bluuebunny/crossref_metadata_2025 huggingface dataset:
The impact of bacterial superinfection during influenza infection of allergic hosts
Authors: Samarasinghe Amali , LeMessurier Kim , Iverson Amy , Rosch Jason , McCullers Jonathan
Published: January 2023

Allergic asthma can be complicated by respiratory pathogens. However, asthmatics suffered less severe morbidity and mortality compared to non-asthmatics during the 2009 influenza A virus (IAV) pandemic. Using a novel mouse model, we determined that IAV infection during allergic airways inflammation protects the host from influenza. Pathogen-pathogen interactions between IAV and Streptococcus pneumoniae (Spn) can lead to fatal outcomes in a healthy host. We hypothesized that allergic hosts will have less morbidity and mortality associated with Spn superinfection after influenza. Infectious IAV was undetectable in the lungs at 72 h after Spn. Similar to animals infected with Spn alone, asthma+Spn mice cleared the bacteria completely by 72 h. Bacterial burden increased in the bronchoalveolar lavage fluid (BALF), lungs, and blood of mice in the IAV+Spn co-infection group. However, bacterial load in animals subjected to the fungal asthma model prior to viral and bacterial infections were significantly reduced in the BALF and absent in the lungs and blood. More leukocytes were found in the BALF of mice infected with IAV. The number of macrophages was similar between all groups, while those in the asthma+IAV, asthma+Spn, asthma+IAV+Spn groups had prominent amounts of eosinophils and CD4+ T cells (30% each) in the airways which was significantly higher than the asthma-only, IAV-only, Spn-only, and IAV+Spn groups. Neutrophils were most abundant in the IAV+Spn controls while the greatest number of CD8+ T cells was present in the IAV-only controls. The lung microbiome was significantly different in asthma+IAV+Spn. Remarkably, the asthma+IAV+Spn group did not have severe morbidity or mortality typically associated with IAV+Spn coinfection.
PGE2 imbalance in diabetic mice impairs dendritic cell maturation and favors methicillin-resistant S. aureus skin infection
Authors: Dejani Naiara , Brandt Stephanie , Pinneros Annie , Glosson-Byers Nicole , Wang Wang , Medeiros Alexandra , Serezani Carlos Henrique
Published: January 2023

Diabetics are more prone to Staphylococcus aureus skin infection than healthy individuals. Neutrophil recruitment and apoptosis are hallmarks of S. aureus infections. Ingestion of infected apoptotic cells (IAC) by dendritic cells (DC) lead to secretion of Th17-related cytokines and prostaglandin E2 (PGE2). We speculate that hyperglycemia inhibits DC migration to the lymph nodes, in a manner dependent on PGE2 levels and impairs Th17 differentiation during MRSA skin infection in type 1 diabetic mice (T1D). Control and T1D mice were subcutaneously (s.c.) infected with 5×106 MRSA CFU. T1D mice showed increased lesion and bacterial load and decreased Th17 population in the skin than nondiabetic mice. We next determined if decreased Th17 correlates with deficient DC migration to draining LN. To track DC migration, mice received s.c. FarRed to stain the skin cells. Our results show that T1D had lower percentage and number of FarRed+DCs+ in the LN than controls.. Impaired DC migration and Th17 differentiation in T1D correlated with decreased skin PGE2 levels. When BMDCs were cultured in high glucose, we observed decreased PGE2 production, migration and maturation after recognition of MRSA-IAC when compared to BMDC kept in normal glucose. Topical treatment with PGE analog (Misoprostol), restored skin DC migration, Th17 differentiation, increased anti-microbial peptides, decreased CFU loads and lesion size in T1D. These data suggest that PGE2 imbalance in hyperglycemia impairs DC migration to LN and fail to mount Th17 responses leading to increased lesions and delayed healing in MRSA skin infections. Misoprostol may be a promising candidate to improve innate and adaptive immune responses in diabetic skin infections.
Regulation of MerTK, GPNMB, and VSIG4 on mouse macrophages
Authors: Del Cid Natasha , Stepniak Dariusz T , Ferrer Allison , Scott Brad , Magana Jonathan , Li Xiaoxiao , Ma Lisa , Miranda Cherlyn , Li Shourong , Schrantz Nicolas , Huang Qiulong , Becker-Catania Sara , Funatake Castle J , Just Peggy
Published: January 2023

We have developed flow cytometry antibodies against three molecules expressed on tissue-resident mouse macrophages: MerTK, GPNMB (aka Osteoactivin, DC-HIL), and VSIG4 (aka CRIg). MerTK, a receptor tyrosine kinase, plays a crucial role in the clearance of apoptotic cells, as well as in the regulation of cytokine secretion and survival. MerTK is highly expressed by splenic red pulp macrophages, which aid in the clearance of apoptotic cells in the blood. GPNMB, a transmembrane glycoprotein, plays a role in negatively regulating cytokine secretion during inflammatory responses and is expressed on tissue-resident macrophages, including microglia. Finally, VSIG4, a B7 family-related complement receptor, negatively regulates T cell-mediated responses and is also expressed on tissue-resident macrophages. The three clones DS5MMER (MerTK), CTSREVL (GPNMB), and NLA14 (VSIG4), were validated using resident and thioglycollate-elicited peritoneal exudate cells. DS5MMER (MerTK) and NLA14 (VSIG4) were also validated by IHC using frozen mouse tissue samples. Here, we have measured surface levels of MerTK, GPNMB, and VSIG4 on mouse bone marrow-derived macrophages by flow cytometry in response to an array of cytokines. We have also assessed the distribution of MerTK- and VSIG4-positive cells in various mouse tissues. Our data show how MerTK, GPNMB, and VSIG4 respond to various pro- and anti-inflammatory conditions in mouse bone marrow-derived macrophages and gives insight into how these receptors may be regulated on mouse macrophages in vivo.
LPS-induced interaction between Cd300b/DAP12 and TLR4/CD14 results in the amplification of inflammation and increased mortality from sepsis.
Authors: Voss Oliver H. , Coligan John E.
Published: January 2023

LPS, present in gram-negative bacteria membranes, causes a strong immune cell activation following detection by TLR4, which initiates a pro-inflammatory cytokine storm that leads to a more severe immunopathology, like septic shock and, subsequently, death. Recently, the myeloid-specific Cd300b receptor was implicated in regulating the immune response to bacterial infection by an unknown mechanism.

