bluuebunny/crossref_metadata_2025 huggingface dataset:
Patrolling ILCs restrain mucosal viral replication through tissue-wide delivery of IFN-γ
Authors: Shannon John P , Reynoso Glennys V. , Kamenyeva Olena , McManus Daniel T , Hickman Heather D.
Published: January 2023

Smallpox, caused by infection with variola virus, led to devastating human pandemics with high mortality until eradication through a global vaccination campaign. Despite eradication, the precise immune mechanisms underlying recovery from infection are incompletely understood. The most common model employed to study smallpox is murine infection with vaccinia virus (VACV), the virus used for human smallpox vaccination. VACV models of smallpox infection commonly employ intranasal or even intravenous routes of infection, with both resulting in widespread viral dissemination. However, variola virus infected humans through the oropharyngeal mucosa, after which oral lesions developed and ruptured, spilling copious infectious virus into the saliva (the route of human-to-human spread). To understand protection in this critical barrier site, we developed a mouse model of lip (labial mucosal) infection. After labial administration, VACV replicates to high titers and two waves of group I innate lymphoid cells (ILCs) are recruited in response to infection. Intravital imaging revealed highly mobile ILCs in the oral mucosa, most of which failed to contact virus-infected cells. Despite the lack of stable contacts, depletion of NK1.1+ cells enhanced viral replication and spread. Instead, ILCs delivered IFN-γ to the mucosal tissue, inducing an antiviral signature in the epithelium. Together, these data illustrate the complex nature of immune protection in a critical yet overlooked tissue during viral pathogenesis.
Interferon-α producing dendritic cells impair viremia control in simian immunodeficiency virus-infected rhesus macaques
Authors: Ko Eunju , Hait Sabrina Helmold , Rahman Mohammad Arif , Enyindah-Asonye Gospel , Hogge Christopher James , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

Dendritic cells (DCs) are potent antigen-presenting cells that orchestrate innate and adaptive immune responses. Interferon (IFN)-α, a cytokine produced by DCs, has both protective and detrimental activities in viral infection. Here we assessed the phenotypes and IFN-α production of mucosal and circulatory DCs in simian immunodeficiency virus (SIV)-infected rhesus macaques during acute and chronic infection to determine their contribution to viremia control. Macaques were initially vaccinated with replicating adenovirus-SIV recombinants, boosted with SIV envelope, and exposed to SIV intravaginally. Those that became infected were divided into high (HVL) and low viral load (LVL) groups based on plasma viremia at 12 weeks post-infection. HVL macaques had ≥ 7.98×104 SIV RNA copies/ml plasma; LVL animals had ≤ 1.17×104. Immunizations increased rectal plasmacytoid DCs (pDCs, CD123+, p=0.0039 in HVL), but SIV infection induced loss of rectal pDCs in the acute (p=0.0039) and chronic phases (p=0.0078). Blood myeloid DCs (mDCs, CD11c+) were increased in the chronic phase (p=0.0195). No differences in HVL and LVL DC frequencies were seen during SIV infection. But higher blood IFN-α+pDCs and mDCs in the acute phase were observed in HVL compared to LVL animals (p=0.042 in both pDCs and mDCs), suggesting blood IFN-α might contribute to SIV persistence and interrupt viremia control. Chronic IFN-α+ rectal pDCs (p=0.015) and acute IFN-α+ blood pDCs (p=0.032) and mDCs (p=0.0058) positively correlated with peak plasma VLs implying IFN-α lessened viral control in SIV infection. Our data suggest that elevated IFN-α+ DC frequencies in acute SIV infection impair SIV viremia control and help maintain higher VLs in SIV-infected rhesus macaques.
CD5+ B-1 cell responses to infection are facilitated by toll-like receptor stimulation
Authors: Smith Fauna Leah , Savage Hannah Pritikin , Kläsener Kathrin , Reth Michael , Baumgarth Nicole
Published: January 2023

B-1 lymphocytes are neonatally-derived, self-reactive, innate-like B cells that produce natural IgM at steady state. B-1 cells also actively respond to infections with induced local IgM production. These two distinct functions of B-1 cells have been linked to the expression CD5, an inhibitor of BCR signaling, with CD5+ cells thought to contribute to steady state IgM, and CD5− cells to local IgM production during infection. The mechanisms underlying such “division of labor” are poorly understood. We aim to test our hypothesis that CD5+ B-1 cells are stimulated by innate signals via TLR, during infection, resulting in their activation and differentiation into CD5− IgM antibody-secreting cells. For that we first cultured CD5+ B-1 cells with TLR9 agonist, ODN-CpG. The cells proliferated, secreted IgM and showed increased Nur77 expression, Akt phosphorylation and interaction of Syk with CD79a, signs of BCR signaling. They lost expression of the BCR-inhibitor CD5 and upregulated the plasma cell marker CD138+. Following influenza infection of mice, CD5+ B-1 cells migrated to the mediastinal lymph nodes (medLN) where they lost CD5 and became IgM secreting cells. Both, global TLR−/− mice and chimeric mice lacking TLR only on B-1 cells had reduced numbers of B-1 derived plasma cells and antibody secreting cells in the medLN compared to controls. Our findings show that TLR-mediated activation of CD5+ B-1 cells results in the rapid reorganization of the BCR-complex, followed by loss of CD5 and that TLR stimulation of CD5+ B-1 is required for differentiation of B-1 cells to infection in vivo. Overall our data suggest that CD5+ and CD5− B-1 cells are not distinct subsets, but rather that CD5 expression indicates the activation state of B-1 cells.
Regulation of antiviral CD8 T cell immunity by CD352/SLAMF6: from patterns to pathways
Authors: van der Heide Verena , Homann Dirk
Published: January 2023

