bluuebunny/crossref_metadata_2025 huggingface dataset:
MiR-23~27~24 clusters control effector T cell differentiation and function
Authors: Lu Li-Fan , Cho Sunglim , Wu Cheng-Jang , Yasuda Tomoharu , Cruz Leilani Ong , Khan Aly Azeem , Lin Ling-Li , Nguyen Duc T , Miller Marina , Lee Hyang-Mi , Kuo Ming-Ling , Broide David H , Rajewsky Klaus , Rudensky Alexander Y
Published: January 2023

Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, here we show that the miR-23~27~24 clusters regulate multiple aspects of T cell biology, particularly Th2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23~27~24 clusters revealed miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, while overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17 and iTreg cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest tight regulation of miR-23~27~24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses.
The RNA binding protein HuR is necessary for IL-2 homeostasis and CD4+ T cell differentiation
Authors: Glascock Jacqueline J , Techasintana Patsharaporn , Ellis Jason S , Ridenhour Suzanne , Atasoy Ulus
Published: January 2023

RNA binding proteins, such as HuR (ELAVL1), fine tune gene expression in activated T cells by interacting with their 3′ UTR AU-rich elements (AREs) via posttranscriptional gene regulation. IL-2 and many Th2 cytokine mRNAs contain 3′ UTR AREs. Thus, we hypothesized that HuR may play a critical role in CD4+ T cell differentiation by regulating IL-2 signaling pathways. We conditionally ablated HuR in CD4+ T cells (distal lck-Cre ROSA HuRfl/fl) to test this hypothesis. HuR KO T cells are able to develop and egress the thymus normally. However, these cells cannot shut off IL-2 production and display defects in Th2 differentiation and cytokine expression. Proliferation defects, decreased p-stat5, and reductions in CD25 and prdm-1 transcription were also observed in the HuR KO T cells. In the absence of HuR, Il2ra (CD25) mRNA recruitment to heavy polysomes is reduced, indicating that HuR plays a critical role in its translation. This suggests that HuR regulates T cell activation and IL-2 homeostasis by controlling CD25 expression. When activated under Th0 conditions, 97% of HuR KO cells expressed IL-2 on day 5 compared to HuR WT cells (50%). HuR KO cells also had large increases in steady state IL-2 mRNA (30-fold) and 7-fold increases in secreted IL-2 protein. Furthermore, HuR KO cells produced scant amounts of the Th2 cytokines at the protein level. These findings led us to investigate the fate of other T cell lineages when HuR is absent. When polarized to Treg, Th17, and Tfh lineages, HuR KO T cells showed reduced levels of the signature transcription factors Foxp3, RoRγt, and Bcl6, respectively, as compared to controls. Thus, we conclude that HuR plays an indispensable role in normal IL-2 homeostasis and CD4+ T cell differentiation.
CD28 signals through tyrosine residues in its cytoplasmic tail to promote CD4+ T cell clonal expansion during infection
Authors: Krueger Peter D , Burbach Brandon , Malhotra Deepali , Miller Jim , Jenkins Marc K
Published: January 2023

CD28 is required for maximal proliferation of CD4+ T cells stimulated through their TCRs. Polyclonal peptide: MHCII-specific CD4+ T cells in mice lacking CD28 or its cytoplasmic tail produced one tenth as many effector and memory cells as wild-type T cells after bacterial infection. However, mutations in the canonical signaling sites, the membrane proximal YMNM and distal PYAP sequences, located in the cytoplasmic tail failed to suppress T cell expansion, suggesting additional sequences are sufficient for signaling. We therefore tested the contribution of the four tyrosine (4Y) residues in the cytoplasmic tail by establishing bone marrow chimeras in which CD28-deficient donor cells were first transduced with either wild-type CD28-CFP or mutated CD28-4Y-YFP and subsequently transferred to irradiated congenically mismatched CD28-sufficient mice. CD28-4Y MHCII-specific CD4+T cells produced one tenth as many effector cells as wild-type CD28-CFP cells after infection with bacterial expressing antigenic peptides. These results implicate that the tyrosine residues located in the cytoplasmic tail are required for CD28 signal transduction in effector CD4+ T cells in vivo.
Regulatory T cells evade restimulation-induced cell death by FOXP3-mediated repression of the SAP promoter.
Authors: Voss Kelsey , Katz Gil , Yan Toria , Kim Yong Chan , Scott David W , Snow Andrew L
Published: January 2023

An efficient adaptive immune response relies on the rapid clonal expansion of effector T cells, paired with timely disposal of those cells in order to avoid damage to host tissues and maintain immune homeostasis. Restimulation-induced cell death (RICD) is one programmed apoptotic pathway that ensures effector T cell expansion remains in check, triggered by repeated stimulation through the T cell receptor (TCR) in the presence of interleukin-2 (IL-2). CD4+ regulatory T cells (Tregs) require IL-2 and experience frequent TCR stimulation, but are highly resistant to RICD. Although the mechanism remains unclear, previous work has shown that RICD resistance in Tregs is dependent on the forkhead box P3 (FOXP3) transcriptional repressor, the master regulator of Treg function. We have previously shown that T cells from patients with X-linked lymphoproliferative disease (XLP-1), caused by null mutations in the signaling adaptor molecule SLAM-associated protein (SAP), are resistant to RICD. In fact, SAP is required for optimal RICD in effector T cells. We now demonstrate that Tregs express very low levels of SAP compared to conventional T cells (Tconv). Using chromatin immunoprecipitation and reporter assays, we show that FOXP3 reduces SAP expression in Tregs by binding to and repressing the SH2D1A (SAP) promoter. Indeed, knockdown of FOXP3 restores SAP-driven RICD sensitivity in Tregs through a FAS ligand-dependent mechanism. Intriguingly, our preliminary data suggest that FOXP3 also confers RICD resistance in Tconv during initial rounds of activation-induced proliferation. Hence our results provide a mechanism by which FOXP3 confers resistance to RICD to help preserve Tregs and protect early effector Tconv cells.
Requirement of EHD family of endocytic recycling regulators for T-cell function.
Authors: Iseka Fany M , Goetz Benjamin , Cypher Luke R , Bielecki Timothy , Storck Matthew , Mosely Lee , Band Vimla , Band Hamid
Published: January 2023