Here, we identified LPS as a ligand for Cd300b and found that wild-type (WT) mice, unlike Cd300b−/− mice, were highly susceptible to septic shock. WT but not Cd300b−/− mice show increased serum levels of pro-inflammatory cytokines (e.g. TNFa) and a reduced level of the anti-inflammatory cytokine, IL-10. Neutralization of IL-10 in Cd300b−/− mice diminishes their survival advantage over WT mice. In vivo depletion and adoptive transfer studies identify Cd300b-expressing macrophages as the key cell type augmenting septic shock, suggesting that Cd300b amplifies the TLR4-LPS induced immune response thereby causing lethal inflammation. Indeed, Cd300b and its adaptor, DAP12, associated with TLR4/CD14 upon LPS binding, promoting MyD88/TIRAP dissociation and the recruitment and activation of Syk and PI3K. This results in the activation of AKT, which subsequently leads to a reduced IL-10 production, via a PI3K/AKT-dependent inhibition of the MEK1/2-ERK1/2 signaling pathway.

In sum, these findings describe a previously unidentified LPS-induced signaling complex consisting of Cd300b/DAP12/TLR4/CD14/Syk/PI3K that effectively amplifies TLR4/CD14-mediated inflammation. Furthermore, our data change the paradigm of how LPS mediates TLR4 signaling in myeloid cells and identify potential targets for future clinical intervention.
Targeting chromatin kinases reveals their role in inducible transcription of inflammatory genes
Authors: Josefowicz Steven , Shimada Miho , Armache Anja , Li Charles , Miller Rand , Lin Shu , Garcia Benjamin , Taunton Jack , Roeder Robert , Allis David
Published: January 2023

The inflammatory response requires selective, rapid regulation of chromatin and induction of transcription for defense against pathogens and environmental insult, and also features abundant kinase activity. We sought to identify the connections between kinase pathway activity and regulation of chromatin through unbiased identification and genomic foot printing of prominent histone phosphorylation events. We identified histone H3 serine 28 phosphorylation (H3S28p) as the principal stimulation-dependent histone modification with enrichment at induced inflammatory genes in mouse macrophages stimulated with bacterial lipopolysaccharide (LPS). Use of chemical inhibitors and genetic rescue identified mitogen- and stress-activated protein kinases (MSK) as the primary mediators of H3S28p in macrophages. Cell-free transcription assays demonstrated that H3S28p directly promotes transcription. Further, we show that MSK can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Indeed, inhibition of MSK in macrophages selectively reduced transcription of stimulation-induced genes. Our results indicate MSK as a nexus of signaling inputs controlling multiple downstream regulators of inducible transcription.
Rapid transcriptional reprogramming of the lipid biosynthesis genes in response to type I interferon
Authors: York Autumn Gabrielle , Williams Kevin J. , Liu Xin , Argus Joseph P. , Tong Ann-Jay , Zhou Quan D. , Smale Stephen T , Bensinger Steven
Published: January 2023

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. In previous studies, we found that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages alone was sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. However, it currently remains unclear how type I IFN signaling rewires lipid metabolism. Here, we show that type I IFN-mediated changes in lipid metabolism occur rapidly at the level of transcription. Systematic studies identified a reduction in primary transcripts of lipid synthesis genes within 30 min post IFN. Mechanistically, we find that the rapid transcriptional reprogramming of lipid synthesis is driven by STAT2, independent of the ability of STAT2 to regulate Ch25h and other interferon stimulated gene (ISG) expression. These observations suggest that STAT2 functions to negatively regulate lipid synthesis genes while concurrently upregulating ISG expression. Thus, we propose a model where type I IFN-mediated activation of STAT2 results in the rapid displacement of the canonical transcriptional regulators of lipid metabolism. As such, activation of STAT2 negatively regulates flux through the lipid biosynthetic pathway. On going studies aim to elucidate the precise changes in promoter occupancy of the lipid synthesis genes in response to type I IFN signaling. In sum, these studies indicate that direct manipulation of lipid homeostasis is intimately intertwined with canonical host defense signaling cascades.
TNF Receptor 2 on CD8+ T cells and TNF receptor 1 on non-CD8+ T cells Contribute Significantly to Chlamydia pneumoniae-Induced Atherosclerosis
Authors: Manam Srikanth , Zafiratos Mark T , Cottrell Jonathan T , Henderson Kyle K , Ramsey Kyle H , Murthy Ashlesh K
Published: January 2023

