bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinated metabolic response of dendritic cells to influenza infection
Authors: Rezinciuc Svetlana , Bezavada Lavanya , Thomas Paul G. , Smallwood Heather S
Published: January 2023

Infection with the influenza virus triggers an innate immune response aimed at limiting viral replication and spread. However, the molecular mechanism(s) underlying the processes of change–in innate immune cell homeostasis are not known. We show here that the influenza virus specifically alters the metabolism of dendritic cells (DCs) to enhance viral biomass production and that the metabolic response of the dendritic cells to viral infection differed from activation via TLR stimulation or infection with inactive virus. Using quantitative mass spectrometry along with pulse chase assay and metabolic flux measurements, we found that metabolic pathway changes dominated after infection, including increased glycolysis and glutaminolysis, and decreased tricarboxylic acid cycle and β-oxidation. This metabolic response to viral infection increased the expression of the c-Myc transcription factor, which indicates that influenza have the potential to induce key metabolic regulatory events. Influenza infection induces a unique metabolic signature in dendritic cells, prior to the canonical innate response to viral infection. Limiting these metabolic changes by metabolite restriction induces massive cell death in infected DCs, suggesting they play a vital role in initiating the innate immune response.
Blocking CCR10 diverts skin-homing T cells to reproductive tract for enhanced establishment of local resident memory T cells
Authors: Zhao Luming , Hu Shaomin , Davila Micha L , Xiong Na
Published: January 2023

Tissue-resident memory T (TRM) cells play an important role in protection against re-occurring infection. Compared with circulating memory T cells, tissue-specific TRM cells have great advantages in mounting faster and more effective responses against local infections. Female reproductive tract (FRT), which is a portal of entry for sexually transmitted infections, is an immunologically restrictive tissue that prevents progenitors of TRM cells entering in the absence of inflammation or infection. Therefore, how to increase number of TRM cells in FRT after vaccination to improve memory response against pathogens becomes an important question. In our present study, we found that a significant portion of T cells in FRT expressed the skin-homing chemokine receptor CCR10 in physiological conditions, and those CCR10+ T cells displayed surface markers typical for TRM cells. Particularly, almost all the CCR10+ CD8+ T cells expressed CXCR3, a homing marker which could help T cell migrate into FRT, suggesting the competition of skin and FRT for CD8+ T cells. After epicutaneous immunization, activated antigen-specific CCR10-knockout CD8+ T cells migrated more efficiently into FRT than CCR10-sufficient T cells. Our finding suggests that blocking CCR10 signals could divert skin-homing T cells into FRT for enhanced establishment of local resident memory T cells.
IL-17 is crucial for psoriatic inflammation, but also initiates an anti-inflammatory feedback loop via signaling into keratinocytes
Authors: Ha Hye-Lin , Wang Hongshan , Ulrich Siebenlist
Published: January 2023

The IL-17 cytokines make crucial contributions to chronic inflammatory skin disease and anti-IL-17 antibodies have proven to be highly efficacious in treating psoriasis. IL-17 cytokines signal via the obligatory adaptor CIKS/Act1. Previously, we have identified critical roles of CIKS-mediated signaling in imiquimod-induced psoriatic inflammation. IL-17/CIKS-mediated signaling into keratinocytes was essential for neutrophil infiltration and contributed to pathology by promoting proliferation and dampening terminal differentiation of keratinocytes. Interestingly, IL-17 signaling into keratinocytes also exerted negative feedback, delimiting continued accumulation of IL-17-producing γδ T cells in skin. We now demonstrate that the negative feedback is mediated in part via IL-19, a member of the IL-10 family of cytokines. IL-19 is strongly induced by IL-17-stimulated keratinocytes and appears to function by suppressing recruitment of IL-17-producing γδ T cells over time, thereby dampening IL-17 production. This stands in contrast to IL-17/CIKS-mediated signaling into non-keratinocytes – specifically dermal fibroblasts - which initially promoted cellular infiltration and enhanced accumulation of IL-17-producing γδ T cells in skin, comprising an early positive feed-forward mechanism. Our data show that CIKS-mediated signaling is dynamic; it is central to the development of both dermal and epidermal hallmarks of psoriasis, but also initiates mechanisms that ultimately mitigate further tissue damage. The latter mechanisms may offer additional avenues for therapeutic intervention.
Memory-phenotype CD4+ T cells spontaneously generated under steady state conditions exert innate Th1-like effector function
Authors: Kawabe Takeshi , Jankovic Dragana , Kawabe Shuko , Huang Yuefeng , Lee Ping-Hsien , Yamane Hidehiro , Zhu Jinfang , Sher Alan , Germain Ronald N , Paul William E
Published: January 2023

CD4+ T cells are composed of naïve, pathogen-specific memory, and pathogen-independent memory-phenotype (MP) cells. Naïve and pathogen-specific memory cells play key roles in adaptive immunity while the homeostatic mechanisms regulating the generation of MP cells and their biological functions are unclear. In this work we show that MP cells are autonomously generated from peripheral naïve cells in the absence of infectious stimulation in a TCR- and CD28-dependent manner. We further demonstrate that MP cells contain a T-bethi subpopulation that is continuously generated by environmental IL-12 and rapidly produces IFN-γ in response to IL-12 in the absence of pathogen recognition. Importantly, these cells can provide nonspecific host resistance against Toxoplasma gondii infection while enhancing the adaptive CD4+ T cell responses. Together, these findings reveal that MP cells are continuously generated from naïve precursors and possess a novel innate immune function by which they produce an early, Th1-like protective response against pathogens.
C-Myc regulation by Notch Signaling Modulates T Cell Differentiation
Authors: Song Jim Jianxun
Published: January 2023