Regulatory mechanisms that ensure effective antigen-specific responses by T-cells without evoking autoimmune disease are of great interest in basic and clinical immunology. While signaling through T-cell receptor (TCR) and positive and negative co-receptors have received great attention, mechanisms that control the dynamic traffic of such receptors between the cell surface and intracellular compartments have received less attention. Based on the well-known ability of the TCR to undergo constitutive internalization and recycling to the cell surface, we hypothesized C-terminal Eps-15 Homology Domain-containing (EHD) proteins may play important roles in TCR-mediated T-cell functions by regulating the recycling of TCR and other receptors on T-cells. EHD proteins (EHD1-4) are key regulators of endocytic recycling of surface receptors, including the MHC-I and MHC-II proteins, but their role in TCR traffic or T-cell function is unknown. We found EHD1, 3 and 4 to be expressed in CD4+T cells. Accordingly, we generated a conditional knockout model in which floxed EHD1, 3 and 4 are deleted in CD4+T cells (CD4-Cre) of mice bearing a myelin oligodendrocyte glycoprotein (MOG)-specific TCR transgene. In vitro, CD4+T-cells from these mice exhibit reduced antigen-driven cell proliferation and IL-2 secretion. In vivo, these mice exhibit reduced severity of experimental autoimmune encephalomyelitis (EAE) upon immunization with antigen. Initial analyses suggest that the recycling of TCR (CD3ɛ) and the surface levels of TCR, CD28, LFA-1 and CD25 on the surface of CD4+ T cells are reduced by EHD1/3/4 knockout. Our studies reveal a novel role of the endocytic recycling proteins of the EHD family in TCR-mediated T-cell activation.
Differences in NAADP-mediated calcium signaling between natural regulatory T cells and naive or effector T cells.
Authors: Wall Katherine Ann , Ali Ramadan A. , Giovannucci David R. , Slama James T.
Published: January 2023

We previously showed that nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent calcium mobilizing second messenger discovered to date, is involved in the initiation and propagation of calcium signals in naive T cells. Combining live-cell imaging and a pharmacological approach using the NAADP antagonist Ned-19, we determined a role for NAADP in proliferation and cytokine production by Th1, Th2, Th17 and CD8 effector T cells. However, natural T regulatory (nTreg) cells showed a different profile. Ned-19 did not inhibit calcium flux in Tregs in response to T cell receptor stimulation. Externally added NAADP produced a significant calcium flux in naive and effector T cells, but not in nTregs. Ned-19 did not inhibit the proliferation of nTregs, which is very low, although it did inhibit proliferation of naive and effector T cells. These results suggest another way in which signaling pathways differ between effector and regulatory T cells that may be exploited to modify immune responses.
Aryl hydrocarbon receptor activation and IL-2 have synergistic effects that enhance human natural killer cell cytotoxicity.
Authors: Li Jian , Doty Andria , Glover Sarah Camile
Published: January 2023

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor. Initial studies of AHR concentrated on its role in the detoxification of xenobiotics. However, recent research has focused on its role in the immune system. AHR has been shown to be involved in the differentiation and maintenance of different immune cell subsets. Little is known about how AHR regulates immune cell effector function. In this study, we were able to show that AHR activation significantly increased IL-2-producing human NK-92MI cell degranulation capacity and cytotoxicity. However, for the FACS-sorted primary human NK cells from blood donor PBMC, AHR activation alone had no influence on NK cell degranulation and cytotoxic functions. We observed similar effector function enhancement of primary NK cells only with the addition of AHR agonist 6-Formylindolo (3,2-b)carbazole (FICZ) in conjunction with IL-2. Moreover, AHR activation significantly increased signaling factor MAPK1 expression in the NK-92MI cells, but not MAPK3 and NF-κB. Surprisingly, FICZ stimulation had no direct impact on human NK cell TNFα production, pre-formed proteins Perforin and Granzyme B production, as well as IL-2 receptor expression, but inhibited IFN-γ production slightly. In summary, AHR agonist FICZ acted synergistically with IL-2 to enhance human NK cell degranulation capacity and cytotoxicity function, possibly through activation of the MAPK1 signaling pathway which is involved in the polarization process of microtubule organizing center(MTOC) and lytic granules, a prerequisite for killing the target. Overall, the modulation of human NK cell response from AHR activation has revealed its potential therapeutic value.
Hemokinin-1 and Substance P provide adjuvancy for T cell activation and survival necessary for innate and adaptive cellular immune responses
Authors: Sumpter Tina L , Tkacheva Olga , Shufesky William , Falo Louis D , Morelli Adrian E , Larregina Adriana T
Published: January 2023