A better understanding of CD8 memory T cell (TM) formation during acute and chronic viral infections is critical for both effective immunotherapy and vaccine design. Signaling lymphocytic activation molecule family receptors (SLAMF) including CD150, CD48, CD229, CD244, CD84, CD352, and CD319 were found to be involved in T cell activation and differentiation, but their contribution to the regulation of antigen-specific T cell immunity remains incompletely understood. Here, we analyzed the dynamics of SLAMF co-expression patterns by antiviral CD8 T cells in response to acute and chronic lymphocytic choriomeningitis virus (LCMV) infection. Our results demonstrate distinct SLAMF levels not only on LCMV-specific CD8 effector (TE) and TM cell subsets, but differential kinetics during acute vs. chronic infection. Importantly, we identified CD352 as a unique marker for long-term CD8 TM potential and capacity with significant upregulation on memory-precursor (MPEC) vs. short-lived effector (SLEC) as well as on central (TCM) vs. effector memory (TEM) CD8 T cells (MPEC>>TCM>TEM≥SLEC). Moreover, in the context of chronic viral infection, we found CD352+ CD8 T cells to be partially resistant to exhaustion as demonstrated by a memory-like phenotype, preserved CD8 T cell function, and better in vivo recall capacity upon adoptive transfer into naïve hosts. Conversely, chronically-infected CD352-deficient mice showed an impaired CD8 T cell response with reduced frequencies of LCMV-specific CD8 T cells, a more exhausted phenotype, lower expression of TM markers, and reduced functional properties. Collectively, our data reveal CD352 as a potential therapeutic target to modify CD8 T cell responses during acute and chronic viral infections.
Dynamics of adaptive natural killer cells in longitudinal analysis of CMV vaccine recipients
Authors: Gyurova Ivayla E , Schlums Heinrich , Bernstein David , Bryceson Yenan , Waggoner Stephen N
Published: January 2023

While cytomegalovirus (CMV) infection poses a considerable burden to public health, an efficacious vaccine is not yet available. Classical vaccine efforts focusing on the induction of B- and T-cell memory have proven ineffective and, therefore, CMV remains a significant public health threat. The incidence of recurrent, severe infections with cytomegalovirus (CMV) and other DNA viruses in patients lacking functional natural killer (NK) cells highlight the importance of these cells in viral control. Therefore, the marked expansion of phenotypically and functionally distinct subsets of NK cells during CMV infection may represent a component of immune control of CMV that should be replicated in vaccines. We interrogated blood leukocytes samples collected longitudinally during a CMV glycoprotein B vaccine trial to ascertain induction or accumulation of these unique CMV-associated subsets of NK cells. We observe tantalizing expansions of CMV-associated subsets of NK cells in some CMV-negative vaccine recipients, that are unexpectedly mirrored by similar expansions in placebo-recipients. Moreover, these subpopulations exhibit marked contractions or patterns of alternating expansion and contraction in other trial participants. Importantly, this unique longitudinal data in healthy CMV-seronegative individuals indicates that NK-cell subsets may react to unknown environmental or inflammatory cues and demonstrate greater flux that appreciated from past cross-sectional studies.
Quantifying the selection on influenza A virus imposed by CD8 T cells
Authors: Li Zheng-Rong Tiger , Zarnitsyna Veronika I , Lowen Anice C , Kohlmeier Jacob E , Antia Rustom
Published: January 2023

An essential question regarding the T cell-inducing influenza vaccine is whether the cellular immunity may drive the virus to rapidly evolve and escape. In our previous modeling study, we have shown an CD8 T cell escape-variant of influenza A virus (IAV) invades extremely slowly given empirical HLA allele frequencies and modest selection pressure. Guided by the model, we aim to quantify the selection from CD8 T cells using a novel digital droplet PCR system under different in vivo settings.

Mice were intranasally primed with HKx31 (H3N2) and challenged with a 1:1 mixture of wild-type and mutant PR8, which harbors an N370Q escaping mutation in its NP366–374 immunodominant epitope, on day 30 post-priming. The area under the in vivo viral growth curve (AUC) is used to measure the fitness of wild-type and mutant viruses. Based on the AUCs of C57BL/6 (H-2b/b) and CF1B6 (H-2b/d), the estimated selective advantage of this mutation is 0.28 [0.20, 0.36] and 0.21 [0.10, 0.34], respectively. Interestingly, although the intramuscularly primed C57BL/6 gave a similar estimate for the advantage (0.22 [0.16, 0.30]), the advantage became much more apparent on day 6 and 8 after PR8 challenge. This implies the lung resident memory CD8 T cells may be the source of selection during the initial stage of IAV infection.