Chlamydia pneumoniae is an important cause of community-acquired pneumonia and has been associated with exacerbation of atherosclerosis. A role of TNF-α in Chlamydia pneumoniae (Cpn) induced atherosclerosis has been reported previously; however, the relative contribution of the two cognate receptors of TNF-α, TNF receptor 1 (TNFR1) and TNFR2 had not been studied. In the current study, C57BL/6J (WT) mice, TNFR1, TNFR2 and TNFR1/2 DKO mice were infected intranasally on days 0, 14 and 28 with 1 × 107 IFU of Cpn (AR39) and were fed a high fat (HF) diet throughout the experiment. One group of WT mice were mock infected with PBS as a control. All Cpn-infected KO mice displayed serum anti-Cpn antibody response, splenic cellular Cpn-specific IFN-γ and TNF-α response, and serum total cholesterol levels comparable to infected WT animals. On day 100 following infection, Cpn infected TNFR1, TNFR2 and TNFR1/2 DKO mice displayed significantly reduced atherosclerotic pathology compared to infected WT mice, and at levels comparable to mock-infected WT animals., suggesting that TNFR1 and TNFR2 play a significant role in Cpn-induced atherosclerosis. We demonstrated recently that CD8+ T cells induce Cpn-mediated atherosclerosis; therefore, we further investigated the role of TNFR1 and TNFR2 on CD8+ T cells in pathogenesis. TNFR2 KO mice, not TNFR1 KO mice, replete with WT CD8+ T cells displayed enhancement of atherosclerotic pathology to levels observed in Cpn-infected WT animals. Collectively, these results demonstrate that TNFR2 on CD8+ T cells and TNFR1 on non-CD8+ T cells contribute significantly to Chlamydia pneumoniae induced atherosclerosis.
Enhanced T cell responses to IL-6 in type 1 diabetes are associated with early clinical disease and increased IL-6 receptor expression
Authors: Hundhausen Christian , Roth Alena , Whalen Elizabeth , Chen Janice , Schneider Anya , Long Alice , Wei Shan , Greenbaum Carla J. , Cerosaletti Karen , Buckner Jane H.
Published: January 2023

IL-6 is a key pathogenic cytokine in multiple autoimmune diseases including rheumatoid arthritis and multiple sclerosis, suggesting that dysregulation of the IL-6 pathway may be a common feature of autoimmunity. The role of IL-6 in type 1 diabetes (T1D) is not well understood. Here we show that STAT3 and STAT1 responses to IL-6 are significantly enhanced in CD4 and CD8 T cells from individuals with T1D compared to healthy controls. The effect is IL-6 specific as it is not seen with IL- 10 or IL-27 stimulation, two cytokines that signal via STAT3. An important determinant of enhanced IL-6 responsiveness in T1D is IL-6 receptor surface expression, which correlated with phospho-STAT3 levels. Further, reduced expression of the IL-6R sheddase ADAM17 in T cells from patients indicated a mechanistic link to enhanced IL-6 responses in T1D. Interestingly, IL- 6- induced STAT3 phosphorylation was inversely correlated with time from diagnosis, suggesting that dysregulation of IL-6 signaling may be a marker of early disease. Finally, whole-transcriptome analysis of IL-6 stimulated CD4+ T cells from patients revealed novel IL-6 targets involved in T cell migration and inflammation, including lymph node homing markers CCR7 and L-selectin. In summary, our study demonstrates enhanced T cell responses to IL-6 in T1D due in part to an increase in IL-6R surface expression. Dysregulated IL-6 responsiveness may contribute to diabetes through multiple mechanisms including altered T cell trafficking and indicates that individuals with T1D may benefit from IL-6 targeted therapeutic intervention such as being currently tested (NCT02293837).
Type I interferon signalling suppresses anti-parasitic CD4+ T cell responses during visceral leishmaniasis
Authors: Kumar Rajiv , Bunn Patrick , Singh Neetu , Sundar Shyam , Engwerda Christian
Published: January 2023

Many pathogens, including viruses, bacteria, and protozoan parasites, suppress cell mediated immune responses through activation of type I Interferon (IFN-1) signalling. However the role of IFN-1 during Leishmania donovani infection causing visceral leishmaniasis (VL) is not well known. Here we report that IFN-1 play an important role in the pathogenesis of VL by impairing parasite clearance and suppressing pro-inflammatory cytokine production. Mice lacking type-1 IFN signalling (B6.IFNαR1−/− mice) had enhanced pro-inflammatory cytokine production and better control of parasite burden in liver as well as spleen compared to wild type C57BL/6 mice. IFN-1 signalling suppressed the CD4+ derived IFN-γ production and prevented Th1 response from controlling parasite replication. Experiment using bone marrow chimeric mice revealed that IFN-1 did not suppress CD4+ T via directly acting to this cell type. Using conventional dendritic cell (cDC) specific IFN-1 deficient mice (CD11c Cre-IFNARfl/fl), we observed that IFN-1 hampered CD4+ T cells response acting via dendritic cells. Studies in VL patients supported these findings and showed enhanced accumulation of mRNA encoding type I IFN signature genes in peripheral blood mononuclear cells (PBMCs) that were reduced following successful drug therapy. Critically, we also showed, using a whole blood assay, that blockade of type-1 IFN signalling enhanced antigen specific IFN-γ production, and that this response was HLA-II restricted. Together, these results identify the type-1 IFN signalling pathways as a potential therapeutic target to treat VL by stimulating anti-parasitic CD4+ T cell responses.
MALT1-deficient CD4 T cells exhibit decreased IL-2 secretion and impaired Th1 and Th17 differentiation
Authors: Kang Sohyeong , Huang Yu-Husan , Wang Xiaoxia , Fung Shan-Yu , Turvey Stuart , Priatel John J
Published: January 2023