Notch signaling is essential for T lineage commitment and further required during early phases of thymocyte differentiation. Active Notch signaling during early stages of T cell development inhibits other lineage potentials, such as B cell and myeloid cell (e.g., dendritic cell) potentials. However, the intracellular signaling pathways by which Notch signaling regulates T cell differentiation remain unknown. Here we show that the transcriptional factor c-Myc is controlled by Notch signaling which regulates T cell differentiation. In a well-established in vitro differentiation of T lymphocytes from stem cells, Notch signaling directly controls c-Myc expression. Overexpression of active c-Myc promotes while dominant-negative (dn) c-Myc inhibits early cell differentiation. Moreover, c-Myc expression mediated by Notch signaling modulates survivin, an inhibitor of apoptosis (IAP) protein, which is crucial for T cell development. Overexpression of active c-Myc increases while dn c-Myc reduces survivin expression, which corresponds to T cell differentiation within the in vitro differentiation system. These results identify c-Myc, together with survivin, as regulators of the differentiation of T cells from Notch signaling.
Bacterial Siderophores Hijack Neutrophil Functions
Authors: SAHA PIU , Yeoh Beng San , Olvera Rodrigo A , Xiao Xia , Awasthi Deepika , Singh Vishal , Dikshit Madhu , Wang Yanming , VIJAY-KUMAR MATAM
Published: January 2023

Neutrophils are primary immune cells that respond to inflammation and eliminate microbial transgression. Yet, E. coli have been reported to bloom during gut inflammation despite the heightened neutrophil activity. The survivability of E. coli in inflamed gut are not completely understood, although their production of enterobactin (Ent; a siderophore) to circumvent inflammation-induced iron scarcity may be one of the potential mechanisms. Herein, we report that Ent is not only an iron chelator, but is an immune-regulator that counter an array of neutrophil functions. We showed that Ent inhibited PMA and LPS induced generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in both mouse and human neutrophils. Ent also impaired the degranulation of primary granules, inhibited phagocytosis and bactericidal activity of neutrophils, but without affecting their migration and chemotaxis. Molecular analysis revealed that Ent can chelate intracellular labile iron that are required for neutrophil oxidative responses. Other siderophores (pyoverdine, ferrichrome, deferoxamine) likewise inhibited ROS and NETs in neutrophils, thus indicating that the chelation of iron may, in part, explain their inhibitory effects. To counter iron theft by Ent, neutrophils rely on the siderophore-binding protein lipocalin 2 (Lcn2) in a ‘tug-of-war’ for iron. The inhibition of neutrophil ROS and NETs by Ent was augmented in Lcn2-deficient than WT neutrophils, but rescued by the exogenous addition of recombinant Lcn2. Taken together, our findings illustrate the novel concept that microbial siderophore’s iron scavenging property may serve as an antiradical defense system, that neutralize immune functions of neutrophils.
Understanding the determinants of CD8+ T cell immunodominance in acute HIV infection
Authors: Warren Joanna A , Xu Yinyan , Costanzo Margaret , Whale Victoria , Liu Michael P.K. , Thomas Rasmi , Tovanabutra Sodsai , Kijak Gustavo , Eller Leigh Anne , Rolland Morgane , Michael Nelson L. , Robb Merlin L. , Eller Michael A. , Goonetilleke Nilu
Published: January 2023

CD8+ T cells detect and clear virus-infected cells. During human immunodeficiency virus-1 (HIV-1) infection, different HIV-specific CD8+ T cell populations are expanded, each targeting a discreet epitope. These different HIV-specific CD8+ T cell populations do not arise equally, and instead establish a hierarchy (immunodominance) based on strength of T cell response to a given viral epitope. Our previous work has shown that relative immunodominance is a major determinant of the rate at which HIV can mutate to escape T cell immune pressure. The precise mechanism of how the first CD8+ T cell response to HIV is generated, and which factors determine T cell dominance is unclear. To investigate determinants of T cell immunodominance in acute HIV-1 infection (~45 days following infection), we determined all primary HIV-1 specific CD8+ T cell responses in 10 individuals in the RV217 early HIV cohort (ECHO). T cell responses were mapped against the unique infecting HIV virus/es in each individual using IFN-γ ELISpot. Across participants, the dominant T cell response mostly targeted variable regions of the HIV-1 genome. We detected a wide (4–13 epitopes) breadth of T cell response across our cohort that was independent of HLA haplotype, with all HIV-1 proteins targeted and different rates of virus escape observed. Ongoing studies are focusing on better understanding how factors, such as T cell receptor repertoire, avidity, antigen presentation and function contribute to T cell immunodominance, which in turn will inform HIV-1 vaccine design.
Control of cytotoxic T lymphocyte memory development by beta2-adrenergic receptor signaling
Authors: Estrada Leonardo Daniel , Agac Didem , Farrar David
Published: January 2023