Chronic inflammatory and autoimmune disorders rely on the generation of efficient T cell responses. The pro-inflammatory neuropeptides, hemokinin-1 (HK-1) and substance P (SP) potentiate cellular immunity by signaling via the neurokinin-1 receptor (NK1R) expressed in leukocytes, including T cells. HK-1 is secreted by leukocytes while SP is released by sensory nerves and tissue resident cells. The role of NK1R signaling in the effector functions of T cells remains ill understood. We investigated the effects of signaling via the NK1R in the population of activated T-cells. In vitro, we compared the effects of HK-1 and SP on the survival and effector functions of wild type (WT) and NK1RKO CD4 and CD8 T cells. In vivo, we compared the effect of NK1R-signaling on T cells by using a delayed-type hypersensitivity (DTH) model in hosts with selective deletion of the NK1R in T cells. We demonstrate that NK1R-signaling is necessary for the generation, and maintenance of the survival and effector functions of CD4 and CD8 T cells. T cells secreted HK-1 and SP to promote self-activation and survival. Signaling via the NK1R resulted in Ca2+-influx in T cells, and that effect was impaired in NK1RKO T cells. Downstream, NK1R signaling activated the Ca2+-dependent NFAT1 and NFAT2 pathways, with the consequent increases in the synthesis and secretion of IL-2 that sustained T cell survival. In addition, NK1R-signaling stimulated the MAPK (c-Fos and c-Jun) and NF-kB pathways resulting in T cell activation. In vivo, DTH assays were significantly compromised in mice with selective deletion of NK1R+ T cells compared to WT. Our results elucidate the role and mechanisms of specific NK1R-T cell signaling in the generation and maintenance of potent T cell responses.
Calcium-dependence of cytotoxic granule endocytosis depends on the Flower protein
Authors: Chang Hsin-Fang , Mannebach Stefanie , Beck Andreas , Schirra Claudia , Pattu Varsha , Flockerzi Veit , Rettig Jens
Published: January 2023

Cytotoxic T lymphocytes (CTLs) kill their targets by secreting perforin and granzymes from cytotoxic granules (CGs) at the interface between CTL and target cells, the immunological synapse (IS). Retrieval of membrane proteins at the IS plays a crucial role in CTL effector function by impacting the serial killing ability of CTLs. However, the cellular and molecular mechanism of CG endocytosis is largely unknown. In neuronal synapses, it has been shown that calcium influx is instrumental to trigger endocytosis, but the identity of calcium channel remains unclear. Flower protein has been reported as a potential candidate to regulate synaptic endocytosis in drosophila. We investigated a potential calcium-dependence of CG endocytosis and whether Flower plays a role in this process. We first confirmed that Flower protein is expressed in WT mouse CD8+ CTLs by western blot. We then generated Flower-deficient mice to further study Flower function. While we observed no change of degranulation and CG secretion in Flower-deficient CTLs, CG endocytosis at the IS was entirely blocked upon target cell contact. Re-introduction of Flower protein or elevation of extracellular calcium rescued the endocytosis defect in Flower-deficient CTLs. Interestingly, live imaging data showed that Flower localizes on intracellular vesicles which polarize to the IS upon target cell contact. We conclude from our data that endocytosis of CGs is calcium-dependent and that the Flower protein is providing the required calcium, probably through release from intracellular organelles.
Use of Twitter in classroom attendance and participation in a Microbiology & Immunology course
Authors: Othman Tesneem , Royan Ayla , Fazal Nadeem
Published: January 2023

The use of social media in education has become increasingly prominent in recent years by educators in an attempt to improve students’ learning and to increase interaction in large classroom lectures. Among the popular social networking websites, Twitter is of particular interest due to its ease of use, micro blogging and hash tag capabilities. The objective of this study was to assess the use of Twitter in classroom attendance among professional first-year students. This study is distinguished by its focus on the pharmacy curricula, and among first year pharmacy students at CSU College of Pharmacy. All students enrolled in Microbiology and Immunology in the spring semester were included in the study. Students were permitted to use mobile devices in class and were recommended to create a Twitter account and add fellow colleagues and the course coordinator to participate in the study. Data from attendance was collected and analyzed according to the number of students that were present at the beginning of lecture, the number of students that were present until the end of lecture, the number of students that tweeted, the number of students that missed the tweet, and the number of students that tweeted their presence but were not actually in class. Since the study was voluntary, a total of 72 students signed a consent form to participate. An average of 59 students were present at the beginning and stayed until the end of lecture for the duration of the study. The analysis of variance (ANOVA) model was used to assess for differences in attendance at the beginning of class and at the end of class. This study demonstrates that the use of Twitter to take classroom attendance is more useful compared to traditional methods of roll call and sign-in sheets and announcements.
Subversion of macrophage autophagy and inflammatory pathways by Coxiella burnetii
Authors: Winchell Caylin G , Graham Joseph G. , Kurten Richard C , Voth Daniel E
Published: January 2023