A child admitted to British Columbia Children’s Hospital displayed a novel clinical presentation of immune dysregulation, exhibiting lymphocytic infiltration and severe inflammation of the intestinal tract and other mucosal surfaces. Whole-exome sequencing revealed that the patient inherited missense MALT1 mutations and her lymphocytes exhibited deficits in MALT1 protein and MALT1-dependent signal transduction. MALT1 plays critical roles in T cell receptor signal transduction through at least two different mechanisms: (1) as a signaling adaptor molecule bringing together CARMA-1 and BCL-10 to form the CBM signalosome complex and (2) as a caspase-like protease (paracaspase) cleaving substrates after arginine residues. Studies on Malt1−/− mice have revealed diminished NF-KB activation and weakened T cell immunity whereas mice lacking paracaspase activity but not MALT1 adaptor function are prone to lethal autoimmunity. Altogether, these mouse findings raise questions regarding the nature of our patient’s mutant MALT1 protein and the role it plays in her proinflammatory phenotype. Consequently, we plan to analyze our patient’s MALT1 mutation by investigating the paracaspase activity, signaling, and function of her T cells. Early studies have found that patient CD4 T cells exhibit defects in Th1 and Th17 differentiation and these impairments could be responsible for inefficient immune responses leading to chronic infections. Finally, a better understanding of MALT1 will provide insight into inflammatory disease and prove invaluable in supporting MALT1-targeted therapies to modulate T cells to treat autoimmune diseases.
Wellness Index: measuring the readiness of our immune system
Authors: Han Jian
Published: January 2023

We performed next generation sequencing on 1000 individuals’ immune repertoires. More than 3 billion T cell receptors beta chain sequences were obtained from peripheral blood samples. Over 75 million unique CDR3 sequences were identified. The majority of the CDR3 sequences (81%) were private and not shared. However, a few thousand of the CDR3 sequences were classified as very public and shared by almost everyone. Taking advantage of this knowledge, we developed a Wellness Index to measure the readiness of our immune system in fighting different diseases. A person’s Wellness Index (WI) equals to the number of the top 1000 very public CDR3s (pCDR3s) found in his/her sample. The more pCDR3s found in a unit blood, the more diversified the immune repertoire, and therefore, the more healthy or more ready to ward off diseases. Although the Wellness Index does not diagnose specific diseases, it provides an accurate and reliable measurement of health status. The Wellness Index may also be associated with cancer patient’s outcomes or responses to different treatments.
The changes of gene expression between pulmonary tuberculosis lesions and normal tissue
Authors: CHUANYOU LI , li wensheng , sun yong
Published: January 2023

Objective

to develop specific genes associated with the formation of these severe tuberculosis lesions by comparing gene expression changes of macrophages between the lesions and normal lung tissue around it

Material and methods

select lesions tissue and surrounding normal lung tissue specimens from three tuberculosis patients, magnetic bead separation method was applied to filter out the macrophages within the organization, extracted RNA and detected with gene chip to contrast the difference of gene expression patterns. between the lesions and surrounding normal lung tissue

Results

there were 332 genes up regulation and 102 genes down regulation to 2 fold in normal lung tissues. making the selecting criteria rising to 4 fold, the number of up regulation and down regulation genes would decrease to 19 and 2. The two down regulation genes were FER1L4 and PPIL5. During the up regulation group. the AREG gene was very special because the up regulation fold of this gene in our three groups were more than 11 times respectively. Moreover, the expression level of four genes which were related with TNF were obviously up regulationd in normal lung tissues

Conclusion

PPIL5, AREG and TNF may be associated with the formation of pulmonary tuberculosis lesions.
High frequencies of Caspase-3-expressing M. tuberculosis-specific CD4 T cells are associated with active tuberculosis
Authors: Adekambi Toidi , Ibegbu Chris , Cagle Stephanie , Ray Susan , Rengarajan Jyothi
Published: January 2023

Background

The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluations and detection of Mycobacterium tuberculosis (Mtb) in the patients’ sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes 3–6 weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that frequencies of Mtb-specific CD4+IFN-g+ T cells expressing caspase-3 would decrease following anti-TB treatment. Caspase-3, a member of cysteine proteases’ family, is highly expressed in CD4 effector T cells and has been shown to regulate T cell activation, cell cycle entry, proliferation and apoptosis.

Methods

Using polychromatic flow cytometry, we evaluated the expression of the active form of caspase-3 on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) (n=22), ATB (n=18), and from ATB patients undergoing anti-TB treatment (n=7).

Results

Frequencies of Mtb-specific IFN-g+CD4+ T cells that expressed active caspase-3 were higher in individuals with ATB compared to those with LTBI suggesting that apoptotic pathways are operant during pulmonary ATB. This marker also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment.

Conclusion

We have identified caspase-3 on Mtb-specific CD4+ T cells as a marker that distinguishes ATB and LTBI and may provide a tool for monitoring treatment response and cure.
Circulating T cells specific for an intestinal bacterial antigen peptide show a distinctive phenotype and potentially proinflammatory gene expression pattern in Crohn’s disease.
Authors: Uchida Amiko M , Boden Elisa , Shows Donna , James Eddie A , Lord James D
Published: January 2023

Background

Crohn’s disease (CD) may be an inappropriate immune reaction to normally well-tolerated intestinal bacterial antigens. Indeed, many CD patients possess abnormally high titers of antibodies specific for intestinal bacterial proteins, such as the outer membrane protein C (OmpC) of E. coli.

Methods

Using MHC-II tetramer-guided epitope-mapping, we identified a specific peptide of OmpC recognized by peripheral T cells from HLA-DR1501+ individuals. HLA-DR1501 tetramers loaded with this OmpC peptide or a known antigenic peptide from influenza virus (Flu) were then used to compare OmpC versus Flu-specific T cells from HLA-DR1501+ healthy and CD donors via flow cytometry and multiplex qPCR.