Cytotoxic T lymphocytes (CTLs) play a critical role in immune responses to intracellular pathogens. CTL responses are initiated by T cell receptor signaling, and they can be modulated by secreted factors, such as cytokines and other extracellular signals. Previous research from our lab demonstrated that the acute effector functions of CTLs are regulated by the neurotransmitter norepinephrine (NE) via beta2-adrenergic receptor (ADRB2) signaling in vitro. We now demonstrate that ADRB2 signaling intrinsically modulates in vivo memory development in response to viral infection using an adoptive transfer model. Expansion of ADRB2-deficient CTLs is unaffected during the initial priming phase in response to the model pathogen, vesicular stomatitis virus. However, ADRB2-deficient CTLs contract rapidly and do not yield any detectable pools of memory cells when compared to wild-type counterparts. Transcriptome analysis revealed a variety of differentially expressed genes, both expressed in resting naïve cells as well as in cells obtained during the primary expansion phase of infection. Among them, we found that the expression of the high affinity IL-2Ra (CD25) was reduced during the activation phase in ADRB2-deficient cells, which was confirmed at the protein level by flow cytometry. While IL-2 responsiveness is a key determinant in programming memory cell development, treatment with IL-2/anti-IL-2 complexes during early phases of infection failed to rescue memory formation of ADRB2-deficient cells, despite a restoration of CD25 expression on day 5 post-infection. Future experiments will dissect the role of ADRB2 signaling during CTL development on the capacity to form memory upon antigen exposure.
Negative regulation of IFNAR1/2 Signaling by USP18 is required for immunity to Listeria monocytogenes
Authors: Flores Ricardo , Mavinkurve Vikram , Muthukumaran Geetha , Cabasal Marian , Nguyen Tammy , Rajan Robin , La-Beck Irene , Paterson Yvonne , Wood Laurence M
Published: January 2023

Listeria monocytogenes (LM) is a bacterial pathogen linked to an increasing number of infections in countries with aging populations and a high mortality rate in immunocompromised patients. The immune response to LM is well-studied due to its proficient induction of T cell responses, a key attribute leading to its development as a vaccine platform. Thus, mechanisms regulating immunity to LM are of significant interest. In this study, we report that ubiqutin-specific protease 18 (USP18), a key regulator of Type I IFN signaling, is induced during LM infection and is necessary for a functional immune response to the pathogen. Our studies demonstrate that USP18 deficiency results in severe susceptibility to primary LM infection and an inability to develop functional adaptive immunity to LM. Increased susceptibility to LM infection in USP18−/− mice was associated with impaired inflammatory cytokine responses, reduced dendritic cell (DC) maturation, blunted splenic T cell function, and elevated immunosuppression. This defective immune response to LM in mice lacking USP18 is due in large part to unregulated Type I IFN signaling. Inhibition of signaling through IFNAR1/2 resulted in a dramatic reversal of LM susceptibility in USP18−/− mice as evidenced by improved LM infection control, DC maturation, T cell function, and induction of protective immunity. Therefore, we find that USP18 deficiency results in an IFNAR1-dependent susceptiblity to LM infection and impairment of protective memory responses; a finding with broad application to disease prevention and vaccine development.
PD-1 signals instruct a critical metabolic switch for maintenance of T cell memory
Authors: Yuzefpolskiy Yevgeniy , Baumann Florian M , Penny Laura A , Kalia Vandana , Sarkar Surojit
Published: January 2023

PD-1 is highly expressed on CD8 T cells during activation, both in acute and chronic viral infections. Inhibitory signaling in cytotoxic T cells through the PD-1 axis is well characterized during chronic infections and has led to the development of clinical checkpoint blockade therapies that restore function to otherwise exhausted CD8 T cells. However, the functional significance of transient PD-1 expression on T cells early after activation during acute infections remains yet to be characterized. It is paradoxical that expression of PD-1 (an inhibitory receptor) during activation temporally coincides with rapid proliferation and production of copious amounts of signature effector cytokines (IFN-γ and TNF-α). Our studies using PD-1−/−antigen-specific CD8 T cells revealed an unexpected lack of an inhibitory role for PD-1 early during priming - PD-1−/−T cells exhibited similar proliferation, accumulation, cytotoxicity and polyfunctionality compared to WT T cells. Surprisingly, PD-1−/−T cells underwent precipitous contraction, leading to near ablation of the memory pool. Mechanistically, PD-1−/−memory Tcells showed a severe defect in antigen-independent homeostatic proliferation in vivo, despite identical expression of common gamma-chain cytokine receptors as WT T cells. Notably, PD-1−/−memory T cells failed to optimally switch the critical utilization of glucose to fatty acid as an energy source, which impaired their homeostatic maintenance and resulted in attrition. These findings show a previously unrecognized role of PD-1 signaling in programming a metabolic switch that is critical for the maintenance of memory CD8 T cells and could make it a novel target for manipulating vaccine-induced memory T cell longevity.
Dynamic modulation of cortical actin at the immunological synapse controls lytic granule secretion in cytotoxic T lymphocytes
Authors: Kapnick Senta , Ritter Alex , Griffiths Gillian , Lippincott-Schwartz Jennifer , Schwartzberg Pamela
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTLs) are critical for eliminating virally infected cells. Upon target recognition, CTLs reorganize their actin cytoskeleton to facilitate the polarized secretion of specialized granules containing lytic proteins that kill cells. Because a single CTL can kill multiple targets, granule secretion must be tightly regulated, ensuring only appropriate cells are killed, and preserving granules for serial killing. However, how CTLs regulate secretion remains unclear. Here, we used live confocal and TIRF microscopy to evaluate how cortical actin affects granule secretion at the membrane. While previous work has shown that clearance of actin at the synapse precedes granule secretion, we find that after granule fusion, cortical actin recovers and no further secretion is observed. Depolymerization of recovered actin led to resumed granule fusion, suggesting that recovered actin acts as a barrier to prevent further lytic granule secretion. We further show that CTLs from mice unable to secrete granules due to a Rab27a mutation that prevents granule fusion, failed to recover cortical actin at the synapse. This suggests that granule fusion itself triggers actin recovery in CTLs. Thus, cortical actin both regulates, and is regulated by, secretion. Finally, using fluorescent reporters, we correlated the clearance and recovery of actin with PIP2. PIP2 binds numerous actin regulatory proteins, suggesting phosphatidylinositol distribution in the membrane as a potential mechanism through which CTLs regulate the density of cortical actin during killing. Our work provides insight into actin-related mechanisms regulating CTL secretion that may serial killing capacity during immune responses.
The Limit of MHC Class II-driven Selection in Germinal Centers
Authors: Yeh Chen-Hao , Nojima Takuya , Kuraoka Masayuki , Kelsoe Garnett H.
Published: January 2023