Coxiella burnetii is the causative agent of human Q fever, a debilitating flu like illness that can progress to chronic disease presenting as endocarditis. After inhalation, C. burnetii is phagocytosed by alveolar macrophages and generates a lysosome-like replication niche known as the parasitophorous vacuole (PV). A type IV secretion system (T4SS) is required for generation of the PV and is one of the pathogen’s few known virulence factors. We previously showed that C. burnetii actively recruits autophagosomes to the PV using the T4SS, but does not alter macroautophagy. Interestingly, the cargo receptor p62 localizes to the PV in a T4SS-dependent manner and co-localizes with LC3. p62 and LC3 interact to select cargo for autophagy-mediated degradation, resulting in p62 degradation and LC3 recycling. In C. burnetii-infected macrophages, p62 is not degraded when cells are starved. We hypothesized that stabilization of p62 was due to activation of the NRF2-KEAP1 pathway, a cytoprotective response to oxidative stress and xenobiotics. Indeed, the NRF2-KEAP1 pathway was activated during infection as evidenced by translocation of NRF2 to the nucleus. A downstream target of the NRF2-KEAP1 pathway is NF-kB, and the role of this inflammatory signaling cascade in C. burnetii pathogenesis is unclear. However, previous studies showed differences in inflammasome activation when macrophages were infected with avirulent or virulent C. burnetii isolates. Therefore, we assessed NF-kB activation in alveolar macrophages infected by avirulent or virulent C. burnetii. Collectively, our studies highlight important host cell signaling pathways exploited by C. burnetii and differences in the innate immune response to differing pathotypes.
Accumulating necrotic cell debris and damaged mitochondria contribute to sepsis-like syndrome during late-phase tularemia.
Authors: Singh Anju , Periasamy Sivakumar , Rahman Tabassum , Ault Jeffrey G. , manella Carmen , Sellati Timothy J.
Published: January 2023

Infection with Francisella tularensis SchuS4 causes a severe pulmonary disease in mice, non-human primates, and patients known as tularemia. F. tularensis subverts host cell death programs to facilitate their unfettered replication within macrophages and other cell types, a common feature of early respiratory infection (≤ 72 h). The mechanism(s) by which the complex process of cell death is initiated remains incompletely-defined as does the impact of F. tularensis on mitochondria, the host cell’s organellar ‘canary in a coal mine’. Herein we reveal that host cells undergo Bax-dependent mitochondrial apoptosis via the intrinsic pathway. These events encompass: i) decreased mitochondrial membrane potential and consequent mitochondrial oncosis, ii) increased mitochondrial superoxide generation, and iii) release of damaged mitochondria and other damage-associated molecular patterns into the lung parenchyma, which contributes to a sepsis-like syndrome. Notably, host recognition of and response to accumulating necrotic cell debris elicits the TNF, IL-1β, IL-6, IL-12p70, and IFN-γ which are absent during early infection, but present during late-phase septic disease (≥ 72 h).
Helminth parasites and cancer-causing tumors condition human monocytes similarly: insights into parallel mechanisms of immune evasion
Authors: Narasimhan Prakash Babu , Akabas Leor , Huda Naureen , Sabzevari Helen , Hofmeister Robert , Nutman Thomas B , Semnani Roshanak Tolouei
Published: January 2023

A number of features of the host-parasite interface are reminiscent of those that are also observed at the host-cancer interface. Both cancer cells and parasites establish a tissue microenvironment that leads to immune evasion and may be related to alterations in function of various innate cells. Here, we investigate how the phenotype and function of human monocytes is altered by exposure to tumors and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi – a causative agent of lymphatic filariasis. After 2 days of culture, cells were harvested, sorted and assessed for mRNA expression, phenotype and function. Monocytes exposed to tumor cell lines showed markedly upregulated expression of M1-associated (TNF-a, IL-1b, IL-8), M2-associated (CCL13, CD206) and Mreg-associated (IL-10, IDO, TGF-b) genes. Similar to cancer cells, but less dramatically mf altered the mRNA expression of IL-1b, CCL13 and IL-8. Similar to the exposure to cancer cell lines, exposure of monocytes to live mf significantly (p<0.05) induced the cell surface expression of the inhibitory ligands PDL1 and PDL2. Finally, exposure of monocytes to cancer cell lines increased the phagocytic ability of these cells, but reduced their ability to induce T cell proliferation, an effect shown to be reversible by PDL1/PD1 blockade. Our data suggest that despite the fact that helminth parasites and tumor cell lines are extraordinarily disparate, they share the ability to alter the phenotype and function of human monocytes and may also share a common mechanism of immune evasion.
The phagosome: Meeting point of the Myddosome, NLRs, and degraded Borrelia burgdorferi
Authors: Cervantes Jorge L. , Benjamin Sarah J. , Chang Yuxiao , Luo Junhong , La Vake Carson J. , Hawley Kelly L. , Caimano Melissa , Vera-Licona Paola , Laubenbacher Reinhard , Ruan Yijun , Radolf Justin , Salazar Juan C.
Published: January 2023

Phagocytosis of the extracellular bacterium Borrelia burgdorferi (Bb) enhances toll-like receptor, NF-κB mediated cytokine responses in human and murine phagocytic cells. TLR adaptor molecule MyD88 is recruited to Bb containing phagosomes, and the spirochetal cargo does not appear to leak from the phagosome into the cytosol. Transcriptome and Network reconstruction analysis identified several master regulators of diverse cellular and molecular processes, including regulation of the TLR-MyD88 pathway, IRFs, cytoskeletal rearrangement, and resolution of inflammation. Surprisingly, genes that code for inflammasome proteins (NLRP3, NOD2 and caspase-1) were also differentially-expressed. We thereby examined the cellular localization of these molecules in Bb-infected WT BMDMs and observed that NLRP3 and NOD2 were observed to colocalize to phagosomes containing degraded spirochetes. NLRP3 also colocalized with MyD88, but only in phagosomes containing degraded spirochetal cargo. Although caspase-1 also colocalized to the phagosome, infection with Bb did not result in an increase in speck formation or secretion of IL-1β.