Results

OmpC-specific T cells more frequently expressed the Th17 marker CD161 (49% v 26%; p=1.1e-18) and the gut-homing integrin α4β7 (43% v 10%; p=1.8e-13) than autologous Flu-specific T cells. Conversely, the Th1 marker CXCR3 was more commonly seen on Flu than OmpC-specific T cells (72% v 61%; p = 0.005). No differences in the frequency or immunophenotype of OmpC-specific T cells were found between healthy and CD patients. However in patients with CD, multiplex PCR of sorted OmpC-specific T cells revealed significantly higher expression of the costimulatory molecule CD226 (p=0.03) compared to healthy controls, with a concomitant trend towards decreased expression of the coinhibitory receptor TIGIT (p=0.1) in CD patients.

Conclusions

OmpC-specific T cells with a gut-tropic, Th17-like phenotype can be found in the peripheral blood, and may have an imbalance in CD226 and TIGIT expression to favor inflammation in CD.
Anatomic partitioning regulates TCR-pMHC interaction and memory T cell development during early immune contraction phase
Authors: Seo Young-Jin , Jothikumar Prithiviraj , Zhu Cheng , Grakoui Arash
Published: January 2023

As virus is cleared, immune cells rapidly undergo contraction; however, how cell function and fate are determined during this phase is not yet fully understood. We unexpectedly discovered that during the early immune contraction phase of an acute infection with lymphocytic choriomeningitis virus, the two dimensional TCR–pMHC effective affinity of TCR transgenic P14 T cells was significantly higher for cells from the splenic red pulp (RP) than the white pulp (WP) compartment. This difference in seeing antigen was governed by FoxP3+ regulatory T cells and TGF-beta. Further, memory precursors from WP preferentially developed into long-term memory cells as compared to cells from RP, despite expression of identical cell surface markers. Our results highlight that intricate regulation of T cell function and fate is determined by anatomic partitioning during the early immune contraction phase. These findings could have an important impact on development of more efficient vaccines and T cell-targeted immunotherapy for infectious diseases, cancers, and autoimmune disorders.
MicroRNA-155 enhances T-cell trafficking and antiviral effector function in a model of coronavirus-induced neurologic disease
Authors: Dickey Laura L. , Worne Colleen , Glover Jessica , Daugherty Joshua , Lane Thomas , O’Connell Ryan M
Published: January 2023

Micro-RNAs (miRNAs) are non-coding RNAs that modulate cellular gene expression at the post-transcriptional level. These molecules are becoming increasingly recognized as important in helping tailor T-cell responses following viral infection. We therefore sought to examine the functional role of miR-155 in a model of viral-induced demyelination. Intracranial injection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in an acute encephalomyelitis followed by an immune-mediated demyelinating disease. Compared to wild-type mice, JHMV-infected mice deficient in miR-155 (miR-155−/−) developed delayed but exacerbated disease concomitant with increased morbidity/mortality, weight loss, and spinal cord demyelination. In addition, miR-155−/− mice had decreased total and virus-specific CD4+ and CD8+ T-cell accumulation within the CNS during the acute phase of disease. Furthermore, IFN-γ and TNF-α production, as well as and cytolytic function, were impaired in CD8+ cells from miR-155−/− mice. These results identify miR-155 as a key mediator of disease development and recovery in a model of virus-induced neurological disease.
Identification of CD112R as a new coinhibitory receptor for CD112
Authors: Zhu Yuwen , Paniccia Alessandro , Schulick Alexander C , Koenig Michelle R , Edil Barish H
Published: January 2023

Cancer immunotherapy has had great success in the clinical setting, with immunomodulating agents targeting immune checkpoints (including CTLA-4 and PD-1) leading the way. However, the overall response rate is still low and its effectiveness on certain tumors is disappointing. Therefore, identifying novel T cell checkpoints is critically needed, for new treatment strategies. TIGIT and CD226 emerge as a novel T cell cosignaling pathway with the potential of cancer immunotherapy. CD226 and TIGIT serve as a costimulatory receptor or a coinhibitory receptor respectively for the ligand CD155 (PVR) and CD112 (PVRL2). In this study, we identified CD112R as a missing coreceptor of this important PVR-like network. CD112R, also a member of the PVR family, is preferentially expressed on T cells and inhibits TCR-mediated signal. We further identified that CD112, widely expressed on antigen-presenting cells and tumor cells, is the ligand for CD112R with high affinity. CD112R competes with CD226 to bind CD112. Disrupting the CD112R/CD112 interaction enhances human T-cell response. Thus, our studies identify CD112R as a novel checkpoint for human T cell, and modulating this pathway will offer new targets for future immunotherapy.
Defining the role of Tim-3 in T cells
Authors: Avery Lyndsay , Kane Lawrence P
Published: January 2023

Chronic viral infection and the tumor microenvironment are two settings in which persistent immune stimulation can occur. Under such conditions, T cell activation becomes downregulated, likely in order to reduce immune pathology, resulting in a phenotype known as T cell ‘exhaustion’. Functionally exhausted T cells are characterized by reduced cytokine production, proliferative capability, and cytotoxic activity, with a corresponding expression of checkpoint receptors, including PD-1, CTLA-4, LAG-3 and Tim-3. While PD-1, CTLA-4, and LAG-3 have known mechanisms for reducing T cell effector function, the intrinsic effects of Tim-3 on T cell activation are largely unknown. In fact, recent evidence reveals divergent effects of Tim-3 in various infectious models. Data from our lab indicate that Tim-3 can enhance acute TCR signaling, while much of the existing literature associates Tim-3 expression with diminished effector function. Current approaches to study Tim-3 require persistent antigen stimulation or ectopic expression. We have developed a novel mouse model where the expression of Tim-3 is induced in vivo by Cre-mediated recombination. We hypothesize that inducible Tim-3 expression may cause T cells to become dysfunctional earlier by enhancing the initial response to stimulation. Using this model, we have found that the presence of Tim-3 enhances T cell activation after acute in vitro stimulation. We are currently testing how induction of Tim-3 will affect responses to a murine model of chronic viral infection, LCMV-clone13. Results from this study will address knowledge gaps about the effects of Tim-3 on T cells and will have practical applications in both chronic viral infection and cancer immunotherapy.
Cbl-b deficiency renders T cells resistant to PD-L1/PD-1 mediated suppression
Authors: Fujiwara Mai , Anstadt Emily J , Clark Robert B
Published: January 2023