B cells undergo iterative rounds of proliferation, hypermutation and selection in the germinal centers (GCs) to generate high affinity, protective antibody. It is now thought that GC B cells with higher affinity BCRs acquire more antigen for processing and presenting in association with MHC class II (MHCII) to T follicular helper (TFH) cells than lower affinity competitors. The resulting increase in T-cell help is hypothesized to be the major factor that drives affinity maturation. In this model for GC B cell selection, the density of peptide/MHCII (pMHCII) complexes available on the B-cell membrane is the major determinant of affinity-dependent selection. To quantify the limits of this model for GC selection, we generated short- and long-term chimeric mice with equal numbers of isogenic, mature B cells that differed by their capacity to express MHCII (MHCII+/+ and MHCII+/−). Separately, both B cell types generated virtually identical humoral responses, including affinity maturation, in response to NP-OVA. In competition, MHCII+/+ B cells were preferentially recruited to early GCs consistent with a critical role for pMHCII density in T:B collaboration. However, once established in GCs, no further competitive advantage was observed during the full course (24 days) of the GC response. During this period, both MHCII+/+ and MHCII+/− GC B cells comparably accumulated VH gene segment mutations and exhibited comparable rates of affinity maturation to the NP hapten. These data indicate that B-cell selection determined by MHCII density is more stringent prior to the establishment of GCs than in the GC itself. This observation may be relevant to our recent observation of the persistence of low affinity GC B cells.
IL-10 downregulates IL-4-induced IgE production through indirect effects on B cells
Authors: Lin Adora A , Nutman Thomas B
Published: January 2023

Control of allergic disease (and of helminth-associated pathology) is associated with decreased antigen-specific IgE and increased antigen-specific IgG4. Although IL-10 appears to contribute to this process, underlying mechanisms are largely unknown. This study explored how IL-10 differentially regulates human IgE and IgG4 production. PBMCs and highly purified total, naïve (CD27-), and memory (CD27+) B cells were cultured with combinations of IL-4, IL-10, and anti-CD40. Quantitative PCR was done for Cε and Cγ4 germline transcripts (GLTs). IgG and IgE isotypes were quantified from culture supernatants using multiplexed immunoassays. IL-4 induced the production of IgE and IgG4 by PBMCs (IgE: geo mean 16.2-fold above baseline; IgG4: geo mean 4.7-fold). IL-4 also increased expression of Cε and Cγ4 GLTs to 54.1-fold and 5.3-fold above baseline, respectively. Addition of IL-10 significantly (p<0.01) reduced IL-4-induced IgE production to 4.6-fold and expression of Cε GLTs to 24.7-fold above baseline, without affecting IgG4 production or expression of Cγ4 GLTs. In cultures of isolated total B cells and B cell subsets, IL-10 had no significant effect on IL-4-induced IgE production; however, IL-10 increased IL-4-induced IgG4 production over 20-fold. IL-4-induced transcription of Cε and Cγ4 GLTs in isolated B cells was not affected by IL-10. Examination of IL-10 receptor (IL-10R) expression on B cell subsets showed increased frequencies of IL-10R+ memory B cells compared to naïve B cells. These results suggest that IL-10 acts indirectly through accessory cells to downregulate IL-4-induced production of IgE. IL-10 can act directly on B cells to upregulate IL-4-induced production of IgG4, likely downstream of germline transcription.
The LFA1 ligand, CD54, is not required for the differentiation of TFH cells or germinal center formation
Authors: Bowlin Marvin Q , Bradley John E , Zajac Allan J , Bullard Daniel C , Randall Troy D
Published: January 2023

In germinal centers (GC), B cells undergo cellular proliferation and affinity maturation, and ultimately differentiate into either high-affinity antibody-secreting cells (ASCs) or memory B cells. GC B cells present antigen to T follicular helper (TFH) cells in order to elicit survival and proliferation signals. The integrin, Lymphocyte Function-Associated Antigen 1 (LFA1), is required for this process, as mice lacking LFA1 or treated with LFA1 blocking antibodies fail to form GCs or TFH cells. We hypothesized that the counter-receptor for LFA1, ICAM-1 or CD54, would be similarly required for GC formation and TFH differentiation. To test this hypothesis, we infected CD54-deficient (KO) and wild-type (WT) mice with influenza virus (PR8) and enumerated ASCs and GC B cells on days 7, 9, and 14. We found that KO mice had more GC B cells than WT mice on days 9 and 14, but observed no difference in the number of ASCs or TFH cells. To determine if this result was intrinsic to the B cell lineage, we generated 50:50 (KO:WT) bone marrow chimeras. Following reconstitution, we infected the mice with PR8 and assayed donor-specific GC B cells and ASCs on days 9 and 14. On day 9, we found that GC B cells were highly enriched for KO cells, whereas the ASCs were highly enriched for WT cells. In contrast, TFH cells were represented equally in WT and KO populations. On day 14, we found that GC B cells were still highly enriched for KO cells, whereas there was no significant difference between WT and KO ASCs at this time. These results lead us to the surprising conclusion that, although the loss of LFA1 leads to the failure of TFH and GC responses, the loss of CD54 does not affect the TFH compartment and actually improves GC B cell responses.
Regulatory T cells facilitate Th17 responses to Bordetella pertussis and aid in resolution of whooping cough in mice.
Authors: Gates Hillary A , Smith Andrew A , Jakoush Tarek A , Silva Elvia , You Hana , Wu Victoria , Westfall Jessica , Segal-Abramson Tzvia
Published: January 2023