Our results show that the phagosome is a crucial cellular location not only for degradation of the spirochete, but where components of the Myddosome and inflammasome are recruited. Our findings are in line with recent studies showing that cytosolic receptors can localize to endosomal membranes, and can potentially have an alternate role in regulating an adequate functionality of the phagosome. Our study evidences the complexity of phagosomal signaling in which MyD88 plays a critical role not only in the generation of inflammatory but inducing regulatory signals in murine macrophages upon Bb infection.
Autophagy promotes Rickettsia australis replication in primary murine macrophages
Authors: Bechelli Jeremy Ray , Smalley Claire , Zhao Xuemei , Hwang Seungmin , Walker David Hughes , Fang Rong
Published: January 2023

Rickettsiae are obligately intracellular bacteria that replicate in the cytoplasm of eukaryotic cells. We found autophagy induction and co-localization of bacteria with LC3 (+) autophagosomes in murine bone marrow-derived macrophages (BMMs) upon infection with R. australis. Autophagy induction by rickettsiae in BMMs of Atg5flox/flox mice was completely abrogated in the counterparts of Atg5flox/flox Lyz-Cre animals. Rickettsial replication was significantly greater in BMMs of Atg5flox/flox mice compared to Atg5flox/flox Lyz-Cre mice, suggesting that Atg5-dependent autophagy promotes rickettsial growth in primary mouse macrophages. Inhibition of mTOR signaling also significantly promoted rickettsial replication in macrophages compared to untreated controls. Furthermore, R. australis-infected Atg5flox/flox macrophages secreted significantly lower levels of pro-inflammatory cytokines including inflammasome-dependent IL-1β compared to Atg5flox/flox Lyz-Cre macrophages. Strikingly, rickettsial growth in Atg5flox/flox macrophages was not significantly different from that in Atg5flox/flox Lyz-Cre macrophages when cells were treated with neutralizing antibodies against IL-1β, suggesting that IL-1β is involved in differential bacterial growth in these cells. In line with the in vitro results, the in vivo rickettsial loads in liver, lung and spleen of infected Atg5flox/flox mice were significantly higher than those in Atg5flox/flox Lyz-Cre mice accompanied by a significantly lower level of IL-β in the serum. Taken together, our data demonstrated the role of autophagy in promoting rickettsial replication in mouse macrophages both in vitro and in vivo.
A role of lamina propria ILC1 in the impaired host antibacterial resistance of chronic alcohol consuming mice
Authors: Ito Ichiaki , Asai Akira , McCalla Claudia T , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Bacterial translocation and subsequent sepsis influenced by ILC1 in lamina propria were studied in chronic alcohol consuming mice (CAC mice) exposed to Enterococcus faecalis oral infection. CAC mice were prepared by an oral gavage of 0.6 ml/day of 20% EtOH for 12 weeks. Control mice received water in the same fashion. All mice were decontaminated and orally infected with 106 CFU/mouse of E. faecalis. The numbers of bacteria in mesenteric lymph nodes (MLNs) and liver of these mice were measured by the colony counting method. Intestines were removed from control and CAC mice, and digested with 100 U/ml of collagenase. Mononuclear cells were isolated from the cell suspension by Percoll density gradient centrifugation. Lineage negative (Lin−) and positive (Lin+) cells were purified magnetically from the mononuclear cells, and analyzed for the population of M1 macrophages (Mϕ) and innate lymphoid cells (ILCs) by flow cytometry. In the results, enterococcal translocation and subsequent sepsis were severely shown in CAC mice, as compared with those of control mice. The number of IFNγ-producing ILC1 was not increased in lamina propria of CAC mice orally infected with E. faecalis, while the large number of ILC1 was isolated from lamina propria of control mice infected with the pathogen. Also, an adequate number of M1Mϕ was isolated from lamina propria of control mice orally infected with E. faecalis, whereas M1Mϕ were not isolated significantly from lamina propria of CAC mice infected with the pathogen. Quiescent Mϕ switch to M1Mϕ in response to IFNγ stimulation. These results suggest that lamina propria ILC1 play an important role in the increased susceptibility of CAC mice to E. faecalis translocation and subsequent sepsis.
The major contribution of IL-1 alpha and caspase-11 in oviduct pathology during Chlamydia infection in the mouse model
Authors: Nagarajan Uma , Allen John , Zhang Yugen , Tripathy Manoj , Montgomery Stephanie
Published: January 2023

