravital PMN labelling, and in vitro assays suggest epithelial dysfunction in EMP2−/− lungs was responsible for deficient transepithelial PMN transit into airspace. We provide evidence that EMP2 supports epithelial surface display of multiple adhesion molecules that are required for cognate interactions with migrating PMNs and that it does so through suppression of epithelial caveolins. Moreover, EMP2 supports epithelial lipid raft mass in a manner dependent on putative cholesterol-binding motifs in its sequence. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular PMN traffic into the alveolus. Our findings identify EMP2 as a potential target for treatment of neutrophilic lung diseases.
Type1 IFN signaling on Tregs modulates the migration of Myeloid Derived Suppressor Cells
Authors: Tanwar Shalini , Metidji Amina , Shevach Ethan M
Published: January 2023

Multiple sclerosis is an autoimmune disorder whose severity is reduced with immune suppressive drugs. Therapeutic intervention with interferon-beta reduces disease exacerbations and delays relapses. The receptor for type 1 interferon, IFNAR, is present on virtually all cell types making it difficult to dissect the mechanisms involved. To address this issue, we induced experimental autoimmune encephalomyelitis (EAE) in mice with a conditional deletion (cKO) of IFNAR in T regulatory (Treg) cells (IFNARfl/flxFoxp3YFP-Cre). cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice appeared to be more activated, the activation status of and effector cytokine production by CD4+T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase, but higher in cKO CD4+T cells in the effector phase. Interestingly, we noted a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs in bone marrow and recruitment to spleen of WT and cKO mice were comparable. Surprisingly, we noted a decrease in the production of monocyte and granulocyte cell recruiting chemokine ligands by CD4+ and CD8+ T cells, B cells and endothelial cells in the dLN during the priming phase in the cKO mice. While modulation of Treg cell number and function by MDSCs has been documented, this study is one of the first to demonstrate that Treg cells modify the lymph node microenvironment by modulating MDSC homing and raises the possibility of a novel role for Treg cells in regulating the kinetics of MDSC recruitment during inflammatory conditions.

This research was supported by the Intramural Research Program of NIAID, NIH.
Limited recirculation of cytotoxic NK cells during normal homeostasis in humans
Authors: Hertwig Laura , Buggert Marcus , Knox James J. , Nguyen Son , Sparrelid Ernesto , Ivarsson Martin A. , Wedemeyer Heiner , Dori Yoav , Itkin Max G. , Betts Michael R , Björkström Niklas K.
Published: January 2023

Natural killer (NK) cells are well known for their cytolytic functions to directly kill virus-infected and malignant cells. Two major NK cell populations exist in humans: Cytotoxic CD56dim and immunoregulatory CD56bright NK cells. Although NK cells are distributed throughout the human body, little is known about which NK cell populations that traffic tissues and egress from tissues to recirculate back to blood. Here, we set outto determine the recirculation patterns of human NK cell subsets during normal homeostasis through access to multiple types of tissues, blood, and thoracic duct lymph (TDL). Whereas cytotoxic NK cells were dominant in peripheral blood, non-lymphoid tissues (liver, uterus, duodenum, adipose tissue) were enriched for immunoregulatory NK cells expressing markers of tissue-residency. Similarly, few cytotoxic NK cells were present in secondary lymphoid organs and efferent lymph (TDL). Instead, afferent and efferent venous blood of human liver was similar in NK cell subset composition as peripheral blood, suggesting that cytotoxic NK cells are retained in vasculature during steady-state. Ongoing mechanistic efforts include assessment of NK cell subsets in patients before and after blocking cellular egress from tissues with FTY-720 (S1PR agonist) treatment. These results offer a revised model for NK cell subset trafficking behavior and suggests that cytotoxic NK cells have limited access to peripheral tissues during normal homeostasis.
TNF targeting small molecule attenuates colonic inflammation in acute DSS-induced colitis mice
Authors: CHO OKKI , Jeong Young-Jin , Heo Tae-Hwe
Published: January 2023

Background

Inflammatory bowel disease (IBD) is a chronic and multifactorial inflammatory disorder which is comprising Crohn’s disease (CD) and ulcerative colitis (UC). Although the incidence of IBD has steady increase in worldwide, the conventional drug therapies with 5-aminosalicylates, corticosteroids, and immunosuppressants are insufficient to maintain remission and can occur side effects. Therefore, other class of drug therapies using specific cytokine inhibitor have recently been focused. In this study, we aimed to investigate that anti-inflammatory effects of TNF targeting small molecule in dextran sodium sulfate (DSS)-induced colitis model.

Methods

C57BL/6 (21–23g) mice were administered 2–2.5% DSS in the drinking water for 5 days to induce acute colitis. Different doses of IA-14069 (20, 40, 80, 160 mg/kg) were orally treated daily for 5–12 days and the mice were sacrificed for the experiments. Normal or DSS control group was given drinking water only or DSS only. Disease severity, body weight change, and colon length were measured, and serum levels of TNF, IFN-g, IL-6, IL-17 and IL-5 were analyzed by ELISA. Colonic epithelial damage was observed by H&E staining of colon tissue.

Results

Compared with normal group, oral administration of 80 mg/kg IA-14069 significantly inhibited the severity of colitis, loss of body weight, and colon shortening of the DSS-induced colitis mice. Moreover, mucosal damages and production of the proinflammatory cytokines were markedly suppressed by treatment of IA-14069.

Conclusion

These findings suggest that IA-14069, a TNF-targeting small molecule, exerts a suppressive effect on DSS-induced colitis and may be used as an IBD therapeutic agent.
Inhibition of ubiquitin-specific processing proteases 10 and 13 modulates IL-10 and TNF-α production in response to LPS in murine macrophages
Authors: Shah Pranav M , Talwar Harvinder , Samavati Lobelia
Published: January 2023

Innate immune response is activated by pathogen-associated molecular patterns (PAMPs) through Toll-like receptors (TLRs). To maintain immune homeostasis, TLR signaling molecules are regulated by a dynamic interplay of various ubiquitination (Ub) and deubiquitylation (DUB) events. Specifically, Lys-63 (K63)-linked ubiquitination on TRAF6 is involved in phosphorylation and activation of downstream MAPKs and IkB kinases. While certain types of ubiquitin modifications lead to activation and stability of proteins, other types cause increased proteasomal degradation and hence deactivation. DUBs are a class of the family of proteases that remove ubiquitin or ubiquitin-like proteins from a variety of protein-substrates. The role of DUBs in regulating the TLR pathway has not been well studied. Through RNA sequencing, we identified that LPS activation of bone marrow derived murine macrophages (BMDMs) leads to significant increase of two DUBs: ubiquitin-specific protease (USP) 10 and 13. To further study their role, we used Spautin-1, an inhibitor of USP10 and USP13. Spautin-1 modulated the LPS-mediated cytokine production and led to upregulation of IL-10, reduction of TNF-α, and had no effect on IL-6 production. However, we found no changes in expression of TRAF-6 or downstream MAPKs, including ERK, JNK, and P38. Alternative pathways were also studied with no effect in pSTAT-1, pSTAT-3, or A20 expression. Altered expression of pro-inflammatory mediators in the presence of Spautin-1 may have clinical importance associated with processes such as acute or chronic inflammatory processes.