The PD-L1/PD-1 pathway is a critical regulator of T cell responses that has become a significant focus in cancer immunotherapy. Cbl-b is an E3 ubiquitin ligase that regulates many aspects of T cell activation. In humans, polymorphisms in the CBLB gene are associated with autoimmunity. Cbl-b deficient (Cbl-b−/−) mice demonstrate spontaneous autoimmunity and enhanced anti-tumor T cell responses. Thus, there is now great interest in manipulating Cbl-b to enhance anti-tumor T cell responses.

We now report that, in contrast to WT T cells, in vitro TCR-stimulated proliferative responses of Cbl-b−/− CD4+ and CD8+ T cells are not suppressed by a recombinant PD-L1 fusion protein (PD-L1 Ig) (% suppression in CD4+: WT 40%, Cbl-b 0%, p<0.01; in CD8+: WT 32%, Cbl-b 5%, p<0.05). Moreover, IFN-γ production of Cbl-b−/− CD4+ T cells is less suppressed by PD-L1 Ig than that of WT cells (% Suppression: WT 68%, Cbl-b 20%, p<0.0001). Importantly, PD-1 expression is comparable between Cbl-b−/− and WT T cells. To confirm this PD-L1 resistance in vivo, we used a model of B16 melanoma in which liver metastases develop only when PD-L1/PD-1 immune regulation is functionally intact. We confirmed that WT mice develop numerous liver metastases which are significantly reduced by anti-PD-1 antibody treatment. Strikingly, Cbl-b−/− mice develop only rare liver metastases even in the absence of anti-PD-1.

In sum, we report for the first time that Cbl-b−/− T cells are resistant in vitro and in vivo to suppression by PD-L1/PD-1. Our finding of Cbl-b−/− T cell resistance to PD-L1/PD-1-mediated inhibition broadens our understanding of Cbl-b’s role in autoimmunity and suggests a new mechanism by which manipulation of Cbl-b function may lead to enhanced anti-tumor T cell responses.
Identifying Fas/CD95 non-apoptotic signaling pathways associated with T cell differentiation
Authors: Thomas Stacy K , Ouyang Claudia , Cruz Anthony C , Klebanoff Christopher A , Yamamoto Tori N , Chan Francis K , Mocarski Edward S , Roback Linda , Restifo Nicholas P , Siegel Richard M
Published: January 2023

Fas (CD95, TNFRSF6), a member of the TNF cytokine superfamily, triggers apoptosis through recruitment of FADD and Caspase 8. However, Fas can also induce other responses besides cell death, including costimulating T-cell activation and promoting tumor cell growth and metastasis. We have found that Fas accelerates the differentiation of naïve T cells into effector memory cells. In vivo, Fas deficiency is known to cause autoimmunity, which has been attributed to a lack of Fas-induced cell death. Interestingly, we observed that palmitoylation-defective Fas C194V transgenic mice, in which Fas cannot induce apoptosis but can signal for differentiation, did not present with autoimmunity. This led us to hypothesize that non-apoptotic Fas signaling plays a role in autoimmunity and investigate the signaling pathways underlying Fas-induced differentiation.

Primary T cells lacking FADD or Caspase 8 and cells treated with caspase inhibitor z-VAD did not differentiate suggesting that Fas-induced T cell differentiation depends on FADD, Caspase 8 and caspase activity. Moreover, we observed that cells undergoing Fas-induced differentiation had increased pAkt and pS6, and that Akt inhibition blocked differentiation. Although Fas does not directly activate Akt, activation of this pathway by Fas in the context of T cell activation is dependent on IL-2, suggesting that instead of directly activating Akt, Fas potentiates the IL-2 initiated PI3K/Akt pathway. Together, our data show that Fas potentiates the induction of PI3K/AKT signaling by IL-2 to accelerate naïve T cell differentiation via a FADD and Caspase 8 dependent mechanism.
Fibroblastic reticular cells of the secondary lymphoid organ suppress T cell activation via COX2 mediated PGE2 secretion.
Authors: Yu Miao , Guo Gang , Xiao Wei , Cui Yan
Published: January 2023

Accumulating evidence demonstrates that fibroblastic reticular cells (FRCs) in the T cell zone of the secondary lymphoid organs (SLO), characterized as CD45− gp38+CD31− cells, play an important role in maintaining T cell tolerance via presenting peripheral tissue–restricted antigens (PTAs) and/or interferon-induced nitric oxide during productive immunity. Here, using FRCs harvested from naïve mice, we revealed that FRCs also inhibit T cell activation via a soluble factor, independent of the aforementioned mechanisms. Specifically, FRCs suppressed CD3/CD28-ligation mediated or antigen induced antigen-specific T cell activation. This FRC-imposed suppression was demonstrated as (1) reduced Zap-70 phosphorylation and Calcium-flux within 30 minutes of TCR-ligation, (2) inhibited upregulation of surface CD69 and CD44 expression and downregulation of CD62L 6–14 hours post-activation, and (3) suppressed T cell effector cytokine production and proliferation. Further studies demonstrated that our observed inhibition of T cell activation by FRC is at least partially contributed by cyclooxygenase-2 (COX-2) mediated production of prostaglandin E2 (PGE2) because COX-2 inhibitor partially reversed FRC mediated inhibition. ELISA results confirmed high production level of PGE2 by FRC (~ 10 – 50ng/ml), at which level T cell activation is greatly suppressed. Taken together, our study illustrated another previously unrecognized mechanism of FRCs in maintaining T cell tolerance, which underscores the crucial role of FRCs in peripheral tolerance and immune regulation. The potential molecular mechanism by which the COX2-PGE2 pathway is activated in FRCs under physiological condition will be discussed.
Novel mechanism in autoimmune regulation by S-nitrosoglutathione
Authors: Won Jeseong , Saxena Nishant , Samuvel Devadoss J. , Choi Seungho , Singh Avtar K. , Singh Inderjit
Published: January 2023