The mouse model of genital Chlamydia trachomatis infection simulates the human chlamydial disease resulting in oviduct damage leading to hydrosalpinx. Using this model, we have shown that signaling by IL-1R is a major contributor of oviduct disease during infection. In the current study, we have investigated the specific contribution of IL-1 alpha and IL-1 beta that signal thorough IL-1R. Further, the contribution of caspase11-mediated cell-death that results in the release of IL-1 alpha and IL-1 beta were investigated. IL-1 alpha KO mice developed significantly less incidence (5%) of hydrosalpinx compared to WT mice (75%) (p=<0.001) upon infection. IL-1 beta KO mice had a reduced incidence in hydrosalpinx than WT mice, but significantly higher than IL-1 alpha KO mice. To determine the mechanism of IL-1 alpha release, infection course and pathology in caspase-1-11 KO mice and caspase-11 KO mice were determined. Both caspase-11 and caspase-1-11 KO mice displayed a reduced incidence of hydrosalpinx. Since the caspase-1 adaptor, ASC KO mice do not show a reduced incidence in pathology, our data suggest that caspase-11 activation is sufficient to drive the pathology. Oviduct pathology is a result of excessive PMN infiltration. Flow cytometric analyses demonstrate significantly reduced PMN infiltration to the genital tracts in IL-1 alpha and caspase-11 KO mice. However, T cell infiltration to the genital tracts is unchanged in both the groups, which reflected in normal infection clearance in IL-1 alpha and caspase-11 KO mice. These data suggest that activation of cell death pathways and local release of IL-1 alpha causes long-term damage to the oviducts during Chlamydia infection.

[Supported by PHS funding NIAID AI067678 (UN)]
Antagonising roles between PACT and influenza A virus polymerase subunits in viral replication and innate immune response
Authors: Chan Chi Ping , Yuen Chun-Kit , Fung Sin-Yee , Kew Chun , Yuen Kwok-Yung , Kok Kin-Hang , Jin Dong-Yan
Published: January 2023

Interplay between host innate immune responses and viral antagonism determines the effectiveness of viral replication and pathogenicity. Influenza A virus (IAV) confronts host antiviral responses including type I interferon production using multiple viral proteins such as NS1. We have previously shown that double stranded RNA-binding protein PACT is an essential coactivator of RIG-I in innate antiviral response. In this study, we have further shown that PACT has dual antiviral activity in both interferon-dependent and interferon-independent manners. We found that antiviral activity of PACT is mediated through not only the activation of RIG-I and interferon production but also the direct suppression of IAV polymerase activity. We found that PACT associates with IAV polymerase subunits, namely PB2, PB1 and PA, and inhibits viral replication. On the other hand, the three polymerase subunits also counteract PACT/RIG-I-mediated production of type-I interferon. Taken together, our findings reveal a new mechanism of virus-host interaction demonstrating the mutual antagonism between PACT and IAV polymerases during IAV infection and replication.

Supported by RGC (HKU1/CRF/11G, N-HKU712/12, T11-707/15-R and C7011-15R), HMRF (12111312, 14130862, HKM-15-M01 and 15140662) and S. K. Yee MRF (2011).
Evolution of host-defense function of C-reactive protein from horseshoe crab to humans
Authors: Pathak Asmita , Singh Sanjay K , Thirumalai Avinash , Armstrong Peter B , Agrawal Alok
Published: January 2023

C-reactive protein (CRP) has been conserved throughout evolution. Human native CRP exhibits calcium-dependent binding specificity for phosphocholine. Human CRP in its non-native structure expresses the capability to bind to deposited and conformationally-altered proteins and which can be achieved by several means including treatment of CRP with acidic pH. The ligand-binding property of human CRP in its non-native structure has implications for toxic and inflammatory conditions and favors the conservation of CRP throughout evolution. It is not known, however, whether CRP from invertebrates exhibits structure-based ligand-binding properties similar to that of human CRP. The aim of this study was to investigate the ligand-binding properties of CRP from the American horseshoe crab Limulus polyphemus. We used oxidized low-density lipoprotein (ox-LDL) immobilized on microtiter plates as a model for deposited and conformationally-altered proteins. We found that Limulus CRP binds to ox-LDL at physiological pH, in contrast to human CRP which requires acidic pH to do so. The binding of Limulus CRP to ox-LDL occurred even in the absence of calcium, suggesting that the binding was not mediated through exposed phosphocholine molecules, if any, on ox-LDL. We conclude that the host-defense function of CRP evolved with the development of the immune system to expose a ligand-binding specificity only when needed, that is, an inflammatory microenvironment would have to be sensed by CRP and that CRP would change its structure to execute its function. Limulus CRP also provides us with a tool to investigate the structure-function relationships of human CRP in animal models of inflammation.
Crystal structure of human tetraspanin CD81 reveals a conserved cholesterol binding cavity
Authors: Zimmerman Brandon , McMillan Brian J. , Seegar Tom C.M. , Kruse Andrew C. , Blacklow Stephen C
Published: January 2023

Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in cell activation, adhesion, migration and proliferation. CD81/Tspan28, the most widely studied member of the family, forms a complex with CD19, CD21 and CD225 to regulate B cell receptor function, and also acts as a receptor or co-receptor for pathogens such as Hepatitis C virus and Plasmodium species. Despite the fundamental importance of tetraspanin proteins in a broad range of physiologic and pathophysiologic processes, structural information about a full-length tetraspanin has remained elusive. Here we present the first crystal structure of a full-length tetraspanin, human CD81. The transmembrane segments of CD81 pack as two largely separated pairs of helices, not as the four-helix bundle predicted previously. The structure is capped by the large extracellular loop 2 (EC2) at the outer membrane leaflet, and the two pairs of helices converge at the inner leaflet to form a shape resembling an inverted teepee. The transmembrane region contains a large pocket suggestive of a ligand-binding site, offering a potential mechanism for binding lipids or other molecules. In support of this idea, we observe electron density consistent with a bound cholesterol molecule in the pocket, and we detect specific binding of cholesterol to this site in vitro. The discovery of the hydrophobic pocket in the core of the transmembrane domain presents a novel therapeutic avenue for targeting CD81 and other tetraspanins in pathogenic infections or malignancies.
B-Cell Selection in Germinal Centers Elicited by Complex Antigens
Authors: Kuraoka Masayuki , Schmidt Aaron G. , Nojima Takuya , Feng Feng , Watanabe Akiko , Kitamura Daisuke , Harrison Stephen C. , Kepler Thomas B. , Kelsoe Garnett
Published: January 2023