In combination with the model prediction, an escape-variant of IAV that carries a mutant immunodominant epitope may need 100 to 500 generations (corresponding to 1 to 5 years if the infection period is assumed to be 4 days) to reach 50% of prevalence given the frequency of cognate HLA alleles is 20%. We concluded that the selection from CD8 T cells may not be sufficient to drive the CD8 T cell epitopes of IAV to evolve rapidly like the antibody epitopes.
Spatial Analysis of CD8+ T cell Activation After Local Viral Replication in the Skin
Authors: Malo Courtney Sarah , Reynoso Glennys V. , Hickman Heather D.
Published: January 2023

The generation of a robust CD8+ T cell response is critical for the elimination of virally infected cells and is dependent on presentation of viral antigen in the draining lymph node. Although we know much about antigen presentation in general, how antiviral T cell activation is precisely accomplished at the spatial level remains incompletely understood. Much of our knowledge of spatial details of viral antigen presentation derives from studies in which virus is subcutaneously injected, which likely differs greatly from presentation after virus replicates in the tissue and naturally drains to the regional lymph node. In order to better understand viral antigen presentation in the lymph node after viral replication in the skin and the impact on the initiation of CD8+ T cell responses, we infected mice in the ear pinna with vaccinia virus (VACV) epicutaneously using a bifurcated needle. VACV replicates to high titers in the skin, and viral antigen and genomes are detectable in the draining cervical lymph node by day 5 post infection, far beyond that after subcutaneous VACV injection. We are currently exploring the spatiotemporal dynamics of T cell activation as well as the antigen presenting cell subsets important for priming after epicutanous VACV infection. These findings are of significance for both our basic understanding of antiviral immunity and for the development of new T cell-based vaccination strategies, where optimization of CD8+ T cell responses is essential.
Protein kinase C-eta (PKCη) is dispensable for the activation and function of CD8+T cells
Authors: Liu Hsin Yu , Pedros Christophe , Canonigo Balancio Ann J , Altman Amnon
Published: January 2023

We previously reported that protein kinase C-eta (PKCη) forms a novel signaling complex with CTLA4 in regulatory T cells (Tregs) and this complex is required for the contact-dependent suppressive activity of Tregs. As a result, PKCη-deficient (Prkch−/−) Tregs fail to suppress anti-tumor immunity. However, given the ubiquitous expression of PKCη, it has remained unclear whether PKCη is also required for the effector functions of conventional T cells, particularly CD8+T cells that play a critical role in anti-tumor immunity. We therefore assessed the effect of genetic PKCη deletion on CD8+T cell-dependent immune functions. We found that Prkch−/− mouse or PRKCH knockdown human CD8+T cells displayed intact, and even enhanced, T cell activation in vitro as measured by proliferation and expression of TNFα, IFNγ, and granzyme B (GzmB) following anti-CD3 plus -CD28 costimulation. Next, we used the acute LCMV infection as an in vivo model to study the characteristics of virus-specific CD8+T cells, which were identified by specific staining with CD8 tetramers. Splenocytes from Prkch−/− mice showed similar frequencies of tetramer+CD8+T cells to those of wild-type (WT) mice, and these cells proliferated normally and expressed normal levels of TNFα and IFNγ. The LCMV-specific Prkch−/−CD8+T cells expressed normal or increased levels of GzmB, and the infected mice displayed greater viral clearance. We conclude that PKCη deficiency does not impact the activation and function of CD8+T cells. Taken together, the selective inhibition of the CTLA4-PKCη signaling pathway in cancer patients is a promising therapeutic strategy to inactivate tumor-promoting Tregs and, thus, enhance anti-tumor immunity, without having adverse effects on CD8+T cells.
Single-cell cloning of untreated HIV-specific natural killer cells in humans
Authors: Lucar Olivier Alexandre , Ghofrani Joshua , Goepfert Paul A , Jost Stephanie , Reeves R. Keith
Published: January 2023

Beyond their ability to eliminate infected cells without the need for prior sensitization, natural killer (NK) cells have been shown to exhibit adaptive immune functions. In particular, our lab demonstrated that antigen-specific memory NK cells responses against SIV can be induced by infection and vaccination in non-human primates. These data suggested that a subset of NK cells in humans may also mediate immunological memory responses to HIV.

To study human HIV-antigen-specific NK cells in more detail, we adapted limiting dilution assays to clonally expand individual peripheral blood NK cells (NKCL) from untreated HIV-infected individuals. Using a calcein acetoxymethyl ester cytotoxicity assay, the ability of each NKCL to lyse MHC-devoid K562 cell lines or autologous B-LCLs pulsed with a pool of MOG- (human self-peptide), HIV Gag- or HIV Env-derived overlapping peptides was assessed.

As expected, all tested NKCL could potently lyse K562 cells (median=56% killing) but did not kill B-LCL pulsed with the MOG control. Strikingly, 37.5% of NKCL displayed anti-HIV Gag cytotoxic activity up to 31% specific lysis (range: 1%–31%), while 25% of NKCL displayed anti-HIV Env cytotoxic activity up to 72% specific lysis (range: 1%–72%). Furthermore, pre-treatment with an anti-NKG2C blocking antibody decreased the specific lysis, thereby confirming the importance of NKG2C receptor in the function of antigen-specific NK cells.