Nitric oxide (NO) plays a pivotal role in pathogenesis and control of autoimmune disease which is caused by abnormal activation of T helper (Th) cells. Under disease conditions of experimental autoimmune encephalomyelitis (EAE), we observed that different subsets of Th cells expressed different levels of NO synthesizing enzymes (nNOS, iNOS, and eNOS). Notably, Th17 cells exclusively expressed high levels of iNOS and its genetic ablation resulted in increased Th17 activation and IL-17 expression but decreased activation of specific subset of regulatory T (CD4+CD25+FOXP− Treg) cells and IL-10 expression in EAE. S-nitrosoglutathione (GSNO) is a physiological NO carrier molecules that mediates variety of NO signaling via trans-S-nitrosylation mechanisms. In EAE, GSNO treatment decreased the disease severity and activation/effector function of Th17 while it increased CD4+CD25+FOXP− Treg activation/effector function. Moreover, the reduced activation/effector function of Th17 by adaptive transfer of GSNO-induced CD4+CD25+FOXP− Tregs suggest that CD4+CD25+FOXP− Treg may be the effector cells antagonizing Th17 in response to iNOS/NO/GSNO. The cellular levels of GSNO is regulated by its catabolic enzyme GSNO reductase (GSNOR). In EAE, both Th17 and of CD4+CD25+FOXP− Treg expressed high levels of GSNOR and pharmacological inhibition of GSNOR or genetic ablation of GSNOR resulted in abrogation of Th17 activation/effector function while activating CD4+CD25+FOXP− Treg. In summary, this study suggest the iNOS/NO/GSNO-inducible CD4+CD25+FOXP− Treg cells as the potential feedback regulator of Th17 and GSNOR as the potential therapeutic target for Th17 and CD4+CD25+FOXP− Treg mediated autoimmune disease.
CD40L signaling from CD8+ T cells confers protection against influenza infection
Authors: Tay Neil Q , Lee Debbie C P , Chua Yen Leong , Prabhu Nayana , Kemeny David Michael
Published: January 2023

CD40L is a principal mediator of a large range of humoral and cellular responses and CD40L-mediated licensing of dendritic cells is known to be necessary for the generation of robust effector and memory CD8+ T cell responses. Although the effects of CD40L when expressed on CD4+ T cells are well studied, its role when expressed on CD8+ T cells remain unclear. We have previously reported that CD40L signaling mediates the induction of IL-12 p70 production in dendritic cells when expressed by CD8+ T cells. However, the role and importance of this mechanism in CD8+ T cell responses during an infection are not known. To investigate the function of CD40L when expressed on CD8+ T cells in an in vivo mouse model, we crossed OT-I CD8+ T cell receptor transgenic mice with CD40L−/− mice to generate OVA-specific CD8+ T cells that are unable to expressed CD40L. By transferring CD8+ T cells from these mice into CD40L-competent mice and using a strain of influenza that expresses the OVA epitope recognized by OT-I CD8+ T cells, we were able to create an influenza mouse model where CD40L signaling is absent only on the responding CD8+ T cells. Our analysis indicates that CD40L signaling by CD8+ T cells confers protection against influenza infection and that this is likely dependent on the ability of CD8+ T cells to induce IL-12 p70 production in a CD40L-dependent manner.
mTOR activity is necessary for cytokine-mediated TBET expression in TLR-stimulated B cells
Authors: Myles Arpita , Naradikian Martin S , Cancro Michael P
Published: January 2023

A recently described subset of B cells – called age associated B cells (ABCs) – is characterized by the increased expression of TBET. While known to be particularly relevant in the settings of aging and autoimmunity, the cell intrinsic signaling pathways leading to the development of the ABC phenotype are not well-characterized. Since the mTOR pathway is a known regulator of TBET expression in other cell types, we examined its role in B cells stimulated with TLR agonists. Our findings indicate that TLR9 (CpG) or TLR7 (Cl097) ligands alone activate mTOR, as evidenced by S6 phosphorylation, but do not yield robust TBET upregulation without concomitant cytokine stimulation. Indeed, IFNγ and IL-21 induced TBET expression is lost in response to rapamycin (mTOR inhibitor) treatment. This indicates that mTOR activity is necessary but not sufficient for TBET expression. The implication that mTOR could be a node for integration of signals downstream of TLR and cytokines to regulate TBET expression in B cells merits further investigation, and might provide novel mechanistic insights into various B cell differentiation programs.
Macrophage lipid accumulation is regulated by substrate stiffness
Authors: Leifer Cynthia A , Gruber Erika , Stelzer Sandra , Erlich Emma , Sinha Siddhartha
Published: January 2023