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (Bp). The prolonged nature of whooping cough has been associated with delayed Th17 responses, which are essential to resolve the infection. Regulatory T cells (Treg) are historically immunosuppressive, but have recently been demonstrated to facilitate proper immune responses during certain infections and to occasionally adapt proinflammatory phenotypes. Because Treg and Th17 cells are considered “brothers in arms,” we evaluated the role of Tregs during immune response to B. pertussis. Here, we test the hypothesis that Tregs are crucial for generating a proinflammatory response that resolves whooping cough. We show that homeostatic lung Tregs acquire a highly proliferative Th17 phenotype during Bp infection in mice and that the frequency of this Th17-like Tresg (T17reg) population raises in parallel with Th17 influx and expansion at late phases of infection. Our data suggests that the frequency of lung T17reg may be modulated by levels of IFNa and IL-2 in the lung. Depleting Foxp3+ cells throughout Bp infection resulted in decreased MHC class II expression on dendritic and endothelial cells, suggesting reduced APC maturation. Additionally, Treg knockout lead to increased lung IL-2 levels (which may diminish Th17 differentiation/accumulation) and compromised IL-17 secretion. Moreover, infected mice lacking Treg had an increased Bp load in the lungs. Our data support Tregs facilitating and assisting Th17 immune responses to Bp to help resolve the infection.
STAT2-mediated regulation of Macrophage Phenotype during Influenza and Bacterial Super-infection
Authors: Gopal Radha , Lee Benjamin , McHugh Kevin J. , Rich Helen E , Ramanan Krishnaveni , Mandalapu Sivanarayana , Clay Michelle E , Manni Michelle L , Robinson Keven M , Rangel-Moreno Javier , Alcorn John F
Published: January 2023

Influenza is a common contagious respiratory illness that affects people worldwide. Secondary bacterial infection is a major complication during influenza infection that causes significant morbidity and mortality. We have previously shown that influenza induces type I interferon (IFN)-mediated inhibition of Type17 immune responses, resulting in exacerbation of bacterial burden during influenza and Staphylococcus aureus super-infection in mice. Here, we investigated the role of the signaling mediator, STAT2, downstream of type I IFN signaling in both influenza infection and bacterial super-infection. Influenza-infected STAT2−/− mice had increased weight loss, increased viral burden, and increased inflammatory responses when compared to influenza-infected WT mice. Despite an exaggerated inflammatory response to influenza infection alone, influenza and methicillin resistant S. aureus (MRSA) super-infected STAT2−/− mice had lower bacterial burden, morbidity, and mortality than WT mice. STAT2−/− mice had an increased expression of markers associated with M1 (classical) and M2 (alternative) macrophages in lung during influenza or influenza-bacterial super infection compared to WT mice. Next, we found neutralization of IFNγ or Arginase1 decreased bacterial clearance in STAT2−/− mice in influenza-bacterial super-infection, suggesting that both M1 and M2 phenotype mediate host defense during influenza-bacterial super-infection. These data demonstrate a clear role for type I IFN induced STAT2 signaling in suppression of macrophage function during influenza, MRSA super-infection. Further, these studies reveal novel mechanistic insight into the roles of macrophage subpopulations in pulmonary host defense.
Blockade of type I interferon signaling reverses HIV-1 induced immune dysfunction and reduces HIV-1 reservoirs
Authors: Cheng Liang , Ma Jianping , Li Jingyun , Li Dan , Li Guangming , Yu Haisheng , Zhang Liguo , Su Lishan
Published: January 2023

Type I interferons (IFN-I) are critical for controlling virus infections, but they also contribute to impaired host immunity and virus persistence. Although IFN-I inhibit HIV-1 replication in vitro and in vivo, persistent IFN-I induction is correlated with immune dysfunction and disease progression in HIV-1 infected patients and in SIV infected monkeys. Moreover, despite efficient suppression of HIV-1 replication with combined antiretroviral therapy (cART), low levels of IFN-I signaling persist in some individuals, which may impede immune recovery and foster viral persistence. We report here studies using a monoclonal antibody to block IFN-α/β receptor (α-IFNAR) signaling in humanized mice (hu-mice) persistently infected with HIV-1. We found that IFNAR blockade rescued human T cells both in number and function despite elevated HIV-1 replication. We further discovered that, in HIV-1-infected mice treated with cART, blocking IFNAR fully reversed HIV-1-induced immune hyper-activation and rescued anti-HIV-1 immune responses. Finally, IFNAR blockade in the presence of cART reduced the size of HIV-1 reservoirs in lymphoid tissues and delayed HIV-1 rebound post-cART cessation. Our results suggest that blocking IFNAR may provide a novel approach to enhance immune recovery and to reduce HIV-1 reservoirs in those who sustain elevated IFN-I signaling during suppressive cART.
Ly6Chi monocytes mediate control of acute alphavirus infection by MAVS-dependent production of type I IFN
Authors: Haist Kelsey Cornell , Burrack Kristina S , Diamond Michael S , Morrison Thomas E
Published: January 2023