Thus, we were able to isolate single NK cells displaying potent HIV-specific killing, within the range of robust cytotoxicity normally found against tumor cells. Together these data suggest that HIV-specific responses mediated by NK cells exist in humans and may have the potential to be harnessed for therapeutic modalities.
MicroRNA-21 is a critical regulator of the antiviral NK cell response
Authors: Geary Clair , Lau Colleen , Zawislak Carolyn , Santosa Endi K , Berrien-Elliott Melissa , Bezman Natalie , Lanier Lewis L , Fehniger Todd A , Sun Joseph Chai-Yuen
Published: January 2023

Natural killer (NK) cells are innate lymphocytes that possess adaptive features, including antigen-specific clonal expansion and memory responses. Global loss of microRNAs (miRNAs) leads to defects in NK cell homeostasis and antiviral responses; however, the role of individual miRNAs in these processes is poorly defined. Here, we investigated miR-21, a highly expressed miRNA in both human and mouse NK cells. While miR-21 was largely dispensable for NK cell development, miR-21-deficient NK cells failed to mediate significant host protection against mouse cytomegalovirus (MCMV) due to defective proliferation. Although activated NK cells upregulated miR-21 early after MCMV exposure, loss of miR-21 did not impact NK cell activation or effector molecule expression during viral infection. Transcriptome analysis of miR-21-deficient NK cells demonstrated dysregulation of cell cycle regulators, including several predicted targets of miR-21. Altogether, our findings demonstrate a novel role for miR-21 as a crucial regulator of adaptive NK cell responses during MCMV infection.
Reevaluating HLA-A2-restricted Lassa epitopes in human Lassa fever survivors
Authors: Sullivan Brian Martin , Sakabe Saori , Hartnett Jessica N , Nho Nhi , Goba Augustine , Momoh Mambu , Sandi John Demby , Kanneh Lansana , Cubitt Beatrice , Garcia Selma D , Ware Brian C , Kotliar Dylan , Robles-Sikisaka Refugio , Gangavarapu Karthik , de la Torre Juan Carlos , Sabeti Pardis C , Andersen Kristian G , Garry Robert F , Grant Donald S , Schieffelin John S , Oldstone Michael B
Published: January 2023

Many factors contribute to the selection of epitope-specific T cells. Often, candidate epitopes are generated through in silico prediction of various biological processes, mainly peptide generation and MHC binding, that are then tested in relevant biological assays. This is especially true of epitopes derived from BSL-4 pathogens or where relevant patient samples are difficult to obtain. Two previous studies identified CD8+ T cell epitopes from the Lassa virus glycoprotein through in silico prediction, experimental MHC binding assays, and epitope generation in HLA-A2 transgenic mice. Using samples from ten HLA-A*02:01 Lassa fever survivors, we tested whether these previously described epitope-specific CD8+ T cells were present and their relation to the broader Lassa virus-specific T cell response. Using overnight stimulation assays, we detected robust LASV-specific responses to the glycoprotein and nucleoprotein, but only one of the three epitopes (GP60–68)shown to be present and protective in mice made a substantial contribution to the overall LASV-specific response. Using a more sensitive proliferation assay, we detected the remaining two epitopes in some individuals at a very low frequency. Overall, this study shows the limitations of epitope discovery through in silico prediction, MHC binding and transgenic mouse models and highlights the complex nature of T cell selection during natural infection of humans.
Illuminating the Mechanism of MHC-I Folding and Antigen Repertoire Selection Using Deep Mutational Scanning and Biophysical Studies
Authors: Sgourakis Nikolaos , McShan Andrew C , Devlin Christine A , Morozov Giora , Procko Erik
Published: January 2023

Chaperones Tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning), and selecting high affinity peptide cargo in the MHC-I groove (editing). While X-ray and cryoEM snapshots of MHC-I molecules prepared in complex with TAPBPR and Tapasin, respectively, have provided important insights into the empty MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Of particular importance is a 16 aa loop region in TAPBPR (corresponding to 11 residues in the sequence of Tapasin), which has been suggested to actively compete with incoming peptides by forming direct contacts with the F-pocket of the empty MHC-I groove. Using a deep mutational scanning functional analysis of TAPBPR, we find that important residues for its chaperoning activity are located on the major interaction surfaces with nascent MHC-I molecules, excluding the loop. However, interactions with properly conformed molecules toward editing of their peptide cargo are influenced by loop mutations, in an MHC-I allele- and peptide-dependent manner, as shown in MHC-I tetramer staining experiments using a TAPBPR library expressed on the surface of yeast. Detailed biophysical characterization by NMR and ITC reveal that the loop does not interact with the empty MHC-I groove to compete with incoming peptides, but instead promotes peptide loading by acting as a kinetic “trap”. Our results suggest that, by utilizing a longer loop, TAPBPR lowers the affinity requirements for peptide selection relative to Tapasin to promote loading under conditions of reduced peptide concentration.
Single cell RNA sequence analysis reveals novel clusters unique to post-infectious myocarditis in the mouse model of Coxsackievirus infection
Authors: Lasrado Ninaad , Arumugam Rajkumar , Jayaraja Sabarirajan , Thibivilliers Sandra , Libault Marc , Starr Timothy K. , Reddy Jay
Published: January 2023

Myocarditis is a common occurrence in children and adolescents that can lead to dilated cardiomyopathy. Since it is difficult to identify the triggers of this complex disease, mechanistic understanding of DCM pathogenesis may create avenues to develop new treatment strategies. We have been engaged in delineating the autoimmune pathways underlying the development of DCM using various mouse models of myocarditis that resemble human disease. One such model is Coxsackievirus B3 (CVB)-induced myocarditis in A/J mice that show two distinct phases of disease, but occur in continuum. While, the acute, infectious phase (up to ~10 days post-infection) is marked by viremia and infiltrations in the hearts, infectious virions become undetected during the chronic phase (~beyond 18 days post-infection), and animals develop DCM. The latter phenotype can occur over a period of ~60 to 90 days, and hitherto, it has been a challenge to determine the underlying mechanisms. To dissect this complex nature of disease, we recently performed single cell RNA sequence analysis on heart infiltrates obtained from CVB-infected mice. Using panels of 15 to 30 marker genes for various immune and non-immune cells, we identified 18 and 26 clusters respectively in healthy and infected hearts that also include few unknown clusters unique for each group. Likewise, transcriptome analyses led us to identify a total of 14,051 genes to be differentially expressed, and of these, 206 and 345 transcripts were unique to healthy and CVB-infected groups, respectively. The data suggest that uniquely expressed genes in the CVB-infected hearts may have a role in the development of post-infectious myocarditis leading to DCM.
Microbiota-driven regulatory T cells suppress experimental autoimmune encephalomyelitis (EAE) in interleukin-17A deficient HLA class-II transgenic mice model of multiple sclerosis (MS)
Authors: Shahi Shailesh K , Freedman Samantha N , Murra Alexandra C , Cady Nicole , Gupta Arnav , Guseva Natalya V , Gibson-Corley Katherine N , Bossler Aaron D , Ganesan Sukirth M , Karandikar Nitin J , Mangalam Ashutosh K
Published: January 2023