Atherosclerosis is characterized by the accumulation of lipid-laden macrophage foam cells within the vessel wall, formation of a plaque, and changes in tissue stiffness from soft near the necrotic core to stiff near the fibrous cap. The mechanisms governing foam cell formation are not well understood, but recent studies demonstrate that cells probe and respond to the physical properties of their surroundings. Stiffness regulates differentiation and function of mesenchymal stem cells and endothelial cells, yet until recently, its role in immune cell function has been largely unexplored. Our lab, and others, has found that substrate stiffness regulates murine macrophage cytokine secretion and phagocytic activity. Because macrophages accumulate near the soft necrotic core where there is abundant lipid, we hypothesized that macrophages on softer surfaces would exhibit enhanced lipid accumulation. To investigate this, we cultured murine macrophages on functionalized tunable polyacrylamide gels that model physiologically relevant stiffnesses. We then used a combination of flow cytometry and immunofluorescence microscopy to show that softer substrate stiffness results in more accumulation of the model lipid, oleic acid, as well as acetylated low-density lipoprotein in macrophages. Transcriptomic analysis identified up- or down-regulated genes in macrophages in response to increased substrate stiffness, including genes implicated in atherosclerosis and lipid metabolism. Together, these data demonstrate that lipid accumulation by macrophages is regulated in part by substrate stiffness and mechanotransduction signaling and reveal a potential link between changes in tissue stiffness and macrophage lipid accumulation.
SHP-1 impacts mitochondrial function in cellular metabolism
Authors: Martinez Hunter , Martinez Ryan , Evavold Brian D
Published: January 2023

SHP-1 is a protein tyrosine phosphatase (PTP) that plays critical roles in many hematopoietic cell signaling pathways. For T cells, initial investigations elucidating the role of SHP-1 show its involvement as a negative regulator by dephosphorylating Lck and ZAP70, two kinases instrumental in early T cell activation. Despite its role as a negative regulator, minimal differences were observed in proliferation of SHP-1 −/− CD4 T cells when compared to WT cells. To investigate this disparity metabolism was studied, as cellular division is highly reliant upon energy allocation. Analysis of T cell glycolysis indicated defects in the absence of SHP-1 and that at maximal respiration SHP-1 plays a role in maintaining energy production and is necessary for optimal mitochondria functionality. Furthermore, in the absence of SHP-1 there is approximately 20% reduction in mitochondrial functionality in both naïve and activated CD4 T cells. This goes in parallel with a noted decrease in the amount of ATP in SHP-1 −/− CD4 T cells. Therefore, SHP-1 plays critical roles in T-cell cellular metabolism in addition to being a key regulatory phosphatase.
Transmembrane TNF–TNFR2 Antagonizes TH17 Differentiation by Promoting Il2 Expression
Authors: McKarns Susan C , Miller Patrick G
Published: January 2023

While IL-17 is critical for host defense, its overabundance promotes autoimmunity. IL-2 represses TH17 differentiation, but a role for tumor necrosis factor (TNF) in this process is not well defined. TNF binds TNF receptor 1 (TNFR1) and TNFR2 to stimulate opposing signaling cascades. Whereas sTNF signals through TNFR1, tmTNF preferentially activates TNFR2. We have previously demonstrated reduced IL-2 production in TNFR1−/− TNFR2−/− double knockout CD4+ T cells. To further explore the mechanism by which TNF regulates IL-2 production, we generated TNFR1−/−, TNFR2−/−, and TNFR1−/− TNFR2−/− 5C.C7 TCR Il2-GFP mice to study Il2 transcription at the single cell level. Our findings indicate that CD4+ T cell–intrinsic tmTNF/TNFR2 stimulates Il2 promoter activity and Il2 mRNA stability. Pharmacological blockade of TNF in wild-type C57BL/6 mice and tmTNF Foxp3-GFP mice also demonstrated tmTNF/TNFR2-mediated augmentation of Il2 expression. We further report elevated expression of TNFR2 on the surface of TH17-polarized CD4+ T cells and show that tmTNF/TNFR2, but not sTNF/TNFR1, inhibits TH17 differentiation. Under TH17-polarizing conditions, elevated IL-17 production by TNFR2-knockout CD4+ T cells correlated with increased STAT3 activity and was prevented by exogenous IL-2. We conclude that increased IL-2 production in response to CD4+ T cell–intrinsic tmTNF/TNFR2 signaling is sufficient to inhibit TH17 differentiation in a Foxp3-independent manner.
The Neurotrophic Factor Neuritin Maintains and Promotes the Function of Regulatory T cells in Autoimmunity and Cancer
Authors: Barbi Joseph J , Vignali Paolo D. A. , Yu Hong , Pan Fan , Pardoll Drew
Published: January 2023

Regulatory T cells (Tregs) enforce immune homeostasis and self-tolerance and inhibit both natural and induced anti-tumor immunity. Consequently, there is considerable interest in therapeutic Treg enhancement or blockade to treat inflammatory/autoimmune diseases and cancer, respectively. Targeting Tregs requires a comprehensive understanding of the factors important for their maintenance, differentiation and function. Here we report that neuritin, a conserved, gpi-anchored molecule important for the development, survival and function of neurons, is highly expressed by induced and natural Tregs. Furthermore, neuritin expression was found to promote the maintenance of the Treg pool by stabilizing Foxp3 expression and promoting the persistence of these cells. Knockout of the neuritin gene in Tregs or anti-neuritin antibody treatment mitigated both Treg-dependent inhibition of colitis and resolution of experimental autoimmune encephalitis. Also, tumors grew poorly in Nrn1−/− mice, which mobilized more robust anti-tumor immunity than their wild type litter mates evidenced by elevated IFNgamma production and reduced PD1 expression. Importantly, we also found that in Tregs, neuritin expression was closely linked to the functional differentiation of Tregs into an activated, peripheral tissue-homing phenotype. As such, neuritin deficiency resulted in an imbalance between “central”- and “effector”-like Treg populations and functions. These findings characterize this neurotrophin as a hitherto unappreciated immunoregulatory molecule and a potential target for therapies aimed at the fine-tuning of Treg function in cancer and inflammatory/autoimmune diseases.