Germinal center (GC) B cells evolve towards increased affinity by a Darwinian process studied intensively in genetically restricted, hapten-specific responses. While experimentally tractable, genetically restricted humoral responses are atypical as antibodies to complex protein antigens represent genetically diverse, polyclonal humoral responses driven by various epitopes arrayed across the antigen. We have developed a single B-cell culture method that supports the proliferation and plasmacytic differentiation of mature and GC B cells. With this tool, we explore the population dynamics of genetically diverse GC responses to two complex antigens – protective antigen of Bacillus anthracis and influenza hemagglutinin – in which B cells compete both intra- and interclonally for distinct epitopes. Our characterizations begin with antigen-binding, mature naïve B cells and follow clonal selection and affinity maturation in GCs. Preferred VH rearrangements among antigen-binding, naïve B cells were similarly abundant in early GCs but, unlike restricted responses to haptens, clonal diversity increased in GC B cells as early “winners” were replaced by rarer, high affinity clones. Despite affinity maturation, half of GC B cells did not detectably bind immunogen but exhibited genetic selection comparable to antigen-binding cells, as determined by VH usage, mutations, and clonal expansion. In GCs elicited by rPA or rHA, interclonal BCR avidities can differ 100-fold and intraclonal avidity by as much as 40-fold. We propose that intraclonal selection in GCs is permissive for a wide range of BCR affinities and that lower affinity/less fit GC B cells may remain viable in GCs for substantially longer periods than generally thought.
Disruption of Folliculin interacting protein-1 results in loss of sIg+ B cells and inhibits autophagy
Authors: Ramirez Julita , Tsang Mark , Margineantu Daciana , Iwata Terri , Hockenbery David M , Iritani Brian M
Published: January 2023

Folliculin interacting protein-1 (Fnip1) was discovered through its interaction with Folliculin (Flcn), a tumor suppressor mutated in the rare disorder Birt-Hogg Dubé syndrome. The Fnip1/Flcn complex interacts with the catabolic regulator AMP kinase. Previous studies have shown that Fnip1 is required for the development of B-cells past the late pre-B stage (Park et al Immunity 2012) and for the development of invariant natural killer T (iNKT) cells (Park et al PNAS 2014). We previously found Fnip1-deficient immune cells exhibited concurrently increased activation of mTORC1 and AMPK. In this study, we used a floxed Fnip1 allele combined with poly inosine:cytosine (poly I:C)-inducible Mx1-Cre to investigate the effects of conditional Fnip1 depletion on immature and mature B cells, and utilized genetic experiments to investigate the mechanism(s) of the B cell developmental block. We show loss of sIg+ B cells in the bone marrow and spleen after conditional Fnip1 depletion. Fnip1-deficient B cells exhibited significantly increased metabolism (oxidative phosphorylation and glycolysis) upon stimulation, possibly as a result of increased mitochondrial number and mTOR activation. Bcl-xL and c-Myc overexpression rescued a small fraction of sIg+ B cells in Fnip1-deficient mice, while p53 deficiency (Trp53−/−) and mTORC1 inactivation (Raptorfl/flmb1-cre) did not. Finally, we show that Fnip1-deficient B cells expressing LC3-GFP have lower mean GFP fluorescence and do not respond as robustly as wild type B cells to induction of autophagy. These results suggest that Fnip1 mediates autophagy in B cells, and that tight regulation of autophagic flux is necessary for development and survival of immature and mature B cells.
Differential apoptosis sensitivity of human effector CD8+ T cells derived from central versus effector memory subsets in response to IL-2 withdrawal
Authors: Larsen Sasha E , Voss Kelsey , Snow Andrew L
Published: January 2023

CD8+ central memory (CM) and effector memory (EM) T cell subsets exhibit well-established differences in proliferative and protective capacity after infectious challenge. However, their relative sensitivity to apoptosis has been largely overlooked, despite the importance of programmed cell death in regulating effector T cell homeostasis. Here we demonstrate that primary human effector T cells derived from the CD8+ EM subset exhibit significantly higher sensitivity to cytokine withdrawal induced cell death (CWID), a critical intrinsic apoptosis program responsible for culling cells once an infection is cleared and interleukin-2 (IL-2) levels diminish. Interestingly, we found no differences in the expression of IL-2 or IL-2 receptor components in cells originating from either subset. Relative to CM-derived effectors, however, EM-derived T cells display more mitochondrial instability and greater basal caspase activation. Indeed, we found that heightened CWID sensitivity in EM-derived effectors is linked to higher expression of the pro-apoptotic Bcl-2 family protein BIM, both at steady state and with de novo induction following withdrawal of exogenous IL-2. These data point to “imprinted” differences in BIM protein regulation preserved between CD8+ CM and EM progeny that govern their relative sensitivity to CWID. These findings offer new insight into why CM-derived T cells display superior effector cell expansion and more persistent memory responses in vivo relative to EM-derived T cells, based on differential apoptosis sensitivity.
Despite KLRG1 expression, highly cytotoxic ThEO/TcEO polarized T cells efficiently establish immunologic memory
Authors: Bartkowiak Todd , Jaiswal Ashvin R , Ai Midan , Budhani Pratha , Ager Casey , Curran Michael A
Published: January 2023