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Despite IL-17A being linked to the immunopathogenesis of MS, the mechanism of IL-17A in disease development and maintenance is unclear. In the present study, we investigated the association of IL-17A and gut microbiota in disease development and severity, utilizing HLA-DR3 transgenic mice lacking IL-17A (IL-17A−/−). We found that HLA-DR3.IL-17A−/− mice had a higher frequency of CD4+CD25+FoxP3+ Treg cells and develop milder EAE compared to IL-17A suffcient HLA-DR3 mice. Utilizing 16S metagenocmic sequencing and analysis of gut microbiota we observed that HLA-DR3.IL-17A−/− mice had an increased abundance of regulatory T cells promoting bacteria in the gut such as Lactobacillus and Clostridia. Fecal transplantation followed by co-housing of HLA.DR3 mice with HLA-DR3.IL17A−/− resulted in development of milder EAE and an increase in CD4+CD25+FoxP3+ Treg cells in HLA-DR3 mice. Further whole-genome shotgun metagenomic analysis showed modulation of pathogen-associated molecular patterns (PAMPs) and O-antigen pathways linked with the host innate immune responses in HLA-DR3.IL-17A−/− mice. Thus our study suggests that gut microbiota changes due to the absence of IL17A induce regulatory T cells and suppresses disease in an animal model of MS by modulating the host-bacterial dynamics via PRR-PAMPS interactions.
Virus-mediated dysbiosis alters immune populations to promote type 1 diabetes onset
Authors: Morse Zachary J , Simister Rachel L , Crowe Sean A , Osborne Lisa C , Horwitz Marc S
Published: January 2023

In combination with genetic determinants, susceptibility to autoimmune diseases such as Type 1 Diabetes (T1D) is established by various environmental factors including infection, microbial dysbiosis, antibiotic use, and vitamin D deficiency. Studies have implicated infection with certain viruses such as coxsackievirus B (CVB) to be an important cofactor associated with diabetes development and pathogenesis. Infections may be an instigating factor to alter the microbiome and this microbial change may be sufficient to skew immune populations and promote autoimmunity. Mucosa-associated invariant T (MAIT) cell populations have been shown to be altered leading up to diabetes onset in patients and mice. These cells are activated by microbial products in the gut to promote intestinal integrity, but they can also take on a more inflammatory phenotype and participate in autoimmune responses in the pancreas. Ultimately, there exists a significant potential for cross-talk between CVB infection, the microbiome, and gut-resident immune cells impacting T1D susceptibility. We have found CVB infection not only promotes onset of T1D in non-obese diabetic (NOD) mice but also causes dysbiosis which resembles that of a spontaneously diabetic NOD mouse. Introducing this new infection-induced microbial composition into naïve mice through the use of fecal microbiome transfers (FMTs) can accelerate T1D onset and alter immune profiles in the gut as well as the pancreas. Furthermore, MAIT populations are altered by this “diabetogenic” microbiome and also respond directly to CVB infection. Together our data highlights the role of virus infection and its ability to affect the gut microbiome and immune homeostasis to contribute to T1D development.
Bacterially-derived indole modulates murine arthritis through alteration of B cell glycosylation enzymes
Authors: Chriswell Meagan , Jubair Widian , Tolbert Briana , Kuhn Kristine
Published: January 2023

Indole, a bacteria-derived immunomodulatory metabolite that binds the aryl hydrocarbon receptor (AhR), is produced by bacterial tryptophanase in the intestinal tract of both mice and humans. Our studies using the murine collagen-induced arthritis (CIA) model of inflammatory arthritis demonstrated that intestinal bacteria influence disease severity through modulation of autoantibody glycosylation, and indole significantly correlated with the development of disease (P<0.0001). Therefore, we hypothesized that indole modulates CIA through modulation of B cell glycosylation enzymes via AhR signaling. Mice with CIA that were protected by treatment with broad-spectrum antibiotics at the time of booster injection (CIA+Abx), lost the protective effect of antibiotics when dosed with indole (arthritis score 3.6±0.75 CIA+Abx+Indole vs. 1±0.00 CIA+Abx, p<0.001). Mice with CIA, compared to CIA+Abx, demonstrated significantly increased expression of β1,4-galactosyltransferase 1 (GalT) and β-galactoside α2, 6-sialyltransferase 1 (St6gal1), two enzymes important in mediating pathogenic antibody glycosylation; this effect was reversed in CIA+Abx+Indole. We further demonstrate that Ramos cells, a human B cell line, upregulate glycosylation enzymes and AhR expression in response to indole exposure, with a fold change expression of 3.315±0.92 of B4GALT5 in indole-treated cells as compared to untreated (p=0.05). While the requirement of AhR and the glycosylation status of antibodies in our studies need to be confirmed, these results suggest that microbiome changes in CIA result in indole signaling on B cells, affecting glycosylation enzymes that modulate autoantibody effector function towards a proinflammatory profile.
Prevotella histicola treatment reduces arthritic pain and partially normalizes gut microbiota and metabolites
Authors: Balakrishnan Baskar , Luckey David , Bodkhe Rahul , Taneja Veena
Published: January 2023