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-borne alphaviruses that cause epidemics of debilitating musculoskeletal disease. Monocytes have been implicated in the pathogenesis of these infections, but their specific roles are less clear. To assess the role of monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice to deplete CCR2+ cells by administering diphtheria toxin (DT). DT-treated CCR2-DTR+ mice showed reduced weight gain and more severe disease following infection and had fewer Ly6Chi monocytes and NK cells in circulation compared to DT-treated WT mice. Also, depletion of CCR2+ cells, but not NK cells alone, restored virulence and increased viral loads in the muscle tissue of mice infected with RRV encoding attenuating mutations in nsP1 to levels seen in monocyte-depleted mice infected with fully virulent RRV. A similar increase in disease severity and viral loads was observed in DT-treated CCR2-DTR+;Rag1−/− mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. In vitro, culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of IFNα gene expression in monocytes that was MAVS-dependent. IFNα expression in monocytes also required new virus particle production from infected cells. Furthermore, monocytes sorted from the circulation and muscle of infected mice had elevated expression of Irf7. Finally, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in the muscle tissue of MAVS−/− mice. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1 counteract this response.
Missense SNP in fcer2 (CD23) results in increased transcription factor activity in human B cells
Authors: Chan Marcia , Gigliotti Nicole , Rezaiekhaligh Mo
Published: January 2023

Asthma is one of the most common chronic conditions in children and more than 6 million children in the US are affected. Furthermore, asthma is the third ranking cause of hospitalizations among children younger than 15. Genetics appears to play a role in both predisposition and treatment resistance of asthma. CD23 is expressed on several cell types and has several roles in IgE-mediated immunity, including antigen focusing and presentation to Th2 cells; mediating production of pro-inflammatory cytokines by monocytes; transporting IgE and allergen-IgE complexes through gastrointestinal epithelium; and participating in both positive- and negative-feedback mechanisms in regulation of IgE synthesis by B cells. Single nucleotide polymorphisms (SNPs) in fcɛr2 encoding the low affinity IgE receptor CD23 (FcɛRII) are associated with increased severe asthma exacerbation and increased serum IgE levels. One SNP (C3750T, rs2228137) encodes a nonsynonymous amino acid change (R62W). Stable human B cell transfectants were established expressing either wild type CD23b-R62 or CD23b-R62W. Stimulation through CD23 caused differences in activation of proteins phosphorylated on tyrosine as well as kinetic differences in transcription factor activity. Peripheral blood was obtained from asthmatic patients genotyped for rs2228137. A comprehensive transcriptome analysis of B cells stimulated through CD23 was performed. Nineteen genes were differentially expressed between subjects homozygous for the wild type allele (cc) and those homozygous for the SNP allele (tt). These data suggest that the rs2228137 SNP may influence asthma severity through prolonged activation of B cells favoring increased IgE production.
The complex allergen house dust mite (HDM) acts directly on macrophages to stimulate actin remodeling
Authors: Keegan Achsah Dorsey , Rennoll Sherri A. , Qi Xiulan , Pedra Joao , Vogel Stefanie N , Gao Hongjuan
Published: January 2023

Asthma is a chronic inflammatory disease of the lungs that results in narrowing of the airways, making it difficult to breathe. Dust is a common trigger of allergy and asthma. House dust mites (HDM) are the main cause of dust allergies. Despite the fact that macrophages are present in abundant amounts within the lung and are one of the first cells exposed to HDM upon inhalation, little is known about the direct effects of HDM on macrophages. Here we demonstrate that HDM can directly act on bone marrow-derived macrophages (BMM) leading to actin remodeling. BMM treated with HDM demonstrated alterations in F-actin (based on Phalloidan staining), changes in shape, and increases in size significantly. We also observed alterations in the phosphorylation of cofilin and slingshot, two proteins that regulate actin polymerization. BMM demonstrated constitutive phosphorylation of cofilin with little phosphorylated slingshot at baseline. However, HDM treatment diminished cofilin phosphorylation while increasing the phosphorylation of slingshot. Since HDM is known to contain LPS, important for HDM-induced allergic inflammation in vivo, we examined the contribution of LPS to actin remodeling. Actin cytoskeleton changes were still evident in BMM treated with HDM in the presence of polymyxin B (PMB) or in Tlr4−/− BMM, although the average cell size was reduced compared to HDM alone. The HDM-induced changes in phosphorylation of cofilin and slingshot were unaffected by PMB or Tlr4 deficiency. These results suggest that HDM induces actin remodeling in macrophages by a mechanism that is largely LPS/TLR4-independent. These observations provide an opportunity to identify novel therapeutic targets for allergic asthma.
IL-33 elicits IL-5 dependent eosinophilia in vivo
Authors: Johansson Kristina Britt Charlotte , Malmhäll Carina , Ramos-Ramírez Patricia , Rådinger Madeleine
Published: January 2023

Introduction

Interleukin (IL)-33 plays important roles in the immunopathogenesis of allergy and asthma. It acts by promoting IL-5 and IL-13 secretion from IL-33 responsive type 2 innate lymphoid cells (ILC2s) or directly activates eosinophils and mast cells. IL-5 is crucial in eosinophilic inflammation by controlling differentiation and survival of eosinophils in the bone marrow as well as release and recruitment of eosinophils to the airways. While ILC2s have emerged as important producers of IL-5 in airways, the cellular source of IL-5 in the bone marrow is less explored. In this study we tested the hypothesis that IL-33 induced airway eosinophilia is an IL-5 dependent process in which ILC2s produce IL-5 locally in the bone marrow.