Targeting immune inhibitory (CTLA-4, PD-1) or stimulatory (OX-40, 4-1BB) co-receptors has proven to be a potent anti-cancer therapeutic strategy, eliciting robust anti-tumor T cell responses and yielding compelling pre-clinical and clinical data in a variety of cancers. We have previously shown in pre-clinical models that 4-1BB agonist antibodies engender strong anti-tumor T cell responses capable of driving regression of established tumors and durable tumor-free survival. This impressive anti-tumor effect is due, in part, to the unique ability of 4-1BB co-stimulation to differentiate a novel subset of T cells that acquires a highly cytotoxic effector profile typically only seen in NK cells. This phenotype- known as ThEO (CD4+) or TcEO (CD8+)-is driven by the T-box transcription factor Eomesodermin, and is further characterized by expression of the inhibitory receptor KLRG1. We show here that, despite high level effector function and expression of several co-inhibitory receptors (PD-1, TIM-3, KLRG1), these ThEO/TcEO phenotype T cells efficiently establish immunologic memory and recall with high frequency upon secondary tumor challenge. Further, they maintain high effector function on recall and can traffic to tissue sites far removed from the original malignancy. These findings provide mechanistic insight into the process of memory formation and give evidence for the capacity of ThEO/TcEO cells to prevent tumor recurrence.
The adaptor molecule BCAP is essential for the formation of CD8+ T cell memory
Authors: Singh Mark Dharam , Hamerman Jessica A , Campbell Daniel J.
Published: January 2023

Generation of effector and memory CD8+ T cells is pivotal for the clearance of intracellular infections and forms the basis for vaccination strategies against a wide range of pathogens and anti-cancer immunotherapy. We have generated novel data which shows that the signaling scaffold protein B cell adaptor for PI3-kinase (BCAP) is upregulated in activated CD8+ T cells, links TCR/CD3 engagement to the PI3K signaling cascade and is essential for CD8+ T cell memory formation. To analyze the cell intrinsic properties of BCAP in CD8+ T cells, we used OT-1+ mice which have a high affinity CD8+ T Cell Receptor (TCR) for the ova peptide. We transferred 5,000 WT OT-1+CD45.1+cells and 5,000 BCAPKO OT-1+CD45.2+ cells into CD45.1+CD45.2+ recipients, then infected with Listeria Monocytogenes expressing the ova peptide. We found that BCAP is essential for the proper clonal expansion of CD8+ T cells and BCAP deficiency in CD8+ T cells resulted in a loss of KLRG1+CD127− Terminal Effector Cells (TECs) in favor of KLRG1− CD127+ Memory Precursor Cells (MPCs) in vivo. Strikingly, we observed an enhanced CD8+ T cell contraction phenotype in BCAP deficient CD8+ T cells which led to a significant loss of antigen-specific memory CD8+ T cells. BCAPKO CD8+ T cells were undetectable in 50% of all mice analyzed at day 100 post infection. Therefore BCAP is critical for the formation of CD8+ T cell memory. We found the greatest loss of cells was confined to the recently described KLRG1+CD27loCXCR3loEomeslo effector memory cells, which have potent cytolytic function and anti-cancer capabilities. Finally we found that the ability of BCAPKO memory CD8+ T cells to respond to secondary infection was dysregulated. To conclude, BCAP is essential for the formation of CD8+ T cell memory.
Control of TH17 and Treg balance by RhoGTPase Cdc42
Authors: Kalim Khalid Wasim , Yang Jun-Qi , Li Yuan , Zheng Yi , Guo Fukun
Published: January 2023

Balanced Th17 and regulatory T (Treg) cell differentiation and function are important for normal immunity and avoidance of autoimmunity. Cdc42 of the Rho GTPase family is an intracellular signal transducer that cycles between an inactive GDP- bound form and an active GTP-bound form under tight regulation. We have recently reported that Cdc42 is required for thymocyte development but suppresses Th1 cell differentiation, with no effect on Th2 cells. In this study, we further examined the role of Cdc42 in Th17 and Treg cell differentiation, using a T cell-specific Cdc42 conditional knockout mouse model. Cdc42 deficiency led to an enhanced Th17 differentiation evidenced by increased IL-17+ cells with increased STAT3 phosphorylation, under in vitro Th17 polarizing condition. In the absence of Cdc42, Foxp3+ cells were drastically reduced under in vitro Treg polarizing condition accompanied by less phosphorylation of Smad2 and Smad3 transcription factors. Functionally, in vitro Treg suppression assay found that Cdc42-deficient Treg cells were impaired in their suppressive activity. In both DSS induced colitis and naïve T cell transfer model of colitis in vivo, Cdc42 deficiency led to an exacerbated disease severity and increased infiltration of Th17 and Th1 cells with less Tregs in spleen, mLN and lamina propria of colon. Finally, Treg cell-specific deletion of Cdc42 resulted in systemic autoimmunity with reduced Treg cells and accordingly spontaneous T cell activation and increased IL-17+IFNγ+ double positive cells. In summary, our data have provided new insights into the regulation of the balance between Th17 and Treg differentiation and identified Cdc42 as a potential molecular target for the treatment of colitis.