We have previously shown that treatment with a novel human commensal, Prevotella histicola, isolated from a duodenal biopsy, can protect HLA-DQ8 mice from collagen induced arthritis (CIA), a model for RA, by modulating the systemic immune response. The present study analyzed the functional and mechanistic impact of P. histicola by comparing pain, motor function, gut microbiota and metabolites in treated and non-treated arthritic DQ8 mice. P. histicola treatment reduced the arthritis-associated pain by improving the motor function when compared to control mice. Analysis of synovium showed that P. histicola treatment increased T regulatory cells and reduced the levels of IL6 in the synovium of arthritic mice which were associated with reduced pain. Treated mice showed P. histicola colonization in the duodenum, a niche from where it was isolated. Further, P. histicola treatment showed varied effects based on circadian rhythm. A longitudinal follow-up of the gut microbial profile in different gut sections suggested that dysbiosis caused due to arthritis in DQ8 is partly normalized to naïve profile after treatment with P. histicola. Treated mice showed an expansion of genus Allobaculum, a butyrate-producing Firmicutes in the guts of DQ8 arthritic mice as compared to pre-treatment. The post-treatment restoration was also reflected in short-chain fatty acids and glucose assimilation rates in T cells. The present study proposes that P. histicola treatment of arthritis in DQ8 mice leads to gut homeostasis. Normalized microbial diversity and metabolites lead to immune regulation resulting in reduced inflammation with improved physical function. The observations suggest P. histicola as a strong candidate for monoclonal bio-therapeutics.
Characterizing the role of Lactobacillus spp. in systemic lupus erythematosus
Authors: Puig Xavier Cabana , Mu Qinghui , Swartwout Brianna , Abdelhamid Leila , Prakash Meeta B. , Reilly Christopher M. , Luo Xin M.
Published: January 2023

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease with no known cure. The role of gut microbiota and its interaction with the immune system can direct tolerance induction to self-antigens not only in the intestinal mucosa but also at the systemic level. Our lab previously showed differences between the gut microbiota in lupus-prone MRL/Mp-Faslpr (lpr) mice compared to healthy controls, with lower Lactobacillaceae levels in lpr mice. Restoring the microbiota with a mix of 5 different Lactobacillus strains, L. reuteri, L. oris, L. johnsonii, L. gasseri, and L. rhamnosus significantly attenuated lupus-liked disease in lpr mice, by increasing regulatory T cells and balancing T helper-17 cells. Interestingly, we found L. reuteri and an uncultured Lactobacillus spp. were present based on 16S rRNA sequencing. To evaluate the effect of the main candidate L. reuteri in attenuating lupus, we treated lpr mice with the single bacterial strain. Surprisingly, L. reuteri did not reproduce the same results as the Lactobacillus mixture. Thus, we tested the remaining strains individually but none was able to recapitulate the effect of the 5 strains. Additionally, we treated lpr mice with the supernatant of the 5 strains containing secreted metabolites, which did not significantly attenuate lupus. These results suggest that communications among different strains of lactobacilli may be required in ameliorating the disease. Probiotic Lactobacillus spp. may be an alternative treatment against SLE, but further investigations are warranted to delineate the mechanisms of how multiple strains of lactobacilli can exert a beneficial effect as a community.
Analysis of the nonobese diabetic mouse islet MHC-II peptidome reveals posttranslationally modified autoantigens
Authors: Lichti Cheryl , Vomund Anthony N. , Peterson Orion J. , Wan Xiaoxiao , Unanue Emil R
Published: January 2023

Autoimmune diabetes results from the destruction of insulin-producing beta cells, a process initiated by CD4 T cells recognizing MHC-II bound beta cell-derived peptides. We identified MHC-II bound peptides in the islets and throughout the peripheral lymphatic system in order to better understand diabetes initiation and progression. Toward this end, we performed unbiased nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis to obtain sequence information for MHC-II peptides isolated from islets, pancreatic lymph nodes and spleens of nonobese diabetic (NOD) mice. Peptides derived from beta cell proteins were further examined for their relative I-Ag7 binding strength and CD4 T cell autoreactivity. For a discussion of the major immunogenic peptides derived from the B chain of insulin and from C peptide, see the abstract of Wan et al.