Methods

IL-5 production by bone marrow ILC2s was analyzed by intracellular flow cytometry in a murine model of IL-33 induced airway eosinophilia.

Results

Intranasal IL-33 administration resulted in eosinophil infiltration in airways and increased eosinophils in bone marrow. It was accompanied by a dramatic induction of eotaxin-2 in airways, indicating eosinophil recruitment to the tissue. IL-5+ ILC2s as well as expression of the IL-33 receptor on ILC2s increased in the bone marrow in response to IL-33 administration. Importantly, airway and bone marrow eosinophils were greatly reduced in mice pre-treated with anti-IL-5 antibodies followed by intranasal IL-33.

Conclusion

Our results demonstrate that IL-33 elicits IL-5 dependent eosinophilia in vivo, where ILC2s represent a local source of IL-5 in the bone marrow.
mTORC1 in B cells regulates antibody responses and promotes mitochondrial and metabolic fitness
Authors: McLetchie Shawna , Raybuck Ariel , Cho Sung Hoon , Lin Jingxin , Boothby Mark Robin
Published: January 2023

B lymphocytes migrate among different micro-anatomic sites for diversification, selection, and eventual differentiation into antibody-secreting plasma cells. Emerging evidence supports the premise that aspects of the nutrient milieu vary within lymphoid micro-environments. However, the role of B cell-intrinsic metabolic programs in regulating B cell differentiation and antibody response quality remain unclear. We now show that the amino acid-sensing mTOR complex 1 (mTORC1) is essential for induction of Bcl6 and IRF4, key transcriptional regulators of germinal center and plasma cell fates. mTORC1 also enhances B cell proliferation upon exposure to antigen, increases the rate of somatic hyper-mutation, and is essential for generating high-affinity class switched antibodies. We also find that AMP-activated kinase (AMPK), an intracellular energy sensor, promotes plasma cell differentiation and antibody production. Collectively, these findings suggest mechanisms by which mTORC1 activity is critical for the germinal center reaction and producing class-switched, high-affinity antibodies, and indicate that AMPK can regulate the development and functional properties of terminally differentiated plasma cells.

Supported by NIH R01 AI113292, HL106812, and NCI T32CA009592-29
HLA-F and receptor function on B cells and monocytes in the inflammatory response.
Authors: Geraghty Daniel , Lee Ni , Wang Kevin L , Ishitani Akiko
Published: January 2023

HLA-F is expressed as an open conformer (OC) independent of bound peptide or β2-microglobulin upon surface upregulation on activated immune cells, binding and stabilizing MHC class I (MHCI) in the open conformer form. Functional roles for HLA-F and MHCI as open conformers include HLA-F and MHCI OC as ligands for KIR3DL2 and activating KIRs, and HLA-F as a cofactor with MHCI in a novel antigen acquisition pathway operating in both HLA class I and II antigen presentation. In addition, our work recently detected a unique conformational form of HLA-F expressed in resting B cells, with HLA-F specific mAb 6A4, distinguishing CD27− and CD27+ B cells. Expression of HLA-F on CD14+ monocytes as detected by 4B4 revealed a second unique conformational form. In addition to the ligand and cofactor roles, a third role for HLA-F as a receptor on B cells and monocytes was established. When purified B cells were subjected to specific mAb treatment, upregulation and secretion of IgA and IgM was observed. A receptor function was also detected upon treatment of purified monocytes with specific mAb, where cytokine and chemokine release marking the M1 and M2 monocyte differentiation pathways was observed. Treatment with mAb also resulted in monycyte differentiation into a macrophage and dendritic cell phenotype. When intracellular glycosylation of HLA-F was examined biochemically, the distinct alternate forms detected by anti-HLA-F mAbs 3D11, 6A4 and 4B4 appeared to mark HLA-F transit in different intracellular pathways that were potentially consistent with the different functional roles for HLA-F. Our results indicate HLA-F may play a heretofore unrecognized but highly central role in related inflammatory immune functions and dysfunctions.
AID phosphorylation is required for base excision repair during class switch recombination
Authors: Matthews Allysia J , Choi Jee Eun , Vuong Bao Q
Published: January 2023

Activation-induced cytidine deaminase (AID) generates dU:dG mismatches in the immunoglobulin heavy chain locus during class switch recombination (CSR). The resolution of these mismatches occurs primarily through the base excision repair (BER) pathway and additionally through the mismatch repair (MMR) pathway. The disruption of either pathway results in a reduction in class switch recombination, whereas ablation of both pathways completely blocks CSR. The phosphorylation of AID at Ser38 (pS38-AID) is required for wild-type levels of CSR, in part through its interaction with APE1, an essential component of the BER pathway. B cells carrying a homozygous knock-in mutation of the AID phosphorylation site (AIDS38A/S38A) have intermediate levels of CSR as compared to wild-type and AID−/− B cells. CSR in AIDS38A/S38AUNG−/− and AIDS38A/S38AMSH2−/− B cells is completely blocked. This data parallels the CSR defect observed in UNG−/−MSH2−/− B cells as compared to UNG−/− or MSH2−/− B cells and genetically confirms the critical role of pS38-AID in the BER repair pathway during CSR.
Inhibition of H3K27me3-specific demethylases promotes plasma cell formation
Authors: Kania Anna , Scharer Christopher , Boss Jeremy M
Published: January 2023

B cell terminal differentiation must be properly regulated to ensure robust humoral immune responses. In additional to the well-established role of key transcription factors, there is a growing interest in the epigenetic regulation of B cell differentiation. However, the epigenetic remodeling that occurs to suppress the B cell and promote the plasma cell fate is still poorly understood. Histone H3 lysine 27 trimethylation (H3K27me3) is a modification associated with a repressive chromatin state and gene silencing. H3K27me3 is established by EZH2 (component of PRC2) and was shown to be essential in the formation and maintenance of germinal centers. In addition, other studies have shown that active removal of silencing marks by H3K27me3-specific demethylases is necessary for T-cell differentiation. To further evaluate the role of H3K27me3 in plasma cell differentiation, we evaluated the effects of pharmacological inhibition of UTX and JMJD3 (H3K27me3-specific demethylases) in B cells stimulated ex vivo with LPS, IL2, and IL5 in the presence of the inhibitor or vehicle control. Our preliminary data suggest that retention of those silencing histone modifications nearly doubles the number of B220+ CD138+ plasma cells after 3-day ex vivo culture as assayed by flow cytometry. We also evaluated viability, gene expression profile, and functionality of the plasma cells treated with or without the inhibitor. Taken together, the collected data provide us with more insight into the role of H3K27me3 dynamics in B cell terminal differentiation.
Expression of immune checkpoint molecules on tumor infiltrating lymphocytes in glioblastoma multiforme
Authors: Park Junsik , Kwon Minsuk , Kim Kyung Hwan , Chang Jong He , Shin Eui-Cheol
Published: January 2023

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and is manifested with poor prognosis. GBM has long been recognized as an immunosuppressive neoplasm characterized by activation of various immune escape mechanisms. Recently, immune checkpoint inhibitors have shown clinically relevant improvements with high response rates and durable tumor remissions in several cancer types, and anti-PD-1, PD-L1 and CTLA-4 blockade are currently tested in clinical trials for GBM. However, the expression and principal role of the checkpoint molecules in GBM have not been well studied. The present study assessed the expression of immune checkpoint molecules in patients with GBM by flow cytometry. Overall levels of PD-1, CTLA-4, Tim-3 and LAG-3 on CD8+ T cells were significantly higher in tumor infiltrating lymphocytes (TILs) compared to those detected on the T cells in peripheral blood mononuclear cells (PBMCs). In the case of CD4+ TILs, PD-1, CTLA-4, Tim-3, LAG-3 and TIGIT were significantly increased. Furthermore, co-expression of the checkpoint molecules was prevalently found in TILs. PD-1+CTLA-4+, PD-1+Tim-3+, and PD-1+LAG-3+ CD8+ T cells and PD-1+CTLA-4+, PD-1+CTLA-4+Tim-3+, and PD-1+CTLA-4+TIGIT+ CD4+T cells were relatively enriched in TILs compared to PBMCs. However, in detail, checkpoint molecules in each individual patient showed various expression patterns. Collectively, our results indicate that GBM induces expression of multiple immune checkpoint molecules on TILs. Further studies are needed to determine whether immune checkpoint blockers can functionally restore these exhausted T cells and which combination of the blockers can optimally restore T cell function in GBM.
Depletion of Treg cells induces an increased activation of T-CD4+ and T-CD8+ cells during colorectal cancer
Authors: Terrazas Luis I , Olguín Efraín J , Medina-Andrade Itzel , Ledesma-Soto Yadira , Saavedra Rafael , Molina Emmanuel , Rodríguez-Sosa Miriam
Published: January 2023

Colorectal Cancer (CAC) is the second most commonly diagnosed cancer in women and the third in men in North America and Europe. CAC is associated with inflammatory responses where intestinal pathology is caused by a T-cell dysregulation that concludes in an imbalance between T activated (Tact) and T regulatory (Treg) cells. Treg cells are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, contributing to the maintenance of immune homeostasis. It has been suggested a tumor-promoting function for Treg cells in CAC, but the kinetics of Treg cells during CAC development is poorly known. Thereby we used a mouse model of CAC induced by azoxymethane and three cycles of dextran sodium sulfate (DSS, 2%). We analyzed the percentage of Treg cells in blood, spleen and mesenteric lymph nodes (MLN) in each cycle. A significant reduction of Treg cells in blood and spleen during the first cycle, but increased percentage of Treg cells during the second and third cycle was observed. We did not observe reduction on Treg cells in the first DSS cycle in MLN, but a significant increase in percentage was detected during the second and third cycle of DSS. A Higher percentage of Treg cells-expressing PD1, CD127 and Tim-3 during the second and third DSS cycle in spleen and MLN was observed, whereas CD25 had no changes. However, we found increasing percentages of CD4+CD25+Foxp3− Tact cells only in the first DSS cycle in blood, spleen, and MLN. These results suggest that a gradually increased number of Treg cells suppress Tact cells during the second and third DSS cycle. Depletion of Treg cells with anti-CD25 PC61 antibody in the second DSS cycle increased percentages of CD4+CD25+Foxp3− Tact and TCD8+ cells, with an impact in tumorigenesis.