Herein we discuss the finding of a deamidated C peptide fragment and our search for fused peptides. We identified the deamidated insulin-1 C peptide fragment Ins1C:51–61, LQTLALEVARE. Evidence in the literature supports this C-terminal deamidation as being spontaneous rather than enzyme-mediated. Deamidation improved I-Ag7 binding compared to the wild type peptide and increased its immunogenicity, demonstrating the biological importance of this modification. The homologous peptide from insulin-2 was not identified, indicating a possible sequence bias for spontaneous deamidation. We identified only one fused peptide bound to I-Ag7, an insulin C peptide-islet amyloid polypeptide hybrid insulin peptide (HIP) that matches the previously reported HIP6.9. We also identified several free fused peptides in crinosomes and posit this as the probable site of their formation.
Toll-like Receptor 2 induces pathogenicity in Th17 cells
Authors: Marks Kathryne E , Flaherty Stephanie , Reynolds Joseph M
Published: January 2023

CD4+ T cells, especially Th17 cells, are instrumental in the development and progression of autoimmune diseases, such as multiple sclerosis (MS), psoriasis, and rheumatoid arthritis. While pathogenic Th17 cells drive inflammation in autoimmunity, the molecular programming underlying their pathogenicity remains insufficiently understood. Furthermore, very little is known regarding the role of the pattern recognition receptor TLR2 in Th17 pathogenicity. We have previously shown that activation of TLR2 with PAM3CSK4 during Th17 differentiation results in increased production of IL-17A and IL-17F and significantly increases the proliferation of Th17 cells in vitro. Herein, we show that TLR2 induces pathogenicity in Th17 cells, similar to what has been shown for IL-23. TLR2 signaling results in increased encephalitogenic migration of Th17 cells, in addition to increased cytokine production. To further investigate the effect of TLR2 activation on Th17 cells, we utilized RNA sequencing and found 390 genes that are upregulated and 355 genes that are downregulated following TLR2 signaling in Th17 cells. Several of these genes we have identified are important regulators that either increase or restrain the inflammatory potential of Th17 cells. We further address the role of TLR2 in Th17-mediated autoimmune disease using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show that encephalitogenic Th17 cells expanded with a TLR2 agonist induced EAE disease to the same extent as those expanded with IL-23. Altogether, this study establishes TLR2 signaling as crucial for induction of pathogenic Th17 cells in autoimmune disease.
Genetic Interactions of the Transcription Factor Ets1 and IL17RA Signaling in Autoimmunity and Immunodeficiency
Authors: Battaglia Michael , Sunshine Alex , Luo Wei , Jin Richard M , Garis Matthew , Stith Alifa , Lindemann Matthew , Miller Lloyd S , Sinha Satrajit , Wohlfert Elizabeth A , Garrett-Sinha Lee Ann
Published: January 2023

Ets1 is a lineage-specific transcription factor that regulates B and T cell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop spontaneous autoimmune disease with high levels of autoantibodies. Naïve CD4+ T cells isolated from Ets1 KO mice differentiate more readily to Th17 cells that secrete IL17, a cytokine extensively implicated in autoimmune disease pathogenesis. To determine if increased IL17 production contributes to the development of autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL17 receptor A subunit (IL17RA KO) to generate double knock out (DKO) mice. We found that the absence of IL17RA signaling did not prevent or ameliorate the autoimmune phenotype of Ets1 KO mice, but rather that DKO animals exhibited worse symptoms with significant increases in activated B cells and secreted autoantibodies. This was correlated with a prominent increase in the numbers of T helper 2 (Th2) and T follicular helper (Tfh) cells. In addition to the autoimmune phenotype, DKO mice also showed aspects of immunodeficiency and developed spontaneous skin lesions colonized by Staphylococcal species. When DKO mice were experimentally infected with Staphylococcus aureus they were unable to clear the bacteria, despite a robust anti-staphylococcal antibody response. Persistent skin infection results in increased immune cell activation and likely contributes to the enhanced autoimmune symptoms of DKO versus Ets1 KO mice. Our studies suggest that targeting IL17RA signaling in autoimmune diseases might in some cases result in increased autoimmunity or to increased susceptibility to opportunistic infections.
GM-CSF mediates development of eosinophil-dominant autoimmune uveitis in the absence of both IFN-γ and IL-17A
Authors: Horai Reiko , Bing So Jin , Silver Phyllis B. , Jittayasothorn Yingyos , Chan Chi-Chao , Caspi Rachel R
Published: January 2023

Autoimmune uveitis is a group of blinding diseases triggered by activated retina-specific T cells. Studies in uveitis patients and experiments in animal models of experimental autoimmune uveitis (EAU) support the notion that Th1 and Th17 cells are both pathogenic effectors and each can elicit ocular autoimmunity independently of the other. Since absence of IFN-γ or IL-17A individually failed to abolish pathology of EAU, we examined EAU development in the absence of both cytokines. Ifng−/−Il17a−/− mice were fully susceptible to EAU and displayed eosinophil-dominant ocular infiltrates, as opposed to mononuclear infiltrates in WT mice. EAU was ameliorated in double-deficient mice when eosinophils were genetically absent or their migration was blocked, supporting a pathogenic role of eosinophils in the absence of both IFN-γ and IL-17A. In Ifng−/−Il17a−/− mice immunized for EAU, ocular infiltrates contained increased GM-CSF-producing CD4 T cells, and supernatants of antigen-recalled splenocytes contained enhanced levels of GM-CSF that contributed to activation and migration of eosinophils in vitro. Systemic or local blockade of GM-CSF ameliorated EAU in Ifng−/−Il17a−/− mice. Neutralization of GM-CSF during the induction phase of EAU decreased eosinophil infiltration to the eye and eosinophil peroxidase levels in the eye and in the serum. These results support the interpretation that, in the concurrent absence of IFN-γ and IL-17A, GM-CSF takes on a major role as a pathogenic effector cytokine and induces an eosinophil-dominant pathology. This may impact therapeutic strategies aiming to target IFN-γ and IL-17A in autoimmune uveitis.
Regulation of necroptosis by targeting tumor suppressor death-associated protein kinase 1
Authors: Lai Ming-Zong , Wu Yung-Hsuan , Chou Ting-Fang , Young Leslie , Hsieh Fu-Yi , Pan Hsuan-Yin , Mo Shu-Ting , Brown Shani Bialik , Chen Ruey-Hwa , Kimchi Adi
Published: January 2023

Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic induction by different stimuli. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1-RIPK3-MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK and AKT, were not regulated by DAPK. In contrast, DAPK selectively promoted MKK3-induced p38 MAPK activation, resulting in enhanced MK2 phosphrylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.
Gamma subunit of complement component 8 is an innate immune suppressor in brain
Authors: Suk Kyoungho
Published: January 2023

The complement system is a part of the innate immune system, which comprises several small proteins activated by sequential cleavages. The majority of these complement components, such as components 3a (C3a) and C5a, are chemotactic and pro-inflammatory. However, in this study, we revealed an inhibitory role of complement component 8 gamma (C8G) in neuroinflammation. Novel findings show that the expression level of C8G increases in inflamed brain, and C8G is mainly localized to brain astrocytes. Experiments using recombinant C8G protein and shRNA-mediated knockdown showed that C8G inhibits microglial activation and neuroinflammation. In acute and chronic animal models of Alzheimer’s disease (AD), which exhibit strong neuroinflammation, C8G attenuated cognitive decline and glial hyperactivation. In these animals and patients with AD, we found higher C8G levels in brain tissue, cerebrospinal fluid, and plasma. Additionally, we identified sphingosine-1-phosphate receptor 2 (S1PR2) as a novel interaction protein of C8G and demonstrated that astrocyte-derived C8G interacts with S1PR2 to antagonize the pro-inflammatory action of S1P in microglia. Taken together, our results reveal a previously unrecognized role of C8G as a neuroinflammation inhibitor and indicate that C8G can be used therapeutically for the containment of neuroinflammation and treatment of related neurological diseases, such as AD.
IEX-1 deficiency in hematopoietic cells mitigates high fat diet-induced obesity and glucose intolerance
Authors: Shahid Mohd , Loya Egwin , Wen Shuguang , Wu Mei X
Published: January 2023

We previously showed that IEX-1 paucity in mice inhibited high fat diet (HFD)-induced obesity and insulin resistance. IEX-1 deficiency induced browning of white adipose tissue (WAT) by promoting alternative activation of macrophages (AAMs), increasing energy expenditure. We now hypothesize that IEX-1 deficiency in macrophages promotes alternative activation of adipose tissue macrophages (ATM) and induces browning to inhibit obesity. We performed reciprocal bone marrow (BM) transplantation between IEX-1 knockout (KO) and wild-type (WT) mice to generate four types of chimeras. We fed these mice with HFD for 20 weeks and monitored their body weight, glucose disposal, and determined ATM phenotype using flow cytometry. While WT+WT-BM control mice (WT mice received WT bone marrow) gained ~80% body weight on HFD, WT+KO-BM mice (WT mice received KO bone marrow) increased their body weight only by 50% (p<0.01). Conversely, KO+WT-BM mice (KO mice received WT bone marrow) exhibited increased weight gain on HFD as compared to KO+KO-BM control mice that remained lean (p<0.05). Likewise, WT+KO-BM mice displayed greater glucose disposal as compared to WT control, and KO+WT-BM mice became glucose intolerant as compared to KO control as determined by glucose tolerance test. Analysis of ATM phenotype in mice fed with HFD for 5 weeks showed that HFD increased the percentage of classically activated macrophages (CAMs) by 3–4-folds (p<0.01), and decreased AAMs by ~30% (P<0.05) in epidydmal and inguinal WAT of WT+WT-BM mice. However, these effects were markedly suppressed in WT+KO-BM mice. Collectively, these data suggest that IEX-1 in hematopoietic cells contributes to HFD-induced obesity and glucose intolerance via inhibiting AAMs polarization.
Phosphatase SHP2-mediated mitochondrial homeostasis for the resolution of inflammation
Authors: Xu Qiang , Guo Wenjie , Wu Xingxin , Sun Yang
Published: January 2023

The negative regulation of inflammation is very important for the resolution of inflammatory diseases. Although many components and mediators in inflammation have been identified, how inflammation is finely regulated remains lack of evidence. Here we show that Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) translocates to mitochondria in the early time of inflammatory stimulation. The RRWFH motif in SH2 domain is essential for the translocation of SHP2. This phosphatase localizes in the mitochondrial matrix with the help of Tom20/Tom40 in outer membrane and Tim23 in inner membrane. Then SHP2 interacts with ANT1, a central molecule controlling mitochondrial permeability transition. This mechanism prevents the collapse of mitochondrial membrane potential, mtDNA release and ROS production, and thus inhibits the hyperactivation of NLRP3 inflammasome. Ablation or inhibition of SHP2 in macrophage causes intensified NLRP3 inflammasome activation, production of IL-1β and IL-18, and increased sensitivity to peritonitis. We are also engaged in seeking the activator of SHP2. We have found that the natural products fusaruside and trichomides A can increase the phosphorylation of SHP2 and some of synthesized compounds can directly activate the phosphatase. These SHP2 activators show a significant anti-inflammatory activity in various disease models. Collectively, our data highlight that SHP2 orchestrates an intrinsic regulatory loop for the mitochondrial homeostasis to limit excessive NLRP3 inflammasome activation, suggesting a possibility to treat inflammatory diseases via maintaining mitochondrial homeostasis.